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PONTIFÍCIA UNIVERSIDADE CATÓLICA DO PARANÁ
CENTRO DE CIÊNCIAS BIOLÓGICAS E DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO EM ODONTOLOGIA
ÁREA DE CONCENTRAÇÃO ESTOMATOLOGIA
CINTIA MUSSI MILANI CONTAR
ANÁLISE CLÍNICA E HISTOLÓGICA DO OSSO ALÓGENO FRESCO CONGELADO
NA RECONSTRUÇÃO DE REBORDO ALVEOLAR MAXILAR
CURITIBA
2008
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CINTIA MUSSI MILANI CONTAR
ANÁLISE CLÍNICA E HISTOLÓGICA DO OSSO ALÓGENO FRESCO CONGELADO
NA RECONSTRUÇÃO DE REBORDO ALVEOLAR MAXILAR
Tese apresentada ao Programa de Pós-Graduação em
Odontologia, da Pontifícia Universidade Católica do
Paraná, como requisito à obtenção do título de Doutor em
Odontologia – Área de Concentração em Estomatologia.
Orientadora: Profa. Dra Maria Ângela Naval Machado
Co-Orientador: Prof. Dr. Jayme Bordini Júnior
CURITIBA
2008
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Dados da Catalogação na Publicação
Pontifícia Universidade Católica do Paraná
Sistema Integrado de Bibliotecas – SIBI/PUCPR
Biblioteca Central
Contar, Cintia Mussi Milani
C759a Análise clínica e histológica do osso alógeno fresco congelado na
2008 reconstrução de rebordo alveolar maxilar / Cintia Mussi Milani Contar ;
orientadora, Maria Ângela Naval Machado ; co-orientador, Jayme Bordini
Júnior. – 2008.
90 f. ; 30 cm
Tese (doutorado) – Pontifícia Universidade Católica do Paraná, Curitiba,
2008
Inclui bibliografia
1. Ossos – enxerto. 2. Processo alveolar. 3. Aumento do rebordo alveolar.
4. Transplante ósseo. I. Machado, Maria Ângela Naval. II. Bordini Júnior,
Jayme. III. Pontifícia Universidade Católica do Paraná. Programa de Pós-
Graduação em Odontologia. IV. Título.
CDD 20. ed. – 617.69
SUMÁRIO
1 ARTIGO EM PORTUGUÊS...................
...................................................................1
PÁGINA TÍTULO....................................... ...................................................................2
RESUMO.................................................. ...................................................................3
INTRODUÇÃO.......................................... ...................................................................4
MÉTODO.................................................. ...................................................................5
RESULTADOS.......................................... ...................................................................7
DISCUSSÃO............................................. ...................................................................9
CONCLUSÃO........................................... .................................................................11
REFERÊNCIAS........................................ .................................................................12
TABELAS.................................................. .................................................................15
FIGURAS.................................................. .................................................................16
2 ARTIGOS EM INGLÊS.......................... .................................................................22
2.1 MANUSCRIPT DRAFT....................... .................................................................23
DECISION LETTER.................................. .................................................................24
TITLE PAGE............................................. .................................................................25
ABSTRACT.............................................. .................................................................26
INTRODUCTION...................................... .................................................................27
METHOD.................................................. .................................................................28
RESULTS................................................. .................................................................29
DISCUSSION........................................... .................................................................30
REFERENCES......................................... .................................................................32
FIGURES.................................................. .................................................................34
2.2 MANUSCRIPT DRAFT....................... .................................................................39
TITLE PAGE............................................. .................................................................40
ABSTRACT............................................... .................................................................41
INTRODUCTION...................................... .................................................................42
METHOD.................................................. .................................................................43
RESULTS................................................. .................................................................44
DISCUSSION........................................... .................................................................45
REFERENCES......................................... .................................................................48
TABLE...................................................... .................................................................50
FIGURES................................................. .................................................................51
3 ANEXOS............................................... .................................................................54
ANEXO 1 - APROVAÇÃO DO COMITÊ
DE ÉTICA.................................................
.................................................................55
ANEXO 2 – ANÁLISE ESTATÍSTICA....... .................................................................56
ANEXO 3 - NORMAS PARA
PUBLICAÇÃO NO PERIÓDICO
JOURNAL OF ORAL AND
MAXILLOFACIAL SURGERY...................
.................................................................60
ANEXO 4 - NORMAS PARA
PUBLICAÇÃO NO PERIÓDICO
INTERNATIONAL JOURNAL OF ORAL
AND MAXILLOFACIAL SURGERY..........
.................................................................62
ANEXO 5 - RESUMO DOS ARTIGOS
USADOS NA DISCUSSÃO.......................
.................................................................69
TERMO DE APROVAÇÃO
CINTIA MUSSI MILANI CONTAR
ANÁLISE E HISTOLÓGICA DO OSSO ALÓGENO FRESCO CONGELADO NA
RECONSTRUÇÃO DE REBORDO ALVEOLAR MAXILAR.
Tese apresentada ao Programa de Pós-Graduação em Odontologia, da Pontifícia
Universidade Católica do Paraná, como parte dos requisitos parciais para a obtenção do
título de Doutor em Odontologia, Área de Concentração em Estomatologia.
Orienta:
Curitiba, 08 de dezembro de 2008.
Rua Imaculada Conceição, 1155 Prado Velho CEP 80215 901 Curitiba Paraná Brasil
Tel.: (41) 3271 1637 Fax: (41) 3271 1405 www.pucpr.br ppgo@pucpr.br
Pontifícia Universidade Católica do Paraná
Centro de Ciências Biológicas e da Saúde
Programa de Pós-Graduação em Odontologia
Orientadora:
DEDICATÓRIA
À memória do meu pai, pelo seu amor incondicional durante todos os dias de sua vida. O destino nos
separou muito cedo, mas seu exemplo de vida e dedicação à família permanecerá comigo para sempre,
guiando meus passos.
Ao meu esposo, Ângelo, companheiro de todas as horas, pelo seu amor, dedicação e paciência. Sem o
seu apoio com certeza eu não teria chegado até aqui.
Aos meus filhos, Guilherme e Beatriz, os maiores presentes de toda minha vida.
AGRADECIMENTO ESPECIAL
À minha orientadora, Profa. Dra. Maria Ângela Naval Machado, em quem descobri muito mais que
uma orientadora espetacular, uma grande amiga. Exemplo perfeito para o significado da palavra
mestre, com toda sua dedicação, disponibilidade, sabedoria, paciência e apoio; que bom seria se as
universidades estivessem repletas de mestres como ela. Faltam palavras para expressar a gratidão que
tenho. Com certeza foi um grande privilégio ser sua orientada. Muito obrigada por tudo.
AGRADECIMENTOS
A Deus, pela minha abençoada vida.
À minha mãe Margaret, meus irmãos Gustavo e Rodrigo e minhas cunhadas Mônica e Geórgia, por
estarem sempre presentes me apoiando nos momentos difíceis e dividindo comigo as alegrias.
Ao meu co-orientador Prof. Dr. Jayme Bordini Júnior, por abrir as portas do Curso de Especialização
em Implantodontia da UFPR para que eu pudesse realizar este trabalho. Obrigada pela amizade,
apoio e confiança.
Ao coordenador dos cursos de Pós-Graduação em Odontologia da PUCPR, Prof. Dr. Sérgio Roberto
Vieira, pela disponibilidade e atenção dispensada sempre que necessário. Exemplo de profissional a
ser seguido, sua garra e dedicação ao ensino me inspiram desde as aulas da graduação. Com toda
certeza sem seu esforço e perseverança este curso possivelmente não existiria.
Ao coordenador do curso de Estomatologia, Prof. Dr. Fernando Henrique Westphalen, pela pronta
disposição em nos atender, em todos os momentos, procurando sempre buscar uma solução para
quaisquer dificuldades que surgissem.
Ao Prof. Dr. Sérgio Aparecido Ignácio, mais um grande exemplo de mestre. Com sua paciência e
sabedoria ao ensinar, consegue fazer a estatística parecer algo simples e fácil de aprender.
Ao Prof. Dr. Antônio Adilson Soares Lima, pelo conhecimento transmitido nas aulas excelentemente
ministradas, pela experiência compartilhada nas clínicas e laboratórios e pelas preciosas sugestões na
correção deste trabalho. É um privilégio para poucos ter a oportunidade de aprender com um mestre
deste porte.
Às Profas. Dras. Luciana Reis Azevedo-Alanis e Paula Cristina Trevilatto pelo tempo disposto e
excelentes sugestões feitas ao trabalho no exame de qualificação. Com certeza é uma honra ser
avaliada por profissionais de tão alto nível.
A todos os professores da Estomatologia e demais áreas conexas, pelo conhecimento transmitido
durante o curso.
Aos todos meus colegas de turma e alunos do mestrado em Estomatologia, pelos bons momentos que
passamos juntos. Fiz aqui amizades que certamente ficarão para o resto da vida.
Aos professores do Curso de Especialização em Implantodontia da Universidade Federal do Paraná,
Prof. Gastão Valle Nicolau, Prof. Francisco Patino, Prof. Hélio Paiva e, em especial, ao Prof. João
Rodrigo Sarot, cuja colaboração foi fundamental para realização deste trabalho.
Ao Professor André Moreira Rodrigues por sua amizade e por me incentivar, desde o início da
graduação, na área de pesquisa.
À secretária Neide Reis Borges, nosso anjo da guarda de todas as horas. Sempre com um sorriso no
rosto e disposta a nos ajudar no que precisássemos.
Ao Sr. Herculano de Souza e funcionárias do Laboratório de Histotecnologia da UFPR, e Ana
Paula Camargo Martins, do Laboratório de Patologia Experimental da PUC-PR, pela colaboração
na realização das lâminas.
1) ARTIGO EM PORTUGUÊS
PÁGINA TÍTULO
Análise Clínica e Histológica do Osso Alógeno Fresco Congelado na Reconstrução de
Rebordo Alveolar Maxilar.
CMM Contar, J Bordini Jr, MAN Machado
Departamento de Estomatologia, Programa de Pós-Graduação em Odontologia,
Pontifícia Universidade Católica do Paraná, Curitiba, Paraná, Brasil
Curso de Especialização em Implantodontia, Universidade Federal do Paraná,
Curitiba, Paraná, Brasil
Endereço para correspondência, rua Imaculada Conceição, 1155 CEP 80215-901 –
Curitiba – Paraná, Brasil – Fone: 41 3271-1637 – Fax: 41-3271-1405
e-mail: [email protected]
Palavras-chave: enxerto ósseo, osso alógeno, colágeno
RESUMO
Análise Clínica e Histológica do Osso Alógeno Fresco Congelado na
Reconstrução de Rebordo Alveolar Maxilar
O enxerto ósseo alógeno surge como uma alternativa ao enxerto autógeno,
apresentando como principais vantagens a diminuição do trauma operatório e o
suprimento praticamente ilimitado de tecido ósseo. Na odontologia, os estudos que
demonstrem clínica e histologicamente a viabilidade do osso fresco congelado em
humanos são escassos. O objetivo deste trabalho foi avaliar a viabilidade de enxertos
ósseos alógenos frescos congelados na reconstrução de rebordo alveolar em
humanos, por avaliação clínica e histológica. A amostra foi composta por 18 indivíduos
com rebordo maxilar atrófico que receberam enxerto ósseo alógeno fresco congelado
em blocos. No período pós-operatório avaliou-se a presença de infecção e exposição
dos blocos. No momento da reabertura, para colocação dos implantes avaliou-se,
clinicamente, a vascularização e a integração dos blocos enxertados. Nesta etapa
amostras do osso alógeno enxertado (teste) e do osso autógeno (controle) do mesmo
indivíduo foram removidas, com broca trefina para análise histológica. As lâminas
foram processadas e coradas com Hematoxilina-eosina, Tricrômico de Masson e
Picrosírius. Os cortes corados com Picrosírius foram analisados sob luz polarizada,
quantificando-se a porcentagem de área e densidade do colágeno maturo e imaturo
dos grupos teste e controle. Ao exame clínico, não foi observada infecção e todos os
blocos estavam bem integrados e vascularizados, não ocorrendo deslocamento de
nenhum deles no momento da instalação dos implantes. Não houve diferença
estatisticamente significante na porcentagem média de área (p=0.36) e densidade
(p=0.21) de colágeno maturo e imaturo quando comparados os grupos teste e
controle. Concluiu-se que o osso fresco congelado é biologicamente viável para
reconstrução de rebordos alveolares maxilares que receberão implantes
osseointegrados, exibindo um padrão de maturação do colágeno similar àquele do
osso autógeno.
INTRODUÇÃO
A utilização dos enxertos ósseos para reconstruir áreas que já não apresentam
volume ósseo suficiente para a colocação de implantes osseointegráveis, tornou-se
um procedimento comum na prática odontológica, com o grande desenvolvimento da
implantodontia nos últimos anos. Além disso, os enxertos também são necessários na
recuperação de áreas após ressecções tumorais, traumas e reconstruções
ortopédicas
1,2
.
O osso autógeno ainda é considerado o gold standard para reconstrução dos
defeitos ósseos por ser o único com propriedades osteogênicas. Seu uso, no entanto,
apresenta algumas desvantagens como: maior morbidade pelo segundo acesso
cirúrgico, quantidade limitada de tecido ósseo disponível, aumento do sangramento e
do tempo operatório
3,4
. Substitutos ósseos como os compósitos minerais, cerâmicas,
biovidros, enxertos alógenos, associados ou não a proteínas ósseas morfogenéticas
(BMPs) e fatores de crescimento vêm sendo utilizados como uma alternativa aos
enxertos autógenos
5
.
O tecido ósseo alógeno é aquele transplantado entre indivíduos da mesma
espécie
6
. O primeiro enxerto ósseo alógeno em humanos foi realizado em 1880 por
um cirurgião escocês que reconstruiu o úmero infectado de uma criança de 4 anos
com a tíbia de outra criança
7
. No período anterior ao estabelecimento dos bancos de
tecido músculo-esqueléticos de grande porte, o osso alógeno era proveniente de
cadáver fresco ou de doador vivo, não havendo tempo para detectar possíveis
contaminações bacterianas
8
. Em 1949, a criação do Banco de Tecidos da Marinha
Americana, com técnicas adequadas de coleta, processamento e armazenamento do
tecido ósseo e completa investigação dos doadores, marcou o início da modernização
dos bancos de tecidos
6
.
Esta alternativa, segundo Hachiya et al.
1
, é o método mais apropriado para a
reconstrução de grandes defeitos ósseos e apresenta como principais vantagens a
diminuição do trauma operatório e o suprimento praticamente ilimitado de tecido
ósseo
9
. Além disso, o uso deste material proporciona uma diminuição da perda
sanguínea, ausência de morbidade da área doadora e redução do tempo
operatório
2,10
. As desvantagens associadas ao uso do osso alógeno são o risco de
transmissão de doenças e a antigenicidade
10
. Os bancos de tecidos seguem rigorosos
protocolos na seleção dos doadores, coleta e armazenamento do osso, para garantir o
suprimento de um tecido seguro
11,12
. O processamento do tecido reduz
significativamente a antigenicidade
12
.
Nos enxertos ósseos alógenos, suas características biomecânicas,
microbiológicas e seu potencial de osteocondução e osteoindução são igualmente
importantes e podem variar dependendo do método de processamento
13
. O osso
alógeno pode ser disponibilizado de quatro formas: fresco, fresco congelado, liofilizado
e desmineralizado
9
.
O osso fresco congelado é coletado cirurgicamente de doadores vivos ou
cadáveres e processado pelo armazenamento do espécime em temperaturas que
variam de -20 a -170°C. Pode ser obtido na forma de anéis cortico-esponjosos ou
particulado esponjoso, cortico-esponjoso ou cortical
9,14
.
Na ortopedia existe, atualmente, uma tendência dos cirurgiões usarem os
enxertos ósseos alógenos em substituição aos autógenos
15
. No entanto, a literatura
sobre o uso deste material em procedimentos buco-maxilo-faciais ainda é bastante
escassa. Diante disso, o objetivo do presente estudo foi avaliar a viabilidade de
enxertos ósseos alógenos frescos congelados na reconstrução de rebordos
alveolares, em humanos, por análise clínica e histológica.
PACIENTES E MÉTODO
O presente estudo foi aprovado com parecer nº 1480 pelo Comitê de Ética em
Pesquisa da Pontifícia Universidade Católica do Paraná.
No período compreendido entre Abril de 2005 a Março de 2006, foram
selecionados 18 indivíduos, 6 homens e 12 mulheres, com idade entre 27 e 61 anos
(média de 41,5 anos), atendidos no Curso de Especialização em Implantodontia da
Universidade Federal do Paraná, que necessitavam de enxerto ósseo previamente a
colocação dos implantes devido à presença de rebordo maxilar atrófico. Destes 3
eram desdentados totais e 15 desdentados parciais.
O diagnóstico de rebordo maxilar atrófico foi estabelecido por exame clínico e
radiográfico. Radiografias panorâmicas e exames laboratoriais foram realizados por
todos os indivíduos e não revelaram nenhuma outra alteração óssea ou sistêmica. Os
exames laboratoriais incluíram hemograma completo, coagulograma, glicemia,
creatinina e pesquisa de anticorpos anti-HIV. Os pacientes que apresentaram
alteração nos mesmos e/ou condição sistêmica (diabetes descompensada, problemas
renais agudos, discrasias sanguíneas, hepatopatias, hipertensão arterial) que contra-
indicassem a cirurgia foram excluídos da amostra.
Em todos os enxertos foram utilizados blocos de anel de tíbia cortico-esponjoso
fresco congelado proveniente do Banco de Tecidos Músculo-Esquelético do Hospital
de Clínicas do Paraná.
Os indivíduos receberam 2 g de amoxicilina (Amoxil, Glaxo SmithKline, Rio de
Janeiro, Brasil) e 8 mg de dexametasona (Decadron, Aché Laboratórios
Farmacêuticos, Guarulhos, São Paulo, Brasil), por via oral, uma hora antes da cirurgia,
conforme protocolo utilizado no Curso de Implantodontia, para minimizar o risco de
infecção e proporcionar uma redução do edema pós-operatório.
O tecido ósseo foi removido da embalagem para descongelamento e imerso
em soro fisiológico uma hora antes do procedimento cirúrgico. Após o
descongelamento o mesmo foi preparado, em blocos, com cinzel e martelo ou disco
diamantado
1
(figura 1).
Sob anestesia local com articaína 4% com epinefrina 1:100.000
2
foi realizada a
incisão em rebordo alveolar e descolamento do retalho mucoperiostal para exposição
das paredes maxilares. Com uma broca esférica nº 1 realizou-se a descorticalização
da área receptora, para provocar um aumento do sangramento medular. Os blocos
foram fixados às paredes maxilares com parafuso de titânio
3
sem deixar mobilidade
nos mesmos e espaço entre o enxerto e a área receptora (figura 2). Quando a
adaptação do bloco não foi perfeita, uma porção de osso proveniente do mesmo anel
de tíbia foi particulada e condensada fortemente nestes espaços. Algumas perfurações
com broca esférica também foram realizadas nos blocos, com o intuito de aumentar a
nutrição para o local. Os retalhos foram suturados com fio de seda 4.0
4
e os pontos
removidos após 7 dias. Uma amostra do osso alógeno que não foi utilizada no enxerto
foi encaminhada para análise histológica.
A medicação pós operatória instituída foi de 1g de amoxicilina a cada 12 horas
por 7 dias, 4 mg de dexametasona a cada 8 horas por 2 dias, 500 mg de paracetamol
com 30 mg de fosfato de codeína (Tylex, Janssen Cilag Farmacêutica Ltda, São
Paulo, Brasil) a cada 6 horas, em caso de dor e bochechos com clorexidina
(Periogard, Colgate-Palmolive, Brasil) duas vezes ao dia, por 15 dias.
No momento da remoção dos pontos os pacientes foram avaliados quanto à
presença de infecção e exposição dos blocos enxertados.
No terceiro mês pós-operatório, uma radiografia panorâmica foi realizada para
avaliar a condição do enxerto (figura 3).
Oito a onze meses após a colocação dos blocos, foi realizada a cirurgia de
reabertura para colocação dos implantes osseointegráveis. Nesta etapa cirúrgica
removeram-se da região enxertada, com broca trefina
5
de 2,5 mm de diâmetro,
1
Disco diamantado – Komet, Fellbach, Alemanha.
2
Mepivacaína 2% - DFL do Brasil Ltda., Rio de Janeiro, RJ, Brasil
3
Parafuso de titânio – Neodent, Curitiba, PR, BR.
4
Fio de seda 4.0 – Ethicon, Inc., Somerville, NJ, USA.
5
Broca trefina 2,5mm – Ind. Tornado, São Paulo, SP
amostras do tecido ósseo alógeno (grupo teste) para avaliação histológica (figura 4).
Estas foram removidas da área de instalação dos implantes, já servindo como preparo
inicial para dos mesmos.
Amostras do osso autógeno, dos mesmos pacientes, foram removidas, com
broca trefina de mesmo diâmetro, de uma região vizinha que não recebeu o enxerto de
osso alógeno, constituindo nosso grupo controle. O grupo teste e o grupo controle
eram, portanto, do mesmo paciente, sendo o teste o osso alógeno enxertado e o
controle o osso autógeno do próprio paciente.
Os espécimes de tecido ósseo coletados foram identificados e fixados em
formalina neutra tamponada a 10% por 48 horas. A descalcificação foi realizada em
ácido tricloroacético a 5%, no laboratório de Histotecnologia da UFPR, por 15 dias. Ao
final deste período, as peças foram desidratadas em álcool, diafanizadas em xilol e
incluídas em parafina. Obtiveram-se cortes no sentido horizontal de 6 µm de
espessura, que foram corados com Hematoxilina-Eosina, Tricrômico de Masson e
Picrosírius para análise histológica.
Os cortes corados com HE e Masson foram utilizados para analisar a
morfologia óssea e aqueles corados com Picrosírius para avaliar a maturação do
colágeno. As imagens dos cortes de osso autógeno e do alógeno coradas com
Picrosírius foram selecionadas e capturadas por uma câmera digital (Sony CCD Íris
®
,
Sony, Japan) acoplada a um microscópio binocular (Olympus BX50
®
; Olympus,
Japan), utilizando objetiva de 10x. Foram capturados dois campos aleatórios de cada
lâmina e no campo selecionado, sob polarização auxiliado pelo sistema de análise de
imagens digitalizadas (Image ProPlus
®
; version 4.5 for Windows, Media Cybernetics
Inc, USA), foi quantificada a porcentagem da área e densidade do colágeno maturo e
imaturo dos grupos teste e controle. A máxima posição de brilho dos feixes de fibras
colágenas foi determinada antes da avaliação na qual fibras mais espessas e
fortemente birrefringentes apresentam uma cor amarelo-vermelha (colágeno maturo),
enquanto as fibras mais finas, esparsas, fracamente birrefringentes, com uma cor
verde (colágeno imaturo)
16,17
.
Os dados foram tabulados e testes estatísticos foram realizados com o SPSS
para Windows 13.0 (SPSS Inc., Chigago, IL, USA). A normalidade dos dados foi
testada usando-se o teste de Kolmogorov-Smirnov, a um nível de significância de 5%.
Em seguida, visando detectar diferenças significativas nas porcentagens de área e
densidade do colágeno maturo e imaturo entre os grupos teste e controle, utilizou-se o
teste t de Student para amostras pareadas.
RESULTADOS
Um total de 39 blocos de anel de tíbia cortico-esponjoso fresco congelado
foram enxertados. O número de blocos que cada paciente recebeu variou de 1 a 4 e o
número de implantes sobre os blocos de 1 a 8. Os indivíduos desdentados totais
receberam 4 blocos e 6 a 8 implantes; para os desdentados parciais o número de
blocos variou de 1 a 2 e o de implantes de 1 a 4.
O período pós-operatório transcorreu sem exposição do bloco ou infecção em
17 indivíduos. Em um caso houve deiscência de sutura, com consequente exposição
de 1 bloco. Neste caso, um segundo procedimento cirúrgico foi realizado para
recobrimento do mesmo. Apesar da exposição, não houve infecção e nenhum
comprometimento do resultado final.
Durante as cirurgias de reabertura, todos os blocos estavam sem mobilidade,
bem integrados e vascularizados, mesmo naqueles casos em que foram realizadas
reconstruções mais extensas (figura 5). A colocação dos implantes sobre os blocos
enxertados demonstrou a funcionalidade e resistência dos mesmos, uma vez que
nenhum bloco soltou-se durante esta etapa.
Não foi necessário enxerto ósseo adicional em nenhum indivíduo. Em todos os
casos os implantes foram posicionados conforme o planejamento inicial, não
ocorrendo em nenhum bloco reabsorção óssea severa que tornasse necessária
mudança no plano de tratamento (Figura 6).
Um total de 58 implantes foram instalados sobre os blocos (figura 7), com um
torque mínimo de 40 Newton; 22 implantes já receberam as próteses. Nenhum dos
implantes foi perdido. O período pós-operatório variou de 8 a 42 meses.
A análise histológica dos cortes corados com HE e Tricrômico de Masson
revelou um osso com típico arranjo lamelar, mostrando os canais de Havers e as
lacunas dos osteócitos, características de osso secundário, em todos os espécimes do
grupo teste (figura 8A). Características histológicas similares foram observadas no
grupo controle, porém o osso autógeno apresentou maior celularidade (figura 8B). Na
periferia dos cortes do grupo teste foram observadas células semelhantes a
osteoblastos e uma maior quantidade de osteócitos nas lacunas (figura 9). Na porção
mais central verificamos uma menor quantidade de osteócitos, porém com a presença
constante de vasos sanguíneos (figura 10).
Na análise, sob polarização, dos cortes corados com Picrosirius observamos,
em ambos os grupos, as fibras colágenas organizadas em lamelas paralelas ou
dispostas em camadas concêntricas, em torno de canais com vasos, formando os
sistemas de Havers, características de osso secundário (figura 11). Com esta análise
foram obtidas as porcentagens médias de área e densidade do colágeno maturo e
imaturo dos grupos teste e controle (tabela 1 e 2). Os resultados do teste Kolmogorov-
Smirnov indicaram que todas as variáveis apresentaram distribuição normal (p= 0.20).
O teste t de Student para amostras pareadas demonstrou não haver diferença
estatisticamente significante tanto na porcentagem de área (p= 0.36), quanto na
porcentagem da densidade (p= 0.21) do colágeno maturo e imaturo quando
comparado os valores médios do grupo teste em relação ao controle.
Quando observamos, sob polarização, o osso alógeno não enxertado,
constatamos a presença exclusiva de áreas de birrefringência amarelas-vermelhas
características do colágeno maturo. Na análise deste mesmo tecido, nove meses após
o enxerto, observamos o surgimento de áreas de birrefringência verde
correspondentes ao colágeno imaturo (figura 12).
DISCUSSÃO
Os enxertos ósseos alógenos são utilizados na ortopedia há muito tempo, com
diversas aplicações, incluindo trauma, cirurgias de coluna vertebral, cirurgias
articulares e pós-ressecções tumorais
10,15,18,19
. Em uma pesquisa prospectiva em
cirurgia de escoliose, os autores concluíram que mesmo na presença de crista ilíaca
autógena adequada, o uso do osso fresco congelado era mais efetivo
10
. O uso dos
enxertos autógenos na ortopedia está se tornando cada vez mais raro, devido à
disponibilidade de ossos alógenos seguros e eficientes
15
. No entanto, na área buco-
maxilo-facial a literatura ainda é escassa e a maior parte dos estudos publicados é em
animal
3,20,21
ou limitada a relatos de caso
22,23,24,25
.
O sucesso da enxertia óssea depende de muitos fatores que incluem o tipo e a
fixação do enxerto, bem como o local e condição da área receptora
26
. Os enxertos
ósseos de qualquer tipo podem regenerar osso por três possíveis mecanismos:
osteogênese, osteoindução e osteocondução
27
. O enxerto alógeno ideal induziria a
formação de um novo tecido ósseo (osteoindução) e proporcionaria um arcabouço
para suportar o tecido ósseo do hospedeiro, em regeneração, que substituiria o
enxerto (osteocondução)
23
.
Apesar das propriedades osteoindutoras dos enxertos alógenos ainda serem
um certo motivo de controvérsia na literatura, alguns autores apontam como principal
vantagem do osso fresco congelado o fato dele não ter suas proteínas ósseas
morfogenéticas destruídas
14,22
. Estudos recentes demonstraram que células
osteoblásticas podem se desenvolver in vitro a partir de espécimes de enxertos
ósseos frescos congelados, após o período de quarentena e que estas são
indistinguíveis daquelas desenvolvidas a partir de enxertos não congelados
28,29
. A
partir dos resultados do presente estudo, podemos sugerir que o osso fresco
congelado apresenta a propriedade de osteocondução e um processo de incorporação
mais lento, devido à presença das lacunas vazias, sem osteócitos, nas porções mais
centrais do enxerto; estudos imunohistoquímicos são necessários para confirmar sua
propriedade de osteoindução.
Outra vantagem atribuída ao osso fresco congelado é o fato dele poder ser
utilizado imediatamente após o descongelamento, com textura e resistência similares
àquelas do osso autógeno, no momento da enxertia
9,22,30
.
Sua principal desvantagem, o potencial risco de transmissão de doenças, tem
sido amplamente estudada e representa um risco mínimo para o paciente
10,25
. O risco
de transmissão de vírus por um enxerto congelado, é, atualmente, inferior a 1:200.000
para o HCV e 1:1.000.000 para o HIV devido aos rigorosos protocolos aplicados aos
doadores dos bancos de tecidos
15
. No Brasil, os bancos de tecidos precisam estar de
acordo com a lei 9.434 publicada pelo Ministério da Saúde em 1997, baseada nas
normas da American Association of Tissue Bank, que determina além de uma extensa
e detalhada anamnese, a realização dos seguintes exames sorológicos em todos os
doadores: VDRL, teste de amplificação e detecção de ácidos nucléicos para HIV e
HCV, anti-HBc, HbsAg, pesquisa de doença de Chagas, anti-HTLV I e II,
toxoplasmose e CMV
31
.
O risco de transmissão de doenças é, virtualmente, não existente para os
enxertos processados
15
, no entanto, o osso liofilizado apresenta propriedades
osteoindutoras, mecânicas e de resistência inferior àquelas do osso fresco
congelado
12,32
. Osso desmineralizado, por sua vez, não suporta carga axial
9
,
apresentando maior utilidade para enxertias dentoalveolares em regiões não
submetidas à força oclusal
33
. Com relação à imunogenicidade, o congelamento
proporciona uma redução da mesma sem alterar as propriedades biomecânicas do
enxerto
12
.
O tecido ósseo é fundamentalmente composto de células, matriz inorgânica e
matriz orgânica
27
. O colágeno corresponde a 90 a 95% do componente orgânico do
osso e é um elemento fundamental no processo de neoformação óssea
23
. Como o
colágeno intersticial apresenta suas moléculas ordenadamente dispostas em uma
orientação paralela, uma birrefringência normal é uma das características clássicas
desta estrutura. As moléculas do colágeno, ricas em aminoácidos básicos, reagem
fortemente com corantes ácidos. Quando o Picrosirius, um corante extremamente
ácido, reage com o colágeno, sua birrefringência normal é aumentada pelo fato das
moléculas do corante se agregarem às fibras colágenas de maneira tal que seus
longos eixos ficam paralelos
16
.
O método de polarização com Picrosirius é um procedimento histoquímico
específico para detecção de colágeno em cortes teciduais, onde o colágeno intersticial
apresenta diferentes cores e intensidade de birrefringência
34
. Áreas birrefringentes
amarelas ao vermelho são indicativas do colágeno maturo e as verdes indicam o
colágeno imaturo, o primeiro tipo a aparecer durante o processo de neoformação ou
remodelação óssea
17
. A cor e a intensidade da birrefringência do colágeno pode
também variar dependendo do diâmetro da fibra, espessura do corte tecidual ou
ambos
35
.
Na instituição aonde o presente estudo foi conduzido, a realização de enxertos
ósseos alógenos em rebordos maxilares aumentou consideravelmente nos últimos três
anos, enquanto os enxertos autógenos diminuíram, indicando a mesma tendência que
ocorre na ortopedia atualmente
15
. O menor tempo operatório, suprimento praticamente
ilimitado e baixa morbidade são algumas das vantagens que estimulam o uso
crescente deste tipo de enxertia.
Os resultados clínicos obtidos com estes 18 indivíduos estão em acordo com
os casos apresentados por outros autores que demonstraram a efetividade dos blocos
de osso alógeno fresco congelado em áreas de implantes dentais
14,22,23,25
. A adoção de
uma técnica cirúrgica adequada e cuidadosa é essencial para obtenção de sucesso
neste tipo de enxertia.
A evidência histológica de neoformação e remodelação óssea em enxertos
alógenos foi previamente relatada por outros autores
23,24
. A análise histológica no
presente estudo pôde corroborar os bons resultados clínicos: uma deposição óssea
orientada e uniforme, com características similares ao osso autógeno, foi observada
em todas as amostras do grupo teste, não ocorrendo necrose do tecido enxertado em
nenhum caso. A presença das áreas de birrefringência verde pode indicar que os
osteoblastos estão sendo estimulados a produzir matriz, o que explicaria a presença
das fibras colágenas imaturas que não apareciam previamente ao enxerto do osso
alógeno.
Apesar do presente estudo ter um período relativamente curto (8-42 meses) de
acompanhamento, um estudo com período de 30-35 anos de acompanhamento em
cirurgias ortopédicas demonstrou que a enxertia óssea alógena é um procedimento
satisfatório e com longa durabilidade no preenchimento de defeitos ósseos
18
.
Sugerimos pesquisas contínuas e estudos clínicos de longo prazo com enxerto
ósseo alógeno fresco congelado. Este material certamente abre uma nova perspectiva
na área de cirurgia buco-maxilo-facial e implantodontia. Um fator importante a ser
estudado é o comportamento deste tipo de enxertia após longos períodos sob carga
oclusal.
CONCLUSÃO
Concluímos que o osso alógeno fresco congelado é biologicamente viável na
reconstrução de rebordos alveolares maxilares, apresentando um padrão de
maturação do colágeno similar ao do osso autógeno.
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allografts. Acta Ortop Bras 11: 220, 2003
3. Shand JM, Heggie AC, Holmes AD et al: Allogeneic bone grafting of calvarial
defects: an experimental study in the rabbit. Internatl J Oral Maxillofac Surg 31:
525, 2002
4. Willians A, Szabo R: Bone Transplantation. Orthopedics 27:488, 2004
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6. Leslie H, Bottenfield S: Donation, banking, and transplantantion of allograft
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7. De Boer H: The history of bone grafts. Clinical Orthopedics and Related
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8. Galea G, Kearney JN: Clinical effectiveness of processed and unprocessed
bone. Transf Med 15: 165, 2005
9. Hardin C: Banked Bone. Craniofac Skel Aug and Replac 27:911, 1994
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11. Friedlander GE: Current concepts review bone-banking. J Bone Joint Surg 64:
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12. Gazdag AR, Lane JM, Glaser D et al: Alternative to autogenous bone graft:
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15. Albert A, Leemruse T, Druez V et al: Are bone autografts still necessary in
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16. Montes GS, Junqueira LCU: The use of the picrosirius-polarization method for
the study of the biopathology of collagen. Mem Inst Oswaldo Cruz 86: 1, 1991
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18. Steinberg EL, Luger E, Zwas T et al: Very long-term radiographic and bone
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TABELAS
Tabela 1. Porcentagem (%) média de área e densidade do colágeno imaturo
(CI) e maturo (CM) obtida no grupo teste (T) e controle (C).
% ÁREA % DENSIDADE
N CI T CI C CM T CM C CI T CI C CM T CM C
1 39,70 33,42 60,30 66,58 60,83 52,64 39,17 47,36
2 52,16 53,44 47,84 46,56 72,08 35,02 27,92 64,98
3 31,06 57,52 68,94 42,48 53,62 55,08 46,38 44,92
4 79,84 68,46 20,16 31,54 74,08 52,03 25,92 47,97
5 53,28 40,29 46,72 59,71 72,08 49,95 27,92 50,05
6 28,32 35,12 71,68 64,88 50,93 60,73 49,07 39,27
7 25,44 39,52 74,56 60,48 55,70 60,28 44,30 39,72
8 65,89 24,78 34,11 75,22 74,46 56,07 25,54 43,93
9 46,58 73,48 53,42 26,52 59,38 78,25 40,62 21,75
10 69,67 63,26 30,33 36,74 85,56 60,38 14,44 39,62
11 51,25 61,96 48,75 38,04 68,69 63,71 31,31 36,29
12 75,66 27,38 24,34 72,62 66,88 64,52 33,12 35,48
13 79,84 61,57 20,16 38,43 77,11 53,07 22,89 46,93
14 23,52 31,74 76,48 68,26 53,19 67,24 46,81 32,76
15 70,58 55,06 29,42 44,94 54,01 51,58 45,99 48,42
16 56,49 77,40 43,51 22,60 42,36 75,04 57,64 24,96
17 72,35 45,13 27,65 54,87 53,56 52,59 46,44 47,41
18 82,54 69,23 17,46 30,77 71,11 60,13 28,89 39,87
N: número de pacientes
Tabela 2. Média e desvio padrão da porcentagem de área e densidade do colágeno imaturo e
maturo do grupo teste e controle
% ACI T ACI C ACM T ACM C DCI T DCI C DCM T DCM C
n 18 18 18 18 18 18 18 18
Média 55.79 51.04 44.21 48.96 63.65 58.24 36.35 41.76
Desvio padrão 19.95 16.71 19.95 16.71 11.45 9.84 11.45 9.84
Valor p 0.36 0.36 0.21 0.21
Teste t de Student para amostras pareadas: p<0.05.
N: número de pacientes
ACI: area de colágeno imaturo; ACM: area de colágeno maturo; DCI: densidade do colágeno imaturo; DCM:
densidade do colágeno maturo.
T: grupo teste; C: grupo controle.
FIGURAS
Figura 1 – A: Preparo do anel ósseo com cinzel e martelo; B: Blocos
preparados para o enxerto.
A B
Figura 2: Bloco fixado à parede maxilar.
Figura 3 – Radiografia panorâmica de controle pós-operatório.
Figura 4 – Amostra do tecido ósseo delimitada pela broca trefina
(seta), para análise histológica.
Figura 5 – Aspecto clínico dos blocos no momento da cirurgia de reabertura.
Observar a excelente integração e vascularização dos mesmos.
Figura 6 – A: Blocos fixados no momento da enxertia; B: Instalação dos
implantes nove meses após o enxerto, evidenciando o baixo grau de
reabsorção óssea dos blocos.
Figura 7 – Radiografia panorâmica de controle pós-operatório evidenciando
os implantes instalados sobre os blocos enxertados.
A
B
A B
Figura 8 – A: osso alógeno; B: osso autógeno. Ambos os tecidos com
características de osso secundário, porém, no osso autógeno observa-se
uma maior quantidade de osteócitos presentes nas lacunas. (HE aumento
original 200X)
Figura 9 – Fotomicrografia do osso alógeno, exibindo a porção mais
periférica do corte, com células sugestivas de osteoblastos (seta) e as
lacunas com osteócitos (HE aumento original 400X).
Figura 10 – Fotomicrografia do osso alógeno exibindo a porção mais
central do corte; lacunas sem os osteócitos e a presença dos vasos
sanguíneos (VS). (A: HE aumento original 200X; B: Tricrômico de
Masson aumento original 200X)
A B
VS
VS
VS
VS
VS
VS
Figura 11 – A: Osso alógeno; B: Osso autógeno. As áreas amarelo-
vermelhas
correspondem ao colágeno maturo e, as verdes, ao imaturo. (Picrosirius
aumento original 100X).
Figura 12 – A: Osso alógeno descongelado, antes do enxerto, evidenciando
a presença exclusiva das áreas birrefringentes amarelo-vermelhas, típicas
do colágeno maturo. B: Nove meses após o enxerto, observa-se a presença
de áreas verdes, indicando a neoformação óssea (Picrosirius aumento
original 200X).
A B
A B
2) ARTIGOS EM INGLÊS
2.1 MANUSCRIPT DRAFT
Observação: Este primeiro artigo em inglês foi enviado para a revista em Março de
2008, quando nossa amostra era de 15 pacientes. Depois do envio do mesmo surgiu a
oportunidade de aumentar a amostra para 18, o que foi feito para o artigo histológico.
É por este motivo que existem diferenças no número de pacientes, idade média dos
mesmos, número de blocos enxertados e tempo de acompanhamento, entre os
artigos.
DECISION LETTER
Your Submission, JOMS-D-08-00201R2, to the Journal of Oral and
Maxillofacial Surgery
De:
Journal of Oral and Maxillofacial Surgery (
j
Enviada:quarta-feira, 3 de dezembro de 2008 19:52:25
Para: [email protected]
Ref.: Ms. No. JOMS-D-08-00201R2
Maxillary Ridge Augmentation With Fresh-Frozen Bone Allografts
Journal of Oral and Maxillofacial Surgery
Dear Ms Contar,
I am pleased to inform you that your paper entitled "Maxillary Ridge
Augmentation With Fresh-Frozen Bone Allografts" has been accepted for
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Journal of Oral and Maxillofacial Surgery
MAXILLARY RIDGE AUGMENTATION WITH FRESH FROZEN BONE
ALLOGRAFTS
Cintia Milani Contar, DDS, MS
6
João Rodrigo Sarot, DDS, MS
7
Jayme Bordini Jr, DDS, PhD
8
Gustavo Holtz Galvão, DDS
9
Gastão Vale Nicolau, DDS, MS
10
Maria Ângela Naval Machado, DDS, PhD
11
1
Postgraduate Student, Center for Health and Biological Sciences, Pontifícia Universidade
Católica do Paraná, Brazil
7
Postgraduate Student, Department of Implantology, Universidade Federal de Santa Catarina,
Brazil
8
Professor and Director of Faculty of Dentistry, Universidade Federal do Paraná, Brazil.
9
Implantologist, Private Practice, Curitiba, Brazil
10
Professor and Chief, Pos-graduation Course of Implantology, Universidade Federal do
Paraná, Brazil
11
Chairman, Center for Health and Biological Sciences, School of Dentistry, Pontifícia
Universidade Católica do Paraná, Brazil
ABSTRACT
Bone resorption in the maxillary ridge frequently results in a knife-edged
deformity, wich complicates implant placement and estabilization. Reconstruction of the
severely atrophic maxilla is still a major challenge for oral and maxillofacial surgeons
and autografting remains the gold standard for replacing bone loss. Rapid incorporation
and consolidation with the lack of immunologic considerations make the bone
harvested from the patient ideal. However, it is limited in supply and has other
drawbacks such as increased operating time and donor site morbidity. The use of bone
allograft provides a reasonable alternative to meet the growing need for primary or
supplementary graft material. The development of tissue banks has allowed for a wider
use of bone allografts, with good results. This study presents 15 cases of maxillary
ridge augmentation with allogenic fresh frozen bone grafts providing effective bone fill
and support for the placement of dental implants.
KEYWORDS: bone resorption, allografts, dental implants
INTRODUCTION
Bone grafting is essential for the repair of bone defects caused by tumors,
trauma, loosening of prosthetic joints and prior dental implant placement in cases of
alveolar ridge resorption.
1-3
It is intended to stimulate bone healing and fill bone
defects, with autologous bone grafting being the standard method of achieving these
goals.
4
Rapid incorporation and consolidation with a lack of immunologic
considerations make bone harvested from the patient ideal.
5
However, limited
supply, donor site morbidity, increased blood loss, operative time, cost and length of
hospital stay are the main drawbacks of bone autografts.
4,5,6
.
The use of allograft bone provides a reasonable alternative to meet the growing
need for primary or supplementary graft material. The first bone allograft was
performed in 1880 by a Scottish surgeon who successfully reconstructed the infected
humerus of a four-year-old boy with a graft obtained from the tibia of a child with
rickets.
7
Transplantation of large, fresh segments of long bone allografts continued and
was expanded over the next ninety years.
8
The establishment of the U.S. Navy Tissue
Bank in 1949 marked the emergence of the modern tissue bank.
9
There is currently an
increasing interest in bone allografts due to the development of bone banks in many
countries.
10
Clinically, the most useful banked allografts are fresh-frozen, freeze-dried and
demineralized bone.
11
Fresh-frozen bone is harvested aseptically from live or cadaver
donors and then frozen. It is available for human recipients after at least six months of
quarantine at -80º C.
12
There is no additional preparation and the osteoinductive
proteins are preserved.
13
Frozen bone is available as cancellous granules,
corticocancellous granules, and cortical granules or chips. Once thawed, it has the
same handling qualities as does fresh bone.
11
The advantages of using bone allografts include convenience for the surgeon,
decreased operative trauma for the patient, an almost unlimited supply of
reconstructive material, decreased blood loss, absence of donor site morbidity and
decreased operative time.
11,13
One of the main concerns with the use of allograft bone
is transmission of infection, most notably hepatitis and acquired immunodeficiency
syndrome (AIDS).
14
Guidelines on donor selection, tissue procurement, tissue
preservation, tissue storage and adequate record-keeping procedures have been
designed by bone banks to ensure the supply of safe allogeneic bone.
15,16
Another
concern with bone allograft is its antigenicity. Either freeze-drying or freezing bone to -
20º C significantly reduces this risk.
6
In the orthopedics one current trend shows surgeons using allografts as
substitutes for autografts.
10
Nevertheless there is limited information about the use of
allografts in maxillofacial procedures. The present investigation clinically and
histologically evaluated the use of fresh-frozen bone in the reconstruction of maxillary
alveolar ridges to confirm the effective bone fill and support for the placement of dental
implants.
PATIENTS AND METHODS
Patient Selection and Reconstructive Surgical Procedure
From April 2005 to March 2006, 15 patients from the post-graduation course in
Implantology at the Universidade Federal do Paraná, Brazil, who had atrophic maxillary
ridge necessitating bone block grafts prior to implant placement, were admitted to this
study. Patients without sufficient compliance to the therapy and patients with systemic
medical conditions were excluded. There were six males and nine females in the group
and the average age was 44 years, ranging from 29 to 61 years. The maxillary
reconstructions were performed with human block grafts of tibia fresh-frozen chips
obtained from the Clinics Hospital Tissue Bank of Universidade Federal do Paraná.
Approval for this study (number 1480) was obtained from the Ethics Committee
in Research at Pontifícia Universidade Católica do Paraná. All subjects signed a
consent form to participate in the study.
The maxillary ridge resorption was diagnosed through clinical and radiological
examination. Panoramic radiographs and routine blood examinations were performed
for all patients and did not reveal any other bone or systemic diseases.
All patients followed an antibiotic regimen of Amoxicillin 2g (Amoxil, Glaxo
SmithKline, Rio de Janeiro, RJ, Brazil) and Dexametason 8mg (Decadron, Aché
Pharmaceutical Laboratories, Guarulhos, SP, Brazil) one hour before surgery.
The bone was thawed for one hour and then the blocks were sculpted with chisels and
rotary instruments. An appropriate local anesthetic (Articaine 4% epinephrine
1:100.000, DFL, Rio de Janeiro, Brazil) was administered and a full-thickness
mucoperiosteal flap was elevated (figure 1). After careful site preparation, which
included decortication of the maxilla in the defect site to enhance marrow space
bleeding, the blocks were perfectly adapted to the maxillary wall without any gap.
Fixation with miniscrews (Neodent, Curitiba, PR, Brazil) was used for stabilization of
the prepared blocks at the recipient site. The miniscrews were placed through the
central portion of the blocks and rested in the palatal portion of the defect to prevent
micro-movement of the graft. The flaps were repositioned without tension and silk
sutures (4.0 Ethicon, Inc., Somerville, NJ, USA) were used for closure. The patients
continued taking Amoxicillin 1 g each 12 hours, for 7 days and Dexametason 4 mg
each 8 hours for 2 days and performed regular chlorhexidine rinses (Periogard,
Colgate-Palmolive Company, Brazil) twice daily starting the day after surgery. The
sutures were removed seven days after surgery, and at this time the patients were
evaluated for infection and wound dehiscence. During the healing period all patients
were seen once a month until the time of implant placement.
In the third postoperative month a panoramic radiograph was done to evaluate
the graft´s incorporation (figure 2). The reopening surgery was carried out an average
of nine months following bone grafts, ranging from 8 to 11 months (figure 3). At this
time one bone core from the graft was removed with a trephine bur from the area
where one implant would be placed for histological analysis. Next, the miniscrews were
removed and titanium implants (Systhex Sistema de Implantes Osseointegrados,
Curitiba, PR, Brazil) were placed (figure 4). Additional grafting was not required at the
time of implant installation for any of the patients, and none of the graft blocks were
dislodged at this time. Panoramic radiographs were routinely performed in the first and
sixth (figure 5) postoperative months to evaluate the implants’ osseointegration.
The bone specimens were routinely processed for serial decalcified sections.
The specimens were fixed in 10% neutral-buffered formaldehyde solution for 48 hours
and demineralized in 5% trichloroacetic acid for 15 days. Subsequently, they were
embedded in paraffin, and 6 μm thin sections were obtained and stained with
hematoxylin-eosin (HE) and Masson trichromic for the histologic analysis. The sections
were examined by light microscope.
RESULTS
Thirty four blocks were placed, and the number of blocks each patient received
ranged from 1 to 4; the number of implants ranged from 1 to 8.
For all patients, the medications prescribed were effective for pain and edema.
The healing period was uneventful for 14 patients. One patient had early exposition of
the block that required a second surgical procedure to cover it. This occurred due to
inadequate flap design in the first surgery. Although its exposition was premature, there
was no infection and no compromise of the final result. None of the other grafts
became exposed or infected.
During the re-entry procedures, all of the grafts were found to be firm in
consistency, well incorporated and vascularized, even in cases involving larger
reconstructions. Some bone resorption was observed in the grafted materials, but all
implants were placed as initially planned (figures 6, 7). There was no excessive bone
resorption in any case that could make changing the treatment plan necessary.
A total of 51 implants were placed over the grafts with a minimum of 40-Newton
torque in all cases. Twenty-two implants were already accompanied by implant-
supported restorations (figures 8, 9). None of the implants were lost. The follow up
period ranged from 24 to 35 months.
The histological analysis of the bone specimens removed during the re-entry
procedures revealed a living bone that showed features characteristic of mature and
compact osseous tissue surrounded by marrow spaces (figure 10). Viable bone was
observed, as well as newly formed bone incorporated with the grafted areas.
DISCUSSION
Transplanting bone from one human to another is an idea that has been with us
for hundreds of years and circumvents the problems with autogenous bone grafting.
5
Allografts have been used in orthopedics for a long time, in many applications
including: trauma, spine fusion, revision arthroplasty, tumor surgery, nonunion.
10,17
In a
prospective trial in scoliosis surgery the authors concluded that even in the presence of
an adequate autogenous iliac crest, the use of frozen bank bone was superior.
6
The
use of autografts in orthopedic surgery is currently becoming rarer, given the
availability of safe and efficient bone allografts.
10
The disadvantages of allografts, such as disease transmission and antigenicity,
have been widely studied and represent a minimal risk to the patient.
6
With the
standard protocols applied by the bone banks the risk of viral transmission by
unprocessed deep-frozen, non-irradiated grafts from screened donors is currently less
than 1:200.000 for HCV and 1:1 million for HIV.
10
It is virtually non existent for
processed bone grafts.
10
Nonetheless, freeze-dried or lyophilized grafts possess
inferior osteoinductive properties, mechanical properties and strength compared with
frozen.
14, 18
Demineralized bone does not tolerate axial loading,
11
having a wide range
of applications in dentoalveolar grafting procedures at low stress areas.
19
As new
pathogens emerge or are discovered, the challenge for tissue banks will be to
continually revise and improve their practices.
8
Bone grafts of any type can only regenerate bone through three possible
mechanisms: direct osteogenesis, osteoconduction, and osteoinduction.
20
The only
material to date that has true osteogenic properties is autograft.
4
The ideal allogeneic
bone graft material would cause new bone to form (osteoinduction) and provide a
scaffold to support the regenerating host bone that will eventually replace the graft
(osteoconduction).
2
The osteoinduction properties of the allografts remain controversial
in the literature. In a recent study, SIMPSON et al
12
showed that osteoblast-related
cells can be grown in vitro from fresh-frozen allograft specimens after the quarantine
period. Other authors confirm that the frozen human bone is prepared so that the
nondemineralized bone matrix and the morphogenetic bone proteins are preserved.
13,21
Bone allograft procedures are very common in the author´s institution. The use
of this kind of graft has clearly increased over the last three years, whereas the use of
autografts decreased indicating the same trend seen in the orthopedics. The minor
operative time, unlimited supply, low morbidity, and painless healing period are some
of the advantages that encourage the growing use of the allografts.
The good results obtained in these 15 patients are in accordance with the cases
presented by other authors
2,13,21,22
, who demonstrated efficacy in using block allografts
in areas of dental implant placement. The only complication presented in this study was
due to an inadequate surgical technique having no relation with the graft material itself.
The histological evidence of new bone deposition in allografts was already
demonstrated in other cases reported in series
2,22
and observed in this study.
Osteocytes encased in a mineral matrix and marrow spaces suggest new bone
incorporation without residual graft material.
Although our follow-up is relatively short here, a very long follow-up study (30-
35 years) for bone allograft indicates it is a satisfactory and durable method for filling
bone defects.
17
The results support the hypothesis that fresh-frozen bone allografts can be
successful as graft material for the treatment of maxillary ridge defects. If adequate
surgical techniques are adopted, this type of bone graft can be safely used in regions
of implant placement as a suitable alternative to autogenous grafts.
Future studies and long-term follow-up studies with fresh-frozen bone allograft
are suggested to learn about the behavior of this material after longer periods with
occlusal loadings.
REFERENCES
1. Hofman A, Konrad L, Hessmann MH, et al: The influence of bone allograft
processing on osteoblast attachment and function. J Orthop Res 23:846, 2005
2. Leonetti J, Koup R: Localized maxillary ridge augmentation with a block
allograft for dental implant placement: case reports. Implant Dentistry 12:217,
2003
3. Hachiya Y, Sakai T, Narita Y, et al: Status of bone bank in Japan. Transpl
Proceed 31:2032, 1999
4. Ilan DI, Ladd AL: Bone graft substitutes. Oper Tech in Plastic and Reconstr
Surg 9:151, 2003
5. Willians A, Szabo R: Bone Transplantation. Orthopedics 27:488, 2004
6. Dodd CAF, Fergusson CM, Freedman L et al: Allograft versus autograft bone in
scoliosis surgery. J Bone Joint Surg 70B:431, 1988
7. De Boer H: The history of bone grafts. Clin Orthop Rel Res 226:292, 1988
8. Tomford W: Bone allografts: past, present and future. Cell Tiss Bank 1:105,
2000
9. Leslie H, Bottenfield S: Donation, banking, and transplantantion of allograft
tissues. Org and Tiss Transplant 24:891, 1989
10. Albert A, Leemruse T, Druez V et al: Are bone autografts still necessary in
2006? A three-year retrospective study of bone grafting. Acta Orthop Belg
72:734, 2006
11. Hardin C: Banked Bone. Craniofac Skel Aug and Replac 27:911, 1994
12. Simpson D, Kakarala G, Hampson K, et al: Viable cells survive in fresh frozen
human bone allografts. Acta Orthop 78:26, 2007
13. Perrot DH, Smith RA, Kabam LB: The use of fresh frozen allogeneic bone for
maxillary and mandibular reconstruction. J Oral Maxillofac Surg 21:260, 1992
14. Gazdag AR, Lane JM, Glaser D et al: Alternative to autogenous bone graft:
efficacy and indications. J Am Acad Orthop Surg 3:1, 1995.
15. Tomford WW, Doppelt SH, Mankin HJ et al: Bone bank procedures. Clin Orthop
174:15, 1983
16. Palmer SH, Gibbons CLMH, Athanasou NA: The pathology of bone allograft. J
Bone Joint Surg 81B:333, 1999
17. Steinberg EL, Luger E, Zwas T et al: Very long-term radiographic and bone
scan results of frozen autograft and allograft bone grafting in 17 patients (20
grafts) a 30- to 35-year follow-up. Cell Tiss Bank 5: 97, 2004
18. Ehrler DM, Vaccaro AR: The use of allograft bone in lumbar spine surgery. Clin
Orthop Rel Res 371:38, 2000
19. Kao ST, Scott DD: A review of bone substitutes. Oral maxillofacial Surg Clin N
Am 19: 513, 2007
20. Marx RE: Bone and bone graft healing. Oral Maxillofacial Surg Clin N Am 19:
455, 2007
21. Accetturi E, Germani KB, Cavalca D: Reconstruction of bone defects in the
maxilla and mandibula through the use of frozen human bone. Transpl Proceed
34:531, 2002
22. Petrungaro PS, Amar S: Localized ridge augmentation with allogenic block
grafts prior to implant placement: case reports and histologic evaluations.
Implant Dentistry 14:139, 2005
FIGURES
Figure 1: Transoperative view of the accentuated maxillary ridge resorption
Figure 2: Panoramic radiograph showing the graft´s incorporation
Figure 3: Transoperative view during the reopening of the area showing
excellent integration of the grafts
Figure 4: The new bone received eight implants simetrically placed.
Figure 5: Panoramic radiograph showing the osseointegrated implants in the
grafted areas.
Figure 6: Transoperative view after fixation with miniscrews of two bone
blocks allografts
Figure 7: Nine months re-entry for implants placement. Notice the bone
remodeling of the grafted blocks.
Figure 8: Final restoration view.
Figure 9: Panoramic radiograph showing the implant-supported restorations.
Figure 10: Mature and compact osseous tissue surrounded by marrow
spaces (Masson Trichromic; original magnification X100).
2.2 MANUSCRIPT DRAFT
TITLE PAGE
Histological Analysis of Fresh-Frozen Bone Allografts in Maxillary Ridge
Augmentation
Running Title: Histological Analysis of Bone Allografts
Cintia Milani Contar
12
João Rodrigo Sarot
13
Maite Barroso da Costa
1
Jayme Bordini Jr
14
Sérgio Aparecido Ignácio
1
Antonio Adilson Soares de Lima
1
Luciana Reis Azevedo Alanis
1
Paula Cristina Trevilatto
1
Maria Ângela Naval Machado
1
Keywords: fresh-frozen bone, allograft, Picrosirius-polarization method
Corresponding author: Cintia Milani Contar
Visconde de Guarapuava Street, 4177 / 1301
Curitiba – PR – Brazil Zip Code 80250-220
Phone: 55-41 30136210
This research was supported by the Brazilian Agency for Scientific and Technological
Development (Conselho Nacional de Desenvolvimento Cientifico e Tecnológico-CNPq)
Process number 475511/2008-5.
12
Center for Health and Biological Sciences, Pontifícia Universidade Católica do Paraná,
Brazil
13
Department of Implantology, Universidade Federal de Santa Catarina, Brazil
14
Department of Stomatology, Universidade Federal do Paraná, Brazil.
ABSTRACT
Bone allograft has become an alternative to autogenous bone due to its decreased
operative trauma and almost unlimited supply of reconstructive material. The aim of the
present study was to histologically evaluate the viability of fresh-frozen bone graft (test
group) used in maxillary ridge augmentation, comparing it to autogenous bone (control
group) using the Picrosirius-polarization method. During the re-entry procedures, nine
months after the fresh-frozen allogeneic bone blocks were placed in the atrophic
maxillary ridges, bone cores were removed with a trephine bur from test and control
group from the same patient. Routine histologic processing using hematoxylin and
eosin and Picrosirius staining was performed. Mature and immature collagen area and
density analysis was carried out for both groups under polarization. The results of
Student’s t-test for paired samples (p>0.05) showed no statistically significant
difference in mature and immature collagen area and density percentage of test and
control groups. Histologically similar bone formation patterns were observed between
both groups. We concluded that fresh-frozen allogeneic bone is viable for maxillary
ridge augmentation, showing a similar collagen pattern to that of autogenous bone.
INTRODUCTION
The
increasing popularity of dental implant surgery has created a heavy
demand for dentoalveolar reconstruction
12
. Insufficient alveolar contours may require
bone grafting procedures to restore adequate bone volume before implant placement in
order to counteract potentially harmful results, such as higher failure rates and
unsatisfactory esthetic results
3,20
.
The sources of bone may come from the host (autograft), a donor (allograft),
and other sources such as xenograft, ceramics and demineralized bone matrix
6
.
Autogenous bone graft remains the gold standard for the reconstruction of bony
defects once it´s the only graft material that contain the four desired properties of bone
graft materials: osteogenesis, osteoinduction, osteoconduction and osteointegration
13
.
Neverthless, there are considerable drawbacks to its use: high morbidity at the donor
site, limited quantity of bone, unpredictable quality of bone, increased blood loss,
increased operative time and donor-site infections
21,25
.
Allogeneic bone is the most commonly used alternative to the autogenous
harvest
16
and the advantages of its use include convenience for the surgeon,
decreased operative trauma and blood loss, absence of donor site morbidity and
greater availability of bone
4,10
.
The properties of the allograft are directly related to the steps taken in
processing the material
13
. Frozen bone is harvested and processed by storing the
sterile specimen at very low temperatures
10
. There is no additional preparation and it is
considered a safe material from immunological and viral points of view
4,19
. Although it is
frequently used by orthopedic surgeons with positive long-term results
23
, some studies
suggest that while meaningful clinical healing does occur with cortical allografts, the
graft is never entirely replaced
24,25
.
Most of the research on fresh-frozen bone allografts has been conducted in the
field of orthopaedic reconstructive surgery; the published oral and maxillofacial
literature is limited in scope. In addition, there was previously no quantitative analysis
of the pattern of collagen fiber organization in fresh-frozen bone grafts used in maxillary
ridge augmentation, as compared to autogenous bone. The aim of the present study
was to histologically evaluate the viability of fresh-frozen bone graft (test group) used in
maxillary ridge augmentation, comparing it to autogenous bone (control group) using
the Picrosirius-polarization method.
PATIENTS AND METHODS
Patient Selection and Reconstructive Surgical Procedure
Approval for this study was obtained from the Ethics Committee in Research at
Pontifícia Universidade Católica do Paraná,
Brazil, number 1480. All subjects signed a
consent form to participate in the study.
From April 2005 to June 2006, 18 patients from the post-graduate course in
Implantology of Universidade Federal do Paraná, who had atrophic maxillary ridge
necessitating bone block grafts prior to implant placement were consecutively admitted
to this study. Patients without sufficient compliance to the therapy and patients with
systemic medical conditions were excluded. There were 6 males and 12 females in the
group and the average age was 41.5 years, ranging from 27 to 61 years. The maxillary
reconstructions were made with human block grafts of tibial fresh-frozen chips obtained
from the Clinics Hospital Bone Bank of Universidade Federal do Paraná.
The maxillary ridge resorption was diagnosed through clinical and radiological
examination. Panoramic radiograph and routine blood examination including
hemogram, coagulogram, creatinine, glycaemia and HIV test were performed for all
patients. Those who presented altered blood tests or any other systemic disease that
would lead to any risk to the surgical procedure were excluded from the study.
Patients were premedicate 1 hour before surgery with amoxicillin (Amoxil, Glaxo
Smithkline, Rio de Janeiro, Brazil) and dexamethasone, 8 mg (Decadron, Aché
Laboratórios Farmacêuticos, Guarulhos, São Paulo, Brazil)
All surgeries followed the same routine: the bone was thawed for 1 hour and
then the blocks were sculpted with chisels and rotary instruments. An appropriate local
anesthetic (Articaine 4% epinephrine 1:100.000, DFL, Rio de Janeiro, Brazil) was
administered and a full-thickness mucoperiosteal flap was elevated. After careful site
preparation, the blocks were perfectly adapted to the maxillary wall and fixed with
miniscrews (Neodent, Curitiba, PR, Brasil). The miniscrews were placed through the
central portion of the blocks and rested in the palatal portion of the defect to prevent
micromovement of the graft (figure 1). The number of blocks that each patient received
ranged from one to four. The flaps were repositioned without tension and silk sutures
(4.0 Ethicon, Inc., Somerville, NJ, USA) were used for closure. One sample of the
allogeneic bone that was not used in the grafting procedure was sent for histological
analysis. Postoperatively, systemic antibiotic (amoxicillin, 1g, twice a day) was
prescribed for 1 week, and paracetamol with codein (one 30mg tablet every 4 to 6
hours for 48 hours) was prescribed for pain control. Dexamethason (4mg three times a
day) was administered for 2 days to minimize edema. Antiseptic mouthwash (0.2%
chlorhexidine gluconate) was used twice daily for 2 weeks. Sutures were removed after
7 days.
The reopening surgery was carried out an average 9 months following bone
grafts, ranging from 8 to 11 months. At this time a bone core from the allogeneic
grafted bone (test group) from the area where implant would be placed, was removed
with a 2.5mm diameter trephine bur, for histological analysis (figure 2). For the control
group a bone core from the autogenous bone of the same patient was removed with a
trephine bur of the same size, from a non-grafted area that would also receive
implants. Titanium implants (Systhex Sistema de Implantes Osseointegrados, Curitiba,
PR, Brazil) where then placed and panoramic radiographs were routinely performed in
the first and sixth postoperative month to evaluate the implant’s osseointegration.
Histological Analysis
The bone specimens were routinely processed for serial decalcified sections.
The specimens were fixed in 10 % neutral buffered formalin solution for 48 hours and
decalcified in 5% trichloroacetic acid for 15 days, and embedded in paraffin. Blocks
were cut to 6-μm-thin slides and stained with hematoxylin-eosin (HE), and Picrosirius.
The sections stained with HE were used to evaluate bone morphology. Those stained
with Picrosirius were viewed with a light microscope (10X at objective 6.3X optovar
1.25), (Olympus BX50; Olympus, Japan), under polarized light coupled to a computer
containing Image ProPlus 4.5 software (Media Cybernetics Inc, USA) for image
analysis of the collagen area and density. The maximally bright position of collagen
fiber bundles was determined before evaluation, where birefringent shades of yellow to
red bands are indicative of mature collagen and a greenish birefringence indicates
immature collagen
8,18
. The percentage of mature and immature collagen area and
density was measured in two fields of each section of test and control groups. The
mean percentage was obtained for each of the examined sections.
Statistical Analysis
All data were tabulated and statistical tests were performed with SPSS for
Windows 13.0 (SPSS Inc., Chicago, IL, USA). The normality of the data was tested
using the Kolmogorov-Smirnov test at a 5% significance level. Student’s t-test (p<0.05)
for paired samples was used to detect significant differences for mature and immature
collagen areas and density percentages in the test and control groups.
RESULTS
A total of 39 fresh frozen bone blocks were grafted and clinical success was
observed in all patients. Healing period was uneventful and there was no infection.
During the reopening surgeries all the blocks were firm in consistency, well
incorporated and vascularized (figure 3). None of the blocks dislodged at the time of
implant placement and additional grafting was not necessary in any case. A total of 58
implants were placed over the grafts with a minimum of 40-Newton torque and 22 were
already accompanied by implant-supported restorations.
The histological analysis of the sections stained with HE revealed typical
lamellar arrangement around Havers canals interspersed with osteocytes in lacunae,
characteristic of secondary bone, in all specimens of the test group. There was no
evidence of acute or chronic inflammatory infiltrate in any of the samples. Similar
histological aspects were observed in control group, however more osteocytes in
lacunae were seen in this group (figure 4).
The margins of the grafted bone revealed osteoblast-like cells and the presence
of a major quantity of osteocytes in lacunae. The central portions of the grafted bone
revealed less osteocytes with a great number of empty lacunae; blood vessels were
always present (figure 5).
Picrosirius-polarization analysis was used to obtain the area and density mean
percentage of mature and immature collagen in test and control group. The
Kolmogorov-Smirnov test revealed that data showed a normal distribution for mature
and immature collagen area and density percentage, when the average values of test
and control group were compared, once p value was 0.20. Student’s t-test revealed no
significant difference in the collagen area (p value 0.36) and density percentage (p
value 0.21) in test and control groups (table 1). In both groups evaluated by
polarization the arrangement of collagen fibers in test and control group did not differ
(figure 6).
The non-grafted allogeneic bone under polarization revealed exclusively the
presence of yellow to red birefringent shades characteristic of mature collagen. Nine
months after the graft, the same tissue presents the greenish birefringent shades
characteristic of immature collagen (figure 7).
DISCUSSION
There are reports of the use of bone allograft from as early as the 1800s;
however, it was the establishment of large-scale tissue banks that facilitated the routine
use of these allografts
7
. The majority of studies of allografts are in the orthopaedic field
where they have been used for a long time with many applications: trauma, spine
fusion, revision arthroplasty, tumor surgery, and nonunion
2,5,15,23
. In the oral and
maxillofacial area there are very few published studies and most of them are in
animals
3,11,21
or limited to case report series
1,4,14,20
. To our knowledge this is the first
study comparing the presence of mature and immature collagen in fresh-frozen block
allografts in maxillary ridge augmentation and autogenous bone in humans under
polarized microscopy.
Fresh-frozen bone can be used immediately on thawing and has texture and
strength characteristics similar to those of autogenous bone at the time of
placement
1,6,10
. It affords decreased immunogenicity without changes in biomechanical
properties
10,25
. The problems of potential disease transmission and antigenicity have
been widely studied and represent a minimal risk to the patient
4,5,19
.
Bone is fundamentally composed of cells, inorganic matrix and organic matrix
16
.
Collagen comprises approximately 90% to 95% of the organic component of bone and
is a fundamental building block in the process of new bone formation
14
. Collagen
molecules, being rich in basic amino acids, strongly react with acidic dyes. Picrosirius
is an elongated dye molecule which reacts with collagen and promotes an
enhancement of its normal birefringence
18
.
The Picrosirius-polarization method is a specific histochemical procedure for
collagen detection in tissue sections, where interstitial collagens display different
interference colours, and intensities, of birefringence
17
. Birefringent shades of yellow to
red bands are indicative of mature collagen, which are typical of mature bone. The
greenish birefringence is indicative of immature collagen, the earliest type to appear
during bone formation and/or renewal
8
. The color and intensity of the collagen
birefringence also varies depending on the fiber diameter, tissue section thickness, or
both
12
.
Others authors
1,9,14,19,20
, have previously demonstrated efficacy in using block
allografts in areas of dental implant placement. The good results obtained in the
present study are in accordance with these previous reports once during the re-entry
procedures, all grafts were firm in consistency, well-incorporated and vascularized.
Furthermore, implant placement in grafted areas demonstrated the functionality and
strength of the regenerated bone as none of the blocks were dislodged at the time and
no additional grafting was necessary.
Bone grafts of any type can regenerate bone through three possible
mecanisms: osteogenesis, osteoinduction and osteoconduction
16
. Although the
osteoinduction properties of the allografts remain controversial in the literature, some
authors indicate the most important advantage of fresh-frozen bone is that the
osteoinductive proteins are not destroyed in the preparation
1,19
. Previous study have
shown that osteoblast-related cells can be grown in vitro from fresh frozen allograft
specimens and these cells were morphologically indistinguishable from those grown
out of freshly harvested trabecular bone
22
. According to the results of the present
study we can suggest the allogeneic bone has an osteoconductive property and a
slower incorporation process due to the presence of many empty lacunae in the central
portion of the graft; immunohistochemical studies are necessary to confirm its
osteoinductive property.
The histological evidence of new bone deposition in allografts was already
demonstrated by other authors
14,20
. The histological analysis in the present study
supports the great clinical results: oriented and uniform bone deposition was observed
and evidence of necrotic bone was not seen in any of the samples of test group.
Histologically similar bone formation patterns were observed between the allograft and
autogenous bone. The presence of a greenish birefringence in the grafted allogeneic
bone that did not appear before the grafting procedure, may indicate that osteoblasts
are being stimulated to produce matrix, demonstrating the presence of a bone renewal
process, which would explain why younger fibers are present.
We can conclude that fresh-frozen bone allograft is biologically viable to
augment maxillary alveolar ridge showing a similar collagen pattern to that of
autogenous bone
Continuous research and clinical studies of this material are suggested as it
certainly opens up a new perspective in the field of oral and maxillofacial surgery.
Acknowledgements
We are deeply grateful to Herculano S. dos Reis Filho and all the employees of
Laboratório de Histotecnologia (UFPR) for their technical assistance in preparing the
slides; Eliene O. N. Romani (FOP-UNICAMP) for cooperation in the photomicrographs.
This research was supported by the Brazilian Agency for Scientific and
Technological Development (Conselho Nacional de Desenvolvimento Cientifico e
Tecnológico-CNPq) Process number 475511/2008-5.
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TABLE
Table 1.
Mean and standard deviation of area and density percentage of immature and mature
collagen of test and control group
% ica T ica C mca T mca C icd T icd C mcd T mcd C
n 18 18 18 18 18 18 18 18
Mean 55.79 51.04 44.21 48.96 63.65 58.24 36.35 41.76
Standard Deviation 19.95 16.71 19.95 16.71 11.45 9.84 11.45 9.84
p value 0.36 0.36 0.21 0.21
Student’s t Test (paired samples): p<0.05.
N: number of patients
ica: immature collagen area; mca: mature collagen area; icd: immature collagen density; mcd: mature collagen
density
T: test group; C: control group.
FIGURES
Figure 1: Fresh frozen bone block allograft fixed to the maxillary ridge
with titanium miniscrews.
Figure 2: Bone core from the allograft (arrow) for histologycal analysis
Figure 3: Transoperative view during the reopening surgery for implant
installation. Notice the excellent graft´s incorporation.
Figure 4: Allogeneic bone (A) Autogenous bone (B). Typical lamellar
arrangement, interspersed with osteocytes in lacunae, characteristic of
secondary bone in both groups. Notice the autogenous bone presents more
osteocytes. (HE, original magnification 20X)
A
B
Figure 5: Allogeneic bone: (A) Margin: notice the presence of osteoblast-
like cells (arrow) and the osteocytes in lacunae (HE, original
magnification 40X); (B) Central portion: empty lacunae and the presence
of blood vessels (BV) (HE, original magnification 20X)
Figure 6: Allogeneic bone (A) Autogenous bone (B). Mature collagen fibers
in yellow-red and immature collagen fibers in greenish color. (Picrosirius-
polarization, original magnification 10X).
Figure 7: Allogeneic bone before grafting (A) showing birefringent shades
of yellow to red bands indicative of mature collagen fibers. Allogeneic
bone nine months after the grafting procedure (B) now showing the
presence of a greenish birefringence, indicating bone renewal
(Picrosirius-polarization, original magnification 20X)
A
BV
BV
BV
A B
A B
3) ANEXOS
ANEXO 1 – APROVAÇÃO DO COMITÊ DE ÉTICA
ANEXO 2 – ANÁLISE ESTATÍSTICA
TABELA 1 – PORCENTAGEM DE AREA E DENSIDADE DO COLÁGENO IMATURO (CI) E
MATURO (CM) DO GRUPO TESTE (T) E CONTROLE (C), NOS CAMPOS 1 e 2.
Campo
1
% ÁREA
% DENSIDADE
Paciente
CI T CI C CM T CM C CI T CI C CM T CM C
1 48,69 34,88 51,31 65,12 55,88 41,67 44,12 58,33
2 72,27 47,58 27,73 52,42 60,63 42,20 39,37 57,80
3 41,84 80,65 58,16 19,35 75,50 81,32 24,50 18,68
4 70,91 73,43 29,09 26,57 58,91 53,39 41,09 46,61
5 61,99 36,78 38,01 63,22 87,27 43,79 12,73 56,21
6 42,96 15,83 57,04 84,17 61,85 44,06 38,15 55,94
7 15,76 21,64 84,24 78,36 35,12 55,22 64,88 44,78
8 88,98 12,05 11,02 87,95 83,93 31,43 16,07 68,57
9 26,49 63,75 73,51 36,25 60,14 81,76 39,86 18,24
10 62,12 66,74 37,88 33,26 85,02 56,60 14,98 43,40
11 48,41 82,77 51,59 17,23 73,73 71,24 26,27 28,76
12 78,23 33,34 21,77 66,66 71,87 67,52 28,13 32,48
13 76,41 45,83 23,59 54,17 65,41 50,13 34,59 49,87
14 18,84 23,68 81,16 76,32 57,58 59,04 42,42 40,96
15 74,60 69,92 25,40 30,08 75,96 59,82 24,04 40,18
16 74,64 89,24 25,36 10,76 41,38 71,49 58,62 28,51
17 73,74 33,01 26,26 66,99 52,72 28,38 47,28 71,62
18 90,00 48,26 10,00 51,74 72,01 50,44 27,99 49,56
Campo
2
% ÁREA
% DENSIDADE
Paciente
CI T CI C CM T CM C CI T CI C CM T CM C
1 30,71 31,96 69,29 68,04 65,79 63,61 34,21 36,39
2 32,04 59,30 67,96 40,70 83,52 27,84 16,48 72,16
3 20,29 34,40 79,71 65,60 31,73 28,83 68,27 71,17
4 88,77 63,49 11,23 36,51 89,26 50,67 10,74 49,33
5 44,56 43,79 55,44 56,21 56,90 56,11 43,10 43,89
6 13,67 54,42 86,33 45,58 40,01 77,41 59,99 22,59
7 35,12 57,39 64,88 42,61 76,28 65,34 23,72 34,66
8 42,81 37,51 57,19 62,49 64,99 80,71 35,01 19,29
9 66,68 83,20 33,32 16,80 58,62 74,73 41,38 25,27
10 77,22 59,78 22,78 40,22 86,10 64,17 13,90 35,83
11 54,09 41,15 45,91 58,85 63,64 56,18 36,36 43,82
12 73,09 21,41 26,91 78,59 61,89 61,53 38,11 38,47
13 83,26 77,30 16,74 22,70 88,81 56,01 11,19 43,99
14 28,20 39,80 71,80 60,20 48,80 75,44 51,20 24,56
15 66,57 40,20 33,43 59,80 32,05 43,33 67,95 56,67
16 38,34 65,56 61,66 34,44 43,33 78,59 56,67 21,41
17 70,97 57,25 29,03 42,75 54,41 76,79 45,59 23,21
18 75,07 90,19 24,93 9,81 70,21 69,83 29,79 30,17
FONTE: Dados da pesquisa
TABELA 2 – PORCENTAGEM MÉDIA DA ÁREA E DENSIDADE DO COLÁGENO
IMATURO (CI) E MATURO (CM) DO GRUPO TESTE (T) E CONTROLE (C).
% ÁREA % DENSIDADE
N CI T CI C CM T CM C CI T CI C CM T CM C
1 39,70 33,42 60,30 66,58 60,83 52,64 39,17 47,36
2 52,16 53,44 47,84 46,56 72,08 35,02 27,92 64,98
3 31,06 57,52 68,94 42,48 53,62 55,08 46,38 44,92
4 79,84 68,46 20,16 31,54 74,08 52,03 25,92 47,97
5 53,28 40,29 46,72 59,71 72,08 49,95 27,92 50,05
6 28,32 35,12 71,68 64,88 50,93 60,73 49,07 39,27
7 25,44 39,52 74,56 60,48 55,70 60,28 44,30 39,72
8 65,89 24,78 34,11 75,22 74,46 56,07 25,54 43,93
9 46,58 73,48 53,42 26,52 59,38 78,25 40,62 21,75
10 69,67 63,26 30,33 36,74 85,56 60,38 14,44 39,62
11 51,25 61,96 48,75 38,04 68,69 63,71 31,31 36,29
12 75,66 27,38 24,34 72,62 66,88 64,52 33,12 35,48
13 79,84 61,57 20,16 38,43 77,11 53,07 22,89 46,93
14 23,52 31,74 76,48 68,26 53,19 67,24 46,81 32,76
15 70,58 55,06 29,42 44,94 54,01 51,58 45,99 48,42
16 56,49 77,40 43,51 22,60 42,36 75,04 57,64 24,96
17 72,35 45,13 27,65 54,87 53,56 52,59 46,44 47,41
18 82,54 69,23 17,46 30,77 71,11 60,13 28,89 39,87
FONTE: Dados da pesquisa.
TABELA 3 – MÉDIA, MEDIANA, DESVIO PADRÃO E ERRO
PADRÃO DA PORCENTAGEM DE ÁREA DO COLÁGENO
IMATURO (ACI) E MATURO (ACM) E DENSIDADE
DO COLÁGENO IMATURO (DCI) E MATURO (DCM)
DO GRUPO TESTE (T) E CONTROLE (C)
FONTE: dados da pesquisa
TABELA 4 – TESTE DE NORMALIDADE
DE KOLMOGOROV-SMIRNOV.
Estatística
N
Valor
p
ACI T 0,145612 18 0,2000
ACI C 0,128998 18 0,2000
ACM T 0,145612 18 0,2000
ACM C 0,128998 18 0,2000
DCI T 0,145005 18 0,2000
DCI C 0,144332 18 0,2000
DCM T 0,145005 18 0,2000
DCM C 0,144332 18 0,2000
FONTE: Dados da pesquisa
LEGENDA:
ACI - % área de colágeno imaturo
ACM - % de área de colágeno maturo
DCI - % da densidade do colágeno imaturo
DCM - % da densidade do colágeno maturo
T – grupo teste
C – grupo controle
N Média Mediana
Desvio
Padrão Erro Padrão
ACI T 18
55,79 54,88
19,95
4,70
ACI C 18
51,04 54,25
16,71
3,94
ACM T 18
44,21 45,12
19,95
4,70
ACM C 18
48,96 45,75
16,71
3,94
DCI T 18
63,65 63,86
11,45
2,70
DCI C 18
58,24
58,10
9,84
2,32
DCM T 18
36,35 36,14
11,45
2,70
DCM C 18
41,76 41,90
9,84
2,32
TABELA 5 – TESTE t de STUDENT – ESTATÍSTICA DAS AMOSTRAS PAREADAS
Média N
Desvio
Padrão Erro Padrão
Par
1 ACI T 55,78 18 19,95 4,70
ACI C 51,04 18 16,70 3,93
Par
2 ACM T 44,21 18 19,95 4,70
ACM C 48,95 18 16,70 3,93
Par
3 DCI T 63,64 18 11,45 2,69
DCI C 58,23 18 9,84 2,32
Par
4 DCM T 36,35 18 11,45 2,69
DCM C 41,76 18 9,84 2,32
FONTE: Dados da pesquisa
LEGENDA: ACI - % área de colágeno imaturo
ACM - % de área de colágeno maturo
DCI - % da densidade do colágeno imaturo
DCM - % da densidade do colágeno maturo
T – grupo teste
C – grupo controle
TABELA 6 – TESTE t DE STUDENT – ESTATÍSTICA DAS AMOSTRAS PAREADAS
Diferenças entre os pares
Média
Desvio
Padrão
Erro
Padrão
95% Intervalo de
confiança
t df
Valor
p
menor maior
Par 1 aci t – aci c 4,7468 21,4 5,0413 -5,8893 15,3829 0,9416 17 0,3596
Par 2 acm t – acm c -4,7468 21,4 5,0413 -15,3829 5,8893 -0,9416 17 0,3596
Par 3 dci t – dci c 5,4070 17,7 4,1771 -3,4058 14,2199 1,2945 17 0,2128
Par 4 dcm t – dcm t -5,4070 17,7 4,1771 -14,2199 3,4058 -1,2945 17 0,2128
FONTE: dados da Pesquisa
LEGENDA: aci - % área de colágeno imaturo
acm - % de área de colágeno maturo
dci - % da densidade do colágeno imaturo
dcm - % da densidade do colágeno maturo
t – grupo teste
c – grupo controle
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Internet resource: International Committee of Medical Journal Editors. Uniform
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[Accessibility verified March 21, 2008]
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ANEXO 5 – RESUMOS DAS REFERÊNCIAS UTILIZADAS NA DISCUSSÃO DOS
ARTIGOS
Acta Orthop Belg. 2006 Dec;72(6):734-40. Links
Are bone autografts still necessary in 2006? A three-year retrospective
study of bone grafting.
Albert A, Leemrijse T, Druez V, Delloye C, Cornu O.
Service d'Orthopédie et de Traumatologie de l'Appareil Locomoteur, Cliniques
Universitaires Saint-Luc, UCL, Brussels, Belgium.
Autograft is considered as the gold standard in bone grafting. However, the
development of tissue banks has allowed for a wider use of bone allografts, with good
results. Demineralised Bone Matrix (DBM) and recombinant human Bone
Morphogenetic Proteins (rh-BMP's) were also introduced to replace the time-honoured
autograft. Is there currently still a place for bone autograft? The authors reviewed the
orthopaedic surgical activity in their institution during the period 2003-2005, and traced
all the surgical procedures in which bone grafting was performed. Tracking forms from
the tissue bank were reviewed to assess the surgical indications. Between 2003 and
2005, the use of autografts decreased from 1.3% to 0.9% of all surgical interventions,
particularly owing to their decreased use in primary fusions, while the use of allografts
increased from 10.7% to 12.7%. Indications for allografts covered all fields of
orthopaedic surgery, including nonunions. Processed allografts represented 90% of all
grafts used. DBM and rh-BMP were used on an exceptional basis. There is currently a
trend for surgeons to use allografts as substitutes for autografts, as processing of the
allografts increases their safety while preserving most of their biological and
mechanical properties. Autografting is now limited to revision operations after failed
fusions, and to combined use at the junction with massive allografts. DBM and rh-BMP
are still controversial but they might replace autografts, even in their currently
remaining indications, if their cost effectiveness and efficiency are established.
J Oral Implantol. 2003;29(3):124-31. Links
A comparison of allogeneic and autogenous iliac monocortical grafts to
augment the deficient alveolar ridge in a canine model. I. Clinical study.
Cranin AN, Demirdjan E, DiGregorio R.
NYU College of Dentistry, New York, NY, USA. [email protected]
A canine model was used to compare autogenous alveolar ridge augmentation bone
grafting with allogeneic grafts. Defects were created by premolar extractions and
measured by radiopaque markers. These markers were used for subsequent
measurements made before and after grafting, and after animal sacrifice to evaluate
the status of the grafted sites. The results were unexpected and disappointing.
J Bone Joint Surg Br. 1988 May;70(3):431-4. Links
Allograft versus autograft bone in scoliosis surgery.
Dodd CA, Fergusson CM, Freedman L, Houghton GR, Thomas D.
Nuffield Orthopaedic Centre, Oxford, England.
The results of a study of the use of autograft versus allograft bone in the surgery of
idiopathic adolescent scoliosis are presented. Two groups of patients, matched for
age, sex, level and angle of curve, received bone grafts, 20 patients having
autogenous bone from the iliac crest and the other 20 having donor bone from a bone
bank. Both groups had otherwise identical posterior fusions and Harrington
instrumentation. There was no difference between the two groups in a blind,
radiographic assessment of bone graft mass at six months, nor in maintenance of the
curve correction over the same period. No major operative complications nor failures
of instrumentation were encountered. There was, however, a marked reduction in
operative time and blood loss in the patients receiving donor bone and also a much
lower incidence of late symptoms relating to the operative sites. We conclude that,
even in the presence of adequate iliac crest, the use of bank bone is superior for
grafting in idiopathic scoliosis surgery.
Cell Tissue Bank. 2008 Mar;9(1):41-6. Epub 2007 Oct 11. Links
Fresh frozen homologous bone in oral surgery: case reports.
D'Aloja E, Santi E, Aprili G, Franchini M.
Negrar, Verona, Italy.
Intraoral bone defects may be treated using autologous grafts, homologous grafts,
heterologous grafts or synthetic products. Autologous bone is now considered the gold
standard for bone grafting procedures. Homologous fresh frozen bone, provided by
bone banks, is frequently utilized by orthopaedic surgeons because it is considered a
safe material from immunological and viral points of view.In the cases reported here,
homologous bone was used to repair some osseous defects without changing the
surgical protocol utilized for autologous bone procedures. The main advantages of this
strategy are low morbidity, shorter surgical times, more comfort and less risk of
infection for the patient as well as the greater availability of bone.
Clin Orthop Relat Res. 2000 Feb;(371):38-45. Links
The use of allograft bone in lumbar spine surgery.
Ehrler DM, Vaccaro AR.
OMNI Orthopaedics, Canton, Ohio, USA.
Bone grafting is an integral part of many lumbar spinal surgeries. The two choices of
bone are autograft and allograft. Each source has its own advantages and
disadvantages. The current study is a literature review of allograft bone use in lumbar
spine surgery. Allograft bone can be procured in greater quantities than autograft. With
standard protocols of harvesting, the risk of disease transfer is negligible. Only fresh-
frozen and freeze-dried products are used. Allografts are incorporated slower and to a
lesser degree than autografts. Fresh-frozen grafts are stronger, more immunogenic
and more completely incorporated than freeze-dried grafts. Allografts used alone or
combined with autografts for posterior lumbar spinal procedures have decreased
fusion rates compared with autografts. If used anteriorly, allografts are well suited for
reconstructive procedures and have good fusion rates, especially if combined with
posterior fusions. If used in the proper situations, allograft bone can be used
successfully in lumbar spine surgeries.
J Craniofac Surg. 2005 Nov;16(6):981-9. Links
Allograft and alloplastic bone substitutes: a review of science and
technology for the craniomaxillofacial surgeon.
Eppley BL, Pietrzak WS, Blanton MW.
Division of Plastic Surgery, Indiana School of Medicine, Indianapolis, Indiana, USA.
Bone healing is a complex and multifactorial process. As such, there are numerous
steps in the process to which intervention can be directed. This has given rise to many
bone graft technologies that have been used to regenerate bone, creating, perhaps, a
bewildering array of options. The options that surgeons have the most familiarity with
are the ones that have been available the longest (i.e., autograft and allograft).
Although useful for the widest spectrum of clinical applications, limitations of these
grafts has prompted the development of new materials. Demineralized bone matrix
formulations and synthetic ceramic materials are now being used with greater
frequency. These biomaterials have demonstrated their usefulness in facial plastic and
reconstructive surgery with their ability to augment and replace portions of the
craniofacial skeleton. The purpose of this article is to describe and discuss the
allograft and alloplastic bone grafting technologies so that the reader can consider
each in the context of the others and gain a better appreciation for how each fits into
the universe of existing and emerging treatments for bone regeneration.
Transfus Med. 2005 Jun;15(3):165-74. Links
Clinical effectiveness of processed and unprocessed bone.
Galea G, Kearney JN.
Tissue Services, Scottish National Blood Transfusion Service, Edinburgh, UK.
Bone allografts have been used clinically for a number of years. Understanding the
biology of bone healing and the impact that bone banking has on this helps to improve
the methodologies used in increasing the quality and safety of banked bone. Banked
bone in its various forms has been used in a variety of surgical procedures, and
although there is no doubt that it is clinically effective, most of the studies have been
retrospective and non-randomized. The review attempts to summarize some of the
data in this area and highlights some of the difficulties encountered in such work.
Although there is no doubt that bone banking is nowadays better controlled, there are
ever-increasing pressures to produce bone that is as safe as possible with the least
impact on its effectiveness. This can only be achieved if the requirements of the
providers and users of bone are better understood.
J Photochem Photobiol B. 2003 May-Jun;70(2):81-9. Links
Low-power laser irradiation improves histomorphometrical parameters
and bone matrix organization during tibia wound healing in rats.
Garavello-Freitas I, Baranauskas V, Joazeiro PP, Padovani CR, Dal Pai-
Silva M, da Cruz-Höfling MA.
Faculdade de Engenharia Elétrica e Computação, Departamento de Semicondutores
Instrumentos e Fotônica, Universidade Estadual de Campinas, Av. Albert Einstein
N.400, 13 083-970 Campinas, SP, Brazil.
The influence of daily energy doses of 0.03, 0.3 and 0.9 J of He-Ne laser irradiation on
the repair of surgically produced tibia damage was investigated in Wistar rats. Laser
treatment was initiated 24 h after the trauma and continued daily for 7 or 14 days in
two groups of nine rats (n=3 per laser dose and period). Two control groups (n=9
each) with injured tibiae were used. The course of healing was monitored using
morphometrical analysis of the trabecular area. The organization of collagen fibers in
the bone matrix and the histology of the tissue were evaluated using Picrosirius-
polarization method and Masson's trichrome. After 7 days, there was a significant
increase in the area of neoformed trabeculae in tibiae irradiated with 0.3 and 0.9 J
compared to the controls. At a daily dose of 0.9 J (15 min of irradiation per day) the 7-
day group showed a significant increase in trabecular bone growth compared to the
14-day group. However, the laser irradiation at the daily dose of 0.3 J produced no
significant decrease in the trabecular area of the 14-day group compared to the 7-day
group, but there was significant increase in the trabecular area of the 15-day controls
compared to the 8-day controls. Irradiation increased the number of hypertrophic
osteoclasts compared to non-irradiated injured tibiae (controls) on days 8 and 15. The
Picrosirius-polarization method revealed bands of parallel collagen fibers (parallel-
fibered bone) at the repair site of 14-day-irradiated tibiae, regardless of the dose. This
organization improved when compared to 7-day-irradiated tibiae and control tibiae.
These results show that low-level laser therapy stimulated the growth of the trabecular
area and the concomitant invasion of osteoclasts during the first week, and hastened
the organization of matrix collagen (parallel alignment of the fibers) in a second phase
not seen in control, non-irradiated tibiae at the same period. The active osteoclasts
that invaded the regenerating site were probably responsible for the decrease in
trabecular area by the fourteenth day of irradiation.
J Am Acad Orthop Surg. 1995 Jan;3(1):1-8. Links
Alternatives to Autogenous Bone Graft: Efficacy and Indications.
Gazdag AR, Lane JM, Glaser D, Forster RA.
Department of Orthopaedics, University of California, Los Angeles, School of
Medicine.
Bone grafting is frequently used to augment bone healing with the numerous
approaches to reconstructing or replacing skeletal defects. Autologous cancellous
bone graft remains the most effective grafting material because it provides the three
elements required for bone regeneration: osteoconduction, osteoinduction, and
osteogenic cells. Autologous cortical bone graft provides these three components to a
limited extent as well and also provides the structural integrity important in
reconstruction of larger defects. However, because autogenous grafting is associated
with several shortcomings and complications, including limited quantities of bone for
harvest and donor-site morbidity, alternatives have been used in a wide range of
orthopaedic pathologic conditions. Grafting substitutes currently available include
cancellous and cortical allograft bone, ceramics, demineralized bone matrix, bone
marrow, and composite grafts. No single alternative graft material provides all three
components for bone regeneration. The clinical applications for each type of material
are dictated by its particular structural and biochemical properties. Composite grafts
consisting of several materials are often used to maximize bone healing, especially
where the grafting site is compromised.
Clin Orthop Relat Res. 1987 Dec;(225):7-16. Links
Natural history of autografts and allografts.
Goldberg VM, Stevenson S.
Case Western Reserve School of Medicine, Department of Orthopaedics, Cleveland,
OH 44106.
The clinical outcome of bone grafting procedures depends on many factors, including
type and fixation of the bone graft as well as the site and status of the host bed. Bone
grafts serve one or both of two main functions, as a source of osteogenetic cells and
as a mechanical support. Autografts, both cancellous and cortical, are usually
implanted fresh and are often osteogenetic, whether by providing a source of
osteoprogenitor cells or by being osteoinductive. The latter is a process whereby the
transplanted tissue induces mesenchymal cells of the recipient to differentiate into
osteoblastic cells. Cortical grafts, whether autogeneic or allogeneic, at least initially act
as weight-bearing space fillers or struts. All bone grafts are initially resorbed, but
cancellous grafts are completely replaced in time by creeping substitution, while
cortical grafts remain an admixture of necrotic and viable bone for a prolonged period
of time. The three-dimensional framework, which supports invasion of the bone grafts
by capillaries and osteoprogenitor cells, termed "osteoconduction", is another
important function of both autografts and allografts. Fresh allografts are more slowly
and less completely replaced by host bones because they invoke both local and
systemic immune responses that diminish or destroy the osteoinductive and
conductive processes. Although freezing or freeze-drying of allografts improves
acceptance, their failure rate is still too high. These processes are also influenced by
the vascularity and composition of the host bed. Thus, the interaction of the host and
the bone graft determines the success of these procedures, which ultimately is to
provide a mechanically efficient support structure.
Otolaryngol Clin North Am. 1994 Oct;27(5):911-25. Links
Banked bone.
Hardin CK.
Wilford Hall United States Air Force Medical Center, Lackland Air Force Base, Texas.
Many forms of banked bone allograft are available to the surgeon. Among the grafts
available are fresh, fresh-frozen, freeze-dried, and demineralized bone. Each one of
these grafts carries risks and has unique limitations and handling properties. In order
to use these materials appropriately, the surgeon must be familiar with the properties
of each and must feel confident that the bone bank providing the graft is supplying a
safe and sterile graft. In the future, allograft bone will become obsolete. In place of
banked bone, surgeons will use synthetically produced bone morphogenic protein that
has been incorporated into an absorbable matrix. These materials will exist in a time-
release form that will allow the graft material to grow and mature with the patient. Until
this goal is achieved and is available clinically, surgeons must be familiar with the
capabilities and limitations of banked bone graft.
Cell Tissue Bank. 2005;6(1):25-31. Links
Detection of living cells in non-processed but deep-frozen bone allografts.
Heyligers IC, Klein-Nulend J.
Department of Orthopaedic Surgery, Skeletal Tissue Engineering Group, Amsterdam
(STEGA), Atrium MC, Heerlen, The Netherlands. IHS01@atriummc.nl
Impacted morselized donor bone is successfully used to treat bone loss in revision
total hip arthroplasties. It is generally thought, but not proven, that the processing and
storage at -80 degrees C of the donor bone kills all cells. Because of the risk of
contamination and to increase our understanding about the process of new bone
formation after revision total hip arthroplasty, the aim of this study was to investigate
whether the donor bone does contain vital cells. Samples from 11 femoral heads were
obtained according to the American and European standards of bone banking, and
tested for their capacity to give rise to proliferating cells, using tissue culture methods.
All bone samples were stored at - 80 degrees C for a minimum of 6 months. Bone
sample cores were morselized and cultured for 6 weeks. Inverted phase contrast
microscopy was used to evaluate cell growth. DNA marker analysis was used to
confirm cellular identity. All bank bone samples gave rise to cell growth. The cell
cultures showed osteoblastic characteristics in that they expressed high levels of
alkaline phosphatase activity. DNA marker analysis showed identical alleles for
cultured cells from frozen bone and freshly obtained buccal cells from the same donor,
indicating that the cells growing from the banked bone were indeed originating from
the donor tissue. It was therefore concluded that -80 degrees C freezing of bone tissue
does not routinely kill cells within the tissue.
Oper Tech Plastic Reconstr Surg
. 2003 9(4):151-60
Bone graft substitutes.
Ilan DI, Ladd AL
Orthopaedics, Stanford University School of Medicine, Palo Alto, CA 94304
Today’s surgeon faces many situations that require bone grafting. Autologous bone is
the traditional standard for treating conditions requiring bone graft. However,
autologous bone grafting has drawbacks such as donor site morbidity and increased
operative time. Graft substitutes for autologous bone are an appealing alternative.
Substitutes include allograft, mineral composites, ceramics, mineral cements, bioactive
glass, proteins and growth factors. The use and availability of these products has
expanded exponentially. The large number of alternatives available and the relative
lack of quality information regarding their indications and effectiveness leave the
surgeon desiring to use the product with a daunting task. This article provides the
surgeon with an overview of the basic concepts of the bone grafting and discusses the
most commonly used bone-graft substitutes and their potential indications.
J Orthop Res. 2004 May;22(3):653-8. Links
Platelet rich plasma and fresh frozen bone allograft as enhancement of
implant fixation. An experimental study in dogs.
Jensen TB, Rahbek O, Overgaard S, Søballe K.
Orthopaedic Research Group, Institute of Experimental Clinical Research, Aarhus
University Hospital, Aarhus Kommunehospital, 8000 Aarhus C, Denmark.
tbj@webspeed.dk
Platelet rich plasma (PRP) is an autologous source of growth factors. By application of
PRP around cementless implants alone or in combination with bone allograft chips,
early implant fixation and gap healing could be improved. We inserted two porous HA
coated titanium implants extraarticularly in each proximal humerus of eight dogs. Each
implant was surrounded by a 2.5 mm gap. Four treatments were block randomized to
the four gaps in each dog: Treatment 1: empty gap, treatment 2: PRP, treatment 3:
fresh frozen bone allograft, treatment 4: fresh frozen bone allograft+PRP. PRP was
prepared from each dog prior to operation by isolating the buffycoat from centrifuged
blood samples. Platelet count in PRP was increased 670% compared to baseline
level. Calcium/thrombin was added to degranulate platelets and form a gel. Three
weeks after surgery, push-out test and histomorphometri was performed. After three
weeks, the non-allografted implants had poor mechanical properties. Bone grafting
significantly increased implant fixation, bone formation in the gap and bone growth on
the implant surface. We found no significant effect of PRP alone or mixed with bone
allograft on implant fixation or bone formation. In conclusion, we showed the
importance of bone allografting on early implant fixation and bone incorporation but we
found no effect of PRP. More studies are needed to investigate the effect and possible
clinical applications of platelet concentrates which are now being commercialised.
Histochemistry
. 1982 74:153-156.
The influence of tissue section tickeness on the study of collagen by the
Picrosirius-polarization method
Junqueira LCU
, Montes GS, Sanchez EM
Laboratories for Cell Biology and Experimental Oncology, University of São
Paulo School of Medicine, São Paulo, Brazil
The influence of tissue section tickness on the color and intensity of birefringence
displayed by collagen in tissue sections studied by means of the Picrosirius-
polarization method, is reported in this paper. When dermal collagen sections of
different ticknesses (ranging from 0.25 to 11μ) were studied by this method, it became
evident that not only did the intensity of birefringence increase proportionally to tissue
section tickness, as was to be expected, but also a gradual shift in color from green
through yellow to red could be observed as tissue section increased. The limitations of
the Picrosirius-polarization method for the localization of collagen types I, II and III in
routinely used histological slides is discussed, showing that this method is useful for
the study of the distribution of the different types of interstitial collagen in normal adult
vertebrate organs.
Oral Maxillofac Surg Clin North Am. 2007 Nov;19(4):513-21, vi. Links
A review of bone substitutes.
Kao ST, Scott DD.
Oral and Maxillofacial Surgery, Medical College of Georgia School of Dentistry,
Augusta, GA 30912, USA. [email protected]du
The use of bone grafts in the repair of defects has a long history of success, primarily
with the use of autologous bone. With increasing technologic advances, researchers
have been able to broaden the spectrum of grafting materials to allografts, xenografts,
and synthetic materials, which provide the surgeon and patient with options, each with
unique advantages. It is with the knowledge of each material that the clinician can
present and suggest the best material and tailor treatment plans to fit each individual.
In this article, we present an overview of the principles of bone grafting, the types of
graft materials available, and an outlook to what the future holds in this area of
medicine and dentistry.
Implant Dent. 2003;12(3):217-26. Links
Localized maxillary ridge augmentation with a block allograft for dental
implant placement: case reports.
Leonetti JA, Koup R.
Autogenous block bone grafts have been highly successful in treating human
periodontal defects, restoring esthetics, and developing adequate bone volume for
dental implant placement. Limitations in available donor bone, the need for an added
surgical procedure, and other potential complications have made the use of allogenic
bone graft materials an important alternative. One patient described in this report
presented with fractured root syndrome of the right maxillary incisor with severe
resorption of the buccal plate. After atraumatic tooth extraction, a staged treatment
approach involving localized ridge augmentation with an allogenic iliac bone block
material and dental implant placement was used. The host bone completely
incorporated the graft with only minor resorption, which enabled the implant to be
placed. The allogenic bone block material used in this study was an effective
alternative to harvesting and grafting autogenous bone for implant site development.
The cases presented in this article clinically demonstrate the efficacy of using a block
allograft in generating effective new bone fill for dental implant placement.
Clin Orthop Relat Res. 2005 Oct;439:144-50. Links
Treatment of giant cell tumors with allograft transplants: a 30-year study.
Mankin HJ, Hornicek FJ.
Orthopaedic Oncology Service, Massachusetts General Hospital, Boston, MA 02114,
USA. [email protected]
We retrospectively reviewed 144 patients with giant cell tumors who had resection and
implantation of cadaveric allografts from 1971-2001. Most procedures were done in
the distal femur, proximal tibia, proximal femur, and proximal humerus. Seventy-eight
percent of patients have retained their grafts and remain functional, but with
limitations. Tumor complications included local recurrences (eight patients, none
required an amputation) and metastases (three patients). None of the patients died.
Allograft fracture occurred in 30 (21%) of the 144 patients, nonunion occurred in 12
(8%) patients, and infection occurred in 12 (8%) patients. Only patients with infection
had a marked decline in outcome. Four patients required amputations, and only two of
the eight patients with infection (17%) retained a functional graft. Eighty of the 144
patients (56%) had no complications, with a 94% success rate for the procedure.
There have been fewer grafts done with the advent of burring, phenolization, and
insertion of polymethylmethacrylate. However, based on our data and the good
outcome for more extensive and destructive tumors, allograft implants can be used for
treatment of patients with aggressive tumors, tumors that have caused fractures, or
tumors that have recurred after conservative treatment.
Oral Maxillofac Surg Clin North Am. 2007 Nov;19(4):455-66, v. Links
Bone and bone graft healing.
Marx RE.
Division of Oral and Maxillofacial Surgery, University of Miami Miller School of
Medicine, 9380 SW 150th Street, Miami, FL 33157, USA. [email protected]
Bone is unique in connective tissue healing because it heals entirely by cellular
regeneration and the production of a mineral matrix rather than just collagen
deposition known as scar. This article discusses the cellular, tissue, and organ levels
in each of the following sections--skeletal embryology, normal bone, examples of
abnormal bone, and bone graft healing--as they relate to the jaws and the craniofacial
skeleton.
Cell Biol Int. 1996 Jan;20(1):15-27. Links
Structural biology of the fibres of the collagenous and elastic systems.
Montes GS.
Laboratory for Cell Biology, University of São Paulo School of Medicine, Brazil.
The different types of fibres of the collagenous and elastic systems can be
demonstrated specifically in tissue sections by comparing the typical ultrastructural
picture of each of the fibre types with studies using selective staining techniques for
light microscopy. A practical modus operandi, which includes the recommended
staining procedures and interpretation of the results, is presented. Micrographs and
tables are provided to summarize the differential procedures. Reticulin fibres display a
distinct argyrophilia when studied by means of silver impregnation techniques, and
show up as a thin meshwork of weakly birefringent, greenish fibres when examined
with the aid of the Picrosirius-polarization method. In addition, electron-microscopic
studies showed that reticulin fibres are composed of a small number of thin collagen
fibrils, contrasting with the very many thicker fibrils that could be localized
ultrastructurally to the sites where non-argyrophilic, coarse collagen fibres had been
characterized by the histochemical methods used. The three different fibre types of the
elastic system belong to a continuous series: oxytalan-elaunin-elastic (all of the fibre
types comprising collections of microfibrils with, in the given sequence, increasing
amounts of elastin). The three distinct types of elastic system fibres have different
staining characteristics and ultrastructural patterns. Ultrastructurally, a characteristic
elastic fibre consists of two morphologically different components: a centrally located
solid cylinder of amorphous and homogeneous elastin surrounded by tubular
microfibrils. An oxytalan fibre is composed of a bundle of microfibrils, identical to the
elastic fibre microfibrils, without amorphous material. In elaunin fibres, dispersed
amorphous material (elastin) is intermingled among the microfibrils.
Mem Inst Oswaldo Cruz
. 1991 86(III):1-11.
The use of the Picrosirius-polarization method for the study of the
biopathology of collagen
Montes GS, Junqueira LCU.
Laboratório de Biopatologia Celular, Faculdade de Medicina da USP, São Paulo, SP,
BR
The histochemical study of collagen hás permitted a better understanding of the
biology of this family of macromolecules in the last decade. Two methods, namely: the
immunohistochemical and the Picrosirius-polarization methods, contributed
significantly to our better knowledge of collagen function and pathology. In this paper
we outline the theoretical basis and applications of the Picrosirius-polarization method
as developed and applied in this laboratory. This short article has not been designed
as a comprehensive reference work: thus, the reader is encouraged to consult a
recent review on the subject for additional information (Montes & Junqueira, 1988)
Int J Oral Maxillofac Surg. 1992 Oct;21(5):260-5. Links
The use of fresh frozen allogeneic bone for maxillary and mandibular
reconstruction.
Perrott DH, Smith RA, Kaban LB.
Department of Oral and Maxillofacial Surgery, University of California, San Francisco.
The use of fresh frozen bone (FFB) alone, or in combination with autogenous bone
(AB), for bony augmentation of the maxilla and mandible in preparation for dental
reconstruction with endosseous implants has been studied. Ten patients received FFB
+/- AB for augmentation of a severely atrophic mandible (n = 6) or for reconstruction of
a jaw defect secondary to trauma or tumor resection (n = 4). Average follow-up was
26.3 +/- 5.4 months. At the time of implant placement, the bone grafts were found to
be firm in consistency, well incorporated, and well vascularized in all 10 patients.
Twenty-nine endosseous implants were placed an average of 8.3 +/- 3.1 months
following bone grafting. One implant failed and was replaced, and one implant remains
buried as a nonfunctional unit. All patients have been restored prosthetically by means
of 28 of the 29 implants. This preliminary study indicates that FFB may be used alone
or in combination with autogenous bone for augmentation or reconstruction of the
atrophic maxilla and mandible. The resultant ridge is adequate to support loaded
endosseous implants. A potential disadvantage is the minimal risk of disease
transmission.
Implant Dent. 2005 Jun;14(2):139-48. Links
Localized ridge augmentation with allogenic block grafts prior to implant
placement: case reports and histologic evaluations.
Petrungaro PS, Amar S.
Institute for Advanced Dental Education Inc., Lake Elmo, Minnesota, Lake Elmo,
Minnesota, USA. [email protected]
The placement of dental implants is based on the amount of alveolar bone present in
the edentulous site to be reconstructed. Insufficient alveolar contours may require
bone grafting procedures to restore an adequate bone volume before implant
placement. Larger osseous defects often require block grafts harvested from the
symphysis or the ramus buccal shelf region. These provide adequate donor sites to
harvest a graft sufficient to restore a significant defect in the osseous structures
planned for implant placements. Autogenous block grafts have been well established
to reconstruct these types of defects prior to implant placement procedures. However,
surgical complications associated with the unfavorable anatomical structures and the
necessity of large donor sites (e.g., symphysis and ramus buccal shelf) have led to the
use of allogenic grafting materials. Recent developments in allogenic grafting
materials have lead to the development of a corticocancellous block graft harvested
from the iliac crest region. This study evaluates the clinical indications of these
allogenic graft materials to replace compromised alveolar bone defects both horizontal
and vertical in nature. The analysis is supported by re-entry procedures and histologic
evaluations to substantiate predictability.
Int J Oral Maxillofac Surg. 2002 Oct;31(5):525-31. Links
Allogeneic bone grafting of calvarial defects: an experimental study in the
rabbit.
Shand JM, Heggie AA, Holmes AD, Holmes W.
Oral and Maxillofacial Surgery Unit, University of Melbourne, School of Dental
Science, Victoria, Australia.
The purpose of this study was to investigate the incorporation of fresh frozen irradiated
membranous allogeneic bone grafts into critical size calvarial defects in the rabbit.
Fifteen rabbits had calvarial defects prepared. Twelve rabbits received allogeneic
grafts and three received autogenous bone grafts. The rabbits were sacrificed at 9 and
12 months postoperatively, and the specimens were examined radiologically,
histopathologically and with fluorescence microscopy. Neovascularization, bone
marrow regeneration and new bone formation was evident throughout the grafts
however revitalization of the entire graft was incomplete at 12 months. This study
revealed that the FFI membranous grafts were well incorporated into rabbit calvarial
defects.
Acta Orthop. 2007 Feb;78(1):26-30. Links
Viable cells survive in fresh frozen human bone allografts.
Simpson D, Kakarala G, Hampson K, Steele N, Ashton B.
Institute of Science and Technology in Medicine, University of Keele, the Robert Jones
and Agnes Hunt Orthopaedic Hospital, Oswestry, Shropshire, UK.
BACKGROUND: Fresh frozen bone allograft is available for human recipients after at
least 6 months of quarantine at -80 degrees C. It is assumed that cryopreservation
without cryoprotectant removes all viable donor cells. METHODS: We studied the in
vitro cell growth from samples of fresh frozen human femoral head allografts after they
had been released for patient use, and compared it with cell growth from a control
group of fresh cancellous bone specimens from excised femoral heads (8 samples in
each group). RESULTS: Cell outgrowths were seen in all of the fresh cancellous bone
specimens (100% of replicates, 48 replicates per specimen) but only in a small
minority of replicates from 4 of the allograft samples (mean 3.1%). Reverse
transcription quantitative polymerase chain reaction (RT-qPCR) investigations
revealed that cell outgrowths from both groups contained mRNA for transcription
factors Runx2 and Osterix, and also for matrix proteins collagen type I, osteocalcin
and bone sialoprotein. This is consistent with the cells being osteoblast-related.
INTERPRETATION: This study confirms that fresh frozen human bone allograft cells
have the potential to grow in vitro, but the significance of this in recipients is currently
unknown.
Cell Tissue Bank. 2004;5(2):97-104. Links
Very long-term radiographic and bone scan results of frozen autograft
and allograft bone grafting in 17 patients (20 grafts) a 30- to 35-year
follow-up.
Steinberg EL, Luger E, Zwas T, Katznelson A.
Department of Orthopaedic Surgery 'B', Tel Aviv Sourasky Medical Center.
In the early 1950s, 48 patients received bone implants from a bone bank in Tel-
Hashomer Hospital that stored frozen autograft and allograft bones at temperatures
less than -17 degrees C. Seventeen (35%) of these patients (20 implants), 10 men
and 7 women, with a mean age of 52.4 (34-69) years were available for follow-up after
a mean period of 32.5 (30-35) years. They underwent clinical examination,
radiographs and bone scans to evaluate their surgical results. Fracture healing, non-
union, graft resorption, osteoporosis and bone sclerosis were used as radiographic
criteria for bone incorporation, and normal, increased and decreased uptake served to
assess the bone scan. Based on the above criteria, the results were satisfactory in 17
(85%) and poor in 3 (15%). The three failures were after shelf operation for hip
dysplasia that used two allografts and one autograft. Allogenous or a combination of
allogenous with autogenous frozen bone grafts proved to be a satisfactory and durable
method for filling bone defects.
Cell Tissue Bank. 2000;1(2):105-9. Links
Bone allografts: past, present and future.
Tomford WW.
Massachusetts General Hospital, Department of Orthopaedic Surgery, Boston, MA
02114, USA.
Bone allograft transplantation has been performed in humans for more than one
hundred and twenty years. During the first one hundred years (1880-1980), the major
problem in bone allograft transplantation was availability. Most of the bone grafts used
during this time were autografts. Allografts were not available due to a lack of
legislation protecting procurers and processers. In addition, surgical procedures
requiring allografts were not being performed. During the next twenty years (1980-
2000), as allografis began to be used, the major issue was safety. Diseases
transmitted during this period included AIDS and hepatitis. Avoidance of disease
transmission became paramount. Sensitive blood tests and extensive efforts by bone
banks to develop ways to clean. bone and clear it of infectious agents helped provide
safe transplants. With concerns of availability and safety receding, the major issue in
the future (2000-? ) will be the efficacy of the transplant. How allograft bone remodels
in the host, how it incorporates and heals to host bone and how it integrates with the
host skeleton will be the most important concerns of bone bankers and tissue
transplant surgeons. Future research efforts will be applied to bone allograft
transplantation to ensure that bone transplants heal quickly and sufficiently to be able
to function as part of the weight-bearing skeletal system.
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