Growth plate, the developmental center of endochondral osteogenesis, can be divided morphologically and functionally into a resting zone, a proliferative zone, a prehypertrophic zone and a hypertrophic zone. Injuries to growth plate often lead to bone growth defects including limb length discrepancy and angulation deformity in children. Currently, their orthopedic corrective surgeries are invasive and limitedly effective and no effective biotherapy has been available. Previous studies on animal models of growth plate damage have investigated the related cellular and molecular events in the repair of damaged growth plates in the 4 distinct inflammatory, fibrogenic, osteogenic and remodeling phases. Related molecules involved in the regulation of the above processes, such as inflammatory cytokines tumor necrosis factor alpha, mitogenic platelet-derived growth factor and bone morphogenetic protein, are found to participate in the regulation of growth plate injury. Exploration of the mechanisms may provide new targets for biotherapy. In addition, development of cartilage tissue engineering, especially application of mesenchymal stem cells, also provides potential interventions for growth plate injury. Key words: Tissue engineering; Wounds and injuries; Bone marrow cells; Growth plate; Chondrogenesis

Objective To investigate connexins43 (Cx43) expression and its effect on chondrogenesis in the cocultures of bone marrow mesenchymal stromal cells (BMSCs)with articular chondrocytes (ACs). Methods The second passage BMSCs and ACs from New Zealand white rabbits were cocultured at the ratio of 3∶1 (BMSCs to ACs) for 21 days in vitro. The coculture patterns included the cell-cell contact coculture in the 24-well plates as experimental group and the cell-cell noncontact coculture through a Transwell chamber as control group. Cx43 inhibitor 18-ɑ glycyrrhetnic acid (GA) 50 μmol/L was supplemented in each group and subsequently cocultured for 21 days. Levels of glycosaminoglycans (GAG) in the medium supernatants of each group were detected by enzyme linked immunosorbent assay (ELISA). Expression of Cx43 and type Ⅱ collagen a1 (COL2a1) in the each group were analyzed using immunocytochemistry staining (ICC) and Western blotting. Results GAG levels of the medium supernatants, expression of Cx43 and COL2a1 were significantly high in the experimental group compared with control group [(327.517±24.612) ng/L vs. (171.612±13.163) ng/L, t=13.683, P 0.05; 0.240±0.028 vs. 0.223±0.026, t=1.086, P>0.05], whereas these all decreased significantly (t=17.541, 6.015, P 0.05). Conclusion Expression of Cx43 increases and Cx43 promotes chondrogenesis in contact coculture of BMSCs with ACs, which plays a major role in the regulation of chondrogenic differentiation. Key words: Bone marrow mesenchymal stem cell; Chondrocyte; Coculture; Connexins 43; Chondrogenesis

Objective To observe the repair effects of bone marrow mesenchymal stem cells (BMSCs) modified by adenovirus mediated B cell lymphoma/leukemia-xL (bcl-xL) gene on the cartilage defect of knee joint in rats. Methods The adenovirus-mediated bcl-xL gene overexpression vector pAdTrack-bcl-xL was constructed. BMSCs were isolated, cultured and amplified, pAdTrack-bcl-xL and blank vector were transfected, and the bcl-xL expression level was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). Fifty SD rats were randomly divided into five groups (n=10 each). The control group received no treatment, and the remaining four groups (model group, BMSCs treatment group, BMSCs treatment group transfected with empty vector and bcl-xL overexpressed BMSCs treatment group) were all used to make models of cartilage defect of the knee joint. Three days after surgery, the animals were treated accordingly. Rats in each group were sacrificed at 6th and 12th week after treatment, and the knee joints were collected and paraffin sections were made. Then the cartilage tissues of the knee joints were stained and observed by hematoxylin and eosin (HE), Masson and saffron-O methods. Results As compared with the empty vector group, the relative mRNA expression of bcl-xL gene in BMSCs of pAdTrack-bcl-xL group was significantly increased (1.07±0.08 vs. 8.15±0.31, t=-52.893, P<0.01), suggesting that BMSCs with stable over-expression of bcl-xL gene were successfully constructed. Pathological staining results showed that, after 6 weeks of treatment, as compared with the model group, in the BMSCs group and the BMSCs treatment group transfected with empty vector, a large amount of chondrogenesis was seen, the arrangement of chondrocytes tended to be orderly, and the number of chondrocytes significantly increased. However, the bcl-xL over-expressed BMSCs treatment group had more chondrogenesis, more orderly arrangement of chondrocytes and significantly greater number of chondrocytes than the BMSCs treatment group transfected with empty vector. As compared with the same group at 6th week, chondrogenesis was significantly increased in each treatment group at 12th week, especially in BMSCs treatment group with bcl-xL overexpression. Conclusion Bcl-xL over-expressed BMSCs can significantly promote the repair of cartilage defect of knee joints in rats, which will lay a theoretical foundation for the clinical treatment of articular cartilage defect. Key words: Bone marrow mesenchymal stem cells; B cell lymphoma/leukemia-xL over-expression; Knee joint; Cartilage defect

Objective To investigate the differentiation of bone mesenchymal stem cells(BMSCs) into chondrocytes by miRNA-206 and its mechanism in osteoarthritis(OA). Methods From January, 2017 to July, 2018, rat BMSCs were isolated, and their CD90 and CD45 were detected by flow cytometry. Transfection of miRNA-206 or miRNA-206 inhibitors into BMSCs using lentiviral vectors, dexamethasone induction for 14 d, then use alician blue staining and type II collagen immunostaining to detect chondrogenic differentiation. MTT assay was used to detect the proliferation of mesenchymal stem cells. Western blot analysis was used to detect the Aggrecan, Col II, Sox9 and Runx2 markers in chondroblast cells. The expression level of the marker gene of Sox9 mRNA in chondroblasts were detected by RT-PCR. OA rat models were treated with lentiviral vectors transfected with miRNA-206 or miRNA-206 inhibitors, and Aggrecan, Col II, Sox9, Runx2 which were the markers of chondrogenesis were detected by Western blot. Results The purity of isolated BMSCs was (80.7±3.9)%. BMSCs transfected with miRNA-206 could promote cell proliferation and increase chondrogenic differentiation. Western blot results showed that the expression of Aggrecan, Col II and Sox9 was increased in the miRNA-206 transfection group, and the expression of Runx2 was down-regulate. Meanwhile, RT-PCR results showed that miRNA-206 can up-regulate the expression of the chondroblast marker gene Sox9 mRNA in BMSCs. Compared with the OA group, miRNA-206 could increase the expression of Aggrecan, Col II and Sox9 signaling proteins in cartilage tissue (P<0.05), and down-regulate the expression level of Runx2(P<0.05). Conclusion The miRNA-206 can positively regulate the differentiation of BMSCs into chondrocytes, increase the ability of cell proliferation, up-regulate the expression of Aggrecan, Col II and Sox9, and down-regulate Runx2. The miRNA-206 increase chondrogenic capacity in rat models of osteoarthritis. Key words: Bone mesenchymal stem cells; Osteoarthritis; Chondrogenic differentiation; miRNA-206; Rat

Natural degeneration or trauma of articular cartilage all can lead to its structural and functional damage. Without blood supply and nerve innervation, chondrocytes in the matrix lacunae obtain essential nutrients and excrete metabolites mainly through osmosis, finally leads to its low metabolic activity and difficulty in self-repair after injury. At present, drug conservative treatment and surgical operation are the main clinical treatment, but both of them can't meet the clinical needs well. The development of cartilage tissue engineering provides a new direction for the repair of articular cartilage injury, in which growth factors plays a very important role. Growth factors, together with seed cells and cell scaffolds, constitute the three elements for the construction of tissue-engineered cartilage. Among them, Growth factors can significantly promote cell proliferation and differentiation and induce their functions. Various growth factors synergistically mediate the differentiation of seed cells into chondrocytes. In recent years, stem cell cartilage tissue engineering developed rapidly, which has opened a new way for repair of articular cartilage damage due to its abundant cell resources, small damage to body itself, strong ability of proliferation and directional differentiation, biological repair and other prominent advantages. Different types of hydrogels and stem cells show different abilities to support chondrogenesis and require different growth factors to induce chondrocyte differentiation. Traditional growth factors for tissue engineering include transcription growth factor β, insulin-like growth factors, bone morphogenetic proteins, fibroblast growth factors and cartilage derived morphogenetic protein. Recently, some scholars found that platelet-rich plasma, platelet-rich fibrin, Kartogenin and Mechano-growth factor can also effectively induce chondrogenic differentiation of stem cells and maintain chondrocyte phenotype. In addition, some synthetic compounds such as dexamethasone and inorganic particles can also promote the differentiation of stem cells into cartilage. This article systematically summarized the new progress of the traditional growth factors, emphatically introduced the new discovered growth factors and some synthetic compounds and inorganic particles, which can induce stem cells into cartilage. Finally classified the different sources of stem cells and its suitable growth factors, and gave an outlook of the next research direction of growth factors.

Objective In order to understand the chondrogenesis differentiation of mesenchymal stem cells in either hydrogel or pellet culture, we applied the two methods and reveal the possible mechanism and for further investigation. Methods In C3H10T1/2 chondrogenic differentiation, we apply extracellular matrix hydrogel mixed the cell suspensions of freshly prepared (including scaling chondroitin sulfate, sodium hyaluronate synthesis and cross-linking agent) co-culture system and high cell density pellet formed by centrifugation. Chondrogenic differentiation of C3H10T1/2 was induced by treatment with TGF-β3 (10 ng/ml), dexamethasone (100 nmol/L), ascorbic acid (50 ug/ml), 1∶100 dilution ITS+Premix and high glucose-DMEM medium with 0.2 volume fraction fetal bovine serum. And high glucose-DMEM medium with 0.2 volume fraction fetal bovine serum is for control group. Histochemistry staining was utilized to identify extracellular proteoglycan and real-time PCR was performed to assess gene expression of SOX9, collagen IIa1/Xa1 and aggrecan for the 1st, 2nd and 3rd week respectively. Results In the hydrogel model for 3 weeks chondrogenic differentiation, the expression of master transcription factor SOX9 was upregulated in both culture models. While the marker genes of collagen IIa1 and collagen Xa1 were all promoted in hydrogel culture, the aggrecan gene expression was peaked in pellet culture. In addition, immunocytochemistry analysis of the hydrogel and pellet for 3 week illustrated the expression of extracellular matrix and more obviously in the hydrogel model. Conclusion In compared with pellet culture, the MSCs in the hydrogel were more likely promoted chondrogenesis leading to the eventual expression of marker genes. And the hydrogel would be applied in regeneration of cartilage injury. Key words: Mesenchymal stem cells; Cellular microenvironment; Chondrocytes

It was initially thought that the growth-promoting effects of GH were exclusively mediated by liver-derived Insulin-like Growth Factor-I (IGF-I). Subsequent studies demonstrated that GH promotes IGF-I synthesis and activity in other organs and in the growth plate. GH has also IGF-I-independent growth-promoting effects. In Igf1 null mice, high circulating GH levels may be responsible for normal chondrocyte proliferation. Furthermore, tibial growth is reduced more in Ghr null mice than in Igf1 null mice, while the body of mice lacking both Ghr and Igf1 is smaller than that of mice lacking Igf1 or Ghr. The increased IGF-II expression in the growth plate in Igf1 null mice suggests that the IGF-I-independent effects of GH may be mediated by IGF-II. The effects of Igf1 receptor (Igf1r) gene deletion in chondrocytes indicate that GH may promote growth directly at the growth plate even when the local effects of IGF-I and IGF-II are abrogated.

Involvement of TGFβ/SMAD2 and Integrin β1/ERK Pathways in Chondrogenesis of CD105-ABBS-Bonded SMSCs Under Weight-Bearing In Vivo

Shear stresses modulate chondrogenic and osteogenic genes expression in human bone marrow derived stem cells

MIR-455-3P and HDACs co-regulate chondrogenesis

We describe the patterns of the limb chondrogenesis and ossification in Bufotes variabilis (Pallas, 1769) by using both serial sections and cleared-and-stained whole mount specimens. The limb morphologies with whole-mount specimens in B. variabilis are compared with those of Bufo bufo and Bufo calamita , whereas the serial sections are compared with those of Rana temporaria and Salamandrella . Based on examination of cleared-and-stained whole mount specimens of B. variabilis , we found that the first ossified limb elements are the femur and tibiofibula (Stage 37) and followed by the humerus and radioulna (Stage 38). Our results clearly show that there are some significant timing differences among bufonids and observations using serial sections are more detailed than whole-mount specimens because of determination of earlier differentiation of limb elements.

The aim of this study was to identify the most appropriate time point for the successful chondrogenic differentiation of adipose tissue-derived mesenchymal stem cells (AMSCs). For this purpose, the expression of some chondrogenesis markers, such as collagen type I, collagen type II, lubricin and RUNX2 have been investigated by immunohistochemical and Western blot analysis at different time points (7, 14, 21 and 28 days). The AMSCs chondrogenic differentiation in the natural self-assembling constructs, called ‘cell pellets’ has been also assessed by the histological (hematoxylin and eosin) and histochemical (alcian blue staining) methods. The results showed that the differentiated chondrocytes, after 21 days of differentiation process, were able to produce increased quantities of collagen type I, collagen type II, and lubricin, suggesting the hyaline cartilage formation, and reduced expression of RUNX2, a protein expressed by the hypertrophic chondrocytes in the late stages of differentiation and normally expressed by osteoblasts. Our study demonstrates that 21 days represents the optimum period for the potential implantation of AMSCs derived chondrocytes for the cartilage defects. This information could be useful for the future development of cell-based therapies for the articular cartilage degenerative diseases.This work was supported by a Grant-in-Aid provided by FIR 2014-2016, (cod. 314509), University of Catania, Italy.

Objective This article is aim to test the safety and the in vitro antibacterial activity of the Lentiviral-medicated humanβ-defensin 3(hβD-3) transfecion synovium-derived mesenchymal stem cells(SMSCs). Methods Tissues with SMSCs were obtained by surgical operations. Cells were explant cultured and purified by magnetic cell separation system. Cells were identified by microscopic observation, immunofluorescence and cell surface markers. Through inducing the cells into fat, osteoblasts and chondroblasts to respectively determine the multi-directional differentiation potential. Construct a hβD-3 contained lentivirus and transfected into SMSCs. Got the cell growth curve and determine the DNA content by using flow cytometry. The NOD/SCID mice were applied to verify the tumorigenicity of SMSCs. Agar diffusion test was applied to doing antibacterial activity test of posttransfecton SMSCs. The rabbit model of knee joint in staphylococcus aureus infections verify its bacteriostatic action in vivo. Results: Purified SMSCs had the structure and surface signatures of MSCs. It had the potential of multi-differentiation. Immunofluorescence had verified that SMSCs transfected by lentivirus could stably express hβD-3. Cell proliferation kinetics, karyotype analysis and Tumorigenic type analysis verified the safety of SMSCs after transfection. The in vivo and vitro antibacterial activity test of the recombinant SMSCs showed that after cell passages, the subcultured SMSCs(P5 cells were used in this article) could express hβD-3 stably and had antibacterial activity.The result of the antibacterial circle showed that low concentration group inhibit bacterial activity while the medium and high group could enlarge compared with the negative and positive control group. Conclusion Lentiviral-medicated hβD-3 gene express SMSCs could successfully subcultured and stably express hβD-3, meanwhile it had apparent in vitro antibacterial activity. Key words: Defensins; Synovial membrane; Mesenchymal stem cells; Lentivirus

In decades, the signaling pathways that regulate chondrogenesis and the differentiation of chondrocytes have been identified. Molecular biological understanding of the commitment of mesenchymal cells to the chondrogenic lineage, which leads to endochondral bone formation, is necessary to understand pathophysiology of bone/cartilage metabolic diseases and cartilage degenerative diseases. In this review, we show how multiple growth factors and signaling molecules are integrated to activate a series of transcription factors in connection with morphological cell features, which regulates chondrogenesis and chondrocyte differentiation.

Objective To observe the CD105- biotin- avidin promoting synovium- derived mesenchymal stem cells(SMSCs)adhesion and selecting CD105- positive cells in the cartilage tissue engineering, and evaluate its effects on cartilage formation. Methods SMSCs were obtained by enzyme digestion.Flow cytometry was used to detect markers on cell surface, including CD29, CD34, CD44, CD45, CD90 and CD105.With divided 3 groups(A:untreated+ cells; B:avidin+ biotin- cells; C:avidin+ CD105- biotin - cells), adhesion cell number was counted by absorbance and random field count at different time point.CD105- positive cells were tested through immunofluorescence. After 3 weeks chondrogenic induction, hematoxylin- eosin(HE), alcian blue staining and immunofluorescence were used to assess chondrogenesis. Results SMSCs showed star or spindle shape with adherent growth. The expression of CD29, CD44 and CD90 was positive, while CD34 and CD45 negative.The positive rate of CD105 was about 42%.Compared with Group A, Group B and C significantly increased cells adhesion at 1 and 2 h(P< 0.05). Although Group B had more cells than C, there was no statistical difference.Immunofluorescence showed CD105- positive cells in Group C were significantly higher than that in Group B and A.After chondrogenic induction, while proteoglycan and typeⅡcollagen were observed in all groups, there was most cartilage specific extracellular matrix in Group C. Con-clusion CD105- biotin- avidin system could not only enhance cells adhesion, but also select CD105 - positive cells, which may promote chondrogenesis better. Key words: CD105; Biotin-avidin; Synovium-derived mesenchymal stem cells; Cartilage tissue engineering

MIR-455 induces chondrogenesis by inhibiting Runx2

Objective To investigate the effect of hyaluronan (HA) on engineered cartilage formation.Methods Human mesenchymal stem cell (hBMSCs) were isolated by density gradient centrifugation and were seeded onto tricalcium phosphate-collagen (TCP-COL) scaffolds.hBMSCs were cultured on the scaffold for 2 weeks in two groups:(1) control group ; (2) HA group (control group culture medium with 100 mg/L HA).The effect of hyaluronan on chondrogenesis was assessed by hematoxylin-eosin (HE) staining,alcian blue staining,anti type Ⅱ collagen immunohistochemical staining,Real-time polymerase chain reaction (RT-PCR) and glycosaminoglycans (GAG) quantification assay.Results The data of hematoxylin-eosin (HE) staining,alcian blue staining,anti type Ⅱ collagen immunohistochemical staining,RT-PCR [Collagen Ⅱ (COL2) (P ＜ 0.01),SRY-related high mobility group-box gene9 (SOX9) (P ＜ 0.01),Aggrecan (P ＜ 0.05)] (8.954 ± 2.612/1.062 ± 0.459,3.512 ± 0.638/1.111 ± 0.595 and 4.588 ± 1.964/1.042 ± 0.335,respectively) and GAG quantification assay (21.741 ± 2.633/16.717 ± 0.595) (P ＜ 0.05) shows HA (100 g/L) could significantly induce the chondrogenic differentiation of hBMSCs on the TCP-COL scaffold.In addition,in the HA group,a significant up-regulation of COL1 (P＜0.05) (3.258 ±1.038/1.127 ±0.678) andCOL10 (P＜0.05) (5.961 ±2.598/1.080 ±0.458) gene were detected compared with the control group,indicating that HA may also promote robust maturation of tissue-engineered cartilage.Conclusion HA not only could significantly induce the chondrogenic differentiation of hBMSCs on the TCP-COL scaffolds,but also could promote maturation of the tissue-engineered cartilage. Key words: Hyaluronic acid; Bone marrow mesenchymal stem cells; Tissue-engineering cartilage

To investigate the effect of estrogen on cell proliferation and expression of proteins of C-type natriuretic peptide (CNP), natriuretic peptides B receptor (NPR-B) and natriuretic peptides C receptor (NPR-C) in ATDC5 cells during chondrogenesis. ATDC5 cells were induced for differentiation with insulin 10 µg/ml (day 0), and were started to be investigated on day 6. They were incubated with: (1) Estradiol (E2) at different concentrations (10(-11)-10(-5) mol/L) for 24 hours (for studying cell proliferation), or for 48 hours (for studying CNP, NPR-B and NPR-C protein expression); (2) E2 (10(-8) mol/L) for 24, 48, 72, 96 and 120 h (for studying cell proliferation), or for 24, 48, 72 and 96 hours (for studying CNP, NPR-B and NPR-C protein expression); (3) E2 (10(-8) mol/L) , and/or ICI 182782 (estrogen receptor antagonist ) (10(-7) mol/L) for 24 hours (for studying cell proliferation). ATDC5 cells proliferation were determined by MTT (OD value). Western-blotting was performed to identify the protein levels of CNP, NPR-B and NPR-C. (1) After incubation with E2 (10(-11)-10(-5) mol/L) for 24 h, ATD5 cell number increased with the increasing E2 concentration, peak in E2 concentrations of 10(-9) and 10(-8) mol/L (0.56 ± 0.06 and 0.52 ± 0.02, P < 0.05 and <0.01, respectively) , while significantly decreased in E2 (10(-5) mol/L) (0.30 ± 0.02) compared with DMSO-control (0.38 ± 0.02) (P < 0.05). After incubation with E2 (10(-11)-10(-5) mol/L) for 48 h, the protein level of CNP, NPR-B and NPR-C increased significantly, with the greatest effect seen at a concentration of 10(-10) mol/L E2 for CNP and NPR-B, 10(-9) mol/L E2 for NPR-C (P < 0.05). (2) After incubation with E2 (10(-8) mol/L) for 24 to 96 hours: (1) The cell number in each of the four time points was significantly increased compared with DMSO-control, with the greatest effect in 48 h (0.030 ± 0.003) (P < 0.05 or <0.01, respectively). While the cell number at 120 h was similar to that in DMSO-control. (2) The protein level of CNP increased significantly at 24 h (P < 0.05), seemed to be increased at 48 h and 72 h and decreased at 96 h. Both NPR-B and NPR-C level seemed to be increased at 24 h (P = 0.060 and 0.055, respectively) and seemed to decrease at 48 h, with decreasing significantly at both 72 h and 96 h (P < 0.05). (3) After incubation for 24 h, there was significant difference among the cell number of the four groups (P < 0.05). Cell number of group E2 (0.470 ± 0.032) was increased compared with group (E2+ICI) (0.410 ± 0.018), both being increased compared with group DMSO-control (0.370 ± 0.011, P < 0.05, respectively). There was no difference in cell number between group ICI 182782(0.360 ± 0.035) and group DMSO-control. E2 promotes the proliferation of ATDC5 cells i.e. chondrogenesis via estrogen receptor mediated mechanism, in both concentration-dependent and time-dependent manner. E2 (10(-11)-10(-8) mol/L) up-regulates protein expression of CNP, NPR-B and NPR-C of ATDC5 cells during chondrogenesis, and regulate the expression of the three proteins mentioned above positively or negatively at different time point, which implied that estrogen is one of the regulators of CNP signaling pathway.

Objective To determine the chondrogenic potential of mesenchymal stem cells (BMSCs) under exposure of extremely low frequency electromagnetic fields (ELF-EMF) and discuss the application prospect of EMF in cartilage tissue engineering.Methods Cell pellets of rat bone marrow-derived BMSCs were cultured in vitro under the addition of fibroblast growth factor 2 (FGF-2) and transforming growth factor-β3 (TGF-β3).Pellet cultures were exposed to sinusoidal ELF-EMF (1.0 mT,50 Hz).After 3 weeks of inductive differentiation culture,chondrogenesis was detected by alcian blue staining,and quantitative real-time polymerase chain reaction (real-time PCR) was used for detection of chondrogenesis related proteins.Glycosaminoglycan (GAG) contents of pellet cultures were determined using the dimethylmethylene blue (DMMB) dye-binding assay.Results With the effects of growth factors,EMF could significantly promote chondrogenic differentiation of BMSCs pellet cultures,and the gene expression of collagen type Ⅱ,X and aggrecan in rat BMSCs was also significantly up-regulated.The gene expression of sex determining region Y box 2 (SOX9) did not show significant difference between EMF exposed groups and nonexposed groups.The glycosaminoglycan (GAG)/DNA ratio of EMF exposed pellet cultures reached 3.108 ±0.341,which was significantly higer than those unexposed pellet cultures.Conclusion Increased expression of collagen type Ⅱ,X and GAG content in pellet cultures under EMF exposure may contribute to the chonderogenic differentiation of rat BMSCs.EMF was able to stimulate and maintain chondrogenic differentiation of rat BMSCs under influence of growth factors.EMF alone,however,could not induce chondrogenic differentiation. Key words: Electromagnetic fields; Mesenchymal stem cells; Pellet culture; Chondrogenic differentiation

Objective To investigate the effect of gap junctional blocker oleamide on growth differentiation factor 5 (GDF-5) induced chondrogenic differentiation.Methods hunan mesenchymal stem cells (hMSCs) were isolated and cultured in vitro,and the cells from Passage 3 were used in this study.hMSCs were randomly grouped into three according to the kinds of induction medium (0,100 μg/L GDF-5,100 μg/L GDF-5 + 2.5 μmol/L oleamide).The proliferation of hMSCs was investigated by methyl thiazol tetrazolium (MTT) assay.The cells were resuspended and the pellets were constructed by centrifugation at a density of 5 × 109/L,and then continued to be kept for two weeks.Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect the expression of mRNA of type Ⅱ collagen,comexin 43 (Cx43)and aggrecan.Western blotting was performed to detect the expression of protein of type Ⅱ collagen,Cx43and aggrecan.Toluidine blue was performed to detect extracellular matrix staining.Results GDF-5 can promote the mRNA expression of type Ⅱ collagen,Cx43 and aggrecan (1.00 ± 0.03 vs.0.40 ± 0.12 ;1.00 ±0.07 vs.0.32 ±0.02; 1.00 ±0.02 vs.0.45 ±0.01,P ＜0.01),and the relative protein expression was also increased (1.50±0.11 vs.0.53 ±0.07; 1.70 ±0.14 vs.0.52 ±0.04; 1.00 ±0.12 vs.0.50±0.06,P＜0.01).While oleamide could up-regulate the expression of mRNA of Cx43 (0.94 ±0.12 vs.0.45 ±0.01,P ＜0.01),but had no effect on type Ⅱ collagen and aggrecan mRNA expression.Oleamide could down-regulate the expression of protein of type Ⅱ collagen and aggrecan,but had no effect onCx43 protein expression(0.42 ± 0.04 vs.0.53 ±0.07; 0.38 ±0.06 vs.0.52 ±0.04,P＜0.01).Toluidine blue staining showed that GDF-5 can promote the synthesis of aggrecan.Conclusion GDF-5 modulation of chondrogenesis involves gap junction-mediated intercellular communication. Key words: Chondrogenic differentiation ; Growth differentiation factor 5 ; Oleamide ; Comexin 43