Native Bone Morphogenetic Protein Research Articles

Osteoinductivity of partially purified bovine, ostrich and emu bone morphogenetic proteins in vitro

The aim of this study was to observe the osteogenic activity of native bone morphogenetic proteins (BMPs) obtained from different species including bovine, ostrich and emu sources in order to compare mammalian and avian BMPs. Rat mesenchymal progenitor marrow stromal cells and pre-osteoblastic C2C12 cell cultures, were exposed to the native BMPs and alkaline phosphatase (ALP) and creatine kinase (CK) levels were determined by assay. The results showed that the ALP activity in C2C12 cultures was elevated by bovine BMP by 2- to 10-fold (p < 0.05-0.001) from day 3 during 14 days. There were no significant differences in avian BMP related elevations of ALP activity except with ostrich BMPs at day 14 (p < 0.05). However, exposure of MSCs cultures to BMPs derived from bovine, ostrich or emu sources resulted in elevated ALP from day 3 (p < 0.05). Bovine BMP resulted in more ALP elevation than with either of the avian BMPs. All of BMPs elevated Creatine kinase (CK) activity from day 1 and climbed until peaking at day 7. Compared with control cultures, CK was elevated more with exposure to emu BMP and was more elevated with greater statistical significance than with bovine and ostrich BMP before day 5. These higher levels remained until day 14 (p < 0.05). The results of this study suggest that both bovine and avian BMPs are able to stimulate osteogenesis in mature osteoblasts in vitro. The strongest synergistic effect on osteogenesis was detected in cells stimulated with bovine BMP. Avian BMPs had lower effects on ALP and CK activity, emu BMP being more effective than ostrich BMP.

The Evaluation of Mineralized Collagen as a Carrier for the Osteoinductive Material COLLOSS®E, In Vivo

In this study, the suitability of mineralized collagen, as a carrier for the native bone morphogenetic protein containing product COLLOSS(®)E, was evaluated in a rat subcutaneous implantation model. Tubular-shaped scaffolds were made from mineralized collagen and were left empty (controls), filled with 40 mg COLLOSS(®)E, or filled with a mixture of COLLOSS(®)E and osteoconductive β-tricalcium phosphate (β-TCP) granules. Six of all the samples were implanted in 18 animals. During the implantation period, macroscopic examination suggested inflammatory reaction. Histological evaluation after 12 weeks confirmed the presence of inflammatory cells. Although the mineralized scaffold and β-TCP granules could be clearly seen, no new bone formation was found. This is in contrast to previous reports in a similar set-up wherein a titanium mesh was used as scaffold material in combination with COLLOSS(®)E. It is concluded, therefore, that the osteoinductive capacity of bone morphogenetic proteins or, more specifically, COLLOSS(®)E is inhibited when the carrier gives rise to a significant inflammatory reaction. Additional studies are necessary to assess the effect of combining COLLOSS(®)E with a dimensionally stable and osteoconductive material like β-TCP.

Endostatin inhibits endochondral ossification

Angiogenesis is essential for the replacement of cartilage by bone during skeletal growth and regeneration. Vascular endothelial growth factor-A (VEGF-A) is a key regulator of angiogenesis whereas endostatin, a potent inhibitor of endothelial cell proliferation and migration, is a natural antagonist of VEGF-A. The regulatory role of these peptides in angiogenesis and bone formation was investigated using adenoviral gene delivery of VEGF-A and endostatin in a mouse ectopic ossification model. Bone formation was induced in the hamstring muscles of adult mice with native bone morphogenetic protein (BMP) extract implemented in gelatine gel together with VEGF-A and endostatin recombinant adenoviral vectors. The mice were sacrificed 1, 2, and 3 weeks after the operation and ectopic bone formation was followed radiographically and histologically. Significant bone formation was induced by BMP extract in all treatment groups. VEGF-A stimulated and endostatin prevented the formation of FVIII-related antigen-positive vessels as well as the number of cartilage-resorbing chondroclasts/osteoclasts. Endostatin alone or in conjugation with VEGF-A reduced bone formation. Excess of VEGF-A stimulated and endostatin reduced bone formation, respectively, at the 3-week time point. Our findings indicate that endostatin retards the cartilage phase in endochondral ossification which subsequently reduces bone formation in our experimental model. We conclude that bone growth and healing, which share features with ectopic bone formation, may be regulated by endostatin.

Heterotopic Bone Formation Around Sintered Porous-Surfaced Ti-6Al-4V Implants Coated with Native Bone Morphogenetic Proteins

Coating endosseous dental implants with growth factors such as bone morphogenetic proteins (BMPs) may be one way to accelerate and/or enhance the quality of osseointegration. The purpose of this study was to investigate in the murine muscle pouch model whether sintered porous-surfaced titanium alloy implants coated with BMPs would lead to heterotopic bone formation around and within the implant surface geometry. Porous-surfaced dental implants were coated with partially purified native human BMPs, with or without a carrier of Poloxamer 407 (BASF Corp., Parsippany, NJ), placed in gelatin capsules and implanted into the hindquarter muscles of mice. Mice were euthanized after 28 days. Sections of retrieved specimens were subsequently prepared for morphometric analysis of bone formation using backscatter electron microscopic images. Human BMPs, either with or without the carrier of Poloxamer 407, led to bone formation within and outside of the sintered porous implant surface. When the sintered implant surface region was subdivided into inner and outer halves, similar levels of bone ingrowth and contact were seen in the 2 halves. Evidence of bone formation to the depth of the solid implant core (i.e., the deepest level possible) also was seen. Sintered porous-surfaced dental implants can be used as substrate for partially purified BMPs in the murine muscle pouch model. With the addition of these osteoinductive factors, the porous implant surface supported bone formation within the surface porosity provided, in some instances, all the way to the solid implant core. The addition of growth factors to a sintered porous surface may be an efficient method for altering locally the healing sequence and quality of bone associated with osseointegration of bone-interfacing implants.

Osteoinductivity of partially purified native ostrich ( Struthio camelus) bone morphogenetic protein: Comparison with mammalian species

Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily. They are capable of inducing ectopic bone formation. Until now, the main interest has been focused on mammalian osteoinductive BMPs, and there are no reports of native BMP extracts of birds. In this study, we isolated and characterized native BMPs of ostrich ( Struthio camelus) and compared them with identically isolated native bovine (cow) and reindeer BMPs with regard to BMP pattern and osteoinductive capacity. The ostrich BMP pattern differed markedly from that of cow and reindeer BMP in non-reduced SDS-PAGE, reduced SDS-PAGE and Western blot. The differences in isoelectric focusing analysis were smaller. However, the ostrich BMP extract had a peak at pH 5.1, clearly differing from the BMPs of cow and reindeer. The osteoinductive capacity and density of ectopic bone, induced by BMP extracts in a mouse thigh muscle pouch, were determined radiographically. The ostrich BMP extract displayed significantly lower osteoinductive capacity and density of induced bone than the bovine and reindeer BMP extracts. In conclusion, our results indicate that the BMP pattern of birds differs considerably from that of mammals, and that the osteoinductive capacity of BMPs and the density of induced bone are lower in birds than in mammals. They also suggest that the bone metabolism of birds is adapted to make light bones suitable for flying.

Gamma Irradiation and Ethylene Oxide in the Sterilization of Native Reindeer Bone Morphogenetic Protein Extract

For human use, it is necessary to sterilize bone morphogenetic proteins (BMPs), in order to reduce the risk of infections and associated complications. We compared the effects of ethylene oxide and gamma irradiation in the sterilization of native reindeer BMP extract with regard to bone induction in the Balb/C mouse thigh muscle pouch model. BMP extract, sterilized with ethylene oxide gas (Steri-Vac 4XL, temperature 29 degrees C, exposure time 4 h, ethylene oxide concentration 860 mg/l), or gamma irradiation at doses of 3.15 MRad was administered in implants containing 5 or 10 mg of BMP extract with collagen carrier. Non-sterilized collagen implants served as controls. New bone formation was evaluated based on the incorporation of Ca45 and radiographically three weeks after implantation. The collagen was not able to induce new bone visible in radiographs. The mean Ca45 incorporation in the gamma sterilized group containing 5 mg of BMP extract was 30% (p = 0.04) and that containing 10 mg of BMP extract was 60% (p = 0.02) higher than seen in the corresponding ethylene oxide sterilized groups. The mean new bone areas were 45% higher in the gamma sterilized groups than in the corresponding ethylene oxide sterilized groups, but the differences were not significant. The mean optical density of new bone in the gamma sterilized group containing 5 mg of BMP extract was 75% (p = 0.00) and in that containing 10 mg of BMP extract was 70% (p = 0.00) higher than seen in the corresponding ethylene oxide sterilized groups. Native reindeer BMP extract is more sensitive to the effects of ethylene oxide gas sterilization than gamma irradiation. These results suggest that gamma irradiation is recommendable for the sterilization of BMP extracts.

Regulation of BMP-induced ectopic bone formation by Ahsg

α2-HS-glycoprotein (Ahsg), also known as fetuin is a serum and bone resident glycoprotein, which binds to TGF-β superfamily members including bone morphogenetic proteins (BMP) and inhibits dexamethasone-induced osteogenesis in bone marrow cultures in vitro. Here we demonstrate that Ahsg reduces cytokine binding to its cognate receptor in HOS osteocyte cells and suppresses intracellular signaling, while in vivo, we test the hypothesis that Ahsg-deficient mice are hyper-responsive to BMP-induced osteogenesis. Human native BMP was implanted into the hindquarter muscles of Ahsg +/+, Ahsg +/− and Ahsg −/− mice and 4 weeks later, ossicle formation was analyzed by radiography, bone density scanning (DEXA) and histomorphometry. Alkaline phosphatase (AP) activity was measured in ossicles as a marker for bone cell differentiation, and was significantly higher in Ahsg −/− versus Ahsg +/− and/or Ahsg +/+ mice. Ectopic ossicle size in the Ahsg +/− mouse was 4-fold greater than that in the wild type (Ahsg +/+), and intermediate to that shown in Ahsg −/− mouse. Bone mineral density (BMD) was lower in the Ahsg −/+ and Ahsg −/− mice compared to Ahsg +/+ littermates. The ratio of cortical to cancellous bone was found to be >2-fold higher in Ahsg −/− mouse in comparison to the Ahsg +/+ mice with no significant change in the Ahsg −/+ mouse. Finally, a significantly higher incidence of satellite ossification; small islands of immature bone, was shown in Ahsg −/− mice as compared to Ahsg +/+ mice. Although Ahsg binds to TGF-β/BMP and blocks receptor signalling, it may also sequester cytokines in matrix, thereby acting as a reservoir of osteoinductive activity when released. This may explain the non-linear relationship between ectopic bone formation characteristics and Ahsg +/+, Ahsg +/− and Ahsg −/− genotypes, although the increase in satellite bone formation might also explain this phenomenon. Our results suggest that Ahsg may be useful for prevention of the heterotopic ossification and the regulation of osteoinductive effects of BMP used with grafts.

Bone tissue induction, using a COLLOSS ®-filled titanium fibre mesh-scaffolding material

Scaffold materials for bone tissue engineering often are supplemented with bone morphogenetic proteins (BMPs). In the current study we aimed to investigate COLLOSS ®, a bovine extracellular matrix product containing native BMPs. Hollow cylindrical implants were made, with a length of 10 mm, a 3 mm inner diameter, and a 5 mm outer diameter, from titanium fibre mesh. The central space of the tube was filled with 20 mg COLLOSS ®. Subsequently, these implants, as well as non-loaded controls, were implanted subcutaneously into the back of Wistar rats, with n = 6 for all study groups. After implantation periods of 2, 8, and 12 weeks, tissue-covered implants were retrieved, and sections were made, perpendicular to the long axis of the tube. Histology showed, that all implants were surrounded by a thin fibrous tissue capsule. After 2 weeks of implantation, the COLLOSS ® material was reduced in size inside the loaded implants, but no bone-like tissue formation was evident. After 8 weeks, in two out of six loaded specimens, new-formed bone- and bone marrow-like tissues could be observed. After 12 weeks, this had increased to five out of six COLLOSS ®-loaded samples. The amount of bone-like tissue did not differ between 8 and 12 weeks, and on average occupied 15% of the central space of the tube. In the non-loaded control samples, only connective tissue ingrowth was observed. In conclusion, we can say that COLLOSS ® material loaded in a titanium fibre mesh tube, showed bone-inducing properties. The final efficacy of these osteo-inductive properties has to be confirmed in future large animal studies.

The effect of different mineral frames on ectopic bone formation in mouse hind leg muscles induced by native reindeer bone morphogenetic protein

Bone morphogenetic proteins (BMPs) require carrier material for slow release and framing material for osteoconduction. The effect of a frame on early bone formation induced by partially purified native reindeer BMP in composite implants containing 3 mg of BMP, type IV collagen and tricalcium phosphate (TCP/Col/BMP) or hydroxyapatite (HA/Col/BMP) or biphasic tricalcium phosphate-hydroxyapatite (TCP/HA/Col/BMP) or biocoral (NC/Col/BMP) was evaluated using a mouse hind leg muscle pouch model. Collagen with native reindeer BMP (Col/BMP) and corresponding implants without native reindeer BMP served as controls. Evaluation was done by incorporation of 45Ca, radiographically and histologically 3 weeks after the implantation. None of the implants without native reindeer BMP were able to induce new bone visible on radiographs. The area of new bone formation in the Col/BMP (p=0.026) and TCP/HA/Col/BMP (p=0.012) groups was significantly greater than in the TCP/Col/BMP group. The optical density of the new bone area was significantly greater in the TCP/HA/Col/BMP group than in the TCP/Col/BMP (p=0.036) or Col/BMP (p=0.02) groups. 45Ca incorporation was many times greater in all the groups containing native reindeer BMP than in the corresponding groups without BMP. In the Col/BMP (p=0.046) and TCP/HA/Col/BMP (p=0.046) groups, 45Ca incorporation was significantly greater than in the TCP/Col/BMP group. No significant differences were found in any parameters between HA/Col/BMP and NC/Col/BMP groups and the other BMP-containing groups. Hydroxyapatite, biocoral and biphasic tricalciumphosphate-hydroxyapatite are equally good as framing material for native reindeer BMP, while tricalciumphosphate is somewhat worse. Osteoinduction of native reindeer BMP works well with collagen alone.

The Osteoinductive Activity of Bone Morphogenetic Protein (BMP) Purified by Repeated Extracts of Bovine Bone

Native bone morphogenetic proteins (BMPs) extracted from bone have been used clinically to stimulate bone regeneration and repair. However, preparation of purified BMP is a laborious process. This study investigated the yield, activity and cost effectiveness of repeatedly extracting the same bone matrix to produce purified BMP.While repeated extraction was able to increase the yield 62% the activity of the partially purified BMP in later extracts decreased both in vitro and in vivo. This decline in activity appears to be due to an increase in non-BMP contaminants, such as collagen, in the extracts. When the first three extracts were combined and processed together activity was equivalent to that of the first extract. A simple analysis based on the cost of reagents used and the time required for purification indicates that separate processing of the extracts is inefficient while combining the first and second extracts and processing them together would result in a small cost saving.Based on this study we would recommend that the demineralized bone matrix be extracted no more than twice and that the extracts be combined for further processing.

Influence of ethylene oxide sterilization on the activity of native reindeer bone morphogenetic protein.

We studied the effects of ethylene oxide sterilization (Steri-Vac 4XL, temperature 29 degrees C, exposure time 4 h 10 min, ethylene oxide concentration 860 mg/l) on the osteoinductivity of partially purified native reindeer bone morphogenetic protein (BMP) in a hind leg muscle pouch model of male NMRI mice. BMP was administered in implants containing 3 mg in a collagen carrier. Implants without sterilization and without BMP served as controls. New bone formation was evaluated based on the calcium yield, radiographic and histological examination 3 weeks after implantation. The implants without BMP were not able to induce new bone visible in radiographs. In the sterilized BMP group, the mean area of new bone was 35% ( p=0.004) and density 32% ( p=0.000) smaller than in the nonsterilized group. Calcium yield was 20% lower in the sterilized group than in the nonsterilized group, but this difference was not significant ( p=0.22). It was many times lower in the group without BMP than in the above-mentioned groups ( p=0,001). We conclude that ethylene oxide gas sterilization reduces the bone-forming activity of native reindeer BMP by one third.

New bone formation induced by injection of native reindeer bone morphogenetic protein extract.

Bone morphogenetic proteins (BMPs) are usually administered with a solid framing material during open surgery. In some instances, percutaneous administration of injectable BMP would be preferable. We tested the new bone-forming activity of injectable native reindeer BMP extract in the Balb/C mouse thigh muscle pouch model. The injectable implants contained 6 mg of native reindeer BMP extract and either physiological saline (NaCl/BMP) or collagen (Gel/BMP). Corresponding implants without BMP served as controls. New bone formation was evaluated based on incorporation of Ca45 and radiographically three weeks after the injection into the mouse thigh muscles. None of the injections without BMP were able to induce new bone visible in radiographs, whereas the injections with BMP induced new bone effectively. There were no significant differences in the area of new bone (p = 0.247) and its density (p = 0.739) between the NaCl/BMP and Gel/BMP groups. Ca-45 incorporation was multifold in the NaCl/BMP and Gel/BMP groups compared to the controls (p = 0.000). No significant differences in Ca-45 incorporation (p = 0.739) between the NaCl/BMP and Gel/BMP groups were observed. Our results suggest that BMP can be administered percutaneously, and that collagen and physiological saline are equally good carriers of injectable implants of native reindeer BMP.

Demineralized bone matrix and native bone morphogenetic protein in orthopaedic surgery.

The recognition that demineralized bone matrix could induce bone formation when placed in mammalian skeletal muscle led to preclinical studies of crude native insoluble bone morphogenetic protein and noncollagenous protein, followed by the clinical application of demineralized bone matrix, chemosterilized autolyzed antigen-extracted allogenic bone, and autolyzed antigen-extracted allogenic bone matrix gelatin. Cultural norms and regulatory agencies influence the availability of different demineralized bone matrix preparations in different parts of the world, but there is continued interest in the biologic structure of native insoluble bone morphogenetic protein and noncollagenous protein aggregates and the applied science of osteoinduction and osteoconduction in reconstructive orthopaedic surgery. Demineralized bone matrix is not widely available in Asia, but tissue processing facilities in the United States distribute demineralized bone matrix materials with different carriers, handling properties, and possibly osteoinductive potential. The purpose of the current study was to review the development and use of various preparations of demineralized bone matrix materials.

INFLUENCE OF ETHYLENE OXIDE STERILIZATION ON NEW BONE FORMATION INDUCED BY BOVINE BONE MORPHOGENETIC PROTEIN

Bone morphogenetic proteins (BMPs) have the capacity to induce and accelerate bone regeneration. Before experimental and clinical settings, BMP must be sterilized. Ethylene oxide (EO) gas sterilizations at different temperatures are commonly used but the effects of that on the osteoinductive capacity of BMP have been the subject of controversy. Here, we investigated the effects of three different EO sterilization methods on the osteoinductivity of partially purified native bovine BMP (bBMP). Gelatin capsules containing 3 mg of bBMP were sterilized as follows: (i) manually inside a dessicator with 12% EO spray (20°C, exposure time 2 h); (ii) with an EO gas sterilizer (Steri-Vac 4XL, temperature 29°C, exposure time 4 h 10 min, ethylene oxide concentration 860 mg/l); (iii) with an EO gas sterilizer (Steri-Vac 5XL, temperature 42°C, exposure time 3 h, ethylene oxide concentration 700 mg/l). The sterilization processes were monitored with samples of Bacillus subtilis (3M, Attest 1264). Osteoinductivity of bBMP was verified by bioassay. After 21 days of implantation of bBMP into the muscle pouches of mice, the animals were killed and new bone formation was measured radiographically and histologically. The EO sterilization techniques used did not significantly decrease the osteoinductive activity of BMP. It is concluded that commercial EO gas equipment sterilization is effective for sterilized BMP and does not decrease the osteoinductive capacity of bovine BMP.

Carriers that concentrate native bone morphogenetic protein in vivo.

It is well known that the native bone morphogenetic protein (BMP) complex is much more active than each of its components: the concentration needed for extraskeletal bone induction is about a thousand times higher for a specific rhBMP than for the native BMP complex as a whole. Since the patient's own body is capable of producing the native BMP complex, the interesting question arises whether it is feasible to develop specific implants that concentrate native BMPs to levels that allow bone induction to take place. Such BMP-concentrating implants differ from carriers that are meant to passively store and deliver rhBMP. The fact that the native BMP complex is much more active than each of its constituent BMP molecules (the required concentration for the native complex is up to a thousand times lower) makes the question even more of interest. Based on the literature, the concept is developed in this article that selected implants, through mineralization in vivo, can concentrate the patient's own native BMP complex.

Rat Skeletal Muscle Myoblasts are Target Cells for the Action of Native Bone Morphogenetic Protein

Bone morphogenetic proteins (BMPs) are multifunctional proteins capable of inducing an osteogenic phenotype in various cell lines and providing alternatives to bone grafts in the field of orthopaedics. Study was carried out here of the specific binding and stimulation of markers of bone metabolism by highly purified native bovine BMP in rat skeletal myoblasts (L6). Binding studies using 125I-labeled BMP and various concentrations of unlabeled BMP showed the existence of a single-class, one-binding-site BMP receptor. The binding was time-, temperature- and dose-dependent in various experiments. In assessing the capacity of BMP to induce myoblasts to differentiate into the osteoblast lineage, osteocalcin production was initiated, alkaline phosphatase activity increased approximately four-fold and calcium uptake almost three-fold compared with control cells during four days culturing with 12.5 μg/ml of BMP. These results indicate that the L6 myoblast is a target cell for the action of native BMP, and under the influence of BMP L6 myoblasts are capable of differentiating in the direction of osteoblasts. This BMP-induced differentiation of myoblasts offers a novel assay system for in vitro detection of osteoinductivity and investigation of the mechanism of ectopic bone formation.

The Calpain–Calpastatin System and Cellular Proliferation and Differentiation in Rodent Osteoblastic Cells

The calpain–calpastatin system, which consists of calpains I and II (two ubiquitously distributedcalcium-activated papain-like cysteine proteases), as well as calpastatin (the endogenous calpain inhibitor), plays an important role in cell proliferation and differentiation in many tissues. However, its contribution to the regulation of osteoprogenitor or pluripotent stem cell proliferation and differentiation into osteoblasts remains poorly defined. In these studies, rat pluripotent mesodermal cells (ROB-C26) and mouse MC3T3-E1 preosteoblasts were induced to differentiate into osteoblasts by long-term culture or in response to bone morphogenetic protein (BMP). The occurrence and distribution of calpain–calpastatin system proteins were determined by immunofluorescent microscopy, measurement of calcium-dependent proteolytic activity, and Western blotting. Treatment of intact MC3T3-E1 cells with an irreversible, membrane-permeable cysteine protease inhibitor attenuated proliferation and alkaline phosphatase upregulation under differentiation-enhancing conditions. Calpain II activity increased during differentiation of MC3T3-E1 cells in postconfluent culture. When ROB-C26 cells were maintained in long-term culture, neutral protease, calpain I, and calpain II activities increased 2- to 3-fold in the absence of BMP. In the presence of partially purified native BMP, neutral protease and calpain I activities also increased similarly, but calpain II activity increased by 10-fold in 3 days. The maximal increase in alkaline phosphatase occurred 4 to 11 days after the calpain II activity had peaked. Induction of differentiation in long-term MC3T3-E1 cultures was associated with higher calpain II and 70- and 110-kDa calpastatin protein levels and lower 17-kDa calpastatin degradation product levels. In conclusion, cysteine protease activity is essential for preosteoblastic proliferation and differentiation. The calpain–calpastatin system is regulated during osteoprogenitor proliferation and differentiation, as it is in other cells, and bone morphogenetic protein is a specific regulator of calpain II.

Treatment with recombinant human macrophage colony-stimulating factor resorbs blood clot and restores osteoclastogenesis in heterotopic bone induced by partially purified native bone morphogenetic protein in osteopetrotic (op/op) mice.

Native bone morphogenetic protein and associated noncollagenous proteins induced the formation of heterotopic bone in the hindquarter muscles of osteopetrotic (op/op) mice and those of their phenotypically normal littermates (+/?). In op/op mice, the heterotopic bone consisted of a disorganized, densely packed mixture of irregular calcified cartilage, osteoid, chondro-osteoid, and fibrous tissue. Injections of recombinant human macrophage colony-stimulating factor initiated bone resorption that began in the peripheral vascularized regions of the metaphyses and continued in central areas of uncalficified avascular chondro-osteoid. On vascularized surfaces, osteoclasts were stained with tartrate-resistant acid phosphatase. In op/op mice treated with macrophage colony-stimulating factor, the osteoclasts were small, with only two or three nuclei, and they did not exhibit tartrate-resistant acid phosphatase staining. In untreated op/op mice, surgical blood clots persisted in the heterotopic sites as late as 3 weeks after the operation, whereas in treated op/op mice, the blood clots were absorbed almost as rapidly as in normal mice. Histologically, recombinant human macrophage colony-stimulating factor restored normal macrophage functions: absorption and organization of blood clot, osteoclastogenesis, synthesis of tartrate-resistant acid phosphatase, bone remodeling, islands of myelopoiesis, and construction of an ossicle complete with a cortex and a medulla filled with functioning hematopoietic bone marrow.

Internalization and Intracellular Processing of Bone Morphogenetic Protein (BMP) in Rat Skeletal Muscle Myoblasts (L6)

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily capable of inducing bone and cartilage formation in ectopic extraskeletal sites and transducing their effects through binding to serine-threonine kinase receptors. In this study, the fate of 125I-labelled native BMP after binding to cell surface receptors on L6-myoblasts was examined with both continuous and intermittent exposure of the ligand. BMP was readily internalized in L6 cells at +37°C, and the internalization reached a plateau in 2 h. Intracellular degradation of 125I-labelled BMP was established, and degradation products were also detected in binding buffer, indicating exocytosis of the processed ligands. BMP receptors were shown to be subject to acute down-regulation by the ligand, and receptors were completely recycled in 3 h. Hence, we conclude that BMP receptors, like receptors for various other polypeptide ligands, have the ability to mediate intracellular delivery and degradation of the ligand.

Composites of bone morphogenetic protein (BMP) and type IV collagen, coral-derived coral hydroxyapatite, and tricalcium phosphate ceramics.

Few studies have been performed to investigate the characteristics of native Bone Morphogenetic Protein (BMP). We derived 5.85 g of native moose BMP from 102 kg of fresh moose bone and performed chromatographic studies to characterise its structure. We found the BMP molecule to have a molecular weight of 11-700 kDa. To investigate the ideal local delivery system for BMP we used porous discs as a carrier. These were implanted into 132 mice. The volume of new bone formed was evaluated by means of histological specimens, radiographs and isotope studies. The best results were obtained by implanting porous discs (coral and ceramic) with type IV collagen and BMP. We suggest that this is the best delivery system for future experiments regarding the acton of BMP.