microRNAs slow translating ribosomes to prevent protein misfolding in eukaryotes.

Slower translation rates reduce protein misfolding. Such reductions in speed can be mediated by the presence of non-optimal codons, which allow time for proper folding to occur. Although this phenomenon is conserved from bacteria to humans, it is not known whether there are additional eukaryote-specific mechanisms which act in the same way. MicroRNAs (miRNAs), not present in prokaryotes, target both coding sequences (CDS) and 3' untranslated regions (UTR). Given their low suppressive efficiency, it has been unclear why miRNAs are equally likely to bind to a CDS. Here, we show that miRNAs transiently stall translating ribosomes, preventing protein misfolding with little negative effect on protein abundance. We first analyzed ribosome profiles and miRNA binding sites to examine whether miRNAs stall ribosomes. Furthermore, either global or specific miRNA deficiency accelerated ribosomes and induced aggregation of a misfolding-prone polypeptide reporter. These defects were rescued by slowing ribosomes using non-cleaving shRNAs as miRNA mimics. We finally show that proinsulin misfolding, associated with type II diabetes, was resolved by non-cleaving shRNAs. Our findings provide a eukaryote-specific mechanism of co-translational protein folding and a previously unknown mechanism of action to target protein misfolding diseases.

Printable oxygen-generating biodegradable scaffold for thicker tissue-engineered medical products.

BACKGROUND: Due to the increasing demand to generate thick and vascularized tissue-engineered constructs, novel strategies are currently being developed. An effective example is the fabrication of a 3D scaffold containing oxygen-releasing biomaterials to solve the limitations of gas diffusion and transport within transplanted tissues or devices. METHODS: In this study, we developed a biodegradable scaffold made of polycaprolactone (PCL) mixed with oxygen-generating calcium peroxide (CPO) to design new structures for regenerative tissue using a 3D printer capable of forming arbitrarily shapes. RESULTS AND CONCLUSION: When osteoblast progenitor cells (MC3T3-E1 cells) were cultured under hypoxic conditions on scaffolds fabricated with this technique, it was shown that cell death was reduced by the new scaffolds. Therefore, the results suggest that 3D-printed scaffolds made from biodegradable oxygen-releasing materials may be useful for tissue engineering and regeneration.

Hydrostatic pressure prevents chondrocyte differentiation through heterochromatin remodeling.

Articular cartilage protects and lubricates joints for smooth motion and transmission of loads. Owing to its high water content, chondrocytes within the cartilage are exposed to high levels of hydrostatic pressure, which has been shown to promote chondrocyte identity through unknown mechanisms. Here, we investigate the effects of hydrostatic pressure on chondrocyte state and behavior, and discover that application of hydrostatic pressure promotes chondrocyte quiescence and prevents maturation towards the hypertrophic state. Mechanistically, hydrostatic pressure reduces the amount of trimethylated H3K9 (K3K9me3)-marked constitutive heterochromatin and concomitantly increases H3K27me3-marked facultative heterochromatin. Reduced levels of H3K9me3 attenuates expression of pre-hypertrophic genes, replication and transcription, thereby reducing replicative stress. Conversely, promoting replicative stress by inhibition of topoisomerase II decreases Sox9 expression, suggesting that it enhances chondrocyte maturation. Our results reveal how hydrostatic pressure triggers chromatin remodeling to impact cell fate and function.This article has an associated First Person interview with the first author of the paper.

Intracellular calcium ion transients evoked by cell poking independently of released autocrine ATP in Madin-Darby canine kidney cells.

The mechanical stimulation induced by poking cells with a glass needle activates Piezo1 receptors and the adenosine triphosphate (ATP) autocrine pathway, thus increasing intracellular Ca2+ concentration. The differences between the increase in intracellular Ca2+ concentration induced by cell poking and by ATP-only stimulation have not been investigated. In this study, we investigated the Ca2+ signaling mechanism induced by autocrine ATP release during Madin-Darby Canine Kidney cell membrane deformation by cell poking. The results suggest that the pathways for supplying Ca2+ into the cytoplasm were not identical between cell poking and conventional ATP stimulation. The functions of the G protein-coupled receptor (GPCR) subunits (G α $\alpha $ q, G ß Î³ $\beta \gamma $ ), ATP-activated receptor and the upstream Ca2+ release signal from the intracellular endoplasmic reticulum Ca2+ store, were investigated. The results show that G α $\alpha $ q plays a major role in the Ca2+ response evoked by ATP-only stimulation, while cell poking induces a Ca2+ response requiring the involvement of both G α $\alpha $ q and G ß Î³ $\beta \gamma $ units simultaneously. These results suggest that GPCR are not only activated by ATP-only stimulation or autocrine ATP release during Ca2+ signaling, but also activated by the mechanical effects of cell poking.

Local Strain Distribution and Increased Intracellular Ca2+ Signaling in Bovine Articular Cartilage Exposed to Compressive Strain.

Articular cartilage is exposed to compressive strain of approximately 10% under physiological loads in vivo, and intracellular Ca2+ signaling is one of the earliest responses in chondrocytes under this physical stimulation. However, it remains unknown whether compressive strain itself evokes intracellular Ca2+ signaling in chondrocytes located within each layer (from surface to deep) in an equal manner with physiological levels of strain. The purpose of this study, therefore, was to determine the distribution of local strain and increased intracellular Ca2+ signaling in layer-dependent cell populations in response to 10% compressive strain loading. For this purpose, the time course of strain was measured in each layer to calculate layer-specific deformation properties. In addition, layer-specific changes in chondrocyte intracellular Ca2+ signals were recorded over time using a fluorescent Ca2+ indicator, Fluo-3, to establish ratios of cells with increased Ca2+ signaling at each depth of cartilage under static conditions or exposed to compression. The results showed that the surface layer was compressed with a larger strain compared with other layers. Few cells with Ca2+ signaling were observed under static conditions. Percentages of responsive cells within compressed cartilage were higher than those within cartilage under static conditions. However, increased intracellular Ca2+ signals were observed in a prominent number of chondrocytes within the deep layer, but not the surface layer, of compressed cartilage. Our results suggest that at a physiological compression level, Ca2+ is upregulated, but the stimulation of Ca2+ signaling in articular cartilage is not simply defined by local deformation.

MicroRNA-23a has minimal effect on endurance exercise-induced adaptation of mouse skeletal muscle.

Skeletal muscles contain several subtypes of myofibers that differ in contractile and metabolic properties. Transcriptional control of fiber-type specification and adaptation has been intensively investigated over the past several decades. Recently, microRNA (miRNA)-mediated posttranscriptional gene regulation has attracted increasing attention. MiR-23a targets key molecules regulating contractile and metabolic properties of skeletal muscle, such as myosin heavy-chains and peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α). In the present study, we analyzed the skeletal muscle phenotype of miR-23a transgenic (miR-23a Tg) mice to explore whether forced expression of miR-23a affects markers of mitochondrial content, muscle fiber composition, and muscle adaptations induced by 4 weeks of voluntary wheel running. When compared with wild-type mice, protein markers of mitochondrial content, including PGC-1α, and cytochrome c oxidase complex IV (COX IV), were significantly decreased in the slow soleus muscle, but not the fast plantaris muscle of miR-23a Tg mice. There was a decrease in type IId/x fibers only in the soleus muscle of the Tg mice. Following 4 weeks of voluntary wheel running, there was no difference in the endurance exercise capacity as well as in several muscle adaptive responses including an increase in muscle mass, capillary density, or the protein content of myosin heavy-chain IIa, PGC-1α, COX IV, and cytochrome c. These results show that miR-23a targets PGC-1α and regulates basal metabolic properties of slow but not fast twitch muscles. Elevated levels of miR-23a did not impact on whole body endurance capacity or exercise-induced muscle adaptations in the fast plantaris muscle.

Enhancing osteogenic differentiation of MC3T3-E1 cells by immobilizing inorganic polyphosphate onto hyaluronic acid hydrogel.

In tissue engineering, precise control of cues in the microenvironment is essential to stimulate cells to undergo bioactivities such as proliferation, differentiation, and matrix production. However, current approaches are inefficient in providing nondepleting cues. In this study, we have developed a novel bioactive hydrogel (HAX-PolyP) capable of enhancing tissue growth by conjugating inorganic polyphosphate chains onto hyaluronic acid macromers. The immobilized polyphosphates provided constant osteoconductive stimulation to the embedded murine osteoblast precursor cells, resulting in up-regulation of osteogenic marker genes and enhanced levels of ALP activity. The osteoconductive activity was significantly higher when compared to those stimulated with free-floating polyphosphates. Even at very low concentrations, immobilization of polyphosphates onto the scaffold allowed sufficient signaling leading to more effective osteoconduction. These results demonstrate the potential of our novel material as an injectable bioactive scaffold, which can be clinically useful for developing bone grafts and bone regeneration applications.

Enhanced chondrogenesis with upregulation of PKR using a novel hydrostatic pressure bioreactor.

In this study, we developed a novel bioreactor to load hydrostatic pressure to promote chondrogenesis of prechondrogenic ATDC5 cells in as little as 3 days. Furthermore, we showed that loading hydrostatic pressure induced the upregulation of PKR, which is known to participate in mechanotransduction in various models.

Bone regeneration in calvarial defects in a rat model by implantation of human bone marrow-derived mesenchymal stromal cell spheroids.

Mesenchymal stem cell (MSC) condensation contributes to membrane ossification by enhancing their osteodifferentiation. We investigated bone regeneration in rats using the human bone marrow-derived MSC-spheroids prepared by rotation culture, without synthetic or exogenous biomaterials. Bilateral calvarial defects (8 mm) were created in nude male rats; the left-sided defects were implanted with MSC-spheroids, ß-tricalcium phosphate (ß-TCP) granules, or ß-TCP granules + MSC-spheroids, while the right-sided defects served as internal controls. Micro-computed tomography and immunohistochemical staining for osteocalcin/osteopontin indicated formation of new, full-thickness bones at the implantation sites, but not at the control sites in the MSC-spheroid group. Raman spectroscopy revealed similarity in the spectral properties of the repaired bone and native calvarial bone. Mechanical performance of the bones in the MSC-implanted group was good (50 and 60% those of native bones, respectively). All tests showed poor bone regeneration in the ß-TCP and ß-TCP + MSC-spheroid groups. Thus, significant bone regeneration was achieved with MSC-spheroid implantation into bone defects, justifying further investigation.

Skeletal muscle adaptation in response to mechanical stress in p130cas-/- mice.

Mammalian skeletal muscles undergo adaptation in response to changes in the functional demands upon them, involving mechanical-stress-induced cellular signaling called "mechanotransduction." We hypothesized that p130Cas, which is reported to act as a mechanosensor that transduces mechanical extension into cellular signaling, plays an important role in maintaining and promoting skeletal muscle adaptation in response to mechanical stress via the p38 MAPK signaling pathway. We demonstrate that muscle-specific p130Cas-/- mice express the contractile proteins normally in skeletal muscle. Furthermore, muscle-specific p130Cas-/- mice show normal mechanical-stress-induced muscle adaptation, including exercise-induced IIb-to-IIa muscle fiber type transformation and hypertrophy. Finally, we provide evidence that exercise-induced p38 MAPK signaling is not impaired by the muscle-specific deletion of p130Cas. We conclude that p130Cas plays a limited role in mechanical-stress-induced skeletal muscle adaptation.

Disruption of skeletal muscle mitochondrial network genes and miRNAs in amyotrophic lateral sclerosis.

Skeletal muscle mitochondrial dysfunction is believed to play a role in the progression and severity of amyotrophic lateral sclerosis (ALS). The regulation of transcriptional co-activators involved in mitochondrial biogenesis and function in ALS is not well known. When compared with healthy control subjects, patients with ALS, but not neurogenic disease (ND), had lower levels of skeletal muscle peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) mRNA and protein and estrogen-related receptor-α (ERRα) and mitofusin-2 (Mfn2) mRNA. PGC-1ß, nuclear respiratory factor-1 (NRF-1) and Mfn1 mRNA as well as cytochrome C oxidase subunit IV (COXIV) mRNA and protein were lower in patients with ALS and ND. Both patient groups had reductions in citrate synthase and cytochrome c oxidase activity. Similar observations were made in skeletal muscle from transgenic ALS G93A transgenic mice. In vitro, PGC-1α and PGC-1ß regulated Mfn1 and Mfn2 in an ERRα-dependent manner. Compared to healthy controls, miRNA 23a, 29b, 206 and 455 were increased in skeletal muscle of ALS patients. miR-23a repressed PGC-1α translation in a 3' UTR dependent manner. Transgenic mice over expressing miR-23a had a reduction in PGC-1α, cytochome-b and COXIV protein levels. These results show that skeletal muscle mitochondrial dysfunction in ALS patients is associated with a reduction in PGC-1α signalling networks involved in mitochondrial biogenesis and function, as well as increases in several miRNAs potentially implicated in skeletal muscle and neuromuscular junction regeneration. As miR-23a negatively regulates PGC-1α signalling, therapeutic inhibition of miR-23a may be a strategy to rescue PGC-1α activity and ameliorate skeletal muscle mitochondrial function in ALS.

Cyclic mechanical strain maintains Nanog expression through PI3K/Akt signaling in mouse embryonic stem cells.

Mechanical strain has been reported to affect the proliferation/differentiation of many cell types; however, the effects of mechanotransduction on self-renewal as well as pluripotency of embryonic stem (ES) cells remains unknown. To investigate the effects of mechanical strain on mouse ES cell fate, we examined the expression of Nanog, which is an essential regulator of self-renewal and pluripotency as well as Nanog-associated intracellular signaling during uniaxial cyclic mechanical strain. The mouse ES cell line, CCE was plated onto elastic membranes, and we applied 10% strain at 0.17 Hz. The expression of Nanog was reduced during ES cell differentiation in response to the withdrawal of leukemia inhibitory factor (LIF); however, two days of cyclic mechanical strain attenuated this reduction of Nanog expression. On the other hand, the cyclic mechanical strain promoted PI3K-Akt signaling, which is reported as an upstream of Nanog transcription. The cyclic mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor wortmannin. Furthermore, cytochalasin D, an inhibitor of actin polymerization, also inhibited the mechanical strain-induced increase in phospho-Akt. These findings imply that mechanical force plays a role in regulating Nanog expression in ES cells through the actin cytoskeleton-PI3K-Akt signaling.

Differentiation-inducing effect of osteoclast microgrooves for the purpose of three-dimensional design of regenerated bone.

In vivo bone remodeling is promoted by the balance between osteoclast and osteoblast activity. Conventional research on bone regeneration has mainly focused on increasing osteoblast activity, with limited studies on the effects of scaffold topography on cell differentiation. Here, we examined the effect of microgroove-patterned substrate with spacings ranging from 1 to 10 µm on the differentiation of rat bone marrow-derived osteoclast precursors. Tartrate-resistant acid phosphatase (TRAP) staining and relative gene expression quantification showed that osteoclast differentiation was enhanced in substrate with 1 µm microgroove spacing compared with that in the other groups. Additionally, the ratio of podosome maturation stages in substrate with 1 µm microgroove spacing exhibited a distinct pattern, which was characterized by an increase in the ratio of belts and rings and a decrease in that of clusters. However, myosin II abolished the effects of topography on osteoclast differentiation. Overall, these showed that the reduction of myosin II tension in the podosome core by an integrin vertical vector increased podosome stability and promoted osteoclast differentiation in substrates with 1 µm microgroove spacing, including that microgroove design plays an important role in scaffolds for bone regeneration. STATEMENT OF SIGNIFICANCE: Reduction of myosin II tension in the podosome core, facilitated by an integrin vertical vector, resulted in an enhanced osteoclast differentiation, concomitant with an increase in podosome stability within 1-µm-spaced microgrooves. These findings are anticipated to serve as valuable indicators for the regulation of osteoclast differentiation through the manipulation of biomaterial surface topography in tissue engineering. Furthermore, this study contributes to the lucidation of the underlying mechanisms governing cellular differentiation by providing insights into the impact of the microtopographical environment.

Effect of Pressure Conditions in Uterine Decellularization Using Hydrostatic Pressure on Structural Protein Preservation.

Uterine regeneration using decellularization scaffolds provides a novel treatment for uterine factor infertility. Decellularized scaffolds require maximal removal of cellular components and minimal damage to the extracellular matrix (ECM). Among many decellularization methods, the hydrostatic pressure (HP) method stands out due to its low cytotoxicity and superior ECM preservation compared to the traditional detergent methods. Conventionally, 980 MPa was utilized in HP decellularization, including the first successful implementation of uterine decellularization previously reported by our team. However, structural protein denaturation caused by exceeding pressure led to a limited regeneration outcome in our previous research. This factor urged the study on the effects of pressure conditions in HP methods on decellularized scaffolds. The authors, therefore, fabricated a decellularized uterine scaffold at varying pressure conditions and evaluated the scaffold qualities from the perspective of cell removal and ECM preservation. The results show that by using lower decellularization pressure conditions of 250 MPa, uterine tissue can be decellularized with more preserved structural protein and mechanical properties, which is considered to be promising for decellularized uterine scaffold fabrication applications.

Cyclic stretch modulates the cell morphology transition under geometrical confinement by covalently immobilized gelatin.

Fibroblasts geometrically confined by photo-immobilized gelatin micropatterns were subjected to cyclic stretch on the silicone elastomer. By using covalently micropatterned surfaces, the cell morphologies such as cell area and length were quantitatively investigated under a cyclic stretch for 20 hours. The mechanical forces did not affect the cell growth but significantly altered the cellular morphology on both non-patterned and micropatterned surfaces. It was found that cells on non-patterns showed increasing cell length and decreasing cell area under the stretch. The width of the strip micropatterns provided a different extent of contact guidance for fibroblasts. The highly extended cells on the 10 µm pattern under static conditions would perform a contraction behavior once treated by cyclic stretch. In contrast, cells with a low extension on the 2 µm pattern kept elongating according to the micropattern under the cyclic stretch. The vertical stretch induced an increase in cell area and length more than the parallel stretch in both the 10 µm and 2 µm patterns. These results provided new insights into cell behaviors under geometrical confinement in a dynamic biomechanical environment and may guide biomaterial design for tissue engineering in the future.

Simultaneous Hydrostatic and Compressive Loading System for Mimicking the Mechanical Environment of Living Cartilage Tissue.

In vivo, articular cartilage tissue is surrounded by a cartilage membrane, and hydrostatic pressure (HP) and compressive strain increase simultaneously with the compressive stress. However, it has been impossible to investigate the effects of simultaneous loading in vitro. In this study, a bioreactor capable of applying compressive stress under HP was developed to reproduce ex vivo the same physical loading environment found in cartilage. First, a HP stimulation unit was constructed to apply a cyclic HP pressure-resistant chamber by controlling a pump and valve. A compression-loading mechanism that can apply compressive stress using an electromagnetic force was implemented in the chamber. The synchronization between the compression and HP units was evaluated, and the stimulation parameters were quantitatively evaluated. Physiological HP and compressive strain were applied to the chondrocytes encapsulated in alginate and gelatin gels after applying high HP at 25 MPa, which induced damage to the chondrocytes. It was found that compressive stimulation increased the expression of genes related to osteoarthritis. Furthermore, the simultaneous application of compressive strain and HP, which is similar to the physiological environment in cartilage, had an inhibitory effect on the expression of genes related to osteoarthritis. HP alone also suppressed the expression of osteoarthritis-related genes. Therefore, the simultaneous hydrostatic and compressive stress-loading device developed to simulate the mechanical environment in vivo may be an important tool for elucidating the mechanisms of disease onset and homeostasis in cartilage.

Translational suppression of atrophic regulators by microRNA-23a integrates resistance to skeletal muscle atrophy.

Muscle atrophy is caused by accelerated protein degradation and occurs in many pathological states. Two muscle-specific ubiquitin ligases, MAFbx/atrogin-1 and muscle RING-finger 1 (MuRF1), are prominently induced during muscle atrophy and mediate atrophy-associated protein degradation. Blocking the expression of these two ubiquitin ligases provides protection against muscle atrophy. Here we report that miR-23a suppresses the translation of both MAFbx/atrogin-1 and MuRF1 in a 3'-UTR-dependent manner. Ectopic expression of miR-23a is sufficient to protect muscles from atrophy in vitro and in vivo. Furthermore, miR-23a transgenic mice showed resistance against glucocorticoid-induced skeletal muscle atrophy. These data suggest that suppression of multiple regulators by a single miRNA can have significant consequences in adult tissues.

Modulation of the long non-coding RNA Mir155hg by high, but not moderate, hydrostatic pressure in cartilage precursor cells.

Osteoarthritis (OA) is the most common joint disease in older adults and is characterized by a gradual degradation of articular cartilage due to decreased cartilage matrix gene expression and increased expression of genes involved in protein degradation, apoptosis and inflammation. Due to the high water content of cartilage, one of the main physical stimuli sensed by chondrocytes is hydrostatic pressure. We previously showed that high pressure above 20 MPa induced gene expression changes in chondrocyte precursor cells similar to what is observed in OA. Micro-RNAs are small non-coding RNAs essential to many physiological and pathological process including OA. As the micro-RNA miR-155 has been found increased in OA chondrocytes, we investigated the effects of high pressure on the expression of the miR-155 host gene Mir155hg. The chondrocyte progenitor cell line ATDC5 was pressurized under hydrostatic pressure up to 25 MPa and the expression of Mir155hg or the resulting micro-RNAs were measured; pharmacological inhibitors were used to identify the signaling pathways involved in the regulation of Mir155hg. We found that Mir155hg is strongly and rapidly up-regulated by high, but not moderate, pressure in chondrocyte progenitor cells. This up-regulation likely involves the membrane channel pannexin-1 and several intracellular signaling molecules including PKC and Src. MiR-155-5p and -3p were also up-regulated by pressure though somewhat later than Mir155hg, and a set of known miR-155-5p target genes, including Ikbke, Smarca4 and Ywhae, was affected by pressure, suggesting that Mir155hg may have important roles in cartilage physiology.

Internal radial perfusion bioreactor promotes decellularization and recellularization of rat uterine tissue.

The advances in infertility treatment technologies such as in vitro fertilization (IVF) help many infertile women to be able to get pregnant. However, these infertility treatments cannot be applied to women who are suffering from absolute uterine factor. Fabrication of functional scaffold in tissue engineering approach is believed to play an important role for uterine regeneration and uterus replacement for treating absolute uterine factor infertility. In this research, we developed an internal radial perfusion bioreactor to promote decellularization and recellularization for fabrication of functional engineered uterine tissue. As a result, the DNA contents of the decellularized uterine tissue with high hydrostatic pressure followed by 7 days internal perfusion washing decreased by 90% compared to native tissue. Collagen and proteoglycan contents in the pressurized uterine tissue with the internal perfusion bioreactor, static (control) and shaking treatment with high hydrostatic pressure showed no significant change compared to the native tissue. The newly developed perfusion bioreactor also enabled to recellularize in the decellularized tissue with statistically significant increase of DNA by 614% compared to non-seeded cell groups. Vimentin and 4',6-diamidino-2-phenylindole (DAPI) was homogeneously expressed in the seeded endometrial stromal cells in the recellularized tissue fabricated using the bioreactor. With the developed internal radial perfusion bioreactor, we are the first group to successfully recellularized uterine tissue in all layers including epithelium, endometrium and myometrium. These results showed that the internal perfusion bioreactor has potential to be utilized for fabrication of functional engineered tissue to promote tissue regeneration.

Correction: Stretching of fibroblast cells on micropatterned gelatin on silicone elastomer.

Correction for 'Stretching of fibroblast cells on micropatterned gelatin on silicone elastomer' by Stefan Müller et al., J. Mater. Chem. B, 2020, 8, 416-425, DOI: .