Assessment of the Healing of Dental Implant Surgical Site Following Low-Level Laser Therapy Using Bioclinical Parameters: An Exploratory Study

Dental implants were invented to extricate patients from the effects of loss of teeth and the surrounding alveolar bone. Successful osseointegration, a prerequisite for loading an implant with a superstructure,¹  depends on the stability of implants. Primary and secondary stability play an important role in implant therapy. Primary stability is influenced by bone quality, bone quantity, implant geometry, and surgical technique. Secondary stability, a biologic phenomenon resulting from secondary bone contact by bone regeneration and remodeling, dictates the time of functional loading.

Various modalities have been used to enhance the process of osseointegration and low-level laser therapy (LLLT) has shown considerable potential in postoperative treatment targeting local bone healing.²  Laser therapy stimulates the maintenance of a cell's redox status to prevent oxidative stress, thus regulating cell growth and division.²  The propensity of LLLT toward densification of bone through osteogenesis has been proven in cases of osteodistraction.³ 

It has been reported that LLLT application accelerates the process of wound healing by enhancing fibroblast proliferation and matrix synthesis as well as by increasing neovascularization.^3,4 

Although many studies have investigated the effect of LLLT on implants, no study has used a biochemical parameter to evaluate the outcomes. Furthermore, these studies do not provide methodical evidence showing how LLLT affects implant stability and success rate in humans.^5–7  Also, satisfactory evidence was not found in a recent systematic review and meta-analysis of existing clinical studies regarding the effects of photobiomodulation therapy on implants.⁸  Hence, in our study we intended to evaluate whether LLLT improves healing of a dental implant surgical site during the osseointegration process using the osteoprotegerin (OPG) marker.

It is important to quantify implant stability at various time points and project a long-term prognosis.⁹  The use of biomarkers is an objective method of estimating the effect of LLLT on the tissues. One such biomarker is OPG, which is a physiologically important regulator in the differentiation and function of osteoclasts.^10–13  It acts to prevent the differentiation of an osteoclast precursor into a mature osteoclast. The receptor activator of nuclear κB ligand (RANKL) protein is expressed by osteoblasts, and the receptor activator of nuclear κB (RSNK) originates on the surfaces of osteoclast precursors and mature osteoclasts. RANKL binds to RANK, and this coaction is necessary for the formation, function, and existence of osteoclasts. OPG acts as a trap receptor for RANKL, and its binding prevents RANKL and RANK from interacting; thus, the function of osteoclasts is hampered.¹⁴  Hence, OPG needs further investigation as a possible biomarker of implant health status.¹⁵ 

This study was centered on the hypothesis that LLLT enhances bone healing around dental implants. Sample size was determined based on power analysis. Thirty patients (13 men and 17 women) older than 18 years were selected and provided informed written consent before enrolling in the study. The study protocol, including recruitment procedures, exclusion/inclusion criteria, and the informed consent, was approved by the Institutional Ethics Committee (registration no. ECR/262/Inst/UP/2013) and followed the principles of the Declaration of Helsinki. We included participants with a single missing tooth, healthy adjacent teeth, Simplified Oral Hygiene Index scores ranging from 0 to 1.2, and implant sites categorized under type A of Misch and Judy classification with adequate height and width of bone not requiring grafting procedures. Exclusion criteria were chronic and debilitating illnesses, treatment with steroids and other medications (eg, bisphosphonates) that alter bone metabolism, pregnancy, bleeding disorders, and history of smoking.

Participants were randomly assigned to 1 of 2 groups using the coin-toss randomization method. The test group consisted of participants who underwent laser biostimulation of the surgical site, and the control group comprised participants who did not undergo any intervention.

The preoperative investigations conducted were orthopantamogram, 3-dimensional cone-beam computed tomography of the edentulous space and routine blood investigations (hemoglobin percent, bleeding time, clotting time, random blood sugar, total leukocyte count, differential leukocyte count, hepatitis B surface antigen, human immunodeficiency virus viral markers).

All the implants were placed by a trained and calibrated investigator to avoid bias. A full-thickness mucoperiosteal flap was raised under local infiltration of 2% lignocaine anesthetic solution (LOX 2% adrenaline; Neon Laboratories Ltd, Mumbai, India), and the osteotomy site was prepared to receive an implant of specific dimensions. A nonsubmerged healing protocol was followed. After implant placement, the gingival former was placed, and the mucoperiosteal flaps were adapted and sutured around it. Postoperative medication included amoxicillin (500 mg/8 hours) for 5 days. Nonsteroidal anti-inflammatory agents were prescribed for postoperative analgesia. All the participants were instructed to rinse with 0.12% chlorhexidine digluconate solution twice daily for 2 weeks.

The neodymium-doped yttrium aluminum garnet (Nd:YAG) laser application was carried out by another investigator who was blinded to the allocation of participants to the test or control groups. The participant was blinded to the intervention. Pulsed Nd:YAG laser beam of wavelength 1064 nm (Fotona lightwalker dental laser system, Ljubljana, Slovenia) was applied over the implant site in a mesial-distal direction for 60 seconds by using an R-24 hand piece (Figures 1 and 2). The pulse-repetition rate was 30 Hz. The laser was used at maximum energy of 2 J with an energy density 0f 7.07 J/cm² over each implant during each session.^5,6,16–20  The total energy applied to each implant at the end of the study was 28 J/cm². The participants allocated to the test group were treated with laser beam irradiation at baseline, 2 weeks, 6 weeks, and 3 months after implant placement. In the control group, the application of laser energy was at a power of 0 J/cm² (no irradiation).

The healing around dental implants was assessed using modified plaque index, modified sulcus bleeding index, probing depth, and radiofrequency analysis²¹  measured after 2 weeks, 6 weeks, and 3 months of implant placement. The outcome evaluation for clinical parameters was performed by a single calibrated investigator. Probing depth was assessed by inserting a standard periodontal probe with a point diameter of 0.5 mm using a probing force of 0.5 N. Wireless radio frequency analysis (Ostell implant stability quotient [ISQ]) was used in this study. A metal rod (peg) was connected to the implant by means of a screw connection (Figure 3). The magnetic attachment on top of this peg was excited by magnetic pulses from a handheld computer.²² 

An unstimulated saliva (5 mL) sample was collected into a pre-labeled sterile container using the resting drooling technique. The samples were centrifuged, and the supernatant was transferred into a sterile Eppendorf tube and appropriately labeled. After processing, all the samples were stored at –80°C until batch analysis. The first saliva sample was collected just before implant surgery to record the baseline data. Subsequent samples were collected at 2 weeks, 6 weeks, and 3 months after implant placement. All the samples were collected between 9 am and 12 pm to minimize diurnal variations associated with saliva sampling.²³ 

The human OPG enzyme-linked immunosorbent assay (ELISA) kit (Qayeebio, Shanghai Qayee Biotechnology Co) was used to estimate OPG in the saliva samples. The depth of color change in the sample mainly depends on the concentration of OPG. The methodology and analytic procedures in the study were reviewed by an independent statistician.

SPSS v20 (SPSS Inc) was used to analyze the data. Paired samples t-test was used to compare observations in the 2 groups. Shapiro-Wilk's test was used to test the null hypothesis that the data are normally distributed.

All the results and the analytic interpretations were reviewed by an independent statistician. Thirty volunteer participants were assigned to the control or the test group. The modified plaque index scores were lower in the test group than the control group, although the difference was not statistically significant (P < .05). The score gradually reduced from 2 weeks to 3 months (Table 1). The probing depth values were significantly lower in the test group at 6 weeks and 3 months as compared to the control group (P < .01). (Table 1).

The test group showed lower modified sulcus bleeding index scores than the control group. The difference was not statistically significant; however, the score was markedly decreased during the 6-week and 3-month follow-up in the test group (Table 1). There was a significant difference in ISQ (P < .01) between the control and test groups at 2 weeks, 6 weeks, and 3 months (Table 1). After 6 weeks of follow-up, 80% of implants in the test group achieved medium stability (60–69 ISQ) and 20% showed high stability (>70 ISQ). On the other hand, all the implants in the control group showed low stability after 6 weeks of follow-up (Table 2).

The OPG level in the control group reached its peak value at 2 weeks compared with the baseline value, followed by a decline at 6 weeks and a statistically significant decline at 3 months (P < .001). In the test group, the salivary OPG levels increased at 2 weeks, followed by a further rise at 6 weeks and a statistically significant (P = .045) peak at 3 months. The OPG levels showed a significant difference (P < .001) between the test group and control group at 3 months, while no difference was observed at baseline, 2 weeks, and 6 weeks (Table 3; Figure 4).

The present study was planned to investigate the healing around the dental implant with LLLT by using OPG as an important biochemical parameter. The results showed that LLLT improved clinical and biochemical parameters.

The quantitative assessment of biomarkers can be done using gingival crevicular fluid (GCF) or saliva samples. Contamination of GCF samples by blood, saliva, or dental plaque affects the accuracy in volume determination and composition of GCF.²⁴  Prolonged time taken in collecting GCF will change the protein concentration of the initial GCF collected.²⁵  A limited quantity of GCF sample collected results in significant error in evaluating the biomarker.²⁶  These limitations can be overcome using saliva as the sample of choice. Owing to its ease of collection, repeatability, and noninvasive sample-collection technique, saliva was chosen to assess the OPG levels in this study.

We used LLLT in the study due to its prominent anti-inflammatory effects on the gingival and periodontal tissue by reducing endothelial activation, inhibiting leukocyte extravasation into the gingival stroma and inactivating bacterial lipopolysaccharide.^27,28 

It is well established that the effect of photobiomodulation depends on various parameters like wavelength, mode, energy density, exposure time, and frequency of treatment.¹⁷  Various animal studies have successfully used laser energy density in the range of 4–7 J/cm².²  Significant improvement in implant stability, osteocyte viability, expression of RANKL, and OPG was observed in studies that used energy density of 6–7 J/cm².^4,6,17  A Nd:YAG laser was not found to be used predominantly in any of these studies. Although many studies have demonstrated successful use of higher densities of laser power, in an attempt to achieve maximum efficacy with lower power, Nd:YAG laser of energy density 7.07 J/cm² was used in our study.

A considerable reduction in the plaque index, sulcus bleeding index, and probing depth scores around the implants corresponds to a significant reduction of peri-implant pathogenic microbes and diminished expression of proinflammatory cytokines with LLLT. In addition, LLLT has been used as an adjunct to mechanical debridement in the treatment of peri-implant diseases, resulting in faster soft-tissue healing.²⁹  The expression of OPG notably increased and was maintained in healing peri-implant tissues with LLLT. Previous studies have also shown that LLLT resulted in a remarkable increase in the expression of OPG and expansion of metabolic activity of bone cells.^17,19 

Higher ISQ in the test group can be associated with the elevated salivary OPG levels in the test group. Hence, LLLT was shown to improve implant stability by enhancing OPG expression. Animal studies have also shown improvement in bone volume, connective tissue density, osseointegration, and reduction in trabecular separation under the influence of OPG.^5,6  High initial stability in the test group may have resulted in reduced micromovement and an accelerated rate of osseointegration. Studies in human participants have shown a positive influence of LLLT on osseointegration in posterior maxilla⁶  and no effect on ISQ in the posterior mandible.⁵  These inconsonant results may be due to the difference in the density and architecture of bone in different areas of maxilla and mandible. Our study was not restricted to a specific area of the jaw. The quality of bone (whether D1, D2, D3, or D4) was not standardized. The influence of LLLT on different areas of the jaw is an independent research problem that is beyond the scope of this study. Hence, further site-specific research should be done to attain decisive results.

The present study suggests that laser treatment of implant sites favorably influences the healing of soft tissue and osseointegration. Improved OPG levels in the test groups might also indicate the early stability and functional loading of implants. Hence, our findings might be a positive impetus to further research in this arena.

Dispute being one of the imperative studies to evaluate the healing of soft and hard tissues under the influence of LLLT, the study has some limitations; it was undertaken in a restricted population confined to a single geographic area. This limits the generalization of the results to a larger diverse population.

A split-mouth study design should have been used to eliminate the confounding factors. As the study was carried out for a short period of 3 months, response of the bone–implant junction to laser therapy in the early loading period was not evaluated. The study did not include participants with medically compromised conditions, such as osteoporosis and diabetes mellitus, which require the assistance of such therapies to improve osseointegration. Hence, studies considering compromised medical situations should be conducted and the advantages of lasers evaluated in these situations. Since the study was not site specific, the variability of bone morphology and microstructure in different areas of the jaw was not considered.

Further long-term studies should be undertaken with larger sample sizes representing varied population to effectively generalize the results of the study in a broad manner. Future studies should explore the efficacy of LLLT based on different powers, energy densities, wavelengths, irradiation times, and dosage. Hence, randomized controlled trials should be conducted to help establish a consistent protocol with different types of lasers used for photobiostimulation. Additionally, the benefits of therapeutic modalities like magnetic energy should be investigated to reduce the duration of healing.

Certain factors impede the application of these study findings to clinical practice. The operator must be familiar with the dental laser system and needs definitive training. Additionally, batch analysis of the saliva samples was done using a 94-well ELISA kit, which is neither economical nor convenient for evaluating salivary OPG levels for a single patient at different follow-up intervals in clinical practice. Fundamentally, research should also be directed toward convenient application of the results attained in these studies to clinical practice.

Within the limitations of the study, it can be suggested that the healing of peri-implant hard and soft tissue may be enhanced with the use of LLLT as an explicit modality during the postoperative period. Enhanced osseointegration and early implant stability can be achieved for loading, thus shortening the time required for conventional loading. The biomarker OPG can be used as a diagnostic assay to determine progress in bone healing at the cellular level. To ascertain the exact effect of laser therapy on healing of peri-implant hard and soft tissues, further large-scale trials will be required on a different population with a sound sample size.

Abbreviations

CBCT:

cone beam computed tomography

ELISA:

enzyme-linked immunosorbent assay

GCF:

gingival crevicular fluid

ISQ:

implant stability quotient

LLLT:

low-level laser therapy

Nd:YAG:

neodymium-doped yttrium aluminum garnet

OPG:

osteoprotegerin

RANK:

receptor activator of nuclear κB

RANKL:

receptor activator of nuclear κB ligand

TNF:

tumor necrosis factor

We thank Dr A. R. S. Bhatt, Dr Pushpa Bharati, and Dr Y. B. Palled (University of Agricultural Sciences, India) for assistance in statistical analysis. We thank Dr S. B. Javali and HealthMinds private limited (KLE Academy of Higher Education & Research, India) and Chetan A. G. for useful discussions and comments.

The authors declare that they have no conflict of interests.