Atelopeptide Type Research Articles

MiR-29a Assists in Preventing the Activation of Human Stellate Cells and Promotes Recovery From Liver Fibrosis in Mice.

The microRNA-29 (miR-29) family is known to suppress the activation of hepatic stellate cells (HSCs) and reversibly control liver fibrosis; however, the mechanism of how miR-29a controls liver fibrosis remains largely unknown. This study was conducted to clarify the mechanism of anti-fibrotic effect of miR-29a and to explore if miR-29a is a promising candidate for nucleic acid medicine against liver fibrosis. Two liver fibrosis murine models (carbon tetrachloride or thioacetamide) were used. MiR-29a mixed with atelocollagen was systemically administered. Hepatic fibrosis was evaluated by histological analysis and the expression levels of fibrosis-related genes. We observed that miR-29a treatment dramatically accelerated the reversion of liver fibrosis in vivo. Additionally, miR-29a regulated the mRNA expression of collagen type I alpha 1 (COL1A1) and platelet-derived growth factor C (PDGFC). We also noted that miR-29a significantly suppressed COL1A1 mRNA expression and cell viability and significantly increased caspase-9 activity (P < 0.05) in LX-2 cells. Pretreatment of miR-29a inhibited activation of LX-2 cell by transforming growth factor beta treatment. MiR-29a exhibited anti-fibrotic effect without cell toxicity in vivo and directly suppressed the expression of PDGF-related genes as well as COL1A1 and induced apoptosis of LX-2 cells. MiR-29a is a promising nucleic acid inhibitor to target liver fibrosis.

Effect of aging on the osteoinductive activity of recombinant human bone morphogenetic protein-2 in rats

BackgroundBone morphogenetic proteins may hold broad potential for use in the reconstruction of bone defects resulting from tumor resection or trauma and in assisting bone healing thanks to methods enabling the synthesis of recombinant human bone morphogenetic protein-2 (rhBMP-2). MethodsrhBMP-2 was implanted with atelopeptide type I collagen as a carrier into the calf muscles of 3-, 8-, and 48-wk-old Wistar/ST male rats. After 21 d, the formation of ectopic neoplastic bone was examined in soft x-ray imaging, and the bone mineral content, bone area, and bone mineral density (BMD) were measured by dual-energy x-ray absorptiometry. In addition, hematoxylin-eosin and von Kossa staining and proliferation cell nuclear antigen immunostaining were performed. ResultsBMD values determined by dual-energy x-ray absorptiometry were 29.40 (standard deviation ±5.47), 24.15 (±2.33), and 19.01 (±2.02) mg/cm2 in the 3-, 8-, and 48-wk-old rats, respectively, demonstrating that BMD significantly decreased with aging (P < 0.05). The von Kossa stain-positive area decreased significantly with aging (P < 0.01). The number of proliferation cell nuclear antigen-positive cells also decreased significantly with aging (P < 0.01). ConclusionsThe capacity of rhBMP-2 to induce ectopic bone formation decreases with aging. These findings will be of considerable benefit in the development of clinical treatments for the regeneration of cranio-maxillofacial bone in elderly patients.

Osteoinduction by bone morphogenetic protein 2-expressing adenoviral vector: application of biomaterial to mask the host immune response.

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.

Effect of FK506 on osteoinduction by recombinant human bone morphogenetic protein-2

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.

Angioneogenesis Around Induced Bone with Recombinant Human BMP-2 in a Latissimus Dorsi Muscle Flap

Bony tissue induced with recombinant human bone morphogenetic protein-2 (rhBMP-2) in the latissimus dorsi muscle flap (LDMF) of rats was investigated in angiography and histological examination. In five rats, rhBMP-2 with atelopeptide type I collagen (CL) as a carrier was implanted in the muscle pocket of LDMF. In five controls, only CL was implanted in the same manner. Three weeks after the implant, contrast medium was injected into the assending aorta. The LDMF was cut off at the base and exposed in soft X-ray. The implant and the surrounding tissue were then histologically examined. In the rhBMP-2-implant group, the vascularity was richer, especially around the implant, and radiated to the induced bony tissue. Contrast medium was observed in the vascular cavities of the marrow inside the trabeculae. In the controls, the vascularity was relatively poor and the remnant CL occupied the whole implant space. This study indicated that rhBMP-2 does not only induce trabeculae in muscle tissue, but also in the marrow, including vessels in the implant and the vascular system around the implant. Then, the induced bony tissue is supported in the circulation by the circulation by the vascular system, as a functional osseous tissue. This phenomenon may be highly beneficial in endogenous tissue engineering and regenerative medicene for skeletal reconstruction using BMP in the future.

ANGIONEOGENESIS AROUND INDUCED BONE WITH RECOMBINANT HUMAN BMP-2 IN A LATISSIMUS DORSI MUSCLE FLAP

Bony tissue induced with recombinant human bone morphogenetic protein-2 (rhBMP-2) in the latissimus dorsi muscle flap (LDMF) of rats was investigated in angiography and histological examination. In five rats, rhBMP-2 with atelopeptide type I collagen (CL) as a carrier was implanted in the muscle pocket of LDMF. In five controls, only CL was implanted in the same manner. Three weeks after the implant, contrast medium was injected into the assending aorta. The LDMF was cut off at the base and exposed in soft X-ray. The implant and the surrounding tissue were then histologically examined. In the rhBMP-2-implant group, the vascularity was richer, especially around the implant, and radiated to the induced bony tissue. Contrast medium was observed in the vascular cavities of the marrow inside the trabeculae. In the controls, the vascularity was relatively poor and the remnant CL occupied the whole implant space. This study indicated that rhBMP-2 does not only induce trabeculae in muscle tissue, but also in the marrow, including vessels in the implant and the vascular system around the implant. Then, the induced bony tissue is supported in the circulation by the circulation by the vascular system, as a functional osseous tissue. This phenomenon may be highly beneficial in endogenous tissue engineering and regenerative medicene for skeletal reconstruction using BMP in the future.

In Vivo Serial Changes in Bone Induction by E. Coli-Derived Recombinant Human BMP-2

At present, several recombinant human bone morphogenetic proteins (rhBMPs) can be produced in mammalian cells. If rhBMPs with a high activity could be produced in bacteria, the bacterial expression system is very useful in clinic. We examined in vivo serial changes in the bone inducing ability of an Escherichia coli-derived rhBMP-2 (ErhBMP-2) variant with N-terminal sequence. Five μg of ErhBMP-2 was mixed with 3 mg of atelopeptide type I collagen (CL) as the carrier, and specimens were implanted into calf muscle pouches of Wistar rats (n = 20). After 3, 7, 14 and 21 days, 5 specimens each were examined. New cartilage was observed 7 days after implantation of ErhBMP-2 with CL. Induced bone was found on the outermost edge of the implant after 14 days. After 21 days, bone formation was associated with much fatty marrow. ALP activity and calcium content gradually increased with time. These changes were similar to those in a study using Chinese hamster ovary cell-derived rhBMP-2. However, these increases were slightly higher than those in the rhBMP-2 study. From these findings, ErhBMP-2 appears to be completely renatured while maintaining its biological activity. ErhBMP-2 with CL may expand by absorbing body fluid in vivo to greater extent than rhBMP-2 with CL, because it lacks heparin-binding sites. A variant ErhBMP-2 showing high activity in vivo, obtained by the bacterial expression system is an important finding, as it should be possible to produce large quantities of ErhBMP-2 at a low-cost for clinical use, in the near future.

IN VIVO SERIAL CHANGES IN BONE INDUCTION BY E. COLI-DERIVED RECOMBINANT HUMAN BMP-2

At present, several recombinant human bone morphogenetic proteins (rhBMPs) can be produced in mammalian cells. If rhBMPs with a high activity could be produced in bacteria, the bacterial expression system is very useful in clinic. We examined in vivo serial changes in the bone inducing ability of an Escherichia coli-derived rhBMP-2 (ErhBMP-2) variant with N-terminal sequence. Five μg of ErhBMP-2 was mixed with 3 mg of atelopeptide type I collagen (CL) as the carrier, and specimens were implanted into calf muscle pouches of Wistar rats (n = 20). After 3, 7, 14 and 21 days, 5 specimens each were examined. New cartilage was observed 7 days after implantation of ErhBMP-2 with CL. Induced bone was found on the outermost edge of the implant after 14 days. After 21 days, bone formation was associated with much fatty marrow. ALP activity and calcium content gradually increased with time. These changes were similar to those in a study using Chinese hamster ovary cell-derived rhBMP-2. However, these increases were slightly higher than those in the rhBMP-2 study. From these findings, ErhBMP-2 appears to be completely renatured while maintaining its biological activity. ErhBMP-2 with CL may expand by absorbing body fluid in vivo to greater extent than rhBMP-2 with CL, because it lacks heparin-binding sites. A variant ErhBMP-2 showing high activity in vivo, obtained by the bacterial expression system is an important finding, as it should be possible to produce large quantities of ErhBMP-2 at a low-cost for clinical use, in the near future.

Recombinant human bone morphogenetic protein-2 promotes osteogenesis within atelopeptide type I collagen solution by combination with rat cultured marrow cells.

We evaluated the combination effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) and cultured rat bone marrow mesenchymal stem cells (MSCs) in atelopeptide type I collagen (AC) solution on osteogenesis in a diffusion chamber (DC) to develop a bone substitute having consistent osteogenic capability for clinical applications. The cultured MSCs were obtained by 10-day primary culture of fresh bone marrow cells of Fischer rats. We prepared three groups of DCs: AC solution with rhBMP-2, AC solution with cultured MSCs, and AC solution with rhBMP-2 and cultured MSCs. The prepared combined solutions were injected into DCs, which were subcutaneously implanted into the backs of syngeneic rats. DCs were harvested after 2, 4, or 8 weeks and analyzed for bone-forming capability by determining histological and osteoblastic biochemical markers. De novo bone formation was observed both inside and outside of the membrane filter of DCs in the group of AC solution with rhBMP-2 and cultured MSCs. The alkaline phosphatase activity and osteocalcin content in the group of AC solution with rhBMP-2 and cultured MSCs were significantly higher than those in the group of AC solution with cultured MSCs at any time. These findings indicate that AC aqueous solution is a useful material not only as a carrier of rhBMP-2 but also as a cell-anchorage for differentiation and proliferation of MSCs. Therefore, this study suggests that clinical repairs of bone defects are feasible using injectable AC solution with rhBMP-2 and cultured MSCs as a bone substitute.

Effect of hyperbaric oxygenation on bone induced by recombinant human bone morphogenetic protein-2

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.

An immuno-light- and electron-microscopic study of the expression of bone morphogenetic protein-2 during the process of ectopic bone formation in the rat

Immunolocalization of endogenous bone morphogenetic protein-2 (BMP-2) was investigated during the process of ectopic bone formation induced by recombinant human BMP-2 (rhBMP-2). Pellets consisting of 5 μg of rhBMP-2 and 6 mg of atelopeptide type I collagen (AC) were implanted into the calf muscles of 6-week-old rats. On days 7, 10, 14, 21 and 28 after implantation, tissue specimens were removed and examined immunohistochemically by light and electron microscopy after incubation with anti-BMP-2 monoclonal antibodies. Immunolocalization by light microscopy showed BMP-2 in chondrocytes at the pellet rim on days 7 and 10, in osteocyte-like cells in the chondroid matrix on day 14, and in osteocytes in the newly formed bone on days 21 and 28 after implantation. Ultrastructurally, on days 7 and 10 after implantation, immunolabelling for BMP-2 was aggregated in vesicle-like matrices released from mature chondrocytes in the chondroid matrix. On day 14, immunolabelling against BMP-2 had accumulated in vesicle-like matrices embedded in the calcified cartilage, in the cytoplasmic vacuoles of chondroclasts absorbing the matrix, and at the resorption surface of the calcified cartilage. On days 21 and 28, BMP-2 immunolabelling was seen in the osteoid layer and osteocyte lacunae. These results suggest that the chondrocytes and osteocytes induced by rhBMP-2 produce endogenous BMP-2. It seems that part of the endogenous BMP-2 that accumulated in the chondroid matrix was absorbed by chondroclasts and then participated in the osteoblastic differentiation of immature mesenchymal cells. This study indicates that, in addition to the implanted exogenous rhBMP-2, endogenous BMP-2 plays an important part in the maintenance of the bone-formation cascade during ectopic osteoinduction.

Bone induction by Escherichia coli -derived recombinant human bone morphogenetic protein-2 compared with Chinese hamster ovary cell-derived recombinant human bone morphogenetic protein-2

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 50μg 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.

Osteogenesis by recombinant human bone morphogenetic protein-2 at skeletal sites.

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.

Ultrastructural study of direct bone formation induced by BMPs-collagen complex implanted into an ectopic site.

Some authors have reported that direct bone formation is ectopically induced by bone morphogenetic proteins (BMPs) independently of cartilage formation when type I collagen is used as a carrier. This study ultrastructurally investigated the mechanism of direct bone formation by BMPs-collagen complex. Partially purified BMPs were combined with atelopeptide type I collagen (AC) and were implanted into the calf muscles of rats (n = 20). Tissue specimens were removed on days 7, 10, 14, and 21 after implantation. Ultrastructurally, several regions near the pellet rim showed evidence of early calcification on day 10. In the uncalcified regions, mitochondrial calcification was seen in mesenchymal cells near AC fibers. The initially calcified regions contained numerous calcified granules deposited in the AC fibers. Some of these granules adhered to the cell membrane of osteogenic cells. In the highly calcified regions, some osteogenic cells secreted uncalcified matrix and deposited needle-like crystals and calcified collagen microfibrils on the AC fibers. This study suggests that the mesenchymal cells invading BMPs-AC complex closely contacted the AC fibers, differentiated into osteogenic cells, and deposited calcified matrix on the AC fibers, resulting in direct osteoinduction without cartilage formation.

Enhancement by recombinant human bone morphogenetic protein-2 of bone formation by means of porous hydroxyapatite in mandibular bone defects.

Hydroxyapatite is osteoconductive and can maintain an original biocompatible form. It is useful, in the reconstruction of bone defects, to enhance the osteoconduction of hydroxyapatite with an osteogenic protein. The aim of this study was to evaluate the bone formation in surgically created defects of rabbit mandibles by a combination of recombinant human bone morphogenetic protein-2 (rhBMP-2), with porous hydroxyapatite and atelopeptide type I collagen used as the carrier for rhBMP-2. A 10-microg rhBMP-2-implanted group (n = 15) and a control group (n = 15), in which only atelopeptide type I collagen and porous hydroxyapatite were implanted, were histologically examined 3, 7, and 21 days after implantation. The alkaline phosphatase activity was also quantitatively analyzed. No new bone formation was observed in either the tested or the control group after 3 days. At 7 days, immature bone tissue was observed in some pores of the rhBMP-2implanted group, while in the control group, immature mesenchymal cells were observed. At 21 days, trabecular bone lined some pore walls. In the central portion, the bone marrow, including angioid tissue, was observed. New trabecular bone formation was observed on portions of the external surface of the hydroxyapatite disk. On the other hand, the control group showed infiltration of immature mesenchymal cells into some pores. Marginal bone formation was found in the pores close to the surface of the disk which opposed mandibular bone. The control group showed a slow, small increase in alkaline phosphatase activity in this study, while the experimental group showed a marked increase at 21 days. This increase was significantly higher in the tested group than in the control group at both 7 and 21 days. The findings indicate that rhBMP-2 accelerated bone formation by osteoconduction from porous hydroxyapatite. The combination of rhBMP-2, atelopeptide type I collagen, and porous hydroxyapatite is suggested to be advantageous for clinical application in reconstructing mandibular bone defects.

Osteoblast progenitor cell responses to characterized titanium surfaces in the presence of bone morphogenetic protein-atelopeptide type I collagen in vitro.

The goal of this study was to investigate the effect of bone cell response to titanium (Ti) surfaces in the presence of bone morphogenetic protein (BMP)-atelopeptide type I collagen mixture. The atelopeptide type I collagen was used as a potential carrier for the BMP. Sterilized 600-grit Ti samples were used as substrates for the cell culture study. X-ray photoelectron spectroscopy indicated the presence of TiO2 on the Ti surface. The in vitro cell culture study was performed using an osteoblast progenitor cell line derived from mice (2T9). At confluency, the cells cultured on Ti surfaces were divided into three groups: unstimulated culture, culture stimulated by BMP-atelopeptide type I collagen (40 ng/mL), and culture stimulated by atelopeptide type I collagen (40 ng/mL). The unstimulated and atelopeptide type I collagen cultures were controls in this study. After 4 days of incubation, protein production, alkaline phosphatase (ALP) activity, and hexosaminidase activity were observed to be the highest for cells exposed to the BMP-atelopeptide type I collagen mixture. Statistical differences in cellular protein production and ALP activity were observed between the controls and the surfaces exposed to the BMP-atelopeptide type I collagen mixture. Similarly, a statistical difference in hexosaminidase activity was observed between unstimulated Ti surfaces and surfaces exposed to BMP-atelopeptide type I collagen mixture. However, no statistical differences in protein production, ALP activity, and hexosaminidase activity were observed between cells exposed to atelopeptide type I collagen solution and the unstimulated surfaces.

Comparison of recombinant and purified human bone morphogenetic protein

Clinically, it would be more convenient to use recombinant than purified preparations of bone morphogenetic protein (BMP). Recently, recombinant human BMP (rhBMP) has attracted the attention of many investigators, but it has not been fully characterized. We examined the bone-inducing activity of rhBMP-2 and compared it with that of purified BMP derived from human bone matrix (phBMP). Two, 10, or 50 μ g of rhBMP-2 or phBMP was mixed with 3 mg of atelopeptide type I collagen (carrier), and specimens were implanted in the calf muscles of Wistar rats (n =5 in each group). Four weeks later, new bone had formed in all the rhBMP-2- and phBMP-implanted muscles and was visible radiographically and histologically. The quantitative analysis indicated that the activity of rhBMP-2 was less than one tenth that of phBMP. It is necessary to find out why rhBMP-2 has fewer activities than phBMP.

Comparative study of intramuscular and intraskeletal osteogenesis by recombinant human bone morphogenetic protein-2

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 μg 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 mm 2 in the intramuscular group and 2.66 mm 2 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.(Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;87:34-8)

Prefabricated muscle flap including bone induced by recombinant human bone morphogenetic protein-2: an experimental study of ectopic osteoinduction in a rat latissimus dorsi muscle flap

SUMMARY.For skeletal reconstruction, a vascularised bone graft is both reliable and useful, but some sacrifice usually occurs at the skeletal donor site. In the search for our alternative, we investigated ectopic osteoinduction by bone morphogenetic protein in a rat muscle flap, to prefabricate a muscle flap that included bone.Right latissimus dorsi muscle flaps were prepared in 16 rats. Recombinant human bone morphogenetic protein-2 (rhBMP-2; 2, 10 and 50 μg) mixed with atelopeptide type I collagen (3 mg) as the carrier was wrapped with the tip of the latissimus dorsi muscle flap. Three weeks later, ectopic osteoinduction in the latissimus dorsi muscle flap was investigated radiologically, histologically and biochemically.A radiological oval shadow was observed in each animal in each rhBMP-2-implanted group. The volume of the oval shadow area in the muscle flap was dependent upon the dosage of rhBMP-2. It indicated that on ectopic osteoinduction by rhBMP-2 in the muscle flap, induced trabeculae were circularly constructed at the margins of the lump. The radio-opacity of the oval shadows was almost equal in all rhBMP-2-implanted groups, and the shadow areas depended on the dosage of rhBMP-2. The biochemical indices of osteogenesis, ALP activity and Ca content in the lump were dependent upon the dosage of rhBMP-2.This study showed that ectopic osteoinduction occurred in the rat latissimus dorsi muscle flap and depended upon the dose of rhBMP-2. Wrapping rhBMP-2 mixed with atelopeptide type I collagen with the tip of a latissimus dorsi muscle flap could create a prefabricated muscle flap that included bone.

コラーゲンを担体とした遺伝子組み換えヒト骨形成因子による骨誘導に関する研究

We studied osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2) with the use of porcine skin-derived atelopeptide type I collagen (CL) as a carrier. Ten μg of rhBMP-2 with 3 mg CL was implanted into the calf muscle of 10-week-old Wistar rats. In the control groups, 3 mg CL only was implanted. Ten days (BMP group I, CL group I; n=5) or four weeks (BMP group II, CL group II; n=5) after implantation, soft X-ray and light microscopic exminations were performed.Soft X-ray findings showed a radiopaque shadow in the implanted muscles in the BMP groups. In BMP group I, a radiopaque shadow was observed partially at the outermost edge of the implanted materials. In BMP group II, a radiopaque shadow was observed at all areas of the implant. In contrast, no such shadows were observed in the CL groups.Histological examination revealed new bone formation in the BMP groups. Woven bone appeared at the outermost edge of the implant material in BMP group I. Trabecular bone and osteoblast-like cells were observed around almost the entire circumference of the implant material in BMP group II. There was no evidence of bone or cartilage in the CL groups. From 10 days to 4 weeks after implantation, osteoinduction was continuously promoted by rhBMP-2 with CL, and the amount of CL decreased in the implant. These results suggest that rhBMP-2 may play an important role in reconstructive surgery for extensive bony defects and augmentation.