International Journal of

Molecular Sciences

Review
The Role of Low-Level Laser Therapy in Bone Healing:
Systematic Review
Micaela Berni 1 , Alice Maria Brancato 1, * , Camilla Torriani 1 , Valentina Bina 2 , Salvatore Annunziata 1 ,
Elena Cornella 1 , Michelangelo Trucchi 1 , Eugenio Jannelli 1 , Mario Mosconi 1 , Giulia Gastaldi 2,3 ,
Laura Caliogna 1 , Federico Alberto Grassi 1,3,4 and Gianluigi Pasta 1

1 Orthopedics and Traumatology Clinic, IRCCS Policlinico San Matteo Foundation, 27100 Pavia, Italy
2 Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy
3 Centre for Health Technologies, University of Pavia, 27100 Pavia, Italy
4 Department of Clinical, Surgical, Diagnostic and Pediatric Sciences, University of Pavia, 27100 Pavia, Italy
* Correspondence: [email protected]

Abstract: Low-level laser therapy (LLLT) is a treatment that is increasingly used in orthopedics
practices. In vivo and in vitro studies have shown that low-level laser therapy (LLLT) promotes
angiogenesis, fracture healing and osteogenic differentiation of stem cells. However, the underlying
mechanisms during bone formation remain largely unknown. Factors such as wavelength, energy
density, irradiation and frequency of LLLT can influence the cellular mechanisms. Moreover, the
effects of LLLT are different according to cell types treated. This review aims to summarize the
current knowledge of the molecular pathways activated by LLLT and its effects on the bone healing
process. A better understanding of the cellular mechanisms activated by LLLT can improve its
clinical application.

Keywords: low-level laser therapy (LLLT); phototherapy; photo-biomodulation; bone regeneration;

bone healing; bone fracture healing; orthopedics treatments

Citation: Berni, M.; Brancato, A.M.;

Torriani, C.; Bina, V.; Annunziata, S.; 1. Introduction
Cornella, E.; Trucchi, M.; Jannelli, E.; Light Amplification by Stimulated Emission of Radiation, commonly known by the
Mosconi, M.; Gastaldi, G.; et al. The acronym LASER, is a technological device developed in the 1960s capable of emitting a
Role of Low-Level Laser Therapy in light beam with precise characteristics of monochromatic, coherence, unidirectionality,
Bone Healing: Systematic Review. Int.
collimance and radiance, which make it a versatile device for various daily areas, including
J. Mol. Sci. 2023, 24, 7094. https://
industrial production, telecommunications, scientific research and in the medical area. In
doi.org/10.3390/ijms24087094
particular, in the latter field, the laser has two main applications: surgery and biostimu-
Academic Editor: Jung Eun Kim lation [1]. In the surgical branch, the use of lasers with a power of over 500 mW, called
high-intensity lasers (HIL), is preferred; these use heat to induce photothermal damage of
Received: 1 March 2023
various intensities [2]. In the biostimulation field, low-level lasers (LLL) are used, with a
Revised: 6 April 2023
power lower than 500 mW and an emission spectrum between red and in the near-infrared
Accepted: 10 April 2023
region because these lasers do not produce heat during their work [3]. Since the 1980s,
Published: 12 April 2023
they have been studied for applications in the biomedical field, but unfortunately, their
mechanisms are still unclear [4]. Several studies of the use of low-level lasers as clinical
therapy (LLLT) have been reported in the literature since they would seem to increase the
Copyright: © 2023 by the authors. speed of mitochondrial electron transport by stimulating the production of ATP by the
Licensee MDPI, Basel, Switzerland. mitochondria, reduce oxidative stress by modulating the reactive species of the oxygen
This article is an open access article and induce several transcription factors, including AP-1, p53, NF-kB and HIF, which allow
distributed under the terms and extracellular matrix deposition, and activation of anti-inflammatory and anti-apoptotic
conditions of the Creative Commons pathways. This effect manifests itself in the clinical setting with a reduction in pain and
Attribution (CC BY) license (https:// inflammation, an improvement in tissue repair, the promotion of regeneration of various
creativecommons.org/licenses/by/ tissues and nerves, and the prevention of tissue damage [5–7]. In vitro studies have shown
4.0/).

Int. J. Mol. Sci. 2023, 24, 7094. https://doi.org/10.3390/ijms24087094 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023, 24, 7094 2 of 13

that the application of LLLT can influence several cellular processes and molecular path-
ways such as the alteration of deoxyribonucleic acid (DNA) synthesis, the gene expression,
the increase of cytokines production and the cellular proliferation [8]. Moreover, interest-
ing in vitro studies have shown that LLLT can stimulate proliferation of several types of
cells such as fibroblasts, endothelial cells and keratinocytes, and it can also modulate the
production of different inflammation factors [9].
Bone regeneration is an optimal physiological process that has four main overlapping
phases: the inflammatory phase, the angio-mesenchymal phase, the bone formation phase
and the bone remodeling phase. Bone infections, major pathological fractures and systemic
diseases can interfere in this process, up to causing failure of repair [10]. In recent decades,
many efforts have been made to understand the regeneration of musculoskeletal tissue
and to identify clinical approaches capable of stimulating and accelerating this process.
The approaches that play a crucial role in this process are: chemical treatments, physical
stimulation including mechanical forces, the use of ultrasound and shock waves and
Scaffold or Electrical or Electromagnetic stimulation, and photostimulation [11]. Several
studies present in the literature confirm a positive influence of laser therapy in the bone
regeneration process; in particular, they show a possible influence of photostimulation at
the level of the molecular mechanisms present in the phase of the process. Since there are
different studies in the literature that report information, sometimes even conflicting, on
the role of laser therapy in bone regeneration, the purpose of this review is to summarize
the main evidence reported in literature, clarify the actual role of photostimulation at the
level of the molecular pathway, and identify a possible clinical use of LLLT.

2. Materials and Methods

This report provides an account of the review of the available literature conducted
in order to achieve the aim of the research. Its reporting is an adaptation of the Preferred
Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [12].
• Identifying the research question
The research question identified for the literature review was first of all to summarize
the current knowledge of the molecular pathways activated by LLLT and its effects on the
bone healing process.
• Identifying relevant studies
A literature search was conducted in order to find all relevant studies on the topic.
These were identified by means of diverse sources.
• Electronic database search
The following electronic databases were searched, taking into consideration the chrono-
logical span between 2002 and 2022: PubMed and Embase
The research strategy was carefully designed, in order to retrieve the most relevant
results. Due to the specificity of the two databases employed, for each one a different search
string was built (1: PubMed search string; 2: Embase search string). In brackets, the number
of the results is provided).
1. ((“low level light therapy” [MeSH Terms] OR (“low level” [All Fields] AND “light”
[All Fields] AND “therapy” [All Fields]) OR “low level light therapy” [All Fields] OR
(“low” [All Fields] AND “level” [All Fields] AND “laser” [All Fields] AND “therapy”
[All Fields]) OR “low level laser therapy” [All Fields]) AND (“bone and bones” [MeSH
Terms] OR (“bone” [All Fields] AND “bones” [All Fields]) OR “bone and bones” [All
Fields] OR “bone” [All Fields] OR (“mesenchymal stem cells” [MeSH Terms] OR
(“mesenchymal” [All Fields] AND “stem” [All Fields] AND “cells” [All Fields]) OR
“mesenchymal stem cells” [All Fields]))) AND ((fft [Filter]) AND (English [Filter])
AND (2002: 2022 [pdat])) [953];
2. (“low level laser therapy”/exp OR “lllt (low level laser therapy)” OR “low-intensity
(therapeutic) laser therapy (lilt)” OR “laser biostimulation” OR “laser therapy, low-
Int. J. Mol. Sci. 2023, 24, 7094 3 of 13

level” OR “low energy laser therapy” OR “low energy laser treatment” OR “low
intensity laser therapy” OR “low intensity laser treatment” OR “low level laser ther-
apy” OR “low level laser treatment” OR “low level light therapy” OR “low power laser
therapy” OR “low power laser treatment” OR “low-level laser therapy” OR “low-level
laser therapy (lllt)” OR “low-level light therapy” OR “photo biomodulation therapy”
OR “photo-bio-modulation therapy” OR “photo-biomodulation (pbm) therapy” OR
“photo-biomodulation therapy (pbmt)” OR “photobiomodulation (pbm) therapy”
OR “photobiomodulation therapy” OR “photobiomodulation therapy (pbm)” OR
“photobiomodulation therapy (pbmt)” OR “soft laser therapy” OR “therapeutic laser
therapy”) AND (“bone”/exp OR “mesenchymal stem cells”) AND [2002–2022]/py
AND [english]/lim [1069].
• Other sources
Twenty studies were also included, starting from a review excluded from the analysis,
since only primary studies were considered according to the inclusion criteria. These
articles were regarded to be relevant, even though they were not identified through the
search strings.
• Study inclusion
Starting from the research question, inclusion and exclusion criteria for the objective
selection of the studies identified were defined. Only studies published in the English
language between 2002 and 2022 were eligible for inclusion. Titles and abstracts and full
texts were screened by the research team—i.e., two authors performed the study selection
and the data extraction independently, and all disagreements were discussed between the
authors.
• Data extraction
A standardized data extraction sheet was prepared, where main information on the
studies was collected (e.g., first author’s name, study title, publication year and DOI).
• Study selection
Via the comprehensive literature search, forty studies were included in this literature
review; twenty of them were identified via database searches and twenty via websites
or citation searching (Figure 1). Figure 1 shows the process of study selection in detail,
covering the number of search records retrieved from the two database searches (n = 2022)
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW
and all other searches (n = 24), the number of screened titles/abstracts (n =4 1828),
of 13
the
number of finally included studies (n = 40).

Figure
Figure1.1.PRISMA
PRISMAflow diagram
flow that
diagram included
that searches
included of databases,
searches registers
of databases, andand
registers other sources
other sources [12].
[12].

3. Results
3.1. Low-Level Laser Therapy (LLLT)
Int. J. Mol. Sci. 2023, 24, 7094 4 of 13

3. Results
3.1. Low-Level Laser Therapy (LLLT)
Laser therapy is a therapeutic approach widely used in various areas of the medical
field, including the biostimulation of different tissues. In this case, the use of a laser with a
power lower than 500 mW and an emission spectrum between 500 and 1000 nm is able to
interfere with the molecular pathways promoting the regeneration and repair of various
tissues and preventing any damage.
LLLT has also recently been used in the orthopedic field to promote the bone healing
process; in particular, the laser used is based on low-density energy, which uses non-thermal
radiant photons from coherent (laser) or non-coherent (LED) light sources. To have effective
treatment in promoting bone regeneration, LLLT must comply with specific dosimetric
parameters; unfortunately, the evidence in the literature is conflicting in highlighting precise
parameters, so it was only possible to identify a range of values that would seem optimal
(Table 1), below which the treatment is not very effective while above it tends to lead to
tissue damage [13].

Table 1. Dosimetry parameters of LED device observed in the literature.

Parameter LED Range

Wavelength (nm) 500–1000
Power density (J/cm2 ) 0.5–30
Energy (mW) 5–100
Irradiation time (s) 3–1440
Repetition rate (d) 1–60
Distance from sample (cm) 0–14

Currently, the most accredited theories hypothesize that LLLT influences various
biological activities: It promotes the enzymatic activity of cytochrome c oxidase (CCO)
through the photodissociation of inhibitory nitric oxide from CCO, with consequent in-
creased mitochondrial respiration and production of adenosine triphosphate (ATP) [14,15].
LLLT also correlates in the modification of the mitochondrial membrane potential, causing
the increase in the redox state of the cells with the consequent increase in the oxidation of
Fe2+ , the inhibition of proline hydroxylases (PHDs) and the deregulation of the factor HIF-
1α. Furthermore, the increase in ROS leads to the alteration of several signaling pathways,
including the cell proliferation, survival, repair and regeneration pathways [5,16,17].

3.2. Molecular Pathways LLT-Activated on Bone Healing

Bone regeneration tends to be an optimal physiological process, but in the presence
of bone infections, blood insufficiency, bone defects, pathological fractures, and systemic
diseases, repair failure is possible [10]. Different clinical approaches have been studied to
stimulate and accelerate this process, including physical stimulation, chemical treatment,
and photo-stimulation [11]. LLLT is used to promote the bone healing process, which is
composed of four main overlapping phases: the inflammatory phase, angio-mesenchymal
phase, the bone formation phase and the bone remodeling phase [18]. The inflammatory
phase is characterized initially by the recruitment and activation of pro-inflammatory sig-
nals and growth factors, including tumor necrosis factor-α (TNFα) and several interleukins,
followed by the recruitment of polymorphonuclear neutrophils (PMNs), macrophages and
platelets, which together with the bone morphogenetic proteins (BMP), already present in
the fracture site, recall mesenchymal stem cells (MSC) [19]. A key modulator of this phase
is Wnt, a signaling pathway capable of activating different molecular pathways, including
Wnt/ß-catenin (canonical), Wnt/Ca2+ , Wnt/planar cell polarity, and Wnt/protein kinase
(non-canonical) [20]. In the angio-mesenchymal phase, characterized by high levels of
VEGF, PDGF and FGF, the invasion and formation of new blood vessels takes place and
the transformation of the avascular cartilage matrix into bone tissue is vascularized. In
Int. J. Mol. Sci. 2023, 24, 7094 5 of 13

particular, VEGF, a transcription factor expressed by hypertrophic osteoblasts and chon-

drocytes, promotes both neo-angiogenesis, inducing the formation of new vessels from
those already present, and revascularization of the fracture site, aiding cell proliferation
and aggregation endothelial stem cells [21]. MSC recruitment marks the initiation of the
bone renewal phase, which is characterized by the differentiation of MSCs into osteoblasts
or chondrocytes for newly forming bone. The transcription factors that play a crucial role
in this phase are: runx-2 and osterix. Runx-2 affects the early phases of osteoblast differenti-
ation by regulating the expression of different bone matrix genes, while osterix intervenes
in the regulation of differentiation in the late phases when runx-2 is down regulated. This
phase of bone regeneration is characterized by several molecular pathways, including
bone morphogenetic protein signaling pathway (BMP), tumor growth factor-β signaling
pathway (TGF-β), Phosphoinositide 3-kinase/Akt/Mammal target rapamycin signaling
pathway (PI3K/Akt/mTOR) and the mitogen-activated protein kinase pathway (MAPK).
At the end of the renewal, mature osteoblasts deposit collagen and calcium at the fracture
site, allowing the formation of callus, also called primary bone [22]. The correct formation
of the bone callus, which is important for a complete healing of the fracture, is guaranteed
by the balance between the osteoblasts and osteoclasts, which respectively allow the for-
mation of new bone or its resorption. This process is regulated by growth hormone (GH),
which through direct stimulation (GH) and indirect (stimulation mediated by the hormone
IGF-1 insulin-like growth factor) has two main functions: to stimulate the proliferation and
activity of osteoblasts to induce the formation of new bone and symmetrically increase the
differentiation and activity of osteoclasts, in order to favor bone resorption. Furthermore,
GH also has the role of inducing the terminal differentiation of the chondrocytes present
in the areas of bone growth [23]. The bone regeneration process is induced by different
pro-inflammatory signals including interleukins, TNFα and interferon-γ [24]. Different ap-
proaches can stimulate and influence this process, including chemical treatments, physical
stimulation (mechanical forces, ultrasound, shock waves, stimulation of different nature),
photostimulation and cell therapies. The latter approach, currently considered the most
effective, consists of using mesenchymal stem cells to increase the bone regeneration pro-
cess, as this cell line has excellent self-renewal and pluripotency properties. MSCs are
easily obtained from several tissues, including bone marrow, adipose tissue, skin, umbilical
cord and placenta, but the best regenerative results have been obtained from bone marrow
MSCs. However, this cellular type also has some negative aspects, among which are high
variability in cell quality and concentration, due to the different tissue sources from which
they were obtained, as well as challenging costs and times. The presence of these limitations
in the use of MSCs has led to the search for new treatments to enhance bone regeneration,
and among these is the LLLT. Photostimulation would seem to influence several biological
activities, including mitochondrial respiration, ATP production and ROS levels in cells,
causing a possible alteration of some cellular biological processes [17]. Therefore, several
studies have been conducted to understand the effects of LLLT, both in vitro, mainly on
BMSC, HUVEC, MSC cells and osteoblasts, and in vivo, especially on rat and rabbit.
Below are some studies, conducted both in vivo and in vitro, showing the possible
effects of LLLT.

3.3. In Vitro Studies

It has been observed that LLLT interacts mainly in the initial phases of bone regenera-
tion process; it stimulates the following signaling pathways (Figure 2 and Table 2):
 BMSC, HUVEC, MSC cells and osteoblasts, and in vivo, especially on rat and rabbit.
Below are some studies, conducted both in vivo and in vitro, showing the possible
effects of LLLT.

3.3. In Vitro Studies

Int. J. Mol. Sci. 2023, 24, 7094 It has been observed that LLLT interacts mainly in the initial phases of bone 6 of 13
regeneration process; it stimulates the following signaling pathways (Figure 2 and Table
2):

Figure 2. Schematic representation of molecular pathways activated by Low-Level Laser Therapy

Figure 2. Schematic representation of molecular pathways activated by Low-Level Laser Therapy
(LLLT).
(LLLT).Abbreviations:
Abbreviations:VEGF
VEGF(vascular
(vascularendothelial growth
endothelial growthfactor); FGFFGF
factor); (Fibroblast Growth
(Fibroblast Factors);
Growth
PDGF (Platelet-Derived
Factors); Growth Factor);
PDGF (Platelet-Derived GrowthTGF-ß (tumor
Factor); growth
TGF-ß factor-
(tumor ß); BMPs
growth factor-(bone morphogenetic
ß); BMPs (bone
morphogenetic
proteins); proteins);
AKT (AKT AKT (AKT Serine/Threonine
Serine/Threonine Kinase 1); PI3K(Phosphatidylinositol-4,5- 3-
Kinase 1); PI3K(Phosphatidylinositol-4,5-Bisphosphate
Bisphosphate
Kinase Catalytic3-Kinase
SubunitCatalytic Subunit(mitogen-activated
Delta); MAPK Delta); MAPK (mitogen-activated protein
protein kinase); ERK kinase); ERK
(extracellular signal-
(extracellular signal-regulated kinase 1/2); SMAD proteins (small mothers against decapentaplegic);
regulated kinase 1/2); SMAD proteins (small mothers against decapentaplegic); runx-2 (runt-related
transcription factor gene); col1 (collagen type 1 gene); ocn (osteocalcin gene); opn (ostepontin gene);
ibsp (bone sialoprotein gene).

Table 2. Summary of in vitro LLLT studies.

Laser Wavelength Laser Power Density Target Effects Bone Phase Target Ref.
WNT-ß-catenin
830 nm 7.64 J/cm2 BMSCs
activation Inflammatory phase [25]

636 nm 5 J/cm2 MSCs PDGF increase Angio-mesenchymal phase [26]

660 nm 4–8 J/cm2 ADSCs NF-kB inhibition Inflammatory phase [27]
660 nm 6 J/cm2 ADSCs FGF increase Angio-mesenchymal phase [28]
808 nm 4.5 J/cm2 BMSC VEGF increase Angio-mesenchymal phase [16,29]
Co-culture of ROS/HIF-1α
808 nm 4.5 J/cm2 BMSC and
activation
Angio-mesenchymal phase [16]
HUVECs
ALP activity
810 nm 2–4 J/cm2 BMSC increase Bone formation phase [30]

940 nm NA MG-63 cell line BMP increase Bone formation phase [31]
Human fetal
808 nm 1.2, 2.4, and 3.6 J/cm2 osteoblasts
BMP crease Bone formation phase [32]
Int. J. Mol. Sci. 2023, 24, 7094 7 of 13

Table 2. Cont.

Laser Wavelength Laser Power Density Target Effects Bone Phase Target Ref.
PI3K/Akt/mTOR
650 nm 1–4 J/cm2 HUVEC
activation Bone formation phase [33]

PI3K/Akt/mTOR
635 nm 0,4 J/cm2 Human osteoblast activation Bone formation phase [34]

MAPK kinase
910 nm 2.85 J/cm2 MC3T3-E1
pathway activation Bone formation phase [11]

3.3.1. Inflammatory Phase

• WNT/β-catenin is a signaling pathway that plays a crucial role in the progression
of bone regeneration; in particular, its activation stabilizes the cytoplasmic β-catenin,
allowing its nuclear translocation and degrades the protein complex formed by axin,
adenomatosis polyposis coli (APC), protein phosphatase 2A (PP2A), glycogen syn-
thase kinase 3 (GSK3) and casein kinase 1 α (CK1α) responsible for the destruction of
ß-catenin [20]. Once activated and translocated to the nucleus, β-catenin interacts with
several DNA-bound proteins, including LEF and TFC, to induce the expression of os-
teogenic markers and the differentiation of pre-osteoblasts [35]. The inflammatory pro-
cess negatively affects this signaling pathway, since the presence of pro-inflammatory
cytokines, including TNF, stimulates the expression of Dickkopf-related protein 1
(DKK-1), an antagonist of the WNT signaling pathway that prevents its activation [36].
Laser treatment, particularity with a wavelength of 830 nm, increases the transloca-
tion of β-catenin from the cytoplasm to the nucleus, increasing the activation of the
pathway and accelerating the bone regeneration process [25].
• NF-kB is a transcription factor present in the cytoplasm, which, following the phos-
phorylation of its inhibitor complex IkBα, can translocate into the nucleus and through
gene transcription, promoting the inflammation process. Treatment, with a red laser
with a wavelength of 660 nm of adipose-derived stem cells (ADSCs), showed a reduc-
tion in phosphorylated IkBα and NF-kB concentration in the nucleus, highlighting
the ability of LLLT to negatively regulate the process of inflammation via NF-kB
inhibition [27].

3.3.2. Angio-Mesenchymal Phase

• VEGF is one of the main regulators of angiogenesis, able to promote the migration
and proliferation of endothelial cells. A subclass of endothelial vessels, termed H-
type vessels due to high expression of CD31 and endomucin, positively influence
osteogenesis by resisting surrounding osteoprogenitors that express high levels of
osterix, a promoter of bone formation. It has been reported through the literature
that irradiation of human bone marrow mesenteric stem cells with an 808 nm laser
increased both VEGF expression and H-type vessel formation, leading to an overall
enhancement of the angiogenic process [16,29].
• FGF contributes to the regulation of several important cellular processes; by acti-
vating the PI3K-AKT signaling pathway, it inhibits cell apoptosis by promoting its
proliferation, while by stimulating the MAPK/ERK pathway it induces osteoblast
differentiation. It is also involved in the process of angiogenesis and wound healing,
and studies in the literature have shown a correlation between irradiation with a
660 nm laser and an increase in FGF expression [28].
• PDGF is a factor that plays a role in the regulation of the mitogenic, angiogenic and
proliferative activity of MSC cells. Thanks to its dimerization and the activation of
several signaling proteins including phospholipase C, the kinases Src, PI3-kinase and
the phosphatase SHP2, it also recalls different growth factors. A significant increase in
this factor was observed when treating MSC cells with a laser with a wavelength of
636 nm [26].
Int. J. Mol. Sci. 2023, 24, 7094 8 of 13

• ROS/HIF-1α pathway activation promotes cell differentiation and endothelial an-

giogenesis, two key mechanisms of bone repair. Recent studies have shown that
the GaAlAs laser treatment with 4.5 J/cm2 of BMSC cells, in simple culture and
more significantly if in co-culture with HUVECs, led to activation of the ROS/HIF-1α
pathway [16].

3.3.3. Bone Formation Phase

• Runt-related transcription factor (runx-2) has a relevant role in the recruitment of
MSCs and in their differentiation into osteoblasts; it also induces the expression of
the bone matrix genes collagen type 1 (col1a1), osteopontin (opn), osteocalcin (ibsp)
and osteocalcin (ocn) [16,35]. Studies in the literature have shown that laser therapy
directs MSCs towards osteogenic differentiation versus adipogenic differentiation [25].
Moreover, an increase in BMSC proliferation and differentiation in osteoblasts was
observed after irradiation with a GaAlAs diode laser with a wavelength of 810 nm
and an energy density between 2 and 4 J/cm2 [30].
• BMP/TGF-β has a key role in the osteogenic process; in particular, TGF- β plays a
crucial role in osteoblast proliferation, while BMP has a remarkable ability in bone
formation as well as being fundamental in the maturation process of osteogenesis [23].
In the literature, different studies reported a correlation between LLLT and the increase
in TGF- β and BMP expression. In particular, it was observed that the irradiation
of osteoblast-like cells (human osteosarcoma cell line MG-63) with LLLT led to an
increase in BMP [31] and promoted osteogenic differentiation through the activation
of ROS, which in turn activated TGF-β 1 [16]. This correlation has also been found in
hypoxic conditions by treating osteoblasts with a GaAlAs laser [32].
• The PI3K/Akt/mTOR signaling pathway, thanks to the activation cascade of its protein
effectors, is one of the main pathways involved in cell survival, regulating cell cycle,
motility, apoptosis, metabolism and cell differentiation, as well as the mechanisms
of transcription and translation. It would also seem to have a correlation with bone
regeneration, and in fact there are some studies in the literature showing a possible
involvement in bone formation and remodeling [23]. Through these studies, it was
observed that exposure to a laser treatment with a wavelength of 650 nm and an energy
density between 1 and 4 J/cm2 of the HUVECs cell line increased the phosphorylation
of PI3K, AKT and mTOR, allowing greater activation and functionality of the signaling
pathway [33]. An interesting study confirmed the role of LLLT on increasing the Akt
expression levels and its activated phosphorylated form on osteoblasts irradiated with
635 nm as compared to untreated cells [34].
• The MAPK kinase pathway plays an important role in signal transduction by con-
trolling several cellular processes, including cell proliferation and differentiation as
well as responses to cellular stress. Furthermore, it also regulates bone formation,
inducing osteogenic differentiation and controlling the vitality and functions of os-
teoblasts. In particular, the use of a laser with a wavelength of 910 nm on the MC3T3-E1
cell line allowed an increase in ERK phosphorylation, increasing the activity of the
pathway [11].

3.4. In Vivo Studies

In the literature, it has been observed also in vivo that LLLT has a relevant role in
bone repair. In vivo studies were conducted mainly on rats and rabbits, and they showed a
significantly accelerated bone healing process after LLLT exposure (Table 3).
Int. J. Mol. Sci. 2023, 24, 7094 9 of 13

Table 3. Summary of in vivo LLLT studies.

Laser Wavelength Laser Power Density Target Effects Bone Phase Target Ref.
Decrease of
pro-inflammatory
830 nm NA rats Inflammation phase [37]
interleukins (IL-1,
IL6, IL8, IL18)
Angio-mesenchymal
830 nm NA rats VEGF increase [37]
phase
PDGF and FGF Angio-mesenchymal
1064 nm 8 J/cm2 rats [38]
increase phase

808 nm 354 J/cm2 rats BMP2, OCN and Bone formation [13]
ALP increase
Human periodontal Radiological
830 nm 16 J/cm2 Bone formation [39]
infra-bony defects evidence
Human surgical
660 nm 2.7 J/cm2 disjunction of the Accelerating of the Bone formation [40]
repair process.
maxilla

Consistent with the results obtained in vitro, in vivo studies have confirmed that laser
therapy facilitates the early stages of bone regeneration [41]. Sixty rats that had irradiation
with an 830 nm laser showed a modulation of the inflammatory process, with a deregulation
of the pro-inflammatory interleukin IL-1, IL-6, IL-8 and IL-18, and anticipated the repair
of the bone defect [37]. In addition, the angio-mesenchymal phase showed improvements
after laser treatment, with greater stimulation of angiogenic genes in damaged tissues; in
particular, the irradiation at 830 nm up-regulates the expression of VEGF [37], while the
use of a laser at 1064 nm increased the expression of PDGF and FGF [38]. In vivo studies
also confirmed more osseointegration of bone implants after treatment with laser therapy,
with an increase in the expression of different bone formation marker proteins, including
BMP2 and OCN, after irradiation with an 808 nm GaAlAs laser [13].
Currently, in the literature, there is only a little evidence about an effective beneficial
effect after the LLLT treatment on patients that agrees on the efficacy of laser therapy,
mainly in the early stages of bone regeneration. These studies showed an increase in
bone formation and mineralization and an acceleration in osteoblast differentiation, after
having subjected patients to laser treatment with a wavelength of 830 nm and an energy
density of 16 J/cm2 [39]. A marked increase in bone regeneration was also confirmed
by a double-blind clinical trial conducted on patients after the surgically assisted rapid
maxillary expansion that has shown an improvement in bone regeneration and a decrease
in complications thanks to a postoperative treatment with GaAlAs laser with a wavelength
of 660 nm and an energy density of 2.7 J/cm2 [40].

4. Discussion
Laser therapy is based on the use of a technological device capable of emitting a light
beam with precise characteristics that make it versatile for various fields, including the
medical one. The choice of a laser with a power lower than 500 mW and an emission
spectrum between red and infrared allows for a therapy that does not generate heat, which
is potentially harmful to the tissues, capable of stimulating and promoting the bone healing
process.
Various studies conducted both in vivo and in vitro, have shown that the effect of
laser treatment increased the bone regeneration process only in the first weeks after the
fracture (2/4 weeks), while after about 60 days there was no advantage observed over
controls [42], indicating that LLLT is effective only in the early stages of the process.
Researches conducted in the literature did not reveal specific physical parameters that make
LLLT optimal but provided a range of values, outside of which the treatment is probably
ineffective. These results suggest that low-level laser stimulation activates a common
conserved response mechanism to physical stimuli. Furthermore, a good influence of LLLT
Int. J. Mol. Sci. 2023, 24, 7094 10 of 13

on regeneration was observed regardless of the cell line considered. Bone regeneration is an
optimal physiological process that, however, can present problems and fail in the presence
of pathological fractures and systemic diseases. Despite having four major overlapping
phases, results in the literature seem to indicate that LLLT affects only the molecular
pathways involved in the first three phases, in particular by activating signaling pathways
involved in the angio-mesenchymal phase and the bone formation phase, and by inhibiting
that characteristic of the inflammatory phase. The inflammatory response, triggered by
blood vessels and bone breakdown, plays an important role in bone healing by enabling
the recruitment of cells required for tissue repair and regeneration [20]. In the literature,
it is reported that the application of LLLT can help modulate the inflammatory response:
in vitro, due to both WNT activation and inhibition of NF-kβ signaling [43], and in vivo,
with a deregulation of the inflammatory pro-interleukin IL-1, IL-6, IL-8 and IL-18, and
has anti-patent bone defect repair [29]. These studies would seem to indicate a negative
regulation of stimuli by LLLT. As regards the angio-mesenchymal phase, characterized by
the formation of new blood vessels and the transformation of the avascular cartilaginous
matrix into vascularized bone tissue, several studies have reported that LLLT treatments
have been able to induce an increase in VEGF, FGF and PDGF in stem cells (hBMSC,
hMSC and hOB), as well as the production of termed H-type vessels, a subcategory of
endothelial vessels that exhibit high levels of CD31. These results were also found in in vivo
studies conducted on rats. Activation of these signaling pathways promoted proliferation,
osteogenic differentiation and angiogenesis [16,26,29]. The recruitment of MSCs and their
differentiation into osteoblasts characterizes the bone formation phase; this phase of the
process also seems to be positively influenced by laser treatment. Both in vitro and in vivo
studies confirm this hypothesis; in fact, an increase in BMP and TGF-beta signaling has
been reported, with the consequent promotion of osteogenic differentiation, by exposing
both stem cells (hMSC, human osteosarcoma cell line MG-63), in rats. However, as far as
the bone remodeling phase is concerned, the studies conducted so far in the literature have
not highlighted a possible role of laser therapy; only one recent in vitro study showed a
possible effect on increasing bone mineralization by irradiation of human osteoblasts with
different types of LLLT [44]. This evidence is supported by few relevant data; therefore, it
would be appropriate to conduct further investigations to validate this hypothesis.

5. Conclusions
All the data reported in the literature show different clinical applications of LLLT but
do not provide accurate knowledge on the stimulation of cellular responses due to the wide
difference in dosimetry parameters (wavelength, power density, energy, irradiation time,
frequency repetition and distance from the sample) used in the different studies. Therefore,
it will be necessary to carry out more accurate analyses such as investigating in vivo cell
survival, apoptosis, epigenetic changes and stress responses to have a broader view of the
entire cellular context and the physiopathology of tissues. However, a better understanding
of the in vitro effects of LLLT on biological systems would be a golden opportunity to
predict in vivo outcomes. Due to the lack of standardized experimental guidelines and the
great heterogeneity of the LLLT dosimetry parameters used both in vitro and in vivo, the
controlled trials produced data that were not comparable and were inconclusive.

Author Contributions: Conceptualization, M.B., A.M.B. and L.C.; methodology, L.C., E.C. and
C.T.; investigation, M.B., A.M.B. and V.B.; writing—original draft preparation, V.B., E.C. and C.T.;
writing—review and editing V.B., S.A. and E.J.; visualization, S.A., E.J., M.T. and M.M.; supervision,
A.M.B., G.G. and M.M.; validation, G.G., F.A.G., M.T. and G.P; project administration, A.M.B., G.P.
and F.A.G. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded on behalf of RAMS, for the project: “Trattamenti innovativi per
le patologie muscolo scheletriche: dal planning virtuale preoperatorio alla medicina rigenerativa
TI-RAMS”, RCR-2021-23671217.
Institutional Review Board Statement: Not applicable.
Int. J. Mol. Sci. 2023, 24, 7094 11 of 13

Informed Consent Statement: Not applicable.

Data Availability Statement: https://pubmed.ncbi.nlm.nih.gov (accessed on 6 February 2023);
https://www.embase.com (accessed on 6 February 2023).
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

ADSCs Adipose-derived stem cells

ATP Adenosine triphosphate
BMP Bone morphogenetic proteins
BMSC Bone marrow Stem Cells
CCO Cytochrome c oxidase
col1a1 Collagen type I gene
DKK-1 Dickkopf-Related Protein 1
FGF Fibroblast Growth Factors
HUVEC Human Umbilical Vein Endothelial Cells
ibsp Bone sialoprotein 2 gene
IkBα Inhibitors of NF-kB
LLLT Low-level laser therapy
MSC Mesenchymal stem cells
NF-kB Nuclear Factor kappa B
ocn Osteocalcin gene
opn Osteopontin gene
PDGF Platelet-Derived Growth Factor
PMNs Polymorphonuclear neutrophils
Runx-2 Runt-related transcription factor gene
TGF-ß Tumor growth factor- ß
TNFα Tumor necrosis factor-α
VEGF Vascular Endothelial Growth Factor
AKT AKT Serine/Threonine Kinase 1
PI3K Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Delta

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