Effects of different combinations of mechanical loading intensity, duration, and frequency on the articular cartilage in mice.

BACKGROUND: Understanding how healthy articular cartilage responds to mechanical loading is critical. Moderate mechanical loading has positive effects on the cartilage, such as maintaining cartilage homeostasis. The degree of mechanical loading is determined by a combination of intensity, frequency, and duration; however, the best combination of these parameters for knee cartilage remains unclear. This study aimed to determine which combination of intensity, frequency, and duration provides the best mechanical loading on healthy knee articular cartilage in vitro and in vivo. METHODS AND RESULTS: In this study, 33 male mice were used. Chondrocytes isolated from mouse knee joints were subjected to different cyclic tensile strains (CTSs) and assessed by measuring the expression of cartilage matrix-related genes. Furthermore, the histological characteristics of mouse tibial cartilages were quantified using different treadmill exercises. Chondrocytes and mice were divided into the control group and eight intervention groups: high-intensity, high-frequency, and long-duration; high-intensity, high-frequency, and short-duration; high-intensity, low-frequency, and long-duration; high-intensity, low-frequency, and short-duration; low-intensity, high-frequency, and long-duration; low-intensity, high-frequency, and short-duration; low-intensity, low-frequency, and long-duration; low-intensity, low-frequency, and short-duration. In low-intensity CTSs, chondrocytes showed anabolic responses by altering the mRNA expression of COL2A1 in short durations and SOX9 in long durations. Furthermore, low-intensity, low-frequency, and long-duration treadmill exercises minimized chondrocyte hypertrophy and enhanced aggrecan synthesis in tibial cartilages. CONCLUSION: Low-intensity, low-frequency, and long-duration mechanical loading is the best combination for healthy knee cartilage to maintain homeostasis and activate anabolic responses. Our findings provide a significant scientific basis for exercise and lifestyle instructions.

IL-6/Stat3 suppresses osteogenic differentiation in ossification of the posterior longitudinal ligament via miR-135b-mediated BMPER reduction.

Interleukin-6 (IL-6) has been reported to induce osteogenic differentiation of mesenchymal stem cells for increasing bone regeneration, while the role of IL-6 in osteogenic differentiation during ossification of the posterior longitudinal ligament (OPLL) remains to be determined. The current study aims to explore the downstream mechanism of IL-6 in cyclic tensile strain (CTS)-stimulated OPLL, which involves bioinformatically identified microRNA-135b (miR-135b). Initially, we clinically collected posterior longitudinal ligament (PLL) and ossified PLL tissues, from which ossified PLL cells were isolated, respectively. The obtained data revealed a greater osteogenic property of ossified PLL than non-ossified PLL cells. The effect of regulatory axis comprising IL-6, Stat3, miR-135b, and BMPER on osteogenic differentiation of CTS-stimulated ossified PLL cells was examined with gain- and loss-of-function experiments. BMPER was confirmed as a target gene to miR-135b. Knockdown of BMPER or overexpression of miR-135b inhibited the osteogenic differentiation of CTS-induced ossification in PLL cells. Besides, IL-6 promoted the post-transcriptional process to mature miR-135b via Stat3 phosphorylation. In conclusion, IL-6 inhibited CTS-induced osteogenic differentiation by inducing miR-135b-mediated inhibition of BMPER through Stat3 activation.

Involvement of mechanosensitive ion channels in the effects of mechanical stretch induces osteogenic differentiation in mouse bone marrow mesenchymal stem cells.

Bone marrow mesenchymal stem cells (BMSCs) can be induced to process osteogenic differentiation with appropriate mechanical and/or chemical stimuli. The present study described the successful culture of murine BMSCs under mechanical strain. BMSCs were subjected to 0%, 3%, 8%, 13%, and 18% cyclic tensile strain at 0.5 Hz for 8 hr/day for 3 days. The expression of osteogenic markers and mechanosensitive ion channels was evaluated with real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blot. The expression of alkaline phosphatase (ALP) and matrix mineralization were evaluated with histochemical staining. To investigate the effects of mechanosensitive ion channel expression on cyclic tensile strain-induced osteogenic differentiation, the expression of osteogenic markers was evaluated with real-time RT-PCR in the cells without mechanosensitive ion channel expression. This study revealed a significant augment in osteogenic marker in BMSC strained at 8% compared to other treatments; therefore, an 8% strain was used for further investigations. The ALP expression and matrix mineralization were enhanced in osteogenic induced BMSCs subjected to 8% strain after 7 and 14 days, respectively. Under the same conditions, the osteogenic marker and mechanosensitive ion channel expression were significantly promoted. However, the loss function of mechanosensitive ion channels resulted in the inhibition of osteogenic marker expression. This study demonstrated that strain alone can successfully induce osteogenic differentiation in BMSCs and the expression of mechanosensitive ion channels was involved in the process. The current findings suggest that mechanical stretch could function as efficient stimuli to induce the osteogenic differentiation of BMSCs via the activation of mechanosensitive ion channels.

Cyclic tensile strain promotes the ECM synthesis of cranial base synchondrosis chondrocytes by upregulating miR-140-5p.

OBJECTIVE: This study aimed to explore the role of miR-140-5p in cranial base synchondrosis chondrocytes (CBSCs) under cyclic tensile strain (CTS). SETTING AND SAMPLE POPULATION: A total of 25 1-week-old Sprague Dawley rats from Shanghai Laboratory Animal Center, Chinese Academy of Sciences, were used. MATERIAL AND METHODS: The second passage of CBSCs was applied with CTS at 10% elongation (1 Hz) for 24 hours. MiR-140-5p levels in CBSCs were detected by qRT-PCR. The role of miR-140-5p in CBSCs was evaluated by transfection of mimics and inhibitor. RNA sequencing and online search of miRNA databases (TargetScan, miRDB and miRanda) were used in prediction of miR-140-5p targets. A luciferase reporter assay was applied to identify the target gene of miR-140-5p. RESULTS: Compared with the control, the expression of Col2a1 and Sox9 was significantly higher after CTS (P < .05). Also, CBSCs demonstrated higher expression of miR-140-5p after CTS loading for 24 hours (P < .05). Overexpression of miR-140-5p promoted ECM synthesis under CTS loading environment, while suppression of miR-140-5p inhibited the effect. Bloc1s2 was a putative target gene of miR-140-5p. CONCLUSIONS: The expression of ECM in CBSCs could be promoted by CTS and miR-140-5p might play a role in this process through targeting Bloc1s2.

The influence of cyclic tensile strain on multi-compartment collagen-GAG scaffolds for tendon-bone junction repair.

Background: Orthopedic injuries often occur at the interface between soft tissues and bone. The tendon-bone junction (TBJ) is a classic example of such an interface. Current clinical strategies for TBJ injuries prioritize mechanical reattachment over regeneration of the native interface, resulting in poor outcomes. The need to promote regenerative healing of spatially-graded tissues inspires our effort to develop new tissue engineering technologies that replicate features of the spatially-graded extracellular matrix and strain profiles across the native TBJ. Materials and Methods: We recently described a biphasic collagen-glycosaminoglycan (CG) scaffold containing distinct compartment with divergent mineral content and structural alignment (isotropic vs. anisotropic) linked by a continuous interface zone to mimic structural and compositional features of the native TBJ. Results: Here, we report application of cyclic tensile strain (CTS) to the scaffold via a bioreactor leads to non-uniform strain profiles across the spatially-graded scaffold. Further, combinations of CTS and matrix structural features promote rapid, spatially-distinct differentiation profiles of human bone marrow-derived mesenchymal stem cells (MSCs) down multiple osteotendinous lineages. CTS preferentially upregulates MSC activity and tenogenic differentiation in the anisotropic region of the scaffold. This work demonstrates a tissue engineering approach that couples instructive biomaterials with cyclic tensile stimuli to promote regenerative healing of orthopedic interfaces.

Effects of cell adhesion motif, fiber stiffness, and cyclic strain on tenocyte gene expression in a tendon mimetic fiber composite hydrogel.

We recently developed a fiber composite consisting of tenocytes seeded onto discontinuous fibers embedded within a hydrogel, designed to mimic physiological tendon micromechanics of tension and shear. This study examined if cell adhesion peptide (DGEA or YRGDS), fiber modulus (50 or 1300 kPa) and/or cyclic strain (5% strain, 1 Hz) influenced bovine tenocyte gene expression. Ten genes were analyzed and none were sensitive to peptide or fiber modulus in the absence of cyclic tensile strain. Genes associated with tendon (SCX and TNMD), collagens (COL1A1, COL3A1, COL11A1), and matrix remodelling (MMP1, MMP2, and TIMP3) were insensitive to cyclic strain. Contrarily, cyclic strain up-regulated IL6 by 30-fold and MMP3 by 10-fold in soft YRGDS fibers. IL6 expression in soft YRGDS fibers was 5.7 and 3.3-fold greater than in soft DGEA fibers and stiff RGD fibers, respectively, under cyclic strain. Our findings suggest that changes in the surrounding matrix can influence catabolic genes in tenocytes when cultured in a complex strain environment mimicking that of tendon, while having minimal effects on tendon and homeostatic genes.

Strain-induced mechanotransduction through primary cilia, extracellular ATP, purinergic calcium signaling, and ERK1/2 transactivates CITED2 and downregulates MMP-1 and MMP-13 gene expression in chondrocytes.

OBJECTIVE: To determine the strain-induced signaling pathways involved in regulating the transactivation of the transcription regulator Cbp/p300 Interacting Transactivator with ED-rich tail 2 (CITED2) and downstream targets in chondrocytes. METHODS: Primary human chondrocytes or C28/I2 chondrocytic cells were subjected to various strain regimes. C57BL/6 mice were subjected to treadmill running. Loss-of-function was carried out using siRNA or inhibitors specific for targeted molecules. mRNA levels were assayed by RT-qPCR, and proteins by western blotting, immunofluorescence, and/or immunohistochemical staining. CITED2 promoter activity was assayed in chondrocytes using wild-type or mutant constructs. RESULTS: Cyclic strain at 5%, 1 Hz induced CITED2 expression and suppressed expression of matrix metalloproteinase (MMP)-1 and -13 at the messenger RNA (mRNA) and protein levels in human chondrocytes. Abolishing primary cilia through knockdown of intraflagellar transport protein (IFT88) attenuated CITED2 gene expression and decreased protein levels. Similar effects were observed with inhibitors of extracellular adenosine triphosphate (ATP) or P2 purinergic receptors, or antagonists of Ca(2+) signaling. Knockdown of IFT88 in articular chondrocytes in vivo diminished treadmill induced-CITED2 expression and upregulated MMPs. Knockdown of hypoxia-inducible factor (HIF)1α, specificity protein 1 (Sp1), or deletion of the shear stress response element (SSRE) in the CITED2 promoter limited cyclic strain-induced transactivation of CITED2. However, the strain induced-transactivation of CITED2 was abolished only on knockdown of HIF1α, Sp1, and SSRE or by loss-of-function of IFT88 or extracellular-signal-regulated kinases (ERK)1/2. CONCLUSIONS: CITED2 transactivation is a critical event in signaling generated by strain and transduced by primary cilia, extracellular ATP, P2 purinergic receptors, and Ca(2+) signaling. Strain-induced CITED2 transactivation requires HIF1α, Sp1, and an intact SSRE and leads to the downregulation of MMPs such as MMP-1 and MMP-13.

Cyclic tensile strain promotes the osteogenic differentiation of a bone marrow stromal cell and vascular endothelial cell co-culture system.

Mechanical stimuli and neovascularization are closely coupled to osteogenic differentiation and new bone formation. The purpose of present study was to detect the effect of cyclic mechanical strain on a co-culture system of bone marrow stromal cells (BMSCs) and vascular endothelial cells (VECs) and to clarify the related mechanisms. Primary BMSCs and VECs were isolated from Sprague-Dawley rats and co-cultured at various ratios (1:0, 1:2, 1:4, 4:1, 2:1, 1:1, and 0:1). To determine optimized loading conditions, the cells were then subjected to various cyclic tensile strains (0%, 3%, 6% and 9%) using a Flexcell 5000 mechanical loading system. A protocol of 6% strain on the co-cultured cells at a 1:1 ratio was selected as the optimized culture conditions based on the best osteogenic effects, which included increased ALP activity, matrix mineralization and the expressions of VEGF, Runx-2 and Col-1. The VEGF-R inhibitor tivozanib was used to analyze the paracrine role of VEGF, and the osteogenesis-promoting effects of 6% tensile strain were abrogated in the co-cultured cells treated with tivozanib. These results demonstrate that cyclic tensile strain promotes osteogenic differentiation in BMSC/VEC co-culture systems, possibly via a VEC-mediated paracrine effect of VEGF on BMSCs.

Moderate mechanical stimulation antagonizes inflammation of annulus fibrosus cells through YAP-mediated suppression of NF-κB signaling.

Intervertebral disc degeneration (IDD) is a leading cause of low back pain. The inflammatory responses caused by aberrant mechanical loading are one of the major factors leading to annulus fibrosus (AF) degeneration and IDD. Previous studies have suggested that moderate cyclic tensile strain (CTS) can regulate anti-inflammatory activities of AF cells (AFCs), and Yes-associated protein (YAP) as a mechanosensitive coactivator senses diverse types of biomechanical stimuli and translates them into biochemical signals controlling cell behaviors. However, it remains poorly understood whether and how YAP mediates the effect of mechanical stimuli on AFCs. In this study, we aimed to investigate the exact effects of different CTS on AFCs as well as the role of YAP signaling involving in it. Our results found that 5% CTS inhibited the inflammatory response and promoted cell growth through inhibiting the phosphorylation of YAP and nuclear localization of NF-κB, while 12% CTS had a significant proinflammatory effect with the inactivation of YAP activity and the activation of NF-κB signaling in AFCs. Furthermore, moderate mechanical stimulation may alleviate the inflammatory reaction of intervertebral discs through YAP-mediated suppression of NF-κB signaling in vivo. Therefore, moderate mechanical stimulation may serve as a promising therapeutic approach for the prevention and treatment of IDD.

Cyclic mechanical strain with high-tensile triggers autophagy in growth plate chondrocytes.

BACKGROUND: Mechanical loading has been widely considered to be essential for growth plate to maintain metabolism and development. Cyclic mechanical strain has been demonstrated to induce autophagy, whereas the relationship between cyclic tensile strain (CTS) and autophagy in growth plate chondrocytes (GPCs) is not clear. The objective of this study was to investigate whether CTS can regulate autophagy in GPCs in vitro and explore the potential mechanisms of this regulation. METHODS: The 2-week-old Sprague-Dawley rat GPCs were subjected to CTS of varying magnitude and duration at a frequency of 2.0 Hz. The mRNA levels of autophagy-related genes were measured by RT-qPCR. The autophagy in GPCs was verified by transmission electron microscopy (TME), immunofluorescence and Western blotting. The fluorescence-activated cell sorting (FACS) was employed to detect the percentage of apoptotic and necrotic cells. RESULTS: In GPCs, CTS significantly increased the mRNA and protein levels of autophagy-related genes, such as LC3, ULK1, ATG5 and BECN1 in a magnitude- and time-dependent manner. There was no significant difference in the proportion of apoptotic and necrotic cells between control group and CTS group. The autophagy inhibitors, 3-methyladenine (3MA) and chloroquine (CQ) reversed the CTS-induced autophagy via promoting the formation of autophagosomes. Cytochalasin D (cytoD), an inhibitor of G-actin polymerization into F-actin, could effectively block the CTS-induced autophagy in GPCs. CONCLUSION: Cyclic mechanical strain with high-tensile triggers autophagy in GPCs, which can be suppressed by 3MA and CQ, and cytoskeletal F-actin microfilaments organization plays a key role in chondrocytes' response to mechanical loading.

Piezo1 and IFT88 synergistically regulate mandibular condylar chondrocyte differentiation under cyclic tensile strain.

OBJECTIVE(S): Mandibular condyle chondrocytes (MCCs) are exposed to various mechanical environments. Primary cilia, as a carrier for ion channels, can sense mechanical signals. Intraflagellar transport protein 88 (IFT88) is crucial for the assembly and function of primary cilia. Piezo1 is a mechanically activated ion channel that mediates mechanical signal transduction. This study aimed to identify the possible synergistic effect between Piezo1 and IFT88 in MCC differentiation during mechanical conduction. MATERIALS AND METHODS: Confocal immunofluorescence staining was used to reveal the Piezo1 localization. Small interfering RNA (siRNA) technology was used to knock down the expression levels of Piezo1 and IFT88. The chondrogenic differentiation ability of MCCs was evaluated by Alcian blue staining, and the early differentiation ability was evaluated by Western blot of SOX9 and COL2A1. RESULTS: Confocal immunofluorescence results showed that Piezo1 localized in the root of primary cilia. Without cyclic tensile strain (CTS) stimuli, Alcian blue staining showed that Piezo1 knockdown had a marginal effect on the chondrogenic differentiation of MCCs, while IFT88 knockdown inhibited the chondrogenic differentiation. The protein levels of SOX9 and COL2A1 decreased significantly with CTS stimuli. However, these protein levels were restored when Piezo1 was knocked down. In addition, IFT88 knockdown decreased the protein level of Piezo1 with or without CTS. CONCLUSION: Piezo1 and IFT88 might play a synergistic role in regulating MCC differentiation under CTS stimuli.

Cyclic tensile strain-induced yes-associated protein activity modulates the response of human periodontal ligament mesenchymal stromal cells to tumor necrosis factor-α.

OBJECTIVES: This study aimed to evaluate the role of yes-associated protein (YAP) in the inflammatory processes induced in human periodontal ligament-derived mesenchymal stromal cells (hPDL-MSCs) by cyclic tensile strain (CTS). DESIGN: hPDL-MSCs from five periodontally healthy individuals were stimulated with 12% CTS and/or TNF-α for 24 h. YAP activity was determined by analyzing the YAP nuclear localization and the target genes expression, using immunofluorescence and qPCR, respectively. Verteporfin was used to inhibit the activation of YAP. The gene expression of interleukin (IL)-6, IL-8, vascular cell adhesion molecule (VCAM)-1, and intercellular adhesion molecule (ICAM)-1 was analyzed by qPCR. RESULTS: In the absence of TNF-α, application of CTS resulted in the nuclear YAP translocation and upregulation of YAP target genes. Verteporfin inhibited the activation of YAP pathway and upregulated the basal expression of IL-6 and IL-8. TNF-α induced the activation of YAP pathway, which was inhibited by verteporfin. However, application of CTS under these conditions diminished TNF-α-induced YAP activation. TNF-α-induced expression of IL-6, VCAM-1, and ICAM-1 was inhibited after the application of CTS. Inhibition of YAP activation by verteporfin diminished TNF-α-induced gene expression of IL-6, VCAM-1, and ICAM-1, and under these conditions no inhibitory effect of CTS on these parameters was observed. CONCLUSIONS: YAP is at least partially involved in the CTS-activated mechanotransduction pathway. The effects of CTS and YAP on the inflammatory responses depend on the inflammatory environment. A better understanding of the inflammatory modulation by mechanical stress may help improve the orthodontic strategies, especially in the patient with periodontitis.

Native extracellular matrix orientation determines multipotent vascular stem cell proliferation in response to cyclic uniaxial tensile strain and simulated stent indentation.

Cardiovascular disease is the leading cause of death worldwide, with multipotent vascular stem cells (MVSC) implicated in contributing to diseased vessels. MVSC are mechanosensitive cells which align perpendicular to cyclic uniaxial tensile strain. Within the blood vessel wall, collagen fibers constrain cells so that they are forced to align circumferentially, in the primary direction of tensile strain. In these experiments, MVSC were seeded onto the medial layer of decellularized porcine carotid arteries, then exposed to 10%, 1 Hz cyclic tensile strain for 10 days with the collagen fiber direction either parallel or perpendicular to the direction of strain. Cells aligned with the direction of the collagen fibers regardless of the orientation to strain. Cells aligned with the direction of strain showed an increased number of proliferative Ki67 positive cells, while those strained perpendicular to the direction of cell alignment showed no change in cell proliferation. A bioreactor system was designed to simulate the indentation of a single, wire stent strut. After 10 days of cyclic loading to 10% strain, MVSC showed regions of densely packed, highly proliferative cells. Therefore, MVSC may play a significant role in in-stent restenosis, and this proliferative response could potentially be controlled by controlling MVSC orientation relative to applied strain.

Effects of cyclic tensile strain and microgravity on the distribution of actin fiber and Fat1 cadherin in murine articular chondrocytes.

Chondrocytes as mechano-sensitive cells can sense and respond to mechanical stress throughout life. In chondrocytes, changes of structure and morphology in the cytoskeleton have been potentially involved in various mechano-transductions such as stretch-activated ion channels, integrins, and intracellular organelles. However, the mechanism of cytoskeleton rearrangement in response to mechanical loading and unloading remains unclear. In this study, we exposed chondrocytes to a physiological range of cyclic tensile strain as mechanical loading or to simulated microgravity by 3D-clinostat that produces an unloading environment. Based on microarray profiling, we focused on Fat1 that implicated in the formation and rearrangement of actin fibers. Next, we examined the relationship between the distribution of Fat1 proteins and actin fibers after cyclic tensile strain and microgravity. As a result, Fat1 proteins did not colocalize with actin stress fibers after cyclic tensile strain, but accumulated near the cell membrane and colocalized with cortical actin fibers after microgravity. Our findings indicate that Fat1 may mediate the rearrangement of cortical actin fibers induced by mechanical unloading.

Mechanical stretch promotes antioxidant responses and cardiomyogenic differentiation in P19 cells.

Accumulating evidence has suggested that mechanical stimuli play a crucial role in regulating the lineage-specific differentiation of stem cells through fine-tuning redox balance. We aimed to investigate the effects of cyclic tensile strain (CTS) on the expression of antioxidant enzymes and cardiac-specific genes in P19 cells, a widely characterized tool for cardiac differentiation research. A stretching device was applied to generate different magnitude and duration of cyclic strains on P19 cells. The messenger RNA and protein levels of targeted genes were determined by real-time polymerase chain reaction and Western blot assays, respectively. Proper magnitude and duration of cognitive stimulation therapy (CST) stimulation substantially enhanced the expression of both antioxidant enzymes and cardiac-specific genes in P19 cells. Sirtuin 1 (SIRT1) played an essential role in the CTS-induced cardiomyogenic differentiation of P19, as evidenced by changes in the expression of antioxidant enzymes and cardiac-specific genes. Mechanical loading promoted the cardiomyogenic differentiation of P19 cells. SIRT1 was involved in CST-mediated P19 differentiation, implying that SIRT1 might serve as an important target for developing methods to promote cardiomyogenic differentiation of stem cells.

Enhanced matrix production by cocultivated human stem cells and chondrocytes under concurrent mechanical strain.

Conventional treatments of osteoarthritis have failed to re-build functional articular cartilage. Tissue engineering clinical treatments for osteoarthritis, including autologous chondrocyte implantation, provides an alternative approach by injecting a cell suspension to fill lesions within the cartilage in osteoarthritic knees. The success of chondrocyte implantation relies on the availability of chondrogenic cell lines, and their resilience to high mechanical loading. We hypothesize we can reduce the numbers of human articular chondrocytes necessary for a treatment by supplementing cultures with human adipose-derived stem cells, in which stem cells will have protective and stimulatory effects on mixed cultures when exposed to high mechanical loads, and in which coculture will enhance production of requisite extracellular matrix proteins over those produced by stretched chondrocytes alone. In this work, adipose-derived stem cells and articular chondrocytes were cultured separately or cocultivated at ratios of 3:1, 1:1, and 1:3 in static plates or under excessive cyclic tensile strain of 10% and results were compared to culturing of both cell types alone with and without cyclic strain. Results indicate 75% of chondrocytes in engineered articular cartilage can be replaced with stem cells with enhanced collagen over all culture conditions and glycosaminoglycan content over stretched cultures of chondrocytes. This can be done without observing adverse effects on cell viability. Collagen and glycosaminoglycan secretion, when compared to chondrocyte alone under 10% strain, was enhanced 6.1- and 2-fold, respectively, by chondrocytes cocultivated with stem cells at a ratio of 1:3.

Cyclic tensile strain facilitates proliferation and migration of human aortic smooth muscle cells and reduces their apoptosis via miRNA-187-3p.

The cardiovascular is a system that contains extremely complex mechanical factors, in which the circulatory flow of blood has rich mechanical laws. Many studies have revealed that mechanical factors play a very important role in the process of revascularization. Hence, it is essential to investigate the mechanical factors in the process of revascularization in depth. A cyclic tensile strain (CTS) was applied to human aortic smooth muscle cells (HASMCs) at a frequency of 1 Hz and amplitudes of 5%, 10% and 15%, respectively. SmallRNA-seq was used to identify differentially expressed miRNAs (DE-miRNAs) responding to CTS in HASMCs. Starbase database predicted the target genes of DE-miRNAs. Metascape was applied for GO and KEGG pathway enrichment analysis and protein-protein interaction network construction. The proliferation and migration of CTS-treated HASMCs were significantly enhanced, and apoptosis were significantly reduced compared to the control group. SmallRNA-seq results demonstrated that 55, 16 and 16 DE-miRNAs were present in 5%, 10% and 15% CTS-treated HASMCs, respectively. Compared to controls, with miR-26a-2-3p and miR-187-3p being the intersection of these DE-miRNAs. Starbase database identified 189 common target genes for miR-26a-2-3p and miR-187-3p. Common target genes are mainly enriched in the basolateral plasma membrane and endocytosis. Further, in vitro experiments exhibited that CTS upregulated miR-187-3p expression, and miR-187-3p enhanced the proliferation and migration of HASMCs and reduced their apoptosis. It is suggested that miR-187-3p may be an important target for CTS participate in the process of cardiovascular disease.[Figure: see text].

Mechanical Stimulation and Diameter of Fiber Scaffolds Affect the Differentiation of Rabbit Annulus Fibrous Stem Cells.

BACKGROUND: Degeneration of the annulus fibrosus (AF), an important structure of the intervertebral disc, is one of the main causes of degenerative disc disease. Fabrication of scaffolds replicating the stratified microstructure of the AF is critical for the successful regeneration of AF. METHODS: In this study, we cultured rabbit AF-derived stem cells (AFSCs) using fabricated electrospun fibrous poly-L-lactic acid scaffolds with different diameters. We applied cyclic tensile strain (CTS) on the scaffolds to regulate the differentiation of AFSCs into specific cell types that resided at the inner, middle, and outer zones of the AF. RESULTS: We found that the morphologies of AFSCs on the smaller-fiber-diameter scaffolds were nearly round, whereas spindle-like cells morphologies were observed on large-diameter scaffolds. CTS enhanced these phenomena and made the cells slender. The expression levels of collagen-I in cells increased as a function of the fiber diameter, whereas collagen-II and aggrecan exhibited opposite trends. Moreover, the application of CTS upregulated the gene expressions of collagen-I, collagen-II, and aggrecan. CONCLUSION: Overlaying the scaffolds with different CTS-stimulated cells could eventually lead to engineered AF tissues with hierarchical structures that approximated the native AF tissue. Thus, the proposed methodologies could be potentially applied for AF regeneration.

The mechano-response of murine annulus fibrosus cells to cyclic tensile strain is frequency dependent.

The intervertebral disk (IVD) is a composite structure essential for spine stabilization, load bearing, and movement. Biomechanical factors are important contributors to the IVD microenvironment regulating joint homeostasis; however, the cell type-specific effectors of mechanotransduction in the IVD are not fully understood. The current study aimed to determine the effects of cyclic tensile strain (CTS) on annulus fibrosus (AF) cells and identify mechano-sensitive pathways. Using a cell-type specific reporter mouse to differentiation NP and AF cells from the murine IVD, we characterized AF cells in dynamic culture exposed to CTS (6% strain) at specific frequencies (0.1 Hz, 1.0 Hz, or 2.0 Hz). We demonstrate that our culture model maintains the phenotype of primary AF cells and that the bioreactor system delivers uniform biaxial strain across the cell culture surface. We show that exposure of AF cells to CTS induces cytoskeleton reorganization resulting in stress fiber formation, with acute exposure to CTS at 2.0 Hz inducing a significant yet transient increase ERK1/2 pathway activation. Using SYBPR-based qPCR to assess the expression of extracellular matrix (ECM) genes, ECM-remodeling genes, candidate mechano-sensitive genes, inflammatory cytokines and cell surface receptors, we demonstrated that exposure of AF cells to CTS at 0.1 Hz increased Acan, Prg4, Col1a1 and Mmp3 expression. AF cells exposed to CTS at 1.0 Hz showed a significant increase in the expression of Acan, Myc, and Tnfα. Exposure of AF cells to CTS at 2.0 Hz induced a significant increase in Acan, Prg4, Cox2, Myc, Fos, and Tnfα expression. Among the cell surface receptors assessed, AF cells exposed to CTS at 2.0 Hz showed a significant increase in Itgß1, Itgα5, and Trpv4 expression. Our findings demonstrate that the response of AF cells to CTS is frequency dependent and suggest that mechanical loading may directly contribute to matrix remodeling and the onset of local tissue inflammation in the murine IVD.

Cyclic tensile strain promotes chondrogenesis of bone marrow-derived mesenchymal stem cells by increasing miR-365 expression.

AIMS: The chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) is critical for cartilage regeneration. Tissues constructed from BMSCs through cartilage tissue engineering still exhibit some histological, morphological, and biomechanical differences from normal cartilage tissues. Cyclic tensile strain (CTS) can increase chondrogenic gene expression and reduce hypertrophic gene expression in chondrocytes. miR-365 has been identified as a mechanoresponsive microRNA and is an important regulator of both chondrocyte hypertrophy and differentiation. Therefore, we hypothesized that CTS may promote the chondrogenesis of BMSCs by upregulating the expression of miR-365. METHODS: BMSCs were subjected to CTS to investigate the effects and mechanism on chondrogenesis. An Agilent miRNA microarray was used to profile miRNAs in the CTS-treated BMSCs and 3D-cultured control BMSCs. miR-365 was shown to interact with HDAC4 mRNA through a luciferase reporter assay. An animal cartilage defect model was constructed and different groups of BMSCs were implanted to investigate their in vivo effect. KEY FINDINGS: CTS promoted BMSC chondrogenesis. miR-365 was significantly upregulated in CTS-treated cells and played an important role in CTS-mediated chondrogenesis. Luciferase assays showed that HDAC4 is a direct target of miR-365. An in vivo study showed that CTS treatment and miR-365 overexpression could promote cartilage regeneration from BMSCs. SIGNIFICANCE: CTS can promote the expression of miR-365, a crucial mechanosensitive microRNA involved in the chondrogenesis of BMSCs, which directly inhibits the expression of HDAC4, in turn, enhancing the chondrogenesis of BMSCs.