Bone-conditioned medium contributes to initiation and progression of osteogenesis by exhibiting synergistic TGF-β1/BMP-2 activity

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.

Activity of two hyaluronan preparations on primary human oral fibroblasts

“…Proliferation rates of HA‐treated HPF or HGF cells were determined using a 5‐bromo‐20‐deoxyuridine (BrdU) incorporation assay (Roche, Basel, Switzerland) as described . After 24 hour of starvation, cells were plated in triplicate at 2 × 10 3 cells/well on 96‐well plates coated with HA at a final concentration of 4 mg/mL in 0.3%FCS/DMEM.…”

Role of hyaluronan in regulating self-renewal and osteogenic differentiation of mesenchymal stromal cells and pre-osteoblasts

“…Total RNA from HA-treated ST2 and MC3T3-E1 cells was isolated using the RNeasy Mini Kit (Qiagen, Basel, Switzerland). RNA, quantified on a NanoDrop 2000c (Thermo Fisher Scientific) instrument, was reverse transcribed, and relative transcripts for the osteogenic differentiation marker genes Col1a1, Col1a2, Spp1, Runx2, Bglap2, Ibsp, and Alpl, normalized to Gapdh, were measured using qRT-PCR as previously described [32]. Additionally, relative transcripts for Bmp2, Bmp4, Bmp7, Tgfb1, Fgf1, Sox2, Yap1, Bmi1, Ctgf, Ccnd1, Dkk1, Apc, and Gsk3b genes, normalized to Gapdh, were measured using FastStart Universal SYBR Green Master ROX (Roche) and the primer sequences listed in Electronic Supplementary Material, Table S1.…”

Effects of hydrogen peroxide on biological characteristics and osteoinductivity of decellularized and demineralized bone matrices

“…35 Moreover, TGF-β1 and BMP-2 could exhibit a strong synergistic activity for promoting the bone formation and ectopic calcification. 36 In this study, both of the two materials displayed the negative staining of VEGF and TGF-β1. It is indicated that the difference of osteoinductive capability of two materials may attribute to the residue of BMP-2.…”

“…A coupled process that caused by the coordination of angiogenesis and osteogenesis could lead to the formation of new bone and HO . Moreover, TGF‐β1 and BMP‐2 could exhibit a strong synergistic activity for promoting the bone formation and ectopic calcification . In this study, both of the two materials displayed the negative staining of VEGF and TGF‐β1.…”