Measuring Effects of Treatment with a Novel Metalloprotease Inhibitor, Extracellular Matrix Protection Factor‐2, on Total Protein Production by Human Gingival Fibroblast Cultures

Abstract

Collagen, produced by fibroblasts, serves an integral role in the structural integrity of the extracellular matrix of the human gingiva, but during periodontal disease, matrix metalloproteases (MMPs) are upregulated and contribute significantly to the degradation of collagen. In this study, we test the effects of the novel MMP inhibitor, Extracellular Matrix Protection Factor‐2 (ECPF‐2) on total protein production by human gingival fibroblasts (HGVFs) reared in serum‐free media. Cells are enzymatically released from gingival tissue isolated during oral surgery and cultures produced from patient samples: normal, non‐inflamed (NTN); inflamed non‐smoker (IFN); inflamed previous smoker (IFP); inflamed current smoker (IFC). Subconfluent cultures were switched to 0.1% fetal bovine serum containing DMEM media overnight and then treated for 24 hours with complete serum‐free DMEM (Control); 5ug ECPF‐2; or 50ug ECPF‐2. Conditioned media was collected and concentrated and the cell layer was extracted with 0.5% CHAPS buffer. Total protein of the samples was quantified using the Pierce Modified Lowry Protein Assay. Regardless of treatment or patient pathology, all cultures tested produced approximately 1.3–1.5 ug/ml/culture of total protein. There was no statistically significant differences between patient samples or within treatment conditions per patient. These data suggest that our culture system allows for viable metabolism in serum‐free medium and treatment with the novel MMP inhibitor, ECPF‐2, does not affect the metabolic activity of HGVF cultures. Therefore, this culture system can be used to measure the therapeutic effects of ECPF‐2 on collagen metabolism associated with periodontal disease.Support or Funding InformationSupport from the PCOM Division of Research