Tracing Immunological Interaction in Trimethylamine N-Oxide Hydrogel-Derived Zwitterionic Microenvironment During Promoted Diabetic Wound Regeneration.

The diabetic wound healing is challenging due to the sabotaged delicate balance of immune regulation via an undetermined pathophysiological mechanism, so it is crucial to decipher multicellular signatures underlying diabetic wound healing and seek therapeutic strategies. Here, this work develops a strategy using novel trimethylamine N-oxide (TMAO)-derived zwitterionic hydrogel to promote diabetic wound healing, and explore the multi-cellular ecosystem around zwitterionic hydrogel, mapping out an overview of different cells in the zwitterionic microenvironment by single-cell RNA sequencing. The diverse cellular heterogeneity is revealed, highlighting the critical role of macrophage and neutrophils in managing diabetic wound healing. It is found that polyzwitterionic hydrogel can upregulate Ccl3+ macrophages and downregulate S100a9+ neutrophils and facilitate their interactions compared with polyanionic and polycationic hydrogels, validating the underlying effect of zwitterionic microenvironment on the activation of adaptive immune system. Moreover, zwitterionic hydrogel inhibits the formation of neutrophil extracellular traps (NETs) and promotes angiogenesis, thus improving diabetic wound healing. These findings expand the horizons of the sophisticated orchestration of immune systems in zwitterion-directed diabetic wound repair and uncover new strategies of novel immunoregulatory biomaterials.

Advances in Material-Based Strategies for Diabetic Bone Regeneration.

Increased bone fragility and poor bone healing are common and serious complications of diabetes, especially in elderly patients. Long-term hyperglycemia often leads to serious infection and nonunion. Diabetes brings changes to bone microenvironment, including imbalanced immunity, disorder of macrophage polarization, deterioration of microvascular system, excessive advanced glycation end products, reactive oxygen species (ROS), local high levels of glucose, and great tendency to infection. The main traditional managements of diabetic bone involve oral medication and systematic drug administration, which exhibit limited therapeutic efficacy and accompanied side effects. Materials-based strategies have recently been potential alternatives for the treatment of diabetic bone diseases. In this review, we highlight the main material-based strategies for diabetic bone repair deficiency, including regulation of macrophages, elimination of excessive ROS, and resistance to bacterial infection. We also describe the future therapeutic designing approaches for smart biomaterials for diabetic bone regeneration, which would provide new ideas to protect bone health in patients with diabetes.

Integrative Analysis Reveals the Diverse Effects of 3D Stiffness upon Stem Cell Fate.

The origin of life and native tissue development are dependent on the heterogeneity of pluripotent stem cells. Bone marrow mesenchymal stem cells (BMMSCs) are located in a complicated niche with variable matrix stiffnesses, resulting in divergent stem cell fates. However, how stiffness drives stem cell fate remains unknown. For this study, we performed whole-gene transcriptomics and precise untargeted metabolomics sequencing to elucidate the complex interaction network of stem cell transcriptional and metabolic signals in extracellular matrices (ECMs) with different stiffnesses, and we propose a potential mechanism involved in stem cell fate decision. In a stiff (39~45 kPa) ECM, biosynthesis of aminoacyl-tRNA was up-regulated, and increased osteogenesis was also observed. In a soft (7~10 kPa) ECM, biosynthesis of unsaturated fatty acids and deposition of glycosaminoglycans were increased, accompanied by enhanced adipogenic/chondrogenic differentiation of BMMSCs. In addition, a panel of genes responding to the stiffness of the ECM were validated in vitro, mapping out the key signaling network that regulates stem cells' fate decisions. This finding of "stiffness-dependent manipulation of stem cell fate" provides a novel molecular biological basis for development of potential therapeutic targets within tissue engineering, from both a cellular metabolic and a biomechanical perspective.

Caspase-3/GSDME-mediated pyroptosis leads to osteogenic dysfunction of osteoblast-like cells.

OBJECTIVE: Cell pyroptosis is implicated in progressive bone loss in dental inflammatory diseases. We induced caspase-3/Gasdermin E (GSDME)-mediated pyroptosis in osteoblast-like cells and evaluated the effects on osteogenesis. MATERIALS AND METHODS: Osteoblast-like cells were treated with various concentrations of sodium butyrate (NaB) to identify the most appropriate for inducing caspase-3/GSDME-mediated pyroptosis. Cells were divided into control, NaB and NaB+Ac-DEVD-CHO (specific caspase-3 inhibitor) groups. Pyroptosis level was evaluated by immunofluorescence, morphological observation, flow cytometry, lactate dehydrogenase (LDH) release assays, mRNA and protein levels of pyroptosis-related markers. Then, inflammation level, osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) expression and osteogenic function were detected. RESULTS: Treatment with 10 mM NaB increased caspase-3 expression, GSDME cleavage, LDH release and the number of pyroptotic cells, with morphologic changes, indicating GSDME-mediated pyroptosis induction. The pyroptosis-related changes were abolished by caspase-3 inhibition. Caspase-3/GSDME-mediated pyroptosis triggered the expression of inflammatory cytokines and RANKL, downregulated alkaline phosphatase (ALP) activity, mineralisation level, mRNA and protein levels of multiple osteogenic markers. These effects were partly reversed by Ac-DEVD-CHO. CONCLUSION: Caspase-3/GSDME-mediated pyroptosis induced by NaB activated the inflammatory response, reduced osteogenic differentiation and disturbed OPG/RANKL axis, leading to osteogenic dysfunction in osteoblast-like cells.

Metformin can mitigate skeletal dysplasia caused by Pck2 deficiency.

As an important enzyme for gluconeogenesis, mitochondrial phosphoenolpyruvate carboxykinase (PCK2) has further complex functions beyond regulation of glucose metabolism. Here, we report that conditional knockout of Pck2 in osteoblasts results in a pathological phenotype manifested as craniofacial malformation, long bone loss, and marrow adipocyte accumulation. Ablation of Pck2 alters the metabolic pathways of developing bone, particularly fatty acid metabolism. However, metformin treatment can mitigate skeletal dysplasia of embryonic and postnatal heterozygous knockout mice, at least partly via the AMPK signaling pathway. Collectively, these data illustrate that PCK2 is pivotal for bone development and metabolic homeostasis, and suggest that regulation of metformin-mediated signaling could provide a novel and practical strategy for treating metabolic skeletal dysfunction.

Advances of Engineered Hydrogel Organoids within the Stem Cell Field: A Systematic Review.

Organoids are novel in vitro cell culture models that enable stem cells (including pluripotent stem cells and adult stem cells) to grow and undergo self-organization within a three-dimensional microenvironment during the process of differentiation into target tissues. Such miniature structures not only recapitulate the histological and genetic characteristics of organs in vivo, but also form tissues with the capacity for self-renewal and further differentiation. Recent advances in biomaterial technology, particularly hydrogels, have provided opportunities to improve organoid cultures; by closely integrating the mechanical and chemical properties of the extracellular matrix microenvironment, with novel synthetic materials and stem cell biology. This systematic review critically examines recent advances in various strategies and techniques utilized for stem-cell-derived organoid culture, with particular emphasis on the application potential of hydrogel technology in organoid culture. We hope this will give a better understanding of organoid cultures for modelling diseases and tissue engineering applications.

The PCK2-glycolysis axis assists three-dimensional-stiffness maintaining stem cell osteogenesis.

Understanding mechanisms underlying the heterogeneity of multipotent stem cells offers invaluable insights into biogenesis and tissue development. Extracellular matrix (ECM) stiffness has been acknowledged as a crucial factor regulating stem cell fate. However, how cells sense stiffness cues and adapt their metabolism activity is still unknown. Here we report the novel role of mitochondrial phosphoenolpyruvate carboxykinase (PCK2) in enhancing osteogenesis in 3D ECM via glycolysis. We experimentally mimicked the physical characteristics of 3D trabeculae network of normal and osteoporotic bone with different microstructure and stiffness, observing that PCK2 promotes osteogenesis in 3D ECM with tunable stiffness in vitro and in vivo. Mechanistically, PCK2 enhances the rate-limiting metabolic enzyme pallet isoform phosphofructokinase (PFKP) in 3D ECM, and further activates AKT/extracellular signal-regulated kinase 1/2 (ERK1/2) cascades, which directly regulates osteogenic differentiation of MSCs. Collectively, our findings implicate an intricate crosstalk between cell mechanics and metabolism, and provide new perspectives for strategies of osteoporosis.