Participation of TNF-α in Inhibitory Effects of Adipocytes on Osteoblast Differentiation.

Mesenchymal stem cells from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) are attractive tools for cell-based therapies to repair bone tissue. In this study, we investigated the osteogenic and adipogenic potential of BM-MSCs and AT-MSCs as well as the effect of crosstalk between osteoblasts and adipocytes on cell phenotype expression. Rat BM-MSCs and AT-MSCs were cultured either in growth, osteogenic, or adipogenic medium to evaluate osteoblast and adipocyte differentiation. Additionally, osteoblasts and adipocytes were indirectly co-cultured to investigate the effect of adipocytes on osteoblast differentiation and vice versa. BM-MSCs and AT-MSCs exhibit osteogenic and adipogenic potential under non-differentiation-inducing conditions. When exposed to osteogenic medium, BM-MSCs exhibited higher expression of bone markers compared with AT-MSCs. Conversely, under adipogenic conditions, AT-MSCs displayed higher expression of adipose tissue markers compared with BM-MSCs. The presence of adipocytes as indirect co-culture repressed the expression of the osteoblast phenotype, whereas osteoblasts did not exert remarkable effect on adipocytes. The inhibitory effect of adipocytes on osteoblasts was due to the release of tumor necrosis factor alpha (TNF-α) in culture medium by adipocytes. Indeed, the addition of exogenous TNF-α in culture medium repressed the differentiation of BM-MSCs into osteoblasts mimicking the indirect co-culture effect. In conclusion, our study showed that BM-MSCs are more osteogenic while AT-MSCs are more adipogenic. Additionally, we demonstrated the key role of TNF-α secreted by adipocytes on the inhibition of osteoblast differentiation. Thus, we postulate that the higher osteogenic potential of BM-MSCs makes them the first choice for inducing bone repair in cell-based therapies.

Nanotopography directs mesenchymal stem cells to osteoblast lineage through regulation of microRNA-SMAD-BMP-2 circuit.

The aim of this study was to investigate if chemically produced nanotopography on titanium (Ti) surface induces osteoblast differentiation of cultured human bone marrow mesenchymal stem cells (hMSCs) by regulating the expression of microRNAs (miRs). It was demonstrated that Ti with nanotopography induces osteoblast differentiation of hMSCs as evidenced by upregulation of osteoblast specific markers compared with untreated (control) Ti at day 4. At this time-point, miR-sequencing analysis revealed that 20 miRs were upregulated (>twofold) while 20 miRs were downregulated (>threefold) in hMSCs grown on Ti with nanotopography compared with control Ti. Three miRs, namely miR-4448, -4708, and -4773, which were significantly downregulated (>fivefold) by Ti with nanotopography affect osteoblast differentiation of hMSCs. These miRs directly target SMAD1 and SMAD4, both key transducers of the bone morphogenetic protein 2 (BMP-2) osteogenic signal, which were upregulated by Ti with nanotopography. Overexpression of miR-4448, -4708, and 4773 in MC3T3-E1 pre-osteoblasts noticeably inhibited gene and protein expression of SMAD1 and SMAD4 and therefore repressed the gene expression of key bone markers. Additionally, it was observed that the treatment with BMP-2 displayed a higher osteogenic effect on MC3T3-E1 cells grown on Ti with nanotopography compared with control Ti, suggesting that the BMP-2 signaling pathway was more effective on this surface. Taken together, these results indicate that a complex regulatory network involving a miR-SMAD-BMP-2 circuit governs the osteoblast differentiation induced by Ti with nanotopography. J. Cell. Physiol. 229: 1690-1696, 2014. © 2014 Wiley Periodicals, Inc.

Poly(vinylidene-trifluoroethylene)/barium titanate composite for in vivo support of bone formation.

In this study, we evaluated the effect of poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane on in vivo bone formation. Rat calvarial bone defects were implanted with P(VDF-TrFE)/BT and polytetrafluoroethylene (PTFE) membranes, and at 4 and 8 weeks, histomorphometric and gene expression analyses were performed. A higher amount of bone formation was noticed on P(VDF-TrFE)/BT compared with PTFE. The gene expression of RUNX2, bone sialoprotein, osteocalcin, receptor activator of nuclear factor-kappa B ligand, and osteoprotegerin indicates that P(VDF-TrFE)/BT favored the osteoblast differentiation compared with PTFE. These results evidenced the benefits of using P(VDF-TrFE)/BT to promote new bone formation, which may represent a promising alternative to be employed in guided bone regeneration.

Autogenous bone combined with anorganic bovine bone for maxillary sinus augmentation: analysis of the osteogenic potential of cells derived from the donor and the grafted sites.

OBJECTIVES: This study aimed to comparatively evaluate the in vitro osteogenic potential of cells obtained from the mandibular ramus (MR, autogenous bone donor site) and from the maxillary sinus (MS) bone grafted with a mixture of anorganic bovine bone (ABB) and MR prior to titanium implant placement (MS, grafted implant site). MATERIAL AND METHODS: Cells were obtained from three patients subjected to MS floor augmentation with a 1 : 1 mixture of ABB (GenOx Inorg(®) ) and MR. At the time of the sinus lift procedure and after 8 months, prior to implant placement, bone fragments were taken from MR and MS, respectively, and subjected to trypsin-collagenase digestion for primary cell culturing. Subcultured cells were grown under osteogenic condition for up to 21 days and assayed for proliferation/viability, osteoblast marker mRNA levels, alkaline phosphatase (ALP) activity and calcium content/Alizarin red staining. ALP activity was also determined in primary explant cultures exposed to GenOx Inorg(®) (1 : 1 with MR) for 7 days. Data were compared using either the Mann-Whitney U-test or the Kruskal-Wallis test. RESULTS: MS cultures exhibited a significantly lower osteogenic potential compared with MR cultures, with a progressive increase in cell proliferation together with a decrease in osteoblast markers, reduced ALP activity and calcium content. Exposure of MR-derived primary cultures to GenOx Inorg(®) inhibited ALP activity. CONCLUSION: These results suggest that the use of GenOx Inorg(®) in combination with MR fragments for MS floor augmentation inhibits the osteoblast cell differentiation at the implant site in the long term.

Progression of osteogenic cell cultures grown on microtopographic titanium coated with calcium phosphate and functionalized with a type I collagen-derived peptide.

BACKGROUND: The functionalization of metallic surfaces aims at promoting the cellular response at the biomaterial-tissue interface. This study investigates the effects of the functionalization of titanium (Ti) microtopography with a calcium phosphate (CaP) coating with and without peptide 15 (P-15), a synthetic peptide analog of the cell-binding domain of collagen I, on the in vitro progression of osteogenic cells. METHODS: Sandblasting and acid etching (SBAE; control) Ti microtopography was coated with CaP, enabling the loading of two concentrations of P-15: 20 or 200 µg/mL. A machined Ti was also examined. Rat calvarial osteogenic cells were cultured on Ti disks with the surfaces mentioned above for periods up to 21 days (n = 180 per group). RESULTS: CaP coating exhibited a submicron-scale needle-shaped structure. Although all surfaces were hydrophobic at time zero, functionalization increased hydrophilicity at equilibrium. Microtopographies exhibited a lower proportion of well-spread cells at 4 hours of culture and cells with long cytoplasmic extensions at day 3; modified SBAE supported higher cell viability and larger extracellular osteopontin (OPN) accumulation. For SBAE and modified SBAE, real-time polymerase chain reaction showed the following results: 1) lower levels for runt-related transcription factor 2 at 7 days and for bone sialoprotein at days 7 and 10 as well as higher OPN levels at days 7 and 10 compared to machined Ti; and 2) higher alkaline phosphatase levels at day 10 compared to day 7. At 14 and 21 days, modified SBAE supported higher proportions of red-dye-stained areas (calcium content). CONCLUSION: Addition of a CaP coating to SBAE Ti by itself may affect key events of in vitro osteogenesis, ultimately resulting in enhanced matrix mineralization; additional P-15 functionalization has only limited synergistic effects.