dental implants have been proven to be a reliable long - term therapy against edentulism [ 13 ] .
however , the reported high success figures of implant therapy have been based on implants inserted using two - stage surgical protocol and conventional loading . the increased demand on implant performance and the broadened treatment indications have led to the development of new moderately rough surfaces .
alterations in both the surface chemistry and topography may contribute to chemical influence on bone tissue , a phenomenon defined as bioactivity .
furthermore , other factors such as surface energy , surface wettability , cellular maturation state , nutrition status , and microstresses alter the degree of bioactivity too .
compared to the previously used turned implants , the bioactively modified implants have demonstrated higher success rate in demanding cases , for example , early functional loading , one - stage surgery , and reconstructive jaw surgery .  
when moderately rough surfaces remain within bone tissue no differences on microbial colonization are observed as compared to minimally rough surfaces .
however , there has been increasing evidence pointing out that as soon moderately rough implants are exposed to the oral milieu the case changes .
a series of studies examining clinical , histological , and radiological aspects of experimental peri - implantitis in a dog model has reported that exposure of the implant surfaces to the oral environment leads to spontaneous progression of experimental peri - implantis [ 911 ] . the same research group has reported that implant surface characteristics affect the possibility to treat experimental peri - implantitis without antimicrobial therapy , thereby influencing the treatment outcome .
additionally , a recent in vitro study has proposed that increased implant roughness promotes bacterial colonization most likely depending on protection of bacteria from shear forces .
even if the referred studies are experimental in nature , the idea of developing an implant that combines the osseoconductive properties of a moderately rough surface with the accessibility for debridement of turned surfaces is intriguing . in order to enhance bone formation ,
laminins are heterotrimeric glycoproteins that bind to integrins , especially 1 and 2 isomers . the n - terminal of laminin-1 has been reported to selectively recruit osteoprogenitors through integrin 1-mediated cell attachment [ 20 , 21 ] and to stimulate production of alkaline phosphatase by osteoblasts .
additionally , recent in vitro studies [ 23 , 24 ] have elucidated the role of laminin as nucleation center and its potential to enhance osteoid formation in a simulated body fluid .
nevertheless , since the in vivo environment is more complex in terms of protein interactions   and desorption of the coating agent , in vivo validation has been imperative . in theory ,
any effects of a protein coating are more pronounced during the early stages of osseointegration .  
the purpose of this in vivo study is to investigate the detailed molecular mechanisms underlying the possible effects of the coating agent laminin-1 on osseointegration and to compare them to histological evaluation methods .
in total , 90 threaded titanium ( grade 4 ) implants with turned surface were used ( diameter : 1.5  mm , length : 2.5  mm , internal hexagonal connection , batch 800101579 , neodent , brazil ) .
half of the implants ( n = 45 ) were coated with laminin-1 in accordance with previous in vitro study   and served as the test group . in brief , laminin-1
( l2020 , sigma - aldrich , stockholm , sweden ) was diluted to a concentration of 100  g / ml in dulbecco 's phosphate - buffered saline ( dpbs ) without cacl2 or mgcl2 ( 14190 - 094 ; gibco , invitrogen corporation , grand island , ny , usa ) .
the implants were subsequently incubated in 48-well plates ( nunclon surface , nunc , roskilde , denmark ) containing 250  l of the laminin-1 solution per well , for 1  h at room temperature . the protein thickness after incubation was estimated by ellipsometry . since the implant surface did not reflect the light beam in a measurable manner , the amount of adsorbed laminin was calculated on optically smooth titanium surfaces produced at the laboratory as described by linderbck et al . . as previously described by bougas et al
, the optically smooth titanium surfaces were fixed in the ellipsometric quvette filled with pbs at room temperature . the ellipsometric angles 0 and 0 were measured at three locations with a rudolph research autoel iii ellipsometer operating in a wavelength of 632.8  nm at a 70 angle of incidence .
subsequently , the quvette was emptied and filled with laminin solution and new angles  and  calculated .
the protein layer thickness was calculated from the ellipsometric angle changes for a protein refractive index of n = 1.465 . by using the mccrackin algorithm for the calculations
, it was concluded that the incubation resulted in protein thickness corresponding to 2.6  nm .
the surface topography of the implants was characterized with an optical interferometer ( microxam , ade phase shift , tucson , az , usa ) operating in wavelength of  = 550  nm . according to the proposed guidelines for implant surface characterization , three implants from each group
were randomly selected and each measured in 9 regions ( 3 thread tops , 3 thread valleys , and 3 flank regions ) .
the following three topographical parameters were evaluated : an amplitude parameter , sa ( m ) = the arithmetic average height deviation from the mean plane ; a spatial parameter , sds ( m ) = the density of summits ; and a hybrid parameter , sdr ( % ) = the developed surface ratio .
the study was approved by the malm / lund , sweden , regional animal ethical committee ( approval number : m253 - 10 ) and included 45 male wistar hannover galas rats with an average weight of 350  g. prior to surgery , the animals were sedated by intraperitoneal administration of a mixture of dormicum 5  mg / ml ( midazolam , roche ) , hypnorm ( fentanylcitrate 0.315  mg / ml and fluanisone 10  mg / ml , janssen pharmaceutical ) and sterile saline 0.9  mg / ml ( braun ) in a dose of 1.52  ml / kg body weight .
the hind legs were disinfected with 70% ethanol and 70% chlorhexidine , and lidocaine hydrochloride ( xylocaine ; astrazeneca ab ) was administrated as the local anaesthetic at each insertion site at a dose of 0.5  ml .
one control implant was operated into the right tibia and one test implant into the left of each animal .
after the operation , buprenorphine hydrochloride ( 0.5  ml temgesic ; reckitt benckiser , slough , uk ) was administered as an analgesic for 3 days .   the animals were divided into three groups and were sacrificed after 3 days ( n = 14 ) , 1 week ( n = 15 ) and 3 weeks ( n = 15 ) with an overdose of carbon monoxide in a gas chamber . the skin above the implants was incised , and 20 implants ( 10 pairs control / test ) for each of the two first groups ( 3 days and 1 week ) were turned out manually . since one implant in the 3-week group did not osseointegrate , 18 implants ( 9 pairs control / test ) were turned out manually .
the removed implants , along with the interface bone tissue , were placed in rnalater solution ( qiagen gmbh , hilden , germany ) , and frozen at 80c until analysis .  
the samples were processed in the tissuelyser instrument ( qiagen gmbh ) together with -mercaptoethanol rneasy lysis - buffer to remove and disrupt the cells attached to the surface of the implant screw .
rna was extracted from the sample mixtures with rneasy micro kit number 74004 ( qiagen gmbh ) according to manufacturer 's instructions , including carrier to minimize decrease in yield due to small sample quantity . during extraction
, all samples were dnase - treated according to manufacturer 's instructions with rnase free dnase set # 1023460 ( qiagen gmbh ) to reduce gdna contamination .
all rna samples were reverse transcribed in single 10  l reactions according to manufacturer 's instructions using tataa rt kit number a103b ( tataa biocenter ab ) to generate cdna .
the controls were analyzed in pools of five , containing 1.5  l of each sample ( total volume 7.5  l ) .
control of rna concentration was not possible due to the presence of carrier in extraction procedure . for each real time rt - pcr ,
10  l mixtures were prepared with 1  l cdna , according to the manufacturer 's recommendations ( applied biosystem , ca , usa ) .
amplification was carried out in 96-well thermal cycle plates on a stepone detection system ( applied biosystems , usa ) according to the manufacturer 's recommendations with custom - designed real - time assays and sybr green detection ( primerdesign ltd , southampton , uk ) ( table 1 ) .
the remaining implants from each group were retrieved en bloc and were immersed in 4% neutral buffered formaldehyde . since one implant from the 3 week group did not osseointegrate possibly due to an incorrect insertion angle , the final numbers of implants processed for histology were ; n = 8 for 3 days , n = 10 for 1 week , and n = 8 for 3 weeks .
all the samples were processed for undecalcified ground sectioning . briefly , after a series of dehydrations and infiltrations in resin , the samples were embedded in light - curing resin ( technovit 7200 vlc ; heraeus kulzer wehrheim , germany ) .
one central ground section was prepared from each implant by using exakt sawing and grinding equipment ( exakt apparatebau , hamburg , germany ) .
the sections were ground to a final thickness of approximately 10  m and histologically stained with toluidine blue mixed with pyronin g. histological evaluations were performed using a light microscope ( eclipse me600 ; nikon , japan ) , and the histomorphometrical data were analyzed by image analysis software ( image j v. 1.43u ; national institutes of health , bethesda , maryland ) .
the bone - implant contact ( bic ) and the bone area ( ba ) percentage along the whole implant were calculated at 10 objective magnification as described previously [ 32 , 33 ] .
the statistical calculations were performed with spss ( version 18 chicago , illinois , usa ) .
the statistical comparison for the mean values of the topographic parameters sa , sds , and sdr was assessed by students t - test . for bic and ba
the nonparametric wilcoxon signed ranks test was used while for relative gene expression , the student 's paired t - test was employed .
the laminin-1 coating increased the density of summits ( sds ) significantly ( p = 0.009 ) . on the contrary ,
the protein coating did neither affect the average height deviation from the mean plane ( sa ) ( p = 0.261 ) nor the developed surface ratio ( sdr ) ( p = 0.446 ) of the implants significantly ( table 2 ) .  
although the gene expression for the osteoprogenitor marker runt - related transcription factor 2 ( runx2 ) was lower for the test than for the control after 3 days , it was doubled at 7 days resulting in statistically significantly higher levels as compared to the control .
after 21 days , the difference in the expression of the gene for runx2 between test and control was evened out ( figure 1(a ) ) .
the second osteoprogenitor differentiation , marker bone morphogenic protein-2 ( bmp-2 ) , did not differ between test and control at any time point ( figure 1(b ) ) .  
the initial gene expression ( 3 days ) of the osteblastic markers osteocalcin ( figure 1(c ) ) , alkaline phosphatase ( alp ) ( figure 1(d ) ) and type i collagen ( figure 1(e ) ) was higher for the control group .
nevertheless , after 7 days the expression of the osteoblastic markers increased for the test group and declined for the control .
this alteration resulted in statistically significantly higher mrna levels of osteocalcin and type i collagen in favour of the test group .
after 21 days no statistically significant differences were detected between test and control in the expression of osteoclacin and alp .
on the contrary , the expression of type i collagen for the control group was enhanced to statistically significantly higher levels as compared to the test group .  
the expression of the osteoclastic marker cathepsin k ( figure 2(a ) ) demonstrated a descending trend for the control group during the observation time .
in contrast to the control group , the expression of cathepsin k remained stable for the test group throughout the observation period . the expressed levels of cathepsin k were statistically significantly lower for the test group at all times ( 3 days , 7 days , and 21 days ) .
the mrna levels of the osteoclastic marker tartrate - resistant acid phosphatase ( trap ) ( figure 2(b ) ) declined with time .
after 3 days , the levels of trap were statistically significantly higher for the control group , whilst no statistically significant differences were detected between test and control at 7 or 21 days .  
the gene expression of integrin 1 ( figure 3(a ) ) for the test group peaked at 7 days . at this time point , the mrna levels for integrin 1 were statistically significantly higher for the test group ( 4.90-fold ) . despite the enhanced levels of integrin 1 mrna expression in the control group after 21 days , no statistically significant differences were detected .  
the proinflammatory cytokines tumor necrosis factor  ( tnf- ) ( figure 3(b ) ) and interleukin 1 ( il-1 ) ( figure 3(c ) ) peaked at 7 days .
the fold of relative mrna expression was 6.65 for tnf- and 51.88 for il-1 in favour of the control group .
the expression of tnf- and il-1 remained stable for the test group at 3 , 7 , and 21 days .
no statistically significant differences were detected between test and control in the expression of the anti - inflammatory cytokine il-10 ( figure 3(d ) ) .  
the values of bic and ba were enhanced after 7 and 21 days as compared to 3 days .
the test group demonstrated higher median bic and ba at all the evaluated time points ( figures 4 and 5 ) .
the differences between the test and control were though not statistically significant ( p > 0.05 ) ( table 2 ) .
in the current study , we have investigated the effects of laminin-1 coating on the early stages of osseointegration .
the implants chosen for this purpose are turned with a smooth surface ( sa = 0.28  m ) .
implants with turned surface have a long history of clinical documentation [ 13 ] and hence may be regarded as the  gold standard .  the rational for rather using a smooth implant than a moderately rough , is based upon the theorem that moderately rough implants enhance osseointegration , thereby possibly concealing any effects of the biochemical coating .
additionally , a turned implant surface was chosen in order to investigate whether a biochemical coating can induce cell responses equivalent to the ones promoted by surface topography modifications [ 35 , 36 ] . according to the surface analysis
since the implant surface has a minimally rough profile , the protein coating may be detected by the interferometer as prominences on the implant surface , hence resulting in elevated peak density of the test implant .  
depending on protein - desorption kinetics , the biochemical coating is theoretically most active during the first days after the implant installation .
for this reason , we have chosen to investigate the early effects of the laminin-1 coating after 3 , 7 , and 21 days .
the early time - points investigated in this study have been previously used in the literature in order to screen the expression of bone - related genes and inflammatory markers in a rat model [ 35 , 37 ] .
since the degree of diffusion of the coating into the surrounding bone is unknown , we have chosen to collect the interface bone tissue from the removed implant instead of retrieving the implant along with the surrounding bone en bloc .
the analysis of the interface bone tissue is further justified by a histological study concluding that implant coating material released by the shear forces during implant insertion residues within the peri - implant space .  
the gene analyses from the interface bone tissue reveal after 7 days significantly higher levels of the transcription factor runx-2 , which is the master gene for osteoblast differentiation and is expressed by the committed osteoprogenitors . at the same time point ,
it has been suggested that laminin stimulates osteoprogenitors by attachment to integrin 1 in vitro [ 20 , 21 ] .
it has also been demonstrated that activation of runx2 by mapk is possible by binding of type i collagen to 2 1 integrins . since collagen type
i is also elevated after 7 days , a possible mechanism of action for laminin-1 could be indirect activation of runx2 by elevating the expression of type i collagen which attaches to integrin 1 .
after 7 days , no significant difference in bmp-2 levels was detected between test and control .
this may imply that laminin-1 has an indirect effect on the existing osteoprogenitors in the peri - implant space , without promoting commitment of the surrounding mesenchymal stem cells to osteoprogenitors via bmp-2 .  
apart from the molecular mechanisms involved in the effect of laminin-1 on osteoprogenitors , our results suggest an additional effect on differentiated osteoblasts .
the gene expression of collagen type i and the marker for mature post - proliferative osteoblasts , osteocalcin , are up - regulated after 7 days .
hence , laminin-1 may contribute to the enrichment of the extracellular matrix in the direct proximity of the implant .
if we additionally take into account the significantly decreased expression of the osteoclastic proteolytic enzyme cathepsin k , the bone remodeling   may be further displaced in favor of osteogenesis . as reported in the literature , low expression of cathepsin k can be correlated to low levels of the proinflammatory cytokines il-1 and tnf- , both considered to be expressed on bone sites with pronounced osteoclastic resorption .
as it concerns the 3-day time point , the expression of both osteogenic and osteoclastic markers is higher for the control group . keeping into consideration that the outcome of the bone metabolism is decided by a coupled mechanism between bone deposition and bone resorption , it is uncertain whether this increased activity will result to more or less new bone .  
a comparison of the findings from the gene analyses to the findings from the histomorphometry reveals some discrepancies .
although there are important differences in gene expression , no differences are detected on bic or ba after 3 and 7 days .
this finding is considered reasonable keeping in mind that bone remodeling is a time - demanding process .
discrepancies between the results from traditional evaluation methods and genetic analysis have been reported previously .
the fact that no differences were detected on bic or ba after 21 days is in agreement with the results from the gene expression .
this result may be explained by the fact that the coating is expected to be more active during the early stages of osseointegration , since it may be gradually desorbed from the interface as demonstrated in vitro .   conclusively , within the limitations of our study , we suggest that it is possible to alter the cell behavior on the implant - bone interface towards the osteogenic direction by coating the implant surface with laminin-1 .
however , the reported changes are not detected by histomorphometry , most likely depending on the fact that this method is not adequately sensitive at the short times of follow - up applied on the present study .
 the aim of this study was to evaluate the early bone response around laminin-1-coated titanium implants . 
 forty - five rats distributed in three equally sized groups were provided with one control ( turned ) and one test ( laminin-1-coated ) implant and were sacrificed after 3 , 7 , and 21 days . 
 real - time reverse - transcriptase polymerase chain reaction was performed for osteoblast markers ( alkaline phosphatase , runt - related transcription factor 2 , osteocalcin , type i collagen , and bone morphogenic protein 2 ) , osteoclast markers ( cathepsin k and tartrate - resistant acid phosphatase ) , inflammation markers ( tumor necrosis factor  , interleukin 1 and interleukin 10 ) , and integrin 1 . 
 bone implant contact ( bic ) and bone area ( ba ) were assessed and compared to the gene expression . 
 after 3 days , the expression of bone markers was higher for the control group . 
 after 7 days , the expression of integrin 1 and osteogenic markers was enhanced for the test group , while cathepsin k and inflammation markers were down - regulated . 
 no significant differences in bic or ba were detected between test and control at any time point . as a conclusion , implant coating with 
 laminin-1 altered gene expression in the bone - implant interface . however , traditional evaluation methods , as histomorphometry , were not adequately sensitive to detect such changes due to the short follow - up time . 
1. Introduction
2. Materials and Methods
3. Statistics
4. Results
5. Discussion