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Biomaterials

Biomaterials - Poster Session E

Poster N14 - Engineering Robust 3D-Printed Tubular Constructs from a Collagen-Microgel Composite Material

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  The material properties of biopolymer networks become highly non-linear under modest strains, which could be useful for engineering biological constructs having tunable and widely variable elasticity. Nonlinear material response is found in reconstituted extracellular matrices and cytoskeletal networks, and inspiration for tunable elastic materials can be drawn from the cell itself; the cell’s elasticity is controlled by cytoskeletal pre-stress generated by molecular motors. However, making tunable elastic materials that rely on molecular motors involves challenges like the stability and spatial distribution of constituent proteins and a supply of ATP. To sidestep these limitations, we leverage materials that hold their configuration after a large strain is applied: packings of soft particles. One of the most useful rheological properties of packed micro-scale hydrogel particles (microgels) is their ability to quickly relax stress after large strains have been imposed, in contrast to elastic networks that store elastic energy and work to return to states of zero strain. In this work, we show how combining the non-linear elasticity of biopolymers with the reconfigurability of microgels enables the development of advanced materials that exhibit dramatic changes in material properties in response to mechanical stimuli. We focus on engineering hollow tube-shaped constructs as a first application area of this new material, demonstrating its compatibility with additive manufacturing.

Materials and

Methods:  To formulate the composite material, polyethylene glycol (PEG) microgels are synthesized using inverse emulsion polymerization. The microgels are collected by centrifugation and serial washing in ethanol, then PBS. The microgel solution is autoclave sterilized, lyophilized, reconstituted in an aqueous buffer, then mixed with 25µM HEPES buffer, PBS, and cold collagen solution at the final desired composition. The material is mixed in a planetary speed mixer and then transferred to ice until experiments are performed. Material properties of the 3mg/mL collagen and 4% (w/w) microgel composites are measured by conducting shear rheology experiments on an Anton Paar MCR 702 rheometer. The composite material is gelled on the rheometer; gelation kinetics are measured under low-amplitude oscillation as the temperature is ramped up to 37⁰C. Sample evaporation is prevented by a solvent trap. Shear moduli of the composite materials are measured under oscillatory shear at 2% strain amplitude over a frequency range of 0.01-10Hz. Stress relaxation and creep compliance measurements are performed in response to step-strains between 2-60% and step-stresses between 0.1-200Pa. The composite materials are 3D printed jammed microgel support media using a custom instrument. Composite microstructure is studied with fluorescence and reflectance confocal microscopy on a Nikon Eclipse Ti-2 microscope. Microgels conjugated with rhodamine B acrylate are imaged using a TRITC fluorescence filter configuration and collagen fibers are imaged using a confocal reflectance filter configuration. To demonstrate the quality of 3D printed structures and the robustness of the structures at the macro-scale, we collect digital photographs, videos, and time-lapse sequences.

Results, Conclusions, and Discussions: Gelation kinetics measurements show that G’ increases by a factor of 20 and plateaus over a time-window of approximately 15 minutes, indicating collagen gelation. Gelled samples are further tested with frequency sweeps, in which we find that G ’ > G”. Both G ’ increases very weakly with increasing frequency, indicating that the composites behave like damped elastic solids over three decades of oscillation frequency. At a representative frequency of 0.1Hz, the elastic modulus of the composite is approximately 350Pa, which is approximately 1/30 the modulus of typical soft tissues. Creep compliance and stress relaxation measurements show that the material strongly strain stiffens, resists persistent stresses, and also “locks in” deformations over long time-scales. For example, the apparent storage modulus under 200 Pa of stress is 750Pa, more than 2x the linear modulus. To demonstrate the robustness of composites, we 3D-printed tubes at three diameters and extracted them from the support material into a PBS bath; we found that the tubes maintained their shape and different orientations and were resistant to applied strains of over 100%. Confocal microscopy images directly showed collagen fiber networks interpenetrating packs of microgel particles. Photography and low-resolution microscopy demonstrated our ability to control the size and shape of the 3D printed structures. (Fig. 1).

Collagen-microgel composite materials can be 3D-printed into robust constructs at low collagen concentrations. Our rheological testing of cast materials demonstrate the robustness of these materials, and our ability to handle and vigorously shear 3D printed constructs confirms that these properties are not eliminated by the additive manufacturing process. However, further quantification is required. Still, constructs made from pure collagen at the same concentrations would totally collapse cell-generated stresses; our rheological tests demonstrate that they will remain mechanically stable under biologically-relevant stresses. These composite materials may find use in biomedical applications; they are made from biocompatible constituents, and we foresee multiple paths forward for seeding them with cells.

Acknowledgements (Optional):