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PubMed1991

[Bone matrix gelatin. Clinical application in 38 cases].

Bone matrix gelatin (BMG) was prepared and used clinically as graft-stuff in 38 patients. It was used to fill the bone defects resulted from excision of benign bone tumors (22 patients) and debridement of chronic osteomyelitis (3), to treat delayed union of fractures (5), and to make posterior spinal fusion (8). Fourty-five days after operation, speckles or flakes of dense shadow appeared in the film at the original defects and fields. In 2 to 6 months, bony union took place in 36 patients (95%). Failure was noted in 2 patients with chronic osteomyelitis. Because of its excellent osteo-inductive property and nonantigenic treat, BMG has been proved to be an ideal substitute for bone graft. It is easy to prepare, safe for storage at room temperature and sound for transportation.

Adult↗
PubMed1990

[Purification and characterization of a growth factor from human bone matrix].

A growth factor was purified from human bone matrix by extraction with EDTA, acetone treatment, gel filtration column chromatography, ion exchange column chromatography, and reversed-phase HPLC. Purified protein migrated as a single band to an area with a molecular weight of about 6,000 on SDS-polyacrylamide gel. The purified protein stimulated dose dependent osteoblast proliferation. The sequence of the first 30 N-terminal amino acids of the protein was identical to that of the human insulin-like growth factor-II (IGF-II). MC3T3-E1 cells reacted immunocytochemically to the monoclonal antibody against IGF-II. The culture medium of MC3T3-E1 cells contained immunoreactive IGF-II, which was measured by an enzyme immunoassay. These results indicate that IGF-II is contained in bone matrix, is effective in osteoblast proliferation, produced by the osteoblasts, and secreted from the osteoblasts. Taken together, these results show that IGF-II is present as a local growth factor in the bone system, playing an important role in bone formation following bone resorption.

Animals↗
PubMed1995

The clinical application of human bone matrix gelatin.

This paper reports the results of 24 cases of bone defect resulting from bone tumor or tumor condition excision, and of posterior spinal fusion, treated by human bone matrix gelatin. The success rate of bone defect repair and spinal fusion is 91.67%. The results suggest that human bone matrix gelatin has excellent osteoinductive effect and is ideal substitute for bone autografts.

Adolescent↗
PubMed1982

Recruitment of osteoclast precursors by purified bone matrix constituents.

The osteoclast, the multinucleated giant cell of bone, is derived from circulating blood cells, most likely monocytes. Evidence has accrued that is consistent with the hypothesis that the recruitment of monocytes for osteoclast development occurs by chemotaxis. In the present study, we have examined the chemotactic response of human peripheral blood monocytes and related polymorphonuclear leucocytes to three constituents of bone matrix: peptides from Type I collagen, alpha 2-HS glycoprotein, and osteocalcin (bone gla protein). The latter two substances are among the major noncollagenous proteins of bone and are uniquely associated with calcified connective tissue. In chemotaxis assays using modified Boyden chambers, Type I collagen peptides, alpha 2HS glycoprotein, and osteocalcin evoke a dose-dependent chemotactic response in human monocytes. No chemotaxis is observed on PMNs despite their ontogenetic relationship to monocytes and their documented sensitivity to a broad range of other chemical substances. Our observations are consistent with the view that osteoclast precursors (monocytes) are mobilized by chemotaxis, and suggest that the chemoattractants responsible for this activity are derived from the bone matrix or, in the case of collagen and osteocalcin; directly from the osteoblasts which produce them.

Bone Matrix↗
PubMed2000

[The effect of estrogen on new bone formation in the implanted bone matrix in aged male rats].

OBJECTIVE: To study the effect of estrogen on new bone formation in the implanted bone matrix in aged male rats and on general bone metabolism. METHODS: The aged male rats were implanted with slices of bone matrix and subcutaneously injected with estrogen, androgen or the solvent for five weeks. The changes in bone histomorphometry, bio-mechanical parameters and urine deoxypyridinoline (Dpd) levels were observed as indexes of bone reabsorption. Calcium, organic phosphate, and alkalphophatase levels in serum and urine were also studied. RESULTS: (1) The areas of new bone formation were significantly larger in estrogen and androgen groups than in the control but without difference between estrogen and androgen groups. (2) The number of osteoclasts was higher in estrogen and androgen groups than in the control (P < or = 0.002) but without difference between estrogen and androgen groups. (3) Urine Dpd/Cr ratio was lower in estrogen and androgen groups than in the control and higher in the control than that obtained 5 weeks ago but without difference between estrogen and androgen groups. CONCLUSION: Estrogen and androgen are able to stimulate new bone formation in the implanted bone matrix and inhibit the reabsorption rate of systemic bones in aged male rats, but there was no difference between estrogen and androgen in these two respects under the conditions of this experiment.

Androgens↗
PubMed2000

Histologic analysis of implant sites after grafting with demineralized bone matrix putty and sheets.

Grafting to restore lost alveolar bone is frequently used to enable placement of endosseous implants and improve cosmesis. Conflicting reports concerning the osteoinductivity of demineralized bone matrix (DBM) and historical use of synthetic bone graft substitutes has limited the use of DBM in oral and maxillofacial applications. Implant placement after bone grafting provides the unique opportunity to biopsy and histologically evaluate new bone formation. Bone grafting of the mandible or maxilla was performed to fill extraction sockets and restore ridge structures in a consecutive series of eight patients. DBM prepared as malleable putty (Grafton DBM Putty) or flexible sheets (Grafton DBM Flex) was used. Biopsies were taken at reentry, and histologic analysis determined the amount and quality of regenerated bone. Extensive new bone formation and minimal residual bone graft matrix were observed at an average of 5 months postoperative. The pattern of new bone maturity and remodeling varied by patient and the time in situ. Putty and Flex regenerated excellent bone height and width for the placement of dental implants, were easy to handle intraoperatively, and readily conformed to bony defects.

Adult↗
PubMed1990

Effect of aluminum on bone matrix inductive properties.

The effect of aluminum on the bone inductive properties of implanted bone matrix was studied in rats. After decalcification femur sections were placed in either 0.1 or 0.01 M AlCl3 or a solution of similar pH without Al for 24 hours. Following 28 days of implantation in subcutaneous pouches the aluminum content was 3232 +/- 1020 and 51 +/- 6 mg/kg in the matrix pretreated with 0.1 and 0.01 M AlCl3. At the same time period following implantation the matrix calcium content was 794 +/- 539 and 3038 +/- 692 mmol/kg in the 0.1 and 0.01 M AlCl3 pretreated groups versus 4252 +/- 579 mmol/kg in the control group (P less than 0.01). In the control group bone histology showed extensive osteoblastic and osteoclastic remodeling, tetracycline labeling and bone formation. In contrast all of these histological features were virtually absent in aluminum treated matrix. Aluminum-induced resistance of bone matrix to collagenase degradation and restoration of bone inductive properties with chelation suggests that aluminum forms intermolecular cross links between collagen fibrils. Aluminum-induced cross links of collagen fibrils and/or its effects on bone inductive proteins present in bone matrix could explain the mechanism by which aluminum induces osteomalacia.

Aluminum↗
PubMed2000

[Experimental study on application of allogeneic bone matrix gelatin in the intervertebral fusion].

OBJECTIVE: To testify the inductive osteogenesis of allogeneic bone matrix gelatin (BMG) in promoting intervertebral fusion. METHODS: The gelatin sponge, allogeneic BMG, decalcified bone matrix (DBM) and alcohol conserved bone were implanted respectively into the intervertebral space of rabbit, whose intervertebral discs were removed before implantation. The intervertebral spaces were evaluated by X-ray and histological examination at 4, 8, and 12 weeks after operation. RESULTS: No obvious immune rejection was observed. Amounts of new bone were formed in the intervertebral spaces at 4 and 8 weeks. And complete infusion of the intervertebral spaces were appeared at 12 weeks. CONCLUSION: Allogeneic BMG can promote bone fusion of intervertebral spaces through osteoinduction, which suggests that allogeneic BMP is an ideal substitute for bone replacement.

Animals↗
PubMed1994

Comparison of various delivery systems for demineralized bone matrix in a rat cranial defect model.

Demineralized bone matrix (DBM) has been successfully used as a substitute for bone grafting. Autogenous bone grafts may cause site morbidity and undergo significant resorption. DBM may overcome these problems, but it has no mechanical stability until bone formation has occurred. We tested various alloplastic implants (i.e., Surgicel, polydioxanone [PDS], porous polyethylene [Medpor], and Gelfoam) in combination with DBM and compared it with DBM alone in a 9 x 9 mm rat cranial defect model. Histological and biomechanical measurements were performed at postoperative month 2. Among the study groups, Gelfoam/DBM inhibited bone formation to varying degrees and was the only group that displayed an inflammatory response. Mechanical pushout tests using a servohydraulic testing frame were conducted. The Medpor/DBM implant displayed the strongest support at 2 months; maximum load was 95% of intact skull. Surgicel/DBM and DBM alone were comparable; maximum load was 66% of intact skull. Gelfoam/DBM and PDS/DBM displayed the weakest support (48% of intact skull). We conclude that, after 2 months of implantation, alloplastic/DBM composites provide osseous structural integration. Gelfoam/DBM is not an effective delivery system for DBM in our model.

Analysis of Variance↗
PubMed2005

Human mesenchymal stem cell derived osteoblasts degrade organic bone matrix in vitro by matrix metalloproteinases.

Some recent studies have suggested that cells of mesenchymal origin might participate in the organic bone matrix dissolution. In the present study, collagen synthesis and degradation by human mesenchymal stem cell (MSC) derived cells were studied at early stage of osteoblast differentiation using a special two-stage in vitro culture model. In this model, cells were cultured on bovine bone slices, which were first resorbed by osteoclasts. Synthesis of type I collagen was markedly enhanced when mesenchymal cells were cultured on bone matrix. After thorough osteoclast removal, MSC derived cells were capable of degrading the organic bone matrix, and caused a release of type I collagen degradation product (ICTP) into the culture medium. This was inhibited by matrix metalloproteinase (MMP) inhibitor, while cysteine proteinase inhibitor or estrogen had no inhibitory effect. Western blot analysis or gelatin zymography confirmed the presence of MMP-2, -8, -13 and -14, but not MMP-1 or -9, in the differentiated cells. 17beta-Estradiol was found to increase the expression of MMP-2 and -14 by these cells. Finally, scanning electron microscopy showed that the differentiating human MSCs were capable of degrading organic bone matrix remnants from the bottom of the resorption lacunae. These data support the hypothesis that collagen cleavage by the same cells that are subsequently responsible for bone formation is MMP mediated process and is an important step coupling bone formation into bone resorption.

Animals↗
PubMed1985

Medullary bone matrix formation, mineralization, and remodeling related to the daily egg-laying cycle of Japanese quail: a histological and radiological study.

A model is presented concerning the remodeling of medullary bone during the egg-laying cycle of hens. This model is partly based on earlier results that showed that the active period of medullary bone resorption that produces part of the calcium needed for the eggshell coincides with the period of medullary bone matrix formation. It was hypothesized that mineralization of this matrix takes place during the subsequent inactive period. The present study examines this model. The medullary bone matrix volume, regardless of the degree of mineralization, is quantified in quail hens at four time points during the egg-laying cycle. No significant changes in the medullary bone volume (i.e., matrix and bone) during the egg-laying cycle were found, implying that matrix formation and bone resorption are kept in balance within a time interval of only a few hours. In the radiological part of this study the whole body retention (WBR) of 99mTc(Sn)MDP is used as a measure of the amount of low calcium medullary bone matrix formed during the egg-laying cycle. This use of 99mTc(Sn)MDP is justified by an autoradiographic experiment that showed that 99mTc(Sn)MDP actually labels newly formed medullary bone. Localization of the radioactive label showed a high positive correlation with the localization of fluorescent tetracycline labels in the medullary bone. From 4 to 22 hours after ovulation, the amount of medullary bone matrix increases linearly in quail hens.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1995

Bone matrix proteins in osteogenesis and remodelling in the neonatal rat mandible as studied by immunolocalization of osteopontin, bone sialoprotein, alpha 2HS-glycoprotein and alkaline phosphatase.

The neonatal rat mandible was used as a model to study bone formation, mineralization, quiescence, and resorption, using immunolocalization and a variety of tissue-processing techniques. Monospecific antibodies for osteopontin (OPN), bone sialoprotein (BSP), alkaline phosphatase (AP) and alpha 2HS-glycoprotein (alpha 2HS-GP) were used on fixed paraffin-embedded tissue, fixed frozen tissue and unfixed frozen tissue. Immunostaining was correlated with mineral content by two procedures, the von Kossa and the morin techniques. Morin fluorescence was used with secondary immunostaining to provide a way of closely correlating bone matrix proteins and matrix mineralization. Co-immunolocalization procedures were used to compare the sites of bone proteins in the matrix. AP was found earliest during osteogenic cell differentiation, appearing in the preosteoblasts, followed by OPN and BSP, which first appeared in osteoblasts. alpha 2HS-GP expression was not observed in cells. The results provide clear evidence for the presence of OPN in osteoid, while BSP and alpha 2HS-GP were confined to the mineralized matrix. Immunostaining of bone proteins is highly technique-dependent: immunolocalization investigations required several methods of approach to ensure adequate demonstration of these proteins in cells and matrix. The results support the contention that osteopontin is multifunctional in bone metabolism, and that alpha 2HS-GP, though produced in the liver, is abundant in bone matrix and may also have a function in bone metabolism.

Alkaline Phosphatase↗
PubMed1980

Solubilized bone morphogenetic protein (BMP) from mouse osteosarcoma and rat demineralized bone matrix.

A selection of proteins including bone morphogenetic protein (BMP) was extracted in a disaggregated form from Dunn osteosarcoma or rat demineralized bone matrix by 4M guanidine hydrochloride (GuHCl) solution without losing its biological activity. The GuHCl extracts of Dunn osteosarcoma were divied into 4 different fractions by cesium chloride (CsCl) density gradients. Under a dissociative condition, the highest new bone yield was obtained in the low dense top one-third fraction, and BMP acitivity declined with increase in the density of each fraction. No BMP potential was observed in the surface-gel fraction under dissociative conditions. Under an associative condition (low GuHCl concentrations), BMP activity appears in the surface-gel fraction, while under a dissociative condition (high concentrations of GuHCl) BMP appears in the fraction below the surface gel. These facts suggest that under associative conditions, BMP aggregates with other low dense proteins in the surface-gel fraction and that this may be the state of aggregation of BMP in cells and matrix in nature. Present observations support the assumption that BMP is a relatively low density protein and excludes the idea of BMP activity in the collagen molecule, per se. A specific protein, with an apparent molecular weight of 63,000 daltons, is present in all fractions that exhibit BMP activity, and absent in fractions that do not exhibit this activity. BMP is not species-specific; rat BMP induces bone formation in mice. CsCl density-gradient centrifugation is an efficient tool for further purification and isolation of BMP.

Animals↗
PubMed1994

The influences of casein phosphopeptides on metabolism of ectopic bone induced by decalcified bone matrix implantation in rats.

The effect of casein phosphopeptides (CPP) on bone metabolism was studied in the ectopic bone induced by the implantation of decalcified bone matrix in rats. Forty-two Wistar male rats of 7 weeks old were fed low calcium diets (0.39% of calcium) with or without supplying 0.50% of CPP, or a control diet (0.91% of calcium) without CPP supplementation. After a 1-week preliminary period, each rat was subcutaneously implanted with 30 mg of demineralized bone matrix powder. Fourteen and 21 days after the implantation, the implants were harvested from 7 rats of each group. Calcium content in the graft was not significantly different among all groups on day 14. Subsequently, the content of calcium rapidly increased in the grafts irrespective of diets given. However, the graft of the CPP- group contained less calcium than the other groups and the calcium content was more in the control rats compared to the CPP+ animals on day 21. Alkaline phosphatase activity (an index of bone and cartilage calcification) was lower in the control group than in the CPP+ group on day 14. The enzyme activity subsequently decreased in the control group but the activity was not changed in the other groups. As a result, the activity of alkaline phosphatase was lower in the control animals than in the other rats on day 21. Tartrate-resistant cap phosphatase activity (an index of bone resorption) was higher in the CPP--group compared to the control on day 14. On day 21, the activity was higher in the CPP--group compared to the others.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1992

Association of giant multinucleated cells with deterioration of osteoinductive potency of bone matrix.

A thirty four intramuscular implants of rat decalcified bone matrix into mice and rats were histologically inspected in order to find out if there is an inverse correlation between the induction of multinucleated giant cell formation and the bone induction activity. It was found that the presence of multinucleated giant cells which avidly resorbed the implanted matrix was almost always combined with the lack of local bone induction. In contrast, the specimens exhibiting intense bone induction were devoided of multinucleated giant cells. Our observation confirm the opinion expressed by Walters and Schneider that multinucleated giant cells induced by mineralized bone chips are not osteoclasts but a type of inflammatory cells, analogous if not identical to the "foreign body cells". It is postulated that formation of multinucleated giant cells is a morphological expression of hyperactivity of mononuclear phagocytes. Hyperactive multinucleated giant cells avidly resorb bone matrix and degrade bone inducing substance(s) like skeletal growth factor and/or bone morphogenetic peptides resulting in the deterioration or lack of bone induction.

Animals↗
PubMed1998

Effects of ketorolac on bone repair: A radiographic study in modeled demineralized bone matrix grafted rabbits.

The effects of ketorolac on bone repair were studied radiographically. The effects of methylprednisolone were also compared. Demineralized bone matrix was grafted into the fractured gap of rabbit's ulna as an experimental bone repair model. The rabbits received ketorolac 2 or 4 mg/kg body weight daily for 6 weeks after transplantation. Serial radiographic studies were performed 2, 4, and 6 weeks after transplantation. Mineralization of grafts and bone union were evaluated as the parameters of bone repair. Comparing the bone repair among groups at individual time points, ketorolac-treated groups showed no statistical differences as compared with the control group in either mineralization or bone union, whereas methylprednisolone significantly suppressed both parameters. However, comparing the mineralization during the healing process in each group, the control group and the group treated with ketorolac 2 mg/kg showed a significant increase during the period from the 2nd to the 4th week of medication, whereas both the group treated with ketorolac 4 mg/kg and the methylprednisolone-treated group showed no significant increase during this period. This poor mineralization of grafted bone in repair process affected by ketorolac suggests that 4 mg/kg of ketorolac might delay the endochondral ossification process during the period from the 2nd to the 4th week of fracture healing.

Animals↗
PubMed2001

Dose-dependent toxicity of a commercially available demineralized bone matrix material.

STUDY DESIGN: A relative risk assessment was performed to determine the safety of three commercially available Grafton demineralized bone matrix quantities used in athymic rats. OBJECTIVE: To evaluate the possible dose-dependent adverse effects of a commercially available demineralized bone matrix containing glycerol. SUMMARY OF BACKGROUND DATA: Commercially available Grafton demineralized bone matrix contains glycerol. The toxic effects of glycerol leading to acute renal failure have been documented. The toxicity of this glycerol-containing substance in higher doses has not been reported. METHODS: Three doses of Grafton putty were implanted in the upper hind limb muscles of athymic nude rats. The rats were observed for adverse effects and early death. Histologic studies were performed. RESULTS: All eight of the rats implanted with the highest dose of Grafton putty (0.008 mL/g) died, five of them within 12 hours of implantation and three in 48 to 72 hours. One rat with the intermediate dose (0.004 mL/g) died within 12 hours of implantation. By 72 hours after implantation, three of the six rats (50%) with the intermediate dose had died. All six of the rats receiving the lowest dose (0.002cc/g) survived. The median lethal dose of Grafton putty in athymic rats was estimated to be 0.00469 mL/g body weight. Histologic analysis of the animals that received the high dose showed acute tubular necrosis, probably secondary to rhabdomyolysis. CONCLUSIONS: In athymic rats, large amounts of Grafton putty lead to death in a dose-dependent manner. Because the median lethal doses of Grafton putty (0.00469 mL/g) and glycerol (0.00442 mL/g) are comparable, a potential source of toxicity is the glycerol contained in the material. The results of this study suggest that high doses have the potential to cause acute renal failure. The authors suggest that clinical usage of Grafton putty in humans should be limited to no more than 2 mL/kg body weight of this material.

Animals↗
PubMed1996

Expression of bone matrix proteins during dexamethasone-induced mineralization of human bone marrow stromal cells.

Glucocorticoids have been shown to induce the differentiation of bone marrow stromal osteoprogenitor cells into osteoblasts and the mineralization of the matrix. Since the expression of bone matrix proteins is closely related to the differentiation status of osteoblasts and because matrix proteins may play important roles in the mineralization process, we investigated the effects of dexamethasone (Dex) on the expression of bone matrix proteins in cultured normal human bone marrow stromal cells (HBMSC). Treatment of HBMSC with Dex for 23 days resulted in a significant increase in alkaline phosphatase activity with maximum values attained on day 20 at which time the cell matrix was mineralized. Northern blot analysis revealed an increase in the steady-state mRNA level of alkaline phosphatase over 4 weeks of Dex exposure period. The observed increase in the alkaline phosphatase mRNA was effective at a Dex concentration as low as 10(-10) M with maximum values achieved at 10(-8)M. In contrast, Dex decreased the steady-state mRNA levels of both bone sialoprotein (BSP) and osteopontin (OPN) over a 4 week observation period when compared to the corresponding control values. The relative BSP and OPN mRNA levels among the Dex treated cultures, however, showed a steady increase after more than 1 week exposure. The expression of osteocalcin mRNA which was decreased after 1 day Dex exposure was undetectable 4 days later. Neither control nor Dex-treated HBMSC secreted osteocalcin into the conditioned media in the absence of 1 ,25(OH)(2)D(3) during a 25-day observation period. The accumulated data indicate that Dex has profound and varied effects on the expression of matrix proteins produced by human bone marrow stromal cells. With the induced increment in alkaline phosphatase correlating with the mineralization effects of Dex, the observed concomitant decrease in osteopontin and bone sialoprotein mRNA levels and the associated decline of osteocalcin are consistent with the hypothesis that the regulation of the expression of these highly negatively charged proteins is essential in order to maximize the Dex-induced mineralization process conditioned by normal human bone marrow stromal osteoprogenitor cells.

Adolescent↗