Tuning thermoresponsive properties of carboxymethyl cellulose (CMC)-agarose composite bioinks to fabricate complex 3D constructs for regenerative medicine.

3D bioprinting has emerged as a viable tool to fabricate 3D tissue constructs with high precision using various bioinks which offer instantaneous gelation, shape fidelity, and cytocompatibility. Among various bioinks, cellulose is the most abundantly available natural polymer & widely used as bioink for 3D bioprinting applications. To mitigate the demanding crosslinking needs of cellulose, it is frequently chemically modified or blended with other polymers to develop stable hydrogels. In this study, we have developed a thermoresponsive, composite bioink using carboxymethyl cellulose (CMC) and agarose in different ratios (9:1, 8:2, 7:3, 6:4, and 5:5). Among the tested combinations, the 5:5 ratio showed better gel formation at 37 °C and were further characterized for physicochemical properties. Cytocompatibility was assessed by in vitro extract cytotoxicity assay (ISO 10993-5) using skin fibroblasts cells. CMC-agarose (5:5) bioink was successfully used to fabricate complex 3D structures through extrusion bioprinting and maintained over 80 % cell viability over seven days. Finally, in vivo studies using rat full-thickness wounds showed the potential of CMC-agarose bulk and bioprinted gels in promoting skin regeneration. These results indicate the cytocompatibility and suitability of CMC-agarose bioinks for tissue engineering and 3D bioprinting applications.

Hyaluronic Acid as Bioink and Hydrogel Scaffolds for Tissue Engineering Applications.

Bioprinting is an additive manufacturing technique that focuses on developing living tissue constructs using bioinks. Bioink is crucial in determining the stability of printed patterns, which remains a major challenge in bioprinting. Thus, the choices of bioink composition, modifications, and cross-linking methods are being continuously researched to augment the clinical translation of bioprinted constructs. Hyaluronic acid (HA) is a naturally occurring polysaccharide with the repeating unit of N-acetyl-glucosamine and d-glucuronic acid disaccharides. It is present in the extracellular matrix (ECM) of tissues (skin, cartilage, nerve, muscle, etc.) with a wide range of molecular weights. Due to the nature of its chemical structure, HA could be easily subjected to chemical modifications and cross-linking that would enable better printability and stability. These interesting properties have made HA an ideal choice of bioinks for developing tissue constructs for regenerative medicine applications. In this Review, the physicochemical properties, reaction chemistry involved in various cross-linking strategies, and biomedical applications of HA have been elaborately discussed. Further, the features of HA bioinks, emerging strategies in HA bioink preparations, and their applications in 3D bioprinting have been highlighted. Finally, the current challenges and future perspectives in the clinical translation of HA-based bioinks are outlined.

Additive manufacturing of peripheral nerve conduits - Fabrication methods, design considerations and clinical challenges.

Tissue-engineered nerve guidance conduits (NGCs) are a viable clinical alternative to autografts and allografts and have been widely used to treat peripheral nerve injuries (PNIs). Although these NGCs are successful to some extent, they cannot aid in native regeneration by improving native-equivalent neural innervation or regrowth. Further, NGCs exhibit longer recovery period and high cost limiting their clinical applications. Additive manufacturing (AM) could be an alternative to the existing drawbacks of the conventional NGCs fabrication methods. The emergence of the AM technique has offered ease for developing personalized three-dimensional (3D) neural constructs with intricate features and higher accuracy on a larger scale, replicating the native feature of nerve tissue. This review introduces the structural organization of peripheral nerves, the classification of PNI, and limitations in clinical and conventional nerve scaffold fabrication strategies. The principles and advantages of AM-based techniques, including the combinatorial approaches utilized for manufacturing 3D nerve conduits, are briefly summarized. This review also outlines the crucial parameters, such as the choice of printable biomaterials, 3D microstructural design/model, conductivity, permeability, degradation, mechanical property, and sterilization required to fabricate large-scale additive-manufactured NGCs successfully. Finally, the challenges and future directions toward fabricating the 3D-printed/bioprinted NGCs for clinical translation are also discussed.

3D bioprinting and photocrosslinking: emerging strategies & future perspectives.

3D bioprinting has enabled the creation of biomimetic tissue constructs for regenerative medicine and in vitro model systems. Large-scale production of 3D structures at the micron-scale resolution is achieved through bioprinting using custom bioinks. Stability and 3D construct compliance play an important role in offering cells with biomechanical cues that regulate their behavior and enable in vivo implantation. Various crosslinking strategies are developed to stabilize the 3D printed structures and new methodologies are constantly being evaluated to overcome the limitations of the existing methods. Photo-crosslinking has emerged as a simple and elegant technique that offers precise control over the spatiotemporal gelation of bioinks during bioprinting. This article summarizes the use of photo-crosslinking agents and methodology towards optimizing 3D constructs for specific biomedical applications. The article also takes into account various bioinks and photo-crosslinkers in creating stable 3D printed structures that offer bioactivity with desirable physicochemical properties. The current challenges of 3D bioprinting and new directions that can advance the field in its wide applicability to create 3D tissue models to study diseases and organ transplantation are also summarized.

Designer DNA biomolecules as a defined biomaterial for 3D bioprinting applications.

DNA has excellent features such as the presence of functional and targeted molecular recognition motifs, tailorability, multifunctionality, high-precision molecular self-assembly, hydrophilicity, and outstanding biocompatibility. Due to these remarkable features, DNA has emerged as a leading next-generation biomaterial of choice to make hydrogels by self-assembly. In recent times, novel routes for the chemical synthesis of DNA, advances in tailorable designs, and affordable production ways have made DNA as a building block material for various applications. These advanced features have made researchers continuously explore the interesting properties of pure and hybrid DNA for 3D bioprinting and other biomedical applications. This review article highlights the topical advancements in the use of DNA as an ideal bioink for the bioprinting of cell-laden three-dimensional tissue constructs for regenerative medicine applications. Various bioprinting techniques and emerging design approaches such as self-assembly, nucleotide sequence, enzymes, and production cost to use DNA as a bioink for bioprinting applications are described. In addition, various types and properties of DNA hydrogels such as stimuli responsiveness and mechanical properties are discussed. Further, recent progress in the applications of DNA in 3D bioprinting are emphasized. Finally, the current challenges and future perspectives of DNA hydrogels in 3D bioprinting and other biomedical applications are discussed.

Reverse engineering of an anatomically equivalent nerve conduit.

Reconstruction of peripheral nervous tissue remains challenging in critical-sized defects due to the lack of Büngner bands from the proximal to the distal nerve ends. Conventional nerve guides fail to bridge the large-sized defect owing to the formation of a thin fibrin cable. Hence, in the present study, an attempt was made to reverse engineer the intricate epi-, peri- and endo-neurial tissues using Fused Deposition Modeling based 3D printing. Bovine serum albumin protein nanoflowers (NF) exhibiting Viburnum opulus 'Roseum' morphology were ingrained into 3D printed constructs without affecting its secondary structure to enhance the axonal guidance from proximal to distal ends of denuded nerve ends. Scanning electron micrographs confirmed the uniform distribution of protein NF in 3D printed constructs. The PC-12 cells cultured on protein ingrained 3D printed scaffolds demonstrated cytocompatibility, improved cell adhesion and extended neuronal projections with significantly higher intensities of NF-200 and tubulin expressions. Further suture-free fixation designed in the current 3D printed construct aids facile implantation of printed conduits to the transected nerve ends. Hence the protein ingrained 3D printed construct would be a promising substitute to treat longer peripheral nerve defects as its structural equivalence of endo- and perineurial organization along with the ingrained protein NF promote the neuronal extension towards the distal ends by minimizing axonal dispersion.

Current standards and ethical landscape of engineered tissues-3D bioprinting perspective.

Tissue engineering is an evolving multi-disciplinary field with cutting-edge technologies and innovative scientific perceptions that promise functional regeneration of damaged tissues/organs. Tissue engineered medical products (TEMPs) are biomaterial-cell products or a cell-drug combination which is injected, implanted or topically applied in the course of a therapeutic or diagnostic procedure. Current tissue engineering strategies aim at 3D printing/bioprinting that uses cells and polymers to construct living tissues/organs in a layer-by-layer fashion with high 3D precision. However, unlike conventional drugs or therapeutics, TEMPs and 3D bioprinted tissues are novel therapeutics and need different regulatory protocols for clinical trials and commercialization processes. Therefore, it is essential to understand the complexity of raw materials, cellular components, and manufacturing procedures to establish standards that can help to translate these products from bench to bedside. These complexities are reflected in the regulations and standards that are globally in practice to prevent any compromise or undue risks to patients. This review comprehensively describes the current legislations, standards for TEMPs with a special emphasis on 3D bioprinted tissues. Based on these overviews, challenges in the clinical translation of TEMPs & 3D bioprinted tissues/organs along with their ethical concerns and future perspectives are discussed.

Key advances of carboxymethyl cellulose in tissue engineering & 3D bioprinting applications.

Carboxymethyl cellulose (CMC) is a water-soluble derivative of cellulose and a major type of cellulose ether prepared by the chemical attack of alkylating reagents on the activated non-crystalline regions of cellulose. It is the first FDA approved cellulose derivative which can be targeted for desired chemical modifications. In this review, the properties along with current advances in the physical and chemical modifications of CMC are discussed. Further, CMC and modified CMC could be engineered to fabricate scaffolds for tissue engineering applications. In recent times, CMC and its derivatives have been developed as smart bioinks for 3D bioprinting applications. From these perspectives, the applications of CMC in tissue engineering and current knowledge on peculiar features of CMC in 3D and 4D bioprinting applications are elaborated in detail. Lastly, future perspectives of CMC for wider applications in tissue engineering and 3D/4D bioprinting are highlighted.

Additive manufacturing of biodegradable porous orthopaedic screw.

Advent of additive manufacturing in biomedical field has nurtured fabrication of complex, customizable and reproducible orthopaedic implants. Layer-by-layer deposition of biodegradable polymer employed in development of porous orthopaedic screws promises gradual dissolution and complete metabolic resorption thereby overcoming the limitations of conventional metallic screws. In the present study, screws with different pore sizes (916 × 918 µm to 254 × 146 µm) were 3D printed at 200 µm layer height by varying printing parameters such as print speed, fill density and travel speed to augment the bone ingrowth. Micro-CT analysis and scanning electron micrographs of screws with 45% fill density confirmed porous interconnections (40.1%) and optimal pore size (259 × 207 × 200 µm) without compromising the mechanical strength (24.58 ± 1.36 MPa). Due to the open pore structure, the 3D printed screws showed increased weight gain due to the deposition of calcium when incubated in simulated body fluid. Osteoblast-like cells attached on screw and infiltrated into the pores over 14 days of in vitro culture. Further, the screws also supported greater human mesenchymal stem cell adhesion, proliferation and mineralized matrix synthesis over a period of 21 days in vitro culture as compared to non-porous screws. These porous screws showed significantly increased vascularization in a rat subcutaneous implantation as compared to control screws. Porous screws produced by additive manufacturing may promote better osteointegration due to enhanced mineralization and vascularization.