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Microscopy of bone cells, bone tissue, and bone healing around implants.

Newer methods of scanning microscopy using both light and electrons are particularly relevant to the study of bone cells, bone matrix organization, matrix mineralization, bone modeling and remodeling, and the adaptation of cells and matrix to implants. Most of such studies are conducted on retrieved implants, at least after the death of the related tissue. Because the retention of the tissue-implant relationship in such preserved tissue is crucial for critical evaluation of the implant, methods based on the study of flat surfaces of embedded tissue blocks are very important. Using electrons, the backscattered electrons in a scanning electron microscope can be employed to evaluate mean atomic number (density) and cathodoluminescence can identify polymers and fluorescent labels. Using light, confocal microscopical techniques permit the examination of layers deep to the block face. Confocal reflected and fluorescence methods allow the study of cell behavior upon both transparent and opaque substrates in the laboratory. Examples of the above are presented and interpretation problems discussed. Current experiments are aimed at enabling the study of bone wound healing and bone adaptation to implanted materials in vivo, through the implantation of optical quality windows and/or newly conceived and designed microscopical objective lenses.

Animals↗

Matrix concentration of insulin-like growth factor I (IGF-I) is negatively associated with biomechanical properties of human tibial cancellous bone within individual subjects.

Insulin-like growth factor-I (IGF-I), abundant in bone matrix, is believed to play an important role during bone development and remodeling. To our knowledge, however, few studies have addressed the relationship between the concentration of IGF-I in bone matrix and the biomechanical properties of bone tissue. In this study, forty-five cylindrical specimens of cancellous bone were harvested from six human tibiae and scanned using micro-computed tomography (microCT). The bone volume fraction (BV/TV) was calculated from three-dimensional (3D) microCT images. Mechanical tests were then performed on a servohydraulic testing system to determine the strength and stiffness of cancellous bone. Following mechanical testing, the concentration of IGF-I in bone matrix was measured by using an enzyme-linked immunoabsorbent assay (ELISA). Within each subject, the concentration of IGF-I in bone matrix had significant (P<0.01) negative correlations with the bone volume fraction, strength, and stiffness of cancellous bone. In particular, the anterior quadrant of the proximal tibia was significantly (P<0.02) greater in IGF-I matrix concentration and marginally significantly lower in strength (P=0.053) and stiffness (P=0.059) than the posterior quadrant. The negative correlations between the cancellous bone matrix concentration of IGF-I and cancellous bone biomechanical properties within subjects found in this study may help us understand the variation of the biomechanical properties of cancellous bone in proximal human tibiae.

Biomechanical Phenomena↗

Phagocytosis of different matrix components by different cell types at bone-forming sites in cultured mouse calvariae.

Sites of bone formation on fragments of parietal bone of fetal-mice cultured for 10 days were examined by electron microscopy after addition of either ruthenium red or ferrocyanide to the postfixation fluid. Osteoclasts, osteoblast-like cells, and macrophages were the principal active cells at these formation sites. The mononuclear cells (osteoblast-like cells and macrophages) in the osteoid tissue showed evidence of having incorporated elements of calcified tissue. Osteoblast-like cells had phagocytized collagen fibrils and calcified bone matrix. This occurred more frequently in the calcifying area. Mononuclear macrophages showed not only phagocytosis and digestion of cellular debris and bone spicules in the osteoid, but also active incorporation of calcified bone matrix that had been detached from its surroundings by its pseudopod-like projections from long cytoplasmic processes. Collagen fibrils were seldom observed within the macrophages. These observations suggest that in our culture system osteoblast-like cells and macrophages at bone formation sites have a phagocytic role in bone remodeling.

Animals↗

Expression of major bone extracellular matrix proteins during embryonic osteogenesis in rat mandibles.

It is not known how bone proteins appear in the matrix before and after calcification during embryonic osteogenesis. The present study was designed to investigate expressions of the five major bone extracellular matrix proteins--i.e. type I collagen, osteonectin, osteopontin, bone sialoprotein and osteocalcin--during osteogenesis in rat embryonic mandibles immunohistochemically, and their involvement in calcification demonstrated by von Kossa staining. Wistar rat embryos 14 to 18 days post coitum were used. Osteogenesis was not seen in 14-day rat embryonic mandibles. Type I collagen was localized in the uncalcifed bone matrix in 15-day mandibles, where no other bone proteins showed immunoreactivity. Osteonectin, osteopontin, bone sialoprotein and osteocalcin appeared almost simultaneously in the calcified bone matrix of 16-day mandibles and accumulated continuously in 18-day mandibles. The present study suggested that type I collagen constitutes the basic framework of the bone matrix upon which the noncollagenous proteins are oriented to lead to calcification, whereas the noncollagenous proteins are deposited simultaneously by osteoblasts and are involved in calcification cooperatively.

Animals↗

An in vitro assay of bone development using fetal long bones of mice: morphological studies.

The purpose of this study was to examine the morphological changes in an in vitro system in which the two elements of bone modelling, formation and resorption, could be studied simultaneously. Pregnant mice were killed on days 15, 16 and 17 of gestation, the fetuses were removed and the radii and ulnae dissected free of soft tissue. The bones were cultured for 6 days in media (BGJ) supplemented with 20% fetal calf serum and 150 micrograms/ml vitamin C. Growth and mineralization were estimated by measuring the total length of the bone, and diaphysis, and by light and transmission electron microscopy (TEM). The results of this study indicate that there is a continuous measurable increase in the total length of fetal mouse long bones over the 6 days of culture. These bones show a continuous growth of periosteal bone, with mesenchymal tissue penetrating into the diaphyseal shaft, and development of bone marrow like tissue. TEM examination showed differentiation of mesenchymal cells to osteoblasts, formation of new bone matrix and bone mineralization similar to that found in developmentally matched controls. In the cartilagenous epiphyses, however, many hydroxyapatite crystals were not associated with matrix vesicles. In addition, some of the chondrocytes of the hypertrophic zone appeared to be dedifferentiating into mesenchymal cells with osteoblast-like features. In spite of the lack of osteoclasts in the 15- and 16-day explants, osteoclasts appeared in the diaphysis after 2 and 4 days in culture. Our results suggest that this system can serve as a good model for the study of bone formation and resorption as they occur, simultaneously, during bone modelling.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Experimental induction of heterotopic bone.

Heterotopic bone can be induced in experimental animals by trauma to the soft tissues, by induction from living cells, or by extracts from bone and teeth. In the first two types, the mechanism of the inductive process is not known, whereas in the latter, a factor isolated from bone matrix induces bone formation. Mesenchymal cells in bone marrow are determined for development into cartilage and bone cells and only an unspecific stimulus, such as trauma or autotransplantation, is sufficient for the development into mature osteogenic tissue. Mesenchymal cells will not differentiate into bone cells unless stimulated by a specific inductive substance, bone morphogenetic protein (BMP). Implanted to a heterotopic site, BMP induces undifferentiated mesenchymal cells into a bone morphogenetic pathway of development, causing heterotopic bone formation. The quantitative inductive response is dependent on the source of the BMP, and the bone formation is also determined by the recruitment of inducible target cells and by the environment at the implantation site. Hence, the environment at the implantation site is of major importance for the amount of bone formed. BMP initiates a cascade of events that is modulated by endocrine and paracrine factors. The heterotopic bone has all the morphologic and biochemical characteristics of orthotopic bone, is subjected to turnover, and even has the intriguing ability to generate the formation of bone marrow. Experimental induction of heterotopic bone has become a most useful method to study osteoneogenesis and has supplied important information on the prerequisites for new bone formation and on the regulation of bone metabolism.

Animals↗

Effects of cyclosporin A on experimental new bone formation in rats.

The effects of the immunosuppressive drug cyclosporin A (CsA) on bone induction by demineralized allogeneic (rat) bone matrix (DABM) and demineralized xenogeneic (rabbit) bone matrix (DXBM) were studied. Growing rats were implanted with three samples each of DABM and DXBM. Groups of eight rats were treated with 0.5 or 2 mg CsA/kg body weight for four weeks and compared with a placebo group. Cyclosporin A treatment enhanced bone induction in DABM implants by 40 to 50% at four weeks, whereas there was no difference from the control group at eight weeks. Demineralized xenogeneic bone matrix induced virtually no bone in control rats at four weeks, whereas the net bone formation increased four to five times in both groups of CsA-treated rats. At eight weeks, DXBM without CsA had induced some bone formation, and the amount was almost equal to that of DABM implants in CsA-treated groups. Also, the mineral accretion rates of DXBM were equal to DABM implants in CsA-treated rats. Cyclosporin A treatment doubled the uptake of 45Ca in the orthotopic skeleton (femora) at four weeks without affecting the mineral content, indicating an increased mineral turnover. Immunologic reactions may inhibit bone induction by DXBM, which can be counteracted by treatment with CsA.

Animals↗

Bacterially induced bone destruction: mechanisms and misconceptions.

Normal bone remodelling requires the coordinated regulation of the genesis and activity of osteoblast and osteoclast lineages. Any interference with these integrated cellular systems can result in dysregulation of remodelling with the consequent loss of bone matrix. Bacteria are important causes of bone pathology in common conditions such as periodontitis, dental cysts, bacterial arthritis, and osteomyelitis. It is now established that many of the bacteria implicated in bone diseases contain or produce molecules with potent effects on bone cells. Some of these molecules, such as components of the gram-positive cell walls (lipoteichoic acids), are weak stimulators of bone resorption in vitro, while others (PMT, cpn60) are as active as the most active mammalian osteolytic factors such as cytokines like IL-1 and TNF. The complexity of the integration of bone cell lineage development means that there are still question marks over the mechanism of action of many well-known bone-modulatory molecules such as parathyroid hormone. The key questions which must be asked of the now-recognized bacterial bone-modulatory molecules are as follows: (i) what cell population do they bind to, (ii) what is the nature of the receptor and postreceptor events, and (iii) is their action direct or dependent on the induction of secondary extracellular bone-modulating factors such as cytokines, eicosanoids, etc. In the case of LPS, this ubiquitous gram-negative polymer probably binds to osteoblasts or other cells in bone through the CD14 receptor and stimulates them to release cytokines and eicosanoids which then induce the recruitment and activation of osteoclasts. This explains the inhibitor effects of nonsteroidal and anticytokine agents on LPS-induced bone resorption. However, other bacterial factors such as the potent toxin PMT may act by blocking the normal maturation pathway of the osteoblast lineage, thus inducing dysregulation in the tightly regulated process of resorption and replacement of bone matrix. At the present time, it is not possible to define a general mechanism by which bacteria promote loss of bone matrix. Many bacteria are capable of stimulating bone matrix loss, and the information available would suggest that each organism possesses different factors which interact with bone in different ways. With the rapid increase in antibiotic resistance, particularly with Staphylococcus aureus and M. tuberculosis, organisms responsible for much bone pathology in developed countries only two generations ago, we would urge that much greater attention should be focused on the problem of bacterially induced bone remodelling in order to define pathogenetic mechanisms which could be therapeutic targets for the development of new treatment modalities.

Alveolar Bone Loss↗

Carboxyterminal telopeptide of type I collagen, ICTP, as a marker of matrix degradation in neonatal mouse calvarial bones, in vitro.

UNLABELLED: Bone resorption, in vitro, is often measured as the release of prelabelled 45Ca from neonatal mouse calvarial bones, or from fetal rat long bones. In this report we describe a technique to measure the breakdown of bone-matrix, in vitro. We also describe a new way to dissect neonatal mouse calvarial bones, in order to obtain large amounts of bone samples. Twelve bone fragments were dissected out from each mouse calvaria and were thereafter cultured in CMRL 1066 culture medium in serum-free conditions in 0.5 cm2 multiwell culture dishes. Matrix degradation after treatment with parathyroid hormone was assessed by measuring the amount of carboxyterminal telopeptide of type I collagen (ICTP) by RIA. The data on matrix degradation was compared to the release of prelabelled 45Ca from neonatal mouse calvarial bones. We found that the dose-responses for parathyroid hormone-induced release of prelabelled 45Ca and ICTP were identical. IN CONCLUSION: RIA-analysis of the ICTP-release is an easy and accurate method to measure degradation of bone-matrix, in vitro. Furthermore, the new dissection technique, described in this report, makes it easy to obtain large amounts of bone samples and thus to perform extensive experiments, e.g. dose-responses for agents that enhance bone resorption.

Animals↗

Age- and gender-related changes in the distribution of osteocalcin in the extracellular matrix of normal male and female bone. Possible involvement of osteocalcin in bone remodeling.

With increasing age, bone undergoes changes in remodeling that ultimately compromise the structural integrity of the skeleton. The presence of osteocalcin in bone matrix may alter bone remodeling by promoting osteoclast activity. Whether age- and/or gender-related differences exist in the distribution of osteocalcin within individual bone remodeling units is not known. In this study, we determined the immunohistochemical distribution of osteocalcin in the extracellular matrix of iliac crest bone biopsies obtained from normal male and female volunteers, 20-80 yr old. Four different distribution patterns of osteocalcin within individual osteons were arbitrarily defined as types I, II, III, or IV. The frequency of appearance of each osteon type was determined as a percent of the total osteons per histologic section. The proportion of osteons that stained homogeneously throughout the concentric lamellae (type I) decreased in females and males with increasing age. The proportion of osteons that lack osteocalcin in the matrix immediately adjacent to Haversian canals (type III) increased in females and males with age. Osteons staining intensely in the matrix adjacent to Haversian canals (type II) increased in females and was unchanged in aging males. Osteons that contained osteocalcin-positive resting lines (type IV) increased in bone obtained from males with increasing age but were unchanged in females. Sections of bone immunostained for osteopontin (SPP-I), osteonectin, and decorin did not reveal multiple patterns or alterations in staining with gender or increasing age. We suggest that the morphology of individual bone remodeling units is heterogeneous and the particular morphologic pattern of osteocalcin distribution changes with age and gender. These results suggest that differences in the distribution of osteocalcin in bone matrix may be responsible, in part, for the altered remodeling of bone associated with gender and aging.

Adult↗

[Immunological comparison of differently treated allografts of bone].

The immunnologic rejection induced by differently treated allografts of bone was compared. Methods Fresh bone (FB), autolyzed antigen-free bone (AAA), bone matrix gelatin (BMG), demineralized bone matrix (DBM) were implanted into the muscle pouch of mice, then, the immunological tests and alkaline phosphatase assay were conducted. Results Allogeneic FB induced the highest level of serum antibody in the host and stimulated lymphocytes proliferation remarkably in vitro; in contrast, AAA, BMG and DBM caused low titer of antibody and inhibited lymphocytes reproduction in vitro. Conclusions. Immunological rejection restrained osteogenesis of the bone implant, whereas the osteoinductive substance of bone suppressed immunological reaction.

Alkaline Phosphatase↗

Immunolocalization of bone extracellular matrix proteins (type I collagen, osteonectin and bone sialoprotein) in human dental pulp and cultured pulp cells.

AIM: To simultaneously analyse the expression of type I collagen, osteonectin and bone sialoprotein (BSP) in human dental pulp of different ages. METHODOLOGY: Cultured dental pulp fibroblasts (FP1 cell line), pulps from dental germs with incomplete root formation (n = 4) and pulps of erupted teeth with total root formation (n = 4) were used. Bone proteins were searched by immunohistochemistry and immunofluorescence using polyclonal antibodies and compared among the three groups assessed. RESULTS: Immunohistochemistry detected the three proteins in dental pulp tissue, as it labelled extracellular matrix, predentine and odontoblasts. The BSP label was weaker, when compared to both type I collagen and osteonectin. The presence of type I collagen was more evident in pulps from erupted teeth, when compared to germ dental pulps. On the other hand, a strong expression of osteonectin in germ dental pulps was observed. CONCLUSIONS: Regardless of the degree of maturation, dental pulps present type I collagen, osteonectin and BSP in the extracellular matrix (ECM) and in the odontoblastic layer. Thus, the results suggest that these proteins are related to the production and mineralization of dentine.

Cell Line↗

An evaluation of human demineralized bone matrices in a rat femoral defect model.

The osteoconductive and osteoinductive potential of two human allogeneic demineralized bone matrix putties were compared in a critical-sized athymic rat femoral defect model. Defects were treated with (1) a demineralized bone matrix in a hyaluronic acid carrier, (2) a demineralized bone matrix in a glycerol carrier, (3) a hyaluronic acid carrier alone, or (4) with no implant. Radiographic examinations and histologic analyses were done at 4, 8, and 16 weeks postoperatively. Eight of the 48 defects treated with a demineralized bone matrix and none of the 36 surgical controls showed complete radiographic healing by 16 weeks and no statistically significant difference between the radiographic scores for the two demineralized bone matrix preparations was found. On histologic review, both preparations of demineralized bone matrix had passive remineralization. The largest foci of endochondral ossification were seen in limbs treated with a demineralized bone matrix in a hyaluronic acid carrier. The 8-mm rat femoral defect allows for stringent assessment of the osteoinductive potential of bone graft substitutes. Hyaluronic acid and glycerol are viable carriers for demineralized bone matrices. As both de-mineralized bone matrices tested provided an adequate osteoconductive matrix and showed some, although limited, osteoinductive capacity, these materials should be used in clinical practice only as bone graft extenders or enhancers.

Aged↗

Collagen: the organic matrix of bone.

Collagen is the principal organic matrix in bone. The triple helical region of the molecule is 1014 amino acids long. In fibrils these molecules are staggered axially by integers of 234 residues or 68 nm (D). This axial shift occurs by self-assembly and can be understood in terms of a periodicity in the occurrence of apolar and polar residues in the amino acid sequence. Because the molecular length L = 4.47 D, there are gaps 1.5 X 36.5 nm regularly arrayed throughout the fibrils. The three-dimensional molecular arrangement is a quasi-hexagonal lattice with three distinct values for the principal interplanar spacings. Analysis of the intensity distribution in the medium-angle X-ray diffraction patterns from tendons has produced the following picture of the molecular arrangement in fibrils (Fraser et al. 1983). The molecular helices have a coherent length of 32 nm and are tilted parallel to a specific place within the lattice. A regular azimuthal interaction exists between these helices. This crystalline region could be the overlap region with a non-crystalline gap region. However, the gap is still regular axially and the molecular helices retain their structure; their lateral packing is perturbed although they retain a 'gap'. Neutron and X-ray scattering experiments have shown that calcium hydroxyapatite crystals occur in the gap and are nucleated at a specific though unknown location within the gap. The c-axis of the apatite crystals is parallel to the fibril axis and its length c = 0.688 nm is close to the axial periodicity in a protein with an extended beta-conformation. If the telopeptides at the end of a collagen molecule do have this conformation they would either have a highly heterogeneous conformation or exist in a folded manner because the overall length of the telopeptides is shorter than a regular collagen repeat of 0.029 nm would allow.

Amino Acid Sequence↗

Cellular activity, matrix proteins, and aging bone.

The extracellular matrix of bone is composed of proteins from both local and exogenous sources. Many of these are known growth factors (e.g., transforming growth factors-beta; insulin-like growth factors; and fibroblast growth factors) which concentrate in mineralized bone and probably contribute to the ability of bone to regenerate itself on injury. With advancing age, human osteoblasts have reduced bone formative properties and in vitro, osteoblasts from fetuses generally have greatly increased proliferative and biosynthetic capacities compared to cells from adult donors. Aside from type I collagen, many noncollagen components are synthesized by osteoblasts and secreted to the bone matrix space. Several of these are cell attachment proteins (e.g., fibronectin, thrombospondin, osteopontin) which greatly influence cytodevelopment and differentiation. They are degraded to lower molecular weight fragments with advancing age, probably deactivating their true bioactivities. It is not know whether the age-related degradation of these proteins affects bone cell function in aged individuals. Several of the bone matrix proteins are also found in platelets and have been implicated in the wound-repair process. One of these, osteonectin, is found in a wide variety of nonbone cell systems, but only in periods of rapid growth and proliferation. Osteonectin production is the highest and is maintained the longest in bone compared to all other tissues of the body. Thus, reduced osteonectin production in aged bone cells may be an important parameter for further study.

Aging↗

Bone extracellular matrix induces homeobox proteins independent of androgens: possible mechanism for androgen-independent growth in human prostate cancer cells.

BACKGROUND: Differences in gene expression in prostate cells are believed to be secondary to epithelial-stromal interactions. We theorized that bone matrix may provide a fertile "soil" for prostate cancer by inducing androgen-dependent genes and allowing for androgen-independent growth. METHODS: Human prostate cancer cells (LNCaP) were grown under different conditions and analyzed for differential expression of mRNA. LNCaP cells were grown in the presence of 10 nM dihydrotestosterone (DHT), on extracellular matrix (ECM) derived from bone cells (without exogenous DHT), and on plastic culture dishes without exogenous DHT. A differential display of mRNA produced by LNCaP cells grown in the above conditions was then analyzed. RESULTS: Multiple unique transcripts were present in cells that were grown in the presence of DHT and on bone ECM (without exogenous DHT), but not on plastic culture dishes without exogenous DHT. Nine of these transcripts were then cloned and analyzed. Many (5/9) of these transcripts were found to contain multiple ATTA motifs in their corresponding 3'-untranslated regions. ATTA motifs have been shown to be homeobox protein-binding sites. Homeobox proteins and their target genes are thought to regulate cellular differentiation. Consistent with this, we demonstrated by reverse transcription polymerase chain reaction (PCR) that homeobox genes were differentially expressed in LNCaP cells when the cells were grown in the presence of DHT and on bone ECM (without exogenous DHT), but not on plastic culture dishes without exogenous DHT. Furthermore, we assayed LNCaP/fetal fibroblast chimeric tumors (n = 8) that were grown in male nude mice. Some of these tumors continued to grow in these mice despite treatment with surgical castration. In blinded studies, we were able to determine which tumor samples were androgen independent by their expression of homeobox genes. All samples that were androgen independent (n = 4) expressed the homeobox genes. Finally, gel retardation assay demonstrated that the homeobox proteins were able to bind to our cloned DNA sequences. Furthermore, footprinting analysis showed that the homeobox proteins bound to the ATTA motif in the 3'-region of our target DNA. CONCLUSIONS: Bone ECM, in the absence of DHT, has the ability to regulate androgen-responsive genes. Furthermore, many of these genes contain homeobox binding sites and the expression of homeobox genes may itself be regulated by bone ECM. If so, this may partially explain the clinical observation that bone provides a fertile "soil" for prostate cancer growth and metastasis.

Adenine↗

[New-bone formation by osteogenic protein-1 and autogenic bone marrow in a critical tibial defect model in sheep].

AIM: Osteogenic Protein-1 (OP-1) is known to be a very potent osteoinductive growth factor. However, experimental studies using critical-size defect models in the weight-bearing lower extremity show non-uniform results. Therefore, we studied the osteoinductivity of OP-1 in a tibial worst-case defect model in sheep. Potential improvement of OP-1 induced new bone formation using a composite graft with autogenous bone marrow was to be investigated. METHOD: In 19 sheep a 5 cm segmental defect of the tibial diaphysis was treated by intramedullary nailing and filled with the following implants: 5 mg OP-1 + inactivated demineralized bone matrix (group 1; n = 6); 5 mg OP-1 + inactivated demineralized bone matrix + 5 ml autogenous bone marrow (group 2; n = 5); autogenous cancellous bone (group 3; n = 4), or inactivated demineralized bone matrix + 5 ml autogenous bone marrow (group 4; n = 4). RESULTS: In total, 3 out of 10 defect sites treated with OP-1 were completely bridged radiographically by 12 weeks. Initially, x-rays showed accelerated new bone formation by use of the composite grafts containing OP-1 and autogenous bone marrow. However, 12 weeks post surgery 3D-CT-volumetry could not detect significant differences of new bone formation within the defect sites treated by OP-1 with or without bone marrow, while new bone formation by autogenous cancellous bone was better than by OP-1. CONCLUSION: In our worst case defect model, the osteoinductive potential of OP-1 is initially accelerated but 12 weeks post surgery not increased when combined with autogenous bone marrow transplantation. So far, critical segmental bone defects of the weight-bearing lower extremity can not be bridged regularly in our model by use of OP-1. Therefore, for the treatment of such critical defects with rotational instability the examined application device of OP-1 can not yet be recommended.

Animals↗

Closure of critical sized defects with allogenic and alloplastic bone substitutes.

PURPOSE: This study evaluates bone regeneration of critical sized cranial vault defects in New Zealand white rabbits using four commercially available bone substitutes: OsteoSet (calcium sulphate pellets), DynaGraft Putty (demineralized bone matrix delivered in a poloxmer excipient), Norian CRS, and Bone Source (two commercially available calcium phosphate cements). MATERIALS AND METHODS: Critical sized defects 15 mm in diameter were created bilaterally in the parietal bones of 30 adult male New Zealand White rabbits. They were divided into three groups with ten animals in each. Bone healing was assessed clinically, radiographically, and histomorphometrically. Group 1 had calcium sulfate bioimplant on one side of the calvarium and an unfilled defect on the contralateral side. Group 2 had DBM putty on one side and Poloxamer gel on the contralateral side. Group 3, the Calcium phosphate cements (CPC), had Norian CRS on one side and Bone Source on the contralateral side. Five animals in each group were killed at 6 weeks and 12 weeks post operatively. RESULTS: All unfilled defects healed with fibrous scar, as did the Plaster of Paris and the poloxamer gel defects. Defects reconstructed with the demineralized bone matrix putty healed with bone throughout the entire defect. This was obvious clinically and radiographically where the defects appeared completely filled with a dense radiopaque tissue. The six-week group displayed new bone formation (87.1%) surrounding the remaining allogeneic particles. Resorption was evidenced by the presence of osteoclastic activity and by the significant decrease in the size of the demineralized bone particles. By 12 weeks, the demineralized bone putty bioimplant was almost completely replaced by new bone (95.5%). Both calcium phosphate cement groups (Norian CRS and Bone Source) had identical patterns of healing. They clinically were visible and firm and uniformly radiopaque with little evidence of new bone formation. Histologically the cement remained unresorbed with little new bone with in the defect at 12 weeks. CONCLUSIONS: The utilization of a demineralized bone matrix putty appeared to allow for complete closure of critical sized calvarial defects in New Zealand white rabbits with viable new bone at 12 weeks.

Analysis of Variance↗