protective effect of a new hyaluronic acid carnosine conjugate on the modulation of the inflammatory response in mice subjected to collagen induced arthritis

Abstract Several studies demonstrated the pharmacological actions of carnosine as well as hyaluronic acid (HA) during joint inflammation. In that regard, the aim of this study was to investigate the protective effect of a new HA -Carnosine conjugate (FidHycarn) on the modulation of the inflammatory response in mice subjected to collagen-induced arthritis (CIA). CIA was induced by two intradermal injections of 100 μl of an emulsion of collagen (CII) and complete Freund's adjuvant (CFA) at the base of the tail on day 0 and 21. At 35 day post CIA induction, the animals were sacrificed. CII injection caused erythema and edema in the hind paws, histological alterations with erosion of the joint cartilage as well as behavioral changes. Oral treatment with FidHycarn starting at the onset of arthritis (day 25) ameliorated the clinical signs, improved behavioral deficits as well as decreased histological and radiographic alterations. The degree of oxidative damage evaluated by inducible nitric oxide synthase (iNOS), nitrotyrosine, poly-ADP-ribose (PAR) expressions and malondialdehyde (MDA) levels, was also significantly reduced in Carnosine+HA association and FidHycarn treated mice. Moreover, the levels of proinflammatory cytokines and chemokines and cyclo-oxygenase COX-2 enzyme were also more significantly reduced by Carnosine+HA and FidHycarn compared to carnosine alone. However, interestingly, in some cases, the effects of FidHycarn were more important than Carnosine+HA association and not statistically different to methotrexate (MTX) used as positive control. Thus, the conjugation of Carnosine with HA (FidHycarn) could represent an interesting therapeutic strategy to combat arthritis disorders

Therapeutic efficacy of intra-articular hyaluronan derivative and platelet-rich plasma in mice following axial tibial loading

<div><p>Objective</p><p>To investigate the therapeutic potential of intra-articular hyaluronan-derivative HYADD^® 4-G and/or platelet-rich plasma (PRP) in a mouse model of non-invasive joint injury.</p><p>Methods</p><p>Non-invasive axial tibial loading was used to induce joint injury in 10-week-old C57BL/6J mice (n = 86). Mice underwent a single loading of either 6 Newton (N) or 9N axial tibial compression. HYADD^® 4-G was injected intra-articularly at 8 mg/mL or 15 mg/mL either before or after loading with or without PRP. Phosphate-buffered-saline was injected as control. Knee joints were harvested at 5 or 56 days post-loading and prepared for micro-computed tomography scanning and subsequently processed for histology. Immunostaining was performed for aggrecan to monitor its distribution, for CD44 to monitor chondrocyte reactive changes and for COMP (cartilage oligomeric matrix protein) as an index for cartilage matrix changes related to loading and cartilage injury. TUNEL assay was performed to identify chondrocyte apoptosis.</p><p>Results</p><p>Loading initiated cartilage proteoglycan loss and chondrocyte apoptosis within 5 days with slowly progressive post-traumatic osteoarthritis (no cartilage degeneration, but increased synovitis and ectopic calcification after 9N loading) at 56 days. Mice treated with repeated HYADD^® 4-G (15 mg/mL) or HYADD^® 4-G (8 mg/mL) ± PRP or PRP alone exhibited no significant improvement in the short-term (5 days) and long-term (56 days) consequences of joint loading except for a trend for improved bone changes compared to non-loaded joints.</p><p>Conclusion</p><p>While we failed to show an overall effect of intra-articular delivery of hyaluronan-derivative and/or PRP in reversing/protecting the pathological events in cartilage and synovium following joint injury, some bone alterations were relatively less severe with hyaluronan-derivative at higher concentration or in association with PRP.</p></div

Synovitis and ectopic ossification.

<p>Enlargement of the synovial lining cell layer and density of the cells in the synovial stroma (red arrows) were individually assessed. The red circle shows a representative ectopic ossification nodule (A). Quantification of synovitis score in each group indicated that synovitis progressed with time but no significant differences were found in the same loading force groups at the same time point. a, b, c, and d: P < 0.01 (B). A representative micro-CT image showing synovial ectopic calcification is shown in which white arrows show the nodules around the injured knee (C). Quantification of ectopic nodules in each group showed higher number of calcified nodules in the 9N group compared to non-loaded knees and knees loaded with 6N force. There were no significant differences observed within the same loading groups. a, b and c P < 0.01 (D). L = lateral side, M = medial side, bars = 100 μm. Error bars represent 95% confidence interval. PBS = phosphate buffered saline, PRP = platelet-rich plasma.</p

CD44 and aggrecan staining.

<p>At day 5, CD44 (green) was mainly seen in the calcified zone of articular cartilage in control knees with only a few CD44-positive signals around hypertrophic chondrocytes (A). CD44 signals were not found at the site of cartilage injury, albeit higher signals were discovered in the chondrocytes immediately around the injured zone (white arrows). There was no difference in the pattern of CD44 expression between different treatment groups. Aggrecan staining (red) was found around the chondrocytes in the intact area and in the lacuna left by the apoptotic chondrocytes in the injured area. Bar = 100 μm. At day 56, CD44 in the loaded knees, same as non-loaded control knees, was mainly seen in the calcified zone of articular cartilage with only a few CD44-positive signals around hypertrophic chondrocytes. No substantial CD44 signals were found in the chondrocytes immediately around the injured zone as day 5. There was no difference in the pattern of CD44 expression between different treatment groups. Aggrecan staining (red) was much weaker in the injured region but remain the same in the intact area of the loaded cartilage. White bars indicate the injured areas (B). Representative images of day 5 (C) and day 56 (D) of injured knees. White dotted boxes indicate the enlarged area. The white arrows in C indicate the higher CD44 signals in the chondrocytes immediately around the injured zone at day 5 while the white arrows in D show the expression of CD44 in the hypertrophic chondrocytes in both loaded and non-loaded knees at day 56. Also, low intensity of aggrecan signal in the injured region at day 56 was noticed. Dotted lines indicate the joint surface. PBS = phosphate buffered saline, PRP = platelet-rich plasma.</p

Loading regimens, treatment options, time points and sample size.

<p>Loading regimens, treatment options, time points and sample size.</p

Micro-CT analysis.

<p>Micro-CT results showed no preventive effect of treatments on BV/TV at day 56 following loading. Trabecular bone was analyzed at the femoral epiphysis at day 5 (top row) and day 56 (bottom row). All images are presented as superior views (A). Quantification of femoral epiphysis trabecular bone volume (BV/TV) showed that there was no significant difference between treatment groups and the PBS at this time point (B). a: P = 0.02. Interestingly, at day 56 (9N group) receiving Hyadd-4G at 15 mg/mL and PRP + HYADD^® 4-G at 8 mg/mL group showed no statistical differences at the 56 days, non-loaded group. Quantification of subchondral bone thickness, showing no preventive effect of treatments on bone loss at 56 days after loading is presented (C). Error bars represent 95% confidence interval. PBS = phosphate buffered saline, PRP = platelet-rich plasma.</p

Study design.

<p>An overview of treatment(s) before and/or after loading, time points of repeated injections and analysis of 6N and 9N loading is shown. X = sacrifice, PBS = phosphate buffered saline, PRP = platelet-rich plasma.</p

Chondrocyte apoptosis and COMP staining.

<p>Representative images of chondrocyte apoptosis (green), COMP (red) and DAPI (blue) immunostaining are shown. At day 5 (A), TUNEL assay identified apoptotic chondrocytes (green) in the injured cartilage area in all loaded groups. COMP (red) was found around the chondrocytes in the intact area and in the lacuna left by the apoptotic chondrocytes in the injured area. At day 56 (B), TUNEL positive chondrocytes in the non-calcified layer of injured cartilage disappeared. No new developed apoptosis was observed. COMP (red) was hardly seen in the injured region and its expression remained the same as day 5 in the adjacent healthy cartilage surface. None of the treatments conferred any protective response on chondrocyte apoptosis or the expression pattern of COMP at day 5 (A) and 56 (B) after loading. White boxes indicate the injured area where enlarged images were taken. Representative images at days 5 and 56 for 9N loading with split channels to show the changes of chondrocyte apoptosis (TUNEL positive cells) and distribution of COMP (red) are also presented (C). Bar = 100 μm, dotted white lines indicate the joint surface. COMP = cartilage oligomeric matrix protein, PBS = phosphate buffered saline, PRP = platelet-rich plasma, TUNEL = terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling.</p

Cartilage injury.

<p>Representative images of cartilage injury of the lateral femoral condyle in each group showing loss of Safranin-O staining and absence of normal nuclear staining are provided for higher (A) and lower (B) magnification. There was no therapeutic effect of HYADD^® 4-G or PRP on cartilage injury and results were not different from PBS treatment. The area between two red lines in each image shows the injury site of each knee. OA scored based on proteoglycan loss on day 56 after loading showed that there was no significant effect of any treatment modality on proteoglycan replacement (C). Bar = 50 μm, ** indicates P < 0.01 compared with other groups. Error bars represent 95% confidence interval. PBS = phosphate buffered saline, PRP = platelet-rich plasma, F = femur, M = meniscus, S = synovium.</p

The Protective Effect of New Carnosine-Hyaluronic Acid Conjugate on the Inflammation and Cartilage Degradation in the Experimental Model of Osteoarthritis

Osteoarthritis (OA) is a disease that currently has no cure. There are numerous studies showing that carnosine and hyaluronic acid (HA) have a positive pharmacological action during joint inflammation. For this reason, the goal of this research was to discover the protective effect of a new carnosine conjugate with hyaluronic acid (FidHycarn) on the inflammatory response and on the cartilage degradation in an in vivo experimental model of OA. This model was induced by a single intra-articular (i.ar.) injection of 25 &micro;L of normal saline with 1 mg of monosodium iodoacetate solution (MIA) in the knee joint of rats. MIA injection caused histological alterations and degradation of the cartilage, as well as behavioral changes. Oral treatment with FidHycarn ameliorated the macroscopic signs, improved thermal hyperalgesia and the weight distribution of the hind paw, and decreased histological and radiographic alterations. The oxidative damage was analyzed by evaluating the levels of nitrotyrosine and inducible nitric oxide synthase (iNOS) that were significantly reduced in FidHycarn rats. Moreover, the levels of pro-inflammatory cytokines and chemokines were also significantly reduced by FidHycarn. Therefore, for the first time, the effectiveness of oral administration of FidHycarn has been demonstrated in an osteoarthritis model. In conclusion, the new FidHycarn could represent an interesting therapeutic strategy to combat osteoarthritis