Dynamic compression can inhibit chondrogenesis of mesenchymal stem cells

The objective of this study was to investigate the influence of dynamic compressive loading on chondrogenesis of mesenchymal stem cells (MSCs) in the presence of TGF-β3. Isolated porcine MSCs were suspended in 2% agarose and subjected to intermittent dynamic compression (10% strain) for a period of 42 days in a dynamic compression bioreactor. After 42 days in culture, the free-swelling specimens exhibited more intense alcian blue staining for proteoglycans, while immunohistochemical analysis revealed increased collagen type II immunoreactivity. Glycosaminoglycan (GAG) content increased with time for both free-swelling and dynamically loaded constructs, and by day 42 it was significantly higher in both the core (2.5 ± 0.21%w/w vs. 0.94 ± 0.03%w/w) and annulus (1.09 ± 0.09%w/w vs. 0.59 ± 0.08%w/w) of free-swelling constructs compared to dynamically loaded constructs. This result suggests that further optimization is required in controlling the biomechanical and/or the biochemical environment if such stimuli are to have beneficial effects in generating functional cartilaginous tissue.     

In vivo chondrogenesis of adult bone-marrow-derived autologous mesenchymal stem cells

The purpose of this study has been to investigate the possible effects of the normal joint cavity environment on chondrocytic differentiation of bone-marrow-derived mesenchymal stem cells (MSCs). Autologous bone marrow was aspirated from the iliac crest of male sheep. MSCs were purified, expanded, and labeled with the fluorescent dye PKH26. Labeled MSCs were then grown on a three-dimensional porous scaffold of poly (L-lactic-co-glycolic acid) in vitro and implanted into the joint cavity by a surgical procedure. At 4 or 8 weeks after implantation, the implants were removed for histochemical and immunohistochemical analysis. The cells labeled with red fluorescent PKH26 in the implants expressed type II collagen and synthesized sulfated proteoglycans. However, the osteoblast-specific marker, osteocalcin, was not detected by immunohistochemistry indicating that the implanted MSCs had not differentiated into osteoblasts by being directly exposed to the normal joint cavity. To investigate the possible factors involved in chondrocytic differentiation of MSCs further, we co-cultured sheep MSCs with the main components of the normal joint cavity, viz., synovial fluid or synovial cells, in vitro. After 1 or 2 weeks of co-culture, the MSCs in both co-culture systems expressed markers of chondrogenesis. These results suggest that synovial fluid and synovium from normal joint cavity are important for the chondrocytic differentiation of adult bone-marrow-derived MSCs.This work was supported by the National Natural Science Foundation of China (nos. 39900036, 20174006, and 20221402), the National Advanced Technology Programs of China (nos. 2003AA744051, 2003AA205041), the Award Foundation for Young Teachers from the Ministry of Education, 973 project (no. G1999054306-03), and the 248 key innovative project of Beijing (no. H010210190123).     

Recapitulation of mesenchymal condensation enhances in vitro chondrogenesis of human mesenchymal stem cells

Mesenchymal condensation is a critical transitional stage that precedes cartilage formation during embryonic development. We hypothesized that "priming" hMSCs to recapitulate mesenchymal condensation events prior to inducing differentiation would enhance their subsequent chondrogenic properties. Our prior studies have suggested that exposing hMSCs to hypoxia (2% O(2)) induces condensation-like effects. We therefore assessed the effect of preconditioning for different time periods on the expression of condensation specific genes by growing hMSCs in expansion medium under different normoxic (20% O(2)) and hypoxic conditions for up to 2 weeks, and subsequently induced chondrogenesis of preconditioned hMSCs. The total cultivation time for each group was 4 weeks and the chondrogenic properties were assessed using gene expression, biochemical analysis, and histological staining. Our results demonstrated the benefits of preconditioning were both time- and oxygen-dependent. Condensation specific genes, SOX-9 and NCAM, were significantly up-regulated in hypoxic conditions at the end of 1 week. COL X and MMP13 expression was also lower than the normoxic samples at this time point. However, this group did not exhibit more efficient chondrogenesis after 4 weeks. Instead, hMSCs preconditioned for 1 week and subsequently differentiated, both under 20% O(2), resulted in the most efficient chondrogenesis. Interestingly, while hypoxia appears to positively enhance expression of chondrogenic genes, this did not produce an enhanced matrix accumulation. The results of this study emphasize the significance of considering the timing of specific cues in developing protocols for stem cell-based therapies and underscore the complexity in regulating stem cell differentiation and tissue formation.     

Fibroblast growth factor-2 primes human mesenchymal stem cells for enhanced chondrogenesis

Human mesenchymal stem cells (hMSCs) are multipotent cells capable of differentiating into a variety of mature cell types, including osteoblasts, adipocytes and chondrocytes. It has previously been shown that, when expanded in medium supplemented with fibroblast growth factor-2 (FGF-2), hMSCs show enhanced chondrogenesis (CG). Previous work concluded that the enhancement of CG could be attributed to the selection of a cell subpopulation with inherent chondrogenic potential. In this study, we show that FGF-2 pretreatment actually primed hMSCs to undergo enhanced CG by increasing basal Sox9 protein levels. Our results show that Sox9 protein levels were elevated within 30 minutes of exposure to FGF-2 and progressively increased with longer exposures. Further, we show using flow cytometry that FGF-2 increased Sox9 protein levels per cell in proliferating and non-proliferating hMSCs, strongly suggesting that FGF-2 primes hMSCs for subsequent CG by regulating Sox9. Indeed, when hMSCs were exposed to FGF-2 for 2 hours and subsequently differentiated into the chondrogenic lineage using pellet culture, phosphorylated-Sox9 (pSox9) protein levels became elevated and ultimately resulted in an enhancement of CG. However, small interfering RNA (siRNA)-mediated knockdown of Sox9 during hMSC expansion was unable to negate the prochondrogenic effects of FGF-2, suggesting that the FGF-2-mediated enhancement of hMSC CG is only partly regulated through Sox9. Our findings provide new insights into the mechanism by which FGF-2 regulates predifferentiation hMSCs to undergo enhanced CG.     

Midazolam inhibits chondrogenesis via peripheral benzodiazepine receptor in human mesenchymal stem cells

Midazolam, a benzodiazepine derivative, is widely used for sedation and surgery. However, previous studies have demonstrated that Midazolam is associated with increased risks of congenital malformations, such as dwarfism, when used during early pregnancy. Recent studies have also demonstrated that Midazolam suppresses osteogenesis of mesenchymal stem cells (MSCs). Given that hypertrophic chondrocytes can differentiate into osteoblast and osteocytes and contribute to endochondral bone formation, the effect of Midazolam on chondrogenesis remains unclear. In this study, we applied a human MSC line, the KP cell, to serve as an in vitro model to study the effect of Midazolam on chondrogenesis. We first successfully established an in vitro chondrogenic model in a micromass culture or a 2D high‐density culture performed with TGF‐β‐driven chondrogenic induction medium. Treatment of the Midazolam dose‐dependently inhibited chondrogenesis, examined using Alcian blue‐stained glycosaminoglycans and the expression of chondrogenic markers, such as SOX9 and type II collagen. Inhibition of Midazolam by peripheral benzodiazepine receptor (PBR) antagonist PK11195 or small interfering RNA rescued the inhibitory effects of Midazolam on chondrogenesis. In addition, Midazolam suppressed transforming growth factor‐β‐induced Smad3 phosphorylation, and this inhibitory effect could be rescued using PBR antagonist PK11195. This study provides a possible explanation for Midazolam‐induced congenital malformations of the musculoskeletal system through PBR.     

Adipose-derived stem cells and chondrogenesis

Cartilage has only a very limited capacity to renew its original structure. Stem cells have been used to repair damaged cartilage, and recent studies have indicated that stem cells from adipose tissue are attractive cell sources that have the capacity of multipotentiality to differentiate into osteogenic, chondrogenic, myogenic, neurogenic and endothelial cells. Adipose-derived stem cells (ASC) have unique characteristics compared with stem cells from BM. At present, ASC have been studied to promote chondrogenesis. This review discusses the application of ASC to cartilage formation.     

Tissue transglutaminase regulates chondrogenesis in mesenchymal stem cells on collagen type XI matrices

Tissue transglutaminase (tTG) is a multifunctional enzyme with a plethora of potential applications in regenerative medicine and tissue bioengineering. In this study, we examined the role of tTG as a regulator of chondrogenesis in human mesenchymal stem cells (MSC) using nanofibrous scaffolds coated with collagen type XI. Transient treatment of collagen type XI films and 3D scaffolds with tTG results in enhanced attachment of MSC and supports rounded cell morphology compared to the untreated matrices or those incubated in the continuous presence of tTG. Accordingly, enhanced cell aggregation and augmented chondrogenic differentiation have been observed on the collagen type XI-coated poly-(L-lactide) nanofibrous scaffolds treated with tTG prior to cell seeding. These changes implicate that MSC chondrogenesis is enhanced by the tTG-mediated modifications of the collagen matrix. For example, exogenous tTG increases resistance to collagenolysis in collagen type XI matrices by catalyzing intermolecular cross-linking, detected by a shift in the denaturation temperature. In addition, tTG auto-crosslinks to collagen type XI as detected by western blot and immunofluorescent analysis. This study identifies tTG as a novel regulator of MSC chondrogenesis further contributing to the expanding use of these cells in cartilage bioengineering.     

Alpha-smooth muscle actin expression and structure integrity in chondrogenesis of human mesenchymal stem cells

The expression of alpha-smooth muscle actin (SMA) by human mesenchymal stem cells (hMSCs) during chondrogenesis was investigated by the use of pellet culture. Undifferentiated hMSCs expressed low but detectable amounts of SMA and the addition of transforming growth factor β1 (TGF-β1) to the culture medium increased SMA expression in a dose-dependent manner. Differentiation in pellet culture was rapidly induced in the presence of TGF-β1 and was accompanied by the development of annular layers at the surface of the pellet. These peripheral layers lacked expression of glycosaminoglycan and type II collagen during early differentiation. Progress in differentiation increased the synthesis of glycosaminoglycan and type II collagen and the expression of SMA in these layers. Double-staining for type II collagen and SMA by immunofluorescence demonstrated the differentiation of hMSCs into cells positive for these two proteins. The addition of cytochalasin D, a potent inhibitor of the polymerization of actin microfilaments, caused damage to the structural integrity and surface smoothness of the chondrogenic pellets. The SMA-positive cells in the peripheral layers of the chondrogenic pellets mimic those within the superficial layer of articular cartilage and are speculated to play a major role in cartilage development and maintenance.This work was supported by grants R92-001-1 and R92-001-2 from the Veterans General Hospital, Taipei, and grant NSC-92-2314-B-075-022 from the National Science Council, Taiwan.     

Wnt inhibitors enhance chondrogenesis of human mesenchymal stem cells in a long-term pellet culture

The long-term effects (~3 weeks) of two Wnt inhibitors (dickkopf [DKK]-1 and secreted frizzled-related protein [sFRP]-1), on the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) was determined. Wnt inhibitors significantly increased the amount of glycosaminoglycan (GAG) in treated pellets (P < 0.05). The gene expression of COL2A1 increased and COL1A1 decreased while the gene expression of SOX-9 and COL10A1 did not change significantly after three weeks of in vitro culture. The protein expression of type II collagen significantly increased (P < 0.05) and that of type I collagen significantly decreased (P < 0.05) while SOX-9 and type X collagen protein expression was unaffected. These findings suggest that Wnt inhibitors promote the chondrogenic differentiation of hMSCs when treated for three weeks.     

The role of mechanical signals in regulating chondrogenesis and osteogenesis of mesenchymal stem cells

It is becoming increasingly clear that mesenchymal stem cell (MSC) differentiation is regulated by mechanical signals. Mechanical forces generated intrinsically within the cell in response to its extracellular environment, and extrinsic mechanical signals imposed upon the cell by the extracellular environment, play a central role in determining MSC fate. This article reviews chondrogenesis and osteogenesis during skeletogenesis, and then considers the role of mechanics in regulating limb development and regenerative events such as fracture repair. However, observing skeletal changes under altered loading conditions can only partially explain the role of mechanics in controlling MSC differentiation. Increasingly, understanding how epigenetic factors, such as the mechanical environment, regulate stem cell fate is undertaken using tightly controlled in vitro models. Factors such as bioengineered surfaces, substrates, and bioreactor systems are used to control the mechanical forces imposed upon, and generated within, MSCs. From these studies, a clearer picture of how osteogenesis and chondrogenesis of MSCs is regulated by mechanical signals is beginning to emerge. Understanding the response of MSCs to such regulatory factors is a key step towards understanding their role in development, disease and regeneration. Birth Defects Research (Part C) 90:75–85, 2010. © 2010 Wiley‐Liss, Inc.     

Apoptosis in chondrogenesis of human mesenchymal stem cells: effect of serum and medium supplements

Apoptosis is an inevitable process during development and is evident in the formation of articular cartilage and endochondral ossification of growth plate. Mesenchymal stem cells (MSCs) can serve as alternative sources for cell therapy in focal chondral lesions or diffuse osteoarthritis. But there are few, if any, studies investigating apoptosis during chondrogenesis by MSCs. The aim of this study was to find the better condition to prevent apoptosis during chondrogenesis by MSCs. Apoptosis were evaluated in MSCs induced in different chondrogenic media by the use of Annexin V, TUNEL staining, lysosomal labeling with lysotracker and immunostaining of apoptotic markers. We found apparent apoptosis was demonstrated by Annexin V, TUNEL staining and lysosomal labeling during chondrogenesis. Meanwhile, the degree of apoptosis was related to the reagents of the defined chondrogenic medium. Adding serum in medium increased apoptosis, however, TGF-β1 inhibited apoptosis. The apoptosis was associated with the activation of caspase-3, the increase in the Bax/Bcl-2 ratio, the loss of lysosomal integrity, and the increase of PARP-cleavage. Pro-inflammatory cytokines, IL-1α, IL-1β and TNFα did not induce any increase in apoptosis. Interestingly, the inhibition of apoptosis by serum free medium supplemented with ITS was also associated with an increase in the expression of type II collagen, and a decrease in the expression of type X collagen, Runx2, and other osteogenic genes, while TGF-β1 increased the expression of Sox9, type II and type X collagen and decreased the expression of osteogenic genes. These data suggest apoptosis occurs during chondrogenesis by MSCs by cell death intrinsic pathway activation and this process may be modulated by culture conditions.     

In vitro chondrogenesis of human synovium-derived mesenchymal stem cells: optimal condition and comparison with bone marrow-derived cells

There are increasing reports that mesenchymal stem cells (MSCs) are present in various tissues other than bone marrow, including synovium. Here we investigated the optimal conditions for in vitro chondrogenesis of human synovium-derived MSCs and compared these cells with bone marrow-derived MSCs, especially in terms of their chondrogenesis potential. Synovium and bone marrow were harvested from six donors during knee operations for ligament injuries. Digested synovium cells or nucleated cells from bone marrow were expanded clonally. A pellet culture system was used for chondrogenesis, and the best combination of up to three cytokines of the seven assessed. Synovium-derived MSCs plated at a lower density expanded more rapidly. Contrary to previous reports, a combination of TGFbeta and dexamethasone was not sufficient to induce chondrogenesis. However, addition of BMP2 to TGFbeta and dexamethasone dramatically increased cartilage pellet size and the synthesis of cartilage matrix. The cartilage pellets were also analyzed by electron microscopy and immunohistology. DNA content per pellet decreased during chondrogenesis, indicating the pellet increased its size through the accumulation of newly synthesized extracellular matrix. Sequential chondrogenic gene expression was demonstrated by RT-PCR. Synovium-derived MSCs looked similar to the bone marrow-derived MSCs in their surface epitopes and proliferation potential; however, cartilage pellets from synovium were significantly larger than those from bone marrow in patient-matched comparisons. We demonstrated that the combination of TGFbeta, dexamethasone, and BMP2 was optimal for in vitro chondrogenesis of synovium-derived MSCs and that the synovium-derived MSCs have a greater chondrogenesis potential than bone marrow-derived MSCs.     

Pre‐activation of retinoid signaling facilitates neuronal differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) can differentiate into neurons in an appropriate cellular environment. Retinoid signaling pathway is required in neural development. However, the effect and mechanism through retinoid signaling regulates neuronal differentiation of MSCs are still poorly understood. Here, we report that all‐trans‐retinoic acid (ATRA) pre‐induction improved neuronal differentiation of rat MSCs. We found that, when MSCs were exposed to different concentrations of ATRA (0.01–100 μmol/L) for 24 h and then cultured with modified neuronal induction medium (MNM), 1 μmol/L ATRA pre‐induction significantly improved neuronal differentiation efficiency and neural‐cell survival. Compared with MNM alone induced neural‐like cells, ATRA/MNM induced cells expressed higher levels of Nestin, neuron specific enolase (NSE), microtubule‐associated protein‐2 (MAP‐2), but lower levels of CD68, glial fibrillary acidic protein (GFAP), and glial cell line‐derived neurotrophic factor(GDNF), also exhibited higher resting membrane potential and intracellular calcium concentration, supporting that ATRA pre‐induction promotes maturation and function of derived neurons but not neuroglia cells from MSCs. Endogenous retinoid X receptors (RXR) RXRα and RXRγ (and to a lesser extent, RXRβ) were weakly expressed in MSCs. But the expression of RARα and RARγ was readily detectable, whereas RARβ was undetectable. However, at 24 h after ATRA treatment, the expression of RARβ, not RARα or RARγ, increased significantly. We further found the subnuclear redistribution of RARβ in differentiated neurons, suggesting that RARβ may function as a major mediator of retinoid signaling during neuronal differentiation from MSCs. ATRA treatment upregulated the expression of Vimentin and Stra13, while it downregulated the expression of Brachyury in MSCs. Thus, our results demonstrate that pre‐activation of retinoid signaling by ATRA facilitates neuronal differentiation of MSCs.