Pellet culture elicits superior chondrogenic redifferentiation than alginate‐based systems

Although pellet culture and encapsulation of chondrocytes into gel‐like biomaterials have lead to major advances in cartilage tissue engineering, a quantitative comparative characterization of cellular differentiation behavior during those cultivation procedures has not yet been performed. Our study therefore aimed at answering the following question: is the redifferentiation pathway of chondrocytes altered by slight changes in the type of alginate biomaterial (pure alginate, alginate‐fibrin, alginate‐chitosan) and how do the cells behave in comparison to biomaterial‐free (pellet) three‐dimensional culturing? Monolayer‐expanded chondrocytes from healthy adult porcine knee joints were cultivated in alginate, alginate‐chitosan, alginate‐fibrin beads and as pellets up to 4 weeks. Quantitative PCR and Immunohistology were used to assess chondrogenic markers. Alginate‐fibrin—encapsulated chondrocytes behaved almost like monolayer chondrocytes. Alginate‐ and alginate‐chitosan encapsulation lead to a low chondrogenic marker gene expression. Although all 3D‐cultured chondrocytes showed a considerable amount of Sox9 expression, only pellet cultivation lead to a sufficient Collagen II expression. This puts the usage of alginate‐cultivated cartilage tissue engineering constructs under question. Fibrin addition is not beneficial for chondrogenic differentiation. Sox9 and Collagen II behave differently, depending upon the surrounding 3D‐environment. © 2009 American Institute of Chemical Engineers AIChE J, 2009     

水凝胶负载干细胞外泌体在组织再生领域的应用研究进展

近年来,间充质干细胞(mesenchymal stem cells,MSCs)衍生的外泌体在组织再生领域引发许多关注。MSCs衍生外泌体作为细胞间通讯的信号分子,具有天然靶向性强、免疫原性低等特点,其通过MSCs旁分泌途径被细胞吸收,参与调控发挥促进细胞或组织再生功能。水凝胶作为再生医学领域的支架材料,具有良好的生物相容性、降解性等特点。将二者制成复合物联合使用后不仅可以提高外泌体在病变位置的滞留时间,且可通过原位注射等方法提高外泌体到达病变位置的剂量,在病变区域治疗效果显著且持续性改善。文中总结了现阶段外泌体与水凝胶复合物材料共同作用促进组织修复、再生的研究结果,以期为未来组织再生领域中的相关研究工作提供借鉴。     

Chondrogenic differentiation and immunological properties of mesenchymal stem cells in collagen type I hydrogel

Allogeneic mesenchymal stem cells (MSCs) are regarded as promising seed cells for engineering cartilage. However, few researches have covered the immune properties of seeded MSCs. Collagen has been considered as good scaffold, whether it has inherent chondrogenic inducibility for MSCs is still in debate. In this study, engineering grafts are constructed by neonatal rabbit MSCs and collagen Type I hydrogel. After periods of culture, the appearance of chondroid tissue in the grafts and the cartilage matrix‐specific genes expressions of seeded cells prove the inducibility of collagen hydrogel, even if the growth factors are absence. With the differentiation, immunological properties of MSCs are changing. The expressions of main histocompatibility complex (MHC) molecules increase and the ability to inhibit the proliferation of activated lymphocytes may be declined. But to a large extent, it keeps the low stimulating to allogeneic lymphocytes and the small absolute value of MHCs. The changes are adverse for avoiding inflammation and rejection. Therefore, suitable scaffold and engineering strategies should be selected. For the grafts based on Collagen I hydrogel and MSCs, a longer culture period might not be necessary. To maintain the immune regulation, a higher initial MSCs density in engineering grafts may be more meaningful. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A Fibrin Matrix Promotes the Differentiation of EMSCs Isolated from Nasal Respiratory Mucosa to Myelinating Phenotypical Schwann-Like Cells

Because Schwann cells perform the triple tasks of myelination, axon guidance and neurotrophin synthesis, they are candidates for cell transplantation that might cure some types of nervous-system degenerative diseases or injuries. However, Schwann cells are difficult to obtain. As another option, ectomesenchymal stem cells (EMSCs) can be easily harvested from the nasal respiratory mucosa. Whether fibrin, an important transplantation vehicle, can improve the differentiation of EMSCs into Schwann-like cells (SLCs) deserves further research. EMSCs were isolated from rat nasal respiratory mucosa and were purified using anti-CD133 magnetic cell sorting. The purified cells strongly expressed HNK-1, nestin, p75^NTR, S-100, and vimentin. Using nuclear staining, the MTT assay and Western blotting analysis of the expression of cell-cycle markers, the proliferation rate of EMSCs on a fibrin matrix was found to be significantly higher than that of cells grown on a plastic surface but insignificantly lower than that of cells grown on fibronectin. Additionally, the EMSCs grown on the fibrin matrix expressed myelination-related molecules, including myelin basic protein (MBP), 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) and galactocerebrosides (GalCer), more strongly than did those grown on fibronectin or a plastic surface. Furthermore, the EMSCs grown on the fibrin matrix synthesized more neurotrophins compared with those grown on fibronectin or a plastic surface. The expression level of integrin in EMSCs grown on fibrin was similar to that of cells grown on fibronectin but was higher than that of cells grown on a plastic surface. These results demonstrated that fibrin not only promoted EMSC proliferation but also the differentiation of EMSCs into the SLCs. Our findings suggested that fibrin has great promise as a cell transplantation vehicle for the treatment of some types of nervous system diseases or injuries.     

自组装IKVAV多肽纳米纤维支架凝胶及其对嗅鞘细胞的作用

旨在观察自组装IKVAV多肽纳米纤维支架凝胶对鼠嗅鞘细胞(OECs)的作用。通过调整IKVAV溶液pH值并加入培养液触发多肽自组装为支架凝胶, 用原子力显微镜检测IKVAV分子可以自组装成编织状纳米纤维(直径为3~5 nm)。采用原代分离培养方法获得OECs单细胞悬液后, 使用差速贴壁法两次纯化OECs且在第12天通过免疫染色计数OECs纯度为85%。将IKVAV多肽纳米纤维支架凝胶与OECs复合培养, 倒置显微镜下观察OECs生长良好, Calcein-AM/PI活、死细胞染色表明活细胞数达95%。CCK-8法间接细胞计数证实IKVAV多肽可促进OECs的黏附, 对OECs增殖没有影响。由此可见IKVAV多肽可以自组装成纳米纤维支架凝胶且对OECs有良好的生物相容性及黏附作用, 可作为神经组织工程支架材料。     

脂肪间质干细胞研究进展

脂肪间质干细胞,是脂肪组织中一类多能性干细胞。其在体外特定的培养条件下,可诱导分化形成脂肪、骨、软骨、肌肉等组织类型细胞。人体脂肪组织十分丰富,用其分离脂肪间质干细胞可避免分离胚胎干细胞所面临的道德伦理问题和获取极少量骨髓分离骨髓间质干细胞时给供者带来极大痛苦等。因此脂肪间质干细胞可作为组织再生工程的干细胞理想的替代资源。本文重点论述脂肪间质干细胞的研究进展,并探讨其临床应用前景。     

Stem cell-biomaterial interactions for regenerative medicine

The synergism of stem cell biology and biomaterial technology promises to have a profound impact on stem-cell-based clinical applications for tissue regeneration. Biomaterials development is rapidly advancing to display properties that, in a precise and physiological fashion, could drive stem-cell fate both in vitro and in vivo. Thus, the design of novel materials is trying to recapitulate the molecular events involved in the production, clearance and interaction of molecules within tissue in pathologic conditions and regeneration of tissue/organs. In this review we will report on the challenges behind translating stem cell biology and biomaterial innovations into novel clinical therapeutic applications for tissue and organ replacements (graphical abstract).     

体外构建组织工程化软骨的初步研究

观察用藻酸钠凝胶在体外长期培养软骨细胞的情况,为求进一步用藻酸钠凝胶在体外构建组织工程化软骨提供初步研究.将传代培养的、细胞终浓度为1×107/mL的软骨细胞复合藻酸钠凝胶,注入圆柱形模具中,将模具分别浸入100、200、300 mmol/L的固化剂氯化钙溶液中,固化15 min使其成形,于体外培养2、4、6周后,行HE染色、阿尔新蓝染色,了解软骨细胞的生长情况.2、4、6周时软骨细胞与藻酸钙凝胶复合良好并能在其中保持活性及分裂能力,且在6周时出现类软骨变化.因此藻酸钠凝胶是可用于体外构建组织工程化软骨的生物材料.     

不同浓度藻酸钙复合软骨细胞体外培养的实验研究

为探寻复合软骨细胞生长的最佳藻酸钙浓度,将传代培养的、细胞终浓度为1×107/mL的软骨细胞复合藻酸钠凝胶,然后滴入浓度分别为100、200、300、400、500mmol/L的氯化钙溶液中,固化15min形成藻酸钙凝珠,于体外培养7d后,行HE染色及Masson’s三色染色,结果显示软骨细胞与固化剂氯化钙浓度为100、200、300mmol/L的藻酸钙凝胶复合良好并能在其中保持活性及分裂能力;而在氯化钙浓度为400mmol/L和500mmol/L的藻酸钙凝胶中,软骨细胞的活性及分裂能力明显下降.因此固化剂氯化钙的浓度可以提高到300mmol/L,该浓度不仅对软骨细胞的生长无影响,而且能适当提高藻酸钙凝胶的机械强度,可以作为一种较理想的软骨组织工程支架材料.     

Engineering bone: challenges and obstacles

Repair of large bone defects is still a challenge for the orthopaedic, reconstructive and maxillo-facial surgeon. Availability of pluripotent stem cells from either autologous or allogenic sources and the potential of inducing the osteogenic phenotype is motivating exploration and development of custom-tailored materials known as "bioengineered bone constructs". In such cases, the clinical scenario involves either expansion of stem cells in monolayer and loading them into a porous scaffold prior to surgery or direct cell expansion within the scaffold, and implanting this novel construct back into the donor patient. In this review, we delineate, from an engineering perspective, the progress that has been made to date and the challenges remaining in successfully translating this promising (but not yet definitively established) approach from bench to the bed site.