| BackgroundsMicrotia is a common disease in the field of plastic surgery,which affects the organ function,appearance and mental health of patients.Autogenous cartilage transplantation is the main method to repair ear cartilage defects,but it brings about some problems such as surgical complications and lack of grafts.Cartilage tissue engineering brings a new strategy for its treatment,which is a technique for constructing cartilage grafts in vitro,by expanding seed cells from organisms,inducing them to chondrocytes and then inoculating them into biological scaffold.The cartilage grafts constructed in vitro can be used in reconstruction of cartilage defects caused by congenital diseases or trauma,as well as in the field of medical cosmetology.One of the main problems is how to obtain enough high-quality chondrogenic seed cells,which directly affects the clinical application of tissue-engineered cartilage.Therefore,it is necessary to identify alternative sources of stem cells with high chondrogenic potential for the treatment of elastic cartilage regeneration.In the early stage,our team found some genes through single cell sequencing of human ear cartilage cells,which may be used as cell surface markers of auricular cartilage derived stem cells.Further identification of these genes which can be used as specific surface markers of cartilage stem/progenitor cell provide reference for obtaining more ideal seed cells for ear cartilage construction.Objective1.To clarify the expression and localization of CD93,CD271,CD202b,CD184 and CD243 in rabbit and human ear cartilage.2.To clarify the CD93,CD271,CD202b,CD184 and CD243 expression in different passages of human ear chondrocytes cultured in vitro.3.CD271 was used as a marker to separate the subpopulations then identified their cytological characteristics.Methods and results1.CD93,CD271,CD202b,CD184 and CD243 expression and tissue localization in rabbit ear cartilage and human residual ear cartilageMethods:The cross-sectional structure of rabbit ear and human ear was clarified by HE staining.The expression and tissue localization of CD93,CD271,CD202b,CD 184 and CD243 in rabbit and human ear cartilage was detected by immunohistochemistry.Results:HE staining in rabbit ear cartilage cross section demonstrated light blue cartilage layer with oval cartilage dark blue chondrocyte,the ventral perichondrium with loose fibrous layer,and the dorsal perichondrium with dense layer and less fibrous.According to immunohistochemical staining results,CD93 was negative in nucleus cartilage,perichondrium and peripheral tissue;the expression of CD243 and CD202b were weak positive in chondrocytes.CD 184 was strong positive in the cell membrane of chondrocytes,but negative in the dorsal,ventral perichondrium and peripheral tissues.CD271 was significantly positive in ear chondrocytes,perichondrium cells and peripheral muscle tissue layer.The results of HE staining in human ear cartilage demonstrated smaller chondrocytes and closer combination of perichondrium and cartilage layer than those of rabbit ear cartilage.Fibrous protrusions were observed in the cartilage layer.The results of immunohistochemical staining showed the negative expression of CD93 protein in chondrocytes,while CD 184 and CD243 were positive,CD202b and CD271 were strongly positive.Besides,CD184,CD243,CD202b and CD271 were positive in vascular endothelium.2.CD93,CD271,CD202b,CD184 and CD243 expression in different passages of human residual chondrocytes cultured in vitro.Methods:Flow cytometry was used to identify the positive rates of the above surface markers in different passages of human remnant ear chondrocytes cultured in vitro.Then CD271 positive and CD271 negative ear chondrocytes populations were sorted by flow cytometry.Results:CD93,CD 184,CD243 and CD202b showed low expression in passage 2 and passage 3 chondrocytes,while the high expression of CD271 in passage 1 chondrocytes and gradually decreased with the passage of culture in vitro.3.Functional verification of different cell subpopulations derived from human residual ear cartilage by CD271.Methods:The proliferation ability of CD271+ and CD271-cells was detected by CCK-8 kit.The migration ability of the two cell groups was detected by cell scratch test.The cell monoclone forming ability of CD271+ and CD271-cells was evaluated by infinite dilution method.The adipogenic ability of CD271+ and CD271-cells was evaluated by oil red O staining.The differentiation of CD271+ and CD271-cells was induced by osteogenic differentiation medium,then detected the osteogenic ability of the two groups by alizarin red staining.CD271+ and CD271-cells populations were induced to forming cartilage in chondrogenic differentiation medium,and then the chondrogenic ability was identified by alcian blue staining.Results:CD271+ and CD271-ear chondrocytes were sorted by flow cytometry and then cultured in dishes.The results showed that CD271-cells had the ability of monoclone formation,while CD271-cells did not.CCK-8 kit was used to compare the proliferation ability of CD271+ and CD271-groups.It was found that CD271+ cells proliferated faster than CD271-cells.CD271+ and CD271-cells were induced to differentiate into adipogenic,osteogenic and chondrogenic lineages.The results showed that CD271+cells had stronger ability to differentiate into three lineages than CD271-cells.Conclusions1.CD271 was highly expressed in the cartilage of rabbit and human remnant ear.CD93 was almost not expressed.CD184,CD243 and CD202b were positive in chondrocytes and perichondral connective tissue.2.There was few expression of CD93,CD 184,CD243 and CD202b in passage 2 and passage 3 ear chondrocytes cultured in vitro.CD271 was highly expressed in passage 1 chondrocytes,while the positive rate gradually decreased with the cell passage in vitro.3.CD271+cells can form monoclones in vitro.Compared to CD271-cell population,CD271+ cell population originated from auricular cartilage demonstrated stronger ability of proliferation,migration and differentiation,which indicates that auricular cartilage derived CD271+ cell population had characteristics of stem/progenitor cells. |
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