Runx1 alleviates osteoarthritis progression in aging mice.

With rates growing quickly with age, osteoarthritis (OA) is the most common cause of chronic disability in aging persons. The discomfort and reduced motion associated with osteoarthritis have a significant impact on quality of life, and there is no known solution. Runt-related transcription factor 1(Runx1) has been shown to play a protective role in the development of osteoarthritis by promoting chondrogenesis. We had created models of ageing mice with osteoarthritis by anterior cruciate ligament transection (ACLT) and analyzed the effects of intra-articular injection of adeno-associated virus/Runx1 (AAV/Runx1) on the models. The results showed that the AAV/Runx1-group maintained better articular cartilage integrity and retained more proteoglycan than the OA group after injection of AAV-Runx1. The markers related to pathological changes in cartilage were downregulated, while the markers related to physiological changes in cartilage were upregulated. This suggests that Runx1 may impede OA progression on the knee joint of ageing mice, potentially playing a protective role in OA and becoming a probable treatment target for osteoarthritis among ageing patients in the future.

Biomimetic Fibers Based on Equidistant Micropillar Arrays Determines Chondrocyte Fate via Mechanoadaptability.

It is recognized that the changes in the physical properties of extracellular matrix (ECM) result in fine-tuned cell responses including cell morphology, proliferation and differentiation. In this study, a novel patterned equidistant micropillar substrate based on polydimethylsiloxane (PDMS) is designed to mimic the collagen fiber-like network of the cartilage matrix. By changing the component of the curing agent to an oligomeric base, micropillar substrates with the same topology but different stiffnesses are obtained and it is found that chondrocytes seeded onto the soft micropillar substrate maintain their phenotype by gathering type II collagen and aggrecan more effectively than those seeded onto the stiff micropillar substrate. Moreover, chondrocytes sense and respond to micropillar substrates with different stiffnesses by altering the ECM-cytoskeleton-focal adhesion axis. Further, it is found that the soft substrate-preserved chondrocyte phenotype is dependent on the activation of Wnt/ß-catenin signaling. Finally, it is indicated that the changes in osteoid-like region formation and cartilage phenotype loss in the stiffened sclerotic area of osteoarthritis cartilage to validate the changes triggered by micropillar substrates with different stiffnesses. This study provides the cell behavior changes that are more similar to those of real chondrocytes at tissue level during the transition from a normal state to a state of osteoarthritis.

FGF19 induces the cell cycle arrest at G2-phase in chondrocytes.

Fibroblast growth factor 19 (FGF19) has appeared as a new possible avenue in the treatment of skeletal metabolic disorders. However, the role of FGF19 on cell cycle progression in skeletal system is poorly understood. Here we demonstrated that FGF19 had the ability to reduce the proliferation of chondrocytes and cause cell cycle G2 phase arrest through its interaction with ß-Klotho (KLB), an important accessory protein that helps FGF19 link to its receptor. FGF19-mediated cell cycle arrest by regulating the expressions of cdk1/cylinb1, chk1 and gadd45a. We then confirmed that the binding of FGF19 to the membrane receptor FGFR4 was necessary for FGF19-mediated cell cycle arrest, and further proved that FGF19-mediated cell cycle arrest was via activation of p38/MAPK signaling. Through inhibitor experiments, we discovered that inhibition of FGFR4 led to down-regulation of p38 signaling even in the presence of FGF19. Meanwhile, inhibiting p38 signaling reduced the cell cycle arrest of chondrocytes induced by FGF19. Furthermore, blocking p38 signaling facilitated to retain the expression of cdk1 and cyclinb1 that had been reduced in chondrocytes by FGF19 and decreased the expression of chk1 and gadd45a that had been enhanced by FGF19 in chondrocytes. Taking together, this study is the first to demonstrate that FGF19 induces cell cycle arrest at G2 phase via FGFR4-p38/MAPK axis and enlarges our understanding about the role of FGF19 on cell cycle progression in chondrocytes.

Stiffened fibre-like microenvironment based on patterned equidistant micropillars directs chondrocyte hypertrophy.

Articular cartilage, composed of collagen type II as a major extracellular matrix and chondrocyte as a unique cell type, is a specialized connective tissue without blood vessels, lymphatic vessels and nerves. This distinctive characteristic of articular cartilage determines its very limited ability to repair when damaged. It is well known that physical microenvironmental signals regulate many cell behaviors such as cell morphology, adhesion, proliferation and cell communication even determine chondrocyte fate. Interestingly, with increasing age or progression of joint diseases such as osteoarthritis (OA), the major collagen fibrils in the extracellular matrix of articular cartilage become larger in diameter, leading to stiffening of articular tissue and reducing its resistance to external tension, which in turn aggravates joint damage or progression of joint diseases. Therefore, designing a physical microenvironment closer to the real tissue and thus obtaining data closer to the real cellular behaviour, and then revealing the biological mechanisms of chondrocytes in pathological states is of crucial importance for the treatment of OA disease. Here we fabricated micropillar substrates with the same topology but different stiffnesses to mimic the matrix stiffening that occurs in the transition from normal to diseased cartilage. It was first found that chondrocytes responded to stiffened micropillar substrates by showing a larger cell spreading area, a stronger enhancement of cytoskeleton rearrangement and more stability of focal adhesion plaques. The activation of Erk/MAPK signalling in chondrocytes was detected in response to the stiffened micropillar substrate. Interestingly, a larger nuclear spreading area of chondrocytes at the interface layer between the cells and top surfaces of micropillars was observed in response to the stiffened micropillar substrate. Finally, it was found that the stiffened micropillar substrate promoted chondrocyte hypertrophy. Taken together, these results revealed the cell responses of chondrocytes in terms of cell morphology, cytoskeleton, focal adhesion, nuclei and cell hypertrophy, and may be beneficial for understanding the cellular functional changes affected by the matrix stiffening that occurs during the transition from a normal state to a state of osteoarthritis.

The role of fibroblast growth factor 7 in cartilage development and diseases.

Fibroblast growth factor 7 (FGF7), also known as keratinocyte growth factor (KGF), shows a crucial biological significance in tissue development, wound repair, tumorigenesis, and immune reconstruction. In the skeletal system, FGF7 directs the cellular synaptic extension of individual cells and facilities functional gap junction intercellular communication of a collective of cells. Moreover, it promotes the osteogenic differentiation of stem cells via a cytoplasmic signaling network. For cartilage, reports have indicated the potential role of FGF7 on the regulation of key molecules Cx43 in cartilage and Runx2 in hypertrophic cartilage. However, the molecular mechanism of FGF7 in chondrocyte behaviors and cartilage pathological process remains largely unknown. In this review, we systematically summarize the recent biological function of FGF7 and its regulatory role on chondrocytes and cartilage diseases, especially through the hot focus of two key molecules, Runx2 and Cx43. The current knowledge of FGF7 on the physiological and pathological processes of chondrocytes and cartilage provides us new cues for wound repair of cartilage defect and therapy of cartilage diseases.

FGF19 increases mitochondrial biogenesis and fusion in chondrocytes via the AMPKα-p38/MAPK pathway.

Fibroblast growth factor 19 (FGF19) is recognized to play an essential role in cartilage development and physiology, and has emerged as a potential therapeutic target for skeletal metabolic diseases. However, FGF19-mediated cellular behavior in chondrocytes remains a big challenge. In the current study, we aimed to investigate the role of FGF19 on chondrocytes by characterizing mitochondrial biogenesis and fission-fusion dynamic equilibrium and exploring the underlying mechanism. We first found that FGF19 enhanced mitochondrial biogenesis in chondrocytes with the help of ß Klotho (KLB), a vital accessory protein for assisting the binding of FGF19 to its receptor, and the enhanced biogenesis accompanied with a fusion of mitochondria, reflecting in the elongation of individual mitochondria and the up-regulation of mitochondrial fusion proteins. We then revealed that FGF19-mediated mitochondrial biogenesis and fusion required the binding of FGF19 to the membrane receptor, FGFR4, and the activation of AMP-activated protein kinase alpha (AMPKα)/peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α)/sirtuin 1 (SIRT1) axis. Finally, we demonstrated that FGF19-mediated mitochondrial biogenesis and fusion was mainly dependent on the activation of p-p38 signaling. Inhibition of p38 signaling largely reduced the high expression of AMPKα/PGC-1α/SIRT1 axis, decreased the up-regulation of mitochondrial fusion proteins and impaired the enhancement of mitochondrial network morphology in chondrocytes induced by FGF19. Taking together, our results indicate that FGF19 could increase mitochondrial biogenesis and fusion via AMPKα-p38/MAPK signaling, which enlarge the understanding of FGF19 on chondrocyte metabolism. Video Abstract.

IL-10 enhances cell-to-cell communication in chondrocytes via STAT3 signaling pathway.

Gap junction intercellular communication (GJIC) allows the transfer of material, message and energy between cells, which influences cell behaviors including cell proliferation, migration, differentiation and apoptosis and determines cell fate. Interleukin-10 (IL-10), a versatile cytokine, attracts more and more attention in the cartilage pathology such as osteoarthritis (OA) due to its potential in anti-inflammation and wound repair. However, whether IL-10 can mediate GJIC in chondrocytes remains elusive. In the current study, we aimed to explore the role of IL-10 on GJIC and its underlying mechanism. We found that IL-10 can promote GJIC in living chondrocytes. IL-10-enhanced GJIC in chondrocytes was dependent on the up-regulation of connexin 43 (Cx43). Knockdown experiment based on siRNA interference then confirmed that IL-10-enhanced GJIC required participation of IL-10 receptor 1 (IL-10R1). IL-10 activated signal transducer and activator of transcription 3 (STAT3) signaling and promoted the nuclear accumulation of p-STAT3 through IL-10 receptor 1. Inhibitor experiment further confirmed the importance of STAT3 signaling in IL-10-mediated GJIC. Taking together, our results provided a thorough process of IL-10-modulated cell-to-cell communication in chondrocytes and established a bridge between inflammatory factor, IL-10, and GJIC, which can increase our understanding about the physiology and pathology of cartilage.

TGF-ß3 enhances cell-to-cell communication in chondrocytes via the ALK5/p-Smad3 axis.

Gap junctional intercellular communication (GJIC) is indispensable for the maintenance of physiological balance in articular cartilage. Transforming growth factor-ß3 (TGF-ß3), an important growth factor of TGF-ß superfamily, is well recognized to play a unique regulatory role in cartilage development and diseases. However, the role of TGF-ß3 in GJIC in adult chondrocytes remains elusive. This work aims to investigate the effect of TGF-ß3 on gap-junction mediated intercellular communication in chondrocytes. We first showed that TGF-ß3 could enhance the synaptic connections between chondrocytes by scanning electron microscopy (SEM) and promote the cell-to-cell communication in living chondrocytes by scrape loading/dye transfer assay. We then confirmed that TGF-ß3 enhanced cell-to-cell communication via up-regulation of connexin 43 (Cx43). We next found that TGF-ß3-enhanced GJIC required the participation of TGF-beta type I receptor ALK5 and depended on the activation of p-Smad3 signalling. Finally, through inhibitor experiments of SB525334 and SIS3, we demonstrated that TGF-ß3-induced functional GJIC in chondrocytes via the axis of ALK5/p-Smad3 signalling. Taking together, these results demonstrate a strong correlation between TGF-ß3 and GJIC in chondrocytes, which provides a new perspective on the importance of TGF-ß3 on cartilage physiology and pathobiology.

PTHrP promotes subchondral bone formation in TMJ-OA.

PTH-related peptide (PTHrP) improves the bone marrow micro-environment to activate the bone-remodelling, but the coordinated regulation of PTHrP and transforming growth factor-ß (TGFß) signalling in TMJ-OA remains incompletely understood. We used disordered occlusion to establish model animals that recapitulate the ordinary clinical aetiology of TMJ-OA. Immunohistochemical and histological analyses revealed condylar fibrocartilage degeneration in model animals following disordered occlusion. TMJ-OA model animals administered intermittent PTHrP (iPTH) exhibited significantly decreased condylar cartilage degeneration. Micro-CT, histomorphometry, and Western Blot analyses disclosed that iPTH promoted subchondral bone formation in the TMJ-OA model animals. In addition, iPTH increased the number of osterix (OSX)-positive cells and osteocalcin (OCN)-positive cells in the subchondral bone marrow cavity. However, the number of osteoclasts was also increased by iPTH, indicating that subchondral bone volume increase was mainly due to the iPTH-mediated increase in the bone-formation ability of condylar subchondral bone. In vitro, PTHrP treatment increased condylar subchondral bone marrow-derived mesenchymal stem cell (SMSC) osteoblastic differentiation potential and upregulated the gene and protein expression of key regulators of osteogenesis. Furthermore, we found that PTHrP-PTH1R signalling inhibits TGFß signalling during osteoblastic differentiation. Collectively, these data suggested that iPTH improves OA lesions by enhancing osteoblastic differentiation in subchondral bone and suppressing aberrant active TGFß signalling. These findings indicated that PTHrP, which targets the TGFß signalling pathway, may be an effective biological reagent to prevent and treat TMJ-OA in the clinic.

Mammalian Sirtuins and Their Relevance in Vascular Calcification.

Cardiovascular diseases are a group of diseases with high morbidity and mortality that affect millions of people each year. Vascular calcification (VC) is an active process that involves the mineral deposition of calcium-phosphate complexes. VC is closely related to cardiovascular diseases, such as hypertension, heart failure, and calcific aortic stenosis, and is a type of ectopic calcification that occurs in the vessel walls. The sirtuins (silent mating-type information regulation 2; SIRTs), are a family of histone deacetylases whose function relies on nicotinamide adenine dinucleotide (NAD+). They have non-negligible functions in the regulation of energy metabolism, senescence, apoptosis, and other biological processes. Sirtuins have important effects on bone homeostasis and VC processes that share many similarities with bone formation. Sirtuins have been confirmed to deacetylate a variety of target proteins related to the occurrence and development of VC, thereby affecting the process of VC and providing new possibilities for the prevention and treatment of cardiovascular diseases. To facilitate the understanding of vascular calcification and accelerate the development of cardiovascular drugs, we reviewed and summarized recent research progress on the relationship between different types of sirtuins and VC.

The roles of Runx1 in skeletal development and osteoarthritis: A concise review.

Runt-related transcription factor-1 (Runx1) is well known for its functions in hematopoiesis and leukemia but recent research has focused on its role in skeletal development and osteoarthritis (OA). Deficiency of the Runx1 gene is fatal in early embryonic development, and specific knockout of Runx1 in cell lineages of cartilage and bone leads to delayed cartilage formation and impaired bone calcification. Runx1 can regulate genes including collagen type II (Col2a1) and X (Col10a1), SRY-box transcription factor 9 (Sox9), aggrecan (Acan) and matrix metalloproteinase 13 (MMP-13), and the up-regulation of Runx1 improves the homeostasis of the whole joint, even in the pathological state. Moreover, Runx1 is activated as a response to mechanical compression, but impaired in the joint with the pathological progress associated with osteoarthritis. Therefore, interpretation about the role of Runx1 could enlarge our understanding of key marker genes in the skeletal development and an increased understanding of Runx1 could be helpful to identify treatments for osteoarthritis. This review provides the most up-to-date advances in the roles and bio-mechanisms of Runx1 in healthy joints and osteoarthritis from all currently published articles and gives novel insights in therapeutic approaches to OA based on Runx1.

Gold standard for nutrition: a review of human milk oligosaccharide and its effects on infant gut microbiota.

Human milk is the gold standard for nutrition of infant growth, whose nutritional value is mainly attributed to human milk oligosaccharides (HMOs). HMOs, the third most abundant component of human milk after lactose and lipids, are complex sugars with unique structural diversity which are indigestible by the infant. Acting as prebiotics, multiple beneficial functions of HMO are believed to be exerted through interactions with the gut microbiota either directly or indirectly, such as supporting beneficial bacteria growth, anti-pathogenic effects, and modulation of intestinal epithelial cell response. Recent studies have highlighted that HMOs can boost infants health and reduce disease risk, revealing potential of HMOs in food additive and therapeutics. The present paper discusses recent research in respect to the impact of HMO on the infant gut microbiome, with emphasis on the molecular basis of mechanism underlying beneficial effects of HMOs.

The alteration of A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) in the knee joints of osteoarthritis mice.

The A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family is gradually being recognized as an important family of mediators that, along with the matrix metalloproteinases (MMPs), control the degradation process in osteoarthritis (OA). The objective of this study was to uncover the detailed alterations of ADAMTS1, ADAMTS2, and ADAMTS5 in the knee joint of OA mice. The OA model was established by anterior cruciate ligament transection (ACLT) on the knee joints of C57BL/6 J mice. The mice showed representative phenotypes of ACLT-induced OA, including obvious deterioration of the cartilage, reductions in the collagen and proteoglycan components in the cartilage matrix of OA mice, and increased inflammation and osteoclast activity. By qPCR, the gene expression levels of Adamts1, -2, and -5 were the top-ranked among Adamts1-5 in cartilage/chondrocytes, osteogenic tissue/osteoblasts, and cortical bone/osteocytes. Moreover, the protein expression levels of ADAMTS1, -2, and -5 were all increased in articular cartilage, the growth plate, and subchondral bone of the knee joint. The results suggest the important roles of ADAMTS1, -2, and -5 in OA disease, which will be helpful in further research on degenerative changes in OA.

Osteoporosis-decreased extracellular matrix stiffness impairs connexin 43-mediated gap junction intercellular communication in osteocytes.

Osteocytes are the main sensitive and responsive cells for mechanical stimuli in bone. The connexin family enables them to communicate with each other via forming functional gap junctions. However, how osteoporosis-impaired extracellular mechanical property modulates gap junction intercellular communication in osteocytes remains elusive. In this study, we established an ovariectomy (OVX)-induced osteoporosis mouse model in vivo and a polydimethylsiloxane (PDMS)-based cell culture substrate model in vitro to explore the influence of extracellular matrix (ECM) stiffness on cell-to-cell communication in osteocytes. Firstly, we established an OVX-induced osteoporosis mouse model by characterizing the changes in radiography, morphology and histochemistry of femurs. Our results showed that osteoporosis decreased the bone matrix stiffness together with the changes including the loss of osteocytes and the decrease of protein markers. Meanwhile, the dendritic process interconnection and channel-forming protein, Cx43, were reduced in osteoporosis mice. Next we mimicked ECM stiffness changes in vitro by using PDMS substrates at ratios 1:5 for normal stiffness and 1:45 for osteoporosis stiffness. Our results showed that the decreased ECM stiffness reduced the number of dendritic processes in a single cell and gap junctions between adjacent osteocytes. We further detected the decreased expression of Cx43, in the substrate with decreased stiffness. Finally, we found that gap junction-based intercellular communication was reduced in living osteocytes in the substrate with decreased stiffness. This study demonstrates the correlation between ECM mechanical property and cell-to-cell communication in osteocytes and might pave the way for further exploration of osteoporosis in terms of biomechanics.

Biomaterial Stiffness Guides Cross-talk between Chondrocytes: Implications for a Novel Cellular Response in Cartilage Tissue Engineering.

The exquisite cartilage architecture maintains an orderly dynamic equilibrium as a result of the interplay between chondrocyte functions and the unique extracellular matrix (ECM) microenvironment. Numerous studies have demonstrated that extracellular cues, including topological, mechanical, and biochemical properties of the underlying substrates, dictate the chondrocyte behaviors. Consequently, developing advanced biomaterials with the desired characteristics which could achieve the biointerface between cells and the surrounded matrix close to the physiological conditions becomes a great hotspot in bioengineering. However, how the substrate stiffness influences the intercellular communication among chondrocytes is still poorly reported. We used polydimethylsiloxane with varied stiffnesses as a cell culture substrate to elucidate a novel cell-to-cell communication in a collective of chondrocytes. First, morphological images collected using scanning electron microscopy revealed that the tunable substrate stiffnesses directed the changes in intercellular links among chondrocytes. Next, fibronectin, which played a vital role in the connection of ECM components or linkage of ECM to chondrocytes, was shown to be gathered along cell-cell contact areas and was changed with the tunable substrate stiffnesses. Furthermore, transmembrane junctional proteins including connexin 43 (Cx43) and pannexin 1 (Panx1), which are responsible for gap junction formation in cell-to-cell communication, were mediated by the tunable substrate stiffnesses. Finally, through a scrape loading/dye transfer assay, we revealed cell-to-cell communication changes in a living chondrocyte population in response to the tunable substrate stiffnesses via cell-to-cell fluorescent molecule transport. Taken together, this novel cell-to-cell communication regulated by biomaterial stiffness could help us to increase the understanding of cell behaviors under biomechanical control and may ultimately lead to refining cell-based cartilage tissue engineering.

Aberrant activation of latent transforming growth factor-ß initiates the onset of temporomandibular joint osteoarthritis.

There is currently no effective medical treatment for temporomandibular joint osteoarthritis (TMJ-OA) due to a limited understanding of its pathogenesis. This study was undertaken to investigate the key role of transforming growth factor-ß (TGF-ß) signalling in the cartilage and subchondral bone of the TMJ using a temporomandibular joint disorder (TMD) rat model, an ageing mouse model and a Camurati-Engelmann disease (CED) mouse model. In the three animal models, the subchondral bone phenotypes in the mandibular condyles were evaluated by µCT, and changes in TMJ condyles were examined by TRAP staining and immunohistochemical analysis of Osterix and p-Smad2/3. Condyle degradation was confirmed by Safranin O staining, the Mankin and OARSI scoring systems and type X collagen (Col X), p-Smad2/3a and Osterix immunohistochemical analyses. We found apparent histological phenotypes of TMJ-OA in the TMD, ageing and CED animal models, with abnormal activation of TGF-ß signalling in the condylar cartilage and subchondral bone. Moreover, inhibition of TGF-ß receptor I attenuated TMJ-OA progression in the TMD models. Therefore, aberrant activation of TGF-ß signalling could be a key player in TMJ-OA development.

Evidence for excessive osteoclast activation in SIRT6 null mice.

SIRT6 is a NAD-dependent histone 3 deacetylase. SIRT6 null mice have been reported suffering osteopenia. However, the role of SIRT6 in bone resorption is still not well understood. In this study, we focused on the role of SIRT6 in osteoclast. We performed histological analysis on the femur, spine, alveolar bone and even tail of mutant mice, and found the bone mass is sharply decreased while the osteoclast activity is significantly increased. These phenotypes were further demonstrated by the osteoclast differentiation in cell-cultures with TRAP staining and Pit Resorption Assay. We next found the proliferation activity of mutant osteoclast precursors was increased, which might account for the enhanced osteoclast formation. The concentration of tartrate-resistant acid phosphatase 5b, a marker of osteoclast differentiation, was significantly higher in the mutant mice than control. Besides, the osteoclastogenic and NF-κB signaling related genes were significantly up-regulated. Moreover, osteoblast/osteoclast co-culture demonstrated that SIRT6 regulated osteoclast mainly through osteoblast paracrine manner, rather than osteoclast-autonomous behavior. Together, the enhanced osteoclast activation in SIRT6 null mice might be regulated by the hyperactive NF-κB signaling and the enhanced proliferation activity of osteoclast precursors through osteoblast paracrine manner at the cellular level.

Bivalent Histone Modifications and Development.

BACKGROUND: Development is an epigenetic regulation dependent event. As one pretranscriptional regulator, bivalent histone modifications were observed to be involved in development recently. It is believed that histone methylation potentially takes charge of cell fate determination and differentiation. The synchronous existence of functionally opposite histone marks at transcript start sequence (TSS) is defined as "Bivalency", which mainly mark development related genes. H3K4me3 and H3K27me3, the prominent histone methylations of bivalency, are implicated in transcriptional activation and transcriptional repression respectively. The delicate balance between H3K4me3 and H3K27me3 produces diverse chromatin architectures, resulting in different transcription states of downstream genes: "poised", "activated" or "repressed". OBJECTIVE: In order to explore the developmental role of bivalent histone modification and the underlying mechanism, we did systematic review and rigorous assessment about relative literatures. RESULT: Bivalent histone modifications are considered to set up genes for activation during lineage commitment by H3K4me3 and repress lineage control genes to maintain pluripotency by H3K27me3. Summarily, bivalency in stem cells keeps stemness via poising differentiation relevant genes. After receiving developmental signals, the balance between "gene activation" and "gene repression" is broken, which turns genes transcription state from "poised" effect to switch on or switch off effect, thus initiates irreversible and spontaneous differentiation procedures. CONCLUSION: Bivalent histone modifications and the associated histone-modifying complexes safeguard proper and robust differentiation of stem cells, thus playing an essential role in development.