OAI ORAL & Implantology CIC Edizioni Internazionali 2017 April-June; 10(2): 151–161. ISSN: 2035-2468
Published online 2017 September 27. doi: 10.11138/orl/2017.10.2.151.

Effects of light-emitting diode (led 640nm) on human gingival fibroblasts: a comparative in vitro study

P. M. MANDRILLO,1 G. FISCHETTO,2 P. ODORISIO,3 F. CURA,4 A. AVANTAGGIATO,5 and F. CARINCIcorresponding author5

1Private practice, Taranto, Italy
2Private practice, Teramo, Italy
3Private practice, Pescara, Italy
4Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy
5Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy

corresponding authorCorresponding author.

Correspondence to: Francesco Carinci, Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Via Luigi Borsari 46, 44121 Ferrara, Italy, Phone/Fax: +39.0532.455582, E-mail: crc@unife.it

SUMMARY

Purpose
The light-emitting diodes (LEDs) have been applied in oral surgery for tissue stimulation and wound healing. Several Authors have highlighted that fibroblasts subjected to phototherapy have an increased viability, proliferation, biomodulation of inflammatory cytokines and genes expression. It remains to be determined which are the best irradiation parameters (energy, wavelength, power) for each type of cell in order to obtain the best bio-stimulation. The aim of this study was to investigate the effects of LED irradiation on primary human gingival fibroblast cells (HGF) on DSP, ELN, HAS1, ELANE, HYAL1, RPL13 genes activation using Real Time PCR. These genes activation is directly connected with elastin protein production and HGF functionality.

Materials and methods
Human gingival tissue biopsies were obtained from three healthy patients during extraction of teeth. The gingival pieces were fragmented with a scalpel and transferred in culture dishes for allow the cells growth. Human gingival fibroblasts at the second passage were seeded on multiple 6-well plates and were stimulated with three different light-emitting diodes (LEDs) fixture. After irradiation, the cells were trypsinized, harvested and lysed for RNA extraction. Genes expression was quantified using Real Time PCR.

Results
We didn’t found significant differences in genes activation of HGF of the three different LEDs. The LED irradiation seems to be directly correlated with the elastin and hyaluronoglucosaminidase 1 genes activation that are directly connected with proteins production and HGF functionality.

Conclusions
HGF show an increased deposition of elastin as well as enhanced expression of collagen type I, which is the main protein related to the synthesis and of the collagen-rich matrix.

Keywords: light-emitting diode, human gingival fibroblasts, primary fibroblasts cell culture, Real Time Polymerase Chain Reaction

Introduction

Light therapy has proven to be effective in reducing the damage oral tissue and promotes healing of tissue and is therefore recommended in surgery and dentistry (1, 2). In particular light-emitting diodes (LEDs) have been applied in oral surgery for tissue stimulation and wound healing (3, 4). LED radiation is monochromatic red-to-near-infrared (NIR) radiation (2). Light in the NIR 600~650 nm and white 400~750 nm range have shown positive effects on cells and tissues. LEDs have many advantages over lasers for use in photo-therapy, in particular in wound healing and for treatment of skin and mucosal ulcers. LEDs are different from low-level laser (LLL) because the LEDs light radiation is not coherent, whereas LLL presents a coherent radiation (2). Concerning the fibroblasts subjected to phototherapy, several Authors have reported encouraging scientific data, such as enhanced cell viability, proliferation (5, 6), bio-modulation of inflammatory cytokines and genes expression (7, 8). Despite these positive results, it remains to be determined what is the optimal intensity and frequency for each cell type and pathway. In other words, it must be determined which are the best irradiation parameters (energy, wavelength, power) for each type of cell in order to obtain the best bio-stimulation.

In clinical situations with oral erosions or ulcers or in post-surgical phase, light should stimulate the epithelium and connective tissue. It has been demonstrated that LEDs irradiation (600~650 nm, 0.3 J/cm2) and white (400~750 nm, 1.26 J/cm2) are capable of propagating through an epithelial barrier and also to stimulate the underlying fibroblast cells (9).

The aim of this study was to investigate the effects of LEDs on primary human gingival fibroblast cells (HGF) on desmoplakin (DSP), elastin (ELN), hyaluronan synthase 1 (HAS1), elastase (ELANE), hyaluronoglucosaminidase 1 (HYAL1), ribosomal protein L13 (RPL13) gene activation using Real Time PCR. These genes activation is directly connected with proteins production and HGF functionality.

Materials and methods

Primary human gingival fibroblast cell (HGF) culture
Human gingival tissue biopsies were obtained from three healthy patients (68-year-old man, 63-years-old woman and 20-year-old men) during extraction of teeth. The gingival pieces were fragmented with a scalpel and transferred in culture dishes containing Dulbecco’s modified Eagle medium (DMEM, Sigma-Aldrich, Inc., St. Louis, Mo) supplemented with 20% foetal calf serum (FBS) and antibiotics, i.e. penicillin 100U/ml and streptomycin 100μg/ml (Sigma Aldrich, Inc.).

Cells were incubated in a humidified atmosphere of 5% CO2 at 37°C. The medium was changed the next day and twice a week. After 15 days, the pieces of gingival tissue were removed from the culture dishes. Cells were harvested after additional 24h of incubation.

LED irradiation on cells cultures
Human gingival fibroblasts at the second passage were seeded on multiple 6-well plates.

The cells stimulation was performed with three different light-emitting diodes (LEDs) fixture.

The LED 1 (Figure 1) was composed of 12 high-brightness red LEDs, type LR.80, fixed on aluminium base plate with a surface area of 12,5 cm × 10 cm. In this device the LEDs were connected in series and they were willing in two rows of six light 2 cm distant to each other. LEDs were designed to run on direct current low voltage (12V), and 150 mA was the absorbed current. The LED 2 (Figure 2) was composed of aluminium base plate with a surface area of 9,5 cm × 9 cm on which was fixed a parabolic reflector of 3 cm diameter with inside 3 high-brightness red LEDs, type LR.80. This device was driven by an electronic circuit that allows the production of red light flashes by peaks of current of 700 mA at 5Hz of frequency with a duration of 100 microseconds each.

Figure 1Figure 1
a) LED 1 composed of 12 high-brightness red LEDs, type LR.80. b) Human gingival fibroblast gene expression profile after LED 1 irradiation. SYBR® Green assay. DSP = 0,33; ELN = 1,39; HAS1 = 0,34; ELANE = 0,69; HYAL1 = 1,31; RPL13 = 1,00.
Figure 2Figure 2
a) LED 2 composed of aluminium base plate with a surface area of 9,5 cm × 9 cm on which is fix a parabolic reflector of 3 cm diameter with inside 3 high-brightness red LEDs, type LR.80. b) Human gingival fibroblast gene expression profile after LED 2 (more ...)

The LED 3 (Figure 3) was composed of 32 RGB LEDs type SMD 5050 (four rows of 9 LED separated 1cm to each other) fixed on aluminium base plate with a surface area of 15 cm × 9 cm. The fixture was setted to emit light at a wavelength of 640 nm. The LEDs were powered with 12 Vdc with an absorbed current of 210 mA.

Figure 3Figure 3
a) LED 3 composed of 32 RGB LEDs type SMD 5050. b) Human gingival fibroblast gene expression profile after LED 3 irradiation. SYBR® Green assay. DSP = 0,74; ELN = 1,38; HAS1 = 0,48; ELANE = 0,24; HYAL1 = 1,38; RPL13 = 1,00.

The LED lamps were placed within 2 cm of the cells and the same cells were exposed to light for 20 minutes once a day for 2 days. A set of untreated cells were used as control.

The cells were maintained in a humidified atmosphere of 5% CO2 at 37°C for other 24h after LED irradiations.

RNA Isolation and Reverse Transcription Polymerase Chain Reaction (RT-PCR)
After irradiation, the cells were trypsinized, harvested and lysed for RNA extraction.

The total RNA was isolated using GenElute™ Mammalian Total RNA Miniprep kit (Sigma-Aldrich, Inc., St. Louis, Mo), following the manufacturer’s instructions. Then, 2,5 μg of total RNA was used for synthesize cDNA using M-MLV Reverse Transcriptase (Sigma-Aldrich, Inc., St. Louis, Mo) in according to the manufacturer’s instructions.

Real Time Polymerase Chain Reaction (Real Time PCR)
The cDNA was amplified by Real Time PCR. The amplification was performed by using Power SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA), and the specific assay was designed for the investigated genes.

SYBR assay reactions were performed in a 20 μl volume using the ABI PRISM 7500 (Applied Biosystems). Each reaction contained 10 μl 2X Power SYBR® Green PCR Master Mix (Applied Biosystems), 400nM concentration of each primer and cDNA.

All experiments performed included non-template controls to exclude contamination of reagents. PCR was performed with two biological replicates.

Expression was quantified using Real Time PCR. The gene expression levels were normalized to the expression of the housekeeping gene Homo sapiens ribosomal protein L13 (RPL13). Quantification was done with the delta/delta calculation method.

Forward and reverse primers for the selected genes were designed using primer express software (Applied Biosystems), and are listed in Table 1.

Table 1Table 1
Primers sequences for SYBR® Green assay.

Results

Figures 1, 2, 3 show the effects of the three different LEDs irradiation on the different genes (HAS1, HYAL1, ELN, ELANE, and DSP) involved in growth, health and elasticity cells.

Specifically, the LED irradiation seems to be directly correlated with the elastin (ELN) and hyaluronoglucosaminidase 1 (HYAL1) genes activation.

Discussion

Despite the promising cell-biostimulatory results obtained with phototherapy, such as modulation of tissue inflammation and stimulation of fibroblast cell metabolism, information about the effects of red LEDs on the HGF metabolism is poor (10). Therefore, the aim of this study was to evaluate the effects of three different LEDs irradiation on HGF.

The use of light therapy for HGF bio-stimulation is an interesting adjuvant treatment for healing during surgical procedures in operative dentistry. Based on the analysis of positive data provided by previous studies, researchers have been encouraged to develop more investigations to establish the most beneficial physical parameters of LEDs or laser irradiation on HGF (11).

Current studies have assessed LEDs energy densities varying from 0.3 J/cm2 and 1.26 J/cm2 LED wavelength. Some Authors (9, 10) recommend power values from 1 to 500 mW and energy densities from 0.04 to 50 J/cm2 for the irradiation of cells in culture with low-intensity therapy parameters. The red parameters of this study were based the indications of previous Authors who have studied biomodulation of HGF subjected to LEDs therapy (12, 13). In the present study, it was shown that red LED irradiation can increase the viability and number of HGF as well as elastin and hyaluronoglucosaminidase 1 genes activation.

Several studies have assessed the viability of HGF exposed to phototherapy (913). Some Authors (10) showed that viability of HGF was enhanced after exposure to one irradiation session with red LED. Therefore, in the present study we decided to evaluate the responses of HGF exposed to irradiation of three different LEDs irradiation.

As determined for cell viability, the number of viable HGF in the present study was also enhanced after irradiation with red LED at the chosen parameters. The ELN and HYAL1 genes expression and bio-stimulation seem to be related to cytochrome C oxidase activation, which enhances levels in the respiratory chain and adenosine triphosphate (ATP), and these biochemical changes led to macroscopic effects such as increased cell proliferation (6). Analysis of these data confirms that LED therapy may increase the viability and stimulate the proliferation of HGF in vitro.

Analysis of these data corroborates previous studies in which HGF (10,11) were subjected to phototherapy. In general, these cells presented increased deposition of elastin and other proteins as well as enhanced expression of hyaluronoglucosaminidase 1, which is the main protein related to the synthesis and of the collagen-rich matrix not only under phototherapy but also with biostimulation substances (1418). The molecular pathways that cause increased production of these specific proteins have not been described thus far. However, this macro effect of phototherapy on HGF suggests that light can be an interesting adjuvant treatment to up-regulate the deposition of collagen-rich matrix by HGF during healing phase. In HGF different energy doses can be the ideal parameter depending on the desired outcome. Overall, in vitro light therapy with red LEDs bio-stimulated all HGF functions assessed in the present study. Therefore, the scientific data obtained in the present study can drive future laboratory investigations or even in vivo studies in animal models to establish irradiation parameters for optimal and friendly clinical phototherapy procedures in HGF tissue regeneration. In recent year medical treatments have successfully used the LEDS in different treatments (healing-resistant wounds and ulcers e.g., chronic diabetic ulcers; in pain management, and in spinal cord and nervous system injuries) when other methods had limited success. Typical applications for LEDs irradiation are ulcerations, including decubitus ulcers and wound healing in the diabetic foot disorders. The LEDs may trigger vasomotor reflexes and enhance the effectivity of the respiratory chain. The LEDs stimulate the blood flow that can help in healing infections and other tissue disorders by accelerating supply with nutrients and increased clearance of metabolites and metabolic waste products. It may also attract cells of the immune system, such as neutrophils and other leukocytes as well as macrophages, because the effectiveness to induce cell migration by light is well established.

However, LED is still not a part of mainstream medicine and dentistry. We have highlighted some important recent developments in dentistry and in studies of cellular and molecular mechanisms behind the clinical findings. Future studies should focus on the efficacy of LED in oral surgery and implant dentistry, in particular in the treatment of peri-implantitis. Tooth replacement with implants is a well-known technique used worldwide in the last forty years (1964). Peri-implantitis can happen with high frequencies in patients affected by periodontal diseases (37, 38, 40, 65102).

In fact, even if the main factor for survival rate of implants is the quality of bone of receiving sites, the bacteria of peri-implantitis may be the main cause of failure of implants (7073, 77, 84, 85, 103105). So another field of application of LED could be peri-implantitis treatment by phototherapy.

Conclusion

In conclusion, there is no difference in genes activation of the three different LEDs. HGF presented increased deposition of elastin as well as enhanced expression of collagen type I, which is the main protein related to the synthesis and of the collagen-rich matrix.

The effect of phototherapy on HGF suggests that light can be an interesting adjuvant treatment to up-regulate the deposition of collagen-rich matrix by HGF during healing phase. Therefore, the scientific data obtained in the present study can drive future laboratory investigations or even in vivo studies in animal models to establish irradiation parameters for optimal and friendly clinical phototherapy procedures in HGF tissue regeneration.

References
1.
Gary N, Wu YCH, TCC. Light-emitting diodes: their potential in biomedical applications. Renewable and Sustainable Energy Reviews. 2010;14:2161–2166.
2.
Whelan HT, Smits RL Jr, Buchman EV, et al. Effect of NASA light-emitting diode irradiation on wound healing. J Clin Laser Med Surg. 2001;19:305–314.
3.
Carroll JD, Milward MR, Cooper PR, et al. Developments in low level light therapy (LLLT) for dentistry. Dent Mater. 2014;30:465–475.
4.
Prindeze NJ, Moffatt LT, Shupp JW. Mechanisms of action for light therapy: a review of molecular interactions. Exp Biol Med (Maywood). 2012;237:1241–1248.
5.
Karu T. Primary and secondary mechanisms of action of visible to near-IR radiation on cells. J Photochem Photobiol B. 1999;49:1–17.
6.
Muili KA, Gopalakrishnan S, Eells JT, et al. Photo-biomodulation induced by 670 nm light ameliorates MOG35-55 induced EAE in female C57BL/6 mice: a role for remediation of nitrosative stress. PLoS One. 2013;8:e67358.
7.
Hartel M, Illing P, Mercer JB, et al. Therapy of acute wounds with water-filtered infrared-A (wIRA). GMS Krankenhhyg Interdiszip. 2007;2:Doc53.
8.
von Felbert V, Hoffmann G, Hoff-Lesch S, et al. Photodynamic therapy of multiple actinic keratoses: reduced pain through use of visible light plus water-filtered infrared A compared with light from light-emitting diodes. Br J Dermatol. 2010;163:607–615.
9.
AlGhamdi KM, Kumar A, Moussa NA. Low-level laser therapy: a useful technique for enhancing the proliferation of various cultured cells. Lasers Med Sci. 2012;27:237–249.
10.
Taoufik K, Mavrogonatou E, Eliades T, et al. Effect of blue light on the proliferation of human gingival fibroblasts. Dent Mater. 2008;24:895–900.
11.
Vinck EM, Cagnie BJ, Cornelissen MJ, et al. Green light emitting diode irradiation enhances fibroblast growth impaired by high glucose level. Photomed Laser Surg. 2005;23:167–171.
12.
Gritsch K, Ponsonnet L, Schembri C, et al. Biological behaviour of buccal cells exposed to blue light. Mater Sci Eng C. 2008;28:805–810.
13.
Gao X, Xing D. Molecular mechanisms of cell proliferation induced by low power laser irradiation. J Biomed Sci. 2009;16:4.
14.
Avantaggiato A, Girardi A, Palmieri A, et al. Comparison of bio-revitalizing injective products: A study on skin fibroblast cultures. Rejuvenation Research. 2015;18:270–276.
15.
Avantaggiato A, Palmieri A, Bertuzzi G, et al. Fibroblasts behavior after N-acetylcysteine and amino acids exposure: Extracellular matrix gene expression. Rejuvenation Research. 2014;17:285–290.
16.
Avantaggiato A, Girardi A, Palmieri A, et al. Bio-Revitalization: Effects of NASHA on Genes Involving Tissue Remodeling. Aesthetic Plastic Surgery. 2015;39:459–464.
17.
Avantaggiato A, Martinelli M, Palmieri A, et al. Hyaluronic acid: the use of its precursor in skin biostimulation. Journal of biological regulators and homeostatic agents. 2015;29:647–654.
18.
Avantaggiato A, Bertuzzi G, Vitiello U, et al. Role of Antioxidants in Dermal Aging: An In Vitro Study by q-RT-PCR. Aesthetic Plastic Surgery. 2014;38:1011–1016.
19.
Rigo L, Viscioni A, Franco M, et al. Overdentures on implants placed in bone augmented with fresh frozen bone. Minerva Stomatol. 2011;60:5–14.
20.
Carinci F, Brunelli G, Franco M, et al. A retrospective study on 287 implants installed in resorbed maxillae grafted with fresh frozen allogenous bone. Clin Implant Dent Relat Res. 2010;12:91–98.
21.
Viscioni A, Rigo L, Franco M, et al. Reconstruction of severely atrophic jaws using homografts and simultaneous implant placement: a retrospective study. J Oral Implantol. 2010;36:131–139.
22.
Franco M, Rigo L, Viscione A, et al. CaPO4 blasted implants inserted into iliac crest homologue frozen grafts. The Journal of oral implantology. 2009;35:176–180.
23.
Viscioni A, Franco M, Rigo L, et al. Implants inserted into homografts bearing fixed restorations. Int J Prosthodont. 2009;22:148–154.
24.
Franco M, Viscioni A, Rigo L, et al. Clinical outcome of narrow diameter implants inserted into allografts. J Appl Oral Sci. 2009;17:301–306.
25.
Viscioni A, Franco M, Rigo L, et al. Retrospective study of standard-diameter implants inserted into allografts. J Oral Maxillofac Surg. 2009;67:387–393.
26.
Carinci F, Brunelli G, Zollino H, et al. Mandibles grafted with fresh-frozen bone: An evaluation of implant outcome. Implant Dentistry. 2009;18:86–95.
27.
Carinci F, Brunelli G, Zollino I, et al. Mandibles grafted with fresh-frozen bone: an evaluation of implant outcome. Implant Dent. 2009;18:86–95.
28.
Franco M, Tropina E, De Santis B, et al. A 2-year follow-up study on standard length implants inserted into alveolar bone sites augmented with homografts. Stomatologija. 2008;10:127–132.
29.
Lucchese A, Carinci F, Saggese V, et al. Immediate loading versus traditional approach in functional implantology. European Journal of Inflammation. 2012;10:55–58.
30.
Traini T, Danza M, Zollino I, et al. Histomorphic-metric evaluation of an immediately loaded implant retrieved from human mandible after 2 years. International Journal of Immunopathology and Pharmacology. 2011;24:31–36.
31.
Scarano A, Murmura G, Carinci F, et al. Immediately loaded small-diameter dental implants: evaluation of retention, stability and comfort for the edentulous patient. European Journal of Inflammation. 2012;10:19–23.
32.
Degidi M, Piattelli A, Carinci F. Clinical outcome of narrow diameter implants: a retrospective study of 510 implants. J Periodontol. 2008;79:49–54.
33.
Degidi M, Piattelli A, Iezzi G, et al. Do longer implants improve clinical outcome in immediate loading? Int J Oral Maxillofac Surg. 2007;36:1172–1176.
34.
Degidi M, Piattelli A, Carinci F. Immediate loaded dental implants: comparison between fixtures inserted in postextractive and healed bone sites. J Craniofac Surg. 2007;18:965–971.
35.
Degidi M, Piattelli A, Iezzi G, et al. Retrospective study of 200 immediately loaded implants retaining 50 mandibular overdentures. Quintessence Int. 2007;38:281–288.
36.
Degidi M, Piattelli A, Iezzi G, et al. Immediately loaded short implants: analysis of a case series of 133 implants. Quintessence Int. 2007;38:193–201.
37.
Degidi M, Piattelli A, Iezzi G, et al. Wide-diameter implants: Analysis of clinical outcome of 304 fixtures. Journal of Periodontology. 2007;78:52–58.
38.
Degidi M, Piattelli A, Gehrke P, et al. Five-year outcome of 111 immediate nonfunctional single restorations. J Oral Implantol. 2006;32:277–285.
39.
Degidi M, Piattelli A, Carinci F. Parallel screw cylinder implants: Comparative analysis between immediate loading and two-stage healing of 1005 dental implants with a 2-year follow up. Clinical Implant Dentistry and Related Research. 2006;8:151–160.
40.
Degidi M, Piattelli A, Gehrke P, et al. Clinical outcome of 802 immediately loaded 2-stage submerged implants with a new grit-blasted and acid-etched surface: 12-month follow-up. Int J Oral Maxillofac Implants. 2006;21:763–768.
41.
Degidi M, Piattelli A, Felice P, et al. Immediate functional loading of edentulous maxilla: a 5-year retrospective study of 388 titanium implants. J Periodontol. 2005;76:1016–1024.
42.
Danza M, Paracchini L, Carinci F. Tridimensional finite element analysis to detect stress distribution in implants. Dental Cadmos. 2012;80:598–602.
43.
Danza M, Grecchi F, Zollino I, et al. Spiral implants bearing full-arch rehabilitation: Analysis of clinical outcome. Journal of Oral Implantology. 2011;37:447–455.
44.
Danza M, Zollino I, Avantaggiato A, et al. Distance between implants has a potential impact of crestal bone resorption. Saudi Dental Journal. 2011;23:129–133.
45.
Carinci F, Danza M. Clinical outcome of implants inserted in piezo split alveolar ridges: A pilot study. Perspectives on Clinical Dentistry. 2011:29–30.
46.
Danza M, Zollino I, Guidi R, et al. Computer planned implantology: Analysis of a case series. Perspectives on Clinical Dentistry. 2011:287–300.
47.
Danza M, Carinci F. Flapless surgery and immediately loaded implants: a retrospective comparison between implantation with and without computer-assisted planned surgical stent. Stomatologija. 2010;12:35–41.
48.
Danza M, Quaranta A, Carinci F, et al. Biomechanical evaluation of dental implants in D1 and D4 bone by Finite Element Analysis. Minerva stomatologica. 2010;59:305–313.
49.
Danza M, Riccardo G, Carinci F. Bone platform switching: a retrospective study on the slope of reverse conical neck. Quintessence Int. 2010;41:35–40.
50.
Danza M, Fromovich O, Guidi R, et al. The clinical outcomes of 234 spiral family implants. J Contemp Dent Pract. 2009;10:E049–056.
51.
Calvo-Guirado JL, Ortiz-Ruiz AJ, Lopez-Mari L, et al. Immediate maxillary restoration of single-tooth implants using platform switching for crestal bone preservation: a 12-month study. Int J Oral Maxillofac Implants. 2009;24:275–281.
52.
Danza M, Guidi R, Carinci F. Comparison Between Implants Inserted Into Piezo Split and Unsplit Alveolar Crests. Journal of Oral and Maxillofacial Surgery. 2009;67:2460–2465.
53.
Danza M, Scarano A, Zollino I, et al. Evaluation of biological width around implants inserted in native alveolar crest bone. Journal of Osseointegration. 2009;1:73–76.
54.
Danza M, Zollino I, Guidi R, et al. A new device for impression transfer for non-parallel endosseus implants. Saudi Dental Journal. 2009;21:79–81.
55.
Andreasi Bassi M, Lopez MA, Confalone L, et al. Clinical outcome of a two-piece implant system with an internal hexagonal connection: a prospective study. J Biol Regul Homeost Agents. 2016;30:7–12.
56.
Danza M, Guidi R, Carinci F. Spiral family implants inserted in postextraction bone sites. Implant Dent. 2009;18:270–278.
57.
Gargari M, Ottria L, Morelli V, et al. Conservative zirconia-ceramic bridge in front teeth. Case report. Oral Implantol (Rome). 2015;7:93–98.
58.
Spinelli D, Ottria L, De Vico GD, et al. Full rehabilitation with nobel clinician® and procera implant bridge®: Case report. ORAL and Implantology. 2013;6:25–36.
59.
Falisi G, Severino M, Rastelli C, et al. The effects of surgical preparation techniques and implant macro-geometry on primary stability: An in vitro study. Medicina Oral, Patologia Oral y Cirugia Bucal. 2017;22:e201–e206.
60.
Pocaterra A, Caruso S, Bernardi S, et al. Effectiveness of platelet-rich plasma as an adjunctive material to bone graft: a systematic review and meta-analysis of randomized controlled clinical trials. International Journal of Oral and Maxillofacial Surgery. 2016;45:1027–1034.
61.
Marrelli M, Pujia A, Palmieri F, et al. Innovative approach for the in vitro research on biomedical scaffolds designed and customized with CAD-CAM technology. International Journal of Immunopathology and Pharmacology. 2016;29:778–783.
62.
Giuca MR, Pasini M, Giuca G, et al. Investigation of periodontal status in type 1 diabetic adolescents. European journal of paediatric dentistry: official journal of European Academy of Paediatric Dentistry. 2015;16:319–323.
63.
Giuca MR, Pasini M, Caruso S, et al. Index of orthodontic treatment need in obese adolescents. International Journal of Dentistry. 20152015
64.
Caruso S, Sgolastra F, Gatto R. Dental pulp regeneration in paediatric dentistry: The role of stem cells. European Journal of Paediatric Dentistry. 2014;15:90–94.
65.
Lauritano D, Martinelli M, Mucchi D, et al. Bacterial load of periodontal pathogens among Italian patients with chronic periodontitis: A comparative study of three different areas. Journal of Biological Regulators and Homeostatic Agents. 2016;30:149–154.
66.
Lauritano D, Scapoli L, Mucchi D, et al. Infectogenomics: Lack of association between vdr, il6, il10 polymorphisms and “red Complex” bacterial load in a group of Italian adults with chronic periodontal disease. Journal of Biological Regulators and Homeostatic Agents. 2016;30:155–160.
67.
Checchi L, Gatto MR, Checchi V, et al. Bacteria prevalence in a large Italian population sample: A clinical and microbiological study. Journal of Biological Regulators and Homeostatic Agents. 2016;30:199–208.
68.
Meynardi F, Pasqualini ME, Rossi F, et al. Correlation between dysfunctional occlusion and periodontal bacterial profile. J Biol Regul Homeost Agents. 2016;30:115–121.
69.
Lombardo L, Carinci F, Martini M, et al. Quantitive evaluation of dentin sialoprotein (DSP) using microbeads - A potential early marker of root resorption. ORAL and Implantology. 2016;9:132–142.
70.
Lauritano D, Cura F, Candotto V, et al. Evaluation of the Efficacy of Titanium Dioxide with Monovalent Silver Ions Covalently Linked (Tiab) as an Adjunct to Scaling and Root Planing in the Management of Chronic Periodontitis Using Pcr Analysis: A Microbiological Study. J Biol Regul Homeost Agents. 2015;29:127–130.
71.
Scapoli L, Girardi A, Palmieri A, et al. Quantitative Analysis of Periodontal Pathogens in Periodontitis and Gingivitis. J Biol Regul Homeost Agents. 2015;29:101–110.
72.
Lauritano D, Cura F, Candotto V, et al. Periodontal Pockets as a Reservoir of Helicobacter Pylori Causing Relapse of Gastric Ulcer: A Review of the Literature. J Biol Regul Homeost Agents. 2015;29:123–126.
73.
Scapoli L, Girardi A, Palmieri A, et al. Interleukin-6 Gene Polymorphism Modulates the Risk of Periodontal Diseases. J Biol Regul Homeost Agents. 2015;29:111–116.
74.
Carinci F, Girardi A, Palmieri A, et al. LAB®-Test 1: Peri-Implantitis and bacteriological analysis. European Journal of Inflammation. 2012;10:91–93.
75.
Carinci F, Girardi A, Palmieri A, et al. LAB®-test 2: Microflora and periodontal disease. European Journal of Inflammation. 2012;10:95–98.
76.
Carinci F, Girardi A, Palmieri A, et al. Lab®-test 3: Genetic susceptibility in periodontal disease. European Journal of Inflammation. 2012;10:99–101.
77.
Scapoli L, Girardi A, Palmieri A, et al. IL6 and IL10 are genetic susceptibility factors of periodontal disease. Dent Res J (Isfahan). 2012;9:S197–201.
78.
Carinci F, Girardi A, Palmieri A, et al. Lab-test 2: microflora and periodontal disease. European Journal of Inflammation. 2012;10:95–98.
79.
Cura F, Palmieri A, Girardi A, et al. Lab-Test((R)) 4: Dental caries and bacteriological analysis. Dent Res J (Isfahan). 2012;9:S139–141.
80.
Roncati M, Lauritano D, Cura F, et al. Evaluation of light-emitting diode (led-835 nm) application over human gingival fibroblast: An in vitro study. Journal of Biological Regulators and Homeostatic Agents. 2016;30:161–167.
81.
Caccianiga G, Rey G, Paiusco A, et al. Oxygen high level laser therapy is efficient in treatment of chronic periodontitis: A clinical and microbiological study using PCR analysis. Journal of Biological Regulators and Homeostatic Agents. 2016;30:87–97.
82.
Lauritano D, Bignozzi CA, Pazzi D, et al. Evaluation of the efficacy of a new oral gel as an adjunct to home oral hygiene in the management of chronic periodontitis. A microbiological study using PCR analysis. J Biol Regul Homeost Agents. 2016;30:123–128.
83.
Carinci F, Palmieri A, Girardi A, et al. Aquolab ® ozone-therapy is an efficient adjuvant in the treatment of chronic periodontitis: A case-control study. Journal of Orofacial Sciences. 2015;7:27–32.
84.
Lauritano D, Cura F, Gaudio RM, et al. Polymerase Chain Reaction to Evaluate the Efficacy of Silica Dioxide Colloidal Solutions in the Treatment of Chronic Periodontitis: A Case Control Study. J Biol Regul Homeost Agents. 2015;29:131–135.
85.
Lauritano D, Petruzzi M, Nardi GM, et al. Single Application of a Dessicating Agent in the Treatment of Recurrent Aphthous Stomatitis. J Biol Regul Homeost Agents. 2015;29:59–66.
86.
Carinci F, Lauritano D, Cura F, et al. Prevention of bacterial leakage at implant-Abutment connection level: An in vitro study of the efficacy of three different implant systems. Journal of Biological Regulators and Homeostatic Agents. 2016;30:69–73.
87.
El Haddad E, Gianni AB, Mancini GE, et al. Implant-abutment leaking of replace conical connection nobel biocare® implant system. An in vitro study of the microbiological penetration from external environment to implant-abutment space. ORAL and Implantology. 2016;9:76–82.
88.
Mancini GE, Gianni AB, Cura F, et al. Efficacy of a new implant-abutment connection to minimize microbial contamination: An in vitro study. ORAL and Implantology. 2016;9:99–105.
89.
Roncati M, Lucchese A, Carinci F. Non-Surgical treatment of peri-Implantitis with the adjunctive use of an 810-nm diode laser. Journal of Indian Society of Periodontology. 2013;17:812–815.
90.
Scarano A, Tripodi D, Carinci F, et al. Biofilm formation on titanium alloy and anatase-Bactercline® coated titanium healing screws: An in vivo human study. Journal of Osseointegration. 2013;5:8–12.
91.
Brunelli G, Carinci F, Zollino I, et al. Sem evaluation of 10 infected implants retrieved from man. European Journal of Inflammation. 2012;10:7–12.
92.
Scarano A, Sinjari B, Di Orio D, et al. Surface analysis of failed oral titanium implants after irradiated with ErCr:ysgg 2780 laser. European Journal of Inflammation. 2012;10:49–54.
93.
Brunelli G, Carinci F, Zollino I, et al. Peri-implantitis. A case report and literature review. European Journal of Inflammation. 2012;10:1–5.
94.
Scarano A, Piattelli A, Polimeni A, et al. Bacterial adhesion on commercially pure titanium and anatase-coated titanium healing screws: An in vivo human study. Journal of Periodontology. 2010;81:1466–1471.
95.
Grecchi F, Zollino I, Candotto V, et al. A case of mandible osteonecrosis after a severe periimplant infection. Dent Res J (Isfahan). 2012;9:S233–236.
96.
Carinci F, Farina A, Zanetti U, et al. Alveolar ridge augmentation: a comparative longitudinal study between calvaria and iliac crest bone grafrs. J Oral Implantol. 2005;31:39–45.
97.
Carinci F, Pezzetti F, Volinia S, et al. Analysis of MG63 osteoblastic-cell response to a new nanoporous implant surface by means of a microarray technology. Clinical Oral Implants Research. 2004;15:180–186.
98.
Oliveira DP, Palmieri A, Carinci F, et al. Osteoblasts behavior on chemically treated commercially pure titanium surfaces. J Biomed Mater Res A. 2014;102:1816–1822.
99.
Andreasi Bassi M, Lopez MA, Confalone L, et al. Hydraulic sinus lift technique in future site development: clinical and histomorphometric analysis of human biopsies. Implant Dent. 2015;24:117–124.
100.
El Haddad E, Lauritano D, Carinci F. Interradicular septum as guide for pilot drill in postextractive implantology: a technical note. J Contemp Dent Pract. 2015;16:81–84.
101.
Azzi L, Carinci F, Gabaglio S, et al. Helicobacter pylori in periodontal pockets and saliva: A possible role in gastric infection relapses. Journal of Biological Regulators and Homeostatic Agents. 2017;31:257–262.
102.
Gargari M, Comuzzi L, Bazzato MF, et al. Treatment of peri-implantitis: Description of a technique of surgical 2 detoxification of the implant. A prospective clinical case series with 3-year follow-up. ORAL and Implantology. 2015;8:1–11.
103.
Carinci F, Girardi A, Palmieri A, et al. Lab-test 1:peri-implantitis and bacteriological analysis. European Journal of Inflammation. 2012;10:91–93.
104.
Carinci F, Scapoli L, Girardi A, et al. Oral microflora and periodontal disease: new technology for diagnosis in dentistry. Ann Stomatol (Roma). 2013;4:170–173.
105.
Scapoli L, Girardi A, Palmieri A, et al. Microflora and periodontal disease. Dent Res J (Isfahan). 2012;9:S202–206.