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Bone fracture healing is a well-orchestrated but complex process that involves numerous regulations at different scales. This complexity becomes particularly evident during the inflammatory stage, as immune cells invade the healing region and trigger a cascade of signals to promote a favorable regenerative environment. Thus, the emergence of criticalities during this stage might hinder the rest of the process. Therefore, the investigation of the many interactions that regulate the inflammation has a primary importance on the exploration of the overall healing progression. In this context, an in silico model named COMMBINI (COmputational Model of Macrophage dynamics in the Bone INjury Immunoresponse) has been developed to investigate the mechano-biological interactions during the early inflammatory stage at the tissue, cellular and molecular levels. An agent-based model is employed to simulate the behavior of immune cells, inflammatory cytokines and fracture debris as well as their reciprocal multiscale biological interactions during the development of the early inflammation (up to 5 days post-injury). The strength of the computational approach is the capacity of the in silico model to simulate the overall healing process by taking into account the numerous hidden events that contribute to its success. To calibrate the model, we present an in silico immunofluorescence method that enables a direct comparison at the cellular level between the model output and experimental immunofluorescent images. The combination of sensitivity analysis and a Genetic Algorithm allows dynamic cooperation between these techniques, enabling faster identification of the most accurate parameter values, reducing the disparity between computer simulation and histological data. The sensitivity analysis showed a higher sensibility of the computer model to the macrophage recruitment ratio during the early inflammation and to proliferation in the late stage. Furthermore, the Genetic Algorithm

Adequate pain management is essential for ethical and scientific reasons in animal experiments and should completely cover the period of expected pain without the need for frequent re-application. However, current depot formulations of Buprenorphine are only available in the USA and have limited duration of action. Recently, a new microparticulate Buprenorphine formulation (BUP-Depot) for sustained release has been developed as a potential future alternative to standard formulations available in Europe. Pharmacokinetics indicate a possible effectiveness for about 72 h. Here, we investigated whether the administration of the BUP-Depot ensures continuous and sufficient analgesia in two mouse fracture models (femoral osteotomy) and could, therefore, serve as a potent alternative to the application of Tramadol via the drinking water. Both protocols were examined for analgesic effectiveness, side effects on experimental readout, and effects on fracture healing outcomes in male and female C57BL/6N mice. The BUP-Depot provided effective analgesia for 72 h, comparable to the effectiveness of Tramadol in the drinking water. Fracture healing outcome was not different between analgesic regimes. The availability of a Buprenorphine depot formulation for rodents in Europe would be a beneficial addition for extended pain relief in mice, thereby increasing animal welfare.