甲壳素/海藻酸钠-纳米羟基磷灰石多孔复合支架材料的实验研究(英文)

目的:制备性能优良的复合支架一直是骨组织工程学研究的重点和难点。比较分析甲壳素对复合支架材料的孔隙率、含水量、降解率及生物力学特性的影响。方法:将甲壳素溶液与海藻酸钠溶液充分混合,然后将一定质量的羟基磷灰石加入混合液。根据甲壳素溶液在混合液中的质量分数不同分为两组:sca1(0%chitin)、sca2(50%chitin)。扫描电镜下观察材料的表面结构以及检测材料的孔径。测量并计算出复合支架材料的孔隙率、降解率、含水量以及生物力学性能。结果:两组支架材料均表现为多孔隙结构,平均孔径大小分别为:121.2±12.6μm、213.3±27.3μm。孔隙率分别为:(90.53±1.62)%、(87.73±1.22)%,统计学分析显示,两组材料孔隙率的差异比较有统计学意义(P0.05)。两组支架材料第6周的降解率分别(:59.12±1.93)%、(22.91±0.953)%,统计学分析显示,两组材料降解率的差异比较有统计学意义(P0.05)。两组含水量分别为:(95.52±1.17)%、(90.42±0.85)%,统计学分析显示,两组材料含水量的差异比较有统计学意义(P0.05)。第二组生物力学特性显著提高。结论:从本实验的实验数据可以看出,甲壳素可以增大材料的孔径,提高材料的降解稳定性,提高材料的生物力学强度。因此,甲壳素在骨组织工程领域具有重要的研究价值,同时为今后的进一步实验提供一定的实验依据。关键词:甲壳素;海藻酸钠;纳米羟基磷灰石;复合支架材料;组织工程     

类人胶原蛋白-透明质酸血管支架的性能及生物相容性

将类人胶原蛋白与透明质酸按不同比例复合,控制透明质酸的终浓度(W/V)分别为0、0.01%、0.05%、0.1%,用京尼平交联,采用真空冷冻干燥方法构建出血管支架材料。通过扫描电镜、XPS分析、拉力测试、压力爆破实验、细胞毒性实验、血管支架细胞种植实验及小鼠皮下植入等方法对其表面超微结构、表面元素组成、力学性能、细胞毒性等级、细胞相容性、组织相容性进行了研究。结果表明:当透明质酸的含量为0.05%时,类人胶原蛋白-透明质酸支架的孔径比较均匀,孔隙率达94.38%,应力为(1000.8±7.9)kPa,爆破压力为(1058.6±8.2)kPa,细胞毒性实验合格,同时具有良好的细胞相容性、组织相容性及降解性能。     

京尼平的微生物转化及其交联特性的测定分析

从土壤中富集筛选获得一株产β-葡萄糖苷酶的菌株,经菌落的形态和18S rDNA鉴定确定为黑曲霉。将筛选出的黑曲霉菌株接种于发酵培养基,利用含有京尼平苷的栀子粉作为底物发酵,通过对发酵条件优化,得到在装液量50/250 mL,栀子粉浓度为10%,转速为180 r/min,发酵时间为96 h时,京尼平的微生物转化率达到最大22%。这种微生物转化法简化了京尼平的生产工艺,大大降低了生产成本。利用微生物转化获得的京尼平交联胶原蛋白材料,研究表明其具有较好的交联特性,是一种在食品、医药等领域都具有应用前景的生物交联剂。     

可控性释放NGF的京尼平- 壳聚糖微球的研究

目的:在体外研究京尼平-壳聚糖微球可控性释放具有生物活性的神经生长因子的可行性。方法:采用"乳化-化学交联"技术制备包埋神经生长因子的京尼平-壳聚糖微球,京尼平为化学交联剂;应用扫描电镜、粒径分布、体外缓释动力学及细胞生物活性分别对微球的性能进行研究。结果:京尼平-壳聚糖微球表面光滑,平均粒径在5.1~50.5μm之间;京尼平的浓度可影响微球在体外释放神经生长因子的速度,经高浓度京尼平交联的微球能减缓并持续释放神经生长因子;此外,从京尼平-壳聚糖微球释放的神经生长因子可维持PC12细胞的生物活性,提高NGF生物利用率。结论:京尼平-壳聚糖微球能有效缓释具有生物活性的NGF超过14天,为神经退行性疾病的治疗提供一种治疗策略。     

天然高分子复合人工肌腱组织细胞外基质的构建与表征

胶原与壳聚糖是2种具有较好生物相容性和一定力学强度的天然高分子,可在肌腱组织工程中用于细胞外基质的构建,但二者单独使用时各有不足.本研究利用二者性能上的互补,在一定的外力场作用下,采用EDC/NHS对2种天然高分子材料进行共价交联,获得具有一定空间取向和力学强度的多孔支架,然后引入细胞黏附因子RGD进行表面修饰,构建了具有较好组织相容性和细胞亲和性及适当降解速率的人工肌腱组织细胞外基质.对基质材料的力学性能、亲水性、体外降解速率等的检测和显微观察,结果显示:所构建的多孔支架材料柔软富有弹性,抗拉强度达:15.0Mpa,相应形变为:7.33%;孔隙率:79.4%;吸水率:772%;保水率:206%;在RPM1640培养液(含10%胎牛血清)和人血清中,3周总降解率分别为4.13%和37.2%,其降解速率可与肌腱修复周期相吻合,RGD修饰后材料对3T3-L1细胞具有较好的亲和性.有望成为理想的人工肌腱组织和人造皮肤细胞外基质,或整形手术的软组织填充材料.     

藻酸盐三维细胞培养在骨组织工程中应用的研究进展

目的综述藻酸盐三维细胞培养系统在骨组织工程中的应用研究进展。方法广泛查阅近年来有关藻酸盐三维细胞培养系统在骨组织工程应用研究的文献进行综述。结果藻酸盐具有良好的生物相容性,无毒、对宿主无免疫原性和生物可降解等独特的物理、化学和生物特性,藻酸盐三维细胞培养系统仍然是迄今理想的骨组织工程支架材料之一。结论藻酸盐三维细胞培养系统不仅将广泛应用于生命科学基础研究,作为一种理想的组织移植的支架材料,有望逐步走向临床应用。     

载荷壳聚糖微球的海藻酸钠水凝胶的制备

目的:在支架材料上引入具有控释行为的微球,旨在通过微球包裹生长因子,通过生长因子的缓慢释放从而促进种子细胞的生长分化。方法:本实验通过在海藻酸钠水凝胶中负载具有控释功能的壳聚糖微球,并通过在微球中包载溶菌酶从而达到控制壳聚糖降解速率的功效。实验研究了不同搅拌速度下壳聚糖微球的形貌及粒径大小,通过扫描电镜对壳聚糖微球及复合支架的形貌进行了观察,通过紫外光吸收法测试了微球的载药量及包封率,并研究了壳聚糖微球在体外的降解行为等。结果:制备的壳聚糖微球表面较光滑,溶菌酶的包封率在25.78%-41.89%之间,载药量在15.20%-24.44%之间。包封溶菌酶的微胶囊在降解9天后壳聚糖分子量下降了70.40%,载荷微球的复合凝胶孔洞增多,孔洞大小均匀。结论:此复合材料有望作为载荷软骨相关生长因子的支架模型,从而解决软骨组织工程中种子细胞匮乏的问题。     

纳米双相磷酸钙陶瓷支架材料肌内降解性能的实验研究

目的:纳米双相磷酸钙陶瓷(Biphasic calcium phosphate nanocomposite,NanoBCP)支架是一种新型支架材料,具有三维立体多孔结构,孔隙率可达60%～80%。本研究观察了纳米双相磷酸钙陶瓷肌内降解情况。方法:将NanoBCP制备为5mm×5mm×1.5mm大小各8块的支架植入SD大鼠腿部肌袋内,相同孔径、孔隙率的羟基磷灰石(Hydroxyapatite,HA)及普通双相磷酸钙陶瓷(Biphasic calciam phosphate,BCP)作为对照,于4、12、24周取材,测定材料降解率(失重率),从大体、组织学观察以了解材料降解情况。结果:材料肌内植入后降解率测定结果:NanoBCP降解率为32%,BCP的降解率为13%,HA的降解率为3%。组织学观察发现,NanoBCP肌内植入24周后,大部分NanoBCP支架已经将解,并且将解的碎片已埋入纤维结缔组织里。结论:NanoBCP与BCP、HA相比有良好的降解性能。     

骨形成蛋白2/珍珠层粉/壳聚糖支架制备及生物性能研究

目的:制备骨形成蛋白2/珍珠层粉/壳聚糖复合多孔支架,观察支架生物性能。方法:采用冷冻干燥法制备骨形成蛋白2/珍珠层粉/壳聚糖多孔支架。用光学显微镜和扫描电子显微镜观察支架表面形貌及孔径大小,用比重瓶法检测支架孔隙率,热重分析探讨支架的热稳定性,用微力试验机进行压缩性能测试,并将支架与兔骨髓间充质干细胞共培养检测细胞黏附性能,将支架埋置大鼠皮下观察其炎症反应。结果与结论:制备的骨形成蛋白2/珍珠层粉/壳聚糖支架孔径大小为100~300μm,孔隙率为91.64%,压缩应力达3.37MPa,与细胞共培养贴附较好,有良好的组织相容性,提示该支架可做为组织工程支架材料应用于临床上骨组织缺损的修复。     

PLGA的不同组成对支架材料性能的影响研究

研究PLGA的不同组成对支架材料的力学性能、降解性能和生物学性能的影响。采用溶液浇注/颗粒沥取法制备出不同组成的PLGA多孔支架,对支架的力学性能和降解速率进行考察,同时将人真皮成纤维细胞接种于不同组成的PLGA支架材料上,培养不同时间后,检测细胞的粘附率和增殖率,以及细胞产生的总胶原含量,并通过扫描电镜观察支架上的细胞形态。结果显示,随PLA比例的增加,支架的力学强度增加,降解速率降低,但都不是线性变化。70:30比例的支架,拉伸强度最高,而70:30和80:20两种比例的支架,其降解速率没有显著性差异。PLGA不同组成的支架,均具有良好的细胞相容性,成纤维细胞粘附率和增殖率在三种比例的支架上没有显著性差异,细胞在支架表面生长良好,分泌大量的细胞外基质,细胞基本铺满整个支架。本文研究发现,PLGA的组成对支架力学性能、降解性能和生物学性能有细小但显著的影响,这将对组织构建选用PLGA支架材料提供有益的帮助。     

Hybrid chitosan–ß‐glycerol phosphate–gelatin nano‐/micro fibrous scaffolds with suitable mechanical and biological properties for tissue engineering

Scaffold‐based tissue engineering is considered as a promising approach in the regenerative medicine. Graft instability of collagen, by causing poor mechanical properties and rapid degradation, and their hard handling remains major challenges to be addressed. In this research, a composite structured nano‐/microfibrous scaffold, made from a mixture of chitosan–ß‐glycerol phosphate–gelatin (chitosan–GP–gelatin) using a standard electrospinning set‐up was developed. Gelatin–acid acetic and chitosan ß‐glycerol phosphate–HCL solutions were prepared at ratios of 30/70, 50/50, 70/30 (w/w) and their mechanical and biological properties were engineered. Furthermore, the pore structure of the fabricated nanofibrous scaffolds was investigated and predicted using a theoretical model. Higher gelatin concentrations in the polymer blend resulted in significant increase in mean pore size and its distribution. Interaction between the scaffold and the contained cells was also monitored and compared in the test and control groups. Scaffolds with higher chitosan concentrations showed higher rate of cell attachment with better proliferation property, compared with gelatin‐only scaffolds. The fabricated scaffolds, unlike many other natural polymers, also exhibit non‐toxic and biodegradable properties in the grafted tissues. In conclusion, the data clearly showed that the fabricated biomaterial is a biologically compatible scaffold with potential to serve as a proper platform for retaining the cultured cells for further application in cell‐based tissue engineering, especially in wound healing practices. These results suggested the potential of using mesoporous composite chitosan–GP–gelatin fibrous scaffolds for engineering three‐dimensional tissues with different inherent cell characteristics. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 163–175, 2016.     

Preparation and Characterization of Collagen–Chitosan–Chondroitin Sulfate Composite Membranes

Collagen (Col)–chitosan (Chi) membrane was modified by a hot dehydrogenation cross-linking method. Carbodiimide was added for further crossing modification. Chondroitin sulfate (CS) was added so that Col–Chi sulfate composite membranes were prepared. The structure of the composite membranes was characterized by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, and its mechanical properties, degradation, and cytotoxicity were characterized. The composite membrane was applied to a full-thickness skin injury in animal experiments performed in rabbits. Strong interactions and good compatibility among Col, Chi, and CS in the composite membrane were present. The good mechanical properties, biocompatibility, digestion resistance, and wound healing promotion of the composite membrane make it a potential wound dressing or skin scaffold for tissue engineering.     

Photoinitiated cross-linking of the biodegradable polyester poly(propylene fumarate). Part II. In vitro degradation

This study investigated the in vitro degradation of both solid PPF networks and porous PPF scaffolds formed by photoinitiated cross-linking of PPF polymer chains. Three formulations of scaffolds of differing porosity and pore size were constructed by varying porogen size and content. The effects of pore size and pore volume on scaffold mass, geometry, porosity, mechanical properties, and water absorption were then examined. Throughout the study, the solid networks and porous scaffolds exhibited continual mass loss and slight change in length. Porogen content appeared to have the greatest effect upon physical degradation. For example, scaffolds initially fabricated with 80 wt % porogen content lost approximately 30% of their initial PPF content after 32 weeks of degradation, whereas scaffolds fabricated with 70 wt % porogen content lost approximately 18% after 32 weeks of degradation. For all scaffold formulations, water absorption capacity, porosity, and compressive modulus were maintained at constant values following porogen leaching. These results indicate the potential of photo-cross-linked PPF scaffolds in tissue engineering applications which require maintenance of scaffold structure, strength, and porosity during the initial stages of degradation.     

载荷壳聚糖微球的海藻酸钠水凝胶的制备

目的：在支架材料上引入具有控释行为的微球,旨在通过微球包裹生长因子,通过生长因子的缓慢释放从而促进种子细胞的生长分化。方法：本实验通过在海藻酸钠水凝胶中负载具有控释功能的壳聚糖微球,并通过在微球中包栽溶茵酶从而达到控制壳聚糖降解速率的功效。实验研究了不同搅拌速度下壳聚糖微球的形貌及粒径大小,通过扫描电镜对壳聚糖微球及复合支架的形貌进行了观察,通过紫外光吸收法测试了微球的载药量及包封率,并研究了壳聚糖微球在体外的降解行为等。结果：制备的壳聚糖微球表面较光滑,溶菌酶的包封率在25．78％41．89％之间,载药量在15．20％-24．44％之间。包封溶茵酶的微胶囊在降解9天后壳聚糖分子量下降了70．40％,载荷微球的复合凝胶孔洞增多,孔洞大小均匀。结论：此复合材料有望作为栽荷软骨相关生长因子的支架模型,从而解决软骨组织工程中种子细胞匮乏的问题。     

Preparation and characterization of chitosan-carbon nanotube scaffolds for bone tissue engineering

In recent years, significant development has been given to chitosan for orthopedic application. In this study, we have prepared scaffolds with the use of low and high molecular weight chitosan with 0.0025%, 0.005% and 0.01% weight of f-multiwalled carbon nanotube (f-MWCNT) by freezing and lyophilization method and physiochemically characterized as bone graft substitutes. Fourier Transform Infrared Spectroscopy, X-Ray Diffraction Analysis, Thermal Gravimetric Analysis, Scanning Electron Microscopy and Optical Microscopy results indicated that the f-MWCNT was uniformly dispersed in chitosan matrix and there was a chemical interaction between chitosan and f-MWCNT. The water uptake ability and porosity of scaffolds increased with an increase the amount of f-MWCNT. The cell proliferation, protein content, alkaline phosphatase and mineralization of the composite scaffolds were higher than chitosan scaffold due to the addition of f-MWCNT. Herewith, we are suggesting that chitosan/f-MWCNT scaffolds are promising biomaterials for bone tissue engineering.     

Preparation and biological properties of a novel composite scaffold of nano-hydroxyapatite/chitosan/carboxymethyl cellulose for bone tissue engineering

In this study, we report the physico-chemical and biological properties of a novel biodegradable composite scaffold made of nano-hydroxyapatite and natural derived polymers of chitosan and carboxymethyl cellulose, namely, n-HA/CS/CMC, which was prepared by freeze-drying method. The physico-chemical properties of n-HA/CS/CMC scaffold were tested by infrared absorption spectra (IR), transmission electron microscope(TEM), scanning electron microscope(SEM), universal material testing machine and phosphate buffer solution (PBS) soaking experiment. Besides, the biological properties were evaluated by MG63 cells and Mesenchymal stem cells (MSCs) culture experiment in vitro and a short period implantation study in vivo. The results show that the composite scaffold is mainly formed through the ionic crossing-linking of the two polyions between CS and CMC, and n-HA is incorporated into the polyelectrolyte matrix of CS-CMC without agglomeration, which endows the scaffold with good physico-chemical properties such as highly interconnected porous structure, high compressive strength and good structural stability and degradation. More important, the results of cells attached, proliferated on the scaffold indicate that the scaffold is non-toxic and has good cell biocompatibility, and the results of implantation experiment in vivo further confirm that the scaffold has good tissue biocompatibility. All the above results suggest that the novel degradable n-HA/CS/CMC composite scaffold has a great potential to be used as bone tissue engineering material.     

Mutually reinforced multicomponent polysaccharide networks

Networks made from chitosan and alginate have been utilized as prospective tissue engineering scaffolds due to material biocompatibility and degradability. Calcium (Ca(2+) ) is often added to these networks as a modifier for mechanical strength enhancement. In this work, we examined changes in the bulk material properties of different concentrations of chitosan/alginate mixtures (2, 3, or 5% w/w) upon adding another modifier, chondroitin. We further examined how material properties depend on the order the modifiers, Ca(2+) and chondroitin, were added. It was found that the addition of chondroitin significantly increased the mechanical strength of chitosan/alginate networks. Highest elastic moduli were obtained from samples made with mass fractions of 5% chitosan and alginate, modified by chondroitin first and then Ca(2+) . The elastic moduli in dry and hydrated states were (4.41 ± 0.52) MPa and (0.11 ± 0.01) MPa, respectively. Network porosity and density were slightly dependent on total polysaccharide concentration. Average pore size was slightly larger in samples modified by Ca(2+) first and then chondroitin and in samples made with 3% starting mass fractions. Here, small-angle neutron scattering (SANS) was utilized to examine mesh size of the fibrous networks, mass-fractal parameters and average dimensions of the fiber cross-sections prior to freeze-drying. These studies revealed that addition of Ca(2+) and chondroitin modifiers increased fiber compactness and thickness, respectively. Together these findings are consistent with improved network mechanical properties of the freeze-dried materials.     

Cell colonization in degradable 3D porous matrices

Cell colonization is an important in a wide variety of biological processes and applications including vascularization, wound healing, tissue engineering, stem cell differentiation and biosensors. During colonization porous 3D structures are used to support and guide the ingrowth of cells into the matrix. In this review, we summarize our understanding of various factors affecting cell colonization in three-dimensional environment. The structural, biological and degradation properties of the matrix all play key roles during colonization. Further, specific scaffold properties such as porosity, pore size, fiber thickness, topography and scaffold stiffness as well as important cell material interactions such as cell adhesion and mechanotransduction also influence colonization.Key words: colonization, pore size, porosity, topography, mechanotransduction, degradation, matrix turnover