Cartilage Glycosaminoglycan Research Articles

Clodronate disodium does not produce measurable effects on bone metabolism in an exercising, juvenile, large animal model.

Bisphosphonates are commonly used to treat and prevent bone loss, but their effects in active, juvenile populations are unknown. This study examined the effects of intramuscular clodronate disodium (CLO) on bone turnover, serum bone biomarkers (SBB), bone mineral density (BMD), bone microstructure, biomechanical testing (BT), and cartilage glycosaminoglycan content (GAG) over 165 days. Forty juvenile sheep (253 ± 6 days of age) were divided into four groups: Control (saline), T0 (0.6 mg/kg CLO on day 0), T84 (0.6 mg/kg CLO on day 84), and T0+84 (0.6 mg/kg CLO on days 0 and 84). Sheep were exercised 4 days/week and underwent physical and lameness examinations every 14 days. Blood samples were collected for SBB every 28 days. Microstructure and BMD were calculated from tuber coxae (TC) biopsies (days 84 and 165) and bone healing was assessed by examining the prior biopsy site. BT and GAG were evaluated postmortem. Data, except lameness data, were analyzed using a mixed-effects model; lameness data were analyzed as ordinal data using a cumulative logistic model. CLO did not have any measurable effects on the skeleton of sheep. SBB showed changes over time (p ≤ 0.03), with increases in bone formation and decreases in some bone resorption markers. TC biopsies showed increasing bone volume fraction, trabecular spacing and thickness, and reduced trabecular number on day 165 versus day 84 (p ≤ 0.04). These changes may be attributed to exercise or growth. The absence of a treatment effect may be explained by the lower CLO dose used in large animals compared to humans. Further research is needed to examine whether low doses of bisphosphonates may be used in active juvenile populations for analgesia without evidence of bone changes.

Screening of poly-beta amino ester coated emulsion of ketorolac for cartilage delivery.

Osteoarthritis (OA) is a prevalent chronic health condition necessitating effective treatment strategies. Globally, there were 86 million people with incident knee osteoarthritis in 2020. Pain management remains the primary approach to OA as the nature of cartilage poses challenges for drug delivery. An emulsion-based delivery system, using a class of positively charged and hydrolysable polymers (poly-beta-amino-esters) to coat oil droplets containing drugs, has been shown to enhance and prolong drug localization in ex vivo cartilage models. As the properties of the polymers used in this technology strongly depend on the monomers used in the synthesis, this study presents the screening of a wide range of PBAEs as droplet coating agents and using ketorolac as a model of nonsteroidal anti-inflammatory drugs. The emulsions prepared with this PBAE library were characterized, and drug localisation and retention were evaluated in both native and glycosaminoglycan (GAG) depleted cartilage ex vivo models. Optimal candidates were identified and tested in an ex vivo model showing the ability to protect chondrocyte cell viability and increase both GAG and collagen contents in cartilage exposed to cytokine (IL-1α) simulating acute cartilage damage. This work demonstrates the potential of PBAE coated emulsion as a delivery system for effective drug delivery in OA treatment.

Age-Related Histologic and Biochemical Changes in Auricular and Nasal Cartilages.

Analyze age-related changes in histologic features and biochemical properties of human auricular cartilage and two subsites of nasal cartilages (quadrangular cartilage and dorsal septal articulation with upper lateral cartilages). Prospective cross-sectional study of nasal and auricular cartilages from seventy-three (73) live donors. Auricular cartilage (AC), quadrangular cartilage (QC), and dorsal septal cartilage articulation (DSA) with the upper lateral cartilage (ULCs) were collected intraoperatively. Histochemical staining was used: Safranin O for glycosaminoglycans (GAGs), Verhoeff's for elastin, and Masson's trichrome for collagen. ImageJ2 software was used to calculate cell count and percent stained for each cartilage type. R studio "ggplot" package was used to visualize age versus cell count or percent stained. Participant ages ranged from 20 to 77 years, average 46.5 years. There was a significant decline in GAGs with age for the DSA subsite, (n = 64, p < 0.001). Significant increase in collagen content with age was observed for DSA subsite (n = 66, p < 0.001) and the QC subsite (n = 64, p < 0.05). There was a statistically insignificant decline in elastin with age (n = 41, p = 0.309) for AC. Cell count declined with age at all cartilage subsites. Our findings confirm that there were age-related decreases in cartilage glycosaminoglycan content, and chondrocyte cell count in both auricular and nasal cartilages. We have also confirmed that collagen content increases with age for both auricular and nasal cartilage. The histologic findings while not statistically significant in all comparisons, provides additional evidence that there is some loss of structural integrity and flexibility in nasal and auricular cartilage with aging. NA Laryngoscope, 134:1220-1226, 2024.

Cationic Carrier Mediated Delivery of Anionic Contrast Agents in Low Doses Enable Enhanced Computed Tomography Imaging of Cartilage for Early Osteoarthritis Diagnosis.

Cartilage tissue exhibits early degenerative changes with onset of osteoarthritis (OA). Early diagnosis is critical as there is only a narrow time window during which therapeutic intervention can reverse disease progression. Computed tomography (CT) has been considered for cartilage imaging as a tool for early OA diagnosis by introducing radio-opaque contrast agents like ioxaglate (IOX) into the joint. IOX, however, is anionic and thus repelled by negatively charged cartilage glycosaminoglycans (GAGs) that hinders its intra-tissue penetration and partitioning, resulting in poor CT attenuation. This is further complicated by its short intra-tissue residence time owing to rapid clearance from joints, which necessitates high doses causing toxicity concerns. Here we engineer optimally charged cationic contrast agents based on cartilage negative fixed charge density by conjugating cartilage targeting a cationic peptide carrier (CPC) and multi-arm avidin nanoconstruct (mAv) to IOX, such that they can penetrate through the full thickness of cartilage within 6 h using electrostatic interactions and elicit similar CT signal with about 40× lower dose compared to anionic IOX. Their partitioning and distribution correlate strongly with spatial GAG distribution within healthy and early- to late-stage arthritic bovine cartilage tissues at 50-100× lower doses than other cationic contrast agents used in the current literature. The use of contrast agents at low concentrations also allowed for delineation of cartilage from subchondral bone as well as other soft tissues in rat tibial joints. These contrast agents are safe to use at current doses, making CT a viable imaging modality for early detection of OA and staging of its severity.

Mass spectrometric investigations of the action of hypochlorous acid on monomeric and oligomeric components of glycosaminoglycans

Hypochlorous acid (HOCl) is a strong non-radical oxidant, which is generated during inflammatory processes under the catalysis of the enzyme myeloperoxidase (MPO). HOCl reacts particularly with sulfhydryl and amino acid residues but affects also many other biomolecules. For instance, the glycosaminoglycans of articular cartilage and synovial fluids (such as hyaluronan) undergo degradation in the presence of HOCl at which the native polysaccharide is fragmented into oligosaccharides in a complex reaction.This is an initial mass spectrometry (MS)-based investigation dealing with the HOCl-induced degradation of glycosaminoglycans and the conversion of the related monosaccharides into chlorinated products. In particular, it will be shown that the reaction between HOCl and hyaluronan is slower than originally assumed and results in the generation of different products (particularly the hyaluronan monosaccharides) by the cleavage of the β-1,3/1,4-glycosidic linkages. The MS detection of chlorinated products is, however, only possible in the case of the monosaccharides. Potential reasons will be discussed.

Chondroitin Sulfate-Tyramine-Based Hydrogels for Cartilage Tissue Repair.

The degradation of cartilage, due to trauma, mechanical load or diseases, results in abundant loss of extracellular matrix (ECM) integrity and development of osteoarthritis (OA). Chondroitin sulfate (CS) is a member of the highly sulfated glycosaminoglycans (GAGs) and a primary component of cartilage tissue ECM. In this study, we aimed to investigate the effect of mechanical load on the chondrogenic differentiation of bone marrow mesenchymal stem cells (BM-MCSs) encapsulated into CS-tyramine-gelatin (CS-Tyr/Gel) hydrogel in order to evaluate the suitability of this composite for OA cartilage regeneration studies in vitro. The CS-Tyr/Gel/BM-MSCs composite showed excellent biointegration on cartilage explants. The applied mild mechanical load stimulated the chondrogenic differentiation of BM-MSCs in CS-Tyr/Gel hydrogel (immunohistochemical collagen II staining). However, the stronger mechanical load had a negative effect on the human OA cartilage explants evaluated by the higher release of ECM components, such as the cartilage oligomeric matrix protein (COMP) and GAGs, compared to the not-compressed explants. Finally, the application of the CS-Tyr/Gel/BM-MSCs composite on the top of the OA cartilage explants decreased the release of COMP and GAGs from the cartilage explants. Data suggest that the CS-Tyr/Gel/BM-MSCs composite can protect the OA cartilage explants from the damaging effects of external mechanical stimuli. Therefore, it can be used for investigation of OA cartilage regenerative potential and mechanisms under the mechanical load in vitro with further perspectives of therapeutic application in vivo.

The effect of chondroitin sulfate concentration and matrix stiffness on chondrogenic differentiation of mesenchymal stem cells.

Chondroitin sulfate (CS), a glycosaminoglycan of native cartilage, has shown its potential in promoting chondrogenesis of mesenchymal stem cells (MSCs), whereas the effect of matrix stiffness in a CS-containing 3D environment on chondrogenesis is still poorly understood. Herein, this study aimed at assessing the effect of CS concentration and stiffness of CS-containing hydrogels on the chondrogenesis of MSCs. Hydrogels composed of 6% (w/v) gelatin methacryloyl (GelMA) and three concentrations, i.e., 4%, 6%, or 10% (w/v), of methacrylated chondroitin sulfate (CSMA) were prepared. The hydrogels of each composition were prepared with two stiffness values (33.36 ± 8.25 kPa vs. 8.42 ± 2.83 kPa). Physical characterization showed similar microporous structures among the six groups, higher swelling ratios and faster degradation in the soft hydrogel groups. MSCs were encapsulated in the six groups of hydrogels and they underwent 28-day chondrogenic differentiation. The cell viability in each group on day 1 was similar and most cells exhibited a round shape without spreading. Afterwards, cellular protrusions in soft hydrogels remained filopodium-like from day 14 to day 28, while most protrusions were lamellipodium-like in stiff hydrogels on day 14 and then transformed into a spherical shape on day 28. The expression of chondrogenic markers analysed by real-time qPCR and immunohistochemical staining demonstrated that the optimal CS concentration for chondrogenesis was 6% (w/v) regardless of the stiffness of hydrogels. In addition, with the same CSMA concentration, the trend was observed that the stiff hydrogels supported superior chondrogenesis of MSCs compared to the soft hydrogel. To summarize, this study presents an advancement in the optimization of CSMA concentration and stiffness of hydrogels for chondrogenesis. In the CSMA/GelMA hydrogel, 6% (w/v) CSMA with an initial Young's modulus around 33 kPa was recommended for cartilage tissue engineering.

A novel whole “Joint-in-Motion” device reveals a permissive effect of high glucose levels and mechanical stress on joint destruction

Osteoarthritis (OA) has recently been suggested to be associated with diabetes. However, this association often disappears when accounting for body mass index (BMI), suggesting that mechanical stress may be a confounding factor. We investigated the combined influence of glucose level and loading stress on OA progression using a novel whole joint-in-motion (JM) culture system. Whole mouse knee joints were placed in an enclosed chamber with culture media and actuated to recapitulate leg movement, with a dynamic stress regimen of 0.5Hz, 8h/day for 7 days. These joints were treated with varying levels of glucose and controlled for osmolarity and diffusion. Joint movement and joint space were examined by X-ray fluoroscopy and microCT. Cartilage matrix levels were quantified by blinded Mankin scoring and immunohistochemistry. Culturing in the JM device facilitated proper leg extension and flexion movements, and adequate mass transport for analyzing the effect of glucose on cartilage. Treatment with higher levels of glucose either via media supplementation or intra-articular injection caused a significant decrease in levels of glycosaminoglycan (GAG) and an increase in aggrecan neoepitope in articular cartilage, but only under dynamic stress. Additionally, collagen II level was slightly reduced by high glucose levels. High levels of glucose and dynamic stress have permissive effects on articular cartilage GAG loss and aggrecan degradation, implicating that mechanical stress confounds the association of diabetes with OA. The JM device supports novel investigation of mechanical stress on the integrity of an intact living mouse joint to provide insights into OA pathogenesis.

Human Adipose- and Amnion-Derived Mesenchymal Stromal Cells Similarly Mitigate Osteoarthritis Progression in the Dunkin Hartley Guinea Pig

Background: Clinical trials are currently underway to investigate the efficacy of intra-articular administration of mesenchymal stromal cells (MSCs) to mitigate osteoarthritis (OA) progression in the knee. Although multiple MSC sources exist, studies have yet to determine whether differences in therapeutic efficacy exist between them. Purpose: To compare the ability of intra-articularly injected adipose-derived MSCs (AD-MSCs) and amnion-derived MSCs (AM-MSCs) to mitigate the progression of knee OA in a small animal model of spontaneous OA, as well as to compare the therapeutic potential of MSCs in hyaluronic acid (HA) and in HA only with saline (OA) controls. Study Design: Controlled laboratory study. Methods: Injections of AD-MSCs or AM-MSCs suspended in HA or HA only were performed in the rear stifle joints of 3-month-old Dunkin Hartley guinea pigs (DHGPs). Repeat injections occurred at 2 and 4 months after the initial injection in each animal. Contralateral limbs received saline injections and served as untreated controls. Subsequently, joints were analyzed for osteoarthritic changes of the cartilage and subchondral bone via histologic and biochemical analyses. To evaluate MSC retention time in the joint space, DHGPs received a single intra-articular injection of fluorescently labeled AD-MSCs or AM-MSCs, and the fluorescence intensity was longitudinally tracked via an in vivo imaging system. Results: No statistically significant differences in outcomes were found when comparing the ability of AD-MSCs and AM-MSCs to mitigate OA. However, the injection of AD-MSCs, AM-MSCs, and HA-only treatments more effectively mitigated cartilage damage compared with that of saline controls by demonstrating higher amounts of cartilage glycosaminoglycan content and improved histological proteoglycan scoring while reducing the percentage of osteophytes present. Conclusion: Intra-articular injection of AD-MSCs, AM-MSCs, or HA only was able to similarly mitigate the progression of cartilage damage and reduce the percentage of osteophytes compared with that of saline controls in the DHGP. However, this study was unable to establish the superiority of AD-MSCs versus AM-MSCs as a treatment to mitigate spontaneous OA. Clinical Relevance: MSCs demonstrate the ability to mitigate the progression of knee OA and thus may be used in a prophylactic approach to delay the need for end-stage treatment strategies.

The Impact of Circular Exercise Diameter on Bone and Joint Health of Juvenile Animals.

Simple SummaryIn many equestrian disciplines, circular exercise is utilized to train, exercise, and compete with horses and can vary in gait, as well as diameter. This study aimed to determine if circular exercise diameter impacts animal health. Calves have previously been used as a terminal skeletal model of juvenile horses, allowing for the collection of musculoskeletal samples that are not acceptable from horses. Calves exercised on a small circle (12-m clockwise), large circle (18-m clockwise), treadmill, or served as non-exercised controls. Exercise was performed at a walking speed, starting at 5 min per day and increasing 5 min weekly until reaching 30 min per day during the 7-week study. The response to exercise was monitored in the forelimb bones and joints. The small circular exercise group was found to have bone diameters that differed between the right and left fused third and fourth metacarpi, and between lateral and medial proximal phalanx bones. Cartilage glycosaminoglycan content was greater in the outside leg of the small circle exercise calves than the inside leg, with no differences noted within other treatments. These differences suggest that altering circular exercise diameter can impact bone and joint health, and that larger diameter circles may prevent asymmetric loading between inside and outside legs.Circular exercise is used in many equestrian disciplines and this study aimed to determine if circle diameter impacts juvenile animal forelimb bone and joint health. On day 0, 24 calves at 9 weeks of age were assigned the following exercise treatments: small circle (12 m clockwise), large circle (18-m clockwise), treadmill, or non-exercised control. Exercise was initiated at 1.1–1.5 m/s for 5 min/d and increased 5 min weekly until reaching 30 min/d. On day 49, synovial fluid was collected from multiple joints, cartilage was collected from the proximal surface of fused third and fourth metacarpi (MC III and IV), and forelimbs underwent computed tomography scans. A statistical analysis (PROC mixed) was performed in SAS 9.4. The inside leg of the small circle treatment had a larger MC III and IV dorsopalmar external diameter than the outside (p = 0.05). The medial proximal phalanx had a greater mediolateral diameter than the lateral proximal phalanx of the small circle treatment (p = 0.01). Fetlock nitric oxide was greater in the large circle and treadmill treatments (p < 0.0001). Cartilage glycosaminoglycan concentration was greater in the outside leg of the small circle exercise treatment than the inside leg (p = 0.03). Even at slow speeds, circular exercise diameter can impact joint and bone health, but faster speeds may have greater alterations.

Bone Biomarkers in Mucopolysaccharidoses

The accumulation of glycosaminoglycans (GAGs) in bone and cartilage leads to progressive damage in cartilage that, in turn, reduces bone growth by the destruction of the growth plate, incomplete ossification, and growth imbalance. The mechanisms of pathophysiology related to bone metabolism in mucopolysaccharidoses (MPS) include impaired chondrocyte function and the failure of endochondral ossification, which leads to the release of inflammatory cytokines via the activation of Toll-like receptors by GAGs. Although improvements in the daily living of patients with MPS have been achieved with enzyme replacement, treatment for the bone disorder is limited. There is an increasing need to identify biomarkers related to bone and cartilage to evaluate the progressive status and to monitor the treatment of MPS. Recently, new analysis methods, such as proteomic analysis, have identified new biomarkers in MPS. This review summarizes advances in clinical bone metabolism and bone biomarkers.

Cyclooxygenase-2 regulates PTHrP transcription in human articular chondrocytes and is involved in the pathophysiology of osteoarthritis in rats.

BackgroundCyclooxygenase-2 (COX-2) inhibitors are prescribed for the management of osteoarthritis (OA)-associated pain and inflammation. However, the role of COX-2 in normal and osteoarthritic articular chondrocytes has not been well investigated. We hypothesize that COX-2 plays a role in articular chondrocytes under normal conditions and during OA progression.MethodsIn vivo COX-2 levels in articular cartilage of normal and papain-induced osteoarthritic rats were compared. The role of COX-2 in human articular chondrocytes (HACs) was tested in vitro by COX-2 overexpression or activity inhibition. The levels of COX-2 and marker gene for normal function or articular cartilage degeneration were evaluated: mRNA by qRT-PCR; proteins by western blotting or immunohistochemistry; and glycosaminoglycan (GAG) by Safranin O–fast green staining. Parathyroid hormone-related protein (PTHrP) promoter activity was detected with luciferase reporter assays.ResultsIn the OA rat study, COX-2 and PTHrP were simultaneously increased in osteoarthritic rat chondrocytes, while increased PTHrP levels were reduced by celecoxib, a COX-2 selective inhibitor. The levels of normal cartilage matrices, GAG and type II collagen decreased, while markers of degeneration, collagen type X and MMP13 were elevated in osteoarthritic articular chondrocytes. Celecoxib rescued the loss of GAG and the increased collagen type X and MMP13 levels. In vitro, COX-2 overexpression in HACs significantly increased Col2a1, Col10a1, PTHrP and MMP13 mRNA expression, which was decreased when COX-2 activity was suppressed. More importantly, COX-2 overexpression upregulated the PTHrP transcription, mRNA expression and protein levels.ConclusionCOX-2 plays a pathophysiological role by preventing terminal differentiation of articular chondrocytes by upregulating PTHrP expression at the early stage of OA progression.The Translational potential of this articleCOX2 up-regulates PTHrP expression in normal and OA articular chondrocytes.

Spaceflight and hind limb unloading induces an arthritic phenotype in knee articular cartilage and menisci of rodents

Reduced knee weight-bearing from prescription or sedentary lifestyles are associated with cartilage degradation; effects on the meniscus are unclear. Rodents exposed to spaceflight or hind limb unloading (HLU) represent unique opportunities to evaluate this question. This study evaluated arthritic changes in the medial knee compartment that bears the highest loads across the knee after actual and simulated spaceflight, and recovery with subsequent full weight-bearing. Cartilage and meniscal degradation in mice were measured via microCT, histology, and proteomics and/or biochemically after: (1) ~ 35 days on the International Space Station (ISS); (2) 13-days aboard the Space Shuttle Atlantis; or (3) 30 days of HLU, followed by a 49-day weight-bearing readaptation with/without exercise. Cartilage degradation post-ISS and HLU occurred at similar spatial locations, the tibial-femoral cartilage-cartilage contact point, with meniscal volume decline. Cartilage and meniscal glycosaminoglycan content were decreased in unloaded mice, with elevated catabolic enzymes (e.g., matrix metalloproteinases), and elevated oxidative stress and catabolic molecular pathway responses in menisci. After the 13-day Shuttle flight, meniscal degradation was observed. During readaptation, recovery of cartilage volume and thickness occurred with exercise. Reduced weight-bearing from either spaceflight or HLU induced an arthritic phenotype in cartilage and menisci, and exercise promoted recovery.

Real-Time Quantification of Cartilage Degeneration by GAG-Targeted Cationic Nanoparticles for Efficient Therapeutic Monitoring in Living Mice.

One of the characterizations of degenerative cartilage disease is the progressive loss of glycosaminoglycans (GAGs). The real-time imaging method to quantify GAGs is of great significance for the biochemical analysis of cartilage and diagnosis and therapeutic monitoring of cartilage degeneration in vivo. To this end, a cationic photoacoustic (PA) contrast agent, poly-l-lysine melanin nanoparticles (PLL-MNPs), specifically targeting anionic GAGs was developed in this study to investigate whether it can image cartilage degeneration. PLL-MNP assessed GAG depletion by Chondroitinase ABC in vitro rat cartilage and intact ex vivo mouse knee joint. A papain-induced cartilage degenerative mice model was used for in vivo photoacoustic imaging (PAI). Oral cartilage supplement glucosamine sulfate was intragastrically administered for mice cartilage repair and the therapeutic efficacy was monitored by PLL-MNP-enhanced PAI. Histologic findings were used to further confirm PAI results. In vitro results revealed that the PLL-MNPs not only had a high binding ability with GAGs but also sensitively monitored GAG content changes by PAI. The PA signal was gradually weakened along with the depletion of GAGs in cartilage. Particularly, PLL-MNPs depicted the cartilage structure and the distribution of GAGs was demonstrated in PA images in ex vivo joints. Compared with the normal joint, a lower signal intensity was detected from degenerative joint at 3 weeks after papain injection, suggesting an early diagnosis of cartilage lesion by PLL-MNPs. Importantly, this PA-enhanced nanoprobe was suitable for monitoring in vivo efficacy of glucosamine sulfate, which effectively blocked cartilage degradation in a high dose manner. In vivo imaging findings correlated well with histological examinations. PLL-MNPs provided sensitive visualization of cartilage degeneration and promising monitoring of therapeutic response in living subjects.

Sprint Exercise of Juvenile Animals Does Not Impact Cartilage Glycosaminoglycan or Synovial Fluid Neopeptide Collagenase Cleavage of Type I and II Collagen Content

Short sprints performed during growth can increase bone strength, mineral density, and cortical widths, but their impact on joint health is not fully understood. Some studies have found joints of young animals are damaged by forced sprints, while others found confinement hindered joint development. This study aimed to determine the impact of short sprints on synovial fluid neopeptide collagenase cleavage of type I and II collagen (C1,2C) and cartilage glycosaminoglycan (GAG) content. Calves were used as a model for young horses in this terminal study. Twenty-four Holstein bull calves were assigned to treatment groups of 1, 3, 5, or 0 days/week of sprinting (n = 6). A sprint consisted of an individual calf being walked from its stall and down a 71-m concrete aisle, at the end of which the calf was released, and encouraged to sprint towards the barn. Calves remained on treatment for 6 weeks and were euthanized on day 42. Immediately posteuthanasia synovial fluid was sampled from the middle carpal joint and cartilage discs sampled from 9 locations on the carpal bones. Analysis was performed in SAS 9.4 with fixed effects of treatment and bone. There were no differences in C1,2C or GAG concentrations between treatments (P = .73 and P = .96, respectively), but there was a difference between individual carpal bone GAG concentration (P = .03). Short sprints for 6 weeks during growth did not impact middle carpal joint C1,2C or carpal bone GAG content, and should be permitted given the benefits to bone health.

The Combination Treatment of Curcumin and Probucol Protects Chondrocytes from TNF-α Induced Inflammation by Enhancing Autophagy and Reducing Apoptosis via the PI3K-Akt-mTOR Pathway.

Osteoarthritis (OA) is a chronic joint disease characterized by cholesterol accumulation in chondrocytes, cartilage degeneration, as well as extracellular matrix (ECM) destruction, and joint dysfunction. Curcumin, a chemical that can reduce cholesterol levels in OA patients, also can inhibit the progression of OA. However, a high concentration of curcumin may also trigger apoptosis in normal chondrocytes. Besides curcumin, probucol that is found can also effectively decrease the cholesterol level in OA patients. Considering that high cholesterol is a risk factor of OA, it is speculated that the combination treatment of curcumin and probucol may be effective in the prevention of OA. To investigate the possible effects of such two chemicals on OA pathophysiology, chondrocyte apoptosis and autophagy behavior under inflammatory cytokine stress were studied, and specifically, the PI3K-Akt-mTOR signaling pathway was studied. Methods. Cell proliferation, colony formation, and EdU assay were performed to identify the cytotoxicity of curcumin and probucol on chondrocytes. Transwell assay was conducted to evaluate chondrocyte migration under TNF-α inflammation stress. Immunofluorescence, JC-1, flow cytometry, RT-PCR, and western blot were used to investigate the signal variations related to autophagy and apoptosis in chondrocytes and cartilage. A histological study was carried out on OA cartilage. Glycosaminoglycan (GAG) release was determined to evaluate the ECM degradation under stress. Results. Compared with a single intervention with curcumin or probucol, a combined treatment of these two chemicals is more effective in terms of protecting chondrocytes from stress injury induced by inflammatory cytokines. The promoted protection may be attributed to the inhibition of apoptosis and the blockage of the autophagy-related PI3K/Akt/mTOR pathway. Such results were also verified in vitro by immunofluorescence staining of OA chondrocytes and in vivo by immunohistochemistry staining of cartilage. Besides, in vivo studies also showed that when applied in combination, curcumin and probucol could block the PI3K-AKT-mTOR signaling pathway; promote COL-II expression; suppress P62, MMP-3, and MMP-13 expression; and inhibit TNF-α-stimulated cartilage degradation. Moreover, the combined medication could help reduce the release of ECM GAGs in OA cartilage and alleviate the severity of OA. Conclusion. A combined treatment of curcumin and probucol could be used to protect chondrocytes from inflammatory cytokine stress via inhibition of the autophagy-related PI3K/Akt/mTOR pathway both in vitro and in vivo, which might be of potential pharmaceutical value for OA prevention and therapy.

Higher aggrecan 1-F21 epitope concentration in synovial fluid early after anterior cruciate ligament injury is associated with worse knee cartilage quality assessed by gadolinium enhanced magnetic resonance imaging 20\u2009years later

BackgroundTo investigate if cartilage related biomarkers in synovial fluid are associated with knee cartilage status 20 years after an anterior cruciate ligament (ACL) injury.MethodsWe studied 25 patients with a complete ACL rupture without subsequent ACL reconstruction or radiographic knee OA. All had a delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) 20 years after the ACL injury, using the T1 transverse relaxation time in the presence of gadolinium (T1Gd) which estimates the concentration of glycosaminoglycans in hyaline cartilage. Synovial fluid samples were aspirated acutely (between 0 and 18 days) and during 1 to 5 follow up visits between 0.5 and 7.5 years after injury. We quantified synovial fluid concentrations of aggrecan (epitopes 1-F21 and ARGS), cartilage oligomeric matrix protein, matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 by immunoassays, and sulfated glycosaminoglycans by Alcian blue precipitation. Western blot was used for qualitative analyses of aggrecan fragments in synovial fluid and cartilage samples.ResultsWestern blot indicated that the 1-F21 epitope was located within the chondroitin sulfate 2 region of aggrecan. Linear regression analyses (adjusted for age, sex, body mass index and time between injury and sampling) showed that acute higher synovial fluid 1-F21-aggrecan concentrations were associated with shorter T1Gd values 20 years after injury, i.e. inferior cartilage quality (standardized effects between − 0.67 and − 1.0). No other statistically significant association was found between molecular biomarkers and T1Gd values.ConclusionHigher acute synovial fluid 1-F21-aggrecan concentrations in ACL injured patients, who managed to cope without ACL reconstruction and were without radiographic knee OA, were associated with inferior knee cartilage quality assessed by dGEMRIC 20 years after injury.

Contrast-enhanced micro-computed tomography of articular cartilage morphology with ioversol and iomeprol.

Non-ionic, low-osmolar contrast agents (CAs) used for computed tomography, such as Optiray (ioversol) and Iomeron (iomeprol), are associated with the reduced risk of adverse reactions and toxicity in comparison with ionic CAs, such as Hexabrix. Hexabrix has previously been used for imaging articular cartilage but has been commercially discontinued. This study aimed to evaluate the efficacy of Optiray and Iomeron as alternatives for visualisation of articular cartilage in small animal joints using contrast-enhanced micro-computed tomography (CECT). For this purpose, mouse femora were immersed in different concentrations (20%-50%) of Optiray 350 or Iomeron 350 for periods of time starting at five minutes. The femoral condyles were scanned ex vivo using CECT, and regions of articular cartilage manually contoured to calculate mean attenuation at each time point and concentration. For both CAs, a 30% CA concentration produced a mean cartilage attenuation optimally distinct from both bone and background signal, whilst 5-min immersion times were sufficient for equilibration of CA absorption. Additionally, plugs of bovine articular cartilage were digested by chondroitinase ABC to produce a spectrum of glycosaminoglycan (GAG) content. These samples were immersed in CA and assessed for any correlation between mean attenuation and GAG content. No significant correlation was found between attenuation and cartilage GAG content for either CAs. In conclusion, Optiray and Iomeron enable high-resolution morphological assessment of articular cartilage in small animals using CECT; however, they are not indicative of GAG content.

Tracking Osteoarthritis Progress through Cationic Nanoprobe-Enhanced Photoacoustic Imaging of Cartilage

A major obstacle in osteoarthritis (OA) theranostics is the lack of a timely and accurate monitoring method. It is hypothesized that the loss of anionic glycosaminoglycans (GAGs) in articular cartilage reflects the progression of OA. Thus, this study investigated the feasibility of photoacoustic imaging (PAI) applied for monitoring the in vivo course of OA progression via GAG-targeted cationic nanoprobes. The nanoprobes were synthesized through electrostatic attraction between poly-l-Lysine and melanin (PLL-MNPs). Cartilage explants with different concentrations of GAGs incubated with PLL-MNPs to test the relationship between GAGs content and PA signal intensity. GAG activity was then evaluated in vivo in destabilization of the medial meniscus (DMM) surgically-induced mouse model. To track OA progression over time, mice were imaged consistently for 10 weeks after OA-inducing surgery. X-ray was used to verify the superiority of PAI in detecting OA. The correlation between PAI data and histologic results was also analyzed. In vitro study demonstrated the ability of PLL-MNPs in sensitively detecting different GAGs concentrations. In vivo PAI exhibited significantly lower signal intensity from OA knees compared to normal knees. More importantly, PA signal intensity showed serial reduction over the course of OA, while X-ray showed visible joint destruction until 6 weeks. A decrease in GAGs content was confirmed by histologic examinations; moreover, histologic findings were well correlated with PAI results. Therefore, using cationic nanoprobe-enhanced PAI to detect the changes in GAG contents provides sensitive and consistent visualization of OA development. This approach will further facilitate OA theranostics and clinical translation. Statement of SignificanceThe study of in vivo monitoring osteoarthritis (OA) is of high significance to tracking the trajectory of OA development and therapeutic monitoring. Here, we developed a cartilage-targeted cationic nanoprobe, poly-l-Lysine-melanin nanoparticles (PLL-MNPs), enhancing photoacoustic imaging (PAI) to monitor the progression of OA. The in vitro study demonstrated the ability of PLL-MNPs to detect different concentrations of GAGs with high sensitivity. We found that the contents of GAGs in vivo steadily decreased from the development of OA initial-stage to the end-point of our investigation via PAI; it reflected the course of OA in living subjects with high sensitivity. These results allow for further development in various aspects of OA research. It has potential for clinical translation and has a great impact on personalized medicine.

Contribution of salidroside to the relieve of symptom and sign in the early acute stage of osteoarthritis in rat model

Ethnopharmacological relevanceThe genus Rhodiola has been used to treat cough, hemoptysis, fever, pain, bruise and other symptoms which are related to injury and inflammation over a thousand years in traditional Tibetan medicine. Salidroside (p-hydroxyphenethyl-β-D-glucoside) is one of the most potent bioactive ingredients of the genus Rhodiola. Aim of studyThe present study aimed to explore whether salidroside could alleviate the clinical symptom and sign in the early acute stage of osteoarthritis (OA) in monosodium iodoacetate (MIA) rat model, and its underlying mechanisms. Materials and methodsOsteoarthritis (OA) was induced in rat knees by intra-articular injection of MIA; simultaneously salidroside was administered by intravenous injection. Pain behaviors were evaluated by knee-bend test, hind limb weight-bearing asymmetry and hind paw mechanical withdrawal threshold. The joint swelling was determined by the difference of knee joint diameter. Inflammatory exudates in synovial fluid were evaluated by leukocyte counting and protein content. Cytokines, chemokines, reactive oxygen species (ROS) and reactive nitrogen species (RNS) markers were determined by Enzyme-linked immunosorbent assay (ELISA) and colorimetric assay in synovial fluid. Pro-inflammatory gene expressions in synovial tissue were detected by quantitative real time RT-PCR (qRT-PCR). Nuclear factor kappa-B (NF-κB) DNA binding assay and western blot were used to determine NF-κB activation and ROS marker protein expression in synovial tissue. Glycosaminoglycan (GAG) content in the cartilage was measured by dimethylmethylene blue method. Hematoxylin and eosin (H&E), Safranin O-fast green and a modified Mankin grading system were used to evaluate the histology of articular cartilage. ResultsSalidroside could alleviate pain and joint swelling in the early acute stage of OA in rat model, reduced the number of leukocytes, total protein content, proinflammatory mediators and ROS/RNS markers in synovial fluid, down regulated the expression of proinflammatory genes in synovium, inhibited the activation of NF- κ B and oxidative stress response in synovium, promoted the synthesis of cartilage GAG, prevented the loss of proteoglycan and chondrocyte degeneration. ConclusionsSalidroside effectively alleviates acute symptom and sign of OA in rat model by its anti-inflammatory and antioxidant affects to inhibit synovial inflammation, which provides a new strategy to prevent the onset and progression of OA.