ASCs结合可降解PLLA/PCL纳米纤维支架构建功能性尿道的可行性研究
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摘要
研究背景与目的
平滑肌是尿道的主要功能成分。平滑肌细胞是一种终末分化细胞,一旦受损后很难再生,而且也缺乏合适可替代的肌体组织,因此修复长段尿道的缺损一直是临床上的难题。有些研究者尝试通过脉管系统活检获得平滑肌细胞,然后在体外培养扩增细胞,制备具有机械性能和收缩功能的组织工程肌肉组织。然而这些成熟的平滑肌细胞在体外增殖能力有限,而且多次分化后,容易失去其收缩能力。最为重要的是,体外培养出来的平滑肌排列无规律,因此细胞收缩时,所有细胞不能朝同一方向收缩,难以实现平滑肌细胞群整体的收缩功能。通过组织工程的方法来制备功能性的尿道,需要解决的两个重要的问题就是寻找到理想的种子细胞和合适支架材料,进而培育出具有方向性排列的平滑肌细胞群。
干细胞是一类处于未分化状态的细胞,它们具有自我更新能力,而且在一定条件下,能向多种成熟细胞类型分化。尽管胚胎干细胞(ESCs, embryonic stem cells)具有多向分化的潜能,但受伦理、宗教、道德和法律的制约,它们的使用受到限制。自成体组织来源的成体干细胞就不受这些因素的制约,而且因为可以从自体组织取材,能避开免疫排斥反应。脂肪干细胞(ASCs, adipose-derived stem cells)就是一种存在于机体脂肪组织的成体干细胞,它们能够比较容易的从脂肪组织提取,而且体外培养时,显示了很强的增殖能力和向多种细胞类型分化的潜能。例如向脂肪、骨、软骨、骨骼肌、平滑肌组织分化。而且ASCs具有易于大量获得、取材方便的优点,因此可作为用于尿道重建组织工程的“种子细胞”。
良好的生物材料支架植入机体后,能够与细胞、机体组织和谐共存。种子细胞与支架材料在体外共培养后,植入体内受损组织部位,能够对机体大块组织缺损进行修补。支架材料分为非可降解材料和可降解材料两类。非可降解材料不能被机体吸收而留在体内,往往不能很好的替代机体组织的功能,如自我更新,新陈代谢及自我优化能力等。可降解材料能对受损组织进行形态、结构及功能上的修复,它们降解为对机体无毒害的CO2和水分子,仅留下支架材料上搭载的细胞与组织融合。本实验所用的PLLA/PCL材料(聚乳酸/聚己内酯)是可降解的聚乳酸(Poly L-lactide, PLLA)和聚己内酯(Polyε-caprolactone, PCL)的共混,通过调整两者的比例可以控制合成材料的柔韧性、伸展性和降解时间,因而制备出组织修复与重建理想的可降解生物支架。
本课题应用PLLA、PCL作为原材料,应用静电纺丝技术制备具有方向性的纳米纤维细胞支架,并采用不同细胞外基质来改良材料的粘附性能,体外将自体ASCs在纳米支架上诱导为平滑肌细胞,以期在体外构建出具有整体收缩功能的平滑肌细胞群移植物。实验中研究了不同组份材料与细胞及组织的生物相容性,细胞外基质对材料细胞亲和力以及ASCs向SMCs分化的影响。而且成功将ASCs诱导为平滑肌细胞(AD-SMCs)后,将带AD-SMCs支架接种于兔尿道缺损模型,比较移植前后兔尿道压变化,为探讨ASCs结合方向性PLLA/PCL纳米纤维支架构建功能性尿道的可行性打下了实验基础。
材料和方法
一. ASCs分离、培养、鉴定和PLLA/PCL纳米纤维支架筛选。
1.ASCs的分离及培养采用从新西兰大白兔(雌、雄各半)腹股沟区皮下脂肪、颈背部皮下脂肪、肾脏周围脂肪组织分离出的ASCs作为实验细胞。ASCs分离技术参照Zuk的方法：将兔不同部位取材的新鲜脂肪组织(约1.5g)置入无菌PBS液冲洗后,在含10%FBS的DMEM中剪成1-2mm碎块；无菌PBS反复洗脱,去除混杂的血细胞和脂肪细胞；置于振荡器内,37℃下0.1%胶原酶消化2小时；等体积含10%胎牛血清的DMEM培养基中止胶原酶反应；250g离心10分钟,取下层细胞团块；含有10%胎牛血清的DMEM培养基分散细胞团块,将细胞浓度调整至1*106/ml后移入无菌培养瓶中；37℃、5%CO2、饱和湿度细胞培养箱中培养。
2.ASCs鉴定流式细胞仪检测体外培养ASCs表面的干细胞标记性分子CD106、CD166；细胞免疫组织化学检测CD31、CD45、CD105。
3.不同部位ASCs体外增殖能力的比较MTT法检测从腹股沟区皮下脂肪、颈背部皮下脂肪、肾脏周围脂肪组织分离出的ASCs第2代细胞第1、2、3、4、5、6、7天的增殖情况,比较三个不同部位来源ASCs增殖能力的差异。
4.PLLA/PCL纳米纤维支架的制备利用静电纺丝的方法制备PLLA/PCL纳米纤维支架。参考Ma的方法,基本步骤如下：取10ml的20%的PLLA/PCL氯仿溶液,加入20ml的注射器,注射器由气泵匀速推注(0.5ml/h),使溶液通过一20G(内径0.21mm)的针头。在针头与接收纳米纤维的铝片之间(距离20-40cm)加高电压(15kv),这样PLLA/PCL氯仿溶液就会从针头高速喷出。在其向接收装置运动的过程中,溶剂氯仿迅速蒸发,铝片上收集到飘落的PLLA/PCL纳米纤维。当接收纳米纤维的铝片改为一匀速旋转的接收装置(铝盘)时,纳米纤维便能以一定的方向排列在铝盘上(图-1)。
铝片上PLLA/PCL纳米纤维丝聚集到一定厚度(0.2-0.4mm)时收集材料。扫描电镜将用于纳米纤维的形态观察。
这一部分主要在我校主校区,在课题合作单位华中科技大学化学系高分子材料实验室完成,合作者提供技术指导和设备、人员的支持。
4.材料的细胞毒性与筛选腹股沟来源ASCs在不同比例PLLA/PCL纳米纤维支架上培养,免疫荧光活死细胞染色(Live/Dead Stain)检测材料的细胞毒性、免疫荧光染色及扫描电镜比较细胞在不同材料上的粘附、增殖差异。
5.材料的组织相容性及材料体内降解检测将不同比例无菌材料种植于SD大鼠鼠背皮下,免疫组化检测不同时间材料种植部位炎症标志因子CD31、CD45、CD68表达情况,评估大鼠对材料的炎症反应以及观察材料的形态变化,了解材料在体内的降解情况。
二.细胞外基质(extracellular matrix, ECM)改良PLLA/PCL纳米纤维支架性能。
1.不同ECM(明胶、胶原、层粘连蛋白(LN)、纤维链接蛋白(FN)、高浓度多聚赖氨酸、低浓度多聚赖氨酸)处理96孔板及1:1 PLLA/PCL纳米纤维支架,MTT法检测不同ECM处理前后,96孔板中细胞增殖能力变化；免疫荧光染色及扫描电镜比较支架材料不同ECM处理前后,细胞粘附能力变化。
2.不同ECM(明胶、胶原、层粘连蛋白(LN)、纤维链接蛋白(FN)、高浓度多聚赖氨酸、低浓度多聚赖氨酸)预处理培养皿后种植ASCs,在平滑肌诱导培养基(smooth muscle inductive medium, SMIM)中诱导ASCs向平滑肌方向分化。RT-PCR和Realtime-PCR检测不同时相,普通培养皿和ECM处理培养皿内诱导细胞平滑肌特异性标记基因(Calponin、SM22、α-SMA、MHC)mRNA的表达及表达量的变化；Western blot检测其蛋白质的表达水平。进而比较不同ECM对ASCs向平滑肌方向分化的影响。
三.方向性PLLA/PCL纳米纤维支架上ASCs成平滑肌分化及兔尿道重建。
1.制备具有方向性的PLLA/PCL纳米纤维支架,扫描电镜检测材料的方向性。
2.ASCs与方向性的PLLA/PCL纳米纤维支架在普通培养基(DMEM)中共培养,免疫荧染色、扫描电镜及MTT法检测材料上细胞的粘附、增殖。
3.ASCs结合使用或不使用LN预处理的PLLA/PCL内米纤维支架在平滑肌诱导培养基(SMIM)中共培养,扫描电镜观察LN处理前后,ASCs的粘附、分化及诱导出的平滑肌(AD-SMCs)的排列。
4.兔尿道狭窄模型的建立及带细胞支架兔尿道修复应用钬激光在直视下对兔尿道行破坏,术后3-4周逆行尿路造影显示尿道狭窄形成。从该兔腹股沟区取脂肪组织,分离出ASCs并在体外培养增殖。将扩增的细胞种植于方向性纤维支架上,普通培养基(DMEM)内培养一周,细胞增殖密集后,换用诱导培养基(SMIM)诱导ASCs向平滑肌分化。成功诱导ASCs为AD-SMCs后,将带细胞支架移植于ASCs来源兔尿道内,不同时间点检测兔尿道压变化(2周、8周),并与尿道狭窄模型建立前,兔尿道压相对比,评估带细胞支架的治疗效果。
结果
1.从兔三个不同部位分离出的细胞都具有脂肪干细胞典型的三角形或梭形外观,流式细胞仪检测细胞表面CD106、CD166阳性率同脂肪干细胞吻合。
2.不同部位ASCs体外增殖能力MTT法检测,差异有显著性(P<0.05)。颈背部增殖能力最强,其次为腹股沟区,腹膜后脂肪来源(肾周脂肪)增殖能力最弱。
3.电镜下观测静电纺丝技术成功制备出具有方向性的纤维支架材料。
4.免疫荧光活死细胞染色(Live/Dead Stain)检测,不同组分PLLA/PCL纳米纤维支架均无明显细胞毒性。免疫荧光活死细胞染色和扫描电镜观测不同时间点(1、2、3天)固定面积视野细胞计数,1：1支架材料细胞粘附能力最强,不同材料上细胞增殖能力没有明显差异(P>0.05)。
5.不同支架材料种植与SD大鼠背部皮下后,第5天、第2、3、4、6、8、12周免疫组化染色。仅仅第5天时,炎性因子CD31、CD45、CD68有阳性表达,第2、3、4、6、8、12周免疫组化检测炎症因子CD31、CD45、CD68均无明显阳性表达,并观察到材料逐渐破碎降解,第12周时,基本看不到组织中材料成分,而且材料种植部位无肉芽组织形成。
6.倒置像差显微镜下,计数不同ECM处理材料(1：1材料)后,相同面积材料上粘附的细胞数与未用ECM处理材料上细胞数对比,ECM处理后各组相同面积材料粘附数与未处理组有明显差异(P<0.05)。
7.不同ECM处理培养皿中细胞增殖能力与未处理培养皿中细胞增殖能力对比,PLL高浓度组、PLL低浓度组、FN组与对照组有明显差异(P<0.05)；明胶、胶原、LN处理组与对照组无明显差异(P>0.05)。
8.不同ECM处理培养皿后接种ASCs,在诱导培养基中诱导6周。Realtime-PCR检测显示不同ECM处理组,平滑肌特异性基因表达水平均随诱导时间延长而升高,6周时各种处理因素平滑肌特异性基因表达水平达到基本一致水平(成功诱导为SMCs)。LN、明胶、胶原能加速平滑肌细胞特异性基因表达。平滑肌细胞Western blot检测显示LN、明胶、胶原组诱导2w检测出calponin;FN、PLL(高、低浓度)、对照组2w未检测到calponin。明胶、胶原、LN、正常对照组细胞诱导3w检测SM22;PLL(高、低浓度)、FN处理组未检测出。PLL(高、低浓度)、FN、胶原、LN、明胶组细胞诱导4w检测出α-actin。PLL(高、低浓度)、明胶、胶原、LN、FN组细胞诱导6w检测出MHC蛋白表达。
9.静电纺丝技术结合电转制备1:1 PLLA/PCL纳米纤维支架,扫描电镜下观察支架上纳米纤维具有一致的方向性。
10.普通培养基中培养一周,免疫荧光染色、扫描电镜检测到1：1方向性PLLA/PCL纳米纤维支架材料上细胞数量逐渐增加。
11.1：1方向性PLLA/PCL纳米纤维支架材料上细胞在诱导培养基(MCDB131)中培养6周时,扫描电镜检测显示支架材料上细胞排列具有方向性。细胞长轴方向与支架材料上,纳米纤维方向相同。
12.钬激光在直视下对家兔尿道进行冲击,术后3-4周兔尿道造影检查可见尿道狭窄形成。将带AD-SMCs的支架植入ASCs来源的兔尿道,第2周与第8周检测兔尿道压,第2周时兔尿道压显示存在尿道狭窄,第8周时,部分兔(2/4)显示正常兔尿道压。
结论
1.腹股沟区来源ASCs取材容易且增殖能力强,是合适的ASCs来源。
2.1:1 PLLA/PCL纳米纤维支架无细胞毒性、细胞粘附性好、组织炎症反应小,是合适的生物支架。
3.ECM能改良材料的粘附性；LN、明胶、胶原能促进ASCs向平滑肌细胞分化。
4.1：1方向性PLLA/PCL纳米纤维支架上能培养出具有方向性的平滑肌细胞。5.带细胞支架移植入兔尿道后,诱导出的平滑肌细胞能存活并且实现了整体的收缩功能。
6. ASCs结合方向性PLLA/PCL纳米纤维支架构建功能性尿道具有可行性。
Smooth muscle cells (SMCs) are the main functional components of urethra. For the limited ability of proliferation, they are hard to recover after damage, this produce great difficulties for urethral reconstruction. Several groups reported the creation of tissue engineered muscle graft with mechanical and contractile functions using in vitro cultured SMCs isolated from vascular tissue biopsies. However, such mature differentiated SMCs have limited ability of proliferation and usually lose their contractile phenotype followed by switching to synthetic ones during in vitro expansion. What's more, the cultured SMCs always in irregular alignment and hard to achieve unitary contractile function. Thus, generating a functional smooth muscle layer through tissue engineering techniques is a prerequisite for successful reconstruction of urethra, which make it necessary to explore alternative cell source and scaffolds for urethral reconstruction since large numbers of functional cells are usually involved.
Stem cells exist in an undifferentiated state, and exhibit both the capacity to self-renew, and the capability to differentiate into more than one type of cells. Although embryonic stem cells seem to exhibit unlimited differentiation potential, they are subject to ethical, legal and political concerns. Stem cells from adult tissue, on the other hand, suffer from few such restrictions. A potential source of stem cells for transplantation is adipose tissue. Adipose-derived stem cells(ASCs) are of mesenchymal origin, can easily be harvested in large quantities, show high proliferation rates in culture and have the capacity to differentiate into diverse cell types, such as dipogenic, osteogenic, chondrogenic, and myogenic lineages.
ASCs located in the widespread fatty tissue of the body, with the advantages of plenty of storage and easily obtained, might to be the excellent "seed cells" for tissue engineering of urethra reconstruct. Favourable biological scaffold can stay in harmony with surrounding cells and tissues of the body, so as to contribute to the recoverage of the damaged tissue. Cocultured the seed cells with scaffolds in vitro to make a manmade graft, then the graft can be implant to the damanged tissue and repair large tissue defects.
Biomaterial can be divided into two general groups:degradable material and non-degradable material.Non biodegradable materials can't be absorbed by the body and stay in the tissue, thus can not substitute the function of biological tissues, such as self-renew, metabolism and self-optimization ability. Degradable materials can repair the damaged tissue in morphology, structure and function,they degraded into non-toxic molecules such as CO2 and water in the body, left the loaded cells fused in the tissue.In this study, we used the coploymers of PLLA and PCL to synthesize our novel scaffolds,through adjust the ratio of PLLA and PCL, we can make grafts with different flexibility, stretching and degradation time.
This study used PLLA and PCL to make directional nanofiber scaffolds through electrospinning technique, cocultured autologous ASCs with the scaffolds and used various ECM to improve the adhesive ability of the scaffolds. Investigated the cell and tissue biocompatibility of the scaffolds and the influence of the ECM to ASCs on its leiomyogenic differentiation.After been successfully induce to AD-SMCs, the cells loaded scaffolds were implanted in the rabbits with urethral defect.We detected the change of pressure of urethra of the rabbits after the scaffolds were implanted, these might give some messages for the feasibility of reconstruct funcitonal urethra through tissue engineering method.
Materials and methods
I. Isolate, culture, identification of ASCs and selecting of PLLA/PCL nanofiber scaffolds.
1.Isolate and culture of ASCs from New Zealand white rabbits (the same nunmber of male and female) inguinal subcutaneous fat area, subcutaneous fat of neck, fat arround the kidney. Using Zuk's method:wash the fresh adipose tissue (about 1.5g) with sterile PBS solution in vivo and cut it into pieces about 1-2mm in size in DMEM(10%FBS contained); sterile PBS repeatedly washed to remove blood cells and fat cells mixed; Enzyme digest the pieces with 0.1% collagenase under 37℃in the oscillator or 2 hours;equal volume of DMEM containing 10% fetal bovine serum medium for suspension of collagenase reaction; 250g centrifugation for 10 minutes, collected the lower cell mass; containing 10% FBS DMEM, scattered cell mass, adjusted the cell concentration to 1*106/ml, moved into sterile culture flasks after; cultured in 37℃,5% CO2 humidified cell incubator.
2. Identification of ASCs Flow cytometry detected the surface cell marker CD106, CD 166 in vitro.
3.Ability of the proliferation of different areas derived ASCs in vitro MTT assay detected the different areas derived ASCs(P2) OD varation on the 1th,2nd,3rd,4th,5th,6th,7th day when cultured in vivo.
4. Preparation of PLLA/PCL nanofiber scaffolds by electrospinning method. Reference to Ma's method [2], the basic steps are as follows:Take 10ml of 20% of the PLLA/PCL chloroform solution, add a 20ml syringe, syringe pump by a uniform injection (0.5ml/h), the solution through a 20G (diameter 0.21mm) needles. And receiving the needle between the nano-fibers of aluminum (from 20-40cm) plus one high voltage (15kv), so that PLLA/ PCL chloroform solution will be ejected from the needle high-speed, in its movement to the receiving device in the process of rapid evaporation of the solvent chloroform, collected aluminum falling PLLA/PCL nanofibers. When receiving aluminum nanofibers into a uniform rotation of the receiver (aluminum plate), the nano-fibers can arrange a certain direction in the aluminum plate (Figure-1).
Collected the PLLA/PCL nanofibers on the Aluminum plant when the nanofibers accumulated to a certain thickness (0.2-0.4mm). Scanning electron microscopy will be used to detect the morphology of nanofibers.
5.Cytotoxicity of material ASCs from inguinal area from cocultured in different proportions of PLLA/PCL nanofiber scaffold, immunofluorescence living and dead cell staining (Live/Dead Stain) detected cytotoxicity of material, immunofluorescence staining and scanning electron microscopy cells can also detected the adhesion and proliferation of ASCs in different materials.
6. Biocompatibility of materials and degradation of material in vivo implanted the scaffolds with different proportion of PLLA/PCL under the dorsal subcutaneous of SD rats, immunohistochemistry detected the expression of inflammatory markers CD31, CD45, CD68 at different times to assess Biocompatibility of materials and degradation of material.
Ⅱ. ECM (extracellular matrix, ECM) modified PLLA/PCL nanofiber scaffolds.
1. Different ECM (gelatin, collagen, laminin, fibronectin, high concentration of polylysine, low concentration of polylysine) pretreated flask and PLLA/PCL nanofiber scaffolds, MTT assay detected the change of the ability of cell proliferation before and after the pretreated with ECM in the culture dish and scaffolds. Immunofluorescence staining and scanning electron microscopy also used to detect the change of cell proliferation ability.
2. Different ECM (gelatin, collagen, LN, FN, high concentration of polylysine, low concentration of polylysine)pretreated flask and PLLA/PCL nanofiber scaffolds, then cocultured the ASCs in SMIM. RT-PCR and Realtime-PCR detected the change of mRNA expression of the smooth muscle-specific marker genes (Calponin, SM22,α-SMA, MHC); Western blot detection the expression levels of according proteins.
Ⅲ. Leiomyogenic differentiation of ASCs on dirrectional PLLA/PCL nanofiber scaffolds and urethral reconstruction of rabbits.
1.Synthesize of directional PLLA/PCL nanofiber scaffolds, scanning electron microscopy detect.
2.ASCs cocultured with the directional PLLA/PCL nanofiber scaffolds in normal medium (DMEM), immune fluorescence staining, scanning electron microscopy and MTT assay detect the cell adhesion and proliferation ability.
3. ASCs cocultured with the directional PLLA/PCL nanofiber scaffolds with/without LN pretreated in the smooth muscle induction medium (SMIM). Scanning electron microscopy detected the adhesion, proliferation changes and the arrangement of smooth muscle (AD-SMCs).
4. Urethral stricture model of rabbit and urethral reconstruct with cells-loaded scaffolds Using the YAG laser distory the urethra of rabbits under direct vision(ureteroscopy),3 to 4 weeks after injured, the urethra can be strictured for scar forming. Retrograde imaging approved it. Isolated ASCs from the inguinal area of the damaged rabbit and cocultured them in directional nanofiber scaffolds with DMEM in vitro. When the ASCs proliferated in a high density, changed the medium with SMIM. After cultured in the smooth muscle induced medium for 6 weeks, ASCs differentiated into smooth muscle cells. Implanted the AD-SMCs loaded scaffolds to the restructed part of ASCs host rabit, detected the urethral pressure change at different time (2 weeks,8 weeks after graft implanted), compared the urethral pressure changes before and after the operation. Investigated the effect of AD-SMCs loaded directional nanofiber scaffolds.
Results
1. Three different areas derived rabbit adipose stem cells have the typical triangular or spindle-shaped appearance, flow cytometry detection show the cell surface marker of CD 105, CD 166 expression the same with other groups reported.
2.MTT assay found different areas derived ASCs had various ability of proliferation in vitro(P<0.05). The most powerful proliferation area was the neck, followed by the inguinal area, retroperitoneal fat sources (kidney around) proliferation of the weakest.
3.Electron microscope scan found the directional nanofiber scaffolds were successfully prepared with Electrospinning method.
4. Immunofluorescence of living dead staining (Live/Dead Stain) showed the nanofiber scaffolds with different components ratio of PLLA/PCL had no significant cytotoxicity. Immunofluorescence staining and scanning electron microscopy detected the cell numbers on various scaffolds at different time points (cells scaffold cocultured for 1,2,3 days) of fixed area of vision, data show 1:1 scaffold had the strongest cell adhesion ability, cell proliferation had no significant difference on different materials (P>0.05).
5.Different scaffolds implanted subcutaneously of the SD rats back, the 5th day, immunohistochemistry found the positive expression of inflammatory factors CD31, CD45, CD68. On the next 2nd,3rd,4th,6th,8th,12th weeks, immunohistochemistry found did not found the expression of such inflammatory factors. IPCM observed the gradual degradation of scaffolds. It almost can not see the of material at 12th week, and there were no granulation tissue appeared around the scaffolds.
6. Compared the number of cells in the same size of area of materials under IPCM in different ECM pretreated materials (1:1 material) with the same area of untreated scaffolds, there were significant differences between the two group (P<0.05).
7. MTT assay was used to detect the infulence of ECM to cell proliferation. Pre treated the dishes with different ECM, cell proliferation ability in the dish treated with PLL of low/high concentration and FN was enhanced, significantly different to the control groups (P<0.05); gelatin, collagen, LN treatment group and control group had no significant difference (P> 0.05).
8. ASCs cultured in induction medium in different ECM pretreated dishes for 6 weeks, RT-PCR, Realtime-PCR and Western blot analysis showed that LN, gelatin, collagen pretreated group detected calponin after been induced for 2w; FN, PLL (high and low concentration), did not detected the expression calponin. Gelatin, collagen, LN and control group dectected the expression of SM22 after been induced for 3w; PLL (high and low concentration), FN group did not detected the expression of SM22. PLL (high and low concentration), FN, collagen, LN, gelatin group detected the positive expression ofα-actin after been induced for 4w. PLL (high and low concentration), gelatin, collagen, LN, FN group detected the positive expression of MHC after been induced for 6w.
9. Electrospinning combined with electroporation prepared 1:1 PLLA/PCL nanofiber scaffolds, scanning electron microscopy found the nanofibers on the scaffold arranged in the same direction.
10. Immunofluorescence staining and scanning electron microscopy detected the cell proliferated on the 1:1 directional PLLA/PCL nanofiber scaffolds gradually when ASCs cultured in DMEM for 1 w.
11.Cocultured ASCs with 1:1 directional PLLA/PCL nanofiber scaffolds in SMIM for 6 weeks, scanning electron microscopy analysis showed that the induced cells arranged in the same direction. The long axis of cells paralleled to the nano-fibers of the scaffold.
12. YAG laser distory the urethra of rabbits under direct vision(ureteroscopy),3 to 4 weeks after injured, the urethra can be strictured for scar forming. Retrograde imaging approved it. The successful induced cells loaded scaffolds implanted into the host rabits urethra, detected the urethral pressure of the rabbits at the first 2 weeks and the first 8 weeks, the urethral pressure showed the presence of urethral stricture at the 2nd week, data showed 2 of 4 rabbits implanted with AD-SMCs loaded scaffolds reached to normal urethral pressure at the 8th week.
Conclusion
1.ASCs derived from inguinal area is the appropriate source of ASCs.
2.1:1 PLLA/PCL nanofiber scaffolds with no cytotoxicity, minimal inflammatory reaction and good cell adhesion character, is the appropriate choice of nanofiber scaffold.
3.ECM can modify the adhesion character of nanofiber scaffolds.what's more, LN, gelatin and collagen can promote the ASCs to differatiate into SMCs.
4.1:1 directional PLLA/PCL nanofiber scaffold can induce the smooth muscle cells arranged in the direction direction.
5.Smooth muscle cells kept alive and accomplished overall contractile function after implanted the AD-SMCs loaded directional scaffold in the rabits urethra.
6.Directional PLLA/PCL nanofiber scaffold loaded with AD-SMCs for functional urethra reconstruction is feasible.
平滑肌是尿道的主要功能成分。平滑肌细胞是一种终末分化细胞,一旦受损后很难再生,而且也缺乏合适可替代的肌体组织,因此修复长段尿道的缺损一直是临床上的难题。有些研究者尝试通过脉管系统活检获得平滑肌细胞,然后在体外培养扩增细胞,制备具有机械性能和收缩功能的组织工程肌肉组织。然而这些成熟的平滑肌细胞在体外增殖能力有限,而且多次分化后,容易失去其收缩能力。最为重要的是,体外培养出来的平滑肌排列无规律,因此细胞收缩时,所有细胞不能朝同一方向收缩,难以实现平滑肌细胞群整体的收缩功能。通过组织工程的方法来制备功能性的尿道,需要解决的两个重要的问题就是寻找到理想的种子细胞和合适支架材料,进而培育出具有方向性排列的平滑肌细胞群。
干细胞是一类处于未分化状态的细胞,它们具有自我更新能力,而且在一定条件下,能向多种成熟细胞类型分化。尽管胚胎干细胞(ESCs, embryonic stem cells)具有多向分化的潜能,但受伦理、宗教、道德和法律的制约,它们的使用受到限制。自成体组织来源的成体干细胞就不受这些因素的制约,而且因为可以从自体组织取材,能避开免疫排斥反应。脂肪干细胞(ASCs, adipose-derived stem cells)就是一种存在于机体脂肪组织的成体干细胞,它们能够比较容易的从脂肪组织提取,而且体外培养时,显示了很强的增殖能力和向多种细胞类型分化的潜能。例如向脂肪、骨、软骨、骨骼肌、平滑肌组织分化。而且ASCs具有易于大量获得、取材方便的优点,因此可作为用于尿道重建组织工程的“种子细胞”。
良好的生物材料支架植入机体后,能够与细胞、机体组织和谐共存。种子细胞与支架材料在体外共培养后,植入体内受损组织部位,能够对机体大块组织缺损进行修补。支架材料分为非可降解材料和可降解材料两类。非可降解材料不能被机体吸收而留在体内,往往不能很好的替代机体组织的功能,如自我更新,新陈代谢及自我优化能力等。可降解材料能对受损组织进行形态、结构及功能上的修复,它们降解为对机体无毒害的CO2和水分子,仅留下支架材料上搭载的细胞与组织融合。本实验所用的PLLA/PCL材料(聚乳酸/聚己内酯)是可降解的聚乳酸(Poly L-lactide, PLLA)和聚己内酯(Polyε-caprolactone, PCL)的共混,通过调整两者的比例可以控制合成材料的柔韧性、伸展性和降解时间,因而制备出组织修复与重建理想的可降解生物支架。
本课题应用PLLA、PCL作为原材料,应用静电纺丝技术制备具有方向性的纳米纤维细胞支架,并采用不同细胞外基质来改良材料的粘附性能,体外将自体ASCs在纳米支架上诱导为平滑肌细胞,以期在体外构建出具有整体收缩功能的平滑肌细胞群移植物。实验中研究了不同组份材料与细胞及组织的生物相容性,细胞外基质对材料细胞亲和力以及ASCs向SMCs分化的影响。而且成功将ASCs诱导为平滑肌细胞(AD-SMCs)后,将带AD-SMCs支架接种于兔尿道缺损模型,比较移植前后兔尿道压变化,为探讨ASCs结合方向性PLLA/PCL纳米纤维支架构建功能性尿道的可行性打下了实验基础。
材料和方法
一. ASCs分离、培养、鉴定和PLLA/PCL纳米纤维支架筛选。
1.ASCs的分离及培养采用从新西兰大白兔(雌、雄各半)腹股沟区皮下脂肪、颈背部皮下脂肪、肾脏周围脂肪组织分离出的ASCs作为实验细胞。ASCs分离技术参照Zuk的方法：将兔不同部位取材的新鲜脂肪组织(约1.5g)置入无菌PBS液冲洗后,在含10%FBS的DMEM中剪成1-2mm碎块；无菌PBS反复洗脱,去除混杂的血细胞和脂肪细胞；置于振荡器内,37℃下0.1%胶原酶消化2小时；等体积含10%胎牛血清的DMEM培养基中止胶原酶反应；250g离心10分钟,取下层细胞团块；含有10%胎牛血清的DMEM培养基分散细胞团块,将细胞浓度调整至1*106/ml后移入无菌培养瓶中；37℃、5%CO2、饱和湿度细胞培养箱中培养。
2.ASCs鉴定流式细胞仪检测体外培养ASCs表面的干细胞标记性分子CD106、CD166；细胞免疫组织化学检测CD31、CD45、CD105。
3.不同部位ASCs体外增殖能力的比较MTT法检测从腹股沟区皮下脂肪、颈背部皮下脂肪、肾脏周围脂肪组织分离出的ASCs第2代细胞第1、2、3、4、5、6、7天的增殖情况,比较三个不同部位来源ASCs增殖能力的差异。
4.PLLA/PCL纳米纤维支架的制备利用静电纺丝的方法制备PLLA/PCL纳米纤维支架。参考Ma的方法,基本步骤如下：取10ml的20%的PLLA/PCL氯仿溶液,加入20ml的注射器,注射器由气泵匀速推注(0.5ml/h),使溶液通过一20G(内径0.21mm)的针头。在针头与接收纳米纤维的铝片之间(距离20-40cm)加高电压(15kv),这样PLLA/PCL氯仿溶液就会从针头高速喷出。在其向接收装置运动的过程中,溶剂氯仿迅速蒸发,铝片上收集到飘落的PLLA/PCL纳米纤维。当接收纳米纤维的铝片改为一匀速旋转的接收装置(铝盘)时,纳米纤维便能以一定的方向排列在铝盘上(图-1)。
铝片上PLLA/PCL纳米纤维丝聚集到一定厚度(0.2-0.4mm)时收集材料。扫描电镜将用于纳米纤维的形态观察。
这一部分主要在我校主校区,在课题合作单位华中科技大学化学系高分子材料实验室完成,合作者提供技术指导和设备、人员的支持。
4.材料的细胞毒性与筛选腹股沟来源ASCs在不同比例PLLA/PCL纳米纤维支架上培养,免疫荧光活死细胞染色(Live/Dead Stain)检测材料的细胞毒性、免疫荧光染色及扫描电镜比较细胞在不同材料上的粘附、增殖差异。
5.材料的组织相容性及材料体内降解检测将不同比例无菌材料种植于SD大鼠鼠背皮下,免疫组化检测不同时间材料种植部位炎症标志因子CD31、CD45、CD68表达情况,评估大鼠对材料的炎症反应以及观察材料的形态变化,了解材料在体内的降解情况。
二.细胞外基质(extracellular matrix, ECM)改良PLLA/PCL纳米纤维支架性能。
1.不同ECM(明胶、胶原、层粘连蛋白(LN)、纤维链接蛋白(FN)、高浓度多聚赖氨酸、低浓度多聚赖氨酸)处理96孔板及1:1 PLLA/PCL纳米纤维支架,MTT法检测不同ECM处理前后,96孔板中细胞增殖能力变化；免疫荧光染色及扫描电镜比较支架材料不同ECM处理前后,细胞粘附能力变化。
2.不同ECM(明胶、胶原、层粘连蛋白(LN)、纤维链接蛋白(FN)、高浓度多聚赖氨酸、低浓度多聚赖氨酸)预处理培养皿后种植ASCs,在平滑肌诱导培养基(smooth muscle inductive medium, SMIM)中诱导ASCs向平滑肌方向分化。RT-PCR和Realtime-PCR检测不同时相,普通培养皿和ECM处理培养皿内诱导细胞平滑肌特异性标记基因(Calponin、SM22、α-SMA、MHC)mRNA的表达及表达量的变化；Western blot检测其蛋白质的表达水平。进而比较不同ECM对ASCs向平滑肌方向分化的影响。
三.方向性PLLA/PCL纳米纤维支架上ASCs成平滑肌分化及兔尿道重建。
1.制备具有方向性的PLLA/PCL纳米纤维支架,扫描电镜检测材料的方向性。
2.ASCs与方向性的PLLA/PCL纳米纤维支架在普通培养基(DMEM)中共培养,免疫荧染色、扫描电镜及MTT法检测材料上细胞的粘附、增殖。
3.ASCs结合使用或不使用LN预处理的PLLA/PCL内米纤维支架在平滑肌诱导培养基(SMIM)中共培养,扫描电镜观察LN处理前后,ASCs的粘附、分化及诱导出的平滑肌(AD-SMCs)的排列。
4.兔尿道狭窄模型的建立及带细胞支架兔尿道修复应用钬激光在直视下对兔尿道行破坏,术后3-4周逆行尿路造影显示尿道狭窄形成。从该兔腹股沟区取脂肪组织,分离出ASCs并在体外培养增殖。将扩增的细胞种植于方向性纤维支架上,普通培养基(DMEM)内培养一周,细胞增殖密集后,换用诱导培养基(SMIM)诱导ASCs向平滑肌分化。成功诱导ASCs为AD-SMCs后,将带细胞支架移植于ASCs来源兔尿道内,不同时间点检测兔尿道压变化(2周、8周),并与尿道狭窄模型建立前,兔尿道压相对比,评估带细胞支架的治疗效果。
结果
1.从兔三个不同部位分离出的细胞都具有脂肪干细胞典型的三角形或梭形外观,流式细胞仪检测细胞表面CD106、CD166阳性率同脂肪干细胞吻合。
2.不同部位ASCs体外增殖能力MTT法检测,差异有显著性(P<0.05)。颈背部增殖能力最强,其次为腹股沟区,腹膜后脂肪来源(肾周脂肪)增殖能力最弱。
3.电镜下观测静电纺丝技术成功制备出具有方向性的纤维支架材料。
4.免疫荧光活死细胞染色(Live/Dead Stain)检测,不同组分PLLA/PCL纳米纤维支架均无明显细胞毒性。免疫荧光活死细胞染色和扫描电镜观测不同时间点(1、2、3天)固定面积视野细胞计数,1：1支架材料细胞粘附能力最强,不同材料上细胞增殖能力没有明显差异(P>0.05)。
5.不同支架材料种植与SD大鼠背部皮下后,第5天、第2、3、4、6、8、12周免疫组化染色。仅仅第5天时,炎性因子CD31、CD45、CD68有阳性表达,第2、3、4、6、8、12周免疫组化检测炎症因子CD31、CD45、CD68均无明显阳性表达,并观察到材料逐渐破碎降解,第12周时,基本看不到组织中材料成分,而且材料种植部位无肉芽组织形成。
6.倒置像差显微镜下,计数不同ECM处理材料(1：1材料)后,相同面积材料上粘附的细胞数与未用ECM处理材料上细胞数对比,ECM处理后各组相同面积材料粘附数与未处理组有明显差异(P<0.05)。
7.不同ECM处理培养皿中细胞增殖能力与未处理培养皿中细胞增殖能力对比,PLL高浓度组、PLL低浓度组、FN组与对照组有明显差异(P<0.05)；明胶、胶原、LN处理组与对照组无明显差异(P>0.05)。
8.不同ECM处理培养皿后接种ASCs,在诱导培养基中诱导6周。Realtime-PCR检测显示不同ECM处理组,平滑肌特异性基因表达水平均随诱导时间延长而升高,6周时各种处理因素平滑肌特异性基因表达水平达到基本一致水平(成功诱导为SMCs)。LN、明胶、胶原能加速平滑肌细胞特异性基因表达。平滑肌细胞Western blot检测显示LN、明胶、胶原组诱导2w检测出calponin;FN、PLL(高、低浓度)、对照组2w未检测到calponin。明胶、胶原、LN、正常对照组细胞诱导3w检测SM22;PLL(高、低浓度)、FN处理组未检测出。PLL(高、低浓度)、FN、胶原、LN、明胶组细胞诱导4w检测出α-actin。PLL(高、低浓度)、明胶、胶原、LN、FN组细胞诱导6w检测出MHC蛋白表达。
9.静电纺丝技术结合电转制备1:1 PLLA/PCL纳米纤维支架,扫描电镜下观察支架上纳米纤维具有一致的方向性。
10.普通培养基中培养一周,免疫荧光染色、扫描电镜检测到1：1方向性PLLA/PCL纳米纤维支架材料上细胞数量逐渐增加。
11.1：1方向性PLLA/PCL纳米纤维支架材料上细胞在诱导培养基(MCDB131)中培养6周时,扫描电镜检测显示支架材料上细胞排列具有方向性。细胞长轴方向与支架材料上,纳米纤维方向相同。
12.钬激光在直视下对家兔尿道进行冲击,术后3-4周兔尿道造影检查可见尿道狭窄形成。将带AD-SMCs的支架植入ASCs来源的兔尿道,第2周与第8周检测兔尿道压,第2周时兔尿道压显示存在尿道狭窄,第8周时,部分兔(2/4)显示正常兔尿道压。
结论
1.腹股沟区来源ASCs取材容易且增殖能力强,是合适的ASCs来源。
2.1:1 PLLA/PCL纳米纤维支架无细胞毒性、细胞粘附性好、组织炎症反应小,是合适的生物支架。
3.ECM能改良材料的粘附性；LN、明胶、胶原能促进ASCs向平滑肌细胞分化。
4.1：1方向性PLLA/PCL纳米纤维支架上能培养出具有方向性的平滑肌细胞。5.带细胞支架移植入兔尿道后,诱导出的平滑肌细胞能存活并且实现了整体的收缩功能。
6. ASCs结合方向性PLLA/PCL纳米纤维支架构建功能性尿道具有可行性。
Smooth muscle cells (SMCs) are the main functional components of urethra. For the limited ability of proliferation, they are hard to recover after damage, this produce great difficulties for urethral reconstruction. Several groups reported the creation of tissue engineered muscle graft with mechanical and contractile functions using in vitro cultured SMCs isolated from vascular tissue biopsies. However, such mature differentiated SMCs have limited ability of proliferation and usually lose their contractile phenotype followed by switching to synthetic ones during in vitro expansion. What's more, the cultured SMCs always in irregular alignment and hard to achieve unitary contractile function. Thus, generating a functional smooth muscle layer through tissue engineering techniques is a prerequisite for successful reconstruction of urethra, which make it necessary to explore alternative cell source and scaffolds for urethral reconstruction since large numbers of functional cells are usually involved.
Stem cells exist in an undifferentiated state, and exhibit both the capacity to self-renew, and the capability to differentiate into more than one type of cells. Although embryonic stem cells seem to exhibit unlimited differentiation potential, they are subject to ethical, legal and political concerns. Stem cells from adult tissue, on the other hand, suffer from few such restrictions. A potential source of stem cells for transplantation is adipose tissue. Adipose-derived stem cells(ASCs) are of mesenchymal origin, can easily be harvested in large quantities, show high proliferation rates in culture and have the capacity to differentiate into diverse cell types, such as dipogenic, osteogenic, chondrogenic, and myogenic lineages.
ASCs located in the widespread fatty tissue of the body, with the advantages of plenty of storage and easily obtained, might to be the excellent "seed cells" for tissue engineering of urethra reconstruct. Favourable biological scaffold can stay in harmony with surrounding cells and tissues of the body, so as to contribute to the recoverage of the damaged tissue. Cocultured the seed cells with scaffolds in vitro to make a manmade graft, then the graft can be implant to the damanged tissue and repair large tissue defects.
Biomaterial can be divided into two general groups:degradable material and non-degradable material.Non biodegradable materials can't be absorbed by the body and stay in the tissue, thus can not substitute the function of biological tissues, such as self-renew, metabolism and self-optimization ability. Degradable materials can repair the damaged tissue in morphology, structure and function,they degraded into non-toxic molecules such as CO2 and water in the body, left the loaded cells fused in the tissue.In this study, we used the coploymers of PLLA and PCL to synthesize our novel scaffolds,through adjust the ratio of PLLA and PCL, we can make grafts with different flexibility, stretching and degradation time.
This study used PLLA and PCL to make directional nanofiber scaffolds through electrospinning technique, cocultured autologous ASCs with the scaffolds and used various ECM to improve the adhesive ability of the scaffolds. Investigated the cell and tissue biocompatibility of the scaffolds and the influence of the ECM to ASCs on its leiomyogenic differentiation.After been successfully induce to AD-SMCs, the cells loaded scaffolds were implanted in the rabbits with urethral defect.We detected the change of pressure of urethra of the rabbits after the scaffolds were implanted, these might give some messages for the feasibility of reconstruct funcitonal urethra through tissue engineering method.
Materials and methods
I. Isolate, culture, identification of ASCs and selecting of PLLA/PCL nanofiber scaffolds.
1.Isolate and culture of ASCs from New Zealand white rabbits (the same nunmber of male and female) inguinal subcutaneous fat area, subcutaneous fat of neck, fat arround the kidney. Using Zuk's method:wash the fresh adipose tissue (about 1.5g) with sterile PBS solution in vivo and cut it into pieces about 1-2mm in size in DMEM(10%FBS contained); sterile PBS repeatedly washed to remove blood cells and fat cells mixed; Enzyme digest the pieces with 0.1% collagenase under 37℃in the oscillator or 2 hours;equal volume of DMEM containing 10% fetal bovine serum medium for suspension of collagenase reaction; 250g centrifugation for 10 minutes, collected the lower cell mass; containing 10% FBS DMEM, scattered cell mass, adjusted the cell concentration to 1*106/ml, moved into sterile culture flasks after; cultured in 37℃,5% CO2 humidified cell incubator.
2. Identification of ASCs Flow cytometry detected the surface cell marker CD106, CD 166 in vitro.
3.Ability of the proliferation of different areas derived ASCs in vitro MTT assay detected the different areas derived ASCs(P2) OD varation on the 1th,2nd,3rd,4th,5th,6th,7th day when cultured in vivo.
4. Preparation of PLLA/PCL nanofiber scaffolds by electrospinning method. Reference to Ma's method [2], the basic steps are as follows:Take 10ml of 20% of the PLLA/PCL chloroform solution, add a 20ml syringe, syringe pump by a uniform injection (0.5ml/h), the solution through a 20G (diameter 0.21mm) needles. And receiving the needle between the nano-fibers of aluminum (from 20-40cm) plus one high voltage (15kv), so that PLLA/ PCL chloroform solution will be ejected from the needle high-speed, in its movement to the receiving device in the process of rapid evaporation of the solvent chloroform, collected aluminum falling PLLA/PCL nanofibers. When receiving aluminum nanofibers into a uniform rotation of the receiver (aluminum plate), the nano-fibers can arrange a certain direction in the aluminum plate (Figure-1).
Collected the PLLA/PCL nanofibers on the Aluminum plant when the nanofibers accumulated to a certain thickness (0.2-0.4mm). Scanning electron microscopy will be used to detect the morphology of nanofibers.
5.Cytotoxicity of material ASCs from inguinal area from cocultured in different proportions of PLLA/PCL nanofiber scaffold, immunofluorescence living and dead cell staining (Live/Dead Stain) detected cytotoxicity of material, immunofluorescence staining and scanning electron microscopy cells can also detected the adhesion and proliferation of ASCs in different materials.
6. Biocompatibility of materials and degradation of material in vivo implanted the scaffolds with different proportion of PLLA/PCL under the dorsal subcutaneous of SD rats, immunohistochemistry detected the expression of inflammatory markers CD31, CD45, CD68 at different times to assess Biocompatibility of materials and degradation of material.
Ⅱ. ECM (extracellular matrix, ECM) modified PLLA/PCL nanofiber scaffolds.
1. Different ECM (gelatin, collagen, laminin, fibronectin, high concentration of polylysine, low concentration of polylysine) pretreated flask and PLLA/PCL nanofiber scaffolds, MTT assay detected the change of the ability of cell proliferation before and after the pretreated with ECM in the culture dish and scaffolds. Immunofluorescence staining and scanning electron microscopy also used to detect the change of cell proliferation ability.
2. Different ECM (gelatin, collagen, LN, FN, high concentration of polylysine, low concentration of polylysine)pretreated flask and PLLA/PCL nanofiber scaffolds, then cocultured the ASCs in SMIM. RT-PCR and Realtime-PCR detected the change of mRNA expression of the smooth muscle-specific marker genes (Calponin, SM22,α-SMA, MHC); Western blot detection the expression levels of according proteins.
Ⅲ. Leiomyogenic differentiation of ASCs on dirrectional PLLA/PCL nanofiber scaffolds and urethral reconstruction of rabbits.
1.Synthesize of directional PLLA/PCL nanofiber scaffolds, scanning electron microscopy detect.
2.ASCs cocultured with the directional PLLA/PCL nanofiber scaffolds in normal medium (DMEM), immune fluorescence staining, scanning electron microscopy and MTT assay detect the cell adhesion and proliferation ability.
3. ASCs cocultured with the directional PLLA/PCL nanofiber scaffolds with/without LN pretreated in the smooth muscle induction medium (SMIM). Scanning electron microscopy detected the adhesion, proliferation changes and the arrangement of smooth muscle (AD-SMCs).
4. Urethral stricture model of rabbit and urethral reconstruct with cells-loaded scaffolds Using the YAG laser distory the urethra of rabbits under direct vision(ureteroscopy),3 to 4 weeks after injured, the urethra can be strictured for scar forming. Retrograde imaging approved it. Isolated ASCs from the inguinal area of the damaged rabbit and cocultured them in directional nanofiber scaffolds with DMEM in vitro. When the ASCs proliferated in a high density, changed the medium with SMIM. After cultured in the smooth muscle induced medium for 6 weeks, ASCs differentiated into smooth muscle cells. Implanted the AD-SMCs loaded scaffolds to the restructed part of ASCs host rabit, detected the urethral pressure change at different time (2 weeks,8 weeks after graft implanted), compared the urethral pressure changes before and after the operation. Investigated the effect of AD-SMCs loaded directional nanofiber scaffolds.
Results
1. Three different areas derived rabbit adipose stem cells have the typical triangular or spindle-shaped appearance, flow cytometry detection show the cell surface marker of CD 105, CD 166 expression the same with other groups reported.
2.MTT assay found different areas derived ASCs had various ability of proliferation in vitro(P<0.05). The most powerful proliferation area was the neck, followed by the inguinal area, retroperitoneal fat sources (kidney around) proliferation of the weakest.
3.Electron microscope scan found the directional nanofiber scaffolds were successfully prepared with Electrospinning method.
4. Immunofluorescence of living dead staining (Live/Dead Stain) showed the nanofiber scaffolds with different components ratio of PLLA/PCL had no significant cytotoxicity. Immunofluorescence staining and scanning electron microscopy detected the cell numbers on various scaffolds at different time points (cells scaffold cocultured for 1,2,3 days) of fixed area of vision, data show 1:1 scaffold had the strongest cell adhesion ability, cell proliferation had no significant difference on different materials (P>0.05).
5.Different scaffolds implanted subcutaneously of the SD rats back, the 5th day, immunohistochemistry found the positive expression of inflammatory factors CD31, CD45, CD68. On the next 2nd,3rd,4th,6th,8th,12th weeks, immunohistochemistry found did not found the expression of such inflammatory factors. IPCM observed the gradual degradation of scaffolds. It almost can not see the of material at 12th week, and there were no granulation tissue appeared around the scaffolds.
6. Compared the number of cells in the same size of area of materials under IPCM in different ECM pretreated materials (1:1 material) with the same area of untreated scaffolds, there were significant differences between the two group (P<0.05).
7. MTT assay was used to detect the infulence of ECM to cell proliferation. Pre treated the dishes with different ECM, cell proliferation ability in the dish treated with PLL of low/high concentration and FN was enhanced, significantly different to the control groups (P<0.05); gelatin, collagen, LN treatment group and control group had no significant difference (P> 0.05).
8. ASCs cultured in induction medium in different ECM pretreated dishes for 6 weeks, RT-PCR, Realtime-PCR and Western blot analysis showed that LN, gelatin, collagen pretreated group detected calponin after been induced for 2w; FN, PLL (high and low concentration), did not detected the expression calponin. Gelatin, collagen, LN and control group dectected the expression of SM22 after been induced for 3w; PLL (high and low concentration), FN group did not detected the expression of SM22. PLL (high and low concentration), FN, collagen, LN, gelatin group detected the positive expression ofα-actin after been induced for 4w. PLL (high and low concentration), gelatin, collagen, LN, FN group detected the positive expression of MHC after been induced for 6w.
9. Electrospinning combined with electroporation prepared 1:1 PLLA/PCL nanofiber scaffolds, scanning electron microscopy found the nanofibers on the scaffold arranged in the same direction.
10. Immunofluorescence staining and scanning electron microscopy detected the cell proliferated on the 1:1 directional PLLA/PCL nanofiber scaffolds gradually when ASCs cultured in DMEM for 1 w.
11.Cocultured ASCs with 1:1 directional PLLA/PCL nanofiber scaffolds in SMIM for 6 weeks, scanning electron microscopy analysis showed that the induced cells arranged in the same direction. The long axis of cells paralleled to the nano-fibers of the scaffold.
12. YAG laser distory the urethra of rabbits under direct vision(ureteroscopy),3 to 4 weeks after injured, the urethra can be strictured for scar forming. Retrograde imaging approved it. The successful induced cells loaded scaffolds implanted into the host rabits urethra, detected the urethral pressure of the rabbits at the first 2 weeks and the first 8 weeks, the urethral pressure showed the presence of urethral stricture at the 2nd week, data showed 2 of 4 rabbits implanted with AD-SMCs loaded scaffolds reached to normal urethral pressure at the 8th week.
Conclusion
1.ASCs derived from inguinal area is the appropriate source of ASCs.
2.1:1 PLLA/PCL nanofiber scaffolds with no cytotoxicity, minimal inflammatory reaction and good cell adhesion character, is the appropriate choice of nanofiber scaffold.
3.ECM can modify the adhesion character of nanofiber scaffolds.what's more, LN, gelatin and collagen can promote the ASCs to differatiate into SMCs.
4.1:1 directional PLLA/PCL nanofiber scaffold can induce the smooth muscle cells arranged in the direction direction.
5.Smooth muscle cells kept alive and accomplished overall contractile function after implanted the AD-SMCs loaded directional scaffold in the rabits urethra.
6.Directional PLLA/PCL nanofiber scaffold loaded with AD-SMCs for functional urethra reconstruction is feasible.
引文
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