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Among various available 3D bioprinting techniques, extrusion-based three-dimensional (3D) bio-printing allows the deposition of cell-laden bio-ink, 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 bio-ink formulation for the extrusion-based 3D bioprinting process. However, due to its low mechanical integrity, hydrogel faces a common challenge in maintaining structural ty. To tackle this challenge, we characterized the rheological properties of a set of hybrid hydrogels composed of cellulose-derived nanofiber (TEMPO-mediated nano-fibrillated cellulose, TONFC), carboxymethyl cellulose (CMC) and commonly used alginate. A total of 46 compositions were prepared using higher (0.5% and 1.0%) and lower percentages (0.005% and 0.01%) of TONFC, 1%–4% of CMC, and 1%–4% of alginate to analyze the rheological properties. The shear thinning coefficients of n and K were determined for each composition from the flow diagram and co-related with the 3D printability. The ability to control rheological properties with various ratios of a nanofiber can help achieve a 3D bio-printed scaffold with defined scaffold architecture.

Due to its inbuilt ability to release biocompatible materials encapsulating living cells in a predefined location, 3D bioprinting is a promising technique for regenerating patient-specific tissues and organs. Among various 3D bioprinting techniques, extrusion-based 3D bio-printing ensures a higher percentage of cell release, ensuring suitable external and internal scaffold architectures. Scaffold architecture is mainly defined by filament geometry and width. A systematic selection of a set of process parameters, such as nozzle diameter, print speed, print distance, extrusion pressure, and material viscosity, can control the filament geometry and width, eventually confirming the user-defined scaffold porosity. For example, carefully selecting two sets of process parameters can result in a similar filament width. However, the lack of availability of sufficient analytical relations between printing process parameters and filament width creates a barrier to achieving defined scaffold architectures with available resources. In this paper, filament width was determined using an image processing technique and an analytical relationship was developed, including various process parameters to maintain defined filament width variation for different hydrogels within an acceptable range to confirm the overall geometric fidelity of the scaffold. Proposed analytical relations can help achieve defined scaffold architectures with available resources.

Due to the three-dimensional nature of the 3D bio-printed scaffolds, typical stagnant cell culturing methods don’t ensure entering medium inside areas or passing through the scaffolds. The bioreactor has frequently provided the required growth medium to encapsulated- and seeded-cells in 3D bio-printed scaffolds. To address this issue, we developed a customized perfusion bioreactor to supply the growth medium dynamically to the cells encapsulated or seeded in the scaffolds. The dynamic supply of fresh growth medium may help improve cell viability and proliferation. Because of its uniform nutrition distribution and flow-induced shear stress within the tissue-engineering scaffold, perfusion bioreactors have been used in a variety of tissue engineering applications. Including a modified setup of our designed bioreactor may improve the in vivo stimuli and conditions, eventually enhancing the overall performance of tissue regeneration. In this paper, we explored the response of fluid flow to certain types of scaffold pore geometries and porosities. We used a simulation technique to determine fluid flow turbulence through various pore geometries such as uniform triangular, square, diamond, circular, and honeycomb. We used variable pore sizes of the scaffold maintaining constant porosity to analyze the fluid flow. Based on the results, optimum designs for scaffolds were determined.

Three-dimensional (3D) bioprinting is a promising technique for creating patient-specific 3D scaffolds of tissues or organs. An appropriate culturing process is critical to confirm encapsulated and seeded cells’ excellent viability and proliferation into scaffolds materials. Traditional stagnant cell culturing methods don’t ensure entering medium inside areas or passing through the scaffolds. To resolve this issue, we developed a customized perfusion bioreactor to supply the growth medium dynamically to the encapsulated or seeded cells. Our custom-designed bioreactor improves the in vivo stimuli and conditions, which may enhance cell viability and proliferation performance. A design of a dual medium tank was utilized allowing the replacement of already-used medium without interrupting perfusion. Accommodating an array of cassettes in a newly designed perfusion chamber allowed a wide range of scaffolds with various size and shapers to hold. In this paper, we explored fluid flow response on scaffolds fabricated with various material compositions with different viscosities. We fabricated scaffolds following a 00–900 deposition pattern with 8% Alginate, 4% Alginate-4% Carboxymethyl Cellulose (CMC), and 2% Alginate-6% CMC incubated, allowing a constant fluid flow for various periods such as 1, 2, 4, and 8 hours. The change of scaffolds fabricated with multiple material compositions was determined in terms of swelling rate, i.e., change of filament width, and material diffusion, i.e., comparison of dry material weight before and after incubation. This comparative study can assist in application-based materials selection suitable for incubating in a perfusion bioreactor.

Three-dimensional (3D) bio-printing is a rapidly growing field attempting to recreate functional tissues for medical and pharmaceutical purposes. The printability of multiple materials encapsulating various living cells can take this emerging effort closer to tissue regeneration. In our earlier research, we designed a Y-like nozzle connector system capable of switching materials between more than one filament with continuous deposition. The device had a fixed switching angle, was made from plastic, and was suitable for one-time use. This paper presents the extension of our previously proposed nozzle system. We considered 30°, 45°, 60°, and 90° angles (vertical and tilted) between the two materials and chose stainless steel as a material to fabricate those nozzle connectors. The overall material switching time was recorded and compared to analyze the effects of those various angles. Our previously developed hybrid hydrogel (4% Alginate and 4% Carboxymethyl Cellulose, CMC) was used as a test material to flow through the nozzle system. These in-house fabricated nozzle connectors are reusable, easy to clean, and sterile, allowing smooth material transition and flow.

Following the success of 3D printing with synthetic polymers like ABS, FLA, Nylon, etc., scientists and researchers have been putting efforts into fabricating bio-compatible materials. It has not only broadened the field of bioengineering and manufacturing but also regenerative medicine. Unlike the traditional 3D printing process, additive bio-manufacturing, also known as 3D bio-printing has a lot of challenges like cell survivability and proliferation, and the mechanical properties of the biomaterials which involve printability and the ability to hold its structural integrity. Proper design of experiments with extensive rheological investigation can help identify useful mechanical property ranges which are directly related to the geometric fidelity of 3D bio-printed scaffolds. Therefore, to investigate the printability of a low viscosity Alginate-Carboxymethyl Cellulose (CMC), multiple concentrations of the mixture were tested maintaining a 8% (w/v) solid content. A set of rheological tests such as the Steady Rate Sweep Test, Three Point Thixotropic Test (3ITT), and Amplitude test were performed. The outcome of those tests showed that the rheological properties can be controlled with the percentage of CMC in the mixtures. The fabricated filaments and scaffolds in the 5 combinations of CMC percentages were analyzed for flowability and shape fidelity. The rheological results and the printability and shape fidelity results were analyzed.

Three-dimensional bioprinting is a rapidly growing field attempting to recreate functional tissues for medical and pharmaceutical purposes. Development of functional tissue requires deposition of multiple biomaterials encapsulating multiple cell types i.e. bio-ink necessitating switching ability between bio-inks. Existing systems use more than one print head to achieve this complex interchangeable deposition, which decreases efficiency, structural integrity, and accuracy. In this research, we developed a nozzle system capable of switching between multiple bio-inks with continuous deposition ensuring the minimum transition distance so that precise deposition transitioning can be achieved. Finally, the effect of rheological properties of different bio-material compositions on the transition distance is investigated by fabricating the sample scaffolds.