A Multi-functional Hybrid System Comprised of Polydopamine Nanobottles and Biological Effectors for Cartilage Repair.

Biological effectors play critical roles in augmenting the repair of cartilage injuries, but it remains a challenge to control their release in a programmable, stepwise fashion. Herein, a hybrid system consisting of polydopamine (PDA) nanobottles embedded in a hydrogel matrix to manage the release of biological effectors for use in cartilage repair is reported. Specifically, a homing effector is load in the hydrogel matrix, together with the encapsulation of a cartilage effector in PDA nanobottles filled with phase-change material. In action, the homing effector is quickly released from the hydrogel in the initial step to recruit stem cells from the surroundings. Owing to the antioxidation effect of PDA, the recruited cells are shielded from reactive oxygen species. The cartilage effector is then slowly released from the nanobottles to promote chondrogenic differentiation, facilitating cartilage repair. Altogether, this strategy encompassing recruitment, protection, and differentiation of stem cells offers a viable route to tissue repair or regeneration through stem cell therapy.

Suppression of MALAT1 promotes human synovial mesenchymal stem cells enhance chondrogenic differentiation and prevent osteoarthritis of the knee in a rat model via regulating miR-212-5p/MyD88 axis.

Osteoarthritis (OA) is one of the most common diseases of the skeleton. Long non-coding RNAs (lncRNAs) are emerging as key players in OA pathogenesis. This work sets out to determine the function of lncRNA MALAT1 in OA and the mechanisms by which it does so. Mesenchymal stem cells isolated from the human synovial membrane are called hSMSCs. The hSMSCs' surface markers were studied using flow cytometry. To determine whether or not hSMSC might differentiate, researchers used a number of different culture settings and labeling techniques. The expression levels of associated genes and proteins were determined using quantitative real-time polymerase chain reaction (RT-qPCR), western blotting (WB), and immunostaining. A dual luciferase reporter experiment and RNA immunoprecipitation (RIP) test demonstrated the direct association between miR-212-5p and MALAT1 or MyD88. MALAT1 was downregulated during the chondrogenic differentiation of hSMSCs, and underexpression of MALAT1 promotes chondrogenesis in hSMSCs. Using dual luciferase reporter and RIP assays facilitated the identification of MALAT1 as a competitive endogenous RNA (ceRNA) that sequesters miR-212-5p. Additionally, the expression of MYD88 was regulated by MALAT1 through direct binding with miR-212-5p. Significantly, the effects of MALAT1 on the chondrogenic differentiation of hSMSCs were counteracted by miR-212-5p/MYD88. Furthermore, our in vivo investigation revealed that the inhibition of MALAT1 mitigated osteoarthritis progression in rat models. In conclusion, the promotion of chondrogenic differentiation in hSMSCs and the protective effect on cartilage tissue in OA can be achieved by suppressing MALAT1, which regulates the miR-212-5p/MyD88 axis.

MiR-143-3p regulates chondrogenic differentiation of synovium derived mesenchymal stem cells under mechanical stress through the BMPR2-Smad signalling pathway by targeting BMPR2.

BACKGROUND: Mesenchymal stem cells (MSCs) derived from the synovium, known as synovium mesenchymal stem cells (SMSCs), exhibit significant potential for articular cartilage regeneration owing to their capacity for chondrogenic differentiation. However, the microRNAs (miRNAs) governing this process and the associated mechanisms remain unclear. While mechanical stress positively influences chondrogenesis in MSCs, the miRNA-mediated response of SMSCs to mechanical stimuli is not well understood. OBJECTIVE: This study explores the miRNA-driven mechano-transduction in SMSCs chondrogenesis under mechanical stress. METHODS: The surface phenotype of SMSCs was analysed by flow cytometry. Chondrogenesis capacities of SMSCs were examined by Alcian blue staining. High throughput sequencing was used to screen mechano-sensitive miRNAs of SMSCs. The RNA expression level of COL2A1, ACAN, SOX9, BMPR2 and miR-143-3p of SMSCs were tested by quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between miR-143-3p and TLR4 was confirmed by luciferase reporter assays. The protein expression levels of related genes were assessed by western blot. RESULTS: High-throughput sequencing revealed a notable reduction in miR-143-3p levels in mechanically stressed SMSCs. Gain- or loss-of-function strategies introduced by lentivirus demonstrated that miR-143-3p overexpression hindered chondrogenic differentiation, whereas its knockdown promoted this process. Bioinformatics scrutiny and luciferase reporter assays pinpointed a potential binding site for miR-143-3p within the 3'-UTR of bone morphogenetic protein receptor type 2 (BMPR2). MiR-143-3p overexpression decreased BMPR2 expression and phosphorylated Smad1, 5 and 8 levels, while its inhibition activated BMPR2-Smad pathway. CONCLUSION: This study elucidated that miR-143-3p negatively regulates SMSCs chondrogenic differentiation through the BMPR2-Smad pathway under mechanical tensile stress. The direct targeting of BMPR2 by miR-143-3p established a novel dimension to our understanding of mechano-transduction mechanism during SMSC chondrogenesis. This understanding is crucial for advancing strategies in articular cartilage regeneration.

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.

Selenium Deficiency Can Promote the Expression of VEGF and Inflammatory Factors in Cartilage Differentiation and Mediates Cartilage Injury.

Selenium plays a crucial role as a micronutrient, primarily exerting its biological functions through selenoproteins. It has been established that selenium deficiency adversely impacts cartilage development, leading to alterations in chondrocyte function. In regions with low selenium intake, endemic osteochondrosis has been documented, characterized by compromised growth plate and articular cartilage formation. Vascular endothelial growth factor (VEGF) stands out as a pivotal angiogenic factor, with elevated levels contributing significantly to vascular invasion into chondrocytes. This VEGF-mediated invasion serves as a key signal, prompting morphological changes in the growth plate and initiating cartilage remodeling. In animal models, the selenium deficiency group exhibited heightened levels of the cartilage damage marker matrix metalloproteinases 13 (MMP13). This resulted in articular cartilage degeneration, accompanied by a substantial increase in VEGF expression within the growth plate and articular cartilage, as compared to the normal group. In a chondrogenic progenitor cell (CPC) differentiation model, insufficient selenium induced chondrocyte damage and upregulated inflammatory factors such as inducible NO synthase (iNOS) and cyclooxygenase-2 (COX2). The selenium-deficient groups showed elevated expressions of VEGF, VEGFR2, MMP13, Collagen X, and Angiopoietin 1, accelerating the degradation of the extracellular matrix (ECM), which further promoted the development of cartilage-related diseases. Taken together, these findings provide novel insights for a better understanding of the role of low selenium in cartilage degeneration and angiogenesis. They shed light on the intricate influence of low selenium levels on the development of articular cartilage, emphasizing the interconnected pathways and processes involved.

Biomimetic kartogenin containing κ-carrageenan hydrogel for nucleus pulposus regeneration.

Intervertebral disc degeneration is a clinical disease that reduces the quality of patient's life. The degeneration usually initiates in the nucleus pulposus (NP), hence the use of hydrogels represents a promising therapeutic approach. However, the viscoelastic nature of hydrogel and its ability to provide biomimetic architecture and biochemical cues influence the regeneration capability. This study focused on tuning the physical nature of a glycosaminoglycan hydrogel (κ-carrageenan) as well as the release kinetics of a chondrogenic factor (kartogenin - KGN) through physical cross-linking. For this, κ-carrageenan was cross linked with 2.5 % and 5 % potassium chloride (KCl) for 15 and 30 min and loaded with KGN molecule at 50 µM and 100 µM. The tight network structure with low water retention and degradation property was seen in hydrogel cross-linked with increased KCl concentration and time. However, optimal degradation along with NP mimicking viscoelastic nature was exhibited by 5 wt% KCl treated hydrogel (H3 hydrogel). All hydrogel groups exhibited burst KGN release at 24 h followed by a sustained release for 5 days. However, hydrogel cross-linked with 5 wt% KCl enhanced chondrogenic differentiation, mainly at lower KGN dose. In summary, this study shows the potential application of biomimetic KGN laden carrageenan hydrogel in NP regeneration.

Mesenchymal stem cells for cartilage regeneration: Insights into molecular mechanism and therapeutic strategies.

The use of mesenchymal stem cells (MSCs) in cartilage regeneration has gained significant attention in regenerative medicine. This paper reviews the molecular mechanisms underlying MSC-based cartilage regeneration and explores various therapeutic strategies to enhance the efficacy of MSCs in this context. MSCs exhibit multipotent capabilities and can differentiate into various cell lineages under specific microenvironmental cues. Chondrogenic differentiation, a complex process involving signaling pathways, transcription factors, and growth factors, plays a pivotal role in the successful regeneration of cartilage tissue. The chondrogenic differentiation of MSCs is tightly regulated by growth factors and signaling pathways such as TGF-ß, BMP, Wnt/ß-catenin, RhoA/ROCK, NOTCH, and IHH (Indian hedgehog). Understanding the intricate balance between these pathways is crucial for directing lineage-specific differentiation and preventing undesirable chondrocyte hypertrophy. Additionally, paracrine effects of MSCs, mediated by the secretion of bioactive factors, contribute significantly to immunomodulation, recruitment of endogenous stem cells, and maintenance of chondrocyte phenotype. Pre-treatment strategies utilized to potentiate MSCs, such as hypoxic conditions, low-intensity ultrasound, kartogenin treatment, and gene editing, are also discussed for their potential to enhance MSC survival, differentiation, and paracrine effects. In conclusion, this paper provides a comprehensive overview of the molecular mechanisms involved in MSC-based cartilage regeneration and outlines promising therapeutic strategies. The insights presented contribute to the ongoing efforts in optimizing MSC-based therapies for effective cartilage repair.

Cytokine Receptor-like Factor 1 (CRLF1) and Its Role in Osteochondral Repair.

BACKGROUND: Since cytokine receptor-like factor 1 (CRLF1) has been implicated in tissue regeneration, we hypothesized that CRLF1 released by mesenchymal stem cells can promote the repair of osteochondral defects. METHODS: The degree of a femoral osteochondral defect repair in rabbits after intra-articular injections of bone marrow-derived mesenchymal stem cells (BMSCs) that were transduced with empty adeno-associated virus (AAV) or AAV containing CRLF1 was determined by morphological, histological, and micro computer tomography (CT) analyses. The effects of CRLF1 on chondrogenic differentiation of BMSCs or catabolic events of interleukin-1beta-treated chondrocyte cell line TC28a2 were determined by alcian blue staining, gene expression levels of cartilage and catabolic marker genes using real-time PCR analysis, and immunoblot analysis of Smad2/3 and STAT3 signaling. RESULTS: Intra-articular injections of BMSCs overexpressing CRLF1 markedly improved repair of a rabbit femoral osteochondral defect. Overexpression of CRLF1 in BMSCs resulted in the release of a homodimeric CRLF1 complex that stimulated chondrogenic differentiation of BMSCs via enhancing Smad2/3 signaling, whereas the suppression of CRLF1 expression inhibited chondrogenic differentiation. In addition, CRLF1 inhibited catabolic events in TC28a2 cells cultured in an inflammatory environment, while a heterodimeric complex of CRLF1 and cardiotrophin-like Cytokine (CLC) stimulated catabolic events via STAT3 activation. CONCLUSION: A homodimeric CRLF1 complex released by BMSCs enhanced the repair of osteochondral defects via the inhibition of catabolic events in chondrocytes and the stimulation of chondrogenic differentiation of precursor cells.

Unlocking Potential: Low Bovine Serum Albumin Enhances the Chondrogenicity of Human Adipose-Derived Stromal Cells in Pellet Cultures.

Bovine serum albumin (BSA) plays a crucial role in cell culture media, influencing cellular processes such as proliferation and differentiation. Although it is commonly included in chondrogenic differentiation media, its specific function remains unclear. This study explores the effect of different BSA concentrations on the chondrogenic differentiation of human adipose-derived stromal/stem cells (hASCs). hASC pellets from six donors were cultured under chondrogenic conditions with three BSA concentrations. Surprisingly, a lower BSA concentration led to enhanced chondrogenesis. The degree of this effect was donor-dependent, classifying them into two groups: (1) high responders, forming at least 35% larger, differentiated pellets with low BSA in comparison to high BSA; (2) low responders, which benefitted only slightly from low BSA doses with a decrease in pellet size and marginal differentiation, indicative of low intrinsic differentiation potential. In all cases, increased chondrogenesis was accompanied by hypertrophy under low BSA concentrations. To the best of our knowledge, this is the first study showing improved chondrogenicity and the tendency for hypertrophy with low BSA concentration compared to standard levels. Once the tendency for hypertrophy is understood, the determination of BSA concentration might be used to tune hASC chondrogenic or osteogenic differentiation.

Electrical Stimulation of Mesenchymal Stem Cells as a Tool for Proliferation and Differentiation in Cartilage Tissue Engineering: A Scaffold-Based Approach.

Electrical stimulation (ES) is a widely discussed topic in the field of cartilage tissue engineering due to its ability to induce chondrogenic differentiation (CD) and proliferation. It shows promise as a potential therapy for osteoarthritis (OA). In this study, we stimulated mesenchymal stem cells (MSCs) incorporated into collagen hydrogel (CH) scaffolds, consisting of approximately 500,000 cells each, for 1 h per day using a 2.5 Vpp (119 mV/mm) 8 Hz sinusoidal signal. We compared the cell count, morphology, and CD on days 4, 7, and 10. The results indicate proliferation, with an increase ranging from 1.86 to 9.5-fold, particularly on day 7. Additionally, signs of CD were observed. The stimulated cells had a higher volume, while the stimulated scaffolds showed shrinkage. In the ES groups, up-regulation of collagen type 2 and aggrecan was found. In contrast, SOX9 was up-regulated in the control group, and MMP13 showed a strong up-regulation, indicating cell stress. In addition to lower stress levels, the control groups also showed a more spheroidic shape. Overall, scaffold-based ES has the potential to achieve multiple outcomes. However, finding the appropriate stimulation pattern is crucial for achieving successful chondrogenesis.

Injectable Bioadhesive Photocrosslinkable Hydrogels with Sustained Release of Kartogenin to Promote Chondrogenic Differentiation and Partial-Thickness Cartilage Defects Repair.

Partial-thickness cartilage defect (PTCD) is a common and formidable clinical challenge without effective therapeutic approaches. The inherent anti-adhesive characteristics of the extracellular matrix within cartilage pose a significant impediment to the integration of cells or biomaterials with the native cartilage during cartilage repair. Here, an injectable photocrosslinked bioadhesive hydrogel, consisting of gelatin methacryloyl (GM), acryloyl-6-aminocaproic acid-g-N-hydroxysuccinimide (AN), and poly(lactic-co-glycolic acid) microspheres loaded with kartogenin (KGN) (abbreviated as GM/AN/KGN hydrogel), is designed to enhance interfacial integration and repair of PTCD. After injected in situ at the irregular defect, a stable and robust hydrogel network is rapidly formed by ultraviolet irradiation, and it can be quickly and tightly adhered to native cartilage through amide bonds. The hydrogel exhibits good adhesion strength up to 27.25 ± 1.22 kPa by lap shear strength experiments. The GM/AN/KGN hydrogel demonstrates good adhesion, low swelling, resistance to fatigue, biocompatibility, and chondrogenesis properties in vitro. A rat model with PTCD exhibits restoration of a smoother surface, stable seamless integration, and abundant aggrecan and type II collagen production. The injectable stable adhesive hydrogel with long-term chondrogenic differentiation capacity shows great potential to facilitate repair of PTCD.

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.

Ocu-miR-10a-5p promotes the chondrogenic differentiation of rabbit BMSCs by targeting BTRC-mediated Wnt/ß-catenin signaling pathway.

MicroRNAs (miRNAs) play an important role in articular cartilage damage in osteoarthritis (OA). However, the biological role of miRNAs in the chondrogenic differentiation of bone marrow mesenchymal stem cell (BMSC) remains largely unclear. Rabbit bone marrow mesenchymal stem cells (rBMSCs) were isolated, cultured, and identified. Afterwards, rBMSCs were induced to chondrogenic differentiation, examined by Alcian Blue staining. Differentially expressed miRNAs were identified in rBMSCs between induced and non-induced groups by miRNA sequencing analysis, part of which was validated via PCR assay. Cell viability and apoptosis were assessed by CCK-8 assay and Hoechst staining. Saffron O staining was utilized to assess chondrocyte hyperplasia. The expression of specific chondrogenic markers, including COL2A1, SOX9, Runx2, MMP-13, Aggrecan, and BMP-2, were measured at mRNA and protein levels. The association between beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC) and miR-10a-5p in the miRNA family from rabbit (ocu-miR-10a-5p) was determined by luciferase reporter assay. A total of 76 differentially expressed miRNAs, including 52 downregulated and 24 upregulated miRNAs, were identified in rBMSCs from the induced group. Inhibition of ocu-miR-10a-5p suppressed rBMSC viability and chondrogenic differentiation, as well as downregulated the expression of ß-catenin, SOX9, COL2A1, MMP-13, and Runx2. BTRC was predicted and confirmed as a target of ocu-miR-10a-5p. Overexpression of BTRC rescued the promoting impacts of overexpressed ocu-miR-10a-5p on chondrogenic differentiation of rBMSCs and ß-catenin expression. Taken together, our data suggested that ocu-miR-10a-5p facilitated rabbit BMSC survival and chondrogenic differentiation by activating Wnt/ß-catenin signaling through BTRC.

Reduced Cell Migration in Human Chondrocyte Sheets Increases Tissue Stiffness and Cartilage Protein Production.

BACKGROUND: Chondrogenic differentiation medium (CDM) is usually used to maintain chondrogenic activity during chondrocyte sheet production. However, tissue qualities remain to be determined as to what factors improve cell functions. Moreover, the relationship between CDM and cell migration proteins has not been reported. METHOD: In this study, the effect of CDM on the behavior of chondrocyte sheets was investigated. Structural analysis, mechanical testing and proteomics were performed to observe tissue qualities. The relationship between CDM and cell migration proteins were investigated using time-lapse observations and bioinformatic analysis. RESULTS: During 48 h, CDM affected the chondrocyte behaviors by reducing cell migration. Compared to the basal medium, CDM impacted the contraction of monolayered chondrocyte sheets. At day 7, the contracted sheets increased tissue thickness and improved tissue stiffness. Cartilage specific proteins were also upregulated. Remarkedly, the chondrocyte sheets in CDM displayed downregulated proteins related to cell migration. Bioinformatic analysis revealed that TGFß1 was shown to be associated with cartilage functions and cell migration. Pathway analysis of chondrocyte sheets in CDM also revealed the presence of a TGFß pathway without activating actin production, which might be involved in synthesizing cartilage-specific proteins. Cell migration pathway showed MAPK signaling in both cultures of the chondrocyte sheets. CONCLUSION: Reduced cell migration in the chondrocyte sheet affected the tissue quality. Using CDM, TGFß1 might trigger cartilage protein production through the TGFß pathway and be involved in cell migration via the MAPK signaling pathway. Understanding cell behaviors and their protein expression would be beneficial for developing high-quality tissue-engineered cartilage.

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.

Electromagnetic field-mediated chitosan/gelatin/nano-hydroxyapatite and bone-derived scaffolds regulate the osteoblastic and chondrogenic phenotypes of adipose-derived stem cells to construct osteochondral tissue engineering niche in vitro.

It is critical to explore the effects of electromagnetic field (EMF) on the construction of functional osteochondral tissue, which has shown certain clinical significance for the treatment of osteochondral injury. At present, there are few studies on the effect of the direction of EMF on cells. This study aimed to investigate the effects of EMF coupling on different parameters to control adipose-derived stem cells (ADSCs) proliferation and specific chondrogenic and osteogenic differentiation at 2D level and 3D level. The proliferation and differentiation of EMF-induced ADSCs are jointly regulated by EMF and space structure. In this study, Cs7/Gel3/nHAP scaffolds were prepared with good degradation rate (86.75 ± 4.96 %) and absorb water (1100 %), and the pore size was 195.63 ± 54.72 µm. The bone-derived scaffold with a pore size of 267.17 ± 129.18 µm was obtained and its main component was hydroxyapatite. Cs7/Gel3/nHAP scaffolds and bone-derived scaffolds are suitable as 3D level materials. The optimal EMF intensity was 2 mT for chondrogenic differentiation and proliferation and 1 mT for osteogenic differentiation and proliferation. It is noteworthy that EMF has a negative correlation with ADSCs proliferation in the vertical direction at 2D level, while it has a positive correlation with ADSCs proliferation at 3D level. EMF mediated 3D osteochondral scaffold provide good strategy for osteochondral tissue engineering construction.

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.

Fabrication and Characterization of Porous PEGDA Hydrogels for Articular Cartilage Regeneration.

Functional articular cartilage regeneration remains an unmet medical challenge, increasing the interest for innovative biomaterial-based tissue engineering (TE) strategies. Hydrogels, 3D macromolecular networks with hydrophilic groups, present articular cartilage-like features such as high water content and load-bearing capacity. In this study, 3D porous polyethylene glycol diacrylate (PEGDA) hydrogels were fabricated combining the gas foaming technique and a UV-based crosslinking strategy. The 3D porous PEGDA hydrogels were characterized in terms of their physical, structural and mechanical properties. Our results showed that the size of the hydrogel pores can be modulated by varying the initiator concentration. In vitro cytotoxicity tests showed that 3D porous PEGDA hydrogels presented high biocompatibility both with human chondrocytes and osteoblast-like cells. Importantly, the 3D porous PEGDA hydrogels supported the viability and chondrogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cell (hBM-MSC)-based spheroids as demonstrated by the positive staining of typical cartilage extracellular matrix (ECM) (glycosaminoglycans (GAGs)) and upregulation of chondrogenesis marker genes. Overall, the produced 3D porous PEGDA hydrogels presented cartilage-like mechanical properties and supported MSC spheroid chondrogenesis, highlighting their potential as suitable scaffolds for cartilage TE or disease modelling strategies.

Evaluation of a Peptide Hydrogel as a Chondro-Instructive Three-Dimensional Microenvironment.

Articular cartilage injuries are inherently irreversible, even with the advancement in current therapeutic options. Alternative approaches, such as the use of mesenchymal stem/stromal cells (MSCs) and tissue engineering techniques, have gained prominence. MSCs represent an ideal source of cells due to their low immunogenicity, paracrine activity, and ability to differentiate. Among biomaterials, self-assembling peptide hydrogels (SAPH) are interesting given their characteristics such as good biocompatibility and tunable properties. Herein we associate human adipose-derived stem cells (hASCs) with a commercial SAPH, Puramatrix™, to evaluate how this three-dimensional microenvironment affects cell behavior and its ability to undergo chondrogenic differentiation. We demonstrate that the Puramatrix™ hydrogel comprises a highly porous matrix permissible for hASC adhesion and in vitro expansion. The morphology and cell growth dynamics of hASCs were affected when cultured on the hydrogel but had minimal alteration in their immunophenotype. Interestingly, hASCs spontaneously formed cell aggregates throughout culturing. Analysis of glycosaminoglycan production and gene expression revealed a noteworthy and donor-dependent trend suggesting that Puramatrix™ hydrogel may have a natural capacity to support the chondrogenic differentiation of hASCs. Altogether, the results provide a more comprehensive understanding of the potential applications and limitations of the Puramatrix™ hydrogel in developing functional cartilage tissue constructs.