title: Absence of substance P and the sympathetic nervous system impact on bone structure and chondrocyte differentiation in an adult model of endochondral ossification.
abstract: OBJECTIVE
  Sensory and sympathetic nerve fibers (SNF) innervate bone and epiphyseal growth plate. The role of neuronal signals for proper endochondral ossification during skeletal growth is mostly unknown. Here, we investigated the impact of the absence of sensory neurotransmitter substance P (SP) and the removal of SNF on callus differentiation, a model for endochondral ossification in adult animals, and on bone formation.
  
  
  METHODS
  In order to generate callus, tibia fractures were set in the left hind leg of wild type (WT), tachykinin 1-deficient (Tac1-/-) mice (no SP) and animals without SNF. Locomotion was tested in healthy animals and touch sensibility was determined early after fracture. Callus tissue was prepared for immunofluorescence staining for SP, neurokinin1-receptor (NK1R), tyrosine-hydroxylase (TH) and adrenergic receptors α1, α2 and β2. At the fracture site, osteoclasts were stained for TRAP, osteoblasts were stained for RUNX2, and histomorphometric analysis of callus tissue composition was performed. Primary murine bone marrow derived macrophages (BMM), osteoclasts, and osteoblasts were tested for differentiation, activity, proliferation and apoptosis in vitro. Femoral fractures were set in the left hind leg of all the three groups for mechanical testing and μCT-analysis.
  
  
  RESULTS
  Callus cells stained positive for SP, NK1R, α1d- and α2b adrenoceptors and remained β2-adrenoceptor and TH-negative. Absence of SP and SNF did not change the general locomotion but reduces touch sensitivity after fracture. In mice without SNF, we detected more mesenchymal callus tissue and less cartilaginous tissue 5 days after fracture. At day 13 past fracture, we observed a decrease of the area covered by hypertrophic chondrocytes in Tac1-/- mice and mice without SNF, a lower number of osteoblasts in Tac1-/- mice and an increase of osteoclasts in mineralized callus tissue in mice without SNF. Apoptosis rate and activity of osteoclasts and osteoblasts isolated from Tac1-/- and sympathectomized mice were partly altered in vitro. Mechanical testing of fractured- and contralateral legs 21 days after fracture, revealed an overall reduced mechanical bone quality in Tac1-/- mice and mice without SNF. μCT-analysis revealed clear structural alteration in contralateral and fractured legs proximal of the fracture site with respect to trabecular parameters, bone mass and connectivity density. Notably, structural parameters are altered in fractured legs when related to unfractured legs in WT but not in mice without SP and SNF.
  
  
  CONCLUSIONS
  The absence of SP and SNF reduces pain sensitivity and mechanical stability of the bone in general. The micro-architecture of the bone is profoundly impaired in the absence of intact SNF with a less drastic effect in SP-deficient mice. Both sympathetic and sensory neurotransmitters are indispensable for proper callus differentiation. Importantly, the absence of SP reduces bone formation rate whereas the absence of SNF induces bone resorption rate. Notably, fracture chondrocytes produce SP and its receptor NK1 and are positive for α-adrenoceptors indicating an endogenous callus signaling loop. We propose that sensory and sympathetic neurotransmitters have crucial trophic effects which are essential for proper bone formation in addition to their classical neurological actions.
authors: Department of Orthopaedic Surgery, University of Regensburg, Germany; Department of Orthopaedic Surgery, Experimental Orthopaedics, Centre for Medical Biotechnology, University of Regensburg, Germany.
authors: Tanja
authors: T
authors: Niedermair
authors: Department of Trauma Surgery, Medical University Innsbruck, Austria.
authors: Volker
authors: V
authors: Kuhn
authors: Department of Orthopaedic Surgery, University of Regensburg, Germany; Department of Orthopaedic Surgery, Experimental Orthopaedics, Centre for Medical Biotechnology, University of Regensburg, Germany.
authors: Fatemeh
authors: F
authors: Doranehgard
authors: Department of Trauma, Hand and Reconstructive Surgery, University Hospital, Münster, Germany.
authors: Richard
authors: R
authors: Stange
authors: Department of Trauma, Hand and Reconstructive Surgery, University Hospital, Münster, Germany.
authors: Britta
authors: B
authors: Wieskötter
authors: Department of Orthopaedic Surgery, University of Regensburg, Germany.
authors: Johannes
authors: J
authors: Beckmann
authors: Department of Trauma Surgery, Medical University Innsbruck, Austria.
authors: Philipp
authors: P
authors: Salmen
authors: Department of Orthopaedic Surgery, University of Regensburg, Germany.
authors: Hans-Robert
authors: HR
authors: Springorum
authors: Laboratory of Experimental Rheumatology and Neuroendocrine Immunology, Department of Internal Medicine I, University of Regensburg, Germany.
authors: Rainer H
authors: RH
authors: Straub
authors: Institute for Molecular Psychiatry, University of Bonn, Germany.
authors: Andreas
authors: A
authors: Zimmer
authors: Department of Orthopaedic Surgery, University of Regensburg, Germany.
authors: Joachim
authors: J
authors: Grifka
authors: Department of Orthopaedic Surgery, University of Regensburg, Germany; Department of Orthopaedic Surgery, Experimental Orthopaedics, Centre for Medical Biotechnology, University of Regensburg, Germany. Electronic address: susanne.graessel@klinik.uni-regensburg.de.
authors: Susanne
authors: S
authors: Grässel
mesh_terms: D000818:Animals; D001696:Biomechanical Phenomena; D002146:Bony Callus / Q000166:cytology / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology*; D019902:Chondrocytes / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D006652:Histological Techniques; D051379:Mice; D018345:Mice, Knockout; D010012:Osteogenesis / Q000502:physiology*; D013373:Substance P / Q000172:deficiency*; D013564:Sympathetic Nervous System / Q000473:pathology*
chemical_list: D013373:Substance P
keywords: Biomechanics; Bone formation; Bone resorption; Bone structure; Endochondral ossification; Fracture callus; Substance P; Sympathetic nerve fibers