Phenotypic screening of seed retention and histological analysis of the abscission zone in Festuca pratensis and Lolium perenne.

BACKGROUND: Seed retention is the basic prerequisite for seed harvest. However, only little breeding progress has been achieved for this trait in the major forage grasses. The aim of this study was to evaluate the potential of plant genetic resources of the important fodder grasses Festuca pratensis Huds. and Lolium perenne L. as source for seed retention in the breeding process. Furthermore, the morphology of the abscission zone, where shattering occurs, was studied on the cell tissue level in different developmental stages of contrasting accessions. RESULTS: 150 and 286 accessions of Festuca pratensis and Lolium perenne were screened for seed retention, respectively. Contrasting accessions were selected to be tested in a second year. We found a great variation in seed retention in Festuca pratensis and Lolium perenne, ranging from 13 to 71% (average: 35%) and 12 to 94% (average: 49%), respectively, in the first year. Seed retention was generally lower in the second year. Cultivars were within the accessions with highest seed retention in Festuca pratensis, but had lower seed retention than ecotypes in Lolium perenne. Field-shattered seeds had a lower thousand grain weight than retained seeds. Cell layers of the abscission zone appeared already in early seed stages and were nested within each other in accessions with high seed retention, while there were two to three superimposed layers in accessions with low seed retention. CONCLUSIONS: Plant genetic resources of Lolium perenne might be a valuable source for breeding varieties with high seed retention. However, simultaneous selection for high seed weight is necessary for developing successful commercial cultivars.

Microdialysis Reveals Anti-Inflammatory Effects of Sulfated Glycosaminoglycanes in the Early Phase of Bone Healing.

Although chronic inflammation inhibits bone healing, the healing process is initiated by an inflammatory phase. In a well-tuned sequence of molecular events, pro-inflammatory cytokines are secreted to orchestrate the inflammation response to injury and the recruitment of progenitor cells. These events in turn activate the secretion of anti-inflammatory signaling molecules and attract cells and mediators that antagonize the inflammation and initiate the repair phase. Sulfated glycosaminoglycanes (sGAG) are known to interact with cytokines, chemokines and growth factors and, thus, alter the availability, duration and impact of those mediators on the local molecular level. sGAG-coated polycaprolactone-co-lactide (PCL) scaffolds were inserted into critical-size femur defects in adult male Wistar rats. The femur was stabilized with a plate, and the defect was filled with either sGAG-containing PCL scaffolds or autologous bone (positive control). Wound fluid samples obtained by microdialysis were characterized regarding alterations of cytokine concentrations over the first 24 h after surgery. The analyses revealed the inhibition of the pro-inflammatory cytokines IL-1ß and MIP-2 in the sGAG-treated groups compared to the positive control. A simultaneous increase of IL-6 and TNF-α indicated advanced regenerative capacity of sGAG, suggesting their potential to improve bone healing.

Impact of Sulfated Hyaluronan on Bone Metabolism in Diabetic Charcot Neuroarthropathy and Degenerative Arthritis.

Bone in diabetes mellitus is characterized by an altered microarchitecture caused by abnormal metabolism of bone cells. Together with diabetic neuropathy, this is associated with serious complications including impaired bone healing culminating in complicated fractures and dislocations, especially in the lower extremities, so-called Charcot neuroarthropathy (CN). The underlying mechanisms are not yet fully understood, and treatment of CN is challenging. Several in vitro and in vivo investigations have suggested positive effects on bone regeneration by modifying biomaterials with sulfated glycosaminoglycans (sGAG). Recent findings described a beneficial effect of sGAG for bone healing in diabetic animal models compared to healthy animals. We therefore aimed at studying the effects of low- and high-sulfated hyaluronan derivatives on osteoclast markers as well as gene expression patterns of osteoclasts and osteoblasts from patients with diabetic CN compared to non-diabetic patients with arthritis at the foot and ankle. Exposure to sulfated hyaluronan (sHA) derivatives reduced the exaggerated calcium phosphate resorption as well as the expression of genes associated with bone resorption in both groups, but more pronounced in patients with CN. Moreover, sHA derivatives reduced the release of pro-inflammatory cytokines in osteoclasts of patients with CN. The effects of sHA on osteoblasts differed only marginally between patients with CN and non-diabetic patients with arthritis. These results suggest balancing effects of sHA on osteoclastic bone resorption parameters in diabetes.

Men who stare at bone: multimodal monitoring of bone healing.

Knowledge of the physiological and pathological processes, taking place in bone during fracture healing or defect regeneration, is essential in order to develop strategies to enhance bone healing under normal and critical conditions. Preclinical testing allows a wide range of imaging modalities that may be applied both simultaneously and longitudinally, which will in turn lower the number of animals needed to allow a comprehensive assessment of the healing process. This work provides an up-to-date review on morphological, functional, optical, biochemical, and biophysical imaging techniques including their advantages, disadvantages and potential for combining them in a multimodal and multiscale manner. The focus lies on preclinical testing of biomaterials modified with artificial extracellular matrices in various animal models to enhance bone remodeling and regeneration.

A stereotaxic breed-averaged, symmetric T2w canine brain atlas including detailed morphological and volumetrical data sets.

Stereotaxic systems and automatic tissue segmentation routines enable neuronavigation as well as reproducible processing of neuroimage datasets. Such systems have been developed for humans, non-human-primates, sheep, and rodents, but not for dogs. Although dogs share important neurofunctional and -anatomical features with humans, and in spite of their importance in translational neuroscience, little is known about the variability of the canine brain morphology and, possibly related, function. Moreover, we lack templates, tissue probability maps (TPM), and stereotaxic brain labels for implementation in standard software utilities such as Statistical Parametric Mapping (SPM). Hence, objective and reproducible, image-based investigations are currently impeded in dogs. We have created a detailed stereotaxic reference frame for dogs including TPM and tissue labels, enabling inter-individual and cross-study neuroimage analysis. T2w datasets were acquired from 16 neurologically inconspicuous dogs of different breeds by 3T MRI. The datasets were averaged after initial preprocessing using linear and nonlinear registration algorithms as implemented in SPM8. TPM for gray (GM) and white matter (WM) as well as cerebrospinal fluid (CSF) were created. Different cortical, subcortical, medullary, and CSF regions were manually labeled to create a spatial binary atlas being aligned with the template. A proof-of-concept for automatic determination of morphological and volumetrical characteristics was performed using additional canine datasets (n = 64) including a subgroup of laboratory beagles (n = 24). Overall, 21 brain regions were labeled using the segmented tissue classes of the brain template. The proof-of-concept trial revealed excellent suitability of the created tools for image processing and subsequent analysis. There was high intra-breed variability in frontal lobe and hippocampus volumes, and noticeable inter-breed corpus callosum volume variation. The T2w brain template provides important, breed-averaged canine brain anatomy features in a spatial standard coordinate system. TPM allows automatic tissue segmentation using SPM and enables unbiased automatic image processing or morphological characterization in different canine breeds. The reported volumetric and morphometric results may serve as a starting point for further research aimed at in vivo analysis of canine brain anatomy and function.

A supplement-free osteoclast-osteoblast co-culture for pre-clinical application.

There is increasing demand for efficient and physiological in vitro cell culture systems suitable for testing new pharmaceutical drugs or for evaluating materials for tissue regeneration. In particular, co-cultures of two or more tissue-relevant cell types have the advantage to study the response of cells on diverse parameters in a more natural environment with respect to physiological complexity. We developed a direct bone cell co-culture system using human peripheral blood monocytes (hPBMC) and human bone marrow stromal cells (hBMSC) as osteoclast/osteoblast precursor cells, respectively, strictly avoiding external supplements for the induction of differentiation. The sophisticated direct hPBMC/hBMSC co-culture was characterized focusing on osteoclast function and was compared with two indirect approaches. Only in the direct co-culture, hPBMC were triggered by hBMSC into osteoclastogenesis and became active resorbing osteoclasts. Bisphosphonates and sulfated glycosaminoglycans were used to examine the suitability of the co-culture system for evaluating the influence of certain effectors on bone healing and bone regeneration and the contribution of each cell type thereby. The results show that the investigated substances had more pronounced effects on both osteoblasts and osteoclasts in the co-culture system than in respective monocultures.

DArT, SNP, and SSR analyses of genetic diversity in Lolium perenne L. using bulk sampling.

BACKGROUND: Lolium perenne L. is the most important forage grass species in temperate regions. It is also considered as a sustainable source of biomass for energy production. However, improvement in biomass yield has been limited by comparison with other major crops. More efficient utilisation of genetic resources and improved breeding schemes are required to advance L. perenne breeding. In an attempt to elucidate the extent of genetic diversity in L. perenne, 1384 DArT, 182 SNP and 48 SSR markers were applied to 297 accessions (Set I) contributed by three German breeding companies and the IPK Genebank. Due to the heterogeneous nature of Lolium accessions, bulk samples were used. Apart from germplasm set I, additional set II and set III was used to determine the reproducibility of marker system and judge the feasibility of bulk strategy in this study. RESULTS: By assessing different bulk sizes, 24 individuals per sample were shown to be a representative number of plants to discriminate different accessions. Among the 297 accessions, all marker types revealed a high polymorphism rate; 1.99, 2.00 and 8.19 alleles, were obtained per locus on average using DArTs, SNPs and SSRs, respectively. The Jaccard distance for DArT markers ranged from 0.00 to 0.73, the Modified Roger's distance (MRD) for SNP markers ranged from 0.03 to 0.52, and for SSR markers from 0.26 to 0.76. Gene diversity for dominant DArT and co-dominant SNP and SSR markers was found to be 0.26, 0.32 and 0.45, respectively. DArT markers showed the highest consistency and reproducibility. CONCLUSION: The resulting data were evaluated using a number of different classification methods, but none of the methods showed a clear differentiation into distinct genetic pools. With regard to hybrid breeding, this will possibly impede substantial progress towards increased biomass yields of L. perenne by utilising heterosis.

Reduced cingulate gyrus volume in Cavalier King Charles Spaniels with syringomyelia and neuropathic pain revealed by voxel-based morphometry: a pilot study.

Objective: Pathomorphological alterations of the central nervous system in dogs, such as syringomyelia and Chiari-like malformation, can cause cranial and cervical hyperesthesia and neuropathic pain. The long-term activity of the pain network can induce functional alteration and eventually even morphological changes in the pain network. This may happen especially in the prefrontal and cingulate cortex, where atrophy of the gray matter (GM) was observed in humans with chronic pain, irrespective of the nature of the pain syndrome. We tested the hypothesis that Cavalier King Charles Spaniels (CKCS) with Chiari-like malformation and associated syringomyelia (SM) and pain show cerebral morphological differences compared to animals without signs of syringomyelia and pain. Methods: Volumetric datasets of 28 different brain structures were analyzed in a retrospective manner, including voxel-based morphometry, using magnetic resonance imaging data obtained from 41 dogs. Results: Volumetric analyses revealed a decrease in GM volumes in the cingulate gyrus (CG) in CKCS with SM and chronic pain when normalized to brain volume. This finding was supported by voxel-based morphometry, which showed a cluster of significance within the CG. Conclusion: GM atrophy in the CG is associated with chronic pain and thus may serve as an objective readout parameter for the diagnosis or treatment of canine pain syndromes.

Lab-in-a-tube: detection of individual mouse cells for analysis in flexible split-wall microtube resonator sensors.

We report a method for the precise capturing of embryonic fibroblast mouse cells into rolled-up microtube resonators. The microtubes contain a nanometer-sized gap in their wall which defines a new type of optofluidic sensor, i.e., a flexible split-wall microtube resonator sensor, employed as a label-free fully integrative detection tool for individual cells. The sensor action works through peak sharpening and spectral shifts of whispering gallery modes within the microresonators under light illumination.

Continuous hemilaminectomy of nine vertebrae can be performed safely in large breed dogs: A case report of a German Shepherd Dog with intervertebral disc extrusion and extensive extradural hemorrhage.

Background: Extended, continuous hemilaminectomy has only been reported in small to medium-sized dogs so far. It remains unclear whether excessive continuous hemilaminectomy can also be performed safely in large breed dogs. Case Description: We describe the surgical treatment and clinical outcome of a 5-year-old German Shepherd Dog that presented with paraplegia and deep pain perception following a short episode of bilateral hind-limb lameness, secondary to jumping off of a car. Computed tomography-myelography revealed that the paraplegia originated from extensive extradural spinal cord compression (Th6-L1), due to intervertebral disc extrusion and associated epidural hemorrhage. The dog was treated with a continuous hemilaminectomy involving nine vertebrae (Th6-L1) and recovered completely with no remaining neurological deficits, within 6 months. Conclusion: The rapid, uncomplicated, and complete functional recovery in the presented case emphasizes the practicability of extensive, continuous hemilaminectomies, also in large breed dogs.

Mineralizing Gelatin Microparticles as Cell Carrier and Drug Delivery System for siRNA for Bone Tissue Engineering.

The local release of complexed siRNA from biomaterials opens precisely targeted therapeutic options. In this study, complexed siRNA was loaded to gelatin microparticles cross-linked (cGM) with an anhydride-containing oligomer (oPNMA). We aggregated these siRNA-loaded cGM with human mesenchymal stem cells (hMSC) to microtissues and stimulated them with osteogenic supplements. An efficient knockdown of chordin, a BMP-2 antagonist, caused a remarkably increased alkaline phosphatase (ALP) activity in the microtissues. cGM, as a component of microtissues, mineralized in a differentiation medium within 8-9 days, both in the presence and in the absence of cells. In order to investigate the effects of our pre-differentiated and chordin-silenced microtissues on bone homeostasis, we simulated in vivo conditions in an unstimulated co-culture system of hMSC and human peripheral blood mononuclear cells (hPBMC). We found enhanced ALP activity and osteoprotegerin (OPG) secretion in the model system compared to control microtissues. Our results suggest osteoanabolic effects of pre-differentiated and chordin-silenced microtissues.

Cyclam with a phosphinate-bis(phosphonate) pendant arm is a bone-targeting carrier of copper radionuclides.

Ligands combining a bis(phosphonate) group with a macrocycle function as metal isotope carriers for radionuclide-based imaging and for treating bone metastases associated with several cancers. However, bis(phosphonate) pendant arms often slow down complex formation and decrease radiochemical yields. Nevertheless, their negative effect on complexation rates may be mitigated by using a suitable spacer between bis(phosphonate) and the macrocycle. To demonstrate the potential of bis(phosphonate) bearing macrocyclic ligands as a copper radioisotope carrier, we report the synthesis of a new cyclam derivative bearing a phosphinate-bis(phosphonate) pendant (H5te1PBP). The ligand showed a high selectivity to CuII over ZnII and NiII ions, and the bis(phosphonate) group was not coordinated in the CuII complex, strongly interacting with other metal ions in solution. The CuII complex formed quickly, in 1 s, at pH 5 and at a millimolar scale. The complexation rates significantly differed under a ligand or metal ion excess due to the formation of reaction intermediates differing in their metal-to-ligand ratio and protonation state, respectively. The CuII-te1PBP complex also showed a high resistance to acid-assisted hydrolysis (t1/2 2.7 h; 1 M HClO4, 25 °C) and was effectively adsorbed on the hydroxyapatite surface. H5te1PBP radiolabeling with [64Cu]CuCl2 was fast and efficient, with specific activities of approximately 30 GBq 64Cu per 1 µmol of ligand (pH 5.5, room temperature, 30 min). In a pilot experiment, we further demonstrated the excellent suitability of [64Cu]CuII-te1PBP for imaging active bone compartments by dedicated small animal PET/CT in healthy mice and subsequently in a rat femoral defect model, in direct comparison with [18F]fluoride. Moreover, [64Cu]CuII-te1PBP showed a higher uptake in critical bone defect regions. Therefore, our study highlights the potential of [64Cu]CuII-te1PBP as a PET radiotracer for evaluating bone healing in preclinical and clinical settings with a diagnostic value similar to that of [18F]fluoride, albeit with a longer half-life (12.7 h) than 18F (1.8 h), thereby enabling extended observation times.

Adjuvant Drug-Assisted Bone Healing: Advances and Challenges in Drug Delivery Approaches.

Bone defects of critical size after compound fractures, infections, or tumor resections are a challenge in treatment. Particularly, this applies to bone defects in patients with impaired bone healing due to frequently occurring metabolic diseases (above all diabetes mellitus and osteoporosis), chronic inflammation, and cancer. Adjuvant therapeutic agents such as recombinant growth factors, lipid mediators, antibiotics, antiphlogistics, and proangiogenics as well as other promising anti-resorptive and anabolic molecules contribute to improving bone healing in these disorders, especially when they are released in a targeted and controlled manner during crucial bone healing phases. In this regard, the development of smart biocompatible and biostable polymers such as implant coatings, scaffolds, or particle-based materials for drug release is crucial. Innovative chemical, physico- and biochemical approaches for controlled tailor-made degradation or the stimulus-responsive release of substances from these materials, and more, are advantageous. In this review, we discuss current developments, progress, but also pitfalls and setbacks of such approaches in supporting or controlling bone healing. The focus is on the critical evaluation of recent preclinical studies investigating different carrier systems, dual- or co-delivery systems as well as triggered- or targeted delivery systems for release of a panoply of drugs.

The influence of different artificial extracellular matrix implant coatings on the regeneration of a critical size femur defect in rats.

Resorbable biomaterials based on artificial extracellular matrices (aECM) represent promising scaffolds for the treatment of large bone defects. Here, we investigated various glycosaminoglycan (GAG) derivatives of varying sulfation degree with respect to their influence on in vivo bone healing. The materials used in this study consisted of GAG-coated degradable polycaprolactone-co-lactide (PCL). Critical size femur defects in rats were filled with autologous bone serving as positive control or the respective coated or uncoated PCL scaffolds. After 2 and 12 weeks, progress in the healing process was investigated by analyzing the new bone matrix formation, the collagen content and hydroxyapatite formation by using micro-computed tomography (µCT), biomechanical testing, nuclear magnetic resonance spectroscopy (NMR) and histology. The sulfated GAG coating contributed substantially to bone regeneration, increased collagen synthesis and initiated mineralization of the organic matrix. Most substantial collagen production was detected in scaffolds coated with chondroitin sulfate. Scaffolds coated with hypersulfated hyaluronan induced formation of new bone volume comparable to what was observed in the positive control. GAG differing in the sugar backbone and degree of sulfation modulate the healing process at different times, eventually leading to improved bone healing.

Adjuvant drug-assisted bone healing: Part I - Modulation of inflammation.

 Critical-size bone defects after compound fractures, infection, or tumor resection are challenging to treat. The same is true for fractures in patients with impaired bone healing due to metabolic diseases and cancer. Despite considerable progress over the last decade in surgical techniques, material design, and dedicated imaging approaches, these scenarios represent unsolved clinical problems. The high socioeconomic burden of such conditions justifies increasing interest in novel osteoinductive drugs for adjuvant therapeutic approaches. There is an increasing body of experimental and clinical literature on potentially promising effects of growth factors, anti-resorptive, and anabolic agents. The true clinical efficacy of these, however, is discussed controversially. Therefore, we aimed to critically examine the hypothesis that targeted adjuvant therapies have the potential to enhance bone regeneration in critical-size bone defects and under systemic conditions that impair bone healing. This first approach to the topic deals with small molecule drugs and compounds that influence the immune response and inflammatory processes. In particular, literature reporting on selective cyclooxygenase-2 inhibitors has been reviewed with respect to their local and systemic mode of action and to stimulate further research on bone healing under critical conditions.

Adjuvant drug-assisted bone healing: Part II - Modulation of angiogenesis.

 The treatment of critical-size bone defects following complicated fractures, infections or tumor resections is a major challenge. The same applies to fractures in patients with impaired bone healing due to systemic inflammatory and metabolic diseases. Despite considerable progress in development and establishment of new surgical techniques, design of bone graft substitutes and imaging techniques, these scenarios still represent unresolved clinical problems. However, the development of new active substances offers novel potential solutions for these issues. This work discusses therapeutic approaches that influence angiogenesis or hypoxic situations in healing bone and surrounding tissue. In particular, literature on sphingosine-1-phosphate receptor modulators and nitric oxide (NO•) donors, including bi-functional (hybrid) compounds like NO•-releasing cyclooxygenase-2 inhibitors, was critically reviewed with regard to their local and systemic mode of action.

Adjuvant drug-assisted bone healing: Part III - Further strategies for local and systemic modulation.

In this third in a series of reviews on adjuvant drug-assisted bone healing, further approaches aiming at influencing the healing process are discussed. Local and systemic modulation of bone metabolism is pursued with use of a number of drugs with completely different indications, which are characterized by a pleiotropic spectrum of action. These include drugs used to treat lipid disorders (HMG-CoA reductase inhibitors), hypertension (ACE inhibitors), osteoporosis (bisphosphonates), cancer (proteasome inhibitors) and others. Potential applications to enhance bone healing are discussed.

Biomaterials in repairing rat femoral defects: In vivo insights from small animal positron emission tomography/computed tomography (PET/CT) studies.

Biomaterials coated with artificial extracellular matrices (aECM) are intended to support the healing of critical size bone defects. This pilot study investigated (i) the feasibility of dual-tracer PET/CT imaging for functional characterization of biomaterial-assisted bone healing in a rat femoral defect model and (ii) the bone healing ability of polycaprolactone-co-lactide (PCL) scaffolds, coated with various aECM consisting of collagen type I (Col) and glycosaminoglycans (GAGs) such as chondroitin sulfate (CS) or polysulfated hyaluronan (sHA3). [18F]FDG and [18F]fluoride PET 4 and 8 weeks after implantation of aECM-coated PCL scaffolds, which provide an in vivo measure of cellular activation and bone mineralization, respectively, combined with CT imaging (in vivo/ex vivo) and histological/immunohistochemical investigations (ex vivo) showed that coating with CS in particular is beneficial for bone healing. The possible involvement of COX-2 and TGase 2, key enzymes of inflammation and ECM remodeling, in these processes offers starting points for targeted adjuvant therapy in the course of various bone healing phases. Our investigations show the feasibility of the selected dual-tracer approach for PET/CT imaging. In principle, this approach can be extended by further PET tracers for the functional characterization of physiological processes such as hypoxia/reperfusion or selected molecular players.

Disruption of the MAP1B-related protein FUTSCH leads to changes in the neuronal cytoskeleton, axonal transport defects, and progressive neurodegeneration in Drosophila.

The elaboration of neuronal axons and dendrites is dependent on a functional cytoskeleton. Cytoskeletal components have been shown to play a major role in the maintenance of the nervous system through adulthood, and changes in neurofilaments and microtubule-associated proteins (MAPs) have been linked to a variety of neurodegenerative diseases. Here we show that Futsch, the fly homolog of MAP1B, is involved in progressive neurodegeneration. Although Futsch is widely expressed throughout the CNS, degeneration in futsch(olk) primarily occurs in the olfactory system and mushroom bodies. Consistent with the predicted function of Futsch, we find abnormalities in the microtubule network and defects in axonal transport. Degeneration in the adult brain is preceded by learning deficits, revealing a neuronal dysfunction before detectable levels of cell death. Futsch is negatively regulated by the Drosophila Fragile X mental retardation gene, and a mutation in this gene delays the onset of neurodegeneration in futsch(olk). A similar effect is obtained by expression of either fly or bovine tau, suggesting a certain degree of functional redundancy of MAPs. The futsch(olk) mutants exhibit several characteristics of human neurodegenerative diseases, providing an opportunity to study the role of MAPs in progressive neurodegeneration within an experimentally accessible, in vivo model system.

Prevalence of syringomyelia in clinically unaffected Cavalier King Charles Spaniels in Germany (2006-2016).

OBJECTIVE: The aim of the study was to investigate the prevalence of syringomyelia in clinically unaffected Cavalier King Charles Spaniels (CKCS) in Germany. MATERIAL AND METHODS: From 2006 to 2016 a total of 339 asymptomatic CKCSs from all over Germany were included. Age ranged from 3 months to 11 years (mean 3.72 years ± 2.17 years). T1- and T2-weighted magnetic resonance images of the head and cervical spine were obtained. RESULTS: Overall, 163 out of 339 (48.1 %) CKCSs showed evidence of syringomyelia. The results (odds ratio of 1.27 per year; p < 0.0001) corroborate the findings of other studies, in which the risk of developing syringomyelia increases with age. CONCLUSION AND CLINICAL RELEVANCE: In conclusion, around half of the dogs within the German CKCS population are affected by syringomyelia. The prevalence of syringomyelia of the German CKCS population is similar to that of the British CKCS population, which is subjected to breeding strategies on the basis of magnetic resonance imaging investigations.