Research On The Regularity Of Vascularization In The Controllable Spherical Porous β-TCP Scaffold

Rapid and effective revascularization is the prerequisite for constructing large tissue-engineered bones. Newborn vessels in the scaffold play several crucial roles: Firstly, vessels can supply cells with nutrient and oxygen for survival in the deep region of the scaffold. Secondly, they are able to transport growth factors, calcium ions and so on into the inner part of the scaffold, to stimulate undifferentiated mesenchymal cells to osteoblasts and to influence new bone formation indirectly. Lastly, it have been reported that the blood flow could regulate the mineral deposition. Tissue-engineered bones are still small ones for vascularization deficiency and unable to meet clinical demands satisfactorily. Therefore, it is of great importance to research on vascularization of scaffold.The researches on the vascularization of tissue-engineered bone have laid emphases on two aspects until now: One is to exert external influences on the scaffold, such as combination with endothelial cells, growth factors or revascularization with microsurgerical methods. The other is to study the internal characteristics of the scaffolds, soughting for the optimal architecture to promote the generation of new vessels. However, the regularity of vascularization within the scaffold remained unknown, especially lacking the concrete quantitative data. Meanwhile, most of scaffolds in previous studies were round lamellar, cuboid or granulo, with irregular external shapes and internal structures, whose pore size varied largely and the interconnection pores were small, therefore leading to the anisotropy in tissue penetration and variability in the results of different section planes observed. So the discrepancies of conclusions were found in the end. The aim of this project were to investigate the regularity of vascularization influenced by 3-dimensional porous architectures and to get the concrete parameters of vascularization, such as the rate, number, diameter and area of regenerating vessels. This will make for the combination of two aspects before-mentioned, such as the improvement of scaffold's architectures and the optimization of the timings or quantities of seeding cells transplanted into scaffolds. It's one of new ideas to obtain the quantitative data of vascularization within scaffolds. Materials of this project were controllable spherical porousβ-TCP scaffolds. Spheres were regular objects without anisotropy so that the tissue penetration and observation of the results were not influenced by directions. The inner microarchitecture of the scaffold was completely controllable, which had identical global pores, smooth pore walls and more than 99% pore connection rate. The size of pores and interconnection pores was defined to an optimal range obtained in the previous comparative studies by our research group. The design and parameters of the spherical scaffold were innovative that haven't been used in other researches. Concerning of methods, it is also another new idea that collecting images on undecalcificated hard sections and then representing 3-dimensional parameters with 2-dimensional data based on the concept of fraction geometry. This method decreased the possible errors in the process of histomorphometry.This project was made up of two parts. In the first part, we qualitatively observed the biological response to the spherical porousβ-TCP scaffold implanted in vivo, in order to know whether this biomaterial would be fit for the research on vascularization in the second part. Methods: Samples were harvested at 1,2,4,8 and 12 weeks postoperatively and observed by HE stain and ponceau red stain on decalcificated paraffin sections, Van Gieson's picric-fuchsin stain on undecalcificated sections and scanning electron microscope (SEM). Result: Cells penetrated into pores 1w postoperatively. Collagen fibers and immature vessel buds were found on the 2nd week and the numbers of them increased highly in 4w. Crescent-shaped neo-bones and osteoids appeared in the peripheral areas of pores in 8w. We detected mature vasculatues and bones in 12 weeks, and the degradation of materials as well.Summary: The spherical porous ?-TCP scaffold demonstrated a good biological performance of cell penetration, vascularization and osteogenesis and was suitable for the research of vascularization. The process of osteogenesis in it was membrane bone formation and the diameter of vascularization was restricted by the interconnection between pores. In the second part, the quantitative analysis of the regularity of vascularization was applied to the same scaffold used in the first part. Spherical porous ?-TCP scaffolds with the same architecture to the first experiment were harvested at 1,2,4,8 and 12 weeks after embedded into muscle-fascia lumbodorsalis pouches in each rabbit separately, and then Van Gieson's stain was made on the undecalcificated sections through the centre of scaffold. Four fields of view on perpendicular directions with a random starting point were chosen in the low power lens (magnification×16). Then 4 successive images in each field mentioned above were gathered in high power lens (magnification×50) for quantificational analysis. The parameters related to blood vessel, such as number, diameter, the depth of tissue penetration(PTP) and the relative vessel area percentage (RVP), were measured in each image and the mean values of those parameters gathered by 16 views represented the corresponding ones for each section. Finally the results were statistically analyzed.Results: Immature vascular buds were seen on the 2nd week. The first vascularization peak could be observed in 4 weeks, characterized by the increase of the number of new blood vessels(P<0.05). In 8w the reforming of vasculature happened with the enlargement of the caliber of new vessels and the increase of the number of vessels (P>0.05). In 12w, the second vascularization peak appeared that the diameter of new vessels increased (P<0.05) and mature vessels had thick walls and little branches, while the numbers of vessels remained stable (P>0.05). Summary: The vascularization in this specific scaffold showed a two-hump mode. The process of vascularization comprised two phases, which were the rapid increase of the number of immature small vessels with a constant diameter of 50~70μm within 4 weeks and the significant enlargement of vessel's caliber to even more than 200μm without noticable change of the number in 12 weeks. The sense of"two-hump"mode of vascularization was that lots of vascular endothelial cells or precursor cells should be combinated to the tissue-engineered bone in the early period and capable of surviving untill 4 weeks in order to generate large amounts of vessel buds. On the other hand, the scaffold should be able to biodegrade appropriately with the increase of the pore and interconnection pore size to accommodate the enlargement of the vessels in 12 weeks.Conclusion: The controllable spherical porous ?-TCP scaffold used in this research demonstrated a good biological performance and was suitable for the research of vascularization. Qualitatively and quantitatively analyses showed a two-hump mode of vascularization in the scaffold. The process of vascularization comprised of two phases: One was the rapid increase of the number of immature small vessels with a constant diameter of 50~70μm within 4 weeks ; the other was the moulding and reforming of vessels in 12 weeks that the caliber increased significantly without noticable change of the number.