Induction Of Chondrogenic Differentiation Research Articles

Regenerated silk fibroin based on small aperture scaffolds and marginal sealing hydrogel for osteochondral defect repair

BackgroundOsteochondral defects pose an enormous challenge without satisfactory repair strategy to date. In particular, the lateral integration of neo-cartilage into the surrounding native cartilage is a difficult and inadequately addressed problem determining tissue repair’s success.MethodsRegenerated silk fibroin (RSF) based on small aperture scaffolds was prepared with n-butanol innovatively. Then, the rabbit knee chondrocytes and bone mesenchymal stem cells (BMSCs) were cultured on RSF scaffolds, and after induction of chondrogenic differentiation, cell-scaffold complexes strengthened by a 14 wt% RSF solution were prepared for in vivo experiments.ResultsA porous scaffold and an RSF sealant exhibiting biocompatibility and excellent adhesive properties are developed and confirmed to promote chondrocyte migration and differentiation. Thus, osteochondral repair and superior horizontal integration are achieved in vivo with this composite.ConclusionsOverall, the new approach of marginal sealing around the RSF scaffolds exhibits preeminent repair results, confirming the ability of this novel graft to facilitate simultaneous regeneration of cartilage–subchondral bone.

In vitro Study of Primary Isolation and Culture of Adipose-Derived Stem Cells and Induction of Chondrogenic Differentiation.

Articular cartilage has constrained potential to restore. The mesenchymal stem cellular remedy has presented new treatment possibilities for this circumstance. The experiment aimed to verify the chondrogenic differentiation capacity of rat adipose tissue-derived mesenchymal stem cells (AD-MSCs) in vitroinside the presence or absence of transforming growth factor-beta (TGF-β1). Rat's subcutaneous adipose tissue minced into a small piece (2-3 mm3) was aseptically collected from the subcutaneous fat under anaesthesia and then digested with collagenase type I (1 mg/mL). Spontaneous chondrogenesis occurred in both AD-MSCs pellet cultures and was similar in both TGF-β1 treated. The untreated pellet cultures were collected after 21 days. Histological assessment for evaluating the level of proteoglycan by alcin blue staining and immunohistochemistry for detecting the presence of collagen type II. A monoclonal antibody directed against collagen type II. Adipose-derived stem cells (AD-MSCs) isolated from rats were immunophenotyped for the expression of MSCs cell surface markers and was performed by Flow cytometer, which demonstrated AD-MSCs highly expressed CD73 (99.69±2.6%), CD90 (98.11±0.3%), and week expression CD44 (17.15±0.3%). The result of histological staining showed the presence of extracellular matrix (ECM) in the hyaline cartilage. This staining indicated a deposit of "acid mucopolysaccharides" in the proximity of the cells. Additionally, most cells are rounded cells stained positive for the presence of the cells encompassed by extracellular matrix (ECM), which were like chondrocytes as seen from the magnified view, lightly pink stained nuclei, and nuclear fast red stain. However, the immunohistochemistry method demonstrated that the presence of TGF-β1 decreased the levels of collagen type I and increased the levels of collagen type II. In conclusion, subcutaneous adipose tissue-derived stem cells can be used in cartilage tissue engineering.

Differentiation of Human Induced Pluripotent Stem Cells (iPSCs)-derived Mesenchymal Progenitors into Chondrocytes.

Induced pluripotent stem cells (iPSCs) generated from human sources are valuable tools for studying skeletal development and diseases, as well as for potential use in regenerative medicine for skeletal tissues such as articular cartilage. To successfully differentiate human iPSCs into functional chondrocytes, it is essential to establish efficient and reproducible strategies that closely mimic the physiological chondrogenic differentiation process. Here, we describe a simple and efficient protocol for differentiation of human iPSCs into chondrocytes via generation of an intermediate population of mesenchymal progenitors. These methodologies include step-by-step procedures for mesenchymal derivation, induction of chondrogenic differentiation, and evaluation of the chondrogenic marker gene expression. In this protocol, we describe the detailed procedure for successful derivation of mesenchymal progenitor population from human iPSCs, which are then differentiated into chondrocytes using high-density culture conditions by stimulating with bone morphogenetic protein-2 (BMP-2) or transforming growth factor beta-3 (TGFβ-3). The differentiated iPSCs exhibit temporal expression of cartilage genes and accumulation of a cartilaginous extracellular matrix in vitro, indicating successful chondrogenic differentiation. These detailed methodologies help effective differentiation of human iPSCs into the chondrogenic lineage to obtain functional chondrocytes, which hold great promise for modeling skeletal development and disease, as well as for potential use in regenerative medicine for cell-based therapy for cartilage regeneration. Key features • Differentiation of human iPSCs into chondrocytes using 3D culture methods. • Uses mesenchymal progenitors as an intermediate for differentiation into chondrocytes.

Comparison of Chondrogenesis Induction by TGF-β3, Kartogenin and Avocado/ soybean Unsaponifiables Between Laboratory and Animal Models

Background and Objectives Cartilage tissue engineering, by using stem cells, scaffolding and appropriate growth factors, seek to produce natural cartilage tissue to replace damaged tissue and solve the problems that exist in the treatment of cartilage damage. This study aims to compare chondrogenic differentiation of adipose-derived stem cells under the influence of the transforming growth factor beta 3 (TGF-β3), Kartogenin, and avocado/soybean unsaponifiables (ASU) on the fibrin scaffold in the laboratory and animal models. Subjects and Methods In this experimental laboratory study, after extraction of stem cells from human adipose tissue, induction of chondrogenic differentiation was done for 14 days on the fibrin scaffold in laboratory. The cells differentiated in the fibrin scaffold were transplanted subcutaneously under the skin of male rats for two weeks. Then, comparison of histological and immunohistochemical studies was performed in both laboratory and animal models. Results A significant increase in the density of Toluidine blue dye accumulation was observed in TGF-β3, Kartogenin and ASU groups in the animal model compared to the laboratory model. Immunohistochemical results for the collagen type X accumulation in the TGF-β3 group showed a significant increase in the animal model compared to the laboratory model. In the Kartogenin and ASU groups, the accumulation of collagen type Χ showed a significant decrease in the animal model compared to the laboratory model. Conclusion The implantation of differentiated cartilage cells in laboratory before subcutaneous transfer to the skin can help mature and complete the characteristics of the constructed cartilage.

Injectable adipose-derived stem cells-embedded alginate-gelatin microspheres prepared by electrospray for cartilage tissue regeneration.

ObjectiveTo prepare adipose-derived stem cells (ADSCs)-embedded alginate-gelatinemicrospheres (Alg-Gel-ADSCs MSs) by electrospray and evaluate their feasibility for cartilage tissue engineering. To observe the efficacy of Alg-Gel-ADSCs MSs in repairing articular cartilage defects in SD rats.MethodsADSCs were isolated and characterized by performing induced differentiation and flow cytometry assays. Alginate-gelatine microspheres with different gelatine concentrations were manufactured by electrospraying, and the appropriate alginate-gelatine concentration and ratio were determined by evaluating microsphere formation. Alg-Gel-ADSCs MSs were compared with Alg-ADSCs MSs through the induction of chondrogenic differentiation and culture. Their feasibility for cartilage tissue engineering was analysed by performing Live/Dead staining, cell proliferation analysis, toluidine blue staining and a glycosaminoglycan (GAG) content analysis. Alg-Gel-ADSCs MSs were implanted in the cartilage defects of SD rats, and the cartilage repair effect was evaluated at different time points. The evaluation included gross observations and histological evaluations, fluorescence imaging tracking, immunohistochemical staining, microcomputed tomography (micro-CT) and a CatWalk evaluation.ResultsThe isolated ADSCs showed multidirectional differentiation and were used for cartilage tissue engineering. Using 1.5 w:v% alginate and 0.5 w:v% gelatine (Type B), we successfully prepared nearly spherical microspheres. Compared with alginate microspheres, alginate gel increased the viability of ADSCs and promoted the proliferation and chondrogenesis of ADSCs. In our experiments on knee cartilage defects in SD rats in vivo, the Alg-Gel-ADSCs MSs showed superior cartilage repair in cell resides, histology evaluation, micro-CT imaging and gait analysis.ConclusionsMicrospheres composed of 1.5 w:v% alginate-0.5 w:v% gelatine increase the viability of ADSCs and supported their proliferation and deposition of cartilage matrix components. ADSCs embedded in 1.5 w:v% alginate-0.5 w:v% gelatine microspheres show superior repair efficacy and prospective applications in cartilage tissue repair.The translational potential of this articleIn this study, injectable adipose-derived stem cells-embedded alginate-gelatin microspheres (Alg-Gel-ADSCs MSs) were prepared by the electrospray . Compared with the traditional alginate microspheres, its support ability for ADSCs is better and shows a better repair effect. This study provides a promising strategy for cartilage tissue regeneration.

Different phenotypes and chondrogenic responses of human menstrual blood and bone marrow mesenchymal stem cells to activin A and TGF-\u03b23

BackgroundDue to its low capacity for self-repair, articular cartilage is highly susceptible to damage and deterioration, which leads to the development of degenerative joint diseases such as osteoarthritis (OA). Menstrual blood-derived mesenchymal stem/stromal cells (MenSCs) are much less characterized, as compared to bone marrow mesenchymal stem/stromal cells (BMMSCs). However, MenSCs seem an attractive alternative to classical BMMSCs due to ease of access and broader differentiation capacity. The aim of this study was to evaluate chondrogenic differentiation potential of MenSCs and BMMSCs stimulated with transforming growth factor β (TGF-β3) and activin A.MethodsMenSCs (n = 6) and BMMSCs (n = 5) were isolated from different healthy donors. Expression of cell surface markers CD90, CD73, CD105, CD44, CD45, CD14, CD36, CD55, CD54, CD63, CD106, CD34, CD10, and Notch1 was analyzed by flow cytometry. Cell proliferation capacity was determined using CCK-8 proliferation kit and cell migration ability was evaluated by scratch assay. Adipogenic differentiation capacity was evaluated according to Oil-Red staining and osteogenic differentiation according to Alizarin Red staining. Chondrogenic differentiation (activin A and TGF-β3 stimulation) was investigated in vitro and in vivo (subcutaneous scaffolds in nude BALB/c mice) by expression of chondrogenic genes (collagen type II, aggrecan), GAG assay and histologically. Activin A protein production was evaluated by ELISA during chondrogenic differentiation in monolayer culture.ResultsMenSCs exhibited a higher proliferation rate, as compared to BMMSCs, and a different expression profile of several cell surface markers. Activin A stimulated collagen type II gene expression and glycosaminoglycan synthesis in TGF-β3 treated MenSCs but not in BMMSCs, both in vitro and in vivo, although the effects of TGF-β3 alone were more pronounced in BMMSCs in vitro.ConclusionThese data suggest that activin A exerts differential effects on the induction of chondrogenic differentiation in MenSCs vs. BMMSCs, which implies that different mechanisms of chondrogenic regulation are activated in these cells. Following further optimization of differentiation protocols and the choice of growth factors, potentially including activin A, MenSCs may turn out to be a promising population of stem cells for the development of cell-based therapies with the capacity to stimulate cartilage repair and regeneration in OA and related osteoarticular disorders.

Synergistic effects of Indian hedgehog and sonic hedgehog on chondrogenesis during cartilage repair.

Sonic hedgehog (Shh) and Indian hedgehog (Ihh) have been shown to control the induction of early cartilaginous differentiation. However, it is unclear whether Ihh and Shh exert synergistic effects on chondrogenesis during articular cartilage repair. Herein, we investigate the effects of chondrogenesis of bone-derived mesenchymal stem cells (BMSCs) following co-transfection with Shh and Ihh via adenoviral vectors in vitro and in vivo. A rotary cell culture system (RCCS) and Cytodex 3 microcarriers were used to create a stereoscopic dynamic environment for cell culture. In the RCCS environment, BMSCs co-transfected with Ihh and Shh displayed stronger chondrogenic differentiation and chondrogenesis than BMSCs transfected with Ihh or Shh alone, and exhibited higher expression levels of Sox 9, ACAN and collagen II, stronger toluidine blue and collagen II immunohistochemical staining. After transplanted into the osteochondral defect at 8weeks, Ihh/Shh co-transfected BMSCs showed a significantly better cartilage repair than BMSCs transfected with Ihh or Shh alone. Ihh and Shh have synergistic effects on the induction of chondrogenic differentiation and chondrogenesis under a microgravity environment, and help to repair damaged cartilage and reverse subchondral defects during the early stages.

Chondrogenic effect of liquid and gelled platelet lysate on canine adipose-derived mesenchymal stromal cells

Osteoarthritis associated with hip dysplasia is one of the most common orthopedic abnormalities in dogs, with an incidence of up to 40% in some breeds. Tissue therapy of cartilage has received great attention, with use of mesenchymal stromal cells and different types of biomaterials. The present study aimed to evaluate the effect of platelet lysate (PL) on the proliferation and differentiation of canine adipose tissue-derived mesenchymal stromal cells (ASCs), in liquid culture or hydrogels. PL was prepared from blood collected from healthy dogs and submitted to freezing-thawing cycles, and hydrogel was formed with canine thrombin. The effect of PL on the proliferation and differentiation of canine ASCs was evaluated in liquid and hydrogel systems, with microscopy, quantification of dsDNA, histology and quantification of glycosaminoglycans. The addition of 5% or 10% PL to the culture medium induced a greater proliferation rate than the presence of 10% fetal bovine serum. The cultivation of ASCs in PL gel, with normal or chondrogenic medium, resulted in maintenance of proliferation level similar to the conventional 2D cultivation, and induction of chondrogenic differentiation, especially in the presence of the chondrogenesis induction medium.

Induction of Chondrogenic Differentiation in Human Mesenchymal Stem Cells Cultured on Human Demineralized Bone Matrix Scaffold under Hydrostatic Pressure.

Articular cartilage damage is still a troublesome problem. Hence, several researches have been performed for cartilage repair. The aim of this study was to evaluate the chondrogenicity of demineralized bone matrix (DBM) scaffolds under cyclic hydrostatic pressure (CHP) in vitro. In this study, CHP was applied to human bone marrow mesenchymal stem cells (hBMSCs) seeded on DBM scaffolds at a pressure of 5MPa with a frequency of 0.5Hz and 4h per day for 1week. Changes in chondrogenic and osteogenic gene expressions were analyzed by quantifying mRNA signal level of Sox9, collagen type I, collagen type II, aggrecan (ACAN), Osteocalcin, and Runx2. Histological analysis was carried out by hematoxylin and eosin, and Alcian blue staining. Moreover, DMMB and immunofluorescence staining were used for glycosaminoglycan (GAG) and collagen type II detection, respectively. Real-time PCR demonstrated that applying CHP to hBMSCs in DBM scaffolds increased mRNA levels by 1.3-fold, 1.2-fold, and 1.7-fold (p < 0.005) for Sox9, Col2, and ACAN, respectively by day 21, whereas it decreased mRNA levels by 0.7-fold and 0.8-fold (p < 0.05) for Runx2 and osteocalcin, respectively. Additionally, in the presence of TGF-β1 growth factor (10ng/ml), CHP further increased mRNA levels for the mentioned genes (Sox9, Col2, and ACAN) by 1.4-fold, 1.3-fold and 2.5-fold (p < 0.005), respectively. Furthermore, in histological assessment, it was observed that the extracellular matrix contained GAG and type II collagen in scaffolds under CHP and CHP with TGF-β1, respectively. The osteo-inductive DBM scaffolds showed chondrogenic characteristics under hydrostatic pressure. Our study can be a fundamental study for the use of DBM in articular cartilage defects in vivo and lead to production of novel scaffolds with two different characteristics to regenerate both bone and cartilage simultaneously.

Downregulation of long noncoding RNA HOTAIRM1 variant 1 contributes to osteoarthritis via regulating miR-125b/BMPR2 axis and activating JNK/MAPK/ERK pathway

Objective: Osteoarthritis (OA) is a leading cause of pain and disability worldwide, which is characterized by degenerative and inflammatory processes affecting joints and surrounding tissues. This study aimed to elucidate the role and possible mechanism of long non-coding RNA HOX antisense intergenic RNA myeloid 1 variant 1 (HOTAIRM1-1) in the development of osteoarthritis (OA).Material and methods: The expressions of HOTAIRM1-1 and miR-125b in OA cartilages and normal articular cartilages were determined. Moreover, human mesenchymal stem cells (MSCs) were cultured in MSC growth medium for 8 days to induce chondrogenic differentiation. The effects of aberrant expression of HOTAIRM1-1 on MSCs viability, apoptosis and differentiation were investigated. Furthermore, the regulatory relationships between HOTAIRM1-1 and miR-125b as well as the target of miR-125b were explored. Besides, the association between HOTAIRM1-1 and JNK/MAPK/ERK pathway was detected.Results: Inverse expression of HOTAIRM1-1 (downregulated) and miR-125b (upregulated) was revealed in OA cartilages. In addition, HOTAIRM1-1 expression increased at 4 days after induction of chondrogenic differentiation and further enhanced after 8 days, while miR-125b had opposite expression changes. Suppression of HOTAIRM1-1 inhibited MSCs viability, induced apoptosis and suppressed cell differentiation. Moreover, HOTAIRM1-1 negatively regulated the expression of miR-125b, and the effects of HOTAIRM1-1 suppression on cell viability, apoptosis and differentiation were achieved by negative regulation of miR-125b. Furthermore, miR-125b regulated MSCs viability, apoptosis and differentiation via targeting bone morphogenetic protein receptor 2 (BMPR2). Besides, overexpression of HOTAIRM1-1 inhibited the activation of JNK/MAPK/ERK pathway.Conclusions: Our results indicate that HOTAIRM1-1 is downregulated in OA cartilages, and its downregulation may inhibit MSCs viability, induce apoptosis and suppress differentiation via regulating miR-125b/BMPR2 axis. JNK/MAPK/ERK pathway may be a possible downstream mechanism to mediate the role of HOTAIRM1-1 in OA development.

MiRNA-101 promotes chondrogenic differentiation in rat bone marrow mesenchymal stem cells.

Effect and related mechanisms of miR-101 on the chondrogenic differentiation of rat bone marrow mesenchymal stem cells (MSCs) were investigated. The expression level of miR-101 was detected during chondrogenic differentiation. Three groups were established to study the potential function between miR-101 and chondrogenic differentiation: miR-NC group (negative control), miR-101 mimics (BMSCs transfected by miR-101 mimics) and mimics + inhibitor (BMSCs transfected by miR-101 mimics and inhibitor), after the induction of chondrogenic differentiation, the cell viability of MSCs and chondrogenic markers were determined, further, the expression level of Sox9 and Runx2 were detected. In our present research, miR-101 was found upregulated during chondrogenic differentiation of MSCs. Compared with the miR-NC group, the cell viability of MSCs was enhanced and the expression level of chondrogenic markers were respectively gained. The expression level of Sox9 was increased but the expression level of Runx2 was decreased by treatment of miR-101 mimics after induction of chondrogenic differentiation. However, these variations of the indicators were reversed by the intervention using the miR-101 inhibitor. Collectively, our research revealed promotion function of miR-101 on chondrogenic differentiation of MSCs, indicating that miR-101 could be a potential therapeutic strategy for the treatment of osteoarthritis (OA).

Chondrogenesis of human adipose derived stem cells for future microtia repair using co-culture technique

Conclusion: In conclusion, these result showed HADSCs could differentiate into chondrocytes-like cells, dependent on signaling induced by TGF-β3 and chondrocytes. This is a promising result and showed that HADSCs is a potential source for future microtia repair. The technique of co-culture is a positive way forward to assist the microtia tissue.Objective: Reconstructive surgery for the repair of microtia still remains the greatest challenge among the surgeons. Its repair is associated with donor-site morbidity and the degree of infection is inevitable when using alloplastic prosthesis with uncertain long-term durability. Thus, human adipose derived stem cells (HADSCs) can be an alternative cell source for cartilage regeneration. This study aims to evaluate the chondrogenic potential of HADSCs cultured with transforming growth factor-beta (TGF-β) and interaction of auricular chondrocytes with HADSCs for new cartilage generation.Methods: Multi-lineages differentiation features of HADSCs were monitored by Alcian Blue, Alizarin Red, and Oil Red O staining for chondrogenic, adipogenic, and osteogenic differentiation capacity, respectively. Further, HADSCs alone were culture in medium added with TGF-β3; and human auricular chondrocytes were interacted indirectly in the culture with and without TGF-βs for up to 21 days, respectively. Cell morphology and chondrogenesis were monitored by inverted microscope. For cell viability, Alamar Blue assay was used to measure the cell viability and the changes in gene expression of auricular chondrocyte markers were determined by real-time polymerase chain reaction analysis. For the induction of chondrogenic differentiation, HADSCs showed a feature of aggregation and formed a dense matrix of proteoglycans. Staining results from Alizirin Red and Oil Red O indicated the HADSCs also successfully differentiated into adipogenic and osteogenic lineages after 21 days.Results: According to a previous study, HADSCs were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen. The results showed HADSCs test groups (cultured with TGF-β3) displayed chondrocytes-like cells morphology with typical lacunae structure compared to the control group without TGF-β3 after 2 weeks. Additionally, the HADSCs test groups increased in cell viability; an increase in expression of chondrocytes-specific genes (collagen type II, aggrecan core protein, SOX 9 and elastin) compared to the control. This study found that human auricular chondrocytes cells and growth factor had a positive influence in inducing HADSCs chondrogenic effects, in terms of chondrogenic differentiate of feature, increase of cell viability, and up-regulated expression of chondrogenic genes.

Inhibition of hyaluronan synthesis alters sulfated glycosaminoglycans deposition during chondrogenic differentiation in ATDC5 cells.

In chondrogenic differentiation, expression and collaboration of specific molecules, such as aggrecan and type II collagen, in extracellular matrix (ECM) are crucial. However, few studies have clarified the roles of hyaluronan (HA) in proteoglycan aggregation during chondrogenic differentiation. We assessed the roles of HA in sulfated glycosaminoglycans deposition during chondrogenic differentiation by means of 4-methylumbelliferone (4-MU), an HA synthase inhibitor, using ATDC5 cells. ATDC5 cells were treated with 0.5 mM 4-MU for 7 or 21 days after induction of chondrogenic differentiation with insulin. Depositions of sulfated glycosaminoglycans were evaluated with Alcian blue staining. mRNA expression of ECM molecules was determined using real-time RT-PCR. The deposition of aggrecan and versican was investigated with immunohistochemical staining using specific antibodies. Effects of 4-MU on HA concentrations were analyzed by HA binding assay. 4-MU suppressed the positivity of Alcian blue staining, although this delay was reversible. Interestingly, stronger positivity of Alcian blue staining was observed at day 21 in cultures with 4-MU discontinuation than in the control. 4-MU significantly increased the mRNA expression of aggrecan, versican, and type II collagen, which was consistent with increased deposition of aggrecan and versican. The HA concentration in ECM and cell-associated region was significantly suppressed with 4-MU treatment. We conclude that the inhibition of HA synthesis slows sulfated glycosaminoglycans deposition during chondrogenic differentiation despite the increased deposition of other ECM molecules. Transient starvation of HA with 4-MU accelerates chondrogenic ECM formation, suggesting its potential to stimulate chondrogenic differentiation with adequate use.

Induction of chondrogenic differentiation of mouse embryonic mesenchymal stem cells through an in vitro pellet model.

This study employed transforming growth factor beta 3 (TGF-β3) induction of the C3H10T1/2 mesenchymal stem cell (MSC) line to construct an in vitro chondrogenic differentiation model for MSCs. A C3H10T1/2 MSC cell line was cultured, amplified, and the seventh generation of cells was centrifuged to construct pellets, which were divided into a non-induced group and an induced group (treated with TGF-β3, vitamin C, dexamethasone, and ITS). Specimens were taken after 7, 14, 21, and 28 days under non-induced and induced culture to compare these two groups by Alcian Blue staining, collagen type II immunohistochemical staining, and transmission election microscopy (TEM) on days 21 and 42. Cell pellets in the non-induced group were smaller than those in the induced group on days 7, 14, 21, and 28 with the pellet morphology of the induced group being more regular and nearly spherical. Alcian blue staining in the induced group was consistently stronger than that in non-induced group across all time points, and type II collagen immunohistochemical IOD values were significantly higher in the induced group over the non-induced group across all time points. On days 21 and 42, TEM revealed that the induced group displayed greater karyokinesis and a higher euchromatin ratio compared to the non-induced group. This specially constructed pellet model treated with TGF-β3-containing chondrogenic medium can effectively promote chondrogenic differentiation of C3H10T1/2 MSC cells in vitro. This in vitro pellet model should be of value in providing a preliminary cell model reference for further studies of the mechanism of chondroblast differentiation of stem cells.

MicroRNA expression profiles in human adipose-derived stem cells during chondrogenic differentiation.

The aim of the present study was to examine the microRNA (miRNA or miR) expression profiles during the chondrogenic differentiation of human adipose-derived stem cells (hADSCs) and identify the potential mechanisms through which miRNAs may affect the process of chondrogenesis. hADSCs were isolated and cultured. The expression levels of chondrogenic markers was detected by FACS analysis and immunohistochemistry. The miRNA expression profiles were then obtained through a miRNA array and confirmed through northern blot analysis. Putative targets of the miRNAs were predicted and validated through a luciferase reporter assay. The comparison of hADSCs following the induction of chondrogenic differentiation with undifferentiated hADSCs revealed 20 miRNAs that were differentially expressed by at least 2-fold, and these miRNAs included 12 upregulated miRNAs and 8 downregulated miRNAs. Northern blot analysis further confirmed the miRNA expression levels. Of these miRNAs, the expression of miR-490-5p was gradually downregulated following the induction of chondrogenic differentiation. The overexpression of miR-490-5p increased the expression of the chondrogenic markers, collagen, type II, alpha 1 (Col2A1), collagen, type X, alpha 1 (Col10A1) and aggrecan. Furthermore, it was confirmed that miR-490-5p directly targets bone morphogenetic protein receptor type 2 (BMPR2). In conclusion, in this study, we identified a set of miRNAs that may play key roles in the regulation of the chondrogenic differentiation of hADSCs. Our results may provide a basis for the further investigations into the molecular mechanisms of action of miRNAs in hADSC chondrogenesis.

Significant increase in Young׳s modulus of ATDC5 cells during chondrogenic differentiation induced by PAMPS/PDMAAm double-network gel: Comparison with induction by insulin

A double-network (DN) gel, which was composed of poly(2-acrylamido-2-methylpropanesulfonic acid) and poly(N,N′-dimethyl acrylamide) (PAMPS/PDMAAm), has the potential to induce chondrogenesis both in vitro and in vivo. The present study investigated the biomechanical and biological responses of chondrogenic progenitor ATDC5 cells cultured on the DN gel. ATDC5 cells were cultured on a polystyrene surface without insulin (Culture 1) and with insulin (Culture 2), and on the DN gel without insulin (Culture 3). The cultured cells were evaluated using micropipette aspiration for cell Young׳s modulus and qPCR for gene expression of chondrogenic and actin organization markers on days 3, 7 and 14. On day 3, the cells in Culture 3 formed nodules, in which the cells exhibited an actin cortical layer inside them, and gene expression of type-II collagen, aggrecan, and SOX9 was significantly higher in Culture 3 than Cultures 1 and 2 (p<0.05). Young׳s modulus in Culture 3 was significantly higher than that in Culture 1 throughout the testing period (p<0.05) and that in Culture 2 on day 14 (p<0.01). There was continuous expression of actin organization markers in Culture 3. This study highlights that the cells on the DN gel increased the modulus and mRNA expression of chondrogenic markers at an earlier time point with a greater magnitude compared to those on the polystyrene surface with insulin. This study also demonstrates a possible strong interrelation among alteration of cell mechanical properties, changes in actin organization and the induction of chondrogenic differentiation.

Evaluation of insulin medium or chondrogenic medium on proliferation and chondrogenesis of ATDC5 cells.

Background. The ATDC5 cell line is regarded as an excellent cell model for chondrogenesis. In most studies with ATDC5 cells, insulin medium (IM) was used to induce chondrogenesis while chondrogenic medium (CM), which was usually applied in chondrogenesis of mesenchymal stem cells (MSCs), was rarely used for ATDC5 cells. This study was mainly designed to investigate the effect of IM, CM, and growth medium (GM) on chondrogenesis of ATDC5 cells. Methods. ATDC5 cells were, respectively, cultured in IM, CM, and GM for a certain time. Then the proliferation and the chondrogenesis progress of cells in these groups were analyzed. Results. Compared with CM and GM, IM promoted the proliferation of cells significantly. CM was effective for enhancement of cartilage specific markers, while IM induced the cells to express endochondral ossification related genes. Although GAG deposition per cell in CM group was significantly higher than that in IM and GM groups, the total GAG contents in IM group were the most. Conclusion. This study demonstrated that CM focused on induction of chondrogenic differentiation while IM was in favor of promoting proliferation and expression of endochondral ossification related genes. Combinational use of these two media would be more beneficial to bone/cartilage repair.

Induced chondrogenic differentiation of parthenogenetic murine embryonic stem cells by insulin-like growth factor 2 treatment in a three-dimensional culture environment

The generation of cartilaginous tissues using pluripotent stem cells is one possible solution for cartilage regeneration, as the mass production of cartilaginous cells is required to cure defects and diseases of cartilage. Parthenogenetic ESCs (PESCs) are a useful stem cell source. The defects in full-term development of this cell type enable researchers to avoid ethical concerns. Moreover, in female patients, if the PESCs are derived from the patient’s own oocytes, the cells will have that patient’s genetic information. Here we present data demonstrating that PESCs can be differentiated into chondrogenic cells with induction medium containing multiple factors such as ascorbic acid, dexamethasone, bone morphogenetic protein-2 (BMP-2), and transforming growth factor-beta (TGF-β), and the induction can be promoted by exogenous insulin-like growth factor 2 (IGF-2), which is silenced in PESCs. Before chondrogenic induction of the PESCs, floating aggregates called embryoid bodies (EBs) were formed in suspension culture for 5 days. In the first series of experiments, the EBs were transferred to chondrogenic induction medium that contained serum, ascorbic acid, dexamethasone, BMP-2, BMP-4 and TGF-β, and the cells were cultured for an additional 21 days using either a two-dimensional (2D) or three-dimensional (3D) culture system. In the second series of experiments, we investigated the effects of different concentrations of IGF-2 supplementation (0, 1, 10 or 100 ng/mL) in a 3D culture system. After induction, the gene expression of the chondrocyte-specific markers, CHORDIN-LIKE 1, COLLAGEN-2, AGGRECAN, DECORIN, MMP13 and PAX-1, was analyzed real-time PCR. In experiment 1, the cells from all experimental groups after chondrogenic induction exhibited morphology characteristic of murine chondrocytes as confirmed by Alcian blue and Safranin O staining. The 3D culture system showed increased induction of chondrogenic differentiation in comparison to the 2D system. In experiment 2, the group supplemented with 100 ng/mL IGF-2 showed the highest induction of chondrogenic differentiation in comparison to other groups. The expression levels of all genes were 7-to 35-fold higher in the group treated with 100 ng/mL IGF-2 in comparison to the IGF-2-free control. The result showed that supplementation with the IGF-2 improves the efficiency of chondrogenic differentiation of murine PESCs in a 3D culture environment. The addition of IGF-2 may re-activate imprinted genes in PESCs that are only expressed in the paternal genome and are normally silent in PESCs. Our findings suggest that compensative supplementation of imprinting factor(s) may improve the efficiency of differentiation to specific lineages in monogenic stem cells.

Induction of chondrogenic differentiation in mesenchymal stem cells by TGF-beta cross-linked to collagen-PLLA [poly(l-lactic acid)] scaffold by transglutaminase 2

Transglutaminase-mediated cross-linking has been employed to optimize the mechanical properties and stability of tissue scaffolds. We have characterized tissue transglutaminase (TG2)-mediated cross-linking as a useful tool to deliver biologically-active TGF to mesenchymal stem cells (MSCs) and direct their differentiation towards a chondrogenic lineage. TGF-β3 is irreversibly cross-linked by TG2 to collagen type II-coated poly(L-lactic acid) nanofibrous scaffolds and activates Smad phosphorylation and Smad-dependent expression of a luciferase reporter. Human bone marrow-derived MSCs cultured on these scaffolds deposit cartilaginous matrix after 14 days of culture at 50 % efficiency compared to chondrogenesis in the presence of soluble TGF-β3. These findings are significant because they suggest a novel approach for the programming of MSCs in a spatially controlled manner by immobilizing biologically active TGF-β3 via cross-linking to a collagen-coated polymeric scaffold.

Cell-sheet technology combined with a thienoindazole derivative small compound TD-198946 for cartilage regeneration

Articular cartilage is a permanent tissue, with poor self-regenerative capacity. Consequently, a tissue engineering approach to cartilage regenerative therapy could greatly advance the current treatment options for patients with cartilage degeneration and/or defects. A successful tissue engineering approach would require not only induction of chondrogenic differentiation, but also suppression of subsequent endochondral ossification and chondrocyte dedifferentiation. We previously reported that direct injection of the thienoindazole derivative, TD-198946, into the knee joints of mice halted the progression of osteoarthritis; the compound induced chondrogenic differentiation without promoting endochondral ossification. In the present study, we applied TD-198946 to a cell-based cartilage reconstruction model, taking advantage of the cell-sheet technology. Cartilaginous cell-sheets were generated by culturing mouse and canine costal chondrocytes and human mesenchymal stem cells with TD-198946 on temperature-responsive dishes. The transplanted cell-sheets were then successfully used to promote the reconstruction of permanent cartilage, with no evidence of chondrocyte hypertrophy in the knee articular cartilage defects created in mice and canines. Thus, TD-198946 is a promising candidate for cell-based cartilage reconstruction therapies, enabling us to avoid any concern surrounding the use of scaffolds or cytokines to stimulate regeneration.