The role of monocyte/macrophage chemokines in pathogenesis of osteoarthritis: A review.

Osteoarthritis (OA) is one of the most common degenerative diseases characterised by joint pain, swelling and decreased mobility, with its main pathological features being articular synovitis, cartilage degeneration and osteophyte formation. Inflammatory cytokines and chemokines secreted by activated immunocytes can trigger various inflammatory and immune responses in articular cartilage and synovium, contributing to the genesis and development of OA. A series of monocyte/macrophage chemokines, including monocyte chemotaxis protein (MCP)-1/CCL2, MCP2/CCL8, macrophage inflammatory protein (MIP)-1α/CCL3, MIP-1ß/CCL4, MIP-3α/CCL20, regulated upon activation, normal T-cell expressed and secreted /CCL5, CCL17 and macrophage-derived chemokine/CCL22, was proven to transmit cell signals by binding to G protein-coupled receptors on recipient cell surface, mediating and promoting inflammation in OA joints. However, the underlying mechanism of these chemokines in the pathogenesis of OA remains still elusive. Here, published literature was reviewed, and the function and mechanisms of monocyte/macrophage chemokines in OA pathogenesis were summarised. The symptoms and disease progression of OA were found to be effectively alleviated when the expression of these chemokines is inhibited. Elucidating these mechanisms could contribute to further understand how OA develops and provide potential targets for the early diagnosis of arthritis and drug treatment to delay or even halt OA progression.

Hypoxia induces DOT1L in articular cartilage to protect against osteoarthritis.

Osteoarthritis is the most prevalent joint disease worldwide, and it is a leading source of pain and disability. To date, this disease lacks curative treatment, as underlying molecular mechanisms remain largely unknown. The histone methyltransferase DOT1L protects against osteoarthritis, and DOT1L-mediated H3K79 methylation is reduced in human and mouse osteoarthritic joints. Thus, restoring DOT1L function seems to be critical to preserve joint health. However, DOT1L-regulating molecules and networks remain elusive, in the joint and beyond. Here, we identified transcription factors and networks that regulate DOT1L gene expression using a potentially novel bioinformatics pipeline. Thereby, we unraveled a possibly undiscovered link between the hypoxia pathway and DOT1L. We provide evidence that hypoxia enhanced DOT1L expression and H3K79 methylation via hypoxia-inducible factor-1 α (HIF1A). Importantly, we demonstrate that DOT1L contributed to the protective effects of hypoxia in articular cartilage and osteoarthritis. Intra-articular treatment with a selective hypoxia mimetic in mice after surgical induction of osteoarthritis restored DOT1L function and stalled disease progression. Collectively, our data unravel a molecular mechanism that protects against osteoarthritis with hypoxia inducing DOT1L transcription in cartilage. Local treatment with a selective hypoxia mimetic in the joint restores DOT1L function and could be an attractive therapeutic strategy for osteoarthritis.

Rutin inhibited the advanced glycation end products-stimulated inflammatory response and extra-cellular matrix degeneration via targeting TRAF-6 and BCL-2 proteins in mouse model of osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is degenerative joint disorder mainly characterized by long-term pain with limited activity of joints, the disease has no effective preventative therapy. Rutin (RUT) is a flavonoid compound, present naturally. The flavonoid shows range of biological activities such as anti-inflammatory and anti-cancer effect. We screened RUT for its activity against osteoarthritis with in vivo and in vitro models of osteoarthritis. METHODS: Animal model of OA was developed using C57BL/6 mice by surgical destabilization of medial meniscus. For in vitro studies the human articular cartilage tissues were used which were collected from osteoarthritis patients and were processed to isolate chondrocytes. The chondrocytes were submitted to advanced glycation end products (AGEs) for inducing osteoarthritis in vitro. Cell viability was done by CCK-8 assay, ELISA analysis for MMP13, collage II, PGE2, IL-6, TNF-α, ADAMTS-5 and MMP-13. Western blot analysis was done for expression of proteins and in silico analysis was done by docking studies. RESULTS: Pretreatment of RT showed no cytotoxic effect and also ameliorated the AGE mediated inflammatory reaction on human chondrocytes in vitro. Treatment of RT inhibited the levels of COX-2 and iNOS in AGE exposed chondrocytes. RT decreased the AGE mediated up-regulation of IL-6, NO, TNF-α and PGE-2 in a dose dependent manner. Pretreatment of RT decreased the extracellular matrix degradation, inhibited expression of TRAF-6 and BCL-2 the NF-κB/MAPK pathway proteins. The treatment of RT in mice prevented the calcification of cartilage tissues, loss of proteoglycans and also halted the narrowing of joint space is mice subjected to osteoarthritis. The in-silico analysis suggested potential binding affinity of RT with TRAF-6 and BCL-2. CONCLUSION: In brief RT inhibited AGE-induced inflammatory reaction and also degradation of ECM via targeting the NF-κB/MAPK pathway proteins BCL-2 and TRAF-6. RT can be a potential molecule in treating OA.

Mitochondria in Injury, Inflammation and Disease of Articular Skeletal Joints.

Inflammation is an important biological response to tissue damage caused by injury, with a crucial role in initiating and controlling the healing process. However, dysregulation of the process can also be a major contributor to tissue damage. Related to this, although mitochondria are typically thought of in terms of energy production, it has recently become clear that these important organelles also orchestrate the inflammatory response via multiple mechanisms. Dysregulated inflammation is a well-recognised problem in skeletal joint diseases, such as rheumatoid arthritis. Interestingly osteoarthritis (OA), despite traditionally being known as a 'non-inflammatory arthritis', now appears to involve an element of chronic inflammation. OA is considered an umbrella term for a family of diseases stemming from a range of aetiologies (age, obesity etc.), but all with a common presentation. One particular OA sub-set called Post-Traumatic OA (PTOA) results from acute mechanical injury to the joint. Whether the initial mechanical tissue damage, or the subsequent inflammatory response drives disease, is currently unclear. In the former case; mechanobiological properties of cells/tissues in the joint are a crucial consideration. Many such cell-types have been shown to be exquisitely sensitive to their mechanical environment, which can alter their mitochondrial and cellular function. For example, in bone and cartilage cells fluid-flow induced shear stresses can modulate cytoskeletal dynamics and gene expression profiles. More recently, immune cells were shown to be highly sensitive to hydrostatic pressure. In each of these cases mitochondria were central to these responses. In terms of acute inflammation, mitochondria may have a pivotal role in linking joint tissue injury with chronic disease. These processes could involve the immune cells recruited to the joint, native/resident joint cells that have been damaged, or both. Taken together, these observations suggest that mitochondria are likely to play an important role in linking acute joint tissue injury, inflammation, and long-term chronic joint degeneration - and that the process involves mechanobiological factors. In this review, we will explore the links between mechanobiology, mitochondrial function, inflammation/tissue-damage in joint injury and disease. We will also explore some emerging mitochondrial therapeutics and their potential for application in PTOA.

Osteoarthritic infrapatellar fat pad aggravates cartilage degradation via activation of p38MAPK and ERK1/2 pathways.

OBJECTIVE: This study aimed to investigate the biochemical effects of osteoarthritic infrapatellar fat pad (IPFP) on cartilage and the underlying mechanisms. METHODS: Human IPFP and articular cartilage were collected from end-stage osteoarthritis (OA) patients during total knee arthroplasty. IPFP-derived fat-conditioned medium (FCM) was used to stimulate human primary chondrocytes and cartilage explants. Functional effect of osteoarthritic IPFP was explored in human primary chondrocytes and articular cartilage in vitro and ex vivo. Activation of relative pathways and its effects on chondrocytes were assessed through immunoblotting and inhibition experiments, respectively. Neutralization test was performed to identify the main factors and their associated pathways responsible for the effects of IPFP. RESULTS: Osteoarthritic IPFP-derived FCM significantly induced extracellular matrix (ECM) degradation in both human primary chondrocytes and cartilage explants. Several pathways, such as NF-κB, mTORC1, p38MAPK, JNK, and ERK1/2 signaling, were significantly activated in human chondrocytes with osteoarthritic IPFP-derived FCM stimulation. Interestingly, inhibition of p38MAPK and ERK1/2 signaling pathway could alleviate the detrimental effects of FCM on chondrocytes, while inhibition of other signaling pathways had no similar results. In addition, IL-1ß and TNF-α instead of IL-6 in osteoarthritic IPFP-derived FCM played key roles in cartilage degradation via activating p38MAPK rather than ERK1/2 signaling pathway. CONCLUSION: Osteoarthritic IPFP induces the degradation and inflammation of cartilage via activation of p38MAPK and ERK1/2 pathways, in which IL-1ß and TNF-α act as the key factors. Our study suggests that modulating the effects of IPFP on cartilage may be a promising strategy for knee OA intervention.

NF-κB inducible miR-30b-5p aggravates joint pain and loss of articular cartilage via targeting SIRT1-FoxO3a-mediated NLRP3 inflammasome.

MicroRNAs (miRNAs) contribute to osteoarthritis (OA) development. Nevertheless, the function and mechanism of miR-30b-5p in OA are unclear. In the present article, we gauged the miR-30b-5p level in OA patients and analyzed its correlation with OA stages. Then, we conducted in-vivo and in-vitro gain-of-function assays to determine the function of miR-30b-5p, silent information regulator 2 homolog 1 (SIRT1) and Fox. Cell counting Kit-8 (CCK-8) assay, BrdU assay and flow cytometry were utilized to gauge cell viability and apoptosis of human chondrocyte (HC-A). The targeting association between miR-30b-5p and SIRT1 was validated through the dual-luciferase reporter assay and RNA immunoprecipitation (RIP) experiment. The results signified that miR-30b-5p was up-regulated in OA patients, OA rats and interleukin-1ß (IL-1ß)-induced chondrocytes. The higher miR-30b-5p expression brought about progressive stages of OA patients and enhanced levels of pro-inflammatory mediators in the synovial fluid. Functionally, overexpressing miR-30b-5p hampered cell viability, aggravated chondrocyte apoptosis and NLRP3 inflammasome activation induced by IL-1ß, while down-regulating miR-30b-5p exerted the reverse effects. The in-vivo experiment exhibited that down-regulating miR-30b-5p improved joint pain and loss of articular cartilage in the rats with restrained inflammation and NLRP3 inflammasome activation. Mechanistically, miR-30b-5p targeted the 3'-non-translated region (3'UTR) of SIRT1, and miR-30b-5p was inducible with NF-κB phosphorylation enhancement. Overexpressing SIRT1 or inhibiting NF-κB relieved miR-30b-5p-induced apoptosis and NLRP3 inflammasome activation by promoting FoxO3a, while down-regulating SIRT1 or FoxO3a reversed miR-30b-5p-in-induced anti-inflammatory and apoptosis-suppressive effects. Collectively, NF-κB-induced miR-30b-5p modulates chondrocyte apoptosis and OA progression by regulating the SIRT1-FoxO3a-mediated NLRP3 inflammasome.

Interleukin 4 promotes anti-inflammatory macrophages that clear cartilage debris and inhibits osteoclast development to protect against osteoarthritis.

OBJECTIVE: Osteoarthritis (OA), the leading cause of joint failure, is characterized by breakdown of articular cartilage and remodeling of subchondral bone in synovial joints. Despite the high prevalence and debilitating effects of OA, no disease-modifying drugs exist. Increasing evidence, including genetic variants of the interleukin 4 (IL-4) and IL-4 receptor genes, implicates a role for IL-4 in OA, however, the mechanism underlying IL-4 function in OA remains unknown. Here, we investigated the role of IL-4 in OA pathogenesis. METHODS: Il4-, myeloid-specific-Il4ra-, and Stat6-deficient and control mice were subjected to destabilization of the medial meniscus to induce OA. Macrophages, osteoclasts, and synovial explants were stimulated with IL-4 in vitro, and their function and expression profiles characterized. RESULTS: Mice lacking IL-4, IL-4Ra in myeloid cells, or STAT6 developed exacerbated cartilage damage and osteophyte formation relative to WT controls. In vitro analyses revealed that IL-4 downregulates osteoarthritis-associated genes, enhances macrophage phagocytosis of cartilage debris, and inhibits osteoclast differentiation and activation via the type I receptor. CONCLUSION: Our findings demonstrate that IL-4 protects against osteoarthritis in a myeloid and STAT6-dependent manner. Further, IL-4 can promote an immunomodulatory microenvironment in which joint-resident macrophages polarize towards an M2 phenotype and efficiently clear pro-inflammatory debris, and osteoclasts maintain a homeostatic level of activity in subchondral bone. These findings support a role for IL-4 modulation of myeloid cell types in maintenance of joint health and identify a pathway that could provide therapeutic benefit for osteoarthritis.

Pretreatment with mechano growth factor E peptide attenuates osteoarthritis through improving cell proliferation and extracellular matrix synthesis in chondrocytes under severe hypoxia.

Osteoarthritis (OA) is characterized by pain and declining gait function associated with degeneration of cartilage. A severe hypoxic environment occurs due to tissue injury in the joint cavity and may aggravate the development of OA. In this study, the effects of severe hypoxia and treatment with mechano growth factor (MGF) E peptide on metabolism of the extracellular matrix (ECM) during the progression of OA were determined. The results showed that cell viability, cell proliferation, and type II collagen expression in chondrocytes were significantly inhibited by cobalt chloride (CoCl2)-simulated severe hypoxia, whereas cell apoptosis and expression levels of hypoxia inducible factor 1 alpha, type I collagen, and matrix metalloproteinases 1/13 were clearly induced. Pretreatment with MGF E peptide reduced the abovementioned adverse effects induced by CoCl2-simulated severe hypoxia in chondrocytes. Pretreatment also upregulated the proliferation of chondrocytes under severe hypoxia through the PI3K-Akt and MEK-ERK1/2 signaling pathways. In a rat model of monosodium iodoacetate (MIA)-induced OA. MIA treatment induced tissue necrosis and cartilage degeneration, and histological score was significantly decreased. The levels of type II collagen and aggrecan were reduced after MIA treatment for 4 or 6 weeks, and abnormal distribution of ECM occurred in the inner epicondyle after 6 weeks. MGF E peptide also reduced the progression of MIA-induced OA by retarding cartilage degeneration, upregulating type II collagen synthesis, and improving ECM distribution after 4 or 6 weeks. Our findings suggest that MGF attenuates the progression of OA, and thus may be applied for the treatment of OA in the clinic.

The p53 status in rheumatoid arthritis with focus on fibroblast-like synoviocytes.

P53 is a transcription factor that regulates many signaling pathways like apoptosis, cell cycle, DNA repair, and cellular stress responses. P53 is involved in inflammatory responses through the regulation of inflammatory signaling pathways, induction of cytokines, and matrix metalloproteinase expression. Also, p53 regulates immune responses through modulating Toll-like receptors expression and innate and adaptive immune cell differentiation and maturation. P53 is a modulator of the apoptosis and proliferation processes through regulating multiple anti and pro-apoptotic genes. Rheumatoid arthritis (RA) is categorized as an invasive inflammatory autoimmune disease with irreversible deformity of joints and bone resorption. Different immune and non-immune cells contribute to RA pathogenesis. Fibroblast-like synoviocytes (FLSs) have been recently introduced as a key player in the pathogenesis of RA. These cells in RA synovium produce inflammatory cytokines and matrix metalloproteinases which results in synovitis and joint destruction. Besides, hyper proliferation and apoptosis resistance of FLSs lead to synovial hyperplasia and bone and cartilage destruction. Given the critical role of p53 in inflammation, apoptosis, and cell proliferation, lack of p53 function (due to mutation or low expression) exerts a prominent role for this gene in the pathogenesis of RA. This review focuses on the role of p53 in different mechanisms and cells (specially FLSs) that involved in RA pathogenesis.

Wogonoside attenuates the articular cartilage injury and the infiltration of Th1/Th2-type cytokines in papain-induced osteoarthritis in rat model via inhibiting the NF-κB and ERK1/2 activation.

OBJECTS: Osteoarthritis is the most common joint disease and a major cause of functional limitation and pain in adults. This study aims to investigate the effect of wogonoside (WOG) on the progression of knee osteoarthritis (KOA) in model rats. MATERIALS AND METHODS: Rats KOA models were established and treated with different doses of WOG (10 mg/kg, 20 mg/kg and 30 mg/kg). The degree of cartilage injury was detected by Mankin scores via HE/Alcian blue staining. The levels of IFN-γ and IL-4 in peripheral blood and synovial fluid and the Th1/Th2 ratio were detected by flow cytometry. The model mice were injected with NF-κB p65 or ERK1/2 inhibitors or activators to further investigate the effect of WOG on KOA. RESULTS: WOG significantly improved cartilage tissue damage and reduced the Mankins score. WOG down-regulated the level of IFN-γ while up-regulated the expression of IL-4, which maintained the balance of Th1/Th2 cells. Further studies showed that the expression of NF-κB p65, phosphorylated p65, cytoplasmic ERK1/2 and nuclear ERK1/2 were all inhibited by WOG. The results of reverse verification experiments showed that the activator of NF-κB p65 and ERK1/2 weakened the protective effect of WOG on KOA, and the inhibitor of NF-κB p65ERK1/2 enhanced the protective effect of WOG on KOA. CONCLUSIONS: WOG inhibited the activation of NF-κB and ERK1/2 to alleviate the articular cartilage injury and Th1/th2 cytokine infiltration in KOA rats.

Decursin alleviates the aggravation of osteoarthritis via inhibiting PI3K-Akt and NF-kB signal pathway.

Osteoarthritis (OA) is a common joint disease that takes joint degeneration or aging as its pathological basis, and joint swelling, pain or dysfunction as its main clinical manifestations. Decursin (DE), the major active component isolated from Angelica gigas Nakai, has been demonstrated to possess anti-inflammatory effect in many diseases. But, the specific physiological mechanism of DE on OA is not clear yet. Therefore, the object of this study was to assess the therapeutic effect of DE on OA, and to explore its potential anti-inflammatory mechanisms. In vitro cell experiments, the inflammatory response in chondrocytes is mediated via interleukin-1ß (IL-1ß), which led to abnormal secretion of pro-inflammatory factors, such as prostaglandin E2 (PGE2), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), cyclooxygenase-2 (COX-2), nitric oxide (NO) and inducible nitric oxide synthase (iNOS). These cytokines were all decreased by the preconditioning of DE in a dose-dependent form of 1, 5, and 10 µM. Moreover, DE could restrain IL-1ß-mediated inflammatory reaction and the collapse of extracellular matrix (ECM) via reducing the secretion of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) and MMPs (matrix metalloproteinases). In short, DE restrained IL-1ß-mediated abnormal excitation of PI3K/AKT/NF-κB axis. Furthermore, molecular docking analysis showed that DE has a strong binding affinity with the inhibitory targets of PI3K. In vivo animal studies, DE treatment could helped to improve destruction of articular cartilage and decreased the serum inflammatory factor levels in an operationally induced mouse OA model. To sum up, these data obtained from the experiment indicate that DE has good prospects for the treatment of osteoarthritis.

Inflammatory signaling sensitizes Piezo1 mechanotransduction in articular chondrocytes as a pathogenic feed-forward mechanism in osteoarthritis.

Osteoarthritis (OA) is a painful and debilitating condition of synovial joints without any disease-modifying therapies [A. M. Valdes, T. D. Spector, Nat. Rev. Rheumatol. 7, 23-32 (2011)]. We previously identified mechanosensitive PIEZO channels, PIEZO1 and PIEZO2, both expressed in articular cartilage, to function in chondrocyte mechanotransduction in response to injury [W. Lee et al., Proc. Natl. Acad. Sci. U.S.A. 111, E5114-E5122 (2014); W. Lee, F. Guilak, W. Liedtke, Curr. Top. Membr. 79, 263-273 (2017)]. We therefore asked whether interleukin-1-mediated inflammatory signaling, as occurs in OA, influences Piezo gene expression and channel function, thus indicative of maladaptive reprogramming that can be rationally targeted. Primary porcine chondrocyte culture and human osteoarthritic cartilage tissue were studied. We found that interleukin-1α (IL-1α) up-regulated Piezo1 in porcine chondrocytes. Piezo1 expression was significantly increased in human osteoarthritic cartilage. Increased Piezo1 expression in chondrocytes resulted in a feed-forward pathomechanism whereby increased function of Piezo1 induced excess intracellular Ca2+ at baseline and in response to mechanical deformation. Elevated resting state Ca2+ in turn rarefied the F-actin cytoskeleton and amplified mechanically induced deformation microtrauma. As intracellular substrates of this OA-related inflammatory pathomechanism, in porcine articular chondrocytes exposed to IL-1α, we discovered that enhanced Piezo1 expression depended on p38 MAP-kinase and transcription factors HNF4 and ATF2/CREBP1. CREBP1 directly bound to the proximal PIEZO1 gene promoter. Taken together, these signaling and genetic reprogramming events represent a detrimental Ca2+-driven feed-forward mechanism that can be rationally targeted to stem the progression of OA.

Repair of articular cartilage defect using adipose-derived stem cell-loaded scaffold derived from native cartilage extracellular matrix.

The purpose of this study was to investigate the feasibility of adipose-derived stem cells (ADSCs) as the seed cells of cartilage tissue engineering. ADSCs were isolated from adipose tissue that was harvested under sterile conditions from the inguen fold of porcines and cultured in vitro. Acellular cartilage extracellular matrix (ACECM) scaffolds of pigs were then constructed. Moreover, inflammatory cells, as well as cellular and humoral immune responses, were detected using hematoxylin and eosin staining staining, immunohistochemical staining, and western blot analysis. The results showed that the cartilage complex constructed by ADSCs and ACECM through tissue engineering successfully repaired the cartilage defect of the pig knee joint. The in vivo repair experiment showed no significant difference between chondrocytes, ADSCs, and induced ADSCs, indicating that ADSCs do not require in vitro induction and have the potential for chondrogenic differentiation in the environment around the knee joint. In addition, pig-derived acellular cartilage scaffolds possess no obvious immune inflammatory response when used in xenotransplantation. ADSCs may serve as viable seed cells for cartilage tissue engineering.

Effect of intra-articular dexmedetomidine on experimental osteoarthritis in rats.

Pharmacological treatment of osteoarthritis is still inadequate due to the low efficacy of the drugs used. Dexmedetomidine via the intra-articular (i.a.) route might be an option for the treatment of osteoarthritis-associated pain. The present study assessed the analgesic and anti-inflammatory effects of dexmedetomidine administered via the i.a. route in different doses in an experimental model of rat knee osteoarthritis induced with monosodium iodoacetate. Rats were allocated to four groups with 24 animals in each group. The OA (osteoarthritis), DEX-1 (dexmedetomidine in dose of 1µg/kg) and DEX-3 (dexmedetomidine in dose of 3µg/kg) groups were subjected to induction of osteoarthritis through injection of monosodium iodoacetate (MIA) via the i.a. route on the right knee; the control group was not subjected to osteoarthritis induction. Clinical assessment was performed on day 0 (before osteoarthritis induction) and then on days 5, 10, 14, 21 and 28 after induction. Treatment was performed on day 7 via the i.a. route, consisting of dexmedetomidine in doses of 1 and 3 µg/kg, while group OA received 0.9% normal saline. The animals were euthanized on days 7, 14, 21 and 28. Samples of the synovial membrane were collected for histopathological analysis, and the popliteal lymph nodes were collected for measurement of cytokines (interleukin [IL] IL-6, tumor necrosis factor alpha [TNF-α]). Dexmedetomidine (1 and 3 µg/kg) significantly reduced the animals' weight distribution deficit during the chronic-degenerative stage of osteoarthritis and improved the pain threshold throughout the entire experiment. Histological analysis showed that dexmedetomidine did not cause any additional damage to the synovial membrane. The TNF-α levels decreased significantly in the DEX-3 group on day 28 compared with the OA group. Dexmedetomidine reduced pain, as evidenced by clinical parameters of osteoarthritis in rats, but did not have an anti-inflammatory effect on histological evaluation.

Systematic Review of Osteochondral Allograft Transplant Immunology: How We Can Further Optimize Outcomes.

Despite the growing success for osteochondral allograft (OCA) transplantation in treating large articular cartilage lesions in multiple joints, associated revision and failure rates are still higher than desired. While immunorejection responses have not been documented, the effects of the host's immune responses on OCA transplantation failures have not been thoroughly characterized. The objective of this study was to systematically review clinically relevant peer-reviewed evidence pertaining to the immunology of OCAs to elucidate theragnostic strategies for improving functional graft survival and outcomes for patients undergoing OCA transplantation. This systematic review of Cochrane Central Register of Controlled Trials, the Cochrane Database of Systematic Reviews, MEDLINE, PubMed, and EMBASE suggests that host immune responses play key roles in incorporation and functional survival of OCA transplants. OCA rejection has not been reported; however, graft integration through creeping substitution is reliant on host immune responses. Prolonged inflammation, diminished osteogenic potential for healing and incorporation, and relative bioburden are mechanisms that may be influenced by the immune system and contribute to undesirable outcomes after OCA transplantation. Based on the safety and efficacy of OCA transplantation and its associated benefits to a large and growing patient population, basic, preclinical, and clinical osteoimmunological studies on OCA transplantation that comprehensively assess and correlate cellular, molecular, histologic, biomechanical, biomarkers, diagnostic imaging, arthroscopic, functional, and patient-reported outcome measures are of high interest and importance.

PLCγ1 inhibition combined with inhibition of apoptosis and necroptosis increases cartilage matrix synthesis in IL-1ß-treated rat chondrocytes.

Osteoarthritis (OA) is an age-related, chronic degenerative disease. With the increasing median age of the population, this disease has become an important public health problem. New, disease-modifying therapies are needed. A potential novel molecular target is phospholipase Cγ1 (PLCγ1), a critical enzyme with important functions including calcium signaling regulation and cell proliferation. In rat chondrocytes treated with IL-1ß (20 ng·mL-1 for 36 h), inhibition of PLCγ1 with U73122 (2 µm for 12 h) increased levels and expression of the cartilage matrix components Collagen2 and Aggrecan. This beneficial effect of PLCγ1 inhibition was counteracted by increased chondrocyte apoptosis and necroptosis, increased cell death, and increase levels of ROS, all potentially negative for OA. Combined treatment of IL-1ß + U73122-treated chondrocytes with inhibitors of apoptosis (Z-VAD, 10 µm) and necroptosis (Nec-1, 30 µm) enhanced the increases in levels and expression of Collagen2 and Aggrecan, and prevented the increases in cell death and ROS levels. These results suggest that PLCγ1 inhibition may be a viable approach for an OA therapy, if combined with targeted inhibition of chondrocyte apoptosis and necroptosis.

Knockdown of Long Non-coding RNA LINC00473 Protects CHON-001 Cells against Interleukin-1ß-Induced Cell Injury.

Osteoarthritis (OA) is a chronic joint disease with high prevalence. However, effective treatment options for OA are still lacking. It was previously reported that LINC00473 was upregulated in patients with OA and upregulated LINC00473 might be associated with the progression of OA; however, the role of LINC00473 in OA remains to be investigated. CHON-001 human chondrocyte cells stimulated with 10 ng/mL interleukin (IL)-1ß were utilized to mimic OA in vitro. Protein expression, cell apoptosis and cell proliferation of CHON-001 cells were investigated by Western blot, Annexin V and propidium iodide (PI) double staining, cell counting-8 kit assay and immunofluorescence staining respectively. The result indicated IL-1ß triggered viability decrease and apoptosis in CHON-001 cells, which was alleviated by LINC00473 knockdown. Meanwhile, IL-1ß-induced upregulation of cleaved caspase 3 and Bax were ameliorated by LINC00473 knockdown. Likewise, IL-1ß-induced downregulation of X-linked inhibitor of apoptosis protein was alleviated by LINC00473 knockdown. In addition, LINC00473 knockdown protected CHON-001 cells against IL-1ß by inhibiting the methylation of LIM mineralization protein (LMP)-1 gene. Moreover, c-Jun N-terminal kinase (JNK)/nuclear factor-kappaB (NF-κB) signaling pathway was proved to be involved in the cell protective effect of LINC00473 knockdown in IL-1ß treated CHON-001 cells. Taken together, LINC00473 knockdown defended CHON-001 cells from IL-1ß induced cell injury via inhibition of the methylation of LMP-1. Thus, LINC00473 might possibly act as a novel therapeutic target for OA.

Th17 lymphocyte-dependent degradation of joint cartilage by synovial fibroblasts in a humanized mouse model of arthritis and reversal by secukinumab.

How T-helper (Th) lymphocyte subpopulations identified in synovial fluid from patients with juvenile idiopathic arthritis (JIA) (Th17, classic Th1, or nonclassic Th1) drive joint damage is of great interest for the possible use of biological drugs that inhibit the specific cytokines. Our objective was to clarify the role of such Th subpopulations in the pathogenesis of articular cartilage destruction by synovial fibroblasts (SFbs), and the effect of Th17 blockage in an animal model. SFbs were isolated from healthy subjects and patients with JIA, and peripheral blood Th lymphocytes subsets were obtained from healthy subjects. Fragments of human cartilage from healthy subjects in a collagen matrix containing JIA or normal SFbs grafted underskin in SCID mice were used to measure cartilage degradation under the effects of Th supernatants. JIA SFbs overexpress MMP9 and MMP2 and Th17 induce both MMPs in normal SFbs, while nonclassic Th1 upregulate urokinase plasminogen activator (uPA) activity. In vitro invasive phenotype of normal SFbs is stimulated with conditioned medium of Th17 and nonclassic-Th1. In the in vivo "inverse wrap" model, normal SFbs stimulated with supernatants of Th17-lymphocytes and nonclassic Th1 produced a cartilage invasion and degradation similar to JIA SFbs. Secukinumab inhibits the cartilage damage triggered by factors produced by Th17.

Complement Expression and Activation in Osteoarthritis Joint Compartments.

Objective: To investigate complement(C) factors(F) and their activation fragments expression in OA joint tissues. Design: Immunohistochemistry and quantitative imaging were performed to analyze C3, C4, and CF (factor) B expression on osteochondral biopsies (43 patients) collected during arthroplasty. Isolated chondrocytes and synoviocytes, cartilage and synovial tissues obtained from surgical specimens of OA patients (15 patients) were cultured with or without IL-1ß. Real time PCR for CFB, C3, and C4 was performed. Culture supernatants were analyzed for C3a, C5a, CFBa, and terminal complement complex (TCC) production. Results: In osteochondral biopsies, C factor expression was located in bone marrow, in a few subchondral bone cells and chondrocytes. C3 was the most expressed while factor C4 was the least expressed factor. Gene expression showed that all C factors analyzed were expressed both in chondrocytes and synoviocytes. In chondrocyte cultures and cartilage explants, CFB expression was significantly higher than C3 and C4. Furthermore, CFB, but not C3 and C4 expression was significantly induced by IL-1ß. As to C activation factors, C3a was the most produced and CFBa was induced by IL-1ß in synovial tissue. TCC production was undetectable in isolated chondrocytes and synoviocytes cell culture supernatants, whereas it was significantly augmented in cartilage explants. Conclusion: C factors were locally produced and activated in OA joint with the contribution of all tissues (cartilage, bone, and synovium). Our results support the involvement of innate immunity in OA and suggest an association between some C alternative pathway component and joint inflammation.

Mechanotransduction and Stiffness-Sensing: Mechanisms and Opportunities to Control Multiple Molecular Aspects of Cell Phenotype as a Design Cornerstone of Cell-Instructive Biomaterials for Articular Cartilage Repair.

Since material stiffness controls many cell functions, we reviewed the currently available knowledge on stiffness sensing and elucidated what is known in the context of clinical and experimental articular cartilage (AC) repair. Remarkably, no stiffness information on the various biomaterials for clinical AC repair was accessible. Using mRNA expression profiles and morphology as surrogate markers of stiffness-related effects, we deduced that the various clinically available biomaterials control chondrocyte (CH) phenotype well, but not to equal extents, and only in non-degenerative settings. Ample evidence demonstrates that multiple molecular aspects of CH and mesenchymal stromal cell (MSC) phenotype are susceptible to material stiffness, because proliferation, migration, lineage determination, shape, cytoskeletal properties, expression profiles, cell surface receptor composition, integrin subunit expression, and nuclear shape and composition of CHs and/or MSCs are stiffness-regulated. Moreover, material stiffness modulates MSC immuno-modulatory and angiogenic properties, transforming growth factor beta 1 (TGF-ß1)-induced lineage determination, and CH re-differentiation/de-differentiation, collagen type II fragment production, and TGF-ß1- and interleukin 1 beta (IL-1ß)-induced changes in cell stiffness and traction force. We then integrated the available molecular signaling data into a stiffness-regulated CH phenotype model. Overall, we recommend using material stiffness for controlling cell phenotype, as this would be a promising design cornerstone for novel future-oriented, cell-instructive biomaterials for clinical high-quality AC repair tissue.