Circulating Fibrocytes: Cell Culture, Identification And Its Effects On The Full-thickness Skin Wound Healing In Diabetic Mice

Objectives:Peripheral blood derived circulating fibrocytes are a newly indentified leukocyte subpopulation that displays fibroblast-like properties.These blood-borne cells rapidly enter sites of tissue injury at the same time as inflammatory cells,suggesting an important role for these cells in wound repair.Circulating fibrocytes are thought to play a role of accelerating wound repair by several mechanisms such as the scretion of extracellular matrix(ECM), cytokine production,antigen presentation,wound constraction,and angiogenesis.But,until now,although its role in wound healing has been described in a number of papers,no any study has provided evidence that circulating fibrocytes could accelerat skin wound repair, especially chronic wound healing as diabitic ulcer.Presently,circulating fibrocytes can be isolated from periheral blood mononuclear cells(PBMCs)via cell culture in vitro.To avoid contamination of other cell types,after 10-14 days of initial isolation,circulating fibrocytes need to be purified by immunomagnetic negative selection.This method could to be improved due to it need special technology and instrument,while the cell yield is lower.For clinic studies on chronic wound healing using this novel cell type,it's necessary to develop a simple and efficient method for cell purification.The aims of this study were(1)to develop a simple and efficient method for circulating fibrocytes purification and enrichment,(2)to identify circulating fibrocytes yielded from above method and(3)to investigate the effects and potential mechanisms of circulating fibrocytes in diabetic mouse full-thickness wound healing.Methods:(1)Mouse parabiotic skin wound healing model that consisted of an unirradiated transgenic GFP⁺ mouse(C57BL/Ka-eGFP)and an unirradiated wild-type mouse originated from same strian were generated by surgical conjunction and wounded on 14 days post parabiosis operation.The blood-borne cells migration in wound sites were observed at 3 days and 7 days after wounding.For harvest the evidence of circulating fibrocytes participated in the early stage of wound healing,immonofluorescence staining was performed to count the migration of blood-derived circulating fibrocytes,endothelia cells at 3 days after wounding. (2)Total mouse peripheral blood mononuclear cells were isolated by density gradient centrifugate with Ficoll/paque and cultured in DMEM supplement with 20%fetal bovine serum for 46 days.No any immunological selection methods were performed except PBS wash before medium replacement.At 5,10,14,21,and 28 days after initial isolation,the dynamic change of cell markers and the percentage of each cell subpopulation were analysised by dual immunofluorescent staing.(3)To identify cultured circulating fibrocytes with their function,at 14,28,35,and 46 days after initial isolation,the relative expression of growth factors(TGF-β1,bFGF,PDGF-A and VEGF-A),chemokines (MIP-1α,MIP-2α,MCP-1),chemokine receptor CXCR4 and ECM protein Col-â… ,α-SMA mRNA were detected by real time PCR and data were compared with the expression of above gene in mouse dermal fibroblasts and PBMCs.Additionally,the effect of cultured cFb on the proliferation of dermal fibroblasts was determined via the in vitro scrape wound repair model. (4)Immediately following the made of dosal full-thickness wound on diabetic mouse,100μl (5.0×10⁵ cells)PKH26 labeled circulating fibrocytes(cFb group)or 100μl PBS were injected into the tail vein.The open wound area was measured at 3,7,10,14,17,and 21 days post wounding.For histology assay,tissue samples were harvested at 3,10 days post wounding, and the labled cFb were be traced under fluorescent microscope.The neutrophil and macrophage recruitment,angiogenesis,and cell proliferation were detected by immunofluorescence staining.Real time PeR was employed to investigate the relative expression of growth factors(TGF-β1,PDGF-A and VEGF-A),chemokines (MIP-1α,MCP-1),and ECM protein Col-â… ,α-SMA mRNA in wound tissue.(5)Data are expressed as(?)±s.Statistical analysis was performed using SPSS for Windows 11.0.Results were determined to be stastically significant if P＜0.05.All stastical diagrams were graphed using Microsoft^?Excel 2004 for Mac.Results:(1)Using the parabiotic mouse full-thickness wound healing model,we determined a numerous blood-derived cells(express GFP⁺)migrated into wound site or wound edge,and the amount of these cells was decreased at 7 days post wounding.After staining with CD45, a-SMA or PECAM-1 antidodies,at 3 days post wound,the major population of these GFP⁺ cell was neutrophils(21.4±4.3%/HPF).Additionally,α-SMA⁺/GFP⁺ cells(7.6±4.4%/HFP) and PECAM-1⁺ cells(4.8±3.1%/HPF)also detected in wound sites at 3 days post wounding. (2)After initial isolation from peripheral blood 3 days,a lot of adherent cells appeared in culture disk.These cells expressed typical spindle shape and formed clone.Identification with CD45/Col-â… and CD11b/Col-â… antibodies,around 6.7±3.1%of CD45/Col-â… double postive cells and 7.5±2.7%of CD11b/Col-â… double postive cells were detected at 5 days after isolation.But the cell markers changed dynamically during cultured in vitro.Rapidly,these cells lost hematopoietic cell marker,CD11b and CD45,but continually expressed Col-â… .At 14 days after isolation,most cells lost hematopoietic markers and the percentage of double positive cells decreased sharply while the percentage of Col-â… ⁺ cells increased(56.8±18.5%). At 28 days after isolation,a higher Col-â… positive cells percentage,96.5±11.5%,was determined by cell count under fluorescent microscope.During the period of cell culture,an increasing percentage ofα-SMA positive cells were detected.(3)Compared with mouse dermal fibroblasts,cultured circulating fibrocytes expressed higher mRNA of various growth factors and chemokins(P＜0.05).As a typical feature of circulating fibrocytes,a higher relative expression of CXCR4(P＜0.05)also was detected in our study by real time PCR assay at 14,28,35,46 days after initial isolation.In contrast,the level of expression of Col-â… andα-SMA were lower than dermal fibroblasts(P＜0.05).(4)Using fluorescent microscope,PKH26 labled circulating fibrocytes were observed on the sections of cFb group wound site,but didn't appear in lung and normal skin.Compared with PBS group,the recruitment of netrophils and macrophages,angiogenesis and cell proliferation were increassed(P＜0.05).The expression of MIP-1α,MCP-1 mRNA in wound tissue also increased at 3 days post wounding(P＜0.05),and the expression of TGF-β1, PDGF-A and VEGF-A mRNA increased at 10 days(P＜0.01).At 3,7,10 days post wounding, wounds in the cFb group had closed fastly than those in PBS group(P＜0.05).By day 21,all the wounds had closed in both groups.Conclusion:According to our studies,we concluded that,(1)Parabiotic mouse skin full-thickness wound model could be employed in the relative studys which explore circulating cells participate in wound repair.Including circulating fibrocytes,endothelial cells could be detected in parabiosis mouse wound tissue via immunofluoresence staining.(2)Using a simple and convenient method,as natural selection by extended culture period in vitro,more admissible purification of circulating fibrocytes which with typical function could be yielded.Although lost the markers of hematopoietic cell,these cells still express higher level of the production of growth factors,chemokines,and the chemokine receptor,CXCR4.(3)Cultured circulating fibrocytes may accelerat the early process of diabetic skin full-thickness wound healing.The most likely mechanism is relat to increasing the inflammatory cells infiltration and the synthesis of growth factors and chemokines.