Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells treated by GSK-3 inhibitors

A study of the cartilage differentiation of mesenchymal stem cells (MSCs) would be of particular interest since one strategy for cell-based treatment of cartilage defects emphasizes the use of cells that are in a differentiated state. The present study has attempted to evaluate the effects of two well-known glycogen synthase kinase-3 inhibitors, including lithium chloride (LiCl) and SB216763 on a human marrow-derived MSC (hMSC) chondrogenic culture. Passaged-3 MSCs were condensed into small pellets and cultivated in the following groups based on the supplementation of chondrogenic medium: transforming growth factor (TGF)-β1, TGF-β1 + LiCl, TGF-β1 + SB216763, TGF-β3, TGF-β3 + LiCl, and TGF-β3 + SB216763. The cultures were maintained for 21 days and then analyzed for expression of Sox9, aggrecan, collagen II, β-catenin, and axin genes. Deposition of glycosaminoglycan (GAG) in the cartilage matrix was also measured for certain cultures. The presence of both LiCl and SB216763 along with TGF-β in the MSC chondrogenic culture led to the up-regulation of cartilage-specific genes. TGF-β3 appeared much better than TGF-β1. Based on our findings, SB216763 was more effective in up-regulation of cartilage-specific genes. These chondrogenic effects appeared to be mediated through the Wnt signaling pathway since β-catenin and axin tended to be up-regulated and down-regulated, respectively. In the culture with SB216763 + TGF-β3, significantly more GAG was deposited (P < 0.05). In conclusion, addition of either SB216763 or LiCl to hMSC chondrogenic culture up-regulates cartilage-specific gene expression and enhances GAG deposition in the culture.     

Molecular and cellular characterization during chondrogenic differentiation of adipose tissue-derived stromal cells in vitro and cartilage formation in vivo

Human adipose tissue is a viable source of mesenchymal stem cells (MSCs) with wide differentiation potential for musculoskeletal tissue engineering research. The stem cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and expanded in vitro easily. This study was to determine molecular and cellular characterization of PLA cells during chondrogenic differentiation in vitro and cartilage formation in vivo . When cultured in vitro with chondrogenic medium as monolayers in high density, they could be induced toward the chondrogenic lineages. To determine their ability of cartilage formation in vivo , the induced cells in alginate gel were implanted in nude mice subcutaneously for up to 20 weeks. Histological and immunohistochemical analysis of the induced cells and retrieved specimens from nude mice at various intervals showed obviously cartilaginous phenotype with positive staining of specific extracellular matrix (ECM). Correlatively, results of RT-PCR and Western Blot confirmed the expression of characteristic molecules during chondrogenic differentiation namely collagen type II, SOX9, cartilage oligomeric protein (COMP) and the cartilage-specific proteoglycan aggrecan. Meanwhile, there was low level synthesis of collagen type X and decreasing production of collagen type I during induction in vitro and formation of cartilaginous tissue in vivo . These cells induced to form engineered cartilage can maintain the stable phenotype and indicate no sign of hypertrophy in 20 weeks in vivo , however, when they cultured as monolayers, they showed prehypertrophic alteration in late stage about 10 weeks after induction. Therefore, it is suggested that human adipose tissue may represent a novel plentiful source of multipotential stem cells capable of undergoing chondrogenesis and forming engineered cartilage.     

Hydrolyzed fish collagen induced chondrogenic differentiation of equine adipose tissue-derived stromal cells

Adipose-derived stromal cells (ADSCs) are multipotent cells which, in the presence of appropriate stimuli, can differentiate into various lineages such as the osteogenic, adipogenic and chondrogenic. In this study, we investigated the effect of transforming growth factor beta 1 (TGF-β1) in comparison to hydrolyzed fish collagen in terms of the chondrogenic differentiation potential of ADSCs. ADSCs were isolated from subcutaneous fat of horses by liposuction. Chondrogenesis was investigated using a pellet culture system. The differentiation medium was either supplemented with TGF-β1 (5 ng/ml) or fish collagen (0.5 mg/ml) for a 3 week period. After the 3 weeks in vitro differentiation, RT-PCR and histological staining for proteoglycan synthesis and type II collagen were performed to evaluate the degree of chondrogenic differentiation and the formation of cartilaginous extracellular matrix (ECM). The differentiation of ADSCs induced by TGF-β1 showed a high expression of glycosaminoglycan (GAG). Histological analysis of cultures stimulated by hydrolyzed fish collagen demonstrated an even higher GAG expression than cultures stimulated under standard conditions by TGF-β1. The expression of cartilage-specific type II collagen and Sox9 was about the same in both stimulated cultures. In this study, chondrogenesis was as effectively induced by hydrolyzed fish collagen as it was successfully induced by TGF-β1. These findings demonstrated that hydrolyzed fish collagen alone has the potential to induce and maintain ADSCs-derived chondrogenesis. These results support the application of ADSCs in equine veterinary tissue engineering, especially for cartilage repair.     

Dose dependent effect of C-type natriuretic peptide signaling in glycosaminoglycan synthesis during TGF-β1 induced chondrogenic differentiation of mesenchymal stem cells

Recent investigations credited important roles to C-type natriuretic peptide (CNP) signaling during chondrogenesis. This study investigated the putative role of CNP in transforming growth factor (TGF)-β1 induced in vitro chondrogenic differentiation of mesenchymal stem cells (MSCs) in pellet culture. MSCs were derived from human trabecular bone and were characterized on the basis of their cell surface antigens and adipogenic, osteogenic, and chondrogenic differentiation potential. TGF-β1 induced chondrogenic differentiation and glycosaminoglycan (GAG) synthesis was analyzed on the basis of basic histology, collagen type II, Sox 9 and aggrecan expressions, and Alcian blue staining. Results revealed that human trabecular bone-derived MSCs express CNP and NPR-B analyzed on the basis of RT-PCR and immunohistochemistry. In pellet cultures of MSCs TGF-β1 successfully induced chondrogenic differentiation and GAG synthesis. RT-PCR analyses of both CNP and NPR-B during this process revealed an activation of this signaling pathway in response to TGF-β1. Similar cultures induced with TGF-β1 and treated with different doses of CNP showed that CNP supplementation at 10^?8 and 10^?7 M concentrations significantly increased GAG synthesis in a dose dependent manner, whereas at 10^?6 M concentration this stimulatory effect was diminished. In conclusion, CNP/NPR-B signaling pathway is activated during TGF-β1 induced chondrogenic differentiation of human trabecular bone-derived MSCs and may strongly be involved in GAG synthesis during this process. This effect is likely to be a dose-dependent effect.     

软骨寡聚基质蛋白过表达对BMP-2诱导骨髓间充质干细胞分化的影响

目的：研究软骨寡聚基质蛋白（cartilage oligomeric matrix protein,COMP）过表达对BMP-2诱导骨髓间充质干细胞成骨及成软骨分化的影响。方法：BMP-2诱导骨髓间充质干细胞分化,通过脂质体转染含人COMP基因的质粒使骨髓间充质干细胞过表达COMP,采用实时定量PCR和Western blotting分析COMP基因过表达、成骨相关基因Ⅰ型胶原、RUNX2、骨钙蛋白以及成软骨相关基因Ⅱ型胶原、SOX9、蛋白聚糖、X型胶原的表达变化;通过茜素红染色观察成骨终末阶段矿化结节的生成情况,阿利新蓝染色观察细胞基质蛋白多糖的合成情况。结果：质粒转染后骨髓间充质干细胞COMP基因蛋白和mRNA表达水平显著提高（P<0.05）。COMP基因过表达后,成骨标记基因RUNX2、Ⅰ型胶原（Col1a1）mRNA水平均显著低于对照组（P<0.05）,RUNX2、骨钙蛋白（Osteocalcin）蛋白表达水平明显低于对照组（P<0.05）,而成软骨标记基因SOX9、蛋白聚糖（Aggrecan）mRNA水平均显著高于对照组（P<0.05）,SOX9、Ⅱ型胶原（Col2a1）蛋白表达均明显多于对照组（P<0.05）。细胞成骨茜素红染色弱于对照组,而阿利新蓝染色强于对照组。过表达组细胞X型胶原（Col10a1）基因表达显著低于对照组（P<0.05）,结论：骨髓间充质干细胞COMP基因过表达可抑制BMP-2诱导其成骨分化,促进骨髓间充质干细胞成软骨分化,并抑制软骨细胞的成熟肥大,为软骨组织工程研究提供新的方向。     

A mutation in COL9A1 causes multiple epiphyseal dysplasia: further evidence for locus heterogeneity

Multiple epiphyseal dysplasia (MED) is an autosomal dominantly inherited chondrodysplasia. It is clinically highly heterogeneous, partially because of its complex genetic background. Mutations in four genes, COL9A2, COL9A3, COMP, and MATR3, all coding for cartilage extracellular matrix components (i.e., the alpha2 and alpha 3 chains of collagen IX, cartilage oligomeric matrix protein, and matrilin-3), have been identified in this disease so far, but no mutations have yet been reported in the third collagen IX gene, COL9A1, which codes for the alpha1(IX) chain. MED with apparently recessive inheritance has been reported in some families. A homozygous R279W mutation was recently found in the diastrophic dysplasia sulfate transporter gene, DTDST, in a patient with MED who had a club foot and double-layered patella. The series consisted of 41 probands with MED, 16 of whom were familial and on 4 of whom linkage analyses were performed. Recombination was observed between COL9A1, COL9A2, COL9A3, and COMP and the MED phenotype in two of the families, and between COL9A2, COL9A3, and COMP and the phenotype in the other two families. Screening of COL9A1 for mutations in the two probands from the families in which this gene was not involved in the recombinations failed to identify any disease-causing mutations. The remaining 37 probands were screened for mutations in all three collagen IX genes and in the COMP gene. The probands with talipes deformities or multipartite patella were also screened for the R279W mutation in DTDST. The analysis resulted in identification of three mutations in COMP and one in COL9A1, but none in the other two collagen IX genes. Two of the probands with a multipartite patella had the homozygous DTDST mutation. The results show that mutations in COL9A1 can cause MED, but they also suggest that mutations in COL9A1, COL9A2, COL9A3, COMP, and DTDST are not the major causes of MED and that there exists at least one additional locus.     

Promotion of embryonic chick limb cartilage differentiation by transforming growth factor-beta

This study represents a first step in investigating the possible involvement of transforming growth factor-beta (TGF-beta) in the regulation of embryonic chick limb cartilage differentiation. TGF-beta 1 and 2 (1-10 ng/ml) elicit a striking increase in the accumulation of Alcian blue, pH 1-positive cartilage matrix, and a corresponding twofold to threefold increase in the accumulation of 35S-sulfate- or 3H-glucosamine-labeled sulfated glycosaminoglycans (GAG) by high density micromass cultures prepared from the cells of whole stage 23/24 limb buds or the homogeneous population of chondrogenic precursor cells comprising the distal subridge mesenchyme of stage 25 wing buds. Moreover, TGF-beta causes a striking (threefold to sixfold) increase in the steady-state cytoplasmic levels of mRNAs for cartilage-characteristic type II collagen and the core protein of cartilage-specific proteoglycan. Only a brief (2 hr) exposure to TGF-beta at the initiation of culture is sufficient to stimulate chondrogenesis, indicating that the growth factor is acting at an early step in the process. Furthermore, TGF-beta promotes the formation of cartilage matrix and cartilage-specific gene expression in low density subconfluent spot cultures of limb mesenchymal cells, which are situations in which little, or no chondrogenic differentiation normally occurs. These results provide strong incentive for considering and further investigating the role of TGF-beta in the control of limb cartilage differentiation.     

Direct Induction of Chondrogenic Cells from Human Dermal Fibroblast Culture by Defined Factors

The repair of large cartilage defects with hyaline cartilage continues to be a challenging clinical issue. We recently reported that the forced expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) can induce chondrogenic cells from mouse dermal fibroblast culture without going through a pluripotent state. We here generated induced chondrogenic (iChon) cells from human dermal fibroblast (HDF) culture with the same factors. We developed a chondrocyte-specific COL11A2 promoter/enhancer lentiviral reporter vector to select iChon cells. The human iChon cells expressed marker genes for chondrocytes but not fibroblasts, and were derived from non-chondrogenic COL11A2-negative cells. The human iChon cells formed cartilage but not tumors in nude mice. This approach could lead to the preparation of cartilage directly from skin in human, without going through pluripotent stem cells.     

Extended passaging, but not aldehyde dehydrogenase activity, increases the chondrogenic potential of human adipose-derived adult stem cells

Adipose-derived adult stem (ADAS) cells represent an abundant population of multipotent mesodermal cells residing in various adipose tissue depots. ADAS cell preparations appear heterogeneous, yet at a clonal level, greater than 50% of these cells exhibit multilineage differentiation potential. To date, there have been few attempts to define prospectively a homogenous population of multipotent cells. In this study, we investigated whether aldehyde dehydrogenase (ALDH) can be used to enrich ADAS cells with increased chondrogenic potential. ALDH has been previously used to isolate primitive hematopoietic progenitors and has been implicated in early neurogenesis. Human ADAS cells were purified based on ALDH activity, and the cells were expanded and induced for chondrogenic differentiation using BMP-6 in a 3-D alginate culture. No significant differences in chondrogenic potential were observed in the ALDH-positive cells compared to unsorted controls. In contrast, significant differences were noted between cells assayed at passage 4 (P4) and cells assayed at passage 9 (P9). Following BMP-6 induction, AGC1 gene expression in P9 cells increased 290-fold over P4 cells. Similarly, COL2A1 expression in P9 cells increased fivefold compared to P4 cells, while COL10A1 levels remained unchanged. Immunohistochemical analysis over 28 days revealed consistent findings at the protein level for collagen II, collagen X, and aggrecan. No changes in telomerase activity were detected across passage, suggesting that ADAS cells retain some level of "stemness" in monolayer culture. These findings suggest that the chondrogenic potential of ADAS cells increases with passage number, although ALDH may not be a suitable marker for chondrogenesis.     

The evaluation of cartilage differentiations using transforming growth factor beta3 alone and with combination of bone morphogenetic protein-6 on adult stem cells

In our quest to standardize our formula for a clinical trial, transforming growth factor-beta3 (TGF-β3) alone and in combination with bone morphogenetic protein-6 (BMP-6) were evaluated for their effectiveness in cartilage differentiation. Bone Marrow Stem Cells (BMSCs) and Adipose Derived Stem Cells (ADSCs) were induced to chondrogenic lineage using two different media. Native chondrocytes served as positive control. ADSCs and BMSCs proved multipotency by tri-lineage differentiations. ADSC has significantly higher growth kinetics compare to Chondrocyte only p ≤ 0.05. Using TGF-β3 alone, BMSC revealed higher expressions for hyaline cartilage genes compare to ADSCs. Chondrocyte has significantly higher early chondrogenic markers expression to ADSCs and BMSCs, while BMSCs was only higher to ADSC at chondroadherin, p ≤ 0.0001. On mature chondrogenic markers, chondrocytes were significantly higher to ADSCs and BMSCs for aggrecan, collagen IX, sry (sex determining region y)-box9, collagen II and fibromodullin; and only to ADSC for collagen XI. BMSC was higher to ADSC for aggrecan and collagen IX, p ≤ 0.0001. The combination of TGF-β3 + BMP-6 revealed increased gene expressions on both BMSCs and ADSCs for early and mature chondrogenic markers, but no significance difference. For dedifferentiation markers, ADSC was significantly higher to chondrocyte for collagen I. Glycosaminoglycan evaluations with both formulas revealed that chondrocytes were significantly higher to ADSCs and BMSCs, but none was significant to each other, p ≤ 0.0001. Combination of 10 ng TGF-β3 with 10 ng of BMP-6 enhanced chondrogenic potentials of BMSCs and ADSCs compare to TGF-β3 alone. This could be the ideal cocktail for either cell’s chondrogenic induction.     

Effects of In Vitro Low Oxygen Tension Preconditioning of Adipose Stromal Cells on Their In Vivo Chondrogenic Potential: Application in Cartilage Tissue Repair

Purpose

Multipotent stromal cell (MSC)-based regenerative strategy has shown promise for the repair of cartilage, an avascular tissue in which cells experience hypoxia. Hypoxia is known to promote the early chondrogenic differentiation of MSC. The aim of our study was therefore to determine whether low oxygen tension could be used to enhance the regenerative potential of MSC for cartilage repair.

Methods

MSC from rabbit or human adipose stromal cells (ASC) were preconditioned in vitro in control or chondrogenic (ITS and TGF-β) medium and in 21 or 5% O₂. Chondrogenic commitment was monitored by measuring COL2A1 and ACAN expression (real-time PCR). Preconditioned rabbit and human ASC were then incorporated into an Si-HPMC hydrogel and injected (i) into rabbit articular cartilage defects for 18 weeks or (ii) subcutaneously into nude mice for five weeks. The newly formed tissue was qualitatively and quantitatively evaluated by cartilage-specific immunohistological staining and scoring. The phenotype of ASC cultured in a monolayer or within Si-HPMC in control or chondrogenic medium and in 21 or 5% O₂ was finally evaluated using real-time PCR.

Results/Conclusions

5% O₂ increased the in vitro expression of chondrogenic markers in ASC cultured in induction medium. Cells implanted within Si-HPMC hydrogel and preconditioned in chondrogenic medium formed a cartilaginous tissue, regardless of the level of oxygen. In addition, the 3D in vitro culture of ASC within Si-HPMC hydrogel was found to reinforce the pro-chondrogenic effects of the induction medium and 5% O₂. These data together indicate that although 5% O₂ enhances the in vitro chondrogenic differentiation of ASC, it does not enhance their in vivo chondrogenesis. These results also highlight the in vivo chondrogenic potential of ASC and their potential value in cartilage repair.     

COL9A3: A third locus for multiple epiphyseal dysplasia

Multiple epiphyseal dysplasia (MED), an autosomal dominant osteochondrodysplasia, is a clinically and genetically heterogeneous disorder characterized by mild short stature and early-onset osteoarthritis. The phenotypic spectrum includes the mild Ribbing type, the more severe Fairbank type, and some unclassified forms. Linkage studies have identified two loci for MED. One of these, EDM1, is on chromosome 19, in a region that contains the cartilage oligomeric matrix protein (COMP) gene. Mutations have been identified in this gene in patients with the Ribbing type, the Fairbank type, and unclassified forms of MED. The second locus, EDM2, maps to chromosome 1, in a region spanning COL9A2. Recently, a splice-site mutation was found in COL9A2, causing skipping of exon 3 in one family with MED. Because of the exclusion of the EDM1 and EDM2 loci in some families, the existence of a third locus has been postulated. We report here one family with MED, evaluated clinically and radiologically and tested for linkage with candidate genes, including COMP, COL9A1, COL9A2, and COL9A3. No linkage was found with COMP, COL9A1, or COL9A2, but an inheritance pattern consistent with linkage was observed with COL9A3. Mutation analysis of COL9A3 identified an A-->T transversion in the acceptor splice site of intron 2 in affected family members. The mutation led to skipping of exon 3 and an in-frame deletion of 12 amino acid residues in the COL3 domain of the alpha3(IX) chain and thus appeared to be similar to that reported for COL9A2. This is the first disease-causing mutation identified in COL9A3. Our results also show that COL9A3, located on chromosome 20, is a third locus for MED.     

Type II Collagen Expression Is Regulated by Tissue-specific miR-675 in Human Articular Chondrocytes

miRNAs have been shown to be essential for normal cartilage development in the mouse. However, the role of specific miRNAs in cartilage function is unknown. Using rarely available healthy human chondrocytes (obtained from 8 to 50 year old patients), we detected a most highly abundant primary miRNA H19, whose expression was heavily dependent on cartilage master regulator SOX9. Across a range of murine tissues, expression of both H19- and H19-derived miR-675 mirrored that of cartilage-specific SOX9. miR-675 was shown to up-regulate the essential cartilage matrix component COL2A1, and overexpression of miR-675 rescued COL2A1 levels in H19- or SOX9-depleted cells. We thus provide evidence that SOX9 positively regulates COL2A1 in human articular chondrocytes via a previously unreported miR-675-dependent mechanism. This represents a novel pathway regulating cartilage matrix production and identifies miR-675 as a promising new target for cartilage repair.     

Novel chondrogenic and chondroprotective effects of the natural compound harmine

A significant number of natural compounds have been shown to regulate the behavior of the cells, in collaboration with cellular proteins. CCN2/connective tissue growth factor (CTGF) has been reported to have essential roles in cartilage development, chondrocyte proliferation and differentiation as well as regulation of the extracellular matrix metabolism. Previous studies demonstrated the capability of CCN2 to regenerate surgical defects in articular cartilage of rat knee. Also, transgenic mice over-expressing cartilage-specific CCN2 were shown to be more resistant to aging-related cartilage degradation. We hypothesized that small molecules that induce CCN2 in chondrocytes could be novel candidates to increase the resistance to aging-related cartilage degradation, or even to correct cartilage degenerative changes incurred in OA. Therefore, this study screened a compound library and identified the β-carboline alkaloid harmine as a novel inducer of CCN2 in human chondrocytic HCS-2/8 cells and osteoarthritic articular chondrocytes. Harmine increased the expression of the cartilage markers aggrecan and COL2α1, as well as that of the master regulator of chondrogenesis, SOX-9. Moreover, harmine notably induced chondrogenesis of prechondrocytic ATDC5 cells in micromass cultures. The chondroprotective effect of harmine was investigated under inflammatory condition by stimulation with TNFα, and harmine was shown to ameliorate TNFα-induced decrease in expression of CCN2 and cartilage markers. These findings uncover novel chondrogenic effects of harmine and indicate harmine as a potential drug for prevention and/or repair of cartilage degradation.     

Ex vivo and in vivo characterization of cold preserved cartilage for cell transplantation

Due to the inconvenient and invasive nature of chondrocyte transplantation, preserved cartilage has been recognized as an alternative source of chondrocytes for implantation. However, there are major concerns, in particular, the viability and quality of the chondrocytes. This study investigated the biochemistry and molecular characterization of chondrocytes isolated from preserved cartilage for purposes of transplantation. Ex vivo characterization was accomplished by storing human cartilage at either 4 or ?80 °C in a preservation medium. Microscopic evaluation of the preserved cartilage was conducted after 1, 2, 3 and 6 weeks. The chondrocytes were isolated from the preserved cartilage and investigated for proliferation capacity and chondrogenic phenotype. Transplantation of chondrocytes from preserved cartilage into rabbit knees was performed for purposes of in vivo evaluation. The serum cartilage degradation biomarker (WF6 epitopes) was evaluated during the transplantation procedure. Human cartilage preserved for 1 week in a 10 % DMSO chondrogenic medium at 4 °C gave the highest chondrocyte viability. The isolated chondrocytes showed a high proliferative capacity and retained chondrogenic gene expression. Microscopic assessment of the implanted rabbit knees showed tissue regeneration and integration with the host cartilage. A decreased level of the serum biomarker after transplantation was evidence of in vivo repair by the implanted chondrocytes. These results suggest that cartilage preservation for 1 week in a 10 % DMSO chondrogenic medium at 4 °C can maintain proliferation capacity and the chondrogenic phenotype of human chondrocytes. These results can potentially be applied to in vivo allogeneic chondrocyte transplantation. Allogeneic chondrocytes from preserved cartilage would be expected to maintain their chondrogenic phenotype and to result in a high rate of success in transplanted grafts.     

Effect of a subset of adipose-derived stem cells isolated with liposome magnetic beads to promote cartilage repair

This study aimed to investigate the ability of CD146⁺ subset of ADSCs to repair cartilage defects. In this study, we prepared CD146⁺ liposome magnetic beads (CD146⁺LMB) to isolate CD146⁺ADSCs. The cells were induced for chondrogenic differentiation and verified by cartilage-specific mRNA and protein expression. Then a mouse model of cartilage defect was constructed and treated by filling the induced cartilage cells into the damaged joint, to evaluate the function of such cells in the cartilage microenvironment. Our results demonstrated that the CD146⁺LMBs we prepared were uniform, small and highly stable, and cell experiments showed that the CD146⁺LMB has low cytotoxicity to the ADSCs. ADSCs isolated with CD146⁺LMB were all CD146⁺, CD105⁺, CD166⁺ and CD73⁺. After chondrogenic induction, the cells showed significantly increased expression of cartilage markers Sox9, collagen Ⅱ and aggrecan at protein level and significantly increased Sox9, collagen Ⅱ and aggrecan at mRNA level, and the protein expression and mRNA expression of CD146⁺ADSCs group were higher than those of ADSCs group. The CD146⁺ADSCs group showed superior tissue repair ability than the ADSCs group and blank control group in the animal experiment, as judged by gross observation, histological observation and histological scoring. The above results proved that CD146⁺LMB can successfully isolate the CD146⁺ADSCs, and after chondrogenic induction, these cells successfully promoted repair of articular cartilage defects, which may be a new direction of tissue engineering.