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.

Emerging perspectives on 3D printed bioreactors for clinical translation of engineered and bioprinted tissue constructs.

3D printed/bioprinted tissue constructs are utilized for the regeneration of damaged tissues and as in vitro models. Most of the fabricated 3D constructs fail to undergo functional maturation in conventional in vitro settings. There is a challenge to provide a suitable niche for the fabricated tissue constructs to undergo functional maturation. Bioreactors have emerged as a promising tool to enhance tissue maturation of the engineered constructs by providing physical/biological cues along with a controlled nutrient supply under dynamic in vitro conditions. Bioreactors provide an ambient microenvironment most appropriate for the development of functionally matured tissue constructs by promoting cell proliferation, differentiation, and maturation for transplantation and drug screening applications. Due to the huge cost and limited availability of commercial bioreactors, there is a need to develop strategies to make customized bioreactors. Additive manufacturing (AM) may be a viable tool to fabricate custom designed bioreactors with better efficiency and at low cost. In this review, we have extensively discussed the importance of bioreactors in functionalizing tissue engineered/3D bioprinted scaffolds for bone, cartilage, skeletal muscle, nerve, and vascular tissue. In addition, the importance and fabrication of customized 3D printed bioreactors for the maturation of tissue engineered constructs are discussed in detail. Finally, the current challenges and future perspectives in translating commercial and custom 3D printed bioreactors for clinical applications are outlined.

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.

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.

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.

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.

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.

Self-assembling tetrameric peptides allow in situ 3D bioprinting under physiological conditions.

We have developed an in situ bioprinting method that allows the printing of cells under true physiological conditions by applying self-assembling ultrashort peptides as bioinks. This method avoids cell stressing methods, such as UV-treatment, chemical crosslinking and viscous bioink printing methods. We further demonstrate that different nanomaterials can easily be synthesized or incorporated in the 3D bioprinted peptide scaffolds which opens up the possibility of functionalized 3D scaffolds.

Epidermal differentiation of stem cells on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanofibers.

Nanomaterials with stem cells have evolved as a promising therapeutic strategy to regenerate various tissues. Tissue engineered grafts with bone marrow derived mesenchymal stem cells (BM-MSCs) can offer a cell-based therapeutic strategy for deep wounds like burns and traumatic ulcers. In this study, we have fabricated poly(3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) nanofibers through electrospinning. The adhesion, proliferation and epidermal differentiation of BM-MSCs on PHBV nanofibers were investigated. Epidermal differentiation media containing epidermal growth factor (EGF), insulin, 3,3',5-triiodo-L-thyronine (T3), Hydrocortisone and 1α, 25-dihydroxyvitamin (D3) were used to trigger differentiation of BM-MSCs on PHBV. The proliferation of BM-MSCs on PHBV was significantly higher than the tissue culture polystyrene (TCPS) control (p < 0.05). Live/dead staining of BM-MSCs on PHBV nanofibers confirmed the change in morphology of BM-MSCs from spindle to polygonal shape indicating their differentiation into keratinocytes. The expression levels of the genes keratin (early), filaggrin (intermediate) and involucrin (late) that are involved in epidermal differentiation were upregulated in a stage-specific manner. Our results demonstrate the potential of PHBV nanofibers in promoting adhesion and differentiation of mesenchymal stem cells. This novel cellular nanofiber construct can be a better alternative to the existing therapies for skin tissue engineering.

Biocompatibility of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanofibers for skin tissue engineering.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) a biodegradable polymer, was electrospun to obtain defect-free nanofibers. The structural similarity of PHBV nanofibers and the extracellular matrix in skin may present well for fibroblast cell adhesion and proliferation. The average fiber diameter of the electrospun fibers was 583 +/- 90 nm. The potential of PHBV scaffolds for human keratinocytes (HaCaT) adhesion, proliferation and gene expression were evaluated. Our results demonstrated that PHBV nanofibers favor HaCaT adhesion and proliferation. After 14 days of culture, loricrin and keratin-1 gene expression were significantly higher when compared to 3 and 7 days (p < 0.05). The expression of genes associated with T lymphocyte activation (HLA-DRB, thymosin beta 10 (h-Tim)) and IL-2 mediated lymphocyte activation genes (h-Tim, Tumour Rejection Antigen (TRA 1), nRap 2) were investigated in human lymphocyte cultured on PHBV nanofibers. T Lymphocyte activation and IL-2 mediated lymphocyte activation genes were down-regulated after 48 and 72 hours of culture. After 24, 48 and 72 hours of culture there was no inflammatory cytokines production by the cultured lymphocytes. Thus, our results confirm the biocompatibility of PHBV nanofibers and suggest that consideration can be given to the use of PHBV nanofibers for skin tissue engineering applications.

Hierarchical mesoporous silica nanofibers as multifunctional scaffolds for bone tissue regeneration.

Mesoporous materials with pore sizes between 2 and 50 nm have elicited widespread interest in catalysis, separation, adsorption, sensors, and drug delivery applications due to its highly ordered pore size along with high hydrothermal stability and easily modifiable surface functionalities. Fabricating these mesoporous materials as continuous fibers offers exciting vistas for biomedical applications especially in tissue engineering. The aim of the present study was to fabricate, characterize, and evaluate the cellular and gene expression of mesoporous silica with a long ordered fibrous morphology to support regeneration of bone tissue. Tetraethyl orthosilicate, polyvinyl pyrrolidone, and the tri-block copolymer P-123 were subjected to electrospinning to fabricate continuous ordered mesoporous silica nanofibers by optimizing solution and operation parameters. Mesoporous silica fibers with an average diameter of 470 nm and mesopores of dimension 5.97 nm were obtained. The combination of micropores, mesopores, macropores, and the nanofibrous morphology imparted excellent bioactivity to the mesoporous silica fibrous scaffolds as demonstrated by the proliferation of human osteoblast-like cells (MG63) and by the maintenance of its phenotype. The upregulation of collagen I, alkaline phosphatase, osteocalcin, osteopontin, and bone sialoprotein signifies the maturation of MG63 cells on the silica scaffold. Hence, these novel scaffolds are promising new biomaterials for orthopaedic applications.

Electrospun nanostructured chitosan-poly(vinyl alcohol) scaffolds: a biomimetic extracellular matrix as dermal substitute.

Electrospinning is a versatile technique to make biomimetic and nanostructured scaffolds for skin tissue engineering. In this study we have electrospun and characterized chitosan (C)-poly(vinyl alcohol) (PVA) blend nanofibers as dermal substitutes and compared with 2D C-PVA films. The in vitro characterization of the C-PVA nanofibers and 2D films were evaluated using mouse 3T3 fibroblast cells and our results demonstrated that the cells adhered and proliferated on the surface of C-PVA nanofibers. In our animal studies, the implantation of C-PVA nanofibers along with topical administration of growth factor R-Spondin 1 on full thickness wounds created on rats showed 98.6% wound closure after two weeks post-surgery. The catalase and superoxide dismutase activity of the healing tissue was significantly higher in the groups treated with topical administration of growth factor and C-PVA nanofibers (p < 0.05). Thus these C-PVA nanofibers along with novel growth factor are promising new biomaterials that could be used as dermal substitutes for accelerated wound healing.

Development of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) fibers for skin tissue engineering: effects of topography, mechanical, and chemical stimuli.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable polyester, was electrospun to form defect-free fibers with high surface-area-to-volume ratio for skin regeneration. Several parameters such as solvent ratio, polymer concentration, applied voltage, flow rate, and tip-to-target distance were optimized to achieve defect-free morphology. The average diameter of the PHBV fibers was 724 ± 91 nm. PHBV was also solvent-cast to form 2-D films, and its mechanical properties, porosity, and degradation rates were compared with PHBV fibers. Our results demonstrate that PHBV fibers exhibited higher porosity, increased ductility, and faster degradation rate when compared with PHBV 2-D films (p < 0.05). In vitro studies with PHBV fibers and 2-D films were carried out to evaluate the adhesion, viability, proliferation, and gene expression of human skin fibroblasts. Cells adhered and proliferated on both PHBV fibers and 2-D films. However, the proliferation of cells on the surface of PHBV fibers was comparable to tissue culture polystyrene (TCPS, control) (p > 0.05). The gene expression of collagen I and elastin was significantly up-regulated when compared with TCPS control, whereas collagen III was down-regulated on PHBV fibers and 2-D film after 14 days in culture. The less ductile PHBV 2-D films showed higher levels of elastin expression. Furthermore, the PHBV fibers in the presence and absence of an angiogenesis factor (R-Spondin 1) were evaluated for their wound healing capacity in a rat model. The wound contracture in R-Spondin-1-loaded PHBV fibers was found to be significantly higher when compared with PHBV fibers alone after 7 days (p < 0.05). Furthermore, the presence of fibers promoted an increase in collagen and aided re-epithelialization. Thus our results demonstrate that the topography and mechanical and chemical stimuli have a pronounced influence on the cell proliferation, gene expression, and wound healing.