A Study Of The Osteogenic Effect Of TGF-β1 With Biomaterials On MC3T3-E1

Background:Biological material to recover the defect and function in bone is a hot point of research of clinical application.But the osteogenetic induction ability of biological material is limited.Transforming growth factor beta-1(TGF-β₁)is one of the major growth factors that regulate the proliferation and differentiation of osteoblast.It stimulates osteoblasts to produce bone matrix,and inhibits the effect of matrix metalloproteinases to prevent protein degradation,so as to promote the osteogenetic process.So we consider joining TGF-β₁ that can promote osteogenesis into biological materials to optimize the material properties.Purpose:Research the biological activity of exogenous TGF-β₁ composite materials in vitro and its effect on signal expression of osteoblast lines(MC3T3-E1).Methods:1 Study on the natural coral and MEDPOR^?as the carrier of exogenous TGF-β₁1.1 Experimental materialsExperimental cell(MC3T3-E1,Japanese cell-bank),prepared to 5×10⁵/ml cell suspension for back-up.Recombined TGF-β₁ concentrate(American R&D Company), prepared to 1ng/ml solution with DMEM medium.Natural square coral(SanYa, HaiNan)and MEDPOR^(?)(American POREX SURGICAL INC,No.9518),cutted into 0.5×0.5×0.5cm³,Co60 disinfected and prewetted for back-up. 1.2 Experimental groupThis experiment is divided into 6 groups,respectively,blank,coral,MEDPOR^?, coral /TGF-β₁,MEDPOR^?/ TGF-β₁,TGF-β₁.Take the natural coral and MEDPOR^? into 24-hole boards,drop the TGF-β₁ solution and then add MC3T3-E1 cell suspension into holes.Take coral and MEDPOR^?as material control,simple MC3T3-E1 cells as blank.The groups are cultured in 37℃,5%C0₂,saturated humidity conditions.1.3 The effects of TGF-β₁ composite materials on cell proliferationAfter 48 hours,add ³H 0.5uCi to each hole and culture for 16h,digest cells by 0.125%trypsin and collect them,and check the value cpm of each hole by Scintillation Counter.1.4 Extracting and testing of osteoblestlike cells mRNAExtract the total RNA of osteoblestlike cells in each group by Trizol,and then reverse transcript for 1h.Carry out half quantitative polymerase reaction(PCR)withβ-actin as internal control.Electrophoresis of PCR products proceed to check TβRâ… ,TβRâ…¡,Smad3, Colâ… ,Colâ…¢,and then scan straps using digital gel image analysis system for straps density.2 Study on fluor-hydroxyapatite(FHA)and fluorapatite(FA)as the carrier of exogenous TGF-β₁2.1 Experimental materialsExperimental cell(MC3T3-E1,Japanese cell-bank),prepared to l×10⁵/ml cell suspension for back-up.Recombined TGF-β₁ concentrate(American R&D Company), prepared to 0.2,1,5ng/ml solution with DMEM medium.FHA and FA powder (Materials science and engineering laboratory of Zhejiang University),mixed with DMEM serum-free medium to get 40μg/ml suspension for back-up.2.2 Experimental groupThis experiment is divided into 12 group,respectively,blank,0.2ng/ml TGF-β₁, 1ng/ml TGF-β₁,5ng/ml TGF-β₁,FHA,FA,FHA/TGF-β₁(0.2ng/ml), FHA/TGF-β₁(1ng/ml),FHA/TGF-β₁(5ng/ml),FA/TGF-β₁(0.2ng/ml), FA/TGF-β₁(1ng/ml),FA/TGF-β₁(5ng/ml).Drop TGF-Pi solution(0.2 ng/ml,1ng/ml, 5ng /ml)and FHA or FA suspension into the medium after that cells have already adhered to bottom.Take simple FHA,FA and TGF-β₁ solution as control group, simple MC3T3-E1 cells as blank.The groups are cultured in 37°C,5%C0₂,saturated humidity conditions.2.3 The effects of TGF-β₁ composite materials on cell proliferationAfter 1d,3d,5d,add Cell Counting Kit-8(CCK-8)solution to each hole and culture for 3h,check the value OD of each hole by Microplate Reader in 450nm wavelength.Results:1 Study on the natural coral and MEDPOR^?as the carrier of exogenous TGF-β₁1.1 The effects of TGF-β₁ composite materials on cell proliferationCells proliferated doubly after adding TGF-Pi(1ng/ml)(p＜0.05),group of MEDPOR^?compounded TGF-β₁ were observed the most significant difference.1.2 Extracting and testing of osteoblestlike cells mRNAThe result of RT-PCR showed that Smad 3 expression was increased significantly in group of pure coral,and TβRâ… ,TβRâ…¡,colâ… ,colâ…¢expression were increased with no statistic difference.Group of pure MEDPOR^?,group of coral compounded TGF-β₁, group of MEDPOR^?compounded TGF-β₁ and group of pure TGF-β₁ displayed significant increase in each expression.Group of MEDPOR^?compounded TGF-β₁ increased with the most significant differece(p＜0.05).MEDPOR^?and TGF-β₁ showed synergistic reaction in TβRâ… and colâ…¢expression.Group of pure TGF-β₁ displayed different expression between TβRâ… and TβRâ…¡.2 Study on FHA and FA as the carrier of exogenous TGF-PiCells of each group proliferate visibly in 3d,and decrease slightly in 5d except the blank,The concentration of 1ng/ml TGF-β₁ has stronger promotion than 0.2ng/ml and 5ng/ml on cell proliferation.Conclusion:The expression of colâ… and colâ…¢is fit to cell proliferation and Smad 3 expression, which verified the associativity of Smad 3 and collagen secretion.MEDPOR^?and TGF-β₁ had synergistic reaction in TβRâ… and colâ…¢expression,1ng/ml TGF-β₁ solution has the strongest promote on the osteoblast proliferation.This experiment reported the esteogenesis influence of biomaterials with TGF-β₁ to proliferation and signal expression of MC3T3-E1,and was beneficial for searching suitable carrier materials.