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.
Osteoarthritis (OA) is a chronic, progressive degenerative joint disease defined by the degradation of articular cartilage, with pathological involvement of periarticular tissues including the synovium and subchondral bone. Existing conservative interventions for OA are limited to symptomatic relief and cannot reverse established cartilage degeneration, underscoring the critical unmet need to address prevailing clinical treatment bottlenecks. Hydrogel microspheres, as an emerging delivery platform integrating drug delivery capabilities with tissue engineering scaffold functions, hold substantial promise for targeted OA therapy and cartilage repair. Their core beneficial properties include minimally invasive injectability, favorable biocompatibility, sustained drug release capacity, and intelligent responsiveness. This review systematically explores the application of hydrogel microspheres in the treatment of OA and provides a detailed summary of material systems and preparation techniques—covering natural materials, synthetic polymers, and composite/hybrid materials, as well as processes such as emulsion cross-linking, microfluidics, electrospray, photolithography, and 3D printing. This paper places particular emphasis on the modulation of mechanical properties, smart responsive release mechanisms, and strategies to enhance bioactivity. Furthermore, it summarizes various therapeutic strategies supported by hydrogel microspheres, including the precise controlled release of small-molecule drugs, the regulation of endogenous stem cell recruitment and directed differentiation, the targeted delivery of gene therapy drugs, and synergistic treatment modalities. By synthesizing core research advances in this field in recent years and elucidating key technological development directions, this review provides a comprehensive reference for advancing the translation of hydrogel microspheres from basic research to clinical applications in the management of OA.
Introducing pre-vascularization in functional full-thickness skin equivalents is critical to improve inosculation upon implantation. This study introduces a mechanically reinforced, bi-layered human skin model comprised of epidermis and pre-vascularized dermis. By combining a collagen type I hydrogel with an electrospun PCL/PLCL nanofibrous membrane coated with fibrin provisional matrix, we developed a composite scaffold that prevents the cell-induced hydrogel contraction and replicates the mechanical heterogeneity of native skin. The model utilizes a simultaneous tri-culture of human endothelial cells, adipose tissue-derived stromal cells, and keratinocytes. A custom 3D-printed, adjustable insert for cultivation at an air-liquid interface facilitates stratification of the dermal and epidermal layers. Our results demonstrate that adipose tissue-derived stromal cells effectively function as pericyte-like stabilizers, driving the formation of dermal capillary-like networks in 3D. This reinforced, pre-vascularized model provides a tool for improving tissue substitutes in vitro and in vivo , and a biomimetic platform for studying dermal-epidermal crosstalk and microvascular formation.
Exogenous electrical stimulation accelerates cutaneous wound closure, but its effect on pathological scarring remains poorly defined. This review reframes the field around scar fate rather than closure speed and applies a three-tier endpoint framework that distinguishes wound closure, tissue-quality surrogates, and validated scar outcomes. We examine endogenous bioelectric signaling and evaluate how electrical stimulation influences electrotaxis, calcium-dependent myofibroblast activation, TGF beta signaling, extracellular matrix remodeling, and immune regulation. Current evidence shows that some platforms reduce collagen I, alpha smooth muscle actin, and myofibroblast activity, whereas other stimulation conditions enhance profibrotic signaling. These divergent effects are strongly dependent on dose, waveform, exposure time, cell source, and biological context. However, validated scar scales, mature scar outcomes, and rigorous fibroproliferative models remain rarely used in current experimental studies. We therefore propose a dosimetry and reporting framework to guide scar-oriented electroceutical design and clinical translation.
Nasoseptal chondrocytes (NCs) are a viable cell source for engineering autologous hyaline cartilage grafts for nasal reconstruction or repairing articular cartilage lesions. Fibroblast growth factor (FGF)-2 and transforming growth factor (TGF)-β1 are commonly used to optimize cell yield and chondrogenic capacity during in vitro culture. However, the comprehensive molecular effects of these factors have yet to be elucidated. Leveraging RNA sequencing, this study characterizes the individual and interactive effects of FGF-2 and TGF-β1 on the NC monolayer transcriptome and subsequent 3D cartilage microtissue qualities, revealing that dual-primed NC transcriptomes converge on an FGF-2-like state. Altogether, our analysis of growth factor interactions at the molecular level provides novel, foundational insights into the regulation of intracellular signaling pathways essential to advancing tissue engineering strategies.
Cryopreservation is essential for mesenchymal stem cell banking, but its effects on adipose-derived stem cell (ADSC) function remain debated. This study evaluated an optimised cryopreservation protocol for goat ADSCs using phenotypic, functional, mitochondrial-associated, conditioned-medium, proteomic, and wound-healing assays. Cryopreserved ADSCs retained morphology, viability, clonogenicity, proliferation, adherence, immunophenotype, and trilineage differentiation, comparable to those of fresh ADSCs. Post-thaw cells showed increased MitoTracker Green signal, JC-1 red/green ratio, ATP content, relative mtDNA abundance, and SOD2-associated immunofluorescence, together with lower H 2 O 2 -induced ROS- and autophagy-associated fluorescence. Conditioned-medium proteomics suggested exploratory shifts toward extracellular matrix-, anti-protease-, and redox-associated proteins. Functionally, cryopreserved ADSCs maintained inflammatory cue-directed migration, modulated macrophage surface marker expression, and reduced oxidative stress-associated injury readouts in goat dermal fibroblasts. In a goat excisional wound model, ADSC treatment improved wound-closure kinetics and supported histological repair. Overall, optimised cryopreservation preserved the functional competence of goat ADSCs and was associated with stress-adapted post-thaw features.
Background In vitro models of the blood-spinal cord barrier (BSCB) are widely utilized for developing therapeutics against neurological diseases such as spinal cord injury (SCI). However, high variability among existing in vitro BSCB models severely limits their predictive power for preclinical research, highlighting the need for a comprehensive synthesis of current model characteristics and performance. Methods We first reviewed in vitro BSCB models under SCI conditions and conducted a comprehensive meta-analysis of mainstream Transwell-based models. The synthesis systematically analyzed critical factors influencing model performance, including cell types, disease modeling strategies ( e.g., hypoxic culture, inflammatory stimuli, oxidative stress), and the inclusion of biomaterial matrices. The meta-analysis quantified differences in key BSCB properties, including permeability, transendothelial electrical resistance (TEER), and junctional/inflammatory protein expression, across healthy, diseased, and treatment contexts. Results The synthesis identified cell composition, disease induction methods, and biomaterial matrix inclusion as core factors determining the ability of in vitro BSCB models to replicate physiological and pathological characteristics. Meta-analysis results revealed significant quantitative differences among three types of BSCB models. Specifically, disease models exhibited consistently higher permeability (measured via FITC-dextran) and lower mean TEER values (97.94 Ω·cm 2 ) compared to healthy models (183.73 Ω·cm 2 ) and treatment groups (146.28 Ω·cm 2 ). Additionally, co-culturing BSCB endothelial cells with glial cells or pericytes was demonstrated to significantly enhance the physiological relevance of the models. Conclusions This review consolidates critical insights into in vitro BSCB model design and performance, providing a clear framework for developing more accurate and reliable models. These findings will facilitate the development of effective therapeutic interventions capable of crossing the BSCB, addressing a key bottleneck in mechanistic research and therapeutic development for treating SCI and other neurological diseases.
Chronic kidney disease (CKD), driven largely by renal fibrosis, lacks effective therapies due to the limited predictive capacity of existing preclinical models. To address this, we developed a human tubuloid-on-a-chip model integrating tubuloids, endothelial cells, and immune cells within a microfluidic system to recapitulate the key pathophysiology of renal fibrosis. Induction of fibrosis with TGF-β1 in this system recapitulated key pathological features, including extracellular matrix deposition, epithelial-mesenchymal transition, and loss of epithelial polarity. Functional assessments revealed impaired tubular reabsorption, including reduced albumin uptake and glucose transport, alongside elevated oxidative stress, mirroring clinical observations in CKD patients. The model’s pharmacological relevance was validated by the therapeutic effects of nintedanib, which attenuated fibrotic phenotypes. Taken together, this tubuloid-on-a-chip platform demonstrates the potential to model complex fibrotic pathologies in vitro and may serve as a useful tool for CKD research and anti-fibrotic drug development, potentially accelerating therapeutic discovery for renal fibrosis.
Tissue engineering (TE) remains a cornerstone of regenerative medicine, aiming to bypass the limitation of organ transplantation through the fabrication of functional tissue substitutes. Traditionally, TE has followed two primary paradigms: the top-down approach, utilising single cells seeded on a scaffold, and the bottom-up approach, employing cell spheroids as building blocks. While top-down offers architectural and structural control, bottom-up promotes self-assembly, native-like extracellular matrix deposition, and intercellular signalling. However, modern techniques increasingly blur this dichotomy, creating a spectrum of cell-based fabrication approaches. This review evaluates the diverse approaches across four major tissue classes: epithelial (pancreas as an example), connective (cartilage), muscle (heart), and nervous (brain) tissues. For each tissue, we examine notable studies to evaluate how different assembly methods recapitulate native tissue properties. By reviewing case studies across diverse tissue types, we highlight the relative strengths and limitations of various fabrication strategies. Although this review is limited by a selective cross-section of literature within a rapidly advancing technological landscape, it provides critical insights into optimising next-generation tissue constructs. In conclusion, we posit that there is no universal fabrication strategy; rather, the future of the field depends on tailoring approaches along this single-cell-to-spheroid spectrum based on the specific architectural and functional demands of the target tissue.
Emerging evidence suggests that the extracellular matrix (ECM) possesses a “memory” that can influence cell physiology and recellularization outcomes. Understanding this memory is essential to allow the use of bioengineered organs derived from diseased ECM, offering a solution to the critical organ shortage. To address this, we investigated whether the memory of ECM derived from metabolic dysfunction-associated steatohepatitis (MASH) livers impacts disease establishment following transplantation. Partial orthotopic transplantation of decellularized MASH-derived ECM was performed in control and MASH recipients. Histological analysis confirmed complete recellularization; however, molecular and metabolomic analyses revealed that MASH ECM stimulated de novo lipogenesis and fibrogenesis, inducing impaired lipid oxidation and mitochondrial dysfunction, which contributed to disease progression by promoting altered lipid turnover and inflammatory signalling. In vitro analysis revealed that MASH-ECM disrupted calcium signalling and promoted the maintenance of a pathological phenotype. Although derived from diseased livers, human ECM can promote cell survival and permissiveness. In conclusion, diseased ECM memory impacts cell physiology, suggesting that the scaffold can drive disease progression independently of the cellular environment. Thus, further studies are needed to develop strategies capable of reversing the pathological memory associated with ECM to allow its use in liver transplantation.
Bone healing is a complex and well-organized process, regulated by various factors ranging from growth factors to hormones, cytokines, mechanical stimuli, and aging. Recently, numerous techniques have been devised to efficiently induce the differentiation of human induced pluripotent stem cells (hiPSCs) to osteoblasts. However, enhancing the efficiency of osteoblast differentiation remains a challenge. Thus, we induced the differentiation of hiPSCs to mesodermal cells through Wnt/BMP signaling based on the generation of hiPSCs. After successful generation of hiPSCs, we induced the differentiation of mesodermal cells to osteoblasts. The results revealed that the runt-related transcription factor 2 ( RUNX2 )-encoding gene was upregulated from the early differentiation stage; hence, the expression of the mature osteoblast marker was higher compared to that observed in other differentiation stages. In addition, the deposition of substrates in mature bones was observed. The results were confirmed via real-time PCR, Alizarin Red Staining, and Von Kossa staining. The coactivation of Wnt and BMP signaling was shown to rapidly and effectively promote the differentiation of osteoblasts. The findings of this study will provide a foundation for future studies on the mechanism of osteoblast development, as well as the biological and pathological investigations of drug screening and bone regeneration tracing.
Intervertebral disc degeneration (IVDD) is a multifactorial and clinically heterogeneous condition involving disturbances in oxidative balance, mitochondrial homeostasis, inflammatory signaling, susceptibility to regulated cell death, extracellular matrix integrity, and biomechanical function. Although biomaterial-based strategies have shown significant promise in preclinical studies, their clinical translation remains hindered by pathological heterogeneity, insufficient mechanistic validation, and frequent reliance on simplified experimental models. These models often fail to fully replicate the chronic, mechanically complex, and clinically diverse nature of human disc degeneration. In addition, current biomaterial strategies are commonly classified according to material composition or isolated molecular targets, potentially obscuring their functional objectives and limiting mechanistic comparisons across therapeutic platforms. This review presents a mechanism-guided framework for interpreting biomaterial interventions in IVDD. Biomaterial strategies are discussed based on their primary pathological targets and therapeutic intentions, including inflammatory regulation, restoration of redox homeostasis, mitochondrial protection, ferroptosis modulation, extracellular matrix preservation, and multifunctional microenvironment-responsive interventions. This framework acknowledges that these pathological processes are interconnected and vary in their relative dominance across clinical and pathological contexts and patient populations. We further analyze current biomaterial strategies according to mechanistic intervention layers, including upstream sensing and initiation-level control, restraint of intracellular amplification, organelle stabilization, regulation of execution checkpoints, and integration with higher-order structural organization. This layered perspective emphasizes that the efficacy of biomaterials depends not only on their composition but also on the pathological context, depth of regulatory influence, mechanistic specificity, and compatibility across intervention layers. Collectively, this review provides an integrated, mechanism-oriented framework for biomaterial design, preclinical evaluation, and translational development in IVDD therapy.
While organoids hold immense promise as in vitro three-dimensional (3D) models, their translational utility is fundamentally constrained by passive diffusion limits (>800 μm), which inevitably trigger necrotic core formation and stochastic structural heterogeneity. This review elucidates how bioprinting shatters these physical bottlenecks by executing a paradigm shift toward spatiotemporal determinism. We systematically decode the mechanobiological evolution of bioinks—charting the transition from exogenous static matrices, which now function as temporal controllers via tunable stress relaxation to direct YAP/TAZ mechanotransduction, to the emerging paradigm of “engineerable living bioinks” driven by endogenous, cadherin-mediated fluid-to-solid jamming transitions. Furthermore, we critically evaluate frontier spatial strategies, highlighting how sacrificial networks and deterministic multi-material assembly establish active convective infrastructures and precise biophysical boundary conditions. By enforcing this rigorous baseline, these technologies definitively rectify pharmacokinetic/pharmacodynamic (PK/PD) distortions—eradicating false-positive noise in high-throughput screening and bridging the post-implantation mass transport vacuum—elevating organoids from stochastic clusters to highly predictive pathophysiological macro-models. Ultimately, we posit that transitioning from isolated morphological fabrication to resolving the inherent systemic metabolic paradoxes of multi-lineage integration is the absolute prerequisite for clinical translation.
Osteogenesis is the process by which mesenchymal stem/stromal cells (MSCs) differentiate into mature osteoblasts, forming a mineralised bone matrix. This process is regulated by soluble factors, mechanical stimuli, and the extracellular matrix (ECM), which together maintain bone and mineral homoeostasis. Mechanotransduction, the conversion of physical cues into intracellular signals, is crucial for MSC fate determination and orchestrates bone matrix remodelling, balancing formation and resorption. Continuous mechanical loading supports optimal osteogenic differentiation, whereas mechanical unloading (sub-physiological mechanical loading) disrupts this equilibrium and increases bone loss risk. These processes involve complex crosstalk among local and systemic factors, immune cells, and osteoblast-osteoclast interactions in response to mechanical cues. This review discusses key biomechanical factors regulating MSC osteogenic differentiation and bone remodelling, and synthesises evidence on skeletal immobilisation and other unloading-associated conditions that contribute to skeletal anomalies. It further emphasises nanovibrational stimulation as a novel approach to enhance MSC osteogenesis and mitigate skeletal anomalies.
Adult articular cartilage chondrocytes have a limited capacity to divide compared to juvenile cells, but the mechanisms behind this decline remain unclear. This study investigates metabolic changes associated with the cessation of chondrocyte proliferation in mouse articular cartilage. Using 5-ethynyl-2'-deoxyuridine (EdU) labeling, the postnatal decline in proliferation was tracked. Label-free fluorescence-lifetime imaging microscopy (FLIM) method, combined with artificial intelligence (AI)-assisted image segmentation, was applied to live cartilage sections to analyze metabolic parameters. Results showed that 1-month-old articular cartilage chondrocytes enter quiescence with significant changes in FLIM fluorescence decay parameters across cartilage zones compared to juvenile chondrocytes. Chondroprogenitors in the superficial zone showed a gradual decrease in citrate synthase content, while glycolytic activity increased with tissue depth. These findings reveal metabolic reprogramming that enables chondrocytes to adapt their metabolism despite limited oxygen availability to meet functional demands. Investigating these chondrocyte adaptations provides key insights for identifying metabolic targets and improving the design of durable, well-integrated tissue-engineered cartilage.
Suboptimal skin regeneration in patients with severe burns leads to significant trauma. Due to their favorable biological properties, fat grafts have been widely used in wound repair. The present study aimed to investigate the regenerative benefits of cold-stimulated fat graft in a contact burn model using C57BL/6J mice. Transplantation of browning fat grafts, with enhanced adipogenic capacity and decreased fibrosis, effectively promoted granulation tissue thickness and re-epithelialization areas. Cold-stimulated fat graft had a significant accumulation of ADSCs, which migrated into wound skin. In adipocyte-deficient mice, the impaired wound-healing phenotype was reversed by browning fat graft. Cold-stimulated fat also exhibited higher levels of damage-associated molecular patterns (DAMPs), which induced the proliferation of ADSCs and promoted ADSC proliferation and differentiation into mature adipocytes. Inhibition of DAMP-related signaling abolished the repair benefits of browning fat graft. In conclusion, transplantation of cold-stimulated fat represents a highly effective strategy for treating burn wounds through promoting DAMP signaling and dermal adipose remodeling.
Alopecia is highly prevalent and debilitating, yet current drugs provide limited, reversible benefit with notable side effects. We established a rapid protocol to generate human induced pluripotent stem cell-derived dermal papilla cells (hiPSC-DPCs) and demonstrated that their conditioned medium (CM) acts as a potent, cell-free hair-regenerative therapy. Transdermal delivery of hiPSC-DPCs or CM accelerated anagen re-entry and hair regrowth in depilated mice, and hiPSC-DPC CM outperformed minoxidil in promoting ex vivo hair-shaft elongation and in vitro proliferation and migration of primary DPCs and keratinocytes. Proteomic and metabolomic profiling revealed enrichment of growth factors, antioxidants, and immunomodulatory metabolites linked to TNF and PI3K-Akt signaling, conferring superior anti-inflammatory and cytoprotective properties relative to primary DPC CM. Moreover, hiPSC-DPC CM mitigated dihydrotestosterone (DHT)-induced pathology by suppressing androgen receptor expression and nuclear translocation. These findings position hiPSC-DPC secretome as a dual-functional, regenerative, and anti-androgenic biologic with translational potential for durable alopecia treatment.
Cell therapy is a promising strategy for tackling neurodegenerative diseases. The most outstanding results with this approach usually involve neuroprotection of damaged neurons at risk of death, but only with limited success. Current therapies are often based on the idea of “one gene, one disease, one drug” for single targets, a concept that limits their actual effectiveness. In contrast, combining different strategies can establish an advanced cell therapy that can slow down neuronal degeneration. In this study, we took advantage of the combination of cell and gene therapy, by transplanting bone marrow stem cells genetically modified to overexpress insulin-like growth factor 1 (IGF1) into a model of selective neurodegeneration, the PCD mouse. This animal is characterized by progressive neuronal loss in the olfactory bulb and alterations in IGF1 levels, among other symptoms. Using different techniques (cell cultures, viral transduction, cell transplants, flow cytometry, qPCR, ELISA, immunohistochemistry, advanced image analysis), our findings showed that neuronal death was virtually blocked, even 130 days after cell transplantation, a result clearly more successful than previous studies. The effects of this transplant are based in part on the regulation of neuroinflammation, increasing the proportion of reactive microglia and reducing that of proinflammatory microglia. In addition, IGF1 overexpression dramatically reduced DNA damage in mutant animals via IGF binding protein 3 pathway: this enhances neuroprotection by complementing the basal effect of cell therapy itself. In summary, our work supports the idea that combining therapeutic approaches and their synergies is a more effective tactic for combating neuronal loss.
Human skeletal muscle models often lack important supportive cell types. Here we developed a co-culture three-dimensional tissue engineered skeletal muscle (3D-TESM) model by combining myogenic progenitors (MPs) with genetically-matched immortalized fibro-adipogenic progenitors (iFAPs). FAPs play a crucial physiological role in myogenesis, tissue remodeling and extracellular matrix (ECM) formation. We demonstrate that co-culture 3D-TESMs effectively recapitulate these processes under controlled conditions, thereby enhancing contractile force, muscle tissue integrity and longevity, as well as improving ECM deposition compared to MP-only 3D-TESMs. Moreover, using pro-fibrotic and pro-adipogenic cell culture compositions we were able to mimic pathological features typically observed in muscular dystrophies: excessive ECM production and the formation of fatty infiltrations. This study provides an advanced skeletal muscle model, with enhanced functional and structural properties, capable of recapitulating pathophysiological processes that require FAPs.
Skeletal muscle (SM) functions both mechanically and as a secretory organ, releasing myokines and extracellular vesicles (EVs) involved in myogenic regulation and inter-tissue communication. While 3D bioengineered SM models are widely used for studying muscle physiology, few have been applied to investigate EV dynamics. This study optimised a 3D SM model to support mature myotube formation and evaluated its utility for SM-EV analysis. Myosin heavy chain expression was reduced at higher Matrigel ® concentrations (40%–60% v/v), highlighting the importance of matrix composition in model design. EVs were successfully isolated using size-exclusion chromatography and ultrafiltration, with yield influenced by cellular differentiation status. Common EV markers (Alix, CD9, CD63) were consistently expressed. Importantly, sarcoplasmic reticulum markers α- and β-sarcoglycan were identified in SM-EV preparations. These findings validate our SM model as a defined platform for studying SM-EV biology and defining molecular cargo.
