Serum‐free Culture of Primary Human Gingival Fibroblasts Demonstrates Differences in Collagen Production Between Smokers and Non‐smokers: A System to Measure Effects of Novel Collagenase Inhibitors

Abstract

Periodontal disease occurs in 50% of the world's population over the age of 30. It is an infectious and inflammatory disease that if left untreated, results in bleeding gums, tooth and soft tissue pain and the inevitable loss of teeth. Current treatments include anti‐infectives that treat the infection but do not necessarily target the host's inflammatory cascade, the underlying perpetuator of the disease progression. We are developing a novel class of collagenase inhibitor that we will test in this serum‐free, primary human gingival fibroblast (HGVF) culture system of which serum‐free conditions will allow us to measure collagen metabolism effectively. To prepare the HGVF cultures, tissue was harvested from two African American (50yo and 57yo) current smoker patients (CS) and two African American (53yo and 60yo) non‐smoking patients (NS). Tissue was dissociated with mild trypsin and collagenase and the released, HGVFs were plated in monolayer T25 flasks 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, at which time, they were switched to low serum (1% FBS) overnight (18 hours), and then into serum‐free media (MEM plus antibiotic, antimycotic and antimycoplasmotic) for 24 and 48 hours. Cell conditioned media, detergent‐extracted cell layer and mRNA were prepared for each condition in at least duplicate wells. Collagen type 1 production (intact and degraded) was measured in the conditioned media and 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/35mm culture CS versus 0.04mg/35mm culture 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 (0.006 mg/35mm culture CS versus 0.052mg/35mm culture NS) including roughly 3 times more collagen type I degradation product (0.041ug/35mm culture CS versus 0.141ug/35mm culture NS). Protein production and mRNA expression for MMP‐8 and −13, inflammatory cytokines, IL1b, IL6 and TNF alpha, will be measured with immunoassay and qRT PCR, respectively. This data supports the notion that serum‐free culture system for primary HGVF can be utilized to measure the actions of novel collagenase inhibitors.Support or Funding InformationThis research was supported with funds from the Division of Research and the Department of Bio‐Medical Sciences at PCOM.