Balancing porosity and mechanical properties of titanium samples to favor cellular growth against bacteria

metals Article Balancing Porosity and Mechanical Properties of Titanium Samples to Favor Cellular Growth against Bacteria Ana Civantos 1,*, Ana M. Beltrán2,* , Cristina Domínguez-Trujillo 2, Maria D. Garvi 3, Julián Lebrato 3, Jose A. Rodríguez-Ortiz 2, Francisco García-Moreno 4, Juan V. Cauich-Rodriguez 5, Julio J. Guzman 2and Yadir Torres 2 1 Micro and Nanotechnology Laboratory, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA 2Departamento de Ingeniería y Ciencia de los Materiales y del Transporte, Escuela Politécnica Superior, Universidad de Sevilla, Virgen de África 7, 41011 Sevilla, Spain; [email protected] (C.D.-T.); [email protected] (J.A.R.-O.); [email protected] (J.J.G.); ytorr[email protected] (Y.T.) 3Grupo TAR, Escuela Politécnica Superior, Universidad de Sevilla, 41004 Sevilla, Spain; [email protected] (M.D.G.); [email protected] (J.L.) 4Institute of Applied Materials, Helmholtz-Zentrum Berlin für Materialien und Energie, Hahn-Meitner-Platz 1, 14109 Berlin, Germany; garcia-mor[email protected] 5 Unidad de Materiales, Centro de Investigaci ó n Cient í fica de Yucat á n A. C., 97205 M é rida, Yucat á n, Mexico; [email protected] *Correspondence: [email protected] (A.C.); [email protected] (A.M.B.) Received: 6 September 2019; Accepted: 22 September 2019; Published: 24 September 2019   Abstract: Two main problems limit the success of titanium implants: bacterial infection, which restricts their osseointegration capacity; and the stiffness mismatch between the implant and the host cortical bone, which promotes bone resorption and risk of fracture. Porosity incorporation may reduce this difference in stiffness but compromise biomechanical behavior. In this work, the relationship between the microstructure (content, size, and shape of pores) and the antibacterial and cellular behavior of samples fabricated by the space-holder technique (50 vol % NH 4 HCO 3 and three ranges of particle sizes) is established. Results are discussed in terms of the best biomechanical properties and biofunctional activity balance (cell biocompatibility and antibacterial behavior). All substrates achieved suitable cell biocompatibility of premioblast and osteoblast in adhesion and proliferation processes. It is worth to highlighting that samples fabricated with the 100–200 µ m space-holder present better mechanical behavior—in terms of stiffness, microhardness, and yield strength—which make them a very suitable material to replace cortical bone tissues. Those results exposed the relationship between the surface properties and the race of bacteria and mammalian cells for the surface with the aim to promote cellular growth over bacteria. Keywords: bone implant; porous titanium; cellular adhesion; bacteria colonization; osseointegration 1. Introduction Longer life expectancy, traumas, and congenital diseases have led to an increase in the use of implants. Bone metal implants are usually made of commercially pure titanium (c.p. Ti) or based on Ti alloys owing to its biocompatibility and mechanical behavior [ 1 ]. C.p. Ti has a Young’s Modulus of 100–110 GPa, which is higher than cortical bone (20–25 GPa), causing stress shielding phenomena and bone resorption [ 2 ]. This problem can be solved using porous material instead of fully-dense one [ 3 – 6 ]. However, in order to achieve a biomechanical balance (stiffness vs. mechanical strength) of the bone tissue to be replaced, an adequate porosity (in terms of size, shape, and distribution of the pores) is Metals 2019,9, 1039; doi:10.3390/met9101039 www.mdpi.com/journal/metals Metals 2019,9, 1039 2 of 16 required. Up to 34 processing routes to fabricate porous materials have been already reported [ 7 – 10 ]. Among them, the use of space-holders stands out. This technique uses particles—such as salt [11,12], sugar [ 13 ], ammonium bicarbonate [ 14 ], or magnesium [ 15 ]—to reproduce a bone-like porosity structure in which the space-holder particles can be removed by thermal process or dissolution. This step can be performed before or during the sintering process. The particle size and morphology are key factors in the design of the pore structure. One remarkable advantage is that the space-holder is a cost-effective and non-toxic method, which does not release any toxic agent which could affect cellular and bacterial behavior. On the other hand, several authors have reported that surface properties such as the topography, roughness, chemistry, and free energy of metallic based biomaterials have a strong influence on the initial adhesion and early differentiation of osteoblast cells [ 16 , 17 ]. In 1987, Gristina was the first author to use the term “the race” for the surface, regarding the competition established between bacteria and cells for the biomaterial surface [ 18 ]. Also, Gristina remarked that if bacteria attached and colonize faster than osteoblast cells—i.e., bacteria wins the race—an infection will take place, which will be almost impossible to remove. This effect is due to the fast process of bacteria to excrete extracellular polymeric substances (EPS) once they are attached. The size is one the most highlighted difference: osteoblasts are bigger in size, ranging from 20–30 µ m, bacteria such as Escherichia coli (E. coli) or Staphylococcus aureus (the most common bacteria presented in titanium dental implants) are around 1–2 µ m in length [ 19 ]. The stiffness, surface topography (i.e., roughness) and surface chemistry can be tailored in order to promote an advanced cell adhesion process. It has been described the high stiffness behavior of bacteria compared to osteoblast cells, which can be a potential factor in bacteria adhesion and colonization [ 20 ]. However, the adhesion mechanisms and the proliferation rate of bacteria are key factors that favor bacteria in the race for the surface. Filia, pili, and the expression of EPS, which constitute the biofilm formation, are some of the mechanisms that participate in the interactions between bacteria and the surface. This EPS covers and protects the bacteria under a biofilm. In this scenario, bacteria becomes more resistant and the antibiotic treatment usually has no effect. On the other hand, osteoblast cells require a more complex mechanism involving integrin receptors and proteins to develop focal adhesion (FA) points. The FA are based of filopodia and lamellipodia cytoplasmatic projections that control and regulate cell to cell interactions and cell surface as well. Therefore, the control of surface properties may reduce or avoid the bacteria attachment resulting in the successful application of the implant on bone regeneration [19]. In a previous work, we reported the manufacture of porous substrates by the space-holder technique which showed a decrease of bacteria attachment [ 21 ]. However, the vast majority of studies which focus on Ti implants made use of either bacteria or celld independently and, thus made it difficult to analyze of what type of surface properties may enhance infections and/or osseointegration. Therefore, here we report for the first time the behavior of bacteria and two murine cell lines (myoblast and osteoblast) on c.p. Ti porous substrates with the aim to design a porous implant able to promote bone regeneration and avoid bacterial infections. In this context, this work examines a detailed study of the influence of microstructural characteristics (size, shape, area, and porosity roughness) on the macro and micro-mechanical behavior, as well as the response of the material in the presence of eukaryotic cell cultures (C2C12-GFP and MC3T3E1) and E. coli strain bacteria (Gram-negative bacteria). 2. Materials and Methods 2.1. Fabrication of Ti substrates The blends of medical grade 4 c.p. Ti were supplied by SE-JONG (Materials Co. Ltd. Gojan-dong Korea). Fully-dense c.p. Ti samples were obtained by conventional powder technology (PM), while porous substrates were fabricated using space-holder technique (50 vol % NH 4 HCO 3 and different ranges of particle size: 100–200 µ m, 250–355 µ m, and 355–500 µ m). The green samples were produced by pressing at 800 MPa by means of an Instron 5505 universal testing machine (Instron, High Wycombe, Metals 2019,9, 1039 3 of 16 UK). Then, the spacer was thermally removed (firstly at 60 ◦ C and, then at 110 ◦ C under low vacuum conditions), both stages of the thermal treatment are carried out for 10–12 h and low vacuum conditions of 10 −2 mbar). Subsequently, the samples (porous and fully-dense) were sintered in a ceramic tubular furnace during 2 h at 1250 ◦C under high vacuum conditions (~10−5mbar). 2.2. Microstructural and Mechanical Characterization Different techniques were employed to characterize the porosity at different levels (superficial and volumetric), as well as the pore distribution, shape, and roughness previously reported [ 3 – 5 , 14 ]. Total porosity was determined by both Archimedes’ method and image analysis (IA), using a Nikon Epiphot optical microscope (Japan) coupled with a Jenoptik Progres C3 camera and Image-Pro Plus 6.2 analysis software. Confocal laser and scanning electron microscopy (SEM, JEOL JSM-6490LV, JEOL, Tokyo, Japan) were used to evaluate the roughness. Considering the importance of pores roughness on bacterial adhesion, X-ray micro-computed tomography (M-CT) was also performed due to its capability to provide three-dimensional (3D) surface roughness data, among other parameters. These measurements were performed using a custom made X-ray scanner composed mainly of a micron focused X-ray source L8121-01 (with a W-target) from Hamamatsu, Japan [ 22 ]. Scans were performed at 100 kV and 100 µ A, with a spot size of 5 µ m and were recorded with a flat panel detector C7943 (120 × 120 mm, 2240 × 2368 pixel), also from Hamamatsu. A 3D reconstruction of the specimen was obtained by acquiring a certain number of X-ray projections during sample rotation over 360 ◦ , followed by software reconstruction of these projections. This method allowed for the qualitative and quantitative exploration of the interior structure of the porous Ti samples, with a pixel size down to 6.4 µ m at a 7.8-fold magnification. Parameters such as equivalent diameter (D eq ), total porosity (P t ), and interconnected porosity (Pi) was determined by M-CT. All these analytical techniques have been implemented, following the procedures described in previous works [3–5,14]. On the other hand, the macro-mechanical behavior of Ti substrates was evaluated by ultrasound technique (dynamic Young’s modulus, E d ) and uniaxial compression test (Young’s modulus, E c ; yield strength, σy ). For mechanical compression testing, the specimen dimensions were fixed to standard recommendation (height/diameter =0.8). The tests were carried out with a universal electromechanical Instron machine 5505 by applying a strain rate of 0.005 mm/mm/min. All tests finished for a strain of 50 pct. The Young’s modulus estimation from the compression stress–strain curves was corrected with the testing machine stiffness (87.9 kN/mm). On the other hand, conventional microhardness studies (Vickers indenter Shimadzu, model HMV-G, Japan) at two different applied loads (HV0.3 and HV1) were performed. All the measures were taken three times per substrate type and applied load. 2.3. In Vitro Cellular Experiments 2.3.1. Analysis of Bacterial Behavior of Porous Substrates E. coli strain Bacterial behavior on porous and fully-dense c.p. Ti substrates is studied, following the protocol summarized in Figure 1. The used E. coli was a lyophilized form from Ielab, Spain (E. coli; ATCC 25922). The culture solution was prepared with Peptone Water (dehydrated culture media, from Panread AppliChem, Spain) and E. coli-Coliforms Chromogenic Medium from Conda Laboratories S.A. The initial concentration of colony-forming units per 100 mL (CFU/100 mL), was determined by membrane filtration, followed by incubation of the membrane on a Petri plate containing E. coli-Coliforms Chromogenic Medium at 37 ◦ C for 24 h. Specifically, the study includes three initial concentrations: 7.8·104(C1), 3.3·103(C2), and 4.3·102(C 3) CFU/100 mL, respectively. Metals 2019,9, 1039 4 of 16 Metals 2019, 9, x FOR PEER REVIEW 4 of 16 Figure 1. Analytical procedures employed to E. coli bacteria analyses. For E. coli bacterial behavior studies, fully-dense and porous substrates were placed in 10 mL test tubes containing each bacterial concentration and these were incubated at 37 °C for 1 h. One tube containing only bacteria was used as reference (R). Afterwards, the bacterial growth was analyzed by three protocols (Figure 1). Protocol 1 (P1) quantified the amount of E. coli per tube and per initial concentration by membrane filtration (pore size of the membrane: 0.45 μm) of the solution of each tube [23]. Protocol 2 (P2) evaluated the E. coli density on the Ti substrate using a sterile swab to smear the total surface of the Ti samples. P2 was applied on the flat surface, meaning the areas of the samples without pores. Then, the swab was applied following a zigzag pattern on a Petri dish containing E. coli-Coliforms Chromogenic Medium and incubated at 37 °C for 24 h. Protocol 3 (P3) analyzed the E. coli proliferation inside the pores by re-immersing the swabbed substrates in test tubes with 10 mL of sterile Peptone water. This solution was mixed using a vortex mixer for 1 min and cooled at 4 °C for 2 h. Tube contents were again mixed with the vortex mixer to detach the bacteria from the cavities and to quantify them using membrane filtration. To guarantee the complete detachment of bacteria from the cavities, the process was repeated three times instead of doing it only once as recommended by ISO 11737:1:2007 standard [24]. The bacterial growth was expressed in total number of CFU for each substrate and concentration. Several measurements were made for each condition (protocol, substrate, and initial concentration). 2.3.2. Evaluation of Cell Adhesion and Proliferation of Eukaryotic Murine C2c12-Gfp Premioblast Cells Attachment and proliferation of C2C12-GFP were analyzed by inverted fluorescence microscopy (Olympus IX51) and CellD Software (Olympus). To that end, C2C12 murine premioblastic cell line was purchased to America Type Culture Collection (ATCC®CRL-1772™) and transfected, via lentivirus, to self-express constitutively green fluorescent protein (GFP). The presence of this GFP group in cells membrane allowed us to detect the cells and follow the adhesion and proliferation process. The routing passage was performed using DMEM (Dulbecco’s modified Eagle medium, Sigma Aldrich), completed with 10% fetal bovine serum (FBS) and 1% of penicillin/streptomycin (100 U/mL, Invitrogen, Germany). For adhesion and proliferation studies, cells were seeded at an initial density of 10,000 cells/cm2, and after 24 h samples were transferred to new 24-well plates and inverted Figure 1. Analytical procedures employed to E. coli bacteria analyses. For E. coli bacterial behavior studies, fully-dense and porous substrates were placed in 10 mL test tubes containing each bacterial concentration and these were incubated at 37 ◦ C for 1 h. One tube containing only bacteria was used as reference (R). Afterwards, the bacterial growth was analyzed by three protocols (Figure 1). Protocol 1 (P1) quantified the amount of E. coli per tube and per initial concentration by membrane filtration (pore size of the membrane: 0.45 µ m) of the solution of each tube [ 23 ]. Protocol 2 (P2) evaluated the E. coli density on the Ti substrate using a sterile swab to smear the total surface of the Ti samples. P2 was applied on the flat surface, meaning the areas of the samples without pores. Then, the swab was applied following a zigzag pattern on a Petri dish containing E. coli-Coliforms Chromogenic Medium and incubated at 37 ◦ C for 24 h. Protocol 3 (P3) analyzed the E. coli proliferation inside the pores by re-immersing the swabbed substrates in test tubes with 10 mL of sterile Peptone water. This solution was mixed using a vortex mixer for 1 min and cooled at 4 ◦ C for 2 h. Tube contents were again mixed with the vortex mixer to detach the bacteria from the cavities and to quantify them using membrane filtration. To guarantee the complete detachment of bacteria from the cavities, the process was repeated three times instead of doing it only once as recommended by ISO 11737:1:2007 standard [ 24 ]. The bacterial growth was expressed in total number of CFU for each substrate and concentration. Several measurements were made for each condition (protocol, substrate, and initial concentration). 2.3.2. Evaluation of Cell Adhesion and Proliferation of Eukaryotic Murine C2c12-Gfp Premioblast Cells Attachment and proliferation of C2C12-GFP were analyzed by inverted fluorescence microscopy (Olympus IX51) and CellD Software (Olympus). To that end, C2C12 murine premioblastic cell line was purchased to America Type Culture Collection (ATCC ® CRL-1772 ™ ) and transfected, via lentivirus, to self-express constitutively green fluorescent protein (GFP). The presence of this GFP group in cells membrane allowed us to detect the cells and follow the adhesion and proliferation process. The routing passage was performed using DMEM (Dulbecco’s modified Eagle medium, Sigma Aldrich), completed with 10% fetal bovine serum (FBS) and 1% of penicillin/streptomycin (100 U/mL, Invitrogen, Germany). For adhesion and proliferation studies, cells were seeded at an initial density of 10,000 cells/cm 2 , Metals 2019,9, 1039 5 of 16 and after 24 h samples were transferred to new 24-well plates and inverted fluorescent images were taken after day 1, 4, 7, and 10 of cell incubation. The complete medium was changed every 2–3 days. This protocol is summarized in Figure 2. Metals 2019, 9, x FOR PEER REVIEW 5 of 16 fluorescent images were taken after day 1, 4, 7, and 10 of cell incubation. The complete medium was changed every 2–3 days. This protocol is summarized in Figure 2. Figure 2. Analytical protocols for the cell studies. 2.3.3. Cell adhesion and Proliferation Studies of Murine MC3T3E1 Osteoblast A bone cell line, MC3T3-E1 (ATCC CRL-2593), was employed to evaluate the effect of porosity in cell viability, cell proliferation, and cell morphology of osteoblast. The subculture routing was performed in MEM (minimum essential medium, Sigma Aldrich, Germany), completed with 10% fetal bovine serum (FBS) and 1% of penicillin/streptomycin (100U/mL, Invitrogen, Germany). 75 cm2 culture flasks were placed in a humid atmosphere with 5% CO 2 at 37 °C. Ti substrates were seeded at 10,000 cells/cm 2 and after 24 h samples were transferred to new 24-well plates. Figure 2 summaries the protocol for this study. Cell Viability of Murine MC3T3E1 Osteoblast Cell metabolic analysis was measured in order to evaluate the cell viability at day 4 of cell incubation. To that end, AlamarBlue assay (DAL1100, Thermofisher, USA) was performed following the manufacturer’s instructions. For this, samples previously seeded with cells were transferred to new 24-well plates and fresh media containing AlamarBlue dye added (10% of culture media volume). After 1.5 h of incubation in darkness at 37 °C, samples were removed and fluorescence signal was read in a microplate reader (Synergy HT, Biotek) using an excitation wavelength of 530 nm and emission wavelength of 590 nm. Fully dense surface was considered control surface as 100% viability. Assays were carried out by triplicate for each sample condition. Cellular Morphology Evaluation of Murine MC3T3E1 Osteoblast After 10 days of cell incubation, osteoblast cells attached to the substrates were washed twice with PBS, and subsequently, fixed using 4% paraformaldehyde (PFA) solution. Fully-dense and porous Ti samples were rinsed carefully with PBS twice, permeabilized with 0.1% (v/v) Triton X-100 and, finally, washed again with PBS. The actin cytoskeleton was stained with Texas Red ® -X phalloidin (Molecular Probes). To that end, the commercial solution was prepared in PBS and left in dark conditions for 20 min at room temperature (RT). Hoechst (Invitrogen, Molecular Probes) was employed as a contrast marker, to detect cell nuclei. Fully-dense and porous substrates were analyzed using an inverted fluorescence microscope (Olympus IX51) with a TRICT filter (λex/λem = 550/600 nm) for Actin, and DAPI filter for Hoechst (λex/λem = 380/455 nm) and images were treated by CellD analysis software (Olympus). Figure 2. Analytical protocols for the cell studies. 2.3.3. Cell adhesion and Proliferation Studies of Murine MC3T3E1 Osteoblast A bone cell line, MC3T3-E1 (ATCC CRL-2593), was employed to evaluate the effect of porosity in cell viability, cell proliferation, and cell morphology of osteoblast. The subculture routing was performed in MEM (minimum essential medium, Sigma Aldrich, Germany), completed with 10% fetal bovine serum (FBS) and 1% of penicillin/streptomycin (100U/mL, Invitrogen, Germany). 75 cm2 culture flasks were placed in a humid atmosphere with 5% CO 2 at 37 ◦ C. Ti substrates were seeded at 10,000 cells/cm 2 and after 24 h samples were transferred to new 24-well plates. Figure 2summaries the protocol for this study. Cell Viability of Murine MC3T3E1 Osteoblast Cell metabolic analysis was measured in order to evaluate the cell viability at day 4 of cell incubation. To that end, AlamarBlue assay (DAL1100, Thermofisher, USA) was performed following the manufacturer’s instructions. For this, samples previously seeded with cells were transferred to new 24-well plates and fresh media containing AlamarBlue dye added (10% of culture media volume). After 1.5 h of incubation in darkness at 37 ◦ C, samples were removed and fluorescence signal was read in a microplate reader (Synergy HT, Biotek) using an excitation wavelength of 530 nm and emission wavelength of 590 nm. Fully dense surface was considered control surface as 100% viability. Assays were carried out by triplicate for each sample condition. Cellular Morphology Evaluation of Murine MC3T3E1 Osteoblast After 10 days of cell incubation, osteoblast cells attached to the substrates were washed twice with PBS, and subsequently, fixed using 4% paraformaldehyde (PFA) solution. Fully-dense and porous Ti samples were rinsed carefully with PBS twice, permeabilized with 0.1% (v/v) Triton X-100 and, finally, washed again with PBS. The actin cytoskeleton was stained with Texas Red ® -X phalloidin (Molecular Probes). To that end, the commercial solution was prepared in PBS and left in dark conditions for 20 min at room temperature (RT). Hoechst (Invitrogen, Molecular Probes) was employed as a contrast marker, to detect cell nuclei. Fully-dense and porous substrates were analyzed using an inverted fluorescence microscope (Olympus IX51) with a TRICT filter ( λ ex/ λ em =550/600 nm) for Actin, and DAPI filter for Hoechst (λex/λem =380/455 nm) and images were treated by CellD analysis software (Olympus). Metals 2019,9, 1039 6 of 16 Statistical Analysis In general, each experiment was performed in triplicate with each bacteria or cell line, media, and sample. Mean values and standard deviation are reported. A p-value of <0.01 was deemed to be statistically significant. For in vitro cells, mean and standard deviation are presented and a one-way ANOVA followed by a post Tukey analysis was performed in which values were be statistically significant when p-value was <0.05. 3. Results 3.1. Microstructural Characterization Results concerning to the microstructural characterization of the porous substrates can be observed in Figures 3and 4. Particularly, Figure 3(top) shows micro-images of the fabricated samples while Figure 3(bottom) displays details about the aspect of the pores, size, and distribution as obtained by IA. So, by the micro-image we have an overview of the fabricated samples, while by IA details about the aspect of the pores, size and distribution can be determined. Figure 4shows data obtained by M-CT which provides three-dimensional information, quite useful to determine the distribution of inner pores as well as the roughness volume percentage of the eroded pore compared to the virtually smoothed pore in dependence of the pore equivalent diameter. The presence of micro-scale roughness pattern within the pores was revealed by M-CT, so total inner surface was calculated analyzing a M-CT representative fraction of total sample volume, taking into account the local deviation of the gray values perpendicular to the contour line within a search distance of 20 µ m. Data obtained by the analyses of the images contained in Figures 3and 4are summarized in Tables 1and 2, such as porosity and parameters to characterize the pores (equivalent diameter of pores, D eq , shape factor, and roughness). Table 1. Total, interconnected, and isolated porosity evaluated by different characterization techniques. Samples PT(%) Pi(%) Archimedes’ Method IA M-CT Archimedes’ Method M-CT Fully-dense 2.3 ±0.1 1.2 ±0.2 – 2.1 ±0.1 – Spacer size (µm) 100–200 44.8 ±0.1 50.3 ±1.3 52.2 ±10.7 43.1 ±0.2 51.3 ±10.6 250–355 45.9 ±0.2 48.7 ±1.9 – 41.0 ±0.1 – 355–500 46.0 ±0.1 47.1 ±4.3 56.4 ±11.1 41.2 ±0.2 55.6 ±10.2 Table 2. Morphological features of the pores: size, shape factor, and roughness of the pore walls. Samples AI M-CT Deq (µm) Shape factor Deq (µm) Roughness,Ra(%) Fully-dense 5.5 ±0.2 0.99 ±0.01 – Spacer size (µm) 100–200 161.1 ±28.5 0.67 ±0.03 191.8 ±6.1 11.3 ±2.5 250–355 261.5 ±9.0 0.67 ±0.01 311.9 ±8.2 7.3 ±2.0 355–500 293.4 ±28.2 0.71 ±0.03 368.4 ±9.1 3.3 ±0.6 Metals 2019,9, 1039 7 of 16 Metals 2019, 9, x FOR PEER REVIEW 7 of 16 Figure 3. Macro-photography of the samples and optical microscopy images of the porous substrates for the different ranges of spacer particles size: 100−200 μm, 250−355 μm, and 355−500 μm. Figure 4. Equivalent diameter determined and interconnected and isolated porosity analyzed by M-CT for two different ranges of spacer particles size: 100−200 μm and 355−500 μm. Figure 3. Macro-photography of the samples and optical microscopy images of the porous substrates for the different ranges of spacer particles size: 100−200 µm, 250−355 µm, and 355−500 µm. Metals 2019, 9, x FOR PEER REVIEW 7 of 16 Figure 3. Macro-photography of the samples and optical microscopy images of the porous substrates for the different ranges of spacer particles size: 100−200 μm, 250−355 μm, and 355−500 μm. Figure 4. Equivalent diameter determined and interconnected and isolated porosity analyzed by M-CT for two different ranges of spacer particles size: 100−200 μm and 355−500 μm. Figure 4. Equivalent diameter determined and interconnected and isolated porosity analyzed by M-CT for two different ranges of spacer particles size: 100−200 µm and 355−500 µm. 3.2. Mechanical Behavior The macro-mechanical (stiffness, yield strength) and micro-mechanical (Vickers hardness) behavior of porous c.p. Ti is summarized in Table 3. Young’s modulus has been measured by ultrasound and uniaxial compression test. These results have been discussed in terms of the advantages and Metals 2019,9, 1039 8 of 16 disadvantages of both techniques. On the other hand, the influence of the indentation load and its relationship with the size of the remaining Ti matrix among pores is evaluated. In this context, it should be noted that, for a fixed percentage of total porosity, an increase in pore size implies an increase in the mean distance between pores (the size of the titanium matrix increases). Table 3. Macro and micro-mechanical behavior of porous substrates. Samples US Uniaxial Compression Test Microhardness Ed(GPa) Ec(GPa) σy(MPa) HV0.3 HV1 Fully-dense 101.2 ±0.3 95 ±1.0 628 ±5 377 ±26 342 ±52 Spacer size (µm) 100–200 20.8 ±0.1 26.0 ±0.9 127 ±21 401 ±42 167 ±81 250–355 22.8 ±0.2 23.1 ±1.0 118 ±14 356 ±35 152 ±72 355–500 20.0 ±0.7 19.7 ±1.2 98 ±18 350 ±36 138 ±70 3.3. Bacteria Behavior The number of CFU of E. coli on titanium substrates as a function of initial bacteria concentration following Protocol 1 is depicted in Figure 5a. It was observed that E. coli initial concentration affected the number of CFU on the substrate i.e., the higher the initial concentration the higher the number of CFU. However, as porosity increased (fully-dense vs. porous c.p. Ti) the number of CFU was significantly increased, especially at the lowest initial bacteria concentration (C3). E. coli bacteria concentration of the medium could influence the medium-substrate interaction. After incubating the samples for 1 h at 37 ◦ C, the number of CFU in the corresponding solutions was determined by membrane filtration. Protocol 2 revealed no bacteria attached onto the flat surface. By Protocol 3, bacteria inside the pores was quantified and its normalization by the surface is shown in Figure 5b,c, respectively. As the size of the spacer increased the number of CFU also increased at both initial bacteria concentrations (Figure 5b). Metals 2019, 9, x FOR PEER REVIEW 8 of 16 3.2. Mechanical Behavior The macro-mechanical (stiffness, yield strength) and micro-mechanical (Vickers hardness) behavior of porous c.p. Ti is summarized in Table 3. Young’s modulus has been measured by ultrasound and uniaxial compression test. These results have been discussed in terms of the advantages and disadvantages of both techniques. On the other hand, the influence of the indentation load and its relationship with the size of the remaining Ti matrix among pores is evaluated. In this context, it should be noted that, for a fixed percentage of total porosity, an increase in pore size implies an increase in the mean distance between pores (the size of the titanium matrix increases). Table 3. Macro and micro-mechanical behavior of porous substrates Samples US Uniaxial Compression Test Microhardness Ed (GPa) Ec (GPa) σy (MPa) HV0.3 HV1 Fully-dense 101.2 ± 0.3 95 ± 1.0 628 ± 5 377 ± 26 342 ± 52 Spacer size (μm) 100 – 200 20.8 ± 0.1 26.0 ± 0.9 127 ± 21 401 ± 42 167 ± 81 250 – 355 22.8 ± 0.2 23.1 ± 1.0 118 ± 14 356 ± 35 152 ± 72 355 – 500 20.0 ± 0.7 19.7 ± 1.2 98 ± 18 350 ± 36 138 ± 70 3.3. Bacteria Behavior The number of CFU of E. coli on titanium substrates as a function of initial bacteria concentration following Protocol 1 is depicted in Figure 5a. It was observed that E. coli initial concentration affected the number of CFU on the substrate i.e., the higher the initial concentration the higher the number of CFU. However, as porosity increased (fully-dense vs. porous c.p. Ti) the number of CFU was significantly increased, especially at the lowest initial bacteria concentration (C3). E. coli bacteria concentration of the medium could influence the medium-substrate interaction. After incubating the samples for 1 h at 37 °C, the number of CFU in the corresponding solutions was determined by membrane filtration. Protocol 2 revealed no bacteria attached onto the flat surface. By Protocol 3, bacteria inside the pores was quantified and its normalization by the surface is shown in Figure 5b and 5c, respectively. As the size of the spacer increased the number of CFU also increased at both initial bacteria concentrations (Figure 5b). Figure 5. Bacteria quantification following protocols described (P1, P2, and P3). ( a ) CFU of E. coli bacteria after membrane filtration (Protocol 1), CFU of E. coli bacteria inside the pores measured following Protocol 3: ( b ) absolute values and ( c ) normalized values by the inner pores surface. Inset: magnification view of the concentration view for the normalized values. Metals 2019,9, 1039 9 of 16 3.4. In Vitro Cell Studies The different studied substrates were tested in vitro conditions using two murine cell lines. In first place, C2C12-GFP premioblastic line was used to evaluate the process of cell adhesion and proliferation after 1, 4, 7, and 10 days of cell culture compiling the results in Figure 6. C2C12-GFP cells were capable to attach and proliferate over the surface and inside the pores, increasing the cell density presented on the samples over time. In the first 24 h, the inverted fluorescence images showed similar cell density between fully-dense and porous substrates. After 4 days, several small green dots which correspond to small alive cell clusters were observed totally dispersed on the surface. Furthermore, at longer incubation periods, cells were able to proliferate, showing an increased cell density over the entire surface. Indeed, at day 10 C2C12 GFP cells were covering the entire porous structure, even filling the pores attaching on pore’s walls and arriving to the edge of the samples (see Figure 7after 10 days, 255–350 µ m sample). Figure 7showed a higher magnification of C2C12-GFP cells growing at 4 days to analyze the cell morphology on c.p. Ti samples. A clear different cell shape was observed between cells growing on flat surfaces and inside pores. Premioblastic cells growing on fully-dense samples showed fusiform morphology, with an elongated cell shape. This cell morphology, most frequent of fibroblastic phenotype, was also observed in flat surfaces of porous substrates independently of pore size (see Figure 7). Metals 2019, 9, x FOR PEER REVIEW 9 of 16 Figure 5. Bacteria quantification following protocols described (P1, P2, and P3). (a) CFU of E. coli bacteria after membrane filtration (Protocol 1), CFU of E. coli bacteria inside the pores measured following Protocol 3: (b) absolute values and (c) normalized values by the inner pores surface. Inset: magnification view of the concentration view for the normalized values. 3.4. In Vitro Cell Studies The different studied substrates were tested in vitro conditions using two murine cell lines. In first place, C2C12-GFP premioblastic line was used to evaluate the process of cell adhesion and proliferation after 1, 4, 7, and 10 days of cell culture compiling the results in Figure 6. C2C12-GFP cells were capable to attach and proliferate over the surface and inside the pores, increasing the cell density presented on the samples over time. In the first 24 h, the inverted fluorescence images showed similar cell density between fully-dense and porous substrates. After 4 days, several small green dots which correspond to small alive cell clusters were observed totally dispersed on the surface. Furthermore, at longer incubation periods, cells were able to proliferate, showing an increased cell density over the entire surface. Indeed, at day 10 C2C12 GFP cells were covering the entire porous structure, even filling the pores attaching on pore’s walls and arriving to the edge of the samples (see Figure 7 after 10 days, 255–350 μm sample). Figure 7 showed a higher magnification of C2C12-GFP cells growing at 4 days to analyze the cell morphology on c.p. Ti samples. A clear different cell shape was observed between cells growing on flat surfaces and inside pores. Premioblastic cells growing on fully-dense samples showed fusiform morphology, with an elongated cell shape. This cell morphology, most frequent of fibroblastic phenotype, was also observed in flat surfaces of porous substrates independently of pore size (see Figure 7). Figure 6. Inverted fluorescence microphotographs of cell adhesion and proliferation of C2C12-GFP cells after 1, 4, 7, and 10 days of cell culture. Common scale bar, 1mm, except for 7 days and pore size 355–500 μm, which a specific scale bar is indicated (200 μm). Figure 6. Inverted fluorescence microphotographs of cell adhesion and proliferation of C2C12-GFP cells after 1, 4, 7, and 10 days of cell culture. Common scale bar, 1mm, except for 7 days and pore size 355–500 µm, which a specific scale bar is indicated (200 µm). Metals 2019,9, 1039 16 of 16 21. Dominguez-Trujillo, C.; Beltr á n, A.M.; Garvi, M.D.; Salazar-Moya, A.; Lebrato, J.; Hickey, D.J.; Rodriguez-Ortiz, J.A.; Kamm, P.H.; Lebrato, C.; Garc í a-Moreno, F.; et al. Bacterial behavior on coated porous titanium substrate for biomedical applications. Surf. Coat. 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