Mechanosensitive channel of large conductance enhances the mechanical stretching-induced upregulation of glycolysis and oxidative metabolism in Schwann cells.

BACKGROUND: Physical exercise directly stretching the peripheral nerve promotes nerve regeneration; however, its action mechanism remains elusive. Our present study aimed to investigate the effects of mechanosensitive channel of large conductance (MscL) activated by mechanical stretching on the cultured Schwann cells (SCs) and explore the possible mechanism. METHODS: Primary SCs from neonatal mice at 3-5 days of age were derived and transfected with the lentivirus vector expressing a mutant version of MscL, MscL-G22S. We first detected the cell viability and calcium ion (Ca2+) influx in the MscL-G22S-expressing SCs with low-intensity mechanical stretching and the controls. Proteomic and energy metabolomics analyses were performed to investigate the comprehensive effects of MscL-G22S activation on SCs. Measurement of glycolysis- and oxidative phosphorylation-related molecules and ATP production were respectively performed to further validate the effects of MscL-G22S activation on SCs. Finally, the roles of phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway in the mechanism of energy metabolism modulation of SCs by MscL-G22S activation was investigated. RESULTS: Mechanical stretching-induced MscL-G22S activation significantly increased the cell viability and Ca2+ influx into the SCs. Both the proteomic and targeted energy metabolomics analysis indicated the upregulation of energy metabolism as the main action mechanism of MscL-G22S-activation on SCs. MscL-G22S-activated SCs showed significant upregulation of glycolysis and oxidative phosphorylation when SCs with stretching alone had only mild upregulation of energy metabolism than those without stimuli. MscL-G22S activation caused significant phosphorylation of the PI3K/AKT/mTOR signaling pathway and upregulation of HIF-1α/c-Myc. Inhibition of PI3K abolished the MscL-G22S activation-induced upregulation of HIF-1α/c-Myc signaling in SCs and reduced the levels of glycolysis- and oxidative phosphorylation-related substrates and mitochondrial activity. CONCLUSION: Mechanical stretching activates MscL-G22S to significantly promote the energy metabolism of SCs and the production of energic substrates, which may be applied to enhance nerve regeneration via the glia-axonal metabolic coupling.

MIR-107/HMGB1/FGF-2 axis responds to excessive mechanical stretch to promote rapid repair of vascular endothelial cells.

The increase of vascular wall tension can lead to endothelial injury during hypertension, but its potential mechanism remains to be studied. Our results of previous study showed that HUVECs could induce changes in HMGB1/RAGE to resist abnormal mechanical environments in pathological mechanical stretching. In this study, we applied two different kinds of mechanical tension to endothelial cells using the in vitro mechanical loading system FlexCell-5000T and focused on exploring the expression of miR-107 related pathways in HUVECs with excessive mechanical tension. The results showed that miR-107 negatively regulated the expression of the HMGB1/RAGE axis under excessive mechanical tension. Excessive mechanical stretching reduced the expression of miR-107 in HUVECs, and increased the expression of the HMGB1/RAGE axis. When miR-107 analog was transfected into HUVECs with lipo3000 reagent, the overexpression of miR-107 slowed down the increase of the HMGB1/RAGE axis caused by excessive mechanical stretching. At the same time, the overexpression of miR-107 inhibited the proliferation and migration of HUVECs to a certain extent. On the contrary, when miR-107 was silent, the proliferation and migration of HUVECs showed an upward trend. In addition, the study also showed that under excessive mechanical tension, miR-107 could regulate the expression of FGF-2 by HMGB1. In conclusion, these findings suggest that pathological mechanical stretching promote resistance to abnormal mechanical stimulation on HUVECs through miR-107/HMGB1/RAGE/FGF-2 pathway, thus promote vascular repair after endothelial injury. The suggest that miR-107 is a potential therapeutic target for hypertension.

Mechanical stretching induces fibroblasts apoptosis through activating Piezo1 and then destroying actin cytoskeleton.

The anatomical positions of pelvic floor organs are maintained by ligaments and muscles. Stress urinary incontinence (SUI) occurs when the pelvic floor tissues are repeatedly stimulated by excessive mechanical tension that exceeds the bearing capacity of ligaments or muscles. Besides, cells respond mechanically to mechanical stimulation by reconstituting the Piezo1 and cytoskeletal system. The aim of this study is to determine how Piezo1 and actin cytoskeleton are involved in the mechanized stretch (MS) induced apoptosis of human anterior vaginal wall fibroblasts (hAVWFs) and the mechanism. A four-point bending device was used to provide mechanical stretching to establish a cellular mechanical damage model. The apoptosis of hAVWFs cells in non-SUI patients was significantly increased by MS, which exhibited apoptosis rates comparable to those of SUI patients. Based on these findings, Piezo1 connects the actin cytoskeleton to the apoptosis of hAVWFs cells, providing an idea for the clinical diagnosis and treatment of SUI. However, the disassembly of the actin cytoskeleton suppressed the protective effect of Piezo1 silencing on MS. Based on these findings, Piezo1 connects the actin cytoskeleton to apoptosis of hAVWFs, providing new insight for the clinical diagnosis and treatment of SUI.

HMGB1/RAGE axis accelerates the repair of HUVECs injured by pathological mechanical stretching via promoting bFGF expression.

AIM: During hypertension-induced endothelial dysfunction, periodic mechanical stretching (MS) activates related inflammatory pathways and leads to endothelial damage, but the underlying mechanisms remain unknown. The present study aimed to determine the injury of HUVECs caused by overstretching and the role of HMGB1-RAGE pathway in HUVECs after injury. MAIN METHODS AND KEY FINDINGS: Human umbilical vein endothelial cells (HUVECs) were cultured and seeded in BioFlex™ plates (six wells). Cells were exposed to 5% (physiological state) and 20% (pathological state) mechanical stretch at 1 Hz for 12 or 24 h in a Flexcell-5000™, with unstretched cells serving as controls. It was found that excessive MS can inhibit cell viability, proliferation, and tube-forming ability resulting in disordered cell arrangement and orientation, slowing cell migration. All these changes cause endothelial damage compared to physiological MS. Endothelial cells (ECs) promote cell migration and self-repair after injury by increasing the High-mobility group box 1 (HMGB1) expression. Experiments and protein prediction networks have shown that HMGB1 can also promote the expression of downstream protein bFGF by binding to receptor for advanced glycation end products (RAGE). Interestingly, VEGF protein expression did not change significantly during this repair process, implying that bFGF replaces the role of VEGF in vascular endothelial repair. SIGNIFICANCE: The present study demonstrates that in the context of endothelial injury caused by excessive MS, the HMGB1/RAGE/bFGF pathway is activated and promotes endothelial repair after injury. Therefore, understanding these mechanisms will help find new therapies for diseases such as hypertension, atherosclerosis, and aneurysm formation.

Suture Cells in a Mechanical Stretching Niche: Critical Contributors to Trans-sutural Distraction Osteogenesis.

Trans-sutural distraction osteogenesis has been proposed as an alternative technique of craniofacial remodelling surgery for craniosynostosis correction. Many studies have defined the contribution of a series of biological events to distraction osteogenesis, such as changes in gene expression, changes in suture cell behaviour and changes in suture collagen fibre characteristics. However, few studies have elucidated the systematic molecular and cellular mechanisms of trans-sutural distraction osteogenesis, and no study has highlighted the contribution of cell-cell or cell-matrix interactions with respect to the whole expansion process to date. Therefore, it is difficult to translate largely primary mechanistic insights into clinical applications and optimize the clinical outcome of trans-sutural distraction osteogenesis. In this review, we carefully summarize in detail the literature related to the effects of mechanical stretching on osteoblasts, endothelial cells, fibroblasts, immune cells (macrophages and T cells), mesenchymal stem cells and collagen fibres in sutures during the distraction osteogenesis process. We also briefly review the contribution of cell-cell or cell-matrix interactions to bone regeneration at the osteogenic suture front from a comprehensive viewpoint.

Puerarin protects fibroblasts against mechanical stretching injury through Nrf2/TGF-ß1 signaling pathway.

INTRODUCTION AND HYPOTHESIS: Stress urinary incontinence (SUI) is the most common form of urinary incontinence in women, which affects women's quality of life worldwide. Mechanical injury of the pelvic floor may disrupt the pelvic supportive tissues and connections via the remodeling of extracellular matrix (ECM), which is supposed to be one of the main pathological mechanisms of SUI. METHODS: The SUI mouse model was established using vaginal distension (VD). Leak point pressure (LPP), maximum cystometric capacity (MCC), collagen, Nrf2 and TGF-ß1 in the anterior vaginal wall were measured in either wild-type or Nrf2-knockout (Nrf2-/-) female C57BL/6 mice with or without puerarin treatment. Then, the mechanical stretching (MS) loaded on L929 cells was generated by a four-point bending device. mTGF-ß1 or LY2109761 (an inhibitor of TGF-ß1) was used to verify the protective effect of puerarin after Nrf2 knockdown or overexpression. RESULTS: The collagen content of the anterior vaginal tissues in VD mice and LPP and MCC was decreased significantly. Besides, the expression levels of Nrf2, TGF-ß1, collagen I and collagen III of MS group were downregulated in L929 cells. Puerarin pretreatment could reverse mechanical injury-induced collagen downregulation and Nrf2/TGF-ß1 signaling inhibition. Moreover, both LY2109761 pretreatment and Nrf2 knockdown could attenuate the protective effect of puerarin in the mechanical injury-induced ECM remodeling, whereas exogenous TGF-ß1 could counteract the effect of Nrf2 downregulation. CONCLUSIONS: Puerarin protected fibroblasts from mechanical injury-induced ECM remodeling through the Nrf2/TGF-ß1 signaling pathway. This might be a new strategy for the treatment of SUI.

Strategies for Controlling the Spatial Orientation of Single Molecules Tethered on DNA Origami Templates Physisorbed on Glass Substrates: Intercalation and Stretching.

Nanoarchitectural control of matter is crucial for next-generation technologies. DNA origami templates are harnessed to accurately position single molecules; however, direct single molecule evidence is lacking regarding how well DNA origami can control the orientation of such molecules in three-dimensional space, as well as the factors affecting control. Here, we present two strategies for controlling the polar (θ) and in-plane azimuthal (ϕ) angular orientations of cyanine Cy5 single molecules tethered on rationally-designed DNA origami templates that are physically adsorbed (physisorbed) on glass substrates. By using dipolar imaging to evaluate Cy5's orientation and super-resolution microscopy, the absolute spatial orientation of Cy5 is calculated relative to the DNA template. The sequence-dependent partial intercalation of Cy5 is discovered and supported theoretically using density functional theory and molecular dynamics simulations, and it is harnessed as our first strategy to achieve θ control for a full revolution with dispersion as small as ±4.5°. In our second strategy, ϕ control is achieved by mechanically stretching the Cy5 from its two tethers, being the dispersion ±10.3° for full stretching. These results can in principle be applied to any single molecule, expanding in this way the capabilities of DNA as a functional templating material for single-molecule orientation control. The experimental and modeling insights provided herein will help engineer similar self-assembling molecular systems based on polymers, such as RNA and proteins.

Polarized M2 macrophages induced by mechanical stretching modulate bone regeneration of the craniofacial suture for midfacial hypoplasia treatment.

The underlying mechanism of the trans-sutural distraction osteogenesis (TSDO) technique as an effective treatment that improves the symptoms of midfacial hypoplasia syndromes is not clearly understood. Increasing findings in the orthopedics field indicate that macrophages are mechanically sensitive and their phenotypes can respond to mechanical cues. However, how macrophages respond to mechanical stretching and consequently influence osteoblast differentiation of suture-derived stem cells (SuSCs) remains unclear, particularly during the TSDO process. In the present study, we established a TSDO rat model to determine whether and how macrophages were polarized in response to stretching and consequently affected bone regeneration of the suture frontal edge. Notably, after performing immunofluorescence, RNA-sequencing, and micro-computed tomography, it was demonstrated that macrophages are first recruited by various chemokines factors and polarized to the M2 phenotype upon optimal stretching. The latter in turn regulates SuSC activity and facilitates bone regeneration in sutures. Moreover, when the activated M2 macrophages were suppressed by pharmacological manipulation, new bone microarchitecture could rarely be detected under mechanical stretching and the expansion of the sutures was clear. Additionally, macrophages achieved M2 polarization in response to the optimal mechanical stretching (10%, 0.5 Hz) and strongly facilitated SuSC osteogenic differentiation and human umbilical vein endothelial cell angiogenesis using an indirect co-culture system in vitro. Collectively, this study revealed the mechanical stimulation-immune response-bone regeneration axis and clarified at least in part how sutures achieve bone regeneration in response to mechanical force.

Transient Receptor Potential Melastatin 7 Promotes Vascular Adventitial Fibroblasts Phenotypic Transformation and Inflammatory Reaction Induced by Mechanical Stretching Stress via p38 MAPK/JNK Pathway.

Remodeling of the arteries is one of the pathological bases of hypertension. We have previously shown that transient receptor potential melastatin 7 (TRPM7) aggravates the vascular adventitial remodeling caused by pressure overload in the transverse aortic constriction (TAC) model. In this study, we sought to explore the functional expression and downstream signaling of TRPM7 in vascular adventitial fibroblasts (AFs) stimulated by mechanical stretching stress (MSS). The expression of TRPM7 was upregulated with a concomitant translocation to the cytoplasm in the AFs stimulated with 20% MSS. Meanwhile, the expression of α-smooth muscle actin (α-SMA), a marker of transformation from AFs to myofibroblasts (MFs) was also increased. Moreover, AF-conditioned medium caused a significant migration of macrophages after treatment with MSS and contained high levels of monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α). Pharmacological and RNA interference approaches using the TRPM7 inhibitor 2-aminoethoxydiphenyl borate (2-APB) and specific anti-TRPM7 small interfering RNA (si-RNA-TRPM7) abrogated these changes significantly. Further exploration uncloaked that inhibition of TRPM7 reduced the phosphorylation of p38 MAP kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) in the AFs stimulated with MSS. Furthermore, inhibition of the phosphorylation of p38MAPK or JNK could also alleviate the MSS-induced expression of α-SMA and secretion of inflammatory factors. These observations indicate that activated TRPM7 participates in the phenotypic transformation and inflammatory action of AFs in response to MSS through the p38MAPK/JNK pathway and suggest that TRPM7 may be a potential therapeutic target for vascular remodeling caused by hemodynamic changes in hypertension.

Stretch-Induced Tenomodulin Expression Promotes Tenocyte Migration via F-Actin and Chromatin Remodeling.

The mechanosensitive gene tenomodulin (Tnmd) is implicated in tendon maturation and repair. However, the mechanism by which mechanical loading regulates Tnmd's expression and its role in tenocyte migration is yet to be defined. Here, we show that Tnmd and migration were upregulated in uniaxial cyclic stress-stimulated tenocytes. The knockdown of Tnmd reduced cell migration in the presence and absence of mechanical loading, suggesting that Tnmd is involved in tenocyte migration. Moreover, the treatment of stress-stimulated tenocytes with the actin inhibitor latrunculin (Lat A), histone acetyltransferase inhibitor anacardic acid (ANA), or histone demethylases inhibitor GSK-J4 suppressed Tnmd expression and tenocyte migration. These results show that actin stress fiber formation and chromatin decondensation regulates Tnmd expression, which might then regulate tenocyte migration. Thus, this study proposes the involvement of the actin and chromatin mechanotransduction pathway in the regulation of Tnmd and reveals a novel role of Tnmd in tenocyte migration. The identification of Tnmd function in tenocyte migration provides insight into the molecular mechanisms involved in Tnmd-mediated tendon repair.

Effect of ILK on small-molecule metabolism of human periodontal ligament fibroblasts with mechanical stretching.

BACKGROUND: Mechanical stimuli can cause periodontal tissue reconstruction. Studies have found that changes in metabolites can be the terminal effect of integrin-mediated mechanical signaling. As a key kinase in integrin regulation, integrin-linked kinase (ILK) mediates mechanical signal transduction, which may contribute to metabolite changes. Defining the components of small-molecule metabolites can optimize mechanical stimuli and periodontal tissue reconstruction. Our purpose is to detect the effect of ILK-mediated mechanical signaling on intracellular small-molecule metabolites (amino acids and organic acids) in human periodontal ligament fibroblasts (HPDLFs). METHODS: Primary HPDLFs were isolated by enzyme digestion method. Tensile stresses were applied on HPDLFs in vitro using a Flexcell system. ILK gene in HPDLFs was knocked down by RNA interference (RNAi). Twenty common amino acids and seven organic acids in HPDLFs were analyzed by gas chromatography/mass spectrometry technique. RESULTS: Five amino acids (ie, alanine, glutamine, glutamate, glycine, and threonine) and three organic acids (ie, pyruvate, lactate, and citric acid) were found to be changed remarkably after mechanical stretching. In addition, baseline levels of four amino acids (ie, glutamate, glutamine, threonine, and glycine) and two organic acids (ie, lactate and citric acid) were significantly different in ILK knockdown compared with wild-type HPDLFs. CONCLUSION: This study suggests that five amino acids (ie, alanine, glutamine, glutamate, glycine, and threonine) and three organic acids (ie, pyruvate, lactate, and citric acid) may act as cellular mediators for mechanical signals in HPDLFs. Among them, four amino acids (ie, glutamate, glutamine, threonine, and glycine) and two organic acids (ie, lactate and citric acid) may be closely linked to ILK.

Mechanical Stretching induces the apoptosis of parametrial ligament Fibroblasts via the Actin Cytoskeleton/Nr4a1 signalling pathway.

The anatomical positions of pelvic floor organs are maintained mainly by ligaments and muscles. Long-term excessive mechanical tension stimulation of pelvic floor tissue beyond the endurance of ligaments or muscles will lead to the occurrence of pelvic organ prolapse (POP). In addition, cytoskeletal reconstitution is a key process by which cells respond to mechanical stimulation. The aim of the present study was to investigate the protective effect of actin cytoskeleton to resist mechanical stretching (MS)-induced apoptosis in parametrial ligament fibroblasts (PLFs) and the underlying mechanisms. MS provided by a four­point bending device could significantly induce apoptosis of PLFs from non-POP patients, which exhibited an apoptosis rate close to that of PLFs from POP patients, and the apoptosis rate was higher following latrunculin A (Lat-A, a potent inhibitor of actin) treatment. In addition, Nr4a1 and Bax expression was increased while Bcl-2 and caspase-3 expression was clearly decreased after treatment with MS and Lat-A. However, the apoptosis induced by MS was reduced when the expression of Nr4a1 was downregulated by siRNA. These outcomes reveal a novel mechanism that links the actin cytoskeleton and apoptosis in PLFs by Nr4a1; this mechanism will provide insight into the clinical diagnosis and treatment of POP.

Effect of Mechanical Stretching of the Right Atrium of Isolated Rat Heart on Dispersion of Repolarization before Fibrillation.

The multi-electrode mapping method was used to analyze electrical activity of isolated rat heart under conditions of standard perfusion, pharmacological stimulation of fibrillation, and mechanical stretching of the right atrium both under normal conditions and before cardiac fibrillation. It was shown that stretching of the right atrium prevented the increase of repolarization dispersion and latency of the electrical signal in the myocardium that were observed before cardiac fibrillation.

MT1-MMP downregulation via the PI3K/Akt signaling pathway is required for the mechanical stretching-inhibited invasion of bone-marrow-derived mesenchymal stem cells.

Mobilization from the bone marrow and the migration of bone-marrow-derived mesenchymal stem cells (BMSCs) through the peripheral circulation to injured tissue sites are regulated by multiple mechanical and chemical factors. We previously demonstrated that mechanical stretching promotes the migration but inhibits the invasion of BMSCs. However, the involved mechanisms, especially the mechanism of stretching-inhibited BMSC invasion, have not been thoroughly elucidated to date. In this study, we found that mechanical stretching with a 10% amplitude at a 1-Hz frequency for 8 hr significantly reduces BMSC invasion and downregulates the expression of membrane type-1 matrix metalloproteinases (MT1-MMP) at both the messenger RNA and protein levels. The overexpression of MT1-MMP restores mechanical stretching-reduced BMSC invasion. Moreover, phosphatidylinositol 3-kinase (PI3K)-dependent Akt phosphorylation in BMSCs was found to be inactivated by mechanical stretching. Pharmacological inhibitors of PI3K/Akt signaling (LY294002 or A443654) reduced the expression of MT1-MMP and impaired BMSC invasion. In addition, the upregulation of Akt phosphorylation by a pharmacological activator (SC79) increased MT1-MMP expression and suppressed mechanical stretching-reduced BMSC invasion. Taken together, our results suggest that mechanical stretching inhibits BMSC invasion by downregulating MT1-MMP expression by suppressing the PI3K/Akt signaling pathway.

Mechanical stress affects methylation pattern of GNAS isoforms and osteogenic differentiation of hAT-MSCs.

Mechanical stress exerts a substantial role on skeletal-cell renewal systems, whereas accumulating evidence suggests that epigenetic mechanisms induce changes and differential gene expression. Although the underlying mechanisms remain to be fully elucidated, our study suggests that the influence of the long term mechanical stimulation elicits epigenetic modifications controlling osteogenic differentiation of human adipose tissue multipotential stromal cells (hAT-MSCs) and contributes to an accelerating in vitro osteogenesis. GNAS imprinting gene acts as a critical regulator of osteoblast differentiation and is implicated in human genetic disorders with pathological formation of ectopic-skeletal bone. Investigating a wide variety of stimuli, we showed that daily mechanical stretch on hAT-MSCs of 7th and 15th days' intervals induced a significant down-regulation in DNA methylation status of critical CpG sites of NESP and GNASXL isoforms, accompanied by up-regulation of the corresponding gene transcripts, and osteogenic differentiation earlier in culture. Importantly, methylation analysis of differentiating bone marrow-derived MSCs revealed similar methylation patterns. Bioinformatic analysis further showed that all CpG islands exhibiting significant methylation alterations encompassed transcriptional repressor CTCF binding sites. We hereby emphasize the need to investigate the epigenetic alterations on hAT-MSCs during environmental mechanical forces and to consider how the knowledge gained through these studies may foster new means of symptoms prevention and management of ectopic bone formation in the clinic.

Autophagy activation facilitates mechanical stimulation-promoted osteoblast differentiation and ameliorates hindlimb unloading-induced bone loss.

Autophagy has been indicated to be involved in regulating bone metabolism. However, little is known about the role of autophagy in mechanical stimulation-influenced osteoblast differentiation and bone formation. In the present study, we first demonstrated that autophagy activation was essential for cyclic mechanical stretching-promoted osteoblast differentiation of bone marrow mesenchymal stem cells. To explore the in vivo role of autophagy in osteoblast differentiation, the hindlimb unloading-induced disuse osteoporosis model was used. Compared to the normal controls, hindlimb unloading led to abundant bone loss as well as lessened autophagy activation of osteoblasts. However, the activation of autophagy by ULK1 overexpression or in the presence of rapamycin significantly increased osteoblast differentiation activity and restored the bone volume. The findings implicate autophagy as a novel mechanosensitive pathway that regulates osteoblast differentiation. The pharmacological activation of autophagy may be an interesting approach for the prevention and treatment of disuse osteoporosis.

Mechanoregulation of osteoblast-like MG-63 cell activities by cyclic stretching.

BACKGROUND/PURPOSE: Mechanical loading plays an important role in regulating bone formation and remodeling. Relevant mechanical stretching can increase the proliferation and differentiation of osteoblastic cells in vitro. However, little is known about the effects of supraphysiological high-level mechanical stretching on the growth and cell cycle progression of osteoblastic cells. METHODS: Osteoblast-like MG-63 cells were seeded onto flexible-bottomed plates and subjected to cyclic mechanical stretching (15% elongation, 0.5 Hz) for 24 and 48 hours in a Flexercell FX-4000 strain unit. Cellular activities were measured by an assay based on the reduction of the tetrazolium salt, 3-[4,5-dimethyldiazol-2-yl]-2,5-diphenyl tetra-zolium bromide (MTT). The number of viable cells was also determined by the trypan blue dye exclusion technique. Cell cycle progression was checked by flow cytometry. mRNA expressions of apoptosis- and cell cycle-related genes (Bcl2, Bax, cdc2, cdc25C, and cyclin B1) were analyzed using an RT-PCR technique. RESULTS: The number of viable cells significantly decreased in osteoblast-like MG-63 cells subjected to cyclic mechanical stretching for 24 or 48 hours. The MTT activity of stretched cells did not change at 24 hours, whereas a significant decrease was noted at 48 hours in comparison to the unstretched controls. The flow cytometry showed that mechanical stretching induced S-phase cell cycle arrest. Furthermore, exposure to mechanical stretching led to apoptotic cell death, as shown by the increase in the hypodiploid sub-G0/G1 cell population. Furthermore, a decreased cdc25C mRNA level was consistently noted in stretched cells. However, the mRNA expressions of Bcl2, Bax, cdc2, and cyclin B1 genes were not significantly altered compared to the unstretched control cells. CONCLUSION: High-level mechanical stretching induced S-phase cell cycle arrest and apoptotic cell death in osteoblastic cells. The results suggest that heavy tensional force is a negative regulator of osteoblastic activities and should, therefore, be minimized if bone formation is attempted during orthodontic/orthopedic treatment.

Construction of chitosan-gelatin polysaccharide-protein composite hydrogel via mechanical stretching and its biocompatibility in vivo.

Natural polysaccharides and protein macromolecules are the important components of extracellular matrix (ECM), but individual component generally exhibits weak mechanical property, limited biological function or strong immunogenicity in tissue engineering. Herein, gelatin (Gel) was deposited to the stretched (65 %) chitosan (CS) hydrogel substrates to fabricate the polysaccharide-protein CS-Gel-65 % composite hydrogels to mimic the natural component of ECM and improve the above deficiencies. CS hydrogel substrates under different stretching deformations exhibited tunable morphology, chemical property and wettability, having a vital influence on the secondary structures of deposited fibrous Gel protein, namely appearing with the decreased ß-sheet content in stretched CS hydrogel. Gel also produced a more homogenous distribution on the stretched CS hydrogel substrate due to the unfolding of Gel and increased interactions between Gel and CS than on the unstretched substrate. Moreover, the polysaccharide-protein composite hydrogel possessed enhanced mechanical property and oriented structure via stretching-drying method. Besides, in vivo subcutaneous implantation indicated that the CS-Gel-65 % composite hydrogel showed lower immunogenicity, thinner fibrous capsule, better angiogenesis effect and increased M2/M1 of macrophage phenotype. Polysaccharide-protein CS-Gel-65 % composite hydrogel offers a novel material as a tissue engineering scaffold, which could promote angiogenesis and build a good immune microenvironment for the damaged tissue repair.

Cyclic Stretching Enhances Angiocrine Signals at Liver Bud Stage from Human Pluripotent Stem Cells in Two-Dimensional Culture.

Angiocrine signals during the development and growth of organs, including the liver, intestine, lung, and bone, are essential components of intercellular communication. The signals elicited during the liver bud stage are critical for vascularization and enhanced during the intercellular communication between the cells negative for kinase insert domain receptor (KDR) (KDR- cells) and the cells positive for KDR (KDR+ cells), which constitute the liver bud. However, the use of a human pluripotent stem cell (hPSC)-derived system has not facilitated the generation of a perfusable vascularized liver organoid that allows elucidation of liver development and has great potential for liver transplantation. This is largely owing to the lack of fundamental understanding to induce angiocrine signals in KDR- and KDR+ cells during the liver bud stage. We hypothesized that mechanical stimuli of cyclic stretching/pushing by the fetal heart adjacent to the liver bud could be the main contributor to promoting angiocrine signals in KDR- and KDR+ cells during the liver bud stage. In this study, we show that an organ-on-a-chip platform allows the emulation of an in vivo-like mechanical environment for the liver bud stage in vitro and investigate the role of cyclic mechanical stretching (cMS) to angiocrine signals in KDR- and KDR+ cells derived from hPSCs. RNA sequencing revealed that the expression of genes associated with epithelial-to-mesenchymal transition, including angiocrine signals, such as hepatocyte growth factor (HGF) and matrix metallopeptidase 9 (MMP9), were increased by cMS in cocultured KDR- and KDR+ cells. The expression and secretions of HGF and MMP9 were increased by 1.98- and 1.69-fold and 3.23- and 3.72-fold with cMS in the cocultured KDR- and KDR+ cells but were not increased by cMS in the monocultured KDR- and KDR+ cells, respectively. Finally, cMS during the liver bud stage did not lead to the dedifferentiation of hepatocytes, as the cells with cMS showed hepatic maker expression (CYP3A4, CYP3A7, ALB, and AAT) and 1.71-fold higher CYP3A activity than the cells without cMS, during 12 day-hepatocyte maturation after halting cMS. Our findings provide new insights into the mechanical factors during the liver bud stage and directions for future improvements in the engineered liver tissue.

Detecting emergence of ruptures in individual layers of the stretched intestinal wall using optical coherence elastography: A pilot study.

We report a new application of compression optical coherence elastography (C-OCE) to monitor the emergence of ruptures in individual layers of longitudinally stretched small-intestine walls using tissue samples (n = 36) from nine minipigs. Before stretching, C-OCE successfully estimated stiffness for each intestine-wall layer: longitudinal muscular layer with serosa, circumferential muscular layer, submucosa and mucosa. In stretched samples, C-OCE clearly visualized initial stiffening in both muscular layers. By 25% elongation, a sharp stiffness decrease for the longitudinal muscular layer, indicated emergence of tears in all samples. With further stretching, for most samples, ruptures emerged in the circumferential muscular layer and submucosa, while mucosa remained undamaged. Histology confirmed the OCE-revealed damaging and absence of tissue damage for ~15% elongation. Thus, C-OCE has demonstrated a high potential for determining the safety tissue-stretching threshold which afterward may be used intraoperatively to prevent rupture risk in intestinal tissues stretched during various diagnostic/therapeutic procedures.