Dedifferentiated Fat Cells Promote Skin Regeneration During Tissue Expansion And Its Mechanistic Study

BackgroundDue to trauma,tumors,and other reasons,large skin and soft tissue defects are very common in the clinic,causing severe problems for patients in appearance,function and psychology.Common treatments include skin grafts,flap transfers,and so on.The operation of skin graft is simple and easy,the survival rate is high,the skin supply is sufficient,and the application is the most common.However,the color of skin graft is not consistent with the wound skin and texture,and wear resistance is poor,and contracture is easy to occur.Flap transfer has more advantages in appearance,texture,and function.It is mostly used for the repair of functional parts or severe wounds.However,the areas of flaps available are limited,and the remote flaps still do not match the skin around the wound with a bloated look.The donor site often cannot be sutured directly and require skin graft coverage,causing secondary damage.Skin soft tissue expansion,referred to as expansion,is a method that a silicon capsule with an injection port is placed under the skin and liquid is injected into the capsule regularly to increase the surface area of the skin which is used for wound coverage.Using skin soft tissue expansion,new skin tissues near the damaged area could be obtained,which are similar to the color,texture,hair distribution and sensory function of affected area.At the same time,the occurrence of scars and deformities in the donor area could be significantly reduced.However,skin and soft tissue expansion has a long expansion period and low expansion efficiency.The saline injection time usually lasts several months.During this period,there are many complications,which brings great pain and inconvenience to the patient.Therefore,it is necessary to construct stable animal models and explore effective intervention methods to improve the expansion efficiency and obtain more new skin in a short time.Previous studies have used papaverine,estriol,growth factors,etc.to promote expansion,but the effect is limited,and the toxic side effects and short half-life of these drugs have limited their application.In recent years,research has found that stem cell transplantation treatments such as bone marrow-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells can significantly increase the efficiency of skin expansion without obvious complications,which has triggered a new round of research boom.However,some of the above stem cells are difficult to obtain,have poor uniformity,and are difficult to obtain in large quantities in a short period.Experimental studies have shown that mature adipocytes,which account for most of the adipose tissue,can be dedifferentiated into stem cells in vitro by a special "ceiling culture method",that is,dedifferentiated fat cells(DFAT).DFAT cells can be obtained in large quantities with high uniformity.The cells have similar surface markers and multidirectional differentiation potential with mesenchymal stem cells,which could be an important source of stem cells.They are expected to have similar effects to increase expansion efficiency and are worthy of corresponding research.Macrophages can polarize into classically activated macrophages(M1)by Th1-related cytokines LPS,etc.,and the characteristic molecular markers are IL-6,IL-12,i NOS,and TNF-α.It plays a pro-inflammatory role in the early inflammatory phase.Macrophages are polarized into alternative activated macrophages(M2)by Th2-related cytokines,such as IL-4,and the characteristic molecular markers include Arg-1,CD206,and IL-10,etc.M2 are mainly involved in the proliferation and repair phase after tissue injury,and have strong anti-inflammatory effects.Skin and soft tissue expansion is a special process of damage repair and regeneration,in which studies have shown that macrophages play a key role.Experiments showed that DFAT cells were similar to mesenchymal stem cells and have anti-inflammatory and immunosuppressive functions,which can significantly promote repair and regeneration of damaged tissues.In conclusion,DFAT cells may have the effect of regulating macrophage polarization to promote expanded skin regeneration and significantly increase the expansion efficiency.ObjectiveThe purpose of this experiment is to construct rat and mouse scalp expansion models,verify the role and mechanism of dedifferentiated adipocytes in promoting skin regeneration during expansion,and explore the regulatory effects of DFAT on macrophage polarization.Methods1)In this experiment,a rat and mouse scalp expansion model were constructed using a custom-made 1 m L micro-expander.The experimental animals were randomly divided into an expansion group and a sham expansion group.The expansion group was implanted with a 1 m L expander subcutaneously at the top of the skull,and saline injection was performed regularly.A 1.5 cm diameter silicone membrane was embedded in the sham expansion group as the control group,and no expansion was performed.The samples were taken on the 7th,14 th,and 35 th days after the operation.Hematoxylin-eosin staining and Masson staining were used for general histological observation.CD31 immunohistochemical staining was used to compare the blood vessel density between the expanded and sham-expanded skin.The cell proliferation ratio and apoptosis were compared between the two groups using proliferating cell nuclear antigen immunohistochemical staining and TUNEL staining.The infiltration of mast cells was observed by Giemsa staining.At the same time,the skin tissue was observed with a transmission electron microscope,and the ultrastructural changes of tissue cells and collagen fibers were compared between these two groups.2)DFAT cells obtained from SD rats and green fluorescent SD rats were cultured using the classic "ceiling culture method",and the morphological changes during the cell culture were observed.The third-generation cells with good growth status were induced to perform multi-directional differentiation.Red O staining and alizarin red staining were used to identify adipogenic and osteogenic differentiation respectively.CD90,CD105,CD34,CD45 and other surface markers were detected by flow cytometry.An SD rat scalp expansion model was constructed,a 1 cm × 1 cm rectangular tattoo was performed on the top of the rat’s head before expander implantation.The animals were randomly divided into 2 groups,which were DFAT group(experimental group)and DMEM group(control group).1 m L of DFAT cells or DMEM culture solution was injected on the 6th day after the expander implantation,and regular quantitative saline injection was started 1 week after the operation.The samples were taken on the 7th,14 th,and 35 th days after the operation.The areas of tattoo on the expanded flap of rats were regularly measured after the operation,and the dynamic curves of the skin area growth of the experimental group and the control group were drawn.The histological changes and the thickness of the epidermis and dermis of the two groups were observed and compared by HE staining.The collagen distribution of the two expanded flaps was compared by Masson staining.PCNA immunofluorescence was used to compare cellular proliferation ratio of the two groups.The blood perfusion of expanded flaps of these two groups was detected by a laser imager,and the blood vessel density was observed at the histological level by immunofluorescence staining.Immunofluorescence double-labeling staining was performed on expanded flaps of the two groups.GFP + DFAT cells were co-stained with vascular endothelial cell marker CD34,fibroblast marker Vimentin,and epidermal cell marker K15 to clarify whether DFAT cells could differentiate into variety kinds of cells.Immunofluorescence staining using macrophage marker CD68,M1 macrophage marker i NOS and M2 macrophage marker CD206 was conducted to observe and analyze the polarization status of macrophages in the expanded flaps.3)DFAT cells of C57 mouse were cultured,adipogenic and osteogenic differentiation ability was identified by Red O staining and alizarin red staining.CD90,CD105,CD34,CD45 and other surface markers were detected by flow cytometry.The RAW264.7 cell line or mouse peritoneal macrophages were divided into six groups for different treatments,including the M0 group: culture without any intervention;the M1group: 100 ng/m L LPS was added to the culture plate;M2 group: 40 ng/m L IL-4 was added to the culture plate;DFAT+M0 group: 0.4 μm well Transwell chamber with DFAT cells was added to the culture plate;DFAT + M1 group: 0.4 μm well Transwell chamber with DFAT cells and 100 ng/m L LPS were added to the culture plate;DFAT + M2 group:0.4 μm well Transwell chamber with DFAT cells and 40 ng/m L IL-4 were added to the culture plate.After a culture of 24 hours,the morphological changes of macrophages were observed;Real-time PCR was conducted to detect the m RNA expression of M1macrophage-related genes TNF-α,i NOS,MCP-1 and M2 macrophage-related genes Arg-1,IL-10 And YM-1.Western Blot was conducted to detect the protein expression of M1 macrophage marker i Nos,M2 macrophage marker Arg-1.CD11 c,CD206 expression were detected by flow cytometry.The concentration of M1 macrophage related gene TNF-α,IL-12(P70)and M2 macrophage related gene IL-10 in the cell culture supernatant was detected by ELISA.Results1)Using custom-made mini-expanders,we successfully constructed the rat and mouse scalp expansion models.The scalp of rats and mice was fully expanded,and the effect was stable and reliable.The expansion did not affect the normal activities of the head and eyelids of the experimental animals.Histological analysis showed that the epidermal layer of the expanded skin was significantly thicker than the unexpanded group(p <0.05),the dermal layer was significantly thinner(p <0.01),the blood vessel density was significantly increased(p <0.01),the proportion of cell proliferation was significantly increased(p <0.01),and the density of apoptotic cells was also greater than that of the unexpanded group(p <0.05).Capsule was formed in both groups,with sparse infiltration of inflammatory cells.Under the transmission electron microscope,it was observed that in the expansion group,some collagen fibers were broken,and fiber bundles of uneven thickness appeared and the arrangement was disordered,while the unexpanded group collagen fibers arranged neatly.In the expansion group,fibroblasts showed a typical proliferative state,such as increased nuclear membrane folds,increased number of organelles like mitochondria,and abundant endoplasmic reticulum in the cytoplasm,while the fibroblasts had a smooth nuclear membrane and a small number of organelles in the sham group.2)Dedifferentiated adipocytes of rats and mice can be successfully isolated and cultured by the classic "ceiling culture method".Dedifferentiation-like structural changes in mature adipocytes can be observed during the culture process.Lipid droplets in a single chamber in adipocytes became small,granular multi-room lipid droplets.Cell bodies protruded from antennae-like structures,and some cells gradually changed from circular to fibrous.Oil red O staining and alizarin red staining confirmed that the dedifferentiated adipocytes have the ability to differentiate into adipocytes and osteoblast.Flow cytometry showed that the cells were positive for CD90 and CD105,and negative for CD34 and CD45.It was found that the area of the expanded flap in the DFAT group significantly increased compared with the control group(p <0.01),and the area after retraction was also significantly larger than that of the control group(p <0.01),the shrinkage rate of the DFAT group was lower(p <0.05).The wet weight(p <0.01)and dry weight(p <0.05)of expanded flap of the DFAT group were higher,and the dry/wet ratio was not significantly changed(P>0.05).HE,Masson staining,and PCNA immunofluorescence staining results showed that after injection of DFAT cells,the dermal layer of the expanded flap thickened(p <0.01),the density of collagen fibers increased,and the proportion of proliferating cells increased significantly(p <0.05).CD34 immunofluorescence staining showed that injection of DFAT cells can significantly increase the density of vessels(p <0.01),laser imaging instrument showed that DFAT cells significantly increased the blood perfusion of expanded flaps(p <0.01).Immunofluorescence double-labeling staining results showed that the injected GFP + DFAT cells were mainly distributed in the subcutaneous and dermal layers,and expressed the vascular endothelial cell marker CD34 and fibroblast marker Vimentin.3)Immunofluorescence staining of macrophage subtype markers on expanded skin specimens revealed that the density of M1 macrophages of DFAT group was significantly reduced,the density of M2 macrophage cells remained at a high level,and the M2 / M1 ratio gradually increased.DFAT cells were co-cultured with macrophages in vitro.It was found that the m RNA expression of M1 macrophage-related genes TNF-α(p <0.01),i NOS(p <0.01),and MCP-1(p <0.01)was significantly reduced,and the expression of M2macrophage-related genes Arg-1(p <0.05),IL-10(p <0.01),YM-1(p <0.01)was significantly increased.Flow cytometry showed that the expression ratio of M1 macrophage marker CD11 c was significantly reduced(p <0.01),and the expression ratio of M2 macrophage marker CD206 was significantly increased after co-culture(p <0.01).Western Blot showed that i NOS expression decreased(p <0.05)and Arg-1 expression increased(p <0.05)after co-culture.The cell culture supernatant was detected by ELISA and found that the expression levels of M1 macrophage-related genes TNF-α(p <0.01)and IL-12(p <0.05)were significantly reduced and the expression of M2 related genes IL-10(p <0.01)was increased after co-culture.ConclusionThe scalp expansion model is stable and reliable,and has no obvious impact on animals.Expansion can cause cell proliferation and damage to some tissue cells.Dedifferentiated adipocytes can be successfully isolated and cultured by the "ceiling culture method".DFATs can promote cell proliferation,increase blood vessel density and blood perfusion,differentiate into fibroblasts and endothelial cells,and inhibit macrophages from polarizing to M1,and promote macrophages to M2.Through these mechanisms,DFATs can significantly promote the regeneration of expanded skin.