Hybrid Bioink Research Articles

3D Bioprinted Chondrogenic Gelatin Methacrylate-Poly(ethylene glycol) Diacrylate Composite Scaffolds for Intervertebral Disc Restoration

Abstract Degenerative spine pathologies, including intervertebral disc (IVD) degeneration, present a significant healthcare challenge due to their association with chronic pain and disability. This study explores an innovative approach to IVD regeneration utilizing 3D bioprinting technology, specifically visible light-based digital light processing (VL-DLP), to fabricate tissue scaffolds that closely mimic the native architecture of the IVD. Utilizing a hybrid bioink composed of gelatin methacrylate (GelMA) and poly(ethylene glycol) diacrylate (PEGDA) at a 10% concentration, we achieved enhanced printing fidelity and mechanical properties suitable for load-bearing applications such as the IVD. Preconditioning rat bone marrow-derived mesenchymal stem cell (rBMSC) spheroids with chondrogenic media before incorporating them into the GelMA-PEGDA scaffold further promoted the regenerative capabilities of this system. Our findings demonstrate that this bioprinted scaffold not only supports cell viability and integration but also contributes to the restoration of disc height in a rat caudal disc model without inducing adverse inflammatory responses. The study underscores the potential of combining advanced bioprinting techniques and cell preconditioning strategies to develop effective treatments for IVD degeneration and other musculoskeletal disorders, highlighting the need for further research into the dynamic interplay between cellular migration and the hydrogel matrix.

Abstract Mo089: Fontan-Associated Liver Disease On A Chip

Introduction: The Fontan procedure is performed in children with single ventricle physiology, and has led to excellent short-term palliation and survival rates but at the cost of long-term complications. Fontan-associated liver disease (FALD) results from hemodynamic changes following the Fontan procedure, leading to elevated central venous pressures, chronic alterations in hepatic perfusion, and resultant hepatic fibrosis. Improved understanding of FALD pathophysiology, which may provide insights for prevention and treatment is challenged by the lack of experimental models. Current in vitro liver models, mostly 2D or suspension hepatic cell cultures, lack biomimicry. This study presents a 3D bioprinted perfusable human liver model for in vitro study of hepatic fibrosis in response to altered flow conditions in FALD patients. Methods: Vascular 3D model of hepatic sinusoid was biofabricated using hybrid bioinks in two distinct peripheral layers and a central lumen. The outer layer was cast with hepatic (HepG2) cells encapsulated in a mixture of gelatin methacrylate (GelMA) and collagen type I. The inner layer consisted of hepatic stellate cells (HSCs) in GelMA. Human endothelial cells (ECs) were seeded onto the central lumen. 3D cellular constructs were cultured in vitro for three weeks in static or dynamic conditions, by circulating the culture media at varying flow conditions to simulate sinusoid pressure characteristic of healthy vs. disease state. Using Volcano system in cath lab, we invasively measured the pressure in mid-channel. Cell viability, proliferation, maturation into functional liver tissue, and tissue stiffness were evaluated via microindentation, flow hemodynamics assays, metabolomic assays, and immunohistochemistry analyses. Results: Bioengineered liver constructs cultured under static and dynamic conditions demonstrated high cell viability and growth and full endothelialization of the sinusoid channel. HepG2 cells exhibited long-term function and maturation, forming clusters. Under FALD flow conditions, there were notable changes in EC-HepG2 cell morphology compared to dynamic flow in the healthy range, accompanied by reduced levels of functional markers. Conclusion: This proof-of-principle study introduces an innovative and highly biomimetic 3D model of hepatic sinusoid, which can serve as a robust platform for in vitro investigations of complex cell-microenvironment interactions in the context of FALD as well as other hepatic disorders.

Nanomaterials-Based Hybrid Bioink Platforms in Advancing 3D Bioprinting Technologies for Regenerative Medicine.

3D bioprinting is recognized as the ultimate additive biomanufacturing technology in tissue engineering and regeneration, augmented with intelligent bioinks and bioprinters to construct tissues or organs, thereby eliminating the stipulation for artificial organs. For 3D bioprinting of soft tissues, such as kidneys, hearts, and other human body parts, formulations of bioink with enhanced bioinspired rheological and mechanical properties were essential. Nanomaterials-based hybrid bioinks have the potential to overcome the above-mentioned problem and require much attention among researchers. Natural and synthetic nanomaterials such as carbon nanotubes, graphene oxides, titanium oxides, nanosilicates, nanoclay, nanocellulose, etc. and their blended have been used in various 3D bioprinters as bioinks and benefitted enhanced bioprintability, biocompatibility, and biodegradability. A limited number of articles were published, and the above-mentioned requirement pushed us to write this review. We reviewed, explored, and discussed the nanomaterials and nanocomposite-based hybrid bioinks for the 3D bioprinting technology, 3D bioprinters properties, natural, synthetic, and nanomaterial-based hybrid bioinks, including applications with challenges, limitations, ethical considerations, potential solution for future perspective, and technological advancement of efficient and cost-effective 3D bioprinting methods in tissue regeneration and healthcare.

Engineering Large-Scale Self-Mineralizing Bone Organoids with Bone Matrix-Inspired Hydroxyapatite Hybrid Bioinks.

Addressing large bone defects remains a significant challenge owing to the inherent limitations in self-healing capabilities, resulting in prolonged recovery and suboptimal regeneration. Although current clinical solutions are available, they have notable shortcomings, necessitating more efficacious approaches to bone regeneration. Organoids derived from stem cells show great potential in this field; however, the development of bone organoids has been hindered by specific demands, including the need for robust mechanical support provided by scaffolds and hybrid extracellular matrices (ECM). In this context, bioprinting technologies have emerged as powerful means of replicating the complex architecture of bone tissue. The research focused on the fabrication of a highly intricate bone ECM analog using a novel bioink composed of gelatin methacrylate/alginate methacrylate/hydroxyapatite (GelMA/AlgMA/HAP). Bioprinted scaffolds facilitate the long-term cultivation and progressive maturation of extensive bioprinted bone organoids, foster multicellular differentiation, and offer valuable insights into the initial stages of bone formation. The intrinsic self-mineralizing quality of the bioink closely emulates the properties of natural bone, empowering organoids with enhanced bone repair for both in vitro and in vivo applications. This trailblazing investigation propels the field of bone tissue engineering and holds significant promise for its translation into practical applications.

Adipose-derived stem cells loaded photocurable and bioprintable bioinks composed of GelMA, HAMA and PEGDA crosslinker to differentiate into smooth muscle phenotype

Developing a polymer-based photocrosslinked 3D printable scaffolds comprised of gelatin methacryloyl (G) and hyaluronic acid methacryloyl (H) incorporated with two molecular weights of polyethylene glycol diacrylate (P) of various concentrations that enables rabbit adipose-derived stem cells (rADSCs) to survive, grow, and differentiate into smooth muscle cells (SMCs). Then, the chemical modification and physicochemical properties of the PGH bioinks were evaluated. The cell viability was assessed via MTT, CCK-8 assay and visualized employing Live/Dead assay. In addition, the morphology and nucleus count of differentiated SMCs were investigated by adopting TRAP (tartrate-resistant acid phosphatase) staining, and quantitative RT-PCR analysis was applied to detect gene expression using two different SMC-specific gene markers α-SMA and SM-MHC. The SMC-specific protein markers namely α-SMA and SM-MHC were applied to investigate SMC differentiation ability by implementing Immunocytofluorescence staining (ICC) and western blotting. Moreover, the disk, square, and tubular cellular models of PGH7 (GelMA/HAMA=2/1) + PEGDA-8000 Da, 3% w/v) hybrid bioink were printed using an extrusion bioprinting and cell viability of rADSCs was also analysed within 3D printed square construct practising Live/Dead assay. The results elicited the overall viability of SMCs, conserving its phenotype in biocompatible PGH7 hybrid bioink revealing its great potential to regenerate SMCs associated organs repair.

3D Superclusters with Hybrid Bioinks for Early Detection in Breast Cancer.

The surface-enhanced Raman scattering (SERS) technique has garnered significant interest due to its ultrahigh sensitivity, making it suitable for addressing the growing demand for disease diagnosis. In addition to its sensitivity and uniformity, an ideal SERS platform should possess characteristics such as simplicity in manufacturing and low analyte consumption, enabling practical applications in complex diagnoses including cancer. Furthermore, the integration of machine learning algorithms with SERS can enhance the practical usability of sensing devices by effectively classifying the subtle vibrational fingerprints produced by molecules such as those found in human blood. In this study, we demonstrate an approach for early detection of breast cancer using a bottom-up strategy to construct a flexible and simple three-dimensional (3D) plasmonic cluster SERS platform integrated with a deep learning algorithm. With these advantages of the 3D plasmonic cluster, we demonstrate that the 3D plasmonic cluster (3D-PC) exhibits a significantly enhanced Raman intensity through detection limit down to 10-6 M (femtomole-(10-17 mol)) for p-nitrophenol (PNP) molecules. Afterward, the plasma of cancer subjects and healthy subjects was used to fabricate the bioink to build 3D-PC structures. The collected SERS successfully classified into two clusters of cancer subjects and healthy subjects with high accuracy of up to 93%. These results highlight the potential of the 3D plasmonic cluster SERS platform for early breast cancer detection and open promising avenues for future research in this field.

Tissue-specific gelatin bioink as a rheology modifier for high printability and adjustable tissue properties.

Decellularized extracellular matrix (dECM) has emerged as an exceptional biomaterial that effectively recapitulates the native tissue microenvironment for enhanced regenerative potential. Although various dECM bioinks derived from different tissues have shown promising results, challenges persist in achieving high-resolution printing of flexible tissue constructs because of the inherent limitations of dECM's weak mechanical properties and poor printability. Attempts to enhance mechanical rigidity through chemical modifications, photoinitiators, and nanomaterial reinforcement have often compromised the bioactivity of dECM and mismatched the desired mechanical properties of target tissues. In response, this study proposes a novel method involving a tissue-specific rheological modifier, gelatinized dECM. This modifier autonomously enhances bioink modulus pre-printing, ensuring immediate and precise shape formation upon extrusion. The hybrid bioink with GeldECM undergoes a triple crosslinking system-physical entanglement for pre-printing, visible light photocrosslinking during printing for increased efficiency, and thermal crosslinking post-printing during tissue culture. A meticulous gelatinization process preserves the dECM protein components, and optimal hybrid ratios modify the mechanical properties, tailoring them to specific tissues. The application of this sequential multiple crosslinking designs successfully yielded soft yet resilient tissue constructs capable of withstanding vigorous agitation with high shape fidelity. This innovative method, founded on mechanical modulation by GeldECM, holds promise for the fabrication of flexible tissues with high resilience.

In Vitro and In Vivo Evaluation of 3D Printed Poly(Ethylene Glycol) Dimethacrylate-Based Photocurable Hydrogel Platform for Bone Tissue Engineering.

This study focuses to develop a unique hybrid hydrogel bioink formulation that incorporates poly(ethylene glycol) dimethacrylate (PEGDMA), gelatin (Gel), and methylcellulose (MC). This formulation achieves the necessary viscosity for extrusion-based three-dimensional (3D) printing of scaffolds intended for bone regeneration. After thorough optimization of the hybrid bioink system with Gel, three distinct scaffold groups are investigated in vitro: 0%, 3%, and 6% (w/v) Gel. These scaffold groups are examined for their morphology, mechanical strength, biodegradation, in vitro cell proliferation and differentiation, and in vivo bone formation using a rat cranial defect model. Among these scaffold compositions, the 3% Gel scaffold exhibits the most favorable characteristics, prompting further evaluation as a rat mesenchymal stem cell (rMSC) carrier in a critical-size cranial defect within a Lewis rat model. The compressive strength of all three scaffold groups range between 1 and 2MPa. Notably, the inclusion of Gel in the scaffolds leads to enhanced bioactivity and cell adhesion. The Gel-containing scaffolds notably amplify osteogenic differentiation, as evidenced by alkaline phosphatase (ALP) and Western blot analyses. The in vivo results, as depicted by microcomputed tomography, showcase augmented osteogenesis within cell-seeded scaffolds, thus validating this innovative PEGDMA-based scaffold system as a promising candidate for cranial bone defect healing.

3D bioprinted multilayered cerebrovascular conduits to study cancer extravasation mechanism related with vascular geometry

Cerebral vessels are composed of highly complex structures that facilitate blood perfusion necessary for meeting the high energy demands of the brain. Their geometrical complexities alter the biophysical behavior of circulating tumor cells in the brain, thereby influencing brain metastasis. However, recapitulation of the native cerebrovascular microenvironment that shows continuities between vascular geometry and metastatic cancer development has not been accomplished. Here, we apply an in-bath 3D triaxial bioprinting technique and a brain-specific hybrid bioink containing an ionically crosslinkable hydrogel to generate a mature three-layered cerebrovascular conduit with varying curvatures to investigate the physical and molecular mechanisms of cancer extravasation in vitro. We show that more tumor cells adhere at larger vascular curvature regions, suggesting that prolongation of tumor residence time under low velocity and wall shear stress accelerates the molecular signatures of metastatic potential, including endothelial barrier disruption, epithelial–mesenchymal transition, inflammatory response, and tumorigenesis. These findings provide insights into the underlying mechanisms driving brain metastases and facilitate future advances in pharmaceutical and medical research.

Development of Plum Seed-Derived Carboxymethylcellulose Bioink for 3D Bioprinting.

Three-dimensional bioprinting represents an innovative platform for fabricating intricate, three-dimensional (3D) tissue structures that closely resemble natural tissues. The development of hybrid bioinks is an actionable strategy for integrating desirable characteristics of components. In this study, cellulose recovered from plum seed was processed to synthesize carboxymethyl cellulose (CMC) for 3D bioprinting. The plum seeds were initially subjected to α-cellulose recovery, followed by the synthesis and characterization of plum seed-derived carboxymethyl cellulose (PCMC). Then, hybrid bioinks composed of PCMC and sodium alginate were fabricated, and their suitability for extrusion-based bioprinting was explored. The PCMC bioinks exhibit a remarkable shear-thinning property, enabling effortless extrusion through the nozzle and maintaining excellent initial shape fidelity. This bioink was then used to print muscle-mimetic 3D structures containing C2C12 cells. Subsequently, the cytotoxicity of PCMC was evaluated at different concentrations to determine the maximum acceptable concentration. As a result, cytotoxicity was not observed in hydrogels containing a suitable concentration of PCMC. Cell viability was also evaluated after printing PCMC-containing bioinks, and it was observed that the bioprinting process caused minimal damage to the cells. This suggests that PCMC/alginate hybrid bioink can be used as a very attractive material for bioprinting applications.

3D printing of high-strength photo-crosslinking flaxseed gum bioink for cartilage regeneration

Three-dimensional (3D) bioprinting provides a new possibility for personal customization of cartilage tissues. Although biocompatible, most natural biopolymer inks have poor mechanical strength to bear repeated extrusion, incomparable with load-bearing cartilage. By utilizing a heteropolysaccharide called flaxseed gum (FG), a strong photo-crosslinked methacrylated FG (FGMA) bioink was synthetized and integrated with stem cells for cartilage defect therapy. As a hybrid bioink, FGMA has favorable 3D printability and shown to exhibit high mechanical strength and superior fatigue resistant ability. 4% FGMA2 (MA substitution = 8.1%) has the modulus of about 41 times of GelMA and could maintain its structure integrity under 60% deformation after 2000 cycles. FGMA2 has a degradation period of about 66 days, similar to that of GelMA. In vitro study shows that FGMA2 has excellent biocompatibility with stem cells and chondrogenic potential, both beneficial to cartilage regeneration in the cartilage lesion model. More importantly, in vivo study demonstrated that the regenerated neo-cartilage tissue by FGMA2 displayed the similar morphology and matchable mechanical strength to the natural cartilage after 8 weeks. In conclusion, FGMA has demonstrated this potential as a high-performance bioink for 3D printing for tissue/organ regeneration.

Printability assessment of modified filament deposition modelling three dimensional bioprinter printer using polymeric formulations

Three dimensional (3D) bioprinting is a rapid prototyping technology that can be used to accurately position living cells and biomaterials called bioink, to fabricate functional living tissue constructs or organs. The bioink are deposited in 3D in a layer-by-layer manner using a bioprinter. However, commercially available 3D bioprinters are expensive, limiting the widespread adoption of this technology in low-resource laboratories. To overcome this limitation the conventional Filament Deposition Modelling (FDM) 3D printer can be modified to a 3D bioprinter by replacing the print head unit. During the makeover, The device has to perform in its most efficient capacity in synergy with the bioink used. Hence it is essential to check the bio-printabililty of bioink in the modified FDM printer. In this study we created certain specific G-codes for the evaluation of hybrid bioinks and authenticated the printability. This study focuses on quantifying the printability of a hybrid hydrogel composed of sodium salt carboxymethyl cellulose (CMC) and gelatin using a 3D bioprinter based on the RepRap prototyper. The 3D design and slicing parameters were generated using opensource software and manually edited for printability evaluation. The results of these experiments indicate the importance of printability evaluation of custom bioprinters and provide some key aspects of how to modify CAD design parameters for printability evaluation. This approach can also be adopted to evaluate the printability of other hydrogels for bio-printing. The codes are created to evaluate the printability of bioink using FDM modified bioprinters. The printability evaluation is limited to high viscous bioink for extrusion bioprinting. A user friendly, simple G-Codes and methodology for evaluation of printability of bioink using FDM modified bioprinters. To authors knowledge, this is the first report on 3D printability evaluation of bioink using FDM modified bioprinters. The study also fulfills an identified need to study printability of bioink in biofabrication by additive manufacturing.

Hepar-on-a-sensor-platform with hybridization chain reaction amplification strategy to intuitively monitor the hepatoxicity of natural compounds.

The irrational use of natural compounds in the treatment of diseases can lead to serious side effects, especially hepatoxicity, and its toxic effects are usually cumulative and imperceptible. Therefore, an accurate sensing platform is urgently needed to monitor the hepatotoxicity of natural compounds. Here, we deposited a thermo-responsive alginate-RGD/Pluronic hydrogel to construct an in vitro three-dimensional(3D) hepar-platform, and a thorough validation was adopted to evaluate the bioprinted hepatic constructs. The engineered hepar-platform was then employed to access its biological response toward Emodin (EM) and Triptolide (TP), two typical hepatotoxic natural compounds. Subsequently, we integrated it with a robust fluorescent sensor based on hybridization chain reaction amplification strategy (HCR) to monitor the early hepatotoxic biomarker - glutathione-S-transferase-alpha (GST-α) secreted by this 3D constructs. Our study was the first attempt to construct an accurate hepar-on-a-sensor platform that could effectively detect GST-α for monitoring the hepatoxic effects of natural compounds. The limit of detection of the platform was 0.3 ng ml-1 and the accuracy of this platform was verified by enzyme linked immunosorbent assay. Furthermore, the variation of GST-α induced by EM and TP was consistent with hepatotoxicity studies, thus providing an important application value for evaluating the hepatotoxicity of natural compounds. STATEMENT OF SIGNIFICANCE: 1. We deposited a thermo-responsive alginate-RGD/Pluronic hydrogel to construct an in vitro three-dimensional(3D) hepar-platform, and elucidated the essential reasons why hybrid bioinks more suitable for 3D extrusion from biomaterials itself. Also, a thorough validation associated with a series of important proteins and genes involved in liver cell metabolism was adopted to evaluate the bioprinted hepatic constructs accurately 2. Glutathione-S-transferase-alpha is a soluble trace biomarker for acute hepatotoxic injury, the hepatotoxic effects of natural compounds on the secretion of GST-α has not been reported to date. We integrated our 3D hepar-platform with recognition molecules-aptamers and HCR amplification strategy to monitor the variation of GST-α, aiming at developing a robust and stable fluorescent biosensing platform to monitor the hepatoxicity of natural compounds.

Tunable metacrylated silk fibroin-based hybrid bioinks for the bioprinting of tissue engineering scaffolds.

Three-dimensional (3D) bioprinting is a powerful technique for the production of tissue-like structures to study cell behavior and tissue properties. A major challenge in 3D extrusion bioprinting is the limited diversity of bioinks, which fulfills the requirements of shear-thinning and strain recovery behaviors and can be solidified by a crosslinking process to retain their shape after printing. Herein, we aimed to develop a natural biopolymer-based formula with dual crosslinking performance to formulate a cell-laden bioink. In this study, methacrylate gelatin (GelMA) and methacrylated silk fibroin (SFMA) with different degrees of methacrylation were fabricated into hybrid bioinks. The GelMA/SFMA bioink of an optimal degree provides excellent rheological properties for extrusion bioprinting, and its hydrogel precursor polymer can form a polymer network at a low temperature and the high shape fidelity of the printed construct through photocrosslinking. Moreover, the hydrogel bioink can encapsulate different types of cells together to create 3D printed constructs that mimic the cellular microenvironment at a microscale level. Human umbilical vein endothelial cells (HUVECs) and rat pheochromocytoma (PC12) cells encapsulated in the 3D printed constructs can maintain high viability and proliferation ability for a long time. Furthermore, the GelMA/SFMA hydrogels were implanted in the subcutaneous tissue of SD rats for the evaluation of biocompatibility and degradability in vivo. Thus, the proposed GelMA/SFMA bioink expands the palette of available bioinks and offers opportunities for biomedical applications such as tissue engineering and soft robotics in clinical applications.

Fluidic integrated 3D bioprinting system to sustain cell viability towards larynx fabrication.

Herein, we report the first study to create a three-dimensional (3D) bioprinted artificial larynx for whole-laryngeal replacement. Our 3D bio-printed larynx was generated using extrusion-based 3D bioprinter with rabbit's chondrocyte-laden gelatin methacryloyl (GelMA)/glycidyl-methacrylated hyaluronic acid (GMHA) hybrid bioink. We used a polycaprolactone (PCL) outer framework incorporated with pores to achieve the structural strength of printed constructs, as well as to provide a suitable microenvironment to support printed cells. Notably, we established a novel fluidics supply (FS) system that simultaneously supplies basal medium together with a 3D bioprinting process, thereby improving cell survival during the printing process. Our results showed that the FS system enhanced post-printing cell viability, which enabled the generation of a large-scale cell-laden artificial laryngeal framework. Additionally, the incorporation of the PCL outer framework with pores and inner hydrogel provides structural stability and sufficient nutrient/oxygen transport. An animal study confirmed that the transplanted 3D bio-larynx successfully maintained the airway. With further development, our new strategy holds great potential for fabricating human-scale larynxes with in vivo-like biological functions for laryngectomy patients.

Blends of gelatin and hyaluronic acid stratified by stereolithographic bioprinting approximate cartilaginous matrix gradients.

Stereolithographic bioprinting holds great promise in the quest for creating artificial, biomimetic cartilage-like tissue. To introduce a more biomimetic approach, we examined blending and stratifying methacrylated hyaluronic acid (HAMA) and methacrylated gelatin (GelMA) bioinks to mimic the zonal structure of articular cartilage. Bioinks were suspended with porcine chondrocytes before being printed in a digital light processing approach. Homogenous constructs made from hybrid bioinks of varying polymer ratios as well as stratified constructs combining different bioink blends were cultivated over 14 days and analyzed by histochemical staining for proteoglycans/collagen type II, cartilage marker expression analysis, and for cellular viability. The stiffness of blended bioinks increased gradually with HAMA content, from 2.41 ± 0.58 kPa (5% GelMA, 0% HAMA) to 8.84 ± 0.11 kPa (0% GelMA, 2% HAMA). Cell-laden constructs maintained vital chondrocytes and supported the formation of proteoglycans and collagen type II. Higher concentrations of GelMA resulted in increased formation of cartilaginous matrix proteins and a more premature phenotype. However, decreased matrix production in central areas of constructs was observed in higher GelMA content constructs. Biomimetically stratified constructs retained their gradient-like structure even after ECM formation, and exclusively exhibited a significant increase in COL2A1 gene expression (+178%). Concluding, we showed the feasibility of blending and stratifying photopolymerizable, natural biopolymers by SLA bioprinting to modulate chondrocyte attributes and to create zonally segmented ECM structures, contributing to improved modeling of cartilaginous tissue for regenerative therapies or in vitro models.

Design and Synthesis of Stem Cell-Laden Keratin/Glycol Chitosan Methacrylate Bioinks for 3D Bioprinting

With the advancements in tissue engineering and three-dimensional (3D) bioprinting, physiologically relevant three-dimensional structures with suitable mechanical and bioactive properties that mimic the biological tissue can be designed and fabricated. However, the available bioinks are less than demanded. In this research, the readily available biomass sources, keratin and glycol chitosan, were selected to develop a UV-curable hydrogel that is feasible for the 3D bioprinting process. Keratin methacrylate and glycol chitosan methacrylate were synthesized, and a hybrid bioink was created by combining this protein-polysaccharide cross-linked hydrogel. While human hair keratin could provide biological functions, the other composition, glycol chitosan, could further enhance the mechanical strength of the construct. The mechanical properties, degradation profile, swelling behavior, cell viability, and proliferation were investigated with various ratios of keratin methacrylate to glycol chitosan methacrylate. The composition of 2% (w/v) keratin methacrylate and 2% (w/v) chitosan methacrylate showed a significantly higher cell number and swelling percentage than other compositions and was designated as the bioink for 3D printing afterward. The feasibility of stem cell loading in the selected formula was examined with an extrusion-based bioprinter. The cells and spheroids can be successfully printed with the synthesized bioink into a specific shape and cultured. This work provides a potential option for bioinks and delivers insights into personalization research on stem cell-laden biofabricated hydrogels in the future.

Engineering Densely Packed Adipose Tissue via Environmentally Controlled In‐Bath 3D Bioprinting

AbstractThe adipose tissue is a crucial endocrine organ that coordinates with other organs, playing a key role in metabolic regulation. However, it remains challenging to recreate the morphology of native adipose tissue with fully packed adipogenic lipid droplets, mainly because of immature adipogenesis caused by the low cell density of current adipose constructs. This study suggests environmentally controlled in‐bath 3D bioprinting to create fully‐mature densely packed adipose tissue (DPAT) in vitro. As a bath suspension, a hybrid bioink composed of 1% alginate and 1.5% adipose‐derived decellularized matrix is developed for selective and compact cell proliferation. In the hybrid bath suspension, the construct printed at a high cell density (>107 cells ml−1) proliferates within the predefined area without unwanted cell migration throughout the bath suspension, forming a densely packed cellular microenvironment. After adipogenesis for 4 weeks, the results demonstrate that selectively proliferated preadipocytes can differentiate into lipid‐accumulating mature adipocytes. The resulting in vivo‐like DPAT is successfully engineered in vitro. The DPAT construct shows the physiological changes associated with obesity under the relevant conditions found in obese patients. The recapitulation of obesity‐induced inflamed adipose tissue through co‐culture with monocytes reveals the potency of the proposed strategy as a promising solution to overcome obesity‐related complications.

3D bioprinted gelatin/gellan gum-based scaffold with double-crosslinking network for vascularized bone regeneration

Three-dimensional (3D) bioprinting holds promise for precise repair of bone defects, but rapid formation of effective vascularized tissue by 3D-printed construct is still a challenge. In this study, deferoxamine (DFO)-loaded ethosomes (Eth) were combined with gelatin methacrylate (GelMA)/gellan gum methacrylate (GGMA) hybrid bioink to fabricate 3D-printed scaffold by photo- and ion-crosslinking. The GelMA/GGMA bioinks showed excellent printability and improved mechanical property through the double-crosslinking method. In vitro experiments showed that Eth-DFO@GelMA/GGMA scaffold had good cytocompatibility while achieved sustained release of DFO, which significantly promoted endothelial cells migration and tube formation, mineralized matrix deposition and alkaline phosphatase expression of osteoblast. In vivo experiments of rat cranial defect model demonstrated that composite scaffold could promote angiogenesis and bone regeneration by activating the hypoxia-inducible factor 1-α (HIF1-α) signaling pathway. In conclusion, this 3D bioprinted Eth-DFO@GelMA/GGMA scaffold can couple angiogenesis and osteogenesis, and will be a promising candidate for the bone defects treatment.

Multiscale Anisotropic Tissue Biofabrication via Bulk Acoustic Patterning of Cells and Functional Additives in Hybrid Bioinks.

Recapitulation of the microstructural organization of cellular and extracellular components found in natural tissues is an important but challenging feat for tissue engineering, which demands innovation across both process and material fronts. In this work, a highly versatile ultrasound-assisted biofabrication (UAB) approach is demonstrated that utilizes radiation forces generated by superimposing ultrasonic bulk acoustic waves to rapidly organize arrays of cells and other biomaterial additives within single and multilayered hydrogel constructs. UAB is used in conjunction with a novel hybrid bioink system, comprising of cartilage-forming cells (human adipose-derived stem cells or chondrocytes) and additives to promote cell adhesion (collagen microaggregates or polycaprolactone microfibers) encapsulated within gelatin methacryloyl (GelMA) hydrogels, to fabricate cartilaginous tissue constructs featuring bulk anisotropy. The hybrid matrices fabricated under the appropriate synergistic thermo-reversible and photocrosslinking conditions demonstrate enhanced mechanical stiffness, stretchability, strength, construct shape fidelity and aligned encapsulated cell morphology and collagen II secretion in long-term culture. Hybridization of UAB is also shown with extrusion and stereolithography printing to fabricate constructs featuring 3D perfusable channels for vasculature combined with a crisscross or circumferential organization of cells and adhesive bioadditives, which is relevant for further translation of UAB toward complex physiological-scale biomimetic tissue fabrication.