Download - Regenerative behavior of biomineral/agarose composite gels as bone grafting materials in rat cranial defects

Regenerative behavior of biomineral/agarose compositegels as bone grafting materials in rat cranial defects

Yoshika Suzawa,1 Takafumi Funaki,2,3 Junji Watanabe,2,4 Soichi Iwai,1 Yoshiaki Yura,1

Takayoshi Nakano,5 Yukichi Umakoshi,5 Mitsuru Akashi2,3,41Department of Oral and Maxillofacial Surgery, Graduate School of Dentistry, Osaka University,1-8 Yamada-oka, Suita, Osaka 565-0871, Japan2Division of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka,Suita, Osaka 565-0871, Japan3BioMedical Technology Hybrid Ltd., Kagoshima University Research and Development Center,1-21-40 Korimoto, Kagoshima 890-0065, Japan421st COE ‘‘Center for Integrated Cell and Tissue Regulation,’’ Osaka University, 2-2 Yamada-oka,Suita, Osaka 565-0871, Japan5Division of Materials and Manufacturing Science, Graduate School of Engineering, Osaka University,2-1 Yamada-oka, Suita, Osaka 565-0871, Japan

Received 19 September 2007; revised 31 July 2008; accepted 15 January 2009Published online 31 August 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32518

Abstract: The main objective of this study was to evaluatethe biological behavior of Hydroxyapatite (HAp)/agaroseand calcium carbonate (CaCO3)/agarose composite gels byan alternate soaking process used for the treatment of surgi-cally produced bone defects in rat cranium. We designedthe following four groups: (i) HAp (HAp/agarose compositegel), (ii) CaCO3 (CaCO3/agarose composite gel), (iii) Agarose(bare agarose gel), and (iv) Defect (no filling materials). Wesubdivided (i) (ii) (iii) into two application types as a (I) Ho-mogenized Group (homogenized materials) and a (II) DiskGroup (disk shaped materials). We assessed samples by ra-diological and histological analyses 0, 4, and 8 weeks afterimplantation. The results indicated that the composite gelsshowed higher radiopacity in microfocus-computed tomog-raphy (lCT) images and showed higher volume in quantita-

tive analyses using Dual Energy X-ray Absorptiometry(DEXA) and Peripheral Quantitative Computed Tomogra-phy (pQCT) than the Agarose and Defect groups. The histo-logical examination showed characteristic images due toeach application form. Consequently, HAp and CaCO3/aga-rose composite gels can be expected to accelerate the speedof producing more new bone associated with osteogenesis.These novel biomaterials play an important role as an alter-native biocompatible and biodegradable bone grafting fillermaterial for autogenous bone. � 2009 Wiley Periodicals, Inc.J Biomed Mater Res 93A: 965–975, 2010

Key words: agarose composite gel; hydroxyapatite (HAp)and calcium carbonate (CaCO3); bone grafting material;application form; rat cranium

INTRODUCTION

The repair of congenital cleft palate and cranialdefects that involve a loss of bone substance causedby infection or tumor resection is of fundamentalimportance in restoring the shape and function of theskull. Bone augmentation is a common procedure toincrease bone volume and allow for proper implantplacement in the alveolar bone defect.1,2 Autogenousbone grafts have been used in the reconstruction of

these defects because they do not cause immunogenicreactions, and because they contain osteogenic cellswhich release growth factors that contribute to therepair of the defects.3–5 The donor sites for autogenousbone frequently include the iliac crest, ramus of themandible, tuberculum mentale, maxillary tubercle,tibia and fibula.6–8 In spite of the advantages offeredby autogenous bone grafts, they sometimes require alonger surgical time and multiple surgical sites.Furthermore, there can be postoperative complica-tions, and there may be an insufficient quantity ofbone. For these reasons, various alternative biomateri-als for bone have been studied.9–12

It is believed that three basic factors are crucial foreffective tissue reconstitution in tissue engineering:the cell, the scaffold, and growth factors. In particu-lar, the development of a scaffold is very important

Correspondence to: M. Akashi; e-mail: [email protected] grant sponsor: The Ministry of Education,

Culture, Sports, Science and Technology, Japan

� 2009 Wiley Periodicals, Inc.

because living cells adhere, proliferate and differentiateon this scaffold during tissue regeneration. In addition,it is necessary to create scaffolds with both biocom-patibility and biodegradability. Moreover, they arerequired to be clinically easy to use for the operation.

We also had the assumption that one of the most im-portant factors to promote bone regeneration by bioma-terial implantation was to transport and control releaseof calcium ions from them into the defect cavity.

In biological systems, various organisms formorganic-inorganic composites, including hydroxyapa-tite (Ca10(PO4)6(OH)2, HAp) and calcium carbonate(CaCO3) as inorganic compounds. For example,bone and teeth consist of 70% HAp and 30% colla-gen. The nacre of mollusk shells and coral reefs haslayered structure of CaCO3 and organic macromole-cules. Particularly, primitive organisms have usedCaCO3 to form unique and hierarchical architecturesfor a variety of biofunctions along with survival.13

Therefore, we focused our attention on HAp andCaCO3, because they are common in nature andhave biological activities such as cell adhesion,proliferation and differentiation properties,14,15 pro-tein-adhesive properties16,17 and good cell compati-bility.7 In addition, we expected that they wouldhave good potential as a possible source to releasecalcium ions as carrier materials. Thus, we usedthem to develop a novel biomaterial, and preparedHAp or CaCO3/agarose composite gel by the alter-nate soaking process.18–20 Specifically, our groupreported the HAp/agarose composite gel wasalready crucial implantable materials in the field oforal and maxillofacial surgery as scaffolds for use inbone tissue engineering.21–23

It is known that CaCO3 has biodegradable andosteoconductive properties.24–26 Ohgushi et al.24 dis-covered that bone formation in CaCO3 is comparableto bioactive HAp in vivo. Furthermore, CaCO3

implants showed better biodegradability than HApimplants. We expected CaCO3 would be absorbedeasily compared to HAp, even they become compos-ite compound with agarose gel, and it had thepotential to be the ideal scaffold for bone regenera-tion. Also, although CaCO3 has great compatibilitywith hard tissue, CaCO3/polymer composite materi-als have not rolled out full as bone regenerative sub-stitutes. Thus, we decided to try to investigate thatthe biological behavior of the CaCO3/agarose com-posite gel as a bone grafting material in vivo andthat whether it would lead to bone formation likeexperiments performed in vitro.27 We proposed anovel approach to evaluate the potential of thesematerials for bone tissue engineering as follows: (1)evaluation of the CaCO3/agarose composite gel as ascaffold in bone regeneration in vivo as a clinicalapplication; (2) comparison of the bone regenerativecapability between HAp/agarose composite gels,

which have already been reported to have osteocon-ductive and hemostatic properties in animal modelsfor tubular bone, and CaCO3/agarose compositegels, by radiological and histological assessments; (3)observation of the differences of the bone regenera-tive process associated with the implanted conditionof the materials.

Therefore, we studied the repair of surgically pro-duced bone defects in rat parietal bone to evaluatethe biological behavior of these materials for bonereconstruction.

MATERIALS AND METHODS

Preparation of HAp or CaCO3/AgaroseComposite Gel

Agarose (NuSieve1) was purchased from Cambrex BioScience Rockland (Rockland, ME). Anhydrous calcium chlo-ride was purchased from Kishida Chemical (Osaka, Japan).Disodium hydrogenphosphate, tris(hydroxymethyl) amino-methane (Tris), tris (hydroxymethyl)aminomethane hydro-chloride (Tris-HCl), 3,3-Bis[N,N-bis(carboxymethyl) amino-methyl]-o-cresolphthalcin (OCPC), sodium hydroxide(NaOH, 1 mol/L), 2-aminoethanol, 8-quinolinol and calciumstandard solution (CaCO3 in 0.1 mol/L HNO3,) were pur-chased from Wako Pure Chemical Industries (Osaka, Japan).Acetic acid (CH3COOH) and boric acid (H3BO3) were pur-chased from Nacalai Tesque (Kyoto, Japan). All reagentswere of extra pure grade, and were used as received. Ultra-pure water was used throughout the experiment.

Agarose gel was obtained by cooling 3 w/v % aqueoussolutions, which were poured into molds of 0.5-mmthickness between two glass slides. The gels were subse-quently punched out into 4-mm diameter discs to use forthe alternate soaking process. The thickness and diameterof the gel disks reflected the implantable range of theanimals used in this study. The preparation of the com-posites was carried out according to our previousreport.11 However, we arranged immersion time becausevolumes of the gel disks were different than before. Wetried various time lengths to detect more crystals effi-ciently formed on the agarose gels and decided it by theresults. Briefly, to make HAp form in the agarose gels,the gel disks were alternately immersed into aqueoussolutions of CaCl2 (pH 7.4, 200 mmol/L) and Na2HPO4

(120 mmol/L) at 48C for 1 h, and were washed withultrapure water after each immersion. The immersioninto each ionic solution and washing was repeated alter-nately for 12 cycles. In a similar way, CaCO3 formationin the agarose gel could be prepared by simply tradingthe Na2HPO4 solution with a Na2CO3 (200 mmol/L) solu-tion and soaked for 5 minutes. The amount of HAp orCaCO3 formed in the agarose gel (mg/gel) was calculatedby gravimetric analysis. The gel was freeze-dried prior toweighing.

The final products were packed and gamma-ray irradia-tioned (25 kGy, about 2 h, Koga Isotope, Shiga, Japan)before implantation.

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Journal of Biomedical Materials Research Part A

Characterization of HAp or CaCO3 in AgaroseComposite Gel

The morphology of the HAp and CaCO3 was observedusing a scanning electron microscope (SEM, JSM-6700FE,JEOL, Tokyo, Japan). The resulting agarose composite gelswere lyophilized and then coated by osmium tetraoxide tosuppress charging for 30 sec. The SEM observation wascarried out using 60003 magnifications.

The swelling ratio of the agarose gel was calculatedusing the following equation:

SwellingRatio ¼ ðWs �WdÞ=Wd

where Ws and Wd are the weights of the gels in theirswollen and freeze-dried states, respectively.

The amount of biomineral formed in the agarose gelwas calculated by the following equation:

ðHApor CaCO3 formedÞðmg=gelÞ ¼ ðfreeze - dried HAp or CaCO3=agarose composite gel after soaking cycles ðmgÞÞ�ðswollen agarose gel=ð1þ Swelling Ratio of agarose gelÞðmgÞÞ

¼ ðfreeze - dried HAp or CaCO3 = agarose composite gel after soaking cycles ðmgÞÞ� ðfreeze - dried agarose gel ðmgÞÞ

The HAp and CaCO3 were also characterized by theirX-ray diffraction patterns (XRD, RINT In Plane UltraX18,Rigaku, Tokyo, Japan). The X-ray source was CuKa, and40 kV and 200 mA were used for the measurement at ascanning angle that ranged from 208 to 508 at 28/min.

The formed HAp and CaCO3 were also evaluated in termsof the total amount of calcium in each gel. First, the HApand CaCO3/agarose composite gels were immersed into1 mL of hydrochloric acid aqueous solution (HCl, 1 mol/L)for over night to completely dissolve the HAp or CaCO3 inthe agarose gels. A small amount of the supernatant wasused for further evaluation, and the concentration of calciumions was evaluated using the OCPC color reagent. Weblended the above six OCPC components, and adjusted thereagent. The fundamental mechanism of the assay was theformation of a chelate compound. Thus, the calcium ions inthe HAp or CaCO3 freed by dissolution were captured bythe reagent. After formation of the chelate compound, thechange in absorbance at 570 nm was monitored. A calciumstandard solution was used for the calibration curve.

Surgical Procedure

Six weeks old male Wistar/ST rats were purchasedfrom Japan SLC (Shizuoka, Japan). They were kept incages in a controlled environment (238C, 12:12 h lightcycle). They had ad libitum access to drinking water andstandard laboratory rat pellet diet. The animals were moni-tored until their date of euthanasia. All of the animal stud-ies were approved by the animal experimental committeeof the Graduate School of Dental, Osaka University.

The rats were anesthetized by an intraperitonealinjection of Nembutal1 (40 mg/kg body weight). Aftershaving the skin, a straight incision was made in the skull,and the periosteum was opened to expose the surface ofthe parietal bones. A bone defect 4 mm in diameter and0.5 mm in depth was produced in each parietal bone witha dental bur under constant irrigation. Each defect wasflushed with saline to remove the bone debris, and thenimplanted with the experimental materials. Two differentapplication forms of the same materials were placed into

the defect as described below, replacing the volume ofbone removed. We show the schematic illustration of theimplant model and photos of implanted materials (Fig. 1).

We defined the groups by the implanted materials:

i. HAp: Implanted HAp/agarose composite gelii. CaCO3: Implanted CaCO3/agarose composite geliii. Agarose: Implanted bare agarose geliv. Defect: Unfilled defect

We further defined the application forms using (i) (ii)(iii) as following group names:

I. Homogenized Group: Some pieces of the prepareddisk shape gel which composite gels (i) (ii) or bareagarose gels (iii) were homogenized like toothpastejust before implantation. Then they were embeddedto the bone defect.

II. Disk Group: Agarose disk shape gels originally werepunched out after molded by using a pair of glassslides. The resulting gels were alternately immersionfor mineralization as composite gels (i) (ii) oruntreated (iii), and then implanted.

The Homogenized Group was much superior to theDisk Group in terms of adhesion property at the bonedefect. The groups Agarose and Defect were designed ascontrols. Both defects were filled with identical materials.

The periosteum and skin were closed in layers withthread sutures. During the experiment, all animals showedan uneventful healing of the area of surgery. No significantreductions in body weights were noted, and no postopera-tive infections were observed. They behaved normally dur-ing the healing phase.

Analytical Methods

The animals were sacrificed and sampled for radiologi-cal and histological analysis at 4 or 8 weeks after implanta-tion. Some of them were also sacrificed immediately aftersurgery, and each calvarium was removed as a sample.

REGENERATIVE BEHAVIOR OF BIOMINERAL/AGAROSE COMPOSITE GELS 967


The samples were fixed in 10% neutral-buffered formalinfor over 2 weeks, and then processed for histology. Thecalvaria of all rats were characterized using X-ray imaging(M-150WE, SOFTEX CORPORATION, Tokyo, Japan). Theirradiation conditions were 30 kV, 1.5 mA, for 8.0 sec andthe focal length was 10 cm. We used X-ray dental film(ISO speed D instant film, size 2) and developed and fixedthem in INSTANT DQE1 (HANSHIN TECHNICAL LAB.LTD., Nishinomiya, Japan). The most important aim of thisanalysis was screening whether the bone defects werefilled with regenerated bone. Judging from the radio-opacity of the defects, all samples were worthy followinganalysis, and therefore we proceeded to the next step.

A small animal microfocus-computed tomography (l-CT) imaging system (SMX-100CT, Shimadzu, Kyoto, Japan)was used to examine bone formation over time in theappropriate individual rats. The specimens were scannedusing a maximum resolution of 42 lm, and the isotropicvoxel size was 600 views/180 degrees. The scanner was setat a voltage of 40 kV and a current of 30 lA. These slicedimages were compiled and analyzed to render 3D imagesand to obtain quantitative architectural parameters. Visualanalyses of the CT data were performed by means of theprovided software (VGStudio, Shimadzu, Kyoto, Japan).

As alternative characterizations, we selected two proce-dures used for the determination of the bone content for thediagnosis of osteoporosis: Dual Energy X-ray Absorptiome-try (DEXA, DCS-600EX-IIIR, ALOKA CO., LTD, Japan) andPeripheral Quantitative Computed Tomography (pQCT,XCT Research SA MDL922011, STRATEC Medizintechnik,Germany), to assess the quantities of the regenerated bone.

DEXA has been the gold standard for the determina-tion of bone density in clinical practice at present. Origi-nally we think it was ideal to analyze all regenerate bonein circular defects. But this was not as easy in practice asin theory. Therefore, in this time, we made the crosssection passing through the center of circular defectrepresent analysis object, we compared the representativevalues.

Specifically, in the case of DEXA, we had an assumptionthat 3 mm square region which matched the center of thecircular defect and we compared bone density (mg/cm2)as the internal bone quantity.

In contrast, pQCT has been to measure the volume den-sity of bone at arbitrary sites. We had an assumption thatrectangular solid region which the middle cross sectionsurface containing it passing through the center of circulardefect in the CT image. Then we compared existingnewly-regenerated bone area (mm2) and the density (mg/cm3) per unit volume. And we estimated the calculatedaverage value of the right and left.

After radiological assessment, the histological specimenswere demineralized by specially prepared solution28 anddehydration was then performed using a graded series ofethyl alcohols and clearing with a xylene substitute. Theywere embedded in paraffin, and the specimens were cutsagittal direction to make histological sections 4-lm thickand stained with hematoxylin-eosin.

RESULTS

Characterization of HAp and CaCO3

in Agarose Gel

First, the morphology of HAp and CaCO3 formedin the agarose gels by alternate soaking wasobserved by scanning electron microscopy (SEM).Figure 2 shows the SEM views of the bare agarosegel, and HAp andCaCO3/agarose composite gelscreated by 12-soaking cycles.

In the case of the bare agarose gel, a smooth andflat surface morphology was observed without anyprecipitates at low magnification. Three-dimensionalnetworks were seen at the higher magnification [Fig.2(a)].

Figure 1. Schematic illustration of the implant model and photos of the two groups of implanted condition. (I) Homoge-nized Group; (II) Disk Group. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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In contrast, on the surface of the mineral compos-ite gels, numerous HAp and CaCO3 particles wereobserved with various sizes of diameter from aboutseveral tens of nanometers to a few micrometers[Fig. 2(b,c)]. These results showed that the biomin-erals were confirmed to form in the gels.

On the CaCO3/agarose composite gels, rhombohe-dral crystals with the specific morphology of calcitewere observed in all areas viewed. Moreover,spherical precipitates were observed in them, andthe crystals could be distinguished from vaterite.Characteristically, in the case of CaCO3, the Figureshows a mixture of small pieces and multiple smallpieces coalesce into large units.

Figure 3 shows the crystallographic structure ofthe HAp and CaCO3 gels studied with X-ray diffrac-tion. HAp is thermodynamically the most stablecalcium phosphate in a natural or basic environment,and therefore was enriched in the agarose gels. TheHAp/agarose composite gel showed the broad peaks

characteristic of HAp. On the other hand, the CaCO3/agarose composite gel showed the typical peaks ofcalcite, which is its most stable crystalline form. Fur-thermore, some peaks attributed to vaterite werepartially observed. The peaks were sharp.

The amount of HAp formation was 1.5 mg/gel,and CaCO3 was 2.0 mg/gel. The gels containedabout 0.97 mg calcium/gel for the HAp/agarosecomposite gel, and about 1.2 mg calcium/gel for theCaCO3/agarose composite gel (Table I).

Radiographic and RadiologicalQualitative Analysis

At 4 and 8 weeks after implantation, we put inscreening all samples by radiographic analysis, andthen we proceeded to the next step to take l-CT scans.

Bone formation in the skull defects were alsoobserved with l-CT images, and were clearer thanthe radiographs. Immediately after the surgery, weobserved only radiolucent circular contours in thedefect, despite the fact that there were numerousparticles of HAp and CaCO3 in the composites,using SEM [Fig. 4(a)].

After 4 weeks, radiolucent areas were still seen forboth materials in the Homogenized Group relativeto the Disk Group. It was impressive that in theDisk Group, there tended to be a characteristically

Figure 2. SEM views of the surface of the gels (36000). (a) Agarose gel; (b) HAp/agarose composite gel; and (c) CaCO3/agarose composite gel.

Figure 3. X-ray diffraction patterns of: (a) Agarose gel;(b) HAp/agarose composite gel; and (c) CaCO3/agarosecomposite gel. [Color figure can be viewed in the onlineissue, which is available at www.interscience.wiley.com.]

TABLE ICharacterization of Implanted Materials

ItemsAgarose

Gel (n 5 9)

HAp/AgaroseCompositeGel (n 5 9)

CaCO3/AgaroseCompositeGel (n 5 9)

Swelling ratio 32.0 6 5.0 3.7 6 0.35 1.2 6 0.39Amount of

mineralformation(mg/gel)

– 1.5 6 0.45 2.0 6 0.30

Quantity ofcalcium(mg/gel)

– 0.97 6 0.31 1.2 6 0.30



observed meshed pattern of radiolucent findings, asif the minerals leaked out of the gel disks into thedefect areas and then they formed new bone [Fig.4(b)].

Finally after 8 weeks, the radio-opaque area wasmuch increased in the defect holes, regardless of thematerials or application forms, except CaCO3 in theDisk Group. In particular, there seemed to be goodbridging between the native bone and the implantsites, and the radio-opaque areas were obviouslyincreased in the Homogenized Group.

In terms of intermaterial comparison, the HAp/agarose gel appeared to induce more osteogenesisthan the CaCO3/agarose gel, especially after 8weeks. The same could be said for borderlinesbetween the regenerative bone and the native bone.HAp/agarose gel was barely a radiolucent line

between them compared to CaCO3/agarose gel[Fig. 4(c)].

In contrast, the Agarose and Defect group clearlyshowed more radiolucent images than the compositegels at 8 weeks after implantation. In the Agarosesubgroup in the Homogenized Group, granular ra-dio-opacities seemed to be dotted or occupied thinlines in the defects. On the other hand, in the DiskGroup, there was some opacities seen regeneratingfrom the borders of the defects. Although the Defectgroup showed partial radio-opacities, it had thehighest radiolucent findings.

The radiological qualitative analysis of the regen-erated bone was then performed by DEXA [Fig. 5(a)]and pQCT [Fig. 6(a)]. Figure 5(b) shows the resultsof the analysis by DEXA. These graphs were classi-fied according to the implantable materials and

Figure 4. l-CT images of the calvaria: (a) Immediately after implantation of the composite gel materials; (b) 4 weeks afteroperation of the HAp and CaCO3 in the Homogenized and Disk group; and (c) 8 weeks after operation of the HAp andCaCO3 in the Homogenized and Disk group, and Agarose in the Homogenized and Disk group with Defect as controls.

Figure 5. Results of the DEXA analysis classified by the implant conditions and the implanted materials at 4 or 8 weeksafter implantation. (a) Schematic diagram; (b) Density (mg/cm2). [Color figure can be viewed in the online issue, which isavailable at www.interscience.wiley.com.]

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implanted forms as compared with the controls.These results indicated that both composite gelsshowed higher bone density than the Agarose andDefect groups after 8 weeks. The HAp/agarose gelin the Disk Group seemed to be already formingbone after 4 weeks as compared to the CaCO3/aga-rose gel. Meanwhile, the CaCO3/agarose gels inboth the Homogenized and Disk Groups showedsignificantly increased bone density after 4–8 weeksas compared with the HAp/agarose gel.

Figure 6(b) shows the results for density from thepQCT analysis (data of area not shown). This graphwas also classified according to the implantablematerials and implanted forms as compared withthe controls, and there was a similar trend to theDEXA analysis. This meant that after 8 weeks, bothimplantable materials were superior to the controls.The CaCO3/agarose gel in both Groups showedhigher growth in terms of area. Amazingly, thetotal area of bone recovered was significantly higherin comparison with the Defect site at 8 weeks.Unlike the radiographs, a comparison of the DiskGroup with the Homogenized Group in both DEXAand pQCT analysis revealed that almost all speci-mens in the Disk Group seemed to be consistentlysuperior. On the whole, using agarose gel compo-sites showed better results than the Agarose andDefect group at 8 weeks after implantation in termsof both area and density.

Histological Observations

Figure 7 shows light microscopic images of sec-tions from each defect border. Mainly, there weresome of characteristic findings after 4 and 8 weeksin particular.

The radiological findings were confirmed with thehistological analysis of new bone formation in bothHAp and CaCO3/agarose gels, as detected withH&E staining. It revealed the presence of calcifiedbone as well as osteoid within the regenerate andosteoblasts lined around them, without any fibrousconnective tissues between the biomaterials and theoriginal bone. In general, the adjacent tissuesshowed no signs of inflammation, and there was noaccumulation of seroma. Halfway through the regen-eration, scattered histiocytes could be observed butwere not severe. There were no signs of acuteinflammation or any foreign-body giant cells.

In particular, in the Homogenized Group after 4weeks, new bone formation derived from the bonyborders was observed directed towards the center ofthe defect in both materials [Fig. 7(a)]. In addition,there was a similar histological appearance of aga-rose gel around the new bone bridges. Many bridg-ings occurred in the former defects with mature orimmature bone. Each new bone growth appeared tostretch, expand and bind to each other from the mar-gins of the injury, and bridged across the implantedmaterials with blood vessels. The size of the bridg-ings in the CaCO3/agarose gel [Fig. 7(a,b)] tended tobe thicker than that of the HAp/agarose gel [Fig.7(a)]. The presence of osteoblasts, blood vessels andosteoid tissue could be seen along the marginal armsof those bridges.

In contrast, in the Disk Group after 4 weeks, therewas almost none of this new bone bridging. In fact,although the HAp/agarose gel in this Group eventu-ally showed osteogenesis up to subperiosteum after8 weeks like in the Homogenized Group, it seemedmuch more like granulation tissue than in theHomogenized Group or the Disk Group for theCaCO3/agarose gel. It looked like normal bone inthe repair phase [Fig. 7(b)].

Figure 6. Results of the pQCT analysis classified by the implant conditions and the implanted materials at 4 or 8 weeksafter implantation. (a) Schematic diagram; (b) Density (mg/cm3). [Color figure can be viewed in the online issue, which isavailable at www.interscience.wiley.com.]



There were new bone formations even in thecontrol specimens with severe curved bone trabecu-lae. Only in the Homogenized Group of Agarose,relatively strong inflammatory reaction was observedlocally.

DISCUSSION

Bone grafts are performed to enhance the healingof mandibular fusions, cystic disease or tumors, andto regenerate bone in osseous defects, malformationsand atrophies.29 There are literatures that addressesthese demands, and approximately 60% of these pro-cedures use autografts, 30% use allogeneic products

such as demineralized bone matrix and 10% usesynthetic alloplastic materials such as calcium phos-phate-based ceramics and pastes, collagen or fibrinpolymers, synthetic polymers, and various combina-tions.30–32 We mentioned the use of autogenous bonegrafts limited source of graft material previously. Incontrast, the allograft and xenograft are good substi-tutes of autogenous bone graft. However, there arepotentials of transferring pathogens and difficultiesof shaping into the desired form. Most of syntheticimplants are not biodegradable and may result instress shielding in the surrounding bone or fatiguefailure of the implant. Now, therefore, the search forimproved materials for repairing bone defects con-tinues.33

Figure 7. Histological results. (a) Homogenized Group of composite gel materials at 4 weeks after implantation;(b) Homogenized and Disk Group of composite gel materials at 8 weeks after implantation. [Color figure can be viewedin the online issue, which is available at www.interscience.wiley.com.]

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In particular, it is well known that calcium phos-phate, which is the main constituent of bone andteeth mineral, exists as HAp. It has been studiedextensively, and clinically used as a bone substitutematerial such as a dental implant, bone graft substi-tute, and a coating material for prosthetic joints,because of its excellent osteoconductivity and bioac-tivity.34 However, highly crystalline HAp is not bio-degradable at all or only to a very small extent.Therefore, its use is of limited clinical application.

The most common chemical form of HAp isCa10(PO4)6(OH)2, but the calcium, phosphate, andhydroxyl ions are often replaced by a variety of ionsor even absent in a real bone.35 It is recognized thatthe crystalline nature of natural bone’s calcium phos-phate is immature, and bones or teeth often includecarbonate in them. It is unclear how these mineralcomponents are involved in bone repair. However,there is one study reporting that during the processof healing a bone fracture, many factors wereinvolved, and the mineral components that formedaround the bone defect appeared in a hematoma atearlier stages of the acute inflammatory phase. As aresult of the XRD analysis, the mineral crystals werefound to be apatite, and they continued growingover time.36 These facts suggest that the bone min-eral components observed during the earlier stagesof bone injury become deeply engaged in the repairprocess. For that reason, our biodegradable materialscould play an important role as a scaffold to furtherpromote reparation by releasing bone minerals suchas calcium or phosphate ions as soon as possible af-ter injury.

The crystallinity of the HAp formed in the agarosegels is relatively low, and their intercrystalline bind-ing strength is not strong at this time. Thus, it appearsto be more similar to natural bone apatite than thepre-existing, high-crystallinity sintered HAp.15 There-fore, in this study, we have developed a bone-likebiomaterial that also has good biodegradability.

In contrast, CaCO3 is also known to have bioactiv-ities such as cell or hard tissue compatibility, andbiodegradable properties. As indicated previously,there is a report that bone formation with CaCO3 iscomparable to bioactive HAp in vivo, and CaCO3

implants showed better biodegradability than HApimplants. Although CaCO3 has excellent capabilities,polymer/CaCO3 composite materials have not beenconsidered for bone regeneration.

In this study, the resulting HAp and CaCO3 par-ticles were not mono-disperse. The change in theparticle size is crucial to the specific surface area.Thus, the dissolution rate might be affected. TheFigure 2(c) shows a mixture of small pieces andmultiple small pieces coalesce into large units. Aboutthis, we think it is of advantage when they areimplanted in vivo. Because varying size particle make

it possible to plug the bony defect more densely. It isfavorable property for bone formation that may giveit high osteoconductivity by easily mixed with factorsof osteogenesis in the bone cavity.

The XRD patterns from the composite gels indi-cated peaks typical of calcite, which is the most sta-ble crystalline form of CaCO3. The peaks were sharp,suggesting that the CaCO3 formed in the agarose gelwas of higher crystallinity than HAp. Furthermore, alarge amount of CaCO3 was deposited in the agarosegel as compared with HAp formation.

We observed that both HAp and CaCO3/agarosecomposite gels could function as a bone graftingmaterial as tested by several kinds of analyses,nevertheless in the present study, differencesbetween the two biomaterials were not dramatic.

There was not much objective difference betweenthe HAp or CaCO3/agarose gels and the controlson the radiographic comparison. Therefore, we usedDEXA and pQCT to further compare the quantitiesof the new bones. As a result of these analyses, thecomposite materials were all better than the Agaroseand the Defect site after 8 weeks. In fact, it isknown that an area of new bone grows andexpands ahead of its achieving the appropriate den-sity during the bony repair process. In this study,the newly-formed bones from the biomaterials wereof superior area to the Agarose and the Defect.Moreover, bone density was higher than the con-trols. This suggested that the HAp and CaCO3/aga-rose gels tended to accelerate the speed of produc-ing more new bone associated with bone regenera-tion. In contrast, in comparing the Defect andAgarose sites, the Agarose tended to be better thanthe Defect. This result seemed to suggest that thepotential for the agarose gel to serve as a scaffoldfor natural repairing process.

We hypothesized that the presence of the agarosegel may be responsible for the difference betweenthe homogenized biomaterial versus the originalpunched-out disk form. In the Homogenized Group,in which the pasted gel was prepared in advance, itwas possible to mix the surrounding cells and mate-rials immediately after implantation, and calciumions were available from the composite gels at once.Meanwhile, in the Disk Group, we expected that thesurrounding cells would use the agarose meshedstructures as a scaffold to form new bone. Therefore,we observed l-CT images like those in Figure 4(b)as if minerals were weeping from the stereoscopicagarose network.

The histological results also seemed to suggestusing agarose gel as the base material made it diffi-cult to proceed into the defect area in the DiskGroups. It was uncertain, but the specimens in theDisk Group seemed to fill the defects with moregranular tissue than the Homogenized Group, spe-



cifically after 4 weeks. This may be due to theremaining pieces of gel, which enabled the newbone bridging to stretch after 4 weeks, and thus thegranulation tissue might invade earlier than theosteogenic cells, as shown by the histology from Fig-ure 7(a). However, the agarose gel disk did performan important function as a scaffold, because after 8weeks the defects were mostly filled with new bonein almost all gels filled cases.

Taking agarose gel as the base material, the reasonwhy the chronic inflammation in the specimens wasscattered with histiocytes may be explained partlyby the fact that the particles from the agarose gelswere decayed and phagocytized. However, thesebio-based agarose gels have good safety, and at 8weeks after implantation, the HAp or CaCO3/aga-rose composite gels were completely absorbed andreplaced by newly-formed bone. In this study, thegels were sterilized by gamma-ray irradiation. Thepolymer backbone of agarose might be broken insome parts; thus, the composite gel would start deg-radation ahead of time.

In the bony defects created in the rats, the agarosecomposite gel formed a cohesive mass when in con-tact with the blood of the animal, a feature thatensured its accommodation in the defect. It alsoseemed to assist in controlling the bleeding due tothe intrinsic properties of the agarose gel. Moreover,it was easily mixed, transferred and packed, andwas well-contained in the defect site. Since it wasnot as solid as calcium phosphate cement, there wassufficient time to shape the biomaterial during theoperation. Thus, we realized that this biomaterialwas easier to handle.

We should reconsider the method used to com-pare HAp with CaCO3, and compare the alterationscaused by regulating the weight percentage of aga-rose gel as the base material. It may be advisablealso to observe the osteogenesis at shorter intervals.However, the important conclusions of the presentstudy are based on a variety of analyses, and suggestHAp and CaCO3/agarose composite gels may playimportant roles as alternative biodegradable bonegrafting filler materials for autogenous bone inhumans. This is the first report to document whathappens when HAp/agarose composite gels areused as a scaffold for membranous bone, andwhether the CaCO3/agarose composite gel would bea valid biomaterial.

CONCLUSIONS

This study revealed that HAp and CaCO3/agarosecomposite gels formed by an alternate soaking pro-cess represent biocompatible and biodegradablebone grafting biomaterials of the filling type. We

designed a rat parietal bone defect model, andimplanted them with two application forms. Wethen studied the repair of these bone defects to eval-uate the biological behavior of these biomaterials.

The results of the quantitative analysis indicatedthat the HAp and CaCO3/agarose composite gelscontribute to increase volume of new bone forma-tion. Moreover, in this study, we also visuallydetermined the difference caused by the applicationforms in connection with agarose gel as the basematerial.

The authors especially thank Dr. Takuya Ishimoto,Graduate School of Engineering, Osaka University for pro-viding expertise and support in radiological analyses.

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