according to the u.s . national institutes of health , up to 80% of human bacterial infections involve biofilm - associated microorganisms 1 . among these , implant - related infections
do still have a tremendous impact in orthopaedics and trauma 2 , with high social and economic costs 3 , 4 , posing challenging diagnostic and therapeutic dilemmas 5 .
in fact , peri - prosthetic joint infection ( pji ) remains one of the most feared complications in orthopaedic surgery and among the first reasons for implant failure 6 .
moreover , given the increasing number of hip and knee arthroplasties performed , the prevalence of this complication is rising , with increasing costs for national health systems and increasing biological costs for the patients , such as loss or reduced joint function and deterioration in their physical and psychological health 7 . according to the widely accepted model of the ' race for the surface ' for pji development ,
host and bacterial cells compete for surface colonization , with a low probability of bacterial attachment if host cells adhere to implant first , and vice versa . in the event of bacterial adhesion to an implant ,
in addition , the matrix protects the biofilm cells from various microbicidal agents and stresses , including dehydration , toxins , ultraviolet light , chemical disinfectants , temperature and osmotic shock , and lead them to increased resistance against antimicrobials 9 , 10 . to address the limited efficacy of existing antibiotics in the treatment of established bacterial biofilms , novel approaches are required to prevent bacterial adhesion and biofilm formation 11 .
adhesion is a necessary first step in microbial colonization and pathogenesis and provides a good theoretical target for new preventive and treatment strategies 12 .
bacterial adhesion to surfaces can be divided into a first , reversible phase and a second , irreversible phase .
once an implant is inserted into the body , it is covered by a conditional protein layer composed of host proteins , such as albumin and complement , that act as a reservoir of several receptors for bacterial adhesive ligands , mediating adhesion of free - floating bacteria to the surface of the biomaterials 14 , 15 ; these first adhesions are , however mechanically and biologically unstable .
few minutes after this first , reversible phase , bacterial clusters attached to the surface starts to express biofilm related genes , produce glycocalyx and form mature biofilm , thus transforming the adhesion from reversible to irreversible 16 .
full - formed biofilm can be found few hours after the first bacterial adhesion 17 .
antimicrobial surface coatings can be based on an anti - adhesive principle that prevents bacteria to adhere and form biofilms 18 .
in fact , some polymer coatings , like the hydrophilic polymethacrylic acid , polyethylene oxide or protein - resistant polyethylene glycol can be applied to the surface of titanium implants and result in significant inhibition of bacterial adhesion 19 - 22 .
hydrophobic and superhydrophobic surface treatment technologies have also shown a great repellent antibacterial effect in preclinical studies 23 - 25 .
however , clinical application of completely novel coating technologies and compounds , not otherwise previously tested in humans , appears particularly challenging 26 .
bacterial colonization can also be blocked by an inhibitor interfering with ligand - receptor interaction for bacterial attachment .
one of these inhibitors could be hyaluronic acid ( ha ) , a glycosaminoglycan made up of glucuronic acid and n - acetylglucosamine disaccharide units .
it is a uniform , linear and unbranched molecule , with highly variable length and molecular weight ( up to 106 da ) .
it is abundant in skin ( up to 56% ) and in connective tissues , with a turnover ranging from several hours to a few days depending on tissues .
hyaluronic acid constitutes one of the main components of extracellular matrices . because of its biological properties , ha has several clinical applications ( aesthetic surgery , dermatology , dentistry , orthopedics and opthalmology ) 27 .
extensive studies on the chemical and physicochemical properties of ha and its physiological role in humans , together with its versatile properties , such as its biocompatibility , non - immunogenicity , biodegradability , and viscoelasticity , have proved that it is an ideal biomaterial for medical and pharmaceutical applications 28 , 29 . among its various properties ,
several studies have recently shown the ability of ha to protect against various infectious agents 30 , depending on ha concentration and molecular weight 31 , 32 , while more recently ha interference on bacterial adhesion and biofilm formation has been extensively investigated 33 .
given its high biocompatibility and well known safety profile and the anti - adhesive capabilities , ha and its composites represent an attractive , non - antibiotic , option to mitigate the impact of biofilm - related infections in various clinical settings including implant - related infections . aim of this review is to provide an update of the current evidence concerning ha ability to reduce / prevent bacterial adhesion and biofilm formation .
nearly two decades ago , pavesio et al . 34 were probably the first to describe the ability of ha to resist bacterial adhesion , with particular reference to staphylococcus epidermidis , and its non - fouling properties 35 , proposing coated polymeric medical devices ( e.g. , intraocular lenses , stents and catheters ) to reduce implant - related infections .
in particular , a hydrophilic ha overlayer , linked to the surface of polymethylmethacrylate intraocular lenses ( iols ) , was shown to be able to prevent fibroblasts adhesion and to greatly reduce staphyloccous epidermidis adhesion to the implant surface 36 .
the impact of slime dispersants and anti - adhesives on in vitro biofilm formation on iols was further investigated by kadry and co - workers 37 , using a staphyloccous epidermidis wild strain , isolated from a patient with endophtalmitis ; the authors reported the ability of hyaluronan to reduce bacterial adhesion to iols to 30% , compared with untreated control cells .
the authors suggested the use of adjuvant therapy such as dispersants or anti - adhesives , in addition to the antibiotics in irrigating solutions for bacterial ocular infections .
more recently , the in vitro antiadhesive and antibiofilm activity of hyaluronic acid towards bacterial species commonly isolated from respiratory infections was investigated by drago et al .
33 . in this study , the interference exerted on bacterial adhesion was evaluated by using hep-2 cells , while the antibiofilm activity was assessed by means of spectrophotometry after incubation of biofilm with hyaluronic acid and staining with crystal violet .
the experimental findings clearly demonstrated how hyaluronic acid is able to interfere with bacterial adhesion to a cellular substrate in a concentration - dependent manner .
moreover , staphylococcus aureus biofilm was found to be more sensitive to the action of ha , compared to that produced by haemophilus influenzae and moraxella catarrhalis . concerning more specifically the antimicrobial activity , ha
has also been shown to exert varied bacteriostatic , but not bactericidal , dose - dependent effects on different microorganisms in the planktonic phase 31 , 38 . in this
regard , radaeva et al . reported the inhibiting activity of ha with respect to some pseudomonas species 39 , while ardizzoni and co - workers 30 investigated the effects of ha on 15 atcc bacterial strains , representative of clinically relevant bacterial and fungal species .
their results showed that different microbial species and , sometimes , different strains belonging to the same species , are differently affected by ha . in particular , staphylococci , enterococci , streptococcus mutans , two escherichia coli strains , pseudomonas aeruginosa , candida glabrata and c. parapsilosis displayed a ha dose - dependent growth inhibition , while no ha effects were detected in e. coli atcc 13768 and c. albicans and s. sanguinis was favoured by the highest ha dose . comparing the potential bacteriostatic effect of some of the most commonly used biomatrix materials ( collagen type i , hyaluronic acid , hydroxyapatite , polylactic acid and polyglycolic acid ) on the growth over the first 12h of exposure of some of the most common orthopaedic bacterial pathogens ( staphylococcus aureus , staphylococcus epidermidis , -hemolytic streptococcus , pseudomonas aeruginosa ) , carlson and co - workers 38 found that ha had the most significant bacteriostatic properties on the studied organisms .
31 investigated the potential bacteriostatic effect of hyaluronic acid in different concentrations and molecular weight on oral and non - oral microorganisms ( staphylococcus aureus , propionibacterium acnes , actinobacillus actinomycetemcomitans , pavotella oris and porphyromonas gingivalis ) with potential application in dentistry surgery ; the results showed that different hyaluronan solutions exerted varied bacteriostatic effects on all the bacterial strains .
the authors concluded that the clinical application of hyaluronan in form of membranes , gels , or sponges during surgical therapy may reduce bacterial contamination of the surgical wound , thereby lessening the risk of postsurgical infection and promoting more predictable regeneration .
concerning possible orthopaedic applications , in 2004 harris and richards 40 showed the visualization and quantification of s. aureus adhering to a variety of different treated / coated titanium surfaces . in their study ,
coating titanium with sodium hyaluronate significantly decreased the density of s. aureus adhering to the surfaces and its potential use in osteosynthesis , orthopaedics or dental applications was suggested out . in a very recent review on polysaccharide - based coatings , that have been proposed over the last ten years to impede biofilm formation on material surfaces exposed to bacterial contamination ,
hyaluronic acid was discussed as one of the most studied , with demonstrated non - fouling properties on glass surfaces 41 ; displaying hydrophilic characteristics ( contact angle of 22 ) , this coating was in fact reported to reduce adhesion of s. epidermidis and e. coli by several orders of magnitude compared to the unmodified glass slide .
similarly , adhesion of s. aureus on ti foils functionalized with hyaluronic acid - catechol was lower than on pristine substrates .
based on ha antiadhesive properties , a novel ha - based hydrogel has been recently proposed , in order to protect implanted biomaterials in orthopaedics , trauma and dental surgery from bacterial colonization 42 ; this fast - resorbable hydrogel coating , composed of covalently linked hyaluronan and poly - d , l - lactide (  defensive antibacterial coating  , dac , novagenit srl , mezzolombardo , italy ) , has been found to have a synergistic antibiofilm activity with various antibacterials and able to be effectively manually spread onto the surface of various biomaterials commonly used in orthopaedics , trauma and dental surgery 43 ( fig .
1 ) . the ability to completely cover even sand - blasted titanium surface and resist scraping has in fact been confirmed by scanning electron microscopy ( sem ) analysis ( cf .
this is an important requirement in order to reduce the exposed surface of a biomaterial , thus creating a uniform coating of the surface and leaving no pores or cracks that could eventually be colonized by planktonic bacteria . in unpublished experiments ( novagenit srl , data on file ) , in order to evaluate dac ability to prevent bacterial adhesion , 200 mg of hydrogel were homogenously spread on the surface of sterile titanium discs .
hydrogel - coated substrates and uncoated substrates ( controls ) were then placed into sterile 6-wells polystyrene plates and overlaid with a standardized inoculum ( 10 cfu / ml ) of bacterial cells for 15 , 30 , 60 and 120 minutes .
the remaining adhered cells were detached by adding a solution of 0.1% w / v dithiothreitol ( dtt ) ( sigma - aldrich , milan , italy ) to each well and stirring for 15 minutes at room temperature .
then , 100 l of each sample were plated onto tryptic soy agar ( tsa ; merck , darmstadt , germany ) and incubated at 37c for 24 hours for cfu counts .
the results showed that the adhesion density of s. aureus on titanium discs pre - treated with dac , was significantly lower than adhesion on untreated controls at each time point ( fig .
2 ) . in particular , reductions of adhered bacteria equal to 86.8% , 80.4% ,
74.6% and 66.7% vs untreated discs were observed after 15 , 30 , 60 and 120 minutes of incubation , respectively , while an increase of adhesion density during time was observed for both control and pre - treated discs ( fig .
further analyses were conducted to show the ability to dislodge previously adhered bacteria ; to this aim , titanium discs were placed into sterile 6-wells polystyrene plates and overlaid with a standardized inoculum ( 10 cfu / ml ) of bacterial cells in order to allow the adhesion of bacterial cells .
afterwards , 200 mg of hydrogel were spread on the surface of contaminated titanium discs in order to remove previously adhered bacteria .
non - adherent bacteria were removed by rinsing with sterile saline , while the remaining adhered cells were detached by adding 0.1% dtt as previously described .
then , 100 l of each sample were plated onto tsa and incubated at 37c for 24 hours for cfu counts .
the results showed that dac hydrogel treatment of discs reduced the amount of adhered bacteria in respect to control discs after 15 , 30 , 60 and 120 minutes of 84.0% , 72.8% , 72.3% and 64.3% , respectively ( fig.4 ) .
once again , an increase of adhesion density during time was observed for both control and treated discs ( fig .
dac hydrogel showed similar or superior in vitro activity , compared to various antibacterials and a synergistic activity when used in combination ( fig .
were grown on chrome - cobalt devices in 6-wells polystyrene plates containing tsb for 24 hours at 37c .
then , growth medium was removed together with non - adherent bacteria and new broth added .
the plates were incubated at 37c in ambient air , until a visible biofilm was obtained .
gentamycin and vancomycin were tested at a final concentration of 20 mg / ml . similarly , when mixed with the hydrogel , 60 mg of gel powder were reconstituted with 1 ml of water for injections containing gentamicin or vancomycin at 20 mg / ml concentration .
amount of biofilm at each time was determined before hydrogel and antibiotic agents addition and after 0.5 , 1 , 2 , 4 , 6 , 24 and 48 hours of incubation by a spectrophotometric assay .
in particular , at each time , broth was removed and biofilm stained with crystal violet .
after elution of the stain from implants with absolute ethanol , the amount of biofilm was quantified by reading optical density ( o.d . ) at a wavelength of 595 nm against blank ( consisting of ethanol ) .
amount of biofilm at each time was compared with that formed on the same type of implant before treatment .
each assay was performed in duplicate and repeated for three times . at each time point , both for gentamycin and vancomycin showed only a partial inhibition of biofilm formation ( ca .
40 - 50% for vancomycin ) , with minor difference between the two studied microorganisms . on the other side ,
50% in comparison to the untreated controls , while a combination of the hydrogel with either antibacterial resulted in a larger reduction of biofilm formation ( approximately 75 to 80% in comparison with untreated controls ) .
both these experimental studies show the ability of the dac hydrogel to significantly reduce bacterial adhesion and biofilm formation of common bacterial pathogens , thus potentially providing an effective protection of the implant ; however , these data also point out how , in the clinical setting , in the absence of an adequate immune response from the host and/or of sufficient local levels of antibiotics , a passive antiadhesive coating 18 like ha can be overcome by the remaining bacteria in a time - dependent manner .
for this reason , any passive antiadhesive coating of implants 44 should probably better be seen as a tool to reduce and delay bacterial adhesion and biofilm formation to a variable degree , also depending on the local environment , the contaminating bacterial species and initial bacterial load ; this may still provide an additional advantage to the host 's cells to first colonize the implanted biomaterial and win the competition with the microorganisms that may eventually be present , thus contributing to reduce the occurrence of implant - related infections .
several clinical local applications of ha to reduce the impact of biofilm - related infections have been reported , in different clinical settings , with favourable results and no adverse events .
45 recently described topical administration of hyaluronic acid in children with recurrent or chronic middle ear inflammations and chronic adenoiditis . in this prospective , single - blind , randomised controlled study , otoscopy , tympanometry and pure - tone audiometry in children which received the daily topical administration of normal 0.9% sodium chloride saline solution ( control group ) or 9 mg of sodium hyaluronate in 3 ml of a 0.9% sodium saline solution was performed .
the final analysis was based on 116 children ( 49.1% boys ; mean age , 62.9  17.9 months ) : 58 in the control group and 58 in the study group . at the end of follow - up ,
the prevalence of patients with impaired otoscopy was significantly lower in the study group ( p value = 0.024 ) compared to baseline but not in the control group . in comparison with baseline ,
the prevalence of patients with impaired tympanometry at the end of the follow - up period was significantly lower in the study group ( p value = 0.047 ) but not in the control group .
the reduction in the prevalence of patients with conductive hearing loss ( chl ) ( p value = 0.008 ) and those with moderate chl ( p value = 0.048 ) was significant in the study group , but not in the control group .
the mean auditory threshold had also significantly improved by the end of treatment in the study group ( p value = 0.004 ) but not in the control group .
several studies have also reported the beneficial effect of topical ha in chronic urinary tract infections ( uti ) .
in contrast to traditional antibiotic therapy , which aims at eradicating pathogens , treatment with ha targets bacterial adherence to the bladder mucosa with the presumption that a damaged glycosaminoglycan mucous layer facilitates bacterial adherence and therefore recurrent uti 46 . among others 47 , 48 ,
lipovac and colleagues evaluated the efficacy of nine ha bladder instillations over 6 months in 20 women with a history of recurrent uti .
their status was assessed prospectively but compared with a retrospective review of patients ' charts .
the number of infections per year per patient was significantly reduced ( from 4.990.92 to 0.560.82 , p>0.001 ) and the mean time to recurrence ( from 76.724.6 to 178.325.5 days , p>0.001 ) was prolonged significantly .
nevertheless 65% of treated patients were free of recurrences until the end of study ( 47.6 weeks ) 49 .
were able to provide a higher level of evidence by reporting a prospective , randomized , double - blind , placebo - controlled study , in which a significant reduction of 77% ( p<0.0002 ) in the uti rate per patient per year versus placebo was observed at the end of the study .
moreover , mean time to uti recurrence was significantly prolonged ( 185.278.7 versus 52.733.4 days , p<0.001 ) after treatment compared with placebo .
overall urinary symptoms and quality of life measured by questionnaires significantly improved compared with placebo 50 .
very recently a multicentre european study confirmed the efficacy of intravesical administration of combined hyaluronic acid and chondroitin sulphate ( cs ) for the treatment of female recurrent urinary tract infections 51 .
a total of 276 adult women received intravesical administration of ha+cs or standard of care ( antimicrobial/ immunoactive prophylaxis/ probiotics / cranberry ) . at follow - up ,
181 patients treated with ha+cs and 95 patients treated with standard of care from 7 centres were available .
the crude and adjusted ors ( 95% ci ) for bacteriologically confirmed recurrence within 12 months were 0.77 ( 0.46 to 1.28 ) and 0.51 ( 0.27 to 0.96 ) , respectively .
studies were also undertaken to determine the effect on clinical variables , sub - gingival bacteria and local immune response brought about by application of hyaluronan - containing gels in early wound healing after scaling and root planing ( srp ) in dentistry 52 , 53 . in the study reported from eick et al .
the exclusion criteria were : antibiotics intake in the 6 months before the study , periodontal treatment received during the previous year , pregnancy , nursing , smoking , chronic diseases such as diabetes mellitus or rheumatoid arthritis , and allergy to ingredients in the drug . in the test group ( n=17 ) , a 0.8% hyaluronan - containing gels ( ha ) was introduced into all periodontal pockets during srp and a 0.2% ha gel was applied by the patients onto the gingival margin twice daily during the following 2 weeks while the control group ( n=17 ) was treated with srp only ; no placebo was used . probing depth ( pd ) and clinical attachment level ( cal )
were recorded at baseline and after 3 and 6 months , and subgingival plaque and sulcus fluid samples were taken for microbiologic and biochemical analysis . in both groups , pd and cal
the changes in pd and the reduction of the number of pockets with pd5 mm were significantly higher in the test group after 3 ( p=0.014 and 0.021 ) and 6 ( p=0.046 and 0.045 ) months .
six months after srp , the counts of treponema denticola were significantly reduced in both groups ( both p=0.043 ) , as were those of campylobacter rectus in the test group only ( p=0.028 ) .
although to date no surface modification has been reported to be able to fully prevent bacterial adhesion and biofilm formation 55 , available data show that hyaluronic acid has a proven in vitro antiadhesive / antibiofilm effect against some of the most common pathogens and it has been used safely , alone or in combination with other polymers , with satisfactory results in different conditions associated with biofilm - related chronic infections .
clinical data in various applications , including dentistry , urology , wound management , dermatology and orthopedics , allow to consider the potential use of ha as a protective coating barrier of implants particularly safe and feasible on a large scale basis .
while antibacterial coatings to mitigate the occurrence of implant- and biofilm - related infections are regarded as one of the most needed technology , currently only few and insufficient options are available for clinical use in orthopedics and trauma surgery 18 .
considering the pathogenesis of implant - related infections , any protection offered by a fully biocompatible antiadhesive barrier , like ha and some of its derivatives , could be extremely useful to reduce the tremendous burden of implant - related infections .
on the other hand , it should be noted that hyaluronic acid as a passive protective barrier has some limits . among others ,
the antiadhesive / antibiofilm effect is limited and may vary , depending on the type of the microorganism , the bacterial load , the local environment , etc .
; moreover , ha protection may be neutralized by the possible ability of some bacteria to produce hyaluronidase , an enzyme that catalyzes the degradation of hyaluronic acid 56 , while collagen and hyaluronan may even become possible ligands for microbial attachment in particular situations 57 , 58 .
to overcome at least some of these limits , possible loading of hyaluronic - based hydrogels with antibiotics is technically feasible and has been proposed by different authors 59 - 62 , being a possible option for future developments and large scale clinical applications , provided that regulatory requirements can be met .
 living in biofilms is probably the most common condition for bacteria and fungi and biofilm - related infections account for the majority of bacterial infectious diseases worldwide.among others biofilm - related infections , those associated with implanted biomaterials have an enormous and still largely underestimated impact in orthopaedics and trauma , cardio - surgery and several other surgical disciplines.given the limited efficacy of existing antibiotics in the prevention and treatment of bacterial biofilms , new strategies are needed to protect implants and host tissues , overcoming the striking ability of the microorganisms to adhere on different surfaces and to immediately protect themselves by forming the biofilm matrix.adhesion is a necessary first step in microbial colonization and pathogenesis and provides a potential target for new preventive and treatment approach.among various polymers , tested as antibacterial coatings , hyaluronic acid and some of its composites do offer a well - established long - term safety profile and a proven ability to reduce bacterial adhesion and biofilm formation.aim of the present review is to summarize the available evidence concerning the antiadhesion / antibiofilm activity of hyaluronic acid and some of its derivatives to reduce / prevent bacterial adhesion and biofilm formation in various experimental and clinical settings . 
Introduction
Anti-adhesive and anti-biofilm properties of hyaluronic acid and its composites
Clinical applications of hyaluronic acid to prevent bacterial adhesion
Conclusion