pla/pga copolymer as a bone substitute in maxillary sinus

SABRINA FERREIRA

PLA/PGA COPOLYMER AS A BONE

SUBSTITUTE IN MAXILLARY SINUS

AUGMENTATION. HISTOLOGICAL, HISTOMETRIC

AND IMMUNOHISTOCHEMISTRY STUDY IN

RABBITS

Araçatuba - SP

2014

1

SABRINA FERREIRA

PLA/PGA COPOLYMER AS A BONE

SUBSTITUTE IN MAXILLARY SINUS

AUGMENTATION. HISTOLOGICAL, HISTOMETRIC

AND IMMUNOHISTOCHEMISTRY STUDY IN

RABBITS

Dissertação apresentada à Faculdade de Odontologia do Campus de Araçatuba - FOA, Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP, como pré-requisito para obtenção do título de “Mestre em Odontologia” - área de concentração em Cirurgia e Traumatologia Buco-Maxilo-Facial.

Orientador: Prof. Adj. Idelmo Rangel Garcia Júnior.

Araçatuba - SP

2014

2

Dedicatória, Agradecimentos

e Epígrafe

3

Dedicatória

Àqueles que são donos do meu coração e fonte de vida.

À mamãe (Carmen das Graças Ferreira), que com amor incondicional é minha

fortaleza, meu porto seguro, minha vida. Meu exemplo de amor, fé imensurável, compaixão,

humanidade, de força, garra,... de mulher. Com carinho, sempre nos dizia que o estudo seria

a herança que nos deixaria e que tudo na vida se conquista com muito esforço e dedicação.

Obrigada por todo apoio sempre. Te amo.

Ao meu irmão (Danilo Douglas Ferreira) que apesar de toda correria e da distância,

sempre torceu e rezou muito por mim. Te amo meu irmão e você sabe que sempre estarei ao

seu lado junto com a mamãe. Que Deus te abençoe sempre.

Dedico a estes meus amores esta conquista que é fruto de muito amor à minha

profissão, mas também é fruto de muito sacrifício como meses longe de casa com coração

apertado por não poder cuidar dos meus quando precisaram, ou sem poder estar presente

num abraço em seus aniversários. São vocês as flores que alegram e fortalecem minha

caminhada.

4

Agradecimentos Especiais

Primeiramente, agradeço a Deus por todas as graças e por sua imensa misericórdia.

Obrigado Senhor por cuidar de mim e de minha família, por ser luz no nosso caminho, por

mostrar a cada dia que entregue em suas mãos podemos seguir com passos firmes e sem

medo. Obrigada senhor pelos dias alegrias e pelos dias de tristeza quando o senhor me

confortou em seus braços e, principalmente, obrigada pela vida. E seja feita sempre, Senhor, a

vossa vontade!

Ao meu orientador, Prof. Adj. Idelmo Rangel Garcia Júnior. Muito obrigada por tudo.

Obrigada por acreditar em mim, por me confiar grandes trabalhos seus onde tive a

oportunidade de crescer muito na pesquisa, e por permitir que mesmo em meio ao medo e

as dúvidas eu pudesse passar a dar meus próprios passos. Obrigada pela disponibilidade em

todos os momentos que precisei, por me escutar quando eu precisei falar, pelo apoio

fundamental que tanto me trouxe segurança e pela paciência. Aprendi muito com o senhor,

mas sua humildade, sua humanidade e sua inigualável serenidade de quem dá um passo de

cada vez vou guardar como maiores aprendizados. Receba toda minha admiração e gratidão,

mestre. Que Deus continue abençoando o senhor e sua família.

A querida Profa. Ass. Dra. Roberta Okamoto que tão docemente sempre me recebeu

com carinho, obrigada pela disponibilidade e pela imensa participação neste estudo em sua

co-autoria e em tantos outros. É um honra poder aprender com a senhora.

Aos queridos professores da Disciplina de Cirurgia e Traumatologia Buco-maxilo-facial,

Profa. Ass. Dra. Alessandra Marcondes Aranega, Profa. Ass. Dra. Ana Paula Farnezi Bassi, Profa.

Ass. Dra . Daniela Ponzoni, Prof. Ass. Dr. Francisley Avila Souza e Prof. Adj. Osvaldo Magro

Filho, pela amizade, confiança, pelo bom convívio, pelo respeito e por todo aprendizado.

Recebam meu carinho e admiração.

5

Ao querido Prof. Titular Wilson Roberto Poi, pelo exemplo de professor e mestre que

tanto admiro e respeito. Já lhe disse uma vez, mas quero deixar aqui registrado, já o admirava

antes de ser sua aluna. Na graduação tive a honra de ter um mestre que o senhor também

ajudou a formar (Prof. Titular Ronaldo Célio Mariano) e que também levou pra sua carreira

seu exemplo de docência. Este mesmo mestre que contava histórias de um professor da

UNESP de Araçatuba com quem ele muito aprendeu, Prof Poi. E, agora, na pós-graduação,

eu tive o prazer de conhecê-lo e a honra de ser sua aluna. Obrigada pelos ensinamentos e

pelos abraços tão confortantes que aliviavam a dor da distância dos que amo.

A minha doce Dirce Maria (Maria Dirce Colli Boatto), o que eu faria se Deus não tivesse

colocado esse anjo no meu caminho? Não tenho palavras pra dizer o quão importante você

foi pra mim. Aprendi muito com você, tanto quanto como com meus mestres. Obrigada por

todo carinho e paciência em me ensinar tudo o que você levou uma vida pra aprender. Mas

obrigada mesmo pelo carinho de mãe que confia, que acredita e que protege e defende. Te

amo muito. Que Deus continue abençoado você, Sérgio e suas “crianças”.

Ao Léo (Leonardo Perez Faverani), mais um dos anjos que Deus mandou pra me

ajudar nesta caminhada. Foi mais que amigo, foi família. Obrigada, querido, por tanto

carinho, atenção, lealdade, companheirismo. Muitos são os momentos de dor, mas poucos

são os que aceitam compartilhar a dor do próximo. É muito fácil querer viver a alegria do

outro, difícil é viver a dor e comer o sal junto. Você viveu todas as minhas alegrias, minhas

dores, angústias, medos, dúvidas, ansiedades, não só como espectador, mas como

participante ativo. Saiba que rezo sempre por você e sua família. E, além disso, também foi

Mestre pra mim. Mestre é aquele que não teme transmitir seu conhecimento e que tem

prazer em fazê-lo, aprendi muito com você, obrigada por tudo.

Ao querido André (André Luis da Silva Fabris), que com tanto carinho me acolheu.

Obrigada por essa amizade incontestável, como o Léo, obrigada por viver minhas alegrias,

mas, principalmente, obrigada por viver minhas dores. Sempre que precisei você estava lá.

Passamos por momentos difíceis e delicados que toda amizade proporciona pela liberdade

6

que temos um com o outro, mas sabemos que o guardaremos para sempre são os

verdadeiros gestos de carinho, atenção, dedicação, companheirismo sem esperar nada em

troca. Saiba que estará sempre em meu coração e em minhas orações. Agradeço também

sua querida mãe, Maria Ivone Fabris, por todo carinho com que sempre me recebeu em sua

casa, pela amizade e por torcer por mim, igualmente agradeço ao Gustavo da Silva Fabris, seu

irmão. Deus abençoe sua família e a conserve sempre unida.

À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), pela

concessão da Bolsa de Mestrado durante todo o curso. Meus sinceros agradecimentos por

promover o apoio financeiro necessário que viabilizou a realização deste sonho.

7

Agradecimentos

À Faculdade de Odontologia de Araçatuba – UNESP, na pessoa da diretora Ana Maria

Pires Soubhia pela oportunidade de realização do curso de Mestrado e por todo carinho com

que fui recebida por esta instituição.

À Coordenação do Programa de Pós-Graduação em Odontologia, da Faculdade de

Odontologia de Araçatuba, da Universidade Estadual Paulista “Julio de Mesquita Filho” na

figura da Profa. Adja. Maria Jose Hitomi Nagata.

Aos queridos funcionários do Departamento de Cirúrgia e Clinica Integrada (Cleide L.

S. Toquetão, Gilmar M. de Oliveira, Odair Vicente, Renato G. de Oliveira, DonaTina, Paulo R

Gratão e Joilson B. Lellis). Muito obrigado pelo carinho e respeito.

As queridas funcionárias Michele, Fran, Paula, Camila, Patrícia, Hélide que com muito

carinho e respeito nos apoiaram durante todo o curso. Obrigada por toda atenção e

empenho.

Ao querido funcionário Sr. Wilson e seu amigo canino Apolo pelo carinho e pelas

conversas. Que Deus os abençoe.

Aos funcionários do Biotério, em especial aos funcionários Sr. Camilo e Sr. João Batista,

que ofereceram suporte para a obtenção dos animais utilizados neste trabalho.

Aos colegas da pós-graduação em Odontologia (Juliana Zorzi Coléte, Patrícia Bermejo,

Bianca Bravim Bomfim, Carlos Alberto Timóteo, Gabriel Ramalho Ferreira, Lamis Meorim

Nogueira, Marial Del Pilal Rodrigues e Juceléia Maciel, Rodrigo Pereira e Giovanna

Francisconi). Pela nossa excelente convivência, pelo companheirismo e aprendizado.

Aos queridos pacientes pela confiança e pela entrega ao permitir nosso aprendizado

por meio de sua dor. Muito obrigada.

8

Epígrafe

“Preparar o futuro significa fundamentar o presente.”

“O Pequeno Príncipe”, Antoine de Saint-Exupéry

9

Abstract

The proposition of this study was evaluate the osteoconductive capability of the solid form of

PLA/PGA copolymer in an experimental model of maxillary sinus grafting. Twenty male white

New Zealand rabbits, each weighing about 3.0kg, were used and were divided into two

groups, according to the sinus filling material, as follows: autogenous bone chips and

PLA/PGA copolymers. Augmented area differ between the groups after 3, 7 and 15 days

(P=0.004). However, the values became similar on day 40 (P=0.458). After 3 and 7 days the

percentage of bone was statistically significant between autogenous bone and PLA/PGA

copolymer (P=0.004 and P=0.004). After 15 and 40 days the values were comparable in the

two periods (P=0.087 and P=0.087). Immunohistochemistry confirms the results on the

histomorphometric data. In conclusion, PLA/PGA copolymer seems to be suitable as

resorbable material able to induce bone growth in bone defects. This observation suggests

that the material have osteoconductive properties also suitable for application in maxillofacial

surgery.

Key-words: polylactic acid, polyglycolyc acid, autogenous bone, sinus lift, rabbit

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Resumo

A proposta deste estudo foi avaliar a capacidade osteocondutora da forma sólida do

copolímero de PLA/PGA em um modelo experimental de enxerto em seio maxilar . Vinte

coelhos machos, branco, da raça Nova Zelândia, cada um pesando cerca de 3,0 kg, foram

utilizados e foram divididos em dois grupos de acordo com o material de preenchimento do

seio, como segue: ósseo autógeno particulado e copolímero PLA/PGA . A área aumentada

diferiu entre os grupos após 3, 7 e 15 dias (p = 0,004) . No entanto, os valores tornaram-se

semelhante aos 40 dias (p = 0,458). Depois de 3 e 7 dias, a porcentagem óssea foi

estatisticamente significativa entre osso autógeno e PLA / PGA copolímero (P = 0,004 e P =

0,004). Após 15 e 40 dias os valores foram semelhantes nos dois períodos (P = 0,087 e P =

0,087 ). Imunohistoquímica confirma os resultados sobre os dados histomorfométricos. Em

conclusão, o copolímero de PLA/PGA parece ser adequado como material reabsorvível capaz

de induzir o crescimento do osso em defeitos ósseos. Esta observação sugere que o material

tem propriedades osteocondutoras também apropriado para aplicação em cirurgia maxilo-

facial.

Palavras- chave: ácido polilático, ácido poliglicólico, osso autógeno, elevação do seio, coelho.

11

Listas e Sumário

12

Lista de Figuras

Figure 1: PLA/PGA copolymer 50/50 saturated with calcium phosphate ..................................... 38

Figure 2:. Autogenous bone collection; (a) tibial metaphysic; (b) and (d) autogenous tissue

samples; (c) bone scraper ..................................................................................................................... 38

Figure 3: Surgical procedure steps for sinus lift. (a) Exposure of the nasal dorsum, (b), (c), (d)

and (e) Preparation of the surgical window, (f) PLA/PGA copolymer graft, (g) bone

graft, (h) graft completed. .................................................................................................................... 39

Figure 4: (a) Nose complex and (b) frontal plane ....................................................................................... 40

Figure 5: Captured magnification: Specimen at 15 days after surgery, X1.0, H.E .......................... 41

Figure 6: Histomorphometric analysis : Mensured area, X2.8, H.E. ...................................................... 42

Figure 7: Selected region for histomorphometric analysis: A: imaginary extensions of the pre-

existing cortical bone at the margins of the window; B: center region and C:

membrane region ..................................................................................................................................... 43

Figure 8: Autogenous bone group, 3days ........................................................................................................ 44

Figure 9: Autogenous bone group, 7days ........................................................................................................ 45

Figure 10: Autogenous bone group, 15days .................................................................................................. 46

Figure 11: Autogenous bone group, 40days .................................................................................................. 47

Figure 12: PLA/PGA copolymer group, 3days ................................................................................................ 48

Figure 13: Autogenous bone group, 3days ..................................................................................................... 49

Figure 14: PLA/PGA copolymer group, 15days ............................................................................................. 50

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Figure 15: PLA/PGA copolymer group, 40days ............................................................................................. 51

Figure 16: Graphic of augmented area, autogenous bone and PLA/ PGA copolymer groups

................................................................................................................................................................................ 52

Figure 17: Graphic of %bone ................................................................................................................................... 52

Figure 18: Graphic of % soft tissue ......................................................................................................................... 53

Figure 19: Pie chart of the mean proportions of new bone and non-calcified soft tissues ..... 53

Figure 20: Autogenous bone group; OC immunoreactivity in (a) 3, (b) 7, (c) 15 and (d) 40

days. ................................................................................................................................................................ 54

Figure 21: PLA/PGA copolymer group; OC immunoreactivity in (a) 3, (b) 7, (c) 15 and (d) 40

days ................................................................................................................................................................... 54

Figure 22: Autogenous bone group; TRAP immunoreactivity in (a) 3, (b) 7, (c) 15 and (d) 40

days ................................................................................................................................................................. 55

Figure 23: Figure: PLA/PGA copolymer group; TRAP immunoreactivity in (a) 3, (b) 7, (c) 15

and (d) 40 days........................................................................................................................................... 55

Figure 24: OC and TRAP values at 3, 7, 15 and 40 days in autogenous bone group .............. 56

Figure 25: OC and TRAP values at 3, 7, 15 and 40 days in PLA/PGA copolymer group ......... 56

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Lista de Tabelas

Table 1: Augmented area – Mean (mm²)............................................................................................................. 57

Table 2: %bone – Mean (%) .......................................................................................................................................... 57

Table 3: % soft tissue – Mean (%) .............................................................................................................................. 58

Lista de Abreviaturas

PLA ........................................................................................................................................... Polylactic acid

PGA ..................................................................................................................................... Polyglycolyc acid

PLA/PGA .................................................................................................................. Poly-Lactide-co-Glycolide

PVP-I ......................................................................................................... Polyvinyl Pyrrolidone Iodine

TRAP ............................................................................................. Tartrate-resistant acid phosphatase

OC .............................................................................................................................................. Osteocalcin

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Sumário

Introduction .......................................................................................................................................................... 17

Proposition ............................................................................................................................................................ 18

Material and methods ...................................................................................................................................... 19

Copolymer PLA/PGA Production ...................................................................................................... 19

Surgical Procedures .................................................................................................................................. 19

Obtaining Autogenous Bone Graft ........................................................................................ . 20

Sinus Lift ................................................................................................................................................... 20

Histologic Procedures .............................................................................................................................. 21

Histological and Histomorphometric Analysis ............................................................................ 21

Immunohistochemical Analysis .......................................................................................................... 22

Statistical assessment ................................................................................................................................ 22

Results .................................................................................................................................................................. 23

Histologic Findings .................................................................................................................................... 23

Experimental group with autogenous bone graft ........................................................... 23

Experimental group with PLA/PGA copolymer ................................................................. 24

Histomorphometric Analysis ................................................................................................................ 24

Augmented area ............................................................................................................................... 24

% bone, % soft tissues ....................................................................................................................... 25

Immunohistochemical Analysis .......................................................................................................... 25

Discussion .............................................................................................................................................................. 26

References ............................................................................................................................................................. 31

Tabelas e Figuras ...................................................................................................................................................... 37

Anexos .......................................................................................................................................................................... 59

16

“PLA/PGA copolymer as a bone substitute in maxillary sinus

augmentation. Histological, histometric and

immunohistochemistry study in rabbits”

17

Introduction

Loss of teeth promotes severe, irreversible resorption of alveolar bone1, 2

. In the

posterior region of the maxilla, the height extending from the floor of the maxillary sinus to the

alveolar crest decreases. Various procedures for maxillary sinus augmentation have been

developed to increase bone volume and height to promote stability of endosseous implants1,

3, 4.

These procedures require the use of graft materials to maintain the augmented space

and to promote osteogenesis. Early studies advocated the use of autogenous bone to ensure

graft survival and bone formation in the augmented space1, 4

. Blomqvist et al5 reported a

success rate of 82% with implants placed into grafted bone in the maxillary sinus from the iliac

crest; Lundgren et al6 reported a success rate of 80% with such implants. Some authors have

even reported successful short-term sinusal bone augmentation and implant outcomes with

only a blood clot and no grafting material7-11

Moy et al12

and Nishibori et al13

found that the

quantity and quality of bone produced by autogenous bone grafts were superior to those of

bone produced by allografts.

Numerous reports on autogenous grafts at various anatomical sites have been

presented14-17

. Although autogenous bone is still often regarded to be the gold standard in

bone augmentation procedures, morbidity at the donor site as well as reports of significant

levels of resorption, whether intra-oral, extra-oral, block or particulated bone is used,

necessitate the consideration of alternative biomaterials18-21

.

The families of polylactic acid (PLA), polyglycolyc acid (PGA) and their co-polymers

(Poly-Lactide-co-Glycolide, PLA/PGA), both biodegradable, are at the cutting edge of bone

reconstruction procedures. These materials are manufactured in a variety of forms to produce

appliances for use in bone surgery such as tissue barriers22

, fixation devices23, 24

, porous solid

graft25-29

, solid or semi-solid carriers for delivery of growth factor (GF), bone morphogenetic

18

proteins (BMPs) and other bioactive molecules30, 31

. These materials may also serve as

scaffolding to promote specific cell adhesion, differentiation and bone formation26, 29, 31

.

The resorption of PLA and PGA acids occurs through hydrolysis and hydrolytic

mechanisms. Implanted polymers are expected to play their supportive role in the healing

process and then to degrade to matrix products, which are eliminated through the normal

excretory routes and are replaced by the neighboring tissues32, 33

. This polymer is gradually

substituted by new bone over time, but its degradation depends on the formulation,

amorphous/crystalline structure, isomeric characteristics, molecular weight and amount of

material used27, 34

. The exact composition and architecture of the polymeric scaffold becomes

fundamental to promote successful bone regeneration.

Although extensively used in several orthopedic appliances35

, PLA/PGA have been

scarcely applied in oro-maxillo-facial applications. PLA-derived devices were implemented to

prevent alveolar osteitis or dry socket with discordant results by Hooley and Golden36

. PLA

and decalcified freeze-dried bone allografts were clinically studied in the treatment of

periodontal intra-osseous defects by Meadows et al.37

. Results showed a limited amount of

tissue regeneration and the persistence of PLA particles surrounded by soft tissue. A new

PLA/PGA (Fisiograft®

, Ghimas, Italy), manufactured as sponge blocks, gel and powders, was

studied in maxillary sinus floor augmentations29, 38

, as space maker, in alveolar ridge

preservation26

, ridge augmentation with split-crest technique, in the treatment of deep

periodontal intra-osseous defect28

, and in critical bone defects in experimental animals25

with

contrasting results.

Proposition

We therefore histologically, histomorphometrically and immunohistochemically

evaluated the osteoconductive capability of the solid form of PLA/PGA copolymer in an

experimental model of maxillary sinus grafting.

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Material and methods

Copolymer PLA/PGA Production

PLA/PGA copolymer consisted of a mixture of lactic and glycolic acid 50/50 saturated

with calcium phosphate, extent that make them gain a solid consistency (Figs. 1a and 1b). The

solution pH was controlled by adding the buffer solution. The method used was the

polymeric or Pechini39

method, is a technique that can be applied to the development of

advanced materials precursors. The process is based on the ability of certain organic acids

such as citric, lactic and glycolic have to form chelates. These chelates may be esterified with a

polyalcohol when heated and polymerized at higher temperatures forming a resin.

Surgical Procedures

All experimental protocols involving animals were used conformed to procedures

described in the Guiding Principles for the Use of Laboratory Animals and the study approved

by the Animal Committee of Dentistry School of University Estadual Paulista Júlio de Mesquita

Filho, UNESP (00724-2012) (Annex 1).

Twenty male white New Zealand rabbits, each weighing about 3.0kg, were used and

were divided into two groups, according to the sinus filling material, as follows: Group 1:

Control, autogenous bone chips and Group 2: PLA/PGA copolymers particles.

All animals underwent surgeries for bilateral sinus lift procedures. The rabbits were

anesthetized with 1% chloridrate of ketamine (Francotar, Virbac Ltda, São Paulo, Brazil), along

with with a sedative, 2% chloridrate of xylazine (Virbaxyl 2%, Virbac Ltda, São Paulo, Brazil), in

the recommended dose, respectively, 60 mg/kg and 5.0mg/kg intramuscularly, and 0.5ml of

1% lidocaine with epinephrine (1:100000) was injected subcutaneously at the midline of the

nasal dorsum and into the tibial metaphysic.

20

Obtaining Autogenous Bone Graft

Linear incision with approximately 40mm was made in the direction of the long axis in

the tibial metaphysic after previous manual trichotomy and antisepsis preoperative in the right

tibia with friction of gauze soaked in 10% Polyvinyl Pyrrolidone Iodine degermante, with 1%

active iodine (PVP-I 10% Riodeine ®, Rioquímica, São José do Rio Preto) associated with the

topic PVP (10% PVP-I aqueous, with 1% active iodine, Riodeine®, Rioquímica, São José do Rio

Preto). Then, the soft tissue overlying the region was elevated in total thickness, exposing the

bone tissue (Fig. 2a). With the aid of scraper autogenous bone tissue samples were collected

for subsequent filling of the maxillary sinus (Fig. 2b, 2c and 2d). Completed sample collection,

the surgical flap was repositioned and sutured in layers.

Sinus Lift

Surgical interventions were performed under strict sterile conditions. The surgical area

was shaved and disinfected with iodine. The sinus lift surgical procedure was performed

according to Asai, Shimizu e Ooya (2002). A midline incision extending for about 50mm was

made, and the skin and periosteum were elevated sufficiently to expose the nasal bone and

nasoincisal suture line (Fig. 3a). A trephine bur with a 5.0mm internal diameter was used to

delineate the diameter of the bone window for the maxillary sinus access (Fig. 3b and 3c). A

circular window was opened in the nasal bone with the use of a round diamond bur nº 1014

under copious irrigation with saline solution (Fig. 3d). The window was located approximately

20mm anterior to the nasofrontal suture line, 10mm lateral to the midline (Fig. 3e). Care was

taken during this procedure to avoid damaging the sinus mucosa. The sinus membrane was

carefully elevated to allow the insertion and condensation of the graft materials. No

membrane was used to cover the bone window. In the experimental groups, the

compartment was filled with PLA/PGA copolymer (Fig. 3f) and autogenous bone graft (Fig.

3g and h). A suture was placed between the periosteum and skin.

21

Histologic Procedures

After 3, 7, 15 and 40 days of the surgery, five animals were sacrificed with an overdose

of anesthetics. After decapitation and dissection, the entire nose complex, including the nose

and sinus cavities (Fig. 4a), was fixed in buffered 10% formaldehyde (Merck, Darmstadt,

Germany) for 48 h and washed in tap water for 24 h, and immersed in buffered 4% EDTA for

demineralization. Specimens from the normal and experimental groups were slowly

decalcified and were cut in a frontal plane (Fig. 4b). The specimens were then dehydrated,

embedded in paraffin, and sliced into sections about 6µm thick. The sections were stained

with hematoxylin and eosin (Merck & Co., Inc., NJ, USA) for the light microscopic examination.

Some sections were reserved for immunohistochemistry reactions.

Histological and Histomorphometric Analysis

Each image of the calcified specimens at the rostrocaudal midpoint of the antral wall

was digitally captured at magnification X1.0, X2.8 and X160 and sent to the Image J Program

– Version 1.47 (National Institutes of Health (NIH), Bethesda, Maryland, USA). The composition

of the total augmented sinus was identified (in mm²). The proportions (in %) of each

composite (new bone - NB and soft tissue - ST) were obtained. To evaluate the distribution of

regenerated bone in the grafted sinus area as a secondary outcome variable, the above-

mentioned parameters were measured in specific standardized areas. The areas were

randomly selected and photomicrographs were taken in original magnification X160. The

window region was selected within the grafted area interfacing the imaginary extensions of

the pre-existing cortical bone at the margins of the window, the center region was at the

middle of the whole augmented sinus and membrane region. The membrane region was

chosen from just above the Schneiderian membrane (Fig. 5, 6 and 7):

http://en.wikipedia.org/wiki/Bethesda,_Maryland

22

Augmented area: defined as the raw surface of the secluded space underneath the

sinus membrane;

% bone, % soft tissues: percentage of the measured augmented area, defined as the

raw surface of the secluded space underneath the sinus membrane.

Immunohistochemical Analysis

Immunohistochemical analysis were performed at 3, 7, 15, and 40 days (n = 5). The

following primary antibodies used for immunohistochemical processing were from Santa Cruz

(Biotechnology, CA): anti-osteocalcin (OC) and tartrate-resistant acid phosphatase (TRAP). As a

secondary antibody, a biotinylated donkey anti–goat antibody (Jackson Immunoresearch

Laboratories, West Grove, PA) was used. The signal of the immunohistochemistry reaction

with an avidin biotin system (Kit ABC-Vectastain Elite ABC–peroxidase standard, reagent A and

B only–PK6100; Vector Laboratories, Burlingame, CA), and diaminobenzidine (Sigma, St. Louis,

MO) was used as chromogen.

Immunohistochemical reactions were controlled to evaluate the specificity of the labels.

Hematoxylin and eosin staining was performed and used as a reference of the

cytoarchitecture of the tissue. Immunohistochemical analysis for consist of a qualitative

examination was stratified by a range of values ranging from 0 to 3 (0 = no protein expression,

1 = protein with little expression, 2 = intermediate protein expression, high protein expression

3 =).

Statistical assessment

All morphometric data were assessed using the Mann-Whitney. The level of statistical

significance was set at 5%.

23

Results

Histologic Findings

Experimental group with autogenous bone graft

After 3 days, the augmented dome-like space formed by the elevated sinus mucosa

was found to be filled with acellular autogenous bone particles and blood clot, osteocytes in

the lacunae disappeared in the grafted bone (Fig. 8).

After 7 days, peripheral newly formed bone trabeculae had irregular surfaces with

numerous osteoblasts and osteoclasts were seen in areas near the sinual membrane and

bone tissue of the nasal complex. Therefore early steps of centripetal new bone formation

were observed progressing from the anterior part of the sinus and the bone walls. Cell

colonization was found everywhere in the cavity, and remnants of clot were sometimes

visible. Remnant acellular autogenous bone particles were seen in the central region of the

space with connective tissue and blood vessels. There was no evidence of acute or chronic

inflammation (Fig. 9).

After 15 days, the newly formed woven bone adjacent to the membrane and bone

tissue of the nasal complex showed osteoclastic resorption and the central region were seen

newly formed woven bone and soft tissue. There were many osteocytes in the newly formed

bone. Woven bone with many osteoblasts was observed around the grafted bone, especially

under the elevated antral membrane (Fig. 10).

After 40 days, the augmented space revealed mature cortical and trabecular bone

with intertrabecular vascularized adipose tissue. The bone chips were completely remodeled

and their remnants were not visible (Fig. 11).

24

Experimental group with PLA/PGA copolymer


was found to be filled with blood clot. Copolymer particles were not observed. Mesenchymal

cell proliferation was only visible at the periphery of the created space, while the blood clot

was still non-invaded by mesenchymal cells in the center of the cavity. No inflammatory cell

infiltration was present (Fig. 12).

After 7 the augmented dome-like space was found to be filled connective tissue with

fibroblasts and blood vessels. Remnant blood clots were seen in the central region of the

space. Peripheral newly formed woven bone was seen. Therefore early steps of centripetal

new bone formation were observed progressing from the anterior part of the sinus and the

bone walls (Fig. 13).

After 15 days, peripheral newly formed bone was seen in areas near the sinual

membrane and bone tissue of the nasal complex. Connective tissue was seen in the central

region of the space with fibroblasts and blood vessels (Fig. 14).


with intertrabecular vascularized adipose tissue. The bone trabeculae showed the minimal

osteoclastic resorption. Some areas near the sinual membrane and bone tissue of the nasal

complex revealed mature cortical and trabecular bone with intertrabecular vascularized

adipose tissue (Fig. 15).

Histomorphometric Analysis

Augmented area

Augmented area differ between the groups after 3, 7 and 15 days(P=0.004). However,

the values became similar on day 40 (P=0.458) (Fig. 16). The comparison of values of 3 and 40

days autogenous bone group and PLA/PGA copolymer group and were also statistically

25

significant (P=0.004 e P=0.004) .Values in mm2 of the augmented area are presented in Table

1.

% bone, % soft tissues

After 3 days, in the autogenous bone group, bone inside the created space under the

membrane constituted 56.67% of the measured augmented area, while in the PLA/PGA

copolymer group, the amount of bone constituted 0%. The percentage of bone was

significantly higher with autogenous bone than with PLA/PGA copolymer (P=0.004).

After 7 days, in the autogenous bone group, bone ingrowths constituted 39.80% of

the measured augmented area, while in the PLA/PGA copolymer group, the amount of bone

ingrowth failed to reach 13.93%. The percentage of bone was significantly higher with

autogenous bone than with PLA/PGA copolymer (P=0.004).

After 15 days, the area occupied by newly formed bone reached, respectively, 43.47%

and 31.63% (P=0.087) for the autogenous bone and the PLA/PGA copolymer groups. The

implanted bone chips could be observed in the central area.

After 40 days, in the autogenous bone and PLA/PGA copolymer groups, the newly

formed bone area increased to 51.63% and 39.87%, respectively. The values were

comparable in the two groups (P=0.087). Detailed results are presented in Table 2 and shows

the correlation between volume changes over time and % bone and % soft tissues within

augmented tissues (Figs 17, 18 and 19) (Tables 2 and 3).


OC immunoreactivity could be seen in all augmented area for the autogenous bone

group in the early periods of this study, 3 days (intermediate protein expression), with

increased at 7 days (high protein expression) and remaining high on 15 and 40 days (high

protein expression) after surgery (Fig. 20). Similarly to autogenous bone group, in the

26

PLA/PGA copolymer group, OC immunoexpressivity was seen intermediate at 3 days, with

increased at 7 days (high protein expression), remaining high on 15 days (high protein

expression). However, declined at 40 days (intermediate protein expression) after surgery (Fig.

21).

In TRAP immunohistochemistry could be seen in autogenous bone group high protein

expression in the early periods, 3 days. However, this declined at 7 days (intermediate protein

expression). At 15 days after surgery increased expression of TRAP (high protein expression)

with decline at 40 days (intermediate protein expression) (Fig. 22). Differently to the

autogenous bone group, in the PLA/PGA copolymer group TRAP immunoexpressivity had

gradual increased: 3 days protein with little expression, 7 days with intermediate protein

expression and 15 days with high protein expression. However, declined at 40 days

(intermediate protein expression) (Figs. 23, 24 and 25).

Discussion

In posterior maxilla restoration using osseointegrated implants, sinus floor elevation is

often also undertaken by an autogenous bone graft. Autogenous bone graft is considered

the ideal graft to satisfy the following criteria: (1) low risk of infection, (2) low antigenicity, (3)

the ability to produce bone by osteoinduction and osteoconduction, and (4) easy

correction40

. However, the preferred autogenous material causes specific problems such as its

limited supply, attendant donor-site morbidity, and the occasional unsuitability for the

proposed reconstruction because of poor tissue quality, or the extremely difficulty in shaping

the graft18, 19

Although bone grafting represents the standard reconstruction procedure, resorbable

polymers have become very useful auxiliary materials to enhance functional and structural

clinical outcome23, 24, 28, 41

. Among these polymers32

, polylactides are deemed to be very

promising resorbable materials. Polylactide copolymers with different mechanical properties

27

and different degradation rates have been used in numerous studies to enhance the healing

of bone defects24, 32, 33

. Some authors have seen in the degradability properties of PLA/PGA

copolymer a chance for tissue neogenesis 24-26

. Our results seems to confirm these

opportunities.

This rabbit study investigated the area stability of sub-sinusal bone regeneration when

autogenous bone chips or PLA/PGA copolymer 50/50 were used as space fillers. Early bone

formation and the continuing behavior of the regenerated bone with these different types of

filling material were also assessed. Rabbits have similar maxillary sinus ventilation to humans

and a well-defined ostium opening to their nasal cavities. The rabbit sinus lift model is

therefore appropriate and well documented for evaluating bone regeneration physiology in

sub-sinusal bone regeneration42, 43

.

Adequate bone healing was observed with the two space fillers used in the present

study and no signs of inflammation could be detected. Similar findings have already been

reported by several authors29, 42, 43

; unfortunately, the early steps of bone regeneration were

poorly studied in these papers.

In this study, in PLA/PGA copolymer cell proliferation was detected after 7 days only

along the bone wall and under the sinusal membrane, while the central part was still

occupied by a dense cluster of red blood cells, in contrast with the autogenous bone group

where the augmented space was completely invaded by mesenchymal cells. A gradient of

maturity of the newly formed tissue from the native bony walls to the center of the cavity

illustrated the centripetal nature of the regenerative process.

At 15 days, in the PLA/PGA copolymer group, non-mineralized fibrous tissue still

occupied the central area. At 40 days, in the PLA/PGA copolymer group, woven bone was

found everywhere in the remaining cavity. Therefore, PLA/PGA copolymer implanted into

sub-sinusal cavities allowed bone formation. Rimondini et al25

reported that the

histomorphometric data demonstrated that the experimental material is able to promote bone

healing even in a large bone defect such as in rabbit femoral condyles. The bone maturation

28

improved from 30 to 90 days. The data concerning the development of the forming bone

showed that during the 30 to 90 days the material allowed a bone remodeling from reticular

toward a trabecular structure.

Fisiograft®

is a synthetic resorbable sponge formed by 50–50 lactide–glycolide

polymer. This copolymer has shown the fastest degradation rate of the d-l lactide/glycolide

biomaterials, with the polymer degrading in about 50–60 days41

. In the previous study37

PLA

granules were still evident at 6-month surgical re-entry.

In the autogenous bone group, the early healing process was characterized by a

particularly high cellular density along the bone chips, even at a distance from the bony walls.

This picture strongly suggests an osteogenic potential provided by autogenous bone chips. At

15 days, the anatomy of the dense woven bone seemed to be influenced by the initial rolled

shape of the bone chips, now remodeled. The remodeling process continued from 15 days to

40 days yielding, in the long term, rarefied mature bone architecture. According to Schlegel et

al.44

, this remodeling process was also observed in sinus lifts performed in humans with

particulated autogenous bone.

New bone formation was already observed at 2 weeks after grafting. It is of interest

that new bone formation was observed in all parts under the elevated antral membrane,

around the grafted bone, and at the superior part of the original sinus floor. The result

indicates that new bone formation may depend not only on

osteoconduction from the recipient site but also on osteoinduction by bone morphogenetic

protein in the grafted bone and osteogenesis by the bone cells that were grafted with the

bone.

Albrektsson45

reported that early revascularization meant early bone remodeling of a

graft in the rabbit tibia; in the present study, blood vessels were already seen in the grafted

bone at 7 days. It is suggested that early revascularization also assisted in the new bone

formation for the maxillary sinus in the present study.

29

Much newly formed bone was noted under the elevated membrane at 40 days after

grafting. Moreover, continuous cortical bone was observed under this membrane. These

results indicate the difference in bone formation between the continuous bone located under

the elevated membrane and continuous bone located elsewhere. In the present study, the

medullary cavity was filled with many fat cells.

In our study, the mean bone fractions in the grafted area were 56.67%, 39.80%,

43.47% and 51.63%, respectively, at 3, 7, 15, and 40 days after grafting. The bone fraction in

the bone biopsy cores was reported in a few studies; Moy et al46

, for example, indicated that

the composition of bone was 59.4% from chin bone graft, and Lundgren et al47

reported the

mean bone fractions in chin bone grafted areas to be 40% and 48%, respectively, at 6 and 12

months after bone grafting.

It is speculated that the period from 2 weeks to 4 weeks in the present study

corresponds to the period from 6 months to 12 months in human beings48

. Previous

investigations have shown that most osteogenic remodeling of the autogenous bone graft in

a rabbit model will occur within the first 6 to 8 weeks postsurgery45, 48-50

. In cortical bone

healing, full graft vascularization was complete by day 30, with the osteogenic phase ending

by day 3545

. Similar results were found by Roberts et al51

, who determined that the resorption

and reversal phase is approximately 1 week in rabbits; while the duration of the bone

formation phase was about 5 weeks.

Immunohistochemistry confirms the results on the histomorphometric data. During the

period of increase of regeneration area, between 3 and 7 days, the OC values also showed an

increase in both experimental groups. Important to note that the lower expression group in

the copolymer shows an early stage of the process when compared to autogenous bone

group. In both groups, the period 7-15 days it is possible to see a tendency to equilibrium in

the processes of bone formation and remodeling, we see a tendency to balance the values of

OC and TRAP. Increased OC shows the mineralization phase in which it is the bone tissue

after 40 days in the autogenous bone group, since the values of TRAP are consistent the

30

period of remodeling of bone trabeculae, characteristic of this period. Unlike the group

copolymer in the same period seems to still keep the balance-training resorption.

The present study showed that the long-term outcome of augmentation of the sinus

was not the maturation of newly formed bone, but conversely atrophy of bone. Indeed, an

increase in adipose tissue in marrow is observed in all conditions that lead to loss of bone,

such as osteoporosis, age-related osteopenia, or immobilisation52

. This apparent reciprocal

relation between reduced bone density and increased fat can be explained by an imbalance

in the production of bone-forming and fat-forming cells53

.

But an excess of adipose tissue in marrow is considered to put at risk the long-term

ability to maintain the mechanical strength of the skeleton54

. Mechanical stress is an essential

factor for the corticalisation of bone55

. Ideal bone grafts should eventually be absorbed and

encourage formation of new bone, which replenishes lost bone permanently.

In conclusion, PLA/PGA copolymer seems to be suitable as resorbable material able to

induce bone growth in bone defects. This observation suggests that the material have

osteoconductive properties also suitable for application in maxillofacial surgery.

31

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37

Tabelas e Figuras

38

Figures

Figure 1: PLA/PGA copolymer 50/50 saturated with calcium phosphate.

Figure 2: Autogenous bone collection; (a) tibial metaphysic; (b) and (d) autogenous tissue

samples; (c) bone scraper.

39

Figure 3: Surgical procedure steps for sinus lift. (a) Exposure of the nasal dorsum, (b), (c), (d)

and (e) Preparation of the surgical window, (f) PLA/PGA copolymer graft, (g) bone graft, (h)

graft completed.

40

Figure 4: (a) Nose complex and (b) frontal plane.

41

Fig

ure

5: C

ap

ture

d m

ag

nifi

catio

n:

Speci

men

at

15

days

aft

er

surg

ery

, X1

.0, H

.E.

42

Figure 6: Histomorphometric analysis : Mensured area, X2.8, H.E.

43

Fig

ure

7: S

ele

cted

reg

ion

fo

r h

isto

mo

rph

om

etr

ic a

naly

sis:

A:

imag

inary

ext

en

sio

ns

of th

e p

re-e

xist

ing

co

rtic

al b

on

e

at

the m

arg

ins

of th

e w

ind

ow

; B: c

en

ter

reg

ion

an

d C

: mem

bra

ne

reg

ion

.

44

Fig

ure

8: A

uto

ge

no

us

bo

ne g

rou

p, 3

days

45

Fig

ure

9: A

uto

ge

no

us

bo

ne g

rou

p, 7

days

46

Fig

ure

10

: Au

tog

en

ou

s b

on

e g

rou

p, 1

5d

ays

47

Fig

ure

11

: Au

tog

en

ou

s b

on

e g

rou

p, 4

0d

ays

48

Fig

ure

12

: PLA

/PG

A c

op

oly

mer

gro

up

, 3d

ays

49

Fig

ure

13

: PLA

/PG

A c

op

oly

mer

gro

up

, 7d

ays

50

Fig

ure

14

: PLA

/PG

A c

op

oly

mer

gro

up

, 15

days

51

Fig

ure

15

: PLA

/PG

A c

op

oly

mer

gro

up

, 40

days

52

35.74 36.13

27.45

32.52

28.36 28.99

35.44

31.49

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

3 Days 7 Days 15 Days 40 Days

MEA

N (

mm

²)

AUGMENTED AREA

Autogenous Bone PLA/PGA Copolymer

56.67

39.80

43.47

51.63

0

13.93

31.63

39.87

0.00

10.00

20.00

30.00

40.00

50.00

60.00


MEA

N (

%)

% Bone


Figure 16: Graphic of augmented area, autogenous bone and PLA/ PGA copolymer

groups.

Figure 17: Graphic of %bone.

53

Figure 19: Pie chart of the mean proportions of new bone and non-calcified soft tissues

43.33

60.40 56.53

48.37

100.00

86.07

68.37

60.13

0.00

20.00

40.00

60.00

80.00

100.00

120.00


MEA

N (

%)

% Soft Tissues


Figure 18: Graphic of % soft tissue.

54

Figure 20: Autogenous bone group; OC immunoreactivity in (a) 3, (b) 7, (c) 15 and (d) 40 days.

Figure 21: PLA/PGA copolymer group; OC immunoreactivity in (a) 3, (b) 7, (c) 15 and (d) 40 days.

55

Figure 22: Autogenous bone group; TRAP immunoreactivity in (a) 3, (b) 7, (c) 15 and (d) 40 days.

Figure 23: Figure: PLA/PGA copolymer group; TRAP immunoreactivity in (a) 3, (b) 7, (c) 15 and (d)

40 days.

56

.

Figure 24: OC and TRAP values at 3, 7, 15 and 40 days in autogenous bone group

Figure 25: OC and TRAP values at 3, 7, 15 and 40 days in PLA/PGA copolymer group.

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50


Autogenous Bone

OC TRAP

0

0.5

1

1.5

2

2.5

3

3.5


PLA/PGA Copolymer

OC TRAP

57

Tables

Table 1: Augmented area – Mean (mm²)

Time Autogenous Bone PLA/PGA Copolymer P-value

3 days 35.74 28.36 P=0.004

7 days 36.13 28.99 P=0.004

15 days 27.45 35.44 P=0.004

40 days 32.52 31.49 P=0.458

Table 2: %bone – Mean (%)


3 days 56.67 0 P=0.004

7 days 39.80 13.93 P=0.004

15 days 43.47 31.63 P=0.087

40 days 51.63 39.87 P=0.087

58

Table 3: % soft tissue – Mean (%)


3 days 43.33 100 P=0.004

7 days 60.40 86.07 P=0.004

15 days 56.53 68.37 P=0.087

40 days 48..37 60.13 P=0.087

59

Anexos

60

Annex 1 - Animal Committee

61

Annex 2 – Article

PLA/PGA copolymer as a bone substitute in maxillary sinus

augmentation. Histological, histometric and immunohistochemistry study in

rabbits

Sabrina Ferreira, DDS1; Roberta Okamoto, DDS, PhD

1; Francisley Ávila Souza, DDS, PhD

1;

Idelmo Rangel Garcia Júnior, DDS, PhD1.

1Division of Surgery and Traumatology Bucco-Maxillo-Facial, Department of Surgery and

General Clinic, Araçatuba Dental School, Univ Estadual Paulista Jºlio de Mesquita Filho –

UNESP, Brazil.

Author responsible for correspondence

Sabrina Ferreira

Telephone: +55 18 3636-3270 / 3636-3237

Rua José Bonifácio, 1193 Vila Mendonça

Department of Surgery and General Clinic

CEP: 16015-050 Araçatuba, SP, Brazil

e-mail: [email protected]

Conflicts of Interest and Source of Funding: none

mailto:[email protected]

62

Abstract

The proposition of this study was evaluate the osteoconductive capability of the solid form of

PLA/PGA copolymer in an experimental model of maxillary sinus grafting. Twenty male white

New Zealand rabbits, each weighing about 3.0kg, were used and were divided into two

groups, according to the sinus filling material, as follows: autogenous bone chips and

PLA/PGA copolymers. Augmented area differ between the groups after 3, 7 and 15 days

(P=0.004). However, the values became similar on day 40 (P=0.458). After 3 and 7 days the

percentage of bone was statistically significant between autogenous bone and PLA/PGA

copolymer (P=0.004 and P=0.004). After 15 and 40 days the values were comparable in the

two periods (P=0.087 and P=0.087). Immunohistochemistry confirms the results on the

histomorphometric data. In conclusion, PLA/PGA copolymer seems to be suitable as

resorbable material able to induce bone growth in bone defects. This observation suggests

that the material have osteoconductive properties also suitable for application in maxillofacial

surgery.

Key-words: polylactic acid, polyglycolyc acid, autogenous bone, sinus lift, rabbit

Introduction

Loss of teeth promotes severe, irreversible resorption of alveolar bone1, 2

. In the

posterior region of the maxilla, the height extending from the floor of the maxillary sinus to the

alveolar crest decreases. Various procedures for maxillary sinus augmentation have been

developed to increase bone volume and height to promote stability of endosseous implants1,

3, 4.

63

These procedures require the use of graft materials to maintain the augmented space

and to promote osteogenesis. Early studies advocated the use of autogenous bone to ensure

graft survival and bone formation in the augmented space1, 4

. Blomqvist et al5 reported a

success rate of 82% with implants placed into grafted bone in the maxillary sinus from the iliac

crest; Lundgren et al6 reported a success rate of 80% with such implants. Some authors have

even reported successful short-term sinusal bone augmentation and implant outcomes with

only a blood clot and no grafting material7-11

Moy et al12

and Nishibori et al13

found that the

quantity and quality of bone produced by autogenous bone grafts were superior to those of

bone produced by allografts.

Numerous reports on autogenous grafts at various anatomical sites have been

presented14-17

. Although autogenous bone is still often regarded to be the gold standard in

bone augmentation procedures, morbidity at the donor site as well as reports of significant

levels of resorption, whether intra-oral, extra-oral, block or particulated bone is used,

necessitate the consideration of alternative biomaterials18-21

.

The families of polylactic acid (PLA), polyglycolyc acid (PGA) and their co-polymers

(Poly-Lactide-co-Glycolide, PLA/PGA), both biodegradable, are at the cutting edge of bone

reconstruction procedures. These materials are manufactured in a variety of forms to produce

appliances for use in bone surgery such as tissue barriers22

, fixation devices23, 24

, porous solid

graft25-29

, solid or semi-solid carriers for delivery of growth factor (GF), bone morphogenetic

proteins (BMPs) and other bioactive molecules30, 31

. These materials may also serve as

scaffolding to promote specific cell adhesion, differentiation and bone formation26, 29, 31

.

The resorption of PLA and PGA acids occurs through hydrolysis and hydrolytic

mechanisms. Implanted polymers are expected to play their supportive role in the healing

process and then to degrade to matrix products, which are eliminated through the normal

excretory routes and are replaced by the neighboring tissues32, 33

. This polymer is gradually

substituted by new bone over time, but its degradation depends on the formulation,

amorphous/crystalline structure, isomeric characteristics, molecular weight and amount of

64

material used27, 34

. The exact composition and architecture of the polymeric scaffold becomes

fundamental to promote successful bone regeneration.

Although extensively used in several orthopedic appliances35

, PLA/PGA have been

scarcely applied in oro-maxillo-facial applications. PLA-derived devices were implemented to

prevent alveolar osteitis or dry socket with discordant results by Hooley and Golden36

. PLA

and decalcified freeze-dried bone allografts were clinically studied in the treatment of

periodontal intra-osseous defects by Meadows et al.37

. Results showed a limited amount of

tissue regeneration and the persistence of PLA particles surrounded by soft tissue. A new

PLA/PGA (Fisiograft®

, Ghimas, Italy), manufactured as sponge blocks, gel and powders, was

studied in maxillary sinus floor augmentations29, 38

, as space maker, in alveolar ridge

preservation26

, ridge augmentation with split-crest technique, in the treatment of deep

periodontal intra-osseous defect28

, and in critical bone defects in experimental animals25

with

contrasting results.

Proposition

We therefore histologically, histomorphometrically and immunohistochemically

evaluated the osteoconductive capability of the solid form of PLA/PGA copolymer in an

experimental model of maxillary sinus grafting.

Material and methods

Copolymer PLA/PGA Production

PLA/PGA copolymer consisted of a mixture of lactic and glycolic acid 50/50 saturated

with calcium phosphate, extent that make them gain a solid consistency. The solution pH was

controlled by adding the buffer solution. The method used was the polymeric or Pechini39

method, is a technique that can be applied to the development of advanced materials

65

precursors. The process is based on the ability of certain organic acids such as citric, lactic and

glycolic have to form chelates. These chelates may be esterified with a polyalcohol when

heated and polymerized at higher temperatures forming a resin.

Surgical Procedures

All experimental protocols involving animals were used conformed to procedures

described in the Guiding Principles for the Use of Laboratory Animals and the study approved

by the Animal Committee of Dentistry School of University Estadual Paulista Júlio de Mesquita

Filho, UNESP (00724-2012).

Twenty male white New Zealand rabbits, each weighing about 3.0kg, were used and

were divided into two groups, according to the sinus filling material, as follows: Group 1:

Control, particulate autogenous bone chips and Group 2: PLA/PGA copolymers particles.

All animals underwent surgeries for bilateral sinus lift procedures. The rabbits were

anesthetized with 1% chloridrate of ketamine (Francotar, Virbac Ltda, São Paulo, Brazil), along

with with a sedative, 2% chloridrate of xylazine (Virbaxyl 2%, Virbac Ltda, São Paulo, Brazil), in

the recommended dose, respectively, 60 mg/kg and 5.0mg/kg intramuscularly, and 0.5ml of

1% lidocaine with epinephrine (1:100000) was injected subcutaneously at the midline of the

nasal dorsum and into the tibial metaphysic.

Obtaining Autogenous Bone Graft

Linear incision with approximately 40mm was made in the direction of the long axis in

the tibial metaphysic after previous manual trichotomy and antisepsis preoperative in the right

tibia with friction of gauze soaked in 10% Polyvinyl Pyrrolidone Iodine degermante, with 1%

active iodine (PVP-I 10% Riodeine ®, Rioquímica, São José do Rio Preto) associated with the

topic PVP (10% PVP-I aqueous, with 1% active iodine, Riodeine®, Rioquímica, São José do Rio

66

Preto). Then, the soft tissue overlying the region was elevated in total thickness, exposing the

bone tissue. With the aid of scraper autogenous bone tissue samples were collected for

subsequent filling of the maxillary sinus. Completed sample collection, the surgical flap was

repositioned and sutured in layers.

Sinus Lift

Surgical interventions were performed under strict sterile conditions. The surgical area

was shaved and disinfected with iodine. The sinus lift surgical procedure was performed

according to Asai, Shimizu e Ooya (2002). A midline incision extending for about 50mm was

made, and the skin and periosteum were elevated sufficiently to expose the nasal bone and

nasoincisal suture line. A trephine bur with a 5.0mm internal diameter was used to delineate

the diameter of the bone window for the maxillary sinus access. A circular window was

opened in the nasal bone with the use of a round diamond bur nº 1014 under copious

irrigation with saline solution. The window was located approximately 20mm anterior to the

nasofrontal suture line, 10mm lateral to the midline. Care was taken during this procedure to

avoid damaging the sinus mucosa. The sinus membrane was carefully elevated to allow the

insertion and condensation of the graft materials. No membrane was used to cover the bone

window. In the experimental groups, the compartment was filled with PLA/PGA copolymer

and autogenous bone graft. A suture was placed between the periosteum and skin.

Histologic Procedures

After 3, 7, 15 and 40 days of the surgery, five animals were sacrificed with an overdose

of anesthetics. After decapitation and dissection, the entire nose complex, including the nose

and sinus cavities, was fixed in buffered 10% formaldehyde (Merck, Darmstadt, Germany) for

48 h and washed in tap water for 24 h, and immersed in buffered 4% EDTA for

demineralization. Specimens from the normal and experimental groups were slowly

67

decalcified and were cut in a frontal plane. The specimens were then dehydrated, embedded

in paraffin, and sliced into sections about 6µm thick. The sections were stained with

hematoxylin and eosin (Merck & Co., Inc., NJ, USA) for the light microscopic examination.

Some sections were reserved for immunohistochemistry reactions.

Histological and Histomorphometric Analysis

Each image of the calcified specimens at the rostrocaudal midpoint of the antral wall

was digitally captured at magnification X1.0, X2.8 and X160 and sent to the Image J Program

– Version 1.47 (National Institutes of Health (NIH), Bethesda, Maryland, USA). The composition

of the total augmented sinus was identified (in mm²). The proportions (in %) of each

composite (new bone - NB and soft tissue - ST) were obtained. To evaluate the distribution of

regenerated bone in the grafted sinus area as a secondary outcome variable, the above-

mentioned parameters were measured in specific standardized areas. The areas were

randomly selected and photomicrographs were taken in original magnification X160. The

window region was selected within the grafted area interfacing the imaginary extensions of

the pre-existing cortical bone at the margins of the window, the center region was at the

middle of the whole augmented sinus and membrane region. The membrane region was

chosen from just above the Schneiderian membrane:

Augmented area: defined as the raw surface of the secluded space underneath the

sinus membrane;

% bone, % soft tissues: percentage of the measured augmented area, defined as the

raw surface of the secluded space underneath the sinus membrane.

http://en.wikipedia.org/wiki/Bethesda,_Maryland

68


Immunohistochemical analysis were performed at 3, 7, 15, and 40 days (n = 5). The

following primary antibodies used for immunohistochemical processing were from Santa Cruz

(Biotechnology, CA): anti-osteocalcin (OC) and tartrate-resistant acid phosphatase (TRAP). As a

secondary antibody, a biotinylated donkey anti–goat antibody (Jackson Immunoresearch

Laboratories, West Grove, PA) was used. The signal of the immunohistochemistry reaction

with an avidin biotin system (Kit ABC-Vectastain Elite ABC–peroxidase standard, reagent A and

B only–PK6100; Vector Laboratories, Burlingame, CA), and diaminobenzidine (Sigma, St. Louis,

MO) was used as chromogen.

Immunohistochemical reactions were controlled to evaluate the specificity of the labels.

Hematoxylin and eosin staining was performed and used as a reference of the

cytoarchitecture of the tissue. Immunohistochemical analysis for consist of a qualitative

examination was stratified by a range of values ranging from 0 to 3 (0 = no protein expression,

1 = protein with little expression, 2 = intermediate protein expression, high protein expression

3 =) (Figs 2 and 3).

Statistical assessment

All morphometric data were assessed using the Mann-Whitney. The level of statistical

significance was set at 5%.

Results

Histologic Findings

Experimental group with autogenous bone graft

69


was found to be filled with acellular autogenous bone particles and blood clot, osteocytes in

the lacunae disappeared in the grafted bone.

After 7 days, peripheral newly formed bone trabeculae had irregular surfaces with

numerous osteoblasts and osteoclasts were seen in areas near the sinual membrane and

bone tissue of the nasal complex. Therefore early steps of centripetal new bone formation

were observed progressing from the anterior part of the sinus and the bone walls. Cell

colonization was found everywhere in the cavity, and remnants of clot were sometimes

visible. Remnant acellular autogenous bone particles were seen in the central region of the

space with connective tissue and blood vessels. There was no evidence of acute or chronic

inflammation.

After 15 days, the newly formed woven bone adjacent to the membrane and bone

tissue of the nasal complex showed osteoclastic resorption and the central region were seen

newly formed woven bone and soft tissue. There were many osteocytes in the newly formed

bone. Woven bone with many osteoblasts was observed around the grafted bone, especially

under the elevated antral membrane.


with intertrabecular vascularized adipose tissue. The bone chips were completely remodeled

and their remnants were not visible (Fig. 1).

Experimental group with PLA/PGA copolymer


was found to be filled with blood clot. Copolymer particles were not observed. Mesenchymal

cell proliferation was only visible at the periphery of the created space, while the blood clot

was still non-invaded by mesenchymal cells in the center of the cavity. No inflammatory cell

infiltration was present.

70

After 7 the augmented dome-like space was found to be filled connective tissue with

fibroblasts and blood vessels. Remnant blood clots were seen in the central region of the

space. Peripheral newly formed woven bone was seen. Therefore early steps of centripetal

new bone formation were observed progressing from the anterior part of the sinus and the

bone walls.

After 15 days, peripheral newly formed bone was seen in areas near the sinual

membrane and bone tissue of the nasal complex. Connective tissue was seen in the central

region of the space with fibroblasts and blood vessels.


with intertrabecular vascularized adipose tissue. The bone trabeculae showed the minimal

osteoclastic resorption. Some areas near the sinual membrane and bone tissue of the nasal

complex revealed mature cortical and trabecular bone with intertrabecular vascularized

adipose tissue (Fig.1).

Histomorphometric Analysis

Augmented area

Augmented area differ between the groups after 3, 7 and 15 days(P=0.004). However,

the values became similar on day 40 (P=0.458). The comparison of values of 3 and 40 days

autogenous bone group and PLA/PGA copolymer group and were also statistically significant

(P=0.004 e P=0.004) .Values in mm2 of the augmented area are presented in Table 1.

% bone, % soft tissues

After 3 days, in the autogenous bone group, bone inside the created space under the

membrane constituted 56.67% of the measured augmented area, while in the PLA/PGA

71

copolymer group, the amount of bone constituted 0%. The percentage of bone was

significantly higher with autogenous bone than with PLA/PGA copolymer (P=0.004).

After 7 days, in the autogenous bone group, bone ingrowths constituted 39.80% of

the measured augmented area, while in the PLA/PGA copolymer group, the amount of bone

ingrowth failed to reach 13.93%. The percentage of bone was significantly higher with

autogenous bone than with PLA/PGA copolymer (P=0.004).

After 15 days, the area occupied by newly formed bone reached, respectively, 43.47%

and 31.63% (P=0.087) for the autogenous bone and the PLA/PGA copolymer groups. The

implanted bone chips could be observed in the central area.

After 40 days, in the autogenous bone and PLA/PGA copolymer groups, the newly

formed bone area increased to 51.63% and 39.87%, respectively. The values were

comparable in the three groups (P=0.087). Detailed results are presented in Table 2 and

shows the correlation between volume changes over time and % bone and % soft tissues

within augmented tissues (Tables 2 and 3).


OC immunoreactivity could be seen in all augmented area for the autogenous bone

group in the early periods of this study, 3 days (intermediate protein expression), with

increased at 7 days (high protein expression) and remaining high on 15 and 40 days (high

protein expression) after surgery. Similarly to autogenous bone group, in the PLA/PGA

copolymer group, OC immunoexpressivity was seen intermediate at 3 days, with increased at

7 days (high protein expression), remaining high on 15 days (high protein expression).

However, declined at 40 days (intermediate protein expression) after surgery.

In TRAP immunohistochemistry could be seen in autogenous bone group high protein

expression in the early periods, 3 days. However, this declined at 7 days (intermediate protein

expression). At 15 days after surgery increased expression of TRAP (high protein expression)

72

with decline at 40 days (intermediate protein expression). Differently to the autogenous bone

group, in the PLA/PGA copolymer group TRAP immunoexpressivity had gradual increased: 3

days protein with little expression, 7 days with intermediate protein expression and 15 days

with high protein expression. However, declined at 40 days (intermediate protein expression)

(Fig. 2).

Discussion

This rabbit study investigated the area stability of sub-sinusal bone regeneration when

autogenous bone chips or PLA/PGA copolymer 50/50 were used as space fillers. Early bone

formation and the continuing behavior of the regenerated bone with these different types of

filling material were also assessed. Rabbits have similar maxillary sinus ventilation to humans

and a well-defined ostium opening to their nasal cavities. The rabbit sinus lift model is

therefore appropriate and well documented for evaluating bone regeneration physiology in

sub-sinusal bone regeneration40, 41

.

Adequate bone healing was observed with the two space fillers used in the present

study and no signs of inflammation could be detected. Similar findings have already been

reported by several authors29, 40, 41

; unfortunately, the early steps of bone regeneration were

poorly studied in these papers.

In this study, in PLA/PGA copolymer cell proliferation was detected after 7 days only

along the bone wall and under the sinusal membrane, while the central part was still

occupied by a dense cluster of red blood cells, in contrast with the autogenous bone group

where the augmented space was completely invaded by mesenchymal cells. A gradient of

maturity of the newly formed tissue from the native bony walls to the center of the cavity

illustrated the centripetal nature of the regenerative process.

At 15 days, in the PLA/PGA copolymer group, non-mineralized fibrous tissue still

occupied the central area. At 40 days, in the PLA/PGA copolymer group, woven bone was

73

found everywhere in the remaining cavity. Therefore, PLA/PGA copolymer implanted into

sub-sinusal cavities allowed bone formation. Rimondini et al25

reported that the

histomorphometric data demonstrated that the experimental material is able to promote bone

healing even in a large bone defect such as in rabbit femoral condyles. The bone maturation

improved from 30 to 90 days. The data concerning the development of the forming bone

showed that during the 30 to 90 days the material allowed a bone remodeling from reticular

toward a trabecular structure.

Fisiograft®

is a synthetic resorbable sponge formed by 50–50 lactide–glycolide

polymer. This copolymer has shown the fastest degradation rate of the d-l lactide/glycolide

biomaterials, with the polymer degrading in about 50–60 days42

. In the previous study37

PLA

granules were still evident at 6-month surgical re-entry.

In the autogenous bone group, the early healing process was characterized by a

particularly high cellular density along the bone chips, even at a distance from the bony walls.

This picture strongly suggests an osteogenic potential provided by autogenous bone chips. At

15 days, the anatomy of the dense woven bone seemed to be influenced by the initial rolled

shape of the bone chips, now remodeled. The remodeling process continued from 15 days to

40 days yielding, in the long term, rarefied mature bone architecture. According to Schlegel et

al.43

, this remodeling process was also observed in sinus lifts performed in humans with

particulated autogenous bone.

New bone formation was already observed at 2 weeks after grafting. It is of interest

that new bone formation was observed in all parts under the elevated antral membrane,

around the grafted bone, and at the superior part of the original sinus floor. The result

indicates that new bone formation may depend not only on osteoconduction from the

recipient site but also on osteoinduction by bone morphogenetic protein in the grafted bone

and osteogenesis by the bone cells that were grafted with the bone.

Albrektsson44

reported that early revascularization meant early bone remodeling of a

graft in the rabbit tibia; in the present study, blood vessels were already seen in the grafted

74

bone at 7 days. It is suggested that early revascularization also assisted in the new bone

formation for the maxillary sinus in the present study.

Much newly formed bone was noted under the elevated membrane at 40 days after

grafting. Moreover, continuous cortical bone was observed under this membrane. These

results indicate the difference in bone formation between the continuous bone located under

the elevated membrane and continuous bone located elsewhere. In the present study, the

medullary cavity was filled with many fat cells.

In our study, the mean bone fractions in the grafted area were 56.67%, 39.80%,

43.47% and 51.63%, respectively, at 3, 7, 15, and 40 days after grafting. The bone fraction in

the bone biopsy cores was reported in a few studies; Moy et al45

, for example, indicated that

the composition of bone was 59.4% from chin bone graft, and Lundgren et al46

reported the

mean bone fractions in chin bone grafted areas to be 40% and 48%, respectively, at 6 and 12

months after bone grafting.

Immunohistochemistry confirms the results on the histomorphometric data. During the

period of increase of regeneration area, between 3 and 7 days, the OC values also showed an

increase in both experimental groups. Important to note that the lower expression group in

the copolymer shows an early stage of the process when compared to autogenous bone

group. In both groups, the period 7-15 days it is possible to see a tendency to equilibrium in

the processes of bone formation and remodeling, we see a tendency to balance the values of

OC and TRAP. Increased OC shows the mineralization phase in which it is the bone tissue

after 40 days in the autogenous bone group, since the values of TRAP are consistent the

period of remodeling of bone trabeculae, characteristic of this period. Unlike the group

copolymer in the same period seems to still keep the balance-training resorption.

The present study showed that the long-term outcome of augmentation of the sinus

was not the maturation of newly formed bone, but conversely atrophy of bone. Indeed, an

increase in adipose tissue in marrow is observed in all conditions that lead to loss of bone,

such as osteoporosis, age-related osteopenia, or immobilisation47

. This apparent reciprocal

75

relation between reduced bone density and increased fat can be explained by an imbalance

in the production of bone-forming and fat-forming cells48

.

But an excess of adipose tissue in marrow is considered to put at risk the long-term

ability to maintain the mechanical strength of the skeleton49

. Mechanical stress is an essential

factor for the corticalisation of bone50

. Ideal bone grafts should eventually be absorbed and

encourage formation of new bone, which replenishes lost bone permanently.

In conclusion, PLA/PGA copolymer seems to be suitable as resorbable material able to

induce bone growth in bone defects. This observation suggests that the material have

osteoconductive properties also suitable for application in maxillofacial surgery.

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humans. Clin Oral Implants Res 2003 Oct;14(5):651-8.

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81

Figure e Table

Fig

ure

1: P

LA/P

GA

co

po

lym

er

at

(a)

3, (

b)

7, (

c) 1

5 a

nd

(d

) 4

0 d

ays

. Au

tog

en

ou

s b

on

e g

rou

p a

t (e

) 3

, (f)

7, (

g)

15

an

d (

h)

40

days

.

82

Figure 2: Autogenous bone group; OC immunoreactivity in (a) 3, (b) 7, (c) 15

and (d) 40 days. PLA/PGA copolymer group; OC immunoreactivity in (e) 3, (f) 7,

(g) 15 and (h) 40 days.

83

Figure 3: Autogenous bone group; TRAP immunoreactivity in (a) 3, (b) 7, (c) 15

and (d) 40 days. PLA/PGA copolymer group; TRAP immunoreactivity in (e) 3, (f)

7, (g) 15 and (h) 40 days.

84

Table 1: Augmented area – Mean (mm²)


3 days 35.74 28.36 P=0.004

7 days 36.13 28.99 P=0.004

15 days 27.45 35.44 P=0.004

40 days 32.52 31.49 P=0.458

Table 2: %bone – Mean (%)


3 days 56.67 0 P=0.004

7 days 39.80 13.93 P=0.004

15 days 43.47 31.63 P=0.087

40 days 51.63 39.87 P=0.087

85

Table 3: % soft tissue – Mean (%)


3 days 43.33 100 P=0.004

7 days 60.40 86.07 P=0.004

15 days 56.53 68.37 P=0.087

40 days 48..37 60.13 P=0.087

86

Anexo 3 – Normas IJOMS

International Journal of Oral & Maxillofacial Surgery

Guide for Authors

Would authors please note that the reference style for the journal has now changed. Please

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." Citations in the text to

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identified..."

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Examples:

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Book/monograph: Costich ER, White RP. Fundamentals of oral surgery. Philadelphia: WB

Saunders, 1971: 201-220.

Book chapter: Hodge HC, Smith FA. Biological properties of inorganic fluorides. In: Simons JH,

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3. One should support all assertion by peer review literature which should be a primary

research or large clinical studies rather than a case report.

4. Please include any financial disclosures at the end of the letter. This would include the

potential conflicts of interest not just related to the specific content of your letter but also the

content of the IJOMS article and other related areas.

5. Please recognize that letters that are essentially in agreement with the author's findings and

offer no additional insights provide little new information for publication. Likewise, letters that

highlight the writer's own research or are otherwise self-promotional will receive a low

publication priority.

6. There may be a need for additional editing. Should editing be required the letter will be

sent back to the author for final approval of the edited version.

7. It is important to use civil and professional discourse. It is not advisable that one adopt a

tone that may be misconstrued to be in anyway insulting.

8. Finally, it is not advisable to provide a letter that is anecdotal. While personal experiences

can have great value in patient care, it is generally not strong evidence to be placed in a letter

to the editor.

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Catalogação na Publicação (CIP) Serviço Técnico de Biblioteca e Documentação – FOA / UNESP

Ferreira, Sabrina. F273e Copolímero PLA/PGA como substituto ósseo em levantamento de seio maxilar. Estudo histológico, histométrico e imunoistoquímico em coelhos / Sabrina Ferreira. - Araçatuba : [s.n.], 2014 31 f. : il. ; tab. + 1 CD-ROM Dissertação (Mestrado) – Universidade Estadual Paulista, Faculdade de Odontologia de Araçatuba Orientador: Prof. Idelmo Rangel Garcia Júnior 1. Ácido Polilático 2. Ácido Poliglicólico 3. Seio Maxilar 4. Imunoistoquímica Black D7 CDD 617.64

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