Development and Characterization of Gelatin-Norbornene Bioink to Understand the Interplay between Physical Architecture and Micro-Capillary Formation in Biofabricated Vascularized Constructs.

The principle challenge for engineering viable, cell-laden hydrogel constructs of clinically-relevant size, is rapid vascularization, in order to moderate the finite capacity of passive nutrient diffusion. A multiscale vascular approach, with large open channels and bulk microcapillaries may be an admissible approach to accelerate this process, promoting overall pre-vascularization for long-term viability of constructs. However, the limited availability of bioinks that possess suitable characteristics that support both fabrication of complex architectures and formation of microcapillaries, remains a barrier to advancement in this space. In this study, gelatin-norbornene (Gel-NOR) is investigated as a vascular bioink with tailorable physico-mechanical properties, which promoted the self-assembly of human stromal and endothelial cells into microcapillaries, as well as being compatible with extrusion and lithography-based biofabrication modalities. Gel-NOR constructs containing self-assembled microcapillaries are successfully biofabricated with varying physical architecture (fiber diameter, spacing, and orientation). Both channel sizes and cell types affect the overall structural changes of the printed constructs, where cross-signaling between both human stromal and endothelial cells may be responsible for the reduction in open channel lumen observed over time. Overall, this work highlights an exciting three-way interplay between bioink formulation, construct design, and cell-mediated response that can be exploited towards engineering vascular tissues.

Visible light mediated PVA-tyramine hydrogels for covalent incorporation and tailorable release of functional growth factors.

The translation of growth factors (GFs) into clinical applications is limited by their low stability in physiological environments. Controlled GF delivery through biomaterial vehicles provides protection from proteases, targeted delivery, and longer term release profiles. However, current methods used to incorporate GFs into biomaterials still present limitations. While direct adsorption and encapsulation result in burst release, covalent incorporation provides a tailorable release profile but generally requires more complicated processes and chemical modification of the GFs. Bioaffinity methods provide long-term release profiles but fail in their specificity, resulting in GF-dependent applicability and release profiles. In the present study, we introduce tyraminated poly-vinyl-alcohol (PVA-Tyr) as a GF-delivery vehicle that can covalently incorporate native GFs through a photo-initiated cross-linking process via formation of bi-phenol bonds. Mass loss and release studies revealed that protein-loaded PVA-Tyr hydrogels had highly tailorable degradation times from 7 to 92 days, during which the covalently incorporated proteins were released in a linear fashion. The incorporation of bovine serum albumin (BSA), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), or brain-derived growth factor (BDNF) resulted in similar incorporation efficiencies and release profiles, demonstrating the low specificity and versatility of the system. Furthermore, functional studies demonstrated that VEGF, bFGF and BDNF released from the PVA-Tyr hydrogels retained the ability to increase the metabolic activity, migration, and 3D vessel formation of endothelial cells and mesenchymal stem cells. Taken together, this demonstrates that PVA-Tyr shows high potential as a highly tailorable GF delivery tool for a range of different regenerative medicine applications.

One-Step Photoactivation of a Dual-Functionalized Bioink as Cell Carrier and Cartilage-Binding Glue for Chondral Regeneration.

Cartilage defects can result in pain, disability, and osteoarthritis. Hydrogels providing a chondroregeneration-permissive environment are often mechanically weak and display poor lateral integration into the surrounding cartilage. This study develops a visible-light responsive gelatin ink with enhanced interactions with the native tissue, and potential for intraoperative bioprinting. A dual-functionalized tyramine and methacryloyl gelatin (GelMA-Tyr) is synthesized. Photo-crosslinking of both groups is triggered in a single photoexposure by cell-compatible visible light in presence of tris(2,2'-bipyridyl)dichlororuthenium(II) and sodium persulfate as initiators. Neo-cartilage formation from embedded chondroprogenitor cells is demonstrated in vitro, and the hydrogel is successfully applied as bioink for extrusion-printing. Visible light in situ crosslinking in cartilage defects results in no damage to the surrounding tissue, in contrast to the native chondrocyte death caused by UV light (365-400 nm range), commonly used in biofabrication. Tyramine-binding to proteins in native cartilage leads to a 15-fold increment in the adhesive strength of the bioglue compared to pristine GelMA. Enhanced adhesion is observed also when the ink is extruded as printable filaments into the defect. Visible-light reactive GelMA-Tyr bioinks can act as orthobiologic carriers for in situ cartilage repair, providing a permissive environment for chondrogenesis, and establishing safe lateral integration into chondral defects.

Growth Factor Delivery Systems for Tissue Engineering and Regenerative Medicine.

Growth factors (GFs) are often a key component in tissue engineering and regenerative medicine approaches. In order to fully exploit the therapeutic potential of GFs, GF delivery vehicles have to meet a number of key design criteria such as providing localized delivery and mimicking the dynamic native GF expression levels and patterns. The use of biomaterials as delivery systems is the most successful strategy for controlled delivery and has been translated into different commercially available systems. However, the risk of side effects remains an issue, which is mainly attributed to insufficient control over the release profile. This book chapter reviews the current strategies, chemistries, materials and delivery vehicles employed to overcome the current limitations associated with GF therapies.