Construction And Evaluation Of 3D OMM Based On Two Biological Scaffold Materials

In recent years,tissue engineering technology has been widely used in the field of stomatology.The key to tissue engineering technology is to achieve 3D culture of cells.Compared with the 2D environment such as culture dishes,the growth of cells in 3D scaffold lasts longer and is closer to the environment of body,which can finally help to achieve the in vitro construction of tissues and organs.In this paper,the bio-scaffold material will be used to achieve the 3D culture of HGF and HOK,and construct the stable 3D OMM in vitro.Objective: To achieve 3D co-culture of HGF and HOK and construct a stable 3D OMM in vitro through comparing the 3D culture effect of the two bio-scaffold materials,so as to lay a foundation for clinical and experimental researches.Methods: Dispase II enzyme separation method combined with tissue block method was used to culture primary HGF,and SV40-T antigen lentiviral vector and hTERT recombinant lentiviral vector were used to achieve the immortalized HOK.HGF and HOK cells were identified by immunofluorescence(Vimentin/CK13)and HE staining.HGF was cultured in bovine acellular dermal matrix and Corning Matrigel matrix for 7 days.The tissue engineered connective tissue model was constructed.The 3D growth of HGF on the two scaffolds was observed by HE and DAPI staining of paraffin section.The problem of paraffin section was solved by designing a Matrigel 3D tissue-adhesive slide that conforms to the internal space of the embedding box and improving the conventional paraffin sectioning procedure.Furthermore,HGF was planted in bio-scaffold material for 7 days to form tissue-engineered connective tissue.HOK was planted on connective tissue and cultured for 7 days to form epithelial layer.After 7 days of air-liquid surface culture,paraffin sectioning of 3D OMM was performed using a modified paraffin section method for Matrigel matrix.The culture effect of 3D OMM constructed by two bio-scaffold materials was observed by HE and immunofluorescence staining.Results:(1)The primary culture of HGF can be achieved by Dispase II enzyme separation method combined with tissue blocks method.The growth of 3rd to 6th passage HGF was good.After the 8th passage,the cell proliferative activity decreased and the morphology changesd.HOK was immortalized and can be passed to the 15 th passage.The cell proliferative activity was still normal,and the cell morphology was stable,which was a single layer of typical paving stones without overlapping each other.In the bovine acellular dermal matrix group,the growth of HGF mainly concentrated on the surface of the scaffold,and the cells were relatively rare in the deep layer of the scaffold.In the Corning Matrigel Matrix group,blue-stained HGF nuclei were scattered throughout the scaffold.(2)Matrigel matrix shrank and curled easily during the process of traditional paraffin sectioning,it also easily separated from Transwell 0.4?m microporous membrane,and even the scaffold material is broken and fragmented.The frozen section method made it easy to form ice crystals inside the Matrigel matrix,and a large number of voids were generated,thereby losing the complete tissue morphology and causing a large loss of HGF inside the tissue.However,by using a modified paraffin section for Matrigel matrix in tissue sectioning,the collagen scaffold material stayed intact and continuous without voids and fragmentation.The 3D connective tissue model also stayed structurally intact.(3)Under HE and immunofluorescence staining,the cells in the bovine acellular dermal matrix group mainly concentrated in the superficial layer of the scaffold.In the deeper layers of the scaffold,the cells were relatively rare.The boundary between the epithelial layer and the subepithelial connective tissue layer of the tissue was unclear.For the Corning Matrigel Matrix group,the cells were evenly distributed throughout the scaffold material,with a continuous strip of epithelial cell layer on the surface,and the nucleus blue-stained fibroblasts were scattered in the subepithelial matrigel.Conclusion:(1)Dispase II enzyme separation method combined with tissue blocks method can successfully achieve primary HGF,and the the problem of low amplification times of HOK was solved through immortalized induction.(2)Compared with traditional paraffin sectioning and frozen sectioning,modified paraffin sectioning method for Matrigel matrix was more suitable for matrigel matrix materials.(3)Compared with bovine acellular dermal matrix,Matrigel matrix is more suitable for constructing 3D tissue engineered connective tissue model and 3D OMM.