Bone resorption in orthotopic and heterotopic bone of dichloromethylene bisphosphonate-treated rats

The effect of the bisphosphonate dichloromethylene bisphosphonate (Cl2MBP) on orthotopic and heterotopic bone, induced by implants of demineralized bone matrix (DBM) in rats, was analyzed, with special reference to bone resorption. The heterotopic bone was formed by induction for 3 weeks; at this time, the rats were given daily subcutaneous injections of 3 mg/kg of body weight of Cl2MBP or saline, until sacrifice. Prior to the start of treatment, the animals were given 45Ca and [3H]proline to label the inorganic and organic components of bone, respectively. Groups of rats were sacrificed at intervals from 1 to 31 days after isotope injection, and the net formation of bone and the elimination rates of the two isotopes were studied in the heterotopic bone, in diaphyseal and metaphyseal bone, and in teeth. The treatment with Cl2MBP caused a doubling of the daily net increase in mineral of the induced heterotopic bone, and a less pronounced increase in the ash content of tibiae. The treatment decreased the elimination rates of both isotopes in the orthotopic and heterotopic bone, showing that decreased bone resorption is the cause of the increased net bone formation.     

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.     

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.     

Immunosuppression with FK506 increases bone induction in demineralized isogeneic and xenogeneic bone matrix in the rat.

The aim of the present study was to investigate a systemic induction of bone formation in rats by immunosuppression with FK506 (1 mg/kg body weight intraperitoneally [ip]) in a model of osteoinduction of isogeneic and xenogeneic demineralized bone matrix (DBM) for a period of 28 days. In particular, alterations of in vitro cytokine synthesis and changes of lymphocyte subsets were studied. DBM was implanted intramuscularly in the abdominal wall of Lewis rats (seven per group). Blood was sampled on days -7, 0, 7, and 28 for determination of in vitro tumor necrosis factor a (TNF-alpha) synthesis and lymphocyte subsets by flow cytometry (CD3+, CD4+, CD8+, CD45+, ED9+, and Ia+ antibodies). Ossicles of de novo formed bone and the tibias were removed on day 28 after double tetracycline labeling for histomorphometric analysis. Immunosuppression with FK506 significantly decreased lipopolysaccharide (LPS)-stimulated in vitro cytokine synthesis after 7 days and 28 days (p < 0.05). Compared with control animals FK506 treatment significantly increased the volume of induced bone in isogeneic (2.1 +/- 0.3 mm3 vs. 10.8 +/- 0.9 mm3) and xenogeneic (O mm3 vs. 4.7 +/- 0.8 mm3) DBM. Bone histomorphometry of the tibias revealed that immunosuppression increased both bone formation and bone resorption, accompanied by a significant reduction in the relative trabecular area (Tb.Ar). FK506 caused a decrease in the counts of CD8+ T cells probably because of destruction or dislocation of these cells. This suggests that the amount of CD8+ cells and the degree of T cell activation in terms of mean fluorescence intensity (MFI) may be associated with bone metabolism. In support of this, statistical analysis revealed a significant positive correlation between parameters of bone formation as well as bone resorption and the CD4+/CD8+ ratio. There was a significant negative correlation between parameters of remodeling of the metaphysis of the tibia and induced bone volume (BV), respectively, and MFI values of CD3+/Ia+ cells. These findings suggest an important role of T lymphocytes in bone formation and bone resorption in vivo. FK506 caused a marked increase of bone formation in DBM. However, the conclusion that immunosuppression increases fracture healing warrants further investigation.     

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.     

Effect of hydrogen peroxide on osteoinduction by demineralized bone

The osteoinductive capacity of demineralized bone matrix (DBM) has led to wide use of this material for surgical reconstruction. Preparation of DBM often includes sterilization with ethylene oxide, disinfection with various chemical agents, or irradiation. Exposure to hydrogen peroxide (H2O2) is used for both sterilization and bleaching of bone, the latter primarily for cosmetic reasons. We investigated the effect of H2O2, on the osteoinductive capacity of DBM. Cortical bone implants prepared from rat femurs were placed into 3% H2O2 solution. Control specimens were not exposed to H2O2. Bones were then lipid-extracted, demineralized, sterilized with ethylene oxide, and freeze-dried in an identical manner. Allografts were implanted into rat hosts for 1 to 3 weeks. Osteoinduction proceeded rapidly in implants not exposed to H2O2, with chondrocytes and new bone appearing in the implant. After 3 weeks, perforations in the implant were largely replaced with new bone. In contrast, osteoinduction did not occur in implants treated with H2O2. Perforations in H2O2-treated implants were filled with vascularized fibrous tissue, but no cartilage or bone. These findings reveal that H2O2 used for disinfection or bleaching of DBM can abolish its osteoinductive capacity in rats.     

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.     

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.     

Bone morphogenetic protein induces bone in the squirrel monkey, but bone matrix does not.

Demineralized bone matrix (DBM) reproducibly induces extraskeletal bone formation in rodents, but its effects in dogs and primates are negative or uncertain. In previous studies on the squirrel monkey, DBM did not induce bone, although the same implants were effective in nude rats. In the present study, the DBM was augmented with recombinant human bone morphogenetic protein-2 (BMP-2). Bone was formed in 10 of 12 monkeys, as verified by histology and calcium content. However, in 4 monkeys, the induced bone mass appeared smaller than the original implant. DBM controls induced microscopic amounts of bone in 2 out of 10 monkeys. In the nude rats, all DBM controls and augmented implants induced bone. The difficulties in achieving bone induction in higher animals may be overcome, at least partially, by using a higher concentration of the inductive protein than is present in DBM.     

Effects of acetylsalicylic acid and naproxen on bone resorption and formation in rats

The influence of acetylsalicylic acid (ASA) (150 mg/kg/12 h) and naproxen (20 mg/kg/12 h) on bone metabolism in young male rats has been studied. The doses were chosen to provide serum concentrations comparable with ordinary anti-inflammatory steady-state levels in humans. After the rats had been prelabeled with collagen- and mineral-tracing radioisotopes the rats received the drugs by gavage twice a day for 9 and 18 days. Bone resorption was measured as loss of carbon-labeled hydroxyproline (collagen) and strontium-85 (minerals). At 9 days ASA had retarded both collagen and mineral resorption in the femur by about 10% compared with controls. The resorption of both collagen and minerals was inhibited. After 18 days' treatment there were no differences regarding bone resorption, but bone formation had decreased by about 10% in the ASA-treated animals, as measured by net increases of collagen and calcium in the femur. Naproxen did not influence bone resorption or formation significantly. The results indicate an inhibitory effect of ASA on bone resorption and formation in growing rats, whereas the effect of naproxen seems negligible.     

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.     

Influence of indomethacin on experimental bone metabolism in rats

The effect of indomethacin on heterotopic and orthotopic bone formation in rats was analyzed with respect to dose and time of initiation of drug administration. Three weeks after implantation of demineralized bone matrix, the ash weight of implants from animals receiving the highest doses of indomethacin was approximately 25% lower than that of controls. The ash weight of implants was only affected in rats receiving indomethacin from the week before implantation or from the time of implantation. In a separate study, no effect on the rate of resorption measured by elimination of 3H-proline and 45Ca or on the amount of ash could be detected. Orthotopic bone remained unaffected by indomethacin treatment. The study showed that in order to inhibit experimental new bone formation, indomethacin must be present before or at the time of implantation of demineralized bone matrix.     

Effects of gallium on bone in the rat.

Gallium nitrate lowers the serum calcium in patients with hypercalcemia caused by malignancy and is available for clinical use. The mechanism for the hypocalcemic action is unknown, however. The present studies were undertaken to determine the effects of gallium on bone metabolism. Normal male rats were implanted subcutaneously with mineralized allogeneic bone matrix. Histomorphometry of the implants and of tibiae was determined after three doses of tetracycline administered at intervals of 1 week. Gallium as nitrate was administered daily by intraperitoneal injection at doses of 0.9, 1.8, and 3.6 mg elemental gallium per kg body weight for 21 days in one study and at 3.5 mg/kg for 33 days in a second study. All the gallium-treated rats gained weight. Rats given gallium at doses of 3.5 mg/kg or more grew at a lower rate than untreated controls (-7 and -10% at doses of 3.5 and 3.6 mg/kg, respectively; p less than 0.05). At a dose of 0.9 mg/kg, gallium did not inhibit bone resorption or lower serum calcium but inhibited bone formation by 32% and bone apposition by 36% at the endosteal surface of the tibia. At a dose of 1.8 mg/kg, gallium produced modest hypocalcemia, prevented a rise in circulating 1,25-dihydroxyvitamin D [1,25-(OH)2D], inhibited bone resorption in implants, and inhibited bone formation by 19% and bone apposition by 18%. At a dose of 3.5 mg/kg, gallium lowered the serum calcium and serum 1,25-(OH)2D, inhibited growth, and accentuated the antiresorptive and antiformative effects seen at the two lower doses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methotrexate effects on heterotopic bone in rats

We studied the effects of high-dose methotrexate on heterotopic bone formation induced by implants of demineralized bone matrix in the abdominal wall of growing rats. Methotrexate induced an arrest in normal weight gain of the animals, more pronounced the younger the animals were. The youngest animals had reduced ash weight and decreased isotope uptake in the tibiae and teeth. However, implants from these animals, given methotrexate 10 days before implantation of bone matrix, had a 33 per cent increase in ash content. When methotrexate was given at, or 10 days after, implantation, heterotopic bone formation was reduced by 40 and 22 per cent, respectively, whereas orthotopic bone was considerably less affected in these older animals. In a second experiment, no difference in elimination rates of 45Ca between methotrexate-treated and control rats in implants, teeth, or tibiae were found. It appears that a less detrimental effect of methotrexate on new bone formation can be expected if the drug is given before, or a substantial period after, surgery requiring bone formation for healing.     

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.     

Ultrastructure, tartrate-resistant acid phosphatase activity and calcitonin responsiveness of osteoclasts at sites of demineralized bone matrix implant-induced osteogenesis

The morphology, ultrastructure, tartrate-resistance acid phosphatase reactivity, and calcitonin responsiveness of osteoclasts induced at sites of demineralized bone matrix (DBM) implant-induced osteogenesis in rats were determined. Osteoclasts at these ectopic sites had a morphologic and ultrastructural appearance similar to osteoclasts normally found in skeletal tissues. When observed by scanning electron microscopy, resorption surfaces on the implants had well-defined resorption pits (Howship's lacunae), indicative of active bone resorption. The osteoclasts stained intensely for tartrate-resistance acid phosphatase, an enzyme that is specific for osteoclasts. In response to human calcitonin, hypocalcemia occurred and osteoclasts lost their ruffled borders, indicating that these cells are responsive to exogenous hormonal stimulation. The osteoclasts induced by subcutaneous implantation of DBM had morphologic and functional characteristics similar to osteoclasts normally found in skeletal tissues.     

The effects of immobilization on bone histomorphometry in rats

To determine whether immobilization acts directly on bone by alteration of mechanical loading or systemically, studies of the effects of immobilization were carried out on histomorphometry of diaphyses of tibiae and on subcutaneous implants of demineralized allogenic bone matrix of rats. The right hind leg of growing rats was denervated by severing the tibial nerve. A sham operation on the right hind leg was performed in control animals. Bone formation at the endosteal and periosteal surfaces was significantly lower in tibiae from limbs with severed nerves as compared to tibiae from the intact limbs of nerve-sectioned rats and from both limbs of sham-operated control rats. Bone formation was decreased at both 3 and 7 weeks after immobilization. The decreased formation resulted in significant reductions in cross-sectional area. At 3 weeks post denervation, the periosteal bone formation rate was lower in tibiae of intact limbs from denervated rats as compared to tibiae from intact limbs of sham-operated animals. This finding was attributed to reduced physical activity of the denervated rats. In the implants, nerve section did not alter the amount of implant matrix resorbed, the amount of bone matrix synthesized, or the amount of calcium in the implant. These findings support the hypothesis that inhibition of bone formation at the tibial diaphysis in response to immobilization resulted from altered mechanical loading and not from the production of substances acting systemically. Whereas the mean medullary area of tibiae was not altered by nerve section, it was decreased in tibiae of all groups compared to the values of basal controls, indicating that bone formation was greater than bone resorption.(ABSTRACT TRUNCATED AT 250 WORDS)     

BMP-2 for intramuscular bone induction:Effect in squirrel monkeys is dependent on implantation site

Demineralized bone matrix (DBM) reproducibly induces extraskeletal bone formation in rodents, but its effects in dogs and primates are negative or uncertain. In previous studies on the squirrel monkey, DBM did not induce bone, although the same implants were effective in rats. DBM implants augmented with recombinant human bone morphogenetic protein-2 (rhBMP-2) induced intramuscular bone formation in squirrel monkeys. However, the amount of induced bone was often minimal and sometimes absent. One explanation of this weak and unpredictable effect could be reactions to cellular components of the allogeneic DBM that was used as carrier. Therefore, we now repeated the experiment, using bovine type I collagen as carrier. 48 collagen discs (10 mm diameter), containing 0, 10, 40 or 200 pg rhBMP-2, were implanted in 6 monkeys and 6 rats. The BMP-2 implants induced dose-dependent amounts of intramuscular bone in rats whereas, in squirrel monkeys, macroscopic bone induction occurred in less than half of the BMP-2 implants. There was no dose-dependency. Whether bone was formed or not was significantly influenced by individual variation among the monkeys, and by implant location within the muscle. Implants close to the muscle aponeurosis more often induced bone than did purely intramuscular ones. In this small series, we could not demonstrate a significant effect of BMP-2, as compared to control implants. Presumably there were too few cells expressing BMP-2 receptors in the only minimally traumatized muscle of these monkeys.     

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.     

Insulin-like growth factor I increases bone formation in old or corticosteroid treated rats

We studied bone induction in subcutaneous implants of demineralized bone matrix with or without insulin-like growth factor I (IGF-I) in aged or corticos-teroid-treated rats. Each rat carried one pair of implants, one control and one experiment implant, containing IGF-I dissolved in a hyaluronan solution for slow release. The rats were killed after 3 weeks and the results were evaluated by measuring the calcium content of implants. Young (6-7 weeks) and old (19-27 months) rats were used. A group of young rats was treated for 1 week with subcutaneous injections of 140 ng/kg dexamethasone daily. Old rats produced only approximately 1 % as much bone as young rats. Local delivery of IGF-I did not increase bone formation in young rats. In old rats, bone formation was increased by IGF-I, 3000 ng/implant. Corticosteroids reduced bone formation in young rats. This effect was partially reversed by local administration of IGF-I.