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BACKGROUND:It can be theoretical y speculated that the combination of titanium, micro-arc oxidation coating and arginine-glycin-aspartic acid (RGD) polypeptide wil show better mechanical and biological properties. OBJECTIVE:To observe the microstructure and cellproliferation of the titanium and micro-arc oxidation coatings modified with RGD polypeptide by different modification methods. METHODS:Ninety specimens of pure titanium and micro-arc oxidation coatings were divided into three groups, with 30 specimens in each group. The first 30 specimens were pure titanium physical y decorated with RGD polypeptide only. The other two groups of specimens were physical and chemical coupling adsorption RGD polypeptide micro-arc oxidation samples, respectively. The fluorescence microscope was used to observe the effects and amounts of grafting RGD polypeptide on each sample. Content of the RGD polypeptide on the surface of the specimens were measured by X-ray photoelectron spectroscopy. The mouse bone marrow stromal cells were cultured on the surfaces of three groups samples, and the adhesion and proliferation of the cells cultured for 3 hours, 12 hours, 24 hours and 3 days were observed by optical microscope respectively. RESULTS AND CONCLUSION:There were green fluorescence spots, with varying size and amount, on the surface of specimens in three groups. In the unit field of view, the fluorescence was the strongest in chemical coupling adsorption RGD polypeptide micro-arc oxidation samples, indicating that these specimens were grafted with many polypeptides. In physical coupling adsorption RGD polypeptide micro-arc oxidation samples, smal amount of polypeptides were found on the surface, and the content of the RGD polypeptide was the highest in chemical coupling adsorption RGD polypeptide micro-arc oxidation samples. No apparent cytotoxicity was observed in three groups. The cellproliferation was the best in chemical coupling adsorption RGD polypeptide micro-arc oxidation samples. Experimental findings suggest that, chemical coupling method can wel fix the RGD polypeptides on the surface of pure titanium samples containing micro-arc oxidation, without any cytotoxicity, which contribute to promote the cellgrowth and proliferation.
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Objective To observe dynamic changes of Nestin and Erythropoietin(EPO)expression after cerebral ischemia and investigate possible mechanism of intrinsic neurogenesis. Methods A total of 36 SpragueDawley rats were divided into a control group(n=4)and an experimental group(n=32)randomly.The experimental group were further divided into 8 subgroups corresponding to the observation time points of 3,6,12,24 hours and 3,7,14,and 21 days after reperfusion.The model of experimental ischemia was made by 4-VO.The specimens were made into paraffin section.The expression of Nestin and EPO were detected by immunohistochemistry method. Results (1)Nestin detection:No Nestin positive cells were observed in hippocampal zone,subventricular zone (SVZ)and contex in the control group.The expression of Nestin started at 3 h in SVZ but not in hippocampal zone and it started to increase at 6 h after cerebral ischemia and reach a peak at 14d after cerebral isehemia in the hippocampal zone,then decreased at 21 d.The difference of Nestin expression among different time points is statistically significant(P<0.05).(2)EPO detection:A few EPO positive cells were observed in hippocampal zone in the control group.The expression of EPO started to increase at 3 h after cerebral ischemia and reach a peak at 24 h after cerebral ischemia,then decreased with time.The difference of EPO expression among the different time points is statistically significant(P<0.05). Conclusion (1)The increased expression of EPO and Nestin after cerebral ischemia might be a beneficial protective response of cells to the ischemic injury.(2)The sequence of expression of EPO and Nestin after cerebral ischemia is relevant.EPO may promote proliferation of NSC.
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BACKGROUND: Recent researches have shown that mature survival brain has been proved possessing regenerative plasticity, namely structural and functional rebuilding. In mature CNS, Gap-43 level can be used as an important index for axonal impairment and regenerative reaction during brain ischemia. Moreover neural functional recovery is proved closely connected with brain plasticity after ischemic stroke.OBJECTIVE: To investigate the influence of earlier rehabilitative training on the expression of neural plasticity related with growth-associated protein Gap-43 and synaptophysin P38 mRNA in ischemic penumbra region of rat.DESIGN: Randomized controlled study.SETTING: Anatomical Department of Affiliated Hospital of Qingdao University.MATERIALS: This study was conducted at Anatomical Laboratory of Affiliated Hospital of Qingdao University from December 1999 to June 2002. Totally 52 healthy female Wistar rats were randomly divided into sham operation group of 4 rats, rehabilitative training group of 24 rats and nature recovery group of 24 rats.METHODS: Thread bolt methods was used to establish Cerebral Middle Artery IR model on rats in rehabilitative group and nature recovery group by inserting an 4-0 nylon thread from external carotid artery, while rats in sham operation group received the same treatment without thread insertion. The experimental models were believed successfully established,if rat displayed left Homer's sign with right forearm flexed and abducted when lifting it up by tails after awakened. Specimen were collected at postoperative 24 hours in sham operative group; while rats in rehabilitative training group received rehabilitative training in MG labyrinth after CMA blocked for 120 minutes and reperfusion for 6 hours, twice a day for 10-20 minutes each time, in avoid of fatigue. Specimen were collected at postoperative 6 hours, 1, 3, 7, 14, 21 days in operative group;specimen were collected at occlusion 120 minutes reperfusion 6 hours, 1,3, 7, 14, 21 days in nature recovery group which was used as control of nature course of disease. They were made into wax slices and underwent Nissl's staining, meanwhile VIDAS 21 microscopic image analysis system was used to detect the products absorbency in ischemic penumbra region (value A), unimpaired callus value A in the same slice was considered as background value, therefore adjusted value A could be obtained by removing background value A. The immuneactivity at ischemic center were not quantitatively analyzed.of growth-associated protein Gap-43 and synaptophysin P38 mRNA at observations at ischemic penumbra region in rehabilitative training group.RESULTS: Totally 52 rats were enrolled in this study and all data sociated protein Gap-43 and synaptophysin P38 mRNA at ischemic penumbra region at various IR time points: Cortical region was lightly stained in sham operation group, and the adjusted value A of Gap-43 and P38 mRNA were 0.37±0.12 and 0.70±0.14 respectively; while the Gap-43 and P38 mRNA expression began increase at postoperative 6 hours and 3 days, reaching to the peak level at 7 days and then gradually descended from onset of 14 days in nature recovery group. The expression of Gap-43 and P38 mRNA in rehabilitative training group were found higher than the corresponding nature recovery group at IR 6 hours, 1, 3, 7, 14 and 21 days, but the difference was proved signifiCell morphological observations in rehabilitative training group: Under low-power microscope, we can observed necrotic nerve cells in ischemic center, with the number of nerve cell decreased and neurons displayed ischemic denaturalization: shrunk cell seemed like triangle, fan and strip with nuclei deeply colored, the nucleolus was unclear and cytoplasm displayed vacuole denaturalization. Cortical neuronal cytoplasm was loose with nuclei slightly metamorphosed and deep colored. Denatured neurons were co-existed with normal neurons. There was great deal of collagenocyte that appeared in ischemic center and penumbra region at postoperative 14 days.CONCLUSION: The expression of Gap-43 and P38 mRNA related to neural plasticity becomes active in post IR penumbra region. Since earlier rehabilitative training can stimulate synaptogenesis from Gap-43 and P38 mRNA neurons, thereby improving the brain plasticity and postischemic limb functional recovery.
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Objective To deliver an exogenous therapeutic gene—hTGF ?1 to rabbit lumbar intervertebral disc in vivo by adenovirus vector and observe the expression of the exogenous therapeutic gene. Methods 20?l of 0 01?mol/L PBS, with or without adenovirus (6?10 6pfu) carrying TGF ?1 gene (Ad/CMV hTGF ?1) which were constructed by our laboratory, was injected directly into nucleus pulposus tissues of lumber discs of mature New Zealand white rabbits. Immunohistochemical staining for human transforming growth factor ?1 was performed on the rabbit disc tissues in different periods after operation. A half quantitation for immunohistochemical staining was performed by VIDAS analysis system. Results Discs injected with Ad/CMV hTGF ?1 exhibited extensive and intense positive immunohistochemical staining for transforming growth factor ?1 from 1 week to 12 weeks after operation, and positive pellets in nucleus pulposus cells in 4th day group, while the control groups(intact group and PBS group) showed negative or weakly positive immunohistochemical staining. The OPTDM was significantly increased in the discs injected with Ad/CMV hTGF?1 compared to the contact discs or the discs injected with only PBS( P