Boosting osteoblast differentiation: enhancing the effects of low-level blue laser therapy on human embryonic stem cell-derived mesenchymal stem cells to improve viability and calcium deposition

Abstract

Mesenchymal stem cells (MSCs) are widely studied for their regenerative capacities in bone tissue repair. Low-level laser therapy (LLLT) has emerged as a promising method to stimulate stem cell proliferation, viability, and differentiation. In this study, we focus on how low-level blue-laser treatment (457 nm) affects human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) at various energy densities, highlighting its potential to enhance osteogenic differentiation for clinical applications in treating osteoporosis. To determine how low-level blue-laser treatment at various energy densities (0.5–5.0 J/cm²) influences the proliferation, viability, migration, and osteogenic differentiation of hESC-MSCs. hESC-MSCs were cultured to near confluence, then irradiated at doses ranging from 0.5 to 5.0 J/cm². Cell proliferation, viability, and migration were assessed at 72 h. Flow cytometry evaluated CD146 expression, and Alkaline phosphate (ALP) activity was measured. Osteogenic gene expression (Runx2, ALP, BMP2, BMP4, and osteonectin) and in vitro mineralization were also examined. Blue-laser treatment at 0.5–3.5 J/cm² significantly increased cell proliferation (p < 0.01) and viability (p < 0.05), while migration was enhanced at 0.5–2.5 J/cm² (p < 0.001). CD146 expression rose at 0.5, 1.0, and 2.0 J/cm², with a 1.9-fold increase in ALP activity at 2.0 J/cm². Osteogenic markers and mineralization were likewise upregulated at 2.0 J/cm², indicating enhanced osteoblast differentiation. These findings indicate that LLLT combined with a blue laser results in changes in various biological processes at the cellular and genetic levels in hESC-MSCs, indicating that these cells are sensitive to blue laser treatment. Thus, these results demonstrate that hMSCs are responsive to blue-laser treatment, which may be used in the clinic to treat osteoporosis.

Clinical trial number

Not applicable

Introduction

Osteoporosis is a progressive bone disease characterized by a decrease in bone mass and density, which can lead to a heightened risk of fractures in the elderly population, resulting in substantial morbidity due to bone fragility [1–3]. Standard treatments for osteoporosis include medications, lifestyle and dietary changes to support bone health. Mesenchymal stem cells (MSCs) have emerged as a promising option in regenerative medicine for treating a range of conditions, including osteoporosis [4, 5]. In clinical settings, MSCs derived from bone marrow or adipose tissue have been used to support bone regeneration due to their ability to differentiate into osteoblasts (OBs) and their trophic effects on bone healing. Human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) may be a superior alternative in the clinic. These cells have several advantages, including their pluripotency, which enables them to differentiate into a broader range of cell types, including OBs.

Low-level laser therapy (LLLT) is used to increase the proliferation of various cell types, such as fibroblasts and epithelial cells, including stem cells [6–10]. LLLT is a fast-growing technology that has been used to treat multiple conditions by increasing the ability of cells to promote wound healing and tissue regeneration [11, 12]. LLLT also has benefits in the field of dentistry, helpful for relieving pain and decreasing inflammation [13–15]. In particular, blue-laser treatment (405 to 480 nm) has been shown to promote tissue repair and the osteogenic differentiation of stem cells [16] via a small increase in reactive oxygen species (ROS) levels. These findings suggest that the tissue-repair effects of a blue-laser may increase stem cell proliferation and viability.

The objectives of the present study were to compare the effects of LLLT at different energy densities on low-level blue-laser (LLBL)-treated hESC-MSCs, with special attention given to differences in cell proliferation, viability, migration and osteoblastic cell differentiation. In our study, we utilized hESC-MSCs as a model for osteoblastic differentiation due to their potential to mimic the regenerative processes of bone tissue. LLBL is a strategic method for promoting osteogenic differentiation by modulating cellular activity and increasing the expression of bone-related markers.

Materials and methods

hESC-MSC culture

The hESC-MSCs were obtained from the Stem Cell Unit, College of Medicine, King Saud University, Riyadh [17]. All media, serum and other reagents used for cell culture were obtained from Gibco (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). All plasticware used during this study was obtained from Thermo (Thermo Fisher Scientific, Waltham, MA, USA). hESC-MSCs were maintained in full culture medium (FCM) with high-glucose DMEM supplemented with 10% fetal calf serum, 1% essential amino acids and 1% antibiotic–antimycotic solution (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 37 °C in a humidified atmosphere containing 5% CO₂. Semiconfluent cells were harvested by incubation with 0.25% trypsin in 0.01% ethylenediaminetetraacetic acid solution at 37 °C for 3–5 min. The trypsinization reaction was stopped by the addition of FCM. The cells were centrifuged at 500 × g for 10 min at room temperature. Then, the cell pellet was resuspended in FCM, after which the cells in the suspension were seeded into 48-well plates at 4 × 10⁴ cells/well and incubated at 37 °C.

Low-level laser irradiation

The hESC-MSCs were divided into two groups: nonirradiated (control) and irradiated groups. The hESC-MSCs in the irradiated groups were irradiated by means of LLBL at different energy densities, such as 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5–5.0 J/cm², for 20 to 200 s, as described in previous publications [18–21]. The output power density of LLBL was 25 mW/cm² during all experiments because it had been optimal for the photobiostimulation of cell proliferation in our laboratory. Briefly, hESC-MSCs were plated in 48-well plates and incubated overnight as described above.

The hESC-MSCs were irradiated only once. Nonirradiated hESC-MSCs were maintained under the same conditions as irradiated hESC-MSCs, such as the replacement of FCM with phosphate-buffered saline (PBS), and room temperature during irradiation. The medium was replaced with sterile PBS to minimize the loss of laser energy due to absorption by the red component of the FCM. Beam has 1 mm diameter. Irradiation area was 10 mm, which is almost equal to surface of each well in the 48-well plate as explained in our previous publications [20, 21]. To avoid the effects of second-order variables, such as quantification and properties, we maintained the cells in all the experimental groups, including the control group, under the same environmental conditions, such as temperature, humidity, and light. After irradiation, the irradiated and nonirradiated hESC-MSCs were maintained in fresh FCM and incubated as described above. All irradiation experiments were performed in triplicate, and the average value for each hESC-MSC group was calculated for analysis.

Cell proliferation analysis

The effect of LLBL irradiation on hESC-MSC viability was assessed by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Specifically, hESC-MSCs were seeded in 48-well plates and incubated overnight. Then, the cells were irradiated and incubated. At the end of 72 h, hESC-MSCs were incubated with 100 µl/well of MTT (0.5 mg/ml in PBS) for 2 h under the same conditions. Then, the MTT solution was replaced with an equal volume of isopropanol for 45 min, and the absorbance at 549 nm was read by a microplate reader (BioTek, USA) [20, 22].

Cell viability rate

The effect of LLBL on hESC-MSC viability was determined by counting the number of viable cells via the trypan blue exclusion assay (Trypan blue solution, Sigma Aldrich Company, Ltd Irvine, Ayrshire, UK). For evaluation of hESC-MSC viability, cells were seeded in 48-well plates and incubated overnight. Then, the cells were irradiated with LLBL and incubated. At the end of 72 h, the hESC-MSCs were washed with PBS and harvested by gentle trypsinization at a constant volume of 0.1 ml, mixed with an equal volume of trypan blue and loaded into a hemocytometer chamber for cell counting. Then, viable (unstained) and nonviable (stained) cells were counted, with three separate counts performed for each well [20]. The numbers of viable and nonviable cells in the irradiated and nonirradiated groups were compared, and graphs showing the cell viability data were generated.

Cell migration assay

The effect of LLBL on hESC-MSC migration was assessed in vitro via a wound healing “scratch” assay [23, 24]. The hESC-MSCs were seeded and incubated overnight. Then, a sterile 200-µl pipette tip was used to create a scratch in the cell monolayer, and the cells were irradiated with LLBL. The cell debris was removed by changing the media, and the cells were further incubated. At 72 h after the scratch was made and irradiation was completed, images were captured to evaluate the number of cells within the scratch zone as an indicator of the level of cell migration. Images were taken using a charge-coupled device camera (IX2-SL, Olympus, Japan) attached to an inverted phase-contrast microscope (Model No. IX51, Olympus, Japan) at a power of 10×. For statistical analyses, images were taken from three separate “scratch areas” at the same magnification. At the end of the experiments, the images were compared to the number of migrated hESC-MSCs in the “scratch area”, and the cell migration data were plotted in graphs. All assays were performed in triplicate on at least two separate occasions.

Analysis of OB differentiation

The characteristics of hESC-MSCs [17] are similar to those of primary hMSCs in terms of their cellular and molecular phenotypes. The hESC-MSCs were cultured in DMEM supplemented with 0.25 mol/l D-glucose, 0.004 mol/l L-glutamine, 0.006 mol/l sodium pyruvate, 10% FBS, 1× penicillin‒streptomycin (pen-strep), and nonessential amino acids (Gibco-Invitrogen, USA). The cells were grown to 70–80% confluence in FCM at a concentration of 50,000 cells/ml. For OB induction, FCM was replaced with OB differentiation medium supplemented with a cocktail of 10% FBS, 1% pen‒strep, 0.003 mol/l L-ascorbic acid (Wako Chemicals, Germany), 0.01 mol/l β-glycerophosphate (Sigma, Germany), 1e8 mol/l calcitriol (1α,25-dihydroxy vitamin D3; Sigma), and 1e-8 mol/l dexamethasone (Sigma). OB differentiation was performed in 48-well plates to induce hESC-MSC differentiation toward the OB lineage. We treated cells with LLBL at an energy density of 0.5, 1.0–2.0 J/cm² with the control. After treatment with LLBL, OB differentiation medium was added to each well of hESC-MSCs, and 48-well plates were incubated for 10 days. The OB differentiation media were replaced three times per week, and the conditions were maintained for 10 days [17]. The differentiation process was monitored by measuring alkaline phosphatase (ALP) activity.

Quantitative ALP activity

To accurately measure ALP activity after OB differentiation, we utilized the ALP Activity Colorimetric Assay Kit from BioVision, Inc. (California, USA) with modifications. On the day designated for analysis, each well was gently washed with PBS to remove any unbound cells or debris. Subsequently, 50 µl of the provided p-nitrophenyl phosphate (pNPP) substrate solution was dispensed into each well. The plates were then shielded from light and incubated for 2 min at room temperature for the enzymatic reaction. After incubation, the optical density (OD) of the resultant product was measured at a wavelength of 405 nm with a spectrophotometer designed for microplate readings.

Flow cytometry

Flow cytometric analysis was conducted with single cells derived from both the LLBL-treated populations and the LLBL-untreated controls. Following LLBL, the cells were dissociated using trypsinization and subsequently neutralized with 10% serum. The cells were then resuspended in fluorescence-activated cell sorting (FACS) buffer (PBS [Invitrogen] supplemented with 1% Fetal bovine serum) at an approximate density of 1 million cells/ml. For the immunophenotyping of each MSC marker, we allocated approximately 100,000 cells for staining with each specific antibody. We utilized a panel of antibodies, including CD14APC, CD29PE, CD31FITC, CD34FITC (from DAKO), CD44PE, CD73PE, and CD146PE. The corresponding isotype control antibodies, IgG1-FITC, IgG1-PE, and IgG-APC, were obtained from BD Biosciences (San Diego). The cells were stained with the indicated antibody at a 1:10 dilution in FACS buffer for 30 min on ice. After two washes in FACS buffer, the cells were resuspended in the same buffer. A minimum acquisition of 10,000 events per sample was achieved using a FACSCalibur flow cytometer from BD Biosciences. Data analysis was carried out with CellQuest Pro^® software.

Quantitative real-time PCR (qRT‒PCR)

Total RNA was isolated from nonirradiated and irradiated hESC-MSCs using an innuPREP RNA Mini Kit (Analytik Jena, Germany, REF No: 845-KS-2040250) according to the manufacturer’s protocol. The concentration and purity of the extracted RNA were assessed with a NanoDrop spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, USA). We synthesized complementary DNA (cDNA) from 1 µg of total RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA), a ProFlex RT PCR system and a Multigene thermal cycler according to the manufacturer’s instructions.

For analysis of mRNA levels, RT‒PCR was performed with cDNA samples utilizing the Applied Biosystems Real-Time PCR Detection System and the Fast SYBR Green PCR Kit (Applied Biosystems, UK), again following the manufacturer’s detailed protocol. Gene expression was standardized against the expression of the housekeeping gene GAPDH. We used the comparative cycle threshold (CT) method to determine relative expression levels, where ΔCT represents the difference in CT values between the target and reference genes. Experimental details used in this study are showing in flowchart Fig. 1.

Fig. 1.

Flowchart showing experimental details used in this study

Statistical analysis

One-way analysis of variance (ANOVA) was used for comparisons of continuous data, which are presented as the mean ± SD, with Tukey’s HSD test used for post hoc analysis (GraphPad Prism, version 4.0, San Diego, CA, USA). A P value < 0.05 was considered to indicate statistical significance. We performed student t-test for FACS & PCR data and graphs were prepared using Microsoft excel.

Results

LLBL irradiation increases the proliferation of cultured hESC-MSCs

For analysis of the effects of LLBL at various energy densities on the biological characteristics of hESC-MSCs, we performed an MTT-based proliferation assay. Our MTT-based analysis of hESC-MSC proliferation revealed a significant increase in hESC-MSC proliferation following irradiation with LLBL at different energy densities. LLBL increased hESC-MSC proliferation by 1.27, 1.31, 1.35, 1.39, 1.35, 1.35, 1.32, 1.03, 0.95 and 0.95-fold at doses of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 J/cm², respectively, compared to that of the control, with the maximal effect obtained following irradiation at 2.0 J/cm² (P < 0.001) (Fig. 2a). However, blue-laser irradiation did not significantly increase the proliferation of hESC-MSCs when the energy density increased from 4.0 to 5.0 J/cm². Post hoc tests revealed that blue laser irradiation at energy densities ranging from 0.5 to 3.5 J/cm² had significant effects, and the lowest P value was < 0.01 (Table 1).

Fig. 2.

a: Effect of low-level blue-laser treatment at a dose of 0.5 to 5.0 J/cm² with no irradiation (control) on the proliferation of human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) after 72 h of irradiation. Dimethylthiazol tetrazolium bromide (MTT) assay was used to measure cell proliferation. b: Effect of low-level blue-laser treatment at doses ranging from 0.5 to 5.0 J/cm² with no irradiation (control) on viability of hESC-MSC after 72 h of irradiation. Trypan blue staining was used to measure cell viability. Cells were obtained from the wells of a culture dish by trypsinization, and the number of living hESC-MSCs was counted under a microscope as explained in methodology section. c: A scratch assay was used to determine the effect of 0.5 to 5.0 J/cm² low-level blue-laser treatment on the migration of hESC-MSCs after 72 h of irradiation. The number of migrated hESC-MSCs was counted in the scratch area. The results of control and treatments were compared and graph was plotted

Table 1.

Statistical data analysis of human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) treated with different energy densities of blue-laser irradiation

Low-level laser therapy dose (J/cm2)	P value
	Proliferation	Viability	Migration
Control vs. 0.5	P < 0.01	P < 0.001	P < 0.001
Control vs. 1.0	P < 0.001	P < 0.001	P < 0.001
Control vs. 1.5	P < 0.001	P < 0.001	P < 0.001
Control vs. 2.0	P < 0.001	P < 0.001	P < 0.001
Control vs. 2.5	P < 0.001	P < 0.001	P < 0.001
Control vs. 3.0	P < 0.001	P < 0.05	P > 0.05
Control vs. 3.5	P < 0.001	P < 0.05	P > 0.05
Control vs. 4.0	P > 0.05	P > 0.05	P > 0.05
Control vs. 4.5	P > 0.05	P > 0.05	P > 0.05
Control vs. 5.0	P > 0.05	P > 0.05	P > 0.05

Significance values obtained from one-way ANOVA of the means of three cell culture experiments examining the proliferation, viability and migration of hESC-MSCs treated with blue-laser irradiation at different low-level laser energies and for controls (no irradiation or 0.0 J/cm2) using one-way ANOVA and the Tukey HSD post hoc test

LLBL therapy increases the viability of cultured hESC-MSCs

Compared with the control cells, the hESC-MSCs exposed to LLBL exhibited increased cell viability in an energy density-dependent manner (Fig. 2b). Compared with the control, blue-laser treatment significantly increased the viability of hESC-MSCs by 2.01, 2.32, 1.92, 1.78, 1.77, 1.5, 1.48, 1.08, 1.05 and 1.04-fold in wells exposed to 0.5, 1.0, 1.5 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 J/cm², respectively, with a maximal and significant effect obtained following 1.0 J/cm² treatment (P < 0.001). Compared with that of the cells in the control group, the number of viable hESC-MSCs treated with blue-laser radiation from 4.0 to 5.0 J/cm² also increased, but the difference was not significant.

Post hoc analysis revealed that blue laser irradiation at energy densities ranging from 0.5 to 3.5 J/cm² significantly increased the viability of the treated hESC-MSCs compared with that of the control hESC-MSCs, and the lowest P value was < 0.05 (Table 1).

LLBL therapy increases the migration of cultured hESC-MSCs

Compared with those in the control group, the number of hESC-MSCs that migrated into the scratch area in the wound healing assay significantly increased from 0.5 to 2.5 J/cm². As shown in Fig. 2c, compared with the control treatment, blue laser treatment increased hESC-MSC migration by 1.65, 1.84, 1.92, 2.0, 1.89, 1.39, 0.98, 0.78, 0.86 and 0.73-fold at energy densities of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 J/cm², respectively, and the maximal effect was observed following treatment at 2.0 J/cm² (P < 0.001). Phase contrast photomicrographs of the wound-healing assay revealed that LLBL significantly increased the migration of hESC-MSCs at an energy density of 2.0 J/cm² (Fig. 3). However, although blue-laser irradiation increased the migration of hESC-MSCs at an energy density of 3.0 J/cm², the difference was not significant, and at higher energy densities ranging from 3.5 to 5.0 J/cm², blue-laser irradiation did not increase the migration of hESC-MSCs relative to that of the controls, suggesting that LLBL at high energy densities was ineffective. One-way ANOVA revealed that the energy densities of the LLBL at 0.5 to 2.5 J/cm² significantly increased the migration of hESC-MSCs, and the P value was < 0.001 (Table 1).

Fig. 3.

Representative phase-contrast images demonstrating hESC-MSC migration to the center of the scratch to close the gap caused by the “scratch” in the control and low-level blue laser-treated hESC-MSC groups. The number of cells that had moved into the wound/scratch area was counted after 72 h irradiation. The treatment and control (non-irradiation a) groups were compared. The energy densities used were 0.5, 2.0–4.0 J/cm² (b, c and d, respectively). The experiments were performed in triplicates, and the data were analyzed by ANOVA

Figures 4 depict the distribution of triplicate samples for the control group and the effects of blue laser radiation ranging from 0.5 to 5 J/cm², specifically concerning cell proliferation (Fig. 4a), viability (Fig. 4b), and migration (Fig. 4c). The data distribution in Fig. 4a adheres to a normality pattern, with the ascending portion of the curve gradually increasing from 0.5 J/cm², peaking at 2 J/cm² before experiencing a gradual decline. In contrast, cell viability, as illustrated in Fig. 4b, reaches its maximum earlier at 1 J/cm², exhibiting a slight rightward skew before declining. Additionally, Fig. 4c demonstrates a normal distribution, showing a consistent increase until 1.5 J/cm², ultimately peaking at 2 J/cm².

Fig. 4.

Normal distribution of human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) treated with low-level blue-laser treatment at a dose of 0.5 to 5.0 J/cm² and no irradiation (control) after 72 h of irradiation. a, b and c representing data for proliferation, viability and migration, respectively

LLBL irradiation increases the OB differentiation of hESC-MSCs

The effect of blue-laser irradiation on hESC-MSC differentiation into OBs was evaluated by determining ALP activity. The resulting data showed that blue-laser irradiation increased differentiation and that this effect gradually increased. Our data demonstrated that 0.5 J/cm² did not affect ALP activity; however, at 1.0 and 2.0 J/cm², ALP activity increased by 1.6- and 1.9-fold, respectively, suggesting that LLBL promoted OB differentiation (Fig. 5).

Fig. 5.

Alkaline phosphatase (ALP) activity of hESC-MSCs treated with low-level blue-laser irradiation at energy densities such as control, 0.5, 1.0 and 2.0 J/cm². The ALP activity levels of the treatment group were compared to those of the control groups. OD: optical density

Flow cytometric analysis

Flow cytometric analysis of untreated hESC-MSCs revealed that key MSC markers were present and highly expressed (Table 2). FACS analysis of hESC-MSCs revealed that treatment with blue laser irradiation at energy densities of 0.5, 1.0 and 2.0 J/cm² reduced the expression of all of the key MSC markers, with the maximal reductions observed at 2.0 J/cm² (Table 3). Table 2; Fig. 6a, show the marker expression on hESC-MSCs under control conditions in the absence of treatment or with the isotype control treatment. The expression levels of the CD (Cluster of Differentiation) markers under control conditions and after treatment with the blue laser are presented in Table 3. Our results showed that blue-laser treatment reduced the expression of CD29 and CD73 compared with that in the control group (Table 3).

Table 2.

FACS analysis of general markers expressed on human embryonic stem cell-derived mesenchymal stem cells under control conditions

Control	Percentage of total cells
CD29	81.70%
CD14	0%
CD73	75.40%
CD34	0%
CD146	55.80%
CD31	0%
CD44	5.30%

Table 3.

Flow cytometric analysis of human embryonic stem cell-derived mesenchymal stem cells treated with blue laser irradiation at different energies (0.5, 1.0 and 2.0 J/cm²⁾

Blue laser	0.5 J/cm2	1.0 J/cm2	2.0 J/cm2
CD29	64.00%	65.70%	60.10%
CD14	0%	0%	0%
CD73	65.70%	64.80%	56.40%
CD34	0%	0%	0%
CD146	52.00%	58.60%	59%
CD31	0%	0%	0%
CD44	6.20%	5.80%	5.60%

Fig. 6.

a: Gating strategy and immunophenotypic analysis of control mesenchymal stem cells (MSCs): This figure illustrates the flow cytometric gating strategy used to identify the immunophenotype of control human MSCs. The top panel displays a forward and side scatter plot (FSC-A vs. SSC-A) demarcating viable cell populations. The lower panels show the FITC and APC fluorescence intensities, with the P2 and P3 gates indicating the expression of specific surface markers. This analysis confirmed the presence of key mesenchymal markers and the exclusion of hematopoietic and endothelial markers, validating the purity and identity of the MSC population under control conditions. b: Flow cytometric analysis of irradiated hESC-MSCs. A series of dot plots representing flow cytometric analysis for the characterization of MSC surface markers under control conditions and upon exposure to varying intensities of blue laser light are shown. Each plot illustrates the cell population gated for the following specific markers: CD29, CD14, CD73, and CD34, which are canonical markers for identifying MSCs and distinguishing them from other cell types. In the control group (left panel), the high expression levels of CD29 and CD73 confirmed the mesenchymal lineage of the cells, as these markers are indicative of MSCs. The low expression of CD14 and CD34 suggested the exclusion of hematopoietic lineage cells, verifying the purity of the MSCs in the control sample. In a comparison of the controls to the samples treated with different blue laser intensities (right panel), the consistent expression patterns of CD29 and CD73 across all conditions indicate the stability of mesenchymal markers despite the laser treatment. The maintenance of low CD14 and CD34 levels reinforces the absence of hematopoietic contamination. The percentages in each quadrant quantify the proportion of cells expressing each marker, providing a precise measure of marker prevalence in the sample

The treatment with blue laser did not significantly alter the expression of CD14 and CD34 markers. However, a slight reduction in CD29 and CD73 expression was observed at 1 J/cm² (Fig. 6b). This suggests that blue laser treatment might influence the expression of certain stem cell markers, potentially affecting the differentiation state of the human ESC-MSCs.

Expression analysis of key osteoblastic genes

Alkaline phosphatase (ALP) gene expression increased when hESC-MSCs were treated with LLBL at doses between 0.5 and 1.0 J/cm², with the maximal effect occurring at 1.0 J/cm². However, irradiation at a higher energy density (2.0 J/cm²) reduced ALP gene expression (Fig. 7a). We then investigated the gene expression of Runx2, a key early OB marker, and observed that LLBL irradiation at only 1.0 J/cm² significantly upregulated Runx2 expression (Fig. 7a). We evaluated BMP2 gene expression and found that blue-laser irradiation did not affect BMP2 gene expression, regardless of the energy density used (Fig. 7a). Furthermore, irradiation at 1.0 J/cm² increased the gene expression of collagen type I (Col1) by approximately 3-fold (Fig. 7a). The expression of osteonectin (ON), a late-stage marker found only in mature OBs, followed the same pattern as that of BMP4 (Fig. 7a). In addition, blue-laser treatment increased matrix mineralization, as demonstrated by Alizarin Red S staining (Fig. 7b). Together, these data show that LLBL significantly increased the biological activities of hESC-MSCs.

Fig. 7.

a: hESC-MSC osteoblasts differentiated under the influence of low-level blue laser irradiation. Total RNA was extracted for qRT‒PCR analysis of osteogenic gene expression after treatment with blue (B) laser at different concentrations (0.5, 1.0 and 2.0 J/cm²) for the following genes: BMP2, ON, ALP, Col1a, Runx2 and BMP4. The fold change refers to the increase in gene expression under control conditions (osteogenic media without a blue laser). b: hESC-MSCs differentiated in osteogenic conditions in the presence of a low-level blue laser. Alizarin Red staining for matrix mineralization of hESC-MSCs differentiated into osteoblasts in the presence of a low-level blue laser. Light microscopy image at 100× magnification

Discussion

The use of hESC-derived MSCs for osteoporosis treatment may be a major advancement. Their consistent differentiation capacity and prolific growth can be particularly advantageous for patients who require an abundant supply of OBs for bone regeneration. The integration of LLLT with hESC-derived MSCs is a novel approach for the treatment of osteoporosis. Previous studies have suggested that LLLT can promote osteogenic differentiation and increase the proliferation and viability of MSCs [25–28]. The therapeutic potential of LLLT in facilitating bone regeneration and repair has been observed in various in vitro and in vivo models [19, 29, 30].

The precise mechanisms by which LLLT exerts its effect on hESC-MSCs warrant further investigation, but the current evidence suggests that a synergistic effect could amplify therapeutic outcomes. Our study lays the groundwork for a transformative treatment paradigm that combines the advanced regenerative properties of hESC-MSCs with the biostimulatory power of LLLT, opening avenues for targeted and effective clinical interventions for osteoporosis treatment.

The initial aim of this study was to evaluate the effects of LLBL irradiation at 0.5 to 5.0 J/cm² on the biological characteristics and differentiation of hESC-MSCs, including cell proliferation, viability and migration. LLLT is a type of photomodulation that uses photons to modulate biological activities without causing any damage to living cells [8, 31–33].

Pinheiro et al., recommended lower doses of LLLT to irradiate mucosa and skin wounds because light absorption and scattering are greater due to the lack of an optical barrier [34]. High doses of LLLT reduce its biostimulatory effects and damage photoreceptors as a result of the inhibition of metabolism and consequent cell death [32, 35]. On the basis of these observations and because our objective was to compare the effects of different blue laser energy densities, hESC-MSCs were treated with blue laser irradiation. We observed that blue laser irradiation at low energy densities (0.5 to 3.5 J/cm²) significantly increased the proliferation and viability of hESC-MSCs, whereas high doses did not increase proliferation or viability. In addition, blue-laser irradiation at 0.5 and 2.5 J/cm² significantly increased the migration of hESC-MSCs. The results of this study suggested that higher doses of LLBL irradiation were ineffective at increasing the proliferation, viability and migration of hESC-MSCs. These results also showed that the effect of low-dose blue-laser treatment of hESC-MSCs was dose dependent. These results are comparable with our previous study, where LLBL irradiation significantly promoted the normal biological process of melanocytes [20].

The MTT assay was used to measure cell proliferation, and trypan blue staining was used to measure cell viability [36]. In the MTT assay, the optical densities in the wells with treated cells were greater than those in the wells with untreated control cells, indicating cell proliferation in the treated wells. For cell viability, trypan blue staining was conducted to differentiate nonviable stained cells from viable unstained cells [24]. Significant differences between irradiated and nonirradiated hESC-MSCs were observed at low energy densities, indicating an increase in the mitochondrial activity and viability of irradiated hESC-MSCs.

We observed that blue-laser irradiation at 0.5 to 2.5 J/cm² significantly increased hESC-MSC migration to close the scratch. Additionally, LLBL at 2.0 J/cm² was more effective at promoting hESC-MSC migration than was LLBL at the other doses evaluated (Figs. 2c and 3; Table 1). Thus, the scratch assay can be used as an alternative for assessing cell migration in vivo. The hESC-MSCs moved toward the center, and this movement was a good indicator of cell proliferation and migration [37]. LLBL irradiation stimulates cellular processes in hESC-MSCs that promote proliferation, viability, and migration and may represent a good strategy for stem cell-based therapy.

The flow cytometric data provided information on the impacts of low-intensity blue-laser therapy at various intensities on the expression of surface markers on hESC-MSCs. Among them, CD29, CD73, and CD146 are well-known markers of hESC-MSCs. The increase in CD29 and CD146 expression after treatment with a dose of 1.0 J/cm² compared to 0.5 J/cm² suggested that moderate laser irradiation may increase the stemness characteristics of hESC-MSCs. Specifically, CD146 is associated with hESC-MSC multipotency and the ability of hESC-MSCs to adhere to plastic, a feature that is critical for their manipulation in vitro. However, at a higher intensity of 2.0 J/cm², a notable decrease in the expression of CD29 and CD73 was observed, indicating that excessive irradiation may be detrimental to the maintenance of MSC characteristics. This finding could be due to the induction of differentiation or cellular stress responses, which may downregulate these markers.

The absence of CD14, CD34, and CD31 expression across all laser intensities is consistent with the characteristics of hMSCs, which typically lack hematopoietic and endothelial markers. This finding suggested that LLBL treatment does not induce the differentiation of hMSCs into hematopoietic or endothelial lineages within the tested parameters.

Finally, the expression of CD44, a cell adhesion molecule involved in cell‒cell interactions and migration, decreased slightly with increasing laser intensity. This finding indicates an effect on the migratory capability of hMSCs at higher laser doses, although the change is relatively small, and its biological importance remains to be further investigated. LLBL therapy at a dose of 1.0 J/cm² appears to support the maintenance of hMSC characteristics, while higher doses may adversely affect the expression of certain stem cell markers. These findings are consistent with previous studies, which highlighted the importance of dosimetry in laser therapy for optimal cell proliferation and viability [18, 19].

A variable response in gene expression across different laser intensities was observed. The blue laser consistently increased ALP activity at different doses. This finding indicates that different wavelengths of light can differentially regulate osteogenic markers. BMP2 and Runx2 are crucial for osteoblastic differentiation and bone regeneration. The results indicate that specific light conditions can significantly increase their expression, with LLBL irradiation at certain doses (0.5 and 1.0 J/cm²) having particularly strong effects on BMP2 expression. This finding suggests the potential for these laser treatments to promote osteogenic induction. The effects on gene expression are dose dependent, as shown by the variable fold changes at different energy densities. For some genes, there appears to be an optimal dose beyond which the effectiveness of the therapy diminishes or becomes inconsistent. The upregulation of these genes under specific light treatments could have important implications for tissue engineering applications, especially in increasing the osteogenic potential of stem cells for bone regeneration. In addition, RT‒PCR was used to evaluate matrix mineralization, and ALP staining confirmed OB differentiation and maturation. However, while the RT‒PCR and staining results show changes in gene expression, further studies are needed to correlate these findings with the results of cell viability and proliferation assays to understand the full biological impact of the laser treatments.

Our findings align with earlier reports indicating that LLLT significantly improves MSC proliferation, viability, and osteogenic differentiation at specific dose ranges [8, 9, 18, 19]. Like Hou et al. and Tuby et al., who observed enhanced proliferation and growth-factor secretion in bone marrow MSCs with moderate-intensity laser exposure, we also noted a clear dose-dependent effect, with optimal responses at lower energy levels (0.5–3.5 J/cm²) [18, 19]. Beyond this threshold, the cells showed diminished or non-significant improvements, which echoes observations from studies suggesting that excessive laser doses can disrupt mitochondrial function and reduce cellular benefits [32, 35]. Thus, our results both confirm and extend existing work by underscoring how a narrow therapeutic window of blue-laser irradiation fosters MSC proliferation and osteogenic differentiation—supporting its promise as a clinical adjunct for bone regeneration.

Conclusion

Lower energy densities of the blue laser significantly augmented the proliferation, viability, and migration of hESC-MSCs, indicating an optimal therapeutic window for increasing cell performance. ANOVA test revealed that blue laser enhanced significantly proliferation, viability and migration at lower doses. Flow cytometric analysis showed that stemness marker CD146 increased after irradiation at 0.5, 1.0 and 2.0 J/cm². The implications of these findings are particularly salient for the advancement of stem cell-based therapies, offering a potential avenue for optimizing cell cultivation techniques and increasing the efficacy of regenerative medicine interventions.

Building on these in vitro findings, future work should investigate blue-laser therapy specifically for alveolar bone loss in animal models of periodontal disease, where the relatively shallow bone depth allows more effective laser penetration. Studies might explore combining LLLT with dental scaffolds or growth factors to accelerate alveolar bone regeneration—an approach supported by past evidence of laser-facilitated healing in periodontal defects. Refining dose parameters (wavelength, energy density, and frequency) will be key to developing targeted clinical protocols. Ultimately, if these preclinical results translate well, blue-laser therapy could help combat periodontal bone loss and establish standardized guidelines for dental regenerative procedures.

Acknowledgment

This work was funded by the National Plan for Science, Technology and Innovation (MAARIFAH) King Abdul-Aziz City for Science and Technology, Kingdom of Saudi Arabia, grant number 13-MED-1390-02.

Data availability

All data supporting the findings of this study are available within the paper and its figures & tables.

Declarations

Ethics approval

There was no involvement of any animal or human being during this study. Therefore, no need of approval from ethics committee/institutional review board.

Competing interests

Authors declare non-financial competing interests.