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The effects of hydroxyapatite coated micromachined substrata on osteogenesis Perizzolo, David 2000

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The Effects of Hydroxyapatite Coated Micromachined Substrata on Osteogenesis. By David Perizzolo B .Sc , The University of British Columbia, 1998 A Thesis Submitted In Partial Fulfilment Of The Requirements For The Degree Of Master Of Science In Dental Science, i n The Faculty of Graduate Studies (Department of Oral Biological and Medical Sciences; Dentistry) We accept this thesis as conforming to the required standard: The University of British Columbia 2000 © David Perizzolo, 2000 in presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of /fird Ss'o/yj 4-J(k>zjf£jf/§CitsiC£f The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract Osteogenes is was studied using rat bone cel ls cultured on hydroxyapatite (HA) or titanium (Ti) coated smooth and grooved substrata. Osteoblast cultures were maintained from 24hrs to 6 weeks in culture medium and supplemented with L-ascorbic ac id-2-phosphate and F3-glycerophosphate to promote mineral ization. The HA coat ings were character ized using X-ray diffraction, surface roughness and scanning electron microscopy (SEM) . The ceramic hydroxyapatite coat ing, was dense and uniform, containing HA crystals and was ~ 1(im thick. Time-lapse c inemicrography of osteoblasts locomotion on HA sur faces revealed osteoblasts moved with the direction of the grooves, that is they exhibited contact guidance. Scann ing electron microscopic observat ions revealed osteoblasts were elongated and orientated on both Ti and HA grooved sur faces. Col lagen fibers, as a s s e s s e d by picro-sir ius staining and polarized light microscopy, were al igned on both HA and Ti grooved sur faces. Although not quantif ied, it appeared that nodule formation was greatest under culture condit ions that produced al igned co l lagen. Os teogenes is was measured by counts of tetracycl ine label led bone-l ike nodules and alkaline phosphatase activity. HA coated surfaces produced i i significantly more mineral ized nodules than Ti sur faces. Sur faces with grooves and narrow gaps produced the highest number of nodules. All grooved substrata produced significantly more nodules than smooth sur faces. These results are consistent with the concept that substrata that form a microenvironment by restricting diffusion increase bone-l ike t issue production. A novel finding in this thesis was that there was a statist ical ly signif icant interaction between topography and chemistry in the formation of mineral ized nodules. A n excel lent correlat ion (r= 0.958) between alkaline phosphatase at 2 weeks and nodule counts at 6 weeks was observed, suggest ing that A lk -P is a good leading indicator of osteogenesis on microfabricated sur faces. Therefore A l k - P might be used to screen surfaces for their nodule production, thus saving time and expense. The results of this study indicated that topography and chemistry can affect os teogenes is on biomater ials, and that interactions between chemistry and topography can occur. i i i Table of Contents Page Abstract ii Table of contents iv List of tables vi List of figures vii Acknowledgements ix I. Introduction 1 1. Overview 2 2. Titanium and Hydroxyapatite Implants 3 3. Bone Matrix 9 4. Effects of Surface Topography on Cell Behaviour 14 5. Proposed Mechanisms of Enhanced Osteogenesis on Micromachined Surfaces 17 6. Conditions Promoting Mineralization 19 7. Techniques used to Identify Osteogenic Tissue 21 8. Objective of the thesis 28 II. Materials and Methods 30 1. Substrates 31 A. Micromachining 31 B. Surface pattern 32 C. Coatings 32 D. Preparation for cell culture experiments 35 2. Cultures of osteogenic cel ls derived from rat calvar ia 36 3. Experimental design 37 A. Culture 37 B. Scanning electron microscopy (SEM) 39 i v C. Polarization microscopy 40 D. Time-lapse cinemicrography 41 E. Tetracycline Incorporation 41 F. Alkaline Phosphatase 42 G . Correlat ion of Tetracycl ine and Alkal ine Phospha tase activity 44 3. Statistics 44 III. Results 45 1. Characterization of Sputter-coated HA surfaces 46 A. X-Ray diffraction 46 B. Surface roughness 46 C. Scanning Electron Microscopy 49 2. Osteogenic cell cultures 49 A. Scanning Electron Microscopy 49 3. Collagen Arrangement 54 A. Picro-Sirius Red Staining 54 4. Nodule Quantification 58 A. Nodule Counts 58 B. % Area of Nodule on Ridge 66 5. Alkaline Phosphatase Activity 66 A. Optimum pH 66 B. Controls 66 C. Alkaline Phosphatase activity 70 6. Correlat ion of Tetracycl ine Incorporation and Alkal ine Phosphatase activity 73 7. Time-Lapse Cinemicrography 77 IV. Discussion 81 V. Future Work 94 VI. Bibliography 100 v List of Tables Page Table 1. Analysis of Variance of the grooved areas 63 Table 2. Analys is of Var iance of nodule counts in the smooth Gaps . . 65 Table 3. Nodule formation on HA & Ti smooth gaps 67 Table 4. Percent of Nodule on ridge 68 v i List of Figures Page: Figure 1. A schematic cross section of grooved surfaces 3 3 Figure 2. A macro-image of the pattern 3 4 Figure 3. A schematic diagram of the experimental design 3 8 Figure 4. Schematic diagram of A lk-P reaction 4 3 Figure 5. X-ray diffraction of HA coated micromachined substrata. 4 7 Figure 6. Surface profiles of substrata 4 8 Figure 7. Scanning electron micrographs of substrata 5 0 Figure 8. S E M of sputtered HA coating 51 Figure 9. Osteoblasts cultured for 24 hour on HA coated substrata. 5 2 Figure 10. Scanning electron micrograph of two week culture 5 3 Figure 11. Nodules on micromachined substrata 5 5 Figure 12. Ref lected polar ized light micrograph of HA surface stained with picro-sirius red 5 6 Figure 13. Orientation of collagen on grooved substrata 5 7 Figure 14. Tetracycline controls 5 9 Figure 15. Tetracycline labelled nodule 6 0 Figure 16. Nodule counts on grooved substrata 6 2 Figure 17. Nodule counts on smooth gaps 6 4 Figure 18. The effects of pH on alkaline phosphatase activity 6 9 v i i Figure 19. Alkaline phosphatase activity 71 Figure 20. Alkal ine phosphatase activity on grooved substrata 7 2 Figure 21. Alkal ine phosphatase activity on smooth gaps 7 4 Figure 22. Correlat ion between A lk -P/Tet racyc l ine (grooves) 7 5 Figure 23. Correlat ion between A lk -P/Tet racyc l ine (smooth gaps) . 7 6 Figure 24. Alk-P/Tetracycl ine labelled nodules 7 8 Figure 25. Time-lapse series 7 9 Figure 26. Development of tetracycline labell ing of a nodule during one week 8 0 v i i i Acknowledgements I wish to thank my supervisor, Dr. Donald M. Brunette for his support, patience and understanding throughout this thesis. He provided guidance in all aspects to help me complete this thesis. I would a lso like to express my appreciation to my thesis committee members, Drs. V. Jukka Uitto, E. Putnins, N. Dorin Ruse and J . Douglas Waterf ield, for their cons t ruc t i ve par t ic ipa t ion . I would like to extend a special thanks to Dr. W.R . Lacefield (University of A l a b a m a , Schoo l of Dentistry) for his col laborat ion in the coating of the substrata with hydroxyapatite. I would a lso like to thank Dr. Babak Chehroudi who assisted in supervising while Dr. Brunette was away. I also thank Mrs. Lesley Weston and Mr. Andre Wong for their technical instructions and help in cell culturing methods, polar izat ion microscopy, electron microscopy and photomicrography. A specia l thanks to Mr. Bruce M c C a u g h e y for his helpful suggest ions regarding the photographic pictures. I would like to acknowledge the U B C Department of Electr ical Engineering and in particular Dr. A l ina Kulpa who fabricated the si l icon micromach ined sur faces . Most importantly I thank my parents and family members for their support, help, and understanding during this Master 's degree. Introduction 1 1. Overview: For an implant to be successfu l it must be non-toxic, cause minimal foreign-body reaction, and integrate with the t issue (Ong et al, 1998). Osseointegrat ion has been defined as contact establ ished between bone and an implant surface without the interposition of non-bone or connect ive t issue, at the light microscopic level (Gross et al, 1997). Bone formation on an implant surface depends upon the metabol ic and secretory activities of only one cell type: the osteoblast (Davies, 1996). The s u c c e s s , that is the longevity, of bone-contact ing implants depends on the amount and rate of bone formation occurring on their sur faces (Massas et al, 1993). The material used plays an important role in the longevity of an implant as it affects the amount of osseointegrat ion (Vercaigne et al, 1998). The more osseointegrat ion that an implant has the better the prognosis (Gross et al, 1997). It is widely recognized that sur face propert ies, most particularly topography and chemistry, are of prime importance in determining cell and t issue responses to a biomaterial because it is the surface that forms the interface with the t issue (Brunette and Chehroud i , 1999). Although there is no agreement on what constitutes the ideal topography, there is some agreement on which materials are biocompatible (Burnette, 1988). The two materials studied in this thesis, Ti and hydroxyapatite (HA), have 2 been shown to be biocompatible (Massas etal, 1993). The sur faces of commercial ly avai lable dental implants vary greatly in their topography and chemical composi t ions (making them hard to compare) (Brunette, 1988; Wennerberg, 1996; Brunette and Chehroudi , 1999; Wie land, 1999). Moreover, there is sparse information on the best surface topography (Wennerberg, 1996), possibly due to the number of surface topographies being used and the lack of a clear understanding of how surfaces interact with t issues (Qu et al, 1996). There is thus a need for studies using precisely control led sur faces. Our laboratory introduced micropatterning and micromachin ing p rocesses , used in microelectronics to biomaterial appl icat ions, and this approach has enabled the investigation of how well def ined features of topography affect cell behaviour in vitro (Brunette, 1986a,b; Brunette et al, 1983; 1991) and in vivo (Chehroudi et al, 1990; 1992). Of particular interest to this thesis is that titanium coated micromachined substrata have been shown to increase the formation of bone-l ike t issue adjacent to both in vitro (Ratkay, 1995) and in vivo implanted sur faces (Brunette etal, 1991, Chehroudi et al, 1992). Al though titanium implants have had cons iderab le s u c c e s s , investigators have also studied calc ium phosphate ceramics , especia l ly hydroxyapatite and tr icalcium phosphate, and found that ceramic implants 3 were assoc ia ted with enhanced mineralization (Ong et al, 1998; Chang et al, 1999). One reason for the interest in hydroxyapatite is that its C a / P ratio are similar to that of natural bone (Sun et a/,1997). Moreover, HA is biocompatible and produces very low (if any) inflammatory response. HA implants have also produced direct contact with bony structures (Sun et a / ,1997) . To date, there has not been any work on hydroxyapatite (HA) coated micromachined substrata. Moreover, there is no agreement on the optimal topography of an implant for bone contacting appl icat ions and commercia l ly avai lable implants vary in this aspect . A s this thesis will examine both titanium and hydroxyapatite sur faces on bone-l ike t issue formation in vitro, a brief introduction of Ti and HA implants will now be g iven. 2. Titanium and Hydroxyapatite Implants: In or thopaedics and dentistry, titanium or titanium al loys are among the materials of cho ice , with cobal t -chromium and sta in less steel being used in orthopaedics to some extent (Ahmad et al, 1999). T h e attract iveness of Ti implants lies in their high strength, relatively low elast ic modulus, high corrosive res is tance and biocompatibi l i ty (Thomas etal, 1987; Sobal le , 1993). Furthermore, Ti implants have been shown to be successfu l (compared to other metals) in short and long term studies 4 for mineral ized matrix production (Ahmad et al, 1999). Bioact ive materials, materials that have been des igned to induce specif ic biological activity such as HA, have been found to result in more bone contact than Ti (Sobal le, 1993). Moreover HA implants support more bone ingrowth than Ti (Sobal le, 1993). Hydroxyapati te, a calc ium phosphate material, is accepted as osteoconduct ive, caus ing bone to grow into areas that it would otherwise not occupy (Jarcho, 1981; Schwartz etal, 1993; Gross etal, 1997; Davies, 1998; Chang et al, 1999). Beckham et al (1971) found that there is an intimate relationship between mineral ized bone and calc ium phosphate ceramic. In 1973, the first indication of a chemical bond between bone t issue and HA was reported (Driskell et al, 1973 cited in de Bruijn et al, 1991). Moreover HA is the only substance shown to elicit a chemical bond with bone when implanted, causing the implant to be more stable. For these reasons HA is very attractive for cl inical appl icat ions (de Bruijn et al, 1991; Kay, 1993; Manley, 1993; Thomas, 1994; Sun etal, 1997; Chang et al, 1999). HA was introduced commercial ly in 1981 for medical appl icat ions in granular form for alveolar ridge augmentat ion and periodontal lesion fill ings (Manley, 1993). A s development cont inued, applications have expanded to include blocks and coat ings, increasing the options for restorative dental and orthopaedic appl icat ions (Manley, 5 1993) . There are several additional advantages to using HA for implants. HA does not elicit an immune response; moreover, synthetic HA is not readily bioresorbable (it does not readily undergo biological resorption by solut ion- or cel l -mediated processes) and is therefore appropriate for long-term clinical use (Manley, 1993; Thomas, 1994). HA is also known to increase the bone-implant shear attachment strength up to five t imes that of uncoated metal sur faces, indicating a more rapid t issue adaptation to the implant sur faces (Thomas, 1994). Cl in ical studies have reported high success rates for implants with HA, nevertheless some controversies exist regarding the effects of HA coatings (Hench et al, 1984; Gross et al, 1997; 1998). Hydroxyapatite has been used as a coating on metall ic substrates (such as T i -6A1-4V alloy and commercial ly pure Ti) and as a dental implant material for many years (Chang etal, 1999; Chou etal, 1999). The most popular method of applying HA onto metallic substrates is a p lasma spray procedure (Whitehead etal, 1993; Cheang and Khor, 1996). This procedure yields a coating mainly consist ing of the bas ic apatitic structure, but an amorphous phase of HA is also formed (Cheang and Khor, 1996). This amorphous phase of HA is found to have less hydroxyl groups (Whitehead etal, 1993; Cheang and Khor, 1996). Furthermore, other 6 calcium phosphate phases have been identified in the coat ings such as (3-tricalcium phosphate (TCP) , a - T C P and oxyhydroxyapati te, as well as non-crystal l ine calc ium phosphate material (Whitehead et al, 1993; Cheang and Khor , 1996). These problems occur because the p lasma flame reaches a high temperature causing the HA to be thermodynamical ly unstable (Wolke, 1997) . Furthermore the HA ceramic is heated to a molten state causing the HA to be partially dehydroxylated and undergo phase transitions (Cheang and Khor, 1996). Therefore care must be taken to achieve appropriate condit ions to yield desirable structure and composi t ion of the HA coating (Cheang and Khor, 1996; Wolke, 1997). A better method is sputter coat ing, specif ical ly R F Magnetron sputtering, which forms a dense and uniform coating (Lacef ield, 1988; Jansen et al, 1993; Lacef ie ld, 1998) . In addition to the R F magnetron sputtering p rocess , heat treating the HA coated substrate has been found to increase the bond strength between HA and the substrate (Lacefield, 1988; Wolke etal, 1997). Other advantages to this method are the high rate of deposi t ion, the ease of sputtering, the production of high-purity f i lms, the extremely high adhesion of fi lms and the excel lent coverage of difficult surface geometr ies (Jansen et al, 1993). The deposited HA films are character ized by methods such as scanning electron microscopy (SEM) and x-ray diffraction (XRD) , to ensure that the coating has a uniform thickness 7 and wel l -crystal l ized calc ium phosphate ceramic (Jansen et al, 1993). By evaluat ing the character ist ics of both amorphous and crystal l ine coat ings through cellular responses, it is thought that a HA coating can be designed that will accompl ish a specif ic purpose in the body (Gross et al, 1997). One problem associated with the use of HA coating is that they are somet imes lost in vitro and in vivo through p rocesses such as delaminat ion, abrasion or dissolution (Bauer, 1993). The dissolution of HA is a function of crystallinity (Morgan et al, 1996). One way of testing dissolution of crystall ine and amorphous HA coat ings is by immersing the HA coated surfaces in Ringer 's solution (Gross etal, 1997). The dissolut ion rate is correlated with the pH of the surrounding environment (Chou etal, 1999). Furthermore crystall ine coatings have been found to be more stable than amorphous coat ings, because crystal l ine HA coat ings show no s igns of degradation except crack ing, which is attributable to release of residual s t resses (Gross et al, 1997). An advantage of highly crystal l ine coat ings is that they are less soluble (Caul ier et al, 1995) and do not alter the pH of the surrounding fluids such as culture medium. Changes in pH can occur as a result of dissolution of the ceramic coatings (Chou et al, 1999) and would be expected to alter experimental results. An except ion to the correlat ion between crystall inity and dissolution was found by Ogiso et al (1998). They suggest that the 8 crystallinity may decrease the binding strength between the HA coating and substrate, and not the dissolution. However, Og iso e r a / (1998) used p lasma sprayed implants that were not addit ionally heat treated, and heat treatment is found to ensure higher crystall inity (Lacef ie ld ,1988; G ross et al ,1998). Cel lu lar responses are also inf luenced by the crystall inity of HA surfaces (Ong et al, 1998). For example HA crystall ite s ize inf luences the express ion of osteoblast character ist ics (Ong et al, 1998). The higher the crystal l inity, the higher the rates of cel l prol i feration and differentiation achieved (Ong etal, 1998; Chou etal, 1999). HA supports the proliferation and differentiation of os teob lasts better then Ti (Massas et al, 1993; Ong et al, 1998). This increase in proliferation and differentiation may be one of the causes of the increased bone formation seen with HA implants (Massas etal, 1993). Furthermore HA is general ly assoc iated with a higher rate of formation and higher amount of bone t issue in contact with the implant compared to titanium implants (Massas etal, 1993; Chang etal, 1999). A brief introduction to the components of bone, the t issue of primary interest in this thesis fol lows. 3. Bone Matrix: Bone consists of inorganic and organic components. Calc i f ied bone 9 was found to be composed of 76 to 77 percent (dry weight) inorganic bone substance, the balance is organic (Ham, 1974). The organic matrix is formed mostly of co l lagen, with col lagen type I, being the major constituent (Ham, 1974; Satomura and Nagayama, 1991). Co l lagen type I in the form of a fibrous network provides the structural integrity of many connect ive t issues, such as bone and dentin (Watt, 1986; Uitto and Larjava, 1991). Moreover col lagen type I directly supports mineral ization by serving as a site of nucleation for the induction of mineral components such as hydroxyapatite crystals (Bel lows et al, 1986; Gl imcher, 1989). There are several other minor co l lagenous components in bone: types VI (cell to col lagen binding), V, X , XI, and XII col lagens (Uitto and Larjava, 1991). Although type V and III col lagens are also found in bone, they are not present in sufficient quantit ies to be measured accurately (Bel lows etal, 1986; Uitto and Larjava, 1991; Ve is , 1993). Moreover there is also evidence of the presence of col lagen type XII; that is assoc iated with type I col lagen and forms cross bridges between col lagen fibrils as well as mediating the binding of other connect ive t issue components such as proteoglygans (Uitto and Larjava, 1991). Proteoglycans exist in bone and are also found in the extracellular matrix (Watt, 1986). The main dermatan sulfate proteoglycans found in bone and carti lage are decorin and big lycan, which share signif icant 1 0 sequence homology (Uitto and Larjava, 1991). Decorin expression is exclus ive to matrix-centred functions, such as regulating cel l growth and col lagen fibril formation (Bianco et al, 1990; Y a m a d a et al, 1999). Biglycan, very similar to decorin, is expressed in a range of specia l ized cell types, including connect ive t issue (skeletal myof ibers, bone) and is directly involved in cel l regulatory funct ions including cel l proliferation and differentiation (Bianco etal, 1990; Inoue etal, 1999; Y a m a d a etal, 1999). Furthermore biglycan is found in developing bone and possible plays a role in bone development (Inoue et al, 1999). There are also famil ies of non-col lagenous proteins present in bone that play a key role in differentiation, activation of bone cel ls , maturation and mineralization of bone matrix (Nefussi et al, 1997). One glycoprotein that is used as a marker of mineral ization, is osteonect in. Osteonect in has affinity to both col lagen type I and HA (Romberg et al, 1985) and appears to mediate the in vitro mineral ization of co l lagen type I (Romberg etal, 1985). Osteonect in synthesis may be increased by active osteoblast ic cel ls in a nodule, or it may be that its accumulat ion within the nodule is related to its associat ion with other matrix components (Bel lows et al, 1986; Nefussi et al, 1997). Another marker for mineral ization is osteocalc in (bone G L A protein) a bone and dentin speci f ic protein. Osteoca lc in is primarily restricted to 1 1 mineral ized t i ssues , in part icular to mineral izat ion fronts (Nefussi et al, 1997). Moreover osteocalc in functions as an osteoclast recruiter (Davies, 1996). Osteoca lc in is synthes ized almost exclusively by osteoblasts (Beresford etal, 1993; Davies, 1996; Okumura etal, 1997). Another family of non-col lagenous proteins related to bone include the sialo (phospho) proteins: osteopontin (secreted phosphoprotein (SPP-1 ) or BSP-I ) and bone sialoprotein (BSP-II) (Veis, 1993). Osteopont in is not specif ic to bone or dentin and is multi-functional (Pinero et al, 1995; Davies, 1996). It is involved in early bone development, as it inf luences the attachment of osteoblasts to sites of bone growth (Pinero et al, 1995). It has also been suggested that during bone remodell ing osteopontin may aid the attachment of osteoclasts to bone (Pinero et al, 1995). Both osteopontin and bone sialoprotein appear to be distributed in osteoids, mineral ized matr ices and mineral izat ion fronts (Pinero et al, 1995) . Osteoblasts secrete bone sialoprotein (Pinero et al, 1995). The assoc ia t ion of amorphous , e lec t ron-dense, granular extra-f ibri l lar materials with BSP-II acts as a nucleation structure involved in the mineral izat ion process (Nefussi et al, 1997). Moreover BSP-II is located at si tes of early mineral izat ion speci f ical ly restricted to a reas at which crystal l i tes are present (Pinero et al, 1995; Nefussi et al, 1997). 1 2 There are numerous other enzymes and growth factors found in bone that appear to have important roles in mineralization. Some growth factors associated with bone matrix formation include transforming factor (TGF)-B and -B (Yamada etal, 1999), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), insulin-like growth factors (IGF) I and II, bone morphogenic proteins (BMP's) and osteo-inductive factor (Hollinger and Leong, 1996; Bostrom, et al, 1999; Strayhorn et al, 1999). However, there is not just one growth factor but numerous growth factors that are working in bone maintenance and repair (Hollinger and Leong, 1996; Bostrom, etal, 1999). Bone matrix associated enzymes include collagenases, proteinases, cathepsins and alkaline phosphatase (Golub, 1996). Alkaline phosphatase activity is a major marker of the osteoblastic phenotype and sites of bone mineralization activity (Ballanti et al, 1995). Bone also contains albumin, which is synthesized in the liver and has widespread distribution (Peel, 1995). Albumin's suggested function in bone is as a regulating carrier for small molecules and ions involved in mineralization (Peel, 1995; Davies, 1996). The other major component of bone is inorganic. The inorganic portion of bone matrix is primarily hydroxyapatite ( C a 1 Q ( P 0 4 ) 6 ( O H 2 ) , which gives bone its hardness and rigidity (Ham,1974). The precise mechanism 1 3 of mineral izat ion, in which the organic matrix becomes calci f ied by inorganic HA is still under investigation. One suggest ion of the underlying phys icochemica l basis and mechanism for the formation of ca lc ium phosphate crystals in bone and other t issues, and in biological mineralization is a heterogeneous nucleation by one or more of the organic const i tuents (Gl imcher , 1989). Bone matrix ves ic les play a role in initial hydroxyapati te formation (Schwartz and Boyan, 1994). They are assoc iated with primary calcif ication and mineral ization fronts in a number of t issues (Boyan et al, 1993). These bone matrix ves ic les are secreted by osteoblasts and contain non-col lagenous proteins, alkal ine phosphatase, A T P - a s e . In time HA crystals form along the inner portion of the matrix ves ic le membrane (Boyan et al, 1993). Furthermore bone matrix ves ic les are thought to be the vehic les used to transport media for triggering nucleat ion of mineral crystals at distant locat ions (Mikuni -Takagaki et al, 1995). After the secret ion of specif ic and non-specif ic bone proteins contained in the bone matrix ves ic les , mineral izat ion of the matrix is initiated by express ion of the membrane glycoprotein alkaline phosphatase (McComb etal, 1979). 4. Effects of Surface Topography on Cell Behaviour: It is known that surface topography inf luences cell behaviour, in vivo and in vitro (Brunette, 1988; Brunette et al, 1991; Brunette and 1 4 Chehroudi , 1999; Chehroudi etal, 1992; Chehroudi and Brunette, 1995; Qu et al, 1996; Chang et al, 1999). More specif ical ly surface topography has affected osteoblast behaviour resulting in enhanced mineral ized t issue formation in vivo and in vitro (Brunette, 1988; Brunette et al, 1991; Brunette and Chehroudi , 1999; Chehroudi etal, 1992; Chehroudi and Brunette, 1995; Qu etal, 1996). One aspect in which surface topography inf luences cell behaviour is by altering cell shape (Brunette 1988). Cel l shape in turn inf luences many aspects of cell behaviour including cell adhesion, locomotion, and gene expression (Watt, 1986; Brunette and Chehroudi , 1999). For cel ls to adhere to a surface cel ls must contact, attach and spread onto the surface (Weiss, 1975). Severa l surface properties inf luence cel l attachment, for example , wettability (Baier, 1986), sur face area (Brunette, 1988) and composit ion (Chang etal, 1999). Wettability is measured by the critical contact angle formed by liquid droplets spreading on a surface (Baier, 1986). A way to increase the wettability is by glow-discharging the surface so that a high-energy surface is formed (Baier, 1986). Sur face area is also a factor affecting cell attachment. At the simplest level a surface with a greater surface area, for example a grooved versus a smooth surface, provides more opportunity for cell attachment (Brunette, 1988). The composi t ion of the surface affects 1 5 attachment. Osteoblasts have been shown to attach and adhere better to HA than Ti implants (Chang et al, 1999), perhaps because osteoblasts produce more extracel lular matrix and expand cytoplasmic extensions (both assoc ia ted with cell adhesion) over the entire HA surface compared to osteoblasts on Ti surfaces (Wilke et al, 1998). Another cel lular function of bone cel ls affected by surface topography is cell locomotion (Chesmel et al, 1995). Contact (topographic) guidance, which refers to the tendency of a cell to be oriented and guided in its direction of motion by the shape of the surface with which it is in contact. This behaviour was observed in some of the earl ier studies of cel ls in culture (Harr ison, 1914). Us ing t ime- lapse c inemicrography (Brunette etal, 1991) it was found that osteoblast- l ike cel ls initially elongate and orient themselves on Ti grooved substrata, and became progressively more oriented to the grooves over time (Qu et al, 1996) . Gene expression is also affected by cell shape which in turn is affected by surface topography. The shape of a cell on a surface will affect it's proliferation and differentiation (Folkman and M o s c o n a , 1978; Watt, 1986; Bas le etal, 1998). Cel ls in a rounded configuration will have their proliferation restricted, which is shown by a reduction in the proportion of cel ls synthesiz ing DNA. Furthermore changing cell shape by 1 6 stretching cel ls by mechan ica l means st imulates prol i feration (Brunette 1984) as can changing substratum adhes iveness (Folkman and Moscona , 1978). Moreover the differentiation of cel ls is affected by cell shape, for example erythroleukemic cel l- f ibroblast hybrids that grow in suspens ion can express haemoglobin, whereas adherent c lones of the cel ls can not (Allan and Harr ison, 1980; Watt, 1986). A brief d iscuss ion on proposed mechanisms of osteogenes is on micro fabr ica ted subs t ra ta fo l lows. 5. Proposed Mechanisms of Enhanced Osteogenesis on Micromachined Surfaces: Attachment , prol i ferat ion, metabol ism, matrix syn thes is and differentiation of osteoblast- l ike cel l l ines in vitro are affected by one or more of the four interrelated propert ies of b iomater ia ls: chemica l composi t ion, surface energy, surface roughness and surface topography (Schwartz and Boyan 1994). One biomaterial, such as Ti , used by Chehroudi e r a / ( 1 9 9 2 ) on grooved substrata proved more effective than smooth surfaces in enhancing mineral ization in vivo. The process by which mineral ization is initiated and regulated by topography in vivo and in vitro is not well understood but Chehroudi et al (1992) suggest three mechanisms. One possibility is that topography can produce a bone-inductive microenvironment. For example grooved sur faces could produce 1 7 a confined environment in which growth factors can ach ieve the concentrat ions required for bone production. A second possibil ity is that the organization of col lagen bundles may be signif icant in the initiation of mineral izat ion (Chehroudi et al, 1992). This suggest ion ar ises from considerat ion of the work of Ve is (1993) who found that the col lagen fibrils in associat ion with phosphoprote ins initiate the nucleation of apatite crystals. In vivo the development of intra-membranous bone t issue fol lows a sequence in which the first step is character ized by a random, felt-like orientation of co l lagen fibrils which is considered as a primary scaffold in vivo (Schenk and Buser, 1998). In the second month col lagen fibrils are packaged into parallel arrays with alternating courses (such as plywood) and this organizat ion gives bone the highest structural strength (Schenk and Buser , 1998). Furthermore Davies and Matsuda (1988b) found that col lagen fibrils were morphological ly more organized into linear bundles on some substrata, for example bioactive g lass, that enhanced mineral izat ion. Therefore it is conceivable that micromachined grooved surfaces may encourage the orientation and arrangement of co l lagen fibrils that promote mineral ized-t issue production (Chehroudi et al, 1992). Orientation and spatial distribution of col lagen can be examined using a histochemical stain that enhances the birefr ingence of col lagen (Dz iedz i c -Goc lawska etal, 1982; 1 8 Pierard, 1989; Montes and Junqueira, 1991; Canham etal, 1999). A third possibil i ty is that cell shape and polarity can inf luence the cel l 's gene express ion , differentiation and function (Watt, 1986). Overal l it appears that micromachined grooved sur faces could therefore promote mineral izat ion through diverse mechan isms by their known property of orienting cel ls (Chehroudi et al, 1992), extracel lular matrix (Qu et al, 1996), and altering cell shape and gene expression (Chou etal, 1999). A brief d iscuss ion on condit ions promoting mineral izat ion fol lows. 6. Condit ions Promoting Mineralization: Osteoblasts are the only cel ls that have the ability to form bone on material sur faces (Davies, 1996). The mineral izat ion p rocess is still under investigation, but some supplements are known to favour mineral izat ion (Bel lows etal, 1991; 1992). One important supplement is ascorb ic acid (Vitamin C) (Bel lows et al, 1991). Ascorb ic acid stimulates the formation and hydroxylat ion of co l lagen, permitt ing suff icient amounts of co l lagenous matrix to be deposi ted for nodule production (Bel lows et al, 1986). Ascorb ic acid is necessary for mineral izat ion, but the mineral ized t issue produced in the presence of ascorb ic acid without organic phosphate is not morphological ly identical to t issue mineral ized in vivo (Tenenbaum and Heersche, 1982; Bel lows etal, 1986; 1991). For mineral izat ion to mimic physio logical bone, in vitro sys tems must often 1 9 be supplemented with a source of organic phosphate (Tenenbaum and Heersche, 1982; Bel lows etal, 1986). The rationale for using organic phosphate is that it provides an additional source of phosphate ions that are made avai lable in p laces where alkaline phosphatase is present (Tenenbaum and Heersche, 1982). Moreover it has been suggested that organic phosphates may be more important for mineral izat ion than circulating inorganic phosphate, both as a source of phosphate ions and as a cel lular regulator of mineralization (Tenenbaum and Heersche , 1982). Although circulating levels of inorganic phosphate in vivo are abundant in serum and bone they play a relatively small role in the initiation of mineral izat ion; never the less, for hydroxyapat i te crysta ls to form some inorganic phosphate is required (Tenenbaum and Heersche, 1982). For in vitro systems to mimic physiological bone it is necessary that osteoblasts produce a mult i- layered structure surrounded by a mineral ized matrix (Davies, 1988a), to ach ieve a three-d imensional structure of mineral ized t issue housing osteocytes (Bel lows et al, 1986). S ince L-ascorbic acid has a very short half life of about 1.5 hours in culture (Feng et al, 1977), many investigators adopted a protocol that entailed refreshing the medium at least three t imes a week. Within the past decade a new longer acting ascorbate analogue, L-ascorbic acid phosphate (Asc-P) , has been developed (Hitomi etal, 1992). This 2 0 ascorbate is more stable and lasts longer than natural L-ascorbic acid (Hitomi et al, 1992; Ja iswal ef al, 1997). Refreshing the medium twice a week with the ascorbate analogue yields comparable results to L-ascorbic acid (Beresford et al, 1993; Ja iswal et al, 1997). In addition L-ascorbic acid phosphate supplementation can produce mineral ized nodules even in the absence of 3-glycerophosphate (Beresford etal, 1993). A s c - P also is capable of inducing rapid osteogenesis as defined by the appearance of osteoblast ic cel l morphology, increased express ion of a lkal ine phosphatase and the formation of a mineral ized extracel lular matrix containing hydroxyapat i te (Ja iswal et al, 1997). A brief introduction of some markers used to identify osteogenic t i ssue fo l lows. 7. Techniques used to Identify Osteogenic Tissue: S o m e simple criteria that have been used to identify osteogenesis in vitro include a) morphology: an aggregation of cel ls with osteoblast ic morphology; b) col lagen deposit ion; and c) mineralization as a s s e s s e d by varies stains (see for example Ratkay, 1995). Based on previous reports as well as preliminary studies, this thesis employed these markers as fol lows a) morphology as a s s e s s e d by reflected light differential interference contrast and scanning electron microscopy; b) col lagen as v isual ized by staining with picro-sir ius red and polar ized light 21 microscopy; and c) mineral izat ion using tetracycl ine label l ing and alkal ine phosphatase activity using naphthol A S - M X phosphate substrate. More detai led information on some of these techniques fol lows. Polar izat ion microscopy is usually used for unstained t issues (Puchtler et al, 1973). There are several dyes that stain col lagen and will show birefr ingence but the most speci f ic is picro-sir ius red F 3 B A . This stain has been used extensively for staining col lagen-contain ing structures and was used in this thesis (Puchtler et al, 1973; Canham et al, 1999) . Sir ius red F 3 B A is a long and fairly linear azo dye that has greater refractivity for light vibrating along its long axis than ac ross it, thus this and similar dyes are know as intrinsically anisotropic dyes (Puchtler et al, 1973). Puchtler e f a / ( 1 9 7 3 ) a lso found that the intensity and colour in polar ized light are related to the concentration of the dye and the thickness of the col lagen. A s more information on this dye became known, Junque i ra et al (1979) real ized that all structures s ta ined with Sir ius red had enhanced birefringency. These areas were known to contain col lagen; however, the stain by itself is not specif ic for col lagen (Junqueira et al, 1979). It is the combination of aqueous picric acid solution and Sirius red that appears to lead to select ive staining of col lagen (Junqueira et al, 1979). S ince col lagen is a basic protein, the sulphonic groups of Sir ius 22 red interact at low pH with the amino groups of lysine and hydroxylysine, and the guanidine groups of arginine in col lagen (Junqueira etal, 1979). The picro-sir ius staining increases the birefringent nature of co l lagen at least 7 0 0 % in light intensity (Junqueira et al, 1979). The increase in the birefringence is because the sirius red molecules attach to the col lagen fibrils in such a way that their long axes are paral lel with the col lagen fibrils that are themse lves intr insical ly birefr ingent (Junquei ra et al, 1982) . One way of observing the increase in birefringence is linear polar ized light microscopy (Wolman, 1970). Us ing linearly polar ized light, col lagen orientation can be demonstrated by compar ing two v iews of the same specimen rotated 45° to each other so that only the col lagen that is parallel to the polarizer will be brightly seen (Wolman, 1970; Boyde et al, 1984). Polar iz ing microscopes, however, do not detect birefringent material if its optic axis is perpendicular to the plane of focus (Frohl ich, 1986) . It has been suggested that polarized light can be used to distinguish different types of col lagen (Junqueira et al, 1982; Montes and Junquei ra, 1991). It was observed that picro-sir ius staining c a u s e s different types of fibres to appear in different colours when seen in polar ized light (Montes and Junqueira, 1991). Col lagen fibres show up in the form of 2 3 thick, brilliant (strongly birefringent) yel low or red f ibres against a dark background when studied by the picro-si r ius-polar izat ion method, whereas reticulin f ibres display a weak birefr ingence and are character ized by their thinness and greenish colour (Montes and Junqueira, 1991). Moreover differences between col lagen types I, II and III are observed but it was subsequent ly real ized that the dif ferences are the result of the th ickness of the fibres and the packing of these different col lagens (Dayan etal, 1989; Andrade etal, 1997). However, some caution in the interpretation of col lagen types by picro-sir ius red staining must be made. Pierard (1989) states that identifying col lagen type by this colour method is erroneous. This is because the colour of col lagen fibers are unrelated to the fiber's molecular nature and staining it may vary according to the orientation of the slide on the stage of the microscope (Pierard, 1989). He suggests that picro-sir ius staining should be limited to the orientation and density of co l l agen . Overal l it is bel ieved that the picro-sir ius polar izat ion method is an extremely s imple, rel iable, relatively speci f ic , sensi t ive and cheap method which provides an easy and precise method of qualitative or quantitative analys is of col lagen (Junqueira etal, 1979; Montes and Junquei ra, 1991; Andrade etal, 1997). 2 4 The picro-sir ius staining method is a reliable method and it has found appl icat ions in pathology in the diagnosis of co l lagenous d iseases (Dz iedz i c -Goc lawska etal, 1982; Montes and Junquei ra, 1991; Trau etal, 1991; Rabau and Dayan, 1994). However for identifying mineralization different markers, such as tetracycl ine and A l k - P activity, are required. Tetracycl ine is a widely used antibiotic in cl in ical medic ine and dentistry (van der Bijl and Pit igoi-Aron, 1995). It has been found that tetracycl ines are taken up and accumulate in newly-formed mineral ized t issues more readily than in old bone (Ibsen and Urist, 1964; Frost, 1968; McClure , 1982; Misra , 1991; van der Bijl and Pi t igoi-Aron, 1995). Furthermore because tetracycline does not bind to old bone as readily as newly-formed bone, tetracycl ine-stained old bone normally does not f luoresce under ultraviolet or blue light (Ibsen and Urist, 1964; Perr in, 1965; Frost, 1968; Davies etal, 1985; Misra, 1991). Moreover s ince tetracycl ine f luoresces, the binding of tetracycl ine and calc ium has been successfu l ly used as f luorescent labels for studying bone metabol ism (Ibsen and Urist, 1964; van der Bijl and Pit igoi-Aron, 1995). Tetracycl ine incorporates preferentially with new bone calc ium because (1) in vivo sites of new bone formation are c loser to the blood supply than is old bone, therefore new bone is richer in tetracycl ine; (2) the bone crystals are smaller, and therefore present a greater surface area for 2 5 incorporat ion; (3) the diffusion rate of tetracycl ines through matrix and minerals in older, denser bone is slower, s ince tetracycl ines are relatively large molecules (=5x bigger than a water molecule) (Ibsen and Ur is t , 1964). Another advantage of tetracycline is that it can be used in vivo and in vitro to observe bone cell behavioral rates such as bone resorption and formation, and population dynamics (Frost, 1968). The effects of tetracycl ine on cell cultures can be either beneficial or inhibitory depending on concentrat ions (Vernil lo and Rifkin, 1998). Inhibition of bone growth occurs with high concentrat ions (50u.g/ml) of tetracycl ine but lower concentrat ions (20u,g/ml) do not impede mineral izat ion (Gruber,1993). Moreover the use of 5u.g/ml of tetracycl ine gives a signal that is readily detectable under UV f luorescence microscopy with little effect on mineral izat ion (Gruber, 1993). A benef ic ial effect of tetracycl ine for some cl in ical condit ions is that it can slow osteoclast-mediated bone resorption directly by reducing the bone-binding capacity of osteoclasts (Keller and Carano , 1995; Verni l lo and Rifkin, 1998). Tetracycl ine inhibits bone resorption in vitro by, in part, reducing co l lagenase activity (Ramamurthy et al, 1990; Verni l lo and Rifkin, 1998). Another marker used to detect mineral izat ion activity is alkal ine phosphatase (Alk-P) which is assoc ia ted with cell membranes and occurs 2 6 in many cell types, including osteoblasts (Kiernan, 1981). A l k -P is bel ieved to be involved in bone formation, and is a characterist ic of bone cel ls producing mineral ized matrix (Farley and Bayl ink, 1986; Mikuni-Takagak i et al, 1995). A l k -P activity is not expressed in any significant amount in osteocytes (Mikuni-Takagaki et al, 1995). Furthermore the presence of A l k -P hinders the extracellular phosphorylat ion of bone proteins by osteocytes possibly through a phosphoprotein k inase pathway (Mikun i -Takagak i et al, 1995). Overal l A l k -P activity is thought to be an excel lent differentiation marker for os teogenic cel ls involved in mineral izat ion (Ballanti et al, 1995). Moreover use of A l k -P as an indicator of mineral izat ion activity cor re la tes wel l with tet racycl ine label l ing in studies of mineral izat ion of osteoblast ic cel l l ines (Ballanti et al, 1993; 1995). Measurements can be made of A l k -P activity using an enzyme-substrate reaction end product (Sabokbar et al, 1994). The substrate is a monobasic sodium salt of a -naphthy l phosphate, the monoester of a -naphthol, and phosphoric acid (Kiernan, 1981). The a-naphthol freed by the hydrolysis is a phenolic compound and can be coupled with a diazonium salt (Kiernan, 1981). Furthermore naphthol A S - M X phosphate, a substrate, has been found to be the most satisfactory for the detection of A lk -P activity compared to other naphthol substrates (Ackerman, 1962). 2 7 Although naphthol A S - M X forms a more intense colour then does a -naphthol, its rate of coupl ing with diazonium salt is s lower (Kiernan, 1981). The reaction between the substrate and A lk -P occurs in an alkaline pH and an insoluble coloured azoic dye is produced, s ince the dye is added to the reaction medium (Kiernan, 1981). The diazonium salt used is Fast Blue R R , thought to be one of the best available for this purpose (Ackerman, 1962; K iernan, 1981; Ballanti etal, 1995). By definition the pH of the medium used in the enzymat ic reaction must be alkaline to detect A lk -P activity (Yabe, 1985). The optimum pH is in the range of 9.6-10.0, with a pH of 9.6 being best for cel ls in contact with HA coatings (Er icson, 1969; Yabe,1985) . The optimum temperature, determined for the detection of human A lk -P was 25°C, thus the A lk -P reaction can be carried out at room temperature (Copeland et al, 1985). 8. Objective of the Thesis: Since micromachining can produce precise and control led topographies, it can be used to investigate the role of substratum topography in mineral izat ion. Hydroxyapati te is a lso thought to increase bone formation, but its interaction, if any, with topography is not known. The aims of this thesis are: A. To confirm that micromachined substrata in the form of grooves of varying depths increase mineral ized t issue formation. 2 8 B. To investigate the effects of smooth, dense HA coat ings on bone-like t i ssue format ion. C. To determine whether the combined effects of HA coatings and topography are addit ive, subtract ive or interactive. D. To determine the contribution of col lagen orientation and microenvironment to the increased bone formation seen on micromachined s u b s t r a t a . 29 M a t e r i a l s & M e t h o d s 3 0 1. Substrates: A. Micromachining: Micromachining is a technique that produces topographic features with precise dimensions in silicon wafers. The particular micromachining techniques used in this study were those developed at the University of British Columbia by Camporese et al (1981) for the fabrication of high quality photomasks for solar cells. Micromachining begins with the production of a master pattern which is decreased to the appropriate dimensions by a step-and-repeat photographic process to produce photomasks, glass plates on which a metallic master pattern is placed. Then the master pattern is transferred onto a silicon wafer by photolithography. A thin silicon oxide layer is produced on the surface of the wafer, which is then covered with a UV sensitive organic polymer, called photoresist. Then the wafer is exposed to UV-radiation through the photomask. After exposure the photoresist is removed chemically, creating a negative copy of the master pattern. Next a chemical reaction removes the silicon oxide from the areas that do not have photoresist and finally the rest of the polymer is removed. To create the desired depth for the silicon oxide pattern it is chemically etched by anisotropic etchants which can create well defined shapes with sharp 31 edges and comers . The result is a positive copy of the master pattern made out of si l icon oxide on the surface of the si l icon wafer. B. Surface Pattern: The pattern used in this study was developed by Khakbaznejad (2000). The pattern was etched into a si l icon wafer, as descr ibed earlier, in Dr. N.A.F. Jaeger 's Laboratory, Department of Electr ical Engineer ing, U B C . The over all d imensions were 1cm by 1.5cm and within this grooves were etched out. The angle of the grooves tapered at 55°. The grooves were etched to three depths (3u,m, 10pm and 30jxm) each having a pitch of 47u,m and a ridge of 5pm. (Figure 1). In the same surfaces, within the grooves, there were smooth gaps etched (interrupting the grooves). There were four different smooth gaps all having the same area of 9 0 0 0 0 p m 2 but differing widths of 50pm, 100pm, 150pm and 200pm. There are ten smooth gaps of each width on each surface depth. (Figure 2). C. Coatings: The wafers were coated with titanium or hydroxyapatite. For titanium wafers they were sputter-coated with 50nm of titanium in Dr. N.A.F. Jaeger 's laboratory. For HA coating si l icon wafers were sent to Dr. W.R . Lacefield at the University of A labama School of Dentistry. There the wafers were c leaned with substrate bias of 100W for 5 minutes prior to sputtering. They were then R.F. Magnetron sputtered C a - P coated at 400W, 3 2 . Pi tch 4 7 p m \ ) A ) 3 p m d e e p g r o o v e s ^ R i d g e S p m ^ Groove **T Depth . Pi tch 4 7 p m \ > B) 1 0 p m d e e p g r o o v e s ^ R i d g e 5 p r r ^ \ / JlOpnN f Groove Depth F i g u r e 1. Schematic cross-sect ions of grooved surfaces. T h e t h ree u s e d in th is t h e s i s . 3 3 F i g u r e 2. A macro-image of the pattern. T h i s pa t te rn is s e e n o n the s u b s t r a t a u s e d in th is s t u d y . N o t i c e the d i f ferent g a p s s i z e s in ter rupt ing the g r o o v e s . 3 4 with 5x10 " 3 mbar working pressure and sputtering rate of 100-150 nm/min for 1 hour (thus « 1u,m thick). Fol lowing the coat ing the wafers were heat treated for 2 hours at 500°C and analyzed by a Thin Film X-ray Diffractometer (Phi l ips X 'Per t , Multi Pu rpose Diffractometer) to characterize the coating of HA. A copper target is used as an x-ray source (wavelength= 0.15406nm) and the incident angle omega was fixed to 1 degree and 2-theta angle was scanned for the thin film x-ray diffraction measurments. This method is very similar to the one used by Wolke et al (1998) . Sur face roughness was measured using a profi lometer (Alpha-step 200, Tencor Instruments, Mountain View, C A , U S A ) . Six samples for each surface type were measured and the average roughness (R ) was quan t i f i ed . D. Preparations for cell culture experiments: The wafers after being coated were cut with a diamond pen so that the overall pattern (1 by 1.5 cm) and smooth pieces were used. In order to retain maximum overall HA coating the wafers were not washed and to ensure that the wafers were sterile they were g low-discharged (Baier, 1986) for 3 minutes in an argon-gas chamber developed by Aebi and Pol lard, (1987). The glow-discharge treatment a lso produces a high surface energy surface that faci l i tates cel l at tachment. 3 5 2. Culture of osteogenic cells derived from rat calvaria: To test the effect of topography on bone formation between HA and Ti coated surfaces newborn rat calvar ia osteogenic cell cultures were used. These cultures have been shown to produce mineral ized t issues on standard culture d ishes (Rao etal, 1977; Bel lows etal, 1986; Bhargava et al, 1988; Q u , et al, 1996; Dubois etal, 1998). The method used for osteoblast isolation was similar to that descr ibed by Hasegawa et al (1986) and Chehroudi et al (1992). Briefly, frontal, parietal and occipital bones (calvaria) of newborn (24-36 hours old) Sprague-Dawley rats were careful ly d issec ted , r insed in plentiful amounts of sterile phosphate buffered sal ine (PBS) and placed in t issue culture medium ( a-min imum essent ia l medium, a - M E M ) with 15% fetal calf serum ( F C S ) and antibiotics (100|ig/ml Penici l l in G , 50u;g/ml Gentamycin sulphate and .3ng/ml Fungizone) added. Eighteen to twenty calvariae were dissected and minced into p ieces = 1 m m 3 . The minced t issue was then incubated in 5ml of a digestion mixture containing 180U/ml of clostridial co l lagenase (type la, S igma, St. Louis, M O , USA) and .5mg/ml trypsin (Gibco, Burlington, Ontario, Canada) in P B S . The suspens ion was digested for 2x-20 minutes at 37°C with stirring in a Pierce "React i -v ia l " (Pierce Chemica l Company, Rockford, IL, U S A ) and the supernatant d iscarded. After a further 20 minutes incubation the 3 6 supernatant was kept and mixed with an equal volume of cold fetal calf serum and centri fuged at 1500rpm. Fol lowing the centrifugation the supernatant was discarded and the cells were resuspended in a - M E M containing 15% F C S and antibiotics. This population of cel ls was designated sub-culture O. The cel ls were placed in a 7 5 c m 2 t issue culture flask (Falcon, Bector Dickinson Labware, Franklin Lakes , N J , U S A ) . 3. Experimental Design: The design of the experiments in this thesis is schemat ical ly il lustrated in figure 3 and the details of the procedures follow. A. Culture: Osteoblasts cel ls were removed from the culture flask by using a trypsin (Gibco) solution (0.25% trypsin, 0 . 1% g lucose, citrate-sal ine buffer, pH 7.8) when required. Subcultures l-lll were used only. Calvar ia l cel ls were electronical ly counted (Coulter Ce l l Counter) and plated in six-well culture plates (35mm diameter each wel l ; Falcon) at a population density of 1.0x 1 0 5 c e l l s / c m 2 . Cultures were incubated in a humid atmosphere of 9 5 % air and 5% C 0 2 at 37°C. When the cells became confluent 7-14 days after plating the medium was further supplemented with 58u.g/ml L-ascorbic acid phosphate magnes ium salt 77-hydrate (Wako 3 7 Experimental Design: Cells Growth to * Supplements added Confluence F e d Fed Fed Fed ^ (7-14 days) 1 1 1 1 1 1 1 1 I I I | | | Day 1 4 A 1 week 2 weeks 3 weeks 4 weeks f t * f t t I SEM I Alk-P Alk-P I SEM Collagen Stain SEM Collagen Stain SEM Collagen Stain N o d u l e C o u n t s Collagen Stain Collagen Stain Collagen Stain Collagen Stain • DIC TI > ; ^ F i g u r e 3. A schematic diagram of the experimental design. S E M , ( s c a n n i n g e l e c t r o n m i c r o s c o p y ) . C o l l a g e n s t a i n , ( P i c r o - s i r i u s red F3B s t a i n ) . A l k - P , ( A l k a l i n e p h o s p h a t a s e act iv i ty) . N o d u l e c o u n t s . T e t r a c y c l i n e w a s a d d e d a l o n g wi th the s u p p l e m e n t s for , four w e e k s a n d the n o d u l e s c o u n t e d u s i n g f l u o r e s c e n c e m i c r o s c o p y . D I C T L , (Di f ferent ia l I n te r fe rence C o n t r a s t T i m e - L a p s e m o v i e s ) . 3 8 Pure Chemica l Industries Ltd., R ichmond, V A , U S A ) and 3.15mg/ml Na-B-Glycerol-phosphate (Sigma, St. Louis, M O , USA) over the next 4 weeks. The medium was changed twice a week. At the end of the culture period the t issue cultures were fixed and processed for microscopy. Fixative used for most cultures was 10% buffered formalin phosphate (Fisher Scienti f ic C o , Fair Lawn, N J , USA) for 1hr, for scanning electron microscopy was 2 .5% Glutaraldehyde ( J B S - C h e m , Dorval, Q U E ) for 30 minutes and for alkal ine phosphatase activity was citrate acetone (Sigma) for 30 seconds . B. Scanning electron microscopy (SEM): To observe the morphology of osteogenic cel ls and mineral ized nodules, some cultures were prepared for scanning electron microscopy (SEM) . Fixed cultures (24hours,1,2 and 4 weeks old) were post-fixed in 2% buffered osmium (Canemco Inc, Lachine, Q U E ) for 30 minutes. After rinsing with .1M phosphate buffer, pH 7.4 fol lowed by disti l led H 2 0 , 2 % tannic acid (Fisher Scientif ic Co) was added as this enhances osmium fixation making it more electron dense. The spec imens were then put back into 2 % buffered osmium for 30 minutes. After fixation they were dehydrated with graded alcohol (50%-100%, 1hr total), crit ical-point dried with C 0 2 ( L A D D Research Ind, Burl ington, VT, U S A ) , sputter-coated (Hummer VI Sputtering Sys tem, Techn ics , A lexandr ia , V A , U S A ) with TOnm of gold and observed in a scanning electron microscope (Cambridge 3 9 Stereoscan 100, Le ica , Canada) . To observe structures underlying the nodule (4 week cultured surfaces were used) cel lophane tape was used to lift the top of the surface, then both tape and the underlying area of the wafer were re-coated with 10nm of gold and re-examined under S E M . C. Polarization microscopy: To observe col lagen and its orientat ion, picro-sir ius red staining and polar ized light microscopy were used. The bireference of col lagen is enhanced with this stain. The method used is similar to the one used by Pearse (1985). Cultures from 24hrs and 1 to 6 weeks were used. At the end of each period they were fixed in 10% buffered formalin for 1 hour. After rinsing in distil led water fol lowed by 9 5 % ethanol the cultures were treated for 10 minutes in alkal ine alcohol at 60°C (alkaline alcohol: 9 5 % ethanol + ammonium hydroxide (Fisher Scientif ic Co. ) ; pH > 8.0). Fol lowing this treatment they were rinsed with disti l led water and put in with picro-sir ius red (100ml Saturated aqueous picric acid + 0.1 g sirius red F 3 B (Gurr, B D H Laboratory Suppl ies, Poo le , England)) for 1 hr at room temperature. Sur faces were rinsed in 1% acet ic acid until acid was clear, dehydrated quickly (75%-100% in repeated stages), c leared in xylene and mounted in Entellan (EM Sc ience, Gibbstown, N J , USA) . The surfaces were examined under a polarising microscope (Car lZe iss Jenapol) at 20x magnification and photographs using Kodak Ektachrome 160T or 4 0 Fuj icolor Pro fess iona l 4 0 0 N P H fi lm. D. Time-lapse cinemicrography: H A - c o a t e d surfaces smooth, 3, 10, 30u.m depths and 5u;m depth-100|nn pitch were used. Individual sur faces were mounted on glass sl ides and placed in a Pentz chamber (Bachofer, Reut l ingen, W. Germany) which was placed in a stage incubator (Bachofer)at 37°C, with perfused 9 5 % air, 5% C 0 2 , and v iewed with reflected-light differential-interference-contrast optics. A pentium I P C computer with Northern Ecl ipse software (Empix, M iss issauga , ON) was used to capture images from a colour video camera (Sony powerHAD 3 C C D ) mounted on a Reichert microscope. A PowerTower Pro 180 computer with Sc ion Image software (1.62) was also used to capture images from a Hamamatsu video camera (model C2400-07) . Both sets of images were edited by Sc ion Image software (1.62) on the PowerTower and made into Quickt ime supplier mov ies . For the movie following nodule development a 5)o,m depth-100|j,m pitch sur face was used , fol lowing four weeks culture with mineral izat ion supplements, tetracycline was added for an addit ional week. The tetracycline becomes incorporated into the nodule and images could be taken of nodules formed on the surface using the Princeton PentaMax 12 bit C C D camera at 20x magnification with UV light (488nm). 41 E. Tetracycline Incorporation: To quantify mineral ized t issue 5u,g/ml of tetracycl ine was added to the media supplements of L-ascorbic acid phosphate and N a - 3 -glycerol-phosphate. At the end of four weeks the sur faces were fixed with 10% buffered formalin for 1 hr at room temperature. After rinsing with disti l led water the sur faces were dehydrated (75%-100), c leared in xylene and mounted in Entellan (EM Science) . Mineral ized t issue was observed and nodules counted under UV light (488nm) at 20x magnification on a Ze iss ASIO-2-Motor ized Stage microscope with a Princeton PentaMax 12 bit C C D camera. The nodules counted were an aggregation of cel ls, producing a multi-layer that were at least 35u,m in diameter and f luoresced. Pictures were also taken of smooth and grooved surfaces and all smooth gaps. In addition the pictures were used to determine the percentage of a nodule's area located on surface features, using NIH Image s o f t w a r e . F. Alkaline Phosphatase: Alkal ine phosphatase activity was measured , using a diazonium coupl ing reaction (Figure 4), for 2 and 3 weeks only, s ince Alk-P is highest at those times (Jaiswal et al, 1997). Procedures were those recommended by S igma diagnostics using Fast Blue R R Salt (Sigma 4 2 OH F i g u r e 4 . Schematic diagram of Alk -P reaction. T h e r e a c t i o n b e t w e e n s u b s t r a t e a n d d i a z o n i u m c a t i o n ( F a s t B l u e R R Sa l t ) ( K i e r n a n , 1 9 8 1 ) . 4 3 diagnostics, St. Louis, M O , USA) and Naphthol A S - M X phosphate alkaline solution was used as the substrate solution from S igma alkal ine phosphatase kit (No. 85L-2). No counter stain was used. The alkal ine-dye mixture was adjusted to pH 9.6 which was found to be optimum for this study. Pictures were taken on a CEI pentium computer with Sony powerHAD 3 C C D colour video camera at 16x magnif ication with Northern ecl ipse software (Empix) and the areas that reacted with the dye were quantified on a Powertower Pro 180 with Sc ion Image software. H. Correlation of Tetracycline and Alk-P activity. Using the method for quantifying mineral ized nodules, 10|iim and smooth, HA and Ti coated micromachining substrata were cultured for 3 weeks with mineralization supplements. At the end of 3 weeks the cultures were stained using the A lk -P activity method and mounted with glycerol. Pictures were taken using U.V. light (488nm) on a confocal laser scanning microscopy (MC80 , Zeiss) with Fujicolor Profess ional 4 0 0 N P H film at 20x magni f icat ion. 3. Stat ist ics: S P S S statistical software was used for the analys is of all data. Ana lys is of var iance (ANOVA) and Student -Newman-Keu ls test to identify condi t ions that differed signif icantly (p< 0.05). 4 4 III. R e s u l t s 4 5 In the following sect ions of this thesis the results are presented in the sequence: the characterizat ion of sur faces, responses of osteogenic cel ls to micromachined sur faces in terms of cell morphology, col lagen organizat ion, bone-l ike nodule product ion, a lkal ine phosphatase activity, and the correlat ion between tetracycl ine incorporat ion and A l k -P activity. 1. Characterization of Sputter coated HA surfaces: A. X-Ray diffraction: X-ray diffraction analysis provided by Dr. W R Lacef ield using techniques for the calc ium phosphate coated substrata indicated that in fact a HA structure was present on the coatings. (Figure 5). Peaks were observed at 24°, 32°, and 33° at 2o that are representative of HA. B. Surface roughness: A profilometer was used to determine roughness of the HA and Ti coated "smooth" surfaces. The mean roughness (R ) values obtained were HA=2.8nm and Ti=1.3nm. However these values were lower than the accepted resolution of the profi lometer (5nm, Wie land personal communication) and thus can be considered as smooth at least as assessed by the usual standards applicable in implantology. Figure 6 shows profilometer printouts of a 10|xm deep HA and Ti coated surfaces. From the profiles of the grooves the depth and pitch can be observed and were determined to be =10>m deep with =47u.m pitch, confirming the design 4 6 counts/s •2Theta F i g u r e 5 . X-Ray diffraction of HA coated micromachined substrata. T h e p e a k s at 2 4 ° , 3 2 ° , a n d 3 3 ° d e g r e e s a r e a r e p r e s e n t a t i v e of H A . ( D e g r e e s 2 o ) . 4 7 n4''14 n2-2R ID # UERT. SOOkfl dftu 10J .2kA k f l . 120 .RuQ 101 .4kfl TIR 0 . fi Ra 0 . fl L 310.Oum R 310.Oum 40 A r e a 0 . 0 0 0 0 SCAN MENU 1 um s-'urn 2000 .2 1 400 1 80 5 25 SCAN f - 8 s e c H I R . < — STYLUS 9mq - 4 0 ! ..ill!.. 2 5 0 0 3 8 0 u m LEUEL. A 05^01 11 :04 IB # • UERT. 200kR my l o i . i m Aug 101 .3kf l TIR 0 . A Ra 0 . A HORIZ lOOum O.OOum L 243.Oum R 243.Oum kA 120 30 40 A r e a 0 . 0 0 0 0 SCAN MENU 1 um S''um 2000 .2 1 400 1 80 5 25 SCAN t~ 8 s e c D I R . < — STYLUS 9mg 0 164um LEUEL -40 "22:5 B •i i" 3 0 0 u m i i : i i : i i : i i : I I : i i ii; :• /' : : : / : ! . . . . ! . . . / ' \ i / ; . . \ : . / I I : i ii V i t_ _ ..«•: (•••{•••' • • ; I I , : I I i i : ; V ,'j \ , i i " ; F i g u r e 6. Surface profiles of substrata. P r o f i l o m e t e r p r in tou ts of a) H A a n d b) T i c o a t e d m i c r o m a c h i n e d s u b s t r a t u m . T h e la rge p e a k s a r e p ro f i l es of the 10>m d e e p g r o o v e s c o a t e d wi th H A a n d T i . D e p t h =10pm a n d p i tch =47|am. N o t e that the s h a p e s of the p ro f i l es d o not c o n f o r m to the t o p o g r a p h y b e c a u s e of the ef fect of s t y l us s i z e (12.5p,m) w h i c h s m o o t h s out the r e c o r d of the t o p o g r a p h y at the g r o o v e e d g e s . 4 8 specif icat ions. Furthermore both Ti and HA had the same topography, thus the HA coating was uniform and had little affect on the pitch or depth of these subst ra ta . C. Scanning Electron Microscopy (SEM): S E M observations of the substrata are given in figure 7. A smal l difference was noticed between the HA and Ti sur faces in ridge width. A s a result of the H A coating the ridges of the H A substrata were 4u.m wide, whereas the Ti sur faces had ridges of 5u.m width. A c ross sect ional view of the substrata was a lso obtained (figure 8) and the H A coating was observed to be uniform, dense and approximately 1u.m thick. 2. Osteogenic cell cultures: A. SEM: After 24hrs of culture, osteoblasts on smooth HA exhibited no preferred orientation. (Figure 9). In contrast osteoblasts on grooves were found to be al igned with the grooves. At 1 week and 2 weeks of culture it was difficult to determine cell orientation because the cel ls had become densely confluent and cell outlines were not clear. Moreover in 2 week cultures aggregat ions of cel ls and globular structures were seen (figure 10), indicating possib le areas of nodule formation. Simi lar results were observed on Ti substrata. Osteoblasts cultured for four weeks were examined for nodule 4 9 A B F i g u r e 7. A , B ) Scanning electron micrographs of substrata. M i c r o g r a p h s pr io r to b e i n g u s e d in cu l tu re . A ) H A s u r f a c e . B ) T i s u r f a c e , no t i ce the s m a l l but d e t e c t a b l e d i f f e rence in r idge t h i c k n e s s . 50 F i g u r e 8. Scanning electron micrograph of a sputtered HA coat ing (<->). D e b r i s p r e s u m a b l y f o r m e d d u r i n g f rac tu re of the s u r f a c e (<-). 51 B F i g u r e 9 . A , B ) Osteoblasts cultured for 24hours on HA coated substrata. A ) O n the s m o o t h s u r f a c e , o s t e o b l a s t s w e r e a r r a n g e d r a n d o m l y (x47) . B) 3 0 p m d e e p s u r f a c e , o s t e o b l a s t s d e m o n s t r a t e d a l i g n m e n t w i th g r o o v e s a n d c e l l s w e r e e l o n g a t e d ( x77 ) . S i m i l a r resu l t s w e r e o b s e r v e d o n T i s u b s t r a t a . 5 2 F i g u r e 10 . S c a n n i n g e l e c t r o n m i c r o g r a p h o f t w o w e e k c u l t u r e . O s t e o b l a s t s c u l t u r e d o n a 3LUTI d e e p H A c o a t e d m i c r o m a c h i n e d s u b s t r a t u m . G l o b u l a r s t r u c t u r e s a r e e v i d e n t in a g g r e g a t e s , p o s s i b l y i n d i c a t i n g a r e a s w h e r e n o d u l e f o r m a t i o n is b e g i n n i n g . ( x153 ) . S i m i l a r resu l ts w e r e o b s e r v e d o n d e e p e r s u b s t r a t a a n d on T i c o a t e d s u b s t r a t a . (<->, r e p r e s e n t s g r o o v e d i r e c t i o n ) . 5 3 production. A representative nodule seen on HA and Ti substrata is shown in figure 11 A. Globular structures, similar to those reported in the literature to be mineral ized (Davies et al, 1988b; Chehroudi et al, 1992; Okumura etal, 1997) were observed and some osteoblasts were attached to these globular structures. (Figure 11 A) . To observe the under structure of the nodules cel lophane tape was attached to the nodule and then stripped from the surface. In the area underneath the removed nodule, globular structures (presumably mineral ized accret ions) were found, and g lobules interdigitated with fibres that were most likely col lagen. (Figure 11B). 3. Collagen Organization: A. Picro-sirius Red Staining: A s the substrata were complex and had the possibil ity of producing artificial optical effects, picro-sir ius red staining was done on the surfaces in the absence of cel ls. No stain was observed on either HA or Ti substrata, but the ridges did show some brightness resulting from reflected light. (Figure 12). Cultures form 24hrs to six weeks were ana lyzed and found to show an increase in col lagen production indicated by the amount of birefringent red stain. Figure 13 shows this increased amount in col lagen after 2 weeks and 6 weeks culture on HA substrata. As depth increased 5 4 F i g u r e 11. N o d u l e s o n m i c r o m a c h i n e d s u b s t r a t a . A , B ) O s t e o b l a s t s c u l t u r e d for 4 w e e k s o n H A c o a t e d m i c r o m a c h i n e d s u b s t r a t a . A ) A nodu le f o r m e d o n the 30 |am d e e p s u b s t r a t u m . M i n e r a l i z e d g l o b u l e s (*) a n d o s t e o b l a s t s (<-) a re e v i d e n t ( x70 ) . B ) A n a r e a of cu l t u re w h e r e part of a n o d u l e h a s b e e n s t r i p p e d off u s i n g c e l l o p h a n e t a p e . M i n e r a l i z e d g l o b u l e s in te rd ig i ta ted wi th p r e s u m p t i v e c o l l a g e n f i b res ( « - ) f o u n d at the b a s e of a n o d u l e ( x 6 9 6 ) . S i m i l a r resu l t s w e r e o b s e r v e d o n a l l d e p t h s a n d s u r f a c e s . 5 5 F i g u r e 1 2 . Reflected polarized light micrograph of HA surface stained with picro-sirius red. H A c o a t e d 1 0 p . m d e e p m i c r o m a c h i n e d s u b s t r a t u m u s e d a s the c o n t r o l . N o c e l l s w e r e p r e s e n t but the s u b s t r a t u m w a s i n c u b a t e d for 1.5 w e e k s w i th cu l t u re m e d i u m . N o s ta i n w a s o b s e r v e d o n th is H A con t ro l s u r f a c e nor w a s t he re s t a i n o n a T i s u r f a c e t rea ted i d e n t i c a l l y . ( x 2 0 ) 5 6 F i g u r e 1 3 . Orientation of collagen on grooved substrata. P i c r o - s i r i u s s t a i n i n g of o s t e o g e n i c c e l l s c u l t u r e d o n H A s u b s t r a t a . A & B ) 2 w e e k cu l tu re , 3 n m d e e p , s h o w i n g l e s s a l i g n m e n t in g r o o v e s a n d l e s s c o l l a g e n than C & D . A) 0° d e g r e e . B ) 4 5 ° d e g r e e rotat ion of s a m e a r e a a s A . C & D ) 6 w e e k cu l tu re , 30p.m d e e p , s h o w i n g m o r e a l i g n m e n t of c o l l a g e n in g r o o v e s (<-) t han in g a p . C ) 0° d e g r e e . D) 4 5 ° d e g r e e rotat ion of s a m e a r e a a s C , a pos i t i on in w h i c h b i re f r i ngen t s t r u c t u r e s a l i g n e d wi th the g r o o v e s a r e e x t i n g u i s h e d . S i m i l a r r esu l t s w e r e f o u n d o n T i s u b s t r a t a . ( x20) 5 7 the orientation of col lagen became parallel to the grooves, as is evident by comparing figures 13A&B and 13C&D. By a 45° degree rotation of the field shown in figure 13A&C, the col lagen that was not evident in figures 13B&D. By the same token col lagen fibres evident in figure 13B&D are not evident in figure 13A&C. Furthermore in the smooth gap areas of the 3u,m deep surfaces, col lagen was found to be oriented perpendicular to the grooves as well as diagonally within the gaps. (Figure 13A&B). The col lagen orientation in the gaps of the 30u.m sur faces was similar to that observed on 3|am deep surfaces. (Figure 13 Gaps) . Similar results were observed on Ti substrata (data not shown). 4. Nodule Quantification: A. Nodule counts: The HA and Ti substrata without cel ls were tested to insure that the light that is emitted from the surfaces was not an optical artifact. The results d isplayed in figure 14 show no f luorescence under U.V. light. The appearance of a mineral ized nodule labelled with tetracycline examined under U.V. light is shown in figure 15. A lso v isual ized (figure 15) are small (< 35u.m) globules label led with tetracycl ine that were too small to be counted as discrete mineral ized nodules. The numbers of nodules on smooth and grooved surface 5 8 B F i g u r e 14 . T e t r a c y c l i n e c o n t r o l s . A & B ) . C e l l s w e r e not p r e s e n t bu t t h e s u b s t r a t a ( 1 0 u m d e e p ) w e r e i n c u b a t e d for 1.5 w e e k s wi th m i n e r a l i z a t i o n s u p p l e m e n t s a n d t e t r a c y c l i n e in the cu l tu re m e d i u m . F l u o r e s c e n c e u n d e r U . V . l ight ( 4 8 8 n m ) w a s not o b s e r v e d . A ) H A s u r f a c e . B) T i s u r f a c e . 59 Figure 15. Tetracycline labelled nodule. Osteoblasts cultured on micromachined substrata for 4 weeks with mineralization supplements and tetracycline. A nodule observed under U.V. light (488nm) on a HA coated surface. Arrows represent globules labelled with tetracycline that were too small to be counted as discrete mineralized nodules. 6 0 topographies are given in figure 16. HA coated grooves were found to produce significantly more mineral ized nodules at all depths than Ti coated grooves. (Table 1). The number of nodules increased with depth of grooves with the 30jxm deep surface was found to produce more nodules then shal lower depths on both HA and Ti surfaces. When the experiment was repeated similar results were obtained. It should be noted that this experiment is a two factor factorial design with the factors being type of surface at two levels (HA & Ti) and the second factor being depth of grooves at four levels (smooth, 3, 10, 30u,m). The dependent variable is the number of nodules. Such a design enables one to test for interactions between the factors, in this experiment the interaction concerns whether the effect of coating (HA & Ti) varies depending on the topography of the surface. The statistical analysis of the data indicated that the effects of coating and depth were both significant (p< 0.01). Moreover the interaction effect was also significant (p< 0.05). (Table 1). The same analysis was applied to the HA coated smooth gaps which produced significantly more nodules then the Ti coated smooth gaps. (Figure 17). Signif icant dif ferences were a lso observed between patterns and between sur faces. (Table 2) but no signif icant interaction effect between pattern and surface was observed. The 50u.m gap and 30u,m depth 61 CM E E tn o 3 T3 O 3 2 3 oH 2 8H 26 A 2 44 2 2H 2 oH 1 8 H 1 6 H 1 4^ 1 2H 1 O H Smooth 3 1 0 Depths (\xm) F i g u r e 16 . Nodule counts on grooved substrata. A s i g n i f i c a n t d i f f e r e n c e in n o d u l e c o u n t s w a s f o u n d b e t w e e n H A a n d T i g r o o v e s , a n d s ign i f i can t d i f f e r e n c e s w e r e a l s o o b s e r v e d b e t w e e n d e p t h s (p<0.05) . O s t e o b l a s t s cu l t u red o n m i c r o m a c h i n e d s u b s t r a t a for 4 w e e k s wi th m i n e r a l i z a t i o n s u p p l e m e n t s a n d t e t r acyc l i ne . N u m b e r of n o d u l e s (± S t d . D e v . ) in the g r o o v e d a r e a s . R e p e a t e x p e r i m e n t s s h o w e d a s im i l a r pa t te rn . 6 2 * * * A N A L Y S I S O F V A R I A N C E NODOOUNT b y DEPTH SURFACE UNIQUE sums o f squares A l l e f f e c t s en t e r ed s i m u l t a n e o u s l y Source o f V a r i a t i o n M a i n E f f e c t s DEPTH SURFACE Sum o f Squares 580011574 464544753 115466821 DF Mean Square 4 145002893.506 3 154848251.014 1 115466820.981 S i g F o f F 80.785 .00 86.270 .00 64.330 .000 2-Way I n t e r a c t i o n s DEPTH SURFACE 18321759 18321759 3 6107253.088 3 6107253.088 3.403 .017 3.403 .017 E x p l a i n e d R e s i d u a l 598333333 7 85476190.470 47.621 .00 2283148148 1272 1794927.789 T o t a l 2881481481 1279 2252917.499 1280 cases were p roces sed . 0 cases (.0 pe t ) were m i s s i n g . T a b l e 1. Analysis of-Variance of the grooved areas. N o d c o u n t ( N o d u l e c o u n t s ) . S u r f a c e , (Ti a n d H A c o a t i n g s ) . D e p t h , ( s m o o t h , 3 , 10 , 30>m d e e p ) . S i g n i f i c a n t d i f f e r e n c e s w e r e o b s e r v e d by t ype of s u r f a c e a n d d e p t h s . T h e r e w a s a s ign i f i can t d i f f e r e n c e in in te rac t ion b e t w e e n s u r f a c e a n d d e p t h . 6 3 E E 5 0 n m -yt— l O O u r - O - - 1 5 0 n r -jgjj 2 0 0 n r S m o o t h 3 * 1 O Depths ( | i m ) - t r " T -S5S 1 I 3 O E E ~c75 _g> 2 0 T 3 O 1 s . . . . j ^ . . - . 5 o \x m — MS— 1 0 0 n r r — - o - • 1 5 0 n r r — (gf— 2 0 0 n r r J - 1 -i 1 o Depths (pm) B I i 3 0 F i g u r e 17 . Nodule counts on smooth gaps. A , B ) O s t e o b l a s t s c u l t u r e d o n m i c r o m a c h i n e d s u b s t r a t a for 4 w e e k s wi th m i n e r a l i z a t i o n s u p p l e m e n t s a n d t e t r a c y c l i n e . N o d u l e s c o u n t e d in the a r e a s of s m o o t h g a p s s u r r o u n d e d by g r o o v e s . A s ign i f i can t d i f f e rence w a s o b s e r v e d b e t w e e n H A a n d T i , b e t w e e n d e p t h s , a n d b e t w e e n g a p s i z e s (50 , 1 0 0 , 1 5 0 , 2 0 0 u m ) (p<0.05) . A ) H A s m o o t h g a p s , the 50um g a p in the 30um d e e p g r o o v e s p r o d u c e d the m o s t n o d u l e s . B ) T i s m o o t h g a p s 5 0 u m g a p in the 30p,m d e e p g r o o v e s p r o d u c e d the m o s t n o d u l e s . R e p e a t e x p e r i m e n t s s h o w e d s im i l a r pa t t e rns , s e e tab le 3. T h e r e w a s c o n s i d e r a b l e va r i a t i on in the n u m b e r of n o d u l e s c o u n t e d in the g a p s . (E r ro r b a r s r e p r e s e n t s t a n d a r d e r ro r s ) . 6 4 * * * A N A L Y S I S O F V A R I A N C E * * * COUNTS b y PATTERN SURFACE UNIQUE sums o f squares A l l e f f e c t s en t e r ed s i m u l t a n e o u s l y Source o f V a r i a t i o n M a i n E f f e c t s PATTERN SURFACE Sum o f Squares 336400694 322555556 13845138 DF Mean Square 13 25876976.432 19.224 12 26879629.627 19.969 1 13845138.091 10.286 S i g o f F .00 .00 .001 2-Way I n t e r a c t i o n s PATTERN SURFACE 19898148 12 1658179.012 1.232 .256 19898148 12 1658179.012 1.232 .256 E x p l a i n e d R e s i d u a l 362509259 1041836420 25 14500370.369 10.773 .000 774 1346041.886 T o t a l 1404345679 799 1757629.135 800 cases were p roces sed . 0 cases (.0 pe t ) were m i s s i n g . Table 2. Analys is of Variance of nodule counts in the smooth gaps. Counts, (nodule counts). Surface, (Ti and HA coatings). Pattern, (gap size at each depth). Significant differences observed by type of surface and pattern, but no significant difference in the interaction between surface and pattern. 6 5 was found to have more nodules than other depths and gap sizes on both HA and Ti surfaces. (Figure 17). Considerably more variation was noted in the data gathered from the gaps, possibly because only relatively small areas were available for counting. When repeated, the experiment showed similar results. (Table 3). B. % Area of Nodule on Ridge: To determine if nodules were being formed preferentially on the ridges or within the grooves, the relative areas of the nodule located on the ridge and groove were measured. Table 4 shows the percentage of a nodule that lies on a ridge. The results showed no significant difference between HA and Ti, nor was there a significant difference found among depths. However the least percentage area of a nodule that was located on the ridge was on deeper grooves. This is what would be expected if the formation of a nodule was favoured by the microenvironment formed as a result of restricted diffusion in the grooves. 5. Alkaline Phosphatase Activity: A. Optimum pH: The optimal pH had to be established for the enzymatic reaction for the cells used in this study. In two experiments the optimum pH was observed to be 9.6. (Figure 18). B. Controls: 66 Nodule Formation on HA & Ti Smooth Ga ) S ( N o d u l e s / m m 2 + S t d . Dev.) Surface Type Smooth Smooth Gaps HA 14.6+2.9 50>m 10(him 150nm 200nm Ti 10.7+1.4 HA 3nm 21.1+9.5 14.4+10.9 14.4+10.3 13.9+8.7 HA 10>m 24.4+12.2 17.8+9.8 13.9±9.5 10.6+8.4 HA 30>m 26.1+11.0 17.8+12.7 13.3+10.6 13.9+8.7 Ti 3[xm 16.1+13.7 12.8+8.3 15.0+10.4 12.2+11.3 Ti 10[im 10.6+9.9 13.3+10.6 10.6+8.4 13.9+12.4 Ti 30^m 16.1+11.1 15.0+9.7 17.2+13.2 10.6+8.4 T a b l e 3 . Nodule formation on HA & Ti smooth gaps. R e p e a t e d e x p e r i m e n t of o s t e o b l a s t s cu l t u red o n m i c r o m a c h i n e d s u b s t r a t a for 4 w e e k s wi th m i n e r a l i z a t i o n s u p p l e m e n t s a n d te t racyc l i ne . N o d u l e s w e r e c o u n t e d in the s m o o t h g a p s . T a b l e s h o w e d the 50\xm g a p 3 0 u m d e e p H A c o a t e d s u r f a c e p r o d u c e d m o r e n o d u l e s / m m 2 . 6 7 % of Nodule on R i d g e (%± std. Dev.) D e p t h H A T i 3Lim 23+9 20+7 10um 22+9 19+8 30| im 20+11 1 8 ± 5 T a b l e 4 . P e r c e n t o f N o d u l e o n r i d g e . O s t e o b l a s t s c u l t u r e d o n m i c r o m a c h i n e d s u b s t r a t a for 4 w e e k s wi th m i n e r a l i z a t i o n s u p p l e m e n t s a n d t e t r acyc l i ne . A r e a s of n o d u l e s l o c a t e d o n the r idge w e r e c a l c u l a t e d u s i n g N I H Image so f twa re . T a b l e s h o w s a t rend that n o d u l e s p re fe r the g r o o v e s o v e r the r i dge , but no s ta t i s t i ca l s i g n i f i c a n c e . (p>0.05) . 6 8 0.1 1 PH F i g u r e 18 . The effects of pH on Alk-P activity. O s t e o b l a s t s w e r e c u l t u r e d o n s m o o t h T i s u b s t r a t a for 2 w e e k s wi th m i n e r a l i z a t i o n s u p p l e m e n t s . A l k - P ac t i v i t y w a s d e t e r m i n e u s i n g naph tho l A S - M X p h o s p h a t e a s the subs t ra te a n d F a s t B l u e R R a s the d i a z o n i u m d y e . % A r e a s of A l k - P a c t i v i t y / m m 2 w e r e c a l c u l a t e d f r om i m a g e s u s i n g N I H I m a g e s o f t w a r e . T h e two e x p e r i m e n t s s h o w resu l t s of p H 9.6 a s o p t i m u m . (E r ro r b a r s r e p r e s e n t s t a n d a r d er ror ) . 6 9 To insure that the reaction is specif ic to A l k -P a number of controls were done including the fol lowing: a) sur faces incubated in media alone (ie. no cel ls) , b) epithelial cel ls on sur faces (epithelial cel ls lack this enzymat ic ability), c) Ti smooth controls plated with osteoblasts but no substrate was added, d) the cel ls were heat treated, and e) the pH reduced to 5.0 to ensure that acid phosphatase was not being detected. No stain was observed in any of these five control condit ions. (Figure 19). Using the reaction solution at a pH of 9.6 on osteoblasts cultured on Ti smooth sur faces, a positive reaction was visible and clear differences between the controls and pH 9.6 were evident. (Figure 19). C. Alk-P activity: Alk -P activity on HA and Ti coated grooved substrata was found to have significantly more % area of posit ive A l k -P act iv i ty /mm 2 on HA than Ti grooved substrata for both 2 week cultures and 3 week cultures. (Figure 20). Furthermore there was a signif icant effect of depth of the grooves but no signif icance in the interaction between depths and surface. Similar to the data for nodule formation, there was an increase in A l k -P activity as depth increased. (Figure 20). It should be noted that the % area of positive A lk -P act iv i ty /mm 2 dec reases from the 2 week to 3 week cultures. When A lk -P activity was examined in the smooth gaps a 7 0 A B E F Figure 19. Alkal ine phosphatase activity. A,B,C,D,E,F) . A&B) 5p.m deep lOO^m pitch HA coated surfaces. A) Control with no cells present; stain was not deposited on HA. B) Control with epithelial cells that do not exhibit Alk-P were cultured on the HA surface for 2.5 weeks, no staining was observed. C,D,E,F) Osteoblasts cultured on Ti smooth substrata. C) Control with no substrate added. D) Control with osteoblasts heat-treated using a steam bath for 10 minutes prior to staining for Alk-P activity. No positive reaction observed. E) Control for pH. Staining for Alk-P activity at a pH of 5.0. No positive reaction observed. F) Staining Alk-P activity at a pH of 9.6. Noticeable positive staining reaction. 71 0.24 1 I Smooth Depths (nm) 1.44 0.24 Smooth B Depths (|im) F i g u r e 2 0 . Alkal ine phosphatase activity on grooved substrata. A , B ) A l k a l i n e p h o s p h a t a s e act iv i ty e x p r e s s e d a s a p e r c e n t a g e of a r e a s t a i n e d pos i t i ve by o s t e o b l a s t s cu l tu red o n m i c r o m a c h i n e d s u b s t r a t a . O b s e r v a t i o n s m a d e o n g r o o v e d a r e a s . A s ign i f i can t d i f f e rence w a s f o u n d b e t w e e n H A a n d T i , a n d s ign i f i can t d i f f e rences w e r e a l s o o b s e r v e d b e t w e e n d e p t h s (p<0.05) . A ) 2 w e e k c u l t u r e s . B) 3 w e e k c u l t u r e s , no t i ce a d e c r e a s e in A I K - P act iv i ty . R e p e a t e x p e r i m e n t s s h o w e d s i m i l a r p a t t e r n s . (E r ro r b a r s r e p r e s e n t s t a n d a r d e r ro r ) . 7 2 significant difference was observed between HA and Ti substrata for both 2 weeks and 3 week cultures. (Figure 21). Furthermore significant dif ferences were observed among the different depths. A s with the nodule data, there was considerably more variation in the data from the gaps but gap s izes. This was observed in both 2 and 3 weeks and on both HA and Ti sur faces. Furthermore, general ly there was an increase in A l k -P activity as the depth of the micromachined grooves increased. (Figure 21). A lso evident from figure 21 was a decrease in A l k -P activity between weeks 2 and 3. 6. Correlation of Tetracycline Incorporation and Alk-P activity: The correlation between A lk -P activity and the nodule counts was also examined. (Figures 22 & 23). Examining the data from the grooved areas revealed an excellent correlation (r=0.958) between the two methods where the A l k -P activity levels at 2 weeks were high but a lower correlation was observed in the 3 weeks data when the A l k -P activity had decreased. (Figure 22). The correlation was not as high for the gaps and the pattern was different in that the 3 week A l k -P cultures demonstrated a higher correlation then the 2 week A lk -P cultures. (Figure 23). To demonstrate the relationship of mineral izat ion and A l k - P activity the A l k -P stain was used on cultures label led with tetracycl ine. most often the 50 pm^gap s ize had more A lk -P activity then all the other 7 3 1.85 1.6 .2 1.35 o 0. Q. 1.1 0.6 0.35 0.1 Smooth 3 i i i 1 0 Depths (nm) - H - i 30 1.85 1.35 o 0. a < 0.85H ^ , 0.35 1.85 1.6 >1.35 o a- 1.1 a. ,0.85 a> < 0.6 0.35 0.1 — A — 50nm -•Jfr- 100nm - - o - - I 50nm —H— 200nm Smooth 3 1 0 Depths (nm) B 1.85 0.35 - A — 5 0 n m lOOnm •O--- 150nm -BS— 20 0nm tr Smooth 3 —i— 1 0 —i— 30 Smooth 3 10 3 0 Depths (nm) Depths (nm) C D F i g u r e 2 1 . Alkal ine phosphatase activity on smooth gaps. A , B , C , D ) A l k a l i n e p h o s p h a t a s e act iv i ty e x p r e s s e d a s a p e r c e n t a g e of a r e a s t a i n e d pos i t i ve by o s t e o b l a s t s cu l tu red o n m i c r o m a c h i n e d s u b s t r a t a . D a t a o b s e r v e d f rom the s m o o t h g a p s l o c a t e d wi th in the g r o o v e s . A s ign i f i can t d i f f e rence w a s f o u n d b e t w e e n H A a n d T i , a n d s ign i f i can t d i f f e r e n c e s w e r e a l s o o b s e r v e d b e t w e e n d e p t h s , a n d b e t w e e n g a p s i z e s (50 , 1 0 0 , 1 5 0 , 2 0 0 n m ) (p<0.05) . A l l s h o w e d that 50|am pa t te rn a n d 3 0 um d e p t h h a d m o r e A l k - P ac t iv i ty . A & B ) H A s u b s t r a t a . A ) C u l t u r e d for 2 w e e k s . B) C u l t u r e d for 3 w e e k s , s h o w i n g l e s s A l k - P ac t iv i ty . C & D ) T i s u b s t r a t a . C ) C u l t u r e d for 2 w e e k s . D) C u l t u r e d for 3 w e e k s , s h o w i n g l e s s A l k - P ac t iv i ty . ( E r r o r b a r s r e p r e s e n t s t a n d a r d e r ro r ) . 7 4 0.25 0.5 0 .75 1 1.25 1.5 % Area Alk-P Positive F i g u r e 22. Correla t ion between A l k - P & Tetracycl ine (grooves). A c o r r e l a t i o n g r a p h of n o d u l e s c o u n t e d wi th % a r e a A l k - P act iv i ty in the g r o o v e d a r e a s . A l o w e r co r re la t i on w a s o b s e r v e d at 3 w e e k c u l t u r e s . R e p e a t e d e x p e r i m e n t s o b s e r v e d s i m i l a r r esu l t s . (E r ro r b a r s r e p r e s e n t s t a n d a r d e r ro r ) . 7 5 1.75 % Area Alk-P Positive F i g u r e 2 3 . Correlat ion between A l k - P & Tetracycl ine (smooth gaps). A c o r r e l a t i o n g r a p h of n o d u l e s c o u n t e d wi th % a r e a A l k - P act iv i ty in the s m o o t h g a p s w i th in the g r o o v e s . L o w e r co r re la t i on t h a n g r o o v e s f igure 2 2 a n d l ower at 2 w e e k s w a s o b s e r v e d . R e p e a t e d e x p e r i m e n t s o b s e r v e d s i m i l a r r esu l t s . (E r ro r b a r s not s h o w n b e c a u s e of the h i g h va r i a t i on ) . 7 6 Since the stain was able to be seen under the same U.V. light (488nm) ev idence of the relationship was il lustrated in figure 24 which showed that the tetracycl ine labell ing could be found in the vicinity of A l k -P a c t i v i t y . 7. Time-Lapse Cinemicrography: This study was done to investigate osteoblast behaviour on HA sur faces using differential interference optics and v ideo microscopy. The videos showed osteoblasts assuming an elongated shape on grooved surfaces, and after one week osteoblasts located in the gaps were al igned with the grooves forming the border of the gaps. (Figure 25). At 2 weeks, however, some cel ls were observed that were not al igned with the grooves as the culture became more dense and multi-layers formed. (Figure 25F). In addition, observat ions were made on cel ls cultured on a 5u.m deep lOOpm pitch HA coated surface to follow the formation of a nodule. The resulting video showed that osteoblasts in the periphery of the nodule apparently exper ienced mitosis more frequently than osteoblasts adjacent to the nodule, but there were insufficient observat ions for statist ical analysis. To confirm that the aggregation of cel ls was an actual nodule, tetracycline labell ing was preformed for an addit ional week. It can be seen that tetracycl ine was incorporated into this nodule figure 26B. 7 7 F i g u r e 2 4 . A l k - P & T e t r a c y c l i n e l a b e l l e d n o d u l e s . O s t e o b l a s t s c u l t u r e d o n m i c r o m a c h i n e d s u b s t r a t a for 3 w e e k s wi th m i n e r a l i z a t i o n s u p p l e m e n t s a n d t e t r a c y c l i n e . S t a i n e d for A l k - P ac t iv i ty . O b s e r v e d u n d e r U . V . l ight ( 4 8 8 n m ) . N o t i c e the n o d u l e s f l u o r e s c i n g a n d the p u r p l e - b l u e s t a i n of A l k - P act iv i ty in the v ic in i ty of t he n o d u l e s (<-). A , B ) H A c o a t e d s u b s t r a t a . A ) S m o o t h . B) 1 0 u m d e e p g r o o v e d . 7 8 A B E F Figure 25. Time-lapse series. Time-lapse differential interference contrast micrographs of osteoblasts cultured on 3\xm deep micromachined substratum. A) 24hrs. B) 4 days. C) 1 week. D) 8 days. E) 10 days. F) 2 weeks. 79 B F i g u r e 2 6 . Development of tetracycline labelling of a nodule during one week. O s t e o b l a s t s cu l t u red o n H A c o a t e d m i c r o m a c h i n e d s u r f a c e , 5 u m d e e p , 1 0 0 | i m p i t ch . A) D i f fe ren t ia l i n t e r f e r e n c e c o n t r a s t t i m e - l a p s e m o v i e m a d e for 4 w e e k s w i th m i n e r a l i z a t i o n s u p p l e m e n t s . B) T h e s a m e a r e a w a s p h o t o g r a p h e d u n d e r U . V . l ight af ter a n add i t i ona l w e e k wi th t e t r acyc l i ne in the m e d i u m . T e t r a c y c l i n e h a s b e e n i n c o r p o r a t e d into the a r e a s of the ce l l a g g r e g a t e i nd i ca t i ng it is a d e v e l o p i n g n o d u l e . 8 0 IV . D i s c u s s i o n 81 This thesis used micromachined surfaces coated with HA and Ti to investigate os teogenes is in vitro. Although the precise mechan isms of mineral izat ion are still under invest igat ion, this thes is provides ev idence to support a role for material surface properties in the production of bone- l ike t issue in cell culture. Many propert ies of biomaterials such as their chemistry, crystall inity, and topography affect cel lular responses in vitro and in vivo (Boyan etal, 1993; Massas etal, 1993; Dubois etal, 1998; Ong etal, 1998; Brunette and Chehroudi , 1999). The topography of the surface affects the production of bone-l ike nodules on Ti surfaces in vivo (Chehroudi et al, 1992), and in vitro (Brunette, 1988; Brunette e r a / , 1991; Boyan etal, 1993; Brunette and Chehroudi , 1999), on cell culture plates (Davies and Matsuda, 1988b) and on HA surfaces (Chang et al, 1999). Moreover it has been found that more nodule formation is assoc ia ted with HA surfaces than with Ti sur faces (Massas et al, 1993). This thesis investigated the effects of well def ined topographies, the use of HA, and their interactions on nodule production in vitro. A summary of the results of this thesis f o l l o w s : 1) A thin, dense coat ing of hydroxyapatite resulted in signif icantly more bone-l ike nodules than were formed on a titanium coat ing. 2) A grooved, micromachined topography produced an increased number of 8 2 bone-l ike nodules. 3) An interaction was found between topography and chemistry. The increase in nodule formation produced using a HA coating was greatest when assoc ia ted with the deepest surface feature of topography. 4) Alkal ine phosphatase, an enzyme thought to be involved in the initiation of mineral ization, may be a good leading indicator of the amount of nodule product ion in vitro. 5) The finding that the deepest surface feature produced the most nodules is consistent with the concept that a development of a suitable microenvironment enhances mineral ized t issue product ion. 6) The results regarding the role of col lagen orientation in formation of nodules were equivocal . More nodules were found on grooved surfaces than on smooth surfaces, and the col lagen was al igned with these grooves. However, similar numbers of nodules were found in smooth gaps on the substrata where there was no obvious alignment of co l lagen. The following components of the study will now be d iscussed : hydroxyapatite coat ing, the effects of topography on cel l orientation, the combined effects of topography and chemistry on mineral ized nodule production, alkal ine phosphatase as a mineral ization marker, and col lagen involvement in minera l izat ion. HA coatings: 8 3 Scann ing electron microscopy and surface roughness measurements indicated that although there was a small (1u.m) increase in ridge width, the depth of surface features did not differ between micromachined Ti and HA surfaces. The topographies of the HA and Ti surfaces were considered to be identical for practical purposes s ince the change in width was small and the depth of feature has consistently been a more important factor than lateral spac ings in determining cell behaviour (Chehroudi and Brunette, 1995). Furthermore the HA coating was found to be uniform and dense, a result confirmed by other studies using sputter coating methods (Jansen etal, 1993; Wolke, 1997; Wolke e r a / , 1998). The degree of crystallinity of HA could not be definitely determined because standards establ ished for thin HA films of known crystall inity are not avai lable (Lacef ie ld ; personal communicat ion) , however, x-ray diffraction clearly demonstrated that HA crystals were present (figure 5). Effects of Topography on cell orientation: It has been shown that surface topography affects cel l orientation (Brunette, 1986; 1988; Brunette etal, 1991; Chehroudi and Brunette, 1995; Ratkay, 1995; Qu et al, 1996). In this thesis the contact-guided cell locomotion seen on HA-coated grooved (figure 25) sur faces was similar to the observat ions of Qu et al (1996), in which the osteoblast- l ike cel ls orientated their long axis with the grooves, and elongated in the direction 8 4 of the grooves on Ti substrata. Orientation of osteoblasts has also been related to the orientation of a naturally occurr ing substrate (Basle et al, 1998). Bas le etal (1998) observed human osteoblast- l ike cel ls , al igned with col lagen fibers on xenogenic bone biomaterial. Thus , my results are in agreement with numerous studies showing that cell orientation is affected by topography (Brunette and Chehroudi , 1999). Effects of topography and chemistry on mineralized nodule product ion: It has been previously observed that microfabricated grooved Ti substrata enhance the production of mineral ized nodules (Brunette, 1988; Brunette etal, 1991; Chehroudi etal, 1992; Chehroudi and Brunette, 1995) a finding that was confirmed in this study. On average, approximately twenty three percent more bone-l ike nodules were found on HA than on Ti coated substrata. The increase in nodules ranged from 12% to 29%, thus HA produced approximately 2.5 times more nodules on the deepest groove relative to a smooth surface. These results are in general agreement with the work by Morgan etal (1996), who found that HA sur faces were assoc ia ted with 8 t imes more mineral formation than sandblasted Ti sur faces. The difference in results could be attributable to their use of a different cell line (UMR 106-01 B S P ) at 2.5 t imes the plating density than that employed in my study. In addition they used a p lasma-sprayed HA 8 5 coating and measured elevated mineral formation rather than nodules. Similarly, Verca igne et al (1998), who also used a p lasma sprayed HA coated Ti implant, found in an in vivo study in goats that the percentage of bone contact was 2.5 times greater on HA implants compared to Ti . These two studies as well as my results, conform to the general rule that HA surfaces increase mineral ization compared to Ti sur faces (Kay, 1993; Soba l le , 1993). Resul ts from this study showed that regardless of coat ing, sur faces with the deepest grooves (30p:m) and narrowest gaps (50u.m) produced more mineral ized nodules than shal lower grooves and wider gaps. Studies by Ratkay (1995) and Chehroudi e r a / (1992; 1997) found similar results. Ratkay, using the same cell populations as used in this study, showed that 30u,m deep grooves on Ti coated micromachined surfaces produced more bone-l ike nodules than smooth surfaces, although she used a plating density twice that used in this thesis. The topographies she used were also different in that the two groove depths were 19u.m and 30u.m with a pitch of 39u.m. Furthermore, she used L-ascorbic acid in the supplements and replaced the media three times a week and used Von K o s s a staining to a s s e s s bone-l ike nodules. An in vivo study in rats, by Chehroudi et al (1992), using Ti coated micromachined implants, found that mineral izat ion t issue (assessed by 86 t ransmission electron microscopy (TEM)) was adjacent to deep grooved surfaces (30(xm) but not with smooth sur faces. Another rat study by Chehroudi et al (1997), also using Ti coated micromachined surfaces and assess ing the bone index by radiographs and computer image processing, found that in the range 30pm to 120|nm deep, the bone-l ike foci formation decreased as the depth of grooves increased. A study by Khakbaznejad (2000, in preparation), used the same topography as this thesis with the same cell populations and found that smooth gaps produced more bone-like nodules than the grooves, and the production of nodules decreased as depth of feature increased. The contradictory results between Khakbazne jad and this thesis could be attributed to the numerous variat ions in methods such as cell density, supplements, feeding frequency, and nodule assessment . Khakbaznejad used twice the plating density than that used in this thesis. Furthermore he used L-ascorbic acid in supplements which were fed three times a week, and bone-like nodules were a s s e s s e d by Von K o s s a staining rather than tetracycl ine label l ing. It, thus, appears that bone-l ike nodule formation in response to topography is very sensi t ive to the specif ic culture condit ions. Nevertheless, the condit ions used in this thesis produced results that mimicked the performance of implants in vivo in that HA increased mineral ized t issue production as did microfabricated grooved topographies. 8 7 A novel finding in this study was that an interaction was found between topography and surface chemistry (p< 0.05). That is, the increase in nodule production on HA surfaces was greater than the amounts expected by adding the effects of HA and groove depth. A s far as is known, an interaction between chemistry and microfabricated topographies in the production of bone-l ike t issue in vitro has not previously been reported. Deep grooves (30u.m) and narrow gaps (50u,m) have confined areas in which diffusion would be expected to be restricted, therefore it is possib le that the concentrat ion of regulatory factors in these areas differs from that found in the bulk medium. Such a situation could be cal led a microenvironment and indeed this possibil i ty was proposed by Chehroud i et al (1992). It is possible that the combination of deep micromachined surfaces and HA would allow regulatory factors to achieve concentrat ions that promote mineral ized t issue formation (Chehroudi et al, 1992; Chehroudi and Brunette, 1995). Another possibil i ty is that the microenvironment found in HA substrata contains higher levels of C a 2 + and P 0 4 " 3 ions which are released from the HA (Morgan et al, 1996; Keller, 1998) . In this thesis the long acting ascorbate analogue, L-ascorbic ac id-2-phosphate (Asc-P) , was used. L-ascorbic ac id-2-phosphate has been 8 8 used in previous studies (Hitomi et al, 1992; Ja iswal et al, 1997). Ja iswal et al (1997), using mesenchymal stem cel ls plated at one hundredth of the density of that used in this thesis on t issue culture plates, found that more mineral ized area (as a s s e s s e d by Von K o s s a staining) was seen with A s c - P than L-ascorbic ac id . These results are in agreement with my prel iminary tests that indicated that L-ascorb ic -2-phosphate produced more mineral ized t issue than L-ascorbic acid (data not shown). Moreover, Ja iswal etal (1997), suggested that the increase in mineral ized area that they observed in cultures using A s c - P , was the result of changing the media only twice a week rather than three t imes. They bel ieved that the less frequent media changes al lowed the cultures to concentrate their soluble products in the media. Alkaline Phosphatase: Alkal ine phosphatase plays a crucial role in the initiation of mineralization but is not needed for the enlargement of bone nodules (Bel lows etal, 1991; 1992). Moreover, there is general ly a greater increase in A l k -P activity on HA surfaces compared to Ti sur faces (Massas et al, 1993; O z a w a and Kasugai , 1996), as was observed in this thesis. It has been suggested that cultures on HA surfaces have greater A lk -P activity than those on Ti , because there is increased growth and differentiation of osteoblasts on HA substrata (Massas et al, 1993; O z a w a 8 9 and Kasuga i , 1996). In this thesis I found that the percent area of A lk -P increased with groove depth on Ti and HA surfaces. The importance of substrata topography in mineral izat ion in vitro was a lso observed by Schwartz et al (1996). Furthermore they have shown that both growth and differentiation are affected by topography. Their study used chondrocyte cell populations at a density ten t imes less than that used in this thesis. They employed solid Ti disks that differed in surface roughness and found that course a grit blasted surface had a higher A l k -P activity than fine grit b lasted. Moreover, the importance of topography in mineral ization in vitro was also observed on ceramic substrata by Dubois etal (1998). They measured the specif ic A l k -P activity in behaviour of bone cel ls on g lass, plastic and ceramic and found that ceramic had higher A l k -P activity. These studies, together with the results in this thes is , show that A l k -P activity is affected by the topography and chemistry of the surface (Kieswetter et al, 1996). It has been suggested by Aubin and Turksen (1996) that A lk -P is found in certain stages of differentiation of the osteogenic l ineage seen in cultures. Although absent in the earliest s tages (mesenchymal stem cel ls and early osteoprogenitor cells) and the final stage (osteocytes), A l k -P is found in the intermediate s tages (late osteoprogeni tor ce l ls , pre-osteoblasts and osteoblasts). A study by Sammons et al (1994), who 9 0 plated similar cell populations as used in this thesis on a dense, irregular coating of HA, found that A l k -P activity increased within two weeks and decreased thereafter. My results agreed, both on the smooth as well as the grooved Ti and HA surfaces. Similarly, a study by M a s s a s etal (1993) who used pure HA substrata also found an increase in A l k -P activity within 2 weeks and a decrease thereafter. In addit ion, Ja iswal et al (1997) who used mesenchymal stem cel ls plated on t issue culture plates also found an increase within 2 weeks fol lowed by a decrease, within 3 weeks, in A l k -P activity. In this thesis a subsequent decrease of A l k -P was seen at three weeks which may result from the onset of mineral izat ion at which time the progression of mineral izat ion proceeds independent ly of A l k -P activity (Bel lows etal, 1991; 1992). Furthermore, the onset of mineral izat ion correlates with the maturation of osteoblasts (Aubin and Turksen, 1996) which leads to a decrease in proliferation and differentiation (Schwartz et al, 1993; 1996; Sammons et al, 1994; Ja iswal et al, 1997). Thus my results conform to what is general ly observed with A l k -P activity in vitro. A lk -P is thought to be involved in the initiation of mineral izat ion by releasing phosphate from organic phosphate (Bel lows etal, 1991; 1992). The activity of A l k -P was measured in this thesis in order to determine whether it correlated with nodule production in vitro. The correlation 91 between A l k -P activity at two weeks and mineral ized nodule production at 6 weeks was highest (r=.958) on grooved areas. Studies by Ballanti et al (1995) a lso found a good correlation between A lk -P activity and bone formation (r=.784), using human transil iac bone biopsies and the same two methods as this thesis (tetracycline for bone labell ing and an azo-d iazonium reaction for A l k -P activity). Moreover A l k - P activity in the vicinity of mineral ized area (figure 24), was a lso noted by Ballanti et al (1993). In these studies, A lk -P seems to be a good leading indicator of subsequent mineral ization on various substrata. Thus , in the future it would be feasible to screen surfaces for their nodule production at 6 weeks by using A lk -P at 2 weeks thus saving time and expense. Collagen involvement in mineralization: Resul ts presented in this thesis demonstrated that col lagen was present on the underside of a mineral ized nodule, and was interdigitated with mineral ized globules, similar to the findings of Dav ies and Matsuda (1988b) who used cel l populations similar to this thesis on bioactive glass. In addit ion, there were col lagen fibers al igned with the grooves on Ti and HA surfaces. The orientation of the col lagen fibers may be important s ince speci f ic orientations of col lagen in vivo are assoc ia ted with certain stages of bone production (Schenk and Buser , 1998). Mineral izat ion is initiated by an extracel luar matrix of which col lagen is 9 2 the major component (Veis, 1993). Surface topography can affect the orientation of col lagen fibers deposi ted by cel ls (Brunette, 1986b; Davies and Matsuda, 1988b; Chehroudi and Brunette, 1995; Bas le etal, 1998). As the structural matrix macromolecules (such as col lagen) define the shape and structure of the mineral izing compartment (Veis, 1993). Sur face topography could inf luence mineral izat ion through its effects on col lagen o r i en ta t i on . It should be noted, however, that in this study col lagen orientation was similar on HA and Ti coated grooved substrata, therefore, other factors must be responsible for the dif ferences in bone nodule formation between the substrata. However, for both HA and Ti substrata, nodule production did roughly correlate with col lagen orientation. That is, most nodules were found where the col lagen was al igned best (i.e. grooves), an intermediate number of nodules were found in the smooth gaps where there was some preferred orientation of col lagen f ibers, and lower numbers of nodules were found on smooth surfaces where the col lagen fibers had no preferred orientation. Thus, my results suggest that col lagen orientation may have some role in promoting nodule formation; but strong support for this possibi l i ty would require the quantif ication of col lagen al ignment so that a quantitative correlat ion could be establ ished. 9 3 V. Future Work 94 My data shows that bone-l ike nodule formation increases with increasing depth of surface feature. One possible explanat ion is that the deep grooves restrict diffusion of regulatory and other molecu les, thus promoting the development of a microenvironment within the grooves that differs from the bulk medium in the culture. Other studies have used the concepts of restricted diffusion (Miura and Shiota, 2000) and microenvi ronment (Schmeiche l et al, 1998) as explanat ions for cellular behaviour in chondrogenic pattern formation (Miura and Shiota , 2000) or in breast epithelial cell phenotypes (Schmeichel et al, 1998). Components of such a microenvironment could be soluble, i.e in the media, or cel l -assoc ia ted or be related to the extracel luar matrix. A limitation of the ability of this thesis to support the concept of microenvironment is that no direct measurements of the contents of the media within the grooves were done. One approach to extend the findings of this thesis, therefore, would be to sample the media within the grooves (where the microenvironment is thought to develop) and compare it to media sampled well above the grooves and to media sampled from close to the smooth sur faces. Of particular interest would be the concentrat ions of molecules assoc ia ted with bone production, such as the growth factors transforming growth factor-3 (TGF-3) and bone morphogenet ic protein-7 (BMP-7) , which have both been studied extensively in the field of bone 9 5 biology (Bostrom et al, 1999). For example, if concentrat ions of T G F - 3 , estimated by using an EL ISA technique, were found to be elevated in the grooves, an increase in bone formation would be expected (Bostrom et al, 1999). Furthermore, by observing the amount of mineral ized t issue produced and comparing the levels of growth factors, it might be possible to determine the regulatory factors that play an important role in mineral izat ion. If a difference in the concentrat ion of growth factors (i.e T G F - 3 and BMP-7) was found, a related approach would be to use such data to alter the media on smooth surfaces so that the levels of growth factors were the same as those found within the grooves, and then determine whether there was a similar increased production of nodules. It would be expected that if concentrat ions of growth factors (i.e T G F - 3 and BMP-7 ) were in the range of 1u,g to 10ng for T G F - 3 and 3.13u,g of B M P - 7 (Bostrom etal, 1999), then production of nodules on smooth surfaces would increase. The difference between the study proposed here and standard assays of growth factor effects is that the concentrat ions of growth factors employed in the proposed study would be specif ied by measurements of levels of regulatory factors in the microenvironment where bone-l ike t issue formation in cultures was enhanced . Another area where more information is required is the effect of substrata topography and chemistry in the differentiation of osteogenic 9 6 cel ls. A s the differentiation sequence of osteogenic cel ls advances from mesenchymal stem cel ls to osteocytes, the cel ls acquire express ions of several known markers (i.e. alkaline phosphatase and osteocalcin) (Aubin and Turksen, 1996). In the Aubin and Turksen (1996) model, markers for mesenchymal stem cel ls and early osteoprogenitor cel ls are not known. However, late osteoprogenitor cel ls express A l k - P , at the pre-osteoblast stage osteopontin and A lk -P are expressed, and at the osteoblast stage A l k -P , osteopontin, osteocalc in, and bone sialoprotein are expressed and finally at the osteocyte stage osteopontin, os teocalc in , and bone sialoprotein are expressed. This model of osteogenic l ineage that Aubin and Turksen (1996) suggested may be examined using grooved and smooth sur faces, as well as HA coated sur faces, to determine whether specif ic s tages of differentiation are affected by substratum topography. The immunolabell ing methods used on fetal rat calvar ia by Aubin and Turksen (1996) on cultures on grooved and smooth surfaces could be used sequential ly over a defined time period to compare the appearance of markers assoc ia ted with bone-l ike t issue production such as osteocalc in , osteopontin and bone sialoprotein. One might expect that differentiation would be accelerated on grooved surfaces, therefore the number of cel ls exhibit ing osteocalc in , for example, would be greater at earl ier t imes of culture on grooves compared to smooth surfaces, possibly leading to an 9 7 early onset of mineral izat ion, resulting in formation of more nodules. Another approach to manipulating the microenvironment would be to alter feeding frequencies (i.e. media changes). By changing media f requencies, the concentrat ions of soluble products would be expected to be altered. For example, an increase in feeding frequency might be expected to disrupt the development of a microenvironment within the grooves, and thus restrict nodule production to similar levels to those found on smooth surfaces. It was a lso found that there was an interaction between topography and chemistry in the formation of mineral ized nodules. However, it is not known if this interaction is speci f ic to the part icular topography employed in this thesis. To investigate this interaction further, one approach would be to change the V-shaped grooves to grooves with vertical walls at various depths using both Ti and HA coat ings. In changing the shape of the grooves, a greater surface area is created, al lowing more attachment area for cel ls (Brunette, 1988). The increased surface area produced by changing groove shape might be expected to have effects similar to increasing surface area by increasing groove depth as in V-shaped grooves. Surface area could also be increased by increasing the number of grooves. Thus, exploring the effects of changing the shape and number of the grooves would allow for the relationship of surface area 9 8 and surface geometry to be investigated. Another aspect of this thesis that warrants further explorat ion is the process of the development of a nodule. With the use of tetracycline and the appropriate blue light (Frost, 1968), differential interference optics and video microscopy, it is possible to observe the growth of a nodule from the early stages up to full development. 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