(B-52) Extrusion-based 3D printed thiolated gelatin hydrogels for fibrocartilaginous enthesis regeneration

Introduction: The enthesis is a highly organized tissue that transitions between the tendon and the bone and transduces strain between them. Rotator cuff injuries occur at this junction and are treated by mechanically attaching the tendon and bone. In the US, approximately 250,000 rotator cuff surgeries are performed annually, resulting in up to 94% of retears, suggesting a need for new treatment approach [1,2]. Previously, we have developed a biomaterial to treat enthesis injuries, featuring a thiolated-gelatin (Gel-SH) hydrogel interface connecting mineralized collagen (bone) and non-mineralized collagen (tendon) porous scaffolds to dissipate localized strain at the interface effectively [3,4]. Yet, new avenues are needed to investigate whether a spatially controlled material would enhance material’s biological performance. Incorporating mesenchymal stem cells (MSCs) may be beneficial for enthesis repair due to their ability to support enthesis fibrocartilage differentiation and modulate the host immune response. Currently, we are seeking to develop 3D printing approaches to spatially control the integration of MSC-laden hydrogels into porous collagen biomaterials, as this technique allows for fabricating constructs with controlled patterns relevant to specific tissues. We employ Gel-SH hydrogels as the basis for the MSC-laden hydrogel, however, current material properties lead to long crosslinking times and vast rheological variations throughout the bioprinting process. This work proposes the development of a printable hydrogel that will allow for the deposition of patterned cells, investigating Gel-SH kinetics using small amplitude oscillatory rheology (SAOS), and estimate effective 3D printing time limits during crosslinking process.


Materials and Methods: Thiolated gelatin (Gel-SH) was synthesized by substituting primary amines on the gelatin backbone for thiols using Traut’s reagent and lyophilized for 7 days. Gel-SH hydrogels were fabricated by creating disulfide bonds using horseradish peroxidase (HRP) as a catalyst reagent to create phenolic radicals on tyramine, which oxidizes sulfhydryl groups on Gel-SH. 2 mL of 7.5 wt.% Gel-SH hydrogel precursor was loaded into an Anton Paar MCR 302 Rheometer after adding HRP crosslinking solution to perform time sweeps at different frequencies, using 25 mm stainless steel plates at 2% strain, 25 °C, and 1 mm gap to differentiate crosslinking kinetics between functionalized gelatin. 5 mL of 7.5 wt. % Gel-SH was printed (1 bar, 16 mm/s) after starting the crosslinking process using a Bioplotter Manufacturer Series (EnvisionTEC, Germany) to study its printability.


Results, Conclusions, and Discussions:

Preliminary results show that Gel-SH has the potential to be an effective printable bioink. We defined gelation time as the cross point where the elastic modulus (G’) was equal to the loss modulus (G’’) on a time sweep (1 rad/s and 2% strain) and estimated a suitable printing time after iterating. The gelation time for 7.5 wt.% Gel-SH hydrogels was 10 minutes, and its effective printing time was 17 minutes, Figure 1a. 3D printed constructs held up to 4 layers of extrusion without external supports, and printed filaments achieved diameters below 600 µm, Figure 1b. Gel-SH hydrogels exhibited great shape fidelity compared to the designed CAD, making Gel-SH a promising material for 3D bioprinting. Current work seeks to understand Gel-SH hydrogel kinetics to adjust printing settings and achieve higher deposition control via SAOS and steady shear rheology. Using this dynamic material, we envision defining a printability parameter describing the strength-to-mass ratio of an effective bioink. Following this study, we aim to predict suitable printing parameters for cell-seeded materials. We expect changes on cell-seeded Gel-SH hydrogel kinetics, however, we are confident that this first study will establish an approach to define suitable printable conditions, decreasing exhausting iterative printing process and amounts of materials needed. Future work will study MSCs seeded hydrogels viability and metabolic activity after 3D extrusion, to understand materials capacity for cellular protection.

Gel-SH hydrogels exhibit great challenges for 3D bioprinting due to their dynamic crosslinking process. However, its ability to form stable tunable materials via enzymatic-catalyzed reaction makes it a promising material for cell-laden hydrogels. The presented methodology will elucidate a novel strategy for printing dynamic materials, avoiding iterative steps and accelerating the material fabrication process. This study will enable us to tune hydrogel properties and printing conditions, such as gelatin degree of functionalization, temperatures, and concentrations of cell-seeded materials, leading to a biomaterial with the potential of regenerating the enthesis environment.