Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

A mechanical and biological evaluation of calcium polyphosphate as a structural bone scaffold in revision… Siggers, Kevin 2007

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_2007-0588.pdf [ 7.43MB ]
Metadata
JSON: 831-1.0078589.json
JSON-LD: 831-1.0078589-ld.json
RDF/XML (Pretty): 831-1.0078589-rdf.xml
RDF/JSON: 831-1.0078589-rdf.json
Turtle: 831-1.0078589-turtle.txt
N-Triples: 831-1.0078589-rdf-ntriples.txt
Original Record: 831-1.0078589-source.json
Full Text
831-1.0078589-fulltext.txt
Citation
831-1.0078589.ris

Full Text

A Mechanical and Biological Evaluation of Calcium Polyphosphate as a Structural Bone Scaffold in Revision Total Hip Replacement by K E V I N SIGGERS B.Sc , The University of British Columbia, 2005 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Materials Engineering) THE UNIVERSITY OF BRITISH C O L U M B I A September 2007 © K e v i n Siggers, 2007 Abstract R e v i s i o n total h ip replacement ( T H R ) c o m m o n l y uses the addi t ion o f morse l l i zed cancellous bone ( M C B ) for f i l l i n g bone defects and to encourage n e w bone growth, however problems associated w i t h allograft include l im i t ed ava i lab i l i ty , disease t ransmission and implant migra t ion . The use o f synthetic scaffolds as a substitute material is attractive due to its ava i lab i l i ty and ease o f s ter i l izat ion, however due to the h igh loads associated w i t h the h ip and the need to promote bone growth the number o f suitable materials is l imi ted . The nove l ceramic c a l c i u m polyphosphate ( C P P ) has been shown to have good mechanica l and b io log ica l properties and m a y be a suitable scaffold material for r ev i s ion T H R . In the first part o f this study particulate C P P is tested i n compress ion and shear and its mechanical properties are compared to that o f the go ld standard graft o f M C B . In addit ion, compress ion and shear tests are carried out o n a number o f spherical particles o f va ry ing size and mater ia l i n order to determine a relat ionship between the part icle and graft bed properties. T h e results show that C P P has s imi la r mechanica l properties to that o f M C B i n both compress ion and shear, m a k i n g it a suitable substitute graft material . Furthermore a relat ionship between the particle modulus and graft bed modulus show m i n i m a l gain i n bed stiffness w i t h increased particle stiffness. The second aspect o f the study is to determine the effect o f substrate mater ia l on M S C expansion and differentiation. C P P , al logeneic bone and hydroxyapat i te / t r ica lc ium phosphate ( H A / T C P ) are seeded w i t h mar row stromal cel ls ( M S C s ) and cul tured i n expansion condi t ions. A t 0, 3, 7, 14 and 21 days ce l l numbers and gene expressions are evaluated. Af te r an in i t i a l drop i n ce l l numbers, C P P and bone supported an increase i n prol iferat ion act iv i ty and show no up-regulat ion o f the mature bone markers on C P P and Bone , w h i c h suggests that these substrates support M S C expans ion rather than differentiation. In contrast, M S C number on H A / T C P decreased w i t h t ime and it showed a d o w n regulat ion o f early osteogenic markers. T h i s a long w i t h a substantial increase i n mature markers indicate that H A / T C P favours M S C differentiation and maturat ion along the osteogenic l ineage. Table of Contents Abstract • i i Table o f Contents i v L i s t o f Tables v i L i s t o f Figures v i i L i s t o f Abbrev ia t ions v i i i Acknowledgement s i x Ded ica t ion x 1 Introduction 1 1.1 B a c k g r o u n d 1 1.2 Scaf fo ld Requirements 5 1.3 Thesis Object ives 8 1.4 References 9 2 M e c h a n i c a l properties o f particulate bone scaffolds i n T H R : a compar i son o f C P P a n d M C B 12 2.1 Introduction 12 2.2 Mater ia l s and Me thods 15 2.2.1 Mate r ia l s 15 2.2.2 Me thods 17 2.2.3 Statist ical A n a l y s i s 20 2.3 Results 20 2.4 D i s c u s s i o n 24 2.5 C o n c l u s i o n 32 2.6 References 33 3 Effect o f bone graft substitute o n mar row stromal c e l l pro l i fera t ion and differentiation 37 3.1 Introduction 37 3.2 Mater ia l s and Me thods 39 3.2.1 Scaf fo ld Mate r ia l s 39 3.2.2 C e l l i so la t ion , seeding and culture 42 3.2.3 C e l l u l a r Prol i fera t ion 43 3.2.4 C e l l u l a r Different ia t ion 44 3.2.5 Scanning e lect ion microscope ( S E M ) 45 3.2.6 Statistics 45 3.3 Results 46 3.3.1 Seeding 46 3.3.2 C e l l u l a r prol i ferat ion 46 3.3.3 C e l l u l a r differentiation 48 3.3.4 S E M 52 3.4 D i s c u s s i o n 52 3.5 C o n c l u s i o n 57 3.6 References 58 4 C o n c l u s i o n '. 61 4.1 Summary and Future W o r k 61 4.2 Reference L i s t 63 A p p e n d i x A : C e l l Cul ture Procedures ." 64 P la t ing M S C ' s 64 W a s h particles: 64 Spl i t t ing C e l l s 64 Seed M S C ' s o n particles 65 Seeding o n plast ic 66 Cu l tu r ing C e l l s 66 M T T 66 L y s i s o f C e l l s o n particles : 67 A p p e n d i x B : Stat is t ical M e t h o d s : 69 Student t-test 69 A N O V A 69 Pearson's Cor re la t ion 71 v List of Tables Table 2.1: M a t e r i a l properties o f particles used i n testing 17 Table 2.2: Construct modulus f rom confined compress ion tests 21 Table 2.3: Resul ts o f shear tests reported as M o h r - C o u l o m b parameters 22 Table 3.1 Gene names, symbols and reference numbers 45 v i List of Figures Figure 1.1: Schematic o f r ev i s ion total h ip replacement 3 Figure 2.1: Part icles used i n testing 16 Figure 2.2: Exper imen ta l setup 19 Figure 2.3: C o n f i n e d compress ion results 21 Figure 2.4: Resul ts o f shear test.Shear strength vs shear displacement 23 Figure 2.5: Resul ts o f shear test. Shear strength vs compressive no rma l stress 24 Figure 2.6: Exper imen ta l construct modulus E c vs particle modulus E p 27 Figure 3.1: Particulates o f substrate materials 41 Figure 3.2: Osteogenic , adiopogenic and chondrogenic differentiation o f M S C s 47 Figure 3.3: C e l l numbers and B r d U incorporat ion 48 Figure 3.4: The temporal expression o f osteogenic genes 50 Figure 3.5: The temporal expression o f chondrogenic genes 51 Figure 3.6: The temporal expression o f adipogenic genes 52 v i i List of Abbreviations T H R Tota l h ip replacement U H M W P E U l t r a h igh molecula r weight polyethylene M C B M o r s e l l i z e d cancellous bone H A Hydroxyapat i te T C P T r i c a l c i u m Phosphate CPP C a l c i u m polyphosphate E c M o d u l u s o f the particulate constructs E P M o d u l u s o f particle MSCs M a r r o w stromal cel ls RUNX -2 Runt related transcription factor 2 A L P A l k a l i n e phosphatase COL-I C o l l a g e n I O N Osteonect in OP Osteopont in BSP B o n e sialoprotein O C Osteoca lc in SOX 9 S R Y - b o x containing gene 9 C O L II C o l l a g e n II A G G A g g r e c a n PPARy Perox i some proliferator-activated receptor g a m m a L P L L i p o p r o t e i n L ipase G A P D H Glycera ldehyde-3 -phosphate dehydrogenase BMPs B o n e morphogenic proteins F G F Fibroblasts growth factor T C Tissue culture plastic PBS Phosphate buffered saline E M E x p a n s i o n med ia O M Osteogenic media T C / E M Tissue culture plastic i n expansion m e d i a T C / O M Tissue culture plastic i n osteogenic m e d i a M T T 3-(4, 5-dimethyl th iazol-2yl) -2 , 5d iphenyl te t razol ium D M S O D i m e t h y l sulfoxide BrdU 5 -bromo-2-deoxyur id ine S E M Scann ing elect ion microscope Acknowledgements Specia l thanks to m y supervior, D r G o r a n Fern lund, w h o has g iven a great deal o f support and guidance through the development and comple t ion o f m y masters. I w o u l d also l ike to thank D r Hanspeter F re i , w h o spent many hours adv i s ing and teaching me i n the f ie ld o f c e l l b io logy . I w o u l d also l i ke to acknowledge the help I received f rom D r Fab io R o s s i and the members o f his group at the B i o m e d i c a l Research Center m y wife M a r i k o . 1 Introduction 1.1 Background A total h ip replacement ( T H R ) is a h igh ly effective procedure for restoring the h ip function o f patients suffering f rom ailments such as osteoarthritis or a fracture o f the h ip due to trauma. The total h ip replacement o r ig ina l ly developed i n the 1950 's by S i r John Charnley consists o f a meta l l i c ba l l jo in t w h i c h is f ixed i n the medul la ry canal o f the femur and an ul tra h i g h molecula r weight polyethylene ( U H M W P E ) cup secured into the socket o f the p e l v i s ' . D u e to an aging popula t ion and improvements to the procedure the incidence o f total h ip replacements is increasing, and i n Canada has j u m p e d f rom 16,500 • • 2 i n 1994 to over 25,000 i n 2004 . A l t h o u g h compl ica t ions over the past two decades have decreased s ignif icant ly there are s t i l l problems associated w i t h T H R . One major concern is femoral osteolysis f o l l o w i n g surgery causing excessive loosening o f the implant . Osteolysis is a degeneration or d issolu t ion o f bone due to a number o f reasons, i nc lud ing aging, stress sh ie ld ing and implant wear particles . In a l l cases a r ev i s ion surgery is required to correct the p rob lem and accounts for between 10 and 2 0 % o f a l l h ip surgeries each year 4 . In severe cases a r ev i s ion total h ip replacement m a y be required. A rev i s ion T H R invo lves the remova l o f the loose implant and the source o f wear particles; the jo in t is then prepared and fitted w i t h a new implant . In the case where severe osteolysis has occurred, a long-stem prosthesis is cemented into an impacted allograft. Impact ion al lograft ing is a method first made popular by G i e i n w h i c h 1 morse l l i zed cancel lous bone ( M C B ) f rom a donor is impacted i n the medul la ry canal o f 3 5 femur w h i l e l eav ing a sma l l canal for the new prosthesis to be inserted ' . A c r y l i c bone cement is then injected into the graft under pressure fo l l owed by the inser t ion o f the new prosthesis, F igure 1.1. T h i s cemented allograft construct has the benefit o f p rov id ing in i t ia l mechanica l support o f the implant as w e l l as being a scaffold for bone ingrowth from surrounding bone 6 . A l t h o u g h impact ion al lograft ing is the o n l y method that has been shown to reverse the loss o f bone stock caused by os teo lys i s 6 there are a number o f problems associated w i t h this technique. Al logra f t material has been k n o w n to transmit pathogens such as hepatitis C and H I V ' . The impac t ion al lograft ing procedure has also been associated w i t h early implant subsidence o f the femoral component as w e l l as a h igh incidence o f intra- and postoperative fracture 9 . Furthermore, due to the increased use o f allograft, the bone bank supply o f cancellous bone may not be sufficient to meet the increasing demand i n the future. 2 Figure 1.1 Schematic of revision total hip replacement A number o f synthetic materials are n o w being investigated for the use as bone graft substitutes. W h e n us ing a biomater ia l for bone tissue engineering a number o f factors must be considered. The material must be biocompat ib le i n that they should not el ici t an inf lammatory response or exhibi t any cyto toxic i ty when p laced i n the body. They should maintain structural support and integrity unt i l the newly formed bone is able to take over the mechanical role. The scaffold material should also encourage the format ion o f bone and have enough v o i d space to a l l o w bone ingrowth as w e l l as vascular iza t ion for 3 maintenance o f the n e w l y formed tissue. A n d f ina l ly they must be easy to use, such that they can be easi ly s ter i l ized and handled dur ing surgery 1 0 . A m o n g the more popular materials be ing studied that meet many o f these design requirements are ceramics o f ca l c ium phosphate, a p r i nc ipa l inorganic constituent o f natural bone. Hydroxyapat i te ( H A ) , t r i ca l c ium phosphate ( T C P ) and a biphasic c a l c i u m phosphate ( H A / T C P ) are the 11 12 most w i d e l y used and have shown to form tight bonds w i t h host bone tissue ' . Hydroxyapat i te has a chemica l compos i t ion o f Caio(PO"4)6(OH)2 or 3Ca3(P04)3 -Ca(OH)2 and spatially it is a tr iangular structure made up o f 3 Ca3(PC>4)3 surrounding a central C a ( O H ) 2 1 3 . T C P has a chemica l compos i t ion o f Ca3(P04)2 that has three po lymorphs P, a, and d. P - T C P is the most c o m m o n l y used for tissue engineering applicat ions and has a structure made up o f two different columnar crystals. The first c o l u m n ( A ) has the form P 0 4 C a 0 3 C a 0 6 P 0 4 and the second c o l u m n ( B ) has the fo rm P 0 4 C a 0 7 C a O g CaOg P 0 4 . E a c h A c o l u m n is surrounded by 6 B columns w h i l e each B is surrounded by 2 A and 4 B c o l u m n s 1 4 . H o w e v e r due to there brittle nature, these ceramics i n porous fo rm have l o w strength and toughness, w h i c h has hindered their use i n c l i n i c a l appl icat ions. A nove l ceramic, c a l c i u m polyphosphate ( C P P ) , currently be ing invest igated for the regeneration o f cartilage has been shown to be b iocompat ib le and to have good mechanical properties and m a y prove to be a suitable graft mater ial for the h i p 1 5 . C P P [Ca(PC»4)2]n is an inorganic po lymer whose structure o f C P P is compr i sed o f 2 element units o f a c i rcular cha in made up o f eight PO4 tetrahedrons. The c i rc le cha in units are thought to be connected through the O a t o m 1 6 . Some o f the features that make C P P part icular ly appeal ing are that C P P can be produced w i t h an interconnected porous 4 1 7 network o f approximate ly lOOum i n suitable for ingrowth o f bone . Porous C P P is reported to have a tensile strength up to 24.1 M P a w h i c h is s imi la r to the properties o f bone (5-10 M P a cancel lous , 80-150 M P a c o r t i c a l ) 1 0 ' 1 5 . The degradation product o f C P P is c a l c ium orthophosphate w h i c h is naturally occurr ing and easi ly metabol ized i n the b o d y 1 5 . Furthermore C P P can be made into particles o f va ry ing size w h i c h can easi ly form to any defect site i n the bone. 1.2 Scaffold Requirements One o f the m a i n requirements o f a scaffold being used as a replacement for M C B i n rev i s ion T H R is that it has adequate mechanica l properties to prevent m i c r o m o t i o n and subsidence o f the implan t i n the h ip . In impac t ion al lograft ing a particulate bed o f bone surrounds the implant and is conf ined by the cy l i nd r i ca l shel l o f the cor t ica l bone. The load from the weight o f the body is transmitted f rom the pe lv i s d o w n through the implant to the graft bed then outwards to the surrounding cor t ical bone. Thus i n order to prevent subsidence o f the implant i n the femur the graft bed must effect ively transfer the load from the implant to the surrounding cort ical bone without significant movement or failure. The mechanica l properties o f M C B have been investigated thoroughly i n an effort to improve its s tabil i ty i n the h ip . A number o f different techniques have been used to improve the graft bed qual i ty i nc lud ing wash ing and d ry ing o f the M C B , us ing different 1 O |Q A impac t ion protocols , and the addi t ion o f extender particles ' ' . In these studies the 5 metrics used to measure any improvements to the graft bed dur ing these procedures are 1 8 0 1 00 compressive modulus and the shear strength ' ' . The compress ive modulus , related to the pack ing density and contact between particles, is thought to be benef ic ia l for in i t i a l implant stabili ty as w e l l as l i m i t i n g the intermittent m o t i o n o f the graft a l l o w i n g for easier integration o f surrounding b o n e 2 3 ' 2 4 . The shear strength o f the graft bed, w h i c h is influenced by the internal f r ic t ion and in ter locking o f the graft part icles, is thought to be 18 21 important i n the prevent ion o f stem subsidence i n the femur ' . Thus i n order to be suitable for the use i n r ev i s ion T H R , the graft bed o f a scaffold mater ial must have a compressive modulus and shear strength s imi la r or better than that o f M C B . Another important requirement o f a scaffold material being used i n r ev i s ion h ip surgeries is that it must provide a suitable environment for bone growth. The process o f bone growth is a c o m p l e x development o f osteogenic cel ls compr ised o f three major stages: ce l l co lon iza t ion and mul t i l ayer ing , in i t ia l intercellular matr ix product ion , and matr ix Of\ minera l iza t ion ' . In the in i t i a l co lon iza t ion stage there is a large recruitment o f osteogenic cel ls f rom the surrounding bone. Th i s group o f cel ls is a heterogeneous popula t ion o f cel ls i n the osteogenic lineage ranging f rom undifferentiated mesenchymal stem cells to fu l ly commi t t ed osteoblasts. E a c h ce l l type i n the osteogenic lineage has a distinct funct ional i ty and a l l are required for the cont inued growth and maintenance o f new bone. Osteoblasts are the p r imary bone forming cel ls and dur ing new bone development they arrange themselves i n layers located c l o s e t o the bone surface. These cells lay d o w n a co l l agen matr ix that they i n turn minera l ize . Once osteoblasts are surrounded by mine ra l i zed bone they become osteocytes w h i c h r emain i n the bone and 6 regulate the transportation o f nutrients and waste. C e l l s that are close to the osteoblast on the side away f rom the n e w l y fo rming bone front are ca l led preosteoblasts. These cel ls are unable to produce bone but they are capable o f ce l l d i v i s i o n and therefore are the supporting cel ls o f the osteoblasts and w i l l differentiate into osteoblasts as the bone front moves toward t h e m 2 7 . The preosteoblasts are i n turn supported by a class o f cel ls k n o w n as osteoprogenitors. These cel ls are h igh ly proliferat ive and are the earliest cel ls commit ted to the osteogenic lineage w h i c h can be induced to differentiate into 25 28 preosteoblasts ' . H o w e v e r , none o f the cel ls i n the osteogenic l ineage have the abi l i ty for se l f renewal . T h i s is a characteristic reserved for a class o f cel ls ca l l ed stem cells . Th is abi l i ty for stem cel ls to se l f renew makes them an important component i n mainta ining tissues throughout the l ifet ime. M e s e n c h y m a l stem cel ls i n particular, are responsible for the maintenance o f mesenchymal tissues i nc lud ing bone and give rise to commit ted cel ls such as osteoprogenitors. A c o m m o n technique be ing used i n tissue engineering is to seed a heterogeneous ce l l popula t ion (acquired f rom bone marrow) containing mesenchymal stem cel ls and osteogenic cel ls onto scaffolds i n order to enhance the bone growth o n the 12 29 30 * scaffolds ' ' . S ignals received f rom both the substrate they are attached to and the surrounding culture m e d i u m , regulate the expansion and differentiation behaviour o f a ce l l popu l a t i on 1 1 . A s these cel ls differentiate d o w n the osteogenic l ineage there are characteristic changes i n the genes they express 3 1 . B y moni to r ing the gene expression along w i t h the c e l l numbers, investigators can determine the osteogenic potential o f a 7 substrate. It order to be a suitable substrate for bone growth a mater ia l must be 32 osteogenic i n that it a l lows the growth and differentiation o f osteogenic cel ls . 1.3 Thesis Objectives The objectives o f this study are to investigate the mechanica l and b i o l o g i c a l feasibi l i ty o f us ing c a l c i u m polyphosphate as a replacement scaffold for mor se l l i z ed cancel lous bone i n rev is ion h ip surgery. In the mechanica l testing we w i l l use conf ined compress ion and direct shear tests to compare the compressive modulus and the shear strength o f C P P to that o f M C B . Furthermore w e w i l l investigate h o w the properties o f the constitute particles such as size, shape and modulus influence the properties o f the entire graft bed. In the b io log i ca l testing we w i l l investigate the effect o f substrate mater ia l o n the growth and differentiation o f bone mar row cel ls , through ce l l popula t ion and gene expression assays. 8 1.4 References 1. Dona ld , S. M . S i r John Charn ley (1911-1982): Inspirat ion to future generations o f orthopaedic surgeons. Scottish MedicalJournal 52[2], 43-6. 2007. 2. Tota l N u m b e r o f H i p and K n e e Replacement Procedures, Canada . Hospital Morbidity Database, CIHI. 10-25-2006. 3. Dattani , R. F e m o r a l osteolysis f o l l o w i n g total h ip replacement. Postgraduate MedicalJournal 83 [979], 312-6. 2007. 4. Mahomed , N . N. , Barret t , J . A . , Ka tz , J . N . , Phi l l ips , C . B., Los ina , E . , Lew , R. A . , Guadagno l i , E . , Har r i s , W . H . , Poss, R., and B a r o n , J . A . Rates and outcomes o f p r imary and rev i s ion total h ip replacement i n the U n i t e d States M e d i c a r e popula t ion . Journal of Bone and Joint Surgery-American Volume 8 5 A [ 1 ] , 27-32. 2003. 5. Fosse, L. , Ronningen, H . , Lund -La rsen , J . , Benum, P., and Grande , L . Impacted bone stiffness measured dur ing construct ion o f morse l l i sed bone samples. Journal Of Biomechanics 37[11], 1757-66. 2004. 6. Toms, A . D., Ba rke r , R. L., Jones, R. S., and Ku ipe r , J . H . Current concepts r ev i ew - Impact ion bone-grafting i n rev i s ion jo in t replacement surgery. Journal of Bone and Joint Surgery-American Volume 8 6 A [ 9 ] , 2050-60. 2004. 7. Con rad , E . U. , Gre tch , D. R., Obermeyer, K . R., Moogk , M . S., Sayers, M . , Wi l son , J . J . , and Strong, M . Transmiss ion o f the Hepa t i t i s -C V i r u s by Tissue-Transplantat ion. Journal of Bone and Joint Surgery-American Volume 7 7 A [ 2 ] , 214-24. 1995. 8. Draenert, G . F. and Del ius, M . The mechanica l ly stable steam ster i l izat ion o f bone grafts. Biomaterials 28[8], 1531-8. 2007. 9. Kn ight , J . L . and He lming , C . Col lar less po l i shed tapered impac t i on grafting o f the femur dur ing rev i s ion total h ip arthroplasty - Pi t fa l l s o f the surgical technique and fo l low-up i n 31 cases. Journal of Arthroplasty 15[2], 159-65. 2000. 10. Temenoff, J . S. and M i k o s , A . G . Injectable biodegradable materials for orthopedic tissue engineering. Biomaterials 21 [23], 2405-12. 2000. 11. H ing , K . A . B i o c e r a m i c bone graft substitutes: Influence o f poros i ty and chemistry. International Journal of Applied Ceramic Technology 2[3] , 184-99. 2005. 9 12. Livingston, T. L., Gordon, S., Archambault, M., Kadiyala, S., Mcintosh, K., Smith, A., and Peter, S. J. M e s e n c h y m a l stem cel l s combined w i t h b iphasic c a l c i u m phosphate ceramics promote bone regeneration. Journal of Materials Science-Materials in Medicine 14[3], 211-8. 2003 . 13. Gutowska, L, Machoy, Z., and Machalinski, B. The role o f b ivalent metals i n hydroxyapat i te structures as revealed by molecula r m o d e l i n g w i t h the H y p e r C h e m software. Journal of Biomedical Materials Research Part A 7 5 A [ 4 ] , 788-93. 12-15-2005. 14. Yashima, M., Sakai, A., Kamiyama, T., and Hoshikawa, A. C rys t a l structure analysis o f beta- t r icalc ium phosphate C a - 3 ( P 0 4 ) ( 2 ) by neutron powder diffraction. Journal of Solid State Chemistry 175[2], 212-1. 2003 . 15. Pilliar, R. M., Filiaggi, M. J., Wells, J. D., Grynpas, M. D., and Kandel, R. A. Porous c a l c i u m polyphosphate scaffolds for bone substitute applications - i n v i t ro characterization. Biomaterials 22[9], 963-72. 2001 . 16. Guo, L. H., Li, H., and Gao, X. H. Phase transformations and structure characterizat ion o f c a l c i u m polyphosphate dur ing sintering process. Journal ' .of Materials Science 39[23], 7041-7. 12-1-2004. 17. Klawitter, J. J., Bagwell, J. G., Weinstein, A. M., Sauer, B. W., and Pruitt, J. R. Eva lua t i on o f B o n e - G r o w t h Into Porous H i g h - D e n s i t y Polyethylene . Journal of Biomedical Materials Research 10[2], 311-23. 1976. 18. Dunlop, D. G., Brewster, N. T., Madabhushi, S. P. G., Usmani, A. S., Pankaj, P., and Howie, C. R. Techniques to improve the shear strength o f impacted bone graft - The effect o f particle size and wash ing o f the graft. Journal of Bone and Joint Surgery-American Volume 8 5 A [ 4 ] , 639-46. 2003 . 19. Cornu, O., Bavadekar, A., Godts, B., Van Tomme, J., Delloye, C , and Banse, X. Impact ion bone grafting w i t h freeze-dried irradiated bone. Part II. Changes i n stiffness and compactness o f morse l i zed grafts - Exper iments i n cadavers. Acta Orthopaedica Scandinavica 74[5], 553-8. 2003 . 20. Blom, A. W., Grimm, B., Miles, A. W., Cunningham, J. L., and Learmonth, I. D. Subsidence i n impac t ion grafting: the effect o f adding a ceramic bone graft extender to bone. Proceedings of the Institution of Mechanical Engineers Part H-Journal of Engineering in Medicine 2 1 6 [ H 4 ] , 265-70. 2002. 21 . Brewster, N. T., Gillespie, W. J., Howie, C. R., Madabhushi, S. P., Usmani, A. S., and Fairbairn, D. R. M e c h a n i c a l considerations i n impact ion 'bone grafting. J.Bone Joint Surg.Br. 81 [1], 118-24. 1999.' 22. Tanabe, Y., Wakui, T., Kobayashi, A., Ohashi, H., Kadoya, Y., and Yamano, Y. De te rmina t ion o f mechanica l properties o f impacted human morse l l i zed 10 cancel lous allografts for rev i s ion jo in t arthroplasty. Journal of Materials Science-Materials in Medicine 10[12], 755-60. 1999. 23. Karrholm, J., Hultmark, P., Carlsson, L., and Malchau, H. Subsidence o f a non-pol i shed stem i n revis ions o f the hip us ing impac t ion allograft - Eva lua t ion w i t h radio stereometry and dual-energy X - r a y absorptiometry. Journal of Bone and Joint Surgery-British Volume 81B[1 ] , 135-42. 1999. 24. Carter, D. and Beaupre, G. Skeletal Func t ion and F o r m . 1-318.2001. Cambr idge , U K , Cambr idge Un ive r s i t y Press. 25. Knabe, C , Berger, G., Gildenhaar, R., Howlett, C. R., Markovic, B., and Zreiqat, H. The functional expression o f human bone-der ived cel ls g rown on rap id ly resorbable c a l c i u m phosphate ceramics. Biomaterials 25[2], 335-44. 2004. 26. Davies, J. E. E a r l y Ext race l lu la r M a t r i x Synthesis by B o n e C e l l s . Dav ies , J . E . The B o n e - B i o m a t e r i a l Interface. [20], 214-28. 1991. Toronto , U n i v e r s i t y o f Toronto Press. 27. Holtrop, M. E. L i g h t and E lec t ron Structure o f B o n e - F o r m i n g C e l l s . H a l l , B . K . The Osteoblast and osteocyte. [1], 1-40. 1990. C a l d w e l l , N J , The Te l fo rd Press Inc. B o n e . 28. Tenenbaum, H. C. C e l l u l a r Or ig ins and Theories o f Different ia t ion o f B o n e -F o r m i n g C e l l s . H a l l , B . K . The Osteoblast and Osteocyte. [2], 41-70. 1990. C a l d w e l l , N J , The Te l fo rd Press Inc. Bone . 29. Zhou, Y. F., Chen, F. L., Ho, S. T., Woodruff, M. A., Lim, T. M., and Hutmacher, D. W. C o m b i n e d mar row stromal cell-sheet techniques and high-strength biodegradable composi te scaffolds for engineered functional bone grafts. Biomaterials 28[5], 814-24. 2007. 30. Rust, P. A., Kalsi, P., Briggs, T. W. R., Cannon, S. R., and Blunn, G. W. W i l l mesenchymal stem cel ls differentiate osteoblasts o n allograft? Clinical Orthopaedics and Related Research [457], 220-6. 2007. 31. Aubin, J. E. B o n e stem cel ls . Journal of Cellular Biochemistry , 73-82. 1998. 32. Bauer, T. W. A n o v e r v i e w o f the his tology o f skeletal substitute materials. Archives of Pathology & Laboratory Medicine 131 [2], 217-24. 2007. 11 2 Mechanical properties of particulate bone scaffolds in THR: a comparison of CPP and MCB 1 2.1 Introduction Impaction allografting is a technique used in revision total hip replacement in which morsellized cancellous bone (MCB) is impacted into the medullary canal of the femur in order to fill bone defects, support a new implant and provide a matrix for bone regeneration ' . This technique has been shown to have positive results in stabilizing the implant 2 ' 3 ' 4 ' 5, however clinical problems such as intra- and post-operative fracture of the femur and stem subsidence have been observed ' ' ' . Additional problems associated with the use of allograft in revision surgeries, such as disease transmission and availability 9 ' 1 0 , have prompted the development of substitute synthetic bone grafts. Synthetic calcium phosphates, such as hydroxyapatite (HA) and tricalcium phosphate (TCP) have been extensively studied for the use as bone scaffolds 1 ' ' l 2 ' 1 3 . Calcium phosphate materials are promising because they are readily available and, being similar to the mineral component of bone, they have shown good osteoconductivity13 ,14. Furthermore the porosity and degradation rates of these bioceramics can be tailored in the manufacturing process. One of the major drawbacks however is their brittleness as well as their poor strength when processed with pore sizes suitable for bone incorporation. Calcium polyphosphate (CPP) is novel calcium phosphate ceramic currently being investigated for the use in cartilage repair 1 5 ' 1 6 , 1 7 . CPP has good mechanical properties as 1 A version of this chapter will be submitted for publication. 12 shown i n a study b y P i l l i a r et al, where C P P rods o f 3 0 - 4 5 % poros i ty were measured to have tensile strengths o f up to 24.1 M P a 1 5 . The b iocompat ib i l i ty o f C P P was examined i n a later experiment by Grynpas et al, where s imi la r rods were surg ica l ly implanted i n the distal femurs o f N e w Zea land white rabbits and after 1 year had bone ingrowth up to 2 5 % o f avai lable pore area and degradation o f 5 9 % 1 6 . C P P has been s h o w n to have suitable b iocompa t ib i l i t y and w i t h better mechanica l properties than many other c a l c i u m phosphate materials it m a y provide a suitable bone graft for r ev i s ion h ip surgeries. In addi t ion to the mater ial properties, the structure o f the graft is important i n determining the mechanica l characteristics o f the graft bed. The use o f particulate materials as bone grafts has a number o f attractive attributes. Part icles are easy to use dur ing surgery, they readily conform to defect sites, and when packed together they inherently fo rm a porous network (important for bone ingrowth and vascular izat ion) . H o w e v e r particulate graft beds are susceptible to shear failure and shear deformation, and i n impac t ion al lograft ing this has been suggested as a major cause for migra t ion o f the implant i n femur 1 8 , 1 9 ' 2 0 . In so i l mechanics , the M o h r - C o u l o m b failure l aw is used to describe the shear strength, if , o f an aggregate g iven by rf = c + c r t a n ^ (2.1) Where a is the appl ied normal stress, c is the cohesion o f the particles, and </> is the internal f r ic t ion between the particles 2 1 . M a n y studies use the M o h r - C o u l o m b criteria as 13 a measure of the graft bed shear capability ' ' . Graft bed compressive stiffness or modulus has also been suggested to be of primary importance for implant stability, progressive vascularization, and replacement by host bone 2 2 ' 2 3 . We believe that by measuring both the shear strength and compressive modulus of the graft beds we can get an overall measure of the graft bed load bearing capability. Our first objective is to determine the mechanical properties of a calcium polyphosphate particulate and compare it to the mechanical properties of the current gold standard for impaction allografting, M C B . The stiffness of the constructs is compared through testing in confined compression and the shear strengths of the materials are compared through direct shear tests. A number of studies have tried to improve the properties of allograft beds through different impaction techniques or adding extender particles such as bioglass or HA/TCP but no one has attempted to determine the maximum properties attainable1 8'2 4'2 5. Thus, our second objective is to establish a relationship between the properties of the particles and the properties of the particulate construct. This is achieved by characterizing the mechanical properties of ideal particulate constructs comprised of spherical balls of varying material and size. 14 2.2 Materials and Methods 2.2.1 Materials The C P P particles used i n this study were p rov ided by D r . P i l l i a r (Univers i ty o f Toronto) , and were formed by ca l c in ing precursor powders o f c a l c i u m phosphate monobas ic monohydrate i n a p l a t i num crucible 1 5 . These powders were then gravi ty sintered i n cy l ind r i ca l tubes w h i c h were later crushed into angular particles o f 1-3mm i n diameter (Figure 2.1a). The resul t ing particles have 30 -45% internal poros i ty w i t h interconnected pores i n the lOOpjm range (Figure 2.1b). The compressive modulus o f the particles is reported to be approximate ly 5 G P a and tensile strengths i n the range o f 5 - 2 4 M P a 1 5 . The M C B used for compar i son to C P P was not tested i n this study but were tested us ing the same experimental setup by A l b e r t et al . B r i e f l y , thir ty-nine femoral heads were morse l l i zed w i t h a Lere bone m i l l (DePuy , Warsaw, I N , U S A ) . The cor t ica l bone from the femoral neck and cartilage were removed pr ior to morse l l i za t ion . The graft was pooled together and r insed i n a saline solut ion to remove fat and m a r r o w tissue. The graft particles ranged i n size between 0.6 and 8 m m , w i t h 5 0 % finer than 2 . 4 m m . The spherical bal ls (Cinco t ta Industrial Componets Inc.) used for the second objective o f this study are compr i sed o f three different materials; steel, glass and n y l o n o f approximate modulus 200, 70 and 2 G P a respectively 2 1 . The steel bal ls consisted o f three different sizes (1.19, 1.54 and, 2 .38mm i n diameter) w h i l e glass and n y l o n balls were 1.54mm i n diameter (Figure 2.1a). A l l balls had a d imens iona l tolerance o f 15 ± 0 . 1 2 7 m m . The geometry and mechanical properties o f a l l particles used i n this study are shown i n Table 2 .1 . Material Particle Diameter (mm) Particle Shape Internal Pore Size (um) Young's Modulus E P (Gpa) Steel 1.19, 1.54, 2.38 Sphere N/A 200 a Glass 1.54 Sphere N/A 70 a Nylon 1.54 Sphere N/A CPP 1-3 Irregular -100 5 b MCB 0.6-8 Irregular ~500c 0.1-1 c Table 2.1: Material properties of particles used in testing "From CRC Material Science and Engineering Handbook27 b From Pilliar et al15 0 From Gibson and Ashby35 2.2.2 Methods The CPP and spherical particles were tested in confined compression using a servohydrolic testing machine (Instron Model 8874, Instron, Canton, Massachusetts). A stainless steel cylinder of 19mm internal diameter, wall thickness of 6mm was filled to a height of ~35mm. The samples were then loaded via a piston at a stress rate of 400 kPa per minute to a maximum stress of 2000 kPa (Figure 2.2a). The stress a is calculated as the applied normal force F n o r m a i divided by the cross-sectional area of the graft bed. The compressive strain s is calculated as s = 1-H/H0 where H 0 is the initial height of the graft bed and H is the instantaneous height. A total of six samples (N=6) were tested for each particle type. The M C B was tested on the same testing equipment however due to its biphasic nature, the test cylinder was prepared with 36 radial holes of 1mm diameter to allow for fluid drainage. The time effects of the M C B also required a different compression test protocol described previously . 17 The particles were tested i n conf ined shear i n a custom designed shear box , w h i c h is s imi lar to the conf ined compress ion setup. The shear box consists o f two cyl inders w i t h internal diameter o f 1 9 m m and w a l l thickness 6 m m . The top cy l inder was r i g i d l y f ixed i n place w h i l e the bot tom cy l inder was free to move a long a l inear guide. The samples were f i l l ed to a height o f ~ 2 5 m m , w i t h h a l f the sample o n either side o f the shear plane. A normal load was appl ied to the sample v i a the material testing machine (Instron M o d e l 8874, Instron, Canton , Massachusetts) , wh i l e a lateral shear load was appl ied to the bottom cyl inder us ing a side mounted actuator (Instron M o d e l A 5 9 1 - 4 , Instron, Canton , Massachusetts) (Figure 2.2b). Three normal stresses were used, 125, 250, 375 k P a , representing a range o f stress seen by allograft material i n a h ip reconstruct ion . The samples were sheared at a rate o f 1 .2mm/min up to a total o f 6 m m . A total o f s ix samples ( N = 6) were sheared for each particle type. The shear stress o f the mater ia l was defined as shear force over the area o f intersection o f the two cyl inders (Equa t ion 2.2). T h i s equation accounts for the decrease i n cross sectional area as the cyl inders sl ide past each other. T(8) = n : ^ : 1 (2.2) (d/2)2[2 c o s _ I ( 8 I d ) - s i n ( 2 cos" 1 (8Id))\ Where FS| i ear is the shearing force, d is the cy l inder diameter and 8 is the displacement o f the bot tom cy l inder relative to the top. T o a l l o w for a direct compar i son between C P P and the M C B , the C P P constructs were impacted pr ior to shear testing. The samples were impacted 20 t imes at 9 0 0 N by the material testing machine at a rate o f 4hz w h i c h represents the impac t ion procedure used c l i n i c a l l y 2 8 . 18 Figure 2.2: Experimental setup: a) confined compression and b) direct shear. The vo lume fraction o f particles i n the graft bed o f the C P P and spherical particles was found by measur ing the amount o f water displaced by the sample i n a cy l inder w i t h an internal diameter o f 1 9 m m and at a height o f 2 5 m m . The v o l u m e fraction o f M C B was measured f rom his to logy by sect ioning the graft bed and measur ing the percent o f the cross-sectional area occup ied by bone 2 6 . Th i s measurement technique counts internal pores as be ing unoccupied space and w i l l therefore result i n a lower v o l u m e fraction than the technique used for the C P P and spherical particles w h i c h do not account for internal pores. 19 2.2.3 Statistical Analysis Pearson correlations were used to determine the l inear correlat ion, R , o f the construct modulus E c w i t h the particle size. Compress ion results were ana lyzed w i t h a student T -Test. Shear strength was compared between the materials w i t h a 2 -way A N O V A (material, no rmal stress). S tudent -Newman K e u l s tests were used for post-hoc analyses, and a signif icance leve l o f 0.05 was used. 2.3 Results The results o f the compress ion tests are shown i n Figure 2.3 as the average compressive stress a o f the samples (N=6) as a function o f strain 8 for each particulate ( C P P and spherical). D u e to the n o n l inear nature o f the stress-strain curves the modulus o f the particulate constructs, E c , was quantified as the secant modulus at a compress ive stress a o f 1100 k P a . The secant modulus represents the effective stiffness o f the construct as it is used c l i n i c a l l y . The resul t ing E c for a l l materials are presented i n Table 2.2, a long w i t h the m o d u l i o f the particles themselves, E p . F igure 2.3 and Table 2.2 show that E c increases w i t h E p and i f l inear correlations are performed E c is also shown to increase l inearly w i t h the particle diameter d for the steel particles ( R = 0.82). Table 2.2 shows that the construct modulus E c is m u c h smaller than the particle modu lus E p . 20 Steel «• 0 • ' 0:02 0.04 0.06 0.08 0.1 ,'" 0.12 0. Compressive Strain £ Figure 2.3: Confined compression results - stress as a function of strain - for spherical and CPP particles. The lines represent an average of six individual tests. Construct modulus E c defined as secant modulus at a compressive stress a = 1100 kPa. Material Size (mm) E P (GPa) Ec (GPa) Vf(%) E P / E C Steel Large (d=2.38) 200 0.310 60.1 ± 0.2 645 Medium (d=1.54) 200 0.237 58.1 ± 0.2 844 Small (d=1.19) 200 0.198 59.5 ± 1.0 1008 Glass Medium (d=1.54) 70 0.093 59.3 ± 0 . 9 751 Nylon Medium (d=1.54) 2 0.031 59.0 ± 0 . 1 64.5 CPP (d=1-3) 5 0.0151 48.9 ± 0 . 9 331 MCB (d=0.6-8) 0.1-1 0.0149 38 (33-45)a 6.71-67.1 Table2.2: Construct modulus Ec measured as the secant modulus of the stress strain curve from confined compression tests at a = 1100 kPa. Volume fraction of particles in the graft bed is the V/%). a Measured from histology as the graft density by Albert26. Reported as median (range) Measurements showed significant difference between CPP and spherical particle volume fractions, 48.9 ± 0.9%, and -59% respectively. The volume fraction of the M C B graft is reported to be approximately 38% . 21 The results of the shear tests for the CPP particles are shown in Figure 2.4 as the average shear stress x of the tests (N=6) at each of the different normal stresses a (125kPa, 250kPa, and 375kPa). The shear strength, if, is commonly defined as the value of the shear stress corresponding to the "plateau" of the shear displacement curve. Since there is no clear plateau for the CPP tests, and to allow for direct comparison with the shear results o f M C B , the shear strength Xf is taken to be the shear stress at a displacement of 8=lmm (approximately 5% cylinder width). For the spherical particles (graphs not shown) the shear strengths were taken to be at displacement equal to particle diameter, which was found to correspond with the plateau of the shear stress. There was an increase in the shear strength with increasing normal stress a for all materials (p<0.01). The results of shear tests are presented in terms of the Mohr-Coulomb parameters cohesion intercept (c) and friction angle (cp) in Table 2.3. Mohr-Coulomb Data ( r f = c + a tanO) Material Size Cohesion c (kPa) Friction Angle <I> (Degrees) • Steel Large 25.9 39.7 Medium 16.6 37.4 ' Small 20.3 29.9 Glass Medium 14.5 38.5 Nylon Medium 28.5 33,9 C P P 380 45.6 M C B 510 29.2 Table 2.3: Results of shear, tests reported as Mohr-Coulomb parameters. Figure 2.5 shows the results of the shear strength Xf as a function of the compressive normal stress a. These results show that there is no significant difference in the shear 22 strengths of CPP and MCB particulates (p > 0.3), there is a significant increase of the shear strength of CPP and MCB compared to the spherical balls (pO.Ol). Furthermore, the spherical ball data yielded no difference in shear strength between different materials of the same size (steel, glass, and nylon of 1.54mm diameter)(p>0.1), but the steel particles of different sizes (1.18mm, 1.54mm and 2.36mm diameter) showed a significant difference between large and small particles at normal stresses of 250 kPa and 375 kPa (p<0.05). 2 3 4 Shear Displacement 5 (mm) Figure 2.4: Results of shear test for CPP particles for normal stress: 125, 250 375 kPa. The lines represent an average of six individual tests. Shear strength, xf> defined as shear stress at a displacement of 1 mm. 23 900 800 Tf = c + o tan<p 0 (d=1.54mm) Steel A Glass • Nylon • 0 50 100 150 200 250 300 Compressive Normal Stress a (kPa) 350 400 450 Figure 2.5: Results of shear test - shear strength as a function of normal stress a (125, 250, 375 kPa). Mohr-Coulomb parameters are defined as shown. Results were offset on the abscissa for clarity. MCB data from Albert26. 2.4 Discussion In the confined compress ion results o f Figure 2.3 a non l inear stress-strain relat ionship is observed i n a l l materials. T h i s effect can be expla ined by contact mechanics models such as the Her tz contact mode l and the de Gennes soft crust mode l w h i c h describe the stress-strain relationship between two particles being forced together 2 9 . B o t h models g ive a relationship o f the f o r m n (2.3) 24 Where a is the appl ied compress ive stress, E p is the modulus o f the part icle, s is the compressive strain, and n is a mode l parameter. In the Her tz m o d e l o f two homogenous spheres the term n is equal to 3/2 whereas the de Gennes m o d e l w h i c h accounts for a softer surface due to irregularit ies o f ox ida t ion results i n an n va lue o f 2. B o t h these models indicate that particles w i t h higher m o d u l i w i l l result i n stiffer constructs and that the secant modulus E c increases w i t h increasing particle modulus E p and strain as s This result is consistent w i t h our compress ion results for the spherical part icles but not i f we compare the C P P results w i t h the n y l o n ba l l results. T h i s is most l i k e l y expla ined by the difference i n shape o f the two particles. It has been shown that i r regular i ty hinders particle mo t ion and their ab i l i ty to reach dense pack ing configurat ions (Table 2.2), resulting i n fewer contact points between particles . D u r a n suggests that a particulate construct becomes more r i g i d w i t h increasing load through a prol i fera t ion o f contact points (wh ich further explains the non-l ineari ty o f the stress-strain curves i n F igure 2.3) . Therefore C P P hav ing a lower pack ing density than n y l o n has fewer contact points and results i n a lower construct stiffness. Ano the r effect o f the part icle shape is a difference i n deformation o f the particles. Irregular particles are more deformable than round particles, because o f more loca l i zed deformation, w h i c h is seen by compar ing cone-to-plane and sphere-to-plane contact i n a Her tz ian contact m o d e l 3 1 . 25 Because spherical part icles represent an ideal particulate it is useful to use the confined compress ion data o f the spherical particles to gain an understanding o f the effect o f particle modulus E p o n the construct modulus E c . The E c and E p data o f Tab le 2.2 plotted on a log- log graph (Figure 2.6) shows a relationship between the construct modulus and particle modulus . The m e d i u m ba l l data is approximately descr ibed by the straight l ine fit EC»C[EPY (2.5) where the constant term C = 0.02 and E c and E p are i n units o f G P a . T h i s non linear result is consistent w i t h the Her tz contact mode l , and the de Gennes soft crust mode l w h i c h can be shown to have a s imi la r relationship w i t h power terms o f % and Vi respectively. 26 C=0.02 Steel (d=2.38mm) (d=1.54mm) • ^ 0 ^ ^ (d=1.19mm) * ^ y ^ ^ Ec=C(E p ) a 4 Nylon (d=1.54mm) Glass (d=1.54mm) , MCB (d=0.6-8mm) ^ C P P (d=1-3mm) 1 10 100 1000 Particle Modulus E p (Gpa) Figure 2.6: Experimental construct modulus E c as a function of particle modulus E p . The points represent an average of six individual tests. Straight line fit to medium ball data. A notable outcome of Equation 2.5 is the constant term (C =0.02), which suggest that the construct modulus E c of a spherical particulate will always be a small fraction of the particle modulus. This result is similar to that of by Abel-Ghani et al who showed a construct modulus of glass ballotini being 0.007-0.03 of the particle modulus . Equation 2.5 can be further refined by defining the construct modulus E c as a function of the applied stress c. A family of curves can be used to show (2.6) 27 where C = 0.002 and E c and E p units o f G P a and a is i n units o f k P a . Equa t ion 2.6 was found to c lose ly describe current experimental data w i t h an appl ied stress between 1000 and 2000 k P a . One o f the requirements o f materials used for bone scaffolds is that they have an interconnected pore structure to a l l o w bone ingrowth and vascular iza t ion. Therefore it is useful to extend Equa t ion 2.6 to include the effects o f internal porosi ty i n the particles. A number o f studies have shown a decrease i n modu lus w i t h increased p o r o s i t y 3 3 ' 3 4 . F o r example the G i b s o n - A s h b y m o d e l 3 5 is g iven by Y o u n g ' s modulus o f the s o l i d material . C o m b i n i n g Equat ions 2.6 and 2.7 results i n a relationship for the modu lus o f a construct o f porous particles A s this relat ionship is based o n a mode l for spherical particles it does not account for the two important features o f irregular particles: increased deformation and decreased pack ing density. The constant ( C = 0.002) i n Equa t ion 2.8 assumes a r andom pack ing density o f approximate ly 6 0 % w h i c h is i n between the loose stack l i m i t for spheres o f 5 6 % and the theoretical m a x i m u m random pack ing o f spheres o f 6 4 % . Since irregular particles have lower p a c k i n g density and increased deformation they w i l l inevitable have a lower construct modulus as is evidenced by the C P P particles. Equa t ion 2.8 predicts EP«E,(I-PY (2.7) Where E the effective part icle modulus , p is the porosi ty o f the part icle and E s is (2.8) 28 that C P P particles o f 4 0 % porosi ty and Y o u n g ' s modulus o f 100% dense C P P o f 48 G P a 3 7 to have a construct modulus o f 0.066 G P a , s ignif icant ly higher than the measured E c o f 0.0151 G P a . Hence w e bel ieve Equa t ion 2.8 represents an approximate upper bound for the construct modulus o f porous particles. The shear results shown i n F igure 2.5 give insight into the effects o f part icle modulus , size, and shape on shear strength. The shear data o f m e d i u m s ized bal ls (d = 1.54mm) shows that particle modu lus does not s ignif icant ly affect the shear strength o f the construct. T h i s observat ion is i n agreement w i t h so i l mechanics theories where it is generally-agreed that f r ic t ional forces and cohesion between particles are the p r imary mechanism i n deve lop ing shear strength o f a particulate c o n s t r u c t 2 1 . There are two types o f fr ict ional forces that resist mo t ion i n a so i l , s l id ing f r ic t ion and translational or in ter locking fr ic t ion. S l i d i n g f r ic t ion develops through the in te r lock ing o f mic roscop ic aspirates on the part icle surface. Inter locking fr ic t ion arises f rom particles be ing required to move f rom their o r ig ina l pos i t ion and ride over adjacent particles. The effect o f these fr ict ional forces is reflected i n the M o h r - C o u l o m b term o f internal f r ic t ion ^ . In the shear data for the steel part icles o f a l l three sizes there is an increase i n the internal f r ic t ion angle w i t h increasing part icle size. Since there is no difference i n the surface morphology o f the part icles this effect is l i k e l y due to the difference i n in te r locking fr ict ion between part icle sizes. The breakdown o f in te r locking is associated w i t h i n increase i n v o l u m e as the particles ride over one another 2 1 . F o r larger particles this vo lume change is greater and thus requires a higher shear force, w h i c h c o u l d exp la in the observed particle size effect. One distinct result f rom the shear data i n F igure 2.5 is the 29 difference i n shear strength o f C P P and M C B compared to that o f the spherical particles. Th i s difference is m a i n l y due to the cohesion (c) o f the particles descr ibed by the ver t ical intercept i n F igure 2.5, w h i c h represents the shear strength under zero appl ied normal stress. The cohes ion is l i k e l y dependent on the particle shape since there is no significant difference i n the cohes ion o f the spherical particles. Spher ica l part icles have poin t - l ike contacts that are mechan ica l ly instable, w h i c h means loca l s l ipp ing and rotat ion can easily o c c u r 3 0 . Irregular particles w i l l have more stable face-to-face contacts w h i c h is superior at resist ing s l id ing and rotation . The effect o f cohes ion far outweighs any gain through internal f r ic t ion as even a large steel spherical part icle construct under a normal load o f 375 k P a w i l l not match the shear strength o f an angular C P P construct w i t h no normal load (Figure 2.5). Ano the r interesting point is that even though the spherical particles had a s ignif icant ly higher vo lume fraction ( V f = 59%) to that o f C P P ( V f = 48%), their shear strengths were m u c h lower . T h i s suggests that i n shear the number o f contact points is not as important as the type o f contact points. T h i s is consistent w i t h the w o r k o f D u n l o p , w h o found no significant difference i n the shear strength o f M C B w i t h vary ing amounts o f part icle grading . W h e n interpreting the results o f this study it is important to note the l imi ta t ions o f the tests. B o t h the.confined compress ion and shear tests were performed i n testing chambers w h i c h are sma l l relative to the A S T M standards 3 8 . B y us ing a sma l l s ize cy l inder the edge effects m a y be significant , but it was felt that a cy l inder o f this size more c lose ly represented the c l i n i c a l si tuation o f a medul la ry canal . Furthermore, shear testing was performed i n a direct shear box w h i c h forces a material to shear a long a prescribed plane 30 rather than a l l o w i n g the mater ial to shear a long the weakest plane as i n a t r i ax ia l shear test. Th i s may lead to ar t i f ic ia l ly h i g h shear strength. H o w e v e r , many studies have used this method o f testing and it is felt to be adequate for the purpose o f compar ing different constructs 1 8 ' 1 9 > 2 0 . F i n a l l y , the measurement o f shear strength is d i f f icul t to define. The A S T M standards define shear failure occurr ing i n the between 10-20% shear T O displacement however there is no indica t ion o f what this represents . The shear strength o f C P P was taken at 5 = 1 m m to a l l o w for direct compar i son w i t h previous results for M C B , whereas the shear strength o f the spherical balls was taken to be at a displacement equal to their diameter. P h y s i c a l l y this represents the hor izonta l displacement one ba l l must travel i n order to m o v e f rom beside an adjacent ba l l to the top o f it. There are a couple m a i n impl ica t ions o f this study. Firs t , even i f a particulate construct is comprised o f very s t i f f part icles, it w i l l have a re la t ively l o w compress ive construct modulus . F o r example , us ing the upper bound solut ion i n F igure 2.6 w e can see that even a construct o f a lumina particles ( E p ~ 400 G P a ) w o u l d result i n a compress ive construct modulus no higher than 0.5 G P a . T h i s is m u c h lower than the modu lus o f P M M A (~3 G P a ) currently used as a bone cement to strengthen and stiffen allograft i n r ev i s ion surgery. T h i s indicates that bone graft made o f particles m a y need to be supplemented w i t h a binder mater ial i n order to achieve an adequate stiffness. Second, shear strength is s ignif icantly increased for irregular shaped particles, however , i rregular shaped particles give a m u c h lower p a c k i n g density w h i c h negatively affects the compress ive construct modulus . Therefore, w h e n m a k i n g a particle construct, a balance between pack ing 31 density and shape must found i n order to op t imize both the construct modu lus and shear strength. 2.5 Conclusion This study showed that c a l c i u m polyphosphate particulate graft beds had s imi l a r mechanical properties both i n terms o f stiffness i n confined compress ion and shear strength to that o f mor se l l i z ed bone. Th i s suggests that C P P , f rom a mechanica l point o f v i e w , is a suitable bone graft mater ial for the use i n rev i s ion surgery. C o n f i n e d compression tests showed that the stiffness o f the construct increased w i t h particle stiffness and appl ied stress. It was also observed that irregular shaped particles had a lower pack ing density and resulted i n lower a construct modulus . Shear tests showed that shear strength increased w i t h increasing normal load and was m u c h higher for irregular shaped particles than spherical particles. The shear strength o f the construct was not affected by the part icle modulus , and on ly was weak ly affected by the size o f the particles. 32 2.6 References 1. Elting, J. J., Mikhail, W. E. M., Zicat, B. A., Hubbell, J. C , Lane, L. E., and House, B. P re l iminary-Repor t o f Impact ion Graf t ing for Exchange Femora l Ar throplas ty . Clinical Orthopaedics and Related Research [319], 159-67. 1995. 2. Halliday, B. R., English, H. W., Timperley, A. J., Gie, G. A., and Ling, R. S. M. F e m o r a l impac t ion grafting w i t h cement i n r ev i s ion total h ip replacement -E v o l u t i o n o f the technique and results. Journal of Bone and Joint Surgery-British Volume 85B[6 ] , 809-17. 2003. 3. Frances, A, Moro, E, Cebrian, J, Marco, F, Garcia-Lopez, A, Serfaty, D, and Lopez-Duran, L. Reconst ruct ion o f bone defects w i t h impacted allograft i n femoral stem rev i s ion surgery. Intrenational Orthopedics (SICOT) 31 [4], 457-64. 2007. 4. Mahomed, N. N., Barrett, J. A., Katz, J. N., Phillips, C. B., Losina, E., Lew, R. A., Guadagnoli, E., Harris, W. H., Poss, R., and Baron, J. A. Rates and outcomes o f p r imary and rev i s ion total h ip replacement i n the U n i t e d States M e d i c a r e popula t ion . Journal of Bone and Joint Surgery-American Volume 85A[1], 27-32. 2003. 5. Ornstein, E., Atroshi, I., Franzen, H., Johnsson, R., Sandquist, P., and Sundberg, M. Resul ts o f h ip rev i s ion us ing the exeter stem, impacted allograft bone, and cement. Clinical Orthopaedics and Related Research [389], 126 -33 .2001 . 6. Knight, J. L. and Helming, C. Col lar less po l i shed tapered impac t ion grafting o f the femur dur ing rev i s ion total h ip arthroplasty - Pi t fa l l s o f the surgical technique and fo l low-up i n 31 cases. Journal of Arthroplasty 15[2], 159-65. 2000. . 7. Eldridge, J. D. J., Smith, E. J., Hubble, M. J., Whitehouse, S. L., and Learmonth, I. D. M a s s i v e early subsidence f o l l o w i n g femoral impac t ion grafting. Journal of Arthroplasty 12[5], 535-40. 1997. 8. Meding, J. B., Ritter, M. A., Keating, E. M., and Faris, P. M. Impact ion bone-grafting before insert ion o f a femoral stem w i t h cement i n rev i s ion total h ip arthroplasty - A m i n i m u m two-year fo l low-up study. Journal of Bone and Joint Surgery-American Volume 7 9 A [ 1 2 ] , 1834-41. 1997. 9. Goodman, J. L. The safety and avai labi l i ty o f b l o o d and tissues - Progress and challenges. New England Journal of Medicine 351 [8], 819-22. 8-19-2004. 33 10. Sugihara, S., van Ginkel, A. D., Jiya, T. U., van Royen, B. J., van Diest, P. J., and Wuisman, P. I. J. M. His topathology o f retr ieved allografts o f the femoral head. Journal of Bone and Joint Surgery-British Volume 81B[2] , 336-41 . 1999. 11. van Haaren, E. H., Smit, T. H., Phipps, K., Wuisman, P. I. J. M., Blunn, G., and Heyligers, I. C. Tr icalc ium-phosphate and hydroxyapat i te bone-graft extender for use i n impac t ion grafting rev i s ion surgery. Journal of Bone and Joint Surgery-British Volume 87B[2] , 267-71. 2005. 12. Temenoff, J. S. and Mikos, A. G. Injectable biodegradable materials for orthopedic tissue engineering. Biomaterials 21 [23], 2405-12. 2000. 13. Livingston-Arinzeh, T. L., Tran, T., Mcalary, J., and Daculsi, G. A comparative study o f b iphasic c a l c i u m phosphate ceramics for human mesenchymal s tem-cel l - induced bone formation. Biomaterials 26[17] , 3631-8. 2005. 14. Baksh, D., Davies, J. E., and Kim, S. Three-dimensional matrices o f c a l c i u m polyphosphates support bone growth i n vi t ro and i n v i v o . Journal of Materials Science-Materials in Medicine 9[12], 743-8. 1998. 15. Pilliar, R. M., Filiaggi, M. J., Wells, J. D., Grynpas, M. D., and Kandel, R. A. Porous c a l c i u m polyphosphate scaffolds for bone substitute applicat ions - i n vi t ro characterization. Biomaterials 22[9], 963-72. 2001 . 16. Grynpas, M. D., Pilliar, R. M., Kandel, R. A., Renlund, R., Filiaggi, M., and Dumitriu, M. Porous c a l c i u m polyphosphate scaffolds for bone substitute applicat ions i n v i v o studies. Biomaterials 23 [9], 2063-70. 2002. 17. Waldman, S. D., Grynpas, M. D., Pilliar, R. M., and Kandel, R. A. Character iza t ion o f cartilagenous tissue formed o n c a l c i u m polyphosphate substrates i n v i t ro . Journal of Biomedical Materials Research 62[3], 323-30. 12-5-2002. 18. Dunlop, D. G., Brewster, N. T., Madabhushi, S. P. G., Usmani, A. S., Pankaj, P., and Howie, C. R. Techniques to improve the shear strength o f impacted bone graft - The effect o f particle size and wash ing o f the graft. Journal of Bone and Joint Surgery-American Volume 8 5 A [ 4 ] , 639-46. 2003 . 19. Brewster, N. T., Gillespie, W. J., Howie, C. R., Madabhushi, S. P., Usmani, A. S., and Fairbairn, D. R. M e c h a n i c a l considerations i n impac t ion bone grafting. J.Bone Joint Surg.Br. 81[1], 118-24. 1999. 20. Tanabe, Y., Wakui, T., Kobayashi, A., Ohashi, H., Kadoya, Y., and Yamano, Y. De te rmina t ion o f mechanica l properties o f impacted human morse l l i zed cancel lous allografts for rev i s ion jo in t arthroplasty. Journal of Materials Science-Materials in Medicine 10[12], 755-60. 1999. 34 21. Yong, R. N and Warkentin, B. P. S o i l Properties and Behav iour . 1975. A m s t e r d a m , E l sev i e r Scient i f ic Pub l i sh ing C o m p a n y . Deve lopments i n Geotechnica l Engineer ing 5. 22. Karrholm, J., Hultmark, P., Carlsson, L., and Malchau, H. Subsidence o f a non-po l i shed stem i n revis ions o f the h ip us ing impac t ion allograft - Eva lua t ion w i t h radiostereometry and dual-energy X - r a y absorptiometry. Journal of Bone and Joint Surgery-British Volume 81B[1] , 135-42. 1999. 23. Malkani, A. L., Voor, M. J., Fee, K. A., and Bates, C. S. F e m o r a l component r ev i s ion us ing impacted morse l l i sed cancellous graft - A b iomechan ica l study o f implant stabil i ty. Journal of Bone and Joint Surgery-British Volume 7 8 B [ 6 ] , 973-8. 1996. 24. Cornu, O., Bavadekar, A., Godts, B., Van Tomme, J., Delloye, C , and Banse, X. Impact ion bone grafting w i t h freeze-dried irradiated bone. Part II. Changes i n stiffness and compactness o f morse l i zed grafts - Exper iments i n cadavers. Acta Orthopaedica Scandinavica 74[5], 553-8. 2003 . 25. Grimm, B., Miles, A. W., and Turner, I. G. O p t i m i z i n g a hydroxyapati te/ t r icalcium-phosphate ceramic as a bone graft extender for impac t ion grafting. Journal of Materials Science-Materials in Medicine 12[10-12], 929-34. 2001 . 26. Albert, C , Masri, B., Duncan, C , Oxland, T., and Fernlund, G. Impact ion al lograft ing - The effect o f impact ion force and alternative compac t ion methods o n the mechanica l characteristics o f the graft. Submitted for publication in JBJS . 2007. 27. Shackelford, J. and Alexander, W. C R C M a t e r i a l Science and Eng inee r ing Handbook . Shackel ford , J . and Alexander , W . T h i r d . 2001 . B o c a Ranton , C R C Press L L C . 28. Fosse, L., Ronningen, H., Lund-Larsen, J., Benum, P., and Grande, L. Impacted bone stiffness measured dur ing construct ion o f morse l l i sed bone samples. Journal Of Biomechanics 37[11], 1757-66. 2004. 29. Duran, J. Sands, Powders , and Grains . 2000. N e w Y o r k , Sp r inge r -Ver l ag N e w Y o r k Inc. Par t i a l ly Ordered Systems. G u y o n , E . , L a m , L . , L a n g e v i n , D . , and Stanley, H . E . 30. Troadec, J. P. and Dodds, J. A. G l o b a l geometiral descr ip t ion o f homogeneous hard sphere packings . B i d e a u , D . and Hansen, A . Di so rde r and Granular M e d i a . [5], 133-63. 1993. Ams te rdam, E l sev ie r Science publishers B . V . R a n d o m Mater ia l s and Processes. Stanley, H . E . and G u y o n , E . 35 31. Cho, G. C., Dodds, J., and Santamarina, J. C. Par t ic le shape effects on pack ing density, stiffness, and strength: Na tura l and crushed sands. Journal of Geotechnical and Geoenvironmental Engineering 132[5], 591-602. 2006. 32. Adams, M. J., McKeown, R., and Whall, A. A mic romechan ica l m o d e l for the conf ined un i -ax ia l compress ion o f an assembly o f elas t ical ly deforming spherical particles. Journal of Physics D-Applied Physics 30[5], 912-20. 3-7-1997. 33. Boccaccini, A. R. and Fan, Z. A new approach for the Y o u n g ' s modulus-porosi ty correla t ion o f ceramic materials. Ceramics International 23 [3], 239-45. 1997. 34. Pabst, W., Gregorova, E., and Ticha, G. E las t i c i ty o f porous ceramics - A cr i t ica l study o f modulus-poros i ty relations. Journal of the European Ceramic Society 26[7] , 1085-97. 2006. 35. Gibson, L. and Ashby, M. F. Cance l lous bone'. C e l l u l a r So l ids Structure and Properties. [11], 316-30. 1988. Oxfo rd , Pe rgamon Press. 36. de Gennes, P. G. Ref lect ions on the mechanics o f granular matter. Physica A-Statistical Mechanics and Its Applications 261 [3-4], 267-93. 12-15-1998. 37. Guo, W., Kim, S., Grynpas, M. D., Pritzker, K. P. H., and , Pilliar R. M. C a l c i u m polyphosphate fibres for composi te biomater ia ls . Degradation studies, 20th Annual Meeting of Soc for Biomaterials . 1994. 38. A S T M D 3 0 8 0 - 0 4 Standard Test M e t h o d for Di rec t Shear Test o f So i l s Unde r Conso l ida ted Dra ined Condi t ions . 331-7. 2004. Wes t Conshohocken , A S T M International. 36 3 Effect of bone graft substitute on marrow stromal cell proliferation and differentiation2 3.1 Introduction M a r r o w stromal cel ls ( M S C s ) have been w e l l established as a source o f progenitor cells for various lineages o f the musculoskele ta l system capable o f differentiating into osteoblasts, adipocytes, chondrocytes, and myoblasts I ' 2 , 3 > 4 . M S C s can be readi ly isolated from bone mar row aspirates by virtue o f their adherence to plast ic and expanded a b i l l i on - fo ld i n culture w h i l e main ta in ing their prol i ferat ion and differentiat ion potential 5 ' 6 . Th is makes M S C s ideally, suited for tissue engineering and regenerative medic ine approaches to repair musculoskele ta l tissue since they w i l l not o n l y contribute to the IRQ regeneration but also main ta in the repaired tissue ' ' . M S C s are be l ieved to conta in a smal l popula t ion o f muli tpotent ia l mesenchymal stem cells . A l t h o u g h their "sternness" is s t i l l debatable they are thought to have the capacity for asymmetr ic d i v i s i o n and give rise to commit ted progenitors o f the different mesenchymal tissues i nc lud ing bone l 0 . The commitment o f mesenchymal stem cells towards the osteoblast l inage can be characterized by different maturat ion stages starting w i t h a mult ipotent ia l stem c e l l , and progressing through osteoprogenitor, preosteoblast, osteoblast and f ina l ly to osteocyte or bone l i n i n g c e l l n > 1 2 ' 1 3 . These differentiation or maturation stages are associated w i t h changes i n expression levels o f osteoblast-2 A version of this chapter will be submitted for publication. 37 associated molecules i nc lud ing c b f a l ( R U N X - 2 ) , a lkal ine phosphatase ( A L P ) , col lagen I (Col - I ) , osteonectin ( O N ) , osteopontin (OP) , bone s ialoprotein ( B S P ) , and osteocalcin ( O C ) 1 1 ' 1 2 . Different ia t ion o f M S C towards the ostogenic lineage can be induced by soluble factors i nc lud ing dexamethasone, ascorbic ac id and P-glycerphosphate or w i t h growth factors such as bone morphogenic proteins ( B M P s ) and fibroblasts growth factor ( F G F ) on a number o f subst ra tes 1 4 ' 1 5 ' 1 6 . C e l l differentiation and prol i fera t ion can also be induced by signals d i rect ly received from the substrate through trans-membrane or cytoplasmic receptors 1 7 . It has been shown recently that differences i n substrate elasticity and surface mic ro structure can lead to different lineage c o m m i t m e n t 1 8 ' 1 9 . H o w e v e r it remains unclear f rom these investigations whether different substrates affect prol iferat ion and differentiat ion o f M S C s under expansion condi t ions . Therefore the a i m o f this study was to determine the effect o f different substrates, currently used for bone regeneration and tissue engineering, on the prol iferat ion and differentiation o f M S C s under expansion condi t ions . The four substrates evaluated i n this study were bone, c a l c i u m polyphosphate ( C P P ) , biphasic hydroxyapat i te / t r ica lc ium phosphate ( H A / T C P ) and tissue culture plast ic ( T C ) . B o n e is currently one o f the most used bone graft materials, w i t h a natural compos i t ion 20 21 and structure it makes an ideal scaffold for bone heal ing and r emode l l i ng ' . C P P is a nove l ceramic currently be ing investigated for its use i n cartilage repair due to its good b iocompat ib i l i ty and mechan ica l properties . H A / T C P is a ceramic that has been studied 23 extensively and has been shown to be suitable for bone ingrowth in vitro and in vivo . 38 The objectives o f this study were to observe the effect o f the different materials on M S C proliferat ion and determine their l inage commitment through gene express ion profi le . 3.2 Materials and Methods 3.2.1 Scaffold Materials The bone particles were harvested f rom the femora and tibiae o f seven rats (Figure 3.1a). The soft tissue was scraped f rom the bones w h i c h were then crushed into particles o f 1-7 m m i n size. Part icles conta ining cart i laginous tissue were r emoved and the remain ing particles were washed several t imes i n 7 0 % ethanol for s ter i l izat ion and to remove any remaining bone mar row. The C P P scaffold mater ial was p rov ided by D r . P i l l i a r (Univers i ty o f Toronto) and was formed by ca l c in ing precursor powders o f c a l c i u m phosphate monobas ic monohydrate i n a p la t inum crucible (Figure 3 .1b) 2 4 . These powders were then gravi ty sintered i n cy l ind r i ca l tubes w h i c h were later crushed into angular particles o f l - 3 m m i n diameter. The resulting particles have an interconnected porous network (30-45% porosi ty) w i t h pore diameter i n the range o f 1 0 0 u m 2 5 . The hydroxyapat i te / t r ica lc ium phosphate ( H A / T C P ) scaffold mater ia l was purchased f rom Berke ley A d v a n c e d Biomater ia l s Inc (Figure 3.1c). The ceramic was manufactured w i t h a compos i t ion o f 2 0 % H A and 8 0 % T C P , pores o f approximate ly 2 5 0 u m , and a 39 particle size o f l - 3 m m . B o t h C P P and H A / T C P particles were r insed several t imes i n 70 % ethanol. 40 Figure 3.1 Particulates o f substrate materials a) Bone b) C P P c) H A / T C P . S E M images o f M S C seeded on bone (e)(h)(k) at days 0, 7, 21 respectively, on C P P (f)(i)(l) at days 0, 7, 21 respectively and on H A / T C P (g)(j)(m) as days 0, 7, 21 respectively. 41 3.2.2 Cell isolation, seeding and culture M S C s were isolated f rom the femora and tibiae o f a 6 weeks o l d transgenic G F P Sprague-Dawley rat ( N B R P , Japan). The bones were crushed and the bone mar row was removed through several washes w i t h phosphate buffered saline ( P B S ) . The bone mar row was then plated on to 15 c m f ibronect in coated tissue culture d i sh i n expans ion med ia ( E M ) containing M e s e n c u l t basal m e d i u m (Cat# 05401 Stem C e l l Technologies , Vancouver ) supplemented w i t h 1 5 % fetal bovine serum (Cat# 06471 Stem C e l l Technologies , Vancouve r ) and 100 U / m l penic i l l in-s t reptomycin . Af t e r 4 days, the non-adherent cells were r emoved and the attached cel ls were expanded to approximate ly 8 0 % confluence. The cel ls were further expanded to passage two then cryopreserved unt i l they were used. Before seeding the cryopreserved M S C were thawed and further expanded to passage five i n E M . The mult ipotent ia l nature o f the M S C popula t ion was conf i rmed b y differentiating the cells a long osteogenic, chondrogenic and adiopogenic lineages (Figure 3.2). P r io r to seeding a l l scaffold materials were repeatedly washed i n 7 0 % ethanol and P B S then incubated i n E M for 48hrs. The particles were placed i n 4 8 - w e l l non-tissue culture plates w i t h an equal mass o f particles i n each w e l l . The M S C s were seeded o n the scaffolds w i t h 800u.l o f a c e l l suspension (1 . O x 10 6 ce l l /ml ) . Af ter an in i t i a l incubat ion per iod o f 5 m i n at 3 7 ° C . t h e plates were centrifuged for 6 m i n ( l , 0 0 0 g , 4 ° C ) and incubated for another 26 6hrs at 3 7 ° C . F o l l o w i n g the 6hr incubat ion per iod the supernatant was removed and the scaffolds were washed w i t h P B S to remove any non-adherent cel ls . The cel ls i n the supernatant and wash were col lected and counted as a measure o f the seeding eff iciency. 42 The scaffolds were transferred to 2 4 - w e l l non-tissue culture plates and incubated at 3 7 ° C i n 2 m l o f culture m e d i u m for up to 21 days, w i t h the m e d i u m changed every two days. In addi t ion to the three particulate substrates, M S C s were plated o n two controls o f tissue culture plastic at density o f 10,000 c e l l s / c m 2 . The plast ic controls ( T C / E M and T C / O M ) were cultured i n E M and i n osteogenic med ia ( O M ) consis t ing o f Mesencu l t , 1 5 % F B S (Stem C e l l Technologies , Cat# 06473) 0.01 u M dexamethasone, 5 0 u g / m l ascorbic ac id and 5 m M ^-Glycerophosphate . A l l experiments were performed i n triplicates at t ime periods o f 0 (direct ly f o l l o w i n g 6hr seeding period), 3, 7, 14 and 21 days. 3.2.3 Cellular Proliferation C e l l numbers at the different t ime intervals were determined by measur ing the metabol ic act ivi ty o f the M S C s w i t h a M T T (3-(4, 5-dimethyl th iazol-2yl) -2 , 5diphenyl te t razol ium bromide) assay. The particles were transferred f rom the 24 w e l l plates to 96 w e l l plates and a vo lume o f 200u.l o f M T T solut ion (5mg/ml i n P B S ) was added and incubated at 3 7 ° C for 3 '/zhrs. The M T T solut ion was aspirated f rom the we l l s and the formazan crystals were d i sso lved i n 160pl o f d imethyl sulfoxide ( D M S O ) for 20 m i n . A l i q u o t s o f 80(0,1 the D M S O solu t ion were then transferred to new we l l s i n the plate and the absorbance was read on a microplate reader (Spectra M a x 190, M o l e c u l a r Dev ices , U n i o n C i t y , C A ) at 570nm. Ce l lu la r prol iferat ion was measured through B r d U (5-bromo-2-deoxyur idine) incorporat ion into the n e w l y synthesized D N A o f repl icat ing cel ls . B r d U was added to 43 the media at a concentrat ion o f l O u M and cultured for 24hrs. A t the different t ime intervals the M S C s were f ixed w i t h 2 % paraformaldehyde, pe rmeab i l i zed w i t h 10% saponin and stained w i t h a B r d U antibody. T o facilitate count ing o f B r d U posi t ive M S C s the. nuc le i were co-stained w i t h Hoechst . Three arbitrary locat ions o n the samples were used to quantify the percent B r d U posi t ive cel ls . 3.2.4 Cellular Differentiation The expression levels o f osteogenic, chondrogenic and adiopogenic markers were determined w i t h q R T - P C R us ing Taqman probes ( A p p l i e d B iosys tems , Foster C i t y , C A , Tabe l 3.1). F o l l o w i n g the culture per iod (0, 3, 7, 14, 2 I d ) total ce l lu lar R N A was extracted us ing T R I Z O L (Invitrogen) and reverse transcribed us ing superscript III (Invitrogen). Target gene expression was normal ized relative to the house keeping gene G A P D H . 44 Gene Name Symbol Ra t Genebank # Taqman # Osteogenic Runt related t ranscript ion factor 2 R U N X 2 X M 346016. M m 0 3 0 0 3 4 9 1 m l C o l l a g e n I C O L I X M 213440 Rn00801649 g l A l k a l i n e Phosphatase A L P NM" "013059 R n 0 0 5 6 4 9 3 1 _ m l Osteonect in O N N M _ 012656 R n 0 0 5 6 1 9 5 5 _ m l Osteopont in O P N M . 012881 R n 0 1 4 4 9 9 7 2 _ m l B o n e Sia lopro te in B S P N M _ _012587 R n 0 0 5 6 1 4 1 4 _ m l Os teoca lc in O C N M _ _013414 R n 0 0 5 6 6 3 8 6 _ g l Chondrogenic S R Y - b o x conta in ing gene 9 S O X 9 X M 343981 RnO 1751069 m H C o l l a g e n II C O L II N M _ 012929 M m 0 0 4 9 1 9 2 6 _ g l A g g r e c a n . A G G N M _ 022190 R n 0 0 5 7 3 4 2 4 _ m l Adipogenic Perox isome proliferator-activated P P A R y N M _ 013124 M m 0 0 4 4 0 9 4 5 _ m l receptor g a m m a L ipop ro t e in L ipase L P L N M _ 012598 R n 0 0 5 6 1 4 8 2 _ m l Housekeeping Glycera ldehyde-3 -phosphate G A P D H N M _ 002046 H s 0 2 7 5 8 9 9 1 _ g l dehydrogenase Table 3.1 Gene names, symbols and reference numbers. The probes for RUNX2, COL II, PPARy and GAPDH are cross-reactive for Mouse and Rat. 3.2.5 Scanning election microscope (SEM) The scaffold materials were prepared for S E M imag in ing by be ing f ixed i n 4 % paraformaldehyde and then were dehydrated i n series o f graded ethanol washings fo l lowed by cr i t ica l point d ry ing . The dried samples were sputter coated w i t h go ld and imaged us ing a scanning electron microscope (S-2600 V P S E M , Hi t ach i ) . 3.2.6 Statistics The effect o f substrate and t ime o n ce l l number, B r d U incorporat ion and m R N A expression was determined w i t h a 2-way A N O V A w i t h the substrate and t ime as factors. 45 Student -Newman K e u l s tests were used for post-hoc analyses at a s ignif icance level o f 0.05. 3.3 Results 3.3.1 Seeding There were no signif icant differences o f in i t ia l M S C attachment to the substrates (p>0.07). The seeding efficiencies, g iven as the number o f cel ls seeded minus the cells i n effluent wash solut ion, were 9 0 . 1 % ± 4 . 4 , 87 .3% ± 5 . 1 , 83 .2% ± 6 . 9 for B o n e , C P P and H A / T C P respectively. N o M S C s were detected i n the effluent w as h o f the plast ic controls and therefore w e assume a seeding eff iciency o f approximate ly 100%. 3.3.2 Cellular proliferation The M T T assay shows a significant in i t i a l decrease i n ce l l numbers o n C P P and B o n e over the first three days w i t h a recovery by D a y 21 (p<0.047) (Figure 3.3). In contrast H A / T C P showed a gradual ly decreasing trend i n c e l l numbers over the entire 21 days. B r d U incorporat ion increased w i t h t ime for a l l scaffolds and was the highest (>30%) i n Bone and C P P (p<0.001) (Figure 3.4). B o n e and H A / T C P had li t t le or no B r d U incorporat ion o n D a y s 3 and 7, but showed a significant increase at D a y 14 (p<0.003) (Figure 3.4). The plast ic controls ( T C / E M and T C / O M ) showed constant increase i n ce l l numbers over the entire 21 day culture per iod (not shown). 46 Figure 3.2 Osteogenic, adiopogenic and chondrogenic differentiation o f M S C s . For the osteogenic and adiopogenic differentiation, 5000 and 50,000 M S C respectively were plated in 96 wel l plates. The osteogenic differentiation media consisted of Mesencult, 15% F B S (Stem Ce l l Technologies, Cat# 06473) 0 . 0 l u M dexamethasone, 50ug/ml ascorbic acid and 5 m M p-Glycerophosphate and the plates were stained with A l izar in Red S after 4 weeks in culture. The adipogenic medium consisted o f E M supplemented with 0.1 u M dexamethasone and 6ng/ml insulin and stained with O i l Red O after 3 weeks in culture. For 47 chondrogenic differentiation 500,000 MSCs were pelleted and grown for 3 weeks in Mesencult and 10% FBS (Stem Cell Technologies Cat# 06471) supplemented with 0.0 l u M dexamethasone, 50ug/ml ascorbic acid and lOng/ml TGF-B1. Cryosection of the pellet was stained with Alcian blue. 1.00 0.00 B O N E CPP HA/TCP I Day 0 a Day 3 • Day 7 • Day 14 • Day 211 70 ^ 6 0 J2 50 a> ^ 4 0 > I 30 a. 3 20 5 10 H 11 B O N E C P P HA/TCP b) ICS Day 3 D D a y 7 Q D a y 14 • Day 21 I Figure 3.3 (a) Cell numbers were determined by an M T T assay for the 21 day culture period, (b) The percent BrdU incorporation into the cells over the 21 days of culture on the different substrates. 3.3.3 Cellular differentiation The results o f the q R T - P C R for the osteogenic, chondrogenic and adipogenic markers are shown i n Figures 3.5, 3.6 and 3.7 respectively. There was a s ignif icant decrease i n C o l - I and A L P expression w i t h i n the first 3 days on a l l scaffold materials (p<0.0002). Af ter the in i t ia l decline, C o l - 1 remained unchanged unt i l D a y 14 and increased s ignif icant ly on Bone and C P P (p<0.02) by D a y 21 . Co l -1 expression was s ignif icant ly higher on bone and C P P compared w i t h H A / T C P (p<0.002). S imi l a r l y , O N in i t i a l ly decreased on a l l 48 particles w i t h i n the first 3 days and then gradually increased un t i l day 2 1 . O n the plastic control , T C / E M , there were significant increases on A L P , C o l - 1 and O N f rom D a y 0 to D a y 21 (p<0.0002) w i t h s ignif icant ly higher expression than T C / O M by D a y 21 (p<0.0002). R U N X 2 showed no significant change o n the B o n e , C P P or T C / E M , however there was a s ignif icant decrease i n expression on H A / T C P (p<0.001) and increase i n expression o n T C / O M by day 21 (p<0.005). Late stage osteogenic markers such as O C and B S P were expressed at very l o w levels close to the detection l i m i t o n a l l substrates, except i n osteogenic m e d i u m where O C arid B S P express ion constantly increased f rom D a y 0 to D a y 21 . The expression o f O P remains constant o n Bone , C P P and T C / E M , however shows a significant increase by D a y 7 o n both H A / T C P and T C / O M (p<0.0002). 49 0.02 x • Q. < 20.015 1 0.01 p 0.005 1 BONE C P P HA/TCP TC/EM TC/OM BONE HA/TCP TC/EM TC/OM 0.0200 H 0.0150 o30 a. CO con C D U Q . X LU 0100 0050 0.0000 ALP 0.0004 0.0002 BONE C P P HA/TCP 25 Q <20 a !15H 10 | 5 BONE C P P HA/TCP 4 0 BONE C P P HA/TCP TC/EM TC/OM d) BONE 0.0016 x • O O , o CD 0012 10.0008 i CC c o $0.0004 H oc 0.00004 0.00002 BONE C P P HA/TCP JS C P P HA/TCP TC/EM TC/OM e) BONE C P P HA/TCP TC/EM TC/OM BONE C P P HA/TCP TC/EM TC/OM I Day 0 H Day 3 O Day 7 E Day 14 • Day 21 Figure 3.4 The temporal expression o f osteogenic genes on the different substrates for 21 days. The osteogenic genes (a) R U N X - 2 , (b) C O L I, (c) A L P , (d) O N , (e) O P , (f) O C are expressed relative to the house keeping gene G A P D H at time points o f 0, 3, 7, 14 and 21 days, for al l substrate materials. A l l values are presented as the means ± standard deviations o f triplicates. Inset panels are magnified views o f expression on scaffold materials, shown for clarity. The highest expression o f a l l chondrogenic markers w i t h an increasing trend was observed o n tissue culture plast ic w i t h expansion m e d i u m ( T C / E M ) . There was a significant d o w n regulat ion o f A G G on a l l other substrates w i t h i n the first 3 days 50 (p<0.001). S O X - 9 in i t i a l l y decreased and then was up regulated on B o n e , C P P at D a y 21 (p<0.006). V e r y l o w levels o f C o l - 2 were expressed on and a l l substrates and culture condit ions. 10.14 -Q SOX9 <0.12 -O 3 0.1 -CD > 10.08 -* 0 . 0 6 -o 10.04 -£ &0.02 -o -a) BONE C P P HA/TCP •a 3 1 0.12 x Q < CD 3 0.08 o > l o . 0 6 a .1 0.04 H a CO 1) a-0.02 LU 0 A G G 0.03 0.02 0.01-0-BONE C P P HA/TCP Fl n i b) BONE C P P HA/TCP TC/EM TC/OM I Day 0 H Day 3 • Day 7 • Day 14 • Day 21 Figure 3.5 The temporal expression o f chondrogenic genes on the different substrates for 21 days. The chondrogenic genes (a) S O X - 9 and (b) A G G are expressed relative to the house keeping gene G A P D H at time points o f 0, 3, 7, 14 and 21 days, for all substrate materials. A l l values are presented as the means ± standard deviations o f triplicates. Inset panel is a magnified view o f expression on scaffold materials, shown for clarity. P P A R y expression constantly increased from D a y 3 to 21 o n H A and T C / O M , and was significant higher compared w i t h B o n e and C P P (p<0.0002). O n Bone , C P P and T C / E M , P P A R y expression increased s l ight ly and then decreased by D a y 2 1 . In contrast to P P A R y , L P L express ion was h igh and increased s ignif icant ly w i t h t ime o n Bone , C P P and T C / O M (p<0.0003), but not on H A and T C / E M w h i c h on ly s l ight ly changed dur ing the entire culture per iod. 51 Q 0.025 £L < 0 0.02 H > 1 0.015 cc g 0.01 I 0.005 P P A R y 0.005-0.004 0.003 0.002 0.001 0 i BONE C P P HA/TCP 1 0.25 I • < 0.2 CD o 10.15 5 « 0.1 g £0.05 UJ 0 LPL a) BONE C P P HA/TCP TC/EM TC/OM b) BONE C P P HA/TCP TC/EM TC/OM I Day 0 Q Day 3 H Day 7 H Day 14 • Day 21 Figure 3.6 The temporal expression o f adipogenic genes on the different substrates for 21 days. The adipogenic genes (a) P P A R y and (b) L L P are expressed relative to the house keeping gene G A P D H at time points of 0, 3, 7, 14 and 21 days, for al l substrate materials. A l l values are presented as the means ± standard deviations o f triplicates. Inset panel is a magnified view o f expression on scaffold materials, shown for clarity. 3.3.4 SEM The S E M images o f the samples at D a y s 0, 7, and 21 are show i n figure 3.1. These images show that the cel ls had sufficiently attached to the substrate after a 6 hour seeding period. The rounded cel ls i n figures 3.1 d, e, f are indicat ive o f d y i n g or dead cel ls . There was also a noticeable decrease i n ce l l numbers at day 7 o n the bone and H A / T C P samples, w i t h a recovery o f cel ls by D a y 21 . In addi t ion, a l l substrates had an increase i n ce l l size by D a y 21 . 3.4 Discussion This comprehensive analysis demonstrated a strong effect o f the c e l l culture substrate on proliferat ion and differentiat ion o f M S C i n expansion condi t ions. M S C s proliferat ion and differentiation o n bone, C P P and H A / T C P , w h i c h are c o m m o n l y used for tissue 52 engineering appl icat ions, were compared to tissue culture plast ic i n both expansion and osteogenic differentiation culture condit ions. U s i n g osteogenic, chondrogenic and adipogenic culture condi t ions w e were able to induce M S C s differentiation into bone nodules, cartilage and fat respect ively (Figure 3.1). Th is conf i rmed the tr i-potency o f the bone mar row der ived heterogeneous ce l l populat ion w h i c h were seeded and evaluated on the different substrates. B y adapting the seeding method proposed by D a r et a l w e achieved h igh seeding efficiencies for bone, C P P and H A / T C P (90 .1% ± 4 . 4 , 87 .3% ± 5 . 1 , 83 .2% ± 6 . 9 respectively) ensuring that sufficient M S C s were attached to the substrates. O f the M S C s that were seeded on bone many died or migrated o f f the particles w i t h i n the first 3 days. There was also a slight drop i n ce l l numbers o n the C P P particles. M o s t l i ke ly this in i t i a l decl ine i n ce l l numbers is due to change o f substrate since there was no drop i n c e l l number observed o n the tissue culture plast ic (e.g. T C / E M and T C / O M ) . The corresponding d o w n regulat ion i n the expression o f C o l - 1 and A G G i n the first three days suggests that the deposi t ion o f col lagen is no longer needed for the M S C s to adhere to the particulate substrates. After an in i t i a l drop i n c e l l numbers, C P P and bone supported an increase i n ce l l numbers and B r d U incorporat ion at D a y 14 and 21 , w h i l e H A / T C P experienced a cont inued decline i n ce l l numbers over the entire 21 day per iod w i t h on ly m i n o r B r d U incorporation. S ince h i g h prol i ferat ing cel ls are typ ica l ly osteoprogenitors and lesser 11 1 2 extent preosetoblast ' , the recovery o f ce l l numbers on C P P and bone may indicate that 53 these two scaffolds mainta ined a immature M S C popula t ion w h i c h conta in a larger number o f osteoprogenitor or preosteoblasts compared w i t h H A / T C P . A l o n g w i t h the prol i fera t ion characteristics, the temporal gene expression helps to elucidate the extent o f differentiation a long the osteoblast l ineage. A number o f genes are k n o w n to be associated w i t h the early stage o f prol i ferat ion and matr ix development. R U N X - 2 , a runt-related transcript ion factor, is c rucia l i n con t ro l l ing osteogenic commitment but is mainta ined dur ing differentiation to other ce l l types such as adipocytes and chondrocy tes 6 ' 2 7 . C o l - 1 is expressed i n the prol iferat ive stage and dur ing the early stages o f matr ix lay d o w n ' ' , whereas A L P is expressed postprol iferat ion during extracellular m a t u r a t i o n 1 1 ' 2 9 . O N is a non-collagenous prote in that is expressed i n preosteoblast a l l the w a y to osteocytes and is thought to give stabil i ty to extracellular matr ix and provides a point o f associat ion between proteoglycans and co l lagen . Late stage markers o f osteogenesis, O P and BS.P, are noncol lagenous proteins associated w i t h the end stages o f mat r ix maturat ion and early stage o f minera l iza t ion w h i l e O C is strongly l i nked w i t h minera l i za t ion o f the extracellular m a t r i x 1 1 ' 1 6 ' 2 8 . The expression o f R U N X - 2 o n the a l l the substrates indicates the predispos i t ion o f the cells on a l l substrates to fo rm bone. The significant decrease i n R U N X 2 o n H A / T C P may suggest the cel ls are differentiating d o w n the osteoblast l ineage. It has been suggested however that R U N X 2 is i n v o l v e d i n osteogenesis i n modula t ion o f ac t iv i ty not through quantitative change i n gene expression, w h i c h may exp la in the va r i ab i l i ty i n expression i n the T C / O M s a m p l e 3 1 . 54 C o m p a r i n g the T C / E M and T C / O M samples there is an obvious difference i n expression levels o f early stage markers and late stage markers. T C / E M shows increasing expression o f early stage markers, C o l - 1 , A L P and O N , over the 21 days whereas T C / O M shows no significant change i n C o l - 1 or A L P after the first 3 days. The express ion o f O N for T C / O M does s ignif icant ly increase by D a y 7 however it is s ignif icant ly lower than E M . In contrast the late stage marker show the opposite trend, w i t h T C / O M showing significant increases i n O P , B S P and O C over the 21 days w h i l e T C / E M has s ignif icant ly lower expression levels o f each gene. The gene expression o n the two controls suggests that T C / E M is main ta in ing an immature ce l l popula t ion w h i l e T C / O M causes differentiation d o w n the osteogenic lineage. T h i s is i n agreement w i t h the fact that the cells expanded i n T C / E M pr ior to seeding maintained their prol i fera t ion and differentiation ab i l i ty w h i l e ce l l cultured i n T C / O M formed bone nodules. S i m i l a r l y w h e n compar ing the different scaffold materials, a l logeneic bone and C P P show and increase i n C o l - 1 and O N f rom D a y 3 to D a y 21 w h i l e H A / T C P shows a decrease i n levels over the culture per iod. Af ter the in i t i a l drop the express ion o f A L P on bone and C P P remains unchanged dur ing the culture per iod but show s ignif icant ly higher levels than H A / T C P at D a y 2 1 . The late stage marker, O P , however showed a significant increase i n expression o n H A / T C P by day 14, and were substantial h igher than bone and C P P . Th i s suggest that bone and C P P l ike T C / E M had ce l l populat ions w i t h a higher number o f immature osteogenic cel ls w h i l e H A / T C P caused differentiation and osteoblast maturation o f the M S C s . A l l three scaffold materials had very l o w expressions o f the late 55 stage markers B S P and O C s imi la r to T C / E M w h i c h suggest that there is not late stage osteoblast maturat ion or minera l iza t ion . The s imi la r i ty between bone and C P P extend into the expression o f chondrogenic and adipogenic genes. B o n e and C P P both have a significant increase o f S O X 9 , the transcription factor for regulat ion o f chondrogenic differentiation, at D a y 21 w h i l e H A / T C P showed no change. Once more the expression o f the scaffold mater ial is paral leled i n the controls w i t h T C / E M showing an increase i n S O X 9 and T C / O M showing no change. T h o u g h this correspondence o f scaffold material and plast ic control is not seen i n the markers for adipogenic differentiation the difference between the gene expression i n bone and C P P to that o f H A / T C P is apparent. B o n e and C P P had increased expression o f L P L and unchanged expression i n P P A R y w h i l e H A / T C P showed the opposite w i t h no change i n L P L and significant increase o f P P A R y . W h i l e it is diff icul t to infer whether the substrates are causing differentiation d o w n the chondrogenic adipogenic lineages it is clear that the substrates have a major influence o n gene expression. Cell-substrate interaction is not fu l ly understood, whether it is the substrate surface chemistry or morpho logy that is causing the prol i ferat ion o f cel ls o n bone and C P P and differentiation on H A / T C P is unclear. P re l iminary S E M images (Figure 3.1) lead us to believe that the morpho logy o f the substrate surfaces may impact c e l l attachment and expansion and that the smooth morphology o f bone and C P P was benef ic ia l for c e l l proliferat ion. Furthermore it is not clear whether the role o f the mat r ix dur ing bone 56 regeneration is to support prol i ferat ion or cause differentiation. H o w e v e r , this study suggests that i n the absence o f soluble induct ion factors bone supports ce l lu lar growth and not differentiation. C P P hav ing very s imi lar influence o n ce l lu lar g rowth and differentiation makes it a p romis ing candidate for a bone graft substitute. H A / T C P i n contrast promotes differentiat ion and although it may a id i n bone growth it does not provide a suitable environment for the maintenance o f immature prol i fera t ing cel ls important for sustained bone regeneration. 3.5 Conclusion In this study, mesenchymal stromal cel ls ( M S C s ) seeded on a l logeneic bone and ca l c ium polyphosphate showed s imi l a r prol i ferat ion characteristics and gene expression. They also showed a s imi la r gene expression to that o f M S C seeded o n plast ic and cultured i n an expansion m e d i a T C / E M , suggesting that bone and C P P support immature proliferating M S C s . In contrast, M S C s seeded on biphasic hydroxyapat i te / t r ica lc ium phosphate showed s imi la r gene expression to M S C s seeded o n plast ic i n an osteogenic media T C / O M , suggesting H A / T C P induces differentiation o f M S C s d o w n the osteogenic l ineage. 57 3.6 References 1. Aubin, J. E. and Herbertson, A. Osteoblast lineage i n experimental animals . Beresford, J . and O w e n , M . M a r r o w Stromal C e l l Cul ture . [6], 88-110. 1998. Cambr idge , U K , Cambr idge Un ive r s i t y Press. 2. Gimble, J. M. M a r r o w stromal adipocytes. Beresford, J . and O w e n , M . M a r r o w stromal c e l l culture. [5], 67-87. 1998. Cambr idge , U K , Cambr idge U n i v e r s i t y Press. 3. Cancedda, R., Cancedda, F. D, and Dozin, B. Chondrocy te Cul ture . Beresford, J . and O w e n , M . M a r r o w stromal ce l l culture. [7], 111-27. 1998. Cambr idge , Cambr idge U n i v e r s i t y Press. 4. Pittenger, M. F., Mackay, A. M., Beck, S. C , Jaiswal, R. K., Douglas, R., Mosca, J. D., Moorman, M. A., Simonetti, D. W., Craig, S., and Marshak, D. R. Mul t i l i neage potential o f adult human mesenchymal stem cells . Science 284[5411] , 143-7. 4-2-1999. 5. Pittenger, M. F., Mackay, A. M., Beck, S. C , Jaiswal, R. K., Douglas, R., Mosca, J. D., Moorman, M. A;, Simonetti, D. W., Craig, S., and Marshak, D. R. Mul t i l i neage potential o f adult human mesenchymal stem cel ls . Science 284[5411] , 143-7. 4-2-1999. 6. Bianco, P., Riminucci, M., Gronthos, S., and Robey, P. G. B o n e m a r r o w stromal stem cel ls : Nature , b io logy , and potential applicat ions. Stem Cells 19[3], 180 -92 .2001 . 7. Livingston-Arinzeh, T. L., Tran, T., Mcalary, J., and Daculsi, G. A comparat ive study o f b iphasic c a l c i u m phosphate ceramics for human mesenchymal s tem-cel l - induced bone formation. Biomaterials 26[17] , 3631-8. 2005. 8. Chen, J. W., Wang, C. Y., Lu, S. H., Wu, J. Z., Guo, X. M., Duan, C. M., Dong, L. Z., Song, Y., Zhang, J. C , Jing, D. Y., Wu, L. B., Ding, J. D., and Li, D. X. In v i v o chondrogenesis o f adult bone-marrow-der ived autologous mesenchymal stem cel ls . Cell and Tissue Research 319[3] , 429-38. 2005. 9. Hoffmann, A., Pelled, G., Turgeman, G., Eberle, P., Zilberman, Y., Shinar, H., Keinan-Adamsky, K., Winkel, A., Shahab, S., Navon, G., Gross, G., and Gazit, D. Neo tendon format ion induced by manipu la t ion o f the Smad8 s igna l l ing pathway i n mesenchymal stem cel ls . Journal of Clinical Investigation 116[4], 940-52. 2006. 10. Naveiras, O . and Daley, G. Q. S tem cells and their niche: a matter o f fate. Cellular and Molecular Life Sciences 63 [7-8], 760-6. 2006. 11. Aubin, J. E. B o n e stem cel ls . Journal of Cellular Biochemistry , 73-82. 1998. 58 12. Holtrop, M. E. L i g h t and E lec t ron Structure o f B o n e - F o r m i n g C e l l s . H a l l , B . K . The Osteoblast and osteocyte. [1], 1-40. 1990. C a l d w e l l , N J , The Te l ford Press Inc. B o n e . 13. Tenenbaum, H. C. C e l l u l a r Or ig ins and Theories o f Different ia t ion o f B o n e -F o r m i n g C e l l s . H a l l , B . K . The Osteoblast and Osteocyte. [2], 41-70. 1990. C a l d w e l l , N J , The Te l fo rd Press Inc. Bone . 14. Baltzer, A. W. A., Lattermann, C , Whalen, J. D., Wooley, P., Weiss, K., Grimm, M., Ghivizzani, S. C , Robbins, P. D., and Evans, C. H. Genet ic enhancement o f fracture repair: heal ing o f an exper imental segmental defect by adenovira l transfer o f the B M P - 2 gene. Gene Therapy 7[9], 734-9. 2000. 15. Knabe, C , Berger, G., Gildenhaar, R., Howlett, C. R., Markovic, B., and Zreiqat, H. The functional expression o f human bone-der ived cel ls g rown o n rap id ly resorbable c a l c i u m phosphate ceramics. Biomaterials 25[2], 335-44. 2004. 16. Hauschka, P. V . G r o w t h Factor Effects i n B o n e - F o r m i n g C e l l s . H a l l , B . K . The Osteoblast and Osteocyte. [4], 103-70. 1990. C a l d w e l l , N J , The Te l fo rd Press Inc. B o n e . 17. Hing, K. A. B i o c e r a m i c bone graft substitutes: Influence o f poros i ty and chemistry. International Journal of Applied Ceramic Technology 2[3] , 184-99. 2005. 18. Engler, A. J., Sen, S., Sweeney, H. L., and Discher, D. E. M a t r i x elastici ty directs stem c e l l l ineage specif icat ion. Cell 126[4], 677-89. 8-25-2006. 19. Graziano, A, d'Aquino, R., Cusella-De Angelis, M. G., Laino, G., Pattelli, A, Pacifici, M, De Rosa, A, and Papaccio, G. Concave p i t -conta in ing scaffold surfaces improve stem cel l -der ived osteoblast performance and lead to significant bone tissue formation. PLoS ONE 2[6]. 2007 . 20. Rust, P. A., Kalsi, P., Briggs, T. W. R., Cannon, S. R., and Blunn, G. W. W i l l mesenchymal stem cel ls differentiate osteoblasts o n allograft? Clinical Orthopaedics and Related Research [457], 220-6. 2007. 21 . Draenert, G. F. and Delius, M. The mechanica l ly stable steam ster i l izat ion o f bone grafts. Biomaterials 28[8], 1531-8. 2007. 22. Pilliar, R. M L , Filiaggi, M. J., Wells, J. D., Grynpas, M. D., and Kandel, R. A. Porous c a l c i u m polyphosphate scaffolds for bone substitute applicat ions - i n vi t ro characterization. Biomaterials 22[9], 963-72. 2001 . 23. Livingston-Arinzeh, T. L., Tran, T., Mcalary, J., and Daculsi, G. A comparative study o f biphasic c a l c i u m phosphate ceramics for human mesenchymal s tem-cel l - induced bone formation. Biomaterials 26[17] , 3631-8. 2005. 59 24. Pilliar, R. M., Filiaggi, M. J., Wells, J. D., Grynpas, M. D., and Kandel, R. A. Porous c a l c i u m polyphosphate scaffolds for bone substitute applications - i n vi t ro characterization. Biomaterials 22[9], 963-72. 2001 . 25. Pilliar, R. M., Filiaggi, M. J., Wells, J. D., Grynpas, M. D., and Kandel, R. A. Porous c a l c i u m polyphosphate scaffolds for bone substitute applications - i n vi t ro characterization. Biomaterials 22[9], 963-72. 2001 . 26. Dar, A., Shachar, M., Leor, J., and Cohen, S. Card iac tissue engineering -O p t i m i z a t i o n o f cardiac ce l l seeding,and dis t r ibut ion i n 3 D porous alginate scaffolds. Biotechnology and Bioengineerin'g 80[3], 305-12. 11-5-2002. 27. Zaidi, M. Skele ta l r emode l ing i n health and disease. Nature Medicine 13[7], 791-8 0 1 . 2 0 0 7 . 28. Simmons, D. J. and Grynpas, M. D. Mechan i sms o f B o n e Fo rma t ion i n V i v o . H a l l , B . K . T h e Osteoblast and Osteocyte. [6], 193-302. 1990. C a l d w e l f N J , The Te l fo rd Press Inc. Bone . 29. Knabe, C , Berger, G., Gildenhaar, R., Howlett, C. R., Markovic, B., and Zreiqat, H. The functional expression o f human bone-der ived cel ls g rown o n rap id ly resorbable c a l c i u m phosphate ceramics. Biomaterials 25[2], 335-44. 2004. 30. Knabe, C , Berger, G., Gildenhaar, R., Howlett, C. R., Markovic, B., and Zreiqat, H. The functional expression o f human bone-der ived cel ls g rown o n rap id ly resorbable c a l c i u m phosphate ceramics. Biomaterials 25[2], 335-44. 2004. 31. Zhou, Y. F., Chen, F. L., Ho, S. T . , Woodruff, M. A., Lim, T . M., and Hutmacher, D. W. C o m b i n e d mar row stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts. Biomaterials 28[5], 814-24. 2007. 60 4 C o n c l u s i o n 4.1 Summary and Future Work In this study, the p re l iminary evaluat ion o f both the mechanica l and b i o l o g i c a l characteristics o f c a l c i u m polyphosphate ( C P P ) was conducted to determine its v i ab i l i t y as a structural scaffold i n r ev i s ion total h ip replacement ( T H R ) . M e c h a n i c a l testing showed that C P P and M C B have s imi lar mechanica l properties i n both conf ined compress ion and i n shear. Further mechanica l testing o f ideal spherical particles showed that al though increasing the particle modulus increases the part icle construct modulus , but the value o f the modulus is s t i l l very l o w compared to that o f the ac ry l i c bone cement currently used to a id i n the structural support o f the implant . The shear tests revealed that the particle modulus has l i t t le influence on the shear strength o f the particle construct wh i l e the shape o f the part icle has a large effect. The b i o l o g i c a l testing showed that C P P had s imi lar osteogenic properties to that o f a l logenic bone, both o f w h i c h supported the growth o f M S C s o n their surface though d i d not cause a differentiat ion M S C s d o w n the osteogenic lineage. In contrast H A / T C P promoted the differentiation d o w n that osteogenic lineage but d i d not support ce l l prol iferat ion. These results suggest C P P is a suitable substitute mater ial for M C B i n T H R procedures, and w o u l d p rov ide adequate structural support w h i l e a l l o w i n g cel lu lar prol i ferat ion to occur w h i c h is v i t a l for sustained bone growth. W h i l e pre l iminary testing y ie lded posi t ive results for the use o f C P P as a structural scaffold, further mechan ica l and b io log i ca l testing is required to con f i rm its feasibi l i ty for 61 appl icat ion i n T H R . The next stage o f mechanica l testing is to load the material dynamica l ly i n a geometry s imi la r to that found i n the h ip . One o f the m a i n differences w i t h C P P and M C B is that M C B is a biphasic visco-elast icplast ic mater ia l that is subject to creep dur ing sustained l o a d i n g 1 . Th i s behaviour is thought to a l l o w for deformation o f the graft post surgery and is a cause for implant subsidence 1 ' 2 . Therefore it w o u l d be useful to measure the subsidence o f the implant i n a graft bed o f both C P P and M C B under sustained c y c l i c load ing to determine the stabil i ty o f the graft over t ime. Further b io log ica l test w o u l d inc lude the cul tur ing o f C P P seeded w i t h M S C s i n a med ia w i t h osteogenic induc t ion factors. A l t h o u g h it was shown that C P P was osteoconductive i n that it a l l owed prol i fera t ion o f cells on its surface it needs to be s h o w n that it is possible to g row bone on the surface. U s i n g a med ia w i t h induct ion factors, the cel ls should have an environment that a l lows for both prol iferat ion and differentiation, leading to sustained bone growth. F o l l o w i n g this test an i n v i v o study i n rats o f the M S C growth o n the various particles is needed to conf i rm the results o f the i n v i t ro tests. 62 4.2 Reference List 1. Giesen, E . B., Lamerigts, N. M., Verdonschot, N., Buma, P., Schreurs, B. W., and Huiskes, R. M e c h a n i c a l characteristics o f impacted morse l l i sed bone grafts used i n r ev i s ion o f total h ip arthroplasty. J.Bone Joint Surg. Br. 81 [6], 1052-7. 1999. 2. Voor, M. J., Nawab, A., Malkani, A. L., and Ullrich, C. R. M e c h a n i c a l properties o f compacted morse l ized cancellous bone graft us ing one-d imens iona l consol ida t ion testing. Journal Of Biomechanics 33[12], 1683-8. 2000. 63 Appendix A: Cell Culture Procedures Plating MSC's -Defrost v i l e o f M S C i n hand -Immediately m i x w i t h 5 m l o f culture m e d i u m ( C M ) -Centrifuge for 5 minutes at 1500 r p m -Aspirate o f f so lu t ion -Re-suspend i n 10-20ml C M -Sp l i t so lu t ion i n h a l f i n separate 15cm tissue culture plates - A d d C M to br ing total so lu t ion i n each plate to 2 0 m l Wash particles - W a s h i n 7 0 % E T O H unt i l no longer c loudy -Leave i n 7 0 % E T O H over night -3x P B S - 1 5 % F B S C M f o r 4 8 h r s Splitting Cells -Aspira te o f f so lu t ion f rom each plate - W a s h w i t h 15ml P B S -8 m l T E i n each plate and incubate 5 m i n - A d d 15ml P B S and wash a couple o f times -Pipette contents into a F a l c o n tube -Centrifuge -Aspira te o f f so lu t ion -Re-suspend i n 10-20ml C M -Spl i t so lu t ion i n h a l f i n separate 15cm tissue culture plates - A d d C M to br ing total so lu t ion i n each plate to 2 0 m l Seed MSCs on particles -Aspira te o f f so lu t ion f rom each plate - W a s h w i t h 15ml P B S -8 m l T E i n each plate and incubate 5 m i n - A d d 15ml P B S and wash a couple o f times -Pipette contents into a F a l c o n tube -Centrifuge -Aspira te o f f so lu t ion and add 1ml C M to each tube - Resuspend cel ls -Pipette 1 Oul o f so lu t ion and dilute w i t h 90ul C M for each fa lcon tube -Count cel ls - U s i n g tweezers s ter i l ized w i t h 7 0 % E T O H place particles i n 48 w e l l plates (1/3 -1/2 full) -Seed ~800,000 o n each sample i n 48 w e l l plates (non-tissue culture) -incubate for 5 m i n -centrifuged at lOOOg for 6 m i n at 4 ° C -Incubate cel ls for s ix hours 65 Seeding on plastic -Seed 10,000 cel ls o n 6 w e l l tissue culture plates Culturing Cells -After 6 hour seeding per iod , pipette out C M ( D O N O T A S P I R A T E ) count the cel ls i n r emoved solut ion - W a s h particles i n P B S (to remove any cells not attached) by pipet t ing, count cells i n wash so lu t ion -Transfer particles to n e w 24 w e l l plate (non-tissue culture) - A d d 2 m l o f C M to each w e l l -Incubate and change C M every other day for the remainder o f the test MTT -Aspira te so lu t ion o f f o f a l l samples - W a s h particles/plastic w i t h P B S (carefully w i t h the plast ic controls) -Aspirate o f f P B S -Transfer particles to 96 w e l l plate - E a c h w e l l f rom the 24 w e l l gets split up into 2 we l l s -Plast ic samples (plastic and osteo) are left i n their o r ig ina l plate 66 - A d d 180ul M T T solu t ion to each w e l l (carefully on the plast ic to be sure not to knock the cel ls off) -Incubate for 3-4 hours -After 3-4 hours aspirate o f f M T T "solution (use a pipette for the plast ic controls) - A d d 160 D M S O to plast ic control we l l s and to the first w e l l for each particle samples -Incubate for 10 minutes -After 10 minutes transfer 160ul D M S O from the first w e l l to the second w e l l for each particle sample -Incubate for another 10 m i n -Pipette the so lu t ion f rom the particle we l l s into a new w e l l (this step prevents bubbles f rom being transferred to the f inal we l l ) -Pipette out 80ul f rom this w e l l into the f inal w e l l (these are what the absorbance are tested on) -Test Absorbance at 570 n m Lysis of Cells on particles -Aspira te so lu t ion o f f o f a l l samples - W a s h particles w i t h P B S -Aspira te o f f P B S -Transfer particles to 1.2ml tubes - A d d 800ul T r i z o l , let stand at r o o m temp for 3 0 m i n - A d d 180ul ch lo ro fo rm 67 -Shake tubes by hand for 15 seconds then let stand for 2-3 m i n at r o o m temp -Centrifuge at samples @ 1 2 0 0 0 G for 15 m i n @ 4 degree -Transfer aqueous phase to new tubes • - A d d 400u l i sopropy l and incubate i n freezer for 2 hours -Centrifuge @ 1 2 0 0 0 G for 10 m i n at 4 degrees - R e m o v e supernatant (carefully- leave a litt le so lu t ion i n the bottom) - W a s h i n 800ul 7 5 % ethanol, m i x by vor texing, then centrifuge at 7 5 0 0 G for 5 m i n at 4 degrees -aspirate o f f so lu t ion (do the last bit with a pipette because the pellet doesn't always stick to the tube and can get sucked up if you try to aspirate all the solution off) and air dry for 5 m i n -redissolve i n 30u l R N A s e free water m i x by pipet t ing -incubate for 10 minutes i n 55 degree water bath 68 Appendix B: Statistical Methods Student t-test The student T test was used to determine the probabi l i ty that the difference between two normal ly distr ibuted populat ions (o f t distribution) are f rom chance. The normal distributions are described by the samples estimates (due to the sma l l sample size) o f standard devia t ion s, its mean r\ and number o f data points. The student t-test is a n u l l hypothesis and therefore returns a value o f 0 i f the distr ibutions are f rom the same populat ion. The t-test is g iven as fo l lows : U s i n g the t value and the number o f degrees o f freedom one can determine the probabi l i ty that the difference i n the populat ions is not due to chance ( in a table o f significance). T y p i c a l l y a p value less that 0.05 is taken to mean the two distr ibutions are from different populat ions. A n a l y s i s o f variance ( A N O V A ) used here was to test the effect o f different treatments on a material or substrate. A N O V A tests the n u l l hypothesis that a l l the popula t ion means are equal. It is calculated through the compar ison o f two estimates o f the variance o f the t = where ANOVA 69 entire popula t ion. The two estimates o f variance are; the variance i n w i t h i n a treatment (mean square error or M S E ) and the variance between the different treatments (mean square between or M S B ) g iven by Ys2 MSE = and MSB = ns2m n Where Sj is the standard devia t ion o f each populat ion, s m is the standard devia t ion o f the means o f each popula t ion relative to the total popula t ion mean, and n is the number o f measurement i n each popula t ion . The ratio o f M S B / M S E gives a value o f 1 i f the n u l l hypothesis is true i n w h i c h case a l l the popula t ion means are equal . I f the ratio is m u c h larger than 1 then the M S B is an inflated estimate o f the variance and therefore the n u l l hypothesis is false. B a s i c a l l y , when M S B is large then the variance between the sample means is large and therefore are l i ke ly not f rom the same popula t ion . U s i n g the ratio k n o w as an f va lue a long w i t h the degrees o f freedom one can determine the probabi l i ty that the difference i n the populations is not due to chance ( in a table o f significance). T y p i c a l l y a p value less that 0.05 is taken to mean the two distributions are f rom different populat ions . A two wa y A N O V A is an extension o f a one w a y A N O V A i n that it contains two independent factors (such as the substrates and the t ime o f culture i n this study). The two way A N O V A gives a n u l l hypothesis i f means o f the first factor are the same, i f the 70 means o f the second factors are the same, or there is no interaction between the two samples. Pearson's Correlation The Pearson's correla t ion g iven as R is a measure o f the l inear i ty between the two variables (such as E c and E p ) . The value o f R ranges f rom -1 to 1, w i t h -1 representing and perfect inverse l inear relat ionship, 1 representing a perfect l inear relat ionship, and 0 representing no relat ionship. The correlat ion is g iven as: R = 71 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0078589/manifest

Comment

Related Items