The present invention first relates to a bi-functional peptidic compound characterized in that it comprises a metal-binding peptide (MBP) domain and a cell- specific peptide (CSP) domain, said MBP and CSP domains being linked by a linker. A bi-functional peptidic compound according to the invention is able to bind to the metallic surface of a biomaterial compound to gingival cells, thereby enhancing the cell- adhesion of said biomaterial compound. The present invention also relates to a composition comprising said bi-functional peptidic compound, and to a biomaterial compound associated with said bi-functional peptidic compound, it relates in particular to a dental implant functionalized by the adsorption on its surface of a bi-functional peptide according to the invention.

Implantable biomaterials may be functionalized by surface modification in order to improve their properties. WO 2010 094212 discloses plasma treated and controllable release antimicrobial peptide coated titanium alloy for surgical implantation. The surface forms antimicrobial layers on the alloy capable of resisting microbial adhesion and proliferation, while allowing mammalian cell adhesion and proliferation when the alloy is implanted to human body. JP 2009007339 discloses an adhesive for dental implant, reinforcing boundability of the dental implants with biological tissues, especially soft tissues such as gingival epithelium. The adhesive for dental implants comprises a ceil adhesive artificial peptide (P). Further, a siiane coupling agent and/or a titanium coupling agent is preferably contained in the adhesive. US 7897163 discloses a bone graft material and a scaffold for tissue engineering applications having an osteogenesis-promoting peptide immobilized on their surface. The shielding membrane and implant comprise ceil adhesion derived peptides including ROD amino acids. Indeed the mostly used bio-active peptide sequence to promote cell adhesion on different surfaces is the RGD (-arginine— glycine— aspartate-) sequence. It is found in most extracellular matrix proteins such as fibronectin, laminin, and vitronectin influencing cell adhesion, mobility, proliferation, and cell survival and has been shown to bind approximately half of the 24 known human integrins. Searching to promote titanium surface endothelization on titanium stents implants Meyer et al., (2007) attached a titanium binding peptide having the sequence SCSDCLKSVDFIPSSLASS (SEQ ID N°9) to the RGD sequence. The authors demonstrated a better adhesion and proliferation of endothelial cells on titanium functionalized with the peptide-RGD binder. The same laboratory team used this new approach for other biomaterials than titanium (Meyer et al., 2008, 2009, 201 1). Yazici H. et al. (2013) bound phage display selected titanium binding peptide (TiBP) to Arg- Gly-Asp-Ser (RGDS) sequence (SEQ ID N° 10), to improve fibroblast cell adhesion on commercial grade Ti surface.

Hence the layer-by-layer (LbL) method has been applied for functionalization of

Ti surfaces and immobilization of bioactive organic molecules on them has been peiformed ( reviewed in Boudou et al., 2010). The LbL film deposition method consists of the alternate adsorption of oppositely charged polyelectrolytes that auto-assemble, leading to the formation of polyelectrolyte multilayer (PEM) films. Chemical cross- li nking, based on gliitaraldehvde chemistry, for inhibition of free polymers diffusion withi n the films was previously proposed as a suitable methodology for increasing fil m s bio-compatibility.

EP 2 385 057 discloses a peptide having the amino acid sequence SVSVGMKPSPRP (SEQ ID N° l) and its binding to Silicon substrates. Estephan et al. (2012 ) also disclose a peptide hav ing the sequence SEQ ID N° l and the large variety of inorganic and organic substrates bound by this peptide.

WO 2006/1 16737 discloses a peptide hav ing the sequence SWELL. YPLRANL (SEQ ID N°4) and its ability to disrupt cell lines adhesion. Devemy and Blaschuk (2009) disclose an antagonist of E- and N-cadherin having the sequence SEQ ID N°4 and its ability to disrupt intercellular adhesive complexes.

None of these documents disclose a bi-functional peptide comprising a peptide metal-binding peptide and a cell-specific peptide linked by a linker.

There is still a need for a simple and reliable method to functionalize the surface of a biomaterial compound, and in particular of an implantable oral biomaterial, to increase its cell adhesion and to improve his clinical application and durability, in particular on oral conditions.

This is the object of the present invention.

Pure titanium (Ti) and titanium alloys (T1 ₆ AI ₄ V) have been the most successful and widespread metals used for oral and maxillofacial implant. The epithelial sealing at the abutment has been identified as the critical factor to prevent periimplant inflammation. Once the epithelial cells have migrated to the implant surface, they adhere directly via basal lamina (< 200 nm) and hemidesmosomes formation. The adhesion must be stable and resistant to various external factors like mechanical constraints and oral bacterial pathogens.

The present invention provides a new bi-functional metal-cell specific peptidic compound, and in a particular a metal-cell specific peptide (MCSP) and a method to functionalize the surface of a biomaterial compound, wherein said bi-functional peptidic compound comprises a metal binding peptide (MBP) domain linked to a cell-specific peptide (CSP) domain to increase the cell adhesion of said functionalized biomaterial compound. The inventors clearly demonstrated the enhanced cell-adhesion force on keratinocytes to surface.

A functionalization method according to the invention is easy-to-use, and the bi- functional peptidic compounds can be synthetized in desired quantities and modified according to the envisaged application by standard methods. The invention goes beyond the state of the art in sensitivity and detection limit due to the ordered array of molecules assured by the peptides.

A bi-functionalization process according to the present invention is able to overcome the problem related to the unwanted polymerization and denaturation of capturing molecules when amine-activation is performed via glutaraldehyde chemistry. A process according to the invention is able to confer biocompatibility and stability to a functionalized biomaterial, and may reduce its toxicity.

The present invention discloses four artificial MCSP among which was selected one best candidate for implant surface functionalization. The present invention will become more fully understood from the detailed description given herein and from the accompanying drawings, which are given by way of illustration only and do not limit the intended scope of the invention.

Each amino acid is herein represented according to the IUPAC amino-acid abbreviation, such as follows:

Table 1

The present invention first relates to a peptidic compound characterized in that it comprises: a) a metal-binding peptide (MBP) domain able to bind to a titanium surface, said MBP domain comprising an amino acid sequence selected from the group consisting of:

• SEQ ID N°l or a sequence having at least 80% identity with the sequence SEQ ID N° 1 , and

• SEQ ID N°2 or a sequence having at least 80% identity with the sequence SEQ ID N°2,

b) and a cell-specific peptide (CSP) domain comprising an amino acid sequence of 10 to 15 amino-acids,

wherein said MBP and CSP are linked by a linker.

By "metal-binding peptide", "metal-binding peptide domain" or MBP domain, it is intended a peptide comprising an amino acid sequence which is able to bind to an inorganic substrate, and preferably to a metallic surface.

The ability of a MBP domain of a peptide according to the invention to bind to a titanium surface is characterized by the affinity of said MBP domain for a titanium surface, and preferably a high affinity of said MBP domain for a titanium surface.

A person skilled in the art of determining the affinity of a peptide for another compound will be able to choose the appropriate method and conditions to determine the ability of a MBP domain of the invention to bind to a titanium surface.

Among methods for determining the affinity of a peptide domain for a surface, the following methods may be cited: in vitro affinity measurement methods, including ELISA and competitive inhibition, biopanning, microscopy, including fluorescence microscopy and atomic force microscopy, spectroscopy, including Fourier transform infrared spectroscopy, spectrometry, including MALDI-TOF mass spectrometry.

In a particular embodiment, a MBP domain according to the invention is also able to bind to metallic surface other than titanium. A metallic surface other than titanium, able to be bound by a MBP domain according to the invention, may be chosen among one of the following: a titanium alloy, gold, carbon, cobalt, platinum, steel, surgical stainless steel, silicone, chrome and a chrome alloy. By "cell-specific peptide", "cell-specific peptide domain" or CSP domain, it is intended a peptide comprising an amino acid sequence which is able to bind or to adhere to cells, preferably to mammalian cells, said mammalian cells being chosen in the group consisting of mice cells, rat cells and human cells, preferably human cells. In a particular embodiment, a CSP domain of a bifunctional peptide according to the invention is able to bind to epithelial cells, and more particularly to keratinocytes or to endothelial cells. In a particular embodiment, a CSP domain according to the invention is able to bind to E-cadherin, to N-cadherin, to a domain thereof or to a combination thereof. In another particular embodiment, a CSP-domain according to the invention is chosen in the group consisting of E-cadherin antagonists, N-cadherin antagonists, or E- and N-cadherin antagonists.

"Amino acid sequence" and "sequence" will be employed indifferently in the present specification. "Amino acid" and "amino acid residue" will also be employed indifferently. The amino acid sequences are read from the N-Terminal extremity to the C-Terminal extremity of said amino acid sequence of the polypeptide. Therefore the amino acid in position 1, i.e. the first amino acid, of a sequence is the amino acid at the N-terminal extremity of the sequence.

Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

The term "peptide" means a polymer of at least about 4, 5, 6, 7, or 8 amino acids. Throughout the specification, standard three letter or single letter designations for amino acids are used. In the art, this term is often used interchangeably with "polypeptide" or "protein". The term "peptidic compound" according to the invention designates a compound comprising a peptide, or comprising two peptide domains linked by a linker, wherein said linker has a peptidic or a non-peptidic nature. In a particular embodiment of the invention, two peptide domains are linked by a peptidic linker and said peptidic compound is a peptide.

As used herein the term "identity" herein means that two amino acid sequences are identical (i.e. on an amino acid basis) over the window of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, in which the amino acid sequence to be compared can contain additions or deletions with respect to the reference sequence for optimal alignment between those two sequences. The percentage of sequence identity is calculated by determining the number of positions at which the identical amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e. the window size) and multiplying the result by 100 to yield the percentage of sequence identity. The percentage of sequence identity of an amino acid sequence can also be calculated using BLAST software with the default or user defined parameter.

The invention also relates to a functional derivative of a bi-functional peptidic compound according to the invention, comprising one or more modifications that do not substantially affect the activity of the bi-functional peptidic compound such as previously defined. Modifications include addition and/or deletion and/or substitution of one or more amino acids, and/or replacement of one or more amino acid side chain by a different chemical moiety, and/or protection of the N-terminus, the C-terminus, or one or more of the side chains by a protecting group, and/or introduction of double bonds, and/or cyclization and/or modification of stereospecificity in the amino acids chain. An amino acid may be substituted by an equivalent amino acid without modifying the peptide structure. For example, leucine, valine and isoleucine may be substituted, or aspartic acid and glutamic acid, or glutamine and asparagine, or asparagine by lysine. The substituting amino acids are not limited to those naturally occurring in proteins, such as L-a-amino acids, or their D-isomers. The peptides can be substituted with a variety of moieties such as amino acid mimetics well known to those of skill in the art. An amino acid mimetic as used here is a moiety other than a naturally occurring amino acid that conformationally and functionally serves as a substitute for an amino acid in a polypeptide of the present invention. The individual amino acids can be incorporated in the peptide by a peptide bond or peptide bond mimetic. A peptide bond mimetic of the invention includes peptide backbone modifications well known to those skilled in the art.

These modifications are well known by a person skilled in the art and modified peptides according to the invention may be easily produced and tested.

In a particular embodiment, the present invention relates to a bi-functional peptidic compound characterized in that it comprises:

· a metal-binding peptide (MBP) domain able to bind to a titanium surface, said MBP-domain comprising an amino acid sequence selected from the group consisting of: SVSVGMKPSPRP (MBP-1, SEQ ID N°l), a sequence having at least 80% identity with the sequence SEQ ID N°l , WDPPTLKRPVSP (MBP-2, SEQ ID N°2) and a sequence having at least 80%> identity with the sequence SEQ ID N°2, and

· a cell-specific peptide (CSP) domain comprising an amino acid sequence selected from the group consisting of: PPFLMLLKGSTRFC (CSP-1, SEQ ID N°3), a sequence having at least 80% identity with sequence SEQ ID N°3, SWELYYPLRANL (CSP-2, SEQ ID N°4) and a sequence having at least 80%> identity with the sequence SEQ ID N°4,

wherein said MBP and CSP are linked by a linker.

In an embodiment, a bi-functional peptidic compound according to the invention comprises a MBP domain comprising a sequence having at least 80% identity, preferably at least 90%> identity with sequence SEQ ID N°l, or having the sequence SEQ ID N°1.

In another embodiment, a bi-functional peptidic compound according to the invention comprises a MBP domain comprising a sequence having at least 80% identity, preferably at least 90%> identity with sequence SEQ ID N°2, or having the sequence SEQ ID N°2.

In another embodiment, the present invention relates to a bi-functional peptidic compound according to the invention comprising a CSP domain comprising an amino acid sequence having at least 80% identity, preferably 90%> identity, with sequence SEQ ID N°3, or having the sequence SEQ ID N°3.

In another embodiment, the present invention relates to a bi-functional peptidic compound according to the invention comprising a CSP domain comprising an amino acid sequence having at least 80%> identity, preferably 90%> identity, with sequence SEQ ID N°4, or having the sequence SEQ ID N°4.

In a particular embodiment, a peptide according to the invention comprises a MPB domain and a CSP domain, wherein said MBP and CSP are linked by a peptide linker. A bi-functional peptide according to this embodiment of the invention is defined as a metal-cell specific peptide (MCSP).

In a particular embodiment, a peptide according to the invention comprises from 20 to 50 amino acids, preferably from 20 to 40 amino acids, preferably from 25 to 35 amino acids, preferably from 25 to 30 amino acids, even more preferably consists of 27 amino acids.

In a particular embodiment, the invention relates to a peptidic compound comprising a metal-binding peptide and a cell-specific peptide, linked by a linker, which is adapted for the functionalization of a biomaterial.

In a particular embodiment, the invention relates to a peptidic compound comprising a MBP-domain and a CSP-domain, linked by a linker, wherein said peptide is modified by any standard biochemical procedure well known by a person skilled in the art, in order to improve its biochemical characteristics, in particular its stability.

In a particular embodiment, a peptide according to the invention comprises a metal-binding peptide and a cell-specific peptide, linked by a peptide linker, wherein said MBP domain has the sequence SEQ ID N°l, or an amino acid sequence having at least 80%> identity, preferably at least 90%> identity with sequence SEQ ID N°l, and said

CSP domain has the sequence SEQ ID N°4, or an amino acid sequence having at least

80% identity, preferably at least 90% identity with sequence SEQ ID N°4.

In a particular embodiment, a peptide according to the invention comprises a metal-binding peptide and a cell-specific peptide, linked by a peptide linker, wherein said MBP domain has the sequence SEQ ID N°l and said CSP domain has the sequence

SEQ ID N°4. In another particular embodiment, a peptide according to the invention comprises a MBP domain and a CSP domain, linked by a peptide linker, wherein said peptide linker comprises, or consists of,a short amino-acid sequence of 1 , 2, 3, 4 or 5 amino acids residues in length. In a bi-functional peptide according to the invention, the linker is designed to facilitate the chemical immobilization of the MBP domain to the inorganic substrate and the binding of the CSP domain to the cells. In a particular embodiment, a bifunctional peptide according to the invention comprises, or consists of, 1, 2, 3, 4 or 5 amino acids chosen among any amino acids. In a particular embodiment,the peptide linker is a flexible linker. In another particular embodiment, the peptide linker is a rigid linker. In a particular embodiment, the peptide linker comprises at least one Gly, Ser, Arg, Leu, Cys, and/or Lys amino acids. In another embodiment the peptide linker comprises Gly and Ser amino acids, or is defined as a glycine-rich linker or a serine -rich linker.

In a more particular embodiment of the invention, a peptide according to the invention comprises a peptide linker which exclusively comprises Gly amino acids. In a particular embodiment, in a bifunctional peptide according to the invention, the linker consists of three Gly amino acids.

In a more particular embodiment, a peptide according to the invention comprises a metal-binding peptide and a cell-specific peptide, linked by a peptide linker, wherein said peptide comprises an amino acid sequence selected from the group consisting of:

- S VS VGMKPSPRP GGGPPFLMLLKGSTRFC (MCSP-1, SEQ ID N°5), and an amino acid sequence comprising at least 80% identity, preferably at least 90%, with the sequence SEQ ID N°5,

- SVS VGMKPSPRP GGGS WELY YPLRANL (MCSP-2, SEQ ID N°6), and an amino acid sequence comprising at least 80%> identity, preferably at least 90%>, with the sequence SEQ ID N°6,

- WDPPTLKRPVSP GGGPPFLMLLKGSTRFC (MCSP-3, SEQ ID N°7), and an amino acid sequence comprising at least 80%> identity, preferably at least 90%>, with the sequence SEQ ID N°7, - WDPPTLKRPVSPGGGSWELYYPLRANL (MCSP-4, SEQ ID N°8), and an amino acid sequence comprising at least 80% identity, preferably at least 90%, with the sequence SEQ ID N°8. The following table 2 summarizes the peptide sequences.

Table 2

In an even more particular embodiment, a bi-functional peptide according to the invention comprises an amino acid sequence selected from the group consisting of: SEQ ID N°5, SEQ ID N°6, SEQ ID N°7 and SEQ ID N°8.

In a more particular embodiment, a bi-functional peptide according to the invention has an amino acid sequence selected from the group consisting of: SEQ ID N°5, SEQ ID N°6, SEQ ID N°7, and SEQ ID N°8.

In a more particular embodiment, a peptide according to the invention has the sequence SEQ ID N°6.

In another aspect, the present invention relates to a composition for the functionalization of a biomaterial compound, said composition comprising at least a bi- functional peptide according to the invention. A composition according to the invention is adapted for the functionalization of a biomaterial intended to be implanted or administered to a host, in particular to a mammal host and more particularly to an animal or a human host.

A composition according to the invention consists preferably in a liquid phase comprising a sterile buffer. In a composition according to the invention, the buffer is chosen among buffers well known by a person skilled in the art, and in particular Dulbecco's Phosphate Buffered Saline (DPBS - Sigma Aldrich) without calcium chloride and magnesium chloride. This buffer has a neutral pH and the following chemical composition: Potassium Phosphate Monobasic 0.20 g/L; Potassium Chloride 0.20 g/L; Sodium Chloride 8.00 g/L; Sodium Phosphate Dibasic [Anhydrous] 1.15 g/L. The buffer was chosen according the instruction for dilution of lyophilizated sterile peptides, like the most available solution for cell culture application.

In a more particular embodiment, a composition according to the invention comprises at least one a peptide comprising a metal-binding peptide and a cell-specific peptide, linked by a peptide linker wherein the peptide concentration is comprised between about 0.001 mM and about 1 mM, more preferably between about 0.01 mM and about 0,2 mM, and more preferably is of about 0,1 mM, wherein "about" designates a 10% variation of said concentration value.

The present invention also relates to an inorganic substrate functionalized by adsorption onto its surface of a peptide according to the invention.

By "functionalization" it is intended the association of said inorganic substrate with a peptide according to the invention resulting in the adsorption of said peptide onto the surface of said inorganic substrate.

The present invention also relates to an inorganic substrate functionalized by adsorption onto its surface of a peptide according to the invention, wherein said inorganic substrate is a biomaterial compound.

By "biomaterial compound" it is intended a material compatible with biological tissues and organisms.

In a particular embodiment, a biomaterial compound functionalized by adsorption onto its surface of at least one peptide according to the invention is chosen in the group consisting of: a microparticle, a nanoparticle, a tube, a nanotube, or a biomaterial incorporating thereof. In a more particular embodiment, a microparticle, a nanoparticle, a tube, a nanotube, which surface has been functionalized with a peptide according to the invention, may be part of a self-assembled structure or of a complex material.

In another particular embodiment, a biomaterial compound according to the invention is a medical device, preferably a medical device for implantation. In a particular embodiment, a biomaterial compound or a medical device according to the invention is a part of a multi-component material.

In a more particular embodiment, a biomaterial compound according to the invention is a medical device chosen in the group consisting of: an implant, a dental implant, an implant for maxilo-facial surgery, a biosensor, a balloon, a stent, a shunt, a catheter, a myocardial plug, a pacemaker lead, a dialysis access graft, a heart valve, a pump, a prosthesis, an electronic device, an optic device, or a functional part thereof.

In a more particular embodiment, a biomaterial compound according to the invention is chosen in the group consisting of: a dental implant, the abutment screw of a two-parts dental implant or the cervical zone, or collar, of a monoblock implant.

In a particular embodiment, the invention relates to a biomaterial compound functionalized by a peptide according to the invention, wherein said biomaterial compound is made of a material chosen in the group consisting of: titanium, a titanium alloy, surgical stainless steel, gold, carbon, cobalt, platinum, silicone, chrome and a chrome alloy.

In a particular embodiment, the invention relates to a biomaterial compound functionalized by a peptide according to the invention, wherein the surface of said biomaterial compound is made of an inorganic material, said inorganic material being preferably chosen in the group consisting of: titanium, a titanium alloy, surgical stainless steel, gold, carbon, cobalt, platinum, silicone, chrome and a chrome alloy.

In a more particular embodiment, the invention relates to a biomaterial compound according to the invention, wherein the surface of said biomaterial compound is made of a titanium alloy, and preferably of T1 ₆ AI ₄ V.

In another particular embodiment, the invention relates to a biomaterial compound according to the invention, wherein the surface of said biomaterial compound is made of silicone (Si), and preferably of porous silicone. In a more particular embodiment, the invention relates to a Si nano-particle or to a Si micro- particle, which surface has been functionalized with a peptide according to the invention, and which exhibit a better solubility, stability and molecular recognition properties than the non-functionalized Si nano-particle or to a Si micro-particle. This functionalized Si nano-particle or to a Si micro-particle may be part of a self-assembled structure, or part of a complex material.

In a particular embodiment, the present invention relates to a dental implant functionalized with a peptide comprising a sequence chosen in the group consisting of: the sequence SEQ ID N°6, a sequence having at least 80% identity with SEQ ID N°6, and a sequence having at least 90% identity with SEQ ID N°6.

In a more particular embodiment, the invention relates to a titanium dental implant, or to a T1 ₆ AI ₄ V dental implant functionalized with a peptide comprising the sequence SEQ ID N°6.

In a more particular embodiment, the invention relates to a biomaterial compound associated with a peptide according to the invention, said biomaterial compound being characterized in that the cell adhesion force on epithelial cells of said biomaterial compound is comprised between about 0,1 and about 10 nN, preferably between about 0,5 and about 5 nN. In a more particular embodiment, the cell adhesion force on keratinocytes of said biomaterial compound is comprised between about 0,1 and about 10 nN, preferably between about 0,5 and about 5 nN, wherein "about" designates a 10%> variation of said value.

In another embodiment, the present invention also relates to a method for the functionalization of a peptide according to the invention with a biomaterial compound, said method comprising a step of contacting said biomaterial compound with a composition comprising a at least one peptide according to the invention. Functionalization of a biomaterial compound with a peptide according to the invention is obtained by adsorption of said peptide onto the surface of said biomaterial. A method for the functionalization of the surface of a biomaterial compound according to the invention may comprise a step of contacting said biomaterial compound according to the invention by any method known by a person skilled in the art, such as, for example spraying, coating or printing a peptide on the surface. This functionalization can be performed for example by dipping said biomaterial in a composition according to the invention for a time sufficient to ensure the adsorption of the peptide onto the surface of said biomaterial, such as for example 2 hours, then washed. Biomaterial peptide functionalization may also be performed by any method known by a person skilled to contact said biomaterial with a phase comprising a peptide according to the invention.

In another aspect, the present invention also relates to the use of a peptide according to the invention for the functionalization of a biomaterial compound. In another aspect, the present invention also relates to the use of a composition according to the invention for the functionalization of a biomaterial compound.

In another embodiment, the present invention relates to the use of a peptide comprising the amino acid sequence SEQ ID N°6, or the use of a composition a peptide comprising the amino acid sequence SEQ ID N°6, for the functionalization of a biomaterial compound.

In a more particular embodiment, the present invention relates to the use of a peptide comprising the amino acid sequence SEQ ID N°6 for the functionalization of a dental implant, or a part of a dental implant.

In another particular embodiment, the present invention relates to a kit for the functionalization of a biomedical compound, said kit comprising: a peptide according to the invention or a composition according to the invention, and at least one compound chosen in the group consisting of: buffers, positive control and instructions for use.

The following examples are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified. LEGENDS OF THE FIGURES

Figures 1A to ID represent the MALDl-TOF spectra of MBP-1 on titanium and on titanium-alloy Ti ₆ Al ₄ V (Figures 1A and IB) and the MALDl-TOF spectra of MBP-2 on titanium and on titanium-alloy Ti ₆ Al ₄ V (Figures 1C and ID).

Figures 2A and 2B represent the force spectroscopy of metal binding peptides affinity. Figure 2A represents the typical adhesion force curve measured with AFM, wherein the force (expressed in nN) is a function of indentation (in microm). The black curve represents the retracting curve (pulling). The inset shows a schematic representation of the peptide-functionalized tip, wherein the biotinylated peptide binds to streptavidin which in turns binds to biotin at the surface of the tip.

Figure 2B represents the measured adhesion force, in pN, of MBP-1 (black box) and MBP-2 (white box) on Ti ₆ Al ₄ V (left part of the figure) and on Ti (right part of the figure).

Figure 3 represents a force-indentation curve showing the typical unbinding of a single keratinocyte from the Ti surface, as measured by AFM, wherein the force (expressed in nN) is a function of indentation. The black curve represents the retracting curve (pulling). Arrows represent some adhesion events preceded by a force plateau. The inset shows an image of a keratinocyte cell adsorbed on the lip-less AFM cantilever.

Figures 4A to 4D represent a single-cell force spectroscopy analysis. Figure 4A represents, from left to right, the single cell adhesion forces on bare and functionalized (MCSP-1, MCSP-2, MCSP-3 and MCSP-4) Ti surfaces, measured with the same cell. Figure 4B represents the single cell adhesion forces on bare and functionalized (MCSP- 1, MCSP-2, MCSP-3 and MCSP-4) Ti ₆ Al ₄ V surfaces, measured with the same cell. Figure 4C represents the influence of the substrate to single cell adhesion forces on non- functionalized and functionalized (MCSP-2 and MCSP-4) Ti and Ti ₆ Al ₄ V surfaces. Figure 4D represents the influence of the substrate to single cell adhesion forces on non- functionalized and functionalized (MCSP-1 and MCSP-3) Ti and Ti ₆ Al ₄ V surfaces. Figure 5 represents variation of surface roughness on metal surfaces, on titanium (clear bars) and Ti ₆ Al ₄ V (dark bars), the RMS is respectively represented, from left to right, for bare surface, functionalized surface with MCSP-1, MCSP-2, MCSP-3 or MCSP-4. Figure 6 represents the results of single cell force spectroscopy analysis before and after BSA, wherein adhesion force (nN) is indicated for the following surfaces, from left to right: Ti, Ti ₆ Al ₄ V, Ti/MCSP-2, Ti ₆ Al ₄ V /MCSP-2, Ti/MCSP-2/BSA, Ti ₆ Al ₄ V /MCSP-2/BSA.

Figure 7 is a histogram representation of cell adhesion on functionalized surfaces in a para-nitrophenil phosphate (pNPP) cellular test. The relative adhesion, expressed in arbitrary units, is represented for Ti (white bars) and Ti ₆ Al ₄ V (black bars) surface, wherein, from left to right, the surface are bare or functionalized with, from left to right, MCSP-1, MCSP-2, MCSP-3 or MCSP-4.

Figures 8 A-D represent Epi-fluorescent microscopy of oral keratinocyte cells labeled with cell-specific peptides; fluorescent microscopy analysis of oral keratinocyte cells labeled with SWELYYPLRANL amino-acid sequence; EABA (Endogenous Actine Biotine Activity) suppressed cells without peptides; Figure 8A represents fluorescent microscopy image (bar = 10(^m);Figure 8B represents black and white (BTW) image of the green channel of the fluorescent image; Figure 8C represents grey color distribution in each particle in the BTW image; Figure 8D represents histograms of the mean grey color distribution in BTW image.

Figures 9 A-D represent Epi-fluorescent microscopy of oral keratinocyte cells labeled with cell-specific peptides; fluorescent microscopy analysis of oral keratinocyte cells labeled with EWMIHYDSALTS amino-acid sequence; Figure 9A represents fluorescent microscopy image (bar = ΙΟΟμιη); Figure 9B represents black and white (BTW) image of the green channel of the fluorescent image; Figure 9C represents grey color distribution in each particle in the BTW image; Figure 9D represents: histograms of the mean grey color distribution in BTW image.

Figures 10 A-D represent Epi-fluorescent microscopy of oral keratinocyte cells labeled with cell-specific peptides; fluorescent microscopy analysis of oral keratinocyte cells labeled with the peptide having the SWTWHFPESPPP amino-acid sequence; Figure 10 A represents fluorescent microscopy image (bar = ΙΟΟμιη); Figure 10 B represent a black and white (BTW) image of the green channel of the fluorescent image; Figure 10 C represents a grey color distribution in each particle in the BTW image; Figure 10 D represents histogram of the mean grey color distribution in BTW image.

Figures 11 A-D represent Epi-fluorescent microscopy of oral keratinocyte cells labeled with cell-specific peptides; fluorescent microscopy analysis of EABA suppressed cells in the absence of peptides; Figure 11 A represents fluorescent microscopy image (bar = ΙΟΟμιη); Figure 11 B represents black and wait (BTW) image of the green channel of the fluorescent image; Figure 11 C represents grey color distribution in each particle in the BTW image; Figure 11 D represents histograms of the mean grey color distribution in BTW image. EXAMPLES

Example 1: Selection and surface affinity characterization of metal binding peptides (MBP)

A. Experimental conditions

Substrate preparation prior cell incubation

Ti and T1 ₆ AI ₄ V discs (d=15 mm. and thickness 1 mm) were polished on carbure disks and using diamond pastes (6 μιη, 1 μιη and 0.25 μιη) with a polishing machine (Escil, Lyon, France). Specimens were finally thoroughly cleaned in sodium dodecyl sulfate 0.1M (Sigma Aldrich), hydrochloric acid 0.1 % (Sigma Aldrich) and finally ultra-clean water (Milli-Q; 18 M cm; Merck Millipore; KGaA, Darmstadt, Germany) at 22°C with an ultrasound bath for 5 min. For cell incubation all surfaces were cleaned in a 70% alcohol bath for 10 min. Finally the samples were washed tree times and stored in PBS (Gibco®, Invitrogen, Carlsbad, CA, USA) under sterile conditions. Polishing and cleanup procedures were repeated before every manipulation. Four bi-functional - metal binding cell specific peptides (MCSPs) were synthetized (MilleGen, Toulouse, France) with purity higher than 80%. Titanium and T1 ₆ AI ₄ V surfaces were incubated in 100 μΜ PBS solutions of MCSPs for 2 hours then washed tree times with PBS (Invitrogen, Carlsbad, CA, USA) before cell incubation.

Oral keratinocyte cells

Cells from non-tumoural, immortalized oral keratinocyte cell line, TERT-2 OKF-6 (BWH Cell Culture and Microscopy Core, USA) were cultivated in defined keratinocyte serum-free medium (KSFM; Gibco®, Invitrogen, Carlsbad, CA, USA), supplemented with: CaC12 (Sigma Aldrich), 0.25 μg Bovine Pituitary Extract (BP A), 0.2 ng/ml epithelial growth factor (EGF; Gibco®, Invitrogen, Carlsbad, CA, USA), 0.3 mM, 10% Pen Strep X 100 (Peniciline - 10 000 Unit/ml, Streptomicine - 10 000 mg/ml) (Gibco®, Invitrogen, Carlsbad, CA, USA). The medium was changed every two days until cells were used. For all experiments cells from 7 to 9 population number were used. After 90%> of cell confluence, the cells were detached with 0.05%> Trypsin-EDTA (Gibco®, Invitrogen, Carlsbad, CA, USA) for 5 min.

Phage display selection of surface binding peptides An Ml 3 bacteriophage library (PhD- 12 PD Peptide Library Kit™) supplied by New England Biolabs (Beverly, USA) in phosphate -buffered saline solution containing 0.1% TWEEN-20 (PBST; Sigma Aldrich) was exposed to Ti and T1 ₆ AI ₄ V samples. After rocking for 1 h at room temperature, the Ti and Ti-alloy surfaces were thoroughly washed with PBST to rinse off unbound phages. Bound phages were then eluted from the surface under acidic conditions (glycine -HC1 pH 2.2, 10 min), which disrupt the interaction between the displayed peptide and the target. Before elution, target wells were changed to prevent elution of phages bound to the plastic walls. After neutralization with Tris-HCL (pH 9.1), the eluted phages were infected into the bacterial host strain Escherichia coli ER2738 and thereby amplified. After three to six rounds of biopanning, monoclonal phage populations were selected and analyzed individually. Finally, ten phages were selected and amplified from each sample, followed by the extraction of their DNA that will define the genetic code of the expressed peptide.

MALDI-TOF/TOF analysis

Ti and T1 ₆ AI ₄ V surfaces were incubated in 100 μΜ solution of MBP-1 peptide (SEQ ID N°l) or MBP-2 peptide (SEQ ID N°2) for 2 hours. Samples were rinsed thoroughly, either with a hydrophilic solution: ultra clean water (Milli-Q; 18 M_ cm; Merck Millipore; KGaA, Darmstadt, Germany), a hydrophobic solution: acetonitrile 100% (Sigma - Aldrich) or an ionic solution (NaCl, 1M; Sigma - Aldrich). The presence of the peptides on the dry surfaces was identified with MALDI TOF/TOF spectrometry. Samples were analyzed using a 4800 Plus MALDI-tandem time-of-flight system (MALDI-TOF/TOF) Proteomics Analyzer (Applied Biosystems, Foster City, USA) in positive reflector ion mode using a 20-kV acceleration voltage. The YAG laser was operated at a 200-Hz firing rate with a wavelength of 355 nm. Mass spectrometry spectra were acquired for each measure using 1500 laser shots. All acquired spectra of samples were processed using 4000 Series ExplorerTM software (Applied Biosystems, Foster City, USA) in default mode.

The peptide was identified by searching in the Swiss-Prot database using Protein Pilot TM 2.0 software (Applied Biosystems, Foster City, USA) or Protein Prospector. The ExPASy database was used to calculate the monoisotopic theoretical mass of the peptide. Atomic force microscopy (AFM)

AFM measurements were carried out with an Asylum MFP-3D head and controller (Asylum Research, Santa Barbara, CA), mounted on an Olympus inverted microscope. Height images were recorded in tapping mode and in liquid at room temperature. Typically, 512 x 512 point scans were taken at a scan rate of 1 Hz per line. Both trace and retrace images were recorded and compared. Force Measurements by Atomic Force Microscopy. Relative binding strengths of peptides onto Ti and T1 ₆ AI ₄ V surfaces were measured in contact mode and in a liquid medium (PBS pH 7.4) with a functionalized tip. Force measurements were taken at constant loading rates (vertical piezo-velocity of 1 μητ/s). The spring constant of the tip was calibrated in the presence of PBS solution by the thermal fluctuation method and found to be about 18pN/nm. For tip functionalization, the ultrasoft AFM cantilever tips (Biolever-Olympus) were rinsed with copious amounts of Milli-Q water and then dried. In the next step, tip functionalization was performed: the AFM cantilever tips were incubated in 1 μg/ml biotinylated bovine serum albumin (BSA; Sigma-Aldrich) solution in PBST, pH 7.0, at room temperature overnight, and then the tip was incubated for 30 min in 100 μg mL-1 streptavidin in PBST, and finally in BSA (1%) for 1 h to block the nonspecific binding sites. Thorough rinsing separated all the steps. Biotinilated peptides were fixed on the tip prior to each measurement. Single-cell force spectroscopy (SCFS): Tipples cantilevers (MikroMasch, Tallinn, Estonia) were used for cellimplant surfaces adhesion measurements. Determination of the spring constant for each cantilever was performed by thermal calibration, resulting in 0.03 N/m. Individual oral keratinocyte cells were attached to a surface activated tipless cantilever using the protocol of Zhang et al. 2006 35 to obtain Concanavalin-A (Con A; Sigma-Aldrich) mediated linkage. All measurements were carried out with the tip and the attached cell immersed in complemented KSF medium (Gibco®, Invitrogen, Carlsbad, CA, USA) at 30°C and within 3 h, allowing us the possibility to compere the force adhesion between different types of surfaces with the same cell. The measurements were taken in five different point of every surface. Force measurements were carried out with the same loading rate οΐΐ ταΐ^,. Specificity test of MCSP-2 cell adhesion

MCSP-2 functionalized surfaces were incubated in bovine serum albumin solution (lmg/ml in PBS) for 10 min. Single cell force spectroscopy measurement on a BSA treated surfaces was accomplished as previously described.

Short time cell adhesion test

Confluent cells were washed with PBS and detached with 0.05% Trypsin-EDTA for 5 min at 37 C°. Cells were then resuspended in supplemented KSFM, and 5X105 cells per well was incubated with the un-functionalized and functionalized Ti and T1 ₆ AI ₄ V surfaces. The samples were incubated at 37 C° under a 5% C02 humidified atmosphere for four hours. At the end of the incubation, the cells were washed three times with PBS and lysed with 500 μΐ, of the acid phosphatase lysing buffer (0.1 M sodium acetate, 0.1% Triton X-100, pH 5.5), supplemented with 1 mg/mL of pNPP (para-ntropheny lphosphate, Sigma- Aldrich). After 1 h incubation at 37 C°, the reaction was stopped by the addition of 50 μΐ _^ of 1 N NaOH (Sigma-Aldrich) for 30 min at room temperature. The yellow colorimetric reaction was measured by a microtiter plate reader (EL-800 Universal Microplate Reader, BioTec Instruments INC Vermont, US.) at 405 nm. To determinate the concentration of adherent cells, a a linear relationship was used between the percentage of adhering cells and the light absorption yellow-coloured solution due to para-nitrophenyl phosphate coloration.

Statistical analysis

Data analysis was performed by means of SAS software. Generalized Linear Model (GLM procedure) was used to test the two main effects: implant material (Ti vs. T1 ₆ AI ₄ V), and surface functionalization with different peptides. Multiple mean comparisons were performed with least square means procedure. Root square transformation was applied to the data to obtain a Gaussian distribution.

B. Results

Selection of metal binding peptides was achieved via phage display technology, by performing three biopanning rounds against T1 ₆ AI ₄ V surface. After the third round the SVSVGMKPSPRP (MBP-1) peptide sequence was expressed by 37.5% of the phages. Table 3 represents the percentage of peptide expression in phage display. Peptides Frequency (round 3) Frequency (round 4)

WDPPTLKRPVSP (SEQ ID N°2) 12.5% 40%

LPSHHTPKWGLS (SEQ ID N°l 1) 12.5% 20%

SVSVGMKPSPRP (SEQ ID N°l) 37.5% 20%

GTLGGDYMKYLS (SEQ ID N°12) 12.5% 20%

LPIDHAATHESR (SEQ ID N°13) 12.5% 0%

HPAWALGKLNVE (SEQ ID N°14) 12,5 % 0 %

Table 3

MBP-1 peptide has a great potential as a linker to functionalize metallic surfaces if specificity is not a key factor. To select a second peptide the 6 bacteriophage clones (every clone have has been amplified separately and a bank of 6 bacteriophages was then created by mixing the clones) were used for the fourth biopanning round. In the fourth round the WDPPTLKRPVSP sequence (MBP-2) was expressed by 40% of the phages. Hence MBP-1 and MBP-2 were further chosen as the metal binding part of the bi-functional peptides.

The affinity of both MBPs to Ti and T1 ₆ AI ₄ V was tested by mass spectrometry and force spectroscopy. After incubation of MBP-1 or MBP-2 peptides with Ti and T1 ₆ AI ₄ V surfaces, their resistance to three different rinsing conditions using hydrophilic, hydrophobic and ionic solutions (see methods section) was evaluated. The presence of peptides on the surfaces after rinsing was identified with MALDI TOF/TOF spectrometry. Spectra after acetonitrile rinsing are showed on Figures 1A to ID. MBP-1 peptide was identified on both Ti and T1 ₆ AI ₄ V surfaces after the three types of rinsing. Table 4 presents the MALDI/TOF-TOF detection of MBP-1 and MBP-2 peptides on Ti and T1 ₆ AI ₄ V surfaces after rinsing with acetonitrile, water and 1M NaCl.

Table 4 The obtained value of mass-to-charge ratio (m/z) for MBP-1 was 1239.6 in agreement with the theoretical mass (table 3) and on both metals was identified a small peak at m/z of 1255.6 corresponding to the oxidized form of the peptide. Table 5 presents the physico-chemical characteristics of the peptides.

Table 5

Characteristics were calculated using compute programs on http://expasy.org, of the selected peptides for Ti and Ti6A141V. a) MW = the theoretical monoisotopic molecular weights; b) pi = the isoelectric point; c) II = the instability index of a peptide: II < 40 predicts a stable, II > 40 predicts an unstable peptide; d) AI = the aliphatic index; e) Hydropathicity = calculated as the sum of hydropathy of all the amino acids, divided by the number of residues in the sequence. The larger the number, the more hydrophobic is the amino acid or the peptide.

The slight differences in the determined peptides mass on the different materials were due to the thickness variation of the samples, which induced small differences in time of flight. The sequence of the MBP-1 peptide presents a hydrophobic first half and a hydrophilic second half. The fact that the peptide remains on the surface after acetonitrile rinsing, known to break the hydrophobic links, suggests alternative binding. The adhesion resists also to high ionic strength solvent indicating that electrostatic interactions are not critical. The second metal binding peptide (MBP-2) was not detected after rinsing with hydrophilic (water) or ionic solutions (1M NaCl). The obtained value of mass-to-charge ratio (m/z) for MBP-2 was 1391.6 and correspond to the theoretical [M + H] + mass (Figures 1C and ID). The MBP-2 peptide contains two positively charged (K, R) amino acids and one negatively charged (D) amino acid, hence his total charge is twofold lower than the charge of MBP-1 (table 3). This may explain its lower electrostatic interaction with the negatively charged metal surfaces indicated by its rinsing with 1M NaCl. The mass-spectrometry spectra of MBP-2 resisting to acetonitrile rinsing (Figure 1C and ID) reveal two peaks at m/z 1406 and m/z 1423.6 suggesting the presence of oxygen ions in a peptide structure. One can remark that in ten sequences with high affinity to the T1 ₆ AI ₄ V, D, C and S amino acids occur more frequently. Previously, general oxide binding motifs -SPS- and -SGS-were proposed trough molecular modeling. Comparing MBP-1 and MBP-2 sequences it is difficult to conclude on a general motif for metal oxide adhesion.

To have a clearer view on MBP-1 and MBP-2 affinity to the Ti metals, force spectroscopy study was conducted by AFM. Monitoring unbinding processes of adsorbed molecules under external stress leads to quantification of adhesion forces. The adhesion forces measured between MBPs and Ti and T1 ₆ AI ₄ V surfaces are presented in Figures 2A and 2B. MBP-1 adhesion force on T1 ₆ AI ₄ V and Ti surfaces are 67.07 ± 1.34 pN and 65.42 ± 2.48 pN, respectively. MBP-2 adhesion force on both surfaces are 109.76 ± 2.62 pN and 134.61 ± 2.65 pN.

MBP-2 peptide reveals statistically higher adhesion forces to both metals. Adhesion of MBP-2 on T1 ₆ AI ₄ V is almost twofold stronger than the adhesion force of MBP-1. For comparison the strength of osteopontine and ανβ3 integrin bonds is 50 ± 2 pN 29, ICAM-1 and Anti-ICAM-1 antibody bond is 100 ± 50 pN30, and cadherin- mediated cell-cell interaction has a minimal binding force in the range of 50 pN. The highly specific and strongest non-covalent bond is the biotin-streptavidin interaction ranging from 250 to 320 pN according to the experimental conditions. Therefore, it can be concluded that the interactions of MBP-1 and MBP-2 with the two metal surfaces are in the range of antigen-antibody forces.

Example 2: Single-cell force spectroscopy (SCFS) on implant surfaces

Metal-cell specific peptides (MCSP) were designed by linking of one MBP sequence and one cell specific peptide (CSP) sequence via tree glycine residues, as summarized in table 2.

Single cell adhesion spectroscopy (SCFS) measurements were performed in atomic force microscope to investigate the capacity of four bi-functional peptides to increase oral keratinocytes adhesion on both Ti and T1 ₆ AI ₄ V metals. Oral keratinocyte cells were attached to tip-less cantilevers by means of biotin-concanavalin-A. A typical force-indentation curve recorded during the SCFS measurements is displayed in Figure 3. The loading rate was set to 2μηι/8. After reaching the maximal loading force, the contact was maintained for 5s (at a constant piezo z position). Small adhesion events were recorded in the retracting curve (black curve) even at long pulling distances. Some of them were preceded by a force plateau that could suggest membrane tethers (arrows). These small events were not taken into account in our analysis. The inset shows an image of a keratinocyte cell adsorbed on the tip-less AFM cantilever. Images of the cantilever were always recorded before and after each set of measurements and compared. No significant difference was observed between them. Scale bar represents 18μιη.

The relatively short contact time of 5 s between cell and substrate means that no information is recorded about how the cell responds to the surface on a longer time scale, thus excluding the effects of, for example of changes in protein expression and limiting our study to the analysis of proteins and protein complexes that are already present at the cell membrane as well as to the study of mechanical properties of the cell. The cell adhesion forces were registered towards the bare Ti surface and after its functionalization with the peptides (Figure 4A). MCSP-2 and MCSP-4 functionalized surfaces presented higher cell adhesion force than MCSP-1 and MCSP-3 peptides. A significant difference was found between cells adhesion against MCSP-2 and MCSP-4 functionalized Ti compared to the non-functionalized Ti surface. Adhesion force values are significantly higher for MCSP-2 and MCSP-4 on both surfaces (Figures 4A and 4B).

In order to directly compare the influence of peptide functionalization on the cell adhesion, force measurements were performed on both bare Ti and T1 ₆ AI ₄ V surfaces and after their functionalization with MCSP-2 and MCSP-4 peptides (Figure 4C). The highest cell adhesion force was found against Ti and T1 ₆ AI ₄ V surfaces functionalized with MCSP-2, without significant difference between the two peptide functionalized metals. This result confirmed that the MCSP-2 peptide assures significantly higher adhesion to both metals compared to the naked implant surfaces. Significantly higher cell adhesion was found between un-functionalized surface and Ti/ MCSP-4, but not between Ti ₆ Al ₄ V and Ti ₆ Al ₄ V / MCSP-4 surfaces. The same experiments were performed using MCSP-1 and MCSP-3 peptides for functionalization of both metals (Figure 4D). Results show a significantly higher adhesion against Ti ₆ Al ₄ V functionalized with MCSP-3 compared to the bare Ti ₆ Al ₄ V surfaces.

The MCSP-1 and MCSP-3 peptides have a common cell specific peptide CSP-2 and different MBP's, but they did not demonstrate the same comportment prior to cell adhesion force to metal surfaces. The combination of MBP-1 and CPS-2 (cadherin binding peptide) in MCSP-2 appears to be the most successful peptide configuration for Ti functionalization to promote oral epithelial cell adhesion. The measured cell adhesion forces are of the same order of magnitude as in previous works, however it should be stressed that measured forces are strongly depending on experimental conditions (contact time, loading rate). Mesendodermal cells adhesion forces on substrates coated with fibronectin range from 0.198 - 0.405 nN, for contact times of 1- 5 s and maximal loading forces of 400 pN37. Adhesion forces of Chinese hamster ovary (CHO) cells against collagen matrix, using a maximum loading force of InN and short contact times (5-10 s) are around several hundred pico-newtons. Single cell adhesion forces between murin calvaria pre -osteoblast cells and partially denatured collagen (free RGD - motif) are close to 1 nN after a contact time of 5 s using a loading force of InN. One can note also a difference in cell adhesion forces depending on the cell type that could be explained by the large variability of membrane characteristics between cells.

As previously suggested surface roughness is one of the factors influencing the score of detachment force. The roughness measured in AFM as a root-mean square (RMS) changes dramatically due to the functionalization: for bare Ti surface it was 5.4 nm (Figures 5 A to 5C), and after functionalization with MCSP-2 it increases to 21 nm. Indeed, AFM height images recorded in tapping mode demonstrates the following: Bare Ti surface Rms: 5.4 nm, Ti/ MCSP-2 surface Rms: 21.3 nm, Bare Ti ₆ Al ₄ V surface Rms: 6.02nm and Ti ₆ Al ₄ V /MCSP-2 surface Rms: 13.8 nm. The RMS of bare Ti ₆ Al ₄ V surface increased from 6.0 nm to 13.8 nm after absorption of MCSP-2. RMS increased also for MCSP-3 functionalized Ti surface. AFM height images of metal surfaces after functionalization with MCSP-2 (Figure 4C for Ti; Figure 4D for ΤΪ6Α14) demonstrated similar peptide deposition patterns with agglomerates of about 200 nm. Increasing of surface roughness after MCSP-2- functionalization could explain its higher adhesion performances over other peptides.

To challenge the adhesion effect of MCSP-2, which proved to be the most promising candidate for functionalization of Ti and T1 ₆ AI ₄ V surfaces, cell adhesion forces before and after bovine serum albumin (BSA) adsorption on functionalized MCSP-2 surfaces were compared. BSA is a widely used blocking agent to nonspecific binding by inhibiting hydrophobic and ionic or electrostatic interactions between proteins. Single cell adhesion forces against naked and MCSP-2- functionalized Ti and T1 ₆ AI ₄ V surfaces before and after BSA treatment were recorded with an oral keratinocyte cell (Figure 6). A statistically significant difference in adhesion forces was found towards the non- and functionalized Ti and T1 ₆ AI ₄ V surfaces. After BSA absorption, the cell adhesion against Ti ₆ Al ₄ V /MCSP-2 surfaces decreases drastically to the level of adhesion against the bare T1 ₆ AI ₄ V surface. Significantly higher cell adhesion on Ti/MCSP-2/BSA surface compared to bare Ti after BSA treatment was found (p=0.0001). The albumin is not a cell-specific peptide. His absorption on MCSP- 2-functionalized alloy surface, decrease the cell adhesion force, blocking the nonspecific sites. Contrarily, a significantly higher cell adhesion on Ti/MCSP-2 surface compared to bare Ti after BSA treatment was found. That could be due to presence of free specific cell adhesion cites of MCSP-2 after the BSA absorption. These results show that functionalization with bi-functional peptides enhances adhesion of human gingival epithelial cells Example 3: Short time cell adhesion test on Ti implants

The number of adherent living cells was compared on functionalized and non- functionalized Ti metal surfaces, after four-hour cell incubation, by means of a colorimetric para-nitrophenil phosphate cell viability test (pNPP). This methodology can be also applied for testing the cell-adhesion or more precisely the mechanical resistance of adherent cell to rinsing procedure, on different surfaces. After eliminating non-adherent cells with rinsing, cells attached to the surface were subjected to the acid phosphatase viability test. These pNPP tests complemented the SCFS measurements where was tested the force between one cell and five different positions on the surface, to obtain information about a great number of adherent cells on the whole surface of the implant, four hours after incubation. The results indicate a higher number of adherent cells on Ti surfaces (with or without functionalization) than on the T1 ₆ AI ₄ V surfaces (Figure 7). Surfaces functionalized with MCSP-2 demonstrate a statistically significant higher concentration of adherent cells in comparison to non-functionalized surfaces. Both cell culture test and SCFS results show that the bi-functional MCSP-2 peptide enables the best oral keratinocyte cell adhesion on Ti and T1 ₆ AI ₄ V surfaces. MCSP-2 contains the MBP-1 metal binding peptide and for the cellular part, the cadherin binding peptide with the CSP-2 sequence. MBP-1 peptide makes strong adhesion on both implant surfaces that resisted to hydrophobic, hydrophilic and ionic rinsing procedures. The peptide H-SWELYYPLRANL-NH2 (CSP-2) was described by Devemy and Blaschuk (2009) as the specific binding peptide of E-cadherin ectodomain with high affinity binding of 9.4 mM.43 Only the combination of these two amino acid sequences resulting the MCSP-2, amongst the four proposed bi-functional peptides, provide a favorable spatial configuration to comply its double adhesion function to the Ti bases metals and to the oral epithelial cells. MCSP-2 peptide assures a cell adhesion that is stable four hours after incubation, resisting to the influence of external factors like BSA adsorption. These results show that adhesion is significantly higher on MCSP-2 functionalized surfaces than bare surfaces (p=0.0001). Adhesion is better in all Ti surface than in Ti alloy surfaces four hours after incubation.

Conclusion

The capability of four bi-functional synthetic peptides to increase oral keratinocyte cell adhesion on Ti and T1 ₆ AI ₄ V implant surfaces was characterized. Single cell force spectroscopy and cell culture short time adhesion tests demonstrate that MCSP-2 (MBP1 + CSP2 sequences) increases oral keratinocyte cells adhesion on the implant surfaces. The strong adhesion force and the high adhesion affinity of oral keratinocyte cell on peptide functionalized implant surface are extremely important properties for its clinical applications. Example 4:

For this experiment we chose three amino acid sequences (peptides) which were described as cell adhesion modulating agents and comprise one or more cell adhesion recognition (CAR) sequences. The (CAR) sequence was presented in WO2006/116737 and is defined by the following formula: W-X-L/I/V/F/M/A, wherein X is an amino acid selected from E, T, Y, Q, M, F, D, or L and wherein L/I/V/F/M/A is Leu or He or Val or Phe or Met or Ala.. This sequence according to the above formula is selected from the group consisting of WEL, WTL, WYI, WEF, WQM, WEV, WTV, WYV, WQV, WTM, WQF, WTF, WMI, WDF, WTI, WFI, WQL, WEI, and WLA.

In WO 2006/116737 the authors described a large group of compounds and methods for modulating cadherin-mediated process, and the ability of the peptides having the amino acid sequence EWMIHYDSALTS and SWELYYPLRANL to disrupt SKOV-3 and MCF-7 cell adhesion was demonstrated. These linear peptides EWMIHYDSALTS and SWELYYPLRANL disrupted MCF-7 and SKOV-3 cell adhesion at 1 mg/ml concentration. The two peptides were considered as good candidates for our experiment. From the (CAR) sequence group, we choose a third amino acid sequence as a negative control. The peptide SWTWHFPESPPP was described like less soluble than SWELYYPLRANL in aqueous solutions (Devemy and Blaschuk, 2009) and in the same time the place of (CAR) sequence in SWTWHFPESPPP was changed. The (-WHF-) sequence was not presented like cell adhesion recognition sequence, although its responds to the presented formula. The three peptides were applied with a) cell line of non-tumoral, immortalized oral keratinocyte cells, TERT-2 OKF-6 (BWH Cell Culture and Microscopy Core, USA). Cells lines described in WO 2006/116737 (MCF-7 and SKOV-3) are carcinoma cell lines. MCF-7 cells are a carcinoma cells isolated from human breast adenocarcinoma with estrogen and cytokeratin sensitivities. SKOV-3 cells are a human ovary cancer cells with epithelial-like morphology. Unlike these two cell lines, oral keratinocyte cells TERT-2 OKF-6 are immortalized human epithelial cells with origin from oral mucosa (Dickson et al.). We chose this cell line since the purpose of our experiment was not to select an amino acid sequence that can be applied as an anticancer treatment as in WO 2006/116737. Our aim was to find an adequate amino acid sequence for specific recognition of oral keratmocyte cell surface. Subsequently this sequence will be used in the construction of bi-functional peptides to link keratmocyte cells and titanium.

Material and methods: Materials

C-terminal biotinilated peptides with purity higher than 80% were purchased from Polypeptide Laboratoires (67100 Strasbourg, France). Avidin (product number: A9275), Biotin (product number: B4639), FITC labeled Avidin (product number: A2050) were purchased from Sigma Aldrich (Sigma Aldrich, St. Louis, USA). Cells from non- tumoural, immortalized oral keratmocyte cell line, TERT-2 OKF-6 (BWH Cell Culture and Microscopy Core, USA) were used for all experiment.

Cell labeling with Peptide-biotin/avidin-FITC complex: Before the labeling, the oral keratmocyte cells were incubated for 12 hours at 37 C° under a 5% C02 humidified atmosphere in a 24 well plates in supplemented keratmocyte serum-free medium (KSFM; Gibco®, Invitrogen, Carlsbad, CA, USA). The cell concentration was 2x105 and the cell population 7-9. For suppression of the endogenous avidin-biotin activity (EABA) we used a protocol previously described by Wood et al. (1981). The protocol contains the following steps: 1) PBS incubation and wash x 10 min; 2) Avidin incubation x 20 min; 3) PBS incubation and wash x 5 min; 4) Biotin incubation x 20 min; 5) PBS incubation and wash x 5 min. The described concentrations of Avidin ranging from 0.1-0.01% and biotin ranging from 0.01-0.001% were adequate for use in EABA suppression. After calibration we used a PBS solution for the Avidin with concentration 200 μg/ml and for the biotin 20μg/ml. 6) Incubation with biotinilated peptide x 30 min in concentration 500μg/ml. 7) Subsequent specific staining with Avidin-FITC ^g/ml in PBS at 4°C x 20 min; 8) PBS incubation and wash x 5 min; 9) Fixation with 4% para- formaldehyde solution in PBS; 10) PBS incubation and wash x 5 min. Cell labeling with each peptide was repeated in triplicate. For control group we used EABA suppressed cells without peptides. The specific cell labeling was observed with epi-fluorescent microscopy.

Epi-fluorescent microscopy was realized with an Eclipse TE2000-E (Nikon) Microscope.

Avidin-FITC fluorophore (Ex/Em wavelength - 540-580 nm; Sigma- Aldrich) was detected by the Y-2E/C filter (Excitation (Ex.) 528-553 nm; Dichromatic Mirror (DM): 595 nm, Barrier filter (BA): 600-660 nm; Nikon). Cell observation was realized using CFI Plan Fluor 40/0.66 dry Nikon objective (Nikon, Tokyo, Japan).

Image analysis: The analysis of fluorescent images was realized by Image J sofwere (NIH, Bethesda, MD, USA, http://rsb.info.nih.gov/ij/). The aim was to measure the degree of fluorescence by measuring the degree of the grey color into the images. Using the function "color - split channels" we isolated the green channel of each image. Each green channel image was transformed in black and wait 8 bit image using the function threshold - BTW (image - adjust - threshold - BTW). The histograms for the grey color distribution was created with the function "analyze -histogram". To illustrate the grey color distribution of each particle into the image we use the function "analyze - image plot". Results and discussion:

The results of this experiment are presented in Figures 8 A-D; 9 A-D, 10 A-D and 11 A- D. For each peptide and the control (8 A-D; 9 A-D, 10 A-D, 11 A-D.) we presented epi- fluorescent microscopy and the intensity of the grey color (respectively the fluorescence intensity) after the image treatment (see methods). On Figures 8 A-D for oral keratinocyte cells labeled with SWELYYPLRANL amino-acid sequence we distinguished well limited, fluorescent cells with a high fluorescent intensity. The mean fluorescent intensity of that image was 29± 16 with maximal value at 248. On the image with EWMIHYDSALTS marked cells (Figure 9 A-D), we found zones with large fluorescent agglomerates and zones with unique fluorescent cells with mean image fiuorescence intensity of 32± 19. The values of the mean fluorescence intensity between images (8 A-D) and (9 A-D) were practically equal, but the maximal value of fluorescent intensity of image 8B was lower. One possible explanation of the largest fluorescent agglomerates in figure (9 A-D) is that these are agglomerates from cells and peptides precipitated.

Contrarily the fluorescent intensity of the cells on the third image (Figure 10 A-D) was almost two fold lower. We could distinguish some low fluorescent cells, but that fluorescence was much more weaker than on the figure 8 A. The mean fluorescent intensity of the SWTWHFPESPPP marked cells was near to the control group (cells without peptide) despite the high concentration of fluorescent peptide for labeling (500 μ^πιΐ).

Conclusion:

Within this experiment, using epi-fluorescent microscopy, we compared three amino- acid CAR sequences that could recognize the cadherin receptors on the surface of oral keratinocyte cells. At a concentration of 500 μg/ml, we found that the peptide SWELYYPLRANL was the most adequate candidate between the three peptides and it was chosen for oral keratinocyte surface cell recognition.

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