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Preparation and characterization of biodegradable chitosan_

Biomaterials25(2004)779–785

Preparation and characterization of biodegradable chitosan/

hydroxyapatite nanocomposite rods via in situ hybridization:

a potential material as internal?xation of bone fracture

Qiaoling Hu*,Baoqiang Li,Mang Wang,Jiacong Shen

Institute of Polymer Composites,Zhejiang University,Hangzhou310027,People’s Republic of China

Received27January2003;accepted14July2003

Abstract

A transparent and slight yellow chitosan(CS)/hydroxyapatite(HA)nanocomposite with high performed,potential application as internal?xation of bone fracture was prepared bya novel and simple in situ hy bridization.The method solves the problem of the nano-sized particle aggregation in polymer matrix.XRD,TEM and SEM were used to determine component and morphology of the composite.Results indicated that nano-HA particles were dispersed well in CS matrix,which can also be proved bythe transparent appearance of composite rod,and that the structure of composite is assembled by CS molecule in the order of layer-by-layer.The mechanical properties of the composite were evaluated by using bending strength and modulus,and compared with some other bone replacement materials such as PMMA and bone cement.The initial mechanical properties of bending strength and modulus of composite are86MPa and3.4GPa,respectively,which is double or triple times stronger than that of PMMA and bone cement.It was found that the bending strength and modulus of CS/HA with ratio of100/5(wt/wt)is slightlyhigher than that of pure CS rod.The addition of HA can also reduce the ratio of water absorption of composite,which postponed the retention of mechanical properties of CS/HA composite under moisture condition.The phenomenon can be predicted with the?t exponential function according the data measured.

Keywords:In situ hybridization;Chitosan;Hydroxyapatite;Nanocomposite

1.Introduction

Metallic implants are widelyused in manytreatments and are fairlysuccessful.However,theydo not provide the optimum therapydue to their shortcomings such as stress shielding during post-healing,chronic in?amma-tion caused bycorrosion,and fatigue and loosening of the implant.As a result,a second surgeryis often required to remove the metallic implant after healing, and it increases the risk of the operation and the expense to the patient.Degradable polymeric implants eliminate the need for a second operation and can prevent some of the problems associated with stress shielding during post-healing,and can also be used simultaneouslyto deliver therapeutic drugs to treat infections or growth factors to accelerate new bone growth.

A desirable material for bioabsorbable implant should provide enough initial mechanical strength,induce or promote new bone formation byosteogenic cell at a required site and posses some bioactive such as osteoinductive.Chitosan(CS),was suggested as an alternative polymer for use in orthopedic applications to provide temporarymechanical support the regeneration of bone cell ingrowth due to its good biocompatible[1], non-toxic,biodegradable,and inherent wound healing characteristics[2,3].CS had been used in various form such as zero dimension microsphere[4],two-dimension membrane[5],three-dimension pin or rod[6].Hydro-xyapatite(HA),Ca10(PO4)6(OH)2,was used in various biomedical?elds such as dental material,bone sub-stitute and hard tissue paste.HA can accelerate the formation of bone-like apatite on the surface of implant [7].Recently,it has been reported that HA can be osteoinductive because theycan induce bone formation when implanted in dogs[8].

CS can be utilized in combination with other bioactive inorganic ceramics,especiallyHA to further enhance tissue regenerative ef?cacyand osteoconductivity[9,10]. Incorporation of HA with CS,the mineral component

*Corresponding author.

E-mail address:huql@https://www.wendangku.net/doc/3118561229.html,(Q.Hu).

of bone,could improve the bioactivityand the bone-bonding abilityof the CS/HA composites[11].Bioma-terial scientists had paid their attention to investigate CS/HA composites with different HA content ranging from50%(wt)to90%(wt),such as quick hardening past for bone repair[12,13],porous CS/HA scaffold for tissue engineering[14]or control release of drug[15].It

was found that the more fragile the composite is,the higher HA content in composite.So the composite still possess characteristics of ceramics such as brittleness and stiffness.CS just plays in a role of adhesive to dissolve the problem of dif?cultyof HA speci?c shape and migration of HA powder when implanted.The conventional method to fabricate CS/HA composite is that HA powder was mixed with CS dissolved in2% acetic acid solution,then the mixture was impressed into mold,?nallywas freezing-dried to make sponge composite.The?nal material is heterogeneous,opaque. We had reported that CS rod with high strength byin situ precipitation method[6].The composite rod of CS/ HA,with opaque and the weak interfacial bonding between HA?ller and CS Matrix resulting in a slightly lower mechanical properties when compared with pure CS,had been reported[16].The aim of this work was to prepare homogenous,transparent and high-strength CS/HA nanocomposite,in which the matrix CS is precipitated and the?ller of HA synthesized simulta-neouslybyin situ hy bridization.

2.Experiments

2.1.Materials

Biomedical grade CS(viscosity-average molecular weight 5.63?105)was supplied bythe Qingdao Huizhong Bioengineering Co.,Ltd(Qingdao,China) with91%degree of the deacetylation.HA A was made in our lab in the presence of2%acetic acid solution. Biochemical grade HA B powder with an mean particle diameter of26m m and analytical grade calcium nitrate tetrahydrate(Ca(NO3)2á4H2O)were purchased from the Chinese PharmacyG roup Shanghai Reagent Company (Shanghai,China).Potassium di-hydrogen Phosphate (KH2PO4)and sodium hydroxide(NaOH)were sup-plied byHangzhou Xiaoshan Chemical reagent Cor-poration(Hangzhou,China).

2.2.Preparation of CS/HA nanocomposite by in situ hybridization

Ca(NO3)2á4H2O and KH2PO4were added into 100ml acetic acid aqueous solution with concentration of2%(v/v).The ratio of CS/Ca(NO3)2á4H2O/KH2PO4 was listed in Table1.The solution was stirred for30min until the calcium salt and phosphate salt were entirely dissolved.The5g of CS powder were added under vigorous agitation and the mixture was stirred for4h at room temperature to obtain a homogeneous polymer solution.The resulting solution was held for6h to remove the air bubbles trapped in viscous liquid. Cylindrical CS/HA rod(4.5mm diameter,80mm length)was prepared as follows.CS solution(appro-ximate10ml)was cast on the internal surface of cylindrical glass mold and the redundant CS solution was poured out,then the mold was soaked in a solution of5%(wt/v)NaOH aqueous solution for2h to precipitate a CS membrane(Fig.1a).The mold?lled with the resulting solution was allowed to soak in5% (wt/v)NaOH aqueous solution for8h to form a CS/HA gel rod,which contains95–97%(v)water.The gel rod was washed with distilled water until the pH of washed water is about7and removed the CS membrane,then air-dried in oven at60 C for24h.A transparent,slight yellow CS/HA rod was prepared after experiencing a large extent of shrinkage in the process of drying(Figs. 1b and5a).

2.3.Preparation of CS/HA B composites by blending method

Cylindrical CS/HA B rod(4.5mm diameter,80mm length)was prepared byblending method as described previously[16].Brie?y,CS solution of5%(wt/v)was prepared bydissolving5g of CS in100ml acetic acid of 2%(v/v).HA B powder with different CS/HA B ratio, listed in Table1,was added to the prepared solution to make a CS/HA B mixture and stirred until HA B powder was thoroughlydispersed in the mixture.The following procedures are the same as that was mentioned in Section2.2.At last,an opaque,white CS/HA B rod was formed(Fig.5b).

2.4.Testing of mechanical properties

The rods were retreated in oven at60 C for2h to remove the moisture remaining in the composite.Three-point bending tests were performed on Shenzhen Reger Company’s the universal materials testing machine,the span length was40mm and loading rate was2mm/min. The ultimate bending strength(s b)and bending modulus(E b)of the CS/HA were calculated according to Eqs.(1)and(2).And the fractures of sample were Table1

The ratio of CS/HA in composites

CS/HA

(wt/wt)

CS(g)HA B(g)Ca(NO3)2á

4H2O(g)

KH2PO4(g)

100/550.250.6250.215

100/1050.50 1.250.43

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recorded bydigital camera(Nikon digital camera E900)

s b?8F max L

p d3

;e1T

E b?4L3

3p d4

D F

D l

;e2T

where F max is the maximum load(N),L is support span (mm),d is diameter of sample(mm),D F=D l is gradient of linear portion of load-displacement curve(N/mm).

2.5.XRD analysis

The crystal structure of HA distributed in CS matrix was determined with powder XRD(Rigaku D/Max—2550PC)using Cu K a radiation generated at40kV and 50mA.The sample was scanned from5 to90 in2y: 2.6.TEM and SEM observations

TEM was used to evaluate the dispersion and particle size of HA in composite.TEM were carried out on the JEM-1200EX(JEOL,Japan).SEM was performed with JSM-5510LV(JEOL),to examine the fracture surfaces after bending test,and the samples were tested directly.

2.7.Water absorption

Water absorption of CS/HA composite with different HA content had been studied to evaluated the effect of HA content on the size stabilityof material.The ratio of water absorption(W a)at time t was calculated using the following equation:

W a%?WtàW0

?100%;e3T

where W t and W0are the weights of sample at time t and the drystate at23 C,respectively.3.Results and discussion

3.1.The mechanism of preparation CS/HA composite by in situ hybridization

The presence of amino group enables CS to exist in a soluble or solid form depending on the pH value of environments.The free amino group of CS(CS–NH2) was protonated to CS–NH3+,when CS was dissolved in an acetic acid(HAc)solution,which was shown as follows.

CS2NH2tHAc"CS2NHt

tAcàepH?4:2T:e4TThe precipitation pH of CS is generallyclose to6.0, and Viala et al.[17]established that the presence of calcium and phosphate ions in the solution allows the soluble form of CS to exist when below pH6.7,while pH of HA formation was more than10.So we can safely draw the conclusion that calcium and phosphate ions existed in form of ions in the resulting solution,and that the CS was precipitated in the presence of HA formation simultaneouslywhen the pH of the environment was more than10

CS2NHt

tOHà-CS2NH2ekTtH2OepH>6:7T;

e5T

10Ca2tt6H2POà

t14OHà

-Ca10ePO4T6eOHT2ekTt12H2OepH>10T:e6TDuring the process of gel-rod formation,a cylindrical CS membrane was used to obstruct the resulting solution and5%NaOH aqueous solution(Fig.2).In case the size of ions are small enough to permeate the CS membrane,ions such as OHà,H+,Acàand so on,will diffuse through the CS membrane owing to the concentration gradient of ions until their concentration became uniform.Ca2+and H2PO4àwere dif?cult to emigrate through membrane due to the presence of the viscous liquid CS resulting solution,while CS molecule cannot emigrate at all.The velocityof migration of OHàand H+were faster than that of the other ions.When OHàare migrating into result solution through CS membrane and encountered the CS–NH3+,Ca2+and

Fig.1.Photos of cylindrical CS/HA(100/5,wt/wt)rod wet gel rod(a),and transparent and slight yellow dry rod(b).

Q.Hu et al./Biomaterials25(2004)779–785781

H 2PO 4à,the CS was precipitated and calcium phosphate simultaneouslyto form the ?rst lay er of gel rod,then formed second layer,and so on.

The diffused speed of OH àtends to reduce due to the resistance of CS layers formed during process of diffusion of OH à.Considering the increase of thickness of gel rod layer,it is dif?cult for OH àto diffuse into the internal of CS resulting solution,which results in the formation of the layer structure.

Bytraditional method of preparation CS/HA com-posite,the process of acid–base neutralization reaction was so rapid that the arrangement of the precipitated CS molecule was irregular.While in the in situ hybridiza-tion,the process of acid–base neutralization reaction was controlled bythe rate of immigration of OH àthrough the CS membrane,which depended on the concentration gradient of OH àand the thickness of precipitated CS/HA gel.So the architecture of the precipitated CS by in situ hybridization is layer-by-layer which was showed in (Fig.3).

While due to the impermeabilityof the HA and CS,anyions trapped during the formation of the CS/HA materials can be easilywashed out,then dried in oven at 60 C.The volumetric shrinkage of gel rod is up to approximate 95–97%(v).Because the process of drying starts from outside to inside of gel rod,the ?rst layer of CS/HA composite shrinkages prior to the other layers.The shrinkage force generated byoutside applied on the inside of gel rod (Fig.4).Therefore,the CS/HA rod can be self-reinforced bybehavior of shrinkage in the process of drying.

3.2.Appearance of CS/HA composite prepared by in situ hybridization

The appearance of CS/HA composite prepared by using different method was shown in Fig.5.The

→Fig.2.The schematic representation of mold of preparation CS/HA rod by in situ hybridization,5%sodium hydroxyl aqueous solution (A),the mold (B),cylindrical CS membrane (C),resulting CS solution containing Ca 2+and H 2PO 4à

(D).

Fig.3.The schematic representation of the mechanism of formation of CS/HA composite byin situ hy

bridization.

shrinkage force shrinkage force

Fig.4.The schematic representation of the effect of shrinkage self-reinforced.

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appearance of CS/HA prepared byin situ hy bridization was transparent and slight yellow,and it is same as that of CS rod.However,the appearance of CS/HA B prepared byblending method was entirelyopaque and white,independent of the ratio of CS and HA.The fact of transparent appearance of CS/HA composite byin situ hybridization indicates the uniformity of HA dispersed in CS matrix,and that the size of HA particles synthesized simultaneously in the presence of CS were smaller than that of wavelength of visible light.In situ hybridization may offer the potential for much better control of particle size,shape and distribution than be achieved bysimple blending method.

3.3.Determination component by XRD

XRD patterns of formed composite and pure HA made in our lab were shown in Fig.6.The wide peak appeared approximate20 was assigned to CS in line(a) and(b).The sharp diffraction characteristic peaks appeared at around25.8 ,which refers to2y?25:8 , and31.86 in the CS/HA composite prepared bythe in situ hybrid method(a)correspond to the peaks of HA powder at25.8 and31.86 made in lab(c).These peaks are similar to that of CS/HA composite prepared by blending method(b).Therefore,the calcium phosphate existing in the formed composite is exactlyHA.

3.4.Morphology of the composite

TEM micrographs of CS/HA=100/5(wt/wt)compo-site were shown in Fig.7a.As can be seen in Fig.7a, nano-sized HA particles(approximate100nm length, 20–50nm width)were dispersed well in CS matrix homogeneously.After bending test photographs of cross-section of sample were observed byusing digital camera(Fig.7b)and SEM.The arrow A in Fig.7b illustrates the layer-by-layer structure of composite prepared byin situ hy bridization in view of the macrostructure.This fact was further identi?ed bythe microstructure of composite shown in Figs.7c and d.we can get the idea that the precipitated CS molecules assemble in the order of layer-by-layer from these facts.This is also an evidence of the mechanism of preparation CS/HA nanocomposite via in situ hybridization.

3.5.Mechanical properties of CS/HA composite

The bending strength and modulus of CS/HA composite prepared byin situ hy bridization and that of some other materials were listed in the Table2. Bending strength and modulus of CS/HA(100/5,wt/wt) composite prepared byin situ hy bridization are86MPa, 3.4GPa,respectively.All of these material properties are2–3times stronger than that of PMMA and bone cement[13],indicating that such a novel bioabsorbable CS/HA composite could be used as internal?xation of bone fracture in the view of application.Meanwhile,the bending strength and modulus of CS/HA is only34% and17%when compared with cortical bone pin made from human femora[18].As we known,incorporation HA into CS matrix via blending method would result in the decrease of mechanical properties of CS/HA material due to the weaker interfacial bonding between HA?ller and CS matrix[16].However,no decrease appeared in aspect of mechanical properties of CS/HA prepared byin situ hy bridization,which is similar to that of pure CS,80MPa,3.9GPa,and much higher than

Fig.6.XRD patterns of CS/HA(100/5,wt/wt)composite prepared by in situ hybrid method(a),by blending(b)and HA made in lab

(c). Fig.5.CS/HA(100/5,wt/wt)rod prepared byin situ hy brid method(a),byblending method(b),pure CS rod(c).

Q.Hu et al./Biomaterials25(2004)779–785783

that of CS/HA prepared byblending method,68MPa,3.2GPa,respectively.

3.6.Water absorption

The behavior of water absorption of CS/HA compo-site was investigated,and results were shown in Fig.8.The water absorption of the CS reaches a highest point of 58%after 24h.However,the use of 5and 10g per 100g CS of HA results in a signi?cant reduction in water absorption of composite,10%and 17%,respectively.The water absorption of CS/HA composite reduce when incorporation HA with CS matrix due to formation of a temporaryHA barrier preventing water permeating into CS,which postponed the attenuation of mechani-cal properties of CS/HA composite under moisture condition.

In order to illustrate the in?uence of HA content on the water absorption quantitatively,the data of water absorption measured were ?t as exponential function in Origin 6.1(Origin Lab Corporation),the relationship between W a (%)and time (h)immersing in the water as follows:

W a ?W s tA exp àt

t 1

ewhen t X 0:25T;e7T

where W a is the water absorption at time t ;W s is saturated water absorption (%).A and t 1are the offset factor of saturated water absorption (%)and time constant (h),respectively.The coef?cients of Eq.(7)were listed in Table 3and the correlation coef?cients (R 2)of the ?t functions were close to 1.It was found that the t 1tend to increase with increasing HA content in

Table 2

Mechanical properties of CS/HA composite and other bone replacement materials Sample

s b (MPa)E b (GPa)Sample

s b (MPa)E b (GPa)Bone cement [13]36.54 1.31CS rod [16]

80712 3.970.5PMMA [13]

41.5 2.21CS/HA B (100/5)rod 6872 3.270.2CS/HA(70/30)[13]19

1.0

CS/HA(100/5)rod

8677

3.470.1

Cortical bone pin [19]

275752

18.371.3

W a t e r A b s o r p t i o n (%)

Time(h)

Fig.8.Dependence of water absorption on time with different CS/HA ratio.

Fig.7.TEM micrographs of CS/HA (100/5,wt/wt)(a:?10K),photographs of CS/HA (100/5,wt/wt)composite (b)and SEM observation at B region (c,d)after bending test.

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784

composite,which indicated that the addition of HA in CS indeed reduces the water absorption.The different between t c and t m at30%water absorption with different samples listed in Table3is verysmall,which implied that the?t function is verysuitable to predict the water absorption.

4.Conclusion

A transparent and slight yellow CS/HA nanocomposite with high strength was prepared byin situ hy bridization. The high mechanical properties are obtained due to the layered structure con?rmed by the SEM photographs of composite.The initial mechanical properties of bending strength and bending modulus of composite are86MPa and3.4GPa,respectively,which is2–3times stronger than that of PMMA and bone ceramics.Nano-size HA particles identi?ed byTEM and XRD was dispersed well in CS/HA composites,which can also be proved bythe transparent apparent of composite rod.The addition of HA can reduce the water absorption,which postponed the retention of mechanical properties of CS/HA composite under moisture condition. Acknowledgements

The authors wish to thank the National Natural Science Foundation of China(NNSFC)(Grant No. 50173023)and Science and TechnologyProject Plane of Zhejiang Provence(2003C31044).

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Table3

The coef?cient equation of W a and time of CS/HA with different ratio

CS/HA W s(%)A(%)t1(h)R2t m(h)t c(h)

100/058.4371.71à53.7171.977.7470.810.99096 5.02 4.93 100/547.7570.75à46.8770.927.8670.410.996977.627.63 100/1040.1270.41à38.7970.508.1470.280.998710.7610.93

t m:time measured when W a is30%of sample.

t c:time calculated when W a is30%of sample.

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