Kunio Takaoka

Evaluation of polylactic acid homopolymers as carriers for bone morphogenetic protein

Polylactic acid (PLA) homopolymers with molecular weights of 105,000; 21,000; 3300; 650; and 160d (PLA105000, PLA21000, PLA3300, PLA650, and PLA160, respectively) were prepared and evaluated as carriers for bone morphogenetic protein (BMP). Composites consisting of 4 mg of water-soluble, semipurified BMP and 100 mg of one of the PLA homopolymers were implanted into the dorsal muscles of mice. PLA105000/BMP, PLA21000/BMP, PLA3300/BMP, and PLA160/BMP composites failed to induce new bone formation; PLA105000, PLA21000, and PLA3300 elicited foreign-body reactions or chronic inflammation (or both), and PLA160 produced tissue necrosis. PLA650/BMP composites induced cartilage formation within one week and induced bone with hematopoietic marrow at three weeks postimplantation. PLA650/BMP composites were completely absorbed and replaced by new bone. The results suggest that within this group of PLA homopolymers, PLA650 may be the only type suitable for use as a BMP carrier.

Immunohistochemical detection of bone morphogenetic proteins in bone and soft-tissue sarcomas

Background. Bone morphogenetic proteins (BMPs) are potent inducers of bone formation. Functional and immunohistochemical studies have identified BMPs in a subset of osteosarcomas. In the present study, the authors extend the analysis of BMP expression to other bone and soft tissue sarcomas.

Expression of bone morphogenetic proteins in human osteosarcoma. Immunohistochemical detection with monoclonal antibody

Background. Bone morphogenetic proteins (BMP) induce ectopic bone formation in vivo and may play a role in normal bone development. In addition, bone morphogenetic activity, as measured in a bone‐forming assay in immunodeficient, athymic nu/nu mice, is present in a proportion of osteosarcomas; this activity, which may be mediated by BMP, is correlated with a poor prognosis.

Polylactic acid-polyethylene glycol block copolymer : a new biodegradable synthetic carrier for bone morphogenetic protein

A new biodegradable polymer, a polylactic acid-polyethylene glycol (PLA-PEG) block copolymer, proved to be an effective and suitable carrier for bone morphogenetic protein (BMP). Composites of semipurified BMP and PLA-PEG consisting of a PLA segment with a molecular weight (MW) of 650 d and a PEG segment with a MW of 200 d (PLA-PEG 650-200) were implanted under the fasciae of the dorsal muscles of mice. Three weeks after implantation, the PLA-PEG 650-200/BMP composites were completely absorbed and replaced by newly induced bone with hematopoietic marrow. The composites induced twice as much bone as composites of BMP and a 650-d PLA homopolymer. Results indicate that of all the biodegradable synthetic polymers the authors have tested, PLA-PEG 650-200 is the most suitable and effective BMP carrier. Composites of PLA-PEG 650-200/BMP and hydroxyapatite powder (HAP) also induced ectopic bone formation. Because PLA-PEG 650-200/BMP is viscous and semiliquid and PLA-PEG 650-200/BMP/HAP is doughy and plastic, the former can be used as an injectable osteoinductive material and the latter as a plastic mold.

Ossification of the ligamentum flavum induced by bone morphogenetic protein : an experimental study in mice

Ossification of the ligamentum flavum and secondary spinal-cord compression were produced experimentally in mice by implanting bone morphogenetic protein (BMP) in the lumbar extradural space. The ligamentum flavum became hypertrophied and ossified, and protruded into the spinal canal. The thickness of the ossified ligament increased gradually with time, leading to compression and deformation of the spinal cord which showed various degrees of degeneration. Demyelination occurred in the posterior and lateral white columns and neuronal loss or chromatolysis in the grey matter. The pathological findings in the experimental animals closely resemble those found in the human disease and suggest that BMP may be a causative factor of ossification of the ligamentum flavum in man. This experimental model may be useful for the study of myelopathy caused by gradual spinal-cord compression.

Gene cloning and expression of a bone morphogenetic protein derived from a murine osteosarcoma

Based on information from partial amino acid sequences of a protein with bone-inducing activity that was purified from a murine osteosarcoma (Dunn type), a cDNA library of the sarcoma was screened to clone a gene complementary to the protein. The cloned cDNA was amplified and transfected into Chinese hamster ovarian (CHO) cells for expression. When the protein produced by the transfected cell line was implanted in combination with pure carrier collagen into allogeneic mice, ectopic ossicles consistently developed at implanted sites within two weeks. The nucleotide sequence of the cDNA and its deduced amino acid sequence were homologous to those of human bone morphogenetic protein-4 (also BMP-2B). In addition, the cDNA and deduced amino acid sequences were identical to those proposed for murine BMP-4 derived from the normal murine fetus. It is postulated that the cloned cDNA encodes the protein responsible for bone formation induced by implantation of devitalized Dunn-type osteosarcoma tissue or cells. The protein product was identified as murine BMP-4, a member of the TGF-beta gene family.

Transfilter bone induction by Chinese hamster ovary (CHO) cells transfected by DNA encoding bone morphogenetic protein-4

This study was undertaken to identify the factor responsible for classical transfilter bone induction by a murine osteosarcoma. Chinese hamster ovary (CHO) cells were transfected with the complementary DNA (cDNA) for bone morphogenetic protein-4 (BMP-4) that was purified from a murine (Dunn) osteosarcoma. Diffusion chambers were filled with the cells expressing the gene for BMP-4 and implanted subcutaneously into the flanks of ICR strain nude mice. Ectopic transfilter bone formation was seen consistently on the outer surfaces of the cellulose acetate membranes of chambers containing transfected cells at three weeks after implantation. Bone was not observed on chambers loaded with nontransfected CHO cells. The transfected CHO cells were inoculated into nude mice to form tumors, which were then homogenized, defatted, and bioassayed also in the ICR, nu/nu mice. The cell-free implants consistently elicited new bone and marrow within three weeks, whereas the control implants consisting of nontransfected tumor were not osteoinductive. These experimental results suggest that BMP-4 is one of the molecules responsible for the transfilter bone induction by vital Dunn osteosarcoma cells reported by Heiple and for the ectopic bone induction after implantation of devitalized, freeze-dried Dunn osteosarcoma tissue described originally by Amitani.

Expression of mRNA of murine bone-related proteins in ectopic bone induced by murine bone morphogenetic protein-4

To determine whether a system of ectopic bone formation induced by osteosarcoma-derived bone-inducing substance (bone morphogenetic protein-4) can be used as a model of developing bone at the molecular level, we studied the expression of bone-related protein mRNAs in the process of ectopic bone formation using non-radioisotopic in situ hybridization. Osteonectin mRNA was detected in fibroblast-like cells, which are similar to periosteal cells from the early to middle stages of bone development. The proportion of osteonectin mRNA-expressing cells was greater than that of osteopontin mRNA-expressing cells in hypertrophic chondrocytes and osteoblast-like cells. In contrast, osteopontin mRNA was localized in a limited population of hypertrophic chondrocytes, a single layer of osteoblast-like cells adjacent to the bone trabeculae in the middle stage of bone formation, and in a limited subset of osteocytes in the late stage. A strong osteocalcin mRNA signal was detected in osteoblast-like cells from the middle to late stages and in a limited subset of osteocytes in the late stage of bone development. Since the sequential gene expression pattern of bone-related proteins in the present system is comparable to that in embryonic osteogenesis, this system may be useful as a model for studying gene expression in osteogenesis.

Preservation of ectopically induced bone in the mouse by estradiol

The effects of estrogen on bone formation and bone resorption were examined in an experimental model of ectopic bone induction. In this experimental model, ectopic ossicles of uniform size were elicited reproducibly in three weeks by implanting pellets containing murine osteosarcoma derived bone morphogenetic protein (BMP), into the muscles of mice. Thereafter the induced ossicles showed a gradual reduction in bone mass due to negative bone balance. To estimate the effects of estrogen on bone formation and bone resorption, beta-estradiol-3-benzoate was administered exogenously to host mice from day 21 to day 41 (0.45 micrograms/g body weight, on alternate days, 10 treatments). Radiologic and histologic analysis confirmed the positive effect of estradiol on preserving bone mass over this period. Quantitative analysis showed an increase in the Ca content in ossicles after 21 days of estradiol treatment (139.1% of the baseline value on day 21). In the control group, the bone mass was reduced (47.4% of that on day 21). Bone resorption was determined by the reduction in 45Ca radioactivity from ossicles prelabeled with this radioisotope before estradiol administration. The rate of loss of the prelabeled 45Ca radioactivity was suppressed by estradiol (68.8% of prelabeled value in estradiol treated group and 87.7% in control group). The effect of exogenous estrogen on the bone forming phase of bone turnover in the ossicles was quantified by incorporation of 45Ca and 3H-proline. These bone formation parameters were increased to 2.2 and 1.9 times higher than those of the control group.(ABSTRACT TRUNCATED AT 250 WORDS)

Periosteal sunburst spiculation in osteosarcoma. A possible role for bone morphogenetic protein

Periosteal sunburst spiculation is a peculiar radiographic feature of osteosarcoma, and it represents a reactive ossification resulting from the action of normal osteoblasts rather than tumor cells. Because bone morphogenetic protein is known to be a potent inducer of ectopic bone formation, the authors hypothesized that bone morphogenetic protein may be involved in the pathogenesis of such reactive bone formation in osteosarcoma. Chinese hamster ovary cells transfected with the bone morphogenetic protein-4 gene were injected into the femurs of athymic nude mice to form experimental bone tumors producing bone morphogenetic protein. Two weeks after intramedullary injection, new bone formation was observed radiographically and histologically within the extraosseous portions of the tumors. This showed a close resemblance to sunburst spiculation in human osteosarcomas. In contrast, the control nontransformed Chinese hamster ovary tumors showed no extraosseous bone formation. Because the induced bone was composed of multiple parallel spicules similar to those found in human bone morphogenetic protein-producing osteosarcomas, these findings suggest that periosteal sunburst spiculation may be the result of bone morphogenetic protein production by osteosarcoma cells.