Experimental Study Of Macrophages In Skin Regeneration During Tissue Expansion

BackgroundReconstruction of large skin defects resulting from different causes remains one of the biggest challenges in reconstructive surgery.Skin grafting,skin flap transplantation,vascularized composite tissue allotransplantation and tissue expansion are currently used for defect repair.However,grafting of the sizeable defects often proves disappointing in terms of skin quality,color,texture and donor site deformity.Skin flap transplantation provide similar but insufficient skin tissues for the reconstruction of large wound.Vascularized composite tissue allotransplantation such as face transplantation and hand transplantation reconstruct the large defect with similar skin tissues,but long-term immunosuppressive and side effect limit its clinical applications.A better and perhaps ideal treatment strategy in such instances is the use of skin soft tissue expansion for wound repair.Skin tissue expansion can harvest enough“extra skin tissue”which is similar in texture,color and flexibility to the lost tissue to reconstruct large skin defects after trauma,tumor excision or infection.However,multiple surgical procedures,low expansion efficiency,and high complication rates limit its applications.Therefore,the mechanism by which skin responds to mechanical strain remains to be elucidated.During the expansion period,the skin undergoes stress and relaxation as well as injury and repair.Immune cells such as macrophages rush to the wounded site to clear debris and repair the damage.Depending on the stages of polarization,macrophages can be subdivided into classically activated M1 or alternatively activated M2 phenotypes.M1macrophages are known to secrete pro-inflammatory cytokines,such as IL-1,IL-6,and TNF-?,and mediate a host defense against debris and degradation of the extracellular matrix,while M2 macrophages have been reported to contribute to tissue repair by producing anti-inflammatory cytokines such as VEGF,TGF-?and IL-10.Recent researches have been shown that macrophages are necessary for liver,kidney and skeletal muscle repair after injury and play a key role to initiate tissue regeneration in highly regenerative animals such as zebrafish and salamander.Currently,the change of skin macrophages and its mechanism during tissue expansion still remain unclear.To investigate the subtype changes of macrophages and its role in skin regeneration during skin expansion are necessary for identifying new targets to reduce clinical complications of tissue expansion and increase expansion efficiency.ObjectiveTo study the dynamic changes and subtype switches of macrophages in skin tissues undergoing expansion,and investigate the role of macrophages and its mechanism during tissue expansion.Methods1)Silicone expanders?10 mL?were implanted subcutaneously into the backs of Sprague-Dawley rats.Then rats were divided into expansion group and sham control group.Tissue specimens were collected at day 1,day 3,day 7,day 14 and day 35.Hematoxylin and eosin?H&E?stain was used to investigate pathological change of expanded skin tissues,and CD68 immunofluorescent staining was used to compare the dynamic changes of macrophages in skin tissues undergoing expansion between expansion group and sham control group.Then we used iNOS⁺CD68⁺to mark M1 cells and CD206⁺CD68⁺to mark M2 cells.The number of M1 and M2 cells in skin and fibrous capsule during expansion were detected at each time point.Furthermore,total RNA was extracted from expanded skin at each time point,isolated RNA was reverse transcribed into cDNA using a cDNA synthesis kit and then qPCR was used to explore the changes of M1 related cytokines such as IL-1?,IL-6,TNF-?and M2 related cytokines IL-10 and TGF-?.2)Macrophages can recognize and phagocytose clodronate liposomes and then apoptosis occurs,we plan to use clodronate liposomes to deplete the macrophages in skin during tissue expansion.First,we developed macrophages depletion skin model and control skin model in one rat during tissue expansion:the border of two symmetrical 1.0cm×1.5 cm rectangle areas on bilateral of the expanded flap were tattooed for tracing the expanding areas,40?L of 50?g/mL PBS liposomes or clodronate liposomes were subcutaneously injected into the left or right side of the tattooed skin area on post-operation days 0,3,and 10.Tissue biopsies for CD68 immunofluorescent staining were collected at days 7,14,and 35,respectively.After the protocol was built,rats were divided into expansion group and sham control group,and both groups received treatment of liposomes.Tissue biopsies were harvested at days 7,14,and 35.The tattooed area of either side of each expanded skin was measured on days 14,28,and 35,respectively,and the relative skin thicknesses of the skin biopsy samples?dermis plus epidermis?collected at days 14 and 35 were calculated by using ImageJ.The Masson staining positive areas were analyzed by Image-Pro Plus 6.0,and the average percentage of the stained area was calculated.Transcutaneous oxygen pressure?TcPO₂?of the expanded skin flap were monitored at days 14 and 35,respectively,following the manufacturer's instruction.PCNA immunofluorescent staining was carried to detect the number of active cells and Isolectin immunofluorescent staining was used to measure subcutaneous vascular density.Last,total RNA was extracted from expanded skin at days 14 and 35,and then qPCR was used to explore the mRNA changes of EGF?bFGF?VEGF and TGF-?in macrophages depletion skin and control skin.3)RAW 264.7 cells were cultured in vitro,we used 100 ng/mL LPS and 25 ng/mL IFN-?to turn macrophages into M1 subtype and we used 40 ng/mL IL-4 to turn macrophages into M2 subtype.Then M1 cells or M2 cells were detected by PE conjugated CD11c or APC conjugated CD206.Total RNA was extracted from both M1 cells and M2cells,isolated RNA was reverse transcribed into cDNA and then qPCR was used to explore the mRNA changes of VEGF?EGF?bFGF and TGF-?in both M1 and M2 cells.Mouse embryonic fibroblasts were isolated and cultured.After 3 passages,cell culture supernatant of M0,M1 and M2 were used to culture fibroblasts for 24 hours.RNA was extracted from fibroblasts of each group and qPCR was used to explore the mRNA changes of Col1A1 in fibroblasts.Results1)We successfully developed the tissue expansion model on the back of rats.The densities of macrophages were significantly higher in the expanded skin tissues on days 14?P<0.01?and 35?P<0.01?,respectively,relative to that of non-expanded tissues.Future more,we found the cellular density of CD68⁺iNOS⁺M1 macrophages dropped significantly at day 14?P<0.01?and day 35?P<0.0001?,respectively,compared to that at day 7.In contrast,the cellular density of M2 macrophages markedly increased during tissue expansion at day 35?P<0.05?relative to that at day 7.We found the proportion of M1 cells in was higher in fibrous capsule at day 1 and day 3 compare to days 7,14 and 35,respectively?P<0.05?.However,there was no significance in the proportion of M2 cells between each time point.We revealed that M1 related cytokines such as IL-1?,IL-6 and TNF-?were higher at day 1 compare to that at other time points and M2 related cytokine IL-10 was higher at day 1 and day 3 compare to that at other time points?P<0.05?.2)We successfully developed a topical macrophages depletion model by using clodronate liposomes.Immunofluorescence staining of the tissue sections with the treatment of clodronate liposomes showed a marked reduction in the macrophage population at days 3,14,and 35,respectively?P<0.05?.We found the skin tissues in the macrophages depleted side showed less expansion area than that of the control side at days14,28 and 35,respectively?P<0.01?.Histopathologic examination revealed that macrophages depleted skin in the expansion group displayed thinner skin thickness at days14 and 35 respectively than that in control skin?P<0.01?.There was no significant difference in skin thickness or expansion area between macrophages depletion side and the control side in the non-expansion sham group.The control expanded skin showed a significant increase in collagen density than that in the macrophages depleted skin at day14 and day 35?P<0.01?through microscopic evaluation of Masson's trichrome stained tissue sections.Moreover,the control expanded skin revealed more PCNA-positive cells per field than that of the macrophages depleted skin at day 14 and day 35?P<0.001?and the macrophages depleted skin had a lower degree of vascularization than that of the control expanded skin at day 14 and day 35?P<0.0001?.The measurements of the transcutaneous oxygen pressure showed a higher oxygen pressure in the control expanded skin at day 35?P<0.05?.The expressions of VEGF,EGF,bFGF,and TGF-?dramatically decreased in the macrophages depleted skin compared to that of the control expanded skin at day 14?P<0.05?.3)By utilizing 100 ng/?L LPS and 25 ng/?L IFN-??M1?or 40 ng/?L IL-4?M2?,we successfully turned 80%-94%unpolarized RAW 264.7 cells into M1 cells and 60%-80%unpolarized RAW 264.7 cells into M2 cells.The expressions of VEGF dramatically increased in M1 cells than that in M0 and M2 cells by using qPCR analysis;and the expressions bFGF and TGF-?were higher in M2 than that in M0 and M1 cells.Moreover,we found the expressions of Col1a1 dramatically increased in mouse embryonic fibroblasts when cultured by cell culture supernatant of M2 compare with unpolarized M0or M1.Conclusion1)Tissue expansion model on the back of rat is a mature and stable animal model.The densities of macrophages in skin increased during tissue expansion and M1 macrophages were dominant following silicone expander implantation.In contrast,the number of M2macrophages maintained in the high-level during tissue expansion and skin regeneration.2)Topical use clodronate liposomes could successfully deplete tissue macrophages in skin.After the macrophages were depleted,the skin regeneration was inhibited,as evidenced by a smaller expansion area,thinner skin layers and decreased collagen synthesis.The mechanism may be related to decreased cell proliferation rate,skin vascularization and growth factors after macrophages were depleted in expanded skin.3)The expressions of growth factors increased after macrophages polarized in vitro.M2 macrophages could induce collagen production in fibroblasts by extracellular secretions.