摘要
本论文课题来源为国家自然科学基金项目“可降解生物材料与内皮细胞界面反应对内皮细胞功能蛋白表达的影响及机理研究”(No．30370411)。
理想的组织工程材料应具有可控的生物降解性。要解决组织生长和材料降解的匹配问题，必须了解材料在使用中的生物学过程，并阐明材料降解与细胞生长和组织构建的相互作用机制。这是一个材料学与生命科学交叉的课题，也是生物材料研究中的难点。血管化问题是组织工程取得成功的关键环节，研究生物材料对组织血管化过程的影响和机理，设计和开发促血管化的支架材料是组织工程对生物材料的挑战。已有研究表明，材料的三维结构和表面性质可显著影响血管形成，但材料降解产物会不会影响以及如何影响血管形成目前尚不清楚。
本论文针对以上问题，借鉴医学领域的研究方法，建立了组织工程材料降解产物血管化功能的表征体系。在此基础上，研究了组织工程材料降解产物的血管化功能，结合对材料降解产物的表征和降解规律的研究，探讨了降解时间及降解产物组分与血管化功能之间的关系。同时还研究了改性对材料／细胞界面上内皮细胞行为的影响。
1．建立了组织工程材料降解产物血管化功能表征体系。
依据血管形成过程及其分子调节机制，选择内皮细胞形成血管的三大步骤(包括增殖、迁移和血管样结构TLS的形成)为细胞水平指标，调节血管形成的VEGF、MMP-2的基因表达和F-actin重组作为分子水平指标，建立了组织工程材料降解产物血管化功能细胞水平和分子水平的的表征体系。新建了细胞迁移定量检测方法CMQM；新建了TLS的定量检测方法TLSQM；选择MTT比色法检测细胞增殖，实时荧光定量RT-PCR技术检测VEGF和MMP-2 mRNA的表达；BODIPYFL标记的鬼笔环肽标记F-actin，在荧光显微镜下观察其组装情况。
2．研究了三类组织工程材料降解产物的血管化功能。
分别以壳聚糖、聚乳酸和掺锶聚磷酸钙为例，以生理盐水为降解介质和对照，采用上述表征体系对天然高分子、合成高分子及生物陶瓷三大类组织工程材料降解产物的血管化功能进行了研究。结果表明，三类材料在生理盐水中均可降解，降解产物随时问累积，改变细胞生长的微环境，并对内皮细胞形成血管的重要步骤和调节血管形成的功能蛋白表达以及细胞骨架重构都有显著影响：可抑制也可促进血管化，取决于材料的种类和降解时间。且细胞水平和分子水平结果基本一致(掺锶聚磷酸钙的研究还表明体内动物实验和体外表征结果一致)。但降解时间的影响以及降解产物中单体所起的作用都各有不同。分述如下：
(1)天然高分子壳聚糖(CS)降解产物的血管化功能
降解时间的影响：在120天的降解时间内，细胞反应受到抑制不是在降解后期，而是在降解中期(20天～60天)。提示，天然高分子材料CS降解时间的延长和降解产物的累积不是影响内皮细胞行为的决定因素。不同时期产生的降解产物可能具有不同的分子结构，从而具有不同的生物活性。
氨基葡萄糖(GS)单体的作用：在120天的降解时间内，CS降解液中GS浓度介于0.05～0.37mmol／L范围内。该浓度下，单纯的GS对内皮细胞增殖和迁移无显著影响，对TLS的作用效果也不同于降解液。提示，CS的降解产物中，影响内皮细胞行为的主要因素不是GS单体及其量的累积，而可能是GS齐聚物或与GS单体的共同作用。
(2)合成高分子聚乳酸(PLA)降解产物的血管化功能
降解时间的影响：降解初期(前7天)，对细胞增殖、迁移和TLS的形成均无不良影响，或有一定促进。但随着降解时间的延长，细胞行为受到抑制：降解30天后显著抑制细胞迁移和TLS形成，降解90天显著抑制细胞增殖。提示，对合成材料PLA，适量的降解产物有促血管化功能，而过量累积则会严重破坏细胞生态环境，抑制其生长。
乳酸(LA)单体的作用：120天的降解时间内，PLA降解液中LA单体浓度介于(16.13～431.32)mmol／L之间，碱水解后LA总浓度介于(63.91～799.91)mmol／L之间。该浓度下单纯的LA已显著抑制细胞行为，但PLA降解液中LA浓度达到190mmol／L时才对细胞增殖有显著抑制。提示，乳酸齐聚物的存在可降低高浓度单分子乳酸对细胞的毒性作用。
(3)生物陶瓷掺锶聚磷酸钙(SCPP)的血管化功能
降解时间的影响：与生理盐水和聚磷酸钙(CPP)比较，SCPP90天的降解产物对内皮细胞行为几乎都有促进作用。
锶元素的作用：单纯的锶元素对细胞迁移无明显影响，但浓度在10nmol／L～1mmol／L范围内可不同程度地提高细胞增殖和形成TLS能力。SCPP90天降解产物中锶元素浓度介于3.92μmol／L～20.65μmol／L之间，对血管化有明显的促进作用。
3．组织工程材料改性对材料／细胞界面上内皮细胞行为的影响
对本论文重点研究的三种生物材料(CS、PLA以及CPP)分别进行改性，研究改性前后材料的细胞毒性和材料／细胞界面反应对内皮细胞行为的影响，结合改性前后材料性质变化探讨了改性对细胞行为影响的机理。结果发现，改性对三种材料的细胞毒性均无不良影响，却显著改变了细胞在材料表面的粘附、铺展以及形成TLS等行为。分述如下：
(1)海藻酸钠(ALG)改性对内皮细胞在CS表面行为的影响：
改性对CS表面性质的影响：AFM和水接触角实验结果表明，海藻酸钠改性能显著降低CS的表面粗糙度，增加表面亲水性；
改性对细胞行为的影响：细胞在CS表面形成TLS结构，与胶原凝胶表面的TLS类似；但改性后的表面具有抗内皮细胞粘附的性能。材料表面亲水性的增加和粗糙度的下降可能导致细胞粘附下降的原因。
(1)磷脂胆碱改性对PLA表面内皮细胞行为的影响：
改性对PLA表面性质的影响：水接触角和摄水率结果表明，磷脂胆碱改性能提高聚乳酸的亲水性，且随着磷脂含量的增加，亲水性增强；XPS分析表明，改性后的材料在水环境发生表面重构，磷脂胆碱基团从材料的本体向表面发生翻转。
改性对细胞行为的影响：与改性前相比，PC／LLA比例为1／46和1／30的材料表面，细胞的粘附和铺展相对滞后，但继续培养，细胞能在材料上完全粘附和铺展，并且增殖。细胞初期粘附铺展滞后可能是材料亲水性增强造成的。而具有生物活性的磷脂官能团向材料表面翻转使材料表面发生重构，可能是使细胞最终实现粘附并增殖的原因。
(3)掺锶改性对CPP表面内皮细胞行为的影响：
改性对CPP表面性质的影响：掺锶能显著影响CPP的微观结构。掺锶后材料晶粒尺寸增大，晶粒间连接紧密，形成更平滑的表面；掺锶能显著减少CPP经培养液浸泡后表面形成的凝胶状物质。
改性对细胞行为的影响：掺锶改性能显著促进ECV304细胞在CPP表面的粘附和铺展，其原因可能是掺锶减少CPP凝胶并降低其表面粗糙度。
本论文首次建立了组织工程材料降解产物血管化功能表征体系，为组织工程材料功能性的表征和评价提供新的思路和方法参考。对三类生物材料的表征发现，材料降解产物可改变细胞的微生态环境，进而显著改变内皮细胞形成血管的关键步骤以及调节血管形成的生长因子和蛋白酶的基因表达。该结果提示要解决组织工程中的血管化问题，必须重视材料降解产物对血管化的影响。对组织工程材料不仅要评价其安全性，还应深入研究其血管化功能。本论文的研究也为解决组织工程血管化问题探索了从非生长因子角度促血管化的可能性。
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 CS
Effects 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.
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