研究背景及意义

      软骨组织的自我修复和再生能力极为有限，由各种因素所致的软骨组织缺损的修复一直是临床面临的巨大挑战。组织工程技术应用生物学和工程学原理，有望为受损软骨组织的再生提供先进的治疗策略，但目前该项技术尚未实现大规模的临床转化应用，主要瓶颈之一在于种子细胞来源问题。间充质干细胞是一类存在于多种组织的成体干细胞，因其具有多向分化潜能以及不涉及伦理问题而备受研究者青睐。其中，脂肪来源干细胞（adipose-derived stem cells，ADSCs）因组织来源丰富、易获取及免疫原性低等优点已成为自体和同种异体干细胞治疗和组织工程重要的种子细胞来源。脂肪组织经酶消化后，得到的是基质血管成分细胞（stromal vascular fraction，SVF）。SVF不但包含ADSCs，还含有一定比例的内皮祖细胞、内皮细胞、造血细胞（包括造血干细胞、粒细胞、单核细胞、淋巴细胞）及周细胞等。相较于ADSCs，分离获取的SVF无需体外培养扩增，可实现一期手术自体回植，节省临床治疗时间。比较两者在体外三维条件下成软骨特性及体内构建可注射弹性软骨再生情况是否具有差异，将为以细胞治疗为基础的软骨组织工程技术提供理论基础，为弹性软骨构建中种子细胞的优化选择提供参考。

研究目的

1、从兔脂肪组织分离获得ADSCs和SVF，比较两者相关生物学特性，并对ADSCs进行鉴定，为体外及体内成软骨研究提供细胞源基础。

2、通过组织学染色、基因表达检测以及组织糖胺多糖（glycosaminoglycan，GAG）定量分析等方面系统比较兔脂肪来源的ADSCs 和SVF成软骨特性的差异，确立基于以上细胞的种子细胞应用策略，同时为组织工程弹性软骨构建中种子细胞的优化选择提供参考。

研究方法

1、应用胶原酶消化法和贴壁培养法分离纯化ADSCs，应用胶原酶消化法获取SVF，显微镜下观察两者的形态学特征；通过Muse^TM细胞分析仪，比较两者的细胞活性；通过流式细胞分析，比较两者相关细胞表型表达差异，包括CD31、CD34、CD45、CD73、CD90及CD105，并应用CD45^-CD34⁺CD31^-标记SVF中的ADSCs亚群，以明确SVF中ADSCs细胞群所占比例。此外，对分离纯化的ADSCs进行成脂、成骨及成软骨诱导，结合细胞表面标志物表达情况等，进行干细胞特性的自我鉴定。

2、应用胰酶结合胶原酶消化法分离兔耳廓软骨细胞（auricular chondrocytes, ACs），将ADSCs、SVF、ACs、ADSCs+ACs及SVF+ACs（共培养组细胞为1:1混合）在体外三维（pellet）培养条件下进行成软骨诱导，4周后，进行大体观察、组织学染色、tunel染色、Real-time PCR检测及GAG含量定量分析，比较ADSCs和SVF体外成软骨潜能。

3、将Pluronic F-127凝胶负载的兔来源的ACs、ADSCs+ACs、SVF+ACs（共移植组细胞为1:1混合）植入裸鼠皮下，8周取材，进行大体观察、湿重检测、组织学染色、Real-time PCR成软骨相关基因检测及GAG定量分析，比较ADSCs和SVF分别与ACs共移植后体内可注射弹性软骨再生情况。

研究结果

1、倒置相差显微镜下观察ADSCs与SVF的细胞形态，可见ADSCs呈纺锤样（长梭形），细胞形态均一； SVF呈悬浮状态，细胞大小不同。Muse^TM细胞活性分析结果表明：ADSCs细胞活性高于SVF（ADSCs：94.87±1.652%；SVF：71.72±2.053%）。细胞流式分析表明：兔来源的ADSCs和SVF相关细胞表型表达存在差异。培养扩增的ADSCs高表达CD73和CD105间充质干细胞标记物（CD73：88.07±5.444%，CD105：88.47±4.113%），低表达CD90间充质干细胞标记物（CD90：23.43±6.647%），阴性表达CD31内皮细胞/内皮祖细胞标记物、CD34干细胞标记物及CD45造血干细胞标记物（CD31：1.822±1.480%，CD34：1.935±0.1334%，CD45：2.105±0.9136%）；分离获取的SVF，CD31、CD34、CD45、CD73、CD90及CD105标记物均有不同程度表达（CD31：33.97±4.246%，CD34：26.72±1.845%，CD45：20.97±3.260%，CD73：31.65±5.279%，CD90：41.98±7.238%，CD105：19.08±1.493%），且SVF中ADSCs细胞群所占比例为9.947±1.072%。分离纯化的ADSCs可成功向脂肪、骨及软骨方向分化。

2、ADSCs、SVF、ACs、ADSCs+ACs及SVF+ACs经过体外三维条件下成软骨诱导4周后，均可形成稳定的pellet。单独诱导组中，ADSCs组和SVF组比较，ADSCs组细胞外基质分泌情况优于SVF组，并且ADSCs组总GAG含量及GAG/DNA含量检测高于SVF组。在与ACs共培养中，组织学染色结果显示ADSCs+ACs组软骨基质分泌情况与SVF+ACs组比较，在pellet表层区无明显差异，而在pellet核心区和中间区，ADSCs+ACs组基质分泌情况明显优于SVF+ACs组。Real-time PCR软骨相关基因表达及GAG含量检测均为ADSCs+ACs组高于SVF+ACs组。Tunel染色结果表明ADSCs+ACs组细胞凋亡率在pellet表层区、中间区及核心区均显著低于SVF+ACs组。

3、Pluronic F-127负载的ACs、ADSCs+ACs及SVF+ACs在裸鼠皮下均可形成软骨样组织，各组标本大小不等，SVF+ACs组湿重小于其余两组。组织学染色结果显示ADSCs+ACs组在标本边缘和中心部位基质分泌情况均优于SVF+ACs组。Real-time PCR结果显示ADSCs+ACs组ACAN和COMP的相对表达量高于SVF+ACs组，但COL2A1的相对表达量，两者无统计学差异。GAG定量检测结果显示ADSCs+ACs组GAG含量高于SVF+ACs组，且差异具有统计学意义。

研究结论

1、兔来源的ADSCs和SVF在细胞活性和相关细胞表型表达方面存在差异。纯化的ADSCs为细胞活性更高的均质性细胞群；分离获取的SVF为异质性细胞群，并且，纯化的ADSCs具有多向分化潜能。

2、体外三维培养条件下，ADSCs、SVF、ACs、ADSCs+ACs及SVF+ACs均可在成软骨诱导液中，向软骨方向分化。ADSCs组的成软骨特性优于SVF组；且在与ACs共培养中，相较于SVF+ACs共培养组，ADSCs+ACs共培养组成软骨情况更佳。

3、可注射材料Pluronic F-127凝胶负载的ADSCs+ACs体内弹性软骨再生情况优于SVF+ACs，可初步认为在与ACs裸鼠体内共移植（或体外共培养）时，相较于SVF，ADSCs可能是更为理想的种子细胞来源。但从应用便捷性等方面考虑，SVF仍然具有一定的临床应用优势。

Background

      The repair of cartilage defects caused by various factors has always been a great challenge in clinic because cartilage has a low spontaneous repair and regeneration capacity. Tissue engineering, based on the principles of biology and engineering, is expected to provide an advanced procedure for the regeneration of damaged cartilage tissue. However, this technology has not been applied in large-scale clinical transformation, and the limited source of seed cells limits its clinical application. Mesenchymal stem cells (MSCs) are a kind of adult stem cells which exist in a variety of tissues and have multipotential differentiation. MSCs are favored by researchers because they do not involve ethical problems and have great differentiation potential. Among them, adipose-derived stem cells (ADSCs) have become an important seed cell source for autologous and allogeneic stem cell based therapies and tissue engineering due to their abundant sources, easy access and low immunogenicity. After digestion of adipose tissue with collagenase, the pelleted stromal vascular fraction (SVF) is obtained. SVF represents a cell population composed of ADSCs, endothelial cells, endothelial progenitor cells, hematopoietic cells (including hematopoietic stem cells, granulocytes, monocytes, lymphocytes) and pericytes, among which there is a certain proportion of stem cells. SVF can not achieve allogeneic stem cell transplantation due to its immunogenicity, however, freshly acquired SVF does not require in vitro culture and expansion, so it can achieve first-stage surgical autologous transplantation and save clinical treatment time. The comparison of chondrogenic characteristics under three-dimensional conditions in vitro and injectable elastic cartilage regeneration in vivo between ADSCs and SVF will provide a theoretical basis for cartilage tissue engineering technology based on cell therapy and provide a reference for optimal selection of seed cells in the construction of elastic cartilage.

Objective

1. Compare biological characteristics of ADSCs and SVF isolated from rabbit adipose tissue and identify ADSCs, so as to provide cell source basis for chondrogenic research in vitro and in vivo.

2. Compare the chondrogenic characteristics of rabbit derived ADSCs and SVF by histology, gene expression and GAG quantitative analysis, and establish the application strategy of seed cells based on the above cells, so as to provide a reference for the optimal selection of seed cells in the construction of tissue engineered elastic cartilage.

Methods

1. ADSCs were isolated and purified by collagenase digestion method and adherence culture method. SVF was obtained by collagenase digestion method. The cell morphological characteristics of ADSCs and SVF were observed under microscope, and the cell viability of the two groups was compared by Muse^TM cell analyzer. Surface marker profiles of ADSCs and SVF, including CD31, CD34, CD45, CD73, CD90 and CD105, were compared by flow cytometry. The ADSCs subsets in SVF were identified as CD45^-CD34⁺ CD31^- to clarify the proportion of ADSCs in SVF. In addition, ADSCs were induced by adipogenesis, osteogenesis and chondrogenesis, and self-identification of ADSC stem cell characteristics was carried out in combination with the expression of ADSC cell surface markers.

2. Rabbit auricular chondrocytes (ACs) were isolated by trypsin and collagenase digestion. In three-dimensional culture conditions in vitro, ADSCs, SVF, ACs, ADSCs+ACs and SVF+ACs (1:1 mixture in co-culture groups) were induced in chondrogenic differentiation medium for 4 weeks and we compared the chondrogenic potential of ADSCs and SVF in vitro by gross observation, histological staining, tunel staining, Real-time PCR analysis and GAG quantitative analysis.

3. Rabbit-derived ACs, ADSCs+ACs and SVF+ACs (1:1 mixture in co-transplantation groups) loaded with Pluronic F-127 gel were subcutaneously implanted into nude mice for 8 weeks, and we compared injectable elastic cartilage regeneration in vivo after co-transplantation of ADSCs and SVF with ACs by gross observation, histological staining, Real-time PCR chondrogenesis related gene detection and GAG quantitative analysis.

Results

1. The cell morphology of ADSCs and SVF was observed under inverted phase contrast microscope. It was found that ADSCs had a spindle-shaped fibroblast-like appearance and the cell morphology was uniform. SVF cells were of different sizes and they were in suspension. Muse^TM cell viability analysis showed that the viability of ADSCs was higher than that of SVF. Flow cytometry showed that there were differences in surface marker profiles between ADSCs and SVF from rabbits. Isolated and expanded ADSCs contained a more homogenous cell population, which were CD31, CD34, CD45 negative (CD31：1.822±1.480%，CD34：1.935±0.1334%，CD45：2.105±0.9136%), but positive for MSC marker like CD73, CD105 (CD73：88.07±5.444%，CD105：88.47±4.113%), and the expression rate of CD90 in ADSCs was 23.43±6.647%. SVF represented a heterogeneous cell population containing not only cells with typical MSC markers (CD73：31.65±5.279%，CD90：41.98±7.238%，CD105：19.08±1.493%), but also CD31⁺ endothelial cells/endothelial progenitors, CD34⁺ stem cells and CD45⁺ hematopoietic cells (CD31：33.97±4.246%，CD34：26.72±1.845%，CD45：20.97±3.260%), and the proportion of ADSCs in SVF was 9.947±1.072%. The isolated and expanded ADSCs were able to differentiate into adipocytes, osteoblasts and chondrocytes.

2. In three-dimensional culture conditions in vitro, ADSCs, SVF, ACs, ADSCs+ACs and SVF+ACs could form pellet after chondrogenesis induced for 4 weeks. Compared with ADSCs group and SVF group, the results of histological staining showed the secretion of extracellular matrix in ADSCs group was better than that in SVF group, and the total GAG quantification and GAG/DNA in ADSCs group were higher than those in SVF group. In coculture with ACs, histological staining results showed that there was no significant difference in cartilage matrix secretion between ADSCs+ACs group and SVF+ACS group in pellet surface region, while the matrix secretion of ADSCs+ACs group was significantly better than that of SVF+ ACs group in pellet core and middle region. The expression of Real-time PCR cartilage related genes and GAG contents in ADSCs+ ACs group were higher than those in SVF+ ACs group. TUNEL staining results showed that cell apoptosis rate of ADSCs+ ACs group was significantly lower than that of SVF+ ACs group in pellet surface region, middle region and core region.

3. ACs, ADSCs+ACs and SVF+ACs loaded with Pluronic F-127 could form cartilage-like tissue in nude mice subcutaneously, and the specimens of each group were different in size, and the specimens in SVF+ACs group were smaller than those of the other two groups. Histological staining results showed that the matrix secretion of ADSCs+ACs group was better than that of SVF+ACs group at the edge and center of the specimen. Real time PCR results showed that the relative expressions of ACAN and COMP in ADSCs+ACs group were higher than those in SVF+ACs group, but there was no significant difference in the relative expression of COL2A1 between the two groups. GAG quantitative results showed that GAG content in ADSCs+ACs group was higher than that in SVF+ACs group, and the difference was statistically significant.

Conclusions

1. There are differences in cell viability and surface marker profiles expression between rabbit-derived ADSCs and SVF. The isolated and purified ADSCs are homogeneous cell population with higher cell viability. The isolated SVF represents a heterogeneous cell population. And, the isolated and purified ADSCs have potential of multi-lineage differentiation.

2. In three-dimensional culture conditions in vitro, ADSCs, SVF, ACs, ADSCs+ACs and SVF+ACs can be successfully induced to differentiate into cartilage. The chondrogenic properties of the ADSCs group are better than those of the SVF group, and compared with SVF+ACs, ADSCs+ACs have stronger chondrogenic characteristics.

3. The regeneration of elastic cartilage in vivo in Pluronic F-127-loaded ADSCs+ACs is better than that in SVF+ACs. It can be preliminarily considered that ADSCs may be a better source of seed cells than SVF in vivo co transplantation with ACs in nude mice (or in vitro coculture with ACs). However, in terms of application convenience, SVF still has some advantages in clinical application.