Bone tissue engineering builds on the understanding of bone structure and aims to develop platforms that effectively regenerate bone defects. Bone derives its unique mechanical properties from an architectural design extending over nanoscale to macroscopic dimensions. A wide range of structural proteins are found in the ECM of bone, including serum-derived proteins, proteoglycans, glycosylated proteins, Gla-containing proteins, and small integrin-binding proteins. Mentioned proteins bind to strands of collagen fibrils (collagen type I, collagen type X, collagen type III, and collagen type V) [89], dominating at the nanometer level with a diameter scale between 35 to 60 nm. The hydroxyapatite crystals are embedded with collagen molecules and increase the rigidity of the bone. Besides, a wide range of proteins is engaged in the regulation of ECM formation [90]. Bone ECM also contains a number of biologically active molecules. For example, BMP2 [12], IGF1 [91], and TGF-β1 play crucial roles in osteoblast-osteoclast development and bone formation. RUNX2, a master transcriptional regulator of osteoblast differentiation [92], responds to numerous extracellular signals, including BMP and TGF-β1, and binds specific DNA sequences with several co-factors to regulate downstream target genes osteoblasts [93]. One such RUNX2 co-factor is SP7, and Sp subfamily member of the Sp/XKLF transcription factor family, leading to the upregulation of type I collagen [94]. SP7 also acts downstream of RUNX2 to regulate osteoblast development [36], exerting its regulatory effects by binding to guanine-rich sequences in specific target genes [95]. Several osteoblast-associated genes/gene products, including RUNX2, SP7, ALPL, COL1A1, IBSP, and BGLAP, are used as markers to demonstrate osteoblastogenesis in a developmental stage-dependent manner [96]. ALPL is a membrane-bound glycosylated enzyme that acts as a phosphatase to release inorganic phosphate from other molecules, increasing the concentration of phosphate required for calcification [97]. As of today, miRNA mimic-NF assemblies with osteogenic activity have been described only in a handful of publications (PubMed) summarizing cell culture models and laboratory animal models. James et al. provided the first report of the potential utility of a miRNA mimic-NF assembly [98]. In this example, electrospun and cross-linked gelatin nanofibers containing miRNA-29a inhibitors were supplied to MC3T3-E1 cells and mouse MSCs. Of the various target candidates tested, Tgfb1 and Igf1 were upregulated by the miRNA-29a inhibitor, leading to increased Sparc in the MC3T3-E1 cells and collagen deposition in MSCs. These NFs (35–50 µm thick) were stable for at least 7 days in the culture medium, and the release of miRNA-29a inhibitor into the medium was found as early as 2 h, gradually increasing and being sustained up to 72 h. To promote the differentiation switch toward osteoblast and accelerate the angiogenesis, osteogenic miR-22 and angiogenic miR-126 were introduced into electrospun PLC NFs [11]. In spite of their almost equal fiber diameter, the NFs derived from miR-22 and miR-26 had less mechanical strength and hydrophobicity. Cell adhesion, proliferation, and osteogenic differentiation of human iPS cells cultured under osteogenic conditions were enhanced by PLC NFs containing miR-22 and miR-26 compared to NFs without these miRNAs. In these cultures, more than 50% of the incorporated miRNAs were released during the first 72 h. Additionally, the 3D nanofiber aerogels containing miR-26a mimic were developed for the regeneration of calvaria bone defects in a rat model. The results of implantations indicate that aerogel- miR-26a mimic promotes bone formation up to 55%, while the aerogel loaded with negative control of miRNA could encourage bone formation up to 20%, as shown in Figure 6. Table 2 represents miRNA mimic-related molecules incorporated into NFs scaffolds for bone regeneration [99].

Two reports have extended our understanding of the effects of miRNA-NFs assemblies on skeletal regeneration in vivo [100,101]. Poly (L-lactic acid) (PLLA)-graft-poly (hydroxyethyl methacrylate) was employed to prepare nanofibrous porous solid microspheres. These solid microspheres with an average diameter between 30 and 60 μM and approximately 15 μM in pore size supported proliferation and chondrogenic differentiation of rabbit MSCs. When rabbit MSCs precultured on solid microspheres carrying antagomir-199a with a hyperbranched polymer vector were implanted into the subcutis of nude mice, and needle puncture-induced lumbar disc degeneration of rabbits, increased chondrogenesis and intervertebral disc regeneration, respectively, were seen. These results were attributed to the antagomir targeting Hif1a, with the subsequent upregulation of Sox9 and other chondrocyte markers. The scaffold carrying hyperbranched polyplex-miR-10a mimic (known as a T regulatory cell [Treg] marker) complexes, in combination with the Treg recruitment factors IL2 and TGFβ (2 mg of microspheres containing 0.8 nmol miR-10a mimic, 1mg IL2, and 2 mg TGFβ protein), served as an injectable scaffold [101]. The nanofibrous scaffold was 60–90 nm in diameter, with multiple pores, and was designed to release IL2, TGFβ, and miR-10a mimic differentially for recruiting Tregs locally and promoting their differentiation effectively. miR-10a was expected to exert its effect on the PI3K-AKT-MTOR pathway by targeting IRS-1 and enhancing Treg differentiation. Since Tregs are a crucial regulator of the immune system in maintaining tolerance to self-antigens and suppressing the activities of other immune cells [102], nanofibrous microspheres carrying the active molecules as above were evaluated in a mouse model of periodontitis, a typical inflammatory disease that leads to gingival recession and alveolar bone defects [103]. Local injection of this assembly into the periodontal margin effectively inhibited the upregulation of gene expression of the primary cytokines of effector T cells in parallel with an increased ratio of Tregs/Th17 cells and concomitantly depressed osteoclastic bone resorption in the foci.

Furthermore, human iPSCs were transduced with miR-2861 mimic, and then the osteogenic differentiation of cells was investigated when cultured on electrospun PLGA nanofibers. Surprisingly, the results indicated that osteogenic functions including RUNX2, ALPL, BGLAP, and SPARK in iPSCs improved when the transfected cells were cultured on the NFs and compared to the tissue culture plate. All these indicate the impact of NFs on osteogenic function [104]. Adipose-derived mesenchymal stem cells culturing on miR-181a/b-1 mimic incorporated with PLGA NFs compared with the same cells cultured on the tissue culture plate. The results revealed that adipose-derived mesenchymal stem cells cultured on NFs containing miR-181a/b-1 mimic expressed significantly more Runx2 and Spark than cells cultured on a tissue culture plate [105].