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Hindawi Publishing CorporationInternational Journal of BiomaterialsVolume 2013 Article ID 620765 14 pageshttpdxdoiorg1011552013620765

Research ArticleTemperature-Responsive Gelation of Type I CollagenSolutions Involving Fibril Formation and Genipin Crosslinkingas a Potential Injectable Hydrogel

Shunji Yunoki Yoshimi Ohyabu and Hirosuke Hatayama

Biotechnology Group Tokyo Metropolitan Industrial Technology Research Institute 2-4-10 Aomi Koto-ku Tokyo 135-0064 Japan

Correspondence should be addressed to Shunji Yunoki yunokishunjiiri-tokyojp

Received 24 May 2013 Revised 9 August 2013 Accepted 24 August 2013

Academic Editor Bruce Milthorpe

Copyright copy 2013 Shunji Yunoki et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

We investigated the temperature-responsive gelation of collagengenipin solutions using pepsin-solubilized collagen (PSC) andacid-solubilized collagen (ASC) as substrates Gelation occurred in the PSCgenipin solutions at genipin concentrations 0ndash2mMunder moderate change in temperature from 25 to 37∘C The PSCgenipin solutions exhibited fluidity at room temperature for atleast 30min whereas the ASCgenipin solutions rapidly reached gel points In specific cases PSCwould be preferred over ASC as aninjectable gel system The temperature-responsive gelation of PSCgenipin solutions was due to temperature responses to genipincrosslinking and collagen fibril formation The elastic modulus of the 05 PSCgenipin gel system could be adjusted in a range of25 to 50 kPa by the PSC and genipin concentrations suggesting that a PSCgenipin solution is a potential injectable gel system fordrug and cell carriers with mechanical properties matching those of living tissues

1 Introduction

Injectable hydrogels (in situ formed hydrogels) are promis-ing substrates for tissue-engineering applications because oftheir ability to provide biodegradable scaffolds with physicalproperties that match those of living tissues and to con-trol drug delivery with minimally invasive operations [1]Thermo- and pH-responsive polymers have been studiedextensively with the aim of achieving physical crosslink-ing that responds to physiological conditions (pH neutraltemperature 37∘C) without using cytotoxic stimulation [2]Such physical crosslinking poses little or no cytotoxicity [3ndash5] In contrast chemical crosslinking employs free radicalsMichael-type addition or Schiff-base reactions [1] A physi-cally crosslinked injectable hydrogel is thus considered to bemore biocompatible

Type I collagen (abbreviated here as ldquocollagenrdquo) has beenused as a physically crosslinked injectable hydrogel Collagenmolecules are stable in acidic solutions at low temperaturesand are capable of forming nanofibrils that respond to bodytemperature and neutral pH [6 7] permitting use of colla-gen as an injectable hydrogel [8 9] Among biocompatible

polymers collagen is one of themost preferred for gel systemsbecause of its many advantages including biocompatibilitybioabsorbability and cell adhesiveness [10] Injectable colla-gen hydrogels have been successfully used for soft-tissue aug-mentation [11 12] drug delivery carriers [13 14] and hard-tissue augmentation [15] Collagen offers an ideal injectablegel system from the biological perspective but the weakmechanical properties of collagen gels limit their practicalapplications The complex modulus of cell-encapsulated col-lagen gel is less than 20 Pa at a collagen concentration of 02[16] much lower than that of living tissues The shear storagemodulus of acellular collagen gel is no more than 150 Pa[17] However conventional chemical crosslinking cannot beused for injectable collagen gel systems because the cytotoxiccrosslinkers are directly exposed to living cells and tissues

Regarding the potential cytotoxicity of chemical cross-linkers natural crosslinkers such as enzymes (eg transg-lutaminase [18] lysyl oxidase [19]) and plant extracts (eggenipin [20ndash26]) have been expected to provide alternativesand reduce cytotoxicity of biomaterials Among them geni-pin shows rapid crosslinking of collagen comparable to glu-taraldehyde [21] Genipin is a natural crosslinker of proteins

2 International Journal of Biomaterials

extracted from gardenia fruit and its cytotoxicity has beenreported to be much lower than that of most commonly usedsynthetic crosslinkers

Mechanical characterization of collagen fibrillar gelscrosslinked by genipin has been firstly reported by Sundara-raghavan et al [24] Macaya and coworkers [25] have recentlyreported an injectable collagen gel system using genipin as acrosslinker and acid-solubilized collagen (ASC) as a sub-strateThemechanical properties of the gels (of storage mod-ulus approximately 250 Pa) were much better than those ofcollagen fibrillar gels without chemical crosslinking [16]

However the fluidity of the collagengenipin solutionat room temperature was unknown We expected that theslow fibril formation of pepsin-solubilized collagen (PSC)compared with ASC [27 28] would contribute to maintainfluidity at room temperature PSC is prepared from ASC orcollagenous tissues by pepsin digestion and lacks nonhelicalterminal regions (telopeptides) while triple-helical confor-mation is intact [29] Conventional biomaterial processinghas employed PSC rather than ASC because of its processing-related [29] and immunological benefits [30] The tempera-ture responsiveness of genipin crosslinking is as important forgelatin properties as that of collagen fibril formation A pre-vious study [21] showed that genipin crosslinking of collagenwas similar in 72 h reaction at temperatures of 25ndash45∘Chowever the temperature responsiveness on second-minutetimescale is still unknown Sol to gel transition of injectablecollagen (ie fibril formation) occurs on such timescale

In the present study we investigated the fluidity of acollagengenipin solution at room temperature and its gela-tion response to physiological temperatures using pepsin-solubilized collagen (PSC) as a substrate We hypothesizedthat an injectable collagen gel system using PSC confers suf-ficient and prolonged fluidity at room temperature We alsoinvestigated the temperature response of genipin crosslink-ingThe potential of PSCgenipin solution as an injectable gelsystem is discussed

2 Materials and Methods

21 Materials Materials comprise PSC from porcine skinin dilute HCl (1 solution Nipponham Japan) ASC fromporcine tendon in dilute HCl (03 solution Nitta GelatinJapan) chitosan powder (Chitosan 500Wako Pure ChemicalIndustries Japan) genipin (Wako Pure Chemical IndustriesJapan) glutaraldehyde (25 solution Wako Pure Chemi-cal Industries Japan) and phosphate-buffered saline (PBS)tablets (Sigma-Aldrich MO USA)

22 Preparation of Solutions The PSC (1) and ASC (03)and solutions were used with no pretreatment Accordingto the manufacturesrsquo data by electrophoresis PSC and ASCcontained 11 wt and a trace amount of type III collagenrespectively PSC solutions of 03 and 06 were preparedby dilution of the 1 PSC solution with pH 3 dilute HCl A1 chitosan solution was prepared by dissolution of chitosanpowder in 100mM acetic acid solution Genipin solutions atconcentrations ranging from 1 to 10mM were prepared by

dissolution of genipin powder in 2times PBS and held for 2 weeksin the refrigerator prior to use for the following examinationsGA solutions at a concentration of 12mM were prepared bydilution of 25 GA solution with 2 times PBS

23 Dynamic Viscoelastic Tests

231 Apparatus Changes in fluidity and gelation processeswere monitored by dynamic viscoelastic tests with a rheome-ter (HAAKE MARS III Thermo Fisher Scientific Inc MAUSA)The temperature was sensitively controlled by a Peltiercontrolled plate Two types of sensors were used a double-cone sensor DC601Ti (diameter of 60mm cone angle of 1∘)and a parallel-plate sensor HPP35S (diameter of 35mm withsurface serrated to avoid slippage of gels during oscillation)The double-cone sensor has a shear surface twice as highas that of a conventional cone plate stabilizing viscoelasticdata of low viscous samples by enhanced sensitivity A lidwas used to minimize drying of the solutions during mea-surement at room temperature enabling precise viscoelasticmeasurements for more than 30min

232 Sample Preparations PSCgenipin and ASCgenipinsolutions were prepared as follows PSC and ASC solutions(3 g) at concentrations of 03 06 or 1 were placed in 15mLpolypropylene tubes and maintained at 4∘C Three millilitersof precooled genipin solution or GA solution (concentrationsof 0ndash10mM) at 4∘C was added to the collagen solution andvortexed immediately to prepare neutral collagen solutioncontaining genipin or GA in 1 times PBSThe pH of the mixturesof collagengenipin and collagenGA mixtures was 72 therewas a slight decrease from the pH of PBS (74) We thereforeemployed the simple gelation protocol on the assumptionthat such a slight decrease in pH hardly affected collagenfibril formation and chemical crosslinkingWe think that PBSis appropriate solvent for in vitro experiments of injectablecollagen because NaCl concentration has a strong effect oncollagen fibril formation The NaCl concentration in PBS(140mM) is equal to those of saline and conventional cell cul-ture media as possible solvents of injectable collagen A smallaliquot of the collagengenipin solution at collagen concen-trations of 015 03 or 05 was placed on the rheometer

As for the chitosangenipin solution 3 g of the acidic chi-tosan solution at a concentration of 1 was placed in a 15mLpolypropylene tube and kept at 4∘C Following mixing withgenipin solution or GA solution according to the proceduredescribed above 05 chitosan solutions containing genipinor GA were obtained

233 Test Conditions Dynamic viscoelastic tests were ini-tiated in 60 s after the collagen and genipin solutions weremixed using the following test conditions

(1) Fluidity TestOscillation at a frequency of 1Hz was appliedat a constant shear stress of 01 Pa and constant temperature of25∘C using the double-cone sensor The condition was in thelinear viscoelastic regime of the collagengenipin solutionsChanges in storagemodulus (G1015840) and lossmodulus (G10158401015840) wereregisteredThe gel point was determined as the point at which

International Journal of Biomaterials 3

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Figure 1 Results of a fluidity test for the 015 PSCgenipin solutions containing various concentrations of genipin (a) 0mM (b) 05mM(c) 1mM or (d) 2mM Temperatures were maintained at 25∘C

G1015840 andG10158401015840 intersected in their time-course change Fluidity ofsolutions was defined as G1015840 lt G10158401015840

(2) Temperature-Responsive Gelation Test Oscillation at afrequency of 1Hzwas applied at a constant shear deformationof 001 using the parallel-plate sensor The temperature waskept at 25∘C for intervals ranging from 10 to 40min increasedfrom 25 to 37∘C over 30 s and then kept at 37∘C for 60min

Changes in G1015840 and G10158401015840 were recorded The oscillation condi-tions were designed to produce a linear viscoelastic regime inthe collagengenipin solutions after the gel points

24 Penetration Test

241 Apparatus Penetration tests of the PSCgenipin gelswere conducted with a mechanical tester (TAXTplus Stable


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Micro Systems UK) A cylindrical stainless probe (5mm indiameter) was used

242 Sample Preparation PSC solutions of 015 and 05containing various concentrations of genipin were preparedaccording to the sample preparation for the dynamic vis-coelastic tests The solutions were degassed by centrifugationfor 30 s after vortexing and aliquots (4mL) were poured into

petri dishes (diameter 55mm) and placed on the surface of awater bath at a temperature of 37∘C After 30min of warmingthe dishes were sealed with a paraffin film to avoid drying ofthe gels moved to an incubator at a temperature of 37∘C andincubated for 72 h to complete gelation

243 Penetration of PSCGenipin Gels Stiffness of thematured PSCgenipin gels was determined by a penetration


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Figure 3 Results of a fluidity test for the 05 ASCgenipin solutions containing various concentrations of genipin (a) 0mM (b) 05mM(c) 1mM or (d) 2mM Temperatures were maintained at 25∘C

test using themechanical testerThe centers of the gels (119899 = 5)in the biological plates were probedwith the cylindrical probeat a cross-head speed of 02mms and stress-strain curveswere obtainedThe sensitivity of themechanical tester was setat 10 PaThe elastic modulus was calculated from the slope ofthe stress-strain curve in its linear region (strain from 0005to 004) in which consolidation of the gels by the biologicalplates did not affect the mechanical data

25 Scanning Electron Microscopy (SEM) Observation Themorphologies of collagen fibrils in the gels were observedby SEM Fixation and drying of the gels were performedfollowing our previous report using glutaraldehyde and t-butyl alcohol [31] In brief the gels prepared as described inSection 242 were fixed with 25 glutaraldehyde solutionand subsequently dehydrated by sequential immersion in20 50 75 90 95 and 99 ethanol The immersion in


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Figure 4 Storagemodulus (G1015840) of the 05 PSC1mM genipin solutionmeasured by a temperature-responsive gelation testThe temperaturewas maintained at 25∘C for (a) 600 (b) 1200 and (c) 1800 s and then increased to 37∘C

99 ethanol was repeated three times The ethanol in thedehydrated gel was thoroughly substituted with t-butanoland subjected to drying in vacuo The interiors of the driedgels were exposed by peeling with tweezers and coated withAu SEM measurements were performed with a MiniscopeTM3000 (Hitachi Ltd Tokyo Japan) at an accelerationvoltage of 5 kV

3 Results

31 Fluidity Tests of PSCGenipin Solutions at Room Temper-ature Figure 1 shows the results of the fluidity test for the015 PSCgenipin solutions containing various concentra-tions of genipin (0ndash2mM) In the absence of genipinG10158401015840 wasgreater than G1015840 over the entire range of timepoints investi-gated (Figure 1(a)) exhibiting a viscous nature Turbidity wasnot observed providing evidence that fibril formation of PSCdid not occur in 60min at 25∘C G1015840 and G10158401015840 showed time-dependent increases in the presence of genipin where the gel

point defined as the time at which G1015840 and G10158401015840 were equalbecame shorter with the increasing concentration of genipin(Figures 1(a)ndash1(d)) The reduced gel points indicated thatgenipin gradually crosslinked collagen monomers The gelpoint was determined to be 2190 plusmn 180 s (mean plusmn SD 119899 = 3)at a genipin concentration of 1mMThe gel points of the 05PSCgenipin solutions were longer than those of the 015PSCgenipin solutions at the same genipin concentrations(Figure 2) The gel point at a genipin concentration of 2mMwas determined to be 2490 plusmn 270 s (mean plusmn SD 119899 = 3) At agenipin concentration of 1mM the solution did not reach agel point within 60min in triplicate experiments

32 Fluidity Tests of ASCGenipin Solutions at Room Tem-perature Fluidity tests were also conducted for 015ASCgenipin solutions (Figure 3) The solution became tur-bid immediately after the ASC solution was mixed with thegenipin solution The immediate increase in G10158401015840 resulted inthe gel points within 60 s (Figures 3(b) and 3(c)) Even when


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Figure 5 Storagemodulus (G1015840) of the 05 PSC1mM genipin solutionmeasured by a temperature-responsive gelation testThe temperaturewas maintained at 25∘C for 1800 s and then increased to (a) 37 (b) 33 or (c) 30∘C

the ASC solution was mixed with PBS containing no genipinthe solution reached a gel point within 60 s (Figure 3(a))These results indicated that the immediate increases in G1015840 of015 ASCgenipin solutions were predominantly because ofactive fibril formation of ASC at neutral pH

33 Temperature-Responsive Gelation of PSCGenipin Solu-tions Temperature-responsive gelation tests were conductedfor the 05 PSC1mM genipin solutions The intervals at25∘C were set at 600 1200 and 1800 s during which thesolutions remained viscous (Figure 2(c)) Sharp increases inG1015840 were observed approximately 100 s after the solutionsreached the target temperature (37∘C) (Figure 4) indicatingthat gelation was triggered by the increase in temperatureto physiological temperature The rates of modulus increasewere not affected by the length of intervals at 25∘C Lowertarget temperatures (33 and 30∘C) also were investigatedFigure 5 shows the results of temperature-response testsemploying target temperatures of 30 33 and 37∘C and an

interval of 1800 s The rate of increase in G1015840 decreased withthe target temperature supporting the hypothesis that this gelsystem is temperature-responsive The turbidity of the gelscorresponding to the actual fibril formation rate tended todecrease with increase in the concentration of genipin

34 SEM Observation Figure 6 shows SEM images of thecross-sections of dried PSCgenipin gels A drying pro-cess using t-butyl alcohol sublimation enabled observationof collagen fibrils in the gels with minimal destructionPSCgenipin gels at genipin concentrations of 05 1 and2mM (Figures 6(b)ndash6(d)) showed networks of collagennanofibrils resembling those obtained from PSC gel contain-ing no genipin (Figure 6(a)) We must note that the apparentfibril density in the SEM images became similar by the alcoholdehydration and sublimation process

35 Effects of Collagen and Genipin Concentrations onTemperature-Responsive Gelation The effect of the genipin


N D58 times10k 10120583m

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Figure 6 SEM images of cross-sections of dried PSCgenipin gels at genipin concentrations of (a) 0mM (b) 05mM (c) 1mM and (d)2mM

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Figure 7 Storage modulus (G1015840) of the 05 PSCgenipin solution measured by a temperature-responsive gelation test The temperature wasmaintained at 25∘C for 600 s and then increased to 37∘C Numbers in figures indicate genipin concentrations


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Figure 8 Storage modulus (G1015840) of PSC1mM genipin solutionsmeasured by a temperature-responsive gelation test PSC concentra-tions were (a) 015 and (b) 05 The temperature was maintainedat 25∘C for 600 s and then increased to 37∘C Numbers in figuresindicate PSC concentrations

concentration on gelation was investigated by temperature-responsive gelation tests for 05 and 015 PSC solu-tions No significant increase in G1015840 was observed in theintervals at 25∘C for all the solutions (Figure 7) The gelpoints were almost independent of genipin concentrationsall the 015 PSC solutions containing 0ndash2mM of genipinpassed through gel points in the increasing temperature from25∘C to 37∘C (Figure 7(a)) As for the 05 PSC solutionscontaining genipin gel points were achieved in 160ndash180 safter the temperatures reached 37∘C (Figure 7(b))The rate ofincrease inG1015840 was proportional to the increase in the genipinconcentration (Figure 7) The genipin concentration depen-dence was similar in 015 and 05 PSC solutions althoughG1015840 values of 05 PSC gels were much higher than thoseof 015 gels The effect of the collagen concentration ongelation was also investigated The rate of increase in G1015840accelerated as the PSC concentration increased from 015 to05 (Figure 8)

36 Temperature Responsiveness of Genipin Crosslinking andCollagen Fibril Formation To evaluate the temperaturedependence of genipin crosslinking without the influenceof collagen fibril formation a 05 chitosan5mM genipinsolution was subjected to the temperature-responsive gela-tion test Chitosan is a suitable substrate for investigating thetemperature responsiveness of genipin crosslinking becauseit has free amino residues and the solutions containing nogenipin are stable at temperatures ranging from 25 to 37∘C(data not shown) Figure 9(a) shows the change in G1015840 ofa 05 chitosan5mM genipin solution with increase intemperature from 25 to 37∘C G1015840 of the chitosan solutionsbegan to increase approximately 60 s after the temperaturereached 37∘C Only a slight increase in G1015840 was observed

when the temperature was kept at 25∘C (Figure 9(b)) Theseresults indicated that genipin crosslinking was activated withincreasing temperature from 25 to 37∘C

The temperature dependence of collagen fibril formationwithout the influence of genipin crosslinking was also evalu-ated Figure 10 shows the change inG1015840 of a 05 PSC solutioncontaining no genipin with increase in temperature from 25to 30 33 and 37∘CThe initial rise in G1015840 decelerated with thetarget temperature At the target temperature of 30∘C only aslight increase in G1015840 (6 Pa) was observed in 60min

37 Comparison of Temperature Responsibility of GA andGenipin Crosslinking The05PSC6mMGAwas subjectedto the fluidity test and temperature-responsive gelation testto compare temperature responsiveness between genipin andGA crosslinking Figure 9(a) shows the change inG1015840 of a 05chitosan6mM GA solution compared with that of a 05chitosan5mMgenipin solutionAlthoughGAconcentrationwas higher than genipin concentration the rate of increasein G1015840 of the chitosanGA solution was lower than that ofthe chitosangenipin solution The fluidity test determinedthe gel points to be 780 plusmn 70 s (mean plusmn SD 119899 = 3) for the05 PSC6mM GA solution and 1000 plusmn 120 s (mean plusmn SD119899 = 3) for the 05 PSC5mM genipin solution indicatingthat GA crosslinking was faster than genipin crosslinking atroom temperature

Representative gelation curves of both the solutionsobtained by the temperature-responsive gelation test areshown in Figure 8 The 05 PSC5mM genipin solutionshowed a higher rate of increase in G1015840 than the 05PSC6mM GA solution whereas the fluidity test of chitosanshowed that crosslinking of 5mM genipin at 25∘Cwas slowerthan that of 6mM GA The difference in the temperature-responsive gelation tests was confirmed by triplicate exper-iments These results indicated that the temperature respon-siveness of genipin crosslinking was higher than that of GAcrosslinking

38 Penetration Tests for Matured PSCGenipin GelsFigure 11 shows the elastic modulus of matured PSCgenipingels obtained by the penetration test as a function of genipinand PSC concentrations Plots of the elastic modulus againstthe genipin concentration are almost linear for both 05and 015 PSCgenipin gels (Figure 11(a)) Only genipinconcentrations higher than 05mM markedly increasedthe stiffness of PSC gels when compared with the stiffnessof gels containing no genipin The elastic moduli of 05and 015 PSCgenipin gels reached 502 plusmn 82 kPa and179 plusmn 20 kPa respectively at a genipin concentration of2mMThe almost linear correlation between elastic modulusand PSC concentration at a constant genipin concentrationof 2mM yielded the following formula for the gels

119884 = 926 sdot 119883 + 36 (1)

where119884 is the elasticmodulus (kPa) and119883 is the PSC concen-tration () Based on the linear correlation a 01 increasein the PSC concentration corresponds to an approximately9 kPa increase in the elastic modulus


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Figure 9 (a) Storage modulus (G1015840) of chitosan solutions measured by a temperature-responsive gelation test Solid line 05 chitosan5mMgenipin solution dotted line 05 chitosan6mM GA solution (b) Storage modulus (G1015840) of the 05 chitosan5mM genipin solutionmeasured by a fluidity test at a constant temperature of 25∘C

39 Time-Dependent Change of Activity of Genipin Crosslink-ing To test time-dependent change of activity of genipincrosslinking temperature-responsive gelation tests were con-ducted for the 025 PSC10mM genipin solutions by usinggenipin solutions prepared 0ndash30 d before the tests Figure 12shows the curves obtained at 37∘C after the intervals of600 s at 25∘C The shapes of gelatin curves were similar forsamples using genipin solution 0 1 2 and 3 d elapsed after thepreparation When the elapsed time increased from 3 to 7 dthe sigmoidal curves changed to J-like curves The slope ofthe curves increased as the elapsed time increased and almoststabilized in the elapsed time of 11 d

4 Discussion

We demonstrated that temperature-responsive gelationoccurred in PSCgenipin solutions at genipin concentrations0ndash2mM under a moderate change in temperature from25 to 37∘C while the solutions exhibited fluidity at roomtemperature for at least 30min In specific cases this stabilityat room temperature could be an important property forinjectable collagen while fluidity of PSCgenipin solutionscan be maintained at lower temperatures In practicalapplications there is a possibility that temperatures ofinjectable collagen increase to room temperature duringoperations depending on environments (especially on roomtemperatures) operation procedures equipment and skillsof operators The fluidity at 25∘C therefore warrants easyhandling of injectable collagen without unexpected gelationduring various operations such as degassing the solutions orincorporating drugs and cells uniformly The rapid gelationbehavior under physiological conditions is beneficial not onlyfor drug and cell entrapment to yield uniform distributions

within the gelledmatrix but also for creating required shapesand sizes in the body A desirable gelation time appears tobe within several minutes for minimizing diffusion of suchcontents collagen and genipin The PSCgenipin gel systemappears to satisfy the requirements for the physical propertiesof an injectable gel system

The temperature-responsive gelation of the PSCgenipinsolutions was because of the temperature responsiveness ofgenipin crosslinking and collagen fibril formation Inves-tigations using the chitosangenipin gel system revealedthat genipin crosslinking itself was rapidly activated withincrease in temperature from 25 to 37∘C Furthermore PSCitself showed well-known gelation by fibril formation inresponse to increase in temperature from 25 to 37∘C ThePSCgenipin gels consisted of collagen fibrils From thesefindings it was evident that not only collagen fibril formationbut also genipin crosslinking contributed to the temperatureresponsiveness of gelation of the PSCgenipin solutions undersuch moderate changes in temperature

The continuous increase in G1015840 of the PSCgenipin gelsafter temperature reached 37∘Cappeared to be predominantlydue to genipin crosslinking according to the findings thatcollagen fibril formation in the absence of genipin wasalmost stabilized in 15min and that genipin crosslinking tochitosan occurred continuously for at least 60min Genipincrosslinking on collagen has been reported to continue for upto 3 days depending on the genipin concentration [25]

Special attention should be given for ASCgenipin systembecause of the rapid loss of fluidity at room temperature theuse of a refrigerator or an ice bath appropriately to maintainlow temperature The loss of fluidity and gelation was pre-dominantly due to collagen fibril formation in which genipincrosslinking would not substantially affect the early stage of


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pera

ture

(∘C)

G998400

(Pa)

(c)

Figure 10 Storage modulus (G1015840) of the 05 PSC solution containing no genipin with increase in temperature The temperature wasmaintained at 25∘C for 600 s and then increased to (a) 37 (b) 33 or (c) 30∘C

the gelation according to our data for genipin crosslinkingto chitosan at 25∘C Rapid fibril formation of ASC comparedwith that of PSC is thought to be because of the existenceof nucleation centers derived from the oligomeric structure[32] and the presence of telopeptide regions important forstabilizing the collagen structure [28] In Macayarsquos pioneerstudy of a collagengenipin gel system [25] ASC from rattail tendon was used and rapid increase in G1015840 was observedapproximately 50 s after the collagengenipin solution wassubjected to rheological tests Those results were reproducedby our experiments using ASC from porcine tendon We rec-ommend that PSC be used for a collagengenipin gel systemto preserve fluidity at room temperature offering sufficienttime to degas the solutions and uniformly incorporate cellsand drugs into the gels

The temperature responsiveness of genipin crosslinkingwas greater than that of conventional GA crosslinking BothGA and genipin crosslink free amino residues of polymersubstrates where the crosslinkers are oligomeric and poly-meric forms [23 24] It appears that the genipin is a preferable

crosslinker for temperature-responsive gelation of collagenwith respect to temperature responsiveness of crosslinking aswell as biological safety

The elastic modulus of the 05 PSCgenipin gel systemcan be adjusted over a range from 25 to 50 kPa by thePSC and genipin concentrations and increases in the PSCconcentration are expected to further increase the elasticmodulus The PSCgenipin gel system allows control of theelastic modulus of injectable collagen gels to match thoseof living tissues brain tissue (approximately 03 kPa) [33]muscle (approximately 10 kPa) [34] and the collagen-basedprecursor to bone (approximately 30 kPa) [35] Furthermorethe mechanical properties of the collagen gel contribute tomaintain the shape of the gel in living tissues Howeverdirect cell exposure to genipin has been reported to poseserious cytotoxicity risks [24ndash26] According to these reportsusing various cell types the genipin concentration limitin injectable gels appears to be below 05ndash10mM exceptfor neural stem cells [25] Based on the above findings ofcytotoxicity of genipin and the fact that wt of collagen had


0

10

20

30

40

50

60

0 05 1 15 2 25

PSC 05

PSC 015

Genipin concentration (mM)

Elas

tic m

odul

us (k

Pa)

(a)

0

10

20

30

40

50

60

0 01 0302 04 05 06PSC concentration (mM)

Elas

tic m

odul

us (k

Pa)

y = 926x + 36

R2 = 099

(b)

Figure 11 Elastic modulus of matured PSCgenipin gels obtained by a penetration test (a) 05 and 015 wt PSC gels containing genipin atconcentrations ranging from 0 to 4mM (b) 05 03 and 015 wt PSC gels containing 2mM genipin The gels were matured by incubationfor 72 h Data are presented as mean plusmn SD 119899 = 3

a substantial effect on the modulus (Figure 8) the elasticmodulus should be controlled by the collagen concentrationrather than the genipin concentration

The elastic modulus determined by penetration tests maybe different from that by dynamic viscoelastic tests becauseof differences in deformation modes and ranges Penetrationtests confer compressive deformation in the gels whileshear deformation is generated by dynamic viscoelastic testsHowever the shearmodulus of thematured PSCgenipin gelscould not be preciselymeasured by dynamic viscoelastic testsWe learned that it is difficult to determine shear modulusprecisely when the matured gels prepared in containers aremoved to a rheometer and subsequently measured Thedifficulty is probably due to the leakage of water from the gelsby continuous stress generated by a plate sensor

The limitation of PSCgenipin gel system appears tobe a spontaneous crosslinking of collagen in PSCgenipinsolutions that occurs even in a refrigerator making it difficultto stock and distribute the mixed solutions In practical usesPSC and genipin solutions should be stocked separately andmixed just before operations We must note that activityof genipin crosslinking increases over a period of a weekafter genipin is dissolved in PBS That is the reason thatwe used genipin solutions stocked in a refrigerator for over2 weeks A possible explanation for the activity change isself-polymerization of genipin in aqueous solution describedby Sung et al [21] Such structural changes may alter theactivity of intermolecular and intramolecular crosslinking orinterference with collagen fibril formation If dried genipin

powder is dissolved in PBS just before operation a time-course change in activity of genipin crosslinking should beinvestigated

This study lacks biocompatibility data of PSCgenipingels which is also the limitation of this study The bio-compatibility of PSC has been well established Howevercytotoxicity of genipin seems to be a complex event because itis probably affected by mobility as well as activity of genipinOur study demonstrated that activity of genipin is affectedby temperature Gelation of PSCgenipin solution woulddecrease the mobility of gelation decreasing cytotoxic effectsof genipin The in vitro and in vivo biocompatibility dataof PSCgenipin system are in progress to demonstrate thecytotoxicity of this injectable gel system

5 Conclusions

We demonstrated that PSCgenipin solutions are a potentialinjectable gel system (1) fluidity was maintained at 25∘C forat least 30min (2) a subsequent increase in temperature from25 to 37∘C caused rapid gelation and (3) an elastic modulusmarkedly higher than that of conventional collagen fibrillargels can be obtained at a genipin concentration of 05mMat which cells other than neural stem cells can survivedirect contact with genipin In specific cases PSC would bepreferred over ASC as an injectable gel system Temperature-responsive gelation of PSCgenipin solutions involves tem-perature responsiveness of both genipin crosslinking andcollagen fibril formation In vivo studies are now in progress


0

50

100

150

200

250

0 300 600 900 1200 1500Time (s)

0d

30d

11d

7d

3d

2d

1d

G998400

(Pa)

Figure 12 Storage modulus (G1015840) of the 025 PSC10mM genipinsolution measured by a temperature-responsive gelation test Thenumbers in the figure indicate the elapsed days after the preparationof genipin solution The curves obtained at 37∘C after the intervalsof 600 s at 25∘C were depicted

to test mechanical properties and cytotoxicity of PSCgenipingel system

Acknowledgments

This research was supported in part by a grant programldquoAdaptable and Seamless Technology Transfer Programthrough target-driven RampD (A-STEP) Exploratory ResearchJSTrdquo The authors would like to thank Enago (httpwwwenagojp) for the English language review

References

[1] H Tan and K G Marra ldquoInjectable biodegradable hydrogelsfor tissue engineering applicationsrdquo Materials vol 3 no 3 pp1746ndash1767 2010

[2] D Schmaljohann ldquoThermo- and pH-responsive polymers indrug deliveryrdquo Advanced Drug Delivery Reviews vol 58 no 15pp 1655ndash1670 2006

[3] H Tan C M Ramirez N Miljkovic H Li J P Rubin and KGMarra ldquoThermosensitive injectable hyaluronic acid hydrogelfor adipose tissue engineeringrdquo Biomaterials vol 30 no 36 pp6844ndash6853 2009

[4] Y-L Chiu S-C Chen C-J Su et al ldquopH-triggered injectablehydrogels prepared fromaqueousN-palmitoyl chitosan in vitrocharacteristics and in vivo biocompatibilityrdquo Biomaterials vol30 no 28 pp 4877ndash4888 2009

[5] J Yan L Yang GWang Y Xiao B Zhang andNQi ldquoBiocom-patibility evaluation of chitosan-based injectable hydrogels forthe culturing mice mesenchymal stem cells in vitrordquo Journal ofBiomaterials Applications vol 24 no 7 pp 625ndash637 2010

[6] B R Williams R A Gelman D C Poppke and K PiezldquoCollagen fibril formation Optimal in vitro conditions and

preliminary kinetic resultsrdquo Journal of Biological Chemistry vol253 no 18 pp 6578ndash6585 1978

[7] D L Helseth Jr and A Veis ldquoCollagen self-assembly in vitroDifferentiating specific telopeptide-dependent interactionsusing selective enzyme modification and the addition of freeamino telopeptiderdquo Journal of Biological Chemistry vol 256no 14 pp 7118ndash7128 1981

[8] Z Ruszczak and R A Schwartz ldquoCollagen uses in dermatologyan updaterdquo Dermatology vol 199 no 4 pp 285ndash289 1999

[9] G Charriere M Bejot L Schnitzler G Ville and D J Hart-mann ldquoReactions to a bovine collagen implant Clinical andimmunologic study in 705 patientsrdquo Journal of the AmericanAcademy of Dermatology vol 21 no 6 pp 1203ndash1208 1989

[10] C H Lee A Singla and Y Lee ldquoBiomedical applications ofcollagenrdquo International Journal of Pharmaceutics vol 221 no1-2 pp 1ndash22 2001

[11] A W Klein ldquoTechniques for soft tissue augmentation an ldquoA toZrdquordquo American Journal of Clinical Dermatology vol 7 no 2 pp107ndash120 2006

[12] M Kimura T Nito K-I Sakakibara N Tayama and S NiimildquoClinical experience with collagen injection of the vocal folda study of 155 patientsrdquo Auris Nasus Larynx vol 35 no 1 pp67ndash75 2008

[13] H Bentz J A Schroeder and T D Estridge ldquoImproved localdelivery of TGF-beta2 by binding to injectable fibrillar collagenvia difunctional polyethylene glycolrdquo Journal of BiomedicalMaterials Research vol 39 no 4 pp 539ndash548 1998

[14] S Griffey N D Schwade and C GWright ldquoParticulate dermalmatrix as an injectable soft tissue replacementmaterialrdquo Journalof Biomedical Materials Research vol 58 no 1 pp 10ndash15 2001

[15] Y Jinno Y Ayukawa Y Ogino I Atsuta Y Tsukiyama and KKoyano ldquoVertical bone augmentation with fluvastatin in aninjectable delivery system a rat studyrdquo Clinical Oral ImplantsResearch vol 20 no 8 pp 756ndash760 2009

[16] R KWillits and S L Skornia ldquoEffect of collagen gel stiffness onneurite extensionrdquo Journal of Biomaterials Science vol 15 no 12pp 1521ndash1531 2004

[17] E A Sander and V H Barocas ldquoBiomimetic collagen tissuescollagenous tissue engineering and other applicationsrdquo in Col-lagen Structure andMechanics P Fratzl Ed pp 475ndash504 2008

[18] R-N ChenH-OHo andM-T Sheu ldquoCharacterization of col-lagen matrices crosslinked using microbial transglutaminaserdquoBiomaterials vol 26 no 20 pp 4229ndash4235 2005

[19] W M Elbjeirami E O Yonter B C Starcher and J L WestldquoEnhancing mechanical properties of tissue-engineered con-structs via lysyl oxidase crosslinking activityrdquo Journal ofBiomedical Materials Research A vol 66 no 3 pp 513ndash5212003

[20] H -W Sung R -NHuang L L HHuang C-C Tsai andC -TChiu ldquoFeasibility study of a natural crosslinking reagent for bio-logical tissue fixationrdquo Journal of BiomedicalMaterials Researchvol 42 no 4 pp 560ndash567 1998

[21] H -W Sung Y Chang I -L Liang W -H Chang and Y -CChen ldquoFixation of biological tissues with a naturally occurringcrosslinking agent fixation rate and effects of pH temperatureand initial fixative concentrationrdquo Journal of Biomedical Mate-rials Research vol 52 no 1 pp 77ndash87 2000

[22] Y Chang C-C Tsai H-C Liang and H-W Sung ldquoIn vivoevaluation of cellular and acellular bovine pericardia fixed witha naturally occurring crosslinking agent (genipin)rdquo Biomateri-als vol 23 no 12 pp 2447ndash2457 2002


[23] H-W SungW-HChang C-YMa andM-H Lee ldquoCrosslink-ing of biological tissues using genipin andor carbodiimiderdquoJournal of Biomedical Materials Research A vol 64 no 3 pp427ndash438 2003

[24] H G Sundararaghavan G A Monteiro N A Lapin Y JChabal J R Miksan and D I Shreiber ldquoGenipin-inducedchanges in collagen gels correlation of mechanical propertiesto fluorescencerdquo Journal of BiomedicalMaterials ResearchA vol87 no 2 pp 308ndash320 2008

[25] D Macaya K K Ng and M Spector ldquoInjectable collagen-genipin gel for the treatment of spinal cord injury in vitrostudiesrdquo Advanced Functional Materials vol 21 no 24 pp4788ndash4797 2011

[26] C Wang T T Lau W L Loh K Su and D-A Wang ldquoCyto-compatibility study of a natural biomaterial crosslinker-genipinwith therapeutic model cellsrdquo Journal of Biomedical MaterialsResearch B vol 97 no 1 pp 58ndash65 2011

[27] D W Bannister and A B Burns ldquoPepsin treatment of avianskin collagen Effects on solubility subunit composition andaggregation propertiesrdquo Biochemical Journal vol 129 no 3 pp677ndash681 1972

[28] K Sato T Ebihara E Adachi S Kawashima S Hattoriand S Irie ldquoPossible involvement of aminotelopeptide in self-assembly and thermal stability of collagen I as revealed by itsremoval with proteasesrdquo Journal of Biological Chemistry vol275 no 33 pp 25870ndash25875 2000

[29] A K Lynn I V Yannas and W Bonfield ldquoAntigenicity andimmunogenicity of collagenrdquo Journal of Biomedical MaterialsResearch B vol 71 no 2 pp 343ndash354 2004

[30] K H Stenzel T Miyata and A L Rubin ldquoCollagen as abiomaterialrdquo Annual Review of Biophysics and Bioengineeringvol 3 pp 231ndash253 1974

[31] S Yunoki and T Matsuda ldquoSimultaneous processing of fibrilFormation and cross-linking improvesmechanical properties ofcollagenrdquo Biomacromolecules vol 9 no 3 pp 879ndash885 2008

[32] K Purna Sai and M Babu ldquoStudies on Rana tigerina skincollagenrdquo Comparative Biochemistry and Physiology B vol 128no 1 pp 81ndash90 2001

[33] B S Elkin E U Azeloglu K D Costa and B Morrison IIIldquoMechanical heterogeneity of the rat hippocampusmeasured byatomic force microscope indentationrdquo Journal of Neurotraumavol 24 no 5 pp 812ndash822 2007

[34] A J Engler M A Griffin S Sen C G Bonnemann H LSweeney and D E Discher ldquoMyotubes differentiate optimallyon substrates with tissue-like stiffness pathological implica-tions for softor stiffmicroenvironmentsrdquo Journal of Cell Biologyvol 166 no 6 pp 877ndash887 2004

[35] A J Engler S Sen H L Sweeney and D E Discher ldquoMatrixelasticity directs stem cell lineage specificationrdquo Cell vol 126no 4 pp 677ndash689 2006

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


extracted from gardenia fruit and its cytotoxicity has beenreported to be much lower than that of most commonly usedsynthetic crosslinkers

Mechanical characterization of collagen fibrillar gelscrosslinked by genipin has been firstly reported by Sundara-raghavan et al [24] Macaya and coworkers [25] have recentlyreported an injectable collagen gel system using genipin as acrosslinker and acid-solubilized collagen (ASC) as a sub-strateThemechanical properties of the gels (of storage mod-ulus approximately 250 Pa) were much better than those ofcollagen fibrillar gels without chemical crosslinking [16]

However the fluidity of the collagengenipin solutionat room temperature was unknown We expected that theslow fibril formation of pepsin-solubilized collagen (PSC)compared with ASC [27 28] would contribute to maintainfluidity at room temperature PSC is prepared from ASC orcollagenous tissues by pepsin digestion and lacks nonhelicalterminal regions (telopeptides) while triple-helical confor-mation is intact [29] Conventional biomaterial processinghas employed PSC rather than ASC because of its processing-related [29] and immunological benefits [30] The tempera-ture responsiveness of genipin crosslinking is as important forgelatin properties as that of collagen fibril formation A pre-vious study [21] showed that genipin crosslinking of collagenwas similar in 72 h reaction at temperatures of 25ndash45∘Chowever the temperature responsiveness on second-minutetimescale is still unknown Sol to gel transition of injectablecollagen (ie fibril formation) occurs on such timescale

In the present study we investigated the fluidity of acollagengenipin solution at room temperature and its gela-tion response to physiological temperatures using pepsin-solubilized collagen (PSC) as a substrate We hypothesizedthat an injectable collagen gel system using PSC confers suf-ficient and prolonged fluidity at room temperature We alsoinvestigated the temperature response of genipin crosslink-ingThe potential of PSCgenipin solution as an injectable gelsystem is discussed

2 Materials and Methods

21 Materials Materials comprise PSC from porcine skinin dilute HCl (1 solution Nipponham Japan) ASC fromporcine tendon in dilute HCl (03 solution Nitta GelatinJapan) chitosan powder (Chitosan 500Wako Pure ChemicalIndustries Japan) genipin (Wako Pure Chemical IndustriesJapan) glutaraldehyde (25 solution Wako Pure Chemi-cal Industries Japan) and phosphate-buffered saline (PBS)tablets (Sigma-Aldrich MO USA)

22 Preparation of Solutions The PSC (1) and ASC (03)and solutions were used with no pretreatment Accordingto the manufacturesrsquo data by electrophoresis PSC and ASCcontained 11 wt and a trace amount of type III collagenrespectively PSC solutions of 03 and 06 were preparedby dilution of the 1 PSC solution with pH 3 dilute HCl A1 chitosan solution was prepared by dissolution of chitosanpowder in 100mM acetic acid solution Genipin solutions atconcentrations ranging from 1 to 10mM were prepared by

dissolution of genipin powder in 2times PBS and held for 2 weeksin the refrigerator prior to use for the following examinationsGA solutions at a concentration of 12mM were prepared bydilution of 25 GA solution with 2 times PBS

23 Dynamic Viscoelastic Tests

231 Apparatus Changes in fluidity and gelation processeswere monitored by dynamic viscoelastic tests with a rheome-ter (HAAKE MARS III Thermo Fisher Scientific Inc MAUSA)The temperature was sensitively controlled by a Peltiercontrolled plate Two types of sensors were used a double-cone sensor DC601Ti (diameter of 60mm cone angle of 1∘)and a parallel-plate sensor HPP35S (diameter of 35mm withsurface serrated to avoid slippage of gels during oscillation)The double-cone sensor has a shear surface twice as highas that of a conventional cone plate stabilizing viscoelasticdata of low viscous samples by enhanced sensitivity A lidwas used to minimize drying of the solutions during mea-surement at room temperature enabling precise viscoelasticmeasurements for more than 30min

232 Sample Preparations PSCgenipin and ASCgenipinsolutions were prepared as follows PSC and ASC solutions(3 g) at concentrations of 03 06 or 1 were placed in 15mLpolypropylene tubes and maintained at 4∘C Three millilitersof precooled genipin solution or GA solution (concentrationsof 0ndash10mM) at 4∘C was added to the collagen solution andvortexed immediately to prepare neutral collagen solutioncontaining genipin or GA in 1 times PBSThe pH of the mixturesof collagengenipin and collagenGA mixtures was 72 therewas a slight decrease from the pH of PBS (74) We thereforeemployed the simple gelation protocol on the assumptionthat such a slight decrease in pH hardly affected collagenfibril formation and chemical crosslinkingWe think that PBSis appropriate solvent for in vitro experiments of injectablecollagen because NaCl concentration has a strong effect oncollagen fibril formation The NaCl concentration in PBS(140mM) is equal to those of saline and conventional cell cul-ture media as possible solvents of injectable collagen A smallaliquot of the collagengenipin solution at collagen concen-trations of 015 03 or 05 was placed on the rheometer

As for the chitosangenipin solution 3 g of the acidic chi-tosan solution at a concentration of 1 was placed in a 15mLpolypropylene tube and kept at 4∘C Following mixing withgenipin solution or GA solution according to the proceduredescribed above 05 chitosan solutions containing genipinor GA were obtained

233 Test Conditions Dynamic viscoelastic tests were ini-tiated in 60 s after the collagen and genipin solutions weremixed using the following test conditions

(1) Fluidity TestOscillation at a frequency of 1Hz was appliedat a constant shear stress of 01 Pa and constant temperature of25∘C using the double-cone sensor The condition was in thelinear viscoelastic regime of the collagengenipin solutionsChanges in storagemodulus (G1015840) and lossmodulus (G10158401015840) wereregisteredThe gel point was determined as the point at which


0001

001

01

1

10

0 1200 2400 3600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(a)

0001

001

01

1

10

0 1200 2400 3600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(b)

0001

001

01

1

10

0 1200 2400 3600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(c)

0001

001

01

1

10

0 1200 2400 3600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(d)





24 Penetration Test



0001

001

01

1

10

0 1200 2400 3600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(a)

0001

001

01

1

10

0 1200 2400 3600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(b)

0001

001

01

1

10

0 1200 2400 3600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(c)

0001

001

01

1

10

0 1200 2400 3600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(d)







0001

001

01

1

10

0 120 240 360 480 600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(a)

0001

001

01

1

10

0 240120 360 480 600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(b)

0001

001

01

1

10

0 240 360120 480 600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(c)





20

25

30

35

40

Tem

pera

ture

(∘C)

2500

2000

1500

1000

500

00 1200 2400 3600

Time (s)

G998400

(Pa)

(a)

20

25

30

35

40

Tem

pera

ture

(∘C)

2500

2000

1500

1000

500

00 1200 2400 3600

Time (s)

G998400

(Pa)

(b)

20

25

30

35

40

Tem

pera

ture

(∘C)

2500

2000

1500

1000

500

00 1200 2400 3600

Time (s)

G998400

(Pa)

(c)



3 Results





20

25

30

35

40

0

2500

2000

1500

1000

500

0 1200 2400 48003600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

2500

2000

1500

1000

500

0 1200 2400 48003600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)

20

25

30

35

40

0

2500

2000

1500

1000

500

0 1200 2400 48003600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(c)








N D58 times10k 10120583m

(a)

(a)

(b)

N D65 times10k 10120583m

(b)

(c)

N D61 times10k 10120583m

(c)

(d)

N D67 times10k 10120583m

(d)


20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

2mM 1mM05mM 0mM

G998400

(Pa)

(a)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

2mM 1mM05mM 0mM

G998400

(Pa)

(b)



05

030015

2000

1500

1000

500

0

40

35

30

25

200 1200 2400 3600

Time (s)

G998400

(Pa)

Tem

pera

ture

(∘C)









119884 = 926 sdot 119883 + 36 (1)



20

25

30

35

40

0

400

200

600

800

0 1200600 1800 2400 3000Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

400

200

600

800

0 1200600 1800 2400 3000Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)



4 Discussion







20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(c)







0

10

20

30

40

50

60

0 05 1 15 2 25

PSC 05

PSC 015


Elas

tic m

odul

us (k

Pa)

(a)

0

10

20

30

40

50

60


Elas

tic m

odul

us (k

Pa)

y = 926x + 36

R2 = 099

(b)







5 Conclusions



0

50

100

150

200

250

0 300 600 900 1200 1500Time (s)

0d

30d

11d

7d

3d

2d

1d

G998400

(Pa)



Acknowledgments


References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0001

001

01

1

10

0 1200 2400 3600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(a)

0001

001

01

1

10

0 1200 2400 3600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(b)

0001

001

01

1

10

0 1200 2400 3600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(c)

0001

001

01

1

10

0 1200 2400 3600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(d)





24 Penetration Test



0001

001

01

1

10

0 1200 2400 3600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(a)

0001

001

01

1

10

0 1200 2400 3600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(b)

0001

001

01

1

10

0 1200 2400 3600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(c)

0001

001

01

1

10

0 1200 2400 3600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(d)







0001

001

01

1

10

0 120 240 360 480 600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(a)

0001

001

01

1

10

0 240120 360 480 600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(b)

0001

001

01

1

10

0 240 360120 480 600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(c)





20

25

30

35

40

Tem

pera

ture

(∘C)

2500

2000

1500

1000

500

00 1200 2400 3600

Time (s)

G998400

(Pa)

(a)

20

25

30

35

40

Tem

pera

ture

(∘C)

2500

2000

1500

1000

500

00 1200 2400 3600

Time (s)

G998400

(Pa)

(b)

20

25

30

35

40

Tem

pera

ture

(∘C)

2500

2000

1500

1000

500

00 1200 2400 3600

Time (s)

G998400

(Pa)

(c)



3 Results





20

25

30

35

40

0

2500

2000

1500

1000

500

0 1200 2400 48003600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

2500

2000

1500

1000

500

0 1200 2400 48003600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)

20

25

30

35

40

0

2500

2000

1500

1000

500

0 1200 2400 48003600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(c)








N D58 times10k 10120583m

(a)

(a)

(b)

N D65 times10k 10120583m

(b)

(c)

N D61 times10k 10120583m

(c)

(d)

N D67 times10k 10120583m

(d)


20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

2mM 1mM05mM 0mM

G998400

(Pa)

(a)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

2mM 1mM05mM 0mM

G998400

(Pa)

(b)



05

030015

2000

1500

1000

500

0

40

35

30

25

200 1200 2400 3600

Time (s)

G998400

(Pa)

Tem

pera

ture

(∘C)









119884 = 926 sdot 119883 + 36 (1)



20

25

30

35

40

0

400

200

600

800

0 1200600 1800 2400 3000Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

400

200

600

800

0 1200600 1800 2400 3000Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)



4 Discussion







20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(c)







0

10

20

30

40

50

60

0 05 1 15 2 25

PSC 05

PSC 015


Elas

tic m

odul

us (k

Pa)

(a)

0

10

20

30

40

50

60


Elas

tic m

odul

us (k

Pa)

y = 926x + 36

R2 = 099

(b)







5 Conclusions



0

50

100

150

200

250

0 300 600 900 1200 1500Time (s)

0d

30d

11d

7d

3d

2d

1d

G998400

(Pa)



Acknowledgments


References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0001

001

01

1

10

0 1200 2400 3600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(a)

0001

001

01

1

10

0 1200 2400 3600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(b)

0001

001

01

1

10

0 1200 2400 3600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(c)

0001

001

01

1

10

0 1200 2400 3600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(d)







0001

001

01

1

10

0 120 240 360 480 600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(a)

0001

001

01

1

10

0 240120 360 480 600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(b)

0001

001

01

1

10

0 240 360120 480 600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(c)





20

25

30

35

40

Tem

pera

ture

(∘C)

2500

2000

1500

1000

500

00 1200 2400 3600

Time (s)

G998400

(Pa)

(a)

20

25

30

35

40

Tem

pera

ture

(∘C)

2500

2000

1500

1000

500

00 1200 2400 3600

Time (s)

G998400

(Pa)

(b)

20

25

30

35

40

Tem

pera

ture

(∘C)

2500

2000

1500

1000

500

00 1200 2400 3600

Time (s)

G998400

(Pa)

(c)



3 Results





20

25

30

35

40

0

2500

2000

1500

1000

500

0 1200 2400 48003600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

2500

2000

1500

1000

500

0 1200 2400 48003600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)

20

25

30

35

40

0

2500

2000

1500

1000

500

0 1200 2400 48003600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(c)








N D58 times10k 10120583m

(a)

(a)

(b)

N D65 times10k 10120583m

(b)

(c)

N D61 times10k 10120583m

(c)

(d)

N D67 times10k 10120583m

(d)


20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

2mM 1mM05mM 0mM

G998400

(Pa)

(a)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

2mM 1mM05mM 0mM

G998400

(Pa)

(b)



05

030015

2000

1500

1000

500

0

40

35

30

25

200 1200 2400 3600

Time (s)

G998400

(Pa)

Tem

pera

ture

(∘C)









119884 = 926 sdot 119883 + 36 (1)



20

25

30

35

40

0

400

200

600

800

0 1200600 1800 2400 3000Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

400

200

600

800

0 1200600 1800 2400 3000Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)



4 Discussion







20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(c)







0

10

20

30

40

50

60

0 05 1 15 2 25

PSC 05

PSC 015


Elas

tic m

odul

us (k

Pa)

(a)

0

10

20

30

40

50

60


Elas

tic m

odul

us (k

Pa)

y = 926x + 36

R2 = 099

(b)







5 Conclusions



0

50

100

150

200

250

0 300 600 900 1200 1500Time (s)

0d

30d

11d

7d

3d

2d

1d

G998400

(Pa)



Acknowledgments


References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0001

001

01

1

10

0 120 240 360 480 600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(a)

0001

001

01

1

10

0 240120 360 480 600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(b)

0001

001

01

1

10

0 240 360120 480 600Time (s)

Shea

r mod

ulus

(Pa)

G998400

G998400998400

(c)





20

25

30

35

40

Tem

pera

ture

(∘C)

2500

2000

1500

1000

500

00 1200 2400 3600

Time (s)

G998400

(Pa)

(a)

20

25

30

35

40

Tem

pera

ture

(∘C)

2500

2000

1500

1000

500

00 1200 2400 3600

Time (s)

G998400

(Pa)

(b)

20

25

30

35

40

Tem

pera

ture

(∘C)

2500

2000

1500

1000

500

00 1200 2400 3600

Time (s)

G998400

(Pa)

(c)



3 Results





20

25

30

35

40

0

2500

2000

1500

1000

500

0 1200 2400 48003600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

2500

2000

1500

1000

500

0 1200 2400 48003600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)

20

25

30

35

40

0

2500

2000

1500

1000

500

0 1200 2400 48003600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(c)








N D58 times10k 10120583m

(a)

(a)

(b)

N D65 times10k 10120583m

(b)

(c)

N D61 times10k 10120583m

(c)

(d)

N D67 times10k 10120583m

(d)


20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

2mM 1mM05mM 0mM

G998400

(Pa)

(a)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

2mM 1mM05mM 0mM

G998400

(Pa)

(b)



05

030015

2000

1500

1000

500

0

40

35

30

25

200 1200 2400 3600

Time (s)

G998400

(Pa)

Tem

pera

ture

(∘C)









119884 = 926 sdot 119883 + 36 (1)



20

25

30

35

40

0

400

200

600

800

0 1200600 1800 2400 3000Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

400

200

600

800

0 1200600 1800 2400 3000Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)



4 Discussion







20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(c)







0

10

20

30

40

50

60

0 05 1 15 2 25

PSC 05

PSC 015


Elas

tic m

odul

us (k

Pa)

(a)

0

10

20

30

40

50

60


Elas

tic m

odul

us (k

Pa)

y = 926x + 36

R2 = 099

(b)







5 Conclusions



0

50

100

150

200

250

0 300 600 900 1200 1500Time (s)

0d

30d

11d

7d

3d

2d

1d

G998400

(Pa)



Acknowledgments


References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




20

25

30

35

40

Tem

pera

ture

(∘C)

2500

2000

1500

1000

500

00 1200 2400 3600

Time (s)

G998400

(Pa)

(a)

20

25

30

35

40

Tem

pera

ture

(∘C)

2500

2000

1500

1000

500

00 1200 2400 3600

Time (s)

G998400

(Pa)

(b)

20

25

30

35

40

Tem

pera

ture

(∘C)

2500

2000

1500

1000

500

00 1200 2400 3600

Time (s)

G998400

(Pa)

(c)



3 Results





20

25

30

35

40

0

2500

2000

1500

1000

500

0 1200 2400 48003600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

2500

2000

1500

1000

500

0 1200 2400 48003600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)

20

25

30

35

40

0

2500

2000

1500

1000

500

0 1200 2400 48003600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(c)








N D58 times10k 10120583m

(a)

(a)

(b)

N D65 times10k 10120583m

(b)

(c)

N D61 times10k 10120583m

(c)

(d)

N D67 times10k 10120583m

(d)


20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

2mM 1mM05mM 0mM

G998400

(Pa)

(a)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

2mM 1mM05mM 0mM

G998400

(Pa)

(b)



05

030015

2000

1500

1000

500

0

40

35

30

25

200 1200 2400 3600

Time (s)

G998400

(Pa)

Tem

pera

ture

(∘C)









119884 = 926 sdot 119883 + 36 (1)



20

25

30

35

40

0

400

200

600

800

0 1200600 1800 2400 3000Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

400

200

600

800

0 1200600 1800 2400 3000Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)



4 Discussion







20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(c)







0

10

20

30

40

50

60

0 05 1 15 2 25

PSC 05

PSC 015


Elas

tic m

odul

us (k

Pa)

(a)

0

10

20

30

40

50

60


Elas

tic m

odul

us (k

Pa)

y = 926x + 36

R2 = 099

(b)







5 Conclusions



0

50

100

150

200

250

0 300 600 900 1200 1500Time (s)

0d

30d

11d

7d

3d

2d

1d

G998400

(Pa)



Acknowledgments


References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




20

25

30

35

40

0

2500

2000

1500

1000

500

0 1200 2400 48003600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

2500

2000

1500

1000

500

0 1200 2400 48003600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)

20

25

30

35

40

0

2500

2000

1500

1000

500

0 1200 2400 48003600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(c)








N D58 times10k 10120583m

(a)

(a)

(b)

N D65 times10k 10120583m

(b)

(c)

N D61 times10k 10120583m

(c)

(d)

N D67 times10k 10120583m

(d)


20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

2mM 1mM05mM 0mM

G998400

(Pa)

(a)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

2mM 1mM05mM 0mM

G998400

(Pa)

(b)



05

030015

2000

1500

1000

500

0

40

35

30

25

200 1200 2400 3600

Time (s)

G998400

(Pa)

Tem

pera

ture

(∘C)









119884 = 926 sdot 119883 + 36 (1)



20

25

30

35

40

0

400

200

600

800

0 1200600 1800 2400 3000Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

400

200

600

800

0 1200600 1800 2400 3000Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)



4 Discussion







20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(c)







0

10

20

30

40

50

60

0 05 1 15 2 25

PSC 05

PSC 015


Elas

tic m

odul

us (k

Pa)

(a)

0

10

20

30

40

50

60


Elas

tic m

odul

us (k

Pa)

y = 926x + 36

R2 = 099

(b)







5 Conclusions



0

50

100

150

200

250

0 300 600 900 1200 1500Time (s)

0d

30d

11d

7d

3d

2d

1d

G998400

(Pa)



Acknowledgments


References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




N D58 times10k 10120583m

(a)

(a)

(b)

N D65 times10k 10120583m

(b)

(c)

N D61 times10k 10120583m

(c)

(d)

N D67 times10k 10120583m

(d)


20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

2mM 1mM05mM 0mM

G998400

(Pa)

(a)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

2mM 1mM05mM 0mM

G998400

(Pa)

(b)



05

030015

2000

1500

1000

500

0

40

35

30

25

200 1200 2400 3600

Time (s)

G998400

(Pa)

Tem

pera

ture

(∘C)









119884 = 926 sdot 119883 + 36 (1)



20

25

30

35

40

0

400

200

600

800

0 1200600 1800 2400 3000Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

400

200

600

800

0 1200600 1800 2400 3000Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)



4 Discussion







20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(c)







0

10

20

30

40

50

60

0 05 1 15 2 25

PSC 05

PSC 015


Elas

tic m

odul

us (k

Pa)

(a)

0

10

20

30

40

50

60


Elas

tic m

odul

us (k

Pa)

y = 926x + 36

R2 = 099

(b)







5 Conclusions



0

50

100

150

200

250

0 300 600 900 1200 1500Time (s)

0d

30d

11d

7d

3d

2d

1d

G998400

(Pa)



Acknowledgments


References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




05

030015

2000

1500

1000

500

0

40

35

30

25

200 1200 2400 3600

Time (s)

G998400

(Pa)

Tem

pera

ture

(∘C)









119884 = 926 sdot 119883 + 36 (1)



20

25

30

35

40

0

400

200

600

800

0 1200600 1800 2400 3000Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

400

200

600

800

0 1200600 1800 2400 3000Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)



4 Discussion







20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(c)







0

10

20

30

40

50

60

0 05 1 15 2 25

PSC 05

PSC 015


Elas

tic m

odul

us (k

Pa)

(a)

0

10

20

30

40

50

60


Elas

tic m

odul

us (k

Pa)

y = 926x + 36

R2 = 099

(b)







5 Conclusions



0

50

100

150

200

250

0 300 600 900 1200 1500Time (s)

0d

30d

11d

7d

3d

2d

1d

G998400

(Pa)



Acknowledgments


References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




20

25

30

35

40

0

400

200

600

800

0 1200600 1800 2400 3000Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

400

200

600

800

0 1200600 1800 2400 3000Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)



4 Discussion







20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(c)







0

10

20

30

40

50

60

0 05 1 15 2 25

PSC 05

PSC 015


Elas

tic m

odul

us (k

Pa)

(a)

0

10

20

30

40

50

60


Elas

tic m

odul

us (k

Pa)

y = 926x + 36

R2 = 099

(b)







5 Conclusions



0

50

100

150

200

250

0 300 600 900 1200 1500Time (s)

0d

30d

11d

7d

3d

2d

1d

G998400

(Pa)



Acknowledgments


References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(a)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(b)

20

25

30

35

40

0

50

100

150

200

0 1200 2400 3600Time (s)

Tem

pera

ture

(∘C)

G998400

(Pa)

(c)







0

10

20

30

40

50

60

0 05 1 15 2 25

PSC 05

PSC 015


Elas

tic m

odul

us (k

Pa)

(a)

0

10

20

30

40

50

60


Elas

tic m

odul

us (k

Pa)

y = 926x + 36

R2 = 099

(b)







5 Conclusions



0

50

100

150

200

250

0 300 600 900 1200 1500Time (s)

0d

30d

11d

7d

3d

2d

1d

G998400

(Pa)



Acknowledgments


References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0

10

20

30

40

50

60

0 05 1 15 2 25

PSC 05

PSC 015


Elas

tic m

odul

us (k

Pa)

(a)

0

10

20

30

40

50

60


Elas

tic m

odul

us (k

Pa)

y = 926x + 36

R2 = 099

(b)







5 Conclusions



0

50

100

150

200

250

0 300 600 900 1200 1500Time (s)

0d

30d

11d

7d

3d

2d

1d

G998400

(Pa)



Acknowledgments


References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0

50

100

150

200

250

0 300 600 900 1200 1500Time (s)

0d

30d

11d

7d

3d

2d

1d

G998400

(Pa)



Acknowledgments


References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials
























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



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Research Article Temperature-Responsive Gelation of Type I ...downloads.hindawi.com/journals/ijbm/2013/620765.pdf · Research Article Temperature-Responsive Gelation of Type I Collagen