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1. Influence of Short Fiber Encapsulated in Hybrid Hydrogel For 3d Bioprinting Process: Aspect of Rheological Properties

   Tuladhar, S.; Clark, S.; & Habib, M. A. (May 2023, Proceedings of the IISE Annual Conference & Expo 2023)

   Babski-Reeves, K; Eksioglu, B; Hampton, D. (Ed.)

   Extrusion-based three-dimensional (3D) bio-printing is one of the several 3D bioprinting methods that is frequently used in current times. This method enables the accurate deposition of cell-laden bio-ink while ensuring a predetermined scaffold architecture that may allow living tissue regeneration. Natural hydrogels are a strong choice for bio-ink formulation for the extrusion-based 3D bioprinting method because they have a combination of unique properties, which include biocompatibility, reduced cell toxicity, and high-water content. However, due to its low mechanical integrity, hydrogel frequently struggles to retain structural stability. To overcome this challenge, we evaluated the rheological characteristics of distinct hybrid hydrogels composed of carboxymethyl cellulose (CMC), a widely used alginate, and nanofibers generated from cellulose (TEMPO-mediated nano-fibrillated cellulose, TONFC). Therefore, to examine the rheological properties, a set of compositions was developed incorporating CMC (1%–4%), alginate (1%–4%), and higher and lower contents of TONFC (0.5%) and (0.005%) respectively. From the flow diagram, the shear thinning coefficients of n and K were calculated, which were later linked to the 3D printability. With the guidance of diverse nanofiber ratios, it is possible to regulate the rheological properties and create 3D bioprinted scaffolds with well-defined scaffold architecture. 

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2. Tuning Shear Thinning Factors of 3D Bio-Printable Hydrogels Using Short Fiber

   https://doi.org/10.3390/ma16020572  

   Tuladhar, Slesha; Clark, Scott; Habib, Ahasan (January 2023, Materials)

   Among various available 3D bioprinting techniques, extrusion-based three-dimensional (3D) bioprinting allows the deposition of cell-laden bioink, ensuring predefined scaffold architecture that may offer living tissue regeneration. With a combination of unique characteristics such as biocompatibility, less cell toxicity, and high water content, natural hydrogels are a great candidate for bioink formulation for the extrusion-based 3D bioprinting process. However, due to its low mechanical integrity, hydrogel faces a common challenge in maintaining structural integrity. To tackle this challenge, the rheological properties, specifically the shear thinning behavior (reduction of viscosity with increasing the applied load/shear rate on hydrogels) of a set of hybrid hydrogels composed of cellulose-derived nanofiber (TEMPO-mediated nano-fibrillated cellulose, TO-NFC), carboxymethyl cellulose (CMC), and commonly used alginate, were explored. A total of 46 compositions were prepared using higher (0.5% and 1.0%) and lower percentages (0.005% and 0.01%) of TO-NFC, 1–4% of CMC, and 1–4% of alginate to analyze the shear thinning factors such as the values of n and K, which were determined for each composition from the flow diagram and co-related with the 3D printability. The ability to tune shear thinning factors with various ratios of a nanofiber can help achieve a 3D bio-printed scaffold with defined scaffold architecture. 

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3. 3D Bio-Printability of Hybrid Pre-Crosslinked Hydrogels

   https://doi.org/10.3390/ijms222413481  

   Nelson, Cartwright; Tuladhar, Slesha; Launen, Loren; Habib, Ahasan (December 2021, International Journal of Molecular Sciences)

   Maintaining shape fidelity of 3D bio-printed scaffolds with soft biomaterials is an ongoing challenge. Here, a rheological investigation focusing on identifying useful physical and mechanical properties directly related to the geometric fidelity of 3D bio-printed scaffolds is presented. To ensure during- and post-printing shape fidelity of the scaffolds, various percentages of Carboxymethyl Cellulose (CMC) (viscosity enhancer) and different calcium salts (CaCl2 and CaSO4, physical cross-linkers) were mixed into alginate before extrusion to realize shape fidelity. The overall solid content of Alginate-Carboxymethyl Cellulose (CMC) was limited to 6%. A set of rheological tests, e.g., flow curves, amplitude tests, and three interval thixotropic tests, were performed to identify and compare the shear-thinning capacity, gelation points, and recovery rate of various compositions. The geometrical fidelity of the fabricated scaffolds was defined by printability and collapse tests. The effect of using multiple cross-linkers simultaneously was assessed. Various large-scale scaffolds were fabricated (up to 5.0 cm) using a pre-crosslinked hybrid. Scaffolds were assessed for the ability to support the growth of Escherichia coli using the Most Probable Number technique to quantify bacteria immediately after inoculation and 24 h later. This pre-crosslinking-based rheological property controlling technique can open a new avenue for 3D bio-fabrication of scaffolds, ensuring proper geometry. 

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4. Rheological Investigation of Pre-Crosslinked Hybrid Hydrogels for 3D Bio-printing Processes.

   Tuladhar, Slesha; Nelson, Cartwright; Habib, Ahasan (May 2022, Proceedings for IISE Annual Conference & Expo 2022)

   Ellis, K; Ferrell, W; Knapp, J. (Ed.)

   Despite being a very popular topic and researched by several scientists, the entire 3D bioprinting process is still subjected to several challenges like geometric fidelity, mechanical complexities, cell viability, and proliferation. Rheological investigations along with the proper design of experiments help to explore the physical and mechanical properties of biomaterials and 3D printed scaffolds that are directly associated with their geometric fidelity. To ensure post-printed structural integrity, viscosity thickeners and crosslinkers were used in this research. Mixtures of Carboxymethyl Cellulose (CMC, viscosity enhancer), Alginate, and CaCl2 and CaSO4 (crosslinkers) were prepared at various concentrations maintaining minimum solid content. For each composition, a set of rheological tests was performed in form of flow, thixotropic, amplitude, and frequency tests. This research presents an overview of controlling the rheological properties of various bio-inks that are viscosity enhancer and pre-crosslinkers dependent, which opens doors to looking at 3D bioprinting in a very different way. 

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5. Rheological Study of Low-Viscous Hydrogels for 3d Bio-Printing Processes

   Tuladhar, Slesha; Nelson, Cartwright; Habib, Ahasan. (July 2021, Proceedings of the 2021 IISE Annual Virtual Conference)

   The promising success of 3D printing technique with synthetic polymers like nylon, ABS, PLA and epoxy motivates the researchers to put efforts into fabricating constructs with biocompatible natural polymers. The efforts have been broadened into various fields such as bioengineering, manufacturing, and regenerative medicine. Additive biomanufacturing commonly known as 3D bioprinting shows a lot of potential in tissue engineering with those natural polymers. Some challenges such as achieving printability, maintaining geometry in post printing stage, comforting encapsulated cells, and ensuring high proliferation are to be resolved to turn this process into a successful trial. Appropriate design of experiments with a detail rheological investigation can identify useful mechanical properties which is directly related to shape fidelity of 3D bio-printed scaffolds. As candidate natural polymers, Alginate-low viscous Carboxymethyl Cellulose (CMC) was used restricting the solid content 8% (w/v). Various rheological tests, such as the Steady Rate Sweep Test, Thixotropic (3ITT), Amplitude, and Frequency test were performed. The result indicated that rheological properties are CMC dependent. Printability and shape fidelity were analyzed of the filaments and scaffolds fabricated with all the combinations. The rheological results were co-related with the printability and shape fidelity result. 

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