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     

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.     

Changes in lipids during matrix: Induced endochondral bone formation

Summary The changes in lipids occurring during the process of endochondral ossification have been characterized by studying the discrete phases of matrix-induced endochondral bone formation in the rat. Calcium-acidic phospholipid-phosphate complexes were shown to increase in concentration during cartilage calcification (day 9) and to peak in content during early bone formation (day 11–13), the times during which the rate of mineral deposition, as indicated by the change in ash weight was greatest. These data support the hypothesis that the calcium-acidic phospholipid-phosphate complexes play a role in thein vivo initiation of hydroxyapatite deposition. The overall lipid composition of the induced matrix newly formed cartilage (days 7–9) was comparable to that of normal cartilage, with the phospholipid composition matching that of chondrocyte plasma membranes. Times of vascular invasion and formation of marrow cavities were marked by elevated total lipid and triglyceride contents.     

Influence of irradiation on the osteoinductive potential of demineralized bone matrix

Summary Samples of demineralized bone matrix (DBM) were exposed to graduated doses of radiation (1–15 Megarad) (Mrad) utilizing a linear accelerator and then implanted into the thoracic region of Long-Evans rats. Subcutaneous implantation of DBM into allogenic rats induces endochondral bone. In response to matrix implantation, a cascade of events ensues; mesenchymal cell proliferation on day 3 postimplantation, chondrogenesis on day 7, calcification of the cartilagenous matrix and chondrolysis on day 9, and osteogenesis on day 11 resulting in formation of an ossicle containing active hemopoietic tissue. Bone formation was assessed by measuring alkaline phosphatase activity, the rate of mineralization was determined by measuring⁴⁵Ca incorporation to bone mineral, and⁴⁰Ca content measured the extent of mineralization; acid phosphatase activity was used as a parameter for bone resorption. The dose of radiation (2.5 Mrad) currently used by bone banks for sterilization of bone tissue did not destroy the bone induction properties of DBM. Furthermore, radiation of 3–5 Mrad even enhanced bone induction, insofar as it produced more bone at the same interval of time than was obtained from unirradiated control samples. None of the radiation doses used in these experiments abolished bone induction, although the response induced by matrix irradiated with doses higher than 5 Mrad was delayed.     

Decreased osteoinductive potential of bone matrix from ovariectomized rats

The effect of estrogen deficiency on matrix-induced bone formation was investigated. Female rats were ovariectomized and given demineralized bone matrix (DBM) intramuscularly 3 weeks before termination. the DBM was taken from previously ovariectomized and from sham-operated on rats. the animals were killed at various times after ovariectomy (6-27 weeks). Implants were processed undemineralized for histologic and biochemical studies.

Normal DBM implanted in ovariectomized or normal rats induced extensive bone formation 6 weeks postovariectomy. the amount of newly formed bone decreased with the age of host rats. Bone matrix taken from ovariectomized rats was incompletely resorbed in both ovariectomized and normal hosts, therefore reducing the extent of osteogenesis andbone-marow formation. Instead, chondrogenesis was intensive, but delayed. the calcium, magnesium, and zinc contents were decreased in implants taken from ovariectomized rats when compared with implants taken from normal animals.

Normal osteoinduction with DBM taken from normal rats and implanted in ovariectomized rats and the absence of osteogenesis with DBM taken from ovariectomized rats indicate that an estrogen-deficient environment is not crucial for altered matrix-induced endochondral bone formation in ovariectomized rats. An altered composition of matrix from ovariectomized rats and a subsequent abnormality in the cell-matrix interaction should be considered responsible.     

Decreased osteoinductive potential of bone matrix from ovariectomized rats

The effect of estrogen deficiency on matrix-induced bone formation was investigated. Female rats were ovariectomized and given demineralized bone matrix (DBM) intramuscularly 3 weeks before termination. The DBM was taken from previously ovariectomized and from sham-operated on rats. The animals were killed at various times after ovariectomy (6-27 weeks). Implants were processed undemineralized for histologic and biochemical studies. Normal DBM implanted in ovariectomized or normal rats induced extensive bone formation 6 weeks postovariectomy. The amount of newly formed bone decreased with the age of host rats. Bone matrix taken from ovariectomized rats was incompletely resorbed in both ovariectomized and normal hosts, therefore reducing the extent of osteogenesis and bone-marrow formation. Instead, chondrogenesis was intensive, but delayed. The calcium, magnesium, and zinc contents were decreased in implants taken from ovariectomized rats when compared with implants taken from normal animals. Normal osteoinduction with DBM taken from normal rats and implanted in ovariectomized rats and the absence of osteogenesis with DBM taken from ovariectomized rats indicate that an estrogen-deficient environment is not crucial for altered matrix-induced endochondral bone formation in ovariectomized rats. An altered composition of matrix from ovariectomized rats and a subsequent abnormality in the cell-matrix interaction should be considered responsible.     

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.     

Cellular events associated with the induction of bone by demineralized bone

Implantation of demineralized bone (DB) in the form of powder or intact segments in extra skeletal sites stimulates new bone formation. Urist and co-workers presented substantial evidence that there is a noncollagenous protein that has the ability to induce bone formation. One aim of this study was to trace the process of bone formation when DB, in the form of perforated rectangular plates, is implanted subcutaneously in 2-month-old rats. A second objective was to determine whether cartilage cells play a role in the formation of bone in this model. Various DB plates with 0.25 mm diameter holes were implanted subcutaneously for 1-4 weeks in rats. One week after implantation, DB plates were covered by vascularized connective tissue that invaded the perforations. Aggregates of chondrocytes were observed within the holes and on periosteal surfaces in only a few specimens. Further cartilage proliferation was not observed, and by the 2nd week there was no evidence of endochondral bone formation. Where these cartilage-like cells were present, a thin layer of mineral was deposited around them; resorption and fibrous tissue infiltration followed. This aborted form of endochondral calcification was not followed spatially by bone formation. Patent vascularized channels were invaded by alkaline phosphatase-positive mononuclear cells and fibroblasts, and became enlarged by the enzymatic action of macrophages. The next step involved the calcification of DB plates adjacent to the wide spaces. Osteoclasts now appeared leading to the resorption of this recalcified matrix. The eroded and now enlarged lacunar surfaces were lined by newly formed bone and osteoblasts. This process continued so that, at the end of 4 weeks following implantation, the original DB plates were replaced by trabecular bone. Biochemical data on calcium and alkaline phosphatase levels in the implants paralleled the morphological observations.     

Osteoinductivity of demineralized bone matrix in immunocompromised mice and rats is decreased by ovariectomy and restored by estrogen replacement

The osteoinduction potential of human demineralized bone matrix (DBM) in females with low estrogen (E2) is unknown. Moreover, the osteoinductivity of commercial human DBM is tested in male athymic rats and mice, but DBM performance in these animals may not reflect performance in female animals or provide information on E2's role in the process. To gain insight, human DBM was implanted bilaterally in the gastrocnemius of twenty-four athymic female mice (10 mg/implant) and twenty-four athymic female rats (15 mg/implant). Eight animals in each group were sham-operated (SHAM), ovariectomized (OVX), or ovariectomized with E2-replacement (OVX+E2) via subcutaneous slow release capsules of 17beta-estradiol. OVX and OVX+E2 animals were pair-fed to SHAM animals. Four animals from each group were euthanized at 35 days and four at 56 days. Animal weight, uterine weight, and blood estrogen levels confirmed that pair feeding, ovariectomy, and E2 replacement were successful. Histological sections of implanted tissues were evaluated qualitatively for absence or presence of DBM, ossicle formation, and new bone or cartilage using a previously developed qualitative scoring system (QS) and by histomorphometry to obtain a quantitative assessment of osteoinduction. OVX mice had a small but significant QS decrease at 35 days compared to SHAM mice, confirmed by quantitative measurement of ossicle, marrow space, and new bone areas. The QS in rats was not affected by OVX but histomorphometry showed decreased new bone in OVX rats, which was restored by E2. The QS indicated that the number of new bone sites was not reduced by OVX in rats or mice at 56 days, but the relative amount of new bone v. marrow space was affected and differed with animal species. Residual DBM was less in OVX animals, indicating that DBM resorption was affected. Cartilage was present in rats but not in mice, suggesting that endochondral ossification was slower and indicating that bone graft studies in these species are not necessarily comparable. These results show the importance of E2 in human DBM-induced bone formation and suggest that E2 may be needed for clinical effectiveness in post-menopausal women.     

Characterization of Matrix-Induced Osteogenesis in Rat Calvarial Bone Defects: II. Origins of Bone-Forming Cells

Two experimental models that separated demineralized bone matrix (DBM) implants from the host bone were utilized to identify the origins of bone-forming cells in the repair of calvarial defects in rats. Rat DBM, Guanadine-HCl (Gdn-HCl) extracted insoluble residue of DBM, and Gdn-HCl extracted insoluble DBM to which the dialyzed Gdn-HCl extract was added back, were implanted in the two models which prevented cells of the adjacent host bone from participating in the repair. In addition, cells in the dura and in the subcutaneous tissue overlying the calvarial defect were locally labeled with ³H-thymidine to identify the origins of those cells that were stimulated to divide and differentiate to osteoblasts. Histological studies of the temporal events that occurred during the healing process in these defect models, combined with ³H-thymidine labeling demonstrated that the osteoblasts induced by DBM were initially derived from undifferentiated mesenchymal stem cells of the dura and later augmented by cells in the overlying connective tissue covering the defect, and not from cells in the cranial bone surrounding the circular defect. The cells of both dura and subcutaneous tissue were stimulated to proliferate and differentiate principally to osteoblasts and to a very much lesser extent to chondroblasts by DBM and by reconstituted components of DBM after Gdn-HCl extraction. Gdn-HCl-extracted insoluble DBM failed to induce bone or cartilage. These results indicate that the cytokines or other factors present in DBM are required to induce bone-forming cells derived from the dura and the overlying connective tissue for the repair of the calvarial defect. Received: 1 January 1999 / Accepted: 13 August 1999     

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.     

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.     

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.     

Effect of demineralized bone matrix on polymorphonuclear leukocyte degranulation.

The potential use of allogenic demineralized bone matrix to augment or treat bone defects or nonunions in animals and humans is currently being investigated. Demineralized bone matrix induces osteogenesis by a multistep cascade of endochondral ossification that is mediated by bone-induction factors. The migration and activation of polymorphonuclear leukocytes appear to be critical in the initiation of the cascade of osteogenesis induced by demineralized bone matrix. This study examined the effects of demineralized bone matrix on the degranulation of polymorphonuclear leukocytes. Demineralized bone matrix stimulated the release of polymorphonuclear leukocyte-specific, but not azurophilic, granules in a time and dose-dependent manner. The ability of the bone matrix to induce this degranulation was independent of its size and species. The mechanism by which this degranulation occurs is not completely understood; however, it is known that it does not occur by means of a receptor that requires guanidine triphosphate-dependent regulatory proteins as does polymorphonuclear-leukocyte degranulation induced by N-formyl peptide. The factor that stimulates degranulation is not type-I collagen but rather appears to be a cytokine that has a heparin-binding domain and a molar mass of 10-70 kDa. Loss of the ability of demineralized bone matrix to induce degranulation of polymorphonuclear leukocytes correlated positively with the loss of its ability to induce bone formation.     

Ultrastructural immunolocalization of fibronectin in epiphyseal and metaphyseal bone of young rats

Fibronectin is a well known glycoprotein of extracellular connective tissue matrices due to a specific amino acid-sequence (RGD) suggested to act as an attachment factor in cell-cell or cell-matrix interactions. Although also present in bone, little is known about the role of fibronectin in this tissue. To obtain data for discussions on function we used ultrastructural immunolocalization techniques to quantitatively examine the distribution of fibronectin in various bone matrix compartments. The study was focused on three different stages of endochondral ossification in growing long bones of young rats. The results show large amounts of fibronectin in mature bone tissue. At a higher magnification, an obvious fibronectin association to individual fibrils of collagen type I was demonstrated. Intracellular labeling was observed in Golgi-related vesicles in some active osteoblasts of metaphyseal bone, indicating local synthesis of fibronectin. In contrast to previous suggestions based on light microscopic observations, the labeling of bone or cartilage matrices facing the surface of all cell types were low. The pattern is clearly different from that of osteopontin and bone sialoprotein, two other bone matrix proteins with the same cell-binding sequence. Our results indicate that fibronectin at these stages of development participates in matrix organization rather than being an important link between cartilage or bone matrix and adjacent cells.     

Monkey bone matrix induces bone formation in the athymic rat, but not in adult monkeys

Demineralized bone matrix from young and adult monkeys was implanted intramuscularly for 6 weeks in athymic rats and adult monkeys. Cartilage and bone induction was evaluated by histology and calcium content. In the athymic rat, most implants induced cartilage or bone. In the monkeys, cartilage was formed only on rare occasions and there was no sign of bone formation. We conclude that (a) adult monkey bone matrix contains bone inductive properties; (b) these properties are not sufficient to induce bone formation in adult monkey muscle sites.     

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.     

Influence of bone‐derived matrices on generation of bone in an ectopic rat model

Most bone regeneration experimental models that test bone‐derived matrices take place in conjunction with the native bone. Here, we compared the relative effectiveness of bone matrix components on bone‐marrow‐directed osteogenesis in an ectopic model. Cortical bone cylinders consisted of diaphysis of DA rat femurs. They were either demineralized (DBM), deproteinized (HABM), or nontreated (MBM). Fresh bone marrow was placed into cylinders and implanted at subcutaneous thoracic sites of 2‐month‐old DA rats. At designated times the cylinders were surgically removed from the animals. Microradiographs of DBM and histology of DBM and MBM cylinders demonstrated progressive increase in mineralized bone volume and its trabecular configuration. Bone filled the inner volume of DBM and MBM cylinders within 4 weeks, while in HABM cylinders mostly granulation tissue developed. In the DBM cylinders cartilage deposited within 10 days, while in the MBM cylinders bone was directly deposited. As early as day 3 after marrow transplantation, marrow cells interacting with DBM increased significantly the genes that express the cartilage and the bone phenotype. In conclusion, organic components of bone are needed for marrow‐directed osteogenesis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:664–670, 2010     

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.     

Demineralized Bone Matrix Injection in Consolidation Phase Enhances Bone Regeneration in Distraction Osteogenesis via Endochondral Bone Formation

Background

Distraction osteogenesis (DO) is a promising tool for bone and tissue regeneration. However, prolonged healing time remains a major problem. Various materials including cells, cytokines, and growth factors have been used in an attempt to enhance bone formation. We examined the effect of percutaneous injection of demineralized bone matrix (DBM) during the consolidation phase on bone regeneration after distraction.

Methods

The immature rabbit tibial DO model (20 mm length-gain) was used. Twenty-eight animals received DBM 100 mg percutaneously at the end of distraction. Another 22 animals were left without further procedure (control). Plain radiographs were taken every week. Postmortem bone dual-energy X-ray absorptiometry and micro-computed tomography (micro-CT) studies were performed at the third and sixth weeks of the consolidation period and histological analysis was performed.

Results

The regenerate bone mineral density was higher in the DBM group when compared with that in the saline injection control group at the third week postdistraction. Quantitative analysis using micro-CT revealed larger trabecular bone volume, higher trabecular number, and less trabecular separation in the DBM group than in the saline injection control group. Cross-sectional area and cortical thickness at the sixth week postdistraction, assessed using micro-CT, were greater in the regenerates of the DBM group compared with the control group. Histological evaluation revealed higher trabecular bone volume and trabecular number in the regenerate of the DBM group. New bone formation was apparently enhanced, via endochondral ossification, at the site and in the vicinity of the injected DBM. DBM was absorbed slowly, but it remained until the sixth postoperative week after injection.

Conclusions

DBM administration into the distraction gap at the end of the distraction period resulted in a significantly greater regenerate bone area, trabecular number, and cortical thickness in the rabbit tibial DO model. These data suggest that percutaneous DBM administration at the end of the distraction period or in the early consolidation period may stimulate regenerate bone formation and consolidation in a clinical situation with delayed bone healing during DO.