Poster V3 - Characterization of Collagen Remodeling in HABP-instructed Endogenous HA Environments

Introduction: Remodeling of Collagen fibrils are regulated by complex events not only modulated by matrix remodeling enzymes,
but also crosslinking reactions with other proteins, proteoglycans and glycosaminoglycans such as Hyaluronic acid
(HA). The interactions between HA and Collagen is unique because the quantity, molecular weight and sequential
order of expression of HA tightly regulates scar formation after wound healing. For instance, embryonic wounds
known to have early deposition of HA with high molecular weights leading to recovery of basket weave pattern in
wounded site [1]. HA binding peptides (HABP) are unique molecules that have high affinity towards HA and we
recently showed the surfaces coated by HABPs have ability to retain endogenous HA produced by fibroblasts. We
then asked if endogenous HA produced by dermal fibroblasts have potential to modulate collagen fibril organization
by the fibroblasts and whether fibroblasts with neonatal origin have differences in Collagen remodeling in elevated
HA environments compared to adult fibroblasts. Towards this goal, we encapsulated primary human neonatal
(nHDF) and adult (aHDF) dermal fibroblasts in collagen gels formed in the presence/absence of HABPs.
Furthermore, using 2-photon confocal microscopy equipped with Second Harmonic Generation (SHG) imaging
capability we quantitatively analyzed collagen remodeling. We wish to understand whether HABP instructed
endogenous HA could tune collagen organization

Materials and

Methods: Preparing Collagen Gels: The collagen gels were created with 10X
PBS, Type I Bovine Collagen Solution, and varying amounts of 0.5 M
NaOH, diH₂O, HABP, Scr-HABP for maintaining a pH score of 7. All
of the components were kept on ice to prevent the gelation process
before placing 250 µl of each mixture into a MatTek Glass Bottom
Culture Dishes. The gels were then left to incubate at 37°C for 2 hours
to solidify. Cell Culturing of ahDF and nhDF: aHDF and nHDF were
obtained from ATCC and used within passages 1-15. Cells were
cultured in an animal-free medium obtained from ATCC. For cell
encapsulation, cells were trypsinized and encapsulated in collagen gels (Control, HABP, Scr HABP) at a 10,000
cell/250 µl gel precursor density. After allowing the gels to solidify for 2 hours culture medium was added.
Fluorescent Labeling Cells were labeled using ActinGreen 488 and NucBlue following procedures provided by
the supplier. 2-photon and Second Harmonic generation imaging of HABP-Collagen hydrogels: Olympus 2-
photon excitation microscope used to capture the fluorescence of the fibroblasts based on the immunostaining.
Whereas, the second harmonic generation microscope was able to capture the second harmonic effect of the collagen
fibers in the samples.

Results, Conclusions, and Discussions: Results and

Discussion: To analyze the results, the z-stack images were stacked using Max Intensity using Fiji
ImageJ. Afterward the images were analyzed in the CT-Fire software to characterize the individual collagen fibers.
The initial results gathered thus far indicate a stronger remodeling ability of collagen in the samples with the
scrambled HABP. In Figure 1, the individual collagen fibers have been highlighted to showcase the remodeling
around the neonatal fibroblast in the sample. In the figure, it can be observed how the collagen fibers organize
around the cell to align their orientation to one another. The experiments are ongoing and the ahDF samples have
yet to be imaged and processed.
Conclusions and Acknowledgements: We observed HABP facilitated microenvironments remodeled differently
than control environments by both aHDF and nHDFs. Investigating this relationship would allow for the potential
ability to in the future study methods of utilizing HABP to find a way to promote remodeling of collagen and HA
to a wound site as well as to speed up the healing process of these wound sites.

Acknowledgements (Optional):

References:
1. Wu, H., Shao, C., Shi, J., Hu, Z., Zhou, Y., Chen, Z., Tang, R., Xie, Z., & Jin, W. (2023). Hyaluronic acid-mediated collagen
intrafibrillar mineralization and enhancement of dentin remineralization. Carbohydrate Polymers, 319, 121174.
https://doi.org/10.1016/J.CARBPOL.2023.121174