Heated Tobacco Products Impair Cell Viability, Osteoblastic Differentiation, and Bone Fracture-Healing.

Abstract

The negative impact of cigarette smoking on bone union has been well documented. However, the impact of heated tobacco product (HTP) use on bone fracture-healing remains unclear. The present study investigated the effect of HTPs on preosteoblast viability, osteoblastic differentiation, and fracture-healing and compared the effects with those of conventional combustible cigarettes. Cigarette smoke extracts (CSEs) were generated from combustible cigarettes (cCSE) and HTPs (hCSE). CSE concentrations were standardized by assessing optical density. Preosteoblast (MC3T3-E1) cells were incubated with normal medium, cCSE, or hCSE. The cell viability was assessed via MTT assay. After osteoblastic differentiation of CSE-exposed cells, alkaline phosphatase (ALP) activity was assessed. To assess the in vivo effects of CSEs, a femoral midshaft osteotomy was performed in a rat model; thereafter, saline solution, cCSE, or hCSE was injected intraperitoneally, and bone union was assessed on the basis of micro-computed tomography (μCT) and biomechanical analysis 4 weeks later. MC3T3-E1 cell viability was reduced in a time and concentration-dependent manner when treated with either cCSE or hCSE. ALP activity after osteoblastic differentiation of cCSE-treated cells was significantly lower than that of both untreated and hCSE-treated cells (mean and standard deviation, 452.4 ± 48.8 [untreated], 326.2 ± 26.2 [cCSE-treated], and 389.9 ± 26.6 [hCSE-treated] mol/L/min; p = 0.002). Moreover, the levels of osteoblastic differentiation in untreated and hCSE-treated cells differed significantly (p < 0.05). In vivo assessment of the femoral midshaft cortical region revealed that both cCSE and hCSE administration significantly decreased bone mineral content 4 weeks after surgery compared with levels observed in untreated animals (107.0 ± 11.9 [untreated], 94.5 ± 13.0 [cCSE-treated], and 89.0 ± 10.1 mg/cm3 [hCSE-treated]; p = 0.049). Additionally, cCSE and hCSE-exposed femora had significantly lower bone volumes than unexposed femora. Biomechanical analyses showed that both cCSE and hCSE administration significantly decreased femoral maximum load and elastic modulus (p = 0.015 and 0.019). HTP use impairs cell viability, osteoblastic differentiation, and bone fracture-healing at levels comparable with those associated with combustible cigarette use. HTP use negatively affects bone fracture-healing to a degree similar to that of combustible cigarettes. Orthopaedic surgeons should recommend HTP smoking cessation to improve bone union.