(K-440) 3D Scaffold Optimization of an Aligned Collagen I Hydrogel for Disease Modeling

The extracellular matrix (ECM) is the microenvironment that provides structure and biochemical cues to cells ¹. ECM are made primarily of collagen I, whose dysregulated maintenance has been linked to the development of various diseases including fibrosis and cancer ^{2 3 4}. Tissue ECM proteins can align during diseases while collagen fiber thickness has been shown to increase with tumor growth and disease development ^{5 6 4}. Currently, there is not a comprehensive model of ECM microarchitecture for diseased microenvironment. The goal of this experiment was to optimize the procedure for a temperature-dependent casting method with aligned fibers to mimic pathological conditions. Our model for damaged tissue uses a collagen I hydrogel that mimics diseased tissue architecture. A custom-made stretching device coupled with cold-casting and pre-stretching gels was used to create thicker aligned fibers. A more accurate disease model will allow researchers to test the response of diseased tissue to varying treatments while decreasing the use of animal testing ^{4 7}.

Collagen I was isolated from rat tails by removing the tendons between each vertebra as previously described ⁸. Tendons were digested in acetic acid for 3 days, and the supernatant was removed and frozen at -20°C for at least 24 hours. The supernatant was lyophilized and resuspended as a 10 mg/mL pregel.

Silicone molds were applied to polydimethylsiloxane (PDMS) sheets and placed in a plasma cleaner for 5 minutes. Molds for stretched samples were placed between two clamps on either side that were pulled apart and held on a stretching device. Non-stretched samples were placed directly into a petri dish. Plasma-treated molds were further modified using polyethyleneimine and glutaraldehyde to promote hydrogel crosslinking as previously established ⁹. M199 media was added to the collagen pregel, the gel was neutralized using sodium hydroxide and/or hydrochloric acid, and phosphate-buffered saline (PBS) was added to make the final pregel concentration of 3 mg/mL. The solution was added to wells of the treated silicone molds.

Cold-casting gels began at 4°C, and the temperature was increased through a step-wise process to 37°C. This experiment varied time spent at 4°C, 22°C, and 37°C to slow polymerization of the collagen, allowing thicker fibers to form. Warm-cast gels were immediately warmed to 37°C for 30 minutes. Stretched gels were removed from the stretcher, and PBS was added to cover the gels to retain hydration during storage.

Collagen fibril network in different gels were visualized using confocal reflectance microscopy. Image analysis was performed to determine fiber thickness and alignment.

Collagen formation at 37°C mimics the structure of healthy tissue through formation of a network of thin fibers ⁶. In contrast, cold-casting collagen I limits the number of smaller fibers formed and increases the thickness of existing fibers through an entropy-driven process ². Longer and thicker fibers mimic the collagen structure found in diseased tissues. Therefore, cold-cast hydrogels can act as a model for disease. By adding uniaxial strain to gels, collagen fibers align perpendicular to the direction of strain, causing alignment seen in diseased tissues ⁵. To optimize the procedure for stretched cold-cast samples, time spent at 4°C/22°C/37°C was varied. Times combinations were initially tested with non-stretched hydrogels and then narrowed down to four combinations: 10/5/15, 10/10/10, 10/15/5, and 10/15/10. These time frames showed longer and thicker fibers not seen in other hydrogels. It is speculated that longer incubation times at 4°C caused the collagen to aggregate, preventing thicker fiber formation. Stretched gels were made using the time combinations, and the 10/15/5 time frame demonstrated the most significant alignment and fiber thickness therefore mimicking ECM microarchitecture for disease. Fiber thickness was measured by stacking images of the gels and using Fiji ImageJ to measure the diameter of individual fibers. 75 fibers were measured per time frame, and the largest measured thickness was 2.59 µm found during the 10/15/5 time frame. Average fiber thickness was found to be 2.10 µm for 10/5/15, 2.34 µm for 10/10/10, and 2.33 µm for 10/15/10. An ANOVA test was performed and followed up with a Tukey Post Hoc test in which it was determined that there was a significant difference between the time frames of 10/5/15 and 10/15/5; however, no other time frame was found to have a significant difference. Since the 10/10/10 method is used within current practice and no other time combination is found to have significantly larger fibers, hydrogels for disease modeling will be made using the 10/10/10 time frame within future work. In the future, aligned cold-cast hydrogels will be used to test treatment for fibrotic scarring seen in fibrosis and traumatic injuries, as well as solid cancers exhibiting desmoplasia.

1.           Hussey, G. S., Dziki, J. L. & Badylak, S. F. Extracellular matrix-based materials for regenerative medicine. Nat. Rev. Mater. 3, 159–173 (2018).

2.           McCoy, M. G., Seo, B. R., Choi, S. & Fischbach, C. Collagen I hydrogel microstructure and composition conjointly regulate vascular network formation. Acta Biomater. 44, 200–208 (2016).

3.           Extracellular Matrix in Kidney Fibrosis: More Than Just a Scaffold - Roman David Bülow, Peter Boor, 2019. https://journals.sagepub.com/doi/full/10.1369/0022155419849388.

4.           Gurrala, R. et al. Quantifying Breast Cancer-Driven Fiber Alignment and Collagen Deposition in Primary Human Breast Tissue. Front. Bioeng. Biotechnol. 9, (2021).

5.           Kim, S. H. et al. Anisotropically organized three-dimensional culture platform for reconstruction of a hippocampal neural network. Nat. Commun. 8, 14346 (2017).

6.           Seo, B. R. et al. Collagen microarchitecture mechanically controls myofibroblast differentiation. Proc. Natl. Acad. Sci. 117, 11387–11398 (2020).

7.           Wilks, B. T. et al. Quantifying Cell-Derived Changes in Collagen Synthesis, Alignment, and Mechanics in a 3D Connective Tissue Model. Adv. Sci. 9, 2103939 (2022).

8. Lewis, M, et al. Neuro-Regenerative Behavior of Adipose-Derived Stem Cells in Aligned Collagen I Hydrogels. Preprint. BioRxiv. (2023).

9.           Verbridge, S. S. et al. Oxygen-controlled three-dimensional cultures to analyze tumor angiogenesis. Tissue Eng. Part A 16, 2133–2141 (2010).