Titanium and its medical-grade alloys, in particular the Ti6Al4V alloy used in this study, represent the materials of choice for realizing a wide range of dental implant components. Due to its excellent biocompatibility and resistance to corrosion, high strength and good mechanical properties [42–44] Ti6Al4V is largely used in dental implantology, especially for transmucosal components where limiting biofilm formation is a crucial factor. Adhesion and biofilm formation at the implant surface due to oral microbiota or pathogenic microorganisms introduced during or after surgery is one of the main causes of dental implants failure [45, 46]. The formation and preservation of an effective mucosal seal around the transmucosal components [47–49] and a quick osseointegration process [49, 50] are essential for limiting the microbial migration to the coronal portion of the implant. Therefore, effective solutions for oral implantology should inhibit microbial cell attachment and biofilm formation at the surface of the implant and of transmucosal components preserving biocompatibility and bioactive properties required for the integration with the peri-implant tissues.
Within the scientific community there is still an open debate about how to address and resolve the problem of peri-implant diseases: some researchers advocate a return to smooth or less rough surfaces [51, 52]. Other authors suggest improving the mucosal seal around implants, being considered the true entrance door for the microorganisms [53, 54]. In addition to this, some scientists believe that a revision of the indications and criteria of modern implantology is needed . Most probably, a multilateral prevention and a structured therapeutic intervention could represent a promising approach. Specific and regular check-ups of the peri-implant tissues, and the evaluation and mitigation of risk factors (e.g. periodontitis, smoking, systemic diseases) are effective precautions [13, 56–58], but still insufficient to have a resolutive impact on the incidence of the disease.
Peri-implantitis is therefore a well-established disease and urges for new strategies, procedures and devices able to counteract or prevent its occurrence. Recently, it has been suggested that new surfaces or coatings with antibacterial or anti-biofilm properties should be developed and tested [59, 60]. Microbial biosurfactants emerged as new of anti-biofilm agents for coating implantable devices preserving their biocompatibility. However, published studies are mainly focused on the evaluation of antimicrobial activity on planktonic cells [61, 62]. A limited number of studies addressed the potential of rhamnolipid biosurfactants in preventing microbial adhesion and biofilm formation on conditioned surfaces [30, 63–66], and to our knowledge, this is the first time that an anti-biofilm rhamnolipid-coating was applied on medical-grade titanium.
Zezzi do Valle Gomes et al.  reported that a polystyrene surface pre-coated with a 1.0% aqueous solution of rhamnolipids produced by P. aeruginosa LBI reduced by 58% and 68% the adhesion of Listeria monocytogenes and S. aureus respectively. The anti-biofilm activity of the rhamnolipid biosurfactant produced by Burkholderia thailandensis E264 was explored by Elshikh et al. against some typical oral colonizers . An inhibition of biofilm formation of 57% for Streptococcus oralis, 70% for Neisseria mucosa and Actinomyces naeslundii and 83% for Streptococcus sanguinis was observed on a polystyrene surface pre-coated with the rhamnolipid at a concentration of 6.25 mg/mL .
In a recent work by Ceresa et al. , a coating with the biosurfactant R89, the same used in our study, has proven to be effective in inhibiting Staphylococcus spp. biofilm formation on medical-grade silicone up to 72 h, with an overall inhibition of 76% for S. aureus and 63% for S. epidermidis. Mass spectrometry analysis of R89BS revealed that the BS crude extract is a mixture composed by homologues of mono-rhamnolipids (75%) and di-rhamnolipids (25%). In addition to anti-biofilm properties, R89BS showed antibacterial activity on Staphylococcus spp. planktonic cells, with a minimal inhibitory concentration (MIC) values of 0.06 mg/mL for S. aureus and 0.12 mg/mL for S. epidermidis . Stemming from these encouraging results, we addressed the possibility of realizing a coating with R89BS on titanium.
In this study, the rhamnolipids-coating was realized by R89BS physical adsorption at the titanium surface, and its anti-biofilm efficacy was investigated on Staphylococcus aureus and Staphylococcus epidermidis up to three days. S. aureus and S. epidermidis were selected as they represent the two major bacterial strains responsible of titanium implant-related infections. They are frequently introduced during implants surgery or in the post-operative period, causing infections that generally involve the formation of an antibiotic-resistant biofilm .
Different quantitative aspects of microbial biofilm were considered in this study, such as total biomass and cell metabolic activity. The designed experimental conditions allowed the reproducible development of a mature and structured staphylococcal biofilm on the titanium surfaces. The R89BS-coating was able to significantly reduce Staphylococcus spp. cells adhesion over time, showing a remarkable effect at 24 h with a biofilm inhibition of about 97% and 61% for S. aureus and S. epidermidis respectively. At more prolonged incubation times, the inhibition decreased, but was still able to guarantee a significantly lower amount of biofilm in coated samples compared to uncoated controls.
The anti-biofilm activity of R89BS can be related to its ability to reduce titanium hydrophobicity, possibly because of the R89BS molecules orientation at the surface, interfering with the hydrophobic interactions responsible of the initial adhesion of the microbial cells to a solid surface. According to Walencka et al. , biosurfactants may affect both the interactions of bacterial cells with each other and with the surface, thanks to their ability to reduce surface tension and to change bacterial cell walls charge. Moreover, at neutral pH condition the carboxylic groups of the fatty acid alkyl chain are mainly in the anionic form, so an electrostatic repulsion is established between the negative charges of the bacterial surface and the negative charges of the biosurfactant molecules on the titanium surface [67, 71].
In this study, the anti-biofilm efficacy of R89BS-coating was also assessed on three representative titanium surfaces used in the manufacturing processes of dental implants and/or implant components (e.g. transmucosal abutments and healing abutment). L-L and RBT disc surfaces, obtained by laser-etching and blasting respectively, are characterized by higher surface roughness compared to the M&P titanium. In literature, it is reported that rougher surfaces support differentiation, growth and attachment of bone cells, and increases mineralization, promoting osseointegration essential for implants success [72–75]. This is one of the reasons why several different morphologies were developed and deployed on the market and different morphologies are often adopted for different parts of the same implant. However, it seems that an increase in surface roughness also enhances bacterial adhesion and biofilm formation [76–78]. The coating with R89BS resulted effective in inhibiting Staphylococcus spp. adhesion on the three tested commercial surfaces, with comparable (for S. aureus) or better (for S. epidermidis) results with respect to those obtained with laboratory prepared surface, highlighting the potential of this biosurfactant in preventing dental implants colonization, irrespective from their surface morphology.
Another important aspect of R89BS-coated titanium is its low cytotoxicity. Ceresa et al.  reported no significant cytotoxicity on eukaryotic cells for R89BS in solution at concentrations less than or equal to 0.2 mg/mL. In this work, further data were provided by using an additional biocompatibility test, closer to the destination of use of the titanium component, by simulating the possible elution of the BS from a titanium device into a liquid medium. No cytotoxic effect was detected when eukaryotic cells were exposed to the eluate from R89BS-coated titanium discs. These data enlarge the body of evidence that support further testing toward in-vivo applications.
Despite the interesting results we obtained, there are some limitations of our study that, if addressed, will provide more accurate data. Although S. aureus ATCC 6538 and S. epidermidis ATCC 35984 are widely recognized as biofilm former strains and positive results with these strains are encouraging, further tests have to be carried out, possibly including biofilm forming strains of relevance for the microbiome of the peri-implant diseases [79, 80]. In that regard, additional investigation could be performed on multi-species biofilms formed on titanium surfaces by commonly initial, early, secondary and late dental colonizers (such as Streptococcus oralis, Veillonella parvula, Fusobacterium nucleatum and Porphyromonas gingivalis) in a protein-rich medium in anaerobic conditions as described by Sanchez et al. .. Some limitations are however present in realizing robust biofilm model in-vitro due to stringent culturing conditions required by the majority of the oral microorganisms. These limitations could be overcome by using a flow chamber system in which biofilms could grow under hydrodynamic conditions and the environment could be carefully controlled and easily modulated . Animal in-vivo studies could possibly provide the best testing conditions and should be considered in future.
A further limitation is represented by the loss of anti-biofilm efficacy in time. Although 72 h could be relevant to protect the implant immediately after surgical placement, a prolonged efficacy is desirable to prevent the onset of the peri-implant diseases at later stages after installation. An explanation of the gradual reduction of R89BS-coating efficacy over time can be found in the nature of the bonds between the titanium surface and the biosurfactant. Physical adsorption is a simple method to coat the titanium for a short time since interactions of the BS molecules with the surface are realized through weak bonds based mainly on hydrophobic and van der Waals interactions. A progressive detachment of R89BS with time is possible when the implant is exposed to an aqueous environment. This gradual loss may generate areas of uneven coating where microbial cells can adhere creating, gradually, thicker biofilms with the consequent observed reduction of activity. Alternative bonding strategies should be assessed in the future to improve durability of R89BS-coating and long-term anti-biofilm efficacy, for example through chemical modification of the titanium surface in order to promote a covalent bonding of the active molecule to the surface.
Eventually, the clinical application of this coating to dental implants, or to titanium transmucosal components, requires several additional tests before being considered safe and effective. Biocompatibility test performed up to now were limited to cytocompatibility assay and cannot guarantee that interaction with bone or soft tissues is not affected by the presence of the R89BS coating. Additional biocompatibility tests with relevant cell lines (e.g.: gingival fibroblasts, osteocytes) have to be considered before animal testing.
A range of possible clinical applications can be envisaged. The R89BS coating can be considered for application not only to the dental implant, but also to other titanium implant components (e.g. titanium transmucosal components). Temporary transmucosal components (e.g. healing abutments) coated by R89BS can also be considered as possible adjuvant in the healing process of an infected gum, having the advantage of being removable and exchanged during the healing process. Moreover, different ways to coat titanium surfaces with R89BS can be considered, ranging from the application of the coating at the end of the component manufacturing process, to the “on site” application realized by the dentist before the implant placement or at implant revision. The surface treatment can be limited to specific areas of the implant component (e.g.: implant shoulder, abutment external surface) by selectively masking other areas before application. This could help preserving peculiar surface characteristics and peri-implant tissue interaction.