Abstract
The aim of the study was to establish an in vitro fracture hematoma (FH) model that mimics the in vivo situation of the human fracture gap in order to assess drug efficacy and effectiveness for the treatment of fracture healing disorders. Human peripheral blood and mesenchymal stromal cells (MSCs) were coagulated to produce in vitro FH models, which were incubated in osteogenic medium under normoxia/hypoxia and analyzed for cell composition, gene expression and cytokine/chemokine secretion. To evaluate the model, we studied the impact of dexamethasone (impairing fracture healing) and deferoxamine (promoting fracture healing). Under hypoxic conditions, MSCs represented the predominant cell population, while the frequencies of leukocyte populations decreased. Marker gene expression of osteogenesis, angiogenesis, inflammation, migration and hypoxic adaptation increased significantly over time and compared to normoxia, while cytokine/chemokine secretion remained unchanged. Dexamethasone favored the frequency of immune cells compared to MSCs, suppressed osteogenic and pro-angiogenic gene expression, and enhanced the secretion of inflammatory cytokines. Conversely, deferoxamine favored the frequency of MSCs over that of immune cells and enhanced the expression of the osteogenic marker RUNX2 and markers of hypoxic adaptation. In summary, we demonstrate that hypoxia is an important factor for modeling the initial phase of fracture healing in vitro and that both fracture-healing disrupting and promoting substances can influence the in vitro model comparable to the in vivo situation. Therefore, we conclude that our model is able to mimic in part the human FH and could reduce the number of animal experiments in early preclinical studies.
Highlights
The healing of approximately 10% of bone fractures is impaired, and this is accompanied by pain and suffering of the affected patients and tremendous socio-economic costs (Gomez-Barrena et al, 2015; Gaston and Simpson, 2007)
3.1 Temporal cellular composition of the in vitro fracture hematoma (FH) models is dominated by long-term survival of mesenchymal stromal cells (MSCs) In order to simulate the first phase of fracture healing in vitro, we generated in vitro FH models consisting of human MSCs and peripheral blood cells
In consideration of mimicking the fracture gap microenvironment characterized by hypoxia and to evaluate its impact on temporal distribution and cell composition, we incubated the in vitro FH models for 6, 12, 24 and 48 h under hypoxic conditions (1% O2, which is the average oxygen level measured in the incubator at 5% CO2 and high humidity flushed with nitrogen) as compared to normoxia (18% O2, which is the average oxygen level measured in the incubator at 5% CO2 and high humidity flushed with room air) (Fig. 1)
Summary
The healing of approximately 10% of bone fractures is impaired, and this is accompanied by pain and suffering of the affected patients and tremendous socio-economic costs (Gomez-Barrena et al, 2015; Gaston and Simpson, 2007). During the process of fracturing, the bone marrow canal is opened and adjacent blood vessels rupture. The cells emerging from the bone marrow (e.g., mesenchymal stromal cells – MSCs, hematopoietic progenitor cells and premature lymphocytes) mix with peripheral blood in the fracture gap, coagulate and form the fracture hematoma (FH). MSCs are considered to play a pivotal role in an adequate healing process, since they are able to differentiate into both chondrocytes and osteoblasts/osteocytes, thereby facilitating bone healing (Knight and Hankenson, 2013). Matrix metalloproteinase 2 (MMP2) and MMP9, which are fundamental for appropriate bone healing (Henle et al, 2005), are induced via the HIF-1 pathway (Luo et al, 2006; O’Toole et al, 2008)
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