带血管肌筋膜包埋脂肪干细胞载体复合物构建血管化组织工程脂肪的实验研究
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摘要
[背景及目的]
外伤、感染或肿瘤切除等各种原因造成机体局部皮肤软组织的缺损,尤其是颜面部肿瘤或乳腺肿瘤的切除常致整个器官的缺失。这种器官的缺失及局部软组织缺损所致的形态畸形和功能障碍,目前临床上多以自体组织(皮瓣或肌皮瓣)及人工材料进行修复或充填,尽管这能有效地改善受区的组织缺损状况,但它们均存在着许多众所周知的缺点和不足。例如:自体组织的来源不足、皮瓣的血供问题以及由于供区损伤带来的继发性畸形等。因此,其临床应用受到极大的限制。此外,近年来还有其它多种多样的方法也被尝试,包括自体真皮移植,自体脂肪游离移植,胶原注射,人工材料充填等等。然而这些方法的治疗效果也不十分理想:例如人工材料的移植排斥反应,游离脂肪移植后的吸收问题给治疗效果带来不可预知性等。尽管修复软组织缺损的方法较多,却难以完全满足临床的需要。组织工程技术的出现为临床上更好的解决组织器官缺损等难题提供了一个全新的思路。
组织工程技术能以少量种子细胞,经体外扩增后与生物材料复合植入体内,修复较大的组织或器官缺损,重建生理功能,为真正实现无损伤修复组织缺损和真正意义上的形态、结构与功能重建开辟了全新的途径。组织工程学的发展需要几个必备的因素,即:(1)可获得的种子细胞,并能控制其生长和组织形成;(2)结构精确的三维支架,以适合再造需要的组织量和形状,而且与细胞具有良好的生物相容性;(3)适宜的微环境,能提供充分的血供和营养,维持细胞增殖和功能。
目前已证实脂肪组织中存在类似骨髓基质细胞的间充质干细胞,且被命名为脂肪组织来源的干细胞(Adipose-derived stem cells,ADSCs)。这种干细胞经证实同样具有体内外多向分化潜能,如能向成熟脂肪细胞、成骨细胞、软骨细胞、肌肉细胞及心肌细胞定向转化,对多种组织的损伤具有良好的修复作用,而且取材方便,获取量大,不易造成供区继发畸形等,有望成为组织工程研究中的重要种子细胞。
组织工程常用的支架材料分为天然材料和人工合成材料,天然材料包括:天然多糖类材料有纤维素、甲壳素、透明质酸等;天然蛋白质类材料有胶原和纤维蛋白等;人工合成的材料有聚乳酸(PLA)、聚乙醇酸(PGA)或两者聚合物聚乳酸-聚乙醇酸(PLGA)、聚四氟乙烯、羟基磷灰石、聚乙烯醇、海藻酸盐等。根据它们各自特点,已被不同程度地应用于组织工程的研究中。由于应用脂肪组织工程技术修复软组织缺损近年才引起学者们的关注,故而用于构建脂肪组织工程有效的生物材料支架研究不多,主要为可降解的多孔泡沫,如PLGA、透明质酸、膨体聚四氟乙烯等。然而,究竟何种材料能成为脂肪组织工程种子细胞的理想载体尚不十分明确;同时关于脂肪组织工程血管化的研究国尚内罕见报道,因此,对这一课题进行广泛深入的研究具有深远意义。
本课题研究了成年兔ADSCs向脂肪细胞、成骨细胞及软骨细胞的体外定向诱导分化能力,同时判断ADSCs与海绵状Ⅰ型胶原支架复合后的生物相容性;进而采用BrdU标记的ADCSs作为种子细胞,以海绵状Ⅰ型胶原蛋白为支架,通过观察带血管肌筋膜包裹方法对移植物种子细胞的成活作用影响,以探讨ADSCs-载体复合物在体内成脂效应的理想途径和构建工程化、血管化的脂肪组织的适宜方法,为临床上自体脂肪组织移植修复软组织缺损及脂肪干细胞的有效移植提供理论依据。
[材料与方法]
1、成年兔脂肪干细胞(ADSCs)的分离培养与鉴定
切取成年兔腹股沟皮下脂肪垫,使用酶消化法进行原代细胞培养及传代培养,观察细胞形态及功能变化,细胞传至第3代供实验使用。采用细胞免疫荧光染色检测细胞表面与干细胞相关的部分分子如CD29、CD34、CD44及CD49d,确定细胞类型;同时在体外对细胞进行向脂肪细胞、成骨细胞及软骨细胞诱导分化,并分别以油红O染色、茜素红染色及阿利辛蓝染色进行定性鉴定。
2、BrdU体外脂肪干细胞标记及观察标记物对细胞生物学性状的影响
BrdU体外标记第3代ADSCs,取P3代细胞以终浓度分别为5、10、15和20μmol/L的BrdU进行标记,分别记为A、B、C、D组;另一组不含BrdU,作为空白对照组E。标记12、24、48和72h后,采用免疫荧光染色法检测各组细胞阳性标记率,以确定BrdU的最佳标记浓度及最佳标记时间;相差显微镜及荧光显微镜观察细胞生长情况及传代后细胞标记率的变化;检测ADSCs标记后向脂肪细胞分化的能力;使用MTT比色分析法检测标记后细胞的增殖及毒性情况。
3、成年兔ADSCs与海绵状Ⅰ型胶原蛋白生物相容性的检测
将标记后的P3代ADSCs与Ⅰ型胶原蛋白海绵支架体外联合培养,采用两种不同的混合方法(细胞直接滴加于支架表面法和细胞注射于支架内部法),测定细胞在支架上的黏附率,相差显微镜及电子显微镜动态观察细胞在支架上黏附、伸展、生长和增殖情况;检测ADSCs与支架复合后向脂肪细胞分化的能力;使用MTT比色分析法检测接种在支架的细胞的增殖情况。
4、带血管肌筋膜包埋ADSCs-载体复合物构建血管化组织工程脂肪的体内研究
经解剖分离成年兔两侧背阔肌肌皮瓣,分离形成带血管蒂的肌筋膜囊,分别包裹成脂诱导前、后的ADSCs与Ⅰ型胶原蛋白载体复合物(A组,n=10和B组,n=10),另将同样大小的复合物植入同一动物右侧臀大肌肌筋膜囊(无血管蒂)作为对照组(C组,n=10);而空支架组(D组)即不含细胞成份的空支架植入兔左侧臀大肌肌筋膜囊,各组实验均采用自身对照。术后8W取材,经油红O染色、HE染色和免疫组织荧光染色确定新生组织的性质;测定三组新生组织的湿重和微血管密度,分析评价带血管肌筋膜包裹方法对促进移植物血管化与种子细胞成活的影响。
[结果]
1.原代培养的ADSCs形态类似于成纤维细胞,具有很强的增殖及多向分化能力。分别在脂肪细胞诱导分化培养基、成骨细胞诱导分化培养基及软骨细胞诱导分化培养基的作用下,能分化出成熟的脂肪细胞、成骨细胞和软骨细胞;分别以油红O染色、茜素红染色及阿利辛蓝染色呈阳性。免疫荧光检测到细胞表面抗原分子CD29、CD44、CD49d表达呈阳性。
2.第3代ADSCs经BrdU标记后进行免疫荧光染色,在荧光显微镜下胞核呈绿色荧光。总的看来,随着标记时间的延长和BrdU剂量的增加,标记率逐渐增高,BrdU终浓度为15μmol/L且标记48h后细胞标记率＞90%,但标记时间继续延长和BrdU浓度继续增加,细胞标记率无明显增高。经统计分析表明BrdU标记ADSCs的最佳终浓度为15μmol/L,最佳标记时间为48h。与正常未标记细胞比较,标记后细胞的形态及生长增殖状况无明显差别,活细胞数＞98%。细胞标记后不影响向脂肪细胞分化的能力。
3.ADSCs可以在海绵状Ⅰ型胶原蛋白支架上生长并逐渐黏附,细胞未经成脂诱导前,ADSCs与支架的混合方式不同,细胞在支架上的黏附率也不同:ADSCs在支架表面滴加组(A组,n=6)的细胞黏附率为79.5%±2.5%;ADSCs在支架内部注射组(B组,n=6)的细胞黏附率92.7%±2.2%,A、B两组采用独立样本t检验比较差异有统计学意义(t=6.965,P=0.002)。而ADSCs成脂诱导后注射于支架内部的黏附率为91.3%±1.5%(C组,n=6),B、C两组采用独立样本t检验比较差异无统计学意义(t=0.889,P=0.424)。ADSCs与支架混合培养后不影响细胞的形态、生长增殖状况,对细胞无毒性反应;细胞与支架复合后不影响向脂肪细胞分化的能力。
4.术后8W取材发现,A、B、C三组于移植物包埋区均可观察到新生组织形成;空白对照组(D组)未见新生组织形成。A组(n=10)新生组织平均湿重为0.0230g±0.0016g;B组(n=10)新生组织平均湿重为0.0251g±0.0019g;C组(n=10)新生组织平均湿重为0.0202g±0.0014g,A组与B组、B组与C组及A组与C组之间湿重比较,差异有统计学意义(P均＜0.05)。HE染色及油红O染色均证实新生组织为成熟脂肪组织,胶原支架已完全吸收降解;免疫荧光染色阳性证实新生组织为外源性。各组新生微血管密度分别为:A组(n=10)28.5±3.7;B组(n=10)31.2±4.5;C组(n=10)19.3±2.6;A与C,B与C之间新生微血管密度比较,差异有统计学意义(P均＜0.05)。
[结论]
1、本实验成功地从成年兔皮下脂肪组织中分离培养出脂肪干细胞(ADSCs)。建立了一整套有关ADSCs分离、培养和鉴定的较简便的方法。ADSCs具有很强的增殖和自我更新能力。ADSCs具有向脂肪细胞、成骨细胞和软骨细胞分化的潜能。ADSCs来源丰富、取材容易,创伤小,是理想的组织工程种子细胞,具有广阔的应用前景。
2、BrdU标记ADSCs的最佳时间为48小时;最佳终浓度为15μmol/L;标记阳性率＞90%。BrdU对细胞无毒副作用,安全性高,对细胞的生长增殖及成脂分化无明显影响。
3、ADSCs可以在海绵状Ⅰ型胶原蛋白支架上逐渐黏附,细胞黏附率高,生长、增殖良好。支架对细胞无毒性反应。胶原蛋白材料与ADSCs之间具有良好的生物相容性。
4、带血管蒂的肌筋膜瓣包裹法能够更好地促进移植物种子细胞的成活及血管化过程。利用带血管肌筋膜包裹ADSCs与支架材料复合物可构建出血管化组织工程脂肪。
5、在所构建的血管化组织工程脂肪中,外源性植入的ADSCs和来源于血管蒂的血管内皮共同参与了新生脂肪组织的血管化过程。
[Background and Objective]
Factors including trauma,infection and tumors result in the defects of skin and soft tissue especially the tumors excision of the facial surface and breasts often to cause defects of the whole organs.At present clinically,autologous transplantation (skin flaps,myocutaneous flaps) and artificial substitutes were used to repair or fill the defects of soft tissue and surface organs.Although with promising prospects and encouraging results,there's limits such as short of autologous tissue,lack of blood supply and possibility of donor site morbidity,which greatly restrict the application of these techniques.Moreover,other methods including autologous dermis transplantation,autologous fat transplantation and collagen injection,filled with artificial material,and so on.However each method carries considerable disadvantages: for example,synthetic materials invariably result in foreign body reactions,and free adipose tissue grafts shrink to an unpredictable extent.Althogh kinds of methods were used for the repair of soft tissue deformities,No methods was proved to be ideal. Emerging tissue engineering strategies represent an innovative potential solution to many clinical challenges.
Tissue engineering involves there items:(1) seed cells,whose growth is needed to be under strict control and high proliferation is alsoessential.(2) Three-dimensional scaffold structure,which is needed to be degradable and to maintain stable volume and shape.Moreover,good compatibility with seed cells is also indispensabile;(3) appropriate micro-environment,which can provide adequate blood supply and nutrition,ensure cell proliferation and functions.
Previous studies have identified a putative stem cell population within adipose stromal compartment.This cell population,termed adipose-derived stem cells (ADSCs),can be isolated from adipose tissue.ADSCs can differentiate toward the multiple lineages like adipocyte,Cartilage cells,muscle cells and cardiomyocyte. ADSCs are easier to obtain,carry a relatively lower donor site morbidity,and are available in large numbers of stem cells at harvest.Thus,the use of adult ADSCs as the seed cell of to engineer adipose tissue seems to be more inspiring.
In general,Scaffolds include synthetic materials and natural materials.Several popular scaffolds like polylactic acid(PLA),polyglycolic acid(PGA) or both poly lactic-co-glycolic acid(PLGA),PTFE,Hydroxyapatite,polyvinyl alcohol,alginate etal were used to perform multiple tissue engineering.Adipose tissue engineering is a new field so only a few scaffolds were tried including the porous biodegradable materials like PLGA,hyaluronic acid and ePTFE.But no consensus was made of which materials are most ideal for adipose tissue engineering.Considering the lack of research about vascularized engineering adipose tissue,so systemic further researches on it may be of great importance.
We investigage the adipogenic,osteogenetic and chondrogenetic differentiation in vitro about the ADSCs which were harvested from adult rabbit's groin subcutaneous fat,and then to assess the biocompatibility of three-dimensional porous type Icollagen sponge scaffolds with ADSCs.Othe other hand,BrdU was used for labeling the ADSCs,then to explore the adipogenic efficacy and the survival improvement of the ADSCs attched scaffold by encapsulated in muscular fasciae with axial pattern blood vessel.This paper's objective is to probe an ideal method of construction of a new vascularized tissue-engineered adipose which could be applied clinically for repairing the defects of soft tissue.
[Material and Methods]
1.Isolation,expansion and differentiation of rabbit ADSCs
The inguinal fat pads were harvested by exairesis from the rabbits(The Southern Medical University institutional ethics committee approved all experiments in advance),and then enzyme digestion method were used for primary culture and observe changes in cell morphology and function of cells.Only cultured cells for 3 passage were used in this study in vivo.The ADSCs were detected the molecular expression which are associated with the surface of the stem cells such as CD29,CD34,CD44 and CD49d by using cellular immunofluorescence method.We make these cells to adipogenic,osteogenic and chondrogenic differentiation in vitro, then Oil Red O staining and Alizarin red staining and Alcian staining to identify the success of differentiation in order to identify the success of multi-directional differentiation.
2.Changes of biological properties of ADSCs labeled with BrdU marker in vitro
The three passage of ADSCs was incubated with BrdU at different concentrations(0,5,10,15 and 20μmol/L) for incubating time(12,24,48 and 72 h),to identify the optimal BrdU concentration and incubating time for cell labeling. Immunofluorescence and trypanblau strain were performed respectively to calculate the labeling index(positive rate) and the cells' activity for different time after BrdU labeling in vitro.Then we compare the difference of adipogenic potentiality before and after BrdU labeling in vitro.MTT colorimetric analysis was used to detect of the rate of cell proliferation and cytotoxicity of BrdU.
3.Biocompatibility of ADSCs attached with typeⅠcollagen sponge scaffold in vitro
ADSCs after BrdU labelling attached to the typeⅠcollagen sponge scaffold to form compounds respectively,then contrast phase microscope was used to observe cells growth,adhesion or expansion.Whereafter,we observed the cells disposition and proliferation under scanning electron microscope(SEM) in vitro.We determine the adhesion rates of ADSCs before and after marked by BrdU.Then we compare the difference of adipogenic potentiality between single ADSCs and ADSCs combine with collagen in vitro.MTT colorimetric analysis is used to detect of the rate of cell proliferation.
4.Experimental research of vascularized tissue-engineered adipose via the
ADSCs-attached scaffolds encapsulated in muscular fasciae with axial pattern blood vessels.
All animals(n=10) procedures were performed in accordance with the guidelines of the Southern Medical University Animal Care and Use Committee. Own control was carried out at all the rabbits(n=10).TypeⅠcollagen sponge (10mm×10mm×5mm) containing ADSCs(1×10~7 cell population and without adipogenic differentiated conditions,group A) was transplanted into the fascia musculares sac with blood vessel pedicle of left latissimus dorsi myocutaneous flaps, and the ADSCs(the same cell population and with adipogenic differentiated conditions,group B) attached same size collagen sponge was transplanted into the fascia musculares sac with blood vessel pedicle of right latissimus dorsi myocutaneous flaps at the same rabbits.Another same size collagen sponge containing adipogenic differentiated ADSCs(the same cell population) were also implanted into the random fascia musculares sac without blood vessel pedicle of right gluteus maximus(group C,Control).The rabbits were fed routinely after operation. Euthanasia was performed at the animals after 8 weeks of the ADSCs with scaffolds transplantation.No animal from either the study or control group died during the study period.The neogenetic tissue were observed macroscopically.Then they were dissected out and weighed their humid weight by electronic balance (fidelity=0.0001g).The tissue were embedded in paraffin for conventional Oil red O stain,HE stain and immunofluorescence staining in order to perform histological assessment.Neovascularization was assessed by measuring the number of capillaries in 10 fields of HE stain slide.To analyze and assess the effectiveness of the method of muscular fasciae with axial pattern pedicle encapsulated ADSCs for enhancing vascularization tissue engineering adipose and survival of the ADSCs.
[Results]
1.The original cultured ADSCs looks like to fibroblasts,but it has strong tendency for high proliferation and multiple differentiation.With the effect of adipogenic, osteogenic and chondrogenic differentiation medium,it is proved to be able to differentiate into mature adipocyte,osteoblast and chondrocyte,respectively.The differentiation can be proved by Oil Red O,Alizarin red and Alcian positive staining respectively.This kind of cell(ADSCs) is also proved to be CD29,CD44 and CD49d positive expression.
2.We perform immunofluorescence staining program after labeled by the BrdU.The ADSCs' nucleus show green fluor under fluorescence microscope.On the whole,The labeling ratio increased gradually with the incubating time and concentration of BrdU. There are more than 90%ADSCs were labelled when the cells incubating 48h and the concentration of BrdU maintaining 15μmol/L at the same time,and the living cell more than 98%.but the labeling ratio does not increased with prolong of the time and the increasing concentration of BrdU.It is indicated that the optimal time for labeling ADSCs is 48h and the most appropriate concentration is 15μmol/L of BrdU.The morphous,growth,proliferation and adipogenic potentiality of the labeled ADSCs not to be influenced compare to the unlabelled ADSCs.
3.ADSC attached to typeⅠcollagen sponge scaffold and normally grew.The adhesion rates different from the mixing means of cells and scaffold.The adhesion rates is 79.5%±2.5%when we drop ADSCs onto the surface of scaffold(n=3),and the adhesion rates is 92.7%±2.2%when we inject ADSCs into the interior of scaffold(n=3).There was significant difference between group A and group B (t=6.965,P=0.002).On the other hand,The adhesion rates is 91.3%±1.5%when the adipogenic-differentiated ADSCs were injected into the interior of scaffold.There has no significant difference between group B and group C(t=0.889,P=0.424).There has no influence on the morphous,growth,proliferation,adipogenic potentiality and no cytotoxicity when ADSCs attached scaffold.
4.Eight weeks after transplantation,the neogenetic tissue formation were observed macroscopically in the operative area of group A,group B and group C.But there has no neogenetic tissue were observed in the operative area of the group D.HE stain and oil red O stain show that the neogenetic tissue are mature adipose tissue,the positive display of immunofluorescence staining make it sure that the tissue was construted with transplanted ADSCs.The mean humid weight of the neogenetic tissue are 0.0230g±0.0016g(n=10),0.0251g±0.0019g(n=10) and 0.0202g±0.0014g(n=10) respectively.There was a significant difference between the group A and group B, group B and group C,group A and group C(all value of P＜0.05).The scaffolds were degraded thoroughly after 8 weeks at the new formed tissue.Histological evaluation of 20 fields of neogenetic adipose tissue showed that capillary density,an index of neovascularization,increased markedly in Group A and Group B.A statistically significant increase in capillary density was observed after 8 weeks in group A and group B as compared to control(group C):28.5±3.7 capillaries in group A versus 31.2±4.5 capillaries in the group B and 19.3±2.6 capillaries in control group(all value of P＜0.05).
[Conclusions]
1.We successfully isolated and cultured the ADSCs from inguinal subcutaneous fat pads of adult rabbits.And then we established a set of convenient methods about isolation,culture and identification for the ADSCs.The cells have great potentiality of proliferation and adipogenic,osteogenic and chondrogenic differentiation in vitro.ADSCs should be used for pluripotent seed cells harvested from adipose tissue.ADSCs are ideal in many aspects:they can be easily and effectively harvested,handled and multiplied non-invasively and abundantly;their pluripotency and proliferative efficiency are not less than those of bone marrow-derived MSCs;and donor morbidity is lower than for MSCs harvested from other sites.So ADSCs have great prospect of clinical application.
2.The labeling index may achieve over 90%when the continually passaged cells of ADSCs were incubated with 10μmol/L BrdU for 48h.So the optimal time for labeling ADSCs is 48h and the most appropriate final concentration for the BrdU is 15μmol/L.BrdU has no obvious cytotoxicity and influence on the growth, proliferation and differentiation to the cells.
3.ADSCs can attach to typeⅠcollagen sponge scaffold,normally grew and proliferated intro.There has a high adhesion rates when the ADSCs were injected into the interior of scaffold.There has no influence on the morphous,growth,proliferation and differentiation and no cytotoxicity when ADSCs attached scaffold in vitro.So it has excellent biocompatibility between ADSCs with typeⅠcollagen sponge scaffold.
4.In this study,conclusion can be drawn that the adipose-derived stem cells which attached scaffold encapsulated in muscular fasciae with axial pattern blood vessel pedicle can increased neovascularization and enhanced tissue engineering adipogenesis.So the engineering vascularized adipose tissue can be constructed by transplanted autologous ADSCs-attached scaffold encapsulated in muscular fasciae with axial pattern blood vessel pedicle.Autologous implanted adipose-derived stem cells can be differentiated into endothelial.Both the ADSCs and and endotheliocytes which derived the axial pattern blood vessel pedicle participate in vascularization and then increase the neovascularization.
外伤、感染或肿瘤切除等各种原因造成机体局部皮肤软组织的缺损,尤其是颜面部肿瘤或乳腺肿瘤的切除常致整个器官的缺失。这种器官的缺失及局部软组织缺损所致的形态畸形和功能障碍,目前临床上多以自体组织(皮瓣或肌皮瓣)及人工材料进行修复或充填,尽管这能有效地改善受区的组织缺损状况,但它们均存在着许多众所周知的缺点和不足。例如:自体组织的来源不足、皮瓣的血供问题以及由于供区损伤带来的继发性畸形等。因此,其临床应用受到极大的限制。此外,近年来还有其它多种多样的方法也被尝试,包括自体真皮移植,自体脂肪游离移植,胶原注射,人工材料充填等等。然而这些方法的治疗效果也不十分理想:例如人工材料的移植排斥反应,游离脂肪移植后的吸收问题给治疗效果带来不可预知性等。尽管修复软组织缺损的方法较多,却难以完全满足临床的需要。组织工程技术的出现为临床上更好的解决组织器官缺损等难题提供了一个全新的思路。
组织工程技术能以少量种子细胞,经体外扩增后与生物材料复合植入体内,修复较大的组织或器官缺损,重建生理功能,为真正实现无损伤修复组织缺损和真正意义上的形态、结构与功能重建开辟了全新的途径。组织工程学的发展需要几个必备的因素,即:(1)可获得的种子细胞,并能控制其生长和组织形成;(2)结构精确的三维支架,以适合再造需要的组织量和形状,而且与细胞具有良好的生物相容性;(3)适宜的微环境,能提供充分的血供和营养,维持细胞增殖和功能。
目前已证实脂肪组织中存在类似骨髓基质细胞的间充质干细胞,且被命名为脂肪组织来源的干细胞(Adipose-derived stem cells,ADSCs)。这种干细胞经证实同样具有体内外多向分化潜能,如能向成熟脂肪细胞、成骨细胞、软骨细胞、肌肉细胞及心肌细胞定向转化,对多种组织的损伤具有良好的修复作用,而且取材方便,获取量大,不易造成供区继发畸形等,有望成为组织工程研究中的重要种子细胞。
组织工程常用的支架材料分为天然材料和人工合成材料,天然材料包括:天然多糖类材料有纤维素、甲壳素、透明质酸等;天然蛋白质类材料有胶原和纤维蛋白等;人工合成的材料有聚乳酸(PLA)、聚乙醇酸(PGA)或两者聚合物聚乳酸-聚乙醇酸(PLGA)、聚四氟乙烯、羟基磷灰石、聚乙烯醇、海藻酸盐等。根据它们各自特点,已被不同程度地应用于组织工程的研究中。由于应用脂肪组织工程技术修复软组织缺损近年才引起学者们的关注,故而用于构建脂肪组织工程有效的生物材料支架研究不多,主要为可降解的多孔泡沫,如PLGA、透明质酸、膨体聚四氟乙烯等。然而,究竟何种材料能成为脂肪组织工程种子细胞的理想载体尚不十分明确;同时关于脂肪组织工程血管化的研究国尚内罕见报道,因此,对这一课题进行广泛深入的研究具有深远意义。
本课题研究了成年兔ADSCs向脂肪细胞、成骨细胞及软骨细胞的体外定向诱导分化能力,同时判断ADSCs与海绵状Ⅰ型胶原支架复合后的生物相容性;进而采用BrdU标记的ADCSs作为种子细胞,以海绵状Ⅰ型胶原蛋白为支架,通过观察带血管肌筋膜包裹方法对移植物种子细胞的成活作用影响,以探讨ADSCs-载体复合物在体内成脂效应的理想途径和构建工程化、血管化的脂肪组织的适宜方法,为临床上自体脂肪组织移植修复软组织缺损及脂肪干细胞的有效移植提供理论依据。
[材料与方法]
1、成年兔脂肪干细胞(ADSCs)的分离培养与鉴定
切取成年兔腹股沟皮下脂肪垫,使用酶消化法进行原代细胞培养及传代培养,观察细胞形态及功能变化,细胞传至第3代供实验使用。采用细胞免疫荧光染色检测细胞表面与干细胞相关的部分分子如CD29、CD34、CD44及CD49d,确定细胞类型;同时在体外对细胞进行向脂肪细胞、成骨细胞及软骨细胞诱导分化,并分别以油红O染色、茜素红染色及阿利辛蓝染色进行定性鉴定。
2、BrdU体外脂肪干细胞标记及观察标记物对细胞生物学性状的影响
BrdU体外标记第3代ADSCs,取P3代细胞以终浓度分别为5、10、15和20μmol/L的BrdU进行标记,分别记为A、B、C、D组;另一组不含BrdU,作为空白对照组E。标记12、24、48和72h后,采用免疫荧光染色法检测各组细胞阳性标记率,以确定BrdU的最佳标记浓度及最佳标记时间;相差显微镜及荧光显微镜观察细胞生长情况及传代后细胞标记率的变化;检测ADSCs标记后向脂肪细胞分化的能力;使用MTT比色分析法检测标记后细胞的增殖及毒性情况。
3、成年兔ADSCs与海绵状Ⅰ型胶原蛋白生物相容性的检测
将标记后的P3代ADSCs与Ⅰ型胶原蛋白海绵支架体外联合培养,采用两种不同的混合方法(细胞直接滴加于支架表面法和细胞注射于支架内部法),测定细胞在支架上的黏附率,相差显微镜及电子显微镜动态观察细胞在支架上黏附、伸展、生长和增殖情况;检测ADSCs与支架复合后向脂肪细胞分化的能力;使用MTT比色分析法检测接种在支架的细胞的增殖情况。
4、带血管肌筋膜包埋ADSCs-载体复合物构建血管化组织工程脂肪的体内研究
经解剖分离成年兔两侧背阔肌肌皮瓣,分离形成带血管蒂的肌筋膜囊,分别包裹成脂诱导前、后的ADSCs与Ⅰ型胶原蛋白载体复合物(A组,n=10和B组,n=10),另将同样大小的复合物植入同一动物右侧臀大肌肌筋膜囊(无血管蒂)作为对照组(C组,n=10);而空支架组(D组)即不含细胞成份的空支架植入兔左侧臀大肌肌筋膜囊,各组实验均采用自身对照。术后8W取材,经油红O染色、HE染色和免疫组织荧光染色确定新生组织的性质;测定三组新生组织的湿重和微血管密度,分析评价带血管肌筋膜包裹方法对促进移植物血管化与种子细胞成活的影响。
[结果]
1.原代培养的ADSCs形态类似于成纤维细胞,具有很强的增殖及多向分化能力。分别在脂肪细胞诱导分化培养基、成骨细胞诱导分化培养基及软骨细胞诱导分化培养基的作用下,能分化出成熟的脂肪细胞、成骨细胞和软骨细胞;分别以油红O染色、茜素红染色及阿利辛蓝染色呈阳性。免疫荧光检测到细胞表面抗原分子CD29、CD44、CD49d表达呈阳性。
2.第3代ADSCs经BrdU标记后进行免疫荧光染色,在荧光显微镜下胞核呈绿色荧光。总的看来,随着标记时间的延长和BrdU剂量的增加,标记率逐渐增高,BrdU终浓度为15μmol/L且标记48h后细胞标记率＞90%,但标记时间继续延长和BrdU浓度继续增加,细胞标记率无明显增高。经统计分析表明BrdU标记ADSCs的最佳终浓度为15μmol/L,最佳标记时间为48h。与正常未标记细胞比较,标记后细胞的形态及生长增殖状况无明显差别,活细胞数＞98%。细胞标记后不影响向脂肪细胞分化的能力。
3.ADSCs可以在海绵状Ⅰ型胶原蛋白支架上生长并逐渐黏附,细胞未经成脂诱导前,ADSCs与支架的混合方式不同,细胞在支架上的黏附率也不同:ADSCs在支架表面滴加组(A组,n=6)的细胞黏附率为79.5%±2.5%;ADSCs在支架内部注射组(B组,n=6)的细胞黏附率92.7%±2.2%,A、B两组采用独立样本t检验比较差异有统计学意义(t=6.965,P=0.002)。而ADSCs成脂诱导后注射于支架内部的黏附率为91.3%±1.5%(C组,n=6),B、C两组采用独立样本t检验比较差异无统计学意义(t=0.889,P=0.424)。ADSCs与支架混合培养后不影响细胞的形态、生长增殖状况,对细胞无毒性反应;细胞与支架复合后不影响向脂肪细胞分化的能力。
4.术后8W取材发现,A、B、C三组于移植物包埋区均可观察到新生组织形成;空白对照组(D组)未见新生组织形成。A组(n=10)新生组织平均湿重为0.0230g±0.0016g;B组(n=10)新生组织平均湿重为0.0251g±0.0019g;C组(n=10)新生组织平均湿重为0.0202g±0.0014g,A组与B组、B组与C组及A组与C组之间湿重比较,差异有统计学意义(P均＜0.05)。HE染色及油红O染色均证实新生组织为成熟脂肪组织,胶原支架已完全吸收降解;免疫荧光染色阳性证实新生组织为外源性。各组新生微血管密度分别为:A组(n=10)28.5±3.7;B组(n=10)31.2±4.5;C组(n=10)19.3±2.6;A与C,B与C之间新生微血管密度比较,差异有统计学意义(P均＜0.05)。
[结论]
1、本实验成功地从成年兔皮下脂肪组织中分离培养出脂肪干细胞(ADSCs)。建立了一整套有关ADSCs分离、培养和鉴定的较简便的方法。ADSCs具有很强的增殖和自我更新能力。ADSCs具有向脂肪细胞、成骨细胞和软骨细胞分化的潜能。ADSCs来源丰富、取材容易,创伤小,是理想的组织工程种子细胞,具有广阔的应用前景。
2、BrdU标记ADSCs的最佳时间为48小时;最佳终浓度为15μmol/L;标记阳性率＞90%。BrdU对细胞无毒副作用,安全性高,对细胞的生长增殖及成脂分化无明显影响。
3、ADSCs可以在海绵状Ⅰ型胶原蛋白支架上逐渐黏附,细胞黏附率高,生长、增殖良好。支架对细胞无毒性反应。胶原蛋白材料与ADSCs之间具有良好的生物相容性。
4、带血管蒂的肌筋膜瓣包裹法能够更好地促进移植物种子细胞的成活及血管化过程。利用带血管肌筋膜包裹ADSCs与支架材料复合物可构建出血管化组织工程脂肪。
5、在所构建的血管化组织工程脂肪中,外源性植入的ADSCs和来源于血管蒂的血管内皮共同参与了新生脂肪组织的血管化过程。
[Background and Objective]
Factors including trauma,infection and tumors result in the defects of skin and soft tissue especially the tumors excision of the facial surface and breasts often to cause defects of the whole organs.At present clinically,autologous transplantation (skin flaps,myocutaneous flaps) and artificial substitutes were used to repair or fill the defects of soft tissue and surface organs.Although with promising prospects and encouraging results,there's limits such as short of autologous tissue,lack of blood supply and possibility of donor site morbidity,which greatly restrict the application of these techniques.Moreover,other methods including autologous dermis transplantation,autologous fat transplantation and collagen injection,filled with artificial material,and so on.However each method carries considerable disadvantages: for example,synthetic materials invariably result in foreign body reactions,and free adipose tissue grafts shrink to an unpredictable extent.Althogh kinds of methods were used for the repair of soft tissue deformities,No methods was proved to be ideal. Emerging tissue engineering strategies represent an innovative potential solution to many clinical challenges.
Tissue engineering involves there items:(1) seed cells,whose growth is needed to be under strict control and high proliferation is alsoessential.(2) Three-dimensional scaffold structure,which is needed to be degradable and to maintain stable volume and shape.Moreover,good compatibility with seed cells is also indispensabile;(3) appropriate micro-environment,which can provide adequate blood supply and nutrition,ensure cell proliferation and functions.
Previous studies have identified a putative stem cell population within adipose stromal compartment.This cell population,termed adipose-derived stem cells (ADSCs),can be isolated from adipose tissue.ADSCs can differentiate toward the multiple lineages like adipocyte,Cartilage cells,muscle cells and cardiomyocyte. ADSCs are easier to obtain,carry a relatively lower donor site morbidity,and are available in large numbers of stem cells at harvest.Thus,the use of adult ADSCs as the seed cell of to engineer adipose tissue seems to be more inspiring.
In general,Scaffolds include synthetic materials and natural materials.Several popular scaffolds like polylactic acid(PLA),polyglycolic acid(PGA) or both poly lactic-co-glycolic acid(PLGA),PTFE,Hydroxyapatite,polyvinyl alcohol,alginate etal were used to perform multiple tissue engineering.Adipose tissue engineering is a new field so only a few scaffolds were tried including the porous biodegradable materials like PLGA,hyaluronic acid and ePTFE.But no consensus was made of which materials are most ideal for adipose tissue engineering.Considering the lack of research about vascularized engineering adipose tissue,so systemic further researches on it may be of great importance.
We investigage the adipogenic,osteogenetic and chondrogenetic differentiation in vitro about the ADSCs which were harvested from adult rabbit's groin subcutaneous fat,and then to assess the biocompatibility of three-dimensional porous type Icollagen sponge scaffolds with ADSCs.Othe other hand,BrdU was used for labeling the ADSCs,then to explore the adipogenic efficacy and the survival improvement of the ADSCs attched scaffold by encapsulated in muscular fasciae with axial pattern blood vessel.This paper's objective is to probe an ideal method of construction of a new vascularized tissue-engineered adipose which could be applied clinically for repairing the defects of soft tissue.
[Material and Methods]
1.Isolation,expansion and differentiation of rabbit ADSCs
The inguinal fat pads were harvested by exairesis from the rabbits(The Southern Medical University institutional ethics committee approved all experiments in advance),and then enzyme digestion method were used for primary culture and observe changes in cell morphology and function of cells.Only cultured cells for 3 passage were used in this study in vivo.The ADSCs were detected the molecular expression which are associated with the surface of the stem cells such as CD29,CD34,CD44 and CD49d by using cellular immunofluorescence method.We make these cells to adipogenic,osteogenic and chondrogenic differentiation in vitro, then Oil Red O staining and Alizarin red staining and Alcian staining to identify the success of differentiation in order to identify the success of multi-directional differentiation.
2.Changes of biological properties of ADSCs labeled with BrdU marker in vitro
The three passage of ADSCs was incubated with BrdU at different concentrations(0,5,10,15 and 20μmol/L) for incubating time(12,24,48 and 72 h),to identify the optimal BrdU concentration and incubating time for cell labeling. Immunofluorescence and trypanblau strain were performed respectively to calculate the labeling index(positive rate) and the cells' activity for different time after BrdU labeling in vitro.Then we compare the difference of adipogenic potentiality before and after BrdU labeling in vitro.MTT colorimetric analysis was used to detect of the rate of cell proliferation and cytotoxicity of BrdU.
3.Biocompatibility of ADSCs attached with typeⅠcollagen sponge scaffold in vitro
ADSCs after BrdU labelling attached to the typeⅠcollagen sponge scaffold to form compounds respectively,then contrast phase microscope was used to observe cells growth,adhesion or expansion.Whereafter,we observed the cells disposition and proliferation under scanning electron microscope(SEM) in vitro.We determine the adhesion rates of ADSCs before and after marked by BrdU.Then we compare the difference of adipogenic potentiality between single ADSCs and ADSCs combine with collagen in vitro.MTT colorimetric analysis is used to detect of the rate of cell proliferation.
4.Experimental research of vascularized tissue-engineered adipose via the
ADSCs-attached scaffolds encapsulated in muscular fasciae with axial pattern blood vessels.
All animals(n=10) procedures were performed in accordance with the guidelines of the Southern Medical University Animal Care and Use Committee. Own control was carried out at all the rabbits(n=10).TypeⅠcollagen sponge (10mm×10mm×5mm) containing ADSCs(1×10~7 cell population and without adipogenic differentiated conditions,group A) was transplanted into the fascia musculares sac with blood vessel pedicle of left latissimus dorsi myocutaneous flaps, and the ADSCs(the same cell population and with adipogenic differentiated conditions,group B) attached same size collagen sponge was transplanted into the fascia musculares sac with blood vessel pedicle of right latissimus dorsi myocutaneous flaps at the same rabbits.Another same size collagen sponge containing adipogenic differentiated ADSCs(the same cell population) were also implanted into the random fascia musculares sac without blood vessel pedicle of right gluteus maximus(group C,Control).The rabbits were fed routinely after operation. Euthanasia was performed at the animals after 8 weeks of the ADSCs with scaffolds transplantation.No animal from either the study or control group died during the study period.The neogenetic tissue were observed macroscopically.Then they were dissected out and weighed their humid weight by electronic balance (fidelity=0.0001g).The tissue were embedded in paraffin for conventional Oil red O stain,HE stain and immunofluorescence staining in order to perform histological assessment.Neovascularization was assessed by measuring the number of capillaries in 10 fields of HE stain slide.To analyze and assess the effectiveness of the method of muscular fasciae with axial pattern pedicle encapsulated ADSCs for enhancing vascularization tissue engineering adipose and survival of the ADSCs.
[Results]
1.The original cultured ADSCs looks like to fibroblasts,but it has strong tendency for high proliferation and multiple differentiation.With the effect of adipogenic, osteogenic and chondrogenic differentiation medium,it is proved to be able to differentiate into mature adipocyte,osteoblast and chondrocyte,respectively.The differentiation can be proved by Oil Red O,Alizarin red and Alcian positive staining respectively.This kind of cell(ADSCs) is also proved to be CD29,CD44 and CD49d positive expression.
2.We perform immunofluorescence staining program after labeled by the BrdU.The ADSCs' nucleus show green fluor under fluorescence microscope.On the whole,The labeling ratio increased gradually with the incubating time and concentration of BrdU. There are more than 90%ADSCs were labelled when the cells incubating 48h and the concentration of BrdU maintaining 15μmol/L at the same time,and the living cell more than 98%.but the labeling ratio does not increased with prolong of the time and the increasing concentration of BrdU.It is indicated that the optimal time for labeling ADSCs is 48h and the most appropriate concentration is 15μmol/L of BrdU.The morphous,growth,proliferation and adipogenic potentiality of the labeled ADSCs not to be influenced compare to the unlabelled ADSCs.
3.ADSC attached to typeⅠcollagen sponge scaffold and normally grew.The adhesion rates different from the mixing means of cells and scaffold.The adhesion rates is 79.5%±2.5%when we drop ADSCs onto the surface of scaffold(n=3),and the adhesion rates is 92.7%±2.2%when we inject ADSCs into the interior of scaffold(n=3).There was significant difference between group A and group B (t=6.965,P=0.002).On the other hand,The adhesion rates is 91.3%±1.5%when the adipogenic-differentiated ADSCs were injected into the interior of scaffold.There has no significant difference between group B and group C(t=0.889,P=0.424).There has no influence on the morphous,growth,proliferation,adipogenic potentiality and no cytotoxicity when ADSCs attached scaffold.
4.Eight weeks after transplantation,the neogenetic tissue formation were observed macroscopically in the operative area of group A,group B and group C.But there has no neogenetic tissue were observed in the operative area of the group D.HE stain and oil red O stain show that the neogenetic tissue are mature adipose tissue,the positive display of immunofluorescence staining make it sure that the tissue was construted with transplanted ADSCs.The mean humid weight of the neogenetic tissue are 0.0230g±0.0016g(n=10),0.0251g±0.0019g(n=10) and 0.0202g±0.0014g(n=10) respectively.There was a significant difference between the group A and group B, group B and group C,group A and group C(all value of P＜0.05).The scaffolds were degraded thoroughly after 8 weeks at the new formed tissue.Histological evaluation of 20 fields of neogenetic adipose tissue showed that capillary density,an index of neovascularization,increased markedly in Group A and Group B.A statistically significant increase in capillary density was observed after 8 weeks in group A and group B as compared to control(group C):28.5±3.7 capillaries in group A versus 31.2±4.5 capillaries in the group B and 19.3±2.6 capillaries in control group(all value of P＜0.05).
[Conclusions]
1.We successfully isolated and cultured the ADSCs from inguinal subcutaneous fat pads of adult rabbits.And then we established a set of convenient methods about isolation,culture and identification for the ADSCs.The cells have great potentiality of proliferation and adipogenic,osteogenic and chondrogenic differentiation in vitro.ADSCs should be used for pluripotent seed cells harvested from adipose tissue.ADSCs are ideal in many aspects:they can be easily and effectively harvested,handled and multiplied non-invasively and abundantly;their pluripotency and proliferative efficiency are not less than those of bone marrow-derived MSCs;and donor morbidity is lower than for MSCs harvested from other sites.So ADSCs have great prospect of clinical application.
2.The labeling index may achieve over 90%when the continually passaged cells of ADSCs were incubated with 10μmol/L BrdU for 48h.So the optimal time for labeling ADSCs is 48h and the most appropriate final concentration for the BrdU is 15μmol/L.BrdU has no obvious cytotoxicity and influence on the growth, proliferation and differentiation to the cells.
3.ADSCs can attach to typeⅠcollagen sponge scaffold,normally grew and proliferated intro.There has a high adhesion rates when the ADSCs were injected into the interior of scaffold.There has no influence on the morphous,growth,proliferation and differentiation and no cytotoxicity when ADSCs attached scaffold in vitro.So it has excellent biocompatibility between ADSCs with typeⅠcollagen sponge scaffold.
4.In this study,conclusion can be drawn that the adipose-derived stem cells which attached scaffold encapsulated in muscular fasciae with axial pattern blood vessel pedicle can increased neovascularization and enhanced tissue engineering adipogenesis.So the engineering vascularized adipose tissue can be constructed by transplanted autologous ADSCs-attached scaffold encapsulated in muscular fasciae with axial pattern blood vessel pedicle.Autologous implanted adipose-derived stem cells can be differentiated into endothelial.Both the ADSCs and and endotheliocytes which derived the axial pattern blood vessel pedicle participate in vascularization and then increase the neovascularization.
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