Repair of critical-sized bone defects with anti-miR-31-expressing bone marrow stromal stem cells and poly(glycerol sebacate) scaffolds

Abstract

The repair of critical-sized defects (CSDs) is a significant challenge in bone tissue engineering. Combining the use of progenitor cells with gene therapy represents a promising approach for bone regeneration. MicroRNAs play important roles in most gene regulatory networks, regulate the endogenous expression of multiple growth factors and simultaneously modulate stem cell differentiation. Our previous study showed that knocking down miR-31 promotes the osteogenesis of bone marrow stromal stem cells (BMSCs). To investigate the therapeutic potential of cells engineered to express anti-miR-31 for CSD repair, lentiviral vectors encoding negative control, miR-31 precursor and anti-sense sequences were constructed and transduced into osteo-inductive BMSCs. The expression of osteogenic-specific genes, alkaline phosphatase activity and Alizarin Red S staining were investigated to evaluate the effects of miR-31 on the cell fate of BMSCs over a 3-week period. In addition, miR-31-modified BMSCs seeded on poly(glycerol sebacate) (PGS) scaffolds were used to repair 8 mm critical-sized calvarial defects in rats. The results showed that miR-31 suppression significantly increased the expression of osteogenic-specific genes in vitro at the mRNA and protein levels, and that robust new bone formation with high local bone mineral density was observed in the anti-miR groups in vivo. Moreover, the PGS scaffolds carrying anti-miR-31-expressing BMSCs exhibited good biocompatibility and a high regeneration rate (~60 %) within in vivo bone defects. Our results suggest that miR-31 gene delivery affects the potential of BMSCs for osteogenic differentiation and bone regeneration and that PGS is a potential substrate for genetically modified, tissue-engineered bone in the repair of large bone defects.

Genetic Engineering of Mesenchymal Stem Cells for Regenerative Medicine

“…in the expression of osteogenic lineage genes in in vitro studies, and bone formation was detected in vivo [143].…”

MiRNA inhibition in tissue engineering and regenerative medicine

“…For example, inhibiting endogenous miR-31 significantly increases the osteogenic potential of bone mesenchymal stem cells (BMSCs) [136]. In one demonstration, Deng et al lentivirally-transfected BMSCs to express anti-miR-31 and observed a 2.5-fold increase in expression of special AT-rich sequence-binding protein 2, a protein involved in osteoblastic differentiation and bone development [137]. This increased expression was maintained in vitro at a relatively constant level between 4 and 21 days after treatment [137].…”

“…In one demonstration, Deng et al lentivirally-transfected BMSCs to express anti-miR-31 and observed a 2.5-fold increase in expression of special AT-rich sequence-binding protein 2, a protein involved in osteoblastic differentiation and bone development [137]. This increased expression was maintained in vitro at a relatively constant level between 4 and 21 days after treatment [137]. To determine whether modulation of miR-31 activity in BMSCs affected CSD repair in vivo , BMSCs were transfected in vitro with anti-miR-31 containing vectors, seeded 24 hours later onto poly(glycerol sebacate) (PGS) scaffolds, and then implanted into 8 mm rat calvarial CSDs.…”

“…Eight weeks after surgery, micro-CT was used to visualize the amount of bone ingrowth into the defects (Figure 4). Overall, incorporation of cells pre-treated with anti-miR-31 into the PGS scaffold significantly increased bone regeneration in a CSD when compared with the PGS alone [137], demonstrating that bone tissue engineered constructs can improve tissue integration and regeneration through incorporation of anti-miR therapeutics.…”

Efeito da membrana de Poli(vinilideno-trifluoretileno)/titanato de bário sobre a formação óssea em defeitos criados em calvárias de ratos

“…O miR-146a e o -146b também são relevantes, uma vez que regulam negativamente a SMAD7 (banco de dados GeneCards e TargetScanhttp://www.genecards.org / http://www.targetscan.org). Quanto aos miRs regulados negativamente pela membrana de P(VDF-TrFE)/BT, o miR-31 é considerado um inibidor da osteogênese (Deng et al, 2014) e possui um papel importante na osteoclastogênese e no controle da reabsorção óssea (Baglio et al, 2013). Os resultados iniciais são promissores e novos experimentos são necessários para a avaliação funcional de miRs que possam estar envolvidos com o potencial osteogênico da membrana de P(VDF-TrFE)/BT.…”