|Year : 2018 | Volume
| Issue : 1 | Page : 17-22
Peri-implant mucosae inflammation during osseointegration is correlated with low levels of epidermal growth factor/epidermal growth factor receptor in the peri-implant mucosae
Marcos Alexandre Fonseca1, Lucas Carneiro Costa1, Aristides Da Rosa Pinheiro2, Telma Regina Da Silva Aguiar2, Valquiria Quinelato1, Leticia Ladeira Bonato1, Fernando Luiz Duarte Almeida3, José Mauro Granjeiro4, Priscila Ladeira Casado5
1 Department of Post Graduation in Dentistry, Fluminense Federa University, Rio de Janeiro, Brazil
2 Department of Clinical Dentistry, Fluminense Federa University, Rio de Janeiro, Brazil
3 Private Studing Practice Collaboration, Rio de Janeiro, Brazil
4 Department of Quality and Technology, National Institute of Metrology; /Cell Therapy Center, Clinical Research Unit and Biology Institute, Federal Fluminense University, Rio de Janeiro, Brazil
5 Department of Clinical Dentistry, Fluminense Federa University; /Cell Therapy Center, Clinical Research Unit and Biology Institute, Federal Fluminense University, Rio de Janeiro, Brazil
|Date of Web Publication||13-Apr-2018|
Dr. Priscila Ladeira Casado
Rua Maário Santos Braga, 28, Centro, 24020.140 Niterói, Rio de Janeiro
Source of Support: None, Conflict of Interest: None
Peri-implant mucosae inflammation during osseointegration period can promote host response imbalance and bone resorption by bacteria infiltration. Aim: To evaluate the association between EGF and EGFR gene expressions in the peri-implant tissue with mucosae inflammation during osseointegration period. Material and Methods: Forty-nine participants, with 59 endosseos implants, were recruited for this study. All participants included were rehabilitated with implants in two stages surgical protocol, presenting favorable bone quality and quantity. Osseointegration was evaluated one month after exposure surgery, wich was performed three months (mandible) and 6 months (maxillary) after implant placement. The criteria to consider the proper osseointegration were: Implant immobility; absence of peri-implant radiolucency; and no clinical signs of inflammation. Based on clinical and radiographic characteristics of peri-implant sites, participants were characterized as (i) having healing without complications (with proper osseointegration without mobility of the implant, and without clinical signs of mucosal inflammation) (control group) or (ii) failure peri-implant healing (with inadequate osseointegration characterized by signs of mucosae inflammation and/or implant mobility) (test group). Gingival biopsies were collected from 49 participants after osseointegration period, during the exposure procedure. Total RNA from gingival samples was isolated using the Trizol® reagent. The reaction of reverse transcription of PCR was performed for the synthesis of complementary DNA from 300ng RNA using Improm-II Reverse Transcription System™. Specific primers for EGF (NM_001963.4) and EGFR (NM_005228.3) were based on the BLAST data. The Livak method (2-ΔΔCT) was used to determine the relative quantification of the expression of EGF and EGFR. The values were normalized by relative expression of β-actin. Results: There was no difference between control and test groups to race, sex, age, alcohol consumption, general medical conditions, current medications, edentulism and periodontal phenotype. All RNA samples revealed proportions A260 nm/A280 nm more than 1.9. EGF and EGFR showed significant lower expression in gingival tissues removed from regions with failure healing (test group). EGF showed mRNA expression with an average of 44.53 ± 79.16 and 01.02 ± 1:33 in the control groups and test, respectively (P = 0.008). Similarly EGFR expression was significantly higher in the control group (102.03 ± 329.57) compared to the test group (7.85 ± 4.16) (P = 0:04). Conclusion: Low levels of EGF and EGFR are associated with inadequate healing of mucosal peri-implant during the osseointegration period.
Keywords: Epidermal growth factor, osseointegration, receptor of epidermal growth factor, wound healing
|How to cite this article:|
Fonseca MA, Costa LC, Da Rosa Pinheiro A, Da Silva Aguiar TR, Quinelato V, Bonato LL, Duarte Almeida FL, Granjeiro JM, Casado PL. Peri-implant mucosae inflammation during osseointegration is correlated with low levels of epidermal growth factor/epidermal growth factor receptor in the peri-implant mucosae. Int J Growth Factors Stem Cells Dent 2018;1:17-22
|How to cite this URL:|
Fonseca MA, Costa LC, Da Rosa Pinheiro A, Da Silva Aguiar TR, Quinelato V, Bonato LL, Duarte Almeida FL, Granjeiro JM, Casado PL. Peri-implant mucosae inflammation during osseointegration is correlated with low levels of epidermal growth factor/epidermal growth factor receptor in the peri-implant mucosae. Int J Growth Factors Stem Cells Dent [serial online] 2018 [cited 2018 Sep 25];1:17-22. Available from: http://www.cellsindentistry.org/text.asp?2018/1/1/17/228980
| Introduction|| |
The osteogenic process requires a careful chronological and spatial coordination of molecular signals leading to proliferation, migration, and differentiation of mesenchymal precursor cells into osteoblasts. During installation of the implant, bone formation around the implant of titanium is activated when the matrix is exposed to the extracellular fluid, releasing noncollagenous proteins and growth factors.,
One of the main factors that have directly influence the osseointegration process is the formation of peri-implant mucosal sealing, which is the only physical barrier able to not allow interference of the external environment in bone formation. Among the molecules responsible for gingival healing, epidermal growth factor (EGF) binding to epidermal growth factor receptor (EGFR), activates a cascade of intracellular events able of stimulating the proliferation of epithelial cells, fibroblasts, and endothelial cells, directly influencing the formation of the junctional epithelium, during osseointegration period.
The absence of adequate peri-implant soft tissue healing can facilitate bacterial penetration, promoting an imbalance in the host response and favoring inflammatory response., Consequently, osteoclast activity increases with active bone resorption and possible loss of the implant.,
The association between the health of the peri-implant soft tissue during the osseointegration process orchestrated by EGF and EGFR has not been studied previously. Therefore, the aim of this study was to evaluate the association between EGF and EGFR gene expressions in the peri-implant tissue with peri-implant mucosae inflammation during osseointegration period.
| Materials and Methods|| |
Forty-nine research participants, with 59 osseointegrated endosseous implants, were recruited for a year to the study in the Specialization in Implantology at the Fluminense Federal University, Rio de Janeiro, Brazil. The clinical procedures were conducted in accordance with the recommendations of the Ethics Committee of the Universidade Federal Fluminense under the registration number 80.627 and were conducted in full accordance with ethical principles, including the World Medical Association Declaration of Helsinki (version 2008 revised in 2010). The free and clear consent term was obtained from all participants. Initial clinical parameters of the study population are presented in [Table 1] and [Table 2], as well as medical and dental history. Exclusion criteria were bisphosphonate use, ingestion of antibiotics and anti-inflammatory agents in the past 3-month pregnancy and/or lactating smokers, absence of preoperative radiographs, and participants with active periodontal disease. All participants included were rehabilitated with minimum dimensions of implant 3.4 mm thick and 7 mm in height in two-stage surgical procedure, showing quality and quantity of bone favorable for installation, making use only of analgesic agents after surgery.
Osseointegration and peri-implant mucosae evaluation were based on Costa et al.'s  criteria. Based on the clinical characteristics of the sites, participants were characterized as (i) having healing without complications (without clinical signs of mucosal inflammation) (control group) or (ii) failure peri-implant mucosae healing (with signs of mucosal inflammation) (test group).
Gingival samples were collected from 49 research participants after the osseointegration period, during the reopening implant procedure. The samples were immediately submerged in the reagent for stabilization of RNA (RNA later, Qiagen, Valencia, CA, USA) and stored at −80°C until RNA extraction.
Total RNA from gingival samples was isolated using the reagent Trizol ® (InvitrogenTM by Life Technologies, NY, USA) according to the manufacturer's protocol. Treatment with DNase to digest the genomic DNA which could lead to false-positive results was performed using DNA–free DNase ® (Ambion by InvitrogenTM by Life Technologies, NY, USA). The RNA integrity was confirmed by electrophoresis, agarose gel 1.2% stained with SYBR Stain ® (InvitrogenTM by Life Technologies, NY, USA). The purity of the RNA was confirmed by the ratio of absorbance at 260/280 spectrophotometer, and the amount of RNA was estimated at 260 nm (Nanodrop ® 1000, ThermoScientific, Wilmington, USA). The reverse transcription polymerase chain reaction (RT-PCR) was performed for the synthesis of complementary DNA (cDNA) from 300 ng RNA using ImProm-II Reverse Transcription System™ (Promega Corporation, Wisconsin, USA), according to the manufacturer's protocol. The background control (RT-PCR without RNA template) and the RT reactions (PCR reactions without reverse transcription) were performed with all the RT-PCRs. The quantitative PCR (qPCR) was performed in software MxPro-Mx3005P (Stratagene/Agilent Technologies, Wilmington, DE, USA) using SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) with 1.5 μl of cDNA in each reaction. qPCR was performed with an activation at 95°C for 10 min, followed by 40 cycles of denaturing and extension (95°C for 15 s and 60°C for 1 min). Specific primers for EGF (forward 5'TGGAACTGCTTGGTGTTCGT-3'/reverse 5'AACTTCACCACCTGCCAACA-3') NM_001963.4 and EGFR (forward 5'CTCTTGCTGCTGGTGGTGG-3'/reverse 5'CTCCACAAGCTCCCTCTCCT-3') NM 005228.3, were made based on the BLAST data (http://blast.ddbj.nig.ac.jp/top-j.html). The Livak method (2−ΔΔCT) was used to determine the relative quantification of the expression of EGF and EGFR. The values were normalized relative to constitutive expression of β-actin (forward 5'-AAT TAC GAG CTG CGT GTG G-3'/reverse 5 '-AGA GCG CAG GTA GGA TAG CA-3'). Two series of experiments were performed for each tissue sample to ensure reproducibility. Data are presented with the relative variation of bending a calibrator (RNA pool from all samples). All reactions were performed in duplicate containing essential information according to Bustin et al.
The sample size required for a study with 80% power was calculated using an online calculator at http://statpages.org/proppowr.html. The nominal variables were expressed by frequencies and percentages. To access the meaning of nominal variables between groups it was performed the Fisher exact Chi-square test. Continuous variables were expressed as a mean and standard deviation. The Shapiro–Wilk test was used to assess the distribution between the variables. Analysis of variance was performed using (ANOVA)/t-test or Mann–Whitney tests to compare means between groups when the variable was in a normal or nonnormal distribution including gene expression analysis after the calculation for 2−ΔΔCT method. P < 0.05 was considered statistically significant. Statistical analyses were performed using Stata 11.1 (Stata Corp, Texas, USA).
| Results|| |
Of the total of 49 individuals evaluated for 6 months, there were 36 (73.4%) women and 13 (26.6%) men, with a mean age 56.54 ± 9.8 years. There was no difference between the control and test groups as to race, sex, age, alcohol consumption, general medical conditions, current medications, tooth loss, and periodontal phenotype [Table 1] and [Table 2].
Considering the peri-implant assessment [Table 3], a month after exposure of the implant, the test group showed a higher prevalence of red mucous membranes, swelling, and pus (P< 0.0001).
However, plaque presence, the implant region, and the loss of the implant were not statistically different between groups.
Participants with peri-implant mucosae inflammation (test group) had a higher prevalence of a history of chronic periodontitis (P< 0.0001). According to the analysis of odds ratio, clinically, chronic periodontitis history was a risk factor for mucosae inflammation. Participants with chronic periodontitis history have 22.5 times more likely to develop inadequate healing than participants with no history of chronic periodontitis (odds ratio: 22.5, confidence interval: 4.1–120.5) [Table 4].
|Table 4: Correlation between history of chronic periodontitis and harmful peri-implant healing|
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Correlation between clinical parameters and the expression of epidermal growth factor and epidermal growth factor receptor
To withstand the potential association between the EGF and EGFR genes with the quality of the endosseous implant osseointegration, the expression of these genes was investigated in 49 gingival discards obtained after the osseointegration process in subjects with and without inflammation in mucosae healing. All RNA samples revealed proportions (A260 nm/A280 nm) of more than 1.9. From the total sample, EGF and EGFR were detected in 42 tissue mRNA (85.7%). EGF and EGFR showed significantly lower expression in gingival tissues removed from regions with inadequate healing (test group). EGF showed mRNA expression with an average of 44.53 ± 79.16 and 01.02 ± 1.33 in control and test groups, respectively (P = 0.008) [Figure 1]. Similarly, EGFR expression was significantly higher in the control group (102.03 ± 329.57) compared to the test (7.85 ± 4.16) (P = 0.04) [Figure 2].
|Figure 1: Epidermal growth factor expression in the Test and Control groups. Note the low expression, considering the average of the mRNA determined by 2−ΔΔCT in the Test group (P = 0.008)|
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|Figure 2: Epidermal growth factor receptor expression in the Test and Control groups. Note the low expression, considering the average of the mRNA determined by 2−ΔΔCT in the Test group (P = 0.04)|
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| Discussion|| |
Based on the potential correlation between inadequate healing of the peri-implant tissue and the development of early peri-implant disease, the aim of this study was to evaluate the association between the expression of EGF and EGFR genes in the peri-implant mucosa, with the quality of peri-implant mucosae healing. Our results clearly show that EGF and EGFR genes are less expressed in the mucous corresponding to regions with inflammation. In contrast, regions with proper healing, without inflammation clinically detectable, and integrity of the peri-implant mucosa showed high expression of the same genes, consolidating the low expression of these genes as a risk factor for the inappropriate tissue regeneration around the implant and therefore the development of possible early peri-implantar disease.
The main function of the gingival epithelium is to protect the underlying structures against physical stress generated during the chewing process and acts as the primary barrier to bacterial penetration. The gingival fibroblasts are the most numerous cells in the gingival connective tissue, responsible for the synthesis of collagen and elastic fibers and especially the formation of granulation tissue during tissue healing.,
In natural teeth, the junctional epithelium provides sealing at the base of the periodontal groove against the penetration of chemical pathogens and bacterial substances. As no cement or fiber insertion is seen in the titanium surface, a mucous sealing provides the main barrier against the spread of pathological insults to the peri-implant deep tissue. This sealing around the endosseous implants is provided by the presence of junctional epithelium, the sulcular epithelium, and connective tissue, which has weak adhesion to the titanium structure made by hemidesmosomes. Destruction of the integrity of perimucosa surface of titanium leads to the extension of pathological bag directly to bone tissue.
Understand the physiological peri-implant characteristic and association with the release of EGF and its receptor, can clarify the association between healing profile, postimplant placement, and the levels of growth factors.
The gene that controls the production of EGF in the human is in the chromosome 4 and its molecule contains 53 amino acids. Specific receptors or EGFR are present in epithelial cells of local with high and low proliferation index and cell differentiation. In oral tissues, EFG receptors are present in all epithelia. In other cells, such as fibroblasts and endothelial cells, EGF also acts as a mitogen. Molecules of EGF are found in the interstitium of the oral submucosal tissue. In saliva, the EGF is present since the salivary glands are epithelial organs. In the repair, the EGF has proved to be important, and the epithelial surfaces stimulate proliferation, differentiation, organization, and keratinization of the superficial layers in the regenerative process of ulceration.
The EGF in the saliva and in the epithelial cells stimulates peri-implant epithelial proliferation and starts the formation of peri implant junctional epithelium. Once that integration occurs between the implant and the epithelium, salivary EGF penetration stops or decreases drastically, and the cell renewal process returns to the normal level. In our study, the levels of EGF/EGFR were measured immediately after the clinically determined implant osseointegration, and this growth factor and its receptor were measured directly in the peri-implant mucosa without being regarded their levels in the saliva or crevicular fluid peri-implantar. Thus, the findings observed during implant reopening may suggest that high levels of EGF/EGFR in the peri-implant mucosa can be correlated with the high regenerative capacity still present on the osseointegration process and maintenance of the peri implantar mucosal sealing in healthy tissue.
Another important issue to consider is that, physiologically, the healing is divided into three phases: inflammation, proliferation, and remodeling. During this process, EGF has a central role being produced by a variety of cells, including endothelial cells and fibroblasts, with regulatory function in the final healing cascade after the formation of the hemostatic plug and its expression is altered by the expression of its receptor EGFR on target cells, being a dose-dependent mitogen for the fibroblasts granulation in the healing remodeling phase. Therefore, studies have explored the importance of studying the EGF receptor along with EGF to better understand the biology of this growth factor, and its level after the period of acute healing phase, technically, occurs after 14 days in the gingival epithelium and 30 days in the bone tissue. Thus, our study looked not only the expression of EGF but also of EGFR and its correlation with the clinical mucosae characteristics observed after the period of peri-implant healing (remodeling) previously established by Brånemark et al.
Despite the EGF/EGFR is associated with the process of peri-implant saucerization  and the etiopathogenesis of cancer, especially squamous cell carcinoma, our study, in contrast, showed that high levels of EGF are associated with health peri-implant and mucosal healing properly. These results, however, have not considered the analysis of peri-implant bone. Deficient EGF mice altered severely the process of ossification and the recruitment of osteoclasts and interfering in bone remodeling. Based on these findings, future studies are needed, including the analysis of bone tissue, to better understand the function of the EGF/EGFR in peri-implant tissue.
However, confirming our findings, several studies ,, identified high levels of EGF and EGFR in cells derived from the junctional epithelium in healthy tissues. It is speculated that EGF/EGFR system participates in osteoblast differentiation, by stimulating the proliferation of osteoblastic progenitors, with high levels at regenerated tissues.EGF negatively regulates ALPase activity in periodontal ligament cells, whereas increasing DNA synthesis and fibronectin  suggesting that EGF may act as a regulator of proliferation and periodontal regeneration.
Based on the apparent contradiction in the literature concerning levels of EGF/EGFR in healthy gingiva, we can consider that these findings were conducted on studies following different clinical protocols, sample collection in different tissues, including mucosa, bone, and saliva, and different times of healing factors which modify the expression of this growth factor and its receptor. In our study, we evaluated the expression of EGF/EGFR in situ, in the peri-implant mucosae, immediately after the period of clinical osseointegration, during tissue remodeling phase. Our findings found biological support and consider the high levels of EGF and EGFR in healthy mucosa as active in tissue homeostasis and maintenance of mucosal sealing.
EGF has been explored for use in regenerative therapies, to increase their levels in regenerating tissues including alveolar bone, root, and periodontal and tooth pulp  and also decreasing the risk of ankylosis and root resorption.
This therapeutic application is extremely relevant, considering that current rates show an increasing incidence of peri-implant disease in rehabilitated participants with endosseous implants. The inadequate implant osseointegration occurs due to the action of external and internal forces, being called biomechanical failures. These factors include bacterial infection, occlusal overload, genetic and early exposure of the implant.
These failures arising from the peri-implant mucosal injury (mucositis) that can spread to the underlying bone (peri-implantitis), triggering bone loss and/or loss of the implant, and affecting about of 65% of the population rehabilitated with endosteal implant. In our study, we consider the failures that occur early and are directly related to the early development of peri-implant disease.
This is the first study demonstrating the association between inadequate mucosae healing during the period of osseointegration and expression of EGF/EGFR. Our results showed that high levels of EGF and EGFR are associated with peri-implant health. One reason may be related to the fact that the expression in healthy peri-implant tissue, in our study, is within the normal range to allow proper healing and formation of mucous sealing. In contrast, the group with mucosal inflammation or inadequate osseointegration may have presented these clinical features for low expression of EGF and EGFR which may have hindered the proper epithelial proliferation and peri-implant remodeling. Thus, further studies are necessary to allow better identify which levels considered “normal” for peri-implant proper healing and which EGF and EGFR levels considered “at risk” for developing injury.
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Conflicts of interest
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]