Cellular and molecular bioengineering最新文献

Introduction: Approximately 15% of dilated cardiomyopathy (DCM) cases are associated with Bcl2-associated athanogene 3 (BAG3) gene mutations, which play a crucial role in myofilament organization and contractile behavior. Previous studies have highlighted the role of dynamic mechanical stress in myofibril alignment in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs). In this study, we employed thermo-responsive shape memory polymers (SMPs) to mimic the dynamic mechanical environment of the extracellular matrix (ECM) and investigated their impact on myofibril assembly in healthy wild-type (WT) and BAG3 knockout (BAG3-/-) hiPSC-CMs.

Methods: We synthesized Tert-Butyl Acrylate (TBA) and Butyl acrylate (BA)-based SMP substrate. hiPSC-CMs were cultured on 30oC on 40% strained (dynamic) and un-strain (static) SMPs for two days before proceeding with polymer recovery at 37oC. Myofibril components of BAG3 knock-out (KO) and WT CMs were evaluated by immunocytochemistry fluorescent images at 5 hours and 24 hours after triggering the shape changes of the SMP substrate. We quantified Z-lines and M-lines of hiPSC-CMs to evaluate sarcomere remodeling on static and dynamic substrates.

Results: Our findings revealed that BAG3-/- hiPSC-CMs exhibited persistent Z-line disruption in sarcomeres compared with WT hiPSC-CMs, but M-line structures were less sensitive to mechanical stress at 5 hours, highlighted a temporal distinction in the assembly and regulation of Z-lines over M-lines. While no significant changes were detected at 5 hours, BAG3-/- CMs exhibited similar impairments in M-line organization as seen with Z-lines. These findings suggest that M-lines in BAG3-/- CMs display heightened sensitivity to dynamic mechanical actuation, but this phenotype emerges only after prolonged culture.

Conclusions: This study highlights the interplay between genetic deficiency and mechanical stress to facilitate disease progression in BAG3-associated DCM.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00880-z.

Background: Rotator cuff injuries are the most common type of tendinopathies affecting up to 10% of young adults and more than 60% of the elderly. Tendons have notoriously limited regenerative capacity which is attributed to their low vascularisation and low cell-to-tissue ratio. That leads to an inefficient repair process resulting in fibrotic scar tissue with poorer mechanical properties. Recent advances in tissue engineering and biofabrication techniques have been anticipated with great hope in the field of regenerative medicine.

Methods: In this review, we discuss the insights gained from immune-based strategies, 3D biofabrication, and in vivo testing in the context of rotator cuff repair. Particular emphasis is placed on in vivo studies that bridge the gap between laboratory innovation and clinical translation, outlining promising avenues for future therapeutic development.

Results: Regardless of the huge progress in in vitro modelling and in vivo healing of RCTs in animals, clinical translation has not yet succeeded because mechanical loading, and chronic inflammation in humans are hard to recapitulate. Biologic variability, regulatory complexity, and poor reproducibility also slow down translation to the clinic.

Conclusion: With a number of encouraging results so far, multidisciplinary research will continue to elucidate the complex biological processes in terms of (1) immune and tendon cell engagement and modulation, natural matrix deposition and remodelling; (2) material mimicry regarding topography, anisotropic, gradient continuity, biomechanical properties; and (3) in vivo behaviour in structural reconstruction and functional performance in the long-term. Such integrative approach is essential to overcome today's limitations in RC repair and to outline next-generation strategies to achieve improved clinical outcomes.

Purpose: Type 1 diabetes (T1D) is an autoimmune disorder that causes selected destruction of insulin-secreting pancreatic beta cells leading to insulin deficiency, hyperglycemia, and long-term complications. T1D has no cure and is primarily self-managed with blood sugar monitoring and exogenous insulin injections, which do not enable proper metabolic control and decreases patient's and caregivers' quality of life. Beta cell replacement through islet transplantation could cure T1D if current limitations such as the need for chronic systemic immunosuppression to prevent rejection and recurrence of autoimmunity are addressed. A potential new treatment addressing these limitations is based on transplantation of donor islets encapsulated in hydrogels with suitable and stable permselectivity and mechanical properties. Specifically, these hydrogel coatings must be (1) permeable to nutrients, insulin and glucose, necessary for coated cell viability and functionality, but impermeable to antibodies, to enable immune isolation, and (2) resistant to degradation, over time.

Methods: This study uses Fluorescence Recovery after Photobleaching (FRAP) and Atomic Force Microscopy (AFM) to determine the diffusion coefficient and Young's modulus of elasticity of individual model beads and primary and pseudoislets conformally coated with polyethylene glycol (PEG) over an extended period of time to evaluate the stability and viability of this novel therapeutic method for beta cell replacement without immunosuppression in T1D.

Results: The conformal hydrogel coatings remained functional and did not deteriorate over the 100-day time period, showing a promising stability to enable long-term immunoisolation of encapsulated islets.

Conclusions: This report demonstrated a novel measurement technique capable of assessing the mechanical and transport properties of individually coated samples, giving a more precise characterization of inherent variabilities within a sample population. Moreover, the approach is adaptable to other therapeutic cell clusters and organoids, supporting broader applications in cell transplantation therapies and offering a robust method for batch release validation in clinical applications.

Purpose: The periodontal ligament (PDL), which contains osteoblasts, is a unique connective tissue that resists mineralization despite being located between mineralized cementum and alveolar bone. The role of physiological mechanical stress, particularly the cyclic hydrostatic pressure (HP) generated during mastication, in regulating osteogenic potential remains poorly understood. This study aimed to compare the effects of static and cyclic HP on osteogenic differentiation and to elucidate the underlying mechanisms.

Methods: A custom-built pressure-loading device was developed to apply either static or cyclic HP to MC3T3-E1 pre-osteoblastic cells. These cells, which are widely used as models for osteogenic differentiation, were subjected to conditions mimicking PDL-relevant mechanical environments. Osteogenic differentiation was evaluated using alkaline phosphatase (ALP) activity assays. Actomyosin contractility was assessed through immunofluorescence staining of focal adhesions using vinculin, along with phosphorylated myosin light chain (p-MLC) to assess myosin activity. To examine the role of actomyosin contractility, cells were treated with blebbistatin, a myosin II inhibitor.

Results: Compared to static HP (5 MPa), cyclic HP (5 MPa, 0.2 Hz) more strongly inhibited ALP activity. HP stimulation enhanced both focal adhesion formation and p-MLC levels, with the latter showing a markedly greater increase under cyclic HP than static HP. Notably, inhibition of p-MLC phosphorylation with 10 μM blebbistatin attenuated these HP-induced effects, indicating that actomyosin contractility plays a critical role in mediating the suppression of osteogenic differentiation.

Conclusions: Cyclic HP more effectively inhibited osteogenic differentiation than static HP, likely through enhanced actomyosin contractility associated with increased p-MLC expression in MC3T3-E1 pre-osteoblastic cells.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00875-w.

Purpose: In solid tumors, cancer cells adapt to hypoxic and nutrient deprived environments to support malignant progression. This study examined whether hypoxic and low glucose conditions enhance malignant behaviors more strongly in highly migratory MG63-R10 cells, which are derived from osteosarcoma MG-63 cells, compared to parental MG-63 cells, and further investigated whether lysophosphatidic acid (LPA) receptor signaling regulates this adaptation.

Methods: MG63-R10 and MG-63 cells were cultured under hypoxic (1% O₂) or normoxic (21% O₂) conditions in media containing 4500, 2000, or 1000 mg/L glucose. LPA receptor expression was analyzed by quantitative real time RT-PCR. Cell growth and motility were assessed, and pharmacological modulators AM966 (LPA₁ antagonist), GRI-977143 (LPA₂ agonist), and (2S)-OMPT (LPA₃ agonist) were used to evaluate receptor specific effects on cell growth and motility.

Results: Under 1% O₂, LPAR2 expression increased in MG63-R10 cells, while LPAR1 and LPAR3 expression decreased. MG63-R10 cells showed lower growth than MG-63 cells under 21% O₂, but higher growth under hypoxia. MG63-R10 cell motility was higher than that of MG-63 cells at 21% O₂ and was further enhanced under 1% O₂. AM966 and GRI-977143 increased MG63-R10 motility, whereas (2S)-OMPT suppressed it. MG63-R10 motility significantly increased in 2000 and 1000 mg/L glucose, whereas MG-63 motility remained unchanged across glucose levels.

Conclusion: These results suggest that, compared to parental MG-63 cells, highly migratory osteosarcoma MG63-R10 cells adapt their malignant cellular functions to hypoxic and low-glucose conditions through LPA receptor signaling, highlighting this pathway as a potential therapeutic target in aggressive osteosarcomas.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00873-y.

Introduction: G protein-coupled estrogen receptor (GPER) is a heptahelix estrogen-binding G protein-coupled receptor, and a potential therapeutic target for estrogen-related cancers and diseases. Recently, GPER has been recognized as a key mechano-regulator, but the effects on cell adhesion, spreading, morphology, migration, and differentiation are inconsistent or even contradicting in literature, due to the variations across cell lines and complex crosstalks with ER, and non-genomic actions of other hormones. Here, we focus on investigating the GPER effect on mesenchymal stem cell (MSC) mechanotransduction and differentiation.

Methods: MSCs treated by synthetic agonist G1 and untreated cells were cultured on fibronectin-coated surfaces. Cell migration was studied by both chemokinesis and chemotaxis experiments. After two-week differentiation, MSC adipogenesis and osteogenesis were evaluated by staining lipid droplets in adipocytes with Oil-Red O and alkaline phosphatase in osteocytes with NBT/BCIP, respectively. In particular, since micropatterns have been widely used to mimic extracellular matrix (ECM) cues, modulate MSC mechanotransduction and differentiation, we investigate the GPER effect on both single-cell and sub-cellular fibronectin microline patterns prepared by microcontact printing.

Results: GPER activation regulates cytoskeleton organization, with reduced cell polarization, thinner ventral stress fibers, and reduced RhoA signaling; reduces MSC migration speed; significantly promotes osteogenesis and inhibits adipogenesis. Cell elongation by micropatterns and the reduction of cell polarization by GPER coexist in a sophisticated interplay.

Conclusions: GPER directly mediates MSC mechanotransduction by RhoA inactivation, while its sustained effect on MSC differentiation promotes osteogenesis and inhibits adipogenesis despite reduced cell polarization and tension, suggesting potential mechanisms other than RhoA signaling. Our findings pave the way towards a deep understanding of GPER's role and its interplay with ECM cues in mechanotransduction and differentiation, which will be important for developing GPER as a new therapeutic target, as well as considering its important effects in stem cell therapies and hormonal therapies.

Purpose: Articular chondrocytes synthesize and maintain the avascular and aneural articular cartilage. In vivo these cells are surrounded by a 3D pericellular matrix (PCM) containing predominantly collagen VI. The PCM protects chondrocytes and facilitates mechanotransduction. PCM stiffness is critical in transmitting biomechanical signals to chondrocytes. Various culture systems with different hydrogels are used to encapsulate chondrocytes for 3D culture, but many lack either the PCM or the in vivo stiffness of the cartilage matrix. This study aimed at establishing a culture system to investigate (a) if chondrocytes cultured in alginate will develop a PCM and (b) study mechanotransduction via metabolic changes induced in 3D agarose-embedded chondrocytes upon mechanical stimulation.

Methods: We cultured primary human and bovine chondrocytes in monolayers or as alginate encapsulated cells in media containing sodium L-ascorbate. PCM expression was analyzed by immunofluorescence and western blots. We further characterized the response of chondrocytes embedded in physiologically stiff agarose to dynamic compression through metabolomic profiling.

Results: We found that primary human and bovine chondrocytes, when cultured in alginate beads with addition of sodium L-ascorbate for 7 days, had a pronounced PCM, retained their phenotype, and synthesized both collagens VI and II. This novel culture system enables alginate-encapsulated chondrocytes to develop a robust PCM thereby creating a model system to study mechanotransduction in the presence of an endogenous PCM. We also observed distinct compression-induced changes in metabolomic profiles between the monolayer-agarose and alginate-released agarose-embedded chondrocytes indicating physiological changes in cell metabolism.

Conclusion: These data show that 3D preculture of chondrocytes in alginate before encapsulation in physiologically stiff agarose leads to pronounced development of pericellular matrix that is sustained in the presence of ascorbate. This model can be useful in studying the mechanism by which chondrocytes respond to cyclical compression and other types of loading simulating in vivo physiological conditions.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00872-z.