Resveratrol protects primary cilia integrity of human mesenchymal stem cells from cigarette smoke to improve osteogenic differentiation in vitro

Abstract

Several studies have explored the negative effects of cigarette smoke on bone healing; however, the complex pathogenesis still remains unclear. One crucial and primary factor determining effective fracture repair is the recruitment and differentiation of mesenchymal stem cells (MSCs) into bone-forming cells. Recently, primary cilia, microtubule-based sensory organelles, have been shown to be critical in lineage commitment and differentiation of MSCs. Our present study indicates that exposure to cigarette smoke extract (CSE 0.1-10%) impaired osteogenic differentiation of human mesenchymal stem cell line (SCP-1) and interestingly, also affected primary cilia distribution and integrity in these cells during the differentiation. Furthermore, significant amounts of free radicals generated by CSE could be causative of primary cilia loss since treatment with 0.01% of hydrogen peroxide, a prime free radical in CSE, destroyed primary cilia in these cells. The debilitated differentiation of CSE-exposed SCP-1 cells also correlated with the significantly reduced expression of transcription factor and target genes of primary cilia-specific hedgehog signalling, a key player in osteogenic differentiation. As a treatment strategy, co-incubation of the CSE-exposed SCP-1 cells with the antioxidant resveratrol (1 µM) had a protective effect as it significantly reduced free radical production, protected the primary cilia and enhanced osteogenic differentiation. The current study shows for the first time that cigarette smoke affects primary cilia in human MSCs during osteogenic differentiation and treatment with resveratrol could reverse the effects and enhance differentiation, thus opening up potential therapeutic alternatives to treat fracture healing in smokers, in particularly, when delayed fracture healing is assumed.

Usefulness of Mesenchymal Cell Lines for Bone and Cartilage Regeneration Research

“…Positive for VKS (increased compared with MSCs) [14], ARS [78], ALP activity [78], and upregulation of osteogenesis-related genes [76] Positive for TBS in pellet culture [14]; ColII and GAG production in 3D printed scaffolds [24] Positive for OROS [14]…”

Primary Human Chondrocytes Affected by Cigarette Smoke—Therapeutic Challenges

“…Cigarette smoke is a toxic and carcinogenic mixture of over 5000 chemicals, including nicotine, tar, heavy metals, and hydrogen cyanide [1]. Several in vitro, in vivo, and clinical studies have shown a positive association between cigarette consumption and musculoskeletal disorders [3,5]. Sporadic studies have evaluated the relationship between cigarette smoke and symptomatic OA, however, these studies showed conflicting results [8,9,36].…”

“…Then the CSE was further diluted (0.1%, 0.5%, 1%, 5%, and 10%) with chondrocyte culture medium. The CSE concentrations corresponded to exposures associated with smoking from 0.01 pack (0.1%) to 1 pack (10%) of cigarettes/day [3].…”

“…Subsequently, they were incubated with 10 µM 2 , 7 -dichlorfluorescein-diacetate (DCFH-DA, Sigma-Aldrich, Darmstadt, Germany) in serum-free culture medium for 30 min at 37 • C. After washing the cells twice with PBS, cells were stimulated with CSE. The fluorescence intensity, representing reactive oxygen species (ROS) levels, was quantified using a plate reader (ex/em = 485/520 nm) [3].…”

“…It is known that over 5000 harmful and toxic chemical compounds, as well as free radicals like reactive oxygen species (ROS) are contained in cigarette smoke [1,2]. Several in vitro and in vivo studies have proven the deleterious effects of cigarette smoke on the musculoskeletal system [3][4][5]. Continuous inhalation of cigarette smoke leads to surgical complications and prolonged hospital stays [5].…”