Fibroblast growth factor 7 protects osteoblasts against oxidative damage through targeting mitochondria.

The pathophysiology of osteoporosis is significantly influenced by the impaired functioning of osteoblasts, which is particularly caused by oxidative stress. Nevertheless, the underlying mechanisms responsible for this phenomenon are still not well understood. The objective of this study was to elucidate the impact of fibroblast growth factor 7 (FGF7) on the behavior of osteoblasts under conditions of oxidative stress. The osteoblast-like MC3T3 cells were pretreated with recombinant FGF7 in the presence of oxidative stress induced by hydrogen peroxide (H2 O2 ). We first provided the evidence that the endogenous FGF7 was significantly increased in osteoblasts in response to the increased H2 O2 levels. Recombined FGF7 demonstrated a remarkable capacity to resist the detrimental effects of H2 O2 -induced oxidative stress, including the increase in cell apoptosis, decrease in osteoblast viability, and impairment in osteogenic differentiation capacity, on osteoblasts. Furthermore, we extensively explored the mechanism underlying these protective effects and discovered a remarkable modulation of reactive oxygen species (ROS) homeostasis in H2 O2 -treated cells following the pronounced expression of FGF7, which significantly differed from the control group. Additionally, we observed that FGF7 exerted partial preservation on both the morphology and function of mitochondria when exposed to oxidative stress conditions. Furthermore, FGF7 exhibited the ability to enhance the activation of the p38/MAPK signaling pathway while concurrently suppressing the JNK/MAPK signaling pathway in response to oxidative stress. These results underscore the promising role and underlying mechanisms of FGF7 in preserving osteoblast homeostasis in the face of oxidative stress.

PDGF-AA promotes gap junction intercellular communication in chondrocytes via the PI3K/Akt pathway.

BACKGROUND: Gap junction intercellular communication (GJIC) plays an important role in cell growth, development and homeostasis. Connexin 43 (Cx43) is an important half-channel protein responsible for gap junction formation. Platelet-derived growth factor AA (PDGF-AA) regulates the proliferation, migration, metabolism, apoptosis and cell cycle of chondrocytes. However, the role of PDGF-AA in gap junction intercellular communication in chondrocytes is not fully understood. In the current study, we performed experiments to explore the effect of PDGF-AA on GJIC and its underlying biomechanical mechanism. METHODS: qPCR was performed to determine the expression of PDGF, PDGFR and connexin family genes in chondrocytes and/or cartilage. A scrape loading/dye transfer assay was used to determine GJIC. Western blot analysis was applied to detect the expression of Cx43 and PI3K/Akt signaling pathway proteins. Immunofluorescence staining was utilized to examine protein distribution. Scanning electron microscopy was used to delineate the morphology of chondrocytes. RESULTS: Expression of PDGF-A mRNA was highest among the PDGF family in chondrocytes and cartilage tissues. PDGF-AA promoted functional GJIC formation in chondrocytes by upregulating the expression of Cx43. Enhanced functional GJIC formation in chondrocytes induced by PDGF-AA occurred through the activation of PI3K/Akt signaling and its nuclear accumulation. CONCLUSION: For the first time, this study provides evidence demonstrating the role of PDGF-AA in cell-to-cell communication in chondrocytes through mediating Cx43 expression.

Osteoblasts induce glucose-derived ATP perturbations in chondrocytes through noncontact communication.

Cartilage and subchondral bone communicate with each other through material and signal exchanges. However, direct evidence provided by experimental studies on their interactions is insufficient. In the present study, we establish a noncontact co-culture model with a transwell chamber to explore the energetic perturbations in chondrocytes influenced by osteoblasts. Our results indicate that osteoblasts induce more ATP generation in chondrocytes through an energetic shift characterized by enhanced glycolysis and impaired mitochondrial tricarboxylic acid cycle. Enhanced glycolysis is shown by an increase of secreted lactate and the upregulation of glycolytic enzymes, including glucose-6-phosphate isomerase (Gpi), liver type ATP-dependent 6-phosphofructokinase (Pfkl), fructose-bisphosphate aldolase C (Aldoc), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), triosephosphate isomerase (Tpi1), and phosphoglycerate kinase 1 (Pgk1). Impaired mitochondrial tricarboxylic acid cycle is characterized by the downregulation of cytoplasmic aspartate aminotransferase (Got1) and mitochondrial citrate synthase (Cs). Osteoblasts induce the activation of Akt and P38 signaling to mediate ATP perturbations in chondrocytes. This study may deepen our understanding of the maintenance of metabolic homeostasis in the bone-cartilage unit.

Lipid metabolism in cartilage and its diseases: a concise review of the research progress.

The homeostasis of the vertebrate body depends on anabolic and catabolic activities that are closely linked the inside and outside of the cell. Lipid metabolism plays an essential role in these metabolic activities. Although a large amount of evidence shows that normal lipid metabolism guarantees the conventional physiological activities of organs in the vertebrate body and that abnormal lipid metabolism plays an important role in the occurrence and deterioration of cardiovascular-related diseases, such as obesity, atherosclerosis, and type II diabetes, little is known about the role of lipid metabolism in cartilage and its diseases. This review aims to summarize the latest advances about the function of lipid metabolism in cartilage and its diseases including osteoarthritis, rheumatoid arthritis, and cartilage tumors. With the gradual in-depth understanding of lipid metabolism in cartilage, treatment methods could be explored to focus on this metabolic process in various cartilage diseases.

[The Roles of RUNX1 in the Proliferation and Osteogenic and Adipogenic Differentiation of Dental Pulp Stem Cells].

OBJECTIVE: To investigate the influence of Runt-related transcription factor 1 (RUNX1) on the proliferation, osteogenic differentiation and adipogenic differentiation of dental pulp stem cells (DPSC) in vitro. METHODS: DPSCs were transfected through lentiviral vector carrying the target gene RUNX1 and green fluorescent protein (GFP). After 48 h, transfection efficiency was determined with the fluorescent marking of GFP and Western blot. The effect of the overexpression of RUNX1 on DPSC proliferation and colony formation was determined with CCK-8 and colony formation assay; cell cycle of DPSC was detected by flow cytometry. RUNX1 siRNA was transfected into the DPSCs. After mineralized induction, the effect of RUNX1 overexpression/silencing on the osteogenetic differentiation of DPSC was tested by alkaline phosphatase (ALP) staining and alizarin red staining. After adipogenic induction, oil red O staining was done in order to observe the effect of overexpression/silencing of RUNX1 on the adipogenic differentiation of DPSC. RESULTS: RUNX1 protein was overexpressed in DPSC after lentiviral transfection. Fluorescent test showed successful transfection of lentiviral transfection and over 70% of the cells showed stable expression of GFP protein. The proliferation and colony-formation efficiency of DPSC was enhanced significantly and the proportion of DPSCs in the S phase was significantly increased in the RUNX1-overexpessed group ( P<0.05). ALP activity and mineralized nodule formation ability increased, while lipid droplets decreased in the RUNX1-overexpessed group ( P<0.05). ALP activity and mineralized nodule formation ability decreased, while lipid droplets increased in the RUNX1 knockdown group ( P<0.05) . CONCLUSION: RUNX1 promotes DPSC proliferation and osteogenic differentiation while it inhibits DPSC adipogenic differentiation.

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.

TGF-ß3 mediates mitochondrial dynamics through the p-Smad3/AMPK pathway.

It is well recognized that mitochondrial dynamics plays a vital role in cartilage physiology. Any perturbation in mitochondrial dynamics could cause disorders in cartilage metabolism and even lead to the occurrence of cartilage diseases such as osteoarthritis (OA). TGF-ß3, as an important growth factor that appears in the joints of OA disease, shows its great potential in chondrocyte growth and metabolism. Nevertheless, the role of TGF-ß3 on mitochondrial dynamics is still not well understood. Here we aimed to investigate the effect of TGF-ß3 on mitochondrial dynamics of chondrocytes and reveal its underlying bio-mechanism. By using transmission electron microscopy (TEM) for the number and morphology of mitochondria, western blotting for the protein expressions, immunofluorescence for the cytoplasmic distributions of proteins, and RNA sequencing for the transcriptome changes related to mitochondrial dynamics. We found that TGF-ß3 could increase the number of mitochondria in chondrocytes. TGF-ß3-enhanced mitochondrial number was via promoting the mitochondrial fission. The mitochondrial fission induced by TGF-ß3 was mediated by AMPK signaling. TGF-ß3 activated canonical p-Smad3 signaling and resultantly mediated AMPK-induced mitochondrial fission. Taken together, these results elucidate an understanding of the role of TGF-ß3 on mitochondrial dynamics in chondrocytes and provide potential cues for therapeutic strategies in cartilage injury and OA disease in terms of energy metabolism.

Mapping the human oral and gut fungal microbiota in patients with metabolic dysfunction-associated fatty liver disease.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a phenotype of liver diseases associated with metabolic syndrome. The pathogenesis MAFLD remains unclear. The liver maintains is located near the intestine and is physiologically interdependent with the intestine via metabolic exchange and microbial transmission, underpinning the recently proposed "oral-gut-liver axis" concept. However, little is known about the roles of commensal fungi in the disease development. This study aimed to characterize the alterations of oral and gut mycobiota and their roles in MAFLD. Twenty-one MAFLD participants and 20 healthy controls were enrolled. Metagenomics analyses of saliva, supragingival plaques, and feces revealed significant alterations in the gut fungal composition of MAFLD patients. Although no statistical difference was evident in the oral mycobiome diversity within MAFLD and healthy group, significantly decreased diversities were observed in fecal samples of MAFLD patients. The relative abundance of one salivary species, five supragingival species, and seven fecal species was significantly altered in MAFLD patients. Twenty-two salivary, 23 supragingival, and 22 fecal species were associated with clinical parameters. Concerning the different functions of fungal species, pathways involved in metabolic pathways, biosynthesis of secondary metabolites, microbial metabolism in diverse environments, and carbon metabolism were abundant both in the oral and gut mycobiomes. Moreover, different fungal contributions in core functions were observed between MAFLD patients and the healthy controls, especially in the supragingival plaque and fecal samples. Finally, correlation analysis between oral/gut mycobiome and clinical parameters identified correlations of certain fungal species in both oral and gut niches. Particularly, Mucor ambiguus, which was abundant both in saliva and feces, was positively correlated with body mass index, total cholesterol, low-density lipoprotein, alanine aminotransferase, and aspartate aminotransferase, providing evidence of a possible "oral-gut-liver" axis. The findings illustrate the potential correlation between core mycobiome and the development of MAFLD and could propose potential therapeutic strategies.

Transforming growth factor-ß1-induced N-cadherin drives cell-cell communication through connexin43 in osteoblast lineage.

Gap junction (GJ) has been indicated to have an intimate correlation with adhesion junction. However, the direct interaction between them partially remains elusive. In the current study, we aimed to elucidate the role of N-cadherin, one of the core components in adhesion junction, in mediating connexin 43, one of the functional constituents in gap junction, via transforming growth factor-ß1(TGF-ß1) induction in osteoblasts. We first elucidated the expressions of N-cadherin induced by TGF-ß1 and also confirmed the upregulation of Cx43, and the enhancement of functional gap junctional intercellular communication (GJIC) triggered by TGF-ß1 in both primary osteoblasts and MC3T3 cell line. Colocalization analysis and Co-IP experimentation showed that N-cadherin interacts with Cx43 at the site of cell-cell contact. Knockdown of N-cadherin by siRNA interference decreased the Cx43 expression and abolished the promoting effect of TGF-ß1 on Cx43. Functional GJICs in living primary osteoblasts and MC3T3 cell line were also reduced. TGF-ß1-induced increase in N-cadherin and Cx43 was via Smad3 activation, whereas knockdown of Smad3 signaling by using siRNA decreased the expressions of both N-cadherin and Cx43. Overall, these data indicate the direct interactions between N-cadherin and Cx43, and reveal the intervention of adhesion junction in functional gap junction in living osteoblasts.

Osteoblasts impair cholesterol synthesis in chondrocytes via Notch1 signalling.

OBJECTIVES: Previous reports have proposed the importance of signalling and material exchange between cartilage and subchondral bone. However, the specific experimental evidence is still insufficient to support the effect of this interdependent relationship on mutual cell behaviours. In this study, we aimed to investigate cellular lipid metabolism in chondrocytes induced by osteoblasts. METHODS: Osteoblast-induced chondrocytes were established in a Transwell chamber. A cholesterol detection kit was used to detect cholesterol contents. RNA sequencing and qPCR were performed to assess changes in mRNA expression. Western blot analysis was performed to detect protein expression. Immunofluorescence staining was conducted to show the cellular distribution of proteins. RESULTS: Cholesterol levels were significantly decreased in chondrocytes induced by osteoblasts. Osteoblasts reduced cholesterol synthesis in chondrocytes by reducing the expression of a series of synthetases, including Fdft1, Sqle, Lss, Cyp51, Msmo1, Nsdhl, Sc5d, Dhcr24 and Dhcr7. This modulatory process involves Notch1 signalling. The expression of ncstn and hey1, an activator and a specific downstream target of Notch signalling, respectively, were decreased in chondrocytes induced by osteoblasts. CONCLUSIONS: For the first time, we elucidated that communication with osteoblasts reduces cholesterol synthesis in chondrocytes through Notch1 signalling. This result may provide a better understanding of the effect of subchondral bone signalling on chondrocytes.

Runx1 protects against the pathological progression of osteoarthritis.

Runt-related transcription factor-1 (Runx1) is required for chondrocyte-to-osteoblast lineage commitment by enhancing both chondrogenesis and osteogenesis during vertebrate development. However, the potential role of Runx1 in joint diseases is not well known. In the current study, we aimed to explore the role of Runx1 in osteoarthritis induced by anterior cruciate ligament transaction (ACLT) surgery. We showed that chondrocyte-specific Runx1 knockout (Runx1f/fCol2a1-Cre) aggravated cartilage destruction by accelerating the loss of proteoglycan and collagen II in early osteoarthritis. Moreover, we observed thinning and ossification of the growth plate, a decrease in chondrocyte proliferative capacity and the loss of bone matrix around the growth plate in late osteoarthritis. We overexpressed Runx1 by adeno-associated virus (AAV) in articular cartilage and identified its protective effect by slowing the destruction of osteoarthritis in cartilage in early osteoarthritis and alleviating the pathological progression of growth plate cartilage in late osteoarthritis. ChIP-seq analysis identified new targets that interacted with Runx1 in cartilage pathology, and we confirmed the direct interactions of these factors with Runx1 by ChIP-qPCR. This study helps us to understand the function of Runx1 in osteoarthritis and provides new clues for targeted osteoarthritis therapy.

Insulin-like growth factor 1 promotes neural differentiation of human stem cells from the apical papilla.

OBJECTIVE: Insulin-like growth factor 1 (IGF1) is one of the vital factors in regenerative endodontics. Previous studies have focused on the role of IGF1 in the mineralization of dental tissues. However, the role of IGF1 in the neural differentiation of dental stem cells was little discussed. DESIGN: IGF1 was overexpressed in human stem cells from the apical papilla (hSCAPs) by lentivirus and knocked down in hSCAPs by small interfering RNA. The neural differentiation level of hSCAPs was investigated histologically by HE staining and Nissl staining after neural induction for 3 days. The expression of proteins was examined by western blot and immunofluorescence. RESULTS: IGF1 promoted neural differentiation of hSCAPs, more cell processes and Nissl-positive body stained cells. IGF1 overexpression could both promote glial differentiation in hSCAPs, characterized by the increase of S100ß and GFAP proteins, and neuronal differentiation, characterized by the increase of ßIII-tubulin and functional GAD67/vGLUT1 proteins. Conversely, IGF1 knockdown suppressed both glial and neuronal differentiation. IGF1 activated AKT to regulate the early neural differentiation of hSCAPs. CONCLUSIONS: The results indicate IGF1 could promote neural differentiation of hSCAPs by activating AKT signaling and provide a cue for the candidate of induced neural seeding cells in regenerative endodontics.

The virulence factor GroEL promotes gelatinase secretion from cells in the osteoblast lineage: Implication for direct crosstalk between bacteria and adult cells.

OBJECTIVE: The aim of this study was to demonstrate the influence of the virulence factor GroEL on osteoblast behavior by characterizing the changes of secreted gelatinases. DESIGN: ELISA was performed to detect GroEL from samples from patients with or without apical periodontitis. An apical periodontitis model was established in rats and the expression of MMP-2, MMP-9 and NF-κB was evaluated by immunofluorescence staining. The primary osteoblasts and osteoblast-like MC3T3 cells were stimulated with recombinant GroEL, and gelatin zymography was used to determine the activity and expression of MMP-2 and MMP-9. Western blot was used to screen signaling pathways, and immunofluorescence staining was performed to confirm the activated signaling. RESULTS: First, we found expression of GroEL to be higher in oral saliva, gingival crevicular fluid and periradicular granulation tissue of patients with apical periodontitis than it was in healthy control patients. We next found that recombinant GroEL could increase the activity of the gelatinases, MMP-2 and MMP-9, which were secreted by both primary osteoblasts and MC3T3 cells. In a rat apical periodontitis model, strong expression of gelatinases was confirmed. Then, we found that GroEL-enhanced gelatinase activity was mediated through activation of NF-κB signaling. Acetylated NF-κB accumulated in the cell nucleus and bound to the promoter of MMP-2 and MMP-9 genes, thus initiating their high expression. CONCLUSION: This study reveals a direct interaction between oral bacteria and adult cells by demonstrating that gelatinase secretion is induced by GroEL, which partially explains bone resorption through gelatinase activation.