Scaffolds of PCL combined to bioglass: synthesis, characterization and biological performance.

Biomaterials may be useful in filling lost bone portions in order to restore balance and improve bone regeneration. The objective of this study was to produce polycaprolactone (PCL) membranes combined with two types of bioglass (Sol-Gel and melt-quenched) and determine their physical and biological properties. Membranes were produced through electrospinning. This study presented three experimental groups: pure PCL membranes, PCL-Melt-Bioglass and PCL-Sol-gel-Bioglass. Membranes were characterized using Scanning Electron Microscopy, Fourier Transform Infrared Spectrophotometry (FTIR), Energy-Dispersive Spectroscopy and Zeta Potential. The following in vitro tests were performed: MTT assay, alkaline phosphatase activity, total protein content and mineralization nodules. Twenty-four male rats were used to observe biological performance through radiographic, fracture energy, histological and histomorphometric analyses. The physical and chemical analysis results showed success in manufacturing bioactive membranes which significantly enhanced cell viability and osteoblast differentiation. The new formed bone from the in vivo experiment was similar to that observed in the control group. In conclusion, the electrospinning enabled preparing PCL membranes with bioglass incorporated into the structure and onto the surface of PCL fibers. The microstructure of the PCL membranes was influenced by the bioglass production method. Both bioglasses seem to be promising biomaterials to improve bone tissue regeneration when incorporated into PCL.

Hidrogel de quitosana com nanofibras de Policaprolactona (PCL) associado a extratos fitoterápicos no metabolismo celular e ação antimicrobiana: estudo in vitro / Evaluation of the chitosan hydrogel with PCL nanofibers associated with phytotherapeutic extracts in cellular and microbiological activity: in vitro study

O objetivo neste estudo foi produzir hidrogel de quitosana (CH) com PCL e fitoterápicos para uso preventivo de úlcera de pressão. Os hidrogéis de CH foram produzidos com glicerofosfato (GP) e com xantana (X), associados ao PCL e foram caracterizados por estereomicroscopio, intumescimento, molhabilidade e MEV. Posteriormente foram submetidos ao teste de viabilidade (MTT) com fibroblastos HFF-1 e queratinócitos HaCat. O hidrogel que apresentou melhor resultado foi escolhido para continuar na pesquisa. Posteriormente, extratos de Pfaffia panculata K, Juglans regia L, Rosmarinus officinalis L, Zingiber officinale, Própolis e Hamamelis foram colocados em contato com cepas de Staphylococcus aureus (S.a) (ATCC 6538), Streptococcus pyogenes (S.p) (ATCC 19615), Staphylococcus epidermidis (S.e) (ATCC 12228), Pseudomonas aeruginosa (P.a) (ATCC 15442), Escherichia coli (E.c) (ATCC 25922) e Klebsiella Pneumoniae (K.p) (ATCC 4352) na forma planctônica nos testes de CIM e CMM. Os dois melhores extratos fitoterápicos foram avaliados quanto ao sinergismo no teste checkerboard e posteriormente associados ao hidrogel anteriormente eleito. A seguir, o comportamento da HaCat e HFF-1 com os hidrogéis foi analisado por MTT, proteína total, ELISA, genotoxicidade e formação de biofilme monotípico com suspensões padronizadas (107 cel/mL) de S.a, S.e, S.p, P.a, E.c e K.p. Na caracterização e viabilidade o hidrogel CHX PCL apresentou os melhores resultados. Os extratos selecionados após CIM, CMM e checkerboard foram gengibre (G) e própolis (P). O extrato G se destacou na CIM com inibição de K. p e P. a. Os extratos de G e P demonstraram ação microbicida para K. p e P. a e somente o extrato P obteve ação microbicida para S. a na CMM. Houve ação aditiva dos extratos associados no checkerboard para S.p e ação aditiva e sinérgica para S. e. Os grupos de hidrogéis foram compostos por: quitosana xantana (CHX), CHX própolis (CHXP), CHX gengibre (CHXG) e CHX própolis e gengibre associados (CHXPG), todos associados ao PCL. Todos os hidrogéis demonstraram viabilidade celular acima de 70% do grupo controle, permitindo metabolismo celular observado na proteína total. Houve quantificação de IL-6 maior no grupo CHX nas duas linhagens de células enquanto a quantificação de IL-10 não exibiu diferença estatística entre os grupos. Todos os hidrogéis promoveram redução acentuada de biofilme de K.p e E.c. Os grupos CHX, CHXP e CHXG reduziram biofilme de S.e. O grupo CHXG reduziu biofilme de S.p. Para S.a e P.a o grupo CHXPG foi mais eficaz reduzindo biofilme. Concluímos que os hidrogéis apresentaram resultados satisfatórios e promissores, trazendo inovação por associação de biopolímeros e associação de extratos fitoterápicos pouco estudados. Os resultados positivos justificam a continuidade dos estudos com esse biomaterial(AU)

The aim of this study was to produce chitosan hydrogel (CH) with PCL and herbal medicines for preventive use of pressure ulcers. The CH hydrogels were produced with glycerophosphate (GP) and xanthan (X), associated with PCL and were characterized by stereomicroscope, swelling, wettability and SEM. Subsequently, they were submitted to a viability test (MTT) with HFF-1 fibroblasts and HaCat keratinocytes. The hydrogel that presented the best result was chosen to continue the research. Subsequently, extracts of Pfaffia panculata K, Juglans regia L, Rosmarinus officinalis L, Zingiber officinale, Propolis and Hamamelis were placed in contact with strains of Staphylococcus aureus (Sa) (ATCC 6538), Streptococcus pyogenes (Sp) (ATCC 19615), epidermidis (Se) (ATCC 12228), Pseudomonas aeruginosa (Pa) (ATCC 15442), Escherichia coli (Ec) (ATCC 25922) and Klebsiella Pneumoniae (Kp) (ATCC 4352) in planktonic form in CIM and CMM tests. The two best herbal extracts were evaluated for synergism in the checkerboard test and subsequently associated with the previously elected hydrogel. Next, the behavior of HaCat and HFF-1 with hydrogels was analyzed by MTT, total protein, ELISA, genotoxicity and monotypic biofilm formation with standardized suspensions (107 cel / mL) of Sa, Se, Sp, Pa, Ec and Kp In the characterization and viability the CHX PCL hydrogel presented the best results. The extracts selected after MIC, CMM and checkerboard were ginger (G) and propolis (P). The G extract stood out in the MIC with inhibition of K. p and P. a. The extracts of G and P showed microbicidal action for K. p and P. a and only the extract P obtained microbicidal action for S. a in CMM. There was an additive action of the associated extracts on the checkerboard for S.p and an additive and synergistic action for S. e. The hydrogel groups were composed of: xanthan chitosan (CHX), CHX propolis (CHXP), CHX ginger (CHXG) and CHX propolis and ginger associated (CHXPG), all associated with PCL. All hydrogels demonstrated cell viability above 70% of the control group, allowing cellular metabolism observed in the total protein. There was a greater quantification of IL-6 in the CHX group in the two cell lines while the quantification of IL-10 did not show statistical difference between the groups. All hydrogels promoted a marked reduction in the biofilm of K.p and E.c. The CHX, CHXP and CHXG groups reduced S.e biofilm. The CHXG group reduced S.p. For S.a and P.a, the CHXPG group was more effective in reducing biofilm. We conclude that the hydrogels presented satisfactory and promising results, bringing innovation through association of biopolymers and association of phytotherapic extracts little studied. The positive results justify the continuity of studies with this biomaterial(AU)

Titanium alloys: in vitro biological analyzes on biofilm formation, biocompatibility, cell differentiation to induce bone formation, and immunological response.

Biological effects of titanium (Ti) alloys were analyzed on biofilms of Candida albicans, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus mutans, and Streptococcus sanguinis, as well as on osteoblast-like cells (MG63) and murine macrophages (RAW 264.7). Standard samples composed of aluminum and vanadium (Ti-6Al-4V), and sample containing niobium (Ti-35Nb) and zirconium (Ti-13Nb-13Zr) were analyzed. Monomicrobial biofilms were formed on the Ti alloys. MG63 cells were grown with the alloys and the biocompatibility (MTT), total protein (TP) level, alkaline phosphatase (ALP) activity, and mineralization nodules (MN) formation were verified. Levels of interleukins (IL-1ß and IL-17), tumor necrosis factor alpha (TNF-α), and oxide nitric (NO) were checked, from RAW 264.7 cells supernatants. Data were statically analyzed by one-way analysis of variance (ANOVA) and Tukey's test, or T-test (P ≤ 0.05). Concerning the biofilm formation, Ti-13Nb-13Zr alloy showed the best inhibitory effect on E. faecalis, P. aeruginosa, and S. aureus. And, it also acted similarly to the Ti-6Al-4V alloy on C. albicans and Streptococcus spp. Both alloys were biocompatible and similar to the Ti-6Al-4V alloy. Additionally, Ti-13Nb-13Zr alloy was more effective for cell differentiation, as observed in the assays of ALP and MN. Regarding the stimulation for release of IL-1ß and TNF-α, Ti-35Nb and Ti-13Nb-13Zr alloys inhibited similarly the synthesis of these molecules. However, both alloys stimulated the production of IL-17. Additionally, all Ti alloys showed the same effect for NO generation. Thus, Ti-13Nb-13Zr alloy was the most effective for inhibition of biofilm formation, cell differentiation, and stimulation for release of immune mediators.

Interações biológicas e microbiológicas da liga Ti-35Nb-7Zr oxidadas e não oxidadas: estudo in vitro / Biological and microbiological interactions of the Ti-35Nb-7Zr alloy it's oxidized and non-oxidized: in vitro study

O objetivo neste estudo foi avaliar in vitro a influência da liga Ti-35Nb-7Zr e de seus elementos básicos, submetidos ou não ao processo de oxidação, na atividade de osteoblastos e na formação de biofilmes monotípicos. As amostras foram confeccionadas com diferentes materiais: a) titânio puro (Ti - controle); b) liga Ti-35Nb-7Zr (L); c) Nb (nióbio); d) Zr (zircônio); e) Ti oxidado (TiO); f) liga Ti-35Nb-7Zr oxidada (LO); g) Nb oxidado (NbO); h) Zr oxidado(ZrO). Previamente ao estudo in vitro, todas as amostras foram caracterizadas por microscopia eletrônica de varredura (MEV) e espectroscopia de dispersão de energia (EDS) para observar a topografia de superfície e a presença dos elementos básicos. Células mesenquimais obtidas de fêmures de rato, após diferenciação em osteoblastos foram cultivadas sobre as amostras. Após períodos pré-determinados, foram realizados os testes de citotoxicidade, atividade de fosfatase alcalina, produção de proteína total, formação e quantificação dos nódulos de mineralização adesão e proliferação celular. Para análise da formação dos biofilmes monotípicos, suspensões padronizadas (106 céls/mL) com micro-organismos de S. aureus, S. mutans, P. aeruginosa e C. albicans foram cultivados por 24 h sobre as amostras e submetidos ao teste de MTT. Todas as amostras permitiram o espraiamento celular. O Nb e Zr exibiram uma adesão celular mais madura com prolongamentos celulares evidenciados. O Ti e a L apresentaram maior viabilidade celular sendo estatísticamente diferente dos demais (p<0,05). O ZrO apresentou menor viabilidade exibindo diferença estatística com o Ti e a Liga. O NbO expressou maior quantidade de proteína total com diferença estatística da L, Zr e LO (p<0,05) enquanto o Zr exibiu o menor resultado com diferença estatística do Ti, Nb, TiO, NbO e ZrO. Na quantificação da atividade de fosfatase alcalina o Zr apresentou o melhor resultado e foi estatísticamente diferente de todos os demais (p<0,05). O Ti demostrou menor atividade de fosfatase alcalina diferindo estatísticamente do Nb, Zr e ZrO. O Ti demonstrou o maior resultado na quantificação dos nódulos de mineralização com diferença estatística do NbO e ZrO. O ZrO exibiu menor resultado sendo semelhante estatísticamente ao Zr e ao NbO. Em todos os grupos foram observadas a proliferação celular onde a LO apresentou o maior número de células proliferadas e o ZrO a menor proliferação sendo que não houve diferença estatística. O elemento Nb, independente da oxidação obteve a menor formação de biofilme para S. aureus e P. aeruginosa com diferença estatística (p<0,05) do Zr, TiO, LO e NbO para S. aureus e Ti para P. aeruginosa. O elemento Ti, oxidado ou não, obteve menor formação de biofilme para C. albicans e S. mutans com diferença estatística de todos para ambos os microrganismos (p<0,05). O Zr apresentou maior formação de biofilme para S. aureus com diferença estatística do Ti, Liga, Nb, LO e ZrO. O ZrO demonstrou maior formação de biofilme para S. mutans com diferença estatística dos demais (<0,05). Para C. albicans a L demostrou maior aderência de biofilme diferindo estatísticamente dos demais. Concluímos que a liga Ti-35Nb-7Zr apresentou influência positiva na atividade osteoblástica nos testes in vitro bem como na avaliação microbiológica quanto a formação de biofilmes monotípicos podendo ser idicada para o uso biomédico. Sugere-se que a diminuição de quantidade de biofilme observada seja devido a ação do elemento básico Nióbio enquanto que o Zircônio pode ter auxiliado na diferenciação celular. Devido a variedade de resultados encontrados nas amostras oxidadas, mais estudos que abordem a influência desta superfície no comportamento celular de osteoblastos e microbiológicos são necessários(AU)

The objective of this study was to evaluate in vitro the influence of the Ti-35Nb- 7Zr alloy and its basic elements, whether or not subjected to the oxidation process, on the osteoblast activity and on the formation of monotypic biofilms. The samples were made with different materials: a) pure titanium (Ti - control); B) Ti-35Nb-7Zr (A) alloy; C) Nb (niobium); D) Zr (zirconium); E) Oxidized Ti (TiO); F) oxidized Ti-35Nb-7Zr (AO); G) Nb oxidized (NbO); H) Oxidized Zr (ZrO). Prior to the in vitro study, all samples were characterized by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) to observe the surface topography and the presence of the basic elements. Mesenchymal cells obtained from mouse femurs after differentiation into osteoblasts were cultured in the samples. After pre-determined periods, cytotoxicity tests, alkaline phosphatase activity, total protein production, formation and quantification of the mineralization nodules, adhesion and cell proliferation were performed. To analyze the formation of monotypic biofilms, standardized suspensions (106 cells / ml) with S. aureus, S. mutans, P. aeruginosa and C. albicans microorganisms were cultured for 24 h on the samples and submitted to the MTT test. All samples allowed for cell spreading. Nb and Zr exhibited more mature cell adhesion with evidenced cell extensions. The Ti and A presented greater cell viability being statistically different from the others (p <0.05). ZrO presented lower viability exhibiting statistical difference with Ti and A. The NbO expressed the highest amount of total protein with a statistical difference of L, Zr, and AO (p <0.05) while Zr showed the lowest result with a statistical difference of Ti, Nb, TiO, NbO, and ZrO. In the quantification of the alkaline phosphatase activity, the Zr presented the best result and was statistically different from all the others (p <0.05). Ti showed lower alkaline phosphatase activity differing statistically from Nb, Zr, and ZrO. Ti showed the highest result in the quantification of mineralization nodules with a statistical difference of NbO and ZrO. ZrO showed lower result being statistically similar to Zr and NbO. In all groups, cell proliferation was observed where AO had the highest number of proliferated cells and ZrO had the lowest proliferation, and there was no statistical difference. The Nb element, independent of oxidation, obtained the lowest biofilm formation for S. aureus and P. aeruginosa with a statistical difference (p <0.05) of Zr, TiO, AO and NbO for S. aureus and Ti for P. aeruginosa. The Ti element, oxidized or not, obtained lower biofilm formation for C. albicans and S. mutans with a statistical difference of all for both microorganisms (p <0.05). The Zr presented higher biofilm formation for S. aureus with a statistical difference of Ti, A, Nb, AO and ZrO. The ZrO showed a higher biofilm formation for S. mutans with a statistical difference of the others (p <0.05). For C. albicans an L showed a higher biofilm adhesion differing statistically from the others. We conclude that the Ti-35Nb- 7Zr alloy had a positive influence on the osteoblastic activity in the in vitro tests as well as on the microbiological evaluation regarding the formation of monotypic biofilms and could be used for biomedical use. It is suggested that the decrease in the amount of biofilm observed is due to the action of the basic element Niobium whereas Zirconium may have aided in cell differentiation. Due to the variety of results found in the oxidized samples, further studies that address the influence of this surface on osteoblast and microbiological cell behavior are required