covalently crosslinked chitosan hydrogel formed at neutral ph and body temperature

Covalently crosslinked chitosan hydrogel formed at neutralpH and body temperature

Yi Hong,1 Zhengwei Mao,1 Hualin Wang,2 Changyou Gao,1 Jiacong Shen1

1Key Laboratory of Macromolecule Synthesis and Functionalization of the Ministry of Education, Departmentof Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, People’s Republic of China2Department of Otorhinolaryngology, The Second Hospital Affiliated to Zhejiang University,Hangzhou 310027, People’s Republic of China

Received 29 July 2005; accepted 21 March 2006Published online 29 August 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30837

Abstract: Water-soluble chitosan having double bonds(CS–MA–LA) was synthesized by sequential grafting ofmethacrylic acid (MA) and lactic acid (LA) via the reactionbetween amino groups and carboxyl groups under thecatalysis of carbodiimide. Its molecular structure was veri-fied by FTIR and 1H NMR characterizations. Elementalanalysis measured grafting ratios of 19% and 10.33% forMA and LA, respectively. CS–MA–LA was readily solublein pure water and did not precipitate till pH 9. Gelation ofthe CS–MA–LA was realized by thermal treatment at bodytemperature under the initiation of a redox system, ammo-nium persulfate (APS)/N, N,N0,N0-tetramethylethylene-diamine (TEMED). The gelation time could be mediated ina wide range, e.g. from 6 to 20 min, by reaction tempera-ture and/or initiator’s concentration. 3T3 fibroblast cultureshowed that the cytotoxicity of the hydrogel extractant was

dependent on the cell seeding number and the initiator’sconcentration. With enough number of cells (>2.5 � 104) andlow initiator’s concentration (5 mM), the cytotoxicity intro-duced by the initiator is very minimal and negligible.Although the hydrogel could cause acute inflammationand foreign body reaction, no tissue necrosis and malig-nant infection were evidenced in vivo, demonstrating thatthe material has better histocompatibility. These featureshave endowed the chitosan with great opportunity as in-jectable biomaterials, which may find wide applications inthe rapidly developed fields such as tissue engineeringand orthopedics. � 2006 Wiley Periodicals, Inc. J BiomedMater Res 79A: 913–922, 2006

Key words: chitosan; injectable; thermal crosslinking; bio-materials; water-soluble

INTRODUCTION

Injectable scaffolds are increasingly attractive alongwith the development of tissue engineering and ortho-pedics since they can support and guide the regenera-tion of damaged tissues or organs in an in vivo cultureenvironment. Clinical implantation can also be per-formed in a minimally invasive way. Hydrogel sys-tems composed of collagen,1 chitosan,2 gelatin,3 algi-

nate4,5 and poly(propylene fumarate) (PPF)6,7 arewidely applied as the injectable scaffolds.8,9 Theycan be easily injected to the wanted sites and solidi-fied in situ. Cells and/or other bioactive componentssuch as cell growth factors can be simultaneously in-corporated into the system at a solubilized or sus-pended state. The solidified hydrogels possess highcontent of water, which favors the exchange of gases,nutrients and metabolites, and their intrinsic structuremimics cell growth environment, i.e. the extracellularmatrix. Since formed and used in vivo, the hydrogelsand their soluble counterparts should be biocompati-ble and biodegradable. Neutral solution pH and mildgelation conditions are also required to maximum sus-tain the viability of contained cells and to reduce theharmfulness to the surrounding tissues.10

Chitosan is a N-deacetylation product of chitin. Ithas been widely applied in the fields of drug deliveryand tissue engineering because of its biodegradabilityand good biocompatibility.11,12 Unmodified chitosanmolecules are only dissolved in acidic solution be-cause of their strong interchain hydrogen bonding. By

Correspondence to: C. Gao; e-mail: [email protected] grant sponsor: Major State Basic Research Pro-

gram of China; contract grant number: 2005CB623902Contract grant sponsor: Ph.D. Programs Foundation of

Ministry of Education of China; contract grant number:20050335035Contract grant sponsor: Natural Science Foundation of

China; contract grant number: 20434030Contract grant sponsor: National Science Fund for Distin-

guished Young Scholars of China; contract grant number:50425311

' 2006 Wiley Periodicals, Inc.

change in pH value, covalently or ionically crosslink-ing, chitosan hydrogel has been formed.13 However,the acidic solubility and gelation methods employedso far will surely limit the application of chitosan as aninjectable hydrogel for tissue regeneration in vivo. Upto present, only two kinds of chitosan hydrogel sys-tems have been developed as injectable scaffolds. Forexample, glycerol-2-phosphate (b-GP) has been usedto adjust the chitosan solution from acidic to neutral,thus chitosan hydrogel are formed at a temperatureclose to 378C.2,14 By incorporation of viable chondro-cytes into the hydrogel system, injection of the hydro-gel into a mouse has formed proteoglycan-rich matrixin vivo. Moreover, mesenchymal stem cells (MSCs)have been mixed with an injectable thermosensitive(water-soluble chitosan-g-poly(N-isopropylacrylamide))hydrogel. In vivo results demonstrate that the MSCscan be differentiated to chondrocytes, and cartilage isformed after culturing for 14 weeks after the cell–hydrogel complex is injected into the submucosallayer of the bladder of rabbit.15

Therefore, modification of chitosan molecule to en-hance its solubility at neutral pH and to develop afriend gelation method is of both practical and tech-nological significance. In the present study, a novelchitosan gelation system is developed. The derivatedchitosan has good solubility at neutral pH, and canbe covalently thermocrosslinked to form hydrogel atbody temperature. For this purpose, methacrylic acid(MA) and lactic acid (LA) are successively graftedonto the chitosan molecules that endow the chitosanwith crosslinkable and water-soluble features, re-spectively. The chitosan hydrogel is then formed atan elevated temperature under the initiation of a redoxsystem, ammonium persulfate (APS) and N,N,N0,N0-tetramethylethylenediamine (TEMED). Its cyto-toxicity is further assessed by in vitro 3T3 fibroblastculture.

EXPERIMENTAL SECTION

Materials

Chitosan (average MZ % 600,000) was obtained from Hai-debei Marine Bioengineering Company, Ji’nan, China. MAand APS were purified via distillation under reduced pres-sure and recrystallization, respectively. 1-Ethyl-3-(3-dime-thylaminopropyl) carbodiimide hydrochloride (WSC) waspurchased from Sigma. LA and TEMED (>98%) were used asreceived.

Synthesis of CS–MA–LA

Eight hundred milligrams of chitosan was dissolved in100 mL water and 420 mL (0.48 mM) MA, to which 930 mg

(0.48 mM) WSC were added. The reaction lasted for 24 hat room temperature under magnetic agitation. The pHvalue of the mixture solution increased from 4 to *7 dur-ing this process owing to the alkaline nature of the result-ant urea. In order to remove the unreacted MA and othersmall molecular weight products, the resultant mixturewas sealed in a membrane with a cut off molecular weightof 10,000 Da and dialyzed in a lager amount of triple-dis-tilled water for 3 days. Finally, MA grafted chitosan (CS–MA) was obtained by freeze-drying.

Half of the CS–MA (*400 mg) was dissolved in 50 mLwater containing 210 mL (0.2 mM) LA overnight, to which460 mg WSC was added. The mixture was stirred for 24 hat room temperature. Following the purification stepsdescribed earlier, the water-soluble and cross-linkable chito-san (CS–MA–LA) was obtained. The yields of both CS–MAand CS–MA–LA were over 90%.

Measurement of water solubility

The water solubility was measured according to litera-ture.16 Briefly, chitosan, CS–MA, and CS–MA–LA weredissolved separately in water (1 mg/mL) with a pH valueof 3. At this pH value, chitosan and its derivatives arecompletely soluble. The pH value of each solution wasgradually raised by addition of 0.1 mol/L NaOH solution,and the pH value at which precipitation occurred wasmeasured using a pH meter. The concentrations of the chi-tosan and its derivatives should not be diluted below 70%of the original concentration (1 mg/mL) upon addition ofNaOH solution.

Gelation of CS–MA–LA

CS–MA–LA aqueous solution was gelated by radical po-lymerization under the initiation of a redox system includingoxidant APS and reducer TEMED. APS and TEMED werepreviously made into 1M solutions, respectively. 1% CS–MA–LA aqueous solution was sequentially mixed with APSand TEMED solutions. The mole ratio of APS and TEMEDwas kept same for all the experiments. Then this mixture wasinjected into a mold by a syringe. The gelation was con-ducted in the mold at a given temperature. The gelation timewas recorded right from the mixing to the state that the mix-ture lost its flow ability. Three to five parallel experiments wereconducted and average data were reported as mean6 standarddeviation. Initiator’s concentration and reaction temperaturewere varied to evaluate the gelation time.

Swelling ratio of the hydrogel

For measuring the swelling ratio, chitosan hydrogelswere prepared by gelation of 1% CS–MA–LA solution anddifferent concentration of APS/TEMED for 24 h at 378C.The chitosan hydrogels were balanced in PBS for 24 h at378C and weighted (W1). Then the hydrogels were dehy-drated under reduced pressure at 358C to constant weights(W2). The swelling ratio of the hydrogel is defined as (W1 �W2)/W2.

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Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a

Crosslinking yield

One percent CS–MA–LA solution was gelated at differ-ent concentration of APS/TEMED for 24 h at 378C. Thechitosan hydrogels were freeze-dried and weighted (W3).The freeze-dried hydrogels were immersed in 3% aceticacid solution for 24 h, followed by extensive washing withdeionized water. Then the hydrogels were freeze-dried againto constant weights (W4). The crosslinking yield is defined as(W4/W3) � 100%.

Cytotoxicity test at different cell seeding densityand APS/TEMED concentration

The cytotoxicity of the chitosan hydrogel was assessed byculture of 3T3 fibroblasts supplemented with the extractantof the hydrogel. APS and TEMED were previously madeinto 1M PBS solutions and then sterilized by membrane fil-tration with a pore size of 0.22 mm, respectively. CS–MA–LAwas sterilized under UV radiation for 3 h and then dissolvedin PBS. Hydrogels were fabricated from a 1% CS–MA–LA/PBS solution and 5 or 10 mM APS/TEMED at 378C. Thehydrogels were treated with Dulbecco’s minimum essentialmedium (DMEM) supplemented with 10% fetal bovine se-rum (FBS) at a ratio of 100 mg hydrogel/mL DMEM for 24 hat 378C. The solutions extracted from the hydrogels gelatedat 5 mM and 10 mM APS/TEMED are designated as E5 andE10, respectively. DMEM supplemented with 10% FBS wasused as a negative control.

Different numbers of 3T3 fibroblasts were seeded onto a96-well culture plate. The cells were cultured in 200 mLDMEM supplemented with 10% FBS at 378C in 5% carbondioxide atmosphere. After 12 h, DMEM was removed and200 mL E5 was added. After incubation at 378C for 24 h, thecytoviability was quantitatively measured by MTT assay.The preserved ratio of cytoviability is defined as A2/A1

� 100%, where A1 and A2 represent the absorbance of thenegative control and the samples, respectively. All datawere averaged from 3 parallel experiments and expressedas mean 6 standard deviation.

Each well of the 96-well culture plate was seeded with1 � 105 3T3 fibroblasts. The cells were cultured in 200 mLDMEM supplemented with 10% FBS at 378C in 5% carbondioxide atmosphere. After 12 h, DMEM was removed and200 mL E5 or E10 was added. The cytoviability was quantita-tively measured by MTT assay after incubation at 378C for1d–4d. The morphology of the cells at 1d and 4d wasobserved under confocal laser scanning microscopy (CLSM,Bio-Rad Radiance 2100). The cells were incubated in 5 mg/mL fluorescein diacetate (FDA)/PBS solution for 10 min. Inthis process, FDA (no fluorescence) could penetrate throughcell membranes and was hydrolyzed into fluorescein by via-ble cells, which was then excited at 488 nm under CLSM.17

MTT assay

After the cells were cultured for a given time, 20 mL MTT(3-(4,5-dimethyl) thiazol-2-yl-2,5-dimethyl tetrazolium bro-mide, 5 mg/mL) was added into each well. The cells werecontinually cultured for another 4 h. During this period, via-

ble cells could reduce the MTT to formazan pigment, whichwas dissolved by 200 mL dimethyl sulphoxide after removalof the culture medium. The absorbance at 570 nm wasrecorded under a microplate reader (Bio-Rad 550).

In vivo inflammatory reaction of thechitosan hydrogel

Amixture of 1% CS–MA–LA/PBS solution and 5 mMAPS/TEMED solution was subcutaneously injected into whitemice, each with 0.5 mL liquid. After implantation for 1d, 3d,and 10d, the mice were sacrificed and anatomized to investi-gate the inflammatory reaction. The skins and hydrogelswere harvested for histological evaluation. The sections ofthe skins and the hydrogels were stained by H&E.

Characterizations

FTIR spectra were recorded on a BRUKER VECOTR22 spec-trometer. 1H NMR spectrum of CS–MA–LA was recordedon an ANAVCE DMX500 with D2O as solvent working at500 MHz. Elemental analysis was performed on an elemen-tal analyzer (Flash EA-1112).

Statistical analysis

Data were analyzed using ANOVA. Significance wasdetermined at a value of p < 0.05.

RESULTS AND DISCUSSION

Synthesis and characterization of CS–MA–LA

Chitosan can be regarded as a copolymer of N-acetyl-glucosamine and N-glucosamine units randomly dis-tributed throughout the molecular chain. It is dis-solved only in acidic solution for its strong intermolec-ular hydrogen bonding. It contains abundant aminogroups, through which both polymerizable (e.g. acry-late) and water-soluble groups can be conveniently in-troduced. In the present work, MA and LA are sequen-tially grafted onto the chitosan chains via the combi-nation between the carboxyl groups and the aminoand/or hydroxyl groups to yield water soluble andpolymerizable CS–MA–LA under the catalysis ofcarbodiimide (Scheme 1). Since MA and LA are bothweak acids and chitosan can be directly dissolved intheir solutions, the reaction is easily proceeded with-out involvement of other acid. The byproducts ofsmall molecular weight and unreacted monomersare then removed by dialysis.18–21

FTIR and 1H NMR characterizations confirmed thestructure of chitosan and its derivatives (Fig. 1). TheIR spectrum of chitosan [Fig. 1(a)] illustrates peaksassigned to the saccharide structure at 1153, 1081,

CHITOSAN HYDROGEL FORMED AT NEUTRAL pH AND BODY TEMPERATURE 915


1029, and 898 cm�1.22,23 The peaks at around 1653and 1598 cm�1 are assigned to amide I band and NH2

group, respectively. Accompanying with the weak-ening of absorbance at 1598 cm�1, new peaks at 1626and 1574 cm�1 emerge in the IR spectrum of CS–MA[Fig. 1(b)], which should be assigned to the C¼¼Cdouble bond and the amide II band, respectively.This result demonstrates that MA has been success-fully grafted. After grafting LA [Fig. 1(c)], the peakintensity at 1574 cm�1 is further increased in the IRspectrum of CS–MA–LA. Moreover, a new peak at1734 cm�1 assigned to ester bond appears. This wouldmean that the carboxylic acid groups of LA react notonly with amino groups, but also with hydroxylgroups of chitosan. That considerable amount of estersis formed after LA grafting is understandable, since at

this moment the absolute amount of NH2 groups hasbeen largely decreased by reaction with MA. Never-theless, the IR spectrum has undoubtedly evidencedthe introduction of LA.

Characterization of CS–MA–LA under 1H NMRconfirms also its molecular structure. Chemical shiftsbelonging to the saccharide structure are assigned asfollows: 1H NMR (D2O) d ¼ 2.79(H2), d ¼ 3.43–3.91(H3–H6), d ¼ 1.91(��NCOCH3).

24 Chemical shiftsat d ¼ 5.64 and d ¼ 5.42 are assigned to H2C¼¼C��(a) of MA, respectively. Chemical shifts at d ¼ 1.84and d ¼ 1.20 are assigned to methyl groups of MA(c) and LA (b), respectively. Moreover, MA and LAsubstitution degrees are 19% and 10.33% as detectedby elemental analysis, respectively (Table I). All theseresults have confirmed that both MA and LA havebeen grafted onto the chitosan chains.

Water solubility

It was shown in Table I that the highest soluble pHincreased from 5 to 9 when the chitosan was graftedwith MA and LA. When the acidic solution of chitosanwas neutralized with NaOH, it was sedimentated atapproximately pH 5, implying that chitosan is notsoluble in water. After grafting of MA, the precipita-tion pH value was raised to 7. However, when im-mersed under pure water, this MA grafted chitosancould be only swollen but not dispersed. By contrast,the CS–MA–LA could be completely dissolved in purewater with low viscosity. This has endowed the poly-saccharide with great opportunity as injectable scaf-fold in situ. The substitution of MA and LA can alterthe regular molecular structure of chitosan, and thusweaken the intermolecular hydrogen bonding. YetMA only is not able to yield grafting product with suf-ficient water solubility, since the H-bonding cannot beadequately screened. It was reported previously thatthe chitosan can become water-soluble when the de-acetylation degree is *50%.25 Yet this deacetylationdegree is hardly controllable. A further introductionof LA may not only make the chitosan molecularstructure more irregular, but also can enhance the

Scheme 1. Synthesis route and molecular structure ofCS–MA–LA.

Figure 1. FTIR spectra of (a) chitosan, (b) CS–MA, (c) CS–MA–LA and (d) chitosan hydrogel. [Color figure can beviewed in the online issue, which is available at www.interscience.wiley.com]

TABLE IThe grafting ratios and maximum soluble pHs

of chitosan, CS–MA and CS–MA–LA

Sample C/N RatioGraftingRatio (%)

MaximumSoluble pHb

Chitosana 6.44 78.00 <5CS–MA 7.20 19.00 <7CS–MA–LA 7.51 10.33 <9

aThe deacetylation degree of chitosan was calculatedaccording to the C/N ratio.

bData represent the highest pH value at which the pre-cipitation occur.

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H-bonding between chitosan and water molecules be-cause of the existence of pendent hydroxyl group in LA.

Hydrogel formation

Different from the before-mentioned gelation sys-tems for chitosan,2,14,15 chitosan hydrogel describedhere is formed via crosslinking reaction between thedouble bonds. For this to occur, a redox system is usedso that polymerization can be performed at body tem-perature. It has been identified that the APS/TEMEDinitiation system is water-soluble and cytocompatibleand thus is used to initiate the polymerization ofPPF.26–28 Figure 2 illustrates the macroscopic gelationprocess. After addition of APS/TEMED and incuba-tion at 378C for a few minutes, liquid CS–MA–LAsolution [Fig. 2(a)] was transferred into transparentchitosan hydrogel [Fig. 2(b)] which could sustain itsmacroscopic shape [Fig. 2(c)]. In the FTIR spectrumof chitosan hydrogel [Fig. 1(d)], the absorbance at1626 cm�1 (C¼¼C double bonds) has disappeared,indicating the occurrence of polymerization. This haspreliminarily demonstrated that the as-synthesized

water-soluble chitosan has the potential capability tobe used as injectable and in situ gelable scaffold.

Gelation time

As an injectable biomaterial in clinical applicationit is required that the polymer solution is stable atroom temperature for a relatively long period, andforms hydrogel at body temperature (378C) rapidly.Therefore, the gelation time was investigated by vary-ing the initiator concentration and the incubation tem-perature (Fig. 3).

The gelation time decreased rapidly along withthe increase of initiator’s concentration as shown inFigure 3(a). When the APS/TEMED concentrationwas set at 2.5 mM, the gelation time was longer than30 min at 378C (the data was not shown in [Fig. 3(a)]).When the concentration was set at 5 and 10 mM,formation of the hydrogel required only *5.5 and*1.5 min, respectively. At still higher concentra-tion, e.g. 15 mM, the gelation was completed within*30 s. It is understandable that with higher concen-tration of initiators larger amount of free radicals will

Figure 2. (a) 1% CS–MA–LA water solution, (b) gelation of (a) in 5 mM APS/TEMED at 378C, and (c) shape persistentbehavior of (b). Images were taken with a digital camera. [Color figure can be viewed in the online issue, which is avail-able at www.interscience.wiley.com]



be created. Consequently the crosslinking polymeriza-tion can proceed in a relatively fast rate.

APS/TEMED initiating system is sensitive to tem-perature.27 Higher temperature can accelerate the gen-eration and diffusion of free radicals, and also im-prove the motion ability of macromolecular chains. Asshown in Figure 3(b), at an APS/TEMED concentra-tion of 5 mM the gelation time decreased from *20 to*5.5 min when the temperature was raised from 25to 378C. As an injectable hydrogel, an appropriategelation time is important. The property of the chito-san solution, i.e. rather stable at room temperaturewhile can be gelated at body temperature, is very prom-ising for clinical application. The longer gelation timeat room temperature benefits for operation, while theshorter gelation time at body temperature can preventfrom the liquid diffusion and favor the shape persist-ence. It is worth noting that no apparent temperatureincrease was measured during the gelation process.

Swelling behavior of the chitosan hydrogels

Figure 4 presents the swelling behavior of the chi-tosan hydrogels formed at 378C with different APS/TEMED concentration. At a lower concentration ofAPS/TEMED (<7.5 mM), the balanced swelling ratioof the hydrogels decreased initially as a function of theinitiator’s concentration (p < 0.05). When the initiator’sconcentration was higher than 7.5 mM, the swellingratio was kept at a relatively low level without signifi-cant difference (p > 0.05). This feature matches inver-sely with the crosslinking yield of the hydrogels, e.g.with higher crosslinking yield the swelling ratio islower. When the crosslinking density is higher, theswelling of the hydrogel is largely restricted. More-over, it is not strange that the crosslinking yield showsa positive correlation with the initiator’s concentration.

Polymerization of the chitosan macromonomers islargely dependent on the encountering probability ofC¼¼C double bonds. Crosslinking can take place only ifa macromolecular radical is close enough to anotherC¼¼C bond. The macromolecular chains have very lowmoving ability and are confined within a limited spa-tial volume. Higher initiator’s concentration will createmore macromolecular radicals at a definite volume.As a result, there will be a higher chance for the mac-romolecular radicals to react with other C¼¼C bonds.Hence, a higher crosslinking yield can be produced.

Cytotoxicity

To assess the toxicity of the chitosan hydrogel, 3T3fibroblasts were cultured in medium of extractant.First, the cytotoxicity under different cell seeding

Figure 3. Gelation time of 1 wt % CS–MA–LA solution as a function of (a) APS/TEMED concentration at 378C, and (b)incubation temperature with APS/TEMED concentration of 5 mM.

Figure 4. Swelling ratio and crosslinking yield of chitosanhydrogel as a function of APS/TEMED concentration. Thehydrogels were immersed under PBS at 378C. [Color figurecan be viewed in the online issue, which is available at www.interscience.wiley.com]

918 HONG ET AL.


number was investigated (Fig. 5). After cultured for24 h, the cytoviability measured by MTT assay in E5medium and in DMEM control was compared as afunction of cell seeding number. The overall cell via-bility in both control and E5 medium increased alongwith the cell seeding number till 5 � 104 (p < 0.05).With still higher cell number, no significant differencewas found (p > 0.05). Compared with the negativecontrol, the cytoviability of E5 was significantly low(p < 0.05) when the cell number was smaller than 2 �104. Above this seeding number, no significant differ-ence was detected. This alteration tendency is moreclearly illustrated by the preserved viability ratio asshown in the inset of Figure 5. Hence, one can concludethat the extractant from the hydrogel has negative effecton the viability of 3T3 cells when the cell number issmall, i.e. some degree of cytotoxicity which should bemainly attributed to the initiators. However, withenough number of cells, the cytotoxicity introduced bythe initiators is very minimal and neglectable.

To further identify the cytotoxicity of the hydrogel,cell culture with an initial seeding number of 1 � 105

in mediums of negative control, E5 and E10 was per-formed. As shown in Figure 6, the cytoviability of boththe E5 and the control was increased as a function ofculture time, indicating that the cells in these culturemediums can normally proliferate. By contrast, thecytoviability of sample E10 was steadily decreased asa function of the culture time, implying that instead ofnormal proliferation part of the seeding cells in thismedium should be dead. The profiles of cell viabilityof E5 and control are very close, although at some cul-ture intervals the cytoviability of E5 is still lower thanthat of the control. Figure 7 compares the morphology

of 3T3 fibroblasts after cultured in mediums of control[Fig. 7(a,d)], E5 [Fig. 7(b,e)] and E10 [Fig. 7(c,f)] for 1d[Fig. 7(a–c)] and 4d [Fig. 7(d–f)]. Confluent cell layershave been formed for the control and the E5 since theculture time of 1d. No apparent difference of cell mor-phology for the control and the E5 can be identified.By contrast, a fewer cells were measured for the E10,particularly after cultured for 4d. Since clinically thecell number (>2 million/mL) is far beyond the highestvalue used in this study, the hydrogel formed at aninitiator’s concentration of 5 mM can be roughlyregarded as nontoxic to cells, or at least that the toxic-ity can be neglected.

In vivo inflammatory reaction

The in vivo inflammatory reaction was assessed bysubcutaneous injection of a mixture of 1% CS–MA–LA/PBS solution and 5 mM APS/TEMED solutioninto white mice. All the mice survived throughoutthe implantation period with normal performance.No malignant infection, tissue necrosis, and abscesswere found in the implanted sites. Histological sec-tions of the skin–hydrogel interfaces and the chitosanhydrogels are shown in Figure 8. After implantationfor 1d, a large number of neutrophils (inflammationcells) infiltrated through the skin–hydrogel interface[Fig. 8(a)] and the hydrogel [Fig. 8(d)], implying thatacute inflammatory reaction occurred at this stage. Atday 3, the inflammation was aggravated. The skin–hydrogel interface became loose, and the exudationand the edema could be observed as well [Fig. 8(b)].Meanwhile, histocytes infiltrated into the hydrogel[Fig. 8(e)]. After implantation for 10d, the large num-

Figure 5. Cytoviability of 3T3 fibroblasts as a function ofcell seeding number. Cells were cultured in (a) controlDMEM medium and (b) E5 medium. Inset is the preservedratio of cytoviability, which is calculated by [(a�b)/a] �100%. [Color figure can be viewed in the online issue,which is available at www.interscience.wiley.com]

Figure 6. Cytoviability of 3T3 fibroblasts as a function ofculture time. Cell seeding number was 1 � 105/well.[Color figure can be viewed in the online issue, which isavailable at www.interscience.wiley.com]



ber of neutrophils had significantly decreased in theinterface [Fig. 8(c)] and the hydrogel [Fig. 8(f)]. In-stead, a large number of histocytes appeared to form aforeign body granuloma in the hydrogel, indicatingthat the acute inflammation has shifted to the foreignbody reaction. As a general rule in evolution of inflam-matory reaction, the granuloma will be finally assimi-lated to eliminate the inflammation. Thus the emer-gence of the granuloma is a positive sign indicatingthat the inflammation caused by the hydrogel is onlytemporary. In conclusion, although the hydrogel couldcause acute inflammation and foreign body reaction,no tissue necrosis and malignant infection are evi-denced in vivo, demonstrating that the material hasbetter histocompatibility.

CONCLUSIONS

Water-soluble and thermocrosslinkable chitosan issuccessfully synthesized via sequentially grafting ofMA and LA under the catalysis of water-soluble car-bodiimide. FTIR, 1H NMR, and elemental analysis

confirm the molecular structure and the substitutiondegree of the as-synthesized CS–MA–LA. Gelation ofthe CS–MA–LA is performed at very mild conditionsby using a redox initiation system, APS/TEMED. At aconcentration of 5/5 (mM) (APS/TEMED) the gelationtimes are *20 and *6 min at 25 and 378C, respec-tively. No apparent temperature elevation is recordedduring the gelation process. The property of the chito-san solution, i.e. rather stable at room temperaturewhile can be gelated at body temperature, is verypromising for clinical application. The swelling ratioof the chitosan hydrogels decreases along with theincrease of the APS/TEMED concentration initially,then reaches a constant value. This matches inverselyto and thus can be explained by the crosslinking yieldof the hydrogels. 3T3 fibroblast culture demonstratesthat with sufficient number of cells, the cytotoxicity in-troduced by the initiators is very minimal and neglect-able. Since clinically the cell number (>2 million/mL)is far beyond the highest value used in this study(1 � 105/200 mL), the hydrogel formed at an initia-tor’s concentration of 5 mM can be roughly regardedas nontoxic to cells, or at least the toxicity can be

Figure 7. CLSM images to show morphology of 3T3 fibroblasts after cultured in mediums of control (a,d), E5 (b,e) andE10 (c,f) for 1d (a–c) and 4d (d–f). Cell seeding number was 1 � 105/well. Viable cells were stained by FDA, thus exhibitbright color. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com]

920 HONG ET AL.


neglected. In vivo test shows that the hydrogel sys-tem can induce foreign body reaction with sufficienthistocompatibility. The study has demonstrated thatthe as-synthesized water-soluble chitosan has the poten-tial capability to be used as injectable and in situ gel-able scaffold.

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Figure 8. Histological evaluation of the skin–hydrogel interfaces (a–c) and the chitosan hydrogels (d–f) after subcutane-ous injection of a mixture of 1% CS–MA–LA/PBS solution and 5 mM APS/TEMED solution into white mice for 1d (a,d),3d (b,e), and 10d (c,f). N and H represent the neutrophils and histocytes, respectively. Magnification of left side and rightside in each image is �100 and �400, respectively. [Color figure can be viewed in the online issue, which is available atwww.interscience.wiley.com]



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covalently crosslinked chitosan hydrogel formed at neutral ph and body temperature

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