BACKGROUND: Chondroprogenitors, with enhanced chondrogenic potential, have emerged to be a promising alternative for cell-based therapy in cartilage repair. Platelet-rich plasma (PRP), widely used for intra-articular treatment, has a short half-life. Freeze-dried PRP (FD-PRP), with an extended half-life and retained growth factors, is gaining attention. This study compares the efficacy of Migratory Chondroprogenitors (MCPs) in gelled PRP and FD-PRP using in-vitro and ex-vivo models, assessing FD-PRP as a potential off-the-shelf option for effective cartilage repair.
METHODS: MCPs were isolated from osteoarthritic cartilage samples (n = 3), characterized through FACS and RT-PCR. For in-vitro analysis, cells were loaded into gelled PRP and FD-PRP scaffolds at a density of 1x106 cells per scaffold. Trilineage differentiation studies and live-dead assays were conducted on MCPs using Calcein AM/Propidium Homodimer-1. In ex-vivo analysis, MCPs of the same density were added to Osteochondral Units (OCU) with chondral defects containing PRP gel and FD-PRP scaffolds, harvested on the 15th and 35th days for histological examination. Controls included cell-free scaffolds.
RESULTS: Our in-vitro analysis demonstrates the robust viability of MCPs in both scaffolds, with no discernible impact on their differentiation capacity. Ex-vivo analysis of the OCU for cartilage repair showed that the chondrogenic potential characterized by the accumulation of extracellular matrix containing glycosaminoglycans and collagen type II production (with no alteration in collagen type X), was observed to be better with the gel PRP and the gel PRP containing MCP groups.
CONCLUSIONS: These findings support the preference for gel PRP as a superior synergistic scaffold for chondroprogenitor delivery.

The curdlan gel is a natural material produced by bacteria. It utilizes chemical cross-linking reactions to form a 3D porous composite hydrogel, increasing its porosity and water content, and improving its mechanical properties. It can be used in tissue repair and regenerative medicine. Curdlan-Poly(vinyl alcohol) (PVA) composite hydrogel can rapidly swell within 1 min due to its porous structure. Compression tests confirmed that it still maintains its original mechanical strength, even after five repeated freeze-thaw (FT) processes, making it suitable for long-term cryopreservation. The purpose of this study is to transplant umbilical cord mesenchymal stem cells (UC-MSCs) on Curdlan-PVA composite hydrogel and observe the chondrocytes on the material. The results of using 4\',6-diamidino-2-phenylindole (DAPI), hematoxylin and eosin (H&E), calcein-acetoxymethyl ester (calcein AM), and Collagen type II-Fluorescein isothiocyanate (FITC) staining, confirmed that UC-MSCs can attach and differentiate into chondrocytes on 3D Curdlan-PVA composite hydrogel.

Physical inactivity is considered a significant risk factor for mortality and morbidity among chronic hemodialysis (HD) patients. Therefore, physical exercise is recommended in the treatment of HD patients. Although the beneficial effects of physical exercise in HD patients are well-described in the literature, the underlying physiological mechanisms still need to be fully understood. Recently, microRNAs (miRNAs) have emerged as potential mediators of the therapeutic effects of physical exercise in healthy individuals. miRNAs are short, single-stranded, noncoding RNAs involved in gene expression regulation. Specifically, upon forming the RNA-induced silencing complex, miRNAs selectively bind to specific miRNAs within cells, reducing gene expression. miRNAs can be secreted by cells in an accessible form or enclosed within exosomes or extracellular vesicles. They can be detected in various body fluids, including serum (circulating miRNAs), facilitating the study of their diverse expression. Currently, there is no available data regarding the impact of physical exercise on the expression of miRNAs involved in osteogenic differentiation, a fundamental mechanism in the development of vascular calcification, for HD patients. Therefore, we have designed an observational and longitudinal case-control study to evaluate the expression of miR-9 and miR-30b in HD patients participating in a 3-month interdialytic physical exercise program. This paper aims to present the study protocol and review the expression of circulating miRNAs in HD patients and their modulation through physical exercise.

OBJECTIVE: This study aims to investigate the effectiveness of arthroscopic autologous matrix-induced chondrogenesis (AMIC) procedure with or without polyglycolic acid-hyaluronic acid (PGA-HA)-based cell-free scaffold (CFS) in Bristol Stage 4 and Stage 5 osteochondral lesion of the talus (OLT) ranging between 1.5 and 3 cm2 .
METHODS: Between March 2018 and March 2021, a total of 47 patients with OLTs (29 males, 18 females; mean age: 22.8±2.3 years; range, 18 to 65 years) were retrospectively analyzed. The patients were divided into two groups based on the procedures applied. Patients in the first group (Group 1, n=23) underwent the AMIC procedure alone (curettage, microfracture, and grafting), while patients in the second group (Group 2, n=24) underwent AMIC procedure with PGA-HA-based CFS. The localization of the lesions was evaluated. All OLTs were diagnosed with preoperative radiography and magnetic resonance imaging (MRI). During the preoperative period, lesion stages were evaluated based on the Bristol staging system, and the postoperative results were evaluated based on the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) scoring system.
RESULTS: The mean follow-up was 36.2±5.6 months. In the early period, the three-month functional scores were comparable between the groups. While a significant increase was observed in the American Orthopaedic Foot and Ankle Society (AOFAS) scores from the mean preoperative of 62.71±4.44 points to the postoperative of 86.00±6.58 points in Group 1, a significant increase in the AOFAS score was observed from 65.28±7.91 points to 95.42±4.41 points in Group 2 at 12-month follow-up (p=0.016, p=0.011, respectively). The functional scores tended to progress after 12 months. Radiologically, a complete defect filling was observed in a mean of 10.5±2.7 months. No graft hypertrophy was recorded in any patients. The AOFAS and MOCART scores in Group 2 were found to be statistically significantly higher than that in Group 1 (p=0.034 for AOFAS 1/AOFAS 2 and p=0.006 for MOCART 1/MOCART 2). Overall, there was a positive, but weak, significant correlation between the final AOFAS scores and MOCART scores (r=0.347, p<0.001).
CONCLUSIONS: Arthroscopic AMIC procedure in deep OLTs between 1.5 cm2 and 3 cm2 can yield a statistically significant improvement both clinically and radiologically; however, the use of a PGA-HA-based CFS in addition to this procedure can improve the clinical and radiological recovery.

Articular cartilage has constrained potential to restore. The mesenchymal stem cellular remedy has presented new treatment possibilities for this circumstance. The experiment aimed to verify the chondrogenic differentiation capacity of rat adipose tissue-derived mesenchymal stem cells (AD-MSCs) in vitro inside the presence or absence of transforming growth factor-beta (TGF-β1). Rat\'s subcutaneous adipose tissue minced into a small piece (2-3 mm3) was aseptically collected from the subcutaneous fat under anaesthesia and then digested with collagenase type I (1 mg/mL). Spontaneous chondrogenesis occurred in both AD-MSCs pellet cultures and was similar in both TGF-β1 treated. The untreated pellet cultures were collected after 21 days. Histological assessment for evaluating the level of proteoglycan by alcin blue staining and immunohistochemistry for detecting the presence of collagen type II. A monoclonal antibody directed against collagen type II. Adipose-derived stem cells (AD-MSCs) isolated from rats were immunophenotyped for the expression of MSCs cell surface markers and was performed by Flow cytometer, which demonstrated AD-MSCs highly expressed CD73 (99.69±2.6%), CD90 (98.11±0.3%), and week expression CD44 (17.15±0.3%). The result of histological staining showed the presence of extracellular matrix (ECM) in the hyaline cartilage. This staining indicated a deposit of \"acid mucopolysaccharides\" in the proximity of the cells. Additionally, most cells are rounded cells stained positive for the presence of the cells encompassed by extracellular matrix (ECM), which were like chondrocytes as seen from the magnified view, lightly pink stained nuclei, and nuclear fast red stain. However, the immunohistochemistry method demonstrated that the presence of TGF-β1 decreased the levels of collagen type I and increased the levels of collagen type II. In conclusion, subcutaneous adipose tissue-derived stem cells can be used in cartilage tissue engineering.

Osteoarthritis (OA) is described as a chronic degenerative disease characterized by the loss of articular cartilage. Senescence is a natural cellular response to stressors. Beneficial in certain conditions, the accumulation of senescent cells has been implicated in the pathophysiology of many diseases associated with aging. Recently, it has been demonstrated that mesenchymal stem/stromal cells isolated from OA patients contain many senescent cells that inhibit cartilage regeneration. However, the link between cellular senescence in MSCs and OA progression is still debated. In this study, we aim to characterize and compare synovial fluid MSCs (sf-MSCs), isolated from OA joints, with healthy sf-MSCs, investigating the senescence hallmarks and how this state could affect cartilage repair. Sf-MSCs were isolated from tibiotarsal joints of healthy and diseased horses with an established diagnosis of OA with an age ranging from 8 to 14 years. Cells were cultured in vitro and characterized for cell proliferation assay, cell cycle analysis, ROS detection assay, ultrastructure analysis, and the expression of senescent markers. To evaluate the influence of senescence on chondrogenic differentiation, OA sf-MSCs were stimulated in vitro for up to 21 days with chondrogenic factors, and the expression of chondrogenic markers was compared with healthy sf-MSCs. Our findings demonstrated the presence of senescent sf-MSCs in OA joints with impaired chondrogenic differentiation abilities, which could have a potential influence on OA progression.

BACKGROUND: Mesenchymal stem cells (MSCs) possess the potential to differentiate into chondrocytes, which makes them an ideal source for healing cartilage defects. Here, we seek to identify the essential genes participating in MSCs chondrogenesis.
METHODS: Human MSCs were induced for chondrogenesis for 7, 14, and 21 days using a high-density micromass culture system, and RNA was extracted for RNA-seq.
RESULTS: A total of 6247 differentially expressed genes (DEGs) were identified on day 7, and 85 DEGs were identified on day 14. However, no significant DEGs was identified on day 21. The top 30 DEGs at day 7, including COL9A3, COL10A1, and CILP2, are closely related to extracellular matrix organization. While the top 30 DEGs at day 14 revealed that inflammation-related genes were enriched, including CXCL8, TLR2, and CCL20. We also conducted protein-protein interaction (PPI) networks analysis using the search tool for the retrieval of interacting genes (STRING) database and identified key hub genes, including CXCL8, TLR2, CCL20, and MMP3. The transcriptional factors were also analyzed, identifying the top 5 TFs: LEF1, FOXO1, RORA, BHLHE41, and SOX5. We demonstrated one particular TF, RORA, in promoting early MSCs chondrogenesis.
CONCLUSIONS: Taken together, our results suggested that these DEGs may have a complex effect on MSCs chondrogenesis both synergistically and solitarily.

UNASSIGNED: Osteoarthritis (OA) is recognized as a degenerative joint disease that leads to chronic pain and low quality of life in animals. Captive elephants, the largest land mammals with a long lifespan, are more prone to develop OA due to restricted spaces and insufficient physical activity. This study aimed to investigate the effect of transforming growth factor-β1 (TGF-β1) and insulin-like growth factor 1 (IGF-1) on elephant chondrogenesis in a scaffold culture of articular chondrocytes.
UNASSIGNED: Elephant chondrocytes-seeded gelatin scaffolds were cultured in chondrogenic media with or without 10 ng/mL of TGF-β1 or IGF-1 alone or 5-10 ng/mL of their combination for up to 21 days. The mRNA expression of cartilage-specific anabolic genes, ACAN and COL2A1, was analyzed using a real-time reverse transcription-polymerase chain reaction. The amounts of sulfated glycosaminoglycans (sGAGs) in conditioned media and contents in cultured scaffolds were determined through dimethylmethylene blue assay. Cell morphology, accumulation of proteoglycans, and details of the cultured scaffolds were determined using hematoxylin-eosin staining, safranin O staining, and scanning electron microscopy (SEM), respectively.
UNASSIGNED: TGF-β1 alone significantly upregulated ACAN gene expression but not COL2A1, while IGF-1 alone did not enhance both ACAN and COL2A1 genes. The combination significantly upregulated both mRNA expression levels of ACAN and COL2A1 gene at day 14. The sGAGs accumulation and contents in the treatment groups, except IGF-1 tended to be higher than the controls, concomitantly with the production of the extracellular matrix, showed the formation of a cartilage-like tissue through histological and SEM analyses.
UNASSIGNED: Together, our results suggest that the single treatment of TGF-β1 has a selective effect on ACAN gene, while the combined growth factors seem to be an advantage on elephant chondrogenesis. This three-dimensional culture model is probably helpful for developing cartilage regeneration in vitro and is further applied in tissue engineering for OA treatment in vivo.

Seeding cells are key factors in cell-based cartilage tissue regeneration. Monoculture of either chondrocyte or mesenchymal stem cells has several limitations. In recent years, co-culture strategies have provided potential solutions. In this study, directly co-cultured rat costal chondrocytes (CCs) and human Wharton\'s jelly mesenchymal stem (hWJMSCs) cells were evaluated as a candidate to regenerate articular cartilage.
Rat CCs are directly co-cultured with hWJMSCs in a pellet model at different ratios (3:1, 1:1, 1:3) for 21 days. The monoculture pellets were used as controls. RT-qPCR, biochemical assays, histological staining and evaluations were performed to analyze the chondrogenic differentiation of each group. The 1:1 ratio co-culture pellet group together with monoculture controls were implanted into the osteochondral defects made on the femoral grooves of the rats for 4, 8, 12 weeks. Then, macroscopic and histological evaluations were performed.
Compared to rat CCs pellet group, 3:1 and 1:1 ratio group demonstrated similar extracellular matrix production but less hypertrophy intendency. Immunochemistry staining found the consistent results. RT-PCR analysis indicated that chondrogenesis was promoted in co-cultured rat CCs, while expressions of hypertrophic genes were inhibited. However, hWJMSCs showed only slightly improved in chondrogenesis but not significantly different in hypertrophic expressions. In vivo experiments showed that all the pellets filled the defects but co-culture pellets demonstrated reduced hypertrophy, better surrounding cartilage integration and appropriate subchondral bone remodeling.
Co-culture of rat CCs and hWJMSCs demonstrated stable chondrogenic phenotype and decreased hypertrophic intendency in both vitro and vivo. These results suggest this co-culture combination as a promising candidate in articular cartilage regeneration.

Osteochondral lesions of the talus (OLTs) are a common cause of post-traumatic ankle pain and disability. Atelocollagen-induced chondrogenesis (ACIC) aims to encourage the development of hyaline cartilage, which is biomechanically superior to fibrocartilage. This single-center, retrospective database study assessed patients who underwent arthroscopic microfracture with or without atelocollagen scaffold augmentation for OLT. Between 2010 and 2019, 87 patients underwent microfracture only and 31 patients underwent ACIC. Propensity score matching was used to match the ACIC group in a 1:2 ratio to a corresponding microfracture-only group using logistic regression. American Orthopaedic Foot & Ankle Society (AOFAS) scores, 100-mm Visual Analog Scale (VAS), Short Form-36 (SF-36), and satisfaction were assessed at preoperative, 3-, 6-, and 12-month intervals. There were no differences in baseline characteristics between groups after matching (P > .05). Both groups had similar improvements to VAS, AOFAS, and SF-36 scores up to 12 months (P > .05). Both groups had significant 1-year improvements to physical functioning, physical limitations in usual role activities, pain, and social functioning domains, but the ACIC group additionally had significant improvements to general health, vitality, and mental health. Patients in the ACIC group were also more satisfied than the microfracture group at all time points. Patients with OLTs who underwent ACIC reported superior satisfaction and improvements to quality of life, although clinical outcomes were similar to those who underwent microfracture alone at 1 year.Level of Evidence: Level III: Cohort study.