丝蛋白应用于牙周组织工程的可行性

背景：丝蛋白是有利于表皮细胞、成纤维细胞、成骨细胞、血管内皮细胞、胶质细胞黏附和生长的一种新型生物材料。 目的：评估丝蛋白作为支架材料应用于牙周组织工程的可行性。 方法：采用组织块法培养人牙周膜细胞,将第5代细胞悬液以2×10⁷ L^-1的浓度接种到丝蛋白支架材料上复合培养,并以1%,10%,50%,100%的丝蛋白支架浸提液培养,观察人牙周膜细胞在丝蛋白上及在丝蛋白浸提液中生长状况,用MTT法测定浸提液培养人牙周膜细胞的活力。 结果与结论：扫描电镜可见人牙周膜细胞在丝蛋白支架上伸展充分,生长旺盛,不同浓度丝蛋白支架浸提液培养对人牙周膜细胞的增殖与碱性磷酸酶活性均无影响。说明丝蛋白材料具有良好的生物相容性、独特的力学性能,可作为人牙周膜细胞黏附生长的理想支架材料较好地应用于牙周组织工程中。     

组织工程引导骨再生膜的体外构建

采用多孔的 PCL/ PL A共聚膜 ,对材料进行亲水性和细胞亲和性表面加工后 ,用作组织工程载体材料。通过将 5× 10 6 / ml成骨诱导 10 d的 MSCs与 PCL/ PL A共同培养 7d,扫描电镜观察细胞在材料中的生长状态。发现材料作为细胞生长的支持结构与细胞的相容性良好 ,细胞可以黏附在材料的表面和孔隙中。实验证明该材料具有良好的生物相容性 ,可以在体外与骨髓间质干细胞共同构建组织工程化的细胞 -材料复合物用于引导性骨再生。     

肝脏组织工程纳米纤维支架材料的比较研究

探讨海藻酸钠、壳聚糖和PLGA纳米纤维支架的机械稳定性、生物相容性及细胞在其表面的生长规律,寻找合适的肝脏组织工程支架材料。用静电纺丝的方法分别制备海藻酸钠、壳聚糖和PLGA纳米纤维支架,观察材料的机械稳定性及肝细胞在材料表面的活性和生长情况。接种后0.5 h内,肝细胞在壳聚糖和海藻酸钠材料表面贴壁,生长良好。第二天起,肝细胞在壳聚糖表面逐渐聚集生长,形成聚集体,而在海藻酸钠材料表面无聚集。第三天后,海藻酸钠材料发生溶胀,纳米结构破坏,而壳聚糖纳米支架保持完好。在观察期间,PLGA材料表面一直没有肝细胞黏附。肝脏细胞不能在PLGA纳米材料表面黏附生长;壳聚糖具有良好的生物相容性,且肝细胞在壳聚糖纳米材料表面能形成球形聚集体;海藻酸钠生物相容性好,但机械稳定性差,容易降解,不能单独作为肝脏组织工程的纳米支架材料。     

壳聚糖-脱细胞真皮三维材料作为骨组织工程支架材料的研究

目的观察MC3T3-El成骨前体细胞在壳聚糖-脱细胞真皮三维支架材料上的黏附情况,并评价其细胞相容性。方法通过冷冻干燥制备壳聚糖-脱细胞真皮三维支架材料,并测试其孔隙率、密度和吸水率,通过扫描电镜分析支架的微观形貌。采用体外培养细胞的方法,将MC3T3-E1细胞直接接种到壳聚糖-脱细胞真皮三维支架材料上,培养2,3,4,5h,各时间点各取3个样品,测定细胞在支架上的黏附率,确定最佳的细胞贴壁时间。将细胞接种到支架上,共培养1,3,5,7,9,11,13d,采用MTS方法绘制细胞增殖曲线,组织化学染色观察细胞形态,并利用材料试验机测试不同时间材料细胞复合物的压缩弹性模量。结果壳聚糖-脱细胞真皮材料具有连通的多孔结构,孔隙率为92.8%,密度为97.96g/L,吸水率为(2169±100)%。细胞相容性实验显示,成骨细胞易于在支架材料上黏附、增殖。结论壳聚糖-脱细胞真皮材料具有连通的孔隙,孔径较均匀,MC3T3-El成骨前体细胞易在壳聚糖-脱细胞真皮三维支架材料上黏附、增殖,表明该支架材料具有良好的细胞相容性。     

聚己内酯/明胶复合纳米材料的细胞相容性

目的:观察真皮成纤维细胞在聚己内酯/明胶(PCL/Gel)复合纳米材料上生长与增殖情况,评价此材料的生物相容性.方法:采用静电纺丝法制备PCL/Gel复合纳米纤维,将原代培养的真皮成纤维细胞接种到材料的表面,扫描电镜观察真皮成纤维细胞在复合纳米材料上的生长情况.实验分两组,材料组和对照组分别在接种后48、72、96 h用MTT法检测细胞的增殖情况.结果:真皮成纤维细胞在PCL/Gel复合纳米材料表面贴附牢固,生长形态良好.MTT检测结果统计显示此材料能够促进真皮成纤维细胞在其表面增殖.结论:PCL/Gel复合纳米材料具有较好的生物相容性,有望作为修复足底软组织创伤的支架材料.     

壳聚糖修饰丝素蛋白与人脂肪间充质干细胞体外构建组织工程脂肪

背景:在保留丝素蛋白原有优点的基础上,采用带正电荷的水溶性壳聚糖对其表面进行修饰,可改善细胞在支架材料上的黏附性。目的:验证壳聚糖表面修饰丝素蛋白支架材料与人脂肪间充质干细胞的生物相容性及两者体外构建组织工程脂肪的可行性。方法:将第3代人脂肪间充质干细胞悬液以1×107 L-1浓度接种于壳聚糖表面修饰丝素蛋白支架材料上作为实验组,以单纯的细胞悬液为对照组,MTT法检测细胞在支架材料上的黏附和增殖能力。将第3代人脂肪间充质干细胞悬液以1×109 L-1浓度接种于壳聚糖表面修饰丝素蛋白支架材料上,分别进行成脂诱导培养与高糖培养基常规培养,14 d后行细胞-支架复合物油红O染色与RT-PCR检测。结果与结论:人脂肪间充质干细胞在壳聚糖表面修饰丝素蛋白支架材料上黏附、增殖良好。成脂诱导14 d后,油红O染色显示壳聚糖修饰丝素蛋白支架材料上有大量脂肪细胞生成,且过氧化物酶增殖物活化受体γ2基因表达阳性。结果表明壳聚糖表面修饰丝素蛋白支架材料具有良好的体外生物相容性,与人脂肪间充质干细胞共培养可被成功诱导为成熟脂肪细胞。     

纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥与骨髓基质细胞的生物相容性*☆

背景：在前期的试验中,通过共沉淀法合成了纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合粉体,并与柠檬酸衍生物溶液调和制备出可生物降解、适当力学性能以及较好黏合强度的骨水泥。 目的：验证纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥材料对体外兔骨髓基质细胞黏附及增殖的影响,了解材料的生物相容性。 方法：应用共沉淀法制备纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合材料作为骨水泥的固相粉体,将柠檬酸衍生物配制成溶液作为液相调和制备黏合性骨水泥。培养兔骨髓基质细胞,传代扩增后接种到材料上,体外继续培养;以细胞加入无材料的培养皿培养为对照。 结果与结论：体外培养的兔骨髓基质细胞2 d后呈梭形成纤维细胞样,生长良好。有材料实验组细胞数显著多于对照组(P < 0.01)。扫描电镜下骨水泥材料具有良好的多孔网状结构,兔骨髓基质细胞伸出多个伪足样突起,紧密贴附在材料表面。两组细胞均保持持续增殖,2,4,6,和8 d实验组增殖均显著快于对照组(P < 0.01)。提示纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥材料具有良好的生物相容性。      

碱性成纤维细胞生长因子对许旺细胞在小肠黏膜下层支架上黏附及增殖的影响

背景:许旺细胞复合小肠黏膜下层是构建人工神经的可行方法,碱性成纤维细胞生长因子有促进许旺细胞增殖的作用。目的:验证碱性成纤维细胞生长因子对许旺细胞在小肠黏膜下层支架材料表面的细胞增殖及黏附状态的影响。方法:将体外分离培养的2代SD乳鼠许旺细胞接种于小肠黏膜下层支架材料上并加入50μg/L的碱性成纤维细胞生长因子复合培养为实验组,以单纯的许旺细胞复合小肠黏膜下层作为对照组,分别用MTT法测定细胞增殖能力,细胞黏附率检测细胞在支架上的黏附情况,用流式细胞仪测定细胞分裂周期,并用苏木精-伊红染色及描电镜观察细胞形态及与材料的贴附情况。结果与结论:MTT显示实验组的细胞增殖吸光度值明显高于对照组(P0.05),实验组的细胞黏附率为(69.47±3.17)%,对照组为(44.58±1.76)%(P0.05),细胞周期显示实验组比对照组的G2/M+S期细胞百分含量高(P0.05),培养7d后的苏木精-伊红染色及扫描电镜显示实验组比对照组材料上聚集的细胞数量多,细胞形态伸展更明显,细胞贴附力更好。因此碱性成纤维细胞生长因子可明显地改善许旺细胞在小肠黏膜下层上的增殖及黏附能力,能促进许旺细胞与小肠黏膜下层支架的复合而构建人工神经导管。     

膨体聚四氟乙烯与人脂肪干细胞的体外生物相容性

背景：膨体聚四氟乙烯多孔高分子聚合材料是临床常用的植入假体,具有良好的生物相容性,不易变形、变质,不产生炎症吸收反应,可允许细胞游走和组织向内生长。 目的：观察人脂肪干细胞与膨体聚四氟乙烯材料的生物相容性。 方法：将第4代人脂肪干细胞与膨体聚四氟乙烯体外复合培养,采用倒置相差显微镜观察细胞在支架上黏附、生长及增殖情况,计算细胞黏附率,MTT比色法检测细胞增殖率。 结果与结论：刚接种的细胞呈圆形透亮,在支架材料表面分布均匀,细胞活性佳,3 h后大量细胞贴壁,24 h后可见少量呈短梭形的脂肪干细胞贴壁,3 d首次换液,细胞清晰可见,低密度生长时呈短梭形或多角形,分布均匀,种植7 d后细胞数量明显增加,极少细胞从支架上掉落,细胞黏附率平均达95.7%,并且细胞仍保持正常的分裂增殖速度。说明膨体聚四氟乙烯材料具有良好的细胞相容性,可作为脂肪组织工程的种子。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;组织工程全文链接：     

筋膜成纤维细胞与纤维蛋白凝胶的体外生物相容性

背景：纤维蛋白凝胶是一种天然可降解的生物支架材料,具备可用于组织工程支架的共性,被越来越多地用做种子细胞载体应用于组织工程修复。 目的：观察兔筋膜成纤维细胞与纤维蛋白凝胶的体外生物相容性。 方法：采用组织块贴壁法培养新西兰大白兔皮下固有筋膜组织成纤维细胞,以胰酶消化法对其进行传代。将第4代成纤维细胞悬液与纤维蛋白凝胶共培养,以倒置相差显微镜动态观察纤维蛋白凝胶表面成纤维细胞的形态及增殖情况;共培养5 d时,以免疫荧光染色激光共聚焦显微镜鉴定成纤维细胞,扫描电镜观察成纤维细胞的生长贴附情况。 结果与结论：倒置相差显微镜下显示,纤维蛋白凝胶表面的成纤维细胞形态与单纯培养成纤维细胞无明显差别;扫描电镜显示,成纤维细胞在纤维蛋白凝胶表面伸展充分,伸出的“伪足”与纤维蛋白凝胶有很好的贴附并分泌基质样物质,可见纤维蛋白凝胶并未改变成纤维细胞的形态学特征;激光共聚焦显微镜显示,成纤维细胞波形蛋白呈阳性表达,证明成纤维细胞种植在纤维蛋白凝胶表面后性质未发生变化,未被诱导分化。表明筋膜成纤维细胞与纤维蛋白凝胶在体外有很好的生物相容性。  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      

Fabrication and characterization of porous alginate/polyvinyl alcohol hybrid scaffolds for 3D cell culture

Porous alginate/polyvinyl alcohol (PVA) hybrid scaffolds as bioartificial cell scaffolds were fabricated to improve cell compatibility as well as flexibility of the scaffolds. The alginate/PVA hybrid scaffolds with different PVA compositions up to 50 wt% were fabricated by a modified freeze-drying method including the physical cross-linking of PVA and the following chemical cross-linking of alginate. The prepared alginate/PVA hybrid scaffolds were characterized by morphology observations using scanning electron microscopy (SEM), the measurements of porosity and average pore sizes and the measurements of compressive strength and modulus. The scaffolds exhibited highly porous, open-cellular pore structures with almost the same surface and cross-sectional porosities (total porosities about 85%, regardless of PVA composition) and the pore sizes from about 290 microm to about 190 microm with increasing PVA composition. The alginate/PVA hybrid scaffolds were more soft and elastic than the control alginate scaffold without significant changes of mechanical strength. The scaffolds were examined for their in vitro cell compatibility by the culture of chondrocytes (human chondrocyte cell line) in the scaffolds and the following analyses by MTT assay and SEM observation. It was observed that the alginate/PVA scaffolds had better cell adhesion and faster growth than the control alginate scaffold. It seems that 30 wt% addition of PVA to alginate in the fabrication of the hybrid scaffolds is desirable for improving their flexibility and cell compatibility.     

Impact of the composition of alginate and gelatin derivatives in bioconjugated hydrogels on the fabrication of cell sheets and spherical tissues with living cell sheaths

Gelatin and alginate derivatives possessing phenolic hydroxyl moieties (gelatin–Ph and Alg–Ph) were dissolved in aqueous solution and conjugated via horseradish peroxidase-catalyzed crosslinking, resulting in hydrogelation. The objective of creating the hydrogels was to prepare cell sheets and spherical tissues wrapped in living cell sheaths. An increase in the gelatin–Ph content in the hydrogel improved cellular adhesion on the hydrogel surface but hindered degradability by alginate lyase. A hydrogel with the desired characteristics was obtained from a solution containing 0.5% (w/v) gelatin–Ph and 1.5% (w/v) Alg–Ph. Human aortic endothelial (HAE) cells and mouse embryo fibroblast 10T1/2 cells grew on the hydrogels and could be harvested as cell sheets by treatment with alginate lyase. 10T1/2 cells enclosed in Alg–Ph/gelatin–Ph microcapsules composed of the conjugate hydrogel elongated on the inner surface of the microcapsules and grew three times faster than those enclosed in Alg–Ph microcapsules. Alg–Ph/gelatin–Ph microcapsules not only supported growth of the enclosed cells into spherical tissues, but also provided a cell adhesive outer surface for the fabrication of an HAE cell layer. Finally, spherical tissues of 10T1/2 cells wrapped in living HAE cell sheaths were obtained by treatment with alginate lyase.     

Analysis of the biological response of endothelial and fibroblast cells cultured on synthetic scaffolds with various hydrophilic/hydrophobic ratios: influence of fibronectin adsorption and conformation

In this study we developed polymer scaffolds intended as anchorage rings for cornea prostheses among other applications, and examined their cell compatibility. In particular, a series of interconnected porous polymer scaffolds with pore sizes from 80 to 110 microns were manufactured varying the ratio of hydrophobic to hydrophilic monomeric units along the polymer chains. Further, the effects of fibronectin precoating, a physiological adhesion molecule, were tested. The interactions between the normal human fibroblast cell line MRC-5 and primary human umbilical vein endothelial cells (HUVECs) with the scaffold surfaces were evaluated. Adhesion and growth of the cells was examined by confocal laser scanning microscopy. Whereas MRC-5 fibroblasts showed adhesion and spreading to the scaffolds without any precoating, HUVECs required a fibronectin precoating for adhesion and spreading. Although both cell types attached and spread on scaffold surfaces with a content of up to a 20% hydrophilic monomers, cell adhesion, spreading, and proliferation increased with increasing hydrophobicity of the substrate. This effect is likely due to better adsorption of serum proteins to hydrophobic substrates, which then facilitate cell adhesion. In fact, atomic force microscopy measurements of fibronectin on surfaces representative of our scaffolds revealed that the amount of fibronectin adsorption correlated directly with the hydrophobicity of the surface. Besides cell adhesion we also examined the inflammatory state of HUVECs in contact with the scaffolds. Typical patterns of platelet/endothelial cell adhesion molecule-1 expression were observed at intercellular boarders. HUVECs adhering on the scaffolds retained their proinflammatory response potential as shown by E-selectin mRNA expression after stimulation with lipopolyssacharide (LPS). The proinflammatory activation occurred in most of the cells, thus confirming the presence of a functionally intact endothelium. Little or no expression of the proinflammatory activation markers in the absence of LPS stimulation was observed for HUVECs growing on scaffolds with up to a 20% of hydrophilic component, whereas activation of these markers was observed after stimulation. In conclusion, scaffolds containing up to 20% hydrophilic monomers exhibited excellent cell compatibility toward human fibroblast cell line MRC-5 and human endothelial cells. Atomic force microscopy confirmed that adsorbed serum proteins such as fibronectin probably accounted for the positive correlation of HUVEC adhesion and surface hydrophobicity.     

壳聚糖与海藻酸钙双相陶瓷骨支架的机械性能及细胞相容性

BACKGROUND: Hydroxyapatite/β-tricalcium phosphate biphasic ceramic bone has good cell compatibility, but its mechanical properties are poor. OBJECTIVE: To construct chitosan/ or calcium alginate/biphasic ceramic bone scaffolds and to detect their mechanical properties and cytocompatibility. METHODS: Different concentrations of chitosan (2%, 4%, 7%, 10%) or calcium alginate (3%, 4%, 5%, 7%) were mixed with biphasic ceramic bone to prepare chitosan/biphasic ceramic bone scaffold and calcium alginate/biphasic ceramic bone scaffold. Their morphology and structure, coagulation time, anti-dissolution properties, shear force, compressive strength and cell compatibility were detected. RESULTS AND CONCLUSION: (1) Coagulation time: with the concentration increase, the initial and final setting time of these two kinds of composite scaffolds were prolonged to some extent. (2) Scanning electron microscopy: these two kinds of composite scaffolds showed porous microstructures with different pore sizes. (3) Anti-dissolution properties: the calcium alginate/biphasic ceramic bone scaffold (3%, 4%, 5%, 7%) and chitosan/biphasic ceramic bone scaffold (7%, 10%) had good anti-dissolution properties in the liquid. (4) Mechanical strength: with the concentration increase, the shear force and compressive strength of the calcium alginate/biphasic ceramic bone scaffold were reduced. (5) Cell compatibility: the cytotoxicity of chitosan/ or calcium alginate/biphasic ceramic bone scaffolds was graded as 0-1 or 2-3, respectively. These results show that the chitosan/biphasic ceramic bone scaffold has better mechanical properties and cell compatibility than the calcium alginate/biphasic ceramic bone scaffold.      

小肠黏膜下层细胞相容性的研究

评价猪小肠黏膜下层与人胚骨膜成骨细胞 (Human embryonic periosteal osteoblasts,HEPOB)、人胚皮肤成纤维细胞 (Hum an em bryonic skin fibroblasts,HESFB)及兔肾血管内皮细胞 (Rabbitrenal vascular endothelialcells,RRVEC)的细胞相容性。制备猪小肠黏膜下层 (Sm all intestinal submucosa,SIS) ,将 HEPOB、HESFB和RRVEC与小肠黏膜下层体外复合培养 ,采用相差显微镜观察细胞的黏附和生长 ,并用 MTT法测定细胞增殖 ,用流式细胞仪检测复合培养细胞在不同培养时间的细胞周期、细胞凋亡。结果表明三种细胞均可在 SIS上黏附生长 ,SIS可促进 RRVEC的增殖 ,SIS对三种细胞的细胞周期和细胞凋亡率无影响。 SIS有良好的细胞相容性 ,其孔径、结构有助于 HEPOB、HESFB和 RRVEC细胞的黏附和生长 ,是良好的组织工程生物衍生材料。     

脱细胞异体真皮基质与人脂肪干细胞的生物相容性

背景：脱细胞异体真皮基质具有优良的生物相容性和组织细胞诱导功能。 目的：评价人脂肪干细胞与脱细胞异体真皮基质的生物相容性。 方法：取健康成年人吸脂术后的脂肪组织分离脂肪干细胞,并行原代与传代培养,传至第3代,将细胞与脱细胞异体真皮基质联合体外培养3,7 d,倒置相差显微镜和扫描电镜观察细胞在支架材料上的黏附、生长及增殖情况,并计算细胞在材料上的黏附率;XTT比色法检测细胞的生长增殖情况。 结果与结论：脂肪干细胞在支架材料上分布均匀,24 h内细胞开始伸展、黏附,二三天完全伸展变形,以梭形为主,呈网状排列;随着培养时间延长,支架上的细胞逐渐增多;人脂肪干细胞细胞与脱细胞异体真皮基质混合培养后平均黏附率为95.03%,并保持正常的生长增殖速度,表明支架对细胞具有良好的黏附性;脱细胞异体真皮基质材料与人脂肪干细胞复合后相容性良好。     

复合胰岛素样生长因子1壳聚糖胶原支架与人牙周膜细胞的增殖

背景：胰岛素样生长因子1具有促进成纤维细胞有丝分裂的作用,同时具有促进牙周细胞生长、分化及合成细胞外基质的作用。 目的：观察负载胰岛素样生长因子1的壳聚糖胶原支架对于人牙周膜细胞增殖的作用。 方法：将人牙周膜细胞分别接种于负载胰岛素样生长因子1的壳聚糖胶原支架与普通胶原支架上,于接种的1 h、24 h及1周检测重组人转化生长因子β1的释放,于第1,7,28天检测两组细胞的黏附和增殖情况。 结果与结论：负载胰岛素样生长因子1的壳聚糖胶原支架组第1,24小时和第1周的重组人转化生长因子β1释放率明显低于普通胶原支架组(P < 0.01)。两组接种第1天的细胞黏附和增殖检测比较差异无显著性意义  (P > 0.05),负载胰岛素样生长因子1的壳聚糖胶原支架组接种第7,28天的细胞黏附和增殖情况优于普通胶原支架组(P < 0.01)。表明负载胰岛素样生长因子1的壳聚糖胶原支架可显著促进人牙周膜细胞的增殖。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;组织工程全文链接：     

A denatured collagen microfiber scaffold seeded with human fibroblasts and keratinocytes for skin grafting

Biomaterial scaffolds are categorized into artificial or natural polymers, or combinations of the two. Artificial polymers often undergo serum protein adsorption, elicit foreign body and encapsulation immune responses post-implantation. Large pore bovine electrospun collagen I was therefore screened as a candidate for human keratinocyte and fibroblast cell scaffolds. Human HaCaT keratinocyte and dermal fibroblasts were seeded on electrospun denatured collagen I microfiber (DCM) scaffolds and after 72 h Livedead(?) assays performed to determine adhesive cell, survival and scaffold penetration. Both keratinocytes and fibroblasts attached to and survived on DCM scaffolds, however only fibroblasts migrated over and into this biomaterial. HaCaT keratinocytes remained largely stationary on the scaffold surface in discrete islands of monolayered cells. For this reason, normal human epidermal keratinocyte (NHEK) scaffold interactions were assessed using scanning and transmission electron microscopy (EM) that demonstrated DCM scaffolds comprised networks of interlocking and protruding collagen fibers with a mean diameter of 2-5 μm, with a mean inter-fiber pore size of 6.7 μm (range 3-10 μm) and scaffold thickness 50-70 μm. After 72 h the keratinocytes and fibroblasts on DCM scaffolds had attached, flattened and spread over the entire scaffold with assembly of lamellapodia and focal adhesion (FA)-like junctions. Using transmission EM, NHEKs and HaCaT keratinocytes assembled desmosomes, lamellapodia and FA junctions, however, neither hemidesmosomes nor basal lamina were present. In long term (21 day) co-culture fibroblasts migrated throughout the scaffold and primary keratinocytes (and to a lesser extend HaCaTs) stratified on the scaffold surface forming a human skin equivalent (HSE). In vivo testing of these HSEs on immunocompetent (BalbC) and immunodeficient (SCID) excisionally wounded model mice demonstrated scaffold wound biocompatibility and ability to deliver human cells after scaffold biodegradation.     

基于同轴细胞打印双网络生物墨水优化及类血管支架的打印

文题释义：双网络生物墨水：生物墨水是指可以用于生物3D打印机的材料,具有类似细胞外基质的理化性质,可用于制造与人体器官相似的组织。双网络生物墨水内部具有两种交联网络,能使体外构建的组织具有良好的机械性能,适用于不同的应用场景。 同轴细胞打印：生物3D打印也叫细胞打印,是指操控细胞生物墨水体外构建活性组织的过程。同轴细胞打印是生物3D打印的延伸和发展,通过结合多层同轴针头可以直接快速制备含有内部连通网络的组织工程支架。 背景：细胞体外培养情况下无法在远离营养物质200 μm以上的区域存活,血管网络构建对组织工程领域厚组织和器官再生至关重要,同轴细胞打印为体外构建类血管通道提供了一种新的方式。 目的：优化生物墨水的同轴细胞打印性能,制备具有类血管结构的组织工程支架。 方法：通过间歇式巴氏灭菌制备无菌海藻酸钠溶液,冷冻保存;以脱胶蚕丝为原料制备无菌丝素蛋白冻干粉,密封保存;将丝素蛋白冻干粉加入解冻的海藻酸钠溶液中,再加入人脐静脉内皮细胞,作为生物墨水;将生物3D打印机的外轴连接生物墨水,内轴连接交联剂,同轴打印类血管支架材料,进行光学相干层析成像扫描、扫描电镜观察;拉伸测试海藻酸钠与丝素蛋白/海藻酸钠同轴打印环形试件(不含细胞)的弹性模量。采用冷冻保存7 d的海藻酸钠溶液与人脐静脉内皮细胞制作同轴打印支架,冷冻保存7 d的海藻酸钠溶液、人脐静脉内皮细胞与密封保存6个月的丝素蛋白冻干粉制作同轴打印支架,培养24 h后死活染色观察细胞存活率。设计打印串联与并联结构的类血管支架,培养1,3,7,10,14 d后检测细胞增殖情况。 结果与结论：①光学相干层析成像扫描显示,该混合生物墨水最高打印高度为9层,整体厚度约为4.4 mm;扫描电镜显示,类血管支架的中空纤维丝外壁呈无规则条状卷曲,存在微米级内部连通孔隙结构,中空纤维丝内壁具有更致密的孔隙结构;②丝素蛋白/海藻酸钠同轴打印环形试件的弹性模量大于单纯海藻酸钠同轴打印环形试件(P < 0.05);③采用保存7 d海藻酸钠溶液制作的支架细胞存活率为(86.7±3.4)%,加入丝素蛋白冻干粉支架的细胞存活率为(98.1±1.2)%,说明冷冻保存7 d的海藻酸钠溶液未染菌,丝素蛋白的保质期可达6个月;④并联结构类血管支架培养7,10,14 d的细胞增殖活性高于串联结构的类血管支架(P < 0.05);⑤结果表明,实验制备的类血管支架材料具有良好的生物相容性与机械性能。 ORCID: 0000-0002-5556-6672(张一帆) 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     

胶原/丝素电纺纳米纤维膜体外培养肝细胞的研究

本实验采用静电纺丝技术制备胶原/丝素纳米纤维支架,对支架上培养的人肿瘤肝细胞HepG2进行形态学观察、细胞功能和代谢功能检测。胶原/丝素纳米纤维支架以六氟异丙醇(HFIP)为溶剂通过静电纺丝技术制备,5种支架材料胶原和丝素配比分别为10∶0、7∶3、5∶5、3∶7、0∶10。扫描电镜结果显示制备的纤维平均直径在550～1100 nm之间,随着丝素含量的增加纤维平均直径增加。细胞培养结果显示HepG2细胞在材料表面生长状态良好并与支架材料紧密结合。随培养时间延长,常规培养组细胞在第5 d后逐渐死亡,失去细胞功能,胶原/丝素纳米纤维支架组细胞在4～9 d内能够维持稳定状态,其尿素合成、蛋白分泌与常规培养组有明显差别,其中丝素含量为50%组的细胞状态和细胞功能高于其它组。实验表明胶原/丝素纳米纤维支架材料细胞相容性良好,较之常规培养细胞增殖效果明显,维持功能表达时间延长,有望用于改善人工肝生物反应器中的细胞活性,维持细胞功能表达。