titanium and its alloys are employed as implant materials because of their desirable mechanical properties and  biocompatibility  . for decades , oral , maxillofacial and orthopedic surgeons have utilized dental implants , screws and plates , and prostheses to substitute lost teeth , to fix bone fragments and to replace joints , respectively . also , many surgical instruments , such as drills and saws , are made with titanium alloys . the  biocompatibility  of titanium has been demonstrated by in vivo studies with both animal models and clinical trials , for more than forty years . several authors studied other different aspect of implant morphology in order to value their impact on implant stability . macro - design is the shape of the prosthesis : some examples are offered by the cylindrical or root form of dental implants . mini - design is the dimension of the threads or the shape of the neck of the dental fixture . micro - design is the shape of the implant surface : an example of which is provided by the grooves and holes  resulting from surface treatments like machination , acid etching and sand blasting procedures . these treatments determine the roughness of the surface and the  holes  have a cellular dimension . nano - design is determined by the molecular composition of the surface , such as those composed of : hydroxyapatite , zirconium and titanium . usually , mechanical proprieties are related to macro- and mini - design , whereas biological properties are related to micro- and nano - design . mechanical properties are responsible for primary implant stability , whereas biological properties act on the osseointegration process . when titanium is exposed to oxygen a stochastic distribution of three titanium - oxide isoforms ( i.e. , rutile , brookite and anatase ) are available on the surface of the prosthesis . these isoforms are responsible for its material biological properties . although previous studies have demonstrated that titanium surfacing is capable of modulating osteoblast gene expression , they failed to separate the effect of roughness ( i.e. , micro - dimension ) from that of composition ( i.e. , nano - dimension ) . effect of the titanium nanotubes layer on osteoblast gene expression has been studied , comparing the expression profiling of osteoblasts ( hob ) cultivated on two type of surface : pure titanium disk ( td ) and nanotubes titanium disk ( ntd ) in order to detect if ntd surface stimulates osteoblast proliferation . the quantitative expression of the mrna of specific genes , like transcriptional factors ( runx2 ) , bone related genes ( spp1 , col1a1 , col3a1 , alpl , and fosl1 ) was examined by means of real time reverse transcription - polymerase chain reaction ( real time rt - pcr ) . 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 . after 3 minutes pickling in a hf ( carlo erba)/hno3 ( carlo erba ) solution , made by a volumetric ratio of 1:3 and diluted in deionized water until to 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 ) , which is generated by a potentiostat / galvanostat solartron 1286 for 3 minutes . 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 . nh4f for 3 hours at 60 v. after the anodization treatment , all the samples are washed in glycole ethylene , left overnight in the dry room , in order to dry them . so to crystallize the tio2 nanotubes , obtained in amorphous form by anodic growth , after a pre - heat treatment at 80c in vacuum for 3 hours , all the samples have been placed in a tubular furnace ( lenton ) for 1 hour at 580c , with a slope of 1c / minute in air , so to be transformed into the anatase phase . fragments of bone derived from skull of an healthy volunteer were sampled during operation and transferred in 75 cm culture flasks containing dmem medium supplemented with 20% fetal calf serum , antibiotics ( penicillin 100 u / ml and streptomycin 100 micrograms / ml - sigma aldrich , inc . st louis , mo , usa ) and amminoacids ( l - glutamine - sigma aldrich , inc . after 15 days , the pieces of bone tissue were removed from the culture flask . reverse transcription to cdna was performed directly from cultured cell lysate using the taqman gene expression cells - to - ct kit ( ambion inc . , austin , tx , usa ) , following manufacturer 's instructions . briefly , cultured cells were lysed with lysis buffer and rna released in this solution . cell lysate were reverse transcribed to cdna using the rt enzyme mix and appropriate rt buffer ( ambion inc . 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 . the gene expression levels were normalized to the expression of the housekeeping gene rpl13a and were expressed as fold changes relative to the expression of the untreated cells . 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 table 1 . primer and probes used in real time pcr all pcr reactions were performed in a 20 l volume using the abi prism 7500 ( applied biosystems , foster city , ca , usa ) . each reaction contained 10 l 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 amplification profile was initiated by 10-minute incubation at 95c , followed by two - step amplification of 15 seconds at 95c and 60 seconds at 60c for 40 cycles . 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 . after 3 minutes pickling in a hf ( carlo erba)/hno3 ( carlo erba ) solution , made by a volumetric ratio of 1:3 and diluted in deionized water until to 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 ) , which is generated by a potentiostat / galvanostat solartron 1286 for 3 minutes . 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 . nh4f for 3 hours at 60 v. after the anodization treatment , all the samples are washed in glycole ethylene , left overnight in the dry room , in order to dry them . so to crystallize the tio2 nanotubes , obtained in amorphous form by anodic growth , after a pre - heat treatment at 80c in vacuum for 3 hours , all the samples have been placed in a tubular furnace ( lenton ) for 1 hour at 580c , with a slope of 1c / minute in air , so to be transformed into the anatase phase . fragments of bone derived from skull of an healthy volunteer were sampled during operation and transferred in 75 cm culture flasks containing dmem medium supplemented with 20% fetal calf serum , antibiotics ( penicillin 100 u / ml and streptomycin 100 micrograms / ml - sigma aldrich , inc . , st louis , mo , usa ) and amminoacids ( l - glutamine - sigma aldrich , inc . after 15 days , the pieces of bone tissue were removed from the culture flask . for the assay , hob were trypsinized upon subconfluence and seeded on ntd and td . reverse transcription to cdna was performed directly from cultured cell lysate using the taqman gene expression cells - to - ct kit ( ambion inc . , briefly , cultured cells were lysed with lysis buffer and rna released in this solution . cell lysate were reverse transcribed to cdna using the rt enzyme mix and appropriate rt buffer ( ambion inc . 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 . the gene expression levels were normalized to the expression of the housekeeping gene rpl13a and were expressed as fold changes relative to the expression of the untreated cells . 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 table 1 . primer and probes used in real time pcr all pcr reactions were performed in a 20 l volume using the abi prism 7500 ( applied biosystems , foster city , ca , usa ) . each reaction contained 10 l 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 amplification profile was initiated by 10-minute incubation at 95c , followed by two - step amplification of 15 seconds at 95c and 60 seconds at 60c for 40 cycles . gene expression in hob cultivated on ntd was compared with that cultivated on td ( control ) in order to check whether ntd is more osteoinducent respect td . after 15 days , hob cultivated on ntd showed the up - regulation of bone related genes sp7 , eng , fosl1 and spp1 and the down - regulation of runx2 , col3a1 , col1a1 , and alpl [ figure 1a ] . relative gene expression of hob cultivated on titanium disks for 15 days ( a ) and 30 days ( b ) after 30 days of treatment , the bone related genes sp7 , eng , fosl1 and runx2 were up - regulated in hob cultivated on ntd , while col3a1 , col1a1 , alpl and spp1 were down - regulated [ figure 1b ] . titanium is well tolerated by the bone and is able to induce osseointegration of the implant . recently titania film have been used to cover prosthetic implants in medical applications such as orthopedic or dental surgery . titania film creates an active surface that is able to improve the biocompatibility of titanium implants . in prostheses made of titanium , the titania film surface is mainly composed of two polymorphs of titanium , rutile and anatase . rutile is considered the stable form of titania while anatase is metastable and converts to rutile at high temperatures . compared to rutile , anatase exhibits stronger interactions between metal and support , and the surface of anatase titania can absorb more oh- and po43- than that of rutile titania in body fluid , which favors the depositing of bone - like apatite . effect of the titanium nanotubes on hob gene expression has been studied , comparing the expression profiling of osteoblasts cultivated on two type of surface : pure titanium disk ( td ) and nanotubes titanium disk ( ntd ) in order to detect if ntd surface stimulates hob proliferation . the quantitative expression of the mrna of specific genes , like transcriptional factors ( runx2 and sp7 ) and bone related genes ( spp1 , col1a1 , col3a1 , alpl , eng and fosl1 ) was examined by means of real time reverse transcription - polymerase chain reaction ( real time rt - pcr ) . after 15 days of treatment the bone related genes sp7 , eng , fosl1 and spp1 were up - regulated in hob cultivated on ntd respect to hob cultivated on td . sp7 is a zinc finger transcription factor that regulates bone formation and osteoblast differentiation in vitro and in vivo and that is expressed in the early stage of osteogenic differentiation . eng is a receptor for tgf-1 and -3 and modulates tgf- signaling by interacting with related molecules , such as tgf-1 , -3 , bmp-2 , -7 , and activin a. it is speculated that these members of the tfg- super family are mediators of cell proliferation and differentiation and play regulatory roles in cartilage and bone formation . ntd also modulate the expression of fosl1 that encodes for fra-1 , a component of the dimeric transcription factor activator protein-1 ( ap-1 ) , which is composed mainly of fos ( c - fos , fosb , fra-1 and fra-2 ) and jun proteins ( c - jun , junb and jund ) . ap-1 sites are present in the promoters of many developmentally regulated osteoblast genes , including alkaline phosphatase , collagen i , osteocalcin ( oc ) . demonstrated that differential expression of fos and jun family members could play a role in the developmental regulation of bone - specific gene expression and as a result , may be functionally significant for osteoblast differentiation . spp1 encodes osteopontin , which is a phosphoglycoprotein of bone matrix and it is the most representative non collagenic component of extracellular bone matrix . osteopontin produced by osteoblasts , show high affinity to the molecules of hydroxylapatite in extracellular matrix and it is chemo - attractant to osteoclasts . after 30 days of treatment these genes , sp7 , eng and fosl1 , continue to be up - regulated in hob cultivated on ntd , indicating the osteodifferentiative properties of titanium nanotubes . in addition another bone related genes , runx2 , was over - expressed in hob cultivated on ntd respect to control . runx2 is a transcriptional factor activated in the first stage of differentiation and plays a fundamental role in osteoblast maturation and homeostasis . our results , demonstrates that ntd can lead to osteoblast differentiation and extracellular matrix deposition and mineralization in dental pulp stem cells by the activation of osteoblast related genes spp1 , these results showed that ntd is more osteoinducent surface compared to td , promoting the osseointegration .
background : titanium is used worldwide to make osseointegrable devices , thanks to its favorable characteristics as mechanical proprieties and biocompatibility , demonstrated by in vivo studies with animal models and clinical trials over a forty - year period . however , the exact genetic effect of the titanium layer on cells is still not well characterized.materials and methods : to investigate how titanium nanotubes stimulate osteoblasts differentiation and proliferation , some osteoblast genes ( sp7 , runx2 , col3a1 , col1a1 , alpl , spp1 and fosl1 ) were analyzed by quantitative real time rt- pcr.results:after 15 days , osteoblasts cultivated on titanium naotube showed the up - regulation of bone related genes sp7 , eng , fosl1 and spp1 and the down - regulation of runx2 , col3a1 , col1a1 , and alpl . 
 after 30 days of treatment , the bone related genes sp7 , eng , fosl1 and runx2 were up - regulated while col3a1 , col1a1 , alpl and spp1 were down-regulated.conclusions:our results , demonstrates that titanium nanotubes can lead to osteoblast differentiation and extracellular matrix deposition and mineralization in dental pulp stem cells by the activation of osteoblast related genes spp1 , fosl1 and runx2 .