Low-level laser therapy improves peri-implant bone formation: resonance frequency, electron microscopy, and stereology findings in a rabbit model

Abstract

Previous studies have reported positive effects of low-level laser therapy (LLLT) on bone healing. This study evaluated the effects of LLLT on peri-implant healing in vivo. Thirty-two rabbits had their mandibular left incisors removed, followed by immediate insertion of a dental implant into the fresh socket. Animals were assigned randomly to four groups: control (non-irradiated) or LLLT at three different doses per session: 5 J/cm 2 , 10 J/cm 2 , and 20 J/cm 2 . A GaAlAs laser (830 nm, 50 mW) was applied every 48 h for 13 days, starting immediately after surgery. The implant stability quotient (ISQ) was measured using resonance frequency analysis upon implant insertion and immediately after death, 30 days after the last application. Tissues were prepared for scanning electron microscopy (SEM) and stereology. Variables measured were bone–implant contact (BIC) and bone neoformation within implant threads at three different sites. The results showed better ISQ for the 20 J/cm 2 group ( P = 0.003). BIC values were significantly higher ( P < 0.05) in the 20 J/cm 2 group, on both SEM and stereology. Bone area values were better in the 10 J/cm 2 ( P = 0.036) and 20 J/cm 2 ( P = 0.016) groups compared to the control group. Under these conditions, LLLT enhanced peri-implant bone repair, improving stability, BIC, and bone neoformation. The findings support and suggest parameters for the design of clinical trials using LLLT after implant placement.

The rationale for the use of low-level laser therapy (LLLT) relies on its ability to exert, at the cellular level, biomodulatory effects on the molecular and biochemical processes that take place during intrinsic tissue repair. Several in vivo and in vitro studies have suggested positive effects of LLLT on the tissue repair process, both in animal models and in culture media. These therapeutic effects include the following: increased epithelial and fibroblast proliferation and enhanced collagen synthesis, thus speeding the process of repair; increased potential for bone remodelling and repair; restoration of nerve function after injury; normalization of hormonal function; immune regulation; reduced inflammation and oedema; modulation and relief of pain; and improved postoperative analgesia. Even though dose is one of the most important parameters of laser therapy, the data available are not sufficient to support the design of clinical studies.

Preclinical studies have suggested that LLLT has beneficial effects on bone repair. Regarding peri-implant bone healing after titanium implant placement, previously published studies have shown more evident bone maturation and increased bone–implant contact (BIC) in LLLT-irradiated bone than in control groups. The main findings reported in the literature are summarized in Table 1 .

Table 1
LLLT protocols described in previous studies evaluating peri-implant effects.
Author Year Type of light Animal model n Wavelength (nm) Power (mW) Total dose (J/cm 2 ) No. of sessions
Dörtbudak et al. 2002 Red Monkey 5 690 100 30 5
Pinheiro et al. 2003 Infrared Rabbit 14 830 10 602 7
Khadra et al. 2004 Infrared Rabbit 12 830 150 270 10
Lopes et al. 2005 Infrared Rabbit 14 830 10 602 7
Jakse et al. 2007 Red Rabbit 12 680 75 12 3
Kim et al. 2007 Infrared Mouse 20 830 96 40.32 7
Lopes et al. 2007 Infrared Rabbit 14 830 10 602 7
Pereira et al. 2009 Infrared Rabbit 12 780 70 367.5 7
Campanha et al. 2010 Infrared Rabbit 30 830 10 602 7
Maluf et al. 2010 Infrared Mouse 24 795 120 48 6

LLLT, low-level laser therapy.

The objective of this study was to assess the local effects of LLLT on the peri-implant healing process after implant placement in the rabbit mandible, immediately after mandibular incisor extraction, based on resonance frequency analysis (RFA), BIC, and bone neoformation area (BA) within implant threads, measured using scanning electron microscopy (SEM) and stereological analysis.

Materials and methods

Animals

The study sample comprised 32 male New Zealand rabbits ( Oryctolagus cuniculus ), weighing 3–4 kg and aged 3 months. The animals were allocated randomly to one of four different groups, with eight in each: three experimental groups treated with LLLT at different energy densities (5 J/cm 2 , 10 J/cm 2 , and 20 J/cm 2 ) and one non-irradiated control group. All animals received a solid diet and water ad libitum throughout the experiment and were housed under normal lighting, humidity, and temperature conditions in a climate-controlled environment. All animals underwent extraction of the mandibular left incisor followed by immediate placement of a dental titanium implant in the fresh socket.

Surgical protocol

Animals were anesthetized by intramuscular injection of ketamine hydrochloride (40 mg/kg) and xylazine hydrochloride (3 mg/kg). The area around the mandibular left incisor was prepared with 2% chlorhexidine digluconate and local infiltration of 0.5 ml lidocaine hydrochloride 2% with epinephrine 1:100,000. The mandibular left incisor was extracted with the aid of #5 paediatric extraction forceps. The fresh extraction socket was then drilled gradually, and a dental implant (3.25 mm diameter × 11.5 mm, NanoTite; BIOMET 3i, Florida, USA) placed in accordance with the manufacturer’s instructions. Implant stability was measured using RFA, followed by placement of a cover screw. The socket was sutured with 4–0 nylon monofilament. While the animal was still under anaesthesia, the site of laser irradiation was shaved and the long axis of the implant marked on the skin with a surgical marker. At the end of the procedure, animals received analgesia and antimicrobial prophylaxis ( Fig. 1 ). Perioperative procedures were performed by a veterinary physician. The authors performed the surgeries and LLLT procedures.

Fig. 1
Experimental surgery, low-level laser therapy (LLLT), and resonance frequency analysis (RFA) procedures. (A) Extraction of the mandibular left incisor. (B) Surgical aspect of the intact socket walls. (C) Implant insertion. (D) Operative view after complete implant insertion. (E) LLLT hand-piece tip held medial and lateral to the long axis of the implant during application. (F) RFA (inset) just after implant placement.

LLLT irradiation

Spot laser irradiation was performed using a gallium–aluminium–arsenide (GaAlAs) active medium infrared diode laser (wavelength 830 nm, power 50 mW), in continuous emission mode (Thera Lase; DMC Equipamentos, São Carlos, SP, Brazil), applied every 48 h over a 13-day intervention period for a total of seven applications. The first session was started immediately after surgery.

Energy density varied among the groups. The laser was applied holding the hand-piece perpendicular to the basal bone of the mandible. Animals in the 5 J/cm 2 experimental group received two spot doses of 2.5 J/cm 2 per session, one point medial and one lateral to the long axis of the implant, as marked previously on the overlying skin, for a total dose of 5 J/cm 2 per session (index dose). Animals in the 10 J/cm 2 group received twice the index dose (5 J/cm 2 per point, for a total 10 J/cm 2 per session), and those in the 20 J/cm 2 group received four times the index dose (10 J/cm 2 per point, for a total 20 J/cm 2 per session).

Non-irradiated animals (control group) underwent sham irradiation, i.e., all the procedures performed in the experimental groups were also performed in the control group, but with the laser device unpowered ( Table 2 ).

Table 2
LLLT parameters.
Parameter/group Control 5 J/cm 2 10 J/cm 2 20 J/cm 2
Average power (mW) 50 50 50
Wavelength (nm) 830 830 830
Pulse parameters CW CW CW
Energy per point (J/cm 2 ) 0 2.5 5 10
Energy density (J/cm 2 ) 0 5 10 20
Irradiation time per point (s) 0 51 101 201
Total dose (J/cm 2 ) 0 35 70 140

CW, continuous wave; LLLT, low-level laser therapy.

Death

On day 45 of the experiment (30 days after the last LLLT session), the animals were sedated (same protocol used for the surgical procedure) and killed with an overdose of 1% propofol (1 ml/kg) and 10% potassium chloride (1 ml/kg) injection. Final implant stability measurements were obtained by RFA. The mandibular halves containing the implants were removed by dissection and fixed in 10% neutral buffered formalin.

Stability measurement

Implant stability was measured using RFA and an Osstell device (Osstell AB, Göteborg, Sweden). The operator held the tip of the hand-held probe perpendicular to a SmartPeg attached to the implant. The device was recalibrated after each measurement. Implant stability was assessed at the time of implant placement (time point 1) and 30 days after the last LLLT session (time point 2), thus providing pre- and post-LLLT implant stability quotient (ISQ) values. ISQ is determined on an ordinal scale of 1–100 units based on the resonance frequency read by the device. The mean of four ISQ measurements (mesial, distal, buccal, and lingual aspects) was used in the analysis.

Scanning electron microscopy (SEM)

Two random samples from each group were dehydrated in a graded ethanol series (50%, 60%, 70%, 80%, and 90%), embedded in heat- and chemically-activated resin, and sliced along the sagittal plane (long axis of the implant) using an annular saw. The resulting specimens were then sanded and buffed to enhance the surface for examination. Images were obtained using a Philips electron microscope (XL-30 FEG EDX; Philips, Eindhoven, the Netherlands) at 250× magnification from three distinct areas of the implant (apical, middle, and cervical thirds).

Stereology

The six remaining samples from each group were dehydrated in a graded alcohol series (70%, 80%, 90%, and 100%), followed by progressive infiltration with heat-cure resin for thin sectioning. Sections were obtained along the long axis of the implant (sagittal plane of the bone specimen) with a precision microtome set to a thickness of 30 μm. The sections were buffed and stained using toluidine blue. Images of three distinct areas of the implant (apical, middle, and cervical thirds) were captured by a light microscope coupled to a digital camera.

BIC and BA analysis

UTHSCA Image Tool 3.0 software (University of Texas Health Science Center at San Antonio, Texas, USA) was used to assess (1) BIC, expressed as the ratio of bone–implant contact to total linear surface area with potential for contact and (2) BA, expressed as the ratio of the area of newly formed bone within the implant threads to total area of possible bone formation. Both variables were measured at each third of the implant (threads 1, 5, and 9), and the mean for each of the three regions was calculated.

Statistical analysis

The Shapiro–Wilk test confirmed the normality of data distribution. Analysis of variance (ANOVA) and Tukey’s post hoc test, at a significance level of 5%, were used to evaluate differences in SEM and stereological variables. ISQ values were calculated using generalized estimating equations (GEE), at the same significance level.

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Jan 17, 2018 | Posted by in Oral and Maxillofacial Surgery | Comments Off on Low-level laser therapy improves peri-implant bone formation: resonance frequency, electron microscopy, and stereology findings in a rabbit model
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