the osseointegration cascade begins immediately after the implant is placed in the bone , where the blood contiguously interacts with the implant surface .
irrespective of the biomaterial , surface topography , or wettability status of the surface , the initial contact with blood will rapidly attract proteins , which in turn will initiate the process of bone formation [ 2 , 3 ] .
in fact , protein adsorption to the implant surface has been suggested to be important for the osteoconduction stage of osseointegration [ 46 ] .
studies have also investigated the significance of the protein - implant interaction phenomenon [ 79 ] , in which some proteins significantly enhance migration , attachment , proliferation , and differentiation at the implant surface .
protein adsorption to biomaterials is intriguing since one specific protein never remains in a single niche for extended periods of time and constantly undergoes alterations depending on its molecular weight .
the so - called  vroman effect  is an indication that proteins play different roles in biological reactions .
for example , the effect of plasma fibronectin has been thoroughly studied along with bone biomechanical properties , and it has been reported to play an important role in the migration and attachment of mesenchymal cells and to be a regulator of bone density [ 11 , 12 ] .
another example is collagen type i , which is the major constituent protein of the bone matrix , assembles in the presence of fibronectin , and is thereby considered indispensible protein for osteogenesis .
hence , intentionally doping implant surfaces with proteins that have direct relationship with osteogenic events such as bone matrix formation may improve both the quality and the quantity of osseointegration [ 8 , 1518 ] .  
when protein doping implant surfaces that are typically textured ( rough ) , further topographical alterations may occur and might therefore affect cell adhesion and differentiation by potentially enhancing the effects of adsorbed protein layers .
in addition , it has been suggested that substrate surfaces possessing nanostructures show a high affinity for protein adsorption [ 2022 ] . as reported by puckett et al .
, intentionally applied nanogrooved surfaces presented higher fibronectin attachment compared to the control surfaces ( no grooves ) .
such surface topography may be suitable for sustaining higher volumes of target proteins and thus may facilitate implant adherence for a longer duration , supposedly owing to the augmented surface area rendered by such length scale texturization .
it has been reported that cell morphology , cytoskeleton and adhesion formation , and then subsequent cell growth and differentiation are altered by nanotopographies , thereby stimulating the osteoprogenitor cell differentiation towards an osteoblastic phenotype .
apart from alterations in cell morphology , it was demonstrated that an increased gene expression of the osteoblastic markers , runx-2 , and osteocalcin was evident when the cells were cultured on rough and grooved implant surfaces as compared to tissue culture plastic . in the present study , we have focused on a potential osteogenesis - enhancing protein , namely , laminin-1 , which is a heterotrimeric glycoprotein that contains an arginine - glycine - aspartic acid ( rgd ) sequence .
rgd is an integrin receptor binder , which is commonly found within the extracellular matrix proteins , and is the most widely occurring cell adhesive motif recognised by about 50% of all known integrins such as 21 , 31 , 51 , v1 , v3 , v5 , and 64 .
it has been reported that when applied to implant surfaces , the rgd sequence - impregnated modification significantly hastens osseointegration [ 27 , 28 ] and upregulates the osteoblast focal adhesion through integrin - mediated mechanisms .
besides the well - known bone forming ability of the rgd sequence , another interesting feature of laminin-1 is that it has the ability to selectively recruit osteoprogenitor cells .   
another reported feature of the laminin-1 is that it may possibly act as a nucleation center for the precipitation of calcium phosphates ( cap ) .
it was shown that in a model using the simulated body fluid ( sbf ) , the titanium surface presented more cap precipitation when laminin-1 was added to the sbf than sbf alone .
thus , it can be hypothesised that this unique protein may have an impact on the initial responses of implant - bone interactions .  
the aim of the present study was to dope an implant surface presenting nanostructures with laminin-1 and observe the biological response at the implant interface .
we hypothesised that the addition of laminin-1 would enhance osteogenic markers in the early stages of osseointegration . to test our hypothesis ,
implants were placed in rabbit tibiae for two weeks ; bone morphology and total mrna were extracted to evaluate the expression of genes involved in the inflammation and bone remodelling processes .
scanning electron microscopy ( sem ) ( leo 440-zeiss , oberkochen , germany ) was used for the assessment of surface morphology .  
the topography of the control implants was characterized using an interferometer ( microxam ; ade phase shift technology inc . ,
the parametric calculation was performed after form errors and waviness were removed with a 50  m  50  m gaussian filter .
the following three - dimensional parameters were selected : sa ( m ) , the arithmetic average height deviation from a mean plane ; sds ( m ) , the density of summits , and sdr ( % ) , the developed surface ratio .
commercially pure titanium ( grade 4 ) implants ( neodent , curitiba , parana , brazil , length , 2  mm ; diameter , 1.5  mm ) were used .
the surface was nanotextured by treating it with a solution consisting of equal volumes of concentrated h2so4 and 30% aqueous h2o2 for 2  h at room temperature under sterile conditions .
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 solution per well for 1  h at room temperature .   to characterize the coated laminin-1 ,
ellipsometry was used in order to estimate the amount of adsorbed laminin-1 on optically smooth titanium surfaces .
in brief , cleaned sio2 surfaces were placed in an evaporation chamber with final pressure below 1  10  torr .
approximately 200  nm of titanium was physical vapour deposited ( pvd ) and spontaneously oxidized at room conditions .
thereafter , the prepared surfaces were fixed in the ellipsometric quvette filled with pbs at room temperature . angles 0 and 0 were measured in three locations with a rudolph research autoel iii ellipsometer operating in a wavelength of 632.8  nm at a 70 angle of incidence .
the quvette was emptied and filled with laminin-1 solution and new angles  and  were calculated .
the thickness of the adsorbed protein was estimated to be 26   by using the maccrackin algorithm .  
nine lop - eared male rabbits ( mean body weight , 4.0  kg ) were used for the study .
one test ( laminin-1-coated ) implant and one control ( noncoated ) implant were inserted into the left and right tibial metaphysis , respectively .
the animal study was approved by the malm / lund , sweden regional animal ethics committee ( approval number : m282 - 09 ) .    before surgery ,
the animals were anaesthetised with intramuscular injections of a mixture of 0.15  ml / kg medetomidine ( 1  mg / ml dormitor ; orion pharma , sollentuna , sweden ) and 0.35  ml / kg ketamine hydrochloride ( 50  mg / ml ketalar ; pfizer ab , sollentuna , sweden ) .
lidocaine hydrochloride ( xylocaine ; astrazeneca ab , sdertlje , sweden ) was administrated as the local anaesthetic at each insertion site at a dose of 1  ml . after the operation , buprenorphine hydrochloride ( 0.5  ml temgesic ; reckitt benckiser , slough , uk ) was administered as an analgesic for 3 days . after 2 weeks
, the rabbits were sacrificed with an overdose ( 60  mg / ml ) of pentobarbital natrium ( apoteksbolaget ab , stockholm , sweden ) .   implants from both groups ( n = 3 ) were removed from the tibiae , cleaned in 4% neutral - buffered formaldehyde solution for 10  min , dehydrated using an ascending series of ethanol , and dried .
the retrieved implant samples were observed using sem under various magnifications . for gene expression analysis , both control and test groups from all 9 rabbits
were removed and the retrieved samples were placed in rna later solution ( qiagen gmbh , hilden , germany ) until analysis . in order to obtain detectable rna , each of the 9 samples in the control and test groups were pooled for rna isolation .
total rna was isolated from the surrounding tissue using trizol reagent ( gibco brl , carlsbad , ca , usa ) and ezna tissue rna isolation kit ( omega bio - tek , norcross , ga , usa ) .
total rna was quantified using a nanodrop spectrophotometer ( thermo scientific nanodrop technologies , wilmington , de ) .  
the reverse transcription reaction test was performed according to the manufacturer 's instructions , using the high capacity cdna archive kit ( applied biosystems , foster city , usa ) .
600  ng total rna was mixed with 100  ul reaction volume of reverse transcriptase ( rt ) buffer , primers , nuclease - free water , and multiscribe rt .
real - time quantitative pcr was conducted under standard enzyme and cycling conditions on a stepone system , using custom - designed real - time assays and sybr green detection ( primerdesign ltd . ,
cdna corresponding to 6  ng of mrna was used in each pcr reaction , and mixtures were prepared according to the manufacturer 's instructions in 10  l triplicates for each target cdna .
amplification was carried out in 96-well thermal cycle plates on a stepone system ( applied biosystems ) .
the sem image for the surface of the nanostructures is presented in figures 1(a)1(c ) , which depicted homogeneous nanostructures covering the entire implant surface .
nanostructures below 50  nm were identified at higher magnification images ( figure 1(c ) ) .
the mean sa  ( sd ) was 0.28  ( 0.07 )  m ; sds  ( sd ) was 195,203 ( 7,871 ) ; sdr  ( sd ) was 8.15 ( 0.53)% .  
figure 1(d ) shows three - dimensional optical interferometry image of the surface . out of the 3 samples of each group , figures 2(a)2(d ) present representative electron micrographs for the samples that remained for 2 weeks in vivo . in both control and test implants ,
the results of real - time rt - pcr are presented in figure 3 . in general , the osteoblast markers that presented higher expression in the case of the test implants were runx-2 ( 1.62-fold ) , osteoclcin ( 1.53-fold ) , alkaline phosphatase ( alp ) ( 1.97-fold ) , and collagen i ( 1.04-fold ) . on the other hand , the expression of igf-1 was low ( 0.84-fold ) .
in the case of the test implants , all osteoclast markers investigated in the present study showed higher expression for the experimental group relative to control , namely , tartrate - resistant acid phosphatase ( trap ) ( 1.67-fold ) , calcitonin receptor ( 1.35-fold ) , and atpase ( 1.25-fold ) .
the inflammatory markers that showed higher expression for the test implants than the control implants were il-10 ( 1.53-fold ) and tnf- ( 1.61-fold ) , whereas il-6 showed lower expression ( 0.59-fold ) .
protein doping is considered one of the promising methods of surface modifications for hastening the early stages of osseointegration both qualitatively and quantitatively [ 8 , 27 , 35 , 36 ] . in most studies concerning protein doping of implant surfaces ,
the beneficial enhancements were primarily restricted to the initial stages of healing and have been shown to have smaller effects when longer periods of experimental time were observed
. such early effect may be related to competitive protein adsorption , and thereby given the protein adsorption desorption dynamics at the implant surface region , protein doping of implant surfaces is indeed expected to be effective in the initial stages of osseointegration .
such an improvement and upregulation of the early bone response is of great clinical importance since it is in both practitioners ' and patients ' interest that implants osseointegrate faster for shortening the treatment period .
the results obtained by rt - pcr showed distinct differences between the nanostructured surface with / without laminin-1-coating .
evaluation of the selected osteoblastogenesis - related markers revealed that most of the markers showed higher expression around laminin-1-coated implants relative to the control implant group .
it is of great interest that the expression levels of alp , runx-2 , and collagen i were higher in the case of the laminin-1-coated implant since these markers are indicators of higher osteoprogenitor and osteoblast precursor activity .
in addition , the higher expression of osteocalcin , the specific marker for bone formation , indicates that the differentiation activity of cells into osteoblasts was upregulated around laminin-1-coated implants . on the other hand ,
lower amounts of igf-1 expression ( reported to promote osteoblast activity   and osteoblast proliferation ) were detected around laminin-1-coated implants , indicating that the proliferation activity at the interface was suppressed . while our results are contradictory concerning the early osteogenic events , osteoblast proliferation and differentiation activity
osteoclast - mediated bone resorption around dental implants plays an important role in bone remodelling and thereby osseointegration establishment and maintenance [ 43 , 44 ] . in the present study ,
it has been reported that osteoblastogenesis and osteoclastogenesis transact and regulate each other through the receptor activator of nuclear factor - kappa b ligand ( rankl)/rank / osteoprotegerin ( opg ) system pathway [ 45 , 46 ] .
thus , we speculate that the higher level of osteoblastic gene expression may have induced higher osteoclastic gene expression . in the present study , the osteoclastic activity may have been highly active due to mutual interactions after surface doping with laminin .
it is well recognized that integrin v3 , which is highly produced by osteoclasts , presents high affinity for the rgd - motif , which is included in many of the extracellular matrix proteins .
although this might be a non - laminin specific mechanism , another integrin molecule , that is , 21 , is also expressed by mammalian osteoclasts and is highly specific for laminin and collagen .
thus , the increase in osteoclast proliferation denoted by higher levels of trap and calcitonin receptor may be laminin specific directly by means of laminin/21 interaction .  
the rt - pcr results also demonstrated that most of the inflammatory factors were upregulated for the laminin-1-doped group .
have reported that laminin-1 localises where inflammation exists , as seen in chronic periodontal inflammatory responses .
since inflammatory reactions are part of the healing process , the induced inflammatory gene expression further supports higher degrees of osteogenesis at the laminin-1-coated implant interface . for example , tnf- has been proven to be necessary for intramembranous ossification   and to increase matrix mineralization and the levels of bone morphogenic protein-2 and alkaline phosphatase in vitro .
these findings are in agreement with the observed increase of osteoclcin and alkaline phosphatase in our study .
a study in knockout il-10 mice has demonstrated decreased gene expression of alkaline phosphatase and osteocalcin in the absence of the il-10 gene .
thus , the elevated levels of il-10 in our study are well correlated to the increased gene expression of those osteoblast markers .
additionally , il-6 has been reported to stimulate osteoclastic bone resorption   hence explaining the enhanced expression of osteoclastic markers .
it is speculated that because of the early time point , the mechanical attachment strength of the bone tissue to the implant surface may be low , and the mineralisation is still in progress .
this statement is further supported by the high gene expression of alp , which indicates that bone is still under maturation .
for this reason , it is potentially like that large segment of immature bony tissues may have detached from the implant interface , rather than development of a fracture within the bone .  
although the results of this study are preliminary , the information motivates further investigation of the novel protein we utilized in the current study as an implant coating .
further , evaluation of gene expression may help capture detailed differences , which may be difficult to detect with the conventional imaging and biomechanical evaluation techniques .
we hypothesised that the addition of laminin-1 would enhance osteogenic markers in the early stages of osseointegration .
compared to the noncoated nanostructured implant surface , the protein - doped nanostructured implant surface presented higher gene expression of typical genes involved in the osseointegration cascade , and therefore the hypothesis of the study was accepted .
 
 aim . this study aimed to observe the morphological and molecular effect of laminin-1 doping to nanostructured implant surfaces in a rabbit model . 
 materials and methods . 
 nanostructured implants were coated with laminin-1 ( test ; dilution , 100  g / ml ) and inserted into the rabbit tibiae . 
 noncoated implants were used as controls . 
 after 2 weeks of healing , the implants were removed and subjected to morphological analysis using scanning electron microscopy ( sem ) and gene expression analysis using the real - time reverse transcriptase - polymerase chain reaction ( rt - pcr ) . 
 results . 
 sem revealed bony tissue attachment for both control and test implants . 
 real - time rt - pcr analysis showed that the expression of osteoblast markers runx-2 , osteocalcin , alkaline phosphatase , and collagen i was higher ( 1.62-fold , 1.53-fold , 1.97-fold , and 1.04-fold , resp . ) for the implants modified by laminin-1 relative to the control . 
 all osteoclast markers investigated in the study presented higher expression on the test implants than controls as follows : tartrate - resistant acid phosphatase ( 1.67-fold ) , calcitonin receptor ( 1.35-fold ) , and atpase ( 1.25-fold ) . 
 the test implants demonstrated higher expression of inflammatory markers interleukin-10 ( 1.53-fold ) and tumour necrosis factor- ( 1.61-fold ) relative to controls . 
 conclusion . 
 the protein - doped surface showed higher gene expression of typical genes involved in the osseointegration cascade than the control surface . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion