原位定量评价组织工程研究中细胞生长状态的新方法

目的 探讨一种能原位定量评定细胞在高分子聚合载体上生长情况的方法。方法 采用CFDA－AM和AM和PI双染结合激光扫描共聚焦显微镜手术，用平均荧光强度代表细胞数量，对组织工程骺软骨中骨骺软骨细胞在聚乳酸载体上1、2及3周时的死活细胞数量进行评价。结果 骨骺软骨细胞在聚乳酸载体上的530nm的平均荧光强度随时间逐渐增加，而603nm的荧光强度在第2周时有所增加，而到第3周时则无明显增加。与细胞在载体上重量增加的结果一致。结论 首次采用的原位定量检测载体上死活细胞的方法是可行的，解决也在不透明载体上即不影响细胞和载体细胞，而可以原位快速定量评定细胞生长的难题。     

聚乳酸载体在组织工程骺软骨研究中的应用

目的 了解聚乳酸是否可以用作骨骺软骨细胞的载体。方法 采用多孔聚乳酸膜片作载体，与骨骺软骨细胞共培养，通过组织学、透射电镜、扫描电镜观察细胞与载体的关系及细胞生长情况；通过凝胶渗透色谱法了解聚乳酸载体肌肉植入后分子量改变情况和局部反应。结果 代表团显微镜、组织学及电镜观察均显示细胞可以在载体上生长，并且分泌基质；聚乳酸载体植入体内后分子量逐渐减少，周围异物细胞较少。结论 首次成功地培养出了组织工程骺软骨；聚乳酸在体内可以不断降解，组织相容性较好；骨骺软骨细胞可以在载体上不断生长。     

软骨组织工程中细胞外基质的研究

目的：分析软骨组织工程中软骨细胞、软骨基质、生长因子之间的相互作用及细胞外基质替代物的开发。 资料来源：应用计算机检索Medline1995-01／2005-12有关软骨组织工程中软骨细胞外基质的文章，检索词为“cartilage；extraeellular matrix”．并限定文章语言种类为English。 资料选择：对资料进行审阅,选取包括软骨组织工程的文章，并查阅全文。纳入标准：①软骨细胞外基质及其受体。②软骨组织重建相关生长因子。③软骨细胞外基质替代物。排除标准：Meta分析、综述文献、重复研究。 资料提炼：共收集到1 179篇关于组织工程软骨及细胞外基质的文献．纳入符合标准的文献29篇。 资料综合：软骨组织无血管，其自我修复及重建能力有限，较小的创伤就会造成严重的软骨损伤及变性。软骨基质在软骨自身动态平衡及软骨修复等方面起着非常重要的作用，软骨细胞合成、分泌软骨基质并受软骨基质的调控。重建软骨组织就必须考虑软骨细胞及其周围环境的相互作用。 结论：重建关节软骨基质、促进软骨复原，研究软骨细胞和其周围环境之间的相互作用是关键。     

软骨组织工程研究中细胞源-软骨细胞和成纤维细胞

目前关节软骨仍是供软骨修复的主要软骨细胞来源,但获得关节面软骨本身是一种有创操作,且细胞的低产量,低有丝分裂率,低生物活性进一步限制了关节软骨作为供体细胞来源的实际临床应用.体内其他作为自体软骨细胞源包括耳骨、鼻中隔、肋软骨、颞颌关节髁突软骨等,而不同的软骨源具有不同的结构、组成和功能,产生具有不同生化、物理和生物力学特性的细胞及细胞外基质.目前用于软骨再生的软骨细胞主要来源于幼稚动物,这些刚出生的和幼体动物的软骨细胞较年老供体的软骨细胞增殖率高,形成软骨的潜能较强.皮肤可为组织工程提供一种侵袭性小、来源丰富的成纤维细胞源.将成纤维细胞直接置于软骨缺损的聚乳酸网格会导致纤维组织增生,然而在恰当的培养条件下,成纤维细胞可以被诱导向软骨细胞表型转化.     

聚乳酸载体在组织工程骺软骨研究中的应用

目的了解聚乳酸是否可以用作骨骺软骨细胞的载体。方法采用多孔聚乳酸膜片作载体,与骨骺软骨细胞共培养,通过组织学、透射电镜、扫描电镜观察细胞与载体的关系及细胞生长情况;通过凝胶渗透色谱法了解聚乳酸载体肌肉植入后分子量改变情况和局部反应。结果倒置显微镜、组织学及电镜观察均显示细胞可以在载体上生长,并且分泌基质;聚乳酸载体植入体内后分子量逐渐减少,周围异物细胞较少。结论首次成功地培养出了组织工程骺软骨;聚乳酸在体内可以不断降解,组织相容性较好;骨骺软骨细胞可以在载体上不断生长。     

软骨组织工程中细胞外基质的研究

目的:分析软骨组织工程中软骨细胞、软骨基质、生长因子之间的相互作用及细胞外基质替代物的开发。资料来源:应用计算机检索Medline1995-01/2005-12有关软骨组织工程中软骨细胞外基质的文章,检索词为“cartilage;extracellularmatrix”,并限定文章语言种类为English。资料选择:对资料进行审阅,选取包括软骨组织工程的文章,并查阅全文。纳入标准:①软骨细胞外基质及其受体。②软骨组织重建相关生长因子。③软骨细胞外基质替代物。排除标准:Meta分析、综述文献、重复研究。资料提炼:共收集到1179篇关于组织工程软骨及细胞外基质的文献,纳入符合标准的文献29篇。资料综合:软骨组织无血管,其自我修复及重建能力有限,较小的创伤就会造成严重的软骨损伤及变性。软骨基质在软骨自身动态平衡及软骨修复等方面起着非常重要的作用,软骨细胞合成、分泌软骨基质并受软骨基质的调控。重建软骨组织就必须考虑软骨细胞及其周围环境的相互作用。结论:重建关节软骨基质、促进软骨复原,研究软骨细胞和其周围环境之间的相互作用是关键。     

马桑内酯诱导神经元微丝表达及其与细胞缝隙连接通道关系的研究

目的:研究马桑内酯(CoriariaLactone,CL)对神经元微丝F-actin的影响及其与缝隙连接通道的关系,以及它们在癫痫发病中的意义。方法:利用培养的神经元,采用直接免疫荧光法,用激光扫描共聚焦显微镜观察细胞骨架F-actin的结构及分布,同时测定F-actin含量。结果:致痫剂量的马桑内酯作用于神经元48h后,F-actin的荧光强度较对照组降低(P<0.01),给予CL+1-庚醇(1-hepatal),CL+1-庚醇+丙戊酸钠,CL+1-庚醇+卡马西平作用于神经元48h后F-actin的荧光强度较对照组明显升高(P<0.01),且以加丙戊酸钠和卡马西平组更显著。结论:CL可引起细胞内F-actin含量的降低,而通道阻滞剂1-庚醇,抗癫痫药丙戊酸钠、卡马西平可引起细胞内F-actin含量的增加,这种变化影响细胞骨架微丝装配和神经元胞间信号传导,可能在癫痫的产生中发挥重要作用。     

让昨天告诉今天：组织工程研究的学术与技术进展

组织工程研究成果按时间年限的导读：1987年10月美国科学基金会在华盛顿举办的生物工程小组会议上首次提出“组织工程”一词，1988年正式定义。1988年，组织工程学创始人美国的Vacanti教授将聚羟基乙酸（PGA）和聚乳酸（PLA）作为支架，复合软骨细胞后植入裸鼠皮下进行体内培养，28d后发现有软骨样组织出现。1989年，美国的Wakitan教授将软骨细胞接种于胶原凝胶内培养获得成形的软骨，     

组织工程化软骨种子细胞的研究与展望

目的：软骨组织的再生能力有限，组织工程软骨的构建对修复软骨缺损有重大的意义。而软骨种子细胞是研究组织工程软骨最为重要的材料，也是目前软骨组织工程研究的重点及难点，因此，探讨软骨种子细胞的来源及其相关的生长因子对软骨组织工程的发展有重要的指导作用。资料来源：应用计算机检索Medline 1994—01／2004—09相关文章，检索词“tissue engineering cartilage，seeding chondmcytes”，并限定文章语言种类为English。同时计算机检索维普医学数据库和http：／／www．wanfangdata．com．cn 2002—01／2004—12相关文章，检索词“组织工程，软骨细胞”，限定文章语言种类为中文。资料选择：纳入条件：①随机对照实验研究。②单一样本研究。排除条件：①重复研究。②综述文献。资料提炼：共收集到35篇关于组织工程软骨的种子细胞的相关文献，其中10篇因系重复研究而被筛除。选取的25篇分别选自19种不同的杂志。资料综合：将25篇文献资料中软骨种子细胞的来源及其相关的生长因子研究的新进展予以综合。结论：随着胚胎干细胞技术、体外大规模培养技术、生长因子的应用及基因修饰等技术的成熟，组织工程软骨的研究将取得突破性的进展。     

两种培养基对铜绿假单胞菌和大肠埃希菌生物被膜厚度的影响

目的探讨营养成分不同的两种培养基对临床分离的铜绿假单胞菌和大肠埃希菌形成生物被膜形态的影响。方法使用电击转化将pSMC21转入临床分离的3株铜绿假单胞菌和3株大肠埃希菌体内，构建表达绿色荧光蛋白的菌株。在Luria-Bertani（LB）和M9葡萄糖基础培养基中以盖玻片为支持表面，进行细菌生物被膜的连续培养。经过12天的培养周期，使用共聚焦激光扫描显微镜检测盖玻片上生物被膜的厚度。使用方差分析检验每个样本5个测量值的重复性，并用配对T检验来分析同一株细菌在两种培养基中生物被膜的厚度变化。结果pSMC21可以在6株临床分离株中稳定表达绿色荧光蛋白；每个样本5个测量值重复性好，其差异无统计学意义（P=0．46）；大肠埃希菌在LB和M9培养基中形成生物被膜平均厚度的差异无统计学意义（t=-0．42，P〉0.7），而铜绿假单胞菌在LB培养基中形成的生物被膜更厚，其差异有统计学意义（t=3．46，P〈0．02）。结论铜绿假单胞菌所形成生物被膜的平均厚度因培养基成分的不同而有显著差异，而大肠埃希菌所形成生物被膜的平均厚度则不受培养基成分不同的影响。     

On‐line monitoring of oxygen as a non‐destructive method to quantify cells in engineered 3D tissue constructs

Regulatory guidelines have established the importance of introducing quantitative quality controls during the production and/or at the time of release of cellular grafts for clinical applications. In this study we aimed to determine whether on‐line measurements of oxygen can be used as a non‐destructive method to estimate the number of chondrocytes within an engineered cartilage graft. Human chondrocytes were seeded and cultured in a perfusion bioreactor, and oxygen levels in the culture medium were continuously monitored at the inlet and outlet of the bioreactor chamber throughout the culture period. We found that the drop in oxygen across the perfused construct was linearly correlated with the number of cells per construct (R² = 0.82, p < 0.0001). The method was valid for a wide range of cell numbers, including cell densities currently used in the manufacture of cartilage grafts for clinical applications. Given that few or no non‐destructive assays that quantitatively characterize an engineered construct currently exist, this non‐invasive method could represent a relevant instrument in regulatory compliant manufacturing of engineered grafts meeting specific quality criteria. Copyright © 2012 John Wiley & Sons, Ltd.     

低强度次声对体外培养的骨样细胞骨架蛋白F-actin表达的动态影响

目的：探讨4Hz／100dB、12Hz／100dB、20Hz／100dB的次声作用后，小鼠成骨样细胞MC3T3的细胞骨架F—actin表达的改变。方法：将MC3T3接种于细胞玻片上，并分为对照组和4Hz／100dB、12Hz／100dB、20Hz／100dB的次声暴露组。对照组无次声输出，其他各组接受次声作用30min／d。第3d于暴露后2h、4h、8h不同时间，对细胞进行F—actin的免疫荧光染色．应用激光扫描共聚焦显微镜．观察细胞F—actin的表达改变，测定单个细胞F—actin的平均荧光强度。结果：对照组细胞大部分荧光物质呈弥漫状态，胞膜荧光较强，胞浆内少量肌动蛋白纤维丝，方向不规则，长短不一；不同频率次声作用后2h．各次声作用组均可看到胞浆中微丝F—actin明显粗大纤长，荧光物质大多为较长的粗大应力丝，沿细胞纵轴排列较多，其中20Hz，100dB组的荧光强度增强较对照组具有显著意义（P〈0．05）；在次声作用后4h和8h．次声作用组的细胞F—actin仍处于较高表达状态．其中12Hz，100dB组和20Hz，100dB组的荧光强度增高较对照组具有显著意义（P〈0．05）。不同频率次声作用组细胞的F—actin变化趋势较一致，各组在各时间点未见明显差异（P〉0．05）。结论：4HZ、12Hz和20Hz的100dB的次声作用30min／d后，可诱导F—actin表达的增强，这种改变在8h后仍未见减弱。     

A novel method to align cells in a cardiac tissue‐like construct fabricated by cell sheet‐based tissue engineering

Fabrication of cardiac tissue from human induced pluripotent stem cell‐derived cardiomyocytes (hiPS‐CMs) has received great interest, but a major challenge facing researchers is the alignment of cardiomyocytes in the same direction to optimize force generation. We have developed a novel method of fabricating a cardiac tissue‐like construct with aligned cells based on the unidirectional stretching of an hiPS‐CM sheet. A square cell sheet was harvested from a temperature‐responsive culture dish and placed on a silicone surface, and an extending force was imposed on the silicone to stretch the cell sheet along one direction. To enable evaluation of cardiomyocyte morphology in vitro, a cell sheet was constructed by coculture of hiPS‐CMs and human adipose‐derived stem cells. In separate experiments, a stretched double‐layered cell sheet constructed from hiPS‐CMs alone was transplanted onto the muscle of an athymic rat, and its features were compared with those of a nonstretched (control) cell sheet. Immediately after stretching, the stretched cell sheet was significantly longer than the control cell sheet. Immunohistological analysis revealed that the cardiomyocytes showed unidirectional alignment in the stretched cell sheet but random directionality in the control cell sheet. Two weeks after transplantation, immunohistology demonstrated that the stretched cell sheet had retained the unidirectionality of its myocardial fibers and had an orientation intensity that was higher than that of the control cell sheet after transplantation or the stretched cell sheet before transplantation. Our technique provides a simple method of aligning an hiPS‐CM‐derived cardiac tissue‐like construct without the use of a scaffold.     

Three-dimensional culture of mouse bone marrow cells on stroma formed within a porous scaffold: influence of scaffold shape and cryopreservation of the stromal layer on expansion of haematopoietic progenitor cells

This study's primary goal was to develop an effective ex vivo expansion method for haematopoietic cells. 3D culture of mouse bone marrow cells was performed in porous scaffolds using a sheet or cube shape. Bone marrow cells were cultured on bone marrow-derived stromal layers formed within the scaffolds and the effect of scaffold shape on the expansion of haematopoietic cells was examined. In some experiments, stromal layers within cubic scaffolds were frozen and then used to culture bone marrow cells after thawing. Results show that after comparison, total cell density and expansion of haematopoietic cells were greater in cultures using the cubic scaffold, suggesting that it was superior to the sheet-like scaffold for expanding haematopoietic cells. When cryopreserved stroma was used, it effectively supported the expansion of haematopoietic cells, and a greater expansion of haematopoietic cells [(erythroid and haematopoietic progenitor cells (HPCs)] was achieved than in cultures with stromal cells that had not been cryopreserved. Expansion of cells using cryopreserved stroma had several other advantages such as a shorter culture period than the conventional method, a stable supply of stromal cells, and ease of handling and scaling up. As a result, this is an attractive method for ex vivo expansion of haematopoietic stem cells (HSCs) and HPCs for clinical use.     

An innovative stand‐alone bioreactor for the highly reproducible transfer of cyclic mechanical stretch to stem cells cultured in a 3D scaffold

Much evidence in the literature demonstrates the effect of cyclic mechanical stretch in maintaining, or addressing, a muscle phenotype. Such results were obtained using several technical approaches, useful for the experimental collection of proofs of principle but probably unsuitable for application in clinical regenerative medicine. Here we aimed to design a reliable innovative bioreactor, acting as a stand‐alone cell culture incubator, easy to operate and effective in addressing mesenchymal stem cells (MSCs) seeded onto a 3D bioreabsorbable scaffold, towards a muscle phenotype via the transfer of a controlled and highly‐reproducible cyclic deformation. Electron microscopy, immunohistochemistry and biochemical analysis of the obtained pseudotissue constructs showed that cells ‘trained’ over 1 week: (a) displayed multilayer organization and invaded the 3D mesh of the scaffold; and (b) expressed typical markers of muscle cells. This effect was due only to physical stimulation of the cells, without the need of any other chemical or genetic manipulation. This device is thus proposed as a prototypal instrument to obtain pseudotissue constructs to test in cardiovascular regenerative medicine, using good manufacturing procedures. Copyright © 2012 John Wiley & Sons, Ltd.     

A novel method for the direct fabrication of growth factor-loaded microspheres within porous nondegradable hydrogels: Controlled release for cartilage tissue engineering

Because of similar mechanical properties to native cartilage, synthetic hydrogels based on poly(vinyl alcohol) (PVA) have been proposed for replacement of damaged articular cartilage, but they suffer from a complete lack of integration with surrounding tissue. In this study, insulin-like growth factor-1 (IGF-1), an important growth factor in cartilage regeneration, was encapsulated in degradable poly(lactic-co-glycolic acid) (PLGA) microparticles embedded in the PVA hydrogels in a single step based on a double emulsion. The release of IGF-1 from these hydrogels was sustained over 6 weeks in vitro. Poly(glycolic acid) (PGA) fiber scaffolds were wrapped around the hydrogels, seeded with chondrocytes, and implanted subcutaneously in athymic mice. The release of IGF-1 enhanced cartilage formation in the layers surrounding the hydrogels, in terms of the content of extracellular matrix components and mechanical properties, and increased integration between the cartilage layers and the hydrogels, according to gross observation of the cross-sections and histology. The compressive modulus of the cartilage-hydrogel constructs without IGF-1 was 0.07 ± 0.02 MPa, compared to 0.17-0.2 MPa for hydrogels that contained IGF-1. The biochemical and mechanical markers of cartilage formation were not different between the low and high concentrations of IGF-1, despite an order of magnitude difference in concentration. This study shows that the sustained release of IGF-1 can enhance tissue formation and points to a possible strategy for effecting integration with surrounding tissue.     

Alteration of the extracellular matrix and alpha‐gal antigens in the rat lung scaffold reseeded using human vascular and adipogenic stromal cells

Regenerated organs are expected to solve the problem of donor organ shortage in transplantation medicine. One approach to lung regeneration is to decellularize the organ and reseed it with selected cells. An advantage of the procedure is reduced immunogenicity, because all cells can be theoretically replaced by autologous cells. However, little is known regarding the extracellular matrix (ECM) damage during decellularization and ECM reconstruction process in the organ regeneration. We aimed to evaluate ECM damage and reconstruction of the decellularized–recellularized rat lung, including the removal of alpha‐gal xenoantigens. Rat lungs were perfused with sodium dodecyl sulfate and Triton X‐100 via the pulmonary artery, after which the decellularized scaffold was reseeded with rat or human endothelial cells and adipose‐derived stem cell (ASCs). The ECM and alpha‐gal antigen were evaluated using immunohistochemistry, western blotting, and a glycosaminoglycan assay. Alcian blue staining revealed increased production of proteoglycan following the addition of ASCs to the rat lung recellularized with rat lung microvascular endothelial cells. Glycosaminoglycan levels decreased in the decellularized lung and increased in the recellularized lung, especially in the ASC‐treated group. Immunohistochemical expression of the alpha‐gal protein was decreased to an undetectable level in the decellularized lung tissue and disappeared after recellularization with human cells. In western blot analysis, the bands of alpha‐gal protein almost disappeared after recellularization with human cells. In conclusion, characteristics of the regenerated ECM might depend on the species and type of cells used for recellularization. Therefore, alpha‐gal antigen might be eliminated after a prolonged culture, when using human cells.     

脂肪干细胞成骨分化及与复合支架结合：在修复骨质疏松症骨缺损中的应用

背景：针对骨质疏松症伴发骨缺损疾病的传统治疗方法,诸如自体骨移植、异体骨移植、生物材料植入均有明显的局限性。以脂肪干细胞为种子细胞,采用再生医学的方法,为骨质疏松症骨缺损的修复提供了一种新的途径。
　　目的：就骨质疏松症的发病机制及其对骨缺损修复的影响、信号通路对脂肪干细胞成骨分化的调控、脂肪干细胞修复骨质疏松症骨缺损的可行性等方面作一总结。
　　方法：应用计算机检索中国期刊全文数据库(CNKI)和PubMed数据库中1998年1月至2014年9月相关文献。在标题和中以“脂肪干细胞,骨质疏松症,骨缺损,成骨分化,骨再生”或“adipose-derived stem cel s, osteoporosis, bone defect, osteogenic differentiation, bone regeneration”为检索词进行检索,重点对77篇文章进行分析。
　　结果与结论：近年来,脂肪干细胞已广泛应用于再生医学的研究。随着有关学科如再生医学、组织工程学、分子生物学、材料学的发展,对脂肪干细胞成骨分化调控机制的研究不断深入,脂肪干细胞复合生物支架构建组织工程骨为解决骨质疏松症骨缺损修复难题,提供了一种新的思路和方法,具有良好的发展前景。     

The use of a novel bone allograft wash process to generate a biocompatible,mechanically stable and osteoinductive biological scaffold for use in bone tissue engineering

Fresh‐frozen biological allograft remains the most effective substitute for the ‘gold standard’ autograft, sharing many of its osteogenic properties but, conversely, lacking viable osteogenic cells. Tissue engineering offers the opportunity to improve the osseointegration of this material through the addition of mesenchymal stem cells (MSCs). However, the presence of dead, immunogenic and potentially harmful bone marrow could hinder cell adhesion and differentiation, graft augmentation and incorporation, and wash procedures are therefore being utilized to remove the marrow, thereby improving the material's safety. To this end, we assessed the efficiency of a novel wash technique to produce a biocompatible, biological scaffold void of cellular material that was mechanically stable and had osteoinductive potential. The outcomes of our investigations demonstrated the efficient removal of marrow components (~99.6%), resulting in a biocompatible material with conserved biomechanical stability. Additionally, the scaffold was able to induce osteogenic differentiation of MSCs, with increases in osteogenic gene expression observed following extended culture. This study demonstrates the efficiency of the novel wash process and the potential of the resultant biological material to serve as a scaffold in bone allograft tissue engineering. © 2014 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons Ltd.