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.

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 student's 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. © 2015 American Association of Anatomists.

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.

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.

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.