异种活性脱细胞肌腱支架的初步构建
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摘要
第一章牛脱细胞肌腱支架制备
目的：采用多次脱细胞循环处理去除牛肌腱腱细胞、利用交联剂交联脱细胞支架增强力学强度、过氧乙酸及冷冻干燥增加空隙率、最后进行灭菌处理制备异种脱细胞肌腱支架,并通过多项检测指标评价制备的脱细胞肌腱支架去除抗原和细胞成分的效果、检测交联程度和力学性能,评价体内外组织相容性,为异种肌腱的应用提供实验依据。
方法：取健康成年牛腱,于-80℃深冻保存1个月,室温复融后进行α-半乳糖苷酶消化及脱细胞处理：PBS缓冲液中浸泡数小时,根据实验设计选择添加a-半乳糖苷酶；胰蛋白酶溶液消化数小时,用含胎牛血清的DMEM培养基终止胰酶消化；于聚乙二醇辛基苯基醚,脱氧胆酸钠混合液中脱细胞,更换脱细胞溶液,重复脱细胞循环数次,PBS反复清洗；EDTA浸泡数小时；DMEM培养基浸泡,PBS反复清洗；利用京尼平交联脱细胞肌腱数小时,PBS反复清洗；DMEM培养基浸泡后置于过氧乙酸-乙醇溶液中,PBS反复清洗；-80℃深冻后冷冻干燥；15kGyγ射线辐照制备脱细胞肌腱支架。取新鲜深冻及不同脱细胞循环处理牛肌腱,HE染色后光学显微镜观察细胞去除情况及肌腱纤维组织形态学特征,荧光显微镜下观察DAPI染色样本DNA和RNA去除情况。取新鲜深冻,脱细胞未经α-半乳糖苷酶处理(1-7个脱细胞循环),α-半乳糖苷酶处理样本,分别进行抗α-半乳糖基抗原ELISA定量：取-20℃保存样本室温消融,分别将组织样本充分剪碎至匀浆。取a-gal ELISA试剂盒室温平衡,分别设空白孔(空白对照不加样本及酶标试剂,其余各步操作相同)、标准孔、待测样本孔。在酶标包被板上标准品准确加样50μl,待测样本孔中先加样本稀释液40μl,然后再加待测样本10μl(终稀释倍数为5),样本加于酶标板孔底,尽量不触及孔壁,轻轻晃动混匀；封板膜封板后置37℃温育30min；揭除封板膜,弃液体,甩干,每孔加满洗涤液,静置30min后弃去,重复5次,拍干；加入酶标试剂,50μl孔,空白除外；37℃温育30min；洗涤液清洗5次,拍干；加显色剂A,50μl/孔,再加显色剂B,50山/孔,震荡混匀,37℃避光显色10min;加终止液终止反应,501μl/孔；以空白孔调零,450nm波长测量各孔吸光度(OD值)。以标准物的浓度为横坐标,OD值为纵坐标绘制标准曲线,根据样本OD值由标准曲线查出相应浓度,乘稀释倍数计算样本实际浓度根据a-半乳糖基浓度确定脱细胞循环次数；扫描电子显微镜(SEM)观察新鲜深冻及脱细胞肌腱支架的脱细胞程度及纤维结构。取新鲜深冻,脱细胞处理(1-7个脱细胞循环)样品,以E.Z.N.A. TM Tissue DNA Kit利用NANODROP2000分光光度计检测样本总DNA：称取实验样本,充分研碎后加TL缓冲液、OB蛋白酶混匀后温浴过夜;加RNA酶后高速离心,吸取上层悬液与BL缓冲液充分混匀,温育后加无水乙醇,强力震荡充分混匀；取HiBind(?) DNA柱放置于收集管内,将之前所制备样本加入DNA柱内,高速离心后弃液体；将DNA柱放入新的收集管内,HB缓冲液冲洗,高速离心后弃液体及收集管；将DNA柱放入原收集管内,用DNA清洗缓冲液冲洗,高速离心后弃液体保留收集管；将DNA柱放入原收集管内,同上清洗；将DNA柱放入原收集管内,最大速度离心后将DNA柱放入无菌离心管内,添加预热洗脱缓冲液,室温静置数分后高速离心,重复洗脱；提取DNA溶液即用或-80℃保存备用。根据残余DNA量确定脱细胞循环次数。综合组织学观察、扫描电镜观察、α-半乳糖基浓度以及残余DNA量确定脱细胞循环次数。采用茚三酮法利用分光光度计在570nm下测量吸光度,测定京尼平交联度：(1)柠檬酸、NaOH和氯化亚锡混合,加去离子水调至25m1,另将茚三酮加入25m1乙二醇甲醚中,再将上述两液体混合搅拌,制成茚三酮溶液,避光保存。(2)称取样品,加入茚三酮溶液,100℃水浴20min,冷却至室温,加异丙醇溶液,用分光光度计在570nm下测量吸光度。以甘氨酸溶液做标准曲线(n/M,n/4M,n/8M,n/16M, n/32M,),并通过此标准曲线查找各样本中自由氨基酸的摩尔数。利用异丙醇替换法计算深冻及脱细胞肌腱支架的孔隙率。对新鲜深冻肌腱和脱细胞肌腱支架进行力学拉伸试验,以抗张强度、断裂延伸率及弹性模量为评价指标,观察力学性能变化。通过倒置显微镜观察,利用CCK-8在450nm波长条件下应用酶标仪测量光吸收值检测细胞增殖情况,利用中性红于540nm波长测量光吸收值检测细胞存活情况,综合评价体外细胞相容性。采用大鼠皮下埋入实验：备皮,用手术刀片于背部做纵形切口,大小10mm左右,钝性分离皮下组织,形成一囊袋,皮下埋入,以新鲜牛腱为阳性对照,胶原海绵为阴性对照,脱细胞肌腱支架为实验组,术后3、7、14、21d进行大体及组织学观察,评价所制备肌腱支架的体内相容性。
结果：HE染色后可见脱细胞肌腱支架腱束呈波浪形紧密规则排列,与深冻肌腱相比腱束间距略有增大,未观察到细胞成分；DAPI染色可见深冻肌腱内大量DNA和RNA,而脱细胞肌腱支架则未发现DNA和RNA；实验同时观察到过度脱细胞处理可致肌腱断裂,胶原变性,腱束间距过大,失去原有三维结构特征。α-半乳糖基抗原检测结果显示经α-半乳糖苷酶处理后样本内未检测到a-半乳糖基抗原的存在；经脱细胞循环处理,α-半乳糖基抗原浓度逐渐降低,经3次处理后,未检测到α-半乳糖基抗原。SEM观察可见深冻肌腱和脱细胞肌腱支架腱束排列规则紧密,脱细胞肌腱支架肌腱束之间距离略有增加,空隙度优于深冻肌腱,未发现细胞成分。样本经脱细胞循环处理后,DNA含量明显下降,经3个脱细胞循环处理,DNA含量约下降至原有含量的1/3,4个脱细胞循环处理,DNA含量约降至原有含量的1/4。综合组织学观察、扫描电镜观察、a-半乳糖基浓度以及残余DNA量确定本研究采用4个脱细胞循环进行脱细胞处理。茚三酮法测定京尼平交联度达57.27%。对孔隙率的测定显示脱细胞肌腱支架(56.800±9.311%)高于深冻肌腱(38.660±2.996%%),二者有显著性差异(t=-4.147,P=0.003)。力学性能检测深冻肌腱抗张强度为22.640±4.504MPa,脱细胞肌腱支架组为29.73±4.62MPa,统计学结果显示差异无统计学意义(t=-1.904,P=0.130)；深冻肌腱断裂延伸率为15.200±4.071%,脱细胞肌腱支架组为13.830±3.424%,统计学结果显示差异无统计学意义(t=0.445,P=0.679);深冻肌腱弹性模量为151.250±12.885MPa,脱细胞肌腱支架组为218.680±23.833MPa,统计学结果显示差异有统计学意义(t=-4.311,P=0.022)。脱细胞肌腱支架抗张强度和弹性模量均较深冻肌腱有增加,断裂延伸率则较之减小。CCK-8检测结果显示,在不同的时间点实验组与阴性对照组的吸光度值有显著性差异(F=578.697,P<0.001),实验组吸光度值高于阴性对照组,差异有显著性意义(F=4.011,P<0.05),组别因素与时间因素之间无交互效应(F=1.650,P>0.05)。同一检测时间点,不同组别间吸光度值无显著性差异(P>0.05)：同一组别内,在不同的检测时间点的吸光度值有显著性差异(P<0.001);2、5、7d细胞增殖率分别为105%、105%和110%,细胞毒性评价均为0级：中性红检测3d细胞存活率为99%,7d为106%。体内组织相容性实验结果显示阳性对照组表现为大量淋巴细胞浸润,实验组和阴性对照组结构相似,阴性对照组更快的被机体所吸收：术后3d,实验组与对照组镜下所见相似,植入物被层状炎性组织包绕,其间可见小血管扩张、充血,白细胞渗出,炎细胞以中性粒细胞和巨噬细胞为主。术后1w,两组植入物仍被光滑的薄层结缔组织包裹,炎症细胞较3d减少；包裹组织外层主要由梭形的纤维细胞和疏松的胶原纤维组成。对照组海绵胶原支架出现降解,实验组可观察到细胞成分沿实验组肌腱边缘向内部长入,几乎无炎细胞浸润,无胶原降解。术后2w,胶原海绵组包裹组织明显缩小,内部仅残存少量胶原支架,支架周围见大量细胞浸润。实验组肌腱周围包裹组织与1w所见相似,边缘肌腱胶原内少量炎细胞浸润,以巨噬细胞为主,伴有轻微胶原溶解,偶见多核巨细胞,细胞向肌腱内部长入增加。术后3w,胶原海绵已经基本吸收,实验组与2w类似,长入肌腱内细胞增多。
结论：采用多步脱细胞处理后经京尼平交联脱细胞肌腱支架,并利用过氧乙酸-乙醇结合γ射线辐照灭菌所制备的肌腱支架具有：(1)无细胞成分及α-半乳糖基抗原；(2)保存了完整的天然肌腱三维支架；(3)良好的力学性能；(4)良好的体内、体外组织相容性。本研究为异种肌腱的实际应用提供初步实验基础。
第二章活性脱细胞支架的初步构建
目的：摸索富含血小板血浆的制备方法,将富含血小板血浆与缓释剂混合并复合脱细胞肌腱支架,初步构建具有缓释能力的活性脱细胞肌腱支架系统。
方法：取SD大鼠,戊巴比妥钠腹腔注射麻醉,用预先抗凝的10ml注射器行心脏采血,采用低密度两步离心法制备富含血小板血浆：以200×g离心后,将全部上清液、中间层(白细胞和血小板层)及少量红细胞移入PE管内,相同条件下再次离心后,吸取上清血浆,以5倍全血血小板浓度重悬制备富含血小板血浆。通过ELISA法检测富含血小板血浆中血小板源性生长因子(PDGF)、血管内皮细胞生长因子(VEGF)、胰岛素生长因子-1(IGF-1)、转化生长因子-β1(TGF-β1)四种生长因子浓度。取4℃保存的ELISA试剂盒,恢复至室温。将标准品及待测样本加入酶标板,混匀后置37℃40min；洗涤液充分洗板,滤纸印干；加蒸馏水和第一抗体工作液各50μl/孔,混匀后置37℃20min:洗板；加酶标抗体工作液,混匀后置37℃10min洗板；加底物工作液,混匀后置37℃暗处10min;以空白孔调零,450nm波长测量各孔吸光度(OD值)。以标准物的浓度为横坐标,OD值为纵坐标绘制标准曲线,根据样本OD值由试剂盒自带软件绘制标准曲线查出相应浓度,乘稀释倍数计算样本实际浓度。利用Ⅰ型胶原溶液激活富含血小板血浆,用注射器将二者混合液注入制备的牛脱细胞肌腱支架中,制备活性肌腱支架。将所制备的缓释系统以4倍蒸馏水稀释,于3,12,24,48h利用ELISA试剂盒检测相应生长因子的浓度,评价缓释功能。对活性脱细胞肌腱支架进行免疫组织化学染色,检测样本是否存在TGF-β和VEGF两种生长因子：取石蜡切片,经二甲苯及逆梯度酒精脱蜡水化；PBS洗涤；过氧化氢处理；PBS洗涤后酶胰酶修复；蒸馏水洗涤；封闭液封闭；一抗过夜；PBS洗涤后添加二抗；PBS洗涤后加SABC; PBS洗涤数次；DAB显色：自来水冲洗后苏木素复染；盐酸分化、返蓝；脱水、透明、封片,光学显微镜下观察。将活性脱细胞肌腱支架与大鼠成纤维细胞共培养,倒置相差显微镜观察细胞生长增殖情况并利用CCK-8检测成纤维细胞增殖情况。
结果：ELISA法检测富含血小板血浆中PDGF浓度为3624.20±1453.62pg/ml、IGF-1浓度为376.64±136.66pg/ml、TGF-β1浓度为1103.45±1149.31pg/ml,未检测出样本中VEGF浓度。缓释检测显示随时间的延长,生长因子逐渐由支架释放,未检测出样本中VEGF浓度,PDGF在48h后的浓度约为12h及24h的3倍,IGF-1在48h后的浓度约为12h及24h的2倍,TGF-1在48h后的浓度约为12h的3倍,24h的2倍。免疫组织化学未发现明显阳性染色结果。将所制备的活性肌腱支架与大鼠成纤维细胞共培养后,可以观察到细胞形态、生长状态良好。CCK-8检测实验组OD值3.280±0.235,对照组OD值2.947±0.168,两组差异有统计学意义(t=-3.987,P=0.001),细胞相对增殖率为111%。
结论：本研究探索了富含血小板血浆的制备方法,初步构建了具有一定缓释功能的活性脱细胞肌腱支架,所构建的活性支架具有一定的促进细胞增殖的功能。
第三章活性肌腱支架修复兔跟腱缺损
目的：建立动物缺损模型评价活性脱细胞肌腱支架的修复能力,为其实际应用提供实验依据。
方法：取日本大耳白兔,用预先抗凝的注射器于兔耳中心动脉取血,按第二章的方法制备富含血小板血浆。按第一章的方法制备脱细胞肌腱支架,并按第二章方法构建活性脱细胞肌腱支架。戊巴比妥钠兔耳缘静脉注射麻醉,后肢小腿后侧备皮,暴露跟腱分离内侧束,切除中段2cm,造成跟腱缺损,保留浅部外侧束和深部束；组1采用相同长度的活性脱细胞肌腱作为植入物,组2用对侧浅内侧束作为移植物修复缺损,组3采用相同长度粗细的异种牛肌腱作为植入物,组4不予修复,直接缝合。石膏绷带固定,单笼饲养,正常饮食,术后连续3d肌注庆大霉素。术后观察动物是否存活,并记录动物饮食、精神状态,有无腹泻、感染,伤口愈合及术侧肢体活动情况。于术后3d、2w、4w、8w、12w,每组随机取3只兔处死,观察大体及组织学修复情况。
结果：术后各实验动物均饮食正常,精神状态佳,无腹泻,固定肢肌肉存在不同程度萎缩,去除石膏后逐渐恢复。组1与组2相似。伤口愈合良好,伤口无红肿及感染；无跟腱断裂,植入腱和宿主腱吻合端结合紧密,未发现移植物与自体肌腱缝合端拉长或断裂,移植物与周围组织有不同程度的粘连,组1和组2相似,组3移植肌腱受到机体排斥,组4断腱未愈合。术后3d,植入腱与宿主腱吻合处可见垂直肌腱长轴生长的肉芽组织,中性粒细胞和巨噬细胞为主,细胞向植入腱内生长,周围纤维包裹,有小血管形成；术后2w,对照组自体植入腱与宿主腱间的肉芽组织成熟,炎性细胞和毛细血管较少,可见大量成纤维细胞,新生的胶原纤维垂直于肌腱长轴,组织结构融为一体,靠近吻合端的植入腱内改建活跃,脱细胞植入腱与宿主腱吻合端所见与对照组相似,连接近端可见新生纤维血管组织、大量成纤维细胞。4w,植入腱与宿主腱间的肉芽组织炎性细胞减少,毛细血管、成纤维细胞增多,脱细胞活性肌腱与自体肌腱相比,炎性细胞和毛细血管略多。术后8w,植入腱与宿主腱间的肉芽组织更加成熟,炎性细胞很少,大量成纤维细胞,缝合口处可见大量沿肌腱长轴走行,排列密集的新生胶原纤维,脱细胞活性肌腱与自体肌腱相比,无明显差别。术后12w胶原纤维排列更加规则,胶原束更为紧密,但与正常肌腱相比,胶原纤维排列及成熟度仍略差,自体植入腱与脱细胞植入腱没有明显差别。Masson染色结果显示随时间延长,新生胶原逐渐增多,并取代陈旧胶原。2w时新生胶原较少,以陈旧胶原为主；4w时仍以陈旧胶原占多数,新生胶原较2w时增多；8w时见大量的新生胶原,陈旧胶原大部分被取代。
结论：采用兔跟腱缺损模型,利用活性脱肌腱支架修复缺损跟腱,移植后机体的免疫排斥反应及体内组织学转归与自体植入腱相似,可有效地修复肌腱缺损,为临床应用提供了动物实验依据。
Chapter one Preparation of decellularizd tendon xenograft
Objective:Multiple decellularization cycles were carried out to remove bovine tenocytes. Then the decellularizd tendon xenograft was cross linked to enhance biomechanical property of the scaffold. The prepared scaffolds were finally sterilized by peracetic-acid ethanol combined with low dose y irradiation. Procedures were performed to determine the decellularization, deprivation of a-gal antigen, changes of biomechanical property, in vitro and in vivo cytocompatibility in order to provide experimental foundation for future application of decellularized tendon xenograft.
Methods:Achilles tendons were harvested aseptically and stored at80℃for one month. Frozen tendons were thawed, and all adhering connective-tissue was removed. Then the tendons were decellularized as followed. Samples were washed with PBS, and then incubated in trypsin solution, which was terminated by DMEM with FBS. This was followed by incubation inTriton-X solution that contained Triton and NA-deoxycholate. This step was repeated. After washed with PBS, samples were incubated in EDTA for and washed by DMEM. The samples were then washed with PBS. After different cycles'washing, samples were treated by a-galactosidase. The samples were then washed with PBS and cross linked by incubation with Genipin solution. The solution was discarded and samples were washed with PBS. Samples were treated with PE solution which contained peracetic acid and ethanol. Samples were washed with PBS until residue of peracetic acid was accepted. After deep frozen, samples were freeze-dried and sterilized with15kGy y irradiation. Fresh-frozen and decellularised tendon scaffolds were processed for histological analysis. The sections were mounted on slides and stained with hematoxylin and eosin as well as4,6-diamidino-2-phenylindole (DAPI). The surface of each tendon was manually evaluated from the tendon sections mounted on glass slides and analyzed by optical microscopy, from which decellularization and3-D structure of tendons were evaluated. Quantity of a-gal antigen from fresh-frozen, a-galactosidase treated and decellularised tendon scaffolds was determined using a commercial available ILISA kit. Scanning electron microscopy was performed on the fresh-frozen tendons and the decellularised tendon scaffolds. Cross-sectional and longitudinal-sectional electron-micrographs were obtained to evaluate decellularization and3-D structure of tendons. Fresh-frozen and decellularised tendon scaffolds were freeze-dried and weighed. Then total DNA of specimens was isolated using E.Z.N.A.TM Tissue DNA Kit and determined using NANODROP2000spectrophometer. According to histological observation, SEM observation, concentration of a-gal antigen and quantity of DNA, the proper decellularization cycle was determined. Ninhydrin assay was performed to assess cross linking of genipin by determine the free amino group content. And the optical absorbance of the solution was recorded with a spectrophotometer at a wavelength of570nm. Using glycine at various known concentrations as standard, concentration of free amino was calculated, from which cross linking of genipin was determined. Isopropanol was used to dertermine porosity of fresh-frozen and decellularised tendon scaffolds. Uniaxial load-to-failure tests were performed with a digital universal testing machine. And tensile strength,%strain at ultimate failure load and elastic tensile modulus were compared to test bio mechanical property of fresh-frozen and decellularised tendon scaffolds. In order to evaluate in vitro cytocompatibility, CCK-8was used to determine the absorbance of the solution measured with an enzyme-linked immunoassay. For the cell viability assay, the neutral red solution was chosen to determine the absorbance measured at540nm using the96-well plate spectrophotometer noted above. The absorbance obtained was directly proportional to the viability of the cell populations and inversely proportional to the cytotoxicity of the material. A sterile surgical procedure was performed to implant the specimens subcutaneously in the dorsum of the rats. Decellularised tendon scaffold were experimental group, while fresh bovine tendon was positive control and collagen sponge was negative control. At3rd,7th,14th and21st day post-implantation, the animals were euthanised and the implants were harvested. In vivo host cell infiltration and inflammatory response were evaluated by hemalaun-eosin (H&E) staining with optical microscopy.
Results:Cellular components were evident in fresh-frozen bovine tendon prior to decellularization after H&E staining. After processing, cellular components were not observed and more inter-fascicular and intra-fascicular space was present. DNA and RNA were evident in fresh-frozen bovine tendon prior to decellularization after DAPI staining, while after processing, DNA and RNA were not observed. Collagenous degeneration, rupture of collagen fibre and changes of natural structure of tendon were observed in over-decellularized samples. Scanning electron microscopy confirmed the dense micro-architecture observed in the histologic sections of the fresh-frozen tendons, as well as an increase in pore size and porosity following oxidative treatment. Quantity of a-gal antigen decreased gradually after decellularization. And a-gal antigen could not be quantified after the third cycle of decellularization. No a-gal antigen was detected in sample processed with a-galactosidase. DNA content of the decellularized/oxidized bovine tendon scaffolds was significantly decreased after decellularized and oxidative treatment when compared to the fresh-frozen tendons. DNA content decreased by2/3after the third cycle of decellularization and dropped3/4after the fourth cycle of decellularization. Combining histological observation, SEM observation and determination of a-gal antigen and DNA content,4-decellularization-cycle was chosen. After fixation, we noted that the color of the tissue fixed with genipin became dark blue. By determine the free amino group content, the rate tissue fixed by genipin was calculated as56.27%. Porosity increased after decellularization and significant difference between control and experimental group was noticed (t=-4.147, P=0.003). The tensile strength of fresh-frozen and decellularised tendon scaffolds was22.640±4.504MPa and29.733±4.623MPa respectively. No significant difference was found between two groups (t=-1.904, P=0.130).%strain at ultimate failure load of fresh-frozen and decellularised tendon scaffolds were15.200±4.071%and13.830±3.424%respectively. No significant difference was found between two groups (t=0.445, P=0.679). And elastic tensile modulus of fresh-frozen and decellularised tendon scaffolds were151.250±12.885MPa and218.680±23.833MPa respectively. Significant difference was noticed between two groups (t=-4.311, P=0.022). The results showed that tensile strength and tensile modulus increased, while%strain at ultimate failure load decreased after decellularization. In CCK-8tests, significant difference was noted between control and experimental group at different time (F=578.697, P<0.001). And the value of OD in experimental group is significantly higher than control group (F=4.011, P<0.05). No interaction effect was noticed betreen groups (F=1.650, P>0.05).The relative growth rate at2nd,5th, and7th day were105%,105%and110%respectively. The rating for cytotoxic reacting grade was0. And the rates of viability of the cell were99%and106%respectively for the3rd and7th day. Experiment for in vivo cytocompatibility, bovine xenograft without any decellularization showed a large volume of lymphocytes surrounding and infiltration. While the decellularized tendon scaffold and collagen sponge demonstrated similar reaction and response from the hosts. And compared to decellularized tendon scaffold, collagen sponge was absorbed more quickly. At the3rd day, grafts were surrounded with granulation tissue, where angiotelectasia, hyperaemia and leukocyte exudation was observed. The inflammatory cells were mainly neutrophil granulocyte and macrophagocyte. At the first week, grafts were still surrounded with granulation tissue, and the inflammatory cells decreased. Fibroblasts and collage fibres increased in the surrounding granulation tissue. In control, absorption of collagen sponge was observed. While in experimental group, infiltration of fibroblasts into grafts was noted. At the2nd week, the absorption of collagen sponge was obvious and only little collagen was found in control. The experimental grafts showed similar responses to the1st week. Slight absorption of graft was noticed in the experimental group. And more cell filtration was observed in the peripheral part of grafted tendon scaffolds. After three weeks, almost no collagen sponge was found. More cells infiltration into grafted scaffolds was observed in experimental group.
Conclusion:The decellularized tendon scaffold was prepared by multiple decellularizations, genipin cross linking and sterilized by peracetic-acid ethanol combined y irradiation. This scaffold characterized by:(1) free of cells and a-gal antigen;(2) the natural structure of tendon was well maintained;(3) well preserved biomechanical property;(4) good cytocompatibility. Experimental foundation was provided for future application of decellularized tendon xenograft.
Chapter two Preliminary construction of bioactive decellularizd tendon xenograft
Objective:To explore the method of preparation for platelet rich plasma. Platelet rich plasma was activated and combined with decellularized tendon xenograft through agent characterized with potential of control release the growth factors in platelet rich plasma.
Methods:Intraperitoneal injection of pentobarbital sodium was carried out to anesthetize SD rats. With the10ml syringe processed with anticoagulant in advance, arterial blood was procured from the heart anesthetized SD rats. Then platelet rich plasma was prepared through two centrifugation steps:The blood was centrifuged with a relative centrifugal force of200g for10minutes at a constant temperature of22℃. With a plastic transfer pipette, the whole supernatant, consisting of blood plasma, leucocytes, and thrombocytes, and also red blood cells above the buffy coat were drawn. The exceeding layer of erythrocytes was discarded. This solution was run at a relative centrifugal force of200g for10minutes. The supernatant was partly discarded and platelet rich plasma which concentration was five times of the whole blood. The concentration of platelet-derived growth factor (PDGF), vascular endothelial growth Factor (VEGF), Insulin growth factor-1(IGF-1), and transforming growth factor-β1(TGF-β1) was determined by ELISA. The standard curve was drawn by standard samples provides with the ELISA kit, and absorbance of the experimental samples were determined by enzyme-linked immunoassay. Then the concentration of samples could be determined from the standard curve. The actual concentration would be calculated by multiplying the dilution ratio. Collagen I was used to activate platelet rich plasma. And the solution of Collagen I and platelet rich plasma was injected into the decellularized tendon xenograft with syringe to prepare the bioactive decellularized tendon scaffold. The bioactive decellularized tendon scaffolds with potential system of control release were then immerge into distilled water with four times volume of the solution of Collagen I and platelet rich plasma. ELISA tests were performed at3rd,12th,24th and48th hour to determine the concentration of VEGF, PDGF, IGF-1and TGF-β1. The control release potential of the bioactive decellularized tendon scaffold was evaluated. The bioactive decellularized tendon scaffold was preceded with immunohistochemical staining to determine the existence of TGF-β and VEGF. The rat fibroblasts were co-cultured with the bioactive decellularized tendon scaffold. Then growth and proliferation of fibroblasts were observed with inverted phase contrast microscope. CCK-8was also used to determine the absorbance of the solution measured with an enzyme-linked immunoassay.
Results:The results of ILISA showed that concentration of PDGF was3624.20±1453.62pg/ml in the prepared platelet rich plasma. And the concentration of IGF-1and TGF-β1was376.64±136.66pg/ml and1103.45±1149.31pg/ml respectively. However, the concentration of VEGF was not detected through ILES A test. The control release tests showed that the concentration of growth factors in the solution increased gradually. For PDGF, the concentration at48h was about three times of concentration at12h and24h. For IGF-1, the concentration at48h was around two times of concentration at12h and24h. For TGF-β1, the concentration at48h was around three times of concentration at12h and two times of concentration at24h. No obvious positive results were observed for immunohistochemical staining of TGF-β and VEGF. After co-cultured with the bioactive decellularized tendon scaffolds, fibroblast grew and proliferated well. CCK-8test showed that the absorbance of experimental group was3.280±0.235, while2.947±0.168for control. There was significant difference between two groups (t=-3.987, P=0.001). And the relative growth rate was111%.
Conclusion:The preparation of platelet rich plasma was explored. The prepared bioactive decellularized tendon scaffolds possessed the potential of control releasing growth factors in platelet rich plasma. The prepared bioactive decellularized tendon scaffolds could improve growth and proliferation of rat fibroblasts.
Chapter three Achilles tendon repair in a rabbit model with prepared bioactive decellularized tendon scaffolds
Objective:Animal tendon repair model was to be founded to evaluate the repairing potential of the bioactive decellularized tendon scaffolds.
Methods:The whole blood was drawn from the rabbit ear central artery with pre-anti-coagulated syringe. Then platelet rich plasma was prepared followed the methods descripted in chapter two. The decellularized tendon scaffolds was prepared with the methods founded in chapter one. And the bioactive decellularized tendon scaffolds was constructed followed the procedure introduced in chapter two. Each rabbit was anesthetized with3%Pentobarbital,30mg/kg. The hind paws were shaved and prepared for sterile operative intervention. A2.5cm longitudinal incision of the skin was performed using a lateral paramedian approach followed by a longitudinal splitting of the crural fascia and the paratenon which surrounds the Achilles tendon complex. Subsequently, the Achilles tendon complex was exposed. The medial M. gastrocnemius tendon was separated from the lateral M. gastrocnemius tendon and the M. flexor digitalis superficialis tendon.2cm excision was created and repaired as study design, graft was sutured to host stumps with5-0 nylon in an "8" way strengthened by interrupted stitched. In group1, the medial M. gastrocnemius tendon was repaired with the bioactive decellularized tendon scaffolds. In group2, the medial M. gastrocnemius tendon was repaired with the opposite medial M. gastrocnemius tendon. In group3, the medial M. gastrocnemius tendon was repaired with the bovine tendon xenograft. For group4, the medial M. gastrocnemius tendon was not repaired. The tendon was reclined and the crural fascia/paratenon closed using1-0silk suture and continuous suture. The subcutaneous tissue was closed using1-0silk suture then the skin using1-0silk suture. Antibiosis was performed with intramuscularly injected gentamicin (20mg/kg) continuously three days post-operative. Immobilization of the animals was conducted for three weeks. The animals were housed individually in a standard rabbit cage and kept at the same conditions of temperature, humidity and light, and subjected to comprehensive veterinary care. Clinical parameters including appetite, diarrhea, infection, wound healing and adhesion were monitored and assessed. On3rd day and at2nd,4th,8th and12th week post-implantation, animals were euthanized and the implants were harvested. Gross and histological evaluation was performed for the repaired Achilles tendon.
Results:All rabbits showed normal appetite, and except for1animal in group2caught infections, there was no evidence of clinical complications such as local infection or diarrhea. In all groups there existed muscle atrophy of immobilized limb, but recovered after immobilization was removed. Group1and group2shared similar macroscopic alterations:no tendon rupture existed and no elongation at the interfere zone of grafts and host tendons were found. At the first few weeks, color of grafts was translucent and dull white, while at12w the color was close to host tendons and similar appearance was observed between grafted and host tendon. Grafts integrated tightly into stumps of host Achilles tendon and slight adhesion with the surrounding tissue was found. In group3, the xenograft was objected by host and no healing of the excised tendon in group4. Therefore the histological observation was compared between group1and group2. On the third day post-operation, granulation tissue was found grew perpendicular to the long axis of tendons. Main cellular components were neutrophils and macrophages and cells infiltration into graft was observed. Fibril vascular capsule formed along grafted and host tendons; meanwhile micro angiogenesis was confirmed at the periphery of the graft. Group1and group2exhibited similar alterations. No much leukocytes infiltration and cellular necrosis existed around grafted tendons. Interface between graft and host Achilles tendon:At2nd week post operation, maturation of granulation tissue, irregularly arranged cells were observed, which included few inflammatory cells, micro vessels and lots of fibroblasts. New disorganized collagen fibrils were perpendicular to the long axis of tendons. Remodeling was active near interface, lots of micro vessels, fibroblasts and macrophages found. At4th week, inflammatory cells inside granulation tissue decreased, while micro vessels and fibroblasts increased. In group1inflammatory cell was a little more and micro vessels were slightly less compared to group2. At8th week, few inflammatory cells existed at interface zone, on contrary lots of fibroblasts scattered in long axis of tendons. New collagen fibrils were dense and paralleled in the direction of long axis of tendons. At12th week, collagen fibrils were denser and more organized paralleled, but still less mature than normal tendon. In the whole observed period, group1and group2presented similar alteration. Grafted tendons:At2nd week, fibril vascular capsule formed along grafted tendons for both group. Angiogenesis and collagen synthesis occurred at periphery of the grafted tendons. For both groups tendons, at remote part from interface or central part of grafted tendons, dense regularly arranged collagen fibrils were observed in sections, and little or no cell infiltration was found. Cell infiltration and angiogenesis increased at4th week. Less fibroblasts and micro vessels were observed in group1. At8th week, remodeling was evident that lots of new collagens were replacing grafted tendons not only at periphery but also in the central part of grafted tendons. New collagen fibrils were small but dense. At12weeks, remodeling was more evident and new collagen fibrils were close to normal tendons. Within observed period, similar alteration occurred for experimental and control group. After Masson staining new collagen fibrils exhibited green, while original ones presented red. It is observed that new collagen fibrils gradually increased and replacing original ones. At2nd week, most of collagen fibrils were stained red, while fibrils at periphery or near interface exhibited green. At4th week, red stained fibrils still were majority, but green stained fibrils increased and were found at inner part of grafted tendons. At8th week, only few fibrils were stained red, most of which were replaced by green stained fibrils.
Conclusion:In the rabbit model, the responses of host to tendon autograft and the bioactive decellularized tendon scaffolds were similar when the two grafts were used to repair the medial M. gastrocnemius tendon of the Achilles tendon complex. And the two grafts also experienced the similar histological alteration. Therefore, and the bioactive decellularized tendon scaffold was believed to repair the excised tendon effectively, which provided experimental support for its future application.
目的：采用多次脱细胞循环处理去除牛肌腱腱细胞、利用交联剂交联脱细胞支架增强力学强度、过氧乙酸及冷冻干燥增加空隙率、最后进行灭菌处理制备异种脱细胞肌腱支架,并通过多项检测指标评价制备的脱细胞肌腱支架去除抗原和细胞成分的效果、检测交联程度和力学性能,评价体内外组织相容性,为异种肌腱的应用提供实验依据。
方法：取健康成年牛腱,于-80℃深冻保存1个月,室温复融后进行α-半乳糖苷酶消化及脱细胞处理：PBS缓冲液中浸泡数小时,根据实验设计选择添加a-半乳糖苷酶；胰蛋白酶溶液消化数小时,用含胎牛血清的DMEM培养基终止胰酶消化；于聚乙二醇辛基苯基醚,脱氧胆酸钠混合液中脱细胞,更换脱细胞溶液,重复脱细胞循环数次,PBS反复清洗；EDTA浸泡数小时；DMEM培养基浸泡,PBS反复清洗；利用京尼平交联脱细胞肌腱数小时,PBS反复清洗；DMEM培养基浸泡后置于过氧乙酸-乙醇溶液中,PBS反复清洗；-80℃深冻后冷冻干燥；15kGyγ射线辐照制备脱细胞肌腱支架。取新鲜深冻及不同脱细胞循环处理牛肌腱,HE染色后光学显微镜观察细胞去除情况及肌腱纤维组织形态学特征,荧光显微镜下观察DAPI染色样本DNA和RNA去除情况。取新鲜深冻,脱细胞未经α-半乳糖苷酶处理(1-7个脱细胞循环),α-半乳糖苷酶处理样本,分别进行抗α-半乳糖基抗原ELISA定量：取-20℃保存样本室温消融,分别将组织样本充分剪碎至匀浆。取a-gal ELISA试剂盒室温平衡,分别设空白孔(空白对照不加样本及酶标试剂,其余各步操作相同)、标准孔、待测样本孔。在酶标包被板上标准品准确加样50μl,待测样本孔中先加样本稀释液40μl,然后再加待测样本10μl(终稀释倍数为5),样本加于酶标板孔底,尽量不触及孔壁,轻轻晃动混匀；封板膜封板后置37℃温育30min；揭除封板膜,弃液体,甩干,每孔加满洗涤液,静置30min后弃去,重复5次,拍干；加入酶标试剂,50μl孔,空白除外；37℃温育30min；洗涤液清洗5次,拍干；加显色剂A,50μl/孔,再加显色剂B,50山/孔,震荡混匀,37℃避光显色10min;加终止液终止反应,501μl/孔；以空白孔调零,450nm波长测量各孔吸光度(OD值)。以标准物的浓度为横坐标,OD值为纵坐标绘制标准曲线,根据样本OD值由标准曲线查出相应浓度,乘稀释倍数计算样本实际浓度根据a-半乳糖基浓度确定脱细胞循环次数；扫描电子显微镜(SEM)观察新鲜深冻及脱细胞肌腱支架的脱细胞程度及纤维结构。取新鲜深冻,脱细胞处理(1-7个脱细胞循环)样品,以E.Z.N.A. TM Tissue DNA Kit利用NANODROP2000分光光度计检测样本总DNA：称取实验样本,充分研碎后加TL缓冲液、OB蛋白酶混匀后温浴过夜;加RNA酶后高速离心,吸取上层悬液与BL缓冲液充分混匀,温育后加无水乙醇,强力震荡充分混匀；取HiBind(?) DNA柱放置于收集管内,将之前所制备样本加入DNA柱内,高速离心后弃液体；将DNA柱放入新的收集管内,HB缓冲液冲洗,高速离心后弃液体及收集管；将DNA柱放入原收集管内,用DNA清洗缓冲液冲洗,高速离心后弃液体保留收集管；将DNA柱放入原收集管内,同上清洗；将DNA柱放入原收集管内,最大速度离心后将DNA柱放入无菌离心管内,添加预热洗脱缓冲液,室温静置数分后高速离心,重复洗脱；提取DNA溶液即用或-80℃保存备用。根据残余DNA量确定脱细胞循环次数。综合组织学观察、扫描电镜观察、α-半乳糖基浓度以及残余DNA量确定脱细胞循环次数。采用茚三酮法利用分光光度计在570nm下测量吸光度,测定京尼平交联度：(1)柠檬酸、NaOH和氯化亚锡混合,加去离子水调至25m1,另将茚三酮加入25m1乙二醇甲醚中,再将上述两液体混合搅拌,制成茚三酮溶液,避光保存。(2)称取样品,加入茚三酮溶液,100℃水浴20min,冷却至室温,加异丙醇溶液,用分光光度计在570nm下测量吸光度。以甘氨酸溶液做标准曲线(n/M,n/4M,n/8M,n/16M, n/32M,),并通过此标准曲线查找各样本中自由氨基酸的摩尔数。利用异丙醇替换法计算深冻及脱细胞肌腱支架的孔隙率。对新鲜深冻肌腱和脱细胞肌腱支架进行力学拉伸试验,以抗张强度、断裂延伸率及弹性模量为评价指标,观察力学性能变化。通过倒置显微镜观察,利用CCK-8在450nm波长条件下应用酶标仪测量光吸收值检测细胞增殖情况,利用中性红于540nm波长测量光吸收值检测细胞存活情况,综合评价体外细胞相容性。采用大鼠皮下埋入实验：备皮,用手术刀片于背部做纵形切口,大小10mm左右,钝性分离皮下组织,形成一囊袋,皮下埋入,以新鲜牛腱为阳性对照,胶原海绵为阴性对照,脱细胞肌腱支架为实验组,术后3、7、14、21d进行大体及组织学观察,评价所制备肌腱支架的体内相容性。
结果：HE染色后可见脱细胞肌腱支架腱束呈波浪形紧密规则排列,与深冻肌腱相比腱束间距略有增大,未观察到细胞成分；DAPI染色可见深冻肌腱内大量DNA和RNA,而脱细胞肌腱支架则未发现DNA和RNA；实验同时观察到过度脱细胞处理可致肌腱断裂,胶原变性,腱束间距过大,失去原有三维结构特征。α-半乳糖基抗原检测结果显示经α-半乳糖苷酶处理后样本内未检测到a-半乳糖基抗原的存在；经脱细胞循环处理,α-半乳糖基抗原浓度逐渐降低,经3次处理后,未检测到α-半乳糖基抗原。SEM观察可见深冻肌腱和脱细胞肌腱支架腱束排列规则紧密,脱细胞肌腱支架肌腱束之间距离略有增加,空隙度优于深冻肌腱,未发现细胞成分。样本经脱细胞循环处理后,DNA含量明显下降,经3个脱细胞循环处理,DNA含量约下降至原有含量的1/3,4个脱细胞循环处理,DNA含量约降至原有含量的1/4。综合组织学观察、扫描电镜观察、a-半乳糖基浓度以及残余DNA量确定本研究采用4个脱细胞循环进行脱细胞处理。茚三酮法测定京尼平交联度达57.27%。对孔隙率的测定显示脱细胞肌腱支架(56.800±9.311%)高于深冻肌腱(38.660±2.996%%),二者有显著性差异(t=-4.147,P=0.003)。力学性能检测深冻肌腱抗张强度为22.640±4.504MPa,脱细胞肌腱支架组为29.73±4.62MPa,统计学结果显示差异无统计学意义(t=-1.904,P=0.130)；深冻肌腱断裂延伸率为15.200±4.071%,脱细胞肌腱支架组为13.830±3.424%,统计学结果显示差异无统计学意义(t=0.445,P=0.679);深冻肌腱弹性模量为151.250±12.885MPa,脱细胞肌腱支架组为218.680±23.833MPa,统计学结果显示差异有统计学意义(t=-4.311,P=0.022)。脱细胞肌腱支架抗张强度和弹性模量均较深冻肌腱有增加,断裂延伸率则较之减小。CCK-8检测结果显示,在不同的时间点实验组与阴性对照组的吸光度值有显著性差异(F=578.697,P<0.001),实验组吸光度值高于阴性对照组,差异有显著性意义(F=4.011,P<0.05),组别因素与时间因素之间无交互效应(F=1.650,P>0.05)。同一检测时间点,不同组别间吸光度值无显著性差异(P>0.05)：同一组别内,在不同的检测时间点的吸光度值有显著性差异(P<0.001);2、5、7d细胞增殖率分别为105%、105%和110%,细胞毒性评价均为0级：中性红检测3d细胞存活率为99%,7d为106%。体内组织相容性实验结果显示阳性对照组表现为大量淋巴细胞浸润,实验组和阴性对照组结构相似,阴性对照组更快的被机体所吸收：术后3d,实验组与对照组镜下所见相似,植入物被层状炎性组织包绕,其间可见小血管扩张、充血,白细胞渗出,炎细胞以中性粒细胞和巨噬细胞为主。术后1w,两组植入物仍被光滑的薄层结缔组织包裹,炎症细胞较3d减少；包裹组织外层主要由梭形的纤维细胞和疏松的胶原纤维组成。对照组海绵胶原支架出现降解,实验组可观察到细胞成分沿实验组肌腱边缘向内部长入,几乎无炎细胞浸润,无胶原降解。术后2w,胶原海绵组包裹组织明显缩小,内部仅残存少量胶原支架,支架周围见大量细胞浸润。实验组肌腱周围包裹组织与1w所见相似,边缘肌腱胶原内少量炎细胞浸润,以巨噬细胞为主,伴有轻微胶原溶解,偶见多核巨细胞,细胞向肌腱内部长入增加。术后3w,胶原海绵已经基本吸收,实验组与2w类似,长入肌腱内细胞增多。
结论：采用多步脱细胞处理后经京尼平交联脱细胞肌腱支架,并利用过氧乙酸-乙醇结合γ射线辐照灭菌所制备的肌腱支架具有：(1)无细胞成分及α-半乳糖基抗原；(2)保存了完整的天然肌腱三维支架；(3)良好的力学性能；(4)良好的体内、体外组织相容性。本研究为异种肌腱的实际应用提供初步实验基础。
第二章活性脱细胞支架的初步构建
目的：摸索富含血小板血浆的制备方法,将富含血小板血浆与缓释剂混合并复合脱细胞肌腱支架,初步构建具有缓释能力的活性脱细胞肌腱支架系统。
方法：取SD大鼠,戊巴比妥钠腹腔注射麻醉,用预先抗凝的10ml注射器行心脏采血,采用低密度两步离心法制备富含血小板血浆：以200×g离心后,将全部上清液、中间层(白细胞和血小板层)及少量红细胞移入PE管内,相同条件下再次离心后,吸取上清血浆,以5倍全血血小板浓度重悬制备富含血小板血浆。通过ELISA法检测富含血小板血浆中血小板源性生长因子(PDGF)、血管内皮细胞生长因子(VEGF)、胰岛素生长因子-1(IGF-1)、转化生长因子-β1(TGF-β1)四种生长因子浓度。取4℃保存的ELISA试剂盒,恢复至室温。将标准品及待测样本加入酶标板,混匀后置37℃40min；洗涤液充分洗板,滤纸印干；加蒸馏水和第一抗体工作液各50μl/孔,混匀后置37℃20min:洗板；加酶标抗体工作液,混匀后置37℃10min洗板；加底物工作液,混匀后置37℃暗处10min;以空白孔调零,450nm波长测量各孔吸光度(OD值)。以标准物的浓度为横坐标,OD值为纵坐标绘制标准曲线,根据样本OD值由试剂盒自带软件绘制标准曲线查出相应浓度,乘稀释倍数计算样本实际浓度。利用Ⅰ型胶原溶液激活富含血小板血浆,用注射器将二者混合液注入制备的牛脱细胞肌腱支架中,制备活性肌腱支架。将所制备的缓释系统以4倍蒸馏水稀释,于3,12,24,48h利用ELISA试剂盒检测相应生长因子的浓度,评价缓释功能。对活性脱细胞肌腱支架进行免疫组织化学染色,检测样本是否存在TGF-β和VEGF两种生长因子：取石蜡切片,经二甲苯及逆梯度酒精脱蜡水化；PBS洗涤；过氧化氢处理；PBS洗涤后酶胰酶修复；蒸馏水洗涤；封闭液封闭；一抗过夜；PBS洗涤后添加二抗；PBS洗涤后加SABC; PBS洗涤数次；DAB显色：自来水冲洗后苏木素复染；盐酸分化、返蓝；脱水、透明、封片,光学显微镜下观察。将活性脱细胞肌腱支架与大鼠成纤维细胞共培养,倒置相差显微镜观察细胞生长增殖情况并利用CCK-8检测成纤维细胞增殖情况。
结果：ELISA法检测富含血小板血浆中PDGF浓度为3624.20±1453.62pg/ml、IGF-1浓度为376.64±136.66pg/ml、TGF-β1浓度为1103.45±1149.31pg/ml,未检测出样本中VEGF浓度。缓释检测显示随时间的延长,生长因子逐渐由支架释放,未检测出样本中VEGF浓度,PDGF在48h后的浓度约为12h及24h的3倍,IGF-1在48h后的浓度约为12h及24h的2倍,TGF-1在48h后的浓度约为12h的3倍,24h的2倍。免疫组织化学未发现明显阳性染色结果。将所制备的活性肌腱支架与大鼠成纤维细胞共培养后,可以观察到细胞形态、生长状态良好。CCK-8检测实验组OD值3.280±0.235,对照组OD值2.947±0.168,两组差异有统计学意义(t=-3.987,P=0.001),细胞相对增殖率为111%。
结论：本研究探索了富含血小板血浆的制备方法,初步构建了具有一定缓释功能的活性脱细胞肌腱支架,所构建的活性支架具有一定的促进细胞增殖的功能。
第三章活性肌腱支架修复兔跟腱缺损
目的：建立动物缺损模型评价活性脱细胞肌腱支架的修复能力,为其实际应用提供实验依据。
方法：取日本大耳白兔,用预先抗凝的注射器于兔耳中心动脉取血,按第二章的方法制备富含血小板血浆。按第一章的方法制备脱细胞肌腱支架,并按第二章方法构建活性脱细胞肌腱支架。戊巴比妥钠兔耳缘静脉注射麻醉,后肢小腿后侧备皮,暴露跟腱分离内侧束,切除中段2cm,造成跟腱缺损,保留浅部外侧束和深部束；组1采用相同长度的活性脱细胞肌腱作为植入物,组2用对侧浅内侧束作为移植物修复缺损,组3采用相同长度粗细的异种牛肌腱作为植入物,组4不予修复,直接缝合。石膏绷带固定,单笼饲养,正常饮食,术后连续3d肌注庆大霉素。术后观察动物是否存活,并记录动物饮食、精神状态,有无腹泻、感染,伤口愈合及术侧肢体活动情况。于术后3d、2w、4w、8w、12w,每组随机取3只兔处死,观察大体及组织学修复情况。
结果：术后各实验动物均饮食正常,精神状态佳,无腹泻,固定肢肌肉存在不同程度萎缩,去除石膏后逐渐恢复。组1与组2相似。伤口愈合良好,伤口无红肿及感染；无跟腱断裂,植入腱和宿主腱吻合端结合紧密,未发现移植物与自体肌腱缝合端拉长或断裂,移植物与周围组织有不同程度的粘连,组1和组2相似,组3移植肌腱受到机体排斥,组4断腱未愈合。术后3d,植入腱与宿主腱吻合处可见垂直肌腱长轴生长的肉芽组织,中性粒细胞和巨噬细胞为主,细胞向植入腱内生长,周围纤维包裹,有小血管形成；术后2w,对照组自体植入腱与宿主腱间的肉芽组织成熟,炎性细胞和毛细血管较少,可见大量成纤维细胞,新生的胶原纤维垂直于肌腱长轴,组织结构融为一体,靠近吻合端的植入腱内改建活跃,脱细胞植入腱与宿主腱吻合端所见与对照组相似,连接近端可见新生纤维血管组织、大量成纤维细胞。4w,植入腱与宿主腱间的肉芽组织炎性细胞减少,毛细血管、成纤维细胞增多,脱细胞活性肌腱与自体肌腱相比,炎性细胞和毛细血管略多。术后8w,植入腱与宿主腱间的肉芽组织更加成熟,炎性细胞很少,大量成纤维细胞,缝合口处可见大量沿肌腱长轴走行,排列密集的新生胶原纤维,脱细胞活性肌腱与自体肌腱相比,无明显差别。术后12w胶原纤维排列更加规则,胶原束更为紧密,但与正常肌腱相比,胶原纤维排列及成熟度仍略差,自体植入腱与脱细胞植入腱没有明显差别。Masson染色结果显示随时间延长,新生胶原逐渐增多,并取代陈旧胶原。2w时新生胶原较少,以陈旧胶原为主；4w时仍以陈旧胶原占多数,新生胶原较2w时增多；8w时见大量的新生胶原,陈旧胶原大部分被取代。
结论：采用兔跟腱缺损模型,利用活性脱肌腱支架修复缺损跟腱,移植后机体的免疫排斥反应及体内组织学转归与自体植入腱相似,可有效地修复肌腱缺损,为临床应用提供了动物实验依据。
Chapter one Preparation of decellularizd tendon xenograft
Objective:Multiple decellularization cycles were carried out to remove bovine tenocytes. Then the decellularizd tendon xenograft was cross linked to enhance biomechanical property of the scaffold. The prepared scaffolds were finally sterilized by peracetic-acid ethanol combined with low dose y irradiation. Procedures were performed to determine the decellularization, deprivation of a-gal antigen, changes of biomechanical property, in vitro and in vivo cytocompatibility in order to provide experimental foundation for future application of decellularized tendon xenograft.
Methods:Achilles tendons were harvested aseptically and stored at80℃for one month. Frozen tendons were thawed, and all adhering connective-tissue was removed. Then the tendons were decellularized as followed. Samples were washed with PBS, and then incubated in trypsin solution, which was terminated by DMEM with FBS. This was followed by incubation inTriton-X solution that contained Triton and NA-deoxycholate. This step was repeated. After washed with PBS, samples were incubated in EDTA for and washed by DMEM. The samples were then washed with PBS. After different cycles'washing, samples were treated by a-galactosidase. The samples were then washed with PBS and cross linked by incubation with Genipin solution. The solution was discarded and samples were washed with PBS. Samples were treated with PE solution which contained peracetic acid and ethanol. Samples were washed with PBS until residue of peracetic acid was accepted. After deep frozen, samples were freeze-dried and sterilized with15kGy y irradiation. Fresh-frozen and decellularised tendon scaffolds were processed for histological analysis. The sections were mounted on slides and stained with hematoxylin and eosin as well as4,6-diamidino-2-phenylindole (DAPI). The surface of each tendon was manually evaluated from the tendon sections mounted on glass slides and analyzed by optical microscopy, from which decellularization and3-D structure of tendons were evaluated. Quantity of a-gal antigen from fresh-frozen, a-galactosidase treated and decellularised tendon scaffolds was determined using a commercial available ILISA kit. Scanning electron microscopy was performed on the fresh-frozen tendons and the decellularised tendon scaffolds. Cross-sectional and longitudinal-sectional electron-micrographs were obtained to evaluate decellularization and3-D structure of tendons. Fresh-frozen and decellularised tendon scaffolds were freeze-dried and weighed. Then total DNA of specimens was isolated using E.Z.N.A.TM Tissue DNA Kit and determined using NANODROP2000spectrophometer. According to histological observation, SEM observation, concentration of a-gal antigen and quantity of DNA, the proper decellularization cycle was determined. Ninhydrin assay was performed to assess cross linking of genipin by determine the free amino group content. And the optical absorbance of the solution was recorded with a spectrophotometer at a wavelength of570nm. Using glycine at various known concentrations as standard, concentration of free amino was calculated, from which cross linking of genipin was determined. Isopropanol was used to dertermine porosity of fresh-frozen and decellularised tendon scaffolds. Uniaxial load-to-failure tests were performed with a digital universal testing machine. And tensile strength,%strain at ultimate failure load and elastic tensile modulus were compared to test bio mechanical property of fresh-frozen and decellularised tendon scaffolds. In order to evaluate in vitro cytocompatibility, CCK-8was used to determine the absorbance of the solution measured with an enzyme-linked immunoassay. For the cell viability assay, the neutral red solution was chosen to determine the absorbance measured at540nm using the96-well plate spectrophotometer noted above. The absorbance obtained was directly proportional to the viability of the cell populations and inversely proportional to the cytotoxicity of the material. A sterile surgical procedure was performed to implant the specimens subcutaneously in the dorsum of the rats. Decellularised tendon scaffold were experimental group, while fresh bovine tendon was positive control and collagen sponge was negative control. At3rd,7th,14th and21st day post-implantation, the animals were euthanised and the implants were harvested. In vivo host cell infiltration and inflammatory response were evaluated by hemalaun-eosin (H&E) staining with optical microscopy.
Results:Cellular components were evident in fresh-frozen bovine tendon prior to decellularization after H&E staining. After processing, cellular components were not observed and more inter-fascicular and intra-fascicular space was present. DNA and RNA were evident in fresh-frozen bovine tendon prior to decellularization after DAPI staining, while after processing, DNA and RNA were not observed. Collagenous degeneration, rupture of collagen fibre and changes of natural structure of tendon were observed in over-decellularized samples. Scanning electron microscopy confirmed the dense micro-architecture observed in the histologic sections of the fresh-frozen tendons, as well as an increase in pore size and porosity following oxidative treatment. Quantity of a-gal antigen decreased gradually after decellularization. And a-gal antigen could not be quantified after the third cycle of decellularization. No a-gal antigen was detected in sample processed with a-galactosidase. DNA content of the decellularized/oxidized bovine tendon scaffolds was significantly decreased after decellularized and oxidative treatment when compared to the fresh-frozen tendons. DNA content decreased by2/3after the third cycle of decellularization and dropped3/4after the fourth cycle of decellularization. Combining histological observation, SEM observation and determination of a-gal antigen and DNA content,4-decellularization-cycle was chosen. After fixation, we noted that the color of the tissue fixed with genipin became dark blue. By determine the free amino group content, the rate tissue fixed by genipin was calculated as56.27%. Porosity increased after decellularization and significant difference between control and experimental group was noticed (t=-4.147, P=0.003). The tensile strength of fresh-frozen and decellularised tendon scaffolds was22.640±4.504MPa and29.733±4.623MPa respectively. No significant difference was found between two groups (t=-1.904, P=0.130).%strain at ultimate failure load of fresh-frozen and decellularised tendon scaffolds were15.200±4.071%and13.830±3.424%respectively. No significant difference was found between two groups (t=0.445, P=0.679). And elastic tensile modulus of fresh-frozen and decellularised tendon scaffolds were151.250±12.885MPa and218.680±23.833MPa respectively. Significant difference was noticed between two groups (t=-4.311, P=0.022). The results showed that tensile strength and tensile modulus increased, while%strain at ultimate failure load decreased after decellularization. In CCK-8tests, significant difference was noted between control and experimental group at different time (F=578.697, P<0.001). And the value of OD in experimental group is significantly higher than control group (F=4.011, P<0.05). No interaction effect was noticed betreen groups (F=1.650, P>0.05).The relative growth rate at2nd,5th, and7th day were105%,105%and110%respectively. The rating for cytotoxic reacting grade was0. And the rates of viability of the cell were99%and106%respectively for the3rd and7th day. Experiment for in vivo cytocompatibility, bovine xenograft without any decellularization showed a large volume of lymphocytes surrounding and infiltration. While the decellularized tendon scaffold and collagen sponge demonstrated similar reaction and response from the hosts. And compared to decellularized tendon scaffold, collagen sponge was absorbed more quickly. At the3rd day, grafts were surrounded with granulation tissue, where angiotelectasia, hyperaemia and leukocyte exudation was observed. The inflammatory cells were mainly neutrophil granulocyte and macrophagocyte. At the first week, grafts were still surrounded with granulation tissue, and the inflammatory cells decreased. Fibroblasts and collage fibres increased in the surrounding granulation tissue. In control, absorption of collagen sponge was observed. While in experimental group, infiltration of fibroblasts into grafts was noted. At the2nd week, the absorption of collagen sponge was obvious and only little collagen was found in control. The experimental grafts showed similar responses to the1st week. Slight absorption of graft was noticed in the experimental group. And more cell filtration was observed in the peripheral part of grafted tendon scaffolds. After three weeks, almost no collagen sponge was found. More cells infiltration into grafted scaffolds was observed in experimental group.
Conclusion:The decellularized tendon scaffold was prepared by multiple decellularizations, genipin cross linking and sterilized by peracetic-acid ethanol combined y irradiation. This scaffold characterized by:(1) free of cells and a-gal antigen;(2) the natural structure of tendon was well maintained;(3) well preserved biomechanical property;(4) good cytocompatibility. Experimental foundation was provided for future application of decellularized tendon xenograft.
Chapter two Preliminary construction of bioactive decellularizd tendon xenograft
Objective:To explore the method of preparation for platelet rich plasma. Platelet rich plasma was activated and combined with decellularized tendon xenograft through agent characterized with potential of control release the growth factors in platelet rich plasma.
Methods:Intraperitoneal injection of pentobarbital sodium was carried out to anesthetize SD rats. With the10ml syringe processed with anticoagulant in advance, arterial blood was procured from the heart anesthetized SD rats. Then platelet rich plasma was prepared through two centrifugation steps:The blood was centrifuged with a relative centrifugal force of200g for10minutes at a constant temperature of22℃. With a plastic transfer pipette, the whole supernatant, consisting of blood plasma, leucocytes, and thrombocytes, and also red blood cells above the buffy coat were drawn. The exceeding layer of erythrocytes was discarded. This solution was run at a relative centrifugal force of200g for10minutes. The supernatant was partly discarded and platelet rich plasma which concentration was five times of the whole blood. The concentration of platelet-derived growth factor (PDGF), vascular endothelial growth Factor (VEGF), Insulin growth factor-1(IGF-1), and transforming growth factor-β1(TGF-β1) was determined by ELISA. The standard curve was drawn by standard samples provides with the ELISA kit, and absorbance of the experimental samples were determined by enzyme-linked immunoassay. Then the concentration of samples could be determined from the standard curve. The actual concentration would be calculated by multiplying the dilution ratio. Collagen I was used to activate platelet rich plasma. And the solution of Collagen I and platelet rich plasma was injected into the decellularized tendon xenograft with syringe to prepare the bioactive decellularized tendon scaffold. The bioactive decellularized tendon scaffolds with potential system of control release were then immerge into distilled water with four times volume of the solution of Collagen I and platelet rich plasma. ELISA tests were performed at3rd,12th,24th and48th hour to determine the concentration of VEGF, PDGF, IGF-1and TGF-β1. The control release potential of the bioactive decellularized tendon scaffold was evaluated. The bioactive decellularized tendon scaffold was preceded with immunohistochemical staining to determine the existence of TGF-β and VEGF. The rat fibroblasts were co-cultured with the bioactive decellularized tendon scaffold. Then growth and proliferation of fibroblasts were observed with inverted phase contrast microscope. CCK-8was also used to determine the absorbance of the solution measured with an enzyme-linked immunoassay.
Results:The results of ILISA showed that concentration of PDGF was3624.20±1453.62pg/ml in the prepared platelet rich plasma. And the concentration of IGF-1and TGF-β1was376.64±136.66pg/ml and1103.45±1149.31pg/ml respectively. However, the concentration of VEGF was not detected through ILES A test. The control release tests showed that the concentration of growth factors in the solution increased gradually. For PDGF, the concentration at48h was about three times of concentration at12h and24h. For IGF-1, the concentration at48h was around two times of concentration at12h and24h. For TGF-β1, the concentration at48h was around three times of concentration at12h and two times of concentration at24h. No obvious positive results were observed for immunohistochemical staining of TGF-β and VEGF. After co-cultured with the bioactive decellularized tendon scaffolds, fibroblast grew and proliferated well. CCK-8test showed that the absorbance of experimental group was3.280±0.235, while2.947±0.168for control. There was significant difference between two groups (t=-3.987, P=0.001). And the relative growth rate was111%.
Conclusion:The preparation of platelet rich plasma was explored. The prepared bioactive decellularized tendon scaffolds possessed the potential of control releasing growth factors in platelet rich plasma. The prepared bioactive decellularized tendon scaffolds could improve growth and proliferation of rat fibroblasts.
Chapter three Achilles tendon repair in a rabbit model with prepared bioactive decellularized tendon scaffolds
Objective:Animal tendon repair model was to be founded to evaluate the repairing potential of the bioactive decellularized tendon scaffolds.
Methods:The whole blood was drawn from the rabbit ear central artery with pre-anti-coagulated syringe. Then platelet rich plasma was prepared followed the methods descripted in chapter two. The decellularized tendon scaffolds was prepared with the methods founded in chapter one. And the bioactive decellularized tendon scaffolds was constructed followed the procedure introduced in chapter two. Each rabbit was anesthetized with3%Pentobarbital,30mg/kg. The hind paws were shaved and prepared for sterile operative intervention. A2.5cm longitudinal incision of the skin was performed using a lateral paramedian approach followed by a longitudinal splitting of the crural fascia and the paratenon which surrounds the Achilles tendon complex. Subsequently, the Achilles tendon complex was exposed. The medial M. gastrocnemius tendon was separated from the lateral M. gastrocnemius tendon and the M. flexor digitalis superficialis tendon.2cm excision was created and repaired as study design, graft was sutured to host stumps with5-0 nylon in an "8" way strengthened by interrupted stitched. In group1, the medial M. gastrocnemius tendon was repaired with the bioactive decellularized tendon scaffolds. In group2, the medial M. gastrocnemius tendon was repaired with the opposite medial M. gastrocnemius tendon. In group3, the medial M. gastrocnemius tendon was repaired with the bovine tendon xenograft. For group4, the medial M. gastrocnemius tendon was not repaired. The tendon was reclined and the crural fascia/paratenon closed using1-0silk suture and continuous suture. The subcutaneous tissue was closed using1-0silk suture then the skin using1-0silk suture. Antibiosis was performed with intramuscularly injected gentamicin (20mg/kg) continuously three days post-operative. Immobilization of the animals was conducted for three weeks. The animals were housed individually in a standard rabbit cage and kept at the same conditions of temperature, humidity and light, and subjected to comprehensive veterinary care. Clinical parameters including appetite, diarrhea, infection, wound healing and adhesion were monitored and assessed. On3rd day and at2nd,4th,8th and12th week post-implantation, animals were euthanized and the implants were harvested. Gross and histological evaluation was performed for the repaired Achilles tendon.
Results:All rabbits showed normal appetite, and except for1animal in group2caught infections, there was no evidence of clinical complications such as local infection or diarrhea. In all groups there existed muscle atrophy of immobilized limb, but recovered after immobilization was removed. Group1and group2shared similar macroscopic alterations:no tendon rupture existed and no elongation at the interfere zone of grafts and host tendons were found. At the first few weeks, color of grafts was translucent and dull white, while at12w the color was close to host tendons and similar appearance was observed between grafted and host tendon. Grafts integrated tightly into stumps of host Achilles tendon and slight adhesion with the surrounding tissue was found. In group3, the xenograft was objected by host and no healing of the excised tendon in group4. Therefore the histological observation was compared between group1and group2. On the third day post-operation, granulation tissue was found grew perpendicular to the long axis of tendons. Main cellular components were neutrophils and macrophages and cells infiltration into graft was observed. Fibril vascular capsule formed along grafted and host tendons; meanwhile micro angiogenesis was confirmed at the periphery of the graft. Group1and group2exhibited similar alterations. No much leukocytes infiltration and cellular necrosis existed around grafted tendons. Interface between graft and host Achilles tendon:At2nd week post operation, maturation of granulation tissue, irregularly arranged cells were observed, which included few inflammatory cells, micro vessels and lots of fibroblasts. New disorganized collagen fibrils were perpendicular to the long axis of tendons. Remodeling was active near interface, lots of micro vessels, fibroblasts and macrophages found. At4th week, inflammatory cells inside granulation tissue decreased, while micro vessels and fibroblasts increased. In group1inflammatory cell was a little more and micro vessels were slightly less compared to group2. At8th week, few inflammatory cells existed at interface zone, on contrary lots of fibroblasts scattered in long axis of tendons. New collagen fibrils were dense and paralleled in the direction of long axis of tendons. At12th week, collagen fibrils were denser and more organized paralleled, but still less mature than normal tendon. In the whole observed period, group1and group2presented similar alteration. Grafted tendons:At2nd week, fibril vascular capsule formed along grafted tendons for both group. Angiogenesis and collagen synthesis occurred at periphery of the grafted tendons. For both groups tendons, at remote part from interface or central part of grafted tendons, dense regularly arranged collagen fibrils were observed in sections, and little or no cell infiltration was found. Cell infiltration and angiogenesis increased at4th week. Less fibroblasts and micro vessels were observed in group1. At8th week, remodeling was evident that lots of new collagens were replacing grafted tendons not only at periphery but also in the central part of grafted tendons. New collagen fibrils were small but dense. At12weeks, remodeling was more evident and new collagen fibrils were close to normal tendons. Within observed period, similar alteration occurred for experimental and control group. After Masson staining new collagen fibrils exhibited green, while original ones presented red. It is observed that new collagen fibrils gradually increased and replacing original ones. At2nd week, most of collagen fibrils were stained red, while fibrils at periphery or near interface exhibited green. At4th week, red stained fibrils still were majority, but green stained fibrils increased and were found at inner part of grafted tendons. At8th week, only few fibrils were stained red, most of which were replaced by green stained fibrils.
Conclusion:In the rabbit model, the responses of host to tendon autograft and the bioactive decellularized tendon scaffolds were similar when the two grafts were used to repair the medial M. gastrocnemius tendon of the Achilles tendon complex. And the two grafts also experienced the similar histological alteration. Therefore, and the bioactive decellularized tendon scaffold was believed to repair the excised tendon effectively, which provided experimental support for its future application.
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