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We evaluated the usefulness of artificial collagen as a new carrier for recombinant human bone morphogenetic protein-2 (rhBMP-2) by comparing it with that of atelopeptide collagen, which is derived from porcine skin, and which we have previously shown to be useful for the induction of bone. rhBMP-2 5μg with either atelopeptide collagen 3mg or artificial collagen 3mg was implanted into the calf muscle of 10-week-old Wistar rats (n=3 in each group). Three rats were given artificial collagen alone and acted as controls (n=3). Radiographic evaluation, histological analysis, and biochemical examinations were made on day 21 after implantation. Soft radiographs (wavelength 10-0.10nm) showed opaque shadows in both groups. Histological analysis showed that new bone had formed in both experimental groups. Endochondral ossification was found at the outermost edge of the implanted collagen in the atelopeptide group. However, there was less ossification in the implanted collagen in the artificial collagen group. On biochemical examination, alkaline phosphatase activity and calcium concentrations in both experimental groups were higher than in the control group, and were higher in the atelopeptide group than in the artificial collagen group. Our results suggest that artificial collagen is useful as a carrier for rhBMP-2 designed to promote the formation of new bone., (Copyright © 2010 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.)

Bone morphogenetic protein (BMP) is a unique cytokine that induces bony tissue in soft tissue. Tissue reactions at and around the implantation of recombinant human BMP-2 (rhBMP-2) into the soft tissue of rats and nonhuman primates were investigated. At the osteoinduced site of rats, massive trabeculae-lined osteoblasts and rich marrow were observed. At and around the nonosteoinduced sites of nonhuman primates, large clear nuclei were observed in reaction to rhBMP-2 implantation. The surrounding area was visually classified into zones 1, 2 and 3. Zone 3 was near the center of the implant. The area of nuclei, the major axis, the minor axis and the ratio of minor axis per major axis were image-analyzed in the histological views. In zones 1, 2 and 3, the nuclear areas were 18.0 (3.1) mean (SD); unit micron2, 33.4 (5.61) and 110.1 (23.7), respectively. The major axes of nuclear ellipses were 7.45 (0.22) (unit micron), 7.76 (0.26), and 13.9 (1.88), respectively. The minor axes were 3.07 (0.53), 5.59 (0.95) and 10.1 (1.35), respectively. The ratios of minor axis per major axis of nuclear ellipses were 0.4 (0.57), 0.72 (0.11) and 0.73 (0.11) in zones 1, 2 and 3, respectively. These results showed that in zones 2 and 3 cell and tissue reactions were marked against rhBMP-2 implantation.

We constructed a human bone morphogenetic protein 2 (BMP-2)-expressing adenoviral vector, AxCABMP-2, which showed osteoinduction in immunosuppressed rats. In immunocompetent rats, new bone was not induced, because of the rapid elimination of transduced cells. Biomaterials such as collagen can be used as carriers for the delivery of DNA vectors, allowing prolonged expression of plasmid DNA in normal animals. We evaluated osteoinduction with AxCABMP-2 and atelopeptide type I collagen in immunocompetent rats. Collagen plus AxCABMP-2 (BMP group), collagen plus AxCALacZ (LacZ group), or collagen alone (CL group) was implanted into calf muscle pouches in immunocompetent rats, or AxCABMP-2 alone (injection group) was injected into the calf muscle. On days 3, 7, 14, and 21 after treatment, osteoinduction was evaluated. In the BMP group, bone formation was not observed on days 3 and 7. On day 14, radiographic formation was seen, but little bone formation was detected histologically. On day 21, new bone formation was observed both radiologically and histologically. In the other groups, osteoinduction was not found at any time. Immunohistochemical analysis on days 3 and 7 revealed decreased immunogenicity in the BMP group compared with the injection group. These findings suggested that collagen was an effective masking material for our vector.

We established the clonal mesenchymal cell line, GFP-C3 (C3), which differentiates into osteoblasts in response to BMP-2 from calvariae of newborn green fluorescence protein (GFP) transgenic mice. This cell line cultured with control medium expressed low levels of alkaline phosphatase (ALP) activity and osterix mRNA and undetectable ALP and osteocalcin mRNA. Incubation of these cells with rhBMP-2 increased ALP activity dose-dependently and induced substantial levels of ALP, osteocalcin and osterix mRNA expression. C3 cells infected with adenovirus vector encoding BMP-2 (AdBMP-2) or Runx2 (AdRunx2) showed greatly increased ALP mRNA expression in a time-dependent fashion. Transduction with AdRunx2-induced expression of ALP and osteocalcin mRNA, but not osterix mRNA by day 3. Transduction with AdBMP-2 induced apparent expression of ALP and osterix mRNA by day 1 after transduction, but induced only weak expression of osteocalcin mRNA day 3 after transduction. Transplantation of C3 cells transduced with AdBMP-2 into back subfascia in wild-type mice with a complex of poly-d,l-lactic-co-glycolic acid/gelatin sponge (PGS) generated ectopic bone formation involving GFP-positive osteoblasts and osteocytes 2 weeks after transplantation. C3 cells transduced with AdRunx2 or AdLacZ failed to induce ectopic bone formation. Transplantation of C3 cells transduced with AdBMP-2 into craniotomy defects in wild-type mice using PGS as a carrier induced bone formation 2 weeks after transplantation, and replaced defects 4 weeks after transplantation. C3 cells transduced with AdRunx2 failed to induce bone repair after transplantation into craniotomy defects. These results indicate that C3 cells retain differentiation potential into osteoblasts in response to BMP-2. They are useful tools for analyzing the process of osteoblast differentiation in vivo after transplantation.

We have previously utilized a human bone morphogenetic protein-2 (BMP-2)-expressing recombinant adenoviral vector (AxCAOBMP-2) for osteoinductive gene therapy in rats. However, immunosuppression is essential for osteoinduction by AxCAOBMP-2 and this is one of the major impediments to its clinical use. Injection of AxCAOBMP-2 together with the immunosuppressant FK506 made it possible to markedly reduce the dose of the immunosuppressive agent and still induce ectopic bone reliably. We injected AxCAOBMP-2 and FK506 into the right calf muscle of rats, while the same number of plaque forming units of AxCAOBMP-2 and the same dose of FK506 placebo (vehicle) were injected into the left calf muscle. At 1, 3, 5, 7, 9 days after injection, BMP-2 mRNA expression was significantly higher in the right calf muscle than in the left calf muscle. At 21 days after injection, significantly more ectopic bone was observed in the right calf muscle than in the left calf muscle. These results indicate that coinjection of FK506 significantly promotes osteoinduction. In addition, local injection of FK506 may also make it possible to prevent a decrease of gene expression with other adenoviral vector.

The objective of this study was to evaluate the effect of hyperbaric oxygenation on bone formation by recombinant human bone morphogenetic protein-2 (rhBMP-2) under unfavourable conditions. The calf muscles of 10 rats with low-blood supply were prepared by ligating and cutting the right femoral artery, and 10 micro of rhBMP-2 was implanted in the calf muscle. Five rats each were randomly assigned to the hyperbaric oxygenation group and the control group (untreated). The rats in the hyperbaric oxygenation group were treated with hyperbaric oxygenation at 2.0 atmospheres absolute for 3 weeks. In the histologic evaluation, the number of osteoblasts in the hyperbaric oxygenation group was greater than that in control group. The area of the trabecular bone induced in the hyperbaric oxygenation group was significantly larger than that in the control group. The values of alkaline phosphatase activity and calcium contents in the hyperbaric oxygenation group were significantly higher than those in the control group. The results of the present study suggest that hyperbaric oxygenation increases the partial oxygen pressure in low blood supply tissue and accelerates the activity and rate of osteoinduction by rhBMP-2. Hyperbaric oxygenation therapy may increase the clinical application of rhBMP-2 to unfavourable condition.

We investigated the osteogenic potential of skin fibroblasts that overexpressed BMP-2 or Runx2 by using adenoviral vectors. In in vitro experiments, skin fibroblasts infected with adenovirus vector encoding BMP-2 (AdBMP-2) released substantial levels of BMP-2 proteins into culture media, and those infected with adenovirus vector encoding Runx2 (AdRunx2) produced its protein. Transduction of BMP-2 or Runx2, respectively, increased alkaline phosphatase (ALP) activity and induced expression of mRNAs of ALP, osteocalcin, and osterix in skin fibroblasts. In in vivo experiments, we investigated the bone induction activity by transplantation of a complex composed of carrier [poly-D,L-lactic-co-glycolic acid/gelatin sponge (PGS)] and skin fibroblasts (PGS/SF complex). Transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdBMP-2-induced ectopic bone formation when transplanted into the subfascia of back muscle, unlike those infected with AdRunx2. Transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdBMP-2 into craniotomy defects induced bone formation from 2 weeks after transplantation, and almost all PGS was replaced by newly synthesized bone at 6 weeks. To investigate the fate of the transplanted cells, we transplanted skin fibroblasts isolated from green fluorescence protein transgenic mice into craniotomy defects. Transplantation of these skin fibroblasts transfected with AdBMP-2 generated green fluorescence protein-positive osteoblasts and osteocytes, indicating that the transplanted skin fibroblasts differentiated into osteoblastic lineage cells during bone repair. In contrast, transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdRunx2 induced a few ALP-positive cells at 1 week after transplantation, but their number decreased depending on time after transplantation. In addition, transplantation of these complexes was insufficient to induce bone repair. Taken together, our results suggest that skin fibroblasts expressing BMP-2 are more suitable for cell-mediated therapy of bone repair than those expressing Runx2.

BMP-3b is a novel BMP-3-related protein and its biological functions are unknown. In order to investigate the biological actions of BMP-3b, we constructed a BMP-3b-expressing recombinant adenoviral vector (AxCAKBMP-3b). We show that over expression of BMP-3b stimulated the induction of differentiation and the osteoinduction activity of a human BMP-2-expressing recombinant adenoviral vector (AxCAOBMP-2). C2C12 cells were infected in vitro with AxCAKBMP-3b, AxCAOBMP-2 and a control vector containing no foreign genes (AxCAwt). Cells infected with AxCAOBMP-2 and AxCAKBMP-3b produced more alkaline phosphatase and secreted more osteocalcin into the culture medium than cells infected with AxCAOBMP-2 and AxCAwt. When AxCAOBMP-2, AxCAKBMP-3b, and AxCAwt were injected into the calf muscles of nude rats (F 344/N Jcl-rnu), the osteoinduction seen with AxCAOBMP-2 and AxCAKBMP-3b was greater than that seen with AxCAOBMP-2 and AxCAwt., (Copyright 2002 Elsevier Science Inc.)

FK506 is an immunosuppressant that is used widely in organ transplantation, and it has recently been recognized as effective for promoting the growth of bone grafts [J. Bone Miner. Res. 15 (2000) 1147]. In this study, we evaluated the influence of FK506 on osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2) using atelopeptide type I collagen as a carrier. We administered FK506 (1 mg/kg/day intramuscularly) on days -2 to 0, -2 to 7, and -2 to sacrifice. rhBMP-2 was implanted into the calf muscle of Wistar rats (thirty per group) and the implant was sampled on days 7, 14, and 21. Radiographic evaluation, histological examination, and biochemical analysis were performed. It was found that FK506 promoted the early stage of osteoinduction after short-term administration. However, long-term administration of this agent accelerated both bone formation and bone resorption. In order to use FK506 effectively for promoting bone growth, we must further examine the appropriate dose, method, and period of administration.

The aim of this study was to examine the potential of immunoselected genetically modified human osteoprogenitors to form bone in vivo on porous PLA scaffolds. Human osteoprogenitors from bone marrow were selected using the antibody STRO-1 utilising a magnetically activated cell separation system. The STRO-1(+) fraction isolated 7% of nucleated marrow cells and increased fibroblastic colony formation by 300% and alkaline phosphatase activity by 190% over unselected marrow cell cultures. To engineer bone tissue, STRO-1(+) culture-expanded cells were transduced with AxCAOBMP-2, an adenovirus carrying the human BMP-2 gene, injected into diffusion chambers containing porous PLA scaffolds, and implanted in vivo. After 11 weeks the presence of bone mineral was observed by X-ray analysis and confirmed for mineral by von Kossa, as well as bone matrix composition by Sirius red staining, birefringence, and type I collagen immunohistochemistry. Bone formation in vivo indicates the potential of using immunoselected progenitor cells and ex vivo gene transfer with biodegradable scaffolds, for the development of protocols for the treatment of a wide variety of musculo-skeletal disorders.

To clarify the effect of recombinant human basic fibroblast growth factor (FGF-2) on the osteoinductive activity of recombinant human bone morphogenetic protein-2 (BMP-2) in vivo, different amounts of FGF-2 (0, 16, 80 and 400 ng, and 2, 10 and 50 micro g: n=10 in each group), BMP-2 (2 micro g) and type I collagen as a carrier were mixed and implanted into rat calf muscles. Three weeks after implantation, compared with the controls, the radiopaque shadows of the implants were increased in the 16, 80 and 400 ng FGF-2-treated groups, but decreased in the 2, 10 and 50 micro g FGF-2-treated groups. In addition, alkaline phosphatase activity was increased in the 16, 80 and 400 ng FGF-2-treated groups but decreased in the 50 micro g FGF-2-treated group. Histological examination revealed increased bone formation in the 16, 80 and 400 ng FGF-2-treated groups. These results show that combined treatment with FGF-2 and BMP-2 has a biphasic effect on osteoinductive activity, i.e. it increases with low doses of FGF-2 and decreases with high doses of FGF-2.

The purpose of this study was to investigate the therapeutic use of recombinant human bone morphogenetic protein-2 (rhBMP-2) in internally deranged temporomandibular joints (TMJ). Defects (2 mm in diameter) were created in the surface of the condylar head. Lyophilized rhBMP-2 with collagen as the carrier was implanted in the defects in different doses: rhBMP-2 15 microg (n = 5); rhBMP-2 3 microg (n = 5); rhBMP-2 0.6 microg (n = 5). In the two control groups, the defects were either filled with collagen alone (n = 5) or left untreated (n = 5). Three weeks postoperatively the sites of defects were examined under light microscopy. In the 15 micromg and the 3 microg groups, new cartilage had filled the defects; endochondral ossification was also found deep within the defect. In the 0.6 microg group, fibrous tissue was proliferating in most areas of the defect, although cartilage was also found in some parts. In the two control groups, there was either soft tissue repair only or no evidence of tissue repair. These findings suggest that BMP-2 could stimulate the repair of defects in the articular cartilage of the mandibular condyle head during the 3 weeks postoperatively. To observe the progress of endochondral ossification in more detail, it may be necessary to extend the experiment for a longer period of time. However, this study supports the contention that BMP-2 may be useful in the regeneration of cartilage in TMJ disease., (Copyright 2002 The British Association of Oral and Maxillofacial Surgeons.)

Heterotopic osteoinduction in a muscle of a medium-sized, non-human primate (Japanese macaque monkey; Macaca fuscata) was investigated with recombinant human bone morphogenetic protein-2 (rhBMP-2) mixed with atelopeptide type I collagen as the carrier. Nine monkeys were divided into three groups of three: groups I (1.25 mg rhBMP-2), II (250 micrograms rhBMP-2) and III (50 micrograms rhBMP-2). Four weeks after implanting into the calf muscle pouch, the implant was examined radiographically and histologically. In one specimen of three in group I, marked radio-opaque shadow, massive chondrogenesis and partial osteogenesis were observed. In the other two specimens, only microscopic calcification signs were recognized. In groups II and III, no findings of heterotopic osteoinduction were radiographically observed; however, nuclei from muscle bundles reacted to rhBMP-2 and were large and round, as in muscle bundles near the site of osteogenesis in group I. A positive control study using rats was carried out in parallel. This was a dose-finding study, with the monkeys in group III acting as a sub-effective dose (placebo) control, and rats acting as an active control, or verum, to show that the techniques are sufficiently sensitive. Bone morphogenetic protein appears to osteoinduce less bony material in soft tissue in primates than in rats.

The aim of this study was to determine the feasibility of adenoviral gene transfer into primary human bone marrow osteoprogenitor cells in combination with biodegradeable scaffolds to tissue-engineer bone. Osteoprogenitors were infected with AxCAOBMP-2, a vector carrying the human BMP-2 gene. Alkaline phosphatase activity was induced in C2C12 cells following culture with conditioned media from BMP-2 expressing cells, confirming successful secretion of active BMP-2. Expression of alkaline phosphatase activity, type I collagen and mineralisation confirmed bone cell differentiation and maintenance of the osteoblast phenotype in extended culture for up to 6 weeks on PLGA porous scaffolds. In vivo implantation of adenoviral osteoprogenitor constructs on PLGA biodegradeable scaffolds, using diffusion chambers, also demonstrated bone cell differentiation and production of bone tissue. The maintenance of the osteoblast phenotype in extended culture and generation of mineralised 3-D scaffolds containing such constructs indicate the potential of such bone tissue engineering approaches in bone repair., ((C)2002 Elsevier Science (USA).)

To clarify the mechanism of osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2), we examined the time-course localization of bone morphogenetic proteins (BMPs) immunostained by an anti-BMP-2 monoclonal antibody after implantation of pellets consisting of rhBMP-2 and collagen in rat calf muscle pouch. On day 3 after implantation, BMP was detected in the entire lump, and the intensity of staining for BMP around the implant on day 7 was weaker than that on day 3. The staining for BMP decreased with time and the region of staining for BMP remained more centralized in the implant. On day 10 after implantation, BMP was observed in part of the newly induced cartilage, especially around chondrocytes. On day 14 after implantation, BMP was localized in the newly induced woven bone. On day 21, BMP staining was found in osteoblasts at the surface of the newly induced bone. Especially, the staining for BMP decreased from day 10 to day 21. These results indicate that the woven bone was replaced with mature lamellar bone from day 14 to day 21. The present findings suggest that rhBMP-2 plays an important role in osteoinduction, especially at the early stage.

We evaluated the time course of osteoinduction by an adenoviral vector, AxCAOBMP-2, in normal rats (Group I) and 2 immunosuppressed groups (Groups II and III). Immunosuppression was induced by 125 mg/kg of cyclophosphamide injected intraperitoneally the day before vector injection. Groups I and III received a high dose of AxCAOBMP-2 (25 microl; 8.75 x 10(8) pfu) and Group II a low dose (5 microl; 1.75 x 10(8) pfu). Each dose of AxCAOBMP-2 was injected into the right calf muscle of rats. On days 7, 14 and 21 postinjection, the osteoinducive activity in each group was investigated radiologically, histologically, immunohistochemically and biochemically. Osteoinduction was observed only in Groups II and III on days 14 and 21. The activity of osteoinduction in Group III was higher than that in Group II. There was little difference in the expression of LacZ between Groups I and III on day 3. However, there was a marked difference in BMP-2 protein expression between Groups I and III on day 7 postinjection. We speculated that the reason for this was that most of the infected cells were eliminated by the immune system of the host from days 3 to 7. These results suggest that gene therapy with AxCAOBMP-2 under transient immunosuppression may be useful for bone reconstruction.

We examined the effect of hyperbaric oxygen (HBO) therapy on the osteoinductive activity of recombinant human bone morphogenetic protein-2 (rhBMP-2), 5mg of which was implanted into the calf muscle of rats using atelopeptide type I collagen as a carrier. Thirty Wistar rats were divided equally to be given HBO or act as controls. New bone formation was measured radiographically, biochemically, and histol ogically 3, 7, and 21 days after implantation. In both groups, new bone formation was found on day 21. However, there was significantly more new bone in the HBO group. In the HBO group, cartilage was present at the outer edge of the implanted material on day 7. On days 7 and 21, the local tissue alkaline phosphatase activity and calcium content in the HBO group were significantly greater than in the control group. These results suggest that HBO accelerated the activity and rate of osteoinduction by rhBM P-2., (Copyright 2001 The British Association of Oral and Maxillofacial Surgeons.)

Background: Bone morphogenetic proteins (BMPs) play important roles in the migration of osteoblast progenitor cells, the proliferation of mesenchymal cells, and their differentiation into chondrogenic and osteogenic cells. However, the optimum procedure to deliver BMPs remains unknown. To examine the effectiveness of a gene transfer procedure for the delivery of BMP-2, we constructed a human BMP-2-expressing replication-deficient adenoviral vector, AxCAOBMP-2, and evaluated its osteoinductive activity in vitro and in vivo., Methods: C2C12 myoblasts were infected in vitro with this viral vector or an Escherichia coli LacZ gene-expressing control adenovirus vector (AxCALacZ). Twenty-four hours after the infection, indirect immunofluorescence was performed. On day 5 after the infection, alkaline phosphatase (ALP) in the cells and osteocalcin in the culture medium were measured. Furthermore, to examine the effectiveness of gene transfer of BMP-2 in vivo, we evaluated osteoinduction by AxCAOBMP-2, under transient immunosuppression with cyclophosphamide, given at a dose of 125 mg/kg intraperitoneally the day before injection of the adenoviral vector. Twenty-five microliters of AxCAOBMP-2 (8.75 x 10(8) plaque-forming units [pfu], Group I) and AxCALacZ (1.75 x 10(8) pfu, control group) and 5 microl of AxCAOBMP-2 (1.75 x 10(8) pfu, Group II) were injected into a right calf muscle of Wistar rats. On day 21, bone formation in each group was investigated radiologically and histologically., Results: Abundant BMP-2 expression in C2C12 cells infected with this viral vector was confirmed by immunofluorescence. C2C12 cells transferred with the BMP-2 gene by this vector produced ALP in the cells and also produced and secreted osteocalcin in the culture medium. Osteoinduction was found only in the AxCAOBMP-2 treated groups with immunosuppression. Osteoinduction activity was higher in Group I than in Group II., Conclusion: This study demonstrated the osteoinductive activity in vitro and in vivo by an adenoviral vector carrying the BMP-2 gene., Clinical Relevance: Gene therapy with AxCAOBMP-2 under transient immunosuppression may be useful for bone reconstruction.

Most recombinant human bone morphogenetic protein (rhBMP) is currently obtained from Chinese hamster ovary (CHO) cells. If rhBMP with more activity could be derived from Escherichia coli (E. coli), a large quantity of rhBMP could be produced at low cost. The bone-inducing ability of an E. coli -derived rhBMP-2 (ErhBMP-2) variant with an N-terminal sequence was examined and compared with CHO cell-derived rhBMP-2 (CrhBMP-2). Two, 10, or 50 microgram of ErhBMP-2 or CrhBMP-2 was mixed with 3mg of atelopeptide type I collagen as the carrier, and specimens were implanted into the calf muscle pouches of Wistar rats (n= 5 in each group). Three weeks later, new bone had formed in all the ErhBMP-2-implanted and CrhBMP-2-implanted muscles. Radiographic and histological examinations showed that the bone induced by ErhBMP-2 had a large hollow bone matrix with more fatty marrow than the bone induced by CrhBMP-2. Quantitative analysis indicated that the activity of ErhBMP-2 was similar to that of CrhBMP-2, so ErhBMP-2 may be useful for inducing bone formation., (Copyright 2000 The British Association of Oral and Maxillofacial Surgeons.)

Background: The mechanism of occurrence of calcification and ossification in pilomatricoma remains unclear., Objectives: To elucidate the pathogenesis of calcification and ossification in pilomatricoma we examined the role of bone morphogenetic protein (BMP)-2, which plays important parts in inducing ectopic bone formation both in vivo and in vitro., Methods: Twenty cases of pilomatricoma were studied immunohistochemically using anti-BMP-2 monoclonal antibody., Results: In normal skin, including hair follicles, there was no BMP-2 expression. In all pilomatricomas, BMP-2 was found exclusively in the cytoplasm of shadow cells but not in basophilic cells. In two cases of bone formation seen in pilomatricoma, osteoblasts in the periosteal area showed a strong positive reaction, while bone trabeculum (bone matrix) showed no reaction., Conclusions: Our findings indicate that shadow cells positive for BMP-2 may play an important part in generating bone formation in pilomatricoma.

Osteogenesis was evaluated in the mandibular bone by combinations of various dosages of recombinant human bone morphogenetic protein-2, atelopeptide Type I collagen, and porous hydroxyapatite (four groups: Group I, 2 micrograms recombinant human bone morphogenetic protein-2, atelopeptide Type I collagen, and porous hydroxyapatite; Group II, 10 micrograms recombinant human bone morphogenetic protein-2, atelopeptide Type I collagen, and porous hydroxyapatite; Group III, 50 micrograms recombinant human bone morphogenetic protein-2, atelopeptide Type I collagen, and porous hydroxyapatite; Control Group, only atelopeptide Type I collagen and porous hydroxyapatite). The prepared materials were implanted in the mandibular bone hole (7 mm in diameter, 2 mm deep). Three weeks later, the alkaline phosphatase activity in the implanted region was determined, and the histologic features of the excised tissue were examined. There were significant differences in histologic and biochemical findings among the four groups. In the recombinant human bone morphogenetic protein-2 implanted groups, osteogenesis increased with the dosage of recombinant human bone morphogenetic protein-2, as assessed by alkaline phosphatase activity and histologic findings. The results suggest that atelopeptide Type I collagen is an effective carrier for recombinant human bone morphogenetic protein-2 and that porous hydroxyapatite would be advantageous for clinical application as a material to maintain its original form after implantation.

To compare the osteoinductive activity of recombinant human bone morphogenetic protein-2 (rhBMP-2) at various sites in rats, 5 microg of rhBMP-2 were implanted into various sites, using atelopeptide type-I collagen (CL) as a carrier (BMP groups). CL implantation was used as a control. Forty Wistar rats were divided into intramuscular, intermuscular, subcutaneous and intrafatty site groups (IrM, IeM, SC and IF, respectively). Bone formation was evaluated radiographically, histologically and biochemically 21 days after implantation. In the BMP groups, the alkaline phosphatase activity and calcium content at all sites were higher than those in the control groups. Among the BMP groups, the new bone formation was highest in the IrM and lowest in the IF radiographically, histologically and biochemically. Five microg of rhBMP-2, a relatively low dose, induced adequate new bone formation in all sites. The variations of osteoinductive activity of rhBMP-2 in various sites may be due to differences in the blood supply.

To examine the effectiveness of gene transfer of bone morphogenetic protein (BMP)-2 in vivo, we evaluated osteoinduction by an adenoviral vector, AxCAOBMP-2, under transient immunosuppression with an immunosuppression drug (cyclophosphamide), which was given at a dose of 125 mg/kg intraperitoneally the day before vector injection. Twenty-five microliters of AxCAOBMP-2 (8.75 x 10(8) pfu, Group I) and AxCALacZ (1.75 x 10(8) pfu, control group) and 5 microliter of AxCAOBMP-2 (1.75 x 10(8) pfu, Group II) were injected into a right calf muscle. On day 21, induced bone in each group was investigated radiologically, histologically, and biochemically. The finding of osteoinduction was only seen in the AxCAOBMP-2-treated groups with immunosuppression. The activity of osteoinduction in Group I was higher than that in Group II. These results suggest that gene therapy with AxCAOBMP-2 under transient immunosuppression may be useful for bone reconstruction., (Copyright 2000 Academic Press.)

To examine the effectiveness of a gene transfer of bone morphogenetic protein (BMP)-2 into C2C12 myoblasts, we constructed a human BMP-2-expressing replication-deficient adenoviral vector, AxCAOBMP-2. C2C12 cells were infected in vitro with either this viral vector or an Escherichia coli LacZ gene-expressing control adenovirus vector. An efficient gene transfer to the C2C12 cells was confirmed with the LacZ gene-expressing vector by X-gal staining. Abundant BMP-2 expression in C2C12 cells infected with this viral vector was confirmed by immunofluorescence and Western blot analysis. C2C12 cells transferred with the BMP-2 gene by this vector produced alkaline phosphatase in the cells and also produced and secreted osteocalcin in the culture medium, demonstrating that a gene transfer of BMP-2 into C2C12 cells in vitro could convert these cells from myoblast to osteoblast lineage., (Copyright 1999 Academic Press.)

An autopsy case of biphasic malignant mesothelioma with osseous and cartilaginous differentiation diffusely involving the peritoneal cavity was confirmed by light microscopic histochemistry, immunohistochemistry, and electron microscopy. A reverse transcription-polymerase chain reaction using specific primers for bone morphogenetic protein-2 (BMP-2) revealed weak positive transcript in normal mesothelial cells and up-regulated expression around bone-forming malignant mesothelioma tissue. However, BMP-2 protein expression was detected only in the marginal zone of bone trabeculae and spindle-shaped mesothelioma cells distributed around bone trabeculae in tumor tissue. The distribution of type IV collagen in tumor tissue was in accordance with the BMP-2 expression. Normal mesothelial cells and tumor cells expressed BMP-2 mRNA, but the BMP-2 protein expression was restricted to the bone-forming area in the malignant mesothelioma.

Objective: The purpose of this study was to compare the osteoinducing activity of recombinant human bone morphogenetic protein-2 (rhBMP-2) at intramuscular and intraskeletal sites in rats., Study Design: Five tg of rhBMP-2 was implanted into the right calf muscle of each of 20 rats and into a hole (4 mm in diameter, 1.5 mm in depth) that was made in the mandibular body of each of 20 other rats, with atelopeptide type I collagen as a carrier. The alkaline phosphatase activity and calcium content were quantitatively analyzed 1, 3, 7, and 21 days after the implantation of rhBMP-2 into either mandibular bone (in the intraskeletal group) or calf muscle (in the intramuscular group). The new bone formation was evaluated histologically 21 days after implantation., Results: On days 1 and 3, the alkaline phosphatase activity and calcium content in the intraskeletal group showed no significant differences from those in the intramuscular group. On the 7th and 21st days after implantation, however, the alkaline phosphatase activity and calcium content in the intraskeletal group were significantly higher than those in the intramuscular group. Histometry of the microscopic views showed that the mean trabecular area was 0.87 mm2 in the intramuscular group and 2.66 mm2 in the intraskeletal group., Conclusions: These results suggest that the new bone formation stimulated by rhBMP-2 in the intraskeletal group was greater than in the intramuscular group.