Thu0336 developing a Novel Rapid Ex Vivo Model of Skin Fibrosis for Systemic Sclerosis Research

Abstract

BackgroundSystemic Sclerosis (SSc) is an autoimmune disease that causes tissue fibrosis in multiple systems. Immune dysfunction and dysregulation are known to play a role in the disease, but the pathophysiology of SSc is not clearly understood.(1) Adipose-derived stromal cells (ADSCs) reside in the dermal white adipose tissue (DWAT) and have reparative and regenerative functions.(2) We have previously shown that ADSCs are lost in SSc and that injecting these along with a survival signal can partially reverse fibrosis.(3) To better understand how ADSCs reverse fibrosis, we wanted to develop an ex vivo skin fibrosis model. As the commonly used in vivo bleomycin-injected fibrosis model takes three to four weeks to prepare, a more rapid ex vivo model would further facilitate mechanistic dissection.ObjectivesThis pilot study aims to establish a novel ex vivo experimental model that can demonstrate fibrotic change in mouse skin and represent ADSC loss within days rather than within weeks needed for current models.MethodsSix B6 mice were sacrificed at between 12-16 weeks of age and skin tissues were obtained from the back by 8mm punch biopsies. Skin samples were immersed in Dulbecco’s Modified Eagle’s Medium containing either bleomycin (5 or 10mU/ml) or PBS as control for one or three days. After harvesting, H&E staining was performed on skin samples to assess phenotypic fibrotic changes. Flow cytometry (FACS Canto) was used to measure the viability, total cell number and ADSC change. A total collagen assay quantified collagen production in samples.ResultsOn H&E staining, compared to controls, samples cultured with 5mU/ml bleomycin for a day showed increased density of dermis and deposition of amorphous pink material, likely representing increased collagen. With 10mU/ml bleomycin, these changes were greater and especially affected the upper dermis. On day 3, samples in 5mU/ml and 10mU/ml bleomycin appeared to have denser fibrosis, broader dermis band, and narrower DWAT layer. The histopathological change was most prominent on day 3 sample in 10mU/ml bleomycin, with the dermis layer 40% thicker and the DWAT layer 37% narrower than the control. However, all the samples on day 3 showed stress, such as epidermis detachment and apoptosis in the epidermis. On the Total Collagen Assay, collagen deposition on day 1 in control sample was 7.7ug/mm, compared to 33.9ug/mm from sample in 10mU/ml bleomycin. On day 3, collagen deposition in 10mU/ml bleomycin was 37ug/mm which was 2.5 to 5 times higher than control. On FACS, the decrease in ADSC level correlated to the increase in concentration of bleomycin and the number of days treated, with the greatest drop in samples in 10mU/ml bleomycin on day 3.ConclusionOur results thus far suggest that we are creating a novel ex vivo model of skin fibrosis that can help better understand how ADSCs can be of therapeutic benefit in SSc. This rapid ex vivo model will complement the existing in vivo model. Our next step will be to investigate the changes of profibrotic cytokines before and after ap...