Delayed healing or non-union of bone fractures are still clinically and economically relevant problems. Bone healing includes pro- and anti-inflammatory phases, which proved crucial for successful healing. Being able to modulate these inflammatory responses could potentially promote bone fracture healing. Dendritic cells play important roles in the immune response after bone fracture, due to their immune regulating abilities via cytokine secretion and antigen presentation. Therefore, DCs are a target for immunomodulatory therapies. One way of modulating cell behaviour can be altering osmolality in cell microenvironments. This thesis investigates in vitro if environmental osmolality can modulate the inflammatory phenotype of monocyte-derived dendritic cells (moDCs) in 2D and 3D microenvironments.
To exclude adverse effects such as cell death by hypo- or hyper-osmotic conditions, a range was established where moDCs maintain high viability and metabolic activity. Hypo-osmotic medium was established by diluting iso-osmotic cell medium with deionized water. Hyper-osmotic medium was established in two ways, by supplementing iso-osmotic cell medium with either ionic sodium chloride (NaCl) or inert polyethylene glycol (PEG). moDCs maintained high viability and metabolic activity between 210-385 mOsm/kg for NaCl and PEG. The conditions for the hypo- iso- and hyper-osmotic media for all cell culture experiments were defined as 220, 281 and 381 mOsm/kg, respectively.
In 2D cell culture, hypo- or hyper-osmolality did not stimulate a significant change in surface marker expression of activation markers CD40, CD80, CD83, CD86, CD197 and HLA-DR compared to the non-activated control. This suggests that osmolality alone was not sufficient to modulate the inflammatory phenotype. Hereafter, the effect of osmolality on maturation with Lipopolysaccharide (LPS) was investigated. Exposure to hypo- or hyper-osmolality in 2D moDC culture with LPS as maturation factor, showed no significant difference in expression of CD40, CD80, CD83, CD86, CD197 and HLA-DR compared to the activated control. However, a trend was observed in hyper-osmotic medium with PEG and NaCl, where moDCs showed a decrease of CD40 and HLA-DR expression in the PEG condition and a decrease of CD197 in the NaCl condition. This suggests that hyper-osmolality could attenuate the activating capacity of LPS and depending on the osmolyte, can potentially decrease T cell activation or migration of moDCs. Functional analyses such as mixed lymphocyte reaction and migration assays may elucidate if hyper-osmolality can affect moDC T cell activation and migration. For cell culture in 3D microenvironment, the mechanical properties of fracture hematoma were modelled using alginate hydrogels to gain an understanding of the behaviour of DCs during bone healing. Low molecular weight alginate was used to mimic the stress relaxation behaviour. Stiffness was tuned trough ionic crosslinking concentration. The resulting gels had a stiffness of 8±0.3 kPa and stress-relaxation half-time of 199.7±10.8 s. In these 3D microenvironments during LPS maturation, hypo- or hyper-osmolality did not show a significant change in expression of CD40, CD80, CD83, CD86, CD197 and HLA-DR compared to the activated control. This suggests that moDCs were not sensitive to osmolality during maturation with LPS in 3D microenvironments. Comparing the outcomes of 3D to 2D cell culture, moDCs appeared less sensitive to changes in environmental osmolality during LPS maturation in 3D than on 2D substrates.

Overall, for both the 2D and 3D studies, a larger cohort of donors is needed to improve confidence in the results. Based on the trends observed, the outcome of the study does not indicate that osmolality could be used in 3D microenvironments to modulate the inflammatory phenotype of moDCs, as activation markers remained largely unaffected. In 2D cell culture, hyper-osmolality could modulate the inflammatory phenotype of moDCs during maturation with LPS through downregulation of activation markers. Osmolality should be considered when designing immunomodulatory treatments, as osmolality might affect DC maturation in 2D cell culture.