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Previous issue date: 2013-03-01 / The regeneration of bone defects with loss of substance remains as a therapeutic challenge in the medical field. There are basically four types of grafts: autologous, allogenic, xenogenic and isogenic. It is a consensus that autologous bone is the most suitable material for this purpose, but there are limitations to its use, especially the insufficient amount in the donor. Surveys show that the components of the extracellular matrix (ECM) are generally conserved between different species and are well tolerated even in xenogenic recipient. Thus, several studies have been conducted in the search for a replacement for autogenous bone scaffold using the technique of decellularization. To obtain these scaffolds, tissue must undergo a process of cell removal that causes minimal adverse effects on the composition, biological activity and mechanical integrity of the remaining extracellular matrix. There is not, however, a conformity among researchers about the best protocol for decellularization, since each of these treatments interfere differently in biochemical composition, ultrastructure and mechanical properties of the extracellular matrix, affecting the type of immune response to the material. Further down the arsenal of research involving decellularization bone tissue represents another obstacle to the arrival of a consensus protocol. The present study aimed to evaluate the influence of decellularization methods in the production of biological scaffolds from skeletal organs of mice, for their use for grafting. This was a laboratory study, sequenced in two distinct stages. In the first phase 12 mice hemi-calvariae were evaluated, divided into three groups (n = 4) and submitted to three different decellularization protocols (SDS [group I], trypsin [Group II], Triton X-100 [Group III]). We tried to identify the one that promotes most efficient cell removal, simultaneously to the best structural preservation of the bone extracellular matrix. Therefore, we performed quantitative analysis of the number of remaining cells and descriptive analysis of the scaffolds, made possible by microscopy. In the second stage, a study was conducted to evaluate the in vitro adhesion of mice bone marrow mesenchymal cells, cultured on these scaffolds, previously decellularized. Through manual counting of cells on scaffolds there was a complete cell removal in Group II, Group I showed a practically complete cell removal, and Group III displayed cell remains. The findings allowed us to observe a significant difference only between Groups II and III (p = 0.042). Better maintenance of the collagen structure was obtained with Triton X-100, whereas the decellularization with Trypsin was responsible for the major structural changes in the scaffolds. After culture, the adhesion of mesenchymal cells was only observed in specimens deccelularized with Trypsin. Due to the potential for total removal of cells and the ability to allow adherence of these, the protocol based on the use of Trypsin (Group II) was considered the most suitable for use in future experiments involving bone grafting decellularized scaffolds / A regenera??o de defeitos ?sseos com perda de subst?ncia permanece um desafio terap?utico na ?rea m?dica. ? consenso ser o osso aut?geno, o material mais adequado para esta finalidade, por?m h? limita??es at? para o seu uso, especialmente a quantidade insuficiente no pr?prio doador. Pesquisas de engenharia tecidual evidenciam que os componentes da matriz extracelular (MEC) s?o geralmente conservados entre as diferentes esp?cies sendo bem toleradas, mesmo em receptores xen?genos. Assim, diversos estudos t?m sido realizados na busca por um arcabou?o substituto do osso aut?geno atrav?s da t?cnica de descelulariza??o. Para a obten??o destes arcabou?os, os tecidos devem passar por um processo de remo??o celular, que cause m?nimos efeitos adversos na composi??o, atividade biol?gica e integridade mec?nica na matriz extracelular remanescente. Entretanto, h? controv?rsias acerca do melhor protocolo de descelulariza??o, j? que cada um desses tratamentos interfere de maneira diferente na composi??o bioqu?mica, ultraestrutura e comportamento mec?nico da matriz extracelular, afetando o tipo de resposta imunol?gica ao material. Ademais o baixo arsenal de pesquisas envolvendo a descelulariza??o de tecidos ?sseos representa mais um obst?culo ? chegada de um consenso protocolar. O presente estudo teve como objetivo avaliar a influ?ncia dos m?todos de descelulariza??o na produ??o de arcabou?os biol?gicos a partir de ?rg?os ?sseos de camundongos, visando sua utiliza??o para enxertia. Trata-se de um estudo laboratorial, sequenciado em duas etapas distintas. Na primeira fase foram avaliadas 12 hemi-calv?rias de camundongos, divididas em tr?s grupos (n=4) e submetidas a tr?s diferentes protocolos de descelulariza??o (SDS [Grupo I], Tripsina [Grupo II], Triton X-100 [Grupo III]). Buscou-se identificar aquele que promove a mais eficiente remo??o celular, simultaneamente a melhor preserva??o estrutural da MEC ?ssea. Para tanto, foi realizada an?lise quantitativa do n?mero de c?lulas remanescentes e an?lise descritiva dos arcabou?os, possibilitadas por microscopia. Na segunda etapa, foi realizado um estudo in vitro para avaliar a ades?o de c?lulas mesenquimais da medula ?ssea de camundongos, cultivadas sobre arcabou?os previamente descelularizados. Atrav?s da contagem manual de c?lulas nos arcabou?os, verificou-se total remo??o celular no Grupo II, remo??o praticamente completa no Grupo I, e perman?ncia de c?lulas e remanescentes no Grupo III. Os achados permitiram observar diferen?a significativa apenas entre os Grupos II e III (p=0,042). Melhor manuten??o da estrutura col?gena foi obtida com o Triton X-100, ao passo que a descelulariza??o com Tripsina foi respons?vel pelas maiores altera??es estruturais nos arcabou?os. Ap?s o cultivo, a ades?o de c?lulas mesenquimais s? foi observada nas calv?rias descelularizadas com Tripsina. Devido ao potencial de remo??o total das c?lulas e ? capacidade de permitir a ades?o destas, o protocolo baseado no uso da Tripsina (Grupo II) foi considerado o mais adequado para uso em experimentos futuros, que envolvam enxertia de arcabou?os ?sseos descelularizados
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/17816 |
| Date | 01 March 2013 |
| Creators | Os?rio Junior, Haroldo Abuana |
| Contributors | CPF:59241144653, http://lattes.cnpq.br/7625795408417124, Barbosa, Carlos Augusto Galv?o, CPF:67269621420, http://lattes.cnpq.br/5004397230198722, Santos, Pedro Paulo de Andrade, CPF:02306394458, http://lattes.cnpq.br/2878739335493429, Silva, Jos? Sandro Pereira da |
| Publisher | Universidade Federal do Rio Grande do Norte, Programa de P?s-Gradua??o em Sa?de Coletiva, UFRN, BR, Sa?de P?blica |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Repositório Institucional da UFRN, instname:Universidade Federal do Rio Grande do Norte, instacron:UFRN |
| Rights | info:eu-repo/semantics/openAccess |
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