Immunoprofiling of fracture patients to determine predictive biomarkers of healing

Background

Delayed healing and potential progression to non-union continues to remain a clinical issue in the context of fracture repair. Although it is currently possible to identify patient groups at risk of healing complications, such as diabetics and smokers, currently it is not possible to identify specific patients at elevated risk of delayed healing. Recent studies have suggested that alterations in the specific populations of immune cells may contributed to inappropriately maintained inflammation that detrimentally affects fracture healing capacity in such patients. These immune cell changes typically appear to boost pro-inflammatory cytokine production, either through increased proportions of effector cells (such as CD8 T cells), or via a failure of regulatory immune cells (including regulatory T- and B- lymphocyte subsets) that seek to diminish effector cell function and cytokine production. However, the majority of these reported studies are hampered by limited sample numbers as well as a failure to thoroughly interrogate the immune cell profiles of these delayed healing patients. In addition, the contribution of intercellular signalling, mediated via microRNA (miRNA) resident in extracellular vesicles, to the diminished healing capacity is currently unknown. This raises the exciting possibility that blood-resident factors could provide early clues regarding diminished fracture healing and could thus, if identified, be used as a therapeutic biomarker to aid early intervention in such patients. 

Goal

To conduct detailed phenotyping of immune cells resident in delayed healing patients compared to normal healing patients, in an attempt to determine the defect(s) in immune cell function contributing to delayed healing. In addition, we seek to determine if miRNA resident in extracellular vesicles present in the blood can identify patients at risk of diminished healing and thus potentially be used as a predictive biomarker for healing outcome.

Results

To date we have focused on obtaining a cohort of suitable patients with which to address our overall aims of the study. Mononuclear cells isolated from the peripheral blood (PBMCs) of patients suffering from delayed healing, as well as PBMCs from patients displaying normal healing capacity, are isolated and are cryopreserved until satisfactory numbers (>10 delayed healing patients) have been recruited. Serum is also collected from these patients, which we will use to analyze the levels of pro- and anti-inflammatory cytokines/chemokines, and from which to isolate the extracellular vesicles containing the miRNA cargoes of interest. Using the obtained data from the immune cell profiling and serum analysis we will compare if delayed healing patients display distinct profiles that differ from an age- and sex-matched cohort of normal healing fracture patients. Should such a potential biomarker be proposed from this series of investigations we will then aim to test the specificity and sensitivity of such a predictive biomarker in future clinical studies.