Mechanical Properties of Biomimetic Tissue Adhesive Based on

Mechanical Properties of Biomimetic Tissue Adhesive Based on the Microbial Transglutaminase-Catalyzed Crosslinking ofGelatinMartin K.McDermott,†Tianhong Chen,‡Christina M.Williams,§Kolleen M.Markley,|andGregory F.Payne*,‡,⊥Division of Mechanics and Materials Science,Office of Science and Technology, Food and Drug Administration,9200Corporate Blvd.,HFZ-150,Rockville,Maryland20850, Center for Biosystems Research,University of Maryland Biotechnology Institute,5115Plant Sciences Building,College Park,Maryland20742,Department of Biomedical Engineering,Texas A&M University, College Station,Texas77843-0100,Department of Biomedical Engineering,Marquette University, Milwaukee,Wisconsin53201-1881,and Department of Chemical and Biochemical Engineering, University of Maryland,Baltimore County,1000Hilltop Circle,Baltimore,Maryland21250 Received December15,2003;Revised Manuscript Received March11,2004Fibrin sealants are a type of soft tissue adhesive that employs biochemical reactions from the late stages of the blood coagulation cascade.Intrinsic to these adhesives are a structural protein and a transglutaminase crosslinking enzyme.We are investigating an alternative biomimetic adhesive based on gelatin and a calcium-independent microbial transglutaminase(mTG).Rheological measurements show that mTG catalyzes the conversion of gelatin solutions into hydrogels,and gel times are on the order of minutes depending on the gelatin type and concentration.Tensile static and dynamic loading of the adhesive hydrogels in bulk form demonstrated that the Young’s modulus ranged from15to120kPa,and these bulk properties were comparable to those reported for hydrogels obtained from fibrin-based p-shear adhesion tests of porcine tissue were performed using a newly published American Society for Testing and Materials(ASTM)standard for tissue adhesives.The gelatin-mTG adhesive bound the opposing tissues together with ultimate adhesive strengths of12-23kPa which were significantly higher than the strength observed for fibrin sealants.Even after failure,strands of the gelatin-mTG adhesive remained attached to both of the opposing tissues.These results suggest that gelatin-mTG adhesives may offer the benefits of fibrin sealants without the need for blood products.IntroductionSoft tissue adhesives are required to perform a variety of functions:stop bleeding,seal leaks,bind tissue,and/or facilitate healing.1,2Typically,these functions are met through the in situ generation of a three-dimensional polymeric network that is bonded to the tissue.There are two chemical approaches commonly used for generating such networks: the in situ polymerization of reactive monomers and the in situ crosslinking of preformed polymers.An example of the former is the cyanoacrylate-based adhesives that undergo rapid in situ polymerization upon contact with the tissue. An example of the latter is the gelatin-based adhesives that are in situ crosslinked by resorcinol and formaldehyde.A disadvantage of both of these adhesives is the toxicity of formaldehyde which is a degradation product of cyanoacry-late adhesives3,4and a reactant in gelatin-resorcinol-formaldehyde adhesives.There has been considerable recent effort to develop soft tissue adhesives that are less toxic and address other limitations of currently marketed adhesives. These efforts include the use of alternative materials(e.g., albumin,polysaccharides,poly(ethylene oxide)s),5-10better crosslinkingchemistries(e.g.,glutaraldehyde,carbodiimide),11-15 and more controllable polymerization reactions(e.g.,photo-initiated polymerization).16-18Biochemical crosslinking approaches could offer a more biocompatible alternative to synthetic crosslinking for the in situ generation of the three-dimensional polymeric network required for adhesion.One biochemical approach is to exploit the water-resistant adhesive that allows mussels to attach to wet and submerged surfaces(i.e.the mussel glue).19,20 Scheme1a shows that the mussel uses an enzyme to oxidize phenolic residues(i.e.,tyrosine or dihydroxyphenylalanine residues)of its adhesive protein.These oxidized residues undergo subsequent uncatalyzed crosslinking reactions to yield the three-dimensional polymeric network required for adhesion.21-24A second biochemical crosslinking approach relies on the reactions that naturally occur during blood coagulation.Scheme1b shows that during coagulation thrombin cleaves the blood protein fibrinogen into fibrin that undergoes subsequent crosslinking by factor XIIIa.As*To whom correspondence should be addressed.Phone:(301)405-8389.Fax:(301)314-9075.E-mail:payne@.†Food and Drug Administration.‡University of Maryland Biotechnology Institute.§Texas A&M University.|Marquette University.⊥University of Maryland,Baltimore County.1270Biomacromolecules2004,5,1270-127910.1021/bm034529a CCC:$27.50©2004American Chemical SocietyPublished on Web04/21/2004illustrated in Scheme 1b,factor XIIIa is a calcium-dependent transglutaminase enzyme.25-27Fibrin sealants are currently used in clinical practice 28,29and are prepared by mixing two solutions,typically one contains thrombin and calcium chloride and the second contains fibrinogen and factor XIII (factor XIII is a zymogen that is activated into factor XIIIa by thrombin and calcium 27).One concern with these bio-chemical approaches is the source of the protein;the mussel’s adhesive protein is difficult to obtain,and components of the fibrin sealant are derived from blood.We propose an alternative adhesive that is analogous to the fibrin sealant.Like the fibrin sealant,Scheme 1c shows that this proposed adhesive exploits the same biochemical crosslinking chemistry as the blood coagulation cascade.The differences are that the proposed adhesive uses gelatin as the “structural”protein (not fibrin)and a calcium-independent microbial transglutaminase as the crosslinking catalyst (not factor XIIIa).An adhesive based on gelatin and microbial transglutami-nase (mTG)should be nontoxic since gelatin is already used in various medical products while mTG is approved for food use.30(It should be noted that mTG’s approval for food use does not necessarily indicate that it will be biocompatible in medical applications.)A gelatin -mTG adhesive should also be simple to produce since both components are commercially available and derived from sources more convenient than blood.Finally,a gelatin -mTG adhesive may be simpler to use and subject to fewer sources of variability since there are fewer components and only one reaction step (as illustrated in Scheme 1b,the fibrin sealant requires twothrombin-catalyzed reactions and one transglutaminase-catalyzed reaction).Here we examined whether the mTG-crosslinked gelatin hydrogels offer appropriate mechanical properties for ap-plications as a soft tissue adhesive.There are two challenges to characterizing the mechanical properties of such adhesives.First,when such an adhesive “sets”,its bulk mechanical properties undergo dramatic changes and these changes are not readily observed using a single measurement method.Typically,“setting”is initiated by mixing the structural and crosslinking components.31As the three-dimensional poly-meric network is being formed,the sample undergoes a characteristic sol -gel transition that yields an elastic gel.Rheology is commonly used to examine the sol -gel transi-tion (i.e.,to measure gel time),monitor the initial evolution of mechanical properties,and evaluate the properties of weak gels.32-36Once the hydrogels become strong,however,rheology becomes less convenient;gels formed outside the rheometer cannot be easily loaded onto the instrument while gels formed within the instrument adhere to the parts (i.e.,to the cone and plate).To characterize the mechanical properties of strong gels,standard tensile tests and dynamic mechanical analysis can be used.37The second challenge in evaluating the mechanical proper-ties of a tissue adhesive is the testing method for establishing its adhesive strength.Numerous ASTM methods exist for industrial adhesives.However,these methods are not ap-propriate for evaluating tissue adhesives because tissues are mechanically compliant,have variable moisture levels,and present chemically complex surfaces.The lack of appropriate methods coupled with an increasing interest in medical adhesives has resulted in the development of a new ASTM method for testing tissue adhesives.This method is based on traditional lap-shear methods but employs porcine skin as the model tissue.38In the current study,we designed fixtures to create lap-shear adhesive joints that could be tested following this ASTM method.Materials and MethodsMaterials.Gelatin (type A from porcine)was obtained at two separate bloom values,175and 300(Sigma-Aldrich,St.Louis,MO).Bloom is a standard industrial measure used to rate gelatin “quality”and is related to the shear modulus of physical gelatin gels.We selected a gelatin prepared by acid treatment (type A)and not base treatment (type B),because base treatment hydrolyzes the amide groups of glutamine residues 39and suppresses enzymatic crosslinking.40The calcium-independent microbial transglutaminase (mTG;Activa TI)was obtained from Ajinomoto and was used without further purification.This enzyme is supplied as a proprietary formulation with a maltodextrin support and is reported by the manufacturer to have a specific activity of 100U/gm (lot number L-04207).The skin tissue was obtained from Brennen Medical (St.Paul,MN)and was nonperforated Porcine Mediskin Xenograft (S-106)that was supplied frozen in 7×9cm 2sections with a thickness of 0.032(0.005cm.The fibrin sealant,BaxTisseel Fibrin Sealant,was kindly supplied by Baxter Healthcare Corp.(Glendale,CA).Scheme1Biomimetic Tissue AdhesiveBiomacromolecules,Vol.5,No.4,20041271A concentrated enzyme solution was prepared for each experiment by mixing0.5gm of mTG per mL of deionized water.This enzyme solution was stored on ice until it was ready for use.A concentrated gelatin solution was prepared by adding50gm of gelatin to100mL of warm deionized water and mixing at50°C until the protein was dissolved. The pH of this concentrated gelatin solution was adjusted to6.0by adding small amounts of1M NaOH.Aliquots from this concentrated gelatin solution were then mixed with deionized water to prepare solutions with differing gelatin concentrations.The gelatin solutions were either used immediately,or stored at4°C for no more than4days. Gelatin solutions that were refrigerated were melted at50°C for no more than1h prior to use.Rheological Testing of Adhesive.Rheological tests were performed to examine the dynamics of gel formation.Gel formation was initiated by adding mTG solution to gelatin solutions to achieve a final enzyme activity of15U/gm-gelatin.41After mixing mTG,the solution was immediately loaded on top of the bottom plate of the rheometer,bubbles were removed from the solution using a pipet,and therheometer’s top plate(i.e.,the cone)was lowered onto the sample.Excess solution that exuded from the gap between the top cone and bottom plate was removed using a paper towel,silicon oil was placed along the edge of top plate to limit sample drying,and the rheometer test was initiated. Less than2min were required from the time mTG was mixed with the gelatin solution until the rheological test was initiated.Rheological tests were performed using a Thermo Haake RheoStress1rheometer(Thermo Electron Corpora-tion,Waltham,MA)at a temperature of37(1°C.A6.0 cm diameter cone-and-plate sensor was used with an angle of1°and a gap distance of0.052mm.The samples were subjected to an oscillatory stress of0.5Pa at a frequency of 0.1Hz.Bulk Mechanical Testing of Adhesive.The bulk me-chanical properties of the gelatin-mTG gels were character-ized by tensile static and dynamic testing.The samples were prepared by mixing mTG with gelatin solutions to a final enzyme activity of15U/gm-gelatin.After adding mTG,the solutions were poured into a Petri dish,sealed with a dampened lid and allowed to undergo crosslinking on a level surface at37°C.After incubating for90min,the crosslinked hydrogel was lifted from the Petri dish and cut into tensile specimens having cross-sectional dimensions of0.95(0.03 cm and0.32(0.01cm and lengths of2to2.5cm.After cutting the specimens and measuring their dimensions at room temperature(which required about20min),the tensile specimens were returned to the humid,37°C incubator.The first sample was tested150min after mixing the enzyme into the gelatin solution.The mechanical tests were performed using a dynamic mechanical thermal analyzer(DMTA)V Instrument(Rheo-metric Scientific,Inc.,Piscataway,NJ).The tensile samples were removed from the incubator and placed in the tensile fixture grips that were separated by a1cm gap.A0.03cm diameter wire thermocouple was inserted into a portion of the sample that protruded above the top grip.After closing the sample chamber,2-5min were required for the sample to reequilibrate to37(1°C.The sample was then loaded at a strain rate of1%/sec to a strain of20%and then unloaded back to0%strain.Initial studies showed that the stress-strain curves were different for the first and second loading/ unloading cycles.Such differences are commonly observed with soft,biological materials.42The stress-strain curves for subsequent loading/unloading cycles resulted in stress-strain curves that were similar to that for the second cycle(we examined4loading/unloading cycles in our initial studies). Thus,we used the stress-strain curve from the sample’s second loading/unloading cycle to determine the Young’s modulus(E).E was calculated as the slope of the linear regression line for data between0and4%strain.Immediately after completing the tensile static test,the sample was dynamically loaded at a peak strain of1.5%and frequency of0.5Hz.These dynamic tests allowed the determination of the complex modulus(E*),storage modulus(E′),loss modulus(E′′),and loss tangent(tanδ)E′′/E′).Lap-Shear Testing of Adhesive p-shear testing was performed following the procedures in ASTM F2255-03(“Test Method for Strength Properties of Tissue Adhesives in Lap-Shear by Tension Loading”).Figure1 shows that0.90(0.04cm2porcine skin tissue samples were attached to the aluminum backing using ethyl cyanoacrylate glue(Krazy Glue,Toagosel Co.,Ltd,Japan).As illustrated in Figure1,about0.15mL of gelatin-mTG adhesive was applied to one tissue surface,and then the two surfaces were lapped together.A47.5(0.3gm lead weight was placed on the adhesive joint directly over the tissues.The adhesive joint with the weight were incubated at37(1°C in enough water to cover the adhesive joint(but not the weight).After 120min,the adhesive joint was removed from the water and attached to the tension fixture of the DMTA.After the sample temperature was equilibrated to37(1°C in1-2 min,the upper and lower grips were separated at a speed of 5mm/min to measure the adhesive shearstrength.p-shear adhesive test.Schematics illustrate how the adhesive joints were formed and tested.Photographs of an adhesive joint alone and after mounting in the mechanical analyzer.1272Biomacromolecules,Vol.5,No.4,2004McDermott et al.Results and DiscussionFormation of Adhesive Hydrogel.Rheological methods were used to characterize the dynamics of gel formation.In these experiments,a gelatin solution was mixed with transglutaminase and immediately loaded onto the rheometer where the samples were subjected to oscillatory stresses.Figure 2shows typical results for samples prepared with gelatins of 175and 300bloom.Figure 2a shows that immediately after mixing mTG with the 175bloom gelatin,the sample behaved as a solution with the elastic modulus (G ′)being less than the viscous modulus (G ′′).Over time,Figure 2a shows that both moduli increased,but G ′increased more rapidly and became equal to G ′′after 28min.The point at which G ′becomes equal to G ′′is typically used as a rheological measure of the gel-point.32,36Figure 2b showsresults for a sample prepared with a 300bloom gelatin.As expected,the higher bloom gelatin had higher initial moduli and gelled more rapidly compared to the lower bloom gelatin of Figure 2a.A convenient comparison of the two samples is summarized in Figure 2c that shows the complex viscosity (η*)increases more rapidly for the higher bloom gelatin.The results in Figure 2show that transglutaminase catalyzed the conversion of gelatin solutions into gels (gelatin solutions lacking transglutaminase do not form gels at 37°C).These gels formed as a result of the covalent crosslinking of the gelatin chains and differ from the thermally reversible physical gelatin gels that are induced by cooling.40,41,43A series of rheological tests were performed with samples containing different gelatin concentrations.Figure 3a shows that the gel time depends on the gelatin bloom with lower bloom gelatin taking longer to gel.Depending on the gelatin concentration,we observed gel times to vary between 10and 29min for the 175bloom gelatin,and 2.7to 5.1min for the 300bloom gelatin.These time frames areconvenientFigure 2.Evolution of mechanical properties for gels formed by the mTG-catalyzed crosslinking of gelatin.Elastic (G ′)and viscous (G ′′)moduli for hydrogels formed from (a)175bloom gelatin and (b)300bloom gelatin.(c)Complex viscosity (η*)of these two samples.Gels were prepared by incubating gelatin (16.5%)with mTG (15U/gm-gelatin)at 37°C while these samples were subjected to oscillatory stresses (0.5Pa at 0.1Hz).Figure 3.Effect of gelatin bloom and concentration on gel formation.(a)Gel time is the time where the elastic and viscous moduli cross (i.e.,G ′)G ′′).(b)Complex viscosity (η*)evaluated at the gel point.(c)Elastic moduli evaluated at the gel point.Tests were performed in triplicate.Biomimetic Tissue Adhesive Biomacromolecules,Vol.5,No.4,20041273in the sense that it allows the gelatin and mTG to be thoroughly mixed before being applied to the tissue.If necessary,it should be possible to adjust the gel time by adjusting the mTG activity or adding other ingredients(not investigated here).For instance,previous studies showed that the gel time is significantly reduced by the inclusion of a high molecular weight polysaccharide(i.e.chitosan)to the solution.41Figure3,parts b and c,shows the complex viscosity(η*) and elastic moduli at the gel point(G′)G′′)for samples prepared at differing gelatin concentrations.These parameters provide a measure of the flow properties and gel strength at a single time,the gel-point,and provide an indication of the ease of mixing and applying the adhesive to the tissue surface.31If an adhesive is too weak or is applied before it has begun to gel,then it will have low viscosities and elasticities.In this instance,the adhesive will be able to flow into and mechanically“interlock”with tissue to improve adhesive-tissue bonding.However,a lower viscosity ad-hesive may lack sufficient cohesive strength to be retained where it is applied and it may flow away.This problem can be particularly important if the adhesive must be applied to wet tissue.In contrast,stronger gels or gels that have been allowed to set longer have greater cohesive strength(i.e., larger G′),retain their shape,but may not effectively penetrate and interlock with tissue.Thus,the adhesive’s flow properties and gel strength are practically important and the desired values will depend on the specific situation(e.g., wet or dry tissue).The adhesive’s flow properties and gel strength can be controlled by the enzyme activity,the gelatin bloom,and the gelatin concentration,whereas the results in Figure2show that these properties are rapidly changing during crosslinking.Bulk Mechanical Properties of Adhesive Hydrogel.The bulk mechanical properties of the mTG-catalyzed gelatin gels were characterized by static and dynamic tensile loading. For these studies,mTG was mixed with a gelatin solution and incubated in a Petri dish to form hydrogels that were subsequently cut into tensile specimens.Approximately 2.5-3h after initiating the reaction,the specimens weremounted in tensile fixtures for analyses.In the first study, the specimen was loaded in tension at a rate of1%/s(0.01 cm/s)to20%strain and then unloaded back to0%strain. Loading and unloading were performed for two cycles,and results from the second cycle were analyzed.The typical stress-strain curve of Figure4a shows that little hysteresis was observed between loading and unloading which indicates that the sample underwent little plastic deformation.This behavior is expected for a crosslinked elastic network.The Young’s modulus(E)was calculated from Figure4a using the slope of the linear regression line for strains between0 and4%.The value of E determined for the sample of Figure 4a is61kPa.Tensile static tests were performed for samples prepared at differing gelatin concentrations.Figure4b shows that the magnitude of E was between15and120kPa.This range is similar to that reported for fibrin-based adhesives containing high fibrin content(15and30kPa for sealants prepared with 30and70mg/mL fibrinogen,respectively).44Figure4b also shows that E tended to increase with increasing gelatin concentration which is also consistent with results from fibrin-based adhesives.44Finally,Figure4b shows that bulk properties of the gelatin-mTG adhesive were significantly dependent on the gelatin bloom with higher bloom gelatin yielding higher values for E.After completing the tensile static tests,each sample was then dynamically loaded at a peak strain of1.5%and a frequency of0.5Hz.These measurements allow the follow-ing properties to be determined;complex modulus(E*), storage modulus(E′),loss modulus(E′′),and tanδ()E′′/ E′).Results from these studies are summarized in Table1. For each of the samples prepared with the300bloom gelatin, the values for E*and E′are nearly the same,whereas the values for tanδare substantially less than0.1.These results indicate that these materials behave as elastic solids.Con-sistent with the results in Figure4b,Table1shows that samples prepared from the175bloom gelatin had lower E* and E′values compared to samples prepared from300bloom Figure4.Tensile static testing of mTG-catalyzed gelatin-based gels. Samples were prepared by reacting gelatin with mTG(15U/gm-gelatin)in a Petri dish to form thin hydrogels.After reaction,the gels were cut into tensile specimens that were mounted in tensile fixtures, loaded to20%strain and then unloaded.(a)Typical stress-strain curve for a sample prepared with300bloom gelatin(16.5%)during its second loading/unloading cycle.(b)Young’s modulus(E)for gelatin-mTG gels prepared with differing gelatin blooms and con-centrations.Tests were performed with5-10replicates.Table1.Bulk Mechanical Properties of Hydrogels Formed by mTG-Catalyzed Crosslinking of Gelatingelatinbloom(%)E(kPa)E*(kPa)E′(kPa)E′′(kPa)tanδ17516.515(38.5(3.27.8(3.3 2.8(1.70.39(0.22 17526.540(732(732(7 2.5(0.90.08(0.04 30016.560(560(560(5 1.9(0.30.03(0.01 30019.977(1272(1472(14 1.9(0.20.03(0.01 30023.191(1399(2499(24 6.8(5.10.06(0.04 30026.5120(22130(20130(20 6.4(4.20.05(0.031274Biomacromolecules,Vol.5,No.4,2004McDermott et al.gelatin.Also,Table 1shows good agreement in results obtained from tensile static testing and dynamic loading of the samples (i.e.,E ≈E *).It is desirable to relate the observed mechanical properties for the mTG-crosslinked gelatin gel to the underlying network structure.A strict chemical approach to assess network structure (i.e.,the types and number of crosslinks)is complicated by the facts that gelatin is not a simple monodisperse starting material,and the crosslinks are difficult to detect/quantify because the linkage is not unique (i.e.,an amide crosslink replaces an amide side chain).An alternative (although approximate)approach to estimate network struc-ture is to employ simplified physical models and use averaged values for gelatin.As outlined in the Appendix,we employed an idealized model for rubber elasticity as suggested by Sperling.45Also,we used an average amino acid composition for type A gelatin obtained from Rose 39and an average molecular weight for our gelatin provided by the supplier.This method indicates that the crosslink density for our gels reaches 8mole crosslink/m 3and 2crosslinks per gelatin chain,but only about 20%of the glutamine and lysine residues undergo crosslinking.Strength of Gelatin -mTG Adhesive.The testing of tissue adhesives generally results in high variability for numerous reasons:the starting tissue can have varying properties (e.g.,moisture levels);alignment of the tissue surfaces prior to contact can be challenging;and the adhesive joints can be damaged in sample handling.To minimize these problems,we constructed the alignment fixture shown in Figure 5that allows seven adhesive joints to be formed rapidly and reproducibly.Initially,skin tissue was bonded to aluminum backings (using cyanoacrylate adhesive)and the tissue was kept moist by covering it with gauze that had been soaked in PBS buffer.Seven backings with tissues attached were placed onto the alignment fixture to create the lower tissue surfaces of the seven adhesive joints.As illustrated in Figure 5,the adhesive (≈0.15mL)was spread onto each upper tissue surface,and then the upper and lower surfaces were contacted to create an adhesive joint.A weight was attached to the upper backing to apply a consistent force to each adhesive joint while it was curing.The sliding spacer bars shown in Figure 5ensured that the upper and lower tissue surfaces were aligned when they were being contacted.These spacer bars were removed immediately after the tissue surfaces were contacted and the adhesive joint was formed.Removing the spacer bars avoids the possibility that any adhesive that is exuded from between the tissue surfaces can form a bond with the surface of the spacer.Any bonds formed between the adhesive joint and other surfaces can make it difficult to remove the adhesive joint from the alignment fixture and risks damage to the adhesive bond formed between the two tissue surfaces.Approximately 3.5min were required to complete the formation of all seven adhesive joints in the alignment fixture.After forming the seven adhesive joints,the alignment fixture was submerged in a water bath at 37°C for 2h while the adhesive was curing.When incubation was complete,the adhesive joints were removed from the alignment fixture,mounted on the DMTA and tested by applying loads to separate the surfaces at a rate of 5mm/min.Figure 6a shows a typical force versus deflection curve for this lap-shear test in which failure was defined as the maximum load achieved (the maximum load for the sample in Figure 6a was 1.6N).When the sample was strained past failure,the force resisting further deflection diminished slowly indicating that failure was not brittle.The adhesive’s toughness results because strands of gel remain connected to the two skin surfaces even after failure.In fact,Figure 6b shows that some strands remain even after the resisting force diminished to zero and the samples were removed from the DMTAgrips.Figure 5.Alignment fixture to assemble adhesive joints.Schematics and photographs show adhesive joints in various stages of formation in the alignment fixture.Seven aluminum backings with tissue were placed in the fixture separated by removable spacer bars.Adhesive (≈0.15mL)was applied to the upper tissue surfaces and the two tissue surfaces were contacted to create the adhesive joint.The spacer bar separating two finished adhesive joints was removed as shown,leaving the adhesive joints aligned and exposed to the environment.The completed adhesive joints in the alignment fixture were immersed in water and allowed to cure at 37°C.Biomimetic Tissue Adhesive Biomacromolecules,Vol.5,No.4,20041275。