The TrkB agonist, 7,8-dihydroxyflavone, impairs fracture healing in mice.

OBJECTIVES: To study the effects of the selective TrkB agonist, 7,8-dihydroxyflavone (7,8-DHF), on fracture healing in mice and on an osteoprogenitor cell line, Kusa4b10, in vitro. METHODS: Mice received unilateral closed mid-shaft tibial fractures and treated for two weeks with vehicle or 5 mg/kg/day DHF and euthanised at 28 days post-fracture. Calluses were analysed by micro-computed tomography (µCT) and three-point bending biomechanical test. Kusa4b10 cells were cultured with 50nM of 7,8-DHF or vehicle for 3-, 7-, 14-days for RT-PCR, and 21 days for mineralization. RESULTS: µCT found 7,8-DHF calluses had decreased tissue volume (p=0.042), mean polar moment of inertia (p = 0.004), and mean cross-sectional area (p=0.042) compared to controls. At 28 days biomechanical analyses showed 7,8-DHF treatment decreased peak force (p=0.011) and stiffness per unit area (p=0.012). 7,8-DHF treatment did not change Kusa4b10 gene expression of Runx2 and alkaline phosphatase at all time points, nor mineralization. CONCLUSIONS: 7,8-DHF treatment had a negative impact on fracture healing at 28 days post-fracture via an unknown mechanism. 7,8-DHF may have had a central role in impairing fracture healing.

Teaching students to explain the pathophysiology of diseases to lay audiences with a scaffold that supports student choice.

Expert educators in science argue that science graduates are often lacking skills in effectively communicating scientific information to lay audiences. To address this, we designed a project, Communicating Disease, for final-year undergraduate human pathophysiology students. Students chose a disease, a relevant nonscientific target audience, and a mode of communication and produced a communiqué designed to educate the audience on the pathophysiology of the disease. Separately, students justified their choice of disease and target audience. Upon completion of the project, students completed an anonymous questionnaire, and their submitted work was analyzed. Our study demonstrated that students thought it was important to learn how to effectively communicate science to a lay audience and felt that the project had supported them in developing knowledge and skills that enabled them to do so. Students were adequately challenged, and most students gave their best effort to the project, indicating a high level of engagement. Evaluation of student performance was consistent with the students' own perceptions and showed that most students communicated the pathophysiology effectively to the target audience and appropriately justified their choice of disease and target audience. Nevertheless, opportunities for improvement with some aspects of communication, production quality, and creativity were evident. This model is suitable for a range of scientific disciplines to engage students in developing their ability to communicate scientific information to lay audiences-a skill that it can be argued is vital for improving the scientific literacy of our community at large.

A novel rat model of heterotopic ossification after polytrauma with traumatic brain injury.

Neurological heterotopic ossification (NHO) is characterized by abnormal bone growth in soft tissue and joints in response to injury to the central nervous system. The ectopic bone frequently causes pain, restricts mobility, and decreases the quality of life for those affected. NHO commonly develops in severe traumatic brain injury (TBI) patients, particularly in the presence of concomitant musculoskeletal injuries (i.e. polytrauma). There are currently no animal models that accurately mimic these combinations of injuries, which has limited our understanding of NHO pathobiology, as well as the development of biomarkers and treatments, in TBI patients. In order to address this shortcoming, here we present a novel rat model that combines TBI, femoral fracture, and muscle crush injury. Young adult male Sprague Dawley rats were randomly assigned into three different injury groups: triple sham-injury, peripheral injury only (i.e., sham-TBI + fracture + muscle injury) or triple injury (i.e., TBI + fracture + muscle injury). Evidence of ectopic bone in the injured hind-limb, as confirmed by micro-computed tomography (µCT), was found at 6-weeks post-injury in 70% of triple injury rats, 20% of peripheral injury rats, and 0% of the sham-injured controls. Furthermore, the triple injury rats had higher ectopic bone severity scores than the sham-injured group. This novel model will provide a platform for future studies to identify underlying mechanisms, biomarkers, and develop evidence based pharmacological treatments to combat this debilitating long-term complication of TBI and polytrauma.

Meta-Analysis of Grainyhead-Like Dependent Transcriptional Networks: A Roadmap for Identifying Novel Conserved Genetic Pathways.

: The Drosophilagrainyhead (grh) and vertebrate Grainyhead-like (Grhl) transcription factors are among the most critical genes for epithelial development, maintenance and homeostasis, and are remarkably well conserved from fungi to humans. Mutations affecting grh/Grhl function lead to a myriad of developmental and adult onset epithelial disease, such as aberrant skin barrier formation, facial/palatal clefting, impaired neural tube closure, age-related hearing loss, ectodermal dysplasia, and importantly, cancers of epithelial origin. Recently, mutations in the family member GRHL3 have been shown to lead to both syndromic and non-syndromic facial and palatal clefting in humans, particularly the genetic disorder Van Der Woude Syndrome (VWS), as well as spina bifida, whereas mutations in mammalian Grhl2 lead to exencephaly and facial clefting. As transcription factors, Grhl proteins bind to and activate (or repress) a substantial number of target genes that regulate and drive a cascade of transcriptional networks. A multitude of large-scale datasets have been generated to explore the grh/Grhl-dependent transcriptome, following ablation or mis-regulation of grh/Grhl-function. Here, we have performed a meta-analysis of all 41 currently published grh and Grhl RNA-SEQ, and microarray datasets, in order to identify and characterise the transcriptional networks controlled by grh/Grhl genes across disparate biological contexts. Moreover, we have also cross-referenced our results with published ChIP and ChIP-SEQ datasets, in order to determine which of the critical effector genes are likely to be direct grh/Grhl targets, based on genomic occupancy by grh/Grhl genes. Lastly, to interrogate the predictive strength of our approach, we experimentally validated the expression of the top 10 candidate grhl target genes in epithelial development, in a zebrafish model lacking grhl3, and found that orthologues of seven of these (cldn23,ppl, prom2, ocln, slc6a19, aldh1a3, and sod3) were significantly down-regulated at 48 hours post-fertilisation. Therefore, our study provides a strong predictive resource for the identification of putative grh/grhl effector target genes.

Treadmill Exercise before and during Pregnancy Improves Bone Deficits in Pregnant Growth Restricted Rats without the Exacerbated Effects of High Fat Diet.

Growth restriction programs adult bone deficits and increases the risk of obesity, which may be exacerbated during pregnancy. We aimed to determine if high-fat feeding could exacerbate the bone deficits in pregnant growth restricted dams, and whether treadmill exercise would attenuate these deficits. Uteroplacental insufficiency was induced on embryonic day 18 (E18) in Wistar Kyoto (WKY) rats using bilateral uterine vessel ligation (restricted) or sham (control) surgery. The F1 females consumed a standard or high-fat (HFD) diet from 5 weeks, commenced treadmill exercise at 16 weeks, and they were mated at 20 weeks. Femora and plasma from the pregnant dams were collected at post-mortem (E20) for peripheral quantitative computed tomography (pQCT), mechanical testing, histomorphometry, and plasma analysis. Sedentary restricted females had bone deficits compared to the controls, irrespective of diet, where such deficits were prevented with exercise. Osteocalcin increased in the sedentary restricted females compared to the control females. In the sedentary HFD females, osteocalcin was reduced and CTX-1 was increased, with increased peak force and bending stress compared to the chow females. Exercise that was initiated before and continued during pregnancy prevented bone deficits in the dams born growth restricted, whereas a HFD consumption had minimal bone effects. These findings further highlight the beneficial effects of exercise for individuals at risk of bone deficits.

The selective TrkA agonist, gambogic amide, promotes osteoblastic differentiation and improves fracture healing in mice.

OBJECTIVES: To study effects of the selective TrkA agonist, gambogic amide (GA), on fracture healing in mice and on an osteoprogenitor cell line in vitro. METHODS: Mice were given bilateral fibular fractures and treated for two weeks with vehicle or 1 mg/kg/day GA and euthanized at 14-, 21-, and 42-days post-fracture. Calluses were analysed by micro-computed tomography (µCT), three-point bending and histology. For RT-PCR analyses, Kusa O cells were treated with 0.5nM of GA or vehicle for 3, 7, and 14 days, while for mineralization assessment, cells were treated for 21 days. RESULTS: µCT analysis found that 21-day GA-treated calluses had both decreased tissue volume (p<0.05) and bone surface (p<0.05) and increased fractional bone volume (p<0.05) compared to controls. Biomechanical analyses of 42-day calluses revealed that GA treatment increased stiffness per unit area by 53% (p<0.01) and load per unit area by 52% (p<0.01). GA treatment increased Kusa O gene expression of alkaline phosphatase and osteocalcin (p<0.05) by 14 days as well as mineralization at 21 days (p<0.05). CONCLUSIONS: GA treatment appeared to have a beneficial effect on fracture healing at 21- and 42-days post-fracture. The exact mechanism is not yet understood but may involve increased osteoblastic differentiation and matrix mineralization.

Roles of neurotrophins in skeletal tissue formation and healing.

Neurotrophins and their receptors are key molecules that are known to be critical in regulating nervous system development and maintenance and have been recognized to be also involved in regulating tissue formation and healing in skeletal tissues. Studies have shown that neurotrophins and their receptors are widely expressed in skeletal tissues, implicated in chondrogenesis, osteoblastogenesis, and osteoclastogenesis, and are also involved in regulating tissue formation and healing events in skeletal tissue. Increased mRNA expression for neurotrophins NGF, BDNF, NT-3, and NT-4, and their Trk receptors has been observed in injured bone tissues, and NT-3 and its receptor, TrkC, have been identified to have the highest induction at the injury site in a drill-hole injury repair model in both bone and the growth plate. In addition, NT-3 has also recently been shown to be both an osteogenic and angiogenic factor, and this neurotrophin can also enhance expression of the key osteogenic factor, BMP-2, as well as the major angiogenic factor, VEGF, to promote bone formation, vascularization, and healing of the injury site. Further studies, however, are needed to investigate if different neurotrophins have differential roles in skeletal repair, and if NT-3 can be a potential target of intervention for promoting bone fracture healing.

Gambogic amide, a selective TrkA agonist, does not improve outcomes from traumatic brain injury in mice.

OBJECTIVES: There is evidence that treatment with nerve growth factor (NGF) may reduce neuroinflammation and apoptosis after a traumatic brain injury (TBI). NGF is thought to exert its effects via binding to either TrkA or p75 neurotrophin receptors. This study aimed to investigate the effects of a selective TrkA agonist, gambogic amide (GA), on TBI pathology and outcomes in mice following lateral fluid percussion injury. METHODS: Male C57BL/6 mice were given either a TBI or sham injury, and then received subcutaneous injections of either 2 mg/kg of GA or vehicle at 1, 24, and 48 h post-injury. Following behavioural studies, mice were euthanized at 72 h post-injury for analysis of neuroinflammatory, apoptotic, and neurite outgrowth markers. RESULTS: Behavioural testing revealed that GA did not mitigate motor deficits after TBI. TBI caused an increase in cortical and hippocampal expression of several markers of neuroinflammation and apoptosis compared to sham groups. GA treatment did not attenuate these increases in expression, possibly contributed to by our finding of TrkA receptor down-regulation post-TBI. CONCLUSIONS: These findings suggest that GA treatment may not be suitable for attenuating TBI pathology and improving outcomes.

Closed head experimental traumatic brain injury increases size and bone volume of callus in mice with concomitant tibial fracture.

Concomitant traumatic brain injury (TBI) and long bone fracture are commonly observed in multitrauma and polytrauma. Despite clinical observations of enhanced bone healing in patients with TBI, the relationship between TBI and fracture healing remains poorly understood, with clinical data limited by the presence of several confounding variables. Here we developed a novel trauma model featuring closed-skull weight-drop TBI and concomitant tibial fracture in order to investigate the effect of TBI on fracture healing. Male mice were assigned into Fracture + Sham TBI (FX) or Fracture + TBI (MULTI) groups and sacrificed at 21 and 35 days post-injury for analysis of healing fractures by micro computed tomography (µCT) and histomorphometry. µCT analysis revealed calluses from MULTI mice had a greater bone and total tissue volume, and displayed higher mean polar moment of inertia when compared to calluses from FX mice at 21 days post-injury. Histomorphometric results demonstrated an increased amount of trabecular bone in MULTI calluses at 21 days post-injury. These findings indicate that closed head TBI results in calluses that are larger in size and have an increased bone volume, which is consistent with the notion that TBI induces the formation of a more robust callus.

Experimental Traumatic Brain Injury Induces Bone Loss in Rats.

Few studies have investigated the influence of traumatic brain injury (TBI) on bone homeostasis; however, pathophysiological mechanisms involved in TBI have potential to be detrimental to bone. The current study assessed the effect of experimental TBI in rats on the quantity and quality of two different weight-bearing bones, the femur and humerus. Rats were randomly assigned into either sham or lateral fluid percussion injury (FPI) groups. Open-field testing to assess locomotion was conducted at 1, 4, and 12 weeks post-injury, with the rats killed at 1 and 12 weeks post-injury. Bones were analyzed using peripheral quantitative computed tomography (pQCT), histomorphometric analysis, and three-point bending. pQCT analysis revealed that at 1 and 12 weeks post-injury, the distal metaphyseal region of femora from FPI rats had reduced cortical content (10% decrease at 1 week, 8% decrease at 12 weeks; p < 0.01) and cortical thickness (10% decrease at 1 week, 11% decrease at 12 weeks p < 0.001). There was also a 23% reduction in trabecular bone volume ratio at 1 week post-injury and a 27% reduction at 12 weeks post-injury in FPI rats compared to sham (p < 0.001). There were no differences in bone quantity and mechanical properties of the femoral midshaft between sham and TBI animals. There were no differences in locomotor outcomes, which suggested that post-TBI changes in bone were not attributed to immobility. Taken together, these findings indicate that this rat model of TBI was detrimental to bone and suggests a link between TBI and altered bone remodeling.

Tibial fracture exacerbates traumatic brain injury outcomes and neuroinflammation in a novel mouse model of multitrauma.

Multitrauma is a common medical problem worldwide, and often involves concurrent traumatic brain injury (TBI) and bone fracture. Despite the high incidence of combined TBI and fracture, preclinical TBI research commonly employs independent injury models that fail to incorporate the pathophysiologic interactions occurring in multitrauma. Here, we developed a novel mouse model of multitrauma, and investigated whether bone fracture worsened TBI outcomes. Male mice were assigned into four groups: sham-TBI+sham-fracture (SHAM); sham-TBI+fracture (FX); TBI+sham-fracture (TBI); and TBI+fracture (MULTI). The injury methods included a closed-skull weight-drop TBI model and a closed tibial fracture. After a 35-day recovery, mice underwent behavioral testing and magnetic resonance imaging (MRI). MULTI mice displayed abnormal behaviors in the open-field compared with all other groups. On MRI, MULTI mice had enlarged ventricles and diffusion abnormalities compared with all other groups. These changes occurred in the presence of heightened neuroinflammation in MULTI mice at 24 hours and 35 days after injury, and elevated edema and blood-brain barrier disruption at 24 hours after injury. Together, these findings indicate that tibial fracture worsens TBI outcomes, and that exacerbated neuroinflammation may be an important factor that contributes to these effects, which warrants further investigation.

Osteoclast formation elicited by interleukin-33 stimulation is dependent upon the type of osteoclast progenitor.

Osteoclasts are bone resorbing multinucleated cells (MNCs) derived from macrophage progenitors. IL-33 has been reported to drive osteoclastogenesis independently of receptor activator of NFκB ligand (RANKL) but this remains controversial as later studies did not confirm this. We found IL-33 clearly elicited functional dentine-resorbing osteoclast formation from human adult monocytes. However, monocytes from only 3 of 12 donors responded this way, while all responded to RANKL. Human cord blood-derived progenitors and murine bone marrow macrophages lacked an osteoclastogenic response to IL-33. In RAW264.7 cells, IL-33 elicited NFκB and p38 responses but not NFATc1 signals (suggesting poor osteoclastogenic responses) and formed only mononuclear tartrate-resistant acid phosphatase positive (TRAP(+)) cells. Since TGFß boosts osteoclastogenesis in RAW264.7 cells we employed an IL-33/TGFß co-treatment, which resulted in small numbers of MNCs expressing key osteoclast markers TRAP and calcitonin receptors. Thus, IL-33 possesses weak osteoclastogenic activity suggesting pathological significance and, perhaps, explaining previous conflicting reports.

Thymosin ß4 administration enhances fracture healing in mice.

Thymosin ß4 (Tß4 ) is a regenerative peptide that we hypothesized would promote healing of fractured bone. Mice received a bilateral fibular osteotomy and were given i.p. injections of either Tß4 (6 mg/kg) or saline. Calluses from saline- and Tß4 -treated mice were analyzed for: (1) biomechanical properties and (2) composition using micro-computed tomography (µCT) and histomorphometry. Biomechanical analysis showed that Tß4 -treated calluses had a 41% increase in peak force to failure (p < 0.01) and were approximately 25% stiffer (p < 0.05) than saline-treated controls. µCT analysis at 21 days post-fracture showed that the fractional volume of new mineralized tissue and new highly mineralized tissue were respectively 18% and 26% greater in calluses from Tß4 -treated mice compared to controls (p < 0.01; p < 0.05, respectively). Histomorphometry complemented the µCT data; at 21 days post-fracture, Tß4 -treated calluses were almost 23% smaller (p < 0.05), had nearly 47% less old cortical bone (p < 0.05) and had a 31% increase in new trabecular bone area/total callus area fraction compared with controls (p < 0.05). Our finding of enhanced biomechanical properties of fractures in mice treated with Tß4 provides novel evidence of the therapeutic potential of this peptide for treating bone fractures.

Transient expression of myofibroblast-like cells in rat rib fracture callus.

BACKGROUND AND PURPOSE: We have previously shown that early fracture callus of rat rib has viscoelastic and contractile properties resembling those of smooth muscle. The cells responsible for this contractility have been hypothesized to be myofibroblast-like in nature. In soft-tissue healing, force generated by contraction of myofibroblasts promotes healing. Accordingly, we tried to identify myofibroblast-like cells in early fibrous callus. ANIMALS AND METHODS: Calluses from rat rib fractures were removed 7, 14, and 21 days after fracture and unfractured ribs acted as controls. All tissues were analyzed using qPCR and immunohistochemistry. We analyzed expression of smooth muscle- and myofibroblast-associated genes and proteins including alpha smooth muscle actin (αSMA), non-muscle myosin, fibronectin extra domain A variant (EDA-fibronectin), OB-cadherin, connexin-43, basic calponin (h1CaP), and h-caldesmon. RESULTS: In calluses at 7 days post-fracture, there were statistically significant increases in expression of αSMA mRNA (2.5 fold), h1CaP mRNA (2.1 fold), EDA-fibronectin mRNA (14 fold), and connexin-43 mRNA (1.8 fold) compared to unfractured ribs, and by 21 days post-fracture mRNA expression in calluses had decreased to levels approaching those in unfractured rib. Immunohistochemistry of 7 day fibrous callus localized calponin, EDA-fibronectin and co-immunolabeling of OB-cadherin and αSMA (thus confirming a myofibroblastic phenotype) within various cell populations. INTERPRETATION: This study provides further evidence that early rat rib callus is not only smooth muscle-like in nature but also contains a notable population of cells that have a distinct myofibroblastic phenotype. The presence of these cells indicates that in vivo contraction of early callus is a mechanism that may occur in fractures so as to facilitate healing, as it does in soft tissue wound repair.

α(1) adrenergic receptor agonist, phenylephrine, actively contracts early rat rib fracture callus ex vivo.

Early, soft fracture callus that links fracture ends together is smooth muscle-like in nature. We aimed to determine if early fracture callus could be induced to contract and relax ex vivo by similar pathways to smooth muscle, that is, contraction via α(1) adrenergic receptor (α(1) AR) activation with phenylephrine (PE) and relaxation via ß(2) adrenergic receptor (ß(2) AR) stimulation with terbutaline. A sensitive force transducer quantified 7 day rat rib fracture callus responses in modified Krebs-Henseliet (KH) solutions. Unfractured ribs along with 7, 14, and 21 day fracture calluses were analyzed for both α(1) AR and ß(2) AR gene expression using qPCR, whilst 7 day fracture callus was examined via immunohistochemistry for both α(1) AR and ß(2) AR- immunoreactivity. In 7 day callus, PE (10(-6) M) significantly induced an increase in force that was greater than passive force generated in calcium-free KH (n = 8, mean 51% increase, 95% CI: 26-76%). PE-induced contractions in calluses were attenuated by the α(1) AR antagonist, prazosin (10(-6) M; n = 7, mean 5% increase, 95% CI: 2-11%). Terbutaline did not relax callus. Gene expression of α(1) ARs was constant throughout fracture healing; however, ß(2) AR expression was down-regulated at 7 days compared to unfractured rib (p < 0.01). Furthermore, osteoprogenitor cells of early fibrous callus displayed considerable α(1) AR-like immunoreactivity but not ß(2) AR-like immunoreactivity. Here, we demonstrate for the first time that early fracture callus can be pharmacologically induced to contract. We propose that increased concentrations of α(1) AR agonists such as noradrenaline may tonically contract callus in vivo to promote osteogenesis.

Early fracture callus displays smooth muscle-like viscoelastic properties ex vivo: implications for fracture healing.

Cells of early, fibrous callus in bone fractures possess much alpha smooth muscle actin. This callus contracts and relaxes; however, active and passive components of its force production have yet to be defined. We aimed to establish whether passive viscoelastic properties of early soft fracture callus are smooth muscle-like in nature. Under anesthesia one rib was fractured in rats and calluses removed 7 days later for analysis. Urinary bladder detrusor muscle and Achilles tendon were also resected and analyzed. Force production in these tissues was measured using a force transducer when preparations were immersed in calcium-free Krebs-Henseleit solution (pH 7.4, 22 degrees C). Viscoelastic responses were measured in each preparation in response to 50 microN increases and decreases in force after achieving basal tissue tension by preconditioning. Callus, bladder, and tendon all displayed varying, reproducible degrees of stress relaxation (SR) and reverse stress relaxation (RSR) (n = 7 for all groups). Hysteresis was observed in callus, with the first SR response significantly larger than that produced in subsequent stretches (p < 0.05). Callus SR responses were greater than tendon (p < 0.001) but less than bladder (p < 0.001). Callus RSR responses were greater than tendon (p < 0.001), but no significant difference was seen between RSR of callus and bladder. We concluded that early, soft callus displayed significant SR and RSR phenomena similar to smooth muscle tissue, and SR and RSR may be important in maintenance of static tension in early callus by promoting osteogenesis and fracture healing.

Early callus of fractured rib of rat contracts and relaxes ex vivo.

PURPOSE: Wound contraction is an essential process in early soft-tissue repair, yet contraction of callus in fracture repair has not been investigated previously. Fracture callus consists of several cell types, many of which may have the capacity to contract. Accordingly, the purpose of the present study was to (i) determine whether early soft fracture calluses contract and relax ex vivo and (ii) identify and locate the contractile protein, alpha smooth muscle actin (alphaSMA) in callus. METHODS: One non-weight-bearing rib was fractured in adult male rats under anaesthesia and 10 calluses were removed 5, 7 and 9 days later for examination. Force production by calluses was measured using a sensitive force transducer when callus preparations were immersed sequentially in solutions known to either contract or relax smooth muscle preparations. Calluses and unfractured rib were analysed for the presence of alphaSMA using Western Blot and immunohistochemical techniques. RESULTS: When immersed in normal Krebs-Henseleit solution (K-H; pH 7.4, 22 degrees C) 7 callus preparations contracted and 3 relaxed. The force response was phasic (3 calluses) or tonic (7 calluses). Subsequent immersion in Ca(2+)-free K-H resulted in no change in force in 4 calluses, a decrease in force (relaxation) in 3 calluses, and an increase in force (contraction) in 2 calluses when compared to the force in the preceding solution (K-H). The final incubation in a solution having a high [K+] (64 mM) partially relaxed 6 calluses, contracted 3 and produced no change in force in 1 callus compared to the final force of the callus in the Ca(2+)-free solution. Collagen (in the form of rat Achilles tendon), the major structural protein in soft fracture callus, relaxed in K-H and continued to relax during exposure to Ca(2+)-free K-H and to solutions having a high [K+]. Western Blot and immunohistochemical studies detected the presence of alphaSMA in calluses and (in particular) in osteoprogenitor cells of fibrous callus respectively, as well as its absence from unfractured rib. CONCLUSIONS: (i) Early, soft fracture callus is capable of contracting and relaxing, (ii) the responses of callus to K-H, Ca(2+)-free and high [K+] solutions are distinctly different from the responses of smooth muscle preparations reported in the literature, (iii) the cell types in callus, particularly osteoprogenitor cells in uncalcified, collagenous matrix, have an essential contractile protein, alphaSMA, to support the observed contraction and relaxation and (iv) the contraction of soft fracture callus may facilitate fracture repair by creating tension within the callus and drawing the fracture ends together.

Expression of galanin and galanin receptor-1 in normal bone and during fracture repair in the rat.

The neuropeptide galanin (GAL) has recognized physiological actions in the nervous system and other tissues, but there is no documented evidence of GAL influencing normal or pathological bone metabolism. GAL expression, however, is upregulated in central and peripheral nerves following axotomy and is known to influence neural regeneration. Thus, severance of skeletal-associated nerves during fracture could similarly increase local GAL concentrations and thereby influence fracture healing. The initial aim of this study was therefore to identify the presence of GAL in normal bone and/or fracture callus by assessing the concentration and cellular localization of GAL in intact and/or fractured rat rib, using radioimmunoassay and immunohistochemistry, respectively. Groups of Sprague-Dawley rats (13 weeks old) had their left sixth ribs surgically fractured or underwent sham surgery and then calluses and nonfractured rib samples were analyzed at 1 and 2 weeks postsurgery (n = 5-6 per group). Low (basal) concentrations of GAL were detected in control ribs, whereas at 1 and 2 weeks postfracture, callus samples contained markedly increased levels of peptide ( approximately 32- and 18-fold increase, respectively, relative to controls; P < 0.01), revealing a strong upregulation during bone healing. Plasma GAL concentrations were also increased at 2 weeks postfracture (P < 0.005). In normal (nonfractured) rib, minimal levels of GAL-like immunoreactivity (LI) were present in cortical bone, periosteum, endosteum, and surrounding skeletal muscle. In costal cartilage plates, intense GAL-LI was present in all chondrocytes of the hypertrophic zone and in a population of chondrocytes in the reserve zone. GAL-LI was not present, however, in chondrocytes in the proliferative zone of costal cartilage or skeletal muscle fibers. In fracture callus, levels of GAL-LI were moderate to intense in osteoprogenitor cells and osteoblasts, in some chondrocytes, and in cartilaginous, osseous, and periosteal matrices. Subsequent studies revealed the presence of galanin receptor-1-like immunoreactivity (GALR1-LI) in most cell types shown to contain GAL-LI, although the distribution of GALR1-LI was more extensive in reserve zone chondrocytes than that of GAL-LI; and GALR1-LI also appeared in late proliferative zone chondrocytes of costal cartilage. In summary, GAL concentrations were significantly increased in fracture callus and plasma of rats that underwent rib fracture. In addition, GAL- and GALR1-LI was also detected in specific cells and structures within costal cartilage, bone, and fracture callus. These results strongly implicate GAL in aspects of cartilage growth plate physiology and fracture repair, possibly acting in an autocrine/paracrine fashion via GALR1.