Expression Of Chondrogenic Markers Research Articles

Senolytic therapy combining Dasatinib and Quercetin restores the chondrogenic phenotype of human osteoarthritic chondrocytes by the release of pro-anabolic mediators.

Cellular senescence is associated with various age-related disorders and is assumed to play a major role in the pathogenesis of osteoarthritis (OA). Based on this, we tested a senolytic combination therapy using Dasatinib (D) and Quercetin (Q) on aged isolated human articular chondrocytes (hACs), as well as in OA-affected cartilage tissue (OARSI grade 1-2). Stimulation with D + Q selectively eliminated senescent cells in both, cartilage explants and isolated hAC. Furthermore, the therapy significantly promoted chondroanabolism, as demonstrated by increased gene expression levels of COL2A1, ACAN, and SOX9, as well as elevated collagen type II and glycosaminoglycan biosynthesis. Additionally, D + Q treatment significantly reduced the release of SASP factors (IL6, CXCL1). RNA sequencing analysis revealed an upregulation of the anabolic factors, inter alia, FGF18, IGF1, and TGFB2, as well as inhibitory effects on cytokines and the YAP-1 signaling pathway, explaining the underlying mechanism of the chondroanabolic promotion upon senolytic treatment. Accordingly, stimulation of untreated hAC with conditioned medium of D + Q-treated cells similarly induced the expression of chondrogenic markers. Detailed analyses demonstrated that chondroanabolic effects could be mainly attributed to Dasatinib, while monotherapeutical application of Quercetin or Navitoclax did not promote the chondroanabolism. Overall, D + Q therapy restored the chondrogenic phenotype in OA hAC most likely by creating a pro-chondroanabolic environment through the reduction of SASP factors and upregulation of growth factors. This senolytic approach could therefore be a promising candidate for further testing as a disease-modifying osteoarthritis drug.

Human umbilical cord mesenchymal stem cells on treating osteoarthritis in a rabbit model: Injection strategies

Human umbilical cord mesenchymal stem cells (UCMSCs) are a novel stem-cell source to treat osteoarthritis (OA). Here we investigated the therapeutic effects of UCMSCs injection strategies on knee OA in a rabbit model. Thirty OA rabbits randomly received normal saline, a single dose of 1 × 106 UCMSCs, or three injections of 1 × 106 UCMSCs at 2, 4, 6 weeks. Articular cartilages were collected after 8 weeks. Macroscopic and histological assessments indicated that intra-articular injection of UCMSCs, both single and multiple injection, significantly reduced the formation of periarticular osteophytes and articular cartilage degeneration when compared with the control. Furthermore, both UCMSCs injections increased the expression of chondrogenic markers in the articular cartilage, and reduced the levels of TNF-α and IL-6 in synovium. Micro-CT showed significant reduction of sub-chondral bone degeneration and osteophytes in the multiple-injection group compared to the control and single-injection group. Taken together, intra-articular injection of UCMSCs for OA treatment is safe and effective. Single and multiple injection of UCMSCs had comparable reparative effect on cartilage lesions, while multiple injection of UCMSCs further exerted effect on enhancing subchondral bone volume.

Aspirin attenuates the detrimental effects of TNF-α on BMMSC stemness by modulating the YAP-SMAD7 axis

BackgroundBone marrow mesenchymal stem cells (BMMSCs) are commonly used for cell transplantation to treat refractory diseases. However, the presence of inflammatory factors, such as tumour necrosis factor-alpha (TNF-α), at the transplantation site severely compromises the stemness of BMMSCs, thereby reducing the therapeutic effect of cell transplantation. Aspirin (AS) is a drug that has been in use for over a century and has a wide range of effects, including the regulation of cell proliferation, multidirectional differentiation, and immunomodulatory properties of stem cells. However, it is still unclear whether AS can delay the damaging effects of TNF-α on BMMSC stemness.MethodsThis study investigated the effects of AS and TNF-α on BMMSC stemness and the molecular mechanisms using colony formation assay, western blot, qRT-PCR, and overexpression or knockdown of YAP and SMAD7.ResultsThe results demonstrated that TNF-α inhibited cell proliferation, the expression of stemness, osteogenic and chondrogenic differentiation markers of BMMSCs. Treatment with AS was shown to mitigate the TNF-α-induced damage to BMMSC stemness. Mechanistic studies revealed that AS may reverse the damage caused by TNF-α on BMMSC stemness by upregulating YAP and inhibiting the expression of SMAD7.ConclusionAS can attenuate the damaging effects of TNF-α on BMMSC stemness by regulating the YAP-SMAD7 axis. These findings are expected to promote the application of AS to improve the efficacy of stem cell therapy.

Efficiently directing differentiation and homing of mesenchymal stem cells to boost cartilage repair in osteoarthritis via a nanoparticle and peptide dual-engineering strategy

Mesenchymal stem cells (MSCs) are expected to be useful therapeutics in osteoarthritis (OA), the most common joint disorder characterized by cartilage degradation. However, evidence is limited with regard to cartilage repair in clinical trials because of the uncontrolled differentiation and weak cartilage-targeting ability of MSCs after injection. To overcome these drawbacks, here we synthesized CuO@MSN nanoparticles (NPs) to deliver Sox9 plasmid DNA (favoring chondrogenesis) and recombinant protein Bmp7 (inhibiting hypertrophy). After taking up CuO@MSN/Sox9/Bmp7 (CSB NPs), the expressions of chondrogenic markers were enhanced while hypertrophic markers were decreased in response to these CSB-engineered MSCs. Moreover, a cartilage-targeted peptide (designated as peptide W) was conjugated onto the surface of MSCs via a click chemistry reaction, thereby prolonging the residence time of MSCs in both the knee joint cavity of mice and human-derived cartilage. In a surgery-induced OA mouse model, the NP and peptide dual-modified W-CSB-MSCs showed an enhancing therapeutic effect on cartilage repair in knee joints compared with other engineered MSCs after intra-articular injection. Most importantly, W-CSB-MSCs accelerated cartilage regeneration in damaged cartilage explants derived from OA patients. Thus, this new peptide and NPs dual engineering strategy shows potential for clinical applications to boost cartilage repair in OA using MSC therapy.

Influence of storage conditions on the viability of spheroids from human chondrocytes

BACKGROUND: The use of tissue-engineered products for treating hyaline cartilage injuries is currently a promising direction for regenerative medicine. The study of storage and transportation conditions of such preparations is essential in product development because it determines the viability and functional activity of the cellular component of the product. In addition, such work should be performed following the requirements of Russian legislation before using the developed drug product in clinical trials. AIM: To characterize the storage conditions for tissue-engineered preparations such as chondrospheres. METHODS: Chondrospheres were formed from human chondrocytes obtained from the biopsy material of the knee joint cartilage of patients diagnosed according to ICD-10 codes M15–M19. After the formation of chondrospheres, cell viability was examined using the metabolic dye PrestoBlue, and the expression of chondrogenic markers (SOX9, aggrecan, collagen type 1 and type 2) was preserved using real-time polymerase chain reaction under various transportation conditions, i.e., at different storage times, medium composition, and temperature. RESULTS: The analysis of the expression of chondrocyte markers confirmed that cells in chondrospheres retain a chondrocyte phenotype. In the cell viability study of tissue-engineered products under various conditions (temperature, solution, and time), the most favorable environment for storing the product was phosphate buffer saline and 0.9% sodium chloride solution (р 0.05). The metabolic activity of the cells that make up the tissue-engineered product was maintained when stored at +4 °C for up to 3 days (р 0.05). CONCLUSION: Isotonic solution and temperature regime +4 °C for not more than 2 days showed the preferable medium and conditions for storing and transporting the prototype cartilage implant as chondrospheres.

Enhanced chondrogenic potential in GelMA-based 3D cartilage model via Wnt3a surface immobilization

Cartilage tissue engineering aims to develop functional substitutes for treating cartilage defects and osteoarthritis. Traditional two-dimensional (2D) cell culture systems lack the complexity of native cartilage, leading to the development of 3D regenerative cartilage models. In this study, we developed a 3D model using Gelatin Methacryloyl (GelMA)-based hydrogels seeded with Y201 cells, a bone marrow mesenchymal stem cell line. The model investigated chondrogenic differentiation potential in response to Wnt3a stimulation within the GelMA scaffold and validated using known chondrogenic agonists. Y201 cells demonstrated suitability for the model, with increased proteoglycan content and upregulated chondrogenic marker expression under chondrogenic conditions. Wnt3a enhanced cell proliferation, indicating activation of the Wnt/β-catenin pathway, which plays a role in cartilage development. GelMA hydrogels provided an optimal scaffold, supporting cell viability and proliferation. The 3D model exhibited consistent responses to chondrogenic agonists, with TGF-β3 enhancing cartilage-specific extracellular matrix (ECM) production and chondrogenic differentiation. The combination of Wnt3a and TGF-β3 showed synergistic effects, promoting chondrogenic differentiation and ECM production. This study presents a 3D regenerative cartilage model with potential for investigating cartilage biology, disease mechanisms, and drug screening. The model provides insights into complex cartilage regeneration mechanisms and offers a platform for developing therapeutic approaches for cartilage repair and osteoarthritis treatment.

Low-density cultured cartilage cells expanded in platelet lysate present distinct features to develop an innovative clinical treatment for diffuse cartilage lesions.

Chondrocyte-based cell therapies are effective for the treatment of chondral lesions, but remain poorly indicated for diffuse lesions in the context of early osteoarthritis (OA). The aim of this study was to develop a protocol to obtain chondroprogenitor cells suitable for the treatment of diffuse chondral lesions within early OA. Cartilage cells were expanded at low density in human platelet lysate (hPL). A test was performed to exclude senescence. The expression of surface cluster of differentiation 146, cluster of differentiation 166, major histocompatibility complex (MHC)-Iand MHC-II and of genes of interest were evaluated, as well as the trophic potential of these cells, by the assessment of lubricin and matrix production. The immunomodulatory potential was assessed through their co-culture with macrophages. Cartilage cells expanded at low density in hPL showed higher proliferation rate than standard-density cells, no replicative senescence, low immunogenicityand expression of lubricin. Moreover, they presented an increased expression of chondrogenic and antihypertrophic markers, as well as a superior matrix deposition if compared to cells cultured at standard density. Cartilage cells induced on macrophages an upregulation of CD206, although a higher increase of CD163 expression was observed in the presence of low-density cells. These findings lay the grounds to explore the clinical usefulness of low-density cultured cartilage cells to treat diffuse lesions in early OA joints for both autologous and allogenic use. Not applicable.

Long noncoding RNA UCA1 promotes the chondrogenic differentiation of human bone marrow mesenchymal stem cells via regulating PARP1 ubiquitination.

Bone marrow mesenchymal stem cells (BMSCs) possess the potential to differentiate into cartilage cells. Long noncoding RNA (lncRNAs) urothelial carcinoma associated 1 (UCA1) has been confirmed to improve the chondrogenic differentiation of marrow mesenchymal stem cells (MSCs). Herein, we further investigated the effects and underlying mechanisms of these processes. The expression of UCA1 was positively associated with chondrogenic differentiation and the knockdown of UCA1 has been shown to attenuate the expression of chondrogenic markers. RNA pull-down assay and RNA immunoprecipitation showed that UCA1 could directly bind to PARP1 protein. UCA1 could improve PARP1 protein via facilitating USP9X-mediated PARP1 deubiquitination. Then these processes stimulated the NF-κB signaling pathway. In addition, PARP1 was declined in UCA1 knockdown cells, and silencing of PARP1 could diminish the increasing effects of UCA1 on the chondrogenic differentiation from MSCs and signaling pathway activation. Collectively, these outcomes suggest that UCA1 could act as a mediator of PARP1 protein ubiquitination and develop the chondrogenic differentiation of MSCs.

Modulation of Canine Adipose-Derived Mesenchymal Stem/Medicinal Signalling Cells with Ascorbic Acid: Effect on Proliferation and Chondrogenic Differentiation on Standard Plastic and Silk Fibroin Surfaces.

Ascorbic acid (AA) plays a crucial role in both the proliferation and chondrogenic differentiation potential of mesenchymal stem/medicinal signalling cells (MSCs); these are both key aspects of their general therapeutic use and their increasing use in veterinary medicine. Current immunomodulatory therapies require efficient expansion of MSCs in the laboratory, while emerging tissue regeneration strategies, such as cartilage or bone repair, aim to use differentiated MSCs and modulate the expression of chondrogenic and hypertrophic markers. Our aim was to investigate whether the addition of AA to the growth medium enhances the proliferation of canine adipose-derived MSCs (cAMSCs) grown on standard plastic surfaces and whether it affects chondrogenic differentiation potential on silk fibroin (SF) films. We assessed cell viability with trypan blue and proliferation potential by calculating population doubling. Chondrogenic induction on SF films was assessed by Alcian blue staining and gene expression analysis of chondrogenic and hypertrophic genes. The results showed that growth medium with AA significantly enhanced the proliferation of cAMSCs without affecting cell viability and modulated the expression of chondrogenic and hypertrophic genes of cAMSCs grown on SF films. Our results suggest that AA may be used in growth medium for expansion of cAMSCs and, at the same time, provide the basis for future studies to investigate the role of AA and SF in chondrogenic differentiation of MSCs.

Expression of Chondrogenic Potential Markers in Cultured Chondrocytes from the Human Knee Joint.

While substantial progress has been made in engineering cartilaginous constructs for animal models, further research is needed to translate these methodologies for human applications. Evidence suggests that cultured autologous chondrocytes undergo changes in phenotype and gene expression, thereby affecting their proliferation and differentiation capacity. This study was designed to evaluate the expression of chondrogenic markers in cultured human articular chondrocytes from passages 3 (P3) and 7 (P7), beyond the current clinical recommendation of P3. Cultured autologous chondrocytes were passaged from P3 up to P7, and quantitative polymerase chain reaction (qPCR) was used to assess mRNA expression of chondrogenic markers, including collagen type I (COLI), collagen type II (COLII), aggrecan (AGG), bone morphogenetic protein 4 (BMP4), transcription factor SOX-9 (SOX9), proteoglycan 4 (PGR4), and transformation-related protein 53 (p53), between P3 and P7. Except for AGG, no significant differences were found in the expression of markers between passages, suggesting the maintenance of chondrogenic potential in cultured chondrocytes. Differential expression identified between SOX9 and PGR4, as well as between COLI and SOX9, indicates that differences in chondrogenic markers are present between age groups and sexes, respectively. Overall, expression profiles of younger and male chondrocytes exhibit conversion of mature cartilage characteristics compared to their counterparts, with signs of dedifferentiation and loss of phenotype within-group passaging. These results may have implications in guiding the use of higher passaged chondrocytes for engineering constructs and provide a foundation for clinical recommendations surrounding the repair and treatment of articular cartilage pathology in both sexes.

Ocu-miR-10a-5p promotes the chondrogenic differentiation of rabbit BMSCs by targeting BTRC-mediated Wnt/β-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 β-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 β-catenin expression. Taken together, our data suggested that ocu-miR-10a-5p facilitated rabbit BMSC survival and chondrogenic differentiation by activating Wnt/β-catenin signaling through BTRC.

Increased Wnt5a/ROR2 signaling is associated with chondrogenesis in meniscal degeneration.

The aim of the present study was to investigate the association between chondrogenic differentiation and Wnt signal expression in the degenerative process of the human meniscus. Menisci were obtained from patients with and without knee osteoarthritis (OA), and degeneration was histologically assessed using a grading system. Immunohistochemistry, real-time polymerase chain reaction (PCR), and Western blot analysis were performed to examine the expressions of chondrogenic markers and of the components of Wnt signaling. Histological analyses showed that meniscal degeneration involved a transition from a fibroblastic to a chondrogenic phenotype with the upregulation of SOX9, collagen type II, collagen type XI, and aggrecan, which were associated with increased Wnt5a and ROR2 and decreased TCF7 expressions. OA menisci showed significantly higher expressions of Wnt5a and ROR2 and significantly lower expressions of AXIN2 and TCF7 than non-OA menisci on real-time PCR and Western blot analysis. These results potentially demonstrated that increased expression of Wnt5a/ROR2 signaling promoted chondrogenesis with decreased expression in downstream Wnt/β-catenin signaling. This study provides insights into the role of Wnt signaling in the process of meniscal degeneration, shifting to a chondrogenic phenotype. The findings suggested that the increased expression of Wnt5a/ROR2 and decreased expression of the downstream target of Wnt/β-catenin signaling are associated with chondrogenesis in meniscal degeneration.

One-step stromal vascular fraction therapy in osteoarthritis with tropoelastin-enhanced autologous stromal vascular fraction gel.

Background: Osteoarthritis (OA) is a debilitating degenerative joint disease, leading to significant pain and disability. Despite advancements, current regenerative therapies, such as mesenchymal stem cells (MSCs), face challenges in clinical efficacy and ethical considerations. This study aimed to evaluate the therapeutic potential of stromal vascular fraction gel (SVF-gel) in comparison to available treatments like hyaluronic acid (HA) and adipose-derived stem cells (ADSCs) and to assess the enhancement of this potential by incorporating tropoelastin (TE). Methods: We conducted a comparative laboratory study, establishing an indirect co-culture system using a Transwell assay to test the effects of HA, ADSCs, SVF-gel, and TE-SVF-gel on osteoarthritic articular chondrocytes (OACs). Chondrogenic and hypertrophic markers were assessed after a 72-hour co-culture. SVF-gel was harvested from rat subcutaneous abdominal adipose tissue, with its mechanical properties characterized. Cell viability was specifically analyzed for SVF-gel and TE-SVF-gel. The in vivo therapeutic effectiveness was further investigated in a rat model of OA, examining MSCs tracking, effects on cartilage matrix synthesis, osteophyte formation, and muscle weight changes. Results: Cell viability assays revealed that TE-SVF-gel maintained higher cell survival rates than SVF-gel. In comparison to the control, HA, and ADSCs groups, SVF-gel and TE-SVF-gel significantly upregulated the expression of chondrogenic markers COL 2, SOX-9, and ACAN and downregulated the hypertrophic marker COL 10 in OACs. The TE-SVF-gel showed further improved expression of chondrogenic markers and a greater decrease in COL 10 expression compared to SVF-gel alone. Notably, the TE-SVF-gel treated group in the in vivo OA model exhibited the most MSCs on the synovial surface, superior cartilage matrix synthesis, increased COL 2 expression, and better muscle weight recovery, despite the presence of fewer stem cells than other treatments. Discussion: The findings suggest that SVF-gel, particularly when combined with TE, provides a more effective regenerative treatment for OA by enhancing the therapeutic potential of MSCs. This combination could represent an innovative strategy that overcomes limitations of current therapies, offering a new avenue for patient treatment. Further research is warranted to explore the long-term benefits and potential clinical applications of this combined approach.

An Advanced Bioconjugation Strategy for Covalent Tethering of TGFβ3 with Silk Fibroin Matrices and its Implications in the Chondrogenesis Profile of Human BMSCs and Human Chondrocytes: A Paradigm Shift in Cartilage Tissue Engineering.

The transforming growth factor-β class of cytokines plays a significant role in articular cartilage formation from mesenchymal condensation to chondrogenic differentiation. However, their exogenous addition to the chondrogenic media makes the protocol expensive. It reduces the bioavailability of the cytokine to the cells owing to their burst release. The present study demonstrates an advanced bioconjugation strategy to conjugate transforming growth factor-β3 (TGFβ3) with silk fibroin matrix covalently via a cyanuric chloride coupling reaction. The tethering and change in secondary conformation are confirmed using various spectroscopic analyses. To assess the functionality of the chemically modified silk matrix, human bone marrow-derived mesenchymal stem cells (hBMSCs) and chondrocytes are cultured for 28 days in a chondrogenic differentiation medium. Gene expression and histological analysis reveal enhanced expression of chondrogenic markers with intense Safranin-O and Alcian Blue staining in TGFβ3 conjugated silk matrices than where TGFβ3 is exogenously added to the media for both hBMSCs and chondrocytes. Therefore, this study successfully recapitulates the native niche of TGFβ3 and the role of the silk as a growth factor stabilizer. When cultured over TGFβ3 conjugated silk matrices, hBMSCs display increased proteoglycan secretion and maximum chondrogenic trait with attenuation of chondrocyte hypertrophy over human chondrocytes.

Mesenchymal Stem Cells Cultured in a 3D Microgel Environment Containing Platelet-Rich Plasma Significantly Modify Their Chondrogenesis-Related miRNA Expression.

The aim of this work is to study the effect of platelet factors on the differentiation of mesenchymal stem cells (MSCs) to hyaline cartilage chondrocytes in a three-dimensional environment. MSCs were cultured in a microgel environment with a chondrogenic medium. The microgel consisted of microspheres that combine gelatin and platelet-rich plasma (PRP). The gelatin/PRP microdroplets were produced by emulsion. The gelatin containing the microdroplets was enzymatically gelled, retaining PRP and, just before seeding the cells, platelets were activated by adding calcium chloride so that platelet growth factors were released into the culture media but not before. Platelet activation was analyzed before activation to rule out the possibility that the gelatin cross-linking process itself activated the platelets. The gene expression of characteristic chondrogenic markers and miRNA expression were analyzed in cells cultured in a differentiation medium and significant differences were found between gelation/PRP microgels and those containing only pure gelatin. In summary, the gelatin microspheres effectively encapsulated platelets that secreted and released factors that significantly contributed to cellular chondrogenic differentiation. At the same time, the microgel constituted a 3D medium that provided the cells with adherent surfaces and the possibility of three-dimensional cell-cell contact.

Involvement of Yes-Associated Protein 1 Activation in the Matrix Degradation of Human-Induced-Pluripotent-Stem-Cell-Derived Chondrocytes Induced by T-2 Toxin and Deoxynivalenol Alone and in Combination.

T-2 toxin and deoxynivalenol (DON) are two prevalent mycotoxins that cause cartilage damage in Kashin-Beck disease (KBD). Cartilage extracellular matrix (ECM) degradation in chondrocytes is a significant pathological feature of KBD. It has been shown that the Hippo pathway is involved in cartilage ECM degradation. This study aimed to examine the effect of YAP, a major regulator of the Hippo pathway, on the ECM degradation in the hiPS-derived chondrocytes (hiPS-Ch) model of KBD. The hiPS-Ch injury models were established via treatment with T-2 toxin/DON alone or in combination. We found that T-2 toxin and DON inhibited the proliferation of hiPS-Ch in a dose-dependent manner; significantly increased the levels of YAP, SOX9, and MMP13; and decreased the levels of COL2A1 and ACAN (all p values < 0.05). Immunofluorescence revealed that YAP was primarily located in the nuclei of hiPS-Ch, and its expression level increased with toxin concentrations. The inhibition of YAP resulted in the dysregulated expression of chondrogenic markers (all p values < 0.05). These findings suggest that T-2 toxin and DON may inhibit the proliferation of, and induce the ECM degradation, of hiPS-Ch mediated by YAP, providing further insight into the cellular and molecular mechanisms contributing to cartilage damage caused by toxins.

MICROENVIRONMENTAL DEPENDENCE OF PROPER CHONDROCYTIC MATURATION

Articular cartilage is a relatively hypoxic tissue with a unique extracellular matrix that is enriched with cations, resulting in an elevated interstitial fluid osmolarity. Several biomechanical and physicochemical stimuli are reported to influence chondrocyte metabolism. For regenerative in vitro applications, increasing the extracellular osmolarity above plasma level to more physiological valuesinduces chondrogenic marker expression and the differentiation of chondroprogenitor cells. Calcineurin inhibitor FK506 modulates the differentiation of primary chondrocytes under such conditions and its effect on cell proliferation, extracellular matrix quality, and BMP- and TGF-β signaling will be described. Supraphysiological osmolarity compromises chondrocyte proliferation, while physosmolarity or FK506 did not. Rather, the combination of the latter increased proteoglycan and collagen expression in chondrocytesin vitro and in situ, affecting expression of TGF-β-inducible protein TGFBI and chondrogenic (SOX9, Col2) as well as terminal differentiation markers (e.g., Col10). Surprisingly, expression of particularly minor collagens (e.g., Col9, Col11) was improved. Physiological osmolarity seems to promote terminal chondrogenic differentiation of progenitor cells through sensitization of TGF-β superfamily signaling at the type I receptor. While hyperosmolarity alone facilitates TGF-β superfamily signaling, FK506 seems to enhance signaling by releasing the FKBP12 break from the type I receptor to improve collagenous marker expression. Our data help explaining seemingly contradictory earlier findings and potentially benefit future cell-based cartilage repair strategies.

ENHANCING CARTILAGE TISSUE FORMATION IN GELMA/ALGINATE-TYRAMINE INTERPENETRATED NETWORKS (IPNS) WITH LOW INTENSITY PULSE ULTRASOUND STIMULATION (LIPUS)

In the native articular cartilage microenvironment, chondrocytes are constantly subjected to dynamic physical stimuli that maintains tissue homeostasis. They produce extra cellular matrix (ECM) components such as collagens (type II mainly, 50-75%), proteoglycans (10-30%) and other type of proteins1 . While collagen offers a large resistance in tension, proteoglycans are the responsible of the viscoelastic response under compression due to the negative charge they confer to the ECM allowing it to entrap a large amount of interstitial fluid. In pathologic states (e.g. osteoarthritis), this ECM is degenerated and the negative charge becomes unbalanced, losing the chondroprotective properties and resulting on an overloaded chondrocytes that further degenerate the matrix.Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) has been used to generate acoustic (pressure) waves that create bubbles that collapse with cells, inducing a stimulus that can modulate cell response2. This mechanical stimulation promotes the expression of type II collagen, type X collagen, aggrecan and TGF-β, appearing as a great strategy to regenerate cartilage. However, current strategies make use of extrinsic forces to stimulate cartilage formation overlooking the physico-chemical properties of the degenerated cartilage, resulting in an excessive load-transfer to chondrocytes and the consequent hypertrophy and degeneration.Here, interpenetrated networks (IPNs) with different compositions were created using methacrylated gelatin (GelMA), to mimic the collagen, and alginate functionalized with tyramine (Alg-tyr) to mimic glycosaminoglycans and to introduce a negative charge in the model. Within the matrix chondrocytes where encapsulated and stimulated under different conditions to identify the ultrasound parameters that enhance tissue formation. Samples with and without stimulation were compared analysing the expression and deposition of collagen II, aggrecan, collagen X and TGF-β. The results suggested that the chondrogenic marker expression of the samples stimulated for 10 minutes per day for 28 days, was two times higher overall in all of the cases, which was correlated to the tissue formation detected.Acknowledgments: The authors would like to thank the Basque Government for the “Predoctoral Training Program for Non-Doctoral Research Staff 2021-2022” (Grant ref.: PRE_2021_1_0403). This work was supported by the RETOS grant PID2020-114901RA-I00 of the Ministry of Science and Innovation (MICINN).

Microtissue Culture Provides Clarity on the Relative Chondrogenic and Hypertrophic Response of Bone-Marrow-Derived Stromal Cells to TGF-β1, BMP-2, and GDF-5.

Chondrogenic induction of bone-marrow-derived stromal cells (BMSCs) is typically accomplished with medium supplemented with growth factors (GF) from the transforming growth factor-beta (TGF-β)/bone morphogenetic factor (BMP) superfamily. In a previous study, we demonstrated that brief (1-3 days) stimulation with TGF-β1 was sufficient to drive chondrogenesis and hypertrophy using small-diameter microtissues generated from 5000 BMSC each. This biology is obfuscated in typical large-diameter pellet cultures, which suffer radial heterogeneity. Here, we investigated if brief stimulation (2 days) of BMSC microtissues with BMP-2 (100 ng/mL) or growth/differentiation factor (GDF-5, 100 ng/mL) was also sufficient to induce chondrogenic differentiation, in a manner comparable to TGF-β1 (10 ng/mL). Like TGF-β1, BMP-2 and GDF-5 are reported to stimulate chondrogenic differentiation of BMSCs, but the effects of transient or brief use in culture have not been explored. Hypertrophy is an unwanted outcome in BMSC chondrogenic differentiation that renders engineered tissues unsuitable for use in clinical cartilage repair. Using three BMSC donors, we observed that all GFs facilitated chondrogenesis, although the efficiency and the necessary duration of stimulation differed. Microtissues treated with 2 days or 14 days of TGF-β1 were both superior at producing extracellular matrix and expression of chondrogenic gene markers compared to BMP-2 and GDF-5 with the same exposure times. Hypertrophic markers increased proportionally with chondrogenic differentiation, suggesting that these processes are intertwined for all three GFs. The rapid action, or "temporal potency", of these GFs to induce BMSC chondrogenesis was found to be as follows: TGF-β1 > BMP-2 > GDF-5. Whether briefly or continuously supplied in culture, TGF-β1 was the most potent GF for inducing chondrogenesis in BMSCs.

Liquid platelet-rich fibrin produced via horizontal centrifugation decreases the inflammatory response and promotes chondrocyte regeneration in vitro.

Objective: Recently, liquid platelet-rich fibrin (PRF), a rich source of concentrated platelets and growth factors, has emerged as a promising agent for stimulating tissue regeneration. However, its specific efficacy in chondrocyte proliferation and cartilage regeneration remains underexplored. To address this question, we investigated liquid PRF obtained through horizontal centrifugation and compared its effects with hyaluronic acid (HA), a high molecular weight glucosamine supplement widely used in clinical practice to safeguard against chondral damage. Materials and Methods: Liquid PRF, produced using horizontal centrifugation (liquid H-PRF) at 500g for 8min, served as our experimental agent. We conducted cell viability and proliferation assays using PRF-conditioned medium. We assessed the chondrocyte phenotype of ATDC5 cells through toluidine blue and alcian blue staining, real-time polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence staining. Furthermore, we examined the expression of genes involved in inflammation through RT-PCR and Western blot analysis. Results: Liquid H-PRF exerted notable effects on chondrocytes, influencing proliferation, inflammatory responses, and chondrogenic differentiation. The H-PRF group displayed significantly higher expression of chondrogenic markers, including Col2a1, compared to HA-treated cells, whereas aggrecan expression was significantly higher in the HA group. PRF also demonstrated the ability to reduce inflammatory levels in chondrogenic ATDC5 cells, and this effect was further enhanced when PRF from the buffy coat zone was added. In comparison, chondrocytes cultured in the HA group produced significantly fewer inflammatory factors than those in the PRF group, as confirmed qualitatively by Western blot analysis. Conclusion: Liquid H-PRF emerged as a potent stimulator for chondrogenesis and a regulator of the inflammatory response, achieving levels similar to HA. Moreover, liquid H-PRF exhibited strong potential for enhancing the production of cartilage extracellular matrix and promoting chondrogenic regeneration with notably increased Col2a1 levels. Future research should encompass animal studies and human trials to further evaluate the comparative effectiveness of liquid PRF versus HA, potentially as an alternative or complementary strategy for future clinical applications.