血管化组织工程骨修复猕猴胫骨缺损的实验研究
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摘要
目的我们曾采用血管化组织工程骨修复大动物(山羊)20mm胫骨缺损，获得了确切的修复效果。为了进一步为临床试验提供更详细、可行的方法，我们选取猕猴为实验动物，在进行解剖观察后，提出了进一步的血管化方法，通过对支架外型的特殊设计，允许血管和筋膜两种血管化方法的并存和协同，这样不仅加快了组织工程骨的血管化进程，而且对成骨质量的提高和时限的缩短等有了明显的作用效果。
本实验目的在于检测猕猴骨髓基质干细胞与β-磷酸三钙的组织相容性，建立可行的组织工程骨修复猕猴胫骨段性缺损的动物模型，检测组织工程骨体内血管化程度和成骨情况，探究血管化程度与成骨质量的关系。
方法
第一部分取传三代猕猴骨髓基质细胞与材料复合培养，将猕猴BMSC与β-TCP复合培养，单纯细胞组作对照。所取样本使用倒置相差显微镜，扫描电镜观察两组细胞形态及增殖情况，MTT法半定量检测细胞增殖情况。
第二部分对一只自然死亡的猕猴进行新鲜解剖，观察其胫骨的形态及周围知名血管束的解剖路径；将11只猕猴双侧胫骨(共22处)制成中段20mm骨-骨膜缺损，随机平均分成2组(实验组和对照组各9侧)，实验组在缺损处填塞由骨髓基质干细胞(BMSCs)和具有特殊外型(侧槽和中空管)的β-磷酸三钙(β-TCP)支架体外构建的复合物，在中空管内移入隐动、静脉束的一段，工程骨外被带蒂深筋膜；对照组只填塞组织工程骨。另外2只猕猴的无填充物胫骨缺损作空白对照。钢板螺钉固定。在4、8、12周时间点分别行放射线检测，墨汁灌注标本，组织学检测及标本大体观察。
第三部分将20只猕猴双侧胫骨(共40处)制成中段20mm骨-骨膜缺损，随机平均分成4组(一个实验组和三个对照组，每组各9侧)，实验组在缺损处填塞由骨髓基质干细胞(BMSCs)和具有特殊外型(侧槽和中空管)的β-磷酸三钙(β-TCP)支架体外构建的复合物，在中空管内移入隐动、静脉束的一段，外被带蒂深筋膜；其余3个对照组分别填塞组织工程骨并包裹筋膜、单纯组织工程骨和单纯支架组。另外2只猕猴的无填充物胫骨缺损作空白对照。钢板螺钉固定。在4、8、12周时间点分别行放射影像学评分和X射线阻射密度分析，以及血管面积和成骨图像分析。得出数据进行统计学分析。
结果
第一部分：猕猴BMSCs体外增殖能力较强，在传3代后的第6天达高峰。细胞经诱导后，在第3天就可见良好的定向分化能力。该支架材料与猕猴BMSCs具有良好的相容性。但材料在加入培养基后会出现崩解，对细胞的初期附着有一定影响。
第二部分：本实验以通过对组织工程骨材料外型的特殊设计，采用血管植入和筋膜包裹的血管化方法修复猕猴双侧胫骨20mm缺损为实验模型，进行了术后大体观察、X线和组织学检测，初步完成了模型的设计和检测工作，认为模型是可行的，为进一步的临床试验提供了参考，但需要更深入的研究。
第三部分：
一般观察术后4周：4组标本无明显差异，均在两接触面、材料后面和钢板侧有部分结缔组织或软骨样组织形成，中空管及侧槽为软组织填塞，材料与骨接触面能折动。A组材料中空管内原植入血管腔内充满墨汁。材料本身无明显吸收。
术后8周：4组标本观察有差异。A组植入物除前面外，其余各面及与骨接触面均有骨样组织包裹，侧槽及中空管也有骨样组织填塞，植入血管观察不清，不易折断，材料1／3吸收；B组在植入物内侧面、前面及中空管内无骨样组织形成，其余各面及与骨接触面仅有少部分骨样组织形成，易折断，材料无明显吸收。C和D组植入物与骨接触面仅有少部分骨样组织形成，易折断，材料无明显吸收。4组标本均有墨染，以A，B两组最明显。
术后12周：4组标本观察有明显差异。A组植入物各面及中央部完全被骨样组织所包裹或替代，坚硬，折不断，材料2／3被吸收；B组和C组植入物于内侧及前面仍有部分材料未被骨样组织所包裹或替代，用力可以折断，材料1／3被吸收。D组成骨罕见，材料无吸收，轻易弯折。
空白组(12 w)缺损处完全被肉芽组织填塞，无骨样组织增生。
显微镜下观察A组术后4周时在移植物边缘、断面及中央有少量新生骨组织——编织骨形成，临近新骨处的材料孔隙内也有新骨的形成和小“肺泡样”墨染结构，植入血管腔内墨染，材料较完整，孔隙未见扩大。8周时新骨形成明显增多，墨染血管样结构明显增多，多靠近外缘及中央，于新骨与材料交接处可见破碎的材料，小颗粒状，植入血管腔内未见墨染。12周时移植物完全被骨样组织所包裹，形成的骨样组织把材料分成几小块，有骨小梁样结构形成。于新骨及剩下的材料中均能见到血管样结构，密度明显增大。新骨中的血管样结构较粗大，走行直，少有分支；材料中的血管样结构多呈“肺泡状”，分支多，串珠样，孔隙之间有交通。于新骨与材料交接处可见较多破碎的材料，小颗粒状，此处的墨染血管样结构最多。
B组术后4周时在移植物边缘、断面有少量新生骨组织——编织骨形成，材料孔隙内也有新骨的形成和小“肺泡样”墨染结构，植入血管腔内墨染，材料较完整，孔隙未见扩大。8周时新骨形成增多，墨染血管样结构明显增多，多靠近外缘，于新骨与材料交接处可见破碎的材料，植入血管腔内未见墨染。12周时形成的骨样组织把材料大部分包被，有骨小梁样结构形成。于材料中央部少见血管样结构。
C组术后4周时在移植物边缘和中央未见新生骨组织，仅在断面处有少量编织骨形成，移植物仅有一面(后面)外缘有小“肺泡样”墨染结构，移植物完整，孔隙未见增大。8周时在移植物边缘、断面有少量新生骨组织形成，仅在新骨与材料交接处可见较多墨染血管样结构，余处少见。12周时可见移植物有少部分吸收，新骨及血管样结构的形成量比同组8周的有所增加，但仍然很稀少。
D组4周时无成骨，移植物断端处可见有少量墨染结构。8周时仅在断端可见少量成骨，墨染结构较4周时增加。12周时移植物断端的成骨情况并未增加，墨染结构也未增加。
血管面积及成骨面积分析各时间点上A组中央部和周围部的血管样结构面积及成骨面积与B、C和D组比较有显著差异(P＜0.01)。而且随时间的推移呈上升趋势。B和C组中央部血管样结构面积比较无统计学意义(P＞0.05)。B组周围部的血管样结构面积与C组比较有统计学意义(P＜0.05)。
放射线观察A组8周显示移植物密度降低，两断面有连续性骨痂形成；12周显示移植物密度明显降低，个别区域低于正常骨，连续性骨痂明显。B和C组8周和12周移植物密度未见降低，断面处有连续性骨痂。D组各时间点无明显变化。
影像评分从正位X线片骨缺损部位阻射密度值中可以看出，4 w各组间无差异，B和C组各时间点间无差异；8、12 w A组与B和C间有差异，A组各时间点间有显著差异(P＜0.01)。A组各时间点阻射密度值呈减少趋势，可能和材料被逐渐吸收有关。
结论
本实验以通过对组织工程骨材料外型的特殊设计，采用血管植入和筋膜包裹的血管化方法修复猕猴双侧胫骨20mm缺损为实验模型，进行了术后大体观察、X线检测及评分和组织学检测及血管密度图像分析，结果显示实验所采用的血管化方法可以加快和提高组织工程骨的成骨作用，为进一步的临床试验提供了参考，但需要更深入的研究。
OBJECTIVE: The study and the clinical application of tissue engineered bonegraft have got more and more recognition, but there are some key problems whichneed solving. Many experiments indicate that the angiogenesis plays a key role in theosteogenesis.An experimental pattern was set up designed to prepare a kind ofvascularized engineered-bone graft for repairing rhesus tibia defects and analyze thecharacters of the angiogenesis and osteogenesis in vivo by rontgenographic andmorphological approaches.
We have ever repaired the 20mm tibia defect of big animals (goats) withvascularized tissue engineered bone, and achieved good results. In order to supplymore detailed and practical approaches for the clinical experiments, we chooserhesuses as experimental animal this time, and put forward the approach of furtherangiogenesis after the anatomical observation, that is, allowing the coexistence andco-growth of both blood vessels and fascia by specially designing the scaffoldconformation can not only accelerate the angiogensis of the tissue engineered bone,but also can greatly improve the quality of the osteogenesis and shorten the healingperiod.This study aims at: detecting the compatibility of BMSCs andβ-TCPscaffold, setting up an animal repairing rhesus tibia defects with engineered bone anddetecting the angiogensis of the tissue engineered bone and the quality of theosteogenesis.
METHODS:
The first part: The third passage of rBMSCs were cultured withβ-TCP withthe cells cultured without the materials as the control. The morphology andproliferation of cells were observed by inverted phase contrast microscope andscanning electron microscope(SEM). MTT assay was used to semiquantitativelyevaluate the cell proliferation.
The second part: A 20 mm tibia defect was made in each of both sides of the 9rhesuses and fixed with a plate. The 18 tibia defects were randomly divided into twogroups equally. The gaps in experiment group were plugged with the engineeredbones constructed in vitro by bone marrow-derived stroma cells(BMSCs) andβ-TCPscaffolds in each one with a slit and a central tunnel. In addition, a correspondingportion of saphenous artery and veins was moved to run through along the centraltunnel in the scaffold with the "gate" help of the slit, and the engineered bone wastotally hugged by a sheet of pedicled deep fascia. The gaps in control group, however,were inserted engineered bones only. The vascularization for each treatment wasassessed by physical, histopathological and X-ray examinations at time intervals of 4,8, 12 weeks after operation.
The third part-The 36 bilateral tibia 20mm defects were randomly divided into4 groups equally. The gaps in experimental groups were plugged with engineeredcomposites which were totally hugged by a sheet of pedicled deep fascia andadditionally a corresponding portion of saphenous artery and veins were moved to runthrough along the central tunnel in the scaffold with the "gate" help of the slit. Thegaps in the three other control groups, however, were inserted with fascia-coatedcompounds, compounds only and pure scaffold only respectively. In addition, tworhesuses were picked up as blanks. The angiogenesis and osteogenesis for eachtreatment was assessed by macroscopical, histological and roentgenographic analyses at time intervals of 4, 8, 12 weeks postoperative.
RESULTS
The first part: Rhesus BMSCs showed a active ability of proliferation and amountthe highest level at 6th day at third passage. Osteogenic differentiation were carriedout at the third day after induction. The scaffold material presented finebiocompatibility with rhesus BMSCs while coculturing together but the disintegrationof material might have certain influence after cells seeding onto the scaffold.
The second part: Through the special design to engineered bone features andapplying double vacularization methods of saphenous bundle insertion and pedicleddeep fascia coated, the experimental animal modal was set up. The samplesharvested from the subjects were analyzed by gross observation, roentgenographicand histologic examination. The modal and its procedure were recognized a feasibleway to get engineered bone revascularizaton but the results needed further researchin order to supply theoretic evidences for final clinical trials.
The third part:
General results
4 weeks postoperatively: There is no significant differences among 4 groupswhich all have some connective or chondroid tissue formation on two contactsurfaces, the back of the material and the side of the plate. The samples presented noabsorption and its contact surface could be easily broken.
8weeks postoperatively: There are differences among 4 groups. In group A,besides what has mentioned before, other surfaces and the contact surfaces with thebone are all wrapped up with bonelike tissues which also fills the slit and the centraltunnel. The implanted blood vessels can clearly be seen, and the bone are not easy tobe broken, 1/3 materials has been absorbed; in group B, no bonelike tissues form onthe medial surface and the front of the implanted material, and the same with the central tunnel. A few bonelike tissues appear on the other surfaces and the contactsurfaces with the bone; in group C, few bonelike tissues form on the contact surfacesbetween the implanted material, which are easy to be broken and the material has notbeen significantly absorbed, in group D, the condition is relatively the same as that ingroup C.
12 weeks postoperatively: There are significant differences among 4 groups. Ingroup A, all the surfaces of the implanted material and the central part are whollywrapped up or replaced by bonelike tissues which are hard and cannot be broken. And2/3 materials have been absorbed; in group B and C, partial materials of the medialsurface and the front have not been replaced by bonelike tissues yet, which can bebroken with force, and 1/3 material has been absorbed; in group D, rare boneliketissues were seen at the contact interfaces, and no material has been absorbed.
Blanks: At 12 week, the defect has been completely filled with granulationtissues, without osteogenesis.
Microscopic observation
Group A: At 4w postoperatively, a few new bonelike tissues, woven bone,form on the edges, the sections and the center of the implanted material, near which,new bone and small alveoli-like ink-stained structures also appear in the holes of thematerial. The implanted blood vessels lumens are stained with ink, and the material isfairly complete, the holes have not dilated. At 8w, new bone and ink-stained bloodvessel structures have significantly increased, most of which are close to the externalmargin and the center. Broken materials, like fine particles, can be seen at thejunctions between the new bone and the material, and the implanted blood vesselshave not been ink-stained. At 12w, the implanted material has been completely coatedwith the bonelike tissues which separate the material into several small parts withbone trabecula structure formation. The density of the blood vessels structures greatly increases in the new bone and the left material. Those in the new bone are quite thick,straight and have less branches, while those in the left material are mostly alveolialike, rosary like and have more branches, with holes connected with each other.Quite a lot broken materials, like small particles, are seen at the junctions between thenew bone and the material, where the amount of ink-stained blood vessels structuresis maximal.
Group B: At 4w postoperatively, a few newborn bone tissues, woven bonetissues form on the edge and sects of the implanted material, and there are also newbone and small alveoli like structures within the holes of the material. The lumen ofthe implanted blood vessel is ink-stained, the material is fairly complete, and theholes have not dilated. At 8w, more new bones form, and much more ink-stainedblood vessels appear. Many of them are near to the external edge, and brokenmaterials are visible at the junctions between the new bone and the material. Thelumens of the implanted blood vessel are not ink-stained. At 12w, most of the materialis wrapped up by the new bone and bone trabeculae have formed. Few blood vesselscan be seen in the center of the material.
Group C: At 4w, no new bone can be seen on the edge and the center of theimplanted material, a few woven bones have formed only on the sections, and smallalveoli like ink-stained structures can be seen only on the external edge of thematerial back. The material is fairly complete and the holes have not dilated. At 8w, afew new bones appear on the edge and the sections of the implanted material, andink-stained blood vessels are seldom seen except that quite a lot appear at thejunctions between the new bone and the material. At 12w, the implanted material hasbeen slightly absorbed. The new bone and the blood vessel have increased comparedwith those at 8w, but still very few.
Group D: No newbone formed at 4 weeks postoperatively, but a few of "ink-staining" structure showed up at interfaces. The "ink-staining" structureincreased quantitatively and a little of bonelike tissue could be seen at interfaces at 8and 12 weeks.
Analyses of angiogenic and osteogenie area
Take the central tunnel and its vicinity within 2mm as the center portion, and theother places as the surrounding portion.There were statistical significance for bothangiogenic and osteogenic areas comparing the vaso-areas of both central andperipheral parts in group A to those of group B, C and D(P＜0.01). No significancecould be seen between group B and C for comparing the angiogenic areas at centralparts (P＞0.05), while it is significant comparing the angiogenic areas at peripheralparts(P＜0.05).
X-rays Observation
At 8w, group A's images show that the density of the implanted materialdecreases, and continual bony callus forms at the interfaces. At 12w, group A's imagespresent obviously decreased density which is lower than that of the normal bone inindividual areas, and the continual bony callus is manifest. At 8w and 12w, group Band C's images show no decreased density and the continual bony callus appear onthe sections. Group D shows no changes at every check intervals.
Roentgenographic scores
From the blocking density values at the bone defects, it can be seen that nodifferences exist among the 4 groups at 4w, and no differences exist between group Band C at all observing time points; At 8w and 12w, group A is different from group Band C, and there are differences between all the time intervals of group A(P＜0.01).The density gradually decreases with the time, which is probably due tothe absorption of the implanted material.
Conclusion
Effects on the rhesus' activities seemed to be rare. This study shows us a feasibleand effective angiogenesis approach which can accelerate osteogenesis in vivo ofengineered bone.
本实验目的在于检测猕猴骨髓基质干细胞与β-磷酸三钙的组织相容性，建立可行的组织工程骨修复猕猴胫骨段性缺损的动物模型，检测组织工程骨体内血管化程度和成骨情况，探究血管化程度与成骨质量的关系。
方法
第一部分取传三代猕猴骨髓基质细胞与材料复合培养，将猕猴BMSC与β-TCP复合培养，单纯细胞组作对照。所取样本使用倒置相差显微镜，扫描电镜观察两组细胞形态及增殖情况，MTT法半定量检测细胞增殖情况。
第二部分对一只自然死亡的猕猴进行新鲜解剖，观察其胫骨的形态及周围知名血管束的解剖路径；将11只猕猴双侧胫骨(共22处)制成中段20mm骨-骨膜缺损，随机平均分成2组(实验组和对照组各9侧)，实验组在缺损处填塞由骨髓基质干细胞(BMSCs)和具有特殊外型(侧槽和中空管)的β-磷酸三钙(β-TCP)支架体外构建的复合物，在中空管内移入隐动、静脉束的一段，工程骨外被带蒂深筋膜；对照组只填塞组织工程骨。另外2只猕猴的无填充物胫骨缺损作空白对照。钢板螺钉固定。在4、8、12周时间点分别行放射线检测，墨汁灌注标本，组织学检测及标本大体观察。
第三部分将20只猕猴双侧胫骨(共40处)制成中段20mm骨-骨膜缺损，随机平均分成4组(一个实验组和三个对照组，每组各9侧)，实验组在缺损处填塞由骨髓基质干细胞(BMSCs)和具有特殊外型(侧槽和中空管)的β-磷酸三钙(β-TCP)支架体外构建的复合物，在中空管内移入隐动、静脉束的一段，外被带蒂深筋膜；其余3个对照组分别填塞组织工程骨并包裹筋膜、单纯组织工程骨和单纯支架组。另外2只猕猴的无填充物胫骨缺损作空白对照。钢板螺钉固定。在4、8、12周时间点分别行放射影像学评分和X射线阻射密度分析，以及血管面积和成骨图像分析。得出数据进行统计学分析。
结果
第一部分：猕猴BMSCs体外增殖能力较强，在传3代后的第6天达高峰。细胞经诱导后，在第3天就可见良好的定向分化能力。该支架材料与猕猴BMSCs具有良好的相容性。但材料在加入培养基后会出现崩解，对细胞的初期附着有一定影响。
第二部分：本实验以通过对组织工程骨材料外型的特殊设计，采用血管植入和筋膜包裹的血管化方法修复猕猴双侧胫骨20mm缺损为实验模型，进行了术后大体观察、X线和组织学检测，初步完成了模型的设计和检测工作，认为模型是可行的，为进一步的临床试验提供了参考，但需要更深入的研究。
第三部分：
一般观察术后4周：4组标本无明显差异，均在两接触面、材料后面和钢板侧有部分结缔组织或软骨样组织形成，中空管及侧槽为软组织填塞，材料与骨接触面能折动。A组材料中空管内原植入血管腔内充满墨汁。材料本身无明显吸收。
术后8周：4组标本观察有差异。A组植入物除前面外，其余各面及与骨接触面均有骨样组织包裹，侧槽及中空管也有骨样组织填塞，植入血管观察不清，不易折断，材料1／3吸收；B组在植入物内侧面、前面及中空管内无骨样组织形成，其余各面及与骨接触面仅有少部分骨样组织形成，易折断，材料无明显吸收。C和D组植入物与骨接触面仅有少部分骨样组织形成，易折断，材料无明显吸收。4组标本均有墨染，以A，B两组最明显。
术后12周：4组标本观察有明显差异。A组植入物各面及中央部完全被骨样组织所包裹或替代，坚硬，折不断，材料2／3被吸收；B组和C组植入物于内侧及前面仍有部分材料未被骨样组织所包裹或替代，用力可以折断，材料1／3被吸收。D组成骨罕见，材料无吸收，轻易弯折。
空白组(12 w)缺损处完全被肉芽组织填塞，无骨样组织增生。
显微镜下观察A组术后4周时在移植物边缘、断面及中央有少量新生骨组织——编织骨形成，临近新骨处的材料孔隙内也有新骨的形成和小“肺泡样”墨染结构，植入血管腔内墨染，材料较完整，孔隙未见扩大。8周时新骨形成明显增多，墨染血管样结构明显增多，多靠近外缘及中央，于新骨与材料交接处可见破碎的材料，小颗粒状，植入血管腔内未见墨染。12周时移植物完全被骨样组织所包裹，形成的骨样组织把材料分成几小块，有骨小梁样结构形成。于新骨及剩下的材料中均能见到血管样结构，密度明显增大。新骨中的血管样结构较粗大，走行直，少有分支；材料中的血管样结构多呈“肺泡状”，分支多，串珠样，孔隙之间有交通。于新骨与材料交接处可见较多破碎的材料，小颗粒状，此处的墨染血管样结构最多。
B组术后4周时在移植物边缘、断面有少量新生骨组织——编织骨形成，材料孔隙内也有新骨的形成和小“肺泡样”墨染结构，植入血管腔内墨染，材料较完整，孔隙未见扩大。8周时新骨形成增多，墨染血管样结构明显增多，多靠近外缘，于新骨与材料交接处可见破碎的材料，植入血管腔内未见墨染。12周时形成的骨样组织把材料大部分包被，有骨小梁样结构形成。于材料中央部少见血管样结构。
C组术后4周时在移植物边缘和中央未见新生骨组织，仅在断面处有少量编织骨形成，移植物仅有一面(后面)外缘有小“肺泡样”墨染结构，移植物完整，孔隙未见增大。8周时在移植物边缘、断面有少量新生骨组织形成，仅在新骨与材料交接处可见较多墨染血管样结构，余处少见。12周时可见移植物有少部分吸收，新骨及血管样结构的形成量比同组8周的有所增加，但仍然很稀少。
D组4周时无成骨，移植物断端处可见有少量墨染结构。8周时仅在断端可见少量成骨，墨染结构较4周时增加。12周时移植物断端的成骨情况并未增加，墨染结构也未增加。
血管面积及成骨面积分析各时间点上A组中央部和周围部的血管样结构面积及成骨面积与B、C和D组比较有显著差异(P＜0.01)。而且随时间的推移呈上升趋势。B和C组中央部血管样结构面积比较无统计学意义(P＞0.05)。B组周围部的血管样结构面积与C组比较有统计学意义(P＜0.05)。
放射线观察A组8周显示移植物密度降低，两断面有连续性骨痂形成；12周显示移植物密度明显降低，个别区域低于正常骨，连续性骨痂明显。B和C组8周和12周移植物密度未见降低，断面处有连续性骨痂。D组各时间点无明显变化。
影像评分从正位X线片骨缺损部位阻射密度值中可以看出，4 w各组间无差异，B和C组各时间点间无差异；8、12 w A组与B和C间有差异，A组各时间点间有显著差异(P＜0.01)。A组各时间点阻射密度值呈减少趋势，可能和材料被逐渐吸收有关。
结论
本实验以通过对组织工程骨材料外型的特殊设计，采用血管植入和筋膜包裹的血管化方法修复猕猴双侧胫骨20mm缺损为实验模型，进行了术后大体观察、X线检测及评分和组织学检测及血管密度图像分析，结果显示实验所采用的血管化方法可以加快和提高组织工程骨的成骨作用，为进一步的临床试验提供了参考，但需要更深入的研究。
OBJECTIVE: The study and the clinical application of tissue engineered bonegraft have got more and more recognition, but there are some key problems whichneed solving. Many experiments indicate that the angiogenesis plays a key role in theosteogenesis.An experimental pattern was set up designed to prepare a kind ofvascularized engineered-bone graft for repairing rhesus tibia defects and analyze thecharacters of the angiogenesis and osteogenesis in vivo by rontgenographic andmorphological approaches.
We have ever repaired the 20mm tibia defect of big animals (goats) withvascularized tissue engineered bone, and achieved good results. In order to supplymore detailed and practical approaches for the clinical experiments, we chooserhesuses as experimental animal this time, and put forward the approach of furtherangiogenesis after the anatomical observation, that is, allowing the coexistence andco-growth of both blood vessels and fascia by specially designing the scaffoldconformation can not only accelerate the angiogensis of the tissue engineered bone,but also can greatly improve the quality of the osteogenesis and shorten the healingperiod.This study aims at: detecting the compatibility of BMSCs andβ-TCPscaffold, setting up an animal repairing rhesus tibia defects with engineered bone anddetecting the angiogensis of the tissue engineered bone and the quality of theosteogenesis.
METHODS:
The first part: The third passage of rBMSCs were cultured withβ-TCP withthe cells cultured without the materials as the control. The morphology andproliferation of cells were observed by inverted phase contrast microscope andscanning electron microscope(SEM). MTT assay was used to semiquantitativelyevaluate the cell proliferation.
The second part: A 20 mm tibia defect was made in each of both sides of the 9rhesuses and fixed with a plate. The 18 tibia defects were randomly divided into twogroups equally. The gaps in experiment group were plugged with the engineeredbones constructed in vitro by bone marrow-derived stroma cells(BMSCs) andβ-TCPscaffolds in each one with a slit and a central tunnel. In addition, a correspondingportion of saphenous artery and veins was moved to run through along the centraltunnel in the scaffold with the "gate" help of the slit, and the engineered bone wastotally hugged by a sheet of pedicled deep fascia. The gaps in control group, however,were inserted engineered bones only. The vascularization for each treatment wasassessed by physical, histopathological and X-ray examinations at time intervals of 4,8, 12 weeks after operation.
The third part-The 36 bilateral tibia 20mm defects were randomly divided into4 groups equally. The gaps in experimental groups were plugged with engineeredcomposites which were totally hugged by a sheet of pedicled deep fascia andadditionally a corresponding portion of saphenous artery and veins were moved to runthrough along the central tunnel in the scaffold with the "gate" help of the slit. Thegaps in the three other control groups, however, were inserted with fascia-coatedcompounds, compounds only and pure scaffold only respectively. In addition, tworhesuses were picked up as blanks. The angiogenesis and osteogenesis for eachtreatment was assessed by macroscopical, histological and roentgenographic analyses at time intervals of 4, 8, 12 weeks postoperative.
RESULTS
The first part: Rhesus BMSCs showed a active ability of proliferation and amountthe highest level at 6th day at third passage. Osteogenic differentiation were carriedout at the third day after induction. The scaffold material presented finebiocompatibility with rhesus BMSCs while coculturing together but the disintegrationof material might have certain influence after cells seeding onto the scaffold.
The second part: Through the special design to engineered bone features andapplying double vacularization methods of saphenous bundle insertion and pedicleddeep fascia coated, the experimental animal modal was set up. The samplesharvested from the subjects were analyzed by gross observation, roentgenographicand histologic examination. The modal and its procedure were recognized a feasibleway to get engineered bone revascularizaton but the results needed further researchin order to supply theoretic evidences for final clinical trials.
The third part:
General results
4 weeks postoperatively: There is no significant differences among 4 groupswhich all have some connective or chondroid tissue formation on two contactsurfaces, the back of the material and the side of the plate. The samples presented noabsorption and its contact surface could be easily broken.
8weeks postoperatively: There are differences among 4 groups. In group A,besides what has mentioned before, other surfaces and the contact surfaces with thebone are all wrapped up with bonelike tissues which also fills the slit and the centraltunnel. The implanted blood vessels can clearly be seen, and the bone are not easy tobe broken, 1/3 materials has been absorbed; in group B, no bonelike tissues form onthe medial surface and the front of the implanted material, and the same with the central tunnel. A few bonelike tissues appear on the other surfaces and the contactsurfaces with the bone; in group C, few bonelike tissues form on the contact surfacesbetween the implanted material, which are easy to be broken and the material has notbeen significantly absorbed, in group D, the condition is relatively the same as that ingroup C.
12 weeks postoperatively: There are significant differences among 4 groups. Ingroup A, all the surfaces of the implanted material and the central part are whollywrapped up or replaced by bonelike tissues which are hard and cannot be broken. And2/3 materials have been absorbed; in group B and C, partial materials of the medialsurface and the front have not been replaced by bonelike tissues yet, which can bebroken with force, and 1/3 material has been absorbed; in group D, rare boneliketissues were seen at the contact interfaces, and no material has been absorbed.
Blanks: At 12 week, the defect has been completely filled with granulationtissues, without osteogenesis.
Microscopic observation
Group A: At 4w postoperatively, a few new bonelike tissues, woven bone,form on the edges, the sections and the center of the implanted material, near which,new bone and small alveoli-like ink-stained structures also appear in the holes of thematerial. The implanted blood vessels lumens are stained with ink, and the material isfairly complete, the holes have not dilated. At 8w, new bone and ink-stained bloodvessel structures have significantly increased, most of which are close to the externalmargin and the center. Broken materials, like fine particles, can be seen at thejunctions between the new bone and the material, and the implanted blood vesselshave not been ink-stained. At 12w, the implanted material has been completely coatedwith the bonelike tissues which separate the material into several small parts withbone trabecula structure formation. The density of the blood vessels structures greatly increases in the new bone and the left material. Those in the new bone are quite thick,straight and have less branches, while those in the left material are mostly alveolialike, rosary like and have more branches, with holes connected with each other.Quite a lot broken materials, like small particles, are seen at the junctions between thenew bone and the material, where the amount of ink-stained blood vessels structuresis maximal.
Group B: At 4w postoperatively, a few newborn bone tissues, woven bonetissues form on the edge and sects of the implanted material, and there are also newbone and small alveoli like structures within the holes of the material. The lumen ofthe implanted blood vessel is ink-stained, the material is fairly complete, and theholes have not dilated. At 8w, more new bones form, and much more ink-stainedblood vessels appear. Many of them are near to the external edge, and brokenmaterials are visible at the junctions between the new bone and the material. Thelumens of the implanted blood vessel are not ink-stained. At 12w, most of the materialis wrapped up by the new bone and bone trabeculae have formed. Few blood vesselscan be seen in the center of the material.
Group C: At 4w, no new bone can be seen on the edge and the center of theimplanted material, a few woven bones have formed only on the sections, and smallalveoli like ink-stained structures can be seen only on the external edge of thematerial back. The material is fairly complete and the holes have not dilated. At 8w, afew new bones appear on the edge and the sections of the implanted material, andink-stained blood vessels are seldom seen except that quite a lot appear at thejunctions between the new bone and the material. At 12w, the implanted material hasbeen slightly absorbed. The new bone and the blood vessel have increased comparedwith those at 8w, but still very few.
Group D: No newbone formed at 4 weeks postoperatively, but a few of "ink-staining" structure showed up at interfaces. The "ink-staining" structureincreased quantitatively and a little of bonelike tissue could be seen at interfaces at 8and 12 weeks.
Analyses of angiogenic and osteogenie area
Take the central tunnel and its vicinity within 2mm as the center portion, and theother places as the surrounding portion.There were statistical significance for bothangiogenic and osteogenic areas comparing the vaso-areas of both central andperipheral parts in group A to those of group B, C and D(P＜0.01). No significancecould be seen between group B and C for comparing the angiogenic areas at centralparts (P＞0.05), while it is significant comparing the angiogenic areas at peripheralparts(P＜0.05).
X-rays Observation
At 8w, group A's images show that the density of the implanted materialdecreases, and continual bony callus forms at the interfaces. At 12w, group A's imagespresent obviously decreased density which is lower than that of the normal bone inindividual areas, and the continual bony callus is manifest. At 8w and 12w, group Band C's images show no decreased density and the continual bony callus appear onthe sections. Group D shows no changes at every check intervals.
Roentgenographic scores
From the blocking density values at the bone defects, it can be seen that nodifferences exist among the 4 groups at 4w, and no differences exist between group Band C at all observing time points; At 8w and 12w, group A is different from group Band C, and there are differences between all the time intervals of group A(P＜0.01).The density gradually decreases with the time, which is probably due tothe absorption of the implanted material.
Conclusion
Effects on the rhesus' activities seemed to be rare. This study shows us a feasibleand effective angiogenesis approach which can accelerate osteogenesis in vivo ofengineered bone.
引文
1 Langer R, Vacanti JP. Tissue engineering. Science, 1993, 260:920-926.
2 Lysaght MJ, Hazlehurst AL. Tissue engineering: the end of the beginning. Tissue Eng, 2004, 10:309-320.
3 Khalil-Marzouk JF. Allograft replacement of the trachea. Experimental synchronous revascularization of composite thyrotracheal transplant. J Thorac Cardiovasc Surg,1993 ,105:242-246.
4 Eiselt P, Kim BS, Chacko B, Isenberg B, Peters MC, Greene KG, Roland WD, Loebsack AB, Burg KJ, Culberson C, Halberstadt CR, Holder WD, Mooney DJ. Development of technologies aiding large-tissue engineering. Biotechnol Prog, 1998, 14:134-140.
5 Vacanti JP, Langer R. Tissue engineering: the design and fabrication of living replacement devices for surgical reconstruction and transplantation. Lancet, 1999, 354:32-34.
6. Bouhadir KH, Mooney DJ. Promoting angiogenesis in engineered tissues. J Drug Target, 2001,9:397-406.
7 Colton CK. Implantable biohybrid artificial organs. Cell Transplant, 1995,4:415-436.
8 Folkman J, Hochberg M. Self-regulation of growth in three dimensions. J Exp Med, 1973,138:745-753.
9 Risau W. Mechanisms of angiogenesis. Nature, 1997, 17;386:671-674.
10 Shalaby F, Ho J, Stanford WL, Fischer KD, Schuh AC, Schwartz L, Bernstein A, Rossant J. A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis.Cell, 1997,89:981-990.
11 Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ, Holash J. Vascular-specific growth factors and blood vessel formation. Nature, 2000,407:242-248.
12 Lee H, Cusick RA, Browne F, Ho Kim T, Ma PX, Utsunomiya H, Langer R, Vacanti JP. Local delivery of basic fibroblast growth factor increases both angiogenesis and engraftment of hepatocytes in tissue-engineered polymer devices. Transplantation, 2002,73:1589-1593.
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1 Langer R, Vacanti JP. Tissue engineering. Science, 1993, 260:920-926.
2 Lysaght MJ, Hazlehurst AL. Tissue engineering: the end of the beginning. Tissue Eng, 2004, 10:309-320.
3 Khalil-Marzouk JF. Allograft replacement of the trachea. Experimental synchronous revascularization of composite thyrotracheal transplant.J rhorac Cardiovasc Surg, 1993, 105:242-246.
4 Eiselt P, Kim BS, Chacko B, Isenberg B, Peters MC, Greene KG, Roland WD, Loebsack AB, Burg KJ, Culberson C, Halberstadt CR, Holder WD, Mooney DJ. Development of technologies aiding large-tissue engineering. Biotechnol Prog, 1998, 14:134-140.
5 Vacanti JP, Langer R. Tissue engineering: the design and fabrication of living replacement devices for surgical reconstruction and transplantation. Lancet, 1999, 354:32-34.
6 Bouhadir KH, Mooney DJ. Promoting angiogenesis in engineered tissues. J Drug Target, 2001,9:397-406.
7 Colton CK. Implantable biohybrid artificial organs. Cell Transplant, 1995,4:415-436.
8 Folkman J, Hochberg M. Self-regulation of growth in three dimensions. J Exp Med, 1973,138:745-753.
9 Risau W. Mechanisms of angiogenesis. Nature, 1997, 17;386:671-674.
10 Shalaby F, Ho J, Stanford WL, Fischer KD, Schuh AC, Schwartz L, Bernstein A, Rossant J. A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis.Cell, 1997,89:981-990.
11 Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ, Holash J. Vascular-specific growth factors and blood vessel formation. Nature, 2000,407:242-248.
12 Lee H, Cusick RA, Browne F, Ho Kim T, Ma PX, Utsunomiya H, Langer R, Vacanti JP. Local delivery of basic fibroblast growth factor increases both angiogenesis and engraftment of hepatocytes in tissue-engineered polymer devices. Transplantation, 2002,73:1589-1593.
13 Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science, 1997,275:964-967.
14 Shi Q, Rafii S, Wu MH, Wijelath ES, Yu C, Ishida A, Fujita Y, Kothari S, Mohle R, Sauvage LR, Moore MA, Storb RF, Hammond WP. Evidence for circulating bone marrow-derived endothelial cells.Blood, 1998,92:362-367.
15 Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney M, Magner M, Isner JM, Asahara T. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med, 1999,5:434-438.
16 Isner JM, Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization.J Clin Invest, 1999, 103:1231-1236.
17 Caplan AI. Mesenchymal stem cells and gene therapy. Clin Orthop Relat Res, 2000,379:67-70.
18 Musgrave DS, Bosch P, Lee JY, Pelinkovic D, Ghivizzani SC, Whalen J, Niyibizi C, Huard J. Ex vivo gene therapy to produce bone using different cell types. Clin Orthop Relat Res, 2000,378:290-305.
19 Oreffo RO, Virdi AS, Triffitt JT. Retroviral marking of human bone marrow fibroblasts: in vitro expansion and localization in calvarial sites after subcutaneous transplantation in vivo. J Cell Physiol, 2001, 186:201-209.
20 Stein I, Neeman M, Shweiki D, Itin A, Keshet E. Stabilization of vascular endothelial growth factor mRNA by hypoxia and hypoglycemia and coregulation with other ischemia-induced genes. Mol Cell Biol, 1995,15:5363-5368.
21 Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, Risau W, Nagy A. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature, 1996,380:435-439.
22 Sage EH, Vernon RB. Regulation of angiogenesis by extracellular matrix: the growth and the glue. J Hypertens Suppl, 1994,12:145-152.
23. Polverini PJ. The pathophysiology of angiogenesis. Crit Rev Oral Biol Med, 1995,6:230-247.
24 Peters MC, Polverini PJ, Mooney DJ. Engineering vascular networks in porous polymer matrices.J Biomed Mater Res, 2002,60:668-678.
25 Alon T, Hemo I, Itin A, Pe'er J, Stone J, Keshet E. Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat Med, 1995,1:1024-1028.
26 Norrby K. Angiogenesis: new aspects relating to its initiation and control. APMIS, 1997,105:417-437.
27 Ferrara N, Alitalo K. Clinical applications of angiogenic growth factors and their inhibitors. Nat Med, 1999,5:1359-1364.
28 Connolly DT, Heuvelman DM, Nelson R, Olander JV, Eppley BL, Delfino JJ, Siegel NR, Leimgruber RM, Feder J. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest, 1989,84:1470-1478.
29 Dvorak HF, Detmar M, Claffey KP, Nagy JA, van de Water L, Senger DR. Vascular permeability factor/vascular endothelial growth factor: an important mediator of angiogenesis in malignancy and inflammation. Int Arch Allergy Immunol, 1995,107:233-235.
30 Hillsley MV, Frangos JA. Bone tissue engineering: the role of interstitial fluid flow. Biotechnol Bioeng, 1994,43:573-581.
31 陈滨，裴国献，王珂，金丹，魏宽海，任高宏．筋膜蒂促组织工程骨体内再血管化的实验研究．解放军医学杂志，2002，27(6)：482-484．
32 金丹，裴国献，陈滨．骨组织工程研究中的血管、神经化问题．现代康复，2001，5(8)： 18-9
33 Albrektsson T. Repair of bone grafts. A vital microscopic and histological investigation in the rabbit. Scand J Plast Reconstr Surg, 1980, 14:1-12.
34 Burchardt H. Biology of bone transplantation. Orthop Clin North Am, 1987, 18:187-96.
35 Prolo DJ, Rodrigo JJ. Contemporary bone graft physiology and surgery.Clin Orthop Relat Res, 1985, 200:322-342.
36 Pelissier P, Villars F, Mathoulin-Pelissier S, Bareille R, hafage-Proust MH, Vilamitjana-Amedee J. Influences of vascularization and osteogenic cells on heterotopic bone formation within a madreporic ceramic in rats. Plast Reconstr Surg, 2003, 111:1932-1941.
37 Perets A, Baruch Y, Weisbuch F, Shoshany G, Neufeld G, Cohen S. Enhancing the vascularization of three-dimensional porous alginate scaffolds by incorporating controlled release basic fibroblast growth factor microspheres.J Biomed Mater Res A, 2003, 65:489-497.
38 Soker S, Machado M, Atala A. Systems for therapeutic angiogenesis in tissue engineering. World J Urol, 2000, 18:10-18.
39 Elcin YM, Dixit V, Gitnick G. Extensive in vivo angiogenesis following controlled release of human vascular endothelial cell growth factor: implications for tissue engineering and wound healing. Artif Organs, 2001, 25:558-565.
40 Frerich B, Kurtz-Hoffmann J, Lindemann N, Muller S. Tissue engineering of vascularized bone and soft tissue transplants. Mund Kiefer Gesichtschir, 2000, 4(Suppl 2):490-495.
41 杨志明，樊征夫，解慧琪，秦廷武，彭文珍．组织工程化人工骨血管化研究．中华显微外科杂志，2002，25(2)：119-122．
42 Cassell OC, Morrison WA, Messina A, et al. The influence of extracellular matrix on the generation of vascularized, endineered, transplantable tissue, inn N Y Acad Sci. 2001 Nov;944:429-42
43 曾宪利，裴国献，金丹，唐光辉，王学明，刘哓霞，曾俊岭，张文高．血管化组织工程骨修复猕猴胫骨缺损模型的建立及初步观察．中华创伤骨科杂志，2005，7(4)：353-357．
44 Cassell OC, Morrison WA, Messina A, Penington AJ, Thompson EW, Stevens GW, Perera JM, Kleinman HK, Hurley JV, Romeo R, Knight KR. The influence of extracellular matrix on the generation of vascularized, engineered, transplantable tissue. Ann N Y Acad Sci, 2001, 944:429-442.
45 Meinhart J, Deutsch M, Zilla P. Eight years of clinical endothelial cell transplantation. Closing the gap between prosthetic grafts and vein grafts. ASAIO J, 1997, 43(5):M515-521.
46 Edelman ER. Vascular tissue engineering: designer arteries. Circ Res, 1999, 85:1115-1117.
47 Huang L, Nagapudi K, Apkarian RP, Chaikof EL. Engineered collagen-PEO nanofibers and fabrics. J Biomater Sci Polym Ed, 2001, 12:979-993.
48 Li WJ, Laurencin CT, Caterson EJ, Tuan RS, Ko FK. Electrospun nanofibrous structure: a novel scaffold for tissue engineering. J Biomed Mater Res, 2002, 60:613-621.
49 Matthews JA, Wnek GE, Simpson DG, Bowlin GL. Electrospinning of collagen nanofibers. Biomacromolecules, 2002, 3:232-238.
50 Shin M, Yoshimoto H, Vacanti JP. In vivo bone tissue engineering using mesenchymal stem cells on a novel electrospun nanofibrous scaffold. Tissue Eng, 2004, 10:33-41.
51 Rouwkema J,de Boer J, Van Blitterswijk CA. Endothelial cells assemble into a 3-dimentional prevascular network in a bone tissue engineering construct. Tissue Eng. 2006, 12(9):2686-2693.
52 Gross TP, Cox QG, Jinnah RH. History and current application of bone transplantation. Orthopedics, 1993,16:895-900.
2 Lysaght MJ, Hazlehurst AL. Tissue engineering: the end of the beginning. Tissue Eng, 2004, 10:309-320.
3 Khalil-Marzouk JF. Allograft replacement of the trachea. Experimental synchronous revascularization of composite thyrotracheal transplant. J Thorac Cardiovasc Surg,1993 ,105:242-246.
4 Eiselt P, Kim BS, Chacko B, Isenberg B, Peters MC, Greene KG, Roland WD, Loebsack AB, Burg KJ, Culberson C, Halberstadt CR, Holder WD, Mooney DJ. Development of technologies aiding large-tissue engineering. Biotechnol Prog, 1998, 14:134-140.
5 Vacanti JP, Langer R. Tissue engineering: the design and fabrication of living replacement devices for surgical reconstruction and transplantation. Lancet, 1999, 354:32-34.
6. Bouhadir KH, Mooney DJ. Promoting angiogenesis in engineered tissues. J Drug Target, 2001,9:397-406.
7 Colton CK. Implantable biohybrid artificial organs. Cell Transplant, 1995,4:415-436.
8 Folkman J, Hochberg M. Self-regulation of growth in three dimensions. J Exp Med, 1973,138:745-753.
9 Risau W. Mechanisms of angiogenesis. Nature, 1997, 17;386:671-674.
10 Shalaby F, Ho J, Stanford WL, Fischer KD, Schuh AC, Schwartz L, Bernstein A, Rossant J. A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis.Cell, 1997,89:981-990.
11 Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ, Holash J. Vascular-specific growth factors and blood vessel formation. Nature, 2000,407:242-248.
12 Lee H, Cusick RA, Browne F, Ho Kim T, Ma PX, Utsunomiya H, Langer R, Vacanti JP. Local delivery of basic fibroblast growth factor increases both angiogenesis and engraftment of hepatocytes in tissue-engineered polymer devices. Transplantation, 2002,73:1589-1593.
13 Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science, 1997,275:964-967.
14 Shi Q, Rafii S, Wu MH, Wi jelath ES, Yu C, Ishida A, Fujita Y, Kothari S, Mohle R, Sauvage LR, Moore MA, Storb RF, Hammond WP. Evidence for circulating bone marrow-derived endothelial cells. Blood, 1998,92:362-367.
15 Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney M, Magner M, Isner JM, Asahara T. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization.Nat Med, 1999,5:434-438.
16 Isner JM, Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization.J Clin Invest, 1999,103:1231-1236.
17 Caplan AI. Mesenchymal stem cells and gene therapy. Clin Orthop Relat Res, 2000,379:67-70.
18 Musgrave DS, Bosch P, Lee JY, Pelinkovic D, Ghivizzani SC, Whalen J, Niyibizi C, Huard J. Ex vivo gene therapy to produce bone using different cell types. Clin Orthop Relat Res, 2000,378:290-305.
19 Oreffo RO, Virdi AS, Triffitt JT. Retroviral marking of human bone marrow fibroblasts: in vitro expansion and localization in calvarial sites after subcutaneous transplantation in vivo. J Cell Physiol, 2001,186:201-209.
20 Stein I, Neeman M, Shweiki D, Itin A, Keshet E. Stabilization of vascular endothelial growth factor mRNA by hypoxia and hypoglycemia and coregulation with other ischemia-induced genes. Mol Cell Biol, 1995,15:5363-5368.
21 Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, Risau W, Nagy A. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature, 1996,380:435-439.
22 Sage EH, Vernon RB. Regulation of angiogenesis by extracellular matrix: the growth and the glue. J Hypertens Suppl, 1994,12:145-152.
23 Polverini PJ. The pathophysiology of angiogenesis. Crit Rev Oral Biol Med, 1995,6:230-247.
24 Peters MC, Polverini PJ, Mooney DJ. Engineering vascular networks in porous polymer matrices. J Biomed Mater Res, 2002,60:668-678.
25 AlonT, Hemo I, Itin A, Pe'er J, Stone J, Keshet E. Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat Med, 1995,1:1024-1028.
26 Dvorak HF, Detmar M, Claffey KP, Nagy JA, van de Water L, Senger DR. Vascular permeability factor/vascular endothelial growth factor: an important mediator of angiogenesis in malignancy and inflammation. Int Arch Allergy Immunol, 1995, 107:233-235.
27 陈滨，裴国献，王珂，金丹，魏宽海，任高宏．筋膜蒂促组织工程骨体内再血管化的实验研究．解放军医学杂志，2002，27(6)：482-484．
28 金丹，裴国献，陈滨．骨组织工程研究中的血管、神经化问题．现代康复，2001，5(8)： 18-9
29 Albrektsson T. Repair of bone grafts. A vital microscopic andhistological investigation in the rabbit. Scand J Plast Reconstr Surg, 1980, 14:1-12.
30 Burchardt H. Biology of bone transplantation. Orthop Clin NorthAm, 1987, 18:187-96.
31 Prolo DJ, Rodrigo JJ. Contemporary bone graft physiology and surgery. Clin Orthop Relat Res, 1985, 200:322-342.
32 汪群力，裴国献，曾宪利，金丹，魏宽海，任高宏。成年恒河猴骨髓基质干细胞的体外培养。中华创伤骨科杂志。2004，6(7)：728-730．
33 裴国献，金丹．骨组织工程学种子细胞研究进展．中华创伤骨科杂志，2003，5： 55-58．
34 陈滨，裴国献，王柯，金丹，魏宽海，任高宏．筋膜瓣促组织工程骨体内再血管化的实验研究．解放军医学杂志，2002，27：482-484．
35 杨志明，主编．组织工程基础与临床．成都：四川科学技术出版社，2002．3．
36 Stevenson S, Goldberg VM, Shaffer J. Histomorphometrical analysis of vascularized and nonvascularized fibular autografts in dogs treated and not treated with cylosporin. Tran Orthop Res Soe, 1998, 13:388-393.
37 Glowacki I. Angiogenesis in fracture repair. Clin Orhtop Relat Res, 1998, 355 (Suppl):82-89.
38 Zwaginga JJ, Doevendans P. Stem cell-derived angiogenic/vasculogenic cells: possible therapies for tissue repair and tissue engineering. Clin Exp Pharmacol Physiol, 2003, 30:900-908.
39 Eckardt H, Bundgaard KG, Christensen KS, Lind M, Hansen ES, Hvid I. Effects of locally applied vascular endothelial growth factor (VEGF) and VEGF-inhibitor to the rabbit tibia during distraction osteogenesis. J Orthop Res, 2003, 21:335-340.
40 阳富春，杨志明，李秀群，周悦婷，林凡，秦廷武，罗静聪，解慧琪．组织工程化骨修复猕猴长段骨缺损的实验研究．中国修复重建外科杂志，2003，17：406-410．
41 曾宪利，裴国献，金丹，唐光辉，王学明，刘哓霞，曾俊岭，张文高．血管化组织工程骨修复猕猴胫骨缺损模型的建立及初步观察．中华创伤骨科杂志，2005，7(4)：353-357．
1 Langer R, Vacanti JP. Tissue engineering. Science, 1993, 260:920-926.
2 Lysaght MJ, Hazlehurst AL. Tissue engineering: the end of the beginning. Tissue Eng, 2004, 10:309-320.
3 Khalil-Marzouk JF. Allograft replacement of the trachea. Experimental synchronous revascularization of composite thyrotracheal transplant.J rhorac Cardiovasc Surg, 1993, 105:242-246.
4 Eiselt P, Kim BS, Chacko B, Isenberg B, Peters MC, Greene KG, Roland WD, Loebsack AB, Burg KJ, Culberson C, Halberstadt CR, Holder WD, Mooney DJ. Development of technologies aiding large-tissue engineering. Biotechnol Prog, 1998, 14:134-140.
5 Vacanti JP, Langer R. Tissue engineering: the design and fabrication of living replacement devices for surgical reconstruction and transplantation. Lancet, 1999, 354:32-34.
6 Bouhadir KH, Mooney DJ. Promoting angiogenesis in engineered tissues. J Drug Target, 2001,9:397-406.
7 Colton CK. Implantable biohybrid artificial organs. Cell Transplant, 1995,4:415-436.
8 Folkman J, Hochberg M. Self-regulation of growth in three dimensions. J Exp Med, 1973,138:745-753.
9 Risau W. Mechanisms of angiogenesis. Nature, 1997, 17;386:671-674.
10 Shalaby F, Ho J, Stanford WL, Fischer KD, Schuh AC, Schwartz L, Bernstein A, Rossant J. A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis.Cell, 1997,89:981-990.
11 Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ, Holash J. Vascular-specific growth factors and blood vessel formation. Nature, 2000,407:242-248.
12 Lee H, Cusick RA, Browne F, Ho Kim T, Ma PX, Utsunomiya H, Langer R, Vacanti JP. Local delivery of basic fibroblast growth factor increases both angiogenesis and engraftment of hepatocytes in tissue-engineered polymer devices. Transplantation, 2002,73:1589-1593.
13 Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science, 1997,275:964-967.
14 Shi Q, Rafii S, Wu MH, Wijelath ES, Yu C, Ishida A, Fujita Y, Kothari S, Mohle R, Sauvage LR, Moore MA, Storb RF, Hammond WP. Evidence for circulating bone marrow-derived endothelial cells.Blood, 1998,92:362-367.
15 Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney M, Magner M, Isner JM, Asahara T. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med, 1999,5:434-438.
16 Isner JM, Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization.J Clin Invest, 1999, 103:1231-1236.
17 Caplan AI. Mesenchymal stem cells and gene therapy. Clin Orthop Relat Res, 2000,379:67-70.
18 Musgrave DS, Bosch P, Lee JY, Pelinkovic D, Ghivizzani SC, Whalen J, Niyibizi C, Huard J. Ex vivo gene therapy to produce bone using different cell types. Clin Orthop Relat Res, 2000,378:290-305.
19 Oreffo RO, Virdi AS, Triffitt JT. Retroviral marking of human bone marrow fibroblasts: in vitro expansion and localization in calvarial sites after subcutaneous transplantation in vivo. J Cell Physiol, 2001, 186:201-209.
20 Stein I, Neeman M, Shweiki D, Itin A, Keshet E. Stabilization of vascular endothelial growth factor mRNA by hypoxia and hypoglycemia and coregulation with other ischemia-induced genes. Mol Cell Biol, 1995,15:5363-5368.
21 Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, Risau W, Nagy A. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature, 1996,380:435-439.
22 Sage EH, Vernon RB. Regulation of angiogenesis by extracellular matrix: the growth and the glue. J Hypertens Suppl, 1994,12:145-152.
23. Polverini PJ. The pathophysiology of angiogenesis. Crit Rev Oral Biol Med, 1995,6:230-247.
24 Peters MC, Polverini PJ, Mooney DJ. Engineering vascular networks in porous polymer matrices.J Biomed Mater Res, 2002,60:668-678.
25 Alon T, Hemo I, Itin A, Pe'er J, Stone J, Keshet E. Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat Med, 1995,1:1024-1028.
26 Norrby K. Angiogenesis: new aspects relating to its initiation and control. APMIS, 1997,105:417-437.
27 Ferrara N, Alitalo K. Clinical applications of angiogenic growth factors and their inhibitors. Nat Med, 1999,5:1359-1364.
28 Connolly DT, Heuvelman DM, Nelson R, Olander JV, Eppley BL, Delfino JJ, Siegel NR, Leimgruber RM, Feder J. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest, 1989,84:1470-1478.
29 Dvorak HF, Detmar M, Claffey KP, Nagy JA, van de Water L, Senger DR. Vascular permeability factor/vascular endothelial growth factor: an important mediator of angiogenesis in malignancy and inflammation. Int Arch Allergy Immunol, 1995,107:233-235.
30 Hillsley MV, Frangos JA. Bone tissue engineering: the role of interstitial fluid flow. Biotechnol Bioeng, 1994,43:573-581.
31 陈滨，裴国献，王珂，金丹，魏宽海，任高宏．筋膜蒂促组织工程骨体内再血管化的实验研究．解放军医学杂志，2002，27(6)：482-484．
32 金丹，裴国献，陈滨．骨组织工程研究中的血管、神经化问题．现代康复，2001，5(8)： 18-9
33 Albrektsson T. Repair of bone grafts. A vital microscopic and histological investigation in the rabbit. Scand J Plast Reconstr Surg, 1980, 14:1-12.
34 Burchardt H. Biology of bone transplantation. Orthop Clin North Am, 1987, 18:187-96.
35 Prolo DJ, Rodrigo JJ. Contemporary bone graft physiology and surgery.Clin Orthop Relat Res, 1985, 200:322-342.
36 Pelissier P, Villars F, Mathoulin-Pelissier S, Bareille R, hafage-Proust MH, Vilamitjana-Amedee J. Influences of vascularization and osteogenic cells on heterotopic bone formation within a madreporic ceramic in rats. Plast Reconstr Surg, 2003, 111:1932-1941.
37 Perets A, Baruch Y, Weisbuch F, Shoshany G, Neufeld G, Cohen S. Enhancing the vascularization of three-dimensional porous alginate scaffolds by incorporating controlled release basic fibroblast growth factor microspheres.J Biomed Mater Res A, 2003, 65:489-497.
38 Soker S, Machado M, Atala A. Systems for therapeutic angiogenesis in tissue engineering. World J Urol, 2000, 18:10-18.
39 Elcin YM, Dixit V, Gitnick G. Extensive in vivo angiogenesis following controlled release of human vascular endothelial cell growth factor: implications for tissue engineering and wound healing. Artif Organs, 2001, 25:558-565.
40 Frerich B, Kurtz-Hoffmann J, Lindemann N, Muller S. Tissue engineering of vascularized bone and soft tissue transplants. Mund Kiefer Gesichtschir, 2000, 4(Suppl 2):490-495.
41 杨志明，樊征夫，解慧琪，秦廷武，彭文珍．组织工程化人工骨血管化研究．中华显微外科杂志，2002，25(2)：119-122．
42 Cassell OC, Morrison WA, Messina A, et al. The influence of extracellular matrix on the generation of vascularized, endineered, transplantable tissue, inn N Y Acad Sci. 2001 Nov;944:429-42
43 曾宪利，裴国献，金丹，唐光辉，王学明，刘哓霞，曾俊岭，张文高．血管化组织工程骨修复猕猴胫骨缺损模型的建立及初步观察．中华创伤骨科杂志，2005，7(4)：353-357．
44 Cassell OC, Morrison WA, Messina A, Penington AJ, Thompson EW, Stevens GW, Perera JM, Kleinman HK, Hurley JV, Romeo R, Knight KR. The influence of extracellular matrix on the generation of vascularized, engineered, transplantable tissue. Ann N Y Acad Sci, 2001, 944:429-442.
45 Meinhart J, Deutsch M, Zilla P. Eight years of clinical endothelial cell transplantation. Closing the gap between prosthetic grafts and vein grafts. ASAIO J, 1997, 43(5):M515-521.
46 Edelman ER. Vascular tissue engineering: designer arteries. Circ Res, 1999, 85:1115-1117.
47 Huang L, Nagapudi K, Apkarian RP, Chaikof EL. Engineered collagen-PEO nanofibers and fabrics. J Biomater Sci Polym Ed, 2001, 12:979-993.
48 Li WJ, Laurencin CT, Caterson EJ, Tuan RS, Ko FK. Electrospun nanofibrous structure: a novel scaffold for tissue engineering. J Biomed Mater Res, 2002, 60:613-621.
49 Matthews JA, Wnek GE, Simpson DG, Bowlin GL. Electrospinning of collagen nanofibers. Biomacromolecules, 2002, 3:232-238.
50 Shin M, Yoshimoto H, Vacanti JP. In vivo bone tissue engineering using mesenchymal stem cells on a novel electrospun nanofibrous scaffold. Tissue Eng, 2004, 10:33-41.
51 Rouwkema J,de Boer J, Van Blitterswijk CA. Endothelial cells assemble into a 3-dimentional prevascular network in a bone tissue engineering construct. Tissue Eng. 2006, 12(9):2686-2693.
52 Gross TP, Cox QG, Jinnah RH. History and current application of bone transplantation. Orthopedics, 1993,16:895-900.