Proteomic analysis for the study of vitality on human rib fractures: a pilot study

Introduction. Blunt force chest injuries with rib fractures are frequent in forensic caseworks. However, the possibility to set chronologically the traumatic mechanism on human bone is currently very limited. This study aims to a) identify the proteomic expression on bone marrow of human ribs that were damaged with fracture (vitality); b) evaluate the proteomic changes over different known survival times; c) assess proteomic differences among resuscitation fractures and other types of rib traumas (e.g., vehicle and train crashes, falling from heights). Material and methods. 15 cases with rib fractures and different survival times, and 7 controls with different causes of deaths but no traumatic injuries. Specifically, the survival times of cases included (4) < 1 hour, (3) = 1 hour, (3) = 2 hours, (3) = 5-10 hours, and (2) = 24-72 hours; ratio men-to-women was 1,14:1 and the mean age 54,06. The selection of the case samples was based on the highest hemorrhagic infiltration which could be detected macroscopically; for controls, sampling was conventionally taken on the 4th right rib. Then, soft tissues around each sample were mechanically removed and frozen at -80°C. Each sample was further processed and dissected with a scalpel to collect the bone marrow (in the fracture foci for cases). Proteomic analyses were performed on bone marrows and results were expressed over logarithmic2 fold changes. Statistical tests included ANOVA applying the false discovery rate (q<0.05). Results and discussion. The trauma group showed high expressions of acute-phase and inflammatory proteins (q<0.001) such as alpha-crystallin B chain, plasminogen, complement component C3, complement factor B and alpha-1 acid glycoprotein 1. Moreover, there was an increasing expression in the cases of both myosins (heavy and light chains) and different extracellular matrix proteins including the bone-specific collagen alpha-1 (XII) chain. Finally, carbonic anhydrase 2 (CA2) was detected as highly expressed and significative (q<0.001) in the trauma group. In the trauma group, inflammatory proteins showed a common increasing trend over the time whereas both myosins and alpha-crystallin B chain decreased progressively. Notably, the expression of CA2 increased linearly with high statistical significance (q<0.05) for all the survival subgroups of trauma. No statistical differences were noted among resuscitation fractures and the other types of rib traumas. These results provide that the bone marrow reacts to the trauma similarly to other tissues by recruiting cells in the fracture foci (evidence of myosins) to start the early inflammatory phase (evidence of inflammatory proteins). However, bone marrow also shows a particular response to trauma by increasing the expression of CA2 which is a very specific enzyme and fundamental for the process of bone remodeling. Therefore, this study provides specific forensic markers of vitality which could help to set chronologically the traumatic mechanism on human bone.