The effect of synthesized cartilage tissue from human adipose-derived mesenchymal stem cells in orthopedic spine surgery in patients with osteoarthritis

Osteoarthritis is a joint disease that causes degeneration of articular cartilage and involvement of subcutaneous bone and inflammation of surrounding tissues. It can affect any joints, but the most common joints are the joints of the hands, feet, knees, thighs, and spine. Osteoarthritis patients need surgery in acute cases. The use of methods that increase the efficiency of this surgery has always been considered by researchers and surgeons. For this purpose, in the current study, the effect of synthesized cartilage tissue from human adipose-derived mesenchymal stem cells was considered in orthopedic spine surgery in patients with osteoarthritis. Thirty patients over the age of 60 who had acute spinal osteoarthritis and required surgery were selected. The pellet culture system of human adipose-derive mesenchymal stem cells of each patient was used to construct cartilage tissue. For 15 of them, in addition to implants, cartilage grafts were transplanted during surgery. All patients were monitored by the Oswestry Disability Index questionnaire, for one year. In general, the results showed that over time, patients with transplanted cartilage tissue and implants were in a better condition than patients who underwent only implant surgery.

Overexpression of Long Non-Coding RNA AP001505.9 Inhibits Dedifferentiation in Human Hyaline Chondrocyte

Background: Autologous chondrocyte implantation (ACI) requires a large number ofhuman hyaline chondrocytes. Unfortunately, human hyaline chondrocytes oftenundergo dedifferentiation in vitro. Long non-coding RNAs (lncRNA) play aregulatory role in gene expression in many pathological and physiological processes.However, their role in human hyaline chondrocyte dedifferentiation remains unclear.This study aimed to investigate the expression profiles of lncRNAs in human hyalinechondrocyte dedifferentiation.Methods: Human hyaline chondrocytes were cultured in vitro and screened for theoccurrence of dedifferentiation using real-time quantitative PCR (qPCR),immunofluorescence, and western blotting. The expression profiles of lncRNAs andmRNAs during dedifferentiation were analyzed by microarray analysis and real-timeqPCR. We used pellet culture to redifferentiate chondrocytes and the expression ofrelated lncRNAs were assessed. The function of lncRNA AP001505.9(ENST00000569966) was determined by overexpression, fluorescence in situhybridization, competing endogenous RNA (ceRNA) analysis, and double luciferaselabeling.Results: We probed human hyaline chondrocytes dedifferentiation and identified 334upregulated and 381 downregulated lncRNAs. The expression of downregulatedlncRNA AP001505.9 in dedifferentiation was reversed by pellet culture. Theoverexpression of AP001505.9 inhibited dedifferentiation by promoting theexpression of SRY-Box transcription factor 9 (SOX-9) and inhibiting the expressionof type I collagen (COL1) both in vitro and in vivo.Conclusion: This study reveals for the first time the expression profiles of lncRNAsin human hyaline chondrocyte dedifferentiation, thereby providing a new perspectivefor exploring the potential mechanism of chondrocyte dedifferentiation.

Chondrogenic Potential of Pellet Culture Compared to High-Density Culture on a Bacterial Cellulose Hydrogel

Cell-based approaches of cartilage lesions use different culture systems to obtain optimal cell quality. Pellet cultures with high cellular density (HD) are the gold standard to keep chondrocytes in a differentiated stage. Bacterial cellulose (BC) hydrogel is discussed to prevent cellular aging and dedifferentiation. The hypothesis of this study was that HD culture on BC hydrogel (HD hydrogel) might reach the chondrogenic potential of pellet culture (pellet). Human articular osteoarthritic (OA) and non-osteoarthritic (non-OA) chondrocytes were cultured for seven days within pellets and compared to HD hydrogel and HD polystyrene. Gene expression analysis and histological assessment were performed. We observed no significant change of COL2A1 expression by the culture system (pellet, HD hydrogel and HD polystyrene) but a significant change of COL2A1/COL1A1-ratio, with the highest ratio in pellets. Chondrocytes on HD hydrogel showed an elevated expression of MMP13 and on polystyrene an increased expression of COL1A1 and MMP13. The patterns of gene expression changes observed in OA and non-OA chondrocytes in reaction to the different culture systems were similar in those two cell groups. Pellet cultures moreover formed a histomorphologically superior neocartilage. Concluding, human chondrocytes kept the potential to express COL2A1 in all HD culture systems. However, pellets excelled in a higher COL2A1/COL1A1-ratio, a higher extracellular matrix deposit and in not developing degeneration and dedifferentiation markers. This underlines the superiority of pellet culture in maintaining the chondrogenic potential of human chondrocytes in vitro.

The effect of the combination of TGF-β1 and BMP2 with high-density pellet cell culture during chondrogenic differentiation of pluripotent stem cells.

Introduction: The osteoarthritis is a serious threat for well-developed and ageing countries. Present techniques of treatment of damaged cartilage are not sufficient. Hence, new strategies should be developed. One of the potential sources for the regeneration of cartilage is pluripotent stem cells (PSC). Aim: The development of an efficient protocol of chondrogenic differentiation using PSC. Material and methods: The human embryonic stem cell line (BG01V) was used in this study. The chondrogenic differentiation was performed using high-density pellet culture in the presence of TGF-β1 (10 ng/ml) and BMP2 (100 ng/ml). After 21 days gene expression analysis of markers related to chondrogenesis was done. Additionally, the histological staining was performed to detect the deposition of proteoglycans and collagens in differentiated pellet culture. Results: Obtained pellets exhibited decreased expression of pluripotent markers. The upregulation of mesodermal marker and type II collagen was observed in differentiated pellets in the presence of applied growth factors. The histological analysis revealed mild deposition of proteoglycans and collagens. Conclusion: The presented approach enables to obtain chondrogenic pellets in their early stages of chondrogenesis.

Proinflammatory Effects of IL-1β Combined with IL-17A Promoted Cartilage Degradation and Suppressed Genes Associated with Cartilage Matrix Synthesis In Vitro

Combinations of IL-1β and other proinflammatory cytokines reportedly promote the severity of arthritis. We aimed to investigate the effects of IL-1β combined with IL-17A on cartilage degradation and synthesis in in vitro models. Cartilage explant degradation was determined using sulfated glycosaminoglycans (S-GAGs) levels, matrix metalloproteinase (MMP13) gene expression, uronic acid, and collagen contents. Cell morphology and accumulation of proteoglycans were evaluated using hematoxylin-eosin and safranin O staining, respectively. In the pellet culture model, expressions of cartilage-specific anabolic and catabolic genes were evaluated using real-time qRT-PCR. Early induction of MMP13 gene expression was found concomitantly with significant S-GAGs release. During the prolonged period, S-GAGs release was significantly elevated, while MMP-13 enzyme levels were persistently increased together with the reduction of the cartilaginous matrix molecules. The pellet culture showed anabolic gene downregulation, while expression of the proinflammatory cytokines, mediators, and MMP13 genes were elevated. After cytokine removal, these effects were restored to nearly basal levels. This study provides evidence that IL-1β combined with IL-17A promoted chronic inflammatory arthritis by activating the catabolic processes accompanied with the suppression of cartilage anabolism. These suggest that further applications, which suppress inflammatory enhancers, especially IL-17A, should be considered as a target for arthritis research and therapy.

Human Articular Chondrocytes Retain Their Phenotype in Sustained Hypoxia While Normoxia Promotes Their Immunomodulatory Potential

Objective To assess the phenotype of human articular chondrocytes cultured in normoxia (21% O2) or continuous hypoxia (2% O2). Design Chondrocytes were extracted from patients undergoing total knee replacement ( n = 5) and cultured in ~21% (normoxic chondrocytes, NC) and 2% (hypoxic chondrocytes, HC) oxygen in both monolayer and 3-dimensional (3D) pellet culture and compared with freshly isolated chondrocytes (FC). Cells were assessed by flow cytometry for markers indicative of mesenchymal stromal cells (MSCs), chondrogenic-potency and dedifferentiation. Chondrogenic potency and immunomodulatory gene expression was assessed in NC and HC by reverse transcription quantitative polymerase chain reaction. Immunohistochemistry was used to assess collagen II production following 3D pellet culture. Results NC were positive (>97%, n = 5) for MSC markers, CD73, CD90, and CD105, while HC demonstrated <90% positivity ( n = 4) and FC ( n = 5) less again (CD73 and CD90 <20%; CD105 <40%). The markers CD166 and CD151, indicative of chondrogenic de-differentiation, were significantly higher on NC compared with HC and lowest on FC. NC also produced the highest levels of CD106 and showed the greatest levels of IDO expression, following interferon-γ stimulation, indicating immunomodulatory potential. NC produced the highest levels of CD49c (>60%) compared with HC and FC in which production was <2%. Hypoxic conditions upregulated expression of SOX9, frizzled-related protein ( FRZB), fibroblast growth factor receptor 3 ( FGFR3), and collagen type II ( COL2A1) and downregulated activin receptor-like kinase 1 ( ALK1) in 3 out of 4 patients compared with normoxic conditions for monolayer cells. Conclusions Hypoxic conditions encourage retention of a chondrogenic phenotype with some immunomodulatory potential, whereas normoxia promotes dedifferentiation of chondrocytes toward an MSC phenotype with loss of chondrogenic potency but enhanced immunomodulatory capacity.