Toll-like receptor activation regulates the paracrine effect of adipose-derived mesenchymal stem cells on reversing osteoarthritic phenotype of chondrocytes.

BACKGROUND: The therapeutic efficacy of intra-articular mesenchymal stem cells (MSCs) injection for patients with osteoarthritis (OA) currently exhibits inconsistency, and the underlying mechanism remains elusive. It has been postulated that the immunomodulatory properties and paracrine activity of MSCs might be influenced by the inflammatory micro-environment within osteoarthritic joints, potentially contributing to this observed inconsistency. METHODS: Adipose-derived MSCs (ADSCs) were isolated from SD rats and pre-treated with Toll-like receptor 3 (TLR3) agonist Poly I:C or Toll-like receptor 4 (TLR4) agonist LPS. The pre-treated ADSCs were then co-cultured with IL-1ß-induced osteoarthritic chondrocytes using a Transwell system to analyze the paracrine effect of ADSCs on reversing the osteoarthritic phenotype of chondrocytes. RESULTS: RT-PCR and Western blot analysis revealed that Poly I:C and LPS pre-treatments up-regulated the expression of IL-10 and IL-6 in ADSCs, respectively. Furthermore, only Poly I:C-preconditioned ADSCs significantly promoted proliferation while inhibiting apoptosis in IL-1ß-treated chondrocytes. Additionally, Poly I:C-preconditioned ADSCs downregulated MMP13 expression while upregulating aggrecan and collagen II expression levels in IL-1ß-treated chondrocytes. CONCLUSIONS: TLR3 activation polarizes ADSCs into an immunomodulatory phenotype distinct from TLR4 activation, exerting differential effects on reversing the osteoarthritic phenotype of chondrocytes; thus indicating that MSCs' paracrine effect regulated by TLRs signaling impacts the efficacy of intra-articular MSCs injection.

Intra-Articular Injection of Interleukin-8 Neutralizing Monoclonal Antibody Effectively Attenuates Osteoarthritis Progression in Rabbits.

OBJECTIVE: Cytokines are implicated in the pathogenesis of osteoarthritis (OA), and this study aims to assess the therapeutic potential of an IL-8 neutralizing monoclonal antibody (mAb) for OA intervention. DESIGN: The study employed a rabbit model of OA induced by anterior cruciate ligament transection (ACLT) surgery to investigate the effects of an interleukin (IL)-8 neutralizing mAb, with hyaluronic acid (HA) used as a positive control. Primary outcomes assessed in the rabbits included cartilage repair, synovitis, joint effusion, changes in footprints, and lower limb loading conditions. RESULTS: Compared to HA, intra-articular injection of the IL-8 neutralizing mAb demonstrated a more pronounced attenuation of OA progression and enhancement of cartilage repair. We observed a reduction in synovitis and joint effusion, indications of bone marrow edema, as well as improvements in lower limb function. In knees treated with the neutralizing IL-8 mAb, there was a significant decrease in IL-8 levels within the synovial tissues. CONCLUSIONS: The IL-8 neutralizing mAb exhibits promising therapeutic potential in the management of OA by attenuating inflammation and facilitating cartilage repair. However, further investigations are warranted to comprehensively elucidate the underlying mechanisms, optimize treatment protocols, and ensure the long-term safety and efficacy of this innovative therapeutic approach.

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.

Macrophage migration inhibitory factor reversed senescent phenotype in human chondrocytes in vitro.

BACKGROUND: The senescence of chondrocytes, which is closely linked to the development of osteoarthritis (OA), has been found to be influenced by the inflammatory environment of joint cavity. However, there remains a lack of comprehensive understanding regarding the specific mechanisms through which cytokine impacts chondrocytes senescence. PURPOSE: To investigate the effects of MIF on the chondrocytes senescence and explore the underlying mechanism. METHODS: Human cytokine array and ELISA were used for the level of MIF in synovium fluid. CCK-8 was used for chondrocytes viability. IF, WB, SA-ß-gal staining and flow cytometry were used for the chondrogenic, apoptotic and senescent phenotype of chondrocytes. RESULTS: The level of MIF was significantly increased in OA patients. MIF significantly reversed the senescent phenotype induced by LPS pretreatment in human chondrocytes. MIF significantly enhanced the expression of Col II, SOX9, and ACAN in LPS pre-treated human chondrocytes. Furthermore, MIF significantly inhibited the apoptosis of LPS-induced senescent chondrocytes. CONCLUSION: Increased level of MIF in osteoarthritic joint cavity might effectively suppress the senescent phenotype and simultaneously improve the chondrogenic phenotype in chondrocytes, the underlying mechanism was likely to be independent of apoptosis.

The influence of inflammation on the characteristics of adipose-derived mesenchymal stem cells (ADMSCs) and tissue repair capability in a hepatic injury mouse model.

BACKGROUND: Mesenchymal stem cells (MSCs) are adult stem cells with self-renewal and multi-directional differentiation potential and possess the functions of immunomodulation, regulation of cell growth, and repair of damage. Over recent years, MSCs have been found to regulate the secretion of inflammatory factors and to exert regulatory effects on various lymphocytes in inflammatory states, and on the subsequent repair of tissue damage caused by inflammation. In the present study, we analyzed the effects of tissue inflammation on the characteristics of MSCs. METHODS: Human fat derived from the infrapatellar fat pad (IPFP) of knees with differing degrees of inflammation was extracted from specimens derived from total knee arthroplasties. HE and immunohistochemical staining was performed to directly observe the evidence and degree of inflammation in human infrapatellar fat pad tissue in order to classify MSCs cells, by their origin, into highly inflamed and lowly inflamed groups, and to study the effect of tissue inflammation on cell acquisition rates via cellular counting data. Flow cytometry assays were performed to investigate the effect of tissue inflammation on MSC surface marker expression. Trilineage differentiation, including osteogenesis, adipogenesis, and chondrogenesis, was performed to assess the effect of tissue inflammation on the ability of MSCs to undergo directed differentiation. The effect of tissue inflammation on the ability of MSCs to proliferate was investigated via clone formation studies. RNA-sequencing was performed to evaluate the transcriptomes of MSCs derived from different areas of inflammation. The effect of tissue inflammation on tissue repair capacity and safety of MSCs was investigated via a murine model of acute liver injury. RESULTS: The results of cell count data indicate that a high degree of tissue inflammation significantly decreases the acquisition rate of MSCs, and the proportion of CD34+ and CD146+ cells. The results of our trilineage differentiation assay show that a higher degree of inflammation decreases osteogenic differentiation and enhances adipogenic and chondrogenic differentiation of MSCs. However, these differences were not statistically significant. Clone formation assays indicate that the degree of tissue inflammation at the MSC source does not significantly affect the proliferative capacity of MSCs. The transcriptomes of MSCs remain relatively stable in fat pad tissues derived from both highly and lowly inflamed samples. The results of acute liver injury investigations in mice indicate that MSCs of high and low inflammatory tissue origin have no significant difference in their tissue repair capability. CONCLUSIONS: High tissue inflammation at the source of MSCs reduces the acquisition rate of MSCs and the percentage of CD34+ and CD146+ cells acquisition. However, source tissue inflammation may not significantly affect trilineage differentiation potential and proliferative capacity of MSCs. Also, MSCs obtained from differing source degrees of inflammation retain stable and similar transcriptomic profile and are both safe and efficacious for tissue repair/regeneration without detectable differences.

Etanercept embedded silk fibroin/pullulan hydrogel enhance cartilage repair in bone marrow stimulation.

Background: Bone marrow stimulation (BMS) is the most used operative treatment in repairing cartilage defect clinically, but always results in fibrocartilage formation, which is easily worn out and needs second therapy. In this study, we prepared an Etanercept (Ept) embedded silk fibroin/pullulan hydrogel to enhance the therapeutic efficacy of BMS. Methods: Ept was dissolved in silk fibroin (SF)-tyramine substituted carboxymethylated pullulan (PL) solution and enzyme crosslinked to obtain the Ept contained SF/PL hydrogel. The synergistical effect of SF/PL hydrogel and Ept was verified by rabbit osteochondral defect model. The mechanism of Ept in promoting articular cartilage repair was studied on human osteoarthritic chondrocytes (hOACs) and human bone marrow mesenchymal stromal cells (hBMSCs) in vitro, respectively. Results: At 4 and 8 weeks after implanting the hydrogel into the osteochondral defect of rabbit, histological analysis revealed that the regenerated tissue in Ept + group had higher cellular density with better texture, and the newly formed hyaline cartilage tissue was seamlessly integrated with adjacent native tissue in the Ept + group. In cellular experiments, Ept treatment significantly promoted both gene and protein expression of type II collagen in hOACs, while decreased the protein levels of metalloproteinase (MMP)-13 and a disintegrin and metalloprotease with thrombospondin motifs 5 (ADAMTS5); alcian blue staining, type II collagen and aggrecan stainings showed that addition of Ept significantly reversed the chondrogenesis inhibition effect of tumor necrosis factor alpha (TNF-α) on hBMSCs. Conclusion: BMS could be augmented by Ept embedded hydrogel, potentially by regulating the catabolic and anabolic dynamics in adjacent chondrocytes and enhancement of BMSCs chondrogenesis.

Tropoelastin improves adhesion and migration of intra-articular injected infrapatellar fat pad MSCs and reduces osteoarthritis progression.

Intra-articular injection of mesenchymal stem cells (MSCs) is a promising strategy for osteoarthritis (OA) treatment. However, more and more studies reveal that the injected MSCs have poor adhesion, migration, and survival in the joint cavity. A recent study shows that tropoelastin (TE) regulates adhesion, proliferation and phenotypic maintenance of MSCs as a soluble additive, indicating that TE could promote MSCs-homing in regenerative medicine. In this study, we used TE as injection medium, and compared it with classic media in MSCs intra-articular injection such as normal saline (NS), hyaluronic acid (HA), and platelet-rich plasma (PRP). We found that TE could effectively improve adhesion, migration, chondrogenic differentiation of infrapatellar fat pad MSCs (IPFP-MSCs) and enhance matrix synthesis of osteoarthritic chondrocytes (OACs) in indirect-coculture system. Moreover, TE could significantly enhance IPFP-MSCs adhesion via activation of integrin ß1, ERK1/2 and vinculin (VCL) in vitro. In addition, intra-articular injection of TE-IPFP MSCs suspension resulted in a short-term increase in survival rate of IPFP-MSCs and better histology scores of rat joint tissues. Inhibition of integrin ß1 or ERK1/2 attenuated the protective effect of TE-IPFP MSCs suspension in vivo. In conclusion, TE promotes performance of IPFP-MSCs and protects knee cartilage from damage in OA through enhancement of cell adhesion and activation of integrin ß1/ERK/VCL pathway. Our findings may provide new insights in MSCs intra-articular injection for OA treatment.

Microconvex Dot-Featured Silk Fibroin Films for Promoting Human Umbilical Vein Endothelial Cell Angiogenesis via Enhancing the Expression of bFGF and VEGF.

Insufficient vascularization of grafts often leads to delayed tissue ingrowth and impaired tissue function in tissue engineering. The surface topography of grafts plays critical roles in angiogenesis. In the present study, we prepared silk fibroin (SF)-based microtopography films with the number of convex dots ranging from 37 to 4835/mm2. The convex dot-featured topography surfaces were characterized by scanning electron microscopy, a Profilm3D optical profilometer, atomic force microscopy, and a contact angle goniometer. The effect of microtopographic films on the proliferation, adhesion, and expression of angiogenic factors of human umbilical vein endothelial cells (HUVECs) was investigated. Our results demonstrated that the SF film surface with 2899 convex dots/mm2 significantly enhanced adhesion, viability, and levels of vascular endothelial growth factors and basic fibroblast growth factors of HUVECs and significantly downregulated the level of α-SMA in human aortic smooth muscle cells, indicating that the microtopographic films could promote angiogenesis. Furthermore, in vitro results showed that HUVEC proliferation was positively correlated with yes-associated protein (YAP) activation, suggesting that the enhanced angiogenesis was mediated via the YAP pathway. Finally, mice subcutaneous embedding model results indicated that the SF film surface with 2899 convex dots/mm2 could significantly enhance angiogenesis in vivo. Altogether, our results showed that the SF film surface with 2899 convex dots/mm2 promoted the angiogenesis of HUVECs and offered a novel angiogenesis-promoting strategy of implant surface design for tissue engineering.

Asiatic acid attenuates hypertrophic and fibrotic differentiation of articular chondrocytes via AMPK/PI3K/AKT signaling pathway.

BACKGROUND: Osteoarthritis (OA), the most common joint disorder, is characterized by a progressive degradation of articular cartilage. Increasing evidence suggests that OA is closely associated with cartilage pathologies including chondrocyte hypertrophy and fibrosis. METHODS: In this study, we showed that asiatic acid (AA) treatment reduced chondrocyte hypertrophy and fibrosis. First, the cytotoxicity of AA (0, 5, 10, and 20 µM) to chondrocytes was evaluated, and 5 µM was selected for subsequent experiments. Then, we detected the gene and protein level of chondrocyte hypertrophic markers including type X collagen (COL-X), matrix metalloproteinase-13 (MMP-13), alkaline phosphatase (ALP), and runt-related transcription factor 2 (Runx2); chondrocyte fibrosis markers including type I collagen (COL-Ι) and alpha-smooth muscle actin (α-SMA); and chondrogenic markers including SRY-related HMG box 9 (SOX9), type II collagen (COL-II), and aggrecan (ACAN). Further, we tested the mechanism of AA on inhibiting chondrocyte hypertrophy and fibrosis. Finally, we verified the results in an anterior cruciate ligament transection (ACLT) rat OA model. RESULTS: We found that AA treatment inhibited the hypertrophic and fibrotic phenotype of chondrocytes, without affecting the chondrogenic phenotype. Moreover, we found that AA treatment activated AMP-activated protein kinase (AMPK) and inhibited phosphoinositide-3 kinase/protein kinase B (PI3K/AKT) signaling pathway in vitro. The results in an ACLT rat OA model also indicated that AA significantly attenuated chondrocyte hypertrophy and fibrosis. CONCLUSION: AA treatment could reduce hypertrophic and fibrotic differentiation and maintain the chondrogenic phenotype of articular chondrocytes by targeting the AMPK/PI3K/AKT signaling pathway. Our study suggested that AA might be a prospective drug component that targets hypertrophic and fibrotic chondrocytes for OA treatment.

Natural ingredients-derived antioxidants attenuate H2O2-induced oxidative stress and have chondroprotective effects on human osteoarthritic chondrocytes via Keap1/Nrf2 pathway.

Osteoarthritis (OA) is the most common disabling joint disease and its pathological process is closely related to oxidative stress. Recent studies have shown that antioxidants allicin, sulforaphane, and lycopene derived from natural ingredients garlic, broccoli, and tomato can reduce the degree of oxidative stress and the expression of inflammatory markers, indicating that theses antioxidants might be helpful for OA treatment. In this study, we investigated the effects of allicin, sulforaphane, and lycopene on H2O2-stimulated human osteochondral samples and osteoarthritic chondrocytes. Our results revealed that allicin, sulforaphane, and lycopene effectively reduced the oxidative stress-induced cell apoptosis, and increased gene expression of antioxidant enzymes. Besides, these natural ingredients-derived antioxidants reduced the expression of inflammatory factors, enhanced the chondrogenic matrix synthesis, and reduced the hypertrophic differentiation of osteoarthritic chondrocytes. These regulations were mainly through the activation of Keap1/Nrf2 pathway. Our findings suggest that these antioxidants might be a potential therapeutic strategy for OA.

Effects of Conditioned Medium From Osteoarthritic Cartilage Fragments on Donor-Matched Infrapatellar Fat Pad-Derived Mesenchymal Stromal Cells.

BACKGROUND: Mesenchymal stromal cell (MSC)-based therapies have emerged as a promising strategy for osteoarthritis (OA) treatment. In particular, infrapatellar fat pad (IPFP)-derived MSCs have become a good option to treat knee OA. PURPOSE: To investigate the influence of the local microenvironment of the knee joint, especially OA cartilage, on the bioactivities of injected/implanted IPFP MSCs. STUDY DESIGN: Controlled laboratory study. METHODS: Conditioned medium (CM) derived from OA cartilage fragments was collected and characterized. Donor-matched IPFP MSCs were treated with control medium (Dulbecco's modified Eagle medium (DMEM)/F-12 or chondrogenic medium), control medium + CM, or CM alone; and a series of behaviors including the viability, migration, chondrogenic and hypertrophic differentiation, and catabolic activity of IPFP MSCs were evaluated among groups. RESULTS: There were 14 cytokines detected in CM. CM treatment improved the viability of IPFP MSCs. CM hindered the migration of IPFP MSCs. In chondrogenic differentiation, the presence of CM increased the expression of chondrogenic markers but also enhanced the state of hypertrophy and catabolism. CONCLUSION: OA cartilage-secreted factors could induce chondrogenic differentiation but also resulted in negative effects including the weakened migration, increased hypertrophy, and catabolism of IPFP MSCs in vitro. CLINICAL RELEVANCE: These findings provide an insight on the fate of IPFP MSCs after intra-articular injections.

miR-100-5p-abundant exosomes derived from infrapatellar fat pad MSCs protect articular cartilage and ameliorate gait abnormalities via inhibition of mTOR in osteoarthritis.

Osteoarthritis (OA) is the most common disabling joint disease throughout the world and its therapeutic effect is still not satisfactory in clinic nowadays. Recent studies showed that the exosomes derived from several types of mesenchymal stem cells (MSCs) could maintain chondrocyte homeostasis and ameliorate the pathological severity of OA in animal models, indicating that MSCs-derived exosomes could be a novel promising strategy for treating OA. In this study, we investigated the role and underlying mechanisms of infrapatellar fat pad (IPFP) MSCs-derived exosomes (MSCIPFP-Exos) on OA in vitro and in vivo. Our data revealed that MSCIPFP could produce amounts of MSCIPFP-Exos, which exhibited the typical morphological features of exosomes. The MSCIPFP-Exos ameliorated the OA severity in vivo and inhibited cell apoptosis, enhanced matrix synthesis and reduced the expression of catabolic factor in vitro. Moreover, MSCIPFP-Exos could significantly enhance autophagy level in chondrocytes partially via mTOR inhibition. Exosomal RNA-seq showed that the level of miR-100-5p that could bind to the 3'-untranslated region (3'UTR) of mTOR was the highest among microRNAs. MSCIPFP-Exos decreased the luciferase activity of mTOR 3'UTR, while inhibition of miR-100-5p could reverse the MSCIPFP-Exos-decreased mTOR signaling pathway. Intra-articular injection of antagomir-miR-100-5p dramatically attenuated MSCIPFP-Exos-mediated protective effect on articular cartilage in vivo. In brief, MSCIPFP-derived exosomes protect articular cartilage from damage and ameliorate gait abnormality in OA mice by maintaining cartilage homeostasis, the mechanism of which may be related to miR100-5p-regulated inhibition of mTOR-autophagy pathway. As it is relatively feasible to obtain human IPFP from OA patients by arthroscopic operation in clinic, MSCIPFP-derived exosomes may be a potential therapy for OA in the future.

Synergistically regulated spontaneous calcium signaling is attributed to cartilaginous extracellular matrix metabolism.

Ca2+ has been recognized as a key molecule for chondrocytes, however, the role and mechanism of spontaneous [Ca 2+ ] i signaling in cartilaginous extracellular matrix (ECM) metabolism regulation are unclear. Here we found that spontaneous Ca 2+ signal of in-situ porcine chondrocytes was [Ca 2+ ] o dependent, and mediated by [Ca 2+ ] i store release. T-type voltage-dependent calcium channel (T-VDCC) mediated [Ca 2+ ] o influx was associated with decreased cell viability and expression levels of ECM deposition genes. Further analysis revealed that chondrocytes expressed both inositol 1,4,5-trisphosphate receptor (InsP3R) and Orai isoforms. Inhibition of endoplasmic reticulum (ER) Ca 2+ release and store-operated calcium entry significantly abolished spontaneous [Ca 2+ ] i signaling of in-situ chondrocytes. Moreover, blocking ER Ca 2+ release with InsP3R inhibitors significantly upregulated ECM degradation enzymes production, and was accompanied by decreased proteoglycan and collagen type II intensity. Taken together, our data provided evidence that spontaneous [Ca 2+ ] i signaling of in-situ porcine chondrocytes was tightly regulated by [Ca 2+ ] o influx, InsP3Rs mediated [Ca 2+ ] i store release, and Orais mediated calcium release-activated calcium channels activation. Both T-VDCC mediated [Ca 2+ ] o influx and InsP3Rs mediated ER Ca 2+ release were found crucial to cartilaginous ECM metabolism through distinct regulatory mechanisms.