Strategies to Promote Vascularization in 3D Printed Tissue Scaffolds: Trends and Challenges.

Three-dimensional (3D) printing techniques for scaffold fabrication have shown promising advancements in recent years owing to the ability of the latest high-performance printers to mimic the native tissue down to submicron scales. Nevertheless, host integration and performance of scaffolds in vivo have been severely limited owing to the lack of robust strategies to promote vascularization in 3D printed scaffolds. As a result, researchers over the past decade have been exploring strategies that can promote vascularization in 3D printed scaffolds toward enhancing scaffold functionality and ensuring host integration. Various emerging strategies to enhance vascularization in 3D printed scaffolds are discussed. These approaches include simple strategies such as the enhancement of vascular in-growth from the host upon implantation by scaffold modifications to complex approaches wherein scaffolds are fabricated with their own vasculature that can be directly anastomosed or microsurgically connected to the host vasculature, thereby ensuring optimal integration. The key differences among the techniques, their pros and cons, and the future opportunities for utilizing each technique are highlighted here. The Review concludes with the current limitations and future directions that can help 3D printing emerge as an effective biofabrication technique to realize tissues with physiologically relevant vasculatures to ultimately accelerate clinical translation.

Design-encoded dual shape-morphing and shape-memory in 4D printed polymer parts toward cellularized vascular grafts.

Current additive manufacturing technologies wherein as-printed simple two-dimensional (2D) structures morph into complex tissue mimetic three-dimensional (3D) shapes are limited to multi-material hydrogel systems, which necessitates multiple fabrication steps and specific materials. This work utilizes a single shape memory thermoplastic polymer (SMP), PLMC (polylactide-co-trimethylene carbonate), to achieve programmable shape deformation through anisotropic design and infill angles encoded during 3D printing. The shape changes were first computationally predicted through finite element analysis (FEA) simulations and then experimentally validated through quantitative correlation. Rectangular 2D sheets could self-roll into complete hollow tubes of specific diameters (ranging from ≈6 mm to ≈10 mm) and lengths (as long as 40 mm), as quantitatively predicted from FEA simulations within one minute at relatively lower temperatures (≈80 °C). Furthermore, shape memory properties were demonstrated post-shape change to exhibit dual shape morphing at temperatures close to physiological levels. The tubes (retained as the permanent shape) were deformed into flat sheets (temporary shape), seeded with endothelial cells (at T < Tg), and thereafter triggered at ≈37 °C back into tubes (permanent shape), utilizing the shape memory properties to yield bioresorbable tubes with cellularized lumens for potential use as vascular grafts with improved long-term patency. Additionally, out-of-plane bending and twisting deformation were demonstrated in complex structures by careful control of infill angles that can unprecedently expand the scope of cellularized biomimetic 3D shapes. This work demonstrates the potential of the combination of shape morphing and SMP behaviors at physiological temperatures to yield next-generation smart implants with precise control over dimensions for tissue repair and regeneration.

A 4D printed nanoengineered super bioactive hydrogel scaffold with programmable deformation for potential bifurcated vascular channel construction.

Four-dimensional (4D) printing of hydrogels enabled the fabrication of complex scaffold geometries out of static parts. Although current 4D fabrication strategies are promising for creating vascular parts such as tubes, developing branched networks or tubular junctions is still challenging. Here, for the first time, a 4D printing approach is employed to fabricate T-shaped perfusable bifurcation using an extrusion-based multi-material 3D printing process. An alginate/methylcellulose-based dual-component hydrogel system (with defined swelling behavior) is nanoengineered with carbonized alginate (∼100 nm) to introduce anti-oxidative, anti-inflammatory, and anti-thrombotic properties and shape-shifting properties. A computational model to predict shape deformations in the printed hydrogels with defined infill angles was designed and further validated experimentally. Shape deformations of the 3D-printed flat sheets were achieved by ionic cross-linking. An undisrupted perfusion of a dye solution through a T-junction with minimal leakage mimicking blood flow through vessels is also demonstrated. Moreover, human umbilical vein endothelial and fibroblast cells seeded with printed constructs show intact morphology and excellent cell viability. Overall, the developed strategy paves the way for manufacturing self-actuated vascular bifurcations with remarkable anti-thrombotic properties to potentially treat coronary artery diseases.

NIR-Responsive Deployable and Self-Fitting 4D-Printed Bone Tissue Scaffold.

The treatment of irregular-shaped and critical-sized bone defects poses a clinical challenge. Deployable, self-fitting tissue scaffolds that can be implanted by minimally invasive procedures are a promising solution. Toward this, we fabricated NIR-responsive and programmable polylactide-co-trimethylene carbonate (PLMC) scaffolds nanoengineered with polydopamine nanoparticles (PDA) by extrusion-based three-dimensional (3D) printing. The 3D-printed scaffolds demonstrated excellent (>99%), fast (under 30 s), and tunable shape recovery under NIR irradiation. PLMC-PDA composites demonstrated significantly higher osteogenic potential in vitro as revealed by the significantly enhanced alkaline phosphatase (ALP) secretion and mineral deposition in contrast to neat PLMC. Intraoperative deployability and in vivo bone regeneration ability of PLMC-PDA composites were demonstrated, using self-fitting scaffolds in critical-sized cranial bone defects in rabbits. The 3D-printed scaffolds were deformed into compact shapes that could self-fit into the defect shape intraoperatively under low power intensity (0.76 W cm-2) NIR. At 6 and 12 weeks postsurgical implantation, near-complete bone regeneration was observed in PLMC-PDA composites, unlike neat PLMC through microcomputed tomography (micro-CT) analysis. The potential clinical utility of the 3D-printed composites to secure complex defects was confirmed through self-fitting of the scaffolds into irregular defects in ex vivo models of rabbit tibia, mandible, and tooth models. Taken together, the composite scaffolds fabricated here offer an innovative strategy for minimally invasive deployment to fit irregular and complex tissue defects for bone tissue regeneration.

Development of silk microfiber-reinforced bioink for muscle tissue engineering and in situ printing by a handheld 3D printer.

Volumetric muscle loss (VML) presents a significant challenge in tissue engineering due to the irreparable nature of extensive muscle injuries. In this study, we propose a novel approach for VML treatment using a bioink composed of silk microfiber-reinforced silk fibroin (SF) hydrogel. The engineered scaffolds are predesigned to provide structural support and fiber alignment to promote tissue regeneration in situ. We also validated our custom-made handheld 3D printer performance and showcased its potential applications for in situ printing using robotics. The fiber contents of SF and gelatin ink were varied from 1 to 5 %. Silk fibroin microfibers reinforced ink offered increased viscosity of the gel, which enhanced the shape fidelity and mechanical strength of the bulk scaffold. The fiber-reinforced bioink also demonstrated better cell-biomaterial interaction upon printing. The handheld 3D printer enabled the precise and on-demand fabrication of scaffolds directly at the defect site, for personalized and minimally invasive treatment. This innovative approach holds promise for addressing the challenges associated with VML treatment and advancing the field of regenerative medicine.

Granular Porous Nanofibrous Microspheres Enhance Cellular Infiltration for Diabetic Wound Healing.

Diabetic foot ulcers (DFUs) are a significant challenge in the clinical care of diabetic patients, often necessitating limb amputation and compromising the quality of life and life expectancy of this cohort. Minimally invasive therapies, such as modular scaffolds, are at the forefront of current DFU treatment, offering an efficient approach for administering therapeutics that accelerate tissue repair and regeneration. In this study, we report a facile method for fabricating granular nanofibrous microspheres (NMs) with predesigned structures and porosities. The proposed technology combines electrospinning and electrospraying to develop a therapeutic option for DFUs. Specifically, porous NMs were constructed using electrospun poly(lactic-co-glycolic acid) (PLGA):gelatin short nanofibers, followed by gelatin cross-linking. These NMs demonstrated enhanced cell adhesion to human dermal fibroblasts (HDF) during an in vitro cytocompatibility assessment. Notably, porous NMs displayed superior performance owing to their interconnected pores compared to nonporous NMs. Cell-laden NMs demonstrated higher Young's modulus values than NMs without loaded cells, suggesting improved material resiliency attributed to the reinforcement of cells and their secreted extracellular matrix. Dynamic injection studies on cell-laden NMs further elucidated their capacity to safeguard loaded cells under pressure. In addition, porous NMs promoted host cell infiltration, neovascularization, and re-epithelialization in a diabetic mouse wound model, signifying their effectiveness in healing diabetic wounds. Taken together, porous NMs hold significant potential as minimally invasive, injectable treatments that effectively promote tissue integration and regeneration.

Emerging 4D fabrication of next-generation nerve guiding conduits: a critical perspective.

The latest advancements in the field of manufacturing for biomedicine, digital health, targeted therapy, and personalized medicine have fuelled the fabrication of smart medical devices. Four-dimensional (4D) fabrication strategies, which combine the manufacturing of three-dimensional (3D) parts with smart materials and/or design, have proved beneficial in creating customized and self-fitting structures that change their properties on demand with time. These frontier techniques that yield dynamic implants can indeed alleviate various drawbacks of current clinical practices, such as the use of sutures and complex microsurgeries and associated inflammation, among others. Among various clinical applications, 4D fabrication has lately made remarkable progress in the development of next-generation nerve-guiding conduits for treating peripheral nerve injuries (PNIs) by improving the end-to-end co-aptation of transected nerve endings. The current perspective highlights the relevance of 4D fabrication in developing state-of-the-art technologies for the treatment of PNIs. Various 4D fabrication/bio-fabrication techniques for PNI treatment are summarized while identifying the challenges and opportunities for the future. Such advancements hold immense promise for improving the quality of life of patients suffering from nerve damage and the potential for extending the treatment of many other disorders. Although the techniques are being described for PNIs, they will lend themselves suitably to certain cases of cranial nerve injuries as well.

Light-Mediated 3D Printing of Micro-Pyramid-Decorated Tailorable Wound Dressings with Endogenous Growth Factor Sequestration for Improved Wound Healing.

Medical dressings play an important role in the field of tissue engineering owing to their ability to accelerate the process of wound healing. Great efforts have been made to fabricate wound dressings with distinctive features for promoting wound healing. However, most of the current synthesis methods either generate dressings of uniform size or involve complex fabrication techniques, thus limiting their commercialization for the personalized dressings. We report here a dressing, which presents a paradigm shift in the design of the dressing from uniform films to a micro-patterned film. The hypothesis driving the design is the ability of the 3D patterns to provide an efficient transient matrix filling the depth of the wound rather than just providing a barrier and slight re-epithelialization. We demonstrate the use of the digital light processing 3D printing technique to generate micro-pyramid-decorated wound healing dressings with individualized design and with bio-compatible gelatin methacryloyl to contact the wounded areas. In addition to providing better adhesion to the migratory cells, the micro-pyramids also enable covalent conjugation of heparin, providing capability to sequester endogenous growth factors (GFs). Based on these advantages, the developed dressing not only adheres strongly to the wound bed but also promotes the treatment of a rat wound model by utilizing the power of endogenous GFs for tissue regeneration. Thus, it is believed that the developed dressing can break through the limitation of traditional wound treatment and be an ideal candidate for wound healing.

Photopolymerized silk fibroin gel for advanced burn wound care.

The future of burn wound treatment lies in developing bioactive dressings for faster and more effective healing and regeneration. Silk fibroin (SF) hydrogels have proven regenerative abilities and are being explored as a burn wound dressing. However, unfavorable gelation conditions limit the processability and clinical application. Herein a white light-responsive photopolymerization technique was adapted for gelation via photooxidation of tyrosine. To render the gel suitable for application to irregular and non-planar burn surfaces, SF gel-incorporated dressing (SFD) was fabricated. The mild gelation conditions using white light afforded the loading of drugs for local delivery. The moisture balance ability of the dressing was confirmed by the favorable measures of swelling capacity (106 ± 1 %) and moisture retention (≈10 h). The in vitro cytocompatibility of the gel was confirmed using HaCaT cells. Finally, in vivo performance of the SFD was tested on a second-degree burn in a rodent model. The gross analysis and histological assessment revealed scarless healing in SFD-treated groups. Overall, the SFD developed in this work is shown to be a promising candidate for advanced burn wound care.

4D Printed Programmable Shape-Morphing Hydrogels as Intraoperative Self-Folding Nerve Conduits for Sutureless Neurorrhaphy.

There are only a few reports of implantable 4D printed biomaterials, most of which exhibit slow deformations rendering them unsuitable for in situ surgical deployment. In this study, a hydrogel system is engineered with defined swelling behaviors, which demonstrated excellent printability in extrusion-based 3D printing and programmed shape deformations post-printing. Shape deformations of the spatially patterned hydrogels with defined infill angles are computationally predicted for a variety of 3D printed structures, which are subsequently validated experimentally. The gels are coated with gelatin-rich nanofibers to augment cell growth. 3D-printed hydrogel sheets with pre-programmed infill patterns rapidly self-rolled into tubes in vivo to serve as nerve-guiding conduits for repairing sciatic nerve defects in a rat model. These 4D-printed hydrogels minimized the complexity of surgeries by tightly clamping the resected ends of the nerves to assist in the healing of peripheral nerve damage, as revealed by histological evaluation and functional assessments for up to 45 days. This work demonstrates that 3D-printed hydrogels can be designed for programmed shape changes by swelling in vivo to yield 4D-printed tissue constructs for the repair of peripheral nerve damage with the potential to be extended in other areas of regenerative medicine.

Development and characterization of gel-in-water nanoemulsion as a novel drug delivery system.

The effective delivery of anti-cancer drugs with minimal side effects and better therapeutic efficacy has remained an active area of research for many decades. Organogels have gained attention in recent years as potential drug delivery systems due to their high bioavailability, no first-pass metabolism and rapid action. Considering this, in the current study an organogel based nanoemulsion was developed aiming to effectively deliver hydrophobic drugs via encapsulation within in situ gellable organogel droplets, termed as gel-in-water (G/W) nanoemulsion. G/W nanoemulsion was prepared using a combination of lipiodol and organogelator 12-hydroxystearic acid (12-HSA) as inner gel phase; dispersed in water by ultrasonication and stabilized with polyoxyethylene hydrogenated castor oil (HCO-60) as a surfactant. The prepared nanoemulsion showed high drug loading efficiency (≈97%) with a mean diameter of 206 nm. Lower polydispersity index (PdI) value (≈0.1) suggests monodispersed nature of G/W nanoemulsion in the continuous phase. G/W nanoemulsion was found stable over six months in terms of particle size, zeta potential and pH at different storage temperatures. There was no cytotoxic effect of prepared G/W nanoemulsion on primary hepatocytes in vitro. In contrast, paclitaxel-loaded G/W showed a significant decrease in melanoma cell growth (*p < 0.05) both in vitro and in vivo. Our results support the hypothesis that organogel based nanoemulsions can be a promising drug delivery system.

Co-culture of mesenchymal stem cells and human umbilical vein endothelial cells on heparinized polycaprolactone/gelatin co-spun nanofibers for improved endothelium remodeling.

Endothelization of a tissue-engineered substrate is important for its application as an artificial vascular graft. Despite recent advancements in artificial graft fabrication, a graft of <5 mm is difficult to fabricate owing to insufficient endothelization that results in thrombosis after transplantation. We aimed to perform a co-culture of adipose-derived mesenchymal stem cells (MSCs) with human umbilical vein endothelial cells (HUVECs) on antithrombogenic polycaprolactone (PCL)/heparin-gelatin co-spun nanofibers to evaluate the role of co-culturing in promoting quick endothelization of vascular substrates without surface modification by growth factors or other ECM proteins that trigger the endothelization process. Using a co-axial electrospinning technique, we attempted to fabricate our scaffold balancing between mechanical properties and biocompatibility. Antithrombogenic characteristics were then imparted to the fabricated nanofiber substrate by grafting of heparin. Finally, we performed a co-culture of MSCs and HUVECs on the fabricated co-spun nanofiber substrate to obtain proper endothelization of our material under the in-vitro culture. Staining for CD-31 at seven days of culture revealed enhanced CD-31 expression under the co-culture condition; actin staining revealed healthy cobblestone HUVEC morphology, suggesting that MSCs can aid in proper endothelization. Hence, we conclude that co-culture is effective for quick endothelization of vascular substrates.