| Objective To observe the vascularization of porous silk fibrion scaffolds(PSFSs)following spinal cord injury(SCI) in rats, and to explore the mechanism of SCI repair intissue engineering so as to lay the foundation for silk fibroin(SF) of central nervoussystem(CNS) repair both theoretically and experimentally.Methods (1) Fabrication of the implants The implants including PSFSs and PVAwith similar pore sizes, porosity and interconnectivity were prepared.The surface structureof the implants was observed with scanning electron microscopy(SEM).(2) Establishmentof animal models,grouping,and transplantation of the implants28female SpragueDawley(S-D) rats, each with a body weight of250~300g, were selected and put into twogroups randomly (A:experimental group, n=16; B:control group,n=12). Following alaminectomy, all the animals received a lateral hemisection at the left of the spinal cord atthe T9–T10level. PSFSs was transplanted into the spinal cord of rats in group A andpolyvinyl alcohol(PVA) into the spinal cord of rats in group B. Then the rats were kept inseparate cages and cared to prevent infection.(3) Detection At6points of time，4d、7d、10d、14d、21d and28d postinjury，2rats from each group were killed following a perfusion.And the spinal cord containing the material was cut for histological andimmunohistological staining in order to observe the inflammatory response,variation,andthe formation of microvessels in the material. Furthermore,the unltrastructure ofmicrovessels formed in the material was detected with transmission electronmicroscopy(TEM) in group A at the time points of14d,28d postoperation.Results (1) The initial shape of PSFSs was a cylinder, like the spinal cord of rats inappearance.PSFSs was cut to be Semicircular.SEM showed PSFSs with proper pore sizes,porosity,and high interconnectivity rate. The structural parameter of PVA was similarto that of PSFSs.(2) The SCI model of the hemisection in rats was successfully established.And a series of effective postoperative caring methods were summarized. All the ratssurvived after the operation, and with few complications.(3) Observation of the materialsA10mm spinal cord containing the material was cut, which was yellowish, well-integratedwith the surrounding spinal cord tissue and with a few connective tissue on the surface ofthe material.(4) Observation of HE staining HE staining showed that the inflammatoryresponse was more moderate, and the degradation of materials more obvious in group Athan that in group B.Lumen-like structure was seen in PSFSs at7d postoperation, but thisdid not occur in PVA. And the number of lumen-like structure increased gradually.(5)Immunohistochemical observation The formation of microvessels was observed byCD34staining,And the microvessels density(MVD) was quantified. The value of MVDwas1.4,3.6,10.6,8.6,8.8respectively at different points of time after the operation in groupA.On the contrary, it was0,2.2,4.8,4.6,4.0respectively in group B. The P value isstatistically significant(P<0.05). The microvessels formed as early as7d in group A, andthe peak appeared at14d postoperation. Then its quantity decreased to a stable level. Nomicrovessel was seen at7d in group B. But The trend of MVD in group B was similar tothat in group A.(6) Observation of TEM The ultrastructure of microvessels was observed byTEM. Endothelial cells around by Pericytes in PFSFs were detected at14d postoperation.Redcells were in the lumen.The ultrastructure of microvessels progressed at28d postoperation. Thisindicated the fomation of perfused, functional microvessels.Conclusion PSFSs can be vascularized in SCI of rats, which is the precondition for cellsurvival and regeneration of neural axons. SF has the potential to be an ideal scaffold for centralnervous system(CNS) repair in tissue engineering. |
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