Laser Therapy Induces Increased Viability and Proliferation in Isolated Fibroblast Cells

Introduction. Laser therapy (LT), which stimulates natural biological processes in the application region, is frequently used in dental treatments. Objective. The aim of this study is to evaluate the effects of LT that could increase wound healing on fibroblast cells in vitro. Methods. Twenty-four hours after preparing the fibroblast cell culture plates, laser irradiation was performed 1, 2, and 3 times according to the test groups using an Nd:YAG (neodymium-doped yttrium aluminum garnet) laser with a power output of 0.5 W, 1 W, 2 W, and 3 W. Cell proliferation analysis was performed by MTT (methylthiazole diphenyl tetrazolium) assay at the twenty-fourth hour following the last laser application. Results. In terms of the laser irradiation power level, the most proliferation was observed in 1 W and 2 W application groups. Although a statistically significant increase was observed, particularly at 0.5 W, the increase at 1 W was greater than at a power output of 0.5 W. In terms of the number of laser irradiation applications, the most proliferation was observed in 2 and 3 application groups. The highest proliferation value was obtained with 1 W of power for 2 applications, and the lowest was with 3 W of power for 3 applications. Conclusions. The findings of this study show LT increased fibroblast cell proliferation, depending on the power output level of the laser and number of applications. In addition to the proliferation and mitotic activity of the fibroblast cells, the results demonstrate that LT could increase wound healing after oral surgery and periodontal treatments.

Fibroblasts, which are found in the bodily organs and tissues with extracellular matrix (ECM) molecules, are the main connective tissue cells.¹ One of the important functions of fibroblasts is the synthesis and hemostatic balance of the ECM in tissues and organs. Fibroblasts are highly metabolically active cells that express and secrete most of the ECM components, such as collagen, proteoglycan, fibronectin, tenascin, laminin, and fibronectin.² Metabolically active cells play a critical role in regulating the ECM, intracellular fluid volume, and pressure in wound healing. If necessary, fibroblasts also can be transformed into other cells, especially osteocytes.^2,3

The use of lasers in dentistry has a wide range of applications from caries diagnoses to soft and rigid tissue procedures.^4-6 Despite the common use of these dental lasers, there is another type of laser that does not cut or ablate the tissues. Laser therapy (LT) applications have been used for many years in the field of dentistry and oral surgery for the treatment of pain relief, wound healing, and overall positive effect on inflammatory processes. The basic principle of LT is based on the biostimulation or biomodulation effect.⁷ The term laser therapy refers to atopic phototherapy released from light sources that emit a low amount of energy. When light hits a living cell, it leaves a small amount of energy in that cell.⁸ The cellular photoreceptors absorb the LT light and can transfer it to the mitochondria to produce adenosine triphosphate (ATP). With the increase in vasodilatation via ATP synthesis, the use of oxygen is increased, and activity of the cytoplasmic enzymes with the nucleic acids stimulates cell mitosis.⁹ 

Laser therapy is frequently used in many different dental treatments, including the healing of chronic and acute wounds and shortening the recovery process of operative areas with minimal pain.¹⁰ Discussion on tissue biostimulation using LT is still ongoing. The absence of a uniform report on the physical and biological variables, such as the type of laser, output power (continuous or vibrating), frequency of shot wavelength, application time and mode, distance of source to tissue applied, histological tissue differences, and absorption characteristics, make the standardization of results difficult. This study evaluated the effects of LT that could increase wound healing on fibroblast cells in vitro.

All experimental procedures were submitted to and approved by the Clinical Research Ethics Committee of Ordu University (Ordu, Turkey). The experimental part of the study was carried out in the Atigencell Cell Culture Laboratory (Trabzon, Turkey).

Cell culture
This study used commercially obtained human fibroblast cells. The cells were grown in T75 tissue culture flasks (Wuxi NEST Biotechnology, Jiangsu, China) supplemented with essential medium alpha (Lonza, Morristown, NJ), 10% fetal bovine serum (Lonza), antibiotics (penicillin and streptomycin), and antimycotics (Fungizone; Geneva Pharmaceuticals, Inc, Princeton, NJ). Incubation was performed at a constant temperature (MCO-17 AI; SANYO Electric Co, Ltd, Osaka, Japan) with 5% carbon dioxide (CO₂) to provide a 95% humidified air mixture at 37°C. The cell culture media were subjected to pH monitoring with daily bacterial and fungal contamination, and the nutrients were renewed every 2 days. When the fibroblast cells reached confluence, the cell monolayer was washed with calcium and magnesium-free phosphate buffered saline followed by trypsin (Lonza) to prepare single cell suspension. After counting the cells using a Thoma cell counting chamber under an inverted microscope, the cell concentration was adjusted to 1 × 10⁵ cells/mL.

Determination of the total cell count using a hemocytometer
The intercellular junctions of the fibroblast cells were removed, and the cells were detached from the sterile culture dishes using 0.25% trypsin-EDTA (Lonza). After centrifugation (1500 rpm for 5 minutes), the liquid remaining in the upper part of the tube was discarded with a Pasteur pipette. The cells collected in the bottom of the tube were resuspended in 1 mL of fresh medium, and 10 µL of the suspension was mixed with 10 µL of trypan blue.¹¹ This mixture was counted by spreading it on a Thoma slide. The total number of cells per mL of suspension was calculated using the Louis and Siegel Formula.¹² 

Laser irradiation procedure
For this study, 6 plates with 96 wells each were used, and the cell culture was prepared by dividing it in each of the 6 fibroblast plates. The cell culture plates were further divided into 3 groups according to the LT application periods (group 1: 1 LT application, group 2: 2 LT applications, and group 3: 3 LT applications). Two plates in each group also were divided into control and laser doses (first plate: 0.5 W LT, 1 W LT, and control; and second plate: 2 W LT, 3 W LT, and control). The cells were seeded at an initial density of 5 × 10⁴ cells/cm² in every well of all plates. 

For the laser applications, a free-running, pulsed-wave neodymium-doped yttrium aluminum garnet (Nd:YAG) laser (wavelength of 1064 nm under air cooling) (SmartFile; DEKA, Calenzano, Italy) with a phototherapy probe was used under the following irradiation parameters: power output of 0.5 W, 1 W, 2 W, and 3 W; energy of 100 mJ; frequency of 5 Hz, 10 Hz, 20 Hz, and 30 Hz; continuous wave mode; time of 0.5 minutes; and distance of 1 cm from the culture media. The nonirradiated control cells were subjected to room light for the same period of time and maintained outside the incubator under the same conditions as the laser-irradiated cells. To prevent interoperator variations, the same author (H.S.) performed all LT applications. A schematic representation of the 96-well plates prepared for cell proliferation and LT applications is shown in the Figure.

Cell proliferation/viability assay
A methylthiazole diphenyl tetrazolium (MTT) assay based on the determination of the metabolic activity was used to assess cell proliferation. At a concentration of 0.5 mg/mL, MTT was added to all wells. Following 3 hours of incubation at 37°C and 5% CO₂, the contents were replaced with dimethyl sulfoxide for 15 minutes at room temperature to extract the formazan crystals from inside the cells. Subsequently, 100 mL of a purple-colored sample solution was transferred from each well into a new 96-well plate, and the optical density was read using a microplate reader (Sunrise; Tecan Group Ltd, Männedorf, Switzerland) at 570 nm. Since the amount of color produced is directly proportional to the number of viable (metabolically active) cells, the relative number of the adhered live cells on the discs could be determined based on the optical absorbance of the sample. The mean values at the 96-hour post-seeding time point were recorded. 

Statistical analysis
Statistical analyses were performed using SPSS (version 11.5 for Windows; SPSS Inc, Chicago, IL). When analyzing the data, a 2-way analysis of variance was used. A value of P ≤ .05 was considered statistically significant.

Generally, cell proliferation rates were higher in the 0.5 W and 1 W LT groups than in the control, 2 W, and 3 W LT groups. However, a lower cell proliferation was detected in those cells irradiated with 2 W and 3 W of power. Also noted, the proliferation rates increased as the number of applications increased when compared with the controls, especially in those cases in which the irradiation was performed 2 or 3 times more. In addition, there was less proliferation in the samples irradiated 3 times with 3 W of power. 

The statistical data obtained as a result of the fibroblast cell proliferation tests are presented in the Table. When the proliferation rates in the test and control groups were compared in terms of the laser irradiation power level, the most proliferation was observed in cells treated with 1 W and 2 W. Although a statistically significant increase was observed in the proliferation levels, particularly at 0.5 W, the increase in the power levels at 1 W was greater than at the 0.5 W power level. When the proliferation rates were compared in terms of the number of laser irradiation applications, the most proliferation was observed in those cells with 2 and 3 applications. Moreover, the proliferation levels increased statistically when compared with the control group in the case of 1 application, but the increases in 2 and 3 applications were even greater. In general, the highest proliferation value was obtained with 1 W of power for 2 applications, and the lowest was with 3 W of power for 3 applications.

In this study, the authors hypothesized that LT could increase the proliferation and mitotic activity of fibroblast cells and wound healing. For this purpose, the cell proliferation values were assessed via MTT assay as a result of LT applied to the fibroblast cells at different powers.

Laser therapy is used in medicine and dentistry for the treatment of impaired microcirculation, wound healing, pain relief, fracture healing, and inflammation.¹³ Wound healing, one of the most studied effects of LT, is a complex process involving many types of cells, enzymes, growth factors, and other substances. The use of LT in wound healing has been shown to be effective in modulating local and systemic responses.¹⁴ In studies investigating the response of fibroblasts to lasers, increased cell division and collagen production have been reported.^15,16 However, in studies supporting the hypothesis that LT may accelerate wound healing, depending on the wavelength, dose, and local conditions in the soft tissues, it is possible its effect on wound healing are not only dependent on the total irradiation dose but also on the irradiation time and mode.^16-18

The beneficial effects of LT on wound healing have been reported in animal studies.^19,20 The most commonly mentioned mechanism for increasing laser wound healing is the effect of laser irradiation on intracellular metabolism and collagen production by fibroblasts.²¹

In studies performed using the tissue culture method, it was stated that the growth and mobility of the applied fibroblasts and collagen production increased. Obradović et al²² reported LT may have a biostimulant effect on periodontal wound healing in patients with type 1 and type 2 diabetes mellitus. 

In an animal model of type 2 diabetes,²³ basic fibroblast growth factor (bFGF) production was evaluated to determine the effects of low-level LT on cutaneous wound healing. They²³ reported a significant improvement in the histology of wound closure and wound healing following treatment at 4 J/cm², and a 3-fold increase in the quantitative analysis of bFGF production in laser-treated diabetic and non-diabetic mice.

In an in vitro study by Saygun et al,²⁴ 685 nm and 830 nm diode lasers in 2 J/cm² energy were applied in 1 dose and 2 doses. The authors²⁴ found low-level laser administration increased the expression of bFGF, insulin-like growth factor-1 (IGF-1), and IGF-1 receptors from human gingival fibroblast (GF) cells. 

Loevschall and Arenholt-Bindslev²⁵ reported ATP production increased with the absorption of laser light in subcellular structures, and thus stimulated cell functions. In several in vitro studies,^25-27 LT showed an increased rate of fibroblast proliferation, collagen synthesis, and release of growth factors from fibroblasts, as well as stimulating cell proliferation.

A series of studies by Hawkins et al^28-32 demonstrate the positive effects LT had on human skin fibroblasts. In their study series, they evaluated the effect of a diode laser at different wavelengths and doses on the proliferation and migration of fibroblasts. The same investigators³⁰ reported that the cumulative effect at low doses (2.5 J/cm² and 5 J/cm²) served as the stimulator, while multiple doses (16 J/cm²) at high doses served as inhibitors. 

In a cell culture study performed in a type 2 diabetic animal model,²³ LT at a wavelength of 632 nm and an energy density of 4 J/cm² was applied on the cutaneous wound surface, and successful results were achieved in wound closure and wound healing. Further, Kajagar et al³³ investigated the effect of LT on wound healing in nonhealing diabetic foot ulcers. In laser-treated ulcers, they observed a statistically significant healing compared with traditional treatment alone.³³ 

In the fields of medicine and dentistry, in addition to the standard disease treatments, the use of different devices has become commonplace in accordance with the developments in technology and research. Lasers have been investigated for this purpose, and it is believed that LT has the potential to stimulate normal cell functions.^9,10 Laser therapy has been reported to increase ATP, accelerate mitosis, improve tissue repair, stimulate bone repair, normalize collagen and elastic fibril accumulation in tissue repair, stabilize fibroblast production, increase peripheral blood flow, and improve anti-inflammatory activity.⁹

The published reports in the literature show varied application ranges and doses of laser applications. Cotler et al³⁴ found it useful to start at close intervals and continue at long intervals. Likewise, Tuner and Hode¹⁰ indicated that, in LT, the optimal therapeutic dose was 2 J/cm² to 4 J/cm² under the skin and 0.5 J/cm² to 1 J/cm² at the open wound.

In an in vitro study by Usumez et al,³⁵ the authors reported that LT had a prominent proliferation effect on human GF cells, but the duration of this effect was limited. Their findings show repeated applications are necessary to achieve a positive laser effect in clinical practice.³⁵ In the present study, cell proliferation rates were higher in the laser-treated cultures than in the control group. When evaluating the cell proliferation levels in terms of application periods, the authors found the proliferation rates increased with an increasing application frequency when compared with the controls, especially for 2 and 3 applications.

In the present study, although the cell proliferation levels were statistically increased in the LT-treated cell cultures at all power levels, the increase in the 1 W power level was found to be greater than in the 0.5 W power level, with the lowest in the 2 W and 3 W samples. In addition, the most proliferation was observed in the cells with 2 and 3 applications. In general, the highest proliferation value was obtained with 2 applications at 1 W of power, and the lowest was with 3 applications at 3 W of power.

Since this is a cell culture study, the authors evaluated the effects of LT in a short time (96 hours) and could not investigate long-term effects. Also, only the Nd:YAG laser was used. Future studies should include a comparison of the effects of different types of lasers in different doses.

The findings of this study have led to the conclusion that LT increased fibroblast cell proliferation, depending on the power output level of the laser and number of applications. In addition to the proliferation and mitotic activity of the fibroblast cells, the findings show LT could increase wound healing. 

Authors: Nihal Kara, PhD, DDS¹; Hilal Selamet, PhD, DDS²; Yasin Atakan Benkli, PhD, DDS³; Meral Beldüz, PhD, DDS⁴; Ceren Gökmenoğlu, PhD, DDS⁵; and Cankat Kara, PhD, DDS⁵

Affiliations: ¹Department of Pedodontics, Faculty of Dentistry, Ordu University, Ordu, Turkey; ²Department of Periodontology, Faculty of Dentistry, Okan University, Istanbul, Turkey; ³Department of Orthodontics, Faculty of Dentistry, Ordu University; ⁴Department of Biology, Faculty of Science, Karadeniz Technical University, Trabzon, Turkey; and ⁵Department of Periodontology, Faculty of Dentistry, Ordu University

Correspondence: Cankat Kara, DDS, PhD, Department of Periodontology, Faculty of Dentistry, Ordu University, Cumhuriyet, Mustafa Kemal Blv. No:478, 52200 Altınordu/Ordu, Turkey; mcankat@hotmail.com 

Disclosure: This study was supported by Ordu University/Turkey Scientific Research Project Unit. The authors disclose no financial or other conflicts of interest.