S-adenosylmethionine (SAM) is considered to be a useful therapeutic agent for degenerative cartilage diseases, although its mechanism is not clear. We previously found that polyamines stimulate expression of differentiated phenotype of chondrocytes. We also found that cellular communication network factor 2 (CCN2) played a huge role in the proliferation and differentiation of chondrocytes. Therefore, we hypothesized that polyamines and CCN2 could be involved in the chondroprotective action of SAM. In this study, we initially found that exogenous SAM enhanced proteoglycan production but not cell proliferation in human chondrocyte-like cell line-2/8 (HCS-2/8) cells. Moreover, SAM enhanced gene expression of cartilage-specific matrix (aggrecan and type II collagen), Sry-Box transcription factor 9 (SOX9), CCN2 and chondroitin sulfate biosynthetic enzymes. Blockade of the methionine adenosyltransferase 2A (MAT2A) enzyme catalyzing intracellular SAM biosynthesis restrained the effect of SAM on chondrocytes. The polyamine level in chondrocytes was higher in SAM-treated culture than control culture. Additionally, alcian blue staining and RT-qPCR indicated that the effects of SAM on the production and gene expression of aggrecan were reduced by the inhibition of polyamine synthesis. These results suggest that the stimulation of polyamine synthesis and gene expression of chondrogenic differentiation factors, such as CCN2, account for the mechanism underlying the action of SAM on chondrocytes.

Focal cartilage defects are a prevalent knee problem affecting people of all ages. Due to its avascular nature, cartilage has limited self-repair capacity, and osteochondral defects can lead to pain and long-term complications such as osteoarthritis. Autologous chondrocyte implantation (ACI) has been a successful surgical approach for repairing osteochondral defects over the past two decades. However, a major drawback of ACI is the de-differentiation of chondrocytes during their in vitro expansion. In this study, we isolated ovine chondrocytes and cultured them in a two-dimensional environment as for ACI procedures. We hypothesised that the 3D scaffolds would support the cells re-differentiation without the need for growth factors and so we encapsulated them into soft collagen and alginate (col/alg) hydrogels. Chondrocytes embedded into hydrogels were viable and proliferated. After 7 days they acquired a rounded morphology and started to aggregate. Gene expression studies showed that the genes associated with chondrogenesis started to be up regulated as early as day one. At 21 days chondrocytes had extensively colonized the hydrogel, forming large cell clusters and started to deposit collagen II and aggrecan with limited collagen type I deposition. These findings highlight the potential of soft col/alg hydrogels to enhance ACI outcomes by creating a favourable microenvironment for chondrocyte reprogramming and re-differentiation, eliminating the dependency on growth factors.

As a chronic disease, osteoarthritis (OA) leads to degradation of both cartilage and subchondral bone, of which the development is related to proinflammatory cytokines like interleukin-1β. In the present study, the anti-inflammatory effect of Specnvezhenide in osteoarthritis and mechanism of it was studied in vitro and in vivo. The results showed that Specnvezhenide decreases interleukin-1β-induced expression of matix-degrading enzymes and reduces the activation of NF-κB and wnt/β-catenin pathways in vitro. Furthermore, Specnvezhenide treatment prevents the degeneration of both cartilage and subchondral bone in rats OA model. As conclusion, to the best of our knowledge, we report firstly that Specnvezhenide decreases interleukin-1β-induced inflammation on rat chondrocytes by inhibiting activation of NF-κB and wnt/β-catenin pathways, and has therapeutic potential in OA treatment.

Glycosphingolipids (GSLs), a subtype of glycolipids containing sphingosine, are critical components of vertebrate plasma membranes, playing a pivotal role in cellular signaling and interactions. In human articular cartilage in osteoarthritis (OA), GSL expression is known notably to decrease. This review focuses on the roles of gangliosides, a specific type of GSL, in cartilage degeneration and regeneration, emphasizing their regulatory function in signal transduction. The expression of gangliosides, whether endogenous or augmented exogenously, is regulated at the enzymatic level, targeting specific glycosyltransferases. This regulation has significant implications for the composition of cell surface gangliosides and their impact on signal transduction in chondrocytes and progenitor cells. Different levels of ganglioside expression can influence signaling pathways in various ways, potentially affecting cell properties, including malignancy. Moreover, gene manipulations against gangliosides have been shown to regulate cartilage metabolism and chondrocyte differentiation in vivo and in vitro. This review highlights the potential of targeting gangliosides in the development of therapeutic strategies for osteoarthritis and cartilage injury and addresses promising directions for future research and treatment.

The modification of proteins by small ubiquitin-related modifier (SUMO) molecules, SUMOylation, is a key post-translational modification involved in a variety of biological processes such as chromosomes organization, DNA replication and repair, transcription, nuclear transport, and cell signaling transduction. In recent years, emerging evidence has shown that SUMOylation regulates the development and homeostasis of the skeletal system, with its dysregulation causing skeletal diseases, suggesting that SUMOylation pathways may serve as a promising therapeutic target. In this review, we summarize the current understanding of the molecular mechanisms by which SUMOylation pathways regulate skeletal cells in the physiological and disease contexts.

High-frequency near-infrared diode laser provides high peak output, low heat accumulation, and efficient biostimulation. Although these characteristics are considered suitable for osteoarthritis (OA) treatment, the effect of high-frequency near-infrared diode laser in in vitro or in vivo OA models has not yet been reported. Therefore, we aimed to assess the biological effects of high-frequency near-infrared diode laser irradiation on IL-1β-induced chondrocyte inflammation in an in vitro OA model. Normal Human Articular Chondrocyte-Knee (NHAC-Kn) cells were stimulated with human recombinant IL-1β and irradiated with high-frequency near-infrared diode laser (910 nm, 4 or 8 J/cm2). The mRNA and protein expression of relevant inflammation- and cartilage destruction-related proteins was analyzed. IL-1β treatment significantly increased the mRNA levels of IL-1β, IL-6, TNF-α, MMP-1, MMP-3, and MMP-13. High-frequency near-infrared diode laser irradiation significantly reduced the IL-1β-induced expression of IL-1β, IL-6, TNF-α, MMP-1, and MMP-3. Similarly, high-frequency near-infrared diode laser irradiation decreased the IL-1β-induced increase in protein expression and secreted levels of MMP-1 and MMP-3. These results highlight the therapeutic potential of high-frequency near-infrared diode laser in OA.

The human degenerative cartilage has low regenerative potential. Chondrocyte transplantation offers a promising strategy for cartilage treatment and regeneration. Currently chondrogenesis using human pluripotent stem cells are accomplished using human recombinant growth factors. Here, we differentiated human induced pluripotent stem cells (hiPSCs) into chondrocytes and cartilage pellet using minicircle vectors. Minicircles are used as a non-viral gene delivery system for gene therapy in various diseases. Non-viral gene delivery can produce growth factors without integrating into the host genome. Minicircle vectors containing bone morphogenetic protein 2 (BMP2) and transforming growth factor, beta 3 (TGFβ3) were successfully generated and delivered to hiPSC-derived outgrowth (OG) cells. Cell pellets generated using minicircle-transfected OG cells successfully differentiated into chondrogenic lineage. Chondrogenic pellets transfected with growth factor-encoding minicircles effectively recovered osteochondral defect in rat models. Taken together, this work shows the potential application of minicircles in cartilage regeneration using hiPSCs.

Bone growth plate abnormalities and skull shape defects are seen in hypophosphatasia, a heritable disorder in humans that occurs due to deficiency of Tissue Nonspecific Alkaline Phosphatase (TNAP, Alpl) enzyme activity. Abnormal development of the cranial base growth plates (synchondroses) and abnormal skull shapes have also been demonstrated in global TNAP-/- mice. To distinguish local vs. systemic effects of TNAP on skull development, we utilized P0-Cre to knockout TNAP only in cranial neural crest derived tissues using TNAP flox mice. Here we show that TNAP deficiency in cranial neural crest leads to leads to skull shape defects and deficient growth of the intersphenoid synchondrosis (ISS). ISS chondrocyte abnormalities included increased proliferation in resting and proliferative zones with decreased apoptosis in hypertrophic zones. ColX expression is increased, indicative of premature differentiation in the absence of TNAP. Sox9 expression is increased in both resting and prehypertrophic zones of mutant mice. Expression of PTHrP and IHH were also increased. Finally, cranial base organ culture revealed that inorganic phosphate (Pi) and pyrophosphate (PPi) have specific effects on cell signaling and phenotype changes in the ISS. Together, these results demonstrate that TNAP expression in growth plate chondrocytes is essential for normal development, and that the mechanism likely involves Sox9, PTHrP, IHH and PPi.

To evaluate the relative importance of IGF-I expression in various cell types for endochon-dral ossification, we quantified the trabecular bone at the secondary spongiosa and epiphysis of the distal femur in 8-12-week-old male mice with a global knockout of the Igf-I gene as well as conditional deletion of the Igf-I gene in osteoblasts, chondrocytes, osteo-blasts/chondrocytes, and their corresponding control wild type littermates. The osteoblast-, chondrocyte- and osteoblast/chondrocyte-specific Igf-I conditional knockout mice were generated by crossing Igf-I floxed mice with Cre transgenic mice in which Cre expression is under the control of Col1α2 or Col2α1 promoter. We found that global disruption of Igf-I resulted in 80% and 70% reduction in bone size, which is defined as total volume, at the secondary spongiosa and epiphysis of the distal femur, respectively. Abrogation of Igf-I in Col1α2-producing osteoblasts, but not Col2α1-producing chondrocytes, decreased bone size by 25% at both the secondary spongiosa and epiphysis while deletion of the Igf-I globally or specifically in osteoblasts or chondrocytes reduced trabecular bone mass by 25%. By contrast, global Igf-I knockout but not conditional knockout of Igf-I in osteoblasts and/or chondrocytes reduced trabecular bone mass in the epiphysis. The reduced trabecu-lar bone mass at the secondary spongiosa in osteoblast- and/or chondrocyte-specific Igf-I conditional knockout mice is caused by reduced trabecular number and increased trabec-ular separation. Immunohistochemistry studies revealed that expression levels of chon-drocyte (COL10, MMP13) and osteoblast (BSP) markers were reduced in the secondary spongiosa and the epiphyses in the global Igf-I knockout mice. Our data indicate that local and endocrine IGF-I actions in bone are pleiotropic and dependent on cell type as well as the bone compartment where IGF-I acts.

Recent improvements within the fields of high-throughput screening and 3D tissue culture have provided the possibility of developing in vitro micro-tissue models that can be used to study diseases and screen potential new therapies. This paper reports a proof of concept study on the use of microvalve-based bioprinting to create laminar MSC-chondrocyte co-cultures as an in vitro model of autologous chondrocyte implantation (ACI), an established cellular therapy for osteoarthritis. Microvalve-based bioprinting uses microvalves to deposit cells suspended in a liquid in a consistent and repeatable manner. In this case MSCs and chondrocytes have been sequentially deposited into an insert based transwell system in order to create a laminar co-culture, with variations in the ratios of the cell types used to investigate the potential for MSCs to stimulate improved repair. Histological and indirect immunofluorescence staining revealed the formation of dense tissue structures within the chondrocyte and MSC-chondrocyte cell co-cultures, alongside the establishment of a proliferative region at the base of the tissue. No stimulatory or inhibitory effect in terms of ECM production was observed through the introduction of MSCs, although the potential for an immunomodulatory benefit remains. This proof-of-concept study therefore provides a novel method to enable the scalable production of therapeutically relevant micro-tissue models that can be used for in vitro research to optimise ACI procedures.

The aim of this study was to investigate the role of DNA methylation in the regulation of in vitro and in vivo cartilage formation. Based on the data of an RNA chip-assay performed on chondrifying BMP2-overexpressing C3H10T1/2 cells, the relative expression of Tet1 (tet methylcytosine dioxygenase 1), Dnmt3a (DNA methyltransferase 3) and Ogt (O-linked N-acetylglucosamine transferase) genes was examined with RT-qPCR in mouse cell-line based and primary micromass cultures. RNA probes for in situ hybridization were used on frozen sections of 15-day-old mouse embryos. DNA methylation was inhibited with 5-azacytidine during culturing. We found very strong but gradually decreasing expression of Tet1 throughout the entire course of in vitro cartilage differentiation along with strong signals in the cartilaginous embryonic skeleton. Dnmt3a and Ogt expressions did not show significant changes with RT-qPCR and gave weak in situ hybridization signals. Inhibition of DNA methylation applied during early stages of differentiation reduced cartilage-specific gene expression and cartilage formation. In contrast, it had stimulatory effect when added to differentiated chondrocytes. Our results indicate that the DNA demethylation-inducing Tet1 is a significant epigenetic factor of chondrogenesis, and inhibition of DNA methylation exerts distinct effects in different phases of in vitro cartilage formation.