Fabrication of Negative Charged Poly (Ethylene glycol)-diacrylate Hydrogel as a Bone Tissue Engineer

世界最新医学信息文摘2016年第16卷第18期●论著●Fabrication of Negative Charged Poly (Ethylene glycol)-diacrylate Hydrogel as a Bone Tissue Engineering scaffoldWANG Ya qi, LIU Jie^, TAN Fei^ ,XIE Wei(Department of Prosthcdontics, The afliated hospital of Qingdao university,Qingdao University,Qingda,Shandong)ABSTRACT : Objective To improve the cell atachment of PEGDA hydrogel, the SMAS small molcule was used to modify the PEGDAhydrogel. The charged hydrogel would show improved cell attachment and enhanced protein adsorption caused by enhancement of electrostaticadsorption. Method In this study, a series of charged hydrogels were produced by adding different concentrations of charged small moleculemonomer into the PEGDA solution. Then, we investigate the physicochemical and biological characteristics of charged hydrogels, includingFTIR, swelling ratio, contact angle, cell attachment. Result The results indicate that the charged monomer had been successfully incorporatedinto PEGDA hydrogel. Meanwhile, the protein adsorption of the hydrogel increased with increasing concentration of charge modification.Moreover, compared to PEGDA hydrogel, the cell atachment signifcantly improved on the charged hydrogel. Conclusion The chargedhydrogel would be a promising scaffold candidate for bone tsse engineering.KEY WORDS : Tissuse engineering; Charged hydrogel; ChargeChinese Library Classification (CLC) : R318Document Code: ADOI: 10.96/j.ssn.1671-3141.2015.18.0020 Introductionbank), HEPES (Beijjing Solarbio Life Science & TechnologyIn recent years, the bone tissue engi neeri ng has beenCo., Ltd), PE GDA (synthesized by Laboratory of Chemicalrapidly developed as the new technology of bone defectEngineering of Wuhan University), D2959photoinitiator (Italyrepairing. In the tissue engineering, the interaction betweenSigma- Aldrich Company), Sodium MethylallyI Sulfonatecells and scaffold material is one of core issues. The ideal(SMAS) (Shanghai Aladdin Reagent Company), Sodium DodecyIscaffold materials for bone tissue engineering should provideSulfonate (SDS) (Shenzhen Y aneng Biological Technology Co.corresponding space for growth of repair tissue, and promoteLtd.), BCA Kit (Nanjing Jiancheng Bioengineering Institute),adhesion of seed cells, thus to speed up the healing andmulti- function micro- plate reader (T ecan Safire' Switzerlandreconstruction of bone tissue'".With the rise of concept of minimal invasive, injectableTecan Company), ultra- violet lamp (48w365nm Jiangsuhydrogel bone tissue engineering scaffold is more and moreK unshan Dongfei Chuangxin E lectronic T echnology Co.,concerned. Poly (ethylene glycol)- diacrylate (PE GDA) is a kindLtd.), attenuated total reflection infrared spectroscopy FTIRof good artificially synthesized scaffold; however, the strong(NICOLET380FT- IR America Nicolet Company), and contacthydrophilicity of material itself makes it difficult to adsorbinstrument (C601 Kino Industry Co, Ltd.)adhesive mediated proteins in the extracellular matrix, thus1.2 Test Methodto block the adhesion of seed cells, and limit its application.1.2.1 Preparation of Electric PEGDA GelSchol, ars home and abroad usually improve the performanceUse 20mM of HEPES buffer solution to prepare PE GDAof materials by the method of using adhesive polypeptide ofgel precursor solution of 25% of mass concentration, 0.8%graft function on gel surface; however, at present, the quantityD2959 photo crosslinker solution and 2mM of SMAS solutionand category of adhesive polypeptide of PE GDA surface graft,respectively, sealed for standby.which did not change the fact that it is difficult for gel toAdd different amount of SMAS respectively into 25wtadsorb proteins'. In view of charging characteristics of natural% of PEGDA precursor solution. With 0.8% D2959 as theproteins in physiological environment, PE GDA gel carryingphotoinitiator, prepare PE GDA gel precursor solution withnegative charge is to be prepared, using the electrostaticabsorption effect to promote the adhesion of adhesive proteins0mM, 2.5mM, 5mM and 10mM as final concentration of SMASon gel surface, thus to improve biological properties of the gel. .respectively,sealed for standby. The preparation method ofIn this research, Sodium Methyl Acrylate Sulfonate (SMAS)precursor solution is shown in Table 1, with single PE GDA gelcarrying negatively charged small molecules is to be graftedas the control group.to main chain of PE GDA, to adjust electricity of gel throughTable 1 The preparation methods of groups of negative chargedadjustment of grafting amount of SMAS, and promote thehydrogel preparationadsorption of adhesion proteins on gel surface, thus to improvePE GDAD2959SMASHEPES Total volumebiol ogical properties of PEGDA gel, and provide theoreticalGroups_(ml(mL)(μ L)(u L)basis for further research on scaffold materials of PE GDAHGPO0.0.509000tissue engineering.HGP2.5 0..51.25898.751 Materials and MethodsHGP5 .2.5897.51.1 Main Materials, Reagents, Instruments and EquipmentHGP10 0.2Mouse osteoblast cell strain MC3T3- E1 (Shanghai cell中国煤化工MHCNMHGAuthor: WANG Y aqi。Corresponding author: LIU Jie,TAN Fei.2_World Latest Medicine Information (Electronic Version) 2016 Vo1.16 No.181.2.2 Preparation of Electric PEGDA Gelin addition, this peak is caused by carboxyl group in SMASAbsorb 1ml of gel precursor solution, drop it on sterilemolecules.glass plate, and cover with another sterile glass plate, with the 2.2 Test on Swelling Ratio of Electric PEGDA Gelspacing of 1mm of slide between two gl ass plates. IluminateTable 2 shows the change in swelling ratio of gel modifiedfor 15min with UV curing light of 365nm; after the gel solutionby different concentrations of SMAS respectively ddH2O andis fully cured, cut it into the size of 1x 1x 1cm with sterilePBS. The result shows that with the increase in modificationblade, for standby.amount of SMAS small mol ecules in ddH2O, the swelling ratio1.2.3 Analysis of Chemical Structure of Electric PEGDA Gelof gel is obviously improved (P<0.05). However, in PBS, smallUse infrared spectrum FTIR to analyze chemical structuremol ecules have little impact on the swelling performance of gelof PEGDA hydrogel. After each group of samples are freeze(P>0.05).dried for 24h, use FTIR spectrometer to carry out scanTable 2 Swelling ratio and zeta potential of hydrogels incorporatedanalysis within the range of 800- 2000cm ', observe the changewith various concentrations of charge monomer (%, x+s)in chemical structure between groups of gel, and analyze thechange in grafing amount of different concentrations of SMASSweling ratio(%6)Groupssmall molecules.ddH20PBSHG(19.45+ 0.5227.50+ 0.72After preparation of PEGDA gel, each group consists ofHG2.529.76土2.15*28.85t 3.89six pieces, respectively immersed in ddH2O and PBS, whichHG534.45+ 3.47*29.10+ 2.82is taken out after immersed at37。C for 24h, wiped dry withfilter paper, and weighed (Ws). Put each group of samples inHG1048.01土3.81*29.43+ 3.28the oven at 50 °C for 24h, and weigh (Wd) it after completely*means significance of difference of experi ment group compared with thedehydrated. Calculate swelling ratio of each group of gelcontrol group (P<0.05)respectively in ddH2O and PBS.2.3 Determination of Contact Angle on Surface of ElectricSwelling Ratio=(Ws- Wa)Ngx 100%PEGDA Gel1.2.5 Determination of Contact Angle on Surface of ElectricTable 3 shows the change in contact angle on surface of gelmodified by different concentrations of SMAS. The result showsAfter preparation of electric PEGDA gel of differentwith the increase in modification amount of small molecules,concentrations of SMAS molecules, wipe dry the surfacethe contact angle on the surface of gel continuously decreases,water with filter paper, and use contact angle tester to test thewhich shows that the hydrophilicity of gel increases. However,contact angle of pure water on material surface.compared with HGO Group, the contact angle on the surface of1.2.6 Observation of Cell Adhesion on Surface of Electricgel of Group HG2.5 did not significantly change (P>0.05).With mouse osteoblast sample MC3T3- E1 cell strain asTable 3 Contact angle (mg, °,x s)seed clls, conduct conventional culture under the conditionscontact angle(° )of a - ME M culture medium containing 10% FBS, 100pg/HGO27.30+ 1.81mL streptomycin, 100U/mL penicillin, 37 C , 5% CO2 andsaturated humidity. Change the solution for every 2- 3 days.20.05+ 1.46*with 1x 105 cells inoculated on the surface of each sample, and18.55土2.95*cell suspension quantity in each hole is 100μ L; add solution*means significance of difference of experiment group compared with thefor every one hour, and supplement full culture medium to3mL along hole wall after 4h; and observe the growth of cells2.4 Observation of Cell Adhesion on Surface of Electricat 2h, 4h and 8h respectively.PEGDA Ge1.3 Statistical AnalysisIn order to confirm the effect of modification by differentThe experimental data were expressed as mean standardconcentrations of small molecules on cell adhesion action,deviation, using SPSS software for statistical analysis (SPSS17). Single factor analysis of variance one- way ANOVA is .of electric gel is observed. The result shows that at 2h, onused to analyze the experimental result, and intergroup t- testthe surface of gel of Group HG0 and HG2.5, most seed cells(Student S- test) is used to analyze intergroup mean differencestill keep spherical (Figure 1A, B). However, it can be seen(P=0.05).that massive seed cells have been preliminarily developed on2 Resultthe surface of gel of two groups- HG5 and HG10, presenting.1 Analysis of Chemical Structure of Electric PEGDA Gelshort and small spindle (Figure 1C, D); However, at 4h, on theThe results of unmodified PE GDA gel and FTIR spectrumsurface of gel of two groups- HGO and HG2.5, it is found thatof electric gel modified by different concentrations of SMASonly few MC3T3- E1 cells have been preliminarily extended,molecules show that SMAS molecules are successfully graftedand a considerabl市国煤化istill presentsto main chain of PE GDA; the spectrum had new peak atspherical (Figure 1化工face of gel of1105cm ', and the peak intensity increases conti nuouslyGroup HG5 and GrdMHC N M H Griously furtherwith the increase in the concentration of SMAS molecules;extended, with short and small pseudopodium outstretched for投稿邮箱sjzxyx88@126.com世界最新医学信息文摘2016年第16卷第18期.massive cells. With the extension of adhesion time, at 8h, ongroup HG10 at 8hthe surface of each group of gel, seed cells have been extended3 Discussionrelatively well. H owever, the cell quantity adhered to theAt present, the mechanism for mutual adhesion betweensurface of gel of Group HG5 and Group HG10 is significantlycells and scaffold materials is not completely clear. However,more than the control group (Figure 1K, L).more and more researches show that interaction of' scaffold-protein medium layer- cell" mode is usedls. After the scaffoldcontacts with internal environment, the protein will adsorbthe material surface in several seconds. Subsequently, theadhesive mediated protein can combine with the correspondingreceptor on the surface of cells, and transfer a series of bio-information of external materials to cells, thus to adjustbiological properties of cells. With constant deepening of theresearch, the impact of strength, surface charge, hydrophilicityand chemical group of biomaterials on biological behavior ofseed cells is more and more concerned".cell adhesion. In physiological environment, natural proteinis in negative and positive ionization state. Only in case ofisoelectric point and the trend of positive and negative ionsof protein dissociation is equal, the net charge of protein iszero. However, almost all isoelectric points of natural proteinis quite different from pH value of physiol ogical environment.Aiming at the natural characteristic of proteins, scholars havestudied the effect of surface charge on the protein adsorption.Scholars found that on the gold surface carrying positivecharge, the adsorption amount of osteopontin was significantlyhigher than gold surface of electric neutrality[5]. Tingfound that polymer el ectrolyte membrane carrying positivecharge could significantly increase the adhesion amount offibrobl ast and epithelium, and show good activity after celladhesion[6]. In this research, it is found that with the increasein modification quantity of small molecules, the charge amountcarried by gel continuously increases, the performance ofprotein adsorption continuously increases, and the adhesion ofcells on the surface of gel is significantly improved.The result of FTIR shows that SMAS small moleculeshave been scessfully grafted into PE GDA hydrogel. Withthe increase in SMAS concentration in precursor solution, thepeak continuously increases at the peak of 1105cm , whichshows more and more SMAS small molecules are combined tomain chain of PE GDA gel.The modification of SMAS small mol ecules makesPEGDA gel change in the swelling performance. The resultshows that compared with control group, the swelling ratioof modified gel in ddH2O significantly increases. This resultand unsaturated bond in PE GDA are competitively occupiedFig.1 Comparison of cell morphology on the hydrogelby SMAS, thus to cause the reduction of crosslinking degreesurface modified with different concentrations of chargeof gel. The reduction of crosslinking degree means polymernetwork of modified gel is relatively loosel); therefore, moreA, B, C, D represent respectively the cell morphology onddH2O enters into the gel, causing the increase in the swellingthe hydrogel surface of group HG0, group HG2.5, group HG5,ratio. However, in PBS, the swelling ratio of electric gel didnot significantly change. This phenomenon may be related togroup HG10 at 2hE,F, G, H represent respectively the cell morphology onosmotic equilibrium of solution inside and outside the gel. Dueto electrostatic attraction of electric gel, electrolyte ion is moreeasily to enter inthe electric gelgroup HG10 at 4hreach the osmotic中国煤化工nd outside theI,J, K, L represent respectively the cell morphology ongel faster. DuringTMYHC N M H Gswelling matrixproperties such as pH, ionic strength and equilibrium ion will4World Latest Medicine Information (Electronic Version) 2016 Vo1.16 No.18affect the swelling ratio8.of physical and chemical properties of the gel show that SMASThe previous results showed that the protein adsorptionproperties of Group HG5 and Group HG10 were significantlychain of the gel, while the charge on the surface of the gelimproved. The fast and stable adsorption of adhesive mediatedcan be adjusted according to modification quantity of smallprotein on the surface of scaffold materials is the basis formolecules. E lectrostatic adsorption force between the geleffective adhesive of material surface; however, the strongsurface charge and protein is used, and adsorption of proteinshydrophilicity of PE GDA gel surface largely limits the stable :on the surface of gel is enhanced, to improve biologicaladsorption of adhesion protein on its surface's. The previousproperties of PE GDA hydrogel.research results showed that the grafting of small molecules ofLiteraturescharge can effectively promote the adsorption of proteins on the[1] Benoit DS, Schwartz MP, Durney AR, et al. Small functional groups forsurface of PEGDA gel, thus to improve biological propertiesof the material. Other scholars found the similar results too.contolled differentiation of hydrogel-encapsulated human mesenchymnal stemPernodet found the negative charge could effectively promotecells. Nat Mater. 2008:7:816-823.the deposition and stretch of fibronectin on the surface of[2] Dadsetan M, Knight AM, Lu L, et al. 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