Berberine regulates bone metabolism in apical periodontitis by remodelling the extracellular matrix.

OBJECTIVES: The objectives of this study were to explore the role and related mechanism of berberine in repairing bone destruction in apical periodontics (AP). MATERIALS AND METHODS: AP was established in 14 of 21 male Wistar rats (four weeks of age; 70-80 g) for 3 weeks. The canals were cleaned and administered berberine (2 mg/ml; n = 7) or calcium hydroxide (100 mg/ml; control; n = 7), followed by glass ionomer cement sealing. After 3 weeks, specimen collection followed by micro-computed tomography (µ-CT) and histological staining was performed, including haematoxylin and eosin staining, Masson's trichrome staining, tartrate-resistant acid phosphatase staining, immunohistochemistry and immunofluorescence histochemistry. RESULTS: µ-CT showed that AP lesion volume reduced in the berberine group. Histopathology showed that berberine decreased the activity and number of osteoclasts but increased the expression of proteins related to osteoblast differentiation, including alkaline phosphatase and osterix. The immune cell, T cell, dendritic cell and macrophage counts were significantly decreased in the berberine group. In the berberine group, the expression of extracellular matrix-degraded proteases, metalloproteinases, was decreased; however, that of extracellular matrix-stable proteases, lysyl oxidases, was increased. CONCLUSIONS: Berberine controlled the inflammatory response and regulated bone metabolism in AP by reducing metalloproteinase expression and increasing lysyl oxidases expression.

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.

The involvement of the ERK-MAPK pathway in TGF-ß1-mediated connexin43-gap junction formation in chondrocytes.

Purposes: Gap junction intercellular communication (GJIC) exhibits a key role in maintaining the homeostasis of articular cartilage. Connexin43 (Cx43) protein is predominant in the structures that form gap junctions. We aim to determine the potential underlying mechanisms of TGF-ß1 (Transforming growth factor-ß1)-regulated cell communication in chondrocytes. Materials and methods: After exposure of chondrocytes to recombinant TGF-ß1, quantitative real-time PCR was used to detect expression levels of Cx43 mRNA. Western blot analysis was used to check Cx43 and mitogen-activated protein kinase (MAPK) family components. Immunofluorescence staining was performed to confirm ERK-MAPK pathway activation and Cx43 protein distribution. MAPK inhibitors (ERK inhibitor U0126, JNK inhibitor SP 600125 and P38 inhibitor SP 203580) were applied to verify the specificity effects of ERK-MAPK pathway. GJIC between chondrocytes were evaluated using Scrape loading/dye transfer (SLDT) assay. Results: It was first found that TGF-ß1modulatedthe Cx43protein expressions and its sub-cellular distribution. TGF-ß1 promoted gap junction intercellular communication (GJIC) formations in chondrocytes, especially in a higher cell intensity. ERK-MAPK signaling pathway was activated in TGF-ß1-mediated gap junctions among chondrocytes. Furthermore, the inhibitor of ERK attenuated the increases of Cx43 expressions and functional gap junction formations induced by TGF-ß1, while cross-talk between ERK-MAPK and Smad signal pathways exists shown in the process. Conclusions: This study provides evidence to show the importance of the ERK-MAPK pathway in TGF-ß1-mediated Cx43 expression and functional gap junction formation.

Extracellular Matrix Elasticity Regulates Osteocyte Gap Junction Elongation: Involvement of Paxillin in Intracellular Signal Transduction.

BACKGROUND/AIMS: Osteocytes can sense and respond to extracellular stimuli, including biochemical factors throughout the cell body, dendritic processes, and cilia bending. However, further exploration is required of osteocyte function in response to substrate stiffness, an important passive mechanical cue at the interface between osteocytes and the extracellular matrix, and the deep bio-mechanism in osteocytes involving mechanosensing of cell behavior. METHODS: We fabricated silicon-based elastomer polydimethylsiloxane substrates with different stiffnesses but with the same surface topologies. We then seeded osteocytes onto the substrates to examine their responses. Methodologies used included scanning electron microscopy (SEM) for cell morphology, confocal laser scanning microscopy (CLSM) for protein distribution, western blot for protein levels, co-immunoprecipitation for protein interactions, and quantitative real-time polymerase chain reaction for gene expression. RESULTS: SEM images revealed that substrate stiffness induced a change in osteocyte morphology, and CLSM of F-actin staining revealed that substrate stiffness can alter the cytoskeleton. These results were accompanied by changes in focal adhesion capacity in osteocytes, determined via characterization of vinculin expression and distribution. Furthermore, on the exterior of the cell membrane, fibronectin was altered by substrate stiffness. The fibronectin then induced a change in paxillin on the inner membrane of the cell via protein-protein interaction through transmembrane processing. Paxillin led to changes in connexin 43 via protein-protein binding, thereby influencing osteocyte gap junction elongation. CONCLUSION: This process -from mechanosensing and mechanotransduction to cell function - not only indicates that the effects of mechanical factors on osteocytes can be directly sensed from the cell body, but also indicates the involvement of paxillin transduction.

[The Effects of NF-kappaB Signalling on the TNF-α induced Ratios of MMPs to TIMPs in Chondrocytes].

OBJECTIVES: To explore the influence of the NF-κB inhibitor (bay11-7082) on tumor necrosis factor-α (TNF-α)-induced different ratios of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in chondrocytes. METHODS: Chondrocytes were isolated from the knee joint of a 1-day old mouse by trypsin digestion method. Hematoxylin-Eosin (HE) stain was used to show the morphology of isolated chondrocytes; Semi-quantitative PCR was applied to analyze the influence of bay11-7082 on gene expressions of TNF-α-induced MMPsand TIMPsin chondrocytes; Zymography was used to elucidate activities of the gelatinases induced by TNF-α and/or bay11-7082. RESULTS: TNF-α up-regulated gene expressiosn of the MMPsand TIMPs(P<0.05). The ratios of MMPs/TIMPswere mostly increased except the part of MMP-1. Bay11-7082 could reduce TNF-α-induced MMPsand TIMPsgene expressions, and could make the increased ratio of MMPs/TIMPsdropped to the normal level of chondrocytes. Similar results were observed at the protein level of the gelatinases by zymography. CONCLUSIONS: TNF-α-induced high ratios of MMPs/TIMPs could partiallyexplain over-degradation of cartilage extracellular matrix in osteoarthritis (OA). Blockage of NF-κB with bay11-7082 might provide a possible therapeutic strategy for the OA deterioration.

The role of TGF-beta3 in cartilage development and osteoarthritis.

Articular cartilage serves as a low-friction, load-bearing tissue without the support with blood vessels, lymphatics and nerves, making its repair a big challenge. Transforming growth factor-beta 3 (TGF-ß3), a vital member of the highly conserved TGF-ß superfamily, plays a versatile role in cartilage physiology and pathology. TGF-ß3 influences the whole life cycle of chondrocytes and mediates a series of cellular responses, including cell survival, proliferation, migration, and differentiation. Since TGF-ß3 is involved in maintaining the balance between chondrogenic differentiation and chondrocyte hypertrophy, its regulatory role is especially important to cartilage development. Increased TGF-ß3 plays a dual role: in healthy tissues, it can facilitate chondrocyte viability, but in osteoarthritic chondrocytes, it can accelerate the progression of disease. Recently, TGF-ß3 has been recognized as a potential therapeutic target for osteoarthritis (OA) owing to its protective effect, which it confers by enhancing the recruitment of autologous mesenchymal stem cells (MSCs) to damaged cartilage. However, the biological mechanism of TGF-ß3 action in cartilage development and OA is not well understood. In this review, we systematically summarize recent progress in the research on TGF-ß3 in cartilage physiology and pathology, providing up-to-date strategies for cartilage repair and preventive treatment.

Microenvironmental Stiffness Directs Chondrogenic Lineages of Stem Cells from the Human Apical Papilla via Cooperation between ROCK and Smad3 Signaling.

Cell-based cartilage tissue engineering faces a great challenge in the repair process, partly due to the special physical microenvironment. Human stem cell from apical papilla (hSCAP) shows great potential as seed cells because of its versatile differentiation capacity. However, whether hSCAP has potent chondrogenic differentiation ability in the physical microenvironment of chondroid remains unknown. In this study, we fabricated poly(dimethylsiloxane) (PDMS) substrates with different stiffnesses and investigated the chondrogenic differentiation potential of hSCAPs. First, we found that hSCAPs cultured on soft substrates spread more narrowly accompanied by cortical actin organization, a hallmark of differentiated chondrocytes. On the contrary, stiff substrates were favorable for cell spreading and stress fiber formation. More importantly, the increased chondrogenic differentiation of hSCAPs seeded on soft substrates was confirmed by characterizing increased extracellular proteoglycan aggregation through Alcian blue staining and Safranin O staining and enhanced markers toward chondrogenic differentiation including SRY-box transcription factor 9 (Sox9), type II collagen (Col2), and aggrecan in both normal α-minimum essential medium (αMEM) and specific chondrogenic medium (CM) culture conditions. Then, we investigated the mechanosensing/mechanotransduction governing the chondrogenic differentiation of hSCAPs in response to different stiffnesses and found that stiffness-sensitive integrin ß1 and focal adhesion kinase (FAK) were essential for mechanical signal perception and were oriented at the start of mechanotransduction induced by matrix stiffness. We next showed that the increased nuclear accumulation of Smad3 signaling and target Sox9 facilitated the chondrogenic differentiation of hSCAPs on the soft substrates and further verified the importance of Rho-associated protein kinase (ROCK) signaling in regulating chondrogenic differentiation and its driving factors, Smad3 and Sox9. By using SIS3, the specific inhibitor of p-Smad3, and miRNA targeting Rho-associated protein kinase 1 (ROCK-1), we finally confirmed the importance of ROCK/Smad3/Sox9 axis in the chondrogenic differentiation of hSCAPs in response to substrate stiffness. These results help us to increase the understanding of how microenvironmental stiffness directs chondrogenic differentiation from the aspects of mechanosensing, mechanotransduction, and cell fate decision, which will be of great value in the application of hSCAPs in cartilage tissue engineering.

The virulence factor GroEL directs the osteogenic and adipogenic differentiation of human periodontal ligament stem cells through the involvement of JNK/MAPK and NF-κB signaling.

BACKGROUND: GroEL, a bacterial metabolite, is an important stimulator of inflammation. The aim of this study is to confirm the effect of the virulence factor GroEL on differentiation potential of periodontal ligament (PDL) stem cells (PDLSCs) and the potential mechanisms. METHODS: PDLSCs were obtained from extracted human premolars. GroEL was administered to osteogenic- and adipogenic-induced hPDLSCs. Alkaline phosphatase (ALP) staining, Alizarin Red staining and Oil Red staining were performed. Gene and protein expression were separately measured by qPCR and Western blotting. The expression and localization of activated signaling factors were confirmed by immunofluorescence staining. The inhibitors of myeloid differentiation factor 88 (MyD88, an adaptor protein of TLRs), JNK/MAPK and NF-κB signaling were used to verify their specific effects. RESULTS: First, we found that GroEL inhibited the osteogenic differentiation and enhanced the adipogenic differentiation of hPDLSCs. Next, we found that GroEL increased the expression of TLR2 and TLR4 and GroEL activated JNK/MAPK and NF-κB signaling, which can be blocked by inhibition of MyD88. Finally, we found that inhibition of MyD88 restored GroEL-induced osteogenic and adipogenic differentiation and blocking JNK/MAPK or NF-κB signaling partly restored GroEL effects. CONCLUSION: In the current study, we revealed a potential interaction between bacteria and host cells by showing that GroEL directs the osteogenic and adipogenic differentiation of hPDLSCs by the involvement of JNK/MAPK and NF-κB signaling. This study provides evidence that bacterial products can influence the differentiation of stem cells and reveals potential effect of GroEL on the context of tissue regeneration.

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.

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.

Stiffness and topography of biomaterials dictate cell-matrix interaction in musculoskeletal cells at the bio-interface: A concise progress review.

Mutually interacted musculoskeletal tissues work together within the physiological environment full of varieties of external stimulus. Consistent with the locomotive function of the tissues, musculoskeletal cells are remarkably mechanosensitive to the physical cues. Signals like extracellular matrix (ECM) stiffness, topography, and geometry can be sensed and transduced into intracellular signaling cascades to trigger a series of cell responses, including cell adhesion, cell phenotype maintenance, cytoskeletal reconstruction, and stem cell differentiation (Du et al., 2011; Murphy et al., 2014; Lv et al., 2015; Kim et al., 2016; Kumar et al., 2017). With the development of tissue engineering and regenerative medicine, the potent effects of ECM physical properties on cell behaviors at the cell-matrix interface are drawing much attention. To mimic the interaction between cell and its ECM physical properties, developing advanced biomaterials with desired characteristics which could achieve the biointerface between cells and the surrounded matrix close to the physiological conditions becomes a great hotspot. In this review, based on the current publications in the field of biointerfaces, we systematically summarized the significant roles of stiffness and topography on musculoskeletal cell behaviors. We hope to shed light on the importance of physical cues in musculoskeletal tissue engineering and provide up to date strategies towards the natural or artificial replication of physiological microenvironment.

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.

Compliant substratum modulates vinculin expression in focal adhesion plaques in skeletal cells.

The biophysical properties of the extracellular matrix (ECM) dictate tissue-specific cell behaviour. In the skeleton system, bone shows the potential to adapt its architecture and contexture to environmental rigidity via the bone remodelling process, which involves chondrocytes, osteoblasts, osteoclasts, osteocytes and even peripheral bone marrow-derived stem/stromal cells (BMSCs). In the current study, we generated stiff (~1 014 ± 56) kPa, Young's modulus) and soft (~46 ± 11) kPa silicon-based elastomer polydimethylsiloxane (PDMS) substrates by mixing curing agent into oligomeric base at 1:5 and 1:45 ratios, respectively, and investigated the influence of substrate stiffness on the cell behaviours by characterizing cell spreading area, cell cytoskeleton and cell adhesion capacity. The results showed that the cell spreading areas of chondrocytes, osteoblasts, osteoclasts, osteocytes and BMSCs were all reduced in the soft substrate relative to those in the stiff substrate. F-actin staining confirmed that the cytoskeleton was also changed in the soft group compared to that in the stiff group. Vinculin in focal adhesion plaques was significantly decreased in response to soft substrate compared to stiff substrate. This study establishes the potential correlation between microenvironmental mechanics and the skeletal system, and the results regarding changes in cell spreading area, cytoskeleton and cell adhesion further indicate the important role of biomechanics in the cell-matrix interaction.

TGF-ß1 promotes gap junctions formation in chondrocytes via Smad3/Smad4 signalling.

OBJECTIVES: Connexin-mediated functional gap junction intercellular communication (GJIC) has a vital role in development, homeostasis and pathology. Transforming growth factor-ß1 (TGF-ß1), as one of the most vital factors in chondrocytes, promotes cartilage precursor cell differentiation and chondrocyte proliferation, migration and metabolism. However, how TGF-ß1 mediates GJIC in chondrocytes remains unclear. This study aims to determine the influence of TGF-ß1 on GJIC in mouse chondrocytes and its underlying mechanism. METHODS: qPCR and mRNA microarray were used to verify the expression of genes in the TGF-ß and connexin families in cartilage and chondrocytes. A scrape loading/dye transfer assay was performed to explore GJIC. Western blot analysis was used to detect connexin43 (Cx43) and Smad signalling components. Immunofluorescence staining was performed to characterize protein distribution. RESULTS: The TGF-ß1 mRNA was the highest expressed member of the TGFß super family in cartilage. TGF-ß1 promoted functional GJIC through increased expression of Cx43. TGF-ß1-mediated GJIC required the participation of TGF-ß type I receptor. TGF-ß1 activated Smad3 and Smad4 signalling to facilitate their nuclear translocation. The Smad3 and Smad4 signalling proteins bound to the promoter of Gja1 and thus initiated Cx43 gene expression. CONCLUSIONS: For the first time, these results revealed a vital role of TGF-ß1 in cell-cell communication in chondrocytes via gap junction formation. We describe the regulatory mechanism, the involvement of TGF-ß type I receptor and the nuclear translocation of Smad3/4.

Transforming growth factor-ß1 up-regulates connexin43 expression in osteocytes via canonical Smad-dependent signaling pathway.

Connexin 43 (Cx43)-mediated gap junctional intercellular communication (GJIC) has been shown to be important in regulating multiple functions of bone cells. Transforming growth factor-ß1 (TGF-ß1) exhibited controversial effects on the expression of Cx43 in different cell types. To date, the effect of TGF-ß1 on the Cx43 expression of osteocytes is still unknown. In the present study, we detected the expression of TGF-ß1 in osteocytes and bone tissue, and then used recombinant mouse TGF-ß1 to elucidate its effect on gap junctions (GJs) of osteocytes. Our data indicated that TGF-ß1 up-regulated both mRNA and protein expression of Cx43 in osteocytes. Together with down-regulation of Cx43 expression after being treated with TGF-ß type I receptor inhibitor Repsox, we deduced that TGF-ß1 can positively regulate Cx43 expression in osteocytes. Thus we next focussed on the downstream signals of TGF-ß and found that TGF-ß1-mediated smads, Smad3 and Smad4, to translocate into nucleus. These translocated signal proteins bind to the promoter of Gja1 which was responsible for the changed expression of Cx43. The present study provides evidence that TGF-ß1 can enhance GJIC between osteocytes through up-regulating Cx43 expression and the underlying mechanism involved in the activation of Smad-dependent pathway.

Compliant Substratum Changes Osteocyte Functions: The Role of ITGB3/FAK/ß-Catenin Signaling Matters.

Bone shows the potential to adapt its architecture to environmental rigidity via the bone remodeling process, which is governed by osteocytes. Once the remodeling process is disrupted, the mechanics of the bone architecture is impaired, and bone disease proceeds. During the process of pathogenesis, how the osteocytes start the process of sensing and transducing a weakened extracellular mechanical signal into a biochemical signal remains elusive. In the current study, using polydimethylsiloxane (PDMS) substrates with varied mechanical stiffnesses, we first showed that the osteocytes can sense the mechanical change and respond by showing different cell spreading areas and cytoskeleton distributions. Osteocytes sense ECM mechanics via integrin αvß3, which interacts with focal adhesion kinase (FAK) to promote the transduction of extracellular mechanical stimuli into intracellular biochemical signals. FAK triggers cytoplasmic ß-catenin signaling and the resultant nuclear translocation. This signaling results in changes in the gap junction and mineralization activity of the osteocytes. This study establishes the correlation between microenvironmental mechanics and osteocyte function by characterizing the interaction between integrin αvß3/FAK signaling and ß-catenin signaling, thus providing a deep understanding of mechanosensing and mechanotransduction in osteocytes and bone pathogenesis.

Effects of parathyroid hormone (1-34) on the regulation of the lysyl oxidase family in ovariectomized mice.

Osteoporosis (OP) is a highly prevalent chronic disease. The anabolic agent parathyroid hormone (PTH) is often prescribed for the treatment of OP to strengthen bone quality and decrease the risk of fracture, although the specific mechanisms are still unclear. Lysyl oxidase (LOX) can stabilize the organic matrix through catalyzing the cross-linking of collagen and elastin. In this study, we established osteoporotic models via ovariectomizing C57BL/6J mice and treating them with PTH. We further aimed to determine the expression changes of the LOX family, impacted by PTH, in ovariectomized mice. We observed that bone mass was reduced and bone microstructure was deteriorative in ovariectomized mice. And PTH attenuated the microstructural damage and accelerated bone remodeling, as confirmed via µCT and HE staining. Serum levels of copper and zinc indirectly proved the results. The expression levels of five members of the LOX family all declined in ovariectomized mice compared to in sham-operated control mice (p < 0.05), and the daily injection of PTH successfully reversed the low expression of LOXs in OP. The current study examined expression changes of LOXs in osteoporotic mice and PTH-treated osteoporotic mice for the first time, and provided an important piece of evidence that the aberrant expression of LOXs had intimate associations with the occurrence and development of OP. And LOXs may act as the downstream effectors of PTH, contributing to unbalanced bone metabolism and damaged bone microstructure. Consequently, LOXs may act as promising therapeutic targets for OP.

The Role of the Lysyl Oxidases in Tissue Repair and Remodeling: A Concise Review.

Tissue injury provokes a series of events containing inflammation, new tissue formation and tissue remodeling which are regulated by the spatially and temporally coordinated organization. It is an evolutionarily conserved, multi-cellular, multi-molecular process via complex signalling network. Tissue injury disorders present grievous clinical problems and are likely to increase since they are generally associated with the prevailing diseases such as diabetes, hypertension and obesity. Although these dynamic responses vary not only for the different types of trauma but also for the different organs, a balancing act between the tissue degradation and tissue synthesis is the same. In this process, the degradation of old extracellular matrix (ECM) elements and new ones' synthesis and deposition play an essential role, especially collagens. Lysyl oxidase (LOX) and four lysyl oxidase-like proteins are a group of enzymes capable of catalyzing cross-linking reaction of collagen and elastin, thus initiating the formation of covalent cross-links that insolubilize ECM proteins. In this way, LOX facilitates ECM stabilization through ECM formation, development, maturation and remodeling. This ability determines its potential role in tissue repair and regeneration. In this review, based on the current in vitro, animal and human in vivo studies which have shown the significant role of the LOXs in tissue repair, e.g., tendon regeneration, ligament healing, cutaneous wound healing, and cartilage remodeling, we focused on the role of the LOXs in inflammation phase, proliferation phase, and tissue remodeling phase of the repair process. By summarizing its healing role, we hope to shed light on the understanding of its potential in tissue repair and provide up to date therapeutic strategies towards related injuries.

Influences of Tumor Necrosis Factor-α on Lysyl Oxidases and Matrix Metalloproteinases of Injured Anterior Cruciate Ligament and Medial Collateral Ligament Fibroblasts.

The anterior cruciate ligament (ACL) fails to heal after injury, even after a primary surgical attempt. In contrast, the medial collateral ligament (MCL) can heal relatively well and restore the full joint function. The difference in intrinsic properties of these ligament cells can be due to their different responses to their local factors. TNF-α is considered to be an important chemical mediator in the wound healing of the ligaments. However, TNF-α-induced expression of lysyl oxidases (LOXs) and matrix metalloproteinases (MMPs) after injury is poorly understood. In this study, we use equi-biaxial stretch chamber to realize 12% stretch, which could mimic the injury to the ACL and MCL fibroblasts in vitro, and aim to determine the intrinsic differences between injured ACL and MCL by characterizing the differential expressions of LOXs and MMPs in response to TNF-α. The methods included Semiquantitative PCR, quantitative real-time PCR, Western blot, and zymography. We found that the mRNAs of LOXs had temporal increases in injured ACL and MCL. Moreover, the increases were higher in injured MCL than those in injured ACL (up to 1.77 ± 0.13-fold in LOX, 1.73 ± 0.21-fold in LOXL-1, 2.23 ± 0.27-fold in LOXL-2, 1.95 ± 0.11-fold in LOXL-3, 1.97 ± 0.28-fold in LOXL-4). On the other hand, the expressions of MMPs in injured ACL were much more prominent than those in injured MCL fibroblasts (up to 2.63 ± 0.20-fold in MMP-1, 3.73 ± 0.18-fold MMP-2, 1.58 ± 0.11-fold MMP-3, 4.23 ± 0.31-fold MMP-12). Similar results were observed at the protein level. The differential expression of LOXs and MMPs between the injured ACL and MCL fibroblasts in this study may help explain the healing abilities of the two different ligaments.

The effects of interleukin-1ß in modulating osteoclast-conditioned medium's influence on gelatinases in chondrocytes through mitogen-activated protein kinases.

Osteoarthritis is recognised to be an interactive pathological process involving the cartilage, subchondral bone and synovium. The signals from the synovium play an important role in cartilage metabolism, but little is known regarding the influence of the signalling from bone. Additionally, the collagenases and stromelysin-1 are involved in cartilage catabolism through mitogen-activated protein kinase (MAPK) signalling, but the role of the gelatinases has not been elucidated. Here, we studied the influence of osteoclastic signals on chondrocytes by characterising the expression of interleukin-1ß (IL-1ß)-induced gelatinases through MAPK signalling. We found that osteoclast-conditioned media attenuated the gelatinase activity in chondrocytes. However, IL-1ß induced increased levels of gelatinase activity in the conditioned media group relative to the mono-cultured chondrocyte group. More specifically, IL-1ß restored high levels of gelatinase activity in c-Jun N-terminal kinase inhibitor-pretreated chondrocytes in the conditioned media group and led to lower levels of gelatinase activity in extracellular signal-regulated kinase or p38 inhibitor-pretreated chondrocytes. Gene expression generally correlated with protein expression. Taken together, these results show for the first time that signals from osteoclasts can influence gelatinase activity in chondrocytes. Furthermore, these data show that IL-1ß restores gelatinase activity through MAPK inhibitors; this information can help to increase the understanding of the gelatinase modulation in articular cartilage.