Cartilage-Specific Gene Expression and Extracellular Matrix Deposition in the Course of Mesenchymal Stromal Cell Chondrogenic Differentiation in 3D Spheroid Culture.

Articular cartilage damage still remains a major problem in orthopedical surgery. The development of tissue engineering techniques such as autologous chondrocyte implantation is a promising way to improve clinical outcomes. On the other hand, the clinical application of autologous chondrocytes has considerable limitations. Mesenchymal stromal cells (MSCs) from various tissues have been shown to possess chondrogenic differentiation potential, although to different degrees. In the present study, we assessed the alterations in chondrogenesis-related gene transcription rates and extracellular matrix deposition levels before and after the chondrogenic differentiation of MSCs in a 3D spheroid culture. MSCs were obtained from three different tissues: umbilical cord Wharton's jelly (WJMSC-Wharton's jelly mesenchymal stromal cells), adipose tissue (ATMSC-adipose tissue mesenchymal stromal cells), and the dental pulp of deciduous teeth (SHEDs-stem cells from human exfoliated deciduous teeth). Monolayer MSC cultures served as baseline controls. Newly formed 3D spheroids composed of MSCs previously grown in 2D cultures were precultured for 2 days in growth medium, and then, chondrogenic differentiation was induced by maintaining them in the TGF-ß1-containing medium for 21 days. Among the MSC types studied, WJMSCs showed the most similarities with primary chondrocytes in terms of the upregulation of cartilage-specific gene expression. Interestingly, such upregulation occurred to some extent in all 3D spheroids, even prior to the addition of TGF-ß1. These results confirm that the potential of Wharton's jelly is on par with adipose tissue as a valuable cell source for cartilage engineering applications as well as for the treatment of osteoarthritis. The 3D spheroid environment on its own acts as a trigger for the chondrogenic differentiation of MSCs.

Enhancement of mesenchymal stem cells' chondrogenic potential by type II collagen-based bioscaffolds.

BACKGROUND: Osteoarthritis (OA) is a common degenerative chronic disease accounting for physical pain, tissue stiffness and mobility restriction. Current therapeutic approaches fail to prevent the progression of the disease considering the limited knowledge on OA pathobiology. During OA progression, the extracellular matrix (ECM) of the cartilage is aberrantly remodeled by chondrocytes. Chondrocytes, being the main cell population of the cartilage, participate in cartilage regeneration process. To this end, modern tissue engineering strategies involve the recruitment of mesenchymal stem cells (MSCs) due to their regenerative capacity as to promote chondrocyte self-regeneration. METHODS AND RESULTS: In the present study, we evaluated the role of type II collagen, as the main matrix macromolecule in the cartilage matrix, to promote chondrogenic differentiation in two MSC in vitro culture systems. The chondrogenic differentiation of human Wharton's jelly- and dental pulp-derived MSCs was investigated over a 24-day culture period on type II collagen coating to improve the binding affinity of MSCs. Functional assays, demonstrated that type II collagen promoted chondrogenic differentiation in both MSCs tested, which was confirmed through gene and protein analysis of major chondrogenic markers. CONCLUSIONS: Our data support that type II collagen contributes as a natural bioscaffold enhancing chondrogenesis in both MSC models, thus enhancing the commitment of MSC-based therapeutic approaches in regenerative medicine to target OA and bring therapy closer to the clinical use.

Pleiotrophin inhibited chondrogenic differentiation potential of dental pulp stem cells.

OBJECTIVE: Studies have shown that the levels of pleiotrophin (PTN) are greatly elevated in the synovial fluid and cartilage in osteoarthritis. Therefore, the purpose of this study was to investigate the effect and mechanism of PTN on the chondrogenic differentiation of DPSCs in inflammatory and normal microenvironments. MATERIALS AND METHODS: A lentiviral vector was used to deplete or overexpress PTN in DPSCs. The inflammatory microenvironment was simulated in vitro by the addition of IL-1ß to the culture medium. The chondrogenic differentiation potential was assessed using Alcian Blue staining and the main chondrogenic markers. A dual-luciferase reporter assay was used to explore the relationship between miR-137 and PTN. RESULTS: The results showed that 0.1 ng/mL IL-1ß treatment during chondrogenic induction greatly impaired the chondrogenic differentiation of DPSCs. Supplementation with PTN and PTN overexpression inhibited chondrogenic differentiation of DPSCs, while PTN depletion promoted chondrogenic differentiation. MiR-137 negatively regulated the expression of PTN by binding to the 3'UTR of its mRNA. Moreover, miR-137 promoted chondrogenic differentiation of DPSCs in normal and inflammatory microenvironments. CONCLUSION: Our results suggest that PTN may play an inhibitory role in the chondrogenic differentiation of DPSCs in normal and inflammatory microenvironments, which is regulated by miR-137.

Long non-coding RNA XIST regulates chondrogenic differentiation of synovium-derived mesenchymal stem cells from temporomandibular joint via miR-27b-3p/ADAMTS-5 axis.

OBJECTIVE: Temporomandibular joint osteoarthritis (TMJOA) is a common degenerative disease in jaw joint, accompanied by articular cartilage destruction. Differentiation of stem cells to cartilage has important therapeutic implications in TMJ cartilage repair. Previous studies revealed that lncRNA XIST participated in various biological processes. However, the effect of XIST on chondrogenic differentiation of synovium-derived mesenchymal stem cells (SMSCs) remains unclear. Our study aimed to investigate the function of XIST in chondrogenic differentiation of human SMSCs from TMJ. METHODS: Alcian blue staining was performed to determine proteoglycan in SMSCs. qPCR, western blotting and immunofluorescence assays were allowed to assess sex determining region Y-box 9 (SOX9), Collagen type II alpha 1 chain (COL2A1) and Aggrecan (ACAN) expression. The direct interaction between miR-27b-3p and XIST or ADAMTS-5 was confirmed by dual luciferase reporter assay or RNA immunoprecipitation (RIP) assay. RESULTS: XIST was remarkably down-regulated in chondrogenic differentiation of SMSCs. Functional analysis demonstrated that XIST silencing promoted chondrogenic differentiation of SMSCs. Dual luciferase reporter and RIP assays identified that XIST acted as a sponge for miR-27b-3p. Moreover, XIST regulated ADAMTS-5 expression by directly binding miR-27b-3p. More importantly, miR-27b-3p/ADAMTS-5 rescued the effects of XIST on chondrogenic differentiation of SMSCs. CONCLUSION: The results suggest that XIST modulates SMSCs chondrogenic differentiation via the miR-27b-3p/ADAMTS-5 axis, which provides new targets for TMJOA treatment.

Effects of combination TGF-B1 transfection and platelet rich plasma (PRP) on three-dimension chondrogenic differentiation of rabbit dental pulp-derived mesenchymal stem cells.

Aim: The aim of this study was to evaluate the effects of standard culture medium and chondrogenic differentiation medium with PRP on chondrogenic differentiation of rabbit dental pulp-derived mesenchymal stem cells (rabbit DPSCs) that are transfected with transforming growth factor-beta 1 (TGF-B1) gene, based on the hypothesis of TGF- B1 and PRP can be effective on the chondrogenesis of stem cells. Materials and Methods: Rabbit DPSCs were characterized by using flow cytometry, immunofluorescent staining, quantitative Real Time Polymerase Chain Reaction (qRT-PCR) and differentiation tests. For the characterization, CD29, CD44 and CD45 mesenchymal cell markers were used. Rabbit DPSCs were transfected with TGF-B1 gene using electroporation technique in group 1; with PRP 10% in group 2; with chondrogenic medium in group 3; with both chondrogenic medium and PRP in group 4. DPSCs were cultured in medium with 10% inactive PRP in group 5, chondrogenic medium in group 6, chondrogenic medium with PRP 10% in group 7. SOX9, MMP13 and Aggrecan gene expression levels were evaluated in 3, 6, 12. and 24. days by qRT-PCR. Results: The expression levels of SOX9, MMP13 and Aggrecan were higher in group 2, 3 and group 7 in 3th day however in 24th day group 7 and group 2 were found higher. The expression levels changed by time-dependent. The extracellular matrix of the cells in experimental groups were positively stained with safranin O and toluidine blue. Conclusion: The combination in culture medium of TGF-B1 gene transfection and 10% PRP accelerates the chondrogenic differentiation of DPSCs.

Chondrogeneic Potential of MSC from Different Sources in Spheroid Culture.

We performed a comparative study of the proliferative potential of human mesenchymal stromal cells (MSC) from three sources (tooth pulp, adipose tissue, and Wharton's jelly) in spheroid culture; human chondroblasts served as the positive control. Histological examination revealed signs of chondrogenic differentiation in all studied cell cultures and the differences in the volume and composition of the extracellular matrix. Spheroids formed by MSC from the tooth pulp and Wharton's jelly were characterized by low content of extracellular matrix and glycosaminoglycans. Spheroids from adipose tissue MSC contained maximum amount of the extracellular matrix and high content of glycosaminoglycans. Chondrocytes produced glycosaminoglycan-enriched matrix. Type II collagen was produced by chondrocytes (to a greater extent) and adipose tissue MSC (to a lesser extent). The results of our study demonstrate that MSC from the adipose tissue under conditions of spheroid culturing exhibited maximum chondrogenic potential.

Integrin α4 mediates ATDC5 cell adhesion to negatively charged synthetic polymer hydrogel leading to chondrogenic differentiation.

Negatively charged synthetic hydrogels have been known to facilitate various cellular responses including cell adhesion, proliferation, and differentiation; however, the molecular mechanism of hydrogel-dependent control of cell behavior remains unclear. Recently, we reported that negatively charged poly(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) gel induces chondrogenic differentiation of ATDC5 cells via novel protein reservoir function. In this study, we identified the cell adhesion molecules binding to PAMPS gels that act as mechanoreceptors. First, we performed a pull-down assay by particle gels using cell membrane proteins of ATDC5, and found that multiple membrane proteins bound to the PAMPS gel, whereas the uncharged poly(N,N'-dimethylacrylamide) gel as control did not bind to any membrane proteins. Western blot analysis indicated differential binding of integrin (ITG) isoforms to the PAMPS gel, in which the α4 isoform, but not α5 and αv, efficiently bound to the PAMPS gel. ITG α4 knockdown decreased cell spreading of ATDC5 on PAMPS gels, whereas the enhanced expression increased the behavior. Furthermore, ITG α4 depletion suppressed PAMPS gel-induced expression of bone morphogenic protein (BMP) 4 contributing to chondrogenic differentiation, in concordance with the reduction of ERK activation. These results demonstrated that membrane protein binding to PAMPS gels occurred in a charge-dependent manner, and that ITG α4 plays a crucial role in cell spreading on PAMPS gels and acts as a mechanoreceptor triggering cellular signaling leads to chondrogenic differentiation.

Cartilage progenitor cells combined with PHBV in cartilage tissue engineering.

BACKGROUND: Bone marrow-derived stem cells (BMSCs) and chondrocytes have been reported to present "dedifferentiation" and "phenotypic loss" during the chondrogenic differentiation process in cartilage tissue engineering, and cartilage progenitor cells (CPCs) are novel seeding cells for cartilage tissue engineering. In our previous study, cartilage progenitor cells from different subtypes of cartilage tissue were isolated and identified in vitro, but the study on in vivo chondrogenic characteristics of cartilage progenitor cells remained rarely. In the current study, we explored the feasibility of combining cartilage progenitor cells with poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) to produce tissue-engineered cartilage and compared the proliferation ability and chondrogenic characteristics of cartilage progenitor cells with those of bone marrow-derived stem cells and chondrocytes. METHODS: These three cells combined with PHBV were cultured in vitro for 1 week without chondrogenic induction and then transplanted subcutaneously into nude mice for 6 weeks. The cell-PHBV constructs were evaluated by gross observation, histological staining, glycosaminoglycan content measurement, biomechanical analysis and RT-PCR. RESULTS: The chondrocyte-PHBV constructs and CPC-PHBV constructs became an ivory-whitish cartilage-like tissue, while the BMSC-PHBV constructs became vascularized 6 weeks after the subcutaneous implantation. Histological examination showed that many typical cartilage structures were present in the chondrocyte group, some typical cartilage structures were observed in the CPC group, while no typical cartilage structures were observed in the BMSC group. CONCLUSIONS: Cartilage progenitor cells may undergo chondrogenesis without chondrogenic induction and are better at chondrogenesis than BMSCs but worse than chondrocytes in the application of cartilage tissue engineering.

Enterococcus faecium L-15 Cell-Free Extract Improves the Chondrogenic Differentiation of Human Dental Pulp Stem Cells.

Hyaline cartilage is a tissue of very low regenerative capacity because of its histology and limited nutrient supply. Cell-based therapies have been spotlighted in the regeneration of damaged cartilage. Dental pulp stem cells (DPSCs) are multipotent and are easily accessible for therapeutic purposes. In human gastrointestinal tracts, Enterococcus faecium is a naturally occurring commensal species of lactic acid bacteria. In this work, the human DPSCs were differentiated into chondrocytes using a chondrogenic differentiation medium with or without L-15 extract. We observed that chondrogenic differentiation improved in an E. faecium L-15 extract (L-15)-treated DPSC group via evaluation of chondrogenic-marker mRNA expression levels. In particular, we found that L-15 treatment promoted early-stage DPSC differentiation. Cells treated with L-15 were inhibited at later stages and were less likely to transform into hypertrophic chondrocytes. In L-15-treated groups, the total amount of cartilage extracellular matrix increased during the differentiation process. These results suggest that L-15 promotes chondrogenic differentiation, and that L-15 may be used for cartilage repair or cartilage health supplements. To our knowledge, this is the first report demonstrating the beneficial effect of L-15 treatment on chondrogenic differentiation.

Chondrogenic differentiation of human scalp adipose-derived stem cells in Polycaprolactone scaffold and using Freeze Thaw Freeze method.

Human adipose tissue has been identified as a viable alternative source for mesenchymal stem cells. SADSCs were isolated from human scalp biopsy and then were characterized by Flow cytometry. SADSCS expressed CD90, CD44, and CD105 but did not express CD45 surface marker. Growth factors were used for chondrogenesis induction. Histology and immunohistology methods and gene expression by real-time PCR 14 days after induced cells have shown the feature of chondrocytes in their morphology and extracellular matrix in both inducing patterns of combination and cycling induction. Moreover, the expression of gene markers of chondrogenesis for example collagen type II aggrecan and SOX9 has shown by real-time PCR assay. Then, SADSCs were seeded alone on polycaprolatone (PCL) and with Freeze thaw Freeze (PCL+FTF) scaffolds and SADSCs differentiated toward the chondrogenic lineage and chondrogenesis induction were evaluated using scanning electron microcopy (SEM) and MTT assay. Our results showed that SADSCs were also similar to the other adipose-derived stem cells. Using TGF-beta3 and BMP-6 were effective for chondrogenesis induction. Therefore using of TGF-beta3 and BMP-6 growth factors may be the important key for in vitro chondrogenesis induction. The bio-composite of PCL+FTF nanofibrous scaffolds enhance the chondroblast differentiation and proliferation compared to PCL scaffolds .Therefore, our model will make it possible to study the mechanism of transition from chondroblast to chondrocyte.

PLGA-PTMC-Cultured Bone Mesenchymal Stem Cell Scaffold Enhances Cartilage Regeneration in Tissue-Engineered Tracheal Transplantation.

The treatment of long-segment tracheal defect requires the transplantation of effective tracheal substitute, and the tissue-engineered trachea (TET) has been proposed as an ideal tracheal substitute. The major cause of the failure of segmental tracheal defect reconstruction by TET is airway collapse caused by the chondromalacia of TET cartilage. The key to maintain the TET structure is the regeneration of chondrocytes in cartilage, which can secrete plenty of cartilage matrices. To address the problem of the chondromalacia of TET cartilage, this study proposed an improved strategy. We designed a new cell sheet scaffold using the poly(lactic-co-glycolic acid) (PLGA) and poly(trimethylene carbonate) (PTMC) to make a porous membrane for seeding cells, and used the PLGA-PTMC cell-scaffold to pack the decellularized allogeneic trachea to construct a new type of TET. The TET was then implanted in the subcutaneous tissue for vascularization for 2 weeks. Orthotopic transplantation was then performed after implantation. The efficiency of the TET we designed was analyzed by histological examination and biomechanical analyses 4 weeks after surgery. Four weeks after surgery, both the number of chondrocytes and the amount of cartilage matrix were significantly higher than those contained in the traditional stem-cell-based TET. Besides, the coefficient of stiffness of TET was significantly larger than the traditional TET. This study provided a promising approach for the long-term functional reconstruction of long-segment tracheal defect, and the TET we designed had potential application prospects in the field of TET reconstruction.

Polyethylene glycol modified PAMAM dendrimer delivery of kartogenin to induce chondrogenic differentiation of mesenchymal stem cells.

Partly PEGylated polyamidoamine (PAMAM) dendrimer was used as the nanocarrier for the cytoplasmic delivery of kartogenin (KGN) to induce chondrogenic differentiation of mesenchymal stem cells (MSCs). Here, KGN was conjugated to the surface of PAMAM and the end group of polyethylene glycol (PEG) to obtain PEG-PAMAM-KGN (PPK) and KGN-PEG-PAMAM (KPP) conjugate, respectively. The effects of PPK and KPP on the in vitro chondrogenic differentiation of MSCs were evaluated. KPP induced higher expression of chondrogenic markers than PPK and free KGN. In particular, after treatment of KPP, CBF ß nuclear localization intensity was significantly increased, indicating enhanced efficacy of chondrogenesis. The fluorescein labeled PEG-PAMAM was capable to persist in the joint cavity for a prolonged time of both healthy and osteoarthritis (OA) rats. Thus, PEG-PAMAM could be a useful nanocarrier for intra-articular (IA) delivery of drug to treat OA.

Enhancement of the chondrogenic differentiation capacity of human dental pulp stem cells via chondroitin sulfate-coated polycaprolactone-MWCNT nanofibers.

Most of the conditions involving cartilaginous tissues are irreversible and involve degenerative processes. The aim of the present study was to fabricate a biocompatible fibrous and film scaffolds using electrospinning and casting techniques to induce chondrogenic differentiation for possible application in cartilaginous tissue regeneration. Polycaprolactone (PCL) electrospun nanofibrous scaffolds and PCL film were fabricated and incorporated with multi-walled carbon nanotubes (MWCNTs). Thereafter, coating of chondroitin sulfate (CS) on the fibrous and film structures was applied to promote chondrogenic differentiation of human dental pulp stem cells (hDPSCs). First, the morphology, hydrophilicity and mechanical properties of the scaffolds were characterized by scanning electron microscopy (SEM), spectroscopic characterization, water contact angle measurements and tensile strength testing. Subsequently, the effects of the fabricated scaffolds on stimulating the proliferation of human dental pulp stem cells (hDPSCs) and inducing their chondrogenic differentiation were evaluated via electron microscopy, flow cytometry and RT‒PCR. The results of the study demonstrated that the different forms of the fabricated PCL-MWCNTs scaffolds analyzed demonstrated biocompatibility. The nanofilm structures demonstrated a higher rate of cellular proliferation, while the nanofibrous architecture of the scaffolds supported the cellular attachment and differentiation capacity of hDPSCs and was further enhanced with CS addition. In conclusion, the results of the present investigation highlighted the significance of this combination of parameters on the viability, proliferation and chondrogenic differentiation capacity of hDPSCs seeded on PCL-MWCNT scaffolds. This approach may be applied when designing PCL-based scaffolds for future cell-based therapeutic approaches developed for chondrogenic diseases.

LncRNA SNHG1 enhances cartilage regeneration by modulating chondrogenic differentiation and angiogenesis potentials of JBMMSCs via mitochondrial function regulation.

BACKGROUND: Cartilage is a kind of avascular tissue, and it is difficult to repair itself when it is damaged. In this study, we investigated the regulation of chondrogenic differentiation and vascular formation in human jaw bone marrow mesenchymal stem cells (h-JBMMSCs) by the long-chain noncoding RNA small nucleolar RNA host gene 1 (SNHG1) during cartilage tissue regeneration. METHODS: JBMMSCs were isolated from the jaws via the adherent method. The effects of lncRNA SNHG1 on the chondrogenic differentiation of JBMMSCs in vitro were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), Pellet experiment, Alcian blue staining, Masson's trichrome staining, and modified Sirius red staining. RT-qPCR, matrix gel tube formation, and coculture experiments were used to determine the effect of lncRNA SNHG1 on the angiogenesis in JBMMSCs in vitro. A model of knee cartilage defects in New Zealand rabbits and a model of subcutaneous matrix rubber suppositories in nude mice were constructed for in vivo experiments. Changes in mitochondrial function were detected via RT-qPCR, dihydroethidium (DHE) staining, MitoSOX staining, tetramethyl rhodamine methyl ester (TMRM) staining, and adenosine triphosphate (ATP) detection. Western blotting was used to detect the phosphorylation level of signal transducer and activator of transcription 3 (STAT3). RESULTS: Alcian blue staining, Masson's trichrome staining, and modified Sirius Red staining showed that lncRNA SNHG1 promoted chondrogenic differentiation. The lncRNA SNHG1 promoted angiogenesis in vitro and the formation of microvessels in vivo. The lncRNA SNHG1 promoted the repair and regeneration of rabbit knee cartilage tissue. Western blot and alcian blue staining showed that the JAK inhibitor reduced the increase of STAT3 phosphorylation level and staining deepening caused by SNHG1. Mitochondrial correlation analysis revealed that the lncRNA SNHG1 led to a decrease in reactive oxygen species (ROS) levels, an increase in mitochondrial membrane potential and an increase in ATP levels. Alcian blue staining showed that the ROS inhibitor significantly alleviated the decrease in blue fluorescence caused by SNHG1 knockdown. CONCLUSIONS: The lncRNA SNHG1 promotes chondrogenic differentiation and angiogenesis of JBMMSCs. The lncRNA SNHG1 regulates the phosphorylation of STAT3, reduces the level of ROS, regulates mitochondrial energy metabolism, and ultimately promotes cartilage regeneration.

Composite Hydrogel Containing Collagen-Modified Polylactic Acid-Hydroxylactic Acid Copolymer Microspheres Loaded with Tetramethylpyrazine Promotes Articular Cartilage Repair.

Articular cartilage defects pose a significant challenge due to the limited self-healing ability of cartilage. However, traditional techniques face limitations including autologous chondrocyte expansion issues. This study aims to investigate the effects of the polylactic acid-glycolic acid (PLGA) and collagen-surface modified polylactic acid-glycolic acid (CPLGA) microspheres loaded with tetramethylpyrazine (TMP) on two cell types and the regeneration potential of articular cartilage. CPLGA microspheres are prepared by Steglich reaction and characterized. They evaluated the effect of TMP-loaded microspheres on HUVECs (Human Umbilical Vein Endothelial Cells) and examined the compatibility of blank microspheres with BMSCs (Bone marrow mesenchymal stromal cells) and their potential to promote cartilage differentiation. Subcutaneous implant immune tests and cartilage defect treatment are conducted to assess biocompatibility and cartilage repair potential. The results highlight the efficacy of CPLGA microspheres in promoting tissue regeneration, attributed to improved hydrophilicity and collagen-induced mitigation of degradation. Under hypoxic conditions, both CPLGA and PLGA TMP-loaded microspheres exhibit inhibitory effects on HUVEC proliferation, migration, and angiogenesis. Notably, CPLGA microspheres show enhanced compatibility with BMSCs, facilitating chondrogenic differentiation. Moreover, the CPLGA microsphere-composite hydrogel exhibits potential for cartilage repair by modulating angiogenesis and promoting BMSC differentiation.

Lysine demethylase 3A promotes chondrogenic differentiation of aged human dental pulp stem cells.

Background/purpose: Aging severely impairs the beneficial effects of human dental pulp stem cells (hDPSCs) on cartilage regeneration. Lysine demethylase 3A (KDM3A) is involved in regulating mesenchymal stem cells (MSCs) senescence and bone aging. In this study, we investigated the role of KDM3A in hDPSCs aging and whether KDM3A could rejuvenate aged hDPSCs to enhance their chondrogenic differentiation capacity. Materials and methods: The cellular aging of hDPSCs was evaluated by senescence-associated ß-galactosidase (SA-ß-gal) staining. Protein levels were determined using Western blot analysis. KDM3A was overexpressed in aged hDPSCs by lentivirus infection. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) were used to determine the mRNA levels of stemness markers. Toluidine blue staining was used to evaluate the effect of KDM3A overexpression on the chondrogenic differentiation of aged hDPSCs. Results: hDPSCs at passage 12 or treated with etoposide exhibited augmented cellular senescence as evidenced by increased SA-ß-gal activity. KDM3A was significantly increased during senescence of hDPSCs. Overexpression of KDM3A did not affect the stemness properties but significantly promoted the chondrogenic differentiation of aged hDPSCs. Conclusion: Our findings indicate that KDM3A plays an important role in the maintenance of the chondrogenic differentiation capacity of aged hDPSCs and suggest that therapies targeting KDM3A may be a novel strategy to rejuvenate aged hDPSCs.

Plasticity Comparison of Two Stem Cell Sources with Different Hox Gene Expression Profiles in Response to Cobalt Chloride Treatment during Chondrogenic Differentiation.

The limited self-repair capacity of articular cartilage is a challenge for healing injuries. While mesenchymal stem/stromal cells (MSCs) are a promising approach for tissue regeneration, the criteria for selecting a suitable cell source remain undefined. To propose a molecular criterion, dental pulp stem cells (DPSCs) with a Hox-negative expression pattern and bone marrow mesenchymal stromal cells (BMSCs), which actively express Hox genes, were differentiated towards chondrocytes in 3D pellets, employing a two-step protocol. The MSCs' response to preconditioning by cobalt chloride (CoCl2), a hypoxia-mimicking agent, was explored in an assessment of the chondrogenic differentiation's efficiency using morphological, histochemical, immunohistochemical, and biochemical experiments. The preconditioned DPSC pellets exhibited significantly elevated levels of collagen II and glycosaminoglycans (GAGs) and reduced levels of the hypertrophic marker collagen X. No significant effect on GAGs production was observed in the preconditioned BMSC pellets, but collagen II and collagen X levels were elevated. While preconditioning did not modify the ALP specific activity in either cell type, it was notably lower in the DPSCs differentiated pellets compared to their BMSCs counterparts. These results could be interpreted as demonstrating the higher plasticity of DPSCs compared to BMSCs, suggesting the contribution of their unique molecular characteristics, including their negative Hox expression pattern, to promote a chondrogenic differentiation potential. Consequently, DPSCs could be considered compelling candidates for future cartilage cell therapy.

Mechanomorphological Guidance of Colloidal Gel Regulates Cell Morphogenesis.

Microstructural morphology of the extracellular matrix guides the organization of cells in 3D. However, current biomaterials-based matrices cannot provide distinct spatial cues through their microstructural morphology due to design constraints. To address this, colloidal gels are developed as 3D matrices with distinct microstructure by aggregating ionic polyurethane colloids via electrostatic screening. Due to the defined orientation of interconnected particles, positively charged colloids form extended strands resulting in a dense microstructure whereas negatively charged colloids form compact aggregates with localized large voids. Chondrogenesis of human mesenchymal stem cells (MSCs) and endothelial morphogenesis of human endothelial cells (ECs) are examined in these colloidal gels. MSCs show enhanced chondrogenic response in dense colloidal gel due to their spatial organization achieved by balancing the cell-cell and cell-matrix interactions compared to porous gels where cells are mainly clustered. ECs tend to form relatively elongated cellular networks in dense colloidal gel compared to porous gels. Additionally, the role of matrix stiffness and viscoelasticity in the morphogenesis of MSCs and ECs are analyzed with respect to microstructural morphology. Overall, these results demonstrate that colloidal gels can provide spatial cues through their microstructural morphology and in correlation with matrix mechanics for cell morphogenesis.

Spheroid Culture System, a Promising Method for Chondrogenic Differentiation of Dental Mesenchymal Stem Cells.

The objective of the present work was to develop a three-dimensional culture model to evaluate, in a short period of time, cartilage tissue engineering protocols. The spheroids were compared with the gold standard pellet culture. The dental mesenchymal stem cell lines were from pulp and periodontal ligament. The evaluation used RT-qPCR and Alcian Blue staining of the cartilage matrix. This study showed that the spheroid model allowed for obtaining greater fluctuations of the chondrogenesis markers than for the pellet one. The two cell lines, although originating from the same organ, led to different biological responses. Finally, biological changes were detectable for short periods of time. In summary, this work demonstrated that the spheroid model is a valuable tool for studying chondrogenesis and the mechanisms of osteoarthritis, and evaluating cartilage tissue engineering protocols.

Pre-culture of human mesenchymal stromal cells in spheroids facilitates chondrogenesis at a low total cell count upon embedding in biomaterials to generate cartilage microtissues.

Material-assisted cartilage tissue engineering has limited application in cartilage treatment due to hypertrophic tissue formation and high cell counts required. This study aimed at investigating the potential of human mesenchymal stromal cell (hMSC) spheroids embedded in biomaterials to study the effect of biomaterial composition on cell differentiation. Pre-cultured (3 days, chondrogenic differentiation media) spheroids (250 cells/spheroid) were embedded in tyramine-modified hyaluronic acid (THA) and collagen type I (Col) composite hydrogels (four combinations of THA (12.5 vs 16.7 mg/ml) and Col (2.5 vs 1.7 mg/ml) content) at a cell density of 5 × 106 cells/ml (2 × 104 spheroids/ml). Macropellets derived from single hMSCs (2.5 × 105 cells, ScMP) or hMSC spheroids (2.5 × 105 cells, 103 spheroids, SpMP) served as control. hMSC differentiation was analyzed using glycosaminoglycan (GAG) quantification, gene expression analysis and (immuno-)histology. Embedding of hMSC spheroids in THA-Col induced chondrogenic differentiation marked by upregulation of aggrecan (ACAN) and COL2A1, and the production of GAGs . Lower THA led to more pronounced chondrogenic phenotype compared to higher THA content. Col content had no significant influence on hMSC chondrogenesis. Pellet cultures showed an upregulation in chondrogenic-associated genes and production of GAGs with less upregulation of hypertrophic-associated genes in SpMP culture compared to ScMP group. This study presents hMSC pre-culture in spheroids as promising approach to study chondrogenic differentiation after biomaterial encapsulation at low total cell count (5 × 106/ml) without compromising chondrogenic matrix production. This approach can be applied to assemble microtissues in biomaterials to generate large cartilage construct. STATEMENT OF SIGNIFICANCE: In vitro studies investigating the chondrogenic potential of biomaterials are limited due to the low cell-cell contact of encapsulated single cells. Here, we introduce the use of pre-cultured hMSC spheroids to study chondrogenesis upon encapsulation in a biomaterial. The use of spheroids takes advantage of the high cell-cell contact within each spheroid being critical in the early chondrogenesis of hMSCs. At a low seeding density of 5·106 cells/ml (2 × 104 spheroids/ml) we demonstrated hMSC chondrogenesis and cartilaginous matrix deposition. Our results indicate that the pre-culture might have a beneficial effect on hypertrophic gene expression without compromising chondrogenic differentiation. This approach has shown potential to assemble microtissues (here spheroids) in biomaterials to generate large cartilage constructs and to study the effect of biomaterial composition on cell alignment and migration.