Aaron Campbell. McDonald

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.

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°C). Viscoelastic responses were measured in each preparation in response to 50 µN 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.

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.

α1 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 α1 adrenergic receptor (α1AR) activation with phenylephrine (PE) and relaxation via β2 adrenergic receptor (β2AR) 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 α1AR and β2AR gene expression using qPCR, whilst 7 day fracture callus was examined via immunohistochemistry for both α1AR and β2AR‐ 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 α1AR antagonist, prazosin (10−6 M; n = 7, mean 5% increase, 95% CI: 2–11%). Terbutaline did not relax callus. Gene expression of α1ARs was constant throughout fracture healing; however, β2AR expression was down‐regulated at 7 days compared to unfractured rib (p < 0.01). Furthermore, osteoprogenitor cells of early fibrous callus displayed considerable α1AR‐like immunoreactivity but not β2AR‐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 α1AR agonists such as noradrenaline may tonically contract callus in vivo to promote osteogenesis.

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.

Practical session assessments in human anatomy: Weightings and performance.

Assessment weighting within a given module can be a motivating factor for students when deciding on their commitment level and time given to study a specific topic. In this study, an analysis of assessment performances of second year anatomy students was performed over four years to determine if (1) students performed better when a higher weighting was given to a set of practical session assessments and (2) whether an improved performance in the practical session assessments had a carry‐over effect on other assessment tasks within that anatomy module and/or other anatomy modules that follow. Results showed that increasing the weighting of practical session assessments improved the average mark in that assessment and also improved the percentage of students passing that assessment. Further, it significantly improved performance in the written end‐semester examination within the same module and had a carry‐over effect on the anatomy module taught in the next teaching period, as students performed better in subsequent practical session assessments as well as subsequent end‐semester examinations. It was concluded that the weighting of assessments had significant influences on a students performance in that, and subsequent, assessments. It is postulated that practical session assessments, designed to develop deep learning skills in anatomy, improved efficacy in student performance in assessments undertaken in that and subsequent anatomy modules when the weighting of these assessments was greater. These deep learning skills were also transferable to other methods of assessing anatomy. Anat Sci Educ 9: 330–336.

The effect of content delivery style on student performance in anatomy: Blended Learning and Assessment in Anatomy

The development of new technologies and ensuing pedagogical research has led many tertiary institutions to integrate and adopt online learning strategies. The authors of this study have incorporated online learning strategies into existing educational practices of a second year anatomy course, resulting in half of the course content delivered via face‐to‐face lectures, and half delivered online via tailored video vignettes, with accompanying worksheets and activities. The effect of the content delivery mode on student learning was analyzed by tailoring questions to content presented either face‐to‐face or online. Four practical tests were conducted across the semester with each consisting of four questions. Within each test, two questions were based on content delivered face‐to‐face, and two questions were based on content delivered online. Examination multiple choice questions were similarly divided and assessed. Findings indicate that student learning is consistent regardless of the mode of content delivery. However, student viewing habits had a significant impact on learning, with students who viewed videos multiple times achieving higher marks than those less engaged with the online content. Student comments also indicated that content delivery mode was not an influence on learning. Therefore student engagement, rather than the mode of content delivery, is a determinant of student learning and performance in human anatomy. Anat Sci Educ.