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Application of messenger RNA (mRNA) for bone regeneration is a promising alternative to DNA, recombinant proteins and peptides. However, exogenous in vitro transcribed mRNA (IVT mRNA) triggers innate immune response resulting in mRNA degradation and translation inhibition. Inspired by the ability of viral immune evasion proteins to inhibit host cell responses against viral RNA, we applied non-structural protein-1 (NS1) from Influenza A virus (A/Texas/36/1991) as an IVT mRNA enhancer. We evidenced a dose-dependent blocking of RNA sensors by NS1 expression. The co-delivery of NS1 mRNA with mRNA of reporter genes significantly increased the translation efficiency. Interestingly, unlike the use of nucleosides modification, NS1-mediated mRNA translation enhancement does not dependent to cell type. Dual delivery of NS1 mRNA and BMP-2 mRNA to murine pluripotent stem cells (C3H10T1/2), promoted osteogenic differentiation evidenced by enhanced expression of osteoblastic markers (e.g. alkaline phosphatase, type I collagen, osteopontin, and osteocalcin), and extracellular mineralization. Overall, these results support the adjuvant potentiality of NS1 for mRNA-based regenerative therapies. mRNA therapy has the potential to improve the efficiency of nucleic acid based regenerative medicine. Up to now, the incorporation of expensive modified nucleotides is a common way to avoid IVT mRNA-induced detrimental immunogenicity. We here introduce co-delivery of Influenza virus immune evasion protein-NS1 coding mRNA as a strategy to suppress RNA sensors for maximizing IVT mRNA expression. An increased osteogenic commitment of pluripotent stem cells was observed after BMP2 mRNA and NS1 mRNA delivery. This study revealed how applying non-modified mRNA with NS1 could be a promising alternative as a therapeutic in bone regeneration. Image, graphical abstract [ABSTRACT FROM AUTHOR]

As a growth repair factor, mechano-growth factor (MGF) and its 24 amino acid peptide analogs corresponding to the unique C-terminal E-domain (MGF-Q24E) positively regulate bone regeneration. MGF-Q24E was introduced into the poly(D, L-lactic acid) (PDLLA) to improve bone regeneration in our previous study. MGF-Ct24E-grafted PDLLA was chemically characterized and showed potential as a biofunctional polymer. In this study, we evaluated the cytocompatibility of MGF-Ct24E chemically grafted and physically blended with maleic anhydride modified PDLLA, relative to maleic anhydride modified PDLLA (MPLA) as the control. The surface properties of these three polymer films were characterized with scanning electron microscopy and X-ray photoelectron spectroscopy. Rat calvarial osteoblasts were seeded on the three polymer films, and cell adhesion, spreading, and proliferation were assessed with an inverted microscope, laser scanning confocal microscope, and a cell counting kit-8, respectively. The alkaline phosphatase activity and extracellular calcium production were exploited to characterize the differentiation and mineralization of rat calvarial osteoblasts on various polymer films. The results revealed that compared with MPLA, MGF-024E-MPLA, and MGF-Q24E/MPLA blends promoted adhesion, spreading, proliferation, and the later differentiation and mineralization process of rat calvarial osteoblasts. In addition, the positive effect of MGF-Q24E-MPLA on rat calvarial osteoblasts was maintained longer than the MGF-Q24E/MPLA blends. In conclusion, MGF-Q24E-MPLA synthesized chemically might be a promising biomaterial for bone tissue engineering. [ABSTRACT FROM AUTHOR]

Nucleic acid-based therapy has shown great promise in accelerating bone regeneration as well as other diseases. Nucleic acids used in gene therapy mainly are either plasmid DNA (pDNA) or RNAs. Although pDNA therapy has been extensively studied for decades with encouraging preclinical and clinical results, side effects, and low efficiency associated with nuclear trafficking are hard to bypass. Unlike pDNA, RNAs (mRNA, siRNA, miRNA) exert their function in the cytoplasm, thereby being more efficient in hard-to-transfect cells such as primary osteoblasts. RNA interference-based gene silencing represents a negative regulation which knockdown the expression of antagonists that impair osteogenesis process. In contrary, mRNA therapy for osteogenesis represents a positive regulation which delivers mRNA encoding growth factors to accelerate bone regeneration. This review presents a comprehensive summary of the mRNA and siRNA-based therapies and the targets for bone regeneration in case of bone defect and osteoporosis. [ABSTRACT FROM AUTHOR]