Ignite Creation Date:
2024-05-06 @ 8:25 PM
Last Modification Date:
2024-10-26 @ 3:27 PM
Study NCT ID:
NCT06379022
Status:
COMPLETED
Last Update Posted:
2024-04-23
First Post:
2024-04-18
Brief Title:
Proteomic Analysis of Newly Restored Single Implants
Sponsor:
Aristotle University Of Thessaloniki
Organization:
Aristotle University Of Thessaloniki
Study Overview
Official Title:
Clinical and Cellular Responses of Tissues at Newly Restored Single Implants Compared to Contralateral Natural Teeth During Their First Year of Functional Loading
Status:
COMPLETED
Status Verified Date:
2024-04
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
In 10 systemically healthy non-smokers free of periodontitis one newly restored implant baseline-T0 and one corresponding tooth were followed over 12 months T1 All implants were screw-retained and platform-switched Oral hygiene was closely monitored during the study Probing pocket depth PPD attachment levels CAL bleeding and plaque indices and crevicular fluid were collected from an implant-site PICF and a tooth-site GCF Total proteomic profiles in PICF and GCF were investigated using label-free quantitative proteomics
Detailed Description:
Study population A total of 10 systemically healthy non-smokers who were previously treated for periodontal disease and received implant therapy were recruited between December 2018 and November 2019 Each subject contributed with a newly restored screw-retained implant 2-4 weeks following functional loading baseline T0 and was closely followed over 12 months T1 All study implants were platform-switched ø41 Straumann Bone Level and were installed following a two-stage surgical placement protocol A contralateral tooth in the same jaw but on the opposite quadrant served as the control Implant placement was planned based on clinical and cone beam computerized tomography evaluations and was performed using a prosthetic guide Implants were submerged and were exposed 6 months later when a healing abutment was adjusted on the fixture followed by a screw-retained crown 1 month later Exclusion criteria included younger than 18 years of age 18 teeth excluding third molars untreated periodontal disease periodontitis Stage III-IV evidence of peri-implantitis Berglundh et al 2018 at other implant sites significant soft tissue pathology untreated carious cavities five or more systemic diseases that require intake of medications medical conditions that contraindicated surgical implant placement the medication that interferes with bone metabolism pregnancy or lactation inadequate dimensions of the alveolar ridge or grafted bone at the surgical site and tooth extraction 3 months prior to the study smoking
Clinical assessments Site-specific and full-mouth clinical indices were assessed at T0 and T1 Oral hygiene instructions using a manual toothbrush and the modified Bass technique were re-iterated at four time points over the 12-month study period T0 3- 6-months and T1 Prophylaxis comprising supragingival ultrasonic debridement and polishing with a rubber cup was carried out at the same time points In detail the following site-specific clinical indices probing pocket depth PPD clinical attachment levels CAL recession REC modified gingival index MGI score range 0-4 Lobene et al 1986 modified dental plaque index mPI1 score range 0-3 and modified sulcus bleeding index mBI1 score range 0-3 were determined at the mesial buccal site of the study implant in a similar manner as PPD CAL REC MGI plaque index PI2 score range 0-3 and sulcus bleeding index SBI3 score range 0-5 at the control tooth site The width of keratinized tissues KTW was determined at the mid-buccal aspect of the test-implantcontrol tooth Moreover on a full-mouth basis PPD CAL and dichotomous supragingival dental plaque PI OLeary et al 1972 and bleeding on probing BOP Ainamo et al 1975 were recorded at six sites per toothimplant present in the mouth Periodontal clinical indices were determined using a manual periodontal probe PCP-UNC 15 Hu-Friedy XP-23QW Hu-Friedy Chicago IL USA to the nearest millimeter
Radiographic examination The VixWin Platinum Gendex software Hatfield PA USA linearly assessed the distance between the implant platform and the bone crest I-BC parallel to the long axis of the implant fixture at T0 and T1 on intra-oral digital radiographs Periapical digital radiographs RadioVisioGraphy Trophy Radiology SA Paris France were obtained using the long cone paralleling technique at a 10 cm distance between the x-ray head and the digital sensor
Collection of crevicular fluid samples Peri-implant crevicular fluid PICF was collected from the mesial-buccal site of the newly restored study implant while gingival crevicular fluid GCF was collected from the mesial-buccal site of a contralateral control tooth at T0 and T1 Before PICFGCF collection the surfaces were gently air-dried and isolated from saliva by placing cotton rolls and using a saliva ejector Then supragingival plaque was carefully removed by sterile curettes and paper strips Periopaper OraFlow Inc Smithtown NY USA were gently placed in the mesial aspect of the peri-implantgingival sulcus until mild resistance was felt and held for 30 sec Care was taken to avoid mechanical trauma when placing the strips and those strips contaminated with blood were discarded The strips were stored in Eppendorf tubes Eppendorf Hamburg Germany at -80C until further processed
Proteomic analysis of crevicular fluid samples PICFGCF The PICF and GCF samples collected from the study implant sites n10 and the control tooth sites n10 respectively at T0 and T1 were pooled and used for proteomics analysis In brief 20 µg of total protein per pooled sample were reduced alkylated trypsinized and purified with single-pot solid-phase-enhanced sample preparation SP3 assisted protein digestion using the PreOmics SP3-iST PreOmics GmbH following the manufacturing protocol for protein extraction and digestion These extracts were concentrated using a Speedvac Thermo Savant SPD121P Thermo Scientific Waltham MA USA and stored at -20 C until further use
The digested samples were first reconstituted with 30 µL of 3 acetonitrile ACN in 01 formic acid then subjected to Fusion Orbitrap Lumos mass spectrometer Thermo Fisher Scientific interfaced to an Easy nano-flow HPLC system Thermo Fisher Scientific for proteomics analysis During proteomics analysis a three-liner gradient of acetonitrile and water was used with 01 formic acid at a 300 nLminute flow rate The gradient progressed from 2 to 30 acetonitrile over 60 minutes then from 30 to 97 acetonitrile in 10 minutes and finally held at 97 acetonitrile for 10 minutes The mass spectrometer was set to operate in a data-dependent manner automatically switching between MS and MSMS using the Xcalibur software package from Thermo Fisher Scientific A mass range of 300-1500 mz was selected for the Orbitrap analyzer
The acquired MS data were processed using the Mascot version 241 Matrix Science search engine against an in-house database containing 10125458 entries which included human accessed on 22nd February 2022 from Uniprot and bacterial accessed on 22nd February 2022 from eHOMD proteins common MS contaminants and reverse sequences for estimating the false discovery rate During the proteomics analysis the precursor ion tolerance and fragment ion tolerance were set to 10 ppm and 06 Da respectively Up to two missed cleavages per peptide were allowed for tryptic digestion The carbamidomethylation modification on cysteine was selected as a fixed modification while deamidation of asparagine and glutamine and oxidation of methionine were chosen as variable modification parameters
The spectrum reports of the search results were generated using Scaffold software version 421 Proteome software and proteins with FDR at 1 minimal 2 unique peptides identified as peptide FDR at 01 were used for relative protein abundance analysis Fold changes in abundance intensity were calculated based on value log2 fold change FC between pairs of consecutive time points using R-software R A Language and Environment for Statistical Computing R Development Core Team Proteins with a log2FC 1 of unique spectrum counts between two conditions or exclusively identified in one condition were considered regulated
Bioinformatics analysis for the regulated proteins The regulated proteins underwent overrepresentation analysis ORA against the Hallmark database using the WebGestalt online tool The analysis conducted on March 25th 2022 aimed to compare the regulation of proteins in teeth and implants between T0 and T1 conditions Similarly on March 29th 2022 the analysis examined the regulation of proteins in both teeth and implants under either the T0 or T1 conditions The top 10 enriched results ranked by p-value using the hypergeometric distribution were reported However only pathways with a p-value 005 were considered as regulated
Clinical assessments Site-specific and full-mouth clinical indices were assessed at T0 and T1 Oral hygiene instructions using a manual toothbrush and the modified Bass technique were re-iterated at four time points over the 12-month study period T0 3- 6-months and T1 Prophylaxis comprising supragingival ultrasonic debridement and polishing with a rubber cup was carried out at the same time points In detail the following site-specific clinical indices probing pocket depth PPD clinical attachment levels CAL recession REC modified gingival index MGI score range 0-4 Lobene et al 1986 modified dental plaque index mPI1 score range 0-3 and modified sulcus bleeding index mBI1 score range 0-3 were determined at the mesial buccal site of the study implant in a similar manner as PPD CAL REC MGI plaque index PI2 score range 0-3 and sulcus bleeding index SBI3 score range 0-5 at the control tooth site The width of keratinized tissues KTW was determined at the mid-buccal aspect of the test-implantcontrol tooth Moreover on a full-mouth basis PPD CAL and dichotomous supragingival dental plaque PI OLeary et al 1972 and bleeding on probing BOP Ainamo et al 1975 were recorded at six sites per toothimplant present in the mouth Periodontal clinical indices were determined using a manual periodontal probe PCP-UNC 15 Hu-Friedy XP-23QW Hu-Friedy Chicago IL USA to the nearest millimeter
Radiographic examination The VixWin Platinum Gendex software Hatfield PA USA linearly assessed the distance between the implant platform and the bone crest I-BC parallel to the long axis of the implant fixture at T0 and T1 on intra-oral digital radiographs Periapical digital radiographs RadioVisioGraphy Trophy Radiology SA Paris France were obtained using the long cone paralleling technique at a 10 cm distance between the x-ray head and the digital sensor
Collection of crevicular fluid samples Peri-implant crevicular fluid PICF was collected from the mesial-buccal site of the newly restored study implant while gingival crevicular fluid GCF was collected from the mesial-buccal site of a contralateral control tooth at T0 and T1 Before PICFGCF collection the surfaces were gently air-dried and isolated from saliva by placing cotton rolls and using a saliva ejector Then supragingival plaque was carefully removed by sterile curettes and paper strips Periopaper OraFlow Inc Smithtown NY USA were gently placed in the mesial aspect of the peri-implantgingival sulcus until mild resistance was felt and held for 30 sec Care was taken to avoid mechanical trauma when placing the strips and those strips contaminated with blood were discarded The strips were stored in Eppendorf tubes Eppendorf Hamburg Germany at -80C until further processed
Proteomic analysis of crevicular fluid samples PICFGCF The PICF and GCF samples collected from the study implant sites n10 and the control tooth sites n10 respectively at T0 and T1 were pooled and used for proteomics analysis In brief 20 µg of total protein per pooled sample were reduced alkylated trypsinized and purified with single-pot solid-phase-enhanced sample preparation SP3 assisted protein digestion using the PreOmics SP3-iST PreOmics GmbH following the manufacturing protocol for protein extraction and digestion These extracts were concentrated using a Speedvac Thermo Savant SPD121P Thermo Scientific Waltham MA USA and stored at -20 C until further use
The digested samples were first reconstituted with 30 µL of 3 acetonitrile ACN in 01 formic acid then subjected to Fusion Orbitrap Lumos mass spectrometer Thermo Fisher Scientific interfaced to an Easy nano-flow HPLC system Thermo Fisher Scientific for proteomics analysis During proteomics analysis a three-liner gradient of acetonitrile and water was used with 01 formic acid at a 300 nLminute flow rate The gradient progressed from 2 to 30 acetonitrile over 60 minutes then from 30 to 97 acetonitrile in 10 minutes and finally held at 97 acetonitrile for 10 minutes The mass spectrometer was set to operate in a data-dependent manner automatically switching between MS and MSMS using the Xcalibur software package from Thermo Fisher Scientific A mass range of 300-1500 mz was selected for the Orbitrap analyzer
The acquired MS data were processed using the Mascot version 241 Matrix Science search engine against an in-house database containing 10125458 entries which included human accessed on 22nd February 2022 from Uniprot and bacterial accessed on 22nd February 2022 from eHOMD proteins common MS contaminants and reverse sequences for estimating the false discovery rate During the proteomics analysis the precursor ion tolerance and fragment ion tolerance were set to 10 ppm and 06 Da respectively Up to two missed cleavages per peptide were allowed for tryptic digestion The carbamidomethylation modification on cysteine was selected as a fixed modification while deamidation of asparagine and glutamine and oxidation of methionine were chosen as variable modification parameters
The spectrum reports of the search results were generated using Scaffold software version 421 Proteome software and proteins with FDR at 1 minimal 2 unique peptides identified as peptide FDR at 01 were used for relative protein abundance analysis Fold changes in abundance intensity were calculated based on value log2 fold change FC between pairs of consecutive time points using R-software R A Language and Environment for Statistical Computing R Development Core Team Proteins with a log2FC 1 of unique spectrum counts between two conditions or exclusively identified in one condition were considered regulated
Bioinformatics analysis for the regulated proteins The regulated proteins underwent overrepresentation analysis ORA against the Hallmark database using the WebGestalt online tool The analysis conducted on March 25th 2022 aimed to compare the regulation of proteins in teeth and implants between T0 and T1 conditions Similarly on March 29th 2022 the analysis examined the regulation of proteins in both teeth and implants under either the T0 or T1 conditions The top 10 enriched results ranked by p-value using the hypergeometric distribution were reported However only pathways with a p-value 005 were considered as regulated
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None