Osteoblast adhesion

Biomaterials 21 (2000) 667}681

Review

Osteoblast adhesion on biomaterials

K. Anselme*

Institut de Recherche sur les Maladies du Squelette, Institut Calot, Rue du Dr Calot, 62600 Berck sur mer cedex, France

Received 11 January 1999; accepted 21 October 1999

Abstract

The development of tissue engineering in the "eld of orthopaedic surgery is now booming. Two "elds of research in particular are

emerging: the association of osteo-inductive factors with implantable materials; and the association of osteogenic stem cells with these
materials (hybrid materials). In both cases, an understanding of the phenomena of cell adhesion and, in particular, understanding of
the proteins involved in osteoblast adhesion on contact with the materials is of crucial importance. The proteins involved in osteoblast
adhesion are described in this review (extracellular matrix proteins, cytoskeletal proteins, integrins, cadherins, etc.). During
osteoblast/material interactions, their expression is modi"ed according to the surface characteristics of materials. Their involvement
in osteoblastic response to mechanical stimulation highlights the signi"cance of taking them into consideration during development
of future biomaterials. Finally, an understanding of the proteins involved in osteoblast adhesion opens up new possibilities for the
grafting of these proteins (or synthesized peptide) onto vector materials, to increase their in vivo bioactivity or to promote cell
integration within the vector material during the development of hybrid materials.

Keywords: Osteoblast; Adhesion; Biomaterial; Tissue engineering

1. Introduction

Cell adhesion is involved in various natural phe-

nomena such as embryogenesis, maintenance of tissue
structure, wound healing, immune response, metastasis
as well as tissue integration of biomaterial. The biocom-
patibility of biomaterials is very closely related to cell
behaviour on contact with them and particularly to cell
adhesion to their surface. Surface characteristics of ma-
terials, whether their topography, chemistry or surface
energy, play an essential part in osteoblast adhesion on
biomaterials. Thus attachment, adhesion and spreading
belong to the "rst phase of cell/material interactions and
the quality of this "rst phase will in#uence the cell's
capacity to proliferate and to di!erentiate itself on con-
tact with the implant.

It is essential for the e$cacy of orthopaedic or dental

implants to establish a mechanically solid interface with
complete fusion between the material's surface and the
bone tissue with no "brous tissue interface. Moreover,
the recent development of tissue engineering in the "eld

* Tel.: #33-0321-892029; fax: #33-0321-892070.
E-mail address: kanselme@hopale.com (K. Anselme)

of orthopaedic research makes it possible to envisage the
association of autologous cells and/or proteins that pro-
mote cell adhesion with osteoconductive material to cre-
ate osteoinductive materials or &hybrid materials'. Thus,
a complete understanding of cell adhesion and parti-
cularly osteoblast adhesion on materials is now essential
to optimize the bone/biomaterial interface at the heart of
these hybrid materials. This includes an understanding
of the molecules involved in bone cell adhesion, parti-
cularly regarding interaction with the materials but also
the need to take into account osteoblastic reaction to the
mechanical constraints which will be applied to im-
planted materials in vivo. The application of non-de-
structive in vitro mechanical constraints to the cell/
biomaterial interface permits understanding of the
e!ects of mechanical stimulation on the synthesis of
adhesion proteins, cell growth and cell di!erentiation
and provides essential information for the development
of hybrid materials.

This paper o!ers a review of present knowledge of

osteoblast adhesion, focused on in vitro adhesion on
orthopaedic biomaterials. The aim of the author is to
highlight useful information for the improvement of pres-
ent biomaterials and the future development of new
biomaterials.

0142-9612/00/$ - see front matter

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2. Description of bone cell adhesion

The term &adhesion' in the biomaterial domain covers

di!erent phenomena: the attachment phase which occurs
rapidly and involves short-term events like physico-
chemical linkages between cells and materials involving
ionic forces, van der Walls forces, etc. and the adhesion
phase occurring in the longer term and involving various
biological molecules: extracellular matrix proteins, cell
membrane proteins, and cytoskeleton proteins which in-
teract together to induce signal transduction, promoting
the action of transcription factors and consequently regu-
lating gene expression. Those various proteins involved
in cell adhesion will be described in this "rst part.

2.1. Proteins involved in osteoblast cell adhesion

2.1.1. Extracellular matrix proteins

The extracellular matrix of bone is composed of 90%

collagenic proteins (type I collagen 97% and type V col-
lagen 3%) and of 10% non-collagenic proteins (NCP)
(osteocalcin 20%, osteonectin 20%, bone sialoproteins
12%, proteoglycans 10%, osteopontin, "bronectin,
growth factors, bone morphogenetic proteins, etc.). All
these proteins are synthesized by osteoblasts and most
are involved in adhesion. In vitro, other proteins such as

"bronectin

or vitronectin have been shown to be in-

volved in in vitro osteoblast adhesion. Some other pro-
teins with a plasmatic origin are also associated with
mineralized bone matrix but their role in osteoblast
adhesion is not fully de"ned (

a2HS glycoprotein, al-

bumin, immunoglobulin, transferrin, etc.) [1].

Some of the bone proteins have chemotactic or adhes-

ive properties, notably because they contain an Arg}
Gly}Asp (RGD) sequence which is speci"c to the "xation
of cell membrane receptors like integrin ("bronectin, os-
teopontin, bone sialoprotein, thrombospondin, type I
collagen, vitronectin) [1,2].

In order to determine the proteins involved in bone cell

adhesion, experiments consisting of bone cell cultures on
dishes coated with extracellular matrix proteins are cur-
rently used. Human osteoblasts adhere preferentially to

"bronectin as compared with type I, type IV collagen,

and vitronectin but weakly to laminin and type V col-
lagen. In contrast, they do not adhere to type III collagen
[3].

Other adhesion experiments use arti"cially synthesized

peptides like GRGDS (Gly}Arg}Gly}Asp}Ser) [2] or
RGDS (Arg}Gly}Asp}Ser) [4]. After mixing the peptide
with culture medium, it was demonstrated that RGDS
peptide partially inhibited rat calvarial bone cell attach-
ment to "bronectin-coated plates in a competitive dose-
dependent manner although RGES control peptide had
a minimal e!ect on cell attachment [4]. A GRGDS
peptide almost completely blocked the attachment to
bone sialoprotein and vitronectin and slightly decreased

the attachment to "bronectin, type I collagen and throm-
bospondin [2]. The addition of the peptide GRGDSP
(Gly}Arg}Gly}Asp}Ser}Pro) to human osteoblast-like
osteosarcoma cells SaOs-2 cells in a serum free medium,
inhibited cell adhesion by 28% on titanium-based alloys
(Ti6Al4V) and by 40% on CoCrMo alloys although
adhesion on glass and plastic was not a!ected. The con-
trol peptide GRADSP (Gly}Arg}Ala}Asp}Ser}Pro) had
no e!ect on cell adhesion [5]. The strength of rat cal-
varial cell adhesion, measured with a radial #ow appar-
atus, was signi"cantly higher on a RGD-peptide-coated
surface compared to a RGE-peptide-coated surface [6].
The role of RGD-peptides conformation on osteo-
progenitor cell adhesion has been highlighted [7]. These
experiments have demonstrated the importance of iso-
lated RGD-sequence containing peptides in promoting
adhesion of bone cells. Biomaterials may be improved by
a preliminary adsorption of these peptides.

2.1.2. Cytoskeleton proteins

The sites of adhesion between tissue cultured cells and

substrate surfaces are called focal contacts or adhesion
plaques. Focal contacts are closed junctions where the
distance between the substrate surface and the cell mem-
brane is between 10}15 nm. This type of junction is rare
in vivo except for endothelial cells in vessels with high
hydrodynamic stress [8]. They also appear to be analo-
gous to sarcolemnal dense plaques of smooth muscle cells
in vivo.

The external faces of focal contacts present speci"c

receptor proteins such as integrins. On the internal face,
some proteins like talin, paxillin, vinculin, tensin are
known mediating interactions between actin "laments
and membrane receptor proteins (integrins) (Fig. 1).
Many proteins colocalize with vinculin and talin in the
adhesion plaque: integrin, cytoskeletal proteins, pro-
teases, protein kinases and phosphatases, signalling
molecules, etc. These proteins are involved in signal
transduction.

The formation of focal contacts occurs essentially in

cells with low motility and is promoted in vitro by ex-
tracellular matrix proteins like "bronectin or vitronectin.
The architecture of the actin cytoskeleton is essential to
the maintenance of cell shape and cell adhesion. If assem-
bled in long bundles, F-actin supports "nger-like protru-
sions of the plasma membrane known as "lopodia; if
assembled in the form of a mesh, it supports sheet-like
protrusions known as lamellipodia. If present in bundles
coupled with adhesion plaques, actin &stress "bers' may
transmit forces to the substrate [9].

2.1.3. Adhesion molecules

Adhesion molecules are characterized by their capacity

to interact with a speci"c ligand. These ligands may be
situated on the membrane of neighbouring cells or may
be extracellular matrix proteins. Adhesion molecules

668

K. Anselme / Biomaterials 21 (2000) 667}681

Table 1
The integrin family of adhesion receptors

Sub-units

Other names

Ligands

Cells

VLA-1

Collagen-I, collagen-IV and laminin

Bone cells, cultured osteoblastic cells, activated
T-cells, monocytes, melanoma cells and smooth
muscle cells

VLA-2, GPIa-IIa,
ECMRII

Collagens-I to IV, laminin and possibly
Fibronectin

Bone cells, cultured osteoblastic cells, B and
T lymphocytes, platelets, "broblasts, endothelial cells
and melanoma cells

VLA-3, VCA-2,
ECMRI, Gapb-3

Fibronectin, Collagen-I, Epiligrin,
Laminin and Nidogen/Entactin

Cultured osteoblastic cells, B-lymphocytes, kidney
glomerulus and most cultured cell lines

VLA-4, LPAM-2

Fibronectin, VCAM-1

Cultured osteoblastic cells, lymphocytes, monocytes,
eosinophils, NK-cells and thymocytes

VLA-5, FNR,
GPIc-IIa, ECMRVI

Fibronectin

Bone cells, cultured osteoblastic cells, memory-
T-cells, monocytes, platelets and "broblasts

VLA-6, GPIc-IIa

Laminin-1, -2, -4 and -5 (only in vitro)

Platelets, lymphocytes, monocytes, thymocytes and
epithelial cells

VLA-7

Laminin-1

Skeletal and cardiac muscle at speci"c stages during
muscle development and melanoma cells

Fibronectin

Tenascin-C, osteopontin

Adult skeletal muscle, visceral smooth muscle, skin
and corneal epithelial cells, hepatocytes

Fibronectin, vitronectin

Cultured osteoblastic cells

LFA-1, CD11a-CD18

ICAM-1 to -3

Leucocytes

Mac-1, CR-3,
CD11b-CD18

C3bi, coagulation factor X,

"brinogen and ICAM-1

Monocytes, macrophages, NK cells and granulocytes

p150, CR-4,
CD11c-CD18

Fibrinogen

Monocytes, macrophages, granulocytes, NK cells
and activated lymphocytes

VNR, CD51

Fibrinogen, "bronectin, Von Willebrand's
factor, vitronectin, thrombospondin,
osteopontin and bone sialoprotein 1

Bone cells, cultured osteoblastic cells, endothelial
cells, some B-cells, platelets and monocytes

aIIb

GPIIb-IIIa, CD41

Fibrinogen, "bronectin, Von Willebrand's
factor and vitronectin

Platelets

Laminin-1

Immature thymocytes, squamous epithelia, subsets
of endothelial cells, Schwann cells and "broblasts
in the peripheral nervous system

aVbS, aVb3b

Vitronectin

Cultured osteoblastic cells, hepatoma cells,

"broblasts and carcinoma cells

Fibronectin

Carcinoma cells

LPAM-1

MadCAM, "bronectin and VCAM-1

Leukocytes (directed to the Peyer's patches of the gut)

aIELb M290 IEL, aH

Mature synapses of central nervous system

!VLA: very late activation antigen, VCA: very common antigen, ECMR: extra cellular matrix receptor, GP: glycoprotein, Gap: galactoprotein, LFA:

leukocyte function associated antigen, M290 IEL: mouse intraepithelial lymphocyte antigen recognized by monoclonal antibody M290, FNR:

"bronectin receptor, ICAM: inter cellular adhesion molecule, LPAM: lymphocyte Peyer's patch HEV adhesion molecule (mouse), VCAM: vascular

cellular adhesion molecule, CR: C3bi receptor, VNR: vitronectin receptor, MAC: macrophage receptor.

belong to di!erent families. The four main classes are
selectins, immunoglobulin superfamily, cadherins and in-
tegrins. Amongst them, only cadherins and integrins have
been described at this time in osteoblastic cells.

Integrins: cell}substrate adhesion. The integrin family is

composed of 22 heterodimers of two types of sub-units
a and b. 16 a sub-units and 8 b sub-units have been
discovered. This diversity of structures is associated with
various ligand-binding possibilities (Table 1). Integrins
are transmembrane heterodimers consisting of non-
covalently associated

a and b sub-units. Each sub-unit is

made up of a large extracellular domain, a transmem-
brane domain and a short cytoplasmic domain. The
integrin spanning the cell membrane acts as an interfacer
between the intra- and extra-cellular compartments and
can translate the attachment of external ligands to inter-
nal information which induces adhesion, spreading, or
cell migration and consequently regulates cell growth
and di!erentiation (Fig. 1).

Recently, the expression of integrin in bone and in

cultured bone cells has been demonstrated. On human
bone sections, all bone cell types expressed

a1 and

K. Anselme / Biomaterials 21 (2000) 667}681

669

Fig. 1. Representation of the cell proteins involved in cell adhesion on biomaterial.

a5 sub-units although a subpopulation of osteoblastic

cells expressed

a2, a7 and a7b3 [10]. Cultured human

osteoblasts expressed high levels of

a1b1, a3b1, a5b1 and

a7b5 and much lower levels of a2b1, a4b1, a7b1 and a7b3

integrins [11].

b1 sub-unit appears to be the predomi-

nant receptor involved in osteoblast adhesion to collagen
and laminin and is partially involved in adhesion to

"bronectin [3]. In cells adhering in the presence of serum,
b3 and a7 integrin were recovered in focal contacts.

a4 and b1 subunits are expressed by primary human

osteoblasts on "bronectin, type I collagen and laminin-
coated control polystyrene dishes.

a2, a6 and a7 are

expressed on control dishes,

a2, a3 and a7 on collagen-

coated dishes and

a6 and a7 on laminin-coated dishes.

b3 subunit is expressed on all surfaces except on

laminin-coated dishes [12]. Adhesion to "bronectin, col-
lagen- and laminin-coated dishes is inhibited by anti-

integrin subunit antibody although anti-

a5 integrin

subunit antibody a!ects adhesion only to "bronectin
[13]. An understanding of the integrin subunits involved
in osteoblast adhesion provides essential information for
biomaterial improvement and notably for de"nition of
the proteins which may be useful to adsorb on materials
before implantation.

Cadherin: cell}cell adhesion. As cell}substrate ad-

hesions are based on integrin-type receptors, adherens
junctions containing cadherins mediate cell}cell ad-
hesion (CAM). Cadherins are transmembrane glycopro-
teins acting with intracellular partners: catenins which
interact with intracellular proteins [14] (Fig. 1). Associ-
ation with

a, b or c-catenin is a prerequisite for the

adhesive function of cadherins. The cadherin family is
composed of numerous types of calcium-dependent mol-
ecules (E, P, N, L, R, 6B, 7, 11, 4). They associate in

a zipper homophilic model of interactions between cad-
herin molecules exposed on the plasma membrane of
adjacent cells [15]. Firstly, osteoblasts have been shown
to express E-cadherins [16] and cadherin-11 (or OB-
cadherin) [17]. More recently, human osteoblasts have
been shown to express mRNA for cadherin-11, N-cad-
herin and low levels of cadherin-4. The expression of
cadherin-4 is modulated by bone morphogenetic pro-
tein-2 (BMP-2) contrary to cadherin-11 and N-cadherin
expression [18]. Cadherin-11 gene is expressed in the
bone marrow and bone cells obtained from rabbits of
various age groups. The relative level of cadherin-11 gene
is greater in mature rabbit marrow than in young or aged
animals [19].

Gap junctions: cell}cell communications. Cell rec-

ognition and adhesion precede and control cell}cell
communication via gap junctions [20]. Intercellular
communications occur through direct exchange of ions
via gap junctions or through signals produced by the
action of CAM.

Gap junctions are constituted by homohexamus de-

rived from a family of proteins called connexins. When
the connexin of one cell (composed of six connexin mol-
ecules) is in register with a similar structure on a neigh-
bouring cell, a transcellular channel is formed [20]. Tight
and adherens junctions or desmosomes provide anchor-
age to surrounding cells and allow direct exchange of
ions or small molecules between cells (Fig. 1).

Osteoblasts express in vitro two connexins, connexin

43 and connexin 45 [21,22]. Connexin 43 expression by
human osteoblastic cells is regulated by retinoic acid and
transforming growth factor-

b1 (TGF-b1) [23] and by

parathyroid hormone (PTH) and prostaglandin E2

(PGE2) [20,24].

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K. Anselme / Biomaterials 21 (2000) 667}681

2.2. Signal transduction

As previously mentioned, integrins, CAM molecules

and gap junctions regulate gene expression by a signal
transduction induced by cell}substrate or cell}cell ad-
hesion. Integrins and cadherins have direct interactions
with the cytoskeleton (actin) via

a-actinin, talin and

catenins. Integrin clustering and occupancy provoke the
recruitment of tensin and focal adhesion kinase (FAK),
their phosphorylation and subsequently the recruitment
of talin, vinculin, and

a-actinin [25] (Fig. 1). Various

other intracellular signalling pathways are activated by
cell adhesion: protein tyrosine phosphorylations, mito-
gen-activated protein kinase (MAP) activation, Ca

2` in-

#ux, pH alterations and inositol lipid turnover.

b-catenin

molecules can interact with the cadherin system but also
with fascin (a molecule involved in the formation of
F-actin bundles) and with transcription factors [14].
Some other molecules like FAK may play a mechanical
role in binding a number of signalling and cytoskeletal
molecules in parallel with its kinase activity (Fig. 1).

Talin, vinculin and

a-actinin link the F-actin

"bers to

the plasma membrane. The rearrangement of F-actin

"bre bundles induces the cell shape changes producing

the "nger-like or sheet-like protrusions ("lopodia or
lamellipodia, respectively). The signals mediated by cell
shape may be processed by the nuclear matrix which is
physically linked to the cytoskeleton via the nuclear
lamins. Subsequently, adhesion plaque activation is es-
sential for signal transduction and then regulation of
gene expression. ECM-mediated changes in cell shape
can modify the nuclear matrix and therefore modify
gene expression. ECM-mediated changes in cell shape
initiate a switch between the proliferative and di!erenti-
ative state. For example, as osteoblasts shift from a
proliferative to a rounded di!erentiated state, a novel
nuclear matrix protein NMP-2 appears. NMP-2 binds to
the osteocalcin gene promoter and induces its expression
[26].

2.3. Adhesion and cell migration

Cell migration requires a dynamic interaction between

the cell, its substrate and its cytoskeleton. Firstly, cells
develop a protrusion of their leading edge to form
a lamellipodium. Secondly, after formation and "xation
of the lamellipodium, cells use adhesive interactions to
generate the traction and energies required for cell move-
ment. The last step of the migratory cycle is the release of
adhesions at the rear of the cell followed by its detach-
ment and retraction. Integrin have been shown to be
involved in cell migration [27]. In general, cells with
a low motility form strong focal adhesions while motile
cells form less adhesive structures. An intermediate level
of attachment force induces a maximal migration rate
[28]. Some antiadhesive extracellular matrix proteins

play a role in cell migration: tenascin, thrombospondin,
laminin, muscin, proteoglycans [29]. Migration tests on
biomaterials with surface grooves demonstrated that cell
migration was faster on materials with deeper grooves
[30].

3. Osteoblast/material interactions

Osteoblast/material interaction depends on the surface

aspects of materials which may be described according to
their topography, chemistry or surface energy. These
surface characteristics determine how biological molecu-
les will adsorb to the surface and more particularly deter-
mine the orientation of adsorbed molecules [31]. They
also determine the cell behaviour on contact. As pre-
viously shown, cells in contact with a surface will "rstly
attach, adhere and spread. This "rst phase depends on
previously described adhesion proteins. Thereafter, the
quality of this adhesion will in#uence their morphology,
and their capacity for proliferation and di!erentiation.

Early in vitro cytocompatibility studies focused on the

morphological aspect, growth capacity and the state of
di!erentiation of cells on materials with various chemical
compositions [32}38]. The diversity of cell responses to
the di!erent materials tested highlighted the capacity of
cells to discriminate between di!erent chemistries. How-
ever, the sensitivity of in vitro biological tests was some-
times too low to distinguish the e!ects of subtle changes
in substratum surface chemistry. No di!erences were
observed in cell colonization of polystyrene dishes
treated by sulphuric acid and gamma-irradiation al-
though X-ray photoelectron spectroscopy (XPS) analysis
demonstrated that the two treatments introduced di!er-
ent chemical groups onto the polymer surface [39].

Surfaces are di!erent from the corresponding bulk of

the material. For thermodynamic reasons they contain
unsaturated bonds which lead to the formation of surface
reactive layers and adsorbed contamination layers. Prep-
aration technique e!ects such as sterilization e!ects have
been studied by several authors. They have demonstrated
the crucial e!ect of the sterilization methods of commer-
cially pure titanium (cpTi) on in vitro subsequent cell
adhesion [40,41]. These e!ects may be related to steriliz-
ation-induced surface chemical modi"cations [42]. Some
clinical surface preparation on titanium-based alloys are
known to considerably modify the surface chemical char-
acteristics. Improper glow discharge plasma treatment (a
frequently used method for cleaning, preparation and
modi"cation of biomaterial) can produce unwanted and
irreproducible results [43]. The surface oxide on tita-
nium implant materials is mainly TiO2 and is about 2 nm

thick. One signi"cant di!erence, as compared to the
machined unalloyed Ti, was that AlOx and no V was

detected on the machined Ti6Al4V surfaces. The concen-
tration of Al on the outermost surface may constitute

K. Anselme / Biomaterials 21 (2000) 667}681

671

a potential risk for Al dissolution in the surrounding
tissues [44]. Consequently, caution must be taken with
the surface preparation of these alloys. The surface oxide
formed on the alloy might play a role in the extent to
which the materials will be accepted. So, the e!ects of
surface preparation on cell cytocompatibility must be
previously veri"ed before surgical use because the biolo-
gical properties of bulk material could be largely modi-

"ed by the process of surface preparation [45].

3.1. Morphological aspects of osteoblast on materials

The comparison of the behaviour of di!erent cell types

on materials shows that they react di!erently according
to surface roughness [30,46}48]. Scanning electron
microscopic examination of bone cells on materials with
various surface roughness generally demonstrated that
cell spreading and continuous cell layer formation was
better on smooth surfaces compared to rough ones
[35,49,50].

The organization of surface roughness is an important

parameter to consider. In vitro, many authors have dem-
onstrated the contact guidance phenomenon using

"broblasts or epithelial cells. Epithelial cells were mark-

edly oriented along the long axis of 10

lm deep grooves

on a titanium-coated implant [51]. The modi"cations of
groove and ridge dimensions of a silicone rubber substra-
ta suggested that a ridge width )4

lm was necessary to

observe rat dermal "broblast orientation along the sur-
face grooves. The modi"cations of groove width or
groove depth did not a!ect the cellular orientation [52].
The contact guidance phenomenon has also been de-
scribed on osteoblastic cells. On smooth surfaces, bone
cells were randomly oriented although they were aligned
parallel to the direction of the grooves in an end-to-end
fashion in 5

lm-deep grooves. In contrast, they

&ignored'

the surface topography on an 0.5

lm grooved surface

[30]. In vivo, it appears that the surface characteristics of
an implant, particularly roughness, may control tissue
healing and therefore subsequent implant success
[53,54]. The hydrophilic and hydrophobic character-
istics of a material are also of great importance for cell
adhesion. Cell adhesion is generally better on hydrophilic
surfaces [55]. Spreading of human skin "broblasts in-
creased along chemically characterized gradient surfaces
going from the hydrophobic to the hydrophilic end [56].
The morphological aspect of neonate rat calvarial os-
teoblasts cultured on a positively or negatively charged
polymer substrata was signi"cantly di!erent. Cells #at-
tened out so closely onto the positively charged substrata
that the ventral cell membrane was not distinguishable
through the transmission electron microscope. On nega-
tively charged substrata, the ventral cell membrane was
readily visible with only focal contacts with the substrata
[57]. Bone calvarial cells randomly plated on materials
with patterned surface chemistry rapidly (t(30 min) or-

ganized on positive charged regions in the presence of
serum. After 30 min, cells started to align and spread
exclusively on these regions. By day 2, cells started to
extend from positive charged regions to negative charged
regions due to lack of surface area available and were
con#uent after 4 days [46].

3.2. Osteoblast adhesion on material

Cells do not interact with a naked material either

in vitro or in vivo. At each step, the material is
conditioned by the biological #uid components [31]. The
pH as well as the ionic composition and strength of
solution, temperature and the functional group of pro-
teins and substrates are the factors determining protein
adsorption.

Surface energy may in#uence protein adsorption and

the structural rearrangement of the proteins on the ma-
terial [31]. Protein adsorption was di!erent on positively
and negatively charged polymer substrates [57]. Protein
from serum containing media adsorbed on surfaces form-
ing multiple molecular layers [30]. Many recent experi-
ments using materials with patterned surface chemistry
obtained by photolithography demonstrated the di!er-
entiated adsorption of serum proteins on the negative
(DMS) or positive (EDS) charged regions. The role of
vitronectin for in vitro cell adhesion has been highlighted
by several authors [36,47,58]. Sera depleted of vitronec-
tin, with or without "bronectin, greatly reduced cell at-
tachment and spreading on patterned surfaces. Thus, the
presence of vitronectin is essential for in vitro spatial
distribution, attachment and spreading of bone-derived
cells in an EDS region [47].

The role of plasma "bronectin (pFN) adsorption in cell

adhesion has been also established [55]. Moreover, it is
clear that not only the nature of adsorbed biological
molecules but also their conformation will in#uence con-
sequent cell adhesion. Changes in pFN conformation
a!ected "bronectin cell binding domain conformations
and then a!ect FN a$nity with its cell surface receptor
[59].

A direct relationship exists between roughness and

surface energy of materials. A systematic study evaluated
cell adhesion on polymethylmetacrylate (PMMA) mate-
rials with various degrees of surface roughness. It was
demonstrated that the apolar component of surface en-
ergy increased signi"cantly with roughness although the
basic component decreased and that cell adhesion en-
hancement was related to the degree of roughness and
the hydrophobicity [60].

In a recent work, the adhesion of human osteoblasts

on Ti6Al4V substrates with "ve di!erent degrees of sur-
face roughness was studied. Cell adhesion was correlated
with roughness parameters and in particular with para-
meters describing the organization of the surface rough-
ness. More so than the roughness amplitude evaluated by

672

K. Anselme / Biomaterials 21 (2000) 667}681

Ra or Rt, the developed surface or Delta parameter
describing the surface organization was the more in#uen-
tial parameters on human osteoblastic cell adhesion to
orthopaedic alloys [50].

Osteoblast adhesion on materials may also be con-

sidered in relation to the expression of the various
adhesion proteins. Numerous studies using immuno-his-
tochemical methods have shown the presence of actin
and vinculin in cultured osteoblasts on various materials.
Cell adhesion was higher on Ti6Al4V'polystyrene'
CoCrMo alloy. The rate of actin and vinculin cyto-
skeletal reorganization was enhanced on Ti6Al4V. Focal
contacts remained peripherally located in cells on
Ti6Al4V and CoCrMo although on polystyrene the focal
contacts quickly became dispersed along actin "laments.
The authors suggested that osteoblast attachment was
greater on Ti6Al4V because cell spreading and cytos-
keletal organization were enhanced [61]. In human
osteoblasts cultured on a titanium alloy with various
degrees of roughness, we showed the presence after 1,
7 and 14 days of short, thin and dense vinculin positive
patches. Logically, the distribution of focal contacts illus-
trated the mode of adhesion of osteoblasts on the di!er-
ent roughnesses. On smooth surfaces, focal contacts were
distributed uniformly on all the membrane surface which
was in contact with the substratum. On rough surfaces,
focal contacts were visible only at the extremities of cell
extensions where cell membranes were in contact with
the substrate [50].

Not only topographical but also physico-chemical

characteristics of the surface in#uence the distribution of
focal contacts. Analysis of focal contacts using speci"c
labelling of actin and vinculin showed their presence only
on electropolished and etched titanium surfaces but not
on sandblasted ones [62]. The F-actin cytoskeleton
reorganization on various materials showed a typical
sequence: at 6 h, the F-actin was generally di!usely dis-
tributed with circumferential banding near the edge of
cells. At 12 h, thin micro"lament bundles were visible. At
24 h, cells were spread and numerous well-de"ned stress

"bres were observed [37]. After 24 h, an actin micro"la-

ment system ran parallel to the long axis of cells [50].
Double localization of actin and vinculin showed that
actin formed numerous stress "bres thoughout the
cytosol which terminated at the cell periphery and that
the vinculin-containing adhesion plaques were situated
at the terminal &ends' of the actin stress "bres [38].

More recently, the integrins mediating osteoblast ad-

hesion on various materials were studied. Some experi-
ments consisted of inhibiting cell attachment to various
materials and of determining the integrin subunits in-
volved in cell adhesion using anti-integrin subunit anti-
bodies. An antibody to the "bronectin receptor

a5b1

signi"cantly inhibited adhesion on Ti6Al4V by 63% and
to CoCrMo by 49%. Serum had no e!ect on the number
of cells that attached to Ti6Al4V and CoCrMo alloys but

did increase the number of cells that attached to glass.
Osteoblast-like cell adhesion on glass contrary to
Ti6Al4V and CoCrMo was not mediated by integrins but
did require the adsorption of vitronectin [5]. Integrin
expression of primary human osteoblasts was analysed
after culture on rough and polished CoCrMo and
Ti6Al4V alloys.

a3 and a6 integrin sub-units were ex-

pressed only on polished Ti6Al4V.

a2, a4, a7 and b1 in-

tegrin sub-units were expressed on all surfaces and
b3 sub-unit was expressed on all surfaces except on rough

CoCrMo [12]. We also observed the expression of
a3 and b1 integrin sub-units by primary human osteo-

blasts but no expression of

a2 integrin sub-units [50].

The anti-

b1 sub-unit antibody revealed thin, short

"laments parallel to the cytoskeleton arrangement on all

the membrane surface. The morphology of

b1 "laments

varied slightly on di!erent substratum roughnesses
[12,50]. It seems that

b1 integrin was the major integrin

sub-unit involved in osteoblast adhesion on biomaterials.

3.3. Osteoblast diwerentiation on materials

As previously described, some proteins can be adsorb-

ed in vitro from the serum containing media or in vivo
from biological #uids. Vitronectin [63] or "bronectin
[50], extracellular matrix proteins synthesized by bone
cells in vitro or adsorbed from serum, are essential for
osteoblast in vitro adhesion [58]. After the attachment
phase, extracellular collagenous and non-collagenous
matrix proteins are synthesized by cells [30,64].
Neonatal rat calvarial osteoblasts expressed mRNA en-
coding osteonectin and osteopontin when cultured on
Ti6Al4V, HA or tissue culture polystyrene [65]. Some
qualitative and quantitative di!erences in bone protein
expression has been observed between various substra-
tes: rat bone marrow stromal cells expressed more os-
teopontin and bone sialoprotein on HA compared to
cpTi or glass-ceramic [66]. SaOs-2 human osteoblast-
like cell line synthesized a higher collagen and non-
collagen protein quantity when cultured on a CoCrMo
alloy compared to glass control discs [5].

Using human osteoblast-like cells (MG63), it was

found that on titanium disks with various degrees of
roughness, proliferation and alkaline phosphatase activ-
ity was reduced when roughness increased. In contrast,
collagen synthesis increased with roughness although
non-collagenous protein synthesis was not a!ected by
roughness [64]. PGE2 and TGF

b1 production was high-

er on rough surfaces than on smooth ones [49]. More-
over, the roughness increased the MG63 cells response to
1

a,25(OH)2D3 vitamin and notably promoted the in-

crease of osteocalcin synthesis and alkaline phosphatase
activity [67]. Adult jaw bone cells had a lower prolifer-
ation on rough-surface (plasma-sprayed) hydroxyapatite
(HA) or titanium substrates compared to smooth surface
(polished), a lower alkaline phosphatase activity but

K. Anselme / Biomaterials 21 (2000) 667}681

673

a higher osteocalcin synthesis [34]. Human bone-derived
cells cultured on titanium-based metallic substrates [68]
or ceramic substrates [69] expressed and translated dif-
ferently mRNAs for speci"c osteoblastic proteins like
alkaline phosphatase, thrombospondin, osteopontin, os-
teocalcin, osteonectin, type I collagen and bone sialo-
protein. This di!erential regulation of proteins occurring
between cells from the same patient implies that human
bone-derived cells respond to small di!erences in the
surface chemistry and or/microcrystallinity.

Bone cells cultured on materials with patterned surface

chemistry during 15 days formed multilayered cell layer
on the entire surface but Von Kossa staining demon-
strated the presence of mineralized tissue consistent with
the underlying chemical pattern. Mineralized tissue was
preferentially localized on positive charged regions [46].

4. Mechanical aspects of osteoblasts/biomaterial
interactions

The role of mechanical load in bone remodelling is well

known. In vitro, osteoblastic cells respond to mechanical
stimuli with alterations in proliferation and/or pheno-
typic expression. Adhesion is also a!ected by mechanical
stimulation.

Firstly, the in#uence of mechanical stimulation on

in vitro osteoblast adhesion, proliferation and di!erenti-
ation will be considered. Secondly, the use of mechanical
strain for cell detachment and quantitative cell adhesion
evaluation will be presented.

4.1. Mechanical ewects on osteoblasts

The osteoblastic cell response to mechanical stimula-

tion has been widely studied using various cell strains
and various methods of load application. Consequently,
heterogenous results were obtained. The mechanism of
cellular response to #ow was demonstrated to be shear
stress independent [70]. In some cases, a dose depen-
dence was observed between the number of responsive
cells and shear stress magnitude [71,72]. On the other
hand, other authors suggest that bone and bone-like cells
respond to mechanical signals in a trigger-like rather
than a dose}response fashion [73].

Generally, cell proliferation was stimulated by mech-

anical stress [74}78]. Optimum magnitude of tensional
forces for osteoblastic cell division was determined
[75,76]. Applications of mechanical strains on human
bone cells caused a signi"cant e!ect on proliferation but
only in a proportion of subjects [78].

Osteoblast di!erentiation generally implies alkaline

phosphatase activity (ALP) and speci"c protein expres-
sion like osteocalcin, osteopontin, type I collagen and
in vitro mineralization capacity. In vitro mechanical
stimulation has shown various e!ects on ALP activity of

cells [73,75,79}81]. Low vacuum pressure depressed
ALP activity in the cultures of rat calvaria bone cells and
mouse calvaria cell line (MC3T3-E1) [73] although inter-
mittent hydrostatic compression upregulated ALP
activity in mouse calvarial bone cells [80]. Rat calvarial
osteoblasts subjected to #uid #ow expressed lower ALP
mRNA notably under pulsatile #uid #ow [82]. These
observations illustrate the signi"cance of strain charac-
teristics and methods used to apply mechanical strain on
bone cells: continued or intermittent strains, applied by
compression, stretch or #uid #ow methods.

Speci"c osteoblastic protein expression is also modi-

"ed

by strains. Osteopontin synthesis is generally in-

creased [75,83}85]. On the other hand, osteocalcin
synthesis is di!erently in#uenced [73,86]. Collagen ex-
pression by cells under mechanical strains is also highly
variable [82,87]. Osteoprogenitor cells reacted di!erently
to osteoblastic cells. Collagen expression of osteoblastic
cells after intermittent hydrostatic compression was
decreased although collagen expression by osteoprogeni-
tor cells was increased under the same conditions [82].
This illustrates the in#uence of the cell line used for the
experiment.

The cytoskeleton is also modi"ed by mechanical

stimulation. Intermittent hydrostatic compression up-
regulated actin expression [82]. Mechanical strain in-
creased formation and thickening of actin stress "bers
[82]. A 250% increase of vinculin was observed following
mechanical stimulation and especially at the peripheral
edges of the cells [88]. However, vinculin appears not to
play a role in mechanical transduction because depletion
of vinculin from focal contacts did not prevent the re-
sponse of cells to mechanical stimulation [33]. The
microtubule role in mechanical force transduction has
been also highlighted [88,89].

Prostaglandin E2 (PGE2) or prostacyclin synthesis by

osteoblasts are systematically increased by mechanical
strains [78,90}95]. PGE2 synthesis is one of the early
responses of osteoblasts and induces a secondary produc-
tion of cAMP which is involved in signal transduction
[91]. In mechanically stimulated osteoblastic cells, cAMP
increase was also largely demonstrated [70,86,91,96].

As previously described, cell adhesion may occur on

RGD-sequence-containing proteins like collagen or os-
teopontin via speci"c membrane receptors called integ-
rins. Some authors focused on expression of

b1 integrin

sub-units and

a2 integrin sub-units by osteoblasts sub-

mitted to mechanical stimulation [97}99]. Using para-
magnetic beads coated with anti-integrin antibodies, it
was possible to apply de"ned physical forces on indi-
vidual integrin receptor sub-units [97,99]. These experi-
ments con"rmed previous results demonstrating the role
of integrin in transmission of mechanical signals into cells
[100,101].

Cell}cell adhesion proteins are also involved in osteo-

blast response to mechanical stimulation. As previously

674

K. Anselme / Biomaterials 21 (2000) 667}681

Table 2
Techniques for the detachment of cells and quantitative evaluation of
cell adhesion

Technique

Cells

Micropipet aspiration

Human blood cells [110,111]
Porcine aorta endothelial cells [112]
Normal and SV-40 transformed human
dermal "broblasts [113]

Centrifugation

SV-40 transformed murine peritoneal
macrophages and human erythrocytes
[114]
Vero Green monkey kidney [115]
Fibroblasts and glioma cells [116]
Normal and SV-40 transformed human
dermal "broblasts [113]

Paramagnetics beads

Fibroblasts [117,118]
Osteoblastic cells [99]
Osteoblast-like cells [107]

Fluid #ow

Mouse macrophage cell line [119]
3T3 "broblasts [120]
Bovine aortic endothelial cells [121]

Enzymatic detachment

Chick embryo aortic explants [118]
Human and chick embryo trabecular
bone explants [122]
Primary human osteoblastic cells [50]

Spinning disk

3T3 "broblasts [123]
Osteoblast-like cells [108,109]

Microplate manipulation

Chick "broblasts [124]

Microcantilever

L929 murine "broblasts [125]

shown, osteoblasts express connexin 43 and 45 [21,22].
Cyclic stretch enhances gap junctional communications
between osteoblastic cells [102]. These gap junctions
are involved in intercellular calcium wave propaga-
tion [103] and then induce mechanosensitive signal
transduction pathways. Many experiments have shown
that substratum stretch, hydrostatic pressure and

#uid shear stress increased inositol

triphosphate (IP3)

in bone cells with IP3 causing the release of calcium
from intracellular stores. The IP3 biochemical pathway is
one of the mediators of the response of bone cells to
mechanical stimulation [71,90]. Mechanosensitive ion
channels have also been demonstrated in osteoblasts
[104,105].

The principle mechanical detection system of cells is

the matrix-integrin mechanosensory protein complex-
cytoskeleton machinery which is linked to a kinase cas-
cade system. Mechanosensory protein complex contains
talin, vinculin, tensin, paxillin, Src and focal adhesion
kinase (FAK) (Fig. 1). The kinetics of response events in
mechanically loaded cells may entail, during milliseconds
to seconds after stimulation, a signalling response involv-
ing mechanically active channels Ca

2`, Ma`, K`, H`,

IP3, cAMP, PGE2, kinases, G proteins, etc. After min-
utes to hours after stimulation, signalling with kinases,
transcription and transduction proceeds, and cyto-
skeletal protein polymerization and focal adhesion
rearrangement occur. After some days, cells migrate, ex-
press and degrade extracellular matrix, divide or die.
A new state of equilibrium is established [106].

4.2. Quantitative evaluation of cell adhesion

Quantitative evaluation of cell adhesion generally im-

plies the detachment of cells. To detach cells, various
techniques may be used (Table 2).

Most experiments attempted to apply external forces

to cells cultured on control surfaces and either to quan-
tify and mathematically create a model of the adhesive
cell/substrate connections or the mechanical deforma-
tions of individual cells. Only recently have studies fo-
cused on measurement of osteoblastic cell adhesion:
some on control surfaces [99,107], others on bioactive
biomaterials [108,109].

The optimal tensional force that stimulates osteoblast

activity was determined [75]. An application of over
10 000

l-strain resulted in a dedi

!erentiation of the os-

teoblasts and a change in cell morphology to become

"broblast like [126]. The proliferation of human osteob-

lasts was increased signi"cantly by 1% strain although
higher strain magnitudes had lesser (non-signi"cant) ef-
fects or decreased the mitotic activity of the cells [76].
A spinning disc device permitted quanti"cation of rat
osteosarcoma cells on various substrates. Cell attach-
ment strength approximated 20 dyn/cm

2 (2 N/m2) on

bioactive glass or borosilicate control glass. If "bronectin

was previously adsorbed on these materials, cell attach-
ment strength was comprised between about 50 and
70 dyn/cm

2 [109].

Optical techniques such as total internal re#ection

#uorescence microscopy (TRIFM) or internal re#ection

microscopy (IRM) permit visual examination of cell/sub-
strate contacts in real time. Additionally, TRIFM allows
quanti"cation of the separation distance of cells from
a biomaterial surface and has been used to evaluate the
adhesion strength on a biomaterial surface of bovine
endothelial cells following exposure to #ow [127].

5. Perspectives: improvement of biomaterials

Biomaterials currently available for clinical use are

known for their good biocompatibility. However, if most
of them have the mechanical properties required for
a de"ned implantation site, they do not all possess the
necessary bioactivity properties for good tissue regenera-
tion, i.e. not only osteoconductive properties but also
osteoinductive properties.

Several means are currently being developed for bio-

materials improvement like the surface modi"cation of
materials by protein adsorption procedures; or bone tis-
sue engineering associating autologous bone cells with
biomaterial.

K. Anselme / Biomaterials 21 (2000) 667}681

675

5.1. Adhesion protein adsorption

The proteins currently being considered for chemical

surface modi"cation of materials are growth factors (or
related proteins) and adhesion proteins (or related peptides).

Among the bone-related growth factors, some are at

the present time being tested for their ability to promote
bone regeneration. Notably, members of the Transform-
ing Growth Factor-

b family are being widely studied:

TGF-

b1, BMP-2, BMP-7 (or osteogenic protein-1 [OP-

1]). BMPs were identi"ed following puri"cation of
bovine bone proteins after it was discovered that de-
mineralized bone segments or extracts of demineralized
bone-induced bone formation in ectopic sites [128,129].
BMPs associated with various carriers have experi-
mentally shown their e$cacy [130}133] and are current-
ly under clinical investigation [134].

Amongst adhesion proteins, as previously described,

RGD-peptides have shown their e$cacy in promoting
osteoblast adhesion. The use of RGD-peptide may be
a future way of improving biomaterial surface. However,
the promotion of adhesion by RGD-peptides was not
associated with a subsequent enhancement of cellular
functions [135]. OP-1 promoted mineralization of os-
teoblast cultured on a RGD-peptide. These results sug-
gest that the combination of selected bioactive agents
and proactive biomaterials may synergistically enhance
clinically desirable cellular functions [136]. Other pep-
tides were explored. One method consists of synthesizing
a 15-residue peptide related to a biologically active por-
tion of type I collagen. This peptide was adsorbed on
inorganic bovine bone mineral particles to encourage
attachment of human dermal "broblasts. Cell attach-
ment on the coated particles increased with augmented
peptide content on the surface of bone particles. The
presence of P-15 promoted the formation of tri-dimen-
sional colonies. P-15 coated bovine bone mineral par-
ticles may be a useful matrix for bone repair [137].

5.2. Bone engineering: osteoblast/biomaterial association

Recently, a new "eld of biological/biomedical research

has developed: cellular and tissue engineering. This is
a consequence of advances in cell isolation and culture
procedures, combined with a growing understanding of
cell physiology. In particular, the previously described
knowledge of osteoblast adhesion on biomaterials may
provide essential information on the development of

&hybrid

materials' containing biocompatible osteocon-

ductive structural materials and autologous bone cells
for a self-cell therapy.

Former studies in this "eld concerned porous calcium

phosphate

ceramics

associated

with

non-cultured

autologous bone marrow cells. They were demonstrated
to produce bone after subcutaneous implantation
[138}140] and after experimental implantation in bone

defects [141}144]. In fact, bone marrow is known to
contain osteogenic precursor cells [145,146]. After in
vivo implantation in di!usion chambers, human os-
teogenic precursor cells developed an osteogenic tissue
consisting of a mineralizing "brous component and carti-
lage [147,148].

More recently, in vitro long-term culture of osteoblasts

in porous materials like ceramics [149}152], collagen
[153,154], commercial bone substitutes [155,156] or
polymer sca!olds [157] have been developed. They have
allowed investigation of how bone marrow cells adhere
to the surface of porous calcium phosphate ceramics
[150,152]. Cell di!erentiation during cultures was
monitored [151,153,156,158] and osteoblastic cell pen-
etration inside porous material and across inter-pore
connections was illustrated [151,158]. The sizes of inter-
pore connections were the most important parameters
for a deep colonization of porous materials after im-
plantation [158].

Some of these works were at the root of the use of

previously cultured bone cells for hybrid material prep-
aration [159}165]. The culture period may allow stimu-
lation of the inherent osteogenic ability of marrow
stromal stem cells in pores of porous hydroxyapatite
using for example a dexamethasone stimulation of cells
[165]. Frozen-preservation of cells could be used because
recultured cells after thawing also formed bone in in vivo
porous ceramic assay [159]. Cells may be cultured before
implantation either in tissue culture dishes before being
mixed with the material [161,162,166] or directly cul-
tured in the material [159,163,165,167].

6. Conclusion

This review has highlighted the complexity of the phe-

nomena occurring in cell/material interactions and parti-
cularly the role of cell adhesion, which conditions
subsequent cell behavior at the interface with the mate-
rial. A complete understanding of cell behavior in contact
with the material is becoming more and more essential in
attaining adequate health safety conditions for clinical
use of these hybrid materials. The development of tissular
engineering techniques in the orthopaedic domain is re-
quiring more and more the consideration of osteoblast
adhesion properties whether for the improvement of the
surfaces of materials by adsorption or grafting of speci"c
adhesion factors, or for the development of hybrid mate-
rials associating autologous bone cells and materials.

Acknowledgements

The author thanks B. NoeKl for the tables and "gures

achievement and Dr. P. Hardouin for critical reading of
the manuscript.

676

K. Anselme / Biomaterials 21 (2000) 667}681

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