Effect of sonic hedgehog/ß-TCP composites on bone healing within the critical-sized rat femoral defect.

The creation of entirely synthetically derived bone substitute materials which are as effective as autologous bone grafts is desirable. Osteogenesis involves the concerted action of several proteins within a signaling cascade. Hedgehog proteins act upstream of this cascade, inducing the expression of various bone morphogenetic proteins (BMPs) and promoting physiological bone healing. Therefore, the hypothesis that hedgehog signaling in bone defects improves bone healing more than BMP signaling alone was tested. Recombinant N-terminal sonic hedgehog protein (N-SHh), BMP-2 or a combination of the two was added to ß-tricalcium phosphate (ß-TCP) and 5-mm femoral midshaft defects in nude rats were filled with these composites. The defects were stabilized with mini-plates. After eight weeks, the animals were sacrificed and the femora were explanted. The radiological evaluation was followed by a three-point bending test and histological examination. BMP-2/ß-TCP composites showed a trend of increased stiffness compared with the controls (ß-TCP without protein). N-SHh/ß-TCP composites had lower stiffness compared with the control group and the N-SHh/BMP-2/ß-TCP composites also had lower average stiffness compared with the controls (all not significant). Histomorphometry, however, revealed abundant cartilage and bone core formation in the N-SHh-composite groups. The sum of the new cartilage and bone was highest in the combination group N-SHh/BMP-2 (not significant). The addition of N-SHh to bone substitute materials appears to delay bone healing at the applied concentration and observation time but also showed a trend for higher amounts of ossifying cartilage.

Effect of the Hedgehog-inhibitor cyclopamine on mice with osteosarcoma pulmonary metastases.

Chemoresistant metastases of osteosarcoma in humans limit survival in approximately one third of patients. Furthermore, aggressive chemotherapy can lead to side effects and occurrence of secondary malignancies in long time survivors. Therefore, supplemental medical strategies are worthwhile. The well-directed manipulation of cancer-signaling-cascades is an appealing approach. Targeting of the Hedgehog-pathway in cancer has led to promising results in vitro as well as in vivo in a number of different tumor types. Recently, the impact of cyclopamine, which inhibits Hedgehog signaling by binding to the receptor Smoothened, was shown in different human osteosarcoma cell lines in vitro and in vivo. In the present study we examined the influence of cyclopamine on early pulmonary metastases in vivo. Murine osteosarcoma cells, OS-50, were injected into the lateral tail vein of young BALB/c mice. Treatment with subcutaneous cyclopamine injections began after three days. Two weeks later, the animals were sacrificed and the number of pulmonary metastases was counted. We could observe a trend towards decreased metastases in the cyclopamine group (~20%). On the other hand, remarkable side effects were caused by the cyclopamine/ethanol/triolein preparation (mainly skin ulcerations).

Changes of femoral periprosthetic bone mineral density 6 years after treatment with alendronate following total hip arthroplasty.

Earlier osteodensitometric results of femoral periprosthetic bone showed that postoperative antiresorptive treatment with alendronate following total hip arthroplasty (THA) reduces the periprosthetic bone loss that commonly occurs in the first months after surgery. However, whether alendronate can prevent periprosthetic bone loss over the long term, or if bone loss occurs after discontinuing alendronate is unknown. Femoral periprosthetic bone mineral density (BMD) was assessed in 49 patients 6 years after cementless total hip arthroplasty using dual energy X-ray absorptiometry. Twenty-nine patients were treated postoperatively with alendronate and 20 control patients received no treatment. All patients were followed up at 12 months after surgery in a prospective randomized study. The bone mineral density was evaluated in 7 regions of interest according to the Gruen protocol. Six years after total hip arthroplasty, no significant changes were detected in femoral periprosthetic BMD when compared with results at 1 year, and the bone loss in patients with postoperative alendronate treatment was still significantly less than those without treatment. These results suggest that the prevention of femoral periprosthetic bone loss following THA achieved by postoperative antiresorptive treatment with alendronate is of long-standing effect, and further bone loss does not occur after the first postoperative year.

Sonic hedgehog protein promotes proliferation and chondrogenic differentiation of bone marrow-derived mesenchymal stem cells in vitro.

BACKGROUND: Sonic hedgehog (Shh) protein is known to be an important signaling protein in early embryonic development. Also, Shh is involved in the induction of early cartilaginous differentiation of mesenchymal cells in the limb and in the spine. METHODS: The impact of Shh on adult stem cells, human bone marrow-derived mesenchymal stem cells (MSCs), was tested. The MSCs were treated either with recombinant Sonic hedgehog protein (r-Shh) or with transforming growth factor-beta 1 (TGF-beta(1)) as a positive control in vitro for 3 weeks. The effects on cartilaginous differentiation and proliferation were assayed. RESULTS: MSCs when treated with either Shh or TGF-beta(1) showed expression of cartilage markers aggrecan, Sox9, CEP-68, and collagen type II and X within 3 weeks. Only r-Shh-treated cells showed a very strong cell proliferation and much higher BrdU incorporation in cell assay systems. CONCLUSIONS: These are the first data that indicate an important role of Shh for the induction of cartilage production by MSCs in vitro.

Seven BMPs and all their receptors are simultaneously expressed in osteosarcoma cells.

Members of the bone morphogenetic protein (BMP) family and their receptors (BMPRs and activin receptors-ActRs) promote the development of bones with a fine regulation of their expression. Mutations in BMPs or BMPRs cause several diseases, as shown in knockout mice, such as skeletal defects, familial primary pulmonary hypertension and neoplasias. Osteosarcoma is the most frequent primary malignant tumor of bone. Due to their importance in bone development, BMPs, BMPRs and ActRs could also play a role in osteosarcoma growth and development. Previous data have shown that the overexpression of the BMPR-II was related to poor prognosis in malignant and metastatic bone tumors. We evaluate by reverse transcription-linked polymerase chain reaction analysis (RT-PCR) the expression pattern of BMPs, BMPRs and ActRs in five different human osteosarcoma cell lines (MG63, G292, HOS, SaOS and U2). Moreover, we performed the mutational screening of the complete BMPR-II mRNA by automated sequencing of the correspondent cDNA to evaluate the presence of point mutations in osteosarcoma cell lines. All the osteosarcoma cell lines studied simultaneously expressed the BMPs, BMPRs and ActRs investigated. No mutations were detected in the BMPR-II cDNA. Our results suggest the presence of a mechanism involving the simultaneous activation of the BMPs and their receptors in osteosarcoma cell lines.