Implantation Of Bone Morphogenetic Protein Research Articles

Long-term safety and efficacy of human bone morphogenetic protein (HBMP) in the treatment of resistant non-unions and failed arthrodesis.

The bone morphogenetic protein (BMP) has emerged as a suitable alternative to autogenous cancellous bone grafting and despite current knowledge about its mechanism; few studies provide evidence about the long-term safety of BMP. The aim of this investigation is to determine if BMP implantation is a safe and effective agent in a long-term setting for the treatment of patients with resistant non-unions and failed arthrodesis. This study is a retrospective case series study that was conducted on 55 patients who had received BMP. Collected data included all related surgical history, and clinical and X-ray data both pre-operatively and post-operatively. All patients were scheduled for follow-up evaluations at one week and 1, 3, 6, and 12 months post-operatively. Seven patients (13%) experienced adverse events related to their surgery with hBMP. Six patients (11%) experienced persistent non-union; five of these underwent further revision surgery. One patient (2%) developed an infected non-union. No patients experienced tumor induction, allergic reaction to hBMP. The remaining 48 patients achieved osseous union within six months of hBMP implantation. This study differs from previous studies that the use of hBMP is a safe and efficacious treatment method for resistant non-unions and failed arthrodesis in the long-term setting.

Canola and hydrogenated soybean oils accelerate ectopic bone formation induced by implantation of bone morphogenetic protein in mice

Canola oil (Can) and hydrogenated soybean oil (H2-Soy) are commonly used edible oils. However, in contrast to soybean oil (Soy), they shorten the survival of stroke-prone spontaneously hypertensive (SHRSP) rats. It has been proposed that the adverse effects of these oils on the kidney and testis are caused at least in part by dihydro-vitamin K (VK) 1 in H2-Soy and unidentified component(s) in Can. Increased intake of dihydro-VK1 is associated with decreased tissue VK2 levels and bone mineral density in rats and humans, respectively. The aim of the present study was to determine the effects of these oils on bone morphogenetic protein (BMP)-induced ectopic bone formation, which is promoted by VK2 deficiency, in relation to the role of VK in the γ-carboxylation of osteocalcin and matrix Gla protein. A crude extract of BMPs was implanted into a gap in the fascia of the femoral muscle in 5-week-old mice maintained on a Soy, Can, or H2-Soy diet. Newly formed bone volume, assessed by three-dimensional X-ray micro-computed tomography and three-dimensional reconstruction imaging for bone, was 4-fold greater in the Can and H2-Soy groups than in the Soy group. The plasma carboxylated osteocalcin (Gla-OC) and total OC (Gla-OC plus undercarboxylated osteocalcin [Glu-OC]) levels were significantly lower in the Can group than in the Soy group (p < 0.05). However, these levels did not significantly differ between the H2-Soy and Soy groups. The plasma Gla-OC/Glu-OC ratio in the Can and H2-Soy groups was significantly lower (in Can; p = 0.044) or was almost significantly lower (in H2-Soy; p = 0.053) than that in the Soy group. In conclusion, Can and H2-Soy accelerated BMP-induced bone formation in mice to a greater extent than Soy. Further research is required to evaluate whether the difference in accelerated ectopic bone formation is associated with altered levels of VK2 and VK-dependent protein(s) among the three dietary groups.

Bone Morphogenetic Proteins: Their History and Characteristics

Bone tissue engineering is expected to be utilized clinically as a patient friendly strategy instead of autogenous bone grafts, and bone morphogenetic protein (BMP) is applying for bone regeneration. BMPs have been found in the 1960s and their gene cloning has been succeeded two decades later, which revealed that BMPs were members of TGFsuperfamily. BMPs have critical functions in embryonic development and tissue generation, and BMPs induce bone formation in mammals though its primary functions are different. Subcutaneous implantation of BMP in rodent reproduces endochondral bone formation occurred in embryogenesis. Recombinant human BMPs are applied for spinal fusion and non-union fracture repair and also utilized in the field of oral and maxillofacial surgery such as sinus floor augmentation. BMP regenerates mandible bone after its segmental resection in non-human primates as a preclinical trial. However, the results were not conclusive in clinical studies especially in elderly patients. A high dose of BMP is required to restore large bone defects, but it may also induce life threatening significant edema. The further studies are necessary for effective and safe application of BMPs to restore large bone defects.

Histological reaction of the subcutaneous tissue of rats following implantation of bone morphogenetic protein (rhBMP-2) in inorganic bone scaffold and stimulation using low-power laser light

Objective: The aim of this study was to assess the histological reaction of the subcutaneous tissue of rats after the implantation of natural inorganic mineral scaffold from a calf femur containing recombinant human bone morphogenetic protein (rhBMP-2) and irradiated with low-power laser light. Material and methods: Sixteen Wistar rats were incised in the torso in a medial-longitudinal orientation, and the subcutaneous tissue of the left and right sides of the incision was pulled apart for implantation of the inorganic bone scaffold containing rhBMP-2. Diode laser light was applied to the right side implant at a dose of 8 J/cm2 for 3 minutes, forming two groups: G1 (control) and G2 (irradiated with laser). Implants and surrounding tissue were removed from four animals on days 7, 21, 40 and 112 for microscopic study. The histological results were assessed by means of grading (0 = absence, 1 = slight presence, 2 = representative and 3 = very representative), considering the following events: formation of osteoid structure, acute inflammation, chronic inflammation, fibrin deposition, neovascularization, foreign body granuloma and fibrosis. Results: The results showed no statistically significant differences in each of the four periods when the two groups were compared (p&gt;0.05 – Mann-Whitney’s test). Conclusion: The natural inorganic scaffold from a calf femur with rhBMP-2 was a biocompatible combination. Under these conditions, the inductive capacity of rhBMP-2 for cell differentiation was inhibited. A slight hastening of tissue healing was shown in the group that was irradiated with low-power laser light.

The effect of carrier for BMP-2 delivery on histological aspects of tissue-engineered bone

Bone morphogenetic protein-2 (BMP-2) that can regenerate bone by recruiting osteo-progenitor cells and inducing osteogenic differentiation has been highlighted as an alternative therapy to treat bone defects. The therapeutic effect of BMP-2 depends considerably on the delivery carriers. Previous studies demonstrated that collagen and fibrin have the potential to serve as a carrier for delivery of BMP-2. The successful bone regeneration by collagen and fibrin with BMP-2 implantation has been shown in animal models. However, those studies were not focused on the effect of the carriers on the quality of the regenerated bone. In this study, we directly compared the quality of regenerated bone when BMP-2 was delivered with collagen or fibrin, both of which are shown to be excellent carrier for BMP-2. The microstructure, surface morphology, BMP-2 release profile was examined in vitro. To evaluate the quality of regenerated bone, both types of BMP-2 carriers were implanted into the dorsal subcutaneous spaces of the mice. Four and six weeks after implantation, the regenerated bone was evaluated by computed tomography, histology, and histomorphometric analysis. The bone regeneration by the collagen with BMP-2 was higher bone density than bone regenerated by the fibrin with BMP-2. The results of this study show that carrier of BMP-2 is an important factor affecting the quality of engineered bone.

Contralateral psoas seroma after transpsoas lumbar interbody fusion with bone morphogenetic protein-2 implantation

Background contextThe lateral transpsoas approach to interbody fusion of the lumbar spine (lateral lumbar interbody fusion [LLIF]) with recombinant human bone morphogenetic protein-2 (BMP-2) augmentation has been increasingly performed in recent years. Potential side effects and adverse sequelae of BMP-2 in the acute setting remain to be fully elucidated. PurposeTo review the literature for reports of complications related to BMP-2 implantation in lumbar spinal surgery and present a case of a contralateral psoas muscle seroma after LLIF with BMP-2 implantation. Study designCase report and literature review. MethodsThe PubMed database was searched for articles related to adverse events to BMP-2 in lumbar spinal surgery. We report the case of a 57-year-old woman who underwent routine right-sided transpsoas approach for LLIF with the use of BMP-2 at our institution and developed a left-sided psoas muscle fluid accumulation 2 weeks postoperatively. ResultsNo reports of complications contralateral to an LLIF approach attributable to an inflammatory response to BMP-2 were identified in the English literature. In the presented patient, a large (4.2×6.5×2.7 cm) left-sided sterile intramuscular psoas fluid collection was seen on a magnetic resonance imaging study obtained on postoperative day 14. At a 6-month follow-up, left-sided L5 radiculopathy resulting in 4/5 foot drop was confirmed by electromyography. The patient reported here represents the only case of a contralateral psoas seroma with suspected association to BMP-2 utilization in LLIF encountered at our institution. ConclusionsA serous psoas muscle fluid accumulation after BMP-2 implantation may rarely occur contralateral to the surgical approach for LLIF. Further characterization of complications related to BMP-2 implantation after lumbar spinal surgery will help guide preoperative informed decision making and the management of this unusual postoperative adverse event.

Bone morphogenetic protein in complex cervical spine surgery: A safe biologic adjunct?

The advent of recombinant DNA technology has substantially increased the intra-operative utilization of biologic augmentation in spine surgery over the past several years after the Food and Drug Administration approval of the bone morphogenetic protein (BMP) class of molecules for indications in the lumbar spine. Much less is known about the potential benefits and risks of the "off-label" use of BMP in the cervical spine. The history and relevant literature pertaining to the use of the "off-label" implantation of the BMP class of molecules in the anterior or posterior cervical spine are reviewed and discussed. Early prospective studies of BMP-2 implantation in anterior cervical spine constructs showed encouraging results. Later retrospective studies reported potentially "life threatening complications" resulting in a 2007 public health advisory by the FDA. Limited data regarding BMP-7 in anterior cervical surgery was available with one group reporting a 2.4% early (< 30 d) complication rate (brachialgia and dysphagia). BMP use in the decompressed posterior cervical spine may result in neurologic or wound compromise according to several retrospective reports, however, controlled use has been reported to increase fusion rates in select complex and pediatric patients. There were no cases of de novo neoplasia related to BMP implantation in the cervical spine. BMP-2 use in anterior cervical spine surgery has been associated with a high early complication rate. Definitive recommendations for BMP-7 use in anterior cervical spine surgery cannot be made with current clinical data. According to limited reports, select complex patients who are considered "high risk" for pseudoarthrosis undergoing posterior cervical or occipitocervical arthrodesis or children with congenital or traumatic conditions may be candidates for "off-label" use of BMP in the context of appropriate informed decision making. At the present time, there are no high-level clinical studies on the outcomes and complication rates of BMP implantation in the cervical spine.

Affinity-based growth factor delivery using biodegradable, photocrosslinked heparin-alginate hydrogels

Photocrosslinkable biomaterials are promising for tissue engineering applications due to their capacity to be injected and form hydrogels in situ in a minimally invasive manner. Our group recently reported on the development of photocrosslinked alginate hydrogels with controlled biodegradation rates, mechanical properties, and cell adhesive properties. In this study, we present an affinity-based growth factor delivery system by incorporating heparin into photocrosslinkable alginate hydrogels (HP-ALG), which allows for controlled, prolonged release of therapeutic proteins. Heparin modification had minimal effect on the biodegradation profiles, swelling ratios, and elastic moduli of the hydrogels in media. The release profiles of growth factors from this affinity-based platform were sustained for 3 weeks with no initial burst release, and the released growth factors retained their biological activity. Implantation of bone morphogenetic protein-2 (BMP-2)-loaded photocrosslinked alginate hydrogels induced moderate bone formation around the implant periphery. Importantly, BMP-2-loaded photocrosslinked HP-ALG hydrogels induced significantly more osteogenesis than BMP-2-loaded photocrosslinked unmodified alginate hydrogels, with 1.9-fold greater peripheral bone formation and 1.3-fold greater calcium content in the BMP-2-loaded photocrosslinked HP-ALG hydrogels compared to the BMP-2-loaded photocrosslinked unmodified alginate hydrogels after 8 weeks implantation. This sustained and controllable growth factor delivery system, with independently controllable physical and cell adhesive properties, may provide a powerful modality for a variety of therapeutic applications.

Bone Regeneration with BMP-2 Gene-modified Mesenchymal Stem Cells Seeded on Nano-hydroxyapatite/Collagen/ Poly(L-Lactic Acid) Scaffolds

In this study, the capacity of bone morphogenetic protein 2 (BMP-2) gene-transfected bone marrow-derived mesenchymal stem cells (MSCs) in combination with nano-hydroxyapatite/collagen/poly(L-lactic acid) (nHAC/ PLA) to improve the repair of bone defects in rabbit was explored. MSCs from New Zealand White rabbits were cultured and injected with pIRES2-EGFPhBMP-2 or pIRES2-EGFP by electroporation. After the transfer efficiency was determined through the expression of EGFP, the MSCs were seeded on scaffolds to generate an in vitro 3D cell/scaffold construct. The adhesion and proliferation of the MSCs cultured in the scaffold was assessed by SEM. The cellular constructs obtained were allografted into the 15 mm critical-sized segmental bone defects in the radius of New Zealand White rabbits for 12 weeks. The bone regeneration was assessed by radiographical and histological analyses. In vitro, nHAC/PLA facilitated MSC adhesion and proliferation on the scaffold, and gene transfer efficiency reached a maximum of 35.5 ± 3.8%. In vivo, the implantation of BMP-2 transfected MSCs/nHAC/PLA construct significantly enhanced the formation of new bone in the segmental defect, compared to the control groups. This novel 3D BMP-2 transfected MSCs/nHAC/PLA construct has the potential for bone repair by genetic tissue engineering approach.

Combined application of vascularized pedicled pectoralis major clavicular periosteal flap and locking plate fixation plus bone morphogenetic protein implantation in the treatment of clavicular defect and nonunion after operation

Objective To investigate the clinical characteristics of clavicular defect and nonunion after operation and evaluate the clinical effects of vascularized pedicled pectoralis major clavicular periosteal flap and locking plate fixation plus bone morphogenetic protein (BMP) implantation. Methods From January 2004 to April 2008, 12 patients (mean age, 42.8 years old) with clavicular defect and nonunion were treated in Zhongshan Hospital. The internal fixators were removed, and then fibrous scars and sclerotic bones were cleared. The clavicular detects were fixed with locking plate, with implantation of the BMP. The wound was covered with vascularized pedicled pectoralis major clavicular periosteal flap. Postoperative functional rehabilitation exercises were performed. Results All patients were followed up for 8-24 months (mean 1.2 years). The clinical results were evaluated by Constant-Murley scoring system. The clavicular defects were healed in all patients within 4-7 months, and the internal fixators were successfully removed from some of the patients. Conclusions Vascularized pedicled pectoralis major clavicular periosteal flap combined with locking plate fixation plus BMP implantation is effective in the treatment of clavicular defect and nonunion. Key words: Clavicle; Fracture fixation; internal; Bone morphogenetic proteins; Periosteal flap

Influence of Residual Bone on Recombinant Human Bone Morphogenetic Protein‐2–Induced Periodontal Regeneration in Experimental Periodontitis in Dogs

Previous studies reported that bone morphogenetic protein (BMP)-2 induced periodontal regeneration in animals. However, the effects of local host factors on bone formation when using recombinant human (rh)BMP-2 are unknown. The purpose of this study was to evaluate local conditions in recipient sites that affected periodontal regeneration following BMP implantation in experimentally induced horizontal defects in dogs. Experimental periodontitis was induced in the maxillary and mandibular premolars of six male beagles. The recipient sites were divided into four quadrants (maxillary buccal, maxillary palatal, mandibular buccal, and mandibular lingual sites). A polymer-coated gelatin sponge (PGS; 3 x 3 x 2 mm) was impregnated with 7.2 mul rhBMP-2 solution. The recipient sites in each quadrant were treated with physiologic saline/PGS and rhBMP-2/PGS (rhBMP-2 at 7.2 mug/7.2 mul). The dogs were sacrificed 12 weeks post-surgery, and healing was evaluated histologically. Bone formation and connective tissue attachment were observed following rhBMP-2/PGS implantation. In the four recipient sites of the BMP-treated group, significant positive correlations were found between the width of residual bone and the height and area of regenerated bone (r = 0.791; r = 0.828; P <0.0001). The height of regenerated bone was also significantly correlated to the area (r = 0.891; P <0.0001). The width of residual bone was one of the clinical host factors that affected bone regeneration following BMP implantation. However, it did not affect connective tissue attachment, cementum regeneration, and downgrowth of junctional epithelium.

Analyses of regenerative wave patterns in adult hair follicle populations reveal macro-environmental regulation of stem cell activity

The control of hair growth in the adult mammalian coat is a fascinating topic which has just begun to be explored with molecular genetic tools. Complex hair cycle domains and regenerative hair waves are present in normal adult (> 2 month) mice, but more apparent in mutants with cyclic alopecia phenotypes. Each hair cycle domain consists of initiation site(s), a propagating wave and boundaries. By analyzing the dynamics of hair growth, time required for regeneration after plucking, in situ hybridization and reporter activity, we showed that there is oscillation of intra-follicular Wnt signaling which is synchronous with hair cycling, and there is oscillation of dermal bone morphogenetic protein (BMP) signaling which is asynchronous with hair cycling. The interactions of these two rhythms lead to the recognition of refractory and competent phases in the telogen, and autonomous and propagating phases in the anagen. Boundaries form when propagating anagen waves reach follicles which are in refractory telogen. Experiments showed that Krt14-Nog mice have shortened refractory telogen and simplified wave dynamics. Krt14-Nog skin grafts exhibit non-autonomous interactions with surrounding host skin. Implantation of BMP coated beads into competent telogen skin prevents hair wave propagation around the bead. Thus, we have developed a new molecular understanding of the classic early concepts of inhibitory "chalone", suggesting that stem cells within the hair follicle micro-environment, or other organs, are subject to a higher level of macro-environmental regulation. Such a novel understanding has important implications in the field of regenerative medicine. The unexpected links with Bmp2 expression in subcutaneous adipocytes has implications for systems biology and Evo-Devo.

Gene Expression Analysis of Ectopic Bone Formation Induced by Electroporatic Gene Transfer of BMP4

Implantation of bone morphogenetic protein (BMP) using a carrier or by BMP gene transfer into rodent muscle can induce bone formation. Implanted BMP becomes bioactive immediately after implantation. In BMP gene transfer, there is a time-lag between the secretion of gene products and bone formation. We analyzed the gene expression of chondrogenic and osteogenic specific markers in the process of ectopic bone formation by using semi-quantitative RT-PCR.A plasmid vector containing mouse BMP4 gene (pCAGGS-BMP4) was transferred into the gastrocnemius muscles of mice using electroporation. Histological examination revealed the process of endochondral bone formation in the pCAGGS-BMP4 transferred muscles. As chondrogenic markers, aggrecan gene maximal expression was detected on day 7 and decreased by day 14, and for collagen X the gene maximal expression was on day 10. As osteogenic markers, osteocalcin (OCN), bone sialoprotein (BSP) and osteopontin (OPN) gene expressions were clearly detected from day 10 and then increased by day 14. In conclusion, the present study proved that ectopic bone formation by BMP4 gene transfer into the muscle induced endochondral ossification that corresponded well with that to that by implantation of demineralized bone matrix.

Enhancement of Crude Bone Morphogenetic Protein-Induced New Bone Formation and Normalization of Endochondral Ossification by Bisphosphonate Treatment in Osteoprotegerin-Deficient Mice

Osteoprotegerin (OPG) is a novel secreted member of the tumor necrosis factor receptor family which plays a crucial role in negative regulation of osteoclastic bone resorption. We investigated both the quantity and quality of heterotopic new bone induced by crude bone morphogenetic protein (BMP) as a means of examining bone metabolism by bisphosphonate administration in OPG-/- mice. Four weeks after implantation of crude BMP, the volume of heterotopic new bone in OPG-/- mice without alendronate was significantly less than in wild-type (WT) mice. Alendronate treatment of OPG-/- mice resulted in enhancement of the volume of heterotopic new bone. Histological findings revealed that WT mice showed normal bone formation with persistent cartilage that was interspersed with islands of bone. In contrast, the cartilage was replaced by trabecular bone and bone marrow adipocytes in OPG-/- mice without alendronate. However, some cartilage was still present in OPG-/- mice with alendronate compared to those without alendronate. All bone formation-related parameters and bone resorption-related parameters were significantly lower in OPG-/- mice with alendronate than in those without alendronate. These findings suggest that in stimulated osteoclastogenesis without OPG, osteoinductive activity induced by crude BMP is inhibited and endochondral ossification induced by crude BMP is accelerated. On the other hand, alendronate treatment of OPG-/- mice caused osteoinductive activity induced by crude BMP to increase and endochondral ossification induced by crude BMP to be decelerated. In conclusion, inhibition of stimulated osteoclastogenesis results in the enhancement of new bone formation and normalization of endochondral ossification.

Osteoprotegerin regulates bone formation through a coupling mechanism with bone resorption.

Deficiency of osteoprotegerin (OPG), a soluble decoy receptor for receptor activator of nuclear factor-kappaB ligand (RANKL), in mice induces osteoporosis caused by enhanced bone resorption, but also accelerates bone formation. We examined whether bone formation is coupled with bone resorption in OPG-deficient (OPG-/-) mice using risedronate, an inhibitor of bone resorption. Histomorphometric analysis showed that bone formation-related parameters (e.g. mineral apposition rate and osteoblast surface/bone surface) in OPG-/- mice sharply decreased with suppression of bone resorption by daily injection of risedronate for 30 d. OPG-/- mice exhibited high serum alkaline phosphatase activity and osteocalcin concentration, both of which were decreased to the levels in wild-type mice by the risedronate injection. Serum levels of RANKL were markedly elevated in OPG-/- mice, but were unaffected by risedronate. The ectopic bone formation induced by bone morphogenetic protein-2 implantation into OPG-/- mice was not accelerated even with a high turnover rate of bone, but attenuation of mineral density from the ectopic bone was more pronounced than that in wild-type mice. These results suggest that bone formation is coupled with bone resorption at local sites in OPG-/- mice, and that serum RANKL levels do not reflect this coupling.

Application of Bone Morphogenetic Proteins in the Treatment of Clinical Oral and Maxillofacial Osseous Defects

Commonly occurring extensive osseous defects in the oral and maxillofacial area are seen following complete or partial resection of the mandible and other facial bones in oncologic surgery or following traumatic injury. Autogenous osseous grafts have been used to restore these defects. Additionally, bone graft substitute materials and autogenous osseous grafts are applied to congenital defects such as cleft palate, facial clefts, and facial asymmetry. We have simulated these types of defects in appropriately aged Macaca fascicularis and Macaca mulatta monkeys to study the efficacy of using bone morphogenetic protein (BMP) as an osseous inductor. The objective of these studies was to obtain information on the feasibility of employing bone inductors to regenerate large continuity critical-sized maxillofacial defects without using bone grafts. In one study, involving eight animals, the body of the mandible was removed, simulating hemi-mandibulectomy defects following traumatic bone loss or oncologic surgery. Recombinant human (rh) BMP-2 (Genetics Institute, Cambridge, Massachusetts) in a collagen carrier (Colla-Tec Inc., Plainsboro, New Jersey) then was placed in the hemi-mandibulectomy defect with use of titanium orthopaedic mesh fixation (Sofamor Danek-Medtronic, Memphis, Tennessee). Entire bone regeneration of the defect was observed 5 and 6 months postoperatively. In another group of subhuman primates, the restored area was functionally stimulated at the 5-month post-BMP implantation level by placement of intraoral titanium implants. The animals were allowed to function for 8 months with these titanium implants. Microscopic results showed increased density, bone volume, and thickness of the trabecular bone pattern. The bone cortex in the restored defect also increased in thickness compared with the nonsurgical areas. To evaluate the effect of rhBMP-2 in aging individuals, a group of six Macaca animals over 20 years of age received the same type of mandibular resection followed by BMP grafting with functional stimulation by mastication on root form implants placed at 5 months after BMP implantation. The entire mandible regenerated as in the younger group of animals; therefore, age did not appear to be a factor in the reparative process. Thus, the number of stem cells supposedly reduced with increasing age did not appear to affect the overall result of BMP-induced bone regeneration. Additionally, in applying the inductor material to younger monkeys (1-1 1/2 years of age), the rhBMP-2 was placed in simulated bilateral cleft palate defects. On one side, the rhBMP-2 was placed with use of the collagen sponge carrier. The autogenous graft most frequently used at present for regeneration of the osseous defects of maxillary clefts is iliac crest particulate cancellous bone. As a control graft on the contralateral side, therefore, autogenous particulate bone and marrow was placed. At the end of 3 months, the cleft side receiving the BMP-2 showed complete osseous restoration of the simulated cleft. The autogenously grafted side exhibited bone repair but incomplete regeneration of the bone defect at the early (3-months postoperative) stage of healing. The results of these three subhuman primate defect studies--(a) mandibular resection defects in middle-aged Macaca fascicularis animals, (b) mandibular resection defects in Macaca fascicularis animals over 20 years of age, and (c) simulated bilateral clefts in Macaca mulatta animals 1 1/2 years of age (comparable with a 5-year-old child)--were very encouraging. Histomorphometric analysis in all of these investigations indicated that the use of rhBMP-2 in bone repair without the use of bone grafting materials will offer a new method of osseous reconstruction in clinical facial bone defects.

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.

SEM study on microvascular changes following implantation of bone morphogenetic protein combined with hydroxyapatite into experimental bone defects.

BMP-HAP complex was implanted into a bone defect in the femur of Wistar-strain rats, and animals were allowed to heal for one to 8 weeks prior to sacrifice. Similar bone defects without BMP-HAP complex served as controls. Osseous healing and microvascular changes, as revealed by plastic microcorrosion castings, were subsequently examined under a scanning electron microscope. One week after implantation, sproutings and congregate sinusoidal capillary plexuses and primary bone trabeculae (woven bone) were observed around and between the BMP-HAP complexes. Control specimens revealed a fine and immature sinusoidal capillary plexus arising from sproutings and elongations of pre-existing blood vessels, but bone formation was not observed. At two weeks, newly-formed trabeculae were observed around and on the surface of the HAPs, and a network of thick, newly-formed vessels was observed in intervening space. At three weeks, networks of newly-formed vessels were observed on the surface of the HAPs, and surrounding newly-formed trabeculae had become thickened. At four weeks, bone defects were filled, and HAP was completely embedded in new bone. At eight weeks, the HAP was fused with new bone, and the boundary between the HAP and new bone was unclear. In comparison with comparable surgically created control bone defects without implanted BMP-HAP complex, BMP apparently stimulates new vascularization. Further, implanted BMP-HAP apparently stimulates undifferentiated mesenchymal cells to differentiate into angioblasts and osteoblasts, or vice versa (dedifferentiation).

Enhancement by bFGF of osteogenesis induced by rhBMP-2 in rats.

Subcutaneous implantation of bone morphogenetic protein (BMP) combined with a fibrous glass membrane (FGM) induces cartilage formation in the entire inner area of the membrane within 2 wk. It has been hypothesized that a tight FGM network (1 microm exclusion size) provides immature cells with spaces for penetrating into the membrane, but not for vascular formation, at least until 2 wk. To test this hypothesis, basic fibroblast growth factor (bFGF), known to be a potent stimulant of capillary formation, was applied to the implant. BMP was combined with FGM in the presence or absence of bFGF, and then implanted subcutaneously into the backs of rats. The bFGF-supplemented implant caused 1.3 times higher alkaline phosphatase activity and 3 times higher calcium contents at 2 wk, whereas type II collagen contents decreased, thus indicating that bFGF enhances bone formation in BMP/FGM implants. These results suggest that bFGF induces faster and stronger invasion of capillaries into the FGM and destroys its tight network, resulting in acceleration of the ossification process.

Protooncogene expression in osteogenesis induced by bone morphogenetic protein.

In this study, changes in the expression of protooncogenes c-fos and c-myc messenger ribonucleic acid were investigated in mice after implantation of bone morphogenetic protein. The expression of c-fos showed a biphasic pattern. The first increase was observed on Day 1 with the aggregation of round cells. The second increase was observed on Day 7 with the appearance of chondroblasts. The amount of c-myc messenger ribonucleic acid showed the sustained high levels from Days 2 to 7. During this period, the proliferation of mesenchymal cells was histologically evident. After Day 11, the expression of c-fos and c-myc decreased and remained at low levels despite the progress in chondroosteogenesis. The protooncogenes c-fos and c-myc appear to increase before calcification in the process of bone morphogenetic protein induced bone and cartilage development.