Titanium nanotubes stimulate osteoblast differentiation of stem cells from pulp and adipose tissue
Identifieur interne : 002B74 ( Pmc/Corpus ); précédent : 002B73; suivant : 002B75Titanium nanotubes stimulate osteoblast differentiation of stem cells from pulp and adipose tissue
Auteurs : Alfonso Pozio ; Annalisa Palmieri ; Ambra Girardi ; Francesca Cura ; Francesco CarinciSource :
- Dental Research Journal [ 1735-3327 ] ; 2012.
Abstract
Titanium is the gold standard among materials used for prosthetic devices because of its good mechanical and chemical properties. When exposed to oxygen, titanium becomes an oxide, anatase that is biocompatible and able to induce osseointegration.
In this study we compared the expression profiling of stem cells cultivated on two types of surface: Pure titanium disk and nanotube titanium disk in order to detect if nanotube titanium instead (NTD) surface stimulates stem cells towards osteoblast differentiation.
Stem cells cultivated on nanotube titanium disks showed the upregulation of bone-related genes RUNX2, FOSL1 and SPP1.
Results demonstrated that nanotube titanium disk surface is more osteo-induced surface compared to titanium disk, promoting the differentiation of mesenchymal stem cells in osteoblasts.
Url:
DOI: 10.4103/1735-3327.109745
PubMed: 23814578
PubMed Central: 3692168
Links to Exploration step
PMC:3692168Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Titanium nanotubes stimulate osteoblast differentiation of stem cells from pulp and adipose tissue</title><author><name sortKey="Pozio, Alfonso" sort="Pozio, Alfonso" uniqKey="Pozio A" first="Alfonso" last="Pozio">Alfonso Pozio</name><affiliation><nlm:aff id="aff1">ENEA, IDROCOMB, C.R. Casaccia, Rome, Italy</nlm:aff></affiliation></author><author><name sortKey="Palmieri, Annalisa" sort="Palmieri, Annalisa" uniqKey="Palmieri A" first="Annalisa" last="Palmieri">Annalisa Palmieri</name><affiliation><nlm:aff id="aff2">Department of Medical-Surgical Sciences of Communication and Behavior, Section of Maxillofacial and Plastic Surgery, University of Ferrara, Ferrara, Italy</nlm:aff></affiliation></author><author><name sortKey="Girardi, Ambra" sort="Girardi, Ambra" uniqKey="Girardi A" first="Ambra" last="Girardi">Ambra Girardi</name><affiliation><nlm:aff id="aff3">Department of Histology, Embryology and Applied Biology, Centre of Molecular Genetics, CARISBO Foundation, University of Bologna, Bologna, Italy</nlm:aff></affiliation></author><author><name sortKey="Cura, Francesca" sort="Cura, Francesca" uniqKey="Cura F" first="Francesca" last="Cura">Francesca Cura</name><affiliation><nlm:aff id="aff3">Department of Histology, Embryology and Applied Biology, Centre of Molecular Genetics, CARISBO Foundation, University of Bologna, Bologna, Italy</nlm:aff></affiliation></author><author><name sortKey="Carinci, Francesco" sort="Carinci, Francesco" uniqKey="Carinci F" first="Francesco" last="Carinci">Francesco Carinci</name><affiliation><nlm:aff id="aff2">Department of Medical-Surgical Sciences of Communication and Behavior, Section of Maxillofacial and Plastic Surgery, University of Ferrara, Ferrara, Italy</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">23814578</idno><idno type="pmc">3692168</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3692168</idno><idno type="RBID">PMC:3692168</idno><idno type="doi">10.4103/1735-3327.109745</idno><date when="2012">2012</date><idno type="wicri:Area/Pmc/Corpus">002B74</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">002B74</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Titanium nanotubes stimulate osteoblast differentiation of stem cells from pulp and adipose tissue</title><author><name sortKey="Pozio, Alfonso" sort="Pozio, Alfonso" uniqKey="Pozio A" first="Alfonso" last="Pozio">Alfonso Pozio</name><affiliation><nlm:aff id="aff1">ENEA, IDROCOMB, C.R. Casaccia, Rome, Italy</nlm:aff></affiliation></author><author><name sortKey="Palmieri, Annalisa" sort="Palmieri, Annalisa" uniqKey="Palmieri A" first="Annalisa" last="Palmieri">Annalisa Palmieri</name><affiliation><nlm:aff id="aff2">Department of Medical-Surgical Sciences of Communication and Behavior, Section of Maxillofacial and Plastic Surgery, University of Ferrara, Ferrara, Italy</nlm:aff></affiliation></author><author><name sortKey="Girardi, Ambra" sort="Girardi, Ambra" uniqKey="Girardi A" first="Ambra" last="Girardi">Ambra Girardi</name><affiliation><nlm:aff id="aff3">Department of Histology, Embryology and Applied Biology, Centre of Molecular Genetics, CARISBO Foundation, University of Bologna, Bologna, Italy</nlm:aff></affiliation></author><author><name sortKey="Cura, Francesca" sort="Cura, Francesca" uniqKey="Cura F" first="Francesca" last="Cura">Francesca Cura</name><affiliation><nlm:aff id="aff3">Department of Histology, Embryology and Applied Biology, Centre of Molecular Genetics, CARISBO Foundation, University of Bologna, Bologna, Italy</nlm:aff></affiliation></author><author><name sortKey="Carinci, Francesco" sort="Carinci, Francesco" uniqKey="Carinci F" first="Francesco" last="Carinci">Francesco Carinci</name><affiliation><nlm:aff id="aff2">Department of Medical-Surgical Sciences of Communication and Behavior, Section of Maxillofacial and Plastic Surgery, University of Ferrara, Ferrara, Italy</nlm:aff></affiliation></author></analytic><series><title level="j">Dental Research Journal</title><idno type="ISSN">1735-3327</idno><idno type="eISSN">2008-0255</idno><imprint><date when="2012">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec id="st1"><title>Background:</title><p>Titanium is the gold standard among materials used for prosthetic devices because of its good mechanical and chemical properties. When exposed to oxygen, titanium becomes an oxide, anatase that is biocompatible and able to induce osseointegration.</p></sec><sec id="st2"><title>Materials and Methods:</title><p>In this study we compared the expression profiling of stem cells cultivated on two types of surface: Pure titanium disk and nanotube titanium disk in order to detect if nanotube titanium instead (NTD) surface stimulates stem cells towards osteoblast differentiation.</p></sec><sec id="st3"><title>Results:</title><p>Stem cells cultivated on nanotube titanium disks showed the upregulation of bone-related genes RUNX2, FOSL1 and SPP1.</p></sec><sec id="st4"><title>Conclusions:</title><p>Results demonstrated that nanotube titanium disk surface is more osteo-induced surface compared to titanium disk, promoting the differentiation of mesenchymal stem cells in osteoblasts.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Carinci, F" uniqKey="Carinci F">F 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Dent Res J (Isfahan)</journal-id><journal-id journal-id-type="iso-abbrev">Dent Res J (Isfahan)</journal-id><journal-id journal-id-type="publisher-id">DRJ</journal-id><journal-title-group><journal-title>Dental Research Journal</journal-title></journal-title-group><issn pub-type="ppub">1735-3327</issn><issn pub-type="epub">2008-0255</issn><publisher><publisher-name>Medknow Publications & Media Pvt Ltd</publisher-name><publisher-loc>India</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">23814578</article-id><article-id pub-id-type="pmc">3692168</article-id><article-id pub-id-type="publisher-id">DRJ-9-169</article-id><article-id pub-id-type="doi">10.4103/1735-3327.109745</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>Titanium nanotubes stimulate osteoblast differentiation of stem cells from pulp and adipose tissue</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Pozio</surname><given-names>Alfonso</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Palmieri</surname><given-names>Annalisa</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Girardi</surname><given-names>Ambra</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Cura</surname><given-names>Francesca</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Carinci</surname><given-names>Francesco</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="corresp" rid="cor1"></xref></contrib></contrib-group><aff id="aff1"><label>1</label>ENEA, IDROCOMB, C.R. Casaccia, Rome, Italy</aff><aff id="aff2"><label>2</label>Department of Medical-Surgical Sciences of Communication and Behavior, Section of Maxillofacial and Plastic Surgery, University of Ferrara, Ferrara, Italy</aff><aff id="aff3"><label>3</label>Department of Histology, Embryology and Applied Biology, Centre of Molecular Genetics, CARISBO Foundation, University of Bologna, Bologna, Italy</aff><author-notes><corresp id="cor1"><bold>Address for correspondence:</bold> Prof. Francesco Carinci, Department of Medical-Surgical Sciences of Communication and Behavior, Section of Maxillofacial and Plastic Surgery University of Ferrara, Corso Giovecca 203, 44100 Ferrara, Italy. <bold>E-mail:</bold><email xlink:href="crc@unife.it">crc@unife.it</email></corresp></author-notes><pub-date pub-type="ppub"><month>12</month><year>2012</year></pub-date><volume>9</volume><issue>Suppl 2</issue><fpage>S169</fpage><lpage>S174</lpage><history><date date-type="received"><month>6</month><year>2012</year></date><date date-type="accepted"><month>8</month><year>2012</year></date></history><permissions><copyright-statement>Copyright: © Dental Research Journal</copyright-statement><copyright-year>2012</copyright-year><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by-nc-sa/3.0"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><abstract><sec id="st1"><title>Background:</title><p>Titanium is the gold standard among materials used for prosthetic devices because of its good mechanical and chemical properties. When exposed to oxygen, titanium becomes an oxide, anatase that is biocompatible and able to induce osseointegration.</p></sec><sec id="st2"><title>Materials and Methods:</title><p>In this study we compared the expression profiling of stem cells cultivated on two types of surface: Pure titanium disk and nanotube titanium disk in order to detect if nanotube titanium instead (NTD) surface stimulates stem cells towards osteoblast differentiation.</p></sec><sec id="st3"><title>Results:</title><p>Stem cells cultivated on nanotube titanium disks showed the upregulation of bone-related genes RUNX2, FOSL1 and SPP1.</p></sec><sec id="st4"><title>Conclusions:</title><p>Results demonstrated that nanotube titanium disk surface is more osteo-induced surface compared to titanium disk, promoting the differentiation of mesenchymal stem cells in osteoblasts.</p></sec></abstract><kwd-group><kwd>Gene expression</kwd><kwd>nanotubes</kwd><kwd>osteoblasts</kwd><kwd>stem cells</kwd><kwd>titanium disks</kwd></kwd-group></article-meta></front><body><sec id="sec1-1"><title>INTRODUCTION</title><p>Pure titanium and titanium alloys are materials widely used in orthopedic and dental surgery because of their desirable mechanical properties, chemical stability and biocompatibility.[<xref ref-type="bibr" rid="ref1">1</xref>] Several authors studied different characteristics of implant morphology in order to value their impact on implant stability.[<xref ref-type="bibr" rid="ref2">2</xref><xref ref-type="bibr" rid="ref3">3</xref><xref ref-type="bibr" rid="ref4">4</xref><xref ref-type="bibr" rid="ref5">5</xref><xref ref-type="bibr" rid="ref6">6</xref><xref ref-type="bibr" rid="ref7">7</xref><xref ref-type="bibr" rid="ref8">8</xref>] Indeed titanium is used to manufacture joint prosthesis for partial and total joint replacement. Moreover, titanium is also used to produce plates and screws for osteo-synthesis of fractures and for dental implants to substitute lost teeth.[<xref ref-type="bibr" rid="ref9">9</xref>]</p><p>The biocompatibility of titanium is closely related to the properties of the surface oxide layer, in terms of its structure, morphology and composition.[<xref ref-type="bibr" rid="ref10">10</xref>] Normally on the surface of the prosthesis made of titanium is present a stochastic distribution of two titanium-oxide (rutile and anatase), which are responsible for the properties of the material.[<xref ref-type="bibr" rid="ref10">10</xref>] In biomedical implants, the anatase phase shows an enhanced ability to induce bone-like apatite in comparison with the rutile phase.[<xref ref-type="bibr" rid="ref10">10</xref>]</p><p>Various physical and chemical treatments of the titanium surface have been proposed with a view to obtaining the most biocompatible one.[<xref ref-type="bibr" rid="ref10">10</xref>]</p><p>We focused our interest on anatase titanium disks. These surfaces have been used as substrate for the nanotubes growth, to implement their characteristics of biocompatibility.</p><p>Tissue replacement by culturing autologous cells onto three-dimensional matrixes that facilitate cell progenitor migration, proliferation and differentiation[<xref ref-type="bibr" rid="ref11">11</xref>] is one of the most promising bone regeneration techniques.</p><p>Stem cells are undifferentiated cells with the capability to regenerate into one or more committed cell lineages. Stem cells isolated from multiple sources have been finding widespread use to advance the field of tissue repair.[<xref ref-type="bibr" rid="ref12">12</xref>]</p><p>Dental pulp is a niche housing neural-crest-derived stem cells that display plasticity and multipotential capability. This niche is easily accessible and there is limited morbidity of the anatomical site after collection of the pulp.[<xref ref-type="bibr" rid="ref13">13</xref>]</p><p>Dental pulp stem cells (DPSCs) display noticeable plasticity that is explained by their neural-crest origin.[<xref ref-type="bibr" rid="ref14">14</xref>] They can extensively proliferate and differentiate into odontoblastic, adipogenic and neural citotype, have a long lifespan and build <italic>in vivo</italic> an adult bone with Havers channels and an appropriate vascularization.[<xref ref-type="bibr" rid="ref15">15</xref>]</p><p>Adipose tissue is another ideal source of autologous stem cells because it is easily obtainable by lipoaspiration, and its mesenchymal stem cells (MSCs) content is adequate for clinical-grade cell manipulation in regenerative medicine.</p><p>These cells, that display a fibroblast-like morphology and lack intracellular lipid droplets seen in adipocytes,[<xref ref-type="bibr" rid="ref16">16</xref>] can be enzymatically digested out of adipose tissue and separated from the buoyant adipocytes by centrifugation.</p><p>A more homogeneous population emerges in culture under conditions supportive of marrow stromal cells growth. This population, termed adipose tissue-derived stem cells (ADSCs), after expansion in culture display a distinct phenotype based on cell surface protein expression and cytokine expression.[<xref ref-type="bibr" rid="ref17">17</xref>]</p><p>In this study we compared the expression profiling of stem cells (DPSCs and ADSCs) cultivated on two type of surface: Pure titanium disk (TD) and nanotube titanium disk (NTD) in order to detect if NTD surface stimulates MSCs towards osteoblast differentiation.</p><p>The quantitative expression of the mRNA of specific genes, like transcriptional factors (RUNX2 and SP7), bone-related genes (SPP1, COL1A1, COL3A1, ALPL, and FOSL1) and MSCs marker (ENG) were examined by means of real-time Reverse Transcription-Polymerase Chain Reaction (real-time RT-PCR).</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><sec id="sec2-1"><title>Titanium nanotubes disks preparation</title><p>Disks of commercially pure grade-1 titanium (Titania, Italy) have been used as substrate for the nanotube growth. The disks have diameter of 30 mm with a thickness of 0.5 mm, and were arranged to show an active area of 3.8 cm<sup>2</sup>. After 3 min. pickling in a HF (Carlo Erba)/HNO<sub>3</sub> (Carlo Erba) solution, made by a volumetric ratio of 1:3 and diluted in deionized water until 100 ml, all the titanium sheets have been set in three-electrode cell, containing a KOH 1 M solution (Carlo Erba) and subjected to a prefixed and optimized density current (1 mA/cm<sup>2</sup>), which is generated by a Potentiostat/Galvanostat Solartron 1286 for 3 min. The counter-electrode is a Platinum sheet, while the reference is a standard calomel electrode (SCE). The growth of the nanotube arrays has been made using a Glycol Ethylene solution with 1 %wt. H<sub>2</sub>O and 0.2%wt. NH<sub>4</sub>F for 3 h at 60 V. After the anodization treatment, all the samples are washed in glycol ethylene, left overnight in the dry room, in order to dry them. So to crystallize the TiO<sub>2</sub> nanotubes, obtained in amorphous form by anodic growth, after a pre-heat treatment at 80°C in vacuum for 3 h, all the samples have been placed in a tubular furnace (Lenton) for 1 h at 580°C, with a slope of 1°C/min. in air, so as to be transformed into the anatase phase.</p></sec><sec id="sec2-2"><title>DPSCs isolation</title><p>Dental germ pulp was extracted from third molars of healthy subjects, following informed consent. Pulp was digested for 1 h at 37°C in a solution containing 3 mg/ml type I collagenase, 4 mg/ml Dispase, in 4 ml phosphate-buffered saline (PBS) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 500 μg/ml clarithromycin. The solution was then filtered with 70 μm Falcon strainers (Sigma Aldrich, Inc., St Louis, Mo, USA). Filtered cells were cultivated in α-MEM culture medium (Sigma Aldrich, Inc., St Louis, Mo, USA) supplemented with 20% FCS, 100 μM 2P-ascorbic acid, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and placed in 75 ml flasks. Flasks were incubated at 37°C and 5% CO<sub>2</sub> and the medium changed twice a week.</p></sec><sec id="sec2-3"><title>ADSCs isolation</title><p>Human ADSCs were isolated from adipose tissue obtained by liposuction of adult volunteer patients. Fat was finely minced with sterile scissors, put in a tube and digested in DMEM supplemented with 1 mg/ml of collagenase type II, in 37°C water bath for 60 min, swirling occasionally.</p><p>Once centrifugated at 3000 rpm for 5 min, the sample was removed from centrifuge, shaken vigorously (to completely separate stromal cells from primary adipocytes) and centrifugated again for 5 min. The oily supernatant (which includes primary adipocytes) was aspirated and discarded, while the stromal fraction at the bottom was washed three times with 10 ml of PBSA 1X and centrifugated again for 5 min. Finally the pellet was resuspended in 10 ml of Alpha MEM medium (Sigma Aldrich, Inc., St Louis, Mo, USA) supplemented with 10% fetal calf serum, antibiotics (Penicillin 100 U/ml and Streptomycin 100 micrograms/ml-Sigma, Chemical Co., St Louis, Mo, USA) and amino acids (L-Glutamine - Sigma, Chemical Co., St Louis, Mo, USA). The medium was replaced after 2-3 days. Characterization of staminality was carried out by flow cytometric analyses.</p></sec><sec id="sec2-4"><title>Flow cytometric analyses</title><p>The purity of cell cultures was determined by analysis of different antigens after staining with fluorochrome (FITC- or PE-) conjugated mAbs anti-human CD14-FITC, CD14-PE, CD34-FITC, CD45-FITC, CD90-PE, CD105-PE (Immunotech, Marseille, France) and analyzed by FACScan. The nonspecific mouse IgG was used as isotype control (Immunotech). To avoid nonspecific fluorescence from dead cells, live cells were gated tightly using forward and side scatter.</p></sec><sec id="sec2-5"><title>Cell culture</title><p>For the assay, ADSCs and DPSCs were trypsinized upon sub-confluence and seeded on NTD and TD.</p><p>The medium was changed every 3 days. The cells were maintained in a humidified atmosphere of 5% CO<sub>2</sub> at 37°C. Cells were collected for RNA extraction at 15 and 30 days.</p></sec><sec id="sec2-6"><title>RNA processing</title><p>For cellular lysis, and subsequent reverse transcription of the RNA released in the solution, the TaqMan Gene Expression Cells-to-Ct Kit (Ambion Inc., Austin, TX, USA) was used. Once cultured cells were lysed with lysis buffer, free RNA was reverse transcribed to cDNA using the RT Enzyme Mix and appropriate RT buffer (Ambion Inc., Austin, TX, USA).</p><p>Finally the cDNA was amplified by real-time PCR using the included TaqMan Gene Expression Master Mix and the specific assay designed for the investigated genes.</p></sec><sec id="sec2-7"><title>Real-time PCR</title><p>Expression was quantified using real-time PCR. After normalization to the expression of the housekeeping gene RPL13A, the gene expression levels were expressed as fold changes relative to the expression of the untreated cells. Quantification was done with the delta/delta calculation method.[<xref ref-type="bibr" rid="ref18">18</xref>]</p><p>Specific forward and reverse primers and probes for the selected genes were designed using primer express software (Applied Biosystems, Foster City, CA, USA) and are listed in <xref ref-type="table" rid="T1">Table 1</xref>.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Primer and probes used in real-time Polymerase chain reaction</p></caption><graphic xlink:href="DRJ-9-169-g001"></graphic></table-wrap><p>All PCR reactions were performed in a final volume of 20 μl containing 10 μl 2X TaqMan Universal PCR master mix (Applied Biosystems, Foster City, CA, USA), 400 nM concentration of each primer and 200 nM of the probe, and cDNA. The amplifications were carried out using the ABI PRISM 7500 (Applied Biosystems, Foster City, CA, USA). After an initial denaturing step at 95°C for 10 min, amplification profile proceeded with two-step of 15 s at 95°C and 60 s at 60°C for 40 cycles. All experiments were performed including non-template controls to check for the presence of contamination.</p></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><p>ADSCs and DPSCs were cultivated on two type of surface NTD and TD. Gene expression in stem cells cultivated on NTD were compared with that cultivated on TD (control) in order to check whether NTD is more osteoinductive with respect to TD.</p><sec id="sec2-8"><title>Gene expression profiling in ADSCs</title><p>After 15 days, ADSCs cultivated on NTD showed the up-regulation of bone-related genes FOSL1, RUNX2, COL1A1, ENG and the down-regulation of SP7, ALPL and SPP1. Expression of COL3A1 was the same in both cells (cultivated on NTD and TD) [<xref ref-type="fig" rid="F1">Figure 1a</xref>].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Relative gene expression of ADSCs cultivated on titanium disks for 15 days (a) and 30 days (b)</p></caption><graphic xlink:href="DRJ-9-169-g002"></graphic></fig><p>The results of real-time PCR showed that in ADSCs cultivated on NTD after 30 days of treatment, the bone-related genes FOSL1, COL3A1, COL1A1, ALPL and SPP1 were up-regulated, while SP7, ENG and RUNX2 were down-regulated [<xref ref-type="fig" rid="F1">Figure 1b</xref>].</p></sec><sec id="sec2-9"><title>Gene expression profiling in DPSCs</title><p>Different results were obtained for DPSCs. After 15 days, stem cells cultivated on NTD showed the up-regulation of FOSL1 and SPP1 and the down-regulation of SP7, ENG, RUNX2, COL3A1, COL1A1 and ALPL [<xref ref-type="fig" rid="F2">Figure 2a</xref>].</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Relative gene expression of DPSCs cultivated on titanium disks for 15 days (a) and 30 days (b)</p></caption><graphic xlink:href="DRJ-9-169-g003"></graphic></fig><p>After 30 days, the bone-related genes ENG, FOSL1, RUNX2, COL3A1 and SPP1 were up-regulated in DPSCs cultivated on NTD, while ENG, FOSL1 and ALPL were decreased [<xref ref-type="fig" rid="F2">Figure 2b</xref>].</p></sec></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>Titanium proved to be a good material for surgical application. Since thirty years it has been used to produce prosthesis for joint replacement and for dental substitution. Recently, titania film have been used to cover prosthetic implants in medical applications such as orthopedic or dental surgery.[<xref ref-type="bibr" rid="ref19">19</xref>] In prosthesis made of titanium, the titania film surface is mainly composed by two polymorphs of titanium, rutile and anatase. Anatase titania is more advantageous for medical applications than rutile.</p><p>In this study, we tested the osteoinductive property of TDs covered with nanotube in order to detect if this surface is better than pure TDs in terms of osseointegration. Osseointegration is primary element to value when considering implant stability.[<xref ref-type="bibr" rid="ref20">20</xref><xref ref-type="bibr" rid="ref21">21</xref><xref ref-type="bibr" rid="ref22">22</xref>]</p><p>To study the osteoinductive properties of NTD, the expression levels of bone-related genes were measured using real-time RT-PCR in mesenchymal stem cells cultivated on TD.</p><p>The study was first conducted in ADSCs. The gene expression levels were measured at two time point: 15 and 30 days.</p><p>The up-regulation of the bone-related genes RUNX2, COL1A1 and FOSL1 in the first 15 days of cells culture indicated that NTD enhance osteo-differentiation and proliferation compared to TD.</p><p>RUNX2 is an important modulator of osteoblast differentiation that is activated in the first stage of differentiation and plays a fundamental role in osteoblast maturation and homeostasis. RUNX2-null mice have no osteoblasts and consequently bone tissue.[<xref ref-type="bibr" rid="ref23">23</xref>] Thus we demonstrated that NTD increase the activity of RUNX2 gene in ADSCs, which is a key point in osteo-differentiation.</p><p>FOSL1 encodes for Fra-1, a component of the dimeric transcription factor activator protein-1 (AP-1). AP-1 sites are present in the promoters of many osteoblast genes, including alkaline phosphatase, collagen I and osteocalcin.</p><p>Type I collagen synthesis is associated with osteoblast differentiation in the early stage, followed by the synthesis of ALP.[<xref ref-type="bibr" rid="ref24">24</xref>]</p><p>In fact ALPL was up-regulated after 30 days in stem cells cultivated on NTD. Increasing in ALPL expression is associated with osteoblast differentiation.[<xref ref-type="bibr" rid="ref25">25</xref>]</p><p>After 30 days SPP1 and collagens were up-regulated too.</p><p>SPP1 encodes osteopontin, which is a phosphoglycoprotein of bone matrix and it is the most representative non-collagenic component of extracellular bone matrix.[<xref ref-type="bibr" rid="ref26">26</xref>]</p><p>The present study demonstrated that NTD strongly influences the behavior of ADSCs <italic>in vitro</italic> by enhancing proliferation, differentiation and deposition of matrix as demonstrated by the activation of osteoblast-related genes RUNX2, COL1A1 and SPP1.</p><p>Osteo-differentiation promoted by NTD was observed in DPSCs too, after 15 and 30 days of treatment.</p><p>After 15 days, the up-regulation of the bone-related genes FOSL1 and SPP1 was observed.</p><p>Their up-regulation continued in the next 30 days of treatment, associated to an increasing of two osteoblasts-related genes, RUNX2 and COL3A1.</p></sec><sec sec-type="conclusion" id="sec1-5"><title>CONCLUSION</title><p>These results demonstrated that NTD surface is more osteo-induced surface compared to TD, promoting the differentiation of mesenchymal stem cells in osteoblasts.</p></sec></body><back><fn-group><fn fn-type="supported-by"><p><bold>Source of Support:</bold> Nil</p></fn><fn fn-type="conflict"><p><bold>Conflict of Interest:</bold> None declared</p></fn></fn-group><ref-list><title>REFERENCES</title><ref id="ref1"><label>1</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Carinci</surname><given-names>F</given-names></name><name><surname>Guidi</surname><given-names>R</given-names></name><name><surname>Franco</surname><given-names>M</given-names></name><name><surname>Viscioni</surname><given-names>A</given-names></name><name><surname>Rigo</surname><given-names>L</given-names></name><name><surname>De 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