自体外周血EPCs与BMSCs联合PDPBB构建微血管化生物骨
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摘要
[研究背景及目的]在整形外科临床工作中,骨缺损严重导致患者外观畸形、功能毁损。中国是拥有世界上最多人口的国家,每年因先天畸形、各类创伤、疾病、交通意外、肿瘤手术等造成的各类骨组织缺损的患者大概在300万例以上。随着人口老龄化范围的扩大,骨缺损患者的数量正在以每年10%的数率增长。随着组织工程的蓬勃发展和研究的不断深入,各种类型的组织工程骨开始逐渐应用于骨缺损修复,但因种子细胞功能单一、多采用异体种子细胞等,导致组织工程骨因血管化不良、排异反应等引发坏死、吸收,不仅使骨缺损修复失败,坏死物更可对机体产生不良损伤,严重阻碍了组织工程骨进入临床研究及运用。因此,构建组织相容性好,无排异反应,血管化良好的组织工程骨,是组织工程骨进行临床修复应用的关键。本课题组拟采用来源于自体的具有优异新生血管及管腔形成能力的自体外周血内皮祖细胞(EPCs)与自体骨髓间充质干细胞(BMSCs)构建联合培养种子细胞,并将该种子细胞复合于课题组制备的脱蛋白生物骨构建自体组织工程生物骨,对BMSCs对EPCs的血管化促进作用、组织工程骨的成骨作用提供体外、体内连续性动态监控。为构建可血管化、无排异组织工程骨的临床应用提供系统化技术支持。
[方法]
(1)抽取新西兰大耳白兔外周血密度梯度离心法加贴壁法提取纯化自体外周血内皮祖细胞,取第三代细胞进行免疫荧光染色检测贴壁细胞CD34、CD133vWF表面标志；
(2)麻醉状态下抽取新西兰大耳白兔骨髓液,密度梯度离心法加贴壁法提取纯化自体骨髓间充质干细胞,取第三代细胞进行免疫荧光染色检测贴壁细胞CD29、CD34、CD90表面标志；
(3)取骨髓间充质干细胞与外周血内皮祖细胞按照：外周血内皮祖细胞、2：1、1：2、1：1、骨髓间充质干细胞的浓度共同培养；使用倒置显微镜对各组细胞的形态变化进行观察；各组细胞的生长增殖由Wst-1法检测并描绘出曲线,比较各组细胞增殖率；
(4)细胞联合培养体系构建后第3天、7天、14天时将最佳比例自体联合培养细胞(外周血EPCs及BMSCs均来源与同一实验动物)、单纯骨髓间充质干细胞、单纯内皮祖细胞进行碱性磷酸酶染色及和茜素红钙染色；倒置显微镜下观察碱性磷酸酶和骨钙素分泌情况；并采用ALP试剂盒及OC试剂盒定量测定各组细胞碱性磷酸酶和骨钙素含量：
(5)于联合培养3、7、14天分别提取最佳比例自体联合培养细胞、单纯骨髓间充质干细胞、单纯内皮祖细胞组总RNA,采用实时荧光定量PCR技术检测成骨和血管化基因的表达,包括VEGF.Osteonectin.Osteopotin和Collagen Type I:
(6)采用新鲜猪椎骨脱钙、脱细胞处理后制备成部分脱蛋白骨,纤维粘连蛋白包被制成脱蛋白生物骨,运用扫描电镜、液体置换法检测该生物骨的孔隙率及孔径；骨髓间充质干细胞和外周血内皮祖细胞接种部分脱蛋白生物骨片,Wst-1法检测吸光度,检测最佳比例自体联合培养细胞、单纯骨髓间充质干细胞、单纯内皮祖细胞组细胞在脱蛋白生物骨上的生长情况,确定最佳移植时间,运用扫描电镜对各个细胞组在PDPBB粘附、增殖情况进行观测；
(7)抽取外周血及骨髓的健康新西兰大耳白兔12只,于其右上肢、双下肢分别进行自体工程骨回植(PDPBB+外周血内皮祖细胞组；PDPBB+骨髓间充质干细胞；PDPBB+最佳比例自体联合培养细胞组：),分别将组织工程骨植于新西兰大耳白兔股肌袋内(自体联合培养细胞组植回原取材动物体内)；硬组织切片免疫组化染色检测CD34、CD105、ZO-1分析新骨微血管化情况。
[结果]
(1)抽取的外周血用密度梯度离心法、贴壁培养法提取纯化并进行扩增,可获得高纯度的EPCs,第三代外周血EPCs抗原CD34、CD133、vWF免疫荧光鉴定均为阳性；
(2)骨髓液用密度梯度离心法、贴壁培养法提取纯化并进行细胞扩增,可获得大量高纯度的BMSCs,第三代BMSCs抗原CD29、CD90免疫荧光染色鉴定均为阳性,CD34为阴性；
(3)体外联合培养细胞1：2组3天许多细胞间出现突触相互连接,自体联合培养细胞各比例组部分细胞融合成团块状,各组混合细胞Wst-1检测吸光度逐渐升高,骨髓间充质干细胞、2：1、1：2、1：1联合培养细胞体系、外周血内皮祖细胞组均在13天时达到高峰,联合培养细胞组1：2浓度吸光度最高；随后各浓度组细胞吸光度持续平台期,差异有统计学意义；
(4)体外培养第3天时ALP染色：外周血内皮祖细胞组及单纯骨髓间充质干细胞组呈阴性反应,自体联合培养细胞组部分细胞阳性：7天时外周血内皮祖细胞组ALP染色仍未见阳性细胞,自体联合培养细胞组可见阳性细胞增多,有小片状细胞阳性,单纯骨髓间充质干细胞组可见散在颜色较淡的阳性细胞；14天时外周血内皮祖细胞组ALP染色仍未见阳性细胞,联合培养细胞组可见阳性细胞增多成大片状,颜色明显加深,单纯骨髓间充质干细胞组可见散在阳性细胞,颜色仍较淡；各组ALP检测量随时间延长逐渐增高,各时间自体联合培养细胞组ALP最高；外周血内皮祖细胞组碱性磷酸酶没有明显变化；ALP含量在其它各组与自体联合培养细胞之间进行两两比较均有统计学意义(P<0.01)。
(5)体外培养第3天时茜素红骨钙检测自体联合培养细胞组部分细胞阳性；7天时茜素红染色外周血内皮祖细胞组仍呈阴性反应,自体联合培养细胞组可见阳性细胞增多,有小片状细胞阳性,单纯骨髓间充质干细胞组可见颜色较淡的散在阳性细胞；14天时外周血内皮祖细胞组茜素红染色仍呈阴性,联合培养细胞组可见阳性细胞细胞增多成大片状,颜色明显加深,单纯骨髓间充质干细胞组可见多量散在阳性细胞,；随时间延长,各组OC检测量逐渐增高,14天时检测量最高,OC含量在其它各组与自体联合培养细胞之间进行两两比较均有统计学意义(P<0.01)。
(6)实时荧光定量PCR检测显示3天、7天、14天时自体联合培养体系VEGF、 Osteonectin、Osteopotin及Collagen Type I的mRNA表达均逐渐上升,单纯骨髓间充质干细胞组恒定表达少量Osteonectin、Osteopotin及Collagen Type I,VEGF表达量较低,单纯EPCs组VEGF表达升高。其中自体联合培养体系四种因子表达量最高。
(7)电镜观察：由猪椎骨制备部分脱蛋白骨由大量的羟基磷灰石纤维编织而成,孔隙分布均匀,纤维蛋白包被的脱蛋白生物骨表面见大量颗粒状、片状纤维蛋白,自体联合培养细胞组的细胞在骨支架上粘附生长较好,并形成团块状分布,产生大量胶原蛋白丝,单纯骨髓间充质干细胞及单纯内皮祖细胞在骨支架上粘附生长较少；
(8)通过Wst-1检测显示,各细胞组细胞在脱蛋白生物骨上吸光度逐渐增高,在第12天达到峰值,其中自体联合培养细胞组明显升高,各细胞组之间差异有统计学意义(P<0.01)：
(9)植入新西兰大耳白兔股肌袋内的组织工程生物骨血管化情况：采用免疫组化染色检测CD105、CD34、及ZO-1的表达,结果显示第2周肉芽组织已经长入组织工程骨孔隙内,联合培养细胞组于2周、4周、8周可见阳性细胞数逐渐增多,可见薄壁的微血管形成,血管腔内可见红细胞；EPCs组见少量阳性细胞及微血管形成,单纯骨髓干细胞组阳性细胞数少,且未见明显血管管腔样结构；各时间段CD105、CD34、及ZO-1阳性表达于联合细胞组均高于单纯BMSCs组及EPCs组,差异具有统计学意义(P<0.01)；
结论：
(1)兔外周血经密度梯度离心分离及贴壁法提纯即可获得纯度较高的EPCs,无需特殊诱导；
(2)兔骨髓液经密度梯度离心法,并结合贴壁法扩增进行分离纯化获得的BMSCs,经免疫荧光染色鉴定为较高纯度的细胞,可在联合培养实验中应用；
(3)外周血内皮祖细胞和骨髓间充质干细胞体外培养下,细胞促进彼此的增殖；在体外,外周血内皮祖细胞能诱导骨髓间充质干细胞成骨分化,而骨髓间充质干细胞可促进外周血内皮祖细胞微血管化,1：2比例自体联合细胞培养可以达到最好效果；
(4)体外,骨髓间充质干细胞能促进外周血内皮祖细胞血管化的能力,自体联合细胞培养体系血管化能力最强；
(5)外周血EPCs可促进BMSCs在骨支架上的附着和生长,自体联合培养细胞在PDPBB支架材料上的增殖优于单纯骨髓间充质干细胞和单纯外周血内皮祖细胞,12天为最佳移植时机：
(6)在动物活体内,自体联合培养细胞组微血管化能力最强,形成微血管管腔,外周血内皮祖细胞能增强组织工程骨血管化能力,形成新生血管,建立微循环,改善组织工程骨的血液供应；
(7)外周血内皮祖细胞与骨髓间充质干细胞联合培养体系复合PDPBB构建的组织工程骨是骨缺损修复的良好微血管化生物活性材料,具有组织相容性好,微血管化好等符合正常骨组织生理特性的优点,适合于临床实验及临床应用。
[Objective and background] Bone defect is a disease with serious appearance and function damage. China is a country with a large population, In china, there are over3million patients with the bone defect of cranium, mandibula and limb caused by disease, athletic injury, trauma, traffic accident and natural calamity, and this number are increasing10%each year with population aging. Recently, as the explosion of the research of tissue engineering, engineering bone has already began to applied to reconstruct the bone defect. But the failure of engineering bone transplantation which induced the necrosis and absorption happened frequently for insufficient angiogenesis and rejection, and the sphacelus may harm the organism in vivo. These shortcomings severely hold back the clinic research and application of engineering bone. To construct an engineering bone with good histocompatibility, low rejection and good vascularization is a keypoint for bone defect repairment with engineering bone application in clinic. In this research, an co-culture system constructed with autoallergic peripheral blood derived endothelial progenitor cells (EPCs) and autoallergic bone marrow stem cells were seeded onto partially deproteinised biologic bone, to construct an autoallergic biological engineering bone. The efficiency of BMSCs on the angiogenesis of peripheral blood EPCs, and the EPCs on the osteogenesis of BMSCs were dynamic monitored both in vitro and in vivo. This research will provide a new method of constructing a new autoallergic engineering bone with no rejection and good vascularization,
[Methods]
(1) Endothelial progenitor cells (EPCs) were isolated from rabbit peripheral blood and cultured in Endothelial cell medium (EGM). The expression of CD34, CD133, vWF on the third generation adherent cells were detected with immunofluorescent staining;
(2) Bone marrow stem cells(BMSCs) were isolated from rabbit bone marrow blood and cultured in medium added1%fetal bovine serum. The expression of CD29, CD34, CD90on the third generation adherent cells were detected with immunofluorescent staining;
(3) EPCs and BMSCs were co-cultured with the ratio of EPCs,2:1,1:2,1:1and BMSCs, the morphology change of cells were observed with inverted microscope, and the proliferation of cells in each group were evaluated by Wst-1;
(4) Alkaline phosphatase (ALP) and Von Kossa staining of the cells in each group were performed on day3,7and14, the content of ALP and osteocalcin(OC) of the cells in each group were detected on day3,7and14with ALP Kit or OC Kit;
(5) The total RNA of autologous co-cultured cells with best ratio, pure BMSCs and EPCs groups were extracted, and the expression of VEGF, Osteonectin, Osteopotin and Collagen Type I were detected with Real-time Fluorescent Quantitation polymerase chain reaction on day3,7and14;
(6) The partially deproteinised biologic bone (PDPB) were prepared with fresh porcine spine bone and formed partially deproteinised biologic bone(PDPBB) with fibronectin modified, the cavity of PDPBB was observed with scanning electron microscope; The EPCs, BMSCs and the co-culture cells with the ratio1:2were seeded separately onto PDPBB materials, the growth and proliferation of cells on the PDPBB in each group were detected with Wst-1and scanning electron microscope, the most proper occasion for transplanting the biological bone back into rabbit was analyzed;
(7)12health Newzealand white rabbit which has extracted peripheral blood and bone marrow few months ago were underwent the surgery of biological bone transplanted back into their body (PDPBB+peripheral blood EPCs; PDPBB+BMSCs; PDPBB+autologous co-culture system with best ratio with best ratio). The engineering bone were respectively transplanted into the muscle of Newzealand white rabbit (autologous co-culture system transplanted back into donor animals); the microvascularizaion were detected with Immunohistochemistry staining as CD34, CD105and ZO-1.
[Result]
(1) Higher purity EPCs could be isolated with Ficoll. The CD34, CD133and vWF of peripheral blood derived endothelial progenitor cells were positive with immunofluorescent staining on week3weeks;
(2) Higher purity BMSCs could be isolated with the Ficoll. The CD29、CD34and CD90of peripheral blood derived endothelial progenitor cells were positive with immunofluorescent staining on week3weeks;
(3) In vitro, there were many synaptics connection between cells in the autoallergic co-culture system group and some cells formed clusters on the day14. The absorbance of each group increased gradually and reached the peak on day12, and the absorbance of1:2group was highest, and there are statistical significant difference when compared with each group (P<0.01);
(4) In vitro, ALP staining of the EPCs and BMSCs group were negative on day3, but some cells in autologous co-cultured group were positive; EPCs group were still negative on day7, some positive cells were observed in BMSCs group and more positive cells were observed in positive in the co-culture group. The ALP content of each group increased gradually and highest in the co-culture group on the day3,7and14, there are statisticly significant different between each group (P<0.01);
(5) Von Kossa staining of the EPCs and BMSCs group were negative on day3, but some cells in autologous co-cultured group were positive; EPCs group were still negative on day7, some positive cells were observed in BMSCs group and more positive cells were observed in positive in the co-culture group. The OC content of each group increased gradually and highest in the co-culture group on the day3,7and14, there are statisticly significant different between each group (P<0.01);
(6) The mRNA expression of VEGF, Osteonectin, Osteopotin and Collagen Type Ⅰ were gradually increased when detected on day3,7and14with Real-time Fluorescent Quantitation PCR. A few OsteonectinN Osteopotin and Collagen Type Ⅰ and lower mRNA were expressed by pure BMSCs and EPCs. The expression of VEGF, Osteonectin, Osteopotin and Collagen Type Ⅰ were highest in autologous in co-culture system, with significant statistic difference when compared with other groups (P<0.01);
(7) The electron microscope detection:There were a large number of hydroxyapatite nets on the PDPB materials made with porcine spine bone, a lot of granular protein crystallizations were observed on the surface of PDPBB prepared with fibronectin. The growth and proliferation of cells in autologous co-culture system on PDPBB were good, and formed cell clusters. A lot of collagens were created by autoallergic co-culture system. The adhesion of pure bone marrow stem cells and EPCs on PDPBB were not as good as cells in autologous co-culture system;
(8) The proliferation of each group on PDPBB were detected with Wst-1. The absorbance of eacg groups gradually increased and reach the peak on day12, the highest was the autologous co-culture cells group with the ratio1:2, which have significantly different when compared with each other groups (P<0.01);
(9) The microvascularization of the engineering biological bone in vivo were observed with immunohistochemistry, and CD105、CD34、及ZO-1were detected respectly. Granulation tissues were grew into the cavity of engineering bone on week2, and the positive cells of autologous co-culture system were gradually increasing on week2,4and8, and formed some microvascular. There were some positive cells and microvascular were observed in group EPCs, fewer positive cells were observed in group BMSCs and on microvascular structures were observed in this group. The expression of CD105、CD34、及ZO-1were highest in co-cultured group with significant different compared with other groups (P<0.01);
[Conclusion]
(1) EPCs isolated from peripheral blood with Ficoll density gradient centrifugation were highly purified and no induction needed;
(2) BMSCs isolated and purified with Ficoll density gradient centrifugation were highly purified and vivid;
(3) In vitro, cells in co-culture system promote the proliferation of each other. The osteogenesis of BMSCs could be promoted by peripheral blood derived EPCs with the premium ratio1:2;
(4) The microvascularization of EPCs can be promoted by BMSCs with the premium ratio1:2;
(5) The proliferation and adhesion of cells on scaffold were promoted by eachother. The capability of cells growth and proliferation of autologous co-culture system were best than pure BMSCs and pure EPCs groups;
(6) In vivo, the microvascularization of autologous co-cultured cells were best and formed lumen of vessels. EPCs could improved the blood supply for engineering bone with good angiogenesis and microcirculation capability;
(7) The biological engineering bone constructed with peripheral blood derived EPCs, BMSCs and PDPBB are biological material with good microvasculaerization, histocompability, and suitable for clinic experiment and application.
[方法]
(1)抽取新西兰大耳白兔外周血密度梯度离心法加贴壁法提取纯化自体外周血内皮祖细胞,取第三代细胞进行免疫荧光染色检测贴壁细胞CD34、CD133vWF表面标志；
(2)麻醉状态下抽取新西兰大耳白兔骨髓液,密度梯度离心法加贴壁法提取纯化自体骨髓间充质干细胞,取第三代细胞进行免疫荧光染色检测贴壁细胞CD29、CD34、CD90表面标志；
(3)取骨髓间充质干细胞与外周血内皮祖细胞按照：外周血内皮祖细胞、2：1、1：2、1：1、骨髓间充质干细胞的浓度共同培养；使用倒置显微镜对各组细胞的形态变化进行观察；各组细胞的生长增殖由Wst-1法检测并描绘出曲线,比较各组细胞增殖率；
(4)细胞联合培养体系构建后第3天、7天、14天时将最佳比例自体联合培养细胞(外周血EPCs及BMSCs均来源与同一实验动物)、单纯骨髓间充质干细胞、单纯内皮祖细胞进行碱性磷酸酶染色及和茜素红钙染色；倒置显微镜下观察碱性磷酸酶和骨钙素分泌情况；并采用ALP试剂盒及OC试剂盒定量测定各组细胞碱性磷酸酶和骨钙素含量：
(5)于联合培养3、7、14天分别提取最佳比例自体联合培养细胞、单纯骨髓间充质干细胞、单纯内皮祖细胞组总RNA,采用实时荧光定量PCR技术检测成骨和血管化基因的表达,包括VEGF.Osteonectin.Osteopotin和Collagen Type I:
(6)采用新鲜猪椎骨脱钙、脱细胞处理后制备成部分脱蛋白骨,纤维粘连蛋白包被制成脱蛋白生物骨,运用扫描电镜、液体置换法检测该生物骨的孔隙率及孔径；骨髓间充质干细胞和外周血内皮祖细胞接种部分脱蛋白生物骨片,Wst-1法检测吸光度,检测最佳比例自体联合培养细胞、单纯骨髓间充质干细胞、单纯内皮祖细胞组细胞在脱蛋白生物骨上的生长情况,确定最佳移植时间,运用扫描电镜对各个细胞组在PDPBB粘附、增殖情况进行观测；
(7)抽取外周血及骨髓的健康新西兰大耳白兔12只,于其右上肢、双下肢分别进行自体工程骨回植(PDPBB+外周血内皮祖细胞组；PDPBB+骨髓间充质干细胞；PDPBB+最佳比例自体联合培养细胞组：),分别将组织工程骨植于新西兰大耳白兔股肌袋内(自体联合培养细胞组植回原取材动物体内)；硬组织切片免疫组化染色检测CD34、CD105、ZO-1分析新骨微血管化情况。
[结果]
(1)抽取的外周血用密度梯度离心法、贴壁培养法提取纯化并进行扩增,可获得高纯度的EPCs,第三代外周血EPCs抗原CD34、CD133、vWF免疫荧光鉴定均为阳性；
(2)骨髓液用密度梯度离心法、贴壁培养法提取纯化并进行细胞扩增,可获得大量高纯度的BMSCs,第三代BMSCs抗原CD29、CD90免疫荧光染色鉴定均为阳性,CD34为阴性；
(3)体外联合培养细胞1：2组3天许多细胞间出现突触相互连接,自体联合培养细胞各比例组部分细胞融合成团块状,各组混合细胞Wst-1检测吸光度逐渐升高,骨髓间充质干细胞、2：1、1：2、1：1联合培养细胞体系、外周血内皮祖细胞组均在13天时达到高峰,联合培养细胞组1：2浓度吸光度最高；随后各浓度组细胞吸光度持续平台期,差异有统计学意义；
(4)体外培养第3天时ALP染色：外周血内皮祖细胞组及单纯骨髓间充质干细胞组呈阴性反应,自体联合培养细胞组部分细胞阳性：7天时外周血内皮祖细胞组ALP染色仍未见阳性细胞,自体联合培养细胞组可见阳性细胞增多,有小片状细胞阳性,单纯骨髓间充质干细胞组可见散在颜色较淡的阳性细胞；14天时外周血内皮祖细胞组ALP染色仍未见阳性细胞,联合培养细胞组可见阳性细胞增多成大片状,颜色明显加深,单纯骨髓间充质干细胞组可见散在阳性细胞,颜色仍较淡；各组ALP检测量随时间延长逐渐增高,各时间自体联合培养细胞组ALP最高；外周血内皮祖细胞组碱性磷酸酶没有明显变化；ALP含量在其它各组与自体联合培养细胞之间进行两两比较均有统计学意义(P<0.01)。
(5)体外培养第3天时茜素红骨钙检测自体联合培养细胞组部分细胞阳性；7天时茜素红染色外周血内皮祖细胞组仍呈阴性反应,自体联合培养细胞组可见阳性细胞增多,有小片状细胞阳性,单纯骨髓间充质干细胞组可见颜色较淡的散在阳性细胞；14天时外周血内皮祖细胞组茜素红染色仍呈阴性,联合培养细胞组可见阳性细胞细胞增多成大片状,颜色明显加深,单纯骨髓间充质干细胞组可见多量散在阳性细胞,；随时间延长,各组OC检测量逐渐增高,14天时检测量最高,OC含量在其它各组与自体联合培养细胞之间进行两两比较均有统计学意义(P<0.01)。
(6)实时荧光定量PCR检测显示3天、7天、14天时自体联合培养体系VEGF、 Osteonectin、Osteopotin及Collagen Type I的mRNA表达均逐渐上升,单纯骨髓间充质干细胞组恒定表达少量Osteonectin、Osteopotin及Collagen Type I,VEGF表达量较低,单纯EPCs组VEGF表达升高。其中自体联合培养体系四种因子表达量最高。
(7)电镜观察：由猪椎骨制备部分脱蛋白骨由大量的羟基磷灰石纤维编织而成,孔隙分布均匀,纤维蛋白包被的脱蛋白生物骨表面见大量颗粒状、片状纤维蛋白,自体联合培养细胞组的细胞在骨支架上粘附生长较好,并形成团块状分布,产生大量胶原蛋白丝,单纯骨髓间充质干细胞及单纯内皮祖细胞在骨支架上粘附生长较少；
(8)通过Wst-1检测显示,各细胞组细胞在脱蛋白生物骨上吸光度逐渐增高,在第12天达到峰值,其中自体联合培养细胞组明显升高,各细胞组之间差异有统计学意义(P<0.01)：
(9)植入新西兰大耳白兔股肌袋内的组织工程生物骨血管化情况：采用免疫组化染色检测CD105、CD34、及ZO-1的表达,结果显示第2周肉芽组织已经长入组织工程骨孔隙内,联合培养细胞组于2周、4周、8周可见阳性细胞数逐渐增多,可见薄壁的微血管形成,血管腔内可见红细胞；EPCs组见少量阳性细胞及微血管形成,单纯骨髓干细胞组阳性细胞数少,且未见明显血管管腔样结构；各时间段CD105、CD34、及ZO-1阳性表达于联合细胞组均高于单纯BMSCs组及EPCs组,差异具有统计学意义(P<0.01)；
结论：
(1)兔外周血经密度梯度离心分离及贴壁法提纯即可获得纯度较高的EPCs,无需特殊诱导；
(2)兔骨髓液经密度梯度离心法,并结合贴壁法扩增进行分离纯化获得的BMSCs,经免疫荧光染色鉴定为较高纯度的细胞,可在联合培养实验中应用；
(3)外周血内皮祖细胞和骨髓间充质干细胞体外培养下,细胞促进彼此的增殖；在体外,外周血内皮祖细胞能诱导骨髓间充质干细胞成骨分化,而骨髓间充质干细胞可促进外周血内皮祖细胞微血管化,1：2比例自体联合细胞培养可以达到最好效果；
(4)体外,骨髓间充质干细胞能促进外周血内皮祖细胞血管化的能力,自体联合细胞培养体系血管化能力最强；
(5)外周血EPCs可促进BMSCs在骨支架上的附着和生长,自体联合培养细胞在PDPBB支架材料上的增殖优于单纯骨髓间充质干细胞和单纯外周血内皮祖细胞,12天为最佳移植时机：
(6)在动物活体内,自体联合培养细胞组微血管化能力最强,形成微血管管腔,外周血内皮祖细胞能增强组织工程骨血管化能力,形成新生血管,建立微循环,改善组织工程骨的血液供应；
(7)外周血内皮祖细胞与骨髓间充质干细胞联合培养体系复合PDPBB构建的组织工程骨是骨缺损修复的良好微血管化生物活性材料,具有组织相容性好,微血管化好等符合正常骨组织生理特性的优点,适合于临床实验及临床应用。
[Objective and background] Bone defect is a disease with serious appearance and function damage. China is a country with a large population, In china, there are over3million patients with the bone defect of cranium, mandibula and limb caused by disease, athletic injury, trauma, traffic accident and natural calamity, and this number are increasing10%each year with population aging. Recently, as the explosion of the research of tissue engineering, engineering bone has already began to applied to reconstruct the bone defect. But the failure of engineering bone transplantation which induced the necrosis and absorption happened frequently for insufficient angiogenesis and rejection, and the sphacelus may harm the organism in vivo. These shortcomings severely hold back the clinic research and application of engineering bone. To construct an engineering bone with good histocompatibility, low rejection and good vascularization is a keypoint for bone defect repairment with engineering bone application in clinic. In this research, an co-culture system constructed with autoallergic peripheral blood derived endothelial progenitor cells (EPCs) and autoallergic bone marrow stem cells were seeded onto partially deproteinised biologic bone, to construct an autoallergic biological engineering bone. The efficiency of BMSCs on the angiogenesis of peripheral blood EPCs, and the EPCs on the osteogenesis of BMSCs were dynamic monitored both in vitro and in vivo. This research will provide a new method of constructing a new autoallergic engineering bone with no rejection and good vascularization,
[Methods]
(1) Endothelial progenitor cells (EPCs) were isolated from rabbit peripheral blood and cultured in Endothelial cell medium (EGM). The expression of CD34, CD133, vWF on the third generation adherent cells were detected with immunofluorescent staining;
(2) Bone marrow stem cells(BMSCs) were isolated from rabbit bone marrow blood and cultured in medium added1%fetal bovine serum. The expression of CD29, CD34, CD90on the third generation adherent cells were detected with immunofluorescent staining;
(3) EPCs and BMSCs were co-cultured with the ratio of EPCs,2:1,1:2,1:1and BMSCs, the morphology change of cells were observed with inverted microscope, and the proliferation of cells in each group were evaluated by Wst-1;
(4) Alkaline phosphatase (ALP) and Von Kossa staining of the cells in each group were performed on day3,7and14, the content of ALP and osteocalcin(OC) of the cells in each group were detected on day3,7and14with ALP Kit or OC Kit;
(5) The total RNA of autologous co-cultured cells with best ratio, pure BMSCs and EPCs groups were extracted, and the expression of VEGF, Osteonectin, Osteopotin and Collagen Type I were detected with Real-time Fluorescent Quantitation polymerase chain reaction on day3,7and14;
(6) The partially deproteinised biologic bone (PDPB) were prepared with fresh porcine spine bone and formed partially deproteinised biologic bone(PDPBB) with fibronectin modified, the cavity of PDPBB was observed with scanning electron microscope; The EPCs, BMSCs and the co-culture cells with the ratio1:2were seeded separately onto PDPBB materials, the growth and proliferation of cells on the PDPBB in each group were detected with Wst-1and scanning electron microscope, the most proper occasion for transplanting the biological bone back into rabbit was analyzed;
(7)12health Newzealand white rabbit which has extracted peripheral blood and bone marrow few months ago were underwent the surgery of biological bone transplanted back into their body (PDPBB+peripheral blood EPCs; PDPBB+BMSCs; PDPBB+autologous co-culture system with best ratio with best ratio). The engineering bone were respectively transplanted into the muscle of Newzealand white rabbit (autologous co-culture system transplanted back into donor animals); the microvascularizaion were detected with Immunohistochemistry staining as CD34, CD105and ZO-1.
[Result]
(1) Higher purity EPCs could be isolated with Ficoll. The CD34, CD133and vWF of peripheral blood derived endothelial progenitor cells were positive with immunofluorescent staining on week3weeks;
(2) Higher purity BMSCs could be isolated with the Ficoll. The CD29、CD34and CD90of peripheral blood derived endothelial progenitor cells were positive with immunofluorescent staining on week3weeks;
(3) In vitro, there were many synaptics connection between cells in the autoallergic co-culture system group and some cells formed clusters on the day14. The absorbance of each group increased gradually and reached the peak on day12, and the absorbance of1:2group was highest, and there are statistical significant difference when compared with each group (P<0.01);
(4) In vitro, ALP staining of the EPCs and BMSCs group were negative on day3, but some cells in autologous co-cultured group were positive; EPCs group were still negative on day7, some positive cells were observed in BMSCs group and more positive cells were observed in positive in the co-culture group. The ALP content of each group increased gradually and highest in the co-culture group on the day3,7and14, there are statisticly significant different between each group (P<0.01);
(5) Von Kossa staining of the EPCs and BMSCs group were negative on day3, but some cells in autologous co-cultured group were positive; EPCs group were still negative on day7, some positive cells were observed in BMSCs group and more positive cells were observed in positive in the co-culture group. The OC content of each group increased gradually and highest in the co-culture group on the day3,7and14, there are statisticly significant different between each group (P<0.01);
(6) The mRNA expression of VEGF, Osteonectin, Osteopotin and Collagen Type Ⅰ were gradually increased when detected on day3,7and14with Real-time Fluorescent Quantitation PCR. A few OsteonectinN Osteopotin and Collagen Type Ⅰ and lower mRNA were expressed by pure BMSCs and EPCs. The expression of VEGF, Osteonectin, Osteopotin and Collagen Type Ⅰ were highest in autologous in co-culture system, with significant statistic difference when compared with other groups (P<0.01);
(7) The electron microscope detection:There were a large number of hydroxyapatite nets on the PDPB materials made with porcine spine bone, a lot of granular protein crystallizations were observed on the surface of PDPBB prepared with fibronectin. The growth and proliferation of cells in autologous co-culture system on PDPBB were good, and formed cell clusters. A lot of collagens were created by autoallergic co-culture system. The adhesion of pure bone marrow stem cells and EPCs on PDPBB were not as good as cells in autologous co-culture system;
(8) The proliferation of each group on PDPBB were detected with Wst-1. The absorbance of eacg groups gradually increased and reach the peak on day12, the highest was the autologous co-culture cells group with the ratio1:2, which have significantly different when compared with each other groups (P<0.01);
(9) The microvascularization of the engineering biological bone in vivo were observed with immunohistochemistry, and CD105、CD34、及ZO-1were detected respectly. Granulation tissues were grew into the cavity of engineering bone on week2, and the positive cells of autologous co-culture system were gradually increasing on week2,4and8, and formed some microvascular. There were some positive cells and microvascular were observed in group EPCs, fewer positive cells were observed in group BMSCs and on microvascular structures were observed in this group. The expression of CD105、CD34、及ZO-1were highest in co-cultured group with significant different compared with other groups (P<0.01);
[Conclusion]
(1) EPCs isolated from peripheral blood with Ficoll density gradient centrifugation were highly purified and no induction needed;
(2) BMSCs isolated and purified with Ficoll density gradient centrifugation were highly purified and vivid;
(3) In vitro, cells in co-culture system promote the proliferation of each other. The osteogenesis of BMSCs could be promoted by peripheral blood derived EPCs with the premium ratio1:2;
(4) The microvascularization of EPCs can be promoted by BMSCs with the premium ratio1:2;
(5) The proliferation and adhesion of cells on scaffold were promoted by eachother. The capability of cells growth and proliferation of autologous co-culture system were best than pure BMSCs and pure EPCs groups;
(6) In vivo, the microvascularization of autologous co-cultured cells were best and formed lumen of vessels. EPCs could improved the blood supply for engineering bone with good angiogenesis and microcirculation capability;
(7) The biological engineering bone constructed with peripheral blood derived EPCs, BMSCs and PDPBB are biological material with good microvasculaerization, histocompability, and suitable for clinic experiment and application.
引文
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