Fibra de carbono ativada: avaliação das interações biológicas in vitro / Activated carbon fiber: evaluation of biological interactions in vitro

Devido a constante necessidade de desenvolver materiais biocompatíveis com propriedades osteocondutores e osteoindutoras, a presente tese conta com o desenvolvimento de dois estudos in vitro com fibra de carbono obtida a partir de fibra PAN têxtil, incorporada com diferentes íons de metais, na osteogênese com vistas à compreensão das necessidades da engenharia tecidual no desenvolvimento desse biomaterial com adequadas propriedades biológicas. As células foram obtidas dos fêmures de 09 ratos machos adultos (Wistar) pesando 300g, com 90 dias.Estudo 1: A partir da preparação da fibras foram obtidos corpos de prova de 4 mm de diâmetro e 2 mm de altura, dos seguintes grupos: fibra de carbono não ativada (FCNA), fibra carbono ativada (FCA) e fibra carbono ativada com prata (FCAAg). Após plaqueamento (n=5) em meio suplementado (MTS) e meio suplementado osteogênico (MTSO) foram analisados: viabilidade celular, conteúdo de proteína total (PT), atividade de fosfatase alcalina (ALP), interaçãocelular e formação de nódulos de mineralização. Foi avaliada a formação de biofilme nos corpos de prova, utilizando cepas de S. aureus, P. aeruginosa e E. coli. Na viabilidade celular, houve diferença estatística entre grupo controle celular (C) e FCA-MTS, FCAAg-MTS e FCAAg-MTSO. Em PT, não houvediferença, na ALP houve diferença entre C-MTS e as fibras, C-MTSO se mostrou semelhante. Em nódulos, houve diferença entre C-MTS e C-MTSO e as fibras do MTSO. Houve redução de formação de biofilme do S. aureus na FCAAg.Estudo 2: Foram obtidos corpos de prova da mesma dimensão do estudo 1 (n=5) dos seguintes grupos: fibra carbono ativada com prata (FCAAg), fibra carbono ativada com ouro (FCAAu), fibra carbono ativada com cobre (FCACu), fibra carbono ativada com paládio (FCAPd) e fibra carbono ativada com platina (FCAPt). Foram quantificadas a proliferação celular, viabilidade celular, formação de nódulos de mineralização, conteúdo de PT e ALP. Todas as amostras mostraram-se semelhantes quanto a proliferação celular, com exceção do grupo FCAAg comparado ao grupo controle (C). Sobre viabilidade celular, C obteve maior viabilidade que os outros grupos, e FCA obteve maior taxa que os grupos FCAAg, FCACu, FCAPt, sendo semelhante aos grupos FCAAu e FCAPd. Já os grupos FCAAu e FCAPd apresentaram diferença aos grupos FCAAg e FCACu. Na análise de expressão de PT apenas houve diferença entre FCA e FCAAu, sendo FCAAu com menor expressão de produção de PT. Na avaliação da ALP os grupos FCAAg e FACu mostraram diferença estatística e inferior com os grupos C, FCAAu, FCAPd e FCAPt, além disso, o grupo FCA mostrou menor taxa que C.Conclusões: As fibras utilizadas de base para a incorporação dos íons demonstraram grande potencial para uso como scaffold para reparação óssea, isso porque em ambos os estudos, na forma ativada e não ativada, as fibras apresentaram viabilidade celular e quantificação de cálcio satisfatórias. Sendo a versão não ativada mais econômica no que diz respeito ao tempo e custo de preparação. Mais estudos devem ser empregados a fim de assegurar sua segurança clínica em relação à citotoxicidade da incorporação de íons de ouro e paládio.(AU)

Due to the constant need to develop biocompatible materials with osteoconductive and osteoinductive properties, this thesis involves the development of two in vitro studies with carbon fiber obtained from textile PAN fiber, incorporated with different metal ions, in osteogenesis with a view to understanding the needs of tissue engineering in the development of this biomaterial with adequate biological properties. The cells were obtained from the femurs of 9 adult male rats (Wistar) weighing 300g, aged 90 days. Study 1: From the fiber preparation, specimens measuring 4 mm in diameter and 2 mm in height were obtained from the following groups: non-activated carbon fiber (FCNA), activated carbon fiber (FCA) and silver-activated carbon fiber (FCAAg). After plating (n=5) in supplemented medium (MTS) and supplemented osteogenic medium (MTSO), cell viability, total protein content (PT), alkaline phosphatase (ALP) activity, cell interaction and formation of mineralization nodules were analyzed. . Biofilm formation was evaluated in the specimens, using strains of S. aureus, P. aeruginosa and E. coli. In cell viability, there was a statistical difference between the cell control group (C) and FCAMTS, FCAAg-MTS and FCAAg-MTSO. In PT, there was no difference, in ALP there was a difference between C-MTS and fibers, C-MTSO was similar. In nodules, there was a difference between C-MTS and C-MTSO and MTSO fibers. There was a reduction in S. aureus biofilm formation on FCAAg. Study 2: Specimens of the same size as in study 1 (n=5) were obtained from the following groups: carbon fiber activated with silver (FCAAg), carbon fiber activated with gold (FCAAu), carbon fiber activated with copper (FCACu), palladium-activated carbon fiber (FCAPd) and platinum-activated carbon fiber (FCAPt). Cell proliferation, cell viability, formation of mineralization nodules, PT and ALP content were quantified. All samples were similar in terms of cell proliferation, with the exception of the FCAAg group compared to the control group (C). Regarding cell viability, C obtained higher viability than the other groups, and FCA obtained a higher rate than the FCAAg, FCACu, FCAPt groups, being similar to the FCAAu and FCAPd groups. The FCAAu and FCAPd groups showed differences to the FCAAg and FCACu groups. In the analysis of PT expression, there was only a difference between FCA and FCAAu, with FCAAu having lower expression of PT production. In the ALP assessment, the FCAAg and FACu groups showed a lower statistical difference compared to the C, FCAAu, FCAPd and FCAPt groups, in addition, the FCA group showed a lower rate than C. Conclusions: The fibers used as the basis for the incorporation of ions demonstrated great potential for use as a scaffold for bone repair, because in both studies, in activated and non-activated form, the fibers showed satisfactory cell viability and calcium quantification. The non-activated version is moreeconomical in terms of preparation time and cost. More studies must be carried out to ensure its clinical safety in relation to the cytotoxicity of the incorporation of gold and palladium ions. (AU)

Bone repair assessment of critical size defects in rats treated with mineralized bovine bone (Bio-Oss®) and photobiomodulation therapy: a histomorphometric and immunohistochemical study.

This study aimed to investigate the effects of administering photobiomodulation therapy (PBM) with bovine bone matrix on critical size defects in rats. Seventy-two adult male rats (albinus, Wistar), 90 days old, were used. Defect of 5 mm in diameter was made in their calvaria. The animals were divided into 4 groups: C-blood clot, B-Bio-Oss®, L-PBM, B+L-Bio-Oss®+PBM. Each group has been subdivided into 07, 30, and 60 days of observation. For PBM, a low GaAlAs energy of 660 nm was irradiated, total energy density of 45 J/cm2 . PBM was conducted in a trans-surgical form once only. For immunohistochemistry, a semi-quantitative analysis was made of expression of osteoprotegerin (OPG), nuclear kappa B-factor ligand receptor activator (RANKL), and tartrate-resistant acid phosphatase (TRAP). All histomorphometric data were statistically analyzed by ANOVA and Tukey test, significance level of 5%. The groups that showed the highest proportion of neoformation were L (0.39% ± 0.13) and C (0.37% ± 0.97), but groups B and B+L had larger defect size (C-1.75 mm2 ± 0.40, B-3.02 mm2 ± 0.63, L-2.45 mm2 ± 0.53, B+L-3.23 mm2 ± 1.01). In immunohistochemistry, groups B and B+L had higher immunostaining scores for OPG and RANKL at 60 days, and TRAP immunostaining increased in all groups at 30 days, but group L was the only one to present specimens with score 0. Although, at 60 days, groups L and C presented the highest proportion of bone neoformation, at 30 days group B+L had more than twice as much bone neoformation as group B, the choice of treatment application should depend on the aim of the treatment.

The Effect of Photobiomodulation Therapy in Different Doses on Bone Repair of Critical Size Defects in Rats: A Histomorphometric Study.

Introduction: Photobiomodulation therapy (PBM) appears to induce osteogenesis and stimulate fracture repair; because of its capacity, it is considered a promising treatment, but the characteristics of response to different radiation doses must be investigated through in vivo studies to establish their safety and effectiveness. Thus, this paper aims to analyze the effects of the PBM at different doses on the repair of critical bone defects through histological and histomorphometric analyses. Methods: Sixty 90-day-old adult rats (Rattus norvegicus, albinus, Wistar) weighing approximately 300 g were used. Critical bone defects of 5 mm in diameter were performed in their calvaria. The animals were randomly separated into 5 groups: C-Blood clot, L15-PBM 15J/cm2, L30-PBM 30 J/ cm2, L45-PBM 45 J/cm2, L60-PBM 60 J/cm2. Each group was subdivided according to observation periods of 30 and 60 days with 6 rats in each subgroup. Low-level gallium aluminum arsenide (GaAlAs) lasers were used at a 660 nm wavelength, 30 mW and 0.04 cm2 in area. The PBM was applied over 5 points; 4 points of application were distributed on the edges while one point of application was located in the center of the bone defect. PBM occurred right after the procedure. In 30 and 60 days, the animals were euthanized by anesthesia overdose and the analyses were performed. The data were analyzed statistically by the ANOVA, together with the Tukey test, whose significance level was 5%. Results: As regards the treatment factor, the highest percentage of bone neoformation was achieved by group L45-60. The group with the highest closure, despite not having a statistically significant difference with the other doses, was 45 J with only 0.49 mm between edges. Conclusion: Thus, the present study allowed concluding that the highest percentage of bone neoformation area was achieved at 45 J/cm2 in 60 days; that is, it was significantly effective in comparison with other doses.