Cigarette smoke inhalation impairs angiogenesis in early bone healing processes and delays fracture union

Abstract

Aims: Cigarette smoking has a negative impact on the skeletal system, causes a decrease in bone mass in both young and old patients, and is considered a risk factor for the development of osteoporosis. In addition, it disturbs the bone healing process and prolongs the healing time after fractures. The mechanisms by which cigarette smoking impairs fracture healing are not fully understood. There are few studies reporting the effects of cigarette smoking on new blood vessel formation during the early stage of fracture healing. We tested the hypothesis that cigarette smoke inhalation may suppress angiogenesis and delay fracture healing.

Methods: We established a custom-made chamber with airflow for rats to inhale cigarette smoke continuously, and tested our hypothesis using a femoral osteotomy model, radiograph and microCT imaging, and various biomechanical and biological tests.

Results: In the smoking group, Western blot analysis and immunohistochemical staining revealed less expression of vascular endothelial growth factor (VEGF) and von Willebrand factor (vWF). The smoking group also had a lower microvessel density than the control group. Image and biochemical analysis also demonstrated delayed bone healing.

Conclusion: Cigarette smoke inhalation was associated with decreased expression of angiogenic markers in the early bone healing phase and with impaired bone healing.Cite this article: Bone Joint Res. 2020;9(3):99-107.

Keywords: Angiogenesis; Bone healing; Cigarette; Fracture union; Smoking.

Fig. 1

Smoking chamber used to expose animals to cigarette smoke. Animals were housed on the upper level. Cigarettes were lit on the lower level. The outflow airway was connected to a vacuum suction system to maintain airflow.

Fig. 2

Cigarette smoke inhalation compromised fracture healing. a) Bone healing analysis with microCT and histological examination eight weeks after fracture revealed the union rate (n = 10). b) MicroCT imaging demonstrated the ratio of bone:tissue volume.( p = 0.006, Mann-Whitney U test) c) The ultimate load of biomechanical three-point bending test showed strength of healing bone. The contralateral femur served as an internal control (100%) (n = 6). (p = 0.013, Mann-Whitney U test) d) One representative sample is shown in each group with radiograph, microCT images, and histological sections with haematoxylin and eosin (H&E) staining. *Statistically significant.

Fig. 3

Cigarette smoke inhalation reduced the expression of vascular endothelial growth factor (VEGF) and von Willebrand factor (vWF) protein in the fracture callus. a) Western blot analysis was used to detect the expression of VEGF and vWF protein in the fracture callus. β-actin was used as an internal control (n = 4). One representative data set obtained from repeated experiments is shown. b) Quantified data of Western blot analysis for the expression of VEGF in the smoking group, two and four weeks after the fracture (n = 4). c) Quantified data of Western blot analysis for expression of vWF in the smoking group, two and four weeks after the fracture (n = 4). Data are shown as the mean (SD) of six experiments. *p < 0.001 (Mann-Whitney U test).

Fig. 4

Immunohistochemical staining of von Willebrand factor (vWF) showed lower expression in the fracture callus both at two and four weeks after the fracture. a) After two weeks of healing, the callus of the control group showed abundant vWF expression around the vessel-like structures, indicated by the white arrows. In the smoking group, there was little expression of vWF protein or new vessel formation. b) The vWF immunohistochemical staining was quantified with image analysis software (Image-Pro Plus 4.5.1 software; Media Cybernetics, Rockville, Maryland, USA ) (n = 4). Data are shown as mean (SD). c) After four weeks of healing, vWF expression was mainly observed around the cartilage area of the soft callus in the control group. However, the smoking group showed little vWF expression or cartilage formation. d) Quantified results of vWF in the smoking group four weeks after fracture (n = 4). Data are shown as the mean (SD). *p < 0.001 (Mann-Whitney U test). Co, control; Sm, smoking.