INVESTIGATION of EXPRESSION of CTXI LEVELS IN SALIVA WITH EARLY and DELAYED LOADING of DENTAL IMPLANTS

Overview

Background: Implants are commonly used as a treatment choice for partially dentate or in edentulous patients. Currently, no specific biomarker to assess the bone status around the dental implants of healthy patients has been reported to further evaluate bone deposition, resorption and stability. CTX (C-terminal telopeptide) is a known specific biomarker for bone resorption. CTX levels evaluation in serum and urine of patients with bisphosphonates treatment has already been reported. However, its levels in saliva of healthy patients with dental implants has not been investigated, so far. Objectives: The aim of this study is to evaluate salivary CTX levels in dental implants with early and delayed loading and to compare it with the values of mechanical stability test (Periotest). Methods: Patients coming to OPD of Oral Surgery department of Dow International Dental College (DIDC) and are willing for placement of implant in the posterior molar area of mandible will be enrolled in the study. Informed consent will be taken from each patient for participation in the study. Dental implants placement will be performed according to the standard protocols. All patients will be assessed for implant stability on the day of surgery, at 1 month, 2 month and 3 months by Periotest assessment. Also, saliva will be collected and stored from same patients. Later, ELISA will be performed for expressional analysis of CTX.

40 Patients coming to OPD of Dow International Dental College for implant placement in posterior mandible (molar area) will be recruited in the study. Dental implants available in the Outpatient Department (OPD) of Dow International Dental College (DIDC) will be placed in patients. Dental implants will be placed according to the standard protocols by Dr. Shaheen Ahmed. Patients will be divided in two groups. Group A patients will be early loaded, and group B will be conventional loaded with prosthesis. Each patient will be evaluated for implant stability after the surgery, at 1 month, 2 months and 3 months with the help of Periotest. Saliva will be collected from patients in both the groups before placement of implant and on the same days as of Periotest evaluations after surgery. Saliva will be processed and immediately stored at -80oC. Later sandwich ELISA will be performed for expressional analysis of CTX. Levels of CTX before implant surgery will be served as reference value in this study. 2.9.1 Saliva Collection Method: Whole unstimulated saliva (WUS) will be collected from both the study groups. The process of saliva collection is as follows: * Salivary samples will be collected between 9-11 am to minimize diurnal variations in salivary flow and changes in the sialo chemistry. * Patients will be asked to refrain from smoking, drinking and chewing at least one hour before saliva collection to avoid interference of water and food with the analysis. * To remove any food debris from mouth, the patients will be asked to wash the mouth with water before saliva collection. * After rinsing thoroughly, an interval of at least 10 minutes will be given before the collection of saliva samples to avoid sample dilution. * Patients will be asked to spit the saliva in a sterilized falcon tube of 15mL. * Approximately 2-5 mL of saliva will be collected from each patient. * Patient will be informed and told not to cough up mucus. * After saliva collection, samples will be immediately taken to the laboratory for further processing. * Salivary samples will be centrifuged at 8000rpm for 15 min at 4˚C to remove any cell debris and supernatant will be collected. * The collected supernatant will be aliquoted into microtubes of 1ml and will be stored at -80°C until further analysis (processing time of saliva will be kept lowest as possible). 2.9.2 Salivary Analysis: Enzyme Linked Immunosorbent Assay (Sandwich Technique): CTX levels in salivary samples of all the groups will be analyzed by sandwich ELISA (Enzyme Linked Immunosorbent Assay) technique. ELISA testing will be performed by using CTX kit according to manufacturer's instruction. The plate pre-coated with antibody specific to Human CTX will be provided with the kit. Standards or samples are added to the micro ELISA plate wells which allows CTX to bind to its specific antibody. After removing unbound sample, biotinylated CTX detection antibody will be added followed by washing with the buffer. After washing, plate will be incubated with Streptavidin-Horseradish Peroxidase (HRP) that will binds to detection antibody. After incubation, unbound Streptavidin-HRP will be washed away. Finally, substrate solution will be added. The intensity of color developed is proportional to the amount of human CTX present in the sample. The reaction will be stopped by the addition of stop solution and absorbance will be measured spectrophotometrically at 450 nm wavelength. The concentration of CTX in samples is obtained by comparing absorbance of samples to the standards. ELISA will be performed by principal investigator of the study with the help of her Co-supervisor in DRIBBS (Dow Research Institute of Biotechnology and Biomedical Sciences).