Regulation of Bone Differentiation Through the Neuropeptides
This dissertation aims to demonstrate immunoregulatory roles of neuropeptides in bone metabolism both in bone and non-bone microenvironments. Substance P (SP) and calcitonin gene-related peptide (CGRP) are sensory neuropeptides that have been associated with various aspects of the pathophysiology of heterotopic ossification (HO) of soft tissues. These aspects include dysregulation of inflammation, mainly governed by macrophages and mast cells, and bone morphogenetic protein (BMP) signaling. The first set of experiments presented here contributes to the understanding of the involvement of the neuropeptides with HO pathophysiology. Results of these experiments illustrate that the delivery of SP, with no exogenous BMP or direct trauma to the tissue, is sufficient to induce inflammation, upregulate BMP2 expression and HO in murine Achilles tendon, while CGRP delivered together with SP can suppress the inflammatory changes and HO induced by SP. This is the first study that involves direct administration of these neuropeptides into the tendon to investigate their direct effects in the induction of HO. This was enabled by the use of biopatterning technology, which introduced spatial control of the tissue microenvironment by using inkjet-based deposition of the neuropeptides onto collagen scaffolds, i.e. Acellular DermaMatrix (ADM) constructs, and implanting these constructs in vivo. Additionally, in vitro studies demonstrated an interaction between SP and CGRP in direct and indirect regulation of BMP2 signaling. One of the main contributions of the in vitro experiments is that CGRP can counteract the augmenting effects of SP on BMP2 signaling and therefore down-regulate BMP2-induced bone differentiation when combined with SP. Moreover, SP, despite its inflammatory potential, can suppress the inhibitory effects of the inflammatory response that is mediated by macrophage activation on BMP2 signaling, and this effect can be mitigated by CGRP. Therefore, this finding provides additional evidence for the contribution of macrophages together with neuropeptide-mediated inflammation to the induction of HO. The final set of experiments explored the use of vasoactive intestinal peptide (VIP) as a therapeutic approach to improve BMP2-induced bone haling during inflammatory conditions. In order to investigate this problem, a critical-defect model was used, where ADM biopattened with bacterial lipopolysaccharide (LPS), to induce inflammation, VIP and BMP2 were delivered into mouse calvaria to induce bone healing in the presence of inflammation. LPS was shown earlier to have inhibitory effects on BMP2-induced bone healing by creating an inflammatory microenvironment. Our findings indicate that VIP can mitigate inhibitory effects of LPS on BMP2-induced bone differentiation in vitro and bone healing in vivo. Overall, the work presented here adds to the understanding of the interaction between the peripheral nervous system and bone metabolism through regulation of the immune responses in musculoskeletal pathologies.