Skeletal muscle injury involves complex interactions among inflammation, myogenic activation, extracellular matrix remodeling, and functional recovery. However, conventional models often fail to reproduce human injury responses and regenerative processes. Recent advances in biomaterials, biofabrication, and microphysiological systems have enabled more physiologically relevant in vitro muscle platforms. This review summarizes the evolution from two-dimensional cultures to engineered three-dimensional muscle tissues and microphysiological systems, highlighting how scaffold composition, tissue architecture, biophysical stimulation, and multicellular integration influence muscle maturation, injury responses, and repair. We distinguish injury from dysfunction, structural recovery from regeneration and functional recovery, and protective interventions from post-injury repair. We discuss current models of chemical, oxidative, mechanical, electrical, inflammatory, metabolic, and drug-induced muscle injury and dysfunction. A staged evaluation framework integrates structural, molecular, metabolic, and functional readouts across baseline characterization, injury or dysfunction assessment, and subsequent recovery. Finally, we present a decision matrix for aligning injury mechanisms with model design and functional assessment to improve reproducibility, interpretability, and translational relevance.
Recent advancements in constructing human neural cells in vitro have unveiled significant therapeutic potential for treating neurological injuries and neurodegenerative diseases, such as traumatic brain injury, stroke, Parkinson’s disease, and Alzheimer’s disease. The pivotal role of stem cell technologies, particularly induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs), is emphasized not only in modeling these disorders but also in facilitating cellular replacement therapies. The integration of gene editing techniques, such as CRISPR-Cas9, along with innovations in tissue engineering, substantially enhances cell survival and functional integration post-transplantation. Concurrently, emerging technologies like optogenetics and chemogenetics offer precise control over cell behaviors, further promoting effective neural circuit reconstruction. Although preclinical studies have demonstrated promising results, challenges including immune rejection, cellular heterogeneity, and long-term viability remain significant barriers to clinical translation. A comprehensive bibliometric analysis reveals a marked increase in research interest in this field, underscoring the urgent need for sustained, rigorous investigation to optimize transplantation protocols and enhance therapeutic efficacy.
Dental stem cells represent an accessible adult stem cell source with neuronal affinity due to their cranial neural crest origin, yet dental-derived neural-like spheroid models often suffer from heterogeneous differentiation, limited organization, and poor reproducibility. Here, we established a stepwise workflow to induce neurogenic differentiation of dental pulp stem cells (DPSCs) and organize them into spatially controlled, depolarization-responsive neural-like spheroids using a micropatterned culture platform. Seven-day neurogenic induction generated Neuro-DPSCs with marked morphological remodeling, neurite-like processes, and increased expression of neural lineage markers including TUJ1, NSE, MAP2, NeuN, and NEFM. In 3D culture, Neuro-DPSCs self-assembled into spheroidal aggregates, while substrate wettability strongly influenced aggregation and outgrowth. Hydrophobic surfaces promoted spheroid formation but limited long-term maintenance due to free-floating aggregation. To improve spatial control, a micropatterned platform was applied to define adhesive contact points, constrain spheroid positioning, and guide inter-spheroid neurite-like network formation. The resulting spheroids showed enrichment of gene networks associated with neurite-like organization, axon guidance, synaptic vesicle biology, neurotransmitter-related pathways, and receptor signaling, together with KCl-induced depolarization-responsive calcium activity. These findings demonstrate that neurogenic priming combined with micropattern-guided assembly provides a controllable workflow for generating spatially organized neural-like DPSC spheroids.
Building functional tissue in vitro shares a common ceiling. Current models span vascular co-development, trophic interaction, vascular-to-neural sensitization, barrier function, and even functional neuromuscular coupling, yet none has reconstituted functional neurovascular (NV) coupling. We argue that, on one specific axis, the dental pulp is the most direct single-source substrate for dissecting this coupling. A single, clinically accessible neural-crest tissue supplies the perivascular compartment together with glial and neurotrophic support for the neural compartment, in a volume amenable to single-cell methods and one in which an activity-dependent NV interaction is already measurable in vivo. This is a competence claim, not a demonstration; no in-vitro pulp model has closed the loop, leaving unresolved the clinical divergence between reversible and irreversible pulpitis. The review identifies the clinical, experimental, and mechanistic questions a pulp model could resolve, yielding a transferable method for other NV-coupled tissues.
Conventional two-dimensional (2D) cell culture relies on a flat adhesive substrate, which not only restricts cellular three-dimensional growth space but also lacks the complex interactions and multiple signaling pathways between cells and the extracellular matrix (ECM), resulting in significant differences in the survival state of cells cultured in vitro compared to that in their natural three-dimensional (3D) microenvironment in vivo, presenting inherent constraints for investigating complex biological questions in vitro. In this study, we introduce the DNA-inspired biomimetic nanomaterial, Janus base nanotubes (JBNTs), designed to simulate the long collagen fibers in natural tissues to promote 3D spheroid formation within a conventional 2D system. Under physiological conditions, the lysine groups in JBNTs confer positive charges to JBNTs, allowing negatively charged cells and proteins to adhere to the material surface via electrostatic interactions, thereby providing rapid anchorage sites for cells and facilitating spontaneous cell aggregation into 3D structures. We believe that this engineered microenvironment triggers cellular self-regulation, upregulating the expression of cell membrane receptor proteins and key extracellular matrix components. Cells engage with these microenvironmental elements to facilitate the formation of stable 3D cellular spheroids. Additionally, the JBNTs-induced spheroids exhibit a cell viability exceeding 90% and can be readily dissociated into single cells using trypsin for subculture, demonstrating excellent cytocompatibility and high cell reusability, which are advantageous for constructing 3D in vitro models. This study presents an innovative approach for constructing 3D microenvironments, offering new pathways for guiding cellular behavior and advancing 3D cell culture techniques.
