International Journal of Biological Macromolecules 47 (2010) 483487

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International Journal of Biological Macromolecules

journa l homepage: www.e lsev ier .com

Charac mbone su

Alina SioFaculty of Chem

a r t i c l

Article history:Received 2 JunReceived in reAccepted 7 JulAvailable onlin

Keywords:CollagenHydroxyapatitBiomaterial

tes (He benis stucom

ions.re stuof Hinfra

spectt widand c

Thermogravimetric results suggested that collagen chains had been embedded with HAp to several com-plexes with different thermal stabilities. SEM was used to observe the morphology and pore size of thesponges. SEM observations showed the sponges of HAp/Col with fully interconective macroporosity.

2010 Elsevier B.V. All rights reserved.

1. Introdu

Reconstrresection oNatural bonin which hycollagen bture [1].

Severalmicrostructbeen devotewith biopolresearch issents the mbe extractean excellentoxic and itthis reason,biomaterialfoams, nanoand dispers

CorresponE-mail add

0141-8130/$ doi:10.1016/j.ction

uction of bone defects caused by trauma or surgicalf a bone tumor is a major issue in orthopedic surgery.e is a complex inorganicorganic composite material,droxyapatite nanocrystals (Ca10(PO4)6(OH)2, HAp) andrils (Col) are well organized into hierarchical architec-

attempts have been made so far to nd material withure comparable to native bone. Increasing efforts haved to the composites of hydroxyapatites in conjugationymers. Themost important biopolymer in biomaterialscollagen. The natural polymer, collagen, that repre-atrix material of bone, teeth and connective tissue cand from animal or human sources. Collagen providest basis for biomaterials as it is readily available, non-s bril architecture is inherent in natural tissues. Forcollagen is regarded as one of the most useful naturals. Collagen can be processed in sheet, tubes spongesbrous powders, eeces, injectable viscous solution,

ions [2,3].

ding author.ress: as@chem.uni.torun.pl (A. Sionkowska).

Synthetic HAp, almost identical to the bioapatite of bone, hasbeen widely used as an articial bone substitute. HAp has attracteda great deal of attention for hard tissue applications due to its bioac-tive, osteoconductive and biodegradable properties [48]. It hasbeen clinically used as sintered bulk ceramic, porous structures,granules and coatings. However, the application of pureHAp is verylimited due to its brittleness. Thus, composite materials made fromHAp and a polymer (natural such as collagen, or synthetic ones)are highly applicable in bone tissue regeneration.

The composites of HAp/col have beenwidely studied during lastdecade [916].Many researches have prepared and reportedmate-rials based on HAp and another biodegradable or bioreabsorbablematerials, such as chitosan [17,18], gelatin [19], hyaluronic acid[1,20] and synthetic polymers, like polylactic acid (PLA) [1,2122],polyglycolic acid (PGA) [1,2,22], poly(caprolactone) (PCL) [2,22] orpoly(vinyl alcohol) (PVA) [23]. The above materials can be pro-cessed using several techniques, such as porogen leaching, gasfoaming, phase separation, ber meshing, supercritical uid pro-cessing, microsphere sintering, and three-dimensional printing togenerate a range of three-dimensional scaffolds with differentporosities and surface characteristics [1]. Porous hydroxyapatite,a promising material in biomedical application, has been used asbone scaffold and drug carriers [24,25].

Theuseofnano-particleofHApandcollagen type I is apromisingapproach for the generation of new bone substitutes to morpho-

see front matter 2010 Elsevier B.V. All rights reserved.ijbiomac.2010.07.002terization of collagen/hydroxyapatite cobstitute

nkowska , Justyna Kozowskaistry, N. Copernicus University, Gagarin 7, 87-100 Torun, Poland

e i n f o

e 2010vised form 3 July 2010y 2010e 15 July 2010

e

a b s t r a c t

Hydroxyapatiteandcollagencomposibones. Their combination should provical resemblance and properties. In thwere used in different proportions astion of corresponding composite solutchemical and physical properties wecomposite sponge made of compositeated total reection Fourier transformelectron microscopy (SEM). Infrared(ATR) spectroscopy is one of the mosanalysis did not indicate shift of the b/ locate / i jb iomac

posite sponges as a potential

Ap/Col)have thepotential inmimickingandreplacingskeletalecial for bone tissue engineering due to their natural biolog-dy, hydroxyapatite and collagen isolated from animal tendonsposites. Sponges were prepared by freezing and lyophyliza-The properties of composite sponges, such as microstructure,died. In the present investigation, a collagen sponge and a

Ap/Col were prepared in our lab and characterized by attenu--red spectroscopy, thermogravimetric analysis and scanningroscopy (IR) in combination with attenuated total reectionely used technique for surface infrared analysis. The ATR-IRorresponding to COO for none of the used HAp/Col ratios.

484 A. Sionkowska, J. Kozowska / International Journal of Biological Macromolecules 47 (2010) 483487

Fig. 1. Porous sponge, made from HAp and Col, in different sizes (the scale showscentimeters).

logical featwork, a 3Dhydroxyapabe potentiagen materiaInourprevion collagenproliferatio

2. Materia

Collagenyoung rats.dissolved inwas freeze-CHRIST, 2tion of 0.5macetic acid.was supplie

The starmixed and twere set foHAp/Col miwere set inwere freeze

A chemigroups of c

Fig. 2. FT-IR spectrum of collagen and hydroxyapatite.

infrared spectroscopy using a Genesis II FTIR spectrophotometer(Mattson, USA) equipped in ATR device (MIRacleTM PIKE Technolo-gies) with zinc selenide (ZnSe) crystal. All spectra were recorded inabsorption mode at 4 cm1 intervals and 64 scans. The sample of

r the FTIR measurement was prepared by grounding a dryarticle sample with KBr into ne powders which were thend into pellets.morphology of collagen sponges and collagen modied

0 and 80wt.% of HAp were studied using Scanning Elec-icroscopy (SEM) (LEO Electron Microscopy Ltd, England).asurements on all of the samples were repeated at differentns.rmogravimetric analysis (TG)was performed on a TA Instru-SDT 2960 Simultaneous TGA-DTA in nitrogen and at heatingC/min and heating programme 25600 C.

Table 1Assignments o

Assignments

Structural OH2O adsorbeCH (B amidCH3 groupH2O adsorbeNH (II amidCO32 , pyrroCH, CO32

COO

CN (III amiPO43 bendPO43 bendPO4 stretchures and mimic the structure of natural bone. In thistype composite materials from collagen and nano-

tite were prepared. Presented sponges of HAp/Col canlly used as bone tissue engineering scaffolds. The colla-ls have been studied by our research group previously.ous studieswehave shown thatbiomaterials performedobtained in our laboratory promotes the adhesion andn of cells [26,27].

ls and methods

was obtained in our laboratory from tail tendons ofAfter washing in distillate water, the tendons were0.1M acetic acid for 3 days at 4 C. Collagen solution

dried to obtain 100% pure collagen (ALPHA 1-2 LDplus,0 C, 100Pa, 48h). Collagen solution with concentra-ass% was prepared from lyophilized collagen in 0.01MHydroxyapatite (nanopowder,

A. Sionkowska, J. Kozowska / International Journal of Biological Macromolecules 47 (2010) 483487 485

3. Results and discussion

Freeze drying of collagen and HAp/Col blends leads to porousthree-dimensional sponge, mimicking the extracelluar matrix ofbone tissue. Samples can be produced in any size or shape. Fig. 1shows the photograph of the obtained sponges. Properties of thisbiomaterial will be described below.

3.1. Spectroscopy results

FT-IR spectra of collagen and hydroxyapatite are shown in Fig. 2and spectra of hydroxyapatite and collagen composites are shownin Fig. 3.

The spectrum of HAp/Col composite sample is characterized byabsorption bands arising from HAp and collagen. We observed thebands typical for collagen such NH stretching at 3305 cm1 forthe amide A, CH stretching at 3081 cm1 for the amide B, C Ostretching at 1635 cm1 for the amide I, and NH deformationat 1545 cm1 for the amide II. As HAp related bands there arephosphate contours. The phosphate bands are located between 900and 1200 cm1 in IR spectra. In the HAp spectrum one can nd thetypical stretching vibration bands of phosphoric groups at 1025and 1047 cm1. There are also CO3 bands located at 1445, 1414and 876 cm1. Moreover, between 450 and 670 cm1 the bands ofPO43 can be found.

In a few papers published so far regarding HAp/Col compos-ites preparations itwas believed that chemical interaction betweenHAp and Col can be evaluated from the infrared spectrum ofHAp/Col composite [28]. In infrared spectrum of HAp/Col wasobserved a shift of the band corresponding to COO stretchingto lower wave number. This fact suggests the formation of a new

Fig. 4. SEM image of collagen sponge (100).

chemical bondbetweenCa2+ ions on theHAp surface andCOO oncollagen [29]. In our study the ATR-IR analysis did not indicate anyalterations in the Col vibration mode for none of the used HAp/Colratios (Table 1).

3.2. Scanning electron microscopy observations

The porous morphologies of the composite sponges were stud-ied by scanning electron microscopy. Fig. 4 shows the SEM imageof the collagen sponge and Fig. 5ad show the SEM images of theHAp/Col composites.

/w), 500, (c) 80/20 (w/w), 100, (d) 80/20 (w/w), 500.Fig. 5. SEM images of HAp/Col composites: (a) 50/50 (w/w), 100, (b) 50/50 (w

486 A. Sionkowska, J. Kozowska / International Journal of Biological Macromolecules 47 (2010) 483487

Fig. 7. T

From SEfully intercof irregulareralmicronsponge revrelatively la

3.3. Therma

Fig. 6 shand HAp/Co

From TGheatingprocess (Tmax)(Table 2). Fo0.5%, proba

Table 2Thermal param

ColHAp/Col (50HAp/Col (80HAp

In the range 130600 C a weight loss is 0.9% due to the decom-position of HAp. In the characteristic DTG curve for collagen innitrogen one can see two peaks. These peaks are representativefor the two-stage sample destruction due to the temperature. Therst stage (tion of wat250 and 50molecular pated. For HAthe loss of wmuch smalratio 80/20peak on the

mpan DTlossprerati

0/50embeermare byyapa

clus

compacids thaFig. 6. TG curve of hydroxyapatite.

is accopeak oweight

Theweightratio 5beenent thstructuhydrox

4. Con

3DaceticteristicG and DTG curves of collagen sponge and HAp/Col composites.

M observations one can see sponges of HAp/Col withonective macroporosity. SEM results show a structureinterconnected pores. The pore sizes ranged from sev-s to about 500m. Themicrostructure of the compositeeals a porous brous collagen matrix embedded withrge particles of HAp.

l properties

ows TG results for HAp. TG and DTG plots of collagenl composites in nitrogen are presented in Fig. 7.curves we determined the mass decrement during thecess. The temperatureof themaximumspeedof thepro-was determined from the maximum on the DTG curver HAp aweight loss from room temperature to 130 C isbly resulting from the dehydration of adsorbed water.

eters of HAp, collagen and HAp/Col composites.

Stage 1 Stage 2 Stage 3

% Tmax (C) (%) Tmax (C) (%) Tmax (C)

11.9 199 62.4 325 /50) 7.5 58 33.1 311 /20) 8.4 123 8.1 289 9.5 397

0.5 62 0.9 336

scaffolds. Cbility, afncandidatesatite is genmacromoleto stiffen thof composiand collage

Acknowled

FinanciaGrant No NSocial Fundedged.

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l support from theMinistry of Science (MNiSW, Poland)N507 349535 and PhD Students Grant of the European(EFS, Poland) and State Budget is gratefully acknowl-

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Characterization of collagen/hydroxyapatite composite sponges as a potential bone substituteIntroductionMaterials and methodsResults and discussionSpectroscopy resultsScanning electron microscopy observationsThermal properties

ConclusionsAcknowledgementsReferences