All-trans Retinoic Acid Improves The Viability Of Ischemic Skin Flaps In Diabetic Rat Models And Promotes Osteogenic Differentiation And Bone Consolidation In A Rat Distraction Osteogenesis Model

Aims: Endothelial progenitor cells(EPCs)play a critical role in neovascularization,which enhances proliferation under all-trans retinoic acid(ATRA)treatment.However,the effects of ATRA on the skin flap survival in diabetic flap ischemia remains unknown.Distraction osteogenesis(DO)has been a useful approach to treat orthopaedic disorders.However,a high rate of complications and discomfort hamper its further application in clinical practice.Here,we investigated the effects of all-trans-retinoic acid(ATRA)on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cell(rBMSCs)and bone consolidation in a rat DO model.Methods: Ischemic random skin flaps were made in 40 diabetic Sprague-Dawley rats with 20 normal rats used as control in this study.At 7 days postoperatively,the surviving area of each skin flap was measured.Immunofluorescence staining was used to analyze capillary density and EPCs recruited to the flaps.The expression of ANG2 and VEGF was determined by Western blotting.Circulating EPC number was determined by flow cytometry.Invitro tube formation experiment was used to analyze the function of EPCs.Different doses of ATRA were used to treat rBMSCs.Cell viability and osteogenic differentiation were assessed using CCK-8 or alkaline phosphatase staining,respectively.The mRNA expression of osteogenic differentiation-genes(including ALP,Runx2,OCN,OPN,OSX and BMP2)and angiogenic genes(including VEGF,HIF-1,FLK-2,ANG-2 and ANG-4)were determined by quantitative real-time PCR analysis.Further,we locally injected ATRA(10 μM)or PBS(100 μl)into the gap in a rat DO model every three days till termination.X-rays,micro-computed tomography,mechanical testing,and immunohischemistry examinations were used to evaluate the quality of the regenerates.Results: The flap survival rate and capillary density of ATRA-treated flap were significantly increased.Fluorescence activated cell sorting(FACS)analysis demonstrated a marked increase in systemic CD34+/Flk-1+ EPCs in ATRA treated rat.The expression of ANG2 and VEGF was increased in diabetic flap tissues under ATRA administration.Furthermore,ATRA administration restored the impaired function of diabetic EPCs in tube formation.ATRA promoted osteogenic differentiation of rBMSCs.Moreover,ATRA elevated the mRNA expression levels of osteogenic differentiation-genes and angiogenic genes.In the rat model,new bone properties of bone volume/total tissue volume and mechanical strength were higher in the ATRA treated group.Immunohischemistry analysis also confirmed more mineralized bone after ATRA treatment.Conclusion: ATRA could notably exert preventive effects against skin flap necrosis and promote neovascularization in diabetic rats,which may partially through elevating the expression of ANG2 and VEGF,and augmenting EPC mobilization.In conclusion,as a readily available and very cost effective bio-source,ATRA might be a novel therapeutic method to enhance bone consolidation in clinic.