In vivo analysis of the half-life of the osteoinductive potential of demineralized bone matrix using diffusion chambers

Summary Subcutaneous implantation of demineralized bone matrix (DBM) from rat initiates a sequence of developmental events that results in endochondral bone formation. This investigation examined the modification of the osteoinductive potential of DBM during the intial stages of this developmental cascade. Diffusion chambers (DC), constructed with filters of known pore size, permitting or excluding cells from entering the chambers, and containing DBM were subcutaneously implanted into Long-Evans male rats for specific time periods (1–7 days). DC were recovered and the osteoinductive potential of the matrix from these chambers was then tested by subcutaneous implantation and assaying the resulting day 11 plaque tissue enzymatically for alkaline phosphatase activity, and histologically for evidence of chondrogenesis and osteogenesis. The possible modification of DBM by local systemic factors (enzymatic degra-dation) or contact by polymorphonuclear leukocytes (PMNs) was also investigated. We have concluded from this study that the osteoinductive potential of DBM has a half-life of 5–7 days following implantation and although the enzymes collagenase, elastase, and trypsin abolished this activity, pepsin significantly enhanced it. Culture of PMNs with matrix prior to its implantation appeared to have little effect. Furthermore, during the initial stages of matrix-induced endochondral bone formation, DBM serves as both the instructive inducer and permissive substratum required in this process.     

Biochemical differences between dystrophic calcification of cross-linked collagen implants and mineralization during bone induction

Summary Ectopic calcification of diseased tissues or around prosthetic implants can lead to serious disability. Therefore, calcification of implants of glutaraldehyde-cross-linked collagenous tissues and reconstituted collagen was compared with mineralization induced by demineralized bone matrix (DBM). Whereas implants of DBM accumulated large amounts of calcium and a bone-specific γ-carboxyglutamic acid protein (BGP or osteocalcin) following implantation in both young and older rats, implants of cross-linked pericardium calcified with only traces of BGP. Glutaraldehyde-cross-linked DBM failed to calcify after implantation in 8-month-old rats for 2–16 weeks. Implants of cross-linked type I collagen exhibited small calcific deposits 2 weeks postimplantation but calcium content eventually dropped to levels equal to those of soft tissues as the implants were resorbed. The calcium content of DBM implanted in 1- and 8-month-old rats reached comparable levels after 4 weeks, but the BGP content was approximately twice as high in the younger animals than in the older ones. Glutaraldehyde-cross-linked implants of DBM, tendon, and cartilage calcified significantly in young but not in old animals. This form of dystrophic calcification was associated with only trace amounts of BGP. Alkaline phosphatase activity was high in implants of DBM and undetectable in implants of cross-linked collagenous tissues. These results show that implants of glutaraldehyde-cross-linked collagenous tissues and reconstituted collagen calcify to different extents depending upon their origin and the age of the host, and that the mechanism of dystrophic calcification differs significantly from the process of mineralization associated with bone induction as reflected by alkaline phosphatase activity and BGP accumulation.     

Influence of magnesium depletion on matrix-induced endochondral bone formation

Summary The effect of magnesium deficiency on bone cell differentiation and bone formation was investigated using in vivo matrix-induced endochondral ossification as a test system. Demineralized bone matrix was implanted subcutaneously in young (35-day-old) male Long-Evans rats that had been fed a semisynthetic Mg-deficient diet (50 ppm Mg) for 7 days. Plasma Mg levels were reduced to 25–30% of control values at that time. Control rats were pairfed the same diet, supplemented to contain 1000 ppm Mg. The implants were harvested 7, 9, 11, 15, and 20 days after implantation and analyzed for Mg and Ca content,⁴⁵Ca incorporation, and alkaline phosphatase levels. At each stage, plaques (implants) removed from Mg-deficient rats showed retardation in cartilage and bone differentiation and matrix calcification. Magnesium content was markedly reduced when compared to the control plaques. Histological appearance of the matrix-induced plaques confirmed the retardation in bone development and mineralization suggested by the chemical indicators. Most marked was the virtual absence of bone marrow in 20-day-old plaques in Mg-depleted rats. These data show that bone cell differentiation can occur in a severely Mg-depleted environment, although the onset of mineralization and bone remodeling was delayed and bone marrow differentiation was impaired.     

Characterization of Matrix-Induced Osteogenesis in Rat Calvarial Bone Defects: I. Differences in the Cellular Response to Demineralized Bone Matrix Implanted in Calvarial Defects and in Subcutaneous Sites

The cellular and biochemical sequences of osteogenesis induced by implanting demineralized bone matrix (DBM) in rat cranial defects and in subcutaneous sites have been studied by histological, histochemical, and biochemical techniques from days 2 to 28 after implantation. In subcutaneous sites, allogenic DBM induced cartilage cells and matrix for approximately the first 10 days which were subsequently resorbed and replaced by bone with little evidence for the classical endochondral sequence of ossification. In sharp contrast, the first cells that differentiated from the mesenchymal stem cells in the cranial defects were alkaline phosphatase (ALP) positively stained osteoblasts that appeared 3 days after implantation followed by synthesis of bone matrix which calcified shortly thereafter. A few clusters of cartilage cells were observed beginning at days 6–7 which were spatially distinct from the new bone and later resorbed. By day 28 the tissue induced in both the subcutaneous and cranial sites consisted almost solely of bone; however, the total amount of new bone in the subcutaneous implants was significantly less than the mass of bone formed in the calvarial defects. Bovine DBM induced bone formation in rat cranial defects to a very much lesser extent than allogenic DBM. A few cartilage cells were induced by bovine DBM in subcutaneous sites and rapidly resorbed and not replaced with bone. These results clearly indicate that the cellular sequence induced by allogenic and xenogenic DBM and the repair tissues synthesized are distinctly different in the cranial defects from those induced in the subcutaneous sites. Received: 1 September 1998 / Accepted: 15 January 1999     

Characterization of Demineralized Bone Matrix-Induced Osteogenesis in Rat Calvarial Bone Defects: III. Gene and Protein Expression

Our previous studies of rat cranial defect repairs after the implantation of demineralized bone matrix (DBM) have demonstrated that healing occurs initially and principally by the direct induction and proliferation of osteoblasts derived principally from resident mesenchymal stem cells of the dura, and to a lesser extent by resident mesenchymal stem cells of the connective tissues beneath the skin flap. A small amount of cartilage is also synthesized after the direct process of ossification occurs. To further confirm the molecular phenotypes of the repair cells in rat cranial defects, the present study evaluated mRNA expression and synthesis of collagens I, II, and X and osteocalcin in the DBM-induced repair tissue by Northern blot analyses, autoradiography after in vivo ³H-proline labeling of collagen, and immunohistochemistry. The results demonstrated that osteocalcin mRNA appeared in small amounts by day 4 and continued to increase over the experimental period. Much lesser quantities of collagen types II and X mRNAs appeared by day 6 and day 8, respectively. Collagen type I mRNA was present at all times examined but its expression significantly increased by day 5. Autoradiographic and immunohistochemical studies showed that type II collagen was not detected whereas type I collagen was synthesized on days 3–5. The data provide definitive molecular evidence confirming that the initial and by far the major pathway of cranial defects repair induced by implantation of DBM is by the direct induction of resident mesenchymal stem cells to osteoblasts and the direct formation of bone, which is spatially and temporarily distinct from the later formation of cartilage. Received: 30 November 1999 / Accepted: 21 March 2000     

The effect of aging on bone formation in rats: Biochemical and histological evidence for decreased bone formation capacity

Summary Ectopic bone formation by subcutaneously implanted demineralized bone matrix powder (DBM) was assessed biochemically and histologically in Fischer 344 rats of different ages. The total calcium accumulated in implants was greatly depressed in older rats, as was the rate of⁴⁵Ca deposition. High alkaline phosphatase activity appeared later in the 10- and 16-month-old rats compared with 1-month-old rats, and the magnitude of the alkaline phosphatase activity was decreased in 16-month-old rats. The accumulation of the bone-specific vitamin K-dependent bone protein (bone gla protein, BGP) was decreased in the implants in older rats. Histological examination of the implants confirms the decreased ability of aged animals to produce bone in response to DBM. Measurements of total calcium, alkaline phosphatase, and BGP at the site of demineralized bone matrix implants clearly demonstrates that bone formation decreases dramatically with increasing age. Significant differences in total calcium can be detected even between 1-month-old and 3-month-old rats. Serum BGP shows a marked decrease (47%) between 1-month- and 3-month-old rats, a decrease not paralleled by a similar decrease in BGP present in calvarial or tibial bone.     

Evaluation of Partially Demineralized Osteoporotic Cancellous Bone Matrix Combined with Human Bone Marrow Stromal Cells for Tissue Engineering: An In Vitro and In Vivo Study

Allogenous demineralized bone matrix (DBM) represents a potential scaffold for bone tissue engineering due to its close relation in structure and function with autologous bone, but its supply is often restricted by donor availability. Thus, an expanded source of human bone is needed. The aim of this study was to evaluate the capacity of partially DBM scaffolds derived from allogenous cancellous bone of osteoporotic femurs to support osteogenesis of human bone marrow stromal cells (BMSCs) in vitro and in vivo in order to assess their potential use in bone tissue-engineering strategies. Human BMSCs of passage 2 were seeded either on osteoporotic bone–derived DBM scaffolds or on normal bone–derived scaffolds and cultured in osteogenic medium for 14 days. To assess the in vitro proliferation potential and osteogenic differentiation of BMSCs on scaffolds, scanning electronic microscopy observation, DNA content assays, and measurements of alkaline phosphatase activity and osteocalcin content were applied; the results displayed no significant differences between the osteoporotic DBM group and the normal DBM group. After 2 weeks of subculture in vitro, the BMSC/DBM composites were subcutaneously implanted into athymic mice for 8 weeks to evaluate their in vivo bone-forming ability. Histological examination showed tissue-engineered bone formation in the DBM pores in both groups, and no significant differences were observed in either the extent or frequency of new bone formation between these two groups. Based on these results, it can be concluded that osteoporotic bone–derived DBM may serve as a promising scaffold for bone tissue engineering.     

Influence of whole body irradiation and local shielding on matrix-induced endochondral bone differentiation

Summary Subcutaneous implantation of demineralized bone matrix into allogeneic rats induces endochondral dochondral bone formation. We have investigated the effects of irradiation on the sequelae of the interaction of collagenous matrix and mesenchymal cells and on cartilage and bone differentiation. Rats were irradiated in a vertical direction with a midline dose of 850 rad. Radiation entered the rats ventrally while a small area of the upper thorax was locally shielded. After irradiation, bone matrix was implanted in shielded and nonshielded sites, and the implants were studied at various stages. On day 3, [³H]thymidine incorporation, an index of cell proliferation, was inhibited by 70% in the nonshielded sites compared to nonirradiated control rats. The degree of inhibition (35%) was less pronounced in shielded sites. Furthermore, there was recovery of cell proliferation in the shielded sites as opposed to the nonshielded contralateral site. A similar pattern was observed on day 7 as assessed by³⁵SO₄ incorporation into proteoglycans during chondrogenesis. Bone formation and mineralization were quantified on day 11 by alkaline phosphatase activity and⁴⁵Ca incorporation. In nonshielded sites, there was a 73% inhibition of alkaline phosphatase activity. In conclusion, radiation impaired progenitor cell proliferation which resulted in decreased cartilage and bone differentiation. These findings imply that local mesenchymal cells proliferate and differentiate into bone in response to implanted collagenous matrix.     

Influence of vitamin A on matrix-induced endochondral bone formation

Summary The influence of retinoic acid on matrix-induced endochondral bone differentiation was determined. Retinoic acid was administered during discrete stages of endochondral bone formation, specifically, mesenchymal cell proliferation, chondrogenesis, bone formation, and mineralization. In retinoic acid-treated rats examined on day 3 following matrix implantation, biochemical markers for mesenchymal cell proliferation were about 50% of the controls. Chondrogenesis on day 7, assessed by³⁵SO₄ incorporation into proteoglycans, was 27% of the control. In addition, dissociative extraction of proteoglycans with 4.0 M guanidine-HCl and chromatography on Sepharose CL-2B revealed the synthesis of a smaller molecular weight proteoglycan when compared to controls which exhibited the cartilage-specific type. Osteogenesis and bone mineralization were monitored by alkaline phosphatase activity and⁴⁵Ca incorporation. On day 11 alkaline phosphatase activity was decreased by 40% and⁴⁵Ca incorporation was 48% of the control. These results revealed the multiple foci of the actions of excess vitamin A.     

Effect of diphosphonates and calcitonin on the chemistry and quantitative histology of rat bone

Young male rats were treated with one of three diphosphonates, disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP), disodium dichloromethylene-diphosphonate (Cl₂MDP), or disodium methane-1-cyclohexyl-1-hydroxy-1,1-diphosphonate (MCHDP), or with porcine calcitonin. Changes in the length weight, calcium, phosphorus, and ash of the femora and in plasma calcium, phosphate and alkaline phosphatase were measured. Bone formation rate was measured histologically in the tibial diaphysis using tetracycline markers. The lower dose (0.01–1mg P/kg/day s.c.) of the phosphonates, which block immobilization osteoporosis and reduce bone turnover measured by⁴⁵Ca kinetics, did not cause marked changes in the composition of the femora or in plasma values. This suggests that, at these doses in intact animals, the diphosphonates similarly reduce both resorption and mineralization rates. Higher doses of EHDP and MCHDP (10 or 30 mg P/kg/day s.c.) impaired bone growth both in length and width and inhibited mineralization of new cartilage and osteoid. The total calcium content of the bones decreased but the plasma values remained unchanged. Cl₂MDP did not impair mineralization at equivalent doses. EHDP, unlike Cl₂MDP, therefore seems to have two effects on bone. At lower doses it reduces bone turnover rate; but at higher doses it also directly prevents the full mineralization of new matrix. This difference between the effects of EHDP and Cl₂MDP may be important.     

Induction of endochondral bone by demineralized bone matrix from diabetic rats

Summary In this investigation we examined the osteoinductive potential of demineralized bone matrix derived from chronically diabetic (streptozotocin-induced) rats. Long-Evans rats (28–31 days) were made diabetic with a single injection of streptozotocin (65 mg/kg) and provided food and waterad lib for 2 months. Diaphyseal shafts of femurs and tibias removed from the diabetic rats and their sibling controls were dehydrated, pulverized, sieved to 74–420 μm particles, and demineralized Matrix was then bioassayed for its ability to induce endochondral bone on day 11 following subcutaneous implantation over the thorax of Long-Evans rats. The resulting plaques of tissue were subjected to histological analysis, determination of alkaline phosphatase activity, and calcium content. Bone matrix derived from diabetic animals proved to be a significantly better inducer of endochondral bone than did control matrix.     

Effects of indomethacin on demineralized bone-induced heterotopic ossification in the rat

Indomethacin inhibits bone formation when treatment is initiated before the implantation of demineralized bone matrix (DBM). For the inhibition of bone induction to occur, indomethacin treatment had to be initiated 6 h or more before implantation of DBM. Initiating the drug treatment at or after the time of DBM implantation had no effects on the amounts of new bone formed. The inhibition by indomethacin is dose related over a range between 0.04 and 4 mg/kg body weight. Recovered day-1 DBM implants, transplanted into indomethacin pre- and posttreated syngeneic rats, formed bone at the same rate as controls did. However, recovered day-1 DBM implants lyophilized before transplantation showed decreased bone formation but significant dystrophic calcification as judged by a lower alkaline phosphatase activity and an elevated calcium content.     

Efficacy of wheat germ lectin-precipitated alkaline phosphatase in serum as an estimator of bone mineralization rate: Comparison to serum total alkaline phosphatase and serum bone Gla-protein

Summary Serum levels of total alkaline phosphatase activity (S-T-AP), wheat germ lectin-precipitated alkaline phosphatase activity (S-L-AP), and bone Gla-protein immunoreactivity (S-BGP) were measured in 26 patients (23 females and 3 males) aged 35–73 years (mean 59 years) with primary hyperparathyroidism (n=7), hyperthyroidism (n=9), and hypothyroidism (n=10) in whom the bone mineralization rate (m) was determined by⁴⁷Ca-kinetics (continuously expanding calcium pool model). A weak positive correlation (r=0.42,P<0.05) was found between S-T-AP and m, which in the range from 0–18 mmol Ca/day could be estimated with a standard error of 4.6 mmol/day. A closer correlation (r=0.65,P<0.001) was found between S-L-AP and m which was estimated with an error of 3.9 mmol Ca/day. The AP activity in the supernatant showed no significant correlation to m (r=0.11,P>0.50). The highest correlation coefficient (r=0.81,P<0.001) was found between S-BGP and m which could be predicted with an error of 3.4 mmol Ca/day. S-BGP showed a closer correlation to S-L-AP (r=0.71,P<0.001) than to S-T-AP (r=0.58,P<0.01). We concluded that S-L-AP predicts bone mineralization at organ level better than S-T-AP in selected metabolic bone disorders and that the supernatant activity shows no relation to bone turnover. We find the assay easy to handle and suitable for large-scale use in the diagnosis and monitoring of metabolic bone disease.     

Dystrophic calcification and mineralization during bone induction: biochemical differences

The calcification of implants of glutaraldehyde-crosslinked collagenous tissues and collagen was studied in young and old rats and compared to bone induction by non-crosslinked osteogenically active demineralized bone matrix (DBM). Glutaraldehyde-crosslinked implants of DBM, tendon, and cartilage calcified in young but not in old animals and accumulated only trace amounts of BGP (Bone Gla protein, osteocalcin). Alkaline phosphatase activity was high in implants of DBM and undetectable in crosslinked implants. To try and understand why bone formation is so significantly reduced in older Fischer-344 rats, we developed a system which consists of cylinders of DBM sealed at the ends with a Millipore filter. Cells originating from 20-day-old embryo donors were introduced into the chambers prior to subcutaneous implantation. After 4 weeks of implantation in 26-month-old rats, the cylinders containing embryonic calvaria or muscle calls were found to be full of bone and/or cartilage.     

Effects of bisphosphonates on the incorporation of calcium-45 and 3H-proline in orthotopic and in demineralized matrix-induced bone in rats

The influence of the two bisphosphonates, 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) and dichloromethylene bisphosphonate (Cl2MBP), on bone formation was studied in demineralized matrix-induced bone in young rats. Cl2MBP was given in doses of 0.3 and 3 mg P/kg per day and HEBP 2,4, and 8 mg P/kg per day starting immediately after implantation of the bone matrix and continued until sacrifice at 3 weeks. Rates of collagen synthesis and mineralization of implants, femurs, and incisor teeth were quantified by assay of 3H-proline and 45Ca injected 24 h before death. New bone formation in implants was also evaluated by histologic examinations. Implants from Cl2MBP-treated animals had wider bone trabeculae than controls but were colonized by normal appearing hematopoietic tissue. Implants and femurs from both Cl2MBP groups had a higher ash content than controls, but uptake of the two isotopes was not affected. These results indicate that Cl2MBP, at these dose levels, inhibits bone resorption without affecting bone formation in rats. The uptake of 45Ca in implants decreased with increasing doses of HEBP; almost no ash or 45Ca activity was encountered with the highest dose. The bone in implants from this group appeared less mature, and only scanty areas of bone marrow were seen. The uptake of 3H-proline was not affected by HEBP. It appears that, in rats, HEBP primarily inhibits mineralization and resorption, whereas matrix formation remains largely unaffected.     

Indomethacin does not inhibit the anabolic effect of parathyroid hormone on the long bones of rats

Summary Chronic administration of parathyroid hormone, hPTH 1-34, increased bone mass in normocalcemic, young rats [6]. Since PTH can stimulate prostaglandin E₂ (PGE₂) production in bonein vitro, and since PGE₂ can stimulate bone formation, the anabolic effect of PTH could be mediated by PGE₂. To test this hypothesis, experiments were done to determine if indomethacin, which blocks endogeneous PG production, would inhibit the anabolic response of bone to PTH. In the first experiment, male Sprague-Dawley rats, 70–100 g, in groups of five, were treated for 12 days with either hPTH 1-34, 8 μg/100g/day; PTH vehicle; indomethacin, 2 mg/kg/day; or a combination of PTH and indomethacin. In the second experiment, groups of 6 rats each were given vehicle or hPTH 1-34, 8 μg/100g/day, in combination with indomethacin, 0, 1, 2, or 4 mg/kg/day. Subcutaneous injections of PTH were given once daily and indomethacin was given orally in divided doses, twice a day. Rats were killed on day 12 in both experiments; their sera were analyzed and the trabecular and cortical bone of distal femurs processed to determine calcium (Ca) and hydroxyproline content and dry weight. PTH and indomethacin had no significant effect on serum Ca, phosphate, alkaline phosphatase, urea nitrogen and creatinine, or systemic and long-bone linear growth. In rats treated with PTH alone or in combination with indomethacin, bone Ca of distal femurs increased by 28–44%; dry weight by 29–41%, and hydroxyproline by 17–45%. Indomethacin alone had no effect on bone growth. Since indomethacin did not block the anabolic effect of hPTH 1-34, we concluded that the anabolic effect of PGHin vivo was unlikely to be mediated by prostaglandins.     

Heterotopic new bone formation causes resorption of the inductive bone matrix

The bone matrix of growing rats was labeled by multiple injections of 3H-proline, and demineralized bone matrix (DBM) was prepared. The DBM was allotransplanted heterotopically into growing rats. New bone formation was induced in and around the implants. The new bone formation was accompanied by a decrease in the content of 3H; 20 and 30 days after implantation, 72% and 46%, respectively, of the activity remained in the implants. Daily injections of indomethacin (2 mg/kg) inhibited calcium uptake by about 20% at 20 and 30 days and inhibited the release of 3H from the DBM to a similar degree. Heterotopic bone induction by DBM is accompanied by matrix resorption, and inhibition of the new bone formation decreases the resorption of DBM.     

同种异体脂肪干细胞复合脱钙骨修复大鼠尺骨缺损

目的探讨同种异体脂肪干细胞修复管状骨缺损的可行性。方法获取SD大鼠的腹股沟处脂肪,分离培养脂肪干细胞（Adipose-Derived Stem Cells,ADSCs）;鼠第3代ADSCs与脱钙骨复合,24 h后进行成骨诱导培养。检测细胞在材料表面的生长及成骨分化能力。建立鼠两侧尺骨缺损模型,分别植入鼠ADSCs-脱钙骨复合物（实验侧）和单纯脱钙骨材料（对照侧）;8周、24周后取样,行DR和组织学检测,观察成骨情况。结果 ADSCs能在脱钙骨上很好地黏附和生长,并维持成骨分化能力。细胞-材料复合物植入24周后,DR显示实验侧有新生骨基质长成,对照侧未见骨组织生成。组织学检测显示,实验侧缺损区被典型的骨组织取代,可见新生骨小梁附着于脱钙骨表面;对照侧只有少量的骨组织和纤维组织充填。结论 ADSCs-脱钙骨材料复合物植入,能成功修复临界大小的管状骨缺损。     

The matrix of endochondral bone differs from the matrix of intramembranous bone

Summary Osseous tissue develops via two distinctly different processes: endochondral (EC) ossification and intramembranous (IM) ossification. The present study tests the hypothesis that each type of osseous tissue contains unique inducing factors for the promotion of cartilage and bone development. Previous work suggests that subcutaneous implants of demineralized EC and IM bone matrices both induce endochondral ossification. Thus, it concludes that the bone growth promotion properties of the respective matrices are very similar. As it was unclear to us why EC and IM bone powders should possess identical osteoinductive properties, we attempted to reproduce these results. We implanted EC (femoral) demineralized bone matrix (DBM), IM (frontal) DBM, or a mixture of the two into the ventral thoracic subcutaneous tissue of 12 to 15-week-old male Sprague Dawley rats. Morphological and radiolabeling techniques in this study demonstrated that implants of EC bone matrix induce bone formation via EC ossification in contrast to implants of IM bone matrix which do not induce EC ossification. Our findings suggest that the matrix of EC bone differs qualitatively from the matrix of IM bone due to their respective abilities to induced cartilage and/or bone formation. These observations differ from those previously reported possibly because our IM DBM preparations were not contaminated with tissues of endochondral origin. In current clinical practice, EC DBM allografts are often used to induce new bone formation in defects involving both IM and EC bone. We conclude that there may be clinical settings in which it would be more appropriate to replace bone originally formed via IM ossification with IM DBM rather than EC DBM.     

Osteogenesis associated with bone gla protein gene expression in diffusion chambers by bone marrow cells with demineralized bone matrix.

Diffusion chambers with rat bone marrow cells and demineralized bone matrix (DBM) were implanted subcutaneously to syngeneic 8-week-old rats and were harvested every week 3-7 weeks after implantation, and histochemical examination, determination of alkaline phosphatase activity, total calcium and phosphorus, the bone-specific vitamin K-dependent gla-containing protein (BGP) content, and detection of BGP mRNA relative to mineralization were performed. Alkaline phosphatase in diffusion chamber implants reached the highest activity at 4 weeks and then decreased. Calcium and phosphorus deposits occurred at 4 weeks after implantation and were followed by marked increases until 7 weeks, which was comparable to the accumulation of BGP. The BGP gene within the diffusion chambers began to be expressed at 5 weeks, and its expression increased markedly at 7 weeks after implantation. At 4-5 weeks after implantation, new bone adjacent to the membrane filters and cartilage toward the center of the diffusion chamber were observed histochemically. Light microscopic and immunohistologic examinations of chambers with marrow cells and DBM revealed production of mineralized matrices, typical of bone characterized by the appearance of BGP and mineralized nodules. In contrast, bone marrow cells alone did not show extensive bone formation and yielded very low values for these biochemical parameters. The present experiments demonstrate the potential of bone marrow cells and DBM to produce not only cartilage formation but also membranous bone formation associated with increasing expression of BGP mRNA during the later stages of bone formation, as well as a marked accumulation of BGP.