Osteochondral defects, characterized by the structural and functional disruption of articular cartilage and subchondral bone, present significant clinical challenges due to the tissue’s limited intrinsic regenerative capacity. Scaffold-based tissue engineering has paved the way for osteochondral defect treatment; however, fully restoring the complex structure and composition of native osteochondral tissue remains challenging. Recent advances in three-dimensional (3D) printing have enabled the fabrication of layered, anisotropic scaffolds designed to biomimetically recapitulate the native tissue’s zonal properties through precise hierarchical design. High-resolution fabrication techniques facilitate the construction of delicate microarchitectures, while advanced bioprinting methods allow for the incorporation of bioactive factors and cells into the scaffold matrix. This review emphasizes the following four scaffold design paradigms: composite gradients, microarchitectural patterning, biochemical gradients, and cellular heterogeneity. Moreover, key properties of multilayered scaffolds are discussed, including mechanical performance, interfacial strength, and degradation behavior. In addition, several obstacles associated with the in vivo scaffold application are discussed, providing insights to guide future clinical translation in osteochondral defects treatment.

Repairing long peripheral nerve gap injuries and reconstructing corresponding functions remain two major challenges in regenerative medicine. The application of nerve conduits, constructed via neural tissue engineering (NTE) strategies, has emerged as a prominent research focus and an essential tool for nerve repair. Among NTE technologies, additive manufacturing (AM), especially bioprinting, represents one of the most promising fabrication approaches for neural conduits. This review systematically analyzes the current research progress on peripheral nerve conduit fabrication, particularly emphasizing how different conduit structures, biomaterials, and AM techniques synergistically influence nerve regeneration outcomes. The review also summarizes the principles and recommendations for selecting appropriate nerve conduit structures for different defect lengths and injury stages, providing a theoretical basis for the design and practical application of conduit structures. Additionally, it focuses on the role of advanced bioprinting technologies in enhancing conduit complexity, cell guidance, and functional recovery. Furthermore, this review highlights emerging trends and discusses critical future directions for integrating structure design, material selection, and printing strategies toward the next generation of nerve conduits. This review aims to provide a comprehensive perspective for advancing peripheral nerve repair by bridging biomaterial engineering, manufacturing innovations, and regenerative medicine needs.

The emergence of organoid technology has bridged critical gaps between conventional two-dimensional cell cultures and in vivo systems by offering self-organized three-dimensional (3D) microtissues that recapitulate organ-specific architecture, cellular heterogeneity, and functional dynamics. However, traditional organoid models face inherent limitations in structural precision, scalability, and physiological relevance, particularly in replicating vascular networks, mechanical microenvironments, and multicellular interactions. Recent advancements in 3D bioprinting have enabled unprecedented spatial control over cellular and extracellular matrix organization, unlocking new frontiers in engineering organoids with enhanced biomimicry and functionality. This review systematically examines the integration of bioprinting technologies with organoid science, spanning biomaterial innovations, vascularization strategies, and dynamic microenvironmental cues that drive functional maturation. By synthesizing interdisciplinary advances in stem cell biology, materials science, and computational modeling, the work highlights applications across regenerative medicine, disease pathophysiology, and personalized drug screening. Key challenges, including immunogenicity, long-term stability, and clinical scalability, are critically evaluated alongside emerging solutions such as four-dimensional bioprinting, organ-on-chip integration, and artificial intelligence-driven bioink optimization. Through a comprehensive analysis of bioprinted organoids for physiology and 3D disease modeling, this review aims to establish a translational roadmap for leveraging spatially programmed organoids to address unmet clinical needs, revolutionize therapeutic development, and advance precision medicine.

Traditional toxicological testing, which relies on animal models and two-dimensional cell cultures, encounters challenges in accurately predicting human-specific responses due to interspecies variability and the inherent limitations of simplified in vitro systems. Three-dimensional (3D) bioprinting has emerged as a transformative approach, facilitating the fabrication of physiologically relevant tissue constructs with precise spatial control over cellular and extracellular matrix components. This review critically examines recent advancements in 3D-bioprinted organ models, such as the liver, kidney, and lung, for toxicological assessments, including their applications in drug safety evaluation, environmental pollutant screening, and nanomaterial risk assessment. We further analyze persistent technical barriers concerning resolution limitations, material biocompatibility, and the simulation of multi-organ interactions. Finally, we propose integrative strategies that combine organ-on-a-chip platforms, artificial intelligence-driven design, and standardized validation protocols, aiming to accelerate the translational potential of bioprinted models in regulatory toxicology.

Sustainable bioprinting is a transformative approach in tissue engineering and regenerative medicine, offering solutions to environmental challenges while advancing functional outcomes. However, achieving true sustainability remains complex, requiring reductions in material waste and energy use, and scalable, resource-efficient fabrication without compromising biological performance. Artificial intelligence (AI) provides a powerful means to meet these demands through data-driven material design, predictive process optimization, and intelligent control systems that improve both efficiency and environmental impact across the bioprinting workflow. This review examines the integration of AI into sustainable bioprinting across four key areas: hydrogel material discovery and development, bioink screening, process parameter optimization, and AI-assisted intelligent printing. AI facilitates the design of eco-friendly hydrogels by predicting molecular interactions and tailoring structural properties. It also improves bioink formulation by optimizing printability, biocompatibility, and mechanical strength, thereby reducing reliance on resource-intensive trial-and-error experimentation. Furthermore, AI algorithms streamline workflows by dynamically adjusting printing parameters to improve fidelity and reduce waste, while advanced AI-assisted systems demonstrate the feasibility of multi-material, contactless bioprinting, aligning with sustainability goals.

In nature, many biological surfaces exhibit inherent bacteriostatic property due to the existence of special microstructures. However, the key factors and underlying mechanisms driving this property remain unclear. A significant challenge lies in the lack of proper techniques for precisely fabricating such microstructures as well as finely tuning their morphological parameters. In this study, we adopted a two-photon 3D printing-based approach to fabricate microstructures on specified surfaces with accurate control over their morphology, enabling the investigation of structural bacteriostasis. Through abstracting the subtle morphology on shark skin, we replicated their bacteriostatic microstructures and were able to regulate their morphology at the micron scale. By culturing Streptococcus mutans on the surface of these microstructures, we validated their bacteriostatic performance and demonstrated that morphological parameters significantly influenced the efficacy of structural bacteriostasis. Other kinds of microstructures such as micro-holes with bacteriostatic property could also be fabricated and investigated utilizing this two-photon polymerization technology. We believe this strategy offers a powerful tool for researching bacteriostatic mechanisms of various microstructures and will inspire their broad applications in both daily and industrial settings.

Tantalum (Ta) holds considerable potential for clinical applications in artificial vertebral bodies (AVBs) owing to its excellent biocompatibility. A novel Ta AVB structure was engineered by combining thin-walled structure topology optimization with lattice structure filling design methods. Three types of Ta AVBs—designated as AVB-1, AVB-2, and AVB-3—were fabricated using selective laser melting. The influence of sidewall curvature on the mechanical properties and deformation behavior of AVBs was investigated through compression tests and finite element analysis. The elastic modulus and yield strength of the Ta lattice structures ranged from 1.75 to 3.21 GPa and 31 to 65 MPa, respectively. Incorporating topologically thin walls enhanced the elastic modulus and yield strength by factors of 2.26-3.77 and 3-3.62, respectively. A decrease in sidewall curvature was associated with an increase in both elastic modulus and yield strength of the AVBs. Specifically, as the sidewall curvature decreased from 0.027 to 0 mm−1, the elastic modulus and yield strength increased by factors of 2.76 and 2.19, respectively. The yield strengths of the AVBs were comparable to those of human cortical bone. Among the three designs, AVB-2 exhibited the highest yield-strength-to-elastic-modulus ratio (0.029), compared to AVB-1 and AVB-3 (0.024 and 0.019, respectively), suggesting that the optimal sidewall curvature is 0.014 mm−1. AVB-2 effectively mitigated the stress shielding effect while maximizing the load-bearing capacity, indicating its significant potential for clinical applications.

Osteoarthritis (OA) is an age-related degenerative joint disease characterized by progressive cartilage deterioration. Chondrocyte senescence is recognized as a key contributor to the onset and progression of OA. Establishing reliable cartilage senescence models is, therefore, essential for elucidating the underlying mechanisms and developing preventive strategies for OA. 3D-bioprinted models offer significant advantages in precisely controlling tissue architecture, enabling spatial delivery of bioactive molecules, and supporting dynamic cell culture. In this study, we employed 3D bioprinting technology to construct cartilage models and subsequently established cartilage senescence models using hydrogen peroxide (H₂O₂). Firstly, gelatin-sodium alginate hydrogel scaffolds provided favorable mechanical strength and porosity, creating a supportive microenvironment for chondrocyte proliferation. Secondly, these scaffolds exhibited excellent biocompatibility and effectively promoted extracellular matrix synthesis and secretion. By comparing H₂O₂-induced 2D chondrocyte senescence models with 3D-bioprinted cartilage senescence models, our results demonstrated that the 3D models more closely mimicked the molecular characteristics of naturally aged human cartilage. Therefore, the 3D-bioprinted cartilage senescence models represent a promising experimental platform for investigating the pathogenesis and prevention of age-related OA.

This study introduces a standardized approach to generating and assembling G-code for biofabrication, ensuring compatibility and convergence across diverse machines and scales. By using vector-based drawing software, such as Adobe Illustrator, shapes are designed as paths and converted into modular G-code blocks (subroutines). This vector-based approach allows for the straightforward design of complex structures, such as organic shapes, by simply drawing them to scale, avoiding the need for labor-intensive construction. These blocks are assembled into a final script with a modified version of Notepad++ that enhances code segmentation and provides real-time visualization. Unlike many commercial slicers, this method offers precise control over the print path—a critical advantage in biofabrication, where anisotropic structures are essential for directed cell growth and orientation-specific mechanical properties needed in biomimetic tissue design. The method’s versatility is demonstrated across techniques from micro-scale applications, such as melt electrowriting, to macro-scale approaches like bioprinting, freeform printing, and in-gel printing. This process streamlines code generation, allowing both simple and complex shapes to be efficiently produced. Although paths are drawn in 2D, stacking layers enables 3D constructs. The method’s standardized, relative G-code format—compatible with most devices—supports easy transfer across machines with clearly marked, machine-specific segments, creating a unified and adaptable codebase for a range of fabrication scales and techniques.

Collagen I is a key extracellular matrix (ECM) component in bone tissue and one of the most important biomaterials for bone tissue engineering applications. However, printing high-resolution mesh scaffold from collagen I remains challenging due to its relatively weak ink shape fidelity. While previous efforts have attempted to improve printability by increasing ink viscosity, such approaches often compromise ink flowability and yield only modest improvements in printing resolution. To solve this issue, we blended oxidized cellulose with collagen I to form a Schiff-base interaction. The resulting hydrogel exhibited lower viscosity but a more apparent linear rheological characteristic, as demonstrated by our large amplitude oscillation sweep results. This enhanced rheological profile enabled the fabrication of scaffolds with a printing resolution approaching 150 μm—one of the highest reported for collagen I-based scaffolds. Scaffolds with this scale of rod diameter and pore size greatly enhanced the proliferation and osteogenic differentiation of mesenchymal stem cells. Correspondingly, the expression of key osteogenic markers, including N-cadherin, HIF-1α, and β-catenin, was upregulated. These findings broaden our understanding of scaffold design and processing optimization of collagen I-based scaffolds and may advance their application in bone tissue engineering.

Congenital heart disease (CHD) has been one of the most serious problems in newborns. For fetal heart health care, 3D modeling and printing technology have been adopted in the diagnosis of CHD during antenatal care. However, the development of 3D printing techniques and their clinical applications have been hindered by the manual processing of ultrasound (US) volume data in clinical practice. To overcome this problem, we present an interactive semi-automatic method based on deep learning that uses manual processing results from expert sonographers for training. The accuracy, interpretability, and variability of the performances were evaluated on the validation set. The results demonstrated that compared with a physician with less than 3 years of experience, a better Faster- region-based convolutional neural network-based threshold was achieved using our proposed fetal heart reconstruction technique (FRT), with enhanced performance based on the outflow tract view and three-vessel view. No significant difference was found among the clinical parameters, in proportion, measured from the model rebuilt using FRT and US volume data. Furthermore, the reconstruction time of the fetal heart blood pool model was reduced from approximately 5 h to 5 min. Our results indicate that deep learning has the ability to process US data accurately, representing an important step towards the reconstruction of the fetal heart digital model, which is critical for advancing clinical diagnosis and treatment of CHD during pregnancy.

Spinal implants are vital for treating spinal disorders, yet wear particle-induced complications threaten their long-term success. Despite this, the direct effects of implant-derived particles on neural cells remain largely unexplored, especially given the limitations of conventional 2D culture models to capture such complex interactions. The current study introduces a novel in vitro platform consisting of a 3D-bioprinted gelatin methacryloyl (GelMA) hydrogel embedded with neural cells (C6 astrocyte-like and NG108-15 neurons) and spinal implant biomaterial particles, designed to model the spinal cord microenvironment with enhanced physiological relevance. As the first of its kind, this cell-particle-laden system supports the evaluation of neural cell responses to spinal biomaterial particles, including polymers, PEEK-OPTIMA™ and polyethylene Ceridust® 3615, zirconia-toughened alumina (ZTA) ceramic, and CoCrMo metal alloy. The bioprinted platform demonstrated excellent compatibility with various neural cell types and particle compositions, enabling a wide range of biological assays. Cell viability within the 3D model was comparable to traditional 2D cultures, affirming its ability to sustain cell survival while offering improved biomimicry. Biological assays assessing cell viability, reactive oxygen species (ROS) production, and DNA damage provided critical insights into material-specific and time-dependent cellular responses. While no significant cytotoxic effects were observed in short-term cultures, distinct variations in ROS production, and viability emerged based on biomaterial type and exposure duration. Overall, this versatile 3D-bioprinted system presents a robust, scalable tool for mechanistic and toxicological studies of spinal implant wear particles under physiologically relevant conditions.

Hydrogels have emerged as promising scaffolds for cartilage tissue engineering due to their structural mimicry of native articular cartilage extracellular matrix. However, conventional hydrogels typically exhibit only nanoscale porosity and poor mechanical properties, which limit nutrient delivery, metabolic waste exchange, and structural fidelity. To address these challenges, we developed an innovative cell-laden porous silk methacryloyl (SilMA) hydrogel system with biomechanical reinforcement using three-dimensional (3D) bioprinting. The porous architecture was created through a water-in-water emulsification strategy employing poly(ethylene oxide) (PEO) as a sacrificial template. This pore-forming process resulted in a remarkable structural modulation, achieving an increase of over 100% in average pore diameter and a 75% enhancement in overall porosity compared to hydrogels without PEO. However, this structural modification compromised the compressive modulus by approximately 50%. Therefore, homogenized electrospun silk fibroin nanofibers (NFs) were incorporated into the bio-ink to improve the mechanical properties and optimize surface topography. The introduction of NFs (1-2 wt%) not only recovered the compressive strength and modulus (close to SilMA hydrogels) but also improved the 3D printability of PEO/SilMA hydrogels. Additionally, the hydrogel demonstrated excellent biocompatibility and markedly upregulated expression of chondrogenic-related genes, including COL2A1, ACAN, and SOX9. Furthermore, the subcutaneous implantation experiments in non-obese diabetic/severe combined immunodeficiency mice further confirmed the potential of PEO/NF/SilMA hydrogels in promoting cartilage formation. Therefore, this study proposes a promising dual-strategy approach for cartilage tissue engineering, integrating NFs reinforcement and PEO-induced porosity.

Achilles tendon injury is a common musculoskeletal disorder, particularly prevalent among athletes and middle-aged/elderly populations. Heterotopic ossification (HO) following Achilles tendon injury is a frequent complication that significantly compromises patients’ quality of life and athletic performance. Conventional conservative treatments and surgical interventions for HO often yield suboptimal outcomes, failing to restore native tendon functionality. Tissue engineering strategies integrating biomaterials and cells offer promising solutions for tendon regeneration and functional recovery. Three-dimensional bioprinting presents unique advantages in fabricating tissue-engineered scaffolds through precise control of architectural geometry and internal microstructure. In this study, we developed a novel silk fibroin (SF)-hydroxypropyl cellulose (HPC)-tendon stem/progenitor cell (TSPC) bioink with exceptional cytocompatibility and rheological properties. This bioink demonstrated superior printability for fabricating porous Achilles tendon scaffolds with high mechanical strength (elastic modulus: 85 MPa), controlled biodegradability, and optimal porosity (91%). In vitro experiments revealed that SF- HPC-TSPCs scaffolds promoted TSPC survival, migration, proliferation, and tenogenic differentiation within the scaffold microenvironment. In vivo assessments demonstrated that the scaffolds exhibited excellent biocompatibility, elicited no systemic inflammatory or immune responses, and effectively prevented HO in rat models of Achilles tendon injury. This study establishes a groundbreaking approach for addressing post-traumatic HO in tendon regeneration.

Extrusion-based three-dimensional bioprinting is a widely used technique for fabricating cell-laden constructs in tissue engineering and regenerative medicine. However, the mechanical stresses experienced by cells during the printing process can negatively affect their viability. This study examines the influence of nozzle geometry—specifically contraction angle and outlet diameter—on stress distribution and its effects on cell survival. Through a combination of experimental analysis and theoretical modeling, the impacts of nozzle design on the balance between shear and extensional stresses during bioprinting are explored. The findings highlight the importance of optimizing nozzle parameters to minimize mechanical damage and enhance post-printing cell viability. The proposed model provides a framework for guiding nozzle design, offering insights into the development of customized bioprinting strategies that enhance construct fidelity and biological functionality. These results contribute to advancing bioprinting techniques for applications in tissue engineering and regenerative medicine.

The skin is the largest organ of the human body and is the primary barrier against external stressors. However, in cases of severe skin damage or pathological conditions, the body’s natural physiological repair mechanisms are often insufficient to support effective skin tissue repair and regeneration. Bioprinting, a form of three-dimensional (3D) printing technology, utilizes various biomaterials and cells to construct complex 3D structures, offering the potential to overcome the limitations of conventional tissue-engineered skin and to develop functional skin substitutes. In this study, we developed a 3D bioprinter with excellent printing performance to fabricate vascularized skin substitutes. Through methacrylic anhydride-mediated modification of gelatin, we synthesized gelatin methacryloyl (GelMA) with varying degrees of substitution. The resulting GelMA hydrogel exhibited excellent mechanical properties, swelling ratio, porosity, and rheological properties. To create a hydrogel-multicellular composite bio-ink, we adjusted the concentration of the GelMA solution and co-cultured human immortalized epidermal cells, human foreskin fibroblasts, and human umbilical vein endothelial cells to optimize biological function. Importantly, by fine-tuning the printing parameters, the 3D extrusion-printed lines successfully fused into a continuous membrane, enhancing interlayer bonding and mechanical integrity. This process enabled the construction of a vascularized skin substitute with distinct reticular and papillary layers. In addition, the 3D-printed vascularized skin was implanted into skin defect models established in BALB/c nude mice and New Zealand rabbits to investigate its regenerative capabilities. These findings hold significant implications for the utilization of 3D-printed vascularized skin for improving skin injury repair, thereby advancing the field of skin tissue engineering.

3D-printed polycaprolactone (PCL) scaffolds are widely used for bone tissue engineering but suffer from deficiencies, such as difficulty in cell adhesion, lack of osteogenic activity, and poor immunomodulatory capacity. Enhancing the biological responsiveness of PCL scaffolds remains a key focus in bone tissue engineering. In this study, the following three types of scaffolds were prepared: (i) PCL, (ii) strontium (Sr)-doped bioactive glass (SrBG)/PCL, and (iii) polydopamine (PDA)/SrBG/PCL. The scaffolds were assayed in vitro for their effect on the expression of osteoinductive differentiation markers (ALP, RUNX2, and COL1), and their influence on macrophage (MP) (CD206, ARG, TNF-α, IL1β, IL-10, and IL-12) behavior was evaluated. Their effect on bone defect repair was assessed in vivo using micro-computed tomography (micro- CT), hematoxylin and eosin (HE) staining, Masson staining, and immunofluorescence staining (iNOS, CD163, BMP-2, and VEGF). The results demonstrated that PDA/SrBG/ PCL scaffolds significantly promoted the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), inhibited the differentiation of MPs to the M1 phenotype, and promoted the differentiation of MPs to the M2 phenotype, resulting in better pro-osteogenic, immunomodulatory, and angiogenic effects in vivo. This observation may be associated with the release of Sr²+ from SrBG, and surface modification with PDA further enhanced the immunomodulation and bone repair ability of the scaffold. The study demonstrated that the PDA/SrBG/PCL scaffolds exhibit excellent bone repair capabilities and hold strong potential for applications in bone tissue engineering.

Current in vitro tumor models often fail to recapitulate the hierarchical vascular architecture and dynamic interactions of the tumor microenvironment (TME), limiting their utility in cancer research. In this study, we present a multiscale vascularized tumor model integrating coaxial bioprinting, inkjet printing, and fused deposition modeling (FDM) to address this challenge. Firstly, coaxial bioprinting enabled the fabrication of dual-layered vasculature with an endothelium layer and a smooth muscle layer. Secondly, tumor spheroids with precise size control (±10 μm) were generated via inkjet printing by modulating Methacrylate Gelatin (GelMA) concentration and valve actuation time. An FDM-printed chip was designed to co-culture these components under perfusion, facilitating the self-organization of a microvascular network around tumor spheroids. After 11 days of dynamic culture, the model demonstrated tumor-driven angiogenic sprouting and early metastatic behavior, validated by the upregulation of metastasis-related genes (CD44, MMP2, N-cadherin) in vascularized cohorts. Drug testing with paclitaxel revealed dose-dependent suppression of tumor proliferation and invasion. This platform not only mimics the structural and functional complexity of the TME but also provides a scalable, physiologically relevant tool for investigating tumor-vascular crosstalk and evaluating anti-cancer therapeutics.

Three-dimensional (3D) printing has emerged as a promising technique for creating in vitro tumor models that replicate the tumor microenvironment, with the potential to reduce or replace the use of experimental animals. The incorporation of 3D decellularized extracellular matrix (dECM) hydrogels significantly enhances cellular responsiveness and functionality in drug screening. However, the limited printability of dECM restricts its application in ex vivo 3D disease models. To address this limitation, researchers have developed a blended bioink composed of dECM, gelatin methacrylate (GelMA), and gelatin, specifically tailored for direct ink writing-based 3D bioprinting. This formulation exhibits favorable shear-thinning behavior, enhanced viscosity, and thermal-sensitive properties, making it suitable for 3D bioprinting. The combination of dECM with GelMA and gelatin not only improves the printability of the bioink but also enhances the resolution of the printed scaffolds. Furthermore, dECM demonstrated positive effects on human hepatocellular carcinoma (HepG2) cells, promoting proliferation, migration, and cell spheroid formation. A 3D liver cancer model was successfully created in vitro by printing HepG2 cells encapsulated in the bioink containing dECM. This model exhibited characteristics akin to in vivo solid tumors, including notable cell proliferation, protein secretion, and substantial cell spheroid formation (up to 78.83 ± 9.41 μm on day 8). Additionally, it showed drug resistance, with 46.23% and 31.34% cell viability observed at 100 μg/mL concentrations of doxorubicin and paclitaxel, respectively. These findings underscore the potential of bioprinted 3D tumor models composed of GelMA, gelatin, and dECM as valuable platforms for the evaluation of anticancer drugs.

Material extrusion using medical-grade biodegradable hydrogel demonstrates significant potential for manufacturing biocompatible scaffolds in regenerative medicine. However, unpredictable geometric variations in the fabricated models, such as swelling or shrinking, impede the development of complex three-dimensional (3D) hydrogel architectures for in vitro-functionalized tissues and organs. A primary cause of structural deformation, such as wrinkling or even collapse, is improper humidity control during the 3D printing process. Therefore, there is a need to investigate the swelling-shrinking behavior of hydrogels under varying ambient humidity and to determine optimal humidity levels for the printing process. This study established a thermal-humidity-multiphase flow coupling field simulation model to numerically investigate the humidity-driven swelling-shrinking behavior of hydrogel filaments. The optimal 3D printing humidity levels were determined for hydrogel filaments with diameters of 0.2, 0.3, and 0.4 mm, which were found to be 90, 80, and 60%, respectively. Using these humidity settings, several structures were fabricated, demonstrating moderated moisture loss of 3D architecture. Notably, a human ear model was successfully printed, achieving an effective size of 20 mm (length) × 10 mm (width) × 10 mm (height). Our research can benefit the future development in tissue engineering and regenerative medicine.

Maintaining bone formation in microgravity/weightless environments remains a major challenge. Under weightless conditions, osteocytes act as mechanosensors to inhibit Wnt canonical signaling and bone formation by secreting sclerostin. This study explores whether osteocytic Wnt7b can counteract microgravity-induced bone loss through Wnt non-canonical signaling. Unlike previous bioprinting studies that focused on structural scaffolds or generic cell types, a novel bioprinted scaffold consisting of polycaprolactone (supportive) and osteocyte (functional) hydrogels was constructed in this study. Osteocytes overexpressing Wnt7b were co-cultured with bone marrow stromal cells (ST2) in a 3D biomimetic weightless biomicroenvironmental system (3D-BWBM) to assess osteogenic and lipogenic differentiation. The results indicated that osteocytic Wnt7b enhanced osteogenic differentiation and mineralization of ST2 cells via the Wnt non-canonical pathway PKCδ, while suppressing the expression of lipogenic markers (Pparg, Cebpa) and adipogenesis. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis revealed elevated expression of Sost and Mef2c, downregulation of the Wnt target gene Opg, and elevated expression of pro-osteoclastogenic cytokine Rankl and pro-inflammatory cytokines Tnfa and Il1b, thus validating the microgravity effect. Unlike conventional 2D culture of RCCS™ cells, the 3D hydrogels were printed with tunnels (500 μm) for efficient nutrient/ metabolite exchange, resulting in good cell growth, high cell viability (97%), and a six-fold increase in proliferative activity within 7 days. Wnt7b osteocytes were still able to maintain the osteogenic differentiation of ST2 cells, as evidenced by elevated alkaline phosphatase activity, mineralization (1.8-fold increase), and a decrease in osteoblast marker genes (Alpl, Runx2, Col1a1). In conclusion, Wnt7b-PCKδ signaling counteracts microgravity-induced bone loss, and further in vivo studies on osteocytic Wnt7b are warranted to confirm this causal relationship.

The development of mechanically tunable and cytocompatible hydrogels is critical for advancing three-dimensional (3D) bioprinting in tissue engineering. Here, we report a composite bioink composed of gelatin methacrylate (GelMA), methylcellulose, sodium alginate, and laponite-RDS. This formulation supports extrusion-based printing without ionic crosslinkers, mimics the extracellular matrix (ECM), and maintains stable viscoelasticity under physiological conditions (37°C, pH 7.4). Electrostatic and hydrogen bonding interactions among the charged polymers enhance pre-gel viscosity, shear-thinning behavior, and print fidelity. To evaluate its potential in disease modeling, patient-derived keloid fibroblasts were encapsulated in 3D-bioprinted constructs using two GelMA-based formulations with different stiffness levels, such as soft (G4A1M1R1, 2.1 kPa) and stiff (G5A1M1R1, 7.9 kPa), chosen to replicate the mechanical properties of normal dermis and keloid tissue, respectively. Both constructs exhibited excellent cell viability after three days, confirming cytocompatibility. Furthermore, matrix stiffness significantly regulated fibrotic gene expression. The stiffer hydrogel induced higher expression of COL1, MMP2, and IL6, suggesting enhanced myofibroblast activation and ECM remodeling. Immunofluorescence staining further confirmed elevated protein levels of α-SMA, FSP1, and actin stress fibers (F-actin) in the stiff construct, consistent with keloid pathology. Taken together, these results demonstrate that the GelMA-based bioink enables stiffness-dependent modulation of fibrotic responses, offering a simplified yet relevant 3D model of fibrotic skin. This platform may provide a useful basis for future studies on keloid progression and preliminary antifibrotic drug screening.