Study On Effect Of BMSCs On PMNs＆Mφ Influx And Their Chemokines Expression In Diabetic Wound

Background:With90million people living with diabetes and1.3million died in2011, china has thelargest diabetes epidemic in the world. More effective treatment was urged for diabeticWound healing. Wound healing is initiated by early inflammatory cells and involvesinteraction and dual directional regulation of many cells and molecules. It was said thatchronic subclinical inflammation in the skin of diabetics inhibits the acute inflammatoryresponse, coursing inadequate and delayed influx PMNs and M, contributing to impaireddiabetic wound healing. Both domestic and abroad researches have shown that treatmentsrestoring PMNs and M influx in diabetic wounds early after injury tend to result inaccelerated wound healing.Bone mesenchymal stem cells (BMSCs) are multipotent stem cells that hold promisefor an expanding list of therapeutic uses. It was well documented that BMSCs can restorediabetic wound healing in angiogenesis, granulation tissue formation and e-epithelialization.But interaction between BMSCs and inflammation is very complicated. BMSCs wereshown in vitro to release Chemokines, like MIP-2, MCP-1, while they were demonstratedto exert anti-inflammatory effect on lymphocytes.Function of BMSCs may change according to microenvironment. When Nitric oxide(NO) synthesis is insufficient, BMSCs showed pro-inflammatory response throughchemokines. Previous studies showed that skin of diabetic mice produce inadequate NOboth before and after injury. In vitro experiments suggest that BMSCs may be able toincrease the expression of vascular endothelial NO. above all, we get a hint that BMSCscan improve local inflammatory chemokine expression to stimulate PMNs and Minfiltration in diabetic wound early after injury for there is not enough NO in the wound,and then inflammation resolution would happen in the later stage of healing by inhibition ofBMSCs mediated by retored NO. Presents studies have focused on wounds at later time points, and very little attention has been given to the dynamics of macrophage responses indiabetic wounds early after injury.Objectives:The objective of this study was to identify the effect of bone marrow mesenchymalstem cells(BMSCs) treatment on infiltration of neutrophil(PMNs) and macrophage(M),and on mRNA expression of macrophage inflammatory protein-2(MIP-2) and monocytechemotactic protein-1(MCP-1) on diabetic wounds，with genetically induced spontaneousdiabetes models，and to provide a clue in study of effect of BMSCs on PMNs and M, aswell as in basic and application study of BMSCs in diabetic wound healing.Methods:1. BMSCs were separated, cultured and purificated by whole bone marrow culturemethod from4weeks old C57BL/6mice. BMSCs of passage10were tested bydifferentiation experiment and flow cytometry.2. Round full-thickness excision skin wounds with diameter of8mm wereproduced on back of C57BLKS/Nju spontaneous mutation diabetic mice (db/dbmice n=40); To set two groups randomly, and on the day when wounds wereproduced (0day), BMSCs group (n=20) was treated with0.1mlsingle-cell suspension (containing about1X106BMSCs in PBS) per wound bysubcutaneous injection and the control group (n=20) treated with0.1ml PBS inthe same way.3. Percentage of Wound healing was collected on postwound day1,3,5,7,10and14with image processing software IPP6.0; To evaluate the wound tissue byHaematoxylin and eosin staining method. To observe the infiltration ofneutrophils(postwound day1,3,5,7and10) and macrophages(postwound day3,5,7,10and14) by immunohistochemical method and Optical density valuemethod; To detect the relative quantitative mRNA expression of MIP-2andMCP-1on postwound day1,3,5and7by RT-qPCR.Results:1. Bone marrow mesenchymal stem cells (BMSCs) were lisolated, purified andcultured successfully. BMSCs of passage10can be induced to osteogenesisdifferentiation and adipogenic differentiation. 2. on the5th,7th10thday after BMSCs local application, the wound healing rate inBMSCs group[（40.15±3.63）%、（67.62±1.58）%、（98.28±1.51）%] wassignificantly higher than those of the PBS control group[（18.52±3.19）%、（49.58±3.39）%、（78.52±6.52）%, P<0.05], and the time for heaing in BMSCsgroup and PBS group is (11.67±0.58)d and (16.33±0.58)d respectively(P<0.05);The histopathologic observation showed better quality of wound healing,accelerated re-epithelization, and promoted formation of granulation in BMSCsgroup.3. Infiltration of neutrophils and macrophage in diabetic wounds early after injurywere promoted in BMSCs group(P<0.01), of which the peak was also earlierthan in PBS group.4. mRNA expression of MIP-2in BMSCs group was Statistically elevated in thepostwound1st、5thand7thday，（30.91±8.30）,（12.37±2.45）and （7.82±2.52）fold to that in PBS group,(P<0.05). mRNA expression of MCP-1in BMSCsgroup was Statistically promoted in the postwound1st、3thand7thday，（（20.94±2.97）、（3.99±1.40）and（19.67±3.70）fold to that in PBS group,(P<0.05).Conclusions:BMSCs might enhance influx of PMNs and M by mechanism involving promotedvolume of MIP-2and MCP-1in wound, which might also contribute to acceleratedconcrescence of diabetic wound when treated with BMSCs.