Characteristics Of Temporomandibular Joint Disc Cells Detected By Atomic Force Microscopy And Study Of The Cytoskeleton

Objective:To study the cell topography and biomechanical property of the temporomandibular joint discs from goats by using JPK Nano Wizard 3 biological atom force microscopy (Bio-AFM). To observe the morphologyof cytoskeleton (CSK) and estimate the fluorescence intensity of CSK proteins of the goat temporomandibular joint disc(TMJ) cells respectively by using laser scanning confocal microscope,and aim to investigate the biomechanics properties of two types of TMJ disc cells and illustrate the changes of CSK protein during TMJ disc cells passage.Methods:1. Primary goats temporomandibular joint disc cells were cultured by monolayer culture method. AFM was used to contact scan the topography of two kinds of TMJ disc cells under the physiological environment. About 20 chondrocyte-like cells and fibroblast-like cells were selected randomly to obtain the force-vs-distance curves in cytoplasm and nucleus. Young’s modulus and adhesion were analyzed by JPK Data Processing.2.Primary(P0)and passage 1,2,3,4(P 1,P2,P3,P4) TMJ disc cells CSK proteins were stained by immunofluorescence staining respectively. The morphology of CSK were observed by LCSM and the fluorescence intensity of CSK proteins were detected quantitatively by the fluorescence intensity software.Results:1. Morphological observation of temporomandibular joint disc cell, spindle shape of the fibra-like cells took up a large proportion of the primary TMJ cells, part of the round and polygonal chondrocyte-like cells, the cells gradually irregular after passaged, polygonal cells increased.2. AFM results remand us that particular triangle shape of the chondrocyte -like cell’s nucleus took up a large proportion of the cell. Cytoskeleton was arranged dendritically on the surface.Pseudopodia were extended from the edge of the cells. Spindle shape of the fibra-like cell’s nucleus was significantly higher than cytoplasm region. The cytoskeleton was arranged regularly. The edges of the cells were smooth with less pseudopodia extended. There were no differences between the surface roughnesses of the two kinds of cells. According to the force-vs-distance curves results, there were no differences between the Young’s modulus of the two kinds of cells, whereas there were differences in the cytoplasm region. There was no statistical difference between the adhesions of the two kinds of TMJ disc cells.3. Filamentous actin mainly distributed at the periphery of membrane in uniform arrangement. The microtubules were radial through whole cytoplasm and interlaced to be loosened and clear networks through the passage. The intermediate filaments of TMJ disc cells were spreaded though whole cytoplasm and fluorescence intensity inhomogeneity. Dense distributions of intermediate filaments were found surrounding the membrane and less in nuclear.The three kinds of CSK became thicker during cell passage, and coarser fiber bundle was formed in P4 generation. Those three proteins fluorescence intensity of the overall trend was increased, no statistical differences within P3 generation, and statistically significant when P4 generation.Conclusion:Comparison of AFM topography and curves analyze. The two kinds of the TMJ disc cells revealed big difference in their topography, but little difference in their mechanical ability, May be in developmental stage of TMJ disc cells there is no difference between the biomechanics. As the cells passaged, the three kinds of CSK changed in morphology. They formed into bundle, and the thickest bundle can be seen in P4. The immunofluorescence expressions were showed increased too, suggested that P4 generation TMJ disc cells may have occurred to differentiation phenomenon,. All this aimed to provide research and a guideline to select seed cells on temporomandibular joint disc tissue engineering.