A tissue engineered 3D printed calcium alkali phosphate bioceramic bone graft enables vascularization and regeneration of critical-size discontinuity bony defects in vivo.

Introduction: Recently, efforts towards the development of patient-specific 3D printed scaffolds for bone tissue engineering from bioactive ceramics have continuously intensified. For reconstruction of segmental defects after subtotal mandibulectomy a suitable tissue engineered bioceramic bone graft needs to be endowed with homogenously distributed osteoblasts in order to mimic the advantageous features of vascularized autologous fibula grafts, which represent the standard of care, contain osteogenic cells and are transplanted with the respective blood vessel. Consequently, inducing vascularization early on is pivotal for bone tissue engineering. The current study explored an advanced bone tissue engineering approach combining an advanced 3D printing technique for bioactive resorbable ceramic scaffolds with a perfusion cell culture technique for pre-colonization with mesenchymal stem cells, and with an intrinsic angiogenesis technique for regenerating critical size, segmental discontinuity defects in vivo applying a rat model. To this end, the effect of differing Si-CAOP (silica containing calcium alkali orthophosphate) scaffold microarchitecture arising from 3D powder bed printing (RP) or the Schwarzwalder Somers (SSM) replica fabrication technique on vascularization and bone regeneration was analyzed in vivo. In 80 rats 6-mm segmental discontinuity defects were created in the left femur. Methods: Embryonic mesenchymal stem cells were cultured on RP and SSM scaffolds for 7d under perfusion to create Si-CAOP grafts with terminally differentiated osteoblasts and mineralizing bone matrix. These scaffolds were implanted into the segmental defects in combination with an arteriovenous bundle (AVB). Native scaffolds without cells or AVB served as controls. After 3 and 6 months, femurs were processed for angio-µCT or hard tissue histology, histomorphometric and immunohistochemical analysis of angiogenic and osteogenic marker expression. Results: At 3 and 6 months, defects reconstructed with RP scaffolds, cells and AVB displayed a statistically significant higher bone area fraction, blood vessel volume%, blood vessel surface/volume, blood vessel thickness, density and linear density than defects treated with the other scaffold configurations. Discussion: Taken together, this study demonstrated that the AVB technique is well suited for inducing adequate vascularization of the tissue engineered scaffold graft in segmental defects after 3 and 6 months, and that our tissue engineering approach employing 3D powder bed printed scaffolds facilitated segmental defect repair.

A quantitative micro-tomographic gut atlas of the lepidopteran model insect Manduca sexta.

The tobacco hornworm is used extensively as a model system for ecotoxicology, immunology and gut physiology. Here, we established a micro-computed tomography approach based on the oral application of the clinical contrast agent iodixanol, allowing for a high-resolution quantitative analysis of the Manduca sexta gut. This technique permitted the identification of previously unknown and understudied structures, such as the crop or gastric ceca, and revealed the underlying complexity of the hindgut folding pattern, which is involved in fecal pellet formation. The acquired data enabled the volume rendering of all gut parts, the reliable calculation of their volumes, and the virtual endoscopy of the entire alimentary tract. It can provide information for accurate orientation in histology uses, enable quantitative anatomical phenotyping in three dimensions, and allow the calculation of locally effective midgut concentrations of applied chemicals. This atlas will provide critical insights into the evolution of the alimentary tract in lepidopterans.

Measuring Young's modulus of single trabeculae in cancellous bone using a two-point bending test.

BACKROUND: Surgical treatment of proximal humeral fractures poses a major challenge, especially in osteoporotic bone. At present, there appears to exist neither a suitable model for research to optimize the osteosynthesis processes nor are the structural data available which are required for developing such a model. Therefore, the aim of this study is to determine the microscopic morphology and Young's modulus of cancellous bone from human humeral heads considering osteoporotic changes. METHODS: Cylindrical samples were taken from ten fresh-frozen human humeral heads and structural analysis was done with µCT. Ten rod-like trabeculae were prepared from five of the humeral heads each which were measured and tested mechanically. For this purpose, the trabeculae were fixed on a slide and rotated axially under a stereo microscope. The sample cross-section and the depending moment of inertia were extracted from the image data. The samples were then loaded in a 2-point bending test and Young's moduli of the samples were determined. RESULTS: It could be shown that with increasing age of the donor, ossified portion of the cancellous bone decreased (p < 0.05). The average degree of mineralization of the bone was 1.24 (±0.06) g/mm3, which decreased with increasing age (p < 0.05). The determined Young's modulus averaged 1.33 (±1.76) GPa. INTERPRETATION: The verified structural parameter showed osteoporotic changes in the examined bone. This study for the first time determined Young's modulus of single trabeculae of cancellous bone of osteoporotically altered human humeral heads. Implementing the non-destructive sample measurement before exposure resulted in a methodical improvement.

High-throughput screening of caterpillars as a platform to study host-microbe interactions and enteric immunity.

Mammalian models of human disease are expensive and subject to ethical restrictions. Here, we present an independent platform for high-throughput screening, using larvae of the tobacco hornworm Manduca sexta, combining diagnostic imaging modalities for a comprehensive characterization of aberrant phenotypes. For validation, we use bacterial/chemical-induced gut inflammation to generate a colitis-like phenotype and identify significant alterations in morphology, tissue properties, and intermediary metabolism, which aggravate with disease progression and can be rescued by antimicrobial treatment. In independent experiments, activation of the highly conserved NADPH oxidase DUOX, a key mediator of gut inflammation, leads to similar, dose-dependent alterations, which can be attenuated by pharmacological interventions. Furthermore, the developed platform could differentiate pathogens from mutualistic gastrointestinal bacteria broadening the scope of applications also to microbiomics and host-pathogen interactions. Overall, larvae-based screening can complement mammals in preclinical studies to explore innate immunity and host-pathogen interactions, thus representing a substantial contribution to improve mammalian welfare.

Activation of Humoral Immunity during the Pathogenesis of Experimental Chronic Lung Allograft Dysfunction.

Alloreactive and autoreactive antibodies have been associated with the development of chronic lung allograft dysfunction (CLAD), but their pathogenic role is disputed. Orthotopic left lung transplantation was performed in the Fischer-344 to Lewis rat strain combination followed by the application of ciclosporine for 10 days. Four weeks after transplantation, lipopolysaccharide (LPS) was instilled into the trachea. Lungs were harvested before (postoperative day 28) and after LPS application (postoperative days 29, 33, 40, and 90) for histopathological, immunohistochemical, and Western blot analyses. Recipient serum was collected to investigate circulating antibodies. Lung allografts were more strongly infiltrated by B cells and deposits of immunoglobulin G and M were more prominent in allografts compared to right native lungs or isografts and increased in response to LPS instillation. LPS induced the secretion of autoreactive antibodies into the circulation of allograft and isograft recipients, while alloreactive antibodies were only rarely detected. Infiltration of B cells and accumulation of immunoglobulin, which is observed in allografts treated with LPS but not isografts or native lungs, might contribute to the pathogenesis of experimental CLAD. However, the LPS-induced appearance of circulating autoreactive antibodies does not seem to be related to CLAD, because it is observed in both, isograft and allograft recipients.

Accuracy of Digital Impression Taking with Intraoral Scanners and Fabrication of CAD/CAM Posts and Cores in a Fully Digital Workflow.

Current intraoral scanners (IOS) enable direct impression taking for computer-aided de-sign/computer-aided manufacturing (CAD/CAM) posts and cores (P+C) with subsequent milling out of monolithic materials. The aim of this in vitro study was to systematically investigate the accuracy of CAD/CAM-P+C in a fully digital workflow, considering different IOS impression methods (Primescan (PRI), Trios4 without (TRI) and with scanpost (TRI+SP)) (Part A), and CAD/CAM milling of zirconium dioxid (ZIR) and resin composite (COM)-P+C (Part B). Five human models were developed in this study. Micro-CT imaging was used as a reference (REF). For Part A, the models were scanned 12 times for each impression method. Then, IOS datasets (n = 180) were superimposed with REF, and scan accuracy was determined using 3D software (GOMInspect). For Part B, one CAD/CAM-P+C (n = 30) was milled for each model, impression method, and material. The triple-scan method was applied using an industrial scanner (ATOS) to determine the accuracy of the fit. Statistical analysis was performed using analysis of variance (ANOVA, p < 0.05). Part A showed for PRI significantly lower accuracy than TRI and TRI+SP (p < 0.05). The data of Part B revealed significantly higher accuracy for ZIR than for COM (p < 0.05). Within the limitations of this study, CAD/CAM-P+C of the ZIR can be recommended for fabrication in a fully digital workflow regarding the accuracy of fit.

Closure times of neurocranial sutures and synchondroses in Persian compared to Domestic Shorthair cats.

Human-directed selective breeding has modified the phenotype of the modern Persian cat towards an extreme brachycephalic phenotype ('peke-face' Persian), which originates from a spontaneous mutation that first appeared in the 1950s in traditional Persian types. It was suggested that the peke-face phenotype results from pathologic skull development and might represent a craniosynostosis of the coronal sutures. We followed this hypothesis and investigated the time dependent status of the neurocranial sutures and synchondroses in an ontogenetic series of doll-faced and peke-faced Persian cats compared to Domestic Shorthair cats (DSHs). Cranial suture closure was assessed by examining an ontogenetic series of formalin-fixed head specimens (n = 55) and dry skulls (n = 32) using micro-computed tomography. Sagittal, metopic, coronal and lambdoid sutures as well as intersphenoidal, spheno-occipital and spheno-ethmoid synchondroses were examined. Logistic regression analysis was performed to test the global effect of age on suture closure within a group of peke-face Persians, doll-face Persians and DSHs and the 50% probability of having a closed suture was calculated and compared between groups. Age was a perfect predictor for the condition of the coronal sutures in peke-face Persians. Coronal sutures were found to be closed at 0-0.3 months. In doll-face and DSHs, coronal sutures were open throughout the lifetime with the exception of a few very old cats. Results of this study confirmed a coronal craniosynostosis that likely causes the extreme brachycephalic skull morphology in the peke-face Persian.

Rounded atelectasis after exposure to refractory ceramic fibres (RCF).

BACKGROUND: Refractory Ceramic fibres (RCF) are man-made mineral fibres used in high performance thermal insulation applications. Analogous to asbestos fibres, RCF are respirable, show a pleural drift and can persist in human lung tissue for more than 20 years after exposure. Pleural changes such as localised or diffuse pleural thickening as well as pleural calcification were reported. RESULT: A 45 years old man worked in high performance thermal insulation applications using refractory ceramic fibres (RCF) for almost 20 years. During a occupational medical prophylaxis to ensure early diagnosis of disorders caused by inhalation of aluminium silicate fibres with X-ray including high-resolution computed tomography (HRCT), bilateral pleural thickening was shown and a pleural calcification next to a rounded atelectasis was detected. Asbestos exposure could be excluded. In pulmonary function test a restrictive lung pattern could be revealed. In work samples scanning electron microscopy (SEM) including energy dispersive X-ray analysis (EDX) classified used fibres as aluminium silicate fibres. X-ray powder diffraction (XRD) and transmission electron microscopy (TEM) showed crystalline as well as amorphous fibres. CONCLUSIONS: A comprehensive lung function analysis and in case of restrictive lung disorders additional CT scans are needed in RCF exposed workers in accordance to the guidelines for medical occupational examinations comparable to asbestos exposed workers.

Resonance vibration interventions in the femur: Experimental-numerical modelling approaches.

MOTIVE: External vibration excitation might be key to many novel non-surgical interventions for pathologies in the musculoskeletal system and in other parts of the human organism. Lack of understanding about vibration patterns, their controllability, and reproducibility are three limitations of ongoing research. This study establishes a bovine vibration model and animal model replacements for future research. METHODS: We used biological samples (n=5) and one polyurethane sample of the bovine femur. Mechanical resonance was measured experimentally and analysed numerically by finite element method. MAIN RESULTS: The experiments obtained 5 distinct mode shapes for the biological sample set, with standard deviation < 7.5%. Finite element analysis of the biological samples can replicate experimental mode shape deflection. The use of polyurethane changes resonance character but results are also good approximations of the biological samples. CONCLUSIONS: A model of the bovine femur with consistent resonance behaviour is presented with alternatives (polyurethane and finite element analysis) that can serve in reducing the number of necessary biological samples. Future work will be to adapt results to human anatomy. Of clinical interest will be to influence bone pathologies such as post-surgical non-union, or bone functionality as part of haematopoiesis and endocrine secretion.

Bone Status in a Mouse Model of Experimental Autoimmune-Orchitis.

Investigations in male patients with fertility disorders revealed a greater risk of osteoporosis. The rodent model of experimental autoimmune-orchitis (EAO) was established to analyze the underlying mechanisms of male infertility and causes of reduced testosterone concentration. Hence, we investigated the impact of testicular dysfunction in EAO on bone status. Male mice were immunized with testicular homogenate in adjuvant to induce EAO (n = 5). Age-matched mice were treated with adjuvant alone (adjuvant, n = 6) or remained untreated (control, n = 7). Fifty days after the first immunization specimens were harvested. Real-time reverse transcription-PCR indicated decreased bone metabolism by alkaline phosphatase and Cathepsin K as well as remodeling of cell-contacts by Connexin-43. Micro computed tomography demonstrated a loss of bone mass and mineralization. These findings were supported by histomorphometric results. Additionally, biomechanical properties of femora in a three-point bending test were significantly altered. In summary, the present study illustrates the induction of osteoporosis in the investigated mouse model. However, results suggest that the major effects on bone status were mainly caused by the complete Freund's adjuvant rather than the autoimmune-orchitis itself. Therefore, the benefit of the EAO model to transfer laboratory findings regarding bone metabolism in context with orchitis into a clinical application is limited.

Large Animal Model of Osteoporotic Defect Healing: An Alternative to Metaphyseal Defect Model.

Osteoporosis is a common metabolic disorder diagnosed by lower bone density and higher risk of fracture. Fragility fractures because of osteoporosis are associated with high mortality rate. Deep understanding of fracture healing in osteoporosis is important for successful treatment. Therefore, the FDA approved the use of small and large animal models for preclinical testing. This study investigated the clinical relevance of a fracture defect model in the iliac crest of the osteoporotic sheep model and its several advantages over other models. The osteoporosis was achieved using ovariectomy (OVX) in combination with diet deficiency (OVXD) and steroid administration (OVXDS). Fluorochrome was injected to examine the rate of bone remodelling and bone mineralization. The defect areas were collected and embedded in paraffin and polymethyl metha acrylate (PMMA) for histological staining. OVXDS showed significantly lower bone mineral density (BMD) and bone mineral content (BMC) at all time points. Furthermore, variations in healing patterns were noticed, while the control, OVX and OVXD showed complete healing after 8 months. Bone quality was affected mostly in the OVXDS group showing irregular trabecular network, lower cortical bone thickness and higher cartilaginous tissue at 8 months. The mineral deposition rate showed a declining pattern in the control, OVX, and OVXD from 5 months to 8 months. One the contrary, the OVXDS group showed an incremental pattern from 5 months to 8 months. The defect zone in osteoporotic animals showed impaired healing and the control showed complete healing after 8 months. This unique established model serves as a dual-purpose model and has several advantages: no intraoperative and postoperative complications, no need for fixation methods for biomaterial testing, and reduction in animal numbers, which comply with 3R principles by using the same animal at two different time points.

Partial cortico-hippocampectomy in cats, as therapy for refractory temporal epilepsy: A descriptive cadaveric study.

Cats, similar to humans, are known to be affected by hippocampal sclerosis (HS), potentially causing antiepileptic drug (AED) resistance. HS can occur as a consequence of chronic seizure activity, trauma, inflammation, or even as a primary disease. In humans, temporal lobe resection is the standardized therapy in patients with refractory temporal lobe epilepsy (TLE). The majority of TLE patients are seizure free after surgery. Therefore, the purpose of this prospective cadaveric study is to establish a surgical technique for hippocampal resection in cats as a treatment for AED resistant seizures. Ten cats of different head morphology were examined. Pre-surgical magnetic resonance imaging (MRI) and computed tomography (CT) studies of the animals' head were carried out to complete 3D reconstruction of the head, brain, and hippocampus. The resected hippocampal specimens and the brains were histologically examined for tissue injury adjacent to the hippocampus. The feasibility of the procedure, as well as the usability of the removed specimen for histopathological examination, was assessed. Moreover, a micro-CT (mCT) examination of the brain of two additional cats was performed in order to assess temporal vasculature as a reason for possible intraoperative complications. In all cats but one, the resection of the temporal cortex and the hippocampus were successful without any evidence of traumatic or vascular lesions in the surrounding neurovascular structures. In one cat, the presence of mechanical damage (a fissure) of the thalamic surface was evident in the histopathologic examination of the brain post-resection. All hippocampal fields and the dentate gyrus were identified in the majority of the cats via histological examination. The study describes a new surgical approach (partial temporal cortico-hippocampectomy) offering a potential treatment for cats with clinical and diagnostic evidence of temporal epilepsy which do not respond adequately to the medical therapy.

Galleria mellonella as an alternative in vivo model to study bacterial biofilms on stainless steel and titanium implants

The purpose of this study was to establish an infection model of Galleria mellonella larvae as an alternative in vivo model for biofilm-associated infections on stainless steel and titanium implants. First, the model was established with sterile implants to evaluate biocompatibility. Titanium or stainless steel implants were implanted without adverse effects over the entire observation period of 5 days compared to controls and even up to the pupae and moth stage. Then, stainless steel and titanium implants contaminated with Staphylococcus aureus were implanted into larvae to mimic biofilm-associated infection. For both materials, pre-incubation of the implant with S. aureus led to significantly reduced survival of the larvae compared to sterile implants. Larvae could not be rescued by gentamicin, whereas gentamicin significantly improved the survival of the larvae in case of planktonic infection with S. aureus without an implant, confirming the typical character­istics of reduced antibiotic susceptibility of biofilm infections. Biofilm formation and various stages of biofilm maturation were confirmed by surface electron microscopy and by measuring bacterial gene expression of biofilm-related genes on contaminated implants, which confirmed biofilm formation and upregulation of autolysin (atl ) and sarA genes. In con­clusion, G. mellonella can be used as an alternative in vivo model to study biofilm-associated infections on stainless steel and titanium implants, which may help to reduce animal infection experiments with vertebrates in the future.

Gelatin-Modified Calcium/Strontium Hydrogen Phosphates Stimulate Bone Regeneration in Osteoblast/Osteoclast Co-Culture and in Osteoporotic Rat Femur Defects-In Vitro to In Vivo Translation.

The development and characterization of biomaterials for bone replacement in case of large defects in preconditioned bone (e.g., osteoporosis) require close cooperation of various disciplines. Of particular interest are effects observed in vitro at the cellular level and their in vivo representation in animal experiments. In the present case, the material-based alteration of the ratio of osteoblasts to osteoclasts in vitro in the context of their co-cultivation was examined and showed equivalence to the material-based stimulation of bone regeneration in a bone defect of osteoporotic rats. Gelatin-modified calcium/strontium phosphates with a Ca:Sr ratio in their precipitation solutions of 5:5 and 3:7 caused a pro-osteogenic reaction on both levels in vitro and in vivo. Stimulation of osteoblasts and inhibition of osteoclast activity were proven during culture on materials with higher strontium content. The same material caused a decrease in osteoclast activity in vitro. In vivo, a positive effect of the material with increased strontium content was observed by immunohistochemistry, e.g., by significantly increased bone volume to tissue volume ratio, increased bone morphogenetic protein-2 (BMP2) expression, and significantly reduced receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) ratio. In addition, material degradation and bone regeneration were examined after 6 weeks using stage scans with ToF-SIMS and µ-CT imaging. The remaining material in the defects and strontium signals, which originate from areas exceeding the defect area, indicate the incorporation of strontium ions into the surrounding mineralized tissue. Thus, the material inherent properties (release of biologically active ions, solubility and degradability, mechanical strength) directly influenced the cellular reaction in vitro and also bone regeneration in vivo. Based on this, in the future, materials might be synthesized and specifically adapted to patient-specific needs and their bone status.

Effect of 'in air' freezing on post-thaw recovery of Callithrix jacchus mesenchymal stromal cells and properties of 3D collagen-hydroxyapatite scaffolds.

Through enabling an efficient supply of cells and tissues in the health sector on demand, cryopreservation is increasingly becoming one of the mainstream technologies in rapid translation and commercialization of regenerative medicine research. Cryopreservation of tissue-engineered constructs (TECs) is an emerging trend that requires the development of practically competitive biobanking technologies. In our previous studies, we demonstrated that conventional slow-freezing using dimethyl sulfoxide (Me2SO) does not provide sufficient protection of mesenchymal stromal cells (MSCs) frozen in 3D collagen-hydroxyapatite scaffolds. After simple modifications to a cryopreservation protocol, we report on significantly improved cryopreservation of TECs. Porous 3D scaffolds were fabricated using freeze-drying of a mineralized collagen suspension and following chemical crosslinking. Amnion-derived MSCs from common marmoset monkey Callithrix jacchus were seeded onto scaffolds in static conditions. Cell-seeded scaffolds were subjected to 24 h pre-treatment with 100 mM sucrose and slow freezing in 10% Me2SO/20% FBS alone or supplemented with 300 mM sucrose. Scaffolds were frozen 'in air' and thawed using a two-step procedure. Diverse analytical methods were used for the interpretation of cryopreservation outcome for both cell-seeded and cell-free scaffolds. In both groups, cells exhibited their typical shape and well-preserved cell-cell and cell-matrix contacts after thawing. Moreover, viability test 24 h post-thaw demonstrated that application of sucrose in the cryoprotective solution preserves a significantly greater portion of sucrose-pretreated cells (more than 80%) in comparison to Me2SO alone (60%). No differences in overall protein structure and porosity of frozen scaffolds were revealed whereas their compressive stress was lower than in the control group. In conclusion, this approach holds promise for the cryopreservation of 'ready-to-use' TECs.

Unilateral and bilateral agenesis of the upper thyroid horns - A morphometric analysis of the larynx.

The agenesis of one or both upper thyroid horns (UTH) cannot be considered rare with an incidence ranging between 0.8-9.4%. Studies so far have mostly described frequencies, yet surveys on its morphology and traumatology are missing. It is unknown to what extent this anatomical variant impacts the overall symmetry of the larynx. For the trauma reconstruction after cervical trauma this knowledge is required as an asymmetric larynx can show a different fracture pattern. In this investigation, larynxes with unilateral (n=21)/bilateral (n=5) agenesis of the upper thyroid horn(s) and a control group with regular larynxes (n=23) were juxtaposed. Various measurements were taken after µ-CT scanning. In total 10 distances/angles for the thyroid cartilage and 8 for the cricoid cartilage were compared. Using intra-individual and matched pair comparisons the symmetry was assessed. The results of this investigation showed significant differences in the intra-individual and pairwise comparison for the opening angle and the length of the thyroid alae although the lateral inclination of the thyroid alae showed no significant difference. Combining these three parameters into one symmetry score, the comparison was not significant. This research demonstrates that neither unilateral nor bilateral agenesis of the UTH is related to a structural disbalance of the larynx. Thus, different fracture patterns or vulnerabilities are not to be expected.

Re-evaluation of Zospeumschaufussi von Frauenfeld, 1862 and Z.suarezi Gittenberger, 1980, including the description of two new Iberian species using Computer Tomography (CT) (Eupulmonata, Ellobioidea, Carychiidae).

The present study aims to clarify the confused taxonomy of Z.schaufussi von Frauenfeld, 1862 and Zospeumsuarezi Gittenberger, 1980. Revision of Iberian Zospeum micro snails is severely hindered by uncertainties regarding the identity of the oldest Iberian Zospeum species, Z.schaufussi von Frauenfeld, 1862. In this paper, we clarify its taxonomic status by designating a lectotype from the original syntype series and by describing its internal and external shell morphology. Using SEM-EDX, we attempt to identify the area of the type locality cave more precisely than "a cave in Spain". The shell described and illustrated by Gittenberger (1980) as Z.schaufussi appears not to be conspecific with the lectotype shell, and is considered a separate species, Z.gittenbergeri Jochum, Prieto & De Winter, sp. n. Zospeumsuarezi was described from various caves in NW Spain. Study of the type material reveals that these shells are not homogenous in shell morphology. The holotype shell of Z.suarezi is imaged here for the first time. The paratype shell, illustrated by Gittenberger (1980) from a distant, second cave, is described as Zospeumpraetermissum Jochum, Prieto & De Winter, sp. n. The shell selected here as lectotype of Z.schaufussi, was also considered a paratype of Z.suarezi by Gittenberger (1980). Since this specimen is morphologically very similar to topotypic shells of Z.suarezi, the latter species is considered a junior synonym of Z.schaufussi (syn. n.). The internal shell morphology of all these taxa is described and illustrated using X-ray Micro Computer Tomography (Micro-CT).

Impact of Fgf10 deficiency on pulmonary vasculature formation in a mouse model of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD), characterized by alveoli simplification and dysmorphic pulmonary microvasculature, is a chronic lung disease affecting prematurely born infants. Pulmonary hypertension (PH) is an important BPD feature associated with morbidity and mortality. In human BPD, inflammation leads to decreased fibroblast growth factor 10 (FGF10) expression but the impact on the vasculature is so far unknown. We used lungs from Fgf10+/- versus Fgf10+/+ pups to investigate the effect of Fgf10 deficiency on vascular development in normoxia (NOX) and hyperoxia (HOX, BPD mouse model). To assess the role of fibroblast growth factor receptor 2b (Fgfr2b) ligands independently of early developmentaldefects, we used an inducible double transgenic system in mice allowing inhibition of Fgfr2b ligands activity. Using vascular morphometry, we quantified the pathological changes. Finally, we evaluated changes in FGF10, surfactant protein C (SFTPC), platelet endothelial cell adhesion molecule (PECAM) and alpha-smooth muscle actin 2 (α-SMA) expression in human lung samples from patients suffering from BPD. In NOX, no major difference in the lung vasculature between Fgf10+/- and control pups was detected. In HOX, a greater loss of blood vessels in Fgf10+/- lungs is associated with an increase of poorly muscularized vessels. Fgfr2b ligands inhibition postnatally in HOX is sufficient to decrease the number of blood vessels while increasing the level of muscularization, suggesting a PH phenotype. BPD lungs exhibited decreased FGF10, SFTPC and PECAM but increased α-SMA. Fgf10 deficiency-associated vascular defects are enhanced in HOX and could represent an additional cause of morbidity in human patients with BPD.

Deletion of nicotinic acetylcholine receptor alpha9 in mice resulted in altered bone structure.

Alterations in bone strength and structure were found in knockout (KO) mouse strains with deletion of several acetylcholine receptors. Interestingly, the expression of the nicotinic acetylcholine receptors (nAChR) subunit α10 was down-regulated in osteogenic differentiated mesenchymal stem cells of patients with osteoporosis whereas the expression of subunit α9 was not altered. Since nAChR subunits α9 and α10 are often combined in a functional receptor, we analyzed here the bone of adult female KO mice with single deletion of either nAChR alpha9 (α9KO) or alpha10 (α10KO). Biomechanical testing showed a significant decrease of bending stiffness and maximal breaking force in α9KO compared to their corresponding wild type mice. Furthermore, an increase in trabecular pattern factor (Tb.Pf) and structure model index (SMI) was detected by µCT in α9KO indicating reduced bone mass. On the mRNA level a decrease of Collagen 1α1 and Connexin-43 was measured by real-time RT-PCR in α9KO while no alteration of osteoclast markers was detected in either mouse strain. Using electron microcopy we observed an increase in the number of osteocytes that showed signs of degeneration and cell death in the α9KO compared to their wild type mice, while α10KO showed no differences. In conclusion, we demonstrate alterations in bone strength, structure and bio-marker expression in α9KO mice which imply the induction of osteocyte degeneration. Thus, our data suggest that nAChR containing the α9 subunit might be involved in the homeostasis of osteocytes and therefore in bone mass regulation.

Evaluating Systolic and Diastolic Cardiac Function in Rodents Using Microscopic Computed Tomography.

BACKGROUND: The use of microscopic computed tomography to assess the key functional parameters of systolic emptying or diastolic filling in small animals has not been previously reported. The aim of the study was to test whether microscopic computed tomography can assess the dynamics of both left ventricle and right ventricle (RV) diastolic filling and systolic emptying in an experimental model of pulmonary arterial hypertension Methods and Results: The Wistar-Kyoto rats were injected subcutaneously with the VEGF (vascular endothelial growth factor)-receptor inhibitor SU5416 (20 mg/kg body weight) and were then exposed to chronic hypoxia (10% oxygen) for 21 days (SU5416-hypoxia) followed by normoxia for an additional 2 weeks. Thereafter, multiphase cine cardiac images were acquired using a microscopic computed tomography scanner in conjunction with a blood-pool iodinated contrast agent. Examination of the 3-dimensional images of SU5416-hypoxia rats confirmed the presence of severe pulmonary arterial hypertension. Functional parameters that describe the dynamics of ventricular systolic ejection and diastolic filling were calculated. RV peak ejection rate was significantly decreased ( P<0.03) in SU5416-hypoxia rats compared with controls. RV peak filling rate had a significant decrease compared with controls ( P<0.03), particularly in the early phase of diastole ( P<0.03). This was accompanied by increased time to peak filling rate ( P<0.03) and total filling time ( P<0.06). Spearman analysis between microscopic computed tomography RV diastolic indices and invasively derived RV end-diastolic pressure indicated excellent correlation. CONCLUSIONS: We developed a method that allows rapid and accurate assessment of cardiac functional indices and that paves the way for more extensive preclinical cardiovascular research.