Dentistry, Faculty of
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UBCV
Ratkay, Julia
Master of Science - MSc
University of British Columbia
Since the use of osseointegrated implants became common in both dentistry and orthopaedics, researchers have been investigating the problem of improving stabilization of the implant. It is widely accepted that the solution lies in improving the biomaterial interface. Implants with various surface designs and chemical compositions have been investigated at the experimental and clinical levels. Micromachining, a technique widely used i n microelectronics, has the capability to produce surface topographies with precise dimensions and high reproducibility. This allows the testing of the effect of implant surface topography on osseointegration in controlled experiments. These experiments aimed to test the ability of micromachined surfaces to promote bone formation in vitro. A recently developed osteogenic culture was optimized and characterized, in which rat calvarial cells were cultured in the presence of ascorbic acid and organic phosphate in the medium. This method provided a reliable model of reproducible mineralized tissue formation. Histochemical methods, TEM, S EM and EDX techniques demonstrated that the mineralized tissue formed after two weeks culturing had an elevated alkalinephosphatase activity, was rich in collagen, and had a structure, similar to the original calvarial bone. The ability of micromachined surfaces to facilitate osteogenesis was studied when these osteogenic cultures were grown on grooved, pitted, and smooth micromachined surfaces. The developed mineralized tissue was stained utilizing von Kossa's technique and its amount quantified with an UltroScan X L Laser Densitometer. The area, shape and orientation of the mineralized bone colonies were measured with a Videoplan Image Analysis System (Zeiss). There was a statistically significant difference of bone formation between the micromachined and smooth surfaces. Bone-like tissue formed to the greatest extent on pitted surfaces, and to a lesser extent on grooves, but few bone nodules were found on the smooth control surfaces. The colonies on the grooved surfaces were elongated and followed the direction of the grooves, while those on the smooth and pitted surfaces were rounder and randomly oriented. The results indicate that bone-like tissue formation is influenced by the substratum surface topography and micromachining may be useful i n determining the ideal surface topography for promoting bone formation on titanium surfaces.
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THE E F F E C T OF MICROMACHINED SURFACES O N OSTEOGENESIS IN VITRO by JUL IA RATKAY D.M.D., Semmelweis University Medical School, Budapest, 1986 A THESIS SUBMITTED IN PARTIAL FULF ILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF(.^^9'E:;;IW'. DENTAL SCIENCE in T H E FACULTY OF GRADUATE STUDIES (Department of Oral Biology) We accept this thesis as conforming tathe required standard THE UNIVERSITY OF BRITISH COLUMBIA April, 1995 © Julia Ratkay, 1995 In presenting t h i s thesis i n p a r t i a l f u l f i l l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Oval (LdoUo^ The University of B r i t i s h Columbia Vancouver, Canada Date A p i / c f i 2.6. . Abstract Since the use of osseointegrated implants became common in both dentistry and orthopaedics, researchers have been investigating the problem of improving stabilization of the implant. It is widely accepted that the solution lies in improving the biomaterial interface. Implants with various surface designs and chemical compositions have been investigated at the experimental and clinical levels. Micromachining, a technique widely used in microelectronics, has the capability to produce surface topographies with precise dimensions and high reproducibility. This allows the testing of the effect of implant surface topography on osseointegration in controlled experiments. These experiments aimed to test the ability of micromachined surfaces to promote bone formation in vitro. A recently developed osteogenic culture was optimized and characterized, in which rat calvarial cells were cultured in the presence of ascorbic acid and organic phosphate in the medium. This method provided a reliable model of reproducible mineralized tissue formation. Histochemical methods, T E M , S E M and EDX techniques demonstrated that the mineralized tissue formed after two weeks culturing had an elevated alkaline-phosphatase activity, was rich in collagen, and had a structure, similar to the original calvarial bone. i i The ability of micromachined surfaces to facilitate osteogenesis was studied when these osteogenic cultures were grown on grooved, pitted, and smooth micromachined surfaces. The developed mineralized tissue was stained utilizing von Kossa's technique and its amount quantified with an UltroScan X L Laser Densitometer. The area, shape and orientation of the mineralized bone colonies were measured with a Videoplan Image Analysis System (Zeiss). There was a statistically significant difference of bone formation between the micromachined and smooth surfaces. Bone-like tissue formed to the greatest extent on pitted surfaces, and to a lesser extent on grooves, but few bone nodules were found on the smooth control surfaces. The colonies on the grooved surfaces were elongated and followed the direction of the grooves, while those on the smooth and pitted surfaces were rounder and randomly oriented. The results indicate that bone-like tissue formation is influenced by the substratum surface topography and micromachining may be useful in determining the ideal surface topography for promoting bone formation on titanium surfaces. i i i Table of contents Abstract i i Table of contents iv List of tables vi List of figures vi i Acknowledgements v i i i I. INTRODUCTION 1 1. Overview 2 2. Characteristics of mineralized tissue 3 3. Culture of osteogenic cell populations 13 4. Effect of surface topography on cell behaviour in vitro 15 5. Surface topography and mineralization 19 6. Objective of the thesis 21 II. MATERIALS AND METHODS : 22 1. Substrates 23 A. Micromachining 23 B. Preparation of replicas 24 C. Surface patterns 24 D. Design and fabrication of substrates containing replicas . . . 25 E. Cleaning and preparation for cell culture experiments 26 2. Cell Cultures 31 A. Calvaria osteogenic cell culture 31 B. Calvaria explant culture 35 3. Qualitative methods 40 A. Tissue processing and microscopic techniques 40 B. Fluorescence microscopy 40 C. Transmission electron microscopy (TEM) 41 D. Scanning electron microscopy (SEM) 42 E. Energy dispersive X-ray (EDX) analysis 42 F. Time-lapse cinemicrography 43 4. Quantitative methods 43 A. Laser densitometry 43 B. Videoplan image analysis 43 B . l . Area analysis 43 iv B.2. Two-dimensional shape analysis 44 B.3. Orientation analysis 44 5. Statistics 44 III. RESULTS 47 1. Characterization of bone formation 48 A. Optimization of culture conditions 48 A. 1. Cell population 48 A. 2. Population density 48 A. 3. Culture passage number 49 A.4. Culture conditions 49 A. 5. Time course 49 B. Nodule formation and mineralization 51 B. l Histochemistry 51 von Kossa's method 51 Alkaline phosphatase activity 51 Van Gieson staining . . 57 B.2 Transmission electron microscopy 57 B.3 Scanning electron microscopy 57 B. 4 Fluorescence microscopy 58 2. Effect of surface topography on bone-like tissue formation 67 A. Surface preparation 67 B. Behaviour of osteoblasts on micromachined surfaces 67 C. In vitro experiments on micromachined surfaces 70 C. l Calvaria osteogenic cell culture 70 C.2 Calvaria explant culture 75 3. Quantitative studies on the effect of surface topography on bone-like tissue formation 87 A. Laser densitometry 87 B. Video image analysis 88 IV. DISCUSSION 94 V. FUTURE WORK 103 VI. BIBLIOGRAPHY 105 v List of Tables Table 1 50 Table 2 91 Table 3 92 Table 4 93 vi List of Figures Figure 1 28 Figure 2 30 Figure 3 34 Figure 4 37 Figure 5 39 Figure 6 46 Figure 7 54 Figure 8 56 Figure 9 60 Figure 10 62 Figure 11 64 Figure 12 66 Figure 13 69 Figure 14 74 Figure 15 78 Figure 16 80 Figure 17 82 Figure 18 84 Figure 19 86 Figure 20 90 vi i Acknowledgement I wish to thank my supervisor, Dr. Donald M. Brunette for his support throughout my graduate studies and for his understanding of the different priorities I had after the birth of my first and second children during these years. He proved to be a reasonable godfather of these "projects". I would like to express my appreciation to my thesis committee members, Drs V.-Jukka Uitto and Bing Wu, for their constructive participation. I am indebted for the help of Dr. Babak Chehroudi, who lent his expertise arising from the related projects in this laboratory, he accomplished. I am especially grateful for his help regarding the scanning electron microscopy work. I also thank Dr. Carol Oakley for the help with fluorescence microscopy. I also thank Mrs. Lesley Weston and Mr. Andre Wong for technical instructions and help in the cell culturing methods, ultramicrotomy, electron microscopy and photomicrography. I would like to acknowledge the assistance and expertise of the Department of Electrical Engineering, UBC and in particular, Mr. Hiroshi Kato who fabricated the silicon micromachined surfaces. I am thankful for the technical work of Mr. Michael Weiss (Department of Zoology , UBC), who is responsible for the Energy Dispersive X-ray analysis. Most importantly I have to thank my whole family for their support, help and survival both during the experiments and the thesis writing. The experiments of this thesis were supported by the Brit ish Columbia Health Care Research Foundation. vi i i I. INTRODUCTION 1. Overview Although implant-like devices have been used since ancient Egyptian times, implantology as a scientific discipline is only a generation old. An implant is a device surgically inserted in the tissue to add or substitute for a function of that tissue. The more frequently employed implants include cardiovascular indications such as heart valves, joint prostheses, artificial lenses and corneas, mammary prostheses and dental implants, each of them exceeding a quarter of a million applications per year (Hench, 1980). Implants used in the oral cavity include subperiosteally, submucosally and endosseally located cylindrical, blade and mandibular shaped implants (Chehroudi, 1991). The dental implants used most commonly are pergingival devices that penetrate through oral epithelium. A successful implant has to be non-toxic, cause minimal foreign-body reaction and integrate into the tissue. This integration is necessary to prevent infection and epithelial downgrowth, the two main causes of percutaneous implant failures. Osseointegrated implants (anchored and integrated in the bone) demonstrate the necessary elements for a mechanically stable device. However, there is insufficient scientific data on what constitutes the optimal surface of an implant for maximal osseointegration and I attempt to approach this problem in this thesis. 2 2. Characteristics of mineralized tissue In the process of osseointegration new mineralized tissue has to be deposited around the implant. This tissue resembles bone, as it has similar components and substructure, however it may not necessarily have the structural features such as osteons or trabecular systems, characteristic of bone. In this thesis I wil l use the terms bone, bony tissue and bone-like tissue as synonyms to describe the mineralized tissue that forms adjacent to the biomaterial surfaces. In the following pages I shortly introduce the components of this bony tissue and the process involved in its formation. Organic Bone Matrix A monolayer of osteoblasts on the surface of the bone is responsible for the deposition of organic matrix. These osteogenic cells are rich in rough endoplasmic reticulum and mitochondria, consistent with active protein synthesis. Once the osteoblasts have secreted bone matrix around themselves, they become entrapped in it and differentiate into osteocytes. The deposited organic matrix - osteoid - calcifies subsequently. About 95% of this organic matrix is collagenous protein, another 4% are proteoglycans, containing chondroitin-4-sulphate and chondroitin-6-sulphate glycosaminoglycans (Sammon & Thomas, 1981), and the remaining 1% consists of non-collagenous proteins. 3 The backbone of the organic matrix is collagen, a structural protein having at least 15 different types (Uitto & Larjava, 1991). Most types consists of three polypeptide chains rich in proline and hydroxyproline. These chains develop a triple helical conformation. As the collagen matures inter- and intramolecular webs of cross-links develop. Transformation of procollagen into collagen is a cell membrane-associated event. Once collagen is secreted, it associates into fibrils extracellularly. Type I collagen is the major constituent of the organic matrix in the form of a fibrous web, providing structural integrity for the matrix. It is suggested to be a nucleator in the formation of hydroxyapatite crystals, which event would start in the gap region of collagen fibres (Sodek et al., 1991). However, there are also several minor collagenous components in the bone: types VI ( cell to collagen binding), V, X, XI, XII (associated with type I, cross bridges between collagen fibrils, binds proteoglycan) (Niyibizi & Eyre, 1989, reviewed by Uitto & Larjava, 1991). Interestingly no type III collagen or fibronectin (a widely distributed non-collagenous matrix adhesion protein) was found in significant amount (Sodek & Mandell, 1982; Linde, 1982, 1989). Proteoglycans exist in bone as two classes of small proteoglycans (byglycan or PGI and decorin or bone PGII; Fisher & Termine, 1985; Stanford & Keller, 1991), differing somewhat in the protein core and the number of attached chondroitin sulphate chains. Immunohistological characterization showed little tissue specificity of PGII (Day et al., 1986), but there are possible functional differences. While these proteoglycans interact the same way with collagen fibrils as other tissue PGs (Triffit, 1987), they appear to be involved in the inhibition of calcification processes (Howells & Pita , 1976). Several families of non-collagenous proteins (NCP) have been described in bone. NCPs are mostly polyanions which bond Ca++ ions strongly, thus they likely participate in the mineralization process, although their exact function is still not clarified (Glimcher, 1989). The best known phosphoprotein in bone is osteonectin (SPARC in dentin), a glycoprotein which has affinity to both collagen and hydroxyapatite and in vitro causes mineralization of type I collagen (Termine et al., 1981a.; Termine et al., 1981b; Romberg et al., 1985; Termine, 1985). However, osteonectin is not bone specific, being produced by fibroblasts (Young et al., 1986) and also found in several types of tissue (Malaval et al, 1987), indicating it might not be involved in mineralization. Sialoproteins, another family of NCPs characterized by their sialic acid content and calcium binding abilities, include osteopontin, renamed secreted phosphoprotein I (SPP-I), and bone sialoprotein (BSP) (Triffitt, 1987; Linde, 1989; Kasugai et al., 1991; Mikuni-Takagaki et al., 1995). Osteopontin/bone SPP-I has been suggested to bridge between bone cells and hydroxyapatite, but the protein was found not to be specific for bone. It seems that tissue specificity is provided through post-translational modification such as sulphation of bone SPP-I (reviewed by Sodek et al, 1991). BSP appears to be mineralized tissue specific and described as a possible nucleator of HA formation (Glimcher, 1989). The bone constituent of y-carboxyglutamic acid (bone Gla protein or osteocalcin) is a vitamin K dependent carboxylation product, with high bone 5 specificity (Gundberg et al., 1984; Price, 1985). It is the most abundant non-collagenous protein contributing 15% of total NCP in rats and immunohistologically it was only detected in osteoblasts, osteocytes and calcified bone matrix. Another Gla-containing protein was characterized in bone matrix in association with bone morphogenetic protein (matrix Gla-protein) (Price et al., 1983; Price & Williamson, 1985). Osteocalcin is suggested to inhibit hydroxyapatite formation and it is necessary for osteoclastic resorption. However, the percentage of osteocalcin found in human bone is low, only 1% of the total NCPs (reviewed by Sodek et al., 1991). Another calcium-binding protein in mineralized tissues is calbindin-D9k Balmain, 1991). Calbindin-D9k is present in osteoblasts and osteocytes and it is found in matrix vesicles. This vitamin-D-dependent protein is suggested to be a participant of mineral nucleation. Bone also contains serum proteins, such as albumin, however it is unlikely that it is produced by osteoblasts (Linde, 1989). Numerous other enzymes and growth factors are found in bone that may have important roles in the mineralization process as nucleator / promoter / inhibitor or cell regulators, chemotactic, differentiation and mitogenic agents (Mohan & Baylink, 1991). However, they wil l not be described in detail here, as they fall outside the scope of this thesis. Without attempting to be comprehensive, growth factors associated with bone matrix formation include transforming growth factor (TGF)-fli and -B2 (Centrella et al, 1994), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), insulin-like growth factors (IGF) I and II, bone morphogenic proteins (BMPs) (Wozney et al., 1988) 6 and osteoinductive factor (Seyedin, 1989; Mohan & Baylink, 1991). TGF-13, the most abundant of these factors in bone matrix stimulates matrix protein synthesis and proliferation of osteoblasts (Bonewald & Mundy, 1990). Bone matrix associated enzymes include collagen matrix degrading collagenases and cathepsins, alkaline phosphatase and proteinase. Messenger RNA of cytokines IL-lfi and IL-6 were also found to be expressed in developing human bone (Doddse^a/., 1994). Inorganic Bone Matrix and Mineralization 70% of the weight of the bone is provided by inorganic matrix, primarily in the form of hydroxyapatite (Caio(P04)e(OH)2). The precise mechanism of the mineralization process, in which the organic matrix becomes calcified by inorganic calcium phosphate deposition is still not clarified. However, it might involve both cellular and physicochemical mechanisms (Glimcher, 1984). Matrix vesicles are secreted by osteoblasts containing amorphous matrix, non-collagenous proteins, alkaline phosphatase and ATP-ase. These vesicles are associated with primary bone formation and calcification and thought to transport their content towards the mineralization line (Boyan el al., 1993). There is a 10-20 ^m unmineralized matrix between the mineralization front and the osteoblasts. Matrix vesicles may be used by the cells to transport material through this distance to the mineralization front, to trigger nucleation of mineral crystals (Fortuna et al, 1980). The anionic non-collagenous peptides attach to the collagen and might serve as nucleating sites by entrapping Ca2+ ions (Linde, 1989). However, matrix vesicles are not necessarily present on the 7 sites of osteoid calcification (Boskey, 1985). A new theory suggests that around the mineralization front in the absence of alkaline phosphatase (inhibitor of phosphorylation) casein kinase II phosphorylates matrix proteins, which might initiate mineralization. This event would be facilitated by newly differentiated osteocytes, which show low alkaline phosphatase and high casein kinase activity (Mikuni-Takagaki et al., 1995). Crystal growth follows, resulting in hydroxyapatite crystals, arranged parallel to the collagen fibres of the matrix with close contact/binding to it. Histochemical techniques used as markers for osteogenic tissue There are markers which can be tested for to determine i f bony tissue is deposited in vivo around an implant or in vitro on the culture surface. A simplified criteria for osteogenesis includes (a) cells showing osteoblastic morphology and function; (b) collagen (type I) deposition in E C M ; (c) mineralization. In preliminary studies I selected and optimized the methods to be used for characterization of osteogenesis in my in vitro experiments. Bone cells produce organic matrix that undergoes a mineralization process to become bone. The primary bone cells participating in bone production are osteoblasts. As osteoblasts do not express any known surface marker molecules which would unequivocally distinguish them from other cell types, their phenotype can be established only by examining a combination of morphological and functional features. Light and electron microscopy shows that osteoblasts are basophilic, cuboidal, rich in rough endothelial reticulum 8 and mitochondria, have a vesicle-rich Golgi apparatus, numerous microtubules and microfilaments, consistent of active protein synthesis (Holtrop, 1990). The primary function of osteoblasts is bone formation. Nevertheless, bone production is not exclusively related to cells of well defined osteogenic lineage. Some cells of other tissues may undergo dedifferentiation and subsequently differentiate into osteogenic cells as a reaction to inducing factors and produce bone-like tissue at tissue locations where bone formation is not normally found (ectopic ossification) (Cole & Cohen, 1990). Characteristically active osteoblasts also exhibit high alkaline phosphatase activity. Alkaline phosphatase (AP), associated with the cell membrane, occurs in many types of cells, including osteoblasts (McComb, 1979). However, AP activity is absent in osteocytes which makes AP an excellent differentiation marker for osteogenic cells (Mikuni-Takagaki et al., 1995). Alkaline phosphatase (AP) was thought to have an activation role in the initial matrix mineralization, however it turned out that AP might actually inhibit phosphorylation of matrix proteins by phosphoprotein kinases (Mikuni-Takagaki et al., 1995). AP activity can be measured by detecting the end product of the enzyme-substrate reaction. A P catalyzes the hydrolysis of phosphoric acid esters with an optimum pH around 8.0 (Kiernan, 1981), thus the monobasic sodium salt of a-naphthyl phosphate can serve as enzyme substrate. In the detection of AP activity the organic product of hydrolysis (a-naphthol) can be trapped by 9 diazonium salts, resulting in an insoluble, coloured azo-dye (diazonium coupling method). A phosphate ester of naphthol-AS can also be used as substrate. Naphthol-AS forms more brightly coloured azo-dyes, albeit more slowly than a-naphthol. In a pilot study to establish the optimal conditions for A P detection by the diazo-coupling method, I examined different tissue preservation techniques (formalin, ethanol and acetone fixation; Gruber, 1988), substrates (Naphthol AS-BI and Naphthol AS-TR phosphates), diazonium salts (Fast Red Violet LB , Fast Red TR, Fast Blue RR, Fast Garnet GBR, Fast Black K, Pararosanilin, and New Fuchsin). For my purposes of AP activity detection in mineralizing tissue cultures I selected a method employing formalin fixation of the cultures and staining with Fast Red Violet LB diazonium salt and Naphthol AS-BI phosphate as enzyme substrate (see Materials and Methods section). Theoretically the most specific way for the detection of collagens could be immunohistology. However, commercially available antibodies against collagen (especially type I) are not necessarily reliable, as they are usually not tested for immunohistochemical use, and may cross-react with other types of extracellular matrix proteins (Coil, 1993). Histochemically collagen can be detected by a mixture of anionic dyes which differentially stain collagen and cytoplasm (Kiernan, 1981). In the trichrome procedure two or more anionic dyes can be combined with heteropolyacid treatment. This results in selective colouring of collagen, however cartilage and secretory granules can stain similarly. This method is 10 highly sensitive for fine collagenous fibres, but complicated and hard to interpret as coloration varies with any slight change in procedure. The scientific background of the mechanism whereby collagen stains specifically is not well understood. Theories of the differential staining of cytoplasm and collagen include permeability differences for different sizes of dyes; heteropolyacids make the collagen negatively charged thus stainable by cationic dyes; or different binding mechanisms of heteropolyacids in cytoplasm and collagen. Unfortunately none of the above ideas stand close scrutiny: the mechanisms are unknown (Kiernan, 1981). In the van Gieson's method two anionic dies can be used in a mixture. An iron-haematoxylin nuclear stain is followed by staining with acid fuchsine in saturated aqueous picric acid. Collagen stains red, cytoplasm yellow, and nuclei brownish black. The staining is specific and easily interpretable. The mechanism of collagen staining is explained by the binding of acid fuchsine to hydroxyl groups of serine, threonine, and hydroxylysine by hydrogen bonds through the nitrogen atoms of the dye (Lillie, 1964). Periodic acid - Schiff method (PAS) can also be applied since collagen is a glycoprotein (Kiernan, 1981). Schiff s reagent stains tissue aldehyde, but the collagen stain would be weaker than with other generally used methods. Additionally PAS is not specific for collagen and would stain similarly other glycoproteins such as serum proteins, cell membranes (glycocalix), and so forth. 11 For the detection of calcification Alizarin Red, GBHA, or von Kossa's method can be used (Riernan, 1981). Alizarin Red S, a synthetic anthraquinone dye is used to demonstrate calcium by forming coloured chelates with it. This dye is also an anionic dye, resulting in a non-specific pink background colour for most of the tissues. I found this background colour unacceptably high which did not allow for the recognition of specific areas of calcium deposition. Before staining, the calcium must be released from its insoluble form for chelation, resulting in some diffusion of the staining. This method is also extremely pH sensitive. Glyoxal-bis-(2-hydroxyanil) (GBHA) is a chelating agent for metals. It is very sensitive for soluble calcium, but insoluble calcified material largely remain unstained. Another great disadvantage is that the tissue has to be freeze-dried, as staining tissue fixed by aqueous reagent results in diffusion. The most widely used stain for calcified materials in tissue is the von Kossa procedure, which is less sensitive than the above stains, but results in sharp, localized and specific staining. The technique actually stains phosphate and carbonate anions, which are associated with calcium in calcified tissue. Silver nitrate is used to treat the tissue and substitute calcium with silver in the calcium phosphate deposits. Light exposure results in reduction to metal silver, showing black coloration. 12 3. Culture of osteogenic cell populations In vivo assays have been developed to detect bone formation of osteoblast like cells, such as cell grafts to chorioallantoic membranes of chick embryos (Nijweide et al., 1982), intramuscular injection of cells (Groot et al., 1983; Moskalewski et al., 1983), and intraperitoneal implantation of cells i n diffusion chambers (Simmons et al., 1982; Ashton et al., 1984; Bab et al., 1984). However, in vitro approaches allow for more controlled study of bone formation. In the in vitro experiments conditions are not so complex, thus evaluation is more reliable; sufficient number of experiments can be accomplished; and intervals between observations may be relatively shorter. Mineralization by osteogenic cells have been described in fetal and adult rat calvaria cell culture (Binderman et al., 1974; Williams et al., 1980), and rabbit bone cell isolates from endosteal surfaces (Anderson et al., 1984). However, reproducible bone formation was first reported only in organic phosphate supplemented culture of chick periosteal cells (Tenenbaum, 1981; Tenenbaum & Heersche, 1982). The osteoid-like tissue (unmineralized matrix) produced by osteoblasts readily mineralizes in the presence of organic phosphate, usually added to the medium as ^-glycerophosphate. This is a substrate for alkaline phosphatase and it is converted to inorganic phosphate which together with the collagenous matrix participates in mineralized matrix development. Further experiments emphasized the need of organic phosphate and ascorbic acid for mineralization in mouse and fetal bovine calvaria cell culture systems (Ecarot-Charrier et al., 1983; Whitson et al., 1984). These findings were confirmed and 13 a reproducible mineralizing osteogenic system was described by Bellows et al. (1986). At present, there are four different techniques which have been proven to be useful in the investigation of osteogenesis in vitro (Davies et al., 1987; Tarrant & Davies, 1987; (Davies et al., 1989) and which I considered for my experiments. The calvarial technique uses neonatal rat calvaria, not older than 2-5 days. After the animals are sacrificed, calvariae are dissected and stored in an appropriately selected tissue culture medium. Then periostea are also removed to eliminate the main source of fibroblasts from the system. This method has been used to study osteoblast migration, colonization and new tissue formation (Jones & Boyde, 1977). The calvarial envelope technique differs from the one described previously in that it leaves the endocranial periosteum partially attached to the underlying bone. The periosteum partially elevated to form an envelope where the substrate is placed (Radder et al., 1994). Osteoblast-like cells can be obtained by sequential enzymatic digestion of animal bones. Fetal or neonatal calvariae of rodents and chickens are suitable for this purpose (Ecarot-Charrier et al., 1983). The harvested cells are capable of forming bone-like tissue (Tenenbaum & Heersche, 1982; Nefussi et al., 1985; Bellows et al, 1986; Bellows et al, 1993). Mechanical removal of cells from bone marrow by washing out femoral bone marrow of rats yields a mixed population of cells (Maniatopoulos et al, 1988). Two major cell groups can be classified: haemopoietic cells and cells of 14 the reticular system of the bone marrow, which consist of fibroblasts, chondroblasts, osteoblasts and stem cells. The reticular cells can survive numerous passages while the haemopoietic cells are destroyed during the culturing procedure. Cultures are capable of forming bony tissue (Davies et al., 1990; Gomi & Davies, 1993). The methods outlined above make it possible to investigate specific phases of bone-formation, by controlling culture conditions. However, the calvarial method is not able to provide an uncontaminated osteoblast population. The size of the substrate is also restricted. Mechanical removal of cells from the bone marrow results in a heterogenetic cell population. Obtaining cells by enzymatic digestion of neonatal rat calvaria is a relatively simple and reproducible method which provides a more homogeneous osteoblast population. Thus it appears to be an appropriate culturing system for the study of cell behaviour related to interactions with substrata having different topographies. 4. Effect of surface topography on cell behaviour in vitro Unti l recently implants were designed to resist different forces and corrosion in the host environment. However, in today's implant planning biocompatibility have risen to the same level of importance. Ratner suggested that the description of biocompatibility as a passive phenomenon should be changed to an active role whereby materials would be engineered to achieve 15 certain tissue reactions based on scientific rather than empirical findings (Ratner, 1993). Generally a number of materials, including titanium and its alloys, hydroxyapatite, aluminum oxide, and certain ceramics have been used in different implants based on their non-toxic, non-irritant and non-corrosive characteristics. The application of different surface topographies as implant surfaces based on scientific findings has considerable potential to fulfil some of the new requirements for biocompatibility. New technologies allow the reproducible engineering of the implants to the most finite details while recent research brought more understanding of the effect of implant surface topography on implant retention (reviewed by Chehroudi & Brunette, 1995, In Press). In vitro studies apply specific surfaces, which are also parts of clinically utilized implants. These include smooth and textured surfaces, the latter consisting of rough, porous, fibre-based, machined and micromachined or microetched surfaces. The intent of the different textured surface designs a priori is to provide greater surface area thus more stable tissue-implant interface by increased cell attachment and tissue ingrowth (Bobyn et al., 1982; Pilliar, 1983). However, there are resulting changes in other cellular behaviours, as well. These changes can influence in both negative and positive directions the stability of an implant, or in vitro a tissue-substrate interface. In vitro studies described different patterns of cell behaviour effected by surface topography. Rough surfaces resulted in increased levels of cell attachment by osteoblasts (Bowers et al., 1992), however this was less pronounced in the case of oral epithelial cells (Chehroudi et al., 1988; 1989). 16 This effect proved to be cell-type dependent, as gingival fibroblasts were found to prefer smooth surfaces for maximal attachment (Kononen et al., 1992). The increased cell attachment was not the result of their increased surface area alone, as it could be shown in experiments employing precision engineered micromachined surfaces which allowed calculation of surface areas (Chehroudi et al, 1988; 1989; 1990). There are other substrate properties influencing cell attachment, such as mechanical rigidity (Maroudas, 1973; Harris, 1982; Harris, 1984), wettability (Baier, 1970; Baier et al., 1984, 1988), and surface charge (Rappaport et al., 1960; Maroudas, 1975). The different response by cell types to surface topography could be exploited by selecting a surface type which is not compatible with the attachment of unwanted cells. For example a rough surface which allowed cell adhesion by osteoblasts in preference to fibroblasts would be desirable for implants contacting bone (Chehroudi, 1991). However the situation can be complex as cells of the macrophage-monocyte system were found to favour rough surfaces, as well (rugophilia - Rich & Harris, 1981). In the clinical environment this might result in a long term inflammatory response against the implant (Perala et al., 1992; Rahal et al., 1993; Labarre et al., 1993). Porous substrata can also selectively promote cell attachment by porous size exclusion (Chvapil et aZ.,1984). Porous surfaces also produce a more stable tissue-implant interface and by reducing tissue-implant friction they promote osseointegration (Pilliar, 1985; 1988; Pill iar et al., 1986; Aspenberg et al., 1992). This could be explained by the significant level of interdigitation of cells attaching to porous material (Meyele et al., 1993; Grinnel, 1978). 17 Grooved surfaces encourage both fibroblasts and epithelial cells to orient with their grooves (Brunette et al, 1986a, 1986b). Grooved substrata can also modify the cell shape. Fibroblasts were taller (Chou et al., 1995 In Press) more elongated and their long axis was parallel to the grooves (Brunette et al., 1988; Oakley & Brunette, 1995 In Press) and epithelial cells were round on micromachined surfaces (Hong & Brunette, 1987). Cell shape can affect cell growth (Folkman & Moscona, 1978.; McDonald et al., 1989), proteinase secretion (Hong & Brunette, 1987; Werb et al, 1986), differentiation (Watt et al, 1988) and gene expression (Ben-Ze'ev, 1987; Ben-Ze'ev, 1991; Chou et al, 1995, In Press) of the cells. Another cellular function effected by surface topography is contact guidance, which is a property of cells to align and migrate in a direction specified by the substratum (Weiss, 1959). It is capable of controlling the cell migration thus with the proper placement of surfaces bearing this feature epithelial downgrowth on implants can be inhibited (Chehroudi, 1991). Contact guidance was observed early in the history of cell culture (Harrison, 1910; 1914; Weiss & Garber, 1952; Weiss and Taylor, 1956; Weiss, 1959). The basic mechanism is not fully understood as yet, but proposed mechanisms include microexudate formation (Weiss, 1958), selective adhesion (Weiss, 1958; Carter, 1965; Ohara, 1979), mechanical properties of the cellular components involved in cell movement (Curtis & Varde, 1964; Dunn & Heath, 1976; Dunn, 1982), and the involvement of cell processes in cell movement (Oakley & Brunette, 1995, In Press). Recent hypotheses have emphasized the role of cytoskeletal components, such as microtubules (Oakley & Brunette, 1995, In Press) and microfilaments (Dunn & Heath, 1976). Dunn & Heath 18 proposed that the shape of the substratum orients the microfilament bundle formation. The contraction of these bundles would then move the cell toward the cell-attachment, along the restrictive substrate shape. However, subsequent work has demonstrated that orientation occurs prior to formation of microfilament bundles, and the behaviour of the components in lamellae might have more important role in contact guidance (Oakley & Brunette, 1993). Adjacent tissue explants tend to form a cellular bridge, connecting the tissues. This so called "two-center" effect is the result of the attached cells exerting force on structures found in their environment (Weiss, 1959; Harris et al, 1981; Harris,1982). 5. Surface topography and mineralization The observation that surface configuration can affect mineralization is not novel and has been observed in a number of systems. Perhaps the earliest example of the possible effects of an implant forming a bone-inductive microenvironment in vivo is the work of Selye et al. (1960), who implanted glass cylinders of various shapes into rats so as to direct tissue ingrowth in desired directions. A striking observation was that bone and cartilage formation occurred, but only in some sizes and shapes of cylinders. Bone formation has been observed in conjunction with a variety of porous and textured surfaces, as well as machined surfaces having microscopic grooves, on a number of dental implant systems (Brunette, 1988). Such textured substrata could act through 19 several not mutually exclusive mechanisms such as by allowing a bone-inductive microenvironment to be established, or by inducing cell polarity that facilitates extracellular matrix secretion, or by selecting for cells with osteogenic properties by their locomotory or attachment properties. Osteoblasts develop tight attachment to titanium and its alloys (Groessner-Schreiber & Tuan, 1992; Gomi & Davies, 1993; Stanford et al., 1994). Rough surfaces promote increased mineralization by osteoblasts, as it has been shown in vitro on sandblasted titanium alloy surface by an embryonic chick calvarial osteogenic cell culture (Groessner-Schreiber & Tuan, 1992). Mineralized matrix deposition was demonstrated on scratched polystyrene by bone marrow cells from rat (Gomi & Davies, 1993). Calcification at the tissue-implant interface also occur on porous surfaces (Selye, 1962; Holund et al., 1979; Woodward, 1981; Cifkova et al., 1987; Rolland et al., 1989; Stoker et al., 1990), provided that the pore size is not smaller than 50-100 \im (Brunette, 1988; Pill iar et al., 1991). Square shaped pores were described as better promoters of mineralization than round ones (Goodman et al., 1993). Plasma-spraying of a porous surface has been claimed to result in faster and more stable osseointegration of implants (Schroeder et al., 1981; Schroeder et al, 1989; Luckey et al., 1992). 20 6. Objective of the thesis One approach to distinguish between possible mechanisms of the effects of surface topography, is to study surfaces which systematically vary in their surface topographies in ways that might be expected to favour certain mechanisms. Because micromachining can produce surfaces with precisely controlled topography that can be varied in shape, depth and spacing, the use of micromachined surfaces could be a valuable method of investigating the topographical control of bone formation on artificial substrata. The aims of this thesis work are the following: A. To establish an in vitro system that allows the quantitative measurement of the effects of surface topography on bone-like tissue formation. B. To obtain information on the effects of surface topography on mineralization that wil l enable us to design surfaces which promote bone-like tissue formation, thus osseointegration of (dental) implants. 21 II. MATERIALS & METHODS 22 1. Substrates A. Micromachining Micromachining is a technique that produces grooves, slots or pits with precise dimensions in silicon or gallium arsenide wafers. This technology has been extensively utilized in diverse applications such as solar cells, high value capacitors, solid state and miniature gas chromatography systems (Kendall 1979). The particular micromachining techniques employed 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 reduced to the desired dimensions by a step-and-repeat photographic process to produce photomasks, glass plates on which a metallic master pattern is placed. The master pattern on the photomask is transferred onto a silicon wafer by photolithography. In brief, a thin silicon oxide layer is produced on the surface of the wafer, which is then covered with a U V sensitive organic polymer, called photoresist. Then the wafer is exposed to U V radiation through the photomask. Following, the exposed photoresist is removed chemically, creating a negative copy of the master pattern. The next chemical reaction removes the silicon oxide from the areas stripped of photoresist and finally the rest of the polymer is removed. The result is a positive copy of the master pattern made out of silicone oxide on the surface of the silicon wafer. We can also create negative copies by using a different photoresist, which is strengthened by U V radiation. The next step of micromachining is chemical etching, which uses the oxide pattern as a mask. The pattern on the silicon wafer is etched to the desired 23 depth by anisotropic etchants which can create well defined shapes with sharp edges and corners. B. Preparation of replicas In my experiments the wafers were not used directly i n tissue cultures, instead epoxy replicas were prepared. This technique provided the possibility to perform electron microscopic studies and the ability to view the tissue cultures with a phase contrast microscope through the whole culture period. Impressions of the micromachined surfaces were made in a vinyl silicone impression material (Perfourm, Miles Inc., Ontario), and the impressions were used to cast replicas of the original patterns in epoxy resin (Epotek 302-3, Epoxy Technology, Billerica, MA, USA). The liquid epoxy resin was left to harden at room temperature on a levelled surface, then placed into a 60 °C oven for 3 days. Baking for such a prolonged period was necessary in order to prevent excessive release of resin compounds, which would have harmful effects on the cells. Only replicas perfectly identical to the original surface were used, those with air-bubbles, scratches or other distortions were discarded. C. Surface patterns The original surface patterns, as described earlier, were etched into a silicon wafer. The depth, shape and repeat spacing of an anisotropically-etched surface can be regulated by the design of the master pattern, the time of etching and the crystalline orientation of the silicon wafer. 24 My studies employed grooved and tapered pitted surfaces. The grooves were V-shaped with walls forming a 55° ± 3° angle with the surface and a depth of 19 \im ± 4 |im SD or 30 |im ± 8 [im SD, and a repeat spacing of 39 |im ± 5 |im. The tapered pits formed a square at the surface, with sides of 270 ^im ± 1 |im SD, and had walls that tapered at an angle of 55° to the surface and were 120 urn ± 1 |im SD deep (Figure 1). These micromachined surfaces were similar to those used in a previous study which produced connective tissue ingrowth and bone formation (Chehroudi et al., 1992). D. Design and fabrication of substrates containing replicas Two different designs were employed to meet the requirements of the particular experiment. The first design was created for the calvaria osteogenic culture (Figures 1-2). It contains all four different surface patterns to be tested, including the smooth surface control. To fabricate it, at first an approximately 2 mm thick, 1" x 1" square base was made from epoxy resin (Epotek). This material was selected over glass coverslips used by Bellows et al. (1986) to allow for subsequent processing and sectioning for E M . Square molds of vinyl silicon (Perfourm) were created with smooth surfaces, filled with 1 ml of liquid resin and left to harden overnight on a levelled surface. The following day the squares were removed from the molds, the sharp corners and edges were carefully trimmed. The surface patterns were mounted on the base by silicon impression squares with 1 cm sides. The replicas were then placed back on a level surface for an additional day, followed by baking in a 60 °C oven for 3 days. At the end of this period the replicas were cooled to room temperature, 25 the impression squares were peeled off and thin ridges between the different patterns were removed. The second design was created for the calvaria explant culture and is discussed under that culture method (Figure 5). E. Cleaning and preparation for cell culture experiments The epoxy replicas were cleaned by ultrasonication for 20 minutes in a detergent specifically formulated for tissue culture (7X, Flow Laboratories, McLean, VA). After being rinsed twenty times with deionized water, they were dried overnight in a tissue-culture laminar-flow hood and stored in a Petri dish. A l l implants were coated with an approximately 50 nm thick titanium layer and treated for 3 minutes in an argon-gas glow-discharge chamber (Baier & Meyer, 1988; fabricated by J.P. Sweeney, this department), then immediately stored in deionized, degassed, sterile distilled water in cleaned, water-tight Teflon containers. The glow-discharge treatment not only sterilized the substrata but also created high surface energy which facilitates cell attachment. 26 22 mm Shallow Grooves Deep Grooves 1 9 ± 4/7m 30 ± 8A/m repeat spacing: 39 ±5£/m repeat spacing: 39 ± 5^m Tapered pits Smooth 120 ± l u m d e e p 270 ± l ( i m wide across top wal ls taper at 55° from top to bottom 10 mm 27 Figure 1 Schematic diagram indicating the layout and the parameters of the substrata. 28 23 Figure 2 Epoxy replica with micromachined and smooth control surfaces. 30 2. Cell cultures To test the effects of implant surface topography on bone formation in a more controlled environment, we decided to use two osteogenic cell culture systems; [A] calvaria osteogenic cell culture (COC) and [B] calvaria explant culture. These culture systems have been shown to produce mineralized tissues on standard culture dishes reproducibly (Bellows et al., 1986; Davies et al., 1989). A. Calvaria osteogenic cell culture (COC) The method used for cell isolation was similar to that described by Hasegawa et al. (1986). In brief, frontal, parietal and occipital bones (calvaria) of newborn or 1-3 day old Sprague-Dawley rats (Figure 3) were carefully dissected and rinsed in copious amounts of sterile phosphate buffered saline (PBS), and placed in tissue culture medium (a minimum essential, a -MEM; Terry Fox Laboratory, Vancouver) supplemented with 15% fetal calf serum (FCS, Boecknek) antibiotics (100 |ig/ml Penicillin G, Sigma; 50 |ig/ml Gentamycin sulphate, Sigma; and 0.3 |ig/ml Fungizone, Flow Laboratories, McLean, VA). Eighteen calvariae were dissected under sterile conditions and minced into pieces approximately 1-2 mm3. The minced tissue was then incubated in 5 ml of a digestion mixture containing 180 U/ml of clostridial collagenase (type la, Sigma, St. Louis, MO. USA), and 0.5 mg/ml trypsin (Gibco, Burlington, Ontario, Canada) in PBS. The suspension was digested for 20 minutes at 37° C with stirring in a Pierce "Reacti-viar (Pierce Chemical 31 Company, Rockford, IL, USA). After 20 minutes the supernatant was harvested with a sterile Pasteur pipette and it was filtered through a mesh to eliminate small pieces of undigested tissue. The filtered cell suspension was then mixed with an equal volume of cold fetal calf serum (FCS) to stop the enzyme reaction, than centrifuged at 150 g for 5 minutes. Then the supernatant was discarded and the cells were resuspended in a -MEM containing 15% FCS and antibiotics. This population of cells was designated population I. The procedure was repeated at 20 minute intervals to yield populations (P) II - IV. The cells were counted electronically (Coulter Cell Counter) and plated at 2 x 10 3 cells/cm2 either in 75 cm 2 tissue culture flasks (if they were to be subcultured later) or into six-well culture plates (35 mm diameter each well). Cultures were incubated in a humid atmosphere of 95% air and 5% CO2 at 37°C. Two days after primary plating of calvarial cells on standard culture dishes, the medium along with the dead cells was discarded, and adherent cells of PII and PHI were trypsinized and replated at 2 X 10 3 cells/cm2 onto titanium-coated replicas of micromachined and smooth control surfaces (Figure 2) which were individually placed into the wells of 6-well plates. When the cells became confluent 5-6 days after plating, the medium was supplemented with 50 pig/ml ascorbic acid and 10 m M Na-B-glycerol-phosphate over the subsequent 30 days. The medium was changed every second day. Na-B-glycerol-phosphate acts as a source of organic phosphate, and ascorbic acid is required for collagen synthesis; both have been shown to be necessary for bone matrix formation and mineralization process in vitro (Tenenbaum 1981; Bellows et al., 1986). 32 Parietal bones X Occipital bone Frontal bones 33 Figure 3 Anatomy of the rat calvaria. Circle outlines the dissected area. 34 At completion of the culture period the tissue cultures were fixed and processed for microscopy. B. Calvaria explant culture (CEO The calvarial explant system used in these experiments was adapted from that described by Davies et al. (1989). Whole calvaria of newborn rats were carefully dissected under sterile conditions, and the periosteum and endosteum was removed by scraping with a sterile spatula. The calvariae were then placed in a 24-well culture dish with the internal (concave) side up (Figure 4). Micromachined test surfaces ( 4 x 2 x 2 mm) as well as smooth control surfaces were replicated in epoxy resin, coated with ~ 50 nm titanium and placed on the calvarial bone (Figure 5). A total of 6 surfaces were placed on the calvaria and cultured for 30 days in a M E M supplemented with FCS 15%, antibiotic, 50 |ig/ml ascorbic acid and 10 mM Na-fi-glycerol-phosphate. The whole calvarial culture system provides a source of osteogenic cells from bone and a source of fibroblastic cells mainly from the sutures between individual bones. It has been speculated that this method may provide an environment which closely approximates in vivo conditions (Davies et al., 1989), where implants are interacting with a mixed population of cells. After 30 days of culture, the entire calvaria and the test surface were fixed in buffered glutaraldehyde and processed for SEM. 35 C A L V A R I A L M E T H O D TEST M A T E R I A L 3G Figure 4 Schematic description of the calvarial explant culture. (Adopted from Davies, 1988.) 37 38 Figure 5 Photograph showing an implant (imp) placed on the inner surface of a calvarial explant (C). Arrows indicate the location of the micromachined or smooth control surfaces. 39 3. Qualitative methods A. Tissue processing and microscopic techniques After 1 month of culture, cell cultures were fixed with buffered 2.5% glutaraldehyde (TEM, SEM), or 10% buffered formalin (light microscopy) for 1 hour at 4°C. Van Gieson staining was employed to demonstrate the presence of collagen. Haematoxylin and Toluidine blue stains were used to demonstrate the light microscopical structure of the developing nodules. Alkaline phosphatase activity was demonstrated by a diazonium coupling reaction. For this purpose, cultures were fixed at 4°C for 4 hours in 10% Ca-acetate buffered formalin. After fixation the cultures were incubated in 1 mg/ml Fast Red Violet LB salt and 1 mg/ml Naphthol AS-BI phosphate as substrate in 0.05 M Tris buffer at pH 9.1 for up to 20 minutes. The presence of Ca mineral deposits was investigated with von Kossa's method after fixation. It was assumed that the intensity of the stain was directly related to the extent of the mineralization in the cultures. B. Fluorescence microscopy Other cultures were labelled with 5 i^g/ml oxytetracycline to obtain fluorescence images of mineralized nodules and viewed under a laser confocal microscope (Zeiss, Oberhausen, Germany) at 488 nm. The tetracycline was given together with media supplements until fixation. 40 C. Transmission electron microscopy (TEM) Because the standard dehydrating agent, alcohol, had been previously found to deform the grooves and undermine the titanium coating, Aquembed (Ladd, Burlington, VT.), a water-miscible resin, was used. Epoxy replica/tissue blocks were dehydrated in graded Aquembed (50%-100%, 1 hr in each change), then infiltrated with graded Aquembed/Epon (J.B. E M Dorval, Canada) (50%-100%, 1 hr i n each change) on the rotator. The specimens stayed on the rotator overnight in fresh Epon. The next day, infiltration was carried on with 3 changes of fresh Epon without accelerator (2 hr in each change), followed by 3 changes of Epon with accelerator (2 hr in each change). Specimens were then placed under vacuum at 4°C overnight and finally embedded in Epon. Sections were taken throughout the length of the epoxy replica using a Sorvall MT2 microtome. Two-|im-thick sections were cut i n an orientation such that the grooves or pits were cross sectioned (parallel to the long axis of the grooves), stained with Toluidine Blue and observed under light microscopy at 500 times magnification. Selected areas of the block were trimmed for transmission electron microscopy (TEM), and sections fifty-to-sixty-nanometer thick were cut with a diamond knife from the tissue/replica interface. The sections were then stained with alcoholic uranyl acetate and aqueous lead citrate, and viewed under an electron microscope. 41 D. Scanning electron microscopy (SEM) To observe the morphology of osteogenic cells and mineralized nodules, some cultures were prepared for scanning electron microscopy (SEM). Glutaraldehyde fixed cultures were post-fixed in 2% buffered osmium for 1 hour, briefly dehydrated with graded alcohol (50%-100%, l h r total), critical-point dried with CO2, sputter-coated (Hummer VI Sputtering System, Technics, Alexandria, Virginia, USA) with palladium or carbon (~ lOnm-thick) and observed in a scanning electron microscope (Cambridge Stereoscan 100, Leica, Canada). Our previous experience and the observations of this study on epoxy replicas indicated that brief dehydration with alcohol limited to one hour did not distort micromachined replicas, nor did it lift the titanium coating. E. Energy dispersive X-rav (EDX) analysis EDX was performed on selected portions of the cultured surfaces using a Link AN5500 system (M. Weiss, Depertment of Zoology, UBC) to identify mineral deposits by studying their chemical composition. To expose the tissue closest to the implant surface, compressed air was used to blow away the superficial cells. Cultures were post-fixed with osmium (see Materials & Methods 3.D), then the tissue samples were again coated with either palladium or carbon. 42 F. Time-lapse cinemicrography Grooved or control flat substrata were mounted on glass slides and placed in a Pentz chamber (Bachofer, Reutlingen, W. Germany) which was placed in a stage incubator (Bachofer), and viewed with reflected-light differential-interference-contrast optics. Timing and exposure of the 16 mm cinefilm (Kodak 2415, Opti-Quip, Highland Mills, N.Y.) was controlled by an Opti-Quip cinemicrographic system. 4. Quantitative methods A. Laser densitometry An UltroScan X L laser densitometer (Pharmacia L K B , Sweden) was used to analyze the total amount of the mineralized nodules formed on the epoxy replicas of micromachined surfaces. Replicas were scanned and the optical density of each surface pattern of a replica, containing micromachined and control smooth surfaces, was mapped separately. A blank replica was used to establish the background level (Figure 6B). B. Videoplan image analysis B.l. Area analysis Epoxy replicas were viewed under a dissecting microscope equipped with a camera lucida at 26 x magnification. Then a digitizing tablet connected to a 43 computer with a Zeiss Videoplan software package was used to trace the mineralized areas on each surface. B.2. Two-dimensional shape analysis The computer system described above was used to measure the shape of the individual mineralized nodules formed on the micromachined and smooth control surfaces using the equation: form PE= 47i*Area/(Perimeter)2. A value of one indicates a circular shape and values <1 indicate an elliptical shape. B.3. Orientation analysis A l l mineralized nodules were outlined as described previously. The angle formed between the longest axis of a nodule and the long axis of grooves was measured. For pitted and smooth surfaces, the orientation angle was measured relative to the border of the individual surface square. Pitted surface replicas were fabricated such that the border of the pits was parallel to the border of the surface square. 5. Statistics Data are presented as mean ± standard deviation. One-way analysis of variance with Tukey HSD multiple comparison tests was calculated using IBM-Systat statistical package. Bartlett's test was applied to check the homogeneity of variance of the data. 44 Figure 6 Epoxy replica of micromachined and smooth (control) surfaces on which osteogenic calvarial cells have been cultured, stained with von Kossa (A), and analyzed by UltroScan X L laser densitometer (B). 46 III. RESULTS 47 1. Characterization of bone formation A. Optimization of culture conditions The optimal conditions for the formation of mineralized nodules on titanium surfaces had to be established. A.l. Cell populations Cell populations isolated from rat embryonic calvaria by sequential trypsin digestion were examined for their ability to form mineralized nodules on the micromachined surfaces. When cultured on tissue-culture plastic, nodules of mineralized tissue were formed in all populations, but the nodules were most frequent in populations II-III with a lesser number being found in populations I and IV. It was established, that the populations, most efficient in forming bone nodules on smooth plastic were forming the most nodules on micromachined substrata, as well. A.2. Population density. Titration of the number of bone-forming nodules as a function of cells added were performed on all smooth and micromachined substrata to determine the number of cells that were required to obtain mineralized nodules (Table 1). 48 A.3. Culture passage number It was determined that rat embryonic calvarial cells of less than two passages had to be used because the production of mineralized nodules per cell plated declined when later passages were used. In addition it was found that cells could be frozen and replated and still form nodules. It was clear that comparisons between substrata would have to be made using cells from the same passage number. A.4. Culture conditions Bellows et al. (1986) work was confirmed, that the culture medium had to be supplemented with ascorbic acid and B-glycerophospate to ensure mineralization. When our cultures were supplemented with ascorbic acid alone, mineralized nodule formation did not occur, although there were clumps of densely aggregated cells. Neither did J3-glycerophospate alone promote nodule formation. Nodules formed only when both of these supplements were added to the cell culture. A.5. Time-course No detectable difference between micromachined and smooth titanium-coated surfaces was noted in the time required for mineralized nodule production when cultures were examined every 2 to 3 days. 49 Variables Conditions tested Result Plating Density (per cm2) 3x l0 2 ; 6x l0 2 ; 9x l0 2 ; 2x103; 5x103 2x l0 3 produced the most nodules. Supplements Three suppls. were added separately or together: - vitamin C, - Na-B-glycerol phosphate, - dexamethasone (Bellows et al, 1989). Vitamin C and Na-13-GP were together necessary for nodule formation, but not dexamethasone. Days in Culture Examined cultures every other day between day 2 - 30. Resulted in longitudinal study of nodule formation. For large, fully mineralized nodules long term (30 day) cultures were most suitable. Digestion Time vs. Population A. sequential digestion after 10, 20, 30, 50, 70 mins. B. sequential digestion after 20, 40, 60, 80 mins. A. Populations: I f ibroblastic; II fibroblast - osteoblast mixture III max. nodules: IV less nodules; V only few cells left B. Populations: I f ibroblastic; II -III osteoblastic, max. nodules; IV few cells left Passage Number Subcultures up to 3 passages. Primary cultures produced the most nodules. Table 1 Optimization of osteogenic culturing conditions. 50 B. Nodule formation and mineralization B. 1 Histochemistry von Kossa's method The process of nodule formation in rat calvarial cell culture is depicted in Figure 7, using von Kossa's method to detect calcification. Upon plating the cells appeared spindle shaped, resembling fibroblasts and remained so, as long as supplements were not added: Shortly following supplementation with ascorbic acid and organic phosphate the cultures showed clear signs of focal nodule formation. By day 5 several aggregates of osteoblast-like polygonal cells were evident in the cultures. Between day 6 to 8 the appearance of nodules was evident in the multilayer, confluent cultures (A). In the presence of ascorbic acid and organic phosphate the cultures had mineralizing foci by day 12 (B), The foci continued to calcify (C) and developed into three-dimensional mineralized tissue (plaques showing structural arrangement above the plane of the multilayer culture having several times the thickness of the surrounding cell layer) by about day 14 (D). In addition to the von Kossa method seen above, further histochemical techniques were used to demonstrate mineralization in these nodules. Alkaline phosphatase activity Alkaline phosphatase enzyme activity is an osteoblast differentiation marker (Rodan & Rodan, 1983; Majeska et al, 1985). The activity of this enzyme, as demonstrated by an azo-coupling reaction, was 51 52 Figure 7 Different stages of mineralized nodule formation in rat calvarial cell culture (von Kossa staining). A. Day 6, a nodule which is elevated from the substratum plane (20x objective, Haematoxylin counterstained). B. Day 10, focal mineralization (5x objective, Toluidine blue counterstained). C. Day 12, mineralization continues (20x objective, Toluidine blue counterstained). D. Day 14 and later, three-dimensional mineralized tissue formation staining intensively with von Kossa's stain for calcified tissue (lOx objective, Haematoxylin counterstained). 54 Sb" Figure 8 Elevated alkaline phosphatase enzyme activity i n a mineralizing nodule in a 7 day old rat calvarial cell culture. Azo-coupling reaction with Fast-Red Violet LB salt and ASBI phosphate as enzyme substrate (lOx objective). 56 highly elevated already in seven day old cultures, (Figure 8). The area of staining was appropriate in shape and size for a mineralizing nodule, shown earlier. Van Gieson staining Collagen production is another characteristic activity in the extracellular matrix of mineralizing tissues. Early bone extracellular-matrix formation starts with the deposition of collagen fibres. This mesh of fibres provides the first foci for hydroxy-apatite crystal deposition. In mature bone, collagen fibres are fully embedded in the mineralized matrix (Glimcher, 1984). Van Gieson staining demonstrated that these nodules were highly positive for collagen (Figure 9). B.2. Transmission electron microscopy The transmission electron microscopic examination of the extracellular matrix in mineralized nodules showed matrix vesicles characteristic to an ongoing mineralization process. Collagen bundles were prominent parallel to and in close juxtaposition with the culture surface (Figure 10). B.3. Scanning electron microscopy Scanning electron microscopic comparison showed great similarities of the mature cell culture to the original bone tissue, the rat calvaria (Figure 11). 57 B.4. Fluorescence microscopy Internal bone markers for bone growth measurement include Alezarin Red, lead, and tetracycline uptake (Harell et al., 1973). Fluorescence microscopy of tetracycline uptake is a commonly used method to detect pathological new-bone formation in vivo (Frost, 1969, Marshall, 1969). The in vitro application of this technique showed mineralization in early nodules on both smooth and micromachined surfaces (Figure 12). 58 Figure 9 Collagen production in and around the mineralized nodules (30 day old culture, van Gieson staining, 40x objective). 60 64 Figure 10 Transmission electron micrographs of the extracellular matrix in a mineralized nodule. Note the matrix vesicles (MV), and the collagen bundles (C). Collagen was detected in close proximity of the culture surface without an intervening amorphous layer. 62 Figure 11 Scanning electron micrographs displaying notable similarities between the surfaces of a mature rat calvarial cell culture (A) and the original rat calvaria (B). Globular bodies of different diameters seem to be connected by bundles of fibres. 64 Figure 12 Tetracycline uptake of early mineralizing nodules after 12 days of culture (fluorescence microscopy). A. smooth culture surface (20x objective), B. tapered pitted titanium surface (25x objective), C. grooved titanium surface (20x objective) 66 2. Effect of surface topography on bone-like tissue formation The aim of this study was to compare nodule formation on flat surfaces and surfaces with grooves or pyramidal depressions prepared by micromachining as described in the Materials & Methods section. A. Surface preparation S E M observations of the test surfaces indicated that micromachined surfaces were replicated with high fidelity to the master pattern on the silicon wafers, and the titanium coating was smooth and continuous with no evidence of cracks. B. Behaviour of osteoblasts on micromachined surfaces It was demonstrated previously that surface topography influences the behaviour of fibroblasts (Brunette, 1986b, 1988). However, the assumption that osteoblasts would exhibit contact guidance similar to fibroblasts had to be validated. Time lapse cinemicrographic experiments were performed on the behaviour of calvarial osteoblasts on titanium-coated grooved micromachined substratum. The selection of groove sizes was based on data previously obtained in this laboratory (Chehroudi et al., 1989, 1990, 1992). Figure 13 demonstrates that these cells became oriented with their long axis parallel to the grooves. Moreover the cells migrated in the direction of the grooves and were not observed crossing from one groove to another. This directed migratory behaviour contrasted with that of osteoblasts cultured on titanium-coated smooth surfaces, where it was found that the cells were oriented and moved 67 Figure 13 Frames from a time-lapse cinefilm demonstrating that osteoblast-like cells assumed an elongated shape on a grooved substratum and migrated in the direction of the grooves. Arrows indicate a cell moving along a groove. Asterisk indicates two cells moving in opposite directions along a groove. Grooves have a repeat spacing of 30 (im and a depth of 3 |jm. 69 with no apparent directional preference. These observations indicate osteoblasts exhibit contact guidance. C. In vitro experiments on micromachined surfaces To test the effects of surface topography on bone formation in a controlled environment two osteogenic cell culture systems were used: calvaria osteogenic cell culture and calvaria explant culture. As it was demonstrated earlier, these culture systems produce mineralized tissues on standard culture dishes reproducibly. C.l. Calvaria osteogenic cell culture Cells from P-II and P-III, the populations associated with the highest frequency of mineralized-nodule production (see Result l . A . l , and Bellows et al., 1986), were cultured on titanium-coated replicas of micromachined and smooth surfaces. At the light-microscopic level the development of mineralized tissues on these surfaces can be readily revealed by using Von Kossa stain (Culling et al., 1985). On surfaces with tapered pits the cells formed a monolayer over most of the surface, and in places the cells formed hammock-like or sail-like sheets that extended across the pits. As noted in the time-lapse studies osteoblast-like cells oriented themselves parallel to the grooves. Mineralized nodules were found on micromachined surfaces and smooth surfaces (Figure 14). 70 74 IX Figure 14 Bone nodules demonstrated by von Kossa. Note nodules (arrows) are oriented along the long axis of the micromachined grooves, 30 |im deep(A, B) and 19 urn deep (C). Nodules formed on smooth surfaces (D) and also at the top (E) or bottom (F) of tapered pitted surfaces did not show a preferred orientation. 74 At the electron-microscopic level, in addition to morphological features, EDX elemental analysis was used to identify mineral deposits. In agreement with Davies et al. (1991) and Lowenberg et al. (1991), S E M observations revealed globular accretions that appeared to be attached to the substratum. However, these microglobules were found to be of two sizes; large globules > 10 urn in diameter, and small globules of < 0.5-3 |im in diameter. Occasionally, large globules were located at the bottom of the tapered pits or grooves (Figure 15, 16A); whereas small globules were observed primarily at the periphery of individual osteoblast/cyte-like cells (Figure 15C,D). Small globular accretions were the only type of mineralization found on the smooth control surfaces during the time period of this study (Figure 16B). EDX analysis of both large and small globules revealed substantial amounts of Ca and P, indicating the mineralized nature of these structures (Figure 17). The osmium peak is a result of tissue processing (see Materials and Methods 3.E). C.2. Calvaria explant culture The implant surfaces remained on the calvarial bone during the experiment and S E M observations indicated that in all cases the surfaces were covered by a lawn of cells that migrated from the calvarial explant. These migrating cells were interspersed with extracellular matrix and on the micromachined surfaces numerous mineralized globules (= 5 |j.m) were embedded within the cells and matrix (Figure 18). EDX analysis of these globules revealed Ca and P peaks as well as a large peak of Os. Globules were 75 76 Figure 15 Scanning electronmicrographs of osteogenic calvarial cells cultured on tapered pitted surfaces. Note large mineralized globules (LG) [A,B], and small mineralized globules (SG) which were most frequently formed at the periphery of osteocyte-like cells (OC) located at the bottom of a pit [C,D]. 78 19 Figure 16 Scanning electronmicrographs of osteogenic calvarial cells cultured on micromachined grooved [A, side view] and smooth control [B] surfaces. Note large globules (LG) and small globules (SG) at the bottom of a groove, as well as small globules at the periphery of an osteocyte-like cell (OC) on the smooth surface. 80 Figure 17 Energy dispersive x-ray (EDX) spectrum of large mineralized globules similar to those shown in Figures 15 and 16, demonstrating major peaks for calcium (Ca), phosphorus (P), Osmium (Os) and a small peak for titanium (Ti). 82 83 Figure 18 Scanning electronmicrographs of a calvarial explant cultured for 4 weeks. Arrows indicate highly mineralized globules. Colonizing cells migrated from a calvarial explant onto the micromachined (A) and smooth control (B) surfaces. Arrows indicate partially mineralized globules that were observed on the grooved surfaces. 84 A 2000 1500 • 1000 oun 500 U 0 0 p 2.5 5 Kev 7.5 Os 1 10 B 8000 1 I 6000 J a o 4000-2000 . 0 0 Ca 2.5 5 Kev 7.5 Os 10 Figure 19 Energy dispersive x-ray (EDX) spectra (counts, full scale = 8000(a) or 2000(b) versus keV) of partially mineralized globules similar to those shown in Figure 18A (A), and mineralized globules formed on the calvarial bone, similar to those shown in Figure 18B (B). 86 also found on the calvarial bone that was not in contact with the implant surface. The amounts of Ca and P were greater in these globules than globules formed adjacent to the micromachined implants (Figures 19A,B). On the smooth surfaces we were unable to identify mineralized globules. Although sporadic nodular accumulations of tissue were noted, they failed to show evidence of calcification when examined by EDX. 3. Quantitative studies on the effect of surface topography on bone-like tissue formation. A. Laser densitometry Figure 20 summarizes the quantitative data obtained from 24 replicas (each with four different surface patterns) of five separate cell cultures. Thirty day old cultures were fixed and stained with von Kossa's method, which yielded a black staining of all mineral deposits in the tissue. The intensity and area of the staining was quantitated by laser densitometry resulting in optical density readings. It was inferred, that the total optical density of each surface is related to the total amount of calcified material. The optical density of the mineralized deposits on the smooth surfaces was significantly less (p<0.05) than the volumes calculated on the micromachined surfaces. 87 B. Video Image analysis Table 2 illustrates the mineralized areas evaluated by computerized morphometric measurement on the same surfaces which were evaluated by laser densitometry for the amount of mineralization. Pits and grooves produced significantly larger mineralization areas than smooth surfaces (p<0.01). Table 3 describes the results of the two-dimensional shape analysis based on data from 587 individual bone nodules formed on micromachined or smooth control surfaces. Nodules formed on smooth surfaces were significantly (p<0.05) rounder, while the shape of the nodules formed on grooved surfaces became elliptical. Table 4 shows the results of the orientation angle analysis of the same mineralized nodules. Nodules formed on grooved surfaces aligned along the grooves in a significantly more parallel (-180°) manner than nodules formed on pitted or smooth control surfaces. 88 Surfaces S 19 |im-deep grooves El 30 |im-deep grooves @j tapered pitted surface • smooth surface 89 Figure 20 Amount of calcified tissue obtained on different surfaces measured by densitometry. In each sample equal areas of the four different surfaces were examined. The amount of calcification is measured on Von Kossa stained material using a laser densitometer. The amount of calcification on a given surface type in a sample is expressed relative to the sum of the optical densities of the four different surfaces on each replica. calcification amountln x=calcification volume (relative %), xln= * 100 X calcification amount 1-4 n=sample number The data are calculated in this way so as to eliminate errors that might result from differences between replicas such as thickness of coating or epoxy. N=24 in each group. Error bars represent standard deviation. Tukey HSD multiple comparison test results after x2 transformation of the heteroscedastic data (Bartlett test) at p<0.05 : tapered pits ~ 30 |im-deep grooves > 19 |im-deep grooves = smooth 90 Type of Surface Mineralized surface area Number of %X±%SD surfaces 1. Grooved a. 19 ± 4 um deep 20.8 ±2.9 24 b. 30 ± 8 urn deep 29.1 ±3.2 24 2. 120 urn-deep tapered pits 34.9 ±2.5 24 3. Smooth 15.3 ± 1.9 24 Table 2 The mineralized surface area is measured as Von Kossa stained areas whose magnitude is determined by tracing the outlines on a digitizing tablet connected to a computer with morphometric software. The mineralized area on a surface type in a sample is expressed as the corresponding percentage of the sum of the four different surface type calcification areas in that sample. mineralized surface area i n x % = mean mineralized surface area xln= * 100 X mineralized surface area (relative %) 1-4 SD = standard deviation. Tukey HSD multiple comparison test p<0.01 : tapered pits ~ 30 um-deep grooves > 19 um-deep grooves ~ smooth 91 Type of Surface Shape Factor X±SD Number of Bone Nodules 1. Grooved a. 19 ± 4 |im deep 0.41 ±0.17 135 b. 30 ± 8 p i deep 0.42 ±0.16 155 2. 120 urn-deep tapered pits 0.46 ±0.15 185 3. Smooth 0.54 ±0.20 112 Table 3 The shape factor of the mineralized nodules formed on the micromachined and smooth control surfaces was calculated. Samples were positioned under a dissecting microscope equipped with a camera lucida at 26x magnification. Then the image of a digitizing tablet was superimposed to the image of the tissue section on the computer screen. The outlines of the individual nodules were delineated with a magnetic pen on the tablet connected to a computer. Data from 3 independent experiments were evaluated. Calcified areas less than 0.1 mm2 were excluded Shape factor = 47t*Area/(Perimeter)2 Where the value of one indicates a circular shape and values < 1 indicate a more elliptical shape. x = mean of shape factors of bone nodules SD = standard deviation The result of Tukey HSD multiple comparison test at p<0.001 : smooth = tapered pits > grooves 92 Type of Surface Orientation Angle (degree) Number of Bone X±SD Nodules 1. Grooved a. 19 ± 4 urn deep 170.8° ± 15.9° 135 b. 30 ± 8 urn deep 170.2° ± 13.9° 155 2. 120 um-deep tapered pits 128.9° ±32.1° 185 3. Smooth 136.3° ±40.9° 112 Table 4 The orientation angle was calculated as described in Table 2, except using the angle made up by the longer diameter of the bone nodule and the x axis, which was chosen to be consequently parallel with the same side of the surface pattern, as well as with the grooves. Data is expressed relative to the orientation angle 180° (parallel to the surface). x = mean of orientation angle of bone nodules SD = standard deviation The result of Tukey HSD multiple comparison test at p<0.001 : grooves > smooth ~ tapered pits 93 IV. DISCUSSION 94 The first goal of my work was to establish and characterize an in vitro system for the study of mineralization/bone formation by osteoblasts on micromachined substrata. Two osteogenic cell culture systems were studied: rat calvaria osteogenic cell culture and rat calvaria explant culture, as these models have been described being reliable mineralization producing cultures (Bellows et al., 1986; Davies et al., 1989). The optimal conditions I found for the rat newborn calvarial cell culture system to produce mineralized tissue on micromachined surfaces included: 1). cell populations II-III, 2). 2x l0 3 cell population density, 3). less than 2 culture passages, 4). addition of ascorbic acid and B-glycerophosphate to culture medium. Under these conditions nodules were formed after about 14 days. These nodules had the characteristic of bone-like tissue. Elevated alkaline-phosphatase activity was measured by an azo-coupling reaction and collagen rich content of the extracellular matrix of these nodules was detected by Van Gieson staining. T E M showed collagen bundles in close proximity of substratum and a parallel orientation to the substratum surface. S E M demonstrated the nodules were structurally comparable to the original calvarial bone. The elevated tetracycline uptake shown by fluorescence microscopy also indicated a mineralization process taking place in these cell cultures. In vitro cell cultures provide a controlled environment, which is very important as a first approach of a scientific investigation. Thus they are useful in determining bone induction in response to different surface topographies. However, there are obvious drawbacks to in vitro experiments, and they should always be complemented by in vivo investigations i f clinical implications are to be made. In vitro osteogenic cell cultures are static, i.e. they can not reproduce the dynamic nature of bony tissue with its continuous deposition, turnover and 95 remodelling which takes place at load-bearing implant interfaces in vivo (Stanford & Keller, 1991). They can not take into account the in vivo inflammatory reaction against a foreign body such as an implant. In vitro cell culture removes cells from their normal environment, including tissue structure, as well. This can result in artificial reactions as the cells have different contacts in a monolayer culture, than in tissue. Moreover, all of them are subjected to the same extent to factors present in culture medium compared to the individual conditions experienced by a cell in vivo, that arise from the cell's position in the tissue (Stanford & Keller, 1991). Osteogenic cells can be isolated freshly as primary cultures or can be derived from permanent or immortal cell lines. Permanent cell lines may consist of transformed osteosarcoma cells such as UMR-6, ROS 17-2 and MC3T3-Ei osteoblastic clones (reviewed by Rodan & Rodan, 1983) or they may be bone cell clones selected from primary cultures (Kodama et al., 1981, Aubin et al., 1982). Each of these experimental models can have advantages depending on the problem to be studied. For my study, which involved quantitative analysis of changes in phenotype, I selected primary cultures, as in this model the possible complication of the transformed phenotype - which likely accompanies immortal cell lines - can be discounted (Wong, 1990). However, this choice resulted in a time consuming optimization of culture conditions and in the necessity of low cell passage numbers, as expression of osteoblastic features decrease with each subculture (Wong & Kohn, 1974). There are four techniques preferentially used to establish primary osteogenic cell cultures: calvarial, calvarial envelope, calvaria cell and bone marrow cell methods (see Introduction 3). I selected two techniques, the rat calvarial and the rat calvaria cell culture systems. Early work by Tenenbaum & Heersche 96 (1982) described the development of an osteogenic culture system in chick using chick periosteum in vitro in which dexamethasone stimulated osteogenesis. Bellows et al. (1986, 1989) adopted its use, but I found no necessity for supplementing osteoblastic rat calvarial cell cultures with dexamethasone, as mineralized nodule formation was quite frequent in the absence of this supplement. However, it was necessary to add organic phosphate and ascorbic acid to confluent cell cultures to induce calcification, in close agreement to Bellows et al. (1986). Mineralized nodule formation did not take place in the absence of these supplements. However, it would be of interest to add these supplements at the time of plating to the cell culture instead of when the cells reached confluence. This approach might produce better separated/delineated nodules on a monolayer culture, instead of large calcified areas on a multilayer culture. I also found that the method of harvesting osteoblastic cells from foetal rat calvaria, described by Bellows et al. (1986), which included the Caesarian sectioning and kill ing of the pregnant mother could be modified by using two to five days old, newborn rats, as their calvaria was only partially calcified. This simplified the experiment and yielded more material. Bellows et al. (1986) used several criteria to characterize their in vitro bone-like tissue, including: high alkaline phosphatase activity associated with the covering osteoblastic layer, collagen content of the bony matrix, showing signs of mineralization, and the presence of osteonectin and osteocalcin in the mineralized tissue. They primarily employed histochemistry and microscopy to show these characteristics, however they applied immunohistochemistry to detect osteonectin and osteocalcin, type I and III collagens and the absence of type II collagen. Immunohistochemistry has been used for the detection of mostly non-collagenous bone matrix proteins in other laboratories, as well 97 (Oliva et al., 1992; Malaval et al., 1994). I considered the histochemical, EDX, light and electron microscopic observations efficient to establish satisfactorily the mineralized nature of the nodules and the osteoblast-like phenotype of the cells involved in their production. Additional immunohistological and biochemical testing of the nodules is beyond the scope of this work which is intended to examine whether certain types of surface topographies promote in vitro mineralization. The main goal of this thesis work was to investigate the effect of micromachined surfaces on mineralized nodule formation. First of all, similar to fibroblasts studied previously at this laboratory (Brunette, 1986b, 1988), the present study showed that osteoblasts exhibited contact guidance on grooved micromachined surfaces. In both culture systems S E M and EDX revealed mineralizing globules with high calcium and phosphorus content on pitted and grooved surfaces but less frequently on smooth surfaces. To quantitate the relative amount of mineralization on the different surface patterns, cell cultures grown on these substrata were stained with von Kossa's method and the amount of mineralized tissue was measured by laser densitometry. This showed an increase in calcified deposits on the grooved surfaces over the smooth surface. Moreover, there was a significant increase in bone-like tissue formation with deeper grooves (Figure 20). Another measure, employed to ascertain the effects of surface topography on the cells was computer assisted morphometric analysis. This method found that the surface area of the mineralized tissue was increased on the micromachined surfaces compared to smooth control surfaces. Shape 98 analysis showed significant alteration with nodules found on grooved surfaces exhibiting a more elliptical shape than those nodules on smooth surfaces. Results of orientation angle measurements underlined the importance of contact guidance by grooved surfaces: nodules were aligned parallel with the grooves, but nodules were not oriented on smooth or pitted surfaces. Branemark established the concept of osseointegration and described it as a direct bone to implant surface connection which involves a structural and functional bone tissue interface and provides long-term implant stability (Branemark, 1983, 1985). There is widespread agreement that surface topography is important in implant performance. However, the design of surface topography varies, resulting in a wide selection of commercial devices with different surfaces (Brunette, 1988). It has also been suggested that surface topography of implant substrate influences mineralization. Groessner-Schreiber & Tuan (1992) demonstrated that the synthesis of bone matrix and subsequent mineralization were enhanced by rough-textured (sandblasted) or porous-coated (plasma-spraying the rough-textured surface with a titanium alloy) titanium substrata in vitro . Moreover, Gomi & Davies (1993) found results similar to mine in that roughened polystyrene dishes promoted mineralized tissue deposition by rat bone marrow cells. However, these surface preparations result in highly uneven, random surface patterns, which does not allow for the systematic study of the effects of surface topography. I used micromachined surfaces as substratum for osteogenic cell cultures. Micromachining can produce surfaces with precisely controlled topography, that can be varied in shape, depth, and spacing. The implementation of micromachined surfaces proved to be a valuable method in these experiments in 99 investigating the topographical control of in vitro bone formation on artificial implant models. The process by which mineralization is influenced by different surface topographies is still not understood. Several mechanisms could be involved and the following possibilities are of particular interest with respect to the micromachined surfaces used in these studies: Micromachined surface topographies could produce a bone-inductive microenvironment. Evans and Potten (1991) have proposed that regulation of stem cell activity in vivo depends upon complex interactions that include restriction of the distribution of regulatory factors. The micromachined surfaces may act by constricting osteogenic cells into a confined volume in which regulatory factors achieve concentrations that promote calcified tissue formation. Such a possibility is given credence by the in vitro experiments of Nijweide (1975) as well as Tenenbaum and Heersche (1982), who demonstrated that periosteal sheets have to be folded to produce a bone inductive microenvironment. Similarly, Nakahara et al (1991) have shown that periosteal cells enzymatically liberated from chick tibia produce bone and hypertrophic cartilage only in a microenvironment produced by cells plated in high density as cells forming skeletal tissue prefer to be piled up on each other before matrix can be secreted. However, in the light of this concept, there is a possibility that the increased population density resulting from cells accumulating in the micromachined grooves could result in more matrix being produced than in standard monolayer cultures. The control smooth surfaces used in my calvarial cell culture system were plated at a low cell density, and there being no surface textures to encourage high cell densities a bone-inductive microenvironment as 100 described above might not develop. The micromass culturing method, in which cells are plated in a droplet and stay in high density has been used to study mineralization in vitro (Boskey et al., 1994) and in the future it would be of interest to compare smooth and micromachined surfaces using this method. Another possibility is that micromachined surfaces produce an arrangement of collagen that promotes mineralization. It is generally agreed that a prerequisite for mineralization is the synthesis and assembly of an extracellular matrix onto which crystals of mineral can be formed (Gerstenfeld 1988). It has been suggested that the spatial arrangement of the collagenous matrix, the major component of the extracelluar matrix of calcified tissue, may play an important role in initiation of mineralization in vivo (Weiss 1988). A first approach to determining whether such a process was operative would be to study the arrangement of collagen fibrils on smooth and micromachined surfaces by performing serial histological sectioning and three-dimensional reconstructions of the implant/tissue interface. A third possibility is that the effects may be mediated by cell shape changes. Alteration of cell shape by surface topography can result in change of gene expression, resulting in the change of phenotype (Watt, 1986; Bissell & Barcellos-Hoff, 1987). Solursh (1989) demonstrated that induction of a round shape in mesenchymal cells promoted chondrogenesis, while induction of a flattened shape on the same cells promoted fibroblast differentiation. Chondrocytes lost their type II collagen expression when cultured attached to substrata (West et al, 1979; von der Mark, 1980). Human fibroblasts produced increased level of fibronectin mRNA when their cell shape changed during culture on grooved surfaces (Chou et al., 1995, In Press). In vitro experiments allow the evaluation of differential selection for cellular phenotype by different 101 implant surfaces. In my experiments I found that grooved surfaces produced shape changes in osteoblasts and increased mineralization. Thus it is possible that there may be a causal relationship between these two phenomenon. It can be concluded that: rat osteogenic cell cultures can be used to study mineralization processes on different culture surfaces, bone-like tissue formation is influenced by the substratum surface topography, micromachining may be useful in determining the ideal surface topography for promoting bone formation on titanium surfaces and there is need for further studies on this topic to determine the mechanisms involved. 102 V. Future Work In this thesis I successfully established an in vitro osteogenic system in the testing of the effect of surface topography on mineralization. I also demonstrated the importance of surface topography in orienting and increasing the mineralization. It is evident from this work, that grooved surfaces and in some extent pitted surface patterns were more effective in promoting calcification, compared to smooth surfaces. These results point towards the need to examine in more detail the different features (depth, angle, form and spacing) of the surface grooves in order to develop the best surface for bone-like, tissue formation based on in vitro experience. We should also examine the mechanism by which these grooved surfaces are able to increase mineralization. How can the structured orientation of osteogenic cells positively effect calcification? What are the important features of topography that produced bone inductive microenvironment? As cell shape can alter gene expression, and topography affects cell shape, do grooved surfaces change the biochemistry and structure of the newly deposited bone? In this work the calvarial explant system was used to establish that osteoblasts migrating to micromachined implant surfaces can produce a mineralizing matrix similar to that of the originating tissue. This system also have a potential in examining the phenomenon of osteoconduction of different surface topographies. This could be demonstrated by placing calvarial explants 103 on test surfaces and examine the presence and the rate of cell migration and bone-like tissue production. A number of studies have recently been published the effects of different cytokines and growth factors on in vitro osteogenesis (Ellies et al., 1992; Hughes et al, 1992). It would be of interest to examine how the effects of these factors and different surface topographies combine, based on the possibility, that certain factors might alter the processes involved in the topographical influence on mineralization. For example one factor that would be of interest is TGF-B. This factor affects cell adhesion and is present in bone, and it is conceivable that it could change the way osteoblasts behave on different surface topographies. TGF-B dependent chemotaxis of osteoblastJike cells also might influence the cytoskeleton, and thus the alignment of cells on the grooved surfaces. Finally, but importantly the surfaces and conditions most effective in vitro should be tested in vivo and in the event that bone production was increased significantly, these surfaces could be tested on implants for clinical use. 104 VI. BIBLIOGRAPHY 105 Albrektsson T, Branemark P-I, Hansson H-A, Kasemo B, Larsson K, Lundstrom I, McQueen DH, Skalak R. 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The effect of micromachined surfaces on osteogenesis in vitro
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