京尼平交联3D打印胶原/壳聚糖支架的表征北大核心

背景:胶原/壳聚糖支架需交联才能达到相应力学性能,有研究表示调节交联剂浓度可以在一定范围内调控支架的理化性能。目的:探究京尼平浓度对胶原/壳聚糖支架理化性能的影响,制备理化性能可调节的组织工程支架。方法:将胶原和壳聚糖粉末分别溶于弱酸后混合均匀,作为打印墨水,利用生物3D打印机低温打印胶原支架与胶原/壳聚糖支架,经冻干、中和处理后分别以1,3,5 mmol/L的京尼平进行交联。检测各组支架的表观结构稳定性、抗拉能力、溶胀性能、降解性能与生物相容性。结果与结论:①将支架在PBS中浸泡3 d后,对比未交联的冻干支架,交联后胶原支架表面维持规则的孔结构,但是支架出现明显变形;交联后胶原/壳聚糖支架表面结构规则,仅1 mmol/L京尼平交联的胶原/壳聚糖支架存在轻微变形。②随着京尼平浓度的增加,各组支架的力学性能增加,并且对应交联浓度下的胶原/壳聚糖支架力学性能好于胶原支架。③随着京尼平浓度的增加,胶原支架的溶胀率下降,胶原/壳聚糖支架的溶胀率无明显变化。④浸泡于胶原酶溶液中后,不同浓度京尼平交联的胶原支架在1 h内被完全降解,胶原/壳聚糖支架的降解速率随京尼平浓度的增加而降低,均呈现先快速后平缓的趋势。⑤将骨髓间充质干细胞接种于各组交联支架3 d后,1,3 mmol/L京尼平交联的胶原/壳聚糖支架(或胶原支架)上的细胞数量明显多于5 mmol/L京尼平交联的胶原/壳聚糖支架(P<0.05)。⑥结果表明,京尼平可在一定范围调节胶原/壳聚糖支架理化性能,其中3 mmol/L京尼平交联的胶原/壳聚糖支架具有较好的力学性能、抗酶解能力与生物相容性。     

The influence of molecular weight of chitosan on the physical and biological properties of collagen/chitosan scaffolds

Biopolymer blends between collagen and chitosan have the potential to produce cell scaffolds with biocompatible properties. However, the relationship between the molecular weight of chitosan and its effect on physical and biological properties of collagen/chitosan scaffolds has not been elucidated yet. Porous scaffolds were fabricated by freeze-drying the solution of collagen and chitosan, followed by cross-linking by dehydrothermal treatment. Various types of scaffolds were prepared using chitosan with various molecular weights and blending ratios. Fourier transform infrared spectroscopy proved that collagen and chitosan scaffolds at all blending ratios contained mainly electrostatic interactions at the molecular level. The compressive modulus decreased with increasing the concentration of chitosan. Equilibrium swelling ratios of approximately 6-8, determined in phosphate-buffered saline at physiological pH (7.4), were found in case of collagen-dominated scaffolds. The lysozyme biodegradation test demonstrated that the presence of chitosan, especially the high-molecular-weight species, could significantly prolong the biodegradation of collagen/chitosan scaffolds. In vitro culture of L929 mouse connective tissue fibroblast evidenced that low-molecular-weight chitosan was more effective to promote and accelerate cell proliferation, particularly for scaffolds containing 30 wt% chitosan. The results elucidated that the blends of collagen with low-molecular-weight chitosan have a high potential to be applied as new materials for skin-tissue engineering.     

3D打印胶原/壳聚糖支架改善大鼠脊髓损伤后神经功能恢复

文题释义：壳聚糖：为一种天然多糖,是虾蟹等低等动物外壳的重要成分,具有一定的机械强度,并且具有良好的生物相容性和抗菌性,在生物工程领域具有较好的应用前景。 3D生物打印：是组织工程中最重要的技术之一。目前常用的三维生物打印方法包括喷墨打印、挤压生物打印和激光生物打印,选择好合适的材料后,在计算机指导下根据所选择的生物材料和细胞类型逐层准确地打印出所设计的结构。 背景：3D打印技术可以根据需求制备出满足脊髓植入形状、大小和表面形态要求的生物支架。 目的：观察3D打印胶原/壳聚糖支架对脊髓损伤大鼠神经功能恢复的影响。 方法：将胶原和壳聚糖按2∶1的质量比混合,采用冷冻干燥法制备普通胶原/壳聚糖支架,采用3D打印机制备3D打印胶原/壳聚糖支架,分别测量两种支架的孔隙率和弹性模量,电镜观察支架形态。将神经干细胞分别与3D打印胶原/壳聚糖支架、普通胶原/壳聚糖支架共培养,进行扫描电镜观察与CCK-8检测。将40只雌性SD大鼠(由中国人民解放军医学科学院军事科学院提供)随机分成4组：假手术组、脊髓损伤组、普通胶    原/壳聚糖支架组和3D打印胶原/壳聚糖支架组,后3组制作脊髓全横断损伤模型,普通胶原/壳聚糖支架组和3D打印胶原/壳聚糖支架组损伤处填充对应的支架材料,术后相应时间点进行后肢功能BBB评分、斜坡实验、神经电生理检测与磁共振平扫。实验方案经天津市神经创伤重点实验室伦理委员会批准。 结果与结论：①扫描电镜显示,3D打印胶原/壳聚糖支架具有互连的多孔结构,普通胶原/壳聚糖支架内部结构紊乱;②神经干细胞在3D打印胶原/壳聚糖支架表面生长良好,完全伸展,且3D打印胶原/壳聚糖支架表面神经干细胞的活性显著高于普通胶原/壳聚糖支架组(P < 0.05);③3D打印胶原/壳聚糖支架的孔隙率与弹性模量均高于普通胶原/壳聚糖支架组(P < 0.05);④3D打印胶原/壳聚糖支架组术后3-8周的BBB评分高于脊髓损伤组、普通胶原/壳聚糖支架组(P < 0.05),术后4,6,8周的斜坡实验角度大于脊髓损伤组、普通胶原/壳聚糖支架组(P < 0.05);⑤3D打印胶原/壳聚糖支架组术后8周的运动诱发电位振幅、体感诱发电位振幅大于脊髓损伤组与普通胶原/壳聚糖支架组(P < 0.05),运动诱发电位潜伏期、体感诱发电位潜伏期短于脊髓损伤组与普通胶原/壳聚糖支架组(P < 0.05);⑥磁共振平扫显示与脊髓损伤组及普通胶原/壳聚糖支架组比较,3D打印胶原/壳聚糖支架组损伤处具有较好的连续性与较多的神经纤维束通过;⑦结果表明,3D打印胶原/壳聚糖支架可促进脊髓损伤大鼠神经功能的修复。 ORCID: 0000-0001-5771-8222(史新宇) 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering

Porous scaffolds for skin tissue engineering were fabricated by freeze-drying the mixture of collagen and chitosan solutions. Glutaraldehyde (GA) was used to treat the scaffolds to improve their biostability. Confocal laser scanning microscopy observation confirmed the even distribution of these two constituent materials in the scaffold. The GA concentrations have a slight effect on the cross-section morphology and the swelling ratios of the cross-linked scaffolds. The collagenase digestion test proved that the presence of chitosan can obviously improve the biostability of the collagen/chitosan scaffold under the GA treatment, where chitosan might function as a cross-linking bridge. A detail investigation found that a steady increase of the biostability of the collagen/chitosan scaffold was achieved when GA concentration was lower than 0.1%, then was less influenced at a still higher GA concentration up to 0.25%. In vitro culture of human dermal fibroblasts proved that the GA-treated scaffold could retain the original good cytocompatibility of collagen to effectively accelerate cell infiltration and proliferation. In vivo animal tests further revealed that the scaffold could sufficiently support and accelerate the fibroblasts infiltration from the surrounding tissue. Immunohistochemistry analysis of the scaffold embedded for 28 days indicated that the biodegradation of the 0.25% GA-treated scaffold is a long-term process. All these results suggest that collagen/chitosan scaffold cross-linked by GA is a potential candidate for dermal equivalent with enhanced biostability and good biocompatibility.     

Chitosan-alginate as scaffolding material for cartilage tissue engineering

Tissue compatibility of chitosan-alginate scaffolds was studied in vitro in terms of cell morphology, proliferation, and functionality using HTB-94 cells. The scaffold has an interconnected 3D porous structure, and was fabricated by thermally induced phase separation followed by freeze drying. Cell proliferation on the chitosan-alginate scaffold was found to be faster than on a pure chitosan scaffold. After cell culture for 2 weeks in vitro, the cells on the chitosan scaffold gradually assumed a fibroblast-like morphology while the cells on the chitosan-alginate scaffold retained their spherical morphology throughout the period of study. SDS-PAGE electrophoresis and Western blot assays for proteins extracted from cells grown on scaffolds indicated that production of cartilage-specific collagen type II, a marker for chondrocytic phenotype, increased from week 2 to week 3 on the chitosan-alginate scaffold but decreased on the chitosan scaffold. This study suggested that chitosan-alginate scaffolds promote cell proliferation, enhance phenotype expression of HTB-94 chondrocytes, and may potentially serve as an improved alternative to chitosan scaffolds for cartilage tissue engineering.     

Characterization and evaluation of chitosan matrix for in vitro growth of MCF-7 breast cancer cell lines

Biodegradable polymer scaffolds were prepared from chitosan with varying degree of deacetylation for in vitro culture of human breast cancer MCF-7 cell lines. These polymers were characterized in terms of functional groups by FTIR and swelling properties. Polymers having high degree of deacetylation showed better swelling properties irrespective of the molecular weight. These polymers were biocompatible and non-toxic towards human epithelial MCF-7 cell lines. Attachment kinetics of MCF-7 cell lines on to polymer scaffold was investigated and it was observed that polymer having high degree of deacetylation favored better cell attachment. In CPIII polymer scaffold having 80% degree of deacetylation, a maximum of 1 millions cells per mg pf polymer were adsorbed within 1h. It appears that high swelling and high degree of deacetylation of chitosan helped in better adsorption of cancer cell lines. The cellular morphology of the attached cells on chitosan matrix was similar to that observed with regular plastic culture with the difference that, cells grew as three-dimensional clumps on chitosan matrix. Polymer having high degree of deacetylation not only favored better adsorption but also showed improved cell growth kinetics. Maximum cell concentration of 6.5 x 10(5) cells/ml was achieved in 5 days culture on CPIII polymer scaffold. The glucose consumption and lactate production pattern of the MCF-7 cell lines on chitosan polymer matrix were similar to that observed on cell growth on tissue culture flask. These results indicate that chitosan scaffold having high degree of deacetylation can be used for three-dimensional growth of MCF-7 cancer cell lines. Such in vitro 3D culture of cancer cells can thus be used as a model for the cytotoxic evaluation of anticancer drugs.     

Preparation and assessment of glutaraldehyde-crosslinked collagen-chitosan hydrogels for adipose tissue engineering

A portfolio of crosslinked chitosan:collagen blends was prepared, and their microarchitecture and water binding capacity were studied to investigate their application for adipose tissue engineering. Glutaraldehyde (GA) concentration had little effect on scaffold morphology or water binding capacity. However, the processing freezing temperature prior to lyophilization affected both. In vitro cytocompatibility of pre-adipocytes (PAs) was assessed for a candidate collagen:chitosan blend using two assays. Results confirm the viability of PAs on GA-crosslinked collagen:chitosan scaffolds. A rat subcutaneous pocket assay was employed to assess PA-seeded scaffolds in vivo. Animal tests proved that PA-seeded scaffolds were biocompatible, could induce vascularization, and form adipose tissue.     

The influence of molecular weight of chitosan on the physical and biological properties of collagen/chitosan scaffolds

Biopolymer blends between collagen and chitosan have the potential to produce cell scaffolds with biocompatible properties. However, the relationship between the molecular weight of chitosan and its effect on physical and biological properties of collagen/chitosan scaffolds has not been elucidated yet. Porous scaffolds were fabricated by freeze-drying the solution of collagen and chitosan, followed by cross-linking by dehydrothermal treatment. Various types of scaffolds were prepared using chitosan with various molecular weights and blending ratios. Fourier transform infrared spectroscopy proved that collagen and chitosan scaffolds at all blending ratios contained mainly electrostatic interactions at the molecular level. The compressive modulus decreased with increasing the concentration of chitosan. Equilibrium swelling ratios of approximately 6–8, determined in phosphate-buffered saline at physiological pH (7.4), were found in case of collagen-dominated scaffolds. The lysozyme biodegradation test demonstrated that the presence of chitosan, especially the high-molecular-weight species, could significantly prolong the biodegradation of collagen/chitosan scaffolds. In vitro culture of L929 mouse connective tissue fibroblast evidenced that low-molecular-weight chitosan was more effective to promote and accelerate cell proliferation, particularly for scaffolds containing 30 wt% chitosan. The results elucidated that the blends of collagen with low-molecular-weight chitosan have a high potential to be applied as new materials for skin-tissue engineering.     

Novel chitosan-based hyaluronan hybrid polymer fibers as a scaffold in ligament tissue engineering

To clarify the feasibility of using novel chitosan-based hyaluronan hybrid polymer fibers as a scaffold in ligament tissue engineering, their mechanical properties and ability to promote cellular adhesion, proliferation, and extracellular matrix production were studied in vitro. Chitosan fibers and chitosan-based 0.05% and 0.1% hyaluronan hybrid fibers were developed by the wet spinning method. Hyaluronan coating significantly increased mechanical properties, compared to the chitosan fibers. Rabbit fibroblasts adhesion onto hybrid fibers was significantly greater than for the control and chitosan fibers. For analysis of cell proliferation and extracellular matrix production, a three-dimensional scaffold was created by simply piling up each fiber. At 1 day after cultivation, the DNA content in the hybrid scaffolds was higher than that in the chitosan scaffold. Scanning electron microscopy showed that the fibroblasts had produced collagen fibers after 14 days of culture. Immunostaining for type I collagen was clearly predominant in the hybrid scaffolds, and the mRNA level of type I collagen in the hybrid scaffolds were significantly greater than that in the chitosan scaffold. The present study revealed that hyaluronan hybridization with chitosan fibers enhanced fiber mechanical properties and in vitro biological effects on the cultured fibroblasts.     

Developing an alginate/chitosan hybrid fiber scaffold for annulus fibrosus cells

A fibrous scaffold made of alginate or alginate/chitosan was fabricated for annulus fibrosus (AF) cell culture using a wet-spinning and lyophilization technique. The scaffolds were evaluated using several in vitro tests. Scanning electron microscopy showed the scaffold fibers generally aligned in one direction with individual fiber diameters varying between 40-100 microm. The alginate/chitosan hybrid scaffold exhibited a slower degradation rate, while both scaffold types did not display any cyto-toxicity to 3T3 fibroblasts and could maintain canine AF cell growth. The AF cells retained their spherical shape within the fibrous scaffold at the beginning of the culture period and formed into cell clusters at later times. Specific extracellular matrix molecules, including collagen I, collagen II, and aggrecan, could be detected in the AF cell clusters. These results demonstrate the feasibility of using this hybrid alginate/chitosan scaffold for AF cell culture, and the potential application for intervertebral disc tissue engineering.     

不同脱乙酰度壳聚糖支架制备及降解性能评价

制备不同脱乙酰度壳聚糖支架,采用SEM观察其表面形貌,检测孔隙率,吸水溶胀率,体内、外降解率。结果表明不同脱乙酰度支架均具有高孔隙三维结构。随脱乙酰度增加,孔隙率分别为93.46%、90.02%和86.71%;溶胀率分别为820%、803%和772%;第4周体外降解率分别为30.44%、22.88%和17.10%;体内降解率为57.48%、40.23%和29.53%。其降解率与脱乙酰度呈负相关,体内降解速率快于体外。可通过控制壳聚糖的脱乙酰度大小为软骨缺损修复提供匹配良好的降解支架材料。     

Growth of osteoblast-like cells on biomimetic apatite-coated chitosan scaffolds

Porous scaffold materials that can provide a framework for the cells to adhere, proliferate, and create extracellular matrix are considered to be suitable materials for bone regeneration. Interconnected porous chitosan scaffolds were prepared by freeze-drying method, and were mineralized by calcium and phosphate solution by double-diffusion method to form nanoapatite in chitosan matrix. The mineralized chitosan scaffold contains hydroxyapatite nanocrystals on the surface and also within the pore channels of the scaffold. To assess the effect of apatite and porosity of the scaffolds on cells, human osteoblast (SaOS-2) cells were cultured on unmineralized and mineralized chitosan scaffolds. The cell growth on the mineralized scaffolds and on the pure chitosan scaffold shows a similar growth trend. The total protein content and alkaline phosphatase enzyme activity of the cells grown on scaffolds were quantified, and were found to increase over time in mineralized scaffold after 1 and 3 weeks of culture. The electron microscopy of the cell-seeded scaffolds showed that most of the outer macropores became sealed off by a continuous layer of cells. The cells spanned around the pore wall and formed extra cellular matrix, consisting mainly of collagen in mineralized scaffolds. The hydroxyproline content also confirmed the formation of the collagen matrix by cells in mineralized scaffolds. This study demonstrated that the presence of apatite nanocrystals in chitosan scaffolds does not significantly influence the growth of cells, but does induce the formation of extracellular matrix and therefore has the potential to serve for bone tissue engineering.     

Thermal dehydration treatment and glutaraldehyde cross-linking to increase the biostability of collagen-chitosan porous scaffolds used as dermal equivalent

A biodegradable scaffold for skin-tissue engineering was designed using collagen and chitosan, which are common materials for biomedical application. The scaffolds containing different amounts of chitosan were prepared by mixing the collagen and chitosan solutions followed by removal of the solvent using a freeze-drying method. The cross-linking treatment of these scaffolds was performed using the dehydrothermal treatment (DHT) method or glutaraldehyde (GA) to increase their biostability. The effect of the chitosan concentration and the cross-linking methods on the morphology of these scaffolds was studied by SEM. The water retention and the biodegradability in vitro of various collagen-chitosan scaffolds were investigated. Finally the biocompatibility of the collagen-chitosan (10 wt% chitosan) scaffold treated with different cross-linking methods was evaluated using a in vivo animal test. A mild inflammatory reaction could be detected in the early stages, and GA treatment can decrease the inflammatory reaction in a long-term implantation. After implantation for four weeks, all kinds of scaffolds, especially the GA-treated scaffolds (Col-GA) were filled with a large number of fibroblasts and were vascularized to a certain extent. These results suggest that the GA-treated scaffold has an increased biostability and excellent biocompatibility. It can be a potential candidate for skin-tissue engineering.     

Three-dimensional nanohydroxyapatite/chitosan scaffolds as potential tissue engineered periodontal tissue

The development of suitable three-dimensional scaffold for the maintenance of cellular viability and differentiation is critical for applications in periodontal tissue engineering. In this work, different ratios of porous nanohydroxyapatite/chitosan (HA/chitosan) scaffolds are prepared through a freeze-drying process. These scaffolds are evaluated in vitro by the analysis of microscopic structure, porosity, and cytocompatibility. The expression of type I collagen and alkaline phosphatase (ALP) activity are detected with real-time polymerase chain reaction (RT-PCR). Human periodontal ligament cells (HPLCs) transfected with enhanced green fluorescence protein (EGFP) are seeded onto the scaffolds, and then these scaffolds are implanted subcutaneously into athymic mice. The results indicated that the porosity and pore diameter of the HA/chitosan scaffolds are lower than those of pure chitosan scaffold. The HA/chitosan scaffold containing 1% HA exhibited better cytocompatibility than the pure chitosan scaffold. The expression of type I collagen and ALP are up-regulated in 1% HA/chitosan scaffold. After implanted in vivo, EGFP-transfected HPLCs not only proliferate but also recruit surrounding tissue to grow in the scaffold. The degradation of the scaffold significantly decreased in the presence of HA. This study demonstrated the potential of HA/ chitosan scaffold as a good substrate candidate in periodontal tissue engineering.     

A healing method of tympanic membrane perforations using three-dimensional porous chitosan scaffolds

Both surgical tympanoplasty and paper patch grafts are frequently procedured to heal tympanic membrane (TM) perforation or chronic otitis media, despite their many disadvantages. In this study, we report a new healing method of TM perforation by using three-dimensional (3D) porous chitosan scaffolds (3D chitosan scaffolds) as an alternative method to surgical treatment or paper patch graft. Various 3D chitosan scaffolds were prepared; and the structural characteristics, mechanical property, in vitro biocompatibility, and healing effects of the 3D chitosan scaffolds as an artificial TM in in vivo animal studies were investigated. A 3D chitosan scaffold of 5 wt.% chitosan concentration showed good proliferation of TM cells in an in vitro study, as well as suitable structural characteristics and mechanical property, as compared with either 1% or 3% chitosan. In in vivo animal studies, 3D chitosan scaffold were able to migrate through the pores and surfaces of TM cells, thus leading to more effective TM regeneration than paper patch technique. Histological observations demonstrated that the regenerated TM with the 3D chitosan scaffold consisted of three (epidermal, connective tissue, and mucosal) layers and were thicker than normal TMs. The 3D chitosan scaffold technique may be an optimal healing method used in lieu of surgical tympanoplasty in certain cases to heal perforated TMs.     

Comparison of the properties of collagen–chitosan scaffolds after γ-ray irradiation and carbodiimide cross-linking

The property of collagen–chitosan porous scaffold varies according to cross-linking density and scaffold composition. This study was designed to compare the properties of collagen–chitosan porous scaffolds cross-linked with γ-irradiation and carbodiimide (CAR) for the first time. Eleven sets of collagen–chitosan scaffolds containing different concentrations of chitosan at a 5% increasing gradient were fabricated. Fourier transform infrared spectroscopy was performed to confirm the success of cross-linking in the scaffolds. The scaffold morphology was evaluated under scanning electron microscope (SEM). SEM revealed that chitosan was an indispensable material for the fabrication of γ-ray irradiation scaffold. The microstructure of γ-ray irradiation scaffold was less stable than those of alternative scaffolds. Based upon swelling ratio, porosity factor, and collagenase degradation, γ-ray irradiation scaffold was less stable than CAR and 25% proportion of chitosan scaffolds. Mechanical property determines the orientation in γ-irradiation and CAR scaffold. In vitro degradation test indicated that γ-irradiation and CAR cross-linking can elevate the scaffold biocompatibility. Compared with γ-ray irradiation, CAR cross-linked scaffold containing 25% chitosan can more significantly enhance the bio-stability and biocompatibility of collagen–chitosan scaffolds. CAR cross-linked scaffold may be the best choice for future tissue engineering.     

复合骨碎补总黄酮的丝素蛋白/壳聚糖支架在兔关节软骨损伤中的应用研究

目的 以丝素蛋白/壳聚糖支架为载体将骨碎补总黄酮应用于兔软骨损伤局部,观察修复效果,为临床提供实验数据。方法 制备丝素蛋白/壳聚糖支架、骨碎补总黄酮缓释微球与负载骨碎补总黄酮缓释微球的丝素蛋白/壳聚糖支架,扫描电子显微镜下观察支架形貌,同时检测该支架的体外缓释能力。24只新西兰大白兔随机分3组,利用电钻在股骨滑车部位构建直径3.5 mm、深1.5 mm的软骨损伤模型,空白组软骨缺损处不植入任何材料,对照组植入单纯的丝素蛋白/壳聚糖支架,实验组植入负载骨碎补总黄酮缓释微球的丝素蛋白/壳聚糖支架,术后12周、24周行标本大体与组织学观察,RT-PCR检测修复组织Sox-9、II型胶原与聚集蛋白聚糖mRNA的表达量,Western blot检测软骨缺损部位II型胶原蛋白表达,分析软骨修复效果。结果 丝素蛋白/壳聚糖支架具有良好的三维孔隙结构,孔洞之间相互联通;制备的载药微球表面较光滑,为较规则的圆球形;载药微球均匀分散于丝素蛋白/壳聚糖支架基质中。丝素蛋白/壳聚糖支架可在体外持续稳定地释放骨碎补总黄酮,实验组软骨损伤修复效果优于对照组,对应的ICRS评分与Wakitani组织学评分高于对照组...     

体外构建三维肝肿瘤模型及药物筛选

背景：体外构建三维肿瘤模型替代现有二维平面肿瘤细胞模型用于药物筛选是肿瘤药筛技术发展的必然趋势。 目的：体外构建三维肝肿瘤模型体系,并用于抗肿瘤药物的敏感性研究。 方法：以人肝癌细胞HepG2作为模型细胞,以壳聚糖/胶原混合材料制备水凝胶支架,体外构建肝(肿瘤)细胞的三维培养体系,表征三维肝(肿瘤)细胞聚集体的形态、生长、细胞骨架分布等,并以二维平面培养的肝肿瘤细胞为对照,研究三维肝肿瘤模型对临床常用的化疗药物的敏感性。 结果与结论：①肝细胞在壳聚糖/胶原水凝胶支架中培养10 d后形成三维的聚集细胞团。②肝细胞在水凝胶支架中生长速度略慢于二维平面培养,但在三维体系下肝细胞能长时间保持细胞活性。③在水凝胶支架中肝细胞三维生长后,纤维蛋白骨架发生重排,结构与在体肝组织更接近。④在水凝胶支架中的三维肝肿瘤细胞模型对化疗药物的敏感性降低。由此可见,在壳聚糖/胶原水凝胶支架中形成的三维肝(肿瘤)模型,其细胞骨架结构更接近体内肝组织,因此可用于体外药筛模型研究。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

Effects of the controlled-released TGF-beta 1 from chitosan microspheres on chondrocytes cultured in a collagen/chitosan/glycosaminoglycan scaffold

The objectives of this study were (1) to develop a three-dimensional collagen/chitosan/glycosaminoglycan (GAG) scaffold in combination with transforming growth factor-beta1 (TGF-beta 1)-loaded chitosan microspheres, and (2) to evaluate the effect of released TGF-beta 1 on the chondrogenic potential of rabbit chondrocytes in such scaffolds. TGF-beta 1 was loaded into chitosan microspheres using an emulsion-crosslinking method. The controlled release of TGF-beta 1, as measured by enzyme-linked immunosorbent assay (ELISA), was monitored for 7 days. The porous scaffolds containing collagen and chitosan were fabricated by using a freeze drying technique and crosslinked using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) in the presence of chondroitin sulfate (CS), as a GAG component. The TGF-beta 1 microspheres were encapsulated into the scaffold at a concentration of 10 ng TGF-beta 1/scaffold and then chondrocytes were seeded in the scaffold and incubated in vitro for 3 weeks. Both proliferation rate and glycosaminoglycan (GAG) production were significantly higher in the TGF-beta 1 microsphere-incorporated scaffolds than in the control scaffolds without microspheres. Extracellular matrix staining by Safranin O and immunohistochemistry for type II collagen were elevated in the scaffold with TGF-beta 1 microspheres. These results suggest that TGF-beta 1 microspheres when incorporated into a scaffold have the potential to enhance cartilage formation.     

Novel 3D collagen scaffolds fabricated by indirect printing technique for tissue engineering

This article reports the mechanical properties and in vitro evaluation of a collagen scaffold fabricated using an indirect 3D printing technique. Collagen scaffolds, featuring predefined internal channels and capillary networks, were manufactured using phase change printing. It was observed that the collagen scaffolds featured internal channels and a hierarchical structure that varied over length scales of 10-400 microm. In vitro evaluation using hMSCs demonstrated that the resultant collagen based scaffolds have the ability to support hMSC cell attachment and proliferation; cells can migrate and survive deep within the structure of the scaffold. The cell numbers increased 2.4 times over 28 days in culture for the lysine treated scaffolds. The cells were spread along the collagen fibers to form a 3D structure and extracellular matrix was detected on the surface of the scaffolds after 4 weeks in culture. The crosslinking treatment enhanced the biostability and dynamic properties of the collagen scaffolds significantly.