研究背景

人体皮肤老化有内源性和外源性因素，其中皮肤受到紫外线照射所致的日光性老化是皮肤老化中重要原因之一。目前治疗皮肤老化，尤其是面部老化的方法包括光电技术、外用生物及合成性药物、肉毒毒素及胶原蛋白等注射类产品。随着脂肪移植技术的普及以及脂肪间充质干细胞（Adipose-derived Stem Cells，ASCs）在再生医学领域的深入研究，应用脂肪组织及其相关移植物促进皮肤组织再生的报道也日益增多。

通过抽吸自体脂肪，术中快速制备可用于填充皮下浅层及真皮层以更好地促进老化皮肤再生的相关脂肪移植物得以开发，包括有Microfat为代表的颗粒脂肪、Nanofat为代表的乳化脂肪和以SVF-gel为代表的乳化脂肪浓缩物。上述三种脂肪移植物直径颗粒均较小，可通过尖细针进行浅层以及精细部位的填充，已应用于面颈部皱纹、痤疮后瘢痕等方面，起到改善瘢痕质地、皮肤弹性和色泽的作用。但体外机械加工处理会使脂肪组织的结构和成分发生较大变化，造成三种脂肪移植物的生物学性状不同，在促进皮肤年轻化的效果可能存在差异。因目前尚无系统性对比研究三种脂肪移植物在促进老化皮肤再生方面的差异及其优缺点，介于此，本研究首先拟通过体外实验观察三种脂肪移植物的结构特点及其对含有的细胞成分进行表型鉴定，对三者含有的ASCs增殖和多向分化能力进行比较；其次通过动物体内实验，以紫外线（Ultraviolet，UV）模拟太阳光照射BALB/c-裸鼠光老化皮肤为模型，将三种脂肪移植物移植于裸鼠背部皮下浅层及真皮内，观察移植物的体内转归以及其对光老化皮肤组织形态学的影响，为临床应用脂肪相关移植物促进光老化皮肤年轻化提供理论依据及优化方案。

研究目的

1. 通过对Microfat、Nanofat及SVF-gel三种脂肪移植物的宏观和微观结构进行观察，对其成分进行鉴定，并对各自含有的ASCs在增殖和多系分化能力方面进行比较，明确三种脂肪移植物的生物学特点；

2. 探索如何通过UVA+UVB模拟太阳光照射建立良好的裸鼠光老化皮肤损伤模型；

3. 探讨Microfat、Nanofat及SVF-gel三种脂肪移植物在改善皮肤光老化损伤的作用效果及其可能机制。

研究方法和结果

1. Microfat、Nanofat、SVF-gel细胞生物学特征

方法：通过临床吸脂获取人体脂肪组织制备Microfat、Nanofat和SVF-gel，计算三者的制备率，通过对上述三种脂肪产物移植物进行钙黄绿素-AM（Calcein-AM）/碘化丙啶（Propidium Iodide, PI）染色、组织切片HE染色、Masson染色、DAPI+Perilipin+vWF免疫荧光染色和扫描电镜观察三种脂肪移植物的活性和形态结构。通过胶原酶消化法提取三种脂肪产物含有的SVF细胞，行：① Muse细胞计数检测细胞活性和产量；② 流式细胞仪分析SVF细胞中ASCs（CD31-CD34+CD45-）和ECs（CD31+CD45-）所占比例；③ 应用CCK8法测定P1代ASCs的增殖能力；④ ASCs体外行成脂、成骨和成软骨诱导培养，通过qPCR(Real-time Quantitative Polymerase Chain Reaction)检测三系分化相关基因的相对表达量。

结果：三种脂肪移植物在制备率上，Microfat与Nanofat明显高于SVF-gel。大体观察Microfat呈淡黄色细颗粒状，Nanofat 呈米白色乳状，SVF-gel介于Microfat与Nanofat之间，三者均能顺利通过27G锐针进行移植。通过AM/PI染色、组织切片免疫组化及荧光染色、电镜等多种方式观察到：Microfat呈颗粒状，具有“葡萄串”样的微观结构，相对保留了完整的脂肪组织结构和具有活性的成熟脂肪细胞；经机械性乳化后，Nanofat的脂肪组织结构完全丧失，成熟的脂肪细胞被完全破坏，细胞外基质成碎片化混悬于大量的油脂中；SVF-gel的脂肪结构破坏较Nanofat轻，保留了少量具有活性的成熟脂肪细胞，经油脂的去除过程浓缩了细胞外基质和SVF细胞。采用胶原酶消化法提取三种脂肪移植物中具有活性的SVF细胞进行计数，其中SVF-gel 组中最多、Microfat组中次之、Nanofat组中最少，三者差异明显；经流式细胞仪鉴定SVF细胞中含有的ASCs亚群比例，Nanofat和SVF-gel组中多于Microfat组，而内皮细胞（Endothelial Cells，ECs）亚群的比例三组并无统计学差异。同时我们考虑经机械乳化过程是否对ASCs的生物学性状产生影响，我们选取ASCs传代培养P1时间点测定了其增殖和三系分化能力，结果显示三组均有良好的成脂、成骨和成软骨分化能力、以及细胞增殖能力，三组之间均无明显差异。

2. 紫外线照射诱导BALB/c-裸鼠皮肤光老化损伤模型的建立

方法：取6周龄BALB/c-雌性裸鼠，装入鼠笼中置于紫外线(Ultraviolet, UV)灯箱下，进行UVA+UVB模拟太阳光联合照射，依据所测辐照强度每次以1个最小红斑剂量（Minimal Erythema Dose，MED）的UVB和10倍于UVB的UVA进行照射，每周照射6次连续12周。采用Bissett视觉褶皱量表进行大体评分，硅橡胶皮肤印模采用Visioline VL650进行拍摄，软件分析计算皱纹平均深度及皱纹总面积；组织切片行HE、Masson及改良Weigert染色观察皮肤组织结构变化、胶原纤维及弹性纤维变化；皮肤组织行羟脯氨酸含量的测定；电镜观察皮肤组织的微观结构变化；行免疫荧光及qPCR检测皮肤组织中MMPs的蛋白表达及其mRNA相对表达量。

结果：BALB/c-裸鼠共行UVA+UVB联合照射12周后成功建立了光老化损伤的皮肤模型。与未UV照射组比较，UV照射组背部皮肤出现了明显的组织病理变化：大体观见皮肤弹性变差、横向皱纹加重、表面粗糙脱屑；Bissett视觉量表评分、皱纹总面积和皱纹平均深度均明显增加；扫描电镜观察胶原纤维排列稀疏紊乱、分布不均匀，弹性纤维及网状纤维成无序状分布，表现为丝状、团块状，各纤维成分相互缠绕；组织学HE、Masson及改良Weigert染色观察真皮层蓝染的成纤维细胞核密度减少，真皮层胶原纤维稀疏、排列紊乱，真皮层中横向排列的成熟弹性纤维断裂、扭曲，堆积于皱纹两侧沟壁，而表皮基底层与真皮乳头层的锚纤维大部分消失，毛囊、汗腺以及皮下脂肪组织轻度萎缩，皮肤层厚度下降；测量羟脯氨酸含量显著降低；通过免疫荧光染色观察MMP-9、MMP-13的蛋白表达量明显升高，qPCR检测MMP-9、MMP-13 mRNA的相对表达量亦明显升高。

3. Microfat, Nanofat, SVF-gel 改善BALB/c-裸鼠皮肤光老化损伤的研究

方法：从健康吸脂患者获取颗粒脂肪制备Microfat、Nanofat及

SVF-gel，以皮肤光老化损伤的裸鼠为模型，将三组脂肪移植物移植于裸鼠背部皮下浅层及真皮层内（27G锐针），移植PBS为对照组。于术后观察裸鼠背部皮肤变化；8周取材，行组织学HE染色观察皮肤结构变化、测量皮肤层厚度、评估炎症细胞浸润及组织纤维化程度；Masson及天狼猩红染色观察真皮层胶原纤维分布情况、I型和Ⅲ型胶原纤维所占皮肤总面积及其比值；测量皮肤中羟脯氨酸含量；改良Weigert染色观察弹性纤维变化；增殖细胞核抗原（Proliferating Cell Nuclear Antigen, PCNA）免疫荧光染色观察皮肤层组织细胞的增殖情况；Perilipin免疫组化染色观察移植脂肪组织脂肪活性情况；通过电镜观察皮肤组织微观结构变化。

结果：移植后观察皮下移植物逐渐变小、颜色变浅；各组皮肤表面粗糙程度及脱屑均有减轻，色泽由灰暗逐渐变为正常，各组皮肤表面观并无明显差异。移植8周后取材观察三组移植物的体积部分吸收，其中Microfat成活较好，Nanofat仅残留少量油脂成分，SVF-gel组中脂肪成活较差。与对照组比较，HE染色观察Microfat, Nanofat 和SVF-gel组中皮肤层均无明显增厚；在皮肤层及移植物内均可见炎性细胞浸润，其中以Nanofat组中最为明显，SVF-gel组次之、Microfat中最少；Nanofat组中移植物纤维化，未见有成活脂肪细胞，可见巨噬细胞吞噬坏死组织后形成大量的泡沫细胞，SVF-gel 组中移植物大多数形成纤维化包块，可见少量具有结构的脂肪细胞，Microfat组可见移植的脂肪组织结构完整，经perilipin染色可观察到大量具有活性的脂肪细胞。Masson及天狼猩红染色观察到对照组中真皮层蓝染的纤维组织较为稀疏，排列不规律，可见断裂及卷曲；Microfat组中真皮层纤维组织致密，成波浪束状样均匀分布；Nanofat组及SVF-gel组中由于深层炎性细胞浸润诱发组织纤维增生，皮肤纤维组织分布致密但失去了正常结构。通过天狼猩红染色在偏振光下分析I型和Ⅲ型胶原纤维总和所占皮肤层面积，Nanofat组与SVF-gel组中最多，Microfat其次，对照组最少，这与各组皮肤中检测的羟脯氨酸含量相一致。但与Microfat组比较，Nanofat组与SVF-gel组中I型胶原纤维所占面积比值相对较少，而Ⅲ型相对较多，I型/Ⅲ型比值降低，表明组织发生了纤维化。通过改良Weigert弹力纤维染色，观察对照组真皮层内仍有较多变性、断裂堆积的弹性物质，而在实验组中均明显减少，可见有新生弹性纤维生成，其中Microfat组真皮层内可见较多锚纤维成树枝状垂直于真皮乳头成或毛囊腺体分布，而Nanofat组及SVF-gel组中在真皮网状层形成大量无序状分布的细小新生弹性纤维。为评估三组移植物对组织细胞的促增殖作用，行PCNA免疫组化染色观察到Microfat组，Nanofat组及SVF-gel组在皮肤层中平均荧光强度均高于对照组；而在移植物内，Nanofat组及SVF-gel组由于慢性炎性细胞浸润及成纤维细胞的异常增殖，DAPI及PCNA荧光呈强阳性表达。

研究结论

Microfat、Nanofat及SVF-gel均可通过细小锐针行皮肤层内注射移植，但三种移植物在大体观、微观结构、含有的细胞成分等方面存在明显差异；

UVB和UVA联合照射可模拟太阳光引起BALB/c-裸鼠皮肤光老化损伤，本研究能为建立BALB/c-裸鼠皮肤光老化损伤的模型提供较为理想的实验方案；

Microfat、Nanofat及SVF-gel均具有促进皮肤增殖作用：Nanofat 和SVF-gel移植后受区炎症反应较重，易致组织纤维化增生；Microfat移植后脂肪成活好，受区炎症反应轻，可通过容量效应及再生效应改善皮肤光老化损伤。

Background

There 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.

Objectives

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.

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.

To explore the role of adipose derived therapeutic products in improving skin photoaging in nude mice.

Methods and Results

1. Biological characteristics of Microfat, Nanofat, and SVF-gel

Methods: 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/propidium 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.It 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 SVF 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 P1generation 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 mice
    Methods: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, type III 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.

Conclusion

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;

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；

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.