Plasticity of the skeleton and skeletal deformities in zebrafish (Danio rerio) linked to rearing density

The teleost zebrafish (Danio rerio), an established model for human skeletal diseases, is reared under controlled conditions with defined parameters for temperature and photoperiod. Studies aimed at defining the proper rearing density have been performed with regard to behavioural and physiological stress response, sex ratio and reproduction. Studies concerning the effect of rearing density on the skeletal phenotype are lacking. This study analyses the response of the skeleton to different rearing densities and describes the skeletal deformities. Wild-type zebrafish were reared up to 30 dpf (days post-fertilization) in a common environment. From 30 to 90 dpf, animals were reared at three different densities: high density (HD), 32 fish l^–1; medium density (MD), 8 fish l^–1 and low density (LD), 2 fish l^–1. Animals at 30 and 90 dpf were collected and whole-mount stained with Alizarin red S to visualize mineralized tissues. The entire skeleton was analysed for meristic counts and 172 types of deformities. The results showed that the rearing density significantly influenced the specimens’ average standard length, which decreased with the increase in the rearing density. Differences in meristic counts among the three groups were not observed. Rearing density–independent malformations affected the ribs, neural arches and the spines of the abdominal region, as well as vertebrae of the caudal complex. The HD group showed the highest number of deformities per specimen, the highest number of observed types of deformities and, together with the MD group, the highest frequency of specimens affected by severe deformities. In particular, the HD group showed deformities affecting arches, spines and vertebral centra in the caudal region of the vertebral column. This study provides evidence of an effect of the rearing density on the development of different skeletal phenotypes.     

bFGF Promotes the Proliferation of Rat Peripheral Blood Mesenchymal Stem Cells Through β-Catenin Signaling

bFGF (basic fibroblast growth factor, bFGF) could promote the proliferation of bone marrow and cord blood mesenchymal stem cells. However, the effect of bFGF on the proliferation of peripheral blood mesenchymal stem cells (PBMSCs) needs further research. This study aimed to investigate the role of bFGF on the culture and expansion of PBMSCs in vitro. Firstly, arterial blood was collected from rats abdominal aorta. After mononuclear cells (MNCs) were separated with Ficoll separation fluid, MNCs were cultured in the DMEM medium without bFGF (served as control group) or with bFGF (10, 20 ng/mL, served as 10 or 20 ng/mL bFGF group). PBMSCs were obtained by adherent culture method. The third passage of PBMSCs was detected for the MSC surface markers and the effect of bFGF on the cell cycle of PBMSCs using flow cytometry. The effects of bFGF on colony formation, cell growth, and the expressions of cyclin D1, cyclin E, p21 and β-catenin were evaluated. PBMSCs showed no difference in morphology among the three groups. PBMSC clonies appeared 14 days after cultivation. Compared with the control group, the cell growth confluence of PBMSCs was obviously increased by 40% and 80% in groups treated with 10 ng/mL bFGF or 20 ng/mL bFGF respectively after culture of 21 days (all P<0.05). Compared with the group treated with 10 ng/mL bFGF, the confluence of PBMSCs in 20 ng/mL group was further increased by 28% (P<0.05). Cells of the third passage were positively stained for CD29 and CD90, while were negative for CD45. These results were consistent with the phenotypic characteristics of MSCs. Compared with the control group, the colony number of PBMSCs in the 10 ng/mL and 20 ng/mL bFGF groups was increased by 51% (P<0.05) and 92% (P<0.05), respectively. Compared with the 10 ng/mL group, the colony number of PBMSCs was further increased in 20 ng/mL group by 14% (P<0.05). The growth curve of PBMSCs showed that after 7 days of culture, the number of PBMSCs in 10 ng/mL bFGF group and 20 ng/mL bFGF group was increased by 41% (P<0.05) and 61% (P<0.05), respectively. Moreover, the cell number had a statistically significant difference between these two groups (P<0.05). Results from flow cytometry cell cycle showed that the numbers of PBMSCs in the G1 phase of experimental groups were significantly decreased as the concentration of bFGF increased when compared with the control group (P<0.05), whereas the number of PBMSCs in the S phase was significantly increased (P<0.05). Immunofluorescence experiments showed that, compared with the control group, bFGF significantly promoted the nuclear translocation and expression of β-catenin in PBMSCs. Compared with the 10 ng/mL group, the PBMSCs in 20 ng/mL bFGF group showed stronger nuclear translocation and expression of β-catenin. Western blot experiments showed that the levels of β-catenin and its target proteins cyclinD1 and cyclinE were significantly increased (all P<0.05), whereas expression of p21 was significantly decreased in PBMSCs in the bFGF groups in a concentration dependent pattern when compared with control group (P<0.05). The study firstly confirms that bFGF promotes the proliferation of PBMSCs by regulating the β-catenin signaling pathway, which may facilitate the aquisition of larger number of PBMSCs for stem cell engineering in vitro.     

A New Computational Efficient Approach for Trabecular Bone Analysis using Beam Models Generated with Skeletonized Graph Technique

Micro-finite element (FE) analysis is a well established technique for the evaluation of the elastic properties of trabecular bone, but is limited in its application due to the large number of elements that it requires to represent the complex internal structure of the bone. In this paper, we present an alternative FE approach that makes use of a recently developed 3D-Line Skeleton Graph Analysis (LSGA) technique to represent the complex internal structure of trabecular bone as a network of simple straight beam elements in which the beams are assigned geometrical properties of the trabeculae that they represent. Since an enormous reduction of cputime can be obtained with this beam modeling approach, ranging from approximately 1,200 to 3,600 for the problems investigated here, we think that the FE modeling technique that we introduced could potentially constitute an interesting alternative for the evaluation of the elastic mechanical properties of trabecular bone.     

Modifcation of the French Azure C Tissue Stain

A staining procedure for monocytes in specimens of blood and bone marrow was developed. The technique was a two step procedure in which unfixed cells were exposed first to a methanolic solution of C.I. basic blue 54. Next, an aqueous alkaline buffered solution of C.I. basic blue 141 was added to the first staining solution. After staining for 10 min in the solution with two stains, slides or coverslips were washed for 5 sec in pH 5.6 phosphate buffer and drained dry. The cytoplasm of monocytes stained intensely deep purple and frequently nuclei were stained red. Similar staining was not found in other types of normal or abnormal blood and bone marrow cells.     

Heparin-functionalized thermoresponsive surface

Temperature-dependent regulation of affinity binding between bioactive ligands and their cell membrane receptors is an attractive approach for the dynamic control of cellular adhesion, proliferation, migration, differentiation, and signal transduction. Covalent conjugation of bioactive ligands onto thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-grafted surfaces facilitates the modulation of one-on-one affinity binding between bioactive ligands and cellular receptors by changing temperature. For the dynamic control of the multivalent affinity binding between heparin and heparin-binding proteins, thermoresponsive cell culture surface modified with heparin, which interacts with heparin-binding proteins such as basic fibroblast growth factor (bFGF), has been proposed. Heparin-functionalized thermoresponsive cell culture surface induces (1) the multivalent affinity binding of bFGF in active form and (2) accelerating cell sheet formation in the state of shrunken PIPAAm chains at 37°C. By lowering temperature to 20°C, the affinity binding between bFGF and immobilized heparin is reduced with increasing the mobility of heparin and the swollen PIPAAm chains, leading to the detachment of cultured cells. Therefore, heparin-functionalized thermoresponsive cell culture surface was able to enhance cell proliferation and detach confluent cells as a contiguous cell sheet by changing temperature. A cell cultivation system using heparin-functionalized thermoresponsive cell culture surface is versatile for immobilizing other heparin-binding proteins such as vascular endothelial growth factor, fibronectin, antithrombin III, and hepatocyte growth factor, etc. for tuning the adhesion, growth, and differentiation of various cell species.     

早期强化步行基本功训练对脑卒中的预后分析

目的：分析早期强化步行基本功训练对脑卒中预后的影响。方法：选取我院2008年10月一2011年10月收治的176例恢复期脑卒中患者,按照随机数字表分为观察组及对照组,各88例,均接受常规药物治疗和康复训练,观察组在此基础上接受早期强化步行基本功训练,对比两组患者的预后。结果：两组患者治疗后FMA—LE、BBS及MBI评分均见上升,观察组较对照组上升程度更为明显（P〈0．05）,T2时期观察组评分亦见升高,而对照组评分与T1时期相比无明显统计学差异（P〉0．05）;两组患者治疗后步长、步宽及步速均见上升,观察组较对照组上升程度更为明显（P〈0．05）,T2时期观察组步态参数亦见升高,而对照组步态参数与T1时期相比无明显统计学差异（P〉0．05）。结论：早期强化步行基本功训练能够有效改善脑卒中患者下肢运动功能和平衡能力,保证治疗后步行功能,避免生活质量下降,该康复方法值得广泛推广。     

Subtractive phage display technology identifies zebrafish marcksb that is required for gastrulation

In the present study, we used a phage display technique to screen differentially expressed proteins from zebrafish post-gastrula embryos. With a subtractive screening approach, 6 types of single-chain Fv fragments (scFvs) were screened out from an scFv antibody phage display library by biopanning against zebrafish embryonic homogenate. Four scFv fragments (scFv1, scFv3, scFv4 and scFv6) showed significantly stronger binding to the tailbud embryos than to the 30%-epiboly embryos. A T7 phage display cDNA library was constructed from zebrafish tailbud embryos and used to identify the antigens potentially recognized by scFv1, which showed the highest frequency and strongest binding against the tailbud embryos. We acquired 4 candidate epitopes using scFv1 and the corresponding genes showed significantly higher expression levels at tailbud stage than at 30%-epiboly. The most potent epitope of scFv1 was the clone scFv1-2, which showed strong homology to zebrafish myristoylated alanine-rich C-kinase substrate b (Marcksb). Western blot analysis confirmed the high expression of marcksb in the post-gastrula embryos, and the endogenous expression of Marcksb was interfered by injection of scFv1. Zebrafish marcksb showed dynamic expression patterns during embryonic development. Knockdown of marcksb strongly affected gastrulation movements. Moreover, we revealed that zebrafish marcksb is required for cell membrane protrusion and F-actin alignment. Thus, our study uncovered 4 types of scFvs binding to zebrafish post-gastrula embryos, and the epitope of scFv1 was found to be required for normal gastrulation of zebrafish. To our knowledge, this was the first attempt to combine phage display technique with the embryonic and developmental study of vertebrates, and we were able to identify zebrafish marcksb that was required for gastrulation.