Influence of Polymerization Conditions on Collagen I, II and III Blend Hydrogels

Collagen hydrogels are used extensively in literature for a wide range of applications. Collagen I hydrogels are the most prevalent type explored due to its availability and lower cost. However, in native tissue, multiple collagen types are often found to work in conjunction to create stable structural support for tissues and provide specific biological cues for cells. Thus, blended hydrogels have been studied, under physiological conditions, for various applications such as cartilage tissue engineering (I/II)¹ and for cardiac and vocal tissue engineering (I/III)² to more accurately recapitulate native tissue. The addition of collagen II and III for these applications resulted in improved biological results which highlights the importance of blended collagen hydrogels. The impact of polymerization temperature on the polymerization of collagen I hydrogels has been explored in literature and has been shown to have a profound impact on the microstructure and resulting mechanical and transport properties of the hydrogels.<br/>This work extends the notion of exploring polymerization temperature to adjust the gelation of blended hydrogels to alter hydrogel microstructure and thereby tune the mechanical and transport properties of the gels. In this work, we studied the effect of various polymerization temperatures (25, 30, and 37 °C) on the polymerization kinetics of blended hydrogels (I/II and I/III) compared to collagen I hydrogels alone, and the corresponding impact on fundamental hydrogel properties. Through turbidity measurements and rheological time sweeps, we found that the addition of collagen II or III slows the polymerization time compared to pure collagen I hydrogels. Additionally, within each hydrogel type, increasing temperature reduced polymerization time. The differences in polymerization kinetics contributed to variable microstructures. Hydrogels polymerized at 37 °C appeared similar across the different polymerization conditions tested. These gels resulted in smaller, more compact fibrils. When polymerization temperatures were lowered to 30 °C, the blended hydrogels began to exhibit differences as compared to collagen I alone. Microstructure differences in the hydrogels led to variations in mechanical properties. Mechanical properties for all gel types were the highest for hydrogels polymerized at 25 °C and lower for both 30 and 37 °C. When exploring the transport properties, the addition of collagen II or III to collagen I altered the diffusion of dextran through the matrix as observed by Transwell macromolecular recovery studies. Finally, the degradation rates of the gels slowed under collagenase with the addition of collagen II or III.<br/>¹Kilmer, C. E. et al. Collagen Type I and II Blend Hydrogel with Autologous Mesenchymal<br/>Stem Cells as a Scaffold for Articular Cartilage Defect Repair. <i>ACS Biomater. Sci. Eng.</i> 6,<br/>3464–3476 (2020).<br/>²Roman, B. et al. A Model for Studying the Biomechanical Effects of Varying Ratios of Collagen Types I and III on Cardiomyocytes. <i>Cardiovasc. Eng. Technol. </i>(2021).