Considerations Regarding Sandblasting of Ti and Ti6Al4V Used in Dental Implants and Abutments as a Preconditioning Stage for Restorative Dentistry Works

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Sandblasting dental implants and abutments prior to their use in restorative dentistry is an important preconditioning stage for several compelling reasons: this process greatly improves the longevity, functionality, and success of dental restorations because it strongly affects surface topography, helps remove unwanted contaminants, aids wettability, and promotes a better mechanical interlocking between the abutments and the restorative works.

Abstract

Sandblasting materials used for dental restoration are a valuable preconditioning technique that enhances the physical properties and promotes osseointegration and cell adhesion. Triplicate groups of Ti medical grade 4 and Ti6Al4V were blasted with 16 series of various naturally occurring and synthetically produced spraying materials of controlled granulometry at three spraying durations each and two spraying pressures, and the results were tested for the determination of the surface roughness taken as an average of 80 points ±5 points for each particular series of operating parameters. SEM analysis and specific tests to see whether or not cell cultures proliferate on the treated materials were also conducted. It was found that in all cases, regardless of the spraying material or working conditions, the roughness profile achieved is a uniformly distributed one. A reduction in the blasting pressure by half led to a decrease in the roughness between 30 and 35%. The use of glass balls as blasting material led to decreased roughness and more uniformly distributed roughness values for Ti as well as for Ti6Al4V, regardless of spraying duration or applied pressure compared to other spraying materials. Blasting with olivine led to increased, as well as uniformly distributed, values, and hence the conclusion that one may control the roughness size by choosing one or another of the above materials without the need to change any other operating parameters. In the case of Ti, the achieved roughness is greater than in the case of Ti4Al6V, regardless of the blasting material; the differences are smaller the softer the sandblasting material due to the fact that Ti alloys have better mechanical properties and increased hardness compared to pure Ti. SEM analysis showed that the use of sintered hydroxyapatite as an additive to the blasting material does not necessarily lead to a substantial deposition of hydroxyapatite on the substrate materials; only traces of it were identified during the analysis. As a general conclusion, this study showed that by sandblasting Ti and Ti6Al4V with different spraying materials, one may control the surface roughness, and this technique may be an attractive method for preconditioning these materials for restorative dentistry.

1. Introduction

Sandblasting is a mechanical process carried out by spraying various-sized particles of controlled known hardness at constant pressure and speed rate (changeable at will) onto the surface of a certain material in order to clean and/or modify its surface roughness [1,2,3,4,5,6,7].

The importance of sandblasting resides in its inherent properties and the envisaged outcome: enhanced surface roughness and increased surface area, essential for mechanical interlocking between the implant and the abutment in the case of restorative dentistry, thorough surface cleanliness, increased wettability, and effective mechanical retention [1,2,3,4,5,8,9,10,11,12,13,14,15].

The recent trend in dental implant and abutment research is focused on the effect of implant surface functionalization as a means to increase success chances. Some researchers concentrated on sandblasting as a preconditioning phase in the modification of the surface of dental implants and abutments to increase osseointegration and biofilm adhesion to them [1,2,3,4,5,6,7,15,16,17,18]. This comes as a direct result of addressing one of the main concerns of the scientific community related to the limitation of the risks associated with implant rejections and the development of peri-implantitis (a pathological condition occurring in tissues around the dental implants, characterized by inflammation in the peri-implant connective tissue and progressive loss of supporting bone) [1,2,3,4,15,16,17,18].

As the challenge for researchers is to develop new technologies or materials with the necessary properties for osseointegration while simultaneously guaranteeing a reduced accumulation of bacteria in the vicinity of the abutment/implant [5], the use of titanium and its alloys as the main material for this appears to be well justified. The techniques developed for surface modification are due to the Ti inherent properties to oxidize naturally to a stable oxide layer of a few nm, thus preventing corrosion and avoiding the associated inflammatory processes, resulting in a positive impact on osseointegration, durability, and wear of invasive or non-invasive dental restorations [5,15,16,18].

Any change in the appearance of the material surface can lead to cell adhesion and multiplication, morphology proliferation, and differentiation of the cells interacting with the implants at the implant/tissue interface, so that obtaining an optimal surface roughness of the implants facilitates osseointegration and cell adhesion from an early stage and determines the viability of the implant [6].

The material surface in the discussion may be processed using various methods (e.g., polishing, sandblasting, and acid etching) in order to obtain a certain desired roughness morphology that would lead to the improvement of the bioactivity of the surface [1,2,4,5,15,16,17,18].

The sandblasting technology is influenced by the type of abrasive material, the shape and size of the grains, the conditions set forth for the deposited material layer, and the process parameters [5,6,7].

The sandblasting process involves the projection of irregular abrasive particles (Al₂O₃, either naturally occurring or synthetically produced) on the substrate surface at very high speeds. After sandblasting, the surfaces are blown with a jet of dry and clean air to remove any particles of dust and traces of the material, degreased, and rinsed again, as the impeccable cleaning of the materials after sandblasting is vital for the viability of the dental implant. The most commonly used material for sandblasting titanium implants and abutments is Al₂O₃, either naturally occurring or synthetically produced [8].

The surface of most implants used in oral implantology is normally microtextured, obtained by sandblasting and subsequent processing using acids, resulting in uniformly distributed microgaps at the implant/abutment interface [9,10,11,12].

Studies by various authors concluded that microlacunae are inevitable and contribute to the onset of peri-implant disease, and even if the healing abutments are inserted for a short period, their interfaces with the implant are sensitive to bacterial colonization [11,13].

Kim et al. studied micro-leakage occurring at abutment interfaces carried out on a large number of implants, some of them being sandblasted, and they showed that the outcome from sandblasting is beneficial in seal maintenance [14].

Understanding the implant morphology and manufacturing technology is very important to explain the impact on micro-leakage, as the characteristics of the particles and the parameters of the sandblasting process are variables that greatly influence the surface roughness [2,15,16,17]. Balza et al. investigated the influence of the sandblasting treatment and the behavior of Al₂O₃ abrasive particles in changing the surface properties of Ti6Al4V samples, contributing to a better understanding of the link between the properties of the material surface and the characteristics of the sandblasting agent used [15].

Other studies reported in the literature correlating the influence of the particle size of the sandblasting material and the surface topography of the abutments/implants showed that sandblasting commercially Ti grade 1 [CpTi] and Ti6Al4V alloy promoted a remarkable soft tissue adhesion [2,17].

Yabe et al. and Supriadi analyzed the effects of sandblasting parameters in the case of CpTi in terms of roughness and material deformations. An in vivo study using a shear adhesion test on mouse dermis was also carried out, and the conclusion was that sandblasting and acid treatment of the sandblasted surface improved cell adhesion on functionalized CpTi [6,18].

There are major differences between epithelial attachment around dental abutments/implants and around natural dentition: the deposition of the thin epithelial layer around implants and abutments attracts, in a large number of cases, the formation of bacterial colonies (mainly in cases of poor hygiene care) and causes inflammation of the tissues, as well as resorption of the dental alveolus, similar to periodontal disease cases [3,7,19].

To prevent this inflammation, one has to identify and eliminate all the risk factors involved in this process [7,8,10]. The complications induced by bacterial infections and associated with dental implants, which eventually lead to peri-implantitis, may be avoided if one succeeds in creating a very good epithelial seal on the abutment/implant surface.

As the roughness of the implant/abutments surface influences the growth of the epithelium (animal tests showed the presence of a peri-implant zone with a width of about 3.5 mm around the untreated titanium abutments), it is advisable to process these surfaces (by sandblasting, acid etching, or chemical functionalization) to increase the chances of a successful implant procedure [6,17,19].

Kohal et al. and Vigolo et al. reported the formation of healthy soft tissue around titanium and zirconium oxide surfaces compared to the surface of gold alloy abutments [20,21].

The long-term success of an implant is conditioned by achieving a good seal around the abutment/implant and a good healing of the tissue around the transmucosal area of the abutment. This is conditioned by the formation in the initial stages of a blood clot and a mild inflammatory process that promotes new healthy tissue formation; the physical and chemical characteristics of the material surfaces directly influence the bacterial profile accumulated on their surface, as well as the quality of the interface between them and the soft tissue [20,21,22,23]. A favorable surface for the above-mentioned process may be achieved by sandblasting the surface of titanium implants and abutments, followed by various chemical treatments, so that the chances of attaching the epithelium or fibroblast cells are improved, obtaining a good tightness of the peri-implant soft tissue [3,18,22,23].

Sandblasted implant surfaces exhibit a micro-roughness morphology, resulting in enhanced surface energy and hydrophilicity with a positive impact on osteoblast activity and bone formation processes, leading to better osseointegration; cells grown on smooth surfaces tended to exhibit higher levels of proinflammatory mediators and peri-implant inflammations compared to moderately rough surfaces obtained by sandblasting [21,22,23].

The research carried out by L. Monsalve-Guil et al. revealed a survival rate of implants of 97.1% after 90 days from the date of applying the abutment, and after 17 years of monitoring, the risk of failure was by up to 80% lower in the case of sandblasted implants compared to normal ones [7].

The purpose of this work was to investigate the effect of various blasting/spraying materials of controlled size distribution and working conditions (spraying pressure, spraying time) on the surface roughness of Ti medical grade 4 and Ti6Al4V and whether or not such blasted surfaces allow gingival cell proliferation. The significance of this work is particularly important as Ti and Ti6Al4V remain, in spite of a series of novel materials such as zirconium and zirconia introduced recently, some of the most used materials in dental restoration for the manufacturing of dental implants and dental abutments.

2. Materials and Methods

The Ti disks used in this study were made of pure Titanium medical grade 4 (TICp4), Signer Titanium AG, Freienbach, Swiss, having a diameter of 6 mm, 1 mm thickness, while Ti alloy disks were made of Ti6Al4V, Dynamet USA, Signer Titanium AG Swiss (Ti max 90%, Al max 6%, V max 4%, Fe max 0.25%, O max 0.2%) with a diameter of 8 mm, 1 mm thickness. The disks were cut using a L20 8M CNC lathe, Citizen Cincom, Esslingen Germany, with 6 linear axes and two rotary axes. The slight difference in the diameter size is due to the fact that the original rods used to cut the disks were available for purchase only in the above-specified diameter dimensions.

A comparison of the physical and mechanical properties of Ti and Ti6Al4V is presented in

Table 1. Physical and mechanical properties of Ti, Medical Grade 4 and Ti6Al4V.

Property	Ti	Ti6Al4V
Density, g/cm3	4.51	4.43
Melting point, °C	1660	1660
Tensile strength, MPa	680	950
Yield strength, MPa	560	850
Poisson’s ratio	0.34–0.40	0.34–0.38
Elastic modulus, GPa	105–120	110–114
Elongation at break, %	23	14
Hardness (Vickers)	250	349

.

All disks intended for sandblasting with the same spraying material were placed on a support specifically constructed for this purpose as a series of triplicates for Ti and triplicates for Ti alloy with double-adhesive tape. The sandblasting equipment was loaded with the required spraying material, and by moving the sliding device through the sandblasting window, all sets of discs (3 Ti CP4 and 3 Ti6Al4V) were sandblasted at the same time, at the same pressures, and for the same durations established in the protocol. Then, the disks and the spraying material were changed, and the procedure was repeated for all the spraying materials, pressures, and blasting durations.

The sandblasting was carried out manually using a Geko SBC 110 booth produced by Gebo Tools SRL, Cluj-Napoca, Romania, provided with multiple nozzle blasting guns (4, 5, 6, 7 mm), using two different working pressures: 3 and 6 bar.

For an easy understanding and codification of samples and working conditions, where it is not specified, normal working pressure means a working pressure of 6 bar, while half working pressure means a working pressure of 3 bar.

The particles used for sandblasting are presented in

Table 2. Blasting material group setting.

Blasting Material	Size Range, [mm]	Working Pressure, [Bar]
1. White electrocorundum F90, mixed with sintered hydroxyapatite with a particle size ≤ 63 µm, in a ratio of 3:1	0.15–0.20	6
2. White electrocorundum F90, mixed with sintered hydroxyapatite with a particle size ≤ 63 µm, in a ratio of 3:1	0.15–0.20	3
3. Fine white electrocorundum mixed with sintered hydroxyapatite with a particle size ≤ 63 µm, in a ratio of 3:1	0.10–0.15	6
4. Fine white electrocorundum mixed with sintered hydroxyapatite with a particle size ≤ 63 µm, in a ratio of 3:1	0.10–0.15	3
5. White electrocorundum F90	0.15–0.20	6
6. White electrocorundum F90	0.15–0.20	3
7. Fine white electrocorundum	0.10–0.15	6
8. Fine white electrocorundum	0.10–0.15	3
9. Glass balls	0.04–0.07	6
10. Glass balls	0.04–0.07	3
11. Olivine	0.1–0.5	6
12. Olivine	0.1–0.5	3
13. Red garnet	0.40–0.80	6
14. Red garnet	0.40–0.80	3
15. Brown electrocorundum	0.120–0.212	6
16. Brown electrocorundum	0.120–0.212	3

.

The digital microscope used to acquire the micrographs was an Optika SFX-91D, Opptika, Ponteranica, Italy.

After preliminary degreasing of the samples in isopropyl alcohol for 5 min at 25 °C in an ultrasonic bath (40 kHz), the sandblasting was carried out manually, at a 90° angle, at 30 mm distance from the abutments, using a 7 mm nozzle, at 3 and 6 bar pressure, the blasting times being 10, 20, and 60 s. Similar cleaning and blasting operating parameters are reported in the literature [7,8,15,16].

In order to establish the influence of the sandblasting materials used as well as the influence of the working parameters, measuring the roughness is required [1,2,16].

The purpose of evaluating the roughness parameters on the surfaces of the samples is to observe possible changes in the surface of the material depending on the process to which they were subjected. In general, roughness is defined by the set of irregularities that form the relief of the real surface, whose steps are relatively small in relation to their depth.

The following roughness parameters were used to evaluate the surfaces:

-

The Ra parameter evaluates the average of the peaks and valleys on the scanned surface within the evaluation length;

-

The Rz parameter evaluates the average height between the five highest peaks, as well as the average depth between the five lowest valleys, on the scanned surface;

-

The Rt parameter evaluates the difference between the height of the highest peak and the depth of the deepest valley within the evaluation length. To determine the surface roughness of the samples and the differences between the processes to which they were subjected, the Hommel-Etamic Nanoscan 855 Jenoptik, Vienna, Austria equipment presented in Figure 1 was used. The Hommel-Etamic Nanoscan 855 has a resolution of 0.6 nanometers on a 20 mm measuring range.

The investigated roughness parameters were evaluated for each individual sample. To collect the profile, the sample was fixed to the table of the equipment and assessed using a probe with a 2 µm tip and a scanning speed of 0.25 mm/s.

The equipment used to perform SEM analyses to provide information on film morphology (shape, size, and size distribution of crystallites) was a TESCAN VEGA, Kohoutovice, Czech Republic, having the following characteristics: energy—200 eV–30 keV, beam current—1 pA to 2 µA, resolution—3 nm at 30 keV, magnification 1×–1,000,000×, Figure 2.

The samples were carefully fixed on the inner microscope Winchester bench without touching their surface. The samples were examined, and the images were acquired using energy settings between 15 and 30 keV.

Before the in vitro experiments, to prevent bacterial and fungal contamination, all sandblasted Ti (TiCP4) and Ti6Al4V alloy disks were individually sterilized in Petri dishes by autoclave for 30 min at 120 °C.

Gingival epithelial cells (Innoprot, Derio, Spain, REF: P10864) were cultured in the specific medium for epithelial cells supplemented with 2% fetal bovine serum (FBS), 1% penicillin-streptomycin (PE/ST), and growth factors for epithelial cells (EpiCGS) in sterile culture dishes, on which poly-L-lysine (2 µg/cm²) was previously deposited by incubation at 37 °C for at least 1 h. The cells were kept in culture in the incubator at 37 °C in a humid atmosphere with 5% CO₂.

Propagation in cell culture when 90% confluence was reached was achieved using trypsin/EDTA solution. Briefly, the cells were washed with saline buffer solution (PBS) without calcium and magnesium ions and treated with a trypsin/EDTA solution for 3–5 min. Later, the detachment solution was removed from the culture vessel, neutralization solution (TNS) was added, and the detached cells were re-suspended by gentle pipetting and moved into a tube containing fetal bovine serum. The cells were then centrifuged at 1000 rpm for 5 min to remove apoptotic or dead cells from the supernatant. The cells were seeded in new sterile culture vessels, on which poly-L-lysine (2 µg/cm²) had previously been deposited at a density of 5000 cells/cm². hGEpiC cells at passage 2 were used in the experiments.

For adhesion and cell morphology experiments, hGEpiC cells were seeded at 1 × 104 cells in 96-well culture microplates, 2 × 104 cells in 48-well culture microplates, 4 × 104 cells in 48-well culture microplates, the 24-well culture, respectively, 2 × 105 cells in 6-well culture microplates that contained the sterilized Ti-based materials or control Coverslip. This cell density was chosen following experiments previously carried out to establish the optimal cell density regarding cell viability and proliferation using the MTS proliferation kit (CelITiter 96^® Aqueous One Solution Proliferation Assay Kit Test, Promega, Madison, WI, USA. In all the experiments performed, hGEpiC cells were cultured in direct contact with the surface of the studied titanium-based materials for a period of 48 h at 37 °C and in a humid atmosphere with 5% CO₂.

In the adhesion and cell morphology experiments, the hGEpiC cells seeded in the appropriate culture microplates in direct contact with the surface of the studied Ti materials were fixed and marked with specific adhesion markers. To label the actin filaments, the samples were incubated with phalloidin conjugated with Alexa Fluor 488 (Invitrogen, Thermo Fischer Scientific, Waltham, MA, USA, dilution 1:100 in 0.5% BSA–PBS solution).

For the sandblasted samples, labeling of vinculin (VNC) was conducted by incubating with primary monoclonal anti-vinculin antibodies (Merck, Buchs, Switzerland, dilution 1:150 in 0.5% PBS–BSA solution) and later with secondary antibodies coupled with the fluorophore Alexa Fluor 594 (Thermo Fischer Scientific, Waltham, MA, USA, 1:400 dilution in 0.5% PBS–BSA solution).

The samples were arranged on the microscope slide, Mounting FluorSave™ Reagent (Merck, Buchs, Switzerland) was added, and later, they were visualized by fluorescence microscopy with the 10× objective using the Zeiss Axiocam ERc5s Apotome microscope Hitech Instruments, Pennsburg, PA, USA, with the ApoTome.2 cursor mode.

3. Results

3.1. Determination of the Roughness Profile and the Parameters of the Blasted Samples

The results of the blasting material investigated using digital micrography are presented in

Table 3. Digital micrographs for used blasting materials.

Blasting Material	Digital Micrographs, 4×
White electrocorundum F90 of particle size 0.15–0.20 mm mixed With sintered hydroxyapatite, 2×
White electrocorundum of particle size 0.10–0.15 mm mixed With sintered hydroxyapatite, 2×
White electrocorundum F90 particle size 0.15–0.20 mm, 2×
White electrocorundum F90 particle size 0.15–0.20 mm, 2×

and

Table 4. Digital micrographs for used blasting materials.

Blasting Material	Digital Micrographs, 4×
Glass balls, 0.04–0.07 mm
Olivine, particle size 0–0.5 mm, 2×
Red garnet, particle size 0.40–0.80 mm, 2×
Brown electrocorundum, particle size 0.120–0.212 mm, 2×

. This was carried out to confirm the actual size distribution after sieving the blasting material. It was found that the actual measurements for individual grains correspond to the actual distribution category they were placed in (actual microscopy measurements are depicted in blue on micrographs). As the scanning speed of the microscope tip is 0.25 mm/s for a profile length of 20 mm, the final value presented as the Ra parameter is obtained as an average of 80 points (+/−5 points), measured on a profile of each disk.

Representative graphs of the effect of the materials used for blasting, as well as the blasting durations versus the depicted roughness profile, as well as the roughness parameters, as they are described in detail in Section 2, are presented in Figure 3, Figure 4, Figure 5 and Figure 6. As the number of experiments is very high (98 roughness profiles and 98 roughness parameters), only these representative samples were explicitly included here; all the roughness profiles are presented in the extensor in the Supplementary Material enclosed in this paper. Figure 3 and Figure 4 depict the roughness profiles for the unblasted Ti and Ti4Al6V; Figure 5 and Figure 6 depict the blasted Ti and Ti6Al4V sample for a 10 s blasting time using white electrocorundum F90, granulometry between 0.15 and 0.20 mm mixed with sintered hydroxyapatite with a granulometry smaller than 63 µm, in a ratio of 3:1, under the normal working pressure and 6 bar blasting conditions.

The resulting roughness parameter, Ra [µm], for all the experiments carried out, deduced from the roughness profiles, has been centralized in Table 4 and

Table 5. Summarized values of Ra, [µm] for various blasting materials and conditions (1-10 to 8-60).

Sample	Ra, Ti6Al4V, [µm]	Ra, Ti, [µm]
Blank, unblasted	0.1354	0.2055
1-10	1.7703	1.0704
1-20	1.3463	1.4513
1-60	1.5043	1.4993
2-10	1.3472	1.1449
2-20	1.1422	1.1645
2-60	1.2295	1.1385
3-10	0.9809	1.0121
3-20	1.1930	1.0399
3-60	1.0652	1.0389
4-10	1.0480	1.1670
4-20	1.0733	1.8941
4-60	1.0252	0.9378
5-10	1.0746	1.0504
5-20	1.5539	1.1124
5-60	1.4036	1.3237
6-10	0.8885	1.8142
6-20	1.3938	0.9965
6-60	1.3708	1.4875
7-10	1.7690	0.9348
7-20	1.3630	0.9550
7-60	1.8821	0.9795
8-10	0.8411	0.8480
8-20	0.8610	0.8960
8-60	1.0868	1.3878

, where the codification represents the following: the first digit is the blasting material and blasting conditions; the second digit is the blasting time in seconds, e.g., blasted Ti, 5–20—blasted with white electrocorundum F90, particle size 0.15–0.20 mm—normal working pressure, 6 bar for 20 s.The blasting times were 10, 20, and 60 s, and the codding for blasting material and blasting conditions are as follows:

• White electrocorundum F90 of particle size 0.15–0.20 mm mixed with sintered hydroxyapatite 3:1, normal working pressure, 6 bar;
• White electrocorundum F90 of particle size 0.15–0.20 mm mixed with sintered hydroxyapatite 3:1, half working pressure, 3 bar;
• White electrocorundum of particle size 0.10–0.15 mm mixed with sintered hydroxyapatite, 3:1, normal working pressure, 6 bar;
• White electrocorundum of particle size 0.10–0.15 mm mixed with sintered hydroxyapatite, 3:1, half working pressure, 3 bar;
• White electrocorundum F90 particle size 0.15–0.20 mm—normal working pressure, 6 bar;
• White electrocorundum F90 particle size 0.15–0.20 mm—half working pressure, 3 bar;
• White electrocorundum of 0.10–0.15 mm particle size—normal working pressure, 6 bar;
• White electrocorundum of 0.10–0.15 mm particle size—half working pressure, 3 bar rate;
• Glass balls for sandblasting 0.04–0.07 mm—normal working pressure, 6 bar;
• Glass balls for sandblasting 0.04–0.07 mm—half working pressure, 3 bar;
• Olivine—particle size 0.1–0.5 mm—normal working pressure, 6 bar;
• Olivine—particle size 0.1–0.5 mm—half working pressure, 3 bar;
• Red garnet—particle size 0.40–0.80 mm—normal working pressure, 6 bar;
• Red garnet—particle size 0.40–0.80 mm—half working pressure, 3 bar;
• Brown electrocorundum—particle size 0.120–0.212 mm—normal working pressure, 6 bar;
• Brown electrocorundum—particle size 0.120–0.212 mm—half working pressure, 3 bar.

Comparing the values of Ra for the sandblasted samples to the blank samples, presented in Table 5 and

Table 6. Summarized values of Ra, [µm] for various blasting materials and conditions (9-10 to 16-60).

Sample	Ra, Ti6Al4V, [µm]	Ra, Ti, [µm]
Blank, unblasted	0.1354	0.2055
9-10	0.2623	0.2522
9-20	0.1886	0.2336
9-60	0.2613	0.2181
10-10	0.2526	0.2791
10-20	0.1833	0.2750
10-60	0.2588	0.2556
11-10	1.6319	1.3071
11-20	1.7691	1.4519
11-60	1.7595	1.5617
12-10	1.2783	1.2256
12-20	1.2737	1.0687
12-60	1.4867	1.5177
13-10	1.4561	2.8206
13-20	1.9282	2.5769
13-60	2.5735	2.4411
14-10	1.9579	2.1483
14-20	1.9228	2.1547
14-60	2.5234	2.5275
15-10	0.9990	0.9317
15-20	1.2356	1.1485
15-60	1.0039	1.0423
16-10	0.8467	1.0445
16-20	0.9462	0.9881
16-60	1.0707	1.0627

, regardless of the operating conditions, the common trend is an increase in the actual value of the surface roughness for Ti medical grade 4 as well as for the Ti4Al6V. This leads to the conclusion that sandblasting, if operated within carefully chosen parameters, is an efficient way of preconditioning the above-mentioned metals by increasing their roughness and, hence, their total active surface area.

3.2. SEM Analysis

SEM analysis was employed to elucidate whether the use of sintered hydroxyapatite as an additive to the blasting material leads to a substantial deposition of hydroxyapatite on the substrate materials.

The results obtained following SEM and EDS analysis are presented in Figure 7, Figure 8, Figure 9 and Figure 10.

3.3. Cell Adhesion

The results regarding the experiments carried out to investigate cell adhesion are presented in Figure 11, Figure 12 and Figure 13.

4. Discussion

It was found that in all cases, regardless of the spraying material or working conditions, the roughness profile achieved is a uniformly distributed one (Figure 4, Figure 5, Figure 6 and Figure 7 and those depicted in the Supplementary Material).

A reduction in the blasting pressure by half led to a decrease in the Ra parameter by approximately 30–35% in most cases, which offers certain guidelines should one want to marginally increase or decrease the surface roughness. These findings are in agreement with those reported in the literature [2,4,15].

When comparing the effect of the spraying duration (10 s, 20 s, 60 s) versus the Ra values in Table 5 and Table 6 for samples of Ti medical grade 4, no consistent correlation may be found in some cases (white electrocorundum F90 of particle size 0.15–0.20 mm mixed with sintered hydroxyapatite 3:1, glass balls for sandblasting 0.04–0.07 mm, red garnet—particle size 0.40–0.80 mm). A minimum value may be found at 20 s spraying duration, which may be explained by the fact that an increase in the spraying time leads to a relative leveling of the obtained roughness, and a further increase in the spraying time leads to more deformations on the surface level and hence an increase in the sample roughness. These may be considered exceptions, as the general rule observed for the remaining samples is that an increase in the spraying duration leads to an increase in the actual values of Ra. The higher the spraying duration (white electrocorundum of 0.10–0.15 mm particle size, olivine—particle size 0–0.5 mm, red garnet—particle size 0.40–0.80 mm, brown electrocorundum—particle size 0.120–0.212 mm—half working pressure, 3 bar), the greater the increase. However, within the experimental errors, the values of Ra for 20 s and 60 s spraying time are marginally similar to those obtained for a spraying duration of 10 s (white electrocorundum of particle size 0.10–0.15 mm mixed with sintered hydroxyapatite, 3:1, half working pressure, 3 bar, olivine—particle size 0–0.5 mm—normal working pressure, 6 bar). This brings us to the conclusion that one may achieve good results within the first 10 s of spraying, saving a lot of time and energy while maintaining the dimensional integrity of the material subjected to the blasting procedure, a very important factor to take into consideration. Similar findings apply for Ti4Al6V, although one may say that there is no replicated pattern for increases or maximum points. These can be interpreted taking into account the differences in the mechanical properties of the two materials considered for blasting substrates. Similar findings were reported in the literature, with an emphasis on the conclusion, as we showed above, that the characteristics of the particles and the parameters of the sandblasting process are variables that greatly influence surface roughness [2,15,17].

When comparing the roughness obtained on Ti substrates vs. Ti4Al6V in Table 4 and Table 5, one may notice, with a few exceptions, that under the same blasting conditions, the roughness obtained on the Ti surface is higher than that obtained on Ti4Al6V. These can be explained by corroborating the results from Table 4 and Table 5 with the physical properties presented in Table 1, where one may see that the tensile strength is 39.7% higher for Ti4Al6V vs. Ti. The hardness is 39.6% higher for Ti4Al6V vs. Ti, and hence, the impact of spraying material leads to deeper indentations in Ti compared to Ti4Al6V.

Considering each spraying material individually, in the case of sandblasting with normal white electrocorundum F90 mixed with hydroxyapatite, a maximum of the Ra parameter can be observed for a sandblasting time of 20 s for both materials, Ti and Ti6Al4V.

Sandblasting with fine electrocorundum and hydroxyapatite produced a nearly uniform roughness for both materials, suggesting that the size of the blasted material is the determinant factor regardless of the material subjected to this treatment.

In the case of samples blasted with normal electrocorundum at a normal working pressure of 6 bar, one may obtain a uniform distribution of Ra values in the sample profile in the case of Ti regardless of the blasting duration, leading to the conclusion that the roughness profile is stabilized within the first 10 s.

The use of glass balls as sandblasting material led to decreased and more uniformly distributed roughness values for both materials regardless of time or applied pressure compared to other spraying materials. This may be easily exploited for practical applications as, according to [7], a higher roughness favors the osseointegration process for implants, and a lower roughness surface has a beneficial effect on the cell adhesion on abutments.

Sandblasting with olivine, however, led to obtaining relatively high and uniform values for the two materials, hence the conclusion that one may control the roughness size by choosing one or another of the above materials without the need to change any other operating parameters.

The red garnet produces a higher Ra for Ti, and it appears to be the material causing the highest roughness compared to any other materials.

Sandblasting with brown electrocorundum led to obtaining a fairly close value for Ra between the two materials, regardless of spraying pressure.

The roughness is clearly influenced by the type of sandblasting material and the shape of the particles, even if their hardness is relatively close in value, which will also influence the cellular adhesion process.

In the case of sandblasting materials where hydroxyapatite was also used, following the SEM microscopic analysis presented in Figure 7, Figure 8, Figure 9 and Figure 10, one may observe that, although calcium and phosphorus are clearly identified for both Ti and Ti alloy, there are minute traces (Figure 8 and Figure 10).

Electron microscopy certified the uniform distribution of chemical compounds in the crystalline-looking groundmass. The elemental composition consists of Ti, Al, and V, elements obviously expected as they form the Ti or Ti alloy samples. The other elements are found in variable percentages depending on blasting/spraying materials, as the case may be, confirming that in the case of sandblasting, there is an inevitable result that traces of the sprayed material are found attached to the blasted materials, implying that an additional cleaning procedure ought to be employed, as the case may be, or the imposed requirements. Their concentration is, however, very small.

Regarding cell culture and proliferation, as can be seen in Figure 11, Figure 12 and Figure 13, the sandblasted Ti and Ti alloy allowed the adhesion of cells 48 h after seeding, the cells being found on the entire available surface of the materials.

Thus, phalloidin labeling of hGEpiC cells adhered to sandblasted Ti alloy surfaces revealed a distribution of actin fibers predominantly towards the periphery of the cell, in contrast to the normal uniform cytoplasmic distribution of actin filaments over the entire cell surface. The same behavior of the gingival epithelial cells is preserved on the surface of the sandblasted Ti alloy.

In hGEpiC epithelial cells, the cytoskeleton protein vinculin is distributed especially in the peripheral areas of contact with the material surface, in the case of disks based on sandblasted Ti, and as a punctate marker in the case of disks based on sandblasted Ti alloy.

This study, although comprehensive, is limited only to sandblasting, and one may envisage as further prospects a combined study of sandblasting and acid etching or electrochemical anodization, as these combined methods may offer better results due to the synergistic effects of these complementary techniques.

5. Conclusions

Sandblasting Ti and Ti4Al6V with different spraying materials and in various conditions proved that it is possible to control the surface roughness by carefully choosing the blasting material as well as the working conditions, especially the blasting duration. There is a distinct difference between sandblasting Ti and sandblasting Ti6Al4V alloy, and the main cause is the difference in their mechanical properties, with Ti being affected more than its alloy. With respect to the blasting material, the softer the spraying material, the better the yield of a uniformly distributed roughness, regardless of the spraying time or applied pressure. Sandblasting with brown electrocorundum generated a similar roughness for the two materials, regardless of the applied pressure. The SEM analysis showed that the use of sintered hydroxyapatite as an additive to the blasting material does not lead to substantial deposition of hydroxyapatite on the substrate materials; only traces of it were identified during the analysis. Both Ti and Ti6Al4V performed well in the experiments regarding cell culture and proliferation, so sandblasting may be an attractive method for preconditioning these materials for restorative dentistry.