The effects of titanium nitride-coating on the topographic and biological features of TPS implant surfaces

The effects of titanium nitride-coating on the topographic and biological features of TPS implant surfaces

Journal of Dentistry, 2011-11-01, Volume 39, Issue 11, Pages 720-728, Copyright © 2011 Elsevier Ltd

Abstract

Objectives

Titanium nitride (TiN) coating has been proposed as an adjunctive surface treatment aimed to increase the physico-mechanical and aesthetic properties of dental implants. In this study we investigated the surface characteristics of TiN-coated titanium plasma sprayed (TiN-TPS) and uncoated titanium plasma sprayed (TPS) surfaces and their biological features towards both primary human bone marrow mesenchymal stem cells (BM-MSC) and bacterial cultures.

Methods

15 mm × 1 mm TPS and TiN-TPS disks (P.H.I. s.r.l., San Vittore Olona, Milano, Italy) were topographically analysed by confocal optical profilometry. Primary human BM-MSC were obtained from healthy donors, isolated and expanded. Cells were seeded on the titanium disks and cell adhesion, proliferation, protein synthesis and osteoblastic differentiation in terms of alkaline phosphatase activity, osteocalcin synthesis and extracellular mineralization, were evaluated. Furthermore, adhesion and proliferation of Streptococcus pyogenes and Streptococcus sanguinis on both surfaces were also analysed.

Results

TiN-TPS disks showed a decreased roughness (about 50%, p < 0.05) and a decreased bacterial adhesion and proliferation compared to TPS ones. No difference ( p > 0.05) in terms of BM-MSC adhesion, proliferation and osteoblastic differentiation between TPS and TiN-TPS surfaces was found.

Conclusions

TiN coating showed to modify the topographical characteristics of TPS titanium surfaces and to significantly reduce bacterial adhesion and proliferation, although maintaining their biological affinity towards bone cell precursors.

Introduction

Titanium (Ti) and its alloys have proved their safety and efficacy as dental implant materials, as demonstrated by the long-term success rate of implant-supported rehabilitations. Osseointegration of dental implants results from the interaction between the titanium surface and the cellular and matrix constituents of the surrounding bone tissue. Topographic and chemical surface modifications of titanium implants may significantly affect osseointegration. In particular, micro-rough surfaces, obtainable by several techniques, such as titanium plasma-spraying, sand-blasting, acid etching and anodic oxidation, have been demonstrated to increase the implant affinity for bone cells and lead to higher values of bone-to-implant contact rate and retention into the bone.

However titanium implants, especially in the commercially pure form, have a low strength and can undergo to physical abrasion in the oral environment, e.g. due to oral prophylaxis procedures. Furthermore, the titanium grey colour can raise aesthetic problems when it is not adequately masked by soft tissue at level of the gingival area.

In order to overcome these problems, the titanium nitride (TiN) coating has been recently introduced also in the field of dental titanium implants. Titanium nitride is a material commonly used to cover a number of metal tools, including surgical instruments, in order to improve their surface properties and aesthetic appeal (thanks to its characteristic golden colour). TiN-coated dental implants show higher physico-mechanical properties, and allow a better camouflage under the gingival tissue, rather than conventional grey titanium implants. However, whether TiN-coating could affect the topographic and the biological features of a dental implant surfaces has not been extensively investigated.

Titanium plasma sprayed implants (TPS) have been largely commercialised in the last decades and were proved to guarantee higher performances compared to turned implants in terms of bone-to-implant contact rate and retention into the bone. However, some studies have showed that the use of such highly rough surfaces couples with higher bacterial plaque retention and marginal bone loss due to perimplantitis.

The purpose of the present study was to investigate if an additional treatment of TiN-coating of conventional titanium plasma sprayed implant surfaces could affect their properties in terms of topographic characteristics, affinity towards bone osteoprogenitors (human bone marrow mesenchymal stem cells), and susceptibility to bacterial colonization ( Streptococcus pyogenes and Streptococcus sanguinis) .

Materials and methods

Products and reagents

All cell culture biologics were purchased from Gibco BRL (Grand Island, NY, USA), and all chemicals were from Sigma Chemical Co. (St. Louis, MO, USA) when not otherwise specified.

Specimen preparation

Two different titanium implant surfaces were analysed: titanium plasma sprayed (TPS) and TiN-coated titanium plasma sprayed (TiN-TPS).

All specimens were prepared by a commercial firm (P.H.I. s.r.l., San Vittore Olona, Milano, Italy) in form of 15-mm wide and 1-mm thick disks of Ti–6Al–4V. The disks were cleaned of surface organic contaminants by ultrasonic agitation in a series of detergent solutions, acetone, ethanol, and deionised water. All specimens were plasma sprayed with grade 4 titanium. Test specimens were additionally TiN coated by a process of physical vapour deposition (PVD). All disks were singularly packed and finally sterilised in a steam autoclave.

For cell culture assays, titanium disks were put on the bottom of 24-well plates. The polystyrene surface of the multiwell plates was used as control.

Surface topographic characterisation

Qualitative and quantitative measurements of implant surfaces were made by a confocal optical profilometer (PLμ 2300, Schaefer Italia srl, Rovigo) on 636 μm × 849 μm areas at ambient conditions. All measurements were performed on 6 different points, randomly distributed over the surface, with at least one scan effected close to the centre and one close to the edge of each specimen. Mean roughness (Sa), root mean square roughness (Sq) and ten-point average roughness (Sz) were calculated as typical surface texture parameters. Such parameters allow to duly characterise surfaces modified by different treatments and procedures, such as the TPS and the TiN-TPS. In particular, Sa is the arithmetic average of the absolute values of the surface height deviations measured from a reference plane. It is the most diffused parameter for measuring surface texture, however it only quantifies the “absolute” magnitude of the surface heights and is insensitive to the spatial distribution of the surface heights. Sa is also insensitive to the “polarity” of the surface texture in that a deep valley or a high peak will result in the same Sa value. Sq, namely the root mean square roughness, is the statistical measure of the magnitude of the height distribution and correlates well with Sa. Also in this case, a series of high peaks or a series of deep valleys of equal magnitude will produce the same Sq value. Sz, namely the ten-point average roughness, is found from the difference between the average maximum peak height of the ten highest peaks and is the average maximum valley depth of the ten lowest valleys found over the complete 3D image. Sz may be used to characterise the extreme features of a surface, being a nominal measure of the “peak-to-valley” range of the surface.

Preparation of a collection of human bone marrow mesenchymal stem cells (BM-MSC)

Ten ml samples of human bone marrow were harvested from two healthy donors. Informed consent was provided according to the Declaration of Helsinki. The research was institutionally approved. BM-MSC cultures were initiated as previously described. Briefly, heparinised bone marrow was diluted 1:5 with complete culture medium consisting of OptiMEM containing 10% (v/v) foetal calf serum (FCS), 100 units/ml penicillin, 100 μg/ml streptomycin and 50 μg/ml sodium ascorbate, and incubated at 37 °C in a 5% CO 2 humidified atmosphere. Although present in a percent extremely low with respect to the total of mononuclear cells originally present in the bone marrow, BM-MSC can be obtained on the basis of their ability to adhere on polystyrene plates, whilst the cells of the haemopoietic lineage remain in suspension and can be easily removed. After 48 h, the medium containing all non-adherent cellular elements was centrifuged 10 min at 800 × g in order to remove the haematopoietic cells and added again to the dish. In 3–4 days, several foci of adherent spindle-like cells appeared and reached the sub-confluence in 2 weeks. The medium was refreshed every 3 days, each time leaving one half of the conditioned medium. The cells harvested from each donor were kept separately and not pooled with other preparations. Cultures between the second and fourth passage were used in our experiments.

Cell adhesion and proliferation

BM-MSC were seeded on implant surfaces and control wells at a density of 15,000 cells/cm 2 in complete culture medium.

Cell adhesion to implant surfaces at 6 h from plating and cell proliferation at 3 days were assessed by MTT vitality assay. The key component of this assay is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Mitochondrial dehydrogenases of living cells reduce the tetrazolium ring, yielding a blue formazan product, which can be measured spectrophotometrically. Cells were washed with phosphate buffered saline (PBS) and incubated with 0.5 mg/ml MTT solution for 4 h at 37 °C. At the end of this time, the liquid was aspirated and the insoluble formazan produced was dissolved in isopropanol–HCl 0.1 M. The optical density was measured at 570 nm, subtracting background absorbance determined at 690 nm.

Cell adhesion and morphology were also evaluated by scanning electron microscopy (SEM). Cells were plated on titanium surfaces as above mentioned. After 6 h cells were rinsed three times with PBS and fixed for 30 min with 2.5% glutaraldehyde. The fixed cell layers were washed in PBS and dehydrated by graded ethanol solutions and critical point drying. Samples were mounted on stubs, coated with Au/Pd alloy and examined by SEM (Quanta 200, FEI Europe Company, the Netherlands).

Osteoblastic differentiation

The effects on cell differentiation was evaluated analyzing the expression of specific markers of the osteoblastic phenotype, namely alkaline phosphatase activity, osteocalcin production and the mineralization of the extracellular matrix.

Alkaline phosphatase specific activity

The alkaline phosphatase (AP) specific activity of BM-MSC grown on the titanium surfaces was evaluated after 7 and 14 days of culture. Once removed the medium, the wells were rinsed with 20 mM TRIS–HCl – 0.5 M NaCl, pH 7.4 (TBS) and the cells lysed with a specific buffer (20 mM Tris/HCl, pH 7.4, 0.5 mM NaCl, 0.25%Triton X-100, 0.5 mM PMSF, 0.5 mM DTT). After 30 min in ice, the cell lysates were centrifuged at 13,000 × g for 5 min, and the supernatants assayed for AP activity. Protein concentration was determined according to the method of Bradford.

AP activity was determined by measuring the release of para-nitrophenol (PNP) from disodium para-nitrophenyl phosphate (PNPP). The reaction mixture contained 10 mM PNPP, 0.5 mM MgCl 2 , diethanolamine phosphate buffer pH 10.5, and 10–30 μg of cell lysate in a final volume of 0.5 ml. After 30 min at 37 °C, the reaction was stopped by adding 0.5 ml of 0.5 M NaOH. PNP levels were measured spectrophotometrically at 405 nm. The AP activity was normalised to the protein content and expressed as units/mg protein, where one unit was defined as the amount of enzyme that hydrolyzes 1 nmol of PNPP/min under the specified conditions.

Osteocalcin synthesis

To evaluate osteocalcin synthesis confluent cultures grown on the different surfaces for 2 weeks were incubated in FCS-free Opti-MEM in the presence of 0.1% bovine serum albumin and 100 nM 1,25-dhydroxycolecalciferol for 48 h. The levels of polypeptide secreted in the medium were measured by means of an immunoenzymatic assay (Biosource International, Camarillo, CA, USA) that utilises monoclonal highly specific antibodies and a peroxidase as conjugated enzyme. The amount of osteocalcin was calculated in ng/ml and then normalised to the protein content.

Extracellular matrix mineralization

The ability of titanium surfaces to promote the extracellular matrix mineralization was tested by quantification of the calcium levels. BM-MSC confluent cultures were incubated for 20 days with an osteogenic medium composed of 100 nM dexamethasone and 10 mM β-glycerophosphate. The calcium levels were measured colorimetrically using arsenazo III, reagent: cells were decalcified with 0.6 N HCl for 24 h and the calcium released in the supernatant was determined at 575 nm using a plate reader and calculated according to a standard solution.

Bacterial cultures

Bacteria cell lines used in this study were S. pyogenes and S. sanguinis . The dry pellet was rehydrated in 6 ml of Luria broth (LB) consisting of 10 g tryptone, 5 g yeast extract, and 5 g NaCl per litre double distilled water with the pH adjusted to 7.4. The bacterial suspension was agitated under standard cell conditions (5% CO 2 /95% humidified air at 37 °C) for 24 h until the stationary phase was reached. The second passage of bacteria was diluted at a ratio of 1:200 into fresh LB and incubated until it reached stationary phase. The next passage was then frozen in one part LB and one part glycerol and stored at −18 °C. All experiments were conducted from this frozen stock. One day before bacterial seeding for experiments, a sterile 10 ml loop was used to withdraw bacteria from the frozen stock and to inoculate a centrifuge tube with 3 ml of fresh LB.

Bacterial adhesion and proliferation

Bacterial growth from 2 day cultures of each strain was harvested and the optical density (OD) in each culture tube was adjusted to 1 × 10 7 bacteria/ml (as estimated by the McFarland scale) by diluting the LB bacteria cultures to an optical density of 0.52 at 562 nm in a spectrophotometer and then further diluted at a ratio of 1:90 in LB Medium. Sterile titanium surfaces were placed individually in 12 well plates and a total of 10 6 cells/ml suspension of each reference strain was added, in a total volume of 2 ml. Plates were incubated for 2, 6, 12 and 24 h at 37 °C. After incubation, each sample was washed twice with PBS; after washing, the foils of each samples were incubated in LB Medium and in 12 well plates for 48 h to the bacterial growth to each surface. After incubation, 1 ml was harvested and the optical density was spectrophotometrically measured at 562 nm.

Bacterial adhesion was also observed using an environmental scanning electron microscope (ESEM Quanta 200, FEI Europe Company, the Netherlands). In particular, titanium samples, after a 2 h-incubation with S. sanguinis , were imaged in ESEM mode at 15 kV, using Peltier Stage and gaseous secondary electron detector (GSED). The chamber parameters were settled to 58 °C temperature and 3.60 Torr pressure.

Statistical analysis

Statistical analysis was performed by a dedicated software (NCSS for Windows, Kaysville, Utah, USA). Data were expressed as the mean ± standard deviation (SD) of relative units (percentage of control) or absolute values. The means of each experimental group were compared by unpaired Student’s t -test, with the value of significance set at p < 0.05.

Results

Surface roughness and topographical characterisation

Images of TPS and TiN-TPS surfaces obtained by confocal optical profilometry are shown in Fig. 1 a . The two samples appear similar, with a comparable extremely rough surface.

Qualitative and quantitative characterisation of implant surfaces. (a) Confocal optical profilometry images of titanium plasma spray (TPS) and titanium-nitride TPS (TiN-TPS) implant surfaces. (b) Roughness values measured on TPS and TiN-TPS surfaces. Data are expressed as mean ± standard deviation (SD). * = p < 0.05; ** = p < 0.01; Sa = mean roughness; Sq = root mean square roughness; Sz = ten-point average roughness.
Fig. 1
Qualitative and quantitative characterisation of implant surfaces. (a) Confocal optical profilometry images of titanium plasma spray (TPS) and titanium-nitride TPS (TiN-TPS) implant surfaces. (b) Roughness values measured on TPS and TiN-TPS surfaces. Data are expressed as mean ± standard deviation (SD). * = p < 0.05; ** = p < 0.01; Sa = mean roughness; Sq = root mean square roughness; Sz = ten-point average roughness.

Sa, Sq and Sz values of the experimental surfaces are reported in Fig. 1 b. Significantly higher values for the uncoated surfaces compared to the TiN-coated one were detected in terms of Sa, Sq ( p < 0.05) and Sz ( p < 0.01).

BM-MSC adhesion and proliferation

Cell adhesion on implant surfaces was evaluated 6 h after plating by SEM analysis and MTT viability test. Both TPS and TiN-TPS surfaces showed a good affinity for BM-MSC, as evidenced by SEM images ( Fig. 2 a) , resulting in comparable cell adhesion values ( Fig. 2 b). On a parallel set of samples, BM-MSC were cultured for 72 h. At the end of the incubation, no difference in terms cell number between the two surface types was detected ( Fig. 2 c).

Fig. 2
Cell adhesion and proliferation on implant surfaces. (a) SEM images of bone marrow mesenchymal stem mesenchymal stem cells (BM-MSC) grown for 6 h on titanium plasma spray (TPS) and titanium-nitride TPS (TiN-TPS) implant surfaces at magnification 20,000×. (b) Adhesion of BM-MSC at 6 h from seeding on TPS and TiN-TPS implant surfaces. (b) Proliferation of BM-MSC at 72 h from seeding on TPS and TiN-TPS implant surfaces. In both cases, cell vitality was spectrophotometrically assessed by MTT assay. Results were obtained from 3 independent experiments. Data are expressed as percentage of those obtained on cells cultured on polystyrene surface (control) ± standard deviation (SD). NS = not significant ( p > 0.05).

Osteoblastic differentiation

Surface typology did not affect the expression of early and late osteoblastic markers. Both TPS and TiN-TPS specimens showed comparable values ( p > 0.05) in terms of alkaline phosphatase activity at both 7 and 14 days from plating ( Fig. 3 a) , osteocalcin synthesis at 14 days ( Fig. 3 b) and extracellular matrix mineralization at 20 days ( Fig. 3 c). Also the overall protein synthesis at 7 and 14 days was comparable ( p < 0.05) between the two experimental groups ( Fig. 3 d).

Fig. 3
Cell differentiation in response to implant surfaces. (a) Alkaline phosphatase specific activity of bone marrow mesenchymal stem cells (BM-MSC) cultured for 7 and 14 days on titanium plasma spray (TPS) and titanium-nitride TPS (TiN-TPS) implant surfaces. (b) Osteocalcin synthesis by BM-MSC at 14 days from seeding on TPS and TiN-TPS implant surfaces. (c) Extracellular matrix mineralization expressed as calcium level produced by BM-MSC cultured for 20 days on the implant surfaces. (d) Overall protein synthesis by BM-MSC at 7 and 14 days from seeding on TPS and TiN-TPS implant surfaces. Results were obtained from 3 independent experiments. Data are expressed as percentage of those obtained on cells cultured on polystyrene surface (control) ± standard deviation (SD). NS = not significant ( p > 0.05).

Bacterial adhesion and proliferation

Significantly different bacterial adhesion and proliferation on TPS and TiN-TPS surfaces was interestingly assessed. In particular, both bacteria cultures ( S. pyogenes and S. sanguinis ) showed a significantly lower adhesion and proliferation over time on TiN-TPS compared to TPS surfaces ( Fig. 4 ). Such results were also confirmed by ESEM analysis ( Fig. 4 a) which clearly showed less dense and numerous bacterial cells on TiN-TPS than on TPS surfaces 2 h after plating.

Bacterial adhesion and proliferation on implant surfaces. (a) Colonies of S. sanguinis were plated on titanium plasma spray (TPS) and titanium-nitride TPS (TiN-TPS) implant surfaces. After 2 h, samples were imaged by environmental scanning electron microscopy (ESEM) (magnification ×5000). (b) and (c) Time course of S. sanguinis (b) and S. pyogenes (c) proliferation at 2, 6, 12 and 24 h from seeding on TPS and TiN-TPS implant surfaces. Bacterial concentration was spectrophotometrically assessed at 562 nm. Results were obtained from 3 independent experiments. Data are expressed as optical density (OD) values ± standard deviation (SD). * = p < 0.05, ** = p < 0.01, *** = p < 0.001 between TPS and TiN-TPS.
Fig. 4
Bacterial adhesion and proliferation on implant surfaces. (a) Colonies of S. sanguinis were plated on titanium plasma spray (TPS) and titanium-nitride TPS (TiN-TPS) implant surfaces. After 2 h, samples were imaged by environmental scanning electron microscopy (ESEM) (magnification ×5000). (b) and (c) Time course of S. sanguinis (b) and S. pyogenes (c) proliferation at 2, 6, 12 and 24 h from seeding on TPS and TiN-TPS implant surfaces. Bacterial concentration was spectrophotometrically assessed at 562 nm. Results were obtained from 3 independent experiments. Data are expressed as optical density (OD) values ± standard deviation (SD). * = p < 0.05, ** = p < 0.01, *** = p < 0.001 between TPS and TiN-TPS.

Discussion

In the present study the biological response of human bone marrow mesenchymal stem cells to TiN-coated/uncoated implant surfaces was analysed. TiN-coating showed to affect both the topographical and biological features of tested implant surfaces, reducing in particular bacterial adhesion, without, however, compromising BM-MSC adhesion, proliferation and differentiation.

Bone marrow mesenchymal stem cells are able to self-renew and to differentiate into precursors of several tissues, including the osteoprogenitor cells. They play a central role in the osseointegration process, being involved in the normal remodelling and reparative mechanisms of bone. The contact of the osteoprogenitor cells with the implant surface represent a central phase of the osteointegration process, being the basis for all the following events, including the deposition of an organised extracellular matrix and its mineralization. The in vitro experimental approach allows investigating in detail this interaction, as well as other phenomena, but many confounding factors need to be controlled. Only a correct assessment and standardization of the in vitro , as well as in vivo and clinical , research parameters may allow some kind of extrapolation of the results “from bench to bed side”. In this sense, the selection of a suitable cell system is crucial. In our study, the biological response of BM-MSC to implant surfaces was investigated using primary human cultures. Primary cells from normal tissues represent, in our opinion, the ideal cellular model for pre-clinical evaluations, because of their closeness to the biological response of real living tissues, with respect to animal-derived or transformed osteoblast cell lines, although their use entails some inter-donor variability. In order to limit this variable and enhance data reliability, two different BM-MSC populations were prepared in our study and both of them gave in all cases comparable response.

Different chemo-mechanical treatments exist which are able to modify the surface topography and roughness of surgical and prosthetic implant components. Titanium plasma spraying represents a recognised method of increasing surface roughness and enlarging the implant surface area. With this additive process, titanium powders are injected into a plasma torch at high temperature. The titanium particles are projected onto the surface of the implants where they condense and fuse together, forming a uniform film.

The result is a highly rough surface, in the 10 μm range, with an isotropic topographical pattern, i.e. without a visible direction in its roughness. As shown by our measurements, the TPS surfaces showed an average Sa value of about 6 μm, that is considerably higher than smooth surfaces (turned, polished, etc.) or moderately rough surfaces (sandblasted, acid etched, etc.) which are in the <0.5 μm and 1–2 μm range of Sa values, respectively, following the classification proposed by Albrektsson and Wennerberg.

TiN coating was applied as an adjunctive treatment to TPS surfaces. Different nitriding processes exist, but the physical vapour deposition represents one of the most efficient and widely used TiN-coating technique in the biomedical field. With this process, the titanium is vaporised in a nitrogen atmosphere by means of a cathodic arc, and the evaporating material and the reactive gas become highly ionised. The result is the formation of a thin layer of titanium nitride which is tightly connected to the substratum by an intermixed zone which is a peculiarity of the cathodic arc technique.

An interesting finding of our investigation is that the TiN-coating process significantly altered the topographic characteristics of TPS surfaces. The roughness values of TiN-TPS disks were significantly lower compared to the uncoated ones (Sa 3 vs. 6 mm). In other words, TiN-coating seemed to switch the TPS roughness from a high-roughness range close to a moderate-roughness one. Being an addictive process, we can suppose that the titanium nitride vaporised onto the implant surface may have filled the TPS “valleys” and pits leading to a more uniform and less rough surface.

Nowadays, there is consensus on the clinical advantages of using moderately rough surfaced implants, rather than highly rough ones. This indication is based not so much on a less advantageous effect of highly rough surfaces on the osseointegration process, but rather on the problems related to their exposition to the oral bacteria. In a recent systematic review the authors show a positive relationship between bone-to-implant contact rate and surface roughness with Sa values from ∼0.5 μm up to ∼8.5 μm, thus including both minimally, moderately and highly rough surfaces. On the contrary, literature data are quite unanimous in considering highly rough surfaces more attractive for plaque formation and more exposed to the occurrence of a perimplantitis process with consequent marginal bone resorption.

It is well known that both chemical and topographical surface characteristics may considerably affect the biological features of a substratum. Bacteria are very sensitive to any surface alteration, as well as to any external substance which they may come in contact, but human cells and bacteria may respond differently to such modifications. For instance, TiN-coating is reported to favour cellular attachment of human gingival fibroblasts but reducing at the same time bacterial adhesion to the surface. In line with those results, our findings showed that TiN surfaces were less attractive for bacteria in terms of adhesion and proliferation through time, although maintaining their positive effect on BM-MSC response, with possible clinical implications in limiting inflammatory signs at level of the transmucosal portion of the implants. Such effects could be related to the chemical modification induced by the TiN treatment, as well as to the alteration of the surface texture, with a significant reduction of the roughness parameters of TiN-coated surfaces.

In the present study, TPS and TiN-TPS surfaces showed to induce a comparable response of BM-MSC cultures in terms of both cell adhesion/proliferation and differentiation. Only few studies have investigated the effect of TiN-coating at level of the bone-implant interface, focusing on the adhesion of bone cells precursors to TiN-coated implant surfaces. In all these cases, the authors showed a comparable cell adhesion and proliferation to TiN-coated and uncoated substrates. Although it is difficult to compare the above mentioned studies amongst them, due to the existing remarkable difference in terms of many variables, such as the typology of TiN-coating applied, the surface topography of the specimens and the cellular model used, our results appear in line with the available literature data, showing comparable results between TiN-coated and uncoated surfaces in terms of cell adhesion at 6 h from plating and short-term proliferation at 3 days. The differentiation of BM-MSC towards the osteoblastic phenotype is a complex process which follows a precise temporal sequence involving several phases. The evaluation of AP activity, osteocalcin and extracellular calcium levels allowed us to follow BM-MSC through the phases of the differentiation process. The only research available in literature which dealt with osteogenic markers in response to TiN surfaces was performed on mature osteoblasts. In that study, cells seeded onto TiN-coated titanium surfaces showed no difference in terms of osteogenic markers (alkaline phosphatase activity and osteocalcin synthesis) with respect to uncoated surfaces.

In our experiments, AP specific activity, as well as the synthesis of osteocalcin and the deposition of calcium into the extracellular matrix, were comparable between TiN coated and uncoated samples. Thus, TiN-coating did not show to hamper BM-MSC differentiation in terms of any of the investigated markers with respect to the uncoated titanium surfaces.

Conclusion

TiN-coating significantly improved the biological properties of TPS surfaces reducing bacterial adhesion and proliferation, with potentially relevant clinical significance in terms of decreased susceptibility to the development of a peri-implant disease, although maintaining the advantageous features of these surfaces towards bone cell precursors.

Acknowledgements

This research was supported by Research Grant from the Second University of Naples, Naples, Italy . The authors would like to thank P.H.I. s.r.l. (San Vittore Olona, Milano, Italy) for kindly providing the titanium specimens used in the experimentation.

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The effects of titanium nitride-coating on the topographic and biological features of TPS implant surfaces Marco Annunziata , Adriana Oliva , Maria Assunta Basile , Michele Giordano , Nello Mazzola , Antonietta Rizzo , Alessandro Lanza and Luigi Guida Journal of Dentistry, 2011-11-01, Volume 39, Issue 11, Pages 720-728, Copyright © 2011 Elsevier Ltd Abstract Objectives Titanium nitride (TiN) coating has been proposed as an adjunctive surface treatment aimed to increase the physico-mechanical and aesthetic properties of dental implants. In this study we investigated the surface characteristics of TiN-coated titanium plasma sprayed (TiN-TPS) and uncoated titanium plasma sprayed (TPS) surfaces and their biological features towards both primary human bone marrow mesenchymal stem cells (BM-MSC) and bacterial cultures. Methods 15 mm × 1 mm TPS and TiN-TPS disks (P.H.I. s.r.l., San Vittore Olona, Milano, Italy) were topographically analysed by confocal optical profilometry. Primary human BM-MSC were obtained from healthy donors, isolated and expanded. Cells were seeded on the titanium disks and cell adhesion, proliferation, protein synthesis and osteoblastic differentiation in terms of alkaline phosphatase activity, osteocalcin synthesis and extracellular mineralization, were evaluated. Furthermore, adhesion and proliferation of Streptococcus pyogenes and Streptococcus sanguinis on both surfaces were also analysed. Results TiN-TPS disks showed a decreased roughness (about 50%, p < 0.05) and a decreased bacterial adhesion and proliferation compared to TPS ones. No difference ( p > 0.05) in terms of BM-MSC adhesion, proliferation and osteoblastic differentiation between TPS and TiN-TPS surfaces was found. Conclusions TiN coating showed to modify the topographical characteristics of TPS titanium surfaces and to significantly reduce bacterial adhesion and proliferation, although maintaining their biological affinity towards bone cell precursors. 1 Introduction Titanium (Ti) and its alloys have proved their safety and efficacy as dental implant materials, as demonstrated by the long-term success rate of implant-supported rehabilitations. Osseointegration of dental implants results from the interaction between the titanium surface and the cellular and matrix constituents of the surrounding bone tissue. Topographic and chemical surface modifications of titanium implants may significantly affect osseointegration. In particular, micro-rough surfaces, obtainable by several techniques, such as titanium plasma-spraying, sand-blasting, acid etching and anodic oxidation, have been demonstrated to increase the implant affinity for bone cells and lead to higher values of bone-to-implant contact rate and retention into the bone. However titanium implants, especially in the commercially pure form, have a low strength and can undergo to physical abrasion in the oral environment, e.g. due to oral prophylaxis procedures. Furthermore, the titanium grey colour can raise aesthetic problems when it is not adequately masked by soft tissue at level of the gingival area. In order to overcome these problems, the titanium nitride (TiN) coating has been recently introduced also in the field of dental titanium implants. Titanium nitride is a material commonly used to cover a number of metal tools, including surgical instruments, in order to improve their surface properties and aesthetic appeal (thanks to its characteristic golden colour). TiN-coated dental implants show higher physico-mechanical properties, and allow a better camouflage under the gingival tissue, rather than conventional grey titanium implants. However, whether TiN-coating could affect the topographic and the biological features of a dental implant surfaces has not been extensively investigated. Titanium plasma sprayed implants (TPS) have been largely commercialised in the last decades and were proved to guarantee higher performances compared to turned implants in terms of bone-to-implant contact rate and retention into the bone. However, some studies have showed that the use of such highly rough surfaces couples with higher bacterial plaque retention and marginal bone loss due to perimplantitis. The purpose of the present study was to investigate if an additional treatment of TiN-coating of conventional titanium plasma sprayed implant surfaces could affect their properties in terms of topographic characteristics, affinity towards bone osteoprogenitors (human bone marrow mesenchymal stem cells), and susceptibility to bacterial colonization ( Streptococcus pyogenes and Streptococcus sanguinis) . 2 Materials and methods 2.1 Products and reagents All cell culture biologics were purchased from Gibco BRL (Grand Island, NY, USA), and all chemicals were from Sigma Chemical Co. (St. Louis, MO, USA) when not otherwise specified. 2.2 Specimen preparation Two different titanium implant surfaces were analysed: titanium plasma sprayed (TPS) and TiN-coated titanium plasma sprayed (TiN-TPS). All specimens were prepared by a commercial firm (P.H.I. s.r.l., San Vittore Olona, Milano, Italy) in form of 15-mm wide and 1-mm thick disks of Ti–6Al–4V. The disks were cleaned of surface organic contaminants by ultrasonic agitation in a series of detergent solutions, acetone, ethanol, and deionised water. All specimens were plasma sprayed with grade 4 titanium. Test specimens were additionally TiN coated by a process of physical vapour deposition (PVD). All disks were singularly packed and finally sterilised in a steam autoclave. For cell culture assays, titanium disks were put on the bottom of 24-well plates. The polystyrene surface of the multiwell plates was used as control. 2.3 Surface topographic characterisation Qualitative and quantitative measurements of implant surfaces were made by a confocal optical profilometer (PLμ 2300, Schaefer Italia srl, Rovigo) on 636 μm × 849 μm areas at ambient conditions. All measurements were performed on 6 different points, randomly distributed over the surface, with at least one scan effected close to the centre and one close to the edge of each specimen. Mean roughness (Sa), root mean square roughness (Sq) and ten-point average roughness (Sz) were calculated as typical surface texture parameters. Such parameters allow to duly characterise surfaces modified by different treatments and procedures, such as the TPS and the TiN-TPS. In particular, Sa is the arithmetic average of the absolute values of the surface height deviations measured from a reference plane. It is the most diffused parameter for measuring surface texture, however it only quantifies the “absolute” magnitude of the surface heights and is insensitive to the spatial distribution of the surface heights. Sa is also insensitive to the “polarity” of the surface texture in that a deep valley or a high peak will result in the same Sa value. Sq, namely the root mean square roughness, is the statistical measure of the magnitude of the height distribution and correlates well with Sa. Also in this case, a series of high peaks or a series of deep valleys of equal magnitude will produce the same Sq value. Sz, namely the ten-point average roughness, is found from the difference between the average maximum peak height of the ten highest peaks and is the average maximum valley depth of the ten lowest valleys found over the complete 3D image. Sz may be used to characterise the extreme features of a surface, being a nominal measure of the “peak-to-valley” range of the surface. 2.4 Preparation of a collection of human bone marrow mesenchymal stem cells (BM-MSC) Ten ml samples of human bone marrow were harvested from two healthy donors. Informed consent was provided according to the Declaration of Helsinki. The research was institutionally approved. BM-MSC cultures were initiated as previously described. Briefly, heparinised bone marrow was diluted 1:5 with complete culture medium consisting of OptiMEM containing 10% (v/v) foetal calf serum (FCS), 100 units/ml penicillin, 100 μg/ml streptomycin and 50 μg/ml sodium ascorbate, and incubated at 37 °C in a 5% CO 2 humidified atmosphere. Although present in a percent extremely low with respect to the total of mononuclear cells originally present in the bone marrow, BM-MSC can be obtained on the basis of their ability to adhere on polystyrene plates, whilst the cells of the haemopoietic lineage remain in suspension and can be easily removed. After 48 h, the medium containing all non-adherent cellular elements was centrifuged 10 min at 800 × g in order to remove the haematopoietic cells and added again to the dish. In 3–4 days, several foci of adherent spindle-like cells appeared and reached the sub-confluence in 2 weeks. The medium was refreshed every 3 days, each time leaving one half of the conditioned medium. The cells harvested from each donor were kept separately and not pooled with other preparations. Cultures between the second and fourth passage were used in our experiments. 2.5 Cell adhesion and proliferation BM-MSC were seeded on implant surfaces and control wells at a density of 15,000 cells/cm 2 in complete culture medium. Cell adhesion to implant surfaces at 6 h from plating and cell proliferation at 3 days were assessed by MTT vitality assay. The key component of this assay is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Mitochondrial dehydrogenases of living cells reduce the tetrazolium ring, yielding a blue formazan product, which can be measured spectrophotometrically. Cells were washed with phosphate buffered saline (PBS) and incubated with 0.5 mg/ml MTT solution for 4 h at 37 °C. At the end of this time, the liquid was aspirated and the insoluble formazan produced was dissolved in isopropanol–HCl 0.1 M. The optical density was measured at 570 nm, subtracting background absorbance determined at 690 nm. Cell adhesion and morphology were also evaluated by scanning electron microscopy (SEM). Cells were plated on titanium surfaces as above mentioned. After 6 h cells were rinsed three times with PBS and fixed for 30 min with 2.5% glutaraldehyde. The fixed cell layers were washed in PBS and dehydrated by graded ethanol solutions and critical point drying. Samples were mounted on stubs, coated with Au/Pd alloy and examined by SEM (Quanta 200, FEI Europe Company, the Netherlands). 2.6 Osteoblastic differentiation The effects on cell differentiation was evaluated analyzing the expression of specific markers of the osteoblastic phenotype, namely alkaline phosphatase activity, osteocalcin production and the mineralization of the extracellular matrix. 2.7 Alkaline phosphatase specific activity The alkaline phosphatase (AP) specific activity of BM-MSC grown on the titanium surfaces was evaluated after 7 and 14 days of culture. Once removed the medium, the wells were rinsed with 20 mM TRIS–HCl – 0.5 M NaCl, pH 7.4 (TBS) and the cells lysed with a specific buffer (20 mM Tris/HCl, pH 7.4, 0.5 mM NaCl, 0.25%Triton X-100, 0.5 mM PMSF, 0.5 mM DTT). After 30 min in ice, the cell lysates were centrifuged at 13,000 × g for 5 min, and the supernatants assayed for AP activity. Protein concentration was determined according to the method of Bradford. AP activity was determined by measuring the release of para-nitrophenol (PNP) from disodium para-nitrophenyl phosphate (PNPP). The reaction mixture contained 10 mM PNPP, 0.5 mM MgCl 2 , diethanolamine phosphate buffer pH 10.5, and 10–30 μg of cell lysate in a final volume of 0.5 ml. After 30 min at 37 °C, the reaction was stopped by adding 0.5 ml of 0.5 M NaOH. PNP levels were measured spectrophotometrically at 405 nm. The AP activity was normalised to the protein content and expressed as units/mg protein, where one unit was defined as the amount of enzyme that hydrolyzes 1 nmol of PNPP/min under the specified conditions. 2.8 Osteocalcin synthesis To evaluate osteocalcin synthesis confluent cultures grown on the different surfaces for 2 weeks were incubated in FCS-free Opti-MEM in the presence of 0.1% bovine serum albumin and 100 nM 1,25-dhydroxycolecalciferol for 48 h. The levels of polypeptide secreted in the medium were measured by means of an immunoenzymatic assay (Biosource International, Camarillo, CA, USA) that utilises monoclonal highly specific antibodies and a peroxidase as conjugated enzyme. The amount of osteocalcin was calculated in ng/ml and then normalised to the protein content. 2.9 Extracellular matrix mineralization The ability of titanium surfaces to promote the extracellular matrix mineralization was tested by quantification of the calcium levels. BM-MSC confluent cultures were incubated for 20 days with an osteogenic medium composed of 100 nM dexamethasone and 10 mM β-glycerophosphate. The calcium levels were measured colorimetrically using arsenazo III, reagent: cells were decalcified with 0.6 N HCl for 24 h and the calcium released in the supernatant was determined at 575 nm using a plate reader and calculated according to a standard solution. 2.10 Bacterial cultures Bacteria cell lines used in this study were S. pyogenes and S. sanguinis . The dry pellet was rehydrated in 6 ml of Luria broth (LB) consisting of 10 g tryptone, 5 g yeast extract, and 5 g NaCl per litre double distilled water with the pH adjusted to 7.4. The bacterial suspension was agitated under standard cell conditions (5% CO 2 /95% humidified air at 37 °C) for 24 h until the stationary phase was reached. The second passage of bacteria was diluted at a ratio of 1:200 into fresh LB and incubated until it reached stationary phase. The next passage was then frozen in one part LB and one part glycerol and stored at −18 °C. All experiments were conducted from this frozen stock. One day before bacterial seeding for experiments, a sterile 10 ml loop was used to withdraw bacteria from the frozen stock and to inoculate a centrifuge tube with 3 ml of fresh LB. 2.11 Bacterial adhesion and proliferation Bacterial growth from 2 day cultures of each strain was harvested and the optical density (OD) in each culture tube was adjusted to 1 × 10 7 bacteria/ml (as estimated by the McFarland scale) by diluting the LB bacteria cultures to an optical density of 0.52 at 562 nm in a spectrophotometer and then further diluted at a ratio of 1:90 in LB Medium. Sterile titanium surfaces were placed individually in 12 well plates and a total of 10 6 cells/ml suspension of each reference strain was added, in a total volume of 2 ml. Plates were incubated for 2, 6, 12 and 24 h at 37 °C. After incubation, each sample was washed twice with PBS; after washing, the foils of each samples were incubated in LB Medium and in 12 well plates for 48 h to the bacterial growth to each surface. After incubation, 1 ml was harvested and the optical density was spectrophotometrically measured at 562 nm. Bacterial adhesion was also observed using an environmental scanning electron microscope (ESEM Quanta 200, FEI Europe Company, the Netherlands). In particular, titanium samples, after a 2 h-incubation with S. sanguinis , were imaged in ESEM mode at 15 kV, using Peltier Stage and gaseous secondary electron detector (GSED). The chamber parameters were settled to 58 °C temperature and 3.60 Torr pressure. 2.12 Statistical analysis Statistical analysis was performed by a dedicated software (NCSS for Windows, Kaysville, Utah, USA). Data were expressed as the mean ± standard deviation (SD) of relative units (percentage of control) or absolute values. The means of each experimental group were compared by unpaired Student's t -test, with the value of significance set at p < 0.05. 3 Results 3.1 Surface roughness and topographical characterisation Images of TPS and TiN-TPS surfaces obtained by confocal optical profilometry are shown in Fig. 1 a . The two samples appear similar, with a comparable extremely rough surface. Fig. 1 Qualitative and quantitative characterisation of implant surfaces. (a) Confocal optical profilometry images of titanium plasma spray (TPS) and titanium-nitride TPS (TiN-TPS) implant surfaces. (b) Roughness values measured on TPS and TiN-TPS surfaces. Data are expressed as mean ± standard deviation (SD). * = p < 0.05; ** = p < 0.01; Sa = mean roughness; Sq = root mean square roughness; Sz = ten-point average roughness. Sa, Sq and Sz values of the experimental surfaces are reported in Fig. 1 b. Significantly higher values for the uncoated surfaces compared to the TiN-coated one were detected in terms of Sa, Sq ( p < 0.05) and Sz ( p < 0.01). 3.2 BM-MSC adhesion and proliferation Cell adhesion on implant surfaces was evaluated 6 h after plating by SEM analysis and MTT viability test. Both TPS and TiN-TPS surfaces showed a good affinity for BM-MSC, as evidenced by SEM images ( Fig. 2 a) , resulting in comparable cell adhesion values ( Fig. 2 b). On a parallel set of samples, BM-MSC were cultured for 72 h. At the end of the incubation, no difference in terms cell number between the two surface types was detected ( Fig. 2 c). Fig. 2 Cell adhesion and proliferation on implant surfaces. (a) SEM images of bone marrow mesenchymal stem mesenchymal stem cells (BM-MSC) grown for 6 h on titanium plasma spray (TPS) and titanium-nitride TPS (TiN-TPS) implant surfaces at magnification 20,000×. (b) Adhesion of BM-MSC at 6 h from seeding on TPS and TiN-TPS implant surfaces. (b) Proliferation of BM-MSC at 72 h from seeding on TPS and TiN-TPS implant surfaces. In both cases, cell vitality was spectrophotometrically assessed by MTT assay. Results were obtained from 3 independent experiments. Data are expressed as percentage of those obtained on cells cultured on polystyrene surface (control) ± standard deviation (SD). NS = not significant ( p > 0.05). 3.3 Osteoblastic differentiation Surface typology did not affect the expression of early and late osteoblastic markers. Both TPS and TiN-TPS specimens showed comparable values ( p > 0.05) in terms of alkaline phosphatase activity at both 7 and 14 days from plating ( Fig. 3 a) , osteocalcin synthesis at 14 days ( Fig. 3 b) and extracellular matrix mineralization at 20 days ( Fig. 3 c). Also the overall protein synthesis at 7 and 14 days was comparable ( p < 0.05) between the two experimental groups ( Fig. 3 d). Fig. 3 Cell differentiation in response to implant surfaces. (a) Alkaline phosphatase specific activity of bone marrow mesenchymal stem cells (BM-MSC) cultured for 7 and 14 days on titanium plasma spray (TPS) and titanium-nitride TPS (TiN-TPS) implant surfaces. (b) Osteocalcin synthesis by BM-MSC at 14 days from seeding on TPS and TiN-TPS implant surfaces. (c) Extracellular matrix mineralization expressed as calcium level produced by BM-MSC cultured for 20 days on the implant surfaces. (d) Overall protein synthesis by BM-MSC at 7 and 14 days from seeding on TPS and TiN-TPS implant surfaces. Results were obtained from 3 independent experiments. Data are expressed as percentage of those obtained on cells cultured on polystyrene surface (control) ± standard deviation (SD). NS = not significant ( p > 0.05). 3.4 Bacterial adhesion and proliferation Significantly different bacterial adhesion and proliferation on TPS and TiN-TPS surfaces was interestingly assessed. In particular, both bacteria cultures ( S. pyogenes and S. sanguinis ) showed a significantly lower adhesion and proliferation over time on TiN-TPS compared to TPS surfaces ( Fig. 4 ). Such results were also confirmed by ESEM analysis ( Fig. 4 a) which clearly showed less dense and numerous bacterial cells on TiN-TPS than on TPS surfaces 2 h after plating. Fig. 4 Bacterial adhesion and proliferation on implant surfaces. (a) Colonies of S. sanguinis were plated on titanium plasma spray (TPS) and titanium-nitride TPS (TiN-TPS) implant surfaces. After 2 h, samples were imaged by environmental scanning electron microscopy (ESEM) (magnification ×5000). (b) and (c) Time course of S. sanguinis (b) and S. pyogenes (c) proliferation at 2, 6, 12 and 24 h from seeding on TPS and TiN-TPS implant surfaces. Bacterial concentration was spectrophotometrically assessed at 562 nm. Results were obtained from 3 independent experiments. Data are expressed as optical density (OD) values ± standard deviation (SD). * = p < 0.05, ** = p < 0.01, *** = p < 0.001 between TPS and TiN-TPS. 4 Discussion In the present study the biological response of human bone marrow mesenchymal stem cells to TiN-coated/uncoated implant surfaces was analysed. TiN-coating showed to affect both the topographical and biological features of tested implant surfaces, reducing in particular bacterial adhesion, without, however, compromising BM-MSC adhesion, proliferation and differentiation. Bone marrow mesenchymal stem cells are able to self-renew and to differentiate into precursors of several tissues, including the osteoprogenitor cells. They play a central role in the osseointegration process, being involved in the normal remodelling and reparative mechanisms of bone. The contact of the osteoprogenitor cells with the implant surface represent a central phase of the osteointegration process, being the basis for all the following events, including the deposition of an organised extracellular matrix and its mineralization. The in vitro experimental approach allows investigating in detail this interaction, as well as other phenomena, but many confounding factors need to be controlled. Only a correct assessment and standardization of the in vitro , as well as in vivo and clinical , research parameters may allow some kind of extrapolation of the results “from bench to bed side”. In this sense, the selection of a suitable cell system is crucial. In our study, the biological response of BM-MSC to implant surfaces was investigated using primary human cultures. Primary cells from normal tissues represent, in our opinion, the ideal cellular model for pre-clinical evaluations, because of their closeness to the biological response of real living tissues, with respect to animal-derived or transformed osteoblast cell lines, although their use entails some inter-donor variability. In order to limit this variable and enhance data reliability, two different BM-MSC populations were prepared in our study and both of them gave in all cases comparable response. Different chemo-mechanical treatments exist which are able to modify the surface topography and roughness of surgical and prosthetic implant components. Titanium plasma spraying represents a recognised method of increasing surface roughness and enlarging the implant surface area. With this additive process, titanium powders are injected into a plasma torch at high temperature. The titanium particles are projected onto the surface of the implants where they condense and fuse together, forming a uniform film. The result is a highly rough surface, in the 10 μm range, with an isotropic topographical pattern, i.e. without a visible direction in its roughness. As shown by our measurements, the TPS surfaces showed an average Sa value of about 6 μm, that is considerably higher than smooth surfaces (turned, polished, etc.) or moderately rough surfaces (sandblasted, acid etched, etc.) which are in the <0.5 μm and 1–2 μm range of Sa values, respectively, following the classification proposed by Albrektsson and Wennerberg. TiN coating was applied as an adjunctive treatment to TPS surfaces. Different nitriding processes exist, but the physical vapour deposition represents one of the most efficient and widely used TiN-coating technique in the biomedical field. With this process, the titanium is vaporised in a nitrogen atmosphere by means of a cathodic arc, and the evaporating material and the reactive gas become highly ionised. The result is the formation of a thin layer of titanium nitride which is tightly connected to the substratum by an intermixed zone which is a peculiarity of the cathodic arc technique. An interesting finding of our investigation is that the TiN-coating process significantly altered the topographic characteristics of TPS surfaces. The roughness values of TiN-TPS disks were significantly lower compared to the uncoated ones (Sa 3 vs. 6 mm). In other words, TiN-coating seemed to switch the TPS roughness from a high-roughness range close to a moderate-roughness one. Being an addictive process, we can suppose that the titanium nitride vaporised onto the implant surface may have filled the TPS “valleys” and pits leading to a more uniform and less rough surface. Nowadays, there is consensus on the clinical advantages of using moderately rough surfaced implants, rather than highly rough ones. This indication is based not so much on a less advantageous effect of highly rough surfaces on the osseointegration process, but rather on the problems related to their exposition to the oral bacteria. In a recent systematic review the authors show a positive relationship between bone-to-implant contact rate and surface roughness with Sa values from ∼0.5 μm up to ∼8.5 μm, thus including both minimally, moderately and highly rough surfaces. On the contrary, literature data are quite unanimous in considering highly rough surfaces more attractive for plaque formation and more exposed to the occurrence of a perimplantitis process with consequent marginal bone resorption. It is well known that both chemical and topographical surface characteristics may considerably affect the biological features of a substratum. Bacteria are very sensitive to any surface alteration, as well as to any external substance which they may come in contact, but human cells and bacteria may respond differently to such modifications. For instance, TiN-coating is reported to favour cellular attachment of human gingival fibroblasts but reducing at the same time bacterial adhesion to the surface. In line with those results, our findings showed that TiN surfaces were less attractive for bacteria in terms of adhesion and proliferation through time, although maintaining their positive effect on BM-MSC response, with possible clinical implications in limiting inflammatory signs at level of the transmucosal portion of the implants. Such effects could be related to the chemical modification induced by the TiN treatment, as well as to the alteration of the surface texture, with a significant reduction of the roughness parameters of TiN-coated surfaces. In the present study, TPS and TiN-TPS surfaces showed to induce a comparable response of BM-MSC cultures in terms of both cell adhesion/proliferation and differentiation. Only few studies have investigated the effect of TiN-coating at level of the bone-implant interface, focusing on the adhesion of bone cells precursors to TiN-coated implant surfaces. In all these cases, the authors showed a comparable cell adhesion and proliferation to TiN-coated and uncoated substrates. Although it is difficult to compare the above mentioned studies amongst them, due to the existing remarkable difference in terms of many variables, such as the typology of TiN-coating applied, the surface topography of the specimens and the cellular model used, our results appear in line with the available literature data, showing comparable results between TiN-coated and uncoated surfaces in terms of cell adhesion at 6 h from plating and short-term proliferation at 3 days. The differentiation of BM-MSC towards the osteoblastic phenotype is a complex process which follows a precise temporal sequence involving several phases. The evaluation of AP activity, osteocalcin and extracellular calcium levels allowed us to follow BM-MSC through the phases of the differentiation process. The only research available in literature which dealt with osteogenic markers in response to TiN surfaces was performed on mature osteoblasts. In that study, cells seeded onto TiN-coated titanium surfaces showed no difference in terms of osteogenic markers (alkaline phosphatase activity and osteocalcin synthesis) with respect to uncoated surfaces. In our experiments, AP specific activity, as well as the synthesis of osteocalcin and the deposition of calcium into the extracellular matrix, were comparable between TiN coated and uncoated samples. Thus, TiN-coating did not show to hamper BM-MSC differentiation in terms of any of the investigated markers with respect to the uncoated titanium surfaces. 5 Conclusion TiN-coating significantly improved the biological properties of TPS surfaces reducing bacterial adhesion and proliferation, with potentially relevant clinical significance in terms of decreased susceptibility to the development of a peri-implant disease, although maintaining the advantageous features of these surfaces towards bone cell precursors. Acknowledgements This research was supported by Research Grant from the Second University of Naples, Naples, Italy . The authors would like to thank P.H.I. s.r.l. (San Vittore Olona, Milano, Italy) for kindly providing the titanium specimens used in the experimentation. References 1. Buser D., Mericske-Stern R., Bernard J.P., Behneke A., Behneke N., Hirt H.P., et. al.: Long-term evaluation of non-submerged ITI implants. 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