Fetal cartilage engineering from amniotic mesenchymal progenitor cells

We determined whether cartilage could be engineered from mesenchymal progenitor cells (MPCs) normally found in amniotic fluid. Mesenchymal amniocytes were isolated from ovine amniotic fluid samples (n = 5) and had their identity confirmed by immunocytochemistry. Cells were expanded and then cultured as micromass pellets (n = 5) in a chondrogenic medium containing transforming growth factor-beta2 (TGF-beta2) and insulin growth factor-1 (IGF-1) for 6-12 weeks. Pellets derived from fetal dermal fibroblasts (n = 4) were cultured under identical conditions. Additionally, expanded mesenchymal amniocytes were seeded onto biodegradable polyglycolic acid scaffolds (n = 5) and maintained in the same chondrogenic medium within a rotating bioreactor for 10-15 weeks. Engineered specimens were analyzed quantitatively and compared with native fetal hyaline cartilage samples (n = 5). Statistical analysis was by the unpaired Student's t-test (p < 0.05). The isolated cells stained positively for vimentin and cytokeratins-8 and -18, but negatively for CD31. Micromass pellets derived from mesenchymal amniocytes exhibited chondrogenic differentiation by both standard and matrix-specific staining. In contrast, these findings could not be replicated in dermal fibroblast-based pellets. The engineered constructs derived from mesenchymal amniocytes similarly displayed histological evidence of chondrogenic differentiation and maintained their original size and three-dimensional architecture. Quantitative assays of the engineered constructs revealed lower concentrations of collagen type II, but similar amounts of glycosaminoglycans, elastin, and DNA, when compared to native fetal hyaline cartilage. We conclude that mesenchymal amniocytes can be used for the engineering of cartilaginous tissue in vitro. Cartilage engineering from the amniotic fluid may become a practical approach for the surgical treatment of select congenital anomalies.     

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。     

三种来源于不同围产期组织间充质干细胞的比较

背景：围产期组织作为间充质干细胞的良好来源已经受到广泛关注。 目的：比较3种来源于不同围产期组织的间充质干细胞的特性。 方法：以“mesenchymal stem cells, fetal blood, umbilical cord, placenta”为检索词,检索PudMed数据库中2002年1月至2012年4月的文献,纳入与脐血、脐带、胎盘来源的间充质干细胞相关的权威性且具有代表性的文献。 结果与结论：计算机初检得到248篇文献,对其中16 篇文献进行综述。主要阐述3种来源于不同围产期组织的间充质干细胞的分离培养方法以及生物学特性等方面内容,相对于脐血、胎盘来源的间充质干细胞,来源于脐带华通胶质的间充质干细胞由于其分离方法简单,分离成功率高,扩增潜能较高,分化潜能较高以及致瘤性低而成为相对优越的间充质干细胞来源。     

In vitro engineered cartilage using synovium-derived mesenchymal stem cells with injectable gellan hydrogels

Synovium-derived mesenchymal stem cells (SMSC), a novel line of stem cells, are regarded as a promising cell source for cartilage tissue engineering. The goal of this study was to investigate rabbit SMSC coupled with injectable gellan hydrogels for in vitro engineered cartilage. SMSC were isolated from rabbit synovial tissue, amplified to passage 4 in monolayer, and encapsulated in injectable gellan hydrogels, constructs of which were cultured in chondrogenic medium supplemented with TGF-β1, TGF-β3 or BMP-2 for up to 42 days. The quality of the constructs was assessed in terms of cell proliferation and chondrocytic gene/protein expression using WST-1 assay, real-time RT-PCR, biochemical analysis, histology and immunohistochemical analysis. Results indicate that the viability of SMSC in hydrogels treated with TGF-β1, TGF-β3 and BMP-2 remained high at culture time. The constructs formed cartilaginous tissue with the expression of chondrocytic genes (collagen type II, aggrecan, biglycan, SOX 9) and cartilaginous matrix (sulphated glycosaminoglycan and collagen) as early as 21 days in culture. Both TGF-β1 and TGF-β3 treated SMSC-laden hydrogels showed more chondrogenesis compared with BMP-2 treated SMSC-laden hydrogels. It demonstrates that injectable SMSC-laden gels, when treated with TGF-β1, TGF-β3 or BMP-2, are highly competent for in vitro engineered cartilage formation, which lays a foundation for their potential application in clinical cartilage repair.     

力学因素在间充质干细胞构建功能性组织工程化软骨中的作用

间充质干细胞具有多向分化潜能,可定向分化为软骨组织,并且取材广泛、体外扩增能力强,是广泛应用于软骨组织工程的理想细胞之一。由于关节软骨具有重要的生物力学功能,需要强调和评估间充质干细胞构建组织工程化软骨组织的力学生物学性能。为更好地了解和认识修复软骨的诱导因素、信号通路与力学特性之间的关系,本文回顾了间充质干细胞在功能性软骨组织工程研究中的力学生物学研究进展,并论述了该领域内目前存在的问题及若干可供探索的途径和新方向。     

In vitro engineered cartilage constructs produced by press-coating biodegradable polymer with human mesenchymal stem cells

Cartilage constructs were fabricated by press-coating D,D-L,L-polylactic acid polymer blocks of 1 x 0.5 x 0.5 cm onto high-density cell pellets of 1.5 x 10(6) human mesenchymal stem cells (mhMSCs) isolated from the femoral head of patients undergoing total hip arthroplasty. Following attachment of the cell pellets to the polymer surfaces, chondrogenesis was induced by culturing the constructs for 3 weeks in a serum-free, chemically defined, chondrogenic differentiation medium supplemented with transforming growth factor beta-1 (TGF-beta1). Histochemical analysis showed that the press-coated pellets formed cell layers composed of morphologically distinct, chondrocyte-like cells, surrounded by a fibrous, sulfated proteoglycan-rich extracellular matrix. Immunohistochemical analysis detected collagen type II and cartilage proteoglycan link protein within the extracellular matrix. Expression of the cartilage-specific marker genes collagen types II, IX, X, and XI, and aggrecan was detected by RT-PCR. Scanning electron microscopy revealed organized and spatially distinct zones of cells within the cell-polymer constructs, with the superficial layer resembling compact hyaline cartilage. The fabrication method of press-coating biodegradable polymers with mhMSCs allows the in vitro production of cartilage constructs without harvesting chondrocytes from intact articular cartilage surfaces. These constructs may be applicable as prototypes for the reconstruction of articular cartilage defects in humans.     

组织工程化软骨细胞和骨髓间充质干细胞用于修复同种异体关节软骨缺损

背景：关节软骨几乎没有自身修复的能力,目前临床大多采用自体或异体软骨移植修复、软骨膜或骨膜移植修复、软骨细胞移植修复。由于自体软骨来源有限,异体软骨又存在慢性免疫排斥反应,最终可能导致预后不佳;软骨膜或骨膜移植修复的软骨易于退化,导致修复效果不佳。 目的：总结组织工程化软骨细胞、骨髓间充质干细胞及两者共培养对同种异体软骨缺损修复作用的研究现状。 方法：应用计算机检索PubMed 数据库及中国期刊网全文数据库1994-01/2012-01有关组织工程化软骨细胞和骨髓间充质干细胞用于修复同种异体关节软骨缺损方面的文章,英文检索词为“cartilage defect,allograft,chondrocyte,mesenchymal stem cells,bone marrow mesenchymal stem cells”,中文检索词为“软骨缺损,同种异体移植,软骨细胞,骨髓间充质干细胞”。排除重复性及非中英文语种研究,共保留35篇文献进行综述。 结果与结论：随着体外细胞培养方法的不断改进,现已能够把软骨细胞从坚韧的软骨中分离出来,并获得大量高纯度的软骨细胞并繁殖出新生软骨细胞。软骨细胞培养增殖能力低,传代培养容易引起老化和去分化;而成体骨髓中骨髓间充质干细胞含量少,随传代次数的增多成软骨潜能明显降低。骨髓间充质干细胞和软骨细胞共培养,两种细胞相互促进增殖和分化,作为种子细胞可减少软骨细胞增殖传代次数并节省软骨细胞数量,与组织工程支架材料复合能有效修复关节软骨缺损。     

Cartilage engineering from ovine umbilical cord blood mesenchymal progenitor cells

We aimed to determine whether three-dimensional (3D) cartilage could be engineered from umbilical cord blood (CB) cells and compare it with both engineered fetal cartilage and native tissue. Ovine mesenchymal progenitor cells were isolated from CB samples (n=4) harvested at 80-120 days of gestation by low-density fractionation, expanded, and seeded onto polyglycolic acid scaffolds. Constructs (n=28) were maintained in a rotating bioreactor with serum-free medium supplemented with transforming growth factor-beta1 for 4-12 weeks. Similar constructs seeded with fetal chondrocytes (n=13) were cultured in parallel for 8 weeks. All specimens were analyzed and compared with native fetal cartilage samples (n=10). Statistical analysis was by analysis of variance and Student's t-test (p<.01). At 12 weeks, CB constructs exhibited chondrogenic differentiation by both standard and matrix-specific staining. In the CB constructs, there was a significant time-dependent increase in extracellular matrix levels of glycosaminoglycans (GAGs) and type-II collagen (C-II) but not of elastin (EL). Fetal chondrocyte and CB constructs had similar GAG and C-II contents, but CB constructs had less EL. Compared with both hyaline and elastic native fetal cartilage, C-II and EL levels were, respectively, similar and lower in the CB constructs, which had correspondingly lower and similar GAG levels than native hyaline and elastic fetal cartilage. We conclude that CB mesenchymal progenitor cells can be successfully used for the engineering of 3D cartilaginous tissue in vitro, displaying select histological and functional properties of both native and engineered fetal cartilage. Cartilage engineered from CB may prove useful for the treatment of select congenital anomalies.     

丝素蛋白/羟基磷灰石材料复合骨髓间充质干细胞构建组织工程化软骨

背景：随着组织工程的兴起,软骨损伤的修复可能性显著地提高,但单一的支架材料均不能符合理想支架,有一定的局限性。 目的：观察骨髓间充质干细胞复合丝素蛋白/羟基磷灰石构建组织工程化软骨的可行性。 方法：体外分离培养骨髓间充质干细胞,并定向诱导成软骨细胞,与丝素蛋白/羟基磷灰石复合培养,构建膝关节胫骨平台全层关节软骨缺损。54只大白兔单侧膝关节全层软骨缺损模型后随机抽签法分为3组,复合组植入细胞-丝素蛋白/羟基磷灰石复合物;材料组植入单纯丝素蛋白/羟基磷灰石,对照组不行任何植入。植入后8,12周CT检查及组织学检查观察软骨缺损修复情况。 结果与结论：植入后8周,复合组关节面不平整,关节间隙增大,形成新生类软骨细胞,基质丰富。材料组关节面塌陷,软骨细胞少量增殖。植入后12周,复合组关节面平整,关节间隙如常。大量软骨细胞出现,与周边软骨色泽一样,支架材料完全降解。材料组关节面不平整,软骨细胞不完全充填,支架材料部分降解。对照组未见修复。提示用骨髓间充质干细胞复合丝素蛋白/羟基磷灰石可形成透明软骨修复动物膝关节全层软骨缺损,显示了丝素蛋白/羟基磷灰石材料作为关节软骨组织工程支架材料的良好生物相容性。     

Comparative analysis of mesenchymal stem cells from bone marrow, cartilage, and adipose tissue

Mesenchymal stem cells (MSCs) isolated from bone marrow (BM), cartilage, and adipose tissue (AT) possess the capacity for self-renewal and the potential for multilineage differentiation, and are therefore perceived as attractive sources of stem cells for cell therapy. However, MSCs from these different sources have different characteristics. We compared MSCs of adult Sprague Dawley rats derived from these three sources in terms of their immunophenotypic characterization, proliferation capacity, differentiation ability, expression of angiogenic cytokines, and anti-apoptotic ability. According to growth curve, cell cycle, and telomerase activity analyses, MSCs derived from adipose tissue (AT-MSCs) possess the highest proliferation potential, followed by MSCs derived from BM and cartilage (BM-MSCs and C-MSCs). In terms of multilineage differentiation, MSCs from all three sources displayed osteogenic, adipogenic, and chondrogenic differentiation potential. The result of realtime RT-PCR indicated that these cells all expressed angiogenic cytokines, with some differences in expression level. Flow cytometry and MTT analysis showed that C-MSCs possess the highest resistance toward hydrogen peroxide -induced apoptosis, while AT-MSCs exhibited high tolerance to serum deprivation-induced apoptosis. Both AT and cartilage are attractive alternatives to BM as sources for isolating MSCs, but these differences must be considered when choosing a stem cell source for clinical application.     

Characterization of multipotential mesenchymal progenitor cells derived from human trabecular bone

The in vitro culture of human trabecular bone-derived cells has served as a useful system for the investigation of the biology of osteoblasts. The recent discovery in our laboratory of the multilineage mesenchymal differentiation potential of cells derived from collagenase-treated human trabecular bone fragments has prompted further interest in view of the potential application of mesenchymal progenitor cells (MPCs) in the repair and regeneration of tissue damaged by disease or trauma. Similar to human MPCs derived from bone marrow, a clearer understanding of the variability associated with obtaining these bone-derived cells is required in order to optimize the design and execution of applicable studies. In this study, we have identified the presence of a CD73(+), STRO-1(+), CD105(+), CD34(-), CD45(-), CD144(-) cell population resident within collagenase-treated, culture-processed bone fragments, which upon migration established a homogeneous population of MPCs. Additionally, we have introduced a system of culturing these MPCs that best supports and maintains their optimal differentiation potential during long-term culture expansion. When cultured as described, the trabecular bone-derived cells display stem cell-like capabilities, characterized by a stable undifferentiated phenotype as well as the ability to proliferate extensively while retaining the potential to differentiate along the osteoblastic, adipocytic, and chondrocytic lineages, even when maintained in long-term in vitro culture.     

Coculture of human mesenchymal stem cells and articular chondrocytes reduces hypertrophy and enhances functional properties of engineered cartilage

Mesenchymal stem cells (MSCs) are being recognized as a viable cell source for cartilage repair; however, it still remains a challenge to recapitulate the functional properties of native articular cartilage using only MSCs. Additionally, MSCs may exhibit a hypertrophic phenotype under chondrogenic induction, resulting in calcification after ectopic transplantation. With this in mind, the objective of this study was to assess whether the addition of chondrocytes to MSC cultures influences the properties of tissue-engineered cartilage and MSC hypertrophy when cultured in hyaluronic acid hydrogels. Mixed cell populations (human MSCs and human chondrocytes at a ratio of 4:1) were encapsulated in the hydrogels and exhibited significantly higher Young's moduli, dynamic moduli, glycosaminoglycan levels, and collagen content than did constructs seeded with only MSCs or chondrocytes. Furthermore, the deposition of collagen X, a marker of MSC hypertrophy, was significantly lower in the coculture constructs than in the constructs seeded with MSCs alone. When MSCs and chondrocytes were cultured in distinct gels, but in the same wells, there was no improvement in biomechanical and biochemical properties of the engineered tissue, implying that a close proximity is essential. This approach can be used to improve the properties and prevent calcification of engineered cartilage formed from MSC-seeded hydrogels with the addition of lower fractions of chondrocytes, leading to improved clinical outcomes.     

丝素蛋白-壳聚糖支架复合骨髓间充质干细胞体内构建组织工程化软骨的生物相容性

文题释义： 生物相容性：是指生命体组织对非活性材料产生的一种性能,一般是指材料与宿主之间的相容性,包括组织相容性和血液相容性。 检测相容性的方法：是将支架材料与种子细胞在体外共培养,检测支架毒性、细胞活性、细胞增殖及细胞与支架的黏附情况等指标,该方法具有客观性强、可重复性强、影响因素相对简单及敏感性高等特点。 背景：课题组前期的研究中发现,丝素蛋白-壳聚糖支架材料复合诱导后骨髓间充质干细胞在兔体内能修复缺损的软骨组织,但对于该组织工程化软骨组织的生物相容性还未进一步研究。 目的：研究丝素蛋白-壳聚糖支架材料复合骨髓间充质干细胞在体内构建组织工程化软骨的生物相容性。 方法：使用丝素蛋白-壳聚糖按1∶1比例混合制备三维支架材料,提取兔骨髓间充质干细胞,将诱导后的骨髓间充质干细胞与丝素蛋白-壳聚糖支架构建修复体,再将修复体移植到兔关节软骨缺损模型中修复软骨组织。实验分为3组,实验组植入诱导后骨髓间充质干细胞+丝素蛋白-壳聚糖支架,对照组植入丝素蛋白-壳聚糖支架干预,空白组未植入修复体。 结果与结论：①实验成功制备丝素蛋白-壳聚糖三维支架材料及提取骨髓间充质干细胞,并构建软骨缺损的修复体,将修复体植入兔体内能成功修复缺损的软骨组织;②建模后2,4,8,12周,3组血常规、降钙素原、血沉、C-反应蛋白结果提示无明显的全身感染征象,3组血常规及肝肾功能各时间段比较差异无显著性意义(P > 0.05);③一般观察、苏木精-伊红染色及扫描电镜观察：建模后12周,相比其他两组,实验组软骨缺损已修复,支架材料已吸收,修复组织周围未见炎性细胞,修复组织已正常组织整合良好;④结果证实,丝素蛋白-壳聚糖支架复合骨髓间充质干细胞在体内构建的组织工程化软骨具有良好的生物相容性。 ORCID: 0000-0002-8139-1175(佘荣峰) 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     

Engineered mesenchymal stem cells for cartilage repair

Healthy cartilage is a highly robust tissue, and is resilient against the stringent mechanical and biological constraints imposed upon it. Cartilage defects are common features of joint diseases, but current treatments can rarely restore the full function of native cartilage. Recent studies have provided new perspectives for cartilage engineering using mesenchymal stem cells (MSCs). However, the sequential events occurring during chondrogenesis must be fully understood before we are able to reproduce faithfully the complex molecular events that lead to MSC differentiation and long-term maintenance of cartilage characteristics. Here, we focus on the potential of MSCs to repair cartilage with an emphasis on the factors that are known to be required in inducing chondrogenesis.     

In vivo fate mapping identifies mesenchymal progenitor cells

Adult mesenchymal progenitor cells have enormous potential for use in regenerative medicine. However, the true identity of the progenitors in vivo and their progeny has not been precisely defined. We hypothesize that cells expressing a smooth muscle α-actin promoter (αSMA)-directed Cre transgene represent mesenchymal progenitors of adult bone tissue. By combining complementary colors in combination with transgenes activating at mature stages of the lineage, we characterized the phenotype and confirmed the ability of isolated αSMA(+) cells to progress from a progenitor to fully mature state. In vivo lineage tracing experiments using a new bone formation model confirmed the osteogenic phenotype of αSMA(+) cells. In vitro analysis of the in vivo-labeled SMA9(+) cells supported their differentiation potential into mesenchymal lineages. Using a fracture-healing model, αSMA9(+) cells served as a pool of fibrocartilage and skeletal progenitors. Confirmation of the transition of αSMA9(+) progenitor cells to mature osteoblasts during fracture healing was assessed by activation of bone-specific Col2.3emd transgene. Our findings provide a novel in vivo identification of defined population of mesenchymal progenitor cells with active role in bone remodeling and regeneration.     

Osmotic selection of human mesenchymal stem/progenitor cells from umbilical cord blood

The isolation of undifferentiated adult stem/progenitor cells remains a challenging task primarily due to the rare quantity of these cells in biological samples and the lack of unique markers. Herein, we report a relatively straightforward method for isolation of human mesenchymal stem cells (MSCs) based on their unusual resistance to osmotic lysis, which we term "osmotic selection" (OS). MSCs can remarkably withstand significant exposure to hypotonic conditions (> 30 min) with only a reversible impairment in cell proliferation and with no loss of stem cell potential after exposure. Comparison of MSCs to other circulating nonhematopoietic cells revealed a time regime, by which purification of these cells would be attainable without considerable cell loss. OS showed a 50-fold enrichment of fibroblast colony-forming units from umbilical cord blood samples when compared to commonly employed techniques. After upstream processing, isolated cells using OS were immunophenotyped to be CD14-, CD34-, CD45-, CD44+, CD105+, and CD106+, and displayed multipotent differentiation. Preliminary investigations to determine mechanisms responsible for osmolytic resistance revealed MSCs to have an ineffective volume of 59%, with the ability to double cell volume at infinite dilution. Disruption of filamentous actin polymerization by cytochalasin D sensitized MSCs to osmotic lysis, which suggests a cytoskeletal element involved in osmolytic resistance.     

滑膜间充质干细胞修复膝关节软骨损伤的应用与进展

背景：膝关节软骨损伤后很难愈合,是临床上亟需解决的重要课题。目的：总结滑膜间充质干细胞在膝关节软骨损伤治疗上的优越性。方法：用英文主题词“Knee Joint, Cartilage, Synovial Membrane, Tissues, Injuries, Repair”在PubMed数据库中检索2005年8月至2015年8月文献总共625篇。采纳并分析滑膜间充质干细胞治疗膝关节软骨损伤文献48篇。排除其他非滑膜间充质干细胞及非膝关节软骨损伤治疗的相关文章,保留35篇文章进行综述。结果与结论：应用滑膜间充质干细胞治疗膝关节软骨损伤,能够获得更加理想的治疗效果,损伤组织本身固有的间充质干细胞是修复损伤组织的最佳种子细胞。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     

Interleukin 17 inhibits progenitor cells in rheumatoid arthritis cartilage

Mesenchymal stem cells are known to exert immunomodulatory effects in inflammatory diseases. Immuneregulatory cells lead to progressive joint destruction in rheumatoid arthritis (RA). Proinflammatory cytokines, such as tumour necrosis factor α (TNF‐α) and interleukins (ILs) are the main players. Here, we studied progenitor cells from RA cartilage (RA‐CPCs) that are positive for IL‐17 receptors to determinate the effects of inflammation on their chondrogenic potenial. IL‐17A/F reduced the chondrogenic potential of these cells via the upregulation of RUNX2 protein and enhanced IL‐6 protein and MMP3 mRNA levels. Blocking antibodies against IL‐17 positively influenced their repair potential. Furthermore, treating the RA‐CPCs with the anti‐human IL‐17 antibody secukinumab or the anti‐TNF‐α antibody adalimumab reduced the proinflammatory IL‐6 protein level and positively influenced the secretion of anti‐inflammatory IL‐10 protein. Additionally, adalimumab and secukinumab in particular reduced RUNX2 protein to promote chondrogenesis. The amelioration of inflammation, particularly via IL‐17 antagonism, might be a new therapeutic approach for enhancing intrinsic cartilage repair mechanisms in RA patients.     

Genetic modification of mesenchymal stem cells for cartilage repair

Reproduction of a native, functional architecture in articular cartilage defects is a major problem in orthopaedic surgery. The elaboration of workable options to heal damaged cartilage might necessitate to involve cellular, molecular and environmental components to allow for the formation of an adequate and stable repair tissue in sites of injury. Strategies based on the transfer of candidate sequences to progenitor cells offer powerful tools to achieve this goal. The aim of this report is to provide an overview of the most recent therapeutic approaches developed in experimental orthopaedic research.