Analysis of Changes Induced In Human
Periodontal Ligament, Dental Pulp, Bone Marrow
and Adipose Stem Cells by Low Level Laser Therapy: A
Review and New Perspectives Volume 4 - Issue 2
1Multidisciplinary Department of Medical-Surgical and Dental Specialties, University of Campania Luigi Vanvitelli, Italy
2Department of Medicine and Surgery, University of Milano Bicocca, Italy
Received: April 24, 2018; Published: May 07, 2018
*Corresponding author: Letizia Perillo, Multidisciplinary Department of Medical-Surgical and Dental Specialties, University of Campania Luigi
Vanvitelli, Via L De Crecchio 6, 80138 Naples, Italy
The biomodulation and the biostimulation of Low Level Laser Therapy (LLLT) promote wound healing, dentine repairing processes and
stimulate the biological activity of human Periodontal Ligament Stem Cells (HPDLSCs), human Dental Pulp Stem Cells (HDPSCs), human Bone
Marrow Stem Cells (HBMSCs) and human Adipose Stem Cells (HADSCs). The aim of the study was to review the literature on the LLLT effects
on DPDLSCs, DDPSCs, DBMSCs and HADSCs. The electronic search was performed collecting articles in the PubMed, Medline, Scopus, Lilacs and
Google Scholar databases from January 1990 to January 2018. LLLT was able to significantly increase HPDLSCs, HDPSCs, HBMSCs and HADSCs
proliferation rate, stimulating osteogenic differentiation and opening new possibilities in the tissue regeneration. However, the research
background on this topic showed high heterogeneity and the in vitro studies cannot always mimic the in vivo conditions. Thus, further clinical studies should be performed to determine the appropriate dose of irradiation in different patients.
Abbreviations: ALS: Amyotrophic Lateral Sclerosis, UMN: Upper Motor Neuron, LMN: Lower Motor Neuron, BMI: Weight Loss or Body
Mass Index, PBP: Progressive Bulbar Palsy, IFC: Informed Consent Form, FOIS Scale: Functional Oral Intake Scale, PEMS:Protein Energetic
Malnutrition Scale
In 1967, Endre Mester, a Hungarian physician, after applying
low-power laser light to shaved mice noticed a faster hair growth
compared to the control group [1,2]. This was a new non-surgical
way of use the laser: instead of incise and coagulate tissues many
researchers started to investigate the enhancing and stimulating
effects of light. Currently the Low-Level Laser Therapy (LLLT),
also called “cold laser therapy” is known for biostimulatory and
biomodulatory effects in vivo and in vitro. The LLLT shows not
a thermal but photochemical effect by triggering biochemical
changes within cells after the application of a light ranging between
10mW-500mW. The result of Low-Level Laser irradiance on human
tissues is a reduction of inflammation, pain relief and accelerated
tissue regeneration. In dentistry, the use of LLLT is increasing in the
last years because it promotes wound-healing, dentine repairing
processes and stimulates the biological activity of mesenchymal
stem cells (MSCs) and human periodontal ligament stem cells
(hPDLSCs) [3-26]. MSCs can differentiate into different specialized
cell types and they can be identified and collected in humans from
dental pulp steam cells (hDPSCs) [11,27-38], adipose tissue [39-
45] and cancellous portions of bones [46,47]. The LLLT effects on
cell proliferation have been investigated by several authors but
the main part of the papers do not focus properly on hPDLSCs
and hDPSCs, which could open new possibilities in regenerative
medicine and dentistry. Therefore, the objective of the present
study was to review the literature about the LLLT effects on the
hPDLSCs and hDPSCs, but also on hBMSCs and hADSCs.
The electronic databases searched for eligible relevant scientific
papers from January 1990 to January 2018 were Medline via PubMed, Scopus, Cochrane Central Register of Controlled Trials via
Cochrane Library, Lilac and Google Scholar databases. There was no
language restriction set. Inclusion criteria were studies evaluating
effects of LLLT on hPDLSCs, hDPSCs, hBMSCs and hADSCs. Papers
that did not provide specifically data about hPDLSCs hDPSCs,
hBMSCs and hADSCs were excluded. Reference lists of the selected
articles were also hand-searched for other relevant papers that may
have been missed by the search engines. The titles and abstracts of
all studies resulting from the search were independently assessed
by two reviewers. Full copies of all apparently relevant studies or
those for which there were insufficient data in the title and abstract
to make decision, were obtained. Any disagreement between the
two reviewers on the eligibility of included studies was resolved
through oral discussion and consensus. Studies that did not match
the inclusion criteria in this second selection phase were excluded.
Mechanism of LllT Action
It has been shown that LLLT can augment the oxidative
phosphorylation (OXPHOS) modifying the redox basal-status of
the whole cell and in particular of mitochondria [6,48]. During
OXPHOS, thanks to electron transport chain (ETC), electrons are
shifted from a reducing agent (even called electron donors) to
oxidizing agent (even called electron acceptors), which in biological
reduction–oxidation (redox) reactions is the oxygen (O2) [49].
These redox reactions free energy that is mainly spent to synthetize
chemical energy: the adenosine triphosphate (ATP) [50-52]. The
mechanism of LLLT is based on the absorption of a specific visible
red and near-infrared wavelengths by biological photoreceptors
located inside human cells, augmenting the production of a
trans-membrane electric and mechanical proton-H+ gradient in
mitochondria necessary for OXPHOS [53-57], and enhancing the
activity of mitochondrial complexes IV in an exposure–response
relationship [58,59]. In addition, there is evidence that LLLT
affects the mastocytes cells localized into endothelium in mucous
membranes and dental pulp [4,60,61].
a) Laser stimulation can induce mast cells degranulation:
although they show a key role in endothelial-leukocyte adhesion
molecules and anaphylaxis, the labrocytes have a great defensive
role too, being engaged in wound healing, angioneogenesis,
protecting from infectious agents and getting involved in blood–
brain barrier action [62-64]. In conclusion, LLLT influences
the biological function of a variety of cell types by stimulating
mitochondrial OXPHOS and modulating inflammatory
responses, exerting a range of several beneficial effects upon cell
proliferation and healing.
Effects 0n HPDLSCs
The periodontium (from περί: peri- “around” and -odont
“tooth”) is a specialized tissue surrounding the teeth composed by
cementum, periodontal ligament (PDL), gingiva and alveolar cortex
[65]. The PDL is a membrane-like connective tissue interposed
between the tooth root and the alveolar bone of which the main
component is represented by collagen fibers. It grants mechanical
resistance during masticatory forces and control alveolar bone
turnover maintaining tissue homeostasis and dissipating forces
during physiological or orthodontic tooth movement [21,66,67].
hPDLSCs has the faculty for self-renewal and the capacity to
differentiate into different cell pools like cement oblasts and
osteoblasts, which contribute to the repair of affected cementum
and alveolar bone leading to a partial or complete periodontal
regeneration [68-71].
For this reason, it is meaningful the possibility to stimulate
hPDLSCs proliferation and differentiation to improve periodontal
tissue regeneration [72]. Moreover LLLT helps pain reliefs through
an increased synthesis of prostaglandin E2, interleukin-1b and
osteocalcin during tooth movement [73,74], and the decreasing
levels of plasminogen activator under mechanical stress [70,75-
80]. Kreisle [81,82] reported that the irradiated test group revealed
a highly significant proliferation rate 24h after diode laser exposure
at a power output of 10 mW in continuous wave modality at energy
fluencies of 1.96-7.84 J/cm2 compared to control group. Similarly
Hakki [4] that observed a significant increase of collagen I mRNA
expression only in the LLLT bio-stimulated group. Wu [21] observed
an augmented proliferation and osteogenic differentiation of
hPDLSCs via cAMP regulation with a low-power GaAlAs 660-nm
at 2 J/cm2, highlighting possible osteogenic inducibility of LLLT
during periodontal tissue regeneration procedures [76]. Soares
[83] showed that a power of 30 mW at 1.0 J/cm2 has stimulating
effects while under that parameters he noticed just a small changing
on the proliferation rate of hPDLSC. In addition, it was observed an
accumulative effect between repeated irradiation. According to
the growth curve, the effect was greater when the test-group cells
were irradiated with the dose of 1.0 J/cm2 compared to 0.5 J/cm2, in
particular at 48h and 72h after the second laser stimulation. Huang
[73] noticed higher viability of hPDLSCs at both 5 and 10J/cm2 than
the control group at day 7.`
Table 1:Main papers of LLLT on HPDLSCs.
The flogosis markers expression like iNOS, COX-2, MMP-3, IL-1
and OC activity showed a remarkable positive variation in irradiated
hPDLSCs at days 1 and 5 in the -100kPa incubator compared to nonirradiated
ones. LLLT irradiation seems to improve periodontal
parameters [84] and hPDLSCs showed good response grown onto
3D scaffold in the osteogenic differentiation [85]. These results
suggested that LLLT and hPDLSCs have a role in the maintenance of
the alveolar bone [23] and they are helpful in healing inflammation
and may, in the future, augment regeneration procedures such as
the tooth movement rate even if it is not yet showed definitively by
other papers [86-91]. The most relevant papers focusing on LLLT
and hPDLSCs are reported in Table 1.
Effects on HDPSCs
The hDPSCs can be collected from the pulp of all permanent teeth
(in particular from third molars) and exfoliated deciduous teeth
[92]. They can be detached and augmented showing multipotential
plasticity [11,27-30,32-38,93]and immunosuppressive activity, that
could be considered an added value during healing, reconstructive
and transplantation procedures [31]. In particular, their most
feasible and promising application is related to bone regeneration
[36]. Ginani [94] founded pulp stem cells from permanent teeth
exhibited higher proliferation analyzed at two times (72h and 96h
after irradiation) when irradiated with wavelength of 660 nm and
the dose of 1.0 J/cm2 compared to the non-irradiated control group
Table 2:Main papers of LLLT on HDPSCs.
In a recent investigation Arany [95] demonstrated that lowpower
laser irradiation (810-nm GaAlAs diode laser) can be used
as a minimally invasive tool promote the activity of an endogenous
latent growth factor complex in the dental pulp, transforming
growth factor-β1 (TGF-β1), that subsequently differentiate human
dental stem cells to promote dentin regeneration. Other authors
like Pinheiro [96] who stimulated for three weeks the hDPSCs
with a low-power red 660 nm laser at 5, 10, and 20 J observed
their potential in bone tissue regeneration in cleft and noncleft
patients. In particular the best pool of hDPSCs seems to be
collected from the exfoliated deciduous teeth as showed by Ginani
who irradiated these cells with a 660 nm laser at 30 mW with a
dose of 1.0 J/cm2 [97]. Recently Staffoli [98], Ching [99], Morsczeck
[100,101], Diomede [92] and Bressel [102] highlighted once again
the encouraging data available in literature, which will lead soon to
personalized therapies, and regenerative approaches dental stem
cells based. The most relevant papers focusing on LLLT and hDPSCs
are reported in Table 2.
Effects on HBMSCs and HADSCs
Other sources of human mesenchymal stem cells investigated
in time for bone regeneration are represented by human bone
marrow-derived stem cells (hBMSCs) and adult adipose-derived
stem cells (hADSCs) from adults. hBMSCs are easily and safely
obtained by means of percutaneous withdrawal from the patient’s
bone marrow, and due to their multilineage potential, they can be
stimulated to generate non-hematopoietic tissue, including bone,
cartilage, tendons, and ligaments [26]. Particularly, bone marrowderived
MSCs differentiate into the osteogenic lineage, if cultured in
presence of dexamethasone, ascorbic acid, and α-glycerophosphate
(osteogenic medium). Soleimani [15] investigated effects of LLLT
on hBMSCs proliferation and differentiation into neuron and
osteoblast using different energy densities.
He found that LLLT promoted hBMSCs proliferation significantly
at all energy densities except for 6 J/cm2 in comparison to control
groups on the seventh day of differentiation. LLLT at energy
densities of 3 and 6 J/cm2 dramatically facilitated the differentiation
of hBMSCs into neurons and also, alkaline phosphatase activity
was significantly enhanced in irradiated hBMSCs differentiated
to osteoblast on the second, fifth, seventh, and tenth day of
differentiation. He concluded that using LLLT at 810 nm wavelength
enhances hBMSCs differentiation into neuron and osteoblast in
the range of 2–6 J/cm2, and at the same time increases BMSCs
proliferation (except for 6 J/cm2).
Leonida [26] reported a significantly increased proliferation
of hBMSCs seeded on a three dimensional scaffold of collagen
compared to control group in the first week of LLLT but no further
effects in the second week. There were no differences concerning
hBMSCs differentiation toward the osteoblastic lineage in the first
week but an exponential increase was observed after 14 days of
laser irradiation, with respect to the control group. hADSCs were
primarily described in 2001 as a population of cells derived from
adipose tissue with the potential of differentiation into a number
of mesenchymal cell types, includingosteoblasts, chondrocytesand
adipocytes [17]. Mvula [103] reported that LLLT at 5 J/cm2 using
636 nm diode laser in combination with EGF increased the viability
and proliferation of hADSCs, assessed using adenosine triphosphate
(ATP) luminescence and optical density at 0, 24 and 48 hours after
irradiation. De Villiers [19] investigated effects of LLLT on hADSCs,
differentiated into smooth muscle cells using retinoic acid, exposed
to a 636 nm diode laser at a 5 J/cm2.
He found that, morphologically, hADSCs did not show any
differences but there was an increase in viability and proliferation
post-irradiation. In another research Mvula [17] investigated the
effect of low level laser irradiation on primary cultures of hADSCs
using a 635-nm diode laser, at 5 J/cm2 with a power output of 50.2
mW and a power density of 5.5 mW/cm2. He reported that cellular
morphology did not appear to change after irradiation, there was
an increase of cellular viability, measured by ATP luminescence
statistically significant at 48 hours, and of proliferation of irradiated cells, measured by optical density, at both time points. He also
reported that Western blot analysis and immunocytochemical
labeling indicated an increase in the expression of stem cell marker
β1-integrin after irradiation. The most relevant papers focusing on
LLLT and hBMSCs and hADSCs are reported in Table 3.
The biological outcome of laser irradiation is influenced by
many variables, like: wavelength, spot diameter, energy and
power density, duration and rate of irradiation, medium or plate
variables, nutritional conditions and the pools of cell irradiated. All
the studies analyzed showed qualitative and quantitative different
parameters and for this reason it is very difficult to compare them
and to identify a univocal protocol. Nearly all papers showed
that LLLT had a positive effect on hPDLSCs, hDPSCs, hBMSCs and
hADSCs proliferation with doses used between 0.5 and 10 J/cm2,
while doses higher than 10 J/cm2 exert or no effects [7] or seems
to be antiproliferative [93] and similar outcomes were found
for LED [104]. In addition, no study showed deleterious effects
of LLLT on these cells [105]. The majority of studies about LLLT
focus on hPDLSCs probably due to the major interest in periodontal
regeneration and speeding up orthodontic treatments [14,66,71].
At the best of our knowledge there is no studies performed
on hPDLSCs and hDPSCs in-vivo and for this reason they cannot
always mimic clinical conditions, so the limitations of these in vitro
studies should be considered but there are some animal studies
highlighting possible indications in the regeneration of smooth,
skeletal muscle cells and infarcted myocardium [106-111].
LLLT seems to be effective in stimulating hPDLSCs, hDPSCs,
hBMSCs and hADSCs proliferation but there is no unique protocol
due to the very high heterogeneity of studies. The synergy between
LLLT and stem cells can open new possibilities in the tissue
regeneration, but until now there are no reliable studies performed
in vivo on humans. For this reason further studies, especially in vivo
on human stem cells, should be performed to validate this promising
therapy and to establish an easy and appropriate standardized
protocol to provide the best clinical advantage for the patients.