Light assisted modulation of stem cell function and secretome production: a systematic review on current status and new avenues for regenerative medicine

Abstract

Stem cells (SC) based therapies are proving to be the mainstay of regenerative medicine. Despite the significant potential, direct grafting or implantation of SCs for regenerative therapy encounters various translational roadblocks such as paucity of implantable cells, decreased potency, cell death post-implantation, cell damage caused by the pre-existing inflammation and immune rejection. Hence, an emerging avenue is cell-free approach; use of SC secretome. Although priming approaches based on pharmacological molecules/chemicals, cytokines and growth factors are being explored to elicit enhanced secretome production, the potential concerns include the need for continuous replenishment and potential chemical contamination during secretome isolation. To alleviate these concerns, various non-pharmacological approaches for invigorating SCs are also being investigated and among these, use of photobiomodulation (PBM) has garnered considerable interest. Notwithstanding the positive outcomes, standardized parameters are yet to be established for reproducible results. Moreover, the mechanisms of PBM based SC stimulation and secretome production are poorly elucidated and significant knowledge gaps exist on influence of cell type, culture conditions on PBM. This review aims to provide insight into the current status of this emerging field emphasizing on novel avenues and potential challenges for clinical translation. We also summarize the studies on PBM based proliferation, differentiation and secretome production according to SC cell type and culture conditions. Further, as a fixed PBM based protocol for SC proliferation, differentiation and secretome is lacking, the knowledge on functional targets and pathways in PBM based SC stimulation needs upgradation. Consequently, putative mechanisms for PBM based SC secretome have been proposed.

Introduction

The rapidly growing elderly population, a significant rise in chronic illnesses and organ failures, coupled with a shortage of sufficient organs for transplantation have propelled the need for alternative therapeutics. Thus arises the need for regenerative medicine, which is an expanding field of medical science aimed at repairing and regenerating damaged tissues to restore normal tissue function [1]. Stem Cell (SC) therapy has emerged as a key player in the field of regenerative medicine because of SCs remarkable ability to undergo multiple lineage specific differentiation, self-renewal and secrete cytokines, and other bioactive factors [1]. However, to date, the challenges include generating a sufficient number of therapeutically relevant cells, ensuring their survival in the harsh diseased environment after implantation, and preserving their potency following prolonged ex-vivo expansion before infusion. As a result, various cell priming techniques have been developed to enhance cell renewal, differentiation, and potency [2].

A growing body of recent experimental evidence indicates that efficacy of SC therapy is majorly linked to the production of various paracrine factors and extracellular vesicles, collectively known as secretome. SC secretome can modulate the niche required for tissue repair, regeneration and does not elicit overt immune reaction or teratoma formation. Therefore, it is increasingly being argued that the major challenges encountered in the direct SC therapy can be circumvented by use of secretome. Consequently, to maximize secretome production, various pharmacological priming strategies such as cytokines, growth factors, and small molecules are being explored [2]. However, the roadblocks using the pharmacological approaches include high production costs of stimulants, the necessity for continuous replenishment, the requirement of downstream purification and the negative effects of chemical residues on host cells or tissues post application. To address these challenges (Fig. 1), recently, use of non-pharmacological approaches has been explored, amongst which photobiomodulation (PBM) has considerable advantages. One of these is induction of pleiotropic effects, at transcriptional, translational and post translational levels, which have been unveiled by numerous studies on various primary cells and tissues. Hence, PBM is fast emerging as a nondestructive and minimally invasive modality of modulating SC function. In addition, PBM provides straightforward clinical translatability and adaptability, and in some cases, the implanted SCs can be further manipulated using light exposure. Further, as the scope and applications of PBM are ever expanding, a lot of efforts have been directed to mechanistic aspects. One of the most acceptable mechanisms of PBM involves visible and infrared light-mediated excitation of mitochondrial respiratory complex IV enzyme Cytochrome C oxidase (COX) [3] triggering a cascade of secondary events such as increased ATP, Nitric Oxide (NO), calcium levels, and a short-term burst of low-level reactive oxygen species (ROS) (Fig. 2) [3]. Another suggested mechanism is light-mediated activation of transient receptor potential (TRP) channels and intracellular calcium release. Recently, Sommer et al. presented a new model of PBM via changes in the viscosity of mitochondrial-bound water [4]. However, in spite of the encouraging evidences and progresses made, PBM primed SCs can encounter several practical challenges. Herein, the secretome derived from PBM-primed SCs can alleviate majority of these issues [5].

Fig. 1.

Challenges of generating clinical grade stem cells in-vitro

Fig. 2.

Possible effects of PBM on stem cells

Priming SCs for enhancing secretome production

Various priming strategies have been explored to modulate SC proliferation, differentiation and enhance secretome production [1, 5]. These priming methodologies can be of pharmacological and non-pharmacological in nature.

Pharmacological approaches

The biochemical approaches for priming of MSCs include inflammatory cytokines, modified growth media, hypoxia, lipopolysaccharide, protein kinase C activators, p38 MAPK inhibitors, ceramides, and culture of SCs in 3-D matrices [1, 5]. However, chemical and inflammatory cytokine priming methods, are costly, might reduce in vitro multipotency of SCs and elicit undesirable side effects. Priming with cytokines might cause upregulation of class I and II HLA molecules. There are drawbacks of genetic and epigenetic approaches include transient expression, insertional mutagenesis and chromosomal instability. At the same time, epigenetic approaches also show instability, destruction by in vivo nucleases and possibility of off-target genetic activation and repression, which limit the outcome in clinical applications [6]. Hence, there is an urgent need to optimize the SC production process for therapeutic applications.

Non pharmacological approaches

In the last decade, several ‘non-pharmacological’ approaches such as low dose of ionizing radiation [7], non-invasive pulsed focused ultrasound [8], and high-intensity focused electromagnetic and radiofrequency have been used to stimulate stem cell function, differentiation, and migration [9]. However, the mechanisms of alterations, elicited by these physical methods are not known and the protocols have not been established.

Utility of PBM for augmentation of secretome production in SC has neither been studied in great detail nor reviewed. Moreover, many of the cellular response obtained post PBM can be influenced by the type of SCs, the culture condition, the protocol used for isolation of the SCs [10]. For instance, the inherent propensity of adipogenic and osteogenic differentiation for umbilical cord and adipose derived SCs are different.

Hence, this review provides an insight in PBM augmented SC proliferation, differentiation and secretome, while bringing to the forefront the importance of origin and culture conditions of SCs, which influence PBM effects on cellular response. Also, we suggest some putative new mechanisms on PBM based proliferation and secretome production.

Methodology

This systematic review was conducted and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). Original research articles investigating the effects of PBM therapy on SCs and in conjugation with the SC secretome for regenerative applications published in English from 2000 to March 2024 were retrieved and used for this review (Fig. 3). Relevant articles were obtained from online databases like PubMed, Web of Science and Science Direct. A systematic search strategy was conducted using Medical Subject Heading (MeSH) terms (stem cells) AND (low-level laser therapy OR photobiomodulation), (photobiomodulation OR low-level laser therapy), and (stem cell secretome OR stem cell-conditioned medium). Inclusion and exclusion criteria were decided prior to searching in accordance with the PRISMA protocol (Fig. 3).

Fig. 3.

PRISMA flow diagram of the selection process of the studies included in the systematic review

These criteria were as follows:

Inclusion criteria:

• – Laser/LED parameters are mentioned.

• – Articles published in English language.

• – Published in the last 20 years.

• – In vitro & in vivo study on SCs proliferation, differentiation and secretome.

Exclusion criteria.

• – SCs are not studied.

• – Laser /LED parameters are not mentioned.

• – Conference and meeting abstracts.

• – Articles written in a language other than English.

Result

Previous studies indicate that therapeutic benefits of PBM may vary according to the origin of cells. Engel et al. studied the effect of PBM therapy in two distinct oral cell types namely, keratinocytes and fibroblasts and found that oral keratinocytes demonstrated increased sensitivity to laser doses as noted by significant decrease in cellular viability of keratinocytes compared to fibroblasts [11]. Varying the light wavelength can also have different effect on proliferation and differentiation of SCs. In the following subsections we have categorized the results according to the functional attributes that PBM elicits; proliferation, differentiation, secretome production and anti-inflammatory signals, in different SC types, light source and varying PBM parameters (Table 1).

Table 1.

Salient PBM parameters, cell types and growth medium used in studies on SC stimulation, differentiation and anti-inflammatory response

Blue-Green light spectrum
Origin	Stem cell type	Wavelength (nm) 415–540 & Light Source	Growth Medium & Serum supplement	Duration of each administration (s)	Energy density (~J/cm 2 )	Power density (~mW/cm 2 ) or Power	Result	Reference
(i) Human adipose tissue	hADMSC	525 (Diode Laser)	DMEM media, 10% FBS	1 min 23s	5 J/cm2	59.66 mW/cm2	Green light promotes cell proliferation, not as significant as NIR-PBM.	[11]
	hADMSC	415 (LED) and 540 (Filtered lamp)	DMEM media, 10% FBS	188s	3 J/cm2	16 mW/cm2	Blue/green light inhibits proliferation by activating TRPV1 and increasing calcium and ROS species.	[12]
	hADMSCs	532 (Diode Laser)	Low glucose DMEM, 10% FBS	7s	44 m J/cm2	6 mW/cm2	Green light in a time dependent manner enhances hADMSCs proliferation with decreased expression of inflammatory cytokines and chemokines.	[13]
	hADMSCs	420 (LED) & 540 (Filtered Lamp)	DMEM media, 10% FBS	188s five times (every two days)	3 J/ cm2	16 mW/cm2	PBM at 420 nm and 540 nm increase osteogenic gene expression through TRP/calcium signaling pathway.	[14]
	hADMSCs	475, 516 (LED)	Endothelial growth media	10 m	40 (close distance) & 6 (far distance) J/cm2	80 mW/cm2	Blue/green light hampers chondrogenesis	[15]
ii. Human dental pulp	hDPSCs	456 (LED)	DMEM media, 10% FBS	1, 2, 3, 4 and 5 min	2, 4, 6, 8 and 10 J/ cm2	32 mW/cm2	TRPV1/Ca2+ is involved in osteogenic differentiation of hDPSC.	[37]
iii Human bone marrow	hBMSC & Saos-2 cell line	405 (LED)	DMEM media, 20% FBS & F-12 Coon’s modification media, 10% FBS	30 s	0.378 J/ cm2	12.59 mW/cm2	No significant response induced by violet-blue PBM.	[26]
Red light spectrum
Origin	Stem cell type	Wavelength (nm) 620–660 & Light Source	Growth Medium & Serum supplement	Duration of each administration (s)	Energy density (~J/cm 2 )	Power density (~mW/cm 2 ) / (~W/cm2 ) or Power	Result	Reference
(i) Human adipose tissue	hADSC	660 (Diode laser)	DMEM media, 10% FBS	188s	3 J/cm2	16 mW/cm2	Red light at 3 J/cm2. stimulates proliferation 2.	[12]
	hADSCs	660 (Diode laser)	DMEM media, 10% FBS	188 s five times (every two days)	3 J/ cm2	16 mW/cm2	660 nm does not significantly increase level of RUNX2 and calcium.	[14]
	hADSCs	635 (LED)	Endothelial growth media	10 m	40 (close distance) 6 (far distance) J/cm2	80 mW/cm2	Red light enhances chondrogenesis.	[15]
	hADSC	660 (GaAlAs laser)	Keratinocyte serum free media & low glucose DMEM media (1:1), 5% FBS	264 s and 528 s	4 and 8 J/cm2	15.17 mW/cm2	PBM suppresses the LPS mediated inflammation of hADSC via NF-kB transcriptional activity.	[17]
	ADSC	635 (Diode Laser)	Low glucose DMEM media, 10% FBS	10/20/40s	0.5, 1, 2	50 mW/cm2	No significant change in ADSC proliferation.	[18]
(ii) Human umbilical cord	hUCMSC	620 (LED)	DMEM media, 10% FBS	15 m every 8 h	2 J/cm2		PBM at 620 nm enhances proliferation and osteogenesis of hUMSCs when cultured in osteogenic medium	[20]
	hUCMSC	660 (InGaAIP laser)	DMEM media, 20% Ham’s F-12 media, 15% FBS	10 s	2.5 J/cm2	125 mW/cm2	PBM + osteogenic medium enhances the osteogenic proliferation and the differentiation of hUCMSCs.	[21]
(iii) Human bone marrow	BMSC	660 (Semiconductor Laser)	DMEM media, 10% FBS	157 s scanning & covering) and 28s (spot)	4 J/cm2	714 mW/cm2 (scanning & spot) and 25 mW/cm2 (covering)	Compared to control, LLLT at ~4 J/cm2 increases the level of osteogenic genes like ALP, OPN, OCN, and Runx2.	[22]
	hBMSC	650 (LED)	Mesenchymal stem cell growth media, 10% FBS	10 s, 20 s, 30 s, and 40 s	2, 4, 6, and 8 J/cm2	200 mW/cm2	PBM at ~6 J/cm2 enhances the osteogenic differentiation and mineralization via Wnt/β-catenin signaling pathway.	[23]
	hBMSC & Saos-2 cell line	635 (Diode Laser)	DMEM media, 20% FBS & F-12 Coon’s modification media, 10% FBS	30 s	0.378 J/ cm2	12.59 mW/cm2	PBM at 660 nm modulates the functionality of osteoblasts and MSCs.	[26]
(iv) Human dental pulp	hDPSC	660 (InGaAlP Diode Laser)	Ham’s F-12 culture media, 15% FBS	1, 4, 7, 14, 21 and 28s	1, 3, 5, 10, 15 and 20 J/cm2	0.714 W/cm2	PBM at ~5 J/cm2 improves cell proliferation under stressful conditions.	[35]
(v) Huam peridontal ligament	hPDLSC	660 (GaAlAs Laser)	Keratinocyte serum free media & low glucose DMEM media (1:1), 5% FBS	66, 132 & 264 s	1, 2 and 4 J/cm2	15.17 mW/cm2	PBM at 660 nm enhances proliferation and differentiation of hPDLSC via cAMP regulation.	[27]
	hPDLSC	660 (InGaAlP Laser)	α-MEM media, 15% FBS	16.5 s ad 33 s	0.5 and 1.0 J/cm2	31.25 mW/cm2	Proliferation observed at a fluence of ~1 J/cm2 after 48 and 72 h versus the other group.	[28]
	hPDLSC	650 (LED)	DMEM media, 10% FBS	5, 10, 15, 20, and 25 s	2, 4, 6, 8, and 10 J/ cm2	400 mW/cm2	PBM suppressed inflammatory reaction induces by TNF-α.	[29]
	hPDLSCs	635, 660 (laser)	DMEM media, 15% FBS	3 to 16 s	1, 1.5, 2.5, and 4 J/cm2	635: 0.33 W/cm2 & 660: 0.25 W/cm2	Cell proliferation is increased with PBM at 635, 660 nm not as significant as NIR PBM groups.	[33]
(vi) Human Buccal Fat Pad	Human Buccal Fat Pad Mesenchymal Stem Cells (BFPMSCs)	635, 660 (Diode Laser)	DMEM media, 10% FBS	635 nm: 3, 5, 7, and 12 s 660 nm: 4, 6, 10 and 16 s	1, 1.5, 2.5, and 4 J/cm2	635 nm: 0.33 W/cm2 660 nm: 0.25 W/cm2	PBM at 635, 660 nm induces proliferation of hBFP stem cells but less in comparison to 808 nm PBM groups.	[34]
NIR Spectrum
Origin	Stem cell type	Wavelength (nm) 808–1064 & Light Source	Growth Medium & Serum supplement	Duration of each administration (s)	Energy density (~J/cm 2 )	Power density (~mW/cm 2 )/ (~W/cm 2) or Power (mW/ W)	Result	Reference
(i) Human adipose tissue	hADMSC	825 (Diode Laser)	DMEM media, 10% FBS	8 min 1s (for 825 nm)	5 J/cm2	10.394 mW/cm2 (for 825 nm)	NIR irradiation enhances cell proliferation, viability and migration while consecutive application of NIR-green irradiation enhances the MMP, proliferation and migration rate of ADMSC over time.	[11]
	hADMSC	810 (Diode Laser)	DMEM media, 10% FBS	188s	3 J/cm2	16 mW/cm2	NIR PBM enhances proliferation at the same 3 J/cm2.	[12]
	hADSCs	810 (Diode laser)	DMEM media, 10% FBS	188s five times (every two days)	3 J/ cm2	16 mW/cm2	No significant increase in level of RUNX2 and calcium at 810 nm.	[14]
	hADSC	830 (GaAlAs Laser)	DMEM media, 10% FBS	15 m	0.05 J/cm2	83.5 mW/cm2	830 nm PBM enhances the proliferation and viability of ADSCs in-vivo.	[16]
	ADSC	809 (Diode Laser)	Low glucose DMEM media, 10% FBS	10/20/40s	0.5, 1, 2 J/cm2	50 mW/cm2	Mineralization increased significantly in the 809 nm laser groups.	[18]
	hADSC	940 (InGaAsP diode Laser)	DMEM media, 10% FBS	3.57s	0.54 J/cm2	0.15 W/cm2	ADSCs differentiate into fibroblastic and chondrogenic phenotypes after irradiation at 940 nm.	[19]
(ii) Human bone marrow and mesenchymal tissue	BMSCs	808 (Diode Laser)	RPMI media, 10% Fetal calf serum (FCS)	60 s (every 24 h for 0, 5, 10 and 15 days)	64 J/cm2	1 W/cm2	PBM at ~64 J/cm2 increases the expression of Runx2, an early marker of osteoblast differentiation. Also, higher-fluence suppressed the synthesis of adipogenic transcription factor (PPARg).	[24]
	hMSC	1064 (Laser)	DMEM media, 15% FBS	2 min everyday	8.8, 17.6, and 26.4 J/ cm2	0.07, 0.14 & 0.22 W/cm2	PBM at ~17.6 J/cm2 reduces Adipogenic marker PPARγ in hMSCs and upregulated SOD2 gene by ~ 20-fold.	[25]
	hMSC & Saos-2 cell line	808 (GaAlAs Diode Laser)	DMEM media, 20% FBS & F-12 Coon’s modification media, 10% FBS	30 s	0.378 J/ cm2	12.59 mW/cm2	NIR irradiation displays modification of cytoskeleton, Runx-2 expressions and mineralization.	[26]
(iii) Human peridontal ligament	hPDLSCs	810 or 940 (Laser)	DMEM media, 15% FBS	3, 8 and 13 s	0.5, 1.5 and 2.5 J/cm2	200 mW/cm2	Increase in viability & proliferation noted with 940 nm PBM at ~2.5 J/cm2 at all the time points compared to other groups.	[30]
	hPDLSCs	940 (Diode Laser)	Low glucose DMEM media, 10% FBS	13 s (96 well plate) & 384 s (6 well plate)	4 J/cm2 continuous wave	303 mW/cm2 (96 well plate) & 9.6 mW/cm2	No significant increase in proliferation by PBM. Increase in osteogenic related genes and alkaline phosphatase activity was reported in irradiated groups.	[31]
	hPDLSCs	1064 (Nd: YAG Laser)	α-MEM media, 10% FBS,	20 s every other day	2, 4, 6, and 8 J/cm2	-	PBM at 2–6 J/cm2 promotes proliferation and osteogenesis while PBM at ~8 J/cm2 suppresses osteogenesis.	[32]
	hPDLSCs	808 and 980 (Laser)	DMEM media, 15% FBS	2.5 to 16 s	1, 1.5, 2.5, and 4 J/cm2	808 nm: 0.4 W/cm2 980 nm: 0.25 W/cm2	Maximum cell viability noted after irradiation by 980 nm laser with energy density of ~4 J/cm2 on day 3.	[33]
(iv) Human buccal fat	BFPSCs	808 and 980 (Diode Laser)	DMEM media, 10% FBS	808 nm: 2, 4, 6 and 10 s 980 nm: 4, 6, 10 and 16 s	1, 1.5, 2.5, and 4 J/cm2	808 nm: 0.4 W/cm2 980 nm: 0.25 W/cm2	Highest proliferation rate of the stem cells at 808 nm with ~2.5 J/cm2. 980 nm wavelength is not suitable for proliferation.	[34]
(v) Human dental pulp	hDPSCs	808 (InGaAIP Laser)	α-MEM media, 10% FCS	60 s	6 J/cm2	0.33 W/cm2	When subjected to lipopolysaccharide model PBM blocks the odontoblastic differentiation of DPSCs.	[36]

PBM induced proliferation, generation of anti-inflammatory signals and differentiation in SCs

PBM induced effects in SCs of different origin

ADSCs

For ADMSCs, consecutive exposure to NIR (810 nm) and green light (525 nm) irradiation at a fluence of ~ 5 J/cm² leads to increased proliferation rate, mitochondrial membrane potential and migration rate of ADMSCs with green light being not so effective [12]. A study reported inhibitory effect of blue (415 nm) and green (540 nm) wavelengths on the proliferation of hADSCs, compared to enhanced proliferation with red/NIR (660 and 810 nm) wavelengths at ~ 3 J/cm² [13]. In contrast, green light (532 nm) enhanced the proliferation rate of hADSCs and decreased the levels of inflammatory cytokines and chemokines [14]. Wang et al. demonstrated that blue (420 nm) and green (540 nm) wavelengths have much pronounced effect on osteogenic differentiation through the TRP/calcium signaling pathway [15]. However, the study did not document the irradiance. In a wavelength-dependent study, hADSCs from donors with low intrinsic chondrogenic potential had enhanced proliferation in form of increased pellet size formation, activation of COL2A1 after exposure to red light (635 nm) PBM, while the shorter wavelengths (475 and 516 nm) led to reduced pellet size, GAG/DNA content [16].

LLLT can enhance proliferation of hADSCs in vitro and in vivo when irradiated at 830 nm [17]. Wu et al. reported that PBM using 660 nm laser at ~ 8 J/cm² suppressed the LPS-mediated inflammatory response in hADSCs [18]. In a study on ADSCs, exposure to 635 and 809 nm resulted in no change in proliferation and differentiation while mineralization increased only in 809 nm laser group [19]. As 809 nm is expected to activate cell membrane photoacceptors and Ca⁺⁺ signaling, this observation requires further studies, as to whether mineralization is wavelength dependent. Karic et al. demonstrated that application of a 940 nm laser diode results in the differentiation of ADSCs into fibroblasts and chondrocyte [20].

UMSCs

LED irradiation at 620 nm alone did not elicit significant increase in hUMSC proliferation and osteogenic differentiation, but, in combination with osteogenic medium, strikingly, it increased proliferation and osteogenic differentiation [21]. Combined effect of PBM and osteogenic differentiation medium shows stimulation of both proliferation and differentiation [22] [Table 1] with highest response observed in combined group, followed by the PBM.

BMSCs

In BMSCs, the biostimulatory effects of PBM (~ 4 J/cm²) is most prominent for covering irradiation compared to spot and scanning irradiation [23]. High intensity (200 mW/cm²) exposure (~2–8 J/cm²) does not alter proliferation, but osteogenic differentiation and mineralization via Wnt/β-catenin signaling pathway activation [24]. In other studies, while exposure to 808 nm diode laser at a high fluence of ~ 64 J/cm² enhanced osteogenic differentiation of BMSCs [25], exposure to 1064 nm at ~ 17.6 J/cm² reduced adipogenic marker PPARγ in hMSCs and upregulated SOD2 gene by ~ 20-fold [26]. Even though a study on potential of PBM using different wavelength shows that red wavelengths can effectively promote or improve bone regeneration [27], the study involves a single fluence. Hence, it is difficult to conclude about an optimum fluence.

PDLSCs

PBM also has been demonstrated to elicit increased proliferation in human periodontal ligament stem cells (PDLSCs). Wu et al. [28] reported that PBM at 660 nm enhanced proliferation and differentiation of hPDLSC via cAMP regulation. Also, Soares et al. [29] have demonstrated that energy density of 1 J/cm² (660 nm) has a positive influence on the in-vitro proliferation of hPDLSC. Yamauchi et al. demonstrated that high-intensity red LED (650 nm) can promote intracellular ATP synthesis on hPDLSCs [30].

Rigi Ladez et al. demonstrated that in case of exposure to ~2.5 J/cm², diode laser of 940 nm instead of 810 nm showed a better proliferation of PDLSCs at 24, 48 and 72 h [31]. Their results are in concurrence with the results obtained in the study on inflamed hPDLSCs which show that exposure to ~ 4 J/cm² leads to increased osteogenic-related genes and alkaline phosphatase [32]. Wang et al. observed enhanced proliferation, osteogenic differentiation via BMP/Smad signaling after 1064 nm exposure at ~ 2–6 J/cm² on day 21 and Etemadi et al. also observed positive effects of 980 nm Laser irradiation (~ 4 J/cm²) on PDLSCs on day 3 [33, 34].

BFPSCs

A comparative study conducted on buccal fat pad SCs using 635, 660, 808 and 980 nm at fluence of ~ 1, 1.5, 2.5 and 4 J/cm² suggest that fluence of ~ 1.5 J/cm² at 808 nm is the best parameter [35].

DPSCSs

Ferreira et al. reported an increase in proliferation and OCT4, Nestin, CD90, and CD105 expression as assessed by RT-qPCR at 48 h, after PBM treatment at ~ 5 J/cm² can improve cell proliferation in an oxidative stress milieu without interfering with the undifferentiated status of MSCs [36]. Laser-irradiated human dental pulp SCs (hDPSCs) could suppress gene expression of TNF and RANKL and prevent odontoblastic differentiation of DPSCs subjected to inflammation via treatment with lipopolysaccharide (LPS) [37]. Interestingly, another study also reported that blue LED irradiation at a fluence of ~ 2–10 J/cm² can induce osteogenesis in hDPSCs via upregulation of transient receptor potential vanilloid 1 (TRPV1), indicating the involvement of TRP channels in differentiation [38].

To date, data on the accelerated cellular response to PBM remains inconclusive, varying with wavelength, type of light source (LED/laser), cell origin, and culture medium.

PBM induced secretome production in SCs

PBM along with conditioned medium (CM) derived from hBMSC leads to increase in cellular viability of human dermal fibroblast (HDF) cultured in high glucose medium [39]. CM derived from ADSCs post-PBM on hypertrophic scar fibroblasts (HSFs) and keloid fibroblasts (KFs) could reverse the fibrosis by downregulating the profibrotic genes TGF β1 and Notch-1 [40]. In streptozotocin induced Type I diabetic rat, the synergistic effect of PBM and CM derived from BM-MSCs shortened inflammatory phase and hastened wound healing along with increased gene expression of bFGF, SDF-1 α, and HIF-1α in the CM + laser group [41]. Combination of CM and PBM could improve wound healing along with an increase in angiogenesis and anti-inflammatory activities in infected wounds of type 1 diabetic rat [42]. Khoshirat et al. demonstrated that the combination of CM + PBM had a protective effect on PC12 cells against oxidative stress [43]. CM from photobiomodulated ADSC spheroids could drive angiogenesis and be used as a paracrine-mediated therapy for wound healing [44]. Very recently, Qashty et al. reported that ADSCs and secretome treatments associated with LLLT expressed high therapeutic potential for temporomandibular joint arthritis (TMJ) arthritis with no significant difference [45]. However, for a specific cell type, there is significant variation in the wavelength and fluence used to derive CM/secretome production following PBM. Additionally, there is no comparative evidence to determine if PBM-induced SC secretome production depends on any particular SC cell type.

PBM effect as a function of wavelength and fluence

Majority of studies till date have used red and NIR light, compared to green and blue light for enhancing proliferation, differentiation and secretome production by SCs. Further, an important inference is that for the wavelength spanning 550–940 nm, in the majority (~ 80%) of the studies, the range of fluence for the increasing rate of proliferation and anti-inflammatory effects lies within a range of ~ 0.5–5 J/cm² (Table 1). However, a considerable percentage (~ 40%) of the studies on differentiation and secretome production have also documented beneficial effect in range > 5 J/cm²(Tables 1 and 2). Therefore, more studies are required to come to the conclusion that a varying fluence range can induce different cellular responses like proliferation or differentiation. Whether using green or red light, a biphasic dose response can be observed in ADMSCs and the threshold fluence for cellular inhibition may be > 10 J/cm². Such fluence dependent dichotomous response in other cell types are not documented.

Table 2.

Salient PBM parameters, study model, source of conditioned medium used for secretome production by SC and related applications

Study Model (2 D in vitro / 3-D in vitro /In vivo)	Source of conditioned medium (CM)	Irradiation Parameter & Light source	Outcome	Reference
Human Dermal Fibroblast (HDF)	hBMSC	632.8 nm (HeNe Laser), ~0.00185 W/cm2, 0.5 (378s) & ~1 J/cm2 (756s)	LLLT + BMSC-CM enhanced viability of HDF cultured in high glucose medium	[38]
Keloid tissue samples, hypertrophic scar tissues, normal skin tissues and fibroblast cells	hADSC	655 & 635 nm (Dual model laser beam), 152 s, ~4 J/cm2	TGF-β1 ↓ and Notch-1 ↓ PBMT-AMSCs-CM demonstrated potential of fibrotic treatment of KFs and HSFs	[39]
T1 DM mice model	hBMSC	890 nm (Laser), 200 s, ~0.2 J/cm2, ~1.08 mW/cm2	CM + Laser treatment group: accelerated wound healing, Neutrophils↓, Fibroblasts↑, angiogenesis ↑	[40]
T1 DM mice model of MRSA infected wound	hBMSC	890 nm (Laser), 200 s, ~0.2 J/cm2	Macrophages ↓, wound healing↑, anti-inflammation & angiogenesis ↑	[41]
PC12 cells	BMSC	890 nm (Laser), 3 times a week, ~1.5 J/cm2, ~1.15 W/cm2	Bcl2 ↑ and Bax ↓ Reduction in oxidative stress	[42]
ADSC spheroid (3-D model)	ADSC	660 nm (LED), 10 min, ~12 J/cm2, ~20 mW/cm2	PBM-spheroid-CM stimulated angiogenesis	[43]
Rat ADSC	ADSC	980 nm (Diode Laser), 60 s, ~38 J/ cm2, ~0.64 W/cm2	LLLT + ADSC or LLLT + ADSC-CM enhanced healing of arthritic TMJs rat model	[44]

PBM using lasers vs. LEDs

Among the studies covered in the review, both laser and LEDs have been used for PBM. Though laser versus LEDs have received some attention, in other cell types but in case of SCs, no study directly compares the effect of laser vs. LED (green/blue/red/NIR) using SC of a particular origin on differentiation and secretome. The earlier studies used lasers or diode lasers for PBM. The wide availability, cost benefit and safety aspect seems to propel the field to use LEDs.

Discussion

Generally, SCs exert their therapeutic effects through homing and migration to the target tissue, differentiation and secretion of various bioactive factors to restore/replace the damaged tissues [1, 2]. However, the direct use of SCs has a number of limitations such as possibility of uncontrolled growth, variable treatment output due to immune reaction and inherent variation in regenerative properties of SCs. To overcome these, secretome is going to be an alternative cell-free strategy.

The existing literature on PBM point out that given a cell type, light wavelength can affect proliferation and differentiation. Further, our analysis presented in this review shows that light fluence used for inducing cell proliferation, differentiation and secretome enhancement in the published studies may be different (Fig. 4B i-iv). Furthermore, possible influence of cell origin, light wavelength as well as fluence on SC secretome production is not clear, as there is no mechanistic evidence.

Fig. 4.

A: Possible avenues of light stimulated stem cell secretome production. B: Fluence used for different studies in stem cells; (i) cell proliferation, (ii) differentiation, (iii) secretome production and (iv) eliciting anti- inflammatory effects

Possible influence of the method of isolation, origin, culture condition on PBM outcome in SCs

For hADMSCs, expression of pluripotent genes such as Oct3/4, Nanog, and Klf4 decrease after culture of cells in stromal vascular fraction (SVF) solution on tissue culture polystyrene dishes, whereas the pluripotent gene expression of the cells in SVF solution is maintained after purification by filtration [46]. Previously, comparative analysis of neonatal MSCs derived from amniotic membrane, umbilical cord, and chorionic plate from same donor, grown under serum free conditions demonstrated that neonatal MSCs exhibit a similar morphology and immunophenotypic pattern, but various mesodermal differentiation potentials [47]. Another study reported that meso and endodermal differentiation of cells cultured in serum supplemented medium is correlated with high activity of Wnt/JNK pathway and blocking Wnt inhibits mesodermal conversion of PSCs. When grown in chemically defined medium without serum supplementation, PSCs differentiate preferentially to ectodermal type. However, further studies are necessary to validate whether differentiation ability, expression of pluripotent gene in ADMSCs, isolated via conventional culture, membrane filtration and membrane migration methods may vary post PBM. Also, the influence of cell origin, culture conditions on SC proliferation and differentiation in context of PBM needs attention [48]. These investigations would lead to optimization of a more reproducible PBM protocol.

Influence of PBM parameters and cell physiological status on PBM response in SCs

Light wavelength can play a determinant role in PBM as the nature of chromophore vary according the wavelength. While red wavelength light excites mitochondrial CCO, NIR can act through membrane photoreceptors. Further, light fluence governs the amount of ROS generated inside the cells following PBM and can determine the cell response. Our presented scheme in Fig. 4B, indicate that PBM induced proliferation and differentiation can be fluence dependent. Apart from light fluence and wavelength, irradiance can have significant influence on PBM outcome using SCs. A previous study shows that, for a particular fluence (~ 2.5 J/cm²) and wavelength, increasing the irradiance from ~ 8 mW/cm² to ~ 40 mW/cm² does not yield different outcome. In contrast, for ~ 5 J/cm², the same irradiance range leads to distinct outcomes [49].

It has been observed that with the same parameters of irradiation, PBM could depend greatly upon the cell type with varying mitochondrial count. For instance, differential PBM response is observed in myoblast and myotubes, containing differing mitochondrial number, exposed to blue (400/450nm) or NIR light (810 nm). Myotubes with high mitochondria number produce high level of ROS after blue light compared to NIR light exposure [50]. Likewise, level of catalase varies in different cell types and this affects the fluence required to achieve a positive or negative outcome in PBM. In case of SCs, similar aspects need to be established, as cell type/origin would determine the wavelength and fluence to be chosen for a given response.

Putative mechanisms of PBM induced secretome production by SCs

Over the last one decade, a handful of studies have shown that in addition to increased proliferation and lineage specific differentiation, PBM may enhance secretome production by SCs. The complete mechanism of such observations are still under investigation. From mechanistic perspective, various previous studies implicate that while low ROS level can elicit SC renewal, a little higher concentration of ROS elicit differentiation response and the very high level of ROS being responsible for oxidative stress. Similarly, NO also functions as a signaling agent. Therefore, ROS generation and photo-dissociation of NO from CCO during PBM, can have differential actions which culminate in activation of various signaling pathways such as growth factor production, MAP K activation (Fig. 5A). These actions are dependent upon the levels of ROS, reactive nitrogen species (RNS) and antioxidants (Fig. 5A). While role of NO signaling in SCs and other cells have been studied to some extent, whether such mechanisms operate post PBM, have to be studied.

Fig. 5.

A: Possible ways by which PBM induced ROS and RNS can activate various signaling pathways in SCs. nM: Nano molar. ONOO⁻: Peroxy nitrite radicals. P: Phosphorylation. Adapted from [49]. B: Proposed mechanisms of PBM mediated ROS function in stem cells, via activation of a redox-state dependent protein known as non-histone high mobility group box 1 (HMGB1). Adapted from [50]

A unique feature of SCs is that, in hypoxic milieu SCs remain in quiescent stage, but can achieve increased self-renewal and differentiation capability by upregulating their own endogenous ROS level through mobilization of intracellular machinery [49]. It can be interpreted that, possibly due to the change of priorities from glycolysis to oxidative phosphorylation, there would be a metabolic switch that enhances differentiation of stem cells. With respect to mechanistic aspects of PBM and current perspective of the review, a new concept can be proposed, based on previous observations. The superoxide radicals (O₂.⁻), H₂O₂ can trigger redox signaling pathways that are recognized by transcription factors like Nuclear factor erythroid derived 2 (NRF2), forkhead box protein O (FoxO) and p53.

These transcription factors are sensitive to oxidative stress and stimulate the expression of genes involved in antioxidant defense mechanisms, thereby aiding in the maintenance of cellular homeostasis. Another redox-dependent protein, playing crucial role in SC biology, but not explored from PBM perspective, is non-histone high mobility group box 1 (HMGB1) protein [51]. HMGB1 level is affected by intra and extracellular ROS levels. Elevated levels of H₂O₂ within the cell result in the partial oxidation of HMGB1, forming a disulfide bridge between Cys23 and Cys45 while reducing Cys106. This process leads to the complete impairment of HMGB1’s nuclear functions [49] (Fig. 5B). While ROS dependent signaling actions leading to HMGB1 and increase in SC proliferation, differentiation, tissue regeneration are well known, whether such responses could be elicited by PBM, in particular, needs to be investigated. Further, production of secretory vesicles by SC post PBM, is an aspect that begs current attention. Moreover, exosomes can alter and enhance the response of their recipient cells such as fibroblasts, keratinocytes present at the application site (Fig. 4A) against oxidative stress. So, this is an emerging avenue as far as PBM assisted SC secretome is concerned.

Future perspective

Numerous studies implicate that while low ROS level elicit SC renewal, a little higher concentration of ROS elicit differentiation response and the very high level of ROS being responsible for oxidative stress (Fig. 5A-B). Considering that ROS generation during the PBM is fluence dependent, it would be expected that SC proliferation, differentiation after PBM follow the fluence. However, in the studies thus far, there seems to be no correlation between fluence and type of response. While, ~ 80% of the reported studies have shown that cell proliferation and anti-inflammatory responses are observed at fluence below ~ 5 J/cm² (Fig. 4B, i & iv), in studies on SC differentiation and secretome much higher fluence in the range of ~ upto 64 J/cm² have been utilized (Fig. 4B, ii & iii). Therefore, a systematic study has to be performed to investigate if self-renewal and differentiation response are indeed PBM fluence dependent. Furthermore, the exact mechanism by which PBM would elicit secretome is not clear. Hence, the relation between PBM fluence and secretome production also needs a clear understanding.

The potential of PBM to enhance the secretion of bioactive factors by SCs is still being explored, and further research is necessary to fully understand the underlying mechanisms. Besides boosting the quantity of secretome production, altering the spectrum of factors within SC-derived secretome is another promising approach to maximize the therapeutic benefits of secretome-based cell-free therapy. In this context, PBM presents distinct practical advantages over chemical mediators or growth factor/cytokine supplementation. As is known, different wavelengths can induce different cell signaling, it is expected to cause different secretome factors production. If so, then more than one light wavelength can be combined.

Another avenue of research is the development of new delivery vehicles for secretome, as the secretory components are mostly proteins, which can be cleaved by MMPs and proteases present in chronic inflammatory conditions. Numerous vehicles have been investigated for delivering the secretome, which is beyond the scope of this review, but the most suitable one can be formulated to deliver the secretome derived from light-stimulated cells. Therefore, the reviewed literature can be summarized as follow:

• (i)Fluence used for cell proliferation, secretome production might not be the same.
• (ii)PBM effect on SC secretome may vary according to cell types.
• (iii)ROS generated during different fluence is different. Hence, secretome induction by PBM must be studied as a function of wavelength and fluence.
• (iv)For practical application, clinical studies on CM derived from SCs subjected to PBM can be considered for applications which require prolonged but multiple treatments.

Lastly, laser/LED assisted PBM on various SCs including ADMSCs, PDLSCs significantly increases proliferation and osteogenesis. In fact, the reviewed literature in Tables 1 and 2 signify a standardized protocol to stimulate SCs to achieve the maximum potency is the need of the hour.

Conclusion

The use of primed SC or SC-based secretome for tissue regeneration has immense potential to overcome some of the major roadblocks associated with SC therapy. For secretome-based therapy, PBM can be used to maximize both the production and achieve enrichment of the secretome. However, studies summarized in this review suggest that there is a diverse range of irradiation parameters and wavelengths for therapeutic applications. Consequently, there are critical unmet needs and issues to be addressed. These are: (1) elucidating the mechanism by which PBM stimulates secretome production and (2) the kinetics of pathway activation and trophic factor production in vitro and in vivo. Further, as no standardized protocols are available regarding use of PBM for SCs, optimization of a fixed PBM protocol for increasing the rate and quantity of secretome along with increased self-renewal and lineage specific differentiation potency will produce both a safe and a cost-effective procedure that may be efficiently translated in vivo for the clinical use of various ailments.

Abbreviations

SC

Stem Cell

LED

Light Emitting Diode

LLLT

Low Level Laser Therapy

PBM

Photobiomodulation

COX

Cytochrome C Oxidase

NO

Nitric Oxide

ROS

Reactive Oxygen Species

TRP

Transient Receptor Potential

hADSC

Human Adipose Derived Stem Cell

hPDLSC

Human Periodontal Ligament Derived Stem Cell

hDPSC

Human Dental Pulp Derived Stem Cell

hUMSC

Human Umblical Cord Derived Stem Cell

hBMSC

Human Bone Marrow Derived Stem cells

LPS

Lipopolysaccharide

TRPV1

Transient Receptor Potential Vanilloid 1

CM

Conditioned Medium

HDF

Human Dermal Fibroblast

HSF

Hypertrophic Scar Fibroblast

KFs

Keloid Fibroblasts

TMJ

Temporomandibular Joint Arthritis

SVF

Stromal Vascular Fraction

RNS

Reactive Nitrogen Species

NRF2

Nuclear factor erythroid derived 2

FoxO

Forkhead Box Protein O

HMGB1

High Mobility Group Box 1

MMP

Matrix Metalloproteinases

Author contributions

Mahima Rastogi: Conceptualization, Methodology, Manuscript-Original draft, Mnauscript-Review & Editing. Khageswar Sahu: Conceptualization, Methodology, Manuscript-Original draft, Review & Editing. Shovan Kumar Majumder: Conceptualization, Manuscript-Review & Editing, Supervision.

Funding

Open access funding provided by Department of Atomic Energy.

This study is funded by Raja Ramanna Centre for Advanced Technology, Department of Atomic Energy, Government of India.

Declarations

Ethical approval

The authors declare that the manuscript is original and has not been submitted elsewhere for publications. The authors further declare that during the preparation of this work the author(s) did not use any AI tool. All the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.

Informal consent of authors

Not applicable.

Competing interests

The authors declare no competing interest.