Biological and microbiological interactions of Ti-35Nb-7Zr alloy and its basic elements on bone marrow stromal cells: good prospects for bone tissue engineering.

BACKGROUND: An effective biomaterial for bone replacement should have properties to avoid bacterial contamination and promote bone formation while inducing rapid cell differentiation simultaneously. Bone marrow stem cells are currently being investigated because of their known potential for differentiation in osteoblast lineage. This makes these cells a good option for stem cell-based therapy. We have aimed to analyze, in vitro, the potential of pure titanium (Ti), Ti-35Nb-7Zr alloy (A), niobium (Nb), and zirconia (Zr) to avoid the microorganisms S. aureus (S.a) and P. aeruginosa (P.a). Furthermore, our objective was to evaluate if the basic elements of Ti-35Nb-7Zr alloy have any influence on bone marrow stromal cells, the source of stem cells, and observe if these metals have properties to induce cell differentiation into osteoblasts. METHODS: Bone marrow stromal cells (BMSC) were obtained from mice femurs and cultured in osteogenic media without dexamethasone as an external source of cell differentiation. The samples were divided into Ti-35Nb-7Zr alloy (A), pure titanium (Ti), Nb (niobium), and Zr (zirconia) and were characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). After predetermined periods, cell interaction, cytotoxicity, proliferation, and cell differentiation tests were performed. For monotypic biofilm formation, standardized suspensions (106 cells/ml) with the microorganisms S. aureus (S.a) and P. aeruginosa (P.a) were cultured for 24 h on the samples and submitted to an MTT test. RESULTS: All samples presented cell proliferation, growth, and spreading. All groups presented cell viability above 70%, but the alloy (A) showed better results, with statistical differences from Nb and Zr samples. Zr expressed higher ALP activity and was statistically different from the other groups (p < 0.05). In contrast, no statistical difference was observed between the samples as regards mineralization nodules. Lower biofilm formation of S.a and P.a. was observed on the Nb samples, with statistical differences from the other samples. CONCLUSION: Our results suggest that the basic elements present in the alloy have osteoinductive characteristics, and Zr has a good influence on bone marrow stromal cell differentiation. We also believe that Nb has the best potential for reducing the formation of microbial biofilms.

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

In Vitro Cytotoxicity of a Ti-35Nb-7Zr-5Ta Alloy Doped with Different Oxygen Contents.

Cp-Ti is the most common material used for dental implants, but its elastic modulus is around five times higher than that of bone. Recently, promising alloys that add Nb, Ta, Zr and Mo to Ti have been developed. The mechanical properties of these alloys are directly related to its microstructure and the presence of interstitial elements, such as oxygen, carbon, nitrogen and hydrogen. In this study, the in vitro cytotoxicity of Ti-35Nb-7Zr-5Ta (TNZT) alloys was analyzed in the as-received condition and after being doped with several small quantities of oxygen on a cultured osteogenic cell. The cell's morphology was also examined by scanning electron microscopy (SEM). The TNZT alloy presented no cytotoxic effects on osteoblastic cells in the studied conditions.