兔血管平滑肌细胞和聚羟基丁酯（PHB）的细胞相容性实验研究

探索血管平滑肌细胞和新型可降解材料聚羟基西酯（PHB）的细胞相容性,为组织人工血管的构建寻找理想的支架材料。将组织法体外培养的兔血管平滑肌细胞种植在PHB膜片和PHB三维微孔支架上,在相差显微镜下观察细胞的粘附和生长情况,用MTT法测定细胞粘附率和细胞增殖指数,复合培养7d后进行扫描电镜观察并用流式细胞仪（FCM）测定细胞周期、DNA指数。结果兔血管平滑肌细胞在PHB膜片上粘附率为77％,细胞增殖符合细胞的生长曲线,在PHB三维微孔支架上生长情况良好,并被证实为二倍体细胞。结论是兔血管平滑肌细胞和聚羟基西酯（PHB）的细胞相容性较好,但细胞与材料间的粘附有待进一步改善。     

以组织工程技术构建活性双层人工皮肤

用冷冻干燥胶原凝胶方法制备胶原海绵,经戊二醛交联处理,作为活性双层人工皮肤的支架;以幼儿包皮为细胞来源获得真皮成纤维细胞和皮肤角质细胞,体外扩增后作为种子细胞;在胶原海绵上接种成纤维细胞和角质细胞,体外构建活性双层人工皮肤。研究结果表明,人工真皮内的成纤维细胞生长良好,并且有人纤粘连蛋白类生物活性物质存在,活性双层人工皮肤表皮层有类似干天然皮肤表皮层的组织结构。     

前交叉韧带组织工程种子细胞筛选的初步研究

为组织工程方法构建前交叉韧带筛选一种理想的种子细胞。用体外分离培养兔前交叉韧带（ACL）、内侧副韧带（MCL）、跟腱（AT）、皮肤成纤维细胞（SK）,比较四种细胞的生长状态、增殖活性及Ⅰ、Ⅲ型胶原分泌量。在含体积分数10％胎牛血清的DMEM培养液中,四种细胞生长良好、细胞形态相似,但以SK细胞生长最快,AT细胞次之,MCL细胞和ACL细胞生长最慢。SK、MCL、ACL细胞均分泌Ⅰ、Ⅲ型胶原,AT细胞只分泌Ⅰ型胶原。SK细胞Ⅰ型胶原染色密度较MCL、ACL、AT细胞高,SK细胞Ⅲ型胶原染色密度较MCL、ACL细胞高。说明SK细胞增殖活性高,分泌Ⅰ、Ⅲ型胶原多,取材简便,是理想的前交叉韧带组织工程种子细胞。     

DiI荧光标记示踪兔皮肤成纤维细胞修复前交叉韧带损伤的研究

体内采用组织工程方法构建前交叉韧带种子细胞筛选研究,提供了一种理想的细胞标记的方法.体外分离培养兔皮肤成纤维细胞(SF),比较皮肤成纤维细胞使用荧光染料CM-DiI体外标记前后细胞的生长状态、增殖活性以及检测SF复合纤维生物蛋白胶植入前交叉韧带上12周后的荧光表达情况.结果表明CM-Dil标记SF效果良好,标记阳性率高于95%.而标记前后SF增殖活性无明显变化,荧光标记在体内实验12周后仍有表达.从而证实CM-DiI是筛选前交叉韧带组织工程种子细胞筛选研究中细胞标记、示踪的良好标记物.     

3D打印PLLA/HA复合组织工程骨支架及性能表征

为了解决羟基磷灰石(HA)骨支架降解难的问题,本研究通过在HA骨支架中加入具有良好降解性能的聚左旋乳酸(PLLA)来调节其降解速度.采用3D打印挤出成型设备制备出PLLA/HA复合多孔骨支架,探究不同PLLA质量分数下复合组织工程骨支架抗压强度、亲水性、降解率以及细胞毒性的变化.实验结果表明:五组不同材料配比的复合骨支...     

CPP/PLLA软骨组织工程支架复合材料制备及其性能表征

采用溶媒浇铸/粒子滤取技术与气体发泡相结合的方法制备了三维、连通、微孔网状结构的CPP/PLLA软骨组织工程支架复合材料.测试了CPP/PLLA软骨组织工程支架复合材料的物理、力学性能并借助扫描电镜对其微结构进行了观察.结果表明,支架复合材料的初始物理、力学性能和微结构满足软骨组织工程对其支架材料的要求.     

纳米HAP/CPP/PLLA骨组织工程支架复合材料降解特性

现有支架材料的降解速率与骨细胞生长、繁殖速率不匹配,在降解过程中支架材料的强度、刚度衰减速率与成骨速率不匹配,支架材料在体内降解的酸性副产物会引起炎症反应.为克服以上困难,采用溶媒浇铸、颗粒滤取与气体发泡相结合的方法制备出纳米HAP/CPP/PLLA骨组织工程支架复合材料;选用生理盐水作为模拟体液进行降解实验,测试该支架复合材料的降解性能,用扫描电子显微镜对其在不同降解时期的微观结构进行观察.结果表明:纳米HAP/CPP/PLLA支架复合材料在降解过程中具有三维、连通、微孔网状结构,并具有良好的降解性能和生物相容性,是比较理想的骨组织工程支架材料.     

新型双层皮肤组织工程支架的构建

模拟天然皮肤的真、表皮结构,以胶原和壳聚糖为主要原料构建皮肤组织工程双层支架.将固含量0.8%(mass)的胶原-壳聚糖溶液冻干后压膜,经戊二醛交联后再冻干形成双层支架的底层,然后注入固含量0.2%(mass)的胶原-壳聚糖溶液,经冻干、交联、再冻干即得结构较稳定的双层复合海绵支架.在双层支架上培养的小鼠成纤维细胞贴壁生长正常.本研究构建的胶原-壳聚糖双层支架具有良好的结构稳定性和细胞相容性.     

胶原支架材料的制备与表征

分别采用真空高温脱水和化学交联剂1-乙基-3-(3-二甲基氨基丙基)-碳化二亚胺处理经冷冻干燥后的胶原海绵材料,制备组织工程支架,讨论反应条件对胶原性能的影响,测定胶原支架的力学强度和降解特性.结果发现,不同的处理方法都能保持胶原海绵的三维多孔结构,孔隙率可达到90%;真空高温脱水获得的胶原海绵材料力学强度较小,但断裂伸长率略大;碳化二亚胺交联胶原海绵材料力学强度相对较高,抗张强度为380 kPa左右.胶原海绵交联后,降解速率显著小于未经交联的胶原材料.细胞培养试验表明成纤维细胞可以在支架材料上正常生长.两种交联方法处理胶原海绵,其细胞生长行为差异无显著性,适合于作为组织工程支架.     

组织工程用聚乳酸梯度支架的制备及分形研究

采用改进的溶液浇铸/粒子沥滤工艺,将聚乳酸（PLLA）与致孔剂粒子粉碎后,制备了双层和多层孔径梯度变化的PLLA泡沫支架。扫描电镜观察表明,材料内部界面处存在一定的扩散现象,且界面处孔的连通性好,通过调整致孔剂粒子的粒径以及PLLA与致孔剂粒子的比例,可有效地调控支架的各项参数（材料大小、孔径、孔隙率）。对材料进行分形研究,表明梯度多孔支架内部无明显界面,孔径过渡平缓。     

Hippocampal Neuron-Derived Extracellular Matrix Coated Nanofibrous Scaffold for Neural Tissue Engineering

The aim of this study is to prepare poly-L-lactide( PLLA) electrospun nanofibrous scaffolds coated with hippocampal neuron-derived extracellular matrix( N-ECM)and construct a novel neural tissue engineering scaffold.Neonatal rat hippocampal neurons were seeded on PLLA nanofibers,and then decellularized to derive a cell-free extracellular matrix loaded N-ECM/PLLA modified scaffolds. The morphology and ingredients of N-ECM/PLLA were observed by scanning electron microscopy( SEM) and immunofluorescence staining respectively, and the cytocompatibility of the composite scaffolds was characterized by cell count kit-8( CCK-8) assay. The N-ECM was clearly identified loading on scaffolds when being imaged via SEM and immunofluorescence staining results showed that the N-ECM was made up of fibronectin and laminin. Most importantly, compared with tissue culture polystyrene and pure scaffolds, N-ECM/PLLA scaffolds could effectively facilitate the proliferation of rat adrenal neuroma cells( PC12 cells),indicating their better cell compatibilities. Based on the combination of N-ECM and PLLA biomaterials,the present study has fabricated a unique and versatile neural tissue engineering scaffold,offering a new thought for future neural tissue engineering.     

Preparation of PLGA-collagen hybrid scaffolds with controlled pore structures for cartilage tissue engineering

Scaffolds used for cartilage tissue engineering should have high mechanical strength and well-controlled pore structure to provide suitable microenvironments for functional tissue regeneration. In this study, hybrid scaffolds which had open and interconnected pore structure and high mechanical property were prepared by hybridization of PLGA mesh and collagen using ice particulates as porogen templates. Embossing ice particulates template was used to form open pore structures on the scaffold surfaces. Free ice particulates were used to generate interconnected bulk pores in the scaffolds. Hybridization with PLGA mesh provided the scaffolds with high mechanical property. Bovine articular chondrocytes were cultured in the hybrid scaffolds. The unique pore structures facilitated the homogeneous distribution of chondrocytes and cartilaginous matrices throughout the scaffolds. Subcutaneous implantation demonstrated cartilage-like tissue regeneration. The hybrid scaffolds should have a high potential for cartilage tissue engineering.     

Dexamethasone-Loaded PLGA Microspheres Incorporated PLLA/PLGA/PCL Composite Scaffold for Bone Tissue Engineering

The combination of micro-carriers and polymer scaffolds as promising bone grafts have attracted considerable interest in recent decades.The poly(L-lactic acid)/poly(lactic-co-glycolic acid)/polycaprolactone(PLLA/PLGA/PCL)composite scaffold with porous structure was fabricated by thermally induced phase separation(TIPS).Dexamethasone(DEX)was incorporated into PLGA microspheres and then loaded on the PLLA/PLGA/PCL scaffoldtopreparethedesiredcompositescaffold.The physicochemical properties of the prepared composite scaffold were characterized.The morphology of rat bone marrow mesenchymal stem cells(BMSCs)grown on scaffolds was observed using scanning electron microscope(SEM)and fluorescence microscope.The resultsshowedthatthePLLA/PLGA/PCLscaffoldhad interconnected macropores and biomimetic nanofibrous structure.In addition,DEX can be released from scaffold in a sustained manner.More importantly,DEX loaded composite scaffold can effectively support the proliferation of BMSCs as indicated by fluorescence observation and cell proliferation assay.The results suggested that the prepared PLLA/PLGA/PCL composite scaffold incorporating drug-loaded PLGA microspheres could hold great potential for bone tissue engineering applications.     

Incorporation of Sphingosine 1-Phosphate Loaded Mesoporous Silica Nanoparticles into Poly( L-lactic acid ) Nanofibrous Scaffolds for Bone Tissue Engineering

Controlled release of the functional factors is the key to improve clinical therapeutic efficacy during the tissue repair and regeneration.The three-dimensional(3D)scaffold can provide not only physical properties such as high strength and porosity but also an optimal environment to enhance tissue regeneration.Sphingosine1-phosphate(S1P),an angiogenic factor,was loaded into mesoporous silica nanoparticles(MSNs)and then incorporated into poly(L-lactic acid)(PLLA)nanofibrous scaffold,which was fabricated by thermally induced phase separation(TIPS)method.The prepared scaffolds were examined by attenuated total reflection Fourier transform infrared spectroscopy(ATR-FTIR),scanning electron microscopy(SEM),and transmission electron microscopy(TEM)and compressive mechanical test.The ATR-FTIR result demonstrated the existence of MSNs in the PLLA nanofibrous scaffold.The SEM images showed that PLLA scaffold had regular pore channel,interconnected pores and nanofibrous structure.The addition of MSNs at appropriate content had no visible effect on the structure of scaffold.The compressive modulus of scaffold containing MSNs was higher than that of the scaffold without MSNs.Furthermore,fluorescein isothiocyanate(FITC)was used as model molecule to investigate the release behavior of S1P from MSNsincorporated PLLA(MSNs/PLLA)nanofibrous scaffold.The result showed that the composite scaffold largely reduced the initial burst release and exhibited prolonged release of FITC than MSNs.Thus,these results indicated that S1P-loaded composite nanofibrous scaffold has potential applications for bone tissue engineering.     

Nanofibrous Scaffold Containing Osteoblast-Derived Extracellular Matrix for the Proliferation of Bone Marrow Mesenchymal Stem Cells

Extracellular matrix( ECM) plays a prominent role in establishing and maintaining an appropriate microenvironment for tissue regeneration. The aims of this study were to construct a tissue engineered scaffold by reconstituting osteoblast cell-derived ECM( O-ECM) on the electrospun nanofibrous scaffold,and further to evaluate its subsequent application for promoting the proliferation of bone marrow mesenchymal stem cells( BMSCs). To engineer a biomimetic scaffold, calvarial osteoblasts and electrospun poly-llactic acid( PLLA) nanofibers were prepared and subjected to decellularize for O-ECM deposition. To evaluate and characterize the O-ECM/PLLA scaffold, the morphology was examined and several specific mark proteins of osteoblasts matrix were evaluated.Furthermore,the cell counting kit-8( CCK-8) assay was used to detect the proliferation of the BMSCs cultivated on the O-ECM/PLLA scaffold. The results indicated O-ECM/PLLA scaffold was loaded with Collagen I, Fibronectin, and Laminin, as the composition of the marrow ECM. After decellularization,O-ECM deposition was observed in O-ECM/PLLA scaffold. Moreover,the O-ECM/PLLA scaffold could significantly enhance the proliferation of BMSCs,suggesting better cytocompatibility compared to the other groups tested. Taken together,a biomimetic scaffold based on the joint use of O-ECM and PLLA biomaterials,which represents a promising approach to bone tissue engineering, facilitates the expansion of BMSCs in vitro.     

Fabrication and characterization of electrospun poly-L-lactide/gelatin graded tubular scaffolds: Toward a new design for performance enhancement in vascular tissue engineering

In this study, a new design of graded tubular scaffolds have been developed for the performance enhancement in vascular tissue engineering. The graded poly-L-lactide (PLLA) and gelatin fibrous scaffolds produced by electrospining were then characterized. The morphology, degradability, porosity, pore size and mechanical properties of four tubular scaffolds (graded PLLA/gelatin, layered PLLA/gelatin, PLLA and gelatin scaffolds) have been investigated. The tensile tests demonstrated that the mechanical strength and also the estimated burst pressure of the graded scaffolds were significantly increased in comparison with the layered and gelatin scaffolds. This new design, resulting in an increase in the mechanical properties, suggested the widespread use of these scaffolds in vascular tissue engineering in order to prepare more strengthened vessels.     

Disc-Electrospun PCL Nanofiber Formed Micro-patterned Structure Scaffold

Disc-electrospinning using a disc as spinneret and a rotary drum as collector is a novel technology to prepare nanofiber which has been applied in tissue engineering scaffolds. In this study, nanofibrous mats with mlcro-patterned structure were fabricated via disc-electrospinning. Poly （ε-eaprolactone） （PCL） was dissolved in trifluoroethanol （TFE） at various concentrations （ 2 %-7 % ） （w/v） for electrospinning and the applied voltage ranged from 40 to 70 kV. Scanning electron microscopy （SEM） was employed to observe the morphology of the nanofibrous scaffolds. SEM images illustrated that the nanofibers with beads formed micro-patterned structure such as triangles and other polygons. The average diameter of nanofibers presented various size with the concentration increased from 2% to 7%. The beads on the nanofibers constructed the vertexes of the polygons, while nanofibers bridged between adjacent vertexes. The concentration of solution and applied voltage may be two dominant factors to influence the topological structure of the nanofibrons scaffolds. Cells cultured on the micro-patterned scaffold spread along the edges of the polygons. The scaffold with patterned structure may have a promising application in tissue engineering.     

3D打印制备HA/PCL复合材料组织工程支架的研究

应用熔融沉积成型技术（FDM）制备羟基磷灰石（HA）/聚己内酯（PCL）组织工程支架,探讨其内部结构和力学性能。以羟基磷灰石和聚己内酯为原料,采用熔融共混技术分别制备HA质量分数为20%的nano-HA/PCL和micro-HA/PCL复合材料,使用自主研发的熔体微分FDM 3D打印机制备HA/PCL复合材料组织工程支架。通过显微镜观察发现,所制备的nano-HA/PCL和micro-HA/PCL组织工程支架具有均匀分布且相互连通的近似矩形的孔隙。nano-HA/PCL和micro-HA/PCL组织工程支架的断面图分析结果表明,nano-HA/PCL组织工程支架中HA粒子分布均匀,而micro-HA/PCL组织工程支架中HA粒子发生了团聚,导致nano-HA/PCL组织工程支架的拉伸强度和弯曲强度均高于micro-HA/PCL组织工程支架。因此,利用熔体微分FDM 3D打印机打印生物活性nano-HA/PCL复合材料组织工程支架在骨组织工程中具有潜在的应用前景。     

CPP/PLLA软骨组织工程支架复合材料降解性能研究

采用溶媒浇铸/粒子滤取技术与气体发泡相结合的方法制备出CPP/PLLA软骨组织工程支架复合材料,测试了该支架复合材料的物理力学性能和降解性能.研究结果表明,CPP/PLLA软骨组织工程支架复合材料具有适宜的孔隙率,良好的降解性能和物理力学性能,以及三维连通、微孔、网状微观结构.因此,该复合材料有望成为软骨组织工程支架材料之一.     

聚乙烯醇-透明质酸-胶原组织工程支架研究

以聚乙烯醇(PVA)、透明质酸(HA)和胶原蛋白(Col)为组分,构建组织工程三维多孔支架.将聚乙烯醇分别与不同质量的胶原和透明质酸复合,测定复合材料的含水率和膨胀率.扫描电镜观察材料横截面的表面形态.结果是不同分子质量的PVA与不同配比的Col和HA复合,得到的复合支架材料含水率为57.97%～81.26%,膨胀率为150.00%～459.20%.不同配比的复合支架材料具有不同的内部孔洞结构.结果表明,PVA-HA-Col复合支架材料具有较高的含水率和膨胀率,内部孔洞网络丰富,可作为组织工程支架材料.