Osteoinduction by repeat plasmid injection of human bone morphogenetic protein-2.

BACKGROUND: Bone morphogenetic protein-2 (BMP-2) is an osteoinductive protein and is considered useful for the treatment of skeletal disorders. Previous studies using BMP-2 in clinical applications have encountered difficulties, including the lack of an efficient, safe, inexpensive and simple delivery system. The gene transfer approach is a promising option for utilizing BMP-2. Although viral vector-mediated gene transfer is efficient, safety concerns prevent its clinical application for common diseases. On the other hand, plasmid-based gene transfer is a safe method and can be harnessed for practical applications. METHODS: A plasmid encoding human BMP-2 (pCAGGS-BMP-2) was used and injected repeatedly (one to eight times) into the skeletal muscle of mice at a divided dose. We compared the capability of osteoinduction in the skeletal muscle of mice after gene transfer by repeat injection. BMP-2 production was assessed via immunohistochemistry, and osteoinduction was evaluated using radiography, histology and biochemical assays. RESULTS: The BMP-2 gene was transferred into the skeletal muscle of mice by repeat injection using pCAGGS-BMP-2. Mature bone was frequently observed in mice injected repeatedly with pCAGGS-BMP-2 at a divided dose. This confirms that, if the total dose is fixed, repeat injection with pCAGGS-BMP-2 at a divided dose causes osteoinduction more frequently in the skeletal muscle of mice. CONCLUSIONS: These results suggest the possibility of the effective clinical use of human BMP-2 gene therapy by direct DNA injection, and facilitate the clinical application of BMP-2 gene therapy.

Osteoinduction by microbubble-enhanced transcutaneous sonoporation of human bone morphogenetic protein-2.

BACKGROUND: Bone morphogenetic protein-2 (BMP-2) is believed to participate in bone healing and regeneration. Previous studies using BMP-2 in clinical applications have encountered difficulties that include the lack of an efficient, safe and simple delivery system, and expensive proteins and matrices. The gene transfer approach is a promising option for utilizing BMP-2. Viral vector-mediated gene transfer is efficient, but safety concerns prevent its clinical application for common diseases. Sonoporation is a simple and inexpensive method that only requires a plasmid and a sonoporation device. METHODS: We used a plasmid-based human BMP-2 construct (pCAGGS-BMP-2) and examined the induction of bone in the skeletal muscle of mice after plasmid transfer by transcutaneous sonoporation. First, an in vitro study was performed to confirm the expression of BMP-2 after gene transfer by sonoporation using pCAGGS-BMP-2. Next, the BMP-2 gene was transferred into the skeletal muscle of mice by transcutaneous sonoporation using pCAGGS-BMP-2. BMP-2 production was assessed via immunohistochemistry, and osteoinduction was verified by radiography, histology and biochemical assays. RESULTS: The presence of human BMP-2, alkaline phosphatase and osteocalcin mRNA and the production of the alkaline phosphatase were observed in vitro. Moreover, mature bone was observed in mice sonoporated with pCAGGS-BMP-2, confirming that transcutaneous sonoporation with pCAGGS-BMP-2 caused osteoinduction in the skeletal muscle of mice. CONCLUSIONS: These results suggest the possibility of the effective clinical use of human BMP-2 gene therapy using transcutaneous sonoporation, and should facilitate clinical applications of BMP-2 gene therapy.

Evaluation of pluripotency in human dental pulp cells.

PURPOSE: Postnatal stem cells have been isolated from various tissues, including bone marrow, neural tissue, skin, retina, and dental epithelium. Recently, adult stem cells have been isolated from human dental pulp. Postnatal stem cells have been isolated from a variety of tissues. Previously, it was generally accepted that the differentiation potential of postnatal stem cells was lineage restricted. MATERIALS AND METHODS: Normal impacted third molars were collected from adults and normal exfoliated deciduous teeth (SHED; stem cells from human exfoliated deciduous teeth) by single-colony selection and magnetic activated cell sorting. RESULTS: BMP-2 treatment groups produced alkaline phosphatase in the cells and also produced and secreted osteocalcin in the culture medium, and were capable of inducing an upregulated expression of Osteocalcin or Sox9, Col 2, and Col X by reverse transcriptase polymerase chain reaction (RT-PCR). For adipogenic differentiation, there is potential for SHED and dental pulp stem cells (DPSC) to express 2 adipocyte-specific transcripts, PPARgamma2 and LPL, in vitro, as do bone marrow mesenchymal stem cells by RT-PCR. CONCLUSION: This study demonstrated that pluripotential cells isolated from the pulp of human teeth expanded in vitro and differentiated into osteoblasts, chondrocytes, and adipocytes. DPSC and SHED are not only derived from a very accessible tissue resource but also capable of providing enough cells for potential clinical applications.

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.

Over expression of bone morphogenetic protein-3b (BMP-3b) using an adenoviral vector promote the osteoblastic differentiation in C2C12 cells and augment the bone formation induced by bone morphogenetic protein-2 (BMP-2) in rats.

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.

The effect of fibroblast growth factor-2 on the osteoinductive activity of recombinant human bone morphogenetic protein-2 in rat muscle.

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.

Pluripotency of mesenchymal cells derived from synovial fluid in patients with temporomandibular joint disorder.

AIMS: Mesenchymal stem cells are an interesting source of material for regenerative medicine. The present study aimed at characterizing the phenotype and differentiation potential of adherent synovial fluid-derived cells from temporomandibular joint (TMJ) disorder patients. MAIN METHODS: Synovial fluid collection takes place during TMJ cavity irrigation arthrocentesis under local anesthesia. The synovial fluid-derived adherent cells were fibroblast-like and spindle-shaped. Ex vivo-expanded synovial fluid-derived cells were shown to express STRO-1 and CD146, previously found to be present in bone marrow mesenchymal stem cells. Further, they were identified as being capable of differentiating into a variety of cell types including osteoblasts, chondrocytes, adipocytes, and neurons. KEY FINDINGS: The present study demonstrates that human pluripotent cells can be isolated from synovial fluid. These synovial fluid-derived cells cannot only be derived from a very accessible resource, but are also capable of providing sufficient cells for potential clinical applications. SIGNIFICANCE: These cells may play a role in the regenerative response during arthritic diseases and are promising candidates for developing novel cell-based therapeutic approaches for postnatal skeletal tissue repair.

Experimental study of osteoinduction using a new material as a carrier for bone morphogenetic protein-2.

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.

Accelerators of osteogenesis by recombinant human bone morphogenetic protein-2.

Bone morphogenetic protein (BMP) appears to be one of the most promising cytokine and for clinical use in reconstructive surgery for bony defects and augmentation. To evaluate the effect of basic fibroblast growth factor (bFGF), FK506, elcatonin, and hyperbaric oxygenation (HBO) on osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2), 2 or 5 µg of rhBMP-2 was implanted into intramuscular sites of rats. At 21 days after implantation, the osteoinductive activity in the treatment group and control group was compared radiographically, biochemically, and histologically. The amount of new bone in the treatment group was significantly greater than that in the control group. The alkaline phosphatase activity and calcium content in the treatment group were significantly higher than those in the control group. These results suggest that bFGF, FK506, elcatonin, and HBO accelerated the activity and rate of osteoinduction by rhBMP2. These results may be useful when BMP is applied clinically in near future.

Thyroxine downregulates Sox9 and promotes chondrocyte hypertrophy.

Thyroid hormones exert a profound effect on development, growth, and metabolism of skeleton. In the present study, we evaluated the effects of thyroxine (T4) and growth hormone (GH) on the terminal differentiation of rib growth plate chondrocytes in three-dimensional pellet culture. T4 (30ng/ml) stimulated the expressions of type II and X collagens, alkaline phosphatase (ALP) activity. On the other hand, the expression of chondrogenic transcription factor Sox9 in the T4 treatment group decreased significantly compared to the control group. T4 downregulates Sox9 and promotes hypertrophy. After day 7, T4 increases dramatically the synthesis of type X collagen mRNA, ALP activity, and cellular hypertrophy. Addition of GH does not modify the action of T4. Thus, T4 acts directly on chondrocytes. In conclusion, we demonstrated that T4 enhances the cellular and molecular events of terminal differentiation and hypertrophy of chondrocytes in the three-dimensional cultures.

A bioactive bone cement containing Bis-GMA resin and A-W glass-ceramic as an augmentation graft material on mandibular bone.

The potential of a bioactive bone cement (BABC) as an onlay graft material for the mandible with and without the periosteum was investigated in rabbits. Its matrix consists of bisphenol-alpha-glycidyl methacrylate (Bis-GMA) and triethylene-glycol dimetacrylate (TEGDMA) and its filler is silane-treated CaO-SiO2-P2O5-MgO-CaF2 glass (A-W glass-ceramic) powder. The BABC was pasted onto the mandible under the periosteum in Group 1, and onto the mandible with the periosteum removed in Group 2 and allowed to set in situ. In both groups, the cement-bone interface was filled by new bone at 4, 12 and 48 weeks, and bone grew from adjacent bone tissue into the cement-soft tissue interface at 12 and 48 weeks. There were no differences in the rate of bone formation between the groups. The shearing strength increased progressively from 0.25+/-0.10 MPa (mean+/-SD) at week 1 to 7.98+/-0.62 MPa at week 48. The results suggest that the BABC has good handling properties, a high bonding strength and good biocompatibility, and that it has potential for clinical application as a substitute material for autogenous bone transplantation.

Expression of bone morphogenetic protein in the course of osteoinduction by recombinant human bone morphogenetic protein-2.

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.

Immunoselection and adenoviral genetic modulation of human osteoprogenitors: in vivo bone formation on PLA scaffold.

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.

Adenoviral BMP-2 gene transfer in mesenchymal stem cells: in vitro and in vivo bone formation on biodegradable polymer scaffolds.

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.