Evaluation of processed bovine cancellous bone matrix seeded with syngenic osteoblasts in a critical size calvarial defect rat model

INTRODUCTION: Biologic bone substitutes may offer alternatives to bone grafting procedures. The aim of this study was to evaluate a preformed bone substitute based on processed bovine cancellous bone (PBCB) with or without osteogenic cells in a critical size calvarial defect rat model. METHODS: Discs of PBCB (Tutobone) were seeded with second passage fibrin gel-immobilized syngenic osteoblasts (group A, n = 40). Cell-free matrices (group B, n = 28) and untreated defects (group C; n=28) served as controls. Specimens were explanted between day 0 and 4 months after implantation and were subjected to histological and morphometric evaluation. RESULTS: At 1 month, bone formation was limited to small peripheral areas. At 2 and 4 months, significant bone formation, matrix resorption as well as integration of the implants was evident in groups A and B. In group C no significant regeneration of the defects was observed. Morphometric analysis did not disclose differences in bone formation in matrices from groups A and B. Carboxyfluorescine-Diacetate-Succinimidylester (CFDA) labeling demonstrated low survival rates of transplanted cells. DISCUSSION: Osteoblasts seeded into PBCB matrix display a differentiated phenotype following a 14 days cell culture period. Lack of initial vascularization may explain the absence of added osteogenicity in constructs from group A in comparison to group B. PBCB is well integrated and represents even without osteogenic cells a promising biomaterial for reconstruction of critical size calvarial bone defects.     

Biomechanical model predicts directional tuning of spindles in finger muscles facilitates precision pinch and power grasp

Humans have a sense of static limb position derived primarily from the output of secondary muscle spindle endings. The features of finger pose these proprioceptors signal best were predicted by singular value decomposition of a kinematic model of the human long finger and the six muscles that actuate it. The analysis indicated that muscle spindles signal the location of the fingertip with less error than they signal angles of individual finger joints. The fingertip displacements for which proprioceptors have greatest sensitivity were also predicted. These fingertip displacements seem to correspond to the fine positioning of an object pinched between the fingertip and distal phalanx of the thumb. The analysis also predicted the directions in which subjects can displace the fingertip most rapidly. The directions seem to correspond to rapid closure of precision pinch or power grasp.     

Visual spectral sensitivity of hatchling loggerhead (Caretta caretta L.) and leatherback (Dermochelys coriacea L.) sea turtles,as determined by single-flash electroretinography

Electroretinographic recordings were made from hatchling loggerhead and leatherback sea turtle eyecup preparations to generate dark-adapted spectral sensitivity curves. Both species were maximally sensitive to wavelengths between 500 and 540 nm, with a secondary peak near 380 nm. The spectral sensitivity curve for leatherbacks was attenuated at the long wavelength end of the spectrum relative to that of the loggerheads. This difference may reflect adaptations to lighting available at the relatively shallow (loggerhead) versus deeper (leatherback) sites where each species forages. The broad spectrum of wavelengths detected by both species (near UV to yellow–orange) indicates that vision is likely mediated by more than one photopigment, potentially rendering these turtles capable of color vision.     

Identification and characterisation of a novel adhesin Ifp in <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis>

Background  

In order to identify new virulence determinants in Y. pseudotuberculosis a comparison between its genome and that of Yersinia pestis was undertaken. This reveals dozens of pseudogenes in Y. pestis, which are still putatively functional in Y. pseudotuberculosis and may be important in the enteric lifestyle. One such gene, YPTB1572 in the Y. pseudotuberculosis IP32953 genome sequence, encodes a protein with similarity to invasin, a classic adhesion/invasion protein, and to intimin, the attaching and effacing protein from enteropathogenic (EPEC) and enterohaemorraghic (EHEC) Escherichia coli.     

Directly auto‐transplanted mesenchymal stem cells induce bone formation in a ceramic bone substitute in an ectopic sheep model

Bone tissue engineering approaches increasingly focus on the use of mesenchymal stem cells (MSC). In most animal transplantation models MSC are isolated and expanded before auto cell transplantation which might be critical for clinical application in the future. Hence this study compares the potential of directly auto‐transplanted versus in vitro expanded MSC with or without bone morphogenetic protein‐2 (BMP‐2) to induce bone formation in a large volume ceramic bone substitute in the sheep model. MSC were isolated from bone marrow aspirates and directly auto‐transplanted or expanded in vitro and characterized using fluorescence activated cell sorting (FACS) and RT‐PCR analysis before subcutaneous implantation in combination with BMP‐2 and β‐tricalcium phosphate/hydroxyapatite (β‐TCP/HA) granules. Constructs were explanted after 1 to 12 weeks followed by histological and RT‐PCR evaluation. Sheep MSC were CD29⁺, CD44⁺ and CD166⁺ after selection by Ficoll gradient centrifugation, while directly auto‐transplanted MSC‐populations expressed CD29 and CD166 at lower levels. Both, directly auto‐transplanted and expanded MSC, were constantly proliferating and had a decreasing apoptosis over time in vivo. Directly auto‐transplanted MSC led to de novo bone formation in a heterotopic sheep model using a β‐TCP/HA matrix comparable to the application of 60 μg/ml BMP‐2 only or implantation of expanded MSC. Bone matrix proteins were up‐regulated in constructs following direct auto‐transplantation and in expanded MSC as well as in BMP‐2 constructs. Up‐regulation was detected using immunohistology methods and RT‐PCR. Dense vascularization was demonstrated by CD31 immunohistology staining in all three groups. Ectopic bone could be generated using directly auto‐transplanted or expanded MSC with β‐TCP/HA granules alone. Hence BMP‐2 stimulation might become dispensable in the future, thus providing an attractive, clinically feasible approach to bone tissue engineering.     

Regression and persistence: remodelling in a tissue engineered axial vascular assembly

In later stages of vasculoangiogenesis a vascular network is going through a metamorphosis for optimal perfusion and economy of energy. In this study we make a quantitative approach to phenomena of remodelling in a bioartificial neovascular network and suggest variance of calibre as a parameter of neovascular maturation. For this study, 18 male Lewis rats were subjected to the AV loop operation in combination with a hard porous biogenic matrix and an isolation chamber. The animals were allocated into three groups for different explantation intervals set to 2, 4 and 8 weeks, respectively. Collective attributes like vascular density, percent fractional area and variance of calibre were evaluated for a predefined region of interest (ROI). Late morphogenesis was evaluated by means of scanning electron microscopy. After the fourth week the absolute number of vessels within the ROI decreased (P < 0.03) whereas, on the contrary, the fractional area of all segments increased (P < 0.02). The variance in calibre was significantly increased in the 8‐week group (P < 0.05). Lymphatic growth after week 4, early pericyte migration as well as intussusceptive angiogenesis were identified immunohistologically. Phenomena of remodelling were evaluated quantitatively in a neovascular network and variance could be proposed as a parameter of net vascular maturation.     

Gene expression analysis of ischaemia and reperfusion in human microsurgical free muscle tissue transfer

The aim of this study was to analyse various gene expression profiles of muscle tissue during normoxia, ischaemia and after reperfusion in human muscle free flaps, to gain an understanding of the occurring regulatory, inflammatory and apoptotic processes on a cellular and molecular basis. Eleven Caucasian patients with soft tissue defects needing coverage with microsurgical free muscle flaps were included in this study. In all patients, the muscle samples were taken from free myocutaneous flaps. The first sample was taken before induction of ischaemia in normoxia (I), another one after ischaemia (II), and the last one was taken after reperfusion (III). The samples were analysed using DNA‐microarray, real‐time‐quantitative‐PCR and immunohistochemistry. DNA‐microarray analysis detected multiple, differentially regulated genes when comparing the different groups (I–III) with statistical significance. Comparing ischaemia (II) versus normoxia (I) educed 13 genes and comparing reperfusion (III) versus ischaemia (II) educed 19 genes. The comparison of reperfusion (III) versus normoxia (I) yielded 100 differentially regulated genes. Real‐time‐quantitative‐PCR confirmed the results of the DNA‐microarrays for a subset of four genes (CASP8, IL8, PLAUR and S100A8). This study shows that ischaemia and reperfusion induces alterations on the gene expression level in human muscle free flaps. Data may suggest that the four genes CASP8, IL8, PLAUR and S100A8 are of great importance in this context. We could not confirm the DNA‐microarry and real‐time‐quantitative‐PCR results on the protein level. Finally, these findings correspond with the surgeon’s clinical experience that the accepted times of ischaemia, generally up to 90 min., are not sufficient to induce pathophysiological processes, which can ultimately lead to flap loss. When inflammatory and apoptotic proteins are expressed at high levels, flap damage might occur and flap loss is likely. The sole expression on mRNA level might explain why flap loss is unlikely.     

Non-invasive Predictors of Human Cortical Bone Mechanical Properties: T2-Discriminated 1H NMR Compared with High Resolution X-ray

Recent advancements in magnetic resonance imaging (MRI) have enabled clinical imaging of human cortical bone, providing a potentially powerful new means for assessing bone health with molecular-scale sensitivities unavailable to conventional X-ray-based diagnostics. To this end, ¹H nuclear magnetic resonance (NMR) and high-resolution X-ray signals from human cortical bone samples were correlated with mechanical properties of bone. Results showed that ¹H NMR signals were better predictors of yield stress, peak stress, and pre-yield toughness than were the X-ray derived signals. These ¹H NMR signals can, in principle, be extracted from clinical MRI, thus offering the potential for improved clinical assessment of fracture risk.     

Filling the gap - COI barcode resolution in eastern Palearctic birds

Background

Many fish species experience long periods of fasting in nature often associated with seasonal reductions in water temperature and prey availability or spawning migrations. During periods of nutrient restriction, changes in metabolism occur to provide cellular energy via catabolic processes. Muscle is particularly affected by prolonged fasting as myofibrillar proteins act as a major energy source. To investigate the mechanisms of metabolic reorganisation with fasting and refeeding in a saltwater stage of Atlantic salmon (Salmo salar L.) we analysed the expression of genes involved in myogenesis, growth signalling, lipid biosynthesis and myofibrillar protein degradation and synthesis pathways using qPCR.

Results

Hierarchical clustering of gene expression data revealed three clusters. The first cluster comprised genes involved in lipid metabolism and triacylglycerol synthesis (ALDOB, DGAT1 and LPL) which had peak expression 3-14d after refeeding. The second cluster comprised ADIPOQ, MLC2, IGF-I and TALDO1, with peak expression 14-32d after refeeding. Cluster III contained genes strongly down regulated as an initial response to feeding and included the ubiquitin ligases MuRF1 and MAFbx, myogenic regulatory factors and some metabolic genes.

Conclusion

Early responses to refeeding in fasted salmon included the synthesis of triacylglycerols and activation of the adipogenic differentiation program. Inhibition of MuRF1 and MAFbx respectively may result in decreased degradation and concomitant increased production of myofibrillar proteins. Both of these processes preceded any increase in expression of myogenic regulatory factors and IGF-I. These responses could be a necessary strategy for an animal adapted to long periods of food deprivation whereby energy reserves are replenished prior to the resumption of myogenesis.     

In vitro cytotoxicity of injectable and biodegradable poly(propylene fumarate)-based networks: unreacted macromers,cross-linked networks,and degradation products

This study evaluates the in vitro biocompatibility of an injectable and biodegradable polymeric network based on poly(propylene fumarate) (PPF) and the cross-linking agent PPF-diacrylate (PPF-DA). Using a methyl tetrazolium (MTT) assay, the effect of the concentrations of PPF and PPF-DA on the cytotoxicity of its unreacted macromers, cross-linked networks, and degradation products was examined. The influence of network structure properties on cell viability and attachment to the cross-linked material was also investigated. The unreacted macromers exhibited a time- and dose-dependent cytotoxic response that increased with more PPF-DA in the mixture. Conversely, the cross-linked networks formed with more PPF-DA did not demonstrate an adverse response because increases in conversion and cross-linking density prevented the extraction of toxic products. Fibroblast attachment was observed on the PPF/PPF-DA networks with the highest double bond conversions. The degradation products, obtained from the complete breakdown of the networks in basic conditions, displayed a dose-dependent cytotoxic response. These results show that there are concerns regarding the biocompatibility of injectable, biodegradable PPF/PPF-DA networks but also sheds light onto potential mechanisms to reduce the cytotoxic effects.