Age-related biological characterization of mesenchymal progenitor cells in human articular cartilage

Adult articular cartilage has a low regeneration capacity due to lack of viable progenitor cells caused by limited blood supply to cartilage. However, recent studies have demonstrated the existence of chondroprogenitor cells in articular cartilage. A critical question is whether these mesenchymal progenitor cells are functionally viable for tissue renewal and cartilage repair to postpone cartilage degeneration. This study was designed to compare the number and function of mesenchymal progenitor cells in articular cartilage collected from human fetuses, healthy adults (aged 28-45 years), and elderly adults (aged 60-75 years) and cultured in vitro. We detected multipotent mesenchymal progenitor cells, defined as CD105+/CD166+ cells, in human articular cartilage of all ages. However, mesenchymal progenitor cells accounted for 94.69%±2.31%, 4.85%±2.62%, and 6.33%±3.05% of cells in articular cartilage obtained from fetuses, adults, and elderly patients, respectively (P<.001). Furthermore, fetal mesenchymal progenitor cells had the highest rates of proliferation measured by cell doubling times and chondrogenic differentiation as compared to those from adult and elderly patients. In contrast, alkaline phosphatase levels, which are indicative of osteogenic differentiation, did not show significant reduction with aging. However, spontaneous osteogenic differentiation was detected only in mesenchymal progenitor cells from elderly patients (with lower Markin scales). The lower chondrogenic and spontaneous osteogenic differentiation of mesenchymal progenitor cells derived from elderly patients may be associated with the development of primary osteoarthritis. These results suggest that measuring cartilage mesenchymal progenitor cells may not only identify underlying mechanisms but also offer new diagnostic and therapeutic potential for patients with osteoarthritis.     

Adult human articular chondrocytes in a microcarrier‐based culture system: expansion and redifferentiation

Expanding human chondrocytes in vitro while maintaining their ability to form cartilage remains a key challenge in cartilage tissue engineering. One promising approach to address this is to use microcarriers as substrates for chondrocyte expansion. While microcarriers have shown beneficial effects for expansion of animal and ectopic human chondrocytes, their utility has not been determined for freshly isolated adult human articular chondrocytes. Thus, we investigated the proliferation and subsequent chondrogenic differentiation of these clinically relevant cells on porous gelatin microcarriers and compared them to those expanded using traditional monolayers. Chondrocytes attached to microcarriers within 2 days and remained viable over 4 weeks of culture in spinner flasks. Cells on microcarriers exhibited a spread morphology and initially proliferated faster than cells in monolayer culture, however, with prolonged expansion they were less proliferative. Cells expanded for 1 month and enzymatically released from microcarriers formed cartilaginous tissue in micromass pellet cultures, which was similar to tissue formed by monolayer‐expanded cells. Cells left attached to microcarriers did not exhibit chondrogenic capacity. Culture conditions, such as microcarrier material, oxygen tension, and mechanical stimulation require further investigation to facilitate the efficient expansion of clinically relevant human articular chondrocytes that maintain chondrogenic potential for cartilage regeneration applications. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:539–546, 2011     

Adult ovine chondrocytes in expansion culture adopt progenitor cell properties that are favorable for cartilage tissue engineering

Human chondrocytes in expansion culture can become progenitor-like in their ability to proliferate extensively and secrete neocartilage in chondrogenic culture. Sheep are used as a large animal model for cartilage tissue engineering, although for testing progenitor-like chondrocytes it is important that ovine chondrocytes resemble human in the ability to adopt progenitor properties. Here, we investigate whether ovine chondrocytes can adopt progenitor properties as indicated by rapid proliferation in a colony-forming fashion, and high levels of neocartilage secretion in chondrogenic culture. In conditions known to promote expansion of mesenchymal stromal cells, ovine chondrocytes proliferated through approximately 12 population doublings in 10 days. Time-lapse imaging indicated rapid proliferation in a colony-forming pattern. Expanded ovine chondrocytes that were seeded into agarose and cultured in chondrogenic medium accumulated neocartilage over 2 weeks, to a greater extent than primary chondrocytes. These data confirm that ovine chondrocytes resemble human chondrocytes in their ability to acquire progenitor properties that are important for cartilage tissue engineering. Given the broad interest in using progenitor cells to heal connective tissues, next we compared proliferation and trilineage differentiation of ovine chondrocytes, meniscus cells, and tenocytes. Meniscus cells and tenocytes experienced more than 13 population doublings in 10 days. In chondrogenic culture, cartilage matrix accumulation, and gene expression were largely similar among the cell types. All cell types resisted osteogenesis, while expanded tenocytes and meniscal cells were capable of adipogenesis. While ovine connective tissue cells demonstrated limited lineage plasticity, these data support the potential to promote certain progenitor properties with expansion.     

Evaluation of ghrelin as a distinguishing marker for human articular cartilage-derived chondrocytes and chondroprogenitors

Purpose of the studyCell-based therapeutics for articular cartilage repair primarily employed bone marrow-derived mesenchymal stem cells and chondrocytes. Research to overcome their limitation of formation of a functionally poor fibro-hyaline type of repair tissue led to the discovery of chondroprogenitors (CPCs), cartilage resident stem cells. These cells isolated by adhesion assay using fibronectin (FAA-CPs) and migration of progenitors from explants (MCPs) display higher chondrogenic and lower terminal differentiation potential. During in-vitro culture, chondrocytes tend to de-differentiate and acquire characteristics similar to stem cells, thus making it challenging to distinguish them from other cell groups. Ghrelin, a cytoplasmic growth hormone secretagogue, has been proposed to play a vital role in chondrogenesis, with reports of its higher expression in chondrocytes than BM-MSCs. The aim of this study was to compare the mRNA expression of Ghrelin between BM-MSCs, chondrocytes, FAA-CPs and MCP and the possibility of it serving as a distinguishing marker.MethodsThe four populations isolated from three human osteoarthritic knee joints were characterised by CD marker expression for positive (CD 90, CD73 and CD105) and negative (HLA-DR, CD34 and CD45) MSC markers and trilineage differentiation (adipogenic, osteogenic and chondrogenic) and subjected to qRT-PCR to assess Ghrelin's gene expression.ResultsThis study showed that all groups exhibited similar expression of CD markers and multilineage potential. Though chondrocytes showed greater expression of Ghrelin, it was not statistically significant to classify it as a distinguishing marker between these cell populations.ConclusionGhrelin does not serve to differentiate the subpopulations in terms of their mRNA expression. Further evaluation using their associated enzymes and receptors could provide valuable information to uncover their potential as unequivocal biomarkers.     

定向诱导间充质干细胞成软骨分化相关活性因子研究进展

关节软骨受损或缺失,是导致关节炎等渐进性疾病的主要原因,严重影响患者生活质量。成熟的透明软骨由于缺乏神经支配和血管供应,且软骨细胞增殖能力差,所以很难自我修复。自体软骨细胞移植尚存在局限性,且操作复杂,阻碍了临床应用。间充质干细胞增殖能力较强,并保留有分化潜力,但向成软骨分化需要一定的条件,如细胞因子、支架材料、培养基等。寻找促进诱导间充质干细胞成软骨分化的活性因子,是目前关节软骨再生的重要研究方向。本文就诱导间充质干细胞向软骨分化的相关活性因子的研究进展进行综述。     

Human myogenic endothelial cells exhibit chondrogenic and osteogenic potentials at the clonal level

We have previously reported the high regenerative potential of murine muscle‐derived stem cells (mMDSCs) that are capable of differentiating into multiple mesodermal cell lineages, including myogenic, endothelial, chondrocytic, and osteoblastic cells. Recently, we described a putative human counterpart of mMDSCs, the myogenic endothelial cells (MECs), in adult human skeletal muscle, which efficiently repair/regenerate the injured and dystrophic skeletal muscle as well as the ischemic heart in animal disease models. Nevertheless it remained unclear whether human MECs, at the clonal level, preserve mMDSC‐like chondrogenic and osteogenic potentials and classic stem cell characteristics including high proliferation and resistance to stress. Herein, we demonstrated that MECs, sorted from fresh postnatal human skeletal muscle biopsies, can be grown clonally and exhibit robust resistance to oxidative stress with no tumorigeneity. MEC clones were capable of differentiating into chondrocytes and osteoblasts under inductive conditions in vitro and participated in cartilage and bone formation in vivo. Additionally, adipogenic and angiogenic potentials of clonal MECs (cMECs) were observed. Overall, our study showed that cMECs not only display typical properties of adult stem cells but also exhibit chondrogenic and osteogenic capacities in vitro and in vivo, suggesting their potential applications in articular cartilage and bone repair/regeneration. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1089–1095, 2013     

Expression of a stable articular cartilage phenotype without evidence of hypertrophy by adult human articular chondrocytes in vitro

Chondrocytes that were isolated from adult human articular cartilage changed phenotype during monolayer tissue culture, as characterized by a fibroblastic morphology and cellular proliferation. Increased proliferation was accompanied by downregulation of the cartilage-specific extracellular matrix proteoglycan, aggrecan, by cessation of type-II collagen expression, and by upregulation of type-I collagen and versican. This phenomenon observed in monolayer was reversible after the transfer of cells to a suspension culture system. The transfer of chondrocytes to suspension culture in alginate beads resulted in the rapid upregulation of aggrecan and type-II collagen and the downregulation of expression of versican and type-I collagen. Type-X collagen and osteopontin, markers of chondrocyte hypertrophy and commitment to endochondral ossification, were not expressed by adult articular chondrocytes cultured in alginate, even after 5 months. In contrast, type-X collagen was expressed within 2 weeks in a population of cells derived from a fetal growth plate. The inability of adult articular chondrocytes to express markers of chondrocyte hypertrophy has underscored the fundamental distinction between the differentiation pathways that lead to articular cartilage or to bone. Adult articular chondrocytes expressed only hyaline articular cartilage markers without evidence of hypertrophy.     

Effect of Cryopreservation and Cell Passage Number on Cell Preparations Destined for Autologous Chondrocyte Transplantation

Autologous chondrocyte transplantation (ACT) is an effective and safe therapy for repairing articular cartilage defects and requires cell preservation and subculture before transplantation. We compared the effects of cryopreservation and passaging on cell viability, proliferation, and maintenance of the function of chondrocytes and synovium-derived mesenchymal stem cells (MSCs) used as sources for ACT. These cells were isolated from the knee joints of rabbits and were cultured, passaged serially, and divided into 2 groups that were either cryopreserved or not. The morphology, viability, gene expression, and differentiation potential of the 2 groups were compared. Maintenance of the potential to undergo chondrogenic differentiation was determined with the use of a 3-dimensional culture method. Passaging and cryopreservation significantly affected the ability of chondrocytes to maintain their morphology, express chondrogenic genes, and differentiate. In contrast, synovium-derived cells were not affected by passaging and cryopreservation. Our results may serve as the foundation for the application of passaged and cryopreserved chondrocyte or other source cells of MSCs in ACT.     

Expression of NG2/human melanoma proteoglycan in human adult articular chondrocytes

OBJECTIVE: NG2 is a transmembrane chondroitin sulfate (CS) rich proteoglycan originally identified in rats. It has recently been shown to be identical to human melanoma proteoglycan (HMPG). In rats NG2 has a limited distribution in adult tissues, being expressed predominantly by neuronal and glial cells whereas during development it is also expressed in developing mesenchyme including cartilage. NG2/HMPG has putative roles in interactions between glial and melanoma cells with extracellular matrix (ECM) molecules. This study was undertaken to assess whether NG2/HMPG was expressed by normal and osteoarthritic human articular chondrocytes. DESIGN: Cryostat sections of human fetal knee joints and normal and osteoarthritic articular cartilage were immunostained with antibodies against rat NG2 (N143.8) and HMPG (M28B5, 9.2.27). Immunoprecipitation and Western blotting was carried out on protein extracts of chondrocytes from normal and osteoarthritic cartilage. Immunofluorescence of NG2 and potential ligands was carried out in vitro on cells from normal and osteoarthritic cartilage. RESULTS: Fetal and both normal and osteoarthritic adult cartilage showed strong immunoreactivity for NG2/HMPG. Western blotting showed a smeared component of molecular weight greater than 400 kDa and a faint band at 250 kDa which became predominant upon digestion with chondroitinase ABC. Immunofluorescence of chondrocytes in vitro showed NG2 to be distributed in a punctate pattern without co-localization of actin or several ECM proteins including fibronectin and type VI collagen. CONCLUSION: NG2/HMPG is expressed by human fetal and adult chondrocytes and in adult articular chondrocytes the core protein is chondroitin sulfated. The function of this molecular in human articular cartilage remains to be defined.     

利用脂肪干细胞构建软骨修复兔膝关节软骨缺损的初步研究

目的 利用兔同种异体软骨脱细胞基质支架和脂肪干细胞体外构建组织工程软骨,探讨其修复关节软骨损伤的可行性.方法 将新西兰大白兔的脂肪干细胞与软骨脱细胞基质支架复合,于软骨细胞方向诱导培养基中培养两周,构建组织工程软骨.兔24只随机分为A、B、C 3组, A组关节软骨缺损处置入经诱导的脂肪源干细胞复合软骨基质支架, B组缺损处只置入软骨基质支架, C组软骨缺损处不做任何处理.分别于术后第12周处死动物,修复处行大体、组织学、Ⅱ型胶原免疫组化染色和透射电镜检测.结果 A组软骨缺损处被类软骨组织填充,修复区表面光滑;Ⅱ型胶原免疫组化染色和甲苯胺蓝染色阳性;电镜下可见软骨陷窝内有细胞结构存在,且有大量均匀颗粒状细胞分泌基质成分存在,细胞周围大量胶原纤维.B组软骨缺损处为纤维组织状物填充,C组软骨缺损处无修复组织填充.结论 脂肪干细胞与软骨脱细胞基质复合并向软骨诱导后可良好地修复关节软骨缺损,具有替代正常软骨的潜力.     

Human mesenchymal stem cells induced to differentiate as chondrocytes follow a biphasic pattern of extracellular matrix production

Regenerative medicine and tissue engineering studies are actively developing novel means to repair adult articular cartilage defects using biological approaches. One such approach is the harnessing of adult human therapeutic cells such as those referred to as mesenchymal stem cells. Upon exposure to chondrogenic signals, these cells differentiate and initiate the production of a complex and voluminous cartilaginous matrix that is crucial to both the structure and function of cartilage. Furthermore, this complexity requires the time‐sensitive activation of a large number of genes to produce the components of this matrix. The current study analyzed the kinetics of matrix production in an aggregate culture model where adult human mesenchymal stem cells were induced to differentiate as chondrocytes. The results indicate the existence of a biphasic mode of differentiation and maturation during which matrix genes and molecules are differentially activated and secreted. These results have important implications for developing novel approaches for the creation of tissue engineered articular cartilage. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1757–1766, 2018.

     

Interaktion zwischen humanen Chondrozyten und extrazellulärer Matrix in vitro

BACKGROUND: Autologous chondrocyte transplantation (ACT) has had reasonable success for repairing small articular cartilage defects. A limiting factor for ACT is, however, the in vitro cultivation of chondrocytes because it leads to dedifferentiation. Therefore, the goal of this work was to optimize the monolayer culture of chondrocytes in vitro. MATERIAL AND METHOD: Human articular chondrocytes were plated on either collagen type II or untreated surfaces. The cells were evaluated morphologically and with immunoblotting. RESULTS: On collagen type II surfaces, a stable chondrogenic phenotype, expression of beta1-integrin, and a significant activation of phosphorylated intracellular proteins and the adaptor protein Shc could be observed up to day 20 in culture. Treatment with beta1 integrin antibody led to a loss of cell adhesion (82%). The results indicate that on collagen type II, beta1-integrin receptors are activated. Through the activation of Shc, these stimulate the Ras-MAPK pathway, which stabilizes the chondrogenic phenotype. CONCLUSION: Our results provide a practical and low-cost solution for improved long-term chondrocyte cultivation, thus providing a new perspective for using ACT on larger or arthrotic cartilage defects.     

Restoration of Injured or Degenerated Articular Cartilage

Intra-articular fractures, ligamentous and meniscal injuries, and articular cartilage breakdown are major causes of degenerative joint disease. Lesions on the articular surface seem to have a limited capacity for repair and often progress inexorably toward osteoarthritis. Recent studies on joint immobilization and cartilage atrophy, however, have shown that repair and remodeling of articular cartilage may be possible. Currently used clinical methods of stimulating cartilage repair and remodeling include alteration of the loading on degenerated joints (primarily by using osteotomies), introduction of new cartilage-forming cells by perforation of subchondral bone, and soft-tissue arthroplasty. These procedures provide temporary relief in selected patients, but they often do not predictably restore long-term joint function. Experimentally, cartilage repair has been stimulated successfully with the use of allografts of periosteum and perichondrium, which serve as sources of cells with chondrogenic potential; introduction of cells grown in culture (stem cells or chondrocytes); stimulation by fibrin clot formation; artificial collagen matrices combined with cell transplants; and chondrogenic growth factors. The long-term success of all these methods has not been explored thoroughly, even in animal studies. Nevertheless, some research results are sufficiently encouraging to suggest that repair of the degenerating articular cartilage may be possible in the future.