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Critical Reviews in Oral Biology and Medicine, 2(1):83101 (1991)

The Concept of Osseointegration and Bone Matrix Expression

Clark M. Stanford and John C. Keller
Dows Institute for Dental Research, University of Iowa, College of Dentistry, Iowa City, IA 52242.

ABSTRACT: Osseointegration has been defined as the direct structural and functional connection between ordered, living bone and the surface of a load-carrying implant. To date, this concept has been described by descriptive histological and ultrastructural criteria but not by biochemical means. This review evaluates the basic science work performed on this concept and then applies the concept to the principle of osseous healing. Specific studies are cited where alterations in the healing response are due to clinical management of implant placement and how studies of surface properties may lead to further insights on implant design and prognosis. In addition, a review of bone expression as a function of in vitro stress applications is given. This is followed by an indepth review of the collagens and noncollagenous proteins, described to date, within isolated bone matrix. It is this collagenous matrix (especially type I) that is described as being close to and oriented with a glycoprotein component next to the implant surface. In turn, the large family of noncollagenous proteins are important in mediating bone proliferation, matrix accumulation, orientation, mineralization, and turnover. This section is followed by a discussion of specific growth factors as they may relate to osseous healing around an implant. KEY WORDS: Osseointegration, matrix, bone, implants, surface properties.

I. INTRODUCTION The development and use of metallic implants for use in clinical dentistry have increased dramatically in North America since the concept of "osseointegration" was presented at the Toronto conference in May of 1982. It was at this conference that descriptive in vivo animal studies and clinical results from human patients treated in Sweden since 1965 was presented.13 This concept of a direct bone-to-metal interface has been described, according to Branemark, as consisting of a highly differentiated tissue making "a direct structural and functional connection between ordered, living bone and the surface of a loadcarrying implant." 4 Unfortunately, this concept has become difficult to evaluate because the fundamental level at which the body is responding to the metal implant is never carefully defined. In other words, can claims of a "direct bone contact" as observed at a histological level be indicative of the lack of a local or systemic re1045-4411/91/$. 50 1991 by CRC Press, Inc.

sponse to that surface? It is therefore proposed that "osseointegration" is not the result of an advantageous tissue response but rather the lack of a negative tissue response. The term osseointegration describes a clinical state that provides for long-term stability of a prosthesis, but, under current thinking, is not a property of any one implant system or metal.5-6 The concept of osseointegration can be defined at multiple levels: clinically,2 anatomically,1 histologically, and ultrastructurally.6 To date, little research has been performed to biochemically evaluate the healing response to the implant surface or to evaluate how the material's characteristics, such as surface preparations, cleaning techniques, or sterilization protocols, may affect the short- and long-term function of these devices. It is recognized that significant logistical problems are present in isolating the critical biochemical events in question using in vivo approaches. Nevertheless, this approach, as well as in vitro work, is important and significant


because it is believed that minor alterations in material composition, surface oxide coatings, surface preparation, and sterilization procedures can affect the short- and long-term stability of the metallo-biological interface.711 This review article will discuss previous work evaluating the biological response to implanted titanium with particular respect to the development of extracellular matrix (ECM) and alterations in the bone response to the implant surface. Integrated with this information will be relevant knowledge from the fields of bone biology and extracellular matrix in an attempt to engage the reader in this interesting area. It is the intent of the authors to critique and present information about the extracellular matrix produced by bone tissue, in vitro around an implant, since it is felt that this natural response to a foreign substance will dictate and be representative of the bioacceptability of an implant modality. It is important to emphasize that we are essentially at the neonatal stage of learning about bone responses to artificial surfaces and only entering adolescence in the areas of bone biology. The next decade will dramatically increase our body of knowledge, which can only help us to better understand biological interactions on artificial surfaces.




II. THE PHILOSOPHY AND RESEARCH APPROACHES TO DEFINE OSSEOINTEGRATION It is important to realize that the work of Branemark has become the standard by which all other clinical implant research is compared. However, following the development of this concept, new basic and clinical research efforts have emerged that have begun to reexamine this paradigm. In discussing and assimilating material on bone matrix expression and the concept of osseointegration, a number of relevant questions

response, can models of bone fracture healing be used as a paradigm for a long-term wound-healing response? In temporal terms, Is osseointegration a static or dynamic term? For example, does a change in the biochemical forces applied to an implant affect the osseointegrated status of an implant over time? Further, when a static histological model is promoted as " p r o o f of integration it ignores the dynamic nature of osseous tissue such that an implant may only be ' 'osseointegrated'' under relative terms that can evolve as rapidly as the healing tissue around the fixture. How would the surface properties of an implant alter the structure and/or composition of the extracellular matrix and parenthetically, how might these surface properties affect the maturation of the healing response over time? A direct corollary would be, Is cellular gene expression altered by interaction with a preformed or autosynthesized ECM? There is now evidence available that gene expression can be altered by the ECM, independent of changes in cellular shape.12 How does characterizing the matrix, formed in vitro or in vivo, help in understanding the complex orchestration of its formation, deposition, remodeling, and turnover? At best this question can only be discussed on a preliminary basis because, although a great deal has been learned about the individual components of isolated bone matrix, comparably little is known or understood about how each component interacts with each other and, hence, how they provide a functional and structural environment for differentiated cellular expression.

Can a clinical and morphological concept be defined in a reductionist manner, such that "osseointegration" be a model for an appropriate matrix response to maintain an implant for an indefinite period of time? If osseointegration is defined as a lack of a

In this work, while the authors will not attempt to specifically address these questions, it is believed these questions are the direction in which future research should be emphasized. We will, however, present the current information regarding the development of the ECM as it potentially relates to implants, and subsequent osseointegration at the implant interface. We will review critical information regarding bone healing and the role of the ECM molecules in this process. We firmly believe that it will be the marriage of the


basic research techniques in the disciplines of cell and molecular biology with clinical research that will help answer these important questions.

III. BONE MATRIX EXPRESSION AND ITS RELATIONSHIP TO OSSEOINTEGRATION The concept of a rigidly fixed implant as a prerequisite for long-term stability has implied that the direct bone contact occurs around the entire region of the root form implant.1 In contrast, other researchers have shown a clinically immobile, and thus successful,13 implant can occur with a range of bone implant contact areas as the fixture penetrates through bone.1417 The fact that a range of bone/implant contact areas has been observed in otherwise clinically successful implants has led to the question of how much macroscopic contact is necessary in order to obtain a successful implant.15 Does the wide range of observed contact areas indicate a state of dynamic osseointegration? It is apparent at this time that neither of these questions have yet been answered. According to the Swedish group, the paradigm of osseointegration centers around the selection, usage, and preparation of commercially pure Titanium (cpTi) as an implant material. Considerable effort has been and continues to be performed to develop other materials for implants, such as alloy systems (i.e., Ti-6A1-4V) and ceramic coatings (particularly hydroxyapatite and tricalcium phosphate). Despite an inadequate understanding of the role that specific surface variables have on biological response, cpTi, at this point, remains the material of choice for dental implant use. The use of coatings on implants, very popular recently, adds an additional level of complexity to the system. Since little is known about the metallic responses, and the fact that 4 'osseointegration" was originally described as a direct metal-bone contact, we have chosen to limit our discussion to only the metal-tissue interfacial reaction. Various advantages have been described for the use of commercially pure titanium metal (>99.0% Ti) as an implant material. The surface is composed of an oxide layer with a reported

nonstoichiometric composition of TiO2, TiO, and Ti2O3 depending, in part, on the history of the implant surface and the methods of preparation.18 The Ti oxides have a high dielectric constant under most conditions and a negative surface charge that may affect the attachment or adhesion of biological molecules of the extracellular matrix.1824 The oxide layer is generally 50 to 100 A thick in the absence of special chemical or thermal treatments and has the ability to repair itself by reoxidation when damaged. The effects of these changes in the oxide over time when implanted are unknown. This is especially true in localized regions of the implant surface where aggressive ions (Cl~, S, OH", H + ) lie within a corrosive environment (pits, blebs, etc.): defects that profoundly alter the normal passivated oxide. The thickness, composition, and microstructure of the oxide appear to be related to fabrication techniques and sterilization procedures.1825 Efforts to understand the role of these factors on biological responses, including cellular attachment and development of an ECM, are currently underway in our laboratory. To date, some work has progressed in evaluating the kinds of matrix produced on an implant surface, although it is usually only characterized at a morphological level. When an implant is placed into a surgically prepared socket, a number of different vascular and immunological events will take place. In time, the space between the fixture and bony crypt will heal with new bone by reparative osteogenesis resulting in clinical fixation of the implant. The events that occur in this situation have been studied using several histological approaches.26"30 Several studies have also been performed using the vital optical bone chamber system.130 Using this system, Winet and Albrektsson described primary bone ingrowth that occurred around the titanium chambers as composed of a fibrovascular tissue in nature and not one containing cartilaginous matrix.30 Further, these authors found a significant healing rate of 85.5 jim/d at the osseous front with a peak of growth determined around the third week.30 These forms of investigations, although valuable in determining the vascular and morphological changes occurring in the healing site, suffer from the inability to biochemically evaluate the cellular response around a fixture. This response at a matrix


and then an intracellular level is important when one considers changes in cell response occurring from alterations in the local orientation and relation of cell-cell contact.3132 Linder and others6 described an ultrastructural relationship between a titanium evaporated film and bone tissue in vivo. After 12 weeks, the titanium coated (120 to 250 nm) polycarbonate samples were removed and prepared for TEM. A 20 to 50 nm gap between the closest collagen fibers and the oxide surface of the titanium was described. The authors further characterized this layer as consisting of a hyaluronidase and chondroitinase sensitive material. 6 Hansson and others33 have described cases of bone attachment to cpTi implants in place for various periods of time. They described the morphological appearance of Haversian systems and collagen fibers in decalcified specimens that paralleled and approached to within 20 nm of the oxide surface.33 McQueen and others34 have also suggested the passivated oxide surface can change over an observed 6-year period in vivo, resulting in an increased thickness (up to 200 nm), which contained increased levels of organic and inorganic (Ca, P, S) material. This would suggest the potential for a surface reactivity not usually associated with titanium and supports the concept of a dynamic definition of osseointegration. More recently, Davies and Matsuda35 have developed an in vitro approach to allow the direct growth of an osteoblast-enriched population of cells onto a test implant surface. Using a combination of SEM, TEM, and polarizing light microscopy, they suggested that bioactive surfaces, such as hydroxyapatite, are coated with a dense collagenous matrix after 4 weeks in culture. This matrix appeared to vary morphologically between different material surfaces, suggesting again that the extracellular response can be affected by the implant surface.35

IV. IMPLANT AND FRACTURE HEALING When an implant is placed within a newly prepared implant site, the healing response must consist of a programmed response. The cascade of potential responses will depend on the ability of bone to heal, as well as the lack of a foreign

body response. When bone is fractured it undergoes a process of degeneration, growth, and remodeling that may be replicated around a healing implant. If an implant was truly bioinert, then it may be logical to consider the fracture model as a paradigm for some of the events occurring in an implant site. Fracture healing follows a well-defined cascade of events that start with the initial disruption in the blood supply to the local area.36 (For detailed review papers on bone biology see References 36 to 44.) This is associated with local hemorrhage, anoxia, and apoptosis of the prepared implant site. 145 A corollary to this process is the degree of local thermal and mechanical damage since it has been shown the implant site is extremely sensitive to thermal damage.46-47 The healing response in a fracture site can be classified into three major periods in which inflammation is followed by cellular proliferation and differentiation. The formation of structural tissue (trabeculae, etc.) and finally the homeostatic period of controlled deposition and resorption follows an uninterrupted healing phase.36 The initial inflammatory reaction, lasting up to 5 d,45 will involve the release of vasoactive amines, a fibrin clot formation, macrophage ingress, and formation of the initial granulation tissue. Urist's work has suggested the local pluripotent stem cells will proliferate from the cambian layer of the periosteum and endosteal layers on the marrow side of the implant site.48'50 In rodent studies, this process appears to start within 8 to 12 h of the implantation.51 The differentiation of osteoblasts from precursors may be regulated by the local synthesis and release of autocrine and paracrine growth factors such as TGF-(3. This proliferative response then leads to a thickening of the periosteal layer referred to as a primary callus.45 This expanding callus is calcified and then replaced by endochondral bone formation over the next 4 weeks.45 The exact length of time that this occurs will vary depending in part on the distance between the implant and the viable osteogenic tissue.52 Other studies have also found bone ingrowth into a porous (100 to 400 jxm) surfaced titanium alloy (Ti-6A1-4V) implant model in beagle dogs. 5354 Histochemically, this region of intense wound healing was characterized by a progres-


sive increase in glycogen, a shift to acidic glycosaminoglycan synthesis, marked increases in alkaline phosphatase levels, and an increase in the number of matrix vesicles prior to calcification.55 The remodeling phase, which consists of a period involving greater than 25 to 50 d, occurred under the influence of a load transfer that, in turn, probably affected and guided the regeneration of osseous tissue around the fixture.145 Extracellular matrix expression has also been evaluated as a model for augmenting bone formation in a demineralized bone powder system implanted in nonskeletal sites.56'60 Using this model, it has been shown that bone formation in this system proceeds through an intermediate cartilagenous stage.56 The ECM produced at various stages elicited a specific cellular response depending on the charge density and composition of the various components (glycoproteins, proteoglycans, etc.).61 Reddi suggested a critical inductive matrix thickness was needed for bone formation (74 to 850 fim) that possessed various physical and biochemical properties.56 This factors) has been described as osteogenin, a heparan sulfate binding glycoprotein molecule, isolated from demineralized bone material.62 The importance of the ECM as both a structural and regulatory substrata for tissue growth and differentiation may lie in its ability to sequester local growth factors that can have either immediate or delayed effects.324357 It is also important to emphasize that the matrix can sequester various factors from the serum as well as locally produced components even though the final effect (i.e., concentration) is still the same. The role of direct cell-to-cell contact cannot be excluded since it is highly possible that local bone regulation is occurring, even by mature osteocytes.63 Once normal healing has occurred and the abutments are connected, the implants will transfer the biomechanical forces of mastication to the bone. This response can potentially take a variable mix of three forms; these being a formative response, a resorptive response, or the lack of a response. While clinical cases are reported where the radiographic appearance of a fixture appears to be surrounded by an increase in the trabeculation of bone,1 little is known regarding the in

vitro or in vivo effects of mechanical forces on bone response. How the load transfer is interpreted by the surrounding bone may affect the regulatory balance between resorption and proliferation. The ability of the tissue to differentiate will be dependent in part on the presence of an intact blood supply, adequate tissue oxygenation, and minimal stress.64 Inadequate fixation or disruption in the blood flow (e.g., excessive heat generation) will lead to a greater tendency for a cartilagenous response rather than bone.65 68 In fracture sites, excessive movement between the fractured ends can lead to increased soft tissue proliferation and a consequent increased callus size.68 It has been suggested by some that mobility will lead to direct local tissue damage, ischemia, and consequent increased cartilage formation.68 If, in contrast, a fracture site can heal with minimal movement and a good blood supply, only minimal callus formation occurs.64 The frequency of loading can also affect mineralization. For instance, Klein-Nulend et al.69 have shown intermittent forces can increase mineralization almost two times as much as a steady continuous load. Rodan70 used a compressive apparatus on isolated chick tibial bone to show that organ cultures will respond to external stress by decreasing glucose consumption and increasing DNA synthesis. This work was later reproduced with cartilage tissue where compressive forces, as applied in Rodan's model system, resulted in up to 60% inhibition of ornithine decarboxylase activity in epiphyseal cartilage cells.71 Work by van Kampen,72 using a high-density cartilage culture system, demonstrated significant increases in proteoglycan synthesis when subjected to physiological intermittent stress levels. Bagi and Burger73 followed on these results by demonstrating increased sulfate incorporation in fetal long bone cultures. Another group used a stretched substratum model to show cultures of growth zone cartilage cells could demonstrate increased cAMP and glycosaminoglycan levels over nonstretched controls.74 They did not describe an increased prostaglandin E2 production as suggested by Somjen et al.75 This could be due to differences in the experimental approaches as well as cell type sensitivity to tension in the substratum. Using a different model system, in which coverslips were elastically deformed, Hasegawa demon-


strated a significant increase in the number of cells entering the cell cycle and a 33% increase in collagen production when rat calvarial cells were grown on the coverslips.76 More recently, attempts to study this process have been performed using a cyclic strain apparatus in vitro, where cultured osteoblasts appear to respond favorably when directly exposed to a flexing silicone substratum. 77 Results from preliminary investigations suggest an increase in the rat of DNA synthesis and a morphological alteration in response to a stretching surface such that alignment of the polar axis of the cells was detected in a perpendicular manner relative to the line of force.77 These in vitro approaches are delivering important insights into how bone cells respond to stress at a cellular level and may potentially act as model systems for evaluating cellular effects of stress-transfer by implants.

long-term effects of a chronic inflammatory response, excessive or parafunctional stress levels, or other pathological events that may affect an implant long after it is in function. It is with this in mind that we intend to review some of the salient features known about collagen and some of the noncollagenous proteins (NCP) present in bone matrix. A. Collagen The trimeric molecule of collagen forms a semirigid and stable scaffold under in vivo conditions (for a recent review see References 39 and 78). Although 12 or more types of collagen have now been described, bone matrix consists primarily of fibrillar type I collagen (>90% of the organic matrix). Recent studies have suggested the potential that some of the more minor forms of collagen (types III, V, XI, and XIII) may be associated with the fibrillar form; however, due to the isolation procedures used and the fact that bone is a heterogeneous tissue, the exact source (e.g., endothelial basement membrane) of the various collagens is still unclear.79 Type I collagen molecules consist of two a 1(1) chains and one a2(l) chain that is both posttranslationally modified (e.g., hydroxyproline, hydroxylysine) and extensively cross-linked between chains.39 It is a glycoprotein, resulting from the sequential additions of galactosyl residues to hydroxylysine.80 In this way, bone-associated collagens appear to differ from other forms of type I collagens (e.g., in the dermal connective tissue) in the forms of glycosylation reactions (galactosyl-hydroxyls instead of glucosyl-galactosyl-hydroxyls) and appears to be less crosslinked.39 It remains to be seen if these differences can be utilized to evaluate the kind of cellular response (differentiated bone matrix vs. a fibroblastic scar tissue) that occurs around an implant. The mechanism of secretion has been studied extensively and appears to be regulated by an ordered deposition of polymers, regulated in part by the proteolytic removal of end terminal propeptide sequences. It is upon removal of these sequences that fibril ordering occurs, resulting in the familiar banding pattern (64 nm periodicity in rotary shadowing). It is interesting that recent

V. EXTRACELLULAR MATRIX: COMPONENTS The extracellular matrix (ECM) that surrounds and envelops cells is a complex mixture of glycoproteins and proteoglycans, often having a very large anionic character that tends to absorb water, serum proteins, hormones, growth factors, and miscellaneous ions. At the present time, over 17 different mesenchymal matrix proteins have been described, principally from guanidine extractable connective tissue samples. In extracts of demineralized bone, these comprise the organic matrix of bone. To date, most of the work in this area has concentrated on the isolation of a factor and subsequently evaluating its function, often in solution, in an effort to understand the potential role it may play in regulating bone physiology. This approach, which literally extracts the potential protein out of its normal functional environment, may potentially cause a loss or even enhancenent of normal functions. On the other hand, studies concerning the in vitro functions of osseous proteins can lend great insight into the potential evaluation of (1) normal bone expression in response to an artificial surface, (2) evaluation of how a potential implant surface can be modified in such a way as to promote one phenotype over another, and (3) determine the


evidence has shown some forms of collagen deposition can be modified by the retention of the c-terminal end in the mature state, as well as substitutions of other collagen chains in place of the normal type I chains.78-81 What this means in terms of regulation is unclear at this time. Type I collagen is then cross-linked, in the extracellular environment, after the posttranslational modification of lysyl to hydroxylysyl followed by aldehyde modification by lysyl oxidase. After subsequent condensation, cross-linking by a disulfide isomerase, a rigid fibrous network is created. On this complex meshwork, various extracellular proteins can bind, either specifically or nonspecifically, to provide a structural and regulative surface on which cells attach, spread, proliferate, and migrate. Collagen-like molecules have been described morphologically at the light and ultrastructural levels near an implant surface both in vivo,6*285 and in vitro.*6 Earlier work has suggested collagen fibers were observed up to about 20 nm from the oxide surface but consisted of 100 to 500 nm gradient of increasingly mineralized tissue.6 How this compares to the normal clinical situation is unclear since these studies used a sputter-coated Ti film on unloaded polycarbonate implants. The in vivo response to these surfaces were evaluated only after 12 weeks in a rabbit tibia. Future work in evaluating collagen typing may be accomplished by the more sophisticated molecular biology techniques now available, such as in situ hybridizations with cDNA probes.

B. Noncollagenous Proteins In the last 2 decades, the role of the more minor (<10%) components of the organic bone matrix have generated great interest since it is believed that they modify and regulate bone function, expression, and turnover. The noncollagenous proteins (NCP) that have been isolated from bone tissue and are believed to play important roles in bone physiology are shown in Table 1. 1. Osteocalcin Osteocalcin (or Bone Gla Protein) has been

one of the most thoroughly studied NCPs in bone matrix (reviewed extensively in References 87 and 88). This calcium binding protein (Kd = 0.83 mM)89 is present in bone, dentin, and other mineralized tissues and appears to increase in concentration as bone matures, relative to other NCPs. It is specifically synthesized by differentiated osteoblasts, suggesting it may be an ideal marker for osteoblast phenotypic expression.87 It undergoes a vitamin-K-dependent posttranslational modification resulting in the gamma carboxylation of three glutamate residues.90'92 In the human sequence, osteocalcin has 49 amino acid residues containing three gamma-carboxy glutamate (Gla) residues at positions 17, 21, and 24.93-94 These three positions are highly conserved between various species, suggesting they are of vital importance to the function of this molecule.87 For instance, it has been shown that these Gla-residues are critical for the ability of osteocalcin to bind calcium.95 Hauschka96 lab has described the secondary structure of osteocalcin, consisting of a series of two a-helices separated by a (3-sheet higher-ordered structure. This results in a threedimensional model where the a-helical domains appear to be positioned with a periodicity of charged amino acids (0.54 nm) identical to the periodicity of the x-y plane of hydroxyapatite.96 This property of osteocalcin may regulate its interaction with the oxide layer of an implant as well as to mineralized tissue. In vivo, osteocalcin appears to be synthesized by osteoblasts and deposited just before mineralization in bone and dentin matrix.97100 The biological role of osteocalcin is as yet unclear, although some studies suggest it may have a role in the resorptive response. For instance, bone from rats that were treated with warfarin (an inhibitor of the posttranslational carboxylation reaction) demonstrated less than 2% Gla residues. This tissue was unable to promote the normal recruitment response of osteoclast progenitor cells when implanted subcutaneously and resulted in significant decrease in the amount of 45Ca released from warfarin-treated bone over noninhibited tissue.101103 Interestingly, upon evaluation of callus strength and histologic morphology in warfarintreated rats, no significant differences were found over control groups, although Gla content was less than 2% in new bone formed during the


TABLE 1 Some Noncollagenous Proteins Isolated from Bone

Molecular weight 5,300 15,000 44,000 57,000 75,000 40,000 75,000 120,000 440,000 CBP (?) Cell attachment Cell attachment, matrix organization (?) Binds PGs (?) Regulate mineralization (?) Mineralization Space?, regulate collagen growth? Multiple cell attachment mitogen, differentiation

Name Osteocalcin (BGP) Matrix Gla protein Osteopontin Bone sialoprotein Bone acidic glycoprotein Osteonectin Proteoglycans (biglycan and decorin) Fibronectin


Thrombospondin Growth factors BMP (TGF-6)


Note: CBP: calcium binding protein; PG: proteoglycan.

treatment period.104 Osteocalcin has also been suggested to be chemotactic for osteoblast precursor cells and monocytes.90'92105 Lucas and Caplan106 used stage 24 chick limb bud mesenchyme and muscle-derived fibroblasts in a Boyden chamber assay to first describe this effect. This was followed by a comparison with the osteosarcoma cell line ROS 17/2.8 in which significant chemotactic (or chemokinetic) effects were seen with osteocalcin.106107 This led to the proposal that matrix proteins, like osteocalcin, may act extracellularly to promote the migration of blast-like cells to the source of release.108 It remains to be seen if this response is merely an in vitro artifact or a normal response for this matrix protein. Alternatively, osteocalcin has also been shown to strongly inhibit the formation of hydroxyapatite in solution and promotes the adsorption of protein to hydroxyapatite.90109110 Osteocalcin has also been found to bind to phospholipid vesicles.111 Taken together, this suggests osteocalcin can potentially regulate the rate and orientation of mineral structure formation. 2. Osteopontin Osteopontin is a 44-kDa (in SDS-Page) bone

sialoprotein synthesized by primary osteoblasts, osteoblast-like ROS 17/2.8 cells, cartilage cells as well as periodontal and gingival fibroblasts.112117 (For a review see Reference 39.) Osteopontin (Bone Sialoprotein I) is approximately 317 amino acids long in which 13 sites for glycosylation have been identified. A repeated sequence of nine aspartic acid residues are present that potentially may lead to mineral binding activity.39 In rat embryonic calvaria, the levels of osteopontin mRNA increases relative to total mRNA levels by day 15 of gestation and peaks, as do osteocalcin levels, by the time of birth.118 It has been isolated, cloned, and found to contain the cell binding motif of RGD (ArgGly-Asp) first described in the fibronectin cell binding domain.112 This cell binding sequence appears to be recognized by an integrin cell surface receptor related to the (av(33) vitronectin receptor. 119120 (For review of the integrin superfamily see References 121 and 122.) In culture, the addition of osteopontin appeared to increase cell attachment and spreading of human gingival fibroblasts in a manner different than fibronectin and independent of protein synthesis.113 It is possible that this protein may even enhance cell attachment to a greater degree than


fibronectin or Bone Sialoprotein II (discussed below).114 The synthesis and release of osteopontin appears to be under both hormonal (e.g., 1,25 (OH)2 vitamin D3 increases its transcription, in contrast to BSP-II, while cortocosteroids and parathyroid hormone decrease it) and local paracrine control (e.g., TGF-(3 increases osteopontin synthesis).118123125 Heinegard and Oldberg have recently suggested a potential role for this protein may lie not so much in the formative stages of the mineral phase, but rather in regulating the resorption of the tissue. This proposal was based on the hormone response data as well as the presence of osteopontin integrin receptors on osteoclasts.126 This intriguing and exciting suggestion awaits further investigation. 3. Bone Sialoprotein (BSP) (BSP) is an acidic phosphoprotein isolated from bone matrix containing a very high concentration of O-glycosidically linked sialic acid residues. It appears to be present only within bone tissue upon Northern blot analysis.127 BSP has a peptide backbone of 33,600 Da and a total weight of approximately 59,000 Da. As with osteopontin, this molecule contains a significant concentration of acid amino acids (32% are Glutamate), including a ten amino acidic repeated sequence of glutamic acid that is thought to convey mineral-binding properties to the molecule.37 Upon sequencing, a RGD sequence was detected that appears to convey a specificity for the vitronectin integrin receptor.119 Therefore, much like osteopontin (BSP-I), bone sialoprotein has cell attachment properties, although apparently with less affinity.114 4. Bone Acid Glycoprotein 75 (BAG-75) BAG-75 is an acidic phosphoprotein isolated from mineralized matrix of rat calvaria.128 It comprises only a small amount of the NCPs (0.5 to 1.0%) of total radiolabeled proteins synthesized in explant cultures.129 It appears to exhibit a 30% sequence homology with osteopontin, although it is a separate gene product. It has a strong binding affinity to hydroxyapatite and is often found

associated with small bone proteoglycans.128 Preliminary evidence suggests this acidic phosphoprotein may mediate osteosarcoma cell attachment in culture leading to the idea that this molecule may mediate cell attachment and growth as well as play a role in mineral formation.130 5. Osteonectin (SPARC) Osteonectin is a 33 kDa (cloned) NCP originally isolated by Termine and others,131 using a 4 M guanidine-HCL extracted demineralized bovine bone powder passed through a gel filtration column and then ion exchanged in a 7 M urea solvent (for review see References 131 and 132). It represents the most abundant NCP found in fetal bone matrix; however, it is not bone specific.132 Osteonectin has a close homology to SPARC (secreted protein acidic and rich in cysteine) found associated with platelets, fibroblasts, and to BM-40 associated with the basement membrane of EHS tumor tissue (see Reference 133). Nomura and others have recently described 5' enhancer regions (GAGA boxes) present upstream of the SPARC promotor, leading to the suggestion this gene may be affected by morphogens such as retinoic acid and steroid hormone control.134 It appears to have multifunctional properties, as do most of the NCPs, since it has been found to bind to denatured type I collagen, 135 mineral ions, 136137 and hydroxyapatite.132137 Osteonectin, in vitro, appears to be upregulated during migration and proliferation of endothelial cells and may mediate the cell-substratum interaction of these cells migrating into a wound site.133 It has the same type of repeated, high concentrations of acidic amino acids that may be the site for hydroxapatite binding.137 Interestingly, sequence analysis has also suggested this matrix protein possesses an EF-hand calcium binding region near the carboxy-terminus of the molecule.138 This has led to the suggestion it may act as nucleation sites for mineralization as osteonectin associates with the collagenous ECM.132139 It appears to strongly bind free calcium, much like calmodulin (3 x 10~7M)UO and, again, like most of the other NCPs, appears to strongly inhibit the formation of hydroxyapatite.


This again suggests NCPs, like osteonectin, play a significant role in mediating mineralization of the forming osseous matrix. & Bone Proteoglycans (Biglycan and Decorin) Bone proteoglycans are composed of a central protein core with covalently linked lateral glycosaminoglycan (GAG) chains of repeated disaccharide sugar groups (for review see Reference 39). The GAG composition of bone proteoglycans appears to differ from the more familiar cartilage molecule with the presence of chondroitin 4-sulfate (Ch4SO4) instead of chondroitin 6sulfate.140 In extracts of human alveolar bone, 94% of the GAG molecules were Ch4SO4 molecules.140 Two forms of proteoglycans are present in bone matrix that appear to share a significant homology (55%), although they are distinguishable immunologically.141 Proteoglycan-I or "biglycan" is a leucine rich, 37,000 Da (SDS-PAGE) protein, while "decorin" or bone proteoglycan-II is rich in Glu/ Gin residues. This later form appears to have molecular weights similar to BSP-I. Neither proteoglycan is found exclusively in bone tissue, although the exact GAG composition will vary from tissue to tissue.142 The bone proteoglycans appear to function in the organization of the matrix with important roles in early development and wound healing.39 They appear to alter the orientation of collagen fibers both in vitro and in vivo.143 Proteoglycans may also play a separate role in regulating the mineralization process, although this is still unclear. For instance, one study demonstrated that addition of chondroitin sulfate to cultures of periosteal chick cells would block mineral nodule formation, even when 10 mM sodium (3-glycerolphosphate was present.144 Various studies have suggested the presence of a 20 nm proteoglycan layer exists against the side of an in vivo implant.6-8283 Linder demonstrated a 20 nm thick layer in TEM preparations from unloaded rabbit tibial implants and described a sensitivity of this layer to hyaluronidase and chrondroitinase.6 Further studies, in culture, have also demonstrated a ruthenium red-staining layer and a unique interfacial layer, observed in

TEM preparations, between the proposed PG layer and the oxide surface of cpTi.86 A criticism of these studies evaluating this 20 nm layer is the lack of more refined biochemical and immunohistochemical approaches. With the availability for PG-specific monoclonal antibodies and the immunogold technique, a more defined picture of the events occurring both in culture and in vivo should be attempted.

7. Fibronectins This family of extracellular proteins has been studied extensively over the last decade with a number of important discoveries made in the characterization of fibronectin. It was in the evaluation of fibronectin's structure that various molecular domains of activity were described. These included a 75 kDa cell binding region, a gelatin binding region, a heparan sulfate binding region, and a fibrin binding region. It was within the cell binding region that the RGD cell binding sequence and a synergistic site were first described, allowing fibronectin to be recognized by the FNreceptor(s). Other sites for cell binding have since been described, including an embryonic form that contains an alternatively spliced CS1 domain that allows lymphocyte and melanoma cell recognition (through a a 4 Pj integrin).145 The most common integrin receptor recognizing fibronectin is the a ^ integrin class, present on many cell types, including osteoblasts. Since fibronectin has a large dimer structure, connected by covalent disulfide linkages toward the C-terminal end, it has been suggested to act as a biological "glue" or bridge between the ligands recognizing its various domains. For extensive reviews, please see References 146 to 151. Fibronectin can be found in the plasma at relatively high levels and therefore, although it can be isolated from bone matrix, the exact source or even function of fibronectin in bone is still unclear.39

8. Thrombospondin (TS) TS is a trimeric 420,000 Da glycoprotein that shares multiple properties with the fibronectin class of matrix proteins.152 It is produced by nu-


merous cell types, including osteoblasts, and is found in the extracellular matrix of bone.153 Like the NCPs already described, thrombospondin has a domain structure with a cell binding GRGDS sequence, like FN, and binds to osteonectin, a molecule with which it often co-precipitates.39 It binds strongly to hydroxyapatite,39 and has a Cterminal region with multiple calcium binding sites. The ability of the molecule to mediate cell attachment appears to be very sensitive to calcium since replacing calcium after EGTA treatment still blocks cell attachment to preadsorbed thrombospondin on tissue culture plastic.152 In vitro, thrombospondin increases bone cell attachment but not spreading.152 The presence of thrombospondin is correlated with spatial and temporal areas of development and potentially plays a role in cell adhesion, proliferation, and migration.154 C. Growth Factors and Osseointegration With the current explosion in the number of new growth factors described, it would be beyond the scope of this review to even start addressing this exciting but complex topic. Current knowledge and descriptions are based, much like the properties of the various noncollagenous proteins, on extracts of various tissues. This approach, although a valuable first step, removes the potential factor from its normal environment, resulting in the potential for artifactual description when assayed in culture. This approach is even worse when isolated cell lines are exposed to a single factor, often at pharmacological doses, in order to describe a certain effect. What's more, these factors, as with the prostanoids, operate in a very local paracine or autocine manner where use of even minute quantities in culture will quickly lead to artifacts as all cells become exposed to the potential factor. With these caveats in mind, we have attempted to list the various factors of potential importance in the osseous wound site and refer the reader to more in-depth review articles where possible.38155 The important growth factors are related to bone physiology and osseointegration are shown in Table 2. In general, the growth factors found associated with the bone matrix are mitogenic in na-

Factor Ref.

Fibroblast growth factors (FGF) 181, 182 Acidic-FGF Basic-FGF Insulin-like growth factor (IGF) 182 IGF-1 IGF-2 Transforming growth factors (TGF) 163, 173, 182, 183 TGF-p, TGF-p2 TGF-P12 TGF-p3

ture (FGF, PDGF) and potentially regulate osteoblast phenotypic behavior (e.g., insulin-like G F ) i56-i6o piatelet-derived growth factor (PDGF) is isolated from various osteosarcoma matrices and is produced and released by aggregated platelets. This can lead to the stimulation of collagen synthesis by osteoblast cultures as well as stimulating an increase in the resorptive response. In fact, on balance, since PDGF appears to have a stronger response on osteoblasts, it may actually be more important as a regulator of the resorptive response rather than a formative one.38 It is possible that growth factors, such as PDGF, may affect the normal osteogenic response around an implant since chronic exposure to excessive stresses may lead to inflammation (along with the chronic exposure to a bacterial plaque) that potentially could lead to a shifting in the homeostatic state from one of formation to one of resorption. Complicating this scenario is the release of osteoclast-activating factors (interferon) and other cytokines in the local environment. 1. Transforming Growth Factor-fl (TGF-

Transforming growth factor-(3(s) is another family of growth-promoting peptides with numerous in vitro effects, including increasing the rate at which osteoprogenitor cells enter the Sphase, as well as increased collagen synthesis (transcriptionally and posttranscriptionally).161162 For an in-depth review, please see References 163 and 181. TGF-p is produced by


osteoblasts (transformed and from primary cultures) and promotes the expression of bone-related alkaline phosphatase and osteonectin production in osteoblast cell lines.164165 It is a significant component of the osseous extracellular matrix that may act as a delayed regulator of bone formation by being released during the resorption of the mineralized matrix during remodeling.166168 It has also been proposed as one of the ' 'couplers" for hormone activation between osteoblasts and bone-resorbing osteoclasts.163169 In long-term cultures of human bone marrow cultures, Chenu et al. described a direct inhibitory effect of TGF-0 on the differentiation of osteoclasts, suggesting the role of this factor is opposed to the effects of PDGF.167 TGF-p has also been shown to regulate the formation of various extracellular matrix proteins already discussed in this article. When added to cultures of osteoblast-like cells (ROS 17/2.8), TGF-P! was shown to increase the level of cytoplasmic osteopontin mRNA and levels of osteopontin protein in a dose-dependent manner.124 TGF-f$ has also been suggested to mediate resorption by increasing prostaglandin release in organ culture.170 In two recent papers, Massague has described a potential unifying theory of TGF-(3's actions: the control of cell adhesion.171172 Massague's group has shown that TGF-pJ can elevate the expression of a-subunits and the (3, integrin subunit at both the transcriptional and translational level in a parallel but independent manner.171 TGF-P! enhanced the rate of heterodimer assembly and exposure of the functional cell surface integrin receptor in fibroblasts. TGF-fi! was then found to elevate the expression of integrin receptors for vitronectin (avfJ3) in various cell types such as various fibroblast and osteosarcoma cell lines. Interestingly, TGF-p, appears to specifically downregulate laminin's a 3 integrin subunit in MG-63 osteosarcoma cells, while up-regulating other a and $l subunits.173 This suggests that growth factors, like TGF-(3 may convey their activity, through both direct actions and through a manipulation of the cells with the matrix present around them. Hence, this is a potential explanation of how growth factors, with numerous effects on multiple cells, may convey a specific action on a particular cell type. It remains to be

seen how TGF-(3 may alter the healing response around an implant site because the types of cells that will be exposed to this factor will be constantly changing, although the effects of TGF-(3 on various cell types emphasizes the importance of cellular competence (as in cell surface receptors) in being able to respond to an extracellular matrix. In the classic work by Urist, demineralized bone matrix was isolated and implanted subcutaneously. This matrix then underwent an endochondral-like mineralization process that has been studied extensively, whereby an osteoinductive component of this matrix has been described as bone morphogenetic protein (BMP).174 More recently, this BMP has been characterized as consisting of at least four different proteins (BMP1, BMP2A, BMP2B, and BMP3). Upon cloning and sequence analysis, the BMP-2A and BMP-2B have been found to have significant structural homology to the TGF-p family.175 BMP-3 has a cDNA structural homology to osteogenin, a heparin binding protein described by Reddi's group.175 The protein has been purified from bovine matrix using chromatographic procedures, arriving at a nonreduced protein about 30 to 40 kDa in weight.176 Osteogenin has been shown to elicit significant mineralization in ectopic matrix implants,177179 and has recently been shown to affect the proliferation and differentiation of the mouse osteoblast-like cell line, MC3T3-E1.180 In this model system, osteogenin, added to the culture medium, appeared to inhibit proliferation and encourage the differentiation of the cell line. Alkaline phosphatases, a series of cell membrane enzymes associated with bonelike expression,39 was increased, as was collagenase digestible material in confluent cultures.180

VI. CONCLUSIONS In this review we have attempted to summarize and review what little is known about bone matrix expression, particularly as it relates to a clinical phenomenon described as "osseointegration". We have then reviewed some of the more important matrix proteins described in bone matrix and how the proper orchestration of the matrix may affect osseointegration. Unfortu-


nately, we are still at a state of development where only lists and individual activities of various proteins can be described. We do not know how many of these factors and proteins actually interact in situ, although it is quite reasonable to assume their individual functions are probably altered to a significant degree in the complex and constantly changing milieu present around a healing implant. What information is known about the extracellular matrix layer is at best only anecdotal morphological observations that cry for a more in-depth biochemical analysis. It is our opinion and bias that in vitro approaches will provide the controlled environment in which the resolution of the matrix composition will be defined. These approaches allow for a defined experimental approach, an identification of potential target cells, and provide the resolution needed for biochemical analysis. Unfortunately, these approaches can also provide artifactual environments because tissues are removed from their normal environment and often lack specific cellcell and cell-matrix interactions that only occurs in vivo. Thus, future work in this area must start with the most understandable level and then with that knowledge in tow, move forward to the next higher level of complexity. This scenario is in contrast to the typical approach used today where cursory histological and histochemical approaches are used to investigate the plethora of new implant devices literally flooding the market.











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