gms | German Medical Science

Objectives: Delayed healing occurs in approximately 5 to 10% of all fractures and is often associated with the patients' aberrant inflammatory status e.g., caused by smoking. Early healing starts with the inflammatory phase accompanied by the migration and differentiation of bone progenitor cells, all controlled by the fracture hematoma formed within the fracture gap. What is often overlooked is the fact that adequate blood vessel reconstruction is also essential for proper fracture healing.

The purpose of this study was to investigate if and how smoking affects early fracture repair, especially the fracture hematoma and its interplay with the vascular system. With an in vitro fracture hematoma modeling smoking and non-smoking conditions, viability, inflammation, osteogenesis, and angiogenesis were evaluated.

Methods: In vitro fracture hematomas were produced by introducing human mesenchymal stem cells (SCP-1) into whole blood clots as described by Pfeiffenberger et al. 2019. For modeling smoking conditions, smokers' blood in combination with SCP-1 cells pre-stimulated with 5% cigarette smoke extract for 7 days were used. The hematomas were analyzed regarding cellular viability by Resazurin conversion, LDH release, ATP content; osteogenic/chondrogenic differentiation by gene expression and ALP activity; and angiogenesis by gene expression, HUVEC tube formation, and proliferation assay. Statistics were made using non-parametric Kruskal-Wallis tests.

Results and conclusion: The hematomas of the smokers had increased mitochondrial activity, LDH release, and diameter but similar ATP content to those of the control group. Whereas inflammation decreased over time in non-smokers, smokers showed significantly increased expression and/or secretion of pro-inflammatory markers TNF-α, CCL2, and IL-6 after 4 and 48 h. Expression of osteogenic and chondrogenic differentiation markers ALP, RUNX2, SOX9, BMP2/4, and Noggin was significantly reduced in smokers' hematomas. This was supported by a significant reduction in ALP activity in smokers' hematomas. Regarding angiogenesis, the supernatants of smokers' hematomas were less capable to promote HUVEC proliferation and tube formation. In line, expression of the pro-angiogenic factors PDGF-BB, MMP9, VEGFA, and Angpt1 was reduced in the smoking group.

Smokers' hematomas showed no differences in viability, but higher stress levels compared to the control group. However, larger clot diameters evidence a less stable fibrin network within smokers' hematomas. The hematomas of the smokers showed an increased inflammatory status, decreased osteogenic/chondrogenic potential, and suppressed angiogenic processes, indicating initial barriers to healing. Our results show that there is a large difference between non-smokers and smokers early in the healing process, which may continue as the healing progresses. Therefore, medical intervention in smokers should be considered as early as possible.