Progress in Photobiomodulation for Bone Fractures: A Narrative Review

Abstract

Objective:

The aim of this article is to examine current concepts and the future direction of implementing photobiomodulation (PBM) for fracture treatment.

Background data:

The effectiveness of PBM for bone regeneration has been demonstrated throughout in vitro studies and animal models. Yet, insufficient clinical trials have been reported on treating fractures with PBM.

Materials and methods:

A narrative review was composed on the basis of a literary search. Inclusion criteria consisted of studies between 2000 and 2019 using animal or human fracture models. Exclusion criteria consisted of studies that did not pertain to complete fractures or used other forms of intervention.

Results:

Ten animal studies on rats and rabbits and four clinical trials were found on using PBM for complete fractures.

Conclusions:

Based on positive outcomes in animal trials, parameter optimization of PBM for human fractures still requires extensive research on factors such as dosage, wavelength, penetration depth, treatment frequency, and the use of pulsed waves.

Introduction

Current clinical approaches to enhance bone regeneration include bone transport by distraction osteogenesis, bone grafting, and biophysical stimulation.¹ Regarding bone transport, defected bones are reconstructed where the bone is resected and fixed by a distraction device. Gradual distraction of the two segmented bones leads to the formation and lengthening of the bone through the body's naturally healing process.

Bone grafting is another commonly used procedure where autografts, allografts, or synthetic bone graft substitutes are surgically implanted in diseased or damaged sites with bone loss. The gold standard for the treatment of nonunions is autologous iliac bone graft procedures. However, donor site morbidity, limited bone quantity, the need for an additional surgical procedure, expense, and surgical and postsurgical complications can be major drawbacks.^2,3

Given the disadvantages of autologous and allogenous methods, synthetic bone graft substitutes that promote osteoinductive, osteoconductive, and osteogenic potential have become an enticing alternative.⁴ Bone tissue engineering has given rise to the design and research of suitable biomaterials, cell-based approaches, and bioactive molecules to optimize a bone scaffold that is representative of the extracellular matrix of the bone. Natural, synthetic, and composite biomaterials have been studied to emulate the mechanical properties, high porosity, and pore interconnectivity of bone.⁵ These biomaterials can be combined with osteogenic cells or bioactive molecules to improve bone regeneration capability.

Cell-based approaches to augment bone formation incorporate the use of mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, adipose derived stem cells, stem cells from human exfoliated deciduous teeth, dental pulp stem cells, or predifferentiated osteoblasts.^6

–10 In contrast, bioactive molecules can consist of growth factors, platelet-rich plasma, peptides from the extracellular matrix, or small molecules that regulate bone mass.^4,11,12

Biophysical stimulation is another point of interest in treating bone disorders, such as nonunions, due to its noninvasive nature. Noninvasive stimulation methods that can promote accelerated bone regeneration consist of pulsed electromagnetic fields (PEMF), photobiomodulation (PBM), capacitive coupled electrical fields, and low-intensity pulsed ultrasound (LIPUS).^13
–15 These devices have been shown to enhance bone healing, reduce pain and inflammation, and improve functionality.¹⁵ However, controversy still exists in the application of these therapies as their efficacy varies based on stimulation parameters and method of application. Heterogeneity between studies and outcomes calls for better standardization of all stimulation modalities.

The modality of interest, PBM, is inclusive of low-level laser therapy (LLLT) or light-emitting diode therapy and herein all relevant forms of light or laser emitting therapy are denoted as PBM. PBM is the application of light or lasers at specified wavelengths, typically red and near infrared light, to deliver therapeutic benefits such as wound healing, bone regeneration, pain reduction, or mitigating inflammation.^16,17

PBM is also applied in orthodontics to accelerate intrusion tooth movement,^18,19 bone healing after tooth extraction,²⁰ and to treat dental sensitivity.²¹ In orthopedics, PBM has been shown to be capable of promoting bone regeneration in fractures, bone defects, and osteoporosis in mostly animal models,^14,16 whereas the number of high-quality clinical trials using PBM for fracture healing is still lacking.²² Acceleration or initiation of fracture healing can be beneficial in decreasing the incidence of delayed healing, nonunions, and fracture reinjury. It can also be advantageous in pediatric cases by reducing pain, inflammation, and recovery time, since perioperative pain and anxiety can cause traumatic long-term impacts toward a child.²³

The theoretical mechanism regarding the effect of PBM on mammalian cells is based on the interaction of photons absorbed by the chromophore, cytochrome C oxidase (CCO), which is the terminal enzyme of the mitochondrial electron transport chain.^24
–26 Upregulation of CCO generates a proton gradient on the mitochondrial membrane that aids the synthesis of adenosine triphosphate (ATP).²⁷ Photosensitization of CCO also modulates the amount of reactive oxygen species, Ca²⁺, nitric oxide, alkalization (pH_i), and fission–fusion homeostasis of the mitochondria.^28
–30 This increase in energy and signal transduction leads to the acceleration of cell proliferation and wound healing.

Regarding osteogenic gene expression and activation, PBM has been shown to enhance the expression of osteogenic transcription factors such as Runt-related transcription factor-2 (RUNX-2) and osterix (OSX). These transcription factors, in turn, upregulate regulatory factors, such as osteocalcin (OCN) and osteopontin (OPN), and collagen type 1 (COL1), a predominant protein in the bone matrix.^31,32 Further, in vitro and animal studies have shown increased expression of several types of bone morphogenic proteins (BMPs) after exposure to PBM, indicating its modulatory effects on the BMP/Smad signaling pathway.^31,33
–35

Given the multiple lines of evidence observed through cellular markers, this narrative review assesses the current progress in applying PBM for fracture treatment in vivo by performing a literary search on recent animal and human studies. Treatment factors regarding dosage, wavelength, continuous or pulsed waves, and adverse effects are discussed.

Materials and Methods

Relevant studies from 2000 to 2019 were searched in PubMed and Google Scholar using search criteria such as (“photobiomodulation” OR “low-level laser therapy” OR “low level light therapy”[MeSH Terms]) AND (“fractures, bone”[MeSH Terms] OR “fracture healing”[MeSH Terms]) in PubMed.

Inclusion criteria included studies that: (1) published between the year 2000 to 2019; (2) researched the effects of PBM on animal or human fresh fractures, delayed fractures, or nonunions; (3) animal studies that induced complete fractures with or without fixation devices; and (4) studies that investigated the reduction of pain, reduction of inflammation, or acceleration or initiation of bone regeneration in fractures. Exclusion criteria included studies that: (1) used co-interventions outside of PBM or additional biomaterials, scaffolds, or grafts for fracture repair; (2) investigated other forms of bone disorders outside of fractures; (3) animal studies where they did not indicate a complete osteotomy model; and (4) articles that only used data from a prior study.

Irradiation parameters were tabulated for each article. Laser manufacturers or related articles were sourced to rectify conflicting or missing parameter information.

Results

Ten animal studies were found using rat and rabbit models with complete osteotomies (Tables 1 and 2).^{36

–45} Nine out of 10 animal trials showed positive outcomes in the form of faster bone regeneration or stronger biomechanical testing results compared to their respective control groups.

Table 1.

Animal Studies on Treating Complete Fractures with Photobiomodulation Between the Years 2000 and 2019

Article	Animal model	Fracture site	Controls	Outcomes
Liu et al.³⁶	Female New Zealand white rabbits	Left, mid-shaft tibia External fixation	Fracture with sham laser	• No significant difference in callus thickness • Slightly higher callus volume in treatment group • Significantly higher bone mineral density for treatment group • Less fibrocartilage, increased trabecular structure in treatment group and vice versa in control group
Shakouri et al.³⁷	Female New Zealand white rabbits	Right tibia 3 mm gap External fixation	Fracture with sham laser	• Treatment group had greater mean callus density after 2, 5, and 8 weeks postoperation • Treatment group had significantly lower mean maximum resistance force
Pinheiro et al.³⁸	Male New Zealand rabbits	Right tibia 5 mm gap Internal fixation	• Fracture with bone loss and internal fixation • Fracture with no bone loss and internal fixation	• Raman spectroscopy revealed significantly higher composition of hydroxyapatite in laser treatment group compared to control groups
Léo et al.³⁹	Wistar rats	Right tibia Intramedullary fixation	• No fracture • Fracture with no treatment	• Using mechanical flexion test, treatment group at 15 J/cm² dosage had higher mean maximum force than fracture group with no treatment • Treatment group at 10 J/cm² dosage had no statistical difference in mean maximum force than fracture control group
Pinheiro et al.⁴⁰	Male New Zealand rabbits	Right tibia Internal fixation (mini plate)	• No fracture • Fracture with fixation	• In laser treatment group, light microscopy revealed: (1) no difference in inflammatory infiltration, (2) greater collagen deposition, (3) greater bone deposition, (4) greater bone resorption compared to control group
Sella et al.⁴¹	Wistar rats	Femur Internal fixation	Fracture with sham laser	• Treatment group with significantly lower inflammatory infiltration response and higher periosteal formation on day 13 and 18 • Higher periosteal formation in treatment group on day 13 and 18 • Significantly greater bone formation on day 8, 13, and 18 in treatment group • Treatment group had greater osteocalcin and osteopontin expression on day 8, greater osteonectin expression on day 13
Mostafavinia et al.⁴²	Female Wistar rats	Tibia Intramedullary fixation	Fracture with no treatment	• Significantly higher bending stiffness, maximum force, energy absorption, and stress load in treatment groups • Significant increase in trabecular bone volume, callus bone volume, and number of osteoblasts
Quirk et al.⁴³	Male Sprague–Dawley rats	Femoral diaphysis Intramedullary fixation	Fracture with no treatment	• 660 nm group with significantly higher radiological score than control and 830 nm group at day 14
Sarvestani et al.⁴⁴	Male New Zealand white rabbits	Right radius 3 mm gap	Fracture with no treatment	• Higher radiological score for bone healing after week 2 and 3, but no statistical significance
Pinheiro et al.⁴⁵	Male New Zealand rabbits	Right tibia Internal fixation	Fracture with no treatment	• Raman spectroscopy revealed higher composition of phosphate hydroxyapatite, carbonated hydroxyapatite, and collagen in irradiated groups with the LED group showing better results

Table 2.

Parameter Table for Articles Researching Photobiomodulation Treatment on Complete Animal Fractures from 2000 to 2019

Article	Type of emitters (laser or LED)	Wavelength and bandwidth (nm)	Pulse mode (CW or Hz, duty cycle)	Irradiance per point at target (mW/cm²)	Radiant exposure per point (J/cm²)	No. of points irradiated	No. of treatment sessions	Total radiant power (mW)	Total radiant energy (J)
Liu et al.³⁶	Laser	830	CW	200	10	4	20	78,400	3920
Shakouri et al.³⁷	Laser	780	CW	13	4	1	28	46^a	3.5^a
Pinheiro et al.³⁸	Laser	790	CW	40	4	4	7	560	56
Léo et al.³⁹	Laser	904	9500 Hz, 60 ns	300	10/15	3	22	1980^a	66/99^a
Pinheiro et al.⁴⁰	Laser	780	CW	100	4	4	7	1400	56
Sella et al.⁴¹	Laser	808	CW	7400	37	2	8	3300	16
Mostafavinia et al.⁴²	Laser	890	80 Hz, 180 μs	1.08/1.15	0.972/1.5	1	18	19.44/20.7	17.5/27
Quirk et al.⁴³	LED	680/830	CW	50	45	1	21	N/A	N/A
Sarvestani et al.⁴⁴	Laser	830	1500 Hz, 180 μs	14	4	3	9	10.6^a	3^a
Pinheiro et al.⁴⁵	Laser/LED	780/850 ± 10	CW	17/79.7	5.1/5.1	4	7	238/1115.8	71.4/71.4

Based on estimated spot size using manufacturer specifications or relevant works.

Out of the 10 animal study articles examined, 6 studies used rabbit models (2 female, 4 male) and 4 used rat models (1 male, 1 female, 2 unspecified). Nine out of 10 studies used laser light, whereas one study implemented a custom-made LED and another study compared LLLT to LEDs. The wavelengths used ranged from 660 to 904 nm with most falling in the infrared range. All experiments had controls consisting of no treatment or a sham laser. Treatment frequency ranged from 7 to 28 sessions. Among the studies that provided sufficient information (with reported spot sizes), the energy density per session for lasers ranged from 0.975 to 37 J/cm², and the total energy of the laser treatments was calculated to range from 16 to 3920 J.

Four clinical trials were conducted using lasers to treat bone fractures (Tables 3 and 4).^46

–49 Fracture types consisted of wrist and hand fractures, tibial fractures, mandible fractures, and pediatric upper and lower limb fractures. The studies used inflammation, pain, functionality, or accelerated bone healing as outcome measurements.

Table 3.

Human Studies on Treating Fractures with Photobiomodulation Between the Years 2000 and 2019

Article	Fracture type	Quality of study	Outcomes
Chang et al.⁴⁶	Wrist and hand	Sham control group, random allocation, triple-blinded: patient, interventionist, physician conducting assessment	Significant improvements in pain (VAS), Quick DASH, grip strength
Chang et al.⁴⁶	Wrist and hand		No difference in radiological healing
Nesioonpour et al.⁴⁷	Tibia	Sham control group, random allocation, double-blinded: patient, author conducting assessment	Improvement in pain reduction (VAS) at 2nd, 4th, 8th, 12th, and 24th hour postoperation with less opioid consumption in treatment group
Lauriti et al.⁴⁸	Mandible	Sham control group, random allocation, patient blinded	Significant improvement in inflammation, mandibular dynamics (laterality, protrusion), bite force, and reduction of postoperative pain (3–5 days postoperation) in treatment group
Ip⁴⁹	Pediatric delayed upper and lower limb	No control group, randomization or blinding	Mean time of 6 weeks to radiological fracture union for upper limb fractures and 10 weeks for lower limb fractures.

DASH, Disabilities of the Arm, Shoulder and Hand; VAS, visual analog scale.

Table 4.

Parameter Table for Articles Researching Photobiomodulation Treatment on Human Fractures from 2000 to 2019

Article	Type of emitters (laser or LED)	Wavelength and bandwidth (nm)	Pulse mode (CW or Hz, duty cycle)	Irradiance at target (mW/cm²)	Radiant exposure (J/cm²)	No. of points irradiated	No. of treatment sessions	Total radiant power (mW)	Total radiant energy (J)
Chang et al.⁴⁶	Laser	830	10 Hz, 75 μs	16.21	9.7	1	10	600	360
Nesioonpour et al.⁴⁷	Laser	808 + 650 combined	CW	300 + 100 and 300 + 100	6 + 3 and 3 + 1	4 and 6–8	1	6000–6800	60–68
Lauriti et al.⁴⁸	Laser	660	CW	108	21.6	10	15	16,200	3240
Ip⁴⁹	Laser	810	CW	20	N/A	N/A	28/42	N/A	N/A

Only two of the clinical studies included radiological outcomes that measured the rate of bone healing, one of which had no control, blinding, or randomization. The other clinical study with radiological assessment found no difference in bone healing rate. Three out of three studies showed better pain reduction in the treatment groups and two out of two studies showed improved function. Three studies used infrared light and one study used red light. Three studies used continuous lasers and one study used a pulsed laser. The energy densities in three of these studies ranged from 9.7 to 21.6 J/cm², and the total energies ranged from ∼60 to 3240 J.

Discussion

Animal models

Rat or rabbit models are typically used to study the dynamics of fracture healing and bone regeneration under PBM. Male rats are more preferable due to possible bone alterations caused by female sexual hormones.³⁹

Out of the laser-based studies, Liu et al.³⁶ consisted of the highest total energy applied at 3920 J, where 10 J/cm² per point of continuous laser at 830 nm was irradiated daily on rabbits over a 4-week period. Their outcome measurements included radiological assessment, tibial bone volume, bone morphology, and bone mineral density. Based on radiological measurements, the authors found no significant difference in the callus thickness surrounding the tibial fracture between the two groups. However, the total volume of the callus size and bone mineral density (gray scale level) was measured to be higher in the laser treatment group. Further, blinded microscopic examination revealed higher callus formation and callus structural content in the treatment group with a faster remodeling process based on fibrocartilage formation compared to the control group.

In comparison with a rabbit study using lower total energy, Pinheiro et al.⁴⁰ irradiated a total of 56 J on the fractured tibia of rabbits over two weeks on alternate days of treatments at a wavelength of 780 nm. This study had a lower energy density of 4 J/cm², yet light microscopy still showed positive outcomes with greater collagen deposition, bone deposition, and bone resorption, although no difference in inflammatory infiltration was observed compared to the control group.

Sella et al.⁴¹ applied the least total energy of 16 J on Wistar rats over eight days using an 808 nm gallium-alluminum-arsenide (Ga-Al-As) laser. This study had a high fluency of 37 J/cm², but with a small spot size of 0.02 cm². On the 13th and 18th day after the first treatment, the irradiation group was observed to have a significantly lower inflammatory infiltration response along with positive outcomes for bone formation. Thus, both higher and lower levels of fluency and total laser energy have shown greater bone formation and deposition. However, the two studies that applied lower total energy had conflicting outcomes on inflammation, possibly due to the implementation of different animal models and laser specifications.

In addition to Liu et al.,³⁶ two other studies used radiological scores to assess the rate of fracture healing. Quirk et al.⁴³ treated femoral diaphysis fractures of rats with custom-made 3.8 × 3.8 cm red or infrared LED panels. The fracture was irradiated daily over 3 weeks at a high fluency of 45 J/cm². The authors found that the group irradiated by red LEDs (660 nm) had a significantly higher radiological score than the infrared LED (830 nm) group and control group.

In contrast, Sarvestani et al.⁴⁴ performed radiological assessment where they irradiated the right radius of rabbits for nine sessions over three weeks using an 830 nm pulsed laser at 1500 Hz and 180 μs pulse width. The authors found higher radiological scores for bone healing after week 2 and 3, but there were no significant differences between the control groups. Thus, two out of three studies did not have significantly better radiological outcomes compared to a control group. The study showing significantly positive outcomes found better results with a red LED array, whereas the two other studies used 830 nm infrared lasers.

In another trial of note, Pinheiro et al.⁴⁵ compared the effects of continuous LLLT to LEDs using an equivalent energy density and total energy. However, the laser had a slightly different wavelength of 780 nm compared to the LED at 850 ± 10 nm. The authors irradiated right tibia fractures of rabbits on alternate days over 15 days and used Raman spectroscopy to determine that LED treated fractures yielded a higher composition of phosphate hydroxyapatite, carbonated hydroxyapatite, and collagen compared to the laser treatment group.

Despite the high heterogeneity in irradiation parameters and delivery methods, PBM treatment of most irradiated fractures in these animal studies resulted in significantly improved bone formation compared to control groups by means of mechanical testing, histology, and radiological assessment. However, certain application methods and measurements did not result in significantly improved outcomes, likely due to differences in experimental conditions and irradiation parameters. Differences between factors such as wavelength, energy density, pulse frequency, or coherency were found. Large-scale animal studies would thus be useful in contributing to the standardization of PBM therapies. Given the efficacy observed in both in vitro experiments and animal trials, there is further incentive in conducting clinical trials to treat bone fractures with PBM.

Clinical trials

Chang et al.⁴⁶ conducted a double-blind, randomized control trial treating closed bone fractures in the wrist and hand of 50 patients with an 830 nm laser for 2 weeks. The pulsed laser had a frequency of 10 Hz, pulse width of 75 μs, fluency of 9.7 J/cm², and total energy of 360 J. Outcome measurements that they assessed included pain with the visual analog scale (VAS), functionality using Quick Questionnaire for Disabilities of the Arm, Shoulder and Hand (Quick DASH), hand and finger grip strength, and radiological bone healing. After treatment, Chang et al. found significant improvements in VAS, Quick DASH, and grip strength. However, the authors found no statistical difference in radiological healing between the treatment group and the placebo group. It should be noted that the laser device they used was designed for pain treatment.

A study by Nesioonpour et al.⁴⁷ on postoperative pain after tibial fracture surgery discovered that PBM therapy could alleviate pain. They conducted a double-blind, randomized control trial on 54 patients using a combination of red and infrared lasers for the treatment group with a total of 10–12 irradiation points. A total of one treatment session was applied at the end of surgery. The infrared laser (808 nm) was applied at a fluency of 6 and 3 J/cm², and the red laser (650 nm) was applied at a fluency of 3 and 1 J/cm². Each leg was irradiated from four sides surrounding the tibial fracture and popliteal fossa. Trigger points on muscles and surgical wounds were also treated at multiple points.

The authors used VAS and the amount of analgesic administered as primary outcomes. Patients would be administered analgesic if VAS was three or greater, which was measured at specified times within 24 h postsurgery. They concluded that the treatment group had significantly improved VAS scores over 24 h postoperation and that the amount of analgesia administered to the treatment group was significantly less than the control group.

Lauriti et al.⁴⁸ used a gallium-aluminum-arsenite (Ga-Al-As) device to deliver 15 treatment sessions over two months of continuous PBM or a placebo to 12 randomly allocated male patients with mandibular fractures. The laser was emitted at 660 nm, a fluency of 21.6 J/cm², and total energy of 3240 J. The authors reported significant improvements compared to the control group in the reduction of postoperative facial swelling, pain relief, and increase in bite force. No radiological outcomes were measured to assess the healing rate of the fracture.

A study by Ip⁴⁹ examined the effects of PBM on pediatric patients with upper and lower limb fractures. The study included 17 patients with delayed fractures, who had their casts taken off after 4–6 weeks. Laser therapy (810 nm) was applied on alternate days over the course of 8 weeks for patients with a fracture on the upper limb and 12 weeks for patients with a fracture on the lower limb. All patients developed a solid union upon treatment. The mean time to radiological union was 6 weeks for upper limb group and 10 weeks for the lower limb group. No randomization, blinding, or control groups were included in this trial, and reports of the irradiation parameters were incomplete.

Based on these trials, outcomes indicate that PBM can reduce pain and inflammation and improve functionality when clinically used to treat fractures, although the quantity and quality of these studies were low. In vitro and animal studies provide strong evidence for bone regeneration in the treatment of fractures. Yet, there are limited clinical trials pertaining to fracture healing or the initiation of osteogenesis in delayed unions or nonunions. Out of the four clinical studies, two measured the rate of fracture healing through radiology. Only one study showed evidence of radiological healing. However, no blinding, randomization, or control group was reported in that trial.

Dosage

Producing optimal healing effects through PBM can be dependent on irradiation parameters, exposure duration, and application method. Relevant parameters include factors such as wavelength (nm), power density (mW/cm²) or energy density (J/cm²), total energy density (J), pulsing frequency (Hz), pulse width (μm), exposure duration, treatment period, and point of application.

The World Association for Laser Therapy (WALT) offers dosage recommendations for various types of tendinopathies and arthritis that can range from 1 to 16 J per treatment depending on the wavelength and application site, which corresponds to 14–224 J of total energy over a daily 2-week period.⁵⁰ However, no recommendations are provided for bone defects or fractures. In comparison, the upper limits of WALT recommended dosages are substantially lower than two of the previously mentioned animal (3920 J)³⁶ and clinical (3240 J)⁴⁸ studies for fracture treatment.

To assess optimal energy densities for bone regeneration, Na et al.⁵¹ performed an in vitro dose analysis of PBM therapy on the viability and cellular activity of bone-related cells using a wavelength of 940 nm. They exposed three cell culture types, MC3T3-E1, MLO-A5, and RANKL-treated RAW264.7 cells, to an infrared LED at various energy densities for 10 min. The authors discovered that a low dose (1 J/cm²) of irradiation did not affect the viability of any of the cells, but promoted osteoblast proliferation, osteoclast differentiation, and osteoclastic resorption. A high dose (5 J/cm²) decreased osteocyte and osteoclast viability, and an even higher dose (7.5 J/cm²) decreased osteoblast viability. Thus, it can be inferred that an upper dosage limit needs to be established when using PBM to accelerate bone regeneration.

It is also necessary to establish a lower bound dosage. In a systematic review by Bjordal et al.⁵² on using PBM for pain in chronic joint disorders, trials that used a lower than recommended dose range were ineffective in reducing inflammation. Their suggested dosage for anti-inflammatory effects was 0.4–19 J and a 5–21 mW/cm² power density. At present, no lower bound dosages have been established for fracture healing.

Among the animal trials discovered for fracture healing, Mostafavinia et al.⁴² used the lowest power densities at 1.08 mW/cm² (0.972 J/cm²) and 1.15 mW/cm² (1.5 J/cm²). For both dosages, the biomechanical properties and bone formation of the irradiated subjects were significantly higher compared with the control group. There is yet to be a clinical trial studying equally low power densities for fracture healing.

Wavelength

Bjordal et al.⁵² also proposed that when applying PBM, 50–90% energy could be lost due to the skin barrier, depending on the laser device used. Further, they postulated that 5% and 10% energy were lost per mm tissue in porcine models for infrared and red lasers, respectively. Following this sentiment, the penetration depth of light is known to reach deeper into bodily tissue in relation to a higher wavelength.⁵³ The depth of penetration is largely dictated by wavelength due to the variable scattering and absorption coefficients of chromophores. As such, an increase in wavelength coincides with an increase in maximum penetration depth. Beam width is also a factor where simulations showed that an increase in beam width of up to 5 mm coincides with an increase in penetration of up to 10 mm.⁵³

It should also be noted that although red and infrared light are commonly used in PBM, a wavelength between the range of 700–780 nm is thought to be ineffective due to a trough in absorption spectrum of CCO.⁵⁴ Three of the animal studies in Table 2 used a wavelength of 780 nm in their trials and still achieved positive results for bone regeneration. No trials used wavelengths in between 700 and 780.

Regarding wavelengths just at the border, Kazem Shakouri et al.³⁷ found that their irradiation group at 780 nm had enhanced bone formation in the initial stages of healing, although the irradiation group also resulted in weaker biomechanical properties. Pinheiro et al.⁴⁰ compared combinations of 780 nm irradiation treatment and biomaterial grafts to a control group. Pertaining to only the irradiation versus control group, end results showed no difference in inflammatory reaction, but greater collagen deposition, bone deposition, and bone resorption based on histomorphometry.

In 2018, Pinheiro et al. used a 780 nm laser in comparison to an 850 nm LED on tibia fractures in rabbits.⁴⁵ The authors found that there was a higher composition of hydroxyapatite and collagen in both irradiated groups compared to control groups. Further, the 850 nm LED treatment group showed better results than the 780 nm laser treatment group. Thus, using a wavelength bordering on the trough in the absorption spectrum of CCO is still an effective wavelength for bone regeneration.

Regarding wavelengths outside of red and infrared, blue and green light is a subject of interest for reducing inflammation, reducing pain, promoting wound healing, limiting bacterial growth, improving cellulite appearance, or reducing swelling.⁵⁵

Wang et al. found that blue light (420 nm) and green light (540 nm) were more effective in stimulating osteoblast differentiation in human adipose-derived stem cells compared to red and infrared light.⁵⁶ Wang et al. also found that green and blue light were capable of increasing intracellular calcium in adipose derived stem cells, but did not find the same results in red and infrared light. However, while possibly more effective, the penetration depth of blue and green wavelengths is not as deep as infrared or red wavelengths as they were better absorbed by hemoglobin.^26,53 Under these circumstances, the osteogenic application of green and blue light may be better suited for orthodontics or for in vitro applications with the purpose of seeding, proliferating, and differentiating osteogenic cells on biomaterial scaffolds.

Continuous or pulsed waves

The efficacy of PBM can also be influenced by the implementation of pulsed waves as opposed to continuous waves. The parameters for pulsed waves include pulse width (s), pulse interval (s), pulse frequency (Hz), duty cycle, and peak or average power (W).⁵⁷ Pulse width indicates the ON time of the light, whereas pulse interval indicates the OFF time, also known as “quench period.” Duty cycle is the ratio of pulse width in relation to one period or one on-and-off cycle. Peak power is the intensity of light in one pulse width or the average power over the duty cycle.

A study by Ueda and Shimizu⁵⁸ researched the effects of pulsed irradiation on osteoblastic cells from fetal rat calvariae. The authors showed that pulsed waves at 1 Hz had a significantly greater effect than continuous waves in osteogenic activity, with greater increases in cell proliferation, alkaline phosphatase activity, and bone nodule formation.

A subsequent study by Ueda and Shimizu⁵⁹ used the same rat calvariae cell model to show that lower frequency (1 or 2 Hz) pulsed waves were more effective than continuous or higher frequency (8 Hz) pulsed waves. Further, the lower frequency pulsed waves required a lower total energy density than continuous and high frequency pulsed waves to be effective for bone nodule formation. Pulsed lasers can be advantageous compared to continuous lasers when performing ablations where tissue heating can be mitigated when using high power density lasers.^17,57,60,61

It is also speculated that pulsed PBM could be used to entrain or synchronize biochemical oscillations as shown in a simulation by Pogodaev et al.,⁶² where an enzymatic reaction network was regulated by irradiation pulses. Pulsed waves are also thought to have higher penetration depth in PBM compared to continuous waves. Pulsed waves can achieve higher penetration depth due to the fact that peak power and peak power density are always higher in pulsed modalities given the same average power.⁵⁷

Despite the theoretical advantage of higher penetration depth, only 3 out of the 10 animal studies^39,42,44 and 1 out of 4 clinical studies⁴⁶ used pulsed waves in their experiment on fractures. Among the animal studies, Léo et al.³⁹ and Mostafavinia et al.⁴² both found significantly better biomechanical properties in their pulsed laser group compared to the control group when treating Wistar rats.

Léo et al.³⁹ used a 904 nm pulsed laser (9500 Hz, 60 ns) to irradiate right tibia fractures of rats. The authors compared the delivery of two energy densities at 10 and 15 J/cm² for 22 sessions on alternate days. Mechanical flexion testing showed that the treatment group with a 15 J/cm² dosage had a significantly higher mean maximum force than the 10 J/cm² group and control group.

Mostafavinia et al. measured multiple biomechanical properties and the bone volume of fractured rat tibia that were treated with pulsed waves at a much lower frequency of 80 Hz, a wavelength of 890 nm, and a pulse width of 180 μs.⁴² The authors irradiated the fracture site at 0.972 or 1.5 J/cm² for 6 weeks at three times per week and discovered significantly higher bending stiffness, maximum force, energy absorption, stress load, trabecular bone volume, callus bone volume, and osteoblast count in the treatment groups.

Sarvestani et al.⁴⁴ found no significant difference in rabbit fracture healing when comparing a pulsed laser treatment group to a control group based on radiological score. Chang et al.⁴⁶ used a pulsed laser to treat human wrist and hand fractures and found significant improvements in pain and functionality, yet no difference in radiological healing compared to the control group.

Safety and adverse effects

Outside of bone regeneration, low-level lasers are also used to treat side effects caused by the toxicity of cancer treatments and, as such, the effects of PBM on precancerous or existing cancer cells have become a topic of interest. Frigo et al.⁶³ found that high irradiance and dosage of 2.5 W/cm² and 1050 J/cm² enhanced melanoma growth in mice, although no effects were evident in normal dosages. Yet, a later study that investigated the long-term safety of irradiating lasers on murine bone marrow found that multiple applications and higher dosages did not cause histological changes or neoplasmic response.⁶⁴

Contrarily, an in vitro study by Kara et al.⁶⁵ discovered that the proliferation of cancerous and precancerous cells was encouraged at certain doses of laser irradiation. The authors irradiated Saos-2 osteoblast-like osteosarcoma cells and A549 human lung carcinoma cells at 0.5, 1, 2, or 3 W for one to three treatment applications. They realized that applying two treatment sessions at 1 W resulted in the highest rate of proliferation. These studies have shown that the risk of PBM on cancer tissue could be dependent on irradiation parameters and dosages, but no confirmed trends are yet apparent across studies.

Three systematic reviews were conducted in 2019 on the safety of PBM in the cancer patients or in the presence of cancerous cells. Examining only human clinical trials, de Pauli Paglioni et al.⁶⁶ concluded that PBM was a safe treatment for treating or preventing complications caused by cancer treatment.

Silveira et al.⁶⁷ found mixed results in both in vitro and in vivo studies on using PBM for preventing and managing oral mucositis in head and neck squamous cell carcinoma patients undergoing treatment. The authors determined existing evidence to be inconclusive, finding both inhibitory effects and increased proliferation of tumor cells among different reports.

The third systematic review by da Silva et al.⁶⁸ examined in vitro studies of tumoral cells and also found that the increase and decrease of cell proliferation varied among studies with varying wavelengths and dosages.

Given that the safety concerns of PBM are mostly directed toward patients with or at risk of cancer, the application of PBM in orthopedics is generally considered to be safe with no side effects. As of now, there are no reported adverse effects on using PBM for bone regeneration or fracture healing. However, none of the selected fracture studies in this article performed measurements to check for adverse events. Under this premise, patients should not receive PBM treatment for bone disorders if they have also been diagnosed with cancer until more conclusive studies are established.

Patient-related factors influencing fracture healing

Patient-related factors and the nature of injury can be influential factors that inhibit bone healing to varying extents, rendering the standardization for optimal PBM treatment a tedious task. Patient-related factors that can negatively affect fracture healing include aging, infection, comorbidities, prescribed medications, or other nonprescribed drugs.^69

–72

Age-related changes can lead to a decrease in stem cell quantity or proliferation, as well as decrease their ability to differentiate.^71,72 In addition, higher pro-inflammatory cytokines in elderly people leave them susceptible to an increased pro-inflammatory status. The elevated inflammatory status along with intrinsic age-related changes that alter the survival and function of macrophages and its aging microenvironment are possible contributors to slow down bone healing.⁷²

Infection can also lead to prolonged inflammation where arachidonic acid metabolites, cytokines, nitric oxide, and BMPs play an influential role as inflammatory mediators.⁷³ Effects of infection, exaggerated immune responses, and bone loss can also be seen in the chronic trauma caused by oral diseases.^74,75 Periodontal diseases can result in persistent and chronic inflammation, which can disrupt bone healing and induction of bone regeneration.^73,76

Comorbidities such as diabetes mellitus, malnutrition, and disturbance in blood flow can also negatively impact bone healing. Similar to aging, pro-inflammatory mediators are thought to increase in patients with diabetes, and the patient's ability to downregulate inflammation is also weakened. Moreover, diabetes can lead to the increase of osteoclast formation and to the decrease of osteoblast formation, quantity, and function.⁷⁷

Malnutrition can hinder bone healing as the body demands additional nutrients for fracture repair. Nutrients such as vitamin C, vitamin D, protein, calcium, and phosphorous all play important roles in healing fractures. Vitamin C may regulate chondrocyte fate determination and stimulate osteoblast differentiation and proliferation. In addition, matrix proteins in the bone are largely made up of collagen, which require vitamin C as a cofactor for collagen synthesis.⁷⁸

Calcium and vitamin D are well known for maintaining bone health. Calcium is largely stored in the bone as hydroxyapatite, acting as a calcium reservoir and providing bone strength. Vitamin D (1,25-dihydroxyvitamin) aids the intestinal absorption of calcium when there is normal or insufficient dietary calcium intake.⁷⁹ Moreover, studies have shown that deficiencies in essential and nonessential amino acids play a large role in fracture repair and also increase the risk of fractures.⁸⁰

Comorbidities that decrease vascularization can inhibit fracture healing and contribute to the issue of nutrition deficiency. Thus, angiogenesis is an essential component to regenerating bone where oxygen and nutrients are delivered to the fracture site. Anemic animal models have been shown to have deficiencies in the delivery of iron and oxygen, impairing fracture healing.⁶⁹

Nonsteroidal anti-inflammatory drugs have been suggested to inhibit fracture repair through the inhibition of prostaglandin synthesis, cyclooxygenase activity, or angiogenesis.^69,81 Corticosteroids have inhibitory effects on the production of insulin-like growth factor-1 and transforming growth factor-beta, which may lead to prolonged fracture repair. Conflicting study results signify that the effects of nonsteroidal anti-inflammatory drugs and corticosteroids on fracture healing are still controversial,^69,81

–84 but should be taken into consideration when conducting studies on fracture repair using PBM.

Nonprescribed drugs such as smoking and alcohol have been shown to inhibit fracture healing. Smoking can lead to atherosclerosis and vasoconstriction, causing a disturbance in blood flow and leading to an insufficiency in nutrients and oxygen. Out of 3500 chemical substances in tobacco, nicotine is the largest antagonist to fracture repair. Nicotine can negatively affect osteoblasts, fibroblasts, macrophages, and red blood cells, as well as increase vasoconstriction and platelet aggregation.⁸⁵ Combustion products can also have damaging effects, such as carbon monoxide, which binds to hemoglobin and impairs tissue oxygenation. Excessive alcohol consumption is also known to impair fracture healing in patients with alcohol-induced osteopenia. In these patients, there is a decrease in the bone remodeling rate resulting in a net increase in bone resorption.⁸⁶

Given the numerous factors influencing fracture healing, physicians naturally advise patients to adopt beneficial practices such as restricting smoking or alcohol consumption and consuming appropriate nutritional supplements. When performing clinical PBM trials, unavoidable patient-related factors should be noted or excluded from the trial altogether.

Future Considerations and Conclusion

PBM has a number of advantages in treating bone fractures. Light irradiation can possibly reduce the incidence of delayed and nonunion fractures, as well as accelerate the healing process. Further, PBM has been shown to mitigate pain and inflammation while improving function. In cases of nonunion fractures, autologous bone graft procedures are the current gold standard, yet it is highly invasive in comparison to PBM. More popular biophysical stimulation therapies for fractures, such as PEMF and LIPUS, can be alternative and better researched noninvasive options, yet were found to be questionable in efficacy based on systematic reviews.^87,88

Other treatment methods for nonunions or bone loss that have undergone extensive research include the engineering of synthetic biomaterials, stem cells, and bioactive molecules implemented as bone scaffolds. These treatment methods can be used in conjunction with PBM to further enhance osteogenic effects. As described by Amini et al.,³ the goal in hard tissue engineering is to: (1) develop scaffolds that are representative of the natural bone extracellular matrix niche; (2) develop osteogenic cells as a foundation for the bone tissue matrix; (3) guide cells to differentiate into the correct phenotype; and (4) promote angiogenesis within the hard tissue. PBM can thus be used as a combinatory method to promote tissue regeneration in implanted bioengineered scaffolds with osteogenic cells or bioactive molecular additives.

Kazem Shakouri et al.³⁷ recommended that PBM only be used in cases of poor bone formation, as in nonunions, due to their finding that laser treatment did not improve biomechanical properties. Yet, other animal studies found improved biomechanical properties of PBM treated fractures with higher energy densities or through the use of pulsed lasers.^39,42 Discrepancies among studies could be attributed to heterogeneity in the quality of the research, dosage, and method of delivery.

As with all stimulation therapies, large-scale high-quality studies should be conducted to standardize PBM to achieve optimal effectiveness in relation to its intentional treatment. Methods of increasing effectiveness can involve applying varying wavelengths, coherency, pulse patterns and frequencies, dosage, number of applications, and the duration of each treatment. Further, wavelengths with higher efficacy that have weak tissue penetration ability, such as green and blue light, may be more viable for in vitro use.

Adverse effects should also be reported, particularly in patients with accompanying malignant conditions. It can be presumed that the risk of enhancing the proliferation of cancerous cells is a possible contraindication at certain dosages in patients with precancerous cells or cancerous tissue, although no adverse effects are yet to be reported in prior clinical trials for bone regeneration. Given the evident or speculative inhibition of fracture repair under various patient-related factors, exclusion criteria in clinical PBM trials should take into consideration age, malnutrition, infection, comorbidities, and medications or drugs.

PBM is a less common biophysical stimulation modality for treating fractures, possibly due to impracticality, as light and low-level lasers have a relatively weak penetration ability. Moreover, other modalities such as PEMF can be applied over a cast. Yet, evidence has shown that PBM is effective in improving functionality and decreasing inflammation and pain along with the potential to accelerate the healing process, making it a stimulation therapy worth investigating under specific circumstances. In such cases, the application of PBM for fractures may be viable for conditions where there is a lesser amount of thick overlaying tissue, such as in facial or mandibular fractures, hand or wrist fractures, and certain pediatric fractures.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

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