| [Objective] 1.Explore the extraction and culture methods of nucleus pulposus mesenchymal stem cells(NPMSCs)and annulus-derived stem cells(AFSCs),and to identify the stem cells.2.To compare the differences of different sections and staining methods of rat caudal intervertebral discs.3.To explore the experimental results of intervertebral disc degeneration with annular Gel MA integrated intervertebral disc scaffolds loaded with different concentrations of AFSCs and NPMSCs.[Methods] 1.NPMSCs and AFSCs,were extracted and cultured by single clone method.After subculture,the histomorphology of P0 and P3 cells were observed under light microscope,the three-line differentiation culture of osteogenesis,adipogenesis and chondrogenesis was carried out.2.The caudal intervertebral discs of SD rats were collected and made into frozen sections and paraffin sections.Hematoxylin-eosin staining(HE staining),fuchsin-fast green staining,Masson staining,toluidine blue staining,type I collagen(COL-Ⅰ),type II collagen(COL-Ⅱ)and glycosaminoglycan(GAG)immunofluorescence staining were used to observe the expression of tissue and protein in different layers of intervertebral disc.3.The multimodal simulation intervertebral disc scaffold was constructed,the inner layer was Gel MA hydrogel of 5% concentration,the middle and outer layer of NPMSCs was loaded with 5%,10% and 15% concentration Gel MA hydrogel,and AFSCs was loaded.The physical and chemical properties of the gel were observed,and the differences of histocompatibility and secretory protein expression of the simulated scaffold at different levels were observed.At the same time,the rat model of intervertebral disc degeneration was established,and the rat tail intervertebral disc was replaced for 9 weeks.HE staining,Masson staining and immunohistochemical staining were performed to observe the morphology and protein secretion of multimodal integrated scaffolds.[Results] 1.NPMSCs and AFSCs can be extracted and cultured efficiently by single clone method.2.In the special staining method,paraffin section HE staining can clearly show all layers of tissue,Masson staining can clearly show collagen fibers,fuchsin-fast green staining and toluidine blue staining show better cartilage layers and subchondral bone structure.In the immunofluorescence staining of frozen sections,the collagen protein in annulus fibrosus was mostly COL-I,and the collagen protein in nucleus pulposus tissue was mostly COL-II,GAG,which was mainly secreted by nucleus pulposus cells and nucleus pulposus-like cells.3.In vitro experiments showed that different concentrations of annular Gel MA multimodal integrated intervertebral disc had good histocompatibility and could stretch NPMSCs and AFSCs.COL-I was secreted more in the inner annulus fibrosus.With the increase of the concentration gradient of Gel MA hydrogel,the expression of COL-I decreased.The expression of COL-II in the inner layer of nucleus pulposus tissue was more,in the outer layer of annulus fibrosus tissue.With the increase of Gel MA hydrogel concentration gradient,the expression of GAG decreased gradually,and the expression of laminin in inner nucleus pulposus-like tissue was higher.Animal experiments have shown that the multimodal integrated intervertebral disc scaffolds loaded with different concentrations of AFSCs and NPMSCs are similar to natural intervertebral discs and can mimic normal intervertebral discs.[Conclusion] 1.NPMSCs and AFSCs can be extracted and cultured efficiently by single clone method.2.In the observation of each layer of intervertebral disc,paraffin section is better than frozen section.When observing the fluorescence expression and distribution of various proteins,frozen section is better than paraffin section 3.Multimodal simulation intervertebral disc scaffold can better simulate the characteristics of intervertebral disc tissue and has better experimental results. |
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