In Vitro Study On The Angiogenic Function Of Degradable Tissue Engineering Scaffolds And The Establishment Of The Characterization Method

The work of this paper originates from the NSFC funded program, named "the study on the effect of interaction between degradable biomaterials and cells on function protein expression of EC and its mechanism (No.30370411)".Biodegradability is the basic characteristic of ideal tissue engineering (TE) scaffold. In order to match the tissue growth, it's necessary to learn about the biological process of degradable scaffold after implantation, and to elucidate the interaction between scaffold degradation and cell growth. However, it's an intersection between materials science and life science, and remains a difficulty in biomaterials research. On the other hand, angiogenesis has emerged as a main obstacle of tissue engineering success. Therefore, it's a big challenge to investigate the influence of biomaterials on angiogenesis, and to design or develop scaffold with aniogenic function for tissue engineering application. It has been proved that the 3-D structure and the surface properties of biomaterials can directly exert influences on angiogenesis during the tissue regeneration. However, little research has been down in whether and how the degradation product of TE scaffold affect the angiogenesis process.Aiming at the problem described above, and using the methods in medicine for reference, the present study has established a characterization system for angiogenic function of degradation products of TE scaffold. Using the system, the angiogenic function of degradation products were studied. Furthermore, how the degradation time and the compositions in degradation products affect the angiogenesis were discussed, according to the results of degradation products and degradation rule analysis. In addition, the effects of modification on the behavior of EC at the material/cell interface were also studied.1. A characterization system for angiogenic function of degradation products of TE scaffold has been established.According to the angiogenesis process and its molecular regulation mechanism, endothelial cells (EC) behaviors, including proliferation, migration and tube-like structure formation (TLS) were selected as cellular parameters of angiogenic characterization. In addition, the mRNA expression of vascular growth factor (VEGF) and metalloproteinase-2(MMP-2) and the reorganization of F-actin were selected and molecular parameters. Furthermore, cell migration quantitative method (CMQM) and TLS quantitative method (TLSQM) have been newly established in this paper. At the same time, MTT assay, real-time RT-PCR and phalloidin label were used to determine cell proliferation, mRNA expression and F-actin reorganization respectively.2. Angiogenic function of 3 kinds of TE biomaterials has been studied.With polylactic acid (PLA), chitosan (CS) and calcium polyphosphate (CPP) as model material respectively, and with the physiological saline as degradation medium and negative control, angiogenic function of three kinds of materials, involving the naturally occurring and synthetic polymers along with the bioceramics were investigated using the system. It indicated that the degradation products could significantly improve or restrain the angiogenesis depending on the kind of materials and the degradation time. Moreover, results at cellular level were consistent with those at molecular level. In addition, the results of CPP implantation showed that the results in vivo experiment was also consistent with those of in vitro experiments. However, the effects of degradation time and the role of monomer of 3 kinds of materials in angiogenic function were different. The results are as follows:(1) Angiogenic function of naturally occurred CSEffects of degradation time. In 120days, the effects of degradation products on angiogenic function various with time. It was not during the middle period but the latter period that cell behaviors were restrained. It suggested that the key reason for angiogenic function is not the simple accumulation of degradation products, but their molecular structure.The effect of glucosamine(GS). In 120 days, the concentration of GS in CS degradation fluid is between 0.001mmol/L and 0.37mmol/L. At this concentration, the single GS solution showed no obvious effects on angiogenesis. It suggested that the key factor in CS degradation that exerted effects on angiogenesis was not the GS monomer but its oligomer or the co-effects of GS and its oligomer.(2) The angiogenic function of synthetic polylactic acid (PLA)Effects of degradation time. At beginning of degradation (before 7 days), the degradation products showed no negative effects on the proliferation, migration and TLS formation of EC. Nevertheless, with the prolonged degradation time and the accumulation of the degradation products, the migration and TLS formation were significantly decreased since 30days, and the proliferation was dramatically reduced since 90days.The effects of lactic acid (LA). In 120days, the LA concentration in PLA degradation fluid was from 16.13mmol/L to 431.32mmol/L. At the concentration, the single LA solution restrained the EC behaviors evidently. Whereas, the proliferation of EC wasn't retrained until the LA concentration in degradation fluid amount to 190mmol/L. It suggested that the LA oligomer in degradation fluid might reduce the cytotoxicity of LA monomer.(3)Angiogenic function of strontium-doped calcium polyphosphate (SCPP).Effect of degradation time. Compared with the physiological saline and calcium polyphosphate, almost all the degradation products of SCPP in 90 days promoted the EC growth.Effects of strontium (Sr). although the Sr doesn't affect the EC migration, Sr (10nmol/L～1mmol/L) could improve the proliferation and TLS formation. The concentration of Sr in SCPP degradation fluid is 3.92μmol/L～20.65μmol/L, and can significantly accelerate angiogenesis.3. Effects of materials modification on cell behaviors at the material/cell interfaces have been studied.CS,PLS and CPP were modified respectively. The effects of modification on EC behaviors have been studied. The results showed that there's no negative effect of modification on cytotoxicity. However, the EC function, including attachment, spreading and TLS formation were evidently changed. The results are as follows:(1) Effects of sodium alginate (ALG) modification on EC behaviors on CS surface.Effects on surface properties. After modification, the surface roughness was evidently decreased, and the hydrophilicity was significantly improved.Effects on EC behavior. Before modification, EC on CS surface spontaneously formed into TLS, which was similar to the TLS on collagen gel. However, this phenomenon disappeared, and the attachment and spreading of EC was sharply down-regulated. It may result from the improved hydrophilicity and the decreased roughness.(2) Effects of hosphorylcholine (PC) modification on EC behaviors on PLA surface.Effects on surface properties. PC modification increased the hydrophilicity of PLA. After modified, the PC groupoverturn from the bulk to the surface of PLA, which lead to the surface reconstruction.Effects on EC behaviors. Compared with PLA, the modified one, whose PC/LLA ratio is 1/46 and 1/30, delayed the attachment and spreading of EC. However, after cultured for longer time, EC adhered to the surface and could fully spread, and finally proliferated. The improved hydrophilicity may account for the delayed attachment, and the surface reconstruct may lead to the final spreading and proliferation of EC.(3) Effect of Sr addition on EC behaviors on CPP surface.Effects on surface properties. Microstructure of CPP was evidently changed by Sr addition. The crystalline grain were enlarged and closely connected, resulting in a smoother surface. And the gel-like floc on the surface of CPP was distinctly decreased.Effects on EC behaviors. After Sr addition, EC closely attached to the surface of scaffold and fully spread, resulted in EC monolayer wrapping the surface of scaffold. The decreased roughness on micrometer scale and the decreased CPP gel on the scaffold surface may account for the improved EC attachment and spreading.A system for angiogenic function characterization of degradation products of TE scaffolds was initially established in the present study. It may provide a new aspect and method reference to the function evaluation of TE scaffold. According to the results of angiogenic function study on 3 kinds of biomaterials for TE, the degradation products could alter the microinviroment of EC, subsequently alter the angiogenic behaviors and growth factor and proteinase expression. It suggests that, to meet the angiogenesis need in TE, it's necessary to take the degradation products of biomaterials in to account and to evaluate the effects on angiogenesis. At the same time, this may propose a new non-growth factor way to solve angiogenesis in TE.