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Type I collagen, is the central component of the extracellular matrix (ECM) and its use in the context of regenerative medicine is expected to mark an important turning point towards the use of biomimetic materials in regenerative medicine. Collagen provides mechanical support and biological cues for cellular adhesion and proliferation making it an ideal choice for fabricating biomimetic scaffolds1. In vivo it presents a hierarchical organization in which collagen triple helices self-assemble into highly ordered fibrils which are necessary for cell adhesion and to in a process called fibrillogenesis. However, these structures are seldom found in vitro since their formation requires extremely high collagen concentrations 2.
Integrating collagen’s intrinsic ability to self-assemble with a bottom-up fabrication technique such as 3D printing could bridge the gap in recreating a new generation of biological tissue analogues, able to reproduce their hierarchical nature. Despite the potential to overcome traditional tissue engineering limitations, this combined approach is extremely difficult to implement 3 due to the dramatic viscosity increase with concentration.
Here, we report a new strategy for formulating highly concentrated collagen bioinks with drastically reduced viscosity able to bypass current limitations in 3D bioprinting type I collagen and ensure an ideal environment to support cellular processes.4
The bioink relies on the interaction between collagen and ATP, a small electrolyte, causing the partition and concentration of collagen molecules inside droplets. Rheological measurements allowed to assess the impact of the coacervation process on printability. The formulation and the rheological properties of the bioink were correlated to the viability, proliferative status, morphology and migratory ability of Normal Human Dermal Fibroblasts (NHDFs) in a collagen matrix during 21 days of culture.
Rheological analysis revealed a ten-fold decrease in dynamic viscosity with respect to collagen in solution at equivalent concentration. Importantly, collagen coacervation does not impair the fibril formation process and allows the formation of ordered collagen motifs.
The biocompatibility was assessed by NHDFs’ metabolic activity in dense collagen matrix at 60 mg/mL. Under the confocal microscope (Figure 1) fibroblasts display physiological characteristics (spindle shape and cytoplasmic projections) and colonized and remodelled the matrix the entire printout volume. Because of the biomimetic nature of the resulting materials the final geometry is constant even up to one month of cell culture, a rare feature since fibroblasts tend to contract sub-physiological collagen matrices.
Our formulation offers an innovative collagen bioink, with drastically reduced viscosity, compatible with extrusion-based bioprinting at physiologically relevant concentrations. Moreover, our bioink differentiates itself from existing literature as the only available alternative to process highly concentrated collagen solutions (60-80 mg/mL) by 3D bioprinting, while encapsulating cells. This work provides a compelling formulation to significantly improve the design of 3D cell culture scaffolds and tissue engineering constructs, enabling a better reiteration of tissue macro and microanatomy and enhancing biological function with extended lifespan.
References: 1Zhang, Nat rev method prime. 2021;1:75. 2Darvish, Mater Today Bio. 2022;15:100322. 3 Nichol, Soft Matter. 2009;5:1312-1319. 4Sarrigiannidis SO. Mater Today Bio. 2021;10:100089. 4Blaga D. submitted, 2025.
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