Gene Expression Variation and Implant Wound Healing Among Smokers and Diabetics

FGF2 Promoter Hypermethylation and Implant Wound Healing Among Smokers and Diabetics

Periodontal wound healing is a complex multifactorial process that involves interactions among various cells, growth factors, hormones and extracellular matrices. Although still poorly understood, these interactions trigger a series of events that lead to new tissue formation. One growth factor that plays an important role in wound healing is fibroblast growth factor 2 (FGF2). Many animal and human studies have shown this protein is effective in periodontal regeneration. Recently, epigenetic modifications, such as DNA methylation, have been associated with changes in patterns of gene expression. Preliminary data suggests that FGF2 gene may be differentially methylated in periodontal tissues. Aberrant gene promoter methylation in smokers and diabetics has also been reported in many studies. However, the role of DNA methylation in wound healing has not yet been investigated.

The investigators hypothesize that the methylation status of FGF2 gene can affect the levels of FGF2 secreted during wound healing phase after dental implant surgery. The investigators also hypothesize there exists a difference in methylation levels of FGF2 gene in healthy, smoking and diabetic patients that can interfere with wound healing. The investigators seek to determine whether DNA methylation plays a role in wound healing and whether the methylation level of FGF2 gene varies among healthy, smoking and diabetic patients.

Study Overview

• Status: Completed
• Conditions: Smoking; Diabetes
• Intervention / Treatment: Procedure: Dental implant surgery

Detailed Description

Periodontal wound healing is a complex multifactorial process that involves interactions among various cells, growth factors, hormones and extracellular matrices. Although still poorly understood, these interactions trigger a series of events that lead to new tissue formation. One growth factor that plays an important role in wound healing is fibroblast growth factor 2 (FGF2). FGF2 is a member of the heparin-binding growth factor family, secreted by macrophages and endothelial cells. During the proliferative healing phase, it stimulates fibroblast proliferation & ECM synthesis, and increases chemotaxis, proliferation and differentiation of endothelial cells. During the bone remodeling phase, FGF2 also stimulates mesenchymal progenitor cell migration. Many animal and human studies have shown FGF2 are effective in periodontal regeneration. In 1999, Murakami showed surgically treated 3-wall intrabony defect in dogs grafted with FGF2 was able to demonstrate significantly greater cementum and bone formation. Four years later, his group again found that topical application of rhbFGF in surgically treated class 2 furcation defects in dogs also showed increase in formation of PDL, cementum and bone. In 2008, Kitamura performed a randomized controlled study in humans with 2- or 3-wall intrabony periodontal defects and found that rhbFGF was able to stimulate alveolar bone growth and PDL regeneration.

Recently, epigenetic modifications, such as DNA methylation, have been associated with changes in patterns of gene expression that do not involve changes in DNA sequence. DNA methylation is characterized by the addition of the methyl group onto cytokines within CpG regions. Methylated CpG regions interfere with the access of transcription factors to the promoter region, thereby silencing the gene. This DNA methylation phenomenon has important regulatory functions in normal and pathological cellular processes. It was recognized that alteration in the methylation states at the promoter regions of tumor suppressor genes are implicated with cancer. A persistent inflammation was also observed to cause DNA methylation, which inactivates suppressors of cytokine signaling and results in exaggerated cytokine production. This makes an individual susceptible to periodontal disease. In our laboratory, the investigators have discovered that periodontal disease is associated with increased DNA methylation of the COX-2 promotor, especially the locus immediately adjacent to the NF-kB in the promoter region. Preliminary data (not shown) suggests that FGF2 may be differentially methylated in periodontal tissues. Aberrant gene promoter methylation in smokers and diabetics has also been reported in many studies. However, the role of DNA methylation in wound healing has not yet been investigated.

We hypothesize that the methylation status of FGF2 can affect the levels of FGF2 secreted during wound healing phase after dental implant surgery. We also hypothesize there exists a difference in methylation levels of FGF2 in healthy, smoking and diabetic patients that can interfere with wound healing. We seek to determine whether DNA methylation plays a role in wound healing and whether the methylation level of FGF2 varies among healthy, smoking and diabetic patients.

• Study Type: Observational
• Enrollment (Anticipated): 44

Contacts and Locations

Study Locations

• United States
  • North Carolina
    • Chapel Hill, North Carolina, United States, 27514
      • Department of Periodontology, UNC School of Dentistry

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years to 70 years (Adult, Older Adult)
• Accepts Healthy Volunteers: Yes
• Genders Eligible for Study: All

• Sampling Method: Non-Probability Sample

Study Population

Patients in the graduate periodontal clinic in the school of dentistry at the University of North Carolina

Description

Inclusion Criteria:

• Adult males or females between the age of 18 and 70 years (inclusive)
• Able and willing to follow study procedures and instructions
• Have read, understood and signed an informed consent form
• In good general health
• Have one or more implant placements as their future treatment needs. The implant placement can be either as one-stage or two-stage, and can be either in an edentulous ridge or an extraction socket
• Qualify for enrollment into one of the three study groups
• Have probing depth ≤ 4 mm for all teeth at the same quadrant of implant placement. Sites with probing depth 5 mm will also be included if bleeding on probing in these sites are absent.

Exclusion Criteria:

• Have a chronic disease with oral manifestations
• Exhibit gross oral pathology
• Use of either antibiotics or NSAIDs within 1 month prior to screening examination
• Chronic treatment (i.e. two weeks or more) with any medication known to affect periodontal status (e.g. phenytoin, calcium, antagonists, cyclosporin, Coumadin) within 1 month prior to screening examination
• Systemic conditions, except smoking and diabetes, that are known to affect the periodontal status
• With active infectious diseases such as hepatitis, HIV or tuberculosis
• Known to be pregnant or breastfeeding

Study Plan

How is the study designed?

Number of groups / cohorts

3

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
Control group; Subjects must have never smoked and must be non-diabetic.	Procedure: Dental implant surgery; Surgery involving placement of one dental implant, of either Astra Tech or Straumann system, is performed in all subjects within 2 weeks of screening examination. Implant placement is 1-stage, but can be either on edentulous ridges or in extraction sockets.This is not a randomized treatment arm/group design. The study is observational with regards to the analysis of tissue samples that are collected prior to the routine placement of implants. The implant choice is based upon patient needs and is not related to any outcome.
Smoking group; Subjects must have had at least 5 pack-years of self-reported smoking history, must be currently smoking and must be non-diabetic.	Procedure: Dental implant surgery; Surgery involving placement of one dental implant, of either Astra Tech or Straumann system, is performed in all subjects within 2 weeks of screening examination. Implant placement is 1-stage, but can be either on edentulous ridges or in extraction sockets.This is not a randomized treatment arm/group design. The study is observational with regards to the analysis of tissue samples that are collected prior to the routine placement of implants. The implant choice is based upon patient needs and is not related to any outcome.
Diabetic group; Subjects must have type 2 diabetes. The condition must be diagnosed subjects must be treated by medications and/or insulin. A HbA1C test result either within past 3 months or performed in the first visit must be available. They must have never smoked.	Procedure: Dental implant surgery; Surgery involving placement of one dental implant, of either Astra Tech or Straumann system, is performed in all subjects within 2 weeks of screening examination. Implant placement is 1-stage, but can be either on edentulous ridges or in extraction sockets.This is not a randomized treatment arm/group design. The study is observational with regards to the analysis of tissue samples that are collected prior to the routine placement of implants. The implant choice is based upon patient needs and is not related to any outcome.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
FGF2 methylation level	Genomic DNA is isolated from the collected gingival tissue samples. Methylation alterations in FGF2 are detected through differential methylation hybridization using the EpiTect® Methyl qPCR single assay.	On the day of implant surgery

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
FGF2 mRNA expression level	RNA is isolated from the collected gingival tissue samples and is then processed for gene expression analysis by quantitative real-time PCR.	On the day of implant surgery (DAY 0)
FGF2 protein level	Gingival crevicular fluid, obtained from the two adjacent sites closest to the implant location, is used to quantify specific FGF2 protein levels by ELISA.	On the day of implant surgery (DAY 0) and 2, 4 and 6 weeks following implant surgery
Implant stability quotient (ISQ)	The degree of implant stability at various time points following the surgery is measured using an Osstell ISQ instrument. An ISQ value, ranged between 1 and 100, is generated for each sample at each time point.	4 and 6 weeks following implant surgery
Wound healing indices (WHI)	The degree of soft tissue healing at various time points following surgery is monitored by WHI.	2, 4 and 6 weeks following implant surgery

Collaborators and Investigators

• Sponsor: University of North Carolina, Chapel Hill
• Collaborators: National Institute of Dental and Craniofacial Research (NIDCR); National Center for Research Resources (NCRR)
• Investigators: Principal Investigator: Silvana Barros, DDS, PhD, MS, UNC Chapel Hill School of Dentistry

Publications and helpful links

General Publications

• Kitamura M, Nakashima K, Kowashi Y, Fujii T, Shimauchi H, Sasano T, Furuuchi T, Fukuda M, Noguchi T, Shibutani T, Iwayama Y, Takashiba S, Kurihara H, Ninomiya M, Kido J, Nagata T, Hamachi T, Maeda K, Hara Y, Izumi Y, Hirofuji T, Imai E, Omae M, Watanuki M, Murakami S. Periodontal tissue regeneration using fibroblast growth factor-2: randomized controlled phase II clinical trial. PLoS One. 2008 Jul 2;3(7):e2611. doi: 10.1371/journal.pone.0002611.
• Kaigler D, Cirelli JA, Giannobile WV. Growth factor delivery for oral and periodontal tissue engineering. Expert Opin Drug Deliv. 2006 Sep;3(5):647-62. doi: 10.1517/17425247.3.5.647.
• Murakami S, Takayama S, Ikezawa K, Shimabukuro Y, Kitamura M, Nozaki T, Terashima A, Asano T, Okada H. Regeneration of periodontal tissues by basic fibroblast growth factor. J Periodontal Res. 1999 Oct;34(7):425-30. doi: 10.1111/j.1600-0765.1999.tb02277.x.
• Murakami S, Takayama S, Kitamura M, Shimabukuro Y, Yanagi K, Ikezawa K, Saho T, Nozaki T, Okada H. Recombinant human basic fibroblast growth factor (bFGF) stimulates periodontal regeneration in class II furcation defects created in beagle dogs. J Periodontal Res. 2003 Feb;38(1):97-103. doi: 10.1034/j.1600-0765.2003.00640.x.
• Barros SP, Offenbacher S. Epigenetics: connecting environment and genotype to phenotype and disease. J Dent Res. 2009 May;88(5):400-8. doi: 10.1177/0022034509335868.
• Gomez RS, Dutra WO, Moreira PR. Epigenetics and periodontal disease: future perspectives. Inflamm Res. 2009 Oct;58(10):625-9. doi: 10.1007/s00011-009-0041-7. Epub 2009 May 8.
• Wilson AG. Epigenetic regulation of gene expression in the inflammatory response and relevance to common diseases. J Periodontol. 2008 Aug;79(8 Suppl):1514-9. doi: 10.1902/jop.2008.080172.
• Ling C, Groop L. Epigenetics: a molecular link between environmental factors and type 2 diabetes. Diabetes. 2009 Dec;58(12):2718-25. doi: 10.2337/db09-1003. No abstract available.
• Krupanidhi S, Sedimbi SK, Vaishnav G, Madhukar SS, Sanjeevi CB. Diabetes--role of epigenetics, genetics, and physiological factors. Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2009 Sep;34(9):837-45.
• Simmons RA. Developmental origins of diabetes: the role of epigenetic mechanisms. Curr Opin Endocrinol Diabetes Obes. 2007 Feb;14(1):13-6. doi: 10.1097/MED.0b013e328013da5b.
• Kuroda A, Rauch TA, Todorov I, Ku HT, Al-Abdullah IH, Kandeel F, Mullen Y, Pfeifer GP, Ferreri K. Insulin gene expression is regulated by DNA methylation. PLoS One. 2009 Sep 9;4(9):e6953. doi: 10.1371/journal.pone.0006953. Erratum In: PLoS One. 2009;4(10) 10.1371/annotation/947a8d4a-3585-4b23-ac84-b47a255a70d9.
• Han W, Wang T, Reilly AA, Keller SM, Spivack SD. Gene promoter methylation assayed in exhaled breath, with differences in smokers and lung cancer patients. Respir Res. 2009 Sep 25;10(1):86. doi: 10.1186/1465-9921-10-86.
• Belinsky SA, Palmisano WA, Gilliland FD, Crooks LA, Divine KK, Winters SA, Grimes MJ, Harms HJ, Tellez CS, Smith TM, Moots PP, Lechner JF, Stidley CA, Crowell RE. Aberrant promoter methylation in bronchial epithelium and sputum from current and former smokers. Cancer Res. 2002 Apr 15;62(8):2370-7.

Study record dates

Study Major Dates

• Study Start: August 1, 2010
• Primary Completion (Actual): January 12, 2016
• Study Completion (Actual): February 23, 2016

Study Registration Dates

• First Submitted: August 1, 2012
• First Submitted That Met QC Criteria: August 8, 2012
• First Posted (Estimate): August 13, 2012

Study Record Updates

• Last Update Posted (Actual): February 12, 2020
• Last Update Submitted That Met QC Criteria: February 11, 2020
• Last Verified: February 1, 2019

More Information

Terms related to this study

• Keywords: Diabetes; Smoking; Wound healing; DNA methylation; Implant; FGF2
• Other Study ID Numbers: 10-1184; 1R01DE021052-01 (U.S. NIH Grant/Contract); 1UL1RR025747 (U.S. NIH Grant/Contract); AWU (Other Identifier: UNC)