Assessing the effects of a novel metalloprotease inhibitor, extracellular matrix protection factor-2, on primary human gingival fibroblast's metabolic viability as determined by total protein production
Location
Philadelphia, PA
Start Date
10-5-2021 12:00 AM
End Date
13-5-2021 12:00 AM
Description
In the pathogenesis of periodontal disease, collagen, produced by gingival fibroblasts, is degraded by upregulated metalloproteases (MMPs) leading to the loss of structural integrity of the extracellular matrix. In this study, we investigated the effects of a novel MMP inhibitor, Extracellular Matrix Protection Factor-2 (ECPF-2), on human gingival fibroblast’s (HGVF) metabolic activity. ECPF-2 reversibly inhibits MMP-8 by blocking its interaction with collagen type I, but the effects of this novel therapeutic on HGVF behavior in culture is unknown. Cells were enzymatically released from samples harvested during oral surgery, expanded to passage 2. Subconfluent cultures were transferred to 0.1% fetal bovine serum containing DMEM media overnight, transferred to serum-free medium (SFM) and reared for 72 hours. Based on total protein, total RNA and collagen type I production, 24 hours in serum-free media alone was optimal. Passage 2 cultures were then treated with 5ug ECPF-2, 50ug ECPF-2, or control serum-free media for 24 hours. After 24 hours treatment with ECPF-2, conditioned media was collected and the cell layer was extracted with 0.5% CHAPS buffer. We measured the total protein production in cultures isolated from normal non-inflamed (NTN); inflamed non-smoker (IFN); inflamed previous smoker (IFP) and inflamed current smoker (IFC) patient samples using the Pierce Modified Lowry Protein Assay. Using this methodology, we found that regardless of patient pathology or treatment conditions, all cultures contained approximately 1.3-1.5ug/ml/culture of total protein. There was no statistically significant difference in measured total protein between the treated or control samples. Based on these findings, we suggest that the defined culture system allows for viable metabolism in a serum-free medium when treated with the novel MMP inhibitor, ECPF-2. This given, the culture system can be used to investigate the potential therapeutic effects of ECPF-2 on human gingival fibroblasts isolated from periodontal diseased tissue.
Embargo Period
6-2-2021
Assessing the effects of a novel metalloprotease inhibitor, extracellular matrix protection factor-2, on primary human gingival fibroblast's metabolic viability as determined by total protein production
Philadelphia, PA
In the pathogenesis of periodontal disease, collagen, produced by gingival fibroblasts, is degraded by upregulated metalloproteases (MMPs) leading to the loss of structural integrity of the extracellular matrix. In this study, we investigated the effects of a novel MMP inhibitor, Extracellular Matrix Protection Factor-2 (ECPF-2), on human gingival fibroblast’s (HGVF) metabolic activity. ECPF-2 reversibly inhibits MMP-8 by blocking its interaction with collagen type I, but the effects of this novel therapeutic on HGVF behavior in culture is unknown. Cells were enzymatically released from samples harvested during oral surgery, expanded to passage 2. Subconfluent cultures were transferred to 0.1% fetal bovine serum containing DMEM media overnight, transferred to serum-free medium (SFM) and reared for 72 hours. Based on total protein, total RNA and collagen type I production, 24 hours in serum-free media alone was optimal. Passage 2 cultures were then treated with 5ug ECPF-2, 50ug ECPF-2, or control serum-free media for 24 hours. After 24 hours treatment with ECPF-2, conditioned media was collected and the cell layer was extracted with 0.5% CHAPS buffer. We measured the total protein production in cultures isolated from normal non-inflamed (NTN); inflamed non-smoker (IFN); inflamed previous smoker (IFP) and inflamed current smoker (IFC) patient samples using the Pierce Modified Lowry Protein Assay. Using this methodology, we found that regardless of patient pathology or treatment conditions, all cultures contained approximately 1.3-1.5ug/ml/culture of total protein. There was no statistically significant difference in measured total protein between the treated or control samples. Based on these findings, we suggest that the defined culture system allows for viable metabolism in a serum-free medium when treated with the novel MMP inhibitor, ECPF-2. This given, the culture system can be used to investigate the potential therapeutic effects of ECPF-2 on human gingival fibroblasts isolated from periodontal diseased tissue.