Self-Assembled Monolayers for Dental Implants

Implant-based therapy is a mature approach to recover the health conditions of patients affected by edentulism. Thousands of dental implants are placed each year since their introduction in the 80s. However, implantology faces challenges that require more research strategies such as new support therapies for a world population with a continuous increase of life expectancy, to control periodontal status and new bioactive surfaces for implants. The present review is focused on self-assembled monolayers (SAMs) for dental implant materials as a nanoscale-processing approach to modify titanium surfaces. SAMs represent an easy, accurate, and precise approach to modify surface properties. These are stable, well-defined, and well-organized organic structures that allow to control the chemical properties of the interface at the molecular scale. The ability to control the composition and properties of SAMs precisely through synthesis (i.e., the synthetic chemistry of organic compounds with a wide range of functional groups is well established and in general very simple, being commercially available), combined with the simple methods to pattern their functional groups on complex geometry appliances, makes them a good system for fundamental studies regarding the interaction between surfaces, proteins, and cells, as well as to engineering surfaces in order to develop new biomaterials.


Introduction
e World Health Organization points out two entities of bacterial origin, caries and periodontitis, which are the most disseminated diseases in human, and both are associated with frequent surgical procedures [1]. ese infectious diseases and other noninfectious diseases such as dentoalveolar trauma and congenital absences are the main causes of edentulism. Several preventives and educational programs are used to avoid or reduce the role of these infectious diseases in the early loss of teeth. However, dentoalveolar trauma has been increased due to human activities such as extreme and contact sports [2][3][4].
Partial edentulism produces deleterious e ects on the balance of the cranio-cervico-facial system because it may a ect soft and hard tissues. Intra-arch changes include missing of interproximal contacts, misalignment, diastema, rotation, inclination, periodontal defects, impactation, and mesial drift displacement. Inter-arch changes have been described as occlusal collapse, premature occlusal contact, infraocclusion, and altered vertical dimension. ese changes are synergic, increasing the bruxism, muscle parafunction, teeth wear, ATM symptom, otologic pain, and craniocervical position [5].
Recovering aesthetics and function is possible using orthodontics when space closing is an alternative feasible while in other cases are required surgical and oral rehabilitation procedures such as implant-supported or removable prostheses. Titanium implant-based therapy appears as the "gold standard," with a record of ∼95% survival rates reported after 5 years [6]. However, other concepts have been introduced such as "the success" (Figure 1), which is di erent from long-term survival because it is focused on integral evaluation in terms of aesthetic, function, and biological response, with less than 0.2 mm of apical migration [7]. e implantology history shows two di erent phases de ned as pre-osseointegration and post-osseointegration eras. During the pre-osseointegration era, blade-and plateform implants were developed using cobalt-chromiummolybdenum and di erent stainless steel types. However, a limited long-term success was achieved. Post-osseointegration era started with Branemark's research a decade before that his research were presented to scienti c community, followed by Albrektsson et al. studies to verify clinically the osseointegration of implants [8].
e osseointegration concept was de ned as a biological phenomenon involving direct contact between bone and Ti surfaces, opening a new paradigm of therapy. A wide revision about this topic is available [8,9]. Implant therapy has high levels of predictability in a short term presenting few contraindications to restore partial and full edentulism. Many factors have been evaluated to predict the short-term e ect including surgical stability, individual in ammatory response, periodontal covering, and blood clot formation. However, long-term predictability has been associated with several aspects such as implant-related designs, surgical procedures, anatomic and osseous conditions, systemic diseases, habits like bruxism, prosthetic design, susceptibility, periodontal status, oral microenvironment, native or augmented bone, two-stage or immediate loading, and adherence to support therapy. A poor prognosis is observed in patients with an insu cient quality and/or quantity of bone; patients exhibiting poor quality of bone (type IV) in the posterior area of the maxilla had a 35% implant failure while patients with types I, II, and III showed only 3% failure [10]. e long-term success of an implant ( Figure 1) largely depends on the balance between occlusal equilibrium, osseointegration, and epithelial/connective tissue attachment. A complete sealing of the soft tissue protects the newly formed bone from bacterial metabolic products originated in the bio lm formed around implant [11].
Several animal and in vitro studies have shown similar epithelial and connective structures between the gingiva and the peri-implant mucosa. e outer surface of the periimplant mucosa is aligned by a continuous strati ed keratinized oral epithelium with a junctional epithelium attached to the Ti surface by a basal lamina and hemidesmosomes. e nonkeratinized junctional epithelium has only a few cell layers in the apical portion and is separated from the alveolar bone by a collagen-rich connective tissue. is 3-4 mm biological barrier, which is formed regardless of the original mucosal thickness, protects the osseointegration zone from factors released by the plaque and the oral cavity [12]. e main di erences between the soft tissues around natural teeth and those around implants are the collagen bre orientation, which run parallel from the implant surface to the crest bone, the low number of broblasts, the reduced vascularization revealed as scar tissue, and the loss of the irrigation system of the periodontal ligament [13].
An osseointegrated implant is not exempt from failure and complications. ey are classi ed as biological, mechanical, material surface, iatrogenic, and patient-related failures. Mobility is a sign of implant failure and can be presented as rotational, lateral or horizontal, and axial or vertical [14]. ere are di erent terms in the literature associated with biological implant failures like peri-implant diseases, mucositis, and periimplantitis (Figure 2), where the rst two are reversible inammatory reactions around a functioning implant while peri-implantitis is a chronic in ammation with a loss of the supporting tissues around the implant induced by bacterial colonization and facilitated by the implant/abutment gap and by the chemistry and surface roughness of screw and restorative components [15].
Bacteria colonize and develop bio lms on the transmucosal abutment of osseointegrated dental implants. Like the gingival crevice around the natural tooth, the peri-implant mucosa covering the alveolar bone is closely adapted to the implant. In partially edentulous subjects, the developing microbiota around implants closely resembles the micro ora of the natural teeth [12]. In addition to the dark-pigmented, Gram-negative anaerobic rods, other bacteria are associated with peri-implant infections (Tannerella forsythia, Fusobacterium nucleatum, Campylobacter rectus, Parvimonas micra, and Prevotella intermedia) [16], and eventually with Staphylococcus spp. and Candida spp. [17]. e surface texture of dental implants a ects the rate of osseointegration [18] and biomechanical xation. Surface roughness may be classi ed as "macro," "micro," and "nano" sized topologies. e "macro" ranges from millimetres to 10 μm and is directly related to implant geometry with threaded screws and macroporous coatings helping the primary stability of the implants during the early phases of implantation. However, high surface roughness may increase peri-implantitis risk compared with moderate roughness (1-2 μm) within "micro" range (1-10 μm), maximizing bone/implant interlocking. Surface pro les in the "nano" range play an important role in protein adsorption and osteoblast adhesion and, thus, in osseointegration [19], but no reproducible surface roughness is currently clinically available.
is review rstly presents a brief overview of di erent coating strategies to increase the osseointegration of titanium and is followed by a detailed description of selfassembled monolayers as a nanoscale approach to modify dental implant surfaces.

Biofunctionalization Strategies Available for Dental Implants
All surface modi cation strategies described below aimed to improve the long-term clinical survival and success of those dental biomaterials without altering their bulk properties (e.g., mechanical and nontoxicity). ese coating strategies are mainly focused to increase the osseointegration than to reduce the bacterial colonization. Bioactive surfaces have been developed to improve the osseointegration of bone with dental materials like titanium through coating strategies with immobilized biomolecules such as cell adhesive peptides having the Arg-Gly-Asp (RGD) sequence or bone morphogenetic proteins (BMPs) that play important roles in bone formation in vivo [20], to promote the adhesion of bone cells (i.e., osteoblasts) and subsequent proliferation and mineralization activities [21,22], to induce alkaline phosphatase activity in broblast [23] or the attachment of osteoblast [24].
Finally, antibacterial coatings have been applied using biocidal substances (e.g., antibiotics and antimicrobial peptides) through two systems: (a) a continuous release system, creating a local e ect around the implant and (b) International Journal of Dentistry a permanent immobilization scheme, acting on local microorganisms that contact the surface.
Furthermore, one promising strategy to enhance tissue integration is to develop a selective biointeractive surface that simultaneously enhances bone cell function while decreasing bacterial adhesion [20,22,28].
A resume of surface modi cation methods and e ects is presented in Table 1.

Basic Aspects of Self-Assembled Monolayers (SAMs)
SAMs are spontaneously formed by solution deposition through the immersion of an appropriate substrate into a solution of an active surfactant in an appropriate solvent (e.g., organic or aqueous) or by aerosol spraying or vapor deposition of the active organic compound onto the solid surface, being immersion the most popular and widely studied method for monolayer formation since it is the easiest and most inexpensive way to be applied to appliances with complex geometries [62][63][64]. In a typical procedure, freshly prepared or clean substrate is immersed in a dilute 1-10 mm solution of surfactant compound(s) in high purity solvent for 12-48 h at room temperature. After this period, the slides are withdrawn, rinsed with solvent, and dried under a stream of nitrogen [62]. e driving force for self-assembly is usually the speci c interaction between the head group of the surfactant and  In vitro Signi cantly improves the osteoblast adhesion, proliferation, and alkaline phosphatase (ALP) activity while retaining high antibacterial e cacy after aging for 21 days in PBS [22] In vivo Signi cant increase in bone formation after 4 weeks in vivo in rat femurs and in a rabbit model [29,30] BMP4

In vitro
Induces signi cant alkaline phosphatase activity in pluripotent C3H10T1/2 cells [24] BMP2 In vitro Only 8% of the immobilized BMP2 seems to be available for interaction with the cells and able to induce the signaling cascade with cytocompatible for C3H10T1/2 cells [31] In vitro Reduces the bacterial adhesion (S. aureus and S. epidermidis) and signi cantly promotes attachment, alkaline phosphatase activity, and calcium mineral deposition of both osteoblast and human bone marrow-derived mesenchymal stem cells [20] In vivo Titanium screw implants with nano-anchored oligonucleotides strands hybridized with conjugated rhBMP2 exhibited enhanced bone ingrowth into the perforations and increased bone implant contact after 1 and 4 weeks compared to controls. No di erence was seen after 13 weeks. Bone density around the outer implant surface did not di er signi cantly at any of the intervals. erefore, rhBMP2 immobilized on the surface of titanium implants through nanoanchored oligonucleotide strands can enhance bone implant contact [32] Antiadhesive Polymer coating PEG In vitro Inhibits salivary protein adsorption and the attachment of S. gordonii and S. mutans bio lm was easier to be detached [25] PLL-g-PEG In vitro Human serum adsorbed was below the detection limit of the optical sensor technique (OWLS) (<1-2 ng/cm 2 ). Reduces brinogen adsorption by 96%. Decreases S. aureus adhesion by 89-93% [33,34] PLL-g-PEG conjugated to catechols groups 1 In vitro Suppresses brinogen adsorption. Resists attachment of the cyanobacterium Lyngbya spp.
for at least 100 days [35] PEG conjugate to catechols groups In vitro Resistant to serum proteins (<1 ng/cm 2 to detection limit of OWLS). Almost free of blood protein adsorption. No cytotoxicity against bone-marrow stem cells. Reduces 95% of serum protein adsorption [36][37][38] -

Metal ion incorporation Silver
In vitro Activity e ects against periodontal and peri-implant pathogens, including P. gingivalis, P.
intermedia, A. actinomycetemcomitans, F. nucleatum, Tannerella forsythia, and S. aureus [40,41] In vitro Limits antibacterial activity against P. gingivalis and A. actinomycetemcomitans, probably due to the formation of silver compounds such as AgCl, Ag 2 O, and Ag 2 S [42,43] In vitro Improves the antibacterial e ect against S. aureus and preserves human gingival broblasts viability [44] In vitro Very strong (greater than 4-log or 99.99% reduction) antibacterial e ect against P. aeruginosa for 24 h. Osteoblast adhesion, spread, and proliferation higher than bare-Ti and so does not cause cytotoxicity [45] Zinc In vitro No antibacterial activity against the periodontal bacteria P. gingivalis and A. actinomycetemcomitans, possibly due to the formation of zinc halogens, oxides, or sulphides [43] Copper

In vitro
Antimicrobial e ect on E. coli and especially on S. aureus, with the lethal concentration for S. aureus of 5 μg/ml [46] In vitro Antibacterial activity (90%) against S. aureus [47] International Journal of Dentistry 5 Chlorhexidine into

In vitro
Releases drug above the MIC and degrades after about 2 weeks in vitro [52] Minocycline and rifampicin

In vivo
In a rabbit model that induced infection by inoculating S. aureus in the femoral medullary canal, the coated implants had an infection rate of 38% compared with 100% for the noncoated [53] -Covalent immobilization of biocidal

Vancomycin
In vitro and in vivo Strong bactericidal activity against S. epidermidis and S. aureus over long periods of time (up to 6 weeks) in vitro. Stable bactericidal activity and reduced infection rates when implanted in an infected rat model [54] Vancomycin conjugated to PEG-anachelin

In vitro
Only dead cells (B. subtilis) were detected on surface [55] Gentamicin and penicillin In vitro e covalently immobilized antibiotics retain the antibacterial properties as indicated by a signi cant reduction in the viability of contacting S. aureus [56] AMP Tet213 In vitro Activity against both Gram-positive (S. aureus) and Gram-negative (P. aeruginosa) bacteria with 10 6 -fold reductions of both bacterial strains within 30 min [57] AMP GL13K In vitro Signi cantly fewer live cells of P. gingivalis than disks coated with a control peptide and uncoated Ti under static culture conditions. is GL13K coating showed to be cytocompatible by an adequate proliferation of osteoblasts and human gingival broblasts.
Kills bacteria and prevents formation and growth of S. gordonii bio lms in a drip-ow bioreactor and under regular mild-agitation conditions, with rupture of the cell wall [58,59] Tet-20 In vitro and in vivo Excellent activity against Gram-negative P. aeruginosa and Gram-positive S. aureus, as well as bio lm resistance in vitro. e coating had no toxicity to osteoblast-like cells and showed insigni cant platelet activation and adhesion, and complement activation in human blood. Protects bacterial infection in vivo (rat model) against infected S. aureus [60] hLf1-11 In vitro Reduction in bacterial adhesion, early-stage bio lm formation, and growth of planktonic of S. sanguinis and L. salivarius [61] 6 International Journal of Dentistry the surface of the substrate. Most surfactants used for monolayer studies consist of three distinctive parts: (i) the surface active head group which binds strongly to the surface, (ii) the terminal group that is located at the monolayer surface and normally determines the interfacial properties of the assembly, and (iii) the alkane chain serves as a linker between the head and the terminal groups and facilitates the packing of the molecules in the monolayer with the Van der Waals interactions between adjacent methylene groups that orient and stabilize the monolayer ( Figure 3) [62][63][64][65][66].
Considering that SAMs surface modi cations are at nanoscale, physical and chemical characterizations appear as an important challenge to develop new market products based on this technology. Currently, several specialized surface analytical techniques are available to characterize SAMs for scienti c approaches. e most commonly used techniques in routine SAMs characterization are as follows: ellipsometry, infrared re ection absorption spectroscopy (IRRAS), X-ray photoelectron spectroscopy (XPS), contact angle measurements, near edge X-ray absorption ne structure (NEXAFS), static time-of-ight secondary ion mass spectrometry (ToF-SIMS), surface imaging techniques such as scanning tunneling (STM) and atomic force microscopy (AFM), and electrochemical methods such as capacitance measurements (with cyclic voltammetry or impedance spectroscopy) and heterogeneous electron transfer (cyclic voltammetry). e general analytical capabilities of some of these techniques are presented in Table 2.
SAMs can be of di erent nature according to the surface described in Table 3. e most widely studied and characterized class of SAMs is alkanethiols on gold, which have been used in model systems for various purposes, including corrosion resistance, protein adsorption, cell adhesion, and biosensors. erefore, taking into account the focus of this chapter, a brief description of alkanethiols-SAMs on gold with bioactive osseointegration, antiadhesive, and antibacterial properties is presented below. (For more details on the structure and assembly, preparation, and characterization of gold-alkanethiol monolayers, see references [62,66,72]).

SAMs on Gold as Model Surface for Biomaterials
SAMs of alkanethiols on gold have been used as model surfaces for modulate cells adhesion, including osteoblasts and broblasts, by the immobilization of speci c ligands or proteins such as RGD peptides and bronectin [80][81][82].
Concerning antiadhesive surfaces, SAMs presenting oligomers of ethylene glycol, commonly prepared using the alkanethiols HS(CH 2 ) 11 (OCH 2 CH 2 ) n OH (EG n , n � 3-7 or OEG), resist the adsorption of several proteins and the adhesion of cells [83][84][85][86][87][88][89][90][91]. OEG-SAMs on gold present low adsorption of several blood proteins and blood cell adhesion as well as adhesion of the gastrointestinal bacterial species H. pylori as reported by us [86,[92][93][94]. is antiadhesive e ect has been explained through theoretical and experimental research [95][96][97][98][99], indicating that water penetrates into the (EG) n OH layers of the SAMs forming a stable interfacial water layer, which prevents the direct contact between the underlying surface and the proteins and/cells. In addition, SAMs that comprise an OEG-terminated thiol with an alkanethiol terminated with either a biological ligand or a reactive site for linking to a biological ligand can present the ligands of interest in a structurally well-de ned manner against a background that resists the nonspeci c adsorption of other biomolecules or adhesion of cells. Moreover, the OEG terminal group also does not compromise receptor function either to promote the attachment and proliferation of eukaryotic cells as osteoblast and broblast for improving osseointegration or to avoid the adhesion and colonization of prokaryotic cells following an antibacterial strategy as described below [62].
Antibacterial SAMs strategies have been performed through the immobilization of biomolecules such as antibodies, antibiotics, and antimicrobial peptides by covalent or a nity binding. One of the strongest noncovalent receptorligand binding interactions known in nature is the biotinavidin/streptavidin system, where both streptavidin and avidin have a very high degree of speci city and a nity to biotin (K d � 10 −13 M) with four equivalent sites for biotin [100]. is high binding a nity and selectivity, the symmetry of the biotin-binding pockets that are positioned in pairs at opposite faces of the protein, and the ease of functionalization of diverse biomolecules (e.g.,, antibodies, peptides, and nucleotides) with biotin make the streptavidin-biotin system extremely useful in a wide range of biotechnological applications such as in a nity separations, in diagnostic assays, and for "tagging" of molecules for imaging or delivery of therapeutics [101]. is a nity system is also applied to immobilize biomolecules/ligands onto SAMs surfaces by using alkanethiol terminally functionalized with a biotin moiety ( Figure 4) [102][103][104]. ese SAMs can bind streptavidin or avidin with high coverage, specicity, and activity in such a way to expose two of its binding sites away from the surface. Secondary molecules modi ed with biotin can then be rapidly and conveniently immobilized on these streptavidin/avidin-activated surfaces with minimal impact on their biological activity (e.g., speci city) [105].  International Journal of Dentistry Biotin-containing SAMs (BTMs) have been used in both antiadhesive and antibacterial surface strategies as discussed in Table 4. However, although this system is successful and widely used, it shares the disadvantages of most immobilization schemes requiring chemical modi cation of a protein: (i) chemical modi cation may lead to denaturation or loss of activity and (ii) the presence of multiple sites on the protein available for modi cation results in loss of control over its orientation after immobilization [106].
On the contrary, immobilization by covalent binding provides an irreversible attachment that is required for some application such as coating of implants or microarrays because the ligand should not dissociate or exchange with other compounds [62,85]. Covalent immobilization of some antibacterial ligands on thiol/gold SAMs is presented in Table 4.

Nanostructured SAMs: Trend for Dental Implants
As described previously, SAMs of alkanethiol on gold are the most used, but the formation of well-ordered and strong alkanethiol monolayers has been extremely limited on metal oxides such as the titanium since alkanethiols generally do not adhere to metal oxides or are easily removed by rinsing. Among the self-assembled organic molecules, organophosphorus compounds are somewhat less often characterized compared to thiols but are becoming of great practical interest because of their ability to produce SAMs on a range of metal oxide surfaces including titanium [127,128]. ey also have attracted interest as an alternative to organosilane compounds in the functionalization of metal oxide surfaces due to the large number of available organophosphorus functional molecules and because the reaction mechanisms are not water sensitive [127,129]. As indicated in Table 3, organophosphorus compounds for SAMs can be of organophosphonates (or phosphonic acids) and organophosphates (or phosphate ester), being structurally identical. An organophosphate has 4 oxygens with an alkyl group connected via a phosphoester bond, while organophosphonates have 3 oxygens with a carbon attached directly to phosphorus ( Figure 5(a)). e lack of a hydrolysable P-O-C linkage makes phosphonate compounds more stable in aqueous solution and easier to make SAMs than organophosphate compounds. Phosphonates and phosphonic acids form SAMs on TiO 2 surfaces by the formation of Ti-O-P bonds [127]. e reaction of long-chain alkylphosphonic acids with metal oxide leads to dense, well-ordered SAMs [76,131] that Analytical capability: ++, excellent; +, good, 0, fair; −, poor; −−, not applied.  [127], and in biomedical eld, particularly in dental biomaterials for implants and orthodontic appliances. Like alkanethiol on gold and phosph(on)ate metal oxide, SAMs form monolayers with a "tail-up" orientation and a tilt angle of the hydrocarbon chains of about 30°with respect to the surface normal [128]. e binding mode of organophosphorus molecules has been proposed to be mono-, bi-, and tridentate ( Figure 5(b)), which is dependent on both the surface (i.e., titanium) and the nature of the organophosphorus compounds (i.e., phosphonate or phosphate), and being bidentate for titanium [132].
Gawalt et al. [133] have reported that self-assembly of alkanephosphonates on the native oxide surface of Ti can be a ected by a simple procedure of aerosol deposition of octadecylphosphonic acid followed by solvent evaporation with subsequent heating at 120°C for 18 h giving strongly surface-bound, ordered lms of the alkanephosphonate species, which resist removal by solvent washing or mechanical peel testing. Hähner et al. [134] studied the adsorption of octadecyl phosphate (ODP) onto several oxide surfaces including titanium, showing densely packed SAMs with the packing density analogous to that of alkanethiols on gold. Clair et al. [135] studied and compared the assembly of dodecylphosphoric acid (DDPA) on polished and on nanotextured titanium disks. After immersion on DDPA, smooth Ti surfaces presented a water contact angle of 88°, demonstrating successful deposition of a hydrophobic molecular lm, and an average thickness of 20Å, suggesting that a monolayer of material was deposited (the theoretical length of straight molecules is 18Å). However, nanotextured Ti surfaces presented a greater hydrophobicity because of its nanoroughness, with contact angle as high as 120°, which is higher than that in an ideally at surface. Due to the difference between the molecular height (2 nm) and the substrate average pit size (20 nm), the binding behavior of DDPA molecules is expected to be similar on smooth and nanotextured surfaces; considering the fact that previous studies showed that alkanephoshoric acid forms only monolayers on titanium, the authors assumed a similarity in the formation of a monolayer on this (nanorough) surface. Infrared spectroscopy for a at surface provides characteristic methylene group peaks (CH2 υasymm 2933 cm −1 and CH2 υsymm 2858 cm −1 ) while the nanotextured surface presented peaks shifted (CH2 υasymm 2924 cm −1 and CH2 υsymm 2854 cm −1 ), with these di erences in frequencies re ecting physical states of the phosphate monolayer on the surface, from a relatively densely packed phase on nanotextured Ti to a low-density disordered lm on at-polished Ti. In addition, some deterioration of the hydrophobic properties of the lms was observed after 20 days in air and 10 days in bu er solution without further degradation after an additional storage for 1 month in ambient air. However, prolonged exposure of the samples (3 weeks) to the bu er solution resulted in a signi cant desorption of the organic lm. e authors emphasize that alkanephosphoric acid lms are relatively resistant to aging in a physiological-like environment when compared to thiol-based SAMs, in which spontaneous desorption occurs within a few days of immersion in various solvents, and that a further increase of their durability should be possible by optimizing lm properties (e.g., by using longer alkyl chain molecules which should produce better molecular packing in the lm). Spori et al. [136] reported the in uence of chain length on phosphate SAMs showing a higher degree of order and packing density within the monolayers with alkyl chain lengths exceeding 15 carbon atoms forming crystalline structures and with an average alkyl chain tilt angle of 30°to the surface normal, similar to thiol/gold system. Lecollinet et al. [137] studied the adsorption of a monolayer of ve bisphosphonates on oxidized surfaces of titanium and   [118] Mixed SAMs (HS-EG6-COOH + HS-EG3-OH) resist to adsorption of cytochrome c and lysozyme [119] Biotin-containing SAMs having an ethylene glycol background improve the selectivity to streptavidin, by avoiding nonspeci c protein adsorption, and to subsequent biotin-labelled molecules with a right surface orientation [105,[120][121][122] A natural direct thrombin inhibitor (boophilin) was successfully expressed, puri ed, biotinylated, and immobilized on biotin SAMs, able to adsorb thrombin in a selective way to delay surface-induced plasma coagulation, and so be used for the development of novel hemocompatible materials for blood-contacting devices [107] 10 International Journal of Dentistry

N-α-Ac-L-Lys-D-
Ala-D-Ala (N-R-Ac-KDADA) Biospeci c interaction of vancomycin with this fragment from the bacterial cell wall [123] Free carboxylic acid 4 (HS-EG6-COOH + HS-EG3-OH) + EDC/NHS chemistry Anti-E. coli O157:H7 SPR biosensor with alkane monothiol surface was demonstrated to be very rapid, sensitive, and speci c for potential application in detection of E. coli O157:H7 [124] Magainin I Reduces by more than 50% the adhesion of bacteria (L. ivanovii, E. faecalis, and S. aureus) at the surface, together with the killing of the bacteria that nonetheless adhered to the surface [125] Biotin Biotin-H. pylori glycan structures Several biotinylated adhesins speci c to di erent strains of H. pylori were bound onto biotin SAMs showing that these immobilized ligands maintain an ability to speci cally bind H. pylori and thus o ering new insights into innovative strategies against H. pylori infection based on the scavenging of bacteria from the stomach using speci c H. pylorichelating biomaterials [126] International Journal of Dentistry 11 stainless steel. e authors highlighted that the molecule having a per uoropolyether linked to a bisphosphonate moiety can resist harsh conditions, such as lasting water immersion at 50°C for 6 months, at di erent pHs, autoclaving at 121°C, and biocorrosion required for dental application.
However, in those studies and others described below for immobilized bioactive molecules [138][139][140][141][142][143][144][145][146][147], the amphiphile adlayers were produced from solutions of alkanephosphonates in organic solvents, which can reduce the biocompatibility of the surface [127,130]. Tosatti et al. [148] applied aqueous phosphates to titanium oxide and titanium metal lms to serve as smooth, at model surfaces, and a special titanium dental implant surface with a rough, highly corrugated surface. XPS showed to form densely packed SAMs onto titanium not only as at surface but also for high surface area materials, such as the SLA dental implant surface, with the phosphate headgroups attaching to the titanium (oxide) surface and the terminal end group (either methyl or hydroxy) pointing toward the ambient environment (air, vacuum, or water). e authors point out that " e technique of spontaneous organization of organophosphate molecules on titanium (oxide) surfaces from aqueous solution is believed to have potential for the modi cation of titanium-based medical implants and devices with the aim of tailoring the surface chemistry (chemical or biological functionalities), including groups such as poly(ethylene glycol), cell-adhesive peptides or growth factors." Complementarily, Zwahlen et al. [149] showed that dodecyl phosphate adsorbed from aqueous solution formed SAMs of comparable quality to those of the longer octadecyl phosphate prepared from organic solvent and those of similar thiol/gold systems. erefore, such mixed SAMs on metal oxide surfaces are of particular interest to the biosensor and biomaterial eld, because they allow tailoring surface properties in a precise manner and may prove to be highly relevant for controlling the interaction between the SAM-modi ed surface and biological systems, such as proteins, antibodies, and cells.

SAMs for Bioactive Osseointegration on Titanium.
Viornery et al. [150] rstly showed the formation of a chemical link between Ti disks and three phosphonic acids in water. e bioactivity of the modi ed Ti disks was evaluated by incubating these disks in a physiological solution (Hank's balanced salt solution (HBSS)) for 1, 7, and 14 days. Modi ed surfaces showed only slightly higher calcium levels in the XPS analysis compared to the reference Ti surface, with the surface modi ed with ethane-1,1,2-triphosphonic acid (ETP) inducing the highest calcium phosphate deposition after 14 days incubation [150]. Afterwards, these same types of phosphonic acid-modi ed titanium disks were evaluated in vitro related to the proliferation, di erentiation, and protein production of rat osteoblastic cells (CRP10/30) [151]. No statistical di erences were found in osteoblast proliferation among the phosphonic acid-modi ed titanium, unmodied titanium, and tissue culture plastic (used as a positive control), indicating that the phosphonic acids used were not cytotoxic to the osteoblasts. For all surfaces evaluated, the alkaline phosphatase activity was comparable as negative control (tissue culture plastic). However, the total amount of protein, and especially the collagen type I synthesis, was sensitive to surface modi cation. On titanium modi ed with ETP, the total amount of synthesized protein was signicantly higher than the titanium control surfaces [151]. en, the authors stated that " e covalently attached phosphonic acid molecules on the Ti-metal surface thus may form a sca old for new bone formation, ultimately leading to bonding of the implant to the host tissue." A di erent strategy to enhance titanium osseointegration was explored by Liu et al. [145] rstly through the introduction of di erent end groups including hydroxyl, carboxylic acid, phosphate, and vinyl via the formation of alkyl-based SAM on a Ti foil. Accordingly, a hydroxyapatite coating was successfully obtained with phosphate and carboxylic acid after soaking the Ti foil in a solution that contained ions at a concentration 1.5 times higher than SBF.
en, these authors [146] investigated hydroxyapatite formation on Ti surfaces with various end groups, demonstrating that carboxylic acid as an end group provided the optimal SAM surface for nucleation and growth of biomimetic crystalline HA. It seems that the a nity of carboxylic acid for nuclei of CaP plays a pivotal role in the promotion of HA crystallization at surface. In addition to the functional groups, alkyl chain length of phosphonic acids should also be considered as a factor that in uences hydroxyapatite crystallization at surface. Next, Wu et al. [147] evaluated the deposition from simulated body uid of CaP onto carboxylic acid-terminated phosphonic acid SAMs with three di erent lengths of an alkyl chain (3, 6, and 16 methylene units) to compare the ability of promoting CaP crystallization. SEM, XPS, and X-ray di raction revealed  Figure 5: (a) Structure of organophosphate and phosphonate compounds. Adapted from Durmaz [127]. (b) Di erent bonding modes of a phosphonate unit to a metal oxide surface [130].
that the formation of PA SAMs accelerates the deposition of poorly crystallized HA in an alkyl chain length-dependent manner, primarily due to the higher surface density of Ca 2+ -attracting carboxylic acids. Among PAs studied here, PA containing a 16-carbon alkyl chain gave rise to the titanium surface most e ective for the deposition of hydroxyapatite. Gawalt et al. [138,139] reported that alkylphosphonic acids (11-hydroxyundecylphosphonic acid) self-assemble on the native oxide surfaces of Ti and Ti6Al4V, followed by a heating step that binds the acids strongly to these surfaces as ordered phosphonate lms. ese SAMs with OH-terminated groups were activated to a maleimide group and then immersed in an aqueous solution of cell-adhesive peptide RGDcysteine. e adhesion and spreading of the osteoblasts on RGD-modi ed Ti surface were quite substantial after 24 h and even more so after 3 days. Indeed, cell proliferation continued unabated throughout the test period on this surface. e morphology and actin cytoskeleton of cells were observed by staining with rhodamine phalloidin, with cells remaining small and rounded with no organized actin cytoskeleton on control substrates. However, more than 90% of cells adherent to RGD-modi ed substrates became well spread and organized their actin laments into robust stress bres. Danahy et al. [140] constructed more complex SAMs from α,ω-diphosphonic acids self-assembled on the native oxide surfaces of Ti and Ti6Al4V and thermally treated to get strongly bonded phosphonate monolayers. Data from infrared and X-ray spectroscopies and water contact angle showed that the lms bind to the surface by one phosphonate unit while the other remains free as a phosphonic acid. en, the SAMs were treated with zirconium tetra(tert-butoxide) to give surface Zr complex species. Finally, these surface-bound alkoxides can be further derivatized with the insertion of maleimide group followed by binding the RGD-cysteine peptide. Surfaces modi ed with RGD were stable to hydrolysis under physiological conditions and mechanically strong and shown to be e ective to promote osteoblast adhesion and proliferation with organized actin laments and vinculinpositive focal adhesions.
Adden et al. [141] used two di erent phosphonic acid monolayer lms for immobilization of bioactive molecule BMP2 on titanium surfaces. Monolayers of (11-hydroxyundecyl) phosphonic acid and (12-carboxydodecyl) phosphonic acid molecules were produced by a simple dipping process and the terminal functional groups on these monolayers were activated (carbonyldiimidazole for hydroxyl groups and N-hydroxysuccinimide for carboxyl groups) to bind amine-containing molecules as the BMP2. e hydroxyl-terminated SAM is better ordered and orientated than the carboxyl-terminated SAM, and the CDIactivated surfaces (OH-terminated SAMs) gave higher amounts of BMP2 bound than the NHS-activated surfaces (COOH-terminated SAMs).
A di erent bioactive SAMs coating strategy was done by Mani et al. [152,153] with the use of OH-terminated phosphonic acid SAM on Ti prepared from aqueous solution followed by the chemical attachment of a model drug ufenamic acid through three di erent methods of esterication (acid chloride esteri cation, dry heat esteri cation, and direct esteri cation). e drug release pro les of TSAMs prepared via acid-chloride esteri cation exhibited large data scatter, probably because the drug molecules were not uniformly attached to SAM-coated metal surfaces while TSAMs prepared by dry heat and direct esteri cation methods showed an initial burst release of the drug followed by a sustained slow release for up to 2 weeks. us, this study suggests "the potential for using SAMs as an alternate system for delivering drugs from coronary stents and other metal implants" [152]. en, Mani et al. [153] used SAMs with ufenamic acid only attached by direct esteri cation to study their interaction with human aortic endothelial cells (HAECs), showing that the adhesion of HAECs on TSAMs was equivalent to that of control metal surfaces and superior to that of plain glass surfaces with the cells continued to proliferate on TSAMs even though the rate of proliferation was slower than plain glass or control-Ti. Moreover, the spreading of HAECs on TSAMs with typical polygonal shape indicated that these surfaces are conducive to endothelialization. e expression of surface adhesion protein (platelet endothelial cell adhesion molecule-1) on TSAMs indicated that the endothelial cells preserved their phenotype on these surfaces.
us, this study demonstrated that TSAMs do not elicit an adverse response from endothelial cells in in vitro conditions.
Recently, Rudzka et al. [154] modi ed cpTi surfaces by producing mixed and patterned SAMs in order to induce hydroxyapatite nucleation and growth for bone tissue engineering. Mixed-SAMs were prepared from aqueous solutions having di erent fractions of 11-hydroxyundecylphosphonic acid (UDPA, -OH terminal group) and 12-phosphonododecylphosphonic acid (PDDPA, -PO(OH) 2 terminal group) and patterned-SAMs from single THF solutions of 16-phosphonohexadecanoic acid (PHDA, -COOH terminal group) and octadecylphosphonic acid (ODPA, -CH 3 terminal group) followed by laser ablation. ese authors have shown that the PHDA-SAMs without laser treatment promote signi cantly the hydroxyapatite formation with smaller clusters, demonstrating that the presence of carboxyl groups on the cpTi surface is more favorable for the hydroxyapatite nucleation and growth in SBF than on the laser-ablated surface.
Rojo et al. [155] used a simple, e ective, and clean methodology through the self-assembly chemisorption onto Ti6Al4V alloy surfaces of alendronate, which is a well-known bisphosphonate commonly used in osteoporosis therapy and treatment of other bone diseases. XPS spectroscopy revealed that an e ective mode of bonding is created between the metal oxide surface and the phosphate residue of alendronate, leading to the formation of homogeneous drug distribution along the surface. In addition, in vitro studies showed that alendronate SAMs induce di erentiation of hMSC to a bone cell phenotype and promote bone formation on modi ed surfaces, as evidenced by upregulation in the expression of early markers of osteogenic di erentiation (Runx2, osteopontin (OPN), alkaline phosphatase (ALP), and BMP2).

SAMs with Antiadhesive and Antibacterial Properties on
Titanium. Byun et al. [156] synthetized a PEG-phosphonic acid terminated with an amino group (PA-C11-EG3-NH2) that is used to make SAMs onto titanium surface by aqueous immersion. is denominated pSAM was sequentially modi ed by EMPSA conjugation through EDC/NHS chemistry to insert a terminal thioester functional group followed by PEGylation through NH2-Cys-PEG. Ellipsometry, goniometry, and XPS unambiguously con rmed the presence of PEGs, which provided nonfouling e ects of surfaces, preventing the biological adhesion of cells as the NIH-3T3 adhered cells were reduced by 92.3% after PEGylation.
Amalric et al. [142,143] developed antibacterial nanocoatings on titanium and stainless steel through the functionalization of phosphonate monolayers of mercaptododecylphosphonic acid (MDPA) with silver nitrate (AgNO 3 ) by a two-step scheme: (i) deposition of a thiol-functionalized monolayer by reaction with MDPA solution and (ii) reaction of the terminating thiol groups with silver nitrate to form silver thiolate species. iolterminated groups are expected to react readily with silver cations to form silver thiolates with high formation constants and, accordingly, the silver thiolate groups should be stable toward hydrolysis, but silver ions can be selectively released by exchange between the silver thiolate groups at the surface of the monolayers and the free thiol groups exposed at the surface of the bacterial membrane proteins, with the reaction of Ag+ ions with thiol groups in the bacterial membrane proteins playing an essential role in bacterial inactivation. FTIR con rmed the binding of MDPA to the surface on both the titanium and the stainless steel, suggesting the formation of moderately ordered monolayers compared to alkylphosphonic acid monolayers deposited on similar substrates. XPS analysis con rmed the e ectiveness of these surface modi cations. Postmodi cation with AgNO 3 resulted in the conversion of most of the terminal thiol groups into silver thiolate species, which represented about 60% of all sulfur species in the nal samples, with the density of silver at the surface estimated to 3.5 ± 1 Ag·nm −2 , corresponding to about 0.6 nmol·Ag·cm −2 .
us, the amount of silver was very low compared to other antibacterial silver-coated materials reported in the literature, such as the silver content in stainless steel or titanium samples modi ed by ion implantation or by physical vapor deposition ranging from 80 to 1700 nmol·Ag·cm −2 . Despite their very low silver content, MDPA + AgNO 3 monolayers strongly decreased the bacterial adhesion of the surface compared to the bare titanium or stainless steel substrates: a 3-to 5-log reduction in the number of viable adherent bacteria was found for the four bacterial strains tested (E. coli, S. aureus, S. epidermidis, and P. aeruginosa). Furthermore, the antibacterial e ciency of MDPA + AgNO 3 monolayers remained excellent even after incubation for 3 days at 37°C in fresh human blood plasma, with a 4-log reduction of the number of viable adherent bacteria on the coated substrate compared to unmodi ed substrate. e MDPA + AgNO 3 coating deposited on titanium or stainless steel also strongly decreased the density of bacterial bio lm formed after incubation for 3 days in a culture of E. coli, S. epidermidis, or P. aeruginosa. In addition, the growth of E. coli bio lm on titanium modi ed by MDPA + AgNO 3 was signi cantly inhibited for about 1 week. Even more, since the release of silver ions by hydrolysis of the silver thiolate groups was negligible, the antibacterial e ect observed in this study could result from the exchange of silver between the thiolate groups at the surface of the MDPA + AgNO 3 monolayers and the free thiol groups exposed at the surface of the bacterial membrane proteins. Antibacterial transitory e ect was obtained for an extremely low Ag content compared to conventional coatings, which is important to avoid any toxicity issue and to minimize the release of silver in the environment, which could facilitate the selection of resistant strains. Rather, the aim of these coatings is to prevent contamination during handling of the implant and surgery, then for the rst few days after the implantation, which are considered critical. en, Tîlmaciu et al. [144] showed that these SAMs on titanium signi cantly inhibited E. coli and S. epidermidis adhesion and bio lm formation in vitro, while allowing attachment and proliferation of MC3T3-E1 preosteoblasts. Moreover, osteogenic di erentiation of MC3T3 cells and murine mesenchymal stem cells was not a ected by the nanocoatings. Sterilization by ethylene oxide did not alter the antibacterial activity and biocompatibility of the nanocoatings. After subcutaneous implantation of the materials in mice, MDPA + AgNO 3 nanocoatings exhibit signi cant antibacterial activity and excellent biocompatibility, both in vitro and in vivo, after postoperative seeding with S. epidermidis.
Vaithilingam et al. [157] immobilized Cipro oxacin ® (an antibacterial drug) to a carboxylic acid-terminated phosphonic acid based self-assembled monolayers (SAMs) adsorbed on a selectively laser melting (SLM) Ti6Al4V structure by immersion deposition method in THF. Cipro oxacin-coated Ti6Al4V surfaces are highly stable under the oxidative ambient laboratory conditions for 1, 2, 4, and 6 weeks. When immersed in 10 mm of Tris-HCl bu er (pH 7.4), the drug was observed to release in a sustained manner with 50% of the drug released after 4 weeks and approximately 40% of the drug remaining after 6 weeks. Antibacterial susceptibility tests revealed that the immobilized drug was therapeutically active upon its release against S. aureus and E. coli.
en, these authors also used the same strategy to immobilize paracetamol, which was stable for over four weeks and then began to desorb from the surface, showing a potential to improve biocompatibility and reduce surgical complications after implant placement [158].

SAMs with Dual Biofunctions.
Kang et al. [28] coated titanium with a nonbiofouling poly(poly(ethylene glycol) methacrylate) (pPEGMA) by surface-initiated polymerization to Br-terminated SAMs of phosphonic acid, and BMP2 was chemically conjugated to the activated pPEGMA lms by DSC/DMAP chemistry. e BMP2-conjugated pPEGMA lms induced adhesion and di erentiation of mesenchymal stem cells.
Moreover, Gnauck et al. [159] synthetized a carboxyterminated oligo (ethylene glycol) alkanephosphate with the OEG for resistance against nonspeci c protein adsorption and cells/bacteria adhesion and the COOH-terminal functional group as a linker for the attachment of speci c bioligands, such as peptides and proteins to be present at the surface. XPS data showed that the monomolecular layer is attached with the phosphate group to the substrate but not fully ordered or taking an all-trans conformation. However, this study did not present any result concerning protein adsorption or cell adhesion with only chemical and structure characterization. Bozzini et al. [160,161] synthesized PEGterminated alkanephosphate that was codeposited with OHterminated alkane phosphates from aqueous solution onto TiO 2 lms. XPS and ellipsometry of the resulting mixed SAMs indicate that the PEG density can be controlled by varying the mole fraction of PEG-terminated phosphates in the solutions used during the deposition process, leading to surfaces with di erent degrees of protein resistance [160] and reduction of bacterial adhesion (S. mutans): As the PEG surface density increased, the protein adsorption and bacterial adhesion considerably decreased when compared to uncoated titanium surfaces, while maintaining osteoblast proliferation up to 7 days of culture in vitro with the greatest levels of metabolic activity at the highest PEG surface concentrations [161].
ese results are extremely promising in view of a potential clinical application in dental implants, where reduction of bacteria adhesion and stimulation of bone formation are both highly desirable.

Conclusions
e present review shows that dental-based implant therapy after 30 years is a predictable short-term treatment to patient with full or partial edentulism. However, long-term success and survival of implant need more research looking for a more stable interface tissue/implant. Nanoscale modications of surface implants have been an active scienti c area, where new approaches such as SAMs are providing strategies to modulate tissue response and microbiota microenviroment in terms of bioactivity, antiadhesion, antibacterial, or combined e ects.
(i) Titanium with SAMs for bioactive osseointegration e ect has been highly studied with several strategies such as exposure of carboxylic terminal to promote calcium phosphate or hydroxyapatite deposition, with immobilized cell adhesive RGD peptides to induce osteoblast attachment, spreading, and proliferation and with immobilized bone morphogenetic proteins (BMPs) to promote bone formation. (ii) Antiadhesive and antibacterial SAMs on titanium have been sparsely worked with few studies, based on monolayers with protruding group of ethylene glycol and in the immobilization of metals ions and molecules with activity against bacteria, respectively. (iii) SAMs on titanium with combined bioactivity and antiadhesion or antibacterial e ect have been little described with a monolayer of ethylene glycol to which pro-osseointegration molecules might be immobilized.

Conflicts of Interest
e authors declare that there are no con icts of interest.