Biofunctionalization of Collagen Barrier Membranes with Bone-Conditioned Medium, as a Natural Source of Growth Factors, Enhances Osteoblastic Cell Behavior.

Abstract

A key principle of guided bone regeneration (GBR) is the use of a barrier membrane to prevent cells from non-osteogenic tissues from interfering with bone regeneration in patients with hard tissue deficiencies. The aim of the study was to investigate whether the osteoinductive properties of bone-conditioned medium (BCM) obtained from cortical bone chips harvested at the surgical site can be transferred to a native bilayer collagen membrane (nbCM). BCM extracted within 20 or 40 min, which corresponds to a typical implant surgical procedure, and BCM extracted within 24 h, which corresponds to BCM released from the autologous bone chips in situ, contained significant and comparable amounts of TGF-β1, IGF-1, FGF-2, VEGF-A, and IL-11. Significant (p < 0.001) quantities of BMP-2 were only detected in the 24-h BCM preparation. The bioactive substances contained in the BCM were adsorbed to the nbCMs with almost 100% efficiency. A fast but sequential release of all investigated proteins occurred within 6-72 h, reflecting their stepwise involvement in the natural regeneration process. BCM-coated nbCM significantly (p < 0.05) increased the migratory, adhesive, and proliferative capacity of primary human bone-derived cells (hBC), primary human periodontal ligament cells (hPDLC), and an osteosarcoma-derived osteoblastic cell line (MG-63) compared to cells cultured on BCM-free nbCM. The high proliferative rates of MG-63 cells cultured on BCM-free nbCM were not further potentiated by BCM, indicating that BCM-coated nbCM has no detrimental effects on cancer cell growth. BCM-coated nbCM caused significant (p < 0.05) induction of early osteogenic marker gene expression and alkaline phosphatase activity, suggesting an important role of BCM-functionalized nbCM in the initiation of osteogenesis. The 24-h BCM loaded on the nbCM was the only BCM preparation that caused significant induction of late osteogenic marker gene expression. Altogether, our data define the pre-activation of collagen membranes with short-term-extracted BCM as a potential superior modality for treating hard tissue deficiencies via GBR.