CN101829359A - 脱细胞韧带支架和种子细胞复合培养方法 - Google Patents
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Abstract
本发明涉及组织工程技术领域，具体涉及一种脱细胞韧带支架和种子细胞复合培养方法。韧带脱细胞支架孔隙率小，体外培养成纤维细胞不能向支架内部生长，这是脱细胞韧带支架在韧带组织工程中应用受限制的主要原因。本发明将脱细胞韧带低温干燥后，通过挤压等物理方法容易使得胶原纤维间隙适当增大，而且干燥的脱细胞韧带便于种子细胞悬液的渗透。这样通过渗透方法直接使种子细胞进入脱细胞韧带支架内部。通过体外培养1周，HE染色见细胞在脱细胞韧带内部贴附着胶原纤维生长。本发明的方法有利于种子细胞随着细胞悬液的渗透进入韧带支架内部进行生长。
Description
技术领域
本发明涉及组织工程技术领域，具体涉及一种脱细胞韧带支架和种子细胞复合培养方法。
背景技术
韧带和肌腱损伤后修复后愈合能力差是临床上常见的问题，这使得多种替代物应用于韧带和肌腱的修复重建。其中异体、异种韧带和肌腱移植物材料支架有韧带和肌腱支架的固有特点，随着脱细胞技术及灭菌技术的发展，宿主对异体、异种韧带支架的免疫排斥和疾病传染已大大降低，使得韧带和肌腱脱细胞支架在临床应用成为可能。目前韧带和肌腱脱细胞韧带支架制备方法往往联合应用物理、化学和酶法行脱细胞，获得的脱细胞韧带具有天然的韧带细胞外基质结构，而且具有良好的生物相容性。
但脱细胞韧带支架目前并不能广泛用于组织工程韧带研究，究其原因如下：首先，获得的韧带脱细胞肌腱具有韧带和肌腱固有的细胞外基质和腱膜屏障，这不利于韧带成纤维细胞生长，体内实验发现，具有完整腱膜的脱细胞肌腱植入体内3周后，腱膜能阻挡炎症细胞和成纤维细胞向支架内部迁移，这虽然有利于维持组织工程肌腱的内部结构稳定，但给种子细胞的植入带来很大困难。体外复合培养时，完整的腱膜屏障作用使得种子细胞不能接触腱膜下结构。其次，种子细胞代谢分裂活跃，如骨髓间充质细胞、成纤维细胞等，其体积大于韧带和肌腱内部代谢不活跃的韧带纤维细胞和肌腱纤维细胞(参见文献：高英茂主编，组织学与胚胎学，北京：人民出版社，2005：37-38.)，因此脱细胞支架内的天然孔隙不利于代谢活跃的种子细胞浸润。(参见文献1.GRATZER PF，HARRISON RD，WOODS T.Matrix alteration and not residualsodium sulfate cytotoxicity affects the cellular repopulation of a decellularizedmatrix.Tissue-Eng.2006；12(10)：2975-83.2.Brown AL，Brook-Allred TT，Waddell JE，White J，Werkmeister JA，Ramshaw JA，Bagli DJ，Woodhouse KA.Bladder acellular matrix as a substrate for studying in vitro bladder smoothmuscle-urothelial cell interactions.Biomaterials.2005；26(5)：529-43.)使用超声波(见图2)、多点注射(见图3)、氧化等方法增加韧带或肌腱脱细胞支架的孔隙，但支架内的种子细胞分布不均匀、量少且局限。这均使得韧带脱细胞支架应用受限制。
胶原、蚕丝等纤维材料制成的韧带支架有足够大的孔隙率，种子细胞可以随着细胞悬液的渗透进入韧带支架内部进行生长。
发明内容
本发明的目的在于提供一种脱细胞韧带支架和种子细胞复合培养方法，该方法有利于种子细胞随着细胞悬液的渗透进入韧带支架内部进行生长。
本发明人发现韧带脱细胞支架孔隙率小，体外培养成纤维细胞不能迁移入支架内部生长，这是脱细胞韧带支架在韧带组织工程中应用受限制的主要原因。本发明人在制备韧带脱细胞支架过程中发现，低温干燥脱细胞韧带支架的胶原纤维是可以分离的，胶原纤维保留完整性，并且具有一定强度。这使得增加脱细胞韧带支架胶原纤维的间隙成为可能。通过γ射线进行消毒，能有效杀灭细菌，使得无菌的脱细胞韧带保持干燥，便于细胞悬液的渗透。
本发明提供了一种脱细胞韧带支架和种子细胞复合培养方法，该方法包括以下步骤：
1)将脱细胞韧带支架低温干燥，温度以-80℃至-40℃为宜，优选-56℃；
2)通过物理方法将冻干的脱细胞韧带结构适当松散，以不影响生物力学性质为前提，增加脱细胞韧带结构的空隙率；
3)脱细胞韧带的消毒：将脱细胞韧带包装后使用γ射线消毒；
4)种子细胞种植：将韧带成纤维细胞消化离心悬液滴在脱细胞韧带表面，韧带成纤维细胞随着悬液的渗透，达到脱细胞韧带支架内部，进行体外复合培养。
上述的物理方法如挤压、锤击、震荡等方法，可以使低温干燥脱细胞韧带支架孔隙率增大，物理方法的使用应不明显破坏脱细胞韧带支架的生物力学为原则。
上述方法，可以应用于组织工程韧带/肌腱的制备。
γ射线进行灭菌，照射总剂量15KGray，能有效灭菌。
低温干燥韧带或肌腱脱细胞支架内部不含水分，通过适当的物理方法(如挤压、锤击、震荡)处理可以使得胶原纤维致密程度降低，使得胶原纤维间的孔隙增大，有利于种子细胞悬液的渗透，细胞进入脱细胞支架内部。
组织学检查表明(见图1)，本方法能使成纤维细胞较均匀分布在脱细胞韧带支架内部，并能贴附在胶原纤维生长。本方法较相关文献报道(见图2、3)种子细胞植入方法，胶原结构破坏小，细胞渗透好，具有有明显优势。
附图说明
图1本发明复合培养一周后HE染色组织切片图(200倍)。
图2是摘自Ingram JH，Korossis S，Howling G，Fisher J，Ingham E.The use ofultrasonication to aid recellularization of acellular natural tissue scaffolds for usein anterior cruciate ligament reconstruction.Tissue-Eng.2007；13(7)：1561-72.(400倍，箭头指的是肌腱细胞)
图3是摘自Tischer T，Vogt S，Aryee S，Steinhauser E，Adamczyk C，Milz S，Martinek V.Tissue engineering of the anterior cruciate ligament：a new methodusing acellularized tendon allografts and autologous fbroblasts.Arch OrthopTrauma Surg 2007；127：735-741
其中c(×25)肌腱细胞培养4天，d(×25)肌腱细胞培养14天。
具体实施方式
下面结合附图及实施例对本发明进行详细描述，但本发明的实施不仅限于此。
实施例：兔组织工程脱脱细胞韧带支架和种子细胞复合培养方法和评价
1、取材：选用新西兰家兔(约3kg，第二军医大学动物实验中心提供，下同)，完整切除髌韧带及其胫腓骨止点，清除韧带外滑膜组织，必须保证韧带外膜完整，无菌PBS液中4℃保存。
2、脱细胞
(1)10mM的低渗tris缓冲液+丝氨酸蛋白酶抑制剂(0.05％苯甲基磺酰氟化物乙醇溶剂，35ml/L 50ml乙醇+25mgPMSF)，持续48h。
(2)0.01％胰蛋白酶+0.02％EDTA 8h
(3)1％脱氧胆酸钠(DCA)24h
(4)Hank′s缓冲液+核酸酶(DNase I 200μg/ml+RNase I 20μg/ml)5h
(5)PBS冲洗48h后-80℃保存
所有步骤均在摇床(150RPM)进行，均加5ml/L青霉素/链霉素溶液(10000U/ml 10000mg/ml)溶液预防感染，完成每一步骤均使用PBS冲洗3边。
3、组织学检查
取脱细胞后两组标本给予石蜡包埋，切片(5μm)、HE染色观察。观察细胞核成分(苏木素染色)，胶原排列形态(伊红染色)，见细胞成分完全脱除。
4、脱细胞韧带处理
脱细胞韧带低温干燥后，使用物理方法将其适当挤压、锤击，使胶原纤维变适当松软，注意保持脱细胞韧带支架外膜完整。注意不同韧带或肌腱处理方式不一样，过度处理容易破坏胶原结构而影响脱细胞韧带力学。
5、脱细胞韧带消毒
将脱细胞韧带支架密封包装，使用γ射线(15KGray)消毒，将消毒的脱细胞韧带加入10％胎牛血清细胞培养液24h后未见菌落生长。
6、种子细胞的培养
髌韧带成纤维细胞的分离：新西兰大白兔(3月)1％戊巴比妥钠静脉麻醉后，在无菌操作下取髌韧带，PBS冲洗，并彻底清除髌韧带滑膜及周围软组织。用胶原酶处理15min(37℃)，PBS冲洗3边后将髌韧带剪成1mm3组织块，间隔3mm铺在培养皿(10×10cm2)上，加少量10％胎牛血清细胞培养液。组织块贴壁后(4h)加10％胎牛血清细胞培养液覆盖组织块，培养液没3-4天更换一次。分离的第三代成纤维细胞用于实验。
7、种植方法
将韧带成纤维细胞消化离心悬液(细胞浓度106/ml)，滴在脱细胞韧带表面，韧带成纤维细胞随着悬液的渗透，达到脱细胞韧带内部。
8、复合培养
将渗透细胞悬液的脱细胞韧带悬空培养4小时，使细胞贴壁，每隔1h加细胞培养液，保持韧带湿润。而后移入培养液中悬浮培养1周。
9、组织学检查
培养1周后取组织工程韧带，在室温下OCT胶浸润1h，而后至于-20℃30min，行冰冻切片，取脱细胞韧带支架中间层面行HE染色。如图1所示，韧带支架表面见种子细胞堆积，支架内部见韧带成纤维细胞贴附胶原纤维表面，生长形态(细胞核呈梭形)良好，胶原纤维结构无明显破坏。
Claims (4)
1.一种脱细胞韧带支架和种子细胞复合培养方法，该方法包括以下步骤：
A)将脱细胞韧带支架低温干燥，温度是-80℃至-40℃；
B)通过物理方法将冻干的脱细胞韧带结构适当松散，以不影响生物力学性质为前提，增加脱细胞韧带结构的空隙率；
C)脱细胞韧带的消毒：使用γ射线消毒；
D)种子细胞种植：将韧带成纤维细胞消化离心悬液滴在脱细胞韧带表面，韧带成纤维细胞随着悬液的渗透，达到脱细胞韧带支架内部，进行体外复合培养。
2.根据权利要求1所述的一种脱细胞韧带支架和种子细胞复合培养方法，其特征在于该方法中步骤A)将脱细胞韧带支架低温干燥，温度是-56℃。
3.根据权利要求1或2所述的一种脱细胞韧带支架和种子细胞复合培养方法，其特征在于其中所述的物理方法是挤压、锤击或震荡。
4.根据权利要求1或2所述的一种脱细胞韧带支架和种子细胞复合培养方法，其特征在于该方法中步骤C)脱细胞韧带的消毒：将脱细胞韧带支架密封包装，使用γ射线15KGray消毒。
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| CN104353115A (zh) * | 2014-10-29 | 2015-02-18 | 南通大学附属医院 | 用于胰腺脱细胞支架的试剂盒、支架的制备及再种植方法 |
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| CN101332316A (zh) * | 2008-07-22 | 2008-12-31 | 广州知光生物科技有限公司 | 生物型鼻梁植入体 |
| CN101332315A (zh) * | 2007-06-29 | 2008-12-31 | 上海国睿生命科技有限公司 | 组织工程肌腱及其体外构建方法 |
| CN101496912A (zh) * | 2008-01-30 | 2009-08-05 | 北京大清生物技术有限公司 | 一种生物衍生肌腱修复材料及其制备方法 |
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| CN101332315A (zh) * | 2007-06-29 | 2008-12-31 | 上海国睿生命科技有限公司 | 组织工程肌腱及其体外构建方法 |
| CN101496912A (zh) * | 2008-01-30 | 2009-08-05 | 北京大清生物技术有限公司 | 一种生物衍生肌腱修复材料及其制备方法 |
| CN101332316A (zh) * | 2008-07-22 | 2008-12-31 | 广州知光生物科技有限公司 | 生物型鼻梁植入体 |
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| CN104353115A (zh) * | 2014-10-29 | 2015-02-18 | 南通大学附属医院 | 用于胰腺脱细胞支架的试剂盒、支架的制备及再种植方法 |
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