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Delayed fracture healing is a burden for patients as well as the health care system. Moreover, 5–10% of all fractures show a delay in healing or even result in non-unions. One of the major risk factors for developing a delay is smoking cigarettes. Fracture healing begins with the formation of a fracture hematoma in the fracture gap, which lays the foundation for appropriate healing. After an initial inflammatory phase, the bone can be rebuilt by invading osteoprogenitors as well as adjacent cells. During fracture repair osteogenesis and angiogenesis are tightly coupled, and a delay in healing is not only associated with impaired osteogenesis but also angiogenesis. This work aimed to develop an in vitro model enabling the analysis of early fracture repair of smokers and non-smokers, with a special focus on the interplay between the fracture hematoma and the vascular system.
As a first step, the in vitro fracture hematomas were exposed to hypoxia with an enzymatic system as well as the hypoxia incubator chamber; the latter proved to be more compatible with the chosen disease model. As suspected, the in vitro fracture hematomas showed an early inflammatory reaction, followed by an increase in their osteogenic and angiogenic potential. The in vitro fracture hematomas were analyzed regarding early fracture repair in smokers. Compared with non-smokers, the smoker’s in vitro fracture hematomas showed a more robust inflammatory status as well as a decreased osteogenic differentiation potential. Further, they showed a general downregulation in gene expression of angiogenic factors, and their supernatant reduced human umbilical cord vein endothelial cell (HUVEC) tube formation and proliferation. Interestingly, the gene expression and secretion of the main angiogenic growth factor vascular endothelial growth factor (VEGF) was not altered in the smoker’s early fracture repair in vitro, but there was dysregulation of the angiopoietin (ANGPT)-type I tyrosine kinase receptors 2 (TIE2) axis. This dysregulation could impair early angiogenic events. In conclusion, the smoker’s in vitro fracture hematomas showed initial signs of developing a delay in healing.
In the next step, a co-culture model comprising in vitro fracture hematomas as well as an angiogenic component was established successfully. The results from the smoker’s and non-smoker’s in vitro fracture hematoma monocultures were confirmed in the co-cultures, but the effects were not that prominent. Additionally, HUVECs in the smoker co-culture showed increased stress levels accompanied by a decrease in inflammatory cytokine and ANGPT2 gene expression. As a possible treatment for impaired healing in smokers, herbal extracts of ginseng roots and maqui berries were tested. Both extracts had only minimal effects on early fracture repair of smokers in vitro. However, the ginseng extract showed a trend to restore the impaired ANGPT-TIE2 signaling.
A 3D in vitro model of early fracture repair in smokers and non-smokers including the fracture hematoma as well as the vasculature could be successfully established. Being aware of its limitations, the 3D culture proved suitable for in vitro screening purposes, as the effects of ginseng and maqui berry extracts could be evaluated. Nevertheless, there is room for further improvements to increase the validity of the system or even to allow transfer to other disease models such as diabetes mellitus.
Taken together, in vitro systems mimicking early fracture repair are not yet able to fully replace animal models, as they still lack the full in vivo complexity, making it difficult to analyze complex tissues or organs as well as cell–organ interactions. Nevertheless, they are still very important tools with great development potential and should be used more often as pre-screening devices to reduce unnecessary animal experiments in the future. |
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