Understanding the cellular dynamics, engineering perspectives and translation prospects in bioprinting epithelial tissues.

The epithelium is one of the important tissues in the body as it plays a crucial barrier role serving as a gateway into and out of the body. Most organs in the body contain an epithelial tissue component, where the tightly connected, organ-specific epithelial cells organize into cysts, invaginations, or tubules, thereby performing distinct to endocrine or exocrine secretory functions. Despite the significance of epithelium, engineering functional epithelium in vitro has remained a challenge due to it is special architecture, heterotypic composition of epithelial tissues, and most importantly, difficulty in attaining the apico-basal and planar polarity of epithelial cells. Bioprinting has brought a paradigm shift in fabricating such apico-basal polarized tissues. In this review, we provide an overview of epithelial tissues and provide insights on recapitulating their cellular arrangement and polarization to achieve epithelial function. We describe the different bioprinting techniques that have been successful in engineering polarized epithelium, which can serve as in vitro models for understanding homeostasis and studying diseased conditions. We also discuss the different attempts that have been investigated to study these 3D bioprinted engineered epithelium for preclinical use. Finally, we highlight the challenges and the opportunities that need to be addressed for translation of 3D bioprinted epithelial tissues towards paving way for personalized healthcare in the future.

Three-dimensional printing in orthopaedic surgery: A review of current and future applications.

Three-dimensional (3D) printing is a form of technology in which 3D physical models are created. It has been used in a variety of surgical specialities ranging from cranio-maxillo-facial to orthopaedic surgery and is currently an area of much interest within the medical profession. Within the field of orthopaedic surgery, 3D printing has several clinical applications including surgical education, surgical planning, manufacture of patient-specific prostheses/patient specific instruments and bone tissue engineering. This article reviews the current practices of 3D printing in orthopaedic surgery in both clinical and pre-clinical settings along with discussing its potential future applications.

A portable micro-nanochannel bio-3D printed liver microtissue biosensor for DON detection.

We investigated a portable micro-nanochannel biosensor 3D-printed liver microtissues for rapid and sensitive deoxynivalenol (DON) detection. The screen-printed carbon electrode (SPCE) was modified with nanoporous anodic aluminum oxide (AAO), gold nanoparticles (AuNPs), and cytochrome C oxidase (COx) to enhance sensor performance. Gelatin methacrylate hydrogel, combined with hepatocellular carcinoma cells, formed the bioink for 3D printing. Liver microtissues were prepared through standardized and high-throughput techniques via bio-3D printing technology. These microtissues were immobilized onto modified electrodes to fabricate liver microtissue sensors. The peak current of this biosensor was positively correlated with DON concentration, as determined by cyclic voltammetry (CV), within a linear detection range of 2∼40 µg/mL. The standard curve equation is denoted by ICV(µA) = = 18.76956 + 0.03107CDON(µg/mL), with a correlation coefficient R2 was 0.99471(n＝3). A minimum detection limit of 1.229 µg/mL was calculated from the formula, indicating the successful construction of a portable micro-nanochannel bio-3D printed liver microtissue biosensor. It provides innovative ideas for developing rapid and convenient instrumentation to detect mycotoxin hazards after grain production. It also holds significant potential for application in the prediction and assessment of post-production quality changes in grain.

Emerging technologies in regenerative medicine: The future of wound care and therapy.

Wound healing, an intricate biological process, comprises orderly phases of simple biological processed including hemostasis, inflammation, angiogenesis, cell proliferation, and ECM remodeling. The regulation of the shift in these phases can be influenced by systemic or environmental conditions. Any untimely transitions between these phases can lead to chronic wounds and scarring, imposing a significant socio-economic burden on patients. Current treatment modalities are largely supportive in nature and primarily involve the prevention of infection and controlling inflammation. This often results in delayed healing and wound complications. Recent strides in regenerative medicine and tissue engineering offer innovative and patient-specific solutions. Mesenchymal stem cells (MSCs) and their secretome have gained specific prominence in this regard. Additionally, technologies like tissue nano-transfection enable in situ gene editing, a need-specific approach without the requirement of complex laboratory procedures. Innovating approaches like 3D bioprinting and ECM bioscaffolds also hold the potential to address wounds at the molecular and cellular levels. These regenerative approaches target common healing obstacles, such as hyper-inflammation thereby promoting self-recovery through crucial signaling pathway stimulation. The rationale of this review is to examine the benefits and limitations of both current and emerging technologies in wound care and to offer insights into potential advancements in the field. The shift towards such patient-centric therapies reflects a paradigmatic change in wound care strategies.

Microvascular network based on the Hilbert curve for nutrient transport in thick tissue.

To address the uneven nutrient distribution within three-dimensional (3D) tissue models and organoids currently used in medical research, this study introduces a microvascular network based on the Hilbert curve. Our aim was to develop innovative solutions for enhancing nutrient supply in thick tissue models in vitro. By using 3D bioprinting, we engineered microvascular networks of varying Hilbert orders and validated their efficacy in enhancing nutrient uniformity through numerical simulations and experiments. These networks facilitated broader and more uniform nutrient distribution throughout the thick tissue models, particularly the 2° Hilbert microvascular structure, which occupies less space and significantly reduces regions of cellular death. Furthermore, we explored the potential of assembling larger tissue constructs using the 2° Hilbert microvascular network, showcasing its applicability in constructing large-scale biological models. The findings suggest that the 2° Hilbert microvascular structure is particularly effective in ensuring adequate nutrient delivery, thus enhancing the viability and functionality of large-volume tissue models. These innovations hold significant promise for advancing the fields of tissue engineering and regenerative medicine by improving nutrient delivery to in vitro thick tissue block models. This provides a robust foundation for future in vitro research and clinical applications, potentially leading to more effective treatments and interventions in the medical field. The development of these microvascular networks represents a crucial step forward in overcoming the limitations of current 3D tissue models and organoids, paving the way for more sophisticated and reliable biomedical research tools.

3D bioprinting of an intervertebral disc tissue analogue with a highly aligned annulus fibrosus via suspended layer additive manufacture.

Intervertebral disc (IVD) function is achieved through integration of its two component regions: the nucleus pulposus (NP) and the annulus fibrosus (AF). The NP is soft (0.3-5 kPa), gelatinous and populated by spherical NP cells in a polysaccharide-rich extracellular matrix (ECM). The AF is much stiffer (~100 kPa) and contains layers of elongated AF cells in an aligned, fibrous ECM. Degeneration of the disc is a common problem with age being a major risk factor. Progression of IVD degeneration leads to chronic pain and can result in permanent disability. The development of therapeutic solutions for IVD degeneration is impaired by a lack of in vitro models of the disc that are capable of replicating the fundamental structure and biology of the tissue. This study aims to investigate if a newly developed suspended hydrogel bioprinting system (termed SLAM) could be employed to fabricate IVD analogues with integrated structural and compositional features similar to native tissue. Bioprinted IVD analogues were fabricated to recapitulate structural, morphological and biological components present in the native tissue. The constructs replicated key structural components of native tissue with the presence of a central, polysaccharide-rich NP surrounded by organised, aligned collagen fibres in the AF. Cell tracking, actin and matrix staining demonstrated that embedded NP and AF cells exhibited morphologies and phenotypes analogous to what is observed in vivo with elongated, aligned AF cells and spherical NP cells that deposited HA into the surrounding environment. Critically, it was also observed that the NP and AF regions contained a defined cellular and material interface and segregated regions of the two cell types, thus mimicking the highly regulated structure of the IVD.

Advancements in Tissue Engineering: A Review of Bioprinting Techniques, Scaffolds, and Bioinks.

Tissue engineering is a multidisciplinary field that uses biomaterials to restore tissue function and assist with drug development. Over the last decade, the fabrication of three-dimensional (3D) multifunctional scaffolds has become commonplace in tissue engineering and regenerative medicine. Thanks to the development of 3D bioprinting technologies, these scaffolds more accurately recapitulate in vivo conditions and provide the support structure necessary for microenvironments conducive to cell growth and function. The purpose of this review is to provide a background on the leading 3D bioprinting methods and bioink selections for tissue engineering applications, with a specific focus on the growing field of developing multifunctional bioinks and possible future applications.

AI-Based Knowledge Extraction from the Bioprinting Literature for Identifying Technology Trends.

Bioprinting is a rapidly evolving field, as represented by the exponential growth of articles and reviews published each year on the topic. As the number of publications increases, there is a need for an automatic tool that can help researchers do more comprehensive literature analysis, standardize the nomenclature, and so accelerate the development of novel manufacturing techniques and materials for the field. In this context, we propose an automatic keyword annotation model, based on Natural Language Processing (NLP) techniques, that can be used to find insights in the bioprinting scientific literature. The approach is based on two main data sources, the abstracts and related author keywords, which are used to train a composite model based on (i) an embeddings part (using the FastText algorithm), which generates word vectors for an input keyword, and (ii) a classifier part (using the Support Vector Machine algorithm), to label the keyword based on its word vector into a manufacturing technique, employed material, or application of the bioprinted product. The composite model was trained and optimized based on a two-stage optimization procedure to yield the best classification performance. The annotated author keywords were then reprojected on the abstract collection to both generate a lexicon of the bioprinting field and extract relevant information, like technology trends and the relationship between manufacturing-material-application. The proposed approach can serve as a basis for more complex NLP-related analysis toward the automated analysis of the bioprinting literature.

3D Bioprinting of Pig Macrophages and Human Cells Discovered the P2Y14 Receptor as a Mediator of Xenogenic Immune Responses.

BACKGROUND: The survival rate of pig lung xenotransplantation (PLXTx) recipients is severely limited by intense xenogenic immune responses, necessitating further insights into xenogeneic immunity and the development of models to study the PLXTx immune response. METHODS: We identified regulators of PLXTx immune response Using Gene ontology analysis. We assessed the metabolic changes and protein levels in 3D4/31 pig alveolar macrophages (PAMs) through flow cytometry and immunoblotting. To induce a xenogenic immune response, we co-cultured 3D4/31-PAMs with A549 human alveolar epithelial cells and evaluated cytokine expression using qRT-PCR. RESULTS: Gene ontology analysis identified STAT1 and alveolar macrophages as contributors to lung autoimmunity and transplant rejection. In 3D4/31-PAMs, phorbol myristate acetate-induced glycogen accumulation and cyclooxygenase-2 expression were inhibited by the P2Y14 inhibitor PPTN. Co-culturing 3D4/31-PAMs with A549 human alveolar epithelial cells via 3D bioprinting resulted in a more pronounced inflammatory response than 2D co-culture, with increased expression of genes related to the P2Y14 cascade and inflammation. This inflammatory gene expression was prevented by PPTN treatment. CONCLUSION: Based on these results, we propose alginate bioprinting as an in vitro model for PLXTx and suggest that P2Y14 is a key regulator of xenogeneic immune responses in PAMs.

A critical review on advances and challenges of bioprinted cardiac patches.

Myocardial infarction (MI), which causes irreversible myocardium necrosis, affects 0.25 billion people globally and has become one of the most significant epidemics of our time. Over the past few years, bioprinting has moved beyond a concept of simply incorporating cells into biomaterials, to strategically defining the microenvironment (e.g., architecture, biomolecular signalling, mechanical stimuli, etc.) within which the cells are printed. Among the different bioprinting applications, myocardial repair is a field that has seen some of the most significant advances towards managing the tissue repairing microenvironment. This review critically assesses the most up-to-date biomedical innovations being carried out in cardiac patch bioprinting, with specific considerations given to the biomaterial design parameters, growth factors/cytokines, biomechanical and bioelectrical conditioning, as well as innovative biomaterial-based "4D" bioprinting (3D scaffold structure + temporal morphology changes) of myocardial tissues, immunomodulation and sustained delivery systems used in myocardium bioprinting. Key challenges include the ability to generate large quantities of cardiac cells, achieve high-density capillary networks, establish biomaterial designs that are comparable to native cardiac extracellular matrix, and manage the sophisticated systems needed for combining cardiac tissue microenvironmental cues while simultaneously establishing bioprinting technologies yielding both high-speed and precision. This must be achieved while considering quality assurance towards enabling reproducibility and clinical translation. Moreover, this manuscript thoroughly discussed the current clinical translational hurdles and regulatory issues associated with the post-bioprinting evaluation, storage, delivery and implantation of the bioprinted myocardial patches. Overall, this paper provides insights into how the clinical feasibility and important regulatory concerns may influence the design of the bioink (biomaterials, cell sources), fabrication and post-fabrication processes associated with bioprinting of the cardiac patches. This paper emphasizes that cardiac patch bioprinting requires extensive collaborations from imaging and 3D modelling technicians, biomaterial scientists, additive manufacturing experts and healthcare professionals. Further, the work can also guide the field of cardiac patch bioprinting moving forward, by shedding light on the potential use of robotics and automation to increase productivity, reduce financial cost, and enable standardization and true commercialization of bioprinted cardiac patches. STATEMENT OF SIGNIFICANCE: The manuscript provides a critical review of important themes currently pursued for heart patch bioprinting, including critical biomaterial design parameters, physiologically-relevant cardiac tissue stimulations, and newly emerging cardiac tissue bioprinting strategies. This review describes the limited number of studies, to date in the literature, that describe systemic approaches to combine multiple design parameters, including capabilities to yield high-density capillary networks, establish biomaterial composite designs similar to native cardiac extracellular matrix, and incorporate cardiac tissue microenvironmental cues, while simultaneously establishing bioprinting technologies that yield high-speed and precision. New tools such as artificial intelligence may provide the analytical power to consider multiple design parameters and identify an optimized work-flow(s) for enabling the clinical translation of bioprinted cardiac patches.

Multidirectional Filamented Light Biofabrication Creates Aligned and Contractile Cardiac Tissues.

Biofabricating 3D cardiac tissues that mimic the native myocardial tissue is a pivotal challenge in tissue engineering. In this study, we fabricate 3D cardiac tissues with controlled, multidirectional cellular alignment and directed or twisting contractility. We show that multidirectional filamented light can be used to biofabricate high-density (up to 60 × 106 cells mL-1) tissues, with directed uniaxial contractility (3.8x) and improved cell-to-cell connectivity (1.6x gap junction expression). Furthermore, by using multidirectional light projection, we can partially overcome cell-induced light attenuation, and fabricate larger tissues with multidirectional cellular alignment. For example, we fabricate a tri-layered myocardium-like tissue and a bi-layered tissue with torsional contractility. The approach provides a new strategy to rapidly fabricate aligned cardiac tissues relevant to regenerative medicine and biohybrid robotics.

Embedded Bioprinting of Tumor-Scale Pancreatic Cancer-Stroma 3D Models for Preclinical Drug Screening.

The establishment of organotypic preclinical models that accurately resemble the native tumor microenvironment at an anatomic human scale is highly desirable to level up in vitro platforms potential for screening candidate therapies. The bioengineering of anatomic-scaled three-dimensional (3D) models that emulate native tumor scale while recapitulating their cellular and matrix components remains, however, to be fully realized. In this focus, herein, we leveraged embedded 3D bioprinting for biofabricating pancreatic ductal adenocarcinoma (PDAC) in vitro models combining gelatin-methacryloyl and hyaluronic acid methacrylate extracellular matrix (ECM)-mimetic biomaterials with human pancreatic cancer cells and cancer-associated fibroblasts to generate in vitro models capable of emulating native tumor size (∼6 mm) and stromal elements. By using a viscoelastic continuous polymeric supporting bath, tumor-scale 3D models were rapidly generated (∼50 constructs/h) and easily recovered following in-bath visible light photocrosslinking. As a proof-of-concept, tissue-scale constructs displaying physiomimetic designs were biofabricated. These models also encompass the incorporation of a stromal compartment to better emulate the cellular components of the PDAC native tumor microenvironment (TME) and its stratified spatial organization. Cell-laden tumor-size constructs remained viable for up to 14 days and were responsive to Gemcitabine in a dose-dependent mode. Cancer-stroma models also exhibited increased drug resistance compared to their monotypic counterparts, highlighting the key role of stromal cells in chemotherapeutic resistance. Overall, we report for the first time the freeform biofabrication of PDAC models exhibiting anatomic scale, different structural complexities, and engineered cancer-stromal compartments, being highly valuable for preclinical screening of therapeutics.

Advances in Tissue Engineering and Its Future in Regenerative Medicine Compared to Traditional Reconstructive Techniques: A Comparative Analysis.

Tissue engineering represents a revolutionary approach in regenerative medicine, offering promising alternatives to traditional reconstructive techniques. This systematic review explores recent advances in tissue engineering, comparing their efficacy, postoperative outcomes, and patient satisfaction to conventional methods. A comprehensive literature search was conducted across PubMed, Cochrane Library, and Google Scholar, covering studies published from 2000 to 2024. Fourteen studies were selected for final analysis based on inclusion criteria focusing on outcomes such as scar quality, postoperative pain, and patient satisfaction. The review demonstrated that tissue engineering techniques consistently provided superior cosmetic outcomes with minimal scarring compared to traditional methods. Patients undergoing tissue-engineered procedures experienced mild-to-moderate postoperative pain with rapid resolution, whereas traditional techniques resulted in moderate to severe pain requiring extended management. Furthermore, patients treated with tissue engineering reported high satisfaction rates due to improved cosmetic and functional outcomes. Despite challenges such as ensuring adequate vascularization, controlling scaffold degradation, and overcoming regulatory and cost barriers, ongoing research and development are essential to fully realize the potential of these innovative therapies. Tissue engineering offers significant advantages over traditional reconstructive techniques and has the potential to profoundly improve patient care in regenerative medicine.

Advancing Cartilage Tissue Engineering: A Review of 3D Bioprinting Approaches and Bioink Properties.

Articular cartilage is crucial in human physiology, and its degeneration poses a significant public health challenge. While recent advancements in 3D bioprinting and tissue engineering show promise for cartilage regeneration, there remains a gap between research findings and clinical application. This review critically examines the mechanical and biological properties of hyaline cartilage, along with current 3D manufacturing methods and analysis techniques. Moreover, we provide a quantitative synthesis of bioink properties used in cartilage tissue engineering. After screening 181 initial works, 33 studies using extrusion bioprinting were analyzed and synthesized, presenting results that indicate the main materials, cells, and methods utilized for mechanical and biological evaluation. Altogether, this review motivates the standardization of mechanical analyses and biomaterial assessments of 3D bioprinted constructs to clarify their chondrogenic potential.

Improving tumor microenvironment assessment in chip systems through next-generation technology integration.

The tumor microenvironment (TME) comprises a diverse array of cells, both cancerous and non-cancerous, including stromal cells and immune cells. Complex interactions among these cells play a central role in driving cancer progression, impacting critical aspects such as tumor initiation, growth, invasion, response to therapy, and the development of drug resistance. While targeting the TME has emerged as a promising therapeutic strategy, there is a critical need for innovative approaches that accurately replicate its complex cellular and non-cellular interactions; the goal being to develop targeted, personalized therapies that can effectively elicit anti-cancer responses in patients. Microfluidic systems present notable advantages over conventional in vitro 2D co-culture models and in vivo animal models, as they more accurately mimic crucial features of the TME and enable precise, controlled examination of the dynamic interactions among multiple human cell types at any time point. Combining these models with next-generation technologies, such as bioprinting, single cell sequencing and real-time biosensing, is a crucial next step in the advancement of microfluidic models. This review aims to emphasize the importance of this integrated approach to further our understanding of the TME by showcasing current microfluidic model systems that integrate next-generation technologies to dissect cellular intra-tumoral interactions across different tumor types. Carefully unraveling the complexity of the TME by leveraging next generation technologies will be pivotal for developing targeted therapies that can effectively enhance robust anti-tumoral responses in patients and address the limitations of current treatment modalities.

Small molecular weight alginate gel porogen for the 3D bioprinting of microvasculature.

In order to recreate the complexity of human organs, the field of tissue engineering and regenerative medicine has been focusing on methods to build organs from the bottom up by assembling distinct small functional units consisting of a biomaterial and cells. This bottom-up engineering requires bioinks that can be assembled by 3D bioprinting and that permit fast vascularization of the construct to ensure survival of embedded cells. To this end, a small molecular weight alginate (SMWA) gel porogen is presented herein. Alginate is a biocompatible biomaterial, which can be easily converted into small porogen gels with the procedure reported in this article. The SMWA porogen is mixed with photo-crosslinkable hydrogels and leached from the hydrogel post-crosslinking to increase porosity and facilitate vascularization. As a proof of concept, this system is tested with the commonly used biomaterial Gelatin Methacryloyl (GelMA). The SMWA porogen-GelMA blend is proven to be bioprintable. Incubating the blend for 20 min in a low concentration phosphate buffered saline and sodium citrate solution significantly reduces the remaining porogen in the hydrogel . The intent to completely leach the porogen from the hydrogel was abandoned, as longer incubation times and higher concentrations of phosphate and citrate were detrimental to endothelial proliferation. Nonetheless, even with remnants of the porogen left in the hydrogel, the created porosity significantly improves viability, growth factor signaling, vasculogenesis, and angiogenesis in 3D bioprinted structures. This article concludes that the usage of the SMWA porogen can improve the assembly of microvasculature in 3D bioprinted structures. This technology can benefit the bottom-up assembly of large scaffolds with high cell density through 3D bioprinting by improving cell viability and allowing faster vascularization.

3D Bioprinting of Liquid High-Cell-Proportion Bioinks in Liquid Granular Bath.

Embedded 3D bioprinting techniques have emerged as a powerful method to fabricate 3D engineered constructs using low strength bioinks; however, there are challenges in simultaneously satisfying the requirements of high-cell-activity, high-cell-proportion, and low-viscosity bioinks. In particular, the printing capacity of embedded 3D bioprinting is limited as two main challenges: spreading and diffusion, especially for liquid, high-cell-activity bioinks that can facilitate high-cell-proportion. Here, a liquid-in-liquid 3D bioprinting (LL3DBP) strategy is developed, which used a liquid granular bath to prevent the spreading of liquid bioinks during 3D printing, and electrostatic interaction between the liquid bioinks and liquid granular baths is found to effectively prevent the diffusion of liquid bioinks. As an example, the printing of positively charged 5% w/v gelatin methacryloyl (GelMA) in a liquid granular bath prepared with negatively charged κ-carrageenan is proved to be achievable. By LL3DBP, printing capacity is greatly advanced and bioinks with over 90% v/v cell can be printed, and printed structures with high-cell-proportion exhibit excellent bioactivity.

Bio-printing method as a novel approach to obtain a fibrin scaffold settled by limbal epithelial cells for corneal regeneration.

Treatment of Limbal Stem Cell Deficiency (LSCD), based on autologous transplantation of the patient's stem cells, is one of the few medical stem cell therapies approved by the European Medicines Agency (EMA). It relies on isolating and culturing in vivo Limbal Epithelial Stem Cells (LESC) and then populating them on the fibrin substrate, creating a scaffold for corneal epithelial regeneration. Such a solution is then implanted into the patient's eye. The epithelial cell culture process is specific, and its results strongly depend on the initial cell seeding density. Achieving control of the density and repeatability of the process is a desirable aim and can contribute to the success of the therapy. The study aimed to test bioprinting as a potential technique to increase the control over LESCs seeding on a scaffold and improve process reproducibility. Cells were applied to 0.5 mm thick, flat, transparent fibrin substrates using extrusion bioprinting; the control was the traditional manual application of cells using a pipette. The use of 3D printer enabled uniform coverage of the scaffold surface, and LESCs density in printed lines was close to the targeted value. Moreover, printed cells had higher cell viability than those seeded traditionally (91.1 ± 8.2% vs 82.6 ± 12.8%). The growth rate of the epithelium was higher in bioprinted samples. In both methods, the epithelium had favorable phenotypic features (p63 + and CK14 +). 3D printing constitutes a promising approach in LSCD therapy. It provides favorable conditions for LESCs growth and process reproducibility. Its application may lead to reduced cell requirements, thereby to using fewer cells on lower passages, which will contribute to preserving LESCs proliferative potential.

3D bioprinting of fish skin-based gelatin methacryloyl (GelMA) bio-ink for use as a potential skin substitute.

Gelatin methacryloyl (GelMA), typically derived from mammalian sources, has recently emerged as an ideal bio-ink for three-dimensional (3D) bioprinting. Herein, we developed a fish skin-based GelMA bio-ink for the fabrication of a 3D GelMA skin substitute with a 3D bioprinter. Several concentrations of methacrylic acid anhydride were used to fabricate GelMA, in which their physical-mechanical properties were assessed. This fish skin-based GelMA bio-ink was loaded with human adipose tissue-derived mesenchymal stromal cells (ASCs) and human platelet lysate (HPL) and then printed to obtain 3D ASCs + HPL-loaded GelMA scaffolds. Cell viability test and a preliminary investigation of its effectiveness in promoting wound closure were evaluated in a critical-sized full thickness skin defect in a rat model. The cell viability results showed that the number of ASCs increased significantly within the 3D GelMA hydrogel scaffold, indicating its biocompatibility property. In vivo results demonstrated that ASCs + HPL-loaded GelMA scaffolds could delay wound contraction, markedly enhanced collagen deposition, and promoted the formation of new blood vessels, especially at the wound edge, compared to the untreated group. Therefore, this newly fish skin-based GelMA bio-ink developed in this study has the potential to be utilized for the printing of 3D GelMA skin substitutes.