Cigarette smoke increases pro-inflammatory markers and inhibits osteogenic differentiation in experimental exposure model

Abstract

Tobacco smokers have slowed bone growth and regeneration and more frequent implant failures than non-smokers, but the effect of cigarette smoking on the host response to bone-dwelling biomaterials is poorly understood. Macrophages and mesenchymal stem cells (MSCs) are essential in the healing response after implant placement. This study examined the effects of an experimental model of cigarette smoke exposure using cigarette smoke extract (CSE) on bone architecture in vivo and differentiation and inflammatory cytokine production on clinically relevant microstructured surfaces in vitro. CSE was prepared by bubbling mainstream smoke from one research cigarette (3R4F) in 1 mL phosphate-buffered saline. For in vivo studies, bone morphometry was examined in femurs isolated from mice injected with diluted CSE for 25 days. For in vitro studies, osteogenic markers and interleukins were measured in human MSCs and murine macrophages cultured on rough or rough-hydrophilic titanium (Ti) surfaces in culture media ± CSE for seven days. In vivo, CSE exposure decreased in bone area, volume, and interconnectivity in a dose-dependent manner. In vitro, macrophages exposed to CSE increased production of pro-inflammatory cytokines, abolishing the increase in anti-inflammatory cytokines typically seen on rough-hydrophilic surfaces. MSCs exposed to CSE had lower mRNA expression of osteoblast differentiation markers, increased levels of pro-inflammatory mRNA, and reduced production of osteogenic proteins. Our results demonstrate that CSE decreases osteogenic differentiation and anti-inflammatory interleukin production while increasing pro-inflammatory interleukin production in macrophages and MSCs, suggesting that compounds in CSE strongly affect stem cell differentiation and may compromise bone formation following biomaterial placement. Statement of SignificanceThe study of implantable materials’ interaction with biological systems occurs nearly exclusively in healthy cell and animal models. However, 15% of the US population smokes cigarettes, which is known to modulate immune response and tissue regeneration. To explore this interaction, we created a method of capturing smoke compounds as CSE for in vivo and in vitro use. We found chronic injection into mice produced an osteoporotic, pro-inflammatory phenotype similar to direct smoke models. Furthermore, CSE attenuated osteogenic differentiation and promoted a pro-inflammatory profile in MSCs and macrophages, respectively, when cultured on titanium surfaces. These results demonstrate that this CSE model may be useful for predicting how chronic tobacco exposure may adversely affect the outcome of biomedical implants in pre-clinical models.