摘要
骨组织工程的最主要障碍是早期中心的血管化不足,营养和氧份不能保证细胞的成活。既往虽然有很多促使血管化的方法应用,如埋植在富血管的机体组织内、植入血管束等,有一定的效果,构建出小块的组织工程骨,但它们的成活主要是依赖组织液的渗透,最多只能达到组织内100~300μm,这种血管化称为“外生性血管化”。与之相对应的是“内生性血管化”,即支架材料预先构建血管化,完成内循环交通系统,再加入成骨细胞等构建成骨。方法之一是体外生物反应器内孵育形成内循环;二是植入动静脉短路环法体内构建内生性血供。该方法早期是整形外科用于皮瓣等构建,发现这种血管环可以高效地诱导血管新生,因手术创伤、炎症反应和血流动力学改变等,激发内皮细胞上调血管内皮生长因子、出芽、再生血管。2006年开始用于骨组织工程。
本课题主要策略是利用动静脉环模型原理,首次选取新西兰兔股动静脉,吻合成动静脉短路环,导入天然珊瑚中,外包裹隔离膜,使珊瑚在一个相对独立的空间内产生内血管化,再加入成骨生长因子,构建出带血管蒂的组织工程骨。其特点在于:1.动静脉短路后形成的局部环可以高效地诱导血管新生;2.隔离膜既可以阻断外来纤维组织的过度长入,又可以自由伸缩,满足复杂的组织形态变化需求;3.珊瑚是良好的骨修复材料和骨组织工程支架材料;4.珊瑚发生了内生性血管化,解决了组织工程骨的内部血供和营养障碍,成骨效果明显加强。
实验比较了动静脉血管环法和常规的动静脉血管束法在珊瑚中诱导血管新生的能力,验证了动静脉环法的优越性;通过血管墨汁灌注、血管铸型、血管内膜电镜等方法,探讨了血管新生的原理主要是内皮出芽生长和动脉生长;进一步在血管环周围和珊瑚表层增加纤维蛋白胶,减小血管所受刺激,优化了实验方案;最后以纤维蛋白胶作为载体,融附骨形成蛋白,加入预血管化的珊瑚中,构建出带血管的组织工程骨。
本实验共分六部分。
实验一天然滨珊瑚无害化处理后形态学和抗压强度的变化
目的:检测天然滨珊瑚经化学和物理方法处理后大体、显微结构以及抗压强度等变化。方法:天然滨珊瑚磨改制备成6×8×10 mm3的椭圆形方块,放入5%次氯酸钠溶液中浸泡两周,煮沸3个周期、超声清洗5个周期,然后烤干,最后高温高压消毒。检测珊瑚抗压强度变化,体视镜下和扫描电镜下观察珊瑚表面和孔隙清洁度、大小等变化。结果:三个阶段珊瑚的抗压强度无明显变化。经浸泡和超声处理后珊瑚表面和孔隙内明显清洁,异物减少,而孔隙大小和结构无明显改变。高温高压消毒后珊瑚表面和孔隙与处理后组一致。结论:天然滨珊瑚经物理、化学处理后,表面和内部异物被清除,外观结构和抗压强度不受影响。
实验二动静脉短路环法和血管束法诱导珊瑚血管化能力的初步研究
目的:用AV环和AV束两种血管载体预构珊瑚支架材料血管化,探索哪种方法有更强的诱导血管新生能力。方法:解剖分离出新西兰兔左侧股静脉和股动脉及其腘动脉分支。A组(n=18)是将腘动脉和股静脉末端切断,近心端行血管端端吻合,形成动静脉短路环,环绕套入天然珊瑚块的侧槽中。B组(n=18)是股动静脉血管束保持血流通畅,不切断,动静脉分开套入珊瑚块的两边侧槽中。植入体外加ePTFE膜包裹隔绝,固定于腹股沟皮下。2、4、6周行标本的墨汁灌注和大体观察,墨汁灌注后珊瑚脱钙、HE染色组织学观察,分析珊瑚孔隙中组织结构和血管新生情况,计数每份标本的平均血管密度作定量统计学分析。结果:珊瑚植入体内后表面和深部有大量纤维血管样组织生长。墨汁灌注显示血管广泛分布在珊瑚表层和间隙内,结构成熟,4周已贯穿珊瑚块的全层。2、4、6周血管密度逐渐加大,A组较B组血管生成密度有明显统计学差别(P<0.01)。结论:AV环方法和AV束方法均可以构建出血管化珊瑚支架,前者诱导血管新生的能力更强,说明这种动物模型是可行的。
实验三两种血管束法诱导珊瑚血管化能力的比较
目的:比较末端结扎和不结扎两种血管束法诱导珊瑚血管化的能力。方法:选择新西兰兔的股动脉和股静脉解剖分离形成血管束,A组(n=9)是血管束保持血流通畅,动静脉分别套入珊瑚支架的两侧槽中。B组(n=9)是末端切断结扎形成动静脉环,环绕套入珊瑚支架的侧槽中。两组均外加ePTFE膜包裹隔绝,植入腹股沟皮下。C组(n=3)是单纯珊瑚块直接埋植于皮下作为空白对照。2、4、6周行大体形态学和组织学检查,分析珊瑚孔隙中组织结构和血管新生情况,血管密度作定量统计学分析。结果:珊瑚侧槽、表层和深层间隙内均有一定程度的血管和纤维结缔组织新生,4周基本充满珊瑚大部分,6周血管化明显,贯穿大部分。组织学和定量分析显示血管通畅组较末端结扎组和空白对照组血管生成更明显,空白组主要是纤维组织增生。结论:两种血管束法也可以不同程度地诱导珊瑚血管化,血管通畅组效果最好,操作简单,可以作为构建血管化组织工程骨的一种方法。
实验四纤维蛋白胶复合珊瑚促进动静脉环血管新生能力的研究
目的:检测纤维蛋白胶复合珊瑚后能否促进AV环的血管生成,从而优化设计AV环模型。方法:同样将新西兰兔腘动脉和股静脉吻合成动静脉短路环。A组(n=15)是将AV环套入天然珊瑚块的侧槽后直接用ePTFE膜包裹隔绝,固定于腹股沟皮下。B组(n=15)是将AV环套入天然珊瑚块的侧槽后,血管环和珊瑚周边均匀喷涂纤维蛋白胶,再用ePTFE膜包裹隔绝,固定于腹股沟皮下。2、4、6周行标本的大体观察和墨汁灌注检查,墨汁灌注后珊瑚脱钙、HE染色、组织学观察,分析珊瑚孔隙中组织结构和血管新生情况,血管密度作定量统计学分析。结果:两组珊瑚植入体内后表面和深部均有大量纤维血管样组织生长。墨汁灌注显示血管广泛分布在珊瑚表层和间隙内,结构成熟,纤维蛋白胶组血管生成更明显,2周已贯穿珊瑚块的全层,胶体已基本吸收。2、4、6周血管密度逐渐加大,2周和4周时,B组较A组血管生成密度有统计学差别(P<0.05),6周无明显差别。结论:纤维蛋白胶复合珊瑚后可能减少了对血管的刺激,早期可促进血管新生,比单纯珊瑚中血管新生能力更强,为优化AV环模型提供了一个途径。
实验五动静脉环法和动静脉束法诱导珊瑚血管新生的机制研究
目的:从形态学上探讨和分析AV环、AV束诱导珊瑚血管新生的机制。方法: A组(n=12)是将新西兰兔腘动脉和股静脉吻合成AV环,环绕套入天然珊瑚块的侧槽中并加ePTFE膜包裹隔绝。B组(n=12)是股动静脉血管束保持血流通畅,不切断,动静脉分开套入珊瑚块的两边侧槽中,外加ePTFE膜环绕包裹隔绝。4、8周行两组标本的珊瑚、血管内膜扫描电镜和血管铸型检查,同时取对侧正常股动静脉内膜作电镜为正常对照,血管铸型标本行大体解剖和腐蚀后血管分布等观察。了解血管与珊瑚的关系,血管内膜的变化,以及血管新生程度和来源。结果:扫描电镜结果显示A组血管化的珊瑚内部和表层有大量的血管结构,伴行在主干动静脉旁偏多,且相互交通,发育成熟;血管内膜凹凸不平,可见内陷发芽孔洞;B组珊瑚内血管明显偏少,主干血管旁也较多;血管内膜无明显变化,排列规则,与正常内膜形态接近。血管铸型显示A组血管环周边和珊瑚表层充满小血管;B组血管明显稀疏。腐蚀铸型显示A组血管环动静脉段芽生和伴行有丰富的小血管,在入口处形成网状结构,并相互吻合交通;B组主干动脉血管无发芽新生血管,仅有周边伴行的部分小血管长入。结论:进一步证实AV环方法和AV束方法均可以促进珊瑚的血管化,前者诱导血管新生的能力更强,两者血管生成的机制完全不同,AV环血管主要是通过血管发芽新生的方式,血管生长效率高,AV束血管主要是伴随性长入,可能是动脉生长的方式。
实验六动静脉环法异位构建血管化组织工程珊瑚骨的初步研究
目的:AV环加珊瑚复合骨形成蛋白(BMP),异位构建出带血管蒂的组织工程珊瑚骨。方法: A组(n=6)同前方法形成兔左侧腘动脉和股静脉吻合的AV环,套入珊瑚块的侧槽中,用ePTFE膜包裹隔绝,关闭伤口,术后4周再解剖开ePTFE膜,珊瑚打孔,表面和中央加入含BMP的纤维蛋白胶,重新封闭固定。B组(n=6):珊瑚中央打孔,表层和孔洞内加入含BMP的生物蛋白胶后直接埋置于大腿肌袋内,关闭伤口,作为对照组。所有标本4周和8周分别经平片和CT、墨汁灌注组织切片,HE染色和三色法等检查,观察珊瑚内成骨情况。结果:两组珊瑚均有一定程度的吸收,A组和B组的放射线检查示珊瑚块有不均匀的密度影,组织学显示A组4周组表层有一定程度的骨和软骨样组织形成,8周骨密度加大,结构更成熟,有的形成板层骨,中央也可见少量骨生成;对照B组组织学显示仅表层有少量骨和软骨样组织形成。结论:AV环珊瑚模型可能存在内生性血管化功能,加入骨诱导因子BMP可促进预血管化的珊瑚成骨,最终异位构建出带蒂组织工程骨。珊瑚的预血管化有一定意义,可作为构建血管化组织工程骨新方法。成骨的生理机制和过程需待进一步研究。纤维蛋白胶可以作为BMP的载体用于骨缺损修复。
One of the major difficulties in bone tissue engineering, particularly in the initial phases of tissue or organ reconstruction, is suboptimal vascularization in the center of large cell-containing constructs. This unreliable oxygen and nutrients supply limits the survival of cells. Several investigators have described some methods of constructs neovascularization. The structures were embedded into rich vascular areas, or implanted vascular bundles. Although some were moderately successful, this technique relied on fluid diffusion (100~300μm) to supply cells and scaffold composites before capillaries growed in. That outer blood supply is termed extrinsic vascularization. An alternative to extrinsic vascularization is an intrinsic vascularization approach, that is, induction of vascularization in biomaterials prior to osteoblast injection. There were two methods currently in use. First, to form an intrinsic circulation through bioreactor in vitro. Second, arteriovenous loop model has been gaining acceptance as a means of initiating and sustaining intrinsic blood supply in tissue engineering constructs in vivo. It first arised from the work of constructing autograft in plastic surgery. The arteriovenous loop(AVL) is thought to be triggering a vivid and rapid angiogenetic response by means of two processes. One is a trauma imposed by the surgical construction of the loop itself with subsequent inflammation. Another is an immense rise in pulsatile shear stress upon the vascular walls, which triggers upregulation of VEGF production from the endothelial cells. New blood sprouts from the main vessels. This model was firstly used in bone tissue engineering in 2006.
In our study, it is the first time to induce coral scaffold intrinsic angiogenesis in an isolated environment using this arteriovenous loop model, and to construct a vascularized tissue engineering bone attached vascular pedicel after delivering osteoinductive molecules (bBMP). Here we present a novel and simple approach: an AV loop formed by rabbit femoral artery and vein, wrapped by ePTFE membrane, could efficiently induce coral angiogenesis. The model was able to fit the complicated clinical demands, and more convenient to further manipulation. In this study, we explored the abilities of inducing coral angiogenesis by three vascular carriers: AVL, AV bundles(flow-through and end-ligated). Further study showed angiogenesis could be improved by fibrin gel adding around the blood vessels. Furthermore, the patterns and mechanisms of newly-formed blood vessels were described through vascular india ink injection, vascular casting and scanning electron microscope. It will be feasible for us to optimize this model of intrinsic vascular supply in bone tissue engineering. There were six experiments in the study.
Experiment 1 Changes of general morphology and compressive strength after natural porites harmless treatment
Objective To test the changes of natural porites general morphology, microstructure and compressive strength after their chemical and physical treatment. Methods The porties were made into oval shape blocks (6×8×10 mm3), and immersed in 5% sodium hypochlorite solution for 2 weeks, then boiled three times with 10 min in each time; and followed by ultrasonication for five times with 10 min in each time. At last, they were dried in the oven (80℃, 24 h) and sterilized under condition of high temperature and high pressure (1.3 MPa, 131℃, 30 min). Evaluation methods included testing the changes of their general morphology, microstructure and compressive strength during the three stages. Results There were no great changes of compressive strength in different stages. After the porites were treated by chemical and physical methods, as well as by sterilization, most of foreign bodies were cleared. The surfaces and interspaces of porites became clean and smooth. Conclusion The foreign bodies of porites surfaces and interspaces could be cleared by chemical and physical methods. There were no great changes about structure and compressive strength during the treatment.
Experiment 2 A preliminary study of inducing coral scaffold angiogenesis by an arteriovenous shunt loop and arteriovenous bundles
Objective To construct an axial vascular tissue-engineering bone scaffold model by two vascular carriers of arteriovenous loop and arteriovenous bundles(no-ligated), and compare their abilities of inducing angiogenesis in coral. Methods 36 adult male New Zealand rabbits were used in this study. In group A (n=18), An arteriovenous loop (AVL) was formed by microsurgically anastomosing at the proximal ends between the femoral popliteal artery and vein, and placed in the circular groove of coral block (6×8×10 mm3). In group B (n=18), a flow-through vessels bundles of both femoral artery and vein were placed in the side groove of coral block. All the implants in two groups were wrapped by a micro-porous expanded-polytetrafluoroethylene (ePTFE) membrane, and fixed subcutaneously in the groin by suturing. Evaluation methods included gross morphological observations, histological examinations and India ink perfusion after 2, 4, 6 weeks. The density of blood vessels was analyzed by SPSS 10.0 statistical soft pocket. Results All the corals were encased by newly formed fibrovascular tissues both in two groups. Ink-stained vessels distributed the surfaces and side grooves, and invaded the interspaces of corals. The degree of vascularization increased over the course of the experiment. Blood vessel density demonstrated a significant, continuous increase between 2 weeks and 6 weeks after implantation in group A. The mean value of blood vessel density in group A was significantly higher than that in group B (P < 0.01). Conclusion A vascularized coral model could be constructed by inserting arteriovenous loop and arteriovenous bundles, the former had more abilities to induce angiogenesis than the latter. The model was expected to be used in vascular bone tissue engineering.
Experiment 3 Comparison of inducing vascularized coral scaffolds by two arteriovenous bundles
Objective To investigate the effects of inducing vascularized natural corals by flow-through and end-ligated arteriovenous bundles. Methods Three months male New Zealand rabbits were used in this study. In group A (n=9), a flow-through vessels between the femoral artery and vein were placed in the side groove of coral. In group B (n=9), a ligated pedicle arteriovenous bundle at the end of femoral artery and vein was placed in the circular groove of coral. The implants were wrapped by an ePTFE membrane and fixed subcutaneously in the groin by suturing. In group C (n=3), the corals were implanted under skin, no vessels were inserted. Evaluation methods included gross morphological observations, histological examination, and quantificational statistics of blood vessel density after 2, 4 and 6 weeks. Results New fibrovascular tissues growed around the surface and deep interspaces of corals, majority of vessels distributed around the coral groove. At 6 week, the coral has been completely encased by new granulation tissue. Histological examination showed new fibrovascular tissue proliferation was evident in group A, and sparse in group B, less in group C. The blood vessel density was higher in group A than that in group B and group C. Conclusion A vascularized coral could also be constructed by inserting arteriovenous bundles. Flow-through vessels had more abilities to induce angiogenesis than an end-ligated vessels and no vessels. The method could also be used in bone tissue engineering.
Experiment 4 A study of fibrin gel stimulating coral scaffold angiogenesis in an arteriovenous loop model
Objective To assess and improve the angiogenic effects of fibrin gel in an arteriovenous loop (AVL) model. Methods An arteriovenous loop (AVL) was formed by microsurgically anastomosing at the proximal ends between the femoral popliteal artery and vein. In group A (n=15), the AVL was directly placed in the circular side groove of coral block. In group B (n=15), after there was injected fibrin gel around the coral and AVL, the AVL vessel was placed in the side groove of coral. All the implants in two groups were wrapped by an ePTFE membrane, and fixed subcutaneously in the groin by suturing. Evaluation methods included gross morphological observations, India ink perfusion and histological examinations after 2, 4, 6 weeks. The density of blood vessels was analyzed by SPSS 10.0 statistical soft pocket. Results All the corals were encased by newly formed fibrovascular tissues both in two groups, which was more evident and earlier in group B than that in group A. Ink-stained vessels distributed the surfaces and side grooves, and invaded the interspaces of corals. The degree of vascularization increased over the course of the experiment. Blood vessel density demonstrated a significant, continuous increase between 2 weeks and 6 weeks after implantation. In 2 and 4 weeks, the mean value of blood vessel density in group B was significantly higher than that in group A (P<0.05). There was no statistically significance in 6 weeks. Conclusion A fibrin gel mixed coral AVL model could be efficiently induced angiogenesis because of its little stimulation to blood vessels. The modified method was expected to be used in this model.
Experiment 5 The mechanisms of inducing coral scaffold angiogenesis by an arteriovenous loop and arteriovenous bundles model
Objective To detect the mechanisms of coral angiogenesis by two vascular carriers of arteriovenous loop and arteriovenous bundle model from morphology. Methods In group A (n=12), An arteriovenous loop (AVL) was formed by microsurgically anastomosing at the proximal ends between the femoral popliteal artery and vein, and placed in the circular side groove of coral block. In group B (n=12), a flow-through vessels bundles both femoral artery and vein were placed in the side groove of coral. After 4 and 8 weeks, the cross-section of coral implant, lumen of AVL vessels and contralateral femoral vessels were scanned with an electron microscopy to explore the newly formed vessels and its sprouting source. At the same time, the rabbits were examinated through abdominal aorta vascular casting. The specimen was corroded and observed under stereoscopy. Results SEM showed, in group A, affluent interconnecting small vessels accompanied with the large vessels both in arterial and venous segments in corals. These small vessels seemed to be mature in structure. ECs aranged randomly and unregularly. Remarkably, the vascular lumen had some minute caves, both in that of artery and vein, which suggested they sprout from the vessels. In group B, vessels lumen ECs were spindle-shaped and arranged regularly, no invaginated lumen existed. Vascular casting showed that in group A, significant blood vessels sprouted from all areas of the loop, especially at the entrance of the AV vascular pedicle where they were mainly dense small tubes and interconnected. In group B, however, there were no blood vessels sprouted from the AV bundles and only some small vessels growing from the entrance and exit. Conclusion A vascularized coral model could be constructed by inserting arteriovenous loop and arteriovenous bundles, the former had more abilities to induce angiogenesis than the latter. The mechanism of vascular regeneration in two groups was completely different. In AVL model, the vascular regeneration sprouted from the large femoral artery and vein (angiogenesis), however, they were no new vessels sprouting from the large vessels in AV bundles, which might be arteriogenesis activities.
Experiment 6 A preliminary study of constructing an ectopia vascularized tissue engineering coral bone by an arteriovenous loop model
Objective To construct an axial vascular ectopia tissue-engineering bone fabricated in coral, sustainedly released bBMP by fibrin gel using an arteriovenous loop model. Methods In group A (n=6), An arteriovenous loop (AVL) was formed, placed in the circular side groove of coral block and wrapped by ePTFE membrane. Four weeks later, the implant was exposed and injected fibrin gel including bBMP, then closed as the manner as the prior. In group B (n=6), the coral block was injected fibrin gel including bBMP, and implanted into thigh muscle pouches, then closed the incision. Evaluation methods included gross morphological observations, X-ray films, CT and histological examinations (HE staining and Masson’s trichrome staining) after 4 and 8 weeks. Results All the corals in both groups were partly absorbed on the surfaces. Radiological examination showed there were uneven X-ray projective image resistance on the coral. Histological examination demonstrated that in group A, a certain degree of bone and cartilage-like tissues were distributed in the surface at 4 week; some lamellar bone formed at 8 week. There were only a small amount of bone and cartilage-like cells on the surface in control group B. Conclusion A prevascularized coral could be induced ectopia osteogenesis by sustainedly released bBMP in fibrin gel using an AVL model. There might exist an intrinsic vascularization in coral. The model was expected to be used in vascular bone tissue engineering. The injectable osteoinductive material with fibrin gel as a carrier compounded with BMP was effective in repairing bone defects.
引文
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