Evaluating the impact of PBM wavelengths on CTGF-driven tenogenic differentiation of ADMSCs through multi-level analyses

Abstract

Evaluating the Impact of PBM Wavelengths on CTGF-Driven Tenogenic Differentiation of ADMSCs Through Multi-Level Analyses” studied the effects of photobiomodulation (PBM) on the tenogenic differentiation of adipose-derived mesenchymal stem cells (ADMSCs) stimulated with connective tissue growth factor (CTGF) using a range of wavelengths (525 nm, 825 nm, and their combination) and fluences (5 and 10 J/cm²). Gene expression (qPCR for COL1A1, COL3A1, TNMD, TNC, BGN, and SMAD3), morphological alterations (Giemsa staining), metabolic activity (ATP test), and cytotoxicity (LDH release) were all evaluated using a multi-level experimental design. On Day 5, ATP production was significantly enhanced without cytotoxicity by using dual-wavelength PBM at 5 J/cm². On the other hand, metabolic activity was decreased but membrane integrity was preserved at a higher fluence of 10 J/cm². By Day 10, the spindle-shaped morphology and cellular alignment were more apparent in Giemsa-stained cells that had been exposed to dual-wavelength PBM. A synergistic impact in promoting tendon-specific lineage commitment was shown by gene expression analysis, which showed substantial elevation of critical tenogenic markers under dual-wavelength PBM at 5 J/cm². Taken together, these results show that PBM works best when the wavelength and dosage are optimized, and they provide credence to the idea that PBM might be a non-invasive way to boost tendon regeneration procedures that use stem cells.

Introduction

Many people experience pain, incapacity, and high social expenses as a result of acute tendon injuries and chronic tendinopathy, which especially affects the elderly²¹. For instance, flexor tendon mid-substance repair is among the numerous tendon surgeries performed on the many open wounds that affect the upper extremities, accounting for one-third of all workplace accidents²³. More than three million restricted activity days and more than 1.5 million missed workdays are caused annually by these injuries to the upper extremities. The results after tendon restoration are very unpredictable, leading to a significant clinical burden, even if surgical approaches and rehabilitation procedures have advanced. After surgically repairing tendons in the hand, ankle, and rotator cuff, there is an alarmingly high incidence of gapping or failure at the treatment site¹⁴.

Inflammation, proliferation, ECM creation, and ECM remodelling are the stages that tendon healing follows surgical repair. At the outset of the healing process, inflammation and the tendency to create disordered scar tissue drive the process. The proliferative and remodelling stages of tendon healing are usually sluggish, leading to only moderately effective recovery of tendon strength¹⁷. This is because tendon is cellular in nature and somewhat avascular. As a result, current treatments aim to stimulate tendon regeneration by administering tenogenic growth factors, increasing the number of endogenous tendon stem cells, or delivering mesenchymal stromal cells (MSCs). MSCs may be easily sourced from body parts that already contain them, such as fat and bone marrow. Because of their abundance and relative ease of procurement, adipose-derived MSCs (ASCs) represent an appealing alternative to bone marrow-derived MSCs¹⁰. Adipocytes, tendon fibroblasts, osteoblasts, and chondrocytes are just a few of the cell types that these cells may develop into. The chemical signals that drive the tenogenesis of MSCs remain unknown, despite the application of growth agents and mechanical stimulation, among other tactics¹².

As a potential stimulant, photobiomodulation (PBM) makes use of visible and near-infrared (NIR) light from coherent or non-coherent sources to activate endogenous chromophores, triggering photochemical and photophysical responses^2,8. Increases tenogenic populations, promotes cell proliferation and differentiation, and speeds up bone matrix production whether used alone or in conjunction with biological and/or chemical osteogenic differentiation inducers²².

Photobiomodulation (PBM) is a non-invasive therapeutic approach that utilizes low-intensity light to modulate cellular activity through photochemical rather than thermal mechanisms. The primary photoacceptor for red and near-infrared wavelengths is believed to be cytochrome c oxidase within the mitochondrial respiratory chain¹. Absorption of photons by this chromophore enhances mitochondrial electron transport, leading to increased adenosine triphosphate (ATP) production, transient reactive oxygen species (ROS) generation, and nitric oxide release. These events activate downstream signaling pathways involved in cell proliferation, migration, survival, and differentiation. PBM has been shown to influence key regenerative pathways, including transforming growth factor-beta (TGF-β), mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase/protein kinase B (PI3K/Akt), and Wnt signaling pathways, thereby regulating stem cell fate and tissue regeneration²³.

The biological effects of PBM are highly dependent on wavelength, as different wavelengths interact with distinct cellular chromophores and penetrate tissues to varying depths. Green light at 525 nm has been associated with modulation of cellular redox status, oxidative signaling, and differentiation-related pathways²³. In contrast, near-infrared light at 825 nm exhibits greater tissue penetration and is strongly absorbed by mitochondrial chromophores, resulting in enhanced mitochondrial respiration and ATP synthesis¹⁶. The selection of 525 nm and 825 nm in the present study was based on their distinct yet potentially complementary biological effects. Evaluating these wavelengths individually and in combination may provide insight into wavelength-specific regulation of CTGF-induced tenogenic differentiation of ADMSCs.

Previous research has shown that certain growth factors, such as connective tissue growth factor (CTGF), transforming growth factor beta (TGF-β), growth and differentiation factor 7 (GDF 7, also known as BMP-12), and GDF5.Gelberman⁹, Thomopoulos²⁵, may enhance the process of tenogenesis in both cultivated MSCs and endogenous tendon stem cells.

Recent in vitro and in vivo studies have shown that CTGF, a protein belonging to the CCN family, is highly desirable for tendon repair. This is because CTGF can stimulate endogenous tendon stem/progenitor cells, which in turn promotes tendon regeneration. This process is likely mediated by FAK/ERK1/2 activity^13,15.

Connective tissue growth factor (CTGF) is a well-established regulator of tenogenic differentiation and extracellular matrix synthesis. Although PBM has demonstrated the ability to enhance stem cell proliferation and differentiation in various cell types, its role in CTGF-mediated tenogenic differentiation remains incompletely understood. Therefore, the present study aimed to investigate whether PBM could enhance the biological effects of CTGF during ADMSC differentiation toward a tenogenic phenotype.

The purpose of this work is to assess the impact of two different fluences (5 and 10 J/cm²) of green and near-infrared PBM (525 nm and 825 nm, respectively) on the tenogenic differentiation of ADMSCs triggered by CTGF. A 2D culture method will be used for the research because it offers a controlled microenvironment for mechanistic probing, keeps costs down, and makes the assays repeatable. At both the day 5 and day 10, multi-level assessments was used to evaluate cell proliferation and cytotoxicity with ATP and LDH assays, qPCR was used to measure gene expression (COL1A1, COL3A1, TNC, BGN) with GAPDH as a reference. In conclusion, our findings provide molecular and cellular support for the idea that CTGF and PBM at different wavelength may have a function in the tenogenic differentiation of ADMSC which in turn lends credence to the idea that CTGF combined with ADMSC might improve tendon repair.

Materials and methods

Cell culture

Immortalized adipose-derived mesenchymal stem cells (ASC52telo; hTERT-immortalized, SCRC-4000, ATCC, Manassas, VA, USA) were cultured in Corning^® cell culture flasks (CLS431080, Sigma-Aldrich) utilizing Dulbecco’s Modified Eagle Medium (DMEM; D5796, Sigma-Aldrich, Johannesburg, South Africa) that was supplemented with 10% foetal bovine serum (FBS; S0615, (Biochrom), Whitehead Scientific, Cape Town, South Africa), 0.5% Penicillin-Streptomycin (P4333, Sigma-Aldrich), and 0.5% Amphotericin B (A2942, Sigma-Aldrich). Cells were maintained at 37 °C in a humidified environment with 5% CO₂ and 85% relative humidity using a Heracell™ 150i CO₂ incubator (51026280, Thermo Fisher Scientific, Johannesburg, South Africa). Until 80%−90% confluency, the cells were seeded in complete media at a density of 600 per well in Nunclon Delta 96-well plates (Thermo Fisher Scientific, 167008) and were allowed to adhere for 24 h. The treatment groups were subsequently modified to include the induction media, which comprised CTGF (20 µl), Ascorbic Acid (125 µl), FBS (2500 µl), Penicillin-Streptomycin (250 µl), and Amphotericin B (250 µl). These groups were then subjected to PBM, while the standard group was maintained in complete media without PBM, as illustrated in Tables 1 and 2. Throughout the experiment, media alterations occurred at two-days intervals. The cells underwent careful observation for alterations in morphology, proliferation rates, and gene expression. A range of assays was utilized to evaluate the cellular responses to PBM, including Adenosine Triphosphate (ATP) assay for Cell Proliferation, lactate dehydrogenase (LDH) Assay for Cytotoxicity, quantitative PCR and inverted light microscopy for Cell Morphology.

Table 1.

Experimental cell culture model (5 J/cm²).

Sl.No	Exp. Groups	Induction type
1	Group 1	Standard + Complete media + 0 nm + 0 J/cm²
2	Group 2	Control + Induction Type1 + 0 nm + 0 J/cm²
3	Group 3	Induction Type 1 + 825 nm + 5 J/cm²
4	Group 4	Induction Type 1 + 525 nm + 5 J/cm²
5	Group 5	Induction Type 1 + 525 nm + 825 nm + 5 J/cm²

Table 2.

Experimental cell culture model (10 J/cm²).

Sl.No	Exp. Groups	Induction type
1	Group 1	Standard + Complete media + 0 nm + 0 J/cm²
2	Group 2	Control + Induction Type1 + 0 nm + 0 J/cm²
3	Group 3	Induction Type 1 + 825 nm + 10 J/cm²
4	Group 4	Induction Type 1 + 525 nm + 10 J/cm²
5	Group 5	Induction Type 1 + 525 nm + 825 nm + 10 J/cm²

Seeding optimization

A cell line may affect the seeding density of cells cultivated on a 96well plates. An ideal seeding density generates a quantifiable signal without causing overcrowding, facilitating further biochemical applications. The ideal cell seeding density for immortalised ADMSCs is 1 × 10^5 cells in 2 mL of complete medium. The formula for determining the volume of cells to inoculate from a suspension is as follows:

Laser treatment (irradiation)

Laser treatment was conducted once, 24 h post cell seeding, utilizing a green 525 nm diode laser (National Laser Centre, EN 60825-1:2007) powered by a 100–240 VAC, 47–63 Hz 5 A laser source (OptoElectronics Tech. Co., Ltd., Changchun, China). Additionally, a near-infrared 825 nm diode laser (National Laser Centre, Pretoria, South Africa) was employed, driven by a 1000 mA source (Model 4210, Arroyo Instruments, CA, USA), in a combined dual-wavelength mode. Each laser group was administered a fluence of 5 J/cm² and 10 J/cm². The output power of the laser, measured in milliwatts (mW), was assessed with a Field Mate Laser Power Meter (1098297, Coherent, Johannesburg, South Africa). Additionally, irradiation durations were calibrated using a PM3 high-sensitivity thermopile sensor (1098336, Coherent) to ensure alignment with the desired fluence. The irradiation setup was optimized to ensure homogeneous delivery of laser energy across the adherent cell monolayer while preventing unintended exposure of neighbouring wells. Laser output power and beam diameter were calibrated before each irradiation session. Three experimental groups were established according to the specified treatment conditions. Group 3 consisted of cells that were cultured in induction medium and subjected to PBM at a wavelength of 525 nm, with fluences of 5 J/cm² and 10 J/cm². Group 4 comprised cells that were subjected to induction medium and irradiated with PBM at a wavelength of 825 nm, with a fluence of 5 J/cm² and 10 J/cm². Group 5 consisted of cells that were subjected to induction medium and subsequently exposed to a combined PBM protocol utilizing both 525 nm and 825 nm wavelengths in succession at 5 J/cm² and 10 J/cm² respectively. For the combined-wavelength groups, cells were irradiated first with 525 nm followed immediately by 825 nm. This sequence was maintained consistently throughout the study to ensure experimental standardization.

Additionally, a standard group was established in which cells were cultured in complete medium without any induction or PBM treatment (group 1), alongside a control group where cells were subjected to the induction medium without PBM exposure (group 2). This study utilized green and near-infrared diode lasers, both functioning in continuous wave mode, as detailed in Table 3. Samples were analysed on day 5 and day 10 following irradiation. The present investigation was not designed to evaluate sequence-dependent effects; therefore, the potential influence of irradiation order warrants further investigation.

Table 3.

Laser irradiation parameters.

Laser parameter	Green	Near-infrared
Light source	Diode laser	Diode laser
Wavelength (nm)	525	825
Power output (mW)	450 and 565	127 and 121
Power density (mW/cm2)	49.54 and 62.20	13.98 and 13.32
Spot size (cm2)	9.62	9.62
Emission	Continuous wave	Continuous wave
Fluence (J/cm2)	5 and 10	5 and 10
Time of irradiation	1 min 40 s and 2 min 40 s	5 min 57 s 11 min 44 s

The formula below was employed to derive the exposure time corresponding to each laser wavelength.

where, mW/cm²= power density, W/cm²=intensity, and s=exposure duration.

Proliferation assay

The ATP - proliferation assay was performed to assess cellular energy levels. The culture media in each well was removed and substituted with 1x PBS (50 µl) and CellTiter-Glo 3D reagent (Promega, G9681) (50 µl). The plate was covered with foil and placed on a shaker for 5 min, followed by a 30-minute incubation at room temperature. After incubation, the supernatant was transferred to a Corning 96-well opaque-walled plate (Sigma- Aldrich, CLS3912), and luminescence was measured using the VICTOR Nivo Plate Reader (Perkin Elmer, HH3522019094).

Lactate dehydrogenase (LDH assay)

The LDH assay served as a method to evaluate the integrity of the cell membrane. Following treatment, 50 µl of culture supernatants were collected from each well and transferred to a flat-bottomed Corning 96-well clear plate (Sigma-Aldrich, CLS3370). Additionally, 50 µl of CytoTox96 reagent (Promega, G1780) was incorporated into the wells. The plate was wrapped in foil and placed in a dark environment at room temperature for a duration of 30 min. The absorbance measurement was conducted at 490 nm utilizing the VICTOR Nivo Plate Reader (Perkin Elmer, HH3522019094), which reflects the degree of cell damage or cytotoxicity as determined by the release of LDH enzyme. An LDH positive control underwent analysis by lysing untreated cells with 10 µl of lysis buffer, followed by incubation at 37℃ for 45 min. Subsequently, 50 µl of the lysed cell solution was transferred to a clear 96 well plate (Sigma-Aldrich, CLS3370), and 50 µl of LDH reagent (Promega, G1780) was added. The mixture was then incubated for 30 min in the dark. The absorbance measurement was conducted at 490 nm utilizing the VICTOR Nivo Plate Reader (Perkin Elmer, HH3522019094), which reflects the efficacy of the LDH reagent.

May-Grünwald/Giemsa staining

The morphology and differentiation of ADMSCs were examined using Giemsa staining, with an emphasis on size, shape, and nuclear-cytoplasmic characteristics. Before being stained with May-Grünwald for 4 min and Giemsa Stain for 6 min, the cells were fixed in absolute methanol for 5 min in a volume of 50 µl. After that, they were rinsed with 50 µl of 1x PBS until they were clear. Various photos were captured using various objective lenses while it was under observation using an inverted light microscope.

Gene expression

The expression levels of immortalized ADSC markers and tenogenic genes in immortalized ADSC differentiated tenocyte were measured using quantitative polymerase chain reaction (PCR). After the cells had differentiated, the Quick-RNATM MiniPrep Plus Kit (R1058) from ZYMO RESEARCH in Johannesburg, South Africa was used to extract total RNA. After that, a universal reverse primer and the LunaScript^® RT SuperMix Kit (New England Biolabs, Pretoria, South Africa, M3010) were used to synthesize cDNA from this RNA. An AriaMx Real-Time PCR System (Agilent Technologies, Johannesburg, South Africa, G8830A) was used to amplify and quantify the target genes, which are Collagen type I alpha 1 (COL1A1), Collagen Type III Alpha 1 Chain (COL3A1), Tenascin-C, Biglycan, Tenomodulin (TNMD), and SMAD3 (Inqaba Biotechnical Industries, Pretoria, South Africa). The Luna^® Universal qPCR Master Mix (New England Biolabs, South Africa, M3003) was also employed. An initial denaturation step of 1 min at 95 °C followed by 40 cycles of 95 °C for 15 s and annealing at 60 °C for 30 s was used to conduct all qPCR reactions in triplicate. The 2−(∆∆Ct) technique was used to measure the relative fold change after normalizing gene expression to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), COL1 and COL3 are responsible for encoding the primary structural collagens found in tendons. TNC functions as a glycoprotein associated with tendon development and remodeling. BGN serves as a proteoglycan essential for collagen fibrillogenesis and the organization of the tendon matrix. TNMD, a type II transmembrane glycoprotein, plays a role in tendon development and differentiation, regulated by TGF-Beta/SMAD signaling. SMAD acts as an intracellular protein that transmits extracellular signals from TGF-Beta ligands to the nucleus.

Statistical analysis

Biochemical experiments were structured with triple biological repetitions and technical duplicates to guarantee precise statistical evaluation. A blank sample was extracted from the relevant data set and utilized in the spectrometry tests. The statistical analysis was conducted using SigmaPlot versions 12 and 14. The median error bars, represented by the standard error of the mean (SEM), indicate a sample size of n = 3. A one-way analysis of variance alongside a student t-test was employed to scrutinize the data. The figures indicate statistical variances across the experimental groups using P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).

Results

Seeding optimization of immortalised ADMSCs

In a 96-well plate format, three seeding concentrations 200, 600, and 1,000 cells per well, were examined to identify the optimal plating density for immortalized adipose-derived mesenchymal stem cells (iADMSCs). Each of them corresponds to around 625, 1,875, and 3,125 cells/cm². At two experimental endpoints, Day 5 and Day 10, the goal was to determine a density that prevented overgrowth while maintaining appropriate cell shape and confluence. There was a decrease in cell-cell interactions and test signal sensitivity because the cells remained sparsely distributed at 200 cells per well. However, on Day 10, premature confluence and possible contact inhibition were seen with 1,000 cells per well. At both time points, the medium density of 600 cells/well ensured perfect confluence and homogeneous dispersion, making for excellent circumstances for cell proliferation and differentiation. Reproducibility for future biochemical and morphological investigations is guaranteed by this seeding density, which is in accordance with recognized protocols for mesenchymal stem cell culture. Hence, for all upcoming studies on photobiomodulation and differentiation, a seeding density of 600 cells per well was determined to be optimal.

Cell proliferation

When subjected to photobiomodulation (PBM) at several wavelengths and fluences during CTGF-induced tenogenic differentiation, ADMSCs were measured for metabolic activity and proliferation using the ATP luminescence assay (Fig. 1). On Day 5, the dual-wavelength group (525 nm + 825 nm) had the highest ATP levels at 5 J/cm², indicating a synergistic impact on cell proliferation. ATP is a critical measure of mitochondrial activity and cellular energy status. However, ATP levels showed a significant decrease in all PBM-treated groups at the higher fluence of 10 J/cm² on both days 5 and 10, suggesting the possibility of photoinhibition or cellular stress caused by the excessive energy exposure. Aligning with the biphasic dosage response (Arndt-Schulz Law), our findings highlight the significance of dose-dependent PBM administration; low-level irradiation stimulates proliferation while higher doses repress it. Previous research has shown that PBM modulates mitochondrial activity by activating cytochrome c oxidase and generating reactive oxygen species (ROS). This modulation in turn affects the behaviour and potential differentiation of downstream cells¹¹.

Fig. 1.

ATP luminescence was measured at 525 nm, 825 nm, or 525 nm + 825 nm with 5 or 10 J/cm² on Days 5 and 10 after PBM. The greatest ATP levels, suggesting improved metabolic activity, were seen on Day 5 at a dual wavelength of 5 J/cm². All groups saw a decrease in ATP when exposed to a high fluence (10 J/cm²). The data appears as the average plus or minus the standard deviation. The significance level is **p**<0.0001.

Interestingly, the standard group exhibited the highest ATP levels compared with the differentiation groups. This observation may reflect differences in cellular metabolic demands between undifferentiated and differentiating cells. While undifferentiated cells primarily allocate energy toward proliferation and maintenance, differentiating cells may utilize ATP for lineage commitment, matrix synthesis, and cytoskeletal reorganization. Additionally, ATP measurements were not normalized to cell number or DNA content, which should be considered when interpreting these findings.

Cytotoxicity

In order to determine whether adipose-derived mesenchymal stem cells (ADMSCs) undergoing CTGF-induced tenogenic differentiation under different photobiomodulation (PBM) settings-maintained cell membrane integrity, the LDH cytotoxicity test was used (Fig. 2). A dependable biomarker of cytotoxicity is the release of lactate dehydrogenase (LDH), which is measured at 490 nm and represents the leaking of cytosolic enzymes caused by membrane damage. There were no notable variations among the groups on Day 5 at 5 J/cm² (Panel A), suggesting that the cytotoxicity was modest at this fluence. A cytoprotective effect of PBM against stress-induced damage was shown by the considerably reduced LDH levels in all PBM-treated groups compared to the standard group at 10 J/cm² (Panel B) (****p < 0.0001). Day 10 revealed similar patterns: the PBM groups showed considerably less cytotoxicity than the standard group at both 5 J/cm² (Panel C) and 10 J/cm² (Panel D) (****p < 0.0001), and there were no significant changes between the different wavelengths, including the combined 525 + 825 nm condition. These findings suggest that PBM does not cause cytotoxicity at the investigated fluences and wavelengths and may even improve membrane stability when ADMSCs are differentiating. This discovery is in line with the latest research that suggests PBM helps stem cells survive oxidative stress and keep their cells viable (Zarei et al. 2023; Rezaei et al. 2022; Beheshti et al. 2021).

Fig. 2.

After PBM, cytotoxicity was evaluated by quantifying LDH release at various wavelengths and fluences. On Days 5 and 10, LDH levels were significantly lower in the PBM-treated groups as compared to the control group, particularly at 10 J/cm², suggesting a cytoprotective impact. Displayed as mean ± SD, the data is statistically significant with a p-value less than 0.0001.

May-Grünwald/Giemsa staining

Brightfield micrographs of CTGF-induced tenogenic differentiation in adipose-derived mesenchymal stem cells (ADMSCs) stained with Giemsa are displayed at two time points (Day 5 and Day 10), two fluences (5 J/cm² and 10 J/cm²), and three different photobiomodulation (PBM) conditions (525 nm, 825 nm, 525 + 825 nm) (Fig. 3). The form, alignment, and confluency of the cells may be qualitatively evaluated after fixing and staining them to reveal their overall morphology and nuclear detail. The 825 nm and dual-wavelength groups showed spindle-like morphology with extended cytoplasm on Day 5 at 5 J/cm², which is indicative of early tenogenic commitment. Day 5, at a greater fluence of 10 J/cm², some cells in the 525 nm and control groups exhibited less elongation, whereas the more aligned morphologies were preserved in the 825 nm and combination groups. On Day 10, there was an improvement in alignment and elongation compared to the control and standard groups in all PBM-treated groups, especially those exposed to 5 J/cm². This indicates that differentiation and cytoskeletal organization are still going on. Under all circumstances, the dual-wavelength group (525 + 825 nm) exhibited the most consistent spindle-like shape. The measurement of the scale bar is 20 μm. These pictures show results from three separate trials.

Fig. 3.

ADMSCs stained with Giemsa under PBM as they differentiate into tenogenic cells. On Days 5 and 10, after PBM treatment, representative pictures of Giemsa-stained ADMSCs were taken at 525 nm, 825 nm, or both wavelengths (525 nm and 825 nm) at 5–10 J/cm². Groups treated with PBM exhibited improved alignment and spindle-shaped morphology, particularly at 5 J/cm² and with combined wavelengths, indicating the enhancement of tenogenic characteristics. The measurement of the scale bar is 20 μm.

Morphological observations indicated the development of elongated and spindle-shaped cellular features characteristic of tenogenic differentiation. However, these findings were assessed qualitatively. Future studies should incorporate quantitative image analysis approaches, including measurements of cell aspect ratio, elongation index, alignment, and orientation, to provide a more objective assessment of morphological changes induced by PBM.

Gene expression

At both Day 5 and Day 10 of CTGF-induced tenogenic differentiation, ADMSCs were subjected to photobiomodulation (PBM) at 525 nm, 825 nm, or a combination of the two at two fluences (5 J/cm² and 10 J/cm²). The qPCR heatmap shows the relative gene expression of important tenogenic and extracellular matrix markers (Fig. 4). The results revealed that COL1A1 expression was significantly increased at 525 nm (7.8-fold) and 525 + 825 nm (6.8-fold) on Day 10 at 10 J/cm², suggesting that collagen deposition was improved. On the other hand, COL3A1 and BGN exhibited modest increases at 825 nm and dual wavelengths, which might be interpreted as supporting matrix remodelling. Day 10 with 5 J/cm² revealed a considerable increase (2.4–3.9-fold) in TNMD, a critical measure of tendon maturation, suggesting the onset of late-stage tenogenic differentiation. Consistent with the structure of the cytoskeleton and the extracellular matrix, TNC expression was elevated in these settings as well. Surprisingly, the stimulation of intracellular tenogenic signalling, which is mediated by SMAD3, was only seen at 525 nm on Day 10 with 10 J/cm², a 4.7-fold increase, suggesting a potential dose-specific activation. Taken together, our results show that low-fluence (5 J/cm²) dual-wavelength PBM successfully increases tenogenic gene expression with balanced ECM output, whereas high-fluence PBM may cause imbalanced or too strong responses. Verified earlier by²⁹ these findings corroborate the ability of PBM to regulate stem cell tenogenic transcriptional programs and extracellular matrix remodelling.

Fig. 4.

A heatmap of tenogenic and TGF-β/SMAD3-associated genes was created using quantitative PCR at Day 5 and Day 10 after PBM therapy. iADMSCs were treated with 525 nm, 825 nm, or mixed wavelengths at fluences of 5 and 10 J/cm². Red colours indicate upregulation, blue shades indicate downregulation, and results are given as fold change from untreated controls. On Day 10, SMAD3 expression was highest at 525 nm and 5 J/cm², whereas COL1A1, TNMD, and BGN were maximally elevated at 10 J/cm² when paired with PBM. TGF-β-linked and matrix-driven pathways seem to modulate tenogenic genes based on the situation.

Discussion

Using different wavelengths (525 nm, 825 nm, and 525 + 825 nm) and fluences (5 J/cm² and 10 J/cm²), this study sought to assess the impact of photobiomodulation (PBM) on the tenogenic differentiation of adipose-derived mesenchymal stem cells (ADMSCs) in the presence of connective tissue growth factor (CTGF).

Although PBM was administered only once during the experimental period, its biological effects were observed up to Day 10. Previous studies have demonstrated that PBM can trigger sustained cellular responses through activation of mitochondrial metabolism and downstream signaling pathways that continue beyond the irradiation period⁶. Increased ATP production, transcriptional activation, and modulation of differentiation-associated signaling cascades may initiate long-term changes in gene expression and cellular phenotype. Consequently, a single PBM exposure may be sufficient to influence subsequent stages of stem cell differentiation and extracellular matrix production⁵.

The effects of PBM were characterized at both the cellular and molecular levels using a multi-level investigation that included morphological labelling, quantitative PCR profiling, ATP proliferation tests, and LDH cytotoxicity experiments. With dual-wavelength (525 + 825 nm) PBM at 5 J/cm² showing up as the most consistently favourable setting for fostering tenogenic phenotype with low cytotoxic stress, the results together suggest a dose- and wavelength-dependent response of ADMSCs to PBM²⁷.

Low-dose PBM (5 J/cm2) significantly increased metabolic activity on Day 5 according to the ATP luminescence test, especially when dual-wavelength therapy was used.Rai et al.¹⁹, This finding supports the idea that PBM stimulates cytochrome c oxidase in the respiratory chain, which in turn increases mitochondrial activity and leads to improved cell viability, proliferation, and ATP generation²⁰. But according to the biphasic dose-response model of PBM, exposure to a greater fluence (10 J/cm²) led to considerably lower ATP levels in all groups, particularly by Day 10, which may indicate photoinhibition or even metabolic weariness²⁸. Because greater energy levels may hinder proliferation during differentiation, our findings highlight the significance of fluence adjustment.

On the other hand, no PBM regimen caused cytotoxicity at either fluence, according to LDH cytotoxicity study. Remarkably, LDH release was continuously lower in all PBM-treated groups than in the control group, with notable decreases of 10 J/cm² on Days 5 and 10. PBM has been shown to have a cytoprotective impact by stabilizing mitochondrial membranes in stem cells and lowering oxidative stress¹⁸ Even at greater fluence, the decrease in LDH release under PBM indicates that cell integrity is maintained even if metabolic activity may decrease with continuous exposure, suggesting a possible pro-differentiation, anti-proliferation change brought on by prolonged PBM exposure.

Giemsa staining shed further light on the morphological alterations brought on by PBM therapy. Cells treated with dual-wavelength PBM at 5 J/cm² showed improved spindle-shaped morphology and alignment at all time periods, which are characteristics linked to tenogenic lineage commitment (Morita et al. 2018). By Day 10, these phenotypic changes were more noticeable in the 525 + 825 nm group, where cell orientation and elongation were noticeable. High-fluence PBM (10 J/cm2), on the other hand, produced comparatively less ordered morphology, which is in line with a possible shift away from proliferative behaviour and a reduction in metabolic activity.

A potential mechanism underlying the observed PBM-induced responses may involve transient ROS signaling, which has previously been associated with activation of mitochondrial and differentiation-related pathways. However, intracellular ROS levels were not directly measured in the present study. Therefore, any interpretation regarding ROS-mediated effects should be considered speculative and requires further experimental validation.

Diverse PBM regimens showed diverse patterns of gene expression at the molecular level, according to qPCR analysis. Interestingly, at Day 10 with 10 J/cm2, COL1A1 expression was most significantly increased in the 525 nm and 525 + 825 nm groups (7.8- and 6.8-fold, respectively), suggesting improved matrix deposition. If not accompanied by other tenogenic markers, overly high COL1 expression may indicate fibrotic or imbalanced ECM synthesis, even if this overexpression may promote structural maturation²⁶ In line with dynamic ECM remodelling during early differentiation, COL3A1, a factor in early matrix remodelling, was modestly elevated (3.7-fold) under 825 nm PBM at Day 5⁴.

In line with improved matrix architecture, Biglycan (BGN), a short leucine-rich proteoglycan implicated in collagen fibrillogenesis, had modest expression increases under 525 nm and 525 + 825 nm PBM on Day 10²⁴. Crucially, the 525 + 825 nm group showed a considerable upregulation (2.4–3.9 fold) in Tenomodulin (TNMD), a particular marker of mature tenocytes, at 5 J/cm² on Day 10, demonstrating strong tenogenic differentiation under these circumstances⁷. SMAD3 (4.7-fold at 525 nm, Day 10, 10 J/cm²) and TNC (Tenascin-C), another indicator of tendon maturation, were also upregulated in conjunction with this, indicating activation of the TGF-β/SMAD signalling axis, which is known to control tendon formation (Hou et al., 2021).

Study limitations

The present study has several limitations that should be considered when interpreting the findings. First, PBM-only treatment groups were not included, preventing assessment of the independent tenogenic effects of PBM in the absence of CTGF stimulation, however the reason for this is that PBM acts exclusively as a bioenergetic booster rather than an independent lineage director, meaning light irradiation alone cannot induce the differentiation of stem cells into tenocytes. Because PBM lacked the inherent chemical or structural signals required to alter definitive cell fate on its own, it functions strictly as a non-invasive adjunct³. Second, intracellular ROS production and temperature changes during irradiation were not directly measured, limiting mechanistic interpretation of PBM-induced responses. Third, morphological assessments were qualitative and were not supported by quantitative image analysis. Fourth, only a single PBM treatment was administered during the differentiation period. Finally, the study utilized immortalized ADMSCs, which may not fully replicate the biological behaviour of primary stem cells. Future investigations should address these limitations while exploring the molecular mechanisms linking PBM, TGF-β signaling, and tenogenic differentiation.

Conclusion

The idea that dual-wavelength PBM at low fluence (5 J/cm²) most efficiently promotes tenogenic differentiation of ADMSCs is supported by the combined interpretation of these findings. This condition produced spindle-like shape, preserved membrane integrity (low LDH), balanced ATP-driven proliferation, and coordinated elevated tenogenic markers such TNMD, TNC, and COL1A1. Higher fluences (10 J/cm²) on the other hand, that were linked to lower ATP levels and less favourable cell morphology, elicited strong ECM gene responses (e.g., COL1A1, SMAD3), and suggested that excessive energy delivery may push cells toward terminal differentiation or even early senescence without promoting continuous functional maturation.

Author contributions

AA, AC - Conceptualization; AA and AC - Writing - Original Draft; AA, HA and AC - Writing - Review & Editing; AA - Visualization; AC and HA - Supervision; AC and HA - Funding acquisition. All authors read and approved the final manuscript.

Funding

This research was funded by the National Research Foundation of South Africa Thuthuka Instrument, grant number TTK2205035996; the South African Medical Research Council (SAMRC) Early Investigators Programme through its Division of Research Capacity Development under the Research Capacity Development Initiative from funding received from the South African National Treasury. The content and findings reported/illustrated are the sole deduction, view and responsibility of the researcher and do not reflect the official position and sentiments of the SAMRC, grant number 57050; the University Research Council, grant number 2024URC00813; the Department of Science and Technology’s South African Research Chairs Initiative (DST-NRF/SARChI), Grant number 98337; the University of Johannesburg Global Excellence and Stature 4.0 (GES 4.0) master’s scholarship award.

Data availability

The data that supports this publication can be requested from the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.

Ethics Approval

Approval to conduct this study has been received from the University of Johannesburg, Faculty of Health Sciences Research Ethics Committee (REC). Approval number: REC-2986-2024 under the guidance of the National Health REC approval number: REC 241112-035.