Abstract
Guided bone regeneration (GBR) often utilizes a combination of autologous bone grafts, deproteinized bovine bone mineral (DBBM), and collagen membranes. DBBM and collagen membranes pre-coated with bone-conditioned medium (BCM) extracted from locally harvested autologous bone chips have shown great regenerative potential in GBR. However, the underlying molecular mechanism remains largely unknown. Here, we investigated the composition of BCM and its activity on the osteogenic potential of mesenchymal stromal cells. We detected a fast and significant (P < 0.001) release of transforming growth factor-β1 (TGF-β1) from autologous bone within 10 min versus a delayed bone morphogenetic protein-2 (BMP-2) release from 40 min onwards. BCMs harvested within short time periods (10, 20, or 40 min), corresponding to the time of a typical surgical procedure, significantly increased the proliferative activity and collagen matrix production of BCM-treated cells. Long-term (1, 3, or 6 days)-extracted BCMs promoted the later stages of osteoblast differentiation and maturation. Short-term-extracted BCMs, in which TGF-β1 but no BMP-2 was detected, reduced the expression of the late differentiation marker osteocalcin. However, when both growth factors were present simultaneously in the BCM, no inhibitory effects on osteoblast differentiation were observed, suggesting a synergistic TGF-β1/BMP-2 activity. Consequently, in cells that were co-stimulated with recombinant TGF-β1 and BMP-2, we showed a significant stimulatory and dose-dependent effect of TGF-β1 on BMP-2-induced osteoblast differentiation due to prolonged BMP signaling and reduced expression of the BMP-2 antagonist noggin. Altogether, our data provide new insights into the molecular mechanisms underlying the favorable outcome from GBR procedures using BCM, derived from autologous bone grafts.
Highlights
Despite the increasing number of new bone-grafting substitutes, autografts remain the gold standard for bone augmentation and reconstruction in oral, maxillofacial and orthopedic surgery due to their excellent and cost-effective combination of biological and mechanical properties.[1,2,3] Autologous bone is the only clinically available bone graft source that contains viable osteogenic precursor cells, releases growth factors capable of inducing new bone formation, and provides a scaffold for the ingrowth of new blood vessels and the migration of osteoprogenitor cells.[4]
Our data demonstrated a rapid release of transforming growth factor-β1 (TGF-β1) from autologous bone chips within 10 min versus a delayed bone morphogenetic protein-2 (BMP-2) release from 40 min onwards, suggesting that transforming growth factor (TGF)-β1 and Bone morphogenic protein (BMP)-2 may act both simultaneously and in a sequential manner to promote ST2 cell differentiation towards the osteoblast phenotype
We found that bone-conditioned medium (BCM) extracted within longer time periods (1, 3, and 6 days), corresponding to the early days after the augmentation procedure, act by promoting the later stages of osteoblast differentiation including matrix mineralization
Summary
Despite the increasing number of new bone-grafting substitutes, autografts remain the gold standard for bone augmentation and reconstruction in oral, maxillofacial and orthopedic surgery due to their excellent and cost-effective combination of biological and mechanical properties.[1,2,3] Autologous bone is the only clinically available bone graft source that contains viable osteogenic precursor cells (osteogenicity), releases growth factors capable of inducing new bone formation (osteoinduction), and provides a scaffold for the ingrowth of new blood vessels and the migration of osteoprogenitor cells (osteoconduction).[4]. The harvesting technique significantly influences the survival of bone cells contained within the autograft,[9] and subsequently alters the release of osteoinductive growth factors.[10] a 24-hour extraction of untreated bone chips with cell culture medium had the potential to affect a variety of cell types implicated in graft consolidation.[11,12] This so-called bone-conditioned medium (BCM) induces osteoclastogenesis in bone marrow cultures[13,14], and improves oral fibroblast cell activity through transforming growth factor (TGF)-β1 signaling.[15,16,17] collagen membranes rapidly adsorb the TGF-β1 activity contained in BCM, provoking changes in the gene expression pattern of oral fibroblasts grown on the membranes.[18] pre-coating DBBM and collagen membranes with biologically active BCM that is extracted from locally harvested autologous bone chips during the surgical procedure has great clinical potential. The goal of the present study is to analyze the TGF-β1 and BMP-2 protein release from autologous bone into BCM that is harvested for short periods (minutes)
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