nanobiomaterials are characterized by constituent 
 particles and/or surface features less than 100  nm 
 in at least one dimension 
 . 
 starting with photolithography and dry etching in the 
 1980 's to high - resolution electron beam lithography and other technologies 
 in the 1990 's
, nanotechnology allows for making surface structures for 
 cell engineering and has led to an increasing application in healthcare over the last decades .
nanolayers are used to enhance the 
 surface biocompatibility of polymeric drug delivery systems , 
 control the release of substances such as antibiotics or growth factors 
 , act as gene - delivery 
 vehicles , or serve as robust light emitters for 
 cellular labeling and tracking 
 [ semiconductor nanocrystals , quantum dots ( qds ) ] 
 . 

nanotechnology is also applied to modify and improve the surface 
 structure in orthopaedic implants to promote their 
 osseous integration . however , there are also side effects of nano- and 
 microparticles in vivo . 

micro- and nanoparticles released by friction of 
 articulating partners from artificial joints are a major 
 reason for aseptic 
 implant loosening in orthopaedic surgery and may lead to severe 
 peri - implant 
 osteolysis ( particle disease ) 
 .
in addition , 
 nanoparticles can induce or promote allergic or inflammatory 
 reactions or 
 influence hemolysis and blood coagulation 
 [ 57 ] .
although the cytocompatibility of a biomaterial 
 is strongly influenced 
 by its chemical composition , surface topography plays a 
 crucial role for cell - surface 

 interactions .
material surface 
 properties have been studied intensively , but 
 still lack from reliable data about cytocompatibility .
especially , the 
 superordinate principles of cellular responses to 
 surfaces with a defined 
 topography are not well known and poorly understood . 
 because many variables 
 influence cellular interactions to surface structures , 
 it is difficult to draw 
 conclusions and formulate general principles for 
 nano- and microstructured 
 surfaces .
this review summarizes recent data of effects by nano- and 
 microstructured biomaterials and particles in vitro designed for 
 orthopaedic application to get a solid 
 framework outlining the critical interactions that govern the 
 cytocompatibility . 
 because biomaterials in orthopaedics are predominantly 
 applied on bone , this review is focussed on 
 the interactions of osteoblasts 
  and bone - marrow - derived 
 cells with structured biomaterials .
osteoblasts and osteoclasts are mainly 
 responsible for the osteointegration of 
 nanostructured biomaterials in orthopaedics . 

osteoblasts derive from 
 mesenchymal progenitor cells which are localized mainly 
 in the bone marrow and 
 periosteum .
they are characterized by cuboidal 
 and flat morphology ( diameter about 
 20  m ) , present a large 
 amount of rough endoplasmatic reticlum and a large golgi apparatus , 
 and are potent to produce osteoid , a collagen i rich matrix 
 . 

in addition , these mononuclear cells are also responsible 
 for osteoid calcification ( hydroxyapatite ) .
typical marker proteins for 
 osteoblasts are cbfa1/runx2 , osteocalcin , osteopontin , osteonectin , bone 
 sialoprotein ( bsp ) , osteoprotegerin 
 ( opg ) , collagen i , and alkaline phosphates 
 ( alp ) .
figure 1 
 gives a brief summary of the expression 
 of several markers during osteoblast differentiation .
osteocytes act in a paracrine and mechanosensory manner , and 
 can activate osetoblasts and osteoclasts . 
 the latter cell type derived from the 
 hematopoietic line , has multiple nuclei and is 
 responsible for bone resorption . 

its ruffled border is flanked 
 by a sealing zone which facilitates local acidification 
 and removal of bony 
 matrix such as ca , h3po4 , and 
 h2co3 
 by endocytosis .
osteoclasts express 
 high levels of tartrate - resistant acid phosphatase 
 ( trap ) and cathepsin k. the 
 interaction between osteoblasts and osteoclasts is complex . during differentiation ,
the ostoblast progenitors express receptor 
 activator of nuclear 
 factor  ligand ( rankl ) and macrophage colony - stimulating factor 
 ( m - csf ) which are strong stimuli for osteoclastogenesis .
in contrast , 
 osteoprotegerin ( opg ) is a potent inhibitor 
 of osteoclasts .
moreover , the 
 interactions between osteoblasts and osteoclasts in vivo are regulated 
 by several hormones and cytokines , including parathyroid hormone ( pth ) , 
 calcitonin , and il-6 .
it is generally accepted that the 
 three - dimensional surface topography 
 ( size , shape , surface texture ) is one of 
 the most important parameters that influence cellular reactions 
 [ 2 , 1119 ] . 

although many studies have investigated 
 cellular reaction to different surface pattern , 
 the significance of macro 
 structure studies on bone cell behavior is 
 questionable since in vivo adhesion 
 structures ( e.g. , cell membranes , 
 basement membranes ) are comprised of much 
 smaller nanometer scale features [ 20 , 21 ] .
the immature bone is characterized by an average 
 inorganic grain size of 
 1050  nm whereas mature bone has an average 
 inorganic grain size of 2050  nm 
 ( 25  nm in diameter ) 
 . 

considering these parameters , modern 
 implants for bone application have been 
 designed with a smooth surface at the 
 nanometer level .
it was surprising that 
 some of these have induced the formation of peri - implant 
 fibrous tissue and 
 implant loosening in vivo , while other implants with a higher 
 degree of roughness showed significant better osteoconductive properties 
 [ 2325 ] .
there are various methods to modify the degree of 
 roughness as well as surface energy 
 and topography in orthopaedic implants
. 
 typically applied techniques to enhance 
 the degree of roughness and promote 
 the osteointegrative properties of 
 biometals ( e.g. , ti , cocrmo , ss ) are chemical 
 etching or anodization and 
 
 also sand - blasting , sputter - coating , and machine - tooling .
the lack of knowledge in cellular reaction to 
 nanostructered biomaterials is based 
 to a great extent on 
 the difficulty in varying surface chemistry and 
 topography independently .
moreover , 
 the use of different cell lineages and culture 
 conditions makes it difficult to 
 compare results from different investigators 
 [ 2631 ] 
 ( table 1 ) . 

there is also a lack of consensus concerning the 
 proper representation of implant surface topography 
 . 

one major misunderstanding is the practice of defining a 
 surface by its manufacturing process instead of concisely defining the 
 topographic measurements [ 17 , 33 ] . 
 considering these limitations for interpretation ,
the 
 following review gives an overview of cellular 
 reactions to surface structures 
 of different orthopaedic biomaterials .
the first step after exposure of any biomaterial 
 to a biological environment results in the rapid 
 adsorption of proteins to its 
 surface . 

the composition , type , amount , and 
 conformation of adsorbed proteins regulate the 
 secondary phenomena such as 
 cellular adherence and protein exchange 
 [ 3537 ] 
 and also following cellular reactions such as migration , 
 proliferation , and differentiation . 

the potency for biomaterials to adsorb 
 proteins is influenced by its physiochemical 

 characteristics such as surface energy or hydrophobicity , 
 and is also dependent on 
 the local environment ( ph , 
 concentration of ions , composition and 
 functional groups of proteins , strength 
 of solution , temperature ) 
 ( vroman effect ) 
 ( figures 2 ,   and 3 ) . for inorganic nanocrystals and microstructured 
 surfaces
there are at least two approaches to change 
 their hydrophobic surfaces : 
 a ligand exchange reaction can replace the original 
 hydrophobic surface with 
 bifunctional coupling molecules or an inorganic 
 coating such as silica ( 1 ) or 
 an encapsulation of nanocrystals in an 
  amphiphile organic coating ( 2 ) .
the first phase of protein adsorption onto a 
 biomaterial 's surface is characterized 
 by the attachment of small rapidly diffusing 

 proteins , followed by a progressive replacement by larger 
 proteins with a high 
 affinity to the substrate . here
, especially proteins 
 with arg - gly - asp ( rgd ) 
 containing sequences such as fibronectin or 
 vitronectin act as cell receptors 
 and have chemotactic or adhesive properties to bone cells . 

in addition , these rgd - peptides 
 also have a strong effect on matrix maturation 
 and biomineralization [ 4648 ] .
after conditioning of a naked biomaterial by protein adsorption , cells attach rapidly 
 on the protein - coated surface . 
 besides the influence of proteins , the 
 cellular attachement to a nanostructed surface 

is also influenced by its physiochemical 
 properties , especially by the outer functional groups 
 [ 30 , 50 , 51 ] .   
schweikl et al .  
 showed on self - assembly monolayers that the 
 osteoblast proliferation on hydrocarbon 
 chains , terminated by ch3 , 
 was as high as on amino groups 
 ( nh2 ) 
 and hydrophilic oxidized surfaces , but significantly lower 
 on fluorocarbon 
 ( cf3 ) 
 groups .
mller et al 
 .  
 showed that 
 3-aminopropyl triethoxysilane ( apts ) 
 presents amine functional groups which allow for 
 grafting rgd tripeptides and 
 that the rgd - apts hybrid promotes cell adhesion , 
 spreading , and cytoskeletal 
 organization . here , the zetal potential ( differences in potentials 
 between the surface of a tightly 
 bounded layer and a diffuse layer ) 
 and the interfacial tension ( wettability ) of 
 a surface is crucial [ 54 , 55 ] .
it was demonstrated for cpti surfaces that the contact angle ( ca ) , 
 parameter for 
 wettability , increases linearly with the average roughness when 
 the angles were 
 higher than 45 , but decreases linearly with roughness 
 when the angle was less 
 than 45 . 

recent data examining osteoblast 
 response to controlled surface chemistries indicate that 
 hydrophilic surfaces 
 ( high number of polar components ) improve cell attachment 
 and matrix synthesis and also the osteogenic 
 potency compared to hydrophobic 
 surfaces [ 5759 ] . 

compared ti alloys and cocr alloys towards protein 
 absorptive properties and cell attachment with an 
 osteoblast precursor cell 
 line .
they found no significant 
 differences between ti alloys and cocr , 
 but significantly greater cell adhesion 
 rates for the ti implants and concluded that cell 
 adhesion is a result of 
 higher hydrophilicity of ti alloys . in 
 contrast
, other data showed that a low degree 
 of wettability promotes protein 
 adhesion and also cellular attachment to a biomaterial 
 , 
 and mller et al .  

found no direct correlation between the 
 wettability of the material surface and the osteoblast attachment and 
 proliferation rate .
also qu et al .  
 found no significant differences of cell attachement 
 on various titanium surfaces with different degrees of wettabilities 
 ( hydrophobic acid - etched , coarse - blasted large 
 grit acid - etched , hydrophilic modified 
 acid - etched , and modified coarse - blasted large grit , 
 acid - etched 
 ) on mg68 cells .
heating ( oxygen / atm ) or peroxide treatment of biometals 
 result in a thicker oxide layer 
 and a more hydrophilic surface .
. 
  showed that 
 heat - treated titanium 
 surfaces changed the wettability ( more hydrophilic ) 
 but does not significantly 
 affect the fibronectin and albumin adsorption as well as 
 the initial osteoblast 
 precursor cell attachment in vitro . 

emphasized that the rate 
 of protein correlates more with changes in chemical 
 composition than 
 with changes in wettability in metal surfaces .
they showed that 
 a preheating of ti6al4v specimen does not only lead to a 
 thicker oxide layer but also results in 
 an enrichment of v and al within 
 the surface oxide .
in contrast , 
 post - treatment with butanol after preheating 
 reduces the content of v , but not in al , 
 and significantly increases the rate 
 of fibronectin adsorption up to 2040% 
 .
compared to the cellular attachment phase , the following adhesion 
 phase lasts longer and 
 involves various proteins and molecules 
 ( figure 2 ) . 
 as a link between cell and 
 biomaterial , the interactions of a surface topography 
 and serum proteins are 
 crucial for the cytocompatibility of a biomaterial . 

especially , the adsorption 
 of adhesion proteins , such as fibronectin and vitronectin , from serum 
 containing solutions and integrin - mediated signaling has been 
 demonstrated to 
 mediate cell adhesion and spreading 
 . it has been shown that nanotube or nanoparticle surfaces 
 created by anodization have 
 promoted osteoblast adhesion up to three times 
 compared to unanodized ti . 

these results were confirmed by the group of webster 
 
 and other investigators [ 6871 ] 
 who demonstrated that the initial 
 attachment of osteoblasts onto the surface of biometals such as cpti , 
 ti6al4v , and cocrmo is enhanced by submicron to nanometer 
 consistent particles compared 
 to metals composed of respective micron particles . 

one possible explanation of 
 this phenomenon is the higher amount of particle binding sites for 
 osteoblast adhesion at the surfaces of nanophase metals 
 compared to micron particle size 
 metals .
the theory of enhanced protein and cell binding capacities 
 by larger 
 surface areas / roughness degrees was also confirmed for porous 
 ha materials .
another example of the significance of surface structures 
 for protein binding and 
 osteoblast attachment is the helical rosette nanotubes 
 ( hrn ) which can build 
 self - assembly surface structures .
it was demonstrated that 
 a significant change 
 of hrn coverage by heating correlated with the protein - binding 
 and osteoblast 
 adhesion potency in titanium surfaces [ 73 , 
 74 ] .
it is evident that not only the surface topography 
 influences protein deposition and 
 cell adherence but also proteins and cells modify the surface 
 properties of a 
 defined surface .
based on a surface analysis of the different 
 biometal specimen 
 before and after cell cultivation , we showed previously 
 
 that a cell attachment and/or protein 
 precipitation increase the roughness in polished biomaterials 
 ( steel , ti6al4v , 
 and cocr ) .
for porous coated cocr surfaces , we found only slight and no 
 relevant changes in roughness whereas cell cultivation onto 
 sandblasted ti6al4v 
 lead to a strong decrease in specimen roughness . 

both , the increase in roughness after 
 cell culturing in the different biometals 
 and the decrease in roughness of sandblasted ti6al4v could be 
 explained by the 
 dense cellular growth and accumulation of debris in depth of 
 the structured surfaces 
 and/or protein deposition as shown by other investigators 
 [ 75 , 76 ] .
in addition , not only the amount but also the type of 
 protein adsorption by a 
 surface is crucial for cellular adherence and following reactions such as 
 migration and differentiation . as an example , ti surfaces ( ra : 
 0.370.01  m ) adsorp 
  fibronectin in higher concentrations 
 compared to albumin , and fibronectin - coated ti surface promoted 
 more osteoblast attachments 
 in comparison to albumin - coated ti surfaces 
 . 

these results correspond to the data of other authors 
 who showed excellent osteoconductive properties after 
 fibronectin adsorption 
 onto a biomaterials ' surface [ 7880 ] . based on irm and tem analysis ,
the closest distance 
 of cells to a surface ( glass ) was 
 found to be approximately 10  nm [ 81 , 
 82 ] .
historically , results from chicken 
 fibroblasts have lead to a classification of three different types of 
 separation .
( 1 ) focal contacts ( fc ) : approximately 
 1015  nm separation from the substrate under the 
 peripheral regions of the leading lamellae 
 ( appearing black in tem ) .
fc act as 
 an interface between intra and extracellular components and occur linearly 
 beneath the associated cytoplasmic stress fibres 
 [ 83 , 
 84 ] . 

they are tenacious adhesion sites that 
 remain attached to the substratum even when cells are forcibly detached , 
 indicating their function as anchorage 
 structures .
( 2 ) close contacts : corresponding to approximately 
 30  nm separation ( broader grey areas 
 in tem ) .
( 3 ) greater separation : corresponding to approximately 
 100140  nm ( white 
 regions in tem ) .
it is evident that not only fc appear soon after cellular attachment but also that ( -catenin - positive ) adherence 
 junctions occur within 14 hours 
 for grooved ti - based substrates . 

these observations underline the high 
 significance of an early intercellular communication soon 
 after adherence to a 
 surface .
the mechanisms of initial cellular adherence to a surface are 
 different from long - term adherence as shown by a lack of statistical 
 correlation between short - term adhesion ( strength of cell attachment 
 and early 
 adhesion ) and long - term adhesion ( strength of cell - matrix interface ) 
 forces [ 14 , 15 , 86 ] . 

. 
 
 showed that the cultivation time has an 
 influence on the long - term adhesion in biometal surfaces according to 
  td 
 ( t ) = atb , 
 a being independent of b ( td : time - dependend 
 adhesion index , a : surface - dependent parameter , b : substrat - independent 
 exponent , 0.5+/-0.03 ) . for polylactides ( plla ) , it was shown on oct-1 osteoblast - like 
 cells that cell 
 adhesion but not the proliferation could be enhanced by nanoscale 
 and microscale 
 roughness compared to smooth surfaces .
in addition , 
 there is evidence that fc 
 show a dynamic behavior which allows for cellular migration and motility . 

linear plla fibres with length scales of 0.52  m , 
 constructed by electrospinning , have shown cellular contact 
 guidance and enhanced 
 osteoblastic differentiation . here , cell morphology revealed that cells 
 grown on fibres had smaller projected areas than those on planar 
 surfaces .
also other polymers such as plga have been shown to 
 be effective in enhancing osteoblast differentiation in vitro 
 .
diener et al .   demonstrated 
 on mg-63 osteoblastic cells 
 that fc adhesion was smaller on ti and ss than on collagen - coated glass 
 coverslips and that all fc showed a mobility of focal adhesions .
however , 
 anselme et al .   found higher 
 adhesions on ti6al4v 
 substrates than on noncollagen - covered glass samples , 
 and emphasized that substrates 
 with various surface compositions 
 but with the same surface topography did not 
 induce significant differences of adhesion .
based on the knowledge of protein adsorption and its effects 
 on cellular attachment and adherence , a selective surface coating 
 of nanostructured 
 surfaces with rgd or collagen proteins offer a promising solution 
 to improve 
 the number of osteoblasts adhered on artificial surfaces 
 [ 53 , 95102 ] .
imprinting 
 surfaces technology with deposition of specific 
 protein - recognition sites can 
 help to promote osteoblastic growth 
 and differentiation [ 103106 ] .
protein - recognition 
 can be based on a protein - ligand binding and/or electron donor - acceptor 
 interactions or other types of binding forces .
integrin 
 51 and 5 
 v3 subunits competitively 
 bind to rgd - sites of fibronectin [ 107 , 
 108 ] .
dependent on the 
 surface topography and 
 chemistry of the 

 biomaterial , fibronectin 
 undergoes changes in structure including 
 modulation in functional activity and 
 shift in integrin binding capacity .
based on the data of self - assembled monolayers , 
 it was shown that integrin subunits 
 show selective binding capacities to different 
 terminal groups .
integrin 51 shows a 
 strong affinity to oh and 
 nh2 surfaces , whereas 
 51 and 
 5v3 bind also to cooh but show poor binding capacities 
 on ch3 
 surfaces [ 109113 ] .
furthermore , some data show that oh 
 and nh2 surfaces can up - regulate 
 osteoblast - specific gene expression but also 
 matrix mineralization compared 
 with cooh and 
 ch3 
 functional groups [ 47 , 112 ] .
cell migration and proliferation is the attachment 
 following phase between the cell 
 and the material surface .
it   is evident for designing 
 nanostructured implants 
 that cells use the nanotopography of a substrate for 
 orientation and migration [ 117119 ] .
although 
 it is known that bone cells 
 align along defined substrate morphologies ( contact 
 guidance ) , the detailed relation between ordered 
 nanotopography and cell 
 behavior remains unknown in detail . 

for the first time , in 1964 it was shown 
 that convex surfaces enhance cellular overlap , while 
 grooves minimize cellular 
 overlap .
as pre - requisite to reach a defined cell colonization during 
 directed tissue formation , structured nanophase surfaces 
 lead to a predictable 
 osteoblast orientation and migration on these surfaces 
 [ 17 , 121 , 122 ] . 

interaction between the ecm and associated 
 changes in the 
 orientation of the cytoskeleton are crucial for cell 
 metabolism of cells and 
 morphology due to actin - myosin tension structures 
 .
anisotropic topographies ( e.g. , 
 topographical grooves , chemically patterned stripes , 
 or curved surfaces of a 
 fibre ) are potent to exert morphological 
 as well as physiochemical 
 features on cells at the same time , indicative for the 
 complex environmental 
 influence on cells .
focal contacts are important structures for cellular 
 adherence onto a surface but may 
 also delay migration and mobility of the cells . 

it was shown that bone - derived cells ( mg63 cells ) respond to 
 a nanoscale roughness by a higher cell thickness and a 
 delayed appearance of 
 focal contacts . 

especially , nanoporous 
 ti - oxide surfaces promote cellular 
 spreading and induce numerous filopods and osteoblastic differentiation 
 [ 124 , 
 125 ] . on
electrochemically 
 microstructured hexagonal 
 pattern , mg63-cells go inside 
 30100  m but not in 10  m 
 cavities .
most authors report a 
 parallel orientation of cells cultured on 
 polished ( smooth ) surfaces [ 57 , 114 , 126 ] 
 ( figure 4 ) .
another method to not only enhance cellular adherence but also to 
 promote osteoblastic differentiation and 
 biomineralization of biometals is a surface anodization , 
 for example , by -glycerophosphate sodium and calcium 
 acetate [ 6671 ] .
cellular adhesion via fc may strengthen the linkage 
 between cell and ecm and 
 also impair the ability to dynamically remodelling 
 the ecm and influence the 
 migration rate . for 
 collagen - coated coverslips , focal adhesion of mg-63 
 osteoblastic cells moved 
 with a speed of 60  nm / min , whereas the speed was 
 reduced in ti and more in ss 
 surfaces .
another study 
 on nb2o5-coated 
 polished cpti samples showed that mc3t3-e1-osteoblast 
 migration was fastest on 
 smooth surfaces ( ra = 7  nm ) , whereas adhesion strength , 
 spreading area , and 
 collagen - i synthesis were promoted by intermediate 
 roughness ( ra = 15  nm ) .
however , 
 it was surprising that higher degrees of roughness 
 ( ra = 40  nm ) were rather 
 peaked and reduced the speed of adhesion process in the 
 same study . besides the surface properties of a biomaterial , 
 the cellular migration rate is 
 dependent on the cell type and its differentiation stage . 

a higher migration 
 rate is associated with a lower level of osteoblast 
 differentiation .
cells with 
 a low motility are characterized by a strong formation of fc 
 while motile cells 
 form less adhesive structures .
it was found that mature 
 osteoblasts spread out 
 and form a greater number of fc when settled on smoother surfaces 
 .
although 
 cellular spreading is higher on 
 smoother surfaces , some data indicate that the 
 alp - expression is higher for 
 rough isotropic surfaces ( electro - erosion , acid - etching , 
 sandblasted ) compared 
 to smoother substrates ( machine tooling , polishing ) 
 . considering recent publications , there 
 is no or only week statistical significance that there 
 is a difference between 
 the initial number of adherent cells and following proliferation of cells 
 cultured onto a biometal or ceramic nano-/microscale surface 
 in vitro .
however , 
 some authors emphasize that 
 the influence of functional chemical groups for cellular migration and 
 proliferation are stronger than general surface 
 properties such as wettability . 

especially a tio2-layer 
 seems to promote 
 cellular growth and proliferation on nanostructured 
 biometals [ 128 , 129 ] .
other examples for a promotion of cell - to - bone contact in vitro 
 and also in vivo are 
 machine - etched ti - surfaces ( e.g. , osteotite ) 
 , defined sand - blasted 
 implants [ 124 , 125 , 131 ] , and hydroxyapatite ( ha ) coatings , 
 for example , by plama - spray techniques [ 132134 ] .
recent studies investigating the response of adherent 
 cells to nanography surfaces 
 indicate that different cell phenotypes have different 
 levels of sensitivities [ 117 , 135137 ] . here
, 
 osteoblasts react to features as 
 low as to the 10 nm dimensions , which is comparable in size to a single 
 collagen fibre .
moreover , the qualitative and quantitative kinetics 
 in gene and protein expression is 
 strongly influenced by topography and physiochemistry 
 of a defined surface .
microporous 
 ha surfaces seem to promote a high number of fc and 
 increased levels of alp but 
 short actin stress fibres compared to nonmicroporous ha surfaces 
 [ 72 , 139 ] . 

there is also evidence that ti and ha 
 surfaces can activate early intracellular signalling 
 pathways as shown by 
 expression of relevant molecules such as 
 - and 1-integrin , fak , erk followed 
 by c - jun and c - fos genes for proliferation and alp for differentiation 
 [ 139 , 140 ] . 

however , hallgren et al .  
 found no significant histomorphometric 
 and biomechanical differences between nanopatterned and control implants . 

.  
 showed that microfabricated 
 discontinuous - edge surfaces ( des ) , 
 repeated open square boxes with a depth of 
 10  m , alter osteoblast adherence and migration 
 but enhance cell multilayering , 
 matrix deposition and mineralization when compared to smooth controls .
in contrast to our data , 
 anselme et al .   found 
 higher proliferation 
 rates on ss compared to ti6al4v .
however , bigerelle et al . 
  demonstrated that neither 
 material composition nor surface roughness amplitude influence cell 
 proliferation , whereas they found a very significant influence 
 on manufacturing 
 process and surface topography for long - term adherence 
 and proliferation in vitro .
our in vitro results   confirm the 
 well known osteogenic in 
 vivo properties of ti implants , which may be based on 
 surface factors observed 
 on its outer tio2-layer 
 [ 143146 ] . 

mller et al .  
 demonstrated the ability of osteoblasts 
 to grow into an open - porous ti implant ( metal foam ) and li et al
. 
 
 also demonstrated that mc3t3-e1 cells 
 attach to and are able to divide well in the inner surface of 
 a highly porous 
 trabecular ti6al4v implant .
some in vitro studies demonstrated an 
 enhanced total protein and collagen production , 
 as well as increased alp 
 activity of osteoblasts cultured on nanoparticulate 
 metals ( cpti , ti6al4v , and 
 cocrmo ) indicating advantages for nanostructured surfaces 
 for osteointegration [ 1 , 149 , 150 ] . based on the 
 data of redey et al . , 
 it can be concluded that the low 
 attachment and collagen production rates are related to a low 
 wettability of a 
 nanosurface .
nanotextured surfaces of ti surfaces 
 prepared by chemical etching 
 have upregulated the expression of bsp and 
 op . 
 as demonstrated by qu et al . , 
 the expression of the bone - associated 
 genes such as alp , oc , type - i - collagen , osteoprotegerin , and 
 glyceraldehyde-3-phosphate - dehydrogenase is promoted 
 by modsla ti surfaces . 
 some data also suggest that fluoride - modified 
 ti surfaces can stimulate 
 osteoblastic differentiation compared to 
 unmodified titanium surfaces [ 151 , 
 152 ] .
. 
  showed in their in vitro experiments that nanophase biometals induce 
 significantly greater calcium and phosphorus 
 deposition by osteoblasts and also allow for 
 calcium and phosphorous precipitation from 
 culture media without osteoblasts in 
 contrast to microphase ti6al4v and cocrmo . 

furthermore , the authors found 
 advantages in mineral precipitation without osteoblast for 
 tial4v but no differences 
 in dependency to the type of ti ( wrought , microphase , 
 or nanophase ) .
it was 
 evident that the increased calcium and phosphorus 
 mineral content correlated to 
 greater amounts of underlying aluminium content on 
 ti6al4v surfaces . although some data indicate 
 that nanostructured ti alloys promote non - cell - mediated ca / po4-mineral 
 deposition from culture media compared to cocrmo substrates , 
 the greatest cell - dependend calcium and 
 phosphorus mineral deposition occurred 
 on nanophase cocrmo .
it is evident that micropattern collagen films or 
 scaffolds promote not 
 only cellular adhesion but also allow for 
 an osteoblastic differentiation and 
 biocalcification in vitro [ 153155 ] . 
 for ha- and dcpp - coated , ti 
 surfaces the ca / p ratio influence the biomineralization 
 rate in vitro .
besides the osteoblast - promoting effects 
 of defined substrates and 
 surface topographies , some data also allocate 
 an inflammatory response induced 
 by nano- or microstructured biomaterials . 

it was shown in many studies that 
 cell - biomaterial interactions can activate macrophages 
 which results in the synthesis of proinflammatory 
 agents such as tnf , ifn , il-1 and -6 , 
 rankl and no [ 157159 ] .
some data have 
 shown proinflammatory effects of different 
 biomaterials which increase with the 
 degree of surface roughness . 
 here ,
macrophage inflammatory protein-1 , tnf , 
 monocyte chemoattractant protein-1 , and 
 members of the interleukine and leukotriene family 
 play a crucial role in 
 biometal - induced inflammations 
 [ 160164 ] .
most studies report about an 
 enhanced expression of pro - inflammatory 
 cytokines and chemokines by cells 
 attached to rougher surfaces .
some data also 
 indicate that anionic and neutral hydrophilic surfaces 
 increase macrophage - monocyte apoptosis and reduce macrophage 
 fusion to modulate 
 inflammatory responses to implanted materials 
 .
however , adverse cellular effects seen with 
 metallic implants may 
 also be attributed to corrosion products or to 
 the separation of metal ions 
 ( fe , cr , ni ) which may have a major impact on cellular survival and 
 differentiation [ 166168 ] . 
 those studies which suggest that a cell - mediated metal ion 
 release by biometals that 
  did not affect 
 the cell viability or proliferation are 
 characterized by short cultivation periods or 
 other conditions which limit the 
 reliability of data [ 169171 ] . up to date , only few authors report about no significant influence 
 of the cellular adherence and expression of osteoblast 
 proteins by different 
 biometals and surfaces such as alp expression 
 [ 172 , 
 173 ] .
in contrast to the great opportunity enhancing 
 biocompatibility and osteogenic potency of surfaces applied on bone by 
 nanotechnology , micro- and nanoscaled particles released by friction of 
 artificial joints can induce severe 
 inflammation and may lead to osteolysis and 
 implant failure [ 174 , 
 175 ] 
 ( figure 5 , 
 table 2 ) . there is a wide 
 range in particles size and morphology produced by 
 simulators for artificial 
 joints .
particles released from metal - metal ( crcomo alloys ) are 
 predominantly chromium oxide particles or cocrmo with 
 varying ratios of co and cr .
they show a round to oval morphology 
 and also a substantial number of needle - shaped 
 particles were found during the first circles . 

emphasize the 
 importance of particle size 
 as a critical factor in osteoblasts proliferation 
 and viability in vitro .
some data indicate 
 that in contrast to ti - surfaces 
 nano- and mircoparticles induce an 
 inflammatory response although titanium is 
 one of the biometals with the highest degree of 
 cytocompatibility . as shown by miyanishi et al . 
 , 
 the release of vegf may play a crucial role in the 
 pathogenesis of ti - induced osteolysis .
some data indicate that 
 phagocytosis of ti particles is not a precondition for 
 an inflammatory response 
 such as a release of tnf or il-6 in 
 cultured macrophages . 

it is evident that a binding of the macrophage cd11b / cd18 ( macrophage 
 mac-1 receptors / receptor of complement cr3bi , 
 can also bind to icam-1 and 
 icam-2 ) by integrin - specific antibodies 
 also increased the release of tnf and il-6 in macrophages . 

this finding also suggests 
 that the complement system plays a role in the pathogenesis 
 of particle - induced 
 inflammation , too .
especially , uhmwpe particles with 
 a size range of 
 0.11.0  m have been shown to be most reactive for 
 macrophage activation and cytokine 
 secretion in bone marrow cells [ 179 , 
 180 ]
. however , not only the particle size but also the particle volume 
 ( number ) is a critical factor for particle - mediated 
 release of cytokines by 
 macrophages .
green et al . 
 demonstrated for pe 
 that the cell - particle ratios of 1 : 100 
 ( size 0.497.2  m ) and 1 : 10 
 ( size : 0.494.2  m ) induced 
 significant stronger release 
 of tnf and il-1 in macrophages . 

the authors conclude that especially 
 particles in the phagocytosable size range of 
 0.310  m appear to be the most 
 biologically active ones .
the latter statement was also confirmed for silicon carbide ( sic ) 
 particles and biometals such as cpti , 
 ti6al4v and uhmwpe [ 184 , 185 ] .
granchi et al .  
 investigated the in vitro 
 effects of al2o3 
 and uhmwpe particles in an 
 osteoblast - osteoclast co - culture system . 

both particles did not affect either 
 cell viability or tnf and gm - csf release , 
 whereas il6 release was dependent on 
 the particle concentration .
uhmwpe particles 
 increased the release of rankl 
 from osteoblasts and induced large amounts 
 of multinucleated trap - positive 
 giant cells in an osteoblast - osteoclast 
 co - culture system .
also , 
 carbon - based particles with low wear 
 factors such as p25-cvd showed a high degree 
 of cytocompatibility in vitro . howling et al . 
 
demonstrated 
 on fibroblasts and monocytes that p25-cvd 
 particles < 100  nm were significantly less cytotoxic 
 to both cell types than 
 cocr metal wear particles . 

while the classical water - suspendable 
 nanoc60 nanocrystal is apparently 
 cytotoxic to various cell lines , the closely related 
 fully hydroxylated , 
 c60(oh)24 
 , is nontoxic , 
 thus producing no cellular 
 response .
also , 
 functionalized single - walled carbon nanotubes are nontoxic 
 to cells in culture [ 198200 ] .
there is evidence that not only particle 
 size and chemical content but also the concentration strongly influence 
 cellular reactions in vitro .
wilke 
 et al .  
 showed a positive correlation between the 
 release of proinflammatory cytokines 
 ( il-6 , -1 , and tnf ) and amounts of 
 ti6al4v - particles 
 ( 10 , 
 10 , 10 , 
 and 10 particles / ml ) 
 by human bone marrow cells over 2 weeks .
some in vitro data also indicate that ti particles induce a stronger 
 fibroblastic differentiation signal than uhmwpe 
 in monocytes and other cells [ 182184 ] .
.  
 showed that particles 
 of high - density polyethylene 
 ( hdp ) and ti6al4v induced significantly more 
 proinflammatory mediators ( il-1 , 
 il-6 , tnf ) and bone resorption compared to 
 al2o3 
 and zro2 in vivo . 

based on these data , 
 it can be assumed that ceramics show a high 
 degree of cytocompatibiltiy . for ha especially , particles with a size 
 < 53  m inhibit cellular proliferation , 
 especially in osteoblasts and lead 
 to a decrease in tgf1 and a significant increase in pge2 and ldh 
 concentration , but did not influence the tnf 
 or alp titer in vitro . 
 it could be concluded that larger ha particles may 
 be compatible with bone cells while smaller - sized 
 ha particles can both activate 
 the osteoclasts and decrease the cell 
 population of the osteoblasts in vitro .
numerous variables influence the biocompatibility and osteogenic 
 potency of nanostructured biomaterials in 
 vitro and in vivo .
besides the locotypical 
 environment in vivo or in vitro , the surface 
 structure and the composition of a 
 biomaterial affects cellular attachment , 
 adherence , proliferation and migration , 
 and also 
 differentiation and survival of defined cell types . 
 here , information about typical 
 parameters such as chemical composition , 
 surface structure ( topography , geometry , 
 roughness , particle size ) , surface energy , 
 hydrophobicity , and the degree of 
 solubility in aqueous solutions of a biomaterial 
 will help to value and grade a 
 defined implant concerning its osteblast promoting potency . considering recent publications , we could assume some general 
 principles of cytocompatiblity and cell - surface 
 interactions in nano- and 
 microstructured surfaces .
( 1 ) wettability of a nanosurface influences significantly 
 protein adsorption , which is a prerequisite 
 of cellular adherence in serum containing 
 solutions .
( 2 ) nanostructured surfaces enhance the surface area of 
 biomaterials and promote cellular adherence . ( 3 ) the chemical outer functional 
 groups of a nanosurface significantly influence cellular 
 migration , proliferation , and differentiation but direct 
 correlations between 
 distinct parameters and cell functions are not entirely cleared .
( 4 ) the formation of fc underly a 
 dynamic process and influence the motility and migration of cells . ( 5 ) a higher degree of differentiation 
 is corresponding to a decreased cellular motility .
( 6 ) phagocytable particles with a size 
 < 10  m induce the strongest 
 cellular response with regard to releasing 
 inflammatory cytokines .
( 7 ) although ti has a high degree of 
 cytocompatibility in vitro , phagocytable 
 ti particles can induce a fibroblastic 
 differentiation .
 cell - surface interactions play a crucial role for biomaterial application in orthopaedics . 
 
 it is evident that not only the chemical composition of solid substances influence cellular adherence , 
 migration , proliferation and differentiation but also the surface topography of a biomaterial . 
 
 the progressive application of nanostructured surfaces in medicine has gained increasing interest 
 to improve the cytocompatibility and osteointegration of orthopaedic implants . 
 therefore , the 
 understanding of cell - surface interactions is of major interest for these substances . in this review 
 , 
 we elucidate the principle mechanisms of nano- and microscale cell - surface interactions in vitro for 
 different cell types onto typical orthopaedic biomaterials such as titanium ( ti ) , 
 cobalt - chrome - molybdenum ( cocrmo ) alloys , stainless steel ( ss ) , as well as synthetic polymers 
 ( uhmwpe , xlpe , peek , plla ) . 
 in addition , effects of nano- and microscaled particles and their 
 significance in orthopaedics were reviewed . 
 the significance for the cytocompatibility 
 of nanobiomaterials is discussed critically . 
1. INTRODUCTION
2. BONE CELLS
3. CYTOCOMPATIBILITY OF MICRO- AND 
NANOSTRUCTURED SURFACES