The demand for new approaches for skeletal injury repair is based on the limitations of traditional therapeutics. Current research focuses on scaffolds or scaffolds containing bioactive molecules, which can provide suitable cues and stimuli for tissue formation. microRNAs are small non-coding RNAs that modulate protein expression by modifying mRNA translation and/or stability. Here, we develop gelatin nanofibers as an extracellular matrix-mimic delivery vehicle for miRNAs. Gelatin nanofibers were fabricated by the process of electrospinning and crosslinked using glutaraldehyde (GA) vapors. The crosslinked fiber matrices were then characterized in terms of degree of crosslinking, percentage swelling, tensile strength, miRNA release and cytocompatibility. The study revealed that as GA concentrations used for crosslinking increased, the number of free amino acids were reduced implying more crosslinking. Increased GA concentrations also reduced the percentage swelling as well as increased the mechanical properties of the matrices. miRNA released from nanofibers exhibited an initial burst release within the first 2 hours, followed by a continuous release for up to 96 hours. miR-29a inhibitors were used as a model miRNA to determine whether miRNAs could be delivered via gelatin nanofibers to enhance extracellular matrix production. The biological activity of the released miR-29a inhibitors was confirmed by the significant increase in osteonectin production by MC3T3-E1 pre-osteoblast cells cultured on miR29a inhibitor loaded gelatin nanofibers. miR-29a inhibitor loaded nanofibers induced TGFβ1 and IGF-1 gene expression after 24 hours in MC3T3-E1 pre-osteoblasts. Additionally, miR-29a inhibitors released from nanofibers significantly increased early osteogenic commitment, assessed by the reporter gene pOB Col 3.6 GFPcyan. The study also revealed enhanced collagen production in bone marrow stromal cells cultured on miR-29a inhibitor loaded gelatin nanofibers after 8 days. A subsequent study investigated the feasibility to develop an osteogenic nanofiber matrix by loading miR-29a mimic, an osteogenic miRNA, in gelatin nanofibers. Conversely, mObI cells and bone marrow stromal cells were seeded on miR-29a mimic gelatin nanofibers and compared to scramble controls, to evaluate their ability to induce osteogenesis. mObI cells seeded on miR-29a mimic loaded nanofibers demonstrated increased dose-dependent alkaline phosphatase activity, and enhanced mineralized matrix deposition. Additionally, bone marrow stromal cells exhibited accelerated osteogenesis evidenced by enhanced mineralization, increased early osteogenic reporter mCherry Osterix expression and upregulation of osteogenic gene expression (Runx2, Col1a1 and β-catenin). In summary, these studies demonstrate the feasibility of developing various miRNA inhibitor and mimic loaded gelatin nanofibers, with the ability to activate multiple signal transduction pathways based on the miRNA of choice, with an enormous potential to serve as a biomaterial that can control cell fate and function for desired therapeutics.
