Biological evaluation of PLDLA polymer synthesized as construct on bone tissue engineering application

In bone tissue engineering, cell-scaffold constructs are used to stimulate complete, functional tissue replacement that does not occur naturally in critical-size defects. In this report, we describe the application potential of poly (L-co-D,L lactic acid)-PLDLA 70/30, synthesized in house as constructs loaded with osteoblast-like cells on bone tissue engineering. In vitro biological results show that the porogen leached PLDLA scaffolds are cytocompatible with osteoblast cells, able to stimulate significant cells growth during the first 14 days of culture, during which the morphology and cell behavior of osteoblasts cultured on the scaffolds were monitored by scanning electron microscopy (SEM). In vivo, PLDLA constructs were implanted in 5 mm bilateral critical-size defects created in rat-calvariae and then evaluated histologically 8 and 12 weeks after implantation. The histological results showed that PLDLA constructs supported the growth of new tissue, with a degradation rate close to that of native bone formation and decrease of inflammatory response over time of implantation. These data provide evidences that the synthesized PLDLA polymer has application potential as construct for bone tissue engineering.192300307FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2012/12081-

Hydroxyapatite coating deposited on grade 4 Titanium by Plasma Electrolytic Oxidation

The present study reports the deposition of coating using Plasma Electrolytic Oxidation (PEO) onto grade 4 titanium to produce novel surface features. Samples were treated in an electrolytic solution of calcium acetate and sodium glycerolphosphate. The temporal evolution of hydroxyapatite coatings with high Ra roughness and a maximum thickness of 120 μm was obtained. X-ray spectra revealed the presence of hydroxyapatite, rutile and calcium phosphate. Cell growth measurement by MTT assay showed that the coatings were not toxic because cells grew on all samples17614271433CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçã

Surface characterization and osteoblast-like Cells culture on collagen modified PLDLA scaffolds

Surface modification techniques based on the grafting of chemical functional groups and immobilization of bioactive molecules have been used to improve biocompatibility and clinical performance of bioabsorbable scaffolds in tissue engineering and medicine regenerative applications. This study aimed at developing and characterizing a biomimetic surface to stimulate bone regeneration by a simple and low-cost method of surface biofunctionalization of the poly (L-co-D,L lactic acid)- PLDLA scaffolds. The method was obtained by grafting reaction of carboxyl groups (-COOH) on their surface via acrylic acid (AAc) polymerization process, followed by immobilization of collagen type I (Col). Such approach resulted in a surface morphology markedly modified after treatment, with increase of pores and roughness on PLDLA-AAc surfaces and a network of fibrillar collagen deposition in nonspecific areas of PLDLA-Col surfaces. The cytocompatibility of collagen-immobilized scaffolds was significantly improved in terms of cellular adhesion, proliferation, collagen synthesis and maintenance of osteoblast-like phenotype, indicating, therefore, the fundamental role of collagen protein over the biological interactions that occur by bio-recognition mimetic mechanisms at biomaterials interface. These results indicate that the surface modification method used here may be useful as a strategy to develop biofunctional scaffolds, which provide a more successful clinical application of biomaterials in the tissue engineering field17615231534FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2012/12081-2; 2007/05089-

Biofunctionalization of PLDLA scaffolds immobilized with cyclic RGDfK peptide and knuckle epitope (73-92) of BMP-2

Orientador: Eliana Aparecida de Rezende DuekTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia MecânicaResumo: A biofuncionalização de scaffolds poliméricos biorreabsorvíveis através da imobilização química de peptídeos bioativos, capazes de serem naturalmente reconhecidas pelo tecido ósseo nativo e então, estimularem a diferenciação osteogênica de células tronco mesenquimais, é uma estratégia bastante promissora na área da engenharia tecidual óssea. No presente trabalho, a superfície de scaffolds de poli (L-co-D,L ácido lático) ¿PLDLA foram biofuncionalizadas com o peptídeo cíclico RGDfK e peptídeo derivado do epítopo knuckle da proteína BMP-2 (73-92) que estão envolvidos, principalmente, na adesão celular e regulação da maturação do tecido ósseo. A imobilização dos peptídeos foi obtida através da pré-ativação da superfície do PLDLA usando o tratamento por plasma de oxigênio, seguido da imobilização covalente dos peptídeos via reação EDC/sulfo-NHS. Os resultados obtidos pela técnica de espectroscopia de fotoelétrons excitados por raios-X (XPS) e espectroscopia de reflectância total atenuada (FTIR-ATR) confirmaram a imobilização dos peptídeos que estabelecem eficiência de ligação na proporção de 1:2, com predominância do peptídeo BMP-2 sobre o cRGD, também confirmada por microscopia confocal a laser (MCL). O tratamento por plasma de oxigênio na condição de potência de 100W resultou na expressiva diminuição da massa molar que é indicativa de degradação, contudo, a propriedade mecânica dos scaffolds não foi afetada. A avaliação biológica dos scaffolds biofuncionalizados com os peptídeos cRGDfK, BMP-2 e cRGDfK/BMP-2 demonstrou que a associação dos peptídeos exerce efeito sinérgico sobre a adesão e diferenciação osteogênica de células-tronco mesenquimais de medula óssea de ratos Wistar, que foi confirmada pelo maior nível de fosfatase alcalina e produção de colágeno encontrados após 7 dias de cultura. Estes resultados são indicativos de que o método de modificação de superfície utilizado no presente estudo pode ser usado no desenvolvimento de arcabouços bioativos e biomiméticos com potencial aplicação na engenharia tecidual óssea e medicina regenerativaAbstract: The biofunctionalization of bioresorbable polymeric scaffolds by chemical immobilization of bioactive peptides capable of being naturally recognized by the native bone tissue and then, stimulate osteogenic differentiation of mesenchymal stem cells (MSCs), is a promising strategy in bone tissue engineering field. In the present study, the surface of poly (L-co-D,L lactic acid) ¿ PLDLA was biofunctionalized with the cyclic RGDfK peptide and peptide derived from the knuckle epitope of BMP-2 (73-92), which are mainly involved in cell adhesion and regulation of bone maturation. The immobilization of peptides was obtained through the pre-activation of PLDLA surface using oxygen plasma treatment, followed by the covalent immobilization of peptides via EDC / sulfo-NHS reaction. The results obtained by X-ray photoelectron spectroscopy (XPS) and attenuated total re?ectance infrared spectroscopy (ATR-FTIR) techniques confirmed the immobilization of peptides which establishes bond efficiency in a 1: 2 ratio with predominance of BMP-2 on cRGDfK peptide, also confirmed by confocal laser microscopy (CLM). The oxygen plasma treatment under power condition of 100W resulted in a significant decrease in molecular weight which is indicative of degradation; however, the mechanical properties of biofunctionalized scaffolds were not affected. Biological evaluation of biofunctionalized scaffolds with the peptides cRGDfK, BMP-2 and cRGDfK / BMP-2 demonstrated that the association of the peptides exerts synergistic effect on adhesion and osteogenic differentiation of Wistar rats bone marrow derived mesenchymal stem cells (MSCs), which was confirmed by a higher level of alkaline phosphatase and collagen production found after 7 days of culture. These results indicate that the surface modification method in the present study can be used in the development of bioactive and biomimetic scaffolds with potential application in bone tissue engineering and regenerative medicineDoutoradoMateriais e Processos de FabricaçãoDoutora em Engenharia Mecânica2012/22593-0FAPES

Collagen imobilization on poly (L-co-D,L lactic acid)

Orientador: Eliana Aparecida de Rezende DuekDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia MecânicaResumo: Polímeros biorreabsorvíveis são amplamente empregados como arcabouços na engenharia tecidual. No entanto, devido à natureza hidrofóbica, técnicas de modificação de superfície embasadas na enxertia de grupos químicos funcionais e imobilização de moléculas bioativas são desenvolvidas no intuito de promover a biofuncionalização da superfície desses polímeros, ocasionando uma melhora significativa da interação célula /polímero. Este estudo teve por objetivo desenvolver e caracterizar uma superfície biomimética em arcabouços do copolímero poli (L-co-D,L ácido lático)- PLDLA, através da enxertia de grupos (-COOH) e imobilização de colágeno tipo I. Os arcabouços de PLDLA 70/30 foram obtidos pela técnica de lixiviação de porógenos e submetidos ao método de fotopolimerização oxidativa da superfície do material, seguido da copolimerização enxertiva do ácido acrílico (PLDLA-AAc) e imobilização do colágeno tipo I (PLDLA-Col). A superfície das amostras foi caracterizada por Microscopia Eletrônica de Varredura (MEV), Microscopia de Força Atômica (MFA), Espectroscopia de Reflectância Total Atenuada (FTIR-ATR), Espectroscopia de Fotoelétrons Excitados por raios-X (XPS) e ângulo de contato. As imagens da MFA e fotomicrografias da MEV demonstraram a formação de poros e rugosidade na superfície dos arcabouços de PLDLA-AAc e, deposição do colágeno com formação de uma estrutura fibrilar em pontos inespecíficos da superfície dos arcabouços do PLDLA-Col, resultando no aumento da rugosidade superficial de 149.5nm (PLDLA puro) para 295nm (PLDLA-Col). Os espectros de FTIR-ATR do PLDLA, PLDLA-AAc e PLDLA-Col confirmaram a presença dos mesmos picos de absorção para todos os tratamentos e, a presença dos picos em 1662 e 1559cm-1, típicos de amida I e II nas amostras de PLDLA-Col. A citocompatibilidade dos arcabouços foi avaliada através do cultivo de células osteoblásticas primárias submetidas aos ensaios de citotoxicidade e adesão celular inicial, síntese de colágeno e observação da morfologia celular, obtidas pela MEV. Os resultados obtidos mostraram que a superfície biomimética dos arcabouços de PLDLA-Col melhora, significantemente, a taxa de adesão e proliferação celular bem como estimula a síntese de colágeno (p<0,01). Estes resultados são indicativos de que o método de modificação de superfície utilizado no presente estudo pode ser usado no desenvolvimento de arcabouços bioativos e biomiméticos com potencial aplicação na engenharia tecidualAbstract: Bioreabsorbable polymers are widely used as scaffolds in tissue engineering. However, due to their hydrophobic nature, modification techniques based on graft of chemical functional groups and immobilization of bioactive molecules have been developed, in order to promote biofuncionalization of material surface, causing better interaction between cell / polymer. This study aimed at developing and characterizing a biomimetic surface on scaffolds of the copolymer poly (L-co-D, L lactic acid) (PLDLA 70/30), by grafting of chemical functional groups and immobilized with collagen type I. The scaffolds were obtained by porogen leaching, and submitted to photo-oxidation method of the material surface followed by grafting polymer polymerization of the acrylic acid solution (PLDLA-Acc) and immobilization of the type I collagen (PLDLA-Col). The sample surfaces were characterized by Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) , Attenuated Total Reflectance Infrared Spectroscopy (ATR-FTIR), X-ray Photoelectron Spectroscopy (XPS) and contact angle. AFM images and SEM electromicrographs exhibited porous formation and roughness on the PLDLA-AAc surfaces and collagen deposition with net-like fibrillar structure in non-specific areas of the PLDLA-Col scaffolds, resulting an increase in surface roughness from 149.5 nm (PLDLA pure) to 295nm (PLDLA-Col). ATR-FTIR spectra of PLDLA, PLDLA-AAc and PLDLA-Col exhibited the same absorption peaks for all samples, as well as peaks of 1662 and 1559cm-1 , typical of amide I and II in PLDLA-Col samples. Scaffolds cytocompatibilty was evaluated by osteoblastic-like cell culture, submitted to cytotoxicity and initial cell adhesion assays, collagen synthesis and observation of cell morphology obtained by SEM. The result showed that biomimetic surface of PLDLA-Col scaffolds significantly improves adhesion and cell proliferation rates, as well as stimulates collagen synthesis (p<0,01). These results indicate that the surface modification method in the present study can be used in the development of bioactive and biomimetic scaffold with potential application in tissue engineeringMestradoMateriais e Processos de FabricaçãoMestre em Engenharia Mecânic

Avaliação e monitoramento de implantes ósseos de arcabouços de PLDLA imobilizados com colágeno, préviamente cultivados com osteoblastos

Introdução: Polímeros biorreabsorvíveis, como PLDLA, são amplamente empregados como arcabouços na engenharia tecidual óssea. Incentiva-se sua interação com macromoléculas naturais da matriz extracelular, como o colágeno, para melhor adesão e reconhecimento do material implantado, e maior neoformação óssea. Objetivo: Analisar e comparar a regeneração óssea através de implantes realizados na calota craniana de ratos. Metodologia: Utilizou-se ratos Wistar de aproximadamente 2 meses, e tempos de sacrifício de 8 e 12 semanas, divididos nos grupos: defeito vazio; PLDLA-Col sem osteoblastos; e PLDLA-Col com osteoblastos. Paralelamente, estudou-se a fluorescência PKH26 em animais de 4 semanas, para avaliar a capacidade dos arcabouços de suportar o crescimento celular in vitro, Resultados: A analise histológica dos tempos de 8 e 12 semanas demonstrou neoformação óssea acentuada e irregular nos implantes de PLDLA-Col com ou sem célula, e fechamento parcial da lesão na maioria dos casos. Além disso, observou-se crescimento ósseo sem relação com as bordas do defeito. A histomorfometria de ambos os tempos apresentou o seguinte resultado decrescente em relação a área óssea neoformada: tratamento > controle positivo > controle negativo. Conclusão: Arcabouços de PLDLA-Col, previamente ou não cultivados com osteoblastos, obtiveram resultados promissores quando comparados ao defeito vazio, confirmando sua importante utilidade na engenharia tecidual

CHARACTERIZATION AND IN VITRO

Carbon nanotubes (CNT) have been studied for biomedical applications due to their unique properties. However, pristine CNT have structural features and impurities that can cause toxicity to biological systems. In this work, we describe a method to purify multiwalled carbon nanotubes (MWCNT) by chemical modification and subsequent attachment of hydroxyl and carboxyl groups to improve dispersion and to decrease toxic effects. Nanocomposites from poly (L-lactic acid) (PLLA) and nanotubes were produced by the solvent casting method and characterized and evaluated for cytocompatibility with Vero cells. The nanocomposite interactions with Vero cells demonstrated that the cells were able to adhere and sustain proliferation and showed favorable cytocompatibility. In vitro studies also revealed an increase in fibroblast cell viability in the nanocomposites, compared with neat PLLA

In vitro and in vivo cell tracking of PKH26-labeled osteoblasts cultured on PLDLA scaffolds

Abstract The importance of monitoring in vivo interaction that occurs between cells /bio/tissue recipient in the understanding of tissue regeneration processes becomes ever greater. This study aims to monitor and evaluate the influence of scaffold implants of poly (L-co-D, L lactic acid) - PLDLA synthesized in the laboratory, previously cultured with primary osteoblastic cells heterologously stained with the fluorescent vital dye, PKH26, on the tissue regeneration process in 8 mm central critical defects of the Wistar rat calvaria. The results obtained by MTT assay and monitoring of cells stained with PKH26 dye over 14 days of culture showed that the dye was cytocompatible with osteoblastic cells and did not exert a negative influence on the growth of unstained cells. In the in vivo study, macroscopic observations made during deployment times corroborate the results in vitro, as no apparent signs of toxicity were observed in the implanted bone defect area. The use of mobile monitoring with the dye, PKH26 in vivo is an effective strategy for the understanding of cell behaviour in the presence of PLDLA polymer