Paulo M. Camargo

The clinical value of salivary biomarkers for periodontal disease.

Periodontal disease is a chronic bacterial infection characterized by persistent inflammation, connective tissue breakdown, and alveolar bone destruction (3). It has been a great challenge in periodontology to determine biomarkers for screening and predicting the early onset of disease (prognostic tests) or evaluating the disease activity and the efficacy of therapy (diagnostic tests). Traditional diagnostic measures, such as periodontal pocket depth, attachment level, plaque index, bleeding on probing and radiographic assessment of alveolar bone loss, are informative to evaluate disease severity (83) but provide few useful determinants of disease activity (2, 29, 30, 61, 80). The presence of bleeding on probing is still the best disease activity predictor available, but it is not specific enough and thus reveals too many false positives. The absence of bleeding on probing on the other hand is a very precise negative predictor of disease activity (59, 60, 73). The identification of susceptible individuals or sites at risk from disease, and the diagnosis of active phases of periodontal disease, represent a challenge for both clinicians and oral health researchers. Indeed, the subjectivity and variability of definitions of the periodontitis phenotype in clinical studies are such that no universally accepted diagnostic criteria exist to define what a case of periodontitis is, thus making outcomes from different studies difficult to compare (11). It has long been established that a simple and non-invasive diagnostic tool that allows rapid screening, provides accurate predictive information and enables reliable evaluation of periodontal disease status would be of great value to both dentists and patients. Saliva is a secretion of the salivary and mucous glands and is of major importance in the maintenance of oral health (22). The fluid is readily accessible via a totally non-invasive collection method, and contains locally produced microbial and host response mediators. For the past two decades, saliva has been increasingly evaluated as a diagnostic fluid for detecting caries risk (12, 13, 62), periodontitis (18), oral cancer (63), breast cancer (104–107), salivary gland diseases (42) and systemic disorders such as hepatitis and the presence of human immunodeficiency virus (HIV) or hepatitis C virus (23, 39, 75, 123). The ease of collecting, handling and testing saliva has led to its use for determining hormone levels, including estradiol, progesterone and testosterone, dehydroepiandrosterone and cortisol (31). Numerous drugs are detectible in oral fluid, and can even be quantified in saliva as a viable substitute for testing in blood, and, as a result, salivary diagnostic technology is currently utilized to test for drugs of abuse, such as cocaine (50, 94), methamphetamines (44, 93) and opiates (20). Additionally, testing of saliva can also be used for therapeutic monitoring of drugs, such as digoxin (19, 108), methadone (17) and some anticonvulsants (34). The biochemical analysis of saliva is particularly important in dentistry. Estimation of the risk of disease onset and severity, monitoring of disease progression and evaluation of therapeutic efficacy for premalignant and malignant oropharyngeal lesions as well as infectious diseases of the oral cavity can be performed by analyzing an array of constituents within saliva. Although there is a large body of literature on gingival crevicular fluid biomarkers, this review limits itself to saliva analysis. Salivary constituents that have been studied as potential diagnostic biomarkers for periodontal disease mainly include locally produced proteins of host and bacterial origin (enzymes, immunoglobulins and cytokines), genetic ⁄ genomic biomarkers such as DNA and

Platelet-rich fibrin and bovine porous bone mineral vs. platelet-rich fibrin in the treatment of intrabony periodontal defects.

BACKGROUND AND OBJECTIVE Bovine porous bone mineral (BPBM) is a xenograft that has been successfully utilized in periodontal regeneration. Platelet-rich fibrin (PRF) is a leukocyte and platelet preparation that concentrates various polypeptide growth factors and therefore has the potential to be used as regenerative treatment for periodontal defects. The purpose of this study was to examine the suitability of autologous PRF as regenerative treatment for periodontal intrabony defects in humans and to examine the ability of BPBM to augment the regenerative effects exerted by PRF. MATERIAL AND METHODS Using a split-mouth design, 17 paired intrabony defects were randomly treated either with PRF or with PRF-BPBM combination. Re-entry surgeries were performed at 6 mo. Primary study outcomes were changes in pocket depth, attachment level and defect fill. RESULTS Preoperative pocket depths, attachment levels and transoperative bone measurements were similar for the PRF and PRF-BPBM groups. Postsurgical measurements revealed a significantly greater reduction in pocket depth in the PRF-BPBM group (4.47±0.78 mm on buccal and 4.29±0.82 mm on lingual sites) when compared with the PRF group (3.35±0.68 mm on buccal and 3.24±0.73 mm on lingual sites). The PRF-BPBM group presented with significantly greater attachment gain (3.82±0.78 mm on buccal and 3.71±0.75 mm on lingual sites) than the PRF group (2.24±0.73 mm on buccal and 2.12±0.68 mm on lingual sites). Defect fill was also greater in the PRF-BPBM group (4.06±0.87 mm on buccal and 3.94±0.73 mm on lingual sites) than in the PRF group (2.21±0.68 mm on buccal and 2.06±0.64 mm on lingual sites). CONCLUSION The results of this study indicate that PRF can improve clinical parameters associated with human intrabony periodontal defects, and BPBM has the ability to augment the effects of PRF in reducing pocket depth, improving clinical attachment levels and promoting defect fill.

A Surgical Reentry Study on the Influence of Platelet-Rich Plasma in Enhancing the Regenerative Effects of Bovine Porous Bone Mineral and Guided Tissue Regeneration in the Treatment of Intrabony Defects in Humans

BACKGROUND The purpose of this study was to evaluate the additional benefits provided by the incorporation of platelet-rich plasma (PRP) into a regenerative protocol consisting of bovine porous bone mineral (BPBM) and guided tissue regeneration (GTR) in the treatment of intrabony defects in humans. METHODS Twenty-three paired intrabony defects were surgically treated using a split-mouth design. Defects were treated with BPBM/GTR/PRP (experimental group) or with BPBM/GTR (control group). The clinical parameters evaluated included changes in probing depth, clinical attachment level, and defect fill as revealed by reentry surgeries at 6 months. RESULTS Preoperative probing depths, attachment levels, and transoperative bone measurements were similar for the two groups. Post-surgical measurements taken at 6 months revealed that both treatment modalities resulted in a significant decrease in probing depth, gain in clinical attachment, and bone fill of the defects compared to baseline. Postoperative differences observed between the two groups were 0.72 +/- 0.36 mm at buccal sites and 0.90 +/- 0.32 mm at lingual sites for probing depth, 0.82 +/- 0.41 mm at buccal sites and 0.78 +/- 0.38 at lingual sites for gain in clinical attachment, and 0.85 +/- 0.36 mm at buccal sites and 0.94 +/- 0.42 mm at lingual sites for defect fill, all favoring the experimental sites. However, none of the differences were statistically significant. CONCLUSION Within the limitations related to using a small sample size, PRP did not significantly augment the effects of BPBM and GTR in promoting the clinical resolution of intrabony defects.

Saliva diagnostics: utilizing oral fluids to determine health status.

Imagine a time where your health status could be available to you without the pain, discomfort and inconvenience of a physical examination. Distant vision of an inconceivable future or impending reality with potentially immeasurable impact? Recent advancements in the field of molecular diagnostics indicate this is not only possible, but closer than we think. Novel discoveries and substantial advancements have revealed that saliva may contain real-time information describing our overall physiological condition. Researchers are now reporting that, like blood and tissue biopsies, oral fluids could be a source of biochemical data capable of detecting certain diseases. What is even more intriguing is that this phenomenon not only applies to local disorders like oral cancer and Sjögrens syndrome, but distant pathologies like autoimmune, cardiovascular and metabolic diseases as well as viral/bacterial infections and even some cancers. These revelations have provided a foundation for the burgeoning field of salivary diagnostics and hence spurred the onset of investigations poised at deciphering the salivary milieu. This paper overviews salivary diagnostics from biomarker development to the multitude of techniques utilized in identifying saliva-based molecular indicators of disease. In doing so, we present oral fluids as an easily accessible noninvasive alternative to traditional diagnostic avenues and not just an essential component of the digestive process. Determining saliva as a credible means of evaluating health status represents a considerable leap forward in health care, one that could lead to enormous translational advantages and significant clinical opportunities.

Periodontal Regeneration – Intrabony Defects: A Consensus Report From the AAP Regeneration Workshop

BACKGROUND Treatment of intrabony defects is an important therapeutic goal of periodontal therapy. The goal of this consensus report was to critically appraise the evidence for the available approaches for promoting periodontal regeneration in intrabony defects. In addition to evaluating the effectiveness of new regenerative approaches for intrabony defects, recommendations for future research were defined for this area. METHODS A systematic review was conducted using computerized searches of PubMed and Cochrane databases, supplemented with screening of references in original reports, review articles, and a hand search in selected journals. All searches were focused on regenerative approaches with histologic evidence of periodontal regeneration (proof of principle), clinical trials, and case reports. For purposes of analysis, change in intrabony defect fill was considered the primary outcome variable, with change in clinical attachment as a secondary outcome. The SORT (Strength of Recommendation Taxonomy) grade was used to evaluate the quality and strength of the evidence. During the consensus meeting, the group agreed on the outcomes of the systematic review, pertinent sources of evidence, clinical recommendations, and areas requiring future research. RESULTS The systematic review, which was conducted for the consensus conference, evaluated the effectiveness of the use of biologics for the treatment of intrabony defects. Enamel matrix derivative (EMD) and recombinant human platelet-derived growth factor-BB (rhPDGF-BB) with β-tricalcium phosphate were shown to be efficacious in regenerating intrabony defects. The level of evidence is supported by multiple studies documenting effectiveness. The clinical application of biologics supports improvements in clinical parameters comparable with selected bone replacement grafts and guided tissue regeneration (GTR). Factors negatively affecting regeneration included smoking and excessive tooth mobility. CONCLUSIONS Periodontal regeneration in intrabony defects is possible on previously diseased root surfaces, as evidenced by a gain in clinical attachment, decreased pocket probing depth, gain in radiographic bone height, and overall improvement in periodontal health. These clinical findings are consistent with available histologic evidence. Clinical improvements can be maintained over long periods (>10 years). Although bone replacement grafts have been the most commonly investigated modality, GTR, biologics, and combination therapies have also been shown to be effective. Future research should emphasize patient-reported outcomes, individual response differences, and emerging technologies to enhance treatment results. CLINICAL RECOMMENDATIONS Early management of intrabony defects with regenerative therapies offers the greatest potential for successful periodontal regeneration. The clinical selection and application of a regenerative therapy or combination of therapies for periodontal regeneration should be based on the clinicians experiences and understanding of the regenerative biology and technology. This decision-making process should take into consideration the potential adverse influence of factors, such as smoking, poor oral hygiene, tooth mobility, and defect morphology, on regeneration. Management should be coupled with an effective maintenance program for long-term success.

Prevalence of human cytomegalovirus and Epstein-Barr virus in subgingival plaque at peri-implantitis, mucositis and healthy sites. A pilot study

This study evaluated the prevalence of human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) in peri-implantitis and mucositis sites and the correlation between herpesvirus and clinical parameters. Fifty-six dental implants (mean time of loading, 4.27±1.6 years) were evaluated (20 peri-implantitis, 18 mucositis, 18 healthy peri-implant sites.) The clinical parameters assessed were: visible plaque index (PI), bleeding on probing (BOP), suppuration (SUP), probing depth (PD). A polymerase chain reaction assay identified HCMV and EBV in subgingival plaque samples. The percent of sites with plaque and BOP was significantly higher around mucositis and peri-implantitis compared with healthy implants (p<0.05). The mean PD around the implants was significantly higher in peri-implantitis, followed by mucositis and healthy implants (p<0.05). HCMV was detected in 13 (65%) and EBV in 9 (45%) of the 20 peri-implantitis sites. HCMV was found in 1 of the 18 (6%) healthy periodontal sites and EBV in 2 (11%). A statistically significant correlation was found between presence of HCMV and EBV subgingivally and clinical parameters of peri-implantitis and healthy sites. These results confirm the high prevalence of HCMV and EBV in subgingival plaque of peri-implantitis sites and suggest the viruses have a possible active pathogenic role in peri-implantitis.

Prostaglandins E2 and F2α enhance differentiation of cementoblastic cells

BACKGROUND The prostaglandins (PG) E2 and PGF2α are important cytokines in periodontal physiology and pathology. PGE2 and PGF2α alter cell function by binding and activating the plasmamembrane G-protein-coupled PG receptors. In this study, we examined the PGE2 and PGF2α effects on the immortalized cementoblastic OCCM cells. METHODS Confluent OCCM cells were treated with PGE2 , PGF2α , specific activators/inhibitors of the EP prostanoid receptors, a specific activator of the FP prostanoid receptor, and direct activators/inhibitors of the protein kinase C (PKC) signaling pathway. Mineral nodule formation was assessed by the von Kossa stain. RESULTS PGE2 and PGF2α significantly increased mineralization of OCCM cells. The EP1 and EP3 PG receptor activators 16,16-dimethyl-prostaglandin E2 and sulprostone, also increased mineralization. In contrast, specific activators of the EP2 or the EP2/EP3/EP4 receptors did not have any effect. Fluprostenol, a specific activator of the FP receptor, significantly increased mineralization of OCCM cells. FP and EP (1 or 3) receptors signal through activation of the protein kinase C (PKC) pathway. Indeed, phorbol 12-myristate 13-acetate (PMA), a direct activator of the PKC pathway, significantly increase OCCM mineralization, while pre-treatment of OCCM cells with the PKC inhibitor GF109203× (bisindolylmaleimide) significantly decreased mineralization. CONCLUSION We conclude that PGE2 and PGF2α exert an anabolic effect on OCCM mineralization through activation of PKC signaling. J Periodontol 2005;76:303-309.

Ligature-induced peri-implantitis in mice.

BACKGROUND AND OBJECTIVE Peri-implantitis has a prevalence of 11-47%, involves destruction of peri-implant bone and may lead to implant loss. A detailed understanding of the pathogenesis of peri-implantitis is lacking. The objective of this study was to develop a murine model of experimental peri-implantitis. MATERIAL AND METHODS Machined, smooth-surface, screw-shaped titanium implants were placed in the healed alveolar bone of the left maxillary molars of C57BL/6J male mice, 8 wk after tooth extraction. Peri-implantitis was induced by securing silk ligatures around the head of the implant fixtures. Implant survival and peri-implant bone levels were analyzed by micro-computed tomography (micro-CT) scans and histology, 12 wk after ligature placement. RESULTS Implant survival was 60% (six of 10) for implants with ligatures and 100% (eight of eight) for controls. Micro-CT revealed significantly greater bone loss around the implants that received ligatures and that survived, compared with controls. The radiographic findings were confirmed via histology and toluidine blue staining. CONCLUSION This study describes a murine model of experimental peri-implantitis around screw-shaped titanium implants placed in the edentulous alveolar bone. This model should be a useful tool to dissect pathogenic mechanisms of peri-implantitis and evaluate potential treatment interventions.

Heritability of periodontal bone loss in mice

BACKGROUND Periodontitis is an inflammatory disease of the periodontal tissues that compromises tooth support and can lead to tooth loss. Although bacterial biofilm is central in disease pathogenesis, the host response plays an important role in the progression and severity of periodontitis. Indeed, clinical genetic studies indicate that periodontitis is 50% heritable. In this study, we hypothesized that lipopolysaccharide (LPS) injections lead to a strain-dependent periodontal bone loss pattern. MATERIAL AND METHODS We utilized five inbred mouse strains that derive the recombinant strains of the hybrid mouse diversity panel. Mice received Porphyromonas gingivalis-LPS injections for 6 wk. RESULTS AND CONCLUSION Micro-computed tomography analysis demonstrated a statistically significant strain-dependent bone loss. The most susceptible strain, C57BL/6J, had a fivefold higher LPS-induced bone loss compared to the most resistant strain, A/J. More importantly, periodontal bone loss revealed 49% heritability, which closely mimics periodontitis heritability for patients. To evaluate further the functional differences that underlie periodontal bone loss, osteoclast numbers of C57BL/6J and A/J mice were measured in vivo and in vitro. In vitro analysis of osteoclastogenic potential showed a higher number of osteoclasts in C57BL/6J compared to A/J mice. In vivo LPS injections statistically significantly increased osteoclast numbers in both groups. Importantly, the number of osteoclasts was higher in C57BL/6J vs. A/J mice. These data support a significant role of the genetic framework in LPS-induced periodontal bone loss and the feasibility of utilizing the hybrid mouse diversity panel to determine the genetic factors that affect periodontal bone loss. Expanding these studies will contribute in predicting patients genetically predisposed to periodontitis and in identifying the biological basis of disease susceptibility.

A Murine Model of Lipopolysaccharide-Induced Peri-Implant Mucositis and Peri-Implantitis

Dental implants are a widely used treatment option for tooth replacement. However, they are susceptible to inflammatory diseases such as peri-implant mucositis and peri-implantitis, which are highly prevalent and may lead to implant loss. Unfortunately, the understanding of the pathogenesis of peri-implant mucositis and peri-implantitis is fragmented and incomplete. Therefore, the availability of a reproducible animal model to study these inflammatory diseases would facilitate the dissection of their pathogenic mechanisms. The objective of this study is to propose a murine model of experimental peri-implant mucositis and peri-implantitis. Screw-shaped titanium implants were placed in the upper healed edentulous alveolar ridges of C57BL/6J mice 8 weeks after tooth extraction. Following 4 weeks of osseointegration, Porphyromonas gingivalis -lipolysaccharide (LPS) injections were delivered to the peri-implant soft tissues for 6 weeks. No-injections and vehicle injections were utilized as controls. Peri-implant mucositis and peri-implantitis were assessed clinically, radiographically (microcomputerized tomograph [CT]), and histologically following LPS-treatment. LPS-injections resulted in a significant increase in soft tissue edema around the head of the implants as compared to the control groups. Micro-CT analysis revealed significantly greater bone loss in the LPS-treated implants. Histological analysis of the specimens demonstrated that the LPS-group had increased soft tissue vascularity, which harbored a dense mixed inflammatory cell infiltrate, and the bone exhibited noticeable osteoclast activity. The induction of peri-implant mucositis and peri-implantitis in mice via localized delivery of bacterial LPS has been demonstrated. We anticipate that this model will contribute to the development of more effective preventive and therapeutic approaches for these 2 conditions.