Laser-induced modulation of root dentin surface for growth factor release and enhanced stem cell response

Abstract

Growth factor release from dentin and subsequent cellular response are critical in endodontic regeneration procedures. This study evaluated the effects of Nd: YAG (1064 nm) and diode lasers (980 nm, 635 nm) at varying energy densities on dentin morphology, Transforming Growth Factor-beta 1 release, and the biological response of stem cells to promote pulp regeneration in immature permanent teeth. Dentin blocks from extracted human teeth were treated with 1.5% NaOCl followed by pulsed Nd: YAG or continuous-wave diode lasers (average energy densities of 24, 60 and 120 J/cm²). Controls included 1.5% NaOCl/17% EDTA group and untreated samples. TGF-β1 release was quantified via ELISA. Cell viability (24–72 h), alkaline phosphatase activity (7 days), and surface morphology were assessed. One-way or two-way ANOVA with post-hoc tests were applied (p < 0.05). SEM analysis revealed that all lasers partially disrupted the smear layer and increased surface roughness, while SHED cells on laser-treated dentin showed flattened morphology with extended filopodia, indicating favorable cell-surface interactions. All laser groups significantly increased TGF-β1 release compared to the negative control (p < 0.05), with Nd: YAG at 60 J/cm² producing the highest levels, exceeding even the positive control (p < 0.05). Laser-treated specimens were non-cytotoxic and significantly increased ALP activity compared to the negative control (p < 0.001), with Nd: YAG at 60 J/cm² showing the highest activity, statistically higher than the NaOCl/EDTA positive control (p < 0.05). Low to moderate energy Nd: YAG and diode lasers enhanced TGF-β1 release and ALP activity without cytotoxicity, matching or exceeding EDTA treatment. Thus, laser-assisted dentin conditioning is a promising adjunctive strategy for regenerative endodontics.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10103-026-04971-9.

Introduction

Regenerative endodontic procedures (REPs) aim to restore the pulp-dentin complex and promote continued root development in immature permanent teeth using tissue engineering principles. Unlike conventional treatments such as apexification, which resolve infection but do not encourage further root maturation, regenerative techniques employ tissue engineering principles-utilizing stem cells, signaling molecules, and scaffolds-to facilitate tissue regeneration and complete root formation [1]. Within this context, transforming growth factor-beta1 (TGF-β1) plays a pivotal role in facilitating cellular activities essential to tissue regeneration. TGF-β1 is entrapped within the dentin matrix and can be released upon specific treatment modalities [2]. Once liberated, it promotes stem cell migration, proliferation, and odontoblastic differentiation through the upregulation of key transcription factors and odontogenic markers such as DSPP and DMP-1. Additionally, TGF-β1 stimulates angiogenesis and extracellular matrix deposition, both essential for the formation of vascularized pulp tissue in regenerative endodontic procedures [3, 4].

Conventional protocols for dentin conditioning, such as using ethylenediaminetetraacetic acid (EDTA) or sodium hypochlorite (NaOCl), have significant limitations. While EDTA chelates calcium to demineralize dentin and release matrix-bound growth factors like TGF-β1 [2, 3], prolonged exposure may degrade these bioactive molecules [5, 6]. Conversely, NaOCl effectively dissolves organic tissue but may denature proteins and reduce the biological activity of released factors [7, 8]. Therefore, there is a need for conditioning strategies that can effectively disinfect, modify the dentin surface, and preserve or enhance its inherent bioactivity in a controlled manner.

Laser irradiation has emerged as a promising modality for dentin modification through a photochemical and photothermal mechanisms that depends largely on the delivered energy density. At lower energy levels, initial photochemical interactions (photobiomodulation (PBM)) with dentin chromophores may alter matrix-bound molecules and promote growth factor release before clear thermal effects appear [9]. Absorption of higher laser energy by dentin’s mineral and organic content and then conducting to surrounding water molecules causes partial water evaporation, thermal denaturation of collagen fibrils, and recrystallization of hydroxyapatite crystals. These changes result in surface modifications such as disruption of smear layer and occlusion of dentinal tubules. At higher energy densities, more intense thermal effects induce melting and vaporization of the organic phase, ablation of collagenous components, and fusion of hydroxyapatite crystals, ultimately increasing surface roughness and irregularities [10, 11].

These physical changes may also influence the release of trapped growth factors from dentin matrix [9, 12]. Also, lasers offer the unique potential to integrate antimicrobial efficacy [13, 14] with controlled structural modification in a single step. Optimal laser settings are essential to prevent excessive thermal damage and also proper parameter selection can yield a rough, retentive surface favorable for regenerative applications.

It seems that a systematic comparison of different laser wavelengths and powers on growth factor release and subsequent stem cell response is lacking. Therefore, this study aimed to systematically evaluate and compare the effects of Nd: YAG (1064 nm) and diode (980 nm, 635 nm) lasers at varying energy densities (24, 60 and 120 J/cm²) on root dentin surface morphology, TGF-β1 release, and the subsequent viability and differentiation potential of stem cells from human exfoliated deciduous teeth (SHED), using conventional 1.5% NaOCl/17% EDTA treatment as a benchmark.

Materials and methods

Ethical approval

The use of extracted human teeth was approved by the Ethics Committee of Tabriz University of Medical Sciences (IR.TBZMED.DENTISTRY.REC.1403.009).

Sample preparation

Freshly extracted human teeth from patients aged 18–30 years with intact roots were collected and immediately stored at -80 °C based on standard protocols from the laboratory. Samples were collected within 6 months before the study. All specimens were single-rooted with no previous endodontic treatments, restorations, fractures, caries, or root canal calcifications. Specimens were cleaned of residual calculus and soft tissues. The crown portion of the teeth was removed using a diamond saw at 1000 rpm under water cooling. The teeth underwent mechanical preparation using the step-back technique, with only sterile saline as the irrigant, without the use of chemical agents to avoid any chemical interaction with the dentin matrix prior to experimental treatments. Instrumentation was performed up to a file size of 100 to ensure adequate canal shaping. Following cleaning, the roots were sectioned perpendicularly to their long axis using a low-speed diamond saw under constant pressured water cooling to minimize thermal damage. The canal lumen and its dentin walls were preserved. From each half, a 5-mm-long dentin block was carefully cut from the middle portion for further treatment (5 × 5 × 2 mm blocks).

Each dentin block was randomly allocated into the experimental and control groups (Fig. 1):

Fig. 1.

Study groups showing divisions into two control groups (positive: NaOCl/EDTA; negative: saline only) and three laser-treated groups (Nd:YAG, diode 980 nm, diode 635 nm) at three energy density levels (24, 60, 120 (J/cm²))

• Negative control group: Samples underwent only mechanical preparation with saline, receiving no laser or chemical treatment.

• Positive control group: Samples treated with 10 ml of 1.5% NaOCl followed by normal saline and 10 ml of 17% EDTA solution each for 5 min.

• NaOCl group: Samples were subjected to irrigation with 10 ml of 1.5% NaOCl for 5 min.

• Laser groups: Samples were subjected to irrigation with 10 ml of 1.5% NaOCl for 5 min followed by laser irradiation.

Laser irradiation protocol

The specimens were irradiated with three different laser types: Nd: YAG (1064 nm) (LAMBDA S.p.a, Italy./054), diode (980 nm), and diode (635 nm) (Tripolo, Medency, Italy). For each laser various settings were applied corresponding to calculated average energy densities of 24, 60, and 120 J/cm² (Table 1). All laser irradiations were performed by a single trained operator using a 200 μm diameter fiber held approximately perpendicular to the dentin surface at a distance of 0.5 mm. Each specimen was exposed in a continuous sweeping motion. Ambient conditions (room temperature, humidity) were maintained throughout the experiments.

Table 1.

The laser characteristics

Laser	Nd: YAG			Diode
Wavelength (nm)		1064		980				635
Operating mode		Pulsed-100 µs		Continuous mode
Power (W)	0.2	0.5	1	0.2		0.5	1	0.2*	0.2		0.2
Average energy density (J/cm2)	24	60	120	24		60	120	24	60		120
Frequency (Hz)		10		-
Fiber tip (µm)		200		200
Fiber tip cross-sectional shape		Flat and Circular		Flat and circular
Exposure duration (s)		30		30			30		75	150

*For 635 nm laser, power remained constant at 0.2 W; exposure time was varied (30s, 75 s, 150 s) to achieve energy densities of 24, 60, and 120 J/cm²

TGF-β1 release and ELISA quantification

After dentin conditioning (n = 5/group), each dentin specimen was immersed in 1.5 ml of sterile phosphate buffer saline and incubated in a shaker incubator at 37 °C and 100 rpm for 24 h to facilitate growth factor release. Supernatants were then collected and TGF-β1 levels were quantified using a sandwich ELISA kit specific for human TGF-β1 (Duo Set^®, DY-240), following the manufacturer’s recommended protocol. All standards, controls, and samples were assayed in triplicate, and all protocols were followed strictly to ensure reproducibility.

SHED cell culture

Stem cells from human exfoliated deciduous teeth (SHED cells) (Pasteur Institute, Iran) were cultured in DMEM F12 medium containing 10% fetal bovine serum (FBS). In all tests after the 3rd passage, cells (5 × 10⁴ cells/well) were seeded on dentin blocks placed in 48-well plate and incubated under standard growth conditions (37 °C, 5% CO₂).

MTT cell viability assay

Cell viability was assessed using MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; MTT). After seeding SHED cells on dentin specimens (n = 3/group), the plates were incubated for 24, 48 and 72 h, after which 25 µL MTT solution (5 mg/ml in PBS) was added to each well. Plates were incubated at 37 °C for 4 h to allow enzymatic reduction of MTT to formazan crystals. The supernatant was carefully removed, and 250 µL DMSO was added to each well. Plates were gently shaken for 15 min until formazan was fully dissolved. Absorbance was measured at 570 nm using a microplate reader.

Alkaline phosphatase activity

After 7 days of culturing SHED cells on dentin samples (n = 3/group), ALP activity was measured as an indicator of early cellular differentiation using the Elabscience^® ALP Activity Assay Kit. Then, cells were lysed using a sonicator, and the supernatant was collected by centrifugation. From each sample, 5 µL of supernatant was placed into 96-well plates, and 50 µL of the prepared substrate working solution (prepared by mixing 25 µL of buffer solution and 25 µL of substrate solution) was added to each well. The plates were incubated at 37 °C for 10 min, followed by the addition of 150 µL of chromogenic agent per well. Absorbance was measured at 520 nm using an Epoch BioTek microplate reader (USA). All measurements were performed in triplicate.

SEM analysis of dentin surface and cell morphology

Both dentin surface structure after conditioning and cell morphology (after 48 h of SHED culture on specimens) were prepared as mentioned above and evaluated using scanning electron microscopy (SEM, (TESCAN, Brno, Czech Republic)). Specimens from each group were randomly selected, fixed, dehydrated serially in alcohol, mounted so that the exposed canal lumen and its surrounding dentin wall faced the electron beam and sputter-coated with gold prior to imaging. Using SEM images (n = 6) at ×2000 magnification smear layer was scored independently by two calibrated examiners using the 5-point Hülsmann scale (1 = no smear layer, tubules completely open; 5 = very heavy smear layer, tubules completely covered). Discrepancies were resolved by consensus, and the mean score for each specimen was used for analysis.

Statistical analysis

Data were analyzed using Graphpad PRISM (Version 9). The distribution of data was analyzed by Kolmogorov-Smirnov test. One-way ANOVA with Games-Howell and Tukey tests were used for ELISA and ALP data, respectively. Two-way ANOVA with Tukey test were applied to MTT data to assess the effects of treatment and time. Hülsmann smear layer scores were analyzed using the Kruskal-Wallis test, followed by Dunn-Q Bonferroni post-hoc multiple comparisons. The agreement amongst endodontists was evaluated using the Kappa agreement coefficient analysis. Significance was set at p < 0.05.

Results

Dentin morphology

Dentin morphology in all treatment groups is observed in Fig. 2. The untreated dentin (negative control) surface appears covered by a dense smear layer, with no visible open tubules. Irrigation with 1.5% NaOCl alone only partially disrupted this layer, leaving most tubules occluded. The application of 1.5% NaOCl and 17% EDTA produced the most effective smear layer removal among all groups, with the greatest open dentin tubule diameter. Treatment with the lasers (Nd: YAG and diode 980) resulted in power-dependent morphological changes. Treatment with Nd: YAG and 980 nm diode lasers at 60 J/cm² resulted in partial smear layer disruption with evidence of peritubular melting and increased surface roughness favorable for cell attachment, without evidence of major thermal damage. At the highest energy density (120 J/cm²), both lasers caused surface melting, resolidification, and occasional microcracks. The 635 nm diode laser, across all power settings, produced only limited smear layer alteration and modest tubule exposure with increased surface roughness at 120 J/cm².

Fig. 2.

SEM images (at 2000× magnification with a scale bar of 20 µm) of dentin in negative control (a), dentin treated with only NaOCl (b), NaOCl/EDTA (c), Nd:YAG/24 (d), Nd:YAG/60 (e), Nd:YAG/120 (f), Diode 980/24 (g), Diode 980/60 (h), Diode 980/120 (i), Diode 635/24 (j), Diode 635/60 (k), Diode 635/120 (l)

Smear layer scoring

Smear layer scores on the Hülsmann 1–5 scale showed significant differences among treatment groups (Kruskal–Wallis, p < 0.05) (Fig. 3). The kappa values for inter-observer agreement for evaluating smear layer was 0.9053. In general, the NaOCl/EDTA group exhibited the lowest score (p < 0.0001 compared to no treatment and NaOCl-only groups), consistent with the most effective smear layer removal, whereas the NaOCl-only and laser settings maintained higher scores, indicating more residual smear layer. Among the laser groups, Nd: YAG/120 J/cm² and diode 980/120 J/cm² presented significant difference with no treatment (p < 0.001 and p < 0.01 respectively) and NaOCl-only groups (p < 0.001 and p < 0.05 respectively) and no difference with NaOCl/EDTA group.

Fig. 3.

Smear layer scores on dentin surfaces after chemical and laser treatments

TGF- β1 release profile

TGF-β1 levels in response to different laser and control treatments are presented in Fig. 4a and supplementary Tables 1, 2 and 3. One-way ANOVA revealed a significant overall effect of treatment condition on TGF-β1 concentration (p < 0.0001). The negative control group (No NaOCl/No EDTA) produced low TGF-β1 release, establishing baseline dentin activity. Irrigation with 1.5% NaOCl alone did not significantly increase TGF-β1 release compared to the negative control (p = 0.641), whereas the positive control group (NaOCl/EDTA) exhibited robust TGF-β1 release (p = 0.002).

Fig. 4.

The amount of TGF-β1 (pg/ml) released from conditioned dentin (a), Cell proliferation on conditioned dentin after 24, 48 and 72 hours (b), ALP activity (U/ml) of SHED cells cultured on conditioned dentin (c) *p < 0.05

All laser groups induced significant TGF-β1 release compared to the negative control and NaOCl-treated samples (p < 0.05). Among all groups, Nd: YAG irradiation at 60 J/cm² produced the highest TGF-β1 release, which was significantly higher than that of the NaOCl/EDTA positive control (p = 0.022).

Compared to the NaOCl/EDTA group, no significant differences were observed for Nd: YAG laser at 24 J/cm² and 120 J/cm², diode 980 at 24, 60 and 120 J/cm², or diode 635 at 24 J/cm² (p > 0.05).

Evaluation of biocompatibility

Cell viability was assessed by MTT assay at 24-, 48-, and 72-hours on treated dentin specimens (Fig. 4b and supplementary Tables 4 and 5). At all times, there was no statistically significant difference between NaOCl-treated group, negative control and NaOCl/EDTA groups (p > 0.05).

At 24 h, Nd: YAG laser at all powers and diode 980 at 24 and 60 J/cm² increased cell viability significantly compared to NaOCl-treated group (p < 0.05). By 48 h, only the Nd: YAG 24 J/cm² maintained significantly greater viability relative to the NaOCl group (p < 0.05), while all other intergroup comparisons remained statistically similar (p > 0.05). At 72 h, SHED cells on dentin conditioned with Nd: YAG laser at 24 and 60 J/cm² diode 980 at 24 and 120 J/cm² and diode 635 at 24 J/cm² demonstrated significantly enhanced cell viability compared to both the NaOCl-only and NaOCl/EDTA positive control groups (p < 0.05).

ALP activity

ALP activity was evaluated 7 days after seeding SHED cells onto dentin specimens treated with lasers or chemical agents (Fig. 4c and supplementary Tables 6, 7 and 8). There was no significant difference between negative control and NaOCl-treated group (p > 0.9999). The positive control group (NaOCl/EDTA) exhibited robust ALP activity compared to negative control group and NaOCl irrigation (p < 0.0001).

All laser groups significantly increased ALP activity in SHED cells compared to the negative control (p < 0.001). The highest ALP activity was observed in the Nd: YAG 60 J/cm² groups, which was statistically higher than the NaOCl/EDTA (p < 0.05).

Visualization of SHEDs on conditioned dentin

SEM images in Figure 5. demonstrates the attachment and morphology of SHED cells cultured on dentin surfaces subjected to different conditioning and laser treatments. In the control groups, cells with rounded morphology and poor spreading are visible. In laser groups, cells appeared flattened with extended filopodia, suggesting a moderately favorable surface topography.

Fig. 5.

SEM images (at 2500× magnification with a scale bar of 10 µm) of SHED culture on dentin in negative control (a), dentin treated with only NaOCl (b), NaOCl/EDTA (c), Nd:YAG/24 (d), Nd:YAG/60 (e), Nd:YAG/120 (f), Diode 980/24 (g), Diode 980/60 (h), Diode 980/120 (i), Diode 635/24 (j), Diode 635/60 (k), Diode 635/120 (l)

Discussion

Previous studies have extensively investigated a wide range of irrigants and intracanal medicaments for REP purposes in mature or immature teeth. Despite all advances, most existing approaches remain limited by cytotoxicity, short-term activity, lack of bioactivity, or the need for multiple procedural steps [1, 15–17]. In this context, the present study was designed to move one step forward by evaluating laser irradiation as an alternative to conventional chemical conditioning for dentin bioactivation.

Based on the American Association of Endodontists guideline, 1.5% NaOCl was used prior to laser irradiation in all groups, since it has been reported that the presence of biofilm is a physical barrier against chelating agents and attenuates their effect in releasing growth factors [18]. This step is essential, as neither NaOCl alone nor subsequent chelators reliably eliminate residual bacteria [19].

In this study, Nd: YAG and diode lasers were selected for their established antibacterial efficacy [13, 20], penetration depth [14], and the ability to induce controlled thermal effects on dentin and surrounding tissues [10, 21], which may alter the surface characteristics and favor cellular responses critical for REPs. Consequently, laser-assisted dentin conditioning may represent a paradigm shift in regenerative endodontics, offering a biologically driven alternative to conventional chemical methods by combining effective decontamination, structural modification, and bioactive signal release within a single protocol. The selected energy densities represent a clinically relevant range for intra-canal laser use, from photochemical to more pronounced thermal effects on dentin, while staying within established safety limits [22, 23].

The distinct responses observed among laser groups likely reflect wavelength, power, and mode-dependent interactions with dentin. However, the experimental design does not allow attribution of the results to a single variable, and relationships between wavelength, fluence, and biological response cannot be determined definitively. In particular, the 635 nm diode groups required longer exposure times to achieve the same energy densities, which introduces an additional thermal-time effect. Thus, the results should be interpreted as the outcome of the combined laser parameters rather than the isolated impact of any individual setting.

The results demonstrated that most laser protocols enhanced dentin bioactivity to some extent, as reflected by increased TGF-β1 release, favorable SHED viability and ALP activity compared with untreated dentin. Notably, low to moderate energy density settings (24 and 60 J/cm²) of Nd: YAG and diode lasers produced biological and morphological outcomes comparable to or in case of Nd: YAG 60 J/cm² exceeding the conventional NaOCl/EDTA treatment.

In this study, the 0.2 W (200 mW) settings for all three lasers (Nd: YAG 1064 nm, diode 980 nm, and diode 635 nm) fall within the power range commonly used for low-level laser therapy (LLLT) or PBM. PBM exerts its biological effects predominantly through photochemical and photobiological mechanisms, with negligible thermal effects when appropriate irradiation parameters are used (9). The 0.2 W groups in this study showed minimal effects on dentin surface morphology and smear layer removal, which requires photothermal effects.

The increased dentin bioactivity following laser irradiation in 60 and 120 J/cm² is mainly attributed to the alterations in dentin morphology and thermal interactions between laser energy and the mineralized matrix with the extent of changes influenced by both power settings and method of application [24–26]. These changes are primarily driven by photothermal effects, including vaporization of water and organic components, followed by localized melting and recrystallization of hydroxyapatite phase [27], which results in partial disruption of smear layer and increased surface roughness (as shown in Fig. 2) [28, 29].

At 60 J/cm², Nd: YAG (1064 nm) produced the highest TGF-β1 release, whereas 980 nm diode at the same energy density did not, which may relate to differences in absorption by the mineral and organic phases and the pulsed delivery of Nd: YAG producing high peak powers that favor controlled matrix exposure without extensive protein denaturation. At this energy density, Nd: YAG and 980 nm diode lasers increase exposure of intertubular dentin, partially disrupt the mineral phase, and reveal the underlying collagen-rich organic matrix without extensive denaturation of embedded proteins such as TGF-β1 [21, 27]. This controlled micro-ablation mimics the effects of chelation, facilitating the release of matrix-bound growth factors into the surrounding medium. However, the relationship between laser parameters and growth factor release is non-linear. In this study, increasing the energy density of the 635 nm diode laser resulted in progressively lower TGF-β1 release, while for Nd: YAG and 980 nm lasers, the highest setting (120 J/cm²) did not significantly differ from the positive control but also did not outperform the moderate setting. Thermal accumulation may lead to protein denaturation or occlusion of tubules by melted peritubular dentin, limiting diffusion of growth factors into the medium [30].

The 635 nm diode laser, typically regarded as a low-power photobiomodulation device, produced comparatively lower TGF-β1 release across all lasers. This wavelength, being absorbed mainly by pigments and less by hydroxyapatite or water, induces minimal photothermal alteration in the mineral matrix, consequently, it does not efficiently expose the collagen-bound growth factors, although it was still sufficient to induce modest smear layer alteration and surface roughness changes at higher fluences, supporting a spectrum of effects from primarily photochemical at low doses to more photothermal at higher doses [31].

In terms of biological response, the laser-treated groups generally performed at least as well as, and in some settings better than, the NaOCl/EDTA protocol in supporting SHED viability and early differentiation. While NaOCl/EDTA produced robust TGF-β1 release and ALP activity, cell viability on these chemically conditioned surfaces remained comparable to the negative control, suggesting that their strong demineralizing effect does not necessarily translate into superior cellular support over time. In contrast, several laser settings, particularly Nd: YAG at 60 J/cm² and selected 980 nm diode protocols, maintained or enhanced cell viability relative to both NaOCl-only and NaOCl/EDTA, while simultaneously increasing ALP activity, indicating that laser-conditioned dentin can provide a biologically favorable surface for pulp-derived stem cells. However, it is important to note that ALP activity is an indirect marker and while the results are promising, they should be interpreted as an early indicator of differentiation potential rather than definitive evidence of odontogenesis [32].

The effects of laser irradiation on dentin bioactivity and especially TGF-β1 release remain inconsistent across studies, highlighting the importance of parameter optimization. Two studies evaluated the effects of low-level laser therapy on dentin bioactivation [9, 12]. García-Guerrero et al. found a synergistic effect when combining EDTA with low-power diode laser (at both 810 nm (3 J/cm²) and 650 nm (1 J/cm²)), where the combined treatment outperformed laser irradiation alone in the release of TGF- β1. Interestingly, 810 nm diode (3 J/cm²) resulted in higher PDGF-BB release compared to either combined strategy or EDTA alone [12]. It should be noted that the application mode and fiber tip handling was completely different with the present study. García-Guerrero et al. used a stationary fiber tip, whereas we employed a continuous sweeping motion across the dentin surface. This methodological difference may partially account for discrepancies in growth factor release profiles between the two studies.

In agreement with the previous study, Malekpour et al. reported that photobiomodulation therapy (PBM) (808 nm diode, 250 mW, 3 and 5 J/cm²), both alone and in combination with EDTA, enhanced the release of TGF-β1 from dentin and improved cell viability and migration of DPSCs [9]. Beside the differences in laser parameters, Malekpour et al. used an indirect cytotoxicity model where dentin extract solutions were added to DPSC cultures, whereas our study seeded SHED cells directly onto laser-treated dentin surfaces, providing a more physiologically relevant assessment of cell-surface interactions.

Martín et al. investigated the effect of dentin conditioning with 17% EDTA alone or combined with 808 nm and 980 nm diode lasers (1.5 W, 45 J/cm²) on the expression of odontoblast-like cell markers by dental pulp stem cells (DPSCs). In contrast to the present study, which evaluated laser irradiation as a standalone treatment following NaOCl irrigation, Martín et al. used lasers as an adjunct to EDTA, applying them during irrigation to activate the chelating solution. They reported that EDTA alone promoted the highest expression of dentin sialophosphoprotein (DSPP) and dentin morphoprotein-1 (DMP-1), two key markers of odontoblastic differentiation. However, when EDTA was combined with either 808–980 nm diode lasers, the expression of these markers was significantly reduced compared to EDTA alone [33].

On the whole, all studies support the potential of laser therapy as an adjunctive or alternative strategy to EDTA for dentin bioactivation, with Malekpour and García-Guerrero et al. highlighting the benefits of low-level PBM for cell viability and migration, and our study demonstrating that higher-energy laser irradiation at optimized parameters can effectively release TGF-β1 and promote stem cell differentiation directly from the dentin surface without the cytotoxic concerns associated with EDTA.

Despite the promising findings of this study, several limitations of this in vitro study including tooth block model, limited sample size and evaluation of one growth factor type (TGF-β1) should be acknowledged. In this study dentin surface analysis was qualitative and no quantitative surface roughness analysis was performed. Also, tooth type heterogeneity may have introduced variability in dentin thickness, tubule density, and morphology and subsequently laser absorption characteristics. Furthermore, laser output power was not verified using a calibrated power meter, and we relied solely on the manufacturer’s specified output values.

On the whole, several directions for future research are proposed. Optimization of laser parameters (wavelength, fluence, pulse structure) should be conducted for different tooth types to account for compositional variability. Future studies should also evaluate the growth factor release from biofilm-cultured dentin. Finally, translation to in vivo models is essential to confirm the regenerative potential of laser-conditioned dentin in a physiological context.

Conclusion

Low to moderate-energy Nd: YAG and 980 and 635 nm diode laser conditioning effectively enhanced TGF-β1 release from root dentin, supported SHED viability, and promoted early odontogenic differentiation (ALP activity) to levels comparable to or exceeding conventional NaOCl/EDTA treatment. While direct laser irradiation partially disrupted the smear layer and increased surface roughness, it did not reliably produce fully patent dentinal tubules. These findings suggest that optimized laser parameters may serve as a viable adjunctive or alternative to chemical chelation for dentin bioactivation.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization: H.M, E.F, AH.S, H.E, S.A, E.B- Data curation: S.A, H.M- Formal analysis: E.F, E.B- Funding acquisition: H.M, AH.S, H.E, E.B- Investigation: E.F, E.B, H.E- Methodology: H.M, E.F, AH.S, H.E, S.A, E.B- Project administration: H.M, E.F, AH.S, H.E, S.A, E.B- Resources: H.M, AH.S, H.E, E.B- Software: E.F, H.M, AH.S, S.A- Supervision: H.M, AH.S- Validation: H.M, AH.S- Writing – original draft: E.B- Writing – review and editing: H.M, E.F, AH.S, H.E, S.A, E.B.

Funding

None.

Data availability

Data available upon request.

Declarations

Clinical trial number

Not applicable.

Competing interests

The authors declare no competing interests.