VEGF-PLA纳米缓释微球与SVFs对游离移植脂肪颗粒及脂肪组织工程胶原支架血管化的研究
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摘要
研究背景
组织工程技术是近来学术界研究热点,细胞疗法和基因疗法是组织工程研究的热点,学术界一直探求一种临床实用性强的安全的辅助组织工程手段。
近年来,随着脂肪抽吸术的广泛开展和应用,自体脂肪颗粒注射移植术已经在国内外得到广泛的重视。自体脂肪颗粒以其无排斥、感染率低、注射后组织相容性好等优点,是目前修复软组织缺损比较理性的填充材料。虽然脂肪组织工程技术理论的发展,为真正实现无损伤修复软组织缺损和真正意义上形态、结构与功能重建开辟了新的途径。但是,脂肪组织工程技术目前还不能实现临床应用,所以目前修复软组织缺损较理想的填充材料是自体脂肪颗粒。
自体脂肪颗粒移植在临床上一直备受关注,尤其是负压抽脂术在临床上得到广泛的应用之后,这种移植术被越来越多的临床医生和患者所接受,并在获取、纯化、注射脂肪和提高其存活率等方面不断得到提高。但是,自体脂肪颗粒移植后吸收率可高达30%-50%,这种高吸收率的问题一直不能得到良好的解决,所以移植脂肪成活率较低极大地限制了其在临床上的广泛应用。脂肪组织不仅是储存能量及内分泌器官,还是软组织填充理想的替代物,而且还被认为是成体干细胞的有效来源,因为脂肪组织当中含有多种细胞成分,包括祖细胞成分,这些祖细胞成分可分化成为其他细胞系。脂肪组织当中主要包含脂肪细胞、脂肪干细胞、血管内皮细胞、壁细胞、成纤维细胞、巨噬细胞及细胞外基质成分等,其中脂肪细胞和脂肪干细胞是近年来研究的热点。脂肪干细胞被认为是一种表现脂肪细胞和血管细胞的祖细胞,它位于脂肪细胞之间、血管周围或细胞外基质中,可促进脂肪组织的转归。脂肪干细胞存在于脂肪组织的间质血管碎片中(stromal vascular fraction,SVF),而SVF是干细胞辅助脂肪移植中最重要的组分。通过胶原酶消化,从脂肪组织中分离的多种细胞混合物形成的细胞团就叫做间质血管碎片(SVF)。间质血管碎片中含有丰富的间充质干细胞,可分化为多种谱系,是再生医学、组织工程等最理想的种子细胞。
选择合适细胞外基质支架,作为妥当的细胞载体是组织工程的关键,是使SVFs中的ADSCs得以增殖、分化的重要条件。脂肪组织本身含有非常丰富的胶原、蛋白聚糖、纤维粘连蛋白等,这些组织是优秀的细胞外基质。这些年来热门的SVFs细胞辅助脂肪转移技术,既解决了移植脂肪中ADSCs含量低,同时又解决了移植的SVFs缺乏细胞外基质两个问题,并简化了SVFs提取的体外培养、多次离心重悬等步骤,为临床上解决移植脂肪吸收率高的难题提供了一种解决方法。
在组织工程材料中加入有活性的生长因子,能够增强工程化组织的血管化,减少吸收率,提高移植组织的成活。血管内皮生长因子(vascular endothelial growth factor,VEGF)又称为血管渗透因子,这种细胞因子具有促进血管渗透的作用,同时还可促进血管内皮细胞增殖和迁移、延长血管内皮细胞寿命、促进血管形成、增强血管通透性和改变细胞外基质等作用。除了对血管的作用,它还与创面愈合、创伤组织修复、炎症和肿瘤发生密切相关。VEGF与VEGF受体之间存在正反馈,VEGF因子局部应用可以诱导血管内皮细胞VEGF及其受体的高表达,导致VEGF效应的放大。但是VEGF在体内应用时因其半衰期短、容易降解等因素使得不能充分发挥其生物学效应。药剂学的缓释技术很好的解决这一难题,它通过微球包埋技术将VEGF包埋于聚乳酸纳米微球中,从而使这种细胞因子能够缓慢的释放,达到对周围环境持续发挥生物学效应的目的。这种缓释技术在脂肪组织工程的研究领域具有很大的研究价值。
近年来,组织工程技术的发展迅速,使得很多临床上的难题获得了一种可能性的解决方案。然而,由于组织工程的血管化问题难以解决,大部分的组织工程技术距离临床应用还有很大距离。胶原蛋白是良好的支架材料,其已被美国FDA批准作为人工皮肤材料。对于种子细胞的选择,大部分研究都选择脂肪干细胞。然而由于脂肪干细胞需要培养周期,且操作复杂,临床应用性不强。SVFs作为种子细胞,具有很多优势。以胶原蛋白作为支架材料,以SVFs作为种子细胞构建工程化的脂肪组织,并且以VEGF-PLA缓释微球作为微环境细胞因子,国内外尚未见相关报道。
研究目的
1.临床指导上：希望通过探讨VEGF-PLA纳米缓释微球与SVFs对游离移植脂肪颗粒及脂肪组织工程胶原支架血管化的影响。探索一种临床应用性强的辅助脂肪移植和脂肪组织工程的细胞疗法和基因疗法手段。为下一步将科学研究应用于临床做好理论基础。
2.学术上：基因疗法的细胞因子缓释系统结合SVFs细胞疗法对游离脂肪移植及组织工程胶原支架血管化的研究,在国内外学术界尚未见报道。本研究希望通过对其机理的探讨,进一步揭示其机理,为临床应用做铺垫。
本研究的创新点是,将SVFs与细胞因子缓释系统相结合,联合SVFs和(或)VEGF-PLA缓释微球体内构建工程化脂肪组织及辅助脂肪移植。同类的研究在学术界尚未见报道。具体的研究目的如下。
1.利用酶消化法从人脂肪组织当中分离提取SVFs,进行形态观察,并进行多向诱导分化,探讨其干细胞特性及作为脂肪组织工程理想种子细胞的优势。
2.体外DiI荧光标记SVFs,荧光显微镜观察细胞标记情况并检测DiI标记对SVFs培养贴壁所得PO代细胞增殖和成脂分化的影响,探讨DiI标记作为细胞示踪标记的优势。
3.体外构建VEGF-PLA聚乳酸纳米微球缓释系统,检测样品包封率和载药量,并检测其体外缓释能力,探讨其应用于辅助脂肪颗粒移植研究的可行性。
4.设计对照组,将SVFs和(或)VEGF-PLA缓释微球联合脂肪颗粒移植进行体内实验,探讨SVFs和(或)VEGF-PLA缓释微球对体内脂肪颗粒移植成活的影响。
5.以Ⅰ型胶原固态支架为载体材料,联合SVFs和(或)VEGF-PLA缓释微球体内构建工程化脂肪组织的影响,检测其体内构建脂肪组织的血管化水平,为脂肪组织工程提供实验依据。
材料与方法
1.SVFs的体外分离培养及其培养所得未传代P0代细胞多向诱导分化鉴定
收集抽脂术患者的脂肪组织,纯化后用Ⅰ型胶原酶消化、过滤、离心、铺皿进行贴壁培养,观察细胞生长状态。用MTT法绘制细胞增殖生长曲线;用相应的定向诱导液对SVFs培养所得未传代PO代细胞向脂肪细胞、骨细胞和软骨细胞方向诱导分化,并用油红O、茜素红和阿新蓝染色进行鉴定。
2. SVFs体外DiI荧光标记及观察标记物对SVFs培养所得未传代P0代细胞生物性状的影响
荧光染料DiI体外标记SVFs,倒置显微镜及荧光显微镜观察细胞生长情况,分别用MTT法和油红O定量检测法检测DiI标记后SVFs培养所得未传代P0代细胞增殖能力和成脂分化能力,并与未标记组进行统计学比较,分析标记后细胞生物性状的改变。
3.体外构建VEGF-PLA缓释微球并检测其载药量、包封率及体外缓释特性
体外用超声乳化法构建VEGF-PLA缓释微球,检测微球载药量和包封率；透射电镜观察微球包被情况并测量直径；用ELISA试剂盒检测VEGF-PLA缓释能力,连续检测21d,分析缓释微球的特性,为下一步实验提供依据。
4.SVFs及VEGF-PLA缓释微球对脂肪颗粒移植影响的体内实验研究
分组设计：第1组移植单纯脂肪颗粒；第2组移植脂肪颗粒+SVFs,第3组移植脂肪颗粒+SVFs+VEGF-PLA缓释微球；体外将SVFs用DiI荧光标记,标记后分别按照分组将SVFs和(或)VEGF-PLA缓释微球与脂肪颗粒混匀,观察混匀后的性状,无菌条件下将混合物通过注射的方式随机注射入裸鼠皮下,每只裸鼠注射3个点。8w后取出移植物,准确测量湿重,记录数据并进行统计学比较,组织切片经HE和CD34及VEGF免疫组化染色分析,观察移植后脂肪颗粒存活情况,并观察CD34和VEGF的表达情况,显微镜下计数三组毛细血管数量,并进行统计学比较,分析SVFs和VEGF-PLA缓释微球对移植物血管化的影响。
5.VEGF-PLA缓释微球促进工程化脂肪组织血管化的影响研究
准备三组移植物,1组为SVFs+胶原支架,未体外成脂诱导。2组为SVFs+胶原支架,3组为SVFs+胶原支架+VEGF-PLA缓释微球,2组和3组体外成脂诱导3天后进行裸鼠体内移植,三组移植同一只裸鼠皮下；分别于4w后取出新生标本,通过大体观察、测量湿重、HE染色观察新生组织结构和血管生长情况,高倍镜下计数新生毛细血管密度,收集数据并进行统计学比较分析。
统计学处理
所得数据均采用SPSS16.0软件包进行统计分析,以均数±标准差(x±s)表示。P<0.05差异有统计学差异。SVFs培养贴壁生长曲线(OD值)采用曲线拟合方法分析。DiI标记对细胞增殖能力影响采用重复测量的方差分析。DiI标记后对SVFs培养所得未传代P0代细胞成脂能力影响采用独立样本T检验。透射电镜和圆二色谱法测缓释微球直径的比较采用独立样本T检验。体内脂肪移植物湿重比较采用单因素方差分析。体内脂肪移植物血管数比较采用单因素方差分析。组织工程新生物湿重比较采用单因素方差分析。组织工程新生物血管数比较比较采用单因素方差分析。结果
1.从抽脂术患者得到的脂肪组织当中成功分离得到SVFs,贴壁生长后形态类似于成纤维细胞,具有很强的分裂增殖能力,3至7天时细胞增殖快速。对所测的OD值进行曲线拟合方法分析,用Quadratic模型,R2=0.965,回归方程检验有统计学意义(F=503.047,P=0.000)。用成脂、成骨、成软骨定向分化诱导液诱导2～3周后,分别用油红O、茜素红、阿新蓝染色呈阳性反应。
2.体外用DiI成功标记SVFs,标记后细胞呈现较强的红色荧光,标记率较高,标记后细胞成活率较高,达(92.31±1.52)%。与正常的SVFs成活率(92.73±0.57)%并无差异。两组进行独立样本T检验。经方差齐性分析,两组样本的方差不齐(F=5.396,P=0.043)。两个样本之间的均值差异无统计学意义(t=-0.631,P=0.550),DiI标记对SVF细胞成活率无影响。
对OD值进行重复测量分析。Mauchly's Test of sphericity检验Mauchly'sW为0.001,P=0.018,7次测量的OD值存在相关性。Test of within subjects Effects(Greenhouse-Geisser校正)见时间因素P=0.000。时间和分组作用P=0.524,F=0.693。可见时间因素的作用有统计学意义,测量指标有随时间变化而变化的趋势,而时间因素的作用不受分组因素影响。Test of Between-Subjects Effects分组作用的F=0.062,P=0.810,可见分组因素不起作用,两组无区别。每个检测点DiI标记组和未标记组OD值差异无统计学意义(每个点P>0.05详见表2-2)。重复测量分析两组OD均数变化趋势,2组几乎重叠(详见图2-3)以上的分析结果可见DiI标记对SVFs贴壁培养的PO代细胞增值能力无影响。DiI标记后对SVFs培养所得未传代P0代细胞成脂能力也没有太大的影响,两组进行独立样本T检验。经方差齐性分析,两组样本的方差齐(F=0.794,P=0.399)。两个样本之间的均值差异无统计学意义(t＝1.562,P=0.157),DiI标记对SVF细胞成活率无影响。
3.体外成功构建VEGF-PLA缓释微球,微球表面光滑、球体大小均匀、形态饱满；微球直径平均为(27.56±4.60)nm；微球样品包封率为(89.24±1.24)%,计算样品的载药量为：{(17.85±0.25)×10-3}%；通过ELISA方法检测微球缓释特性,21d内微球可缓慢释放VEGF,第21d累计释药率为80.35%。将微球稀释后在透射电镜下测量其直径为(28.30±4.64)nm。与利用圆二色谱法测量微球直径法比较。两组进行独立样本T检验。经方差齐性分析,两组样本的方差齐(F=0.033,P=0.860)。两个样本之间的均值差异无统计学意义(t=0.253,P=0.253),两种测量方法无差异。
4.将3组混合物移植裸鼠体内8w后,裸鼠全部成活,移植物清晰可见。1组、2组和3组平均湿重分别为：(0.077±0.119)g；(0.159±0.016)g；(0.182±0.120)g。3组数据方差齐性检验,P=0.946,方差具有齐性。3组数据有显著性差异,3组间比较,F=167.678,P=0.000,3组数据差异具有统计学意义。第3组湿重高于第2组(P=0.001)。第2组湿重高于第1组(P=0.000)组织切片HE染色显示第3组脂肪细胞成活率最高,纤维性成分最少,第2组次之,第1组内部纤维性成分最多；免疫组化结果同样是第3组表达最强,第2组次之,第1组最少。血管密度计数经统计学比较分析,第1、2、3组平均新生血管数量分别为：(7.25±1.83)个/HP;(12.65±1.69)个/HP；(14.75±2.02)个/HP；3组数据方差齐性检验,Levene statistic为0.256,P=0.775,方差具有齐性。组间比较,F=87.048,P=0.000,3组数据有显著性差异。第3组血管数多于第2组(P=0.000)。第2组血管数多于第1组(P=0.001)。
5.三组移植物移植裸鼠体内4w后,动物全部成活,第2组和第3组成功构建出脂肪组织。新生物湿重测定,术后4w时三组湿重分别为：(23.28±3.86)mg,(31.16±2.19)mg,(36.67±1.60)mg,运用SPSS软件进行单因素方差分析,首先进行方差齐性检验,Levene statistic为3.044,P=0.069,方差具有齐性。三组湿重比较,差异具有显著性(F=48.768,P=0.000)。1组比2组轻,差异具有显著性(P=0.000)。2组比3组轻,差异具有显著性(P=0.001)。4w时第1组、2组和3组新生毛细血管密度平均分别为(1.81±0.83)个/HP,(2.62±0.89)个／HP,(3.44±1.09)个/HP。Levene statistic检验,方差具有齐性(P=0.318)。Levene statistic为1.177,方差具有齐性(P=0.318)。三组比较F=11.846,差异具有显著性(P=0.000)。1组比2组血管少,差异具有统计学意义(P=0.019)。2组比3组血管少,差异具有统计学意义(P=0.019)。可见VEGF-PLA缓释微球联合SVFs能够明显促进胶原支架体内成脂和血管化水平。
结论
1.抽脂术获得的脂肪组织能够成功分离提取大量的多能干细胞,经形态学、及多向分化鉴定证实为干细胞成分。该细胞容易获取、来源丰富、在相应的诱导条件下能够分化成为目的细胞,可作为脂肪组织工程理想的种子细胞。
2.DiI标记是一种较好的细胞示踪方式,标记后细胞成活率较高,不会影响SVFs培养所得未传代P0代细胞的增殖和成脂分化的能力。
3.将VEGF包被于PLA微球中,可以使微球中VEGF缓慢释放进入局部微环境,从而达到VEGF对周围细胞持续发挥作用的目的。
4. SVFs和VEGF-PLA缓释微球体内能够明显促进脂肪颗粒的成活率,同样能够明显促进脂肪颗粒移植血管化的水平,是一种辅助脂肪颗粒移植理想的种子细胞和细胞因子缓释剂。
5.VEGF-PLA缓释微球联合SVFs体内能够明显促进新生组织的血管化,与胶原支架混合移植能够构建出成熟的脂肪组织。
BACKGROUND
In recent years, with the development and application of liposuction, the autologous fat particle injection has been become more and more popular. Autologous fat transplantation has a lot of advantages, such as no rejection, low infection, good compatibility. It has been seen as an ideal way o repair soft tissue defects. As the development of adipose tissue engineering, a new way has been opened up in the field of repairing soft tissue defects without damage and in the true sense of morphological, structural and functional reconstruction completely. However, engineered adipose tissue can not be used in clinical medicine, autologous fat transplantation is still the ideal material to repair soft tissue defects.
Autologous fat transplantation has always been of clinical concern, especially the negative pressure liposuction has been widely used in clinical practice. The autologous fat transplantation is accepted by more and more clinicians and patients, and the technique in obtaining, purified, injection of fat are improved, the survival rate is also high than before. However, the absorption rate of transplanted fat particles can be as high as30%to50%, there is no good way to solve the high absorption rate, the lower survival rate of transplanted fat limits its clinical application greatly. Adipose tissue is not only the organs to save energy and endocrine, it is also the ideal alternative material of soft tissue material. Adipose tissue has been believed that the effective source of adult stem cells, it has a variety of cell composition, including progenitor cell composition, the progenitor cells can be differentiated into other cell lines. Adipose tissue mainly consists of fat cells, adipose stem cells, endothelial cells, parietal cells, fibroblasts, macrophages and extracellular matrix components, among the cell compositions, adipose cells and adipose stem cells are the research focus in recent years. The adipose-derived stem cells is considered to be a manifestation of the fat cells and vascular progenitor cells, which is located between the fat cells, perivascular or extracellular matrix can promote the outcome of the adipose tissue. Adipose-derived stem cells exist in adipose tissue stromal vascular fractions(stromal vascular fractions, SVF), while the SVF cells are the most important components in autologous fat transplantation. The mixture of a variety of cells isolated from adipose tissue by collagenase digestion, the formation of cell clusters called stromal vascular fraction(SVF). Stromal vascular fraction contains a lot of mesenchymal stem cells which can differentiate into multiple lineages, it is the ideal seed cells for regenerative medicine and tissue engineering.
Select the appropriate cell carrier as the extracellulat matrix is an important condition for SVFs and ADSCs proliferation and differentiation. Adipose tissue is rich in collagen, fibronectin, proteoglycans, and so on, it can provide the extracellular matrix. The technology of cell auxiliary fat transfer was proposed in recent years, while addressing the two issues of the transplanted fat of ADSCs content and lack of transplant SVF extracellular matrix. And at the same time, it simplified the SVFs extracted in vitro and repeatedly centrifugal, weight hanging steps on. It provides a solution to solve the problem of the transplanted fat absorption rate in the clinical.
It can enhance the vascularization of the engineered tissue, reducing the rate of absorption and improving the survival of the transplanted tissue if there are active growth factor in the tissue engineering materials. Vascular endothelial growth factor(VEGF), also known as vascular permeability factor, can promote vascular permeability, endothelial cell proliferation and migration, angiogenesis, and prolong the life of vascular endothelial cells, enhance blood vessel through permeability and change the role of the extracellular matrix and scar tissue healing, it is closely related to inflammation and tumorigenesis. There is a positive feedback between VEGF and its receptors, it can stimulate the endothelial cells high expression of VEGF and its receptors, and thus enlarge the effects of VEGF. However, VEGF can not give full play to its biological effects, because of its short half-life, degradable easily and other factors. The sustained release technology is likely to be a solution to the problem. It could entrapped VEGF in polylactide nanospheres though the technology of entrapped microspheres, so that the cytokine could release to the surrounding environment continuously and exert its biological effects. The slow release technique has great research value in adipose tissue engineering.
In recent years,with the rapid development of tissue engineering technology, people pay more attention to using tissue engineering technology to clinical practice. However, Vascularization becomes the biggest obstacle to the clinical application.Collagen scaffolds is a good choice to tissue engineering. It has been approved by FDA as artificial skin materials. It is well known that Adipose Derived Stem Cells is a good choice as a kind of ideal seed cells. However, Adipose Derived Stem Cells needs a period of time.So it is inconvenient to clinical application. Now more and more scientists have realized that SVFs as seed cells has many advantages. The research which uses collagen protein as scaffold material, SVFs as seed cells, and combines VEGF-PLA slow-release microspheres has not been found.
OBJECTIVES
1. Isolated SVFs from human adipose tissue using the method of enzyme digestion method, The cells were induced for three-line differentiation. Study its characteristics of stem cells and advantages as the ideal seed cells in adipose tissue engineering;
2. Labeled the SVFs with DiI fluorescence in vitro, observed cells'condition with fluorescence microscope, detected the effects of Dil to PO cells coming from SVFs proliferation and adipogenic differentiation. Study the advantages of DiI as the cells'tracking markers;
3. Constructed VEGF-PLA polylactide nanospheres in vitro. Detected its sample encapsulation efficiency and drug loading, and detected the sustained release capabilities. Explore the feasibility of applied assisted fat particles transplantation research;
4. Design the control group, the SVFs and (or) of VEGF-PLA sustained-release microspheres combined with fat particles transplantation in vivo experiments, explore the impact of the SVFs and (or) of VEGF-PLA sustained release microspheres to survival rate of fat particles transplantation in vivo;
5. Using the solide-state type I collagen as carrier material, with SVFs and (or) VEGF-PLA release microspheres to bulid engineered adipose tissue in vivo. Detected the level of the vascularized of engineering adipose tissue in vivo. So as to provided experimental basis to adipose tissue engineering.
1. Isolation of SVFs from adipose tissue and multilineage differentiation
SVFs were isolated from subcutaneous tissue of healthy donors who had undergone liposuction through the method of collagenase digestion. The SVFs were used to next experimental study. Draw cells proliferation curve using the method of MTT. Induced SVFs to adipogenic, osteogenic and chondrogenic differentiation with the corresponding inductive culture medium, oil O, alizarin red and alcian blue staining correspondingly.
2. Labeled the SVFs with Dil fluorescence in vitro, observed cells'condition with fluorescence microscope, detected the effects of Dil to PO cells coming from SVFs proliferation and adipogenic differentiation.
Labeled SVFs with Dil in vitro, observed cells growth condition with inverted microscope and fluorescence microscope. Detected Dil-labeled PO cells coming from SVFs proliferation ability and adipogenic differentiation capacity with MTT assay and Oil Red O quantitative assay, and comparied the data with unlabeled groups statistically, analysis it's biological traits change.
3. Construct VEGF-PLA polylactide nanospheres and investigated the effect of VEGF-PLA release nanospheres on proliferation and adipogenic induction of adipose-derived stem cells in vitro
Constructed VEGF-PLA sustained release microspheres though the method of ultrasonic emulsification in vitro. Detected the microspheres drug loading rate and encapsulation rate and observed the condition of encapsulation with transmission electron microscope, measured the diameter of microspheres under microscope. Detected the VEGF-PLA release capability using ELISA kit.
4. Effects of SVFs and VEGF-PLA release microspheres to fat particles transplantation in vivo.
Group design:group1transplantation of pure fat particles; group2transplantation of fat particles+SVFs; group3transplantation of fat particles+ SVFs+VEGF-PLA sustained release microspheres. Labeled SVFs with Dil in vitro, after that mixed SVFs and (or) VEGF-PLA release microspheres and fat particles respectively in the sterile condition. Transplanted the mixture though injection into nude mice in random, each mice injected three points. At eight weeks after the injections, the grafts were explanted, weighted, and fixed with10%paraformaldehyde, recorded data and statistical comparison. Staining the tissue section with HE and immunohistochemical for CD34and VEGF. Analysis the survival rate of fat particles and observed the expression of CD34and VEGF, counted the capillary number under microscope for three sets, and sataistical comparison with the software SPSS16.0. Analysis the effects of SVFs and VEGF-PLA release microspheres to vascularization of the transplantation.
5. Effects of VEGF-PLA sustained release microspheres to vascularization of adipose tissue engineering in vivo
Prepared three groups of grafts:group1was SVFs+collagen scaffold; group2were SVFs+collagen scaffold; group3were SVFs+collagen scaffold+VEGF-PLA sustained release microspheres. For2and3group, adipogenic induction3days in vitro. After preparation, the grafts were implanted in nude mice subcutaneously, three groups transplanted to the same mice.4weeks after implantation, the grafts were explanted and gross observation, measured the wet weight. Observed new organizational structure and vascular formed condition through HE staining, counted the new capillary density under high magnification, and statistical analysis the data collected.
STATISTICAL ANALYSIS
The data obtained was represent by mean±standard deviation (x±s), using SPSS16.0software package for statistical analysis. P＜0.05represent significant differences. We used Regression, one-way ANOVA,Independent-sample T Test,General Linear Model(Repeated measures).
RESULTS
1. SVFs was obtained successfully from adipose tissue of liposuction patients, had strong proliferate ability, similar to fibroblasts in morphology. Regresson(Quadratic), R=0.982,R2=0.965.Rgression ANOVA, F=503.047,P=0.000.It was statistically significant.After2-3weeks induction in the adipogenic, osteogenic and chondrogenic medium, stained with oil red O, alizarin red and alcian blue respectively, the results were positive.
2. Labeled SVFs with Dil in vitro successfully, the cells showed strong red fluorescence under fluorescence microscope after labeled. The rate of labeled SVFs was high, the survival rate after labeled was high too, up to (92.31±1.52)%.Comparing to the unlabeled group(92.73±0.57)%,t=-0.631, p=0.550. We used General Linear Model(Repeated measures).Mauchly's W (Mauchly's Test of sphericity)was0.001, P=0.018. Using Test of Between-Subjects Effects,F=0.062, p=0.810, Dil had no significant impact on cells proliferation, cells proliferation curve was similar before and after labeling, there was no statistical difference; Dil markers had no significant impact on P0cells coming from SVFs adipogenic capacity, the statistical comparison before and after labeled showed t=1.562, P=0.157.there was no significant differences.
3. Constructed VEGF-PLA sustained release mierospheres in vitro successfully, the surface of mirospheres was smooth, the sphere size was uniform; the average diameter of mierospheres were (27.56±4.60) nm, the rate of encapsulation was (89.24±1.24)%, the mierospheres drug loading rate was{(17.85±0.25)×10－3}%; Detected the VEGF-PLA releasing capability using ELISA kit. The miｃrospheres could release VEGF slowly within21days. At last, the cumulative release rate was80.35%.
4.8weeks after transplantation in nude mice, the mice was survived all, the grafts were visible clearly. The wet weight are (0.077±0.119) g.(0.159±0.016) g、(0.182±0.120) and comparison of three groups of P values were:among three groups P=0.000,F=167.67;group1to group2P=0.000; group2to group3P=0.001, the wet weight of group3was much higher than the other two groups, and group2was higher than group1. Tissue sections stained with HE showed that the group3had the highest survival rate, the fibrous components was the least than the other two groups, group1had the most fibrous component in the graft. The immunohistochemical findings had the similar results as HE staining, the group3had the strongest expression, then was group2, and group1had the least expression.The vessel density was:(7.25±1.83)个／HP;(12.65±1.69)个/HP;(14.75±2.02)个/HP.The statistic analysis of vessel density was:among three groups P=0.000,F=87.048; group１to group2P=0.001, group2to group3P=0.000. Group3had the highest level of blood vessel than the other groups, and group1had the least level.
5.4weeks after transplantation in nude mice, the mice was survived all, the grafts were visible clearly, and group2and group3had formed engineered adipose tissue. The wet weight are (23.28±3.86) mg.(31.16±2.19) mg、(36.67±1.60) mg. The wet weight comparison of three groups of P values were:among three groups P=0.000,F=48.768;group lto group2P=0.000; group2to group3P=0.001. The vessel density was:(1.81±0.83)个／HP;(2.62±0.89)个/HP;(3.44±1.09)个/HP.The statistic analysis of vessel density was:among three groups P=0.000,F=１1.846; group1to group2P=0.019; group2to group3P=0.019. We could see from the results above, VEGF-PLA sustained release microspheres could promote the adipogenic vascularization level of SVFs and college scaffold in vivo significantly.
CONCLUSIONS
1. SVFs were isolated from subcutaneous tissue of healthy donors who had undergone liposuction through the method of collagenase digestion. This method of isolation and cultivation of SVFs was convenient and easier to carry out. The SVFs isolated had the capabilities of pluripotential differentiation, we could had enough ADSCs for implantation in short time. Thus, SVFs could be used as ideal seed cells for adipose tissue engineering.
2. The Dil mark was suitable as SVFs tracing, the survival rate of labeled cells were high, and it did not influence cells proliferation and adipogenic differentiation ability.
3. Constructed VEGF-PLA sustained release microspheres in vitro successfully, the microspheres could release VEGF slowly, so it could affect surrounding cells continuingly.
4. SVFs and VEGF-PLA sustained release microspheres could promote the survival rate of fat particles significantly in vivo, and could promote adipose granule transplant vascularization level too. So the SVFs and VEGF-PLA sustained release microspheres could as the ideal seed cells and cutokine release agent in adipose tissue engineering.
5. VEGF-PLA sustained release microspheres combined with SVFs could promote engineered tissue vascularization significantly in vivo, and mixed transplantation with collagen scaffold could construct mature adipose tissue in vivo.
组织工程技术是近来学术界研究热点,细胞疗法和基因疗法是组织工程研究的热点,学术界一直探求一种临床实用性强的安全的辅助组织工程手段。
近年来,随着脂肪抽吸术的广泛开展和应用,自体脂肪颗粒注射移植术已经在国内外得到广泛的重视。自体脂肪颗粒以其无排斥、感染率低、注射后组织相容性好等优点,是目前修复软组织缺损比较理性的填充材料。虽然脂肪组织工程技术理论的发展,为真正实现无损伤修复软组织缺损和真正意义上形态、结构与功能重建开辟了新的途径。但是,脂肪组织工程技术目前还不能实现临床应用,所以目前修复软组织缺损较理想的填充材料是自体脂肪颗粒。
自体脂肪颗粒移植在临床上一直备受关注,尤其是负压抽脂术在临床上得到广泛的应用之后,这种移植术被越来越多的临床医生和患者所接受,并在获取、纯化、注射脂肪和提高其存活率等方面不断得到提高。但是,自体脂肪颗粒移植后吸收率可高达30%-50%,这种高吸收率的问题一直不能得到良好的解决,所以移植脂肪成活率较低极大地限制了其在临床上的广泛应用。脂肪组织不仅是储存能量及内分泌器官,还是软组织填充理想的替代物,而且还被认为是成体干细胞的有效来源,因为脂肪组织当中含有多种细胞成分,包括祖细胞成分,这些祖细胞成分可分化成为其他细胞系。脂肪组织当中主要包含脂肪细胞、脂肪干细胞、血管内皮细胞、壁细胞、成纤维细胞、巨噬细胞及细胞外基质成分等,其中脂肪细胞和脂肪干细胞是近年来研究的热点。脂肪干细胞被认为是一种表现脂肪细胞和血管细胞的祖细胞,它位于脂肪细胞之间、血管周围或细胞外基质中,可促进脂肪组织的转归。脂肪干细胞存在于脂肪组织的间质血管碎片中(stromal vascular fraction,SVF),而SVF是干细胞辅助脂肪移植中最重要的组分。通过胶原酶消化,从脂肪组织中分离的多种细胞混合物形成的细胞团就叫做间质血管碎片(SVF)。间质血管碎片中含有丰富的间充质干细胞,可分化为多种谱系,是再生医学、组织工程等最理想的种子细胞。
选择合适细胞外基质支架,作为妥当的细胞载体是组织工程的关键,是使SVFs中的ADSCs得以增殖、分化的重要条件。脂肪组织本身含有非常丰富的胶原、蛋白聚糖、纤维粘连蛋白等,这些组织是优秀的细胞外基质。这些年来热门的SVFs细胞辅助脂肪转移技术,既解决了移植脂肪中ADSCs含量低,同时又解决了移植的SVFs缺乏细胞外基质两个问题,并简化了SVFs提取的体外培养、多次离心重悬等步骤,为临床上解决移植脂肪吸收率高的难题提供了一种解决方法。
在组织工程材料中加入有活性的生长因子,能够增强工程化组织的血管化,减少吸收率,提高移植组织的成活。血管内皮生长因子(vascular endothelial growth factor,VEGF)又称为血管渗透因子,这种细胞因子具有促进血管渗透的作用,同时还可促进血管内皮细胞增殖和迁移、延长血管内皮细胞寿命、促进血管形成、增强血管通透性和改变细胞外基质等作用。除了对血管的作用,它还与创面愈合、创伤组织修复、炎症和肿瘤发生密切相关。VEGF与VEGF受体之间存在正反馈,VEGF因子局部应用可以诱导血管内皮细胞VEGF及其受体的高表达,导致VEGF效应的放大。但是VEGF在体内应用时因其半衰期短、容易降解等因素使得不能充分发挥其生物学效应。药剂学的缓释技术很好的解决这一难题,它通过微球包埋技术将VEGF包埋于聚乳酸纳米微球中,从而使这种细胞因子能够缓慢的释放,达到对周围环境持续发挥生物学效应的目的。这种缓释技术在脂肪组织工程的研究领域具有很大的研究价值。
近年来,组织工程技术的发展迅速,使得很多临床上的难题获得了一种可能性的解决方案。然而,由于组织工程的血管化问题难以解决,大部分的组织工程技术距离临床应用还有很大距离。胶原蛋白是良好的支架材料,其已被美国FDA批准作为人工皮肤材料。对于种子细胞的选择,大部分研究都选择脂肪干细胞。然而由于脂肪干细胞需要培养周期,且操作复杂,临床应用性不强。SVFs作为种子细胞,具有很多优势。以胶原蛋白作为支架材料,以SVFs作为种子细胞构建工程化的脂肪组织,并且以VEGF-PLA缓释微球作为微环境细胞因子,国内外尚未见相关报道。
研究目的
1.临床指导上：希望通过探讨VEGF-PLA纳米缓释微球与SVFs对游离移植脂肪颗粒及脂肪组织工程胶原支架血管化的影响。探索一种临床应用性强的辅助脂肪移植和脂肪组织工程的细胞疗法和基因疗法手段。为下一步将科学研究应用于临床做好理论基础。
2.学术上：基因疗法的细胞因子缓释系统结合SVFs细胞疗法对游离脂肪移植及组织工程胶原支架血管化的研究,在国内外学术界尚未见报道。本研究希望通过对其机理的探讨,进一步揭示其机理,为临床应用做铺垫。
本研究的创新点是,将SVFs与细胞因子缓释系统相结合,联合SVFs和(或)VEGF-PLA缓释微球体内构建工程化脂肪组织及辅助脂肪移植。同类的研究在学术界尚未见报道。具体的研究目的如下。
1.利用酶消化法从人脂肪组织当中分离提取SVFs,进行形态观察,并进行多向诱导分化,探讨其干细胞特性及作为脂肪组织工程理想种子细胞的优势。
2.体外DiI荧光标记SVFs,荧光显微镜观察细胞标记情况并检测DiI标记对SVFs培养贴壁所得PO代细胞增殖和成脂分化的影响,探讨DiI标记作为细胞示踪标记的优势。
3.体外构建VEGF-PLA聚乳酸纳米微球缓释系统,检测样品包封率和载药量,并检测其体外缓释能力,探讨其应用于辅助脂肪颗粒移植研究的可行性。
4.设计对照组,将SVFs和(或)VEGF-PLA缓释微球联合脂肪颗粒移植进行体内实验,探讨SVFs和(或)VEGF-PLA缓释微球对体内脂肪颗粒移植成活的影响。
5.以Ⅰ型胶原固态支架为载体材料,联合SVFs和(或)VEGF-PLA缓释微球体内构建工程化脂肪组织的影响,检测其体内构建脂肪组织的血管化水平,为脂肪组织工程提供实验依据。
材料与方法
1.SVFs的体外分离培养及其培养所得未传代P0代细胞多向诱导分化鉴定
收集抽脂术患者的脂肪组织,纯化后用Ⅰ型胶原酶消化、过滤、离心、铺皿进行贴壁培养,观察细胞生长状态。用MTT法绘制细胞增殖生长曲线;用相应的定向诱导液对SVFs培养所得未传代PO代细胞向脂肪细胞、骨细胞和软骨细胞方向诱导分化,并用油红O、茜素红和阿新蓝染色进行鉴定。
2. SVFs体外DiI荧光标记及观察标记物对SVFs培养所得未传代P0代细胞生物性状的影响
荧光染料DiI体外标记SVFs,倒置显微镜及荧光显微镜观察细胞生长情况,分别用MTT法和油红O定量检测法检测DiI标记后SVFs培养所得未传代P0代细胞增殖能力和成脂分化能力,并与未标记组进行统计学比较,分析标记后细胞生物性状的改变。
3.体外构建VEGF-PLA缓释微球并检测其载药量、包封率及体外缓释特性
体外用超声乳化法构建VEGF-PLA缓释微球,检测微球载药量和包封率；透射电镜观察微球包被情况并测量直径；用ELISA试剂盒检测VEGF-PLA缓释能力,连续检测21d,分析缓释微球的特性,为下一步实验提供依据。
4.SVFs及VEGF-PLA缓释微球对脂肪颗粒移植影响的体内实验研究
分组设计：第1组移植单纯脂肪颗粒；第2组移植脂肪颗粒+SVFs,第3组移植脂肪颗粒+SVFs+VEGF-PLA缓释微球；体外将SVFs用DiI荧光标记,标记后分别按照分组将SVFs和(或)VEGF-PLA缓释微球与脂肪颗粒混匀,观察混匀后的性状,无菌条件下将混合物通过注射的方式随机注射入裸鼠皮下,每只裸鼠注射3个点。8w后取出移植物,准确测量湿重,记录数据并进行统计学比较,组织切片经HE和CD34及VEGF免疫组化染色分析,观察移植后脂肪颗粒存活情况,并观察CD34和VEGF的表达情况,显微镜下计数三组毛细血管数量,并进行统计学比较,分析SVFs和VEGF-PLA缓释微球对移植物血管化的影响。
5.VEGF-PLA缓释微球促进工程化脂肪组织血管化的影响研究
准备三组移植物,1组为SVFs+胶原支架,未体外成脂诱导。2组为SVFs+胶原支架,3组为SVFs+胶原支架+VEGF-PLA缓释微球,2组和3组体外成脂诱导3天后进行裸鼠体内移植,三组移植同一只裸鼠皮下；分别于4w后取出新生标本,通过大体观察、测量湿重、HE染色观察新生组织结构和血管生长情况,高倍镜下计数新生毛细血管密度,收集数据并进行统计学比较分析。
统计学处理
所得数据均采用SPSS16.0软件包进行统计分析,以均数±标准差(x±s)表示。P<0.05差异有统计学差异。SVFs培养贴壁生长曲线(OD值)采用曲线拟合方法分析。DiI标记对细胞增殖能力影响采用重复测量的方差分析。DiI标记后对SVFs培养所得未传代P0代细胞成脂能力影响采用独立样本T检验。透射电镜和圆二色谱法测缓释微球直径的比较采用独立样本T检验。体内脂肪移植物湿重比较采用单因素方差分析。体内脂肪移植物血管数比较采用单因素方差分析。组织工程新生物湿重比较采用单因素方差分析。组织工程新生物血管数比较比较采用单因素方差分析。结果
1.从抽脂术患者得到的脂肪组织当中成功分离得到SVFs,贴壁生长后形态类似于成纤维细胞,具有很强的分裂增殖能力,3至7天时细胞增殖快速。对所测的OD值进行曲线拟合方法分析,用Quadratic模型,R2=0.965,回归方程检验有统计学意义(F=503.047,P=0.000)。用成脂、成骨、成软骨定向分化诱导液诱导2～3周后,分别用油红O、茜素红、阿新蓝染色呈阳性反应。
2.体外用DiI成功标记SVFs,标记后细胞呈现较强的红色荧光,标记率较高,标记后细胞成活率较高,达(92.31±1.52)%。与正常的SVFs成活率(92.73±0.57)%并无差异。两组进行独立样本T检验。经方差齐性分析,两组样本的方差不齐(F=5.396,P=0.043)。两个样本之间的均值差异无统计学意义(t=-0.631,P=0.550),DiI标记对SVF细胞成活率无影响。
对OD值进行重复测量分析。Mauchly's Test of sphericity检验Mauchly'sW为0.001,P=0.018,7次测量的OD值存在相关性。Test of within subjects Effects(Greenhouse-Geisser校正)见时间因素P=0.000。时间和分组作用P=0.524,F=0.693。可见时间因素的作用有统计学意义,测量指标有随时间变化而变化的趋势,而时间因素的作用不受分组因素影响。Test of Between-Subjects Effects分组作用的F=0.062,P=0.810,可见分组因素不起作用,两组无区别。每个检测点DiI标记组和未标记组OD值差异无统计学意义(每个点P>0.05详见表2-2)。重复测量分析两组OD均数变化趋势,2组几乎重叠(详见图2-3)以上的分析结果可见DiI标记对SVFs贴壁培养的PO代细胞增值能力无影响。DiI标记后对SVFs培养所得未传代P0代细胞成脂能力也没有太大的影响,两组进行独立样本T检验。经方差齐性分析,两组样本的方差齐(F=0.794,P=0.399)。两个样本之间的均值差异无统计学意义(t＝1.562,P=0.157),DiI标记对SVF细胞成活率无影响。
3.体外成功构建VEGF-PLA缓释微球,微球表面光滑、球体大小均匀、形态饱满；微球直径平均为(27.56±4.60)nm；微球样品包封率为(89.24±1.24)%,计算样品的载药量为：{(17.85±0.25)×10-3}%；通过ELISA方法检测微球缓释特性,21d内微球可缓慢释放VEGF,第21d累计释药率为80.35%。将微球稀释后在透射电镜下测量其直径为(28.30±4.64)nm。与利用圆二色谱法测量微球直径法比较。两组进行独立样本T检验。经方差齐性分析,两组样本的方差齐(F=0.033,P=0.860)。两个样本之间的均值差异无统计学意义(t=0.253,P=0.253),两种测量方法无差异。
4.将3组混合物移植裸鼠体内8w后,裸鼠全部成活,移植物清晰可见。1组、2组和3组平均湿重分别为：(0.077±0.119)g；(0.159±0.016)g；(0.182±0.120)g。3组数据方差齐性检验,P=0.946,方差具有齐性。3组数据有显著性差异,3组间比较,F=167.678,P=0.000,3组数据差异具有统计学意义。第3组湿重高于第2组(P=0.001)。第2组湿重高于第1组(P=0.000)组织切片HE染色显示第3组脂肪细胞成活率最高,纤维性成分最少,第2组次之,第1组内部纤维性成分最多；免疫组化结果同样是第3组表达最强,第2组次之,第1组最少。血管密度计数经统计学比较分析,第1、2、3组平均新生血管数量分别为：(7.25±1.83)个/HP;(12.65±1.69)个/HP；(14.75±2.02)个/HP；3组数据方差齐性检验,Levene statistic为0.256,P=0.775,方差具有齐性。组间比较,F=87.048,P=0.000,3组数据有显著性差异。第3组血管数多于第2组(P=0.000)。第2组血管数多于第1组(P=0.001)。
5.三组移植物移植裸鼠体内4w后,动物全部成活,第2组和第3组成功构建出脂肪组织。新生物湿重测定,术后4w时三组湿重分别为：(23.28±3.86)mg,(31.16±2.19)mg,(36.67±1.60)mg,运用SPSS软件进行单因素方差分析,首先进行方差齐性检验,Levene statistic为3.044,P=0.069,方差具有齐性。三组湿重比较,差异具有显著性(F=48.768,P=0.000)。1组比2组轻,差异具有显著性(P=0.000)。2组比3组轻,差异具有显著性(P=0.001)。4w时第1组、2组和3组新生毛细血管密度平均分别为(1.81±0.83)个/HP,(2.62±0.89)个／HP,(3.44±1.09)个/HP。Levene statistic检验,方差具有齐性(P=0.318)。Levene statistic为1.177,方差具有齐性(P=0.318)。三组比较F=11.846,差异具有显著性(P=0.000)。1组比2组血管少,差异具有统计学意义(P=0.019)。2组比3组血管少,差异具有统计学意义(P=0.019)。可见VEGF-PLA缓释微球联合SVFs能够明显促进胶原支架体内成脂和血管化水平。
结论
1.抽脂术获得的脂肪组织能够成功分离提取大量的多能干细胞,经形态学、及多向分化鉴定证实为干细胞成分。该细胞容易获取、来源丰富、在相应的诱导条件下能够分化成为目的细胞,可作为脂肪组织工程理想的种子细胞。
2.DiI标记是一种较好的细胞示踪方式,标记后细胞成活率较高,不会影响SVFs培养所得未传代P0代细胞的增殖和成脂分化的能力。
3.将VEGF包被于PLA微球中,可以使微球中VEGF缓慢释放进入局部微环境,从而达到VEGF对周围细胞持续发挥作用的目的。
4. SVFs和VEGF-PLA缓释微球体内能够明显促进脂肪颗粒的成活率,同样能够明显促进脂肪颗粒移植血管化的水平,是一种辅助脂肪颗粒移植理想的种子细胞和细胞因子缓释剂。
5.VEGF-PLA缓释微球联合SVFs体内能够明显促进新生组织的血管化,与胶原支架混合移植能够构建出成熟的脂肪组织。
BACKGROUND
In recent years, with the development and application of liposuction, the autologous fat particle injection has been become more and more popular. Autologous fat transplantation has a lot of advantages, such as no rejection, low infection, good compatibility. It has been seen as an ideal way o repair soft tissue defects. As the development of adipose tissue engineering, a new way has been opened up in the field of repairing soft tissue defects without damage and in the true sense of morphological, structural and functional reconstruction completely. However, engineered adipose tissue can not be used in clinical medicine, autologous fat transplantation is still the ideal material to repair soft tissue defects.
Autologous fat transplantation has always been of clinical concern, especially the negative pressure liposuction has been widely used in clinical practice. The autologous fat transplantation is accepted by more and more clinicians and patients, and the technique in obtaining, purified, injection of fat are improved, the survival rate is also high than before. However, the absorption rate of transplanted fat particles can be as high as30%to50%, there is no good way to solve the high absorption rate, the lower survival rate of transplanted fat limits its clinical application greatly. Adipose tissue is not only the organs to save energy and endocrine, it is also the ideal alternative material of soft tissue material. Adipose tissue has been believed that the effective source of adult stem cells, it has a variety of cell composition, including progenitor cell composition, the progenitor cells can be differentiated into other cell lines. Adipose tissue mainly consists of fat cells, adipose stem cells, endothelial cells, parietal cells, fibroblasts, macrophages and extracellular matrix components, among the cell compositions, adipose cells and adipose stem cells are the research focus in recent years. The adipose-derived stem cells is considered to be a manifestation of the fat cells and vascular progenitor cells, which is located between the fat cells, perivascular or extracellular matrix can promote the outcome of the adipose tissue. Adipose-derived stem cells exist in adipose tissue stromal vascular fractions(stromal vascular fractions, SVF), while the SVF cells are the most important components in autologous fat transplantation. The mixture of a variety of cells isolated from adipose tissue by collagenase digestion, the formation of cell clusters called stromal vascular fraction(SVF). Stromal vascular fraction contains a lot of mesenchymal stem cells which can differentiate into multiple lineages, it is the ideal seed cells for regenerative medicine and tissue engineering.
Select the appropriate cell carrier as the extracellulat matrix is an important condition for SVFs and ADSCs proliferation and differentiation. Adipose tissue is rich in collagen, fibronectin, proteoglycans, and so on, it can provide the extracellular matrix. The technology of cell auxiliary fat transfer was proposed in recent years, while addressing the two issues of the transplanted fat of ADSCs content and lack of transplant SVF extracellular matrix. And at the same time, it simplified the SVFs extracted in vitro and repeatedly centrifugal, weight hanging steps on. It provides a solution to solve the problem of the transplanted fat absorption rate in the clinical.
It can enhance the vascularization of the engineered tissue, reducing the rate of absorption and improving the survival of the transplanted tissue if there are active growth factor in the tissue engineering materials. Vascular endothelial growth factor(VEGF), also known as vascular permeability factor, can promote vascular permeability, endothelial cell proliferation and migration, angiogenesis, and prolong the life of vascular endothelial cells, enhance blood vessel through permeability and change the role of the extracellular matrix and scar tissue healing, it is closely related to inflammation and tumorigenesis. There is a positive feedback between VEGF and its receptors, it can stimulate the endothelial cells high expression of VEGF and its receptors, and thus enlarge the effects of VEGF. However, VEGF can not give full play to its biological effects, because of its short half-life, degradable easily and other factors. The sustained release technology is likely to be a solution to the problem. It could entrapped VEGF in polylactide nanospheres though the technology of entrapped microspheres, so that the cytokine could release to the surrounding environment continuously and exert its biological effects. The slow release technique has great research value in adipose tissue engineering.
In recent years,with the rapid development of tissue engineering technology, people pay more attention to using tissue engineering technology to clinical practice. However, Vascularization becomes the biggest obstacle to the clinical application.Collagen scaffolds is a good choice to tissue engineering. It has been approved by FDA as artificial skin materials. It is well known that Adipose Derived Stem Cells is a good choice as a kind of ideal seed cells. However, Adipose Derived Stem Cells needs a period of time.So it is inconvenient to clinical application. Now more and more scientists have realized that SVFs as seed cells has many advantages. The research which uses collagen protein as scaffold material, SVFs as seed cells, and combines VEGF-PLA slow-release microspheres has not been found.
OBJECTIVES
1. Isolated SVFs from human adipose tissue using the method of enzyme digestion method, The cells were induced for three-line differentiation. Study its characteristics of stem cells and advantages as the ideal seed cells in adipose tissue engineering;
2. Labeled the SVFs with DiI fluorescence in vitro, observed cells'condition with fluorescence microscope, detected the effects of Dil to PO cells coming from SVFs proliferation and adipogenic differentiation. Study the advantages of DiI as the cells'tracking markers;
3. Constructed VEGF-PLA polylactide nanospheres in vitro. Detected its sample encapsulation efficiency and drug loading, and detected the sustained release capabilities. Explore the feasibility of applied assisted fat particles transplantation research;
4. Design the control group, the SVFs and (or) of VEGF-PLA sustained-release microspheres combined with fat particles transplantation in vivo experiments, explore the impact of the SVFs and (or) of VEGF-PLA sustained release microspheres to survival rate of fat particles transplantation in vivo;
5. Using the solide-state type I collagen as carrier material, with SVFs and (or) VEGF-PLA release microspheres to bulid engineered adipose tissue in vivo. Detected the level of the vascularized of engineering adipose tissue in vivo. So as to provided experimental basis to adipose tissue engineering.
1. Isolation of SVFs from adipose tissue and multilineage differentiation
SVFs were isolated from subcutaneous tissue of healthy donors who had undergone liposuction through the method of collagenase digestion. The SVFs were used to next experimental study. Draw cells proliferation curve using the method of MTT. Induced SVFs to adipogenic, osteogenic and chondrogenic differentiation with the corresponding inductive culture medium, oil O, alizarin red and alcian blue staining correspondingly.
2. Labeled the SVFs with Dil fluorescence in vitro, observed cells'condition with fluorescence microscope, detected the effects of Dil to PO cells coming from SVFs proliferation and adipogenic differentiation.
Labeled SVFs with Dil in vitro, observed cells growth condition with inverted microscope and fluorescence microscope. Detected Dil-labeled PO cells coming from SVFs proliferation ability and adipogenic differentiation capacity with MTT assay and Oil Red O quantitative assay, and comparied the data with unlabeled groups statistically, analysis it's biological traits change.
3. Construct VEGF-PLA polylactide nanospheres and investigated the effect of VEGF-PLA release nanospheres on proliferation and adipogenic induction of adipose-derived stem cells in vitro
Constructed VEGF-PLA sustained release microspheres though the method of ultrasonic emulsification in vitro. Detected the microspheres drug loading rate and encapsulation rate and observed the condition of encapsulation with transmission electron microscope, measured the diameter of microspheres under microscope. Detected the VEGF-PLA release capability using ELISA kit.
4. Effects of SVFs and VEGF-PLA release microspheres to fat particles transplantation in vivo.
Group design:group1transplantation of pure fat particles; group2transplantation of fat particles+SVFs; group3transplantation of fat particles+ SVFs+VEGF-PLA sustained release microspheres. Labeled SVFs with Dil in vitro, after that mixed SVFs and (or) VEGF-PLA release microspheres and fat particles respectively in the sterile condition. Transplanted the mixture though injection into nude mice in random, each mice injected three points. At eight weeks after the injections, the grafts were explanted, weighted, and fixed with10%paraformaldehyde, recorded data and statistical comparison. Staining the tissue section with HE and immunohistochemical for CD34and VEGF. Analysis the survival rate of fat particles and observed the expression of CD34and VEGF, counted the capillary number under microscope for three sets, and sataistical comparison with the software SPSS16.0. Analysis the effects of SVFs and VEGF-PLA release microspheres to vascularization of the transplantation.
5. Effects of VEGF-PLA sustained release microspheres to vascularization of adipose tissue engineering in vivo
Prepared three groups of grafts:group1was SVFs+collagen scaffold; group2were SVFs+collagen scaffold; group3were SVFs+collagen scaffold+VEGF-PLA sustained release microspheres. For2and3group, adipogenic induction3days in vitro. After preparation, the grafts were implanted in nude mice subcutaneously, three groups transplanted to the same mice.4weeks after implantation, the grafts were explanted and gross observation, measured the wet weight. Observed new organizational structure and vascular formed condition through HE staining, counted the new capillary density under high magnification, and statistical analysis the data collected.
STATISTICAL ANALYSIS
The data obtained was represent by mean±standard deviation (x±s), using SPSS16.0software package for statistical analysis. P＜0.05represent significant differences. We used Regression, one-way ANOVA,Independent-sample T Test,General Linear Model(Repeated measures).
RESULTS
1. SVFs was obtained successfully from adipose tissue of liposuction patients, had strong proliferate ability, similar to fibroblasts in morphology. Regresson(Quadratic), R=0.982,R2=0.965.Rgression ANOVA, F=503.047,P=0.000.It was statistically significant.After2-3weeks induction in the adipogenic, osteogenic and chondrogenic medium, stained with oil red O, alizarin red and alcian blue respectively, the results were positive.
2. Labeled SVFs with Dil in vitro successfully, the cells showed strong red fluorescence under fluorescence microscope after labeled. The rate of labeled SVFs was high, the survival rate after labeled was high too, up to (92.31±1.52)%.Comparing to the unlabeled group(92.73±0.57)%,t=-0.631, p=0.550. We used General Linear Model(Repeated measures).Mauchly's W (Mauchly's Test of sphericity)was0.001, P=0.018. Using Test of Between-Subjects Effects,F=0.062, p=0.810, Dil had no significant impact on cells proliferation, cells proliferation curve was similar before and after labeling, there was no statistical difference; Dil markers had no significant impact on P0cells coming from SVFs adipogenic capacity, the statistical comparison before and after labeled showed t=1.562, P=0.157.there was no significant differences.
3. Constructed VEGF-PLA sustained release mierospheres in vitro successfully, the surface of mirospheres was smooth, the sphere size was uniform; the average diameter of mierospheres were (27.56±4.60) nm, the rate of encapsulation was (89.24±1.24)%, the mierospheres drug loading rate was{(17.85±0.25)×10－3}%; Detected the VEGF-PLA releasing capability using ELISA kit. The miｃrospheres could release VEGF slowly within21days. At last, the cumulative release rate was80.35%.
4.8weeks after transplantation in nude mice, the mice was survived all, the grafts were visible clearly. The wet weight are (0.077±0.119) g.(0.159±0.016) g、(0.182±0.120) and comparison of three groups of P values were:among three groups P=0.000,F=167.67;group1to group2P=0.000; group2to group3P=0.001, the wet weight of group3was much higher than the other two groups, and group2was higher than group1. Tissue sections stained with HE showed that the group3had the highest survival rate, the fibrous components was the least than the other two groups, group1had the most fibrous component in the graft. The immunohistochemical findings had the similar results as HE staining, the group3had the strongest expression, then was group2, and group1had the least expression.The vessel density was:(7.25±1.83)个／HP;(12.65±1.69)个/HP;(14.75±2.02)个/HP.The statistic analysis of vessel density was:among three groups P=0.000,F=87.048; group１to group2P=0.001, group2to group3P=0.000. Group3had the highest level of blood vessel than the other groups, and group1had the least level.
5.4weeks after transplantation in nude mice, the mice was survived all, the grafts were visible clearly, and group2and group3had formed engineered adipose tissue. The wet weight are (23.28±3.86) mg.(31.16±2.19) mg、(36.67±1.60) mg. The wet weight comparison of three groups of P values were:among three groups P=0.000,F=48.768;group lto group2P=0.000; group2to group3P=0.001. The vessel density was:(1.81±0.83)个／HP;(2.62±0.89)个/HP;(3.44±1.09)个/HP.The statistic analysis of vessel density was:among three groups P=0.000,F=１1.846; group1to group2P=0.019; group2to group3P=0.019. We could see from the results above, VEGF-PLA sustained release microspheres could promote the adipogenic vascularization level of SVFs and college scaffold in vivo significantly.
CONCLUSIONS
1. SVFs were isolated from subcutaneous tissue of healthy donors who had undergone liposuction through the method of collagenase digestion. This method of isolation and cultivation of SVFs was convenient and easier to carry out. The SVFs isolated had the capabilities of pluripotential differentiation, we could had enough ADSCs for implantation in short time. Thus, SVFs could be used as ideal seed cells for adipose tissue engineering.
2. The Dil mark was suitable as SVFs tracing, the survival rate of labeled cells were high, and it did not influence cells proliferation and adipogenic differentiation ability.
3. Constructed VEGF-PLA sustained release microspheres in vitro successfully, the microspheres could release VEGF slowly, so it could affect surrounding cells continuingly.
4. SVFs and VEGF-PLA sustained release microspheres could promote the survival rate of fat particles significantly in vivo, and could promote adipose granule transplant vascularization level too. So the SVFs and VEGF-PLA sustained release microspheres could as the ideal seed cells and cutokine release agent in adipose tissue engineering.
5. VEGF-PLA sustained release microspheres combined with SVFs could promote engineered tissue vascularization significantly in vivo, and mixed transplantation with collagen scaffold could construct mature adipose tissue in vivo.
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