In Vitro Bone Cell Behavior on Porous Titanium Samples: Influence of Porosity by Loose Sintering and Space Holder Techniques

Metals 2020, 10, 696; doi:10.3390/met10050696 www.mdpi.com/journal/metals Article In Vitro Bone Cell Behavior on Porous Titanium Samples: Influence of Porosity by Loose Sintering and Space Holder Techniques Ana Civantos 1, *, Mercè Giner 2, *, Paloma Trueba 3 , Sheila Lascano 4 , María-José Montoya-García 5 , Cristina Arévalo 3 , María Ángeles Vázquez 5 , Jean Paul Allain 1,6 and Yadir Torres 3 1 Department of Nuclear, Plasma and Radiological Engineering, College of Engineering, University of Illinois at Urbana-Champaign, Urbana 61801, IL, USA; [email protected] 2 Departamento de Citología e Histología Normal y Patológica, Universidad de Sevilla, Avda. Dr.Fedriani s/n, 41009 Sevilla, Spain 3 Departamento de Ingeniería y Ciencia de los Materiales y del Transporte, Escuela Politécnica Superior, Calle Virgen de África 7, 41011 Seville, Spain; [email protected] (P.T.); [email protected]; [email protected] (C.A.); [email protected] (Y.T.) 4 Departamento de Ingeniería Mecánica, Universidad Técnica Federico Santa María, Avda. Vicuña Mackenna 3939, Santiago de Chile 8940000, Chile; s[email protected] 5 Medicine Department, University of Seville, Avda. Dr. Fedriani s/n, 41009 Sevilla, Spain; [email protected] (M.-J.M.-G.); [email protected] (M.Á.V.) 6 The Ken and Mary Alice Lindquist Department of Nuclear Engineering, Pennsylvania State University, State College 16802, PA, USA * Correspondence: [email protected] (A.C.); mgi[email protected] (M.G.); Tel.: +217-766-0951 (A.C.); +34-627-217-746 (M.G.) Received: 15 April 2020; Accepted: 19 May 2020; Published: 25 May 2020 Abstract: A great variety of powder metallurgy techniques can produce biomimetic porous titanium structures with similar mechanical properties to host bone tissue. In this work, loose sintering and space holder techniques, two frequently used metallurgical techniques, are compared to evaluate the influences of porosity (content, size, morphology and wall roughness), mechanical properties (stiffness and yield strength) and in-vitro cellular responses (adhesion and proliferation of myoblasts and osteoblasts). These comparisons are made to achieve the best balance between biomechanical and bifunctional behavior of a partial porous implant for cortical bone replacement. Cell adhesion (filopodia presence) and spreading were promoted on both porous surfaces and fully dense substrates (non-porous control surfaces). Porous scaffold samples designed using 50 vol.% NaCl space holder technique had an improved bioactive response over those obtained with the loose sintering technique due to higher roughness and scaffold pore diameter. However, the presence of large and heterogeneous pores compromises the mechanical reliability of the implant. Considering both scenarios, the substrates obtained with 40 vol.% NH 4 HCO 3 and pore size ranges between 100 and 200 μm provide a balanced optimization of size and strength to promote in-vitro osseointegration. Keywords: porous titanium; space holder; loose sintering; mechanical behavior; cell adhesion; surface roughness 1. Introduction For many decades, the emphasis of human biomechanics has been on the partial or total replacement of bone tissue with synthetic implants that the body will, in time, integrate as functional parts. Clinical success is achieved when the osteoimplant maximizes the osseointegration and Metals 2020, 10, 696 2 of 21 regeneration of the bone, improving patient health quality. The strategy is for the osteoimplant to provide structural support while reproducing the original stress/strain functionality of the patient’s bone under normal usage. Among all other metallic biomaterials used for bone replacement, titanium (Ti) and its alloys have been recognized as the materials with the best in-vivo and in-vitro performance due to their high mechanical strength, fracture toughness, good corrosion resistance and excellent biological properties [1–7], such as biocompatibility and osteoconductivity (the ability to grow bone tissues over the scaffold surface) [8–10]. However, these materials have shown important drawbacks that compromise the reliability of implants: stress-shielding phenomena [11– 13] and poor osseointegration [14,15]. The stress-shielding effect is caused by the stiffness mismatch between the implant and the surrounding bone (105–110 GPa vs. 2–25 GPa) [16,17]. As a consequence, the implant does not transfer the entire applied load to the bone, which finally promotes bone resorption and implant loosening [18]. In addition, the inert biological character of titanium surfaces results in a poor cellular interaction between Ti and host bone tissue—an outcome that can affect the proper reconstruction of bone, resulting in chronic failure of the implant [19]. For these reasons, the development of a biofunctional Ti implant with enhanced osseointegration for bone tissue replacement remains a challenge to be addressed. The design and manufacturing of implants with lower stiffness are presented as a solution to stress shielding [20–22] and several preliminary efforts have been carried out to achieve implants with a suitable balance between mechanical and biofunctional behavior [23–27]. The reduction of the implant Young’s modulus can be addressed by the design of a porous architecture for titanium-based systems. Among the fabrication options for porous titanium, loose sintering (LS) and space holder (SH) techniques have been identified as low-cost routes [28,29]. However, both techniques result in different porous titanium substrates in terms of mechanical properties and pore design, key factors in bone-implant integration. Furthermore, it is well-known that bone ingrowth with porous structures can vary with the specific pore size of the porous material. Osteoblastic cells are able to attach and produce bone matrix inside pores that preferentially present at least a 100 μm average pore size [26,29]. The introduction of porosity and a rough surface into a biomaterial broadens the scope for possible applications in the biomedical field, because the roughness, higher surface contact and surface free energy have been considered important factors that affect cell adhesion, migration and differentiation [30]. It has been reported that the pores obtained by the space-holder technique and loose sintering have an intrinsic surface roughness, which improves the cell adhesion and inhibits the bacterial attachment [31,32]. Although rough and porous surfaces have positively influenced the fixation and long-term stability of Ti implants [20,33–36], finding the right balance between Young’s modulus and cell interaction remains elusive. Therefore, an excellent approach to improve the reliability of titanium implants and increase clinical success is to design porous titanium implants with an equilibrium of mechanical properties and favorable bioactivity of titanium porous materials [37,38]. In our group, the influences of the spacer particle content (NaCl) on mechanical properties and pore geometry have been studied before [24,39] and we have made a comparison with the LS technique [26]. In addition, the cellular responses of macrophages and osteoblast growing on NH 4 HCO 3 porous scaffolds have been also evaluated, including a chemical surface modification, but limited knowledge is related to the in-vitro cellular response to LS and SH porous substrates. In that sense, we have not investigated the comparison of pore size and morphology, or the effects from total and interconnected porosity that both powder metallurgy (PM) routes have on cell responses. In this work, porous titanium samples obtained by two different powder metallurgy routes (LS, and SH) were fabricated. The influences of content, size range, surface roughness and morphology of the porosity on the mechanical behavior (stiffness and yield strength), and cell adhesion and proliferation, were investigated in detail. 2. Materials and Methods Commercially pure titanium (c.p. Ti) powder produced by a hydrogenation/dehydrogenation process has been used as the starting powder (SE-JONG Materials Co. Ltd., Incheon, Korea). The Metals 2020, 10, 696 3 of 21 titanium powder was equivalent to c.p. Ti Grade IV (ASTM F67-00). Sodium chloride, NaCl (Panreac Química S.A.U., Barcelona, Spain, purity > 99.5%), and ammonium bicarbonate, NH 4 HCO 3 (Cymit Química S.L, Barcelona, Spain, with a purity of 99.9%) were employed as spacers. Two routes were implemented to obtain porous titanium samples: (1) conventional powder metallurgy with no applied pressure (loose sintering, LS) and (2) space-holder technique (SH) with different content and size range of spacer particles. The particle size distributions and the main aspects of the manufacturing routes are summarized in Table 1. In the LS technique, the Ti powder was poured and vibrated into a cylindrical mold of alumina for 2 min and then sintered in a CARBOLYTE STF (Derbyshire, UK) 15/75/450 ceramic furnace with a horizontal tube at two different temperatures (1000 and 1100 °C), for 2 h, under high vacuum conditions (~10 −5 mbar). On the other hand, in the SH route, the mixture of c.p. Ti powder and spaceholder particles (NaCl and NH 4 HCO 3 ) was homogenized using a Turbula ® T2C mixed for 40 min [39,40]. Then the mixture was pressed at 800 MPa in an INSTRON 5505 machine (Instron, MA, USA). Afterward, the elimination of the NaCl spacer was carried out by dissolution on distilled water [39] and removed by thermal evaporation in the case of NH 4 HCO 3 [40]. Finally, the samples were sintered in these cases at 1250 °C, during 2 h and under high vacuum conditions (~10 −5 mbar). Table 1. Manufacturing parameters of porous titanium cylinders. Route Size Distribution, d[10], d[50], d[90] (μm) Compaction Pressure (MPa) Spacer Removal Procedure Sintering Temperature (°C) Titanium Powder Spacer Particles Loose Sintering 9.7, 23.3, 48.4 None 0 None 1000 1100 Space Holder NaCl 30 vol.% 183, 384, 701 800 Distilled water, without stirring, 50 °C, during 16 h 1250 50 vol.% NH 4 HCO 3 30 vol.% 73, 233, 497 Two steps: 60 °C and 110 °C. Both in low vacuum conditions (~10 −2 mbar) and 12 h 50 vol.% 40 vol.% 100-200 119, 184, 286 355-500 355, 424, 564 2.1. Microstructural and Macro-Mechanical Behavior The porosity and mechanical properties of porous Ti samples were characterized using different techniques (at least three specimens have been tested in all cases): (1) Archimedes’ method (ASTM C373-88) which allows the evaluation of the density, and the total and interconnected porosity (P T and P i , respectively). (2) Image analysis (IA), performed with an optical microscope Nikon Epiphot (Nikon, Tokyo, Japan) coupled with a Jenoptik Progres C3 camera (Jenoptik, Jena, Germany), and processed in the software Image-Pro Plus 6.2, Mediacibernetic, Bethesda, MD, USA. IA was evaluated with 10 pictures of 5× and 20× for each processing condition. This technique provides the analysis of (i) the total porosity percentage (P IA ), (ii) equivalent diameter (D eq ) and (iii) pore shape factor (F f ). (3) The uniaxial compression test (Standard ASTM E9-89A and ISO 13314: 2011) allowed the determination of yield strength (σ y ) and Young’s modulus (E c ). (4) The ultrasound technique, which was used to study the dynamic Young’s modulus (E d ) [40,41]. 2.2. In-Vitro Cell Experiments Metals 2020, 10, 696 4 of 21 In order to study the cell viability, adhesion, proliferation and cell morphologies of myoblast and osteoblasts growing in the different porous substrates, cross-sections were prepared from the titanium porous cylinders. The different tests and techniques used to evaluate cellular response, are described in the following scheme—Figure 1. Figure 1. Diagram of in-vitro experimental design. Two murine cell lines, myoblasts (C2C12-GFP) and osteoblasts (MC3T3E1) were used to analyze the biological response to c.p. Ti fully dense and porous substrates. 2.1.1. Cell Adhesion and Proliferation of Myoblast Cells C2C12 cell line (mouse myoblast cell line, CRL-1772 obtained from American Type Culture Collection, ATCC, Manassas, VA, USA) was used to follow cell adhesion and proliferation processes on porous titanium samples. This cell line was transfected previously to constitutively express GFP (green fluorescent protein); thus, samples were evaluated by inverted fluorescent microscope. Cells passaged were carry out using Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% of fetal bovine serum (FBS) plus antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin sulfate) (Invitrogen). Samples were sterilized in an autoclave and placed in a 24-well plate, seeded with 10,000 cells /cm 2 . After 24, 48 and 72 h of incubation, samples were inverted to observe the presence of cells (green fluorescence) attached to the bottom on each well and the porous surfaces using an inverted fluorescent microscope (Olympus IX51). 2.1.2. In-Vitro Evaluation of Osteoblast Response MC3T3E1, a murine pre-osteoblast cell line (CRL-2593, from ATCC, Manassas, VA, USA ), was utilized to analyze the porosity’s effect on cell metabolism and viability during cell adhesion and proliferation process. Routine passaging of the cell line was performed on 25 cm 2 flasks with minimum essential medium (MEM), containing 10% fetal bovine serum plus antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin sulfate) (Invitrogen). Autoclaved porous titanium samples were carefully placed into a 24-well plate, and trypsinized osteoblasts cells were seeded at a cellular density of 30,000 cells/cm 2 per sample. Afterward, 800 μL of prewarmed culture medium was added, Metals 2020, 10, 696 5 of 21 and culture plates were kept at 37 °C in a humidified 5% CO 2 atmosphere. Triplicate blank and TCP (tissue culture plastic) were used as negative and positive controls in the same plate for each time period. Two types of in-vitro experiments were tested: (i) short time experiments to evaluate cell adhesion, proliferation and morphology at 24, 48 and 72 h, and (ii) long-term studies to analyze the cell proliferation and cell morphology of differentiated osteoblasts. These latter experiments were carried out at 1, 4, 7, 14 and 21 days of culturing osteoblasts with osteogenic media (α-MEM medium supplemented with 10 mM ascorbic acid (Merck, Germany) and β-glycerophosphate (StemCell Technologies, Vancouver, BC, Canada) 50 μg/mL). For the evaluation of cell viability and metabolism of osteoblast, AlamarBlue ® reagent (Invitrogen, Waltham, MA, USA) was selected. Briefly, at the end of each culture period, samples were collected to a new 24-well plate to avoid counting non-attached or attached cells on the well plate. New fresh media (800 μL) and 80 μL of AlamarBlue ® reagent were added and the plate was incubated during 1 h 30 min at 37 °C in dark conditions. After that, samples were removed and culture medium was read by fluorescence signal (Biotek FL-600) and absorbance at 570 nm (TECAN, Infinity 200 Pro) for short and long-term studies, respectively. Cell differentiation studies based on alkaline phosphatase (ALP) enzyme quantification were analyzed according to the manufacturer’s protocol (Alkaline Phosphatase Assay kit Colorimetric, Abcam ab83369, Cambridge, UK) of osteoblast cultured on osteogenic media at 4 and 21 days. Basically, ALP kit measures the conversion of a colorless p-nitrophenyl phosphate to a colored p-nitrophenol and the lecture of the absorbance at 405 nm is measured in a 96-well microplate reader. The ALP activity was calculated from a standard curve also measured in the same plate at the same time. Cytoskeletal organization was studied at 72 h using Texas red phalloidin (Molecular Probes) and Hoechst (Thermofisher) as a contrast staining for cell nuclei. In short, cells were rinsed twice with PBS, fixed in 4% paraformaldehyde and permeabilized in 0.1% buffered Triton X-100, and 5 μL/mL from a solution of actin (Texas Red Pahlloidin) in PBS was used for visualization the F-actin for 20 min. After the incubation period, samples were washed with PBS, and images were obtained in an Olympus IX51 microscope. DAPI staining was used to quantify cell nuclei using ImageJ 2.0. software (National Institutes of Health and the Laboratory for Optical and Computational Instrumentation (LOCI, University of Wisconsin, Madison, WI, USA). Finally, scanning electron microscopy (SEM) was performed to evaluate the cell morphology at short (1, 2 and 3 days) and long-term points (7, 14 and 21 days). The samples were fixed in 10% formalin, followed by a dehydration step with ethanolic solutions and coated by gold-coating using a sputter coater (Pelco 91000, Ted Pella, Redding, CA, USA). All micrographs were obtained using a Jeol JSM-6330F scanning electron microscope (Jeol, Tokyo, Japan), and the acceleration voltage was 10 kV for SEM images. 2.1.3. Statistical Analysis All in-vitro experiments were performed in triplicate, with n = 3 for each studied condition. Results were expressed as means and standard deviations to perform a two-way ANOVA followed by a Tukey’s post-test using OriginPro 2019 software (OriginLab, Northampton, MA, USA). Significance was considered at p values of p < 0.05 (*) and p < 0.01 (**). 3. Results Significant progress has been made toward the development of porous c.p. titanium scaffolds for orthopedic and dental applications. Two different porous titanium routes (LS and SH) and two spacer holder particles (NaCl and NH 4 HCO 3 ) were tested to evaluate their influences on the development of porous scaffolds with suitable porosity and mechanical properties. These sintering routes were performed following the Materials and Methods’ description and the ASTM and ISO guidelines. 3.1. Porosity and Mechanical Behavior Table 2 shows Archimedes’ method, image analysis, the compression test and ultrasonic technique results from the samples fabricated using both routes (LS and SH) and different Metals 2020, 10, 696 6 of 21 manufacturing conditions (see Table 1). The porosity evaluation was studied in detail. A representation of the values from both methods is shown in Figure 2. Figure 2. Comparative analysis of the porosity values (P T , P i and P IA ) found in LS and SH porous Ti substrates. The highest porosity values were found in the 50 vol.% NaCl porous substrates. The resultant porosity (P IA ) by IA was similar to the values obtained by Archimedes’ method. Total porosity achieved by the LS route was lower when the temperature was increased (P T = 44.1% at 1000 °C, and P T = 34.3% at 1100 °C). On the other hand, for the SH route, the porosity values found in NaCl were higher, despite the fact it was the same content of spacer as in NH 4 HCO 3 (i.e., for 30 vol.% of NaCl, the P T was 28.5% versus the same percentage, 30 vol.%, of NH 4 HCO 3 that had a P T of 27.8%). This effect was also observed in 50 vol.% of both porous substrates which is explained by the space-holder particle size (see Table 1). Concerning the topographical parameters of the designed porosity, the results demonstrate that: (1) The equivalent diameter (D eq ) was increased when the temperature and/or spacer content increased. This result is associated with the pore coalescence. Additionally, the values of equivalent diameter are consistent with the average sizes of the spacers used. (2) The pore sizes found in SH samples do not allow bone growth towards the interior of the implant, >100 μm being the size reference. (3) The pore size factor (F f ) parameter is a measure of the roundness of the pores (greater when are closer to 1.0). In general, these are greater when the SH method is used. The roundness increases with the sintering temperature (decreases the surface tension), while an increase of the spacer content generates more irregular pores. Regarding the mechanical behavior, the values from the elastic limit and Young’s modulus are consistent with the contents and pore sizes fabricated. In this context, larger pores and in greater proportion decrease yield strength and stiffness, respectively. The loss of mechanical resistance may also be related to the poor quality of the sintering necks (both for low compaction pressures and temperatures) and the increase of the stress concentrator in more irregular pores. Moreover, the reliability of mechanical behavior depends on the population width of the spacer size; it is noted that this generally increases for narrower populations. In general, in samples obtained with 40 vol.% NH 4 HCO 3 and range size of 100–200 μm, obtained by SH (235 MPa and 45.3 GPa), provide a better scenario for the replacement of cortical bone ( y = 150–180 MPa and E = 20–25 GPa). Metals 2020, 10, 696 7 of 21 Table 2. Values obtained from Archimedes’ method, image analysis (IA) and estimated mechanical behavior. Physical and Mechanical Properties Loose Sintering Space Holder NaCl NH 4 HCO 3 1000°C 1100°C 30 (vol.%) 50 (vol.%) 30 (vol.%) 40 (vol. %) 50 (vol.%) 100– 200 μm 355– 500 μm Archimedes’ method P T (%) 44.1 ± 1.7 34.3 ± 0.5 28.5 ± 1.0 47.2 ± 0.9 27.8 ± 0.9 40.2 ± 0.6 40.8 ± 0.5 44.7 ± 0.8 P i (%) 42.1 ± 5.1 31.0 ± 4.8 20.5 ± 0.8 44.1 ± 0.9 22.4 ± 0.9 27.9 ± 0.8 32.9 ± 0.7 38.5 ± 0.8 Density (g/cm 3 ) 2.5 ± 0.2 2.8 ± 0.2 3.2 ± 0.2 2.2 ± 0.2 3.6 ± 0.2 2.69 ± 0.02 2.67 ± 0.02 2.50 ± 0.2 Image analysis P IA (%) 43.0 ± 1.2 36.9 ± 4.0 28.4 ± 4.6 47.9 ± 3.1 29.1 ± 3.8 41.6 ± 3.3 43.8 ± 2.4 44.0 ± 4.0 D eq (μm) 16 ± 17 19 ± 16 370 ± 244 392 ± 260 230 ± 202 226 ± 178 295 ± 287 245 ± 223 F f 0.76 ± 0.11 0.85 ± 0.09 0.81 ± 0.13 0.70 ± 0.14 0.83 ± 0.11 0.71 ± 0.12 0.67 ± 0.06 0.79 ± 0.08 Uniaxial Compression E c (GPa) 13.4 ± 6 21.0 ± 4 6.6 ± 5 5.2 ± 5.0 15.9 ± 4.1 - 8.5 ± 7.0 σ y (MPa) 128 ± 6 206 ± 5 323 ± 10 117 ± 12 298 ± 8 235 ± 6 190 ± 5 149 ± 9 Ultrasound E d (GPa) 42.2 ± 1.0 52.1 ± 1.2 58.9 ± 0.9 39.5 ± 1.3 59.8 ± 1.2 45.9 ± 0.7 45.3± 0.8 41.7 ± 1.0 3.2. Cell Adhesion and Proliferation Porous titanium samples fabricated by LS and SH result in a variety of reduced Young´s moduli as a function of porosity size and distribution (interconnected, total and pore density). It is wellknown that surface parameters affect the cell interactions at the bio-interface. Therefore, the study of cellular response correlated to the mechanical properties of porous titanium should be assessed. First, porous c.p. Ti substrates were seeded with C2C12-GFP cells in order to evaluate cell adhesion, distribution and proliferation during 24–72 h of cell incubation; see Figure 3. Images of fluorescence microscopy were taken every day to compare the main differences in cell growth in fully dense, LS and SH (30 vol.% and 50 vol.% NaCl) substrates. It can be observed that C2C12-GFP cells were attached in all titanium substrates at 24 h. The cell density increased over time, highlighting the suitable biocompatibility of both sintering porous routes. These micrographs demonstrated differences in cell distribution that were even noticeable at 24 h of study. A fully dense surface presented a dense cellular monolayer located in the center of the sample. Cells growing on LS and SH substrates were highly disperse, covering almost the entire surface area. This cell behavior was highlighted at 72 h of cell proliferation in which an increased cell density was reached on LS and SH_50%NaCl surfaces compared to SH_30%NaCl and fully dense. Metals 2020, 10, 696 8 of 21 Figure 3. Inverted fluorescence micrographs of C2C12-GFP cells seeded onto fully dense and porous titanium samples fabricated by LS and SH using NaCl spacer particles at 24, 48 and 72 h. An increase in cell attachment is observed in LS and SH_50%NaCl over the entire surface which contrasts to the cells just located at the center of fully dense samples, confirming lower cell migration at 72 h. Figure 4 showed, at higher magnification, images of C2C12-GFP cells growing at 24, 48 and 72 h of cell incubation. These images corroborate the cell behavior described above and point out the differences between pores and flat surfaces. All porous substrates presented higher surface contact area due to the porosity designed compared to non-porous control samples. Indeed, 50% of total porosity surfaces showed higher cell attachment and cell migration compared to lower porosity levels, 30%, and control Ti surfaces. These factors, roughness and porosity promoted the increased cell adhesion on titanium scaffolds. Moreover, an irregular pore morphology with higher interconnected porosity (P i ) facilitates surface exposure to cells during the adhesion process, as SH_50%NaCl revealed. Metals 2020, 10, 696 9 of 21 Figure 4. Inverted fluorescence micrographs of C2C12-GFP cells seeded onto fully dense and porous titanium samples fabricated by LS and SH using NaCl spacer particles at 24, 48 and 72 h. An increase of cell adhesion and cell density is observed through the cell incubation time, as expected for cells that are well attached and in a proliferation stage. It is well-known that the osseointegration process of titanium implants is influenced by the surface properties, the topography and the chemical nature of the material. This osseointegration has been improved by the development of rough surfaces or porous substrates, or by implementing bioactive coatings [42–44]. Commercial implants have been subjected to different surface modification treatments to produce rough surfaces using different methods (mechanical, chemical, sol-gel, anodic oxidation, physical and biochemical) described elsewhere [37,45,46]. While porous substrates offer a higher surface contact area, rough surfaces facilitate the cell anchorage and adhesion process. Indeed, many research studies have reported the relationship between roughness (Sa) and cell adhesion [44,45]. In this study, all specimens were polished to a mirror-like surface— reaching Sa values of 0.7 μm on flat surfaces found in fully dense and on porous substrates, while the internal walls of the pores revealed higher Sa values (i.e., 5 μm in SH_50%NH 4 NCO 3 ) [20]. Besides the roughness difference, the pore geometry has an impact on cellular attachment. Under this Metals 2020, 10, 696 16 of 21 μm) achieved similar ALP level to fully dense surfaces, but ALP increased at 21 days on the porous scaffolds and lower enzymatic activity was observed on the fully dense surface. Figure 10. Cell differentiation of osteoblasts on porous scaffolds SH_NH 4 HCO 3 -40%. In-vitro evaluation of alkaline phosphatase enzyme (ALP) activity measured at 4 and 21 days of cell culture in osteogenic media. Statistical differences are indicated at p < 0.05 *. This increased cell differentiation was contrasted through SEM analysis in all studied specimens. Figure 11 shows the SEM micrographs of osteoblasts growing on Ti substrates. These SEM images showed the osteoblast cell expansion over all surfaces for 7, 14 and 21 days of cell culture. It was observed that osteoblastic cells were well attached and spread onto the surfaces and inside porous in all samples. Detailed observations of the interactions, cell–cell and osteoblasts–surfaces, demonstrated the interconnectivity of the matrix (marked with yellow arrows and asterisks in Figure 11). The cytoplasmatic extensions created bridges across the pores and enlarged filopodia and lamellipodia protrusions which are presented on porous surfaces. The increased cell density on porous samples compared to fully dense ones is remarkable, which at 14 days, still revealed empty areas in the surface with no attached cells. However, at 21 days all surfaces appeared to be completely covered by MC3T3E1 cells (covering pores) and surrounded by bone matrix. Metals 2020, 10, 696 17 of 21 Figure 11. SEM micrographs of the 7, 14 and 21 days of osteoblast culture growing on porous SH_NH 4 HCO 3 -40%, 100–200 μm and 355–500 μm. Cell morphology and osteoblast proliferation are shown on SH surfaces. The pores covered by osteoblasts at 7 days in SH substrates should be noticed. Cell–cell interaction (white arrow), a cell-surface junction (yellow arrow) and hydroxyapatite (red asterisks) are indicated in the images. The number of cells detected was greater in the sample of SH_NH 4 HCO 3 -40% (100–200 μm) since there are pores completely covered by cells at 14 days. The cells adhered through filopodia (fine cellular projections) marked by the white arrow in Figure 11, and lamellipodia (wider extensions), demonstrating the connection with the biomaterial. Fine extensions allowing the cell–cell connection are also distinguished. From the 14th day of cell culture, the elevated presence of vesicles on the cell surface was highlighted. They are produced by polarized sprouting from the surface of the osteoblast, suggesting the development of a nutrient bone ECM. Many vesicles were observed due to the medium containing β-glycerophosphate (organic phosphate), a substrate of alkaline phosphatase that induces an increase of inorganic phosphates in the medium that will transform into hydroxyapatite marked with a red asterisk in Figure 11 [54,55]. On the 21st day, fully dense Ti and SH_NH 4 HCO 3 -40% (350–500 μm) discs showed hexagonal structures that suggest a possible nucleation of hydroxyapatite (see Figure 11, red asterisks). In the SH_NH 4 HCO 3 -40% (100–200 μm) samples, a cell monolayer of mature osteoblast was observed. In previous studies, we have demonstrated that osteoblasts in the samples of 40% space-holder (SH_NH 4 HCO 3 -40%) were well attached to peaks inside the pores or among depressions, which appear separated by a length that is similar to osteoblast diameter (~20 μm), and the samples with the highest space-holder contents (60 and 70 vol.%) did not show such proper shape of well-attached cells; therefore, the large sizes of pores also could be negative for the osteoblast proliferation [47]. Our results agreed with other authors; they have reported that different ranges of porosity can promote different osteoblast functions. Conway et al. showed that Ti scaffolds with a pore range between 45 Metals 2020, 10, 696 18 of 21 and 106 μm presented the best microarchitecture for early stages of osteoblast attachment, whereas a pore size higher than 300 μm exhibited the most favorable conditions for cell proliferation [56]. 4. Conclusions In this study, we have correlated the porosity designed by two different routes, LS and SH, and the mechanical and cellular behavior of porous titanium substrates towards bone osseointegration. In summary, both techniques are simple, economical and easily reproducible for fabricating porous materials as potential substitutes for cortical bone tissue. However, the sizes and contents of the pores obtained using the LS route are insufficient (D eq = 19 μm) to guarantee the bone ingrowth (>100 μm) and to reach appropriate stiffness values (E = 52 GPa versus E Cortical bone = 20–25 GPa), while the use of large and heterogeneous (wider populations) spacers compromises the reliability mechanical ( y = 180 MPa) of the implant. In-vitro tests show a successful adhesion and spreading of the C2C12-GFP and MC3T3 cell lines (on a flat surface and inside pores of porous samples) confirming the biocompatibility and non-toxicity of both sintering routes. Moreover, both methods enhanced cell adhesion, spreading, proliferation and differentiation of osteoblasts compared to fully dense. However, the use of SH provides bigger pore size, with an irregular pore geometry and higher interconnected porosity, factors that influence cell differentiation of osteoblast. In that sense, SH_NH 4 HCO 3 -40% (100–200 μm) reached a higher cellular metabolic activity and increased alkaline phosphatase levels at 21 days compared to fully dense and SH_NH 4 HCO 3 -40% (355–500 μm). In this context, the substrates obtained with 40 vol.% NH 4 HCO 3 and ranges size of 100–200 μm, are those that offer suitable biomechanics (E = 45.3 GPa and  y = 235 MPa) and biofunctional behavior balance to promote in-vitro osseointegration and solve the stress shielding phenomenon, key factors for the improvement of clinical success of titanium implants. Author Contributions: Conceptualization, project administration, supervision and methodology, M.-J.M.-G., J.P.A. and Y.T.; investigation, formal analysis and validation A.C., M.G., P.T., S.L., C.A. and M. Á .V.; discussion and writing — original draft preparation, all the authors. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the regional government from Andalusia through FEDER-Junta de Andalucía Research Project, reference US-1259771 (modeling and implementation of the freeze casting technique: gradients of porosity with a tribomechanical equilibrium and electro-stimulated cellular behavior). Acknowledgments: The authors thank to laboratory technicians Jesus Pinto and Mercedes Sánchez for their technical support and the Tissue Engineering Group (TEG) from University Complutense of Madrid (UCM) for kindly providing the cell line C2C12-GFP. Conflicts of Interest: The authors declare no conflict of interest. References 1. Brunette, D.M.; Tengvall, P.; Textor, M.; Thomsen, P. Titanium in Medicine: Materials Science, Surface Science, Engineering, Biological Responses and Medical Applications; Springer: Berlin, German, 2001. 2. Van-Noort, R. Titanium: The implant material of today. J. Mater. Sci. 1987, 22, 3801. 3. Williams, D.F. On the mechanism of biocompatibility. Biomaterials 2008, 29, 1–13. 4. He, G.; Liu, P.; Tan, Q. Porous titanium materials with entangled wire structure for load-bearing biomedical applications. J. Mech. Behav. Biomed. Mater. 2012, 5, 16–31. 5. Dizlek, M.E.; Guden, M.; Turkan, U.; Tasdemirci, A. Processing and compression testing of Ti6Al4V foams for biomedical applications. J. Mater. Sci. 2009, 44, 1512. 6. Zhao, X.; Sun, H.; Lan, L.; Huang, J.; Zhang, H.; Wang, Y. Pore structures of high-porosity NiTi alloys made from elemental powders with NaCl temporary space-holders. Mater. Lett. 2009, 63, 2402. 7. Ryan, G.E.; Pandit, A.S.; Apatsidis, D.P. Fabrications Methods of porous metals for use in orthopaedic applications. Biomaterials 2006, 27, 2651–2670. 8. Bram, M.; Stiller, C.; Buchkremer, H.P.; Stover, D.; Baur, H. High-porosity titanium, stainless steel, and superalloy parts. Adv. Eng. Mater. 2000, 2, 196–199. 9. Dunand, D.C. Processing of titanium foams. Adv. Eng. Mater. 2004, 6, 369–376. Metals 2020, 10, 696 19 of 21 10. Wen, C.E.; Yamada, Y.; Shimojima, K.; Chino, Y.; Asahina, T.; Mabuchi, M. Processing and Mechanical Properties of Autogenous Titanium Implant Materials. Mater. Sci. Mater. Med. 2002, 13, 397–401. 11. Singh, R.; Lee, P.D.; Dashwood, R.J.; Lindley, T.C. Titanium foams for biomedical applications: a review. Mater. Technol. 2010, 25, 127–136. 12. Niinomi, M.; Nakai, M.; Hieda, J. Development of new metallic alloys for biomedical applications. Acta. Biomater. 2012, 8, 3888–3903. 13. Gepreel, M.A.-H.; Niinomi, M. Biocompatibility of Ti-alloys for long-term implantation. J. Mech. Behav. Biomed. Mater. 2013, 20, 407–415. 14. Stamboulis, A.G.; Boccaccini, A.R.; Hench, L.L. Novel biodegradable polymer/bioactive glass composites for tissue engineering applications. Adv. Eng. Mater. 2002, 4, 105–109. 15. Domínguez-Trujillo, C.; Ternero, F.; Rodríguez-Ortiz, J.A.; Heise, S.; Boccaccini, A.R.; Lebrato, J.; Torres, Y. Bioactive coatings on porous titanium for biomedical applications. Surf. Coat. Tech. 2018, 349, 584–592. 16. Hsu, H.-C.; Hsu, S.-K.; Wu, S.-C.; Wang, P.-H.; Ho, W.-F. Design and characterization of highly porous titanium foams with bioactive surface sintering in air. J. Alloys Compd. 2013, 575, 326–332. 17. Ye, B.; Dunand, D.C. Titanium foams produced by solid-state replication of NaCl powders. Mater. Sci. Eng., A 2010, 528, 691–697, doi:10.1016/j.msea.2010.09.054. 18. Li, J.; Jansen, J.A.; Walboomers, X.F.; van den Beucken, J.J. Mechanical aspects of dental implants and osseointegration: A narrative review. J. Mech. Behav. Biomed. Mater. 2019, 103, 103574. 19. Chen, Y.; Frith, J.E.; Dehghan-Manshadi, A.; Kent, D.; Bermingham, M.; Dargusch, M. Biocompatible porous titanium scaffolds produced using a novel space holder technique. J. Biomed. Mater. Res. Part B 2018, 106, 2796–2806. 20. Civantos, A.; Domínguez, C.; Pino, R.J.; Setti, G.; Pavon, J.J.; Martínez-Campos, E.; Garcia, F.J.G.; Rodriguez, J.A.; Allain, J.P.; Torres, Y. Designing bioactive porous titanium interfaces to balance mechanical properties and in vitro cells behavior towards increased osseointegration. Surf. Coat. Technol. 2019, 368, 162–174. 21. Schmidutz, F.; Agarwal, Y.; Müller, P.; Gueorguiev, B.; Richards, R.; Sprecher, C. Stress-shielding induced bone remodeling in cementless shoulder resurfacing arthroplasty: a finite element analysis and in vivo results. J. Biomech. 2014, 47, 3509–3516. 22. Dabrowski, B.; Swieszkowski, W.; Godlinski, D.; Kurzydlowski, K.J. Highly porous titanium scaffolds for orthopaedic applications. J. Mech. Behav. Biomed. Mater. B: Appl. Biomater. 2010, 95, 53–61. 23. Herrera, A.; Yánez, A.; Martel, O.; Afonso, H.; Monopoli, D. Computational study and experimental validation of porous structures fabricated by electron beam melting: A challenge to avoid stress shielding. Mater. Sci. Eng. C 2014, 45, 89–93. 24. Muñoz, S.; Castillo, S.; Torres, Y. Different models for simulation of mechanical behaviour of porous materials. J. Mech. Behav. Biomed. Mater. 2018, 80, 88–96. 25. Arabnejad, S.; Johnston, B.; Tanzer, M.; Pasini, D. Fully porous 3D printed titanium femoral stem to reduce stress-shielding following total hip arthroplasty. J. Orthop. Res. 2017, 35, 1774–1783. 26. Lascano, S.; Arévalo, C.; Montealegre-Melendez, I.; Muñoz, S.; Rodriguez-Ortiz, J.A.; Trueba, P.; Torres, Y. Porous Titanium for Biomedical Applications: Evaluation of the Conventional Powder Metallurgy Frontier and Space-Holder Technique. Appl. Sci. 2019, 9, 982. 27. Pavón, J.J.; Trueba, P.; Rodríguez-Ortiz, J.A.; Torres, Y. Development of new titanium implants with longitudinal gradient porosity by space-holder technique. J. Mater. Sci. 2015, 50, 6103–6112. 28. Miao, X.; Sun, D. Graded/Gradient Porous Biomaterials. Materials 2010, 3, 26–47. 29. Torres, Y.; Lascano, S.; Bris, J.; Pavón, J.; Rodriguez, J.A. Development of porous titanium for biomedical applications: A comparison between loose sintering and space-holder techniques. Mater. Sci. Eng. C 2014, 37, 148–155, doi:10.1016/j.msec.2013.11.036. 30. Subramani, K.; Mathew, R.T.; Pachauri, P. Titanium surface modification techniques for dental implants— from microscale to nanoscale. In Emerging Nanotechnologies in Dentistry; Elsevier: Amsterdam, The Netherlands, 2018; pp. 99–124. 31. Domínguez-Trujillo, C.; Ternero, F.; Rodríguez-Ortiz, J.A.; Pavón, J.J.; Montealegre-Meléndez, I.; Arévalo, C.; García-Moreno, F.; Torres, Y. Improvement of the balance between a reduced stress shielding and bone ingrowth by bioactive coatings onto porous titanium substrates. Surf. Coat. Technol. 2018, 338, 32–37. Metals 2020, 10, 696 20 of 21 32. Domínguez-Trujillo, C.; Beltrán, A.M.; Garvi, M.D.; Salazar-Moya, A.; Lebrato, J.; Hickey, D.J.; RodríguezOrtiz, J.A.; Kamm, P.H.; Lebrato, C.; García-Moreno, F. Bacterial behavior on coated porous titanium substrates for biomedical applications. Surf. Coat. Technol. 2019, 357, 896–902. 33. Takemoto, M.; Fujibayashi, S.; Neo, M.; So, K.; Akiyama, N.; Matsushita, T.; Kokubo, T.; Nakamura, T. A porous bioactive titanium implant for spinal interbody fusion: an experimental study using a canine model. J. Neurosurg. Spine. 2007, 7, 435. 34. Scislowska-Czarnecka, A.; Menaszek, E.; Szaraniec, B.; Kolaczkowska, E. Ceramic modifications of porous titanium: effects on macrophage activation. Tissue Cell 2012, 44, 391–400. 35. Borjas, S.; Gil, E.J.; Cordero, L.; Pavón, J.J.; Rodriguez-Ortiz, J.A.; Boccaccini, A.R.; Torres, Y. Electrophoretic deposition and characterization of chitosan/45S5 bioactive glass composite coatings on porous titanium for biomedical applications. Key Eng. Mater. 2015, 654, 189–194. 36. Tobin, E.J. Recent coating developments for combination devices in orthopedic and dental applications: a literature review. Adv. Drug Deliv. Rev. 2017, 112, 88–100. 37. Civantos, A.; Allain, J.P.; Pavón, J.J.; Shetty, A.; El-Atwani, O.; Walker, E.; Arias, S.L.; Gordon, E.; Rodríguez-Ortiz, J.A.; Chen, M., et al. Directed Irradiation Synthesis as an Advanced Plasma Technology for Surface Modification to Activate Porous and “as-received” Titanium Surfaces. Metals 2019, 9, 1349. 38. Wang, M.; Tang, T. Surface treatment strategies to combat implant-related infection from the beginning. J. Orthop. Transl. 2019, 17, 42–54. 39. Torres, Y.; Pavón, J.; Rodríguez, J. Processing and characterization of porous titanium for implants by using NaCl as space holder. J. Mater. Process. Technol. 2012, 212, 1061–1069. 40. Torres, Y.; Rodríguez, J.A.; Arias, S.; Echeverry, M.; Robledo, S.; Amigó, V.; Pavón, J.J. Processing, Characterization and biological testing of porous titanium obtained by space-holder technique. J. Mater. Sci. 2012, 47, 6565–6576. 41. Kikuchi, M.; Takahashi, M.; Okuno, O. Elastic moduli of cast Ti–Au, Ti–Ag, and Ti–Cu alloys. Dent. Mater. 2006, 22, 641–646. 42. Schwarz, M.L.; Kowarsch, M.; Rose, S.; Becker, K.; Lenz, T.; Jani, L. Effect of surface roughness, porosity, and a resorbable calcium phosphate coating on osseointegration of titanium in a minipig model. J. Biomed. Mater. Res. A 2009, 89, 667–678. 43. Le Guéhennec, L.; Soueidan, A.; Layrolle, P.; Amouriq, Y. Surface treatments of titanium dental implants for rapid osseointegration. Dent Mater. 2007, 23, 844–854. 44. Civantos, A.; Martinez-Campos, E.; Ramos, V.; Elvira, C.; Gallardo, A.; Abarrategi, A. Titanium coatings and surface modifications: toward clinically useful bioactive implants. ACS Biomater. Sci Eng. 2017, 3, 1245– 1261. 45. Martin, J.; Dean, D.D.; Cochran, D.L.; Simpson, J.; Boyan, B.; Schwartz, Z. Proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63) cultured on previously used titanium surfaces. Clin Oral Implants Res. 1996, 7, 27–37. 46. Izman, S.; Abdul-Kadir, M.R.; Anwar, M.; Nazim, E.; Rosliza, R.; Shah, A.; Hassan, M. Surface Modification Techniques for Biomedical Grade of Titanium Alloys: Oxidation, Carburization and Ion Implantation Processes. In Titanium Alloys–Towards Achieving Enhanced Properties for Diversified Applications; Nurul Amin, A.K.M., Ed.; Books on Demand: Rijeka, Croatia, 2012; pp. 201–228. 47. Muñoz, S.; Pavón, J.; Rodríguez-Ortiz, J.A.; Civantos, A.; Allain, J.P.; Torres, Y. On the influence of space holder in the development of porous titanium implants: Mechanical, computational and biological evaluation. Mater. Charact. 2015, 108, 68–78. 48. do Prado, R.F.; de Oliveira, F.S.; Nascimento, R.D.; de Vasconcellos, L.M.R.; Carvalho, Y.R.; Cairo, C.A.A. Osteoblast response to porous titanium and biomimetic surface: In vitro analysis. Mater. Sci. Eng. C 2015, 52, 194–203. 49. Li, G.; Wang, L.; Pan, W.; Yang, F.; Jiang, W.; Wu, X.; Kong, X.; Dai, K.; Hao, Y. In vitro and in vivo study of additive manufactured porous Ti6Al4V scaffolds for repairing bone defects. Sci. Rep. 2016, 6, 34072. 50. Romero-Gavilan, F.; Sánchez-Pérez, A.M.; Araújo-Gomes, N.; Azkargorta, M.; Iloro, I.; Elortza, F.; Gurruchaga, M.; Goñi, I.; Suay, J. Proteomic analysis of silica hybrid sol-gel coatings: a potential tool for predicting the biocompatibility of implants in vivo. Biofouling 2017, 33, 676–689. 51. Vilardell, A.; Cinca, N.; Garcia-Giralt, N.; Dosta, S.; Cano, I.; Nogués, X.; Guilemany, J. Osteoblastic cell response on high-rough titanium coatings by cold spray. J. Mater. Sci. Mater. Med. 2018, 29, 19. Metals 2020, 10, 696 21 of 21 52. Caparros, C.; Guillem-Martí, J.; Molmeneu, M.; Punset, M.; Calero, J.; Gil, F. Mechanical properties and in vitro biological response to porous titanium alloys prepared for use in intervertebral implants. J. Mech. Behav. Biomed. Mater. 2014, 39, 79–86. 53. Otsuki, B.; Takemoto, M.; Fujibayashi, S.; Neo, M.; Kokubo, T.; Nakamura, T. Pore throat size and connectivity determine bone and tissue ingrowth into porous implants: three-dimensional micro-CT based structural analyses of porous bioactive titanium implants. Biomaterials 2006, 27, 5892–5900. 54. Anderson, H.C. Molecular biology of matrix vesicles. Clin. Orthop. Relat. Res. 1995, 266–280. 55. Anderson, H.C.; Garimella, R.; Tague, S.E. The role of matrix vesicles in growth plate development and biomineralization. Front. Biosci. 2005, 10, 822–837. 56. Torres-Sanchez, C.; Al Mushref, F.; Norrito, M.; Yendall, K.; Liu, Y.; Conway, P.P. The effect of pore size and porosity on mechanical properties and biological response of porous titanium scaffolds. Mater. Sci. Eng., C 2017, 77, 219–228. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). View publication statsView publication stats