In the current study, all implants were placed crestally, they were platform switched of a single implant system, prosthesis was placed on average 5 weeks after uncovering surgery and healing abutment connection. Implant sulci were probed 2–4 weeks following crown placement and were then probed at 12 months. There was no evidence of early crestal bone loss that could be attributed to bone remodeling following the surgical phase26 and/or the prosthetic phase.27–28 In contrast to previous findings, no apical displacement of the labial soft-tissue margin was observed,29 to suggest buccal bone resorption.28 Evidence shows that osseous changes occur rapidly following abutment connection and before crown placement (time-interval of about 1 month) and stabilize thereafter29–30 with the latter time-period coinciding with that of the current study. However, at 12 months, probing depth, and attachment level -in absence of recession- tended to increase at the implant-site by 0.6 mm without any overt clinical signs of inflammation. At contralateral teeth, PPD and CAL remained at similar levels during the study-period with no signs of gingivitis. Similarly, no significant alterations in the width of keratinized tissues were observed at implants and teeth over 12 months.
Increased probing noted at an implant-site might be due to presence of mild inflammation within the soft-tissues at the implant–abutment connection level.31 The study-participants were closely monitored for oral hygiene and demonstrated low plaque-, inflammation- and bleeding- indices. Previous evidence in dogs demonstrates that the location of the interface between a submerged implant and the connected abutment appeared to play a role in bone levels with increased loss as the interface was placed more apically.30, 32 This finding might be attributed to proliferation of the epithelium apical to the connection interface to isolate the microbial plaque challenge and establish a biological width 2 mm apical to the micro-gap. It might also be that the epithelium could attach onto the implant fixture which is stable rather than to the abutment which is expected to show micromovement and similarly form the biological width 2 mm apical to the micro-gap. Another possibility might be that the blood supply of the periosteum and connective tissue was severed at the time of abutment connection. It should be also noted that improved soft-tissue height is expected to occur at the proximal site of an implant 12 months following prosthetic restoration29,33–34 in motivated patients for oral hygiene. Herein, the interproximal papilla (mesial-buccal) of the implant-site unsurprisingly had not yet established its final configuration at baseline, which might partially account for the tendency of PPD to increase with time.33
There is scarce evidence in the literature regarding the PICF proteome composition in health and disease compared to GCF.35–37 A recent study followed peri-implantitis sites in comparison with tooth- and/or other implant- sites free of disease, when present, and showed distinct biological processes related to local immune responses in disease over health.38 The PICF proteomic expression profile within a peri-implantitis site has been shown to identify responses of implant loss or implant survival.39
The proteins identified herein were predominantly of human origin with only a few having bacterial sources. The GCF collection is known to also absorb cells from the adjacent plaque deposits but has not been found to reflect the composition of subgingival plaque in the same manner as subgingival paper-point samplingdoes.40 This study analyzed by ORA all regulated and exclusive human proteins (see Appendix for the intersection of regulated proteins with previously identified biomarkers from proteomic studies). Interestingly, it appeared that at the protein level during the first year of functional loading, “interferon-alpha response” comprised the only over-represented pathway in PICF. Interferons are cytokines produced mainly in response to pathogens and their products, especially during viral and bacterial infections.41 They modulate functions of the immune system, upregulate major histocompatibility complex molecules, and increase immunoproteasome activity.42 The enrichment of the "interferon-alpha response" in PICF was attributed to the regulation of the proteasome, as evidenced by the proteasome subunit-beta type-8 (P28062), proteasome activator complex subunit-1 (Q06323), and proteasome subunit-beta type-9 (P28065), and of two additional proteins, complement C1s subcomponent (P09871) and interferon-induced GTP-binding protein Mx1 (P20591). The proteasomes degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds, in the cytoplasm of cells exposed to oxidative stress and pro-inflammatory stimuli.43 These protein complexes have been associated with peri-implantitis progression.44 The complement subcomponent C1when activated, initiates the classical complement pathway by the binding of antigen-antibody complexes to the C1q protein. This protein has been shown to selectively promote the migration and survival of inflammatory infiltrates into sites of osseointegrated implants.45 The interaction between the titanium surface of a dental implant and the oral micro-environment results in the release of implant degradation products (titanium particles) that are difficult to be effectively removed by mechanical instrumentation and can trigger biological responses.46 These particles activate the complement complex and are considered to elicit pro-inflammatory responses leading to cell death and osteoclastogenesis47 and these phenomena can be amplified in the presence of microbial deposits. It is also known that the complement component C1q increases with age, having an impact on signaling and differentiation of osteoclasts and osteoblasts leading to reduced bone remodeling that may influence osseointegration of dental implants.48 These findings suggest that at the protein level in PICF during the first year of function, low level of inflammation within the peri-implant tissues was not revealed by clinical and radiographic observations. These early findings necessitate the importance of following up implants without overt clinical signs of inflammation.
The current study showed metabolic processes of “estrogen response” and “glycolysis and gluconeogenesis” being regulated at the gingival crevice of teeth at 12months from baseline. There is emerging evidence in the bone-glucose axis to demonstrate that various bone associated growth/transcription factors, which mediate the function of osteoblasts and osteoclasts, intersect with glucose metabolism49 and this could apply during tissue remodeling processes in absence of infection. Estrogen plays an important role in the growth and maturation of bone as well as in the regulation of bone turnover in adults. It has been shown that estrogen modulates the expression of receptor activator of NF-κB ligand (RANKL), an essential cytokine for bone resorption by osteoclasts. RANKL can be produced by a variety of hematopoietic (e.g., T- and B-cells) and mesenchymal (e.g., osteoblasts) cell types.50 However, the cellular mechanisms by which estrogen acts on bone are still a matter of controversy. Evidence has implicated the periodontium as a target tissue for steroid hormones and various possible mechanisms have been suggested to describe how androgens, estrogens, and progestins affect gingiva by interacting with microbial organisms, the vasculature, the immune system, and specific cells in the periodontium.51–52 “Early estrogen response” and “late estrogen response” also comprised nicotinamide adenine dinucleotide phosphate (NADPH) which is an essential electron donor that functions as a reactive oxygen species (ROS) scavenger in cellular antioxidation systems.53 Interestingly, glucose is a main source of NADPH production and high glucose conditions have been shown to increase NADPH production in addition to ROS generation by periodontal ligament stem cells having adverse effects on their osteogenic differentiation potential.54 Current data reflect hormonal pathways that prevail in the gingival crevice of teeth and might negatively affect tissue homeostasis in patients successfully treated for mild/moderate forms of periodontitis.
An interesting finding was the regulation of the “interferon-alpha response” and “allograft rejection” in pooled PICF versus pooled GCF at baseline. Notable proteins involved in these process networks included complement C1 and proteasome in addition to matrix metallopeptidase-9 (MMP-9) and complement C2. Metallopeptidase-9 may be used as a potential target biomarker for peri-implantitis and has been considered as a potential drug target for peri-implantitis.55–56 Complement C2 is part of the classical and the lectin pathways of complement activation and is involved in the first line of defense against microbial infection.57 At 12 months, the "p53 pathway" was the only significantly enriched Hallmark pathway between teeth and implants. It is worth mentioning that the next two enriched terms, although not reaching the study standards of significance, were "allograft rejection," which includes complement C2, and "early estrogen response." Importantly, these pathways were also represented at baseline. Of significance, the “p53 pathway” responds to stresses that can disrupt the fidelity of DNA replication and cell division. A stress signal results in the activation of the “p53” protein as a transcription factor that initiates a program of cell cycle arrest, cellular senescence, or apoptosis.58 Evidence suggests that “p53” plays a pivotal role in PDL cell homeostasis and seems to be upregulated in oral inflammatory diseases.59 Taken together, the current data reveal cellular unique pathways of inflammatory processes in peri-implant and periodontal tissues that are clinically free of signs of disease. These findings corroborate that recently restored implants appear to have a protein profile that may reflect inflammatory responses to bacterial infection or tissue reaction similar to that of a foreign body reaction.60 It can be argued whether the protein responses identified here reflect a long non-resolving inflammatory response rather than inflammatory processes needed for implant integration. Of interest, protein profiles of PICF in contrast to GCF samples demonstrated significant divergence over the study-period, indicating major shifts in proteins expressed at implant-sites possibly due to tissue maturation.
The current study followed a well-defined cohort of 10 non-smokers that was well characterized. Study limitations include difficulties in eluting crevicular fluid and conducting meaningful analysis due to its small volume, particularly in health. The protein concentrations in the PICF samples exhibited substantial variation across different subjects and time-points. This variability poses challenges for quantitative proteomic analyses due to normalization and alignment difficulties. While pooling samples may result in loss of variation in protein quantification and dilution of low-abundance proteins, it can also unveil unique hits specific to the pooled samples, as observed previously using GCF samples.25 Furthermore, different clusters of gene-expression signatures may represent distinct states of health or disease.61 Consequently, the identification and quantification of the crevicular proteome may not fully reveal the spectrum of regulated proteins or their significance in tissue homeostasis.25 The small sample size of the study limits generalizability of current findings to larger populations.