A maternal brain hormone that builds bone.

In lactating mothers, the high calcium (Ca2+) demand for milk production triggers significant bone loss1. Although oestrogen normally counteracts excessive bone resorption by promoting bone formation, this sex steroid drops precipitously during this postpartum period. Here we report that brain-derived cellular communication network factor 3 (CCN3) secreted from KISS1 neurons of the arcuate nucleus (ARCKISS1) fills this void and functions as a potent osteoanabolic factor to build bone in lactating females. We began by showing that our previously reported female-specific, dense bone phenotype2 originates from a humoral factor that promotes bone mass and acts on skeletal stem cells to increase their frequency and osteochondrogenic potential. This circulatory factor was then identified as CCN3, a brain-derived hormone from ARCKISS1 neurons that is able to stimulate mouse and human skeletal stem cell activity, increase bone remodelling and accelerate fracture repair in young and old mice of both sexes. The role of CCN3 in normal female physiology was revealed after detecting a burst of CCN3 expression in ARCKISS1 neurons coincident with lactation. After reducing CCN3 in ARCKISS1 neurons, lactating mothers lost bone and failed to sustain their progeny when challenged with a low-calcium diet. Our findings establish CCN3 as a potentially new therapeutic osteoanabolic hormone for both sexes and define a new maternal brain hormone for ensuring species survival in mammals.

Engineered bone marrow as a clinically relevant ex vivo model for primary bone cancer research and drug screening.

Osteosarcoma (OS) is the most common primary malignant bone cancer in children and adolescents. While numerous other cancers now have promising therapeutic advances, treatment options for OS have remained unchanged since the advent of standard chemotherapeutics and offer less than a 25% 5-y survival rate for those with metastatic disease. This dearth of clinical progress underscores a lack of understanding of OS progression and necessitates the study of this disease in an innovative system. Here, we adapt a previously described engineered bone marrow (eBM) construct for use as a three-dimensional platform to study how microenvironmental and immune factors affect OS tumor progression. We form eBM by implanting acellular bone-forming materials in mice and explanting the cellularized constructs after 8 wk for study. We interrogate the influence of the anatomical implantation site on eBM tissue quality, test ex vivo stability under normoxic (5% O2) and standard (21% O2) culture conditions, culture OS cells within these constructs, and compare them to human OS samples. We show that eBM stably recapitulates the composition of native bone marrow. OS cells exhibit differential behavior dependent on metastatic potential when cultured in eBM, thus mimicking in vivo conditions. Furthermore, we highlight the clinical applicability of eBM as a drug-screening platform through doxorubicin treatment and show that eBM confers a protective effect on OS cells that parallel clinical responses. Combined, this work presents eBM as a cellular construct that mimics the complex bone marrow environment that is useful for mechanistic bone cancer research and drug screening.

Muscle satellite cells and fibro-adipogenic progenitors from muscle contractures of children with cerebral palsy have impaired regenerative capacity.

AIM: To evaluate the mechanosensitivity of muscle satellite cells (MuSCs) and fibro-adipogenic progenitors (FAPs) in cerebral palsy (CP) and the efficacy of the drug verteporfin in restoring cells' regenerative capacity. METHOD: Muscle biopsies were collected from six children with CP and six typically developing children. MuSCs and FAPs were isolated and plated on collagen-coated polyacrylamide gels at stiffnesses of 0.2 kPa, 8 kPa, and 25 kPa. Cells were treated with verteporfin to block mechanosensing or with dimethyl sulfoxide as a negative control. MuSC differentiation and FAP activation into myofibroblasts were measured using immunofluorescence staining. RESULTS: Surprisingly, MuSC differentiation was not affected by stiffness; however, stiff substrates resulted in large myonuclear clustering. Across all stiffnesses, MuSCs from children with CP had less differentiation than those of their typically developing counterparts. FAP activation into myofibroblasts was significantly higher in children with CP than their typically developing peers, but was not affected by stiffness. Verteporfin did not affect differentiation or activation in either cell population, but slightly decreased myonuclear clustering on stiff substrates. INTERPRETATION: Cells from children with CP were less regenerative and more fibrotic compared to those of their typically developing counterparts, with MuSCs being sensitive to increases in stiffness. Therefore, the mechanosensitivity of MuSCs and FAPs may represent a new target to improve differentiation and activation in CP muscle.

Skeletal muscle progenitors are sensitive to collagen architectural features of fibril size and cross linking.

Muscle stem cells (MuSCs) are essential for the robust regenerative capacity of skeletal muscle. However, in fibrotic environments marked by abundant collagen and altered collagen organization, the regenerative capability of MuSCs is diminished. MuSCs are sensitive to their extracellular matrix environment but their response to collagen architecture is largely unknown. The present study aimed to systematically test the effect of underlying collagen structures on MuSC functions. Collagen hydrogels were engineered with varied architectures: collagen concentration, cross linking, fibril size, and fibril alignment, and the changes were validated with second harmonic generation imaging and rheology. Proliferation and differentiation responses of primary mouse MuSCs and immortal myoblasts (C2C12s) were assessed using EdU assays and immunolabeling skeletal muscle myosin expression, respectively. Changing collagen concentration and the corresponding hydrogel stiffness did not have a significant influence on MuSC proliferation or differentiation. However, MuSC differentiation on atelocollagen gels, which do not form mature pyridinoline cross links, was increased compared with the cross-linked control. In addition, MuSCs and C2C12 myoblasts showed greater differentiation on gels with smaller collagen fibrils. Proliferation rates of C2C12 myoblasts were also higher on gels with smaller collagen fibrils, whereas MuSCs did not show a significant difference. Surprisingly, collagen alignment did not have significant effects on muscle progenitor function. This study demonstrates that MuSCs are capable of sensing their underlying extracellular matrix (ECM) structures and enhancing differentiation on substrates with less collagen cross linking or smaller collagen fibrils. Thus, in fibrotic muscle, targeting cross linking and fibril size rather than collagen expression may more effectively support MuSC-based regeneration.

Tunneling nanotubes mediate the expression of senescence markers in mesenchymal stem/stromal cell spheroids.

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is limited by acquired senescence following prolonged culture expansion and high-passage numbers. However, the degree of cell senescence is dynamic, and cell-cell communication is critical to promote cell survival. MSC spheroids exhibit improved viability compared with monodispersed cells, and actin-rich tunneling nanotubes (TNTs) may mediate cell survival and other functions through the exchange of cytoplasmic components. Building upon our previous demonstration of TNTs bridging MSCs within these cell aggregates, we hypothesized that TNTs would influence the expression of senescence markers in MSC spheroids. We confirmed the existence of functional TNTs in MSC spheroids formed from low-passage, high-passage, and mixtures of low- and high-passage cells using scanning electron microscopy, confocal microscopy, and flow cytometry. The contribution of TNTs toward the expression of senescence markers was investigated by blocking TNT formation with cytochalasin D (CytoD), an inhibitor of actin polymerization. CytoD-treated spheroids exhibited decreases in cytosol transfer. Compared with spheroids formed solely of high-passage MSCs, the addition of low-passage MSCs reduced p16 expression, a known genetic marker of senescence. We observed a significant increase in p16 expression in high-passage cells when TNT formation was inhibited, establishing the importance of TNTs in MSC spheroids. These data confirm the restorative role of TNTs within MSC spheroids formed with low- and high-passage cells and represent an exciting approach to use higher-passage cells in cell-based therapies.

Winning hearts and minds: ECG reporting in the first seizure clinic.

BACKGROUND AND AIMS: An electrocardiogram (ECG) is a mandatory test for anyone presenting with loss of consciousness. Many referrals to the first seizure clinic (FSC) are caused by syncope. We assessed the sensitivity of neurologists' ECG reporting in detecting rhythm abnormalities including some potentially life-threatening cardiac conditions. METHODS: We audited patients referred to a FSC in Glasgow over 4 years. All ECGs were interpreted by the attending neurologist as standard practice. Subsequently, two cardiologists reviewed the ECGs independently. RESULTS: Of 160 consecutive patients, 92 patients (58%) were diagnosed as having seizures, 43 (27%) as syncope, and 25 (16%) were unclassified. Twenty eight ECGs thought to be normal by the neurologist were considered abnormal by the cardiologist, including three with long corrected QT interval. The proportion of abnormal ECGs and disparity in reporting between neurologists and cardiologists persisted independent of the underlying diagnosis. CONCLUSION: Reporting of ECGs by non-cardiologists may not be adequately sensitive in picking up potentially life threatening cardiac conditions. Cardiologist input into FSCs is recommended to enhance the diagnostic yield.

Functionally graded biomaterials for use as model systems and replacement tissues.

The heterogeneity of native tissues requires complex materials to provide suitable substitutes for model systems and replacement tissues. Functionally graded materials have the potential to address this challenge by mimicking the gradients in heterogeneous tissues such as porosity, mineralization, and fiber alignment to influence strength, ductility, and cell signaling. Advancements in microfluidics, electrospinning, and 3D printing enable the creation of increasingly complex gradient materials that further our understanding of physiological gradients. The combination of these methods enables rapid prototyping of constructs with high spatial resolution. However, successful translation of these gradients requires both spatial and temporal presentation of cues to model the complexity of native tissues that few materials have demonstrated. This review highlights recent strategies to engineer functionally graded materials for the modeling and repair of heterogeneous tissues, together with a description of how cells interact with various gradients.

Viva la VOSCE?

BACKGROUND: The COVID-19 pandemic lockdown precluded face-to-face final Objective Structured Clinical Examinations (OSCE) in the UK. RESULTS: In response, we rapidly developed and then successfully implemented a novel Virtual Objective Structured Clinical Examination (VOSCE). CONCLUSIONS: In this article we both describe and reflect on our experience as well as discuss the implications for future undergraduate assessment as the situation evolves.

Hypoxic Preconditioning of Mesenchymal Stem Cells with Subsequent Spheroid Formation Accelerates Repair of Segmental Bone Defects.

Cell-based approaches for musculoskeletal tissue repair are limited by poor cell survival and engraftment. Short-term hypoxic preconditioning of mesenchymal stem cells (MSCs) can prolong cell viability in vivo, while the aggregation of MSCs into spheroids increases cell survival, trophic factor secretion, and tissue formation in vivo. We hypothesized that preconditioning MSCs in hypoxic culture before spheroid formation would increase cell viability, proangiogenic potential, and resultant bone repair compared with that of individual MSCs. Human MSCs were preconditioned in 1% O2 in monolayer culture for 3 days (PC3) or kept in ambient air (PC0), formed into spheroids of increasing cell density, and then entrapped in alginate hydrogels. Hypoxia-preconditioned MSC spheroids were more resistant to apoptosis than ambient air controls and this response correlated with duration of hypoxia exposure. Spheroids of the highest cell density exhibited the greatest osteogenic potential in vitro and vascular endothelial growth factor (VEGF) secretion was greatest in PC3 spheroids. PC3 spheroids were then transplanted into rat critical-sized femoral segmental defects to evaluate their potential for bone healing. Spheroid-containing gels induced significantly more bone healing compared with gels containing preconditioned individual MSCs or acellular gels. These data demonstrate that hypoxic preconditioning represents a simple approach for enhancing the therapeutic potential of MSC spheroids when used for bone healing. Stem Cells 2018;36:1393-1403.

Multifactorial Experimental Design to Optimize the Anti-Inflammatory and Proangiogenic Potential of Mesenchymal Stem Cell Spheroids.

Mesenchymal stem cell therapies promote wound healing by manipulating the local environment to enhance the function of host cells. Aggregation of mesenchymal stem cells (MSCs) into three-dimensional spheroids increases cell survival and augments their anti-inflammatory and proangiogenic potential, yet there is no consensus on the preferred conditions for maximizing spheroid function in this application. The objective of this study was to optimize conditions for forming MSC spheroids that simultaneously enhance their anti-inflammatory and proangiogenic nature. We applied a design of experiments (DOE) approach to determine the interaction between three input variables (number of cells per spheroid, oxygen tension, and inflammatory stimulus) on MSC spheroids by quantifying secretion of prostaglandin E2 (PGE2 ) and vascular endothelial growth factor (VEGF), two potent molecules in the MSC secretome. DOE results revealed that MSC spheroids formed with 40,000 cells per spheroid in 1% oxygen with an inflammatory stimulus (Spheroid 1) would exhibit enhanced PGE2 and VEGF production versus those formed with 10,000 cells per spheroid in 21% oxygen with no inflammatory stimulus (Spheroid 2). Compared to Spheroid 2, Spheroid 1 produced fivefold more PGE2 and fourfold more VEGF, providing the opportunity to simultaneously upregulate the secretion of these factors from the same spheroid. The spheroids induced macrophage polarization, sprout formation with endothelial cells, and keratinocyte migration in a human skin equivalent model-demonstrating efficacy on three key cell types that are dysfunctional in chronic non-healing wounds. We conclude that DOE-based analysis effectively identifies optimal culture conditions to enhance the anti-inflammatory and proangiogenic potential of MSC spheroids. Stem Cells 2017;35:1493-1504.

Nondestructive fluorescence lifetime imaging and time-resolved fluorescence spectroscopy detect cartilage matrix depletion and correlate with mechanical properties.

Tissue engineers utilize a battery of expensive, time-consuming and destructive techniques to assess the composition and function of engineered tissues. A nondestructive solution to monitor tissue maturation would reduce costs and accelerate product development. As a first step toward this goal, two nondestructive, label-free optical techniques, namely multispectral fluorescent lifetime imaging (FLIm) and time-resolved fluorescence spectroscopy (TRFS), were investigated for their potential in evaluating the biochemical and mechanical properties of articular cartilage. Enzymatic treatments were utilized to selectively deplete cartilage of either collagen or proteoglycan, to produce a range of matrix compositions. Samples were assessed for their optical properties using a fiber-coupled optical system combining FLIm and TRFS, their biochemical and mechanical properties and by histological staining. Single and multivariable correlations were performed to evaluate relationships among these properties. FLIm- and TRFS-derived measurements are sensitive to changes in cartilage matrix and correlate with mechanical and biochemical assays. Mean fluorescence lifetime values extracted from FLIm images (375-410 nm spectral band) showed strong, specific correlations with collagen content (R2 = 0.79, p < 0.001) and tensile properties (R2 = 0.45, p = 0.02). TRFS lifetime measurements centered at 520 nm (with a 5 nm bandwidth) possessed strong, specific correlations with proteoglycan content (R2 = 0.59, p = 0.001) and compressive properties (R2 = 0.71, p < 0.001). Nondestructive optical assessment of articular cartilage, using a combination of FLIm- and TRFS-derived parameters, provided a quantitative method for determining tissue biochemical composition and mechanical function. These tools hold great potential for research, industrial and clinical settings.

A phase 2, randomized, double-blind, placebo- controlled study of chemo-immunotherapy combination using motolimod with pegylated liposomal doxorubicin in recurrent or persistent ovarian cancer: a Gynecologic Oncology Group partners study.

BACKGROUND: A phase 2, randomized, placebo-controlled trial was conducted in women with recurrent epithelial ovarian carcinoma to evaluate the efficacy and safety of motolimod-a Toll-like receptor 8 (TLR8) agonist that stimulates robust innate immune responses-combined with pegylated liposomal doxorubicin (PLD), a chemotherapeutic that induces immunogenic cell death. PATIENTS AND METHODS: Women with ovarian, fallopian tube, or primary peritoneal carcinoma were randomized 1 : 1 to receive PLD in combination with blinded motolimod or placebo. Randomization was stratified by platinum-free interval (≤6 versus >6-12 months) and Gynecologic Oncology Group (GOG) performance status (0 versus 1). Treatment cycles were repeated every 28 days until disease progression. RESULTS: The addition of motolimod to PLD did not significantly improve overall survival (OS; log rank one-sided P = 0.923, HR = 1.22) or progression-free survival (PFS; log rank one-sided P = 0.943, HR = 1.21). The combination was well tolerated, with no synergistic or unexpected serious toxicity. Most patients experienced adverse events of fatigue, anemia, nausea, decreased white blood cells, and constipation. In pre-specified subgroup analyses, motolimod-treated patients who experienced injection site reactions (ISR) had a lower risk of death compared with those who did not experience ISR. Additionally, pre-treatment in vitro responses of immune biomarkers to TLR8 stimulation predicted OS outcomes in patients receiving motolimod on study. Immune score (tumor infiltrating lymphocytes; TIL), TLR8 single-nucleotide polymorphisms, mutational status in BRCA and other DNA repair genes, and autoantibody biomarkers did not correlate with OS or PFS. CONCLUSIONS: The addition of motolimod to PLD did not improve clinical outcomes compared with placebo. However, subset analyses identified statistically significant differences in the OS of motolimod-treated patients on the basis of ISR and in vitro immune responses. Collectively, these data may provide important clues for identifying patients for treatment with immunomodulatory agents in novel combinations and/or delivery approaches. TRIAL REGISTRATION: Clinicaltrials.gov, NCT 01666444.

Hydrogel biophysical properties instruct coculture-mediated osteogenic potential.

Cell-based approaches for bone formation require instructional cues from the surrounding environment. As an alternative to pharmacological strategies or transplanting single cell populations, one approach is to coimplant populations that can establish a new vasculature and differentiate to bone-forming osteoblasts. Mesenchymal stem/stromal cells (MSCs) possess osteogenic potential and produce numerous angiogenic growth factors. Endothelial colony-forming cells (ECFCs) are a subpopulation of endothelial progenitor cells capable of vasculogenesis in vivo and may provide endogenous cues to support MSC function. We investigated the contribution of the carrier biophysical properties to instruct entrapped human MSCs and ECFCs to simultaneously promote their osteogenic and proangiogenic potential. Compared with gels containing MSCs alone, fibrin gels engineered with increased compressive stiffness simultaneously increased the osteogenic and proangiogenic potential of entrapped cocultured cells. ECFCs produced bone morphogenetic protein-2 (BMP-2), a potent osteoinductive molecule, and increases in BMP-2 secretion correlated with gel stiffness. Coculture of MSCs with ECFCs transduced to knockdown BMP-2 production abrogated the osteogenic response to levels observed with MSCs alone. These results demonstrate that physical properties of engineered hydrogels modulate the function of cocultured cells in the absence of inductive cues, thus increasing the translational potential of coimplantation to speed bone formation and repair.

Cell Migration and Bone Formation from Mesenchymal Stem Cell Spheroids in Alginate Hydrogels Are Regulated by Adhesive Ligand Density.

The adhesion and migration of cells entrapped in engineered materials is regulated by available adhesive ligands. Although mesenchymal stem cell (MSC) spheroids injected into damaged tissues promote repair, their transplantation in biomaterials which regulate cell migration from the aggregate may further enhance their therapeutic potential. Alginate hydrogels were modified with Arginine-Glycine-Aspartic acid (RGD) at increasing concentrations, and osteogenically induced human MSC spheroids were entrapped to assess cell migration, survival, and differentiation. Cell migration was greater from MSC spheroids in alginate modified with low RGD levels, while the osteogenic potential was higher for spheroids entrapped in unmodified or high RGD density gels in vitro. Upon ectopic implantation, microCT and immunohistochemistry revealed extensive osteogenesis in unmodified and high RGD density gels compared to low RGD density gels. These data suggest that restriction of MSC migration from spheroids correlates with enhanced spheroid osteogenic potential, representing a novel tool for bone tissue engineering.

Detection and Characterization of Xanthomonas vasicola pv. vasculorum (Cobb 1894) comb. nov. Causing Bacterial Leaf Streak of Corn in the United States.

Bacterial leaf streak of corn (Zea mays) recently reached epidemic levels in three corn-growing states, and has been detected in another six states in the central United States. Xanthomonas vasicola was identified as the causal agent of this disease. A multilocus sequence alignment of six housekeeping genes and comparison of average nucleotide identity from draft genome sequence were used to confirm phylogenetic relationships and classification of this bacteria relative to other X. vasicola strains. X. vasicola isolates from Nebraska and South Africa were highly virulent on corn and sugarcane and less virulent on sorghum but caused water-soaking symptoms that are typical of X. vasicola infection on the leaves of all three hosts. Based on host range and phylogenetic comparison, we propose the taxonomic designation of this organism to X. vasicola pv. vasculorum ( Cobb 1894 ) comb. nov. Polymerase chain reaction-based diagnostic assays were developed that distinguish X. vasicola pv. vasculorum and X. vasicola pv. holcicola from each other and from other Xanthomonas spp.

Extracellular Matrix-Coated Composite Scaffolds Promote Mesenchymal Stem Cell Persistence and Osteogenesis.

Composite scaffolds of bioactive glass and poly(lactide-co-glycolide) provide advantages over homogeneous scaffolds, yet their therapeutic potential can be improved by strategies that promote adhesion and present instructive cues to associated cells. Mesenchymal stem cell (MSC)-secreted extracellular matrix (ECM) enhances survival and function of associated cells. To synergize the benefits of an instructive ECM with composite scaffolds, we tested the capacity of ECM-coated composite scaffolds to promote cell persistence and resultant osteogenesis. Human MSCs cultured on ECM-coated scaffolds exhibited increased metabolic activity and decreased apoptosis compared to uncoated scaffolds. Additionally, MSCs on ECM-coated substrates in short-term culture secreted more proangiogenic factors while maintaining markers of osteogenic differentiation. Upon implantation, we detected improved survival of MSCs on ECM-coated scaffolds over 3 weeks. Histological evaluation revealed enhanced cellularization and osteogenic differentiation in ECM-coated scaffolds compared to controls. These findings demonstrate the promise of blending synthetic and natural ECMs and their potential in tissue regeneration.

Diagnostic Exercise: Circling and Behavioral Changes in a Cat.

A 4-year old spayed male domestic shorthair cat was presented with a history of circling and behavioral changes. Neurologic examination showed mild proprioceptive deficits. The lesion was localized in the forebrain, and magnetic resonance imaging revealed the presence of a large midline intracranial mass extending from the frontal lobe to the tentorial region of the brain. Euthanasia was elected due to poor prognosis. Histopathologic evaluation confirmed the presence of a mass composed by sheets and aggregates of large round/polygonal cells and multinucleate cells associated with deposits of cholesterol clefts, scattered hemorrhages and hemosiderin-laden macrophages. Immunohistochemistry showed that the round/polygonal cells and multinucleate cells were strongly positive for major histocompatibility complex class II antigen, variably positive for CD18, and occasionally positive for S100. Subsets of spindle cells showing variable expression of vimentin, S100, and neuron-specific enolase were also present. The final diagnosis was cholesterol granuloma. Differential diagnosis with meningioma is discussed.

The General Practitioner and the military veteran.

OBJECTIVES: To assess the knowledge of members of the Royal College of General Practitioners (RCGP) on veteran's health issues, assess present support, and establish what support is required for GPs when treating veterans. METHODS: An electronic survey of RCGP members across selected faculties. RESULTS: Forty-seven per cent of respondents were 'unsure' or 'didn't know' how many veterans they were responsible for. However, many GPs replied that they had seen a veteran in the last month. Only 7.9% of respondents used the unique Read Code for veterans. Disappointingly, 75% of GPs indicated that they had not seen the RCGP leaflet on veterans' health, and less than 2% had used the RCGP On-Line e-learning resource. CONCLUSIONS: Surveyed GPs had little understanding of how many veterans were registered with their practice, and only a few had accessed learning resources available. GPs requested more information on how to assess veterans and where they could be referred. Further work is required to identify the true size of the problem, while continuing to provide proactive guidance and support to GPs on the health needs of veterans.