| Background:Recently,the incidence rate of cervical spondylosis is increasing year by year in the spine surgery,and the proportion of young patients are increasing.The conservative treatment is not ideal treatment for patients who require surgical treatment.The complications that the traditional anterior cervical decompression and fusion therapy bring are close joint accelerated degeneration and the donor site pain, which can be overcome by the artificial cervical disc replacement surgery and retain the physiological activity of the cervical spine. In the past decade, it has been accepted by the spine surgeons and researchers and got a rapid development. China’s current uses of cervical artificial disc in spinal surgery are all monopolized by European and American products, which are more expensive and there are some other shotage. In this situation, Shenzhen Research Institute of Tsinghua University associated a number of institutions for cooperation, and committed to invent the new artificial cervical disc in accordance with physiological characteristics of the Chinese people. The material of upper and lower endplate of the disc is titanium, which has good biocompatibility, but also weakness of poor biological activity, low strength of combination with bone and easily release of metal ions under physiological conditions.Objects:To design and development a kind of new type artificial cervical disc to meet Chinese people’s physical characteristics and dimensions, we used different modified coatings of disc endplate, and observed the cell compatibility and hemocompatibility of the three kinds of coatings in vitro, and evaluated the biocompatibility of the three kinds of different coatings in vitro of new type artificial cervical disc, and then try to provide experimental evidences for the latter part of the experimental study and clinical application.Methods:1, The three different coatings of new type artificial cervical disc are observed by field emission scanning electron microscopy and detected by X-ray spectrometer;2, Isolating BMSCs from the femur, tibia bone marrow of Wistar rat. BMSCs were passaged and purificated by adherent culture method, and passaged by the proportion of1:3. We can got another generation after four to five days, and the third-generation rat BMSCs were identified. The new type of artificial cervical disc compatibility of different coatings were tested in vitro experiments;3,The rat BMSCs were co-cultured with3different coated samples, observing adhesion and differentiation ability of rat BMSCs on different samples surface, and detecting cytotoxicity and ALP secretion to evaluate the cytocompatibility of different coatings in vitro;4,Collecting three groups of fresh blood from the rat abdominal aortic:the first and second group of fresh blood were touched with the clean, dry surface of different samples, and then added a certain amount of distilled water or physiological saline respectively, and incubated for a certain of period. The solutions were collected to evaluate the dynamic coagulation time and hemolysis of the different samples using a spectrophotometer; the third group of fresh blood was centrifugaled and collected platelet-rich plasma. Different samples were immersed in platelet-rich plasma for certain of period, and then the non-adherent platelets were washed off. After fixation, dehydration, replacement and sprayed gold processions, platelet adhesion proportion was observed by scanning electron microscopy. The measured data were expressed by x±s,using SPSS18.0software for statistical analysis of the experimental data, P<0.05was considered statistically significant.Results:1The result of scanning electron microscopy showed that three kinds of samples have honeycomb porous surface, but there are more uniform gaps on HA and Ti+HA coatings; X-ray spectrometer analyzed that HA, Ti+HA coatings are mainly composed of Ti, Ca, P, Si, O, Celements, and the Ti coating is mainly composed of Ti element.2BMSCs were cultured after separation from rat bone marrow by adherent method, the cells grew well. More than80%of the bottom was covered by BMSCs eight days after primary culture. The third generation appeared fusiform growth, forming a uniform, vigorous growth. The third generation rat BMSCs were identified by flow cytometry and the surface antibody CD44, CD106positive expression rate were95.0%and81.1%respectively. While the hematopoietic cell surface marker CD45negative expression rate was5.0%, in line with the basic criteria as the experimental cells.3Cell compatibility test results in vitro:3.1Rat BMSCs and3different kinds of samples were co-cultured for24,48h respectively. After24h, the result of scanning electron microscope showed that few BMSCs adhesion on bare titanium and the cells were round, while the cells on HA and Ti+HA coating surfaces were fusiform or polygonal; after48h, cells on HA and Ti+HA coating surfaces began to spread, especially polygonal cells on the Ti+HA coating surface, and the outstretched slender pseudopodia extended into the material within the pores, and tight adhesion on surface of the material.3.2Rat BMSCs and3different kinds of samples were co-cultured for2-7d. The amount of ALP secretion was upward trend and there was statistically significant between the three time points in the same group(P<0.05);3kinds of materials have the characteristic of promoting ALP secretion, and there was no statistically significant between the inducer group and Ti coating group(P>0.05); it was the same between HA and Ti+HA coating groups(P>0.05); HA,Ti+HA coating groups pair-wise compared with inducer, Ti coating groups, and there was statistically significant(P<0.05).3.3Rat BMSCs and3different kinds of samples were co-cultured for2-7d.50%,100%soak extract group absorbance values at each time point pair-wise compared with the control group, and there was no significant difference between Ti coating group and the control group in2d and7d, P>0.05; there was significant difference between the100%soak extract group and the control group in4d. P<0.05; the relatively cell proliferation rate on the material surface was80-96%, and material toxicity was level1; HA coating group compared with control group, and there was no significant difference in the first2d, P>0.05; in the4d and7d, there was significant difference between them, P<0.05; the relatively cell proliferation rate on the material surface was89-106%, and material toxicity was level0-1; Ti+HA coating group compared with the control group, and there was no significant difference in the first2d, P>0.05; in the4d and7d, there was significant difference between them, P<0.05; the relatively cell proliferation rate on the material surface was91-109%, and material toxicity was level0-1.4Hemocompatibility test results in vitro:4.1Dynamic coagulation time test, from the line chart drawn from the absorbance values at each time point, we can see that HA, Ti+HA coating groups anticoagulant effect are better than Ti coating group. In the first period of30min, the advantages are more obvious. After30min, the differences between them is slightly narrowing, but the HA, Ti+HA coating groups anticoagulant effects are still better than the Ti coating group.4.2Hemolysis rate detection, from the histogram drawn from the absorbance values at each time point, we can see that the hemolysis rate of HA, Ti+HA coating groups are less than Ti coating group, and the hemolysis rates of them are<1%, in line with the requirement of medical implant hemolysis rate<5%.4.3Platelet adhesion rate detection, platelet-rich plasma was collected from rat anti-coagulation blood after centrifugation.3kinds of samples were co-incubated with platelet-rich plasma for1h. After treatment, the result of scanning electron microscope showed that Ti coating surface had a certain amount of platelets, and the pseudopodia of the platelet outstreched; while on the HA, Ti+HA coating surface the platelet amount was seldom, disc-shaped, and the pseudopodia of the platelet cannot be seen nearly; HA, Ti+HA coatings have good hemocompatibility.Conclusion:The new type artificial cervical disc endplate surface are coated with HA, Ti+HA, which have honeycomb porous surface, and the gaps are uniform; Rat BMSCs are good bone tissue engineering cells, with the advantages of low-cost, easily obtained, and easy amplification, etc; the HA,Ti+HA coatings have good cell compatibility and hemocompatibility in vitro than the Ti coating, this experiment provided some data for the prosthesis coating initial selection and improvement; in the later period, other coatings will also be prepared and selected in order to getting optimum coating for clinical application. |
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