Carboxymethyl cellulose-agarose hydrogel in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nanofibers: A novel tissue engineered skin graft.

Healing chronic and critical-sized full-thickness wounds is a major challenge in the healthcare sector. Scaffolds prepared using electrospinning and hydrogels serve as effective treatment options for wound healing by mimicking the native skin microenvironment. Combining synthetic nanofibers with tunable hydrogel properties can effectively overcome limitations in skin scaffolds made only with nanofibers or hydrogels. In this study, a biocompatible hybrid scaffold was developed for wound healing applications using poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanofibers embedded with hydrogel made of 2 % carboxymethyl cellulose (CMC) blended with 1 % agarose. Hybrid scaffolds, characterized for surface morphology, swellability, porosity, and degradation, were found to be suitable for wound healing. Furthermore, the incorporation of CMC-agarose hydrogel into nanofibers significantly enhanced their mechanical strength compared to PHBV nanofibers alone (p < 0.05). Extract cytotoxicity and direct cytotoxicity tests showed that the hybrid scaffolds developed in this study are cytocompatible (>75 % viability). Furthermore, human adult dermal fibroblasts (HDFa) and human adult immortalized keratinocytes (HaCaT) adhesion, viability, and proliferation studies revealed that the hybrid scaffolds exhibited a significant increase in cell proliferation over time, similar to PHBV nanofibers. Finally, the developed hybrid scaffolds were evaluated in rat full-thickness wounds, demonstrating their ability to promote full-thickness wound healing with reepithelialization and epidermis closure.

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.

Efficient dual crosslinking of protein-in-polysaccharide bioink for biofabrication of cardiac tissue constructs.

Myocardial infarction (MI) is a lethal cardiac disease that causes most of the mortality across the world. MI is a consequence of plaque in the arterial walls of heart, which eventually result in occlusion and ischemia to the myocardial tissues due to inadequate nutrient and oxygen supply. As an efficient alternative to the existing treatment strategies for MI, 3D bioprinting has evolved as an advanced tissue fabrication technique where the cell-laden bioinks are printed layer-by-layer to create functional cardiac patches. In this study, a dual crosslinking strategy has been utilized towards 3D bioprinting of myocardial constructs by using a combination of alginate and fibrinogen. Herein, pre-crosslinking of the physically blended alginate-fibrinogen bioinks with CaCl2 enhanced the shape fidelity and printability of the printed structures. Physicochemical properties of the bioinks such as rheology, fibrin distribution, swelling ratio and degradation behaviour, were determined post-printing for only ionically crosslinked & dual crosslinked constructs and found to be ideal for bioprinting of cardiac constructs. Human ventricular cardiomyocytes (AC 16) exhibited a significant increase in cell proliferation on day 7 and 14 in AF-DMEM-20 mM CaCl2 bioink when compared to A-DMEM-20 mM CaCl2 (p < 0.05). Furthermore, myocardial patches containing neonatal ventricular rat myocytes (NVRM) showed >80 % viability and also expressed sarcomeric alpha actinin & connexin 43. These results indicate that the dual crosslinking strategy was cytocompatible and also possess the potential to be used for biofabrication of thick myocardial constructs for regenerative medicine applications.

Carboxymethyl cellulose-agarose-gelatin: A thermoresponsive triad bioink composition to fabricate volumetric soft tissue constructs.

Polysaccharide based hydrogels have been predominantly utilized as ink materials for 3D bioprinting due to biocompatibility and cell responsive features. However, most hydrogels require extensive crosslinking due to poor mechanical properties leading to limited printability. To improve printability without using cytotoxic crosslinkers, thermoresponsive bioinks could be developed. Agarose is a thermoresponsive polysaccharide with upper critical solution temperature (UCST) for sol-gel transition at 35-37 °C. Therefore, we hypothesized that a triad of carboxymethyl cellulose(C)-agarose(A)-gelatin(G) could be a suitable thermoresponsive ink for printing since they undergo instantaneous gelation without any addition of crosslinkers after bioprinting. The blend of agarose-carboxymethyl cellulose was mixed with 1% w/v, 3% w/v and 5% w/v gelatin to optimize the triad ratio for hydrogel formation. It was observed that a blend (C2-A0.5-G1 and C2-A1-G1) containing 2% w/v carboxymethyl cellulose, 0.5% or 1% w/v agarose and 1% w/v gelatin formed better hydrogels with higher stability for up to 21 days in DPBS at 37 °C. Further, C2-A0.5-G1 and C2-A1-G1hydrogels showed higher storage modulus 762 ± 182 Pa & 2452 ± 430 Pa, higher porosity of 96.98 ± 2% & 98.2 ± 0.8% and swellability of 1518 ± 68% & 1587 ± 25% respectively. To evaluate the in vitro potential of these bioink formulations, indirect and direct cytotoxicity were determined using NCTC clone 929 (mouse fibroblast cells) and HADF (primary human adult dermal fibroblast) cells as per the ISO 10993-5 standards. Importantly, the printability of these bioinks was confirmed using extrusion bioprinting by successfully printing different complex 3D patterns.

Advances in electrospinning and 3D bioprinting strategies to enhance functional regeneration of skeletal muscle tissue.

Skeletal muscles are essential for body movement, and the loss of motor function due to volumetric muscle loss (VML) limits the mobility of patients. Current therapeutic approaches are insufficient to offer complete functional recovery of muscle damages. Tissue engineering provides viable ways to fabricate scaffolds to regenerate damaged tissues. Hence, tissue engineering options are explored to address existing challenges in the treatment options for muscle regeneration. Electrospinning is a widely employed fabrication technique to make muscle mimetic nanofibrous scaffolds for tissue regeneration. 3D bioprinting has also been utilized to fabricate muscle-like tissues in recent times. This review discusses the anatomy of skeletal muscle, defects, the healing process, and various treatment options for VML. Further, the advanced strategies in electrospinning of natural and synthetic polymers are discussed, along with the recent developments in the fabrication of hybrid scaffolds. Current approaches in 3D bioprinting of skeletal muscle tissues are outlined with special emphasis on the combination of electrospinning and 3D bioprinting towards the development of fully functional muscle constructs. Finally, the current challenges and future perspectives of these convergence techniques are discussed.

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.

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.

Recent advancements in cardiovascular bioprinting and bioprinted cardiac constructs.

Cardiovascular diseases (CVDs) are the leading causes of mortality globally that demand the application of tissue engineering strategies to repair damaged tissues. Conventional tissue engineering approaches such as particulate leaching, hydrogels, gas foaming, solvent casting and electrospinning based strategies aim to develop extracellular matrix analogues to promote the regeneration of functional cardiac tissues. However, poor cell seeding efficiency with the non-uniform distribution of cells across thicker scaffolds (>5 mm) limits the clinical potential. The advent of 3D bioprinting offers layer-by-layer cellular integration and facilitates the recapitulation of cellular heterogeneity and intricate hierarchical structural organization. Although the success of 3D bioprinting of cardiac specific tissues has been demonstrated in varying degrees, maintaining unique architecture, cellular heterogeneity and cardiac functions demands the search for cardiac-specific bioinks. Hence, this review outlines the various bioinks explored in the printing of cardiac tissues and the essential properties such as rheological and electromechanical characteristics necessary for the functional restoration. This review further describes the application of 3D bioprinting for the fabrication of several cardiac tissues such as heart valves, coronary arteries, cardiac patches and whole heart. Finally, this review summarizes the existing limitations, unmet technical challenges and potential future focus on the expansion of bioprinting technique to cardiovascular medicine.