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 1mL 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 Significance

The 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.

中文翻译：

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在实验暴露模型中，香烟烟雾增加促炎标记并抑制成骨分化

吸烟者比不吸烟者减慢了骨骼的生长和再生，并且植入失败的频率更高，但是，吸烟对宿主对留有骨的生物材料反应的影响知之甚少。巨噬细胞和间充质干细胞（MSCs）在植入植入物后的愈合反应中至关重要。本研究香烟烟雾暴露的实验模型的使用对骨架构香烟烟雾提取物（CSE）的影响在体内和分化，炎症性细胞因子的产生在临床上相关的微结构化表面在体外。通过将一根研究香烟（3R4F）中的主流烟雾鼓泡在1mL磷酸盐缓冲盐水中来制备CSE。对于体内研究显示，从注射稀释的CSE 25天的小鼠的股骨中检查骨形态。为了进行体外研究，在人MSC中测量了成骨标记物和白介素，并在培养基±CSE中在粗糙或粗糙亲水的钛（Ti）表面上培养了7天的鼠巨噬细胞。在体内，CSE暴露的骨面积，体积和互连性呈剂量依赖性降低。体外，暴露于CSE的巨噬细胞增加了促炎细胞因子的产生，从而消除了通常在粗糙亲水表面上看到的抗炎细胞因子的增加。暴露于CSE的MSC的成骨细胞分化标志物的mRNA表达降低，促炎性mRNA的水平升高以及成骨蛋白的生成减少。我们的结果表明，CSE可减少成骨细胞分化和抗炎性白细胞介素的产生，同时增加巨噬细胞和MSC中炎性白细胞介素的产生，这表明CSE中的化合物强烈影响干细胞的分化，并可能损害生物材料放置后的骨形成。

重要声明

对可植入材料与生物系统相互作用的研究几乎只发生在健康的细胞和动物模型中。但是，美国人口中有15％吸烟，这已知会调节免疫反应和组织再生。为了探索这种相互作用，我们创建了一种在体内和体外捕获烟雾化合物作为CSE的方法。采用。我们发现向老鼠进行长期注射会产生类似于直接烟雾模型的骨质疏松，促炎表型。此外，当在钛表面上培养时，CSE分别减弱了MSC和巨噬细胞的成骨分化并促进了促炎作用。这些结果表明，该CSE模型可用于预测长期烟草暴露如何可能对临床前模型中的生物医学植入物产生不利影响。