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10 Volume 2 | Issue 1/2016 Journal of Oral Science & Rehabilitation S ta ph yloc oc c us a ureus and pe r i i mplant di se ase months, with adjacent healthy teeth, but pre- sentingsignsofperiimplantdiseaseaccordingto Mombelli and Décaillet.13 The patients were in- vitedtoparticipateandwereenrolledafterbeing given a detailed explanation of the study proto- col. Written informed consent was obtained for each patient. All of the patients were recruited fromthe Department ofOralSurgery, University of Valencia, Spain, between September and De- cember 2013. The investigation was conducted accordingtothe principles outlined inthe Decla- rationofHelsinkiof1975forbiomedicalresearch involving human subjects, as amended in 2008. All patients were evaluated clinically and radi- ographically, and their medical histories were recorded. Bone volumes were analyzed using periapicalradiographs. Theinclusioncriteriawere: –presence of periimplant disease with avertical bone defect of > 3 mm after implant integra- tionaccordingtoMombelliandDécaillet13 –age> 18 –norelevantmedicalconditions. Theexclusioncriteriawere: –pregnancyorlactation –known systemic disease or metabolic disor- ders (e.g., HIV) treated with medication detri- mentalto soft tissue and/or bone healing (e.g., high-dosesteroidtherapy,systemictreatment with tetracycline ortetracycline analogs, bone therapeutic levels of fluorides, bisphospho- nates, medication affecting boneturnover, an- tibioticsformorethansevendaysoranyinves- tigationaldrug)—topicalapplicationofsteroids and steroid application through inhalation werenotexclusioncriteria –malignant diseases or other diseases treated withradiotherapyorchemotherapeuticagents (chemotherapy)duringthepastfiveyears –a history of head and neck radiation treatment owingtocertainmedicalconditions –a suspected allergyorincompatibilitywith any of the bone graft substitute components (cal- ciumphosphates,PLGA,NMP) –inability to comply with the protocol require- ments,includingseverealcoholordruguser –involvementinanyotherclinicaltrialduringthe course ofthe presenttrial, orwithin a period of 30 days prior to its beginning or after its com- pletion –acute abscesses localized in the proximity of theprospectivesurgicalfield. After full screening, 16 patients were to be ex- cluded: 13 had taken systemic antibiotics during the three months prior to the microbiological sampling,twowerepregnant,andonerefusedto participate. The final sample consisted of 35 in- dividuals (20 male, 15 female) and 63 affected dentalimplants. Mi cro bi o lo gi cal sampli ng Samples for microbiological analysis were ob- tained from four sites in each patient in the fol- lowing order: (1) the periimplant sulcular fluid (PISF) of each implant; (2) the gingival sulcus (GS)oftheadjacentteeth,usedascontrolgroup; (3) the inner portions of the implant connection and the abutment of each implant (IIP); and (4) the oropharyngeal complex (OF). In all of the groups, the microbiological samples were taken using the GUIDOR Perio-Implant Diagnostic Kit (Sunstar Iberia, Sant Just Desvern, Spain), con- sisting offive sterile absorbent papertips and an emptysterile2 mlmicrotube. Priortocollectionofthesubgingivalplaque, supragingival plaque was eliminated from im- plants andteeth using a cottontip,without pen- etrating the GS. OptraGate (Ivoclar Vivadent, Schaan, Liechtenstein) was used to retract the lips and cheeks completely and to ensure rela- tive isolation.Afterlight drying ofthe areawith an air syringe, papertips were inserted into the periimplant or periodontal sulci for 30 s. The samples from the inner surfaces of the im- plant–abutment complex were obtained after careful removal of both the restorations and the abutments, seeking to avoid contamina- tion. One drop of RNA- and DNA-free water (Water Molecular Biology Reagent, W4502, Sigma-Aldrich, St. Louis, Mo., U.S.) was placed inside the implant connection and three paper tips were inserted for 30 s. The inner surface of the abutment was wet with a drop of RNA- and DNA-free water and smeared with two paper tips. The papertips were placed into the micro- tubes and sent for microbiological analysis to the Institut Clinident laboratory (Aix-en- Provence, France) in the provided mailing en- velopes. Finally, an oral environment analysis was performed using sterile cotton swabs col- lected from the cheeks, tongue, throat and pharynx of each patient. After sample collection, the inner part of the implants and the abutment–restoration complex were cleaned with a 5% chlorhexidine