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In vivo and mechanical evaluation of Calcium Polyphosphate for bone regeneration in revision total hip… Comeau, Patricia Ann 2009

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In vivo and Mechanical Evaluation of Calcium Polyphosphate for Bone Regeneration in Revision Total Hip Replacements by PATRICIA ANN COMEAU B.Sc., University of Alberta, 2007 (Chemical and Materials Engineering)  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF APPLIED SCIENCE  in THE FACULTY OF GRADUATE STUDIES (Metals and Materials Engineering)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) October 2009 ©Patricia Ann Comeau, 2009  Abstract Revision total hip replacements (THRs) commonly involve the cementing of a femoral component into a compressed bone graft such as morsellized cancellous allograft in order to encourage restoration of the living bone stock that has been lost in a primary THR. However, current problems associated with allograft include risk of disease transmission, limited availability, reproducibility, and cost. To address these issues synthetic scaffolds have been proposed as a substitute material due to their availability and ease of preparation and sterilization. Calcium polyphosphate (CPP) is one novel synthetic scaffold that has shown good mechanical properties and biocompatibility, and is evaluated biologically and mechanically in this study.  The in vivo study observed how marrow stromal cell (MSC) proliferation and differentiation was affected by the substrate material. Here CPP, morsellized cancellous bone (MB), and hydroxyapatite/tricalcium phosphate (HA/TCP) were seeded with MSCs and implanted subcutaneously under the back skin of NOD/Scid mice. At 0, 7, 14, and 28 days the samples were harvested and the proliferation characteristics and gene expression were analyzed. CPP, HA/TCP and MB were all shown to have similar proliferation characteristics and gene expression.  In the mechanical study CPP was combined with PLA to form particulate composites and with PMMA to form solid composites. These composites were then tested in un-compacted and pre-compacted states in confined compression. The results show that the solid composites had the greatest improvement over MB in terms of construct stiffness, with the particulate composites having the next greatest improvement when pre-compacted.  ii  The study confirmed that CPP has potential as a synthetic scaffold for replacing MB in the revision THR procedure.  iii  Table of Contents Abstract............................................................................................................................... ii Table of Contents................................................................................................................iv List of Tables ......................................................................................................................vi List of Figures................................................................................................................... vii List of Abbreviations ....................................................................................................... viii Acknowledgements.............................................................................................................ix 1.0 General Introduction ................................................................................................1 1.1 Total Hip Replacements ......................................................................................1 1.1.1 Primary Total Hip Replacements.................................................................1 1.1.2 Revision Total Hip Replacements ...............................................................3 1.2 Basic Bone Biology & Regeneration...................................................................7 1.2.1 Bone Mechanics...........................................................................................7 1.2.2 Bone Formation ...........................................................................................9 1.2.3 Graft Incorporation ......................................................................................9 1.2.4 Factors Affecting Bone Remodeling .........................................................11 1.2.5 Current Bone Regeneration Strategies.......................................................12 1.3 Scaffold Requirements.......................................................................................13 1.3.1 Ideal Scaffold.............................................................................................13 1.3.2 Advantages and Limitations of Current Materials.....................................16 1.4 Thesis Objectives & Scope................................................................................19 1.5 References..........................................................................................................21 2.0  In vivo Evaluation of MSC Proliferation and Differentiation on Synthetic Scaffold Material..................................................................................................................27 2.1 Introduction........................................................................................................27 2.1.1 MSC Biology .............................................................................................27 2.1.2 MSC Proliferation......................................................................................29 2.1.3 MSC Differentiation into Bone .................................................................29 2.1.4 MSC Culturing, Seeding and Identification ..............................................31 2.1.5 In vitro and In vivo Study of MSCs ...........................................................32 2.1.6 Clinical Application...................................................................................33 2.2 Materials & Methods .........................................................................................35 2.2.1 Scaffold Materials......................................................................................35 2.2.2 Cell Isolation, Culturing, Seeding, and Implantation ................................36 2.2.3 Sample Harvesting.....................................................................................39 2.2.4 Cellular Proliferation .................................................................................40 2.2.5 Cellular Differentiation..............................................................................41 2.2.6 Statistics.....................................................................................................43 2.2.7 Analytical Flowsheet .................................................................................43 2.3 Results................................................................................................................45 2.3.1 Seeding ......................................................................................................45 2.3.2 Cellular Proliferation .................................................................................46 2.3.3 Cell Isolation Post-Harvest ........................................................................47 2.3.4 mRNA Isolation and Reverse Transcription..............................................49 2.3.5 Cellular Differentiation..............................................................................51 iv  2.4 Discussion..........................................................................................................54 2.4.1 Seeding ......................................................................................................55 2.4.2 Cellular Proliferation .................................................................................55 2.4.3 Cell Isolation Post-Harvest ........................................................................56 2.4.4 mRNA Isolation and Reverse Transcription..............................................57 2.4.5 Cellular Differentiation..............................................................................57 2.5 Conclusion .........................................................................................................60 2.6 References..........................................................................................................61 3.0 Mechanical Evaluation of Synthetic Scaffolds for Use with Revision THR.........63 3.1 Introduction........................................................................................................63 3.1.1 Material Selection ......................................................................................64 3.1.1.1 Impaction Allografting Materials ..............................................................64 3.1.1.2 Composite Graft Bed .................................................................................66 3.1.2 Testing Technique .....................................................................................67 3.2 Materials & Methods .........................................................................................69 3.2.1 Materials ....................................................................................................69 3.2.2 Methods .....................................................................................................73 3.2.3 Statistics.....................................................................................................75 3.3 Results................................................................................................................75 3.3.1 Particulate Composites ..............................................................................79 3.3.2 Solid Composites .......................................................................................81 3.3.3 Impact of Sample Conditioning.................................................................82 3.4 Discussion..........................................................................................................85 3.4.1 Particulate Composites ..............................................................................85 3.4.2 Solid Composites .......................................................................................86 3.4.3 Impact of Sample Conditioning.................................................................88 3.5 Conclusion .........................................................................................................89 3.6 References..........................................................................................................90 4.0 Thesis Conclusion..................................................................................................93 4.1 Summary and Future Work ...............................................................................93 4.2 Reference ...........................................................................................................96 APPENDIX I - In vivo Study Steps ...................................................................................97 APPENDIX II – Further Supporting Evidence for the In vivo Analysis .........................103 APPENDIX III – Stress versus Strain Curves .................................................................107 APPENDIX IV – UBC Animal Care Certificate.............................................................109  v  List of Tables Table 1: Mechanical Characteristics of Bone......................................................................8 Table 2: Properties for the Ideal Synthetic Scaffold Material ...........................................14 Table 3: Advantages and Limitations of Different Scaffold Materials .............................17 Table 4: Statistical Significance Chart for In vivo study. ..................................................54 Table 5: Properties of Materials Used in Testing ..............................................................71 Table 6: Mechanical Test Matrix.......................................................................................74 Table 7: Results Obtained with Confined Compression....................................................78 Table 8: Volume Fraction in Un-compacted Particulate Composites of CPP and PLA. ..79 Table 9: Statistical Significance Chart for the Mechanical Study. ....................................84 Table 10: Statistical Significance Chart for Sample Conditioning....................................85  vi  List of Figures Figure 1: Total Hip Replacement Schematic.......................................................................3 Figure 2: Graft Incorporation.............................................................................................10 Figure 3: Sequence of Cell Differentiation Along the Osteogenic Lineage. .....................30 Figure 4: (a) Morsellized Bone Chips, (b) Calcium Polyphosphate Particles, and (c) Biphasic Hydroxyapatite/Tricalcium Phosphate Particles ...........................................36 Figure 5: (a) Mice Following Euthanization, and (b) A Mouse Following Hair Removal Prior to Sample Extraction.................................................................................................40 Figure 6: Flowsheet Representing the Steps of the In vivo Implantation of the Scaffolds. ...........................................................................................................................44 Figure 7: Method Flowsheet Showing How BrdU and qRT-PCR Analysis was Completed..........................................................................................................................45 Figure 8: (a) Seeding Efficiency, and (b) Final Successful Seeding Count on the Scaffold Material. ..............................................................................................................46 Figure 9: BrdU Incorporation into the GFP+ Cells During In vivo Implantation. ............47 Figure 10: (a) Collagenase/Dispase GFP+ Cell Count, and (b) Magnetic Sorting GFP+ Cell Count..........................................................................................................................48 Figure 11: GFP+ Cell Recoveries as a Fraction of the Initial Cell Count for the Collagenase/Dispase Reaction and the Magnetic Sorting Technique. ..............................49 Figure 12: (a) mRNA, and (b) cDNA Measured at Each In vivo Endpoint.......................50 Figure 13: Expression of Osteogenic Genes on the Different Substrates Over a Period of 28 Days Relative to GAPDH.........................................................................................52 Figure 14: ALP Expressions Relative to GAPDH for Each Mouse ..................................53 Figure 15: PLA Particles (Left), CPP Particles (Right).....................................................71 Figure 16: 100% PMMA Solid Constructs........................................................................71 Figure 17: Images of the CPP:PMMA (25:75) Solid Composites Sectioned....................72 Figure 18: Images of the CPP:PMMA (50:50) Solid Composites Sectioned....................72 Figure 19: SEM Images at 60x Magnification of PLA (Left), HA/TCP (Middle), and CPP (Right) Particles.........................................................................................................73 Figure 20: SEM Images at 80x Magnification of PMMA (Left), 25:75 CPP:PMMA (Middle), and 50:50 CPP:PMMA (Right) Samples...........................................................73 Figure 21: Experimental Setup for Confined Compression. .............................................74 Figure 22: Stress-strain Curve for a Sample of Un-compacted 100% PLA Construct. ....76 Figure 23: Stress-strain Curve for a Sample of Pre-compacted 100% PLA Construct. ....76 Figure 24: Ec of All Composites Tested. ...........................................................................79 Figure 25: Ec Results for (a) Un-compacted Particulate Composites, and (b) Pre-compacted Particulate Composites........................................................................80 Figure 26: Ec Results for Solid Composites. .....................................................................81  vii  List of Abbreviations ALP……………………… BrdU…………………….. BSP……………………… C/D……………………… cDNA…………………… CJRR……………………. CM……………………… Col-I…………………….. CP.……………………… CPP …………………….. DDW ………………….. FACS...………………….. FBS……………………… FDA……………………... GAPDH…………………. GFP……………………… HA ……………………… HBMSC…………………. HCA…………………….. IA ………………………. ISO……………………… MB ……………………… mRNA…………………… MS………………………. MSCs ……………………. NOD/Scid……………….. OC……………………….. OP……………………….. PBS……………………… PLA……………………... PMMA…………………... PMTs……………………. qRT-PCR ……………….. SEM……………………... TCP……………………… TE……………………….. THR……………………... WBC……………………..  Alkaline Phosphatase 5-bromo-2-deoxyuridine Bone Sialoprotein Collagenase/Dispase complimentary Deoxyribonucleic acid Canadian Joint Replacement Registry Culture Medium Collagen I Calcium Phosphate Calcium Polyphosphate Distilled Deionized Water Fluorescence Activated Cell Sorting Fetal Bovine Serum Food & Drug Administration (US) Glyceraldehyde-3-phosphate dehydrogenase Green Fluorescent Protein Hydroxyapatite Human Bone Marrow Stromal Cells Hydroxycarbonate apatite Impaction Allografting International Organization for Standardization Morsellized Cancellous Bone messenger Ribonucleic acid Magnetic Sorting Marrow Stromal Cells Non-Obese Diabetic/Severe combined immunodeficiency Osteocalcin Osteopontin Phosphate Buffer Solution Poly-(L-lactic acid) Poly-(methyl methacrylate) Photo Multiplier Tubes quantitative Reverse Transcription-Polymerase Chain Reaction Scanning Electron Microscopy Tricalcium phosphate Tissue Engineering Total Hip Replacement White Blood Cell  viii  Acknowledgements I would like to thank Dr. Goran Fernlund for his guidance during my M.A.Sc. research. My research project provided a great opportunity to extend my knowledge and improve my technical skills. To Chiming Yang, Dr. Fabio Rossi and members of the Rossi lab group at the Biomedical Research Center (UBC) I am grateful for their support during my in vivo study. Special thanks to Dr. Hanspeter Frei for providing the initial inspiration for my project. For their assistance with my mechanical study I would also like to thank Dr. Chad Sinclair, Roger Bennet, and David Marechal. They were each instrumental in setting up my confined compression testing apparatus. Mary Fletcher was also of great help with Scanning Electron Microscope imaging. To my family and friends I am grateful for your continuing love and support. I could not have taken this journey without either. I gratefully acknowledge the Nadeau family and NSERC for their financial support.  ix  1.0  General Introduction  The number of total hip replacements (THRs) has been steadily increasing since the early 1990’s (Peppas et al., 2005; Latham et al., 2004). Currently, the average age for a primary THR is around 65 (Speirs et al., 2005; CJRR, 2009). The life expectancy of a 65-year old patient is approximately 17.9 years and, as the longevity of an implant is currently 12-15 years, it is not surprising that an individual will require at least one revision surgery (Peppas et al., 2005; Latham et al., 2004; Zhang et al., 2009; Medline Plus Website).  The hip is a sturdy synovial ball and socket joint that is capable of various movements (Martini, 2006). Cartilage covers the articulating surfaces and ligaments that hold the joint together and cushions the ends of the bones to enable them to move more easily, while the surrounding synovial membrane also makes a small amount of fluid that lubricates the joint and eliminates friction at the hip joint (Medline Plus Website; Fernlund, 2007). Damage to this cartilage can lead to end-stage arthritic conditions that rob patients of their quality of life, with chronic hip pain and disability, and create a need for THR (Sinha, 2002). THR potentially improves an individual’s quality of life in a highly cost-effective medical intervention (Mahomed et al., 2003; Rasanen et al., 2007).  1.1  Total Hip Replacements  1.1.1  Primary Total Hip Replacements  Since the first recorded THR took place in 1938 in Germany, there have been numerous implant designs consisting of various materials, shapes, sizes, and forms that have been introduced into the orthopaedic prosthesis market (Latham et al., 2004, Sinha, 2002). The majority of these THRs are based on Sir John Charnley’s low-friction THR design from the 1960’s (Donald, 2007; Hench, L., 2000; Yamada et al., 2009). In designing a durable and 1  efficient hip implant, several factors such as the patient’s age and mobility, bone aging, material and biological parameters, and implantation techniques need to be considered (Latham et al., 2004).  A femoral component of a hip implant consists of three main regions: head, neck and stem. Figure 1 shows a schematic for the design of a total hip replacement implant. There are two distinct stem design paths for THR fixation: cemented and cementless stems (Sinha, 2002). The current design for a cementless stem commonly consists of a porous coating or textured surface over a portion of the stem to allow for bone ingrowth, while that of the cemented stem does not have this coating and involves the use of special biocompatible cement for immediate stability of the prosthesis.  The surgical procedure takes a few hours and starts with the surgeon first removing the damaged cartilage and bone by reaming the acetabulum, cutting off the head and neck of the femur, and reaming the inside of the femoral cavity (Medline Plus Website; Bozic et al., 2004). Next, the acetabular component and the femoral stem are inserted into their respective cavities, the artificial femoral head is attached, the stem is impacted, and the whole artificial joint is connected back together (Medline Plus Website; Bozic et al., 2004). For a cemented stem, the cement is mixed and pressurized in the femoral canal before the femoral stem is inserted. All other steps remain the same between the two stem designs.  2  Typical Osteolysis Sites  Figure 1: Total Hip Replacement Schematic (Adapted from MIT OCW, 2007)  1.1.2  Revision Total Hip Replacements  Due in part to an increasing demand for THRs in younger and more active patients, an increase in the number of surgeons willing to perform such surgeries, the higher demand these individuals will place on their replacements, and the limited longevity of the current THR design, the need for revision THR has dramatically increased (Toms et al., 2004; Bolland et al., 2007; Bolland et al., 2007-2; Hamadouche et al., 2000).  Normal bone maintenance depends on the balance of bone formation and resorption, and there are several mechanisms by which bone loss after joint replacement can occur. Five of these mechanisms deal with ageing, adaptive bone remodelling or stress shielding, mechanical factors such as implant migration, fluid pressure, and particulate debris (Dattani et al., 2007). Genetic disorders, tumors and sudden trauma may also lead to a loss in the surrounding bone stock (Babis et al., 2005). As host bone is lost, the implant loosens and fails (Toms et al., 2004; Bolder et al., 2003; Nickelsen et al., 2008). Aseptic loosening due to focal osteolysis is often asymptomatic and not detected until it compromises component 3  stability and leaves massive bone defects which limits implant survival (Leopold et al., 2000; Wilkinson et al., 2003; Duisabeau et al., 2004; Dattani et al., 2007; Jones et al., 2003). With further removal of bone possible as the failing implant is removed, replacement of this bone stock has become important, particularly in younger patients. Figure 1 shows the typical location of focal osteolysis around a primary implant.  The main biomechanical aims of a revision THR are to achieve immediate fixation and longterm stability, as well as to stop deterioration of bone and restore the immediate bone stock (Nickelsen et al., 2008). However, the loss of bone can reduce the areas into which new prosthesis can be attached as the bone is not strong enough to support loads. In addition, after the removal of the failed primary implant a fibrocellular membrane and a fairly smooth endosteal surface remains causing the micro-interlock between bone and cement to be poor and early loosening of the revision to occur (Brewster et al., 1999). Various techniques are available for revision after bone loss, such as a porous-coated uncemented implant or a cemented long-stem femoral component, but only two are used with the aim of reconstituting the lost bone: impaction allografting (IA) and the use of structural allograft (Toms et al., 2004; Babis et al., 2005; Lewis et al., 2002; Piccaluga et al., 2002; Brewster et al., 1999). Of the two solutions, impaction grafting with particulates is of greatest interest in our studies. IA provides bone support and stable fixation for the implant after removal of the primary THR and allows for subsequent bone ingrowth and remodeling of the allograft as the living bone stock is restored (Bolland et al., 2007).  IA was originally described by Sloof and colleagues in Nijmegen, The Netherlands in the late 1970s for use in the acetabulum and was later adapted for use in the femur by Ling, Gie and colleagues in Exeter, UK in 1987 (Babis et al., 2005; Bavadekar et al., 2001; Board et al., 2006; Bolland et al., 2007; Bolland et al., 2006; Nelissen et al., 1995). It involves the 4  cementing of a femoral component into compressed bone graft, such as morsellized allograft, in order to create a four-layer composite of implant, cement, allograft bone, and host bone. Ideally, bone remodeling will proceed from the live host bone into the bone graft and replace the impacted graft with a new continuous cancellous lattice over time (Heiner et al., 2005).  Impaction bone grafting requires that morsellized allograft be adequately compacted in the femoral cavity to provide initial stability for the prosthesis. With adequate compaction, early massive subsidence can be prevented and bone remodelling induced (Bolland et al., 2007). Impaction grafting has gained popularity as it is a biological solution that can restore the patients bone stock through a creeping substitution of graft with host bone, similar to the bone remodelling process (Speirs et al., 2005; Halliday et al., 2003; Frei et al., 2004; Stevens et al., 2007). Long-term outcome analysis has shown a wide variation in results with some studies reporting 100% survival at 10.4years, while others report a 28% survival at 15.3years (Board et al., 2006; Dunlop et al., 2003; Halliday et al., 2003; Ornstein et al., 2001; Piccaluga et al., 2002; Fosse et al., 2006). As many as 30-60% of allograft implants, in some reports, have encountered some form of complication that could lead to failure within ten years (Khan et al., 2008). Many factors are responsible for the variability in success and may include the cause of primary THR failure, the degree of osteolysis, implant choice, surgical technique, and quality of bone graft. It also appears that where the study of success rate takes place also has some bearing as results from the center of origin have been excellent, while those from other centers have not been as positive (Bolland et al., 2007). Problems such as intraoperative fracture (11-18%), early massive subsidence (11-21%), cement mantle fracture (12%) and late catastrophic stem breakage have been encountered (Knight et al., 2000; Albert et al., 2008). Additional issues such as costly operations, higher complication rate, longer operative time, potential for significant blood loss and an inferior outcome to primary THR 5  have also been associated with IA (Bolland et al., 2007; Bolland et al., 2007-2). In considering intraoperative fracture, which is fracture that occurs to the host bone during the operation, it is important to acknowledge that both the fragile osteolytic femoral bone for which IA is indicated and technical problems associated with the procedure itself can contribute (Knight et al., 2000; Meding et al., 1997). However, though fractures from excessive graft impaction lead to additional operating room time, with follow-up they appear to have no negative impact on the final outcome.  Meanwhile, the morsellized allograft itself is associated with some issues including limited availability and a risk of disease transmission (Sugihara et al., 1999; Goodman, 2004). Bone banking is a complex and regulated process with several documentation steps that can be very costly and take considerable time (Draenert et al., 2007; Hing, 2005). The many disinfection and sterilization techniques have several unsolved or inevitable problems such as low mechanical stability and limited osseointegration. Such reasons as given previously are why there is such widespread interest in finding an appropriate synthetic biomaterial bone scaffold to replace morsellized bone in impaction grafting. Since 1991 there has been a 100% increase in revision THR due to implant failure, with a further increase expected over the next 30 years or so (Bolland et al., 2007). In 2006-2007 86.4% of the THRs reported to the Canadian Joint Replacement Registry (CJRR) were primary, while 8.9% were first revision and ~3% were of second or greater revision (CJRR, 2009). As more and more young patients require primary and subsequent revision THRs, restoring rather than replacing bone stock will be the chosen implant path (Bolland et al., 2007; Hing, 2004). Currently, more than two million bone graft procedures are done world-wide each year with roughly 90% of these using natural bone (i.e. autograft or allograft) and only 10% with synthetic materials (Will et al., 2008). 6  1.2  Basic Bone Biology & Regeneration  1.2.1  Bone Mechanics  The skeleton is designed to protect vital body organs and to provide a frame for the movement of the musculoskeletal system (Mistry et al., 2005; Hing, 2004; Hing, 2005). It is also responsible for maintaining calcium homeostasis and providing a source of haematopoietic stem cells (Hing, 2004; Rogel et al., 2008; Hing, 2005). Bone consists of a dense matrix that contains collagen, deposits of calcium salts and bone cells and has narrow passageways through it that allow for the exchange of nutrients, waste products, and gases (Mistry et al., 2005).  Calcium phosphate (CP; Ca3(PO4)2) is an inorganic bone component that accounts for approximately two-thirds of bone weight, collagen fibers for most of the remaining third, and cells account for approximately 2% (Martini, 2006; Hutmacher et al., 2007). However, the structure and proportion of these components vary with age, site and history, such that bone from different sources can exhibit markedly different mechanical and functional characteristics (Hing, 2004). Inorganic CP interacts with calcium-hydroxide to form hydroxyapatite (HA; Ca10(PO4)6(OH)2) crystals which are very hard, inflexible, and brittle with dimensions of 20-80nm in length and 2-5nm thick typical, and are themselves considered a class of CP (Martini, 2006; Zhang et al., 2009). These HA crystals are able to withstand compression, but can shatter when bending, twisting, or a sudden impact occurs. By contrast, collagen fibers are composed of water and intertwined proteins arranged in a fibrillar structure such that they are strong, flexible, and tough (Martini, 2006; Rogel et al., 2008). These collagen fibers can readily withstand twisting, bending, and tension but offer little resistance in compression.  7  The wall of the femur’s diaphysis that surrounds the marrow cavity, consists of hard and dense compact bone while the epiphysis at the ends of the femur consists of spongy, porous bone. The composition of compact and spongy bone is the same, with collagen fibers providing an organic framework on which the HA crystals form. This results in the properties of bone being intermediate between these organic and inorganic components (Hutmacher et al., 2007; Rogel et al., 2009). Compact bone has greater compressive and tensile strength than spongy bone and thus provides mechanical support, but it also takes longer than spongy bone to revascularize (Mistry et al., 2005; Babis et al., 2005). The femur can withstand 10-15 times the body weight without breaking but a much smaller force applied to the side of the shaft can break the femur (Martini, 2006). Table 1 shows some of the mechanical properties of both compact (also known as cortical) and spongy (also known as cancellous) bone. Specifically, stiffness (E), ultimate tensile strength (σUTS), ultimate compressive strength (σUCS), fracture toughness (KIC), strain to failure (εfailure), and apparent density (ρ) are reported. As a result of the many factors that affect bone and its properties, which will be discussed in section 1.2.4, there is a range in the mechanical characteristics reported.  Table 1: Mechanical Characteristics of Bone* Compact  Spongy  E (GPa)  14-20  0.1-1.0  σUTS (MPa)  50-150  10-20  170-193  7-10  KIC (MPa*m )  2-12  0.1  εfailure (%)  1-3  5-7  1.8-2.0  0.1-1.0  σUCS (MPa) 1/2  ρ (g/cm3)  *Table 1 is compiled from: Murugan et al., 2005; Gibson et al., 1988.  8  1.2.2  Bone Formation  There are two major forms of bone formation: 1) endochondral and 2) intramembranous ossification. Endochondral ossification sees bone replacing the present cartilage and intramembranous ossification sees bone develop directly from mesenchyme or fibrous connective tissue. Basically, intramembranous ossification skips the intermediary formation of a cartilaginous tissue found with endochondral ossification (Mistry et al., 2005, Martini, 2006). In the case of endochondral ossification, the cartilage enlarges and becomes calcified. Next, the outer shell of the bone forms, with blood vessels going through it to supply nutrients to the inner cells of the structure, and the calcified tissue breaks down (Martini, 2006). Finally, osteoblasts act to form woven bone that will, in time, be remodelled to spongy or compact bone as the site requires it. Eventually, osteoclasts arrive to assist in this remodelling and, in the case of long bones, will create a marrow cavity. The mechanical stability and biological potential of bone and its surrounding soft tissue are crucial for bone regeneration and fracture healing – change in one influences the other (Babis et al., 2005).  1.2.3  Graft Incorporation  Graft incorporation is a term used to “describe the biological interactions between graft material and host site that result in bone formation leading to adequate mechanical properties” (Bauer et al., 2000). Figure 2 depicts the basic idea of graft incorporation with bone growth proceeding from the host bone towards and into the graft material. It should be noted here that “graft material” is synonymous for “scaffold material” in terms of incorporation.  9  Bone Direction  Implant Graft Bed  Host Bone  Figure 2: Graft Incorporation.  During the biological events of incorporation, the following steps are involved: (Step 1) hematoma formation with release of cytokines and growth factors, (Step 2) inflammation, migration, and proliferation of marrow stromal cells and development of fibrovascular tissue in and around the graft, (Step 3) invasion of vessels into the graft, (Step 4) focal osteoclastic resorption of graft surfaces, and (Step 5) intramembranous and/or endochondral ossification on graft surface (Bauer et al., 2000; Busenlechner et al., 2008). The initiation of vascular proliferation as a result of the inflammatory response is necessary to provide the graft with nutrients and cells. Steps 4 and 5 are termed creeping substitution and allow the graft to become replaced by bone. The cellular events involved with bone healing consist of chemoattraction, migration, proliferation, and differentiation, with marrow stromal cells (MSCs) being the fundamental ancestor of this cellular creation (Kraus et al., 2006).  As reported by Bauer et al. (2000) the type of graft material, the tissues at the margin of the graft site, and the systemic physiologic state of the host all affect the rate and extent of graft incorporation. Chronic foreign body response (sustained inflammation and fibrosis) should be avoided as it impedes tissue regeneration (Chan et al., 2008). In terms of bone regeneration, an unstable graft material will likely see the formation of granulation tissue and fibrosis at the interface between graft and host, and limit graft incorporation. In time, and allowing for significant graft incorporation and resorption, the bone remodels by both 10  reshaping and reorganizing the collagen fibers, and by forming strong lamellar bone that will eventually achieve its original, pre-fracture strength (Laurencin, 2003; Busenlechner et al., 2008).  1.2.4  Factors Affecting Bone Remodeling  The organic and mineral components of bone are always being recycled and renewed as a result of bone remodeling. Approximately one-fifth of the adult skeleton is recycled and replaced every year, with older mineral deposits removed from bone and released into circulation at the same time that circulating minerals are being resorbed and deposited (Fernlund, 2007; Hing, 2005). It is thought that the action of various peptides or proteins (also known as signaling molecules), that are released by osteocytes and other sensitive cells, trigger the remodeling process (Hing, 2005). Remodeling allows bone to adapt to new stress and their shapes hence reflect the forces applied on them. It also allows bone to maintain an optimal balance between function and form throughout life (Hing, 2005). As a result, regular exercise is a necessary stimulus for maintaining normal bone structure (Martini, 2006). Meanwhile, as we age bones become thinner and weaker as a normal part of the aging process. Normal bone growth and maintenance are also affected by a combination of nutritional and hormonal factors, genetics, gender, and level of drug or alcohol abuse. Calcium is the most abundant mineral in the human body and it plays a variety of physiological roles such that, in order to prevent damage to essential physiological systems, calcium ion homeostasis must be maintained by the opposing activity of parathyroid and calcitonin hormones (Martini, 2006). As a living tissue bone requires a constant supply of oxygen and nutrients.  11  One prevalent theory for bone maintenance is that as the osteocytes are compartmentalized within the matrix they can sense and respond to changes in fluid flow caused by stress, strain or pressure, though how they sense these changes is not known (Zaidi, 2007). Meanwhile, the terminally differentiated osteoclast, which is derived from the hematopoietic stem cell, maintains mineral homeostasis as it resorbs the extensive surface of mainly cancellous bone. Due to various cellular interactions, remodeling feedback loops are established among osteocytes, osteoclasts, and osteoblasts (Hing, 2005). Bone loss occurs when osteoblastic bone formation uncouples from osteoclastic bone resorption.  1.2.5  Current Bone Regeneration Strategies  Skalak et al., (1988) originally defined tissue engineering (TE) as “the application of the principles and methods of engineering and life sciences towards the fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and the development of biological substitutes to restore, maintain, or improve functions” (Hutmacher et al., 2007). The roots of TE extend as far back as the 1970’s with several independent attempts using natural scaffolds and has recently progressed to synthetics (Place et al., 2009). Our interest in the progressive TE field is in its aim to combine engineering technology with the principles of biological science to develop bone repair and regeneration strategies. These strategies exist in three categories: (1) cell-based, (2) growthfactor based, and (3) scaffold-based. Bone TE strategies that are cell-based involve the transplantation of osteogenic cells into a defect and may be useful for treatment of diabetic, osteoporotic, or aged patients in addition to fracture healing (Mistry et al., 2005). Growthfactor based strategies are based on a carrier that will localize growth factor release to the site of injury and achieve maximum activity of these factors when it is directly implanted in or around the affected tissue (Mistry et al., 2005). 12  Though cell-based and growth-factor based strategies potentially provide the necessary osteogenic and osteoinductive components for treating cases of severe bone injury or loss, there is still a need for the optimization of these two strategies. Urist is given credit for outlining the mechanism of osteoinduction and laying the foundation for further development of both biological and synthetic scaffolds as far back as 1965 (Schieker et al., 2006). The use of scaffolds for TE is necessary to fill critically sized defects and to act as a carrier for the cells and/or growth factors needed to heal the defect (Mistry et al., 2005). Cell colonization, migration, growth and differentiation should be supported by the scaffold in order to effectively guide bone regeneration (Hutmacher et al., 2007). TE scaffolds are structures that can be loaded with information that can direct cell function, rather than just serve as passive cell carriers (Place et al., 2009). In this study the bone regeneration strategy of interest is that involving synthetic scaffolds.  1.3  Scaffold Requirements  As mentioned previously, the limitations associated with allograft bone, such as limited availability and risk of disease transmission, have encouraged the search for a more suitable synthetic scaffold material. In addition, there is a greater ability to control the material properties and to tailor performance with respect to tissue response when synthetic materials are used (Causa et al., 2007).  1.3.1  Ideal Scaffold  There are several important requirements that should be met when a scaffold material is developed for use with impaction grafting as described in Table 2.  13  Table 2: Properties for the Ideal Synthetic Scaffold Material** PROPERTIES Biocompatibility Osteo-compatability  Mechanical Properties  Degradation Characteristics  Material Structure & Composition  Preparation  Final Product  Surgical Requirements  Additional  IDEAL CRITERIA -should evoke no unresolved inflammatory response -should evoke no extreme immunogenicity or cytoxicity -should be osteoconductive -should be capable of hosting osteoinductive &/or osteogenic factors -should enable efficient cell proliferation and differentiation -should match that of host bone as closely as possible -some important properties to consider are noted in Table 1 -should be able to withstand cyclic stresses associated with various activities (range of which varies per person) -should degrade at a rate that allows for proper initial support & the creation of replacement bone that is similar to the original -should not release any hazardous byproducts -should be similar enough to bone to allow for acceptable bone growth & vascularization within the material -pore size: 300-800um -porosity: >40-60% -surface properties should be favourable for cell attachment and migration of the necessary cells to promote osteogenesis -should consider optimal porosity, pore size distribution, permeability & interconnectivity in combination -processing techniques should be carefully chosen to obtain the optimal structure & degradation characteristics -to prevent infection it should be easily sterizable -sterilization method should be carefully chosen in order to not interfere with the material's bioactivity or chemical composition -should be able to produce irregular shapes to match that of the defect in the patient’s bone -it may be necessary to include cell seeding or biomolecule incorporation in the manufacturing process -should improve upon that of donor bone in that it is readily reproducible on a large scale, has a long shelf life, & is easily available to surgeons in a sterile operation -should set in several minutes if injectable -any associated temperature change should be minimal to reduce damage to surrounding tissue -viscous properties should match the site requirements -should be available at a reasonable cost & easily handled -should be commercially producible to the required standards (ISO & FDA) -should consider any other needs like the potential for delivery of antibiotic and chemotherapeutic agents  **Table 2 is Compiled from: Lieberman et al., 2005; Cleynenbreugel et al, 2006; Goldberg et al., 2004; Murugan et al., 2005; Pilliar et al., 2001; Jones et al., 2003; Rogel et al., 2009; Will et al., 2008; Hutmacher et al., 2007; Zhang et al., 2007; Jones, 2009; Kasemo et al., 1994; Khan et al., 2008; Christenson et al., 2007; Schieker et al., 2006; Stevens et al., 2007; Causa et al., 2007; Hing, 2004; Temenoff et al., 2000; Hing, 2005.  In relation to biocompatibility, it is important that the criteria stand for both the material as a whole and degradation by-products. In addition, porosity, permeability, and 14  interconnectivity should all be as high as possible to facilitate diffusion within the scaffolds, improving nutrient supply and waste removal, and thus increasing viability of cells at the center of the construct (Cleynenbreugel et al, 2006; Goldberg et al., 2004). Microporosity may also influence bone regeneration via protein adsorption on a larger surface area (Hing, 2005). However, the optimal combination of such material characteristics with themselves and others, such as sufficient mechanical strength, for use with revision THR is still being studied.  Bone formation and repair is always preceded by vascular network formation, which itself is strongly influenced by the degree of structural interconnectivity between pores (Hing, 2005). It has been reported that cancellous bone and cortical bone take 3-6 months and 6-12 months, respectively, to remodel, and thus achieving stable biomechanical conditions and vascularization is critical (Hutmacher et al., 2007). After 12-18 months the scaffold matrix should start to lose its mechanical properties in a controlled manner and be metabolized by the body without a foreign body reaction (Hutmacher et al., 2007; Jones et al., 2003).  The impact of adding cells and/or growth factors, and that of time in storage and/or in the body, should also be considered (Hutmacher et al., 2007). A further desirable property is to form a scaffold with an outer shape that fits the defect properly, plus one that has radiopaque qualities that differ from bone and will allow for radiographic discrimination in postoperative care (Schieker et al., 2006). Most scaffold based strategies require interaction between osteogenic, osteoinductive, and osteoconductive elements (Mistry et al., 2005). Osteogenic components include cells capable of bone production, osteoinductive factors include bioactive chemicals that induce recruitment, differentiation and proliferation of cells at site of injury, and osteoconductive structures provide the necessary support for bone  15  growth to occur. Hence, the biomaterial scaffold should serve as the osteconductive component.  Regardless of the scaffold strategy used, the material chosen and the corresponding material characteristics are very important to the success of bone regeneration. The following section discusses the potential scaffold materials that have been or are currently of interest.  1.3.2  Advantages and Limitations of Current Materials  Although, to date, no single synthetic scaffold material meeting all the criteria in Table 2 has been found, there are several classes of materials that have shown potential. These materials are calcium sulfate, bioactive glass, calcium phosphates, polymers, and composites and are briefly summarized in Table 3 for their use as a scaffold in bone regeneration.  16  Table 3: Advantages and Limitations of Different Scaffold Materials*** Material Autograft  Allograft  Calcium Sulfate  Bioactive Glass  Calcium Phosphate Hydroxyapatite (HA)  Calcium Phosphate Tri-CP (TCP) Calcium Phosphate Biphasic (HA/TCP)  Calcium Phosphate CP Cement  Calcium Phosphate Calcium Polyphosphate (CPP)  Non-resorbable Polymers  Polymethyl methacrylate (PMMA) Resorbable Polymers Polylactic acid (PLA) Polyglycolic acid (PGA)  Advantages Natural -histocompatible, does not transplant disease, & retains viable osteoblasts -mostly histocompatible, & not restricted by donor-site morbidity Synthetic -bioabsorbable ceramic, & easy to shape, prepare & sterilize, -inexpensive, & has an indefinite shelf life -bioactive ceramic -forms HCA crystals on surface that influences osteoblast behaviour, & may not need osteogenic component added to have bone form -shares similarities with bone, & is biocompatible, bioactive, & osteoconductive -fairly controllable % porosity to allow for the appropriate osteoblastic interaction -less crystalline than HA (but is also similar to bone), is biocompatible, & is 10-20 times more soluble than HA -very similar to bone & is biocompatible, is more rapidly degradable than HA alone, & has a bioreactivity inversely proportional to HA/TCP ratio -biocompatible, osteoconductive & shows good osteoblast interaction -can be injected to fit site -is strong in compression -similar to bone, is biocompatible, is osteoconductive, shows good osteoblast interaction, shows promising degradation characteristics, & can achieve initial strength similar to bone -biocompatible, & is used to fill defects & help reconstruct complex fractures -biocompatible, supports osteoblastic adhesion & growth, -can be tailored to degrade safely (biodegradable) with minimal inflammation  Limitations -risk of donor-site morbidity, limited availability, & increased operative blood loss & time -risk of disease transmission, & sterilization affects collagen component of bone -its stiffness, brittleness, & rapid resorption limit it to filling small bone defects (no structural support) -brittle -slow to resorb (typically >1year) -may form particulate debris -use limited to medium sized defects -stiff, brittle, low fracture toughness -low ability to resorb due to a high crystallinity -pore structure remains difficult to reproduce -fairly stiff, brittle, has low fracture toughness, & cannot be used for structural support alone -fairly stiff, brittle, low KIC -incomplete resorption  -hard to control mechanical properties & porosity -increased surgical time possible -limited to treatment of fracture & defects not weight-bearing -still need to obtain optimal combination of all biological and mechanical properties -still much need for further study  -cannot be readily replaced by bone (will not resorb) -lacks mechanical strength -possibility of some inflammation due to degradation by-products if buffering capacity of biological fluids is not sufficient -limited to wound & fracture repair, & bone fixation pins  ***Table 3 is compiled from the material-specific research papers in section 1.5.  17  For the purposes of meeting the criteria in Table 2, most of the materials studied so far in the search to find a suitable scaffold for bone regeneration (as listed in Table 3 along with their respective advantages and limitations) are not currently adequate. While the first four Calcium Phosphates (CPs) mentioned (i.e. HA, TCP, HA/TCP and CP cement) meet some of the needs for bone replacement, they are limited by their inherent stiffness, brittleness and low fatigue properties relative to bone such that they must be shielded from loading forces until bony ingrowth has occurred, and are generally not fully resorbed during bone remodelling (Baksh et al., 1998; Lieberman et al., 2005). Degradation for this class of ceramics typically takes a minimum of 6 months, though some may take longer than 1 year, and occurs via dissolution and osteoclastic resorption (Khan et al., 2008; Bauer, 2007). Calcium Polyphosphate (CPP) is a relatively new solution to the problems faced by other CPs as a biodegradable inorganic polymeric biomaterial. As a result of a low Ca:P ratio, the atomic structure of CPP is chain-like and allows the material to be classed as an inorganic polymer. This chain-like structure, and ease of hydrolysis at the ‘bridging’ oxygen atoms between the monomers, is ideal for the degradation of CPP into the naturally occurring and readily metabolized calcium orthophosphate (Pilliar et al., 2001). The stable roomtemperature form is monoclinic β-CPP (Porter et al., 2001). CPP has shown strong dissolution properties in a biological environment, similar chemistry to bone, potential to achieve good mechanical properties, and a wide diversity in chemical composition (Qiu et al., 2006). An earlier study by Pilliar et al. (2001) showed porous CPP to have a maximum tensile strength of 24.1MPa which is well within the range of bone and highly suitable for THRs. To date, a few different methods have been used to create CPP scaffolds such as PU sponge method, solid freeform fabrication, and gravity sintering. These studies have observed bone growth, as well as crystallinity and some particle morphology effects on strength and degradation (Baksh et al., 1998; Porter et al., 2001; Pilliar et al., 2001; Qiu et al., 18  2006; Grynpas et al., 2002). In summary, CPP has been shown to be biocompatible, able to support bone growth, able to achieve an initial strength similar to bone, and have suitable initial degradation characteristics. In addition, it has been shown that both amorphous particles and the sintering of fine powders to create CPP particles strongly influence the strength and degradation characteristics of CPP (Porter et al., 2001; Pilliar et al., 2001). Porter et al. (2001) showed that crystalline CPP had greater bending strength and toughness than amorphous CPP, though each suffered mechanically upon in vitro degradation with a slightly more pronounced impact on amorphous CPP. In addition, amorphous CPP was found to degrade four times faster than crystalline CPP, as the dissolution of the latter was limited primarily to the surface (Porter et al., 2001). Particles made from sintering of fine powder (105-150um) were similarly shown to undergo greater densification and were approximately four times stronger than the coarse powder (150-250um) samples (Pilliar et al., 2001). However, particles made from either powder exhibited a 67% decrease in strength after thirty days of in vitro ageing in a 0.1 M tris-buffered solution (pH 7.4). While preliminary studies have shown that CPP has potential for use as a synthetic bone scaffold, further study is needed to verify the results in a more rigorous and realistic testing set-up and to obtain optimal combinations of the various properties required.  1.4  Thesis Objectives & Scope  The overall goal of this study is to investigate the biological and mechanical feasibility of using a synthetic calcium polyphosphate material instead of morsellized cancellous bone in revision THR surgery. The objective of the first part of this study is to determine whether calcium polyphosphate is able to support the proliferation and maturation of seeded marrow stromal cells when implanted in vivo. The study of these cells and their behaviour when seeded on scaffold material is a necessary early study for determining the success of bone 19  growth on and into the synthetic scaffold as part of our tissue engineering strategy. The objective of the second part of this study is to determine if sample conditioning via precompaction and varying the composition of particulate and solid composites can improve upon the confined compressive stiffness of calcium polyphosphate or morsellized cancellous bone when in a confined graft bed. The CPP composites that have a significantly higher confined compressive stiffness than morsellized bone and are closer to that of bone cement will be identified. The particulate composites will consist of calcium polyphosphate and poly-(L-lactic acid), while the solid composites will consist of calcium polyphosphate and poly-(methylmethacrylate). The study of pre-compaction and composite composition influence on construct stiffness is necessary for observing how stable and structurally reliable an implant would be within such a graft when loaded. 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Pilliar, R.M., Filiaggi, M.J, Wells, J.D., Grynpas, M.D., and Kandel, R.A. “Porous Calcium Polyphosphate Scaffolds for Bone Substitute Applications – In Vitro Characterization”, Biomaterials. 22: 963-972, 2001. Place, E.S., George, J.H., Williams, C.K., and Stevens, M.M. “Synthetic polymer scaffolds for tissue engineering”, Chemical Society Reviews. 38: 1139-1151, 2009. Porter, N.L, Pilliar, R.M, and Grynpas, M.D. “Fabrication of Porous Calcium Polyphosphate Implants by Solids FreeForm Fabrication: A Study of Processing Parameters and In Vitro Degradation Characteristics”, Journal of Biomedical Materials Research. 56: 504-515, 2001. Presentation: Bozic, K.J., Rubash, H.E., and Berry, J. “Modes of Failure in Revision Hip and Knee Replacement”. 2004. Qiu, K., Wan, C., Zhao, C., Chen, X., Tang, C., and Chen, Y. “Fabrication and Characterization of Porous Calcium Polyphosphate Scaffolds”, Journal of Materials Science. 41: 2429-2434, 2006. Rasanen, P., Paavolainen, P., Sintonen, H., Koivisto, A., Blom, M., Ryynanen, O., and Roine, R. “Effectiveness of hip or knee replacement surgery in terms of qualityadjusted life years and costs”, Acta Orthopaedica. 78:1, 108-115, 2007. Rogel, M.R., Qiu, H., and Ameer, G.A. “The role of nanocomposites in bone regeneration”, Journal of Materials Chemistry. 18: 4233-4241, 2008. Ruhaimi, K.A. “Effect of Calcium Sulphate on the Rate of Osteogenesis in Distracted Bone”, International Association of Oral and Maxillofacial Surgeons. 30: 228233, 2001. Sakamoto, M., Nakasu, M., Matsumoto, T., and Okihana, H. “Development of Superporous Hydroxyapatites and Their Examination with a Culture of Primary Rat Osteoblasts”, Journal of Biomedical Materials Research. 82A: 238-242, 2007. Schieker, M., Seitz, H., Drosse, I., Seitz, S., and Mutschler, W. “Biomaterials as Scaffold for Bone Tissue Engineering”, European Journal of Trauma. 2:114-122, 2006. 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Zhang, L., and Webster, T.J. “Nanotechnology and nanomaterials: Promises for improved tissue regeneration”, Nano Today. 4: 66-80, 2009.  26  2.0  In vivo Evaluation of MSC Proliferation and Differentiation on Synthetic Scaffold Material1  2.1  Introduction  To promote the repair and regeneration of tissue, such as bone, the most common tissue engineering concept is to combine living cells that respond to their environment, biologically active molecules that provide instructional and molecular cues, and a structural scaffold that serves to anchor, deliver and orient the cells (Zhou et al., 2007; Caplan et al., 2001; Xie et al., 2007). An additional requirement for cell therapy is to meet histocompatability needs between donor cells and the recipient patient (Naveiras et al., 2006). As marrow stromal cells (MSCs) have a multi-lineage potential, are sensitive to specific signaling molecules, and are relatively easy to harvest and expand in vitro, they are attractive cell population for tissue engineering. It has been shown that pre-seeding of MSCs onto suitable matrices prior to implantation could potentially improve the healing process of large bone defects (Xu et al., 2009).  2.1.1  MSC Biology  The fact that bone formation takes place during embryonic development, growth, remodeling, fracture repair, and when induced experimentally suggests that there is a large supply of cells in the body that are capable of osteogenesis many times throughout an individuals lifetime (Aubin, 1998). German pathologist Cohnheim first suggested the presence of nonhematopoietic stem cells in bone marrow 130 years ago (Chamberlain et al., 2007). MSCs are believed to contain a small population of multi-potential mesenchymal stem cells. Evidence that these mesenchymal stem cells were in fact multipotential, exist in  1  A version of this chapter will be submitted for publication. Comeau, P., Frei, H., Rossi, F., and Fernlund, G. In vivo Evaluation of MSC Proliferation and Differentiation on Synthetic Scaffold Material.  27  the normal adult, and could differentiate into osteoblasts, chondrocytes, adipocytes, and even myoblasts was demonstrated by Friedenstein and colleages (Bolland et al., 2007; Chamberlain et al., 2007; Naveiras et al., 2006; Aubin, 1998). Friedenstein’s observations have since been further confirmed by other groups (Chen et al., 2005; Chamberlain et al., 2007; Livingston et al., 2003; Livingston-Arinzeh et al., 2005; Kraus et al., 2006; Engler et al., 2006; Bianco et al., 2001). In fact, mesenchymal stem cells have been found at a frequency of ~1/100,000 nucleated cells in bone marrow and can be isolated and expanded into billions of cells (Livingston et al., 2003; Caplan et al., 2001; Livingston-Arinzeh et al., 2005).  In order to replenish tissue, the stem cell must undergo one or more of the three types of mitotic divisions: (i) replicating division (i.e. proliferation), (ii) differentiating division, and (iii) self-renewal or asymmetric division, the latter of which is unique to stem cells in the adult (Naveiras et al., 2006; Nijweide et al., 1986). A population balance of replicative and differentiative divisions, also known as sustained asymmetric division, is necessary for the homeostatic balance of stem cells, MSCs, and tissue populations to be maintained (Naveiras et al., 2006). In asymmetric stem cell division, the stem cell gives rise to one daughter stem cell similar to itself and another daughter cell that undergoes differentiation. MSCs secrete specific growth factors and cytokines and it is believed that modulation of the synthesis of these secreted molecules, as well as the regulation of other proteins found in the lineage path, is involved with the induction of the cells into each differentiation pathway (Caplan et al., 2001; Aubin, 1998).  28  2.1.2  MSC Proliferation  As previously mentioned, MSC proliferation is a necessary step the cells must undergo for bone tissue to be replenished or regenerated. The golden standard for detecting cellular proliferation involves the in vivo labeling of the cells with BrdU (5-bromodeoxyuridine) when using animal models (BD Biosciences Website, 2009). BrdU is a thymidine analog that will become incorporated into the DNA during the DNA synthesis phase of cell division. After sample harvesting and following membrane permeabilization the cellular incorporation of BrdU can be detected by anti-BrdU specific antibodies using flow cytometry. Two in vivo labeling methods for rodent cells include subcutaneous injection and doping the drinking water.  2.1.3  MSC Differentiation into Bone  MSCs give rise to a hierarchy of cell populations within bone such that the developmental continuum can be artificially separated into a series of developmental stages including MSC, determined osteoprogenitor cell, preosteoblast, osteoblast, and lastly osteocyte as shown in Figure 3 (Bolland et al., 2007). Cells progress from an early progenitor to a fully functional matrix synthesizing osteoblast as a result of the biological periods of cellular proliferation, cellular maturation and focal mineralization, each associated with characteristic changes in gene expression (Knabe et al., 2004; Aubin, 1998). The determination of MSC to become an osteoprogenitor constitutes the initial step in osteoblastogenesis and coincides with high levels of various receptors being expressed such that the cells begin to proliferate (Zaidi, 2007).  29  Marrow Stromal Cells Osteoprogenitors (MSCs)  Preosteoblasts  Osteoblasts  Osteocyte  Figure 3: Sequence of Cell Differentiation Along the Osteogenic Lineage.  Osteoblasts are known to synthesize and secrete Collagen-I (Col-I), Alkaline Phosphatase (ALP) and other non-collagenous extracellular bone-matrix proteins such as Osteocalcin (OC), Osteopontin (OP), and bone sialoprotein (BSP) (Knabe et al., 2004; Hing, 2004). ColI is prevalent during the initial period of proliferation and extracellular-matrix biosynthesis, followed by ALP which shows up during the post-proliferative period of extracellular-matrix maturation. Mature mineralizing osteoblasts become embedded into the secreted matrix and differentiate terminally to become osteocytes. These osteocytes do not express ALP, but will express OP and other bone matrix proteins. Thus, OP, OC, and BSP appear during the third period of extracellular matrix mineralization. OC and BSP are bone specific and their relative composition within bone appears to be self-regulating through the feedback effect between non-collagenous proteins and osteoblasts (Hing, 2004). Expression of the osteoblast-associated genes has been shown to be asynchronously acquired and/or lost as the progenitor cells differentiate (Aubin, 1998). For one thing ALP has been shown to increase then decrease when mineralization is well progressed, while OP peaks twice during proliferation and again prior to certain other matrix proteins such as BSP and OC (Aubin, 1998).  To measure the expression levels of these genes relative to a housekeeping gene of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Hs02758991_gl), the messenger RNA (mRNA) of the cells must first be isolated, mRNA reverse transcripted into complimentary DNA (cDNA), and the cDNA further amplified by PCR in order for it to be 30  detectable. Lastly the cDNA is labeled with Taqman Probes, which will seek out and attach to the expressed genes, and the Real-Time PCR System reports those genes present (Real Time PCR Tutorial Website, 2006).  2.1.4  MSC Culturing, Seeding and Identification  The ability to maintain differentiation and proliferation capacity following culture expansion and cryopreservation suggests that MSCs may be valuable as a readily available and abundant source of cells in the tissue engineering field (Kraus et al., 2006). When the cells are cultured at a sufficiently low density, the MSCs adhere to the tissue-culture treated flask surface. Similar to how these cells are attached to their surrounding extracellular matrix, it is believed that they will attach to the flask and later the scaffold material, via transmembrane integrin receptors found at the cell surface (Stevens et al., 2005). Changes in medium serve to remove the hematopoietic stem cells and other nonadherent cells and to concentrate the MSCs (Pittenger et al., 1999; Zaidi, 2007; Bianco et al., 2001; Kraus et al., 2006). The cells will continue to be passaged, expanding the cell population, until a pure culture is produced (Kraus et al., 2006). The doubling time of the dividing cells depends on the donor and the initial plating density (Chamberlain et al., 2007).  There are two general seeding patterns: static or dynamic conditions (Schieker et al., 2006). Under static conditions, the cell suspension is applied to the scaffold in order to load the cells. With dynamic conditions, the cells are given more time and opportunity to adhere as the complicated circuit ensures media flow across the scaffold multiple times. Thus, although static conditions are simpler, the dynamic conditions may lead to a greater seeding efficiency.  31  On the surface of every cell in the body are various types of specialized proteins (also known as receptors) that are able to selectively bind or adhere to other signaling molecules (Stem Cell Information Website, 2009). These receptors are used by the cell to communicate with other cells and to perform proper functions in the body, but may also be used by researchers in the form of cell markers. Each cell type has markers which are distinct from other kinds of cells. In this way, scientists can use the chemical properties of certain compounds to tag or “mark” the cells. To identify the presence or absence of cells markers researchers use “+” or “-“ symbols, respectively.  2.1.5  In vitro and In vivo Study of MSCs  Following seeding, the tissue engineered construct can then be studied with various models. One popular model is the subcutaneous implantation of cell-matrix constructs which allows for simple and rapid testing and analysis. However, as there is no biomechanical influence exerted on the construct at the ectopic implantation site, only defined implant properties such as osteoinductivity can be evaluated. Orthotopic defect models, though complicated and time-consuming, can overcome such ectopic model limitations (Schieker et al., 2006).  Limitations of animal models, such as in mice or rats, include that: (i) rodent bone composition and structure differs from other species and humans, (ii) the metabolic rate is inversely proportional to the animal size such that the repair rate is higher for smaller animals, (iii) rodents have little or no secondary haversian remodeling in their cortical bone which differs from what is found with humans (Frei et al., 2004). However, in the case of this study which is interested in bone regeneration with revision THR, the different stages of graft incorporation and remodeling are believed to be similar in rats and humans even though the speed of these will be different. This is sufficient cause for rodent models to be used. 32  2.1.6  Clinical Application  As culture preparation per patient increases production time and costs and in turn, cost to the patient and waiting time between MSC collection and treatment, the utility of a tissue engineered construct (i.e. scaffold material + cells) will ultimately depend upon the expedient delivery and cost-effectiveness of the design (Livingston et al., 2003; Kraus et al., 2006). An attractive solution is an allogenic approach in which the MSCs are obtained from the donor, expanded and cryopreserved until use. This approach is particularly appealing as a low immunogenicity allows the MSCs to avoid rejection upon allogenic implantation. However, it is well recognized that donor heterogeneity (i.e. age, gender, and disease history), may influence the yield and proliferative capacity of these cells or their osteogenic capacity (Ciapette et al., 2006).  Impaction allografting (IA) is one revision THR technique used to restore the bone stock that has been lost prior to the failure of the primary THR. However, limitations such as intraoperative and post-operative fracture, as well as the limited availability and risk of disease transmission associated with MB, has restricted the successful application of IA. Of interest in our study is how a synthetic Calcium Polyphosphate (CPP) material can be used in place of MB under similar revision THR conditions and requirements, and meet as many of the requirements in Table 2 (Chapter 1). Synthetic materials are of particular interest for this application due to their availability and ease of preparation and sterilization. An earlier in vitro study by Siggers(2007) suggested that CPP and morsellized cancellous bone (MB) scaffolds will support MSC expansion rather than differentiation, while biphasic Hydroxyapatite/tricalcium phosphate (HA/TCP) scaffolds will be more favourable towards MSC differentiation and maturation along the osteogenic lineage.  33  In order for MSCs to play a role in IA, it is necessary to determine if these cells can survive the impaction process. A study by Mushipe showed that cells (of the human osteosarcome line) seeded onto bovine allograft could survive a limited number of impactions (Bolland et al., 2007). Korda and colleages similarly concluded that the addition of MSC to allograft could survive normal impaction forces in IA, but recommended avoiding excessively high impaction forces (Bolland et al., 2007). Further studies by Bolland and colleagues (2006, 2007 & 2008) have shown that human bone marrow stromal cells (HBMSC) seeded on washed morsellized bone graft or PLA could withstand forces equivalent to a standard femoral impaction bone grafting and that the increased shear strength provided by an allograft-HBMSC composite could allow not only early mobilization of patients but also improved graft incorporation. However, it is possible that the balance between bone resorption and bone regeneration seen in fracture healing and graft incorporation could change when the living composite is subjected to loading and the host’s own cells respond.  This chapter details the in vivo study in which three different substrate materials seeded with MSCs were implanted subcutaneously and the proliferation and differentiation profiles of the cells analyzed. Proliferation analysis is necessary to determine if the original cells performed a replicating division and increased in number, while differentiation analysis is necessary to determine what tissue is present and at what stage. Both profiles are needed in order to properly analyze the ability of the materials to support bone growth. The three substrates evaluated in this study were MB, HA/TCP and CPP. If not for the limitations previously mentioned, MB would be the ideal graft material for bone regeneration due to its natural composite structure. HA/TCP, though shown to be mechanically weak in compression, is a synthetic material that has received a significant amount of attention for use with bone grafting due to its similar composition to bone and promising early in vitro and in vivo 34  studies. CPP is a novel biodegradable inorganic polymeric biomaterial that has shown promise in tissue regeneration and remodeling.  2.2  Materials & Methods  The method flowsheets for in vivo implantation and analysis post-harvest are shown in Figures 6 and 7, respectively. Appendix I has a more detailed procedure for each step of the in vivo experiment. 2.2.1  Scaffold Materials  MB chips were harvested from the femora and tibiae of seven white female rats. The soft tissue was removed from the bones using a scalpel and tweezers, and the bone was then crushed into 1-7mm size particles. To remove any remaining tissue or blood and sterilize the bone, the particles were washed several times in 70% ethanol. The removal of soft tissue has been shown to reduce the immunological load to the patient and risk of disease transmission, and also increase the frictional resistance with greater contact between particles (Board et al., 2006; Dunlop et al., 2003; Bavadekar et al., 2001). As a result, a stronger and stiffer compacted graft can be created which is more resistant to shear failure.  The CPP material used in this study was provided by Dr. Bob Pilliar’s research group at the University of Toronto (Pilliar, R.M. et al., 2002). It was formed by first calcining the precursor powder of calcium phosphate monobasic monohydrate (CPMM) in a platinum crucible at 500ºC for 10 hours. The reaction that occurred as a result of this calcining is as follows: nCa(H2PO4)2*H2O  [Ca(PO3)2]n + 3nH2O CPMM  CPP powder  35  The CPP powder was then melted at 1100ºC to produce an amorphous glass, poured directly into distilled water to form an amorphous frit and then dried in 100% ethanol. Next, the powders were gravity sintered (i.e. pressure-less sintered) in cylindrical platinum tubes at approximately 970ºC for 2hours and crushed into angular particles of 1-3mm in diameter. The resulting particles have an interconnected porous network of 30-45 vol% porosity and an internal pore size in the range of 100um (Pilliar, R.M. et al., 2002). In addition, upon X-ray diffraction analysis Pilliar et al. (2001) confirmed that the gravity sintering of this amorphous frit in the aforementioned technique created stable room-temperature mono-clinic β-CPP of a crystalline nature.  HA/TCP was purchased from Berkeley Advanced Biomaterials Inc. and consisted of a 20% HA and 80% TCP composition. The particles were 1-3mm in size and had pores approximately 250um in diameter. In Figure 4, the three materials studied are shown courtesy of Siggers (2007).  Figure 4: (a) Morsellized Bone Chips, (b) Calcium Polyphosphate Particles, and (c) Biphasic Hydroxyapatite/Tricalcium Phosphate Particles  2.2.2  Cell Isolation, Culturing, Seeding, and Implantation  To obtain MSCs, the femora and tibiae of two 6-week old transgenic GFP (GreenFluorescent Protein positive) Sprague-Dawley rats were crushed and the bone marrow was removed. The bone marrow was then plated onto 15cm tissue culture treated plates (i.e. 36  Vacuum gas plasma treated) and mixed with a culture medium consisting of mesencult basal medium (Cat #05401 Stem Cell Technologies, Vancouver), 15% fetal bovine serum (FBS; Cat #06471 Stem Cell Technologies, Vancouver), and 100 U/mL penicillin-streptomycin (P/S). Mesencult MSC basal medium forms the base solution for the expansion of our GFP+ rat MSCs, FBS comes from the blood of an unborn bovine fetus and provides the necessary growth factors, while P/S is an antibiotic that prevents bacterial growth (Stem Cell Technologies Website, 2006). After four days, the non-adherent cells were washed off and the attached cells were expanded to roughly 80% confluence. The cells were further expanded to passage six then cryopreserved until needed. Before seeding the cryopreserved MSC were thawed and further expanded in the culture medium to passage nine for the in vivo samples, and passage eight for the 0-day in vitro samples. These GFP+ cells are ideal for our ectopic (or subcutaneous) model as the GFP marker on the cells allows us to identify them from all mice cells, mice tissue and any debris present when observing the scaffold in a Zeiss fluorescent microscope after harvesting.  Prior to seeding, the particulate material was repeatedly washed and left in 70% Ethanol to sterilize for a minimum of 48hours, and then rinsed and held in phosphate buffer solution (PBS) for 24hours. PBS is a buffer solution that helps to maintain a constant pH and has solute and ion concentrations similar to that within a mammal. It is non-toxic to cells. Lastly, to acclimatize the particles to the culturing media, the particles were divided into the wells of the 24-well non-tissue culture treated plate with an equal mass in each and roughly 1mL of culture media was added. The culture plate was then incubated for the remaining hours (24-48) prior to seeding. A non-tissue culture treated plate is necessary for seeding of our scaffolds as it minimizes the likelihood that the cells will adhere to the plate instead.  37  The MSCs were seeded on the scaffolds with a cell suspension of 1.0 x 106 cells/mL of culture medium. Following a brief 37ºC incubation period of 5 minutes, the plates were centrifuged at 1500RPM and 4ºC for 6 minutes and then incubated for another 6 hours at 37ºC. After 6 hours, the supernatant was removed from the plate and the scaffolds were washed twice with 500uL of PBS in order to remove any cells that did not adhere. Seeding efficiencies were taken as the number of MSC’s effectively seeded on the scaffold materials as a fraction of the total number of cells provided to the scaffold. Using a hemocytometer (VWR scientific cat#: 15170-208), the cells were counted in the initial suspension provided prior to seeding and in the wash solution put aside after the 6 hour seeding period. To ensure survival of the cells attached to the particles until time of in vivo implantation, after the second wash of PBS, 1mL of chilled PBS was added to each well.  The plates and the necessary surgical equipment were then taken to the air-filtered animal unit where the NOD/Scid (Non-Obese Diabetic/Severe combined immunodeficiency) mice were housed. Although allogenic MSCs are not believed to trigger any immune response, the implantation of a MSC + scaffold construct could itself trigger a sustained immune response as a foreign body. Thus, to avoid rejection of the implanted construct NOD/Scid mice were used. The mice were first anesthetized with Isofluorane until they were comfortably in a surgical plane and then transferred to the anesthetizing tube in order to keep them anesthetized for the duration of the surgical procedure. Next, the hair was clipped on the backs of the mice and the exposed skin was surgically prepared with alcohol pads and iodine. Using sterile tweezers and scissors, the back skin of the mice was cut and the scaffolds placed under the skin. As shown in Figure 6, the scaffolds were randomly placed in each of the three surgical sites chosen, with one for each material. To close the incision, Michel clips were used and iodine was spread liberally to minimize risk of infection. Once the third 38  scaffold had been implanted and the incision closed, the mouse was given oxygen for 12minutes and then transferred to a clean paper towel placed on top of the bedding in the clean animal cage. A heat lamp further assisted in the recovery of the mice from anesthesia and surgery. A typical surgery for a single mouse lasted 20-25minutes, with 5-15minutes of anesthesia recovery time following. All in vivo experiments were performed in triplicates at time periods of 7, 14, and 28-days. For the proliferation analysis with BrdU, a fourth mouse, similarly implanted with seeded scaffolds, was used as a BrdU-negative control. For the differentiation analysis with qRT-PCR, a fourth mouse was implanted with non-seeded scaffolds to serve as a control against the impact of mouse cell adherence to scaffold. This differentiation analysis control was useful in observing how effective the techniques to isolate the GFP+ rat cells were. Lastly, a 0-day qRT-PCR analysis was done immediately following the 6 hour seeding which did not involve any mice implantation. Twenty-four mice were successfully implanted with scaffolds in this study.  2.2.3  Sample Harvesting  Once the endpoint of 7, 14 or 28 days post-implant was reached, the mice were euthanized using CO2, the hair around the implant sites removed using Veet, and the scaffold samples extracted. Figure 5 shows five of the mice following euthanization and one following hair removal with Veet prior to sample extraction. To separate the cells from the material, the samples were placed in 15mL falcon tubes with approximately 1mL of a collagenase/dispase + CaCl2 mixture and kept in a 37ºC warm room for 1 hour and 45minutes. Collagenase/dispase provides a combination of collagenolytic (digestion of collagen) and proteolytic (digestion of proteins) enzymes necessary for tissue degradation to occur and liberate the cells (Roche Applied Science Website, 2007). CaCl2 increases the cell membrane permeability of the collagen connections. After 1 hour, the samples were quickly 39  rinsed with culture medium, filtered (with the filtrate put aside in ice), crushed, incubated with another 1mL of fresh collagenase/dispase + CaCl2 mixture, and put in the warm room for the remaining 45minutes. The samples were then rinsed again with the culture medium and the resulting filtrate mixed with the first filtrate. This combined filtrate was then centrifuged, the supernatant aspirated off, and the cells re-suspended in FACS buffer. The FACS buffer consisted of PBS + 2mM EDTA + 2% FBS, with the EDTA present to prevent aggregation of the cells in the sample.  (a)  (b)  Figure 5: (a) Mice Following Euthanization, and (b) A Mouse Following Hair Removal Prior to Sample Extraction. 2.2.4  Cellular Proliferation  BrdU (5-bromo-2-deoxyuridine) incorporation into the newly synthesized DNA of replication cells was used to measure cellular proliferation of the GFP+ rat MSCs on the scaffolds. To allow for this BrdU incorporation, each of the mice was injected with 100uL of a 1mg BrdU/mL mixture with PBS at each implantation site. In addition, the mice were given 0.8mg BrdU/mL mixture doped water. Fresh injections and BrdU-doped water were given each of the three days prior to the series endpoint. After harvesting the samples, collecting the cells from the scaffold, and re-suspending in the FACs buffer, the cells were stained with anti-CD45 (anti-rat; BD cat# 554876, BD Biosciences) and streptavadin-PE (Caltag cat# SA1004-4, Caltag Laboratories). CD45 is a surface marker found on white 40  blood cell (WBC) progenitors and so, in this study, anti-CD45 was used to detect what fraction of rat blood cells remained with the MSCs when cultured and were seeded to our scaffold. Here our secondary anti-body was streptavadin which itself was linked with a fluorescent PE marker and attached to the anti-CD45 already attached to the WBCs of the rats. As part of the BrdU protocol the cells were then fixed and intracellularly stained with anti-BrdU-APC (BD cat# 550891, BD Biosciences, and then analyzed with the FACS machine (BD Biosciences). The FACS, or ‘fluorescence-activated cell sorting’, machine passed the cell samples through a laser. A scatter profile representative of the size and granularity of the cells was produced, and as the fluorochromes attached to the cells became excited the corresponding specific wavelengths were emitted (Johnson, 2008 - course). Photo Multiplier Tubes (PMTs) captured these emissions and created an electronic pulse, which was then interpreted and displayed by the computer.  2.2.5  Cellular Differentiation  The expression levels of various osteogenic markers were determined with qRT-PCR using Taqman probes (Applied Biosystems, Foster City, CA). The osteogenic genes studied were Bone Sialoprotein (BSP; Taqman#: Rn00561414_ml), Collagen I (Col I, Taqman#: Rn00801649_gl), Alkaline Phosphatase (ALP, Taqman#: Rn00564931_ml), Osteopontin (OP, Taqman#: Rn01449972_mL), and Osteocalcin (OC, Taqman#: Rn00566386_gl).  After harvesting the samples, collecting the cells from the scaffold, and re-suspending in the FACs buffer, the cells were stained with anti-CD44 (anti-rat; BD cat# 554869, BD Biosciences), anti-IgG2a-PE (Caltag cat# M30004-4), and anti-PE magnetic beads (cat# 130048-801, Miltenyi Biotec), and then magnetically sorted to isolate the GFP+ rat cells using the autoMACS machine (Miltenyi Biotec). CD44 is a marker found on the surface of bone 41  cells (i.e. committed osteoprogenitors, pre-osteoblasts, and osteoblasts) and the anti-CD44 antibody was used to identify the fraction of GFP+ rat MSCs that were still present after harvest. Anti-IgG2a is a secondary antibody that attaches to anti-CD44 and is labeled with a PE fluorescent marker which, when further connected to anti-PE magnetic beads, allows for the rat cells to be separated from mice cells and debris upon application of a magnetic field. Magnetic sorting allows for sample purification with the selected cell fraction remaining (i.e. the GFP+ rat cells) and most of the unwanted fraction removed in the process waste (i.e. mice cells and particulate debris). The selected cell fraction was then further treated in the remaining steps of analysis.  Next, the selected samples from the autoMACS were centrifuged, underwent mRNA isolation using the mRNAse-easy kit (Qiagen) and a thermocycler (Biometra), and were measured for mRNA on the Nano-drop machine (ND-1000, NanoDrop Technologies). After determining how much mRNA was present, the samples (i.e. their mRNA) were reverse transcribed using superscript III (Invitrogen) and a thermocycler to get cDNA, and were measured for cDNA on the Nano-drop machine. Lastly, the samples were analyzed for gene expression with the taqman probes (PE biosystems) on the Real-Time PCR system (AB7900HT, Applied Biosystems). To measure the mRNA or cDNA levels in the samples, a 1uL sample was pipetted directly onto the pedestal, the pedestal arm was closed (forming a sample column), and UV-Vis spectroscopy was applied by the machine. The thermocycle heated and cooled the samples accordingly to allow for denaturation and reverse transcription. The Real-Time PCR system involved the use of a laser scan to excite the fluorescent dyes attached to each Taqman probe (which themselves had already attached to their corresponding osteogenic genes) (Applied Biosystems, 2009). A spectrograph and a charge-coupled device camera spectrally resolved and collected the fluorescence emission 42  from each sample analyzed. In this study, target gene expression was normalized relative to the housekeeping gene GAPDH. Each scaffold was analyzed in duplicate so that, for each material at each endpoint, there were 6 samples (i.e. 2 per mouse x 3 mice).  2.2.6  Statistics  The impact of substrate and implantation time on BrdU incorporation and gene expression was analyzed with a 2-way ANOVA in which substrate and time were factors. The post-hoc analysis of BrdU incorporation, mRNA, cDNA, gene expression, seeding efficiency and final cell count successfully seeded was done using Student-Newman Keuls tests at a significance level of 0.05. Table 4 shows a chart of statistical significance between materials and endpoints for the proliferation and differentiation analysis. ‘Yes’ indicates that the difference was statistically significant (p < 0.05), while the ‘+’ shows that the material-time 1 had a greater value than material-time 2 and ‘-‘ shows that it had a lower value than material-time 2.  2.2.7  Analytical Flowsheet  Figure 6 shows the flowsheet for the in vivo implantation of the scaffolds, while Figure 7 shows a method-by-method representation of the analysis of these scaffolds post-harvesting (i.e. after euthanization and removal of the samples from the mice). Further details for each of these steps were given in sections 2.2.3 – 2.2.5.  43  GFP+ Rats  Rat Bone Marrow Collection & MSC Isolation and Expansion to P6  P6 Cryogenation  Thawing and further culture to P8/P9 & Seeding on Material for 6hours  Nonadherent cells are rinsed off & Scaffolds are implanted in NOD/SCID mice Materials 1, 2, & 3 respectively  Figure 6: Flowsheet Representing the Steps of the In vivo Implantation of the Scaffolds. Materials 1, 2, and 3 are CPP, HA/TCP, and MB.  44  BrdU (Proliferation)  On each of 3 days prior to harvest: • Inject mice with BrdU/PBS • Change water to BrdU-doped DDW  Harvest Samples & Perform Collagenase/Dispase + CaCl2 Enzymatic Reaction  qRT-PCR  BrdU  (Differentiation)  (Proliferation)  Stain with:  Stain with:  • • •  • •  anti-CD45-biotin (anti-rat) Streptavadin-PE  anti-CD44 (anti-rat) anti-IgG2a-PE  anti-BrdU-APC  Magnetic Sorting (anti-PE) FACS Analysis  RNA Isolation & cDNA transcription Tag with Taqman probes AB7900 Machine: Reference Gene GAPDH  Figure 7: Method Flowsheet Showing How BrdU and qRT-PCR Analysis was Completed.  2.3  Results  2.3.1  Seeding  The average seeding efficiency and final seed count are shown in Figure 8. For CPP, HA/TCP, and the MB, the seeding efficiencies were 94.87 ±3.31%, 97.05 ±1.11%, 94.59 ±2.86%, respectively. The final cell count was found to be 1.26x106±0.53x106, 45  1.28x106±0.51x106, and 1.25x106±0.52x106 for CPP, HA/TCP, and MB, respectively. There was no significant difference in the seeding efficiency or in the final cell count between material scaffolds.  100% 90% 80%  CPP HA/TCP MB  % Efficiency  70% 60% 50% 40% 30% 20% 10% 0% 1 Material  (a) 2.00E+06  Final Seeding Count  1.80E+06 1.60E+06  CPP HA/TCP MB  1.40E+06 1.20E+06 1.00E+06 8.00E+05 6.00E+05 4.00E+05 2.00E+05 0.00E+00 1 Material  (b) Figure 8: (a) Seeding Efficiency, and (b) Final Successful Seeding Count on the Scaffold Material. All Values are Presented as the Mean ± One Standard Deviation.  2.3.2  Cellular Proliferation  BrdU incorporation 7days following implantation was found to be nil, with an increase at 14days, and a subsequent drop at 28days for all scaffold materials (Figure 9). A 2-way ANOVA showed that the degree of BrdU incorporation was not significantly different 46  between materials for any given endpoint, but that it was significantly different between end points for any given material. At Day 14, BrdU incorporation was highest for CPP and HA/TCP, each showing an incorporation of roughly 30%.  50.00% 45.00%  % of GFP+ with BrdU  40.00% 35.00% 30.00% 25.00% 20.00% 15.00% 10.00% 5.00% 0.00% CPP  HA/TCP  MB  Material 7-Day  14-Day  28-Day  Figure 9: BrdU Incorporation into the GFP+ Cells During In vivo Implantation. All Values are Presented as the Mean ± One Standard Deviation.  2.3.3  Cell Isolation Post-Harvest  The effectiveness of the magnetic sorting (MS) step was measured by counting the number of GFP+ cells present as a fraction of total cell+debris count prior to magnetic sorting and that following it (Figure 10). The count prior to magnetic sorting was also used to determine the effectiveness of the collagenase/dispase (C/D) enzymatic digestion in removing the cells from the scaffold materials post-harvest as shown in Figure 10. The fraction of GFP+ cells recovered from the collagenase/dispase process was shown to be 26.64 ±8.46%, 14.46 ±8.57%, and 14.07 ±3.28% for CPP, HA/TCP, and MB, respectively. Meanwhile, for magnetic sorting the recoveries were found to be 79.51 ±12.05%, 25.64 ±3.65%, and 73.78 ±23.54% for CPP, HA/TCP and MB, respectively. The percentage recoveries of the GFP+ cells following the C/D reaction as well as the magnetic sorting step are shown in Figure 11. As MS follows C/D, the percentage of GFP+ cells recovered after MS is determined as a 47  fraction of GFP+ cells that remained after C/D (i.e. prior to MS). Following the magnetic sort, the GFP+ cell count ranged from 1.09x105 – 5.07x105 for CPP, 1.33x104 – 1.04x105 for HA/TCP, and 5.20x104 – 3.51x105 for MB.  Collagenase/Dispase Counts  GFP+ Cell Count  2.50E+06  2.00E+06  1.50E+06  1.00E+06  5.00E+05  0.00E+00  CPP  HA/TCP  MB  Material 0-Day Prior to C/D 14-Day Prior to C/D  0-Day Post C/D 14-Day Post C/D  7-Day Prior to C/D 28-Day Prior to C/D  7-Day Post C/D 28-Day Post C/D  (a) Magnetic Sorting Counts 2.50E+06  GFP Cell Count  2.00E+06  1.50E+06  1.00E+06  5.00E+05  0.00E+00  CPP  HA/TCP  MB  Material 0-Day Pre-sort 14-Day Pre-sort  0-Day Post-sort 14-Day Post-sort  7-Day Pre-sort 28-Day Pre-sort  7-Day Post-sort 28-Day Post-sort  (b) Figure 10: (a) Collagenase/Dispase GFP+ Cell Count, and (b) Magnetic Sorting GFP+ Cell Count. Solid Bars Represent the GFP+ Cell Count Pre-technique and the Striped Bars are for the Count Post-technique as Seen in a Fluorescent Microscope.  48  % of Cells Recovered with Each Technique  % of GFP+ Cells Recovered  100.00% 90.00% 80.00% 70.00% 60.00% 50.00% 40.00% 30.00% 20.00% 10.00% 0.00%  CPP  HA/TCP  MB  Material % Recovered with C/D  % Recovered with MS  Figure 11: GFP+ Cell Recoveries as a Fraction of the Initial Cell Count for the Collagenase/Dispase Reaction and the Magnetic Sorting Technique. All Values are Presented as the Mean ± One Standard Deviation.  2.3.4  mRNA Isolation and Reverse Transcription  As can be seen in Figure 12, the amount of mRNA (ng/ul) derived from the HA/TCP scaffolds was the lowest of the three scaffolds. However, the corresponding cDNA does not show a similar trend.  49  mRNA measured with Nanodrop 120.00  mRNA (ng/uL)  100.00  80.00  60.00  40.00  20.00  0.00  CPP  HA/TCP  MB  Material 0-Day  7-Day  14-Day  28-Day  (a) cDNA measured with Nanodrop 600.00  cDNA (ng/uL)  500.00  400.00  300.00  200.00  100.00  0.00  CPP  HA/TCP  MB  Material 0-Day  7-Day  14-Day  28-Day  (b) Figure 12: (a) mRNA, and (b) cDNA Measured at Each In vivo Endpoint. All Values are Presented as the Mean ± One Standard Deviation.  For the mRNA measured, both CPP and MB were found to be significantly different than HA/TCP at 14 days in vivo (p < 0.04). In addition, mRNA measured at 0 and 14 days in vivo was found to be significantly different for both CPP and MB (p < 0.03). For the cDNA measured, there were no statistically significant differences between materials at any given endpoint. However, between 0 and 7 days in vivo, CPP, HA/TCP, and MB were each found to be significantly different (p < 0.007). CPP also showed a significant difference between 0 and 28 days as well as between 7 and 14 days in vivo for cDNA measured (p < 0.025).  50  2.3.5  Cellular Differentiation  The results of the qRT-PCR analysis of the ALP, Col-I, OP, OC and BSP osteogenic markers are shown in Figure 13. There is no definite trend in the ALP expression levels. For 7-day and 14-day samples, HA/TCP had the lowest expression of the three materials, but at 28-days it had the highest. However, there was no statistically significant difference between materials at any given endpoint, or for any material when comparing the different in vivo endpoints. Col-I increased from 7-Day to 14-Day for CPP and HA/TCP with a sudden drop down to the 28-day expression, while the maximum expression was at 28-day for MB. The difference between MB and CPP, as well as MB and HA/TCP at 28-days was significant (p < 0.042). In addition CPP at 0-days and 14-days (p < 0.025), and MB at 28-days compared to 0, and 14-days (p < 0.044) were all statistically different pairs. For 0-day and 7-day analysis, HA/TCP was shown to have the highest OP and BSP expression, whereas at 14-day and 28day MB has the highest. However, neither 2-way ANOVA nor the post-hoc analysis revealed any results that were statistically significant for either gene. With the exception of the 14-day levels, MB expression of OC is the highest of the three materials followed by CPP, and lastly HA/TCP. At 0-day MB was statistically different from CPP, as well as from 7-day MB (p< 0.035). However, aside from these two exceptions, OC levels were not significantly different between materials for any given endpoint, nor between endpoints for any given material.  51  ALP  Col I 3.00E+01  Expression relative to G AP DH  E xpression relative to G A PD H  3.00E-03  2.50E-03  2.00E-03  1.50E-03  1.00E-03  5.00E-04  2.50E+01  2.00E+01  1.50E+01  1.00E+01  5.00E+00  0.00E+00  0.00E+00 CPP  HA/TCP  CPP  MB  HA/TCP  Material 0-Day  7-Day  14-Day  28-Day  0-Day  OP  7-Day  14-Day  28-Day  OC 2.50E-04  Exp ressio n relative to G A P D H  6.00E-02  Expression relative to G AP DH  MB  Material  5.00E-02  4.00E-02  3.00E-02  2.00E-02  1.00E-02  0.00E+00  2.00E-04  1.50E-04  1.00E-04  5.00E-05  0.00E+00  CPP  HA/TCP  MB  CPP  HA/TCP  Material 0-Day  7-Day  14-Day  MB  Material 28-Day  0-Day  7-Day  14-Day  28-Day  BSP  Expression relative to GA PD H  2.50E-04  2.00E-04  1.50E-04  1.00E-04  5.00E-05  0.00E+00 CPP  HA/TCP  MB  Material 0-Day  7-Day  14-Day  28-Day  Figure 13: Expression of Osteogenic Genes on the Different Substrates Over a Period of 28 Days Relative to GAPDH. All Values are Presented as the Mean ± One Standard Deviation.  The standard deviation of most samples, as shown in Figure 13, is high. To determine the source of this variance, all of the data points were plotted as shown in Figure 14 for ALP, with the exception of those for Day 0 as no expression was detected at this endpoint. The mice are distinguished according to numbers 1 through 3, while the replicate analysis (i.e. 2 52  samples analyzed per material implanted in each mouse) is labeled according to ‘A’ or ‘B’. To ease the interpretation of the graphs, all of the data points for each material are connected by a line, one for each of the three materials. The graphs corresponding to Figure 14 for the other genes (i.e. Col I, OP, OC and BSP) are shown in Appendix II.  ALP- 7Day  ALP- 14Day 3.50E-03  Expression relative to G AP DH  Exp ressio n relative to G A P D H  3.00E-03  2A  2.50E-03  2.00E-03  '1A 1.50E-03  '1A  2B 1B  2A  2B  3A  1.00E-03  3B 2A  5.00E-04  '1A  2B  1B 1B  0.00E+00  3A  3B  3A  3B  3.00E-03  1B  2.50E-03  '1A  2.00E-03  2B  1.00E-03  '1A  1B  HA-7Day  3A 3A  2A  5.00E-04  2B  '1A  1B  0.00E+00  Data Point CPP-7Day  3A  2A  1.50E-03  2A  3B 3B 3B  2B  Data Point MB-7Day  CPP-14Day  HA-14Day  MB-14Day  ALP- 28Day  Expression relative to G AP DH  1.80E-03  3B  1.60E-03 1.40E-03 1.20E-03 1.00E-03 8.00E-04  2A  2B  6.00E-04 4.00E-04  '1A '1A  2.00E-04  1B 1B 1B  3B 2A 2A  '1A  2B 2B  3A 3A  3B  3A  0.00E+00  Data Point CPP-28Day  HA-28Day  MB-28Day  Figure 14: ALP Expressions Relative to GAPDH for Each Mouse (1, 2, and 3). Letters 'A' and 'B' Represent the Replicate Analysis Taken From Each Mouse.  From Figures 14, it can be concluded that the large standard deviation comes largely from variability associated with using different mice, and not the replicate analysis made per mouse.  53  Table 4: Statistical Significance Chart for In vivo study. ‘Yes’ Signifies that the Pair was Statistically Significant. Material-Time 1 is Compared to Material-Time 2 using StudentNewman Keuls (p < 0.05). With Respect to Material-Time 2 (+) Shows that Material-Time 1 has a Greater Value, While (-) Shows that it has a Lower Value. MaterialTime 1 CPP-0  CPP-7  CPP-14  CPP-28 HA/TCP-0  HA/TCP-7  HA/TCP-14 HA/TCP-28 MB-0  MB-7 MB-14  2.4  MaterialTime 2 CPP-7 CPP-14 CPP-28 HA/TCP-0 MB-0 CPP-14 CPP-28 HA/TCP-7 MB-7 CPP-28 HA/TCP-14 MB-14 HA/TCP-28 MB-28 HA/TCP-7 HA/TCP-14 HA/TCP-28 MB-0 HA/TCP-14 HA/TCP-28 MB-7 HA/TCP-28 MB-14 MB-28 MB-7 MB-14 MB-28 MB-14 MB-28 MB-28  BrdU  mRNA  cDNA  ALP  Col I  OP  OC  BSP  Yes(-) Yes(-)  Yes(-) Yes(-) Yes(-)  Yes(-)  Yes(+)  Yes(+) Yes(+)  Yes(-) Yes(-)  Yes(-)  Yes(+)  Yes(+) Yes(-) Yes(-) Yes(-)  Yes(+)  Yes(-) Yes(-) Yes(-)  Yes(-)  Yes(+)  Yes(+)  Yes(-) Yes(-)  Discussion  This study demonstrated that the scaffold material chosen does have an impact on proliferation and differentiation of MSC in an in vivo setting. It also verified the effectiveness of several analytical techniques in gaining the results. The proliferation and differentiation of MSC on CPP was compared to that on HA/TCP and MB.  54  2.4.1  Seeding  The MSCs were cultured and seeded on the scaffold material in an osteogenic medium and then implanted under the back skin of NOD/Scid mice. By adapting the seeding method proposed by Dar et al, and later used by Siggers(2007) in an in vitro study involving the same scaffold material, we achieved very high seeding efficiencies for CPP, HA/TCP, and MB (94.87% ±3.31, 97.05% ±1.11, 94.59% ±2.86 respectively). These high seeding efficiencies ensured that sufficient MSCs were attached to the substrates. The final seed count prior to implantation averaged over 1.2 million MSC successfully seeded per sample. As the techniques used to remove cells from the scaffold and separate the GFP+ rat cells from the mice cells post-harvest were not expected to have a 100% recovery of cells, the greater number of cells seeded the more cells available for the final analytical technique.  2.4.2  Cellular Proliferation  As the amount of BrdU incorporation was found to be nil 7 days following implantation of the MSC seeded scaffolds, it is possible that the cells entered a quiescent stage following implant. One possible reason for this first quiescent stage is that the transferring of the scaffolds+cells from an osteogenic media in a culture plate rich in growth factors to an in vivo subcutaneous site, not as high in these factors initially, ‘shocked’ the cells. Once the cells had acclimatized to the body of the mice, and their site of implant, the cells recovered and were able to continue growing and proliferating. At Day 14, the degree of BrdU incorporation was greatest for CPP and HA/TCP, with a slightly lower incorporation found with MB. Meanwhile, at Day 28, the BrdU incorporation dropped from that found at Day 14 for all scaffold materials and was lowest for HA/TCP. The difference in BrdU incorporation at any endpoint between the materials was not significant. This signifies that CPP is as good as both MB and HA/TCP in terms of cellular proliferation of our GFP+ cells. As the drop in 55  BrdU incorporation from Day 14 to Day 28 was found to be significant for each of the materials, it is expected that the cells are entering a stage of differentiation. The cells appear to no longer be proliferating at some point between Day 14 and Day 28 and the BrdU incorporation starts to decrease. However, between Day 7 and Day 14 there should be a significant number of highly proliferating cells, such as osteoprogenitors and preosteoblasts (Aubin, 1998), present as the incorporation increased.  2.4.3  Cell Isolation Post-Harvest  To remove the cells from the scaffold material post-harvest, a collagenase/dispase + CaCl2 reaction was used. The recovery of GFP+ cells was found to be fairly low for CPP, but even lower for HA/TCP and MB (26.64 ±8.46%, 14.46 ±8.57%, and 14.07 ±3.28% respectively). However, with a final seeding count greater than 1.2 million pre-implantation a lower recovery from a C/D step still resulted in more than 45,000 GFP+ cells being available for the next step of the analysis in either BrdU or qRT-PCR streams.  The fraction of GFP+ cells recovered using Magnetic Sorting with anti-PE magnetic beads following the C/D step in the qRT-PCR stream was greatest for CPP and MB, and significantly lower for HA/TCP (79.51% ±12.05, 73.78% ±23.54, and 25.64% ±3.65, respectively). In breaking up the particles during the C/D step to increase the surface area available for the enzymatic reaction, the HA/TCP material became very fine and some of it travelled through the filters with the cells. As a result it is thought that the finely ground HA/TCP still present during magnetic sorting may have impeded the selection process of the anti-PE magnetic beads and, hence, a lower fraction of GFP+ cells recovered compared to the other two materials. Even with this lower recovery, the number of GFP+ cells available for the final steps of the qRT-PCR analysis still numbered above 10,000 for each material. 56  2.4.4  mRNA Isolation and Reverse Transcription  The amount of mRNA measured, as one of the steps of the qRT-PCR stream following magnetic sorting, was also found to be lowest with HA/TCP and could similarly be due to the interference of the finely ground material. However, the resulting cDNA measured following reverse transcription did not show a similar trend for the scaffold materials. For the cDNA measured, there were no statistically significant differences between materials at any given endpoint. As the same volume of primers is added to the samples of isolated RNA for reverse transcription (and the creation of cDNA) to occur there is not expected to be any difference between materials at each endpoint for the cDNA measured.  2.4.5  Cellular Differentiation  In addition to an analysis of the proliferation characteristics, an analysis of gene expression helps to determine the extent of differentiation along the osteblast lineage. Cells progress from an early progenitor to a fully functional matrix synthesizing osteoblast as a result of the biological periods of cellular proliferation, cellular maturation and focal mineralization, each associated with characteristic changes in gene expression (Knabe et al., 2004; Aubin, 1998). Col-I is prevalent during the initial period of proliferation and extracellular-matrix biosynthesis, followed by ALP which shows up during the post-proliferative period of extracellular-matrix maturation. Mature mineralizing osteoblasts become embedded into the secreted matrix and differentiate terminally to become osteocytes. These osteocytes do not express ALP, but OP and other bone matrix proteins. Thus, OP, BSP, and OC appear during the third period of extracellular matrix mineralization.  Col-I increased from 7-Day to 14-Day for CPP and HA/TCP with a drop down to the 28-day expression, while the maximum expression was at 28-day for MB. The difference between 57  MB and CPP, as well as MB and HA/TCP at 28-days was significant. In addition, CPP at 0days and 14-days, and MB at 28-days compared to 0 and 14-days were all statistically different pairs. With the exception of the 28-day analysis of MB, this Col-I analysis corresponds to that achieved with the proliferation study – Day 14 appears to be the time point with the greatest number of proliferating cells (i.e. immature osteogenic cells), but after this point the cells are maturing. Meanwhile, with ALP there is no definite trend in expression levels, nor any statistically significant differences observed. For 7-day and 14day samples, HA/TCP has the lowest ALP expression of the three materials, but at 28-days it conversely had the highest. For 0-day and 7-day analysis, HA/TCP was shown to have the highest OP and BSP expression, whereas at 14-day and 28-day MB has the highest. As neither the OP nor the BSP analysis revealed statistically significant results, this suggests that each of the three materials are similar in the number of immature osteogenic cells which are present during the period of in vivo study. With the exception of the 14-day levels, MB expression of OC is the highest of the three materials followed by CPP, and lastly HA/TCP. At 0-day, MB was statistically different from CPP, as well as from 7-day MB. However, aside from these two exceptions, OC levels were not significantly different between materials for any given endpoint, nor between endpoints for any given material. The qRT-PCR analysis done in this study indicates that each of the three scaffold materials had very low expression levels of the late stage markers BSP and OC. This suggests that minimal late stage osteoblast maturation or mineralization occurred. In addition, as a drop in proliferation did not correspond with an increase in differentiation, it is possible that the MSCs seeded on the scaffolds became quiescent following some initial proliferation and required more signal (such as provided by properly harvested allograft/autograft) to undergo differentiation. As the standard deviation of most samples was high, an analysis was done to determine the source of this variance – whether due to replicate data taken from each scaffold per mouse, or 58  to factors associated with separate mice. It was concluded that the large standard deviation comes largely from variability associated with using different mice, and not the replicate analysis made per material implanted in each mouse.  As cell-substrate interaction is still not fully understood, it is unclear what component of the scaffolds surface causes the proliferation and differentiation on CPP, HA/TCP, or MB. However, as this study showed that MSC-seeded scaffolds underwent proliferation but did not show an increasing degree of differentiation with a proliferation decrease, a recommendation for the addition of inductive factors is included in this report. At the minimum, the scaffold defines the ultimate shape of the new bone and cartilage (Causa et al., 2005). It is likely that two quiescent stages occurred in the 28 days of cell-substrate implantation. The first followed directly after implant of the cell-substrates but was overcome as the BrdU incorporation dramatically increased at Day 14. Meanwhile, the second was not so easily overcome as the drop in proliferation following Day 14 did not correspond to an increase in differentiation such that it is recommended that additional factors may be needed to stimulate this transference. Synthetic biomaterials that are endowed with matricellular cues and are able to deliver morphogenic factors are emerging today to guide various tissue regeneration processes (Causa et al., 2007; Stevens et al., 2007). CPP had a similar impact on cellular growth and differentiation, in general, as both MB and HA/TCP which makes it a promising candidate for a synthetic bone graft substitute. This differs slightly from what was found in the in vitro study by Sigger et al. (2007) in which it was found that CPP and MB were equally able to support MSC expansion rather than differentiation, and that HA/TCP instead favoured MSC differentiation and maturation along the osteogenic lineage. In my in vivo study the three materials (CPP, MB and HA/TCP) were found to be equally able to support both proliferation and differentiation to a point along the 59  osteogenic lineage. One possible reason for the difference in in vitro and in vivo results is that the environment in the in vitro osteogenic media does not fully match what is seen by the cells in their in vivo subcutaneous site.  2.5  Conclusion  In this study, marrow stromal cells seeded on morsellized bone chips, calcium polyphosphate, and biphasic hydroxyapatite/tri-calcium phosphate were shown to have similar proliferation characteristics and osteogenic gene expression when implanted subcutaneously in mice for a period of 7, 14, and 28 days. Each of the scaffold materials was shown to be able to support immature proliferating MSCs and induce some differentiation of MSCs down the osteogenic lineage. As both properties are necessary for sustained bone regeneration and growth, CPP has been shown in this study to be a promising bone graft substitute candidate.  60  2.6  References  Applied Biosystems Website (Accessed July 2009): www.appliedbiosystems.com AB7900HT Product Information Sheets. Aubin, J.E. “Bone Stem Cells”, Journal of Cellular Biochemistry Supplement. 30/31: 73-82, 1998. BD Biosciences Website (Accessed July 2009): www.bdbiosciences.ca Bianco, P., Riminucci, M., Gronthos, S., and Robey, P.G. “Bone Marrow Stromal Stem Cells: Nature, Biology, and Potential”, Stem Cells. 19: 180-192, 2001. Bolland, B., Tilley, S., New, A., Dunlop, D., and Oreffo, R. “Adult mesenchymal stem cells and impaction grafting: a new clinical paradigm shift”, Expert Rev. Med. Devices. 4: 393-404, 2007. Caplan, A., and Bruder, S. “Mesenchymal stem cells: building blocks for molecular medicine in the 21st century”, Trends in Molecular Medicine. 7: 259-263, 2001. Causa, F., Netti, P., and Ambrosio, L. “A multi-functional scaffold for tissue regeneration: The need to engineer a tissue analogue”, Biomaterials. 28: 5093-5099, 2007. Causa, F., Nettie, P., Ambrossio, L., Ciapette, G., Pagani, B., Martini, D., and Giunti, A. “ Poly-eta-caprolacton/hydroxyapatite composites for bone regeneration: in vitro characterization and human osteoblast response”, Journal of Biomedical Materials Research Part A. 151-162, 2005. Chamberlain, G., Fox, J., Ashton, B., and Middleton, J. “Concise Review: Mesenchymal Stem Cells: Their Phenotype, Differentiation Capacity, Immunological Features, and Potential for Homing”, Stem Cells. 25: 2739-2749, 2007. Chen, J., Wang, C., Lu, S., Wu, J., Guo, X., Duan, C., Dong, L., Song, Y., Zhang, J., Jing, D., Wu, L., Ding, J., and Li, D. “In vivo chondrogenesis of adult bone-marrow-derived autologous mesenchymal stem cells”, Cell Tissue Res. 319: 429-438, 2005. Ciapette, G., Ambrosio, L., Marletta, G., Baldini, N., and Giunti, A. “Human bone marrow stromal cells: In vitro expansion and differentiation for bone engineering”, Biomaterials. 27: 6150-6160, 2006. Engler, A., Sen, S., Sweeney, H., and Discher, D. “Matrix Elasticity Directs Stem Cell Lineage Specification”, Cell. 126: 677-689, 2006. Frei, H., Mitchell, P., Masri, B., Duncan, C., and Oxland, T. “Allograft impaction and cement penetration after revision hip replacement – A histomorphometric analysis in the cadaver femur”, The Journal of Bone & Joint Surgery”. 86-B: 771-776, 2004. Hing, K.A. “Bioceramic Bone Graft Substitutes: Influence of Porosity and Chemistry”, Applied Ceramic Technology. 2: 184-199, 2005. Hing, K.A. “Bone repair in the 21st century: biology, chemistry or engineering”, Philosophical Transactions: Mathematical, Physical and Engineering Sciences. 362: 2821-2850, 2004. Johnson, A. “FACS Course”, UBC Biomedical Research Center. 2008. Knabe, C., Berger, G., Gildenhaar, R., Howlett, C.R., Markovic, B., and Zreiqat, H. “The functional expression of human bone-derived cells grown on rapidly resorbable CP ceramics”, Biomaterials. 25: 335-344, 2004. Kraus, K.H., and Kirker-Head, C. “Mesenchymal Stem Cells and Bone Regeneration”, Veterinary Surgery. 35: 232-242, 2006. Livingston, T., Gordon, S., Archambault, M., Kadiyala, S., McIntosh, K., Smith, A., and Peter, S. “ Mesenchymal stem cells combined with biphasic calcium phosphate 61  ceramics promote bone regeneration”, Journal of Materials Science: Materials in Medicine. 211-218, 2003. Livingston-Arinzeh, T., Tran, T., Mcalary, J., and Daculsi, G. “A comparative study of biphasic calcium phosphate ceramics for human mesenchymal stem-cell-induced bone formation”, Biomaterials. 26: 3631-3638, 2005. Naveiras, O., and Daley, G. “ Visions & Reflections: Stem Cells and their niche: a matter of fate”, Cellular and Molecular Life Sciences. 63: 760-766, 2006. Nijweide, P., Burger, E., and Feyen, J. “Cells of Bone: Proliferation, Differentiation, and Hormonal Regulation”, Physiological Reviews. 66: 855-886, 1986. Pilliar, R.M., Filiaggi, M.J., Wells, J.D., Grynpas, M.D., and Kandel, R.A. “Porous calcium polyphosphate scaffolds for bone substitute applications – in vitro characterization.” Biomaterials 22-9, p.2063-2070. 2002. Pittenger, M., Mackay, A., Beck, S., Jaiswal, R., Douglas, R., Mosca, J., Moorman, M., Simonetti, D., Craig, S., and Marshak, D. “Multilineage Potential of Adult Human Mesenchymal Stem Cells”, Science. 284: 143-147, 1999. Real Time PCR Tutorial Website (Accessed July 2009): http://pathmicro.med.sc.edu/pcr/realtime-home.htm Roche Applied Science Website (Accessed August 2009): www.roche-applied-science.com Collagenase/Dispase Product Information. Schieker, M., Seitz, H., Drosse, I., Seitz, S., and Mutschler, W. “Biomaterials as Scaffold for Bone Tissue Engineering”, European Journal of Trauma. 2:114-122, 2006. Siggers, K. University of British Columbia M.A.Sc. Thesis, 2007. Stem Cell Information – The National Institutes of Health resource for stem cell research Website (Accessed August 2009). http://stemcells.nih.gov/ Stem Cell Technologies Website (Accessed July 2009): http://www.stemcell.com/ Stevens, M. and George, J. “Exploring and Engineering the Cell Surface Interface”, Science. 310: 1135-1138, 2005. Stevens, B., Yang, Y., Mohandas, A., Stucker, B., and Nguyen, K.T. “A Review of Materials, Fabrication Methods, and Strategies Used to Enhance Bone Regeneration in Engineered Bone Tissue”, Journal of Biomedical Materials Research Part B: Applied Biomaterials. 573-582, 2007. Xie, H., Yang, F., Deng, L., Luo, J., Qin, T., Li, X., Zhou, G., and Yang, Z. “The performance of a bone-derived scaffold material in the repair of critical bone defects in a rhesus monkey model”, Biomaterials. 28: 3314-3324, 2007. Xu, J., Qin, H., Wang, X., Zhou, Q., Luo, F., Hou, T., and He, Q. “Repair of large segmental bone defects using bone marrow stromal cells with demineralized bone matrix”, Orthopaedic Surgery. 1: 34-41, 2009. Zaidi, M. “Skeletal remodeling in health and disease”, Nature Medicine. 13: 791-801, 2007. Zhou, Y., Chen, F., Ho, S., Woodruff, M., Lim, T., and Hutmacher, D. “Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts”, Biomaterials. 28: 814-824, 2007.  62  3.0  Mechanical Evaluation of Synthetic Scaffolds for Use with Revision THR2  3.1  Introduction  Impaction Allografting (IA) requires that the graft be adequately compacted to provide the initial stability, to prevent early massive subsidence, and to induce bone remodeling and graft incorporation. Without bone remodeling and graft incorporation occurring, the graft will eventually collapse and cause the implant to fail (Bolland et al., 2007-2; Board et al., 2006). Even if distal migration (also termed ‘subsidence’) does not lead to failure, it has been associated with high levels of thigh pain, with a reduction in a patient’s ‘quality of life’ and is clinically undesirable. Unfortunately, there is currently no specific indicator or guideline to enable the surgeon to decide when the graft is adequately compacted and concern regarding fracture may lead to under-compaction of the graft during surgery and subsequent prosthesis subsidence.  There are many factors in vivo that affect the subsidence of allograft including: graft preparation, particle morphology and size, graft composition, graft quality, impaction techniques, postoperative loading, and various host variables such as immune response and interface of host bone-graft (Blom et al., 2002; Frei et al., 2005-2; Heiner et al., 2005; Karrholm et al., 1999). Of interest in this mechanical study are the graft materials chosen, the degree of sample conditioning, impaction techniques, and the resultant mechanical properties.  2  A version of this chapter will be submitted for publication. Comeau, P., Siggers, K., and Fernlund, G. Mechanical Evaluation of Synthetic Scaffolds for Use with Revision Total Hip Replacements.  63  3.1.1  Material Selection  3.1.1.1 Impaction Allografting Materials IA currently consists of cementing the femoral component of the implant into a compressed bone graft bed and creating a layered composite of implant, cement, allograft bone, and host bone. Poly-(methyl methacrylate) (PMMA) has been widely used in orthopaedic surgery as the traditional bone cement and is one material component of IA. Some in vitro studies indicate that cemented IA techniques should be used instead of un-cemented as the cement produces a solid layer of graft-cement composite that greatly reduces the potential number of graft shear planes, and results in a greater initial stability than obtained using un-cemented techniques (Board et al., 2006). Frei et al (2005 and 2004) determined that cement penetration to the endosteal cortex would be beneficial for the clinical stability of the IA construct, even if this penetration limits the degree of allograft and cortical revascularization and remodeling. Excessive cement penetration, however, will prevent revascularization of the graft layer in patients and could lead to further necrosis of the surrounding bone (Bolder et al., 2003). In addition, in the presence of cement, a fibrous capsule may form around the implant if the cement composition is such that there is a lack of biocompatibility, toxic monomers can leach out, and heat will be released during the exothermic curing reaction (Ginebra et al., 2004). Due to these disadvantages, and in cases where the bone stock deficiency of the proximal femoral bone is sufficiently severe, the use of a cementless longstemmed implant is recommended in combination with allograft bone (Meding et al., 1997; Nickelsen et al., 2008).  Morsellized cancellous bone (MB) is a biphasic material consisting of bone particles and a fluid phase, and is the other material component of IA. It has a relatively low stiffness and exhibits time-dependent material behaviour of a viscoelastic-viscoplastic nature which is 64  believed to contribute to implant migration in IA (Albert et al., 2008). Morsellized allograft for IA is obtained from two or three femoral heads retrieved during primary THA (Blom et al., 2002; Bolder et al., 2003; Cornu et al., 2003). These femoral heads are then either fresh frozen at -80ºC or freeze dried and stored at room temperature. The bone preparation techniques have been shown to affect both the biological and mechanical properties of the graft, as well as the likelihood of a successful outcome (Tanabe et al., 1999). Freeze dried bone reduces the immunogenic load and risk, but it has also been shown to have inferior mechanical properties (i.e. less strong and stiff, but more brittle) in comparison with freshfrozen bone (Bolland et al., 2007; Cornu et al., 2003). In addition to infection antigenicity, other problems associated with MB include limited availability, reproducibility, and cost. The use of synthetic scaffold materials, either in whole or mixed with the morsellized allograft, could reduce some of these issues and potentially improve the biomechanics of the graft.  As mentioned in Chapter 1, there have been several materials considered for use as a synthetic scaffold in orthopaedic applications where graft incorporation and bone regeneration would be ideal. Unfortunately, very few are currently recommended for loadbearing applications. Synthetic scaffold materials used with revision THR must initially provide sufficient mechanical support for the prosthesis and must also provide a framework onto which the host bone and vascular network can regenerate and heal. Mechanical properties to consider include strength (i.e. compressive, tensile and bending), strain to failure, fracture toughness, and material stiffness. Calcium Polyphosphate (CPP) is a synthetic material that has shown promise for use with revision THRs and is of interest in this study.  65  3.1.1.2 Composite Graft Bed Karrholm et al. (1999) proposed that the most critical success factor for revision THR may be the graft material. This can be related to basic soil mechanics studies which form the basis for the behaviour of bone graft under load (Bolland et al., 2008). A compacted aggregate that is resistant to shear loading should have a graft that is rigidly contained, has a well-graded particle-size distribution, is porous enough to allow fluid to escape (which serves to minimize any pore-fluid and pressure generation), is sequentially layered and well-mixed when compacted, uses large impaction energies, and may need vibration during compaction (Dunlop et al., 2003; Bolland et al., 2007-2). Particle washing has also been found to improve the shear strength of the graft (Bolland et al., 2007).  Studies have shown that mixing of MB with a synthetic ceramic material such as HA/TCP can improve the uniaxial mechanical behavior and initial stability of the construct as it decreases the strain, increases the confined modulus, decreases creep rate, and increases the graft’s resistance to subsidence (Voor et al., 2004; Blom et al., 2002; Bolder et al., 2002; Bolder et al., 2003). This increased initial stability would allow earlier and greater load bearing of the patients post-operatively and could potentially stimulate osteogenesis that much sooner. In fact, a study by Coathup et al. (2008) showed that there was no significant difference between a 50:50 mixture of HA and allograft and a 90:10 mixture in IA when the degree of new bone formation was considered using an experimental sheep model. Other studies have similarly shown that the combination of bioactive ceramics with polymers may also improve the mechanical properties of the scaffolds used in IA (Hutmacher et al., 2007).  In this study composites of particulate CPP and Poly-(L-lactic acid) (PLA) were considered with the idea that PLA would improve upon the confined compression behaviour of CPP alone. It is hypothesized that the softer polymer will increase the number of contact points in 66  the graft bed and allow for a greater fraction of the applied load to be transferred, thus increasing the graft bed stiffness. If a graft bed stiffness closer to that of PMMA, and significantly better than the ‘golden standard’ of MB, can be achieved with the use of synthetic particles, then there will be yet further support for the use of cementless revision THR.  To better understand the mechanical behaviour of CPP, composites of CPP with PMMA were also tested in this study. In particular, this study is interested in observing how the presence of CPP particles within the solid PMMA matrix will alter its performance as earlier studies have shown that the presence of PMMA alone enhances the structural integrity of the IA construct.  3.1.2  Testing Technique  The principal loading of the graft is confined compression (radially and axially) and shear (axially). A recent study has shown that the compressive modulus of the graft typically plays a bigger role than shear strength for implant stability (Albert, 2009). Thus, the focus of this study is on the compressive behaviour of the graft. The uniaxial confined compression model used in this study is designed after the femur cavity in which the graft bed and femoral implant will be inserted during IA.  Albert et al. (2008) concluded that the density of the MB graft bed may depend on the impaction forces as well as on the grading. They showed that increasing the impaction force increased the density of the graft bed by 41%, with a corresponding stiffness and shear strength increase of 93 and 164%, respectively. Albert et al. (2008) also realized the significance of maximizing impaction forces and suggested that, to achieve the necessary force during surgery, the use of femur reinforcement could be beneficial in reducing the risk 67  of intra-operative fracture. One possible femur reinforcement method is the use of strut grafts (Orthodynamics UK Website). Unfortunately, the IA technique is highly operatordependent and the forces that are applied to the graft can vary significantly between surgeons.  In this study, the concept of increasing impaction force to increase graft bed density is extended to the idea of sample conditioning. Samples with no prior conditioning (termed ‘un-compacted’) and those that have previously been loaded and pre-compacted (termed ‘pre-compacted’) are compared using the confined compression model. The construct modulus (or sample stiffness) is interpreted as the secant modulus at a stress of 1100kPa, as was done in an earlier study by Siggers (2007), and is representative of the effective stiffness as it is used clinically. It is hypothesized that the pre-compacted samples will have a greater construct modulus as this prior conditioning will have increased the number of contact points within the graft bed (i.e. the graft bed density) and allow for greater load transference when they are tested in confined compression.  Another point of interest in this study is how the material stiffness (Ep) and the construct stiffness (Ec) compare. Siggers (2007) showed that construct stiffness increases with material stiffness but that the construct stiffness is generally at least two orders of magnitude less than the material stiffness. Thus, the particulate graft is a very inefficient way to create a stiff construct. In this study, I will determine whether it is possible to achieve a lower Ep/Ec ratio, such that the graft bed will better utilize the mechanical properties of the particles. The lower the Ep/Ec ratio and the closer it is to unity, the greater the graft bed effectivness of the construct. This ratio is essentially taken as a measure of graft bed effectiveness.  68  The objective of this mechanical study is to determine how the construct stiffness of particulate and solid composites is affected by prior sample conditioning when tested in confined compression. The results are compared to confined constructs of MB and HA/TCP that have been tested under similar conditions. In this study the analysis of the confined compressive stiffness is comparative and should not be treated as reportable material values. 3.2  Materials & Methods  3.2.1  Materials  The CPP material used in this study was provided by Dr. Bob Pilliar’s research group at the University of Toronto (Pilliar, R.M. et al., 2002). It was formed by first calcining the precursor powder of calcium phosphate monobasic monohydrate (CPMM) in a platinum crucible at 500ºC for 10 hours. The reaction that occurred as a result of this calcining is as follows:  nCa(H2PO4)2*H2O  [Ca(PO3)2]n + 3nH2O CPMM  CPP powder  The CPP powder was then melted at 1100ºC to produce an amorphous glass, poured directly into distilled water to form an amorphous frit and then dried in 100% ethanol. Next, the powders were gravity sintered (i.e. pressure-less sintered) in cylindrical platinum tubes at approximately 970ºC for 2hours and crushed into angular particles of 1-3mm in diameter. The resulting particles have an interconnected porous network of 30-45 vol% porosity and an internal pore size in the range of 100um (Pilliar, R.M. et al., 2002). In addition, upon X-ray diffraction analysis Pilliar et al. (2001) confirmed that the gravity sintering of this amorphous frit in the aforementioned technique created stable room-temperature mono-clinic β-CPP of a  69  crystalline nature. Pilliar et al. (2002) reported that the compressive modulus of the formed CPP particles was approximately 5GPa and the tensile strength 5-24MPa. A Poly-(L-lactic acid) (PLA) polymer was provided by NatureworksTM and was used in combination with CPP to study the impact of particulate composites on the mechanical properties observed through compression testing. The PLA particles were roughly 4-5mm in diameter. Similarly, PMMA (Lecoset, Leco Corporation, USA) was used as a model binder system in combination with CPP to form solid composite cylinders of 30-50mm in height and 16-17mm in diameter.  HA/TCP particles (Berkeley Advanced Biomaterials Inc.) consisted of a 20% HA and 80% TCP composition. The particles were 1-3mm in size and consisted of pores approximately 250um in diameter. This material provided the synthetic standard as it has been proven in prior studies to be successful for use with bone grafting in sites of limited loading.  Morsellized bone (MB) chips that were analyzed by Siggers (2007) provided the “golden standard” to compare all the un-compacted samples to, while those analyzed by Albert (2009) provided the standard for the pre-compacted samples. Table 5 provides relevant properties of the materials analyzed in this study. Figure 15 shows PLA in comparison to CPP and Figure 16 shows 100% PMMA solid constructs. Figures 17 and 18 show the solid composites of CPP and PMMA in 25:75 and 50:50 mixtures, respectively. The HA and MB particles that were mechanically tested and reported in this chapter are as depicted in Figure 4 (Chapter 2).  70  Table 5: Properties of Materials Used in Testing Material CPP HA/TCP MB PLA PMMA a b c d e f  Particle Diameter (mm) 1-3 1-3 0.6-0.8 4-5 --  Particle Shape Irregular Irregular Irregular Elongated Sphere --  Young’s Modulus Ep (GPa) 5.00a 1.80 – 4.20b 0.10 – 1.00c 2.70d 1.80 – 3.10e  Tensile Strength (MPa) 5.00 - 24.00a 0.01 – 4.03f 10.00 -20.00d 50.00d 59.00d  From Pilliar et al., 2001 (measured using diametral compression testing). From Miranda et al., 2007. From Gibson et al., 1988. From Murugan et al., 2005. From Matbase website, 2009. The tensile strength of this material was calculated from the de Groot equation of σT(MPa) = 220exp(-20Vm), where Vm is the volume fraction of microporosity, for traditional CP ceramics (Hench, 1991). The range of microporosity is taken as 20-50%.  Figure 15: PLA Particles (Left), CPP Particles (Right).  Figure 16: 100% PMMA Solid Constructs. 71  Figure 17: Images of the CPP:PMMA (25:75) Solid Composites Sectioned.  Figure 18: Images of the CPP:PMMA (50:50) Solid Composites Sectioned.  Scanning electron microscope (SEM) images were obtained for all samples tested in my study (i.e. all but MB). The samples were mounted on metal pedestals, sputtered with gold in a vacuum argon atmosphere with a Denton Vacuum (Desk II, USA), and then imaged with an SEM machine (HITACHI, Berkshire). Figure 19 shows PLA, HA/TCP and CPP, and Figure 20 shows 100% PMMA solid constructs, as well as the solid composites of CPP and PMMA in 25:75 and 50:50 mixtures.  72  500um  500um  500um  Figure 19: SEM Images at 60x Magnification of PLA (Left), HA/TCP (Middle), and CPP (Right) Particles.  200um  200um  200um  Figure 20: SEM Images at 80x Magnification of PMMA (Left), 25:75 CPP:PMMA (Middle), and 50:50 CPP:PMMA (Right) Samples.  3.2.2  Methods  The particulate and solid cylindrical composites were tested in confined compression using a servohydraulic testing machine (Instron Model 8872, Instron, Canton, Massachusetts) as shown in Figure 21. The particulate mixtures filled the stainless steel cylinder (19mm inner diameter and 6mm wall thickness) to a height of roughly 35mm, while the cylinders were 3050mm high. The pre-compacted samples were first loaded to a maximum stress of 10MPa and then fully unloaded to a near-zero stress. The testing of all samples (whether precompacted or not) consisted of loading the samples via a piston at a waveform rate of 1.0mm/min and a sample rate of 5.0 Hz to a maximum stress of 2MPa. Three samples (N=3) were tested for each sample type. Test matrices are shown in Table 6. The stress was calculated as the applied normal force divided by the cross sectional area of the graft bed (the latter of which was constant). The compressive strain was defined as [instantaneous height 73  displacement]/H0, where H0 is the initial height of the graft bed. From a compressive stress of 1100kPa and the corresponding strain measured, the secant modulus was determined as is shown at the beginning of section 3.3 (Figures 22 and 23).  Table 6: Mechanical Test Matrix Test No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18  Material 1 HA/TCP (100%)  -  Material 2  Scaffold Type Particulate  CPP (100%)  -  Particulate  CPP (80%)  PLA (20%)  Particulate  CPP (50%)  PLA (50%)  Particulate  CPP (20%)  PLA (80%)  Particulate  PLA (100%)  -  Particulate  PMMA (100%)  -  Solid  CPP (25%)  PMMA (75%)  Solid  CPP (50%)  PMMA (50%)  Solid  Conditioning None Pre-compacted None Pre-compacted None Pre-compacted None Pre-compacted None Pre-compacted None Pre-compacted None Pre-compacted None Pre-compacted None Pre-compacted  Figure 21: Experimental Setup for Confined Compression.  74  To determine the volume fraction of material within the confined model cylinder for the particulate composites the mass was recorded of each material in the composite before placing the particles in the cylinder of the model. The referenced density was then used to determine what apparent volume each material actually filled. Volume fraction was then calculated by adding the apparent volume of Material 1 and 2, and dividing this total occupied volume by the total available construct volume. The density of PLA was taken as 1.25 g/cm3 (Plastics Technology website, 2009).  3.2.3  Statistics  The effect of material and sample conditioning on construct stiffness (Ec) was analyzed using Student t-tests at a significance level of 0.05. Tables 9 and 10 show the results from the statistical analysis using Student t-tests, with ‘Yes’ indicating that the pair being compared was significantly different. With respect to Material 2, a ‘+’ indication in Table 9 states that Material 1 had a greater value while ‘-‘ states that Material 1 had a lower value.  3.3  Results  As mentioned previously, to determine the construct modulus (Ec) of the various testing conditions, the secant modulus was determined at a stress of 1100kPa on the stress-strain plots. This secant modulus is representative of the effective construct stiffness and was used in a prior study by Siggers (2007) for analysis of confined compression in the same set-up. The following figures depict this secant analysis for a sample of un-compacted 100% PLA (Figure 22) and pre-compacted 100% PLA (Figure 23).  75  Un-compacted PLA (100%) 2000 1800 1600  Stress (kPa)  1400 1200 1000 800 600 400 200 0 0.00  0.50  1.00  1.50  2.00  Strain (%)  Figure 22: Stress-strain Curve for a Sample of Un-compacted 100% PLA Construct. The Secant is Taken at 1100kPa to Represent the Construct Modulus of the Sample Under Confined Compression.  Pre-Compacted PLA (100%)  10000  Stress (kPa)  8000  6000  4000  2000  0 0.00  1.00  2.00  3.00  4.00  5.00  6.00  Strain (%)  Figure 23: Stress-strain Curve for a Sample of Pre-compacted 100% PLA Construct. The Secant is Taken at 1100kPa to Represent the Construct Modulus of the Sample Under Confined Compression.  As shown in Figures 22 and 23, the secant is taken at 1100kPa to represent the construct modulus of the sample under confined compression. The secant “zero” point for precompaction analysis is taken at the strain where the stress returns to approximately 0kPa after reloading. Table 7 reports the results obtained from this secant analysis. The stress-strain 76  curves for all samples tested are shown in Appendix III. As an approximate measure of the effective material modulus (Ep) for the composites, the volume weighted average modulus of the constituents is used. Neither of the MB construct moduli values given in the table were determined in this study. The Ec for the un-compacted MB was taken from the study by Siggers (2007), while that for the pre-compacted MB is obtained from Albert (2009). Albert (2009) performed 20 impactions at a frequency of 4Hz in a confined compression model and the corresponding construct moduli at the end of this impaction was extrapolated to our precompaction stress of 10MPa.  77  Table 7: Results Obtained with Confined Compression. Material MCB* (100%) MCB** (100%)  Conditioning  Size  Ep (Gpa)  Ec(Gpa)  Ep/Ec  None  (d=0.6-0.8mm)  0.10 – 1.00  0.015  6.71 – 67.11  0.10 – 1.00  0.038  2.63 – 26.35  3.00  0.004  729.95  3.00  0.334  8.99  5.00  0.018  275.48  Pre-comp.  5.00  0.258  19.38  None  4.54  0.011  428.41  Pre-comp.  4.54  0.462  9.83  None  3.85  0.015  257.17  Pre-comp.  3.85  0.332  11.60  None  3.16  0.033  94.86  Pre-comp.  3.16  0.301  10.49  2.70  0.055  81.40  2.70  0.235  19.17  2.001  1.22  0.924  2.65  1.727  1.83  0.845  3.74  1.182  3.11  0.635  5.79  Pre-comp.  Test No. 1 2 3 4 5 6 7 8 9 10 11 12  13 14 15 16 17 18  Particulate Composites HA/TCP (100%) HA/TCP (100%) CPP (100%) CPP (100%) CPP:PLA (80:20) CPP: PLA (80:20) CPP: PLA (50:50) CPP: PLA (50:50) CPP: PLA (20:80) CPP: PLA (20:80) PLA (100%) PLA (100%) PMMA (100%) PMMA (100%) CPP:PMMA (25:75) CPP:PMMA (25:75) CPP:PMMA (50:50) CPP:PMMA (50:50)  None  (d=1-3mm)  Pre-comp. None  None  (d=1-3mm)  (d=4-5mm)  Pre-comp.  None Pre-comp. None Pre-comp. None Pre-comp.  Solid Composites ~50mm High Cylinder 2.45 ~50mm High Cylinder 2.45 ~40mm High Cylinder 3.16 ~40mm High Cylinder 3.16 ~30mm High Cylinder 3.68 ~30mm High Cylinder 3.68  Note: Ep values for the composites are determined by the equation Ep = Vf1*Ep1 + Vf2*Ep2 with 1 and 2 representing Materials 1 and 2, respectively, as outlined in Table 6. *Ec value for un-compacted MB is from Siggers, 2007. **Ec value for pre-compacted MB is from Albert, 2009.  The volume fraction of material within the particulate composites is shown in Table 8.  78  Table 8: Volume Fraction in Un-compacted Particulate Composites of CPP and PLA. These Values are Reported as Mean ± One Standard Deviation. Material 1 CPP (100%) CPP (80%) CPP (50%) CPP (20%) a  Material 2 PLA (20%) PLA (50%) PLA (80%) PLA (100%)  Vf (%) 48.90 ± 0.90a 55.16 ± 0.07 59.26 ± 0.11 61.77 ± 0.04 62.48 ± 0.11  From Siggers, 2007.  From Table 7, the following plot (Figure 24) shows all the Ec values determined in this study alongside the MB constructs tested in prior referenced studies.  3.50  3.00  Ec (GPa)  2.50  2.00  MB* HA/TCP 100% CPP CPP:PLA (80:20) CPP:PLA (50:50) CPP:PLA (20:80) 100% PLA 100% PMMA CPP:PMMA (25:75) CPP:PMMA (50:50)  1.50  1.00  HA/TCP  0.50 HA/TCP MB*  MB*  0.00 Un-compacted  Pre-compacted  Figure 24: Ec of All Composites Tested. All Values are Presented as the Mean ± One Standard Deviation. MB Values are Referenced from Siggers (2007) and Albert(2009).  3.3.1  Particulate Composites  Ec values were plotted in Figure 25 to determine the impact of particulate composite composition on the construct modulus. 79  0.08  Un-compacted Samples 0.07 0.06  Ec (GPa)  0.05 0.04 0.03 0.02 0.01  C  PL A 10 0%  (2 0: 80 ) A PL  PP : C  PP :  PL  A  PL A CP P:  (5 0: 50 )  (8 0: 20 )  CP  P  P 10 0%  A/ TC H  M B*  0.00  (a) 0.80  Pre-compacted Samples 0.70 0.60  Ec (GPa)  0.50 0.40 0.30 0.20 0.10  PL A 10 0%  (2 0: 80 ) LA  C PP :P  A  (5 0: 50 )  (8 0: 20 ) PP :P L C  LA  C PP C PP :P  10 0%  A /T C P H  M B **  0.00  (b) Figure 25: Ec Results for (a) Un-compacted Particulate Composites, and (b) Pre-compacted Particulate Composites. All Values are Presented as the Mean ± One Standard Deviation. Note the Difference in Scale Between (a) and (b). MB Values are Referenced from Siggers (2007) and Albert(2009).  Construct modulus increases for the un-compacted samples as the volume fraction of soft PLA particles increases, while the opposite trend is seen for pre-compacted samples. The trend is statistically significant for the un-compacted particulate samples, but not for the precompacted. Similarly, HA/TCP is significantly different from the un-compacted particulate composite samples, but not from the pre-compacted samples. Both un-compacted and pre-  80  compacted particulate samples, however, are significantly different from MB with two exceptions (un-compacted 50:50 CPP:PLA and pre-compacted PLA).  3.3.2  Solid Composites  Ec values were plotted as shown in Figure 26 to determine the effect of a PMMA binder on the construct modulus of CPP.  3.50  3.00  MB* HA/TCP 100% PMMA CPP:PMMA (25:75) CPP:PMMA (50:50) 100% CPP  Ec (GPa)  2.50  2.00  1.50  1.00 HA/TCP 0.50 MB*  MB* HA/TCP 0.00 Un-compacted  Pre-compacted  Figure 26: Ec Results for Solid Composites. All Values are Presented as the Mean ± One Standard Deviation. MB Values are Referenced from Siggers (2007) and Albert(2009).  Figure 26 shows that as the fraction of PMMA decreases, Ec decreases, whether the samples are pre-compacted or not. This trend is not found to be statistically significant for either the un-compacted or the pre-compacted samples due to the large variability in data. However, both sets of un-compacted and pre-compacted solid constructs are found to be significantly different from MB with the exception of 50:50 CPP:PMMA. The un-compacted solid constructs are also shown to be significantly different from HA/TCP, though this was not 81  similarly found with the pre-compacted solid constructs. The exception for the HA/TCP comparison with the un-compacted samples is the 50:50 construct of CPP:PMMA and, for the pre-compacted samples, it is the 25:75 construct of CPP:PMMA.  Table 8 also shows that as the amount of PLA increases, the volume fraction of material increases (or that fraction of pores decreases). The packing density of the particulate composites is affected by the amount of PLA present.  3.3.3  Impact of Sample Conditioning  The impact of prior sample conditioning has been shown in Figures 24, 25, and 26. For the un-compacted samples (Figure 25a), the mean Ec values are all below 0.06GPa for the particulate composites. Pre-compaction of these particulate composites to 10 MPa increases the construct stiffness by roughly one order of magnitude. These results are statistically significant for all particulate constructs with the exception of 100% PLA. Pre-compaction decreases construct stiffness slightly for all solid constructs due to crack and damage formation. However, this latter outcome is not statistically significant due to the large variability in the results. It is also shown that material choice has an effect on un-compacted samples but little or no effect on pre-compacted samples. Very little statistical significance was observed in the pre-compacted data compared to the un-compacted. However, the composite samples tested in this study were shown to have greater construct stiffness than the 100% MB constructs referenced from studies by Siggers (2007) and Albert (2009) with four exceptions (un-compacted 50:50 construct of CPP:PLA, pre-compacted construct of PLA, and the un-compacted and pre-compacted constructs of 50:50 CPP:PMMA).  The Ep/Ec ratio, or measure of the graft bed effectiveness, is shown for all samples in Table 7. Solid composites most effectively utilize the mechanical properties of the individual 82  components and are affected little by pre-compaction. Pre-compaction of the particulate composite samples, however, improved the graft bed effectiveness by roughly one order of magnitude.  83  Table 9: Statistical Significance Chart for the Mechanical Study. Material 1 is Paired with Material 2 Using Student t-tests (p < 0.05). ‘Yes’ Signifies that the Pair Compared was Statistically Significant. With Respect to Material 2, ‘+’ Shows That Material 1 has a Greater Value, While ‘-‘ Shows that it has a Lower Value. Material 1 MB (100%)  Material 2  Un-compacted  Pre-compacted  HA/TCP (100%)  Yes (+)  Yes (-)  CPP (100%)  Yes (-)  Yes (-)  CPP:PLA (80:20)  Yes (+)  Yes (-)  CPP:PLA (50:50)  Yes (-)  CPP:PLA (20:80)  Yes (-)  Yes (-)  PLA (100%)  Yes (-)  PMMA (100%)  Yes (-)  Yes (-)  CPP:PMMA (25:75)  Yes (-)  Yes (-)  CPP:PMMA (50:50) HA/TCP  CPP (100%)  Yes (-)  CPP:PLA (80:20)  Yes (-)  CPP:PLA (50:50)  Yes (-)  CPP:PLA (20:80)  Yes (-)  PLA (100%)  Yes (-)  PMMA (100%)  Yes (-)  CPP:PMMA (25:75)  Yes (-)  Yes (-)  CPP:PMMA (50:50) CPP (100%)  CPP:PLA (80:20)  Yes (+)  CPP:PLA (50:50)  Yes (+)  CPP:PLA (20:80)  Yes (-)  PLA (100%)  Yes (-)  PMMA (100%)  Yes (-)  CPP:PMMA (25:75)  Yes (-)  Yes (-)  CPP:PMMA (50:50) CPP:PLA (80:20)  CPP:PLA (50:50)  Yes (-)  CPP:PLA (20:80)  Yes (-)  PLA (100%)  Yes (-)  CPP:PLA (20:80)  Yes (-)  PLA (100%)  Yes (-)  CPP:PLA (20:80)  PLA (100%)  Yes (-)  PMMA (100%)  CPP:PMMA (25:75)  CPP:PLA (50:50)  CPP:PMMA (50:50) CPP:PMMA (25:75)  CPP:PMMA (50:50)  84  Yes (+)  Table 10: Statistical Significance Chart for Sample Conditioning. Un-compacted and Precompacted Samples of the Same Material are Paired Using Student t-tests (p < 0.05). ‘Yes’ Signifies that the Pair Compared was Statistically Significant. Material  Sample Conditioning  MB (100%)  Yes*  HA/TCP (100%)  Yes  CPP (100%)  Yes  CPP:PLA (80:20)  Yes  CPP:PLA (50:50)  Yes  CPP:PLA (20:80)  Yes  PLA (100%) PMMA (100%) CPP:PMMA (25:75) CPP:PMMA (50:50) *Tested by Albert (2009).  3.4  Discussion  In designing the testing conditions, it was hypothesized that each sample would make a stiffer construct than the golden standard of morsellized cancellous bone (MB) alone. In order to be suitable for bone regeneration in revision total hip replacements, the synthetic structural scaffold material must be able to meet the necessary requirements as listed in Table 2 and ideally meet or exceed the properties listed for spongy (or cancellous) bone in Table 1. Earlier studies have shown that the use of synthetic materials such as ceramics, when mixed with bone or polymers, can improve the mechanical properties of the construct when confined in a femur (Hutmacher et al., 2007).  3.4.1  Particulate Composites  In this study, it was found that as the volume fraction of the soft PLA in a CPP:PLA particulate composite increased, the construct modulus increased for un-compacted samples but decreased for pre-compacted samples. In addition, both un-compacted and pre-compacted 85  particulate constructs were found to be significantly stiffer than the referenced samples of MB (Siggers (2007) and Albert(2009), respectively). As shown in Figures 15 and 19, the CPP and PLA particles have markedly distinct morphologies. CPP is more irregular, while PLA is smoothly contoured and almost twice the size of CPP. Siggers (2007) proposed that the differences in the construct moduli values in his testing of constructs made of single materials is most likely explained by such differences. Irregularly shaped particles are limited in their ability to move and to achieve a dense packing configuration. The number of contact points between the CPP particles is thus small and will result in lower construct stiffness compared to more smoothly contoured particles. As CPP has a lower packing density than PLA, un-compacted CPP will have a lower construct modulus and this is confirmed in this study. For un-compacted samples, as the volume fraction of PLA in the particulate composite is increased, the volume fraction of material in the construct and the number of contact points correspondingly increased within the construct and the construct stiffness increased. For samples that have been pre-compacted, it is possible that the more deformable irregular CPP particles have developed enough contact points within the construct during pre-compaction such that the stiffness when measured in the confined compression model is closer to that found with PLA. The slight decreasing trend observed with the pre-compacted samples as PLA content is increased could be due to the lower modulus of PLA particles compared to CPP particles.  3.4.2  Solid Composites  The construct moduli of the un-compacted solid composites of CPP and PMMA were shown to be almost two orders of magnitude greater than MB under confined compression, whereas for the pre-compacted solid composites the moduli were only one order of magnitude greater. In general, the solid constructs were found to be significantly different from the reference 86  stiffness values for MB. Whether the samples were pre-compacted or not, as the volume fraction of CPP in the solid composite with PMMA increased, the construct modulus decreased. A possible reason for this decreased modulus is the increased degree of interconnected porosity of the construct as the volume fraction of CPP is increased. As shown in Figure 20 the PMMA does not fully penetrate the CPP particles within the CPP:PMMA solid composites and voids are created within the PMMA phase. The un-filled pores of the CPP phase combined with the voids in the PMMA phase have a negative effect on the construct stiffness. The fraction of un-filled pores in the CPP phase and voids in the PMMA phase appears to increase as the fraction of CPP increases. In a study of CP cement, it was shown that increased porosity reduced some mechanical properties such as strength and stiffness (Barralet et al., 2002). Since the pores and voids present in the cement acted as points of stress, the greater the volume fraction of CPP, the greater the number of stress points and the lower the construct modulus. It is also possible that the testing of the solid composites created cracks within the constructs. The presence of cracks in the precompacted samples could explain the lower stiffness compared to the un-compacted solid composite samples. Although the degradation profile of CPP, particularly when mixed with PMMA, is unknown, it is possible that as the CPP degrades it will allow for a greater degree of creeping bone substitution into the construct and bone regeneration will then be more successful. In this way, the strength and stability of the construct will be sufficient to allow for some initial support of the prosthesis, but over time, as bone is regained, the interconnected porosity of the construct increases. However, this is not an ideal solution in a biological sense as PMMA will itself not degrade and be removed from the IA site. It is possible that the large standard deviation in the analysis of the solid composites could be due to the precision or accuracy of the confined compression testing of these composites. In order to place the cured solid composites into the stainless steel cylinder of the model, there 87  was a slight mis-match in sample-cylinder interface. The circumferential gap between model cylinder and composite sample of roughly 1mm could have led to some initial inconsistencies in the data retrieved. This was particularly the case for the un-compacted solid composites. However, as the analysis of these samples was comparative the data collected is still considered acceptable for this study.  3.4.3  Impact of Sample Conditioning  The pre-compacted particulate composites had construct moduli almost an order of magnitude greater than those un-compacted and the statistical difference between these two sets of samples was found to be significant. The graft bed effectiveness was similarly improved by pre-compaction of the particulate composite samples. Sample conditioning did not appear to have as great an impact on solid constructs, though pre-compacted Ec values were roughly half of those obtained for un-compacted samples. As a result, the hypothesis that sample conditioning would have an effect on construct stiffness was supported by the particulate composites, but not by the solid composites. For particulate composites, the precompaction likely increased the number of contact points such that further loading resulted in greater construct moduli. A study by Albert et al. (2008) obtained similar results to that of the particulate composites in this study, whereby an increased impaction force increased the MB graft bed density and resulted in a greater stiffness.  In considering the results and the statistical analysis, it has been shown in this study that solid composites of CPP:PMMA showed the greatest improvement over MB constructs, while precompaction of the particulate composites of CPP:PLA can be considered the next best improvement.  88  3.5  Conclusion  The objective of this study was to identify CPP composites that have a significantly higher confined compression modulus compared to that of morsellized bone. The study showed that solid composites of CPP and PMMA had significantly improved construct stiffness under confined compression when compared to MB. Un-compacted solid composites were approximately two orders of magnitude greater than MB, while pre-compacted were only one order of magnitude greater. Pre-compacted particulate composites of CPP and PLA were shown to have the next greatest improvement over MB with a significantly increased construct stiffness that was one order of magnitude greater. The presence of the soft PLA particles in the un-compacted particulate composites improved the construct modulus slightly, but was not shown to be as effective as pre-compaction. From a mechanical standpoint, both solid composites and pre-compacted particulate composites may be suitable for use in revision THR surgery. However, though solid composites give the highest stiffness, compacted particulate grafts also showed an improvement over allograft and will likely be much better from a biological point of view.  89  3.6  References  Albert, C., Masri, B., Duncan, C., Oxland, T., and Fernlund, G. “Impaction Allografting – the effect of impaction force and alternative compaction methods on the mechanical characteristics of the graft”, Journal Biomed Mater Res Part B: Appl Biomater. 87B: 395-405, 2008. Albert, C. University of British Columbia, PhD Thesis. 2009. Barralet, J.E., Gaunt, T., Wright, A., Gibson, I., and Knowles, J. “Effect of porosity reduction by compaction on compressive strength and microstructure of calcium phosphate cement”, Journal Biomed Mater Res (Appl Biomater). 63:1-9, 2002. Blom, A., Grimm, B., Miles, A., Cunningham, J., and Learmonth, I. “Subsidence in impaction grafting: the effect of adding a ceramic bone graft extender to bone”, Proc Instn Mech Engrs. 216-Part H: 265-270, 2002. Board, T., Rooney, P., Kearney, J., and Kay, P. “Impaction allografting in revTHR”, The Journal of Bone & Joint Surgery. 88-B: 852-857, 2006. Bolder, S., Schreurs, B., Verdonschot, N., van Unen, J., Gardeniers, J., and Sloof, T. “Particle size of bone graft and method impaction affect initial stability of cemented cups – Human cadaveric and synthetic pelvic specimen studies”, Acta Orthopaedica. 74: 652-657, 2003. Bolland, B., Kanczler, J., Ginty, P., Howdle, S., Shakesheff, K., Dunlop, D., and Oreffo, R. “The application of human bone marrow stromal cells and poly(DL-lactic acid) as a biological bone graft extender in impaction bone grafting”, Biomaterials. 29: 32213227, 2008. Bolland, B., New, A., Madabhushi, S., Oreffo, R., and Dunlop, D. “Vibration-assisted bonegraft compaction in impaction bone grafting of the femur”, The Journal of Bone & Joint Surgery. 89-B: 686-692, 2007-2. Bolland, B., Tilley, S., New, A., Dunlop, D., and Oreffo, R. “Adult mesenchymal stem cells and impaction grafting: a new clinical paradigm shift”, Expert Rev. Med. Devices. 4: 393-404, 2007. Coathup, M., Smith, N., Kingsley, C., Buckland, T., Dattani, R., Ascroft, P., and Blunn, G. “Impaction grafting with a bone-graft substitute in a sheep model of revision hip replacement”, The Journal of Bone & Joint Surgery. 90-B, 246-253, 2008. Cornu, O., Bavadekar, A., Godts, B., Van Tomme, J., Delloye, C., and Banse, X. “Impaction bone grafting with freeze-dried irradiated bone. Part II. Changes in stiffness and compactness of morselized grafts”, Act Orthop Scand. 74: 553-558, 2003. Dunlop, D., Brewster, N., Madabhushi, S., Usmani, A., Pankaj, P., and Howie, C. “Technique to Improve the Shear Strength of Impacted Bone Graft: The Effect of Particle Size and Washing of the Graft”, The Journal of Bone & Joint Surgery. 85: 639-646, 2003 Frei, H., Mitchell, P., Masri, B., Duncan, C., and Oxland, T. “Allograft impaction and cement penetration after revision hip replacement – A histomorphometric analysis in the cadaver femur”, The Journal of Bone & Joint Surgery”. 86-B: 771-776, 2004. Frei, H., Mitchell, P., Masri, B., Duncan, C., and Oxland, T. “Mechanical characteristics of the bone-graft-cement interface after impaction allografting”, Journal of Orthopaedic Research. 23: 9-17, 2005-2. Frei, H., O’Connell, J., Masri, B., Duncan, C., and Oxland, T. “Biological and mechanical changes of the bone graft-cement interface after impaction allografting”, Journal of Orthopaedic Research. 23: 1271-1279, 2005. 90  Gibson, L., and Ashby, M.F. “Cancellous bone. Cellular Solids Structure and Properties”. 316-330, 1988. Oxford, Pergamon Press. Ginebra, M., Driessens, F., and Planell, J. “Effect of the particle size on the micro and nanostructural features of a calcium phosphate cement: a kinetic analysis”, Biomaterials. 25: 3453-3462, 2004. Heiner, A., Callaghan, J., and Brown, T. “A laboratory simulation of morselized bone graft fusion: apparent modulus under operatively based femoral impaction kinetics”, Journal of Biomechanics. 38: 811-818, 2005. Hench, L. “Bioceramics: From Concept to Clinic”, J.Am.Ceram. Soc. 74: 1487-1510, 1991. Hench, L. “Millenium challenge mini-symposium – the challenge of orthopaedic materials”, Current Orthopaedics. 14: 7-15, 2000. Hutmacher, D.W., Schantz, J.T., Lam, C.F., Tan, K.C., and Lim, T.C. “State of the art and future directions of scaffold-based bone engineering from a biomaterials perspective”, Journal of Tissue Engineering and Regenerative Medicine. 1:245-260, 2007. Karrholm, J., Hultmark, P., Carlsson, L., and Malchau, H. “Subsidence of a non-polished stem in revisions of the hip using impaction allograft”, The Journal of Bone & Joint Surgery. 81-B: 135-142, 1999. Material Properties Website (Accessed August 2009): www.matbase.com Meding, J., Merrill, A., Ritter, E., Keatin, M. and Faris, P. “Impaction bone-grafting before insertion of a femoral stem with cement in revision THA. A minimum 2-year followup study”, The Journal of Bone and Joint Surgery. 79: 1834-41, 1997. Miranda, P., Pajares, A., Saiz, E., Tomsia, A., Guiberteau, F. “Mechanical properties of calcium phosphate scaffolds fabricated by robocasting”, Journal of Biomedical Materials Research Part A. 218-227, 2007. Murugan, R., and Ramakrishna, S. “Development of Nanocomposites for Bone Grafting”, Composites Science and Technology. X: 1-22, 2005. Nickelsen, T., Erenbjerg, M., Retpen, J., and Solgaard, S. “Femoral revision with IA and an uncemented femoral component”, Hip International. 18:278-285, 2008. Oakley, J., and Kuiper, J. “Factors affecting the cohesion of impaction bone graft”, The Journal of Bone & Joint Surgery. 88-B: 828-831, 2006. Orthodynamic UK Website (Accessed August 2009): http://www.orthodynamics.co.uk/ Pilliar, R.M., Filiaggi, M.J, Wells, J.D., Grynpas, M.D., and Kandel, R.A. “Porous Calcium Polyphosphate Scaffolds for Bone Substitute Applications – In Vitro Characterization”, Biomaterials. 22: 963-972, 2001. Pilliar, R.M., Filiaggi, M.J., Wells, J.D., Grynpas, M.D., and Kandel, R.A. “Porous calcium polyphosphate scaffolds for bone substitute applications – in vitro characterization.” Biomaterials 22-9, p.2063-2070. 2002. Plastics Technology Website (www.ptonline.com) – Article by Leaversuch, R. “Materials: Renewable PLA Polymer Gets ‘Green Light’ for Packaging Uses.” Siggers, K. University of British Columbia M.A.Sc. Thesis, 2007. Speirs, A., Oxland, T., Masri, B., Poursartip, A., and Duncan, C. “Calcium phosphate cement composites in revision hip arthroplasty”, Biomaterials. 26: 7310-7318, 2005. Tanabe, Y., Wakui, T., Kobayashi, A., Ohashi, H., Kadoya, Y., and Yamano, Y. “Determination of mechanical properties of impacted human morsellized cancellous allografts for revision joint arthroplasty”, Journal of Materials Science: Materials in Medicine. 10: 755-760, 1999. van Haaren, E., Smit, T., Phipps, K., Wuisman, P., Blunn, G., and Heyligers, I. “Tricalciumphosphate and hydroxyapatite bone-graft extender for use in impaction grafting 91  revision surgery – An in vitro study on human femora”, The Journal of Bone and Joint Surgery. 87-B: 267-271, 2005. Verdonschot, N., van Hal, C., Schreurs, B., Buma, P., Huiskes, R., and Slooff, T. “Timedependent mechanical properties of HA/TCP particles in relation to morsellized bone grafts for use in impaction grafting”, Journal Biomed Mater Res (Appl Biomater). 58: 599-604, 2001. Voor, M., White, J., Grieshaber, J., Malkani, A., and Ullrich, C. “Impacted morselized cancellous bone: mechanical effects of defatting and augmentation with fine hydroxyapatite particles”, Journal of Biomechanics. 37: 1233-1239, 2004.  92  4.0  Thesis Conclusion  4.1  Summary and Future Work  The biological and mechanical evaluation of CPP in this study was done to determine the feasibility of using CPP particles as a synthetic scaffold for use with bone regeneration in revision THR.  The purpose of the in vivo biological testing was to observe the proliferation and differentiation profiles of GFP+ rat MSCs when seeded on CPP, HA/TCP and MB and implanted subcutaneously in mice. The intermediate techniques of cell seeding, collagenase/dispase, and magnetic sorting were all found to be effective in allowing for a sufficient number of cells to be available for the final analytical techniques of BrdU and qRT-PCR. CPP, HA/TCP and MB were shown to have similar proliferation characteristics and gene expression. Each of the substrates was equally able to support MSC development on their surface.  Mechanical testing in this study involved measuring the construct stiffness of particulate and solid composites under confined compression and studying the effect of material and prior sample conditioning. Here again, MB and HA/TCP were treated as the golden and synthetic standard, respectively, to determine if the new composite constructs improved the mechanical characteristics. Solid composites of CPP and PMMA were shown to have dramatically improved construct stiffness, particularly when compared to MB. Pre-compacted particulate composites of CPP and PLA also improved the stiffness of the graft bed, though to a lesser degree than solid composites. These results show that CPP is a promising synthetic structural scaffold for replacing MB in the revision THR procedure.  93  This feasibility study confirmed that CPP was able to provide adequate structural support when mixed to form composites and able to support cellular proliferation and differentiation when seeded with MSCs. Both sets of mechanical and biological characteristics are necessary for bone growth to be sustained on the scaffold material.  In performing the statistical analysis with a sample size of 3 and a null hypothesis of equal means it is possible that the interpretation inadvertently incorporated either Type I or Type II errors. Type I error is referred to as the significance level or error rate of the statistical test and is the rejection of a true null hypothesis (Steel et al., 1997). Type II error is the acceptance of the null hypothesis when it is not true. The probability that either rejection has occurred is known as the power of the test. As the sample size of this study was low in both in vivo and mechanical studies the power of the test will be high. However, as in most cases the graphical analysis agrees with that obtained using statistical tools and the data is generally analyzed qualitatively not quantitatively, the results from my study can still be considered representative of the material behaviour.  Although this study further encourages the use of CPP as a synthetic scaffold, additional mechanical and biological testing is required to confirm its potential for use with revision THR. Proposed mechanical testing could involve confined compression of MB or HA/TCP as a particulate composite with CPP and PLA, confined compression of solid composites of CPP or PLA with a bioresorbable cement, and testing under various loading conditions (i.e. cyclic loading and shear). Additional biological testing could involve subcutaneous implantation of the material for a longer time period, implantation of the material in holes drilled in the leg bone, and implantation of the particulate and solid composites. Further study of whether a well-compacted, well-graded, graft bed will limit the degree to which revascularization and graft incorporation can occur is recommended. In addition, it would be 94  interesting to see how the material, when seeded with MSCs, behaves mechanically after various periods of implantation.  95  4.2  Reference  Steel, R., Torrie, J., and Dickey, D. “Principles and procedures of statistics”, McGraw-Hill Companies, Inc. Pages 90-91. 1997.  96  APPENDIX I - In vivo Study Steps Part A: Making Culture Medium (CM) - Collect MSC Basal Medium and warm - Collect FBS and warm - Collect P/S and warm - Add 15% FBS (67.5mL) and 5mL P/S to Medium - Put in fridge (4ºC) to store Part B: MSC Isolation - Remove femur and tibiae from GFP+ rats - Place bone in 50mL Falcon tubes - Clean and rinse bone with EtOH and PBS - Add fresh PBS - Morsellize bone - Strain morsellized bone with 70um cells strainers - Rinse strainer with PBS to wash remnants - Centrifuge the tubes - Add 15mL of CM to each 150x25mm culture-treated tissue plate - Aspirate off solution in tubes - Re-suspend the cells in the tubes with fresh CM - Add 5mL of this MSC+CM mixture to each of the plates - Put all plates in incubator - After ~2 days refresh CM - After another 2-4 days split plates each into three new plates - To reach a passage of 6 the two prior steps were repeated another four times - Once a passage 6 was reached the cells were cryopreserved until needed Part C: Plating MSC’s - Remove vials of cells from cryogenation keg - Defrost vial of MSC in hand - Immediately mix with 5mL of CM and move to new Falcon tubes - Centrifuge tubes for 5minutes at 1500 rpm - Aspirate off solution - Re-suspend cells in 20mL CM per tube - Transfer each tube into its own culture-treated tissue plate - Put plates into incubator (37ºC and 5% CO2) Part D: Splitting Cells - Aspirate off the old CM - Wash with 10mL fresh CM - Aspirate off CM - Wash with 12mL PBS - Aspirate off the PBS - Add 8mL T/E to each plate and swirl around plate - Remove 6.5mL T/E to leave just enough T/E to cover plate - Incubate plates for 5minutes - Lightly bang plates to loosen cells 97  -  Re-suspend in 10mL fresh CM and wash plate at an angle Split solution in half (~6mL solution in each plate) Add fresh CM to bring each culture plate to a total volume of 20mL  Part E: Collecting Morsellized Cancellous Bone - Wet rat with EtOH in surgical areas - Make an incision through the skin at the ankle - Make length-wise incision to pull back the skim from leg - Detach muscles - Remove foot by breaking joint at ankle - Break joint at knee and at hip to separate femur and tibiae - Put femur and tibiae into Falcon tubes Part F: Wash Particles - Wash in EtOH until no longer cloudy - Leave in EtOH for 48hours minimum - Rinse in PBS 3x - Leave in PBS for 24hours - Divide particles into the 24 well non-tissue culture treated plate and incubate with ~1mL CM per well for 24-48 hours Part G: Seed MSC’s on Particles - Aspirate off solution from each plate of MSCs - Wash with 10mL fresh CM and aspirate off - Wash with 10mL PBS and aspirate off - Add 8mL T/E to each plate and swirl around plate - Remove 6.5mL T/E and leave just enough to cover the plate - Incubate for 5 minutes - Bang plates lightly to loosen cells - Add 10mL fresh CM and transfer to new Falcon tubes - Centrifuge tubes at 1500rpm for 5 minutes - Aspirate off solution - Re-suspend in fresh CM (1mL per plate that was transferred to the tube) - Pipette out 10uL and count cells using a hemacytometer - Dilute the solution in the Falcon tubes with fresh CM in order to reach a 1million MSC/mL solution concentration - Collect the prepared substrate material and aspirate off the CM from the wells - Add the determined volume of MSC-mix to each well so that roughly the same number of cells is in each well (aim for ~1 million per well) - Centrifuge at 1000rpm for 6minutes at 4ºC - Incubate for 6hours - Pipette out CM from the wells (DO NOT ASPIRATE) and put aside to count any unattached cells - Wash particles with PBS twice and add this wash to CM already put aside - Use hemacytometer to count this wash solution - Add chilled PBS to each well to preserve cell condition during surgery  98  Part H: Subcutaneously Implanting Material+Cells - Prepare for surgery in filtered animal unit - Check Isofluorane and O2 tank levels - Turn on O2 tanks - Anesthetize 1 mouse at a time for surgery with Isofluorane - Once mouse is in surgical ‘plane’ remove mouse from anesthetizing box and place on anesthetizing tube - Shave hair off back of mouse - Use anti-septic gauze to wet-down surgical area - Lightly cover surgical area with iodine - Use tweezers and scissors to make a single length-wise incision - Grip one side of incision with tweezers and use scissors to open up skin (i.e. make a ‘pocket’ for the material being implanted) - Implant seeded material (one type of material per incision site) - Use tweezers to grasp incision and close - Use Michel stapler to close incision - Cover surgical area and staples with iodine - Repeat incision and implantation steps for the remaining two materials (a total of 3 incision sites per mouse) - Turn off Isoflourane and let mouse recover - Transfer to new mouse cage and monitor until completely recovered - Repeat steps for remaining mice - Check mice every two days to ensure enough water and food is available to them Part I: Euthanizing Mice and Harvesting Samples - Collect mice and move to euthanasia box - Turn on CO2 and leave on for a few minutes until mice are no longer breathing - Turn off CO2 and remove mice from box - Remove Michel staples - Use scissors and tweezers to remove the sample - Plase each sample into its own labeled Falcon tube and cover sample with chilled PBS - Dispose of mice bodies - Mix 1mL collagenase/dispase II with 10uL CaCl2 for each sample (i.e. per each tube) - Add the collagenase/dispase II + CaCl2 mixture to the material+cells in the tubes - Place in warm room (37ºC) for a total of 1hour and 45minutes - After 1hour vortex, filter with 70um strainers and fresh CM, put filtrate aside in ice, and add fresh collagenase/dispase + CaCl2 mixture to the material+cells - After another 45minutes in the warm room filter samples with 70um strainers and fresh CM - Add this second filtrate to new Falcon tubes - Centrifuge strained cells at 1500rpm for 5minutes - Aspirate off solution - Re-suspend in 200uL FACS solution in each Falcon tube - Collect a 96-v-well plate - Place 125uL from first filtrate and 150uL from second filtrate into a v-well per sample - Centrifuge at 1500rpm for 3minutes - Now on to BrdU or qRT-PCR analysis!!! 99  Part J: BrdU Protocol - On each of three days prior to mice euthanasia and sample harvesting the mice were injected with 100uL of a 10:90 mixture of BrdU: PBS per implantation site and given drinking water consisting of 1mL BrdU and 11.5mL DDW per cage - After centrifuging v-well with samples + FACS buffer, aspirate off supernatant - Re-suspend in anti-CD45 (1:100 dilution; anti-rat) with the exception of the CD45 negative controls - Incubate in fridge (4ºC) for 25minutes - Centrifuge at 1500rpm for 3minutes - Aspirate off supernatant - Re-suspend in streptavadin-PE (1:400 dilution) - Incubate in fridge (4ºC) for 25minutes - Centrifuge at 1500rpm for 3minutes (this is also known as “pelleting” the cells) - Aspirate off - Re-suspend in 100uL PBS (even BrdU- controls) - In ice bucket place PBS/BSA/Azide buffer, Permeabilization buffer, and DNAse - Add 100uL 4% PFA and mix well - Let the cell stand at room temperature in dark under aluminum foil for 20minutes - Pellet the cells - Wash the cells with 200uL ice-cool PBS/BSA/Azide buffer - Pellet the cells - Wash the cells with 200uL ice-cool PBS/BSA/Azide buffer (again) - Pellet the cells - Re-suspend in 150uL Permeabilization buffer. Mix gently - Incubate cells at room temperature for 10minutes under aluminum foil - Pellet the cells - Suspend cells in 100uL of PBS/BSA/Azide buffer containing 300ug/mL DNAse - Incubate cells for 1hour at 37C in dark - Pellet the cells - Resuspend in 25uL per well of PBS/BSA/Azide buffer containing 1uL anti-BrdUAPC antibody per sample - Incubate cells in room temperature for 30minutes in the dark - Pellet the cells - Wash with 200uL of PBS/BSA/Azide buffer - Pellet the cells - Wash with 200uL of PBS/BSA/Azide buffer (again) - Pellet the cells - Suspend cells in 200uL FACS buffer and transfer to FACS tubes - Add another 800uL FACS buffer - Take for FACS analysis - Controls for BrdU Analysis are: Control 1 (BrdU-, CD45-), Control 2 (BrdU-, CD45+), Control 3 (BrdU+, CD45-), and Control 4 (BrdU+, CD45+) - As there is no GFP- cell control the cells were compensated using the FACS program in order to account for this  100  Part K: qRT-PCR Protocol (i) mRNA Isolation - After centrifuging v-well with samples + FACS buffer, aspirate off supernatant - Re-suspend in anti-CD44 (1:200 dilution; anti-rat) - Incubate in fridge (4ºC) for 25minutes - Centrifuge at 1500rpm for 3minutes - Aspirate off supernatant - Re-suspend in anti-IgG2a-PE (1:400 dilution) - Incubate in fridge (4ºC) for 25minutes - Centrifuge at 1500rpm for 3minutes - Aspirate off supernatant - Re-suspend in 150uL FACS buffer and transfer to 15mL Falcon tubes - Add 1mL FACS buffer - Add 10uL anti-PE magnetic beads - Incubate in fridge (4ºC) for 25minutes - Centrifuge at 1500rpm for 3minutes - Aspirate off supernatant - Re-suspend in 2mL FACS buffer - Use autoMACS to positively select the fraction of sample which were my GFP+, CD44+ cells - Centrifuge positively selected samples at 1500rpm for 5minutes - Collect mRNAeasy Kit (Qiagen) - Re-suspend cells in 350uL RLT and transfer to safelock tubes - Vortex for at least 20s - Add 350uL filtered 70% EtOH - Mix by pipetting - Apply 700uL of sample to spin column placed in a 2mL collection tube - Close tube and centrifuge for 20s at 10,000rpm - Discard flow through (i.e. bottom clear part is thrown out) - Add 700uL RW1 to column - Close tube and centrifuge for 20s at 10,000rpm - Discard flow through - Transfer to a new 2mL collection tube - Add 500uL RPE onto column - Close tube and centrifuge for 20s at 10,000rpm - Discard flow through - Add another 500uL RPE onto column - Close tube and centrifuge for 2minutes at 10,000rpm to dry silica gel membrane - Discard flow through - Place column into a new 2mL collection tube - Close tube and centrifuge for 1minute at maximum speed - Discard flow through - Transfer to a new 1.5mL collection tube - Pipet 20uL RNAse-free H2O directly onto silica gel membrane - Close tube and centrifuge for 1minute at 10,000rpm - Discard column (i.e. top part) and keep bottom/safelock tube - Perform Nano-drop analysis to measure mRNA  101  (ii) cDNA Reverse Transcription - Move the mRNA to new PCR tubes - Add 1uL random hexamers - Add 1uL dNTP - Put PCR tubes in Thermocycler and set program “40 AJRT1” - To PCR tubes add 4uL of first strain buffer mix - Add 1uL of 0.1M DTT, 0.5uL RNAse-out inhibitor, and 1uL of Superscript III RT - Put PCR tubes in Thermocycler and set program “41 AJRT2HEX” - When done pick up PCR tubes - Perform Nano-drop analysis to measure cDNA (iii) Gene Expression Analysis with Real-Time PCR System - Collect 96-well plate for qRT-PCR analysis - Put 1uL cDNA, 0.5uL taqman probe (corresponding to each gene), 5uL TaqMan 2x Universal PCR Master Mix, and 3.5uL H2O into new PCR tubes - Centrifuge at 1200rpm for 2minutes - Analyze the 96-well plates with the Real Time PCR System - Gene expression is measured relative to the housekeeping gene GAPDH  102  APPENDIX II – Further Supporting Evidence for the In vivo Analysis (Figure 14) Col I - 0Day Per Mouse  Col I- 7Day Per Mouse  2.50E+00  2.00E+01 2A 3A  2.00E+00  3B  1.50E+00  1.00E+00  5.00E-01  Expression relative to GA PDH  Expression relative to GA P DH  1.80E+01 3A  1.60E+01  2A  3A  1.40E+01 1.20E+01  2B  '1A 2A  1.00E+01 8.00E+00 6.00E+00  2B 2B  '1A  '1A  4.00E+00  1B 1B 1B  2.00E+00 0.00E+00  '1A '1A  1B 1B 1B  2A 2A 2A  CPP-0Day  2B 2B 2B  HA-0Day  3A 3A  3B 3B  3A  MB-0Day  CPP-7Day  '1A  1B  2.00E+01 1.50E+01  3A  '1A '1A  2A 2A 1B 1B  2A  2B 2B 2B  3A  3B 3B  3A  3B  0.00E+00  Expression relative to GA P DH  Expression relative to GA P DH  MB-7Day  3.00E+01  2.50E+01  5.00E+00  HA-7Day  Col I - 28Day Per Mouse  3.50E+01  1.00E+01  3B  0.00E+00  Col I - 14Day Per Mouse  3.00E+01  3B 3B  2.50E+01  1B  2.00E+01 2A  1.50E+01 '1A  HA-14Day  2B 1B  1.00E+01  3B 3B  5.00E+00  0.00E+00  CPP-14Day  '1A  MB-14Day  '1A  1B  2A 2A  2B  3A 3A  2B  3B 3A  CPP-28Day  HA-28Day  MB-28Day  Figure 14-i: Col-I Expressions Relative to GAPDH for Each Mouse (1, 2, and 3). Letters 'A' and 'B' Represent the Replicate Analysis Taken From Each Mouse.  103  OP - 0Day Per Mouse  OP - 7Day Per Mouse 3.00E-02 3A  6.00E-02  3B  5.00E-02 4.00E-02 3.00E-02 2.00E-02 1.00E-02 0.00E+00  '1A '1A '1A  1B 1B 1B  3A 2A 2A 2A  CPP-0Day  2B 2B 2B  HA-0Day  3A  Expression relative to GA P DH  Expression relative to GA P DH  7.00E-02  '1A '1A  2.50E-02  2.00E-02  1.50E-02  1B  1.00E-02  1B 2A  5.00E-03 '1A  3B  MB-0Day  CPP-7Day  2B 2B  3A 3A 3A  3B 3B 3B  HA-7Day  MB-7Day  OP - 28Day Per Mouse 4.00E-03  1.80E-03  2B  1.60E-03 1.40E-03 1.20E-03 3B  1B  1.00E-03  2A  8.00E-04  '1A  6.00E-04  '1A  2A  2B  3A  1B 3B 3B  4.00E-04 2.00E-04 '1A  3A 3A  1B 2A  CPP-14Day  2B  HA-14Day  Expression relative to GA P DH  2.00E-03  Expression relative to GA P DH  2A  0.00E+00  3B  OP - 14Day Per Mouse  0.00E+00  2A 1B  3.50E-03  1B  3.00E-03 2.50E-03  '1A  2.00E-03  2A  2B  3A  2A 2A  2B 2B  3A 3A  1.50E-03 1.00E-03 5.00E-04 0.00E+00  MB-14Day  '1A '1A  1B 1B  CPP-28Day  HA-28Day  3B 3B 3B  MB-28Day  Figure 14-ii: OP Expressions Relative to GAPDH for Each Mouse (1, 2, and 3). Letters 'A' and 'B' Represent the Replicate Analysis Taken From Each Mouse.  104  OC - 0Day Per Mouse  OC - 7Day Per Mouse 6.00E-05 3A  Expression relative to GA P DH  Expression relative to GA P DH  3.00E-04  2.50E-04  2.00E-04  1.50E-04 1B  1.00E-04 2B  5.00E-05  '1A '1A '1A  0.00E+00  1B 1B  CPP-0Day  3A 2A 2A 2A  2B 2B  HA-0Day  3A  3B 3B 3B  1B  5.00E-05 '1A  4.00E-05  3.00E-05 3A  2.00E-05 2B  1.00E-05  '1A  0.00E+00  '1A  MB-0Day  '1A  3A  2.00E-05  3A  3B  1.50E-05 1.00E-05  1B '1A  '1A  1B  CPP-14Day  2A 2A 2A  2B  3A  2B 2B  HA-14Day  3B 3B  Expression relative to GA P DH  Expression relative to GA P DH  3A 2B 2B  HA-7Day  3A  3B 3B 3B  MB-7Day  4.50E-05 1B  2.50E-05  0.00E+00  2A 2A  OC - 28Day Per Mouse  3.50E-05  5.00E-06  1B 1B  CPP-7Day  OC - 14Day Per Mouse  3.00E-05  2A  4.00E-05  '1A  3.50E-05 3A  3.00E-05 2.50E-05 2.00E-05  1B  1.50E-05 1.00E-05  1B '1A 2A  2B 2B  5.00E-06 0.00E+00  MB-14Day  '1A  1B  CPP-28Day  2A 2A  3B 3A  2B  HA-28Day  3A  3B  MB-28Day  Figure 14-iii: OC Expressions Relative to GAPDH for Each Mouse (1, 2, and 3). Letters 'A' and 'B' Represent the Replicate Analysis Taken From Each Mouse.  105  BSP - 7Day Per Mouse  BSP - 14Day Per Mouse  2.50E-04  7.00E-06 '1A  2.00E-04  1.50E-04  1.00E-04  5.00E-05  0.00E+00  '1A '1A  1B 1B  CPP-7Day  2A 2A 2A  2B2B  3A 3A  2B  3A  HA-7Day  3B 3B  2B  6.00E-06  Expression relative to GA P DH  Expression relative to GA P DH  1B 3A  5.00E-06 4.00E-06 3.00E-06 2.00E-06 1.00E-06 '1A '1A '1A  0.00E+00  MB-7Day  1B1B 1B  CPP-14Day  2A 2A 2A  2B 2B  HA-14Day  3A 3A  3B 3B 3B  MB-14Day  BSP - 28Day Per Mouse  Expressio n relative to G A P DH  1.20E-05 1B  1.00E-05  8.00E-06 '1A  6.00E-06  4.00E-06  2.00E-06  0.00E+00  '1A '1A  2A 2A 2A  1B 1B  CPP-28Day  2B 2B 2B  HA-28Day  3A 3A 3A  3B 3B 3B  MB-28Day  Figure 14-iv: BSP Expressions Relative to GAPDH for Each Mouse (1, 2, and 3). Letters 'A' and 'B' Represent the Replicate Analysis Taken From Each Mouse.  106  APPENDIX III – Stress versus Strain Curves Part A: Particulate Scaffold Type CPP: PLA Particulate Composites Un-compacted 2000  100% CPP CPP:PLA (80:20) CPP:PLA (50:50) CPP:PLA (20:80) 100% PLA  1800  1600  Stress (kPa)  1400  1200  1000  800  600  400  200  0 0.00  2.00  4.00  6.00  8.00  10.00  12.00  14.00  16.00  18.00  Strain (%)  CPP: PLA Particulate Composites Pre-compacted 2000  100% CPP CPP:PLA (80:20) CPP:PLA (50:50) CPP:PLA (20:80) 100% PLA  1800  1600  Stress (kPa)  1400  1200  1000  800  600  400  200  0 0.00  0.10  0.20  0.30  Strain (%)  107  0.40  0.50  0.60  Part B: Solid Scaffold Type CPP:PMMA Solid Composites Un-compacted 2000  100% PMMA CPP:PMMA(25:75) CPP:PMMA (50:50) 100% CPP  1800  1600  Stress (kPa)  1400  1200  1000  800 600  400  200  0 0.00  2.00  4.00  6.00  8.00  10.00  12.00  14.00  Strain (%)  CPP:PMMA Solid Composites Pre-compacted 2000  100% PMMA CPP:PMMA (25:75) CPP:PMMA (50:50) 100% CPP  1800 1600  Stress (kPa)  1400 1200  1000  800  600 400  200 0 0.00  0.10  0.20  0.30  Strain (%)  108  0.40  0.50  APPENDIX IV – UBC Animal Care Certificate  109  110  111  112  

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