{"paper_id":"037f3dab-b487-4573-b0ee-0fae777ce3b3","body_text":"1Scientific  RepoRts  |          (2019) 9:9744  | https://doi.org/10.1038/s41598-019-46278-6\nwww.nature.com/scientificreports\nEffect of neurokinin-1-receptor \nblockage on fracture healing in rats\nMartijn Hofman1, Frederik Rabenschlag1, Hagen Andruszkow1, Julia Andruszkow2, \nDiana Möckel3, Twan Lammers3, Aneta Kolejewska1, Philipp Kobbe1, Johannes Greven1, \nMichel (Paul Johan) Teuben4, Martijn Poeze5 & Frank Hildebrand1\nNeurologic injury and selective blockage of sensory nerve endings is associated with impaired fracture \nhealing, however, the role of specific neurotransmitters has not been sufficiently investigated. Our \naim was to investigate the impact of specific Substance P-receptor blockage on fracture healing, since \nthe neuropeptide Substance P has both neurogenic and osteogenic activity. After intramedullary \nstabilization, an isolated femur fracture was induced in 72 Sprague-Dawley rats. In the NK1-R group, \nthe neurokinin-1-tachykinin receptor for substance P was blocked by a specific antagonist (SR140333) \nfor the first two weeks after fracture induction. The control group only received vehicle. Gene-\nexpression, histology, micro-computed tomography, and biomechanical tests were performed. NK1-\nreceptor blocking suppressed osteocalcin expression at one week, collagen 1A2 expression at one and \ntwo weeks and collagen 2A1 expression at 2 weeks after fracture induction. Biomechanical testing \nrevealed a significant reduction in maximal load to failure in the NK1-R group at 6 weeks (69.78 vs. \n155.45 N, p = 0.029) and at 3 months (72.50 vs.176.33 N, p = 0.01) of fracture healing. Blocking the NK1-\nreceptor suppresses gene expression in and reduces biomechanical strength of healing bone. Therefore, \nwe assume a potential therapeutic relevance of Substance P in cases of disturbed fracture healing.\nFractures are frequently associated with either central (e.g., traumatic brain injury [TBI]) or peripheral neu-\nrological injuries, and these injuries are well known to affect fracture healing. This phenomenon has partly \nbeen explained by the close anatomical relationship between bones and nerves (e.g., innervation of perios -\nteum with close contact between nerve endings and bone cells). It also reflects the chronological association \nbetween callus formation, bone remodelling, and the regeneration of damaged nerve endings and ingrowth into \nnewly formed bone\n1,2. Disturbances of this re-innervation have therefore been associated with the incidence of \nnon-unions3. Hukkanen and colleagues provided the first evidence for a role of nerve-mediated bone forma-\ntion in fracture healing by showing that a complete denervation of a leg by sciatic nerve section had negative \neffects on the mechanical integrity of the bony callus after fracture\n4. Similarly, Offley and colleagues found that \ncapsaicin-sensitive neurons contribute to cancellous bone integrity and bone homeostasis in the uninjured bone. \nThey concluded that osteoclast numbers, osteoblast activity, bone formation, and bone strength were mediated by \ntransmitters released from efferent sensory nerve endings in bone tissue\n5.\nFurther, complete peripheral nerve transection6–8 and selective blockade of sensory nerve endings9 can impair \nfracture healing, as demonstrated by previous studies utilizing either combined motoric, sensory, and autonomic \ndenervation\n7,8 or complete sensory nerve ending blockade5,9.\nThe exact pathophysiologic mechanism underlying this interaction between fracture healing and nerve inju-\nries is not known. Neuropeptides are produced by nerve endings after TBI and fractures, so neuropeptides poten-\ntially have major relevance in the process of fracture healing\n1,2,10,11. However, no precise role has been established \nfor specific neuropeptides in bone healing12,13.\nOne neuropeptide of particular relevance to bone healing is Substance P , which shows simultaneous effects at \nthe neurogenic and osteogenic activities. For example, Apel et al. showed that sensory denervation, which shut \ndown the transmission of both calcitonin gene-related peptide (CGRP) and Substance P , resulted in an impaired \n1Department of Orthopedic t rauma and Reconstructive Surgery, University of Aachen Medical center, Aachen, \nGermany. 2institute of Pathology, University of Aachen Medical c enter, Aachen, Germany. 3institute for \nexperimental Molecular i maging, center for Biohybrid Medical Systems, University of Aachen Medical center, \nAachen, Germany. 4Department of t rauma and Harald t scherne Laboratory, University Hospital Zurich, Zurich, \nSwitzerland. 5Department of Surgery, Division of t raumasurgery, Maastricht University Medical center, Maastricht, \nthe netherlands. Martijn Poeze and frank Hildebrand jointly supervised this work. correspondence and requests for \nmaterials should be addressed to M.H. (email: mhofman@ukaachen.de)\nReceived: 29 January 2019\nAccepted: 12 June 2019\nPublished: xx xx xxxx\nopeN\n\n\n2Scientific  RepoRts  |          (2019) 9:9744  | https://doi.org/10.1038/s41598-019-46278-6\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nupregulation of collagen I and II in fracture callus and a negative influence on osteoclast function and mechanical \nstrength and maturation of fracture callus9. Neurotransmitters were therefore postulated to play a central role in \nthe interaction between the nerves and the osseous system8,14, and especially substance P seems to have a signif-\nicant involvement in bone metabolism, formation, and resorption, as well as in the osteogenic activity of bone \nmarrow stromal cells and osteogenic cell lines\n15–23. However, the relevance of specific neurotransmitters remains \nunclear in these previous studies. and the specific effects of Substance P on the process of fracture healing have \nnot been elucidated. Therefore, the aim of this study was to investigate the effects of specific Substance P-receptor \nblockage on gene expression, callus formation, and the mechanical strength of healing fractures in an isolated \nfemur fracture model in rats. We hypothesize that specific substance P receptor blockage impairs mechanical \nstrength of healing fractures, decreases gene-expression in the early fracture healing stages and reduces callus \nquantity and quality.\nResults\nAnimal Mortality. At the beginning of the experiments, all animals were in good general health, according \nto the ‘score sheet’ and ‘Body condition scoring’ described in the methods section. No rats died during our study \ndue to impacts of the operative procedure or its sequelae. In the course of the experiments, 2 rats (2.78%, planned \nfor histological analysis) had to be excluded according to our exclusion criteria; one animal had to be removed \ndue to wound dehiscence and the other animal due to anaesthetic complications. In total, this left 70 rats (97.22%) \nfor inclusion in the final analyses.\nGene Expression Analysis (Collagen 1A2- and 2A1- and Osteocalcin-mRNA). Osteocalcin expression \nwas significantly impaired at 1 week in the NK1-R group compared to the Control group (2.6 ± 0.1 fold vs. 13.5 ± 0.4 \nfold, p = 0.0002 for α = 0.05). Collagen 1A2 also showed a significant depression of expression at 1 week (59.5 ± 2.5 fold \nvs. 228.2 ± 33.45 fold, p = 0.00148 for α  = 0.05), as well as at the 2-week time point (86.2 ± 7.3 fold vs. 170.3 ± 3.2 fold, \np = 0.00028 for α  = 0.05). Collagen 2A1 expression showed a significant reduction only after 2 weeks (1.9 ± 0.4 fold vs. \n106.9 ± 2.1 fold, p = 0.000041 for α  = 0.05) (Fig. 1).\nHistological Analysis. The haematoxylin-eosin staining 4 weeks after fracture did not reveal any significant \ndifferences in terms of vascularity or angiogenesis. The number and diameter of vessels were similar in both \ngroups. We performed three microscopical measurements in each animal, but neither in trabecular diameter nor \nin callus diameter significant differences between both study groups were found (Fig. 2).\nMicro-CT Scanning. The micro-computed tomography analyses conducted six weeks after fracture induc-\ntion showed an almost completed process of ossification and remodelling of the fracture site in the Control \ngroup, whereas the healing process in the NK1-R group was still in progress, based on the visual analysis of the \nµCT-scanning images, preceding the quantitative computer tomography analyses. In the quantitative computer \ntomography analyses, six weeks after healing, the total bone volume did not significantly differ between the frac-\ntured side with callus formation and the unfractured side without callus formation in both study groups. This \nimplies that the comparability of the two study groups is warranted. The bone density was significantly lower \nfor the fractured femur compared to the femur on the unfractured side in both groups (NK1-R group, p < 0.01; \nControl group, p < 0.05). Furthermore, a significant difference was noted in the transverse diameter between the \nfractured side (callus) and the unfractured side in both groups (NK1-R group, p < 0.01; Control group, p < 0.005). \nNo significant differences were observed between the Control and the NK1-R groups (Fig. 3).\nThe callus formation analysis did not reveal any significant differences between the two groups in terms of the \nvolume, diameter, length, or density of the callus (Fig. 3).\nBiomechanical tests. Both study groups demonstrated a decreased load to failure of the fractured femora \ncompared to the unfractured side at 6 weeks and 3 months after fracture. By contrast, biomechanical analy-\nses at 6 weeks (Control group 251.84 ± 8.87 N vs. NK1-R group 254.48 ± 9.95 N) and 3 months (Control group \n247.00 ± 28.66 N and NK1-R group 224.67 ± 19.10 N) after fracture showed no significant differences in the load \nto failure for the unfractured side in both groups. The fractured side showed a significant (p = 0.029) difference \nin the load to failure between the NK1-R group (69.78 ± 8.51 N) and the Control group (155.45 ± 9.32 N) after \n6 weeks. This difference was even greater at 3 months after fracture (NK1-R group: 72.50 ± 12.09 N vs. Control \ngroup: 176.33 ± 13.44 N, p = 0.010) (Fig. 4).\nSummary of study results. Our main results can be summarized as follows:\n (1) Blocking of the NK1-receptor for Substance P was associated with a reduced expression of different oste-\nogenic proteins (osteocalcin, collagen 1A2, and collagen 2A1) in the early phase (the first two weeks) of \nfracture healing.\n (2) NK1-receptor blocking impaired the normal improvement in biomechanical strength of the bone in the \nlate phase (6 weeks and 3 months) after fracture.\n (3) Since the quantitative callus extensions showed no significant differences, but the biomechanical strength \nof the healing bone was impaired, NK1-receptor blocking decreased the quality of the callus formed.\n\n3Scientific  RepoRts  |          (2019) 9:9744  | https://doi.org/10.1038/s41598-019-46278-6\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nDiscussion\nFracture healing is well recognized as a complex process that is still not completely understood. However, the \nrelationship between fracture healing and neurologic injury is well described and implies an influence of neuro-\nlogical neurotransmitters\n12,13. Among the various neurotransmitters, Substance P in particular is believed to play \na central role in bone metabolism. In this context, Substance P has been shown to stimulate bone formation by \nosteoblastic cells through interaction with the NK1-receptor\n24, whereas blockage of the NK1-receptor enhanced \nwidespread osteoporotic processes18. In the present study, our aim was to investigate what influence blocking the \nNK1-receptor for substance P might have on fracture healing.\nThe important role of Substance P in bone metabolism was especially supported in the present study by the \ndemonstration, for the first time, that selective blockage of Substance P activity by a NK1-receptor antagonist \nimpairs the gene expression, biomechanical strength, and callus quality of a healing fracture.\nThe decreased gene expression observed in the present study for osteocalcin, collagen 1A2, and collagen 2A1 \nin the early phase of callus formation after Substance P receptor blockade underscores the relevance of Substance \nP in fracture healing. These results are in line with previous RT-PCR studies that showed a similarly impaired \nexpression of osteocalcin and collagen during fracture healing in tachykinin-deficient mice\n15, in sensory dener-\nvated rats9, and in rat osteoblastic cells24.\nFigure 1. Comparison of fold changes in gene expression. The fold changes of osteocalcin (a), collagen 1A2 \n(b), and collagen 2A1 (c) mRNA expression were determined by the reverse transcription polymerase chain \nreaction (RT-PCR) at 3 days, 1 week, and 2 weeks after fracture induction.\n\n4Scientific  RepoRts  |          (2019) 9:9744  | https://doi.org/10.1038/s41598-019-46278-6\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nHowever, beside this role in early fracture healing, Substance P seems also to have relevance for the later \nstages of fracture healing and remodelling of bone. In this context, we were able to show an association between \nNK1-receptor blockade in the first two weeks of fracture healing and a significant decrease in bone strength \nat 6 weeks and even at 3 months after fracture. In previous studies, Niedermair and colleagues showed that \ntachykinin-1-deficient mice (Substance P knockout mice) have a reduced mechanical strength of callus after 3 \nweeks of fracture healing, while Apel and colleagues showed that sensory denervated rats have a reduced mechan-\nical strength of callus after 6 weeks of fracture healing\n9,15. However, none of these studies analysed the biome-\nchanical strength at the end of the remodelling phase, as we did after 3 months. If these effects of NK1-receptor \nblockage are exerted in the remodelling phase or are a result based on the earlier effects of NK1-receptor blockage \non fracture healing has to be explored in future studies.\nContrary to the results presented by Apel and colleagues, our findings did not indicate any significant histo-\nlogical differences in trabecular or callus diameter. We also did not find any significant radiological differences \nin quantitative callus dimensions in the µ CT-analyses between our two study groups, although Apel and col-\nleagues did report significant radiological differences. Our results agree with the observations of Niedermair and \ncolleagues, who found no radiological differences in callus dimensions between tachykinin-deficient mice and \na control group\n15. These comparable quantitative callus dimensions between our study groups, in combination \nwith the decrease in biomechanical strength of the bone in the NK1-R group, can only occur if the blockage of \nthe NK1-receptor for Substance P impairs the quality of the soft and hard callus formed, although we could not \nsubstantiate this presumption with histological proof. In this context, the quality of callus is determined by the \nbiomechanical strength of the callus and later of the remodelled bone, because the objective of fracture healing is \nto restore the original strength of the bone. Since Apel and colleagues performed a denervation that blocked both \nCGRP and Substance P , this might indicate that CGRP has a greater influence on the amount of callus formed, \nwhile Substance P regulates the strength and quality of callus.\nThe findings presented here demonstrate the indispensability of Substance P for normal and appropriate callus \nformation and fracture healing. Nevertheless, clarifying the exact role of Substance P in fracture healing and bone \nformation will require further research. Additional research is also required to assess previously postulated mech-\nanisms of action. For instance, Niedermair and colleagues proposed crucial trophic effects of neurotransmitters \non bone healing via an endogenous callus signalling loop in which chondrocytes producing Substance P and its \nNK1-receptor play an important role\n15. They consider that the absence of Substance P results in a net decrease in \nbone formation, leading to the observed decrease in the quality of the callus.\nAnother interesting theory introduced by Davis and colleagues assumed that bone morphogenetic proteins \n(BMPs) released at the fracture site enter peripheral neurons through the damaged blood–nerve barrier. There, \nthey induce a neuroinflammation with a release of substance P , as well as a subsequent release of osteoprogenitor \ncells in cases of heterotopic ossifications\n25. The results of our study could also point to a possible lead for further \nresearch into enhanced fracture healing in patients with concomitant TBI13,26–28, because Substance P is released \nearly following acute injury to the CNS. This promotes a neurogenic inflammatory response that is characterized \nby an increase in the permeability of the blood–brain barrier29\nOur study also has some limitations. As our findings were derived from an animal model, they are not directly \ntranslatable to humans. We chose a rat model because the blockage of a neurotransmitter can only be performed \nin laboratory animals and because adequate blockage of the substance P-receptor with the NK1-receptor antago-\nnist SR140333 has been previously demonstrated in this species\n30. Furthermore, the fracture model in rats is well \nFigure 2. Microscopic imaging of callus tissue. Callus tissue (arrows) between muscle cells (above) and \nbone (below) in control group (a; calibration = 500 µm) and NK1-R group (b; calibration = 500 µm) in \n5× magnification using haematoxylin-eosin staining. Callus tissue in control group (c; calibration = 100 µm) \nand NK1-R group (d; calibration = 100 µm) in 20× magnification using haematoxylin-eosin staining.\n\n5Scientific  RepoRts  |          (2019) 9:9744  | https://doi.org/10.1038/s41598-019-46278-6\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nFigure 3. Micro-CT imaging shows the process of ossification and remodelling of the fracture site in the \ncontrol group and NK1-R group. Micro-CT-imaging of the unfractured control left femur as 2D cross-sectional \nimage in sagittal plane as well as 3D volume renderings resulting in a spatial resolution of 35 µm voxel side \nlength before (a) and after segmentation of the bone (blue) (b); micro-CT-imaging of the fractured right femur \nas 2D cross-sectional image in sagittal plane as well as 3D volume renderings resulting in a spatial resolution of \n35 µm voxel side length before (c) and after segmentation of the bone (blue), callus (green) and K-Wire (red) \n(d). 2D cross-sectional images in sagittal plane for one unfractured and fractured side of both study groups \n(yellow line as example for callus length and diameter) (e). Micro-CT-based quantification of the total bone \nvolume (f) and bone density (g) of the whole femur in both the fractured side and the control side of both study \ngroups. Diameter at the fracture side compared to the diameter at the correspondence level of the unfractured \nside (h). Micro-CT-based quantification of the total callus volume (i), callus density (j), callus length (k) and \ncallus diameter (l) in the fractured side of both study groups. Significance: *p < 0.05, **p < 0.01, ***p < 0.005.\n\n6Scientific  RepoRts  |          (2019) 9:9744  | https://doi.org/10.1038/s41598-019-46278-6\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\ndocumented in the existing literature, and the influence of NK1-receptor blockage on the complex, multifactorial \nprocess of fracture healing cannot be simulated in an in vitro experiment.\nMoreover, the volume measurements conducted using µ CT analyses might be inaccurate due to obscuring \nby the intramedullary Kirschner wire, which in turn would reduce the volume measurement in the fractured \nbones. Therefore, the segmentation of the bone, the K-wire and the fracture callus regions was performed \nwith a very advanced interactive method (Software Imalytics Preclinical) to minimize the error range. Even \nif we had removed the K-wire shortly before the µCT analysis, this removal would have left an empty space, \nwhich would not be filled with new callus tissue and therefore would probably lead to the same results. \nConversely, our histology findings concerning trabecular and callus dimensions conform to our µ CT find-\nings, indicating that these results are probably reliable. Another possible limitation, was that we did not follow \nup the blocking capacity of SR130444 after finishing the administration after two weeks of fracture healing. \nWe assume that the effect of SR130444 is depleted > 24 hours after application, but we did not verified it with \nimmuno-histological tests.\nIn conclusion, our study findings show that blocking the NK1-receptor for substance P suppresses gene \nexpression of important proteins in the early phases of fracture healing, decreases the quality of callus \nformed, and lessens the biomechanical strength of bone in the late phases of fracture healing. Therefore, \nbased on our results, further research should be focused on the exact mechanism of action of Substance P in \nfracture healing to provide a better understanding of its potential therapeutic relevance in cases of disturbed \nfracture healing.\nFigure 4. Biomechanical testing of the femora. Significant decrease in maximal load to failure (Nmax) of the \nfemora in the NK1-R group at (a) 6 weeks (*p = 0.029) and (b) 3 months (*p = 0.01) after fracture induction.\n\n7Scientific  RepoRts  |          (2019) 9:9744  | https://doi.org/10.1038/s41598-019-46278-6\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nMethods\nAll methods were performed with the approval of the institutional animal committee and of the regulat-\ning authority (LANUV) North Rhine-Westphalia, Recklinghausen, Germany (AZ 84-02.04.2015.A078). All \nanimal experiments were performed in accordance with the guidelines and regulations of the Federation of \nEuropean Laboratory Animal Science Associations (FELASA) and the German Society of Laboratory Animals \n(GV-SOLAS).\nHousing. Our study cohort consisted of 72 adult, female Sprague-Dawley rats, weighing approximately 250 g, \nobtained from Harlan Industries (Indianapolis, Indiana, USA). The animals were housed and the experiments \nwere performed in facility approved by the Federation of European Laboratory Animal Science Associations \n(FELASA) and the German Society of Laboratory Animals (GV-SOLAS). The animals were housed under con-\nditions of controlled temperature (20 ± 2 °C) and air humidity (45–65%), with a 12 h light-darkness cycle and a \nlight intensity of <200 lux. Food and water were offered ad libitum. Prior to study inclusion, all animals were kept \nin groups for one week in the laboratory premises to allow acclimatization. Throughout the entire experiment, all \nanimals underwent physical examinations according to a ‘score sheet’ documentation and the ‘Body condition \nscoring’ according to Hickman\n31 to obtain the general health of the animals.\nBecause of the protective effects of female hormones in cases of inflammatory stimuli, all animals were con-\nfirmed to be in the same ‘metestrus’ phase of the menstrual cycle, as this phase is characterized by low estrogen \nand progesterone levels\n14,32. The menstrual cycle phase was identified by the assessment of vaginal swabs accord-\ning to Marcondes and colleagues33.\nExperimental design and power analysis. In all animals an intramedullary pin was inserted and \na standardized femoral fracture was induced. The animals were then randomly assigned to 2 groups (36 rats/\ngroup), according to their order number (odd or even). The first group underwent a selective blockage of the \nNK1-receptor (NK1-R group). The second group served as the control group and only received vehicle (Control \ngroup). Depending on the experimental subgroup to which an animal was randomly assigned, the femur was \nharvested and the following analyses were executed at different time points: gene expression analysis, histological \nanalyses, micro-CT scanning, and biomechanical testing (Fig. 5).\nOur primary outcome parameter was the load to failure of the rat femora; therefore, our power analysis was \nbased on an average load to failure of 131.3 ± 4.9 N for normal rat femora, as described by previous studies\n9,34. \nOur calculation indicated that a minimum of 4 rats per group was needed to achieve a 95% power for detecting a \ndifference of 10 N with α = 0.05. Therefore, a sample size of six animals per group was chosen to compensate for \nany potential loss of animals. The power analysis was performed with G*Power. Secondary outcome parameters \nwere fold change in gene expression of osteocalcin and collagen 1A2 and 2A1 and callus-volume, -density and \n-diameter in micro-CT-analysis.\nReasons for excluding animals were: death from anaesthetic complications, open fracture, comminuted frac-\nture, implant failure, wound dehiscence or infection, gross technical failure, inadequate RNA for analysis, and \nunintended displacement of the femur during biomechanical testing.\nAnaesthesia and pain management. Thirty minutes preoperatively, the animals received 0.03–0.05 mg/\nkg buprenorphine hydrochloride s.c. as pain medication. The operative procedures were performed under \ngeneral anaesthesia induced with ketamine (100 mg/kg i.p.) and xylazine (2%; 10 mg/kg i.p.) and if necessary, \nextended with 2–2.5 Vol.% isoflurane inhalation. The toe pinch reflex was used to assure adequate anaesthe-\nsia. Post-operative analgesia was assured with buprenorphine hydrochloride (0.03–0.05 mg/kg s.c.) every \n6 h for the first 24–48 h. Subsequently, buprenorphine hydrochloride was given twice daily during the first 3 \nweeks. Furthermore, in the first postoperative week, the drinking water was supplemented with metamizole \n(1 ml/300 ml). The animals were evaluated postoperatively three times per day for signs of acute deterioration.\nFigure 5. Subgroup division and time points of analyses. Subdivision of study cohort in two study groups \n(NK1-R group and Control group) and subsequent division of animals according to performed analysis at \ndifferent time points. Every analysis is performed in 6 animals of both study groups at every time point. At DPO \n(days post-operative) 42 both µCT imaging and biomechanical testing was performed in the same animals.\n\n8Scientific  RepoRts  |          (2019) 9:9744  | https://doi.org/10.1038/s41598-019-46278-6\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nStandardized femoral fracture. After anaesthesia induction, the animals were placed on a heated pad \n(37 °C) and their eyes were covered with moistening ointment. The right rear leg was shaved, disinfected, and \ndraped. Then, a para-patellar incision was made, the patella was everted laterally and a 1.0 mm stainless-steel \nintramedullary Kirschner wire was inserted in a retrograde manner. Its placement was confirmed by fluoroscopy. \nThe K-wire was cut flush with the intercondylar notch and the proximal end was bent over the greater trochanter, \ncut, and hidden subcutaneously. The patella was repositioned and the wounds were closed in layers. Fracture \ninduction was performed with a blunt guillotine according to the method of Bonnarens and Einhorn35. A fluoro-\nscopic evaluation of the fracture site was performed.\nSubstance P receptor blockage.  Substance P activity was blocked through selective blockage of the \nneurokinin-1-tachykinin-receptor (NK1-R) by subcutaneous administration of SR140333 once daily for the first \n14 days, beginning 30 min preoperatively. This period was chosen because it is the most vulnerable period in the \nfracture healing process in rats in terms of gene expression and the influence of cytokines, chemokines, growth \nfactors, etc\n36,37. SR140333 is a non-peptide antagonist of tachykinin NK1 receptors that potently, selectively, and \ncompetitively inhibits substance P binding to NK1 receptors38. SR140333 was administered at a concentration of \n1 mg/kg dissolved in a vehicle of 10 μl DMSO (dimethyl sulfoxide) and 0.2 ml of sterile water30,39. The animals of \nthe control group received the same amount of vehicle for the same period.\nGene expression.  The up-regulation of most genes associated with fracture healing in rats takes place in \nthe first two weeks after fracture induction 36,37; therefore, gene expression was measured at 3, 7, and 14 days \npost-operative (DPO).\nAt each time point, 6 animals from each group (NK1-R group and control group) were euthanized with an \noverdose isoflurane and subsequent cervical dislocation. The intramedullary K-wire was removed and bone and \ncallus at 0.5 cm on each side of the fracture was harvested, frozen in liquid nitrogen, pulverized, and used for RNA \nextraction. The fold changes of osteocalcin, collagen 1A2, and collagen 2A1 mRNA expression were determined \nby the reverse transcription polymerase chain reaction (RT-PCR). The cDNA was transcribed from total RNA \nusing a Maxima H Minus cDNA synthesis kit (Thermo Scientific, US). Real-time quantitative RT-PCR was per-\nformed with Power SYBR Green Master Mix (Applied Biosystems, US) using the StepOnePlus\n™ Real-Time PCR \nSystem (Invitrogen, CA, USA). The 2−ΔΔC T method (a method to analyse the relative changes in gene expression \nfrom real-time qPCR experiments) was used to calculate gene expression with peptidylprolyl cis-trans isomerase \n(PPIA) as an internal housekeeping gene reference. The NK1-R and Control groups were compared for their \nexpression of collagen-1A2, collagen-2A1, and osteocalcin mRNA.\nHistological analysis.  At DPO 28, 10 animals underwent histological analysis (6 of the α NK1-R group \nand 4 of the Control group, due to drop out of 2 animals, see Results  section). The animals were euthanized, \nthe Kirschner wire was removed, and the femora were harvested and fixed in 4% neutral buffered formalin for \n48 hours. Decalcification was performed with ethylenediaminetetraacetic acid (EDTA) and dehydration with \nalcohol. The tissue was embedded in paraffin and 10μm sagittal sections of the callus area and the surrounding \nsoft tissues were cut. The sections were stained with haematoxylin eosin (HE) and assessed with an Olympus BHS \nSystem Microscope. Images were viewed at 40× and 200× magnification, and measurements of the bone were \nperformed.\nMicro-computed tomography (μ CT). Micro-CT imaging was obtained at DPO 42 in 6 euthanized \nanimals from each group using a dual-energy gantry-based flat-panel microcomputed tomography scanner \n(TomoScope 30 s Duo, CT Imaging, Erlangen, Germany). The dual-energy X-ray tubes of the μCT were operated \nat voltages of 40 and 65 kV , with currents of 1.0 and 0.5 mA, respectively. Coverage of the entire leg of the rats was \nachieved by performing three sub-scans; each sub-scan acquired 720 projections with 1,032 × 1,012 pixels during \none full rotation, with durations of 90 s. After acquisition, volumetric data sets were reconstructed using a modi-\nfied Feldkamp algorithm with a smooth kernel at an isotropic voxel size of 35 μm. The bone, K-wire, and fracture \ncallus regions were segmented using an automated segmentation method with interactive correction of segmen-\ntation errors (Software Imalytics Preclinical\n40). (Fig. 2) The total bone volume, callus volume, bone density, callus \ndensity, and transverse diameter of the fracture callus were analysed quantitatively. The mid-part of the femoral \nshaft of the unfractured side of the animals from the Control group were taken for reference.\nBiomechanical testing. Biomechanical testing was performed at two time points in the healing process to \nanalyse the strength of the callus and/or newly formed bone. The first test was performed at DPO 42 in 6 animals \nof both groups; the second test was performed at DPO 84 in 6 animals of both groups. These time points were \nchosen because normal callus formation is completed after 6 weeks and the remodelling process is advanced or \ncompleted after 3 months. The fractured right as well as the unfractured left femur were tested in all animals. \nAfter euthanizing the animals and removing the K-wires, both ends of the femora were embedded, closely to \nthe callus/fracture site in a two-component resin (Technovit\n® 3040 powder + Technovit® Universal Liquid)41,42, \nwhich quickly hardens at low temperature. The embedded femora were tested in the biomechanical testing device \n(Retroline from Zwick Roell AG, Germany), over two cardan yokes, by which the force was conducted perpen-\ndicular to the femur axis. The biomechanical traction test was performed with a traction rate of 1 mm/s = 0.1 N/s \nand a measurement interval of 0.1 s. Digital set-up and control was performed using TestXpert II software (Zwick \nRoell AG, Germany), which enabled real-time measurement of the traction force. The obtained parameters were \nused for the calculation of average load to failure in Newton (N).\nData analysis. The data were analysed using the Statistical Package for the Social Sciences (SPSS; version 22; \nIBM Inc., Somers, NY , USA) and GraphPad Prism 5.0 (San Diego, CA, USA). Continuous data are presented as \n\n9Scientific  RepoRts  |          (2019) 9:9744  | https://doi.org/10.1038/s41598-019-46278-6\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nmean ± standard deviation, while incidences are presented as counts and percentages. Differences between the \ngroups were evaluated with a two-tailed unpaired Student’s t-test and with analysis of variance (ANOV A with post \nhoc Tukey) for continuous data. In general, Pearson’s χ2-test was used for categorical values. The gene expression \nand biomechanics data were analysed with the Mann–Whitney U test as a non-parametrical test. Potential statis-\ntical associations were evaluated with Pearson’s correlation. In general, a two sided p-value < 0.05 was considered \nstatistically significant.\nProofreading. Scribendi Inc. (Chatham, Ontario N7M 0N3 Canada) performed the proofreading of our \nmanuscript.\nData Availability\nThe datasets generated during and/or analysed during the current study are available from the corresponding \nauthor on reasonable request.\nReferences\n 1. Cherruau, M., Facchinetti, P ., Baroukh, B. & Saffar, J. L. Chemical sympathectomy impairs bone resorption in rats: a role for the \nsympathetic system on bone metabolism. Bone 25, 545–551 (1999).\n 2. Lerner, U. H. Neuropeptidergic regulation of bone resorption and bone formation. Journal of musculoskeletal & neuronal interactions \n2, 440–447 (2002).\n 3. Santavirta, S. et al. Immunologic studies of nonunited fractures. 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J.A.: performed histological analyses. \nD.M. and T.L.: performed and analyzed μCT-imaging; A.K.: performed biomechanical analyses. P .K.: Study design \nand interpretation of data. J.G.: performed anesthesia, operations, gene expression studies, and biomechanical \nanalyses; M.T.: performed anesthesia, operations, M.P . and F .H.: revised the paper. 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