Osteoarthritis of the knee (OAK) is a chronic degenerative disease and progresses with an imbalance of cytokines and macrophages in the joint. Studies regarding the use of platelet-rich plasma (PRP) as a point-of-care treatment for OAK have reported on its effect on tissue repair and suppression of inflammation but few have reported on its effect on macrophages and macrophage polarization. Based on our clinical experience with two types of PRP kits Cellaid Serum Collection Set P type kit (leukocyte-poor-PRP) and an Autologous Protein Solution kit (APS leukocyte-rich-PRP), we investigated the concentrations of humoral factors in PRPs prepared from the two kits and the effect of humoral factors on macrophage phenotypes. We found that the concentrations of cell components and humoral factors differed between PRPs purified using the two kits; APS had a higher concentration of M1 and M2 macrophage related factors. The addition of PRP supernatants to the culture media of monocyte-derived macrophages and M1 polarized macrophages revealed that PRPs suppressed M1 macrophage polarization and promoted M2 macrophage polarization. This research is the first to report the effect of PRPs purified using commercial kits on macrophage polarization.

We previously conducted a first-in-human clinical study of articular cartilage repair using autologous chondrocyte sheets and confirmed the regeneration of hyaline-like cartilage in all eight patients. However, regenerative medicine with autologous chondrocyte sheets requires the harvesting of tissue from healthy regions, and the quality of this tissue varies between individuals. To overcome such limitations, allogeneic transplantation is a promising treatment method, particularly for articular cartilage repair. In this study, we investigated the characteristics of polydactyly-derived chondrocyte sheets fabricated from the chondrocytes of young polydactyly donors. Polydactyly-derived chondrocyte (PD) sheets were fabricated from the tissue obtained from eight polydactyly donors (average age = 13.4 months). To create these PD sheets, chondrocytes at passage 2 or 3 were seeded on temperature-responsive culture inserts and cultured for 2 weeks. For comparison, adult chondrocyte sheets were fabricated from tissue obtained from 11 patients who underwent total knee arthroplasty (TKA; average age = 74 years). To create these TKA sheets, chondrocytes and synovial cells were cocultured, and the chondrocyte sheets were triple-layered according to the protocol from our previous clinical study. Cell count, cell viability, cell surface markers, cell histology, and humoral factors secreted by the sheets were characterized and compared between the PD sheets and TKA sheets. Polydactyly-derived chondrocytes proliferated rapidly to establish a layered structure with sufficient extracellular matrix and formed sheets that could be easily manipulated without tearing. Similar to TKA sheets, PD sheets expressed aggrecan and fibronectin at the protein level and the surface markers CD44, CD81, and CD90, which are characteristic of mesenchymal cells. PD sheets also produced significantly higher levels of transforming growth factor beta-1 and lower levels of matrix metalloproteinase-3 than those produced by TKA sheets, suggesting that young polydactyly-derived chondrocytes have advantages as a potential cell source. PD sheets exhibited characteristics thought to be important to chondrocyte sheets as well as proliferative capacity that may facilitate provision of a stable supply in the future.

Chondrocyte sheet transplantation is a novel and promising approach to treating patients who have cartilage defects associated with osteoarthritis. Hyaline cartilage regeneration by autologous chondrocyte sheets has already been demonstrated in clinical research. In this study, the efficacy of polydactyly-derived chondrocyte sheets (PD sheets) as an allogeneic alternative to standard chondrocyte sheets was examined using an orthotopic xenogeneic transplantation model. In addition, the expression of genes and the secreted proteins in the PD sheets was analyzed using a microarray and a DNA aptamer array. The efficacy of PD sheets with respect to cartilage defects was assessed using histological scores, after which the expressions of genes and proteins exhibiting a correlation to efficacy were identified. Enrichment analysis of efficacy-correlated genes and proteins showed that they were associated with extracellular matrices, skeletal development, and angiogenesis. Eight genes (ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, and PDGFA) exhibited a positive correlation with the efficacy of PD sheets, and three genes (RARRES2, APOE, and PGF) showed a negative correlation for both transcriptomic and proteomic analyses. Among these, MIA, DKK1, and GREM1 involved in skeletal development pathways and ESM1 involved in the angiogenesis pathway exhibited a correlation between the amount of secretion and efficacy. These results suggest that these secreted factors may prove useful for predicting PD sheet efficacy and may therefore contribute to hyaline cartilage regeneration via PD sheets.

Osteoarthritis (OA) of the knee is a disease that significantly decreases the quality of life due to joint deformation and pain caused by degeneration of articular cartilage. Since the degeneration of cartilage is irreversible, intervention from an early stage and control throughout life is important for OA treatment. For the treatment of early OA, the development of a disease-modifying osteoarthritis drug (DMOAD) for intra-articular (IA) injection, which is attracting attention as a point-of-care therapy, is desired. In recent years, the molecular mechanisms involved in OA progression have been clarified while new types of drug development methods based on gene sequences have been established. In addition to conventional chemical compounds and protein therapeutics, the development of DMOAD from the new modalities such as gene therapy and oligonucleotide therapeutics is accelerating. In this review, we have summarized the current status and challenges of DMOAD for IA injection, especially for protein therapeutics, gene therapy, and oligonucleotide therapeutics.

In the knee joint, articular cartilage injury can often lead to osteoarthritis of the knee (OAK). Currently, no point-of-care treatment can completely address OAK symptoms and regenerate articular cartilage to restore original functions. While various cell-based therapies are being developed to address OAK, exosomes containing various components derived from their cells of origin have attracted attention as a cell-free alternative. The potential for exosomes as a novel point-of-care treatment for OAK has been studied extensively, especially in the context of intra-articular treatments. Specific exosomal microRNAs have been identified as possibly effective in treating cartilage defects. Additionally, exosomes have been studied as biomarkers through their differences in body fluid composition between joint disease patients and healthy subjects. Exosomes themselves can be utilized as a drug delivery system through their manipulation and encapsulation of specific contents to be delivered to specific cells. Through the combination of exosomes with tissue engineering, novel sustained release drug delivery systems are being developed. On the other hand, many of the functions and activities of exosomes are unknown and challenges remain for clinical applications. In this review, the possibilities of intra-articular treatments utilizing exosomes and the challenges in using exosomes in therapy are discussed.

Cartilage regeneration is a promising therapy for restoring joint function in patients with cartilage defects. The limited availability of autologous chondrocytes or chondrogenic progenitor cells is an obstacle to its clinical application. We investigated the existence and chondrogenic potential of synovial membrane-derived multilineage-differentiating stress-enduring (Muse)-like cells as an alternative cell source for cartilage regeneration. Cells positive for stage-specific embryonic antigen-3 (SSEA-3), a marker of Muse cells, were isolated from the synovial membranes of 6 of 8 patients (median age, 53.5 years; range 36-72 years) by fluorescence-activated cell sorting. SSEA-3-positive cells were cultured in methylcellulose to examine their ability to form Muse clusters that are similar to the embryoid bodies formed by human embryonic stem cells. Muse clusters were expanded and chondrogenic potential of M-cluster-derived MSCs examined using a pellet culture system. Chondrogenic differentiation was evaluated by proteoglycan, safranin O, toluidine blue and type II collagen staining. To evaluate the practicality of the procedure for isolating Muse-like cells, we compared chondrogenic potential of M-cluster derived MSCs with expanded cells derived from the clusters formed by unsorted synovial cells. Synovial membranes contained SSEA-3-positive cells that after isolation exhibited Muse-like characteristics such as forming clusters that expressed NANOG, OCT3/4, and SOX2. In the pellet culture system, cell pellets created from the M-cluster-derived MSCs exhibited an increase in wet weight, which implied an increase in extracellular matrix production, displayed metachromasia with toluidine blue and safranin O staining and were aggrecan-positive and type II collagen-positive by immunostaining. Unsorted synovial cells also formed clusters in methylcellulose culture, and the expanded cell population derived from them exhibited chondrogenic potential. The histological and immunohistochemical appearance of chondrogenic pellet created from unsorted synovial cell-derived cells were comparable with that from M-cluster-derived MSCs. Muse-like cells can be isolated from the human synovial membrane, even from older patients, and therefore may provide a source of multipotent cells for regenerative medicine. In addition, the cluster-forming cell population within synovial cells also has excellent chondrogenic potential. These cells may provide a more practical option for cartilage regeneration.