(I-349) Development of High-Density Fluorescent Collagen Gels for Tracking Cellular Turnover of Collagen During Hierarchical Fiber Formation

Introduction::
Hierarchical organized collagen fibers are the primary source of strength in connective tissue throughout the body, particularly in tendons and ligaments. Injuries to these tissues disrupt collagen organization, resulting in loss of function and over 1.6 million surgeries a year in the US.^1,2 Current treatment strategies fail to restore the structure or function of native tissue. It remains a challenge to form the hierarchical collagen fibers essential for long-term mechanical success in engineered replacements and in vivo after injury.²









Previously, we developed a novel culture system that guides cells in high-density collagen gels to develop aligned collagen fibrils by 2 weeks, that mature into native-sized fibers and fascicles by 6 weeks (Fig 1A).² In this system we start with a collagen hydrogel, and after 6 weeks of culture it is unclear how much is reorganized collagen from the original hydrogel versus new collagen produced by the cells. Having a way to track collagen turnover will allow for better understanding of how cells reorganize and develop collagen fibers.^3,4 The objective of this study was to develop fluorescently labeled rat-tail collagen gels for use in our culture system so to track collagen turnover during hierarchical fiber formation. We hypothesize that the cells will metabolize the fluorescently labeled collagen and release FITC into the media during culture, providing a means to track collagen turnover. We further hypothesize that with time in culture cells will produce de novo unlabeled collagen, resulting in a mixture of labeled and non-labeled organized collagen by 6 weeks.




Materials and Methods::
To produce fluorescent collagen stocks, collagen was reconstituted from rat-tail tendons and stained with 1 mg/mL fluorescein isothiocyanate (FITC).³ Both high and low FITC:collagen molar ratios were evaluated to assess collagen stability. Similar to previous work,⁴ we found the low ratio (1.2 FITC:collagen) yielded more stable collagen, similar to unlabeled collagen, and it was used to make constructs. To evaluate whether cells could still organize and turnover labeled collagen, constructs were created as previously described.² Briefly, neonatal bovine ACL fibroblasts were mixed with FITC collagen to form 20 mg/mL sheet gels at 5x10⁶ cells/mL.² Rectangles (8x30 mm) were cut from the sheet gels and cultured in our clamping device for up to 6 weeks. Cell-free FITC constructs, denoted FITC (-)cells, were produced to evaluate fluorescent fade with time in culture verse cell-mediated reduction in fluorescence. Non-labeled cell-seeded constructs, denoted (-)FITC (+)cells, served as positive controls to determine whether FITC disrupts cellular interaction with collagen gels. Throughout culture, fluorescence of constructs and media were measured at each media change at 495/519 nm ex/em and normalized to constructs at day 1 or fresh media, respectively, to track collagen turnover. Post-culture, organization, composition, and mechanics were analyzed. Confocal reflectance was performed to analyze collagen organization and fluorescence.² Tissue mechanics were analyzed by tensile tests at 0.75% strain/sec to failure.² DNA, glycosaminoglycans (GAGs), and collagen content were measured via Picogreen, DMMB, and hydroxyproline (Hypro) assays.² All data are mean±SD. Statistical analysis was performed by 1- and 2-way ANOVA with Tukey’s post-hoc (p< 0.05 significant).




Results, Conclusions, and Discussions::
Both FITC and non-labeled cell-seeded constructs contracted similarly with time in culture, indicating FITC does not impede cellular interaction with collagen (Fig 1B). Fluorescence measurements taken throughout culture revealed constructs maintained fluorescence through 6 weeks and cells turnover FITC collagen, resulting in significant accumulation of FITC label in the media with time in culture (Fig 1C). Further, FITC did not restrict cell-driven fiber formation, with FITC constructs having similar fiber organization as non-labeled constructs by 6 weeks. Specifically, FITC cell-seeded constructs formed aligned collagen fibrils primarily composed of fluorescent collagen by 2 weeks (data not shown), which matured into larger fibers by 6 weeks with additional de novo collagen (Fig 1D). Collectively, we found cells release a large amount of FITC in the first 2-3 weeks as they are producing aligned fibrils, and have reduced turnover of collagen later in culture as they build larger fibers, shedding light on cellular regulation of collagen during fiber formation. Ongoing work is quantifying collagen in the media with time in culture to further evaluate turnover of original FITC gel vs cell produced collagen.









Mirroring fiber organization, FITC and non-labeled cell-seeded constructs had similar significant increases in tissue tensile mechanics by 6 weeks (Fig 1E). Further, FITC did not affect cell-driven changes in ECM composition. Interesting, FITC constructs had significantly higher DNA compared to non-labeled controls at 2 and 6 weeks. However, despite differences in DNA, FITC and non-labeled constructs had similar concentrations of GAG and collagen.








Overall, we have fluorescently labeled high-density collagen gels, without disrupting fibril formation or cellular interaction with the collagen, and have shown the constructs maintain fluorescence over 6 weeks with little-to-no fading. Further, cells were able to produce aligned hierarchical collagen fibers in FITC collagen, similar to non-labeled collagen. This system allows us to track collagen turnover, providing a better understanding of how cells develop hierarchical fibers. Having a means to evaluate when cells start to produce their own collagen during hierarchical fiber formation will aid in determining cues that drive fiber formation so to better regenerate tendons and ligaments throughout the body.


Acknowledgements (Optional): : This work is supported by the VCU Undergraduate Research Opportunity Program and the VCU Center for Clinical Translational Research (CCTR)

References (Optional): :
[1] Patel+ JOR 2018; [2] Puetzer+ Biomat. 2021; [3] Grunert+ Acta Biomat. 2015; [4] Siadat+ Macromolecular Biosci. 2022