Introduction

Mesenchymal stem cell-based therapy is known to have the potential to induce angiogenesis, primarily through the secretion of angiogenic growth factors. Furthermore, it has been shown that the paracrine properties of MSCs can improve collateral vessel growth in ischemic tissue, bone regeneration, cardiovascular repair after myocardial infarction, and wound healing [1,2,3]. Human mesenchymal stem cells can be retrieved from different sources, such as adipose tissue, cartilage, cord blood, dental pulp, gut, perichondrium, salivary glands or tendons [4]. In vitro, mesenchymal cells have lineage differentiation potential after induction (e.g., into adipocytes after induction with dexamethasone, indomethacin, insulin, methylbutylxanthine or thiazolidinedione; into chondrocytes after induction with ascorbate, bone morphogenic protein 6, dexamethasone or transforming growth factor β; and into osteoblasts after induction with ascorbate, bone morphogenetic protein, dexamethasone, or 1.25 dihydroxy vitamin D3) [4].

However, there are still some limitations regarding the clinical application of MSCs. To overcome them, some methods that improve cell viability (cell death and survival rates), proliferation, differentiation, and migration, have been explored, including MSC preconditioning, genetic modification, and optimization of culture conditions [5].

Generally, photomodulation/photobiomodulation (PBM) or low-power laser refers to non-invasive, non-toxic phototherapy with wavelengths ranging from 600 to 1000 nm, that is, light in the red-near-infra red region of the spectrum [6]. The definition also applies to the word photoactivation. It has been observed that, while red (660 nm) or near-infrared (810 nm) light stimulates proliferation, blue (415 nm) and green (540 nm) light inhibits it, for example, in stem cells derived from human adipose tissue [7]. PBM is biologically attributed to the absorption of light by internal photoreceptors of the respiratory chain located in the mitochondria, which induce mitochondrial activation within cells [6]. The photons absorbed by mitochondria cause an increase in adenosine triphosphate [8]. Another proposed mechanism for PBM action relies on ion channels' sensitivity, allowing calcium to enter the cell [9].

Scientific research on PBM started about 50 years ago [10]. To date, the beneficial effects of PBM have been verified in a variety of diseases and physiological processes, in which the reduction of inflammation or the stimulation of lesion repair has been observed in vivo and in vitro [11], as well as other effects, such as the reduction of hypoxic damage or brain degeneration [12]. However, PBM has not been widely accepted yet, mainly due to the uncertainty regarding its molecular, cellular, and tissue mechanisms of action [13].

Several experiments have shown that administering PBM to MSC cultures accelerated the repair process of skin lesions in normal and ischemic simulations, improved the viability of MSCs, and promoted the release of cytokines in normal and ischemic organs [14, 15]. That is why PBM can be used as MSC preconditioning to improve their regenerative capacity [11, 16]. Furthermore, stem cells and progenitor cells appear to be particularly susceptible to PBM[13]: Its potential to promote MSC growth factor proliferation, differentiation and secretion has already been shown [12]. In several animal models, MSCs were conditioned with PBM to stimulate neoangiogenesis and showed improvements in tissue healing [12, 17].

The PBM devices used are diverse: Helium–Neon (He–Ne) gas lasers; gallium arsenide (GaAs), neodymium-doped yttrium aluminum garnet (Nd:YAG), gallium aluminum arsenide (GaAlAs) and indium-gallium-aluminum-phosphide (InGaAlP) lasers; non-thermal, non-ablative carbon dioxide (CO2) lasers; light-emitting diode (LED) arrays, and visible light [6]. It is well established that the biostimulatory effects of lasers are influenced by parameters such as wavelength, energy density, output power, frequency, or irradiation duration [18]. In addition to the different devices used in the studies carried out with MSCs, a great diversity of parameters had been set, resulting in multiple treatment protocols with different—and sometimes even contradictory—results [7].

Our aim was to conduct a review of published articles carried out in human MSCs with in vitro PBM preconditioning, and identify any possible tendency that might highlight the most suitable procedures.

Methods

A search in English of the PubMed database was carried out using the following search criteria: photobiomodulation or photoactivation or photomodulation, and mesenchymal cells (https://www.ncbi.nlm.nih.gov/pubmed/?term=(photobiomodulation+OR+photoactivation+OR+photobiomodulation)+AND+mesenchimal+cells).

All articles found (including reviews) were listed in an Excel file and arranged by author, year, title, and abstract. Title and abstract were checked for coherent inclusion in the starting list. After a first screening, study duplicates arriving from more than one review were identified and removed. Once the list of studies was available, main text, procedure, results, and conclusions were checked for minimum requirement meeting.

Study Inclusion Criteria

To be included in this review, three criteria had to be met: studies had to be carried out on human MSCs, cell PBM had to be performed in vitro, and its effects had to be analyzed in vivo or ex vivo. Light sources could differ in each study, but the used wavelengths had to range from 600 to 1000 nm. Data of studies that also used other wavelengths were collected as well.

The main registered parameter was wavelength (nm), and secondary parameters were irradiance (W/cm2), dose/fluence (J/cm2), power output (mW), duration of treatment (seconds), frequency of treatment (number of times the treatment was carried out and the period), and cumulative dose (sum of all individual doses). Studies had to assess at least one of the following variables: viability, proliferation, differentiation, or migration.

Other parameters, such as irradiation mode (continuous, fractioned, or punctual) or spot size (cm2), were collected, although they were not included in the analysis. Likewise, variables, such as apoptosis, adherence and secretion of growth factors, or blood vessel count, were included in this review when collected by the studies, though excluded from its conclusions, for that analysis would exceed the focus of this work.

Studies were grouped by wavelengths as follows: Group I (620–625 nm, yellow-orange-green); Group II (625–740 nm, red); Group III (740–1000 nm, near infra-red); and Group IV (studies in which wavelength combinations were applied). Procedures with different wavelengths within the same study were allocated to their corresponding group.

Results

The search yielded a total of 47 articles: 42 single-study articles and five reviews [6, 19,20,21,22]. These reviews contained a total of 67 articles. Of them, 43 were discarded: nine (13.4%, n = 9/67) were excluded because PBM procedures were conducted in vivo; 20, because they were not conducted in human cells (29.9%, n = 20/67); five (7.5%, n = 5/67) were discarded for not including 600–1000 nm wavelengths; and 9 (13.4%, n = 9/67) for being duplicates. This resulted in a total of 24 articles selected, published between 2005 and 2019. Of the 42 single-study articles found, a total of 18 (42.6%, n = 18/42) were discarded because they did not meet the criteria of this review: five (27.8%, n = 5/18) performed PBM procedures in vivo; six studies were not conducted on human cells (33.3%, n = 6/18); in one study (5.6%, n = 1/18), wavelengths did not fit the range; and the last six (33.3%, n = 6/18) were narratives about PBM but did not describe any research study. This resulted in a total of 24 single-study articles selected for this review, published between 2013 and 2020 (Fig. 1).

Fig. 1
figure 1

Flow diagram showing review of literature to identify clinical research papers relating to PBM

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All articles (n = 48) were put together and again, checked for duplicates. Eleven (22.9%, n = 11/48) were discarded, resulting in 37 articles selected for this review. For a general timeframe reference of the 37 articles included in this review: 18 articles (48.6%, n = 18/37) were published between 2016 and 2020; 13 (35.1%, n = 13/37) were published between 2010 and 2015; and six (19%, n = 6/37) were published between 2005 and 2009. In addition to this, six (16.2%, n = 6/37) articles contained several PBM protocols with different wavelengths, amounting to a total of 43 procedures (Fig. 1). These 43 procedures constituted the final n of this review, and were grouped as follows: (a) Group I (620–625 nm), three procedures (7.0%, n = 3/43) [7, 23,24,25] (Table 1); (b) Group II (625–740 nm), 26 procedures (60.5%, n = 26/43) [7, 8, 12, 26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47] (Table 2); (c) Group III (740–1000 nm), 13 procedures (30.2%, n = 13/3) [7, 27, 31, 32, 48,49,50,51,52,53,54,55,56] (Table 3); and (d) Group IV (combinations of wavelengths), one procedure (2.3%, n = 1/43) [27] (Table 4).

Table 1 Group I: studies with wavelengths from 600 to 625 nm (yellow-orange-green)
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Table 2 Group II: Studies with wavelengths from 625 to 740 nm (red)
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Table 3 Group III: Studies with wavelengths from 740 to 1000 nm (near infra-red)
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Table 4 Group IV: Studies in which wavelength combinations were applied
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Results on Viability

Fourteen procedures assessed cell viability (32.6%, n = 14/43) (Table 5). Wavelengths ranged from 630 nm to 808 nm and one protocol combined 630 + 810 nm. Ten procedures (71.4%, n = 10/14) showed an increase in viability; in four (28.6%, n = 4/14), there was no effect. No procedure showed a decrease on viability.

Table 5 Studies procedures that assessed the effect of photobiomodulation on viability
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Results on Proliferation

Thirty four procedures assessed cell proliferation (79.1%, n = 34/43) (Table 6), with wavelengths ranging from 620 to 940 nm. In 27 procedures (79.4%, n = 27/34), there was an increase in proliferation; in three procedures (8.8%, n = 3/34), there was a decrease, and in three procedures (8.8%, n = 3/34), no effect was witnessed. In one study (2.9%, n = 1/34), the effect varied depending on the dose irradiated.

Table 6 Studies procedures that assessed the effect of photobiomodulation on proliferation
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Results on Differentiation

Sixteen procedures assessed cell differentiation (37.2%, n = 16/43) (Table 7). Wavelengths ranged from 620 to 940 nm. In 13 procedures (81.25%, n = 13/16), there was an increase in differentiation; and in three (18.8%, n = 3/16), no effect was reported.

Table 7 Studies procedures that assessed the effect of photobiomodulation on differentiation
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Results on Migration

Three procedures assessed cell migration (7.0%, n = 3/43) (Table 8). Wavelengths ranged from 625 to 660 nm. Two procedures (66.7%, n = 2/3) showed an increase in cell migration and one (33.3%, n= 1/3) reported no effect.

Table 8 Studies procedures that assessed the effect of photobiomodulation on migration
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Discussion

The studies included in this review showed great diversity in protocol design, not only due to the type of device and wavelength applied, but also to important differences in energy parameters, such as dose, exposure time, or light source-sample distance, among others. Furthermore, retrieval location of MSCs varied (adipose tissue, dental pulp, umbilical cord, bone marrow). This diversity made it difficult to draw solid conclusions towards the identification of the best PBM protocols for cell viability, proliferation, differentiation, and migration. Our goal with this review was to confirm PBM´s action on MSCs and, if possible, identify trends in the way cells responded. Thus, further studies are mandatory and should tackle and compare procedure effectiveness.

In the procedures where viability was assessed, 630 nm [26, 27], 635 nm [31, 32], 636 nm [31, 33, 57], 660 nm [36, 43, 44], 668 nm [47], and 808 nm [49]; the most significant increase was found in procedures with 630 nm [27] and 635 nm[31] wavelengths. One study [27] compared different wavelengths (630 nm, 810nm), and again, the most significant increase in viability was seen at 630 nm. On the other hand, the study of Tani et al. [32] did not show viability changes at 635 nm, and the study of Andrade et al. [36] found no differences between compared groups. These findings could be explained by: (i) different cell line, (ii) different exposure time, and, most probable, (iii) different energy density. As a matter of fact, the procedures that reported an increased viability applied lower energy densities: less than 5 J/cm2. In general, studies that reported no effect applied higher energy densities (Table 3).

Proliferation was the most tested variable in these procedures. Most of the procedures that ranged from 620 to 660 nm wavelengths (Tables 2 and 4) showed cell proliferation increase [7, 12, 24, 26, 28,29,30,31,32,33,34,35,36,37,38,39, 43, 44, 46, 49, 57, 58], while a decrease was observed in one procedure [58]. In spite of this positive trend, some results are still difficult to understand: the roles that other variables may play towards cell proliferation remain hidden and a challenge for future studies. The procedures performed with wavelengths ranging from 808 to 980 nm showed the following: in four of them[7, 51, 54, 56], there was an increase; in three [31, 32, 51], no effect was observed; and in one [55], proliferation decreased. In a study carried out by Soleimani et al. [51] at 810 nm, proliferation increased or was not affected, depending on the energy density applied to cells: 2.4 J/cm2 and 3J/cm2 increased proliferation, while a density of 6 J/cm2 had no effect [51].

When wavelengths ranged from 620 to 940 nm (Tables 2, 3 and 4), most authors found an increase in differentiation [24, 25, 28, 31, 35, 48, 50, 51, 53, 56, 59],

In studies assessing migration, Yin K et al. found an increase [46], while Ong et al. did not find any effect at 625 nm [23].

Discussion would be enriched with the presentation of trends, regarding the most effective and popular protocols used, for the reported outcomes to be achieved, however, it has been challenging to find standard protocols of generalized use in humans. For example, in a review about photobiomodulation in bone repair [60], authors found only one article in humans about a clinical case [61]. They included in this review this comment “A lack of persistence in the standardization of methodology employed by authors was observed, with instances of absence of important data, such as output power, energy density and application time, a pattern also observed in reviews relating PBMT to other types of lesions”. The same concern is observed in other reviews, such as review on nerve regeneration, where the authors mentioned regarding the lack of standardization in relation to the application protocols [62]. Another study about photobiomodulation of human osteoblast-like cells in vitro by low-intensity-pulsed LED light cannot compare their results with a previous report because the experimental setups were not identical [63]. With these examples, we want to remark PBM's utility and the importance of focused the research in finding the best variable values to develop standard protocols for each objective that will be of great value to analyze and compare different studies data.

The limitations of the study may include: (1) the fact that it is not a systematic review, since only the PubMed database has been consulted; (2) the great variability of the protocols described in the articles reviewed has made it difficult to trace clear and contrasted trends, although it has been possible to pinpoint a few; and (3) although all the cells studied were human mesenchymal cells, they were grafted from several locations, such as orbital fat, umbilical cord, dental pulp, or bone marrow, among others.

Despite the methodological difficulties already stated, some relationships/trends could be witnessed: (i) blue and green light tended to inhibit the proliferation of hADSCs, (ii) red and NIR light tended to favor cell proliferation and differentiation, (iii) red light tended to favor cell viability, (iv) yellow-orange-green and red light seemed to increase migration, and (v) so far, the combination of any two wavelengths was usually less effective than the most effective of them alone [64,65,66].

Conclusions

As a general conclusion, it can be stated that PBM is an extremely promising way to trigger and stimulate cell metabolic paths that may impact on viability, differentiation, proliferation, or migration, and that might ultimately lead to an enhancement in the cellular regenerative capacity. To determine accurately the clinical potential of PBM and develop efficient and appropriate treatment protocols, future controlled in vivo studies should be performed.