Anterior Cruciate Ligament Transection Model Research Articles

Disease-Modifying Effects of Lenvatinib, a Multiple Receptor Tyrosine Kinase Inhibitor, on Posttraumatic Osteoarthritis of the Knee.

Angiogenesis and vascular endothelial growth factor (VEGF) are involved in osteoarthritis (OA). We previously reported the inhibitory effect of bevacizumab in a rabbit model of OA. In the current study, we investigated the effects of lenvatinib, an angiogenesis inhibitor targeting the VEGF and fibroblast growth factor receptors, on synovitis, osteophyte formation, and cartilage degeneration in a rabbit OA model. Posttraumatic OA was induced by anterior cruciate ligament transection (ACLT) on one knee of each rabbit. Rabbits were placed into four groups according to the following lenvatinib doses: untreated control (n = 12), L0.3: 0.3 mg/kg/day (n = 15), L1.0: 1.0 mg/kg/day (n = 14), and L3.0: 3.0 mg/kg/day (n = 13) groups. We evaluated limb pain using the weight distribution ratio measured with an incapacitance tester, macroscopic osteophyte formation, and femoral condyle synovium and cartilage histology. For cartilage evaluation, the following distal sites of the femur were evaluated separately: femoral-tibial (FT), femoral-patellar (FP), and femoral corner (between FP and FT). The weight distribution ratio at 12 weeks after surgery was higher in the L0.3 and L1.0 groups than in the control group. Osteophyte formation and synovitis scores were significantly lower in the L0.3, L1.0, and L3.0 groups than in the control group. The Osteoarthritis Research Society International scores of the FT, corner, and FP sites in the L0.3 group were lower than in the control group. The cartilage thickness ratio at the FT and corner sites was significantly lower in the L0.3 group than in the control group. Krenn's grading system of cartilage synovitis showed that all lenvatinib-administered groups had significantly lower scores than the control group. MMP3 expression level in cartilage tissue was significantly lower in the L3.0 group compared with the other three groups. ADAMTS5 expression was lower in the L3.0 group compared with the control and L0.3 groups. Oral administration of lenvatinib inhibited synovitis, osteophyte formation, and cartilage degeneration and reduced pain in a rabbit ACLT model. Lenvatinib is an oral VEGF inhibitor that is easier to administer than other VEGF inhibitors and may have potential as a treatment of posttraumatic OA.

Casticin ameliorates osteoarthritic cartilage damage in rats through PI3K/AKT/HIF-1α signaling

Knee osteoarthritis (KOA) is a chronic, disabling knee joint lesion in which degeneration and defects in articular cartilage are the most important features. Casticin (CAS) is a flavonoid extracted from the Chinese herb Vitex species that has anti-inflammatory and antitumor effects. The aim of this study was to investigate the therapeutic and mechanistic effects of CAS on cartilage damage in KOA. A KOA rat model was established by anterior cruciate ligament transection (ACLT), and cartilage morphological changes were assessed by histological analysis and micro-CT scans. Subsequently, chondrocytes were treated with 10 ng/mL IL-1β to establish an OA model. CCK-8 assays and EdU assays were performed to assess the viability of CAS-treated chondrocytes. Western blotting, flow cytometry and Hoechst 33342/PI Double Stain were used to detect chondrocyte apoptosis. Western blotting, qRT‒PCR and ELISA were used to detect changes in inflammatory mediators. In addition, cartilage matrix-related indices were detected by Western blotting, qRT‒PCR and immunofluorescence (IF) analysis. Immunohistochemistry (IHC) and Western blotting were performed to detect the expression of p-PI3K, p-AKT and HIF-1α in vivo and in vitro. Micro-CT, pathological sections and related scores showed that CAS improved the alterations in bony structures and reduced cartilage damage and osteophyte formation in the ACLT model. In vivo, CAS attenuated IL-1β-induced cartilage matrix degradation, apoptosis and the inflammatory response. In addition, CAS inhibited the expression of the PI3K/AKT/HIF-1α signaling pathway in the ACLT animal model and IL-1β cell model. CAS may ameliorate cartilage damage in OA by inhibiting the PI3K/AKT/HIF-1α signaling pathway, suggesting that CAS is a potential strategy for the treatment of OA.

Protective Effects of Extracorporeal Shockwave Therapy on the Degenerated Meniscus in a Rat Model

Background: Loss of meniscal function in association with degenerative changes affects the development and progression of knee osteoarthritis, for which there is currently no effective treatment. Extracorporeal shockwave therapy (ESWT) is an established treatment for musculoskeletal disorders. However, the therapeutic effect of ESWT on meniscal degeneration remains unclear. Purpose: To evaluate the therapeutic effect of ESWT on the degenerated meniscus in an anterior cruciate ligament transection (ACLT) model. Study Design: Controlled laboratory study. Methods: Twelve-week-old male Wistar rats were randomly assigned to 3 groups (normal, ESWT–, and ESWT+). Unilateral ACLT of the right knee was performed in the latter 2 groups. At 4 weeks after ACLT, the ESWT+ group received 800 shockwave impulses at an energy flux density of 0.22 mJ/mm2 in a single session. Histological changes were examined in the posterior portion of the medial meniscus after ESWT (n = 15 per group). Real-time polymerase chain reaction (PCR) was performed after ESWT (n = 5 per group) to analyze the expression of connective tissue growth factor/CCN family member 2 (CTGF/CCN2), sex determining region Y–box 9, vascular endothelial growth factor α, aggrecan, collagen type 1 alpha 2, and collagen type 2 alpha 1 (Col2α1). Immunohistochemistry was used to analyze the expression of CTGF/CCN2 and Ki-67 (n = 5 per group) after ESWT. Results: The meniscal histopathological score at 4 weeks after ACLT was significantly higher than that in the normal group, and the score in the ESWT+ group was significantly lower than that in the ESWT– group at 4 and 12 weeks after ESWT. Real-time PCR revealed that the mRNA expression of CTGF/CCN2 and Col2α1 decreased 4 weeks after ACLT. In the ESWT+ group, real-time PCR revealed that the mRNA expression of CTGF/CCN2 increased 24 hours after ESWT, and the expression of Col2α1 increased 4 weeks after ESWT (all significant data were P < .05). The ratio of CTGF/CCN2-positive cells and Ki67-positive cells was significantly higher in the ESWT+ group after ESWT. Conclusion: The present study revealed that ESWT might suppress ACLT-induced meniscal degeneration by stimulating cartilage repair factors and inducing collagen type 2. Clinical Relevance: ESWT can be an effective treatment to protect the degenerated meniscus in a rat model of ACLT.

α2-Macroglobulin Promotes Chondrocyte Proliferation and Cartilage Matrix Synthesis via Inducing PCNA.

α2-Macroglobulin (A2M) can prevent cartilage degeneration by blocking many types of cartilage-degrading enzymes, but the mechanism remains to be clarified. This study aimed to test that A2M protects against cartilage degeneration by promoting chondrocyte proliferation and cartilage matrix synthesis via inducing proliferating cell nuclear antigen (PCNA). The cartilage degeneration of the anterior cruciate ligament transection (ACLT) model was evaluated by Safranin O-fast green staining, and articular cartilage degeneration was graded using the Osteoarthritis Research Society International (OARSI)-modified Mankin criteria. The chondrocyte proliferation was detected by 5-Bromodeoxyuridinc (BrdU), MTT, and Cell Counting Kit-8 (CCK8) methods. The chondrocyte apoptosis was detected by lactate dehydrogenase (LDH) assay and Annexin PI staining with the flow cytometer. The glycosaminoglycan (sGAG) and aggrecan in culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to analyze the type II collagen and aggrecan mRNA expression. The PCNA protein expression was analyzed by western blot and immunofluorescent staining. A2M can attenuate cartilage degeneration in ACLT rats. The OARSI scores for cartilage degeneration in the A2M group were lower than those in the phosphate-buffered saline (PBS) group. A2M can promote chondrocyte proliferation and inhibit chondrocyte apoptosis, promote the cartilage matrix synthesis in chondrocytes (type II collagen and aggrecan), and culture supernatant (sGAG and aggrecan). At the same time, it also up-regulated the PCNA protein expression in chondrocytes. A2M can promote chondrocyte proliferation and cartilage matrix synthesis via inducing PCNA expression.

Markers of Pain in 3 Different Murine Models of Posttraumatic Osteoarthritis: A Comparison Using Gait Analysis, Imaging, Histology, and Gene Expression

Background: Animal models play an important role in studying posttraumatic osteoarthritis (PTOA) disease progression. Different models exist, such as destabilization of the medial meniscus (DMM), anterior cruciate ligament (ACL) surgical transection (ACLs), and noninvasive ACL rupture. Purpose: To study the effects of PTOA on nociception in 3 different murine models and to relate these findings to macroscopic and microscopic changes in joint tissues. Study Design: Controlled laboratory study. Methods: A total of 42 male C57BL/6 mice, 12 weeks old, were randomly assigned to 4 groups: intact control (n = 10), DMM (n = 10), ACLs (n = 11), and closed ACL rupture (ACLc; n = 11) groups. Gait analysis was performed on 5 mice from the DMM group and 6 mice from ACLs and ACLc groups at 0, 1, 2, 4, 8, and 12 weeks after injury. At the 12-week time point, all mice underwent radiographs and then either micro–computed tomography imaging followed by histology and immunohistochemistry or gene expression analysis of the dorsal root ganglion and tibialis anterior muscle. Results: The peripheral and central pain markers were expressed at significantly higher levels in the synovium of both ACL injury groups when compared with the DMM group. Muscle atrophy genes were significantly upregulated in the ACL injury groups. Pain-related gait behavior started at 4 weeks for the ACL rupture groups and at 12 weeks for the DMM group. High-resolution radiographic imaging and histology demonstrated divergent changes in bone microstructure between the ACLs and DMM groups, suggesting different mechanical loading environments in these models. Conclusion: The principal finding of this study is the presence of markers of nociception at both the gene and the protein levels, with earlier expression in the ACL injury groups when compared with the DMM group. The second finding of this study is that the noninvasive ACL rupture model demonstrated changes comparable with those of the commonly used surgical ACL transection model, supporting use of this clinically realistic model in future studies of PTOA. Clinical Relevance: Quantitative clinical outcomes (imaging, pain scale, gait changes) related to osteoarthritis severity in an animal study, allowing for better understanding of clinical outcomes of osteoarthritis progression after ACL injuries in humans.

L-carnitine alleviates synovitis in knee osteoarthritis by regulating lipid accumulation and mitochondrial function through the AMPK-ACC-CPT1 signaling pathway

BackgroundKnee osteoarthritis (KOA) is a disability-associated condition that is rapidly growing with the increase in obesity rates worldwide. There is a pressing need for precise management and timely intervention in the development of KOA. l-carnitine has been frequently recommended as a supplement to increase physical activity in obese individuals due to its role in fatty acid metabolism, immune disorders, and in maintaining the mitochondrial acetyl-CoA/CoA ratio. In this study, we aimed to investigate the anti-inflammatory effects of l-carnitine on KOA and delineate a potential molecular mechanism.MethodsLipopolysaccharide-stimulated primary rat fibroblast-like synoviocytes (FLS) were treated with an AMP-activated protein kinase (AMPK) inhibitor or siRNA and carnitine palmitoyltransferase 1 (CPT1) siRNA to examine the synovial protective effects of l-carnitine. An anterior cruciate ligament transection model of rats was treated with an AMPK agonist (metformin) and CPT1 inhibitor (etomoxir) to define the therapeutic effects of l-carnitine.Resultsl-carnitine displayed a protective effect against synovitis of KOA in vitro and in vivo experiments. Specifically, l-carnitine treatment can reduce synovitis by inhibiting AMPK-ACC-CPT1 pathway activation and showed an increase in fatty acid β-oxidation, a lower lipid accumulation, and a noticeable improvement in mitochondrial function.ConclusionsOur data suggested that l-carnitine can mitigate synovitis in FLS and synovial tissue, and the underlying mechanism may be related to improving mitochondrial function and reducing lipid accumulation via the AMPK-ACC-CPT1 signaling pathway. Therefore, l-carnitine may be a potential treatment strategy for KOA.

A glucuronated flavone TMMG spatially targets chondrocytes to alleviate cartilage degeneration through negative regulation of IL-1β

Chondrocytes are the only resident cell types that form the extracellular matrix of cartilage. Inflammation alters the anabolic and catabolic regulation of chondrocytes, resulting in the progression of osteoarthritis (OA). The potential of TMMG, a glucuronated flavone, was explored against the pathophysiology of OA in both in vitro and in vivo models. The effects of TMMG were evaluated on chondrocytes and the ATDC5 cell line treated with IL-1β in an established in vitro inflammatory OA model. An anterior cruciate ligament transection (ACLT) model was used to simulate post-traumatic injury in vivo. Micro-CT and histological examination were employed to examine the micro-architectural status and cartilage alteration. Further, serum biomarkers were measured using ELISA to assess OA progression. In-vitro, TMMG reduced excessive ROS generation and inhibited pro-inflammatory IL-1β secretion by mouse chondrocytes and macrophages, which contributes to OA progression. This expression pattern closely mirrored osteoclastogenesis prevention. In-vivo results show that TMMG prevented chondrocyte apoptosis and degradation of articular cartilage thickness, subchondral parameters, and elevated serum COMP, CTX-II, and IL-1β which were significantly restored in 5 and 10 mg.kg−1day−1 treated animals and comparable to the positive control Indomethacin. In addition, TMMG also improved cartilage integrity and decreased the OARSI score by maintaining chondrocyte numbers and delaying ECM degradation. These findings suggest that TMMG may be a prospective disease-modifying agent that can mitigate OA progression.

Chrysin ameliorates synovitis and fibrosis of osteoarthritic fibroblast-like synoviocytes in rats through PERK/TXNIP/NLRP3 signaling.

Objective: Synovitis and fibrosis are common pathological features of knee osteoarthritis (KOA). The interaction of synovitis and fibrosis can promote KOA progression. Chrysin (CHR), a natural flavonoid, may treat inflammation and prevent fibrosis. However, the effect and mechanism of CHR in KOA synovitis and fibrosis remains unclear. Methods: The KOA model was established in male SD rats by anterior cruciate ligament transection (ACLT), and histological analysis was used to evaluate synovitis and fibrosis. IL-6, IL-1β and TNF-α mRNA expression in synovial tissue was measured by qRT‒PCR. Immunohistochemistry (IHC) was performed to detect GRP78, ATF-6 and TXNIP expression in vivo. Synovial fibroblasts (SFs) were treated with TGF-β1 to stimulate the inflammatory response and fibrosis. CCK-8 assays were used to detect the viability of CHR-treated SFs. The IL-1β level was detected by immunofluorescence analysis. Coimmunoprecipitation (Co-IP) and double immunofluorescence colocalization were used to detect the physiological interaction between TXNIP and NLRP3. The expression of fibrosis-related mediators and PERK/TXNIP/NLRP3 signaling molecules was detected by western blotting and qRT-PCR. Results: Four weeks after CHR treatment, pathological sections and associated scores showed that CHR improved synovitis and fibrosis in the ACLT model. In vitro, CHR attenuated the TGF-β1-induced inflammatory response and fibrosis in SFs. Moreover, CHR suppressed the expression of synovial fibrosis markers and PERK/TXNIP/NLRP3 signaling molecules in the synovial tissue of rats with ACLT and cultured SFs. More importantly, we found that CHR inhibited TXNIP-NLRP3 interactions in TGF-β-induced SFs. Conclusion: Our findings indicate that CHR can ameliorate synovitis and fibrosis in KOA. The underlying mechanism may be related to the PERK/TXNIP/NLRP3 signaling pathway.

MiR-760 targets HBEGF to control cartilage extracellular matrix degradation in osteoarthritis

The present study was developed to explore whether microRNA (miR)-760 targets heparin-binding EGF-like growth factor (HBEGF) to control cartilage extracellular matrix degradation in osteoarthritis. Both miR-760 and HBEGF expression levels were analysed in human degenerative cartilage tissues and in interleukin (IL)-1β/tumour necrosis factor (TNF)-α-treated chondrocytes in vitro. A series of knockdown and overexpression assays were then used to gauge the functional importance of miR-760 and HBEGF in OA, with qPCR and western immunoblotting analyses. Bioinformatics assays were used to identify putative miR-760 target genes, with these predictions then being validated through RNA pulldown and luciferase reporter assays. A murine anterior cruciate ligament transection model of OA was then established to prove the in vivo relevance of these findings. These experiments revealed that human degenerative cartilage tissues exhibited significant increases in miR-760 expression with a concomitant drop in HBEGF levels. IL-1β/TNF-α-treated chondrocytes also exhibited significant increases in miR-760 expression with a concomitant drop in HBEGF expression. When chondrocytes were transfected with either miR-760 inhibitor or HBEGF overexpression constructs, this was sufficient to interfere with degradation of the extracellular matrix (ECM). Moreover, miR-760 was confirmed to control chondrocyte matrix homeostasis by targeting HBEGF, and the overexpression of HBEGF partially reversed the effects of miR-760 mimic treatment on the degradation of the cartilage ECM. When OA model mice were administered an intra-articular knee injection of an adenoviral vector encoding a miR-760 mimic construct, cartilage ECM degradation was aggravated. Conversely, the overexpression of HBEGF in OA model mice partially reversed the effects of miR-760 overexpression, restoring appropriate ECM homeostasis. In summary, these data indicated that the miR-760/HBEGF axis plays a central role in orchestrating the pathogenesis of OA, making it a candidate target for therapeutic efforts in OA.

Inhibition of CaMKK2 Decreases Progression of Post-traumatic Osteoarthritis in a Rabbit ACL Transection Model

Background and Hypothesis:Post-traumatic osteoarthritis (PTOA) is a multifactorial degenerative disease of the joint affecting 20-50% of all joint injuries with a total annual cost of $15 billion. There are no current disease-modifying therapies for PTOA. Mechanical stress due to ligament tear or impact injury triggers the release of inflammatory mediators in the joint. Resulting collagen damage, loss of proteoglycans, and cell death triggers further release of inflammatory mediators and reactive oxygen species. This cycle of inflammation leads to PTOA. We hypothesize that inhibition of Ca2+/CaM dependent protein kinase kinase 2 (CaMKK2), a kinase associated with the inflammatory effects in PTOA, will mitigate the disease-propagating mechanisms.&#x0D; Methods:We utilized a rabbit model of PTOA which involved surgical transection of the anterior cruciate ligament (ACL) to generate joint instability. Rabbits were then treated tri-weekly with either STO-609 (CaMKK2 inhibitor, 0.033 mg/kg) or saline (control) for 16 weeks. Rabbits were sacrificed at 16 weeks post-surgery. Tibiofemoral joints were harvested for staining with safranin O fast green (SO) and PTOA grading via Osteoarthritis Research Society International (OARSI) guidelines. Apoptosis was assessed with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). RNA isolation of cartilage and subchondral bone tissue was conducted for qRT-PCR. Gene expression of MMP-13, IL-6, IL-1B, ACAN, COL2A1 was quantified and normalized to GAPDH.&#x0D; Results:Histology and gross morphology showed increased PTOA severity in saline controls compared to STO-609 treated rabbits. There was no significant difference in chondrocyte apoptosis in STO-609 treated rabbits compared to saline controls based on TUNEL staining. Gene expression analyses are in progress.&#x0D; Potential Impact:This study addresses the unmet clinical need for novel disease-modifying therapeutics for PTOA. Preliminary results show that inhibition of CaMKK2 has the potential to decrease cartilage degradation after joint injuries.

Therapeutic effect of induced pluripotent stem cell -derived extracellular vesicles in an in vitro and in vivo osteoarthritis model.

Osteoarthritis (OA) is a multifactorial joint disease associated with the deterioration of chondrocytes and inflammation. Treatment of OA is only aimed at reducing pain and improving joint function. Recently, extracellular vesicles (EVs) secreted from stem cells have emerged as a cell regenerative tool in several degenerative diseases, including OA. We hypothesised that induced pluripotent stem cell (iPSC)-derived EVs would be beneficial for regenerating chondrocytes and OA therapy. Therefore, we aimed to investigate iPSC-EVs' effects on chondrocyte behaviour in an interleukin 1 beta (IL-1β)-induced in vitro OA model and anterior cruciate ligament transection (ACLT)-induced in vivo OA model of rabbit articular cartilage. The iPSC-EVs were isolated by sequential ultracentrifugation from a 48-h-incubated conditional medium of iPSC. The isolated iPSC-EVs were characterised by transmission electron microscopy, western blot analyses, and dynamic light scatter. The effects of iPSC-EVs on the viability of human primary chondrocytes and cell senescence were analysed. Premature senescence of cells was induced by long-term incubation with low doses of hydrogen peroxide. To investigate the therapeutic effect of iPSC-EVs on OA chondrocytes in vitro, IL-1β was used to induce chondrocyte damage. Inflammatory macrophages were activated from THP-1 monocytes to observe the impact of iPSC-EV on macrophage polarisation. The phenotypes of the macrophages exposed to iPSC-EVs were evaluated by ELISA and western blot analyses. The primary chondrocytes were co-cultured with different phenotypes of macrophages to observe the expression of collagen II and catabolic enzymes in chondrocytes. iPSC-EVs were injected intraarticularly into the rabbit with an ACLT-induced OA model. The progression of lesions was assessed through macroscopic and histopathological studies. We showed that iPSC-EVs significantly stimulated the proliferation of primary human chondrocytes and suppressed cell senescence by regulating the expression of p21 and collagen II. iPSC-EVs reduced matrix degradation enzymes and IL-6 expression and attenuated IL-1β-mediated cell death of chondrocytes. Furthermore, iPSC-EVs modulated macrophage polarisation, resulting in the rescue of damaged chondrocytes in an inflammatory microenvironment. In the rabbit ACLT model, the OA-like lesions, including inflammation, subchondral bone protrusion, and articular cartilage destruction, were ameliorated by iPSC-EV. A histopathological study consistently revealed that iPSC-EVs attenuated ACLT-mediated alteration of MMP13 and ADAMTS5 and collagen II expression. iPSC-EVs protected chondrocytes by enhancing cell proliferation, suppressing premature senescence, and maintaining homeostasis of collagen II synthesis and matrix degradation enzymes such as matrix metalloproteinases (MMPs) and ADAMTS5. iPSC-EVs also reduced cell death in IL-1β-mediated chondrocyte cell damage. In the rabbit ACLT-induced OA model, iPSC-EV injection reduced cartilage destruction, as indicated by the upregulation of collagen II and down-regulation of MMP13 and ADAMTS5. Overall, our results suggest that iPSC-EVs possess therapeutic potential and may be used as an OA treatment option. This study highlights the potential of iPSC-EVs as a therapeutic option for chondrocyte regeneration and OA treatment.

Melatonin Prevents Chondrocyte Matrix Degradation in Rats with Experimentally Induced Osteoarthritis by Inhibiting Nuclear Factor-κB via SIRT1.

Osteoarthritis (OA) is a common degenerative joint disease characterized by an imbalance of cartilage extracellular matrix (ECM) breakdown and anabolism. Melatonin (MT) is one of the hormones secreted by the pineal gland of the brain and has anti-inflammatory, antioxidant, and anti-aging functions. To explore the role of MT in rats, we established an OA model in rats by anterior cruciate ligament transection (ACLT). Safranin O-fast green staining showed that intraperitoneal injection of MT (30 mg/kg) could alleviate the degeneration of articular cartilage in ACLT rats. Immunohistochemical (IHC) analysis found that MT could up-regulate the expression levels of collagen type II and Aggrecan and inhibit the expression levels of matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), and ADAM metallopeptidase with thrombospondin type 1 motif 4 (ADAMTS-4) in ACLT rats. To elucidate the mechanism of MT in protecting the ECM in inflammatory factor-induced rat chondrocytes, we conducted in vitro experiments by co-culturing MT with a culture medium. Western blot (WB) showed that MT could promote the expression levels of transforming growth factor-beta 1 (TGF-β1)/SMAD family member 2 (Smad2) and sirtuin 2-related enzyme 1 (SIRT1) and inhibit the expression of levels of phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibi-tor (p-p65) and phosphorylated IκB kinase-α (p-IκBα). In addition, WB and real-time PCR (qRT-PCR) results showed that MT could inhibit the expression levels of MMP-3, MMP-13, ADAMTS-4, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) in chondrocytes induced by interleukin-1β (IL-1β), and up-regulate the expression of chondroprotective protein type II collagen. We found that in vivo, MT treatment protected articular cartilage in the rat ACLT model. In IL-1β-induced rat chondrocytes, MT could reduce chondrocyte matrix degradation by up-regulating nuclear factor-kB (NF-κB) signaling pathway-dependent expression of SIRT1 and protecting chondrocyte by activating the TGF-β1/Smad2 pathway.

Eugenol protects chondrocytes and articular cartilage by downregulating the JAK3/STAT4 signaling pathway.

Osteoarthritis (OA) is a chronic degenerative bone and joint disease common in middle-aged and elderly people. Currently, there is no satisfactory pharmacological treatment. Eugenol is a phenolic compound that has been shown to exert biological anti-inflammatory, antioxidant, and antiapoptotic effects in multiple systems and organs of the human body. However, its therapeutic effect on OA is unclear. This study examined the effect of eugenol on OA using an anterior cruciate ligament transection (ACLT) model in mice and its related signaling pathways in interleukin-1β (IL-1β)-stimulated human chondrocytes. A certain concentration of eugenol inhibited the decrease in cell viability induced by IL-1β or carbonyl cyanide 3-chlorophenylhydrazone (CCCP). In vitro, eugenol effectively inhibited CCCP-induced chondrocyte apoptosis and mitochondrial membrane potential changes and inhibited the expressions of ADAMTS4 and MMP13 upregulated by IL-1β. In vivo, ACLT induced destruction of the articular cartilage and subchondral bone of the mouse tibial plateau, while eugenol effectively protected the cartilage and subchondral bone from such damage. At the same time, eugenol reduced the ACLT-induced upregulation of ADAMTS4 and MMP13 and the downregulation of type II collagen (COLII) and aggrecan in the mouse knee cartilage. Eugenol also inhibited the increased expression of cartilage metabolism signaling molecules such as C-telopeptides of COLII (CTX-II) in ACLT-induced mouse serum. Consistent with the specific changes in the messenger RNA chip, eugenol inhibited the phosphorylation of JAK3 and STAT4 induced by IL-1β. Together, these results suggest eugenol as an effective new drug for the prevention and treatment of OA.

Quantitative high-resolution 7T MRI to assess longitudinal changes in articular cartilage after anterior cruciate ligament injury in a rabbit model of post-traumatic osteoarthritis

ObjectiveTo demonstrate an ultra-high field (UHF) 7 ​T delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) protocol for quantitative post-traumatic osteoarthritis (PTOA) detection and monitoring in a rabbit anterior cruciate ligament transection (ACLT) model. DesignACL transections were performed unilaterally in 5 rabbits (33-weeks-old, 3.5 ​± ​0.5 ​kg) to induce PTOA. MRI exams were performed at 7 ​T prior to and 2, 4, 7 and 10-weeks after ACLT using a modified dGEMRIC protocol. Voxel-based T1 and T2 maps were created over manually drawn femoral cartilage ROIs from the center of the tibial plateau to the posterior meniscus. Femoral, tibial, and patellar epiphyses were harvested 10-weeks post-surgery and processed for μCT imaging and histology. ResultsQuantitative analysis revealed a 35% and 39% decrease in dGEMRIC index in the medial ACLT knee compartment 7- and 10-weeks post-surgery, respectively (p ​= ​0.009 and p ​= ​0.006) when compared to baseline. There was no significant change in the lateral ACLT compartment or in either compartment of the control knees. Visual inspection of histology confirmed PTOA in the ACLT knees. Osteophytes were found only in ACLT knees (osteophyte volume in femur: 94.53 ​± ​44.08 ​mm3, tibia: 29.35 ​± ​13.79 ​mm3, and patella: 3.84 ​± ​0.92 ​mm3) and were significantly larger in the medial compartments of the femur than lateral (p ​= ​0.0312). ConclusionThe dGEMRIC technique quantitatively applied at 7 ​T UHF-MRI demonstrates site-specific cartilage degeneration in a large animal PTOA model. This should encourage further investigation, with potential applications in drug and therapeutic animal trials as well as human studies.

THE KINEMATIC ANALYSIS OF ANTERIOR CRUCIATE LIGAMENT-TRANSECTION MODEL AND RE STABILIZED MODEL

Purpose: Joint instability is a substantial contributor to cartilage degeneration and induce osteoarthritis (OA) of the knee. Besides, mechanical stress alters physiology or pathophysiology of articular cartilage and contribute the OA progression. Anterior cruciate ligament-transection (ACLT) model is widely used as knee OA model and induce OA during a few weeks. Previous reports showed that regaining joint stability after ACLT by controlled abnormal joint movement (CAM) delay the progression of OA via suppress inflammatory cytokines.On this model, the kinematically change by knee stabilization may affect the change of mechanical stress to cartilage.

SUGGESTION OF NEW PTOA MICE MODEL INDUCED BY MECHANICAL STRESS WITHOUT SURGERY

Purpose: Knee osteoarthritis (OA) is characterized by articular cartilage degeneration, synovitis, and subchondral bone changes and early intervention are emphasized because of irreversible joint deformation. Various factors are involved in OA progression, especially abnormal mechanical stress by joint instability recognized as one of the most exacerbating factors causes OA. So far, a lot of studies that use animal model to elucidate OA onset have been reported and the most common PTOA model used in these studies is anterior cruciate ligament-transection (ACL-T) model.

Injectable recombinant block polymer gel for sustained delivery of therapeutic protein in post traumatic osteoarthritis

Protein-based biomaterials offer several advantages over synthetic materials, owing to their unique stimuli-responsive properties, biocompatibility and modular nature. Here, we demonstrate that E5C, a recombinant protein block polymer, consisting of five repeats of elastin like polypeptide (E) and a coiled-coil domain of cartilage oligomeric matrix protein (C), is capable of forming a porous networked gel at physiological temperature, making it an excellent candidate for injectable biomaterials. Combination of E5C with Atsttrin, a chondroprotective engineered derivative of anti-inflammatory growth factor progranulin, provides a unique biochemical and biomechanical environment to protect against post-traumatic osteoarthritis (PTOA) onset and progression. E5C gel was demonstrated to provide prolonged release of Atsttrin and inhibit chondrocyte catabolism while facilitating anabolic signaling in vitro. We also provide in vivo evidence that prophylactic and therapeutic application of Atsttrin-loaded E5C gels protected against PTOA onset and progression in a rabbit anterior cruciate ligament transection model. Collectively, we have developed a unique protein-based gel capable of minimally invasive, sustained delivery of prospective therapeutics, particularly the progranulin-derivative Atsttrin, for therapeutic application in OA.

Intra-Articular Adeno-Associated Virus-Mediated Proteoglycan 4 Gene Therapy for Preventing Posttraumatic Osteoarthritis.

Lubricin, a glycoprotein encoded by the proteoglycan 4 (PRG4) gene, is an essential boundary lubricant that reduces friction between articular cartilage surfaces. The loss of lubricin subsequent to joint injury plays a role in the pathogenesis of posttraumatic osteoarthritis. In this study, we describe the development and evaluation of an adeno-associated virus (AAV)-based PRG4 gene therapy intended to restore lubricin in injured joints. The green fluorescent protein (GFP) gene was inserted the PRG4 gene to facilitate tracing the distribution of the transgene product (AAV-PRG4-GFP) in vivo. Transduction efficiency of AAV-PRG4-GFP was evaluated in joint cells, and the conditioned medium containing secreted PRG4-GFP was used for shear loading/friction and viability tests. In vivo transduction of joint tissues following intra-articular injection of AAV-PRG4-GFP was confirmed in the mouse stifle joint in a surgical model of destabilization of the medial meniscus (DMM), and chondroprotective activity was tested in a rabbit anterior cruciate ligament transection (ACLT) model. In vitro studies showed that PRG4-GFP has lubricin-like cartilage-binding and antifriction properties. Significant cytoprotective effects were seen when cartilage was soaked in PRG4-GFP before cyclic shear loading (n = 3). Polymerase chain reaction and confocal microscopy confirmed the presence of PRG4-GFP DNA and protein, respectively, in a mouse DMM (n = 3 per group). In the rabbit ACLT model, AAV-PRG4-GFP gene therapy enhanced lubricin expression (p = 0.001 vs. AAV-GFP: n = 7-14) and protected the cartilage from degeneration (p = 0.014 vs. AAV-GFP: n = 9-10) when treatments were administered immediately postoperation, but efficacy was lost when treatment was delayed for 2 weeks. AAV-PRG4-GFP gene therapy protected cartilage from degeneration in a rabbit ACLT model; however, data from the ACLT model suggest that early intervention is essential for efficacy.

Assessment of osteoarthritis functional outcomes and intra-articular injection volume in the rat anterior cruciate ligament transection model.

The rat surgical anterior cruciate ligament transection (ACLT) model is commonly used to investigate intra-articular osteoarthritis (OA) therapies,and histological assessment is often the primary outcome measure. However, histological changes do not always correlate well with clinical outcomes. Therefore, this study evaluated functional outcomes in the rat surgical ACLT model and compared intra-articular injection volumes ranging from 20 to 50 μl. Unilateral ACLT was surgically inducedand static weight-bearing, mechanical allodynia, motor function, and gait were assessed in four groups of male, Sprague-Dawley rats (n = 6 per group). Intra-articular injections of 20 µl Dulbecco's phosphate-buffered saline (DPBS), 50 µl DPBS, or 50 µl of synthetic biomimetic boundary lubricant were administered once weekly for 3 weeks postoperatively. Structural changes were evaluated histologically at 20 weeks. Rat cadaver knees were injected with 20, 30, 40, or 50 µl of gadolinium solutions and were imaged using magnetic resonance imaging (MRI). Static weight-bearing, mechanical allodynia, and gait parameters in ACLT groups revealed differences from baseline and naïve controls for 4 weeks post-ACLT; however, these differences did not persist beyond 6 weeks. Different intra-articular DPBS injection volumes did not result in functional or histological changes; however, peri-articular leakage was documented via MRI following 50, 40, and 30 µl but not 20 µl gadolinium injections.Statement of clinical significance:Differences in functional parameters were predominantly restricted to early, postoperative changes in the rat surgical ACLT model despite evidence of moderate histologic OA at 20 weeks. Injection volumes of 20-30 µl are more appropriate for investigating intra-articular therapies in the rat knee.

Abrine promotes cell proliferation and inhibits apoptosis of interleukin-1β-stimulated chondrocytes via PIM2/VEGF signalling in osteoarthritis

BackgroundOsteoarthritis (OA), a common joint disorder with an increasing incidence worldwide, severely affects the quality of life of patients. In Chinese herbal medicine, Abrus cantoniensis Hance is considered to exert protective effects on the liver and to have beneficial effects on the gallbladder; additionally, it has antibacterial and anti-inflammatory properties, as well as the ability to enhance immunity, scavenge free radicals, regulate smooth muscle function, and improve endurance. Abrine extracted from A. cantoniensis Hance has been reported as a main functional compound capable of treating chronic inflammation. PurposeIn this study, we explored the effect of abrine on OA progression. Study DesignBioinformatics analysis was performed on abrine and its potential targets in OA, using the Comparative Toxicogenomics Database, GSE1919 dataset from the Gene Expression Omnibus database, Gene Set Enrichment Analysis, and docking interaction analysis. MethodsThe effect of abrine in vitro was analysed by Cell Counting Kit 8 assays, colony formation assays, enzyme-linked immunosorbent assay, flow cytometry analysis, quantitative real-time PCR, and western blotting using human transformed chondrocyte cell line C28/I2. The effect of abrine was evaluated in vivo using the anterior cruciate ligament transection (ACLT) Sprague-Dawley rat OA model. ResultsAbrine enhanced the proliferation of interleukin (IL)-1β-stimulated C28/I2 cells in a dose-dependant manner. Expression of pro-inflammatory cytokines was induced by IL-1β treatment, whereas abrine treatment repressed the induction of C28/I2 cells in a dose dependant manner (p < 0.05). Abrine induced cell proliferation and inhibited apoptosis in IL-1β-stimulated C28/I2 cells (p < 0.05). Abrine also inhibited Proviral Integrations of Moloney virus 2 (PIM2) expression in IL-1β-stimulated C28/I2 cells (p < 0.05). The expression of vascular endothelial growth factor (VEGF), p-VEGFR2, and p-eNOS was induced by IL-1β treatment in C28/I2 cells, while abrine inhibited this induction in a dose dependant manner. Treatment with abrine decreased the expression levels of PIM2 and VEGF in IL-1β-stimulated C28/I2 cells (p < 0.05). Overexpression of PIM2 induced cell proliferation and inhibited apoptosis in IL-1β-stimulated C28/I2 cells, while VEGF silencing reversed this effect (p < 0.05). Finally, abrine prevented cartilage degradation in the ACLT model. ConclusionWe demonstrated that abrine promoted cell proliferation and inhibited apoptosis in IL-1β-stimulated C28/I2 cells through PIM2/VEGF signalling. These findings indicate PIM2 to be a potential drug target. Moreover, abrine has potential applicability as a therapeutic agent against OA.