A estrutura de um biomaterial para regeneração óssea é fator chave para seu sucesso clínico. Não existe um único biomaterial usado para reparo e regeneração óssea capaz de preencher todos os requisitos para um enxerto ósseo ideal. Assim, optou-se por utilizar neste estudo dois biomateriais largamente utilizados na engenharia de tecido ósseo, a policaprolactona (PCL) e cerâmica bifásica (BCP). Neste estudo, compósitos porosos tridimensionais de PCL:BCP (3:1 e 1:1 p:p) foram fabricados utilizando a combinação das técnicas de evaporação do solvente e lavagem de partículas de sal com ou sem a adição de Angiotensina (Ang)-(1-7). Os compósitos de PCL:BCP com alta porosidade e interconectividade obtidos foram analisados usando microtomografia computadorizada (µ-CT). A viabilidade dos compósitos com e sem a adição da Ang-(1-7) foram analisados utilizando osteoblastos de cultura primária nos períodos de 3 e 7 dias utilizando o teste de MTT e secreção de Fosfatase Alcalina. Diferenças significativas de todas as variáveis foram testadas através de análises multivariáveis (p<0,05). Demonstrou-se uma estrutura mais uniformemente distribuída quando a razão PCL/BCP foi aumentada. A conectividade dos poros foi mensurada através do índice de fragmentação (FI) demonstrando estruturas desconexas nas amostras analisadas possuindo valores de FI próximos. Quanto maior a razão PCL/BCP, maior a dimensão fractal (FD). Análises in vitro utilizando o teste do MTT demonstraram que as amostras contendo ou não Ang-(1-7) possuíram efeitos citotóxico médio e moderado, respectivamente. A análise da atividade de fosfatase alcalina não monstrou diferença entre as amostras no período de 3 dias decrescendo sua atividade nas amostras contendo Ang-(1-7) no período de 7 dias. Dessa forma, utilizando a combinação das técnicas de evaporação do solvente e lavagem do sal, obtiveram-se estruturas porosas tridimensionais, desejável para defeitos ósseos tendo capacidade para sistema de transporte de drogas podendo ser avaliada em futuros testes in vivo.

Highly porous three-dimensional biodegradable scaffolds was obtained from beta-tricalcium phosphate-hydroxyapatite bioceramic (BCP), PCL, and Angiotensin-(1-7). We used the solvent casting and particulate leaching methods (SC/PL). The processed scaffolds were characterized by X-ray microtomography (µ-CT). Biocompatibility tests in vitro were performed during three and seven days using MTT and Alkaline Phosphatase Activity (APA) assays. Both the MTT activity and APA were evaluated using a one-way ANOVA test. The µ-CT results showed that the increase of the PCL:BCP weight ratio leads to structures with lower pore sizes. The pore interconnectivity of the processed scaffolds was evaluated in terms of the fragmentation index (FI). We observed that the obtained composites present poorly connected structures, with close values of FI. However, as the polymer phase is almost transparent to the X-rays, it was not taken into consideration in the µ-CT tests. The MTT activity assay revealed that scaffolds obtained with and without Angiotensin-(1-7) present mild and moderate cytotoxic effects, respectively. The APA assay showed that the rat osteoblasts, when in contact for three days with the PCL composites, presented an APA similar to that observed for the control cells. Nevertheless, for an incubation time of seven days we observed a remarkable decrease in the alkaline phosphatase activity. In conclusion, using the solvent casting and salt leaching method we obtained 3D porous that are composites of PCL, BC and Ang-(1-7), which have suitable shapes for the bone defects, a high porosity and interconnect pores. Furthermore, the viability in vitro showed that the scaffolds have potential for drug delivery system and could be used in future in vivo tests.

Chlorhexidine (Cx) augmented with beta-cyclodextrin (β-cd) inclusion compounds, termed Cx:β-cd complexes, have been developed for use as antiseptic agents. The aim of this study was to examine the interactions of Cx:β-cd complexes, prepared at different molecular ratios, with sterol and yeast membranes. The Minimal Inhibitory Concentration (MIC) against the yeast Candida albicans (C.a.) was determined for each complex; the MICs were found to range from 0.5 to 2 µg/mL. To confirm the MIC data, quantitative analysis of viable cells was performed using trypan blue staining. Mechanistic characterization of the interactions that the Cx:β-cd complexes have with the yeast membrane and assessment of membrane morphology following exposure to Cx:β-cd complexes were performed using Sterol Quantification Method analysis (SQM) and scanning electron microscopy (SEM). SQM revealed that sterol extraction increased with increasing β-cd concentrations (1.71 × 10³; 1.4 × 10³; 3.45 × 10³, and 3.74 × 10³ CFU for 1:1, 1:2, 1:3, and 1:4, respectively), likely as a consequence of membrane ergosterol solubilization. SEM images demonstrated that cell membrane damage is a visible and significant mechanism that contributes to the antimicrobial effects of Cx:β-cd complexes. Cell disorganization increased significantly as the proportion of β-cyclodextrin present in the complex increased. Morphology of cells exposed to complexes with 1:3 and 1:4 molar ratios of Cx:β-cd were observed to have large aggregates mixed with yeast remains, representing more membrane disruption than that observed in cells treated with Cx alone. In conclusion, nanoaggregates of Cx:β-cd complexes block yeast growth via ergosterol extraction, permeabilizing the membrane by creating cluster-like structures within the cell membrane, possibly due to high amounts of hydrogen bonding.