Metallic Scaffold with Micron-Scale Geometrical Cues Promotes Osteogenesis and Angiogenesis via the ROCK/Myosin/YAP Pathway.

The advent of precision manufacturing has enabled the creation of pores in metallic scaffolds with feature size in the range of single microns. In orthopedic implants, pore geometries at the micron scale could regulate bone formation by stimulating osteogenic differentiation and the coupling of osteogenesis and angiogenesis. However, the biological response to pore geometry at the cellular level is not clear. As cells are sensitive to curvature of the pore boundary, this study aimed to investigate osteogenesis in high- vs low-curvature environments by utilizing computer numerical control laser cutting to generate triangular and circular precision manufactured micropores (PMpores). We fabricated PMpores on 100 µm-thick stainless-steel discs. Triangular PMpores had a 30° vertex angle and a 300 µm base, and circular PMpores had a 300 µm diameter. We found triangular PMpores significantly enhanced the elastic modulus, proliferation, migration, and osteogenic differentiation of MC3T3-E1 preosteoblasts through Yes-associated protein (YAP) nuclear translocation. Inhibition of Rho-associated kinase (ROCK) and Myosin II abolished YAP translocation in all pore types and controls. Inhibition of YAP transcriptional activity reduced the proliferation, pore closure, collagen secretion, alkaline phosphatase (ALP), and Alizarin Red staining in MC3T3-E1 cultures. In C166 vascular endothelial cells, PMpores increased the VEGFA mRNA expression even without an angiogenic differentiation medium and induced tubule formation and maintenance. In terms of osteogenesis-angiogenesis coupling, a conditioned medium from MC3T3-E1 cells in PMpores promoted the expression of angiogenic genes in C166 cells. A coculture with MC3T3-E1 induced tubule formation and maintenance in C166 cells and tubule alignment along the edges of pores. Together, curvature cues in micropores are important stimuli to regulate osteogenic differentiation and osteogenesis-angiogenesis coupling. This study uncovered key mechanotransduction signaling components activated by curvature differences in a metallic scaffold and contributed to the understanding of the interaction between orthopedic implants and cells.

Mechanical loading attenuated negative effects of nucleotide analogue reverse-transcriptase inhibitor TDF on bone repair via Wnt/ß-catenin pathway.

The nucleotide analog reverse-transcriptase inhibitor, tenofovir disoproxil fumarate (TDF), is widely used to treat hepatitis B virus (HBV) and human immunodeficiency virus infection (HIV). However, long-term TDF usage is associated with an increased incidence of bone loss, osteoporosis, fractures, and other adverse reactions. We investigated the effect of chronic TDF use on bone homeostasis and defect repair in mice. In vitro, TDF inhibited osteogenic differentiation and mineralization in MC3T3-E1 cells. In vivo, 8-week-old C57BL/6 female mice were treated with TDF for 38 days to simulate chronic medication. Four-point bending test and µCT showed reduced bone biomechanical properties and microarchitecture in long bones. To investigate the effects of TDF on bone defect repair, we utilized a bilateral tibial monocortical defect model. µCT showed that TDF reduced new bone mineral tissue and bone mineral density (BMD) in the defect. To verify whether mechanical stimulation may be a useful treatment to counteract the negative bone effects of TDF, controlled dynamic mechanical loading was applied to the whole tibia during the matrix deposition phase on post-surgery days (PSDs) 5 to 8. Second harmonic generation (SHG) of collagen fibers and µCT showed that the reduction of new bone volume and bone mineral density caused by TDF was reversed by mechanical loading in the defect. Immunofluorescent deep tissue imaging showed that chronic TDF treatment reduced the number of osteogenic cells and the volume of new vessels. In addition, chronic TDF treatment inhibited the expressions of periostin and ß-catenin, but increased the expression of sclerostin. Both negative effects were reversed by mechanical loading. Our study provides strong evidence that chronic use of TDF exerts direct and inhibitory impacts on bone repair, but appropriate mechanical loading could reverse these adverse effects.

MiR-27b regulates podocyte survival through targeting adenosine receptor 2B in podocytes from non-human primate.

MicroRNAs are a group of small non-coding RNAs that play key roles in almost every aspect of mammalian cell. In kidney, microRNAs are required for maintaining normal function of renal cells, disruption of which contributes to pathogenesis of renal diseases. In this study, we investigated the potential role of miRNAs as key regulators of podocyte survival by using a primary cell culture model from non-human primates (NHPs). Through microRNA profile comparison in glomeruli from mouse, rat and NHP, miR-27b was found to be among a list of glomeruli-enriched miRNA conserved across species. In NHP primary podocyte culture, significant downregulation of miR-27b was observed during treatment of puromycin aminonucleoside (PAN), a classic nephrotoxin. Overexpression of miR-27b enhanced PAN-induced apoptosis and cytoskeleton destruction in podocytes while its inhibition had a protective effect. Target identification analysis identified Adora2b as a potential direct target of miR-27b. Ectopic expression of miR-27b suppressed both Adora2b mRNA and protein expression, whereas inhibition of miR-27b increased the transcript and protein expression levels of Adora2B. Dual luciferase assay further confirmed Adora2b as a direct target of miR-27b. Furthermore, knockdown of Adora2b by siRNAs enhanced PAN-induced apoptosis, similar to the phenotypes we had observed with miR-27b overexpression. In addition, stimulating the adenosine signaling by an Adora2b agonist, NECA, improved podocyte survival upon PAN treatment. Taken together, our data identified a novel role of miR-27b-adora2b axis in primary podocyte survival upon injury and suggested a critical role of adenosine signaling pathway in podocyte protection.