| BackgroundThere are endogenous and exogenous factors in human skin aging,among which solar aging due to UV radiation plays an important role in skin aging.Currently,there are various ways to treat human skin aging,especially facial skin aging,including photoelectric technology,topical biological and synthetic drugs,botulinum toxin,collagen and other injectable products.With the popularization of fat grafting in clinical practice and the extensive research on adipose-derived stem cells(ASCs)in the field of regenerative medicine,basic research and clinical reports are increasing on the application of adipose tissue and its derivatives to promote aging skin regeneration.In order to prepare adipose derived therapeutic products from lipoaspirate for filling the superficial subcutaneous and intradermal layers for better regeneration of aging skin,scholars have proposed granular fat represented by Microfat,emulsified fat represented by Nanofat,and concentrated emulsified fat represented by SVF-gel.These three types of products are all small in diameter particles,and can be used for superficial and fine site grafting by sharp and tiny needles,and can improve the scar texture,skin elasticity and color in applications such as facial and neck wrinkles and acne scarring.However,at present,the structure and composition of adipose tissues have changed considerably after mechanical processing in vitro,making their biological properties and their effectiveness in promoting skin rejuvenation different.There is no side-to-side comparative study on the differences of the three products on aging skin rejuvenation and their respective advantages and disadvantages.In this study,firstly,the structural characteristics of the three products and the identification of the cellular components were observed,and the proliferation and multilineage differentiation abilities of the ASCs contained in the three products were comparedin vitro.Secondly,using ultraviolet(UV)irradiation of photoaged skin in nude mice as a model,the three products were grafted into the subcutaneous and dermal layers of the dorsum.We observed the changes of the grafts in vivo and the histomorphological changes of the photoaged skin to provide a theoretical basis and an optimum solution for the clinical application of adipose derived therapeutic products to promote the regeneration of aged skin.Objectives1.To clarify the biological characteristics of the microfat,nanofat and SVF-gel by observing their macroscopic and microscopic structures,identifying their components and comparing the ability of ASCs in terms of proliferation and multilineage differentiation.2.To explore how to establish a qualified photoaging skin model of nude mice by UV irradiation,so as to provide some modeling experience for research and treatment of photoaging skin.3.To explore the role of adipose derived therapeutic products in improving skin photoaging in nude mice.Methods and Results1.Biological characteristics of Microfat,Nanofat,and SVF-gelMethods:Human adipose tissue was obtained by liposuction to prepare Microfat,Nanofat and SVF-gel.The preparation ratio of grafts was calculated.The viability and morphological structure of the three fat grafts were observed by the staining of the calcein-AM/propi dium iodide(AM/PI),HE,Masson and DAPI+Perilipin+vWF immunofluorescence,and electron microscopy.SVF cells contained in the three adipose derived therapeutic products were extracted by collagenase digestion and then examined as follows:muse cell count to detect cell activity and yield;flow cytometry to analyze the proportion of ASCs(CD31-CD34+CD45-)and ECs(CD31+CD45-)in SVF cells;CCK8 method to determine the proliferative capacity of ASCs at P1;ASCs were cultured in vitro to induce adipogenesis,osteogenesis and chondrogenesis,and the relative expression of differentiation-related genes was detected by qPCR.Results:The preparation ratio of microfat and nanofat was significantly higher than that of SVF-gel.Macroscopic observation showed that microfat was a yellow-granule,Nanofat was a beige-emulsion,and SVF-gel was between microfat and nanofat in color and shape.The three grafts all can be smoothly injected through a 27G sharp needle.lt was observed by AM/PI staining,immunohistochemistry and immunofluorescence staining of tissue sections,and electron microscopy that microfat had a "grape bunch-like" microstructure and relatively retained intact adipose tissue structure and viable mature adipocytes.After mechanical emulsification,the structure of adipose tissue in nanofat was completely lost,the mature adipocytes were all destroyed,and the extracellular matrix was fragmented and suspended in a large amount of oil.The destruction of adipose structure in SVF-gel was slighter than that of nanofat,and a small amount of viable mature adipocytes were retained.The extracellular matrix and S VF cells were concentrated by the process of oil removal.Considering the important role of SVF cells in tissue regeneration,we extracted the viable SVF cells from three groups by collagenase digestion The number of viable SVF cells among three groups differ significantly,with the most in SVF-gel group,followed by the microfat group and the least in the nanofat group,The proportion of ASCs subsets in SVF cells identified by flow cytometry was higher in nanofat groupand SVF-gel group than in microfat group,but there was no difference in the proportion of endothelial cell(ECs)subsets.In order to evaluate effect of the process of mechanical emulsification on the biological propertiesof ASCs,the proliferation and multilineage differentiation of ASCs at the P1 generation were measured,no significant differences were detected among the three groups.2.Establishment of skin photoaging model of BALB/c-nude mice induced by ultraviolet radiation.Methods:the 6-week-old BALB/c-nude mice were placed in a cage box and placed under the ultraviolet light box.According to the measured radiation intensity,the nude mice were irradiated with UVA and UVB 6 times a week for 12 weeks according to the one minimumerythema dose each time.The dorsum of nude mice was photographed by Visioline VL650 and the wrinkle was analyzed by software;Bissett visual wrinkle scale was used for general score;tissue sections were stained with HE staining,Masson staining and modified Weigert staining to observe the changes of skin tissue structure,collagen fiber and elastic fiber;the content of hydroxyproline was determined;and the microstructure of skin tissue were observed by electron microscope.The relative expression of MMPs gene in skin tissue was detected by qPCR.Results:The skin photoaging model of BALB/c-nude mice was successfully established after UVA and UVB irradiation for 12 weeks.Compared with the non-UV irradiated group,obvious histopathological changes were observed in the skin of the dorsum of nude mice irradiated with UV:Macroscopic observation showed that the skin elasticity decreased,the transverse wrinkles aggravated,the skin surface was rough and desquamated.The score of Bissett visual scale,the total area of wrinkles and the average depth of wrinkles were significantly increased.Scanning electron microscope showed that the collagen fibers were sparse and disordered and the distribution of elastic fibers and reticular fibers were also disordered and showed filamentous and clumpy,all kinds of fiberswere intertwined with each other.Histological staining of HE,Masson and modified Weigert showed that the nuclear density of dermis blue-stained fibroblasts decreased,and the dermis collagen fibers became sparse and disordered.The mature elastic fibers arranged transversely in the dermis denatured,broken and twisted,and accumulated in the groove walls of the wrinkles.The oxytalan network in the papillary dermis disappeared,the hair follicles and sweat glands decreased,and the subcutaneous adipose tissue atrophied.The thickness of the skin became thinner.The content of hydroxyproline decreased significantly.The protein expression of MMP-9 and MMP-13 in the skin increased significantly by immunofluorescence staining.The relative expression of MMP-9 and MMP-13 genes were significantly increased by qPCR.3.Microfat,Nanofat and SVF-gel promote the regeneration of skin photoaging of BALB/c-nude miceMethods:The fat was harvested from healthy patients who received liposuction and then prepared into microfat,nanofat and SVF-gel.Three type of adipose derived therapeutic products were injected into the subcutaneous and intradermal planes of nude mice with skin photoaging withsharp needle(27G),PBS was used as control group.The changes of skin and fat on the dorsum of nude mice were observed after operation.After 8 weeks,histological HE staining was performed to observe the changes of skin structure,the thickness of skin layer was measured,and the infiltration of inflammatory cells and the degree of tissue fibrosis were evaluated.The distribution of collagen fibers and the ratio of type I to type III collagen fibers were observed by Masson and Sirius red staining,and the changes of elastic fibers were observed by modified Weigert staining.The content of hydroxyproline was measured.The proliferation of tissue cells was observed by proliferating cell nuclear antigen(PCNA)and the adipose viability was observed by perilipin.The microstructure changes of skin were observed by electron microscope.Results:After transplantation,the subcutaneous graft became smaller and the color became lighter,the roughness and desquamation of the skin surface in each group were alleviated,and the skin color gradually changed from gray to normal.There was no significant difference in the skin surface between the control group and the experimental group.After 8 weeks,the nudemice were sacrificed,and the volume of the grafts in the three groups was partially absorbed,in which microfat survived well,only a small amount of oil cyst remained in nanofat,and the fat survival was poor in SVF-gel.Compared with the control group,there was no differences of skin thickness in the three groups Inflammatory cell infiltration was observed in the skin and the grafts,which was most obvious in the nanofat group,followed by the SVF-gel group and least in the microfat group.In the nanofat group,the grafts were fibrotic and no viable adipocytes were found,and a large number of foam cells were formed by macrophages after phagocytosis of necrotic tissues;most of the grafts in the SVF-gel group formed fibrotic masses,and a few structural adipocytes were seen;in the microfat group,the structure of the grafted fat was structurally intactunder the sarcolemma,and the viable adipocytes were observed by perilipin staining.Masson staining showed that the blue-stained fibers in the dermis were sparse and irregular,with breakage and curl in the control group;in the microfat group,the dermis fibers were dense and uniformly distributed in wave bundles;in the nanofat group and SVF-gel group,the proliferation of fibrous tissue was induced by the infiltration of deep inflammatory cells,and the fiber distribution was dense but lost the normal structure.The ratio of the total area of type I and type III collagen fibers to the skin area analyzed by Sirius red staining under polarized light was highest in the nanofat group and SVF-gel group,followed by microfat and least in the control group,which was consistent with the hydroxyproline content detected in each group.However,compared with the microfat group,the ratio of area occupied by type Ⅰ collagen fibers was relatively less in the nanofat group and SVF-gel group,and on the contrary,typeⅢ was relatively more,the type Ⅰ/Ⅲ ratio decreased,which indicated that fibrosis occurred in the skin tissue.Modified Weigert staining showed that there were still many denatured,fragmentized elastic substances in the dermis of the control group,but significantly decreased in the experimental group and new elastic fibers were formed.In the microfat group,many oxytalan fibers were dendritic perpendicular to the dermis and epidermis junction(DEJ)or hair follicles and sweat glands,however,in nanofat groupand SVF-gel group,a large number of disordered small new elastic fibers were formed in the reticular layer.In order to evaluate the proliferative effect of grafts on histiocytes,PCNA immunofluorescence staining showed that the average fluorescence intensity in skin layer of microfat group,nanofat group and SVF-gel group was higher than that of control group,while in grafts,DAPI and PCNA fluorescence expression was strongly positive in nanofat group and SVF-gel group due to chronic inflammatory cell infiltration and abnormal proliferation of fibroblasts.Conclusion1.Microfat,Nanofat and SVF-gel can be injected into the skin layer by small sharp needle,but there are significant differences among them in macrostructure,microstructure and composition;2.UVB combined with UVA irradiation can simulate the skin photoaging injury induced by sunlight in BALB/c-nude mice.This study can provide an ideal experimental protocol for the establishment of skin photoaging model in BALB/c-nude mice;3.Microfat,Nanofat and SVF-gel all have the effect of promoting skin proliferation:nanofat and SVF-gel can lead to tissue fibrosis in the recipient area,while microfat has a good survival and the inflammatory reaction in the recipient area is mild,which can improve the skin photoaging through volume effect and regeneration effect. |
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