1、臍血來源VECs與ADSCs聯合培養移植修復頜骨缺損研究（一） 中文摘要目的：為解決組織工程骨血管化緩慢，新骨生長遲緩等問題，在構建組織工程骨時不僅需要植入有成骨潛能的干細胞，而且同時植入加速血管形成的內皮細胞。血管內皮細胞（Vascular Endothelial Cells，VECs）可以有力的支持脂肪干細胞（Adipose-Derived Stromal Cells，ADSCs）向成骨方向轉變，還可以加快血管的發生為干細胞的成骨提供營養支持。有血管內皮細胞參與的聯合培養的骨組織工程種子細胞越來越成為有希望在臨床上應用。本實驗研究在體、外情況下，在聯合培養體系中血管內皮細胞對脂肪干細胞的增

2、殖、成骨分化的影響；確定聯合培養ADSCs和VECs作為種子細胞的生物工程骨修復骨缺損能力。方法：分離大鼠臍血單個核細胞，血管內皮誘導液培養分化，于3周、6周進行免疫熒光染色檢測貼壁細胞CD34、CD133 、Flk-1、vWF表面標志。用4種不同類型的血清培養基分離培養大鼠脂肪干細胞，免疫熒光法檢測培養細胞CD34、CD133 、Flk-1表面標志；成骨誘導液誘導分化，于14天進行堿性磷酸酶和鈣染色檢驗各細胞成骨分化特性。取脂肪干細胞與臍血源血管內皮細胞按照血管內皮細胞、3:1 、1:3 、1:1 、脂肪干細胞的濃度共同培養；分別倒置顯微鏡下觀察聯合細胞的形態變化；MTT法描出各濃度細胞的生

3、長曲線，比較各組細胞生長曲線差異；7、14天各組ALP試劑盒測定細胞內堿性磷酸酶活性和飽和茜素紅鈣染色；倒置顯微鏡下觀察堿性磷酸酶和骨鈣分泌情況；4、7、14天定量測定各組細胞堿性磷酸酶和骨鈣素含量。用豬椎骨制備部分脫蛋白骨，纖維粘連蛋白修飾制成部分脫蛋白生物骨，掃描電鏡觀察；脂肪干細胞和血管內皮細胞接種部分脫蛋白骨和部分脫蛋白生物骨片，MTT法檢測吸光度，評價纖維粘連蛋白脂肪干細胞在PDPB生長影響及三種不同細胞組在PDPBB生長情況，分析移植最佳時機，掃描電鏡觀察不同細胞組在PDPBB生長情況。分別用BrdU標記的 血管內皮細胞、脂肪干細胞和聯合培養細胞體外復合部分脫蛋白生物骨，植于SD大

4、鼠兩側股部肌袋處；流式細胞儀檢測外周血CD3、CD4、CD8因子情況，根據CD4/CD8分析外周血T淋巴亞群情況來分析機體植入組織工程骨的免疫排斥情況；硬組織切片計算比較新骨成骨量；硬組織切片中Brdu抗體免疫熒光染色，觀察植入的種子細胞在機體內的增殖分化情況。取健康SD大鼠60只，隨機分為5組（A組：PDPBB血管內皮細胞組；B組;PDPBB脂肪干細胞組；C組：PDPBB1:1聯合培養細胞組；D組：單純PDPBB組；E組：空白組），在下頜骨體部椎板咬骨鉗制造0.5×0.4臨界骨缺損，分別將組織工程骨植于SD大鼠下頜骨骨缺損處；E組做空白對照； X線檢查及硬組織切片分析新骨面積成骨量

5、。結果：培養3周臍血單個核細胞CD34、CD133 、Flk-1、vWF抗體免疫熒光檢測均為陽性；六周免疫熒光染色可見CD34、Flk-1、vWF為陽性，CD133部分為陽性。脂肪干細胞培養新生牛血清組貼壁單個核細胞形態單一，呈梭形生長，免疫熒光檢測可見CD34、CD133 、Flk-1染色均為陰性；成骨誘導后堿性磷酸酶陽性；茜素紅染色可見大量紅色鈣結節影；胎牛血清組大量多角細胞呈巢狀生長，形成鋪路石樣形態，間或有梭形細胞存在；多角形細胞CD34、Flk-1均為陽性，CD133部分為陽性，梭形細胞均為陰性；成骨誘導后多角狀細胞死亡，形成空白區，梭形細胞呈堿性磷酸酶和鈣結節陽性。體外聯合培養細胞

6、1:1和3:1組14天細胞之間出現許多突觸相互連接，部分細胞融合成團塊狀，其余組細胞未見細胞團出現。生長曲線可見各組混合細胞吸光度逐漸升高，脂肪干細胞、3:1 、1:3 、1；1濃度組12天時處于高峰，1:1濃度組吸光度最高；血管內皮細胞組第10天吸光度達到高峰；隨后各濃度組細胞吸光度逐漸下降，差異有統計學意義。體外培養第7天時ALP染色脂肪干細胞、3:1、血管內皮細胞組呈陰性反應，1:3和1:1組混合培養細胞部分細胞陽性；茜素紅骨鈣檢測各組均未發現紅色陽性細胞。14天時ALP染色脂肪干細胞、血管內皮細胞組仍呈陰性反應，3:1、1:3和1:1組混合培養細胞可見片狀陽性細胞；茜素紅骨鈣檢測3:1

7、、1:3和1:1組混合培養細胞極少數陽性細胞，其余各組未發現陽性細胞；各組ALP檢測量隨時間延長逐漸增高，各時間1:1組ALP最高；脂肪干細胞組和血管內皮細胞組ALP基本沒有變化；1:1組和其它各組之間兩兩比較均有顯著統計學意義（P0.01）；各組OC檢測量隨時間延長逐漸增高，4天時1:3組OC最高； 7天和14天時1:1組OC最高； 1:1組和其它各組之間兩兩比較均有顯著統計學意義（P0.01）。豬椎骨制備部分脫蛋白骨孔隙分布均勻，由大量的羥基磷灰石纖維編織而成；部分脫蛋白生物骨表面可見大量顆粒狀蛋白結晶；細胞在部分脫蛋白骨上增殖曲線呈“S”形，在部分脫蛋白生物骨增殖曲線失去“S”形，第10

8、天均達到高峰,各分組之間差異有統計學意義（P0.01）；各細胞組在PDPBB上吸光度逐漸增加，第10天均達到高峰，1:1混合細胞組最高，各細胞組之間差異有統計學意義（P0.01）；10天聯合培養細胞細胞組大量細胞與PDPBB附著，呈巢狀分布。異位成骨第2周肉芽組織已經長入組織工程骨孔隙內，4周大量多核巨大破骨細胞位于支架材料周圍長入材料內部，破壞、吸收支架材料；混合細胞組支架材料邊緣開始出現均質、無細胞的類骨物質，孔隙內大量滋養血管形成；12周各組支架材料部分吸收，邊緣模糊，易碎裂；各組邊緣均可見均質的類骨物質出現，單純PDPBB組較少，混合細胞組最多；部分切片上材料周邊骨化中心；各組未見大量

9、淋巴細胞侵潤；新生骨形成聯合細胞組最多，單純PDPBB組較少, 復合聯合細胞組與各組之間差異有統計學意義（P0.01）。外周血T淋巴亞群CD4/CD8變化曲線：單純PDPBB組大鼠外周血CD4/CD8在1-8周期間逐漸升高，第8周時位于最高點，之后逐漸降低，與空白組比較有統計學意義（P0.05）；復合VECs組第一周最高，1-3周逐漸降低，3-12周基本成直線狀；復合ADSCs組、復合混合細胞組和空白組基本成直線狀，與空白組比較差異均無統計學意義（P0.05）。Brdu免疫熒光染色各組植入種子細胞開始呈分散狀分布，細胞逐漸增殖呈巢狀，血管內皮細胞組和聯合培養細胞組植入內皮細胞增殖形成大量新生血

10、管；未標記BrdU空白組未見陽性細胞出現。修復骨缺損第2周各組大量纖維組織包繞植入組織工程骨；空白組纖維組織已經封閉骨缺損；第4周各組植入材料不能移動，PDPBB復合血管內皮細胞組周圍肌肉及纖維組織明顯增厚，把組織工程骨和下頜骨連接成一體，組織工程骨血供較其他組豐富；第8周PDPBB復合聯合培養細胞組組織工程骨空隙已經消失，與下頜骨骨性連接，表面部分材料已皮質化；第12周各組均已和下頜骨骨性連接，單純PDPBB組、PDPBB復合脂肪干細胞和復合血管內皮細胞組材料部分吸收，材料大體形態還保留有原來形態；PDPBB復合聯合培養細胞組形態大體和正常下頜骨相似，材料表面已皮質化；空白組骨缺損未被修復，

11、骨斷裂部分已經被骨皮質封閉，形成半圓形骨缺損區。X線檢查PDPBB復合血管內皮細胞組2周支架材料與下頜骨缺損之間的縫隙較寬，周圍有新生骨痂生成；4-8周骨痂逐漸增多，12周時支架材料與下頜骨缺損之間的縫隙較前明顯減淡，但支架材料大體形態依然存在。 PDPBB復合脂肪干細胞組2周骨缺損周圍骨痂開始生成， 12周組織工程骨骨密度與正常骨質相比已基本相同； PDPBB復合聯合培養細胞組4周-12周骨缺損周圍骨痂生成隨時間延長逐漸增加，8周已經骨性聯合，骨縫已經消失；12周骨密度明顯增加，骨形態已經基本恢復正常；單純PDPBB組12周時支架材料骨密度較前減輕，與正常骨質對比明顯；空白組8周和12周圖像

12、骨缺損面積減小，骨皮質已經封閉缺損端，僅剩下半圓或三角形缺損。組織血檢查新生骨形成混合細胞組最多，復合血管內皮細胞組次之，單純PDPBB組較少, 混合細胞組與各細胞組之間差異有顯著差異（P0.01）；復合血管內皮細胞組與復合ADSCs比較有統計學意義（P0.05）；和單純PDPBB組比較差異有顯著差異（P0.01）。結論：大鼠臍血來源的單個核細胞在體外誘導3周可以分化為EPCs，6周部分細胞分化為成熟血管內皮細胞。新生牛血清培養脂肪干細胞可以得到較純的細胞；脂肪干細胞培養胎牛血清培養含有大量的血管內皮細胞。血管內皮細胞和脂肪干細胞體外培養下，細胞相互促進增殖；血管內皮細胞能在體外誘導脂肪干細胞

13、向成骨細胞方向分化，1:1比例細胞聯合培養可以達到最好效果。纖維粘連蛋白能促進細胞在PDPB支架材料上的增殖。1:1混合細胞在PDPBB支架材料上的增殖優于單種細胞，10天為最佳移植時機。聯合培養細胞組異位成骨能力最強，血管內皮細胞能增強組織工程骨成骨能力；各復合細胞組織工程骨機體內未引起明顯免疫排斥反應。組織工程骨植入種子細胞在體內能大量增殖，參與新組織和新生血管的形成。聯合培養細胞組修復骨缺損能力最強，血管內皮細胞能增強組織工程骨成骨能力。關鍵詞：血管內皮細胞 脂肪干細胞 聯合培養 組織工程骨 骨缺損（二）英文摘要Experimental Study on Repair of Mandib

14、ular Defects 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,

15、 it need 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

16、engineering 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

17、to repair 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 su

18、rface 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

19、special 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

20、:1 、1:3 、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 osteocalc

21、in(OC) 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 PD

22、PBB materials 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 obs

23、erved 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 cytome

24、try to 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

25、 vivo.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 bo

26、ne defect in 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 osteob

27、lasts. Result: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 ser

28、um(NBCS) group 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，

29、we can find that 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 c

30、ells; A great 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 d

31、ay in vitro, 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 differenc

32、es had statistical 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 VE

33、Cs group and 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 cha

34、nges on the ADSCs and VECs group; multiple comparison of the value showed remarkable increase between the 1:1 group and other groups (P<0.01);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 a

35、nd 14th day, multiple 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 crystallizati

36、on in the surface 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 incr

37、eased gradually and 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 sho

38、wed a nestlike distribution .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 in

39、ternal, destructed, 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

40、break;the bonelike 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

41、 in each group.New 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 (P<0.01). According to the CD4/CD8 curve of T lymphocytes subgroup in peripheral bl

42、ood we could see :It 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); In the 1st week it was the highest and decreased gradually between the 1s

43、t week and the 3rd week in the VECs Group,its shape almost changed into a straight line in the consecutive nine weeks；The shape of the ADSCs group, the co-cultured cell group and control group was almost a straight-line, it was no statistically significant compared with control group (P>0.05); Br

44、du immunofluorescent staining shows that the implanted cells of each group were scattering distribution, 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 pos

45、itive cells in the control group.The first two weeks of repairing bone defects, a large number of 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

46、 in each group, the muscle and fibrous tissue markedly thickened around PDPBB composited vascular 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

47、bone constructed by PDPBB composited the co-cultured cells has disappeared, the surface of some 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 g

48、roup and the vascular endothelial cells group , part of the materialhave been absorbed, but, generally 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

49、the control group have not been repaired, the surface of bone defects has been closed and formed 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 call

50、us formation around bone defects; in 4th-8th week the callus gradually increased, in the twelfth 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 a

51、dipose-derived stromal cells group begin to generate in the second week, in the twelfth week, the 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.,

52、the gap have disappeared; in the 12th week,the bone density was significantly increased, the shape 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

53、defect area gradually decreased in the control group in the eighth week and the twelfth week, the 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