Systemic Treatment with the Sphingosine-1-Phosphate Analog FTY720 Does Not Improve Fracture Healing in Mice

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1 Systemic Treatment with the Sphingosine-1-Phosphate Analog FTY720 Does Not Improve Fracture Healing in Mice Aline Heilmann, 1 Thorsten Schinke, 2 Ronny Bindl, 1 Tim Wehner, 1 Anna Rapp, 1 Melanie Haffner-Luntzer, 1 Astrid Liedert, 1 Michael Amling, 2 Anita Ignatius 1 1 Institute of Orthopaedic Research and Biomechanics, Center of Musculoskeletal Research, University of Ulm, Ulm, Germany, 2 Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Received 26 March 2013; accepted 6 June 2013 Published online 1 July 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI /jor ABSTRACT: Sphingosine-1-phosphate (S1P) has recently been recognized as a crucial coupling molecule of osteoclast and osteoblast activity provoking osteoanabolic effects. Targeting S1P receptors could, therefore, be a potential strategy to support bone formation in osteopenic diseases or in fracture repair. Here we investigated whether systemic treatment with the S1P analog FTY720 (Fingolimod) could improve fracture healing. Twelve-week-old, female C57BL/6 mice received an osteotomy of the femur, which was stabilized using an external fixator. The mice received a daily subcutaneous injection of either FTY720 (6 mg/kg) or vehicle from the third postoperative day. Fracture healing was evaluated after 10 and 21 days using biomechanical testing, m-computed tomography, and histomorphometry. Because FTY720 is supposed to influence osteoclast recruitment, osteoclasts were identified in the fracture callus by staining for tartrate resistant acid phosphatase (TRAP). There were no significant differences in callus mechanical properties, tissue composition and osteoclast number between the groups, suggesting that systemically applied FTY720 did not influence bone regeneration in this model of regular fracture healing. Even if further studies should test the potency of FTY720 under unfavorable healing conditions, we conclude that the effect of systemically applied FTY720 on fracture healing might be inferior compared to other anabolic treatments. ß 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: , 2013 Keywords: fracture healing; sphingosine-1-phosphate; FTY720 Conflicts of interest: none. Grant sponsor: German Research Foundation (DFG) (Research Unit FOR793); Grant numbers: IG18/7-2, SCHI 504/5-2. Correspondence to: Anita Ignatius (T: þ ; F: þ ; anita.ignatius@uni-ulm.de) # 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. Bone remodeling is regulated by the finely tuned interaction of bone-forming osteoblasts and bone-resorbing osteoclasts. One crucial molecular mechanism of osteoblast and osteoclast coupling is the receptor activator of nuclear factor kappa B (NF-kB) ligand (RANKL)/ osteoprotegerin (OPG) system. The cytokine RANKL induces osteoclast formation and activity. OPG, a soluble decoy receptor for RANKL, protects bone from resorption by blocking the RANKL effect. Both RANKL and OPG are produced by osteoblasts. 1,2 Vice versa, osteoclasts may direct osteoblast activity by secreting specific factors, among them the lipid mediator sphingosine-1- phosphate (S1P), which has recently attracted attention as a possible osteoanabolic factor. 3,4 S1P is an autocrine and paracrine small signaling molecule that regulates diverse fundamental cellular processes, such as migration, proliferation, differentiation, and apoptosis in many tissue types. 5,6 It is generated by phosphorylation of sphingosine by sphingosine kinase (Sphk). Two mammalian isoforms, Sphk1 and Sphk2, and five high-affinity G protein-coupled S1P receptors (S1Pr1 S1Pr5) have been identified to date. 6 S1pr1, S1pr2, and S1pr3 are expressed by osteoblasts 7 and S1pr1 and S1pr2 by osteoclast precursor cells. 8,9 Osteoclast-derived S1P can promote the migration, differentiation and survival of osteoblasts. 7,10 13 S1P might also induce the egress of osteoclast precursors from the bone marrow 8 and modulate osteoclast differentiation by influencing osteoblast RANKL expression. 7 These data suggest that S1P might be a crucial molecule in osteoblast osteoclast coupling, which provokes osteoanabolic effects. Targeting S1P receptors could, therefore, be a potential strategy to support bone formation in osteopenic diseases or fracture repair. The sphingosine structural analog FTY720 (also known as fingolimod) non-selectively binds to S1P receptors, and is phosphorylated in vivo by Sphk2 to generate the active moiety FTY720-phosphate. 14,15 Indeed, several authors have demonstrated that FTY720 could provoke osteoanabolic effects. FTY720 enhanced osteoblast differentiation in vitro 11 and prevented ovariectomy-induced bone resorption in mice after systemic application by decreasing the attachment of mature osteoclasts to the bone surface. 8 The local release of FTY720 from polymer scaffolds significantly increased vessel formation and bone regeneration within cranial bone defects. 16,17 Similar effects were observed after the implantation of FTY720-coated, devitalized bonegrafts in femoral defects in rats. 18 The authors suggested that locally released FTY720 might attract endothelial progenitor cells to the injury site, thus inducing neovascularization and bone regeneration. Currently, it is not known whether FTY720, through its anabolic effect on bone metabolism, is able to support bone healing after systemic application. Here we investigated the hypothesis that systemic treatment with FTY720 improves diaphyseal fracture healing in mice. METHODS All experiments were performed according to international regulations for the care and use of laboratory animals and were approved by the local ethical committee (No. 1026, 1845

2 1846 HEILMANN ET AL. Regierungspräsidium Tübingen,Tübingen, Germany). Twentynine female C57BL/6 mice (12 weeks old) were provided by the University Medical Center Hamburg-Eppendorf, Germany. The mice were housed in cages in groups of up to four animals on a 14 h light and 10 h dark rhythm with water and food available ad libitum. All mice received the same standard diet (R/M-H, V , Ssniff Spezialitäten GmbH, Soest, Germany). Surgery Surgery was performed under general anesthesia with 2% isoflurane (Forene, Abbott, Wiesbaden, Germany). The fracture healing model was previously described in detail by our group. 19 Briefly, a standardized osteotomy gap was created at the mid-shaft of the right femur using a 0.4 mm Gigli wire saw (RISystem, AO Research Institute, Davos, Switzerland) and stabilized by an external fixator (axial stiffness 3.0 N/mm, RISystem, AO Research Institute). The mice received as analgesia 25 mg/l tramalhydrochloride (Tramal, Gruenenthal GmbH, Aachen, Germany) in the drinking water 1 day preoperatively until 3 days postoperatively. After surgery, the mice were divided into two groups, and received either FTY720 (6 mg/kg bodyweight, Cayman Chemical Company, MI) or vehicle, respectively. FTY720 was dissolved in phosphate buffered saline (PBS) containing 1% dimethyl sulfoxide (DMSO) and 30% fatty acid-free bovine serum albumin (BSA). FTY720 or only the vehicle was subcutaneously injected daily starting on day 3 postsurgery until day of sacrifice. Mice were sacrificed 10 and 21 days post-surgery (n ¼ 6 9). Biomechanical Testing Twenty-one days post-surgery, biomechanical testing of the intact contra-lateral femurs and the osteotomized femurs was performed after removing the fixator to determine the mechanical properties of the callus. The flexural rigidity was evaluated using a non-destructive three-point-bending test. 19 Briefly, the proximal end of the femur was fixed in an aluminum cylinder using a two-component adhesive (i-cem 1 Self-Adhesive, Heraeus Kulzer, Hanau, Germany). The cylinder was fixed in a materials testing machine (Z10, Zwick Roell, Ulm, Germany), serving as the proximal support for the bending test, whereas the femoral condyle rested unfixed on the distal bending support. The bending load was applied to the mid-shaft of the femur up to a maximum of 4 N and continuously recorded versus sample deflection. The flexural rigidity was calculated from the linear elastic region of the testing curve. 19 Micro-Computed Tomography (mct) After completing biomechanical testing, all the femurs were scanned using a mct device (Skyscan 1172, Kontich, Belgium) at a resolution of 8 mm and a voltage of 50 kv and 200 ma. Within each scan, two phantoms with a defined density of hydroxyapatite (250 and 750 mg/cm 3 ) were scanned to determine bone mineral density (BMD). The region of the former osteotomy gap and the mid-shaft in intact contra-lateral femurs were defined as the regions of interest (ROI) (Fig. 1). A grey value corresponding to mg hydroxyapatite/cm 3 was used as a global threshold to distinguish between mineralized and non-mineralized tissue. 20 By segmentation of the former osteotomy gap, total volume (TV), bone volume (BV), bone volume fraction (BV/ TV), and moment of inertia (I x ) were evaluated. Figure 1. Regions of interest (ROI) for mct and histomorphometrical analyses. The osteotomy gap and the periosteal callus between the inner two pins were defined as the ROI after 10 days of healing. The former osteotomy gap was used as ROI after a healing period of 21 days. BM, bone marrow; C, corticalis; OG, osteotomy gap; P, pin hole. Histomorphometry The fracture calli harvested 21 days postoperatively were processed for undecalcified histology. After fixation of the femurs in buffered 4% formalin for a minimum of 48 h, they were dehydrated in increasing ethanol concentrations and embedded in methyl methacrylate. Femurs were cut in the anterior posterior direction into 7-mm slices and stained with Giemsa for tissue quantification. Femurs of mice sacrificed 10 days after surgery were used for decalcified histology. After fixation in buffered 4% formalin for a minimum of 48 h, decalcification was performed using 20% EDTA (ph ). Then femurs were embedded in paraffin, and 7-mm slices were prepared and stained with Safranin O. The femurs were evaluated using light microscopy (Leica DMI6000B; Software MMAF Version MetaMorph 1, Leica, Heerbrugg, Switzerland) under 50-fold magnification. In the undecalcified femurs the former osteotomy gap was defined as the ROI according to mct analysis, whereas in decalcified femurs the region of the osteotomy and the periosteal callus between the inner pins of the fixator were determined as the ROI (Fig. 1). The relative amount of total osseous tissue (TOT), cartilage (Cg) and fibrous tissue (FT) were assessed in all ROI. Osteoclasts were identified using histochemical staining for tartrate-resistant acid phosphatase (TRAP) as previously described. 21 Cells with three or more nuclei, residing on the bone surface and positive for TRAP staining were regarded as osteoclasts. The slices were analyzed under light microscopy at 200-fold magnification. The total number of osteoclasts per bone surface in the callus (OcN/BS) was evaluated and averaged for all the mice of each group. Statistical Analysis The results are presented as mean standard deviation (SD). Statistics software IBM SPSS Statistics 19.0 (SPSS, Inc., Chicago, IL) was used. Differences between the groups were determined using a one-sided Student s t-test. The level indicating significance was p 0.05.

3 FTY720 TREATMENT IN FRACTURE HEALING 1847 Figure 2. Flexural rigidity of the intact (A) and the osteotomized (B) femurs of mice treated with FTY720 or with vehicle (controls) after a healing period of 21 days. n ¼ 6 9, one-sided Student s t-test, level of significance p RESULTS The application of FTY720 did not significantly influence the flexural rigidity of the intact femurs or osteotomized bones (Fig. 2). The flexural rigidity of the osteotomized femurs attained approximately 55% of the bending stiffness of the intact contralateral femur in the FTY20-treated group and of the control group. The mct data revealed a similar BMD in intact femurs and osteotomized femurs of both groups (Table 1). TV, BV, BV/TV, and I x of the fracture callus were not significantly altered by FTY720 treatment. The histomorphometrical evaluation confirmed these results. No significant differences between the treated and control animals were found after 10 and 21 days post-surgery with respect to cartilage and bone formation in the fracture calli (Table 2, Fig. 3). We evaluated osteoclast numbers in Table 1. mct Analyses of Intact and Osteotomized Femurs of Mice Treated With FTY720 or With Vehicle (Controls) After a Healing Period of 21 Days Vehicle FTY720 BMD Intact in 1, , mg HA/cm 3 BMD Callus in mg HA/cm 3 TV in mm BV in mm BV/TV in % I x in mm BMD, bone mineral density; TV, total callus volume; BV, bone volume; BV/TV, bone volume/total volume; I x, moment of inertia. Data are presented as the mean SD. n ¼ 6 9, one-sided Student s t-test, level of significance p the callus, because it was indicated that FTY720 could influence osteoclast recruitment. 8 However, no significant differences in osteoclast number were found between the two groups (Table 2). DISCUSSION Based on the evidence that S1P might provoke osteoanabolic effects 8,10,12 and improved bone defect healing, 22 we investigated whether systemic treatment with the S1P analog FTY720 increased diaphyseal fracture healing in mice. In contrast to our hypothesis, FTY720 did not significantly influence bone repair in the mouse model used, as demonstrated by biomechanical, mct and histomorphometrical evaluations. Our results suggest that S1P receptor stimulation by systemic application of FTY720 might not be an effective strategy to support bone formation in fracture healing. Several studies reported significantly improved bone formation after the sustained release of S1P 22 or FTY from carriers, which were locally implanted into bone defects. Because the authors found enhanced microvascular network formation and arteriolar expansion in the treated bone defects, and because S1P is known to be a potent chemokine for endothelial precursors, 23 they suggested that bone regeneration was mainly improved due to an increased chemoattraction of these cells along the concentration gradient of FTY720 to the site of injury In contrast to these studies, we applied FTY720 systemically during fracture healing. Therefore, FTY720 could not operate as a local chemoattractant for precursor cells. Nevertheless, we had expected a positive effect of FTY720 on bone formation because of the positive influence of S1P on osteoblast activity. Several studies have demonstrated, albeit in vitro, that S1P receptor

4 1848 HEILMANN ET AL. Table 2. Histomorphometrical Analysis of the Fracture Callus of Mice Treated With FTY720 or With Vehicle (Controls) After a Healing Period of 10 and 21 Days Vehicle FTY Days 21 Days 10 Days 21 Days Callus area in mm TOT in % Cg in % FT in % OcN/BS in mm Relative amount of newly formed bone (TOT), cartilage (Cg) and fibrous tissue (FT) and number of osteoclast per bone surface (OcN/BS). Data are presented as mean SD. n ¼ 6 9, one-sided Student s t-test, level of significance p Figure 3. Representative histological sections through the fracture callus of mice treated with FTY720 (right column) or with vehicle (controls, left column) after a healing period of 10 and 21 days. (A and B) Decalcified histology after a healing period of 10 days, Safranin O staining; (C and D) Undecalcified histology after a healing period of 21 days, Giemsa staining; (E and F) Osteoclasts in the peripheral callus after a healing period of 10 days, staining for tartrate-resistant acid phosphatase (TRAP); (G and H) Osteoclasts in the peripheral callus after a healing period of 21 days, TRAP staining. n ¼ 6 9. activation could enhance osteoblast migration, differentiation and survival. 7,10 13 However, we did not find any influence of systemically applied FTY720 on bone healing. The amount of newly formed bone in the fracture callus was not significantly altered, as demonstrated by histomorphometrical and mct analysis, indicating that osteoblast activity was clearly not sufficiently stimulated to improve fracture healing. We also did not observe any effect of FTY720 on the contralateral intact femur after a treatment period of 3 weeks. This was confirmed by a paper reporting that 4 weeks of systemic treatment with FTY720 prevented the ovariectomy-induced bone loss in mice but had no detectable effects in non-ovarectomized mice. The attachment of mature osteoclasts to the bone surface, which was increased after ovariectomy, was partially restored to normal levels by FTY Because the number of osteoclast precursor cells in the blood was concurrently increased, the authors suggested that systemic FTY720 might promote the recirculation of osteoclast precursor cells from the bone surface, thereby preventing bone erosion. 8 To elucidate a potential effect of FTY720 on osteoclast recruitment to the fracture callus, we identified osteoclasts using TRAP staining. Our results revealed that the number of osteoclasts was not affected by FTY720, not even in the late fracture-healing phase, which is characterized by extensive osteoclast recruitment when the bony callus starts to remodel. 24 Taken together, our data indicate that S1P-receptor stimulation by FTY720 treatment did not affect fracture healing either by influencing bone formation or erosion. Whereas in bone remodeling osteoblast and osteoclast activity are closely coupled thereby S1P being regarded as an important anabolic clastokine, 12 in fracture repair the generation of new bone does not necessarily depend on osteoclast activity and is orchestrated by many potent cytokines and growth factors, such as bone morphogenetic proteins (BMPs). 25 Limitations of our study might be the use of young, 12-week-old mice exhibiting a good regenerative capacity and the semi-rigid fracture stabilization allowing regular fracture healing. However, because several

5 FTY720 TREATMENT IN FRACTURE HEALING 1849 anabolic treatments, for example parathyroid hormone or estrogen, were proven to be effective in comparable mouse fracture models, we believe that our model is generally suitable to prove anabolic effects. However, further studies are required to answer the question whether FTY720 would be effective in models with a delayed healing capacity, such as aged or ovariectomized animals. FTY720 is also known as a potent immunosuppressive agent, preventing the emergence of lymphocytes from lymph nodes. 14,15 Therefore, it has been clinically approved for the treatment of multiple sclerosis. 14 As bone healing starts with an inflammatory phase characterized by the invasion of immune cells into the fracture hematoma, 30 FTY720 may influence fracture healing by modulating early inflammatory processes. Indeed, a lower number of CD45-positive leucocytes were observed in bone defects locally treated with FTY720, suggesting that the depression of inflammatory reactions might be one possible mechanism of improved bone regeneration in these studies. 17,18,22 In our study, we did not administer FTY720 immediately after the fracture, rather starting from the third postoperative day. In mice, lymphocytes are already abundantly present in the fracture callus at this time point and the inflammatory phase starts to progress to the proliferative repair phase. 31,32 Therefore, our study did not answer the question, whether FTY720, if applied immediately after fracture, could influence bone healing by altering the recruitment of inflammatory cells to the fracture hematoma. In conclusion, the results of this study demonstrated that systemic treatment with the S1P analog FTY720 was not effective in improving standard fracture healing. Further studies are necessary to prove the potency of FTY720 under unfavorable healing conditions or its immune modulatory effect on early bone healing. However, the effect of systemically applied FTY720 on bone repair might be inferior compared to other anabolic treatments. ACKNOWLEDGMENTS The authors appreciate the technical assistance of Ursula Maile, Marion Tomo, and Sevil Essig. Each author in this manuscript has not and will not receive benefits in any form from a commercial party related directly or indirectly to the content of this manuscript. REFERENCES 1. Boyce BF, Xing L Functions of RANKL/RANK/OPG in bone modeling and remodeling. Arch Biochem Biophys 473: Teitelbaum SL, Ross FP Genetic regulation of osteoclast development and function. Nat Rev Genet 4: Boyce BF Sphingosine-1 phosphate: a new player in osteoimmunology. Dev Cell 16: Kollet O, Dar A, Lapidot T The multiple roles of osteoclasts in host defense: bone remodeling and hematopoietic stem cell mobilization. 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