Serum-free Culture of Primary Human Gingival Fibroblasts Demonstrate Differences in Collagen Production Between Smokers and Non-smokers: A System to Measure Effects of the Novel, Collagenase Inhibitor, Extracellular Matrix Protection Factor -2

Periodontal disease occurs in 50% of the world’s population over the age of 30 and if left untreated results in loss of teeth. We are developing a novel therapeutic, Extracellular Matrix Protection Factor-2 (ECPF-2), that targets the collagenase actions of MMP-8, one of the main causes of the inflammatory cascade in gingival fibroblasts. Tissue was harvested from two, 50yo African American current smoker patients (CS) and two African American (53&60 yo) non-smoking patients (NS), dissociated with mild trypsin and collagenase and released, primary gingival fibroblasts (HGVF) were plated in monolayer and fed with complete media (10% FBS, antimycotic, antibiotic, antimycoplasmotic and MEM media). Passage 2 and 3 HGVFs were plated into 6 well (35mm) tissue culture plates and fed with complete media until subconfluent, switched to low serum (1% FBS) for 18 hours and then into serum-free media (MEM plus antibiotic, antimycotic and antimycoplasmotic) for 24 and 48 hours. Collagen type 1 production (intact and degraded) was measured in the conditioned media and detergent-extracted cell layer fractions . Following 24 hours in serum-free media, HGVF produced relatively equivalent amounts of intact and degraded collagen type I when compared to complete media control indicating that serum-free culturing was not detrimental to the production of collagen. However, HGVF isolated from the current smoker patients secreted approximately double the amount of intact collagen type 1 into the conditioned media than cultures isolated from the non-smoker patients (0.075mg CS; 0.04mg NS). Conversely, HGVF cultures isolated from non-smoking patients incorporated roughly 10 times the amount of intact collagen type I into the extracellular matrix cell layer (0.052mg NS; 0.006 mg CS) including roughly 3 times more collagen type I degradation product (0.041ug CS ; 0.141ug NS). Serum-free culture of primary HGVF can be utilized to measure ECPF-2 alterations in collagen production and degradation.