Poster O19 - Collagen scaffold to investigate contraction, remodeling, and immunomodulatory events in hypermobile Ehlers Danlos Syndrome

Introduction: Ehlers Danlos Syndrome (EDS) is a group of hereditary connective tissue disorders that clinically manifest as joint hypermobility, skin hyperelasticity, atrophic scarring, and blood vessel fragility. There is currently no cure for EDS, only symptom management, and it is underdiagnosed. The most common subtype of EDS, hypermobile EDS (hEDS), is estimated to affect 1 in 500 to 1 in 5000 people and is the only clinical EDS subtype for which the genetic basis and primary affected protein is unknown. The lack of an identified genetic defect makes a hEDS physiological definition more important. To address this need, we have developed a non-mineralized collagen scaffold material with dermal characteristics that maintain fibroblasts. Fibroblasts contribute to the maintenance of connective tissues through secretions of collagen proteins, creating structural support and connecting tissues or organs in the body. Fibroblasts also play a critical role in wound healing and have previously been identified as cells of note in EDS. Here we describe the development of a collagen scaffold that can be used to characterize biophysical response differences in dermal fibroblast samples from patients with and without hEDS. Further work will study not only the mechanical characteristics of the disease, but also the psycho/social factors that may contribute to its severity such as the impact of sex-based differences and stress on cellular function. Studying the impact of these factors on dermal fibroblast behavior and the mechanics of matrix remodeling will help close the knowledge gap, ultimately allowing for improved treatments and more informed diagnoses.

Materials and

Methods: Normal Human Lung Fibroblasts (NHLFs) were used for optimization experiments to identify the minimum number of cells required to reliably measure cell-mediated contraction, before turning to patient derived samples. Skin punch biopsies were collected from non-sun exposed skin from age-matched female EDS positive and EDS negative patients and dermal fibroblasts were cultured. Participants were evaluated clinically using the hEDS criteria and were collected under protocol #21CRU3373 supervised by the Carle Hospital Institutional Review Board. Non-mineralized collagen scaffolds were fabricated through the homogenization and subsequent lyophilization of a type 1 bovine collagen and chondroitin-6-sulfate precursor solution to form porous scaffolds. Biopsy punches were used to create scaffolds of either 6mm or 8mm diameter. Two crosslinking methods were employed to modify the stiffness of the scaffolds without altering the physical structure: a chemical, carbodiimide-based (EDAC) method and a dehydrothermal-based (DHT) method. Chemical crosslinking took place at either a 5:2:1 or 1:1:5 ratio of EDAC:NHS:COOH while DHT was run at 105oC for 24 hours. All scaffolds were hydrated with phosphate buffered saline (PBS) prior to cell seeding. NHLFs were cultured on scaffolds for 14 days and response was assessed through metabolic activity (alamarBlue), cell number (DNA isolation), and gene expression via RT-qPCR. Contraction was quantified via imageJ analysis of scaffold images.

Results, Conclusions, and Discussions: Non-crosslinked scaffolds showed greater contraction than the chemically crosslinked (5:2:1) scaffolds for both dimensions (Fig 1A). This is in line with previous work showing increased material stiffness with increased crosslinking, resulting in an increased force required to contract the matrix. A significant difference was found in the metabolic activity of NHLFs between each crosslinked and non-crosslinked condition. However, all groups demonstrated an overall increase in metabolic activity over the 14-day culture with the crosslinked groups showing the greatest increase over time (Fig 1B). The overall cell number increased over the culture period in the crosslinked scaffolds but decreased in the non-crosslinked in a size independent manner (Fig 1C). The combined metabolic activity and cell number data show the non-crosslinked scaffolds to be resulting in reduced cellular viability and proliferation over time. However, further experiments must be conducted to identify whether the matrix environment is causing this cell death or if the NHLFs are redirecting their energy towards contracting the non-crosslinked scaffolds. Ongoing work looks to see if an optimized crosslinking and cellular seeding density will promote increased cell viability and proliferation while still maintaining quantifiable scaffold contraction. This will allow for an investigation into the mechanisms of fibroblast mediated extracellular matrix contraction and remodeling.

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