Abstract
Host inflammatory immune response comprises an essential element of the bone healing process, where M2 polarization allegedly contributes to a favorable healing outcome. In this context, immunoregulatory molecules that modulate host response, including macrophage polarization, are considered potential targets for improving bone healing. This study aims to evaluate the role of the immunoregulatory molecules VIP (Vasoactive intestinal peptide) and PACAP (Pituitary adenylate cyclase activating polypeptide), which was previously described to favor the development of the M2 phenotype, in the process of alveolar bone healing in C57Bl/6 (WT) mice. Experimental groups were submitted to tooth extraction and maintained under control conditions or treated with VIP or PACAP were evaluated by microtomographic (µCT), histomorphometric, immunohistochemical, and molecular analysis at 0, 3, 7, and 14 days to quantify tissue healing and host response indicators at the healing site. Gene expression analysis demonstrates the effectiveness of VIP or PACAP in modulating host response, evidenced by the early dominance of an M2-type response, which was paralleled by a significant increase in M2 (CD206+) in treated groups. However, despite the marked effect of M1/M2 balance in the healing sites, the histomorphometric analysis does not reveal an equivalent/corresponding modulation of the healing process. µCT reveals a slight increase in bone matrix volume and the trabecular thickness number in the PACAP group, while histomorphometric analyzes reveal a slight increase in the VIP group, both at a 14-d time-point; despite the increased expression of osteogenic factors, osteoblastic differentiation, activity, and maturation markers in both VIP and PACAP groups. Interestingly, a lower number of VIP and PACAP immunolabeled cells were observed in the treated groups, suggesting a reduction in endogenous production. In conclusion, while both VIP and PACAP treatments presented a significant immunomodulatory effect with potential for increased healing, no major changes were observed in bone healing outcome, suggesting that the signals required for bone healing under homeostatic conditions are already optimal, and additional signals do not improve an already optimal process. Further studies are required to elucidate the role of macrophage polarization in the bone healing process.
Highlights
Bone is a dynamic tissue that provides mechanical support and protection to organs and tissues, and serves as a mineral reservoir to body physiological activities
The VEGFA (7–14 days) expressions were downregulated in the VIPand PACAP-treated groups in relation to the control group, and the same was observed in the VEGFB expression (7 days) only on VIP-treated group
Considering the immunological markers analyzed the expression of IL-1b, IL-6, TNF, and iNOS were downregulated in the VIP and PACAP groups (3–7 days), while TGF-b1, IL-10, ARG, and Fizz were upregulated in the analyzed periods in the treated groups
Summary
Bone is a dynamic tissue that provides mechanical support and protection to organs and tissues, and serves as a mineral reservoir to body physiological activities. Bone properties are closely related to its high remodeling capacity, which allows its adaptation to growth and mechanical stimuli, the regulation of mineral components storage/mobilization depending on physiological demand, and confers to the bone tissue a significant healing capacity [1,2,3]. Bone cells are susceptible to the influence of numerous endocrine and immune system derived factors, which can influence bone homeostasis and healing capacity [1,2,3]. Regarding the interaction between bone and immune systems, bone healing allegedly depends on a transitory inflammatory process, where innate immune system cells migrate to the injury site to remove cell or matrix debris and to regulate tissue healing via the production of inflammatory mediators and growth factors [4]. The original definition of M1 and M2 macrophage derives from the effects of prototypic Th cytokines [8, 9]; the M1 differentiation is driven by IFN-g and results in a pro-inflammatory phenotype; while M2 regulatory and pro-reparative phenotype is induced by IL-4 [6, 7, 10,11,12]
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