Healing of rat femoral segmental defect with bone morphogenetic protein-2: a dose response study.

OBJECTIVE: Use of recombinant human bone morphogenetic protein-2 (rhBMP-2) is becoming a common clinical approach to enhance bone repair. There is little or no information in the literature on the dose of rhBMP-2 required for effective healing of critical-sized defects such as those associated with trauma. In this study, we used a segmental defect model to assess the dose response of rhBMP-2 using quantitative and qualitative endpoints. METHODS: Femoral defects in rats were replaced with absorbable collagen sponges carrying rhBMP-2 (0, 1, 5, 10 or 20 µg; N=5). At 4-weeks new bone formation was assessed using quantitative (radiography and microcomputed tomography) and qualitative (histology and backscattered-SEM) endpoints statistically compared. RESULTS: rhBMP-2 showed increased bridging in the gap. Quantitative evaluation presented a bi-phasic dose response curve. Histological assessment revealed that with rhBMP-2 the defect showed the presence of spongy bone with the trabeculae layered with active osteoblasts and osteoclasts. The density and compactness of the bone varied with the dose of rhBMP-2. CONCLUSIONS: Our findings revealed that all doses of rhBMP-2 result in new bone formation. However, there is an optimum dose of 12 µg of rhBMP-2 for bone repair in this model, above which and below which less stimulation of bone occurs.

Osteogenic differentiation of rat bone marrow stromal cells by various intensities of low-intensity pulsed ultrasound.

Bone growth and repair are under the control of biochemical and mechanical signals. Low-intensity pulsed ultrasound (LIPUS) stimulation at 30mW/cm(2) is an established, widely used and FDA approved intervention for accelerating bone healing in fractures and non-unions. Although this LIPUS signal accelerates mineralization and bone regeneration, the actual intensity experienced by the cells at the target site might be lower, due to the possible attenuation caused by the overlying soft tissue. The aim of this study was to investigate whether LIPUS intensities below 30mW/cm(2) are able to provoke phenotypic responses in bone cells. Rat bone marrow stromal cells were cultured under defined conditions and the effect of 2, 15, 30mW/cm(2) and sham treatments were studied at early (cell activation), middle (differentiation into osteogenic cells) and late (biological mineralization) stages of osteogenic differentiation. We observed that not only 30mW/cm(2) but also 2 and 15mW/cm(2), modulated ERK1/2 and p38 intracellular signaling pathways as compared to the sham treatment. After 5 days with daily treatments of 2, 15 and 30mW/cm(2), alkaline phosphatase activity, an early indicator of osteoblast differentiation, increased by 79%, 147% and 209%, respectively, compared to sham, indicating that various intensities of LIPUS were able to initiate osteogenic differentiation. While all LIPUS treatments showed higher mineralization, interestingly, the highest increase of 225% was observed in cells treated with 2mW/cm(2). As the intensity increased to 15 and 30mW/cm(2), the increase in the level of mineralization dropped to 120% and 82%. Our data show that LIPUS intensities lower than the current clinical standard have a positive effect on osteogenic differentiation of rat bone marrow stromal cells. Although Exogen™ at 30mW/cm(2) continues to be effective and should be used as a clinical therapy for fracture healing, if confirmed in vivo, the increased mineralization at lower intensities might be the first step towards redefining the most effective LIPUS intensity for clinical use.