Cultura de células osteogênicas primárias a partir de osso de baixa densidade e análise do reparo ósseo periimplantar em ratas osteoporóticas em função da texturização de superfície por meio da oxidação por plasma eletrolítico
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Data
2019-01-25
Autores
Silva, William Phillip Pereira da [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O objetivo deste estudo foi avaliar um novo método de texturização por PEO com incorporação de Ca e P na superfície do Ti-6Al-4V em ossos de baixa densidade, por meio de avaliação in vitro, ex-in vivo e in vivo, em função de parâmetros topográficos e reparacionais. 57 ratas Wistar (Rattus novergicus), sendo 38 ratas com 6 meses de idade (Grupos OXV - submetidas à ovariectomia e SHAM - cirurgia fictícia) e 19 ratas senis (18 meses de idade: Grupo SENIL), foram divididas para realização do estudo ex-in vivo (n=9) e in vivo (n=48). Os grupos para análise ex-in vivo foram submetidos à eutanásia e os fêmures foram removidos e transportados em meio de cultura contendo meio essencial mínimo modificação alfa (α- MEM) suplementado com 500 µg/mL de gentamicina e 3 µg/mL de fungisona. As células-tronco mesenquimais de medula óssea (CTMs-MO) dos fêmures, foram isoladas e cultivadas em meio de crescimento para manterem-se como CTMs. Após alcançar a subconfluência, as células foram cultivadas em 3 superfícies de discos de Ti-6Al-4V, grupo CONTROLE (superfície usinada) grupo AC (superfície tratada por Ataque Ácido e Jateamento) e grupo PEO (superfície tratada por Oxidação de Plasma Eletrolítico com associação de Cálcio e Fosforo). Para avaliação das respostas celulares foram realizados ensaios de viabilidade celular, expressão gênica de marcadores osteoblásticos, imunolocalização de sialoproteina óssea (BSP) e osteopontina (OPN), atividade da fosfatase alcalina (ALP) e formação de matriz mineralizada. Os dados foram submetidos ao teste ANOVA 1 fator ou Kruskal-Wallis (p < 0,05). Nos experimentos in vivo, após 90 dias, foi instalado um implante em cada tíbia, sendo um implante pertencente ao grupo PEO e o outro implante do grupo AC. Após 42 dias da instalação dos implantes, 8 animais de cada grupo foram submetidos à eutanásia e suas tíbias passaram pela descalcificação, para a análise histológica e imunoistoquímica (OPG, RANKL, OC e TRAP). As demais ratas, após a eutanásia, tiveram suas tíbias coletadas e analisadas em microtomografia computadorizada (BV.TV, Tb.Th, Tb.N, Tb.Sp e Po(tot)) e, em seguida os implantes submetidos ao torque reverso em torquímetro digital (N.cm). A outra metade das tíbias foram processadas com inclusão em resina fotopolimerizadas para cortes calcificados e assim, a análise por microscopia confocal (calceina e alizarina) e em seguida, análise histométrica. Os dados foram submetidos ao teste ANOVA 1 fator ou Kruskal-Wallis, seguido de pós teste Tukey; p < 0,05). A análise da viabilidade celular mostrou que em todos os grupos testes de CTMs-MO para SHAM, OVX e SENIL apresentaram um crescimento progressivo nos diferentes tempos de 3, 7 e 10 dias. Avaliação da expressão gênica através dos genes Runx2, SP7/Osterix, ALP, BSP, OC e OPN e analise pela imunofluorecência apresentaram uma leve tendência de melhores respostas nas CTMs-MO SHAM para o grupo AC, CTMs-MO OVX para o grupo PEO e CTMs-MO SENIS características semelhantes nos grupos AC e PEO. A atividade da fosfatase alcalina ocorreu maior expressão na superfície PEO no grupo SHAM, maior expressão na superfície CONTROLE no grupo OVX e no grupo SENIL houve um equilíbrio em todas as superfícies. A superfície PEO apresentou maior formação de nódulos de mineralização (21º dia) em todos os grupos. Nos experimentos in vivo, as análises histológicas mostrou maior neoformação óssea no grupo PEO quando comparado ao grupo AC nos grupos SHAM e OVX, e no grupo SENIL foram similares os resultados. A avaliação imunoistoquímica demonstrou um equilíbrio em todos os grupos na comparação de superfícies na proteína TRAP, para o processo de remodelação (OPG e RANKL) e mineralização (OC) nos grupos SHAM e SENIL (p>0,05), ocorrendo uma diminuição no grupo OVX, no qual os resultados do PEO foram mais favoráveis que AC nessa conformação. Na análise microtomográfica, BV.TV os resultados foram semelhantes em ambos os grupos, porém em OVX AC mostrou menor porcentagem de volume ósseo e uma maior porosidade (Po(tot)). A análise biomecânica por torque-reverso (N.cm) mostrou que os maiores valores pertenciam ao grupo PEO. A dinâmica do tecido ósseo representada pelo turnover ósseo, observado através dos fluorocromos (Calceína e Alizarina) mostrou-se similares nos grupos experimentais. As superficies PEO E AC nesse estudo demonstram que possuem uma grande capacidade de promoção da formação óssea independente dos tipos ósseos experimentais (SHAM, OVX e SENIL), tanto na área de contato osso e implante (ELCOI), quanto para a área de osso neoformado (AON). Diante das limitações do estudo in vitro e in vivo, os resultados foram esclarecedores para acreditar que o método de texturização aqui testado, por meio da Oxidação por Plasma Eletrolítico (PEO), favoreceu à formação óssea, principalmente nos ossos mais críticos (OVX), inclusive evidenciando maior maturação óssea nos períodos mais tardios aqui analisados.
The objective of this study was to evaluate a new PEO texturing method with Ca and P incorporation on the Ti-6Al-4V surface in low bone density, by means of in vitro, ex vivo and in vivo evaluation through topographic and repairment parameters. 57 Wistar rats (Rattus novergicus), being 38 at 6 months of age (OXV Groups - submitted to ovariectomy and SHAM surgery) and 19 senile rats (18 months of age: SENIL Group) were divided into three subgroups: ex-in vivo (n = 9) and in vivo (n = 48). The Groups for ex-in vivo analysis were euthanized and femurs were removed and transported in culture medium containing minimal alpha modification (α- MEM) medium supplemented with 500 μg / ml gentamicin and 3 μg / ml fungizone. The mesenchymal stem cells from bone marrow (MSC-M) of the femur were isolated and cultured in growth medium to remain as MSCs. After reaching the subconfluence, the cells were grown on 3 surfaces of Ti-6Al-4V discs, CONTROL group (machined surface) group AC (surface treated by etched-acid) and PEO group (surface treated by Electrolytic Plasma Oxidation with the association of Calcium and Phosphorus). Cell viability assays, gene expression of osteoblastic markers, bone sialoprotein (BSP) and osteopontin (OPN), alkaline phosphatase (ALP) activity, and mineralized matrix formation were performed to evaluate cellular responses. Data were submitted to ANOVA 1 factor test or Kruskal-Wallis test (P <0.05). In the groups for the in vivo study, after 90 days, an implant was installed on each tibia, one implant to the PEO group and another implant of the AC group. After 42 days of implant implantation, eight animals from each group underwent euthanasia and their tibiae underwent decalcification for histological and immunohistochemical analysis (OPG, RANKL, OC, and TRAP). The other rats, after euthanasia, had their tibiae collected and analyzed in computerized microtomography (BV.TV, Tb.Th, Tb.N, Tb.Sp and Po (tot)), and then implants submitted to reverse torque in Digital torque wrench (N.cm). Another half of the tibiae were processed with inclusion in photopolymerized resin for calcified cuts and thus the analysis by confocal microscopy (calcein and alizarin) and then histometric analysis. Data were submitted to 1-factor ANOVA or Kruskal-Wallis test, followed by Tukey test; p <0.05). The cell viability analysis showed that in all groups, MOH tests for SHAM, OVX, and SENIL showed a progressive growth in the different times of 3, 7 and 10 days. Evaluation of gene expression through the Runx2, SP7 / Osterix, ALP, BSP, OC and OPN genes and immunofluorescence analysis showed a slight tendency for better responses in the CTMs-MO SHAM for the AC group, CTMs-MO OVX for the PEO group and CTMs-MO SENIS features similar in AC and PEO groups. The alkaline phosphatase activity was higher on the PEO surface in the SHAM group, the greater expression on the CONTROL surface in the OVX group and the SENIL group showed a balance on all surfaces. The PEO surface presented a higher formation of mineralization nodules in all groups (21st day). For the in vivo analyzes, the histological analysis showed greater bone neoformation in the PEO group when compared to the AC group in the SHAM and OVX groups, and in the SENIL group, the results were similar. The immunohistochemical evaluation showed a balance in all groups in the comparison of surfaces in the TRAP protein, for the remodeling process (OPG and RANKL) and mineralization (OC) in the SHAM and SENIL groups (p> 0.05). OVX group, in which PEO results were more favorable than AC in this confirmation. In the microtomographic analysis, BV.TV the results were similar in both groups, but in OVX AC showed a lower percentage of bone volume and a higher porosity (Po (tot)). Biomechanical analysis by torque-reverse (N.cm) showed that the highest values belonged to the PEO group. The dynamics of the bone tissue represented by the bone turnover observed through the fluorochromes (Calcein and Alizarin) were similar in the experimental groups. The PEO and AC surfaces in this study demonstrate that they have a great ability to promote bone formation independent of experimental bone types (SHAM, OVX, and SENIL), both in the area of bone and implant contact (ELCOI) and in the area of newly formed bone (AON). Considering the limitations of the in vitro and in vivo study, the results were enlightening to believe that the texturing method tested here, through Electrolytic Plasma Oxidation (PEO), favored bone formation, mainly in the most critical bones (OVX), including evidence of increased bone maturation in the later periods analyzed here.
The objective of this study was to evaluate a new PEO texturing method with Ca and P incorporation on the Ti-6Al-4V surface in low bone density, by means of in vitro, ex vivo and in vivo evaluation through topographic and repairment parameters. 57 Wistar rats (Rattus novergicus), being 38 at 6 months of age (OXV Groups - submitted to ovariectomy and SHAM surgery) and 19 senile rats (18 months of age: SENIL Group) were divided into three subgroups: ex-in vivo (n = 9) and in vivo (n = 48). The Groups for ex-in vivo analysis were euthanized and femurs were removed and transported in culture medium containing minimal alpha modification (α- MEM) medium supplemented with 500 μg / ml gentamicin and 3 μg / ml fungizone. The mesenchymal stem cells from bone marrow (MSC-M) of the femur were isolated and cultured in growth medium to remain as MSCs. After reaching the subconfluence, the cells were grown on 3 surfaces of Ti-6Al-4V discs, CONTROL group (machined surface) group AC (surface treated by etched-acid) and PEO group (surface treated by Electrolytic Plasma Oxidation with the association of Calcium and Phosphorus). Cell viability assays, gene expression of osteoblastic markers, bone sialoprotein (BSP) and osteopontin (OPN), alkaline phosphatase (ALP) activity, and mineralized matrix formation were performed to evaluate cellular responses. Data were submitted to ANOVA 1 factor test or Kruskal-Wallis test (P <0.05). In the groups for the in vivo study, after 90 days, an implant was installed on each tibia, one implant to the PEO group and another implant of the AC group. After 42 days of implant implantation, eight animals from each group underwent euthanasia and their tibiae underwent decalcification for histological and immunohistochemical analysis (OPG, RANKL, OC, and TRAP). The other rats, after euthanasia, had their tibiae collected and analyzed in computerized microtomography (BV.TV, Tb.Th, Tb.N, Tb.Sp and Po (tot)), and then implants submitted to reverse torque in Digital torque wrench (N.cm). Another half of the tibiae were processed with inclusion in photopolymerized resin for calcified cuts and thus the analysis by confocal microscopy (calcein and alizarin) and then histometric analysis. Data were submitted to 1-factor ANOVA or Kruskal-Wallis test, followed by Tukey test; p <0.05). The cell viability analysis showed that in all groups, MOH tests for SHAM, OVX, and SENIL showed a progressive growth in the different times of 3, 7 and 10 days. Evaluation of gene expression through the Runx2, SP7 / Osterix, ALP, BSP, OC and OPN genes and immunofluorescence analysis showed a slight tendency for better responses in the CTMs-MO SHAM for the AC group, CTMs-MO OVX for the PEO group and CTMs-MO SENIS features similar in AC and PEO groups. The alkaline phosphatase activity was higher on the PEO surface in the SHAM group, the greater expression on the CONTROL surface in the OVX group and the SENIL group showed a balance on all surfaces. The PEO surface presented a higher formation of mineralization nodules in all groups (21st day). For the in vivo analyzes, the histological analysis showed greater bone neoformation in the PEO group when compared to the AC group in the SHAM and OVX groups, and in the SENIL group, the results were similar. The immunohistochemical evaluation showed a balance in all groups in the comparison of surfaces in the TRAP protein, for the remodeling process (OPG and RANKL) and mineralization (OC) in the SHAM and SENIL groups (p> 0.05). OVX group, in which PEO results were more favorable than AC in this confirmation. In the microtomographic analysis, BV.TV the results were similar in both groups, but in OVX AC showed a lower percentage of bone volume and a higher porosity (Po (tot)). Biomechanical analysis by torque-reverse (N.cm) showed that the highest values belonged to the PEO group. The dynamics of the bone tissue represented by the bone turnover observed through the fluorochromes (Calcein and Alizarin) were similar in the experimental groups. The PEO and AC surfaces in this study demonstrate that they have a great ability to promote bone formation independent of experimental bone types (SHAM, OVX, and SENIL), both in the area of bone and implant contact (ELCOI) and in the area of newly formed bone (AON). Considering the limitations of the in vitro and in vivo study, the results were enlightening to believe that the texturing method tested here, through Electrolytic Plasma Oxidation (PEO), favored bone formation, mainly in the most critical bones (OVX), including evidence of increased bone maturation in the later periods analyzed here.
Descrição
Palavras-chave
Oxidação, Osteoporose, Cultura primária de células, Células mesenquimais estromais, Implantes dentários, Oxidation, Osteoporosis, Primary cell culture, Stromal mesenchymal cells, Dental implants