Chemically modified light-curable chitosans with enhanced

Chemically modi?ed light-curable chitosans with enhanced potential for bone tissue repair

Yongzhi Qiu,1Ning Zhang,1,2Qian Kang,3Yuehuei An,3Xuejun Wen1–3

1Clemson-MUSC Bioengineering Program,Department of Bioengineering,Clemson University,Charleston,

South Carolina29425

2Department of Cell Biology and Anatomy,Medical University of South Carolina,Charleston,South Carolina29425

3Department of Orthopedic Surgery,Medical University of South Carolina,Charleston,South Carolina29425

Received9September2007;revised12December2007;accepted8February2008

Published online8May2008in Wiley InterScience(https://www.360docs.net/doc/d06335125.html,).DOI:10.1002/jbm.a.32017

Abstract:Light curable polymers are showing great potential in overcoming some of the current obstacles in bone repair.With stereolithography,they can be fabricated into scaffolds with desired topography for repairing bone defects in irregular shapes.In this study,we developed a series of light curable chitosans,which have photocurabil-ity and good solubility in organic solvent.Through adjust-ing the feeding ratio of the raw components,the content of photocurable groups in the backbone of chitosan can be controlled.The light curable chitosans were characterized through FTIR–ATR and1H NMR,which also con?rmed their controllable photocurability.NIH3T3?broblasts were co-cultured with the photocured samples for7days and the results indicated that these photocured chitosans are cyto-compatible.Scaffolds with interconnected pores, fabricated using salt leaching and photocuring and seeded with fetal bovine osteoblasts,were implanted subcutane-ously in the backs of athymic rat.Histological study on the implants at6weeks showed that the photocured chitosans have good osteoconductivity.The results indicated the light curable chitosans have good biocompatibility and osteoconductivity.ó2008Wiley Periodicals,Inc.J Biomed Mater Res89A:772–779,2009

Key words:light curable;chitosan;osteoconductivity;bone tissue engineering

INTRODUCTION

Every year in the United States,there are 900,000 hospitalizations due to bone defect and trauma.1 Bone tissue engineering provides a clinically applica-ble strategy for bone repair,among which biocom-patible materials and implants play an important role in supporting bone regeneration.For these materials,biocompatibility,chemical structure,pore topography and size,surface topography and me-chanical properties are all important factors in?uenc-ing the?nal success in bone repair.Studies on cur-rently used synthetic materials both in vitro and in vivo indicate that synthetic materials show great potential as scaffolding materials in bone repair.2–6 However,currently used biomaterials have limita-tions in some bone repair scenarios,such as repair-ing irregularly shaped defects in either open or closed forms.Light curable polymers are good can-didates for these repairs.

Light-curable polymers can undergo photopolyme-rization upon light exposure,which shows many advantages compared with chemical polymerization, including a high polymerization rate that overcomes the oxygen inhibition and solvent effects in normal polymerization,good temporal and spatial control and resolution,ambient temperature operation and low energy consumption.7Light-curable polymers may be used for in situ scaffold forming,which makes minimally invasive surgery possible.More-over,the light-curable materials may be used for computer-aided fabrication through rapid prototyp-ing which allows fabricating scaffolds with custom-ized shape and reproducible microarchitecture on a large scale.

Chitosan is a natural biopolymer that has been widely used in medical applications because of its biocompatibility and biodegradability.8–10Many studies have indicated that chitosan is a good candi-date for orthopedic applications.1,6,11–14In cartilage tissue engineering,chitosan was shown to promote chondrocyte attachment and proliferation and main-

Correspondence to:X.Wen;e-mail:xjwen@https://www.360docs.net/doc/d06335125.html, Contract grant sponsor:AO Foundation

Contract grant sponsors:National Institutes of Health, National Center for Research Resources;contract grant number:C06RR018823

ó2008Wiley Periodicals,Inc.

tain chondrogenic phenotype,which may be due to its structural similarity to various glycosaminogly-cans(GAGs)found in articular cartilage.8,11,15,16Chi-tosan has also been extensively used in bone tissue engineering as it has been shown to facilitate growth and biomineralization by osteoblasts.14Modi?ed light curable chitosans may have great potential for use in fabricating scaffolds with desired shape and pore topography for bone tissue engineering and in overcoming some obstacles in current bone repair. Many types of chemical modi?cations of chitosan have been employed to broaden its applications, however,there has been little endeavor in develop-ing photocurable chitosans.

In this study,we improved a synthesis method used by Badawy et al.17and synthesized a series of photocurable chitosans.These chitosan derivatives possess very good solubility in organic solvents and light curability as well.Moreover,the light curability is controllable through adjusting the feeding ratio of raw components during synthesis.The chemical structures of these photocurable chitosans were con-?rmed by FTIR–ATR and1H NMR.The cytocompati-bility of these light-cured chitosans was tested in vitro. To further explore the potential for bone repair,these photocurable chitosans were photocured into scaf-folds with interconnected pores,seeded with osteo-blasts,and the bone formation was evaluated in vivo.

MATERIALS AND METHODS Materials

Chitosan with high molecular weight and more than 85%deacetylation and dimethyl sulfoxide(DMSO)were both obtained from Sigma–Aldrich.(St.Louis,MO).Meth-ane sulfonic acid was purchased from Acros Organics (Geel,Belgium).Benzoyl chloride,methacryloyl chloride and Ammonium hydroxide water solution(5N)were obtained from Fisher Scienti?c.Iragure2959was kindly donated by Ciba Specialty Chemicals.

Synthesis of light-curable chitosans

The light-curable chitosans were synthesized via a method similar to that described by Badawy et al.17 (Fig.1).Brie?y,1g chitosan powder was dissolved in 15mL methane sulfonic acid,and the mixture was stirred until the chitosan was completely dissolved.Acyl chloride at?ve times the M ratio of the glucosidic unit was added dropwise into the solution over a30-min period.The reac-tion was continued for another3h and the solution was stored at2208C overnight.After thawing,the solution was added dropwise into excess water to obtain a precipitate that was then?ltered and stirred in4%amine water solu-tion overnight.The?nal product was obtained after repeated?ltering,rinsing,and vacuum drying until con-stant weight was achieved.For the modi?cations of chito-san,two acyl chloride compounds,benzoyl chloride and methacryloyl chloride,were used.The modi?ed chitosans with benzoyl chloride and methacryloyl chloride were named benzoyled chitosan(BC)and methacrylated chito-san(MC),respectively.We also mixed benzoyl and meth-acryl chloride together to react with chitosan.The amounts of benzoyl and methacryloyl chloride were varied to give ?nal ratios of1:3,1:1,and3:1.The?nal products were named BMC13,BMC11,and BMC31,respectively.(B stands for benzoyl groups,M for methacrylate groups,C for chitosan and the numbers for the feeding ratio of ben-zoyl and methacryloyl chlorides).

Characterizations

The chemical structure of the photocurable chitosans was characterized using the FTIR–ATR(Research Series 100,Madison Instrument,Madison,WI)and1H NMR (Varian INOVA-400(9.4T),Varian,Walnut Creek,CA) operated at400MHz proton with d-DMSO as solvent. Fabrication of light-cured?lm and

3D porous scaffolds

An ultraviolet light generator(American Ultraviolet Company,Santa Ana,CA)was used to generate the UV light to initiate polymerization.The light intensity was800 mW/cm2at a wavelength of365nm.Irradiation from the generator was transmitted through a quartz optical guide ?ber(diameter:5mm;length:1m)to the pre-polymers. All of the pre-polymers before light cured were dissolved in DMSO at15wt%except for MC,which was dissolved at10wt%owing to its lower solubility in DMSO.0.05wt %of the photoinitiator,Irgacure2959,was added to the solution and mixed homogeneously.For the photocured ?lms,the solution was cast on a glass slide and exposed to UV light.The UV cured?lms were stored in DI water for further use.Salt leaching with photocuring were employed to fabricate the porous scaffolds.First,the mold(4mm in diameter and2.5mm in height)was?lled with salt crystals (NaCl)and then the polymer solution was added dropwise on the top of the salt.The chitosan solution

in?ltrated Figure1.Synthesis scheme of photocurable chitosans.

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downwards to the bottom and ?lled the interspaces between salt crystals then the ?lled mold was exposed to UV for the appropriate time period (the distance between UV source and the top of mold is about 1.5cm).After cur-ing,the discs were taken out and immersed in DI water to eliminate the remaining solvent and salt.DI water was changed frequently to ensure that all of the solvent and salt was driven out from the porous scaffolds.Scanning electron microscopy (SEM,Hitachi,Japan)was used to visualized the morphology of the scaffolds.

In vitro cytocompatibility test

The photocured chitosan ?lms were removed from DI water and sterilized with 70%ethanol for 30minutes,then air-dried in the cell culture hood.NIH 3T3?broblasts were used for cytocompatibility testing.After counting,5000NIH 3T3?broblasts were seeded in each well of a 6-well cell culture plate.The sterilized chitosan ?lms were put into the wells and co-cultured with cells.Every other day,alamar blue solution was used to test cytocompatibility.Brie?y,after the culture media was removed,10%v/v ala-mar blue in fresh culture medium was added and incu-bated for 4h.Then the alamar blue solution was removed and the absorbance was measured at 570nm and 600nm.The difference between treated and control cells was calcu-lated and expressed as percentage reduction.

In vivo osteoconductivity test

The porous scaffolds were freeze-dried and sterilized with ethylene oxide.Primary fetal bovine osteoblasts were propagated in monolayer culture until con?uence.The har-vested cells were then loaded onto porous disc scaffolds at a concentration of 53106cells/scaffold.PLGA discs were used as control.Under general anesthesia and sterile con-ditions,the scaffolds were subcutaneously implanted into the backs of 4–6-week-old male,athymic (nu/nu)rat.Six rats were used for each sample type.After 6weeks,the

rats were sacri?ced and the implants were harvested.Each implant was processed for histology and stained with H&E and Anderson’s rapid bone stain counterstained with acid fuchsin.

RESULTS

Synthesis,fabrication and characterizations Solvent solubility

Because of its semicrystalline structure,raw chito-san has very poor solubility in organic solvents and can only be dissolved in some acids,such as acetic acid.After modi?cation,MC has low solvent solubil-ity,becoming cloudy when the concentration reaches 10%in DMSO.However,BMC13,BMC11,and BMC31have very good solubility in many organic solvents (up to 50%),including dimethylformamide (DMF),DMSO,dimethylacetamide (DMAC),acetone,methane chloride,and so forth,which facilitates the fabrication of scaffolds with desired structures.FTIR–ATR

From the IR spectra of each sample (Fig.2),char-acteristic peaks at around 1720cm 21show the exis-tence of ester carbonyl groups,indicating that the acyl chloride groups have reacted with the hydroxyl groups in the chitosan.The presence of amino group peaks at around 1600cm 21,with no peaks at 1670cm 21(amide I)and 1536cm 21(amide II),indicates that the amino groups were protected during the reaction due to the protonation of the amino groups when reacting with methanesulfonic acid.17,18More-over,the peak at 710cm 21indicates the existence of the aromatic ring belonging to the benzoyl

group.

Figure 2.FTIR–ATR characterization of photocurable chi-tosans.[Color ?gure can be viewed in the online issue,which is available at

https://www.360docs.net/doc/d06335125.html,.]Figure 3.1H NMR characterization of photocurable chito-sans (a)BC (b)MC (c)BMC31(d)BMC11(e)BMC13.

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The intensity of this peak decreased when more methacryloyl chloride was used during synthesis.

Conversely,the intensity increased for the peaks belonging to methacrylate groups at about950cm21

and1300cm21.Thus,during synthesis,by adjusting the feed ratio of benzoyl and methacryloyl chloride,

we can control the ratio of curable groups in the ?nal products.

1H NMR

In1H NMR(Fig.3),the chemical shift d52.8–5.2

ppm is assigned to the protons in the pyranose ring. The signals for the protons on the benzoyl group appear at d57.5–8.2ppm,and for the protons on

the methylene in the ole?n,the signal appears at d55.7–6.3ppm.To determine the practical ratio of grafted benzoyl and methacryloyl groups,the peak area of benzoyl and methacryloyl groups was inte-

grated and the ratio was calculated according to Eq.(1).

Practical ratio?ePeak area benzolyl groups=5T:

ePeak area methacryloyl groups=2Te1T

The practical ratio of benzoyl and methacryloyl groups after integration of the peak area is shown in Table I.The results show that the reactivity of ben-zoyl chloride is much higher than methacryloyl chlo-ride as the practical ratio of benzoyl groups is always higher than its feeding ratio.So the content of methacryloyl groups is in?uenced not only by the feeding ratio but also by the reactivity of these two acyl chlorides.Fabrication of light cured chitosan?lms and porous scaffolds and structure study using SEM

As expected,the time scale for light curing is deter-mined by the content of methacryloyl groups in the back-bone.More methacryloyl groups result in a faster curing rate.For example,BMC13(30s)is light cured much faster than BMC31(120s),and BMC11has an intermediate rate. In fabrication of scaffolds,we used salt crystals ranging from125to210l m as the porogen.SEM images(Fig.4)show that the size of the pores is the same as the porogen,and there is no marked differ-ence in morphology between different photocured chitosans.Furthermore,SEM images show that these pores are interconnected.

In vitro cytocompatibility tests

In this in vitro test,photocured chitosan was co-cul-tured with NIH3T3?broblasts to determine whether photocured chitosans release toxic substances which in?uence cell https://www.360docs.net/doc/d06335125.html,tex was used as a positive control and PLGA as a negative control.Alamar blue was used to test cytocompatibility of photocured chi-tosans by measuring the proliferation of the co-cul-tured cells.Speci?cally,alamar blue incorporates an oxidation-reduction(REDOX)indicator that both?uo-resces and changes color in response to chemical reduction of growth medium resulting from cell growth.The percent difference in reduction between cells on experimental groups and control groups was calculated and plotted in Figure5.From the plot,it can be seen that photocured chitosans and PLGA showed no toxicity during the7-day culture period. However,latex showed strong toxicity,with the num-ber of?broblasts in the wells cultured with latex being less than20%of the control(PLGA)and the photocured chitosans.This test indicated that photo-cured chitosans are cytocompatible.

In vivo osteoconductivity test

Osteoconductivity of photocured chitosans was studied over a6-week period.Porous discs seeded

TABLE I

The Theoretical and Practical ratios of Benzoyl and

Methacrylate Groups

BMC31BMC11BMC13

Theoretical ratio3:11:11:3

Practical ratio 4.5:1 1.5:1

1:2

Figure4.SEM of photocured chitosans(A)BMC13(B)BMC11(C)BMC31.

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with fetal bovine osteoblasts were implanted subcu-taneously in the backs of athymic rats.Implants were harvested and processed for paraf?n embed-ding.Specimens in paraf?n blocks were cut into 5

um sections and stained with H&E and Anderson’s rapid bone stain counterstained with acid fuchsin (Fig.6).Anderson’s rapid bone stain stained bone matrix to red and soft tissue to blue.In the PLGA control,extensive amount of ?brotic tissues were found even though some bone matrixes were also observed.The fragmentary bone matrixes in PLGA scaffolds were surrounded by connective tissue.However,low amount of ?brotic tissues were found in the pores of photocured chitosans scaffolds.Instead,extensive amount of bone matrixes were found in the scaffolds (stained into red after applied with Anderson’s rapid bone stain;[Fig.6(E,I,L)].At the same time,many cartilaginous cells with rounded shape were also observed in the pores,which can be seen in H&E staining [Fig.6(D,E,G,H,J,K)].The ECM of these cartilaginous cells is surrounded by or connected with bone matrix [Fig.6(E,I,L)],which indicates that the

endochondral

Figure 5.Cytocompatibility test of photocured chitosans in 7days

culture.

Figure 6.Histological pictures of scaffolds with H&E staining and Anderson’s rapid bone stain counterstained with acid fushcin (A)PLGA,H&E;(B)PLGA,H&E;(C)PLGA,Anderson’s (D)BMC31,H&E;(E)BMC31,H&E;(F)BMC31,Ander-son’s;(G)BMC11,H&E;(H)BMC11,H&E;(I)BMC11,Anderson’s;(J)BMC13,H&E;(K)BMC13,H&E;(L)BMC13,Ander-son’s (In H&E staining,black arrows point to new bone matrix and white arrows point to cartilaginous cells.And in Anderson’s staining,the bone matrix was stained red and cartilaginous cells were blue or light blue.).(Bar in the pictures is 200l m).

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ossi?cation was taking place in the scaffolds.These ?ndings indicate the photocured chitosans have bet-ter osteoconductivity than PLGA.In addition,from the pictures,we also can notice that there is less BMC31remained in the explants than BMC11and BMC13.This might be due to lower crosslinking density and faster degradation of BMC31.

DISCUSSION

Chitosan is a partially de-acetylated derivative of chitin,the second most abundant natural polymer found in shells of marine crustaceans and cell walls of fungi.13,19,20It is a linear polysaccharide composed of a-(1?4)-linked2-amino-2-deoxy-b-D-glucopyra-nose with hydroxyl and amino side groups.Because of its chemical structure,chitosan shows high bio-compatibility leading to its being extensively applied in biomedical areas,for such uses as surgical sutures,dental implants,arti?cial skin,and so forth.9 In recent research,chitosan has been widely used as a scaffolding material for tissue engineering.15,21–27 Many studies indicate that chitosan is suitable for or-thopedic applications.1,6,11–14Chitosan’s amino side groups endow it with a cationic environment that makes it electrostatically interact with anionic mole-cules,such as glycosaminoglycans(GAGs),proteo-glycans and other negatively charged molecules. Because a large number of cytokines/growth factors could be linked to GAGs,the interaction between chitosan and GAGs may retain and concentrate both the endogenous tropic factors secreted by colonizing cells,as well as the exogenous factors that may be loaded inside the scaffold.8This might be one reason that chitosan shows favorable results in cartilage tis-sue engineering.Another reason maybe due to its structural similarity to various GAGs found in artic-ular cartilage.10Moreover,chitosan has also been extensively used in bone tissue engineering because it has been shown to facilitate growth and mineral rich matrix deposition by osteoblasts in culture.14 However,chitosan has very poor solubility because of the intramolecular or intermolecular hydrogen bond formed between amino and acyl groups,which makes it soluble only in aqueous acid solutions,thus greatly limiting its application.19For example,chitosan can be only dissolved into aque-ous solution of acetic acid at a low concentration, around2wt%.The modi?ed chitosans with good solubility in many common organic solvents are believed to possess the abilities not only in further modi?cation by introducing active groups and chemically combining with other polymers to improve mechanical properties but also for process-ing into scaffolds with desired microstructure or gross shape.Various efforts have been made to pre-pare functional chitosan derivatives via chemical modi?cation,but few increased the solubility of chi-tosan in organic solvents.17,18,28–31Nishimura et al. modi?ed chitosan with phthalic anhydride,produc-ing a?nal product that can be easily dissolved into general organic solvents,such as DMSO,DMF,and DMAC.17,18,28–31Badawy et al.developed a method to improve the solubility of chitosan via the grafting of benzoyl groups onto the backbone.17,18,28–31

In this study,we modi?ed chitosan with photocur-able groups(methacrylate groups)and benzoyl groups.The modi?ed chitosan shows great solubility in many common organic solvents.The method we used is similar to that employed by Badawy.17We mixed methacryloyl chloride with benzoyl chloride, and we postulate that methacrylate groups can com-pete with benzoyl groups in grafting on the chitosan backbone.As expected,both groups are grafted onto the backbone.Benzoyl groups provide great solubil-ity in general organic solvents,and methacrylate groups provide photocurability.However,too much methacrylate group will result in decreased solubil-ity.For example,a solution of10%MC in DMSO is cloudy.The mixtures BMC31,BMC11,and BMC13 can reach a concentration as high as50%in DMSO, which may be due to the aromatic rings in the back-bone.Moreover,the content of methacrylate groups in modi?ed chitosan,which provide light curability, can be adjusted by changing the feeding ratio of methacryloyl and benzoyl chlorides,meaning that we can adjust the crosslinking density of the?nal cured network.In addition,we found that more methacryloyl groups in chitosan backbone resulted in a faster curing rate.

The main purpose of this study is to obtain func-tional scaffold materials for bone tissue engineering. From the point of view in bone tissue engineering,a good scaffolding material should have osteocompati-bility and osteoconductivity.For example,it can induce bone repair in the wound that cannot heal if left untreated and facilitate the bone cells growth in the scaffold to form bone.And it will be perfect if the materials have osteoinductivity.32To some extent,these bioactivities are determined by the chemical structure or components.Chitosan is a lin-ear polysaccharide and has been proven to be a can-didate of scaffolding material for bone tissue engi-neering.Borah et al.33found that N-acetyl-chitosan induced calci?cation better than the control.And many other chitosan derivatives have been indicated to have good osteoconductivity,such as N-carboxyl-butyl-chitosan,34imidazole-chitosan,35and6-oxylchi-tin.36In addition,osteoinductivity was also observed with a chitosan coated hydroxyapatite nail.37These results suggested that raw chitosan or modi?ed chi-tosan could be a series of good scaffolding materials

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for bone tissue engineering.To this end,we devel-oped the photocurable chitosans for repairing bone defects.Owing to the advantages of photopolymeri-zation,the photocurable chitosans can be fabricated into scaffolds with desired shape and pore topogra-phy.In this study,we used salt leaching coupled with photocuring to fabricate scaffolds with intercon-nected pores as a model tested the feasibility.For bone tissue engineering,the pore size of the scaffold is a major factor affecting the regeneration of bone. Small pore size prevents cells from penetrating into the scaffold.However,pores which are too large cannot be effectively occupied by cells.The optimal pore size of scaffolds for bone tissue engineering is around100–300l m.The salt crystals used in this study range in size from125to210l m.From the SEM images,it can be seen that after salt leaching, the pores in the scaffolds are in the optimal size range and they are interconnected.The6-week osteoconductivity test in which we implanted cell-seeded photocured modi?ed chitosan scaffolds into the backs of athymic rats showed very good osteo-conductivity of the photocured chitosans.This fur-ther veri?ed that photocurable chitosans are suitable for use as scaffolding materials for bone tissue engi-neering.However,the control,PLGA porous scaf-folds,contains large amount of?brotic tissues and few bone tissues,which may be due to its faster deg-radation rate(loss of porous structure after6weeks from[Fig.6(A–C)]and the acidic degraded products induce in?ammation reaction and reduce the bone formation.Because the photocurable chitosans have the potential to be fabricated into desired shapes, fabricating larger scaffolds with irregular shapes and pore topography through stereolithography is the future target.In addition,the photocurable chitosans can be coupled with other synthetic materials through chemical reactions,which might result in improved mechanical properties.

CONCLUSIONS

A series of photocurable chitosans with great solu-bility in organic solvents have been synthesized.The content of the photocurable groups(methacryloyl groups)in the backbone can be controlled by adjust-ing the feeding ratio of the acyl chloride.The struc-tures of these photocurable chitosans were con-?rmed by FTIR–ATR and1H NMR measurements. Moreover,from1H NMR,we found that the benzoyl chloride has much higher reactivity than methacry-loyl chloride in this reaction.The modi?ed chitosans can be fabricated into porous scaffolds with desired pore size by salt leaching and photocrosslinking, which means that the modi?ed chitosans can be more easily fabricated into scaffolds than raw chito-san.In vitro cytocompatibility testing showed that the photocured chitosans are cytocompatible.Porous photocured discs were also subcutaneously im-planted into the backs of athymic rats.After6weeks of implantation of osteoblasts seeded scaffolds,histo-logical analysis indicated that photocured chitosans have good osteoconductivity.

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Journal of Biomedical Materials Research Part A