聚己内酯静电纺丝纳米纤维支架用于小鼠诱导多能干细胞培养

干细胞联合生物支架材料体外构建功能性组织与器官,成为当前组织再生研究的重要策略,而探求具有良好生物相容性的支架材料是其关键.本研究采用扫描电镜、噻唑蓝（MTT）法、荧光显微染色等方法检测小鼠诱导多能干细胞（murine induced pluripotent stem cells, miPSCs）在聚己内酯（poly ε-caprolactone, PCL）静电纺丝纳米纤维支架上的粘附、增殖等生物学特性,探究聚己内酯纳米纤维支架与miPSCs的生物相容性. 结果显示,miPSC在PCL纳米纤维支架上具有良好粘附性并呈集落样生长,其增殖能力及干性标记物（Oct4-GFP+）的表达均不亚于标准对照组;扫描电镜显示,miPSC在PCL纳米纤维支架材料上呈现出绒毛状突起的表面结构.上述结果表明,PCL纳米纤维支架可促进miPSCs的粘附、自我增殖以及干性维持,两者具有良好的生物相容性,为下一步联合生物支架材料与干细胞构建功能性组织奠定了基础.     

可降解组织工程血管支架成功用于犬股动脉移植

目的采用可降解的聚己内酯接枝肝素材料,负荷b-FGF(碱性成纤维细胞生长因子),体外构建的小口径组织工程血管,完成犬的股动脉移植动物实验。方法利用可降解的聚己内酯接枝肝素材料,电纺丝技术制备组织工程血管支架,并对支架负荷b-FGF生长因子,并进行材料的内皮细胞粘附实验。将体外构建的小口径组织工程血管,完成犬的股动脉移植动物实验,观察通畅率和移植术后组织工程血管的改变。结果可降解聚己内酯接枝肝素材料支架,负荷细胞生长因子(b-FGF),利于内皮细胞粘附。构建的组织工程血管进行体外动物实验构建,3个月移植物通畅率好,移植后取材,有新生内膜迁移和胶原纤维浸入。结论利用可降解聚己内酯接枝肝素材料构建小口径支架,初步符合构建组织工程血管支架的要求。     

Ⅰ型胶原/聚己内酯/凹凸棒石复合支架材料体外诱导成骨的研究

目的:研究Ⅰ型胶原(ColⅠ)/聚己内酯(PCL)/凹凸棒石(ATP)复合支架材料的生物相容性及体外骨诱导性。方法:采用溶液浇铸-粒子滤沥法制备三种不同ATP含量(0% wt、10% wt、30% wt)的ColⅠ/PCL/ATP复合支架材料;将D1细胞与三种支架材料共培养,扫描电镜、鬼笔环肽和H&E染色、CCK-8法评价支架材料的生物相容性;D1细胞复合三种支架材料培养7天、14天、21天后RT-qPCR检测其成骨相关基因(Runx-2、Osterix、ALP、Col I、OPN、OC)的相对表达量,分别评价比较三种支架材料的成骨诱导效应。结果:SEM、鬼笔环肽和H&E染色显示D1细胞在三种支架材料表面均呈现良好的黏附;CCK-8结果显示,细胞在ATP含量30% wt的支架材料上增殖率显著高于其他两组,RT-qPCR检测结果显示,与0% wt、10% wt ATP相比,30% wt ATP组的Runx-2相对表达量在7天时显著升高, 14天、21天降低;ALP相对表达量在14天时显著升高,21天时显著降低;Osterix、Col I、OPN、OC的相对表达量随时间和ATP剂量的增加显著上调(P<0.05)。结论:ColⅠ/PCL/ATP复合支架材料具有良好的生物相容性及骨诱导性,有望成为一种理想的骨组织工程支架材料。     

综述——纳米材料与药物输递：聚乙二醇/聚己内酯嵌段共聚物温敏水凝胶及其在局部药物递送中的应用

近年来,温敏水凝胶被广泛用于药物递送、组织工程等生物医用领域．其中,由聚乙二醇与脂肪族可降解聚酯合成的两亲性聚合物的自组装胶束形成的温敏水凝胶是一种重要的温敏凝胶材料．本文针对聚乙二醇(PEG)与聚己内酯(PCL)形成的两亲性嵌段聚合物温敏水凝胶体系,综述了聚合物分子质量、嵌段序列结构,亲疏水段分子质量与比例、疏水段化学结构等因素对温敏行为的影响,以及该温敏水凝胶在局部药物递送方面的研究进展．     

聚乳酸基热固性聚氨酯及其形状记忆性能

通过聚乳酸二元醇和聚乳酸-聚己内酯共聚物二元醇与六亚甲基二异氰酸酯(HDI)三聚体交联反应合成了一系列生物基热固性聚氨酯(Bio-PUs)。利用傅里叶红外(FTIR)、差示扫描量热分析(DSC)、热失重分析(TGA)、万能拉伸机和细胞毒性等测试方法对获得的聚乳酸基聚氨酯进行了表征。结果表明,与聚乳酸二元醇相比,聚乳酸-聚己内酯共聚物二元醇降低了生物基热固性聚氨酯的玻璃化温度(Tg),提高了热固性聚氨酯的热稳定性;且聚乳酸-聚己内酯型聚氨酯的力学性能和形状记忆性能更为优异。其中,聚乳酸-聚己内酯共聚物二元醇分子量为3 000时得到的热固性聚氨酯(Bio-PU2-3000)的杨氏模量为277.7 MPa,伸长率为230%;聚乳酸-聚己内酯共聚物二元醇分子量为1 000得到的热固性聚氨酯(Bio-PU2-1000)在人体体温下的形变回复时间仅为93 s。另外,通过显微镜观察到细胞在含聚乳酸基热固性聚氨酯的培养液中生长状态良好,表明制备得到的生物基聚氨酯无细胞毒性。     

利用聚己内酯/羟基磷灰石复合支架修复犬胸骨缺损

目的：探讨利用生物可降解支架修复动物胸骨缺损,为临床手术治疗提供新的可行性方法。方法：对于12只比格犬进行手术切除部分胸骨,并利用聚己内酯/羟基磷灰石（PCL/HA）复合支架,并制备出与临床相似的胸骨缺损模型。实验动物分成2组,分别是：空白对照组和PCL/HA支架组。分别于术后第4、12周进行胸部CT扫描,并对胸廓进行三维重建,观察胸骨缺损部位的修复情况,并在第12周取胸骨缺损部位组织进行硬组织切片,苦味酸-品红染色,观察缺损部位的骨组织修复情况,并利用软件进行骨组织比率分析,评估修复情况。结果：通过检查发现空白对照组的胸骨缺损部位未见明显骨连接,胸廓的骨性结构有明显畸形,PCL/HA支架组能很好地维持胸廓的完整性,组织学检查发现PCL/HA支架组的缺损部位有明显新生骨形成,通过软件分析可发现支架组的骨组织比率较空白组的高（P〈0.05）。结论：这些结果表明采用PCL/HA复合材料支架能很好地修复胸骨缺损。     

基于组织工程研究的可降解支架材料选择策略

目前,器官或组织移植是治疗器官衰竭或大范围组织缺损唯一长期有效的方法,但存在供体短缺、免疫排斥等问题。组织工程技术作为一种潜在的替代治疗方法,支架材料的选择是其中具有决定意义的组成部分。组织工程支架材料按其来源可分为天然及其改性修饰材料、人工合成与复合支架材料3种。组织工程目的就是修复临床上的病损组织或器官,并达到较理想的结构和功能的恢复。因此组织工程支架也必须从基本性质上具有一定的仿生化结构及功能,即"活"支架,这样才能彻底代替病损组织或器官。通过多种支架材料的优化组合(即材料的复合),对材料进行表面改性、制备工艺优化及添加细胞因子缓释微球等技术,模拟病损器官组织的特性及周围环境,有望打开组织工程的新局面。理想的组织工程支架应当以临床需要为根本目的,依靠材料学、分子生物学、工程学等多学科间的交叉研究,取各家之长,优化配比组合,达到仿生的目的。本课题组前期工作已经将骨髓间充质干细胞体外诱导分化为胆管上皮样细胞,并设计出左旋聚乳酸/聚己内酯共聚物(PLCL)胆道支架,内部混有包含生长因子的纳米缓释微球,供细胞因子的远期释放,支架内表面涂有基质胶/胶原混合层,且胶内加入bFGF、EGF,提供诱导因子的早期释放。将诱导细胞与PLCL胆道支架复合,制备组织工程胆管。文中综述了现存各类支架材料的研究状况,简单介绍了制备工艺、表面修饰等影响支架性能的因素,力求探索组织工程支架材料的选择策略。     

仿生聚己内酯支架用于乳房组织工程的可行性研究

目的探讨聚己内酯（PCL）乳房形态支架用于组织工程乳房的构建的可能性。 方法通过熔融沉积3D打印制备形态仿生的PCL支架,测量其机械性能,并使用新西兰大白兔动物模型,皮下植入该PCL支架12周和18周后,利用核磁共振成像（MRI）观察支架内部新生组织分布情况,在组织学（HE、Masson及EVG染色）上评估支架内部的脂肪、纤维及血管的分布情况,并进一步使用qRT-PCR检测了12周时PCL支架内部组织的成脂相关基因（PPAR-γ、C/EBP-β、AP-2）、炎症相关基因TNF-α及巨噬细胞标记物F4-80的表达情况,同时使用凝胶渗透色谱法分析了PCL植入体内后平均分子量的变化。2组间均数比较采用独立样本t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验,配对设计的均数比较采用配对t检验。 结果制备的PCL支架孔隙率为（85.30±1.12）%,压缩模量为（8.18±1.39）MPa,植入新西兰大白兔动物模型皮下12周后,MRI影像学显示脂肪组织已由支架周围向内部侵入,HE、Masson及EVG染色同样在该支架边缘观察到部分新生脂肪组织及血管,而支架内部则以疏松排列的纤维组织为主;与原生脂肪比较,12周PCL支架内组织的基因表达分析成脂相关基因C/EBPβ表达水平（2.32±0.28比1.00±0.02）升高,而巨噬细胞标记物F4/80表达（0.80±0.12比1.00±0.03）降低（P均< 0.01）;18周后,HE染色证实支架内部已充满脂肪组织。基因表达证实,与原生脂肪比较,支架内部组织C/EBP-β （3.30±0.63比1.00±0.02）,PPAR-γ （1.81±0.71比0.99±0.02）及AP-2表达水平（1.38±0.16比1.01±0.01）升高（P均< 0.01）;而TNF-α（0.50±0.15比1.00±0.01）及F4/80表达水平（0.52±0.09比1.00±0.03）均降低（P均< 0.001）。而植入体内PCL支架的分子量（M_n）在18周内变化不大[（65.04±2.24）kDa比（64.20±4.09） kDa]。 结论PCL支架具有较好的生物相容性,可用于组织工程乳房的构建,该新西兰大白兔动物模型的建立有利于乳房组织工程的进一步临床转化。     

锶纳米纤维能促进骨组织再生

为了探究锶纳米纤维在骨组织再生中的作用机制,本研究通过静电纺丝来制备聚合物纳米复合材料工程支架,促进骨组织再生。将碳酸锶纳米粒子(nSrCO3)以10%和15%重量比添加到聚己酸内酯(PCL)中,做成纤维直径在300～500 nm范围内的纳米复合材料纤维支架(PCL+10%SrCO3和PCL+15%SrCO3),掺入nSrCO3后降低了纤维支架的结晶度和弹性模量,复合PCL+15%SrCO3支架可在4 d内释放出高达58 ppm的Sr2+离子。细胞研究证实,体外使用的含有15%n Sr CO3的复合支架增强了人间充质干细胞的增殖。在PCL+15%SrCO3中,最小沉积量显著增加达约4倍,促进了骨形成。在PCL+15%SrCO3纤维中,成骨标志物如BMP-2和Runx2的mRNA和蛋白的高表达也可以证明锶纳米纤维能促进骨形成。本研究为研究锶纳米纤维在促进骨组织再生中的运用提供了一定的参考价值。     

膜性高分子材料在兔眼部的生物相容性

目的评价聚羟基脂肪酸(polyhydroxyalkanoates,PHA)、聚乳酸(polylacetic acid,PLA)和聚己内酯(polycaprolactone,PCL)三种膜性高分子材料在兔眼部的生物相容性。方法将24只新西兰兔随机分为4组,每组6只。PHA、PLA、PCL为实验组,材料植入兔右眼结膜下。假手术组结膜下钝性分离,但不植入任何高分子材料。使用裂隙灯显微镜观察并记录植入后不同时间手术眼的反应并评分。裂隙灯下观察材料的吸收时间。术后4周和16周取眼球,行HE染色、Masson染色和天狼猩红染色分别定性观察组织结构和炎症细胞、胶原纤维和胶原纤维的亚型与排列方向。结果术眼刺激性评分等级各组均不高于"轻度刺激性"。结膜下吸收时间PHA、PLA和PCL组分别是16周,12周和大于16周。组织学观察术后4周PHA、PLA和PCL组均形成材料包裹囊腔,囊壁以纤维组织为主,伴有毛细血管形成和炎性细胞浸润,以中性粒细胞为主。胶原纤维染色与假手术组无明显差异,以Ⅰ型和Ⅲ型为主,大致呈平行排列。术后16周PHA和PLA组材料已不可查及,包裹囊腔结构不规则,而PCL材料整体可查及,包裹囊腔规则。各组未见毛细血管,偶见淋巴细胞浸润。胶原纤维与假手术组无明显差异,以Ⅰ型和Ⅲ型为主,仍大致呈平行排列。结论 PHA、PLA和PCL三种膜性高分子材料在兔眼具有较好的生物相容性,结膜下吸收时间分别是16周,12周和大于16周。     

PCL-Based Composite Scaffold Matrices for Tissue Engineering Applications

Biomaterial-based scaffolds are important cues in tissue engineering (TE) applications. Recent advances in TE have led to the development of suitable scaffold architecture for various tissue defects. In this narrative review on polycaprolactone (PCL), we have discussed in detail about the synthesis of PCL, various properties and most recent advances of using PCL and PCL blended with either natural or synthetic polymers and ceramic materials for TE applications. Further, various forms of PCL scaffolds such as porous, films and fibrous have been discussed along with the stem cells and their sources employed in various tissue repair strategies. Overall, the present review affords an insight into the properties and applications of PCL in various tissue engineering applications.     

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。     

基于天然水凝胶的生物材料在骨组织工程中的应用*

天然水凝胶是指原材料来自于天然生物材料的水凝胶。由于这种天然的聚合物含有构成生物体的天然成分,与天然组织具有生物学和化学相似性,而受到特别关注。天然水凝胶由于其与细胞外基质高度的相似性被认为是骨组织工程中优良的仿生基质材料。而针对天然水凝胶机械性能差、成骨诱导性能弱等缺陷,通常需要对天然水凝胶进行改性、引入其他材料或生物活性因子,以此来获得更适用于骨组织工程支架材料。对近年来基于天然水凝胶的生物材料在骨组织工程的应用,与其不同的应用形式(可注射水凝胶、多孔水凝胶支架、3D生物打印水凝胶支架等)进行了概述,以期对这类基于天然水凝胶的生物材料在未来骨组织工程中的应用提供参考。     

Fabrication of porous polycaprolactone/hydroxyapatite (PCL/HA) blend scaffolds using a 3D plotting system for bone tissue engineering

For tissue engineering and regeneration, a porous scaffold with interconnected networks is needed to guide cell attachment and growth/ingrowth in three-dimensional (3D) structure. Using a rapid prototyping (RP) technique, we designed and fabricated 3D plotting system and three types of scaffolds: those from polycaprolactone (PCL), those from PCL and hydroxyapatite (HA), and those from PCL/HA and with a shifted pattern structure (PCL/HA/SP scaffold). Shifted pattern structure was fabricated to increase the cell attachment/adhesion. The PCL/HA/SP scaffold had a lower compressive modulus than PCL and PCL/HA scaffold. However, it has a better cell attachment than the scaffolds without a shifted pattern. MTT assay and alkaline phosphatase activity results for the PCL/HA/SP scaffolds were significantly enhanced compared to the results for the PCL and PCL/HA scaffolds. According to their degree of cell proliferation/differentiation, the scaffolds were in the following order: PCL/HA/SP > PCL/HA > PCL. These 3D scaffolds will be applicable for tissue engineering based on unique plotting system.     

Repair and regeneration of osteochondral defects in the articular joints

People suffering from pain due to osteoarthritic or rheumatoidal changes in the joints are still waiting for a better treatment. Although some studies have achieved success in repairing small cartilage defects, there is no widely accepted method for complete repair of osteochondral defects. Also joint replacements have not yet succeeded in replacing of natural cartilage without complications. Therefore, there is room for a new medical approach, which outperforms currently used methods. The aim of this study is to show potential of using a tissue engineering approach for regeneration of osteochondral defects. The critical review of currently used methods for treatment of osteochondral defects is also provided. In this study, two kinds of hybrid scaffolds developed in Hutmacher's group have been analysed. The first biphasic scaffold consists of fibrin and PCL. The fibrin serves as a cartilage phase while the porous PCL scaffold acts as the subchondral phase. The second system comprises of PCL and PCL-TCP. The scaffolds were fabricated via fused deposition modeling which is a rapid prototyping system. Bone marrow-derived mesenchymal cells were isolated from New Zealand White rabbits, cultured in vitro and seeded into the scaffolds. Bone regenerations of the subchondral phases were quantified via micro CT analysis and the results demonstrated the potential of the porous PCL and PCL-TCP scaffolds in promoting bone healing. Fibrin was found to be lacking in this aspect as it degrades rapidly. On the other hand, the porous PCL scaffold degrades slowly hence it provides an effective mechanical support. This study shows that in the field of cartilage repair or replacement, tissue engineering may have big impact in the future. In vivo bone and cartilage engineering via combining a novel composite, biphasic scaffold technology with a MSC has been shown a high potential in the knee defect regeneration in the animal models. However, the clinical application of tissue engineering requires the future research work due to several problems, such as scaffold design, cellular delivery and implantation strategies.     

Electrospun poly (ɛ-caprolactone)/silk fibroin core-sheath nanofibers and their potential applications in tissue engineering and drug release

One of the key tenets of tissue engineering is to develop scaffold materials with favorable biodegradability, surface properties, outstanding mechanical strength and controlled drug release property. In this study, we generated core-sheath nanofibers composed of poly (?-caprolactone) (PCL) and silk fibroin (SF) blends via emulsion electrospinning. Nanofibrous scaffolds were characterized by combined techniques of scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), differential scanning calorimetry (DSC), contact angle and tensile measurements. An in vitro FITC release study was conducted to evaluate sustained release potential of the core-sheath structured nanofibers. We found that the conformation of SF contained in PCL/SF composite nanofibers was transformed from random coil to β-sheet when treated with methanol, leading to improved crystallinity and tensile strength of nanofibrous scaffolds. The hydrophobicity and diameter of nanofibers decreased when we increased the content of SF in PCL/SF composite nanofibers. Furthermore, we evaluated the potential of fabricated PCL/SF composite nanofibers as scaffold in vitro. The results confirmed that fabricated PCL/SF scaffolds improved cell attachment and proliferation. Our results demonstrated the feasibility to generate core-sheath nanofibers composed of PCL and SF using a single-nozzle technique. The produced nanofibrous scaffolds with sustained drug release have potential application in tissue engineering.     

Open-Source Selective Laser Sintering (OpenSLS) of Nylon and Biocompatible Polycaprolactone

Selective Laser Sintering (SLS) is an additive manufacturing process that uses a laser to fuse powdered starting materials into solid 3D structures. Despite the potential for fabrication of complex, high-resolution structures with SLS using diverse starting materials (including biomaterials), prohibitive costs of commercial SLS systems have hindered the wide adoption of this technology in the scientific community. Here, we developed a low-cost, open-source SLS system (OpenSLS) and demonstrated its capacity to fabricate structures in nylon with sub-millimeter features and overhanging regions. Subsequently, we demonstrated fabrication of polycaprolactone (PCL) into macroporous structures such as a diamond lattice. Widespread interest in using PCL for bone tissue engineering suggests that PCL lattices are relevant model scaffold geometries for engineering bone. SLS of materials with large powder grain size (~500 μm) leads to part surfaces with high roughness, so we further introduced a simple vapor-smoothing technique to reduce the surface roughness of sintered PCL structures which further improves their elastic modulus and yield stress. Vapor-smoothed PCL can also be used for sacrificial templating of perfusable fluidic networks within orthogonal materials such as poly(dimethylsiloxane) silicone. Finally, we demonstrated that human mesenchymal stem cells were able to adhere, survive, and differentiate down an osteogenic lineage on sintered and smoothed PCL surfaces, suggesting that OpenSLS has the potential to produce PCL scaffolds useful for cell studies. OpenSLS provides the scientific community with an accessible platform for the study of laser sintering and the fabrication of complex geometries in diverse materials.     

Polycaprolactone scaffolds fabricated with an advanced electrohydrodynamic direct-printing method for bone tissue regeneration

Electrohydrodynamic (EHD) direct writing has been used in diverse microelectromechanical systems and various supplemental methods for biotechnology and electronics. In this work, we expanded the use of EHD-induced direct writing to fabricate 3D biomedical scaffolds designed as porous structures for bone tissue engineering. To prepare the scaffolds, we modified a grounded target used in conventional EHD direct printing using a poly(ethylene oxide) solution bath, elastically cushioning the plotted struts to prevent crumbling. The fabricated scaffolds were assessed for not only physical properties including surface roughness and water uptake ability but also biological capabilities by culturing osteoblast-like cells (MG63) for the EHD-plotted polycaprolactone (PCL) scaffold. The EHD-scaffolds showed significantly roughened surface and enhanced water-absorption ability (400% increase) compared with the pure rapid-prototyped PCL. The results of cell viability, alkaline phosphatase activity, and mineralization analyses showed significantly enhanced biological properties of the scaffold (20 times the cell viability and 6 times the mineralization) compared with the scaffolds fabricated using RP technology. Because of the results, the modified EHD direct-writing process can be a promising method for fabricating 3D biomedical scaffolds in tissue engineering.