Johannes A. Schuijers

Immunohistochemical localization of nerve growth factor in fractured and unfractured rat bone

We detected nerve growth factor (NGF) by immunohistochemical localization in both fractured and unfractured rat rib. In unfractured bone, periosteal mesenchymal osteoprogenitor cells appeared to be the only skeletal cells which stained for NGF. Adjacent skeletal muscle fibers exhibited NGF staining both in fractured and unfractured bone. Fracture callus periosteal osteoprogenitor cells, marrow stromal cells, osteoblasts, young osteocytes and endothelial cells of new capillaries had moderate to heavy staining for NGF at 1 and 3 weeks after fracture. Deeply positioned osteocytes and osteoclasts showed no NGF staining. Most chondrocytes of fracture calluses stained for NGF, however, some chondrocytes did not stain which may indicate that NGF is produced at particular stages of chondrocytic differentiation. In calluses, periosteal matrix stained heavily for NGF when juxtaposed to cartilage and less obviously when associated with new bone at both 1 and 3 weeks post-fracture. However, other fibrous, cartilaginous and osseous matrices did not stain for NGF at any time. At 6 weeks post-fracture, NGF staining was largely confined to periosteal osteoprogenitor cells. The detection of NGF in periosteal osteoprogenitor cells of unfractured rib points to these cells having a role in nerve maintenance in intact bone. Furthermore, the localization of NGF in osteoprogenitor cells, marrow stromal cells, osteoblasts, certain chondrocytes, endothelial cells, periosteal matrix of the fracture callus and skeletal muscle may mean that these entities participate in fracture innervation. The presence of NGF in the callus may also indicate a direct, as yet undefined action of this neurotrophin on skeletal cell metabolism.

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.

Glucocorticoid responses to stress in castrate and testosterone-replaced rams.

Stress is an important contributor to cardiovascular disease and to reduced immunity and fertility. As the role of androgens in stress is uncertain, we investigated the effects of testosterone (T) on hormonal responses to stress in conscious Romney Marsh wethers. Six T-treated sheep and six control sheep were stressed by exposure to a psychological and a metabolic stimulus. Baseline glucose levels were significantly lower in the treated animals compared with controls (p=0.002). T treatment significantly attenuated ACTH (p<0.01) and cortisol (p<0.05) responses to metabolic stress. Following psychological stress, ACTH responses were significantly lower in treated sheep compared with controls (p<0.05), but differences in mean cortisol responses did not reach significance. There were no significant differences in epinephrine or norepinephrine responses following either stressor. We conclude that T replacement in wethers lowers glucose and attenuates responses to metabolic and psychological stress. While the implications of these results for human physiology require further studies, they suggest that male hypogonadism may play a role in determining the risk of cardiovascular disease and diabetes.

Thymosin β4 administration enhances fracture healing in mice

Thymosin β4 (Tβ4) 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 Tβ4 (6 mg/kg) or saline. Calluses from saline‐ and Tβ4‐treated mice were analyzed for: (1) biomechanical properties and (2) composition using micro‐computed tomography (µCT) and histomorphometry. Biomechanical analysis showed that Tβ4‐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 Tβ4‐treated mice compared to controls (p < 0.01; p < 0.05, respectively). Histomorphometry complemented the µCT data; at 21 days post‐fracture, Tβ4‐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 Tβ4 provides novel evidence of the therapeutic potential of this peptide for treating bone fractures.

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 TGFβ boosts osteoclastogenesis in RAW264.7 cells we employed an IL-33/TGFβ 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.

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.

The effectiveness of separating theory and practicum as a conduit to learning physiology

Many conventional science courses contain subjects embedded with laboratory-based activities. However, research on the benefits of positioning the practicals within the theory subject or developing them distinctly from the theory is largely absent. This report compared results in a physiology theory subject among three different cohorts of students: those taking the theory subject alone, those taking it concurrent with a physiology practicum subject, and those who previously took the subject when it had practicums embedded within the one subject. The path model shows that students taking both physiology theory and physiology practicum attained a significantly higher result in online tests compared with those who took the theory subject alone (P < 0.05) and that this translated to a significantly higher result in the end-of-semester examination. Similarly, students taking both physiology theory and the physiology practicum attained a significantly higher end-examination result compared with those who took the physiology subject in previous years when the practicums were embedded within the theory subject (P < 0.05). In both cases, this increase was largely attained in components that tested critical thinking and deep learning (short theory application questions and extended written questions). We conclude that students undertaking both physiology theory and the physiology practicum likely performed better in the theory subject due to better problem-solving skills and a more developed understanding of theoretical content. We suggest that consideration be given in all science curricula to the separation of theory and practicum by developing two subjects with clearly defined different learning outcomes.

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.

91 Ciprofloxacin Reduces Proteoglycan Synthesis In Equine Tendon Explants

Introduction Ciprofloxacin, a commonly prescribed fluoroquinolone antibiotic, has been shown to have a deleterious effect on the tendon extracellular matrix. The aims of this study were to determine the effects of short term exposure of ciprofloxacin on 1) tenocyte metabolism, 2) glycosaminoglycan content, and 3) mRNA expression levels of extracellular proteoglycans, and to correlate these changes with the production of newly synthesised proteoglycans in explant cultures of equine tendon. Methods Normal superficial digital flexor tendon from 6 yr old Thoroughbred horses were incubated for 4 days in DMEM alone (control), or DMEM containing up to 300 µg/ml ciprofloxacin (CPX). The effect of CPX on the metabolism of tenocytes was determined by lactate production and Alamar blue assay and glycosaminoglycan content was determined by a dimethylene blue assay. Levels of gene expression of aggrecan, versican, decorin, biglycan and fibromodulin were determined by quantitative PCR. The production of newly synthesised proteoglycans was determined by 35S-sulfate incorporation at the end of the treatment period. Results Higher levels of CPX (≥100 µg/ml) reduced tenocyte metabolism by 30–40% as measured by lactate production and Alamar blue assay. There was no change in glycosaminoglycan content nor mRNA expression levels of aggrecan, versican, decorin, biglycan or fibromodiulin in CPX-treated tendon explants compared with control. However, CPX suppressed the synthesis of both 35S-labelled large and small proteoglycans in a dose dependent manner. Discussion The suppression of newly synthesised proteoglycans in tendon explants treated with CPX suggests that these changes are driven by changes in metabolism rather than expression of its associated genes.