bone repair of critical size defects treated with mussel powder associated or not with bovine bone...

lable at ScienceDirect

Journal of Cranio-Maxillo-Facial Surgery xxx (2013) 1e6

Contents lists avai

Journal of Cranio-Maxillo-Facial Surgery

journal homepage: www.jcmfs.com

Bone repair of critical size defects treated with mussel powderassociated or not with bovine bone graft: Histologic andhistomorphometric study in rat calvaria

Daniel Rizzo Trotta, Clayton Gorny Jr., João César Zielak, Carla Castiglia Gonzaga,Allan Fernando Giovanini, Tatiana Miranda Deliberador*

Positivo University, Curitiba, PR, Brazil

a r t i c l e i n f o

Article history:Paper received 26 April 2013Accepted 4 November 2013

Keywords:RatsBone regenerationCritical defectsBone transplantationPernaMytilus edulis

* Corresponding author. Professor Pedro Viriato PariComprido, 81280-330 Curitiba, PR, Brazil. Tel.: þ55 413082.

E-mail address: [email protected] (T.M. Del

1010-5182/$ e see front matter � 2013 European Asshttp://dx.doi.org/10.1016/j.jcms.2013.11.004

Please cite this article in press as: Trotta DR,bone graft: Histologic and histomorphometj.jcms.2013.11.004

a b s t r a c t

The objective of this study was to evaluate the bone repair of critical size defects treated with musselpowder with or without additional bovine bone. Critical size defects of 5 mm were realized in thecalvaria of 70 rats, which were randomly divided in 5 groups e Control (C), Autogenous Bone (AB),Mussel Powder (MP), Mussel Powder and Bovine Bone (MP-BB) and Bovine Bone (BB). Histological andhistomorphometric analysis were performed 30 and 90 days after the surgical procedures (ANOVA eTukey p < 0.05). After 30 days, the measures of remaining particles were: 28.36% (MP-BB), 26.63% (BB)and 8.64% (MP) with a statistically significant difference between BB and MP. The percentage of osseousmatrix after 30 days was, AB (55.17%), 23.31% (BB), 11.66% (MP) and 10.71% (MP-BB) with statisticallysignificant differences among all groups. After 90 days the figures were 25.05% (BB), 21.53% (MP-BB) and1.97% (MP) with statistically significant differences between MP-BB and MP. Percentages of new boneformation after 90 days were 89.47% (AB), 35.70% (BB), 26.48% (MP-BB) and 7.37% (MP) with statisticallysignificant differences between AB and the other groups.

Within the limits of this study, we conclude that mussel powder, with or without additional bovinebone, did not induce new bone formation and did not repair critical size defects in rat calvaria.

� 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rightsreserved.

1. Introduction

The repairing of major bone losses is still a great challenge formodern Regenerative Medicine, especially in craniomaxillofacialand orthopaedic surgery. Despite the great potential of the bonetissue for repair, in some situations, according to the size of thedefect, regeneration cannot be completely achieved because thedefect may be invaded by surrounding connective tissue, which hasa faster speed of cellular proliferation and migration than those ofbone tissue (Melcher, 1976). Thus, the use of bone grafts should beconsidered, in conjunction with bone repair.

Bone grafts are classified into autogenous (the same individualis the donor and receptor of the graft), allogenous (donor and re-ceptor individuals of the same species), xenogenous (donor andreceptor individuals of different species) or alloplastic (synthetic)(Castro-Silva et al., 2009).

got de Souza St, 5300, Campo99764948; fax: þ55 413317

iberador).

ociation for Cranio-Maxillo-Facial

et al., Bone repair of critical siric study in rat calvaria, Journ

Although autogenous bone has been considered the first choiceor “gold standard” for bone grafts because of its osteogenic,osteoinductive, and osteoconductive potentials, its availability islimited and their use results in greater morbidity (increased sen-sibility) to the patients. Therefore, the predictability of the clinicalresults with the use of allogenous, xenogenous and alloplastic bonegrafts leads to consideration of these as valid options for tissuerepair, mainly because of the lack of volume resorption, one sur-gical site, and decreased post-operative morbidity (Daelemanset al., 1997).

Xenografts have been produced for more than twenty years bybiomaterial companies around the world, who have invested in thedevelopment of biocompatible materials capable of increasing and/or accelerating the bone repairing (Castro-Silva et al., 2009). Themost common application of xenografts in Dentistry has been inthe treatment of periodontal defects (Richardson et al., 1999), post-extraction sites (Gonçalves et al., 2009), maxillary sinus augmen-tation (Gonçalves et al., 2009) and alveolar ridge augmentation(Gonçalves et al., 2005). These grafts guide the new bone formation,that is, they have been considered as osteoconductive grafts.

Surgery. Published by Elsevier Ltd. All rights reserved.

ze defects treated with mussel powder associated or not with bovineal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/

mailto:[email protected]

www.sciencedirect.com/science/journal/10105182

http://www.jcmfs.com

http://dx.doi.org/10.1016/j.jcms.2013.11.004



D.R. Trotta et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (2013) 1e62

Xenografts of bovine origin, properly processed, biocompatibleand osteoconductive have a separate role in helping bone repair. In aretrospective study conducted in humans, Block et al. (2012) per-formed surgery for horizontal augmentation of the anterior regionof the maxilla with a particulate bovine xenograft and membrane.Through tomographic analyses, Block et al. (2012) concluded thatthe use of particulate bovine bone graft associated with membranewas efficient in the horizontal augmentation of the anterior maxilla,which was stable during the study period of 500 days.

Among the bovine commercially available xenografts, Bio-Oss�

(Geistlich Biomaterials, Wolhusen, Switzerland) is the mostcommonly used. In Brazil, Orthogen� (Genius, Baumer, São Paulo,Brazil) is one of the viable options for particulate xenografts ofbovine origin. In addition to the bovinexenografts, other sources canbe used to obtain the grafts, such as porcine (Suckow et al., 1999),equine (Di Stefano et al., 2009), marine algae (Buser, 2009) or biocoral grafts (Yukna and Yukna,1998; Shafiei-Sarvestani et al., 2012).

Roux et al. (1988) used coral fragments as bone substitutes incranial surgery. Of the 167 grafts implanted, 150 were used to filldefects of 10 mm of diameter performedwith burs; fivewere largerimplants (length of 20e40 mm) to repair cranial defects and 12were coral blocks to reconstruct the floor of the anterior nasal fossa.According to the authors, the coral implants are biocompatible andtheir resorption occurs while new bone tissue is formed. Roux et al.(1988) concluded that corals are promising biomaterials for the usein cranial reconstructive surgeries. Currently few studies areavailable on the use of corals as bone substitutes.

Similar to the corals, mussels have been considered as calciumsources, because 96% of its chemical composition is composed ofcalcium oxide (CaO). As far as we could find out there are no studieson theuseofmussels as analternativebiomaterial forbone repairing.

This present study aimed to assess the bone repairing of defectsof critical size created in the rat calvaria, treated with musselpowder graft with or without bovine bone graft histologically.

2. Material and methods

This present study was submitted and approved by the EthicalCommittee in Research of the Positivo University, under protocolsnumber 015/2011 and 34/2011. Seventy male rats (Rattus norvegi-cus, albinus, Wistar) were used. They had a mean age of 7 monthswith weight ranging from 365 to 480 g. The animals were randomlydivided into five groups according to Table 1.

2.1. Anaesthetic protocol

For the experimental surgical procedures, the animals werepositioned inside a campanula individually and anaesthesia wasinduced with oxygen and isoflurane (Cristália, Itapira, SP, Brazil)followed by an intramuscular injection on the posterior part of thethigh with 2.3 g xylazine (0.52 mg/kg) (Vetbrands, Paulínia, SP,Brazil) and 1.16 g ketamine (1.04 mg/kg) (Vetbrands, Paulínia, SP,Brazil). Anaesthesia was maintained with isoflurane vaporization(Cristália, Itapira, SP, Brazil) by facial mask if necessary.

Table 1Distribution of groups: C (Control); AB (Autogenous bone); MP (Mussel powder);MP-BB (Mussel powder þ Bovine bone graft) and BB (Bovine bone graft).

Groups 30 days 90 days Treatment

GROUP C 07 animals 07 animals Without treatmentGROUP AB 07 animals 07 animals Autogenous boneGROUP MP 07 animals 07 animals Mussel powderGROUP MP-BB 07 animals 07 animals Mussel powder þ

bovine bone graftGROUP BB 07 animals 07 animals Bovine bone graft

Please cite this article in press as: Trotta DR, et al., Bone repair of critical sbone graft: Histologic and histomorphometric study in rat calvaria, Journj.jcms.2013.11.004

2.2. Surgical procedures

After induction of anaesthesia, shaving and antisepsis of theareas to be operated (calvaria) was carried out. After antisepsis, a“U” shaped incisionwas performedwith the aid of a size 15c scalpelblade for surgical access to the calvaria area and a flap was raisedposteriorly.

A critical size defect (CSD) of 5 mm of diameter (Fig. 1) (Schmitzand Hollinger, 1986) was created with a trephine bur (Neodent,Curitiba, PR, Brazil) mounted in a contra-angle handpiece forimplant (20:1, Kavo, Joinville, SC, Brazil), under copious irrigationwith sterile saline solution.

With the aid of a millimetric probe (PCPUNC 15, Hu-Friedy,Chicago, IL, USA) and a size 701 tapered bur mounted in straighthandpiece, two “L-shape” marks were performed at 2 mm towardsanterior direction and 2 mm towards posterior direction to thesurgical defect margins. The long axis of each “L”was localized on alongitudinal imaginary line which divided the surgical defect byhalf and the marks were then filled with dental amalgam (Permitee SDI, Victoria, Australia). These marks were created to identify themiddle of the original surgical defect during the laboratorial pro-cessing and to locate the original bone margins of the surgicaldefects during the histological and histomorphometric analyses.

In group C, the surgical defect was filled with only the blood clotas a negative control. In group AB, the defect was filled with tritu-rated autogenous bone graft as a positive control. The autogenousbonewas gathered from the calvaria portion that had been removedto create the surgical bone defect. The bonewas ground upwith theaid of a pestle-type bone grinder (Kopp, Curitiba, PR, Brazil).

In group MP, the defects were filled with mussel powder. Thiswas obtained from Perna mussels or brown mussel, which is agenus of freshwater mussel (family MYTILIDAE, class BIVALVIA).The molluscum was removed from its shells, which were washedwith neutral detergent and cleaned with a brush. The shells werethenwashed in running water and immersed into water for 30 minand again washed individually. Next, the shells were stored in arefrigerator at 3 �C up to trituration.

The shells were initially ground up in a hand grinder (Kopp,Curitiba/PR, Brazil), resulting in a powder, so-called dirty powder.Next, the dirty powder was ground up in an electrical motor(Polidora, Knebel, Porto Alegre/RS, Brazil) resulting in a cleanpowder. Then, the clean powder was manually sieved through 60meshes resulting in themussel powder used to fill the bone defects.The mussel powder was placed onto a Petri plate, wrapped intosurgical paper and sterilized in autoclave (Cristófoli, CampoMourão/PR, Brazil). This powder was then inserted into the bonedefects with the aid of a Molt surgical curette.

Fig. 1. A critical size defect (CSD) of 5 mm of diameter in rat calvaria.

ize defects treated with mussel powder associated or not with bovineal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/

D.R. Trotta et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (2013) 1e6 3

In Group BB, the defects were filled with bovine bone graft(Orthogen�) and in Group MP-BB the defects were filled withmussel powder and bovine bone graft (Orthogen�) at 1:1 ratio.

After filling the defects, the soft tissues were repositioned andthe wounds closed with silk sutures (4-0 Silk, Ethicon, Johnson &Johnson, São José dos Campos, SP, Brazil).

To control the post-operative pain, the animals were givenmorphine sulphate (3 mg/kg) (União Química, Jabaquara, SP,Brazil), intramuscularly at the end of the surgery. The analgesia wasmaintained with 20 drops of paracetamol (200 mg/kg) diluted into400 ml of water placed into water drinker for 3 days.

2.3. Euthanasias

The groups were divided into 2 subgroups (n¼ 7) for euthanasiaat 30 and 90 days after the surgical procedure. To perform theeuthanasia, the rats were placed into a gas chamber (CO2) and keptfor 10 min.

2.4. Tissue processing

The original surgical defect area and the surrounding tissueswere removed in blocks. The pieces were fixed in neutral 10%formalin, washed in running water and decalcified in 20% formicacid solution. After the decalcification, each piece was sectioned inhalf, parallel to the sagittal suture. Longitudinal serial cuts, with5 mm of thickness, starting from the centre of the original surgicaldefect were obtained. The cuts were stainedwith haematoxylin andeosin for analyses through light microscopy (HE).

2.5. Histological and histomorphometric analysis

Two histological cuts from each animal was selected repre-senting the centre of the original surgical defect, for histologicaland histomorphometric analyses. The analyses were performed bya single trained operator. For the histological analysis, the imageswere analysed using an optical microscope (021/3 Quimis, Dia-dema, SP, Brazil) and the following parameters were verified: theclosing of the bone defect, the type of newly formed bone, thecharacteristics of the conjunctive tissue, the presence of the osteoidmatrix, the presence of the chronic or acute inflammatory infiltrate,the progression of the repair type present in the surgically createddefect and the thickness of the newly formed tissues in relation tothe original bone from the calvaria. The closing of the defect wasconsidered as complete when all its extension was filled by newlyformed bone. Still, the presence of the remaining particles of themussel powder and bovine bone graft was also evaluated.

To conduct the histomorphometric analysis, the laminas wereserially photographed with the aid of a digital camera coupled to amicroscope with �40 magnification, then grouped together andimported into in Microsoft Powerpoint� software (Microsoft Cor-poration, WA, USA), to result in a single and continuous imagecomprising the two borders of the defect. With the aid of ImageJ�

software version 1.6.0 (Wayne Rasband (NIH), Bethesda, MA, USA)the following histomorphometric measurements were executed:

1. Total Area (TA): measurement of the total area of the defectsurgically created. This measurement was performed fromborder to border of the original defect including the variation ofthe thickness of the calvaria of each animal.

2. Area of Remaining Particles (RP): measurement of the remain-ing particles of the biomaterials used (mussel powder and/orbovine bone graft).

3. Area of Osteoid Matrix (OM): measurement performed for theareas of newly formed bone.

Please cite this article in press as: Trotta DR, et al., Bone repair of critical sibone graft: Histologic and histomorphometric study in rat calvaria, Journj.jcms.2013.11.004

The Total Area (TA) was measured in mm2 and was consideredas 100% of the area to be analysed. The areas of RP and OM werealso measured in mm2 and calculated as a percentage of TA, for-mulas according to the following formulas: RP (mm2)/TA(mm2) � 100 and OM (mm2)/TA (mm2) � 100.

2.6. Statistical analysis

The normality of the data was confirmed (ShapiroeWilk test(p > 0.05)). To evaluate if there were significant statistically dif-ferences among the groups, the data were analysed using analysisof variance (ANOVA), followed by Tukey test. The level of signifi-cance adopted was p < 0.05.

3. Results

3.1. Histological analysis

3.1.1. Group Control (C)Group C e 30 and 90 days post-operative e At these two times,

the results of the histological analysis were similar. The artificialdefect was mostly filled by collagen fibres disposed parallel to thewound surface (Fig. 2A and B).

3.1.2. Group autogenous bone (AB)Group AB e 30 days post-operative e Complete bone closure of

the surgical defect was not seen at this period. The extent of thedefect was mainly occupied by connective tissue, richly cellularizedwith areas of compact newly formed bone, which mimicked Ha-versian bone (Fig. 2C).

Group AB 90 days post-operative e Complete closure of the sur-gical defect was seen in 2 of 7 specimens assessed. In the otheranimals of the group, the defect was mostly occupied by compactHaversian bone tissue, in which areas of discrete medullarymaturation was present (Fig. 2D).

3.1.3. Group mussel powder (MP)Group MP 30 and 90 days post-operative e The defect extension

was mostly occupied by collagen fibres within the few remainingparticles of the grafted material (mussel powder), with few areas ofosteoid matrix (Fig. 2E and F).

3.1.4. Group mussel powder þ bovine bone graft (MP-BB)Group MP-BB 30 and 90 days post-operative e The extension of

the defect was mostly occupied by fibrous tissue with sparsecollagen fibres and loose conjunctive tissue within the remainingparticles of the grafted material (mussel powder and bovine bonegraft) and with a few areas of osteoid matrix (Fig. 2G). At 90 dayspost-operative, we saw the maintenance of the remaining particlesof bovine bone graft and the absorption of the particles of musselpowder in comparison with the findings at 30 days post-operative(Fig. 2H).

3.1.5. Group bovine bone graft (BB)Group BB e 30 and 90 days post-operative e The extension of the

defect was mostly occupied by dense and organized collagen fibreswithin the remaining particles of the grafted material (bovine bonegraft) with areas of osteoid matrix close to both the borders of thedefects and the remaining particles, indicating the osteoconductiveactivity of the material (Fig. 2I and J).

3.2. Histomorphometric analysis

The results of the histomorphometric analysis of all groups at 30post-operative days are seen in Table 2. Regarding the remaining


Fig. 2. A and B (Group C e 30 and 90 post-operative days respectively) the artificial defect was filled in its mostly part by collagen fibres disposed parallely to the wound surface. C(Group AB e 30 post-operative days) the defect extension, mostly, was occupied by connective tissue richly cellularized with areas of compact newly formed bone, which mimicsthe formation of the Haversian bone (stars). D (Group AB e 90 post-operative days) 2 of 7 specimens it was identified the complete bone closing. E and F (Group MP e 30 and 90post-operative days respectively) the defect extension was mostly occupied by collagen fibres (stars) within few remaining particles (arrows) of the grafted material (musselpowder), with few areas of osteoid matrix (triangle). G (Group MP-BB e 30 post-operative days) the extension of the defect, mostly, was occupied by fibrous tissue (stars) withsparse collagen fibres and loose conjunctive tissue within remaining particles of the grafted material (mussel powder (blue arrows) and bovine bone graft (black arrows)) with fewareas of osteoid matrix. H (Group MP-BB 90 post-operative days) the extension of the defect, mostly, was occupied by fibrous tissue (stars) and it could be observed that themaintenance of the remaining particles of bovine bone graft (black arrows) and the absorption of the particles of mussel powder. I and J (Group BB e 30 and 90 post-operative days)it was observed dense and organized collagen fibres (stars) within the remaining particles of the bovine bone graft (arrows) with areas of osteoid matrix (triangle).


particles, the groups MP-BB and BB showed the greatest amount ofremaining material. For the osteoid matrix measurement, thegroup AB exhibited the greatest amount of newly formed bonewithstatistically significant differences among the other groups. GroupC did not show osteoid matrix formation at 30 and 90 days post-operative.

The results of the histomorphometric analysis of all groups at 90days post-operative are seen in Table 3. Regarding the remainingparticles, the results were similar to those at 30 days post-operative. For the osteoid matrix measurement, group AB showedthe greatest amount of newly formed bone with statistically


significant differences to the other groups. The group BB showedthe second greatest amount of newly formed bone with a statisti-cally significant difference to the other groups. For the groups MP-BB and BB, the amount of newly formed was similar, without nostatistical difference to the other groups. The group MP exhibitedthe smallest amount of osteoid matrix.

4. Discussion

The constant search for new biomaterials that may fill or evenrepair large bone defects; be biocompatible with little or no acute


Table 2Mean and standard deviation (SD) percentage of the measurement of RP and OMamong groups at 30 post-operative days.

Group RP measurement (%) SD (%) OM measurement (%) SD (%)

C 0 c 0 0 d 0AB 0 c 0 55.17 a �4.35MP 8.64 b �3.35 11.66 c �4.54MP-BB 28.36 a �4.58 10.71 c �4.75BB 26.63 a �5.97 23.31 b �2.97

RP¼ remaining particles; OM¼ osteoid matrix; C¼ control; AB¼ autogenous bone;MP¼mussel powder; MP-BB¼mussel powder and bovine bone; BB¼ bovine bone.The letters (a, b, c) were considered as statistically significant differences (p < 0.05).For all groups analysed, the significant statistical differences values revealedp < 0.01.

D.R. Trotta et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (2013) 1e6 5

inflammatory reaction; and deliver results similar to autogenousbone has resulted in the development of several synthetic or nat-ural bone substitutes (Dorozhkin, 2011). The aim of this study wasto evaluate the repair capacity of a new xenograft (mussel powder)associated with or without bovine bone graft.

Marine algae and bio corals have been used as xeno-bone graftsbecause they are calcium sources and stimulate the osteoid matrixformation, and are considered as osteoconductive. As far as we areaware there are no studies on the use of mussel as a xenogenousbone graft. Most part of the composition of brown mussel(employed in this study) is calcium oxide. Additionally, the musselpowder is easy to obtain and process with good availability and lowcost. In this study the group MP did not result in an inflammatoryprocess, demonstrating the biocompatibility of the mussel powdergraft. Despite this it did not demonstrate osteoconductive potentialas only 7.37% of the surgical defects were filled with osteoid matrixat 90 post-operative days (Table 3).

This study also aimed to compare the results of mussel powderwith those of another xenogenous biomaterial (bovine bone graft),Orthogen�. We chose Orthogen�, available in particles of lyophi-lized bone, because it was a biomaterial of recent development,with little information in the literature regarding to its use in clin-ical and histological studies. In this study, the group BB and MP-BBshowed that the bovine bone graft was biocompatible, which cor-roborates with the study of Silva et al. (2008) who also demon-strated the biocompatibility of the graft type. In group BB, at 90 dayspost-operative, we saw a little osteoconductive activity of thismaterial with the presence of the osteoid matrix surrounding theremaining particles which were within collagen fibres. This resultconfirms those of recent studies which also used bovine bone graftto achieve bone repair (Silva et al., 2008; Develioglu et al., 2009).

The study of Silva et al. (2012) demonstrated that Bio-Oss�

(bovine bone graft) favoured the formation of 13.64% of non-vitalmineralized tissue, 6.31% of vital mineralized tissue and 81.41% ofnon-mineralized tissue in the repairing of the bone defects in rabbitcalvaria. In this study, a similar result was found in group BB with35.70% of formation of osteoid matrix at 90 days post-operative.

Table 3Mean and standard deviation (SD) percentage of the measurement of RP and OMamong groups at 90 post-operative days.

Group RP measurement (%) SD (%) OM measurement (%) SD (%)

C 0 c 0 0 d 0AB 0 c 0 89.47 a �8.66MP 1.97 b �2.39 7.37 c �4.30MP-BB 21.53 a �11.00 26.48 b �9.73BB 25.05 a �6.75 35.70 b �8.57

RP¼ remaining particles; OM¼ osteoid matrix; C¼ control; AB¼ autogenous bone;MP¼mussel powder; MP-BB¼mussel powder and bovine bone; BB¼ bovine bone.The letters (a, b, c, d) were considered as statistically significant differences(p < 0.05). For all groups analysed, the significant statistical differences valuesrevealed p < 0.01.

Please cite this article in press as: Trotta DR, et al., Bone repair of critical sibone graft: Histologic and histomorphometric study in rat calvaria, Journj.jcms.2013.11.004

Because they are larger particles and exhibit a porous architec-ture, the group BB acts as a scaffold for bone deposition, which leadsto a greater formation of osteoidmatrix,with statistically significantdifferences between groups C, MP and MP-BB at 30 days post-operative. Despite this it was not possible to confirm whetherthere was satisfactory and complete bone repair through new bonein any of the groups and times evaluated, when compared withgroup AB, in which the formation of the osteoid matrix was signif-icantly greater than that of the other groups at both post-operativetimes (Tables 2 and 3), agreeing with some authors (Silva et al.,2008; Paulo et al., 2011) and confirming that the autogenous bonegraft is the gold standard for bone grafting. A determining factor forthis difference regarding to the formation of the osteoid matrixbetween groupsMPandBB could have been the sizes of the particlesand their uniformity. The particles of Orthogen� are larger andmoreheterogeneous, ranging from 91.2 to 497.8 mm (Galia et al., 2011),while the particles of the mussel powder are very small (about10 mm) and homogeneous, which could have disadvantaged thebone deposition due to the fast absorption of the particles withoutan osteoconductive action, despite it being a source of calcium(Jardelino et al., 2009; Wang et al., 2011). According to Wang et al.(2011), a biomaterial with nano and macro particles and withdifferent sizes of porous favours the apposition, migration andpenetration of the bone cells, resulting in high rates of cellularproliferation and consequently more new bone formation. The as-sociation of mussel powder with bovine bone graft (group MP-BB)demonstrated an osteoid matrix formation similar to that of groupBB at 90 post-operative days, showing that the bovine bone graftand not the mussel powder graft accounts for this result.

Regarding the remaining particles, the results of this researchshowed that the bovine bone graft particles, both at 30 and 90 dayspost-operative, were still present (group BB and MP-BB), with verylittle resorbed over time, characterizing a biomaterial of slow ab-sorption. According to Macneill et al. (1999), the graft materialsrequiring longer time periods for their complete resorption reducethe total amount of newly formed bone, because of their continuedpresence. On the other hand, resorption of the particles wasobserved in the groupMP from 30 to 90 days post-operative (Fig. 2Eand F and Tables 2 and 3), showing that the mussel powder graft isan absorbable graft material over time. This is also evident in groupMP-BB, in which the reduction of the number of particles is visibledue to the resorption of the particles of the mussel powder.

Some biomaterials have been considered as capable of con-ducting bone regeneration; so the resorption of these materials,and consequently of the new bone formation, should occursimultaneously; however, the mechanism of this replacement isnot still clear, but the difference in the resorption rates may have aneffect on the amount of newly formed bone (Aaboe et al., 2000). Ingroup MP, although the material had been resorbed, it could not beverified whether this resorption contributed to the bone repair ofthe defect.

The calvarium has been very used for more than a century instudies on substitutes for autogenous bone grafts (Takagi and Urist,1982; Bosch et al., 1998; Furlaneto et al., 2007; Messora et al., 2007;Mariano et al., 2010; Nagata et al., 2010). In this study, the calvariumwas selected because of its similarity to bone in the maxillofacialregion and its limited regenerative capacity (Frame, 1980; Dahlinet al., 1991).

The critical size defect (CSD) is defined as the smallest intrabonywound, in a given bone and animal species, which does not heal byitself (Schmitz and Hollinger, 1986). Thus, an experimental bonedefect used to evaluate the bone repair should be large enough toavoid the spontaneous closing by bone tissue, with the formation ofthe fibrous conjunctive tissue instead. Therefore, the potential forbone repairing of a graft or implant can be demonstrated (Frame,



1980). Bosch et al., in 1998, conducted a study on rat calvaria whereit was established that the size of a CSD in these animals is 5 mm, asthe defects did not show complete bone healing after 6 and 12months post-operative. In this study, this was the type of healingfound in group C at 30 and 90 days post-operative.

5. Conclusion

Within the limits of this study, mussel powder graft with orwithout a bovine bone graft cannot be considered as a biomaterialwith osteoconductive potential because it did not induce the for-mation of an osteoid matrix and did not induce the repair of acritical size defect. The size of the particles seemed to be a pre-dominant factor for the reduced formation of osteoid matrix.Further studies with this material are necessary, employing largerand heterogeneous particles to assess the possible osteoconductivepotential of this material.

References

Aaboe M, Schou S, Hjorting-Hansen E, Helbo M, Vikjaer D: Osseointegration ofsubperiosteal implants using bovine bone substitute and various membranes.Clin Oral Implants Res 11(1): 51e58, 2000

Block MS, Ducote CW, Mercante DE: Horizontal augmentation of thin maxillaryridge with bovine particulate xenograft is stable during 500 days of follow-up:preliminary results of 12 consecutive patients. J Oral Maxillofac Surg 70(6):1321e1330, 2012

Bosch C, Melsen B, Vargervik K: Importance of the critical-size bone defect intesting bone-regenerating materials. J Craniofac Surg 9: 310e316, 1998

Buser D: 20 years of guided bone regeneration in implant dentistry, 2nd edn.Quintessence Publishing Co86e87, 2009

Castro-Silva II, Zambuzzi WF, Granjeiro JM: Current overview of the use of xenograftin dentistry. Innov Implant J Biomater Esthet 4(3): 70e75, 2009

Daelemans P, HermansM, Godet F, Malevez C: Autologous bone graft to augment themaxillary sinus in conjunction with immediate endosseous implants: a retro-spective study up to 5 years. Int J Periodontics Restor Dent 17(1): 27e39, 1997

Dahlin C, Alberius P, Linde A: Osteopromotion for cranioplasty. An experimentalstudy in rats using a membrane technique. J Neurosurg 74: 487e491, 1991

Develioglu H, Saraydin SU, Kartal U: The bone-healing effect of a xenograft in a ratcalvarial defect model. Dent Mater J 28(4): 396e400, 2009

Di Stefano DA, Artese L, Iezzi G, Piattelli A, Pagnutti S, Piccirilli M, et al: Alveolar ridgeregeneration with equine spongy bone: a clinical, histological, and immunohis-tochemical case series. Clin Implant Dent Relat Res 11(2): 90e100, 2009

Dorozhkin SV: Biocomposites and hybrid biomaterials based on calcium ortho-phosphates. Biomatter 1(1): 3e56, 2011

Frame JW: A convenient animal model for testing bone substitute materials. J OralSurg 38(3): 176e180, 1980

Furlaneto FAC, Nagata MJH, Fucini SE, Deliberador TM, Okamoto T, Messora MR:Bone healing in critical-size defects treated with bioactive glass/calcium sulfate.A histologic and histometric study in rat calvaria. Clin Oral Implants Res 18:311e318, 2007


Galia CR, Lourenço AL, Rosito R, Macedo CAS, Quaresma LM: Caracterização físico-química do enxerto de osso bovino liofilizado. Rev Bras Ortop 46(4), 2011

Gonçalves F, Granjeiro JM, Cestari TM, Taga R, Oliveira RC, Zanetti RV, et al: Recu-peração de rebordo alveolar com enxerto xenogênico composto Gen-Tech�.Relato de caso clínico. ImplantNews 2(5): 491e497, 2005

Gonçalves F, Hohn A, Granjeiro JM, Silva IIC, Taga R, Cestari TM, et al: Regeneraçãoóssea em odontologia com a utilização do substituto ósseo composto xen-ogênico Gen Mix. ImplantNews 6(4): 373e379, 2009

Jardelino CL, Takamori ER, Santos SRA, Rossi AM, Granjeiro JM: Biocompatibility ofbovine anorganic xenograft. Key Eng Mater 396e398: 3e6, 2009

Macneill SR, Cobb CM, Rapley JW: In vivo comparison of synthetic osseous graftmaterials. A preliminary study. J Clin Periodontol 26: 239e245, 1999

Mariano R, Messora M, de Morais A, Nagata M, Furlaneto F, Avelino C, et al: Bonehealing in critical-size defects treated with platelet-rich plasma: a histologicand histometric study in the calvaria of diabetic rat. Oral Surg Oral Med OralPathol Oral Radiol Endod 109(1): 72e78, 2010

Melcher AH: On the repair potential of periodontal tissue. J Periodontol 47(5): 256e260, 1976

Messora MR, Nagata MJ, Mariano RC, Dornelles RC, Bomfim SR, Fucini SE, et al: Bonehealing in critical-size defects treated with platelet-rich plasma: a histologicand histometric study in rat calvaria. J Periodontal Res 43(2): 217e223, 2008Apr

Nagata MJ, Furlaneto FA, Moretti AJ, Bouguot JE, Ahn CW, Messora MR, et al: Bonehealing in critical-size defects treated with new bioactive glass/calcium sulfate:a histologic and histometric study in rat calvaria. J Biomed Mater Res B ApplBiomater 95(2): 269e275, 2010

Paulo AO, Castro-Silva II, Oliveira DF, Machado MEL, Bonetti-Filho I, Granjeiro JM:Repair of critical-size defects with autogenous periosteum-derived cells com-bined with bovine anorganic apatite/collagen: an experimental study in ratcalvaria. Braz Dent J 22(4), 2011

Richardson CR, Mellonig JT, Brunsvold MA, McDonnell HT, Cochran DL: Clinicalevaluation of Bio-Oss�: a bovine-derived xenograft for the treatment of peri-odontal osseous defects in humans. J Clin Periodontol 26(7): 421e428, 1999

Roux FX, Brasnu D, Loty B, George B, Guillemin G: Madreporic coral: a new bonegraft substitute for cranial surgery. J Neurosurg 69(4): 510e513, 1988

Schmitz JP, Hollinger JO: The critical size defect as an experimental model forcraniomandibulofacial nonunions. Clin Orthop 205: 299e308, 1986

Shafiei-Sarvestani Z, Oryan A, Bigham AS, Meimandi-Parizi A: The effect ofhydroxyapatite-hPRP, and coral-hPRP on bone healing in rabbits: radiological,biomechanical, macroscopic and histopathologic evaluation. Int J Surg 10(2):96e101, 2012

Silva IIC, Pimentel-Soares S, Bittencourt RC, Granjeiro JM: Natural bovine anorganicapatite and collagen presents osteoconductivity and contribute to bone repairof rat calvaria critical size defect. Key Eng Mater 249: 396e398, 2008

Silva MO, Pelegrine AA, Silva AAP, Júnior LRM, Oliveira RM, França SG, et al:Xenograft enriched with autologous bone marrow in inlay reconstructions: atomographic and histomorphometric study in rabbit calvaria. Int J Biomater2012, 2012 Article ID 170520, 7 p., accepted 23 July 2012

Suckow MA, Voytik-Harbin SL, Terril LA, Badylak SF: Enhanced bone regenerationusing porcine small intestinal submucosa. J Invest Surg 12(5): 277e287, 1999

Takagi K, Urist MR: The reaction of the dura to bone morphogenetic protein (BMP)in repair of skull defects. Ann Surg 196(1): 100e109, 1982

Wang S, Falk MM, Rashad A, Saad MM, Marques AC, Almeida RM, et al: Evaluationof 3D nano-macro porous bioactive glass scaffold for hard tissue engineering.J Mater Sci Mater Med 22(5): 1195e1203, 2011

Yukna RA, Yukna CN: A 5-year follow-up of 16 patients treated with corallinecalcium carbonate (BIOCORAL�) bone replacement grafts in infrabony defects.J Clin Periodontol 25: 1036e1040, 1998


http://refhub.elsevier.com/S1010-5182(13)00303-X/sref1
























































































































bone repair of critical size defects treated with mussel powder associated or not with bovine bone...

Documents