静电纺丝制备小直径血管支架及其血液相容性的研究

以甲酸为溶剂,将具有特定信号识别功能的RGD重组蛛丝蛋白(pNSR32)与聚己内酯(PCL)和壳聚糖(CS)共混,采用静电纺丝技术制备复合纳米纤维小直径血管支架,扫描电子显微镜(SEM)观察支架材料的微观结构,并通过溶血率、动态凝血时间、血小板黏附以及复钙化凝血时间实验评价支架材料的血液相容性.研究表明,(1) 所制备的pNSR32/PCL/CS复合纳米纤维支架内径为4mm,长度可达8cm,并且纤维连续,孔隙相通,孔隙率达85%以上,支架具有三维多孔网状结构,有利于种子细胞在支架上的黏附、增殖和迁移;(2)复合支架材料的溶血率＜5%,黏附的血小板少且不变形,复钙化凝血时间达293s,与单独PCL相比延长94s,说明该支架材料具有良好的血液相容性,有望成为一种新型的小直径组织工程血管支架.     

静电纺丝制备聚己内酯/壳聚糖复合纤维膜

采用静电纺丝技术制备聚己内酯(PCL)/壳聚糖(CS)复合纤维膜,利用扫描电子显微镜(SEM)观察材料配比、纺丝电压和接收距离对复合纤维形态的影响,利用接触角测试仪研究CS含量对复合纤维膜亲水性能的影响。结果表明:CS的加入,有利于提高PCL/CS混合溶液的成纤能力;随着纺丝电压增加和收集距离减小,复合纤维平均直径减小,且直径分布均匀性提高。与PCL纤维相比,PCL/CS复合纤维亲水性得到了较大的提高,使其有望在组织工程中得到潜在的应用。     

双层蛛丝蛋白血管支架的体外降解研究

血管支架的制备成为治疗心血管疾病的必需条件。应用静电纺丝技术制备了(pNSR16/PCL/CS)/(pNSR16/PCL/Gt)双层血管支架,并研究其在磷酸盐缓冲液(pH值=7.4)和多酶降解液中浸泡不同时间的体外降解情况。于2、4、8、12周分别取材,测试失重率、吸水率、降解液的pH值、力学性能和分子量的变化,并进行扫描电镜观察。结果表明,(pNSR16/PCL/CS)/(pNSR16/PCL/Gt)双层血管支架的初始抗弯强度和初始分子量大于(PCL/CS)/(PCL/Gt)支架,并且前者的降解速度、分子量和失重率减少速度快于后者,pNSR16的添加促进了血管支架的降解。在降解过程中,浸泡液pH值呈弱酸性和中性,前期迅速降低并在后期呈现稳定的趋势。支架在酶解液中的降解速度快于在水解液中。     

静电纺丝制备小直径血管支架

静电纺丝技术是一种简便易行的新型小直径血管支架制备方法,能够形成与天然血管细胞外基质类似的纳米级三维结构———仿生天然血管细胞外基质,促进细胞黏附和增殖。介绍了静电纺丝技术制备小直径血管支架的原理以及对采用不同材料(生物高分子材料、可降解人工合成材料及复合材料)静电纺丝制备的小直径血管支架的理化性能和生物学特征进行了描述与比较,指出了静电纺丝技术制备小直径血管支架研究的热点与不足。     

壳聚糖/羟基磷灰石表面修饰聚己内酯多孔骨支架的制备及性能

采用选择性激光烧结技术构建多孔聚己内酯(PCL)骨支架,用原位合成的方法制得壳聚糖/羟基磷灰石(CS/HA)悬浮液,并采用真空浸泡、低速离心和冷冻凝胶的方法使CS/HA黏附在PCL支架的表面,以改善骨支架的生物相容性和细胞增殖活性。通过X射线衍射(XRD)和扫描电子显微镜(SEM)观测复合支架的物相和形貌,测量支架的压缩强度和杨氏模量,测量支架表面的水接触角,并通过体外细胞实验研究复合支架的生物学性能。实验结果表明,原位合成的方法制得了羟基磷灰石(HA);CS/HA凝胶与PCL骨支架表面黏附良好;CS/HA改善了PCL支架表面的亲水性,提升了骨支架的生物相容性和细胞增殖活性。     

皮/芯结构丝素/聚己内酯超细纤维的包覆率调控及性能

本实验分别采用六氟异丙醇(HFIP)、HFIP-甲酸(体积比 8∶2)以及HFIP-二氯甲烷(体积比 8∶2)作为聚己内酯(PCL)的溶剂,采用HFIP作为丝素(SF)的溶剂,利用同轴静电纺丝技术制备皮/芯结构的SF/PCL(C-SF/PCL)纤维。透射电子显微镜的测试结果显示,所制备的纤维网中具有C-SF/PCL纤维。X射线能谱仪的分析结果表明,当芯层PCL采用HFIP-二氯甲烷(体积比 8∶2)作为溶剂时,纤维网中含有较多的C-SF/PCL纤维。拉伸测试结果表明,C-SF/PCL纤维网的拉伸断裂强度和断裂伸长率都较纯SF纤维网有明显提高。以纤维网作为支架培养EA.hy926细胞,结果表明,C-SF/PCL纤维网和SF纤维网相似,细胞都能良好黏附和增殖。以上研究结果说明,C-SF/PCL纤维网可能有潜力应用于血管组织工程支架。     

有序PCL支架的制备与对细胞的定向生长作用

利用静电纺丝技术制备了有序和无序聚己内酯(PCL)纤维支架。通过扫描电镜(SEM)、X射线衍射(XRD)和万能试验机对支架结构与形态、结晶性能及力学性能进行了表征;将体外培养的细胞接种至支架表面,用激光共聚焦显微镜(LSCM)和SEM观察了细胞在支架表面的生长情况。研究结果表明,电纺丝得到的纤维直径均一,有序化纤维支架中86.14%的纤维沿着确定方向有序排列;有序纤维拉伸强度为(27.63±0.75)MPa,比无序纤维的拉伸强度增大了232%。弹性模量基本不变,断裂伸长率明显降低;有序纤维对细胞的定向生长具有良好的引导作用。     

激光熔融静电纺丝法制备PLLA/PCL/nHA复合纤维支架及细胞相容性评价

利用激光熔融静电纺丝技术制备了PLLA/PCL及PLLA/PCL/nHA复合纤维,热压后形成层压复合纤维支架。利用扫描电镜对纤维支架进行了表征,同时对其进行了亲水性的测试,最后通过倒置荧光显微镜和MTT实验对复合纤维支架的细胞相容性进行了评价。研究结果表明,层压复合纤维支架的直径和孔结构具有多样性,nHA能够提高PLLA/PCL层压纤维支架的亲水性,改善支架的细胞相容性,增加细胞的附着能力,提高细胞的存活率。     

磷酸钙相组成对类骨单位复合骨支架的影响

从组成上看, 自然骨是一种无机与有机的复合材料; 从结构上看, 致密骨的基本结构单位为内壁血管化的骨单位。本研究基于组成与结构仿生的原理制备组织工程化支架, 模拟具有复杂结构的密质骨的基本单位——骨单位。为此, 通过静电纺丝和双螺杆挤出相结合的两步制造法, 制备一种具有双层结构的聚己内酯/磷酸钙(PCL/CaP)复合骨支架, 其内层是由电纺纳米纤维组成的空心管, 可贴附内皮细胞层形成与哈佛氏管相类似的结构; 其外层是具有高孔隙率的螺旋状PCL/CaP微丝, 可复合前成骨细胞以模拟骨单位结构中的外层骨样组织。为进一步探索材料组成对于支架生物功能的影响, 分别设计了外层为PCL, PCL/双相磷酸钙(PCL/BCP)和PCL/β-磷酸三钙(PCL/β-TCP)的复合支架, 比较了材料组分变化对外层微丝结构及前成骨细胞(MC3T3-E1)活性的影响。相比于PCL和PCL/β-TCP, PCL/BCP微丝更能显著增强细胞的生长和钙的沉积, 并成功获得可精确调控不同细胞的空间分布的双层复合支架, 实现对复杂结构骨单位的模拟构建, 显示出很好的应用前景。     

壳聚糖/聚氧乙烯复合纺丝液性能对静电纺丝的影响

为了研究壳聚糖/聚氧乙烯复合纺丝液性能对静电纺丝的影响,利用质量分数为3％的壳聚糖(CS)与聚氧乙烯(PEO)以不同的质量比溶解在浓度为50％的冰乙酸水溶液中制备了CS/PEO复合纺丝液,采用静电纺丝技术制备了CS/PEO复合纳米纤维.用扫描电子显微镜(SEM)对制备出的CS/PEO复合纳米纤维进行表征,并测试了CS/PEO复合纺丝液的溶液性能.从复合纺丝液性能对静电纺纤维成型的影响机理角度对实验结果进行了分析.分析结果表明,在其他静电纺丝参数一定时,纺丝液黏度影响射流的稳定性,从而影响纤维的形貌和直径.只要纺丝液电导率在合适的范围内,对静电纺的影响不大.从泰勒的临界公式中得出了纺丝液临界电压与纺丝液表面张力最佳值的一一对应关系,并与本实验中的实验数据相吻合.     

浸没凝胶相分离法制备聚己内酯多孔支架

为了制备结构和性能满足骨组织工程支架要求的聚己内酯(PCL)多孔支架材料,采用浸没凝胶相分离法,以冰醋酸和丙酮为混合溶剂,水为凝固剂,壳聚糖(CS)颗粒为添加剂制得一系列PCL多孔支架。探讨了溶剂组成、PCL浓度、CS添加量对PCL多孔支架结构和性能的影响。结果表明:添加CS颗粒有利于形成多孔三维支架,随着CS含量的增加,孔隙率略微下降,抗压强度提高。随着PCL质量分数的增加,孔隙率明显下降,但抗压强度增大。当溶剂组成中丙酮含量为50 wt%~60 wt%,PCL质量分数不高于10 wt%时,通过改变CS用量,可制得孔隙率和力学性能满足骨组织工程要求的相互贯通的三维多孔支架材料。     

Preparation and characterization of nano-hydroxyapatite/polymer composite scaffolds

Polycaprolactone/chitosan (PCL/CS) porous composite scaffolds were prepared by solution phase separation method, and the scaffolds were further enhanced by filling with nano-hydroxyapatite/polyvinyl alcohol (n-HA/PVA) composite slurry to prepare n-HA-PVA/PCL-CS composite porous scaffolds through slurry centrifugal filling technique. The morphology, microstructure, component, porosity and mechanical property of the scaffolds were characterized using scanning electron microscope, X-ray diffraction, Fourier transform infrared spectroscope, elemental analyzer and material test machine. The results show that PCL/CS scaffolds have mutual transfixion porous structure just like honeycombs. The porosity of the scaffolds can achieve 60-80%. As the content of CS increases, the porosity increases while the compressive strength decreases. After filled with HA/PVA composite slurry, the porosity of n-HA/PCL-CS composite scaffolds decreases, but still greater than 60%, while the compression modulus can increase to 25.7 MPa.     

壳聚糖/聚己内酯-聚乳酸多孔支架制备和表征

为调控骨组织工程支架的力学性能和降解性能,采用相分离方法,以冰醋酸-水为共溶剂配制聚合物溶液,以NaOH溶液为凝固剂,以CS为添加剂制备壳聚糖(CS)/聚己内酯(PCL)-聚乳酸(PLA)三维多孔支架,研究了聚合物质量比对支架结构、形貌、孔隙率、力学性能和降解性能的影响。实验结果表明,CS和基体存在相互作用,CS有利于形成三维相互贯通的微孔结构,但CS的存在会使基体中各组分的熔点降低。随着PCL和PLA用量比例的改变,孔径范围和微孔形貌发生了一系列的变化。当PCL∶PLA为2∶4和3∶3时,所制备的支架孔隙率均大于90%,当进一步增大PCL质量比时,孔隙率迅速下降。抗压测试表明,所制备的支架弹性模量为0.8～8.0 MPa。降解性能分析表明,4周以后,当PCL∶PLA为3∶3时,质量损失率最大,达到5.94%。该分析表明采用相分离法,通过调节PCL和PLA的质量比可制备形貌、孔隙率、降解速率和力学性能满足要求的三维多孔支架材料,有望应用在软骨组织工程上。     

A comparative study of the chondrogenic potential between synthetic and natural scaffolds in an in vivo bioreactor

Abstract

The clinical demand for cartilage tissue engineering is potentially large for reconstruction defects resulting from congenital deformities or degenerative disease due to limited donor sites for autologous tissue and donor site morbidities. Cartilage tissue engineering has been successfully applied to the medical field: a scaffold pre-cultured with chondrocytes was used prior to implantation in an animal model. We have developed a surgical approach in which tissues are engineered by implantation with a vascular pedicle as an in vivo bioreactor in bone and adipose tissue engineering. Collagen type II, chitosan, poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) were four commonly applied scaffolds in cartilage tissue engineering. To expand the application of the same animal model in cartilage tissue engineering, these four scaffolds were selected and compared for their ability to generate cartilage with chondrocytes in the same model with an in vivo bioreactor. Gene expression and immunohistochemistry staining methods were used to evaluate the chondrogenesis and osteogenesis of specimens. The result showed that the PLGA and PCL scaffolds exhibited better chondrogenesis than chitosan and type II collagen in the in vivo bioreactor. Among these four scaffolds, the PCL scaffold presented the most significant result of chondrogenesis embedded around the vascular pedicle in the long-term culture incubation phase.     

Characterisation of blends between poly(ε-caprolactone) and polysaccharides for tissue engineering applications

In this work, bioartificial binary blends between poly(ε-caprolactone) (PCL) and a polysaccharide (chitosan (CS) or starch (S)) with different contents of the natural polymer (5–30 wt.%) were produced. Melt-mixing and double-precipitation were the methods used for the obtainment of PCL/S and PCL/CS blends, respectively. Tubular scaffolds were produced from bioartificial blends by melt-extrusion. Physico-chemical characterisation was performed by differential scanning calorimetry analysis (DSC), thermogravimetry (TGA), scanning electron microscopy (SEM), infrared analysis (FTIR-ATR and micro-ATR mapping), atomic force microscopy (AFM) and stress–strain tests. Blends were not miscible, phase-separated systems, showing a homogeneous composition and morphology only at low polysaccharide content (≤ 10 wt.%). The biocompatibility of bioartificial guides was investigated by culturing NIH-3T3 mouse fibroblasts. Cells response showed the following order: PCL/S > PCL > PCL/CS. For each blend type, biocompatibility increased with decreasing the polysaccharide content. In vitro cell tests using S5Y5 neuroblastoma cells, carried out on the most biocompatible blends, assessed their absence of cytotoxicity towards these model cells of the nervous tissue. Results showed that blends with a low chitosan or starch content (≤ 10 wt.%) are promising for the regeneration of tissues requiring tubular scaffolds, such as the peripheral nerves.     

Hardystonite improves biocompatibility and strength of electrospun polycaprolactone nanofibers over hydroxyapatite: A comparative study

The aim of this study was to compare physico-chemical and biological properties of hydroxyapatite (HA) and hardystonite (HS) based composite scaffolds. Hardystonite (Ca₂ZnSi₂O₇) powders were synthesized by a sol–gel method while polycaprolactone–hardystonite (PCL–HS) and polycaprolactone–hydroxyapatite (PCL–HA) were fabricated in nanofibrous form by electrospinning. The physico-chemical and biological properties such as tensile strength, cell proliferation, cell infiltration and alkaline phosphatase activity were determined on both kinds of scaffolds. We found that PCL–HS scaffolds had better mechanical strength compared to PCL–HA scaffolds. Addition of HA and HS particles to PCL did not show any inhibitory effect on blood biocompatibility of scaffolds when assessed by hemolysis assay. The in vitro cellular behavior was evaluated by growing murine adipose-tissue-derived stem cells (mE-ASCs) over the scaffolds. Enhanced cell proliferation and improved cellular infiltrations on PCL–HS scaffolds were observed when compared to HA containing scaffolds. PCL–HS scaffolds exhibited a significant increase in alkaline phosphatase (ALP) activity and better mineralization of the matrix in comparison to PCL–HA scaffolds. These results clearly demonstrate the stimulatory role of Zn and Si present in HS based composite scaffolds, suggesting their potential application for bone tissue engineering.     

Active ingredient-containing chitosan/polycaprolactone nonwoven mats: Characterizations and their functional assays

This study demonstrates a facile method developed to generate a chitosan/polycaprolactone (CS/PCL) nonwoven mat. All nonwoven mats are composed of microfibers with an average diameter of 2.51 ± 0.69 μm. The X-ray photoelectron spectroscopy data indicate that positively charged nitrogen was generated on the surface of the mats after undergoing CS coating. By using a non-contacting electrostatic voltmeter, we determined that the nonwoven mats exhibited a positive potential and the charge density of the CS/PCL nonwoven mat was in proportion to the thickness of the CS overlayer. Moreover, platelet aggregation and anti-bacterial ability were enhanced by the CS/PCL nonwoven mat as compared to that of PCL nonwoven mat alone. The enhancements of the CS/PCL nonwoven mat on platelet aggregation are further promoted by incorporating a 1 mM calcium ion in its CS overlayer. We also find that the addition of tea tree oil in the CS overlayer significantly inhibited LPS-induced nitrite formation in Raw 264.7 macrophages. In conclusion, our CS/PCL nonwoven mat possesses pharmacological effects including an increase of platelet aggregation, anti-bacterial, anti-adhesive, and anti-inflammatory activities. The performance of this CS/PCL nonwoven mat can be further promoted by incorporating active compounds to exert therapeutic effects in wound healing.     

Effect of testosterone incorporation on cell proliferation and differentiation for polymer–bioceramic composites

In the current study, we characterized the polycaprolactone (PCL), poly(lactic acid-co-glycolic acid) (PLGA), and biphasic calcium phosphate (BCP) composites coated with testosterone propionate (T) using Fourier transform infrared spectroscopy (FTIR) and powder X-ray diffraction (XRD). Osteoblastic cells were seeded with PCL/BCP, PCL/BCP/T, PLGA/PCL/BCP and PLGA/PCL/BCP/T scaffolds, and cell viability, proliferation, differentiation and adhesion were analyzed. The results of physic-chemical experiments showed no displacements or suppression of bands in the FTIR spectra of scaffolds. The XRD patterns of the scaffolds showed an amorphous profile. The osteoblastic cells viability and proliferation increased in the presence of composites with testosterone over 72?h, and were significantly greater when PLGA/PCL/BCP/T scaffold was tested against PCL/BCP/T. Furthermore alkaline phosphatase production was significantly greater in the same group. In conclusion, the PLGA/PCL/BCP scaffold with testosterone could be a promising option for bone tissue applications due to its biocompatibility and its stimulatory effect on cell proliferation.     

静电纺丝天然-人工合成聚合物复合纳米纤维及其对细胞黏附、增殖和迁移的影响

生物材料组成成分对细胞生物功能有不同的影响。利用静电纺丝技术制备了基于聚己内酯(PCL,polycaprolactone)的不同天然蛋白、多糖(丝素蛋白(SF,silk fibroin)、透明质酸(HA,hyaluronicacid))的混合组分纳米纤维,采用了扫描电镜和接触角对纳米纤维进行基础表征。同时,进一步考察了纳米纤维作为组织工程支架的可行性。研究结果表明SF组分能增加材料的可纺性,有利于细胞的前期黏附,并能够促进细胞增殖。HA组分可以改善材料的亲水性,增加细胞伪足并促进细胞迁移。重要的是,PCL/SF/HA纳米纤维能同时结合SF和HA的优点,有望在组织工程领域得到应用。