Joumal of Wuhan University of Technology-Marer. Sci Ed. Oct 2008617DOI 10.1007/s1 1595007-5617-%*Rheologieal and Biological Characteristies of Hyalu-ronic Aeid Derivative Modified by Polyethylene GlyeolCHEN Jinghual", CHEN Jingtao', XU Zheng'°(1 College of Materials and Technology, Tongji University, Shanghai 200092, China;2 College of Publishing and Printing, University of Shanghai for Science and Technology, Shanghai 2003, China)Abstraet: Hyaluronic acid (HA) was chemically modified by polyethylene glycol. Meanwhile,the dynamic mechanics properties of HA derivative and its viscoelastic changes were measured on3ARES3 Rheometer (Japan) at 25 C. Dried cross-linked films of 10X 10 mm2 were immersed inphosphate buffered saline(PBS: pH 7.4) at 37 C with dfiferent time periods to measure its watercontent and in viro degradation. Moreover, cell cultured solutions, which were in the different culti-vation vesse with 1 mg/mL Solution of HA derivative as doing experimental sample for 2 d, 4 d and 7d, were observed, respectively, by an inverted discrepancy microscope. The cell relative growth ratewas analyzed with the SPSS10.0 mathematic statistic software. Based on the above experiments,structure-modified HA derivative can meet the requirements of biomaterials in view of rheological anddegradation in vitro and cytotoxicity charactereistics from clinical medical aspect under this experi-ment conditions.Keywords: hyaluronic acid; viscoelasticity; water content; degradation; cytotoxicity1 IntroductionIn theory, the four function groups of HA molecule,hydroxide, N-acetylamide, carboxyl and end group, canA great deal of researches on hyaluronic acid (HA) all be modified respectively to be changed as a newer HAhas been conducted since it was discovered by Meyer and derivative. As the previous research was mainly on thePalmer in 1934 from the vitreous humour of cattle eyes. modification of HA's hydroate and carboxyl, there stillHA is glycosaminoglycan copolymer of D-glucuronic exists much more studying space for much newer HAacid and N-acetyl-D. glucosamine. Due to its unique derivative producing route. Trying to identify the aboveviscoelastic properties and biocompatibility in body, HA potential routes, with reference of protein' s modificationcan be used widely in clinical applications such as vis- mechanism croslinked with PEG, in this paper, HAcosupplementation for arthritis, wound healing, ocular derivative, modifying its N acetylamide by polyethylenesurgery and plastic surgery. HA is also known to playa glycol, was measured and discussed its rheologicalrole in promoting cell motility and proliferation. Un- properties, the water content of HA derivative film, itsmodified HA has high capacity for lubrication, water vitro degradation and cytotoxicity.sorption and water retention, but natural HA is easilyBased on all the above analysis, we may know HAsolubled in water and is not resistant to enzymatic deg- derivative hydrogel with water content as low as 60 wt%radation. Thus its wider application as a type of bio- has a slow degradation rate. Moreover, the struc-medical materials were hindered2. So chemical modi- ture-changed HA derivative retained equal fine biocom-fications are required for HA to try to improve its bio- patibilities and rheological properties of natural HA, anddegradation characteristics and keep its viscoelastic without toxicity or stimulative to human being body.properties.中國煤化工(Received: June 18, 2007; Acepled: April 7, 2008)2.1 MCNMHGtionofHACHEN Jinghua(陳景華); Assco.Prof; Ph D; E-mail:cjhshl@ 163.comAll the materials used to obtain HA derivativeCorespondingauthor: XU Zheng(徐政): Prof; E milxzheng11718cross-linked by polyethylene glycol (PEG) may be@yahoo.com.cnFunded by the 863 Projet (No.2002AA205091)known in Ref.[3], the process of the resulting solution of618Vol.23 No.5 CHEN Jinghua et al: Rheological and Biological Charactistics..HA derivative modified by PEG was also shown in enhanced with the increment of the mass fraction of so-Ref.[31lution, that is, elasticity and viscosity of solution were in2.2 Rheology of HA derivativedirect ratio to its mass fraction. But elastic module in-With different non-ion aqueous solution with mass creased and viscous module decreased with the incrementfraction of 0.5%，1% and 1 .5%, the dynamic mechanics of shearing frequencyl4".properties of the HA derivative and its viscoelastic100changes were measured on 3ARES3 Rheometer (Japan)mass fractionat 25 C. Furthermore, the dynamic change from viscos-+15ity to elasticity with the change of vibration frequency totwo different fluids, HA and HA derivative, were ana-lyzed and compared.2.3 Water eontent of HA derivative films0.1-Several pieces of dried cross-linked films of 10 mmX 10 mm were weighed and immersed in phosphate0.01 L1000000buffered saline(PBS: pH 7.4) at 37 C for 24 h. AfterRate/rad sremoved from the saline solution, they were placed be-Fig.1 Elastic module and shearing frequency of HA derivativetween two pieces of filter paper to wipe the excess solu-tion, followed by weighing the water-swollen films. They100 [were again dried in a vacuum oven for6 hat 60 C and國then weighed to estimate their sol fraction and watercontent according to the following equationsSol fraction=1 - (Wd2/Wd)(1)Water content (%)=[(Ws- Wd)/Ws]2)0.1 tWhere Wd; and Wd2 are the weight of dried filmsbefore and after water swelling, and Ws is the weight of.0100.1swollen films.2.4 In vitro degradation of HA derivativeFig.2 Viscosity module and shearing frequencyThe cross-linked HA derivative fims were im-of HA derivativemersed in phosphate buffered saline(PBS: pH 7.4) at 37 CThe conversion relationship between viscosity withafter measuring the weight. The films were taken out at elasticity of HA derivative solution with vibrated fre-predetermined intervals and placed between two dryquency changing is shown in Fig.3. The conversion frompieces of filter paper to wipe the excess solution from the low vibrated frequency to high vibrated frequency forfilms. The wet films were weighed, dried in a vacuumsolution equaled from viscosity to elasticity, becauseoven for6 h at 60 C and then weighed again to estimate molecule could untie twist each other at low vibratedits in vitro degradation. .frequency. Its main character was viscosity, but when2.5 Cyloxitoeity of HA derivativevibrated frequency exceeded fixed value, molecule couldConfect 1 mg/mL alleviation solution of HA de-not untie twist each other without enough time, so elas-rivative as experimental sample, and monitore its cyto-ticity was its most character. This fixed value was itstoxicity of cell cultured solution in different cultivationintersection point from viscosity to elasticity. With thevessels with 50% experimental sample with different increment of solution concentration, the position ofcultivation period, respectively as 2, 4, and 7 days bycrossing point turned to be lower and lower. This showedinverted discrepancy microscope, and analyze cell rela- increasing HA derivative concentration reduced crossingtive growth rate with SPSS 10.0 mathematic statisticfrequency and enhance solution's elasticity5.o1.software.The crossing point frequency of HA and its deriva-tive was shown in Figs.3 and 4. Under the same concen-3 Results and Diseussiontration,中國煤化工from viscosity toelasticiof HA derivative.3.1 Rheology eharacteristieThis wlYHc N M H Ging degree betweenRelationship between viscous and elastic modulusdifferent molecule chains for different HA and HA de-are shown in Figs.1 and 2. From Figs.1 and 2, elastic rivative, the latter overlapping was more difficult to bemodulus and viscous modulus of HA derivative solutionopened.Joumal of Wuhan University of Technology-Mater. Sci. Ed. Oct 2008619cross-linked HA derivative hydrogel films with watercontents as low as 60 wt%, although it took 5 days to1.0I 0.obtain such films. It was likely that the optimal pH of thei 0.8t0.2casting solution for HA cross-inked by PEG was around0.6-.4號8, so that the PEG concentration was higher than around( 0.4-0.6出1%7.84.0.2|0.:3.3 In vitro degradation of HA derivativeDegradation of cross-linked HA derivative films0.0L0.001 0.01 0.1 101001000was studied in aqueous media in vitro. The degradation ofFreq/rad scross-linked HA derivative films in vitro was conductedFig.3 Trend of HA derivative from viscosity to elasticityby placing them in PBS (pH 7.4) at 37 C for 2d,5 d and10 d. The weight percent of HA derivative flms after1.2| 1Swaterimmersion in PBS was shown in Fig.6. According to1.04E63wacy40.0Fig.6, the film cross-inked at pH 4.7 shows the fastest, 0.80.degradation rate among the three films, regardless of the愛0.6PEG concentration. The HA derivative film cross-linkedg 0.40.5出at pH 8.0 with 3.6% PEG exhibited practically no weightchange for at least 10 d in PBS(pH 7.4) at 37 C. Fig.70.2-shows a plot of film weight remaining after immersion inJ1.00.00 0.01 0.1100PBS for different time against the water content ofcross-inked HA derivative films. In spite of data scatter,it seemed probable that in viro degradation of HA de-Fig.4 Trend of HA from viscosity to elasticityrivative films cross-linked with PEG was virtually gov-120,ermned by the water content of films, which, in turn, de-00pended on the pH of the casting solution used for pre-paring coss-inked HA derivative films!9!.503.4 Cytotoxieity of HA derivativeAfter incubated for 2 d, 4 d and 7 d, cell tssue mor-3phology having respectively in negative control group,positive control group and experimental control groupcould be investgated by Nikon eclipse TE 2000-U modelPEG concentration/mol%inverted microscope(Fig.8). Growth, differentiation andFig.5 The water content of HA films crosslinked withproliferation of experimental group cells always went on,different concentraionof PEG at Ph 4.7, 6.0 and 8.0and cell quantities gradually increased, cell shape wasfor 120h(◆pH 4.7;▲: pH6.0;■: pH8.0)well and stick wall was all right along with time after3.2 Water content of HA derivative flmincubated 2 d, 4 d and7 d. L-929 cells seemed like shut-Fig.5 shows the water content of HA derivative tle or iregular triangle, and very satiey. But positivefilms obtained by casting HA derivative solutions of control group cells did not grow well, and there werepH 4.7, 6.0 and 8.0 containing dfferent concentrations of some bright spots appeared around them, which indicatedPEG. From Fig.5, we may clearly know that the water there had a few of withering cells and suspended deceasecontent of the HA derivative films subjected to water cllslol..swelling at 37 C after cross-linking was higher thanCell suspended liquid placed on the hematimeter90 wt% if the PEG concentration was lower than ap- board could be counted by microscope, and analyzed cellproximately 0.5%. The sol fraction was very low even relative growth rate was analyzed with Microsoft Excelwhen the water content of films was as high as 95 wt%，2000 statistic program and SPSS10.0 mathematic satisticprobably because the sol fraction could not be determined softw:| 中國煤化工grow rales ofexprecisely due to difficult extraction of water-soluble HA perim0.24、9.30士 1.93molecules from the cross-linked HA hydrogels with andI:TYHC N M H Gorded with differentlower than 90 wt%, the apparent sol fraction was always cultivated period of2 d, 4 d and 7 d, indicating that cellsclose to zero. According to Fig.5, we knew that utilizing grew and divided better, and cell quantity gradually in-cross-linking method of solution could produce creased with time. Moreover, they were higher than that.620Vol.23 No.5 CHEN Jinghua et al: Rheological and Biological Characteristics..20)1201120 1100g88(80言606(60二404020至2010i55isTime/dTimc/d(a)Fig.6 In vitro degradation of HA films cross linked immersed in PES(pH7.4) at different PEG concentrationand pH.▲:0.8%; :2.0%;◆:3.6% (a) pH 4.7; (b) pH 6.0; (c) pH 8.0250 ,140，180元oA。0↑望140150=8905680|A)多201。弋e20 406080100122040608010012040 60 80 1o0Water conten/wt%(b)(C)Fig.7 In viro degradation of HA flms cross-linked immersed in PBS(pH7.4) at dffent immersed days against the water contentof HA cosinked at dfferent pH: 0: 0.8%;▲: 2.0%;■: 3.6%. (a)2d; (b)5d;(e) I0da)(bc)d)(eand 7 dX200; de f: positive group2 d, 4 d and 7 dX200)Tab 1 Relative growth rate in the different experimental groups(n=5, %, xxs)Second dayFourth daySeventh dayGroupsARGRNegative control1.78士0.21100.0010.0土1.中國煤化工:1.02100.0Experimental1.68土0.24"94.389.30士1MYHCNMHG141”99.83Positive control0.96土0.2453.936.45士0.4063.997.01士0.7061.17* Compared with positive control group, P <0.05Joumal of Wuhan University of Technology-Mater. Sci. Ed. Oct 2008621of positive control group, difference (P<0.01) had statis-tic meaning, and there was not obvious difference withI Referencesnegative control group. In Table 1, average relativegrowth rate(RGR) of experimental group cells with dif-[1] Laurent T C, Laurent U B, Fraser J R. The Structure andferent incubated periods, respectively,2 d, 4 d and 7 dFunction of Hyaluronan: An Overview[J]. lmmunol Cellwas 95.49%， its cytotoxicity was estimated 1 grade,Biol, 1996, 74:A1-A5which completely accorded with requirement to tissues [2] Kim B S, Mooney D J. Development of Biocompatible Syn-engineering biomaterials. Cell relative growth rate ofthetic Extracellular Matrices for Tissue Engineering[].positive control group was evidently lower than that ofTrends Biotechnol, 1998, 16(5):224-227experimental group, every spot had its statistic [3] Chen Jinghua, Xu Zheng, Gu Qisheng, et al. Research onsense(P<0.05)，, average RGR was 59.69%, having someRheologyical Property of Hyaluronic Acid DerivativeCross-linked by Polyethylene Glycol[]. J. of Tongji Uni-versity, 2006, 34(1):34-38(in Chinese)4 Conclusions[4] Ambrosio L A, Borachiello P A, Netti P A, et al. Propertiesof New Materials: Rheological Study on Hyaluronic Acida) HA was successfully modified by polyethyleneand Its Derivative Solutions[J]. JMS-Pure and Appliedglycol. Elastisity and viscosity of HA derivative solutionChemistry, 1999, A36 (718):991995was in direct ratio to its mass fraction. And elastic mod- [5] Wik B H, Wik O. Rheology of Hyaluronan and Its Derivutive.ule increased and viscous module decreased with theThe Chemistry, Biology and Medical Applications of Hyalu-increment of shearing frequency.ronan and lts Derivatives[M]. London: Portland Press, 1998, 45:b) The conversion from low vibrated frequency to25-29high vibrated frequency for solution equaled from vis-[6] Balazs E A. The Viscoelastic Interellular Marix and Con-cosity to elasticity, and with the increment of solutiontrol of Cell Function by Hyaluronan. The Chemistry, Biologyconcentration, the position of crossing point changed toand Medical Application of Hyaluronan and lts Deriva-be lower and lower. Under the same concentration, thetives[M]. London: Portland Press, 1998;185-188changing point frequency from viscosity to elasticity for [7] Kenji Tomihata, Yoshito Ikada. Preparation of Cos-linkedHA was higher than for HA derivative.Hyaluronic Acid Films of Low Water Content[J]. J. Bioma-c) Utilizing cross-linking method of solution couldterials, 1997, 18(3):189-194produce cross-linked HA derivative hydrogel films with [8] Tomihata K, Burczak K, Shiraki K. et al. Cross-linking andwater contents as low as 60 wt%.Biodegradation of Native and Denatured Collagen|C]. Ind) In vitro degradation of HA derivative filmsPolymers of Biological and Biomedical Significance,ed. S Wcross-linked with PEG was virtually governed by theShalaby Y Ikada, R, Lannger and J Williams. Americanwater content of films, which, in turn, depended on theChemical Society, Washington DC, 1994:275-278pH of the casting solution used for preparing cross-linked [9] Kobayashi H, Shiraki K and Ikada Y. Toxicity Test of Bio-HA derivative films. And the optimal pH of the castingdegradable Polymers by Implantation in Rabbit Corneal[]. Jsolution for HA cross-linked by PEG was around 8.Biomed. Mater. Res, 1992, 26:1 463-1 466e) Growth, differentiation and proliferation of ex- [10] Li Yanlin, Yang Zhiming, Xie Huiqi. et al. Research on Cellperimental group cells including HA derivative solutionBiocompatibility in Vitro of Bio-derivative Materials. Chi-always went on, and cell quantities gradually increased,nese J. Reparative and Reconstructive Surgery, 2001, 15(4):cell shape was all right along with time after incubated 2 d,227-231(in Chinese)4d and7d.[1] Sung Hsing-wen, Huang Dong nan and Huang lynl H,et al. Inf) Average relative growth rate(RGR) of experi-Viro Evaluation of Cytotoxicity of a Natrally Ourringmental group cells with different incubated periods, re~Cross linking Reagent for Biological Tissue Fixation[J]. J. Bio-spectively,2 d, 4 d and 7 d was 95.49%, and its cyto-mater. Sci. Polymer Edn, 1999, 10(1):63-67(in Chinese)toxicity was estimated as 1 grade.[12] Lv Xiaoying, Kappert HF. New Method of Esimated CytotocityHA derivative modified by polyethylene glycolof Dentistry Materials (MTT experiment)[]. Chinese J. ofmeets requirement of biomaterials and can be safely usedNornasaliry Medicine, 1995, 30(5);337-341 (in Chinese)in medical field in terms of its rheology properties, invitro degradation and cytotoxicity.中國煤化工MYHCNMHG