WO2013131994A2

WO2013131994A2 - Coating for the diagnosis of inflammatory tissues in dental applications

Info

Publication number: WO2013131994A2
Application number: PCT/EP2013/054571
Authority: WO
Inventors: Falko Schlottig; Matthias Schnabelrauch; Lorenz Meinel; Ralf Wyrwa
Original assignee: Thommen Medical AG
Current assignee: Thommen Medical AG
Priority date: 2012-03-08
Filing date: 2013-03-07
Publication date: 2013-09-12
Anticipated expiration: 2014-09-08
Also published as: WO2013131994A3

Description

TITLE

Coating for the diagnosis of inflammatory tissues in dental applications

TECHNICAL FIELD

The present invention relates to a coating, in particular for a dental implant, for the diagnosis of inflammatory tissues in dental applications.

PRIOR ART

A recent consensus meeting has concluded that peri-implantitis occurs around 12 - 40% of implanted teeth. This renders peri-implantits a true threat in today's advancement of implant reliability and performance.

Arguably, the state of the art for diagnosing peri-implant infection and peri-implantitis (most advanced stage of infectious peri-implant complications) followed by most practitioners is bleeding on probing (BOP). The underlying assumption is that bleeding on probing is indicative for inflammation and advanced tissue degradation and, therefore, seen by many as indicative for peri-implantitis in the context of implants. This view is too simplistic. It has been demonstrated that only repeatedly observed absence of bleeding has a high negative predictive value (98%; i.e. the tooth and surrounding tissue are healthy) while presence of bleeding was not prognostic (positive prediction 6%; i.e. useless for prediction of disease state of teeth and surrounding tissue).

Novel tests are available, allowing point of care (PoC) assessment of collagenase expression, including systems such as MMP-8. It has been established, that MMP-8 detected within sulcus fluid is predictive to stratify healthy subjects from patients with inflammatory tissues, i.e. a high prognostic power has been demonstrated for MMP-8. Furthermore, MMP-8 levels return to normal following successful medical intervention, allowing monitoring of therapeutic success within two weeks after treatment had commenced and within the specific context of implant inflammation.

The point of care or PoC technology of measuring MMP-8 levels from sulcus fluid is complex. A test stripe, soaked with sulcus fluid collected by the dentist from the site of interest, is transferred into a complex device within which an ELISA like reaction is facilitated leading to a read-out on MMP-8 activity - in essence, a complex, expensive and difficult to handle system.

There are quite a number of scientific articles dealing with the presence and the levels of metalloproteinase activity as a function of the degree of inflammation of tissue in the mouth. Reference is for example made to Tschesche, Journal of protein chemistry 17 (6) 41, 1998, 549-551; Pozo et al in Journal of periodontal research 40 (3), 2005, 199-207; Kahrmaran et al in Disease markers, 30 (6), 2011, 299-305 as well as Barnett et al in Journal of periodontology, 79 (5), 2008, 819-826.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a new approach for the detection of inflammatory tissue in the dental field.

This and other objects are achieved by the claimed subject matter.

Specifically the present invention in particular relates to a diagnostic coating for identifying the presence of inflammatory tissues in the mouth, in particular in or adjacent to the mandible, the maxilla, an implant or the teeth of a user. In accordance with the present invention, this coating comprises at least one base material or particles embedded and/or attached to said base material; at least one element, such as for example a molecule, attached to said coating base material or particles embedded and/or attached, for the generation of a change in the coating directly detectable (i.e. without additional analytical tools or the like so basically by using at least one of the five senses, i.e. sight - ophthalmoception, hearing - audioception, taste - gustaoception, smell - olfacoception, and touch - tactioception, or a combination thereof) by the patient. In accordance with the invention, the element generates the change upon direct or indirect contact with a marker which is released by inflammatory tissue in response to bacterial mediators. The diagnostic coating is applied to a fixed dental device, in particular it is provided for a dental implant, for a dental abutment, for a dental abutment screw and or for a dental crown. It is applied to this device prior to the insertion of the device into the patient's mouth, and it is there to stay after implantation for the diagnostic purposes thereof.

There are quite a number of prior art documents, reference is made for example to Rios et at in Journal of clinical peridontology 36 (12), 2009, 1011-1017 and Ki vela-Raj amaki et al in clinical oral implants research 14, 2003, 158-165, where certain markers are analysed present in the mouth, however for the analysis purposes body fluid from the mouth has to be taken out and analysed outside of the body. On the other hand WO 02/45661 discloses a mould making composition for forming three-dimensional negative images of dental patients which can be provided with diagnostic additives. The mould making composition is however, in line with its purpose, solid composition which is clearly not suitable, let alone proposed to be used as a chewing gum.

In contrast to prior art approaches, the proposed systems here allow for patient self- monitoring. The chain in this proposal is as follows: In response to tissue degradation, (i) a change for example in the form of a strong bitter taste is released by the system and (ii) this bitter taste can be reported by the patient, (iii) Based on this report, a diagnose can be made and (iv) can lead early on therapy if appropriate.

As pointed out above, peri-implantitis is a true threat in today's advancement of implant reliability and performance. The proposal is addressing this challenge by deploying the human senses, in particular for example the taste / gustatory system, for surveillance of connective tissue degradation, which marks the borderline between gingivitis/mucositis and periodontitis (also called pyorrhea, parodontitis or paradontitis) / peri-implantitis (see also Figure 1). This radically new and easy to use diagnostic tool, identifies and stratifies subjects at risk for development of peri-implantitis, opening a new window of opportunity for medical risk assessment and, therefore, possible intervention at an early stage. This early on detection allows pre-emptive, successful, non-complex and well tolerated treatment. The strategy followed here is disruptive in terms of shifting current point-of- care (PoC; i.e. the practitioner's office) diagnosis to self-monitoring, allowing consultation of one's dentist in diseases stages which are clinically unapparent and within which relatively moderate therapeutic intervention suffice to prevent further destruction of the implant and surrounding tissues and in contrast to more radical interventions necessary at later stages (see also Figure 1).

A completely new diagnosing platform has thus been developed by means of gaining access to the advanced technological capabilities of several contributors.

The disease course of peri-implant infection and its most severe form, peri-implantitis, commences from microbial challenge and biofilm formation. The microbial challenge triggers a defensive host response (release of cytokines and other signals) in response to bacterial mediators e.g. lipopolysaccharides (LPS; component of the cell wall of gram- bacteria), leading to massive infiltration of macrophages (Φ). It is these Φ which are capable of releasing various proteolytic enzymes, including MMPs. Among many other MMPs tested, MMP-8 - also known as type II collagenase - has demonstrated impressive prognostic power to predict clinical progression through enhanced pocket formation, attachment loss, bone resorption, gingival recessions, increased tooth/dental implant mobility and finally tooth dental implant loss. MMP-8 is disrupting the dense tissue collagen network thereby allowing efficient Φ infiltration as a prerequisite for bacteria removal - in other words, MMP-8 activity is directly linked to the first and clinically fully reversible stage of gingival connective tissue destruction (see also Figure 1, second and third boxes from top). MMP-8 upregulation in peri-implant infection is massive as compared to other inflammatory dental disease. For example, MMP-8 levels in sulcus fluid aspirates from healthy sites arbitrarily set to 1 U (pocket depth 3.3 mm; gingival index (GI) 0.6), were elevated in moderate chronic periodontitis to 80 U (pocket depth 5.6 mm; GI 1.6) and 78 U for severe chronic periodontitis (pocket depth 5.9 mm; GI 2.0). In contrast, MMP-8 was upregulated to more than 950 U in patients with peri-implantitis (pocket depth 5.0 mm; GI 2.0) compared to healthy implants (pocket depth 2.4 mm; GI 1.0). Therefore, MMP-8 is an ultra-sensitive, prognostic biomarker for sensing peri-implant infection long before more severe disease states are attained, such as peri-implantitis. The challenge addressed in this proposal is to enable the patient for self-monitoring of MMP-8 activity using the system (conceptually presented in Figure 1) and functionality as outlined in Figure 2.

According to a preferred embodiment of the present invention therefore, the marker inducing the change is a proteolytic enzyme released or upregulated by inflammatory tissue, preferably by macrophages.

The marker inducing the change in accordance with yet another preferred embodiment is most preferably matrix metalloproteinase (MMP), which is preferably activated. Preferably matrix metalloproteinase-8 (MMP-8) activated matrix metalloproteinase-8 (aMMP-8), matrix metalloproteinase-2 (MMP-2), activated matrix metalloproteinase-2 (aMMP-2), matrix metalloproteinase-9 (MMP-9), activated matrix metalloproteinase-9 (aMMP-9), or a combination thereof is the maker of interest triggering the change perceivable the patient carrying the implant.

The gustatory system principally has four primary taste submodalities recognizing sweet, sour, salty, and bitter. Maximal sensitivity is provided for bitter taste and bitter taste can be calibrated for control of inter-patient variability using methods outlined in the European Pharmacopoeia. Sweet taste is more difficult to calibrate among patients but used as a strategy as well in spite of potential challenges for individual calibration of sweet perception. The human tongue offers a fascinating range of sensitivity for tasting sweetness and bitterness, covering five orders of magnitude. Quinine sulfate (bitter) is sensed down to 0.0004 mM, rivaling even our most advanced analytical detectors available today. The artificial sweetener, saccharin, is recognized down to 0.02 mM by the average human. Within the context of this application it is important, that certain short peptides can be typically sensed down 0.05 to 6 mM and this insight is deployed by designing peptide sequences for the system which result in bitter taste following cleavage. By this strategy, the coupling of a flavoring substance can be avoided as the cleaved peptide sequence itself mediates a bitter sensation recognized by the affected patient.

In conclusion, in particular the MMP-8 (System 1) protease sensitive system provides the necessary power to the dentist for early detection and continuous surveillance of peri- implant diseases. The MMP-8 sensitive system recognizes early connective tissue damage. The system provides radically new, easy to use tools to the dentist and patient for early on monitoring of peri-implant diseases with immediate relevance on patient oral health.

The proposed system aims at broad application, including sensing of oral and mucosal alterations following the principle outlined here within for peri-implant infection.

The system is thus radically shifting monitoring of peri-implant infection from assessments involving complex machinery to self -monitoring using the human tongue or the human eye of the user as a sensitive detector. Instead of restricting the monitoring of the oral health status to visits at the dentist, the approach supports frequent self-monitoring such that in case of positive signal, the subject can visit the dentist's office to get a thorough diagnosis. The system (see also Figure 2) is the mode by which clinically unapparent oral diseased tissue becomes detected early on. The main innovation aspects are the following:

• The deployment of human taste sensing / gustatory system for monitoring of inflammatory or other diseases is radically new.

• Connective-tissue damage sensing dental implant system providing a perceivable response for monitoring of earliest signs of peri-implant disease have not been described.

β Coatings with diagnostic features are known but not those which respond to the upregulation of an activated enzyme prognostic for the development of oral diseases.

· Selection of MMP-8 sensitive systems from known sequences which provide the necessary performance to preferentially sense MMP-8 over other proteases in peri-implant disease.

• The correlation of peri-implant disease with MMP-8 elevation as done here controls the information available in literature by data sets to provide a reliable basis for fine-tuning and adapting our system's sensitivity and selectivity to a targeted disease state of the peri-implant zone.

β Modern coupling techniques are deployed to decorate the plastic (spheric system) and/or titanium (implant) surfaces with the peptide sequences, respectively.

The coupled flavoring substances may reduce their potential for (bitter) taste once attached to the peptide sequence. This risk can be mitigated by selecting different coupling sites at the (bitter) tasting molecule, by selecting different (bitter) tasting molecules and by designing bitter tasting peptide sequences, which are known to induce a bitter taste.

Correspondingly therefore in accordance with yet another preferred embodiment the element can on the one hand be a molecule or molecular assembly which, upon direct or indirect contact with the marker undergoes a color change perceivable by the naked eye of the user, and which is embedded or attached to the base material or to particles embedded and/or attached to said base material.

On the other hand the element can be a flavor molecule releasably, preferably releasably covalently, attached to the base material or to particles embedded and/or attached to said base material of the coating.

The flavor molecule can be (releasably) attached to the base material or to particles embedded and/or attached to said base material of the coating by means of a hydrogen bonding or by means of a molecular chain cleavable under direct or indirect contact with the marker. Release is for example possible by lysis of the molecular chain by the marker itself, it is however also possible by means of mechanisms such as agglomeration or attachment of the marker to the linker or another element close by, inducement of a conformational change or the like, corresponding reduction of the binding constant of the flavor molecule to the support and release of the molecule to generate the corresponding taste sensation.

The molecular chain can for example be a polypeptide chain or a sugar chain or a combination thereof. In case of a polypeptide chain, this is preferably constituted of 2-15, most preferably of 3-7 amino acids, and preferably the linker molecule is either directly or indirectly, via an anchoring element, attached to the base material or to particles embedded and/or attached to said base material.

Possible linker sequences to be used as molecular chain cleavable under direct or indirect contact with the marker for the attachment of e.g. a bitter flavor molecule such as quinine, caffeine, theobromine, naringin, sucralose or neotame are given by the following systems, where the cleavage site by the MMP-8 system is indicated by a slash:

Gly-Pro-Gln-Gly/Ile-Ala-Gly-Gln (SEQ-ID1);

Gly-Asn-Gln-Gly/Ile-Ala-Gly-Gln (SEQ-ID 2);

Gly-Leu-Gln-Gly/He-Ala-Gly-Gln (SEQ-ID 3);

Gly-Pro-Asp-Gly/Ile-Ala-Gly-Gln (SEQ-ID 4);

Gly-Pro-Leu-Gly/Ile-Ala-Gly-Gln (SEQ-ID 5);

Gly-Pro-Gln-Glu/Ile-Ala-Gly-Gln (SEQ-ID 6);

Gly-Pro-Gln-Phe/Ile-Ala-Gly-Gln (SEQ-ID 7);

Gly-Pro-Gln-Arg/Ile-Ala-Gly-Gln (SEQ-ID 8);

Gly-Pro-Gln-Gly/Leu-Ala-Gly-Gln (SEQ-ID 9);

Gly-Pro-Gln-Gly/Tyr-Ala-Gly-Gln (SEQ-ID 10);

Gly-Pro-Gln-Gly/Ile-Phe-Gly-Gln (SEQ-ID 11);

Gly-Pro-Gln-Gly/IIe-Glu-G!y-Gln (SEQ-ID 12);

Ala-Ser-Gln-Lys-Arg-Pro-Ser-Gln-Arg-His-Gly-Ser-Lys-Tyr/Leu-Ala-Thr-Ala-Ser (SEQ- ID 13);

Ala-Ser-Gln-Lys-Arg-Pro-Gln-Arg-Ser-Lys-Tyr/Leu-Ala-Thr-Ala-Ser (SEQ-ID 14);

Ac-Pro-Leu-Gly-(2-mercapto-4-methyl-pentanoly)-Leu-Gly-OEt (SEQ-ID 15);

Ac-Pro-Leu-Gly-S-Leu-Leu-Gly-OC2H5 (SEQ-ID 16);

Ac-Pro-Leu-Gly-S-CH2CH(CH3)CH2CH3 (SEQ-ID 17);

Ac-Pro-Leu-Gly-Leu-Leu-GIy-OC2H5 (SEQ-ID 18);

DNP-Pro-Leu-Gly-Ile-Ala-Gly-Gln-D-Arg-OH (SEQ-ID 19);

Lys-Pro-Leu-Gly-Leu-Lys (Dnp)-Ala-Arg-NH2 (SEQ-ID 20);

(Gly-Pro-Hyp)5-Gly-Pro-Lys-Gly-Pro-Gln-Gly/Leu-Arg-Gly-Gln-Lys(Dnp)-Gly-Val-Arg-

(Gly-Pro-Hyp)5-NH2 (SEQ-ID 21);

(Gly-Pro-Hyp)5-Gly-Pro-Ser-Gly-Ala-Glu-Gly-Pro-Lys(-Gly-Pro-Gln-Gly/Leu-Arg-Gly- Gln-Lys(Dnp)-Gly-Val-Arg-(Gly-Pro-Hyp)5-NH2 (SEQ-ID 22);

(Gly-Pro-Hyp)4-Gly-Pro-Lys-Gly-Pro-Gln-Gly/Leu-Arg-Gly-Gln-Lys(Dnp)-Gly-Val-Arg- Gly-Leu-Hyp-Gly-Gln-Arg-Gly-Glu-Arg-(Gly-Pro-Hyp)4-NH2 (SEQ-ID 23).

The flavor molecule upon release preferably triggers the gustatory system of the user, preferably by stimulating a sweet and/or bitter taste.

The flavor molecule together with the linker can be a peptide itself, so e.g. one of a combination of the following systems, where the part right of the slash, if released, provides for the gustatory sensation:

Gly-Thr-Pro-Gly-Pro-Gln-Gly/Ile-Ala-Gly-Gln-Arg (SEQ-ID 24);

Gly-Thr-Ala-Gly-Pro-Pro/Gly-Thr-Pro-Gly-Pro-Gln-Gly (SEQ-ID 25);

Generally speaking the following bitter sequences can be combined with the above mentioned linker peptides, taking the part left of the slash thereof only:

..../Ile-Ala-Met-Glu-Lys ((SEQ-ID 26);

.../Leu-Leu-Gly-Ala-Ile-Leu (SEQ-ID 27);

.. JIle-Ala-Gly-Ile-Phe-Pro (SEQ-ID 28);

.. JIle-Ala-Phe-Pro (SEQ-ID 29);

.. JIle-Ala-Gly-Ile (SEQ-ID 30);

.. Jlle-Phe-Phe-Pro (SEQ-ID 31);

.. JHe-Phe-Ile-Phe (SEQ-ID 32);

.. JIle-Phe-Val-Leu (SEQ-ID 33);

.. JHe-Phe-Trp-Ile (SEQ-ID 34);

.. JIle-Phe-Trp-Tyr (SEQ-ID 35);

.../Tyr-ne-Phe-Pro-Lau-Val (SEQ-ID 36);

.. JIle-Trp-Gly-Gln (SEQ-ID 37);

.. JIle-Trp-Ile-Phe (SEQ-ID 38);

.. JIle-Trp-Gly-Pro (SEQ-ID 39);

.. JIle-Ala-Gly-Gln-Ile-Tyr-PiO-Ile (SEQ-ID 40);

.../Ile-Ser-Pro-Pro-Pro-Gly (SEQ-ID41);

.. JIle-Ala-Gly-Gln-Val-Val-Val (SEQ-ID 42);

... /Gly-Pro-Phe-Pro-Val-Ile (SEQ-ID 43);

.../Phe-Ala-Leu-Pro-Glu-Tyr-Leu-Lys (SEQ-ID 44);

.../Leu-Ile-Tyr-Pro-Ile (SEQ-ID 45);

Ethylation or acetylation just ahead of the cleavage site can be used to reduce the bitterness. E.g:

OEt-Gly-Pro-Leu-Gly/... (SEQ-ID 46);

CH3(0)C-Gly-Pro-Leu-Gly/... (SEQ-ID 47).

Generally speaking, combinations of Tyr, Be, Tyr, Phe, Pro, Leu, Val as well as combinations of Tyr, He, Tyr, Phe, Pro, Leu, Val can be used for the bitter part sequences. The change upon direct or in direct contact with the marker is, in accordance with yet another preferred embodiment, triggered when a minimum marker concentration in saliva or in the tissue surrounding the coated portion of the implant is reached. Preferably the change upon direct or indirect contact with the marker is triggered when a minimum matrix metalloproteinase marker concentration in saliva or the tissue surrounding the coating of the user is reached, in particular matrix metalloproteinase-8 (MMP-8) or activated matrix metalloproteinase-8 (aMMP-8), said minimum marker concentration in saliva for the generation of a change in the coating directly detectable by the user being above 1 ng/ml, preferably above 5 ng/ml, more preferably above 8 ng/ml.

The change upon direct or in direct contact with the marker is preferably triggered when a minimum marker concentration in saliva or the tissue surrounding the coating of the user is reached, wherein preferably the marker is matrix metalloproteinase, in particular matrix metalloproteinase-8 (MMP-8) or activated matrix metalloproteinase-8 (aMMP-8), and wherein the minimum marker concentration in saliva for the generation of a change in the coating directly detectable by the user is in the range of l-6Q00ng/ml, preferably in the range of 5-4000ng/ml, and most preferably in the range of 8-2000 ng/ml.

The minimum marker concentration for the detection of periodontitis is typically a factor of 10, preferably a factor of 100, and more preferably a factor of 500 smaller than the minimum marker concentration for the detection of peri-implantitis, and wherein even more preferably based on this critical concentration difference the generation of a change in the coating directly detectable by the user is differentiated between periodontitis and peri-implantitis.

The element can be attached to a particle with a size in the range of 5-300μτη, preferably in the range of 20-250μηι, wherein preferably the particle is based on a polymer or copolymer or a (co)polymer mixture or (co)polymer blend, preferably on a polymer or copolymer selected from the group consisting of polystyrene, poly(methylmethacrylate), polyethylene, polypropylene, poly(vinylchloride), polycarbonate, polyamide, polysulfone, poly(ethersulfone), polyether, poly(ether-ketone), poly(ether-ether-ketone), poly(tetrafluoroethylen), poly(vinylidenefluoride), polyester, poly(hydroxyalkanoate), polyurethane, polyimide, poly(ether-imide), poly(butadiene), poly(vinylbutyral), polyanhydride, poly(amino acid), poly(organosiloxane), cellulose, chitin or a mixture or blend thereof. The systems can form a three-dimensional matrix, e.g. due to cross-linking processes. The three-dimensional matrix can be based on carboxy groups, amino groups, thiol groups or combinations thereof. Preferably poly(methylmethacrylate) with a three- dimensional carboxy group matrix or another three-dimensional matrix comprising a functional group like amino groups or thiol groups is used.

Preferably the element, or an interlinked anchoring element, is attached to the particle or to the base material, so basically in any kind to the coating, in particular to the coating surface, by means of conventional coupling techniques, preferably by disulfide coupling by implementation of esterified bromine compounds with sodium azide or by coupling via dicarboxylic acid, diisocyanate or diepoxide. The element, or an interlinked anchoring element, can be attached to the particle by amide formation using peptide coupling methods, disulfide coupling, ester formation using procedures like carbodiimide-activated esterifications, urethane, urea or isothiourea formation generated by reaction with diisocyanates or diisothiocyanates, ether formation by reaction with epoxy group containing molecules like diepoxides or activated haloalkyl derivatives, reaction with dialdehydes followed by reductive amination, Michael-type addition reaction as e. g. performed by reaction of an acrylated reaction partner with a thiol-modified one or by known Click Chemistry coupling protocols like the Cu(I)-promoted azide-alkyne [3+2] cycloaddition.

It is also possible that such a coating is provided with a covering additional coating for protection purposes and which only dissolves or biodegrades after a certain time or under the influence of inflammation induced changes in the surrounding, for example pH changes, marker presence, etc.

Oral and/or mucosal alterations due to inflammatory tissue normally induce the change, in particular at least one of the following inflammatory states: gingivitis, mucositis, periodontitis, peri-implantitis, or a combination thereof.

In addition to that the present invention relates to a fixed dental device provided with a coating as outlined above. Specifically, the invention relates to an implant, dental abutment, dental abutment screw or dental crown with a coating as outlined above, wherein preferably in case of a dental implant the coating is located at least at or adjacent to a threaded and/or a non-threaded portion of the dental implant or at a transition region between said portions. According to a preferred embodiment said coating is provided at an axially facing surface, forming and bordering a potential radial slot between the proximal end portion of the dental implant and the outer distal end portion of the abutment during use.

Furthermore the present invention relates to the use of a coating as outlined above for the detection of inflammatory tissue in the mouth, in particular in or adjacent to the coated implant, dental abutment, dental abutment screw or dental crown of a user.

Further embodiments of the invention are laid down in the dependent claims. BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,

Fig. 1 schematically shows the diagnostic approach followed in this application for early diagnosis of risk factors for the development of peri-implant infections, wherein the system (box 5) is sensing matrix metalloproteinase (MMP) regulation indicative for connective tissue degradation as present at the borderline of gingivitis/mucositis and periodontitis/peri-implantitis; today complications are typically recognized once clinical signs appear (box 4) a stage at which the disease course may be irreversible and lasting complications may prevail; today, single assessments can be done in the dentist's office using point of care systems which measure preferentially MMP-8 from sulcus fluid samples collected around the affected implant, wherein naturally, these single assessments provide limited information; the proposed systems are designed to allow on-demand, self-monitoring, so therefore the patient is providing continuous monitoring and based on this the dentist can diagnose complications early on;

Fig. 2 shows in (A) how the functionality is linked to the MMP-8 protease sensitive peptide sequence (center portion) located in between an anchor (left) and a flavoring substance (right); in (B) how the sequence is linked to particles or the surface of the coating, so the base material directly, referred to as 'system' in this application; upon contact with MMP-8 at specific levels, the peptide sequence is cleaved and the off-coming flavoring substance triggers an intensive taste recognized by the patient; in (C) a schematic illustration of a peri-implantitis lesion - an inflammatory cell infiltrate (ICT) is typically located as highlighted by the rectangular box; in (D) schematically how the he peptide sequence sensitive to MMP-8 is covalently linked to the region of the implant where the ICT is typically located to provide most sensitive self-monitoring; when activated, a dentist must perform a thorough diagnosis to identify the root cause of the positive signal as tasted by the patient; and in (E) schematically a perspective view of an implant indicating the surface regions where the diagnostic coating can be applied; and

Fig. 3 shows different embodiments, wherein in a) and b) a first embodiment is shown, in which the linker sequence attached to a substrate/the coating or an anchor, to which initially (a) the flavoring substance is attached, is cleaved for the release of the flavoring substance (b), in c) and d) a second embodiment is shown, in which the linker sequence attached to the coating or an anchor, to which initially (c) the flavoring substance is attached by a non-covalent bonding, for example a coordinative bonding, is conformationally changed such as to release the flavoring substance (d), and in e) and f) a third embodiment is shown, in which a substance attached via linker to the coating (e) and showing a color change upon contact with the trigger molecule.

DESCRIPTION OF PREFERRED EMBODIMENTS

Figure 2 e) in a perspective view shows an implant 8 for the purpose of illustrating possible regions for the application of the diagnostic coating to an implant according to the invention. Generally such an implant 8 comprises a threaded portion 9, at the distal end of which there are provided special axial grooves for at least partial cutting into the bone/tissue during insertion of the implant. On the proximal end of this threaded portion 9 there is typically a cylindrical or conical portion 10, which is unthreaded but which is also surface modified in the sense that it has a rough, microstructured/macrostructured and/or porous surface. The corresponding microstructured/macrostructured surface modification leads to the matte appearance as visible in Figure 2 e, and it allows for better growing in/bioadhesion of the implant when implanted. Due to this microstracture/macrostructure in the surface and the corresponding roughness this is also the region where biofilm formation can take place and where inflammation and thus peri-implantitis can start. On the proximal end of the rough cylindrical region 10 without threading typically follows a smooth machined and/or polished region 11, which can be cylindrical as in the example given in figure 2 e, but which can also be conically converging towards the distal end of the implant. On the proximal side of this smooth region 11 there is provided an axial surface 12 facing along axis 15 of the implant which will face and form a slot between the abutment firmly attached to the implant and the implant. The abutment (not shown) has a corresponding opposite distal axial surface coming into contact or almost into contact with the proximal surface 12 of the implant. During use this slot will, depending on the implant design and its precision, not always be firmly closed but due to the load on the abutment and/or the crown will slightly open and close repeatedly. Therefore there is always a certain flow of body fluid in and out of this slot, the implant side of which is formed by surface 12.

In this particular embodiment of an implant on the surface 12 there is provided a circumferential axially protruding ridge 13, however this ridge, while having many advantageous properties, is not absolutely necessary. On the inner side of this ridge 13 and on the proximal side the implant comprises a blind hole opening with an interior threading (not visible) for attachment of the abutment and correspondingly the crown. The abutment is fixed to the implant by means of an abutment screw which is located in a through bore of the abutment, and which has an outer threading for interacting with the inner threading of the opening 14 in the implant 8. On the abutment the crown forming the tooth replacement is mounted.

The diagnostic coating can be applied in region 9 and/or 10 of the implant 8, since this is the region where, due to the rough surface, there is an enhanced likelihood of inflammation formation. The corresponding coating can be applied by dip coating or the like. Due to the fact that regions 9 and/or 10 are deeply buried in the bone and/or tissue of the patient, it can sometimes however be difficult to make sure that the corresponding diagnostic reaction can be perceived by the user, so that the corresponding taste release actually may migrate to the saliva in a concentration sufficiently elevated for detection.

It is therefore also possible to selectively only or in addition to the above-mentioned coating of regions 9 and 10 coat region 11 with the diagnostic coating. This is a region which is closer to the saliva and is during use at least partially accessible to saliva fluid. One particularly preferred region for the application of the diagnostic coating is the axial surface 12. As mentioned above, this axial surface forms the radial slot between abutment and implant, a slot which will during use open and close due to the load on the crown and which will therefore, similar to a pump, suck in and push out liquid/saliva entering the slot. It therefore, also due to the fact that it is well protected from mechanical stress which might remove the coating, is a particularly suitable place for long time "storage" and availability of the coating. The surface 12 may even be slightly modified for keeping the coating, so it is possible that in this surface a surface structure is provided for taking up the coating material. Also possible is the provision of a coating on the surface of the circumferential ridge 13, in particular its radial surface.

The coating according to the invention can also be applied to the abutment screw, and also to the abutment as such. In case of applying the coating to the abutment in particular the axial surface facing surface 12 of the implant during use is suitable for the application of the diagnostic coating. It is also possible to coat the crown as such in particular in the region which is close to and adjacent to region 11.

Figure 3 shows different possibilities for attachment of releasable flavor substances 1 (a)- (d) or for the attachment of a colorant susceptible to change color upon interaction with MMP, in particular MMP-8, present in saliva or the tissue surrounding the coating.

In the first embodiment as illustrated in figure 3 a, on a substrate 3, which can for example be a particle or the base material of the coating, the flavoring substance molecule or complex 1 is attached via a linker element 2, normally a short and cleavable polypeptide chain. The flavoring substance molecule 1 may itself be a polypeptide or protein, and it may just be the extension of the linker element 2. Upon contact of the marker 4 present in saliva or the surrounding tissue with the linker element 2, the latter is cleaved due to proteolytic interaction, releasing the flavoring substance into the free state 5, inducing the taste sensation (see figure 3b).

As illustrated in figures 3c) and d), the interaction between the trigger/marker 4 present in saliva/the surrounding coating is not necessarily a direct interaction in the sense of a proteolytic interaction between the MMP trigger and the linker element, it can also be and indirect interaction for example in the sense that the MMP trigger attaches or forms a complex with the substrate in the vicinity of the linker element, induces some change, for example a change in the hydrogen bonding structure, and thereby releases the flavoring molecule or flavoring complex 5 into the surrounding. In this case there is normally no chemical bond between the linker element 2 and the flavoring substance 1, however it is also possible that the MMP trigger attaches and itself triggers a proteolytic system mounted on the substrate 3 or the linker element 2 leading to a proteolytic cleavage for the release of the flavoring substance.

Figure 3e) and f) show the situation where the MMP trigger does not induce release of flavoring substance but a color change. To this end upon contact of the MMP trigger 4 with the corresponding colorant substance 6, the latter is transformed into a second, differently colored state 7 leading to a visual signal perceptible to the user and indicative offer a sufficient level of MMP trigger in saliva. It is not only possible that, as illustrated in figure 3e) and f), the color change takes place in a situation where the colorant 6 remains to be fixed to the coating, it is also possible that the colorant 6 is released upon contact with the MMP trigger leading to a color change in saliva or in mouth tissue perceivable to the user.

Experimental section:

In a first step peptide sequences composed of (i) anchor coupled to (ii) sensitive peptide sequence coupled to (iii) flavoring substance using solid phase chemistry (Figure 2), were synthesized by using a solid phase synthesis of MMP-8 sensitive systems.

Three components of the system were synthesized (from C-N Terminus): (i) anchor coupled to (ii) sensitive peptide sequence coupled to (iii) flavoring substance.

30 systems with different protease sensitive protein sequences (as a platform from which sequences can be selected with best MMP-8 selectivity and tailored sensitivity to MMP-8 cleavage) were synthesized. Absolute MMP-8 selectivity is unlikely as proteases typically have broad substrate specificity such that systems are optimized for preferential (strict MMP-8 exclusivity is perceived impossible) MMP-8 reporting. Some cross-sensitivity of the MMP-8 responsive system and particularly to MMP-1 or MMP-3 must be tolerated and was tested. Conceptually and diagnostically, this cross-sensitivity is unproblematic, as MMP-1, MMP-3 and particularly MMP-8 are concomitantly upregulated by infiltrating Φ towards bacteria residing in host tissue, i.e. the same physiological mechanism is sensed by all three proteases. Nevertheless, a relative rate (MMP-8 cleavage over MMP-1 or MMP-8 cleavage over MMP-3) of at least 50:1 is a specification , as is e.g. the case for the sequences SEQ-IDs 2, 6 and 10. More sensitive protein sequences and their relative rate compared to MMP-1 hydrolysis are selected. Synthesis is performed on an in-house, automated solid phase peptide synthesis (SPPS) platform. Synthesis (C to N-terminus) is following established protocols by coupling the carboxyl group of one amino acid to the amino group of another and by adequate use of protecting groups to avoid unintended reactions. Finally, the flavoring substance is attached as outlined below. The taste of the cleaved peptide sequence fragment coupled to the flavoring substance was tested by human volunteers and feedback from these tests helped to further modify the peptide sequence and flavoring substance for optimized bitter taste.

Bitterness in oligopeptides is closely related to hydrophobicity. In fact and for screening purposes, one can proceed by assaying the Q value of peptides (a measure of the average hydrophobicity of a peptide, with Q > Γ400 cal/mol being a threshold for possible bitter taste). Based on this approach, a selection of the following MMP-8 cleavage products suggests bitterness or not: the first five sequences (sequences #1-5) of Table 2 of NAGASE, H. & FIELDS, G. B. 1996; Human matrix metalloproteinase specificity studies using collagen sequence-based synthetic peptides. Biopolymers, 40, 399-416, show a Q* in cal/mol of approximately 1400-1700 and e.g. the fourth sequence shows bitter taste. If the cleaved peptide sequence is sufficiently bitter for reliable recognition, the coupling of the flavoring substance is sometimes not necessary.

Sequences # 1-5 are a selection of MMP-8 sensitive sequences from Nagase loc. cit, with #4 being predicted as bitter and # 2 being predicted as not bitter based on Q value calculations. Modification of # 2 by decoration with another isoleucin and/or leucine at the C-terminus leads to a prediction of bitterness and has been optimized for bitterness in terms of presenting leucine at the N- and C-terminal ends, respectively. Inconsistencies have been reported when linking Q values and bitterness, thus sensory tests are important to confirm the Q-based prediction. Typically, sensory recognition thresholds for peptides are between 0.05 and 6.0 mmol/L, i.e. less than what is sensed for quinine (about 0.0004 mmol/L). Quinine and other strong taste substances are coupled as well and as an alternative to using the bitter taste of cleaved products as shown in this table.

Coupling of quinine (bitter for gustatory detection and fluorescent for easy testing of cleavage, particularly when the spherical system is deployed) or aspartame, an artificial sweetener to the N-terminal end of the 'anchor-protease sensitive peptide' sequence can be performed while still on solid phase (see Figure 2). Bifunctional linkers can be used to couple quinine to the N-terminal end of the anchor-protease sensitive protein sequence. To avoid rapid cleavage of the linker from quinine in vivo, hydrolytically or enzymatically less sensitive linkages can be installed. In a first approach, a diisocyanate linker like hexamethylene diisocyanate is treated with the free secondary OH-group of quinine forming an urethane bond followed by coupling the linker via its remaining isocyanate group to the N-terminal end of the peptide, forming an urea bond. A second approach consists in the reaction of the quinine double bond with a bis-epoxide (e. g. 1,4-butanediol diglycidyl ether) leading to an ether linked quinine which can be subsequently coupled to solid phase attached protein by N-alkylation. Aspartame, or if necessary a N-protected aspartame, can be coupled via its carboxyl group to the peptide N-terminus by conventional peptide synthesis. After coupling the flavoring molecules to the peptide, the formed conjugates can be cleaved from solid phase, purified and characterized using common analytical methods (FT-IR, NMR, MS).The strategy regarding the anchor is outlined below.

Anchoring of sensitive peptide sequence-flavoring substance conjugates to dental implant surfaces.

The fixed dental device, so the dental implant, dental abutment, or dental abutment screw can be based on a ceramic or metallic material. In case of a ceramic material it is preferably based on zirconium oxide, to which possibly additionally is added yttrium oxide and hafnium oxide, and/or containing aluminium oxide, possibly additionally containing silicium dioxide, ferric (III) oxide, and/or sodium oxide, and/or containing silicium nitride, possibly additionally containing silicium dioxide, ferric (III) oxide and/or sodium oxide, and or containing titanium oxide and/or being formed of mixtures of said materials. In case of a metallic material is preferably based on titanium or a titanium-based alloy. The crown is typically based on a ceramic or polymeric material.

Conventional dental implants made from pure titanium (cp-Ti) or titanium alloy (e. g. TiA14V6) are supplied. To immobilize sensitive peptide sequence-flavoring substance conjugates to the metallic implant surfaces, in a first step the titanium surface is treated by a common activation process like water vapour plasma followed by coating of the activated surface with an adhesion promoter containing a reactive functionality to bind to the titanium surface and another functional group to link the protein-flavoring substance conjugate. Suitable adhesion promoters are aminosilanes (e.g. 3- aminopropyltriethoxysilane, aminotitanates (e. g. isopropyl tri(N-ethylene diamino)ethyl titanate), or aminoalkyl phosphonates (e. g. (l-aminohexyl)phosphonic acid). In all cases where peptide-flavoring substance conjugates without free amino groups within the peptide chain and the flavoring substance are used coupling to the adhesion promoter can be performed directly to the C-terminus of the peptide sequence by using conventional peptide formation protocols and coupling agents like carbodiimides. In cases where the conjugates contain interfering functional groups (e. g. free NH₂-groups) a cystein moiety can be attached the C-terminus of the peptide sequence during peptide solid phase synthesis. Then, coupling can be readily performed using disulfide coupling protocols for example with N-succinimidyl-3-(2-pyridyldithio)-propionate as Afunctional coupling agent. Finally, purification procedures are elaborated to ensure removal of unreacted material and by-products from the surfaces. Before using implant devices, the whole immobilization procedure is established on flat titanium samples possessing the same surface characteristics like the implant materials. Each activation and coupling step is monitored by appropriate analytical methods (XPS, measurement of contact angles and zeta potentials, determination of amino groups, etc.) and optimization of the different reaction steps by variation of reagents and reaction conditions is performed. Optimized protocols are used then for the immobilization of protein-flavoring substance conjugates to implant surfaces.

To prepare peptide-flavoring substance conjugate-bearing spheres to be embedded in the coating base material or two form a surfacial layer of the coating, poly(methylmethacrylate) (PMMA) carriers (particle diameter: 17 to 30 μπι) with a three- dimensional carboxy group matrix are used. The conjugates synthesized as outlined above are immobilized to the PMMA spheres by conventional peptide formation protocols (e. g. by using water-soluble carbodiimides to activate the carboxyl groups of the PMMA matrix). In cases where conjugates with interfering functional groups are used the disulfide coupling method can be employed as described above.

For the coupling of peptides to build up the sensitive peptide sequence the following method was used:

Manual coupling of amino acids: After swelling the resin for 30 min in DMF and removing 1 mL 40% piperidine/DMF is added and incubated for 3 min. Then, after removing by vacuum filtration 1 mL 20% piperidine/DMF is added and incubated for 10 min. After removing the resin is washed 6 times with DMF (1 mL, 1 min each). The amino acid (5 eq) is dissolved in 410.90 μL· 0.5 M HOBt in DMF and afterwards transferred to the N-terminal-deprotected peptidyl resin. 31.81 (8 eq) of DIG is added to the reaction mixture and gently shaked for 1 h. After removing the reaction mixture by vacuum filtration the resin is washed 6 times with DMF (1 mL, 1 min each) and 6 times with DCM (1 mL, 1 min each). After the cleavage of peptides by using MMP. The monoisotopic masses have to be checked with MALDI-MS. Preparative purification by high-pressure liquid chromatography (HPLC) is carried with a Phenomenex C18 column (21.2-mm internal diameter, 250-mm length, 7-mm particle size) with eluent A (0.2% TFA in water) and eluent B (0.2% TFA in 1:4 water-acetonitrile). The peptides have to be purified with a gradient of 29 to 54% eluent B in 50 min.or the coupling of the flavoring substance (quinine, caffeine, theobromine, naringin, sucralose or neotame) to the sensitive peptide sequence the following specific methods can be used:

Modification of hydroxygroup-containing flavor molecules with anchor groups:

Example 1:

1.5 mmol of the flavor molecule is dissolved in dichloromethane and consecutively 3 mmol of adipic acid, 3 mmol of Ν,Ν-dicyclohexylcarbodiimide and 3 mmol of 4-(N,N- dimethylamino)pyridine are added. The mixture is stirred for 24 hours at room temperature. Then, the reaction mixture is washed thoroughly with saturated NaHC03 solution, 2N HCl solution and water. The organic phase is isolated, dried over MgS04, and evaporated to dryness under vacuum. The obtained raw material is purified by flash chromatography using a silica gel column and chloroform/methanol as eluent.

Example 2:

3 mmol dodecanedioic acid are stirred at room temperature with 3 mmol of 2,4,6- trichlorobenzoyl chloride and 10 mmol of triethylamine in toluene. After 3 hours stirring, 3 mmol of quinine and 3 mmol of 4-(N,N-dimethylamino)pyridine are added and the mixture is stirred for another 20 hours. The reaction mixture is washed thoroughly with saturated NaHC03 solution, and water, and the aqueous phase is washed twice with ethyl acetate. The organic phases are unified and dried over MgS04. After evaporation of the solvent under vacuum, the obtained raw material is purified by flash chromatography using a silica gel column and chloroform: methanol = 3 : 1 as eluent. Yield: 47 %, brown oil. IR (ATR, cm-1): 2923, 2852, 1738, 1623, 1590, 1505, 1476, 1433, 1357, 1305, 1229, 1157, 1090, 1033, 995, 914, 852, 829, 762, 719.

Example 3:

Step 1: A mixture of 1 mmol of quinine, 1 mmol of 11-bromo-undecanoic acid, 1 mmol of Ν,Ν-dicyclohexylcarbodiimide and 1 mmol of 4-(N,N-dimethylamino)pyridine in dry dichloromethane are stirred for 24 h at room temperature. Then, the reaction mixture is washed thoroughly with saturated NaHC03 solution, 2N HCl solution and water. The organic phase is isolated, dried over MgS04, and evaporated to dryness under vacuum. The product is further purified by flash chromatography using a silica gel column and methanol as eluent XXX. Yield: 20%, yellow oil. IR (ATR, cm-1): 3323, 2924, 2852, 2119, 1738, 1695, 1619, 1571, 1509, 1452, 1357, 1310, 1223, 1167, 1086, 1029, 990, 914, 852, 833, 719, 647. Step 2: 1 mmol of the flavor molecule containing a bromo group is dissolved in DMF and an excess of sodium azide (3 mmol) is added. The mixture is stirred for 20 h at room temperature. After addition of water the reaction mixture is extracted three times with ethyl acetate. The received raw product is further purified by flash chromatography using a silica gel column and chloroform: methanol = 3 : 1 as eluent.

Example 4:

2.5 mmol of the flavor molecule are dissolved in dichloromethane and 0.025 mmol of dibutyltin dilaurate followed by 5 mmol of hexamethylene diisocyanate dissolved in dicloromethane are added. The mixture is stirred for 24 hours at room temperature. After evaporation of the solvent the isocyanate-containing flavor molecule is used without further purification in the next step.

Example 5:

2.5 mmol of the flavor molecule and 5 mmol of poly(ethylene glycol)-diepoxide (molecular weight: 2000 Da) are dissolved in DMSO (20 ml) followed by the addition of 5 mmol KOH. After stirring for 3 hours at room temperature water is added and the mixture is extracted with chloroform. The organic phase is dried over MgS04 and after evaporation of the solvent, the resulting product is used without further purification.

Modification of peptides with anchor groups:

Example 6

1 mmol of the peptide is dissolved in a dioxane/water mixture (1:1) and 2M NaOH is added until the pH reached 9-10. Under a nitrogen atmosphere, 1.1 mmol 3-butyn-l-yl- chloroformate is added and the mixture is allowed to stir for 18 hours. The product is lyophilised and purified by FCPC using an n-BuOH/H20 system. After purification the product is obtained as a white solid.

Coupling of the carboxy group-containing flavor molecule with the peptide:

Example 7:

A mixture of 1 mmol of the carboxy group-containing flavor molecule from example 1 or 2, 1 mmol of the peptide, 1 mmol of N,N-dicyclohexylcarbodiimide and 1 mmol of 4- (N,N-dimethylamino)pyridine in dry dichloromethane are stirred for 24 h at room temperature. Then, the reaction mixture is washed thoroughly with saturated NaHC03 solution, 2N HC1 solution and water. The organic phase is isolated, dried over MgS04, and evaporated to dryness under vacuum. The product is further purified using preparative HPLC. Example 8:

0.5 mmol of the triple bond containing peptide from example 6 and 0.5 mmol of the azide group-containing flavor molecule from example 3 are dissolved in 20 ml of DMF. After addition of the catalyst copper-I-bromide/pentamethyldiethylenetriamine (0,05 mmol), the mixture is stirred for 24 hours at room temperature. After addition of water (150 ml), the mixture is extracted three times with chloroform. The unified chloroform extracts are washed with saturated NaHC03 solution, 2N HC1 solution and water. The organic phase is dried over MgS04 and after evaporation of the solvent the peptide-coupled flavor molecule is obtained as a light yellow solid.

Example 9:

1 mmol of isocyanate-terminated flavor molecule is dissolved in dichloromethane (10 ml) followed by the addition of 0.005 mmol dibutyltin dilaurate. After addition of 1 mmol of the peptide the mixture is stirred for 24 hours at room temperature. The reaction mixture is diluted by adding 10 ml of dichloromethane and washed with saturated NaHC03 solution, 2N HC1 solution and water. The organic phase is dried over MgS04 and after evaporation of the solvent, the resulting raw product is purified using preparative HPLC.

Example 10:

1 mmol of the epoxide-modified flavor molecule and 1 mmol of the peptide are dissolved in DMSO (20 ml). 2 mmol KOH are added and the mixture is stirred for 6 hours at room temperature. Water is added and the reaction mixture is extracted several times with chloroform. The organic phase is washed with saturated NaHC03 solution, 2N HC1 solution and water. The chloroform extracts are dried over MgS04 and after evaporation of the solvent under vacuum, the remaining material is purified using preparative HPLC. Pretreatment of titanium implant surfaces for coupling processes, Example 11:

The titanium surface is activated using the Pyrosil® technology, a combustion chemical vapor deposition (CCVD) process depositing a reactive SiOx layer in the nanometer range onto the titanium surface. The following parameters are used: carrier gas mixture: propane/air: 50/1000 1/min, precursor: hexamethyldisiloxane 15% in isopropanol, burner- substrate distance: 20 mm.

After CCVD treatment, the implants are dipped for 10 sec into a sol of (3- aminopropyl)triethoxysilane, air-dried for 30 min at ambient temperature and further 30 min at 160 °C. The process is repeated twice. The sol is prepared by mixing a solution of ethanol (0.0686 mol), and acetic acid (0.0034 mol) in 1.85 g water with a solution of (3- aminopropyl)triethoxysilane (0.01218 mol, 5%) with 3.16 g Ethanol.

Coupling of flavour molecule-modified peptide to to titanium implant surfaces, Example

12:

A titanium implant sample pre-treated as described in Example 11 or by another common pre-treatment method is placed in a Petri dish filled with a solution of 5 mmol of the flavour molecule-modified peptide in dry DMF. 5 mmol of N,N-dicyclohexylcarbodiimide and 5 mmol of 4-(N,N-dimethylamino)pyridine both dissolved in dry DMF are added. The titanium implant sample is stored for 24 h at room temperature in the Petri dish. Afterwards it is removed and washed twice with DMF.

Example 12a:

The same procedure as described in the context of example 12 was carried out but using a titanium implant without pretreatment according to example 11.

Example 12b:

The same procedure as described in the context of example 12 was carried out, but using a titanium implant pretreated with NaOH solution as follows:

The titanium surface is dipped for 10 sec into 0.05M NaOH solution of distilled water, dried.

For the coupling of flavor molecule-modified peptide to polymer particles the following methods can be used:

Example 13:

The amino group-containing polymer particles (100 mg) are suspended in dichloromethane and a solution of 1 mmol of the peptide in dry dichloromethane is added. After 5 min of stirring, 1 mmol of N,N-dicyclohexylcarbodiimide and 1 mmol of 4-(N,N- dimethylamino)pyridine dissolved in dichloromethane are added and the mixture is stirred 24 h at room temperature. The particles are isolated and washed twice with dichloromethane, ethanol, and water.

Example 14:

The amino group-containing polymer particles (100 mg) and the flavor molecule- containing peptide (lmmol) are suspended in phosphate buffer saline (10 mg/ml, pH = 5- 6). After 5 min, 1 mmol l-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.6 mmol of N-hydroxysuccinimide are added and the mixture is stirred for 4 hours at room temperature. The polymer particles are isolated, washed with deionized water and purified by dialysis against deionized water for 36 hours at room temperature. Suitable purification routines for the spheres have been established as well as analytical techniques to characterize them (particle size after immobilization, mechanical integrity, load capacity).

Collection of sulcus fluid from peri-implant diseased patients in different disease stages and from healthy peri-implant pockets and medical examination and determination of MMP-8 levels from samples obtained, definition of disease-stage specific threshold MMP- 8 concentrations by correlation of measured MMP-8 concentrations with clinical diagnosis: Collection of sulcus fluid and MMP-8 levels:

Patients with peri-implant disease and healthy subjects carrying an implant were examined during clinic visit. After informed consent had been obtained, all subjects answered a questionnaire, followed by a radiological investigation and oral examination. The oral examination included periodontal parameters such as probing pocket depths, clinical attachment level, and recession at the index implant, as well as overall oral hygiene and bleeding index.

Sulcus fluid were collected from the index peri-implant zone using standardized MMP-8 collection strips, which are placed into the periodontal pockets for 30 seconds. The aMMP- 8 was immediately eluted from the strips for 30 seconds and quantitatively assessed with the Dento Analyzer (Dentognostics GmbH, Jena, Germany). The Dento Analyzer is a validated CE marked PoC machine, automatically conducting the entire assay process within 12 minutes chair-side. The assay allows assessment within a range of 2 ng/ml aMMP-8 in eluate up to 200 ng/ml aMMP-8 in eluate.

The MMP-8 concentrations in sulcus fluid were correlated with the clinical examination such that threshold values could be defined as a function of disease scoring.

In vitro testing of system sensitivity to MMP challenge:

The system was challenged to different MMPs and 5 systems are selected with optimized specificity and selectivity for MMP-8 cleavage.

Profile system cleavage as in Figure 2B, D (coupled to spherical carrier or that coupled spherical carrier formulated into a coating layer or directly coupled to the coating layer base material) when exposed to MMPs. MMP-1, 2, 3, 7, 8, 9, 13 was purchased. Enzyme assays were conducted to determine kcat/KM values (as substrate concentration is high, the enzyme is saturated and reaction kinetics are, therefore, controlled by kcat) and relative rate for sequence specificity (MMP-1, MMP-2 and MMP- 13 over MMP-8). Cleavage was assessed through conventional HPLC methods with UV-VIS detection and fluorescence detection where appropriate (fluorescence for e.g. quinine modified systems). Triple stage LC-MS/MS was used for analysis and characterization of fragments. As fragments are below 1500 amu (m z), the LC-MS MS deployed at UWU is capable of robustly assessing these cleavage products with high sensitivity while concomitantly collecting structural data by means of tandem mass spectrometry (MS/MS) for enhanced identification and confirmation.

Testing of at least 30 spherical and 10 implant systems in vitro for MMP-8 sensitivity and selectivity at concentrations as provided from above and differentially against other proteases, proof of principle experiment of the MM8 system that is linked to transmucosal dental implants:

Test of implant in in vivo minipig model:

Miniature pigs have oral and maxillofacial structures similar to those of humans in terms of anatomy, physiology, and disease development. In female minipigs (Ellegaard Goettinger Minipigs, Dalmose, Denmark) tooth extractions of the second and third premolars and the first molar on both sides in the mandible were performed. Following eight weeks of healing, an open full-thickness mucoperiosteal flap was performed as access surgery to the alveolar crest. According to standard surgical protocols, three transmucosal implants decorated with the MMP-8 sensitive system (verum; Figure 2F) were placed in a randomized order and on each side and healing caps were mounted. The MMP8 system was deposited by the surgeon into the peri-implant tissue close to the suspicious implants allowing 7/24 monitoring at those sites (Figure 2D). Subsequently, the minipigs were divided into an experimental and a control group. Slight peri-implant infections (mucositis) were promoted using ligatures and in association with bacterial inoculations in the experimental group. Test specimen was placed in both groups and after a defined storage time (based on the preliminary in-vitro tests), sulcus fluid samples were collected to detect the anchor and sensitive peptide sequence as well as the separated flavoring substance using different approaches (LC-MS, TOF-SIMS).

In vivo pattern demonstrated for implant system

Block sections of peri-implant hard and soft tissue structures were retrieved and all sites histologically and immunohistologically analyzed. Total height of the peri-implant soft tissue, length of junctional epithelium, height of connective tissue and levels of the marginal bone were measured using the light microscopic sections. The same sections were used for morphometric analyses of the percentage of various cells and extracellular matrix compounds as fibroblasts, collagen, blood vessels, inflammatory cells and others. Qualitative evaluation of ground histology sections was performed for osseointegration, bone remodeling activity, stage of bone formation, calcification and signs of peri-implant bone resorption or loss. Special emphasis was placed on the bone-biomaterial interface. Fluorochrome integration (calcein green, xylenol orange, tetracycline) was studied to visualize bone remodeling activity. CT scans, radiographs and microradiographs were assessed for bone density, signs of bone/material resorption and/or sclerosis within or adjacent to the original implant sites.

LIST OF REFERENCE SIGNS

1 flavoring substance 8 implant

2 linker element 9 threaded section of 8

3 coating base 10 unthreaded rough section of 8 material/substrate and/or 11 unthreaded smooth section of anchor 8

4 MMP trigger 12 axially facing surface of 8 5 released flavoring substance 13 circumferential (optional) 6 colorant substance in first ridge on 12

state 14 opening for

colorant substance in second abutment/abutment screw coloured state 15 axis of 8

Claims

1. A coating for a fixed dental device, in particular for a dental implant, for a dental abutment, for a dental abutment screw and/or for a dental crown, for identifying the presence of inflammatory tissues in the mouth, in particular adjacent to said dental device, comprising

a base material or particles (3) embedded and/or attached to said base material; an element (1, 5-7) attached to said base material and/or said particles for the generation of a change in the coating directly detectable by the user;

wherein the element (1, 5-7) generates the change upon direct or indirect contact with a marker (4) which is released by inflammatory tissue in response to bacterial mediators.

2. The coating according to claim 1, wherein the marker (4) inducing the change is a proteolytic enzyme released by inflammatory tissue, preferably by macrophages.

3. The coating according to claim 1 or 2, wherein the marker (4) inducing the change is a matrix metalloproteinase (MMP), in particular an activated matrix metalloproteinase (aMMP), preferably selected from the group consisting of: matrix metalloproteinase-8 (MMP-8), activated matrix metalloproteinase-8 (aMMP-8), matrix metalloproteinase-2 (MMP-2), activated matrix metalloproteinase-2 (aMMP-2), matrix metalloproteinase-9 (MMP-9), activated matrix metalloproteinase-9 (aMMP-9), or a combination thereof.

4. The coating according to any of the preceding claims, wherein the element (1, 5- 7) is a molecule or molecular assembly which, upon direct or indirect contact with the marker (4) undergoes a color change perceivable by the naked eye of the user, and which is embedded or attached to the base material (3) or to particles (3) embedded and/or attached to said base material.

5. The coating according to any of the preceding claims, wherein the element (1, 5- 7) is a flavor molecule (1, 5) releasably, preferably releasably covalently, attached to the base material (3) or to particles (3) embedded and/or attached to said base material.

6. The coating according to claim 5, wherein the flavor molecule (1) is attached to the base material or to particles (3) embedded and/or attached to said base material by means of a molecular chain (2) cleavable under direct or indirect contact with the marker (4).

7. The coating according to claim 6, wherein the molecular chain (2) is a polypeptide chain, preferably of 2-15, most preferably of 3-7 amino acids, either directly or indirectly, via an anchoring element, attached to the base material (3) or to particles (3) embedded and/or attached to said base material, wherein the molecular chain (2) is preferably selected from the group consisting of the sequences SEQ-ID1 - SEQ-ID23, or a combination of a linker fraction thereof with one of the sequences selected from the group consisting of SEQ-ID24-SEQ- ID44.

8. The coating according to any of the preceding claims, wherein the flavor molecule (1, 5) upon release triggers the gustatory system of the user, preferably by stimulating a sweet and/or bitter taste.

9. The chewing gum according to any of the preceding claims, wherein the flavor molecule (1, 5) is a polypeptide chain, preferably of 2-15, most preferably of 3-7 amino acids, either directly or indirectly, via an anchoring element, attached to the base material (3), and wherein preferably the flavor molecule (1, 5) is a polypeptide chain with one of the sequences selected from the group consisting of SEQ-ID24-SEQ-ID44.

10. The coating according to any of the preceding claims, wherein the change upon direct or indirect contact with the marker (4) is triggered when a minimum marker concentration in saliva or the tissue surrounding the coating of the user is reached, wherein preferably the marker (4) is matrix metalloproteinase, in particular matrix metalloproteinase-8 (MMP-8) or activated matrix metalloproteinase-8 (aMMP-8), and wherein the minimum marker concentration in saliva for the generation of a change in the coating directly detectable by the user is above lng/ml, preferably above 5 ng/ml, more preferably above 8 ng/ml, or wherein the minimum marker concentration in saliva for the generation of a change in the coating directly detectable by the user is in the range of l-6000ng/ml, will preferably in the range of 5-4000ng/ml, and most preferably in the range of 8-2000 ng/ml, wherein the minimum marker concentration for the detection of periodontitis is preferably a factor of 10, more preferably a factor of 100, and even more preferably a factor of 500 smaller than the minimum marker concentration for the detection of peri- implantitis, and wherein even more preferably based on this critical concentration difference the generation of a change in the coating directly detectable by the user is differentiated between periodontitis and peri-implantitis.

11. The coating according to any of the preceding claims, wherein the element (1, 5- 7) is attached to a particle with a size in the range of 5-300 μ, preferably in the range of 20-250 μ, wherein preferably the particle is based on a (co)polymer, (co)polymer mixture or (co)polymer blend, preferably a (co)polymer selected from the group consisting of: polystyrene, poly(methylmethacrylate), polyethylene, polypropylene, poly(vinylchloride), polycarbonate, polyamide, polysulfone, poly (ethersulf one), polyether, poly(ether-ketone), poly(ether-ether- ketone), poly(tetrafluoroethylen), poly(vinylidenefluoride), polyester, poly(hydroxyalkanoate), polyurethane, polyimide, poly(ether-imide), poly(butadiene), poly(vinylbutyral), polyanhydride, poly(amino acid), poly(organosiloxane), cellulose, chitin, preferably with a three-dimensional matrix, in particular a three-dimensional carboxy, thiol or amino group matrix or mixtures thereof and wherein preferably the element (1, 5-7), or an interlinked anchoring element, is attached to the particle by means of coupling techniques, preferably disulfide coupling.

12. The coating according to any of the preceding claims, wherein the element (1, 5- 7) is attached to the base material of the coating in that the element (1, 5-7), or an interlinked anchoring element, is attached by means of coupling techniques, preferably selected from the following group of coupling techniques consisting of: amide formation using peptide coupling; disulfide coupling; ester formation preferably using carbodiimide-activated esterifications; urethane formation; urea formation; isothiourea formation, preferably by reaction with diisocyanates or diisothiocyanates; ether formation, preferably by reaction with epoxy group containing molecules, in particular diepoxides, or with activated haloalkyl derivatives; reaction with dialdehydes followed by reductive animation; Michael- type addition reaction preferably as performed by reaction of an acrylated reaction partner with a thiol-modified one; Click Chemistry coupling protocols, preferably by Cu(I)-promoted azide-alkyne [3+2] cycloaddition.

13. The coating according to any of the preceding claims, wherein oral and/or mucosal alterations due to inflammatory tissue induce the change, in particular at least one of the following inflammatory states: gingivitis, mucositis, periodontitis, peri-implantitis, or a combination thereof.

14. Dental implant, dental abutment, dental abutment screw or dental crown with a coating according to any of the preceding claims, wherein preferably in case of a dental implant the coating is located at least at or adjacent to a threaded (9) and/or a non-threaded (10,11) portion of the dental implant or at a transition region between said portions (9-11).

15. Dental implant or dental abutment according to claim 14, wherein said coating is provided at an axially facing surface (12), forming and bordering a radial slot between the proximal end portion of the dental implant (8) and the outer distal end portion of the abutment during use.

16. Use of a coating according to any of the preceding claims for a fixed dental device, in particular for a dental implant, for a dental abutment, for a dental abutment screw and/or for a dental crown for the detection of inflammatory tissue in the mouth, in particular in or adjacent to the mandible, the maxilla, an implant or the teeth of a user.