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This study was designed to investigate effects of heat on the bone-inducing activity of recombinant human bone morphogenetic protein (rhBMP)-2. rhBMP-2 samples were heated at 50, 70, 90, or 100 degrees C for 15 min, or 1, 2, 4, or 8 h, or autoclaved at 120 degrees C for 15 min. The bone-inducing activity of the rhBMP-2 before and after heating was assayed in in vivo and in vitro systems. For the in vivo assay, 5 microg rhBMP-2 samples were impregnated into porous collagen disks (6 mm in diameter, 1 mm thickness), freeze dried, and implanted into the back muscles of ddY mice. Three weeks later, the implant was harvested from the host and examined for ectopic new bone tissue by radiography. The new bone mass was quantified by single-energy X-ray absorptiometry. The in vitro activity of the rhBMP-2 was assayed by adding the BMP sample at a concentration of 100 ng/ml to cultures of MC3T3-E1 cells. After 48 h, the alkaline phosphatase activity was measured. After heating at 50 degrees or 70 degrees C, no significant reduction in bone-inducing activity was noted in either in vivo or in vitro assay systems unless the protein was exposed to sustained heat at 70 degrees C for 8 h, based on in vitro assay data. However, heating above 90 degrees C and for longer periods led to a decrease in the biological activity of the rhBMP-2 in a time- and temperature-dependent manner. rhBMP-2 was rendered inactive when exposed to temperatures at or in excess of 120 degrees C.

We investigated the enhancement of new bone |formation elicited ectopically by recombinant human bone morphogenetic protein-2 (rhBMP-2), using parathyroid hormone (PTH) and a phosphodiesterase inhibitor (PDEi), pentoxifylline (PTX), in an animal model. Collagen sponge sheet discs containing rhBMP were implanted onto the back muscles of mice. PTX alone (200 mg/kg body weight [BW]), PTH(1-34) (10 microg/kg BW), PTX plus PTH (200 mg/kg BW and 10 microg/kg BW, respectively), or vehicle (control) were injected subcutaneously daily for 3 weeks after implantation. At the end of this period, rhBMP-2-induced ectopic ossicles were harvested from each group of animals. Ossicles from the PTX-treated group were significantly larger in size, with unchanged bone mineral density (BMD), as compared with the ossicles from the controls. In contrast, the ossicles from the PTH-treated group had significantly higher BMD, but showed no difference in size when compared with those from the control animals. The ossicles of the PTX + PTH treatment group were significantly larger than those of the control and PTH treatment groups. In addition, the BMD of the harvested tissues from the PTX + PTH treatment group was signifi-cantly higher than that of tissues from the control and PTX treatment groups. Although the calcium content of ossicles was significantly higher in the PTX-, PTH-, and PTX + PTH-treated groups than in the control group, the Ca content of ossicles from the PTH + PTX-treated group was highest (two times that of controls), followed by the PTH- and PTX-treated groups.

Unlabelled: The mechanism of ectopic bone formation has not been clear. After BMP-2 implantation into the back muscles of 198 mice, expression of BMPR-1A, -2, and Noggin was increased during the early phase of the reaction. The results suggest that positive and negative feedback mechanisms modulate ectopic osteogenesis induced by this growth factor., Introduction: The expression of bone morphogenetic protein receptors (BMPRs) and Noggin during ectopic bone formation after implantation of BMP-2 into the back muscles of adult mice was investigated in this study., Methods: One hundred ninety-eight male ddy mice were divided into groups and received either collagen disks containing BMP-2, collagen disks alone, or sham surgery with no disk implantation. Changes in the temporal and spatial expression profiles of BMPRs and Noggin were examined by Northern blotting, in situ hybridization, Western blotting, and immunohistochemistry., Results and Conclusions: In the BMP group, expression of BMPR-1A, -2, and Noggin mRNA and protein was enhanced 2-4 days after implantation in undifferentiated mesenchymal cells and regenerating muscle fibers located close to the BMP-retaining implants. On day 7, the expression was also observed in cartilage cells, and after day 14, in the osteoblastic cells around bone tissue. The level of expression peaked at day 4 after implantation and persisted at a much lower level during the bone forming process. No significant expression of BMPR-1B was detected at the mRNA and protein levels during the bone-forming reaction. In the BMP free control groups, a mild enhancement of BMPR-2 expression was also noted around the implant, but this was not observed for BMPR-1A, -1B, or Noggin. Upregulated expression of BMPR-1A, -2, and Noggin in undifferentiated mesenchymal cells and regenerating muscle fibers occurs during the early phase of BMP-2-induced bone formation. The coordinate expression of these positive and negative regulators of BMP signaling points to a potential regulatory mechanism for bone induction.

Bone morphogenetic protein-4 (BMP-4), a member of the transforming growth factor-beta superfamily, is capable of initiating differentiation of uncommitted mesenchymal cells into a chondro/osteogenic pathway. This study reports the effects of pentoxifylline (PTX), a nonspecific inhibitor of phosphodiesterases (PDEs), that causes elevation of the intracellular cyclic adenosine monophosphate (cAMP) level on the BMP-4-induced chondro/osteogenic differentiation of a mesenchymal cell line, C3H10T1/2; a bone marrow stromal cell line, ST2; and an osteoblastic cell line, MC3T3-E1. It was found that PTX enhanced BMP-4-induced chondro/osteogenic differentiation in C3H10T1/2 and ST2 cells. Similar effects were observed when adding dibutyryl-cAMP and forskolin. These results indicate that cAMP may potentiate the action of BMP-4 on osteoprogenitor cells, highlighting the possibility that PDE inhibitors could be used as therapeutic agents to enhance bone formation through this effect., (Copyright 2002 Elsevier Science Inc.)

Collagen sponge disks (6 mm diameter, 1 mm thickness) were impregnated with recombinant human bone morphogenetic protein-2 (rhBMP-2) (5 microg/disk) and implanted onto the back muscles of mice. Ten or 20 mg/kg per day of Rolipram, a selective inhibitory agent to phosphodiesterase type 4 (PDE-4), or vehicle, was injected subcutaneously into the host mice for 3 weeks. After treatment, rhBMP-2-induced ectopic ossicles were harvested and examined by radiographic and histologic methods to determine the size, bone quality, and mineral content of the ossicles. The ossicles from a group treated with 20 mg/kg per day Rolipram were significantly larger in size and higher in bone mineral density (BMD) and bone mineral content (BMC) than the control samples. No significant differences were noted in mice treated with 10 mg/kg per day of Rolipram. Histologically, ossicles from the high-dose (20 mg/kg per day) Rolipram-treated group showed densely packed, thicker trabeculae when compared with those from the control group. These experimental results indicate that the PDE-4 inhibitor, Rolipram, may enhance the bone-inducing capacity of BMP-2 in mesenchymal cells. This in turn may result in increased responsiveness to BMP-2 and point to a potential use of PDE-4 inhibitors for the promotion of rhBMP-dependent bone repair.

Bone morphogenetic protein (BMP) is a bone-derived growth factor capable of promoting the differentiation of mesenchymal cells into osteogenic lineage pathways. Recently, immunosuppressants were reported to cause a moderate increase in osteoblastic differentiation in a rat osteoblast-like osteosarcoma cell line. If immunosuppressants can induce osteoblastic differentiation, it will be useful for bone tissue transplantation. We assessed the effect of immunosuppressants with or without BMP-4 on inducing osteoblastic differentiation in osteoblast-like and other mesenchymal cells. FK506, an immunosuppressant often used clinically, induced a dose- and time-dependent increase in alkaline phosphatase (ALP) activity, one of the markers of osteoblast differentiation, in cells derived from mesenchyma. In the presence of BMP-4, ALP activity, mRNA levels of ALP and osteocalcin increased. FK506 was found to not only stimulate osteoblastic differentiation, but also to enhance BMP-4 induced osteoblastic differentiation. These results suggest that FK506 promotes differentiation of osteoblastic cells., (Copyright 2002 Academic Press.)

Porous collagen disks (6 mm diameter, 1 mm thickness) were impregnated with recombinant human bone morphogenetic protein-2 (rhBMP-2) (5 microg/disk) and implanted onto the back muscles of mice. Pentoxifylline (PTX), which is a methylxanthine-derived inhibitor of phosphodiesterases (PDEs), or vehicle, was injected (5, 25, 50, 100, 200, and 300 mg/kg body weight/day) into the mice subcutaneously once a day for 3 weeks from the day of implantation of the bone morphogenetic protein (BMP)-laden disks. The rhBMP-2-induced ectopic ossicles were harvested and examined using radiographic, histological, and biochemical methods to determine size, bone quality, and calcium content. When compared with controls, ossicles from mice treated with >50 mg/kg per day of PTX were significantly larger in size and had a greater calcium content. However, no differences were noted in mice treated with lower doses (5 and 25 mg/kg per day) of PTX. The temporal sequence of the bone-forming process was unchanged by PTX based on histological examination. The histology of the ossicles from high- and low-dose PTX-treated mice was essentially identical to that observed in the control mice. These experimental results indicate that PTX enhanced the bone-inducing capacity of BMP-2. The underlying mechanism of action most likely involves the inhibition of intracellular phosphodiesterases and a resulting elevation of the intracellular content of cyclic nucleotides. Further studies are warranted to understand how BMP-induced bone formation is pharmacologically modified by PTX.