The Effects Of Nicotine On The Biological Characteristics Of Rat Patellar Tendon-Derived Stem Cells In Vitro

Objective:To explore the effects of nicotine on cell viability, early cell apoptosis and expressions of tendon-related markers of rat patellar tendon-derived stem cells (TDSCs) and to illuminate the pathogenesis of cigarette smoking on tendon injury and delayed tendon healing.Methods:The patellar tendons of 8-week SD rats were collected, nucleated cells were isolated after type Ⅰ collagenase digestion and plated at optimal cell density (50 cells/cm2) in culture dishes for colonies. The colonies were mixed as primary TDSCs. The multi-differentiation potential of TDSCs(P3) was identified by osteogenic, adipogenic and chondrogenic differentiation assays. Cells were sub-cultured and expanded to three passages in basic culture medium and were divided into two groups. In experimental group, basic culture medium was supplemented with different nicotine concentrations (10-7M, 10-6M,10-5M,10-4M and 10-2M) and basic culture medium was unchanged as control group. Cell viability was detected by MTT assay at 6h,24h,48h and 72h when TDSCs were treated in two different groups. Early cell apoptosis was measured with Annexin V-FITC/PI assay and expressions of tendon-related markers were detected by qRT-PCR after 72h culture.Results:Primary rat patellar TDSCs formed colonies; slender fibroblast-like and polygonal cells were seen after initial washing. Homogeneous fibroblast-like and star-shaped cells were observed at P3. The positive results were seen for alizarin red staining at 7 days after osteogenesis, for Oil red O staining at 10 days after adipogenesis and for H&E staining at 14 days after chondrogenesis. In comparison to the control group, cell viability of the experimental group decreased in a time- and dose- dependent manner. Significant decreasing was observed in 10"2M nicotine concentrations.(p<0.05) The percentage of early apoptotic TDSCs(Annexin V+/PI-) of the experimental group increased significantly in a dose-dependent manner. (p< 0.05) Expressions of Collal and Scx of the experimental group decreased and significance were seen in 10-5M,10-4M and 10’2M nicotine concentrations.(p<0.05)Conclusion:TDSCs could be isolated from rat patellar tendons and possess proliferative and multi-differentiation potential. Nicotine could inhibit cell viability and expressions of tendon-related markers, and lead to early cell apoptosis of TDSCs in vitro. This might be the potential pathogenesis of tendon injury and delayed tendon healing in smokers.