Study Of Constructing A Vascularized Tissue Engineering Bone By An Arteriovenous Loop Model

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 treatmentObjective 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 bundlesObjective 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 bundlesObjective 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 modelObjective 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 modelObjective 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 modelObjective 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.