三维多肽支架中前成骨细胞MC3T3-E1成骨分化的初步研究

在多肽EAK16水凝胶支架上接种小鼠前成骨细胞MC3T3-E1,采用倒置显微镜观察细胞形态,CCK-8(细胞计数试剂盒)检测细胞增殖情况。细胞在诱导培养基中培养1周后,观察不同时间段细胞碱性磷酸酶的分泌活性。采用ALP染色和茜素红-S染色作为定性实验研究MC3T3-E1向成骨方向的分化情况。结果表明,MC3T3-E1细胞在水凝胶支架EAK16上有较好的黏附和增殖能力,诱导培养后细胞有较高水平的碱性磷酸酶表达和矿化基质沉积。多肽水凝胶支架对前成骨细胞MC3T3-E1具有较好的生物相容性。     

钛(Ⅳ)配合物催化合成Ti-P聚乳酸材料的亲水性、降解率和细胞毒性

以双(烷氧-亚胺芳氧)基钛(IV)配合物(BAIP-Ti(IV))为催化剂催化D,L-丙交酯开环聚合合成的Ti-P聚乳酸,用静态水接触角、吸水率及降解实验考察其理化性能,以小鼠颅顶前骨细胞(MC3T3-E1)考察其在材料表面的增值和黏附铺展情况。结果表明:Ti-P聚乳酸比Sn-P聚乳酸有较低的亲水性和较高的抗水解性;MC3T3-E1细胞在Ti-P聚乳酸材料表面显示出的较好的增值活力以及细胞黏附与铺展能力;在Ti-P聚乳酸中残存的微量Ti(IV)金属配合物对MC3T3-E1细胞无细胞毒性,不影响MC3T3-E1细胞在其材料表面上生长。     

丝素/纳米纤维素晶须/壳聚糖多孔复合支架的应用

以冷冻干燥法制备多孔丝素(SF)支架,利用层层自组装将纳米纤维素晶须(CNW)和壳聚糖(CS)交替组装到多孔SF支架上得到SF/CNW-CS多孔复合支架。对SF/CNW-CS多孔复合支架的形貌和机械性能进行了表征。以MG-63细胞进行体外培养评估SF及SF/CNW-CS多孔复合支架的细胞相容性,MTT比色法和荧光图像的测试结果表明,与SF多孔支架相比,MG-63细胞在SF/CNW-CS多孔复合支架上的增殖、粘附和分化功能最高。因此,SF/CNW-CS支架有望成为骨组织工程的理想材料。     

磷酸镁/PBS/小麦蛋白复合骨修复材料

采用共沉淀法合成磷酸镁, 将磷酸镁(MP)、聚丁二酸丁二醇酯(PBS)和小麦蛋白(WP)进行复合, 制备出MP/PBS/WP复合骨修复材料。通过体外降解、生物活性以及细胞培养等实验对复合材料的理化性能及细胞相容性进行了研究。结果显示: MP/PBS/WP复合材料在Tris-HCl缓冲液中浸泡10 d后, 溶液pH从7.4上升至7.51, 浸泡12 w后, 其降解率达到58.43wt%; 复合材料在模拟体液中浸泡10 d后, 其表面形成磷灰石层; 复合材料能促进MC3T3-E1细胞的增殖与分化。结果表明: MP/PBS/WP复合材料具有优良的降解性、生物活性和细胞相容性, 有望成为一种新型骨修复材料。     

羟基丁酸与羟基辛酸共聚体骨组织工程支架的初步研究

采用粒子滤出/冷冻干燥复合法制备羟基丁酸与羟基辛酸共聚体[P(HB-HO)]多孔支架,并进行扫描电镜观察、孔隙率和力学性能检测以及体外降解和细胞相容性实验.结果表明,P(HB-HO)多孔支架孔隙分布均匀,连通性好,孔隙率为50%～90%时,抗压强度在1.7～6.2MPa之间,十二周体外降解率约为20%;与P(HB-H0)复合培养的小鼠成骨样细胞MC3T3-E1黏附率高,生长状态良好.证明P(HB-HO)具有良好的理化性能和细胞相容性,有望成为一种具有临床应用价值的骨组织工程支架材料.     

壳聚糖修饰纳米羟基磷灰石/聚酰胺复合多孔支架的制备及性能研究

利用相转移法制备了纳米羟基磷灰石/聚酰胺66(n-HA/PA66)复合多孔支架.用不同浓度(1%、3%、5%)的壳聚糖(CS)溶液对多孔支架进行了表面修饰.用扫描电镜(SEM)和材料力学试验机对多孔支架修饰前后的形貌和力学性能进行了表征.研究了经CS修饰的n-HA/PA66复合多孔支架在磷酸盐缓冲溶液(PBS)中的浸泡行为,并初步研究了其与MG63细胞的细胞相客性.结果显示,多孔支架具有较为理想的孔隙结构和贯通性,经CS修饰后,其力学强度有显著提高.体外浸泡结果显示,随着漫泡时间的增加,支架表面微结构变得粗糙和多孔化.细胞实验表明该支架有利于细胞在表面的粘附、铺展、生长和增殖.     

微米级煅烧羟基磷灰石/壳聚糖复合膜的制备及性能

将猪松质骨脱脂脱蛋白、煅烧后再进行球磨制备羟基磷灰石(HA), 与壳聚糖(CS)共混制备成HA/CS复合膜。通过正交设计考察了球磨条件对HA粒径的影响, 在复合膜上接种MC3T3-E1前体成骨细胞, 采用SEM及四甲基偶氮唑盐(MTT)方法检测细胞在膜表面的形态及增殖, 评价了材料的细胞相容性。结果表明, 所制备HA为粒径较均一的微米级球形粒子, 中值粒径D₅₀为1.21~1.67 μm。共混膜内HA在基质中分布均匀, 二者结合紧密, 复合膜具有较好的力学性能, MC3T3-E1细胞能在复合膜上很好地黏附生长。细胞增殖结果表明, 复合膜制备条件煅烧温度为1000 ℃, 球配比4∶4∶2, 球磨速率为230 r·min^-1, 球磨时间为2.5 h, HA∶CS =5∶5 (质量比)时最利于细胞增殖。     

羟基磷灰石表面形貌对人成骨肉瘤细胞生物学性能的影响

本文研究了羟基磷灰石(HA)表面形貌对人成骨肉瘤细胞(MG-63)生物学性能的影响。通过单轴压片技术与粒子占位法相结合控制陶瓷表面孔尺度、形态及分布, 从而获得具有不同表面孔结构的HA陶瓷材料。将材料与MG-63共培养, 通过扫描电子显微镜(SEM), MTT检测法表征材料表面形貌对细胞的黏附和增殖影响, 并通过碱性磷酸酶活性(ALP)检测和实时荧光定量(RT-PCR)技术探讨了HA陶瓷材料的表面结构对MG-63成骨分化的诱导作用。结果表明, 大孔结构(孔径大于200 μm)更有利于细胞的黏附和增殖, 而小孔结构(孔径小于100 μm)能促进细胞的成骨分化。孔形貌和孔分布也能影响细胞的生物功能, 相同尺度的孔径, 不规则蜂窝状的多级微孔结构比光滑孔壁的浅孔结构更能诱导细胞的成骨分化。     

表面改性对介孔硅酸钙镁/聚醚醚酮复合材料性能的影响

实验制备了介孔硅酸钙镁/聚醚醚酮复合骨修复材料, 采用砂纸打磨及喷砂对其表面进行改性。结果表明: 表面改性明显提高了复合材料表面的粗糙度和亲水性(水接触角降低), 喷砂在复合材料表面暴露出大量的介孔硅酸钙镁, 形成了多孔结构, 粗糙度和亲水性提高最大。表面改性复合材料在模拟体液中浸泡7 d后, 表面都形成了大量磷灰石; 表面改性促进了MC3T3-E1细胞在复合材料表面粘附、增殖和分化。喷砂比砂纸打磨更明显地提高了复合材料的生物学性能。     

β-磷酸三钙/壳聚糖复合支架的制备及其对成骨细胞生长的影响

采用冷冻萃取和冷冻凝胶法制备了β-磷酸三钙/壳聚糖(-βTCP/CS)复合支架,并采用SEM、XRD、FTIR和万能材料试验机对其性能进行表征。研究结果表明,复合支架的孔隙率85%,且随-βTCP含量的增加而减小。力学性能的测试结果显示,当壳聚糖含量为30%时,复合支架的抗压强度最大(1.73MPa)。同时SEM结果表明壳聚糖与-βTCP的比值为30∶70时,复合支架的孔径在200～500μm之间。将成骨细胞MC-3T3-E1接种于-βTCP/CS复合支架上,培养7d后,发现细胞在-βTCP/CS复合支架上能够很好地粘附、生长和分化,即所制备的复合支架具有良好的细胞相容性。     

静电纺丝天然-人工合成聚合物复合纳米纤维及其对细胞黏附、增殖和迁移的影响

生物材料组成成分对细胞生物功能有不同的影响。利用静电纺丝技术制备了基于聚己内酯(PCL,polycaprolactone)的不同天然蛋白、多糖(丝素蛋白(SF,silk fibroin)、透明质酸(HA,hyaluronicacid))的混合组分纳米纤维,采用了扫描电镜和接触角对纳米纤维进行基础表征。同时,进一步考察了纳米纤维作为组织工程支架的可行性。研究结果表明SF组分能增加材料的可纺性,有利于细胞的前期黏附,并能够促进细胞增殖。HA组分可以改善材料的亲水性,增加细胞伪足并促进细胞迁移。重要的是,PCL/SF/HA纳米纤维能同时结合SF和HA的优点,有望在组织工程领域得到应用。     

β-CaSiO_3/丝素蛋白复合支架材料结构与性能的研究

利用冷冻干燥法制备了β-CaSiO_3/丝素蛋白复合支架材料,经XRD和FTIR分析表明复合支架中丝素的结构主要以β-折叠为主;SEM分析显示材料孔隙分布均匀,孔连通性较好,孔径尺寸约为100～300μm.对支架的孔隙率和机械强度等性能进行了表征,研究表明复合支架的孔隙率为83%～87%,机械强度有较大提高.应用模拟体液浸泡实验研究了复合支架的体外生物活性,并用XRD、FESEM和EDS对试样表面进行了表征;结果显示,样品经模拟体液浸泡3天后,表面都能沉积出类骨羟基磷灰石(HA)层,β-CaSiO_3的加入能加快复合支架表面沉积类骨HA的速度.研究结果显示β-CaSiO_3/丝素蛋白复合支架材料有望作为强度较好的生物活性硬组织修复材料.     

“一步法”制备纳米相丝素蛋白/羟基磷灰石生物复合材料

系统分析了羟基磷灰石（HA）的制备方法和丝素蛋白纤维（SF）的溶解方法,提出一种制备纳米丝素蛋白/羟基磷灰石生物复合材料（SF/HA）的新型反应复合方法——“一步法”。并对由“一步法”制得的SF/HA分别进行了钙磷比测定、红外光谱测试、透射电镜观察和X射线衍射测试。结果表明：SF/HA中的钙磷比是1.6692,与标准HA中的钙磷比1.67一致;SF/HA中同时含有SF和HA中各自的官能团;SF/HA的晶粒横向尺度小于100 nm,SF/HA呈针状或柱状晶粒, SF和HA能够形成复合; SF/HA的晶型属于六方晶系, 当SF在SF/HA中所占质量分数为10%时,晶胞参数a=b=9.0319 A,c=7.0148 A,沿c轴方向平均晶粒尺寸是230.7645 A。“一步法”制备SF/HA具有合理性和可行性。     

Preparation,characterization and biological test of 3D-scaffolds based on chitosan,fibroin and hydroxyapatite for bone tissue engineering

This work describes the preparation and characterization of porous 3D-scaffolds based on chitosan (CHI), chitosan/silk fibroin (CHI/SF) and chitosan/silk fibroin/hydroxyapatite (CHI/SF/HA) by freeze drying. The biomaterials were characterized by X-ray diffraction, attenuated total reflection Fourier transform infrared spectroscopy, thermogravimetric analysis, differential scanning calorimetry, scanning electron microscopy and energy dispersive spectroscopy. In addition, studies of porosity, pore size, contact angle and biological response of SaOs-2osteoblastic cells were performed. The CHI scaffolds have a porosity of 94.2 ± 0.9%, which is statistically higher than the one presented by CHI/SF/HA scaffolds, 89.7 ± 2.6%. Although all scaffolds were able to promote adhesion, growth and maintenance of osteogenic differentiation of SaOs-2 cells, the new 3D-scaffold based on CHI/SF/HA showed a significantly higher cell growth at 7 days and 21 days and the level of alkaline phosphatase at 14 and 21 days was statistically superior compared to other tested materials.     

Deposition behavior and properties of silk fibroin scaffolds soaked in simulated body fluid

Porous 3D silk fibroin (SF) scaffolds were prepared directly from the SF solution with the addition of methanol and glutaraldehyde by a freeze-drying method. The scaffolds were then soaked in the simulated body fluid (SBF) for the deposition of hydroxyapatite (HA) crystals. The XRD and FTIR results showed that the SF were in β-sheet structure, resulting in the high thermal stability and mechanical properties of scaffolds. The XRD and AAS data revealed that the SF scaffolds could induce the continuous growth and enrichment of HA crystals onto the scaffolds with the extension of soaking time. The mechanical properties of scaffolds increased first with the HA-deposition within 3 d of soaking, then it declined. During the full soaking period, no significant change was observed on the porosity and water-binding ability, which were kept at about 84% and 800%, respectively. The cell cultivation results showed that the scaffolds have the satisfied cell biocompatibility, which was promoted after the HA-deposition. This work suggests that the porous 3D SF scaffolds may be a potential candidate in the bone engineering.     

Modification of human cancellous bone using Thai silk fibroin and gelatin for enhanced osteoconductive potential

The modification of human cancellous bone (hBONE) with silk fibroin/gelatin (SF/G) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccini-mide (NHS) crosslinking was established. The SF/G solutions at a weight ratio of 50/50 and the solution concentrations of 1, 2, and 4 wt % were studied. SF/G sub-matrix was formed on the surface and inside pore structure of hBONE. All hBONE scaffolds modified with SF/G showed smaller pore sizes, less porosity, and slightly lower compressive modulus than unmodified hBONE. SF/G sub-matrix was gradually biodegraded in collagenase solution along 4 days. The hBONE scaffolds modified with SF/G, particularly at 2 and 4 wt % solution concentrations, promoted attachment, proliferation, and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (MSC), comparing to the original hBONE. The highest cell number, ALP activity and calcium production were observed for MSC cultured on the hBONE scaffolds modified with 4 wt % SF/G. The mineralization was also remarkably induced in the cases of modified hBONE scaffolds as observed from the deposited calcium phosphate by EDS. The modification of hBONE with SF/G was, therefore, the promising method to enhance the osteoconductive potential of human bone graft for bone tissue engineering.     

Fabrication and characterization of bioactive silk fibroin/wollastonite composite scaffolds

Composite scaffolds of silk fibroin (SF) with bioactive wollastonite were prepared by freeze-drying. X-ray diffraction (XRD) and Fourier transform infrared (FT-IR) spectroscopy analysis showed that random coil and β-sheet structure co-existed in the SF scaffold. The mechanical performance, surface hydrophilicity and water-uptake capacity of the composite scaffolds were improved compared with those of pure SF scaffold. The bioactivity of the composite scaffold was evaluated by soaking in a simulated body fluid (SBF), and formation of a hydroxycarbonate apatite (HCA) layer was determined by FT-IR and XRD. The results showed that the SF/wollastonite composite scaffold was bioactive as it induced the formation of HCA on the surface of the composite scaffold after soaking in SBF for 5 days. In vitro cell attachment and proliferation tests showed that the composite scaffold was a good matrix for the growth of L929 mouse fibroblast cells. Consequently, the incorporation of wollastonite into the SF scaffold can enhance both the mechanical strength and bioactivity of the scaffold, which suggests that the SF/wollastonite composite scaffold may be a potential biomaterial for tissue engineering.     

Novel silk fibroin/hydroxyapatite composite films: Structure and properties

Novel composite films of Bombyx mori silk fibroin (SF) and hydroxyapatite (HA) composite films, with glycerin as an additive, were fabricated by means of co-precipitation, where the theoretical HA content was varied from 2 (w/w)% to 31 (w/w)%. The structure and properties of the composite films were investigated by SEM, XRD, AFM, TGA and tensile testing. The results showed that the composite films were smooth and transparent with the uniform distribution of HA into the composites when the final HA content was lower than 21 (w/w)%. XRD and TGA data showed that the silk fibroin in the composites was predominantly in a β-sheet crystalline structure, which was induced not only by the addition of glycerin, also by the HA crystal growth during the composite fabrication, leading to the thermal stable composite films. On the other hand, the HA crystals had the anisotropic growth with high extent of lattice imperfection and the preferential orientation along c-axis, probably promoted by the silk fibroin. The mechanical testing results showed that both break strain and stress were declined with the increase of HA content in the composites, presumably due to the original brittleness of HA compound.     

Interactions of Fibroblasts with Different Morphologies Made of an Engineered Spider Silk Protein

Spider silk has been investigated for decades due to the intriguing mechanical and also biomedical properties of the silk fibers. Previously, it has been shown that recombinant silk proteins can also be processed into other morphologies. Here, we characterized scaffolds made of the recombinant spider silk protein eADF4(C16) concerning their surface interactions with fibroblasts. Studies of BALB/3T3 cells on hydrogels and films made of eADF4(C16) showed low cell adhesion without observable duplication. Electro‐spun non‐woven scaffolds made of eADF4(C16), however, enabled both their adhesion and proliferation. Since eADF4(C16) lacks specific motifs for cell attachment, fibroblasts cannot generate focal adhesions with the material's surface, and, therefore, other cell–interface interactions such as topographical anchorage or cell attachment mediated by adhesion of extracellular matrix proteins are discussed in this paper. On non‐woven meshes protrusion of filopodia and/or lamellipodia between individual fibers increase the surface contact area, which depends on the diameter of the fibers of the non‐woven meshes. In contrast, at flat (film) or microstructured surfaces (hydrogels) such interactions seem to be precluded.     

Tissue response and biodegradation of composite scaffolds prepared from Thai silk fibroin,gelatin and hydroxyapatite

This work aimed to investigate tissue responses and biodegradation, both in vitro and in vivo, of four types of Bombyx mori Thai silk fibroin based-scaffolds. Thai silk fibroin (SF), conjugated gelatin/Thai silk fibroin (CGSF), hydroxyapatite/Thai silk fibroin (SF4), and hydroxyapatite/conjugated gelatin/Thai silk fibroin (CGSF4) scaffolds were fabricated using salt-porogen leaching, dehydrothermal/chemical crosslinking and an alternate soaking technique for mineralization. In vitro biodegradation in collagenase showed that CGSF scaffolds had the slowest biodegradability, due to the double crosslinking by dehydrothermal and chemical treatments. The hydroxyapatite deposited from alternate soaking separated from the surface of the protein scaffolds when immersed in collagenase. From in vivo biodegradation studies, all scaffolds could still be observed after 12 weeks of implantation in subcutaneous tissue of Wistar rats and also following ISO10993-6: Biological evaluation of medical devices. At 2 and 4 weeks of implantation the four types of Thai silk fibroin based-scaffolds were classified as “non-irritant” to “slight-irritant”, compared to Gelfoam^® (control samples). These natural Thai silk fibroin-based scaffolds may provide suitable biomaterials for clinical applications.