PTH1-34 improves devitalized allogenic bone graft healing in a murine femoral critical size defect.

The treatment of large segmental defects of long bones resulting from trauma, infection, or bone tumor resections is a major challenge for orthopedic surgeons. The reconstruction of bone defects with acellular allografts can be used as an osteoconductive approach. However, devitalized allografts are associated with high rates of clinical failure as a result of poor intrinsic osteoinduction properties and a lack of further remodeling. Nevertheless, evidence suggests that due to its anabolic properties, teriparatide (PTH1-34) could be effective as an adjuvant therapy for massive allograft healing. Therefore, our goal was to investigate in a murine critical-sized defect model whether the intermittent administration of PTH1-34 improves the incorporation and revitalization of acellular structural bone allografts. Thus, a 2.5-mm critical-sized defect was established in the right femur of C57BL/6 mice, followed by the reconstruction with a devitalized cortical structural allograft. A titanium micro locking plate was applied to the anterior femoral surface and secured in place with self-tapping locking screws. Subsequently, daily doses of PTH1-34 (30, and 40 µg/kg) or saline were administered to the mice for 14 days after surgery. The mice were maintained without PTH1-34 therapy for an additional 7 days before being euthanized at 3 weeks post-surgery. Bone graft consolidation was assessed on radiographic images and by histomorphometric analysis. Additionally, to determine the frequency of osteoprogenitor cells in the bone marrow and their in vitro osteogenic capacity, stromal cells were isolated from the bone marrow of animals treated with 30 or 40 µg/kg/day of PTH1-34 following the same protocol used for the experimental animals. Our results suggest that intermittent PTH1-34 treatment at 30 µg/kg/day after femoral allograft reconstruction surgery accelerated the healing process as evidenced by new bone formation induced on endosteal and periosteal surfaces, enhanced revitalization of allogeneic graft, and increased frequency and osteogenic capacity of bone marrow stromal cells (BMSC). These findings should encourage further studies aimed at investigating the potential therapeutic use of intermittent PTH1-34, specifically with regards to the optimal dosing regimen in clinically challenging orthopedic scenarios.

Patient-derived osteosarcoma cells are resistant to methotrexate.

Osteosarcoma is the most common primary bone tumor in children and young adults. The median survival of osteosarcoma patients has not significantly improved since 1990, despite administration of different classes of chemotherapy agents, such as methotrexate, cisplatin and doxorubicin. Cancer stem cells (CSCs) are responsible for the resistance of osteosarcoma to chemotherapy and OCT4, SOX2 and SSEA4 have been used to identify CSCs in osteosarcoma. Here, we used low-passage patient-derived osteosarcoma cells and osteosarcoma cells directly isolated from patients before and after chemotherapy treatments to evaluate the effects of chemotherapy on stem cell markers expression. We demonstrate that primary osteosarcoma cells are resistant to methotrexate treatment and sensitive to cisplatin and doxorubicin in vitro. We also verified that cisplatin and doxorubicin reduce the expression of SOX2 and OCT4 in primary osteosarcoma cells whereas methotrexate does not alter SOX2 and OCT4 expression, however it increases SSEA4 expression in primary osteosarcoma cells. Finally, we found that, although the combination treatment cisplatin plus doxorubicin inhibited the in vivo growth of osteosarcoma cells in NOD-SCID gamma mice subcutaneously injected with SaOs2, the combination treatment cisplatin plus doxorubicin plus methotrexate did not inhibit the in vivo growth of these cells. These observations may provide an explanation for the poor response of osteosarcomas to chemotherapy and point to the need of reevaluating the therapeutic strategies for human osteosarcomas.