| Objective:Temporomandibular joint (TMJ) disc fibrochondrocytes from goats were encapsulated in alginate gel in this study, setting up an in vitro3D culture model for fibrochondrocytes. Various continuous and intermittent short period compressive loading were applied to the cells embedded in alginate gel to investigate the effects of extracellular matrix (ECM) metabolism on mRNA signal level of Aggrecan and Collagen type I (CI).Methods:1. TMJ discs were aseptically excised from one-month old healthy goats, the primary fibrochondrocytes were obtained by cutting discs into pieces, enzymatic treatment with0.2%collagenase I, then seeded in culture flasks and passaged to passage-two (P2). The inverted phase contrast microscope was used to observe the morphological changes and attachment efficiency of fibrochondrocytes, and the images of cell growth were collected by fluorescence microscopy. The fibrochondrocytes were identified with Toluidine blue staining, the qualified P2TMJ cells with normal phenotype were mixed with low viscosity (1.5%w/v) alginate solution at a density of5×105cells/ml. The cells in alginate beads were observed dynamically under inverted phase contrast microscope, and the Safranin O staining for Aggrecan and immunohistochemical staining for Collagen type I were performed to characterize ECM.2. The alginate beads were randomly divided into7groups.6groups were exposed to six kinds of various continuous or intermittent compressive loading within24h, and one nonloading (NL) negative control group was designed. After compressive loading, the viability of cells in alginate gel were evaluated by fluorescence staining using Calcein Acetoxymethylester (calcein-AM) and Propidium Iodide (PI); The TMJ disc cells were recovered from the rest alginate beads dissolved in special liquid, and the mRNA signal level expression of Aggrecan and CI were conducted by using real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. Results:1. The TMJ disc cells obtained by enzyme digestion were mainly spindle-shaped and partly were polygon-shaped, showed favorable growth and function character, kept phenotype expression of fibrochondrocytes steadily for P2. Toluidine blue staining for fibrochondrocytes exhibited positive results.2. Observation under inverted phase contrast microscope, the fibrochondrocytes embedded in alginate gel maintained the spherical morphology. The tissue section staining showed that there was an increase of ECM deposited around cells as time dragged on. After compressive loading, the images of live/dead staining of cells in alginate were collected by fluorescence microscopy, indictating that above92%cells were alive. Compared to the control group, the expression of Aggrecan mRNA relative to GAPDH was significantly up-regulated in stimulated groups of C3, C6and16(P<0.05), particularly in C3and16groups, signal level increased obviously by4.4-and5.1fold respectively. While in group13, Aggrecan mRNA was down-regulated by0.34fold (P<0.05); On the contrary,13increased CI mRNA signal levels by2.57fold (P<0.05), whereas gene expressed lower to0.67fold in C6(P<0.05).Conclusion:1. The collagenase I enzyme digestion method used in the study for isolation of fibrochondocytes is simple and feasible. The cells cultured in vitro maintain the fibrochondrocytes phenotype in P0, P1, P2passages, which is true to most experimental researches. Alginate gel also maintains the spherical phenotype of TMJ disc cells as well as favorable viability, which suggests that alginate is favored for TMJ disc fibrochondrocytes growth.2. The results of this study suggest that short period compressive loading definitely modulate the metabolism of TMJ disc cells, and the metabolic extent depends on the different loading regimes. However, the mechanism is still not clear, many more compressive loading regimes need to be designed to fulfill the demand of optimizing matrix synthesis of fibrochondrocytes. |
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