MicroRNA-26a-modified adipose-derived stem cells incorporated with a porous hydroxyapatite scaffold improve the repair of bone defects

Abstract

Tissue-engineered bone substitutes are frequently used to repair bone defects. Adipose-derived stem cells (ASCs) are a promising source of cells for repairing bone tissue, however, insufficient osteogenic potency remains the main obstacle for their application. The present study aimed to enhance the osteogenic potency of ASCs by transfection of microRNA (miR)-26a, a novel osteogenic and angiogenic promoting miRNA. An inverted fluorescence microscope was used to observe transfection efficiency, while a scanning electron microscope was used to detect morphological alterations. Cell proliferation was monitored continuously for 7 days using a Cell Counting kit-8 assay. Osteogenic differentiation was determined by reverse transcription quantitative polymerase chain reaction, alkaline phosphatase (ALP) staining, collagen secretion and extracellular matrix (ECM) mineralization. ASCs were incorporated with a porous hydroxyapatite (HA) scaffold to create a novel tissue-engineered bone substitute and inserted into the critical tibia defect of rats. New bone formation was evaluated by hematoxylin and eosin and Masson's trichrome staining. The results demonstrated that miR-26a was successfully delivered into the cytoplasm, while the morphology and proliferation of ASCs were not significantly altered. Osteogenic-associated genes were markedly upregulated and ALP production, collagen secretion and ECM mineralization were all increased following transfection of miR-26a. Histological evaluation demonstrated that the modified cells accompanied with a porous HA scaffold markedly promoted new bone formation within the defective area. In conclusion, miR-26a transfection significantly improved the osteogenic potency of ASCs suggesting that modified ASCs incorporated with a HA scaffold may be used as a potential bone substitute.

Scaffold‐Based microRNA Therapies in Regenerative Medicine and Cancer

“…Cationic lipids are among the most commonly utilized nonviral vectors for gene therapy applications. The use of commercial cationic lipids in biomaterial-based approaches to deliver miRNA therapeutics is exemplified by the widespread use of Lipofectamine 2000 [53][54][55][56] along with Lipofectamine RNAiMAX [57] and SiPORT NeoFx. [58] Generally, cationic lipids complex with the miRNA cassettes through electrostatic interaction, and the overall positive charge of the complex dictates the cellular uptake, although neutrally charged liposomes can also encapsulate the cargo within their core.…”

“…[107] Many distinct scaffold types have been tuned to serve the purpose of miRNA delivery, including several hydrogels, [58,92,106,[108][109][110][111][112][113][114] electrospun fibers, [63,[115][116][117][118] and more prolifically porous or spongy scaffolds. [46,47,50,54,55,96,112,[119][120][121][122][123][124][125][126][127][128][129][130] Before reviewing the details of their applications, summarized in Table 2 and described further in the next section, we will discuss the key characteristics making these materials amenable to miRNA delivery. First very general qualities must be fulfilled: biocompatibility, bioresorbability and ease of fabrication.…”

“…[140] β-TCP scaffolds were used by Deng et al in preclinical studies of lentiviral miR-31 inhibition, [45,120] and HA/TCP scaffolds were utilized, in combination with Lipofectamine 2000, by Eskildsen et al to deliver miR-138 inhibitors in vitro, [54] and more recently by Wang and co-workers to deliver miR-26a mimics in vivo. [55] Similarly, nHA/PCL and carboxy-methyl-cellulose/zinc-doped nHA composite scaffolds have been combined with Lipofectamine 2000 and X-treme Gene transfection reagent respectively. [56,125] The common denominator of this body of work is the minimal intervention of the scaffold in the delivery of the miRNA, as the cells are transfected ex situ prior to scaffold seeding.…”

“…[58,63,146] Li et al [58] documented the first report in 2013 using BM-MSCs, HyStem-HP hydrogel, and miR-26a mimic. They demonstrated almost full repair of the defect 12 weeks postimplantation and significantly enhanced vascularization confirming that miR-26a had the capacity to regulate endogenous angiogenic-osteogenic coupling for enhanced bone regeneration.…”

“…They demonstrated almost full repair of the defect 12 weeks postimplantation and significantly enhanced vascularization confirming that miR-26a had the capacity to regulate endogenous angiogenic-osteogenic coupling for enhanced bone regeneration. Wang et al [146] employed an alternative approach with ASCs, miR-26a mimic and Lipofectamine 2000 on porous HA scaffolds. Following 12 weeks implantation, histological assessment showed markedly enhanced bone formation.…”