Effect of Static Load on the Nucleus Pulposus of Rabbit Intervertebral Disc Motion Segment in Ex vivo Organ Culture

Background：The development of mechanically active culture systems helps increase the understanding of the role of mechanical stress in intervertebral disc （IVD） degeneration.Motion segment cultures allow for preservation of the native IVD structure,and adjacent vertebral bodies facilitate the application and control of mechanical loads.The purpose of this study was to establish loading and organ culture methods for rabbit IVD motion segments to study the effect of static load on the whole disc organ.Methods：IVD motion segments were harvested from rabbit lumbar spines and cultured in no-loading 6-well plates （control conditions） or custom-made apparatuses under a constant,compressive load （3 kg,0.5 MPa） for up to 14 days.Tissue integrity,matrix synthesis,and the matrix gene expression profile were assessed after 3,7,and 14 days of culturing and compared with those of fresh tissues.Results：The results showed that ex vivo culturing of motion segments preserved tissue integrity under no-loading conditions for 14 days whereas the static load gradually destroyed the morphology after 3 days.Proteoglycan contents were decreased under both conditions,with a more obvious decrease under static load,and proteoglycan gene expression was also downregulated.However,under static load,immunohistochemical staining intensity and collagen Type Ⅱ alpha 1 （COL2A 1） gene expression were significantly enhanced （61.54 ± 5.91,P =0.035） and upregulated （1.195 ± 0.040,P =0.000）,respectively,compared with those in the controls （P 〈 0.05）.In contrast,under constant compression,these trends were reversed.Our initial results indicated that short-term static load stimulated the synthesis of collagen Type Ⅱ alpha l;however,sustained constant compression led to progressive degeneration and specifically to a decreased proteoglycan content.Conclusions：A loading and organ culture system for ex vivo rabbit IVD motion segments was developed.Using this system,we were able to study the effects of mechanical stimulation on the biology of IVDs,as well as the pathomechanics of IVD degeneration.

Chinese Scientists Working on Diseases of Cultured Organisms in Seawater

Considerable research efforts are being made in China to understand the pathogeny of major marine cultured organisms and their resistance to diseases, which has been listed in the national development program for major basic research projects. The program, also called "973," is in effect a national plan focused on the leading basic

High-frequency magnetic stimulation attenuates beta-amyloid protein 1-42 neurotoxicity in organotypic hippocampal slices

Repetitive transcranial magnetic stimulation （rTMS） has been utilized as a therapeutic tool for neurodegenerative disorders including Alzheimer＇s disease. However, the precise mechanisms of its clinical effects remain unknown. β-amyloid （Aβ） exhibits direct neurotoxic effects and is closely related to neuronal degeneration in Alzheimer＇s disease. Therefore, it has been hypothesized that the neuroprotective effects of rTMS are related to the mechanisms of protection against Aβ neurotoxicity. Organotypic hippocampal slices were prepared from 8-day old, Sprague Dawley rats. The tissue slices were exposed to 100 μmol/L Al3142 since day 12 in vitro with and without high-frequency （20 Hz） magnetic stimulation. Magnetic stimulation efficacy was evaluated by measuring neuronal nuclei （NeuN） protein expression and by observing cultures following propidium iodide fluorescence staining and bromodeoxyuridine （BrdU） immunohistochemistry. Lactate dehydrogenase activity was detected in the culture media to evaluate hippocampal neuronal damage. Our results demonstrated that high-frequency magnetic stimulation significantly reversed the reduction of NeuN protein expression because of Aβ1-42 exposure （P 〈 0.05） and significantly reduced the number of damaged cells in the hippocampal slices （P 〈 0.05）. However, lactate dehydrogenase levels and anti-BrdU staining results did not reveal any statistical differences These findings indicate that high-frequency magnetic stimulation might have protective effect on hippocampal neurons from Aβ1-42 neurotoxicity.

Studies on Biological Characteristics of Ginkgo Hairy Root Culture

The comparative studies on properties of growth and cultivated conditions of seven transformed ginkgo hairy root clones were reported. Different clones display various phenotypes characterized by growth rate.The results show that the suitable inoculum is benefical to the growth of ginkgo hairy root.NH + 4/NO - 3, pH ,sucrose, and inositol have important effects on the growth of ginkgo hairy root.

Effect of Zinc on Bone Metabolism in Fetal Mouse Limb Culture

Objective To determine the effects of zine-deficiency and zine-excess on hone metabolism. Methods We developed the culture model of fetal mouse limbs (16th day) cultivated in self-made rotator with continuing flow of mixed gas for six days in vitro. The cultured limbs were examined by the techniques of 45Ca tracer and X-roentgenography. Results The right limbs cultivated had longer bone length, higher bone density than the left limbs uncultivated from the same embryo; and histologically, the right limbs had active bone cell differentiation, proliferation, increased bone trabecula. clearly calcified cartilage matrix, and osteogenic tissue. Compared with the control group, the zinc-deficient group and zine-excess (Zn2+ l20) μmol/L) group contained less osteocalcin (BGP) and 45Ca content, and lower AKP activity; whereas zine-normal (Zn2+ 45 μmol/L and Zn2+ 70 μmol/L) groups contained more BGP and 45Ca contents, and higher AKP (alkaline phosphatase) activity. Conclusion Both zine-deficiency and zine-excess can alter bone growth and normal metabolism. The results indicate that the culture model of fetal mouse limbs (16th day) in vitro can be used as a research model of bone growth and development.

Clinical application of a shape-preserving rapid corneal donor dehydrater

AIM:To describe the design and clinical application of a corneal donor dehydrator which can quickly dehydrate corneas and keep its original shape.METHODS:The corneal donor material is placed on stainless steel beads with different diameters in the dehydrating box to make the cornea the same shape as the steel ball.Then,the cornea is placed inside the dehydrater for rapid dehydrating using the internal cleaning and ventilation system.Totally 83 eyes underwent deep anterior lamellar keratoplasty(DALK)using corneal donor tissue preserved with corneal dehydrater,and 60 patients(60 eyes)received DALK by the same surgeon using corneal donor tissue preserved with glycerol were included in the control group.The best corrected visual acuity(BCVA),the thickness and transparency of the corneal buttons were recorded.RESULTS:After the completion of dehydrating,all the donor corneas maintained a normal shape without any shrinkage or distortion,and the average intraoperative rehydration time was 43.3±12.1 s during operation.The mean BCVA of the dehydrater group was 0.30±0.18 at 1 wk and 0.32±0.16 at 1 mo,which were statistically better than that of the control group(P<0.001).The score of corneal buttons transparency were lower than that of the control group with statistical difference(P<0.001).The thickness of corneal buttons at 1 wk and at 1 mo in the dehydrater group was significantly better than that of the control group respectively(P<0.001).One week after operation,no corneal button turbidity or edema was observed in both groups.CONCLUSION:The dehydrater can quickly dehydrate the corneal material in a clean and airtight environment and maintain the original shape of the corneal donor during the dehydrating process.This dehydrater is recommended for long-term high-quality preservation in areas where corneal materials cannot be used within a reasonable time period.

INVASION OF SPHEROID OF MURINE LUNG ADENOCARCINOMA (LA_(795)) CELLS INTO EMBRYONIC CHICK HEART FRAGMENTS

An in vitro spheriod invasion model of tumor cell was established by using murine lung adenocarcinoma cell line (LA795) and precultured embryonic chick heart fragment (PHF). The spheroid of LA795 cells were prepared by incubating a suspension of trypsinized LA795 cells on a gyratory shaker. Spheroid aggregates of LA795 cells in diameter of 0. 2 mm were selected and confronted with PHF (diameter of 0. 4 mm) on semi- solid medium for 3 - 4 hours, then, individual confronting pain were transferred into fluid medium for further co-culture on gyratory shaker. After 1, 3, 5 and 7 days, multiple confronting pairs were processed for histological and ultrastructural study. The Invasive capacity and the invasion process of LA795 cells were examined and observed. The results demonstrated that LA795 cell line has a high capacity of invasion and high malignancy in vitro. This spheroid Invasion model is very useful for studying mechanism of Invasiveness of tumor cells in vitro.

A Three-Dimensional (3D) Environment to Maintain the Integrity of Mouse Testicular Can Cause the Occurrence of Meiosis

Adhesions between different cells and extracellular matrix have been studied extensively in vitro, but little is known about their functions in testicular tissue counterparts. Spermatogonia and their companion somatic cells maintain a close association throughout spermatogenesis and this association is necessary for normal spermatogenesis. In order to keep the relative integrity of the testicular tissues, and to detect the development in vitro, culture testicular tissues in a three- dimensional （3D） agarose matrix was examined. Testicular tissues isolated from 6.5 d postpartum （dpp） mouse were cultured on the top of the matrix for 26 d with a medium height up to 4/5 of the 3D agarose matrix. The results showed that in this 3D culture environment, each type of testicular cells kept the same structure, localization and function as in vivo and might be more biologically relevant to living organisms. After culture, germ cell marker VASA and meiosis markers DAZL and SCP3 showed typical positive analysed by immunofluorescence staining and RT-PCR. It demonstrated that this 3D culture system was able to maintain the number of germ cells and promote the meiosis initiation of male germ cells.