Speaker
Description
The development of innovative bioinks and bioprinting strategies is critical for advancing tissue engineering and regenerative medicine. Collagen, a major structural protein in the extracellular matrix, is widely used in bioink formulations due to its biocompatibility and ability to support cell growth and differentiation. However, a primary challenge with collagen-based inks is their low viscosity, which can lead to undesirable spreading during the printing process and insufficient structural integrity of the printed constructs. To address these challenges, we developed a novel support bath for the (3D) bioprinting of collagen-based inks. This support bath not only prevents the spreading of low-viscosity inks, but also promotes collagen fiber formation and alignment, which is crucial to many tissue engineering applications.
The support bath was developed by modifying a previously designed buffer containing phosphate buffer and polyethylene glycol in distilled water. To increase the viscosity of this buffer, we added either nanofibrillated cellulose (NFC) or microfibrillated cellulose (MFC) at different concentrations. Cellulose was selected primarily due to its minimal impact on collagen fiber retention within the solution and its role as a versatile thixotropic agent. To evaluate the efficacy of these viscosity enhancers, we prepared three groups—2% MFC, 10% NFC, and 15% NFC—and compared their effects on final solution opacity and viscosity. As a test ink, we used solubilised articular cartilage extracellular matrix (ECM), which is rich in type II collagen.
Initial rheological data indicated that the 15% NFC solution exhibited the highest viscosity and stability at the application temperature of 37°C, making it the most promising candidate for the support bath. Furthermore, at higher concentrations, NFC produced a clearer bath than MFC, which could be beneficial for specific printing applications. We evaluated the baths’ suitability with 10 mg/ml and 50 mg/ml ECM-based inks to confirm its effectiveness with both low-viscosity and more viscous inks. Among the three groups, only the 15% NFC supportive bath demonstrated adequate support for both high- and low-viscosity inks during and after the printing process.
To create stable constructs using this 3D printing process, we subjected them to freeze-drying and cross-linking using dehydrothermal (DHT) treatment post-printing. The printed constructs were successfully retrieved by solubilizing the NFC bath in distilled water, demonstrating compatibility with collagen-rich inks. To assess fiber alignment, we used histological analysis with Picrosirius Red staining and scanning electron microscopy (SEM). Both methods confirmed enhanced collagen fiber alignment in the constructs printed within the 15% NFC support bath. Furthermore, SEM demonstrated the successful formation of collagen fibers with D-band periodicity.
In conclusion, our novel supportive bath containing 15% NFC significantly improves the stability and printability of collagen-based inks, while promoting fiber alignment in a programmable manner. It also supported the formation of collagen fibers with D-band periodicity. This support bath has potential for various tissue engineering applications, offering a robust solution for 3D printing complex collagen constructs.
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