【作者】 张乃丽；

【导师】 李宝兴；

【作者基本信息】 南方医科大学 ， 人体解剖与组织胚胎学， 2013， 博士

【摘要】 第一章脂肪酶用于骨移植材料脱脂的可行性研究目的：应用脂肪酶对骨组织进行脱脂,检测脱脂处理骨材料的脂肪含量、表面结构、对MC3T3-E1小鼠成骨前体细胞增殖影响、细胞相容性及组织相容性,评价脂肪酶用于骨移植材料脱脂过程的可行性。方法：取新鲜猪股骨,置于-76℃深低温冰箱中冻存两周,去除软组织,锯成骨条或骨小块,超声清洗48h。平均分成四组进行脱脂处理,分别为：脂肪酶处理组：40℃条件下,PH=10的1%浓度的脂肪酶溶液中浸泡4h；NaHCO3/Na2CO3处理组：PH=10的NaHCO3/Na2CO3溶液中浸泡4h；丙酮处理组(阳性对照)：丙酮溶液中浸泡24h,再用无水乙醇浸泡36h去除丙酮；纯化水处理组(阴性对照)：在纯化水中浸泡4h。脱脂过程结束后再超声清洗12h,冷冻干燥。索式提取法浸提各组骨材料,根据浸提前后重量差计算各组骨材料中脂肪含量。扫描电子显微镜观察各组骨材料表面结构,组织学切片,HE和Masson三色染色,观察材料内部结构。取各组骨材料,辐照灭菌,H-DMEM培养基为浸提介质,按0.2g骨材料/ml培养基比例,在37℃条件下浸提72h,制备各组骨材料浸提液,以H-DMEM培养基为试剂对照、无毒性聚乙烯片为阴性对照、苯酚为阳性对照。取指数生长期MC3T3-E1小鼠成骨前体细胞调整浓度为1×104个/ml,按100μ L/孔接种到96孔细胞培养板,培养箱中培养24h,换用各组浸提液继续培养。分别在浸提液培养的第1,4,7d,每孔加入CCK-8试剂10μ L,培养箱中孵育6h后,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。取各组骨材料,置于6孔培养板,取指数生长期MC3T3-E1小鼠成骨前体细胞,并调整细胞浓度至2×105个/ml,按每块材料500μ L细胞悬液接种到各组骨材料,培养箱内培养3h后,加培养基至足量,培养7d后,取出骨材料-细胞复合物,扫描电子显微镜观察细胞在各组骨材料上的生长情况。取各组骨材料分别埋入大鼠脊柱两侧皮下,分别于术后的第1,4,12周随机选取3只动物处死,取材,组织学切片,HE染色,光镜下观察组织学变化。结果：脱脂处理后,脂肪酶、丙酮脱脂及Na2CO3/NaHCO3处理组骨材料表面清洁,无油渍,纯化水浸泡骨材料表面可见油渍。辐照灭菌后,脂肪酶和丙酮处理组骨材料颜色洁白,Na2CO3/NaHCO3处理组骨材料呈微黄色,纯化水清洗的骨材料颜色更黄。脂肪酶处理组骨材料脂肪含量(0.46±0.16%)与丙酮处理组(1.11±0.13%)相近,差异无统计学意义(P=0.065),均低于Na2CO3/NaHCO3处理组(3.46±0.69%),差异有统计学意义(P<0.001)；纯化水组骨材料脂肪含量(8.88±0.18%)高于其他组别,差异有统计学意义(P<0.001)。扫描电镜观察,脂肪酶及丙酮处理组骨小梁表面结构保持完整,仅有少量细胞碎片附着；Na2CO3/NaHCO3处理组骨材料表面有小脂滴及细胞碎片；纯化水处理组骨材料可见大量脂肪滴覆盖在骨小梁表面。组织学观察,脂肪酶处理组在材料浅层及中心部位骨小梁间隙内均未见残留骨髓组织及细胞碎片；Na2CO3/NaHCO3处理组骨材料深层的骨小梁间隙内可见少量残留细胞碎片；丙酮处理组骨材料在中心部位偶见细胞碎片；纯化水处理组骨材料骨小梁间隙内可见残留骨髓组织,部分残留组织连接成片。Masson三色染色显示各组骨材料的骨小梁中的胶原纤维排列有序,未见胶原纤维损伤断裂。对MC3T3-E1小鼠成骨前体细胞的细胞毒性评级结果显示,脂肪酶处理组及丙酮处理组骨材料在第1,4,7d的细胞毒性为0级或1级；Na2CO3/NaHCO3处理组骨材料在各个时间点均为1级；纯化水处理组骨材料的细胞毒性为2～4级。扫描电镜下观察骨材料与MC3T3-E1细胞的相容性,结果显示,MC3T3-E1细胞在脂肪酶及丙酮处理组骨材料骨小梁上附着生长,呈梭形或多角形,几乎完全融合,覆盖骨小梁；Na2CO3/NaHCO3处理组骨材料上仅有部分区域聚集成片生长,但保持梭形或多角形形态；在纯化水处理组骨材料上偶见细胞生长,呈圆形或卵圆形。皮下植入实验结果显示,在植入后第1,4,12周各组材料均无明显免疫排斥反应及其他不良反应。结论：脂肪酶可有效去除骨组织中的脂肪,脱脂过程时间短,不损伤骨组织表面结构,且无毒性物质残留,应用脂肪酶对骨移植材料进行脱脂是改进其制备过程的可行方法。第二章同种骨胶原的提取制备及其理化性质研究目的：脂肪酶法脱脂、稀盐酸脱钙、胃蛋白酶低温条件下酶解骨组织提取骨胶原,检测不同提纯阶段骨胶原的粘度、BMP含量、分子结构等理化性质及细胞相容性,以制备满足可塑形同种骨移植材料粘性载体性能要求的骨胶原。方法：无菌操作取大鼠四肢长骨,深低温冰箱中冻存两周,去除软组织,超声清洗、脂肪酶脱脂、冷冻干燥、粉碎成粒径小于1mm的骨粉、0.6mol/L的HCl脱钙。4℃条件下,浸泡在胃蛋白酶浓度为2.5g/L,PH值为2的乙酸溶液中,持续搅拌酶解72h。15000r/min离心20min,取上清,并取出1/3,标记为组1(粗提骨胶原)。剩余的上清液中加入NaCl晶体10g,过夜沉淀。再次离心,取沉淀加入100ml0.5mol/L的乙酸溶液,过夜溶解,相同条件下再次离心,取上清,并取出1/2,标记为组2(初步纯化骨胶原)。剩余骨胶原溶液应用5mol/L的NaOH溶液调整PH到7,加入NaCl晶体约10g,过夜沉淀。再次离心,取沉淀,加入50ml0.5mol/L乙酸溶液,过夜溶解,再次离心,取上清,标记为组3(纯化骨胶原)。将3组骨胶原溶液装入透析袋中,置于20mmol/L的Na2HPO4溶液中透析8h,灭活胃蛋白酶,换用0.5mol/L醋酸溶液继续透析48h,以上操作均在4℃下进行。将收获的3组骨胶原溶液冷冻干燥。0.5mol/L的乙酸溶液溶解,分别配制浓度为0.2%的3组骨胶原溶液,37℃条件下,检测3组骨胶原溶液粘度。分别配制浓度为1%的3组骨胶原溶液,按大鼠BMP ELISA试剂盒说明书操作,检测各组骨胶原中BMP含量。配制浓度为0.2%的3组骨胶原溶液,SDS-PAGE电泳法检测其分子质量分布情况。配制浓度为0.4%的3组骨胶原溶液,以大鼠Ⅰ型胶原标准品溶液为对照,紫外可见分光光度计进行光谱分析。将3组骨胶原辐照灭菌,以L-DMEM培养基为浸提介质,按1.25cm2骨胶原膜表面积/ml培养基比例,在37℃条件下浸提72h制备各组骨胶原浸提液,以L-DMEM培养基为试剂对照。取对数生长期L929小鼠成纤维细胞,CCK-8法检测细胞毒性,计算细胞相对增殖率并进行细胞毒性评级。另取制备的骨胶原膜,平铺在6孔培养板底,将L929小鼠成纤维细胞以1×105个/ml浓度接种在骨胶原膜上,细胞培养箱中培养,倒置相差显微镜观察细胞形态变化。结果：酶解后骨胶原混合物为无色、透明、粘稠液体,经过盐析、离心纯化变得更加透明,冷冻干燥的骨胶原呈蓬松海绵状,颜色洁白。粘度检测结果显示3组骨胶原均具有良好的粘度,分别为2383.33±202.07mPa.s,1643.33±80.21mPa.s,1480.00±80.0。粗提骨胶原粘度高于其余两组纯化骨胶原,差异有统计学意义(P<0.01)。3组骨胶原中BMP含量分别为336.356+7.627ng/g,295.308±5.444ng/g,284.111±4.291ng/g,粗提骨胶原中BMP含量高于另外两组,差异有统计学意义(P<0.001),初步纯化骨胶原中BMP含量也高于纯化骨胶原,差异有统计学意义(P<0.05)。SDS-PAGE电泳结果显示提取的骨胶原主要由分子量约100KDa大小的胶原蛋白分子αl链和α2链,约200KDa大小的胶原蛋白分子β链,以及少量分子量超过200KDa的多聚体组成。光谱分析检测显示,各组骨胶原吸收光谱与标准大鼠Ⅰ型胶原蛋白相似,最大吸收波长为232nm左右,符合Ⅰ型胶原吸收光谱特征,粗提骨胶原吸收峰最多,初步纯化骨胶原、纯化骨胶原吸收峰明显减少。细胞毒性检测结果显示,各组骨胶原在不同时间点细胞相对增殖率均在95%以上,细胞毒性评级均为1级。形态学观察,在3组骨胶原材料上培养的L929细胞均与正常L929细胞无明显差异。结论：应用脂肪酶脱脂、稀盐酸脱钙、胃蛋白酶低温酶解提取的骨胶原细胞毒性轻微,具有较高粘度,还含有一定量的BMP,具备成为可塑形同种骨修复材料粘性载体条件。只经过酶解离心粗提骨胶原与盐析纯化骨胶原细胞相容性相似,但粘度及BMP含量均高于纯化骨胶原,因此选择粗提骨胶原作为可塑形同种骨修复材料的粘性载体较好。第三章可塑形同种骨修复材料的制备及其生物相容性研究目的：按不同比例将同种DBM颗粒与粗提同种骨胶原复合制备可塑形同种骨修复材料,并进行可塑形能力、抗离散能力检测确定两者最佳复合比例,评价其细胞毒性和组织相容性,初步探讨应用同种DBM颗粒复合粗提同种骨胶原制备可塑形同种骨修复材料的可行性。方法：按第二章中方法制备并筛取粒径为250～750μ m的DBM颗粒及粗提同种骨胶原,辐照灭菌。在超净工作台上,加入无菌过滤的0.5mol/L的乙酸,分别配置浓度为0.75%、1.5%、3.0%、4.5%、6%的骨胶原溶液,使用时力5mol/L的NaOH调整PH值为7。分别在各浓度组骨胶原溶液中按每毫升加入450mg DBM颗粒比例将两者复合,混匀,观察成形情况及可塑形能力。取相同体积各组可塑形同种骨修复材料,捏制成球形,放入六孔培养板中,加无菌PBS平衡液10ml,置入37℃恒温箱中,在不同的时间点观察各组材料的离散程度,检测各组材料抗离散能力,筛选DBM颗粒与骨胶原最佳复合比例。将筛选的最佳复合比例的可塑形同种骨修复材料按0.1g/mL培养基比例,37℃条件下在无血清L-DMEM培养基浸提72h制备浸提液,以L-DMEM培养基为试剂对照,应用L929小鼠成纤维细胞CCK-8法检测细胞毒性。将DBM颗粒及可塑形同种骨修复材料植入大鼠皮下,以无毒聚乙烯片为阴性对照,进行组织相容性评价。结果：各浓度组骨胶原与450mg DBM颗粒复合制备的可塑形同种骨修复材料均可成形,具有一定的可塑形能力。0.75%骨胶原浓度组可塑形同种骨修复材料较为松散,易碎；1.5%骨胶原浓度组材料可塑形能力有一定的提高,但仍较松散；3%、4.5%骨胶原浓度组材料DBM颗粒间粘合紧密,可塑形能力强；6%骨胶原浓度组材料,DBM颗粒间粘合紧密,但硬度较大,可塑形能力稍差。各组可塑形同种骨修复材料在37℃的PBS平衡液中均有一定的抗离散能力。2h时,0.75%骨胶原浓度组材料完全离散；4h时,1.5%、3%骨胶原浓度组材料完全离散；6h时所有组别材料均已离散。根据可塑形能力及抗离散能力检测结果,确定每毫升浓度为4.5%骨胶原与450mgDBM颗粒,即DBM/骨胶原质量比为10/1,为两者最佳复合比例。可塑形同种骨修复材料在第1,4,7d细胞相对增殖率均在95%以上,细胞毒性级为1级。组织相容性评价,植入皮下后1周、4周及12周,DBM颗粒组、可塑形同种骨修复材料组的组织学反应均与阴性对照组相差不大,未见明显免疫排斥反应及其他不良反应。植入后4周,DBM颗粒组及可塑形同种骨修复材料组均可见DBM颗粒被吸收,周围有间充质细胞生长。12周时,DBM颗粒大部分被吸收降解,残存DBM被排列成行的间充质细胞包绕。结论：将DBM颗粒与骨胶原以10：1比例复合制备的可塑形同种骨修复材料具有良好的可塑形能力及抗离散能力,细胞毒性轻微且具有良好的组织相容性和生物可降解性。第四章可塑形同种骨修复材料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,植入制备的可塑形同种骨修复材料,通过组织学评价(HE染色和Masson三色染色)、钙黄绿素荧光标记、Micro-CT扫描评价其修复骨缺损的能力,为其将来在临床上的应用提供实验基础。方法：在超净工作台上,配制浓度为4.5%的同种骨胶原溶液,每毫升加入450mg同种DBM颗粒,充分混匀,制备可塑形同种骨修复材料。取体重为250g大鼠48只,随机分成3组,腹腔注射麻醉,无菌操作暴露颅盖骨,锯取颅盖骨,造成约6×6mm2骨缺损。按组别分别植入各组材料填充骨缺损,实验分组为：DBM组,骨缺损区域植入粒径为250～750μmDBM颗粒；可塑形同种骨修复材料组：骨缺损区域植入可塑形同种骨修复材料；空白对照组,为阴性对照组,骨缺损区域不植入任何材料。分别于术后第4,8,12周每组随机选取4只大鼠,处死取材,固定、脱钙、石蜡包埋、组织学切片,HE染色和Masson三色染色,光镜下观察骨缺损区域的修复情况。术后11周时,每组随机选取4只大鼠,肌肉注射钙黄绿素溶液,标记在此时间点新骨生成情况,1周后,处死取材、固定,塑料包埋,硬组织切片制作厚度为10μm的组织片,荧光显微镜下观察。术后12周取材时所取标本,每组随机选取其中1个样本,固定48h,高分辨率Micro-CT扫描并三维重建。扫描结束后,对标本进行组织学观察。结果：在取材时,DBM组可见部分DBM颗粒从骨缺损区域扩散移出,可塑形同种骨修复材料组也有少量材料从骨缺损区域扩散移出,但绝大部分仍保持在植入部位。空白对照组骨缺损区域被软组织填充。HE染色,光学显微镜下观察,术后4周,DBM组,骨缺损区域可见散在分布的DBM颗粒,间充质细胞环绕DBM颗粒排列,DBM颗粒间有纤维结缔组织生成。可塑形同种骨修复材料组,骨缺损区域可见紧密排列的DBM颗粒,DBM颗粒周围及间隙均有大量间充质细胞分布,可见少量新生骨组织生成。空白对照组,骨缺损区域被纤维结缔组织填充。术后8周,DBM组,骨缺损区域可见大量间充质细胞沿DBM颗粒排列生长,DBM颗粒部分被吸收降解,有少量新生骨组织生成,DBM颗粒间隙仍可见纤维结缔组织。可塑形同种骨修复材料组,骨缺损区域被新生骨组织与DBM颗粒填充,新生骨组织包绕在残存DBM颗粒周围,DBM颗粒部分被吸收降解,周围有大量间充质细胞。空白对照组,骨缺损区域被纤维结缔组织填充。术后12周,DBM组,骨缺损区域可见散在分布的DBM颗粒与新生骨组织,DBM颗粒与新生骨组织间隙可见纤维结缔组织。可塑形同种骨修复材料组,骨缺损区域被大量成熟板层骨及尚未完全降解吸收DBM颗粒填充,可见新生骨髓腔。空白对照组,骨缺损区域仍被纤维结缔组织填充,宿主骨边缘可见少量新生骨组织。Masson三色染色,光学显微镜下观察,术后4周,DBM组,骨缺损区域可见被染成橙黄色的DBM颗粒散在分布,周围可见大量被染成绿色的疏松结缔组织,DBM颗粒边缘部分吸收降解,染成红色；可塑形同种骨修复材料组,骨缺损区域可见橙黄色的DBM颗粒周围及间隙均有大量橙黄色间充质细胞密集分布；空白对照组,骨缺损区域被大量染成绿色的纤维结缔组织填充。术后8周,DBM组,骨缺损区域染成橙黄色的DBM进一步被吸收降解,染成红色区域增多,周围仍可见被染成绿色的纤维结缔组织；可塑形同种骨修复材料组,骨缺损区域可见橙黄色DBM颗粒大部分被吸收降解,染成红色,周围有大量被染成绿色的新生骨组织；空白对照组,骨缺损区域仍被染成绿色的纤维组织填充。术后12周,DBM组,骨缺损区域可见橙黄色的DBM颗粒被进一步吸收降解,可见被染成绿色的新生骨组织；可塑形同种骨修复材料组,骨缺损区域可见橙黄色的DBM颗粒被进一步吸收降解,周围被大量染成淡红色的有序成层排列成熟板层骨包绕；空白对照组,骨缺损区域仍被染成绿色的纤维组织填充。术后12周,钙黄绿素标记的标本,硬组织切片,荧光显微镜下观察,DBM组,骨缺损区域仅有少量被钙黄绿素标记新生骨组织散在分布,发出绿色荧光点；可塑形同种骨修复材料组,骨缺损区域新生骨组织数量较多,部分区域可见连续绿色荧光带；空白对照组,骨缺损区域仅在宿主骨边缘有少量绿色荧光点。术后12周,高分辨率Micro-CT扫描并三维重建,DBM组,骨缺损区域缩小,但大部分仍未完成骨修复,骨缺损边缘可见宿主骨自我修复,宿主骨延伸区域外也可见高密度矿化新生骨组织。可塑形同种骨修复材料组,骨缺损区域大部分被高密度矿化骨组织填充,但仍未完全骨修复,缺损区域可见多个矿化骨组织点,均匀分布于骨缺损区域。空白对照组,骨缺损区域也有一定程度的缩小,在宿主骨边缘可见高密度矿化骨组织,其余区域未见高密度矿化点。可塑形同种骨修复材料组骨缺损区域的骨组织量、矿化骨量、骨矿密度、矿化组织量、矿化组织密度和矿化骨体积分数均优于DBM颗粒组和空白对照组。结论：本研究中所制备的可塑形同种骨修复材料具有良好的修复骨缺损能力,良好的生物相容性及生物可降解性能。更多还原

【Abstract】 Chapter1Study on the Feasibility of Defatting Bone Graft Material with LipaseObjective:To evaluate the feasibility of deffatting bone graft material with lipase, lipids content, surface structure, microstructure, effects on the proliferation of osteogenic precursor cells, and biocompatibility of processed bone graft were investigated.Method:The fresh pig femurs were frozen at-76℃for two weeks. The soft tissues were removed from the bone. Then the bones were sawed into pieces and cleaned with ultrasonic waves for48h in the deionized water,divided into four groups for degreasing. Lipase treated group:the bone materials were soaked in the lipase solution with concentration of1%for4h, the PH value was adjusted to10. NaHCO3/Na2CO3treating group:the bone materials were soaked in NaHCO3/Na2CO3buffer solution with PH=10for4h. Acetone treating group (positive control group):the bone materials were extracted in acetone for24h and soaked in absolute alcohol for another36h to remove acetone. Deionized water treating group (negative control group):the bone materials soaked in the deionized water for4h. All the degreasing treatments were performed at40℃. After degreasing, the bone materials were cleaned for another12h. After the bone materials were lyophilized, the lipids content of each group of treated bone materials was calculated according to the weight difference before and after the Soxhlet extraction. The surface structure of each group of bone materials was observed by scanning electron microscope. The histological sections of bone materials were prepared, stained with HE and Masson trichrome stain. After being sterilized by irradiation, the extract liquids of the different groups were prepared respectively at a concentration of0.2g of bone samples per ml of H-DMEM medium and incubated at37℃for72h. Besides the experimental group, nontoxic polyethylene disks was referred as the negative control group,toxic phenol as the positive control group and medium as the reagent control group. MC3T3-E1cells at the exponential phase were seeded on a96-well culture plate with100μl of medium with1×104cells per ml. Extract liquids were changed after incubated in a humidified atmosphere of5%CO2at37℃for24h. The cell viability was determined using CCK-8assay at1st,4th and7th day after treatment respectively. At the end of the culture period,10μ1of the CCK-8solution was added to each well of the culture plate. After6h incubation, absorbance at450nm was measured with a micro-plate reader. Cell relative proliferation rate was calculated and the cytotoxic grade was evaluated. Different groups of bone materials were put in the6-well tissue culture plate and the exponential phase of MC3T3-E1cells were seeded on the bone materials at a concentration of2×105/ml with500μL cell suspension per material. After3h co-culturing incubation the medium was added to be sufficient. At7th day after co-culturing, the bone material-cell complexes were harvested and observed by scanning electron microscopy. Different groups of bone materials were embedded in rat subcutaneous. At the1nd,4th,12th week after implantation, three rats were sacrificed randomly respectively. The implanted materials and surrounding tissue were harvested. The histological sections were prepared and stained with HE. Then the histological changes were observed under light microscope to evaluate histocompatibility.Results:After treatment, the surfaces of bone materials defatted with lipase, acetone or Na2CO3/NaHCO3were clean, while there was some grease on the surface of bone materials treated with deionized water. After being sterilized by irradiation, bone materials defatted with lipase and acetone appeared white, the bone material treated with Na2CO3/NaHCO3solution was slightly yellow and bone materials treated with deionized water turned to be deep yellow. There was no significant difference between the lipids content of the bone materials treated by lipase (0.46±0.16%) and the bone materials treated with acetone (1.11±0.13%)(P=0.065). Both of them were lower than that of the bone materials treated with Na2CO3/NaHCO3(3.46±0.69%), the difference was of statistical significance (P<0.001). The lipids content of the bone materials treated with deionized water (8.88±0.18%) was higher than that of other groups, the difference was of statistical significance (P<0.001). Observed under scanning electron microscope, on the surface of bone materials defatted with lipase or acetone, the collagen fibers were intact and oriented in a classical fern-like organization, only a little amount of cell debris attached on the trabecular. On the surface of bone materials treated with Na2CO3/NaHCO3solution, there were some small lipid droplets and cell debris attached on the trabecular. On the surface of bone materials treated with deionized water, the lipid droplets bulked over the surface of the trabecula with the bone matrix unexposed. Observed from sections of materials with HE staining, no cells and cellular debris in the center of the bone cubes defatted with lipase were left. Bone treated with acetone was similar with that with lipase. Bone marrow residues, such as cell debris could be observed in the bone materials treated with Na2CO3/NaHCO3. There were some bone marrow residues in the medullar cavities of the bone grafts processed by water. On images of sections with Masson’s trichrome staining, the bone collagen of all groups of materials was stained red. The bone matrix kept the intact collagen which appeared organized in classical lamellar-like. The result of CCK-8assay showed the cytotoxic grade of the bone materials defatted with lipase or acetone was0or1at the1st,4th,7th day after treatment; The cytotoxic grade of the bone materials defatted with Na2CO3/NaHCO3was1at all three time points; The cytotoxic grade of the bone materials treated with deionized water was grade2～4. Observed under the scanning electron microscope, MC3T3-E1cells attached and grew on bone trabecular of the bone materials defatted with lipase or acetone. The cells appeared fusiform or polygonal, fusing together and almost covered the bone trabecular; Only a few cells were observed on the bone processed by Na2CO3/NaHCO3. On the surface of bone materials treated with deionized water, cells dispersed, globular in shape. The histocompatibility evaluation in vivo showed that all the bone materials had no obvious immune rejection and other adverse reactions at the1st,4th,12th week after implantation. Conclusion:The effect of lipase on defatting the bone tissue is obvious and advisable since the defatting procedure has no damages on the surface structure of bone tissue and no toxic residue. Therefore it is a potential choice to use lipase for bone graft material defatting.Chapter2Collagens Extracted from Allogeneic Bone and their Physicochemical PropertiesObjective:To prepare viscous carrier for the malleable allogeneic bone reconstructive material, the bones were defatted by lipase and digested with pepsin at 4℃to extract the bone collagen, followed by that, physicochemical properties, viscosity, BMP content, molecular structure, cytocompatibility of the bone collagen at different extracting stages were measured.Methods:The fresh bone of rat limbs were frozen at-76℃for two weeks. The soft tissues were removed from the bone. Then the bones were ultrasonically cleaned, degreased by lipase, lyophilized, and crushed to particles. After being decalcified by0.6mol/L HCL, the bone particles were immersed in acetic acid solution with the pepsin concentration of2.5g/L at4℃for72h. After the enzymolysis, the bone collagen mixture was centrifuged at a speed of15000rounds per minute for20minutes and the supernatant was harvested. One third of the supernatant was taken out and marked as group1(the crude bone collagen). NaCl was added in the supernatant to precipitate collagen overnight. Centrifugated again, the precipitate was harvested and dissolved by acetic acid solution. Then the bone collagen was centrifuged again and the supernatant was harvested. Half of the supernatant was taken out and marked as group2(the initial purified bone collagen). The PH value of the collagen solution was adjusted to7with the NaOH solution. NaCl was added in the supernatant to precipitate collagen overnight. Centrifuged again, the precipitate was harvested and dissolved by acetic acid solution. Then the bone collagen was centrifuged again. The supernatant was harvested and marked as group3(the purified bone collagen). All three groups of collagen solution were dialyzed in Na2HPO4solution to inactivate the pepsin for8h. Then the collagens were dialyzed in acetic acid solution for another48h. All operations above are carried out at4℃. At last, all three groups of collagen solution were lyophilized. Collagen solution with a concentration of0.2%of the three groups was prepared and their viscosity was measured respectively at37℃. Collagen solution with a concentration of1%of the three groups was prepared1%, and BMP content in bone collagen was detected respectively by the rat BMP ELISA kit. Collagen solution with a concentration of0.2%of the three groups was prepared and the molecular weight distribution of them was detected respectively by SDS-PAGE electrophoresis. Collagen solution with a concentration of0.4%of the three groups was prepared and analyzed respectively by the UV-Vis spectrophotometer. The standard solution of Ⅰ-type collagen was used as the control. The collagens were sterilized by irradiation. The extract liquids of the three groups of collagen were prepared respectively according to the ratio of1.25cm2collagen membrane surface area to1ml L-DMEM medium at37℃for72h. L929cells at the exponential phase were seeded on a96-well tissue culture plate with100μl of medium with1×104cells per ml. Extract liquids were changed after incubated in a humidified atmosphere of5%CO2at37℃for24h. L-DMEM medium was used as reagent control. The cytotoxicity was detected respectively by CCK-8assay at1st,4th and7th day after treatment. The collagen sponge membranes of the three groups were put in the6-well tissue culture plate. Then the exponential phase of L929cells were seeded on collagen sponge membranes at a concentration of2×105/ml and incubated in the incubator.The morphological changes of L929cells were observed under phase contrast microscope.Results:The bone collagen mixture solution was colorless, transparent, viscous liquid. It turned to be more transparent after purification. The lyophilized bone collagen appeared a white fluffy sponge. The viscosity of collagen solutions with a concentration of0.2%was2383.33±202.07mpa.s,1643.33±80.208mpa.s,1480±80.00mpa.s respectively. The viscosity of the crude bone collagen was higher than the collagen of other two groups, with a significant difference (P<0.01). The BMP content of bone collagens were3.364±0.076ng/g,2.953±0.054ng/g,2.841±0.043ng/g respectively. The BMP content of the crude bone collagen is higher than the other two groups, the difference was statistically significant (P<0.001). The BMP content of the initial purified bone collagen is higher than that of the purified bone collagen, the difference was statistically significant (P<0.05). Electrophoresis of the bone collagens revealed that the bone collagens were composed of several proteins with apparent molecular weights of about100kDa and200kDa. Spectral analysis showed, absorption spectrum of the bone collagens were similar with the standard rat type I collagen, the maximum absorption of the bone collagen was about232nm. The crude bone collagen had the most absorption peaks. The absorption peaks of the initial purified bone collagen and the purified bone collagen were less. CCK-8assay showed the relative proliferation rate of the bone collagen at different extracting procedure was more than95%, cytotoxic grades were1at1st,4th and7th day. Morphology of L929cells cultured on the bone collagen membranes appeared no significant difference compared with the normal L929cells.Conclusion:The bone collagens extracted from allogeneic bone with pepsin enzymolysis have slight cytotoxicity, high viscosity, and contain some BMP. They are the potential choices as viscous carrier of the malleable allogeneic bone reconstructive materials. The cytocompatibility of the crude bone collagen is similar with the initial purified bone collagen and the purified bone collagen, while the viscosity and the BMP content of the crude bone collagen was higher than that of purified collagen. So the crude bone collagen was chosen as viscous carrier for the preparation of malleable allogeneic bone reconstructive materials.Chapter3Preparation and Biocompatibility of the Malleable Allogeneic Bone Reconstructive MaterialsObjective:To prepare malleable allogeneic bone reconstructive materials by compounding the allogeneic DBM with the bone collagen, the DBM particles and the bone collagen compounded at the different ratios and then the malleable ability and disintegration of these composites were tested to determine the optimal ratio of the DBM particles and the bone collagen, finally the cytotoxicity and biocompatibility of composites were evaluated.Methods:DBM particles with the size of250～750μm and the crude bone collagen were prepared according to the method in the chapter2and sterilized by irradiation. The bone collagen solutions with concentration of0.75%,1.5%,3%,4.5%,6%were prepared respectively with sterile acetic acid in a sterile environment. The pH value of the bone solutions were adjusted to7by NaOH solution. Then the DBM particles were compounded with the bone collagen at a ratio of450mg DBM particles to1ml bone collagen solutions. The malleable ability and disintegration of the composites were tested. Each composite for the same volume were immersed in the sterile PBS solution in the6-well tissue culture plate at37℃. The disintegration was observed at different time points. The optimal ratio of the DBM particles and the bone collagen in the composites was determined by the malleable ability and disintegration of the composites. The extract liquid of optimal composite was prepared according to the ratio of O.lg composite to1ml L-DMEM medium at37℃for72h. Then the cytotoxicity of the composite was detected by using L929cells with CCK-8assay at1st,4th and7th day after treatment. The DBM particles and optimal composite were embedded in rat subcutaneous with non-toxic polyethylene film as negative control. At the1nd,4th,12th week after implantation, three rats were sacrificed randomly respectively. The implanted materials and surrounding tissues were harvested. The histological sections were prepared and stained with HE. Then the histological changes were observed under light microscope to evaluate histocompatibility.Results:All the composites prepared with DBM particle and different concentrations of bone collagen were malleable and could be formed to certain shape. The composite prepared with the bone collagen at a concentration of0.75%was loose and friable; The plasticity of the composite prepared with the bone collagen at a concentration of1.5%was improved; The composites prepared with the bone collagen at a concentration of3%or4.5%had the best malleable ability. The DBM particles in the composite bonded tightly. The composite prepared with the bone collagen at a concentration of6%turned to be hard and less malleable. All the composites can keep integrated in the PBS solution at37℃in a certain time. The composite prepared with the bone collagen at a concentration of0.75%was fully disintegrated at2nd hour. The composite prepared with the bone collagen at a concentration of1.5%or3%was fully disintegrated at4th hour. All the composites were fully disintegrated at6th hour. According to the malleable ability and disintegration of the composites the optimal ratio of the DBM particles and the bone collagen in the composites was450mg DBM particles to1ml the bone collagen with a concentration of4.5%. So the optimal ratio of DBM/collagen was10/1. CCK-8assay showed the relative proliferation rate of the composite was more than95%, cytotoxic grades were1at1st,4th and7th day. The histocompatibility evaluation in vivo showed that DBM particles and the composite had no obvious immune rejection and other adverse reactions at the1st,4th,12th week after implantation. The DBM particles single or compounded was absorbed and the mesenchymal cells appeared around the DBM particles from the4th week.Conclusion:The malleable allogeneic bone reconstructive material prepared by the allogeneic DBM compounding with the bone collagen has ability of good plasticity, disintegration, slight cytotoxicity, good biocompatibility and biodegradability. The optimal ratio of the DBM particles and the bone collagen in the composites was10/1. Chapter4Study on Bone Regeneration in Rat Calvarial Defects with the Malleable Allogeneic Bone Reconstructive MaterialObjective:To investigate the bone regeneration of the malleable allogeneic bone reconstructive material, the rat calvaria bone defect model was established and implanted with the malleable allogeneic bone reconstructive material. The bone regeneration of the defects was evaluated by histological observation (HE staining and Masson trichrome staining), calcein fluorescence labeling, and Micro-CT scanning.Method:The malleable allogeneic bone reconstructive material was prepared by compounded DBM particles with the bone collagen solution at concentration of4.5%at a ratio of450mg to1ml in a sterile environment. Forty-eight SD rats (250g) were used to investigate the bone regeneration of the malleable allogeneic bone reconstructive material in this study. The animals were divided into3groups randomly and anesthetized. The animals were prepared using a sterile technique in a sterile environment. An anterior-posterior midline incision was made through the skin and muscle to expose cranium. Then a full-thickness6×6mm2parietal defect was created and filled respectively with material according to group. The experimental groups:The DBM Group, bone defect filled with the DBM particles with size of250～750μm; The malleable allogeneic bone reconstructive material group:bone defect filled with the malleable allogeneic bone reconstructive material; The blank control group (the negative control group), nothing was implanted into the bone defect. Four rats of each group were sacrificed randomly at the1st,4th,12th week after surgery. The cranial vault with the attached tissue was removed from each animal and fixed, decalcified, and embedded in paraffin. The sections were made and stained with HE and Masson trichrome stain for light microscopy. Four rats were chosen randomly to label new bone formation with intramuscular injection of calcein solution at11th week after surgery. The labeled animals were sacrificed and1week later. The cranial vault with the attached tissue was removed, fixed, embedded in methylmethacrylate.10μm of histological sections were obtained using a special microtome and analyzed by fluorescent microscopy. At12th week after surgery, one specimen of each group was selected randomly after fixation and scanned in the Micro-CT scanner. Following micro-CT analysis, the specimens were taken for histological sections.Results:Gross observation showed that the DBM particles removed obviously from the bone defect in the DBM group. Bulk of the malleable allogeneic bone reconstructive material remained at the site of implantation except a small amount of that removed in the malleable allogeneic bone reconstructive material group. The bone defects in the blank control group were soft to palpation. Histological observation showed that, at the4th week after surgery, the mesenchymal cells arranged around the scattered DBM particles, fibrous tissue also appeared at interspace of DBM particles in the bone defects of the DBM group. In the malleable allogeneic bone reconstructive material group, a large number of mesenchymal cells arranged around or at the interspace of DBM particles and a small amount of new bone was also seen the bone defect while the bone defect was filled with fibrous tissue in the blank control group. At the8th week after surgery, DBM particles were partially absorbed companied by a large number of mesenchymal cells attaching the DBM particles, a small amount of new bone appearing and fibrous tissue still visible in the bone defects of the DBM group. In the malleable allogeneic bone reconstructive material group, the bone defect filled with woven bone and DBM particles, the woven bone surrounded with the absorbed DBM particles. The bone defect was still filled with fibrous tissue in the blank control group. At the12th week after surgery, woven bone and absorbed DBM particles scattered in bone defect, fibrous tissue was still seen in the bone defects of the DBM group. In the the malleable allogeneic bone reconstructive material group, the bone defect was filled with the mature lamellar bone and the DBM particles which had not yet fully degraded, new bone marrow cavity appeared. The bone defects in the blank control group were still filled with fibrous tissue, a small amount of new bone formed at the edge. Histological observation of section stained with Masson trichrome stain showed that at the4th week after surgery, the bone defects were filled with DBM particles and loose connective tissue fibers in the DBM group. In the malleable allogeneic bone reconstructive material group, a large amount of mesenchymal cells distributed around and interspace of the DBM particles in the bone defects. In blank control group, the bone defects were filled with the loose connective tissue. At the8th week after surgery, the DBM particles were further degraded, the dyed red areas of them increased, the dyed green loose connective tissue was still seen in the defects of the DBM group. In the malleable allogeneic bone reconstructive material group, the bone defects were filled with woven bones which were dyed green and absorbed DBM. In blank control group, the bone defects were still filled with the loose connective tissue. At the12th week after surgery, the defects of the DBM group were filled with the DBM particles, loose connective tissue, and a small mount of woven bone. In the malleable allogeneic bone reconstructive material group, the bone defects were filled with absorbed DBM and the lamellar bones which were dyed red. In the blank control group, bone defects were still filled with fibrous tissue. At the12th week after surgery, observed under the fluorescence microscopy, a small amount of green fluorescence appeared in the defects of the DBM group. The visible green fluorescence bands were seen in the defects of the malleable allogeneic bone reconstructive material group. Only little green fluorescent appeared at the edge of host bone in the bone defect of the blank control group. At the12th week after surgery, high resolution Micro-CT detection and3D reconstruction revealed new bone formation in all groups. High density of mineralized new bone tissue appeared at the edge and center of the defect of the DBM group, but the majority of the bone defect had not yet been completely bone repaired. Multiple mineralized bone tissue distributed in the bone defect of the malleable allogeneic bone reconstructive material group, but the bone defect had been not completely bone repaired. The high density point of mineralized bone tissue was also seen at the edge of host bone in the bone defect of the blank control group. Volume of bone, bone mineral content, bone mineral density, tissue mineral content, tissue mineral density and bone volume fraction in the bone defect of the malleable allogeneic bone reconstructive material group were higher than that of the DBM group and the blank control group.Conclusion:The malleable allogeneic bone reconstructive material prepared in this study has the ability of good bone regeneration, good biocompatibility and biodegradability.更多还原