脐血来源VECs与ADSCs联合培养移植修复颌... - 云南

1、脐血来源VECs与ADSCs联合培养移植修复颌骨缺损研究（一） 中文摘要目的：为解决组织工程骨血管化缓慢，新骨生长迟缓等问题，在构建组织工程骨时不仅需要植入有成骨潜能的干细胞，而且同时植入加速血管形成的内皮细胞。血管内皮细胞（Vascular Endothelial Cells，VECs）可以有力的支持脂肪干细胞（Adipose-Derived Stromal Cells，ADSCs）向成骨方向转变，还可以加快血管的发生为干细胞的成骨提供营养支持。有血管内皮细胞参与的联合培养的骨组织工程种子细胞越来越成为有希望在临床上应用。本实验研究在体、外情况下，在联合培养体系中血管内皮细胞对脂肪干细胞的增

2、殖、成骨分化的影响；确定联合培养ADSCs和VECs作为种子细胞的生物工程骨修复骨缺损能力。方法：分离大鼠脐血单个核细胞，血管内皮诱导液培养分化，于3周、6周进行免疫荧光染色检测贴壁细胞CD34、CD133 、Flk-1、vWF表面标志。用4种不同类型的血清培养基分离培养大鼠脂肪干细胞，免疫荧光法检测培养细胞CD34、CD133 、Flk-1表面标志；成骨诱导液诱导分化，于14天进行碱性磷酸酶和钙染色检验各细胞成骨分化特性。取脂肪干细胞与脐血源血管内皮细胞按照血管内皮细胞、3:1 、1:3 、1:1 、脂肪干细胞的浓度共同培养；分别倒置显微镜下观察联合细胞的形态变化；MTT法描出各浓度细胞的生

3、长曲线，比较各组细胞生长曲线差异；7、14天各组ALP试剂盒测定细胞内碱性磷酸酶活性和饱和茜素红钙染色；倒置显微镜下观察碱性磷酸酶和骨钙分泌情况；4、7、14天定量测定各组细胞碱性磷酸酶和骨钙素含量。用猪椎骨制备部分脱蛋白骨，纤维粘连蛋白修饰制成部分脱蛋白生物骨，扫描电镜观察；脂肪干细胞和血管内皮细胞接种部分脱蛋白骨和部分脱蛋白生物骨片，MTT法检测吸光度，评价纤维粘连蛋白脂肪干细胞在PDPB生长影响及三种不同细胞组在PDPBB生长情况，分析移植最佳时机，扫描电镜观察不同细胞组在PDPBB生长情况。分别用BrdU标记的 血管内皮细胞、脂肪干细胞和联合培养细胞体外复合部分脱蛋白生物骨，植于SD大

4、鼠两侧股部肌袋处；流式细胞仪检测外周血CD3、CD4、CD8因子情况，根据CD4/CD8分析外周血T淋巴亚群情况来分析机体植入组织工程骨的免疫排斥情况；硬组织切片计算比较新骨成骨量；硬组织切片中Brdu抗体免疫荧光染色，观察植入的种子细胞在机体内的增殖分化情况。取健康SD大鼠60只，随机分为5组（A组：PDPBB血管内皮细胞组；B组;PDPBB脂肪干细胞组；C组：PDPBB1:1联合培养细胞组；D组：单纯PDPBB组；E组：空白组），在下颌骨体部椎板咬骨钳制造0.50.4临界骨缺损，分别将组织工程骨植于SD大鼠下颌骨骨缺损处；E组做空白对照； X线检查及硬组织切片分析新骨面积成骨量。结果：培养

5、3周脐血单个核细胞CD34、CD133 、Flk-1、vWF抗体免疫荧光检测均为阳性；六周免疫荧光染色可见CD34、Flk-1、vWF为阳性，CD133部分为阳性。脂肪干细胞培养新生牛血清组贴壁单个核细胞形态单一，呈梭形生长，免疫荧光检测可见CD34、CD133 、Flk-1染色均为阴性；成骨诱导后碱性磷酸酶阳性；茜素红染色可见大量红色钙结节影；胎牛血清组大量多角细胞呈巢状生长，形成铺路石样形态，间或有梭形细胞存在；多角形细胞CD34、Flk-1均为阳性，CD133部分为阳性，梭形细胞均为阴性；成骨诱导后多角状细胞死亡，形成空白区，梭形细胞呈碱性磷酸酶和钙结节阳性。体外联合培养细胞1:1和3:

6、1组14天细胞之间出现许多突触相互连接，部分细胞融合成团块状，其余组细胞未见细胞团出现。生长曲线可见各组混合细胞吸光度逐渐升高，脂肪干细胞、3:1 、1:3 、1；1浓度组12天时处于高峰，1:1浓度组吸光度最高；血管内皮细胞组第10天吸光度达到高峰；随后各浓度组细胞吸光度逐渐下降，差异有统计学意义。体外培养第7天时ALP染色脂肪干细胞、3:1、血管内皮细胞组呈阴性反应，1:3和1:1组混合培养细胞部分细胞阳性；茜素红骨钙检测各组均未发现红色阳性细胞。14天时ALP染色脂肪干细胞、血管内皮细胞组仍呈阴性反应，3:1、1:3和1:1组混合培养细胞可见片状阳性细胞；茜素红骨钙检测3:1、1:3和1

7、:1组混合培养细胞极少数阳性细胞，其余各组未发现阳性细胞；各组ALP检测量随时间延长逐渐增高，各时间1:1组ALP最高；脂肪干细胞组和血管内皮细胞组ALP基本没有变化；1:1组和其它各组之间两两比较均有显著统计学意义（P0.01）；各组OC检测量随时间延长逐渐增高，4天时1:3组OC最高； 7天和14天时1:1组OC最高； 1:1组和其它各组之间两两比较均有显著统计学意义（P0.01）。猪椎骨制备部分脱蛋白骨孔隙分布均匀，由大量的羟基磷灰石纤维编织而成；部分脱蛋白生物骨表面可见大量颗粒状蛋白结晶；细胞在部分脱蛋白骨上增殖曲线呈“S”形，在部分脱蛋白生物骨增殖曲线失去“S”形，第10天均达到高峰

8、,各分组之间差异有统计学意义（P0.01）；各细胞组在PDPBB上吸光度逐渐增加，第10天均达到高峰，1:1混合细胞组最高，各细胞组之间差异有统计学意义（P0.01）；10天联合培养细胞细胞组大量细胞与PDPBB附着，呈巢状分布。异位成骨第2周肉芽组织已经长入组织工程骨孔隙内，4周大量多核巨大破骨细胞位于支架材料周围长入材料内部，破坏、吸收支架材料；混合细胞组支架材料边缘开始出现均质、无细胞的类骨物质，孔隙内大量滋养血管形成；12周各组支架材料部分吸收，边缘模糊，易碎裂；各组边缘均可见均质的类骨物质出现，单纯PDPBB组较少，混合细胞组最多；部分切片上材料周边骨化中心；各组未见大量淋巴细胞侵润

9、；新生骨形成联合细胞组最多，单纯PDPBB组较少, 复合联合细胞组与各组之间差异有统计学意义（P0.01）。外周血T淋巴亚群CD4/CD8变化曲线：单纯PDPBB组大鼠外周血CD4/CD8在1-8周期间逐渐升高，第8周时位于最高点，之后逐渐降低，与空白组比较有统计学意义（P0.05）；复合VECs组第一周最高，1-3周逐渐降低，3-12周基本成直线状；复合ADSCs组、复合混合细胞组和空白组基本成直线状，与空白组比较差异均无统计学意义（P0.05）。Brdu免疫荧光染色各组植入种子细胞开始呈分散状分布，细胞逐渐增殖呈巢状，血管内皮细胞组和联合培养细胞组植入内皮细胞增殖形成大量新生血管；未标记B

10、rdU空白组未见阳性细胞出现。修复骨缺损第2周各组大量纤维组织包绕植入组织工程骨；空白组纤维组织已经封闭骨缺损；第4周各组植入材料不能移动，PDPBB复合血管内皮细胞组周围肌肉及纤维组织明显增厚，把组织工程骨和下颌骨连接成一体，组织工程骨血供较其他组丰富；第8周PDPBB复合联合培养细胞组组织工程骨空隙已经消失，与下颌骨骨性连接，表面部分材料已皮质化；第12周各组均已和下颌骨骨性连接，单纯PDPBB组、PDPBB复合脂肪干细胞和复合血管内皮细胞组材料部分吸收，材料大体形态还保留有原来形态；PDPBB复合联合培养细胞组形态大体和正常下颌骨相似，材料表面已皮质化；空白组骨缺损未被修复，骨断裂部分已

11、经被骨皮质封闭，形成半圆形骨缺损区。X线检查PDPBB复合血管内皮细胞组2周支架材料与下颌骨缺损之间的缝隙较宽，周围有新生骨痂生成；4-8周骨痂逐渐增多，12周时支架材料与下颌骨缺损之间的缝隙较前明显减淡，但支架材料大体形态依然存在。 PDPBB复合脂肪干细胞组2周骨缺损周围骨痂开始生成， 12周组织工程骨骨密度与正常骨质相比已基本相同； PDPBB复合联合培养细胞组4周-12周骨缺损周围骨痂生成随时间延长逐渐增加，8周已经骨性联合，骨缝已经消失；12周骨密度明显增加，骨形态已经基本恢复正常；单纯PDPBB组12周时支架材料骨密度较前减轻，与正常骨质对比明显；空白组8周和12周图像骨缺损面积减

12、小，骨皮质已经封闭缺损端，仅剩下半圆或三角形缺损。组织血检查新生骨形成混合细胞组最多，复合血管内皮细胞组次之，单纯PDPBB组较少, 混合细胞组与各细胞组之间差异有显著差异（P0.01）；复合血管内皮细胞组与复合ADSCs比较有统计学意义（P0.05）；和单纯PDPBB组比较差异有显著差异（P0.01）。结论：大鼠脐血来源的单个核细胞在体外诱导3周可以分化为EPCs，6周部分细胞分化为成熟血管内皮细胞。新生牛血清培养脂肪干细胞可以得到较纯的细胞；脂肪干细胞培养胎牛血清培养含有大量的血管内皮细胞。血管内皮细胞和脂肪干细胞体外培养下，细胞相互促进增殖；血管内皮细胞能在体外诱导脂肪干细胞向成骨细胞方

13、向分化，1:1比例细胞联合培养可以达到最好效果。纤维粘连蛋白能促进细胞在PDPB支架材料上的增殖。1:1混合细胞在PDPBB支架材料上的增殖优于单种细胞，10天为最佳移植时机。联合培养细胞组异位成骨能力最强，血管内皮细胞能增强组织工程骨成骨能力；各复合细胞组织工程骨机体内未引起明显免疫排斥反应。组织工程骨植入种子细胞在体内能大量增殖，参与新组织和新生血管的形成。联合培养细胞组修复骨缺损能力最强，血管内皮细胞能增强组织工程骨成骨能力。关键词：血管内皮细胞 脂肪干细胞 联合培养 组织工程骨 骨缺损（二）英文摘要Experimental Study on Repair of Mandibular D

14、efects with Tissue Engineering Bone Constructed by the Co-culture System Composed of Adipose-Derived Stromal Cells and Vascular Endothelial CellsObjective: In order to further solved the problems that the slow revascularization of tissue engineering bone and the retardation of new bone growth, it ne

15、ed implant not only the stem cell that have differentiation potential to osteoblasts, but also VECs to promote angiogenesis. VECs have the ability of enhancing ADSCs to ossify and new blood vessel can provide nutrition for the ossification of the stem cell. The co-culture seed cells of tissue engine

16、ering are increasingly acknowledged that it is the promising seed cells to be used in tissue engineering. The purpose of this study was to analyze the influence on ossification and proliferation of ADSCs affected by VECs in the system of co-culture in vitro or vivo ; To determine the capacity to rep

17、air bone defects of the co-cultured ADSCs and VECs as seed cells in bone bioengineering.Methods: To isolate mononuclear cells from umbilical cord blood, they were cultured in conditioned medium.The immunofluorescent staining were used to observe expressions and distributions of some special surface

18、marker, such as CD34、CD133 、Flk-1 and vWF on adherent cells after 3 weeks and 6 weeks.To isolate and culture rat adipose mesenchymal stem cells(ADSCs) cultured by four types of blood serum culture-medium, the immunofluorescence method were used to observe expressions and distributions of some specia

19、l surface marker, such as CD34、CD133 and Flk-1 on adherent cells ; They are induced by osteoinductive culture medium, alkaline phosphatase (ALP) and Von Kossa staining were done on the 14th day to test the characteristic of ossification. To co-culture VECs and ADSCs by the proportion of VECs、3:1 、1:

20、3 、1:1 and ADSCs, morphological changes were observed by inverted microscope；The adhesive and proliferation conditions of bone marrow stromal stem cells were evaluated by MTT；Alkaline phosphatase (ALP) and Von Kossa staining were done respectively on the 7th day and 14th day, ALP and osteocalcin(OC)

21、 were detected respectively on the 4th day, 7th day and 14th day. The PDPB materials were made with porcine spine bone .It was modified by fibronectin to made partially deproteinised biologic bone(PDPBB), the surface information of PDPBB was observed with a scanning electron microscope; The PDPBB ma

22、terials were inoculated separately with VECs, ADSCs and the co-culture cells according to the ratio of 1:1 ,the absorbance was tested by MTT automated for assessment the adhesive and proliferation conditions of three different cell on PDPBB, the most proper occasion was analyzed and it was observed

23、with a scanning electron microscope. VECs, ADSCs and co-cultured cells marked by BrdU were compounded separately with the PDPBBs in vitro, the tissue engineering bones were transplanted into both sides of femoral muscles bags in SD rats; The CD3, CD4, CD8 factors were detected with flow cytometry to

24、 analyze the CD4/CD8 of T lymphocytes subgroup in peripheral blood； It was to compare the new bone mass with hard tissue slices;The immunofluorescence staining of Brdu antibody was separately carried on in order to observate the proliferation and differentiation of the seeds implanted cells in vivo.

25、Take 60 healthful SD rats, randomly divided into 5 groups (A group: PDPBB + vascular endothelial cells; B group; PDPBB + adipose-derived stromal cells; C Group: PDPBB +1:1 co-cultured cells group; D group: simple PDPBB group; E groups: blank control group), to make 0.5 0.4cm critical bone defect in

26、mandibular body used bone rongeur, respectively, every tissue engineering bone group were grafted into the mandibular defect models in rats; E group was blank control group; To execute X-ray examination and histological examination by hard tissue biopsy and analysis the amount of osteoblasts. Result

27、:The results of the immunofluorescent staining were positive on CD34、CD133、Flk-1 and vWF after 3 weeks, but their results were only positive on CD34、Flk-1 and vWF after 6 weeks, the part of adherent cells were positive on CD133 . It is thus clear that the cell shape of new-born calf serum(NBCS) grou

28、p is single in culturing ADSCs , presenting fusiform; the results of the immunofluorescent staining were negatively on CD34、CD133 and Flk-1; The ALP staining of majority cells and red calcium nodus were positive after osteogenic induction;But that of fetal bovine serum(FBS) was complex，we can find t

29、hat a great quantity polyhedral cells offer nest growth and took on a cobblestone morphology，the fusiform cells is existing near here; the results of polyhedral cells were positive on CD34 and Flk-1, the part of polyhedral cells were positive on CD133, it is negatively on the fusiform cells; A great

30、 quantity polyhedral cell was died after osteogenic induction, forming blank; The ALP staining of majority cells and red calcium nodus were positive in some fusiform cells There were many synaptics connection in the 1:1 and 3:1 group and part of the cell form cell clusters on the 14th day in vitro,

31、no cell clusters observed in other groups; According to growth curvature, the absorbance of every group increased gradually,it reached the peak in ADSCs、3:1 、1:3 and 1:1 ratio group on 12th day and the highest was the 1:1 group, but the time was 10th day in the VECs group, the differences had statis

32、tical significance.In vitro,ALP staining of the ADSCs,3:1 and VECs group were negative on 7th day, but part of the cell were positive in the 1:3 and 1:1 group; Von Kossa staining of every group was negative on 7th day; on the 14th day , ALP staining was also negative in the ADSCs and VECs group and

33、it was positive in the 3:1,1:3 and 1:1 group , Von Kossa staining was positive in the little cell of the 3:1,1:1 and 3:1 group and other groups were negative.The ALP value of every group increased gradually, it was highest in the 1；1 group on the 4th ，7th and 14th day, there were no changes on the A

34、DSCs and VECs group; multiple comparison of the value showed remarkable increase between the 1:1 group and other groups (P0.01);The OC value of every group increased gradually, it was highest in the 1:3 group on the 4th day, but the OC value of the 1:1 group was highest on the 7th and 14th day, mult

35、iple comparison of the value showed remarkable increase between the 1:1 group and other groups （P0.01）. The PDPB materials made with porcine spine bone had homogeneous pore distribution and formed with a large number hydroxyapatite nets，there were many granular protein crystallization in the surface

36、 of PDPBB materials by fibronectin . The growth curve of cell on the PDPB had a S shape, but that of on the PDPBB had no S shape. The proliferation of every groups reach tip on tenth day, there was significantly different between every group（P0.01）.The absorbance of every group increased gradually a

37、nd it reach tip on 10th day,the highest was the co-culture cells group according to the ratio of 1:1,it have significantly different when every group was compared each other（P0.01）; On 10th day,a great deal of cells adhere on the PDPBB materials in the co-culture cells group and showed a nestlike di

38、stribution .In ectopic osteogenesis,the granulation tissue had grew into the pore space of the tissue engineering bone after two weeks ; The four weeks we could see a large number of multinucleated osteoclasts located in the surround of the scaffold materials and evolved into its internal, destructe

39、d, absorpted the scaffold materials; Homogeneous, non-cell bonelike material began to appear in the edge of scaffold materials; Many vasa vasorums took shape in the pore space of the tissue engineering bone; After 12 weeks the scaffold materials began to absorb , edge blur and easy break;the bonelik

40、e material began to appear in the edge of scaffold materials in every group, the largest was the co-culture group and the less was the PDPBB group; there are ossification centers in the surrounding of matertials in part of slices, we could see no considerable lymphocyte infiltration in each group.Ne

41、w bone formation of the co-culture cell group was the largest ,the less was the PDPBB group , it is statistical significance that the differences between other groups and the co-culture cell group (P0.01). According to the CD4/CD8 curve of T lymphocytes subgroup in peripheral blood we could see :It

42、gradually increased during 1-8 weeks in the PDPBB group, reached the highest point after 8 weeks and then gradually reduced, there was statistical significance campared with the control group (P 0.05); Brdu immunofluorescent staining shows that the implanted cells of each group were scattering distr

43、ibution, the cells gradually proliferated and formed “nestlike” , the implanted vascular endothelial cells in VECs group and the co-cultured cells group formed large quantity new vessels,but it was no positive cells in the control group.The first two weeks of repairing bone defects, a large number o

44、f fibrous tissue enveloped the implanted tissue-engineered bone in each group; bone defects had been closed by fibrous tissue in the control group；In the fourth week implanted materials could not be moved in each group, the muscle and fibrous tissue markedly thickened around PDPBB composited vascula

45、r endothelial cells, link up tissue engineering bone with mandible, blood supply of this group was richer than other groups; In the eighth weeks , the gap between mandible bone and the tissue engineering bone constructed by PDPBB composited the co-cultured cells has disappeared, the surface of some

46、materials have been corticalization; In the twelfth week ,the tissue engineering bone have been connected with mandibular bone in each group. In the simple PDPBB Group, the adipose-derived stromal cells group and the vascular endothelial cells group , part of the materialhave been absorbed, but, gen

47、erally the materials have retained the original form; The shape of the co-cultured cells group was similar to normal mandible , the surface of some materials have been corticalization; the bone defect of the control group have not been repaired, the surface of bone defects has been closed and formed

48、 a semi-circular bone defect area.In X-ray examination ,the gap between the tissue engineering bone of the vascular endothelial cell group and mandibular defects was wide in the second week, new bone callus formation around bone defects; in 4th-8th week the callus gradually increased, in the twelfth

49、 week, we could see that the gap between scaffold material and mandibular defects gradually become vague, but the general form of scaffold materials still exist.The callus around the bone defects of the adipose-derived stromal cells group begin to generate in the second week, in the twelfth week, th

50、e density of tissue-engineered bone was similar to normal bone; The callus of the co-cultured cells group gradually increased from the 4th week to the 12th week, the eighth week obtained osseous fusion., the gap have disappeared; in the 12th week,the bone density was significantly increased, the sha

51、pe of the tissue engineering bone has basically returned to normal ; Scaffold materials density of the simple PDPBB group reduced significantly compared with the normal bone in the twelfth week; The bone defect area gradually decreased in the control group in the eighth week and the twelfth week, th

52、e surface of bone defects has been closed and formed a semi-circular or triangle bone defect shape. In histologic diagnosis ,new bone formation of the co-cultured cell group was the largest , the vascular endothelial cells group come second,the simple PDPBB group was the lowest, it is statistical significance tha