research article evaluation of a thiolated chitosan...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 878930 10 pageshttpdxdoiorg1011552013878930

Research ArticleEvaluation of a Thiolated Chitosan Scaffold forLocal Delivery of BMP-2 for Osteogenic Differentiation andEctopic Bone Formation

In-Ho Bae1 Byung-Chul Jeong12 Min-Suk Kook3 Sun-Hun Kim23 and Jeong-Tae Koh123

1 Department of Pharmacology and Dental Therapeutics School of Dentistry Chonnam National UniversityGwangju 500-757 Republic of Korea

2 Research Center for Biomineralization Disorders School of Dentistry Chonnam National UniversityGwangju 500-757 Republic of Korea

3 Dental Science Research Institute School of Dentistry Chonnam National University Gwangju 500-757 Republic of Korea

Correspondence should be addressed to Jeong-Tae Koh jtkohchonnamackr

Received 4 April 2013 Revised 12 July 2013 Accepted 15 July 2013

Academic Editor Joshua R Mauney

Copyright copy 2013 In-Ho Bae 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

Thiolated chitosan (Thio-CS) is a well-established pharmaceutical excipient for drug delivery However its use as a scaffold forbone formation has not been investigated The aim of this study was to evaluate the potential of Thio-CS in bone morphogeneticprotein-2 (BMP-2) delivery and bone formation In vitro study showed that BMP-2 interacted with the Thio-CS and did not affectthe swelling behaviorThe release kinetics of BMP-2 from theThio-CS was slightly delayed (70) within 7 days compared with thatfrom collagen gel (Col-gel 85) which is widely used in BMP-2 deliveryThe BMP-2 released fromThio-CS increased osteoblasticcell differentiation but did not show any cytotoxicity until 21 days Analysis of the in vivo ectopic bone formation at 4 weeks ofposttransplantation showed that use of Thio-CS for BMP-2 delivery induced more bone formation to a greater extent (18 fold)than that of Col-gel However bone mineral density in both bones was equivalent regardless of Thio-CS or Col-gel carrier Takentogether Thio-CS system might be useful for delivering osteogenic protein BMP-2 and present a promising bone regenerationstrategy

1 Introduction

Current approaches for bone regeneration such as autograftsand allografts face significant limitations [1] Various factorsincluding limited supply risk of immune rejection andchronic immune responses have prompted interest in bonegraft substitutes Many growth factors for bone formationhave been reported Bone morphogenetic protein-2 (BMP-2)is generally acknowledged due to its superior activity It hasbeen used in dental and orthopedic biomaterials to promotebone formation because of its strong osteogenic activityBMP-2 induces bone formation in vivo [2ndash7] presumablyby stimulating mesenchymal stem cell differentiation intoan osteoblast lineage and by increasing the number ofdifferentiated osteoblasts capable of forming bone [8] Thisstimulatory effect of BMP-2 on osteoblastic differentiation

is of major importance during bone healing Despite itsstrong osteoinductive activity the systemic delivery of BMP-2 can be impractical and undesirable because it may haveuncontrolled adverse effects such as unwanted ectopic boneformation In addition clinical use of BMP-2 has beenlimited by the lack of suitable delivery systems Systemsevaluated as carriers to localize BMP-2 include poroushydroxyapatite (HA) [9] absorbable collagen [10] polylacticacid [11] polylactic-co-glycolic acid [12] demineralized bonepowder and bovine collagen type sponges [13] AlthoughHA is a biocompatible material it is not biodegradableTherefore it remains at the defect site Collagen gel (Col-gel) can be immunogenic and demineralized bone powdersuffers from insufficient supply and poor characterization asa delivery system Thus an efficacious delivery system (iescaffold) is still required to localize BMP-2 at the desired site

2 BioMed Research International

Natural biomaterials are widely used for scaffold fabricationin tissue engineering because they facilitate cell attachmentand maintenance of the differentiation function Chitosan(CS) obtained by alkaline deacetylation of chitin is one ofthe most abundant polysaccharides in nature It has receivedconsiderable attention in a variety of areas such as phar-maceutics [14] tissue engineering [15] antimicrobial agents[16] and chromatography [17] because of its propertieswhich include enzymatic biodegradability nontoxicity andbiocompatibility even when used in human and animalmodels [18ndash20] However CS suffers from limited solubilityat physiological pH and causes presystemic metabolism ofdrugs in the presence of proteolytic enzymes [21] Theseinherent drawbacks of CS have been overcome by formingderivatives such as carboxylated CS [22] adding variousconjugates [23] thiolated CS [24] or acylated CS [25] Amongthese various CS derivates thiomer technology has a range ofadvantages for drug delivery such as sustained drug release[26] and high stability [24] The usefulness of thiolatedchitosan (Thio-CS) as a scaffold for controlled drug releasehas been demonstrated by means of model drugs suchas clotrimazole [27] salmon calcitonin [28] insulin [29]and tobramycin [30] However most of the research hasfocused on systemic drug delivery such as neural tissue [31]peroral peptide delivery [32] and nasal administration [33]Despite the advantages ofThio-CS for tissue engineering thepotential application of this material for bone tissue has notbeen investigated The aim of this study was to evaluate thephysicochemical properties of Thio-CS for BMP-2 deliveryand bone formation in vitro and in vivo

2 Materials and Methods

21 Fabrication of Thio-CS To obtain a 1 (wv) solution500mg of CS (average molecular mass 400 kDa FlukaGmbH Buchs Switzerland) was dissolved in 50mL of 1acetic acid by stirring the mixture for 1 h Trautrsquos reagent (2-iminothiolane-HCl 2-IT) was used for the immobilizationof thiol groups to primary amino groups of proteins and themodification of CS We have previously reported the optimalconditions for fabricating Thio-CS [34] In brief the pH of amixture containing a 1 solution of CS and 01mgmL of 2-IT was adjusted ranging from 4 to 12Themixtures were thenincubated for 30min at room temperature To investigate thetime effect of the air oxidation on disulfide bond formationthe samples were incubated for 3-day intervals at pH 7 understirring To remove unreacted agent the resulting mixturewas dialyzed with several exchanges of the dialyzing solutionTo prevent more oxidation of samples 5mM or 04mM ofHCl solution was used as a dialyzing solution depending ondialysis step The samples were freeze-dried at minus80∘C and001mbar (Christ Beta 1ndash8K Germany) and stored at 4∘Cuntil further use

22 Determination of the Thiol Group and Disulfide BondEllmanrsquos reagent (3mgof 551015840-dithiobis (2-nitrobenzoic acid))(Sigma St Louis MO USA) was used to quantify theamount of thiol groups on the modified CS as described

previously [34] Briefly 5mg of the freeze-dried samples wasdissolved in 25mL of demineralized water Then 250120583Lof the samples 250120583L of 5M phosphate-buffered saline(PBS pH 80) and 500 120583L of Ellmanrsquos reagent dissolvedin 10mL of 05M PBS (pH 80) were reacted in the sametube The reaction was allowed to proceed for 2 h at roomtemperature After removal of the precipitated polymerby centrifugation (24000timesg 5min) 300 120583L of the super-natant was transferred to a microtitration plate and theabsorbance was immediately measured at 450 nm (Bio-TekInstruments Winooski VT USA) The amount of thiolmoieties was calculated from a standard curve of absorbanceobtained from solutions with increasing concentrations of L-cysteine hydrochloride hydrate (Sigma-Aldrich SteinheimGermany) Disulfide bond content within precipitate orreacting solutionwas determined after reductionwithNaBH

4

and addition of Ellmanrsquos reagent as described by Habeeb[35] The degree of cross-linking of such materials is usuallydetermined by a chemical analysis method using 246-trinitrobenzenesulfonic acid (TNBS) labeling of residualamine groups [36] To 03mL of sample 03mL of NaHCO

3

(4) and 03mL of TNBS (01) were added The solutionwas allowed to react for 2 h at 40∘C and then 03mL ofsodium dodecyl sulfate (10) and finally 017mL HCl (1M)were addedThe absorbance of the resulting solutionwas readphotometrically at 335 nm against a blank but with 03mL ofH2O instead of the sample

23 Evaluation of the Swelling Behavior To understand theeffect of the molecular transport of liquids into Thio-CS thewater-absorbing (ie swelling) capacity was determined bygravimetric methods Tomeasure the weight ofThio-CS afterswelling 01 g of Thio-CS was placed in trans-well (CorningInc Corning NY USA)The trans-well was then placed intoa 24-well culture dish containing a physiological solution of1mL of PBS (pH 70) and incubated at room temperatureTheswelling ratio was measured by comparing the change in theweight of Thio-CS before and after incubating The percent-age of swelling ratio was calculated by the following formula

Swelling ratio () =(119882

119908minus119882

119894)

119882

119894

times 100 (1)

where119882119908is the weight of the swollen Thio-CS and119882

119894is the

initial weight of the Thio-CS

24 Scanning Electron Microscopy The morphologies of thesamples were examined using scanning electron microscope(SEM) (Hitachi Tokyo Japan) As moisturized materialscannot be detected by SEM the samples were lyophilizedPrior to imaging the samples were fixed and dehydratedThe Thio-CS was soaked in a primary fixative of 25glutaraldehyde (Sigma) for 2 hThe samples were dehydratedby replacing the buffer with increasing concentrations ofethanol (from 40 to 100) for 10min each They were thendried at room temperature for 24 h and subjected to SEMat voltages ranging from 5 to 15 kV after the samples weresputter coated in white gold

BioMed Research International 3

25 Delivery of BMP-2 Using Thio-CS The freeze-driedand sponge-shaped Thio-CS was placed in 01mgmL ofBMP-2 (RampDSystemsMinneapolisMNUSA) solutionThemixture of Thio-CS and BMP-2 was gelated within a few ofminute at the room temperature and we designated it asThio-CS-B2 Type I collagen gel (Col-gel) which is widelyused as a drug delivery system was employed as a control forBMP-2 delivery Col-gel was prepared from acid solubilizedtype I collagen stock solution which is extracted from rattail tendon (BD Biosciences San Jose CA USA) Accordingto the recommendation of manufacturer the stock solutionwas adjusted to final 3mgmL of collagen solution containing01mgmL of BMP-2 and it was gelated at 37∘C (Col-gel-B2)For the evaluation of BMP-2 kinetics Thio-CS-B2 or Col-gel-B2 was placed in trans-well and subsequently the trans-well was assembled with 24-well plate filling with alpha-minimum essential medium (120572-MEM Gibco GaithersburgMD USA) Both gels were incubated for the designated timeat 4∘Cwhile shaking gentlyThe amount of BMP-2 inmediumwas measured by using the BMP-2 enzyme-linked Immuno-sorbent assay kit (Invitrogen Carlsbad CA USA)

26 Cell Culture and Proliferation Assay PreosteoblastMC3T3-E1 cells (1 times 104 cellscm2) were cultured in 120572-MEMcontaining the BMP-2 released from scaffolds as describedpreviously 10 fetal bovine serum (FBS) 100UmL ofpenicillin and 100120583gmL of streptomycin (Gibco) in humid-ified air containing 5 carbon dioxide at 37∘C In caseof inducing osteoblast differentiation 50 120583gmL of ascorbicacid and 5mM of 120573-glycerophosphate were added and theculture media was changed every 3 days To investigate thecytotoxicity of the Thio-CS on MC3T3-E1 the XTT assaywas performed by using an EZ-cytox cell viability assay kit(Daeil lab service Co Seoul Republic of Korea) for 1 47 14 and 21 days Briefly 10 120583L of EZ-cytox reagent wasadded to the cell culture dish By the action of mitochondrialdehydrogenases XTT was metabolized to form a formazandye which was spectrophotometrically determined by mea-suring the absorbance at 450 nm using a microplate reader(Bio-Tek Instruments Winooski VT USA) The amount offormazan salt formed corresponds to the number of viablecells contained in each well

27 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophore-sis and Western Blotting The structural integrity of BMP-2 in the Thio-CS was detected by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) In briefBMP-2 containing Thio-CS was degraded by chitosanase(10mUmL) for 1 h at 37∘C The samples were then mixedwith the loading buffer without a reducing agent The SDS-PAGE was performed with 15 separating gel at a constantvoltage mode (100V) Finally the gel was stained with 1Coomassie brilliant blue solution and was destained withan aqueous solution of 10 methanol and 10 acetic acidTo detect the BMP-2 Western blot analysis was performedThe samples were underwent 15 SDS-PAGE and wereelectrotransferred onto polyvinylidene fluoride membranes

Theblotswere blockedwith a buffer containing 005Tween-20 and 5 skimmed milk and reacted sequentially withprimary and secondary antibodies The primary antibodyagainst BMP-2 (Santa Cruz Biotechnology) and horseradishperoxidase-conjugated secondary antibodies (KPL Gaithers-burg MD USA) were used at 1 1000 and 1 2000 dilutionrespectivelyThe antigen-antibody complexes were visualizedusing the enhanced chemiluminescence image analyzer LAS4000 mini (Fuji Film Tokyo Japan)

28 Measurement of Alkaline-Phosphatase Activity andCalcium Mineral Deposition The MC3T3-E1 cells werecultured for 7 14 and 21 days as described above The cellswere lysed and the lysates were then used to measure thealkalinephosphatase (ALP) activity at 1 2 and 3 weeksIn brief the cell homogenates reacted with the ALP assaymixtures containing 01M 2-amino-2-methyl-1-propanol(Sigma St Louis MO USA) 1mM MgCl

2 and 8mM p-

nitrophenyl phosphate disodium After 10min incubation at37∘C the reaction was stopped with 01 N NaOH andthe absorbance of the resulting solution was measuredphotometrically at 405 nm Quantitative double-strandedDNA in the solution wasmeasured using a picogreen dsDNAquantification kit (Molecular Probes Inc Eugene OR USA)as instructed by the manufacturer To measure the level ofcalciummineral deposition alizarin-red staining (AR-S) wasperformed After 3 weeks in culture the cells were fixed with70 ethanol rinsed five times with deionized water treatedfor 10min with 40mM of AR-S solution at pH 42 and thenwashed with 1 timesPBS for 15min with gentle agitation Stainedsamples were photographed followed by a quantitativeeluting procedure using 10 (wv) cetylpyridinium chloridein 10mM sodium phosphate (pH 70) for 15min at room tem-perature The AR-S concentration was determined bycomparing it to an AR-S standard curve with an opticaldensity of 540 nm

29 Animal Preparation The ethics committee of ChonnamNational University approved the animal study for thisresearch The effects of the Thio-CS scaffold on ectopicbone formation induced by BMP-2 were studied in mice(C57BL6 8 weeks of age obtained from Damool ScienceDaejeon Republic of Korea) Before transplantation themicewere anesthetized with a mixture of rumpun (20mgkg)and ketamine hydrochloride (20mgkg) intramuscularlyThearea of transplantation at the dorsum of the mice was shavedand disinfected Either aThio-CS-B2 or a Col-gel-B2 scaffoldwas subcutaneously transplanted into the dorsumof themiceTransplantations of Col-gel and Thio-CS scaffolds withoutBMP-2 were used as controls After transplantation of thescaffolds (119899 = 6 per group) the mice were given access tofood and water Six weeks after injection the animals weresacrificed by intracardiac injection of KCl and the implantswere isolated and fixed in 10 formaldehyde solution forsubsequent analysis

210 Evaluation of Ectopic Bone Formation Isolated sampleswere subjected to microcomputed tomography (microCT


Lyophilized CS

SEMThio-CSLyophilized

(a)

Free

thio

l gro

up at

conj

ugat

ed

Free

thio

l gro

up at

supe

rnat

ant

Disu

lfide

bon

d at

conj

ugat

ed

00

05

10

15

0 1 2 3

4

3

2

1

0

200

160

120

80

40

0

Air-oxidation time (days)

minus1

(120583

mol

g)

(120583

mol

g)

(120583

mol

g)

(b)

00

01

02

03

04

05

3 4 5 6 7 8 9 10 11 12 13pH

minus01Con

tent

of t

hiol

gro

ups (120583

moL

g)

(c)

CSThio-CSThio-CS-B2

0

100

200

300

400

500

Swel

ling

ratio

s (

)

0 60 120 18010 240Time (min)

(d)

Figure 1 Physicochemical properties ofThio-CS (a) Photograph of lyophilized CS andThio-CS (left panel) and its SEMmorphologies (rightpanel 100x) (b) determination of free thiol or disulfide bond according to the air-oxidation time (c) influence of the pH on the formation ofdisulfide bonds and (d) effect ofThio-CS containing BMP-2 on swellingThe amount of the thiol groups in the supernatant or the pellet aftercentrifugation was determined spectrophotomertically using Ellmansrsquos reagent to quantify the free thiol groups as described in Section 2Thevalues indicated are means plusmn SD (119899 = 3)

Skyscan Kontich Belgium) and histological analysis Thebone volume (BV) and the bone mineral density (BMD) ofthe isolated implants were determined by using microCTin the cone-beam acquisition mode The X-ray source wasset at 50 kV and 200120583A with a pixel size at 1709120583m Theexposure time was 12 sec Four hundred fifty projectionswere acquired over an angular range of 180∘ (angular stepof 04∘) The tomographic acquired images were transformedinto sliced volumetric reconstruction using the Nreconprogram (Skyscan) and analyzed using 3D CT analyzersoftware (CTAN Skyscan) The BMD of the isolated sam-ples was calibrated from Hounsfield units (HU) of 025and 075mgcm3 of the hydroxyapatite density phantomFor histological analysis isolated specimens were seriallysliced decalcified in 8 formic acid and embedded inparaffin wax Five micrometer-thick sections were stainedwith hematoxylin and eosin (HampE) for histological assess-ment

211 Statistical Analysis The difference between theThio-CSand the Col-gel in the release kinetics of BMP-2 was statis-tically compared by using the ANOVA test The statisticaldifferences in biocompatibility ALP activity and AR-S wereanalyzed by the Studentrsquos t-test The results are expressedas the mean plusmn standard deviation (SD) from three or moreseparate experiments A value of lowast

119875

lt 005 lowastlowast119875

lt 0005

was considered statistically significant

3 Results and Discussion

31 Fabrication of Thio-CS and Physicochemical PropertiesThe reagent 2-IT has been widely used for the immobilizationof thiol groups to primary amino groups of proteins [37]Since moisturized materials cannot be detected by SEMunmodified CS and Thio-CS were lyophilized As shown inFigure 1(a) SEM images of Thio-CS showed honeycomb-like pores structure It assumed that the pore of Thio-CS


72

26

17

10

43

M

rhBMP-2

ChitosanaseThio-CS

minus

minus minus

minus minus+

+ +

+

+

+

+

(a)

Stacking gel area

Separating gel area

M

minus

minus minus

minus minus+

+ +

+

+

+

+

(b)

Col-gelThio-CS

120

Rel

cum

ulat

ive r

hBM

P-2

rele

ase (

)

Incubation time (days)

100

80

60

40

20

00 7 14 21 28

lowast

lowastlowast

lowastlowast

(c)

Figure 2 Evaluation of the interaction between BMP-2 andThio-CS The interaction was evaluated by SDS-PAGE (a) and Western blotting(b) (c) In vitro cumulative release of BMP-2 from the Thio-CS and the Col-gel The arrow indicates BMP-2 which detected by the BMP-2antibody M is a molecular weight marker Black dotted line is the border line between the stacking and the separating gel The indicatedvalues are means plusmn SD (119899 = 3) lowast

119875


lt 0005 as compared with those of the Col-gel at the same time point

CS onlyThio-CS

15

10

05

000 7 14 21

Cultivation time (days)

Cel

l pro

lifer

atio

n (A

450)

lowastlowast

(minus)CTR

Figure 3 Biocompatibility of Thio-CS by XTT assay The notreatment (ie cell only) group was used as a control The valuesindicated are means plusmn SD (119899 = 3) lowast

119875

lt 005 as compared with thatof the control at the same time point

occurred by increasing molecular weight of CS throughdisulfide bonding each otherThis is consistent with previousreport that pore size of substance has direct relationshipwith the molecular weight of materials [38] As we reportedpreviously the optimum ratio between 2-IT and CS for theformation of disulfide bonds is 01mgmL and 1 (wv) [34]

However this ratio does not take account of the air-oxidationtime which can affect the formation of disulfide bondsbetween CS polymers Therefore in this study we investi-gated the variation of the disulfide bond and the free sulfateat the conjugate or the supernatant according to the air-oxidation time As shown in Figure 1(b) the free thiol contentin the supernatant was decreased with the air-oxidation timeHowever the free thiol and disulfide groups in the conjugatewere increased Moreover the amount of residual aminogroups in CS was decreased in the Trinitrobenzene sulfonateassay (data not shown) These results suggest that the thiolmoiety of 2-IT was transferred to the amino group of CSthat it can be formed by cross-linking between CS polymersand that these phenomena depend on the air-oxidation timeThe cross-linking of the polymeric chains such as inter- orintramolecular disulfide bonds might result in high stabilityfor drug delivery systems Moreover the formation of thedisulfide bond was increased with pH and was saturatedat pH 7 (Figure 1(c)) The swelling property of the scaffoldplays crucial roles in cell growth cell adhesion nutrientperfusion and tissue regeneration [39] Many researchershave attempted to measure the swelling ratio of materialsusing the gravimetric method [40] As the purpose of thisstudy was to evaluate the potential use ofThio-CS for BMP-2delivery the swelling property ofThio-CSwas comparedwiththat of Thio-CS-B2 which contained BMP-2 UnmodifiedCS was used as a negative control The weight of the Thio-CS increased significantly up to 35 times within 10mincompared with its initial weight and this was maintainedcontinuously A similar phenomenon was observed withThio-CS-B2 although the rate was slightly lower than thatof the Thio-CS However the weight of unmodified CS was


Thio-CSThio-CS-B2Col-gel-B2

ALP

activ

ity (u

nitm

g D

NA

)

4

3

2

1

01 2 3

Cultivation time (weeks)

lowast

lowastlowast

(a)

AR-

S (m

M)

06

04

02

00

10

08

12

lowastlowast

lowastlowast

Thio-CS Thio-CS-B2 Col-gel-B2

(b)

Figure 4 Effects of Thio-CS-B2 on in vitro ALP activity (a) and the level of calcium mineral deposition (b) The level of calcium depositionin 3-week culture was evaluated by AR-S The values indicated are means plusmn SD (119899 = 3) lowast

119875


lt 0005 as compared with that ofThio-CS

lower (approximately 65 at 60min) than that of the others(Figure 1(d))These results suggest that BMP-2 does not affectthe swelling property Thio-CS

32 Interaction betweenThio-CS and BMP-2 As the swellingproperty is insufficient to explain the interaction between theThio-CS and BMP-2 the structural integrity was investigatedThio-CS-B2 was incubated with chitosanase and the reactantwas subjected to SDS-PAGE and Western blotting It wasdifficult to assayThio-CS without chitosanase because its vis-cosity was too high to subject to SDS-PAGE (data not shown)The results pointed to several bands in the chitosanase andthe Thio-CS groups (Figure 2(a)) Basically Thio-CS is apolysaccharide polymer but it may contain other impuritiesHowever BMP-2-like molecules were not observed in theThio-CS This was confirmed by Western blotting whichdetected the specific BMP-2 antibody (Figure 2(b)) indicat-ing that Thio-CS does not contain BMP-2-like moleculesThe Western blot analysis showed that the BMP-2 signal wasobserved only in the BMP-2 and the Thio-CS-B2 group Thesize of the control BMP-2 peptide that was detected wasapproximately 25 kDa However the signal at the lane ofThio-CS-B2 was detected in the stacking gel area indicatingthat it was not mobilized In other words if there wasnot any interaction between BMP-2 and Thio-CS BMP-2should be mobilized to the separating gel area These resultssuggested that BMP-2 interacted with Thio-CS although themechanism is unknown and that BMP-2was located inThio-CS It is likely that this interaction between BMP-2 andThio-CS may affect delaying the release of BMP-2 from Thio-CSrather than simply absorbing

33 In Vitro Release of BMP-2 Based on previous physic-ochemical properties of Thio-CS (Figure 1 and [34]) it isexpected to delay the velocity of the release of BMP-2Therefore the release kinetics of BMP-2 from Thio-CS weremeasured for 28 days and comparedwith those of the Col-gelwhich is widely used in the delivery of BMP-2 (Figure 2(c))As expected the release velocity of BMP-2 delayed in theThio-CS group (70within 7 days) comparedwith that of theCol-gel (85 within 7 days) The cumulative BMP-2 releasedfrom the Thio-CS and the Col-gel almost reached a plateauat 21 and 14 days respectively These results suggest that therelease velocity of the Thio-CS group showed a sustainedpattern of release compared with that of the Col-gel

34 Biocompatibility ofThio-CS Biocompatibility ofThio-CSor CS was evaluated with an XTT assay The assay systemwas based on the absorbance of cell lysate and widely usedto measure cell viability or proliferation because it relies onthe reagent binding only to the mitochondria of living cellsFigure 3 shows viability of the osteoblast cells which werecultured with Thio-CS or CS gel for 21 days No treated cellswere used as a control Cell population in the control wascontinuously increasedwith the cultivation timeAndpatternof cell proliferation inThio-CS groupwas similar to that in thecontrol However cell proliferation in CS-treated group wasdecreased after 14 days The decreases seem to be related tothe gelation or degradation property of CS gel because theCS gel was more soluble or fragile in the culture mediumthan Thio-CS gel Although CS gel itself negatively affectedcell viabilityThio-CS gel did not Based on the results in vivostudy progressed withThio-CS


2D

3D

Thio-CS Col-gelCol-gel-B2 Thio-CS-B2

(a)

10

15

20

0

5

lowastlowast

lowastlowast

Bone

vol

ume (

mm3)

Thio-CSCol-gel Col-gel-B2 Thio-CS-B2

(b)

03

02

01

0

lowastlowastlowastlowast

Bone

min

eral

den

sity

(gc

m3)


(c)

Figure 5 Ectopic bone formation with Thio-CS-B2 Ectopic bone formation was visualized by 2D soft X-Ray and 3D microCT analysis (a)Bone volume (BV) (b) and bone mineral density (c) of the isolated ectopic bones were analyzed by Nrecon and CT analyzer software ofSkyscan The arrows indicate newly formed bone The values indicated are means plusmn SD (119899 = 3) lowastlowast

119875

lt 0005 as compared with control

35 Induction of Osteoblast Differentiation with BMP-2 Deliv-ery Using Thio-CS The ALP activity and the level of cal-cium deposition are important considerations for evaluatingosteoblast differentiation ALP activity which is widely usedas a marker for early differentiation of osteoblastic cells andgenerally expressed beforemineralization [41] wasmeasuredafter 1 2 and 3 weeks in theMC3T3-E1 cell culture withThio-CS-B2TheThio-CS and theCol-gel-B2were used as controlsAs shown in Figure 4(a) the ALP activity was significantlyincreased in the cells cultured with the Thio-CS-B2 and theCol-gel-B2 compared with that ofThio-CS onlyThese resultssuggest that BMP-2 in Thio-CS still retains its biologicalability to enhanceALP activity Calciummineral deposition isa marker of late differentiation of osteoblastic cells [42] Thelevel of calcium mineral deposition after 3 weeks in culturewas investigated by AR-S The results showed that calciumdeposition in Thio-CS-B2 treated group was increased 42fold compared with that of Thio-CS (Figure 4(b)) ThusBMP-2 which delivered into the Thio-CS has a stimulatoryeffect on the differentiation of osteoblastic cells and onmatrixmineralization

36 Induction of In Vivo Ectopic Bone Formation by Thio-CS-B2 BMP-2 is well known to produce ectopic bone formationThis study also determined whether Thio-CS-B2 producesectopic bone in vivo in 8-week-old C57BL6 mice Col-gelcontaining BMP-2 was used as a comparison group WhenThio-CS-B2was separately implanted in left and right sides ofthe dorsum in mice newly formed ectopic bones were obser-ved in both implant sites after 6 weeks The new bones inThio-CS-B2 group were big and each side bone was fused toone Total bone volume inThio-CS-B2 group was higher (18fold) than that in the Col-gel-B2 (Figures 5(a) and 5(b)) Theenhanced bone formation in Thio-CS-B2 might result frommore superior property ofThio-CS than Col-gel for bone for-mation In our previous report [34] Col-gel showed the limi-ted swelling property with scant physical change of theporosity suggesting limiting the capacity to absorb BMP-2The present study also showed that Col-gel-B2 has the burstrelease pattern of BMP-2 compared withThio-CS-B2 On theother handThio-CSproduced the sustained release of BMP-2due to interaction between the gel and the protein (Figure 2)These findings represent thatThio-CS has some advantage for


(a) (b) (c)

Figure 6 Representative histological sections with HampE staining after 4-week transplantation (a) Thio-CS only (b) Col-gel-B2 and (c)Thio-CS-B2 Original magnification is 20x The arrows indicate newly formed bone tissue and the triangles indicate residual scaffolds

bone formation compared to Col-gel in that Thio-CS couldabsorb and maintain a greater amount of BMP-2 Howeverthe bonemineral density of the ectopic bone formed byThio-CS-B2 was not significantly different from that by the Col-gel-B2 (Figure 5(c)) The result suggests that bone qualitybetween both groups would not be different To yield moreaccurate observation for bone formation histological analysiswas performed with HampE stain There was less ectopicbone formation in the Thio-CS group (ie without BMP-2Figure 6(a)) However new bone with residual scaffolds wasobserved in Col-gel-B2 and Thio-CS-B2 group (Figures 6(b)and 6(c))

4 Conclusions

In this study we developed Thio-CS scaffold for BMP-2delivery and bone formation The Thio-CS was made bythe modification of CS with 2-IT The 2-IT contributed toin situ gel formation of CS via disulfide bonding betweenthe 2-IT-derived thiol groups of the CS polymers and thisdisulfide bonding was affected by the air-oxidation time andthe pH The degree of swelling was not affected by BMP-2additionMoreover SDS-PAGEandWestern blotting analysisrevealed an interaction between BMP-2 and Thio-CS Thisinteraction may contribute to delaying the release of BMP-2 from Thio-CS Due to the aforementioned properties ofThio-CS the release velocity of BMP-2 from Thio-CS wasslightly delayed compared with that of the Col-gelThe BMP-2 released fromThio-CS induced osteoblastic differentiationof MC3T3-E1 And the activity of ALP and the level ofcalcium mineral deposition were also increased The Thio-CS study did not show any cytotoxicity in vitro in XTTassay studies in MC3T3-E1 osteoblastic cells Based on ourresults in vivo bone formation studies were performed andthe results showed that BMP-2 containing Thio-CS inducedectopic bone formation to a much greater extent than eitherthe BMP-2 containing Col-gel or the control (no BMP-2)Collectively these results suggest that the Thio-CS deliverysystem might be useful for delivering osteogenic proteinBMP-2 as a biocompatible synthetic polymer and that it

may represent a promising application in bone regenerationstrategies

Conflict of Interests

The authors declare no conflict of interests

Acknowledgment

This study was supported by the National Research Founda-tion of Korea (NRF) Grants funded by the Korea government(MSIP) (nos 2011-0010666 2011-0030757 and 2012K001392)

References

[1] T W Bauer and G F Muschler ldquoBone graft materials anoverview of the basic sciencerdquoClinical Orthopaedics and RelatedResearch no 371 pp 10ndash27 2000

[2] A J Celeste J A Iannazzi R C Taylor et al ldquoIdentification oftransforming growth factor 120573 family members present in bone-inductive protein purified from bovine bonerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 87 no 24 pp 9843ndash9847 1990

[3] E A Wang ldquoBone morphogenetic proteins (BMPs) therapeu-tic potential in healing bony defectsrdquo Trends in Biotechnologyvol 11 no 9 pp 379ndash383 1993

[4] E Ozkaynak D C Rueger E A Drier et al ldquoOP-1 cDNAencodes an osteogenic protein in the TGF-120573 familyrdquo EMBOJournal vol 9 no 7 pp 2085ndash2093 1990

[5] T K Sampath J E Coughlin R M Whetstone et al ldquoBovineosteogenic protein is composed of dimers of OP-1 and BMP-2Atwomembers of the transforming growth factor-120573 superfamilyrdquoJournal of Biological Chemistry vol 265 no 22 pp 13198ndash132051990

[6] E Ozkaynak P N J Schnegelsberg D F Jin et al ldquoOsteogenicprotein-2 A new member of the transforming growth factor-120573 superfamily expressed early in embryogenesisrdquo Journal ofBiological Chemistry vol 267 no 35 pp 25220ndash25227 1992

[7] K Elima ldquoOsteoinductive proteinsrdquoAnnals of Medicine vol 25no 4 pp 395ndash402 1993


[8] A H Reddi and N S Cunningham ldquoInitiation and promotionof bone differentiation by bone morphogenetic proteinsrdquo Jour-nal of Bone and Mineral Research vol 8 no 2 pp S499ndashS5021993

[9] I Ono T Ohura M Murata H Yamaguchi Y Ohnuma andY Kuboki ldquoA study on bone induction in hydroxyapatite com-bined with bone morphogenetic proteinrdquo Plastic and Recon-structive Surgery vol 90 no 5 pp 870ndash879 1992

[10] A W Yasko J M Lane E J Fellinger V Rosen J M Wozneyand E A Wang ldquoThe healing of segmental bone defectsinduced by recombinant human bone morphogenetic protein(rhBMP-2) A radiographic histological and biomechanicalstudy in ratsrdquo Journal of Bone and Joint Surgery vol 74 no 5pp 659ndash670 1992

[11] H D Zegzula D C Buck J Brekke J M Wozney and J OHollinger ldquoBone formation with use of rhBMP-2 (recombinanthuman bone morphogenetic protein-2)rdquo Journal of Bone andJoint Surgery vol 79 no 12 pp 1778ndash1790 1997

[12] R Kenley LMarden T Turek L Jin E Ron and JOHollingerldquoOsseous regeneration in the rat calvarium using novel deliverysystems for recombinant human bone morphogenetic protein-2 (rhBMP-2)rdquo Journal of Biomedical Materials Research vol 28no 10 pp 1139ndash1147 1994

[13] Z Schwartz A Somers J TMellonig et al ldquoAddition of humanrecombinant bone morphogenetic protein-2 to inactive com-mercial human demineralized freeze-dried bone allograftmakes an effective composite bone inductive implant materialrdquoJournal of Periodontology vol 69 no 12 pp 1337ndash1345 1998

[14] G Haipeng Z Yinghui L Jianchun et al ldquoStudies on nervecell affinity of chitosan-derivedmaterialsrdquo Journal of BiomedicalMaterials Research A vol 52 no 2 pp 285ndash295 2000

[15] J L Drury and D J Mooney ldquoHydrogels for tissue engineeringscaffold design variables and applicationsrdquo Biomaterials vol 24no 24 pp 4337ndash4351 2003

[16] S N Kulikov I A Tiurin R S Fassakhov and V P VarlamovldquoAntibacterial and antimycotic activity of chitosanmechanismsof action and role of the structurerdquo Zhurnal mikrobiologiiEpidemiologii i Immunobiologii no 5 pp 91ndash97 2009

[17] Y Furusho A Sabarudin L Hakim K Oshita M Oshimaand S Motomizu ldquoAutomated pretreatment system for thespeciation of Cr(III) and Cr(VI) using dual mini-columnspackedwith newly synthesized chitosan resin andME-03 resinrdquoAnalytical Sciences vol 25 no 1 pp 51ndash56 2009

[18] L Richert P Lavalle E Payan et al ldquoLayer by layer buildup ofpolysaccharide films physical chemistry and cellular adhesionaspectsrdquo Langmuir vol 20 no 2 pp 448ndash458 2004

[19] L Illum ldquoChitosan and its use as a pharmaceutical excipientrdquoPharmaceutical Research vol 15 no 9 pp 1326ndash1331 1998

[20] A Lahiji A Sohrabi D S Hungerford and C G Fron-doza ldquoChitosan supports the expression of extracellular matrixproteins in human osteoblasts and chondrocytesrdquo Journal ofBiomedical Materials Research A vol 51 no 4 pp 586ndash5952000

[21] A Montembault C Viton and A Domard ldquoPhysico-chemicalstudies of the gelation of chitosan in a hydroalcoholic mediumrdquoBiomaterials vol 26 no 8 pp 933ndash943 2005

[22] F Cui F Qian Z Zhao L Yin C Tang and C Yin ldquoPrepa-ration characterization and oral delivery of insulin loaded car-boxylated chitosan grafted poly(methylmethacrylate) nanopar-ticlesrdquo Biomacromolecules vol 10 no 5 pp 1253ndash1258 2009

[23] Y-C Lin F-J Tan K G Marra S-S Jan and D-C Liu ldquoSyn-thesis and characterization of collagenhyaluronanchitosan

composite sponges for potential biomedical applicationsrdquo ActaBiomaterialia vol 5 no 7 pp 2591ndash2600 2009

[24] A Bernkop-Schnurch M Hornof and D Guggi ldquoThiolatedchitosansrdquo European Journal of Pharmaceutics and Biopharma-ceutics vol 57 no 1 pp 9ndash17 2004

[25] M-S Kim Y-J Choi I Noh and G Tae ldquoSynthesis and char-acterization of in situ chitosan-based hydrogel via grafting ofcarboxyethyl acrylaterdquo Journal of Biomedical Materials ResearchA vol 83 no 3 pp 674ndash682 2007

[26] A Bernkop-Schnurch M Hornof and T Zoidl ldquoThiolatedpolymersmdashthiomers synthesis and in vitro evaluation ofchitosan-2-iminothiolane conjugatesrdquo International Journal ofPharmaceutics vol 260 no 2 pp 229ndash237 2003

[27] C E Kast C Valenta M Leopold and A Bernkop-SchnurchldquoDesign and in vitro evaluation of a novel bioadhesive vaginaldrug delivery system for clotrimazolerdquo Journal of ControlledRelease vol 81 no 3 pp 347ndash354 2002

[28] D Guggi AH Krauland andA Bernkop-Schnurch ldquoSystemicpeptide delivery via the stomach in vivo evaluation of anoral dosage form for salmon calcitoninrdquo Journal of ControlledRelease vol 92 no 1-2 pp 125ndash135 2003

[29] L Yin J Ding C He L Cui C Tang and C Yin ldquoDrug per-meability and mucoadhesion properties of thiolated trimethylchitosan nanoparticles in oral insulin deliveryrdquo Biomaterialsvol 30 no 29 pp 5691ndash5700 2009

[30] J Hombach H Hoyer and A Bernkop-Schnurch ldquoThiolatedchitosans development and in vitro evaluation of an oraltobramycin sulphate delivery systemrdquo European Journal ofPharmaceutical Sciences vol 33 no 1 pp 1ndash8 2008

[31] L M Y Yu K Kazazian and M S Shoichet ldquoPeptide surfacemodification ofmethacrylamide chitosan for neural tissue engi-neering applicationsrdquo Journal of Biomedical Materials ResearchA vol 82 no 1 pp 243ndash255 2007

[32] M Werle B Loretz D Entstrasser and F Foger ldquoDesign andevaluation of a chitosan-aprotinin conjugate for the peroraldelivery of therapeutic peptides and proteins susceptible toenzymatic degradationrdquo Journal of Drug Targeting vol 15 no5 pp 327ndash333 2007

[33] D-W Lee S A Shirley R F Lockey and S SMohapatra ldquoThio-lated chitosan nanoparticles enhance anti-inflammatory effectsof intranasally delivered theophyllinerdquo Respiratory Researchvol 7 article 112 2006

[34] I-H Bae W G Jang H-P Lim et al ldquoMorphological propertyand in vitro enzymatic degradation of modified chitosan as ascaffoldrdquo Macromolecular Research vol 19 no 12 pp 1250ndash1256 2011

[35] A F S A Habeeb ldquoA sensitive method for localization of disul-fide containing peptides in column effluentsrdquo Analytical Bio-chemistry vol 56 no 1 pp 60ndash65 1973

[36] R B Sashidhar A K Capoor and D Ramana ldquoQuantitationof 120576-amino group using amino acids as reference standards bytrinitrobenzene sulfonic acid A simple spectrophotometricmethod for the estimation of hapten to carrier protein ratiordquoJournal of Immunological Methods vol 167 no 1-2 pp 121ndash1271994

[37] H J Schramm and T Dulffer ldquoSynthesis and application ofcleavable and hydrophilic crosslinking reagentsrdquo Advances inExperimental Medicine and Biology vol 86 pp 197ndash206 1977

[38] H Nazar D G Fatouros S M Van Der Merwe et al ldquoTher-mosensitive hydrogels for nasal drug delivery the formulationand characterisation of systems based on N-trimethyl chitosan


chloriderdquo European Journal of Pharmaceutics and Biopharma-ceutics vol 77 no 2 pp 225ndash232 2011

[39] K Kataoka Y Suzuki M Kitada et al ldquoAlginate enhanceselongation of early regenerating axons in spinal cord of youngratsrdquo Tissue Engineering vol 10 no 3-4 pp 493ndash504 2004

[40] P Prang R Muller A Eljaouhari et al ldquoThe promotion oforiented axonal regrowth in the injured spinal cord by alginate-based anisotropic capillary hydrogelsrdquo Biomaterials vol 27 no19 pp 3560ndash3569 2006

[41] B G Keselowsky L Wang Z Schwartz A J Garcia andB D Boyan ldquoIntegrin 1205725 controls osteoblastic proliferationand differentiation responses to titanium substrates presentingdifferent roughness characteristics in a roughness independentmannerrdquo Journal of BiomedicalMaterials Research A vol 80 no3 pp 700ndash710 2007

[42] J J J P van den Beucken X F Walboomers O C Boerman etal ldquoFunctionalization ofmultilayeredDNA-coatings with bonemorphogenetic protein 2rdquo Journal of Controlled Release vol 113no 1 pp 63ndash72 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


Natural biomaterials are widely used for scaffold fabricationin tissue engineering because they facilitate cell attachmentand maintenance of the differentiation function Chitosan(CS) obtained by alkaline deacetylation of chitin is one ofthe most abundant polysaccharides in nature It has receivedconsiderable attention in a variety of areas such as phar-maceutics [14] tissue engineering [15] antimicrobial agents[16] and chromatography [17] because of its propertieswhich include enzymatic biodegradability nontoxicity andbiocompatibility even when used in human and animalmodels [18ndash20] However CS suffers from limited solubilityat physiological pH and causes presystemic metabolism ofdrugs in the presence of proteolytic enzymes [21] Theseinherent drawbacks of CS have been overcome by formingderivatives such as carboxylated CS [22] adding variousconjugates [23] thiolated CS [24] or acylated CS [25] Amongthese various CS derivates thiomer technology has a range ofadvantages for drug delivery such as sustained drug release[26] and high stability [24] The usefulness of thiolatedchitosan (Thio-CS) as a scaffold for controlled drug releasehas been demonstrated by means of model drugs suchas clotrimazole [27] salmon calcitonin [28] insulin [29]and tobramycin [30] However most of the research hasfocused on systemic drug delivery such as neural tissue [31]peroral peptide delivery [32] and nasal administration [33]Despite the advantages ofThio-CS for tissue engineering thepotential application of this material for bone tissue has notbeen investigated The aim of this study was to evaluate thephysicochemical properties of Thio-CS for BMP-2 deliveryand bone formation in vitro and in vivo

2 Materials and Methods

21 Fabrication of Thio-CS To obtain a 1 (wv) solution500mg of CS (average molecular mass 400 kDa FlukaGmbH Buchs Switzerland) was dissolved in 50mL of 1acetic acid by stirring the mixture for 1 h Trautrsquos reagent (2-iminothiolane-HCl 2-IT) was used for the immobilizationof thiol groups to primary amino groups of proteins and themodification of CS We have previously reported the optimalconditions for fabricating Thio-CS [34] In brief the pH of amixture containing a 1 solution of CS and 01mgmL of 2-IT was adjusted ranging from 4 to 12Themixtures were thenincubated for 30min at room temperature To investigate thetime effect of the air oxidation on disulfide bond formationthe samples were incubated for 3-day intervals at pH 7 understirring To remove unreacted agent the resulting mixturewas dialyzed with several exchanges of the dialyzing solutionTo prevent more oxidation of samples 5mM or 04mM ofHCl solution was used as a dialyzing solution depending ondialysis step The samples were freeze-dried at minus80∘C and001mbar (Christ Beta 1ndash8K Germany) and stored at 4∘Cuntil further use

22 Determination of the Thiol Group and Disulfide BondEllmanrsquos reagent (3mgof 551015840-dithiobis (2-nitrobenzoic acid))(Sigma St Louis MO USA) was used to quantify theamount of thiol groups on the modified CS as described

previously [34] Briefly 5mg of the freeze-dried samples wasdissolved in 25mL of demineralized water Then 250120583Lof the samples 250120583L of 5M phosphate-buffered saline(PBS pH 80) and 500 120583L of Ellmanrsquos reagent dissolvedin 10mL of 05M PBS (pH 80) were reacted in the sametube The reaction was allowed to proceed for 2 h at roomtemperature After removal of the precipitated polymerby centrifugation (24000timesg 5min) 300 120583L of the super-natant was transferred to a microtitration plate and theabsorbance was immediately measured at 450 nm (Bio-TekInstruments Winooski VT USA) The amount of thiolmoieties was calculated from a standard curve of absorbanceobtained from solutions with increasing concentrations of L-cysteine hydrochloride hydrate (Sigma-Aldrich SteinheimGermany) Disulfide bond content within precipitate orreacting solutionwas determined after reductionwithNaBH

4

and addition of Ellmanrsquos reagent as described by Habeeb[35] The degree of cross-linking of such materials is usuallydetermined by a chemical analysis method using 246-trinitrobenzenesulfonic acid (TNBS) labeling of residualamine groups [36] To 03mL of sample 03mL of NaHCO

3

(4) and 03mL of TNBS (01) were added The solutionwas allowed to react for 2 h at 40∘C and then 03mL ofsodium dodecyl sulfate (10) and finally 017mL HCl (1M)were addedThe absorbance of the resulting solutionwas readphotometrically at 335 nm against a blank but with 03mL ofH2O instead of the sample

23 Evaluation of the Swelling Behavior To understand theeffect of the molecular transport of liquids into Thio-CS thewater-absorbing (ie swelling) capacity was determined bygravimetric methods Tomeasure the weight ofThio-CS afterswelling 01 g of Thio-CS was placed in trans-well (CorningInc Corning NY USA)The trans-well was then placed intoa 24-well culture dish containing a physiological solution of1mL of PBS (pH 70) and incubated at room temperatureTheswelling ratio was measured by comparing the change in theweight of Thio-CS before and after incubating The percent-age of swelling ratio was calculated by the following formula

Swelling ratio () =(119882

119908minus119882

119894)

119882

119894

times 100 (1)

where119882119908is the weight of the swollen Thio-CS and119882

119894is the

initial weight of the Thio-CS

24 Scanning Electron Microscopy The morphologies of thesamples were examined using scanning electron microscope(SEM) (Hitachi Tokyo Japan) As moisturized materialscannot be detected by SEM the samples were lyophilizedPrior to imaging the samples were fixed and dehydratedThe Thio-CS was soaked in a primary fixative of 25glutaraldehyde (Sigma) for 2 hThe samples were dehydratedby replacing the buffer with increasing concentrations ofethanol (from 40 to 100) for 10min each They were thendried at room temperature for 24 h and subjected to SEMat voltages ranging from 5 to 15 kV after the samples weresputter coated in white gold







2 and 8mM p-





Lyophilized CS


(a)

Free

thio

l gro

up at

conj

ugat

ed

Free

thio

l gro

up at

supe

rnat

ant

Disu

lfide

bon

d at

conj

ugat

ed

00

05

10

15

0 1 2 3

4

3

2

1

0

200

160

120

80

40

0


minus1

(120583

mol

g)

(120583

mol

g)

(120583

mol

g)

(b)

00

01

02

03

04

05

3 4 5 6 7 8 9 10 11 12 13pH

minus01Con

tent

of t

hiol

gro

ups (120583

moL

g)

(c)

CSThio-CSThio-CS-B2

0

100

200

300

400

500

Swel

ling

ratio

s (

)

0 60 120 18010 240Time (min)

(d)




119875


lt 0005





72

26

17

10

43

M

rhBMP-2

ChitosanaseThio-CS

minus

minus minus

minus minus+

+ +

+

+

+

+

(a)

Stacking gel area

Separating gel area

M

minus

minus minus

minus minus+

+ +

+

+

+

+

(b)

Col-gelThio-CS

120

Rel

cum

ulat

ive r

hBM

P-2

rele

ase (

)


100

80

60

40

20

00 7 14 21 28

lowast

lowastlowast

lowastlowast

(c)


119875



CS onlyThio-CS

15

10

05

000 7 14 21


Cel

l pro

lifer

atio

n (A

450)

lowastlowast

(minus)CTR


119875






ALP

activ

ity (u

nitm

g D

NA

)

4

3

2

1

01 2 3


lowast

lowastlowast

(a)

AR-

S (m

M)

06

04

02

00

10

08

12

lowastlowast

lowastlowast


(b)


119875








2D

3D


(a)

10

15

20

0

5

lowastlowast

lowastlowast

Bone

vol

ume (

mm3)


(b)

03

02

01

0


Bone

min

eral

den

sity

(gc

m3)


(c)


119875





(a) (b) (c)



4 Conclusions





Acknowledgment


References






















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials









2 and 8mM p-





Lyophilized CS


(a)

Free

thio

l gro

up at

conj

ugat

ed

Free

thio

l gro

up at

supe

rnat

ant

Disu

lfide

bon

d at

conj

ugat

ed

00

05

10

15

0 1 2 3

4

3

2

1

0

200

160

120

80

40

0


minus1

(120583

mol

g)

(120583

mol

g)

(120583

mol

g)

(b)

00

01

02

03

04

05

3 4 5 6 7 8 9 10 11 12 13pH

minus01Con

tent

of t

hiol

gro

ups (120583

moL

g)

(c)

CSThio-CSThio-CS-B2

0

100

200

300

400

500

Swel

ling

ratio

s (

)

0 60 120 18010 240Time (min)

(d)




119875


lt 0005





72

26

17

10

43

M

rhBMP-2

ChitosanaseThio-CS

minus

minus minus

minus minus+

+ +

+

+

+

+

(a)

Stacking gel area

Separating gel area

M

minus

minus minus

minus minus+

+ +

+

+

+

+

(b)

Col-gelThio-CS

120

Rel

cum

ulat

ive r

hBM

P-2

rele

ase (

)


100

80

60

40

20

00 7 14 21 28

lowast

lowastlowast

lowastlowast

(c)


119875



CS onlyThio-CS

15

10

05

000 7 14 21


Cel

l pro

lifer

atio

n (A

450)

lowastlowast

(minus)CTR


119875






ALP

activ

ity (u

nitm

g D

NA

)

4

3

2

1

01 2 3


lowast

lowastlowast

(a)

AR-

S (m

M)

06

04

02

00

10

08

12

lowastlowast

lowastlowast


(b)


119875








2D

3D


(a)

10

15

20

0

5

lowastlowast

lowastlowast

Bone

vol

ume (

mm3)


(b)

03

02

01

0


Bone

min

eral

den

sity

(gc

m3)


(c)


119875





(a) (b) (c)



4 Conclusions





Acknowledgment


References






















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




Lyophilized CS


(a)

Free

thio

l gro

up at

conj

ugat

ed

Free

thio

l gro

up at

supe

rnat

ant

Disu

lfide

bon

d at

conj

ugat

ed

00

05

10

15

0 1 2 3

4

3

2

1

0

200

160

120

80

40

0


minus1

(120583

mol

g)

(120583

mol

g)

(120583

mol

g)

(b)

00

01

02

03

04

05

3 4 5 6 7 8 9 10 11 12 13pH

minus01Con

tent

of t

hiol

gro

ups (120583

moL

g)

(c)

CSThio-CSThio-CS-B2

0

100

200

300

400

500

Swel

ling

ratio

s (

)

0 60 120 18010 240Time (min)

(d)




119875


lt 0005





72

26

17

10

43

M

rhBMP-2

ChitosanaseThio-CS

minus

minus minus

minus minus+

+ +

+

+

+

+

(a)

Stacking gel area

Separating gel area

M

minus

minus minus

minus minus+

+ +

+

+

+

+

(b)

Col-gelThio-CS

120

Rel

cum

ulat

ive r

hBM

P-2

rele

ase (

)


100

80

60

40

20

00 7 14 21 28

lowast

lowastlowast

lowastlowast

(c)


119875



CS onlyThio-CS

15

10

05

000 7 14 21


Cel

l pro

lifer

atio

n (A

450)

lowastlowast

(minus)CTR


119875






ALP

activ

ity (u

nitm

g D

NA

)

4

3

2

1

01 2 3


lowast

lowastlowast

(a)

AR-

S (m

M)

06

04

02

00

10

08

12

lowastlowast

lowastlowast


(b)


119875








2D

3D


(a)

10

15

20

0

5

lowastlowast

lowastlowast

Bone

vol

ume (

mm3)


(b)

03

02

01

0


Bone

min

eral

den

sity

(gc

m3)


(c)


119875





(a) (b) (c)



4 Conclusions





Acknowledgment


References






















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




72

26

17

10

43

M

rhBMP-2

ChitosanaseThio-CS

minus

minus minus

minus minus+

+ +

+

+

+

+

(a)

Stacking gel area

Separating gel area

M

minus

minus minus

minus minus+

+ +

+

+

+

+

(b)

Col-gelThio-CS

120

Rel

cum

ulat

ive r

hBM

P-2

rele

ase (

)


100

80

60

40

20

00 7 14 21 28

lowast

lowastlowast

lowastlowast

(c)


119875



CS onlyThio-CS

15

10

05

000 7 14 21


Cel

l pro

lifer

atio

n (A

450)

lowastlowast

(minus)CTR


119875






ALP

activ

ity (u

nitm

g D

NA

)

4

3

2

1

01 2 3


lowast

lowastlowast

(a)

AR-

S (m

M)

06

04

02

00

10

08

12

lowastlowast

lowastlowast


(b)


119875








2D

3D


(a)

10

15

20

0

5

lowastlowast

lowastlowast

Bone

vol

ume (

mm3)


(b)

03

02

01

0


Bone

min

eral

den

sity

(gc

m3)


(c)


119875





(a) (b) (c)



4 Conclusions





Acknowledgment


References






















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





ALP

activ

ity (u

nitm

g D

NA

)

4

3

2

1

01 2 3


lowast

lowastlowast

(a)

AR-

S (m

M)

06

04

02

00

10

08

12

lowastlowast

lowastlowast


(b)


119875








2D

3D


(a)

10

15

20

0

5

lowastlowast

lowastlowast

Bone

vol

ume (

mm3)


(b)

03

02

01

0


Bone

min

eral

den

sity

(gc

m3)


(c)


119875





(a) (b) (c)



4 Conclusions





Acknowledgment


References






















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




2D

3D


(a)

10

15

20

0

5

lowastlowast

lowastlowast

Bone

vol

ume (

mm3)


(b)

03

02

01

0


Bone

min

eral

den

sity

(gc

m3)


(c)


119875





(a) (b) (c)



4 Conclusions





Acknowledgment


References






















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a) (b) (c)



4 Conclusions





Acknowledgment


References






















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

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MaterialsJournal of


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research article evaluation of a thiolated chitosan...

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