CD146阳性亚群有作为软骨组织工程种子细胞的应用潜力

文题释义：CD146：也称为MUC18,MCAM,Mel-CAM或S-Endo1,是一种跨膜糖蛋白,同时属于免疫球蛋白超级家族的一员。在人体组织中,CD146阳性细胞被认为是微血管的壁细胞,其具有较强的增殖、迁移及自我更新能力。 组织工程种子细胞：组织工程包括3个关键要素——种子细胞、支架和活性因子,其中种子细胞应具有良好的细胞活性、增殖能力、分化及合成基质等生物功能。 背景：再生医学的发展、组织工程技术的出现为软骨缺损重建提供了新的解决思路。在组织工程中,间充质干细胞是应用广泛的种子细胞,然而干细胞作为一个异质性的细胞群体其不同亚群发挥着不同的功能。因此,应用间充质干细胞关键功能亚群进行软骨修复具有广泛应用前景。 目的：从人脂肪间充质干细胞中分离出CD146阳性亚群细胞,验证其生物学特性及其作为软骨组织工程种子细胞的潜力。 方法：人脂肪间充质干细胞由浙江金时代生物技术有限公司提供,通过流式细胞术对人脂肪间充质干细胞表面标志物进行鉴定,应用免疫磁珠分选方法从人脂肪间充质干细胞中分选出CD146阳性表达的细胞亚群。通过基因芯片检测技术及生物信息学分析技术揭示2种细胞的分子特性;体外诱导2种细胞成软骨分化并进行鉴定;冻存复苏前后检测2种细胞的细胞活性及凋亡情况。 结果与结论：①人脂肪间充质干细胞表达高水平干细胞相关标志物CD73、CD90,不表达造血干细胞相关标志物CD34、CD45、HLA-DR;②生物信息分析结果表明CD146阳性亚群细胞与脂肪间充质干细胞相比在炎症通路及骨骼肌肉系统疾病有不同功能;③CD146阳性亚群细胞能够成球软骨分化,并且其成软骨分化能力要优于人脂肪间充质干细胞;④CD146阳性亚群细胞复苏后凋亡情况和活性均要优于人脂肪间充质干细胞;⑤结果表明,CD146阳性亚群细胞具有良好的成软骨分化潜力,是一种具有前景的软骨组织工程种子细胞。 ORCID: 0000-0003-4210-4708(眭翔) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

脂肪来源干细胞在骨／软骨／椎间盘／肌腱组织修复中的应用

骨、软骨、椎间盘、肌腱等组织的自体细胞在体外培养、扩增困难,因此很难真正用于组织的缺损修复。干细胞具有自我更新和向多种组织定向诱导分化的特性,近年来干细胞研究的不断发展为其应用于矫形外科组织再生修复提供了可能。由于脂防来源问充质细胞（ASC）来源丰富、取材容易,近来备受研究者青睐,已被越来越多地应用于干细胞和组织工程领域。主要针对ASC在矫形外科骨,软骨／椎间盘／肌腱等组织修复中的应用作一综述。最近的ASC体内缺损修复结果为其应用于临床矫形外科组织修复的潜能提供了研究依据：     

膀胱组织工程中平滑肌种子细胞来源的研究进展

近年来,组织工程学的蓬勃发展为膀胱替代提供了崭新的思路.其中种子细胞是组织工程膀胱构建及应用研究中的首要环节和基本要素之一.构建膀胱组织工程平滑肌的种子细胞有平滑肌细胞、平滑肌祖细胞、胚胎干细胞和各种组织来源的成体干细胞如骨髓来源干细胞、羊水来源干细胞、脂肪来源干细胞等.对膀胱组织工程学种子细胞来源的研究现状进行回顾,并对其研究前景作一展望.     

膀胱组织工程中平滑肌种子细胞来源的研究进展

近年来,组织工程学的蓬勃发展为膀胱替代提供了崭新的思路.其中种子细胞是组织工程膀胱构建及应用研究中的首要环节和基本要素之一.构建膀胱组织工程平滑肌的种子细胞有平滑肌细胞、平滑肌祖细胞、胚胎干细胞和各种组织来源的成体干细胞如骨髓来源干细胞、羊水来源干细胞、脂肪来源干细胞等.对膀胱组织工程学种子细胞来源的研究现状进行回顾,并对其研究前景作一展望.     

转化生长因子β3诱导脂肪间充质干细胞向纤维软骨细胞的分化

背景：颞下关节盘软骨缺损修复在口腔临床上仍然是较大的挑战,具有多向分化潜能的脂肪间充质干细胞为成纤维软骨类组织修复带来了希望。目前使用转化生长因子β3诱导脂肪间充质干细胞向纤维软骨细胞分化的研究很少。 目的：观察转化生长因子β3对脂肪间充质干细胞生长形貌及向成纤维软骨分化的影响。 方法：采用转化生长因子β3诱导SD大鼠脂肪间充质干细胞,观察成纤维软骨细胞分化的细胞形态,组织学和免疫荧光染色等方法检测脂肪间充质干细胞产生的细胞外基质Ⅰ,Ⅱ型胶原和蛋白多糖表达情况,评价脂肪间充质干细胞作为纤维软骨组织工程种子细胞的可行性。 结果与结论：倒置荧光显微镜观察结果显示脂经转化生长因子β3生长因子诱导之后,细胞有明显的聚集生长现象,形态呈多角形、多边形,细胞外基质分泌增多。阿利新蓝染色结果表明,经转化生长因子β3诱导脂肪间充质干细胞显示明显的深蓝色,表明脂肪间充质干细胞合成了大量的糖胺聚糖。免疫染色结果表明,在转化生长因子β3 诱导下,脂肪间充质干细胞合成Ⅰ,Ⅱ型胶原细胞外基质。提示转化生长因子β3可诱导脂肪间充质干细胞向成纤维软骨样细胞分化,也意味着脂肪间充质干细胞具有作为工程化纤维软骨种子细胞的潜能。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

组织工程技术修复关节软骨:如何构建新型复合模式?

背景:关节软骨缺损自我修复能力有限,传统手段难以修复,关节软骨组织工程为修复大面积关节软骨缺损提供了崭新的方法和途径。目的:综述组织工程技术修复关节软骨的现状、问题及前景。方法:检索PubMed数据库1982至2015相关文献,以"Articular cartilage,repair,tissue engineering"为检索词进行检索。纳入与关节软骨组织工程修复相关的文献,排除重复性研究。根据纳入标准,排除无关联或重复的文献,最终保留39篇文献进行综述。结果与结论:优良的种子细胞具有软骨细胞分化潜能,目前用于关节软骨修复的种子细胞主要有间充质干细胞、胚胎干细胞、脂肪源干细胞及前软骨干细胞等4大类。生长因子趋向于联合应用,生长因子具有诱导宿主实质细胞在体内生长,提高种植的细胞稳定性,加快组织再生的作用。复合支架材料有利于促进细胞接种和细胞的空间分布,促进细胞增殖,是研究的热点。如何优化种子细胞、支架材料和细胞因子的选择和匹配,建立新型复合模式,以获得最佳的关节软骨修复效果,是未来的重要发展方向。     

成纤维细胞的生物学特性及分化潜能

文题释义： 多向分化潜能：指细胞具有自我更新、持续增殖分裂并可以在不同微环境影响下向相应方向分化为多种组织的能力，即经过不同细胞因子的诱导，能够分化为脂肪、骨、软骨、肌肉、血管内皮、肝、神经等细胞系类型，一般为干细胞或类干细胞所具备的能力。 组织工程种子细胞：应用组织工程方法再造组织和器官所用的各类细胞，其中包括分化成熟的成体细胞和具有分化潜能的干细胞，应满足以下几点：采用微创或无创手段即可获取的组织，分裂增殖能力强，功能旺盛，无或仅有极微弱的免疫排斥反应，能够连续传代，且传代培养后不发生形态、功能及遗传物质的改变。 背景：组织工程技术的出现可以从根本上解决组织、器官的修复与重建问题，其中种子细胞的选择和应用则成为当前研究的热难点，成纤维细胞是各组织工程研究的热门选择。 目的：总结分析成纤维细胞的生物学特性、自身分化潜能以及对干细胞分化增殖的影响。 方法：运用计算机检索中国期刊全文数据库(2015至2019年)和PubMed数据库(2005至2019年)中有关于成纤维细胞生物学特性以及多向分化潜能的相关文献，并进行系统的归纳、总结和分析，对成纤维“干性”的研究新进展进行全面阐述。 结果与结论：成纤维细胞代谢旺盛、增殖能力强，具有合成和分泌蛋白质的功能，在不同微环境中可分化为不同细胞，拥有与干细胞一样强大的多向分化潜能，因此常运用于转分化、细胞培养、损伤修复以及组织工程，其中促进干细胞增殖及诱导分化的作用尤为显著，今后应最大化利用成纤维细胞生物学优势与干细胞共培养，为组织工程学提供种子细胞，为临床中创伤的修复提供更佳思路。 ORCID: 0000-0002-4261-361X(杨桂然) 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程     

脂肪干细胞在软骨组织工程中的研究进展

近年来脂肪干细胞（ADSC）的成软骨潜能备受瞩目,其作为种子细胞在软骨组织工程应用方面前景广阔,然而目前并没有一种高效的诱导方法被世界公认。外源性生长因子、基因修饰、共培养、生长因子与共培养联合、支架材料等多种手段均可诱导ADSC向软骨细胞方向分化,但各方法的优劣,诱导分化时的信号通路及新生软骨的性状并未见详细的分析和讨论。文中介绍了以上多种诱导方法的研究现状及问题,并讨论了化学因素、供体年龄、组织获取部位等对其的影响,以期为ADSC的临床应用提供一定的理论参考。     

一种新的种子细胞——滑膜间充质干细胞

滑膜间充质干细胞作为一种新的间充质干细胞在组织工程中有着广阔的应用前景。滑膜间充质干细胞能够分化为软骨、骨、脂肪、骨骼肌，具有取材方便，供区并发症少等优点。因此，滑膜间充质干细胞是软骨、骨、脂肪及骨骼肌的种子细胞的理想来源。介绍了滑膜间充质干细胞的分离、获取以及其生物学特性，并对其在组织工程中应用的研究进展做一回顾。     

骨髓间充质干细胞于软骨组织工程的应用

关节软骨缺损在骨科临床十分常见,目前临床修复软骨缺损的方法很多,但是由于各自固有的缺陷难以达到满意的临床效果,因此探索软骨缺损的修复方法一直是人们不断深入研究的课题。软骨组织工程的发展为软骨缺损的修复提供了新的途径。种子细胞和支架材料是软骨组织的两个基本要素,根据近年来软骨组织工程的研究进展和方向,从骨髓间充质干细胞的诱导方式、诱导机制及研究进展方面进行探讨,证明骨髓间充质干细胞作为种子细胞构建组织工程软骨的优越性。     

Chondrogenic differentiation of adipose-derived adult stem cells by a porous scaffold derived from native articular cartilage extracellular matrix

Adipose-derived adult stem cells (ASCs) have the ability to differentiate into a chondrogenic phenotype in response to specific environmental signals such as growth factors or artificial biomaterial scaffolds. In this study, we examined the hypothesis that a porous scaffold derived exclusively from articular cartilage can induce chondrogenesis of ASCs. Human ASCs were seeded on porous scaffolds derived from adult porcine articular cartilage and cultured in standard medium without exogenous growth factors. Chondrogenesis of ASCs seeded within the scaffold was evident by quantitative RT-PCR analysis for cartilage-specific extracellular matrix (ECM) genes. Histological and immunohistochemical examination showed abundant production of cartilage-specific ECM components-particularly, type II collagen-after 4 or 6 weeks of culture. After 6 weeks of culture, the cellular morphology in the ASC-seeded constructs resembled those in native articular cartilage tissue, with rounded cells residing in the glycosaminoglycan-rich regions of the scaffolds. Biphasic mechanical testing showed that the aggregate modulus of the ASC-seeded constructs increased over time, reaching 150 kPa by day 42, more than threefold higher than that of the unseeded controls. These results suggest that a porous scaffold derived from articular cartilage has the ability to induce chondrogenic differentiation of ASCs without exogenous growth factors, with significant synthesis and accumulation of ECM macromolecules, and the development of mechanical properties approaching those of native cartilage. These findings support the potential for a processed cartilage ECM as a biomaterial scaffold for cartilage tissue engineering. Additional in vivo evaluation is necessary to fully recognize the clinical implication of these observations.     

Comparison of the proliferation, viability, and differentiation capacity of adipose-derived stem cells from different anatomic sites in rabbits

Tissue engineering is clinically promising for missing and damaged tissues. Adipose-derived stem cells (ASCs), a type of mesenchymal stem cells, represent a reliable source of seed cells for tissue engineering with multiple merits such as minimal invasion, abundant yield, little immunity, low morbidity, easy isolation, and rapid expansion. However, because the properties of adipose tissue-derived cells differ depending on the fat depot from which they are derived, we compared the ASCs from three anatomic sites of New Zealand white rabbits: subcutaneous inguinal (SI), subcutaneous dorsocervical (SD), and retroperitoneal perinephric (RP) regions. We investigated cellular behaviors including proliferation, viability, and differentiation. The ASCs of the subcutaneous regions (SI and SD) had higher performances in all assessments compared to those of the RP region. Moreover, the SI and SD ASCs had significant differences, with SI ASCs having better properties than SD ASCs. We conclude that the different anatomic distributions of fat contribute to the different behaviors of ASCs. The SI region offers the most applicable cell source reservoir for ASC tissue engineering.     

Efficient transfer of human adipose-derived stem cells by chitosan/gelatin blend films

Adipose-derived stem cells (ASCs) are a potential source of abundant mesenchymal stem cells and represent a promising cell-based therapy for tissue damage or degeneration conditions. Previous investigations have demonstrated enhanced therapeutic effects of ASCs in a three-dimensional spheroid culture formulation. In this study, we hypothesize that a composite membrane made of chitosan/gelatin (C/G) is beneficial to facilitate transfer of human ASCs in spheroids. Increasing chitosan content within the blends enhanced the mechanical properties of the sample, including tensile strength and elongation-at-break ratio. Although ASC spheroids developed shortly after seeding on pure chitosan films, increasing gelatin proportion in the C/G blends promoted cell adhesion onto the membranes. We also found that ASCs did not proliferate on chitosan films, but C/G blends of different ratios supported ASC proliferation in the first 4 days of culture. However, ASCs on all C/G blends started to detach from the films to form spheroids after day 4, while ASCs on pure gelatin films remained attached and continued to grow. Gradual gelatin release from the C/G blend films, leading to enriched chitosan content in the blends, probably encouraged ASC detachment and spheroid formation. We placed porous collagen matrix on ASC-seeded C/G blends to simulate the application of ASC-seeded C/G films onto injured tissue and found that a C/G film composed of 75% chitosan could facilitate significantly more cell transfer into the overlying collagen sponge. Therefore, a blend film containing 75% chitosan and 25% gelatin showed promising results to serve as a biomaterial for human ASC-based cell therapy.     

Peptide-surface modification of poly(caprolactone) with laminin-derived sequences for adipose-derived stem cell applications

Human adipose tissue has been recognized as a source of adult stem cells for tissue engineering applications such as bone, cartilage, and soft tissue repair. For the success of these tissue-engineering approaches, a cell delivery vehicle such as a hydrogel or scaffold is required to position the stem cells at the site of need. Surface modification techniques have been instrumental in the development of scaffolds that promote cell-surface interactions. In this study, poly(caprolactone) (PCL), surfaces were modified in order to promote the attachment and proliferation of adipose-derived stem cells (ASCs). RGD, YIGSR, and IKVAV peptide sequences derived from the extracellular matrix protein laminin were each covalently attached to an aminated polymer surface using carbodiimide chemistry. The surface was characterized using scanning electron microscopy (SEM), goniometry and X-ray photoelectron spectroscopy (XPS). The attachment and proliferation of ASCs was assessed on the different peptide-treated surfaces. XPS analysis confirmed the presence of the peptide sequences on the surface of the polymer as indicated by the increase in the nitrogen/carbon ratio on the surface of the polymer. Among all peptide sequences tested, IKVAV-treated surfaces had a significantly greater number of ASCs bound 2 and 3 days after cell seeding. SEM confirmed differences in the morphology of the cells attached to the three peptide-treated surfaces. These results indicate that IKVAV is a suitable peptide sequence for use in surface modification techniques aimed at improving the attachment of ASCs to a tissue-engineered scaffold.     

Comparison of adipose stem cells sources from various locations of rat body for their application for seeding on polymer scaffolds

Adipose tissue yields adult adipose stem cells (ASCs) in large quantities via less-invasive methods. These cells are of interest owing to their modulating properties and paracrine activities, which can be harnessed in regenerative medicine. Many studies on the use of rat fat tissue in an autologous animal model have been conducted; however, the different locations to obtain stromal vascular fraction of rat fat depots have not been fully characterized. The purpose of the current study was to identify optimal source of ASC from various locations of rat body. Animal experiments in vitro revealed that fat depots from cervical fat are an optimal ASC source. A high ASC yield facilitates subsequent studies on autologous transplantation in rats. The secondary objective was to compare the efficiency of osteoinductive media composition and evaluate of osteogenic potential of ASCs for seeding on scaffolds for bone repair. Scaffolds were assessed in vitro, using rat adipose stem cells and three-dimensional (3D) scaffolds comprising polycaprolactone (PCL) or polycaprolactone covered with tricalcium phosphate (PCL + 5%TCP). Seeded ASCs adhere to the surface and migrate to the scaffolds. Upon staining and determining alkaline phosphatase levels, PCL + 5%TCP scaffolds performed better than PCL scaffolds. Furthermore, growth factors such as BMP2 and FGF2 significantly increased ASC mineralization and induced osteogenesis (p?<?0.05). Our results may help select and develop pre-clinical animal model for confirming the use of ASC, alone or in association with appropriate biomaterials for bone repair.     

The use of ASCs engineered to express BMP2 or TGF-β3 within scaffold constructs to promote calvarial bone repair

Calvarial bone healing is difficult and grafts comprising adipose-derived stem cells (ASCs) and PLGA (poly(lactic-co-glycolic acid)) scaffolds barely heal rabbit calvarial defects. Although calvarial bone forms via intramembranous ossification without cartilage templates, it was suggested that chondrocytes/cartilages promote calvarial healing, thus we hypothesized that inducing ASCs chondrogenesis and endochondral ossification involving cartilage formation can improve calvarial healing. To evaluate this hypothesis and selectively induce osteogenesis/chondrogenesis, rabbit ASCs were engineered to express the potent osteogenic (BMP2) or chondrogenic (TGF-β3) factor, seeded into either apatite-coated PLGA or gelatin sponge scaffolds, and allotransplanted into critical-size calvarial defects. Among the 4 ASCs/scaffold constructs, gelatin constructs elicited in vitro chondrogenesis, in vivo osteogenic metabolism and calvarial healing more effectively than apatite-coated PLGA, regardless of BMP2 or TGF-β3 expression. The BMP2-expressing ASCs/gelatin triggered better bone healing than TGF-β3-expressing ASCs/gelatin, filling ≈86% of the defect area and ≈61% of the volume at week 12. The healing proceeded via endochondral ossification, instead of intramembranous pathway, as evidenced by the formation of cartilage that underwent osteogenesis and hypertrophy. These data demonstrated ossification pathway switching and significantly augmented calvarial healing by the BMP2-expressing ASCs/gelatin constructs, and underscored the importance of growth factor/scaffold combinations on the healing efficacy and pathway.     

Stemness and transdifferentiation of adipose-derived stem cells using l-ascorbic acid 2-phosphate-induced cell sheet formation

Cell sheet technology has emerged as an important tissue engineering approach. Adipose-derived stem cells (ASCs) have valuable applications in regenerative medicine, but their stemness and differentiation capabilities in the cell sheet format have not been well investigated. In this study, we found that l-ascorbate 2-phosphate (A2-P), a stable form of ascorbic acid, significantly enhanced ASC proliferation and induced ASC sheet fabrication in 7 days with abundant extracellular matrix deposition. Importantly, A2-P treatment significantly enhanced expression of pluripotent markers Sox-2, Oct-4 and Nanog, but treating ASCs with antioxidants other than A2-P revealed no stemness enhancement. Moreover, ASC treatment with A2-P and a collagen synthesis inhibitor, L-2-azetidine carboxylic acid or cis-4-hydroxy-d-proline, significantly inhibited the A2-P-enhanced expression of stemness markers. These findings demonstrated that A2-P enhances stemness of ASCs through collagen synthesis and cell sheet formation. We also showed that A2-P-stimulated collagen synthesis in ASCs may be mediated through ERK1/2 pathway. By culturing the ASC sheets in proper induction media, ASC transdifferentiation capabilities into neuron and hepatocyte-like cells were significantly enhanced after cell sheet formation, while adipogenic and osteogenic differentiation capacities were still maintained. Using a murine model of healing-impaired cutaneous wound, faster wound healing was noted in the group that received ASC sheet treatment, and we observed significantly more engrafted ASCs with evidence of differentiation toward endothelial and epidermal lineages in the cutaneous wound tissue. Therefore, A2-P-mediated ASC sheet formation enhanced ASC stemness and transdifferentiation capabilities, thereby representing a promising approach for applications in regenerative medicine.     

In vitro concurrent endothelial and osteogenic commitment of adipose-derived stem cells and their genomical analyses through comparative genomic hybridization array: novel strategies to increase the successful engraftment of tissue-engineered bone grafts

In the field of tissue engineering, adult stem cells are increasingly recognized as an important tool for in vitro reconstructed tissue-engineered grafts. In the world of cell therapies, undoubtedly, mesenchymal stem cells from bone marrow or adipose tissue are the most promising progenitors for tissue engineering applications. In this setting, adipose-derived stem cells (ASCs) are generally similar to those derived from bone marrow and are most conveniently extracted from tissue removed by elective cosmetic liposuction procedures; they also show a great potential for endothelization. The aim of the present work was to investigate how the cocommitment into a vascular and bone phenotype of ASCs could be a useful tool for improving the in vitro and in vivo reconstruction of a vascularized bone graft. Human ASCs obtained from abdominoplasty procedures were loaded in a hydroxyapatite clinical-grade scaffold, codifferentiated, and tested for proliferation, cell distribution, and osteogenic and vasculogenic gene expression. The chromosomal stability of the cultures was investigated using the comparative genomic hybridization array for 3D cultures. ASC adhesion, distribution, proliferation, and gene expression not only demonstrated a full osteogenic and vasculogenic commitment in vitro and in vivo, but also showed that endothelization strongly improves their osteogenic commitment. In the end, genetic analyses confirmed that no genomical alteration in long-term in vitro culture of ASCs in 3D scaffolds occurs.     

Concise review: human adipose-derived stem cells: separating promise from clinical need

Human adipose-derived stem cells (ASCs) have become an increasing interest to both stem cell biologists and clinicians because of their potential to differentiate into adipogenic, osteogenic, chondrogenic, and other mesenchymal lineages, as well as other clinically useful properties attributed to them, such as stimulation of angiogenesis and suppression of inflammation. ASCs have already been used in a number of clinical trials, and some successful outcomes have been reported, especially in tissue reconstruction. However, a critical review of the literature reveals considerable uncertainty about the true clinical potential of human ASC. First, the surgical needs that ASC might answer remain relatively few, given the current difficulties in scaling up ASC-based tissue engineering to a clinically useful volume. Second, the differentiation of ASC into cell lineages apart from adipocytes has not been conclusively demonstrated in many studies due to the use of rather simplistic approaches to the confirmation of differentiation, such as the use of nonspecific histological dyes, or a small number of molecular markers of uncertain significance. Third, the ASC prepared from human lipoaspirate for different studies differ in purity and molecular phenotype, with many studies using cell preparations that are likely to contain heterogeneous populations of cells, making it uncertain whether ASC themselves are responsible for effects observed. Hence, while one clinical application already looks convincing, the full clinical potential of ASC awaits much deeper investigation of their fundamental biology.