丝素蛋白来源的一体化纤维环-髓核支架的制备与评估

目的 评估以丝素蛋白为材料构建的一体化纤维环-髓核双相支架作为组织工程椎间盘支架的可行性。方法 以丝素蛋白溶液为原料, 分别采用石蜡球致孔法和相分离法制备三维多孔一体化纤维环-髓核支架。采用体视显微镜、 扫描电镜观察支架内部结构, 测定双相支架纤维环相和髓核相的孔径、 孔隙率及一体化压缩弹性模量; 分离培养兔纤维环细胞和髓核细胞, 接种至双相支架的相应部位, 体外培养 48 h, 扫描电镜、 死活 （Live/dead） 细胞染色评价支架与细胞的生物相容性; CCK-8 检测细胞的增殖活性。结果 体视显微镜和扫描电镜可见双相支架纤维环相和髓核相均呈相互连通的多孔结构, 孔隙高度连通, 纤维环髓核交接区域结合紧密; 纤维环相孔径为 （220.0±23.1）μm, 髓核相孔径为 （90.0±17.8） μm; 孔隙率分别为 91%和 93%; 一体化支架压缩弹性模量为 （150.7±6.8） kPa。扫描电镜可见均匀地黏附在支架表面, 细胞周围有细胞外基质分泌; Live/dead 染色显示细胞在支架上活性良好, 无死细胞;CCK-8 增殖分析显示纤维环细胞和髓核细胞均具有良好的增殖活性。结论 以天然丝素蛋白构建一体化纤维环髓核双相支架, 具有良好的孔径、 孔隙率和细胞相容性, 一体化支架两部分结合紧密, 并且具有优越的力学性能, 是构建组织工程椎间盘的理想支架载体。     

聚己内酯/海藻酸钠/壳聚糖材料制备 组织工程椎间盘双相支架

摘要：目的 以聚己内酯（PCL）、海藻酸钠、壳聚糖为材料,研制椎间盘双相支架,并评估其作为组织工程椎间盘 的可行性。方法 聚己内酯作为原料,采用熔融电纺法制备取向性多孔纤维环支架,将海藻酸钠/壳聚糖水凝胶注入 到中空的纤维环（AF）支架中央合成双相椎间盘支架。通过体式显微镜、扫描电镜观测双相支架的结构、孔径、孔隙 率;人脐带干细胞复合双相支架体外培养7 d,用死活细胞染色法评价生物相容性,CCK-8实验测定细胞增殖情况,力 学加载仪器测量双相支架的压缩弹性模量。结果 体式显微镜和扫描电镜可见纤维环相成菱形多孔结构,髓核相 （NP）呈不规则多孔结构;纤维环相和髓核相孔径分别为（225.6±3.9）μm、（205.5±5.2）μm,孔隙率分别为（74.17± 0.39）%、（85.52±0.48）%,支架扫描电镜可见细胞黏附在支架表面,周围有细胞外基质分泌;死活细胞染色显示无死 细胞;CCK-8检测结果显示人脐带干细胞具有良好的增殖活性,压缩弹性模量（173.24±44.93）kPa。结论 以聚己内 酯、海藻酸钠、壳聚糖为材料制备的椎间盘双相支架,具有良好的孔径、孔隙率和细胞相容性,支架间结合紧密,具有 三维网络结构,优良的力学特性,是构建组织工程椎间盘理想载体。     

仿生圆周取向微米纤维支架构建组织工程椎间盘纤维环

摘要： 目的 探讨仿生圆周取向微米纤维支架作为组织工程椎间盘支架的可行性。方法 以聚己内酯溶液为原料, 采用湿法纺丝技术模拟天然纤维环结构, 制备三维圆周取向的微米纤维支架。通过体视显微镜观察支架宏观结构; 扫描电镜、 Micro-CT对支架微观结构、 孔隙率、 纤维直径进行测定; 兔纤维环细胞复合微纤维支架体外培养14 d, 运用扫描电镜、 Live/dead染色评价支架的细胞生物相容性; DiI标记细胞膜和鬼笔环肽染色观察细胞在支架上的长入和生长情况; H&E染色、 番红O染色、 Ⅰ型免疫组化和免疫荧光染色对细胞在支架上的行为进行组织学评价。结果 体视显微镜显示, 纤维环支架呈类似天然纤维环的圆环结构; 扫描电镜和Micro-CT显示, 支架呈3D圆周取向纤维结构, 纤维之间无粘连。纤维环支架的孔隙率为69.33%±6.67%, 纤维直径为 （16.13±2.77） μm。扫描电镜和鬼笔环肽染色见细胞呈长梭形并沿纤维方向铺展, DiI标记细胞膜显示培养14 d后纤维环细胞长入支架内部。组织学染色显示纤维环细胞和分泌的细胞外基质均可沿微米纤维呈取向性分布。结论 以聚己内酯为材料, 采用湿纺成形法制备的微米纤维支架可诱导纤维环细胞取向性生长, 并诱导细胞外基质取向分布, 是构建组织工程椎间盘的理想支架载体。     

取向性丝素蛋白仿生软骨支架的制备及其生物相容性评估

目的 利用定向结晶技术制备具有仿生取向微孔结构的丝素蛋白支架,探讨其作为软骨支架的可行性。方法 以丝素蛋白为原料,采用定向结晶技术制成具有仿生取向微孔结构的支架,扫描电镜观察支架结构,测量支架的孔径、孔隙率和力学性能。从兔颈背部分离出脂肪干细胞,培养后获取第3 代细胞,接种到支架上。CCK 8 检测支架上细胞增殖情况;HE染色和扫描电镜观察细胞在支架上的黏附能力;LIVE/DEAD染色观察支架上的细胞活性。结果 扫描电镜显示,支架纵切面可见平行排列的微管样结构,具有较为统一的方向性,横切面可见椭圆或圆形孔隙结构,横截面微孔结构的平均孔径为（112.43± 12.65）μ m ,孔隙率高达（90.25± 2.05）% ,支架压缩弹性模量为（52.48± 5.78）kPa;复合脂肪干细胞培养,HE染色和扫描电镜可见细胞均匀黏附在支架表面及孔隙内,分泌大量沿微 孔结构取向分布的细胞外基质;CCK 8 检测结果显示细胞在支架上增殖良好;LIVE/DEAD染色显示细胞保持良好的活性,未见死细胞。结论 取向性丝素蛋白支架具有均匀的仿生取向微孔结构,具有合适的孔径、孔隙率和生物相容性,力学强度良好,可作为软骨组织工程的支架载体。     

电纺参数对PLGA/HA复合支架纤维形貌和直径的影响

目的通过静电纺丝的方法制备PLGA/HA复合支架,探讨电纺参数对复合支架纤维形貌和直径的影响。方法以三氯甲烷和N,N-二甲基甲酰胺为混合溶剂制备PLGA/HA纺丝液,通过调节PLGA的浓度、电压、接收距离,制备具有不同表面形貌的PLGA/HA复合纤维,采用SEM观察PLGA的浓度、电压、接收距离对纤维形貌和直径的影响。结果复合纤维的直径随PLGA浓度的增加而增加;随电压的增加而增加;随接收距离的增加先减小后增加。结论制备PLGA/HA复合支架较合适的电纺参数为：PLGA浓度25%,电压20KV,接收距离15cm。静电纺丝法制得的PLGA/HA复合支架有可能作为骨组织再生的支架在组织工程领域发挥作用。     

透室壁性心肌血运重建生物可降解支架的制备与优化

目的:制备心肌内生物可降解性支架,优化支架制备方式,评估其保持心肌孔道通畅的可行性.方法:以聚己内酯(PCL)为材料,聚乳酸-聚乙醇酸共聚物(PLGA)为药物载体,分别通过静电纺丝法、溶液浇注-溶剂挥发法和熔融挤出法制备成生物可降解性支架.采用万能材料测定仪测定支架的力学性能,制备猪慢性心肌局部缺血模型,在体评估该支架在透室壁性心肌血运重建术(TMR)后对心肌孔道的作用效果.结果:熔融挤出法制备的PCL支架具有较好的力学性能,支架压缩80%时承受的应力为1.7 MPa,在TMR后可保持心肌孔道通畅.结论:成功制备出了心肌内生物可降解性药物缓释支架,熔融挤出法制备出的支架能承受心肌压力,可维持TMR后的心肌孔道通畅.     

电纺PLGA/HA复合支架的细胞相容性研究

目的通过静电纺丝的方法制备PLGA/HA复合支架并对其细胞相容性进行表征。方法先通过高压静电纺丝的方法制备PLGA/HA复合生物支架,再将大鼠的骨髓间充质干细胞接种于复合支架上培养,采用SEM观察细胞在支架上的形貌、MTT法分析细胞的增殖情况。结果 PLGA/HA复合支架是由超细纤维构成的三维多孔结构,与天然的细胞外基质结构相似,骨髓间充质干细胞在其表面附着、铺展良好,HA纳米颗粒的加入明显提高了细胞的粘附率。结论静电纺丝法制得的PLGA/HA复合支架有可能作为骨组织再生的支架在组织工程领域发挥作用。     

负载马桑叶提取物纳米纤维膜的制备及其研究

目的 本研究主要对制备的新型负载马桑叶提取物的静电纺丝膜进行性能评价,为该敷料的进一步开发研究奠定基础。方法 采用静电纺丝技术制备了负载马桑叶提取物的PCL纳米纤维膜,观察了纳米纤维表面形貌和结构,并评价其抗菌性及其生物相容性。结果 负载马桑提取物的纳米纤维复合膜的制备符合要求并显示良好的形貌;负载马桑提取物的PCL/马桑叶提取物纳米纤维膜对L929细胞无毒害作用。结论 负载马桑叶提取物的PCL纳米纤维膜具有良好的抗菌性,可作为一种新型伤口敷料。     

静电纺丝PLGA纳米纤维支架修复猪胆管损伤

目的 观察不同质量比聚乳酸-羟基乙酸(PLGA)胆总管支架在体外胆汁中降解情况,实验探索可降解支架修复胆道损伤的可行性.方法 用扫描电镜观察不同质量比的PLGA纳米纤维膜微观形貌,并测定其短期体外胆汁中降解率.观察其在胆总管Ⅰ期缝合、胆总管切开置入支架和胆管缺损修补手术中的作用.术后观察动物黄疽和胆漏情况和动物自身术前和术后肝酶(ALT、AST、ALP)和血胆红素的变化.进行Masson染色和平滑肌肌动蛋白(α-SMA)、增殖细胞核抗原(PCNA)免疫组化观察肝脏及胆管损伤部位组织病理学改变.结果 PLGA 75/25和90/10两种比例的纤维在胆汁中没有发生明显的降解和变形.胆管缺损修补手术的猪胶原增生程度轻,排列相对有序,α-SMA免疫组化评分最低,PCNA免疫组化示胆管缺损区域有新生胆管上皮细胞形成.结论 可降解静电纺丝PLGA纳米纤维支架能有效支撑和修复胆管缺损.     

多孔细菌纤维素/聚乳酸-羟基乙酸聚合物/羟基磷灰石复合纤维支架生物相容性研究

目的探讨多孔细菌纤维素/聚乳酸-羟基乙酸聚合物/羟基磷灰石（BC-PLGA-HA）复合支架的生物相容性。方法采用溶液浇铸/粒子沥滤法进行BC-PLGA-HA多孔复合支架的制备,测定支架抗张强度和孔隙率,作细胞MG-63培养为对照组,对支架对MG-63细胞增殖、黏附活性以及对碱性磷酸酶（ALP）活性的影响进行评价。结果多孔BC-PLGA-HAP支架孔隙率及抗拉强度分别为（64.3±5.6）%、（8.925±0.913）N/mm2,BC-PLGA-HAP支架对成骨样细胞具有较高的增殖及ALP活性。结论多孔BC-PLGA-HAP复合支架作为一种新型支架材料,具良好的生物相容性及孔隙率。     

基于原子力显微镜的损伤星形胶质细胞弹性模量变化的研究

目的 探讨星形胶质细胞受到损伤刺激后弹性模量的变化。方法 分离、 提取新生 2 d SD 大鼠星形胶质细胞, 通过胶质纤维酸性蛋白 （GFAP） 抗体免疫荧光染色对其进行鉴定。实验分为对照组和损伤组, 损伤组为应用细胞损伤仪损伤后 6 h 的星形胶质细胞, 对照组不予损伤。应用原子力显微镜在液相下测试各组细胞的弹性模量, 并对两组结果进行比较、 分析。结果 大鼠星形胶质细胞纯化率达 95%以上。损伤后 6 h 的星形胶质细胞形态紊乱,部分细胞胞体可见肿胀。获得了星形胶质细胞的力学地形图和力压痕曲线。损伤组星形胶质细胞弹性模量较对照组显著增加［（1 689±693） Pa vs.（724±283） Pa, P＜0.01］。结论 损伤刺激会造成星形胶质细胞弹性模量增大, 为进一步从细胞水平了解创伤性脑损伤后的颅内物理微环境提供理论基础。     

Design and development of nanocomposite scaffolds for auricular reconstruction

Auricular reconstruction using sculpted autologous costal cartilage is effective, but complex and time consuming and may incur donor site sequelae and morbidity. Conventional synthetic alternatives are associated with infection and extrusion in up to about 15% of cases. We present a novel POSS-PCU nanocomposite auricular scaffold, which aims to reduce extrusion rates by mimicking the elastic modulus of human ears and by encouraging desirable cellular interactions. The fabrication, physicochemical properties (including nanoscale topography) and cellular interactions of these scaffolds were compared to Medpor®, the current synthetic standard. Our scaffold had a more similar elastic modulus (5.73 ± 0.17 MPa) to ear cartilage (5.02 ± 0.17 MPa) compared with Medpor®, which was much stiffer (140.9 ± 0.04 MPa). POSS-PCU supported fibroblast ingrowth and proliferation; significantly higher collagen production was also produced by cells on the POSS-PCU than those on Medpor®. This porous POSS-PCU nanocomposite scaffold is therefore a promising alternative biomaterial for auricular surgical reconstruction.From the Clinical EditorIn this paper, a novel POSS-PCU nanocomposite auricular scaffold is described to reduce extrusion rates by having a much closer elastic modulus of human ears than the currently available synthetic standard. Enabling desirable cellular interactions may lead to the successful clinical application of these novel scaffolds.     

A Three-Dimensional Polycaprolactone Scaffold Combined with a Drug Delivery System Consisting of Electrospun Nanofibers

A new three-dimensional (3D) scaffold containing a functional drug delivery system (DDS) consisting of electrospun micro/nanofibers is proposed. In the DDS scaffold, a core-shell laminated, structured, electrospun mat of hydrophobic polycaprolactone (PCL) and hydrophilic poly(ethylene oxide) (PEO)/rhodamine-B fibers was embedded in the normal 3D PCL scaffold, which was fabricated by a melt-plotting system. Rhodamine release from the scaffold was controlled physically by the thickness change of the PCL layer, and initial burst in drug release was eliminated by an appropriate thickness of the PCL layer. This simple technique may be useful in fabricating DDS-functional scaffolds for the clinical areas not only of bone and skin regeneration, but also of other tissue regeneration areas, regardless of the degradation rate of the structural scaffold.     

Controlled-release implant system formulated using biodegradable hemostatic gauze as scaffold

A unique polymer-based sustained-release implant was formulated using biodegradable hemostatic gauze as the scaffold. A piece of commercial gauze, Surgicel was coated with a poly(lactic-co-glycolic) acid (PLGA) solution in which drugs were loaded, followed by evaporating the solvent. Drug release kinetics from the PLGA coating was examined using phenol red (PhR, a hydrophilic dye) and carmustine (BCNU, a hydrophobic anti-tumor agent) as model drugs. With an additional drug-free PLGA over-layer coated on top of the drug-loaded PLGA coating, nearly zero order release was archived for both of the model drugs for a period of 12-14 days when the implants were incubated in PBS buffer at 37 degrees C. However, the drug release rate was independent of types of PLGA polymers such as lactide/glycolide ratio. A degradation study showed that the hydrophilic Surgicel scaffold itself degraded in 3 days of the release incubation regardless of the thickness of the polymer coating on top of it, suggesting that the loaded drug may be released through the diffusion channels created by the scaffold degradation. Characterization of this formulation using XRD and DSC indicated that the drug, BCNU, was distributed in the PLGA matrix in amorphous state. Images of scanning electron microscope showed that PLGA was coated on the outer and inner surfaces of the porous Surgicel.     

Paclitaxel-loaded polyester nanoparticles prepared by spray-drying technology: in vitro bioactivity evaluation

Paclitaxel (PTX), an antimicrotubular agent used in the treatment of ovarian and breast cancer, was encapsulated in nanoparticles (NPs) of poly(lactide-co-glycolide) (PLGA) and poly(ε-caprolactone) (PCL) polymers using the spray-drying technique. Morphology, size distribution, drug encapsulation efficiency, thermal degradation and drug release were characterized. MCF7 cells were employed to evaluate the efficacy of the systems on cell cycle and cytotoxicity. The particle size was in the range 0.8-1?μm. The incorporation efficiency of PTX was more than 80% in all NPs obtained. In?vitro drug release took place during 35 days, and drug release rates were in the order PCL?>?PLGA 50:50?>?PLGA 75:25. Unloaded NPs showed to be cytocompatible at MCF7 cells. PTX-loaded NPs demonstrated the release of the drug block cells in the G2/M phase. All PTX-loaded formulations showed their efficacy in killing MCF7 cells, mainly PTX-loaded PLGA 50:50 and PLGA 75:25 that cause a decrease in cell viability lower than 20%.     

Formulation, characterization, and evaluation of ketorolac tromethamine-loaded biodegradable microspheres

Ketorolac tromethamine has to be given every 6 hr intramuscularly in patients for acute pain, so to avoid frequent dosing and patient inconvenience we found it to be a suitable candidate for parenteral controlled delivery by biodegradable microspheres for the present study. Ketorolac tromethamine-loaded microspheres were prepared by o/w emulsion solvent evaporation technique using different polymers: polycaprolactone, poly lactic-co-glycolic acid (PLGA 65/35), and poly lactic-co-glycolic acid (PLGA 85/15). To tailor the release profile of drug for several days, blends of PLGA 65/35 and PLGA 85/15 were prepared with polycaprolactone (PCL) in different ratios. The results revealed that microspheres made with 1:3 (PLGA65/35: PCL) blend released 97% of the drug in 5 days as compared with 97% in 30 days in with pure PLGA65/35 microspheres. Microspheres made with 1:1 (PLGA65/35:PCL) and 3:1 (PLGA65/35:PCL released 98% of the drug in 30 days. In microspheres made with 1:3 (PLGA85/15:PCL), almost the entire drug was released in a week whereas in batches made with pure PLGA85/15 and 3:1 (PLGA 85/15:PCL) more than 80% of the drug was released in 60 days as compared with 96% in 60 days in 1:1 (PLGA85/15:PCL). Higher encapsulation efficiency was obtained with microspheres made with pure PLGA 65/35. These formulations were characterized for particle size analysis by Malvern mastersizer that revealed particle size in range of 12-15 micron and 12-22 micron for microspheres made with polymer blends of PLGA 65/35:PCL and PLGA85/15:PCL, respectively. In with pure PLGA65/35 and PLGA85/15, particle size was 28 micron and 8 micron, respectively. Surface topography was studied by scanning electron microscopy that revealed a spherical shape of microspheres. From our study it we concluded that with careful selection of different polymers and their combinations, we can tailor the release of ketorolac tromethamine for long periods.