In vivo models for evaluating antibacterial activity and osseointegration of dual-biofunctional implant coatings: A systematic review

Abstract

This systematic review assesses the necessity of in vivo models in determining whether these novel dual-biofunctional coating could induce osseointegration and exhibit antibacterial activity by providing a comprehensive biological response that in vitro models cannot replicate. This systematic review included in vivo studies involving intraoral implantation. A comprehensive search of original research published in English up to December 2024 was conducted across Medline, Scopus, and Web of Science. From the screened studies, 16 studies (1 clinical trial and 15 animal studies) met the inclusion criteria. The clinical trial involved 15 patients and reported 4-month outcomes. A large variance was observed among the protocol of animal studies, comprising five species (rats, rabbits, dogs, pigs, and goats). Six studies utilized peri-implantitis models to evaluate osseointegration outcomes under disease challenge. The reviewed studies highlighted various osseointegration-promoting components, such as inorganic compounds (e.g., calcium phosphate), peptides, and metal-based materials, as well as antimicrobial agents including metals, metal oxides, and polymers. By synthesizing in vivo findings, this review seeks to optimize dual-biofunctional coatings that simultaneously eradicate pathogenic bacteria and induce osseointegration within clinically relevant environments. Future work should aim to standardize in vivo protocols and new in vitro methods to reduce heterogeneity.

1. Introduction

Tooth loss is a significant global health concern. According to the World Health Organization, its prevalence reaches 7 % in adults aged 20 years or older and this rate increases dramatically to 23 % among individuals aged 60 or older [1]. Since introduction in the 1960s, titanium dental implants have become a widely-adopted treatment for missing teeth [2]. In spite of their widespread use, there remains a pressing need for improved implant materials that enhance long-term performance while minimizing complications and morbidity [3], [4], [5].

A study pointed out that 1–2 % of patients experience implant failure in the early stage due to inadequate osseointegration, while 5 % experience failure in the late stage due to peri-implantitis among the five million dental implants placed annually in the United States [6]. Furthermore, the weighted survival rate of the failed implants has a low survival rate at ∼86 %, indicating an approximate 14 % secondary failure rate [7]. The additional cost, procedure, and surgical trauma might also deteriorate patients' quality of life [4].

Upon implantation, various types of cells, such as osteoblast and osteoclast, will approach the material to initiate the osteogenesis process [8]. Simultaneously, bacteria may compete with cells for surface colonization, initiating a competitive "race for the surface" phenomenon commonly observed in biomaterial implantation [9]. These challenges highlight the critical need for approaches that either induce osseointegration or reduce the pathogenic factors related to failure [10].

Dual-biofunctional coatings, which are designed to simultaneously induce bone integration and prevent infection, have emerged as a promising solution to these challenges [11], [12]. Although numerous dual-biofunctional coatings have been developed to mitigate dental implant failure, current evidence remains predominantly limited to in vitro studies [13], [14], [15]. While in vitro systems provide distinct advantages, including simplified cell sourcing, controlled culture conditions, and standardized experimental parameters, they are inherently reductionist and lack the physiological complexity required for reliable extrapolation to clinical outcomes [16], [17]. Richard et al. emphasized that cytocompatibility is the appropriate term for in vitro assessments, whereas biocompatibility can only be conclusively evaluated through in vivo studies [18].

Crucially, osseointegration, defined as the direct structural and functional connection between living bone and a load-bearing implant surface [19], is a process inherently requires in vivo models or clinical trials to accurately assess bone-implant interactions [20]. Given the unique microbial and mechanical challenges of the oral environment, dual-biofunctional coatings for dental implants must be validated specifically within the oral cavity [21], [22]. Testing in alternative anatomical regions (e.g., the tibia or femur) fails to account for site-specific factors such as salivary composition and biofilm dynamics, thereby limiting translational relevance [23], [24].

Despite extensive research on antibacterial coatings for dental implants, no comprehensive review has systematically evaluated the efficacy of dual-biofunctional materials using in vivo evidence specific to the oral cavity. This gap in the literature hinders clinical translation, as the biological performance of implant coatings necessitates validation in physiologically relevant models [25], [26]. To address this limitation, this systematic review critically evaluates existing in vivo studies, with particular emphasis on experimental design and surgical protocols. The objectives of this work are twofold: (1) to conduct a comparative analysis of antibacterial materials employed in dental implants, and (2) to assess their osseointegration potential under both healthy conditions and peri-implantitis models. By synthesizing in vivo findings, this review seeks to optimize dual-biofunctional coatings that simultaneously eradicate pathogenic bacteria and induce bone regeneration within clinically relevant environments.

2. Methods

2.1. Protocol registration

This systematic review was conducted and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines. The protocol for this review was registered in Open Science Framework platform (osf.io/4me3y)

2.2. Search strategy

This review was aimed to search literature and evidence on the recent development of dual-biofunctional dental implant coating published till December 2024. We searched three medical databases (Medline, Scopus, and Web of Science) using the following specific search terms: (osteoblast OR osseointegration OR osteogenesis OR bone) AND (antimicrobial OR bacterial OR infection OR biofilm OR microbial) AND dental implant coating AND in vivo. The searching strategy of each database was shown in the Table S1 (Supplementary file). The publications were screened based on the following inclusion and exclusion criteria:

The inclusion criteria:

(a) Population (P): Human or animal subjects

(b) Intervention (I) and control (C): Dental implant coatings designed to enhance osseointegration and antibacterial properties.

(c) Outcome (O): Demonstrated promotion of osseointegration and inhibition of bacterial growth on implant surfaces.

(d) Study design (S): No restriction.

The exclusion criteria:

(a) Non-English publications.

(b) Studies not focused on titanium implants (e.g., zirconia and polyetheretherketone).

(c) Studies reported only in vitro results.

(d) Not used for osseointegration (e.g., crown and abutment)

(e) The in vivo studies not performed in oral cavity.

(f) No access for the full-text.

(g) Not used as the dual-biofunctional implants (promotion of osseointegration and inhibition of bacterial growth).

(h) Review articles

Records from each database were imported into EndNote 2021 (Clarivate Analytics), and duplicates were removed automatically. Two reviewers (J. Z. and Y. Z.) independently screened the titles, abstracts, and full texts based on the eligibility criteria. The inter-agreement reliability between the two reviewers (J. Z. and Y. Z.) was evaluated using the Cohen’s Kappa score [27]. Handsearching was performed by reviewing the references of the included studies. Any discrepancies were resolved through discussion with a third reviewer (J. T.) to reach consensus.

2.3. Data extraction

Data were independently collected by two reviewers (Y. Z. and J. C.) from each of the included studies. A third reviewer (J. T.) reviewed the extracted data to resolve discrepancies and disagreements. For the analysis of coating materials, the following data were extracted: type of coating material, form, and underlying mechanisms. Regarding the efficacy of the coating materials, data were stratified based on study design (clinical trial or animal study). In clinical studies, information on the number of patients and implants, follow-up duration, and survival rates was recorded. For animal studies, results from micro-CT measurements and histomorphometric analyses were documented. In addition, the peri-implantitis model was emphasized to evaluate the potential of coating materials in preventing peri-implantitis.

2.4. Quality assessment

This step was performed by two reviewers (Y. Z. and J. T.) using different criteria if the included study was clinical trial or animal studies. The quality assessment of the clinical trial was performed using the Risk of Bias 2 tool [28].

Quality assessments for animal studies were performed in accordance with the SYRCLE’s risk of bias tool [29]. The additional signaling questions are included to assist judgment. “Yes” indicates low risk of bias; “no” indicates high risk of bias; and “unclear” indicates an unclear risk of bias. If one of the relevant signaling questions is answered with “no,” this indicates high risk of bias.

3. 3. Results

3.1. Searching strategy

The initial database search retrieved 270 records. Following duplicate removal (n = 100), the titles and abstracts of the remaining 170 articles were screened against predefined inclusion-exclusion criteria. Subsequent full-text evaluation resulted in the inclusion of 16 studies for qualitative synthesis (Fig. 1). The inter-rater agreement scores between the two reviewers were 0.82 for titles and abstract screening, and 0.89 for full-text screening. The final cohort comprised 15 animal studies (93.8 %) and 1 clinical trial (6.2 %).

Fig. 1.

Flowchart of search in databases and screening.

3.2. Risk of bias assessment

The clinical trial was assessed by the RoB 2 tool and was rated as “high risk of bias” (Fig. 2A). The methodological quality of included animal studies revealed substantial limitations. Insufficient methodological detail was reported across most evaluated domains, resulting in an overall risk of bias ranging from unclear to high (Fig. 2B).

Fig. 2.

The risk of bias assessment of the in vivo studies (A: clinical trial; B: animal studies).

3.3. Descriptive analysis

Among the included 16 literatures, 15 literatures of dual-biofunctional coating materials were utilized the animal model while only one clinical trial.

3.3.1. The clinical trial

Carinci et al. conducted a randomized clinical trial to evaluate the efficacy of a novel dual-biofunctional implant coating (namely, PIXIT) composed of polysiloxane oligomers and chlorhexidine gluconate [30]. The study cohort comprised 15 systemically healthy patients (60 implants; 9 females, 6 males; mean age: 53 years, range: 45–61 years) without medical histories and smoking habits. Participants were scheduled for bilateral fixed prostheses or crown restorations supported by implant fixtures. The authors did not report the timing of teeth extraction. The patients were randomly allotted for each group test and control. At the 4-month postoperative follow-up, no implant failures or local inflammatory reactions were observed. Antibacterial efficacy was assessed indirectly via PCR analysis and quantification of bacterial colonization on healing caps. Results demonstrated a statistically significant reduction in bacterial load on PIXIT-coated implant healing caps compared to the uncoated control group.

While this study represents the sole clinical trial identified in our literature search, the long-term efficacy of the PIXIT coating warrants further investigation through extended follow-up periods exceeding four months. In addition, future research could employ more methodologies to evaluate antibacterial activity (e.g. probing pocket depth, bleeding and/or suppuration on probing as well as soft tissue recession [31]) and osseointegration parameters (e.g. marginal bone loss and long term survival rate [32]) to enhance clinical relevance.

3.3.2. The animal models

The 15 animal studies exhibited substantial methodological heterogeneity across three key domains: animal selection, surgical protocols, and peri-implantitis disease induction protocols. The animal models of the included in vivo studies were summarized in Table 1.

Table 1.

The animal models included in this systematic review.

Authors (year)	Coating materials	Animal	Location	Position	Peri-implantitis model	In vitro tests
López-Esteban et al. (2014) [33]	Bioactive glass	Dogs	Mandible	Premolars and molars	No	Antibacterial tests (E.coli)
Qiao et al.(2015) [34]	Silver	Dogs	Mandible	Premolars and molars	No	NA
Godoy-Gallardo et al.(2016) [35]	Silver	Dogs	Premolar	Premolar to molars	Yes	NA
Cheng et al. (2016) [36]	Halogenated furanone	Dogs	Mandible	Molars	Yes	NA
Zhang et al.(2019) [37]	Tantalum pentoxide	Dogs	Mandible	Premolar to molars	No	Antibacterial tests (F.nucleatum and P. gingivalis)
Rahmati et al.(2020) [38]	Doxycycline	Dogs	Mandible	Premolar to molars	No	NA
Mistry et al.(2021) [39]	Zninc and hydroxyapatite	Goats	Mandible	Between premolar and incisor teeth	No	NA
Vandamme et al.(2021) [40]	Silicon dioxide	Pigs	Mandible	Premolar and molar	No	NA
Yin et al.(2021) [41]	Zinc and stronitium	Dogs	Mandible	Anterior teeth	Yes	Antibacterial tests (S.mutans and P.gingivitis) Cellular tests (BMSC)
Wu et al.(2021) [42]	N-halamine polymeric	Rabbits	Mandible	Not report	Yes	Antibacterial tests (S. aureus and P. gingivalis) Cellular tests (MC3T3-E1 preosteoblasts)
Yao et al.(2022) [43]	Silver and stronitium	Rabbits	Maxilla	Anterior teeth	No	Antibacterial tests (E.coli and S.aureus) Cellular tests (MC3T3-E1 preosteoblasts)
Wen et al.(2023) [44]	Zinc dioxide	Rats	Maxilla	Molars	Yes	Antibacterial tests (P. gingivalis and A. actinomycetemcomitans) Cellular tests (BMSC)
Xiao et al.(2024) [45]	Titanium dioxdie	Rabbits	Mandible	Anterior teeth	No	Antibacterial tests (E.coli and S.aureus) Cellular tests (MC3T3-E1 preosteoblasts)
Aboelmahasen et al.(2024) [46]	Silver/ hydroxyapatite or zinc oxide	Rabbits	Maxilla	Anterior teeth	No	NA
Liu et al.(2024) [47]	Anionic casein phosphopeptides, amorphous calcium phosphate and cationic poly (L-lysine)	Rabbits	Mandible	Anterior teeth	Yes	Antibacterial tests (P. gingivalis and A. actinomycetemcomitans) Cellular tests (MC3T3-E1 preosteoblasts)

3.3.2.1. Animal selection

The animal models identified in this systematic review comprised five animal species: rats (1 study [44]), rabbits (5 studies [42], [43], [45], [46], [47]), dogs (7 studies, [33], [34], [35], [36], [37], [38], [41]), pigs (1 study [35]) and goats (1 study [39]).

3.3.2.2. Implantation sites

Wen et al. conducted in vivo studies using rat maxillary molars [44]. Among the five rabbit model studies, two evaluated osseointegration outcomes in the maxilla [43], [46], while three focused on mandibular implantation [42], [45], [47]. Four rabbit studies specified anterior teeth as the implantation site, whereas one study omitted site documentation. Dog models predominated in the reviewed literature, with all seven dog studies utilizing mandibular implantation [33], [34], [35], [36], [37], [38], [41]. Most implants of the dog models were placed in premolar/molar regions, with the exception of Yin et al., who targeted the canine region [41]. Both pigs and goats models used mandibular implantation but differed in site selection: pigs studies utilized premolar/molar sites [40], whereas goats implants were positioned between premolar and incisor teeth [39]

3.3.2.3. The peri-implantitis model

Six animal studies evaluated osseointegration outcomes using peri-implantitis models [35], [36], [41], [42], [44], [47]. Four studies induced peri-implantitis by placing non-resorbable silk ligatures around implant necks [35], [36], [41], [42]. Cheng et al. further exacerbated peri-implant infection post-ligation through a high-sucrose dietary [35], [36], [41]. Wen et al. [44] and Liu et al. [47] established peri-implantitis models via bacterial contamination of implants. While Wen et al. [44] utilized a monospecies inoculum (P. gingivalis), Liu et al. [47] applied a polymicrobial solution containing both P. gingivalis and A. actinomycetemcomitans. Critical methodological parameters, including bacterial concentration and contamination duration, were inconsistently reported across these studies.

3.3.2.4. Surgical protocol

Fig. 3 summarizes the experimental timelines of the surgical protocols with the implantation date serving as the baseline. Substantial methodological variability was observed across studies.

Fig. 3.

The surgical timeline and procedure of the animal models included in this systematic review with the implantation date serving as the baseline.

3.4. Healing period I (tooth extraction to implant placement)

Eleven studies placed implants after teeth extraction [33], [34], [35], [36], [37], [38], [40], [41], [44], [45], [47], while four utilized edentulous sites without teeth extraction [39], [42], [43], [46]. The mean healing duration pre-implantation was 6.55 ± 5.73 weeks. Immediate implantation (no healing interval) was performed in four studies [36], [44], [45], [47], whereas seven allowed socket healing for 4–12 weeks prior to implantation [33], [34], [35], [37], [38], [40], [41].

3.5. Healing period II (implant placement to terminal endpoint)

The mean post-implantation observation period was 7.96 ± 4.30 weeks. Mistry et al. [39] and Godoy-Gallardo et al. [35] reported extended 24-week observation periods. Wu et al. conducted a phased protocol: osseointegration was assessed at 4 weeks post-implantation, followed by ligature-induced peri-implantitis with an additional 4-week monitoring period [42]. While most studies utilized single terminal time points, three incorporated serial euthanasia to evaluate temporal healing dynamics [39], [45], [47].

4. Dual-biofunctional coating materials

4.1. Coating materials for inducing osseointegrtion

The dual-biofunctional coating used in dental implant for improving osseointegration could be roughly categorized as inorganic compounds, peptides, metal-based materials (Table 2).

Table 2.

The dental coating materials for inducing osseointegration.

Materials	Form	Mechanisms
Inorganic compounds	Calcium phosphate and hydroxyapatite [39], [46], [47]	Enhances osteoblast adhesion by surface roughness Initiates apatite formation by calcium and phosphate ionic release Activates osteogenic pathways
Peptides	Casein phosphopeptides [47]	Stabilizes amorphous calcium phosphate. Release more calcium ions at lower pH for osseointegration
Metal and metal oxides	Tantalum pentoxide [37]	Promotes selective adhesion of bone mesenchymal stem cells. Stimulates the expression of bone-forming proteins.
	Strontium [41], [43]	Enhances the adhesion, proliferation and osteogenic differentiation.
	Zinc [39] and zinc oxide [44], [46]	Promotes osteoblast attachment and reduces osteoclastic resorption.

Among the reviewed studies, three demonstrated the efficacy of calcium-based materials in enhancing osseointegration [39], [46], [47]. Mistry et al. reported that hydroxyapatite-coated implants exhibited accelerated BIC compared to uncoated controls in a caprine mandibular model, with histological analysis confirming superior osteogenic activity [39]. Similarly, Liu et al. observed enhanced osteogenesis in rabbit models, where hydroxyapatite-modified titanium surfaces significantly increased alkaline phosphataseactivity and trabecular bone density relative to bare titanium [47]. Aboelmahasen et al. incorporated silver nanoparticles into hydroxyapatite coatings, outperforming both uncoated and pure hydroxyapatite groups in the maxilla of rabbits [46].

Some metal ions, including tantalum (Ta), strontium (Sr), and zinc (Zn), have demonstrated significant potential as dual-biofunctional coating materials. Zhang et al. reported that tantalum pentoxide (Ta₂O₅) coatings promoted bone-to-implant contact in canine models by simultaneously upregulating osteogenic markers (e.g., RUNX2 and OCN) [37]. Yao et al. engineered a Sr-Ag co-doped coating that synergistically enhanced osseointegration (P < 0.05 vs. control) [43]. In rabbit models, Sr-coated implants achieved approximately double the pull-out forces of uncoated counterparts (81.1 ± 20.2 N vs. 43.1 ± 12.1 N). Yin et al. co-deposited Sr and Zn ions on titanium surfaces, demonstrating accelerated osseointegration in canine mandibular models under periodontitis conditions (P < 0.01 vs. control) [41]. The peptide materials, such as the casein phosphopeptide, a milk-derived phosphorylated peptide was utilized by Liu et al. to enhance the osseointegration [47].

4.2. Coating materials for preventing infection

The antimicrobial components used in the included literature could be roughly categorized into antibiotics, antiseptics, metal and metal oxides, inorganic compounds, proteins, polymers and others (Table 3).

Table 3.

The coating materials for preventing infection.

Materials	Form	Mechanisms
Antibiotics	Doxycycline [38]	Targets specific bacterial metabolic processes
Antiseptics	Chlorhexidine [30], [40]	Disrupts of bacterial membrane
Metal and metal oxides	Silver [34], [39], [43], [46] Zinc [39], [41] Zinc oxide [44] Tantalum pentoxide [37]	Disrupts of bacterial membrane Generate reactive oxygen species. Interaction with cellular components
Organic compounds	Casein phosphopeptides [47]	Release more calcium ions at lower pH and inhibit the bacterial adhesion
	N-halamine polymer [42]	Release of oxidative halogen species that oxidize and kill bacteria
	Halogenated furanone compound [36]	Acts as N-acyl homoserine lactone antagonists disrupts of bacterial quorum sensing, inhibiting microbial colonization
Inorganic compounds	Bioactive glass [33]	Release of ions, like calcium, sodium and aluminum ions
Others	Titanium peroxide [45]	In situ antibacterial activity of titanium peroxide using near-infrared light irradiation.

To mitigate bacterial adhesion around implants, antibiotics and antiseptics have been widely utilized. Rahmati et al. investigated the effects of doxycycline-coated titanium implants in dog models, demonstrating that such coatings did not harm host immune responses [48]. Carinci et al. conducted a clinical trial involving 15 healthy patients to evaluate the efficacy of the PIXIT implant, which was coated with an alcoholic solution containing 1 % chlorhexidine gluconate [30]. The findings revealed a significant reduction in bacterial loading on the treated implants compared to untreated controls, with no local evidence of inflammation observed.

Metal and metal oxides are among the most extensively utilized antibacterial materials in the literature. Qiao et al. demonstrated that titanium implants functionalized with silver nanoparticles exhibited significantly enhanced antimicrobial properties, alongside improved osseointegration in a canine model [34]. Similarly, Godoy-Gallardo et al. observed reduced vertical bone resorption in silver-coated implants compared to uncoated controls in a peri-implantitis model, attributing these favorable outcomes to the material’s capacity to inhibit bacterial colonization on the implant surface [35]. A comparative study by Aboelmahasen et al. reported the superior antibacterial efficacy of silver coatings in a rabbit model [49]. Wen et al. reported that zinc oxide nanoparticle coatings effectively inhibited key periodontal pathogens while simultaneously promoting bone regeneration in a rat model [50]. However, Mistry et al., observed that zinc-coated implants exhibited direct bone-implant contact but paradoxically demonstrated increased bacterial adherence and coating instability [51].

Organic compounds, including casein phosphopeptide, N-halamine polymer, and halogenated furanone derivatives, have been explored as antibacterial agents for dental implant coatings. Liu et al. utilized casein phosphopeptide as an antibacterial strategy, which functions by binding to bacterial surfaces to inhibit adhesion, growth, and plaque formation [47]. Wu et al. developed a long-lasting renewable antibacterial coating based on N-halamine polymer, capable of releasing active chloride ions to rapidly eradicate bacteria [42]. This coating demonstrated long term antimicrobial efficacy in vitro (12–16 weeks). Halogenated furanones, originally derived from red algae, exhibit structural similarity to bacterial N-acyl homoserine lactone, a key signaling molecule in bacterial quorum sensing. Cheng et al. reported that a halogenated furanone coating effectively combated peri-implant infections in canine models, highlighting its potential for clinical applications [36].

Inorganic compounds, such as bioactive glass, have been investigated for their antibacterial capacity. López-Esteban et al. reported that bioactive glass coatings exhibited significant biocide effects against E. coli in vitro, alongside favorable osseointegration following implantation in canine mandibles [33]. The authors attributed the antibacterial activity of bioactive glass to the high concentration of calcium ions released from the material, which disrupts the electrochemical potential of bacterial membranes. Xiao et al. developed a nanostructured titanium peroxide coating [45]. This coating achieved significant antibacterial efficacy against E. coli and S. aureus under near-infrared irradiation, highlighting its potential for advanced implant applications.

5. The osseointegration efficacy of the dual-biofunctional materials

5.1. The osseointegration efficacy without peri-implantitis models

The results of the osseointegration efficacy without peri-implantitis models were summarized in the Table 4.

Table 4.

The results of the osseointegration efficacy without peri-implantitis models.

Authors(year)	Micro-CT measurement	Histomorphometric measurement	Osseointegration results
López-Esteban et al.(2014)	No	No	Coated implants ≈uncoated implantsa
Qiao et al.(2015)	Yes	Yes	Coated implants > uncoated implants (p < 0.05)
Zhang et al.(2019)	Yes	Yes	Coated implants > uncoated implants (p < 0.05)
Rahmati et al.(2020)	Yes	Yes	Coated implants ≈uncoated implants (p > 0.05)
Mistry et al.(2021)	Yes	Yes	Coated implants > uncoated implants (p < 0.05)
Vandamme et al.(2021)	Yes	Yes	Coated implants ≈uncoated implants (p > 0.05)
Yao et al.(2022)	Yes	Yes	Coated implants > uncoated implants (p < 0.05)
Xiao et al.(2024)	Yes	Yes	Coated implants > uncoated implants (p < 0.05)
Aboelmahasen et al.(2024)	Yes	Yes	Coated implants > uncoated implants (p < 0.05)

a, This study did not perform statistical alnalysis; no p-value was reported.

Compared to uncoated implants, all included studies reported that dual-biofunctional materials exhibited osseointegration effects that were either comparable to or superior than those of the uncoated group. López-Esteban et al. conducted radiological analyses, which revealed no signs of osseointegration loss around implants coated with dual-biofunctional materials [33]. However, this study lacked quantitative comparisons between the dual-biofunctional coating group and the uncoated control group.

The remaining 14 studies employed micro-CT and histomorphometric measurements to comprehensively evaluate the osseointegration efficacy of dual-biofunctional implant coatings, supported by statistical analyses. Micro-CT analyses provided quantitative data on bone volume to tissue volume ratio (BV/TV), bone mineral density, trabecular thickness, and trabecular number. Histomorphometric measurements further quantified the percentage of BIC and bone density within the threads (BDwt%). Among these studies, three reported no significant difference in osseointegration outcomes between the dual-biofunctional coating group and the uncoated group (P > 0.05), while 12 studies demonstrated significantly enhanced osseointegration in the dual-biofunctional coating group (P < 0.05).

5.2. The osseointegration efficacy in peri-implantitis models

Six studies investigated the osseointegration efficacy of dental implant coatings in peri-implantitis models [35], [36], [41], [42], [44], [47]. Due to significant variability in study design, direct comparisons between the results were challenging. Most studies reported significantly enhanced osseointegration around coated implants (P < 0.05) whease Godoy-Gallardo et al. observed similar outcomes between the uncoated group (probing depth = 2.0 ± 0.8 mm) and the silver-coated group (2.1 ± 0.8 mm) [35] (P > 0.05).

Cheng et al. compared clinical measurements of osseointegration, bone-implant contact rate, and ultimate interfacial strength in three groups: halogenated furanone-coated implants, uncoated implants, and a positive control group (minocycline hydrochloride ointment) [36]. The halogenated furanone-coated group demonstrated improved osseointegration, with a probing depth of 2.65 ± 0.26 mm, approximately half that of the uncoated control group (4.50 ± 0.41 mm) and comparable to the positive control group (2.78 ± 0.73 mm), highlighting its potential to promote osseointegration. Yin et al. reported that the zinc and strontium-coated group exhibited a lower probing depth (4 mm) compared to the uncoated group (7 mm) [41]. Radiological analysis further revealed significantly better alveolar bone height maintenance around the coated implants. Wu et al. demonstrated that N-halamine polymer-coated implants significantly enhanced bone height, BV/TV ratio, and maximum removal torque at all evaluated time points [42]. Similarly, Liu et al. observed increased BV/TV and BIC with anionic casein phosphopeptides-amorphous calcium phosphate coatings [47].

6. Discussion

Based on the search protocol, we identified 270 papers, of which 45 were limited to in vitro studies, and 79 in vivo studies involved implantation in non-oral sites such as tibia, femora, or subcutaneous incisions. After applying the inclusion and exclusion criteria, ultimately 16 papers were included in this systematic review, comprising 15 animal studies and 1 clinical trial.

The concept of osseointegration originated from observations of titanium implants in animal models and is defined as the direct structural and functional connection between living bone tissue and the implant surface. This biological process is inherently complex and cannot be fully replicated in vitro. However, recent initiatives by the National Institutes of Health (NIH) to reduce animal studies primarily aim to address ethical concerns, foster scientific innovation, and enhance research efficiency [52]. While in vitro systems offer notable advantages, such as standardized cell sourcing, controlled culture conditions, and reproducible experimental parameters, their translational validity remains limited due to the absence of critical physiological variables present in vivo [16], [53]. Consequently, in vivo models remain indispensable for dual-biofunctional implants, particularly in evaluating their ability of both osseous and soft tissue integration under peri-implantitis challenges.

Unlike implants placed in other parts of the body, dental materials are immediately coated with salivary components upon insertion into the oral cavity [54]. Saliva contains a high concentration of microorganisms, approximately 10⁹ colony-forming units per mL, and its continuous flow supplies proteins to bacteria, accelerating microbial metabolism and biofilm formation [55], [56]. Therefore, the performance, bioactivity and safety of dental implant coatings with dual-biofunctional coatings cannot be fully validated in the non-oral anatomical regions (e.g., the tibia or femur).

After analyzing the osseointegration components utilized in dual-biofunctional coatings, it is evident that the mechanism of inducing osseointegration is predominantly linked to their direct or indirectly ability to enhance calcium signaling pathways and improve the osseointegration ability of the implant surface. Extracellular calcium ions excreted from the coatings or CaP mineralized layer act as coupling factors between osteoclasts and osteoblasts, influencing the expression of specific calcium-channel isoforms on osteoblasts [57], [58], [59]. Higher concentrations of calcium ions stimulate the migration of pre-osteoblasts to resorption sites, where they differentiate into mature osteoblasts responsible for bone formation [54], [60]. Calcium signaling further promotes osteogenesis through key pathways, including BMP, TGF-β, and Wnt/β-catenin [61]. Previous studies have demonstrated that casein phosphopeptides, derived from milk proteins, can bind and chelate calcium ions, stabilizing amorphous calcium phosphate and therefore enhancing osseointegration [62], [63]. Tantalum, another promising implant coating material, exhibits excellent biological properties that facilitate osteoblast adhesion and proliferation, improving osseointegration [63]. Similarly, strontium and zinc, essential trace elements for bone and cartilage tissue formation, have been shown to enhance osseointegration in dental implants [64]. In this systematic review, studies conducted in canine and rabbit models by Zhang et al. [37], Yao et al. [43], and Yin et al. [41] demonstrated that coatings containing tantalum, strontium, and zinc can significantly enhance osseointegration, even in peri-implantitis models.

In this systematic review, metal and metal oxide-based materials are among the most widely used antimicrobial components in dual-biofunctional coatings. These coatings exert their antibacterial effects primarily through the catalytic generation of reactive oxygen species and excessive free radicals, which damage bacterial cellular components and impair DNA repair mechanisms [65], [66]. The antibacterial efficacy of these materials is particularly pronounced against Gram-negative bacteria due to their thinner cell wall structure, composed of lipopolysaccharides, compared to the thicker peptidoglycan layer of Gram-positive bacteria [13], [67]. This structural vulnerability enhances the effectiveness of metal ions in targeting peri-implantitis-related pathogens [68], [69]. Interestingly, unlike the consistently favorable antibacterial performance observed with silver coatings, zinc-coated implants have shown controversial results. Mistry et al. observed this phenomenon in their study of zinc-doped hydroxyapatite applied via plasma spraying [39]. They attributed the inconsistent outcomes to increased surface roughness resulting from the coating process. This finding highlights the significant influence of implant surface topography on bacterial adhesion and suggests that the antibacterial efficacy of the coating’s chemical components may be compromised due to changes in physical parameters [70], [71].

This systematic review includes one clinical trial that evaluated the efficacy of chlorhexidine gluconate-coated dental implants in 15 healthy patients (60 implants) scheduled for bilateral fixed prostheses or crown restorations supported by implant fixtures [30]. Patients were recalled at 4 months post-implantation, and no adverse effects or implant failures were reported. However, the study was limited by the absence of long-term follow-up and a detailed quantitative comparison of osseointegration parameters, which restricts the ability to draw definitive conclusions about the coating's long-term efficacy and performance.

Among the animal studies included in this review, five animal models were utilized: rats, rabbits, dogs, pigs, and goats. To better mimic the oral cavity environment, Schwarz et al. recommended the use of nonhuman primates, as they possess oral structures similar to humans, naturally occurring bacterial plaque biofilms, and a susceptibility to gingivitis [72]. However, none of the included studies employed nonhuman primates as an animal model. In addition, one of the included studies utilized rats, as reported by Wen et al. [44], which is not recommended for dental implant research due to significant differences in bone structure between rats and humans, as well as the size limitations that preclude the simultaneous testing of multiple implants [73].

A large variance was observed in the healing times across the animal studies, including the period following tooth extraction (6.55 ± 5.73 weeks) and the period after implantation (7.96 ± 4.3 weeks). Among the included studies, four involved immediate implant placement following tooth extraction [36], [44], [45], [47]. Passoni et al. investigated the influence of immediate versus delayed implant placement in dog mandibles and found that the immediate implant groups demonstrated significantly better BIC scores (P < 0.05) [74]. Regarding the healing period after implantation, Bonfante et al. measured buccal bone loss and BIC at 2 and 4 weeks post-implantation. Their results indicated a 20–25 % increase in BIC between 2 and 4 weeks (P < 0.05), while buccal bone loss nearly doubled during the same period (P < 0.05) [75]. The authors concluded that histomorphometric parameters, when evaluated in implants placed immediately after tooth extraction, vary significantly over time. These findings highlight the importance of standardizing surgical protocols to ensure accurate comparisons of the efficacy of dual-biofunctional coatings.

Osseointegration outcomes is the prerequisite of dental implants to achieve stable anchorage within the jawbone [20]. Therefore, it is essential to present data demonstrating the safety and efficacy of dual-biofunctional dental implant coating materials before considering clinical application [76]. Given concerns regarding the biocompatibility of these coatings, especially their antimicrobial components [77], [78], [79], both clinical trials and animal studies reported no adverse effects on osseointegration in groups with dual-biofunctional coatings in this systematic review. Godoy-Gallardo et al. attributed the favorable performance of dual bio-functional coatings to their ability to reduce bacterial colonization on the implant surface while indirectly promoting osteoblast activity [35].

Since peri-implantitis affects approximately 10–45 % of dental implants, the ability of dual-biofunctional coatings to preserve bone tissue around implants under peri-implantitis conditions has been a major focus of research [80], [81], [82]. In this systematic review, six in vivo studies evaluated osseointegration outcomes using peri-implantitis models [35], [36], [41], [42], [44], [47]. Most studies reported enhanced BIC and reduced probing depths in the coated groups, suggesting that dual-biofunctional coatings hold significant promise in preventing peri-implantitis and maintaining bone integrity [83]. The oral cavity harbors a vast number of bacteria, and once these bacteria colonize the surface of a dental implant, osseointegration can be compromised [84], [85]. Dual-biofunctional implant coatings can act as a barrier to prevent bacterial colonization while simultaneously supporting osseointegration, providing a beneficial strategy for patients at higher risk of infection [86], [87].

However, factors such as salivary flow, masticatory forces, pH fluctuations, and polymicrobial biofilms can significantly influence the long-term durability and antimicrobial effectiveness of implant coatings [88], [89]. For example, the oral environment poses risks of electrochemical corrosion, such as saliva contains aggressive anions like chlorides that can dissolve protective oxide layers, leading to the degradation of coating materials [90]. Consequently, extended longitudinal studies are essential to evaluate how these coatings perform and withstand the complex conditions within the oral cavity over time.

This systematic review has several limitations. First, only one clinical trial was included, and its follow-up period was limited to four months, which is insufficient to provide long-term evidence on the efficacy and safety of dual-biofunctional dental implant coatings. Second, the 15 animal studies included exhibited significant variability in study protocols and surgical procedures. This heterogeneity hampers direct comparison of results and reduces the overall reliability of the findings, also limiting the capacity to perform meaningful meta-analyses. To address these issues, future research should aim to standardize in vivo study designs, or even better to develop a standardized in vitro model that could mimic in vivo responses. Finally, potential biases should be acknowledged. There may be language bias, as only studies published in English were considered, possibly excluding relevant research published in other languages. Publication bias remains a concern, as studies with positive outcomes are more likely to be published, which could lead to an overestimation of the coating effectiveness.

7. Conclusion

Based on the findings of the included in vivo studies (1 clinical trial and 15 animal studies), the following conclusions were drawn:

• 1.The reviewed studies highlighted various osseointegration-promoting components, such as inorganic compounds (e.g., calcium phosphate), peptides, and metal-based materials (tantalum, strontium and zinc).
• 2.The antimicrobial agents including metals and metal oxides (e.g., silver, zinc, zinc oxide, and tantalum pentoxide) as well as polymers.
• 3.All included dual-biofunctional coatings showed no adverse effects on osseointegration and promising potential in combating peri-implantitis.
• 4.Variability in study designs and short follow-up periods in vivo studies highlights the need of standardized protocol to reduce heterogeneity.

Declaration of Generative AI and AI-assisted technologies in the writing process

During the preparation of this work the authors did not use AI and AI assisted technologies in the writing process.

Declaration of Competing Interest

The authors declare no competing interests.

Appendix A. Supplementary material

Supplementary material