Effect of Low-Level Laser Therapy on Bone Regeneration During Osseointegration and Bone Graft

Abstract

Background: The effect of low-level laser therapy (LLLT) on bone regeneration during osseointegration and bone graft is very controversial. Despite many positive reports of in vitro and in vivo studies and more than 50 randomized clinical trials claiming a positive effect of photobiomodulation (PBM), many reports found no significant effect of lasers. Objective: The aim of this study was to evaluate studies correlating PBM and bone regeneration and to assesses parameters that produce positive results based on dose and output power used. Materials and methods: Four electronic databases were used: PubMed, Springer, Google Scholar, and Cochrane. Results: The research yielded 230 articles. The full texts of all articles were evaluated and scored using eligibility criteria adapted from Cericato et al. After evaluation, only 19 articles met the inclusion criteria. Conclusions: A positive effect of low-level laser energy on bone regeneration within a certain relationship between dose and output power was found. LLLT stimulates cellular metabolism, increasing protein synthesis and subsequent bone regeneration. A high dose combined with low power or a low dose combined with high power appears to produce a positive effect.

Introduction

Phototherapy is the use of light to influence tissue, and it can be accomplished through two distinct techniques: Combining light with a photosensitizing dye in a procedure called photodynamic therapy or use of the light alone in a procedure called low-level laser therapy (LLLT).¹ In recent years, the term photobiomodulation (PBM) is preferred since it suggests influence on cellular metabolism as a result of applied photonic energy.

De Freitas and Hamblin² state that LLLT refers to the use of light in the red or near-infrared region (NIR) with wavelength usually in the range from 600 to 700 nm and from 780 to 1100 nm. A wavelength range between 700 and 780 nm has been found to be ineffective as it coincides with a trough in the absorption spectrum of cytochrome c oxidase.³ Moreover, red/NIR wavelengths are chosen because the penetration through tissue is maximal in this range, due to lower scattering and absorption by tissue chromophores.

The laser or light emitting diode (LED) used for this application typically has an irradiance or power density between 5 and 50 mW/cm². Continuous wave or gated energy has been used at a relatively low energy density (0.04 − 50 J/cm²), and output power can vary widely from 1 to 500 mW.²

In PBM, energy density or irradiance is lower than in other laser uses such as ablation and thermal coagulation.⁴

Application of incorrect parameters of fluence (J/cm²), irradiance (mW/cm²), and delivery time or repetition rate can lead to ineffective treatment. A biphasic dose response or hormesis phenomenon follows the Arndt-Schultz Law, which states that weak stimuli slightly accelerate vital activity; stronger stimuli raise it further until a peak is reached; and even stronger stimuli suppress it until a negative response is achieved.⁵ Studies suggest that the biological effects of laser energy and LED energy are the same when the wavelength is properly chosen.⁶

Phototherapy at the cellular level can be classified into primary, secondary, and tertiary light-induced effects.^7,8

-A primary effect is restricted to photon absorption. Photons emitted from the laser reach the mitochondria of cells, are absorbed by chromophores (cytochrome, porphyrins, and flavoproteins), and are finally converted into chemical energy in the cells. A cascade of signals between mitochondria, nucleus, and oxidative metabolism leads to an increase in adenosine triphosphate (ATP) production, causing pain relief and wound healing.⁹

– A secondary effect is the result of photonic stimulation, amplifying the primary effect and increasing calcium production.

– A tertiary effect is systemic and occurs at a distance from the stimulus.⁸ Thus, laser energy applied to one lesion can stimulate the healing of both the treated lesion and other lesions at a distant location.

There is no agreement for a mechanism of action of PBM. However, a molecular mechanism of PBM proposed by De Freitas and Hamblin in 2016 is attractive.²

Molecular mechanism of PBM

Cytochrome c oxidase (Complex IV of the mitochondrial membrane) is the first photo-acceptor for red and NIR wavelengths.¹ The absorption spectra for light that produce a biological response are very similar to the absorption spectra of cytochrome c oxidase.³ COX is the terminal enzyme of the electron transport chain mediating the electron transfer from cytochrome c to molecular oxygen. Ox acts as a photo-acceptor and transducer of photo signals in the red and NIR regions of the light spectrum.² PBM increases the mitochondrial membrane potential, increases the level of adenosine triphosphate, c-AMP (cyclic adenosine monophosphate), and ROS (reactive oxygen species). In addition, PBM increases the availability of electrons for the reduction of molecular oxygen in the catalytic center of COX.

This suggests that light is absorbed by the chromophore (cytochrome c oxidase), followed by a photo-dissociation of NO (nitric oxide) from COX. As a consequence, the activity of the mitochondria is inhibited and reversed due to the excessive NO binding (NO photodissociates from the heme iron and copper center of COX).²

The mitochondrial alteration function allows the oxygen to come to the site and, because there is communication between mitochondria and nucleus, this will cause an increase in enzymatic activity, electron transport, and ATP production due to alteration of mitochondrial activity.²

Numerous signaling effects occur due to the change in mitochondria, including: an increase in ATP, an alteration in CA concentration, and an increase in c-AMP as a direct consequence of the rise in ROS that leads to the activation of NF-κB. (NF-κB has an important function in the nucleus as it combines with the DNA and the production of NO.)

Activation of transcription factors

(1) NF-κB is a protein complex that is responsible for DNA transcription. An increase in NF-κB leads to enhanced cell transcription that leads to cell proliferation and reduced cell death.²

(2) RANKL (receptor activator of nuclear factor Kappa-B ligand) is a transmembrane protein that is involved in bone regeneration and remodeling, and it also acts as a ligand for osteoprotegerin (OPG). OPG is a secretory soluble receptor and an inhibitor of the RANK receptor.

The RANKL/OPG ratio is an important factor that is used for studying or determining whether bone is removed or formed. During the remodeling process, there is an increase in RANKL production by osteoblasts, which bind to RANK receptors and cause osteoclast progenitor expansion, activation, and fusion into multinucleated osteoclasts.

(3) Hypoxia-inducible factor (HIF-1α): This type of protein is involved in cellular adaptation to hypoxia. It is oxygen dependent and, during PBM, this protein is rapidly activated through two mechanisms. First, it is possible that this protein is mediated by MarK (mitogen-activated protein kinase) and PI3K (phosphatidyinositol 3 kinase). Second, light activation of CX causes oxygen depletion followed by a rapid activation of HIF-1α.

(4) AKt/GSK3β/β: Protein Kinase is activated by PBM, causing a decrease in the activity of GsK 3 (glycogen synthase kinase 3) and inhibiting Bax translocation and the pro-survival action of β catenin.

Other transcription factors that are activated by LLLT are as follows:

(5) AKt/m TOR/cycling pathways F-ERK/FOXM1G-PPaR: H-RUNX2

The effect of LLLT on bone regeneration during osseointegration and bone graft is still not clear. Despite many positive reports of in vitro and in vivo studies and more than 50 randomized clinical trials claiming a positive effect of PBM, many reports found no significant laser effect.

The aim of this study was to evaluate studies correlating PBM and bone regeneration during either osseointegration or bone graft and to assess parameters that produce positive results based on dose and output power used.

Materials and Methods

The present systematic review was conducted by following PRISM (Preferred Reporting Items of Systematic reviews and Meta-analysis).

Research strategy for article identification

Research was conducted on the following electronic databases: Springer, PubMed, Google Scholar, and Cochrane Databases.

Keywords used: LLLT and bone regeneration, LLLT, PBM, LLLT and osseointegration, LLLT and bone graft, and LLLT and cells.

After collecting the data, the titles, abstract, and conclusion were read and unrelated and obviously biased articles were excluded. Also, all case reports and literature reviews were excluded. Only studies dating from 2007 to 2016 were included. Reasons for exclusion are shown in Table 3.

Table 3.

Criteria for Exclusion After Initial Review

Reason for exclusion	PubMed	Springer	Google scholar	Cochrane	Total
Literature and/or systematic review	10	8	11	6	35
Article in language other than English	—	—	15	—	15
Letter from the editor, opinion articles	—	—	8	—	8
Energy density not mentioned	3	2	30	10	45
Use of high energy density	4	2	8	8	22
Article not mentioning either power or power density	5	2	24	6	37
Other (book chapter, appendix, bibliography, index)	—	—	4	2	6
Total exclusion	22	14	100	32	168

Evaluations of articles were performed by two reviewers independently. Fig. 1 shows how the final group of 19 studies was selected.

FIG. 1.

Flowchart of article selection.

Assessment of the studies

After obtaining the full texts, all articles were evaluated and scored by following the checklist described in Table 1. Articles with scores from 0 to 5 points were considered to be of a low quality and were excluded; those with scores from 6 to 8 were considered to be of a moderate quality; and those with scores from 9 to 11 were considered to be of a high quality. Main characteristics of included studies are shown in Table 2.

Table 1.

Eligibility Criteria Adapted from Cericato et al. ⁶ for Inclusion of Studies

Adequate study design	3 points
Keyword inserted in Mesh	1 point
Objective of the study is clear and precise	1 point
Adequate sample selection process and sample losses	1 point
Adequate sample size	1 points
A statistical test is described	1 point
p values are calculated	1 point
Objective and clear results discussion	1 point
Method used for error analysis	1 point

Table 2.

Main Characteristics of Articles Included in the Study

Article	Score	Sample	Fluence (J/cm ² )	Power (mW)
Lopes et al.¹³	10	14 Rabbits	21.5	10
De Almeida et al.¹⁴	9	60 Rats	4.9	35
Gomes et al.¹⁵	10	32 Rabbits	5, 10, 20	50
Mandic et al.¹⁶	10	12 Patients	6.26	42
Maluf et al.¹⁷	10	24 Mices	8	120
Menezes et al.¹⁸	10	20 Dogs	4	40
Marques et al.¹⁹	10	24 Rats	16, 3.7	50
Garcia et al.²⁰	10	8 Patients	92	86
Kim et al.²¹	10	13 Rabbits	49.8 mJ/cm²	100
Rasouli Ghahroudi et al.²⁹	9	12 Rabbits	4	300
Pinheriro et al.³⁰	10	24 Rats	4	40
Soares et al.³¹	9	24 Rabbits	4	70
de Vasconcellos et al.³²	10	56 Rats	4	40
Carneiro et al.³³	10	48 Rats	4	40
Marquez-Martinez et al.³⁴	10	24 Rats	4	40
Gerbi et al.³⁵	10	48 Rats	4	40
Pinheiro et al.³⁶	10	54 Rats	4	40
Fekrazad et al.³⁷	10	58 Rabbits	4	200
Kim et al.³⁸	10	14 Rabbits/14 rats	344	3 W

Introduction to LLLT and Osseointegration

Osseointegration is a direct structural and functional contact between a loaded implant surface and bone at the light microscopic level.¹⁰ An implant is osseointegrated when there is an absence of progressive movement between the bone and the implant.¹¹ Early failure of the implant occurs before placement of the restoration. Possible reasons are improper wound healing, infection, or mobility. Late failure occurs after prosthetic treatment and may be due to overloading or infection.¹²

The effect of LLLT on osseointegration is still controversial. Some studies assert a positive contribution of laser energy on bone, whereas others claim no effect. All the studies were divided into four groups: High dose/low power, low dose/high power, high dose/high power, and low dose/low power. More than 16 J/cm² per session is considered high dose, whereas more than 35 mW is considered high power.

Each study will be described in some detail since parameters and research protocols varied widely. Knowledge of these differences is vital to understanding the summary tables.

Review of LLLT and osseointegration

Lopes et al.¹³ assessed through Raman spectroscopy the effect of diode laser 830 nm on the osseointegration of implants in 14 rabbits: All received titanium implants with 6 acting as controls, whereas 8 were irradiated with laser energy by using the following parameters: 10 mW, 21.5 J/cm² per point, 86 J per session, spot area 0.0028 cm², and seven sessions at 48 h intervals. The animals were sacrificed at 15, 30, and 45 days and were prepared for Raman spectroscopy and scanning electron microscopy (SEM). Results showed a significant difference in the concentration of calcium hydroxyapatite CHAin irradiated and the control group (p = 0.001). It was concluded that infrared laser irradiation improves bone healing.

De Almeida et al.¹⁴ performed an in vitro study in rats to evaluate the influence of LLLT on osseointegration. Titanium implants were placed in the tibia of 60 rats. Of these, 30 underwent laser treatment before implant placement by using a GaAlAs (Gallium-Aluminum-Arsenide) laser: 660 nm, 35 mW, CW, and energy density of 4.9 J/cm², for 4 sec. Ten animals from each group were euthanized after 15, 30, and 60 days. Histological and immunohistochemical analysis was performed by using optical microscopy. Histological and quantitative analysis shows an increased amount of multinucleated tartrate-resistant acid phosphatase-positive cells in the lased group (p < 0.05). It was concluded that LLLT accelerated the osseointegration via stimulation of both osteoblastic and osteoclastic activity.

Gomes et al.¹⁵ evaluated the effect of LLLT on peri-implant healing. A total of 32 rabbits received immediate dental implantation after mandibular incisor extraction. The animals were divided into four groups: a control group or one of three LLLT groups at different doses: 5, 10, and 20 J/cm² by using a semiconductor GaAlAs 830 nm, 50 mW every 48 h for 13 days for a total of five sessions. Resonance frequency analysis (RFA) and SEM showed that the bone implant contact (BIC) was significantly higher in the 20 J/cm² group, with significant improvement in implant stability quotient (ISQ). Bone area values were better in the group receiving 10 J/cm² (p = 0.036) and 20 J/cm² (p = 0.0016).

Mandic et al.,¹⁶ using a split-mouth design, placed self-tapping implants in 12 patients to investigate the influence of LLLT osseointegration on self-tapping implants placed into low-density bone. For each patient, one jaw randomly received LLLT and the other side of the same patient was treated as a placebo. The laser used was a 637 nm GaAlAs, with an output power of 40 mW, CW, and a total irradiation of 6.26 J/cm² per implant. Laser treatment was performed immediately after implant placement and repeated every day for 7 days. The outcome was assessed for implant stability, early implant success, and ALP (alkaline phosphatase) weekly from the first to the sixth week. Throughout this period, irradiated implants achieved higher stability than nonirradiated ones. However, the difference was not significant until the fifth postoperative week.

Maluf et al.¹⁷ randomly divided mice into control and laser groups. In the laser group, the bone-implant interface was subjected to LLLT by using a semiconductor laser 795 nm, CW, power of 120 mW, 8 J/cm² per point, six laser applications with an interval of 2 days, and a total dose of 48 J/cm². The result suggested faster and better bone-implant integration than the control.

Menezes et al.¹⁸ assessed the PBM action of LLLT on peri-implant bone healing of dental implants in the tibia of dogs. Ten dogs served as controls and received implants only in the tibia, with the other 10 dogs receiving implants with laser therapy: GaAlAs 830 nm, power of 40 mW, irradiation interval of 48 h for 2 weeks, 4 J/cm² at five points, CW, Thera laser, which applied 20 J/cm² per session distributed at 5 points of 4 J/cm², and for a total dose of 140 J/cm². After 15 and 30 days, all the specimens were removed and submitted to EDXRF tests (energy-dispersive X-ray fluorescence) to assess the concentration of calcium. At 30 days, the lased group showed a significant increase in calcium concentration compared with the control group (p < 0.05).

Marques et al.¹⁹ proposed and compared a new protocol of LLLT for bone regeneration on 45 rats. After creating a bone defect of 8 mm in the skull, they divided the animals into three groups of 15 rats. Group I was treated with LLLT: transcutaneous applications into four points around the defect, fluence 16 J/cm², power of 50 mW, duration of application 9 sec, and 1 mm distance, applied every 48 h for 15 days. Group II received LLLT by using three applications—during the surgery before closing with sutures, directly into the defect in four points (same points as Group I), and directly to the skin. The parameters used were as follows: fluence 3.7 J/cm², power 50 mW, and time of application 3 sec; then, two other applications were made after 48 and 96 h transcutaneously. Group III was the control group and was not submitted to laser irradiation. Histomorphometric analysis, an immunohistochemistry study, and microscopic evaluation showed significant differences between the treated and untreated groups. It was concluded that laser irradiation supports bone regeneration, and that LLLT works better in the early stages of bone formation.

Garcia et al.²⁰ performed a randomized clinical study to assess the LLLT effect on implant stability by means of RFA. Thirty implants were distributed bilaterally in the mandible of eight patients. In each patient, one side acted as a control group not receiving laser irradiation. The other group received GaAlAs 830 nm (Thera laser), 86 mW, 0.25 J, 92 J/cm², energy of 0.25 J per point, contact mode, total energy of irradiation 5 J, 3 sec/point at 20 points, and spot size of 0.0028 cm². Irradiation was initiated immediately after implant placement and repeated every 48 h for 14 days. RFA was used to determine the stability of dental implants. After 10 days, the increase in ISQ value in the irradiated group was not significantly different than the control. It was concluded that the laser has little impact macroscopically and that LLLT did not have any effect on the stability of implants.

Kim et al.²¹ investigated the LLLT effect on healing and attachment of titanium implant in the bone. Thirteen rabbits received femur implantation and were divided into control and LLLT groups by using GaAlAs 808 nm, with 100 mW output power, 830 mW/cm², and 60 sec, applied to the tissue surrounding the implants daily for 1 week. Rabbits were sacrificed in the 6th (six rabbits) and 12th weeks. Histopathologic results showed that the LLLT group, after 12 weeks, had more bone matrix and collagen around implants. Histomorphometric analysis showed that there was no significant difference in BIC between the groups, although the laser group showed higher average values. Torque tests showed significantly higher removal torque values for the lased group compared with the control (p ≤ 0.05), but only after 12 weeks. Likewise, ISQ values were significantly different between the two groups only after 12 weeks. All tests showed no statistically significant differences after 6 weeks. It was concluded that LLLT might promote implant osseointegration, but there was no statistically significant difference.

Results

Out of nine articles, using diode laser, eight found positive results with significant difference (p < 0.05). This review found, in Table 4, that when high fluence (greater than16 J/cm²) was combined with low power (less than 35 mW) and when high power (greater than 35 mW) was combined with low fluence (less than 16 J/cm²) per session, all studies showed positive results. However, when high fluence (greater than16 J/cm²) was combined with high power (greater than 35 mW), the results showed no significant difference between the irradiated and control groups.^14,15

Table 4.

Review of the Effect of Laser Irradiation on Implant Osseointegration

Author (year)	Laser type, wavelength (nm)	Subject groups	Power (mW)	Power density	Fluence	Application protocol	Assessment	Outcome	Result
Low power (<35 mW) low fluence (<16 J/cm²)
Low power (<35 mW) high fluence (>16 J/cm²)
Lopes et al. (2007)¹³	Diode (GaAlAs), 830 nm CW	Rabbits	10		21.5 J/cm² (per point)	7 Sessions at 48 h intervals 4 points/session	Raman spectroscopy SEM	Difference in the concentration of CHA improved bone healing	Positive p < 0.001
High power (>35 mW) low fluence (<16 J/cm²)
De Almeida et al. (2013)¹⁴	Diode (GaAlAs), 660 nm CW	In vitro 60 rats	35		4.9 J/cm²	4 Sec	Histological analysis	Increased amount of multinucleated TRAP	Positive p < 0.05
De Almeida et al. (2013)¹⁴	Diode (GaAlAs), 660 nm CW	In vitro 60 rats	35		4.9 J/cm²	4 Sec	Quantitative analysis	Increased amount of multinucleated TRAP	Positive p < 0.05
Gomes et al. (2015)¹⁵	Diode (GaAlAs), 830 nm CW	32 Rabbits	50		5 J/cm²	Every 48 h or 13 days	Resonance frequency SEM	BIC and ISQ was significantly higher in the 20 J/cm² group	Positive for 10 J/cm² p = 0.036)20 J/cm² (p = 0.016)
					10 J/cm²
					20 J/cm²
Mandic et al. (2015)¹⁶	Diode (GAALAs), 637 nm CW	12 Patients	40		6.26 J/cm²	Immediately and repeat every day for 7 days	Implant stability early implants success ALP (alkaline phosphatase)	Higher stability for lased group	Positive stability (p < 0.05)
Mandic et al. (2015)¹⁶	Diode (GAALAs), 637 nm CW	12 Patients	40		6.26 J/cm²	Immediately and repeat every day for 7 days		No difference in ALP activity	ALP (p > 0.05)
Maluf et al. (2010)¹⁷	Diode (GAALAS), 795 nm CW	24 Mice	120		8 J/cm²	Six laser application every 2 days	Digital torque machine	Faster and better bone-implant integration	Positive p < 0.05
Menezes et al. (2016)¹⁸	Diode (GaAlAs), 830 nm CW	20 Dogs	40		4 J/cm² each at five points	every 48 h for 2 weeks	CA Concentration (EDXRF test)	Higher CA concentration	Positive p < 0.05
Marques et al. (2015)¹⁹	Diode (GAALAS), 830 nm CW	24 Rats	50		16 J/cm²	Q 48 h for 15 days 3 sec	Histomorphometric analysis	Gr I: higher OP	Positive for Gr II p < 0.05
Marques et al. (2015)¹⁹	Diode (GAALAS), 830 nm CW	24 Rats	50		3.7 J/cm²	Q 48 h for 15 days 3 sec	Immunohistomorphometric analysis	GrII: New bone formation, higher OC, primary bone highly vascularized	Positive for Gr II p < 0.05
High power (>35 mW) high fluence (>16 J/cm²)
Garcia et al. (2012)²⁰	Diode (GaAlAs), 830 nm CW	8 Patients	86		92 J/cm²	Immediately after implant placement Q 48 h for 14 days	Implant stability ISQ value	Increase ISQ in irradiated group with no sig. dif.	(−) No difference between group (p > 0.05)
Kim et al. (2016)²¹	Diode (GaAlAs), 808 nm CW	13 Rabbits	100	830 mW/cm²	49.8 mJ/cm²	60 sec daily for 1 week	Torquing test Histomorphometric analysis	After 6 week: no difference between groups	Positive only after 12 weeks (p > 0.05)
Kim et al. (2016)²¹	Diode (GaAlAs), 808 nm CW	13 Rabbits	100	830 mW/cm²	49.8 mJ/cm²	60 sec daily for 1 week	Torquing test Histomorphometric analysis	After 12 week: higher values of torquing and ISQ for laser group	Positive only after 12 weeks (p > 0.05)

BIC, bone implant contact; EDXRF, energy-dispersive X-ray fluorescence; ISQ, implant stability quotient; TRAP, tartrate-resistant acid phosphatase.

Altan et al.²² concluded that when using 50 mW, 20 J/cm² on osseous expansion, treatment was not effective whereas when using 50 mW, 5 or 6.4 J/cm² the results showed a significant effect.

It must be noted that, using the exclusion criteria of Cericato et al.,⁶ there are no studies of either high or moderate quality that explore the low power (<35 mW) low fluence (<16 J/cm²) scenario. This shortcoming prevents drawing conclusions under these conditions.

Other studies that did not meet inclusion criteria provide further insight into the possible effects of very divergent parameters.

Schwartz-Filho et al.²³ did not find effective results when using 35 mW/cm² power density and 25, 77, and 135 J/cm² fluence. Very low energy density (less than 1 J/cm²) combined with high power density was also not effective.²⁴

Mayer et al.²⁵ as well as Massoti et al.²⁶ found that 20 J/cm², 50 mW showed better results compared with 5 and 10 J/cm², which contradicts the conclusion of this article. However, their spot area of 0.0028 cm² was much different than other studies, which could affect results.

It is suggested that LLLT increases the cellular metabolism. Photonic energy is transmitted to the nucleus, resulting in DNA and RNA synthesis, which causes protein synthesis. This could lead to bone neo-formation and resorption, which increase osseointegration.

Introduction to LLLT and Bone Graft

Bone loss is a major problem in medical and dental specialties and may be caused by physiologic, pathologic, or iatrogenic conditions. Although bone has a regenerative capacity, this capacity is limited if there is mechanical instability, deficient blood supply, or competition with highly proliferative tissue. As a consequence, a bone defect may be created that makes prosthetic rehabilitation impossible.²⁷

There are different methods to overcome this problem, including the use of a graft. This may be autologous bone, a xenograft, or the combination of graft material with an overlying membrane in guided bone regeneration.²⁸

An investigation of the influence of PBM on the success of bone grafting procedures was performed. As earlier, all studies were divided into four groups: high dose/low power, high power/low dose, high dose/high power, and low dose/low power. More than 16 J/cm² per session is considered high dose, whereas more than 35 mW is considered high power.

Again, each study will be described in some detail since parameters and research protocols varied widely. Knowledge of these differences is vital to understanding the summary tables.

Review of LLLT and bone graft

Rasouli Ghahroudi et al.²⁹ conducted a double-blind experiment studying the effect of LLLT and Bio-Oss graft material on the osteogenesis process in rabbit calvarium. They prepared 10 mm identical cranio-caudal incisions and then created four identical circular defects on each rabbit. One defect was used as an untreated control, a second was filled with Bio-Oss, a third was treated with laser irradiation only using diode laser 810 nm, 300 mW, CW, and 4 J/cm², and a fourth was treated with Bio-Oss and laser by using the same laser parameters.

In histological and histomorphometrical evaluation, after 4 weeks, the control group and the “Bio-Oss only” group showed the most inflammation. The “laser only” group showed the least inflammation, and the combination of “laser with Bio-Oss” ranked between the previous groups. After 8 weeks, the control group demonstrated the greatest inflammation and the laser with Bio-Oss was next in order.

The “laser plus Bio-Oss” group showed greater new bone formation than the “laser only” group or the “Bio-Oss only” group.

Pinheriro et al.³⁰ conducted an in vitro study on 24 rats that were divided into three groups. After exposing the femur, they created 3 mm² cavities in the bone. For group I, the periosteum was repositioned and sutured. In groups II and III, the cavities were filled with inorganic bovine bone and sutured. Group III was submitted to seven sessions of LLLT at 48 h intervals (wavelength 830 nm, 40 mW, CW). A dose of 4 J/cm² was applied, giving a total dose of 16 J/cm² to four points around the cavities. Animals were killed after 15, 20, and 30 days; samples were taken, decalcified with nitric acid, and stained with heat shock factor (HSF). Results of the study showed that new bone formation was seen after 21 days in groups II and III and collagen fiber was seen in group III after 30 days.

In this study, the major finding was the increased amount of collagen fiber in the lased group, which indicated a positive effect of LLLT on bone healing.

Soares et al.³¹ conducted a study on rabbits to assess the influence of LLLT on BIC and bone volume around implants inserted in a block of bovine xenograft or autologous bone graft. They used 780 nm, 4 J/cm² per point, 0.5 cm², repeated every other day for 2 weeks. The result showed a higher percentage of new bone formation in the autograft-irradiated groups in comparison to irradiated xenograft without a significant difference (p > 0.05). A comparison of the irradiated and nonirradiated grafts showed that the irradiated group increased the formation of bone on both types of graft, with a significant difference (p = 0.05).

de Vasconcellos et al.³² assessed the effect of the bone repair process with titanium scaffold on rat femur. Fifty-six rats were divided as follows: Group1-SHAM (simulated ovariectomy) animals received the titanium scaffold; Group2-SHAM animals received scaffold and were subjected to LLLT; Group3-ovariectomized animals received scaffold; and Group4-ovariectomized animals received scaffold and LLLT (the ovariectomized rats showed bone characteristics similar to those found in postmenopausal woman). The LLLT source was GaAlAs 780 nm, CW, through an optic fiber, 40 mW, duration of 100 sec, immediately after surgery was repeated every 48 h for 6 weeks, spot diameter of 0.69 cm, and 4 J/cm² applied to four points for a total of 16 J/cm² per session. Results showed that statistically greater new bone formation occurred in the irradiated group. It was concluded that LLLT improves bone repair within a titanium scaffold in both ovariectomized and healthy rats.

Carneiro et al.³³ assessed the effect of using an AlGaAs 830 nm laser on the repair of surgical defects created in the femur of rats. Forty-eight rats were divided into four groups of 12 each: In the control group, the periosteum was repositioned and sutured in place with no other treatment. The other three groups had this same initial treatment. The second group had laser only applied, the third group received hydroxyapatite+ membrane, and the fourth group received HA+ membrane+ laser treatment. Both laser groups had energy applied at 40 mW, CW, 16 J/cm² per session, four points of 4 J/cm², and spot diameter of 0.6 mm. Laser irradiation was repeated seven times every 48 h. Rats were sacrificed after 15, 21, and 30 days. It was found that, after 15 and 21 days, defects submitted to laser therapy healed more quickly, but at 30 days, the level of repair was similar. Laser therapy associated with hydroxyapatite and a biological membrane increased the biomodulation effect on the bone.

Marquez-Martinez et al.³⁴ assessed the effect of PBM on the repair of surgical defects in the femur of rats. Twenty-four rats were divided into three groups of eight each. The first was used as a control, the second received only bovine bone graft, and the third received graft with PBM by using GaAlAs 830 nm, 40 mW, CW, 4 J/cm² applied to four points for a total of 16 J/cm² per session, and spot diameter of 0.6 mm for four sessions. Animals were killed on days 15, 21, and 30. A histological exam showed an increased amount of collagen fiber, osteoblastic activity, and bone trabeculae formation on the 30th day in the lased group. It was concluded that there was a positive effect on bone healing with the use of PBM.

Gerbi et al.³⁵ assessed the effect of laser PBM on the repair of surgical defects; 48 rats were divided into four groups of 12 each: The first one was used as a control, the second group received LLLT, the third group received bone morphogenic proteins (BMPs)+organic bovine bone graft, and the fourth group received BMPs+organic bovine bone+LLLT. The laser used was 830 nm, 40 mW, CW, 0.6 mm fiber diameter, 16 J/cm² per session divided over four points, and seven irradiations every 48 h. The results, assessed histologically after 15, 21, and 30 days, showed an increased deposition of collagen fiber (at 15 and 21 days) and an increased amount of organized bone trabeculae at 30 days in the irradiated groups. It was concluded that combining LLLT with BMPs and bovine bone increased the biomodulating effect of the laser.

Pinheiro et al.³⁶ histologically studied the effect of LLLT on the repair of defects created surgically on 54 rat femurs. Rats were divided into four groups: The first group served as the control, the second group received LLLT, the third group received HA, guided bone regeneration, and the fourth group received HA, GBR, and LLLT.

The irradiated group was subject to GaAlAs, 830 nm, 40 mW, CW, and spot diameter 0.6 mm, immediately after placing the suture; 4 J/cm² per point; session dose was 16 J/cm²; and total treatment dose 112 J/cm² was applied every other day for 15 days. A histological study on day 15 showed that, in the control group, the defect was filled with medullary tissue without osteoblastic activity. At the end of the experiment, the cortical plate was still thinner than the area not being operated. The irradiated group LLLT at day 15 was filled with medullary tissue, but it progressed faster from day 15 until day 21 when new bone formation was seen. When HA and GBR were used, cortical repair, spongy bone, and giant cells were seen at day 15; in group 4 (HA, GBR, and LLLT), cortical repair and a thin plate at the wounded site with spongy bone, osteoblastic activity, and giant cells were seen. At the end of the experiment, for the entire treated group, the cortical plate was similar to the area not being operated. It was concluded that LLLT could be effective in early wound healing.

Fekrazad et al.³⁷ evaluated the effect of LLLT and mesenchymal stem cells on bone regeneration. Fifty-eight rabbits were included and divided into four groups: C = control, L = LLLT, Sc = filled with mesenchymal cells, and SCL = LLLT+mesenchymal cells. The laser application was initiated the day of the surgery and continued every other day for 3 weeks by using GaAlAs 810 nm, CW, 200 mW, 4 J/cm², 0.2 W/cm², spot size of 1 cm², a distance of 0.5 cm, and 20 sec of treatment. All animals were sacrificed and subjected to histological and histomorphometric analysis at 21 days. Results showed a statistically significant increase (p < 0.05) of new bone formation in the group treated with LLLT compared with the two other experimental groups. When comparing the other three groups (C, SC, and SCL), no significant difference between them (p > 0.05) was found. Histological evaluation showed that groups SC and SCL demonstrated a significantly higher level of inflammation compared with the other groups. It was concluded that combining LLLT with mesenchymal cells does not have a synergistic effect on bone formation.

Kim et al.³⁸ investigated the effect of high-power pulsed laser on bone repair, by using an NdYAG Q-switched pulsed laser. Using 14 rats and 14 rabbits, bilateral defects (5 m rats) and (8 mm rabbits) were created along the sagittal suture. All the rabbits were implanted by using a collagen scaffold and divided into two groups: One group was subjected to 0.75 W output power; the other was subjected to 3 W, by using the same fluence (344 J/cm²). For the 14 rats, bilateral defects were either left empty (n = 7) or implanted by using a collagen scaffold (n = 7). All were irradiated at the same fluence (344 J/cm²) and 0.75 W output power. The Q-switched pulsed NdYAG was applied with high peak power, 320 μm diameter, nonfocused at a distance of 1–2 cm, and with a repetition rate of 100 pulses per second every 2 days for 2 weeks. The spot diameter matched defect size (rats = 5 mm, rabbits = 8 mm). Histomorphometric analysis after 4 weeks showed that the NdYAG laser, using high-intensity pulse, high-power laser irradiation was effective in bone formation in rats when the defects were left empty or filled. In rabbits, both 0.75 and 3 W enhanced bone formation, with no significant difference between the two power levels.

Results

Table 5 summarizes the effect of laser irradiation on bone grafting. Most of these studies used 4 J/cm² per point into four points and found a positive response. One study using higher dose and irradiance did not find a significant effect between the control and laser groups. Finally, one study used Nd: YAG rather than diode laser irradiation.

Table 5.

Review of the Effect of Laser Irradiation on Bone Grafting

Author (year)	Laser type, wavelength	Study type, graft type	Power	Fluence	Application protocol	Assessment	Outcome	Result
Low power (<35 mW) low fluence (<16 J/cm²)
Low power (<35 mW) high fluence (>16 J/cm²)
High power (>35 mW) low fluence (<16 J/cm²)
Rasouli Ghahroudi et al. (2014)²⁹	Diode (GaAlAs), 810 nm CW	Scaffold	300 mW	4 J/cm²	7 Session 48 h intervals	Histological analysis	4 Weeks: inflammation control>biooss>laser+biooss>laser	Positive initial inflammation new bone formation
Rasouli Ghahroudi et al. (2014)²⁹	Diode (GaAlAs), 810 nm CW	Scaffold	300 mW	4 J/cm²	7 Session 48 h intervals	Histomorphometrical analysis	8 Weeks: control>laser+biooss>laser>biooss -New bone formation (4 and 8 weeks) laser+biooss>bioss>laser>control	Positive initial inflammation new bone formation
Pinheriro et al. (2003)³⁰	Diode (GaAlAs), 830 nm CW	Inorganic bovine bone	40 mW	4 J/cm²per point, 4 points	7 Session 48 h intervals	Microscopic analysis	Increased amount of collagen fiber + bone healing	Positive
Soares et al. (2013)³¹	Diode, 780 nm CW	Rabbit, xenograft, and autologous		4 J/cm² per point, 0.5 cm²	7 Session 48 h intervals	BIC	The result shows higher % of new bone formation in the irradiated group (sig. dif.)	Positive p < 0.05
Soares et al. (2013)³¹	Diode, 780 nm CW	Rabbit, xenograft, and autologous		4 J/cm² per point, 0.5 cm²	7 Session 48 h intervals	Bone volume		Positive p < 0.05
de Vasconcellos et al. (2016)³²	Diode (GaAlAs), 780 nm CW	Rats, titanium scaffold	40 mW	4 J/cm² per point, 4 points	100 Sec 48 h for 6 weeks	Histologic histomorphometric	LLLT improves bone repair within the scaffold in both ovarectomized and healthy rats (regardless the presence of estrogen)	Positive p > 0.05
Carneiro et al. (2016)³³	Diode (GaAlAs), 830 nm CW		40 mW	4 J/cm² per point, 4 points	7 Times every 48 h	Histological exam	Day 15 and 21: bone repaired more quickly	Positive p > 0.05
Carneiro et al. (2016)³³	Diode (GaAlAs), 830 nm CW		40 mW	4 J/cm² per point, 4 points	7 Times every 48 h	Histological exam	Day 30: same level of bone repair	Positive p > 0.05
Marquez-Martinez et al. (2008)³⁴	Diode (GaAlAs), 830 nm CW	24 Rats, bovine bone	40 mW	4 J/cm² per point, 4 points	4 Sessions 16 J/cm² per session	Histological exam	30 Days increased collagen fiber, osteoblastic activity and bone trabeculae	Positive
Gerbi et al. (2008)³⁵	Diode (GaAlAs), 830 nm CW	48 Rats, organic bovine bone	40 mW	4 J/cm² per point, 4 points	7 Session 48 h intervals	Histological exam	Increased deposition of collagen fiber (at 15 and 21 days) also increased the amount of organized bone trabeculae at 30 days	Positive p > 0.05
Pinheiro et al. (2009)³⁶	Diode	Rats (n = 54), HA, and guided bone Reg.	40 mW	4 J/cm² per point, 4 points	7 Session 48 h intervals	Histological exam	Lased group: Thin plate at the wounded site with spongy bone +osteoblastic activity + giant cell was	Positive
Fekrazad et al. (2015)³⁷	Diode (GaAlAs), 810 nm CW	Rabbit (n = 58), mesenchymal cells	200 mW	4 J/cm² Spot size, 1 cm²	0.5 cm; duration 20 sec	Histologic histomorphometric	A-High level of bone formation	Positive p > 0.05
Fekrazad et al. (2015)³⁷	Diode (GaAlAs), 810 nm CW	Rabbit (n = 58), mesenchymal cells	200 mW	4 J/cm² Spot size, 1 cm²	0.5 cm; duration 20 sec	Histologic histomorphometric	B-No synergistic effect on bone formation when combining LLLT with mesenchymal	Positive p > 0.05
High power (>35 mW) high fluence (>16 J/cm²)
Kim et al. (2015)³⁸	Nd YAG Q switch pulsed	14 Rabbits	3 W	344 J/cm²	100 Sec without rests every 2 days for 2 weeks	Histomorphometric	New bone formation no sig. dif.	Negative p > 0.05
Kim et al. (2015)³⁸	Nd YAG Q switch pulsed	14 Rats	3 W	344 J/cm²	100 Sec without rests every 2 days for 2 weeks	Histomorphometric	New bone formation no sig. dif.	Negative p > 0.05

LLLT, low-level laser therapy.

Studies showed that when combining LLLT with bone graft, the results were much better than when using graft alone or irradiation alone but the improved healing was more effective at the early stage of the healing process.

Discussion

The parameters of most importance in PBM are power density (irradiance) measured in watts/cm² and energy density (fluence) measured in J/cm². Most of the studies discussed here and, indeed, most seen in research literature are based on the misleading parameter of laser output in watts. Depending on the area irradiated by this beam of photons, the power density and the cellular effect produced will be very different.

As an example, one watt delivered through a 400 μm diameter optical fiber will produce a power density of 796 W/cm² whereas the same one watt delivered through a 8 mm diameter therapy handpiece will produce a power density of only 2 W/cm².

Energy density is often reported in research literature, but the spot area at the tissue is routinely omitted. This error makes it impossible to verify the given findings or to see how the vital energy density information was calculated. Inconsistency in reporting these parameters is a major source of contradictory research outcomes, and it has done much to slow the understanding of any PBM effect.

It must be noted that, using the established exclusion criteria of Cericato et al., there are no studies of either high or moderate quality that explore some application scenarios. Neither low power (<35 mW)/low fluence (<16 J/cm²) nor low power (<35 mW)/high fluence (>16 J/cm²) studies were found. This shortcoming prevents drawing any strong conclusions.

Conclusions

On reviewing the studies evaluated here, it is apparent that each study has subtle or major differences in experimental protocol that could have a significant effect on outcome. The complete reading of any study is vital to evaluating its significance since summary tables can be misleading.

According to this review, we can conclude that:

• LLLT increases cellular metabolism.

• Photonic energy is transmitted to the nucleus, resulting in DNA and RNA synthesis, which causes protein synthesis. This will lead to bone neo-formation and resorption, which increase osseointegration.

• There is no fixed value of dose that produces positive PBM response and bone regeneration.

• A higher dose combined with low power or a low dose combined with high power appears to produce a positive result of PBM.

• Higher dose combined with high power may have an inhibitory effect on PBM.

• Proper reporting of vital laser parameters is essential to understanding the effects of PBM on both soft tissue and bone.

Footnotes

Acknowledgments

This article has received no financial assistance or grants from any organization or individual.

Author Disclosure Statement

No competing financial interests exist.

References

Huang

, Sharma

, Carroll

, Hamblin

. Biphasic dose response in low level laser. Dose Response, 2011; 9:602–618.

De Freitas

, Hamblin

. Proposed mechanism of photobiomodulation or low–level lighttherapy. IEEE J Sel Top Quantum Electron, 2016; 22:7000417.

Karu

. Photobiology of low power laser effect. Health Phys, 1989; 56:691–704.

Salehpour

, Rasta

, Mohaddes

, Sadigh-Eteghad

, Salarirad

. Therapeutic effect of 10 Hz pulsed wave laser in rat depression model: a comparison between near infrared and red wavelength. Lasers Surg Med, 2016; 48:695–705.

Martius

. Das Amdt-Schulz Grundgesetz. Munch Med Wschr, 1923; 70:1005–1006.

Cericato

, Bittencourt

, Paranhos

. Validity of the assessment method of skeletal maturation by cervical vertebra: a systematic review and metananlysis. Dentomaxillofacial Radiol, 2015; 44:20140270.

Schindi

, Schindi

, Pernerstorfer-Schon

, Kerschan

, Knobler

, Schindi

. Diabetic neuropathic foot ulcer: successful treatment by low intensity laser therapy. Dermatology, 1999; 198:314–316.

Dyson

. Primary, secondary and tertiary effects of phototherapy: a review. Proc SPIE, 2006; 6140–614005.

Karu

. Primary and secondary mechanisms of action of visible to near-IR radiation on cells. J Photochem Photobiol, 1999; 49:1–17.

10.

Branemark

P-I

, Hasson

, Adell

, et al. Osseointegrated implants in treatment of the edentulous jaw. Experience from a 10-year period. Scand J Plast Reconstr Surg Suppl, 1977; 16:1–132.

11.

Albrektsson

, Johansson

. Osteoinduction, osteoconduction and osseointegration. Eur Spine J, 2001; 10:96–101.

12.

Mouhyi

, Dohan Erenfest

, Alberktsson

. The peri-implantitis: implant surfaces, microstructure, and physicochemical aspects. Clin Implant Dent Relat Res, 2012; 14:130–183.

13.

Lopes

, PinheiroA

, Sathaiah

, Da Silva

, Salagado

. Infrared laser photobiomodulation (λ830) on bone tissue around dental implants: a Raman spectroscopy and scanning electronic microscopy study in rabbits. Photomed Laser Surg, 2007; 25:96–101.

14.

De Almeida

, Faleiros

, Teodoro

, et al. LLLT on bone repair in osseointegration of implants installed in the tibia of rats: histological andimmunohistochemical evaluation. Clin Oral Implants Res, 2014; 15:336–340.

15.

Gomes

, Mayer

, Massoti

, et al. Laser therapy improves periimplant bone formation: resonance frequency, electron microscopy and stereology findings in rabbit model. Int J Oral Maxillafac Surg, 2015; 44:245–251.

16.

Mandic

, Lazic

, Markovic

, et al. Influence of postoperative low-level laser therapy on the osseointegration of self-tapping implants in the posterior maxilla: a 6 week split-mouth clinical study. Vojnosanit Pregl, 2015; 72:233–240.

17.

Maluf

, Maluf

, Brito

, Franca

FMG

, de Brito

. Mechanical evaluation of the influence of low-level laser therapy in secondary stability of implants in mice shinbones. Lasers Med Sci, 2010; 25:693–698.

18.

Menezes

, Araujo

, Carneiro

VSM

, et al. Assessment of the effect of laser photobiomodulation on periimplant bone repair through energy dispersive Xray fluorescence: a study of dogs. Proc SPIE, 2016; 9695:96950.

19.

Marques

, Holqado

, Francischone

, Ximenez

, Okamoto

, Kinoshita

. New LLLT protocol to speed up the bone healing process histometricandimmunohistochemical analysis in rat calvarial bone defect. Lasers Med Sci, 2015; 30:1225–1230.

20.

Garcia Morales

JMI

, Tortamano-Neto

, Todescan

, De Andrada

Jr , Marotti

, Zezell

. Stability of dental implants after irradiation with an 830 nm low-level laser: a double blind randomized clinical study. Lasers Med Sci, 2012; 27:703–711.

21.

Kim

, Kim

, Park

, Kim

. Low-level laser therapy affects osseointegration in titanium implants resonancefrequency, removal torque and histomorphometric analysis in rabbits. J Korean Assoc Oral Maxillofac Surg, 2016; 42:2–8.

22.

Altan

, Bicackci

, Avunduk

, Esen

. The effect of dosage on the efficiency of LLLT in new bone formation at the expanded suture in rats. Lasers Med Sci, 2015; 30:255–262.

23.

Schwartz-Filho

, Reimer

, Marcantonio

Jr , Marcantonio

. Effect of low level laser therapy 685 nm at different doses in osteogenic cell cultures. Lasers Med Sci, 2011; 26:539–543.

24.

Khadra

, Lynqstadaas

, Haanaes

, Mustafa

. Determining optimal dose of laser therapy for attachment and proliferation of human oral fibroblasts cultured on titanium implant material. J Biomed Mater Res, 2005; 73:55–62.

25.

Mayer

, Gomes

, Carlsson

, Gerhardt-Oliveira. Histologic and resonance frequency analysis of peri-implant bone healing after low level laser therapy: an in vivo study. Int J Oral Maxillofac Implants, 2015; 30:1028–1035.

26.

Massotti

, Gomes

, Mayer

. Histomorphometric assessment of the influence of low-level laser therapy on peri-implant tissue healing in the rabbit mandible. Photomed Laser Surg, 2015; 33:123–128.

27.

Sheridan

, Decker

, Plonka

, Wang

. The role of occlusion in implant therapy: a comprehensive updated review. Implant Dent, 2016; 25:829–838.

28.

Barbos Pinheiro

, Limeira Junior Fde

, Marquez Gerbi

, et al. Effect of 830 nm laser light on the repair of bone defectsgrafted with inorganic bovine bone and decalcified cortical osseousmembrane. J Clin Laser Med Surg, 2003; 21:383–388.

29.

Rasouli Ghahroudi

, Rokin

, Kalhori

, et al. Effect of low level irradiation and biooss graft material on the osteogenesis process in rabbit calvarium defect: a double blinded experimental study. Lasers Med Sci, 2014; 29:925–932.

30.

Pinheriro

, Limeira Junior Fde

, Gerbi

, Ramalho

, Marzola

, Ponzi

. Effect of low level laser therapy on the repair of bone defected grafted with inorganic bovine bone. Braz Dent J, 2003; 14:177–181.

31.

Soares

, Magalhaes

, Ferreira

, Marques

, Pinheiro

. New bone formation around implants inserted on autologous and xenografts irradiated or not with IR laser light: a histomorphometric study in rabbit. Braz Dent Journal, 2013; 24:218–223.

32.

de Vasconcellos

, Barbara

, da Silva Rovai

, et al. Titanium scaffold osteogenesis in healthy and osteoporotic rats is improved by the use of low level-laser therapy (GaAlAs). Lasers Med Sci, 2016; 31:899–905.

33.

Carneiro

SMV

, Gerbi

EMM

, Santos–Neto

APd

, et al. Diode λ 830nm laser associated with hydroyapatite and biological membrane: bone repair in rats. Proc SPIE, 2016; 9692:96920L.

34.

Marquez-Martinez

, Pinheiro

, Ramalho

. Effect of infrared laser photobiomodulation on the repair of bone defects grafted with organic bovine bone authors. Laser Med Sci, 2008; 23:313–317.

35.

Gerbi

, Marques

, Ramalho

, et al. Infrared laser further improves bone healing when associated with bone morphogenicproteins: an in vivo study in a rodent model. Photomed Laser Surg, 2008; 26:55–60.

36.

Pinheiro

, Martinez Gerbi

, de Assis Limeira

, et al. Bone repair following bone grafting hydroxyapatite guided bone regeneration and infra-red laser photobiomodulation: a histological study in a rodent model. Laser Med Sci, 2009; 24:234–240.

37.

Fekrazad

, Sadeghi Ghuchani

, Elsaminejad

, et al. The effect of combined low level laser therapy and mesenchymal stem cells on bone regeneration in rabbit calvarial defect. J Photochem Photobiol, 2015; 151:180–185.

38.

Kim

, Kim

, Cho

, Seo

, Hwanq

. High intensity Nd:YAG laser accelerates bone regeneration in calvarial defect models. J Tissue Eng Regen Med, 2015; 9:943–951.