Modulatory effects by neodymium-doped yttrium aluminum garnet laser on fibroblast attachment to single rooted tooth surfaces following ultrasonic scaling and root planning: An in vitro study.

CONTEXT: One of the most important goals of periodontal therapy is connective tissue reattachment to previously diseased root surfaces. In the recent years, laser therapy has been considered as an important tool in improving the treatment of periodontal disease. AIMS: To evaluate the neodymium-doped yttrium aluminum garnet (Nd: YAG) lasers effects on root surfaces affected by periodontal disease and compare this treatment with scaling and root planning (SRP) in terms of fibroblast attachment.
MATERIALS AND METHODS: A sample of 30 single-rooted human teeth extracted because of advanced periodontal disease was used in this study. Sixty specimens obtained by longitudinal sectioning were randomly divided in three groups. Group A control (untreated); Group B SRP; Group C laser (Nd: YAG) and ultrasonic scaling. All specimens were incubated with fibroblast suspension and then fixed and observed under scanning electron microscope.
RESULTS: With a median of 8, the control group (Group A) exhibited the least number of total fibroblasts among all the three groups. The laser and scaling - treated group (Group C) showed the highest number of fibroblasts (median = 49, mean ± standard deviation [SD] = 48.28 ± 17.18), followed by SRP only (Group B, median = 22, mean ± SD = 22.24 ± 8.67).
CONCLUSIONS: Nd: YAG laser irradiation at specific energy densities can be used as a useful tool to condition the root surfaces, enhancing fibroblast attachment. Hence aiding in re-establishment of the connective tissue attachment to the root surfaces of previously diseased teeth.

RCT Entities:   Population Interventions Outcomes

INTRODUCTION

Rapid strides of development in science and technology have revolutionized basic outlook and approach to the problems of dental diseases. For many years, it has been assumed that periodontal problems were invariably progressive and the morbid effects increasing in severity with passage of time. Dental laser research and application has grown steady during the past three decades from its modest, unheralded beginning to a state of development that portends dramatic implications into the nature of clinical dental practice in the future. The ultimate goal of periodontal therapy is predictable regeneration of the periodontium at the site of previous periodontitis. As the periodontal microbiota and bacterial endotoxins contaminate the root surfaces in periodontal pockets, it inhibits migration and attachment of fibroblast.[12] Mechanical instrumentation causes the root surfaces to be covered with a smear layer resulting in obliteration of the orifices of the dentinal tubules, which contain microbiota, bacterial endotoxins, and residual, contaminated root cementum which could in turn compromise periodontal healing and regeneration of connective tissue attachment.[3] To regain such attachment several methods have been utilized which includes barrier membranes, osseous grafts, and root bio-modification using citric acid, ethylenediaminetetraacetic acid (EDTA). Latest being the lasers, because of its ability to condition hard tissues.[4]

The use of neodymium-doped yttrium aluminium garnet (Nd: YAG) laser in the soft tissue surgeries has encouraged the thoughts of researchers to apply this technology in pretreatment of root, which they believe will hold a promising future in periodontal regeneration. The Nd: YAG laser with an energy ranging from 150 to 87.5 mJ/pulse has shown a bactericidal effect suppressing and eradicating putative periodontal pathogens from periodontal pockets as well as from dental hard tissues.[5] Although studies on laser application as an adjunctive tool in the treatment of periodontal disease has been reported, several questions still remain unresolved. These include maximum energy level in order to avoid damaging tissues and the ability of fibroblasts to migrate and attach to root surfaces after laser irradiation. The elimination of calcific deposits, microorganisms and microbial products from the periodontally diseased root surface is considered necessary to produce a biologically acceptable root surface. The effectiveness of scaling and root planning (SRP) in this regard is universally accepted. However, this mechanical instrumentation always produced a smear layer that obliterated the orifices of the dentinal tubules, which contains microbiota, bacterial endotoxins and residual contaminated root cementum compromising the periodontal healing and regeneration of connective tissue attachment.[6789] It is possible that this smear layer may contain the cytotoxic and inflammatory mediators which may affect healing.[2] Therefore, the aim of the present study was to evaluate the Nd: YAG lasers effects following ultrasonic scaling on root surfaces affected by periodontal disease and compare this treatment with ultrasonic SRP alone in terms of fibroblast attachment.

MATERIALS AND METHODS

This in vitro study consists of 30 extracted periodontally compromised single-rooted teeth with a hopeless prognosis suitable to the selection criteria, obtained from the Department of Oral Maxillofacial Surgery, Tamil Nadu Government Dental College and Hospital. Inclusion Criteria included 30 single-rooted human teeth extracted because of extensive loss of periodontal supporting tissues along with evidence of calculus deposits were used in this study. Exclusion criteria included the presence of caries or filling materials or hypoplastic defects, patients with ahistory of systemic diseases, antibiotic therapy 4 months prior to the study and patients who have undergone periodontal debridement in the last 6 months prior to the study.

Sample preparation

Following extraction of the teeth, blood, saliva, and soft tissue debris were removed by light scrubbing with a sterile scrub brush and by rinsing with sterile saline solution. From each tooth, two specimens cut with a sterile diamond disk running at low speed with sterile water coolant were obtained. The first section was 2 mm apical to the cementoenamel junction; the second section was 2-3 mm coronal from the root apex. A longitudinal bucco-lingual section was then cut to expose the pulpal wall which was separated from the remaining outer portion of the root dentin by a bur at low speed to avoid contamination from the pulp. The specimens were transported in a sterile container with distilled water to the laboratory.

Experimental design

Sixty specimens thus collected were randomly assigned to one of three groups:

Group A: Control group: Ten specimens rinsed with sterile saline solution

Group B: SRP group: 25 specimens scaled with ultrasonic instruments and root planed with hand curettes until all visible calculus were removed

Group C: Nd: YAG laser group: 25 specimens were treated with Nd: YAG laser system (wavelength = 1064 nm), used at 80 mJ/pulse in addition to SRP (ultrasonic and hand instrumentation).

The specimens were then transported in a sterile container with distilled water to the laboratory.

Laser treatment

The Nd: YAG laser (Quanta Laser System, Italy) was used at a measured power of 4 W, emitting a pulsed light at a wavelength of 1064 nm, energy per pulse 80 mJ, pulse repetition rate 50 Hz. The distance from the point of convergence to the surface of the specimen was 5 cm, the angle of incidence was kept at a constant 90°. Light was focused and applied directly to each tooth specimen. Time of radiation spent on each specimen was 70 s.

Fibroblast culture and incubation

For this study the continuous mouse fibroblast cell line L-929, which readily adheres and spreads on to most substrates, was used. Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 μg/ml streptomycin, 100 U/ml penicillin, 2.5 μg/ml amphotericin B, and 2 mmol/l glutamine was the cell culture used. The cells were cultured at 37°C in a humidified incubator with 5% carbon dioxide in the air. Fibroblast were harvested by means of a sterile trypsin - EDTA solution, re-suspended in the experimental cell culture medium and diluted to 1 × 105 cells/ml. Then 5 ml of the cell suspension was seeded into the tissue culture polystyrene containing root samples and incubated for 3 days. Cells on the samples were rinsed with Dulbecco's phosphate buffered saline (DPBS) at the end of this period and fixed by DPBS solution containing 4% glutaraldehyde.

Preparation for scanning electron microscopic study

Freshly prepared 4% glutaraldehyde in DPBS solution (pH: 7.2) at room temperature was used to fix specimens for 2½ h and washed trice with DPBS for 10 min each. The specimens were then dehydrated in a graded series of aqueous ethanol (50, 70, 80, 95 and 100%) for 10 min at each concentration. After the last passage in absolute ethanol, dehydration was completed by a 30 min immersion in hexamethyldisilazane. The specimen were then air dried, mounted in scanning electron microscope (SEM) stubs and sputter coated with approximately 200 A° of gold using a sputter coater (Hitachi E-1010 Ion Sputter) for SEM viewing using SEM (Hitachi S-3400 N) operated at an accelerated voltage of 15-20 kV. All specimens were viewed at magnification values ranging from ×100 to ×3500. Briefly three photomicrographs were taken of each root specimen and were considered to be representative of the total surface area. Photomicrographs were taken with a positive angle of 15° at three nonoverlapping points along a diagonal line. The data collected was statistically analyzed.

Statistical analysis

The average of the three readings per sample was entered in the master chart created in Microsoft Excel 2007 (Microsoft Corporation, California USA), and the data entry was verified twice by the same operator. The descriptive statistics (mean, standard deviation [SD], median, minimum and maximum) were calculated for the flat, round and total number of fibroblasts in each of the three groups. The difference between the three groups was evaluated with Kruskal–Wallis analysis of variance (ANOVA), and pair-wise comparison of groups was performed with Mann–Whitney U-test. The alpha level was set at 0.05 for all tests and the data analysis was performed in Statistical package for Social Sciences (SPSS) version 19 (SPSS Inc., an IBM Company, Somers, NY, www.spss.com).

In the present study because three outcome measures were tested against four hypothesized predictors, a Bonferroni-adjusted significance level of 0.00625 was calculated to account for the increased possibility of Type-I error. The Bonferroni correction is based on the idea that if an experimenter is testing n dependent or independent hypotheses on a set of data, the probability of Type I error is offset by testing each hypothesis at a statistical significance level 1/n times what it would be if only one hypothesis were tested.

RESULTS

The observed surface characteristics of specimens within each treatment group were quite consistent, and there were little intra group variations. Thus, the described SEM features are applicable to each specimen within its specified treatment group. Attached fibroblasts observed under SEM in the study specimens (root) were either flat or round in morphology. Flat fibroblasts were found to be tightly attached to specimens by numerous lamellipodia [Figures 1 and 2], occasionally coalesced to form a confluent monolayer [Figures 1 and 3] and the round fibroblast were poorly attached to specimens with few attachment processes [Figure 4] and Table 1.

Figure 1

Scanning electron microscope photograph of root surface (neodymium-doped yttrium aluminium garnet laser/scaling group) with well-spread flat fibroblasts (×3.50 k)

Figure 2

Scanning electron microscope photograph of root surface (neodymium-doped yttrium aluminum garnet laser/scaling group) with flat fibroblasts depicting well-developed lamellipodia (×3.00k)

Figure 3

Scanning electron microscope photograph of root surface (neodymium-doped yttrium aluminum garnet laser/scaling group) with a higher number of flat fibroblasts (×356)

Figure 4

Scanning electron microscope photograph of root surface (SRP group) with immature round fibroblasts (×2.00 k)

Table 1

Master chart of fibroblast type for the various groups (A, B and C)

The Group-wise summary statistics (mean, SD, median, minimum and maximum) for the total number of fibroblasts and each of the two types is presented in Table 2. The frequency distribution of the number of flat and round fibroblasts in all the three groups is presented in Tables 3 and 4, respectively.

Table 2

Group wise descriptive statistics

Table 3

Frequency of distribution of flat fibroblast

Table 4

Frequency of distribution of round fibroblast

The laser and scaling - treated group (Group C) showed the highest number of fibroblasts (median = 49, mean ± SD = 48.28 ± 17.18) [Figures 1–4] followed by SRP only (Group B, median = 22, mean ± SD = 22.24 ± 8.67) [Figures 5 and 6]. With a median of 8, the control group (Group A) exhibited the least number of total fibroblasts among all the three groups [Table 2] [Figures 7 and 8]. The number of flat fibroblasts in these groups also presented a similar trend, with Group C showing the highest (median = 36, mean = 32.56) and Group A, the least (median = 0, mean = 0.2). As for the number of round fibroblasts, Groups A and B were nearly comparable (medians of 7.5 and 7 respectively), and Group C exhibited better result than the other two (median = 13, mean = 15.72). The results of Kruskal–Wallis ANOVA revealed that the inter-group differences for all the three parameters evaluated (flat, round and total number of fibroblasts) were statistically significant at P < 0.001 [Table 5].

Figure 5

Scanning electron microscope photograph of root surface (neodymium-doped yttrium aluminum garnet laser/scaling group) with the highest number of fibroblasts (×500)

Figure 6

Scanning electron microscope photograph of root surface (SRP group) with round and flat fibroblasts (×3.50 k)

Figure 7

Scanning electron microscope photograph of root surface (SRP group) with few flat fibroblasts (×3.00 k)

Figure 8

Scanning electron microscope photograph of root surface (untreated control) with few round fibroblasts (×3.00 k)

Table 5

Group comparison with Kruskal-Wallis ANOVA of flat and round fibroblast

Pair-wise comparisons with Mann–Whitney U-tests indicated that the laser and scaling - treated group consistently produced better results than both the SRP only and the no - treatment groups (P < 0.01 for Group A vs. Group C and P < 0.001 for all other comparisons involvling Group C). Although the flat and total number of fibroblasts were significantly higher in Group B than in Group A (P < 0.001), the difference in the number of round fibroblasts between these groups failed to reach statistical significance at P < 0.05 [Table 6]. SEM analysis of the laser treated specimens revealed no damage to the root surfaces.

Table 6

Pair-wise comparisons with Mann-Whitney tests

DISCUSSION

Over the years, several methods have been used to condition the root for regeneration the latest of them being the lasers owing to its versatile application in the field of medicine and dentistry. Extensive claims have been made for the pulse Nd: YAG laser, which include efficacy in calculus removal,[10] pocket curettage,[1112] pocket depth reduction[13] and treatment of dentinal hypersensitivity. Furthermore, laser treatment has been shown to reduce the amount of the bacterial population in the periodontal pocket according to a study by Ben Hatit et al.[14] Yamaguchi et al.[15] have also shown in their study that laser therapy removes lipopolysaccharide from root surfaces. These studies support the hypothesis that the laser represents a useful tool in periodontal treatment. In contrast to the favorable effects of laser treatment shown in these studies, several questions remains unresolved such as the ability of the fibroblasts to attach to and grow on laser – treated root surfaces, resulting in periodontal healing. The present study has been conducted to evaluate the fibroblast attachment to root surfaces treated by Nd: YAG laser.

Scanning electron microscope observation was analyzed for the fibroblast attachment and could be a measure of biocompatibility of laser – treated surfaces.[1617] Different methods have been used for in vitro assessment of fibroblastic attachment to the root surfaces which includes the qualitative method for the presence of attached cells like staining the root surfaces with different dye as trypan blue[1819] Giemsa stain or methylene blue.[20] The quantitative method involves cell counting after trypsinization and counting the attached cells on photomicrographs produced by SEM[17] which was utilized in the present study. Flat and round fibroblast cells were counted separately and also combined for the total number of fibroblasts. The flat fibroblast with their tightly attached lamellipodia represents the healthy fibroblast while the round fibroblasts with fewer attachment processes were the immature fibroblasts.[21] The results of the present study showed that the lowest numbers of fibroblast attachment were observed in the control group, which could be explained by the presence of contaminating bacterial endotoxins on the control specimens. This was in accordance to the studies by Aleo et al.[18] who demonstrated in their in vitro study that the diseased root portions had minimal cell attachment when compared to the uninvolved root areas. In the SRP group, significantly more fibroblasts were found compared to the control samples. Furthermore, the number of flat fibroblasts was significantly more than in the control group. The reason may be most probably due to a reduction in the amount of bacteria and bacterial endotoxins on the root surfaces obtained with mechanical debridement alone. Although mechanical debridement alone reduces the amount of bacteria and its endotoxins, it leaves the root surfaces covered with a smear layer which could compromise regeneration of the connective tissue attachment, hampering healing as stated by Blomlöf et al.[3] In the present study, there is a significantly higher number of fibroblasts attachment on the Nd: YAG laser/scaled treated root samples when compared with the control group and the SRP group. Again the number of flat fibroblasts was higher than either the SRP or the control group suggesting that the laser treatment combined with ultrasonic scaling could enhance fibroblast attachment on the root surfaces better than SRP treatment alone. The findings of the present study are in accordance with the previous study by Crespi et al.[22] who conducted a similar study with CO2 lasers and concluded that defocused, pulsed mode CO2 laser treatment combined with mechanical instrumentation constitutes a useful tool to condition the root surface and increase the fibroblast attachment. In the present study, the laser treated root surfaces has higher fibroblast attachment which was in agreement with the study by Crespi et al.[22] on erbium YAG (Er: YAG) laser where in the authors indicated that number of attached cells to the Er: YAG laser treated specimens was significantly higher than the control and the ultrasonically treated surfaces.

The mean output of 4 W, 80 mJ/pulse, 50 Hz used for this study was decided on the basis of the studies done by Tewfik et al.[23] where in the authors concluded that the energy level of 4 W, 1 s resulted in the modification of the cementum surfaces that supported cell growth and a study by Qadri et al.[24] who used Nd: YAG laser at 4 W, 80 mJ/pulse and reported a significant improvement in the treatment of periodontal inflammation in combination with SRP. A study by White et al.[25] observed that powers ranging from 0.3 to 3.0 W should not cause a damaging rise in the intrapulpal temperatures. Furthermore, Spencer et al.[26] stated that 4 W for Nd: YAG laser is safe and does not have damaging effects on the root surfaces. Thus, these reports suggest that Nd: YAG laser treatment with an adequate power setting could condition root surfaces without inducing damages or morphological alterations resulting in connective tissue attachment, which was also noted in the present study. Studies by Israel et al.[27] Spencer et al.[26] and Yamaguchi et al.[15] have come to a conclusion that treatment with Nd: YAG laser and ultrasonic scaling resulted in significant improvements in clinical parameters as compared to the baseline values. Several authors have pointed out the bactericidal effects of Nd: YAG laser and its application to detoxify root canal walls as well as the cementum root surfaces. Cobb et al.[5] in their study exhibited that Nd: YAG laser, with an energy ranging from 150 to 87.5 mJ/pulse showed a bactericidal effect suppressing and eradicating putative periodontal pathogens. Furthermore Moritz et al.,[28] Ben Hatit et al.[15] have shown that mechanical instrumentation associated with laser treatment of periodontally involved root surfaces could suppress periodontal pathogens such as Actinobaccillus actinomycetemcomitants, Porpyromonas gingivallis and Prevotella intermedia. Yamaguchi et al.[15] showed that Er: YAG laser treatment could effectively remove LPSs from the root surface. The above mentioned studies on bactericidal effects of Nd: YAG laser support the hypothesis that laser irradiation is a useful tool in significantly reducing bacterial endotoxins enhancing connective tissue attachment which was also the case in the present study. The results of this study is in contrast to the previous study by Trylovich et al.[17] who reported a significant decrease in the number of fibroblasts in the laser treated. Specimens compared with nonlased and control group. The authors of this study found that Nd: YAG laser treatment modifies biocompatibility of the root surfaces and reduced the number of attached fibroblasts when compared to the untreated controls and a endotoxin treated group. This difference between the present study and Trylovich et al. study can be explained on the basis of different experimental protocols followed by the two studies. As Trylovich et al.[17] evaluated Nd: YAG laser treatment on unerupted third molars, the root segments did not show any of the root surfaces modifications associated with naturally occurring periodontal disease. This could also be the reason for the reduction in the absorption of Escherichia coli endotoxin to the cemental surfaces used by the authors to contaminate the root surfaces. In addition, this endotoxin did not cause any significant alteration of the fibroblast attachment. In the present study, the experimental specimens were collected from periodontally diseased teeth which were naturally contaminated by the bacteria and their endotoxins. In a study by Trylovich et al.[17] the SEM analysis showed surface alterations such as charring, crater formation which could be due to the higher energy levels used, also the experimental root segments were not exposed to any mechanical instrumentation after the laser treatment resulting in altered biocompatibility due to possible contaminants, which was in contrast to the present study showing no damages to the root surfaces. Furthermore, the specimens were scaled after laser treatment to remove any possible contaminants on the root surface. The results of this study demonstrated fibroblast attachment to the root cementum surface suggesting that laser treatment combined with mechanical instrumentation could be considered as a useful tool to condition the root surfaces thus enhancing connective tissue reattachment. However, since this study was carried out in vitro in controlled environment further more extensive in vivo studies with our study parameters are necessary to confirm and validate these in vitro observations as laser therapy has been considered an important tool in improving the treatment of periodontal disease.

CONCLUSION

Hence within the limits of the present study it can be concluded that Nd: YAG laser irradiation at 4 W, 80 mJ/pulse can be used as a useful tool to condition the root surfaces, enhancing fibroblast attachment and there by aiding in re-establishment of the connective tissue attachment to the root surfaces of previously diseased teeth. In conclusion, further in vivo studies are to be done focusing on the increase in sample size with laser instruments capable of generating minimal energy levels with special delivery tips and calibrated devices to standardize the angle and constant laser exposure on the sample. Further research and clinical experimentation must be undertaken to substantiate the findings of the present study, which may in the future determine a significant place for lasers in periodontal surgery as being effective or more effective than conventional instrumentation.