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.

Expression of BMP II by human osteoblasts cultivated on dense or porous titanium / Expressão da BMP II por osteoblastos humanos cultivados sobre titânio denso ou poroso

Objective: Modifications of titanium have been described as an important tool improving bone repair and boneimplant contact. The aim of this research was to quantified the expression of the morphogenetic bone protein II (BMP II) produced by human cells with osteoblast differentiation, after cultured over dense or porous samples of pure titanium grade II. Material and Methods: The experimental groups were: control group, dense titanium, porosity of 33.79% and porosity of 41,79% (n=36). The samples were produced by powder metallurgy technique. Mesenquimal steam cells isolated from alveolar bone of healthy donors were stimulated to differentiate, assuming an osteoblastic phenotype, by supplemented medium and plated over the samples. After 7 and 14 days, the RNA was collected to perform reverse transcriptase polymerase chain reaction (RT-PCR) in real time. Data was analysed by t-Student and ANOVA tests. The porosity, the pore morphology and interconnection were evaluated by Scanning Electron Microscopy (SEM). Results: Total porosity (obtained after apply dimensions and density formulas) and surface porosity (SEM) presented significant differencesamong the groups. For the group of total porosity of 33.79%, the superficial porosity was 32.5% (± 7.74%) and for the group of 41.79%, the superficial porosity was 37.4% (± 7.95%), significantly lower. The expression of BMP II was similar in all groups. Conclusion: The present study demonstrated that powder metallurgy has a reduced ability to standardize the porosity in the samples and that the porosity does not interfere in the cellular response of BMP II production, an important inducer of osteoblastic differentiation. (AU)

Objetivo: As modificações do titânio são descritas como importantes ferramentas na melhora do reparo ósseo no contato osso implante. O objetivo deste estudo foi quantificar a expressão da proteína óssea morfogênica II (BMP II) por células humanas com diferenciação osteoblastica, quando cultivadas sobre amostras de titânio puro grau II, denso ou poroso. Material e Métodos: Os grupos experimentais foram: controle, titânio denso, titânio de maior porosidade e titânio de menor porosidade, sendo que, as amostras foram confeccionadas pela técnica da metalurgia do pó. As células isoladas de doadores saudáveis foram plaqueadas sobre as amostras. Após 7 e 14 dias, o RNA foi extraído das células. A qualidade e integridade do RNA foram analisadas qualitativamente por eletroforese e quantitativamente por espectrofotômetro. O cDNA foi confeccionado e a foi utilizada técnica de reação em cadeia da polimerase (PCR) em tempo real. Os dados foram utilizados para quantificação relativa, e o gene constitutivo foi a BetaActina. A morfologia e a interligação dos poros foram comprovadas por Microscopia Eletrônica de Varredura (MEV). Resultados: A porosidade superficial (MEV) teve diferença significativa em relação a porosidade obtida analisando-se volume e massa das amostras. Para o grupo 3,79%, a superficial foi de 32,5% (±7,74%) e para o grupo 41,79% a porosidade superficial foi de 37,4% (±7,95%), significativamente menor. A expressão da BMP II foi semelhante em todos os grupos. Conclusão: Concluiu-se a metalurgia do pó tem reduzida capacidade de padronização da porosidade das amostras por ela confeccionas e que a porosidade não interfere na resposta celular de produção da BMP II, importante indutor de diferenciação osteoblastica.(AU)

Titanium scaffold osteogenesis in healthy and osteoporotic rats is improved by the use of low-level laser therapy (GaAlAs).

The present study aimed to assess the effects of low-level laser therapy (GaAlAs) on the bone repair process within titanium scaffolds in the femurs of healthy and osteoporotic rats. Fifty-six rats were divided into four groups: group Sh: SHAM animals that received scaffolds; group LSh: SHAM animals that received scaffolds and were subjected to laser therapy; group OV: ovarietomized (OVX) animals that received scaffolds; and group LOV: OVX animals that received scaffolds and were subjected to laser therapy. Thirty days following ovariectomy or sham surgery, scaffolds were implanted in the left femurs of all animals in the study. Immediately after opening the surgical site, the inner part of the surgical cavity was stimulated with low-level laser (GaAlAs). In addition to this procedure, the laser group was also subjected to sessions of low-level laser therapy (LLLT) at 48-h intervals, with the first session performed immediately after surgery. The rats were sacrificed at 2 and 6 weeks, time in which femur fragments were submitted for histological and histomorphometric examination, and skin tissue above the scaffold was submitted to histological analysis. At the end of the study, greater bone formation was observed in the animals submitted to LLLT. At 2 and 6 weeks, statistically significant differences were observed between LSh and Sh groups (p = 0.009 and 0.0001) and LOV and OV (p = 0.0001 and 0.0001), respectively. No statistical difference was observed when assessing the estrogen variable. On the basis of our methodology and results, we conclude that LLLT improves and accelerates bone repair within titanium scaffolds in both ovariectomized and healthy rats, when compared to animals not subjected to radiation.

Healing of normal and osteopenic bone with titanium implant and low-level laser therapy (GaAlAs): a histomorphometric study in rats.

The study investigates the influence of low-level laser therapy (LLLT) on bone healing in the femur of osteopenic and normal rats with titanium implants. Ovariectomy and control group were randomly submitted to LLLT, which was applied by gallium-aluminum-arsenium (GaAlAs) laser at the surgical site before and after placing the implant, for seven times. Histomorphometric and statistical analysis were performed. Most irradiated groups showed higher values than the nonirradiated groups. The GaAlAs infrared diode laser may improve the osseointegration process in osteopenic and normal bone, particularly based on its effects in the initial phase of bone formation.

Evaluation of bone ingrowth into porous titanium implant: histomorphometric analysis in rabbits.

A porous material for bone ingrowth with adequate pore structure and appropriate mechanical properties has long been sought as the ideal bone-implant interface. This study aimed to assess in vivo the influence of three types of porous titanium implant on the new bone ingrowth. The implants were produced by means of a powder metallurgy technique with different porosities and pore sizes: Group 1 = 30 % and 180 µm; Group 2 = 30% and 300 µm; and Group 3 = 40% and 180 µm;. Six rabbits received one implant of each type in the right and left tibiae and were sacrificed 8 weeks after surgery for histological and histomorphometric analyses. Histological analysis confirmed new bone in contact with the implant, formed in direction of pores. Histomorphometric evaluation demonstrated that the new bone formation was statistically significantly lower in the group G1 than in group G3, (P = 0.023). Based on these results, increased porosity and pore size were concluded to have a positive effect on the amount of bone ingrowth.

Evaluation of bone ingrowth into porous titanium implant: histomorphometric analysis in rabbits

A porous material for bone ingrowth with adequate pore structure and appropriate mechanical properties has long been sought as the ideal bone-implant interface. This study aimed to assess in vivo the influence of three types of porous titanium implant on the new bone ingrowth. The implants were produced by means of a powder metallurgy technique with different porosities and pore sizes: Group 1 = 30 percent and 180 µm; Group 2 = 30 percent and 300 µm; and Group 3 = 40 percent and 180 µm;. Six rabbits received one implant of each type in the right and left tibiae and were sacrificed 8 weeks after surgery for histological and histomorphometric analyses. Histological analysis confirmed new bone in contact with the implant, formed in direction of pores. Histomorphometric evaluation demonstrated that the new bone formation was statistically significantly lower in the group G1 than in group G3, (P = 0.023). Based on these results, increased porosity and pore size were concluded to have a positive effect on the amount of bone ingrowth.

Porous titanium for biomedical applications: an experimental study on rabbits.

OBJECTIVE: The aim of this study was to carry out an in vivo assessment of bone ingrowth in two different types of porous titanium -the first being completely porous, and the second with a porous surface and dense nucleus, manufactured by powder metallurgy- and to evaluate their mechanical properties. STUDY DESIGN: Ten scaffolds from each group were submitted to metallographic analysis and compression tests. Next, two scaffolds of each type were inserted into 14 rabbits, which were sacrificed 8 weeks after surgery. The samples were submitted for histological examination. RESULTS: Metallographic analysis revealed interconnected pores, and the average interconnected pore diameter was about 360 mm, with 36% total porosity. The totally porous titanium samples and the titanium samples with porous surface and dense nucleus showed an average compressive strength of 16.19 MPa and 69.27 MPa, respectively. After 8 weeks, the animals showed bone ingrowth, even into the most internal pores. CONCLUSIONS: The pore morphology was effective in permitting bone ingrowth in both groups. Titanium scaffolds with a porous surface and dense nucleus showed the best mechanical properties and most adequate interface.

Histomorphometric analysis of pure titanium implants with porous surface versus rough surface

The purpose of this study was to analyze the bone repair around commercially pure titanium implants with rough and porous surface, fabricated using powder metallurgy technique, after their insertion in tibiae of rabbits. Seven male rabbits were used. Each animal received 3 porous-surface implants in the left tibia and 3 rough-surface implants in the right tibia. The rabbits were sacrificed 4 weeks after surgery and fragments of the tibiae containing the implants were submitted to histological and histomorphometric analyses to evaluate new bone formation at the implant-bone interface. Means ( percent) of bone neoformation obtained in the histomorphometric analysis were compared by Student's t-test for paired samples at 5 percent significance level.. The results of the histological analysis showed that osseointegration occurred for both types of implants with similar quality of bone tissue. The histomorphometric analysis revealed means of new bone formation at implant-bone interface of 79.69 ± 1.00 percent and 65.05 ± 1.23 percent for the porous- and rough-surface implants, respectively. Statistically significant difference was observed between the two types of implants with respect to the amount new bone formation (p<0.05). In conclusion, the porous-surface implants contributed to the osseointegration because they provide a larger contact area at implant-bone interface.

O propósito deste estudo foi avaliar a reparação óssea ao redor de implantes de superfície porosa comparados com implantes de superfície rugosa, ambos confeccionados de titânio puro grau 2 por meio da técnica de metalurgia do pó. Os implantes foram inseridos em tíbias de coelhos. Foram utilizados neste estudo 7 coelhos machos, sendo que cada um recebeu 3 implantes de superfície porosa na tíbia esquerda e 3 implantes de superfície rugosa na tíbia direita. Os animais foram sacrificados 4 semanas após a cirurgia e os fragmentos das tíbias contendo os implantes foram submetidos à análise histológica e histomorfométrica, visando analisar a neoformação óssea na interface osso-implante. As médias ( por cento) obtidas na análise histomorfométrica foram avaliadas por meio do teste estatístico t-student de amostras pareadas com nível de significância de 5 por cento. Os resultados da análise histológica mostraram que a osseointegração foi obtida nos dois tipos de implantes com similar qualidade de tecido ósseo. Na análise histomorfométrica, verificaram-se médias de neoformação óssea na interface osso-implante de 79,69 por cento ± 1,00 e 65,05 ± 1,23 para os implantes de superfície porosa e rugosa, respectivamente, e foi observada diferença estatisticamente significante entre os dois tipos de implantes com relação à quantidade de neoformação óssea. Concluiu-se que os implantes de superfície porosa contribuíram para a osseointegração devido à sua maior superfície de contato na interface osso-implante.

Histomorphometric analysis of pure titanium implants with porous surface versus rough surface%Análise histomorfométrica de implantes de titânio puro com superfície porosa versus superfície rugosa

O propósito deste estudo foi avaliar a reparação óssea ao redor de implantes de superfície porosa comparados com implantes de superfície rugosa, ambos confeccionados de titânio puro grau 2 por meio da técnica de metalurgia do pó. Os implantes foram inseridos em tíbias de coelhos. Foram utilizados neste estudo 7 coelhos machos, sendo que cada um recebeu 3 implantes de superfície porosa na tíbia esquerda e 3 implantes de superfície rugosa na tíbia direita. Os animais foram sacrificados 4 semanas após a cirurgia e os fragmentos das tíbias contendo os implantes foram submetidos à análise histológica e histomorfométrica, visando analisar a neoformação óssea na interface osso-implante. As médias ( por cento) obtidas na análise histomorfométrica foram avaliadas por meio do teste estatístico t-student de amostras pareadas com nível de significância de 5 por cento. Os resultados da análise histológica mostraram que a osseointegração foi obtida nos dois tipos de implantes com similar qualidade de tecido ósseo. Na análise histomorfométrica, verificaram-se médias de neoformação óssea na interface osso-implante de 79,69 por cento ± 1,00 e 65,05 ± 1,23 para os implantes de superfície porosa e rugosa, respectivamente, e foi observada diferença estatisticamente significante entre os dois tipos de implantes com relação à quantidade de neoformação óssea. Concluiu-se que os implantes de superfície porosa contribuíram para a osseointegração devido à sua maior superfície de contato na interface osso-implante

Histomorphometric analysis of pure titanium implants with porous surface versus rough surface.

The purpose of this study was to analyze the bone repair around commercially pure titanium implants with rough and porous surface, fabricated using powder metallurgy technique, after their insertion in tibiae of rabbits. Seven male rabbits were used. Each animal received 3 porous-surface implants in the left tibia and 3 rough-surface implants in the right tibia. The rabbits were sacrificed 4 weeks after surgery and fragments of the tibiae containing the implants were submitted to histological and histomorphometric analyses to evaluate new bone formation at the implant-bone interface. Means (%) of bone neoformation obtained in the histomorphometric analysis were compared by Student's t-test for paired samples at 5% significance level.. The results of the histological analysis showed that osseointegration occurred for both types of implants with similar quality of bone tissue. The histomorphometric analysis revealed means of new bone formation at implant-bone interface of 79.69 +/- 1.00% and 65.05 +/- 1.23% for the porous- and rough-surface implants, respectively. Statistically significant difference was observed between the two types of implants with respect to the amount new bone formation (p<0.05). In conclusion, the porous-surface implants contributed to the osseointegration because they provide a larger contact area at implant-bone interface.