胶原支架的制备及与血小板血浆复合的相关研究
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摘要
第一章大鼠鼠尾Ⅰ型胶原制备及理化性质
目的：利用酸溶、盐析提纯法从大鼠鼠尾肌腱中提取Ⅰ型胶原,检测液态胶原的吸光度值及分子量,并对冻干的胶原膜进行表面结构的观察,通过对L-929小鼠成纤维细胞增殖、细胞相容性的影响,评价大鼠鼠尾Ⅰ型胶原用于生物组织工程材料的可行性。
方法：取新鲜SD大鼠鼠尾,抽取肌腱,尽量去除肌腱腱鞘、腱周膜及血管等杂质,置于1：1000新洁尔灭溶液中浸泡10分钟,取出后用0.9%盐水反复冲洗后剪成1mm3组织块,放入大烧杯中加入0.5M乙酸溶液,置于4℃冰箱中提取4天,并间断振摇。将混合溶液于4℃下高速离心25分钟,去除离心管底部杂质收集上清液,缓慢加入NaCL晶体分析纯,边持续搅拌,此时有絮状沉淀出现,再次离心去除上清液收集沉淀,用蒸馏水浸泡沉淀3次以尽量中和NaCL,吸净蒸馏水后再次用0.5M乙酸溶液4℃下过夜溶解沉淀,加入3mol/L的NaOH溶液,调整PH到7,将胶原溶液装入透析袋中,置于10倍体积的蒸馏水中透析2天,去除胶原溶液中的Na+和CL-,每天更换透析液2次,得到的即为纯化的鼠尾Ⅰ型胶原。留取一部分胶原溶液其余冷冻干燥成胶原膜,均置于4℃冰箱中储存。以大鼠鼠尾Ⅰ型胶原标准品溶液作为对照,用紫外可见分光光度计进行光谱分析,用十二烷基磺酸钠-聚丙烯酰胺凝胶(SDS-PAGE)电泳法检测其分子量及各亚型条带的分布情况,扫描电子显微镜观察胶原膜的表面结构特征。取液态胶原溶液及胶原膜,经Y射线辐照灭菌后,L-DMEM培养基为浸提介质,按0.5cm2/ml培养基比例,在37℃条件下浸提48h制备浸提液,以L-DMEM培养基为阴性对照。取指数生长期L-929小鼠成纤维细胞调整浓度为4×104个/ml,按100μl/孔的量接种到96孔细胞培养板,置于37℃5%CO2培养箱中孵育24h,换用浸提液继续培养,阴性对照继续用L-DMEM培养基。分别于1,3,7天,每孔加入0.5%MTT(5mg/ml)溶液20μl,培养箱中继续孵育4小时后,吸弃MTT溶液,每孔加入DMSO溶液150μl,震荡10分钟后用酶标仪检测各孔吸光度值,计算细胞增殖率并评价细胞毒性级别。
结果：经酸溶法提取的大鼠鼠尾肌腱胶原溶液为半透明、粘稠液体,经过盐析纯化后变得更加透明,呈胶冻状。冷冻干燥后的胶原膜呈颜色洁白蓬松海绵状,具有一定的弹性及伸展性。紫外可见分光光度计进行光谱分析显示：经过酸溶、盐析提纯获得的大鼠鼠尾肌腱胶原溶液的最大吸收峰为299nm,而大鼠鼠尾Ⅰ型胶原标准品溶液(Sigma, USA)最大吸收峰为300nm,符合Ⅰ型胶原的特征。SDS-PAGE电泳结果显示自提胶原溶液主要有3个条带组成：分子量大约为210KDa的胶原蛋白分子β链、120KDa和110KDa的α1链和α2链。与鼠尾Ⅰ型胶原标准品溶液电泳特征完全符合,表明白提胶原为Ⅰ型胶原蛋白而且纯度亦很高。扫描电镜观察,可见纤维网格状结构,孔径在100-250μ m之间。经过γ射线辐照灭菌后,液性胶原有部分交联表现,即凝结成块状,而冻干的胶原膜无明显的变化。通过对L-929小鼠成纤维细胞的细胞毒性评级结果显示,液性胶原组及冻干胶原膜组在1,3,7天的细胞生长及形态均正常,细胞毒性为0级或1级,与对照组相比差异没有统计学意义(P>0.05)。
结论：经过酸溶盐析提纯法可以从大鼠鼠尾肌腱中提取纯度较高的Ⅰ型胶原,而且不论胶原溶液还是冻干的胶原膜,均无明显的细胞毒性,具有良好的细胞相容性,为Ⅰ型胶原构建组织工程支架提供了理论依据。
第二章不同条件下京尼平交联胶原海绵的体外初步研究
目的：探讨不同温度及京尼平浓度交联Ⅰ型胶原海绵支架后的各种理化性质的改变。通过对交联后的胶原海绵支架的微观结构、溶胀率、交联度、力学特性、体外细胞毒性及胶原酶降解等理化性质及细胞相容性的检测,来评估京尼平交联后的胶原海绵支架作为骨软骨组织工程材料的可行性。
方法：将冻干的胶原海绵重新用0.5M乙酸彻底溶解后调整PH到7。取48孔细胞培养板,每孔注入胶原溶液800u l并冻干。用PBS配制0.1、0.3、0.5wt%的京尼平溶液,待充分溶解后将胶原支架浸入10ml京尼平溶液中,分别于4、20、37℃条件下交联24h。蒸馏水反复冲洗浸泡交联后的支架以去除未与胶原分子结合的京尼平。再次冻干后即得到9组京尼平交联后的胶原支架：0.1%4℃组；0.3%4℃组：0.5%4℃组；0.1%20℃组；0.3%20℃组；0.5%20℃组；0.1%37℃组：0.3%37℃组和0.5%37℃组,以未交联的胶原支架作为对照组,将各组密封于塑封袋内待用。先行各组材料的大体观察后制作扫描电镜标本,用双面导电胶固定样本于铜台上,喷金后在镜下观察各组胶原支架的形态特征。使用Instron5540材料疲劳测试机进行力学测试,检测材料的抗压缩强度,以1.5mm/min的速率于垂直方向使支架发生形变直至失效。载荷-形变曲线上的最大载荷与材料横截面积的比即为最大压缩强度。将各组材料称重后(w0)室温下浸入PBS溶液中3h,用滤纸吸干表面水分后再次称重(w),增加的重量与原始重量的比率即为材料的溶胀率,以未交联的支架作为对照。使用茚三酮分析来评估各组材料的交联度,称重后的每个材料(7mg)加入4ml茚三酮溶液,于100℃水浴中加热20分钟,待冷却到20℃后加入5m150%异丙醇,紫外可见分光光度计于570nm检测吸光度,用不同浓度(1.0-5.Omg/ml)的甘氨酸溶液作为标准,并绘制标准曲线。用以下公式计算交联度(CD):CD=(未交联样本中的自由氨基摩尔数-交联样本中的自由氨基摩尔数)／未交联样本中的自由氨基摩尔数。体外胶原酶降解试验在4℃条件下进行,将每个样本浸入到2ml含有200μg Ⅰ型胶原酶的PBS溶液中,12h后加入200μ1的0.2M EDTA终止反应,于冰浴中冷却混合物,上清液加入6M HCl于110℃水解24h,水解完成后加入活性炭以去除色素,再次过滤后取滤液2ml,加入2ml柠檬酸缓冲液和2m10.05M氯胺T,室温氧化10分钟后加入高氯酸2ml,放置10分钟后最后加入2ml对二甲氨基苯甲醛,65℃下显色10分钟,冷却后紫外可见分光光度计于570hm检测羟脯氨酸吸光度,用不同浓度(0-5.0mg/ml)的羟脯氨酸溶液作为标准,并绘制标准曲线。交联样本中与未交联样本中的羟脯氨酸含量比即为降解率。按照1.25cm2胶原支架表面积/ml培养基比例,在37℃条件下浸提48h制备各组交联胶原支架的浸提液,以L-DMEM培养基为试剂对照。取对数生长期L-929小鼠成纤维细胞,MTT法检测细胞毒性,计算细胞相对增殖度并进行细胞毒性评级。另外取3周龄SD大鼠的关节软骨和剑突,用酶消化法获取软骨细胞,培养至第二代后以3×105个/ml接种于各组支架,于接种后的第一天和第三天用扫描电镜观察软骨细胞对支架的粘附及生长情况。
结果：京尼平交联后的胶原支架大体形状无明显变化,外观呈现蓝色,颜色的深浅随着不同的交联条件而发生变化,温度越高、京尼平的浓度越高则颜色越深。各组支架内部横截面的结构特征表现为三维、内部互相连通的多孔状结构,随着交联温度及京尼平浓度的升高,由未交联组的纤维网格状结构向交联组的板层状结构过渡,尽管孔径(100-250μm)没有发生改变,但是板层状结构的排列越来越紧密,而且胶原的丝状纤维在交联组彻底消失不见。交联组支架的抗压缩强度亦随着交联温度、京尼平浓度的增高而加大,与未交联组相比差异有统计学意义(P<0.05)。交联支架的溶胀率明显低于未交联组,0.3%370C、0.5%20℃和0.5%37℃组明显低于0.1%4℃组(P<0.05)。在0.1%京尼平溶液中交联,所测得的交联度为6.90-26.48%,0.3%和0.5%组中交联度明显高于0.1%组。细胞毒性检测结果显示,在不同时间点的细胞相对增殖度均在80%以上,细胞毒性评级均为0-1级。京尼平交联24h后,各组支架的抗胶原酶降解能力明显增强,0.3%20℃、0.3%37℃组及0.5%各组与0.1%4℃及0.3%4℃组相比具有统计学意义(P<0.05)。软骨细胞接种于京尼平交联支架第一天,软骨细胞只是粘附于支架内部,到第三天,软骨细胞与支架粘附较紧密。
结论：京尼平可以用作Ⅰ型胶原海绵支架的交联,尽管产生蓝色色素,但是交联后支架的各项理化特征均令人满意。基于我们的数据,理想的交联条件为0.3%京尼平浓度和37℃下交联,支架拥有良好的溶胀率、抗压缩强度、交联度、抗胶原酶降解能力以及较低的细胞毒性,因而具备作为骨软骨组织工程支架的条件。
第三章Ⅰ型胶原/富血小板血浆支架的制备及其体外生长因子释放的初步研究
目的：将二步离心法提取的富血小板血浆(PRP)分别用牛凝血酶和Ⅰ型胶原激活,按一定比例将激活后的PRP与大鼠Ⅰ型胶原复合冻干制备Ⅰ型胶原/富血小板血浆(COL/PRP)支架,通过对血小板血浆成分的分析、支架的结构特征、力学强度、细胞毒性检测,以及测定血小板四种生长因子持续体外释放的含量,来初步探讨Ⅰ型胶原/血小板血浆支架用于骨修复材料的可行性。
方法：SD大鼠心脏取血,根据体重的不同每只可采6-8ml,用动物血液分析仪检测全血中的白细胞、红细胞及血小板的含量,然后将全血于1500rmp下离心10分钟,血液样本即分为三层：上层透亮为血清,中间薄层为血小板及白细胞,下层为红细胞,小心吸取中上层的血清、白细胞、血小板和与中间层相连的少量红细胞(这样可以收集较多的血小板),再次以3000rmp离心10分钟,上层为贫血小板血浆,下层为富血小板血浆和细胞层。弃去上层贫血小板血浆,并用1ml注射器吸除最下面的红细胞,吹打均匀后可以得到500μl左右的PRP,再次用动物血液分析仪检测样本中的白细胞、红细胞及血小板的含量以比较提取PRP过程前后的细胞数量变化。将PRP置于-80℃保存待用。将冻干的胶原海绵用0.5M乙酸彻底溶解后调整PH为7,取干净的48孔细胞培养板,并将PRP常温下复融,分为3组：凝血酶激活组为300μl PRP+30μl (30IU)牛凝血酶,胶原激活组为300u1PRP+300u1I型胶原溶液,空白组为600μlⅠ型胶原溶液。激活10分钟后,凝血酶组每孔加入300μl的1型胶原溶液,各组混合均匀后冻干。扫描电镜观察支架的形态特征,用材料疲劳测试机进行力学强度测试。剩余支架经过环氧乙烷消毒后按照第二章方法制备浸提液,以L-DMEM培养基为试剂对照,应用L-929小鼠成纤维细胞MTT法检测细胞毒性。将消毒后的凝血酶激活组和胶原激活组支架放入消毒的青霉素小瓶内,每个小瓶加入2ml的L-DMEM培养基,置于37℃5%CO2培养箱中浸提,于浸提后的第1、4、7、10天吸取1ml的浸提液,然后再补充lml的L-DMEM培养基。收集的浸提液密封于1.8ml的冻存管置于-80℃保存,待全部时间点的浸提液收集完毕后,用Elisa法检测浸提液中的间隔释放的大鼠转化生长因子β1(TGF-β1)、大鼠成纤维细胞生长因子(FGF)、大鼠血小板衍生生长因子(PDGF)和大鼠血管内皮生长因子(VEGF)的含量。结果：冻干后的COL/PRP支架外观为淡红色,与单纯胶原支架相比质地稍脆,具有一定的弹性。扫描电镜下观察,可见比较均匀一致的多孔结构,孔隙之间互相连通,孔径在50-80μ m之间。离心后的PRP中血小板含量是全血中的3.91±0.98倍,而白细胞和红细胞含量分别是全血中的1.11±0.40倍和0.31±0.20倍。COL/PRP支架的抗压缩强度近似于单纯胶原支架组,组间差异没有统计学意义(P>0.05)。通过MTT法对L-929小鼠成纤维细胞的细胞毒性评级结果显示,COL/PRP支架无细胞毒性,而且细胞增殖率于第一和第三天高于阴性对照组。经ELISA法检测COL/PRP支架释放的四种生长因子含量结果表明：TGF-β1含量在第4天达到高峰,然后2激活组组于第7天和第10天浓度逐渐下降,但是与对照组相比,均能维持相对较高的浓度；FGF和PDGF含量随着时间的延长而呈现逐步下降的趋势,但是凝血酶组在各个时间点的FGF和PDGF释放量均明显高于胶原组；VEGF含量随着时间的延长呈现波浪式释放趋势,第1天至第4天呈现上升趋势,第7天出现下降,而第10天又出现上升,并且2组在各个时间点的VEGF释放量均相当。
结论：经过冻干法制备的COL/PRP支架拥有良好的孔隙率、抗压缩强度、以及无细胞毒性。释放的四种生长因子可以维持较长的时间和较高的浓度,为其作为骨修复材料的应用提供了理论依据。
第四章COL/PRP支架修复大鼠颅盖骨缺损的实验研究
目的：建立大鼠颅盖骨缺损模型,植入制备的COL/PRP支架,通过组织学(HE染色和Masson三色染色)、Micro-CT扫描评价骨缺损的修复情况,为其将来在临床上的应用提供理论基础。
方法：制备酶激活的COL/PRP支架和单纯胶原支架(同第三章),经过环氧乙烷灭菌后待用。取体重为250g的大鼠48只,随机分成3组,0.3%苯巴比妥钠腹腔注射麻醉,无菌操作暴露颅盖骨,用直径5mm环形钻头于单侧颅盖骨造一圆形缺损区,边钻边滴注生理盐水,防止损伤下面的硬脑膜。将手术动物分为三组：COL/PRP组、单纯COL支架组和空白组,植入材料前用生理盐水冲洗切口以去除组织碎屑,然后按组别应用相对应的材料填充缺损区。分别于术后的4,8,12周每组随机选取4只大鼠,处死后取材,高分辨率Micro-CT扫描标本并三维重建。然后标本脱钙进行组织学切片,行HE染色和Masson三色染色,光镜下观察缺损区域的组织修复情况。
结果：术后动物饮食、活动均正常,切口部位无红肿、渗液及化脓等,均为一期愈合。于4周取材时,COL/PRP组与胶原组无显著区别,缺损区域均有一薄层膜状组织覆盖,空白组虽然也有膜状组织,但是更为透明。术后8周,COL/PRP组覆盖缺损区域的膜状组织相对较厚,而胶原组和空白组依次薄于前者,12周时情况愈发明显,各组均未见组织坏死情况的发生。术后4周,COL/PRP支架组骨缺损区域未见明显的新生骨组织形成,主要由纤维组织填充,可见大量的成纤维细胞,新生毛细血管较丰富。胶原支架组骨缺损区域可见纤维结缔组织填充,其间仍然可见未被完全降解的经Masson染色染成淡蓝色的胶原纤维,亦可见少量的新生血管形成,宿主骨边缘未见新骨生成。空白对照组骨缺损区域被纤维结缔组织填充,与另外二组相比相对较薄,纤维结缔组织内几乎无血管长入,宿主骨边缘未见新生骨组织生成。术后8周,COL/PRP支架组骨缺损区域可见相对不成熟的板层骨出现,与纤维结缔组织一起填充缺损区域。胶原支架组骨缺损区域被纤维结缔组织填充,其间及宿主骨周围未见新生骨组织,Masson染色亦可见淡蓝色的胶原纤维。空白对照组骨缺损区域被纤维结缔组织填充,其间及宿主骨周围未见新生骨组织,与4周时相比变化不大。术后12周,COL/PRP支架组于骨缺损区域可见相对成熟的板层骨,可以填充大部分缺损区域,余下则被纤维结缔组织填充。胶原支架组骨缺损区域可见少量新生骨组织及大量纤维结缔组织填充,新生骨组织开始于宿主骨的周围,Masson染色染仍然可见淡蓝色的胶原纤维。空白对照组仍被纤维结缔组织填充,而且其厚度无明显增加,其间及宿主骨边缘未见新生骨组织。高分辨率Micro-CT扫描并三维重建结果显示,术后4周COL/PRP支架组骨缺损区域未见明显缩小,宿主骨边缘有小量的骨修复发生,8周时缺损区域开始变得不规则,由宿主骨向外延伸区域可见密度较高的矿化新生骨组织,12周时缺损区域进一步缩小,大部分为新生的骨组织覆盖,密度接近于正常的宿主骨。由Micro-CT自带分析软件可以检测骨缺损区域的各项成骨指标,随着时间的延长,大部分指标有逐渐增高的趋势,从而表明COL/PRP支架有诱导成骨的能力。胶原支架组术后在Micro-CT上表现为,宿主骨边缘有少量的成骨反应,8周和12周时缺损区域似乎有缩小的趋势,但是不如COL/PRP组明显,分析软件得出8周和12周的各项成骨指标也高于4周,表明胶原或许存在一定的骨诱导能力。空白组术后在Micro-CT上表现无明显的变化,虽然宿主骨边缘有少许的成骨反应,但是整个缺损区域的变化不明显,尽管8周和12周的各项成骨指标均高于4周,但是影像学表明空白组的修复能力是最差的。
结论：本研究中所制备的COL/PRP支架具有良好的骨诱导和修复骨缺损的能力。
Chapter1Preparation and Physico-Chemical Property of Rat Tail Type I Collagen
Objective:To evaluate the feasibility of rat tail collagen using in biomaterials, the absorbance value and molecular weight of collagen solution, microstructure of collagen film, cytotoxicity and biocompatibility of collagen were investigated.
Method:The fresh tendons were extracted from SD rat tails. The peritendineum and blood vessel were carefully removed. After immerging in0.1%benzalkonium chloride solution for10min and washing by0.9%saline, the tendons were cut into small fragments. Then, the tendon fragments were lysed in0.5M ethanoic acid solution for4days at4℃.The mixture was discontinuously pendulated during the process of solvation. The mixed solution was centrifuged for25min at high-speed ratio and4℃.The powders of sodium chloride were added into collected supernate and the mixture was stired continuously. When the flocculation occurred, the mixture was centrifuged once again. The sediments were collected and washed by distilled water for3times. Following dissolve the sediments by ethanoic acid solution, the PH value of collagen solution was adjusted to7by NaOH solution. The depurated collagen solution could be obtained after the process of dialysis. A slight amount of solution was left for SDS-PAGE analysis and spectral analysis. The remaining proportion was freeze-dried. Spectral analysis was determined by a ultraviolet-uisible spectrophotometer using rat tail type I collagen (Sigma) as standard. The molecular weight and isoforms distribution were detected by SDS-PAGE analysis. The structural feature of freeze-dried films was observed by SEM. The cytotoxicity of samples was assessed by the MTT colorimetric assay. Collagen solutions and films were sterilized by gamma irradiation and immersed in low glucose Dulbecco's modified Eagle's medium (L-DMEM) at37℃for48h. L-929mouse fibroblast cells were seeded in96well plates at a density of4.0×104cells/well in0.1ml L-DMEM supplemented with10%fetal bovine serum (FBS), cultured at37℃and5vol%CO2for24h and then treated by leaching liquor of samples. The cells were treated with a normal culture media as control group. After1,3, and7days of culture, the medium was then removed and the cells were washed once with PBS, then20μl of0.5%MTT solution was added to each well followed by incubation for4h at37℃and5vol%CO2. Subsequently, the MTT solution was removed and150μl DMSO was added to each well. The96well plates were placed on a shaker for10min and the optical density (OD) value of each well was measured at490nm using an ELISA reader.
Results:The extracted collagen solution was translucent and viscous. It turned to be more transparent and be similar to gelatum after purification. The color of collagen film was white and the sponge-like structure of collagen film possessed elasticity and extensibility. Compared with the standerd rat tail type I collagen, the maximum absorption peak of the extracted collagen solution was about299nm, while the former was300nm. SDS-PAGE analysis indicates that the extracted and purified collagen from rat tail tendons is mainly of type I, which was characterized by the presence of two alpha chains (al,a2) and a beta component. Their molecular weights were approximately120,110and210kDa, which resembles control. The fiber-and lattice-like structure of collagen film with pore size ranged from100to250μm could be observed by SEM. The collagen liquors could be coagulated after irradiation while the collagen film had nothing to change. MTT assay showed that the cytotoxic grades were0or1at1st,3th and7th day. There is no statistical significance when compared with the control (p>0.05).
Conclusion:The purified type I collagen can be extracted from rat tails. Our results suggest that both collagen solution and film have no cytotoxicity to L-929fibroblast cells. Due to their excellent cell compatibility, they can be used in tissue engineering.
Chapter2The Effects of Different Crossing-Linking Conditions of Genipin on Type Ⅰ Collagen Scaffolds:An In-Vitro Evaluation
Objective:The purpose of this chapter was to analyze the properties of fabricating rat tail type Ⅰ collagen scaffolds cross-linked with genipin under different conditions and to assess the feasibility of these scaffolds using in osteochondral tissue engineering. The morphologies, mechanical properties, cross-linking degree, swelling ratio, in vitro degradation, biocompatibility and cytotoxicity of the scaffolds were evaluated.
Method:The collagen films were lysed in0.5M ethanoic acid solution and adjusted PH value to7. They were transferred into48-well microplates (800μL/well) and freeze-dried. Scaffolds were divided into nine groups and cross-linked by immersion in10ml of PBS containing different concentrations (0.1,0.3and0.5wt%) of genipin for24h at different temperatures (4℃,20℃,37℃). The non-cross-linked scaffolds were set as control. After being cross-linked, scaffolds of different groups were washed with distilled water to remove any residual genipin that might still be present. Afterwards, the3D scaffolds were freeze-dried again and sealed into plastic bags. After general observation, the matrices were fixed by mutual conductive adhesive tape on copper stubs and covered with gold using a sputter coater. The morphology of the scaffolds prepared was observed by a scanning electron microscope. The mechanical test was carried out using a material testing machine (Instron5540, USA) by compression in the vertical direction at a deformation rate of1.5mm/min until failure at20℃. The compressive strength was calculated by Q=Fmax/S, where Fmax is the maximum load on the load-deformation curve and S is the cross-sectional area of each sample. The dry scaffolds were weighed (w0) and then hydrated in PBS for3h at room temperature. After carefully removing the excess surface water with filter paper, the wet scaffolds were weighed (w) again. The swelling ratio of the scaffolds was defined as the wet weight increase (w-w0) to the initial weight (w0). The non-cross-linked collagen sponges were set as the controls. The cross-linking degree (CD) of the different groups was determined by ninhydrin assay. The scaffolds were weighed and a7mg sample from each different group was heated to100℃in a water bath with4ml NHN solution for20min. The solution was then cooled down to20℃, diluted with5ml50%isopropanol, and the optical absorbance of the solution at570nm was measured with a spectrophometer using glycine at various concentrations (1.0-5.0mg/mL) as standard. The equation was used for testing the cross-linking degree of the sample as follows: CD=(NHN reactive eamine)fresh-NHN reactive eamine)fixed/(NHN reactive eamine)fresh×100%. The 'fresh' element means the mole fraction of free NH2in non-cross-linked samples while 'fixed' indicate the mole fraction of free NH2remaining in cross-linked samples. The biodegradability of the type Ⅰ collagen scaffolds was determined by incubating each sample in2mL PBS (pH7.4) containing 200μg collagenase type I (sigma, USA) at4℃for12h. Afterwards, the reaction was discontinued by adding200μL0.2Methylenediaminetetraacetic acid (EDTA) and cooling the commixture in an ice bath immediately. The supernatant of the mixture was hydrolyzed in6M Hcl at110℃for24h. The pigment of solution was eliminated by absorbite and filtrated by filtration membrane. The mixture of2ml filtrate,2ml citrate buffer solution and2ml0.05M chloramine T were oxidated at room temperature for10min. After that,2ml perchloric acid solutions were added in the mixture above.10min later,2ml paradimethylaminobenzaldehyde solutions were added in the mixed solution for coloration at65℃for10min. The ultraviolet spectroscopy absorbance of hydroxyproline was examined by a spectrophometer using hydroxyproline at various concentrations (0-5.0mg/mL) as standard. The biodegradation degree is defined as the proportion of hydroxyproline content in the cross-linked samples to that in non-cross-linked ones. The extract liquids of different groups were prepared respectively at a concentration of1.25cm2of surface area of scaffolds per milliliter of L-DMEM medium and incubated at37℃for48h. The L-DMEM medium was reffered as the control group. The cytotoxicity was determined using MTT assay. The primary chondrocytes were isolated from the joint cartilage and xiphoid process of SD rat (3week) by enzymatic digestion. The second passage chondrocytes suspension were seeded on per scaffold (3×105cells/scaffold). Cell morphology and adherence is evaluated on the cross-linked collagen scaffolds, at1day and3day, by SEM.
Results:The general shape of cross-linked collagen scaffolds were not changed obviously. The collagen scaffolds cross-linked with genipin produced blue pigment. The color appearance of collagen scaffolds changed with the different genipin cross-linking conditions. It seemed that the higher concentration of GP and cross-linking temperature increased the intensity of blue. The cross-section of the cross-linked structures was analyzed by scanning electron microscopy (SEM). All scaffolds presented a three-dimensional interconnected porous structure. Non-cross-linked collagen scaffold with pore size ranged from100to250μm presented the fiber-and lattice-like structure. However, the morphologies of the genipin cross-linked scaffolds undergo a sheet-like structural transition. Although the pore sizes have not been changed markedly, it seems that the sheet-like framework closely aligned with the higher genipin concentration and cross-linking temperature. In addition, the fibers of non-cross-linked collagen scaffold are totally not seen in all cross-linked groups. The compressive strength augments greatly with the increased genipin concentration and cross-linking temperature compared with control (p<0.05). The swelling ratio of each cross-linked scaffold was much lower than that of the control (non-cross-linked)(P<0.05). The swelling ratios of the scaffolds which in0.3%37℃,0.5%20℃and0.5%37℃groups were significantly lower than that of in0.1%4℃(P<0.05). The cross-linking degree ranged from6.90to26.48%when cross-linked by0.1%genipin. The cross-linking degree of scaffolds which in0.3%and0.5%groups were obviously higher than that of in0.1%groups(P <0.05). MTT assay showed that the ratios of cell proliferation were above80%and cytotoxic grades were0or1at1st,3th and7th day. After being treated with genipin for24h, the anti-degradation ability of collagen scaffolds increased remarkably. There is statistical difference between0.3%20℃,0.3%37,0.5%groups and0.1%4℃and0.3%4℃group. The chondrocytes maintain a round shape in day1indicating that they just adhere to the scaffold. At day3, the cells have adhered to the scaffold closely. So, these scaffolds can be used in osteochondral tissue engineering.
Conclusion:Genipin could be used in cross-linking type I collagen scaffolds. Although the producted blue pigment, the properties of fabricating rat tail type I collagen scaffolds cross-linked with genipin were satisfactory. Based on our data, the optimization of process conditions demonstrated that successful cross-linking with genipin could be achieved at0.3%genipin concentrations and37℃. The scaffolds possessed exceptional swelling ratios, biodegradation degrees, cross-linking degrees, compressive strength and lower cytotoxicity under this condition.
Chapter3Preparation and Study on Growth Factor Release of COL/PRP Scaffolds
Objective:The extracted PRP were activated respectively by thrombin and type I collagen solution. Then, the col/prp scaffolds could be obtained by freeze-drying the mixture of activated PRP and type I collagen solution according to a certain proportion. To evaluate the feasibility of these scaffolds using in osteochondral tissue engineering, the analysis of PRP fraction, structural feature, mechanics strength, cytotoxicity of scaffolds and contents of growth factor release were investigated.
Method:6-8ml of blood could be extracted from each SD rat heart. The contents of leucocytes, erythrocytes and platelets of whole blood were measured by an automated animal blood counter. After the blood samples were centrifugated at1500rmp for10min, the blood separated into three phases:platelet-poor plasma (top), the platelet-rich plasma containing leucocytes and platelets (middle), and erythrocytes (bottom). The top and middle layers were transferred to new tubes and centrifuged again at3000rmp for10min. The supernatant plasma was discarded, and the remaining500μL of plasma containing precipitated platelets was blended evenly and designated as PRP. Care was taken to remove erythrocytes by a lml syringe to minimize their interference as far as possible. Samples of PRP were analyzed again by an automated animal blood counter and stored at-80℃. The collagen films were lysed in0.5M ethanoic acid solution and adjusted PH value to7. Samples of PRP were thawed at room temperature and poured into48-well microplates (300μL/well). The mixture of300μL PRP+30μL thrombin (30IU) was defined as thrombin-activated group,300μL PRP+300μL collagen solution was collagen-activated group,600μL collagen was blank group.10min later,300μL collagen solutions were added in each well of thrombin group. The mixed solutions were stired uniformly and freeze-dried. The morphology of the scaffolds prepared was observed by a scanning electron microscope. The mechanical test was carried out using a material testing machine. The rest of scaffolds were sterilized by ethylene oxide. The extract liquids of different groups were prepared respectively according to the method in chapter2. The L-DMEM medium was reffered as the control group. The cytotoxicity was determined using MTT assay. The sterilized scaffolds in thrombin-activated group and ollagen-activated group were placed into penicillin phials.2ml of L-DMEM medium was poured into each phial and incubated at37℃and5vol%CO2. Interval release of TGF-β1, PDGF, FGF and VEGF from each scaffold was measured at1,4,7and10days. At each time point, one ml of media was aspirated from around each sample and replaced with one ml of fresh media. Media samples were stored in1.8ml cryovials in a-80℃freezer until all samples were collected. Concentrations of rat PDGF, TGF-β1, FGF and VEGF were determined using the commercially available Quantikine colorimetric sandwich ELISA kits.
Results:The color of freeze-dried COL/PRP scaffolds was pink. Though it possesed comparative elasticity, the texture of COL/PRP scaffolds was more brittle than the COL ones when compared with the latter. The COL/PRP scaffolds were analyzed by scanning electron microscopy (SEM) and presented a three-dimensional interconnected porous structure with pore size ranged from50to80μm. The platelet content in PRP was about3.91±0.98times more than in whole blood after centrifugation. While leukocytes and erythrocytes content was about1.11±0.40and 0.31±0.20times more than in whole blood respectively. The compressive strength of COL/PRP scaffolds was comparable with COL scaffolds. There was no statistical significance between them (p>0.05). There was no cytotoxicity to L-929fibroblasts in COL/PRP groups. The ratio of cellular proliferation was higher than control at day1and3. The results of four growth factors released by COL/PRP scaffolds as follow:The content of TGF-β1was highest at day4. Although the content fell off at day7and10, the higher concentration could be maintained in each group. The content of FGF and PDGF fell off with time gradually. The concentration of two growth factors in thrombin group was higher than collagen one at each time point. As far as VEGF, the content went up from day1to day4and fell off at day7. While at day10, it went up once again. The concentration of VEGF in two groups was similar at each time point.
Conclusion:The COL/PRP scaffolds possessed exceptional porosity, strength and no cytotoxicity. The four growth factors released from scaffolds could be maintained for a long period and higher concentration. Thus, it is possible that these scaffolds can be used in osteochondral tissue engineering.
Chapte4Study on Rat Calvarial Bone Regeneration of COL/PRP Scaffolds
Objective:The purpose of this chapter was to analyze the bone regeneration of COL/PRP scaffolds by implanting them into rat calvarial defect and to assess the feasibility of these scaffolds using in osteochondral tissue engineering. The histology and Micro-CT scanning were carried out.
Method:The thrombin-activated COL/PRP scaffolds and collagen scaffolds were fabricated according to chapter3and sterilized by ethylene oxide.48Sprague-Dawley rats weighing250g were divided into three groups randomly: COL/PRP group, collagen group and blank group.0.3%phenobarbital sodium was intraperitoneally injected to induce general anesthesia. The rat cranium was exposed under sterile conditions. Five-millimeter-diameter trephine defects were created unilaterally in the calvaria of Sprague-Dawley rats under constant irrigation and with care to avoid injury to the underlying dura. Each defect was flushed with saline solution to remove bone debris. The scaffolds were implanted into calvarial defect. The blank group had no scaffolds.4Animals in each group were sacrificed after4,8and12weeks respectively, and calvaria were harvested for high-resolution microCT analysis. Decalcified samples were embedded in paraffin, and sections were stained with hematoxylin-eosin (H&E) and Masson-Goldner trichrome stain. The information of tissue repair in defect region was observed by light microscope.
Results:The diet and activity of animals were normal after surgery. The incisions were healed primarily without red swelling, exudate and suppuration. There was no difference between COL/PRP group and collagen group at4weeks. The defect area was covered by a thin membrane. It seemed that the membrane in blank group was more transparent. The membrane became thick in COL/PRP group at8weeks, while collagen and blank group was thinner than former in turn. This situation was more obvious at12weeks. There was no tissue necrosis in all groups. The bone regeneration could not be seen at the defect region in COL/PRP group at4weeks. The defect region was filled mainly by fibrous tissue with abundant fibroblasts and new capillaries. The same thing happened in collagen group. The undegradative collagen fiber and slight new capillaries could be seen in fibrous connective tissue. The new bone could not be found at the edge of host bone. The fibrous connective tissue in blank group was thinner than former groups. New capillaries and bone regeneration could not be seen in the fibrous tissue and at the edge of host bone respectively. The immature lamellar bone occurred in COL/PRP group and filled in defect area together with fibrous connective tissue. Like4weeks, there was no bone regeneration in collagen or blank group. The defect only was filled with fibrous connective tissue. At12weeks, the defect area could be filled with much mature lamellar bone and fibrous connective tissue in COL/PRP group. Slight new bone and a great quantity of fibrous connective tissue could be seen at the defect in the collagen group. Bone regeneration formed at the edge of host bone. The bluish collagen fiber stained by Masson staining could be seen in the fibrous connective tissue. In blank group, the defect area was filled with fibrous connective tissue which thickness had not been augmented. There was no bone regeneration around host bone or in the fibrous tissue. The images of bone regeneration were obversed by high-resolution microCT. The defect region in the COL/PRP group had not been decreased at4weeks. Slight bone repair happened at the edge of host bone. While at8weeks, the defect area became irregular and the newly mineralized bone with higher density could be seen at the extension area of host bone. The most defect area was covered by bew bone and became further decreased. The density of bew bone was closed to the host bone. The ossification indexes of defect area could be evaluated by software of the microCT. Most indexes increased gradually with times. This indicated that COL/PRP scaffolds possesed the ability of osteoinduction. Although the slight ossification could be found in collagen group and defect area became decreased with times, but this trend was not obvious when compared with COL/PRP group. The collagen maybe possesed the ability of osteoinduction as the ossification indexes of8and12weeks were higher than4weeks. There was no difference in blank group at different times. Although the slight ossification could be found at the edge of host bone, the whole defect area changed unobviously. The images of microCT showed that the repair ability of blank group was worst in spite of the ossification indexes of8 and12weeks were higher than4weeks.
Conclusion:The COL/PRP scaffolds possesed favourable ability of osteoinduction and repairing bone defect.
目的：利用酸溶、盐析提纯法从大鼠鼠尾肌腱中提取Ⅰ型胶原,检测液态胶原的吸光度值及分子量,并对冻干的胶原膜进行表面结构的观察,通过对L-929小鼠成纤维细胞增殖、细胞相容性的影响,评价大鼠鼠尾Ⅰ型胶原用于生物组织工程材料的可行性。
方法：取新鲜SD大鼠鼠尾,抽取肌腱,尽量去除肌腱腱鞘、腱周膜及血管等杂质,置于1：1000新洁尔灭溶液中浸泡10分钟,取出后用0.9%盐水反复冲洗后剪成1mm3组织块,放入大烧杯中加入0.5M乙酸溶液,置于4℃冰箱中提取4天,并间断振摇。将混合溶液于4℃下高速离心25分钟,去除离心管底部杂质收集上清液,缓慢加入NaCL晶体分析纯,边持续搅拌,此时有絮状沉淀出现,再次离心去除上清液收集沉淀,用蒸馏水浸泡沉淀3次以尽量中和NaCL,吸净蒸馏水后再次用0.5M乙酸溶液4℃下过夜溶解沉淀,加入3mol/L的NaOH溶液,调整PH到7,将胶原溶液装入透析袋中,置于10倍体积的蒸馏水中透析2天,去除胶原溶液中的Na+和CL-,每天更换透析液2次,得到的即为纯化的鼠尾Ⅰ型胶原。留取一部分胶原溶液其余冷冻干燥成胶原膜,均置于4℃冰箱中储存。以大鼠鼠尾Ⅰ型胶原标准品溶液作为对照,用紫外可见分光光度计进行光谱分析,用十二烷基磺酸钠-聚丙烯酰胺凝胶(SDS-PAGE)电泳法检测其分子量及各亚型条带的分布情况,扫描电子显微镜观察胶原膜的表面结构特征。取液态胶原溶液及胶原膜,经Y射线辐照灭菌后,L-DMEM培养基为浸提介质,按0.5cm2/ml培养基比例,在37℃条件下浸提48h制备浸提液,以L-DMEM培养基为阴性对照。取指数生长期L-929小鼠成纤维细胞调整浓度为4×104个/ml,按100μl/孔的量接种到96孔细胞培养板,置于37℃5%CO2培养箱中孵育24h,换用浸提液继续培养,阴性对照继续用L-DMEM培养基。分别于1,3,7天,每孔加入0.5%MTT(5mg/ml)溶液20μl,培养箱中继续孵育4小时后,吸弃MTT溶液,每孔加入DMSO溶液150μl,震荡10分钟后用酶标仪检测各孔吸光度值,计算细胞增殖率并评价细胞毒性级别。
结果：经酸溶法提取的大鼠鼠尾肌腱胶原溶液为半透明、粘稠液体,经过盐析纯化后变得更加透明,呈胶冻状。冷冻干燥后的胶原膜呈颜色洁白蓬松海绵状,具有一定的弹性及伸展性。紫外可见分光光度计进行光谱分析显示：经过酸溶、盐析提纯获得的大鼠鼠尾肌腱胶原溶液的最大吸收峰为299nm,而大鼠鼠尾Ⅰ型胶原标准品溶液(Sigma, USA)最大吸收峰为300nm,符合Ⅰ型胶原的特征。SDS-PAGE电泳结果显示自提胶原溶液主要有3个条带组成：分子量大约为210KDa的胶原蛋白分子β链、120KDa和110KDa的α1链和α2链。与鼠尾Ⅰ型胶原标准品溶液电泳特征完全符合,表明白提胶原为Ⅰ型胶原蛋白而且纯度亦很高。扫描电镜观察,可见纤维网格状结构,孔径在100-250μ m之间。经过γ射线辐照灭菌后,液性胶原有部分交联表现,即凝结成块状,而冻干的胶原膜无明显的变化。通过对L-929小鼠成纤维细胞的细胞毒性评级结果显示,液性胶原组及冻干胶原膜组在1,3,7天的细胞生长及形态均正常,细胞毒性为0级或1级,与对照组相比差异没有统计学意义(P>0.05)。
结论：经过酸溶盐析提纯法可以从大鼠鼠尾肌腱中提取纯度较高的Ⅰ型胶原,而且不论胶原溶液还是冻干的胶原膜,均无明显的细胞毒性,具有良好的细胞相容性,为Ⅰ型胶原构建组织工程支架提供了理论依据。
第二章不同条件下京尼平交联胶原海绵的体外初步研究
目的：探讨不同温度及京尼平浓度交联Ⅰ型胶原海绵支架后的各种理化性质的改变。通过对交联后的胶原海绵支架的微观结构、溶胀率、交联度、力学特性、体外细胞毒性及胶原酶降解等理化性质及细胞相容性的检测,来评估京尼平交联后的胶原海绵支架作为骨软骨组织工程材料的可行性。
方法：将冻干的胶原海绵重新用0.5M乙酸彻底溶解后调整PH到7。取48孔细胞培养板,每孔注入胶原溶液800u l并冻干。用PBS配制0.1、0.3、0.5wt%的京尼平溶液,待充分溶解后将胶原支架浸入10ml京尼平溶液中,分别于4、20、37℃条件下交联24h。蒸馏水反复冲洗浸泡交联后的支架以去除未与胶原分子结合的京尼平。再次冻干后即得到9组京尼平交联后的胶原支架：0.1%4℃组；0.3%4℃组：0.5%4℃组；0.1%20℃组；0.3%20℃组；0.5%20℃组；0.1%37℃组：0.3%37℃组和0.5%37℃组,以未交联的胶原支架作为对照组,将各组密封于塑封袋内待用。先行各组材料的大体观察后制作扫描电镜标本,用双面导电胶固定样本于铜台上,喷金后在镜下观察各组胶原支架的形态特征。使用Instron5540材料疲劳测试机进行力学测试,检测材料的抗压缩强度,以1.5mm/min的速率于垂直方向使支架发生形变直至失效。载荷-形变曲线上的最大载荷与材料横截面积的比即为最大压缩强度。将各组材料称重后(w0)室温下浸入PBS溶液中3h,用滤纸吸干表面水分后再次称重(w),增加的重量与原始重量的比率即为材料的溶胀率,以未交联的支架作为对照。使用茚三酮分析来评估各组材料的交联度,称重后的每个材料(7mg)加入4ml茚三酮溶液,于100℃水浴中加热20分钟,待冷却到20℃后加入5m150%异丙醇,紫外可见分光光度计于570nm检测吸光度,用不同浓度(1.0-5.Omg/ml)的甘氨酸溶液作为标准,并绘制标准曲线。用以下公式计算交联度(CD):CD=(未交联样本中的自由氨基摩尔数-交联样本中的自由氨基摩尔数)／未交联样本中的自由氨基摩尔数。体外胶原酶降解试验在4℃条件下进行,将每个样本浸入到2ml含有200μg Ⅰ型胶原酶的PBS溶液中,12h后加入200μ1的0.2M EDTA终止反应,于冰浴中冷却混合物,上清液加入6M HCl于110℃水解24h,水解完成后加入活性炭以去除色素,再次过滤后取滤液2ml,加入2ml柠檬酸缓冲液和2m10.05M氯胺T,室温氧化10分钟后加入高氯酸2ml,放置10分钟后最后加入2ml对二甲氨基苯甲醛,65℃下显色10分钟,冷却后紫外可见分光光度计于570hm检测羟脯氨酸吸光度,用不同浓度(0-5.0mg/ml)的羟脯氨酸溶液作为标准,并绘制标准曲线。交联样本中与未交联样本中的羟脯氨酸含量比即为降解率。按照1.25cm2胶原支架表面积/ml培养基比例,在37℃条件下浸提48h制备各组交联胶原支架的浸提液,以L-DMEM培养基为试剂对照。取对数生长期L-929小鼠成纤维细胞,MTT法检测细胞毒性,计算细胞相对增殖度并进行细胞毒性评级。另外取3周龄SD大鼠的关节软骨和剑突,用酶消化法获取软骨细胞,培养至第二代后以3×105个/ml接种于各组支架,于接种后的第一天和第三天用扫描电镜观察软骨细胞对支架的粘附及生长情况。
结果：京尼平交联后的胶原支架大体形状无明显变化,外观呈现蓝色,颜色的深浅随着不同的交联条件而发生变化,温度越高、京尼平的浓度越高则颜色越深。各组支架内部横截面的结构特征表现为三维、内部互相连通的多孔状结构,随着交联温度及京尼平浓度的升高,由未交联组的纤维网格状结构向交联组的板层状结构过渡,尽管孔径(100-250μm)没有发生改变,但是板层状结构的排列越来越紧密,而且胶原的丝状纤维在交联组彻底消失不见。交联组支架的抗压缩强度亦随着交联温度、京尼平浓度的增高而加大,与未交联组相比差异有统计学意义(P<0.05)。交联支架的溶胀率明显低于未交联组,0.3%370C、0.5%20℃和0.5%37℃组明显低于0.1%4℃组(P<0.05)。在0.1%京尼平溶液中交联,所测得的交联度为6.90-26.48%,0.3%和0.5%组中交联度明显高于0.1%组。细胞毒性检测结果显示,在不同时间点的细胞相对增殖度均在80%以上,细胞毒性评级均为0-1级。京尼平交联24h后,各组支架的抗胶原酶降解能力明显增强,0.3%20℃、0.3%37℃组及0.5%各组与0.1%4℃及0.3%4℃组相比具有统计学意义(P<0.05)。软骨细胞接种于京尼平交联支架第一天,软骨细胞只是粘附于支架内部,到第三天,软骨细胞与支架粘附较紧密。
结论：京尼平可以用作Ⅰ型胶原海绵支架的交联,尽管产生蓝色色素,但是交联后支架的各项理化特征均令人满意。基于我们的数据,理想的交联条件为0.3%京尼平浓度和37℃下交联,支架拥有良好的溶胀率、抗压缩强度、交联度、抗胶原酶降解能力以及较低的细胞毒性,因而具备作为骨软骨组织工程支架的条件。
第三章Ⅰ型胶原/富血小板血浆支架的制备及其体外生长因子释放的初步研究
目的：将二步离心法提取的富血小板血浆(PRP)分别用牛凝血酶和Ⅰ型胶原激活,按一定比例将激活后的PRP与大鼠Ⅰ型胶原复合冻干制备Ⅰ型胶原/富血小板血浆(COL/PRP)支架,通过对血小板血浆成分的分析、支架的结构特征、力学强度、细胞毒性检测,以及测定血小板四种生长因子持续体外释放的含量,来初步探讨Ⅰ型胶原/血小板血浆支架用于骨修复材料的可行性。
方法：SD大鼠心脏取血,根据体重的不同每只可采6-8ml,用动物血液分析仪检测全血中的白细胞、红细胞及血小板的含量,然后将全血于1500rmp下离心10分钟,血液样本即分为三层：上层透亮为血清,中间薄层为血小板及白细胞,下层为红细胞,小心吸取中上层的血清、白细胞、血小板和与中间层相连的少量红细胞(这样可以收集较多的血小板),再次以3000rmp离心10分钟,上层为贫血小板血浆,下层为富血小板血浆和细胞层。弃去上层贫血小板血浆,并用1ml注射器吸除最下面的红细胞,吹打均匀后可以得到500μl左右的PRP,再次用动物血液分析仪检测样本中的白细胞、红细胞及血小板的含量以比较提取PRP过程前后的细胞数量变化。将PRP置于-80℃保存待用。将冻干的胶原海绵用0.5M乙酸彻底溶解后调整PH为7,取干净的48孔细胞培养板,并将PRP常温下复融,分为3组：凝血酶激活组为300μl PRP+30μl (30IU)牛凝血酶,胶原激活组为300u1PRP+300u1I型胶原溶液,空白组为600μlⅠ型胶原溶液。激活10分钟后,凝血酶组每孔加入300μl的1型胶原溶液,各组混合均匀后冻干。扫描电镜观察支架的形态特征,用材料疲劳测试机进行力学强度测试。剩余支架经过环氧乙烷消毒后按照第二章方法制备浸提液,以L-DMEM培养基为试剂对照,应用L-929小鼠成纤维细胞MTT法检测细胞毒性。将消毒后的凝血酶激活组和胶原激活组支架放入消毒的青霉素小瓶内,每个小瓶加入2ml的L-DMEM培养基,置于37℃5%CO2培养箱中浸提,于浸提后的第1、4、7、10天吸取1ml的浸提液,然后再补充lml的L-DMEM培养基。收集的浸提液密封于1.8ml的冻存管置于-80℃保存,待全部时间点的浸提液收集完毕后,用Elisa法检测浸提液中的间隔释放的大鼠转化生长因子β1(TGF-β1)、大鼠成纤维细胞生长因子(FGF)、大鼠血小板衍生生长因子(PDGF)和大鼠血管内皮生长因子(VEGF)的含量。结果：冻干后的COL/PRP支架外观为淡红色,与单纯胶原支架相比质地稍脆,具有一定的弹性。扫描电镜下观察,可见比较均匀一致的多孔结构,孔隙之间互相连通,孔径在50-80μ m之间。离心后的PRP中血小板含量是全血中的3.91±0.98倍,而白细胞和红细胞含量分别是全血中的1.11±0.40倍和0.31±0.20倍。COL/PRP支架的抗压缩强度近似于单纯胶原支架组,组间差异没有统计学意义(P>0.05)。通过MTT法对L-929小鼠成纤维细胞的细胞毒性评级结果显示,COL/PRP支架无细胞毒性,而且细胞增殖率于第一和第三天高于阴性对照组。经ELISA法检测COL/PRP支架释放的四种生长因子含量结果表明：TGF-β1含量在第4天达到高峰,然后2激活组组于第7天和第10天浓度逐渐下降,但是与对照组相比,均能维持相对较高的浓度；FGF和PDGF含量随着时间的延长而呈现逐步下降的趋势,但是凝血酶组在各个时间点的FGF和PDGF释放量均明显高于胶原组；VEGF含量随着时间的延长呈现波浪式释放趋势,第1天至第4天呈现上升趋势,第7天出现下降,而第10天又出现上升,并且2组在各个时间点的VEGF释放量均相当。
结论：经过冻干法制备的COL/PRP支架拥有良好的孔隙率、抗压缩强度、以及无细胞毒性。释放的四种生长因子可以维持较长的时间和较高的浓度,为其作为骨修复材料的应用提供了理论依据。
第四章COL/PRP支架修复大鼠颅盖骨缺损的实验研究
目的：建立大鼠颅盖骨缺损模型,植入制备的COL/PRP支架,通过组织学(HE染色和Masson三色染色)、Micro-CT扫描评价骨缺损的修复情况,为其将来在临床上的应用提供理论基础。
方法：制备酶激活的COL/PRP支架和单纯胶原支架(同第三章),经过环氧乙烷灭菌后待用。取体重为250g的大鼠48只,随机分成3组,0.3%苯巴比妥钠腹腔注射麻醉,无菌操作暴露颅盖骨,用直径5mm环形钻头于单侧颅盖骨造一圆形缺损区,边钻边滴注生理盐水,防止损伤下面的硬脑膜。将手术动物分为三组：COL/PRP组、单纯COL支架组和空白组,植入材料前用生理盐水冲洗切口以去除组织碎屑,然后按组别应用相对应的材料填充缺损区。分别于术后的4,8,12周每组随机选取4只大鼠,处死后取材,高分辨率Micro-CT扫描标本并三维重建。然后标本脱钙进行组织学切片,行HE染色和Masson三色染色,光镜下观察缺损区域的组织修复情况。
结果：术后动物饮食、活动均正常,切口部位无红肿、渗液及化脓等,均为一期愈合。于4周取材时,COL/PRP组与胶原组无显著区别,缺损区域均有一薄层膜状组织覆盖,空白组虽然也有膜状组织,但是更为透明。术后8周,COL/PRP组覆盖缺损区域的膜状组织相对较厚,而胶原组和空白组依次薄于前者,12周时情况愈发明显,各组均未见组织坏死情况的发生。术后4周,COL/PRP支架组骨缺损区域未见明显的新生骨组织形成,主要由纤维组织填充,可见大量的成纤维细胞,新生毛细血管较丰富。胶原支架组骨缺损区域可见纤维结缔组织填充,其间仍然可见未被完全降解的经Masson染色染成淡蓝色的胶原纤维,亦可见少量的新生血管形成,宿主骨边缘未见新骨生成。空白对照组骨缺损区域被纤维结缔组织填充,与另外二组相比相对较薄,纤维结缔组织内几乎无血管长入,宿主骨边缘未见新生骨组织生成。术后8周,COL/PRP支架组骨缺损区域可见相对不成熟的板层骨出现,与纤维结缔组织一起填充缺损区域。胶原支架组骨缺损区域被纤维结缔组织填充,其间及宿主骨周围未见新生骨组织,Masson染色亦可见淡蓝色的胶原纤维。空白对照组骨缺损区域被纤维结缔组织填充,其间及宿主骨周围未见新生骨组织,与4周时相比变化不大。术后12周,COL/PRP支架组于骨缺损区域可见相对成熟的板层骨,可以填充大部分缺损区域,余下则被纤维结缔组织填充。胶原支架组骨缺损区域可见少量新生骨组织及大量纤维结缔组织填充,新生骨组织开始于宿主骨的周围,Masson染色染仍然可见淡蓝色的胶原纤维。空白对照组仍被纤维结缔组织填充,而且其厚度无明显增加,其间及宿主骨边缘未见新生骨组织。高分辨率Micro-CT扫描并三维重建结果显示,术后4周COL/PRP支架组骨缺损区域未见明显缩小,宿主骨边缘有小量的骨修复发生,8周时缺损区域开始变得不规则,由宿主骨向外延伸区域可见密度较高的矿化新生骨组织,12周时缺损区域进一步缩小,大部分为新生的骨组织覆盖,密度接近于正常的宿主骨。由Micro-CT自带分析软件可以检测骨缺损区域的各项成骨指标,随着时间的延长,大部分指标有逐渐增高的趋势,从而表明COL/PRP支架有诱导成骨的能力。胶原支架组术后在Micro-CT上表现为,宿主骨边缘有少量的成骨反应,8周和12周时缺损区域似乎有缩小的趋势,但是不如COL/PRP组明显,分析软件得出8周和12周的各项成骨指标也高于4周,表明胶原或许存在一定的骨诱导能力。空白组术后在Micro-CT上表现无明显的变化,虽然宿主骨边缘有少许的成骨反应,但是整个缺损区域的变化不明显,尽管8周和12周的各项成骨指标均高于4周,但是影像学表明空白组的修复能力是最差的。
结论：本研究中所制备的COL/PRP支架具有良好的骨诱导和修复骨缺损的能力。
Chapter1Preparation and Physico-Chemical Property of Rat Tail Type I Collagen
Objective:To evaluate the feasibility of rat tail collagen using in biomaterials, the absorbance value and molecular weight of collagen solution, microstructure of collagen film, cytotoxicity and biocompatibility of collagen were investigated.
Method:The fresh tendons were extracted from SD rat tails. The peritendineum and blood vessel were carefully removed. After immerging in0.1%benzalkonium chloride solution for10min and washing by0.9%saline, the tendons were cut into small fragments. Then, the tendon fragments were lysed in0.5M ethanoic acid solution for4days at4℃.The mixture was discontinuously pendulated during the process of solvation. The mixed solution was centrifuged for25min at high-speed ratio and4℃.The powders of sodium chloride were added into collected supernate and the mixture was stired continuously. When the flocculation occurred, the mixture was centrifuged once again. The sediments were collected and washed by distilled water for3times. Following dissolve the sediments by ethanoic acid solution, the PH value of collagen solution was adjusted to7by NaOH solution. The depurated collagen solution could be obtained after the process of dialysis. A slight amount of solution was left for SDS-PAGE analysis and spectral analysis. The remaining proportion was freeze-dried. Spectral analysis was determined by a ultraviolet-uisible spectrophotometer using rat tail type I collagen (Sigma) as standard. The molecular weight and isoforms distribution were detected by SDS-PAGE analysis. The structural feature of freeze-dried films was observed by SEM. The cytotoxicity of samples was assessed by the MTT colorimetric assay. Collagen solutions and films were sterilized by gamma irradiation and immersed in low glucose Dulbecco's modified Eagle's medium (L-DMEM) at37℃for48h. L-929mouse fibroblast cells were seeded in96well plates at a density of4.0×104cells/well in0.1ml L-DMEM supplemented with10%fetal bovine serum (FBS), cultured at37℃and5vol%CO2for24h and then treated by leaching liquor of samples. The cells were treated with a normal culture media as control group. After1,3, and7days of culture, the medium was then removed and the cells were washed once with PBS, then20μl of0.5%MTT solution was added to each well followed by incubation for4h at37℃and5vol%CO2. Subsequently, the MTT solution was removed and150μl DMSO was added to each well. The96well plates were placed on a shaker for10min and the optical density (OD) value of each well was measured at490nm using an ELISA reader.
Results:The extracted collagen solution was translucent and viscous. It turned to be more transparent and be similar to gelatum after purification. The color of collagen film was white and the sponge-like structure of collagen film possessed elasticity and extensibility. Compared with the standerd rat tail type I collagen, the maximum absorption peak of the extracted collagen solution was about299nm, while the former was300nm. SDS-PAGE analysis indicates that the extracted and purified collagen from rat tail tendons is mainly of type I, which was characterized by the presence of two alpha chains (al,a2) and a beta component. Their molecular weights were approximately120,110and210kDa, which resembles control. The fiber-and lattice-like structure of collagen film with pore size ranged from100to250μm could be observed by SEM. The collagen liquors could be coagulated after irradiation while the collagen film had nothing to change. MTT assay showed that the cytotoxic grades were0or1at1st,3th and7th day. There is no statistical significance when compared with the control (p>0.05).
Conclusion:The purified type I collagen can be extracted from rat tails. Our results suggest that both collagen solution and film have no cytotoxicity to L-929fibroblast cells. Due to their excellent cell compatibility, they can be used in tissue engineering.
Chapter2The Effects of Different Crossing-Linking Conditions of Genipin on Type Ⅰ Collagen Scaffolds:An In-Vitro Evaluation
Objective:The purpose of this chapter was to analyze the properties of fabricating rat tail type Ⅰ collagen scaffolds cross-linked with genipin under different conditions and to assess the feasibility of these scaffolds using in osteochondral tissue engineering. The morphologies, mechanical properties, cross-linking degree, swelling ratio, in vitro degradation, biocompatibility and cytotoxicity of the scaffolds were evaluated.
Method:The collagen films were lysed in0.5M ethanoic acid solution and adjusted PH value to7. They were transferred into48-well microplates (800μL/well) and freeze-dried. Scaffolds were divided into nine groups and cross-linked by immersion in10ml of PBS containing different concentrations (0.1,0.3and0.5wt%) of genipin for24h at different temperatures (4℃,20℃,37℃). The non-cross-linked scaffolds were set as control. After being cross-linked, scaffolds of different groups were washed with distilled water to remove any residual genipin that might still be present. Afterwards, the3D scaffolds were freeze-dried again and sealed into plastic bags. After general observation, the matrices were fixed by mutual conductive adhesive tape on copper stubs and covered with gold using a sputter coater. The morphology of the scaffolds prepared was observed by a scanning electron microscope. The mechanical test was carried out using a material testing machine (Instron5540, USA) by compression in the vertical direction at a deformation rate of1.5mm/min until failure at20℃. The compressive strength was calculated by Q=Fmax/S, where Fmax is the maximum load on the load-deformation curve and S is the cross-sectional area of each sample. The dry scaffolds were weighed (w0) and then hydrated in PBS for3h at room temperature. After carefully removing the excess surface water with filter paper, the wet scaffolds were weighed (w) again. The swelling ratio of the scaffolds was defined as the wet weight increase (w-w0) to the initial weight (w0). The non-cross-linked collagen sponges were set as the controls. The cross-linking degree (CD) of the different groups was determined by ninhydrin assay. The scaffolds were weighed and a7mg sample from each different group was heated to100℃in a water bath with4ml NHN solution for20min. The solution was then cooled down to20℃, diluted with5ml50%isopropanol, and the optical absorbance of the solution at570nm was measured with a spectrophometer using glycine at various concentrations (1.0-5.0mg/mL) as standard. The equation was used for testing the cross-linking degree of the sample as follows: CD=(NHN reactive eamine)fresh-NHN reactive eamine)fixed/(NHN reactive eamine)fresh×100%. The 'fresh' element means the mole fraction of free NH2in non-cross-linked samples while 'fixed' indicate the mole fraction of free NH2remaining in cross-linked samples. The biodegradability of the type Ⅰ collagen scaffolds was determined by incubating each sample in2mL PBS (pH7.4) containing 200μg collagenase type I (sigma, USA) at4℃for12h. Afterwards, the reaction was discontinued by adding200μL0.2Methylenediaminetetraacetic acid (EDTA) and cooling the commixture in an ice bath immediately. The supernatant of the mixture was hydrolyzed in6M Hcl at110℃for24h. The pigment of solution was eliminated by absorbite and filtrated by filtration membrane. The mixture of2ml filtrate,2ml citrate buffer solution and2ml0.05M chloramine T were oxidated at room temperature for10min. After that,2ml perchloric acid solutions were added in the mixture above.10min later,2ml paradimethylaminobenzaldehyde solutions were added in the mixed solution for coloration at65℃for10min. The ultraviolet spectroscopy absorbance of hydroxyproline was examined by a spectrophometer using hydroxyproline at various concentrations (0-5.0mg/mL) as standard. The biodegradation degree is defined as the proportion of hydroxyproline content in the cross-linked samples to that in non-cross-linked ones. The extract liquids of different groups were prepared respectively at a concentration of1.25cm2of surface area of scaffolds per milliliter of L-DMEM medium and incubated at37℃for48h. The L-DMEM medium was reffered as the control group. The cytotoxicity was determined using MTT assay. The primary chondrocytes were isolated from the joint cartilage and xiphoid process of SD rat (3week) by enzymatic digestion. The second passage chondrocytes suspension were seeded on per scaffold (3×105cells/scaffold). Cell morphology and adherence is evaluated on the cross-linked collagen scaffolds, at1day and3day, by SEM.
Results:The general shape of cross-linked collagen scaffolds were not changed obviously. The collagen scaffolds cross-linked with genipin produced blue pigment. The color appearance of collagen scaffolds changed with the different genipin cross-linking conditions. It seemed that the higher concentration of GP and cross-linking temperature increased the intensity of blue. The cross-section of the cross-linked structures was analyzed by scanning electron microscopy (SEM). All scaffolds presented a three-dimensional interconnected porous structure. Non-cross-linked collagen scaffold with pore size ranged from100to250μm presented the fiber-and lattice-like structure. However, the morphologies of the genipin cross-linked scaffolds undergo a sheet-like structural transition. Although the pore sizes have not been changed markedly, it seems that the sheet-like framework closely aligned with the higher genipin concentration and cross-linking temperature. In addition, the fibers of non-cross-linked collagen scaffold are totally not seen in all cross-linked groups. The compressive strength augments greatly with the increased genipin concentration and cross-linking temperature compared with control (p<0.05). The swelling ratio of each cross-linked scaffold was much lower than that of the control (non-cross-linked)(P<0.05). The swelling ratios of the scaffolds which in0.3%37℃,0.5%20℃and0.5%37℃groups were significantly lower than that of in0.1%4℃(P<0.05). The cross-linking degree ranged from6.90to26.48%when cross-linked by0.1%genipin. The cross-linking degree of scaffolds which in0.3%and0.5%groups were obviously higher than that of in0.1%groups(P <0.05). MTT assay showed that the ratios of cell proliferation were above80%and cytotoxic grades were0or1at1st,3th and7th day. After being treated with genipin for24h, the anti-degradation ability of collagen scaffolds increased remarkably. There is statistical difference between0.3%20℃,0.3%37,0.5%groups and0.1%4℃and0.3%4℃group. The chondrocytes maintain a round shape in day1indicating that they just adhere to the scaffold. At day3, the cells have adhered to the scaffold closely. So, these scaffolds can be used in osteochondral tissue engineering.
Conclusion:Genipin could be used in cross-linking type I collagen scaffolds. Although the producted blue pigment, the properties of fabricating rat tail type I collagen scaffolds cross-linked with genipin were satisfactory. Based on our data, the optimization of process conditions demonstrated that successful cross-linking with genipin could be achieved at0.3%genipin concentrations and37℃. The scaffolds possessed exceptional swelling ratios, biodegradation degrees, cross-linking degrees, compressive strength and lower cytotoxicity under this condition.
Chapter3Preparation and Study on Growth Factor Release of COL/PRP Scaffolds
Objective:The extracted PRP were activated respectively by thrombin and type I collagen solution. Then, the col/prp scaffolds could be obtained by freeze-drying the mixture of activated PRP and type I collagen solution according to a certain proportion. To evaluate the feasibility of these scaffolds using in osteochondral tissue engineering, the analysis of PRP fraction, structural feature, mechanics strength, cytotoxicity of scaffolds and contents of growth factor release were investigated.
Method:6-8ml of blood could be extracted from each SD rat heart. The contents of leucocytes, erythrocytes and platelets of whole blood were measured by an automated animal blood counter. After the blood samples were centrifugated at1500rmp for10min, the blood separated into three phases:platelet-poor plasma (top), the platelet-rich plasma containing leucocytes and platelets (middle), and erythrocytes (bottom). The top and middle layers were transferred to new tubes and centrifuged again at3000rmp for10min. The supernatant plasma was discarded, and the remaining500μL of plasma containing precipitated platelets was blended evenly and designated as PRP. Care was taken to remove erythrocytes by a lml syringe to minimize their interference as far as possible. Samples of PRP were analyzed again by an automated animal blood counter and stored at-80℃. The collagen films were lysed in0.5M ethanoic acid solution and adjusted PH value to7. Samples of PRP were thawed at room temperature and poured into48-well microplates (300μL/well). The mixture of300μL PRP+30μL thrombin (30IU) was defined as thrombin-activated group,300μL PRP+300μL collagen solution was collagen-activated group,600μL collagen was blank group.10min later,300μL collagen solutions were added in each well of thrombin group. The mixed solutions were stired uniformly and freeze-dried. The morphology of the scaffolds prepared was observed by a scanning electron microscope. The mechanical test was carried out using a material testing machine. The rest of scaffolds were sterilized by ethylene oxide. The extract liquids of different groups were prepared respectively according to the method in chapter2. The L-DMEM medium was reffered as the control group. The cytotoxicity was determined using MTT assay. The sterilized scaffolds in thrombin-activated group and ollagen-activated group were placed into penicillin phials.2ml of L-DMEM medium was poured into each phial and incubated at37℃and5vol%CO2. Interval release of TGF-β1, PDGF, FGF and VEGF from each scaffold was measured at1,4,7and10days. At each time point, one ml of media was aspirated from around each sample and replaced with one ml of fresh media. Media samples were stored in1.8ml cryovials in a-80℃freezer until all samples were collected. Concentrations of rat PDGF, TGF-β1, FGF and VEGF were determined using the commercially available Quantikine colorimetric sandwich ELISA kits.
Results:The color of freeze-dried COL/PRP scaffolds was pink. Though it possesed comparative elasticity, the texture of COL/PRP scaffolds was more brittle than the COL ones when compared with the latter. The COL/PRP scaffolds were analyzed by scanning electron microscopy (SEM) and presented a three-dimensional interconnected porous structure with pore size ranged from50to80μm. The platelet content in PRP was about3.91±0.98times more than in whole blood after centrifugation. While leukocytes and erythrocytes content was about1.11±0.40and 0.31±0.20times more than in whole blood respectively. The compressive strength of COL/PRP scaffolds was comparable with COL scaffolds. There was no statistical significance between them (p>0.05). There was no cytotoxicity to L-929fibroblasts in COL/PRP groups. The ratio of cellular proliferation was higher than control at day1and3. The results of four growth factors released by COL/PRP scaffolds as follow:The content of TGF-β1was highest at day4. Although the content fell off at day7and10, the higher concentration could be maintained in each group. The content of FGF and PDGF fell off with time gradually. The concentration of two growth factors in thrombin group was higher than collagen one at each time point. As far as VEGF, the content went up from day1to day4and fell off at day7. While at day10, it went up once again. The concentration of VEGF in two groups was similar at each time point.
Conclusion:The COL/PRP scaffolds possessed exceptional porosity, strength and no cytotoxicity. The four growth factors released from scaffolds could be maintained for a long period and higher concentration. Thus, it is possible that these scaffolds can be used in osteochondral tissue engineering.
Chapte4Study on Rat Calvarial Bone Regeneration of COL/PRP Scaffolds
Objective:The purpose of this chapter was to analyze the bone regeneration of COL/PRP scaffolds by implanting them into rat calvarial defect and to assess the feasibility of these scaffolds using in osteochondral tissue engineering. The histology and Micro-CT scanning were carried out.
Method:The thrombin-activated COL/PRP scaffolds and collagen scaffolds were fabricated according to chapter3and sterilized by ethylene oxide.48Sprague-Dawley rats weighing250g were divided into three groups randomly: COL/PRP group, collagen group and blank group.0.3%phenobarbital sodium was intraperitoneally injected to induce general anesthesia. The rat cranium was exposed under sterile conditions. Five-millimeter-diameter trephine defects were created unilaterally in the calvaria of Sprague-Dawley rats under constant irrigation and with care to avoid injury to the underlying dura. Each defect was flushed with saline solution to remove bone debris. The scaffolds were implanted into calvarial defect. The blank group had no scaffolds.4Animals in each group were sacrificed after4,8and12weeks respectively, and calvaria were harvested for high-resolution microCT analysis. Decalcified samples were embedded in paraffin, and sections were stained with hematoxylin-eosin (H&E) and Masson-Goldner trichrome stain. The information of tissue repair in defect region was observed by light microscope.
Results:The diet and activity of animals were normal after surgery. The incisions were healed primarily without red swelling, exudate and suppuration. There was no difference between COL/PRP group and collagen group at4weeks. The defect area was covered by a thin membrane. It seemed that the membrane in blank group was more transparent. The membrane became thick in COL/PRP group at8weeks, while collagen and blank group was thinner than former in turn. This situation was more obvious at12weeks. There was no tissue necrosis in all groups. The bone regeneration could not be seen at the defect region in COL/PRP group at4weeks. The defect region was filled mainly by fibrous tissue with abundant fibroblasts and new capillaries. The same thing happened in collagen group. The undegradative collagen fiber and slight new capillaries could be seen in fibrous connective tissue. The new bone could not be found at the edge of host bone. The fibrous connective tissue in blank group was thinner than former groups. New capillaries and bone regeneration could not be seen in the fibrous tissue and at the edge of host bone respectively. The immature lamellar bone occurred in COL/PRP group and filled in defect area together with fibrous connective tissue. Like4weeks, there was no bone regeneration in collagen or blank group. The defect only was filled with fibrous connective tissue. At12weeks, the defect area could be filled with much mature lamellar bone and fibrous connective tissue in COL/PRP group. Slight new bone and a great quantity of fibrous connective tissue could be seen at the defect in the collagen group. Bone regeneration formed at the edge of host bone. The bluish collagen fiber stained by Masson staining could be seen in the fibrous connective tissue. In blank group, the defect area was filled with fibrous connective tissue which thickness had not been augmented. There was no bone regeneration around host bone or in the fibrous tissue. The images of bone regeneration were obversed by high-resolution microCT. The defect region in the COL/PRP group had not been decreased at4weeks. Slight bone repair happened at the edge of host bone. While at8weeks, the defect area became irregular and the newly mineralized bone with higher density could be seen at the extension area of host bone. The most defect area was covered by bew bone and became further decreased. The density of bew bone was closed to the host bone. The ossification indexes of defect area could be evaluated by software of the microCT. Most indexes increased gradually with times. This indicated that COL/PRP scaffolds possesed the ability of osteoinduction. Although the slight ossification could be found in collagen group and defect area became decreased with times, but this trend was not obvious when compared with COL/PRP group. The collagen maybe possesed the ability of osteoinduction as the ossification indexes of8and12weeks were higher than4weeks. There was no difference in blank group at different times. Although the slight ossification could be found at the edge of host bone, the whole defect area changed unobviously. The images of microCT showed that the repair ability of blank group was worst in spite of the ossification indexes of8 and12weeks were higher than4weeks.
Conclusion:The COL/PRP scaffolds possesed favourable ability of osteoinduction and repairing bone defect.
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