Biofilms and oral health: nanotechnology for biofilm control.

The human oral cavity hosts a diverse community of microorganisms, with over 700 species identified to date [ 264 ]. Such a complex ecosystem includes various habitats such as the teeth, gingival sulcus, tongue, cheeks, hard and soft palates, and tonsils, all colonized by bacteria, archaea, viruses, fungi, and protozoa [ 93 , 303 ]. Together, these microorganisms, along with those found in contiguous extensions up to the distal oesophagus form the human oral microbiome [ 50 , 121 ]. Many oropharyngeal infections are polymicrobial, often requiring the cooperation of multiple organisms. However, these microorganisms can also engage in competitive and antagonistic interactions to maintain the oral microecology, which plays a crucial role in determining overall oral health [ 190 , 269 , 345 ]. Within these mixed biofilm communities, communication happens through metabolites or cell contact signals, enabling them to adjust population density, alter gene expression, modulate host responses, and promote disease [ 297 ]. Microorganisms from the oral microbiome have been linked to a range of oral infections, including caries, periodontitis, endodontic infections, alveolar osteitis, and tonsillitis [ 45 , 287 , 291 , 329 ]. Additionally, emerging research has associated these microorganisms with systemic conditions such as cardiovascular diseases, pneumonia, diabetes, stroke, and even certain types of cancer, highlighting the broader health impact of oral microbiota imbalances [ 33 , 198 , 322 , 342 , 356 , 379 ]. Therefore, this section explores the complexity of the microbial community that plays a fundamental role in both oral and overall health. Porphyromonas gingivalis ( P. gingivalis ) is a Gram-negative, anaerobic bacterium that plays a significant role in the development of periodontitis, a chronic inflammatory condition affecting the tissues supporting the teeth (periodontium) [ 118 ]. Chronic periodontitis can lead to severe consequences such as soft tissue degradation, bone loss, and eventually tooth loss [ 178 , 325 ]. As sugars are limited in deep periodontal pockets, P. gingivalis relies on fermenting amino acids such as glutamate, aspartate, valine, leucine, and cysteine for nutrition [ 58 , 349 ]. P. gingivalis and other periodontal pathogens such as Prevotella intermedia and Porphyromonas endodontalis act as late colonizers forming microcolonies on existing biofilms [ 395 ]. The colonization by P. gingivalis is facilitated by a range of virulence factors such as lipopolysaccharides, fimbriae, cysteine proteinases (gingipains), capsular polysaccharides, heme binding proteins, Type IX secretion system (T9SS), peptidylarginine deiminase and outer membrane vesicles that contribute to its pathogenicity [ 44 , 99 , 296 , 325 ] (Fig.  1 , Table  1 ). Fig. 1 Virulence factors of P. gingivalis . Type IX secretion system (T9SS) is responsible for secreting gingipains and other effector molecules. Gingipains contribute to the degradation of host proteins, while fimbriae facilitate adhesion to oral epithelial surfaces and other bacterial species. P. gingivalis also produces additional virulence factors, including the capsule, lipopolysaccharides (LPS), hemagglutinins, peptidylarginine deiminase (PPAD), and outer membrane vesicles (OMVs), all of which contribute to bacterial attachment and virulence [ 325 ] Table 1 Virulence factors and associated infections of oral pathogens Species Diseases caused Site of infection Symptoms Virulence factors Existing treatment options References Porphyromonas gingivalis Dental caries, periodontitis, and peri-implantitis Teeth, gingiva and periodontal tissue Inflammation of periodontal tissue, redness, swelling, and bleeding of gingiva,, formation of periodontal pockets and halitosis Gingipains, Fimbriae, LPS, Collagenase, Aminopeptidase, Capsule, PPAD Mechanical: Supragingival and subgingival scaling, root planing (SRP) Antibiotic classes: Tetracycline, Macrolides, Lincosamides, Nitroimidazoles, β-lactams Antiseptic: Chlorohexidine mouthwashes and gels Surgical Intervention: Flap surgery, Bone/Tissue grafts, and Laser Therapy Aoki et al. [ 31 ], Bostanci and Belibasakis [ 58 ], Gerits, Verstraeten, and Michiels [ 124 ], Haque, Yerex, Kelekis-Cholakis, and Duan [ 142 ], How, Song, and Chan [ 149 ], Jan van Winkelhoff [ 161 ], Jia et al. [ 162 ], Keestra, Grosjean, Coucke, Quirynen, and Teughels [ 173 ], McBain et al. [ 227 ], Persson and Renvert [ 268 ], Ray [ 287 ] Candida albicans Candidiasis subtypes: Pseudomembranous candidiasis, Acute erythematous candidiasis, Chronic erythematous candidiasis, and Chronic hyperplastic or nodular candidiasis Tongue, inner cheek, enamel, dentin and cementum Creamy white lesions on tongue, buccal mucosa, roof of mouth and tonsils, Angular cheilitis, Median rhomboid glossitis, Soreness, difficulty in swallowing Cell wall proteins (Hwp1, Als3), Protein kinases (Ire1, Cbk1), β-1,3-glucans, SAP, Phospholipase, Hemolysin, Transcription regulators( Efg1, Rob1, Ndt80, Bcr1, and Tec1) Antibiotic: Nystatin Antifungal agents: Amphotericin B, Clotrimazole, Miconazole, Ketoconazole, Fluconazole or itraconazole Caprylic acid capsules and tablets Akpan and Morgan [ 10 ], Alnuaimi, Wiesenfeld, O’Brien-Simpson, Reynolds, and McCullough [ 18 ], Bae and Rhee [ 37 ], Chevalier, Ranque, and Prêcheur [ 76 ], Nett and Andes [ 249 ], Rodríguez-Cerdeira et al. [ 293 ], Rosenblatt et al. [ 295 ], Salehi et al. [ 301 ], Talapko et al. [ 350 ], Wibawa [ 373 ] Fusobacterium nucleatum Oral squamous cell carcinoma, dental pulp infection, and periodontitis Gingiva, periodontal pockets, teeth Soreness, swelling, white/red patches on the tongue or gingiva, bleeding, painful swallowing, lumps in the neck or back of the throat Adhesins(Aid1, CmpA, Fap2, FomA, FadA and RadD), OMVs, LPS, serine proteases and butyric acid Mechanical: Flossing, dental cleanup to remove stubborn plaques and tartar buildup Antibiotics: FP 100 (Hygromycin A), Metronidazole, Clindamycin, Amoxicillin, Resveratrol, Cefoxitin, Ampicillin-sulbactam, Tetracyclines Antiseptic: Chlorohexidine mouthwashes and gels Phage therapy Chen et al. [ 75 ] Dabija‐Wolter, Al‐Zubaydi, Mohammed, Bakken, and Bolstad [ 88 ], He et al. [ 145 ], Machuca, Daille, Vinés, Berrocal, and Bittner [ 213 ], McIlvanna, Linden, Craig, Lundy, and James [ 228 ], Yakar et al. [ 378 ], Zhao et al. [ 391 ] Streptococcus oralis Dental caries, gingivitis Tooth surfaces, gingiva, tongue Dark spots, discoloration, pits, or holes in the teeth, tooth sensitivity, swollen gingiva, bad breath SRRPs, IgA1 protease, AsaA, glucosyltransferases, hydrogen peroxide, auto-inducer 2 Mechanical: Flossing, regular brushing Berberine Chloride + Antibiotics: Amoxicillin, ampicillin, erythromycin, oxacillin, clindamycin, linezolid and tetracycline Chu, Trager, Chen, and Shum, [ 80 ], Dziedzic, Wojtyczka, and Kubina [ 104 ] Okahashi et al. [ 257 ], Ren et al. [ 289 , 290 ] Streptococcus mutans Dental caries Tooth enamel, gingiva, pits and fissures of molars and premolars Toothache, swollen gingiva, tooth sensitivity, bad breath Glucosyltransferase(GtfB, GtfC, GtfD), Glucan-binding proteins(GbpA, GbpB, GbpC, GbpD), PacI, Cnm Mechanical: Scaling and polishing, Pit and fissure fillings Antimicrobials: Fluoride, chlorhexidine, xylitol, triclosan Nanoparticles: tannic acid-mediated gold nanoparticles Phage therapy Ben-Zaken et al. [ 43 ], Bowen and Koo [ 59 ], Brady et al. [ 61 ], Gao et al. [ 122 ], Lynch et al. [ 211 ], Selvaraj, Venkatesan, Ganapathy, and Sathishkumar, [ 311 ] Sharma, Ghambir, Gupta, and Singh [ 320 ] Aggregatibacter actinomycetemcomitans Dental caries, periodontitis, and localized aggressive periodontitis (LAP) Gingival tissues, teeth Loose teeth, inflamed or bleeding gingiva, soft tissue abscess in mandibular area, diastema Adhesins (EmaA), Pili(F1p), EPS, leukotoxin A, Cytolethal distending toxin, LPS, hydrogen peroxide, heat shock protein (GroEL) Antibiotics: Metronidazole, amoxicillin, azithromycin, clarithromycin, ciprofloxacin, and moxifloxacin Adha, Ervina, and Agusnar [ 7 ], Benso [ 46 ] Gholizadeh et al. [ 126 ], Lai and Walters, [ 188 ], Rams, Freedman, Chialastri, and Slots [ 283 ], Tsaousoglou, Nietzsche, Cachovan, Sculean, and Eick [ 358 ] Zambon, Haraszthy, Hariharan, Lally, and Demuth [ 388 ] Treponema denticola Acute pericoronitis, Acute necrotizing ulcerative gingivitis, Periodontitis Periodontal pockets Specific symptoms are not yet identified Dentilisin, OMVs, outer sheath proteins, the major sheath protein (Msp), lipoproteins, trypsin like enzymes Mechanical: A full mouth debridement combined with tetracycline therapy Antibiotics: Doxycycline, minocycline, azithromycin, and erythromycin Chi, Qi, and Kuramitsu [ 78 ] Lee and Fenno [ 194 ], Okamoto-Shibayama, Sekino, Yoshikawa, Saito, and Ishihara, [ 259 ], Pisani, Pisani, Arcangeli, Harding, and Singhrao [ 272 ], Sela [ 310 ], Setubal, Reis, Matsunaga, and Haake [ 314 ] Virulence factors of P. gingivalis . Type IX secretion system (T9SS) is responsible for secreting gingipains and other effector molecules. Gingipains contribute to the degradation of host proteins, while fimbriae facilitate adhesion to oral epithelial surfaces and other bacterial species. P. gingivalis also produces additional virulence factors, including the capsule, lipopolysaccharides (LPS), hemagglutinins, peptidylarginine deiminase (PPAD), and outer membrane vesicles (OMVs), all of which contribute to bacterial attachment and virulence [ 325 ] Virulence factors and associated infections of oral pathogens Mechanical: Supragingival and subgingival scaling, root planing (SRP) Antibiotic classes: Tetracycline, Macrolides, Lincosamides, Nitroimidazoles, β-lactams Antiseptic: Chlorohexidine mouthwashes and gels Surgical Intervention: Flap surgery, Bone/Tissue grafts, and Laser Therapy Antibiotic: Nystatin Antifungal agents: Amphotericin B, Clotrimazole, Miconazole, Ketoconazole, Fluconazole or itraconazole Caprylic acid capsules and tablets Mechanical: Flossing, dental cleanup to remove stubborn plaques and tartar buildup Antibiotics: FP 100 (Hygromycin A), Metronidazole, Clindamycin, Amoxicillin, Resveratrol, Cefoxitin, Ampicillin-sulbactam, Tetracyclines Antiseptic: Chlorohexidine mouthwashes and gels Phage therapy Mechanical: Flossing, regular brushing Berberine Chloride + Antibiotics: Amoxicillin, ampicillin, erythromycin, oxacillin, clindamycin, linezolid and tetracycline Mechanical: Scaling and polishing, Pit and fissure fillings Antimicrobials: Fluoride, chlorhexidine, xylitol, triclosan Nanoparticles: tannic acid-mediated gold nanoparticles Phage therapy Mechanical: A full mouth debridement combined with tetracycline therapy Antibiotics: Doxycycline, minocycline, azithromycin, and erythromycin The bacterium’s cysteine proteinases, known as gingipains, include arginine-specific gingipains (RgpA and RgpB) and lysine-specific gingipain (Kgp) can degrade a wide range of host components. These include the basement membrane of periodontal tissue, host proteins, and extracellular matrix elements such as laminin, fibronectin, collagen, and elastin [ 58 ]. Additionally, these gingipains degrade important proteins present in the gingival crevicular fluid, including immunoglobulins, transferrin, and albumin [ 27 , 53 , 89 , 325 ]. The enzymatic activity of gingipains also triggers the release of thrombin and prothrombin, increasing vascular permeability, while the degradation of fibrinogen contributes to increased bleeding at the periodontal site [ 154 ]. Beyond their destructive role, arginine and lysine gingipain complexes also facilitate the adhesion of P. gingivalis to gingival fibroblasts and epithelial cells, further enhancing its colonization and persistence within the host [ 58 ]. The acquisition of heme, which is crucial for the virulence of P. gingivalis , is mediated through specific gingipains, hemophore-like proteins (HmuY, HusA), and outer membrane receptors (HmuR, HusB, IhtA) [ 260 ]. In the early stages of infection, P. gingivalis sources heme from saliva and gingival crevicular fluid, and from erythrocytes during later stages when bleeding occurs [ 333 ]. Gingipains, along with other key effectors such as hemin-binding protein 35, are secreted through the Type IX Secretion System (T9SS) [ 325 ]. Lipopolysaccharides (LPS) of P. gingivalis triggers proinflammatory immune response and bone resorption [ 251 ]. LPS triggers the release of cytokines like Interleukin-1 β (IL-1β), IL-6, and IL-8, leading to damage in periodontal tissues. Furthermore, LPS inhibits vital factors involved in new bone formation, such as collagen type 1 Alpha 1, osteocalcin, and alkaline phosphatase activity, as well as mineralisation in periodontal ligament stem cells (PDLSCs). PDLSCs are crucial for osteoblastic differentiation and periodontal tissue regeneration [ 170 , 313 ]. The capsule (CPS or K antigen) of P. gingivalis makes it resistant to phagocytosis and triggering IgG-mediated immune responses [ 58 ]. Encapsulated strains are more virulent and associated with more spreading infections than non-encapsulated strains [ 63 , 326 ]. Outer membrane vesicles (OMVs) of P. gingivalis contain virulence factors including various C-terminal domain proteins, peptidyl arginine deiminase and gingipains. They help in coaggregation for the formation of polymicrobial biofilms and cause destruction of periodontal tissue by inducing acute inflammation hindering wound healing [ 137 , 363 ]. P. gingivalis expresses two types of fimbriae: FimA (long fimbriae or fimbrillin) and Mfa (short or minor fimbriae). These facilitate bacterial attachment to proline-rich proteins, oral epithelial cells, fibrinogen, and other bacterial species like S. gordonii and Streptococcus oralis ( S. oralis ). Short fimbriae play a role in microcolony formation and biofilm maturation [ 22 , 106 , 183 ]. Whereas, long fimbriae are mainly involved in proinflammatory immune responses and also act as adhesins [ 58 ]. P. gingivalis peptidyl arginine deiminase (PPAD), an enzyme secreted, is responsible for the citrullination of proteins, a post-translational modification that converts arginine residues to citrulline. Citrullinated proteins can travel through the bloodstream to synovial joints, where they act as autoantigens, triggering the production of anti-citrullinated protein antibodies (ACPAs). Citrullinated proteins bound to ACPAs in joints triggers an auto-immune response accompanied by inflammation of joints causing rheumatoid arthritis [ 47 , 123 , 132 , 138 ]. Though the exact involvement of P. gingivalis in atherosclerosis is unknown, citrullinated proteins have been identified in atherosclerotic plaques [ 261 ]. Additionally, periodontitis is considered a risk factor for other conditions, including Alzheimer’s disease, orodigestive cancers, and diabetes mellitus [ 261 , 262 , 315 ]. Candida albicans ( C. albicans ) is a common fungal microorganism in the human oral cavity, typically existing in a harmless yeast form [ 226 ]. Under certain conditions, such as reduced immune competence or an imbalance in bacterial microflora, C. albicans becomes an opportunistic pathogen which leads to oral candidiasis and even systemic candidiasis, involving life-threatening bloodstream infections [ 393 ]. Individuals with compromised immune systems, such as HIV-positive individuals, newborns, the elderly, or those undergoing cancer treatment, are particularly susceptible to C. albicans infections [ 24 , 34 , 174 , 381 ]. Conditions such as xerostomia (dry mouth) and neutropenia further increase susceptibility to C. albicans infection [ 350 ]. Xerostomia, characterized by low salivary flow, creates an environment that favors intense colonization by C. albicans and other fungi [ 65 , 357 ]. In patients with neutropenia, a condition involving reduced neutrophil count, immune defense against C. albicans is weakened, leading to a higher risk of both oral and systemic candidal infections [ 232 ]. Biofilm formation is essential to the virulence of C. albicans , and it contains a mix of yeast, pseudo-hyphal, and hyphal cells encased in an extracellular matrix (ECM) [ 87 ]. A key factor in the pathogenicity of C. albicans is its ability to switch from yeast to hyphal form [ 209 ]. Mutants that cannot make this switch are often less virulent [ 168 , 206 ]. Biofilm formation by C. albicans follows a multi-step process. Initially, yeast-form cells attach to and colonize a surface. These cells then grow and proliferate, forming a basal layer that provides primary stability. As the biofilm matures, the yeast cells transition into hyphal cells, which expand, spread, and contribute to the biofilm's structural complexity. Biofilm maturation typically takes 24 h in in vitro systems, with mature biofilms being characterized by a dense ECM [ 70 ]. The ECM serves to protect the biofilm from physical disruption and enhances resistance to xenobiotics [ 389 ]. In the final stage, hyphal cells disperse from the biofilm, exhibiting heightened virulence and adhesion, enabling them to colonize new sites and establish systemic infections [ 360 , 361 ]. These hyphal cells can invade host tissues by breaking through epithelial and endothelial layers, facilitating the spread of infection and causing tissue damage (Fig.  2 ) [ 136 ]. Fig. 2 Stages of C. albicans colonization on dental surfaces. C. albicans initially adheres to the dental surface and transitions into its more virulent hyphal form. As the biofilm matures, cells can disperse and colonize new sites. Various adhesins play crucial roles at each stage, facilitating adhesion to tooth surfaces and interactions with bacterial species such as S. oralis and S. gordonii [ 14 ] Stages of C. albicans colonization on dental surfaces. C. albicans initially adheres to the dental surface and transitions into its more virulent hyphal form. As the biofilm matures, cells can disperse and colonize new sites. Various adhesins play crucial roles at each stage, facilitating adhesion to tooth surfaces and interactions with bacterial species such as S. oralis and S. gordonii [ 14 ] Saliva plays a vital role in regulating the colonization of C. albicans in the oral cavity. It contains essential ions like sodium, potassium, and chloride, which contribute to its buffering capacity, as well as proteins and glycoproteins that aid microbial adhesion [ 148 , 230 , 302 ]. Certain salivary components, including mucin, fibronectin, and secretory IgA promote microbial aggregation, helping to clear pathogens from the mouth, while antimicrobial peptides such as histatins, defensins, and statherin limit the growth of pathogens [ 110 , 158 ]. Statherin can even revert C. albicans from its pathogenic hyphal state back to its yeast form, potentially reducing the severity of infection, while histatin 5 (Hst5) has notable antifungal properties that help maintain low Candida counts [ 197 , 396 ]. Additionally, components like defensins, cysteine-rich peptides produced as part of innate immunity, can disrupt fungal cell walls through membrane permeabilization, helping protect against infection [ 274 ]. The hyphal cells have specific adhesins, such as Als (agglutinin-like sequence) and Hwp (Hyphal wall protein), which help them bind to oral bacteria like S. gordonii , S. oralis , and P. gingivalis [ 39 , 102 ]. This interaction is crucial for biofilm formation and oral mucosal colonization. Early adhesion genes upregulated in C. albicans include ALS1 , ALS2 , ALS3 , ALS4 , EAP1 , MSB2 , PGA6 , SIM1 , ORF19.2449 , and ORF19.5126 . Seven of these are specifically upregulated in adhered cells, suggesting their role in surface attachment, while three ( MSB2 , ALS3 , and ORF19.2449 ) show surface-independent expression. Later-stage adhesion genes, such as HYR1 , FAV2 , IFF4 , IFF6 , PGA32 , PGA55 , ORF19.3988 , ORF19.4906 , ORF19.5813 , and ORF19.7539.1 are upregulated during biofilm maturation [ 14 , 115 ]. Key master regulators of biofilm formation include Efg1, Rob1, Ndt80, Bcr1, Brg1 and Tec1, which also control hyphal formation, except for Bcr1 [ 252 , 253 ]. Rlm1 and Zap1 regulate extracellular matrix production [ 250 , 254 ], while Ume6, Pes1, and Nrg1 control cell dispersal from the biofilm [ 360 , 361 ] (Fig.  2 ). PES1 , in particular, enhances cell dispersal by regulating the transition from hyphal to yeast form [ 321 ]. C. albicans forms complex biofilms associated with bacteria, particularly species of Streptococcus [ 179 ]. The transcription regulator Efg1, which controls hyphal morphology in C. albicans , is necessary for coaggregation with streptococci . For example, S. oralis increases EFG1 expression in C. albicans during the later stages of biofilm formation, which boosts ALS1 production and enhances biofilm growth [ 376 ]. These interactions between fungal and bacterial cells demonstrate the intricate microbial dynamics in the oral cavity (Table  1 ). Fusobacterium is a genus of anaerobic, Gram-negative bacteria essential for oral biofilm formation and associated with various oral diseases [ 141 ]. Among its species, Fusobacterium nucleatum stands out for its significant role in initiating dental biofilm formation. It adheres to oral surfaces, such as teeth and gingival tissues, through several virulence factors, and acts as a bridging organism within the biofilm community, facilitating interactions between early and late colonizers [ 270 , 300 ]. F. nucleatum employs multiple adhesins (Aid1, CmpA, Fap2, FomA, FadA and RadD) and surface proteins to mediate attachment and biofilm formation, which are critical to its pathogenicity. FadA (Fusobacterium adhesin A) binds specifically to E-cadherin in host epithelial cells, while FomA (major outer-membrane protein) promotes adhesion to both bacterial species and human epithelial cells [ 77 , 134 ]. The YadA-like protein enhances binding to fibronectin and collagen in the salivary pellicle and extracellular matrix [ 134 , 204 , 239 ]. CmpA (coaggregation-mediating protein) facilitates the interaction between F. nucleatum and Streptococcus gordonii [ 202 ]. Additionally, proteins such as RadD and Aid1, enable F. nucleatum to co-aggregate with early colonizers like Streptococcus spp. and late colonizers such as P. gingivalis and Treponema denticola [ 176 , 306 ]. This co-aggregation supports the maturation and stability of multispecies biofilms, which is essential for biofilm persistence [ 355 , 390 ]. Furthermore, F. nucleatum releases outer membrane vesicles (OMVs) that trigger inflammation by inducing the production of proinflammatory cytokines. These OMVs carry components such as LPS, DNA, adhesins, and enzymes, which function as a delivery system for virulence factors [ 74 , 119 ]. Antigenic components within F. nucleatum OMVs can activate Toll-like receptors (TLRs) on epithelial and immune cells, leading to NF-κB pathway activation and the subsequent release of proinflammatory cytokines, contributing to chronic inflammation and tissue damage (Fig.  3 ) [ 107 , 270 ]. Serine protease degrades ECM proteins and immune components like IgA, aiding immune evasion [ 35 ]. Its metabolite, butyric acid, promotes ROS production in osteoblasts, leading to bone destruction and impaired repair [ 71 ] (Table  1 ). Fig. 3 A Biofilm formation of S. mutans on dental surfaces. In sucrose-mediated adhesion, Glycosyltransferases synthesize glucans that promote bacterial attachment. Additional surface proteins involved in adhesion include Gbps, Cnm, and PacI adhesin [ 384 ]. B Virulence factors of F. nucleatum . F. nucleatum serves as a bridging organism between early and late colonizers in dental biofilms, facilitated by RadD and FomA adhesins. Its LPS activates Toll-like receptors, triggering NF-κB pathway and cytokine release, leading to inflammation and tissue damage [ 19 ] A Biofilm formation of S. mutans on dental surfaces. In sucrose-mediated adhesion, Glycosyltransferases synthesize glucans that promote bacterial attachment. Additional surface proteins involved in adhesion include Gbps, Cnm, and PacI adhesin [ 384 ]. B Virulence factors of F. nucleatum . F. nucleatum serves as a bridging organism between early and late colonizers in dental biofilms, facilitated by RadD and FomA adhesins. Its LPS activates Toll-like receptors, triggering NF-κB pathway and cytokine release, leading to inflammation and tissue damage [ 19 ] Beyond bacterial interactions, F. nucleatum also co-aggregates with Candida species, aiding their colonization and supporting polymicrobial biofilms [ 56 ]. In the oral cavity, Candida species produce alcohol dehydrogenase, an enzyme that converts alcohol into acetaldehyde, a known carcinogen [ 56 ]. This interaction suggests that F. nucleatum may indirectly increase oral cancer risk by enhancing acetaldehyde exposure. Additionally, oral leukoplakia, a precancerous condition, is associated with elevated levels of F. nucleatum [ 25 ]. Cooperative interactions between F. nucleatum and P. gingivalis may further promote neoplastic changes by inducing chronic inflammation. A study has demonstrated that the combination of F. nucleatum and P. gingivalis significantly stimulated the proliferation of human Oral squamous cell carcinoma cells in vitro [ 120 ]. Additionally, a study involving colorectal cancer (CRC) patients revealed that F. nucleatum strains from CRC and saliva samples were identical, suggesting an oral origin and a potential role in carcinogenesis [ 177 ]. Furthermore, F. nucleatum was significantly more abundant in faecal samples of CRC patients compared to healthy controls [ 125 , 386 ]. Other Fusobacterium species, such as F. periodonticum , F. necrophorum , and F. varium , also play roles in periodontal diseases and peri-implant infections [ 29 , 193 , 355 ]. Beyond oral health, F. nucleatum has been implicated in systemic conditions, including chronic otitis media [ 103 ], cerebral abscesses [ 140 ], inflammatory bowel disease [ 181 ], ulcerative colitis [ 343 ], sinusitis [ 169 ], adverse pregnancy outcomes [ 370 ], and Lemierre's syndrome [ 233 ]. This is attributed to its ability to disseminate from oral biofilms into the bloodstream, leading to infections in other parts of the body. Streptococcus oralis is a Gram-positive bacterium that belongs to the oral mitis group of streptococci and is highly prevalent in the oral cavity [ 257 ]. It plays a significant role in the early stages of plaque formation [ 176 ]. One of its primary virulence factors is the production of serine-rich repeat protein (SRRP) adhesins, which contain Sialic acid-binding immunoglobulin-type lectin (Siglec)-like domains. These domains allow S. oralis to bind to sialic acid-containing glycan epitopes on platelets, saliva, and oral epithelial cells [ 84 , 273 , 294 , 312 , 327 ]. The bacterium also binds to oral mucins MUC5B and MUC7/SAG via glycans like Leb, LNT, and SLex-like structures on their surface, utilizing sortase A-dependent proteins such as AsaA (associated with sialic acid adhesion) [ 67 , 331 ]. Once attached, S. oralis acts as an early colonizer, facilitating the adhesion of other microbial species. Its receptor polysaccharides interact with lectin-like adhesins on species like Actinomyces and Veillonella, promoting coaggregation and biofilm formation [ 68 , 150 ]. To evade the host immune system, S. oralis produces Immunoglobulin A1 protease(IgA1), a metalloproteinase that cleaves immunoglobulin A (IgA), weakening mucosal defenses and promoting invasiveness [ 82 , 275 ]. The bacterium also synthesizes glucans through glucosyltransferases (GTFs) to promote biofilm development and releases hydrogen peroxide (H 2 O 2 ), which kills immune cells like macrophages, enhancing its persistence in the oral cavity [ 257 , 258 ]. Furthermore, S. oralis forms heterotypic communities to enhance its survival and colonization in multispecies biofilms. This is achieved by binding P. gingivalis fimbriae (Mfa1 and FimA) to streptococcal surface antigens (SspA/B) and surface-expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [ 49 , 372 ]. This interaction triggers the expression of protein tyrosine phosphatase Ltp1, which modulates P. gingivalis pathogenic potential and strengthens biofilm formation [ 139 , 184 , 214 ]. Additionally, the production of AI-2 by S. oralis helps regulate multispecies biofilms and influences the composition of bacterial communities [ 85 ]. The biofilm community formed by S. oralis can significantly impact oral health, contributing to chronic inflammation and periodontal diseases such as gingivitis and periodontitis. In immunocompromised individuals, S. oralis can enter the bloodstream and interact with heart valve tissues, leading to serious conditions like bacteraemia and infective endocarditis [ 69 , 97 , 246 ]. Furthermore, studies show that S. oralis enhances the virulence of C. albicans , promoting tissue invasion and upregulating pro-inflammatory markers in epithelial tissues. This synergy between the two organisms disrupts epithelial barrier integrity by activating calpain-1, a calcium ion-dependent cysteine protease that cleaves E-cadherin [ 221 , 375 ] (Table  1 ). Interestingly, despite its association with virulence, S. oralis plays a protective role in tissue health by reducing the secretion of pro-inflammatory cytokines like IL-6 in fibroblasts and IL-8 in human gingival epithelial cells. This helps protect tissues as long as the mucosa-implant seal remains intact, indicating that S. oralis modulates immune responses to promote homeostasis in peri-implant tissues. Additionally, S. oralis maintains supragingival biofilms by suppressing S. mutans through the production of bacteriocins, further emphasizing its complex role in oral health and disease [ 155 , 354 ]. Streptococcus mutans ( S. mutans ) is a Gram-positive, facultative anaerobe primarily responsible for dental caries and is also linked to periodontitis [ 83 , 144 ]. S. mutans establish biofilms on dental surfaces through both sucrose-dependent and sucrose-independent mechanisms [ 9 ]. These biofilms are critical to its role in tooth decay, as they create a protective matrix that enhances bacterial adherence to teeth and supports its survival within the oral cavity. In the sucrose-dependent mechanism, S. mutans synthesize two main components: glucosyltransferases (GTFs) and glucan-binding proteins (Gbps) [ 40 ]. The bacterium synthesizes three types of glucosyltransferases (GtfB, GtfC, and GtfD) that are incorporated into the salivary pellicle and facilitate the conversion of sucrose to glucan. These enzymes break the glycosidic bonds in sucrose, yielding glucose and fructose. The glucose is then incorporated into a growing glucan polymer [ 234 , 240 ]. The produced glucans facilitate bacterial adhesion between tooth surfaces and other bacterial cells, supporting microcolony formation and enhancing the stability of the biofilm structure [ 59 , 152 ]. Each glucosyltransferase has a unique role in glucan synthesis. GtfB produces water-insoluble glucans rich in α-1,3-glucosidic linkages, which promote bacterial aggregation and facilitate binding with other microorganisms, like Actinomyces viscosus , leading to microcolony formation [ 59 , 180 ]. GtfC synthesizes a mixture of insoluble and soluble glucans (with mostly α-1,6 glucosidic linkages), contributing to the development of the extracellular matrix. Finally, GtfD synthesizes soluble glucans containing α-1,6-glucosidic linkages, which serve as primers for GtfB’s production of insoluble glucans [ 180 ]. S. mutans produces four types of glucan binding proteins including GbpA, GbpB, GbpC and GbpD [ 224 , 307 , 316 ]. GbpA, a key player in biofilm architecture, binds to exopolysaccharides and proteins, enhancing S. mutans adherence to tooth surfaces [ 211 , 223 , 225 ]. GbpB is involved in cell wall construction, supporting peptidoglycan synthesis and cell division, which are essential for maintaining cell integrity within the biofilm [ 118 ]. GbpC, uniquely anchored to the cell wall, plays a vital role in the initial stages of biofilm formation by supporting early bacterial adhesion and colony establishment. In later stages, GbpA and GbpD become more active; GbpD, in particular, binds dextran to mediate adhesion to tooth surfaces and has lipase activity [ 229 , 316 ]. In the sucrose-independent adhesion mechanism, S. mutans utilizes specific surface proteins to adhere to dental surfaces, facilitating initial colonization [ 61 ]. The primary adhesin, Ag I/II (also known as P1, PAc, SpaP, or AgB), binds to salivary agglutinin (gp340), which is a key regulator in S. mutans accumulation within the oral cavity [ 182 ]. Mutants deficient in Ag I/II show a significant reduction around 65% in biofilm formation, underscoring the importance of this adhesin in colonization [ 266 ]. In addition, S. mutans expresses a collagen-binding protein (Cnm), that attaches to type I collagen, a key structural component of dentin, leading to the formation of lesions in dentin [ 6 , 346 ] (Fig.  3 ). In severe cases of dental caries or periodontal bleeding, S. mutans can enter the bloodstream, where it may colonize heart valves and increase the risk of infective endocarditis [ 255 ]. Beyond this, the Cnm protein has been shown to promote S. mutans’ invasion of human coronary artery endothelial cells, indicating a potential role in cardiovascular infections [ 6 ] (Table  1 ). This highlights S. mutans ability not only to impact oral health but also to contribute to systemic health risks. Aggregatibacter actinomycetemcomitans ( A. actinomycetemcomitans ) is a Gram-negative, microaerophilic bacterium critical in oral biofilm formation, particularly associated with juvenile periodontitis [ 332 ]. Its adaptability to both aerobic and anaerobic conditions enable it to survive above and below the gingiva, enhancing its persistence within the oral cavity [ 42 , 374 ]. Initial colonization begins with reversible attachment to dental and gingival surfaces, facilitated by a range of adhesins, fimbriae, and pili, including Fimbrial low-molecular-weight protein (F1p) pili, Extracellular matrix protein adhesin A (EmaA), and micro-vesicles, which aid in the recognition and binding to host tissues [ 112 , 167 , 351 ]. Additionally, A. actinomycetemcomitans produces an exopolysaccharide, poly-β-1,6-N-acetyl-D-glucosamine (PGA), which shields the bacterium from phagocytic killing, and is synthesized through the coordinated action of the pgaABCD operon [ 159 , 364 ] (Table  1 ). Within biofilms, A. actinomycetemcomitans benefit from histone-like nucleoid structuring proteins (H–NS) that regulate gene expression and pili formation, promoting biofilm development and enhancing interspecies interactions [ 41 ]. The bacterium’s quorum sensing (QS) system, primarily regulated by Autoinducer-2 (AI-2) and mediated by the luxS gene along with receptors LsrB and RbsB, supports biofilm integrity, iron acquisition, and virulence [ 4 , 36 , 114 , 318 ]. In polymicrobial biofilms, A. actinomycetemcomitans utilizes hydrogen peroxide (H 2 O 2 ) produced by Streptococcus sanguinis through the catalase encoded by the katA gene, promoting the survival and biomass of P. gingivalis and fostering interspecies communication with S. mutans through QS signaling [ 348 , 394 ]. The pathogenicity of A. actinomycetemcomitans is further marked by its ability to induce osteoclast formation and stimulate interleukin-6 (IL-6) secretion from human gingival fibroblasts (HGFs), contributing to bone resorption [ 172 ]. It also produces Cytolethal distending toxins (CDTs) that cause DNA damage and arrest cell cycles in immune cells like T cells and macrophages, weakening host defenses [ 26 , 334 ]. LPS present on outer membrane of the bacterium trigger inflammatory pathways leading to tissue degradation and bone loss, while leukotoxin A (LtxA) directly targets immune cells, linking A. actinomycetemcomitans to both periodontal disease and rheumatoid arthritis [ 185 ]. During infections, A. actinomycetemcomitans respond to environmental changes by producing heat shock proteins (HSPs), such as GroEL, which can stimulate epithelial cell proliferation. However, at elevated levels, these proteins may induce cytotoxic effects, further exacerbating periodontal damage [ 133 , 208 , 347 ].

Biofilm formation is a critical factor in implant failure, as the establishment of bacterial colonies on implant surfaces significantly hinders the integration of implants into bone tissue [ 175 ]. This process can begin as soon as half an hour after implant placement and persists until the biofilm matures, potentially leading to chronic infections that last indefinitely if not managed effectively. The key factors influencing biofilm formation include surface topology, surface energy, surface chemistry, and wettability of the implant material. Surface roughness is one of the primary contributors to bacterial adhesion, with implants exhibiting a higher roughness average (Ra > 0.2 μm) being more prone to bacterial colonization. Conversely, surfaces with lower Ra values (< 0.088 μm) tend to reduce bacterial attachment. For instance, polished zirconium surfaces with low surface roughness (Ra reduced from 0.12 to 0.06 μm) inhibit bacterial adhesion and promote better tissue integration, whereas sandblasted titanium surfaces with increased roughness (Ra from 0.2 to 0.3 μm) facilitate bacterial colonization, thereby increasing the risk of implant-associated infections [ 238 ]. In addition to surface roughness, the surface energy and surface chemistry of implant materials play a pivotal role in adhesion properties. Materials with higher surface free energy, such as titanium, tend to attract more bacterial populations compared to materials such as zirconium possess lower surface free energy [ 96 ]. This is because higher surface energy increases the thermodynamic favorability of bacterial attachment [ 238 ]. Furthermore, the wettability of the implant surface, characterized by its hydrophilic or hydrophobic nature, is intrinsically linked to surface energy and adhesion behavior. Hydrophilic surfaces (high surface energy) typically enhance protein adsorption and cell adhesion, which is beneficial for tissue integration but may also promote bacterial adhesion for instance, S. epidermidis adherence was significantly higher to hydrophobic surfaces than to hydrophilic surfaces [ 371 ]. In addition to Ra, hydrophobic surfaces (low surface energy) also promote bacterial attachment; for instance, S. sanguinis adherence was significantly higher to hydrophilic surfaces than to hydrophobic surfaces [ 371 ]. Therefore, achieving an optimal balance between surface roughness, surface energy, surface chemistry, and wettability is essential to minimize bacterial colonization while promoting tissue integration [ 341 ]. Once the attachment is established, the biofilm layer acts as a protective barrier for bacteria, shielding them from both host immune agents (such as complement proteins like C3b) and antibiotic treatments (such as amoxicillin). The extracellular polymeric substances (EPS) within the biofilm matrix create a dense, hydrated network that limits the penetration of antibiotics, reducing their local concentration at the infection site [ 282 , 359 ]. Additionally, bacteria embedded within biofilms exhibit altered metabolic states and reduced growth rates, rendering many antibiotics, which typically target actively dividing cells, less effective. The biofilm environment also facilitates horizontal gene transfer, enhancing bacterial resistance to antimicrobial agents [ 113 ]. This multifaceted protection enables bacteria to evade eradication, often resulting in chronic and recurrent infections that compromise implant functionality despite aggressive therapeutic interventions. For example, in patients receiving bisphosphonate (BP) therapy, implant failure was reported even after proper antibiotic administration and repeated surgical debridement. This failure was attributed to the persistent biofilm formation, where colonization by Streptococcus viridans and Fusobacterium at the periodontal margin led to chronic infection and poor treatment outcomes [ 387 ]. Implant failure can be categorized into early and late failures. Early failure occurs before osseointegration, while late failure happens after the implant has become osseointegrated. Regardless of the timing, both types of failure are often associated with biofilm-induced infections. Although no single organism is solely responsible for these infections, effective infection control remains crucial for implant success. Early failures may be prevented with prophylactic antibiotics like amoxicillin, although infections remain a significant risk [ 279 ]. Biofilms also contribute to corrosion and surface damage of dental implants. For instance, S. mutans colonizing the implant surface generates surface pitting and rusting through acid production, which increases corrosion and the risk of peri-implantitis [ 340 ]. This biofilm-related corrosion can lead to implant failure within the first year, underlining the importance of addressing biofilm formation. Dental implants, like other prosthetics, are prone to developing peri-mucositis and peri-implantitis, both of which are directly related to biofilm development. Peri-implant mucositis is a reversible soft tissue inflammation affecting a significant portion of implants, while peri-implantitis leads to irreversible bone loss and more advanced stages of implant failure. Biofilms contribute to infections in up to 65% of implants, and periodontal disorders further raise the risk of peri-implantitis [ 191 ]. Studies show that peri-implant mucositis affects approximately 30.7% of implants, while peri-implantitis is observed in about 21% of implants within two years [ 324 ]. In some cases, implant failure due to infection has been reported in 17 cases, with 97% of cultures from 30 lost implants testing positive for pathogens such as Streptococcus milleri and F. nucleatum . These failures are often linked to fixture instability, with peri-implant radiolucent zones frequently observed. To enhance success rates, it is recommended to place the implant fixtures immediately after prosthesis loading [ 189 ] (Table  2 ). Table 2 Current strategies and limitations in preventing biofilm-induced implant failures Implant type Implant materials Biofilm associated implications on implant failure Existing corrective and preventive measures Limitations of existing preventive measures References Single Tooth Implant or Dental crown Ceramic, porcelain, zirconia, composite resin, and stainless steel Biofilm accumulation, particularly by S. mutans , exacerbates dental crown failure through chemical hydrolysis of adhesive monomers, leading to microleakage and increased acid production, compromising restoration integrity Surface modification of delta crown by antibacterial/bactericidal coating Incorporation of chemical antiplaque agents into dentifrices or mouth rinse can eliminate the risk of crown failure Surface modifications in dental crowns face limitations such as inadequate adhesion, potential for corrosion, challenges in achieving desired biocompatibility, and variability in long-term performance under physiological conditions Emergence of multi-drug-resistant bacterial strains [ 57 ], Kumar, Birru, and Muthu [ 186 ], Murari, Maurya, Nahak, and Pratap [ 241 ], [ 271 , 352 , 366 ] Endosteal implants Aluminium, titanium, and zirconium oxide Biofilms formed by bacteria like S. aureus and gram-negative anaerobes such as P. gingivalis on implant surfaces dominate the "race for the surface", causing peri-implantitis characterized by inflammation, tissue damage, and implant failure Use of antimicrobial peptides or metallic nanoparticles (e.g., silver) decreases biofilm formation, sandblasting, acid etching, or adding antimicrobials facilitates resistance to bacterial adhesion Micro-roughness from sandblasting or acid etching may harbour bacteria, increasing peri-implantitis risk. Conditions like uncontrolled diabetes can restrict healing and infection resistance Amarnath, Muddugangadhar, Tripathi, Dikshit, and Ms [ 23 ], Esquivel‐Upshaw et al. [ 108 ], Gkioka and Rausch-Fan [ 129 ], Reddy and Tanneeru [ 288 ], Teulé-Trull, Altuna, Arregui, Rodriguez-Ciurana, and Aparicio [ 353 ] Subperiosteal implants Chromium, cobalt, and molybdenum alloys Biofilm accumulation on subperiosteal implants, majorly by P. intermedia and P. gingivalis , stimulates peri-implantitis, inflammation and bone loss compromise implant stability, biofilm's protective matrix reduces antimicrobial effectiveness, exacerbating failure Flat and ovate pontics reduce biofilm retention; Patient-specific models minimize bacterial accumulation Risk of mucosal recession, implant exposure, and infection due to surgical invasiveness, fabrication errors or poor fit can lead to soft tissue damage and failure; Hassle in cleaning metal frameworks increases bacterial accumulation and infection risk Ayhan and Öztürk Muhtar [ 32 ], Herce-López et al. [ 147 ], Jia and Yang [ 163 ], Łoginoff, Majos, and Elgalal [ 207 ], Nandakumar, Chittaranjan, Kurian, and Doble [ 247 ] Zygomatic implants Polyetheretherketone(PEEK), titanium, cobalt-chrome alloy, zirconia Biofilm accumulation around zygomatic implants can lead to peri-implantitis, characterized by inflammation and bone loss, potentially resulting in implant failure Due to the complex anatomy and extended length of zygomatic implants, they are susceptible to biofilm accumulation. Regular professional cleaning and maintenance are essential to prevent peri-implant infections; Applying antimicrobial coatings to implant surfaces can inhibit bacterial adhesion and biofilm formation, thereby reducing the risk of infection The extended length and anatomical positioning of zygomatic implants complicate effective biofilm removal, potentially diminishing the efficacy of standard preventive measures; While antimicrobial coatings aim to reduce bacterial adhesion, achieving consistent application and long-term stability on zygomatic implants remains challenging Dhaliwal et al. [ 94 ], Heboyan, Lo Giudice, Kalman, Zafar, and Tribst [ 146 ] Mini dental implants Titanium alloys, cobalt chromium alloys, austenitic Fe–Cr–Ni–Mo steels, tantalum, niobium, zirconium alloys Biofilm formation causes instability and mobility, leading to implant failure; Scanning electron microscopy shows significant bacterial colonization on failed implants Modifying the surface characteristics of mini-implants, such as reducing surface roughness, can decrease bacterial adhesion and subsequent biofilm formation; Coating mini implants with antimicrobial agents can provide a protective barrier against bacterial colonization, thereby preventing biofilm development The reduced size of mini implants offers limited surface area for antimicrobial coatings, potentially decreasing their effectiveness in preventing biofilm formation Ferreira et al. [ 111 ], Li, Yin, Cheng, and Lin [ 199 ], Ma et al. [ 212 ], Sana and Manjunath [ 305 ] Current strategies and limitations in preventing biofilm-induced implant failures Surface modification of delta crown by antibacterial/bactericidal coating Incorporation of chemical antiplaque agents into dentifrices or mouth rinse can eliminate the risk of crown failure Surface modifications in dental crowns face limitations such as inadequate adhesion, potential for corrosion, challenges in achieving desired biocompatibility, and variability in long-term performance under physiological conditions Emergence of multi-drug-resistant bacterial strains Several factors influence the success rates of implants, including the surgical technique, timing, implant position, stability, and sterilization methods. Saliva contamination during surgery can lead to bacterial adhesion and biofilm formation, hindering osseointegration and implant success. Similar to orthopedic implants, surgical intervention complicates the prognosis and is likely to worsen the outcome. The diverse bacterial species in the oral cavity add additional challenges in controlling infection around dental implants [ 164 ]. Corrosion may also result from the presence of bacterial biofilms on dental implants. For example, S. mutans produce acidic metabolic products that erode the titanium oxide layer of the implant. Micromotion and acidic media at the implant junctions can lead to fretting-crevice corrosion, a recurrent causative factor in implant failure [ 339 ]. In addition, bone and soft tissue loss, especially around the gingival margin, caused by inflammatory processes like gingivitis, can contribute to implant failure. Inflammation around the implant, known as peri-implantitis, threatens both bone tissue and the ability of the implant to osseointegrate. Surgical difficulties, anatomical factors, and bacterial infections may also contribute to this failure [ 216 ]. Biofilm formation is a key contributor to implant failure, significantly impacting the integration of implants with bone tissue and promoting chronic infections. The surface characteristics of the implant influence bacterial colonization, while the protective nature of biofilms hinders immune response and antibiotic efficacy. The persistent challenges posed by biofilms, including peri-implantitis, tissue inflammation, and implant corrosion, highlight the urgent need for advanced materials, improved surgical techniques, and effective infection control strategies to enhance implant success rates and longevity. The investigation of implant failures, particularly those associated with biofilm formation, requires experimental models that can closely mimic the complex biological environment of the oral cavity. While in vitro studies provide valuable insights into the initial stages of bacterial adhesion and biofilm development, they fail to replicate the dynamic interactions between bacterial communities, host immune responses, and the mechanical and physiological factors present in living organisms. Therefore, animal models are essential for bridging this gap, as they offer a controlled yet physiologically relevant platform to study the multifactorial processes leading to implant failure, including host-bacteria interactions, inflammatory responses, and tissue remodeling. To examine the effect of biofilm formation on dental implants, researchers created a rat model using bacteria typically present in the human oral microbiome. The study involved implanting endosseous dental implants into female Sprague Dawley rats, with one group receiving continuous antibiotics and the other exposed to S. oralis , F. nucleatum , and P. gingivalis to promote biofilm growth. The results showed successful biofilm formation on the implant surfaces, with the bacteria-exposed group displaying mild to moderate edema around the implants. In contrast, the antibiotic-treated group maintained healthy gingival tissues. These findings highlight the importance of this model in testing implant material modifications aimed at reducing biofilm formation and improving implant success rates [ 52 ]. In another study, a novel animal model was developed to investigate the host response to A. actinomycetemcomitans biofilm on titanium implants, focusing on biofilm-related inflammatory osteolytic oral infections such as periodontitis and peri-implantitis. Biofilms were pre-formed on titanium implants in vitro for one to three days before being inserted into the oral cyst cavities of rats. The results revealed that biofilm-inoculated implants triggered significant inflammation, tissue degradation, and osteoid material loss, with A. actinomycetemcomitans identified via PCR and culture-based assays. Microcomputed tomography confirmed a reduction in bone volume around the biofilm-inoculated implants, demonstrating the model's effectiveness in studying biofilm-related infections and evaluating anti-biofilm interventions [ 116 ]. A canine model was also used to simulate the early stages of peri-implant disease by examining the impact of plaque accumulation on peri-implant tissues. In this study, Beagle dogs had implants placed in their hemi-mandibles for 17 weeks, with plaque accumulation left undisturbed for 16 weeks. Control implants were brushed daily, while test implants were left unbrushed to allow plaque formation. Clinical measurements revealed significant differences between the groups, including increased probing depth, bleeding on probing, plaque accumulation, and clinical attachment loss around the test implants, all indicative of peri-implant infection. Microbiological analysis showed a higher bacterial load in the test group. However, no significant histological or radiological evidence of bone resorption was observed. This model is particularly useful for studying the early stages of peri-implantitis and understanding the etiological factors contributing to biofilm-induced bone loss without mechanical interference [ 220 ]. Together, these investigations underscore the critical role of biofilm formation in the onset and progression of peri-implant infections. By employing diverse animal models—such as rats inoculated with human oral bacteria, titanium implants with A. actinomycetemcomitans biofilms, and dogs mimicking plaque-induced peri-implant disease—researchers have effectively illustrated the inflammatory responses, tissue degradation, and microbiological shifts associated with biofilms. These models provide a strong foundation for evaluating biofilm-reducing materials, targeted therapies, and preventive strategies to minimize implant-related complications.

The review highlights the significant role of oral biofilms in causing dental diseases and the promising potential of nanoparticles in combating these biofilms. Nanoparticles such as silver, zinc oxide, and titanium dioxide have shown effectiveness in disrupting biofilm formation and enhancing the antimicrobial properties of dental materials. The surface functionalization of nanoparticles further improves their efficacy, making them a viable option for preventing biofilm-related infections and enhancing oral health. However, usage poses several inherent challenges related to safety, toxicity, scalability, stability, and regulatory hurdles. For instance, the well studied AgNP was known to cause discoloration of dental materials, which is aesthetically undesirable for patients [ 230 , 231 ]. In addition, production of AgNPs with consistent size and shape can be expensive and technically demanding, affecting their widespread use in dental applications [ 377 ]. Furthermore, the release of AgNPs into the environment during manufacturing or disposal can have negative ecological effects, raising concerns about their sustainability [ 153 ]. Sustained efforts on the development of novel nanoparticles with enhanced antimicrobial properties and biocompatibility is essential for sustainable use of nanoparticles for medical applications. This includes (but not limited to) exploring new scalable nanomaterials (eg. graphene, graphene oxide, and graphdiyne) and appropriate surface functionalization techniques to improve their effectiveness against a broader range of oral pathogens. In addition, designing multifunctional nanoparticles that not only possess antimicrobial properties but also promote tissue regeneration and healing could further make NPs more appealing. This could also lead to more comprehensive solutions for oral health care. Furthermore, implant manufacturers should address regulatory and safety concerns associated with the use of nanoparticles in dental applications and ensure that the newly developed materials meet stringent safety standards to protect patient health. In summary, by focusing on development of novel therapeutic nanoparticles the future of dental care can be significantly improved, leading to better prevention and treatment of biofilm-related oral diseases.

Dental biofilms are complex microbial communities that can be found on surfaces of oral cavity [ 286 , 299 ]. Such biofilms play a crucial role in the development of various oral diseases such as dental caries, periodontitis, and peri-implantitis [ 168 ]. These biofilms consist of bacteria, fungi, viruses, and other microorganisms that interact with each other and lead to the formation of a structured and resilient community [ 218 ]. In addition, microbial communities produce a sticky extracellular matrix and establish a firm attachment to the surfaces in the mouth such as teeth and gingiva [ 48 , 60 ]. In particular, biofilm-associated bacterial cells often metabolize sugars and produce acids as byproducts, which eventually damage the tooth enamel, leading to the formation of dental plaques and cavities [ 237 ]. Given these problems associated with dental biofilms, it is very crucial to understand their dynamics and to develop antimicrobial strategies to mitigate risks. Despite advances in dental care, biofilm-related infections remain a persistent challenge, necessitating a comprehensive review of current knowledge and emerging strategies to manage and prevent these infections effectively. While numerous studies have explored the composition, structure, and pathogenicity of dental biofilms, there are still significant gaps in our understanding of their complex interactions and resistance mechanisms. Existing literature often focuses on specific aspects of biofilms, such as individual microbial species or particular stages of biofilm development, without providing a holistic view of the biofilm ecosystem. Additionally, many studies rely on in-vitro models that may not accurately replicate the dynamic environment of the oral cavity. These limitations highlight the need for a more integrated approach that considers the multifaceted nature of dental biofilms and their interactions with the host immune system and other environmental factors. Therefore, this review article aims to address these gaps by providing a comprehensive overview of dental biofilms, including their formation, composition, and impact on oral and systemic health. In addition, the article also provides exclusive molecular-level details on various virulence factors associated with species-specific attachment of biofilms on oral surfaces and their long-term impact on dental implants. Furthermore, this article presents the latest advancements in biofilm management strategies, such as antimicrobial agents, nanoparticles, and novel therapeutic approaches. In summary, this article will provide a contemporary understanding of the pathogenesis of dental biofilms and ways to prevent them using novel antimicrobial coatings.

Biofilm control strategies integrate physical, chemical, biological, and nanotechnology-based approaches to prevent or disrupt microbial biofilms, which contribute to implant-associated infections and failures. Physical strategies such as ultrasound, electric and magnetic fields, plasma treatments, and irradiation disrupt biofilm integrity [ 203 ], while chemical agents, including antibiotics, quorum sensing inhibitors, and enzymatic disruptors, target biofilm structure and metabolic processes [ 319 ]. Biological approaches, such as bacteriophage therapy and probiotics, use natural antimicrobial mechanisms to inhibit biofilm formation [ 130 , 368 ]. However, nanoparticles (NPs) have emerged as an effective strategy by combining antimicrobial, antibiofilm, and drug delivery properties to enhance implant longevity and prevent microbial colonization [ 20 ]. Nanoparticles fewer than 100 nm in size possess unique antimicrobial properties and are considered for medicine and dentistry. Nanomaterials are increasingly studied for their active surface area, biological activity, and chemical reactivity, which is dramatically greater than the micrometer-sized particles [ 16 , 86 ]. These particles gained attention for their bactericidal properties and high-surface-to-volume ratio for surface functionalization, which enhances their biocompatibility, strain specific interaction, and antimicrobial properties [ 236 ]. In addition, these particles present itself in closer proximity to the microbial membranes followed by release of antimicrobial molecules. The oral microbiome accommodates a wide range of microbes such as bacteria and yeast associated with oral infections signifies that both host microbiome and infections were polymicrobial in nature. The use of nanoparticles can possibly target and control the oral pathogens [ 219 ], effective for coating prosthetic surfaces, enhancing their ability to inhibit biofilm formation and infections in dental prosthetics such as crowns, implants, dentures, and veneers [ 3 , 28 , 55 , 285 ]. The antibacterial mechanisms of nanoparticles can be grouped into three types, though not yet fully understood: interaction with the bacterial cell wall and membrane causing lysis; disruption of protein synthesis by targeting bacterial proteins; and interference with DNA to prevent replication [ 66 , 73 , 235 , 336 ]. NPs such as silver (AgNPs), zinc oxide (ZnONPs), and titanium dioxide (TiO 2 NPs) generate reactive oxygen species (ROS) [ 101 , 284 ], including hydroxyl radicals (OH) and superoxide ions (O 2 − ), which induce oxidative stress, damaging bacterial membranes, proteins, and DNA, ultimately leading to cell death [ 201 , 245 , 385 ]. Additionally, silver ions accumulate within bacterial vacuoles and disrupt metabolic functions by releasing potassium ions, further impairing bacterial survival [ 245 ]. Beyond ROS-mediated toxicity, NPs also compromise bacterial membrane integrity. Cationic nanoparticles, including chitosan, silver, and copper oxide, interact with negatively charged bacterial membranes, increasing permeability and causing cellular leakage. Gold nanoparticles (AuNPs) and AgNPs fluidize bacterial membranes, facilitating nanoparticle entry into the cytosol, where they destabilize membrane proteins (e.g., efflux pumps), cytoplasmic proteins (e.g., actin), and enzymes (e.g., oxidoreductases), impairing metabolism [ 281 , 317 , 369 ]. They also interact with amino, carboxyl, and mercapto groups of proteins and nucleic acids, inhibiting cellular functions. By disrupting the electron transport chain, they impair ion exchange, destabilize membranes, and reduce metabolic activity, ultimately leading to bacterial cell death and biofilm elimination [ 165 , 281 ]. Quorum sensing inhibition represents another crucial NP-based strategy to prevent biofilm formation. AgNPs and selenium nanoparticles (SeNPs) interfere with quorum sensing molecules such as acyl-homoserine lactones (AHLs), disrupting bacterial communication and attenuating virulence [ 276 ]. Zinc oxide nanoparticles (ZnO–NPs) downregulate quorum sensing regulatory genes ( lasB , pqsA ), thereby reducing exopolysaccharide production and biofilm maturation [ 12 ]. Additionally, silver-titanium nanocomposites (AgCl–TiO 2 NPs) degrade signaling molecules such as homoserine lactone (HSL), effectively disrupting bacterial communication and biofilm maturation [ 244 , 280 ]. Furthermore, nanoparticles facilitate extracellular polymeric substance (EPS) degradation, weakening the biofilm matrix that protects bacteria from antimicrobial agents. Enzyme-functionalized nanoparticles, such as ciprofloxacin-loaded poly(lactic-co-glycolic acid) nanoparticles functionalized with DNase I (Cip-PLGA-DNase I NPs), provide a combine controlled antibiotic release with extracellular DNA degradation, disrupting biofilm matrices [ 38 ]. Mesoporous silica nanoparticles (MSNs) functionalized with lysostaphin, serrapeptase, and DNase I work synergistically to degrade proteins, hydrolyze extracellular DNA, and lyse bacterial cells, demonstrating significant potential against persistent infections [ 92 ]. Polymeric nanoparticles (PNPs) further decontaminate titanium implants by disaggregating biofilms and modifying surface hydrophilicity [ 143 ]. Additionally, NPs act as targeted drug delivery systems, ensuring localized and sustained antimicrobial release at implant sites while minimizing systemic toxicity. Polymeric and mesoporous silica nanoparticles provide controlled drug release, enhancing therapeutic efficacy. For instance, doxycycline-doped polymeric nanoparticles (Dox–NPs) applied to sandblasted, acid-etched titanium discs reduce bacterial adhesion and biofilm vitality [ 64 ]. Biodegradable disulfide-bridged mesoporous silica nanoparticles (Ag–MSNs@CHX) co-deliver silver nanoparticles and chlorhexidine through redox and pH-responsive mechanisms, effectively degrading biofilm matrices and inhibiting bacterial growth [ 210 ] (Fig.  4 ). Fig. 4 Process of biofilm degradation by reactive oxygen species (ROS) generation: A Carboxymethyl-dextran-coated iron oxide nanoparticles attach to the bacterial biofilm on the tooth surface. B In the presence of hydrogen peroxide (H 2 O 2 ) and an acidic environment, the iron oxide core catalyzes the generation of ROS. ROS initiate oxidative stress, leading to bacterial cell damage and biofilm disruption. As a result, the biofilm matrix degrades, causing the release of dead bacterial cells and reducing microbial colonization on the tooth surface Process of biofilm degradation by reactive oxygen species (ROS) generation: A Carboxymethyl-dextran-coated iron oxide nanoparticles attach to the bacterial biofilm on the tooth surface. B In the presence of hydrogen peroxide (H 2 O 2 ) and an acidic environment, the iron oxide core catalyzes the generation of ROS. ROS initiate oxidative stress, leading to bacterial cell damage and biofilm disruption. As a result, the biofilm matrix degrades, causing the release of dead bacterial cells and reducing microbial colonization on the tooth surface Surface-modified nanoparticles further enhance implant performance by improving osseointegration and minimizing bacterial adhesion. Techniques such as sandblasting, plasma spraying, anodization, acid etching, and micro-arc oxidation integrate antimicrobial nanoparticles (e.g., TiO 2 , hydroxyapatite, silver, zinc oxide) to modify implant surface roughness, wettability, and bioactivity. These modifications alter surface charge and microtopography, reducing microbial colonization while promoting osteoblast adhesion and proliferation. Thus, by incorporating nanoparticle-based coatings, dental prosthetics achieve enhanced antibacterial properties, preventing biofilm formation and implant-related infections [ 217 ]. Metals such as silver, copper, gold, zinc, and titanium have been used as antimicrobials for centuries. Interestingly, these metals in chemical complexes such as zinc-acetate or citrate (micro-sized molecules) were incorporated in the commercial toothpastes to control the dental biofilms. In addition, titanium dioxides were also used as a teeth whitener in toothpastes. These commercial products evidence the antimicrobial properties of nanomaterials in oral health [ 128 ]. Additionally, silver nanoparticles were proven to inhibit the growth of periodontal pathogens such as Streptococcus mutans , Staphylococcus aureus , Lactobacillus acidophilus, Micrococcus luteus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and C. albicans [ 105 ]. These nanoparticles disrupt bacterial cell structures and functions, inhibiting their growth and reducing their pathogenic impact. By penetrating bacterial cell walls and altering membrane integrity, silver nanoparticles ultimately lead to bacterial cell death [ 383 ]. In addition to silver nanoparticles, other types, such as liposomes and polymeric nanoparticles, are being investigated for their potential in periodontal therapy. These nanoparticles can be encapsulated in biocompatible polymers, which could release them gradually, ensuring sustained antimicrobial activity and prolonged therapeutic effects [ 8 ]. Among these nanoparticles, silver [ 335 ] and copper [ 81 ] have gained more attention due to its biocompatibility. Studies focus on the incorporation of nanomaterials into base materials such as PMMA and hydrogels [ 54 , 196 ]. Early studies on nanoparticle size and antimicrobial activity have shown that the size of the nanoparticle was inversely proportional to the antimicrobial activity, i.e., the smaller-sized Ag 2+ particles between 1 and 10 nm possess more antimicrobial activity or toxic to bacteria than large-sized Ag 2+ particles [ 337 , 365 ]. In addition, studies on the shapes of nanoplates have shown that truncated triangular AgNPs are more toxic to bacteria than spherical and rod-shaped particles. This represents that active facets were to be considered for their antimicrobial properties. Also, bacteria are far more likely to acquire resistance to metal nano-particles than the narrow antibiotics [ 263 ]. Several metallic nanoparticles, including silver (Ag), gold (Au), zinc oxide (ZnO), and titanium dioxide (TiO 2 ), have been explored for oral applications. Silver nanoparticles (AgNPs), typically ranging from 10 to 100 nm in size, exhibit strong antibacterial properties, making them useful in preventing dental plaque and infections [ 215 ]. However, concerns arise regarding their cytotoxicity, as AgNPs can induce oxidative stress and DNA damage in oral epithelial cells [ 397 ]. Gold nanoparticles (AuNPs), usually sized between 1 and 50 nm, are widely researched for their non-toxic nature and ability to enhance drug delivery in periodontal treatments [ 109 ]. Studies suggest that AuNPs interact minimally with cellular components, reducing inflammatory responses compared to other metallic nanoparticles [ 160 ]. Zinc oxide nanoparticles (ZnO–NPs) have demonstrated strong antimicrobial effects against cariogenic bacteria while promoting osteogenesis, making them valuable in dental restorations and bone regeneration. However, ZnO–NPs (10–50 nm) may release reactive oxygen species (ROS), potentially leading to cytotoxic effects at high concentrations [ 278 ]. Titanium dioxide nanoparticles (TiO 2 –NPs), with an average size of 20–200 nm, are commonly used in dental implants due to their excellent biocompatibility and mechanical properties. Studies indicate that TiO 2 –NPs enhance cell adhesion and proliferation without significant toxicity, although concerns remain regarding their long-term accumulation in tissues [ 2 ]. Despite their potential, the safety of nanoparticles in oral health applications requires further evaluation. Factors such as nanoparticle size, concentration, surface charge, and exposure duration influence their cytotoxicity and biocompatibility. Additionally, long-term clinical studies are needed to assess their systemic effects and accumulation risks. Developing biofunctionalized nanoparticles with controlled release properties and enhanced biocompatibility can pave the way for safer and more effective oral healthcare solutions. The covalent attachment of biomolecules to nanoparticles involves the formation of chemical bonds between the nanoparticle surface and functional groups such as amines, carboxyls, and thiols. This functionalization enhances interactions with bacterial cell membranes and enables penetration into biofilms. Additionally, these attachments allow for various biomedical applications, including drug delivery, drug carriers, and cell tracking using fluorophore-conjugated nanoparticles [ 380 ]. The process begins with the activation of the nanoparticle surface, which is typically composed of materials such as gold, silica, or polymers. For example, hydroxyl (–OH) groups can be introduced onto the surface of silica nanoparticles through a silanization process (Fig.  5 c). Next, a linker molecule with reactive ends is employed to bridge the nanoparticle surface and the desired functional group. In the case of gold nanoparticles, thiol (–SH) groups are commonly used as linkers due to their ability to form strong covalent bonds with gold [ 195 ] (Fig.  5 d). Following linker attachment, the functional biomolecule—such as an antibody, peptide, drug molecule, or polymer—is covalently bonded to the nanoparticle. This biomolecule must contain a reactive end capable of forming a covalent bond with either the linker molecule or the activated nanoparticle surface [ 91 ]. The resulting functionalized nanoparticles exhibit enhanced stability and specificity in biomedical applications. Fig. 5 a The surface coating with zwitterionic materials possesses a neutral surface charge on dental materials and potentially shows a reduced Streptococcus mutans, Staphylococcus aureus, Klebsiella oxytoca , and Klebsiella pneumoniae colonization, b glass nanoparticles on the titanium implants reduces the porous membrane structures which eliminate bacterial adhesion. c , d the hydroxyl and thoil side chain molecules were used as linker molecules between the nanoparticles and protein of interest to combat microbial infections a The surface coating with zwitterionic materials possesses a neutral surface charge on dental materials and potentially shows a reduced Streptococcus mutans, Staphylococcus aureus, Klebsiella oxytoca , and Klebsiella pneumoniae colonization, b glass nanoparticles on the titanium implants reduces the porous membrane structures which eliminate bacterial adhesion. c , d the hydroxyl and thoil side chain molecules were used as linker molecules between the nanoparticles and protein of interest to combat microbial infections Carbon nanomaterials (CNMs), including fullerenes and carbon nanotubes (CNTs), possess unique properties that make them valuable in biomedical applications, particularly in dentistry. Fullerenes are spherical carbon molecules, typically composed of 60 carbon atoms (C60), arranged in a structure resembling a soccer ball. These molecules can be chemically modified to interact with biological tissues, enabling applications such as tissue engineering and drug delivery. Carbon nanotubes (CNTs) are cylindrical structures formed by rolling graphene sheets into seamless tubes. They are classified into two types: single-walled nanotubes (SWNTs), composed of a single graphene layer, and multi-walled nanotubes (MWNTs), which consist of multiple concentric graphene layers. CNTs exhibit high electrical conductivity and mechanical strength, allowing them to penetrate cellular membranes and effectively deliver drugs or therapeutic agents [ 171 ]. Their nanoscale size and structural rigidity allow them to penetrate cellular membranes, enabling them to deliver drugs or other therapeutic agents effectively. CNMs act as carriers for a range of therapeutic molecules, including drugs, proteins, and nucleic acids. The delivery mechanism often relies on noncovalent interactions such as, hydrogen bonding (stabilizes molecules on the CNM surface), Van der Waals forces (facilitates stacking of biological molecules like DNA or RNA), electrostatic interactions (enables attachment of oppositely charged therapeutic agents), and hydrophobic interactions (hydrophobic drugs to bind to CNMs, enhancing their solubility in biological environments) [ 367 ]. These interactions ensure that drugs are securely attached to CNMs and released in a controlled manner at the target site. CNMs have demonstrated strong antibacterial properties, primarily through contact-mediated biocidal effects CNMs interact with bacterial membranes, disrupting their structural integrity. Also, electrostatic attraction by positively charged nanoparticles causes bacterial membrane permeability. This leads to leakage of intracellular components, effectively killing the bacteria. Both mechanisms are particularly advantageous as it reduce the chances of developing bacterial resistance compared to traditional antibiotics. CNMs can be coated on biomimetic dental implants to facilitate wound healing and promote integration with the surrounding bone tissue [ 367 ]. The coating serves as a drug-releasing system, delivering anti-inflammatory and antibacterial agents directly to the site. This targeted delivery accelerates the healing process and ensures better implant stability. When combined with graphene, CNMs further enhance their therapeutic properties. The addition of graphene improves the bioactivity and reduces the cytotoxicity of the nanomaterials. This synergy also enables better delivery of bioactive peptides and nucleic acids for tissue regeneration and repair. In recent years, nanocomposites have been extensively studied for their enhanced durability and mechanical properties. Research has shown that various nanoparticle shapes, such as regular and spherical nanocomposites embedded in a matrix, improve bonding and adhesive strength. In restorative dentistry, these composite matrix compounds help mitigate leakage between fillings and teeth while reducing material shrinkage. The incorporation of additional layers (up to two) in nanocomposites aids in maintaining adhesive strength, making them more reliable in clinical applications. This advancement significantly enhances dental restoration. Biocompatible materials, often incorporating nanoparticles, are essential for the success of dental implants, as they promote osseointegration, ensuring the long-term stability and functionality of the implant [ 72 ]. Dental restorative materials, including fillings and crowns, utilize nanomaterials to improve mechanical properties, biocompatibility, and aesthetic appeal. Additionally, nanoparticles are used to deliver therapeutic agents such as antibiotics or anti-inflammatory drugs to specific sites within the oral cavity, enhancing treatment efficacy while minimizing side effects. Nanoparticles with antimicrobial properties can be incorporated into dental materials to inhibit bacterial growth and prevent infections. Among these, calcium phosphate nanoparticles—especially nano-hydroxyapatite (nHA)—play a crucial role in improving dental health [ 277 ]. Since nHA closely resembles natural tooth enamel, it facilitates remineralization and strengthens teeth against decay. Moreover, metal nanoparticles, particularly gold nanoparticles, exhibit strong antibacterial properties, making them valuable for medical disinfection [ 200 ]. These nanoparticles generate reactive oxygen species (ROS) on oxide surfaces, enhancing resistance against a broad spectrum of microorganisms. Various other nanomaterials are also incorporated into toothpaste formulations, each contributing unique properties to improve oral care [ 5 ]. Nanotechnology has revolutionized toothpaste formulations, offering innovative approaches to oral health. Due to their nanoscale size, silica and titanium dioxide nanoparticles penetrate deep into interdental spaces, effectively removing plaque and stains while providing UV protection [ 17 ]. Calcium carbonate nanoparticles further enhance remineralization by interacting extensively with the tooth surface [ 15 ]. Similarly, nanosized sodium trimetaphosphate strengthens enamel and combats bacterial growth [ 51 ]. Additionally, nanoparticles such as selenium, chitosan, and tricalcium phosphate offer a multi-functional approach, exhibiting antimicrobial, anti-inflammatory, and remineralizing properties. These advancements contribute to a healthier oral environment by preventing cavities, reducing tooth sensitivity, and maintaining overall oral health [ 95 ]. Recent advancements in dental implants and prosthetics have incorporated 3D printing technology to enhance surface functionalization. Dental implants—including retainers, dentures, crowns, bridges, and occlusal splints—are highly susceptible to bacterial colonization and biofilm formation [ 392 ]. To address this, 3D-printed prostheses have been developed with patient-specific designs, offering cost-effective solutions made from durable materials that provide extended protection against microbial growth [ 292 ]. Traditionally, polymethyl methacrylate (PMMA) has been widely used for dental implants due to its ability to mimic natural teeth [ 292 ]. However, PMMA is prone to microbial contamination, facilitating biofilm formation on its surface [ 157 ]. While conventional 3D printing with antimicrobial-resistant molecules has limitations—such as porous surface structures that promote bacterial colonization—recent studies have focused on modifying the surface charge of dental implants to repel proteins and bacterial adhesion. For example, PMMA doped with zwitterionic materials such as sulfobetaine methacrylate and 2-methacryloyloxyethyl phosphorylcholine (MPC) has demonstrated a reduced ability for bacterial colonization compared to standard PMMA dental materials. These modified materials significantly inhibit the growth of Streptococcus mutans , Staphylococcus aureus, Klebsiella oxytoca , and Klebsiella pneumoniae (Fig.  5 a), without compromising their durability over time [ 187 ] (Fig.  5 b). In addition, previous studies have shown that the process involves synthesizing bioactive nanoparticles, such as silica-based nanoparticles or bioactive glass nanoparticles, using the sol–gel method to achieve uniform composition and high surface area to address porous structures in teeth using nanoparticles [ 265 ]. The tooth surface is first cleaned to remove plaque and debris, ensuring a suitable substrate for nanoparticle adhesion. A stable nanoparticle suspension is prepared in a biocompatible medium to prevent agglomeration and ensure even distribution. This suspension is then applied to the tooth surface through techniques such as dip-coating, spraying, or brushing. Following application, the nanoparticles undergo a self-assembly process, often through evaporation-induced self-assembly (EISA), to create a highly ordered porous structure that mimics the natural mineral matrix of teeth. The coating is dried under controlled conditions, and calcination may be performed to stabilize the nanoparticle layer [ 131 ]. In studies involving bioactive glass nanoparticles, the osseointegration process demonstrated significant improvements, with accelerated mineralization and enhanced formation of mature bone tissue in close contact with the treated surface [ 30 ]. This approach has also been successfully adapted for commercial applications, such as titanium implants, using optimized coating techniques. The nanotopography of the coating, combined with the inherent chemical bioactivity of the bioactive glass nanoparticles, significantly enhances the formation of bone-like apatite in vitro. It also promotes the osteogenic differentiation of stem cells, even in the absence of additional osteogenic supplements [ 248 ]. These combined properties result in improved osseointegration, demonstrated by the accelerated formation of mature bone tissue in direct and intimate contact with the modified surface within a short implantation period. The method not only fills porous structures but also reinforces the treated surface, enabling durable integration and potential remineralization, thereby supporting both clinical efficacy and long-term prevention from biofilm formation. Orthodontic archwires play a critical role in dental correction by applying controlled forces to align teeth. These wires, typically made from stainless steel, nickel-titanium (NiTi), or beta-titanium alloys, ensure the movement and retention of teeth during orthodontic treatment [ 344 ]. One critical aspect of orthodontic mechanics is the friction generated at the interface of the wire and bracket during tooth movement. This friction, caused by surface contact and movement, resists the desired motion and is influenced by factors such as surface smoothness, roughness, reactivity, applied pressure, and lubrication. Minimizing friction can accelerate tooth movementx, significantly shortening treatment times [ 344 ]. A major challenge in orthodontics is the friction generated at the wire-bracket interface during tooth movement. This friction, influenced by surface smoothness, reactivity, and applied pressure, can hinder treatment efficiency. Reducing friction allows for faster tooth movement and shorter treatment durations. Recent advancements have introduced nanoparticles as an innovative solution. For instance, inorganic fullerene-like tungsten sulfide nanoparticles (IF–WS 2 ) serve as effective dry lubricants, forming self-lubricating coatings on stainless steel wires to enhance sliding efficiency [ 21 ]. Additional approaches include modifying wire dimensions, adjusting bracket designs, and applying friction-reducing coatings to stainless steel and NiTi wires. Orthodontic devices also pose a significant risk of microbial colonization, as their increased surface area facilitates bacterial plaque accumulation. Within six weeks of treatment, bacterial populations—including Streptococcus mutans, Staphylococcus aureus , and Lactobacilli —can increase nearly 30-fold, leading to enamel decalcification and hygiene challenges. To mitigate this, AgNPs measuring between 1 and 10 nm, have been incorporated into orthodontic components such as elastomeric modules, brackets, and wires. AgNPs exhibit strong antibacterial properties, preventing biofilm formation and reducing enamel demineralization. Research indicates that smaller AgNPs effectively prevent Streptococcus mutans adhesion, outperforming conventional orthodontic materials such as stainless steel and NiTi wires [ 1 ]. These advancements in nanoparticle coatings provide dual benefits—reducing friction and enhancing antibacterial properties—ultimately improving the efficiency and hygiene of orthodontic treatments for both patients and practitioners (Table  3 ). Table 3 Nanoparticles composites and antimicrobial activity Nanoparticle Biomolecule Composition Features Antimicrobial activity References Rose-bengal NPs (60 ± 20 nm) Chitosan functionalization using N-ethyl-N0-(3-dimethyl aminopropyl) carbodiimide (EDC 5 mM) and N-hydroxysuccinimide (NHS 5 mM) Structural integrity 0.3 mg/mL conc. Inhibits biofilms Shrestha, Hamblin, and Kishen [ 323 ] AgNPs (2.7 nm) 5% of dimethylamino dodecyl methacrylate (DMADDM) Primer and adhesive AgNPs with DMADDM complex showed higher antibacterial and antibiofilm effect on lactic acid producing bacterial species Nadar, Khan, Patching, and Omri [ 243 ] Quaternary ammonium polyethylenimine NPs (0.01—6 um) Zirconia-silica particles Polymer Resin It have shown 71% reduction in the bacterial viability and reduced the biofilm thickness to 30 um (approx) Chladek, Barszczewska-Rybarek, Chrószcz-Porębska, and Mertas [ 79 ] Glass nanopowders 58S, 63S, 72S (20–90 nm) Data not available Used for root canal studies In vitro studies have shown that MIC, 50 and 100 mg/mL of 58S and 63S inhibited E. coli and S. aureus . 100 mg/mL of 63S inhibited S. typhi , and P. aeruginosa Samiei, Farjami, Dizaj, and Lotfipour [ 304 ] AgNPs (< 5 nm) Resin composite Prevention of enamel demineralization After 24 h, no significant effect to S. mutans, S. sabrinus Al-Ansari, Al-Dahmash, and Ranjitsingh [ 11 ] AgNPs (100–200 nm) Data not available Oral tissue conditioner 1% of AgNPs prevented the growth of the S. mutans, C. albicans, and S. aureus Safari, Barani, and Sadrmohammadi [ 298 ] Nanoparticles composites and antimicrobial activity Studies have shown that micro-movements at the implant-abutment connection can increase the wear of the inner surfaces, leading to the release of metal ions and micro- and nanoparticles into surrounding tissues [ 62 ]. This process, known as tribocorrosion, results from a combination of biofilm adhesion, chemical contact, and mechanical wear. Titanium, commonly used in dental implants due to its biocompatibility, corrosion resistance, and high tensile strength, is particularly affected by this degradation process, leading to the accumulation of titanium nanoparticles in peri-implant tissues [ 256 ]. Upon release, these nanoparticles integrate into tissues and cells through endocytic vesicles, extracellular matrix components, erythrocytes, and plasma. Once inside cells, these nanoparticles induce oxidative stress by increasing ROS levels, leading to mitochondrial dysfunction [ 62 ]. The resulting oxidative imbalance disrupts antioxidant defense mechanisms by reducing superoxide dismutase activity, glutathione levels, and catalase capacity. This process triggers an abnormal activation of macrophages, causing excessive inflammation while simultaneously suppressing innate immune responses [ 100 , 151 ]. Furthermore, increased levels of pro-inflammatory cytokines such as IL-6, IL-1β, TNF-α, and GM-CSF contribute to an inflammatory environment that negatively affects osteogenesis. This environment suppresses osteogenic markers, promotes adipogenic differentiation of mesenchymal stem cells (MSCs), reduces bone regeneration capacity, and enhances osteoclast-mediated bone resorption. These combined effects compromise bone stability, leading to peri-implantitis and ultimately implant failure [ 98 ]. Studies indicate that around 29.48% (implant-based) and 46.83% (subject-based) of dental implants suffer from peri-implant mucositis and around 9.25% (implant-based) and 19.83% (subject-based) develop peri-implantitis [ 192 ]. Beyond localized effects, titanium NPs have been detected in distant organs such as the liver, lungs, spleen, and kidneys, suggesting systemic distribution. This raises concerns regarding potential long-term health impacts, particularly for individuals predisposed to hypersensitivity reactions. Delayed hypersensitivity reactions mediated by T-cells have been observed in genetically susceptible individuals, exhibiting as localized edema, erythematous maculae, vesicles, erosions, and oral lichenoid contact reactions [ 308 , 362 ]. Moreover, silver NPs, despite their antimicrobial properties, exhibit cytotoxicity toward eukaryotic cells, including fibroblasts and osteoblasts. This cytotoxic effect can impair osseointegration, reducing the implant's long-term success. Additionally, metal nanoparticles contribute to peri-implant mucositis and reactive lesions such as pyogenic granuloma and peripheral giant cell granuloma, further complicating implant stability and patient outcomes [ 205 , 222 ]. The clinical application of a product signifies that it has successfully undergone all essential stages required to develop a marketable invention, each dependent on advancements in scientific research. Based on our market-oriented development framework and findings, stagnation in clinical application may primarily stem from unresolved preclinical challenges. This presents an intriguing paradox: while the increasing number of publications in this field highlights extensive efforts by academic researchers in preclinical studies, no product has yet reached the market. An extensive review on antimicrobial coatings reveals an overwhelming focus on in-vitro studies, with only a few clinical investigations. This suggests that limited progress at the preclinical stage has hindered the transition to subsequent phases of the evidence-based research hierarchy [ 90 ]. Notably, fewer studies have been conducted on clinical applications. For instance, Fuchs et al. developed a gentamicin-loaded coating for implants in a clinical study. However, the absence of control groups raises doubts about the reliability of the positive data [ 117 ]. Many clinical studies involve small patient cohorts, limiting the statistical power and generalizability of the findings. The absence of proper control groups in some studies makes it challenging to draw reliable conclusions about the effectiveness of nanoparticle coatings compared to standard dental materials. Similarly, Scoccianti et al. demonstrated that silver-coated prostheses significantly reduced post-surgical infections. However, the lack of control groups and the detection of significant levels of silver in blood and urine samples for two years highlight potential concerns [ 309 ]. Most clinical studies on nanoparticle-coated dental materials have short follow-up periods, making it difficult to assess their long-term safety, durability, and efficacy. Potential adverse effects, such as delayed toxicity or degradation of coatings, may only become evident after extended use. These clinical case studies emphasize the importance of further research into the dosage levels of new particles to mitigate metal accumulation in blood and urine samples. The long-term impact of nanoparticles on human tissues, including potential cytotoxicity, immune reactions, and systemic distribution, remains insufficiently studied. Additionally, control groups are crucial for accurately comparing the effects of metal-coated implants. Long-term follow-ups—extending up to five years in clinical settings—would be valuable in addressing research gaps Fig.  6 a and providing a more comprehensive illustration were provided for better understanding and troubleshooting of the clinical implications Fig.  6 b. Fig. 6 Translational Research Process and Challenges in Clinical Studies: A The translational research pathway from preclinical to clinical research, highlighting key study types such as in vitro studies, in vivo (animal model) studies, case reports, case–control studies, cohort studies, and randomized controlled trials. The transition from promising preclinical results to clinical product development is marked by a gap that requires strategic planning. B A network diagram illustrating clinical study limitations (red nodes) and corresponding strategies to overcome them (green nodes). Blue arrows indicate direct relationships between limitations, while green arrows show solutions addressing specific challenges. Key limitations include lack of long-term clinical data, ethical and patient safety concerns, toxicity, regulatory challenges, and limited sample sizes. Strategies such as enhancing biocompatibility testing, increasing sample sizes, and addressing regulatory issues are proposed to mitigate these challenges. The solutions highlighted in ( B ), such as enhancing biocompatibility testing, improving cost-effectiveness, and ensuring ethical compliance, are critical for bridging the gap in ( A ), ensuring that innovative products successfully navigate the complexities of clinical research and reach patients effectively Translational Research Process and Challenges in Clinical Studies: A The translational research pathway from preclinical to clinical research, highlighting key study types such as in vitro studies, in vivo (animal model) studies, case reports, case–control studies, cohort studies, and randomized controlled trials. The transition from promising preclinical results to clinical product development is marked by a gap that requires strategic planning. B A network diagram illustrating clinical study limitations (red nodes) and corresponding strategies to overcome them (green nodes). Blue arrows indicate direct relationships between limitations, while green arrows show solutions addressing specific challenges. Key limitations include lack of long-term clinical data, ethical and patient safety concerns, toxicity, regulatory challenges, and limited sample sizes. Strategies such as enhancing biocompatibility testing, increasing sample sizes, and addressing regulatory issues are proposed to mitigate these challenges. The solutions highlighted in ( B ), such as enhancing biocompatibility testing, improving cost-effectiveness, and ensuring ethical compliance, are critical for bridging the gap in ( A ), ensuring that innovative products successfully navigate the complexities of clinical research and reach patients effectively The approval process for nanoparticle-based dental materials is significantly influenced by stringent regulations imposed by agencies such as the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA). These regulatory bodies ensure that nanomaterials used in dental applications meet the highest standards for safety, efficacy, and biocompatibility before reaching the market. The FDA classifies these materials under medical devices or drug-device combinations, depending on their intended function. Products with nanoparticle coatings that exhibit antibacterial or bioactive properties often require Premarket Approval (PMA) due to their novel characteristics, necessitating extensive in vitro and in vivo studies [ 338 ]. If the material is substantially equivalent to an existing product, it may receive 510(k) clearance, a faster route to market, but this pathway remains challenging for nanomaterials due to concerns over toxicity and systemic nanoparticle migration [ 13 ]. In contrast, the EMA enforces the Medical Device Regulation (MDR) of the European Union, which requires nanoparticle-based dental materials to comply with ISO 10993 standards for biological safety assessments [ 166 ]. The CE marking certification is essential for commercialization in Europe, but regulatory scrutiny over nanotoxicity, genotoxicity, and immunogenicity has slowed approvals [ 127 ]. Despite the regulatory barriers, ongoing clinical trials and preclinical studies are addressing key concerns surrounding nanoparticle-based dental materials [ 382 ]. These studies help determine the optimal particle size, coating techniques, and release kinetics for safe long-term use. Animal model studies have also demonstrated promising results in terms of bone regeneration and bacterial inhibition, particularly for silver and hydroxyapatite nanoparticle coatings [ 330 ]. However, the translation from preclinical findings to human clinical trials remains slow due to variability in patient response, lack of standardized protocols, and ethical concerns over prolonged exposure to nanoparticles [ 156 ]. Several clinical trials have investigated the effectiveness of silver (Ag) and copper (Cu) nanoparticles in restorative materials, particularly in reducing secondary caries and bacterial adhesion [ 215 ]. Silver nanoparticles have shown strong antibacterial properties against Streptococcus mutans , a primary contributor to dental caries, while copper nanoparticles have been explored for their broad-spectrum antimicrobial activity and potential to enhance dentin remineralization [ 135 ]. Similarly, zinc oxide (ZnO) nanoparticles have been incorporated into dental composites due to their antifungal and anti-inflammatory properties, with some clinical studies indicating improved plaque control and gingival health [ 328 ]. Titanium dioxide (TiO 2 ) nanoparticles, commonly used in whitening and antimicrobial coatings, have been tested in trials to evaluate their long-term biocompatibility and potential risks of systemic absorption [ 267 ]. However, challenges such as small sample sizes, lack of long-term follow-ups, and inconsistent control groups limit the generalizability of findings. To address these regulatory and clinical challenges, efforts are being made to develop standardized testing protocols for nanoparticle-based dental materials. Regulatory bodies are increasingly advocating for longitudinal studies to assess the long-term effects of nanoparticles in oral environments. Furthermore, advancements in computational modeling and in vitro simulations are being explored to predict nanoparticle behavior and toxicity before conducting large-scale human trials [ 267 ]. Multi-center randomized clinical trials with larger cohorts and extended follow-up periods will be essential in overcoming regulatory bottlenecks and demonstrating the real-world applicability of these materials. As the field of nanotechnology in dentistry continues to evolve, the regulatory landscape must adapt to balance innovation with patient safety, ensuring that nanoparticle-based dental materials are not only effective but also biocompatible and safe for long-term use [ 242 ].