脱细胞脊髓基质支架的制备及生物特性

背景：以脱细胞脊髓基质材料为构架的脊髓生物支架,已被证实可恢复或部分恢复受损的脊髓神经功能。 目的：介绍脱细胞脊髓基质支架的制备方法和部分生物特性,对近年来其在脊髓组织工程中的应用及进展作一概述。 方法：应用计算机检索 CNKI 和 PubMed 数据库中 2005年1月至2014年10月关于脱细胞脊髓基质支架材料在脊髓损伤中应用的文章,在主题和摘要中,中文以“脱细胞脊髓,支架材料,脊髓损伤,组织工程学”为检索词检索,英文以“acellular spinal cord;engineering tissue;spinal cord injury;scaffold”为检索词进行检索。 结果与结论：脱细胞脊髓基质支架具有较低的抗原性、优良的生物相容性及类似脊髓的三维支架结构,但存在力学性能差及结构不稳定等缺点。通过京尼平、戊二醛等交联剂改性后可明显提高支架的生物性能。目前国内外已对脱细胞脊髓基质支架在神经修复再生方面的应用做了一些探索,为脊髓组织工程学打下了基础。由于脱细胞脊髓基质支架的诸多优点,脱细胞脊髓基质材料有望成为脊髓组织工程学的理想材料。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     

心脏组织工程中细胞和生物支架的研究热点

文题释义：心脏组织工程：是基于组织工程的技术和原理,利用合适来源的细胞和生物支架制造心脏移植物,用于代替受损心脏组织或促进心肌细胞增殖,以恢复或改善心脏功能的技术。 生物支架：是组织工程技术中用于对细胞成分起支撑作用的移植物,其构成成分类似于细胞外基质成分,部分支架具有孔隙等允许血液中氧气及营养物质通过。 背景：心脏组织工程技术的出现和发展,为心血管疾病尤其是心肌梗死的治疗提供了新的选择。 目的：通过对心脏组织工程的2个核心要素即细胞和生物支架的研究进展进行综述,以期为心脏组织工程技术应用于心血管疾病治疗提供参考及依据。 方法：通过检索PubMed 数据库及中国知网数据库2010至2019年期间心脏组织工程相关文章,以“cardiac tissue engineering,cardiomyocytes differentiation,cardiac tissue engineering,cardiomyocytes differentiation,bone marrow derived stem cells,human embryonic stem cells,induced pluripotent stem cells,menstrual blood stem cells,biological scaffolds”为英文检索词,以“心脏组织工程,心肌细胞分化,干细胞,生物支架”等为中文检索词,最终选择78篇英文文献纳入研究。 结果与结论：多种来源的细胞(包括心肌细胞、骨骼肌细胞、心脏成纤维细胞、骨髓来源干细胞、胚胎干细胞、诱导多能干细胞、月经血干细胞)和生物支架(包括水凝胶、脱细胞支架、细胞片及心脏芯片)都可应用于心脏组织工程,但心脏组织工程仍然存在诸多需要解决的问题,如合适的细胞来源、新型支架材料的研发、诱导分化技术的优化,植入时机及途径的优化。 ORCID:0000-0003-2763-5535(王萍) 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     

脱细胞猪膀胱支架的构建

文题释义： 脱细胞：生物移植或修补材料制备过程中常用到脱细胞方法,脱细胞能很好的保留组织的成分,降低移植或修补后的免疫原性,同时保持良好的机械性能,在临床上得到较为广泛的应用。 猪膀胱支架：利用不同的脱细胞方法去除猪膀胱组织中的细胞后制备为猪膀胱支架,既保留了细胞外基质的完整性,又可以降低移植后排斥反应,为进一步的对组织工程支架材料的研究奠定了可行的基础。 背景：膀胱修补术是目前临床上治疗膀胱缺损的主要方法之一,同源性组织因各种因素的影响来源较少,组织工程膀胱脱细胞基质受到人们越来越多的关注。猪膀胱脱细胞外基质来源广泛,具备天然的细胞外支架结构,成为组织工程膀胱替代材料研究的热点。 目的：研究脱细胞猪膀胱作为组织工程支架材料的可行性。 方法：取新鲜的猪膀胱,联合液氮冻融、十二烷基硫酸钠、胰蛋白酶脱细胞方法制猪膀胱无细胞基质。按不同脱细胞方法分组：①正常对照组：不做任何处理;②实验组：0.6%胰蛋白酶+5%十二烷基硫酸钠(pH 8.0);③脱细胞对照组：分别用0.75%胰蛋白酶(pH 8.0)、1%胰蛋白酶(pH 8.0)、5%十二烷基硫酸钠(pH 7.6)或10%十二烷基硫酸钠(pH 7.6)处理。通过苏木精-伊红染色和VG染色、DNA定量、α-Gal抗原检测,观察猪膀胱脱细胞效果。 结果与结论：苏木精-伊红染色显示实验组猪膀胱细胞成分已基本去除;VG染色显示实验组完整保留了猪膀胱组织的细胞外基质成分;实验组的DNA残留量仅为(49.84±30.13) μg/g,显著低于几个脱细胞对照组(P < 0.05);同时其α-Gal抗原残留也明显低于脱细胞对照组。提示：应用0.6%胰蛋白酶+5%十二烷基硫酸钠处理猪膀胱,在完整保留猪膀胱组织细胞外基质的同时,可有效去除其细胞成分,为构建脱细胞猪膀胱支架提供一定参考价值。 ORCID: 0000-0003-1004-7390(李芹) 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     

去细胞化基质材料修复椎间盘的研究与进展

文题释义：去细胞化：是指利用物理、化学和酶学方法裂解细胞，去除细胞内容物，保留细胞外基质、组织天然构架。通常联合运用以上多种方法去除同种异体或异种细胞成分，理论上可利用由此获得的细胞外基质制造出具备合适生物力学和生物相容性的最低免疫原性支架。 椎间盘：是由位于中央的髓核(蛋白聚糖和Ⅱ型胶原组成的集中高度水化的纤维胶状核)及环绕其周围分层排列的纤维环(主要由Ⅰ型胶原组成的多层纤维软骨环)和上下软骨终板构成的复杂结构，是无血运器官，自身代谢缓慢，损伤后的自我修复、愈合能力极其有限，一般的物理修复与手术治疗无法从根本上修复椎间盘，效果欠佳，容易复发，椎间盘去细胞化组织工程为之提供了新思路。 背景：随着组织工程学的发展，椎间盘组织的修复与再生成为可能，将组织去细胞后的细胞外基质作为修复材料是进行椎间盘组织工程再生的重要手段。 目的：总结近年来用于椎间盘再生去细胞化基质材料的制备工艺、质量控制、应用效果，并进行展望。 方法：检索PubMed、Web of Science和CNKI数据库等收录的有关去细胞化方法及去细胞基修复椎间盘的文章，英文检索词为“Intervertebral disc，Decellularization，Extracellular matrix，Scaffold material，Tissue engineering”，中文检索词为“椎间盘，去细胞化，细胞外基质，支架材料，组织工程”。根据纳入与排除标准对所有文章进行初筛后，保留相关性较高的文章进行综述。 结果与结论：目前的椎间盘去细胞化组织工程旨在最大程度地保留生理相关的生物活性物质，提高力学性能和生物相容性，降低免疫原性。去细胞化基质材料能够模拟椎间盘内细胞外基质的天然微环境，作为细胞载体能对种子细胞起到良好的诱导分化用，其修复椎间盘已经取得了一定进展，但材料的孔隙率、免疫排斥反应、植入体内方式及效果评估等问题还有待进一步的深入研究。 ORCID: 0000-0003-3225-3611(金筱妤) 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     

半月板损伤或退变治疗：干细胞、人工聚合物支架形成组织工程体系的作用与功能

文题释义：半月板：是位于膝关节胫骨平台与股骨内外髁之间的新月型纤维软骨组织,形态上呈“内C外O”,起到了承载负荷、吸收震荡以及稳定关节的重要作用,其损伤或切除容易造成膝关节软骨磨损从而引发早期骨关节炎。脱细胞支架：由经化学和物理的方法去除异体或异种组织中的细胞,形成无免疫原性或低免疫原性的材料构建的组织工程支架。 背景：半月板作为膝关节的重要解剖结构,损伤或者手术切除等均有可能导致早期骨关节炎。因此,利用组织工程技术寻求一种可完全模仿正常生理半月板的取代物尤为重要。 目的：总结近年来半月板组织工程的种子细胞、支架、刺激物的研究进展。 方法：检索PubMed数据库、Web of science、CNKI中国期刊全文数据库、万方数据库收录的文献。英文检索词为：“meniscus;tissue engineering;mesenchymal stem cell;scaffold;Synthetic polymer scaffolds; polycaprolactone;hydrogel;ECM component scaffold;tissue;small intestine submucosa;decellularized;growth factor;dynamic compression;tensile;cyclic hydrostatic pressure”,中文检索词包括：“半月板、组织工程、干细胞、支架、人工聚合物支架、水凝胶、细胞外基质组分支架、小肠黏膜下层、脱细胞技术、生长因子、动态压缩试验、抗拉伸负荷、循环静水压试验”,最终纳入61篇文献进行总结。结果与结论：种子细胞、支架以及生物与物理刺激物是组成半月板组织工程的重要三元素。间充质干细胞仍然是当今组织工程中最为常用的种子细胞,其可种植于人工聚合物支架或者天然支架如细胞外基质组分支架与组织衍生支架,也可通过水凝胶包裹,在体内外生长因子与力学刺激作用下,进而形成再生半月板。现如今半月板组织工程更倾向于系统性综合种子细胞、支架、刺激物成一个体系来研究。因此,如何探索出一套最符合半月板解剖结构、生理功能以及生物相容性的组织工程体系已成为目前组织工程的研究热点。 ORCID: 0000-0001-8259-4540(江宗睿) 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     

牛肌腱冻干脱细胞支架的生物力学特性

背景：目前的脱细胞方法在去除细胞的同时对细胞外基质存在一定的损伤,降低了脱细胞支架的生物力学性能。 目的：分析冻干牛肌腱脱细胞支架的生物力学特性。 方法：取新鲜小牛趾伸屈肌腱,去除小牛肌腱表面的滑膜、腱膜及软组织,双蒸水冲洗干净后低压冻干,通过物理方法制备肌腱纤维束60个,随机均分为两组,实验组于无菌操作下置入丝氨酸蛋白酶抑制剂,室温下持续24 h,无菌PBS冲洗后,再移入低浓度胰酶+乙醇混合溶液中,在不破坏细胞外基质的情况下去除细胞壁,室温下持续5 h,再将纤维束移入脱氧核糖核酸酶溶液中持续5 h,最后将已完成脱细胞步骤的支架使用PBS冲洗48 h,无菌室内室温下干燥;对照组不做处置。检测两组材料的弹性模量、耐久性及最大应力。 结果与结论：两组耐久性相似,但实验组在相同位移处的应力小于对照组;两组弹性模量比较差异无显著性意义,但实验组最大应力低于对照组(P < 0.01)。说明冻干脱细胞支架能够在一定程度上模仿牛肌腱的生物力学功能。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     

自组装多肽纳米纤维水凝胶在细胞三维培养中的应用及特征

背景：自组装多肽类材料因其独特的设计及良好的生物相容性和可降解性在众多三维支架材料中脱颖而出。 目的：综述RADA类离子互补型自组装多肽支架材料的结构和功能化设计,从细胞三维培养方面探讨多肽类材料作为细胞载体材料在细胞治疗中的应用前景。 方法：由作者通过PubMed、Web of science数据库及CNKI数据库检索有关自组装多肽水凝胶的相关文献,检索词为“self-assembly peptide, tissue engineering;自组装多肽,组织工程”,检索文献量总计224篇,纳入包含多肽材料设计、功能化多肽材料、多肽材料用于细胞三维培养方面的研究,最终纳入48篇。 结果与结论：从物理结构角度讲,多肽材料可以在生理环境中自组装成具有纳米级纤维和较高孔隙率的水凝胶,最大程度上模拟细胞外基质的结构,保障细胞生存在一个真正的三维环境中。从生物功能角度讲,多肽材料可以根据不同需求复合特异性的生物活性短肽片断,赋予材料一定的细胞特异性,可以促进细胞的黏附、增殖或分化。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：     

壳聚糖及其衍生物在软骨组织工程中的应用

背景：作为生物型支架,壳聚糖因其独特的多孔三维结构、易于改性的特征及良好的生物相容性成为了软骨组织工程支架材料的研究热点。 目的：就壳聚糖及其衍生物的设计、改性及在软骨组织工程中的应用作一综述。 方法：应用计算机检索PubMed数据库和CNKI数据库,中文关键词为“壳聚糖,壳聚糖衍生物,支架材料,组织工程,软骨组织”,英文检索词为“chitosan;chitosan derivatives;scaffold;tissue engineering;cartilage”,检索文献时间范围为1990年1月至2015年1月。 结果与结论：壳聚糖是一种天然的生物多糖,通过化学改性、共混改性等方法可以改变壳聚糖的溶解度、机械强度、生物活性甚至生物降解性等自身特性,从而制成更为合适的生物支架材料。进一步研究表明,将壳聚糖与种子细胞进行共同体外培养可以获得正常形态的软骨细胞并能合成特异性的细胞外基质成分,在动物体内,壳聚糖支架与种子细胞所构建的组织工程软骨能够修复软骨损伤,形成与周围正常软骨相似的组织。壳聚糖及其衍生物支架材料在软骨组织工程中有较为广阔的研究前景。  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     

脱细胞脂肪组织制备：能否成为异体注射或原位成脂的软组织填充物

文题释义： 细胞外基质(extracellular matrix,ECM)：是由动物细胞合成并分泌到胞外、分布在细胞表面或细胞之间的大分子,主要是一些多糖和蛋白,或蛋白聚糖。这些物质构成复杂的网架结构,支持并连接组织结构、调节组织的发生和细胞的生理活动。细胞外基质是动物组织的一部分,不属于任何细胞,它决定结缔组织的特性,对于一些动物组织的细胞具有重要作用。 脱细胞脂肪组织：脂肪组织移植已成为整形外科及修复重建领域重要的手段,广泛用于软组织缺损的修复和以美容为目的的软组织填充。来源于脂肪组织由细胞外基质组成的脱细胞基质已成为脂肪医学一个新的发展方向,展示出良好的应用前景。脂肪组织脱细胞基质的产生原因可以分为物理性、化学性、酶学以及多种方法的联合,不同的方法对脂肪组织清除细胞的效果不同,对细胞外基质的影响也不同,最终会使宿主对移植的脱细胞材料产生的反应不同,进而影响脱细胞产品的安全性和有效性。 背景：通过脱细胞脂肪组织构建无种子细胞的组织工程脂肪为当前软组织填充的研究热点。 目的：探讨近年来脱细胞脂肪组织的制备方法对其移植后诱导脂肪再生效果的影响,并展望其临床应用前景。 方法：检索1971年1月至2018年12月 PubMed数据、Elsevier数据库相关文献,英文检索词为“adipose tissue engineering;adipose tissue extracellular matrix;soft tissue repair;angiogenesis;adipogenic induction”。阅读近年国内外与脱细胞脂肪组织制备和移植相关的文献,从脱细胞脂肪组织制备方法的改良,交联细胞因子和生物材料等方面进行总结归纳。 结果与结论：当前研究表明,脂肪组织细胞外基质可作为软组织填充的理想支架材料,植入皮下可募集宿主干细胞并诱导期增殖和成脂分化。但现有的脱细胞方案会导致细胞外基质蛋白和结构的损失,这极大的影响了脱细胞脂肪组织植入体内的脂肪再生能力,但通过超临界二氧化碳设备脱油、机械力预处理、交联细胞因子或生物材料等手段可以减少脱细胞脂肪组织制备过程中细胞外基质蛋白的损失和或补充具有促进组织再生功能的蛋白,最终提高脱细胞脂肪组织移植后的血管和脂肪新生能力。脱细胞脂肪组织由于其天然的成脂诱导能力,在脂肪组织工程中具有强大的应用前景,若能克服其制备流程中细胞外基质蛋白损耗或在安全可控的前提下,有望成为可异体注射并原位成脂的理想软组织填充物。 ORCID: 0000-0002-9784-2874(聂佳莹) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

组织工程技术在下尿路修复重建中的应用

背景:下尿路损伤、炎症、肿瘤以及先天性畸形等原因常可造成下尿路组织的严重缺损。传统手术对于下尿路修复重建存在诸多弊端。组织工程利用脱细胞基质与复合种子细胞的生物支架构建组织器官,为下尿路的临床修复与重建开辟了新的治疗思路。目的:就组织工程技术在男性下尿路修复重建中成熟技术做简单概述,并指出现阶段组织工程在临床应用之不足。方法:以"tissue engineering,urethra engineering,bladder;urethra,testicle"为英文检索词;以"组织工程;尿道重建;膀胱;尿道;睾丸"为中文检索词。应用计算机检索1994-01/2009-06 PubMed数据库和CNKI数据库相关文章,检索到文献72篇,排除重复性研究,最终纳入29篇文献进行分析。结果与结论:目前用膀胱替代材料重建膀胱的实验主要有两种模型,即脱细胞基质支架模型和复合种子细胞的生物支架模型。脱细胞基质支架材料主要有膀胱细胞外基质、尿道细胞外基质、小肠黏膜下基质等。复合种子细胞的生物支架则是将种子细胞接种与相应的支架上,经过相应的处理再回植入体内。这两种模型目前正处于实验与临床研究中。组织工程化尿道方面的应用技术与组织工程化膀胱类似。睾丸组织工程研究目前还处于萌芽阶段。目前组织工程"替代品"还无法与正常的组织器官相媲美,还有一定的缺陷。     

IN VITRO RESPONSE OF v-Ha-ras TRANSGENIC MOUSE LYMPHOCYTES AFTER IN VIVO TREATMENT WITH ALCOHOL

Oncomouse is a transgenic mouse carrying an activated v-Ha-ras oncogene under the control of the mouse mammary tumor virus promoter. The objective of this paper was to learn if the in vitro secretion of IL-2 and IFN-γ and the release of sIL-2R by Oncomice splenocytes and thymocytes depended on the presence of the oncogene product, on the in vivo pretreatment with alcohol, or on the in vitro treatment with cocaine or morphine. Oncomice thymocytes released less sIL-2R than FVB thymocytes. Alcohol did not increase sIL-2R release in Oncomice as it did in FVB mice thymocytes. Oncomice thymocytes secreted more IFN-γ than FVB thymocytes, their secretion was downregulated by in vivo treatment with alcohol, while it was upregulated in FVB thymocytes. IFN-γ secretion was lower in Oncomice splenocytes from animals receiving alcohol. Oncomice thymocytes and splenocytes responded in a nearly opposite fashion to their FVB counterparts. Therefore, the in vivo treatment with alcohol modified the in vitro response to cocaine or morphine in an oncogene-dependent and -independent manner. Hence, our results further emphasize the role of v-Ha-ras oncogene in defining the host immune response, and of alcohol in modulating such response.     

Increase of Natural Killer Activity of Mouse Lymphocytes Following in vitro and in Vivo Treatment with Lithium

The in vivo and in vitro influence of lithium lactate on mouse natural killer activity was investigated. In vitro exposure of effector-target mixture to graded concentrations of lithium did not substantially modify the natural killer activity of mouse splenocytes, untreated or pretreated with cyclophosphamide. However in vitro treatment of effector splenocytes increased the frequency of NK-percursor cells.

The in vivo treatment with lithium lactate greatly increased the natural killer activity in intact mice, whereas it did not improve this cytotoxic function in host immunodepressed by cyclophosphamide.

These data suggest that lithium salts produce a modulation of natural killer activity of mouse spleen cells, probably through a mechanism involving the increase of the number of NK-precursors in hosts not subjected to cytotoxic chemotherapy.     

Stimulation of mouse macrophage antigen presentation by cocaine

Cocaine has been demonstrated to have multiple effects on the immune system. Here, we determined the effects of cocaine on macrophage antigen presentation, using an in vitro antigen presentation assay after macrophages were treated with cocaine both in vitro and in vivo. Our results showed that in vitro treatment of macrophages with cocaine significantly enhanced macrophage's ability to present ovalbumin (OVA) and the enhancement was also demonstrated in the macrophages of cocaine-injected mice. The presentation of an OVA-derived antigenic peptide (OVA_323-339), however, was not affected. In vitro cocaine treatment neither affected antigen uptake nor major histocompatibility complex (MHC) II expression and the expression of co-stimulatory molecules B7. These results suggest that cocaine may act on an early event in the antigen handling by accessory cells.     

Pidotimod Stimulates Natural Killer Cell Activity and Inhibits Thymocyte Cell Death

Experiments were performed to analyze the possible effect of the immunomodulating agent Pidotimod (3-L-pyroglutamyl-L-thiazolidine-4-carboxylic acid) on mouse Natural Killer (NK) cell activity and glucocorticoid hormone(GCH)-induced thymocyte apoptosis. The results indicate that in vivo treatment with Pidotimod (200 mg/Kg ip for 5 days) causes a significant increase in NK activity and in vitro treatment produces a significant reduction of dexamethasone-induced thymocyte apoptosis. This inhibition appears to be dose-dependent and is also evident against TPA or Ca⁺⁺ionophore-induced apoptosis.     

In vivo validation of signaling pathways regulating human monocyte chemotaxis

Identification of novel signal transduction pathways regulating monocyte chemotaxis can indicate unique targets for preventive therapies for treatment of chronic inflammatory diseases. To aid in this endeavor we report conditions for optimal transfection of primary human monocytes coupled with a new model system for assessing their chemotactic activity in vivo. This method can be used as a tool to identify the relevant signal transduction pathways regulating human monocyte chemotaxis to MCP-1 in the complex in vivo environment that were previously identified to regulate chemotaxis in vitro. MCP-1-dependent chemotaxis of monocytes is studied in an adoptive transfer model where human monocytes transfected with mutant cDNAs are transferred to mice followed by initiation of peritonitis. Harvesting peritoneal cells at 24 h diminishes the contribution of immunologic responses to the cross-species transfer. Validation of relevant regulatory molecules in vivo is critical for understanding the most relevant therapeutic targets for drug development.     

IN VITRO RESPONSE OF v-Ha-ras TRANSGENIC MOUSE LYMPHOCYTES AFTER IN VIVO TREATMENT WITH COCAINE

Oncomouse is a transgenic mouse carrying an activated v-Ha-ras oncogene under the control of the mouse mammary tumor virus promoter. The objective of this paper was to learn if the in vitro secretion of IL-2 and IFN-γ and the release of sIL-2R by Oncomice spleen and thymus cells depended on the presence of the oncogene product, on the in vivo pretreatment with cocaine, or on the in vitro treatment with cocaine or morphine. Oncomice thymocytes from different experimental groups released less sIL-2R than FVB thymocytes. Oncomice thymocytes secreted more IFN-γ than FVB thymocytes. Oncomice thymocytes cultured in the presence of Con A and cocaine showed a diminished release of sIL-2R and a lower secretion of IFN-γ, a phenomenon not observed in FVB thymocytes. IFN-γ secretion was lower in Oncomice splenocytes. In general, Oncomice thymocytes and splenocytes responded in a nearly opposite fashion to their FVB counterparts. In this study, the in vitro response to mitogens, cocaine or morphine depended on genetic background and not on the in vivo pretreatment with cocaine. Our results emphasize the role of the v-Ha-ras oncogene in defining the host immune response.     

In situ assessment of cell viability within biodegradable polylactic acid polymer matrices

Efforts to expand treatment options for articular cartilage repair have increasingly focussed on the implantation of cell polymer constructs. Primary cells cultured from perichondrium, a chondrogenic tissue, were found to survive in vitro within a biodegradable porous polylactic acid matrix. The novel application of an in situ fluorescent double-stain protocol to cell polymer constructs was supported by increased ³H-thymidine uptake and the ability of cell seeded polylactic acid to form first passage explant cultures. This in situ viability staining technique allowed for rapid determination of cell viability and, in conjunction with confocal microscopy, assessment of cellular distribution within a biodegradable scaffold. Advantages of using this method over histological and electron microscopic analysis include in situ observation, absence of distortion in scaffold architecture due to polymer dissolution and disruption during processing, and obtaining a viability assessment within 30 min. Potential applications of this protocol as a screening tool for laboratory engineered tissues and in the evaluation of cellular injury in natural tissues are discussed.     

Pharmacological stimulation reveals recombinant human nerve growth factor-induced increases of in vivo hippocampal cholinergic function measured in rats with partial fimbrial transections.

The present study determined the effects of chronic recombinant human nerve growth factor administration [1 μg given intracerebroventricularly q.i.d. (every other day) for three weeks] on in vivo hippocampal cholinergic function in adult rats with unilateral partial fimbrial transections. Partial fimbrial transections did not significantly alter the levels of endogenous acetylcholine or [²H₄]acetylcholine in the hippocampus due to functional compensation by surviving cholinergic terminals. In animals chronically treated with nerve growth factor, the levels of endogenous choline, endogenous acetylcholine, [²H₄]choline and [²H₄]acetylcholine accumulated in the hippocampus on the lesioned side were not significantly different from those on the contralateral unlesioned side or from values measured in animals treated with cytochrome c, a control protein. However, changes in cholinergic parameters induced by the partial lesions or recombinant human nerve growth factor treatment became manifest when animals were challenged using pharmacological agents such as pentylenetetrazole or pilocarpine given after lithium chloride pretreatment. First, in nerve growth factor-treated animals administered the general stimulant pentylenetetrazole (10 mg/kg) 2 min prior to measuring in vivo cholinergic parameters, we observed a significant increase in the hippocampal content of [²H₄]choline in both lesioned and unlesioned hippocampi. The magnitude of the increase was significantly higher on the lesioned compared to the unlesioned side. Although chronic recombinant human nerve growth factor treatment induced increases of hippocampal [²H₄]choline levels, there were no concomitant increases in the level of [²H₄]acetylcholine. Second, in nerve growth factor-treated animals administered lithium chloride (3 mmol/kg) 20 h prior to pilocarpine (30 mg/kg), we observed a significant enhancement of the content of endogenous acetylcholine in the hippocampus of the lesioned side. Partial fimbrial transections also reduced in vitro cholinergic parameters reflecting endogenous acetylcholine levels in hippocampal slices. The content of endogenous acetylcholine in the slices was decreased by approximately 50% and chronic nerve growth factor treatment significantly elevated this value to approximately non-lesioned control values. Similarly, reductions in spontaneous and veratridine-evoked release of endogenous acetylcholine induced by partial fimbrial transections were counteracted by recombinant human nerve growth factor treatment.

These findings demonstrate that chronic recombinant human nerve growth factor treatment effectively enhances the in vivo and in vitro synthesis, storage and release of endogenous acetylcholine. The results from the in vivo studies suggest that recombinant human nerve growth factor-induced differences in functional performance of hippocampal neurons may only be manifest during behavioral and/or pharmacological stimulation.     

Effects of Inactivated Streptococci (OK-432) on macrophage functions in Mice

The effects of inactivated streptococci (OK-432) on murine macrophage functions were investigated, in viva treatment of peritoneal macrophages with OK-432 augmented the direct cytotoxic activity against TU5 tumor cells in a 48 h tritiated thymidine release assay. OK-432 also stimulated the rapid (6 h, ⁵¹Cr release) macrophage-mediated killing of Actinomycin D-sensitized WEHI 164 sarcoma cells. Moreover, the expression of la antigens on peritoneal macrophages was found to be greatly enhanced after in vivo treatment with OK-432. The immunomodulatory effects of OK-432 on macrophages functions may contribute to the antitumor activity of inactivated streptococci.     

Engineering of extensor tendon complex by an ex vivo approach

Engineering of extensor tendon complex remains an unexplored area in tendon engineering research. In addition, less is known about the mechanism of mechanical loading in human tendon development and maturation. In the current study, an ex vivo approach was developed to investigate these issues. Human fetal extensor tenocytes were isolated, expanded and seeded on polyglycolic acid (PGA) fibers that formed a scaffold with a shape mimicking human extensor tendon complex. After in vitro culture for 6 weeks, 7 cell-scaffold constructs were further in vitro cultured with dynamic mechanical loading for another 6 weeks in a bioreactor. The other 14 constructs were in vivo implanted subcutaneously to nude mice for another 14 weeks. Seven of them were implanted without loading, whereas the other 7 were sutured to mouse fascia and animal movement provided a natural dynamic loading in vivo. The results demonstrated that human fetal cells could form an extensor tendon complex structure in vitro and become further matured in vivo by mechanical stimulation. In contrast to in vitro loaded and in vivo non-loaded tendons, in vivo loaded tendons exhibited bigger tissue volume, better aligned collagen fibers, more mature collagen fibril structure with D-band periodicity, and stronger mechanical properties. These findings indicate that an extensor tendon complex like structure is possible to generate by an ex vivo approach and in vivo mechanical loading might be an optimal niche for engineering functional extensor tendon.