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Bio-ink

Bio-ink, often referred to as bioink, is a biocompatible material formulation used in three-dimensional (3D) bioprinting to fabricate engineered tissue constructs by precisely depositing living cells within a supportive matrix, typically in the form of hydrogels or cell suspensions.^[1] These materials enable the layer-by-layer assembly of complex, hierarchical structures that mimic native tissues, combining cellular components with biomaterials to support cell viability, proliferation, and differentiation.^[2] Bio-inks are primarily composed of living cells—such as stem cells, fibroblasts, or endothelial cells—integrated into natural polymers like alginate, collagen, or gelatin; synthetic polymers such as polyethylene glycol (PEG) or polycaprolactone (PCL); or hybrid combinations thereof, sometimes including decellularized extracellular matrix (dECM) for enhanced bioactivity.^[3] Natural bio-inks, derived from biological sources, offer superior biocompatibility and mimicry of the extracellular environment, while synthetic variants provide tunable mechanical properties and greater control over degradation rates.^[1] Hybrid bio-inks blend these advantages, and scaffold-free options rely on cell aggregates or spheroids that self-assemble post-printing.^[2] For effective use in bioprinting techniques like extrusion, inkjet, or laser-assisted methods, bio-inks must exhibit specific rheological properties, including shear-thinning behavior for smooth extrusion and appropriate viscosity (ranging from 10 mPa·s for droplet-based printing to up to 6×10^7 mPa·s for extrusion), alongside mechanical strength to maintain structural integrity after deposition.^[2] Essential biological attributes include high cytocompatibility to ensure over 85% cell viability^[1], non-toxic crosslinking mechanisms (e.g., via calcium ions for alginate or UV light for gelatin methacryloyl), and biodegradability that aligns with tissue remodeling timelines.^[3] These properties are critical for achieving high shape fidelity and functional outcomes in printed constructs.^[1] Bio-inks play a pivotal role in advancing tissue engineering and regenerative medicine, facilitating applications such as the creation of vascular networks, cartilage, skin, and organ models for drug testing and transplantation.^[2] Despite progress, challenges persist in scaling production, ensuring long-term functionality in vivo, and developing cost-effective, tissue-specific formulations to address organ shortages.^[1]

Fundamentals

Definition and Composition

Bio-ink is defined as a specialized formulation consisting of living cells embedded within a hydrogel or viscous biomaterial matrix, often incorporating bioactive molecules, that serves as the printable medium in 3D bioprinting processes to fabricate tissue-like constructs.^[2] This material is designed to mimic the extracellular matrix (ECM) of native tissues, providing a supportive environment that maintains cell viability during extrusion, deposition, or other printing techniques while enabling precise spatial organization of biological components.^[1] The core components of bio-ink include living cells, such as stem cells or fibroblasts, which are the functional elements; polymers, either natural or synthetic, that form the hydrogel structure; growth factors to promote cell signaling and differentiation; and solvents to achieve the desired consistency.^[2]^[1] Polymer concentrations typically range from 3% to 20% by weight, balancing mechanical stability, printability, and biocompatibility without compromising cell survival.^[2]^[4] Bio-inks are categorized into cell-laden formulations, which directly encapsulate viable cells within the matrix for immediate tissue mimicry, and acellular variants, which act as structural supports that can be populated with cells post-printing.^[1]^[4] In extrusion-based bioprinting, these formulations generally exhibit shear-thinning viscosities ranging from 0.03 to 60,000 Pa·s, allowing controlled flow through nozzles while retaining shape upon deposition.^[2]^[5] In tissue engineering, bio-ink facilitates layer-by-layer deposition to build hierarchical scaffolds or organoids, enabling the recreation of complex tissue architectures that support cellular adhesion, proliferation, and functional maturation.^[1]^[2] This approach addresses limitations of traditional scaffolding by integrating living components from the outset, promoting regenerative outcomes in applications such as organ repair.^[4]

Historical Development

The origins of bio-ink trace back to the late 1980s and 1990s, when researchers began adapting early 3D printing technologies—initially invented by Charles Hull in 1984 for non-biological materials—to fabricate hydrogel-based scaffolds for tissue engineering.^[6] These foundational efforts involved basic extrusion of biocompatible polymers, such as polylactic acid modified with polyethylene oxide, to support cell adhesion and growth, laying the groundwork for printable biological constructs without live cell encapsulation.^[7] A pivotal advancement occurred in 2003, when Thomas Boland at Clemson University introduced the first cell-encapsulating bio-ink using alginate hydrogel, enabling inkjet bioprinting of viable mammalian cells with over 80% survival rates post-printing.^[8] This innovation, patented and detailed in early publications, shifted the field from acellular scaffolds to functional tissue printing. In 2010, the Wake Forest Institute for Regenerative Medicine, led by Anthony Atala, biofabricated a 3D microscale liver tissue analog using bioprinting techniques, advancing the fabrication of functional organ models.^[9]^[7] In 2012, the introduction of sacrificial bio-inks, such as gelatin-based formulations, allowed for the creation of temporary supports in complex prints, facilitating vascular channel formation after removal, as pioneered in early embedded printing techniques.^[10] The 2010s saw rapid progress in multi-material bio-ink systems, with contributions from the Mirkin group at Northwestern University advancing nanoscale patterning for precise cell placement in hybrid formulations.^[11] Post-2020 developments, including as of 2025, have emphasized hybrid bio-inks combining natural and synthetic components for organ-scale printing, such as vascularized heart and liver models, along with innovations like 4D bioprinting and cryogenic techniques to improve mechanical stability, biocompatibility, and cell viability for regenerative applications. In 2021 and 2022, Wake Forest teams secured first and second place in NASA's Vascular Tissue Challenge using bioprinted vascularized liver and kidney tissues.^[12]^[13] Notable patents, including formulations for tunable bio-ink rheology, have supported these evolutions, though regulatory milestones like FDA approvals for bio-ink-derived scaffolds remain emerging as of 2025.^[14]^[15]

Key Properties

Printability

Printability refers to the capacity of a bio-ink to be precisely deposited during 3D bioprinting processes, such as extrusion or photocrosslinking, while preserving structural integrity and shape fidelity immediately after deposition and over time. This property ensures that printed filaments or layers replicate the intended design from computer-aided models, with typical resolutions ranging from 50 to 400 μm depending on the bioprinting technique and hardware configuration. Shape fidelity is particularly critical for creating complex architectures, as poor printability can lead to filament spreading, merging, or collapse, compromising the overall construct geometry.^[16]^[17] Central to printability are several key physical and mechanical parameters that govern the bio-ink's behavior during and post-deposition. Viscosity is paramount, exhibiting shear-thinning characteristics where the material maintains high viscosity at rest to support shape retention but decreases under applied shear stress for smooth extrusion through the nozzle. Yield stress, typically exceeding 100 Pa, provides the necessary resistance to deformation, preventing filament sagging or spreading under gravitational or self-weight forces. Crosslinking mechanisms further enhance stability by rapidly solidifying the deposited material, either through physical processes like ionic interactions or chemical methods such as photocrosslinking, enabling layer-by-layer stacking without distortion. These parameters must balance to achieve reliable deposition while considering trade-offs with biocompatibility, such as minimizing shear forces that could harm embedded cells.^[16]^[18]^[19] Testing printability involves standardized rheological and geometric assessments to quantify these attributes. Rheological analysis, primarily through oscillatory shear tests, measures properties like storage modulus (indicating elasticity), loss modulus (indicating viscosity), and the transition to non-linear behavior, which correlates with filament stability. A common quantitative metric is the printability index, often calculated as Pr = \frac{L^2}{16A}, where L is the perimeter and A is the area of printed pores or filaments; values near 1 denote high fidelity, while deviations signal collapse (Pr < 1) or over-solidification (Pr > 1). These evaluations are complemented by direct printing trials, analyzing filament diameter and aspect ratio to validate performance under operational conditions.^[16]^[20]^[21] Several operational factors influence printability by modulating the bio-ink's flow and solidification dynamics. Temperature control is essential, often maintained around 37°C to optimize viscosity without premature gelation, though it varies by formulation to ensure consistent extrusion. Nozzle diameter, commonly 100–500 μm, directly impacts resolution and shear rates, with smaller sizes enabling finer features but requiring lower-viscosity inks to avoid clogging. Printing speed, typically 5–20 mm/s, affects filament uniformity and layer adhesion; slower speeds enhance fidelity but may increase exposure to shear, while faster rates risk inconsistencies in deposition. Optimizing these parameters through iterative testing ensures robust printability across diverse bioprinting setups.^[16]^[22]

Biocompatibility and Rheology

Biocompatibility of bio-inks is defined as their capacity to maintain non-toxicity toward encapsulated cells, enabling high cell viability, adhesion, proliferation, and function without eliciting adverse responses. A key metric is achieving greater than 80% cell viability immediately post-printing, assessed through live/dead assays that differentiate viable cells (via calcein-AM staining) from compromised ones (via ethidium homodimer).^[23] This threshold aligns with standards like ISO 10993, where a reduction below 70% indicates potential cytotoxicity from bio-ink components or printing stresses.^[23] Rheological properties critically influence biological performance by ensuring structural integrity during cell encapsulation and post-deposition. The storage modulus (G') must exceed the loss modulus (G'') to confer gel-like stability, with typical G' values ranging from 1 to 100 kPa for bio-inks that support cell-laden constructs.^[24] Swelling ratios and degradation rates—via enzymatic or hydrolytic mechanisms—further modulate nutrient access and matrix remodeling, preventing excessive expansion that could disrupt cell organization or premature breakdown that hinders tissue maturation.^[24] Cell-specific factors in bio-inks emphasize tailored environments for sustained viability and functionality. Porosity levels of 50-90% promote nutrient and oxygen diffusion, essential for metabolic support in dense constructs, as described by the Stokes-Einstein equation for the diffusion coefficient D = \frac{kT}{6\pi\eta r}, where k is Boltzmann's constant, T is temperature, \eta is viscosity, and r is the diffusing particle radius.^[25] Mechanical stiffness tuned to 0.1-50 kPa mimics native tissue mechanics, guiding cell differentiation and migration while minimizing immune responses through low-immunogenic materials that reduce inflammation upon implantation.^[25]^[18] Evaluation techniques include MTT assays to quantify proliferation via metabolic activity and confocal microscopy to visualize extracellular matrix (ECM) deposition, confirming long-term bio-integration.^[23] Rheological optimization complements printability by facilitating gentle cell encapsulation without compromising flow behavior.^[24]

Classification

Structural Bio-inks

Structural bio-inks are specialized formulations in 3D bioprinting designed to provide long-term mechanical support and integrity to engineered tissue constructs without dissolving post-printing, making them essential for fabricating load-bearing structures such as bone and cartilage tissues.^[1] Unlike temporary supports, these bio-inks maintain their scaffold role throughout tissue maturation, enabling the creation of durable architectures that mimic the extracellular matrix of hard tissues.^[26] They typically consist of cell-laden hydrogels reinforced for stability, ensuring precise deposition and shape fidelity during extrusion or other bioprinting modalities.^[27] Key characteristics of structural bio-inks include high mechanical strength, with compressive strengths often ranging from 10 to 100 MPa in reinforced composites to withstand physiological loads, as seen in collagen-hydroxyapatite scaffolds enhanced with microfibrillated cellulose achieving over 28 MPa.^[26] Degradation profiles are tunable over weeks to months to align with tissue regeneration timelines, for instance, decellularized extracellular matrix-based inks degrading over several weeks in vitro.^[26] Minimal swelling in optimized formulations like dual ionically cross-linked hydrogels prevents distortion of printed geometries and supports long-term stability.^[26] These properties are achieved through crosslinking strategies, including ionic methods (e.g., Ca²⁺ for alginate) or photochemical UV exposure for materials like gelatin methacryloyl (GelMA).^[1] Examples of structural bio-inks encompass natural polymers like alginate, which provides baseline ionic crosslinking for stability, often hybridized with synthetics such as polycaprolactone (PCL) to yield tensile moduli around 15 MPa for bone scaffolds.^[26] Synthetic hybrids, including PEG-based gels with breaking stress up to 9.6 MPa, further exemplify tunable reinforcement for cartilage-like constructs.^[26] In contrast to sacrificial bio-inks that dissolve after printing to create voids, structural variants offer permanent support for sustained tissue development.^[1] These bio-inks excel in shape retention and mechanical robustness, facilitating high cell viability (over 90% in many cases) and effective integration in hard tissue engineering applications like osteochondral defect repair.^[27] However, potential cytotoxicity from chemical crosslinkers, such as UV initiators, poses a challenge, requiring careful optimization to balance stability with biocompatibility.^[1] Overall, their advantages in providing enduring scaffolds outweigh drawbacks when tailored for specific load-bearing needs, advancing clinical translation in regenerative medicine.^[26]

Sacrificial and Support Bio-inks

Sacrificial and support bio-inks are temporary, dissolvable materials used in 3D bioprinting to form channels, such as for vasculature, or to provide structural support for overhangs and complex geometries, which are removed after printing through mechanisms like temperature change, dissolution, or perfusion.^[28] These materials enable the fabrication of intricate architectures that would otherwise collapse during the printing process, complementing permanent structural bio-inks by allowing the creation of voids and perfusable networks essential for nutrient delivery in tissue constructs. Examples include carbohydrate-based glasses and poly(vinyl alcohol) for additional options in void formation.^[29] Key characteristics of sacrificial and support bio-inks include low viscosity to facilitate extrusion and post-print removal, high water solubility to ensure complete dissolution, and non-cytotoxic residues that preserve high cell viability in surrounding matrices.^[28] For example, Pluronic F127 exhibits thermoreversibility, forming a gel above 10°C for print stability and liquefying at 4°C for easy extraction without harsh chemicals.^[28] These properties balance printability with biocompatibility, minimizing damage to embedded cells during the removal phase.^[29] Sacrificial bio-inks, such as gelatin, serve primarily as templates for vascular channels and are fully removed to generate hollow structures, while support bio-inks like agarose provide temporary scaffolding for overhanging features in complex designs and can be selectively dissolved.^[28] Gelatin, for instance, gels below 30°C and melts at 37°C, enabling physiological-temperature removal, whereas agarose supports gelation at 35–40°C and dissolution above 90°C.^[28] Removal efficiencies for these materials are high post-printing, allowing high-fidelity void formation without residual occlusion.^[28] Techniques for removing sacrificial and support bio-inks often involve perfusion, where a fluid medium flushes the construct to accelerate dissolution and clear debris from channels.^[28] The dissolution rate of these materials can be controlled to optimize removal parameters.

Natural Bio-inks

Polysaccharide-Based

Polysaccharide-based bio-inks are derived from natural carbohydrates obtained from plant or bacterial sources, offering biocompatibility, low cost, and ease of processing for 3D bioprinting applications.^[30] Common examples include alginate from brown algae, agarose from red algae, and gellan gum produced by bacterial fermentation, which form hydrogels through physical crosslinking mechanisms without requiring harsh chemical conditions.^[30] These materials are particularly valued for their renewability and minimal immunogenicity, making them suitable as foundational components in tissue engineering scaffolds.^[31] These bio-inks generally exhibit ionic or thermal gelation, with viscosities in the range of 10-50 Pa·s under shear rates relevant to extrusion printing, enabling smooth flow through nozzles while maintaining shape fidelity post-deposition.^[32] Degradation primarily occurs via hydrolysis in physiological environments, allowing controlled breakdown over weeks to months depending on crosslinking density and environmental factors.^[30] Their biocompatibility supports cell encapsulation with viabilities often exceeding 90%, though inherent limitations such as limited cell adhesion motifs necessitate modifications for enhanced bioactivity.^[33] Alginate, a linear copolymer of guluronic and mannuronic acids, undergoes rapid ionic gelation upon exposure to divalent cations like Ca²⁺, forming stable networks at concentrations of 1-5% (w/v) that provide excellent printability for complex geometries.^[34] However, its bioinert nature results in poor cell adhesion, and pure forms can exhibit brittleness under mechanical stress.^[30] Gellan gum, available in low- and high-acyl variants, demonstrates shear-thinning behavior that facilitates extrusion at concentrations of 0.75-3% (w/v), with gelation triggered by cooling below 60°C or ionic addition, yielding hydrogels with tunable stiffness for supporting cell proliferation.^[35] Agarose gels thermally at approximately 35°C upon cooling from a molten state, used effectively at 2-4% (w/v) for sacrificial supports due to its reversible sol-gel transition and structural stability, though it shares alginate's challenge of low bioadhesiveness.^[36]^[37] To address drawbacks like mechanical fragility and insufficient cell interaction, polysaccharide bio-inks are often modified through blending with other natural polymers or functionalization, such as incorporating RGD peptides into alginate or combining gellan gum with cellulose for improved elasticity and degradation profiles.^[30] These strategies enhance overall mechanics without compromising printability, though challenges such as inconsistent gelation in complex architectures persist.^[31]

Protein-Based

Protein-based bio-inks, derived from animal sources such as collagen hydrolysis or blood components, serve as highly biocompatible materials that mimic the extracellular matrix (ECM) to promote cell attachment and signaling in 3D bioprinting applications.^[38] Common examples include gelatin, obtained through partial hydrolysis of collagen using enzymes, acids, or alkalis; collagen, extracted from animal tissues like skin and tendons; and fibrin, formed from fibrinogen isolated from blood plasma via centrifugation and cryoprecipitation.^[38] These proteins exhibit excellent biocompatibility, supporting cell adhesion, proliferation, and migration while providing a natural environment for tissue engineering.^[38] Key properties of protein-based bio-inks include temperature-sensitive gelation, typically occurring between 25°C and 37°C, which allows for easy handling during printing and solidification post-extrusion.^[38] They often contain RGD motifs that facilitate integrin-mediated cell adhesion, and their degradation is primarily enzymatic, mediated by matrix metalloproteinases (MMPs) or plasmin, enabling controlled remodeling by encapsulated cells.^[38] Gelatin bio-inks, affordable and widely available due to their natural abundance, are commonly formulated at 5-10% w/v concentrations to balance printability and cell viability.^[39] Methacrylated gelatin (GelMA), a photocrosslinkable derivative, undergoes rapid polymerization under UV or visible light with a photoinitiator, enhancing mechanical stability and shape fidelity for complex constructs.^[39] Collagen type I bio-inks self-assemble into fibrils at pH 7.4 and 37°C, yielding hydrogels with stiffness in the range of 0.1-1 kPa that closely replicate soft tissue mechanics and support high cell viability, such as 90% for NIH 3T3 fibroblasts.^[40] Fibrin bio-inks, polymerized through thrombin-induced clotting in seconds to minutes, are particularly suited for soft tissues like skin or cardiac models, offering rapid gelation and promotion of angiogenesis.^[41] Modifications such as crosslinking with genipin, a natural agent that reacts with primary amines to improve elasticity and prevent premature gelation, are employed to enhance stability in formulations like fibrin/gelatin blends.^[38] However, protein-based bio-inks face limitations including batch-to-batch variability in extraction processes, which can affect molar mass, viscosity, and reproducibility, necessitating standardized purification protocols.^[38]

Synthetic and Hybrid Bio-inks

Synthetic Polymers

Synthetic polymers serve as key components in bio-ink formulations for 3D bioprinting, offering engineered precision, reproducibility, and multifunctionality that surpass the variability often seen in natural counterparts. These lab-synthesized materials, including polyethylene glycol (PEG), Pluronics, and polycaprolactone (PCL), enable the creation of customizable scaffolds with defined architectures for tissue engineering applications. Unlike tissue-derived options such as decellularized extracellular matrix, synthetic polymers provide consistent batch-to-batch properties and ease of sterilization, though they typically require modifications to enhance cell interactivity.^[42]^[43] A hallmark of these polymers is their photocrosslinkability, particularly in UV-initiated systems, which allows rapid solidification post-extrusion to maintain structural fidelity. For instance, PEG derivatives like PEG diacrylate (PEGDA) undergo photopolymerization under physiological conditions, forming stable hydrogels with gelation times on the order of seconds upon light exposure. This property is tunable through the degree of acrylation and photoinitiator concentration, enabling precise control over network density and resulting mechanics. Additionally, these polymers exhibit adjustable mechanical properties, with compressive moduli ranging from 1 kPa for soft, cell-friendly environments to 1 MPa for stiffer constructs mimicking bone, achieved by varying molecular weight, chain length, or crosslinking extent. Controlled degradation, primarily hydrolytic, further supports their utility; PEG-based inks degrade slowly without enzymatic involvement, while PCL offers prolonged stability over months.^[44]^[45]^[42] Pluronics, particularly F127, stand out for their thermoreversible behavior, transitioning from sol to gel states based on temperature, which facilitates shear-thinning during printing and self-supporting structures afterward. At concentrations of 20-25% w/v, Pluronic F127 exhibits a critical micelle temperature of 20-30°C, gelling rapidly at body temperature (around 5 minutes at 37°C) and liquifying upon cooling for easy removal in sacrificial applications. This makes it ideal for creating temporary vascular channels or supports in complex prints. In contrast, PEGDA provides rapid gelation but is inherently inert, lacking native cell-adhesive motifs; bioactivity is imparted through functionalization with peptides like RGD or integration with growth factors. PCL, a thermoplastic, contributes high mechanical strength with a melting point of approximately 60°C, enabling extrusion at low temperatures while maintaining shape fidelity, though it often requires blending with hydrogels for cell encapsulation due to its non-aqueous nature.^[46]^[47]^[48] Hybrids incorporating nanoparticles, such as PEG-clay nanocomposites, enhance these properties by improving rheological stability and nutrient diffusion without compromising printability. Recent advancements as of 2025 include hybrid bioinks combining synthetic polymers with natural components for bioprinting complex vascular structures, such as artery models using embedded 3D printing techniques.^[49]^[42]^[43]^[50] Overall, synthetic polymers excel in scalability and sterilizability—allowing autoclaving or gamma irradiation—facilitating large-scale production and clinical translation. However, their lower inherent biocompatibility, stemming from the absence of bioactive cues, necessitates surface modifications or co-formulation with natural components to promote cell adhesion and proliferation effectively.

Decellularized Extracellular Matrix

Decellularized extracellular matrix (dECM) bio-inks are derived from animal tissues and organs through a decellularization process that removes cellular components while preserving the native extracellular matrix, providing a biomimetic scaffold for 3D bioprinting in tissue engineering.^[51] These bio-inks retain key structural and bioactive elements of the native tissue microenvironment, including collagens, glycosaminoglycans (GAGs), elastin, fibronectin, and growth factors such as vascular endothelial growth factor (VEGF), enabling enhanced cell adhesion, proliferation, and differentiation compared to purely synthetic alternatives.^[52] Sources commonly include decellularized organs like porcine liver and heart, as well as tissues such as porcine skin and human cadaver adipose or dermal matrices, allowing for tissue-specific bioactivity in printed constructs.^[53] The processing of dECM bio-inks typically begins with detergent-based decellularization protocols using agents like sodium dodecyl sulfate (SDS) or Triton X-100 to eliminate cells while minimizing damage to the matrix, followed by rinsing, enzymatic treatments if needed, and sterilization with peracetic acid or ethanol.^[51] The resulting acellular tissue is then lyophilized into a powder form and solubilized through pepsin digestion in an acidic environment (e.g., pH 2 with hydrochloric acid) to create a printable precursor solution, often neutralized and combined with cell suspensions prior to extrusion or other bioprinting methods.^[52] This solubilization step disrupts higher-order structures for injectability but allows self-assembly into hydrogels upon neutralization and incubation at physiological temperatures.^[53] dECM bio-inks exhibit tissue-specific mechanical properties, such as storage moduli of 10-20 kPa for cardiac-derived formulations, which mimic the stiffness of native myocardium and support cardiomyocyte alignment and contractility.^[52] They promote cell-specific differentiation, for instance enhancing hepatocyte functionality in liver-derived dECM by recapitulating cues that induce albumin secretion and cytochrome P450 activity.^[51] Gelation occurs rapidly at 37°C due to pH neutralization and thermal crosslinking of matrix proteins, forming stable hydrogels with shear-thinning behavior suitable for bioprinting.^[53] Despite their advantages, dECM bio-inks face challenges including potential immunogenicity from residual xenogeneic antigens, particularly in porcine-derived materials, which may elicit immune responses in human applications.^[52] Scalability remains a hurdle due to variability in tissue sourcing, batch-to-batch inconsistencies in decellularization efficiency, and the need for standardized protocols to ensure reproducibility.^[51] In practice, these bio-inks have been employed in organoid printing, such as fabricating liver sinusoid models with improved vascularization or cardiac patches that integrate with host tissue for myocardial repair.^[53]

Applications and Challenges

Biomedical Applications

Bio-inks have enabled significant advancements in tissue engineering, particularly for skin and cartilage regeneration. In skin tissue engineering, collagen-alginate-based bio-inks have been used to fabricate multilayered grafts that mimic the dermal and epidermal layers, supporting fibroblast and keratinocyte proliferation for wound closure. For instance, a sodium alginate/gelatin/collagen (SA/Gel/C) bio-ink was bioprinted to create bionic skin models, demonstrating biocompatibility and structural integrity suitable for grafting applications. These constructs have shown promise in preclinical models, with the first clinical trials initiating in 2025, such as a Phase I trial at the Concord Burns Unit using patient-derived cells applied to donor sites in burn patients, achieving significant wound closure in animal studies, with complete healing observed by 4 weeks in porcine models.^[54]^[55] In cartilage engineering, agarose-based bio-inks have facilitated the printing of chondrocyte-laden scaffolds with high cell viability exceeding 80% post-printing, promoting cartilage-specific gene expression such as aggrecan and collagen type II. These agarose-alginate blends exhibit compressive moduli of 25-70 kPa, supporting in vitro cartilage formation and gene expression, though lower than native cartilage (500-1500 kPa).^[56] Organ printing applications leverage bio-inks to construct functional vascular networks and organ models for drug screening. Sacrificial Pluronics, such as Pluronic F127, have been pivotal in creating perfusable vascular channels within tissues, with a 2018 study demonstrating multi-nozzle bioprinting of hierarchical networks using Pluronic as a fugitive material to form endothelium-lined vessels. This approach marked a milestone in achieving interconnected vascular trees up to several millimeters in scale, supporting nutrient delivery in thick constructs. For liver and kidney models, decellularized extracellular matrix (dECM) bio-inks derived from porcine tissues have been employed to bioprint lobule-like structures and proximal tubules, respectively, incorporating hepatocytes, endothelial cells, and stellate cells to replicate native microenvironments. These dECM-based models enable accurate hepatotoxicity and nephrotoxicity assessments for drugs like acetaminophen and dapagliflozin, providing human-relevant responses that reduce reliance on animal testing by offering scalable, physiologically accurate alternatives. Beyond core tissue engineering, bio-inks support wound healing through printed patches that deliver bioactive factors and cells directly to injury sites. For example, gelatin methacryloyl (GelMA) and silk fibroin-based bio-ink patches have accelerated acute wound closure by promoting angiogenesis and re-epithelialization in rodent models. Personalized implants are another key application, where patient-derived cells are incorporated into bio-inks to create autologous constructs, minimizing immunogenicity and enabling customized geometries for defects like bone or cartilage loss. A notable case study is the 2019 FDA rare pediatric disease designation for AuriNovo, the first 3D-bioprinted ear implant using patient-derived auricular chondrocytes in a collagen-based bio-ink, targeted for microtia reconstruction; the product is currently in Phase 1/2a clinical trials as of 2025. Despite these advances, vascularization remains a limitation, with current bio-printed constructs viable at millimeter scales due to oxygen diffusion constraints beyond 200-300 μm, hindering progression to centimeter-sized organs.

Fabrication Challenges and Future Directions

One of the primary challenges in bio-ink fabrication is achieving effective vascularization in printed constructs, as the diffusion limit for oxygen and nutrients in avascular tissues is typically less than 200 μm, leading to necrosis in larger structures.^[57] This limitation hinders the development of clinically relevant thick tissues, where heterogeneous cell distribution and uneven nutrient supply further exacerbate viability issues in large-scale constructs.^[27] Additionally, regulatory hurdles, including compliance with Good Manufacturing Practice (GMP) standards for reproducibility and safety, pose significant barriers to translating bio-inks from laboratory prototypes to clinical applications.^[58] Fabrication processes face critical issues such as sterilizing bio-inks without compromising cell viability, as methods like ethylene oxide exposure can leave carcinogenic residues that damage encapsulated cells.^[59] Scalability remains a key obstacle, with high production costs—often exceeding $1000 per cm³—and the need for automated systems to bridge the gap from bench-scale experiments to clinical volumes.^[60] Looking ahead, multi-nozzle bioprinting systems enable the precise deposition of hybrid bio-inks, allowing for the creation of complex, multi-material constructs that mimic native tissue architectures.^[61] AI-optimized formulations are emerging to accelerate bio-ink design by predicting rheological properties and printability, reducing trial-and-error in development.^[62] Incorporation of nanomaterials, such as MXenes, into bio-inks enhances electrical conductivity, supporting applications in neural tissue engineering where bioelectric signaling is essential.^[63] Emerging trends include in vivo bioprinting via robotic delivery systems, which enable direct deposition of bio-inks onto injury sites for real-time tissue repair, overcoming ex vivo size limitations.^[64] Sustainable sourcing is also gaining traction, with plant-based alternatives to animal-derived decellularized extracellular matrix (dECM) offering renewable, cost-effective options that reduce ethical and environmental concerns.^[65]

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[26]
A Guide to Polysaccharide-Based Hydrogel Bioinks for 3D ... - NIH
This review portrays the most relevant advances in polysaccharide-based hydrogel bioinks for 3D bioprinting, focusing on the last five years.
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Recent advances in 3D bioprinting of polysaccharide-based bioinks ...
May 16, 2024 · This review encompasses recent advancements of polysaccharide-based hydrogels and bioinks for bioengineered tissue regeneration and reconstruction.
[28]
Bioink properties before, during and after 3D bioprinting - IOPscience
Sep 23, 2016 · The bioink properties before, during and after gelation are essential for its printability, comprising such features as achievable structural ...
[29]
Recent advances in 3D bioprinted polysaccharide hydrogels for ...
Jan 15, 2025 · This review comprehensively examines the fabrication methods, characteristics, and applications of polysaccharide hydrogel-driven 3D bioprinting
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https://pmc.ncbi.nlm.nih.gov/articles/PMC9223682/
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https://pubs.rsc.org/en/content/articlehtml/2024/me/d4me00001c
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https://iopscience.iop.org/article/10.1088/1758-5090/8/3/032002
[33]
Systematic evaluation of agarose- and agar-based bioinks for ... - PMC
Nov 21, 2022 · In general, agar bioinks gelled and melted at higher temperatures (T gel, mean = (37.0 ± 0.8)°C and T melt, mean = (92.5 ± 0.9)°C) than agarose ...
[34]
Current Trends on Protein Driven Bioinks for 3D Printing - PMC - NIH
Gelation time, stiffness and degradation rate, can be easily manipulated by varying the concentrations of HRP and H2O2 [108]. The proteins self-assembly ...
[35]
Advances in gelatin bioinks to optimize bioprinted cell functions - PMC
In this review, we summarize the progress in gelatin bioinks for 3D bioprinting, and critically analyze the compositions and crosslinking techniques.
[36]
Collagen as Bioink for Bioprinting: A Comprehensive Review - PMC
This review provides the latest evidence on advances of 3D bioprinting with collagen in the field of tissue engineering.
[37]
Fibrin-based Bioinks: New Tricks from an Old Dog - PMC
New tissue-specific bioinks with good printability, shape fidelity, and biocompatibility are based on “old” (well-known) biomaterials, particularly fibrin.
[38]
A comprehensive review on hydrogel-based bio-ink development for ...
The main objective of this article is to discuss the various strategies for preparing bio-inks to mimic native tissue's extracellular matrix environment.Missing: seminal | Show results with:seminal
[39]
Synthetic Polymers for Organ 3D Printing - PMC - PubMed Central
The commonly used biodegradable synthetic polymers include PLA, PGA, PU, PLGA and PCL, which have taken a priority role in hard tissue and organ 3D printing due ...
[40]
Bioprinting the future using light: A review on photocrosslinking ...
Light-based bioprinting uses light to control biomaterial formation, using photocrosslinking reactions and photoinitiators to create bonds between molecules.
[41]
Recent Advances and Perspective for Tissue Engineering - PMC - NIH
In this brief review, we discuss the recent advancements in using poly(ethylene glycol) diacrylate (PEGDA) hydrogels for tissue engineering applications.
[42]
Pluronic F127 Hydrogel Characterization and Biofabrication in ...
Pluronic F127 hydrogel at 25%w/v has fast gelation (5 min at 37°C), suitable viscoelastic properties, and fast viscosity recovery after shearing.
[43]
Study of sacrificial ink-assisted embedded printing for 3D perfusable ...
The objective of this study is to investigate the printability of sacrificial inks and the creation of perfusable channels in a cross-linkable yield-stress ...
[44]
Biomaterials / bioinks and extrusion bioprinting - ScienceDirect
Extrusion bioprinting is an emerging technique to apply or deposit these bioinks or biomaterial solutions to create three-dimensional (3D) constructs.
[45]
3D Bioprinting Photo-Crosslinkable Hydrogels for Bone and ... - NIH
The bio-ink A, composed of a PEG-clay nanocomposite crosslinked hydrogel, was used to prepare 3D-bioprinting and effectively deliver oxygen and nutrients to ...<|separator|>
[46]
Recent Trends in Decellularized Extracellular Matrix Bioinks for 3D ...
This review discusses the uses of decellularized ECM bioinks and argues that they represent the most biomimetic bioinks available.
[47]
Recent Advances in Decellularized Extracellular Matrix-Based ... - NIH
Apr 18, 2023 · We conducted this review to discuss the current status of and perspectives on dECM-based bioinks for bioprinting in tissue engineering.
[48]
Organ-Derived Decellularized Extracellular Matrix: A Game Changer ...
Mar 1, 2018 · Organ-Derived Decellularized Extracellular Matrix: A Game Changer for Bioink Manufacturing? ... Full text access. Highlights. Many individual ...Missing: text | Show results with:text
[49]
Bioprinted vascular tissue: Assessing functions from cellular ... - PMC
High cell viability as a prerequisite for vascular functions. The diffusion limit of oxygen and nutrients in nonvascular tissues is generally 100–200 μm ...
[50]
3D bioprinting patient-specific grafts for tendon/ligament ... - PMC - NIH
Aug 22, 2025 · 3.4 Challenges faced To achieve clinical application, reproducible, cost-effective, and scalable production compliant with Good Manufacturing ...
[51]
Bioinks adapted for in situ bioprinting scenarios of defect sites
Mar 3, 2023 · Residues from ethylene oxide sterilization are carcinogenic and affect the survival of encapsulated cells in bioinks, so the use of this ...<|control11|><|separator|>
[52]
Three-Dimensional Bioprinting of Human Organs and Tissues - NIH
Sep 7, 2023 · Various factors contribute to this, including the cost of bioinks, the complexity of the printing process, and the requirement for specialized ...
[53]
Multi-Nozzles 3D Bioprinting Collagen/Thermoplastic Elasto-Mer ...
Apr 2, 2025 · In this paper, a versatile multi-materials printing method for hybrid scaffolds was developed, enabling the composite printing of collagen ...
[54]
Bioink Formulation and Machine Learning-Empowered Bioprinting ...
Jun 13, 2022 · In this review, we explore current trends in bioink formulation and how ML has been used to accelerate optimization and enable real-time error detection.Missing: Patent 8236237
[55]
Innovative MXene/SilMA-Based Conductive Bioink for Three ...
Feb 7, 2025 · Conductive bioinks, integrated with 3D bioprinting and electrical stimulation, are essential for advancing neural tissue engineering.
[56]
Optimizing the value of bioinks and robotics to advance in vivo ...
In vivo bioprinting using robotic-assisted technologies. Robotic-assisted technology can increase the capabilities of delivering a bioink to a desired target.
[57]
Scaffolds Bioink for Three-Dimensional (3D) Bioprinting - PMC - NIH
Plant-based bioinks are renewable and biodegradable, which minimizes their environmental impact, and are also an inexpensive and abundant source of protein, ...Animal-Based Bioinks · Plant-Based Bioinks · Marine-Based Bioinks