Contents lists available at ScienceDirect Bone Reports journal homepage: www.elsevier.com/locate/bonr Relaxin 2 carried by magnetically directed liposomes accelerates rat midpalatal suture expansion and subsequent new bone formation Hiroyuki Kamimoto, Yukiho Kobayashi ⁎ , Keiji Moriyama Maxillofacial Orthognathics, Department of Maxillofacial Reconstruction and Function, Division of Maxillofacial/Neck Reconstruction, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan ARTICLE INFO Keywords: Suture expansion Relaxin Liposome Drug delivery system In vivo imaging ABSTRACT Relaxin (RLN) is an insulin-like peptide hormone that enables softening and lengthening of the pubic symphysis and uterine cervix. Here, we analyzed the effects of RLN2 on the expansion of rat midpalatal suture (MPS) using a magnetically directed liposome-based drug delivery system. Thirty-six male rats were divided into three groups: control (MPS was not expanded), lipo (expanded for 1 week with vehicle liposomes encapsulating ferric oxide and Cy5.5), and RLN-lipo (expanded for 1 week with the liposomes coated with RLN2). Rats were sacri- ficed after 1 week of expansion or after 2 weeks of retention. To accumulate RLN2-liposomes, a magnetic sheet was fixed to the palatal mucosa of the MPS. In vivo imaging showed magnetically controlled accumulation of liposomes in the MPS for 72 h. Immunohistochemistry revealed RLN2 expression in the MPS after expansion and relaxin receptor (RXFP) 2 expression at the osteogenic front (OF) in the RLN-lipo group; all groups expressed RXFP1 in the MPS. MPS expansion and bone formation were significantly accelerated at the OF in RLN-lipo group compared with the other groups. In the RLN-lipo group, significantly accelerated serrate bone deposition and elevated periostin (POSTN), iNOS, and MMP-1 levels were observed in the MPS. Sclerostin (SOST) ex- pression was significantly reduced in newly formed bone in the RLN-lipo group. Our data revealed that RLN2 enhanced suture expansion via MMP-1 and iNOS secretion in the sutural fibroblasts and new bone formation via POSTN expression in osteoblasts at the OF. These properties may be useful for developing a new less-invasive orthopedic treatment aiming at sutural modification of cranio- and maxillofacial deformity patients. 1. Introduction Relaxin (RLN) is a pleiotropic hormone of the insulin-like peptide hormone family that is well known to facilitate parturition by inducing the softening and lengthening of the pubic symphysis and softening of the cervix during the peripartum period (Lu et al., 2005). Among members of this family, RLN, insulin-like peptide (INSL) 3, and INSL5 interact with relaxin family peptide receptors (RXFPs) 1–4(Bathgate et al., 2013; Bathgate et al., 2005; Bathgate et al., 2006). The anti- fibrotic effects of human-gene 2 (H2) relaxin (serelaxin), which is structurally related to INSL3, promote the secretion of collagen-de- grading MMPs via RXFP1/ERK1/2 signaling in fibroblasts and myofi- broblasts following kidney injury in rats and in rat renal myofibroblasts (Mookerjee et al., 2009). In osteoblast progenitor cells, RXFP2/INSL3 signaling induces al- kaline phosphatase (ALP) activity, extracellular matrix mineralization, and mitogen-activated kinase (MEK) and ERK1/2 activation (Ferlin et al., 2011). Ferlin et al. reported that 64% of young men with mutated RXFP2 (T222P) had significantly lower bone mass density (Ferlin et al., 2008). Moreover, RXFP2-deficient mice showed decreases in bone mass, mineralizing surface, bone formation (Ferlin et al., 2008), thus, INSL3/RXFP2 signaling was found to be involved in bone metabolism. In our previous study, we reported the expression pattern of Rxfp1 and Rxfp2 mRNAs during mouse craniofacial bone and tooth development (Duarte et al., 2014b) and found that RLN inhibited collagen deposition by inhibiting ColIa1 expression and inducing MMPs secretion into the culture medium of MC3T3-E1 through Rxfp2 using siRNA targeting Rxfp1 and Rxfp2, and concluded that administration of RLN enhanced osteoblastic differentiation, mineralization, and extracellular matrix degradation in vitro through RXFP2 (Duarte et al., 2014a). Moon et al. showed that RLN enhanced bone morphogenetic protein (BMP) 2-in- duced bone formation and osteoblast differentiation by upregulation of runt-related transcription factor 2 (Runx2) expression and activity (Moon et al., 2014). Liposomes have a lipid bilayer with an internal aqueous cavity and have been investigated as a drug delivery system because of their lack https://doi.org/10.1016/j.bonr.2019.100202 Received 6 November 2018; Received in revised form 6 February 2019; Accepted 11 March 2019 ⁎ Corresponding author. E-mail addresses: h-kamimoto.mort@tmd.ac.jp (H. Kamimoto), yukiho-kobayashi.mort@tmd.ac.jp (Y. Kobayashi), k-moriyama.mort@tmd.ac.jp (K. Moriyama). Bone Reports 10 (2019) 100202 Available online 14 March 2019 2352-1872/ © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). T