Osteoporosis and osteoblasts cocultured with adipocytes inhibit osteoblast differentiation by downregulating histone acetylation.

Osteoporosis is characterized by decreased bone mass and adipocyte accumulation within the bone marrow that inhibits osteoblast maturation, leading to a high risk of fractures. Thus, we hypothesized that osteoblasts, besides being negatively affected by interacting with adipocytes, reduce the differentiation of neighboring osteoblasts through the same mechanisms that affect osteoblasts under osteoporotic conditions. We investigated the effect of osteoporosis on osteoblast differentiation and the effect of the conditioned medium of osteoblasts cocultured with adipocytes on the differentiation of other osteoblasts. Osteoporosis was induced by orchiectomy in rats and bone marrow mesenchymal stromal cells (MSCs) were differentiated into osteoblasts. Also, the bone marrow and adipose tissue MSCs were obtained from healthy rats and differentiated into osteoblasts and adipocytes, respectively. Messenger RNA expression, in situ alkaline phosphatase activity, and mineralization confirmed the inhibitory effect of osteoporosis on osteoblast differentiation. This harmful effect was mimicked by the in vitro model using the conditioned medium and it was demonstrated that osteoblasts keep the memory of the negative impact of interacting with adipocytes, revealing an unknown mechanism relevant to the osteoporotic bone loss. Finally, we showed the involvement of acetyl-histone 3 (AcH3) in bone homeostasis as its reduction induced by osteoporosis and conditioned medium impaired osteoblast differentiation. The AcH3 involvement was proved by treating osteoblasts with Trichostatin A that recovered the AcH3 expression and osteoblast differentiation capacity in both situations. Together, our findings indicated that AcH3 might be a target for future studies focused on epigenetic-based therapies to treat bone diseases.

Mesenchymal Stromal Cells Derived from Bone Marrow and Adipose Tissue: Isolation, Culture, Characterization and Differentiation.

Since their discovery, mesenchymal stromal cells (MSCs) have received a lot of attention, mainly due to their self-renewal potential and multilineage differentiation capacity. For these reasons, MSCs are a useful tool in cell biology and regenerative medicine. In this article, we describe protocols to isolate MSCs from bone marrow (BM-MSCs) and adipose tissues (AT-MSCs), and methods to culture, characterize, and differentiate MSCs into osteoblasts, adipocytes, and chondrocytes. After the harvesting of cells from bone marrow by flushing the femoral diaphysis and enzymatic digestion of abdominal and inguinal adipose tissues, MSCs are selected by their adherence to the plastic tissue culture dish. Within 7 days, MSCs reach 70% confluence and are ready to be used in subsequent experiments. The protocols described here are easy to perform, cost-efficient, require minimal time, and yield a cell population rich in MSCs.

The Wnt/ß-catenin signaling pathway is regulated by titanium with nanotopography to induce osteoblast differentiation.

Wnt/ß-catenin signal transduction is involved in the homeostatic control of bone mass. It is well established that a titanium surface with nanotopography (Ti-Nano) favors osteoblast differentiation by modulating different signaling pathways. However, few studies have investigated the participation of the Wnt/ß-catenin pathway in the osteogenic effect of nanoscale topographies. In this study, we aimed to determine whether the Wnt/ß-catenin signaling pathway is involved in the elevated osteogenic potential of Ti-Nano. MC3T3-E1 cells were cultured on Ti-Nano and machined Ti (Ti-Control) for evaluation of the expression of Wnt/ß-catenin signaling pathway-related genes. Based on the results to real-time PCR, the Wnt receptor Fzd4 was selected and silenced by CRISPRi. The resulting cells were cultured on both Ti surfaces, and several events involved in osteoblast differentiation were evaluated. The results revealed that Fzd4 gene silencing, corresponding to negative modulation of Wnt/ß-catenin, inhibits expression of the osteoblast phenotype. It is worthy of note that this inhibitory effect on osteoblast differentiation was more pronounced in cells grown on Ti-Nano compared with those grown on Ti-Control. By disrupting Fzd4 gene expression, we have shown that the elevated osteogenic potential of Ti-Nano is due to activation of the Wnt/ß-catenin signaling pathway, which reveals a new mechanism to explain osteoblast differentiation induced by nanotopography. Such an understanding of the intracellular machinery involved in surface guiding of osteoblast fate may contribute to the development of smart biomaterials to modulate the process of implant osseointegration.