BMP-2 (and partially GDF-5) coating significantly accelerates and augments bone formation close to hydroxyapatite/tricalcium-phosphate/brushite implant cylinders for tibial bone defects in senile, osteopenic sheep.

Bilateral defects (diameter 8 mm) in the medial tibial head of senile, osteopenic female sheep (n = 48; 9.63 ± 0.10 years; mean ± SEM) were treated with hydroxyapatite (HA)/beta-tricalcium phosphate (ß-TCP)/dicalcium phosphate dihydrate (DCPD; brushite) cylinders coated with BMP-2 (25 or 250 micrograms) or growth differentiation factor (GDF)-5 (125 or 1250 micrograms; left side); cylinders without BMP served as controls (right side). Three, 6, and 9 months post-operation (n = 6 each group), bone structure and formation were analyzed in vivo by X-ray and ex vivo by osteodensitometry, histomorphometry, and micro-computed tomography (micro-CT) at 3 and 9 months. Semi-quantitative X-ray evaluation showed significantly increasing bone densities around all implant cylinders over time. High-dose BMP-2-coated cylinders (3 and 9 months) and low-dose GDF-5-coated cylinders (3 and 6 months) demonstrated significantly higher densities than controls (dose-dependent for BMP-2 at 3 months). This was confirmed by osteodensitometry at 9 months for high-dose BMP-2-coated cylinders (and selected GDF-5 groups), and was again dose-dependent for BMP-2. Osteoinduction by BMP-2 was most pronounced in the adjacent bone marrow (dynamic histomorphometry/micro-CT). BMP-2 (and partially GDF-5) significantly increased the bone formation in the vicinity of HA/TCP/DCPD cylinders used to fill tibial bone defects in senile osteopenic sheep and may be suitable for surgical therapy of critical size, non-load-bearing bone defects in cases of failed tibial head fracture or defect healing.

Systematic Postoperative Assessment of a Minimally-Invasive Sheep Model for the Treatment of Osteochondral Defects.

To assess the clinical course of a sheep stifle joint model for osteochondral (OC) defects, medial femoral condyles (MFC) were exposed without patella luxation using medial parapatellar skin (3-4 cm) and deep incisions (2-3 cm). Two defects (7 mm diameter; 10 mm depth; OC punch) were left empty or refilled with osteochondral autologous transplantation cylinders (OATS) and explanted after six weeks. Incision-to-suture time, anesthesia time, and postoperative wound or impairment scores were compared to those in sham-operated animals. Implant performance was assessed by X-ray, micro-computed tomography, histology, and immunohistology (collagens 1, 2; aggrecan). There were no surgery-related infections or patellar luxations. Operation, anesthesia, and time to complete stand were short (0.5, 1.4, and 1.5 h, respectively). The wound trauma score was low (0.4 of maximally 4; day 7). Empty-defect and OATS animals reached an impairment score of 0 significantly later than sham animals (7.4 and 4.0 days, respectively, versus 1.5 days). Empty defects showed incomplete healing and dedifferentiation/heterotopic differentiation; OATS-filled defects displayed advanced bone healing with remaining cartilage gaps and orthotopic expression of bone and cartilage markers. Minimally-invasive, medial parapatellar surgery of OC defects on the sheep MFC allows rapid and low-trauma recovery and appears well-suited for implant testing.

The old sheep: a convenient and suitable model for senile osteopenia.

INTRODUCTION: Existing osteoporosis models in sheep exhibit some disadvantages, e.g., challenging surgical procedures, serious ethical concerns, failure of reliable induction of substantial bone loss, or lack of comparability to the human condition. This study aimed to compare bone morphological and mechanical properties of old and young sheep, and to evaluate the suitability of the old sheep as a model for senile osteopenia. MATERIALS AND METHODS: The lumbar vertebral body L3 of female merino sheep with two age ranges, i.e., old animals (6-10 years; n = 41) and young animals (2-4 years; n = 40), was analyzed concerning its morphological and mechanical properties by bone densitometry, quantitative histomorphometry, and biomechanical testing of the corticalis and/or central spongious region. RESULTS: In comparison with young sheep, old animals showed only marginally diminished bone mineral density of the vertebral bodies, but significantly decreased structural (bone volume, - 15.1%; ventral cortical thickness, - 11.8%; lateral cortical thickness, - 12.2%) and bone formation parameters (osteoid volume, osteoid surface, osteoid thickness, osteoblast surface, all - 100.0%), as well as significantly increased bone erosion (eroded surface, osteoclast surface). This resulted in numerically decreased biomechanical properties (compressive strength; - 6.4%). CONCLUSION: Old sheep may represent a suitable model of senile osteopenia with markedly diminished bone structure and formation, and substantially augmented bone erosion. The underlying physiological aging concept reduces challenging surgical procedures and ethical concerns and, due to complex alteration of different facets of bone turnover, may be well representative of the human condition.

Maternal psychosocial stress during early gestation impairs fetal structural brain development in sheep.

Maternal stress, especially during early pregnancy, predisposes offspring to neuropsychiatric disorders. We hypothesized that maternal psychosocial stress (MPS) during pregnancy affects fetal structural neurodevelopment depending on the gestational age of exposure. Fetal sheep brains were harvested at 130 days gestation (dG, term 150 dG) from ewes frequently isolated from flock-mates during early gestation (first and second trimester; n = 10) or late gestation (third trimester; n = 10), or from control flock-mates (n = 8). Immunohistochemistry for formation of neuronal processes, myelination, synaptic density, cell proliferation and programed cell death was performed on brain tissue sections. Sections of the cortical gray matter, the hippocampal CA3 region and the superficial, subcortical and deep white matter were examined morphometrically. Stress effects depended on the brain region and time of exposure. Stress during early gestation but not during late gestation reduced the amount of neuronal processes in the cerebral cortex and hippocampus by 36.9 ± 10.1% (p < 0.05, mean ± SEM) and 36.9 ± 15.8% (p < 0.05), respectively, accompanied by a decrease in synaptic density in the cerebral cortex and hippocampus by 39.8 ± 23.1% (p < 0.05) and 32.9 ± 13.4% (p < 0.01). Myelination was decreased in white matter layers on average by 44.8 ± 11.7% (p < 0.05) accompanied by reduced (glial) cell proliferation in the deep white matter by 83.6 ± 12.4% (p < 0.05). In contrast, stress during the third trimester had no effect in any brain region. Chronic MPS during the first and second trimester induced prolonged effects on neuronal network and myelin formation which might contribute to disturbed neurobehavioral, cognitive and motor development in offspring of stressed mothers.Lay summaryMany women are exposed to stressful events during pregnancy. Maternal stress especially during early pregnancy predisposes for offspring's neuropsychiatric disorders. In our sheep study, we show that disturbance of fetal brain development is a potential mechanism and is worst during 1st and 2nd trimester.

Assessment of MR imaging during one-lung flooding in a large animal model.

OBJECTIVE: Magnetic resonance imaging (MRI) of the lung remains challenging due to the low tissue density, susceptibility artefacts, unfavourable relaxation times and motion. Previously, we demonstrated in vivo that one-lung flooding (OLF) with saline is a viable and safe approach. This study investigates the feasibility of OLF in an MRI environment and evaluates the flooding process on MR images. METHODS: OLF of the left lung was performed on five animals using a porcine model. Before, during and after OLF, standard T2w and T1w spin-echo (SE) and gradient-echo (GRE) sequences were applied at 3 T. RESULTS: The procedure was successfully performed in all animals. On T1w MRI, the flooded lung appeared homogenous and isointense with muscle tissue. On T2w images, vascular structures were highly hypointense, while the bronchi were clearly demarcated with hypointense wall and hyperintense lumen. The anatomical demarcation of the flooded lung from the surrounding organs was superior on T2w images. No outflow effects were seen, and no respiration triggering was required. DISCUSSION: OLF can be safely performed in an MR scanner with highly detailed visualization of the pulmonary structures on T2w images. The method provides new approaches to MRI-based image-guided pulmonary interventions using the presented experimental model.

Comparison of Near-Infrared Spectroscopy with Needle Indentation and Histology for the Determination of Cartilage Thickness in the Large Animal Model Sheep.

The suitability of near-infrared spectroscopy (NIRS) for non-destructive measurement of cartilage thickness was compared with the gold standard needle indentation. A combination of NIRS and biomechanical indentation (NIRS-B) was used to address the influence of varying loads routinely applied for hand-guided NIRS during real-life surgery on the accuracy of NIRS-based thickness prediction. NIRS-B was performed under three different loading conditions in 40 osteochondral cylinders from the load-bearing area of the medial and lateral femur condyle of 20 cadaver joints (left stifle joints; female Merino sheep; 6.1 ± 0.6 years, mean ± standard error of the mean). The cartilage thickness measured by needle indentation within the region analyzed by NIRS-B was then compared with cartilage thickness prediction based on NIRS spectral data using partial least squares regression. NIRS-B repeat measurements yielded highly reproducible values concerning force and absorbance. Separate or combined models for the three loading conditions (the latter simulating load-independent measurements) resulted in models with optimized quality parameters (e.g., coefficients of determination R2 between 92.3 and 94.7) and a prediction accuracy of < 0.1 mm. NIRS appears well suited to determine cartilage thickness (possibly in a hand-guided, load-independent fashion), as shown by high reproducibility in repeat measurements and excellent reliability compared with tissue-destructive needle indentation. This may provide the basis for non-destructive, intra-operative assessment of cartilage status quo and fine-tuning of repair procedures.

Evidence for Electronically Driven Ferroelectricity in a Strongly Correlated Dimerized BEDT-TTF Molecular Conductor.

By applying measurements of the dielectric constants and relative length changes to the dimerized molecular conductor κ-(BEDT-TTF)_{2}Hg(SCN)_{2}Cl, we provide evidence for order-disorder type electronic ferroelectricity that is driven by the charge order within the (BEDT-TTF)_{2} dimers and stabilized by a coupling to the anions. According to our density functional theory calculations, this material is characterized by a moderate strength of dimerization. This system thus bridges the gap between strongly dimerized materials, often approximated as dimer-Mott systems at 1/2 filling, and nondimerized or weakly dimerized systems at 1/4 filling, exhibiting a charge order. Our results indicate that intradimer charge degrees of freedom are of particular importance in correlated κ-(BEDT-TTF)_{2}X salts and can create novel states, such as electronically driven multiferroicity or charge-order-induced quasi-one-dimensional spin liquids.

Underlying mechanism of subcortical brain protection during hypoxia and reoxygenation in a sheep model - Influence of α1-adrenergic signalling.

While the cerebral autoregulation sufficiently protects subcortical brain regions during hypoxia or asphyxia, the cerebral cortex is not as adequately protected, which suggests that regulation of the cerebral blood flow (CBF) is area-specific. Hypoxia was induced by inhalation of 5% oxygen, for reoxygenation 100% oxygen was used. Cortical and subcortical CBF (by laser Doppler flowmetry), blood gases, mean arterial blood pressure (MABP), heart rate and renal blood flow were constantly monitored. Low dosed urapidil was used for α1A-adrenergic receptor blockade. Western blotting was used to determine adrenergic receptor signalling mediators in brain arterioles. During hypoxia cortical CBF decreased to 72 ± 11% (mean reduction 11 ± 3%, p < 0.001) of baseline, whereas subcortical CBF increased to 168±18% (mean increase 43 ± 5%, p < 0.001). Reoxygenation led to peak CBF of 194 ± 27% in the subcortex, and restored cortical CBF. α1A-Adrenergic blockade led to minor changes in cortical CBF, but massively reduced subcortical CBF during hypoxia and reoxygenation-almost aligning CBF in both brain regions. Correlation analyses revealed that α1A-adrenergic blockade renders all CBF-responses pressure-passive during hypoxia and reoxygenation, and confirmed the necessity of α1A-adrenergic signalling for coupling of CBF-responses to oxygen saturation. Expression levels and activation state of key signalling-mediators of α1-receptors (NOSs, CREB, ERK1/2) did not differ between cortex and subcortex. The dichotomy between subcortical and cortical CBF during hypoxia and reoxygenation critically depends on α1A-adrenergic receptors, but not on differential expression of signalling-mediators: signalling through the α1A-subtype is a prerequisite for cortical/subcortical redistribution of CBF.

PERKAT RV: first in vivo data of a novel right heart assist device.

AIMS: Mechanical right ventricular (RV) support offers a treatment option for critically ill patients with RV failure (RVF). We developed an assist device for rapid percutaneous implantation. The aim of the present study was to investigate the implantation procedure, haemodynamic performance and possible side effects of the novel right ventricular assist device - PERKAT RV - in an animal model. METHODS AND RESULTS: The PERkutane KATheterpumptechnologie RV (PERKAT RV) device consists of a nitinol chamber covered by foil containing inflow valves. An outlet tube is attached to its distal part. The system is designed for 18 Fr percutaneous implantation. The chamber is unfolded in the inferior vena cava while the outlet tube bypasses the right heart with the tip in the pulmonary trunk. An IABP balloon is placed inside. Balloon deflation generates blood flow into the chamber; during inflation, blood is guided into the pulmonary arteries. Acute RVF was induced by venous injection of Sephadex in seven sheep for evaluation of the device. The PERKAT RV was able to improve haemodynamics immediately generating a median increase in cardiac output of 59%. Longer pump support was evaluated in a second study. Four sheep were supported for eight hours without any problems. CONCLUSIONS: The percutaneous implantation and explantation of the PERKAT RV device was possible in the designed way. The sheep studies proved beneficial haemodynamic effects in acute RVF. The system offers easy and safe treatment in acute RVF.

Impact of chronic maternal stress during early gestation on maternal-fetal stress transfer and fetal stress sensitivity in sheep.

Acute stress-induced reduction of uterine blood flow (UBF) is an indirect mechanism of maternal-fetal stress transfer during late gestation. Effects of chronic psychosocial maternal stress (CMS) during early gestation, as may be experienced by many working women, on this stress signaling mechanism are unclear. We hypothesized that CMS in sheep during early gestation augments later acute stress-induced decreases of UBF, and aggravates the fetal hormonal, cardiovascular, and metabolic stress responses during later development. Six pregnant ewes underwent repeated isolation stress (CMS) between 30 and 100 days of gestation (dGA, term: 150 dGA) and seven pregnant ewes served as controls. At 110 dGA, ewes were chronically instrumented and underwent acute isolation stress. The acute stress decreased UBF by 19% in both the CMS and control groups (p < .05), but this was prolonged in CMS versus control ewes (74 vs. 30 min, p < .05). CMS increased fetal circulating baseline and stress-induced cortisol and norepinephrine concentrations indicating a hyperactive hypothalamus-pituitary-adrenal (HPA)-axis and sympathetic-adrenal-medullary system. Increased fetal norepinephrine is endogenous as maternal catecholamines do not cross the placenta. Cortisol in the control but not in the CMS fetuses was correlated with maternal cortisol blood concentrations; these findings indicate: (1) no increased maternal-fetal cortisol transfer with CMS, (2) cortisol production in CMS fetuses when the HPA-axis is normally inactive, due to early maturation of the fetal HPA-axis. CMS fetuses were better oxygenated, without shift towards acidosis compared to the controls, potentially reflecting adaptation to repeated stress. Hence, CMS enhances maternal-fetal stress transfer by prolonged reduction in UBF and increased fetal HPA responsiveness.

The GDF5 mutant BB-1 enhances the bone formation induced by an injectable, poly(l-lactide-co-glycolide) acid (PLGA) fiber-reinforced, brushite-forming cement in a sheep defect model of lumbar osteopenia.

BACKGROUND CONTEXT: Targeted delivery of osteoinductive bone morphogenetic proteins (eg, GDF5) in bioresorbable calcium phosphate cement (CPC), potentially suitable for vertebroplasty and kyphoplasty of osteoporotic vertebral fractures, may be required to counteract augmented local bone catabolism and to support complete bone regeneration. The biologically optimized GDF5 mutant BB-1 may represent an attractive drug candidate for this purpose. PURPOSE: The aim of the current study was to test an injectable, poly(l-lactide-co-glycolide) acid (PLGA) fiber-reinforced, brushite-forming CPC containing low-dose BB-1 in a sheep lumbar osteopenia model. STUDY DESIGN/ SETTING: This is a prospective experimental animal study. METHODS: Bone defects (diameter 5 mm) were generated in aged, osteopenic female sheep and were filled with fiber-reinforced CPC alone (L4; CPC+fibers) or with CPC containing different dosages of BB-1 (L5; CPC+fibers+BB-1; 5, 100, and 500 µg BB-1; n=6 each). The results were compared with those of untouched controls (L1). Three and 9 months after the operation, structural and functional effects of the CPC (±BB-1) were analyzed ex vivo by measuring (1) bone mineral density (BMD); (2) bone structure, that is, bone volume/total volume (BV/TV) (assessed by micro-CT and histomorphometry), trabecular thickness (Tb.Th), and trabecular number (Tb.N); (3) bone formation, that is, osteoid volume/bone volume (OV/BV), osteoid surface/bone surface (OS/BS), osteoid thickness, mineralizing surface/bone surface (MS/BS), mineral apposition rate, and bone formation rate/bone surface; (4) bone resorption, that is, eroded surface/bone surface; and (5) compressive strength. RESULTS: Compared with untouched controls (L1), CPC+fibers (L4) and/or CPC+fibers+BB-1 (L5) significantly improved all parameters of bone formation, bone resorption, and bone structure. These effects were observed at 3 and 9 months, but were less pronounced for some parameters at 9 months. Compared with CPC without BB-1, additional significant effects of BB-1 were demonstrated for BMD, bone structure (BV/TV, Tb.Th, and Tb.N), and bone formation (OS/BS and MS/BS). The BB-1 effects on bone formation at 3 and 9 months were dose dependent, with 100 µg as the potentially optimal dosage. CONCLUSIONS: BB-1 significantly enhanced the bone formation induced by a PLGA fiber-reinforced CPC in sheep lumbar osteopenia. A single local dose as low as 100 µg BB-1 was sufficient to augment middle- to long-term bone formation. A CPC containing the novel GDF5 mutant BB-1 may thus represent an alternative to the bioinert, supraphysiologically stiff polymethylmethacrylate cement presently used to treat osteoporotic vertebral fractures by vertebroplasty and kyphoplasty.

Fetal Sheep Mesenteric Resistance Arteries: Functional and Structural Maturation.

BACKGROUND: Fetal blood pressure increases during late gestation; however, the underlying vascular mechanisms are unclear. Knowledge of the maturation of resistance arteries is important to identify the mechanisms and vulnerable periods for the development of vascular dysfunction in adulthood. METHODS: We determined the functional and structural development of fetal sheep mesenteric resistance arteries using wire myography and immunohistochemistry. RESULTS: Media mass and distribution of myosin heavy-chain isoforms showed no changes between 0.7 (100 ± 3 days) and 0.9 (130 ± 3 days) gestation. However, from 0.7 to 0.9 gestation, the resting wall tension increased accompanied by non-receptor-dependent (potassium) and receptor-dependent (noradrenaline; endothelin-1) increases in vasocontraction. Angiotensin II had no contractile effect at both ages. Endothelium-dependent relaxation to acetylcholine and prostaglandin E2 was absent at 0.7 but present at 0.9 gestation. Augmented vascular responsiveness was paralleled by the maturation of sympathetic and sensory vascular innervation. Non-endothelium-dependent relaxation to nitric oxide showed no maturational changes. The expression of vasoregulator receptors/enzymes did not increase between 0.7 and 0.9 gestation. CONCLUSION: Vascular maturation during late ovine gestation involves an increase in resting wall tension and the vasoconstrictor and vasodilator capacity of the mesenteric resistance arteries. Absence of structural changes in the tunica media and the lack of an increase in vasoregulator receptor/enzyme expression suggest that vasoactive responses are due to the maturation of intracellular pathways at this gestational age.

Low-dose BMP-2 is sufficient to enhance the bone formation induced by an injectable, PLGA fiber-reinforced, brushite-forming cement in a sheep defect model of lumbar osteopenia.

BACKGROUND CONTEXT: Bioresorbable calcium phosphate cement (CPC) may be suitable for vertebroplasty/kyphoplasty of osteoporotic vertebral fractures. However, additional targeted delivery of osteoinductive bone morphogenetic proteins (BMPs) in the CPC may be required to counteract the augmented local bone catabolism and support complete bone regeneration. PURPOSE: This study aimed at testing an injectable, poly (l-lactide-co-glycolide) acid (PLGA) fiber-reinforced, brushite-forming cement (CPC) containing low-dose bone morphogenetic protein BMP-2 in a sheep lumbar osteopenia model. STUDY DESIGN/ SETTING: This is a prospective experimental animal study. METHODS: Bone defects (diameter 5 mm) were generated in aged, osteopenic female sheep and filled with fiber-reinforced CPC alone (L4; CPC+fibers) or with CPC containing different dosages of BMP-2 (L5; CPC+fibers+BMP-2; 1, 5, 100, and 500 µg BMP-2; n=5 or 6 each). The results were compared with those of untouched controls (L1). Three and 9 months after the operation, structural and functional effects of the CPC (±BMP-2) were analyzed ex vivo by measuring (1) bone mineral density (BMD); (2) bone structure, that is, bone volume/total volume (assessed by micro-computed tomography [micro-CT] and histomorphometry), trabecular thickness, and trabecular number; (3) bone formation, that is, osteoid volume/bone volume, osteoid surface/bone surface, osteoid thickness, mineralizing surface/bone surface, mineral apposition rate, and bone formation rate/bone surface; (4) bone resorption, that is, eroded surface/bone surface; and (5) compressive strength. RESULTS: Compared with untouched controls (L1), CPC+fibers (L4) and/or CPC+fibers+BMP-2 (L5) significantly improved all parameters of bone formation, bone resorption, and bone structure. These effects were observed at 3 and 9 months, but were less pronounced for some parameters at 9 months. Compared with CPC without BMP-2, additional significant effects of BMP-2 were demonstrated for bone structure (bone volume/total volume, trabecular thickness, trabecular number) and formation (osteoid surface/bone surface and mineralizing surface/bone surface), as well as for the compressive strength. The BMP-2 effects on bone formation at 3 and 9 months were dose-dependent, with 5-100 µg as the optimal dosage. CONCLUSIONS: BMP-2 significantly enhanced the bone formation induced by a PLGA fiber-reinforced CPC in sheep lumbar osteopenia. A single local dose as low as ≤100 µg BMP-2 was sufficient to augment middle to long-term bone formation. The novel CPC+BMP-2 may thus represent an alternative to the bioinert, supraphysiologically stiff polymethylmethacrylate cement presently used to treat osteoporotic vertebral fractures by vertebroplasty/kyphoplasty.

In situ foamable, degradable polyurethane as biomaterial for soft tissue repair.

Degradable foams which can be inserted endoscopically as liquid or pasty mixtures into soft tissue defects possess a promising potential for the surgical treatment of such defects. The defects can be sealed under in situ foaming and simultaneous material expansion. We developed an in situ foamable (l-lactide-co-ε-caprolactone)-based, star-shaped prepolymer by ring opening polymerization of l-lactide and ε-caprolactone in the presence of meso-erythritol as starter. By conversion of the terminal hydroxyl groups of the formed oligoester with lysine diisocyanate ethyl ester (LDI) an isocyanate-endcapped, reactive prepolymer has been received. Foaming can be initiated by addition of 1,4-diazabicyclo[2,2,2]octane (DABCO), water, LDI and DMSO. By varying the composition of these additives, the foaming and curing time could be varied within a clinically acceptable range. A porosity of approximately 90%, and an average tensile strength of 0.3MPa with elongations of 90% were determined for the foams. In vitro cytotoxicity on cured foams was assayed on 3T3 fibroblasts and demonstrated an excellent cytocompatibility. This was also confirmed in an in vivo study using an established rat model, where prefabricated foams and in situ hardening material were inserted into subdermal skin incisions in parallel. The feature of chronic inflammation was only weakly developed in both groups and slightly more pronounced and persisted for longer time in the group of in situ foamed material. In both groups the foreign materials were vascularized, degraded and substituted by connective tissue. The results encourage to proceed with trials where the materials are used to fill more heavily loaded defects.

GDF5 significantly augments the bone formation induced by an injectable, PLGA fiber-reinforced, brushite-forming cement in a sheep defect model of lumbar osteopenia.

BACKGROUND CONTEXT: Biodegradable calcium phosphate cement (CPC) represents a promising option for the surgical treatment of osteoporotic vertebral fractures. Because of augmented local bone catabolism, however, additional targeted delivery of bone morphogenetic proteins with the CPC may be needed to promote rapid and complete bone regeneration. PURPOSE: In the present study, an injectable, poly(l-lactide-co-glycolide) acid (PLGA) fiber-reinforced, brushite-forming cement (CPC) containing the bone morphogenetic protein GDF5 was tested in a sheep lumbar osteopenia model. STUDY DESIGN/SETTING: This is a prospective experimental animal study. METHODS: Defined bone defects (diameter 5 mm) were placed in aged, osteopenic female sheep. Defects were treated with fiber-reinforced CPC alone (L4; CPC+fibers) or with CPC containing different dosages of GDF5 (L5; CPC+fibers+GDF5; 1, 5, 100, and 500 µg GDF5; n=5 or 6 each). The results were compared with those of untouched controls (L1). Three and 9 months postoperation, structural and functional effects of the CPC (±GDF5) were assessed ex vivo by measuring (1) bone mineral density (BMD); (2) bone structure, that is, bone volume/total volume (assessed by micro-computed tomography and histomorphometry), trabecular thickness, and trabecular number; (3) bone formation, that is, osteoid volume/bone volume, osteoid surface/bone surface, osteoid thickness, mineralized surface/bone surface, mineral apposition rate, and bone formation rate/bone surface; (4) bone resorption, that is, eroded surface/bone surface; and (5) compressive strength. RESULTS: Compared with untouched controls (L1), both CPC+fibers (L4) and CPC+fibers+GDF5 (L5) numerically or significantly improved all parameters of bone formation, bone resorption, and bone structure. These significant effects were observed both at 3 and 9 months, but for some parameters they were less pronounced at 9 months. Compared with CPC without GDF5, additional significant effects of CPC with GDF5 were demonstrated for BMD and parameters of bone formation and structure (bone volume/total volume, trabecular thickness, and trabecular number, as well as mineralized surface/bone surface). The GDF5 effects were dose-dependent (predominantly in the 5-100 µg range) at 3 and 9 months. CONCLUSIONS: GDF5 significantly enhanced the bone formation induced by a PLGA fiber-reinforced CPC in sheep lumbar osteopenia. The results indicated that a local dose as low as ≤100 µg GDF5 may be sufficient to augment middle to long-term bone formation. The novel CPC+GDF5 combination may thus qualify as an alternative to the bioinert, supraphysiologically stiff poly(methyl methacrylate) cement currently applied for vertebroplasty/kyphoplasty of osteoporotic vertebral fractures.

Redistribution of Cerebral Blood Flow during Severe Hypovolemia and Reperfusion in a Sheep Model: Critical Role of α1-Adrenergic Signaling.

BACKGROUND: Maintenance of brain circulation during shock is sufficient to prevent subcortical injury but the cerebral cortex is not spared. This suggests area-specific regulation of cerebral blood flow (CBF) during hemorrhage. METHODS: Cortical and subcortical CBF were continuously measured during blood loss (≤50%) and subsequent reperfusion using laser Doppler flowmetry. Blood gases, mean arterial blood pressure (MABP), heart rate and renal blood flow were also monitored. Urapidil was used for α1A-adrenergic receptor blockade in dosages, which did not modify the MABP-response to blood loss. Western blot and quantitative reverse transcription polymerase chain reactions were used to determine adrenergic receptor expression in brain arterioles. RESULTS: During hypovolemia subcortical CBF was maintained at 81 ± 6% of baseline, whereas cortical CBF decreased to 40 ± 4% (p < 0.001). Reperfusion led to peak CBFs of about 70% above baseline in both brain regions. α1A-Adrenergic blockade massively reduced subcortical CBF during hemorrhage and reperfusion, and prevented hyperperfusion during reperfusion in the cortex. α1A-mRNA expression was significantly higher in the cortex, whereas α1D-mRNA expression was higher in the subcortex (p < 0.001). CONCLUSIONS: α1-Adrenergic receptors are critical for perfusion redistribution: activity of the α1A-receptor subtype is a prerequisite for redistribution of CBF, whereas the α1D-receptor subtype may determine the magnitude of redistribution responses.

Surgical access via right thoracotomy facilitates tricuspid valve surgery in sheep.

In quadrupeds, the three-dimensional orientation of the heart with respect to the thorax is fundamentally different from that in humans. In this study, we assessed the best surgical approach to the tricuspid valve in sheep. Firstly, different surgical access sites to the tricuspid valve were tested in sheep cadavers, the anatomy was analyzed, and the optimal surgical approach to the tricuspid valve was determined. Secondly - along with cardiopulmonary bypass and cardioplegic arrest -the chosen approach was tested in six adult sheep in vivo. Anatomical analyses revealed that a left thoracotomy provided optimal access to the aorta and left heart. However, visualization of the right heart was significantly impaired. In contrast, a right thoracotomy provided good access to the right heart, but the ascending aorta was difficult to approach. Therefore, in the in vivo studies, arterial cannulation was performed through a carotid (n = 4) or femoral (n = 2) artery. In conclusion, a right-sided thoracotomy allows good visualization of all components of the tricuspid valve complex in sheep, but not of the ascending aorta. Consequently, peripheral vessels are preferred for arterial cannulation. This work may stimulate the investigation of pathomechanisms and/or novel treatment options for tricuspid valve pathologies.

Enhanced bone formation in sheep vertebral bodies after minimally invasive treatment with a novel, PLGA fiber-reinforced brushite cement.

BACKGROUND CONTEXT: Injectable, brushite-forming calcium phosphate cements (CPC) show potential for bone replacement, but they exhibit low mechanical strength. This study tested a CPC reinforced with poly(l-lactide-co-glycolide) acid (PLGA) fibers in a minimally invasive, sheep lumbar vertebroplasty model. PURPOSE: The study aimed to test the in vivo biocompatibility and osteogenic potential of a PLGA fiber-reinforced, brushite-forming CPC in a sheep large animal model. STUDY DESIGN/SETTING: This is a prospective experimental animal study. METHODS: Bone defects (diameter: 5 mm) were placed in aged, osteopenic female sheep, and left empty (L2) or injected with pure CPC (L3) or PLGA fiber-reinforced CPC (L4; fiber diameter: 25 µm; length: 1 mm; 10% [wt/wt]). Three and 9 months postoperation (n=20 each), the structural and functional CPC effects on bone regeneration were documented ex vivo by osteodensitometry, histomorphometry, micro-computed tomography (micro-CT), and biomechanical testing. RESULTS: Addition of PLGA fibers enhanced CPC osteoconductivity and augmented bone formation. This was demonstrated by (1) significantly enhanced structural (bone volume/total volume, shown by micro-CT and histomorphometry; 3 or 9 months) and bone formation parameters (osteoid volume and osteoid surface; 9 months); (2) numerically enhanced bone mineral density (3 and 9 months) and biomechanical compression strength (9 months); and (3) numerically decreased bone erosion (eroded surface; 3 and 9 months). CONCLUSIONS: The PLGA fiber-reinforced CPC is highly biocompatible and its PLGA fiber component enhanced bone formation. Also, PLGA fibers improve the mechanical properties of brittle CPC, with potential applicability in load-bearing areas.

Breakdown of Hooke's law of elasticity at the Mott critical endpoint in an organic conductor.

The Mott metal-insulator transition, a paradigm of strong electron-electron correlations, has been considered as a source of intriguing phenomena. Despite its importance for a wide range of materials, fundamental aspects of the transition, such as its universal properties, are still under debate. We report detailed measurements of relative length changes ΔL/L as a function of continuously controlled helium-gas pressure P for the organic conductor κ-(BEDT-TTF)2Cu[N(CN)2]Cl across the pressure-induced Mott transition. We observe strongly nonlinear variations of ΔL/L with pressure around the Mott critical endpoint, highlighting a breakdown of Hooke's law of elasticity. We assign these nonlinear strain-stress relations to an intimate, nonperturbative coupling of the critical electronic system to the lattice degrees of freedom. Our results are fully consistent with mean-field criticality, predicted for electrons in a compressible lattice with finite shear moduli. We argue that the Mott transition for all systems that are amenable to pressure tuning shows the universal properties of an isostructural solid-solid transition.

Flooded Lung Generates a Suitable Acoustic Pathway for Transthoracic Application of High Intensity Focused Ultrasound in Liver.

Background: In recent years, high intensity focused ultrasound (HIFU) has gained increasing clinical interest as a non-invasive method for local therapy of liver malignancies. HIFU treatment of tumours and metastases in the liver dome is limited due to the adjacent ultrasound blocking lung. One-lung flooding (OLF) enables complete sonography of lung and adjoining organs including liver. HIFU liver ablation passing through the flooded lung could enable a direct intercostal beam path and thus improve dose deposition in liver. In this study, we evaluate the feasibility of an ultrasound guided transthoracic, transpulmonary HIFU ablation of liver using OLF. Methods: After right-side lung flooding, ultrasound guided HIFU was applied transthoracic- transpulmonary into liver to create thermal lesions in three pigs. The HIFU beam was targeted five times into liver, two times at the liver surface and three times deeper into the tissue. During autopsy examinations of lung, diaphragm and liver located in the HIFU path were performed. The focal liver lesions and lung tissue out of the beam path were examined histologically. Results: Fifteen thermal liver lesions were generated by transpulmonary HIFU sonication in all targeted regions. The lesions appeared well-demarcated in grey color with a cigar-shaped configuration. The mean length and width of the superficial and deeper lesions were 15.8 mm (range: 13-18 mm) and 5.8 mm (range: 5-7 mm), and 10.9 mm (range: 9-13 mm) and 3.3 mm (range: 2-5 mm), respectively. Histopathological, all liver lesions revealed a homogeneous thermal necrosis lacking vitality. There were no signs of damage of the overlying diaphragm and lung tissue. Conclusions: Flooded lung is a suitable pathway for applying HIFU to the liver, thus enabling a transthoracic, transpulmonary approach. The enlarged acoustic window could enhance the ablation speed for targets in the hepatic dome.