KGN Induced Chondrogenic Differentiation Of Human Umbilical Cord Mesenchymal Stem Cells In Vitro

Objective:Study on the induction of chondrogenic differentiation of human umbilical cord mesenchymal stem cells in vitro by a new small molecular compound,Kartogenin(KGN)in vitro;the preparation and optimization of the carboxymethyl chitosan / sodium alginate hydrogels with self-healing self crosslinking.The feasibility of the application of this hydrogel in the field of cartilage tissue engineering scaffold;Study of the effect of KGN on the induction of chondrogenic differentiation of human umbilical cord mesenchymal stem cells in a three dimensional hydrogel environmentMethod:1.Cell pellet culture was used to evaluate the effect of KGN induced differentiation of human umbilical cord stem cells into cartilage.Human umbilical cord mesenchymal stem cells were centrifuged into cell clones by suspension.The chondrogenic induction fluid of different components(KGN was used as experimental group,TGF-?3 as positive control group,DMSO as blank control group,KGN and TGF-?3 combined group ?TK group?).After 21 days of inducing human umbilical cord mesenchymal stem cells,HE and AB were qualitatively stained by immunohistochemistry.The immunofluorescence staining of cartilage protein and collagen and expression of cartilage related genes were performed to analyze the chondrogenic differentiation effect of KGN on human umbilical cord mesenchymal stem cells.2.The Schiff base reaction with the aldehyde oxidized sodium alginate and carboxymethyl chitosan in the preparation of amino,hydrogel with different crosslinking ratio,by scanning electron microscopy,gel rheological analysis,swelling ratio,degradation rate and in vitro cartilage adhesion evaluation of different cross-linking hydrogel physicochemical properties,application of hydrogel materials in the repair of cartilage regeneration can be explored.3.Human umbilical cord mesenchymal stem cells were loaded into hydrogels by using carboxymethyl chitosan / alginate hydrogel as a three dimensional system.The biocompatibility of hydrogels was studied by scanning electron microscopy,cell proliferation(CCK8 detection)and Live/Dead staining.The chondrogenic induction fluid with different components(KGN as the experimental group,TGF-?3 as the positive control group,DMSO as the blank control groupand KGN and TGF-?3 combined group ?TK group?)were used to induce the differentiation of human umbilical cord mesenchymal stem cells into chondrocytes.After 21 days induction,HE staining,cartilage protein and collagen immunofluorescence staining and expression of cartilage related genes were used to evaluate KGN induced chondrogenic differentiation in human umbilical cord mesenchymal stem cells in three-dimensional environment.Results:1.It was identified by cell flow analysis that the cultured cells were human umbilical cord mesenchymal stem cells.Cells were induced in vitro in the form of pellets for 21 days.Histological sections,immunohistochemistry and cartilage related gene detection of chondrocytes showed that KGN enhanced the differentiation effect of TGF-?3,and KGN alone had no significant effect on chondrogenic differentiation.2.With different amino aldehyde crosslinking ratio / preparation of components of hydrogel,through evaluation of the physicochemical properties of gel components found when amino and aldehyde ratio was 1:1,scanning electron microscopy showed that the uniform and dense porous structure;gelling time can be controlled within 2min;colloid and cartilage tissue adhesion force 146.20 + 5.87 Pa was significantly higher than that of amino / aldehyde ratio of 2:1 colloid(adhesion force is 112.64 + 7.34Pa);the swelling rate and the degradation rate was lower than the ratio of amino aldehyde / colloidal stability 2:1.3.Preparation of carboxymethyl chitosan / oxidized sodium alginate hydrogel(=1:1 / amino aldehyde)has good biocompatibility,hydrogel of cell adhesion,growth and value-added effect;the cell gel complex after 21 days of culture,TK group and TGF-group in the 3 beta protein polysaccharide and collagen content was significantly higher than that of the total KGN group and blank control group;histological staining and immunohistochemical analysis and detection of cartilage related genes showed that KGN induced human umbilical cord mesenchymal stem cells into cartilage differentiation effect is not obvious in the 3D environment,TGF-?3 has obvious cartilage induction effect,and KGN can enhance TGF-?3 chondrogenic differentiation ability.Conclusion:1.In the two-dimensional cell culture environment,the effect of KGN alone on chondrogenesis of human umbilical cord mesenchymal stem cells is not significant.Combined application of KGN and TGF-?3 can enhance cartilage differentiation by synergistic effect,which can be used as a new method for cartilage tissue engineering in the future.2.When the amino aldehyde oxidation of sodium alginate and carboxymethyl chitosan in the crosslinking ratio of 1:1,the degree of crosslinking of hydrogels were prepared with stable suitable gelling time,good swelling and self-healing properties,adhesion force,and a relatively stable rate of cartilage tissue and the degradation rate is expected to as a kind of biomimetic scaffolds for cartilage tissue repair and regeneration.3.Carboxymethyl chitosan/oxidized sodium alginate hydrogel has good biocompatibility,can load the human umbilical cord mesenchymal stem cells for cartilage tissue engineering scaffold in vitro,pure KGN group of three-dimensional environment in human umbilical cord mesenchymal stem cells into cartilage differentiation effect is not obvious,the KGN and TGF-?3 application,has a synergistic effect on chondrogenic differentiation,further proved that KGN and TGF-?3 joint shall be used for the feasibility of repair and regeneration of cartilage tissue in vivo,lay the foundation for later.