The Experimental Study Of Transplantation Of Bone Marrow Mesenchymal Stem Cells For Wound Healing In The Absence Of Inductive Factor

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The in vitro culture,expansion,purification and identification of BMSCs of rats.Objective To establish an effective method for separation,purification and culture of BMSCs of rats,and provide enough number of cells for the next ongoing experiment.Methods 1.3-weeks male SD rats were sacrificed,and the BMSCs from the bone marrow of femur and tibia were separated from the non-adherent cells and then cultured until the three passages.2.The morphology of BMSCs were observed under light microscopy and the particular CD antigen of BMSCs were identified by using FCMs.3.The expression of Oct4 and Klf4 of BMSCs were evaluated by using immunofluorescence staining.Results 1.After 72hs of culture,the primary BMSCs exhibited the growth style with adherence,showing the unique shape of spindle and multiple pseudopod.7-10 days later,the cells showed classic "fibroblast" shape with "wave".2.The particular CD antigen(CD44?CD90?CD31?CD45)of BMSCs demonstrated their pluripotency as 84.95%?88.49%,0.01%,4.22%.3.The positive expression of Oct4 and Klf4 of BMSCs were confirmed by immunofluorescence staining.Conclusions Our method of using separation of cells from the non-adherent cells is easy and effective to obtain enough number of cells and satisfy with the requirement of the ongoing experiment.Part ? Induction of mesenchymal stem cell differentiation in the absence of soluble inducer for cutaneous wound regeneration by a chitin nanofibers-based hydrogelObjective To explore novel therapy using a chitin nanofibers(CNFs)hydrogel as a directive cue to induce BMSCs differentiation for enhancing cutaneous wound regeneration in the absence of cell-differentiating factors.Methods A new physical method to form the hydrogel using "salt induce gelation"was utilized to fabricate the GFP transfected BMSC-chitin nanofiber hydrogel.Then the BMSC-scaffold were transplanted into the full skin defect wound model of SD rats.Western blotting,RT-PCR and immunofluorescence staining were used to assess the relationship between the differentiation of BMSCs and the wound healing effect of BMSC-scaffold.Results First,a "green" fabrication of CNFs hydrogels encapsulating green fluorescence protein(GFP)transfected rat BMSCs was performed via in-situ physical gelation without chemical cross-linking.Without soluble differentiation inducers,CNFs hydrogels decreased the expression of BMSCs transcription factors(Oct4 and Klf4)and concomitantly induced their differentiation into the angiogenic cells and fibroblasts,which are indispensable for wound regeneration.In vivo,rat full-thickness cutaneous wounds treated with BMSCs-hydrogel exhibited better viability of the cells than did local BMSC injection-treated wounds.Similar to that of In vitro result,CNFs hydrogels induced BMSCs to differentiate into beneficial cell types,resulting in accelerated wound repair characterized by granulation tissue formation.Our data suggest that three-dimensional CNFs hydrogel may not only serve as a "protection" to improve the viability of exogenous BMSCs,but also provide a functional scaffold capable of enhancing BMSCs regenerative potential to promote wound healing.This may help to overcome the current stem cell therapy limitations faced by the field of wound regeneration.Conclusions A cell-encapsulated CNFs hydrogel fabricated through a physical and,,green" pathway without chemical modification was used to induce the differentiation of BMSCs for wound healing in the absence of soluble inducers.In addition to the excellent biocompatibility,CNFs hydrogel protected BMSCs from elimination by the harsh wound microenvironment and induced BMS Cs to differentiate directionally into skin regeneration related cells,leading to the enhanced wound healing.Thus,we believe that this approach provides opportunities to improve the effectiveness of stem cell therapy for the treatment of wounds with large defects.More modification of the CNFs biomaterial to reinforce its properties should further advance BMSC-based construct therapy.Part ? Enhancement of Bone-Marrow-Derived Mesenchymal Stem Cell Angiogenic Capacity by NPWT for a Combinatorial Therapy to Promote Wound Healing with Large DefectObjective To investigate whether this combinatory therapy could accelerate angiogenesis in wounds.Methods BMSCs were obtained from SD rats and cultured until three passages.And then the cells were cultured under NPWT to observed the morphological change and the enhanced angiogenesis capacity of BMSCs.In vivo,after the full skin defect wound model were established on SD rats,the combined therapy of using BMSCs and NPWT was used for treating the animal model,and Western blotting,RT-PCR and immunofluorescence staining were used to assess the relationship between the enhanced angiogenesis capacity of BMSCs and the accelerated wound healing.Results In vitro,after 9-day culture,BMSCs proliferation significantly increased in NPWT group.Furthermore,NPWT induced their differentiation into the angiogenic related cells,which are indispensable for wound angiogenesis.In vivo,rat full-thickness cutaneous wounds treated with BMSCs combined with NPWT exhibited better viability of the cells and enhanced angiogenesis and maturation of functional blood vessels than did local BMSC injection or NPWT alone.Expression of angiogenesis markers(NG2,VEGF,CD31,and a-SMA)was up regulated in wounds treated with combined BMSCs with NPWT.Our data suggest that NPWT may act as an inductive role to enhance BMSCs angiogenic capacity and this combinatorial therapy may serve as a simple but efficient clinical solution for complex wounds with large defects.Conclusions These findings demonstrate that the capacity of NPWT to enhance BMSC angiogenic property for cutaneous wound healing potentially by preserving the viability of the cells,stimulating,and inducing them to differentiate into the desired cells that are beneficial for angiogensis within wounds.This combinatory strategy is superior to either of cells transplantation and NPWT therapy alone,leading to the accelerated wound healing.Further optimization of the applicable parameter of NPWT and BMSCs may provide more benefits to clinical application.