A free running pulsed, 1064 nm wavelength-specific ND:YAG laser (Light walker AT)* with an attached 320 µm diameter optical glass fiber attached to a metallic hand piece with a flexible tip, which will be set at 3:4 -watt average power, 20 HZ, and a 100-600 µs pulse duration

Sub-gingival dental plaque biofilm will be collected using size 50 paper point after ensuring a good isolation of the operating field, and sample will be placed in sterile microcentrifuge tubes containing phosphate buffered saline to be transferred immediately to the Microbiology Laboratory of Alexandria University Hospitals. Microcentrifuge tubes will be vortexed for 5 minutes then 200 ul of resulting suspension will be subjected to DNA extraction using QIAamp DNA Minikit (Qiagen, Germany). Specific PCR primers targeting gingival plaque-associated oral microbiota (Treponima denticola Td) will be used in SYBER green real-time PCR. Amplification of the 16SrRNA gene will be used as the denominator against which the amplification of other bacteria will be estimated. The bacterial relative quantification will be calculated automatically by Rotor-gene software and expressed as relative fold difference.

Sub-gingival dental plaque biofilm will be collected using size 50 paper point after ensuring a good isolation of the operating field, and sample will be placed in sterile microcentrifuge tubes containing phosphate buffered saline to be transferred immediately to the Microbiology Laboratory of Alexandria University Hospitals. Microcentrifuge tubes will be vortexed for 5 minutes then 200 ul of resulting suspension will be subjected to DNA extraction using QIAamp DNA Minikit (Qiagen, Germany). Specific PCR primers targeting gingival plaque-associated oral microbiota (Prevotella intermedia Pi) will be used in SYBER green real-time PCR. Amplification of the 16SrRNA gene will be used as the denominator against which the amplification of other bacteria will be estimated. The bacterial relative quantification will be calculated automatically by Rotor-gene software and expressed as relative fold difference.

The gingival index (Löe and Silness,1967) will be used to assess the degree of gingival inflammation. Each tooth is examined and scored (0-3), where 0 = normal gingiva; 1 = mild inflammation: slight change in color, slight edema, no bleeding on probing; 2 = moderate inflammation: redness, edema, and glazing, or bleeding on probing; 3 = severe inflammation: marked redness and edema, tendency toward spontaneous bleeding and ulceration