Effects Of Cartilage Oligomeric Matrix Protein Gene Multilayers On The Differentiation Of Mesenchymal Stem Cells

Background:Biological coatings have been applied to acquire ideal surface properties of medical devices in different situations. Immobilization of bioactive agents and tuning mechanical properties of substrates can efficiently direct cell behavior and fate. Deposit of gene components to modify substrates, resulting in a long-term efficiency, is one of the valued strategies. As such, genes encoding growth factors such as vascular endothelial growth factor (VEGF) and BMP2are utilized to promote osteogenic differentiation and bone formation. However, gene-functionalized substrates for physiological ligament-bone healing have had lack of attention. As an important non-collagenous protein of the extracellular matrix (ECM) in fibrous cartilage, COMP plays a vital role in cell differentiation and the maintenance of cartilage phenotype.Part I The construction of HA/DNA multilayered filmsAims:To investigate the stability and biological activity after the construction and characterization of HA/DNA multilayered films. Methods:Multilayered gene films were constructed via LBL technique. Contact angle measurement was used to detect the the hydrophilic properties of the outermost layers; UV-vis absorption at260nm was performed to monitor the build-up process by UV-vis spectrophotometer; The morphology of multilayered films was characterized by scanning electron microscopy; PBS buffer was chosen as the model of the physiological condition for the stability of (HA/DNA)6films; The release of DNA was detected when multilayered films were immersed in the trypsin solution; The expression of GFP was counted after three days transfection of HEK293T. Results:(1) The contact angle of the following layers undulated from30°to10°as alternate deposit of the HA and DNA layers.(2) The continuous increase of the UV-vis absorbance appeared with the growth of bilayer number.(3) The multilayered films displayed a quite rough facade, which was different from the smooth and uniform appearance on the bare surface.(HA/DNA)4films exhibited loose fragmental surfaces while (HA/DNA)8films showed compact particle-like microstructures.(4) There was hardly any decrease of the amount of DNA in the films even after10days incubation in PBS. The DNA was released from (HA/DNA)6films to the incubation buffer, leading to a decrease of absorbance of the films and a remarkable increase of absorbance of the incubation buffer at day one in the trypsin solution.(5) The GFP expression indicated efficient transfection by the DNA films. Also the expression of GFP on (HA/DNA)6films was significantly higher (p<0.05) than those on (HA/DNA)2according to the different fluorescence counts. Conclusion: Successful construction with rough morphology of the HA/COMP multilayered films was monitored. The multilayered films were stable in physiological condition and the plasmid could be easily released in enzymatic condition. The multilayered films could transfect cells and the transfection efficiency could be easily regulated by tuning the layer number. The properties of multilayered films provided the basis of wide application in medical devices.Part Ⅱ Effects of COMP multilayered films on cell differentiation of MSCsAims:To investigate the effects of HA/COMP multilayered films on the osteogenic and chondrogenic differentiation of MSCs. Methods:HA/COMP multilayered gene films were constructed via LBL technique. HA/COMP films were acted as experimental group and HA/pIRES (pIRES-hrGFP-la) films were acted as negative control. MSCs were cultured with multilayered films and RT-PCR of osteogenic gene markers and chondrogenic gene markers were conducted. Cell stainings were also performed evaluate the process of osteogenesis and chondrogenesis. Results:RT-PCR assay presented significantly higher (p<0.01) COMP expression of MSCs cultured with HA/COMP multilayered films. Compared with control groups, the osteogenic gene expression levels of MSCs with HA/COMP multilayered films were down-regulated while the chondrogenic gene expression levels were up-regulated. Similarly, the alkaline phosphatase (ALP) staining and Alizarin red S staining of MSCs with HA/COMP films were weakened while the alcian blue staining was enhanced. Conclusion:HA/COMP multilayered films could inhibit osteogenic differentiation and promote chondrogenic differentiation of MSCs, which might provide new insight for physiological ligament-bone healing.