Stimulation with bone morphogenetic protein-2 (BMP-2) enhances bone-tendon integration in vitro.

PURPOSE: Preclinical studies have reported that bone morphogenetic protein (BMP)-2 promotes bone-tendon healing following anterior cruciate ligament reconstruction. We examined the region-specific effects of BMP-2 on osteoblast and fibroblast differentiation in a highly standardized murine in vitro co-culture model of bone-tendon integration. MATERIALS AND METHODS: We used quantitative PCR to measure the dose- and time-dependent influence of BMP-2 on the expression of alkaline phosphatase, osteocalcin, collagen type 1 (alpha 1 chain), runt-related transcription factor 2, osteopontin, collagen type 1 (alpha 2 chain), collagen type 5 (alpha 1 chain), decorin, fibromodulin, mohawk homeobox, bone morphogenetic protein receptor, type 1A, bone morphogenetic protein receptor, type 2, and Noggin in the osteoblast, interface, and fibroblast regions of a co-culture model of the murine preosteoblast cell line MC3T3-E1 and the fibroblast cell line 3T6. RESULTS: Stimulation with BMP-2 resulted in a significant upregulation of alkaline phosphatase (p < 0.001), osteocalcin (p < 0.001), collagens (p < 0.001), runt-related transcription factor 2 (p < 0.05), and osteopontin (p < 0.001) expression in the osteoblast region. In the interface region, BMP-2 exposure led to dose- and time-dependent upregulation of alkaline phosphatase (p < 0.001), osteocalcin (p < 0.001), osteopontin (p < 0.001), runt-related transcription factor 2 (p < 0.001), and markers of extracellular matrix production (p < 0.001). Both BMP receptors showed a significant BMP-2-dependent upregulation at the interface region, and Noggin was downregulated at the osteoblast and interface region following BMP-2 exposure. CONCLUSIONS: Exposure to BMP-2 upregulated the expression of genes associated with bone-tendon integration in vitro, suggesting the stimulation of transdifferentiation processes at the interface and fibroblast regions as well as the induction of positive feedback mechanisms. Further studies will be needed to establish BMP-2 dose and treatment algorithms following tendon reinsertion and reconstruction.

Bone morphogenetic protein-7 enhances bone-tendon integration in a murine in vitro co-culture model.

PURPOSE: Bone-tendon healing following anterior cruciate ligament reconstruction is reportedly enhanced by bone morphogenetic protein (BMP)-7. To improve our understanding of the underlying biologic processes, we examined the effects of BMP-7 on region-specific gene expression in vitro. METHODS: A murine in vitro co-culture model simulating the osteoblast, interface and fibroblast regions was established. The dose- and time-dependent region-specific effects of BMP-7 exposure on gene expression of Alpl, Bglap, Col1a1, Runx2 and Spp1 were analysed by quantitative PCR. RESULTS: At the osteoblast region, BMP-7 significantly increased Alp, Bglap, Col1a1, and Runx2 expression, while Spp1 expression was suppressed. At the interface region, BMP-7 exposure resulted in a trend towards increased expression rates of Alpl and Col1a1, whereas Bglap (P < 0.001) and Runx2 (P < 0.01) were significantly upregulated without any detectable effect on Spp1 expression. At the fibroblast region, BMP-7 increased Alpl (P < 0.001), Bglap (P < 0.001) and Runx2 (P < 0.001) expression, but no significant effects were seen on Col1a1 or Spp1. Exposure to BMP-7 (100 ng/ml) had its most pronounced biologic impact on day ten. CONCLUSION: BMP-7 stimulation showed beneficial region-specific effects on bone-tendon healing in vitro, such as enhanced expression of parameters for ossification and fibroblast transdifferentiation, both key processes during successful graft integration.