Bovine Bone Morphogenetic Protein Research Articles

Tibial segmental bone defect treated with bone plate and cage filled with either xenogeneic composite or autologous cortical bone graft

Tibia segmental defect healing in sheep were clinically, radiographically and histologically evaluated. Twelve young sheep aged four to five months were divided into two groups, G1 and G2. A 3.5 cm long segmental defect was created in the right tibial diaphysis with maintenance of the periosteum. The bone defects in both groups were stabilized with a bone plate combined with a titanium cage. In G1 the cage was filled with pieces of autologous cortical bone graft. In G2 it was filled with a composite biomaterial which consisted of inorganic bovine bone, demineralized bovine bone, a pool of bovine bone morphogenetic proteins bound to absorbable ultra-thin powdered hydroxyapatiteand bone-derived denaturized collagen. Except for one G1 animal, all of them showed normal limb function 60 days after surgery. Radiographic examination showed initial formation of periosteal callus in both groups at osteo-tomy sites, over the plate or cage 15 days postoperatively. At 60 and 90 days callus remodeling occurred. Histological and morphometric analysis at 90 days after surgery showed that the quantity of implanted materials in G1 and G2 were similar, and the quantity of new bone formation was less (p = 0.0048) and more immature in G1 than G2, occupying 51 +/- 3.46% and 62 +/- 6.26% of the cage space, respectively. These results suggest that the composite biomaterial tested was a good alternative to autologous cortical bone graft in this experimental ovine tibial defect. However, additional evaluation is warranted prior to its clinical usage.

Clinical Evaluation of Association of Bovine Organic Osseous Matrix and Bovine Bone Morphogenetic Protein Versus Autogenous Bone Graft in Sinus Floor Augmentation

The purpose of the present study is to evaluate clinically and radiographically the behavior of the association between bovine organic osseous matrix (BOM) and bovine bone morphogenetic protein (bBMP) versus autogenous bone graft in promoting bone healing in maxillary sinus lifting procedures. Ten nonsmoking and systemically healthy patients who needed bilateral maxillary sinus grafting for the placement of osseointegrated implants were treated. In all patients, 1 side was selected as test (bBMP + BOM) and the other as control using autogenous particulated bone graft from mandibular ramus. After a period varying from 6 to 11 months, the implants were placed. The radiographic analysis was realized by panoramic-rx. The clinical analysis consisted of transoperative evaluation by visual observation of bone formation of grafted area and initial stability of implants. Radiographically, the images were evaluated observing radio-opaque aspect. Clinically, a better bone formation pattern of the grafted area in the side grafted with autogenous bone could be observed. The initial stability of the implants in the side grafted with autogenous bone was better than in the test side. The placement torque of the implants in the test side was more than 30 N-cm. In the present study, the association of bovine organic osseous matrix and bBMP as a growth factor showed different results when compared with autogenous graft. It was observed in the absence of an radio-opaque image, making it difficult to select the implants. Clinically, the bone formation was partial and showed the presence of the fibrous tissue in the grafted area when using the association of bovine organic osseous matrix and bBMP, and the placement torque of implants was less than the control side.

Role of Bovine Bone Morphogenetic Proteins in Bone Matrix Protein and Osteoblast-Related Gene Expression During Rat Bone Marrow Stromal Cell Differentiation

Bone morphogenetic proteins (BMPs) are known to promote osteogenesis, and clinical trials are currently underway evaluating the ability of certain BMPs to promote bone graft and fracture healing. To observe the mechanism of osteoinductive and bone formation, 100 microg of bovine BMP was tested during osteogenic differentiation of rat bone marrow stromal cells (MSCs) and C2C12 line culture for 14 and 28 days. We examined alkaline phosphatase (ALP) by assay, immunohistochemical studies for bone matrix proteins, and mRNA expression of bone matrix proteins and osteoblast-related analysis by reverse-transcription polymerase chain reaction. ALP activity in MSC cultures was elevated by bovine BMP by two to fivefold (P < 0.05-0.001). DNA and protein content increased over 14 days. BMP significantly increased the mRNA expression of type I collagen, ALP, osterix, osteocalcin, osteopontin, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)-A, and parathyroid hormone receptor time dependently during the osteoblastic differentiation. There was no markedly enhanced mRNA expression of bone sialoprotein (BSP) and glyceraldehyde-3-phosphate dehydrogenase compared with that of control. Immunohistochemical results also showed BMP increased immunoreactive positivity of type I collagen, osteocalcin, osteonectin, osteopontin, and BSP during the C2C12 differentiation. These data indicated that BMP enhances our ability to stimulate the differentiation of osteoblast-like cells and increases osteoinductivity, bone matrix protein formation and mineralization, angiogenesis, and chondrogenesis during osteoblast progenitor cell differentiation in vitro and that the role of chondrogenic is weak.

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.

Clinical and histological evaluation of the association between bovine organic osseous matrix and bone morphogenetic protein (BMP) in maxillary sinus augmentation

Clinical and histological evaluation of the association between bovine organic osseous matrix and bone morphogenetic protein (BMP) in maxillary sinus augmentation

Lack of repair of rat skull critical size defect treated with bovine morphometric protein bound to microgranular bioabsorbable hydroxyapatite

The ability of a pool of bovine bone morphogenetic proteins bound to synthetic microgranular hydroxyapatite (BMPb-HA) to stimulate bone repair was determined in rat critical size defects. An 8-mm diameter defect was created in the calvaria of 25 rats. In 15 rats, the defects were filled with BMPb-HA homogenized with blood (experimental group), and in 10 rats the defects were filled only with blood clots (control). The calvariae of experimental rats were collected 1, 3 and 6 months after surgery and of the control rats at the end of surgery and 6 months thereafter. The morphometric results obtained in the radiographs showed an absence of new bone formation at 1 and 3 months post-surgery and, histologically, the defects were filled with fibrous connective tissue and numerous foci of a foreign body-type granulomatous reaction around hydroxyapatite agglomerates. At the end of 6 months, the number and size of the granulomatous foci decreased and the area of the defects was reduced by 22% compared to the 0-hour control due to the formation of new bone at their borders, although the mean area was similar to the 6-month control. We conclude that the use of BMPb-HA in the treatment of critical size bone defects of the rat skull leads to the formation of a foreign body-type granulomatous reaction that markedly inhibits new bone formation, suggesting that synthetic microgranular hydroxyapatite does not represent a good carrier for BMP-induced bone formation.

Development of a porous poly(L‐lactic acid)/hydroxyapatite/collagen scaffold as a BMP delivery system and its use in healing canine segmental bone defect

A hydroxyapatite/collagen (HAC) composite was produced to mimic the natural extracellular matrix of bone, with the collagen serving as a template for apatite formation. A three-dimensional highly porous scaffold was developed by mixing HAC with poly(L-lactic acid) (PLA) using a thermally induced phase separation technique. Naturally derived bovine bone morphogenetic protein (bBMP) was incorporated into the porous HAC-PLA scaffolds, and the composite then was implanted in diaphyseal defects (2 cm in radius) of adult beagle dogs. Controls were implanted with scaffolds without BMP. The dogs were sacrificed at 6 months, at which time biocompatibility, biodegradability, and osteoinduction were evaluated by histologic and radiologic examination and by bone mineral density (BMD) measurements. All defects healed after treatment with BMP combined with HAC-PLA, and BMD at the site of the defect was higher than the BMD of the intact radius. Fibrous union developed in the control group animals. Histologic observation indicated that the presence of BMP not only promoted osteogenesis but that it also accelerated degradation of the biomaterials. Optimized design parameters of a three-dimensional porous biomaterial would give full scope to the role of BMP as an osteoinductive growth factor.

Linkage mapping of the bovine bone morphogenetic protein receptor‐1B (BMPR1B ) to chromosome 6

Linkage mapping of the bovine bone morphogenetic protein receptor‐1B (<i>BMPR1B </i>) to chromosome 6

The effects of bovine BMP associated to HA in maxillary sinus lifting in rabbits

This study examines the efficacy of using bone morphogenetic protein of bovine origin associated with other biomaterials in maxillary sinus floor augmentation procedures in rabbits (Oryctolagus cuniculus). Various approaches have been attempted to obtain sinus lifting with pre-existing tissue, but all of them have been considered inadequate, because such tissue offers very low bio-quality, requiring additional materials to stimulate bone neo-formation. For this purpose, autogenous bone is considered to be the best grafting material, but harvesting it results in great discomfort for the patient. Biomaterials from human beings or other animals are used as a substitute. In this study, four different grafting materials were used: lyophilized bovine bone powder, absorbable collagen flakes, natural hydroxyapatite (nHA) and synthetic hydroxyapatite (sHA), in nine animals divided into two groups: (A) control group (left sinus)--using just graft material, and (B) BMP group (right sinus)--using graft material with 0.5 mg bovine bone morphogenetic protein (BMPb). The observation periods were of 8 and 12 weeks duration and sequential bone neo-formation polyfluorochrome tracers (alizarin complex, calcein, and tetracycline) were subcutaneously injected, to evaluate the periods of new bone deposition. After the animals were sacrificed, the material was obtained and examined under a fluorescent microscope and also activated by UV light and the conclusion reached that the newly formed bone increase was of 33.34% when compared to the control group at 8 weeks At 12 weeks, the bone deposition in the "BMP" group was not significant while in the "control" group there was continuous growth. This difference showed that the BMP stimulated bone formation during the early periods of healing (8 weeks), although it altered the normal cycle of bone deposition over the longer period (12 weeks). The graft material showed increasing reabsorption, but the natural HA did not show significant alterations. The results of this new animal model indicated that the BMPb used, although facilitating osteoinduction, might not be sufficient to promote qualitative and quantitative bone neo-formation, which could guarantee better prognoses. The BMPb material studied may possibly become an alternative to autogenous grafts causing less discomfort for the patient.

Overview of bone morphogenetic proteins.

A literature review was conducted. To review the discovery of the bone morphogenetic proteins and describe the bone morphogenetic protein products that will or may be available for clinical use. Bone morphogenetic proteins comprise the osteoinductive component of several tissue engineering products in late-stage development as replacements for autogenous bone graft, and for bone augmentation and repair. The literature on bone morphogenetic proteins was reviewed. Bone morphogenetic proteins were discovered originally on the basis of their presence in osteoinductive extracts of bone matrix. Molecular cloning of bone morphogenetic proteins demonstrated that they are a family of related differentiation factors, each capable of inducing the formation of new bone tissue when implanted. Two of the molecules in clinical use, recombinant human bone morphogenetic protein-2 and recombinant human bone morphogenetic protein-7 (OP-1) are produced in a biotechnology process using recombinant deoxyribonucleic acid technology that offers unlimited supply and substantial control over purity and reproducible activity. A third material, bovine bone morphogenetic protein extract, is extracted from bone, and contains a mixture of bone morphogenetic protein molecules. Each of these molecules, although osteoinductive in vivo, has different physiologic roles and biologic activities in vivo and in vitro. Successful development of a product for use in spinal fusion involves selecting the osteoinductive molecule, the amount of the bone morphogenetic protein required, and the method of delivery, as well as conducting subsequent preclinical and clinical studies to evaluate its efficacy and safety. On the basis of the data provided in this issue of Spine, some of these bone morphogenetic protein-based products provide for revolutionary therapies in orthopedic practice.

Composite implant of native bovine bone morphogenetic protein (BMP) and biocoral in the treatment of scaphoid nonunions--a preliminary study.

Bone morphogenetic protein (BMP) has been shown to induce bone formation and union in long bone defects and nonunions. We report a preliminary study of a composite implant consisting of a biocoral frame, carrier collagen and bovine BMP in the treatment of scaphoid nonunions. Two proximal and eight waist area scaphoid nonunions were treated using BMP/coral implant combined with either the Matti-Russe procedure (2 cases) or an interpositional bone graft fixed with screws or compression fixation pins (8 cases). In two cases only a one piece BMP/coral implant was used as an interpositional graft and in other cases interpositional autograft was used with granular BMP/coral implant placed between the fragments and the graft. Only two wrists resulted in complete union. These preliminary results suggest that composite implant of BMP, as used in the present study, may not solve the problems encountered in the treatment of scaphoid nonunions. Poor vascular conditions in scaphoid may not provide enough mandatory osteogenic cells for BMP to function properly. In avascular conditions coral does not resorb edequately and implants may also work as a sequester between the bone graft and the scaphoid bone and therefore actually inhibit the healing process.

Bovine bone implant with bovine bone morphogenetic protein in healing a canine ulnar defect.

Xenograft is considered an alternative material for bone transplantation, but its bone healing capacity is inferior compared to that of autografts and allografts. Here, we tested whether bone morphogenetic protein (BMP) addition enhances the suitability of demineralized xenogeneic bovine bone for bone grafting in dogs, and whether xenogeneic bone is a suitable carrier material for BMPs. The capacity of demineralized bovine bone implants, with and without native partially purified bovine BMP, to heal a 2-cm ulnar defect was determined in six dogs over a follow-up time of 20 weeks. No instances of bone union were seen, but there was slightly more bone formation in the xenografts with BMP, though the difference was not statistically significant. The ulnas treated with an implant with BMP were also mechanically stronger, but the difference was not significant. Computed tomography scans showed no differences in the implant area in bone density, bone mineral content, or bone cross-sectional area. It is concluded that native, partially purified BMP does not sufficiently improve the suitability of bovine demineralized xenografts as a bone substitute material for dog. Demineralized xenogeneic bone does not seem to be a feasible carrier material for BMP.

Enhancement of the in vivo osteogenic potential of marrow/hydroxyapatite composites by bovine bone morphogenetic protein

Enhancement of the in vivo osteogenic potential of marrow/hydroxyapatite composites by bovine bone morphogenetic protein

Influence of Ethylene Oxide Sterilization on New Bone Formation Induced by Bovine Bone Morphogenetic Protein

Influence of Ethylene Oxide Sterilization on New Bone Formation Induced by Bovine Bone Morphogenetic Protein

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.

Endogenous BMP-4 and BMP-7 Gene Expression in CD-1 Mouse During Chondrogenesis Induced by Bovine Bone Morphogenetic Protein Implantation

The osteoinductive activity of bone morphogenetic proteins can be demonstrated by implantation into the muscle pouch in the mouse to induce ectopic cartilage and bone. To study endogenous BMP expression after implantation of 2 mg of native bovine BMP, cDNA clones of the chondroinductive factors BMP-4 and BMP-7 were constructed and used as probes. The investigation concentrated on the early phase of regeneration three and seven days after implantation. Expressions of the endogenous BMP-4 and BMP-7 genes during chondrogenesis was observed. Both BMP-4 and BMP-7 genes were significantly expressed as early as seven days after implantation. The results suggest that ectopic implanted bovine BMP can induce chondrogenesis and that BMP-4/7 participated in this process. This observation could be applied clinically to the repair of damaged cartilage.

Age and magnetic effects on ectopic bone formation induced by purified bone morphogenetic protein

A mixture of heparin–Sepharose-purified bovine bone morphogenetic protein (BMP) and type I atellocollagen was implanted in the subcutaneous tissues of 4-week, 10-month and 18-month-old rats. The implants were removed at 7, 14 and 21 days after implantation. The effects of rat age on ectopic bone formation were evaluated on the explants using H&E staining, morphometric analysis, alkaline phosphatase (ALP) activity and calcium (Ca) content determination, as well as immunohistochemical staining of type IV collagen present in the basement membrane of blood vessels. On day 14 and 21, bone was observed in 4-week and 10-month-old rats but the amount of bone formed in the later was less than in the former. In 18-month-old rats, bone was first found focally in very limited regions of the explants on day 21 and the amount of bone was much less than in 4-week-old rats. At all periods ALP activity was higher in younger rats. On day 7, there were more blood vessels in the explants of 4-week-old rats than in those of 10- or 18-month-old rats. On day 14 and 21, more blood vessels were found in the central regions of the explants in 4-week-old rats than in the same regions in 10- or 18-month-old rats. The findings in the present study indicated that the rate and quantity of ectopic bone formation were reduced, and that the difference in blood vessel distribution might be related to the reduction in ectopic bone formation in aged rats, and suggest that the difference in blood vessel distribution is related to ectopic bone formation. Magnetism can stimulate ectopic bone formation induced by BMP.

Hydroxyapatite coated with heaptocyte growth factor (HGF) stimulates human osteoblasts in vitro.

We have studied in vitro the effect of a hydroxyapatite (HA) tricalcium phosphate material coated with hepatocyte growth factor (HA-HGF) on cell growth, collagen synthesis and secretion of metalloproteinases (MMPs) by human osteoblasts. Cell proliferation was stimulated when osteoblasts were incubated with untreated HA and was further increased after exposure to HA-HGF. The uptake of [3H]-proline was increased after treatment with HA. When osteoblasts were exposed to HA-HGF, collagen synthesis was increased with respect to HA. The secretion of MMPs in control cells was undetectable, but in HA and HA-HGF cells MMP 2 and MMP 9 were clearly synthesised. Our results suggest that HA can promote osteoblast activity and that HGF can further increase its bioactivity.

Age effects on ectopic bone formation induced by purified bone morphogenetic protein

A mixture of heparin-Sepharose-purified bovine bone morphogenetic protein and type I atellocollagen was implanted in the subcutaneous tissues of 4-week, 10-month and 18-month-old rats. The implants were removed at 7, 14 and 21 days after implantation. The effects of rat age on ectopic bone formation were evaluated on the explants using haematoxylin-eosin staining, morphometric analysis, alkaline phosphatase activity and calcium content determination, as well as immunohistochemical staining of type IV collagen present in the basement membrane of blood vessels. On day 14 and 21, bone was observed in 4-week and 10-month-old rats, but the amount of bone formed in the latter was less than in the 4-week-old rats. In 18-month-old rats, bone was first found focally in very limited regions of the explants on day 21 and the amount of bone was much less than in 4-week-old rats. At all periods, akaline phosphatase activity was higher in younger rats. On day 7, there were more blood vessels in the explants of 4-week-old rats than in those of 10-month or 18-month-old rats. On day 14 and 21, more blood vessels were found in the central regions of the explants in 4-week-old rats than in the same regions in 10-month or 18-month-old rats. The findings in the present study indicate that the rate and quantity of ectopic bone formation are reduced in aged rats, and suggest that the difference in blood vessel distribution is related to this reduction in ectopic bone formation.

Bone Morphogenetic Protein in Bone Neoplasms: Comparison of Different Detection Methods

The presence of bone morphogenetic proteins (BMPs) in 13 primary bone neoplasms was investigated with conventional bioassay method and immunohistochemically using antiserum against highly purified mixture of bovine BMPs as antibody. In conventional bioassay after implantation of lyophilized bone tumor tissues into mouse muscle pouches 9/13 samples turned out positive by radiography and 10/13 histologically. By immunohistochemical staining, using the avidin-biotin-peroxidase method, signs of BMPs could be verified in all cases investigated. Microscopical scoring showed the local concentration of BMPs to be especially high in sections from giant cell tumors when compared to other bone neoplasms.