Mechanical stretching boosts expansion and regeneration of intestinal organoids through fueling stem cell self-renewal

Intestinal organoids,derived from intestinal stem cell self-organization,recapitulate the tissue structures and behav-iors of the intestinal epithelium,which hold great potential for the study of developmental biology,disease modeling,and regenerative medicine.The intestinal epithelium is exposed to dynamic mechanical forces which exert profound effects on gut development.However,the conventional intestinal organoid culture system neglects the key role of mechanical microenvironments but relies solely on biological factors.Here,we show that adding cyclic stretch to intestinal organoid cultures remarkably up-regulates the signature gene expression and proliferation of intestinal stem cells.Furthermore,mechanical stretching stimulates the expansion of SOX9+progenitors by activating the Wnt/β-Catenin signaling.These data demonstrate that the incorporation of mechanical stretch boosts the stemness of intestinal stem cells,thus benefiting organoid growth.Our findings have provided a way to optimize an organoid generation system through understanding cross-talk between biological and mechanical factors,paving the way for the application of mechanical forces in organoid-based models.

Effect of mechanical stretching and substrate stiffness on the morphology,cytoskeleton and nuclear shape of corneal endothelial cells

Due to the limited capacity of corneal endothelial cells(CECs)division,corneal endothelial diseases have become a great challenge.The cornea is subjected to various mechanical stimuli in vivo,which may have a positive or negative influence.Thus,it is significant to gain an insight into the mechanism of mechanobiology of CECs for seeking more possible treatment.The purpose of this study was to determine the impacts of mechanical stretch and substrate stiffness on the morphology and fundamental cell behavior of CECs.Rabbit corneal endothelial cells(RCECs)were subjected to a 5%mechanical stretch or cultured on substrates of different stiffness.The impacts of mechanical stimulus on cell area,aspect ratio,circularity,cell density,nuclear shape,cytoskeleton,and cell viability were investigated.The expressions of the corneal endothelium-related markers ZO-1 and Na^(+)/K^(+) ATPase were also evaluated by confocal immunofluorescence microscopy in the stiffness group.Our results suggested that mechanical stretch promoted the rearrangement of the cytoskeleton while decreasing the cell circularity,nuclear area,and cell density as well as cell viability.RCECs cultured on 10 kPa substrates,which was close to the physiological stiffness of rabbit Descemet's membrane(DM),showed better cell morphology,more stable actin cytoskeleton assembly,and more robust expression of the functional marker compared with other softer or stiffer substrates.In summary,mechanical stretch and substrate stiffness have profound influences on the morphology and function of CECs,which may have implications for the understanding and possible treatment of corneal endothelial diseases.

The Effect of Cyclic Stretching on Matrix Production, Mineralization and Differentiation of Osteoblasts

A four-point bending apparatus is used to investigate the effects of stretching on collagen synthesis, mineralization and differentiation of osteoblasts. Cells are stretched at 1500 με for 24 hours. The responses of osteoblasts to mechanical signal of physiological stretching are evaluated from three aspects: collagen production, extracellular inorganic calcium secretion and ALP activity. The results show that osteoblasts decrease the collagen synthesis, calcium secretion and ALP activity compared to the control cells (65.82%,73.51%,48.10% respectively), confirming that cyclic stretching at 1500 με inhabits the physiological activity of osteoblasts.

Mechanical stretch induces mitochondria-dependent apoptosis in neonatal rat cardiomyocytes and G_(2)/M accumulation in cardiac fibroblasts

Heart remodeling is associated with the loss of cardiomyocytes and increase of fibrous tissue owing to abnormal mechanical load in a number of heart disease conditions. In present study, a well-described in vitro sustained stretch model was employed to study mechanical stretch-induced responses in both neonatal cardiomyocytes and cardiac fibroblasts. Cardiomyocytes, but not cardiac fibroblasts, underwent mitochondria-dependent apoptosis as evidenced by cytochrome c (cyto c) and Smac/DIABLO release from mitochondria into cytosol accompanied by mitochondrial membrane potential (△ψ_m) reduction, indicative of mitochondrial permeability transition pore (PTP) opening. Cyclosporin A, an inhibitor of PTP, inhibited stretch-induced cyto c release, △ψ_m reduction and apoptosis, suggesting an important role of mitochondrial PTP in stretch-induced apoptosis. The stretch also resulted in increased expression of the pro-apoptotic Bcl-2 family proteins, including Bax and Bad, in cardiomyocytes, but not in fibroblasts. Bax was accumulated in mitochondria following stretch. Cell permeable Bid-BH3 peptide could induce and facilitate stretch-induced apoptosis and △ψ_m reduction in cardiomyocytes. These results suggest that Bcl-2 family proteins play an important role in coupling stretch signaling to mitochondrial death machinery, probably by targeting to PTP. Interestingly, the levels of p53 were increased at 12 h after stretch although we observed that Bax upregulation and apoptosis occurred as early as 1 h. Adenovirus delivered dominant negative p53 blocked Bax upregulation in cardiomyocytes but showed partial effect on preventing stretch-induced apoptosis, suggesting that p53 was only partially involved in mediating stretch-induced apoptosis. Furthermore, we showed that p21 was upregulated and cyclin B1 was downregulated only in cardiac fibroblasts, which may be associated with G_2/M accumulation in response to mechanical stretch.

Effects of Acute Mechanical Stretch on the Expression of Mechanosensitive Potassium Channel TREK-1 in Rat Left Ventricle

To explore the role of mechanosensitive potassium channel TREK-1, Western blot analysis was used to investigate the expression changes of TREK-1 in left ventricle in acute mechanically stretched heart. Forty Wistar rats were randomly divided into 8 groups （n=5 in each group）, subject to single Langendorff perfusion for 0, 30, 60, 120 min and acute mechanical stretch for 0, 30, 60, 120 min respectively. With Langendorff apparatus, an acute mechanically stretched heart model was established. There was no significant difference in the expression of TREK-1 among single Langendorff perfusion groups （P〉0.05）. As compared to non-stretched Langendorff-perfused heart, only the expression of TREK-1 in acute mechanically stretched heart （120 min） was greatly increased （P〈0.05）. This result suggested that some course of mechanical stretch could up-regulate the expression of TREK-1 in left ventricle. TREK-1 might play an important role in mechanoelectric feedback, so it could reduce the occurrence of arrhythmia that was induced by extra mechanical stretch.

In vitro cell stretching devices and their applications:From cardiomyogenic differentiation to tissue engineering

Mechanical stretch plays an important role in the control of cardiomyocyte behavior,as well as in the study of the mechanisms of cardiovascular function and pathology.The complexity involved in biological systems in vivo has created a need for better in vitro techniques,thus a variety of cell stretching devices have been developed for a deeper understanding of cellular responses to strain.In this review,we introduce the design,functionality,and characteristics of multiple types of cell stretching devices from two and three dimensions,then discuss the research progress of promoting cardiomyogenic differentiation of stem cells by external stretching and its application in cardiac tissue engineering.

Mechanotransduction of liver sinusoidal endothelial cells under varied mechanical stimuli

Liver sinusoidal endothelial cells(LSECs)are the gatekeeper of liver to maintain hepatic homeostasis.They are formed into the highly specialized endothelium between vascular lumen and the space of Disse and are mechanosensitive to respond varied microenvironments.Shear stress and mechanical stretch induced by blood perfusion and substrate stiffness enhancement derived from deposition of extracellular matrix(ECM)are major mechanical stimuli that surround LSECs.This review introduces how LSECs respond to the external forces in both physiological and pathological cases and what is the interplay of LSECs with other hepatic cells.Molecular mechanisms that potentiate LSECs mechanotransduction are also discussed.

S100 calcium-binding protein A9 promotes skin regeneration through toll-like receptor 4 during tissue expansion

Background:In plastic surgery,tissue expansion is widely used for repairing skin defects.However,low expansion efficiency and skin rupture caused by thin,expanded skin remain significant challenges in promoting skin regeneration during expansion.S100 calcium-binding protein A9(S100A9)is essential in promoting wound healing;however,its effects on skin regeneration during tissue expansion remain unclear.The aim of the present study was to explore the role of S100A9 in skin regeneration,particularly collagen production to investigate its importance in skin regeneration during tissue expansion.Methods:The expression and distribution of S100A9 and its receptors-toll-like receptor 4(TLR-4)and receptor for advanced glycation end products were studied in expanded skin.These character-istics were investigated in skin samples of rats and patients.Moreover,the expression of S100A9 was investigated in stretched keratinocytes in vitro.The effects of S100A9 on the proliferation and migration of skin fibroblasts were also observed.TAK-242 was used to inhibit the binding of S100A9 to TLR-4;the levels of collagen I(COL I),transforming growth factor beta(TGF-β),TLR-4 and phospho-extracellular signal-related kinase 1/2(p-ERK1/2)in fibroblasts were determined.Furthermore,fibroblasts were co-cultured with stretched S100A9-knockout keratinocytes by siRNA transfection and the levels of COL I,TGF-β,TLR-4 and p-ERK1/2 in fibroblasts were investigated.Additionally,the area of expanded skin,thickness of the dermis,and synthesis of COL I,TGF-β,TLR-4 and p-ERK1/2 were analysed to determine the effects of S100A9 on expanded skin.Results:Increased expression of S100A9 and TLR-4 was associated with decreased extracellular matrix(ECM)in the expanded dermis.Furthermore,S100A9 facilitated the proliferation and migration of human skin fibroblasts as well as the expression of COL I and TGF-βin fibroblasts via the TLR-4/ERK1/2 pathway.We found that mechanical stretch-induced S100A9 expression and secretion of keratinocytes stimulated COL I,TGF-β,TLR-4 and p-ERK1/2 expression in skin fibroblasts.Recombined S100A9 protein aided expanded skin regeneration and rescued dermal thinning in rats in vivo as well as increasing ECM deposition during expansion.Conclusions:These findings demonstrate that mechanical stretch promoted expanded skin regeneration by upregulating S100A9 expression.Our study laid the foundation for clinically improving tissue expansion using S100A9.

Effects of tensile forces on serum deprivation-induced osteoblast apoptosis： expression analysis of caspases, Bcl-2, and Bax

Background Apoptosis is involved in the adaptive responses of bone to mechanical loading. The purpose of this study was to investigate the effects of tensile forces on osteoblast apoptosis and the related mechanism by analyzing the expression of caspases, Bcl-2, and Bax. Methods Primary osteoblasts were harvested from neonatal rat calvaria and were subjected to cyclic tensile forces for 72 hours using Flexcell 4000 strain unit in Minimum Essential Medium （MEM） with 10% fetal calf serum （FCS） or with serum deprivation. Apoptosis was tested by flow cytometry using annexin V/PI staining. Caspase-3 activity was analyzed via Elisa. The gene expression of caspase-8, -9, Bcl-2, and Bax was quantified by reverse transcription （RT）-PCR. Results In 10% FCS condition, no significant difference in cell apoptosis was found between the stretched and non-stretched osteoblast cultures. Serum withdrawal resulted in higher apoptosis rate in the osteoblasts with increased caspase-3 activity, and elevated expression of caspase-9 and Bax. Six-percent elongation of stretch attenuated the cell apoptosis induced by serum starvation, concurrent with a decrease in caspase-3 activity, a decline of caspase-8 expression, and an elevation of Bcl-2 level. On the contrary, 12% elongation of stretch increased caspase-3 activity and promoted the apoptosis with an elevated expression of caspase-8 and Bax. No significant change of caspase-9 expression was identified upon force application. Conclusions These results suggested that tensile forces regulate cell apoptosis of primary rat osteoblasts through caspase-3 and caspase-8 signaling cascade. Light forces rescue the cells from serum deprivation-induced apoptosis by elevating Bcl-2 expression, while heavy forces promote the apoptotic insult by inducing Bax expression.

Enhanced photogalvanic effect in the two-dimensional MgCl_(2)/ZnBr_(2) vertical heterojunction by inhomogenous tensile stress

The photogalvanic effect (PGE) occurring in noncentrosymmetric materials enables the generation of a dc photocurrent at zero bias with a high polarization sensitivity, which makes it very attractive in photodetection. However, the magnitude of the PGE photocurrent is usually small, leading to a low photoresponsivity, and therefore hampers its practical application in photodetection. Here, we propose an approach to largely enhancing the PGE photocurrent by applying an inhomogenous mechanical stretch, based on quantum transport simulations. We model a two-dimensional photodetector consisting of the wide-bandgap MgCl_(2)/ZnBr_(2) vertical van der Waals heterojunction with the noncentrosymmetric C_(3v) symmetry. Polarization-sensitive PGE photocurrent is generated under the vertical illumination of linearly polarized light. By applying inhomogenous mechanical stretch on the lattice, the photocurrent can be largely increased by up to 3 orders of magnitude due to the significantly increased device asymmetry. Our results propose an effective way to enhance the PGE by inhomogenous mechanical strain, showing the potential of the MgCl_(2)/ZnBr_(2) vertical heterojunction in the low-power UV photodetection.

Nano-hydroxyapatite(n-HA)involved in the regeneration of rat nerve injury triggered by overloading stretch

Damage of axon and glial scars formation both inhibit nerve regenerative growth during nerve injury.In addition,mechanical stretch at high displacement rates of 10%tensile strain can cause marked nerve injury,it is important for finding a proper nano biomaterial to repair nerve injury.Nano-hydroxyapatite(n-HA)has excellent biocompatibility and high bioactivity,which is a good candidate for biomedical engineering applications.But the certain mechanism of n-HA on the injured nerve is seldom reported.In this study,we determined the role of n-HA on the mechanical stretch-induced nerve injury at adult rat spine.Mechanical stretch under strain 10%at displacement rates of 60 mm/min can cause marked broken vessels and edema in spinal cord and dorsal root ganglion tissue in haematoxylin-eosin(HE)staining.However,n-HA application can reverse hemorrhage and edema triggered by high rates of 60 mm/min stretch.Moreover,n-HA can promote positive staining of Netrin-1 increase significantly in spinal cord and dorsal root ganglion tested by immunohistochemistry(IHC)staining.In general,our study indicated that n-HA can repair mechanical stretch-induced nerve injury,it may provide a new approach to block injury and accelerate nerve regeneration in future.