Photoluminescent Self-Healable Waterborne Polyurethane/Mo and S Codoped Graphitic Carbon Nitride Nanocomposite with Bioimaging and Encryption Capability.

Creating polymers that combine various functions within a single system expands the potential applications of such polymeric materials. However, achieving polymer materials that possess simultaneously elevated strength, toughness, and self-healing capabilities, along with special properties, remains a significant challenge. The present study demonstrates the preparation of S and Mo codoped graphitic carbon nitride (g-C3N4) (Mo@S-CN) nanohybrid and the fabrication of self-healing waterborne polyurethane (SHWPU)/Mo@S-CN (SHWPU/NS) nanocomposites for advanced applications. Mo@S-CN is an intriguing combination of g-C3N4 nanosheets and molybdenum oxide (MoOx) nanorods, forming a complex lamellar structure. This unique arrangement significantly improves the inborn properties of SHWPU to an impressive degree, especially mechanical strength (28.37-34.11 MPa), fracture toughness (73.65-140.98 MJ m-2), and thermal stability (340.17-348.01 °C), and introduces fluorescence activity into the matrix. Interestingly, a representative SHWPU/NS0.5 film is so tough that a dumbbell of 15 kg, which is 53,003 times heavier than the weight of the film, can be successfully lifted without any significant crack. Remarkably, fluorescence activity is developed because of electronic excitations occurring within the repeating polymeric tris-triazine units of the Mo@S-CN nanohybrid. This fascinating feature was effectively harnessed by assessing the usability of aqueous dispersions of the Mo@S-CN nanohybrid and photoluminescent SHWPU/NS nanocomposites as sustainable stains for bioimaging of human dermal fibroblast cells and anticounterfeiting materials, respectively. The in vitro fluorescence tagging test showed blue emission from 365 nm excitation, green emission from 470 nm excitation, and red emission from 545 nm excitation. Most importantly, in vitro hemocompatibility assessment, in vitro cytocompatibility, cell proliferation assessment, and cellular morphology assessment supported the biocompatibility nature of the Mo@S-CN nanohybrid and SHWPU/NS nanocomposites. Thus, these materials can be used for advanced applications including bioimaging.

Photo-Polymerizable Autologous Growth-Factor Loaded Silk-Based Biomaterial-Inks toward 3D Printing-Based Regeneration of Meniscus Tears.

Meniscus tears in the avascular region undergoing partial or full meniscectomy lead to knee osteoarthritis and concurrent lifestyle hindrances in the young and aged alike. Here they reported ingenious photo-polymerizable autologous growth factor loaded 3D printed scaffolds to potentially treat meniscal defects . A shear-thinning photo-crosslinkable silk fibroin methacrylate-gelatin methacrylate-polyethylene glycol dimethacrylate biomaterial-ink is formulated and loaded with freeze-dried growth factor rich plasma (GFRP) . The biomaterial-ink exhibits optimal rheological properties and shape fidelity for 3D printing. Initial evaluation revealed that the 3D printed scaffolds mimic mechanical characteristics of meniscus, possess favourable porosity and swelling characteristics, and demonstrate sustained GFRP release. GFRP laden 3D scaffolds are screened with human neo-natal stem cells in vitro and biomaterial-ink comprising of 25 mg mL-1 of GFRP (GFRP25) is found to be amicable for meniscus tissue engineering. GFRP25 ink demonstrated rigorous rheological compliance, and printed constructs demonstrated long term degradability (>6 weeks), GFRP release (>5 weeks), and mechanical durability (3 weeks). GFRP25 scaffolds aided in proliferation of seeded human neo-natal stem cellsand their meniscus-specific fibrochondrogenic differentiation . GFRP25 constructs show amenable inflammatory response in vitro and in vivo. GFRP25 biomaterial-ink and printed GFRP25 scaffolds could be potential patient-specific treatment modalities for meniscal defects.

Current advances in engineering meniscal tissues: insights into 3D printing, injectable hydrogels and physical stimulation based strategies.

The knee meniscus is the cushioning fibro-cartilage tissue present in between the femoral condyles and tibial plateau of the knee joint. It is largely avascular in nature and suffers from a wide range of tears and injuries caused by accidents, trauma, active lifestyle of the populace and old age of individuals. Healing of the meniscus is especially difficult due to its avascularity and hence requires invasive arthroscopic approaches such as surgical resection, suturing or implantation. Though various tissue engineering approaches are proposed for the treatment of meniscus tears, three-dimensional (3D) printing/bioprinting, injectable hydrogels and physical stimulation involving modalities are gaining forefront in the past decade. A plethora of new printing approaches such as direct light photopolymerization and volumetric printing, injectable biomaterials loaded with growth factors and physical stimulation such as low-intensity ultrasound approaches are being added to the treatment portfolio along with the contemporary tear mitigation measures. This review discusses on the necessary design considerations, approaches for 3D modeling and design practices for meniscal tear treatments within the scope of tissue engineering and regeneration. Also, the suitable materials, cell sources, growth factors, fixation and lubrication strategies, mechanical stimulation approaches, 3D printing strategies and injectable hydrogels for meniscal tear management have been elaborated. We have also summarized potential technologies and the potential framework that could be the herald of the future of meniscus tissue engineering and repair approaches.

Asymmetric Hard Domain-Induced Robust Resilient Biocompatible Self-Healable Waterborne Polyurethane for Biomedical Applications.

The synthesis of eco-friendly and biocompatible waterborne polyurethanes (WPUs) through judicious molecular engineering with supreme mechanical strength, good shape recoverability, and high self-healing efficiency is still a formidable challenge because of some mutually exclusive conflicts among these properties. Herein, we report a facile method to develop a transparent (80.57-91.48%), self-healable (efficiency 67-76%) WPU elastomer (strain 3297-6356%) with the highest reported mechanical toughness (436.1 MJ m-3), ultrahigh fracture energy (126.54 kJ m-2), and good shape recovery (95% within 40 s at 70 °C in water). These results were accomplished by introducing high-density hindered urea-based hydrogen bonds, an asymmetric alicyclic architecture (isophorone diisocyanate-isophorone diamine), and the glycerol ester of citric acid (a bio-based internal emulsifier) into the hard domains of the WPU. Most importantly, platelet adhesion activity, lactate dehydrogenase activity, and erythrocyte or red blood corpuscle lysis demonstrated the hemocompatibility of the developed elastomer. Simultaneously, the cellular viability (live/dead) assay and the cell proliferation (Alamar blue) assay of human dermal fibroblasts corroborated the biocompatibility under in vitro conditions. Furthermore, the synthesized WPUs showed melt re-processability with retention of mechanical strength (86.94%) and microbe-assisted biodegradation. The overall results, therefore, indicate that the developed WPU elastomer might be used as a potential smart biomaterial and coating for biomedical devices.

3D Bioprinted Silk-Based In Vitro Osteochondral Model for Osteoarthritis Therapeutics.

3D bioprinting of osteochondral tissue offers unique opportunities for enabling precise pharmacological interventions in osteoarthritis (OA). The current study investigates the screening potential of anti-inflammatory drugs using bioprinted inflamed human osteochondral units. The biomimetic hierarchical geometry is bioprinted using silk-based bioinks encapsulating pre-differentiated stem cells, creating an in vitro model. Inflammation is stimulated in the model, using tumor necrosis factor-alpha and Interleukin-1 beta pro-inflammatory cytokines. The resultant degeneration, akin to OA, is flagged by key markers like sulfated glycosaminoglycan, collagen, alkaline phosphatase, and downregulation of osteochondral transcript levels. In the next step, the screening of anti-inflammatory drugs is validated using celecoxib and rhein. Consequently, in the inflamed constructs, the initial upregulation of the key inflammatory mediators (nitric oxide, Prostaglandin E2), is subsequently downregulated, post-drug treatment. In addition, catabolic markers (matrix metalloproteinases and aggrecanase-1), indicative of hypertrophic and apoptosing chondrocytes, are significantly downregulated in the treatment groups; while the transcript and protein levels required for osteochondral health are attenuated. Therefore, the in vitro model mimicks the inflammation in the early stages of OA, and corroborates a potential high-throughput platform for screening novel anti-inflammatory drugs in OA therapeutics.

Application of 2,4,5-tris(2-pyridyl)imidazole as 'turn-off' fluorescence sensor for Cu(II) and Hg(II) ions and in vitro cell imaging.

The 2,4,5-tris(2-pyridyl)imidazole (L) molecule has been evaluated as a probe for dual sensing of Hg2+ and Cu2+ ions in EtOH/HEPES buffer medium (5 mM, pH = 7.34, 1:1, v/v). Probe L shows a good sensitive and selective turn-off response in the presence of both Hg2+ and Cu2+ ions, which is comprehensible under long UV light. The probe can detect Cu2+ ion in the pH range 3-11 and Hg2+ ion in pH 6-8. The limit of detection for Cu2+ (0.77 µM) is well under the allowable limit prescribed by the United States Environmental Protection Agency. Two metal (Cu2+ /Hg2+ ) ions are needed per L for complete fluorescence quenching. The probe shows marked reversibility on treatment with Na2 EDTA, making the protocol more economical for practical purposes. Paper strip coated with the L solution of EtOH can detect the presence of Cu2+ and Hg2+ ions in the sample using visible quenching of the fluorescence intensity. Density functional theory-time-dependent density functional theory (DFT-TDDFT) calculations support experimental observations, and d-orbitals of Cu2+ /Hg2+ provide a nonradiative decay pathway. Cell imaging study using HDF and MDA-MB-231 cells also supported the viability of L in detecting Cu2+ and Hg2+ ions in living cells.

3D bioprinting of photo-crosslinkable silk methacrylate (SilMA)-polyethylene glycol diacrylate (PEGDA) bioink for cartilage tissue engineering.

Articular cartilage damage poses huge burden on healthcare sector globally due to its extremely weak inherent regenerative ability. Three-dimensional (3D) bioprinting for development of cartilage mimic constructs using composite bioinks serves as an emerging perspective. However, difficulty in development of suitable bioink and chemical crosslinking associated inherent toxicity hamper widespread adoption of this technique. To circumvent this, a photo-polymerizable hydrogel-based bioink which helps in recapitulation of the complex cartilage microenvironment is pertinent. Herein, a photo-crosslinkable bioink containing different concentrations of silk methacrylate (SilMA) and polyethylene glycol diacrylate (PEGDA) was mixed with chondrocytes for biofabrication of 3D bioprinted cartilage constructs. The rheological properties, printability of bioink and physico-chemical characterization of printed hydrogel constructs were examined along with cartilaginous tissue formation. The printed SilMA-PEGDA hydrogel constructs possessed proper internal porous structure and demonstrated most reliable rheological properties, printability along with good mechanical, and degradation properties suitable for cartilage regeneration. Live/dead staining showed cytocompatibility of the 3D-bioprinted SilMA-PEGDA constructs. Moreover, a marked increase in cell number and DNA content was observed within the cartilaginous tissue as indicated by cell viability and DNA content quantitation. Biochemical evaluation confirmed the neocartilage formation within SilMA-PEGDA bioprinted constructs as revealed by enhanced deposition of cartilage specific extracellular matrix-sulphated GAG (sGAG) and collagen type II (>2-fold increase, p < 0.001) with time. Finally, immunohistochemical analysis indicated expression of collagen type II and aggrecan which corroborated with cartilaginous tissue formation. Taken together, we conclude that SilMA-PEGDA bioink could be suitable candidate for bioprinting chondrocytes to support cartilage tissue repair and regeneration.

Silk-Based Bioengineered Diaphyseal Cortical Bone Unit Enclosing an Implantable Bone Marrow toward Atrophic Nonunion Grafting.

Postnatal fracture healing of atrophic long bone diaphyseal nonunions remains a challenge for orthopedic surgeons. Paucity of autologous spongiosa has potentiated the use of tissue engineered bone grafts to improve success rates of bone marrow engraftment used in plate reosteosynthesis. Herein, the development and in vitro validation of a "sandwich-type" biofabricated diaphyseal cross-sectional unit, with an outer mechanically robust bioprinted cortical bone shell, encompassing an engineered bone marrow, are reported. Channelized silk fibroin blend sponges derived from Bombyx mori and Antheraea assama help in developing compartmentalized endosteum, exhibiting specialized osteoblasts (endosteal niche) and discontinuous endothelium (vascular niche). The cellular cross-talk between these two niches triggered via integrin-mediated cell adhesion, enables in preserving quiescence state of CD34+ /CD38- hematopoietic stem cells and their recycling in the engineered marrow. The outer cortical bone strut is developed through multimaterial microextrusion bioprinting strategy. Osteogenically primed mesenchymal stem cells-laden silk fibroin-nano-hydroxyapatite bioink is bioprinted alongside paramagnetic Fe-doped bioactive glass-polycaprolactone blend thermoplastic ink, reinforcing it for mechanical stability. Pulsed magnetic field actuation positively influences the osteogenic commitment and maturation of the bioprinted constructs via mechanotransductory route. Therefore, the assembled engineered marrow and bioprinted cortical shell hold promise as potential orthobiologic substitutes toward atrophic nonunion repairs.

Engineering Microsphere-Loaded Non-mulberry Silk-Based 3D Bioprinted Vascularized Cardiac Patches with Oxygen-Releasing and Immunomodulatory Potential.

A hostile myocardial microenvironment post ischemic injury (myocardial infarction) plays a decisive role in determining the fate of tissue-engineered approaches. Therefore, engineering hybrid 3D printed platforms that can modulate the MI microenvironment for improving implant acceptance has surfaced as a critical requirement for reconstructing an infarcted heart. Here, we have employed a non-mulberry silk-based conductive bioink comprising carbon nanotubes (CNTs) to bioprint functional 3D vascularized anisotropic cardiac constructs. Immunofluorescence staining, polymerase chain reaction-based gene expression studies, and electrophysiological studies showed that the inclusion of CNTs in the bioink played a significant role in upregulating matured cardiac biomarkers, sarcomere formation, and beating rate while promoting cardiomyocyte viability. These constructs were then microinjected with calcium peroxide and IL-10-loaded gelatin methacryloyl microspheres. Measurements of oxygen concentration revealed that these microspheres upheld the oxygen availability for maintaining cellular viability for at least 5 days in a hypoxic environment. Also, the ability of microinjected IL-10 microspheres to modulate the macrophages to anti-inflammatory M2 phenotype in vitro was uncovered using immunofluorescent staining and gene expression studies. Furthermore, in vivo subcutaneous implantation of microsphere-injected 3D constructs provided insights toward the extended time frame that was achieved for dealing with the hostile microenvironment for promoting host neovascularization and implant acceptance.

A three-dimensional printed silk-based biomimetic tri-layered meniscus for potential patient-specific implantation.

Employing tissue engineering principles aided by three-dimensional (3D) printing strategies to fabricate meniscus tissue constructs could help patients with meniscus injury regain mobility, improve pain management and reduce the risk of development of knee osteoarthritis. Here we report a 3D printed meniscus scaffold that biomimics the internal and bulk architecture of the menisci. A shear-thinning novel silk fibroin-gelatin-based bioink with high print fidelity was optimized for the fabrication of scaffolds to serve as potential meniscus implants. Physicochemical characterization of the fabricated scaffolds shows optimum swelling, degradation and mechanical properties. Further, the scaffolds were seeded with meniscus fibrochondrocytes to validate their bioactivity. Fibrochondrocytes seeded on the scaffolds maintained their phenotype and proliferation, and enhanced glycosaminoglycan and total collagen synthesis was observed. Gene expression profile, biochemical quantification and histological studies confirmed the ability of the scaffolds to form meniscus-like tissue constructs. The scaffolds were found to possess amenable immunocompatibility in vitro as well as in vivo. Due to their excellent biological and physicochemical characteristics, these 3D printed scaffolds may be fine-tuned into viable alternatives to the present clinical treatment approaches to meniscus repair.

3D Bioprinting Using Cross-Linker-Free Silk-Gelatin Bioink for Cartilage Tissue Engineering.

Cartilage tissue is deprived of intrinsic self-regeneration capability; hence, its damage often progresses to a chronic condition which reduces the quality of life. Toward the fabrication of functional tissue substitutes, three-dimensional (3D) bioprinting has progressed vastly over the last few decades. However, this progress is challenged by the difficulty in developing suitable bioink materials as most of them require toxic chemical cross-linking. In this study, our goal was to develop a cross-linker-free bioink with optimal rheology for polymer extrusion, aqueous, and nontoxic processing and offers structural support for cartilage regeneration. Toward this, we use the self-gelling ability of silk fibroin blends (Bombyx mori and Philosamia ricini) along with gelatin as a bulking agent. Silk and gelatin interact with each other through entanglement and physical cross-linking. The ink was rheologically and structurally optimized for printing efficiency in printing grid-like structures. The printed 3D constructs show optimal swelling capability, degradability, and compressive strength. Further, the construct supports the growth and proliferation of encapsulated chondrocytes and formation of the cartilaginous extracellular matrix as indicated by the increased sulfated glycosaminoglycan and collagen contents. This was further corroborated by the upregulation of chondrogenic gene expression with minimal hypertrophy of chondrocytes. Additionally, the construct demonstrates in vitro and in vivo biocompatibility. Notably, the ink demonstrates good print fidelity for printing anatomical structures such as the human ear enabled by optimized extrudability at adequate resolution. Altogether, the results indicate that the developed cross-linker-free silk-gelatin polymer-based bioink demonstrated high potential for its 3D bioprintability and application in cartilage tissue engineering.

3D Printing/Bioprinting Based Tailoring of in Vitro Tissue Models: Recent Advances and Challenges.

Prodigious progress in the past decade has pronounced 3D printing as one of the most promising technique for assembling biological materials in a complex layout that mimics native human tissues. With the advent of technology, several improvements in printing techniques have facilitated the development of intricate strategies and designs that were imaginably distant due to the conventional top-down approaches. Most of these advanced strategies generally follow a thorough coordination and an elaborate biomimetic blueprint due to which it is now possible to fabricate in vitro tissue models with ease. However, much remains to be accomplished at several forefronts for utilizing this technology to its full potential. With several printing strategies at the lead, it has now become essential to systematically analyze and learn from several endeavors such that shortcomings can be understood and future efforts can be made toward negating them. Taking account of all the recent tissue specific developments in this field, this review serves as a framework for bringing together in discussion several strategies and constraints in developing small scaled in vitro tissues. Highlighting the growing popularity of the organ and body on chip platforms and their easy scale up using 3D printing, latest advancements, and the challenges in this field are also discussed.