3D Bioprinting Approaches Research Articles

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.

Novel 3D bioprinting approach for spinal cord injury repair using neural stem cells and TGF-β1 monoclonal antibody

ObjectiveTo investigate the repair of spinal cord injury (SCI) in rats using 3D bioprinted decellularized extracellular matrix (dECM) hydrogel, neural stem cells (NSCs), and TGF-β1 monoclonal antibody. MethodsThe spinal cord-derived dECM hydrogel (SC-dECM-gel) was optimized for 3D printing. Rheological tests established a 3 ​% concentration as optimal. In vitro tests assessed the effects of TGF-β1 monoclonal antibody on NSC viability and differentiation. A rat SCI model was treated with the printed gel, and recovery was monitored using the Basso-Beattie-Bresnahan test and evoked potentials. ResultsThe 3 ​% SC-dECM-gel showed superior rheological properties. TGF-β1 monoclonal antibody enhanced NSC survival and differentiation in vitro. In vivo, the 3D bioprinted gel significantly improved motor function and promoted neuronal regeneration. Conclusion3D bioprinted SC-dECM-gel loaded with NSCs and TGF-β1 monoclonal antibody effectively restores motor function and promotes regeneration in SCI rats, offering a promising approach for SCI treatment.

3D Bioprinting Using Universal Fugitive Network Bioinks.

Three-dimensional (3D) bioprinting has emerged with potential for creating functional 3D tissues with customized geometries. However, the limited availability of bioinks capable of printing 3D structures with both high-shape fidelity and desired biological environments for encapsulated cells remains a key challenge. Here, we present a 3D bioprinting approach that uses universal fugitive network bioinks prepared by loading cells and hydrogel precursors (bioink base materials) into a 3D printable fugitive carrier. This approach constructs 3D structures of cell-encapsulated hydrogels by printing 3D structures using fugitive network bioinks, followed by cross-linking printed structures and removing the carrier from them. The use of the fugitive carrier decouples the 3D printability of bioinks from the material properties of bioink base materials, making this approach readily applicable to a range of hydrogel systems. The decoupling also enables the design of bioinks for the biological functionality of the final printed constructs without compromising the 3D printability. We demonstrate the generalizable 3D printability by printing self-supporting 3D structures of various hydrogels, including conventionally non-3D printable hydrogels and those with a low polymer content. We conduct preprinting screening of bioink base materials through 3D cell culture to select bioinks with high cell compatibility. The selected bioinks produce 3D constructs of cell-encapsulated hydrogels with both high-shape fidelity and high cell viability and proliferation. The universal fugitive network bioink platform could significantly expand 3D printable bioinks with customizable biological functionalities and the adoption of 3D bioprinting in diverse research and applied settings.

Engineering Conductive Hydrogels with Tissue‐like Properties: A 3D Bioprinting and Enzymatic Polymerization Approach

Hydrogels are promising materials for medical devices interfacing with neural tissues due to their similar mechanical properties. Traditional hydrogel‐based bio‐interfaces lack sufficient electrical conductivity, relying on low ionic conductivity, which limits signal transduction distance. Conducting polymer hydrogels offer enhanced ionic and electronic conductivities and biocompatibility but often face challenges in processability and require aggressive polymerization methods. Herein, we demonstrate in situ enzymatic polymerization of π‐conjugated monomers in a hyaluronan (HA)‐based hydrogel bioink to create cell‐compatible, electrically conductive hydrogel structures. These structures were fabricated using 3D bioprinting of HA‐based bioinks loaded with conjugated monomers, followed by enzymatic polymerization via horseradish peroxidase. This process increased the hydrogels’ stiffness from about 0.6 to 1.5 kPa and modified their electroactivity. The components and polymerization process were well‐tolerated by human primary dermal fibroblasts and PC12 cells. This work presents a novel method to fabricate cytocompatible and conductive hydrogels suitable for bioprinting. These hybrid materials combine tissue‐like mechanical properties with mixed ionic and electronic conductivity, providing new ways to use electricity to influence cell behavior in a native‐like microenvironment.

Precision improvement of robotic bioprinting via vision-based tool path compensation

Robotic 3D bioprinting is a rapidly advancing technology with applications in organ fabrication, tissue restoration, and pharmaceutical testing. While the stepwise generation of organs characterizes bioprinting, challenges such as non-linear material behavior, layer shifting, and trajectory tracking are common in freeform reversible embedding of suspended hydrogels (FRESH) bioprinting, leading to imperfections in complex organ construction. To overcome these limitations, we propose a computer vision-based strategy to identify discrepancies between printed filaments and the reference robot path. Employing error compensation techniques, we generate an adjusted reference path, enhancing robotic 3D bioprinting by adapting the robot path based on vision system data. Experimental assessments confirm the reliability and agility of our vision-based robotic 3D bioprinting approach, showcasing precision in fabricating human blood vessel segments through case studies. Significantly, it minimizes the printing layer width disparity to just 0.15 mm compared to the 0.6 mm in traditional methods, and it decreases the average error for curved filaments to 7.0 mm2 from the previous 12.7 mm2 in conventional printing. While these results underscore the significant potential of our innovation in creating precise biomimetic constructs, further investigation is necessary to tackle challenges such as accurately distinguishing closely stacked layers using a vision system, especially under varying lighting conditions. These limitations, coupled with issues of computational complexity and scalability in larger-scale bioprinting, emphasize the importance of enhancing the reliability of the vision-based approach across various conditions. Nonetheless, our innovation demonstrates substantial promise in creating precise biomimetic constructs and paves the way for future advancements in vision-guided robotic bioprinting, including the integration of multi-material printing techniques to enhance versatility.

Advances in soft tissue wound care: A Comprehensive Review of Facial traumatic injuries

Soft tissue wound care, particularly in the context of facial traumatic injuries, has undergone significant progress propelled by interdisciplinary collaborations between medical professionals and engineers. This comprehensive review explores the evolution of traditional and advanced techniques for managing soft tissue wounds, emphasizing innovations in scar prevention and the integration of cutting-edge technologies. This review covers the immediate assessment and management of facial traumatic injuries, traditional wound care approaches, and the latest advancements in soft tissue wound healing. Notably, collaborative efforts in biomechanical engineering have led to the development of bioengineered scaffolds, innovative dressings, and customizable implants that promise to revolutionize wound closure and tissue regeneration. The integration of robotics into soft tissue wound care procedures has shown promise in improving precision, reducing invasiveness, and enhancing surgical interventions. Additionally, regenerative technologies, including tissue engineering, 3D bioprinting, and regenerative medicine approaches, offer novel strategies for harnessing the body's natural healing processes. Looking ahead, the future of soft tissue wound care holds exciting prospects, including advancements in technology and imaging for more precise diagnostics, personalized medicine approaches tailored to individual patient profiles, and continued collaboration between medicine and engineering to develop innovative solutions. These future directions are poised to redefine the landscape of soft tissue wound care, offering improved outcomes and quality of life for individuals with facial traumatic injuries and other complex wounds.

Development of a microfluidic-assisted open-source 3D bioprinting system (MOS3S) for the engineering of hierarchical tissues

The engineering of new 3D bioprinting approaches has shown great promise in the field of tissue engineering and disease modelling. However, the high cost of commercial 3D bioprinters has limited their accessibility, especially to those laboratories in resource-limited settings. Moreover, the need for a 3D bioprinting system capable of dispensing multiple materials is growing apace. Therefore, the development of a Microfluidic-assisted Open Source 3D bioprinting System (MOS3S) for the engineering of hierarchical tissues is needed to progress in fabricating functional tissues, but with a technology accessible to a wider range of researchers. The MOS3S platform is designed to allow the deposition of biomaterial inks using microfluidic printheads or coaxial nozzles for the in-situ crosslinking and scaffolds fabrication. The coupling of 3D printed syringe pumps with the motion control system is used for driving the tunable extrusion of inks for the fabrication of centimeter scale hierarchical lattice constructs for tissue engineering purposes. MOS3S performance have been validated to fabricate high-resolution structures with coaxial microfluidic technology, opening to new frontiers for seminal studies in pre-clinical disease modelling and tissue regeneration.

Towards optimized tissue regeneration: a new 3D printable bioink of alginate/cellulose hydrogel loaded with thrombocyte concentrate.

Autologous platelet concentrate (APC) are pro-angiogenic and can promote wound healing and tissue repair, also in combination with other biomaterials. However, challenging defect situations remain demanding. 3D bioprinting of an APC based bioink encapsulated in a hydrogel could overcome this limitation with enhanced physio-mechanical interface, growth factor retention/secretion and defect-personalized shape to ultimately enhance regeneration. This study used extrusion-based bioprinting to create a novel bioink of alginate/cellulose hydrogel loaded with thrombocyte concentrate. Chemico-physical testing exhibited an amorphous structure characterized by high shape fidelity. Cytotoxicity assay and incubation of human osteogenic sarcoma cells (SaOs2) exposed excellent biocompatibility. enzyme-linked immunosorbent assay analysis confirmed pro-angiogenic growth factor release of the printed constructs, and co-incubation with HUVECS displayed proper cell viability and proliferation. Chorioallantoic membrane (CAM) assay explored the pro-angiogenic potential of the prints in vivo. Detailed proteome and secretome analysis revealed a substantial amount and homologous presence of pro-angiogenic proteins in the 3D construct. This study demonstrated a 3D bioprinting approach to fabricate a novel bioink of alginate/cellulose hydrogel loaded with thrombocyte concentrate with high shape fidelity, biocompatibility, and substantial pro-angiogenic properties. This approach may be suitable for challenging physiological and anatomical defect situations when translated into clinical use.

Biofabrication of nanocomposite-based scaffolds containing human bone extracellular matrix for the differentiation of skeletal stem and progenitor cells

Autograft or metal implants are routinely used in skeletal repair. However, they fail to provide long-term clinical resolution, necessitating a functional biomimetic tissue engineering alternative. The use of native human bone tissue for synthesizing a biomimetic material ink for three-dimensional (3D) bioprinting of skeletal tissue is an attractive strategy for tissue regeneration. Thus, human bone extracellular matrix (bone-ECM) offers an exciting potential for the development of an appropriate microenvironment for human bone marrow stromal cells (HBMSCs) to proliferate and differentiate along the osteogenic lineage. In this study, we engineered a novel material ink (LAB) by blending human bone-ECM (B) with nanoclay (L, Laponite®) and alginate (A) polymers using extrusion-based deposition. The inclusion of the nanofiller and polymeric material increased the rheology, printability, and drug retention properties and, critically, the preservation of HBMSCs viability upon printing. The composite of human bone-ECM-based 3D constructs containing vascular endothelial growth factor (VEGF) enhanced vascularization after implantation in an ex vivo chick chorioallantoic membrane (CAM) model. The inclusion of bone morphogenetic protein-2 (BMP-2) with the HBMSCs further enhanced vascularization and mineralization after only seven days. This study demonstrates the synergistic combination of nanoclay with biomimetic materials (alginate and bone-ECM) to support the formation of osteogenic tissue both in vitro and ex vivo and offers a promising novel 3D bioprinting approach to personalized skeletal tissue repair.Graphic abstract

Upconversion 3D Bioprinting for Noninvasive In Vivo Molding.

Tissue engineered bracket materials provide essential support for the physiological protection and therapeutics of patients. Unfortunately, the implantation process of such devices poses the risk of surgical complications and infection. In this study, an upconversion nanoparticles (UCNPs)-assisted 3D bioprinting approach is developed to realize in vivo molding that is free from invasive surgery. Reasonably designed UCNPs, which convert near-infrared (NIR) photons that penetrate skin tissues into blue-violet emission (300-500 nm), induce a monomer polymerization curing procedure in vivo. Using a fused deposition modeling coordination framework, a precisely predetermined trajectory of the NIR laser enables the manufacture of implantable medical devices with tailored shapes. A proof of the 3D bioprinting of a noninvasive fracture fixation scaffold is achieved successfully, thus demonstrating an entirely new method of in vivo molding for biomedical treatment.

Engineering peptide-modified alginate-based bioinks with cell-adhesive properties for biofabrication.

Alginate (ALG) and its oxidised form alginate-dialdehyde (ADA) are highly attractive materials for hydrogels used in 3D bioprinting as well as drop-on-demand (DoD) approaches. However, both polymers need to be modified using cell-adhesive peptide sequences, to obtain bioinks exhibiting promising cell-material interactions. Our study explores the modification of ALG- and ADA-based bioinks with the adhesive peptides YIGSR (derived from laminin), RRETEWA (derived from fibronectin) and IKVAV (derived from laminin) for 3D bioprinting. Two coupling methods, carbodiimide and Schiff base reactions, were employed to modify the polymers with peptides. Analytical techniques, including FTIR and NMR were used to assess the chemical composition, revealing challenges in confirming the presence of peptides. The modified bioinks exhibited decreased stability, viscosity, and stiffness, particularly-ADA-based bioinks in contrast to ALG. Sterile hydrogel capsules or droplets were produced using a manual manufacturing process and DoD printing. NIH/3T3 cell spreading analysis showed enhanced cell spreading in carbodiimide-modified ADA, Schiff base-modified ADA, and PEG-Mal-modified ADA. The carbodiimide coupling of peptides worked for ADA, however the same was not observed for ALG. Finally, a novel mixture of all used peptides was evaluated regarding synergistic effects on cell spreading which was found to be effective, showing higher aspect ratios compared to the single peptide coupled hydrogels in all cases. The study suggests potential applications of these modified bioinks in 3D bioprinting approaches and highlights the importance of peptide selection as well as their combination for improved cell-material interactions.

Robust Generation of ASC Spheroids for Use as 3D Cultures and in Bioprinted Tissue Models.

Three-dimensional (3D) cell culture techniques have become a valuable tool to mimic the complex interactions of cells with each other and their surrounding extracellular matrix as they occur in vivo. In this respect, 3D spheroids are widely acknowledged as self-assembled cellular aggregates that can be generated from a variety of cell types without the need for exogenous material while being highly reproducible, easy to handle, and cost-effective. Furthermore, due to their capacity to be developed into microtissues, spheroids represent potential building blocks for various tissue engineering applications, including 3D bioprinting approaches for tissue model development. Adipose-derived stromal/stem cells (ASCs), due to their ease of isolation, multipotent nature, and secretory capacity, represent an attractive cell source employed in numerous tissue engineering studies and other cell-based therapy approaches. In this chapter, we describe two procedures for robust spheroid generation, namely the liquid overlay technique, either using agarose-coated 96-well plates or employing agarose-cast micromolds. Furthermore, we show, in principle, the generation of ASC spheroids with subsequent adipogenic differentiation and the spheroid generation using adipogenically differentiated ASCs, as well as the morphological characterization of generated spheroids.

Multifunctionalised skin substitute of hybrid gelatin-polyvinyl alcohol bioinks for chronic wound: injectable vs. 3D bioprinting.

Chronic wounds are challenging to heal and increase global mortality. The effectiveness of skin graft is limited by rejection, fibrosis, and inadequate donor site. Multifunctionalised-hydrogel skin substitutes promoted higher wound healing by maintaining the moisture microenvironment and permit gas exchange/nourishment in prolong cell viability/activity. The purpose of this study was to evaluate a skin substitute using two strategies; via injectable and 3D bioprinting technique. New hydrogel formulations that composed of gelatin (GE) and polyvinyl-alcohol (PVA) were constructed using a pre-mix crosslinking approach with genipin (GNP) to generate the biodegradable and biocompatible skin substitute with reduced secondary traumatic wound. GPVA5_GNP (6% GE: 5% PVA crosslinked with GNP) was the most stable hydrogel for wound healing application with the longest enzymatic degradation and stable hydrogelfor absorption of excess wound exudates. Primary human dermal fibroblasts (HDFs) migrated extensively through 3D bioprinted hydrogels with larger average pore sizes and interconnected pores than injectable hydrogels. Moreover, 3D bioprinted GPVA hydrogels were biocompatible with HDFs and demonstrated > 90% cell viability. HDFs maintained their phenotype and positively expressed collagen type-I, vinculin, short and dense F-actin, alpha-smooth muscle actin, and Ki67. Additionally, the presence of GNP demonstrated antioxidant capacity and high-ability of angiogenesis. The utilisation of the 3D bioprinting (layer-by-layer) approach did not compromise the HDFs' growth capacity and biocompatibility with selected bioinks. In conclusion, it allows the cell encapsulation sustainability in a hydrogel matrix for a longer period, in promoting tissue regeneration and accelerating healing capacity, especially for difficult or chronic wound.

Bio-electrospraying 3-D Organotypic Human Skin Cultures.

Organotypic 3D tissue models have greatly contributed to understand a wide range of molecular and cellular characteristics within a functional or diseased tissue. Human skin reconstructs which act as models are most useful for a wide range of investigations, ranging from tissue engineering and regenerative medicine, drug development, screening, and discovery to name a few. There are many approaches for reconstructing 3D skin tissue models, however, to date there have been very few that are able to generate organotypic 3D constructs with a single technology having minimal processing steps to finally scalability. The many manifestations of 3D bioprinting have contributed to this endeavor, having said that, the technology's limitations have tempered those reconstructed models, as they are known to contain low cell numbers/concentrations to those having damaged/dead molecules/cells within the reconstructed tissue, which are not desirable, for exploring as tissues models. Contrary to 3D bioprinting approaches, bio-electrosprays have been demonstrated to possess the ability to handle large concentrations of cells and molecules to whole fertilized embryos without damaging them from a molecular level upwards. Consequently, this article demonstrates, for the first time, bio-electrospray's capacity to reconstruct skin-like structures in vitro and its potential in reconstructing full-thickness 3D organotypic human skin tissues.

Sacrificial scaffold-assisted direct ink writing of engineered aortic valve prostheses

Heart valve disease has become a serious global health problem, which calls for numerous implantable prosthetic valves to fulfill the broader needs of patients. Although current three-dimensional (3D) bioprinting approaches can be used to manufacture customized valve prostheses, they still have some complications, such as limited biocompatibility, constrained structural complexity, and difficulty to make heterogeneous constructs, to name a few. To overcome these challenges, a sacrificial scaffold-assisted direct ink writing approach has been explored and proposed in this work, in which a sacrificial scaffold is printed to temporarily support sinus wall and overhanging leaflets of an aortic valve prosthesis that can be removed easily and mildly without causing any potential damages to the valve prosthesis. The bioinks, composed of alginate, gelatin, and nanoclay, used to print heterogenous valve prostheses have been designed in terms of rheological/mechanical properties and filament formability. The sacrificial ink made from Pluronic F127 has been developed by evaluating rheological behavior and gel temperature. After investigating the effects of operating conditions, complex 3D structures and homogenous/heterogenous aortic valve prostheses have been successfully printed. Lastly, numerical simulation and cycling experiments have been performed to validate the function of the printed valve prostheses as one-way valves.

Biomimetic 3D bioprinting approaches to engineer the tumor microenvironment

With the increasing incidence and mortality rates, cancer remains a major health challenge in the world. Despite advances in therapies and clinical programs, the efficacy of anti-cancer drugs often fails to translate from pre-clinical models to patient clinical trials. To date, pre-clinical cancer models, including two-dimensional cell cultures and animal models, have limited versatility and accuracy in recapitulating the complexity of human cancer. To address these limitations, a growing focus has fostered the development of three-dimensional (3D) tumor models that closely resemble the in vivo tumor microenvironment and heterogeneity. Recent efforts have leveraged bioengineering technologies, such as biofabrication, to engineer new platforms that mimic healthy and diseased organs, aiming to overcome the shortcomings of conventional models, such as for musculoskeletal tissues. Notably, 3D bioprinting has emerged as a powerful tool in cancer research, offering precise control over cell and biomaterial deposition to fabricate architecturally complex and reproducible functional models. The following review underscores the urgent need for more accurate and relevant 3D tumor models, highlighting the advantages of the use of biofabrication approaches to engineer new biomimetics platforms. We provide an updated discussion on the role of bioengineering technologies in cancer research and modeling with particular focus on 3D bioprinting platforms, as well as a close view on biomaterial inks and 3D bioprinting technologies employed in cancer modeling. Further insights into the 3D bioprinting tissue-specific modeling panorama are presented in this paper, offering a comprehensive overview of the new possibilities for cancer study and drug discovery.  

Assessing non-synthetic crosslinkers in biomaterial inks based on polymers of marine origin to increase the shape fidelity in 3D extrusion printing

In the past decade, there has been significant progress in 3D printing research for tissue engineering (TE) using biomaterial inks made from natural and synthetic compounds. These constructs can aid in the regeneration process after tissue loss or injury, but achieving high shape fidelity is a challenge as it affects the construct’s physical and biological performance with cells. In parallel with the growth of 3D bioprinting approaches, some marine-origin polymers have been studied due to their biocompatibility, biodegradability, low immunogenicity, and similarities to human extracellular matrix components, making them an excellent alternative to land mammal-origin polymers with reduced disease transmission risk and ethical concerns. In this research, collagen from shark skin, chitosan from squid pens, and fucoidan from brown algae were effectively blended for the manufacturing of an adequate biomaterial ink to achieve a printable, reproducible material with a high shape fidelity and reticulated using four different approaches (phosphate-buffered saline, cell culture medium, 6% CaCl2, and 5 mM Genipin). Materials characterization was composed by filament collapse, fusion behavior, swelling behavior, and rheological and compressive tests, which demonstrated favorable shape fidelity resulting in a stable structure without deformations, and interesting shear recovery properties around the 80% mark. Additionally, live/dead assays were conducted in order to assess the cell viability of an immortalized human mesenchymal stem cell line, seeded directly on the 3D printed constructs, which showed over 90% viable cells. Overall, the Roswell Park Memorial Institute cell culture medium promoted the adequate crosslinking of this biopolymer blend to serve the TE approach, taking advantage of its capacity to hamper pH decrease coming from the acidic biomaterial ink. While the crosslinking occurs, the pH can be easily monitored by the presence of the indicator phenol red in the cell culture medium, which reduces costs and time.

Bioprinted hASC-laden cell constructs with mechanically stable and cell alignment cue for tenogenic differentiation.

3D bioprinting is a technology that enables the precise and controlled deposition of cells and an artificial extracellular matrix (ECM) to create functional tissue constructs. However, current 3D bioprinting methods still struggle to obtain mechanically stable and unique cell-morphological structures, such as fully aligned cells. In this study, we propose a new 3D bioprinting approach that utilizes a high concentration of bioink without cells to support mechanical properties and drag flow to fully align cells in a thin bath filled with cell-laden bioink, resulting in a hybrid cell-laden construct with a mechanical stable and fully aligned cell structure. To demonstrate the feasibility of this approach, we used it to fabricate a cell-laden construct using human adipose stem cells (hASCs) for tendon tissue engineering. To achieve appropriate processing conditions, various factors such as the bioink concentration, nozzle moving speed, and volume flow rate were considered. To enhance the biocompatibility of the cell-laden construct, we used porcine decellularized tendon ECM.In vitrocellular responses, including tenogenic differentiation of the fabricated hybrid cell structures with aligned or randomly distributed cells, were evaluated using hASCs. In addition, the mechanical properties of the hybrid cell-laden construct could be adjusted by controlling the concentration of the mechanically reinforcing strut using methacrylated tendon-decellularized extracellular matrix. Based on these results, the hybrid cell-laden structure has the potential to be a highly effective platform for the alignment of musculoskeletal tissues.

3D bioprinting of tyramine modified hydrogels under visible light for osteochondral interface

Recent advancements in tissue engineering have demonstrated a great potential for the fabrication of three-dimensional (3D) tissue structures such as cartilage and bone. However, achieving structural integrity between different tissues and fabricating tissue interfaces are still great challenges. In this study, an in situ crosslinked hybrid, multi-material 3D bioprinting approach was used for the fabrication of hydrogel structures based on an aspiration-extrusion microcapillary method. Different cell-laden hydrogels were aspirated in the same microcapillary glass and deposited in the desired geometrical and volumetric arrangement directly from a computer model. Alginate and carboxymethyl cellulose were modified with tyramine to enhance cell bioactivity and mechanical properties of human bone marrow mesenchymal stem cells-laden bioinks. Hydrogels were prepared for extrusion by gelling in microcapillary glass utilizing an in situ crosslink approach with ruthenium (Ru) and sodium persulfate photo-initiating mechanisms under visible light. The developed bioinks were then bioprinted in precise gradient composition for cartilage-bone tissue interface using microcapillary bioprinting technique. The biofabricated constructs were co-cultured in chondrogenic/osteogenic culture media for three weeks. After cell viability and morphology evaluations of the bioprinted structures, biochemical and histological analyses, and a gene expression analysis for the bioprinted structure were carried out. Analysis of cartilage and bone formation based on cell alignment and histological evaluation indicated that mechanical cues in conjunction with chemical cues successfully induced MSC differentiation into chondrogenic and osteogenic tissues with a controlled interface.

Development of a biomimetic arch-like 3D bioprinted construct for cartilage regeneration using gelatin methacryloyl and silk fibroin-gelatin bioinks

In recent years, engineering biomimetic cellular microenvironments have been a top priority for regenerative medicine. Collagen II, which is arranged in arches, forms the predominant fiber network in articular cartilage. Due to the shortage of suitable microfabrication techniques capable of producing 3D fibrous structures, in vitro replication of the arch-like cartilaginous tissue constitutes one of the major challenges. Hence, in the present study, we report a 3D bioprinting approach for fabricating arch-like constructs using two types of bioinks, gelatin methacryloyl (GelMa) and silk fibroin-gelatin (SF-G). The bioprinted SF-G constructs displayed increased proliferation of the encapsulated human bone marrow-derived mesenchymal stem cells compared to the GelMA constructs. Biochemical assays, gene, and protein expression exhibited the superior role of SF-G in forming the fibrous collagen network and chondrogenesis. Protein-protein interaction study using Metascape evaluated the function of the proteins involved. Further GeneMANIA and STRING analysis using Col 2A1, SOX 9, ACAN, and the genes upregulated on day 21 in RT-PCR, i.e. β-catenin, TGFβR1, Col 1A1 in SF-G and PRG4, Col 10A1, MMP 13 in GelMA validated our in vitro results. These findings emphasized the role of SF-G in regulating the Wnt/β-catenin and TGF-β signaling pathways. Hence, the 3D bioprinted arch-like constructs possess a substantial potential for cartilage regeneration.