An in vitro model for osteoarthritis using long-cultured inflammatory human macrophages repeatedly stimulated with TLR agonists.

Osteoarthritis (OA) is characterized by an abundance of inflammatory M1-like macrophages damaging local tissues. The search for new potential drugs for OA suffers from the lack of appropriate methods of long-lasting inflammation. Here we developed and characterized an in vitro protocol of long-lasting culture of primary human monocyte-derived macrophages differentiated with a combination of M-CSF+GM-CSF that optimally supported long-cultured macrophages (LC-Mϕs) for up to 15 days, unlike their single use. Macrophages repeatedly stimulated for 15 days with the TLR2 ligand Pam3CSK4 (LCS-Mϕs), showed sustained levels over time of IL-6, CCL2, and CXCL8, inflammatory mediators that were also detected in the synovial fluids of OA patients. Furthermore, macrophages isolated from the synovia of two OA patients showed an expression profile of inflammation-related genes similar to that of LCS-Mϕs, validating our protocol as a model of chronically activated inflammatory macrophages. Next, to confirm that these LCS-Mϕs could be modulated by anti-inflammatory compounds, we employed dexamethasone and/or celecoxib, two drugs widely used in OA treatment, that significantly inhibited the production of inflammatory mediators. This easy-to-use in vitro protocol of long-lasting inflammation with primary human macrophages could be useful for the screening of new compounds to improve the therapy of inflammatory disorders.

Particulate cartilage and platelet-rich plasma treatment for knee chondral defects in sheep.

PURPOSE: Articular cartilage is vulnerable to multiple types of damage and it has limited reparative and regenerative capacities due to its absence of vascularity. Although a large number of therapeutic strategies exist to treat chondral defects, they have some limitations, such as fibrocartilage formation. Therefore, the goal of the present study was to evaluate the chondrogenic regenerative properties of an autologous-made matrix of particulated cartilage and platelet-rich plasma (PACI + PRP) implantation for the treatment of full-thickness chondral defects in sheep. METHODS: A full-thickness 8 mm diameter cartilage defect was created in the weight-bearing area of the medial femoral condyle in both knees of 16 sheep. The right knees of all animals were treated with particulated autograft cartilage implantation and platelet-rich plasma, while the left knees were injected with Ringer's lactate solution or hyaluronic acid. The sheep were killed 9 or 18 months after surgery. Macroscopic evaluations were performed using three different scoring systems, and histopathological evaluations were performed using a modified scoring system based on different scoring systems. RESULTS: The PACI + PRP groups showed statistically significant differences in the percentage of defect repair and chondrocytes in the newly formed cartilage tissue at 18 months compared to 9 months. CONCLUSIONS: The results suggest that macroscopic appearance, histological structure and chondrocyte repair were improved when using PACI + PRP treatment for chondral defects, producing an outcome similar to the surrounding healthy cartilage. PACI + PRP is a totally autologous, easy, and unexpensive treatment that can be performed in one-step procedure and is useful as a therapeutic option for knee chondral defects.

Proteoglycans in Articular Cartilage and Their Contribution to Chondral Injury and Repair Mechanisms.

Proteoglycans are vital components of the extracellular matrix in articular cartilage, providing biomechanical properties crucial for its proper functioning. They are key players in chondral diseases, specifically in the degradation of the extracellular matrix. Evaluating proteoglycan molecules can serve as a biomarker for joint degradation in osteoarthritis patients, as well as assessing the quality of repaired tissue following different treatment strategies for chondral injuries. Despite ongoing research, understanding osteoarthritis and cartilage repair remains unclear, making the identification of key molecules essential for early diagnosis and effective treatment. This review offers an overview of proteoglycans as primary molecules in articular cartilage. It describes the various types of proteoglycans present in both healthy and damaged cartilage, highlighting their roles. Additionally, the review emphasizes the importance of assessing proteoglycans to evaluate the quality of repaired articular tissue. It concludes by providing a visual and narrative description of aggrecan distribution and presence in healthy cartilage. Proteoglycans, such as aggrecan, biglycan, decorin, perlecan, and versican, significantly contribute to maintaining the health of articular cartilage and the cartilage repair process. Therefore, studying these proteoglycans is vital for early diagnosis, evaluating the quality of repaired cartilage, and assessing treatment effectiveness.

Immunohistochemical Analysis of Knee Chondral Defect Repair after Autologous Particulated Cartilage and Platelet-Rich Plasma Treatment in Sheep.

This study performs an analysis that will enable the evaluation of the quality, durability, and structure of repaired cartilaginous extracellular matrix tissue using an autologous-based particulated autograft cartilage and platelet-rich plasma treatment (PACI + PRP). A single-blind controlled experiment was conducted on 28 sheep to evaluate the efficacy of the PACI + PRP treatment for cartilage defects. Full-thickness 8 mm diameter defects were created in the weight-bearing area of both knees. The right knees received PACI + PRP. The left knees were treated with Ringer's lactate solution (RLS) or hyaluronic acid (HA) injections. Sheep were euthanized at 9- or 18-months post-surgery. An extensive immunohistochemical analysis was performed to assess collagen types (I, II, III, V, VI, IX, X, XI) and aggrecan positivity. A semiquantitative scoring system provided a detailed evaluation of immunostaining. Collagens and aggrecan scores in the PACI + PRP groups were similar to healthy cartilage. Significant differences were found in collagens associated with matrix maturity (II and V), degradation (IX), structure and mechanics (VI), and hypertrophy (X) between healthy cartilage and RLS- or HA-repaired cartilage. The PACI + PRP treatment advanced the repair cartilage process in chondral defects with mature hyaline cartilage and enhanced the structural and mechanical qualities with better consistent cartilage, less susceptible to degradation and without hypertrophic formation over time.

Anti-dsDNA Antibodies Increase the Cardiovascular Risk in Systemic Lupus Erythematosus Promoting a Distinctive Immune and Vascular Activation.

Objective: Systemic lupus erythematosus (SLE) is associated to boosted atherosclerosis development and a higher cardiovascular disease risk. This study aimed to delineate the role of anti-double stranded DNA (anti-dsDNA) antibodies on the molecular profile and the activity of immune and vascular cells, as well as on their enhanced cardiovascular risk. Approach and Results: Eighty SLE patients were included. Extensive clinical/analytical evaluation was performed, including cardiovascular disease parameters (endothelial function, proatherogenic dyslipidemia, and carotid intima-media thickness). Gene and protein expression profiles were evaluated in monocytes from patients diagnosed positive or negative for anti-dsDNA antibodies by using NanoString and cytokine arrays, respectively. NETosis and circulating inflammatory profile was assessed in both neutrophils and plasma. Positivity and persistence of anti-dsDNA antibodies in SLE patients were associated to endothelial dysfunction, proatherogenic dyslipidemia, and accelerated atherosclerosis. In parallel, anti-dsDNA antibodies were linked to the aberrant activation of innate immune cells, so that anti-dsDNA(+) SLE monocytes showed distinctive gene and protein expression/activity profiles, and neutrophils were more prone to suffer NETosis in comparison with anti-dsDNA(−) patients. Anti-dsDNA(+) patients further displayed altered levels of numerous circulating mediators related to inflammation, NETosis, and cardiovascular risk. In vitro, Ig-dsDNA promoted NETosis on neutrophils, apoptosis on monocytes, modulated the expression of inflammation and thrombosis-related molecules, and induced endothelial activation, at least partially, by FcR (Fc receptor)-binding mechanisms. Conclusions: Anti-dsDNA antibodies increase the cardiovascular risk of SLE patients by altering key molecular processes that drive a distinctive and coordinated immune and vascular activation, representing a potential tool in the management of this comorbidity.

Main and Minor Types of Collagens in the Articular Cartilage: The Role of Collagens in Repair Tissue Evaluation in Chondral Defects.

Several collagen subtypes have been identified in hyaline articular cartilage. The main and most abundant collagens are type II, IX and XI collagens. The minor and less abundant collagens are type III, IV, V, VI, X, XII, XIV, XVI, XXII, and XXVII collagens. All these collagens have been found to play a key role in healthy cartilage, regardless of whether they are more or less abundant. Additionally, an exhaustive evaluation of collagen fibrils in a repaired cartilage tissue after a chondral lesion is necessary to determine the quality of the repaired tissue and even whether or not this repaired tissue is considered hyaline cartilage. Therefore, this review aims to describe in depth all the collagen types found in the normal articular cartilage structure, and based on this, establish the parameters that allow one to consider a repaired cartilage tissue as a hyaline cartilage.

Associations of Semen Quality with Seminal Non-essential Heavy Metals in Males from the Canary Islands.

Semen quality and levels of non-essential metals such as strontium (Sr), aluminum (Al), lead (Pb), nickel (Ni), and vanadium (V) were measured. Metals were determined by ICP-OES (inductively coupled plasma - optical emission spectrometry) in semen samples from 102 men who were recruited in a Reproduction Unit in the Canary Islands. The presence of each metal was as follows: Sr: 56.9%, Al: 73.5%, Pb: 45.1%, Ni: 15.7%, and V: 79.4% of the samples. No significant differences were found in the relationship between the spermiogram, the sperm motility, and the concentration of spermatozoa levels of non-essential metals. It is noteworthy that Ni levels tend to be lower in patients with oligozoospermia (t (46.4) = 1.84; p = 0.070). Between lifestyle and non-essential metals, there was a significant relationship between the level of occupational exposure to metals and Ni (χ2(2) = 13.91; p = 0.001). We did not find significant differences in non-essential seminal metal content and smoking status but, there were differences between drinkers and the concentration of V in semen (t (100) = -1.99; p = 0.050). The occupational exposure to metals and place of residence have effects on Al and V levels in semen. Regarding obesity, significant differences were found in Pb levels (t (18.0) = 2.34; p = 0.031). Obese patients have a lower Pb level, and the percentage of progressive sperm motility was lower in obese men (t (98) = 2.14; p = 0.035). The detection of metals in semen opens a new field in the study of male infertility with the possibility of performing treatments aimed at correcting these possible anomalies.