Abstract
Degenerative bone conditions, such as osteoporosis, place a considerable strain on healthcare systems worldwide due to their widespread occurrence and detrimental impact on quality of life (Lems and Raterman, 2017). Current treatments mainly aim to alleviate symptoms rather than fully regenerate damaged bone tissue (Lohiya et al., 2023). This limitation highlights the need for new treatment approaches, such as regenerative medicine using stem cells (SCs), to transform care options and potentially address a wide range of conditions (Velikic et al., 2024; Mahla, 2016). Stem cells play a crucial role in these advancements due to their ability to differentiate into multiple cell types, supporting tissue repair and regeneration (Jin et al., 2023). Among stem cells, adipose-derived mesenchymal stem cells (ADMSCs) are especially promising because they are readily accessible, available in large quantities, and possess strong differentiation potential (Mazini et al., 2019). However, ADMSCs tend to favour adipogenic rather than osteogenic pathways, making it essential to use differentiation inducers (DIs) to steer them toward bone-forming lineages (Zhang et al., 2020). Even with the application of DIs, ADMSCs may still preferentially differentiate into adipogenic lineages under specific conditions. This highlights the necessity for a comprehensive strategy that integrates DIs with biomechanical and biophysical methods, such as a three-dimensional (3D) hydrogel matrix and photobiomodulation (PBM), to achieve targeted differentiation (Bikmulina et al., 2022; Calis et al., 2022).
The use of 3D cell culture systems can greatly enhance the osteogenic differentiation and maturation of ADMSCs by creating an environment that more closely resembles physiological conditions (Kapałczyńska et al., 2018). This method can help connect in vitro models with in vivo applications, providing detailed insights into cellular interactions and metabolic processes, thereby advancing research in stem cells (Jensen and Teng, 2020). Moreover, PBM is acknowledged for its ability to enhance cellular proliferation and differentiation (Arany, 2016). Research indicates that PBM treatment of ADMSCs fosters their growth, development, and differentiation into various cell lineages (Hamblin, 2017). The effects of PBM
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on ADMSCs are affected by parameters such as wavelength and fluence, which can either stimulate or inhibit cellular responses (Soleimani et al., 2012). For example, wavelengths ranging from 660 to 850 nm and fluences between 5 and 10 J/cm² have been linked to increased cell proliferation (Escudero et al., 2019), while wavelengths from 495 to 570 nm promote cell differentiation (Wang et al., 2016). However, further studies are needed to establish standardized protocols that optimize the effectiveness of PBM in stem cell therapies.
This in vitro study was conducted to explore the effects of osteogenic DIs, dextran hydrogel matrices, and PBM using specific wavelengths and fluences. The study utilized near-infrared (NIR) light at 825 nm, green (G) light at 525 nm, and a combination of both NIR and green treatments (825 nm and 525 nm) to assess their influence on the proliferation and differentiation of immortalised ADMSCs into functional osteoblasts and osteocytes. Fluencies of 3 J/cm², 5 J/cm², and 7 J/cm² were applied to evaluate changes in cellular behaviour. The differentiation process for the immortalized ADMSCs began with culture in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS Superior), 1% antibiotics (including 0.5% Penicillin-Streptomycin and 0.5% Amphotericin B), along with an induction medium (IM) composed of 50 nM dexamethasone, 10 mM β-glycerol phosphate disodium, and 0.2 mM ascorbic acid. Additionally, the cells were encapsulated in a 10 μL dextran hydrogel disc at a density of 1 x 10⁴ cells per well within treated 96-well microplates and incubated for 7 days. After changing the media, the cells were exposed to the NIR 825 nm Diode Laser, the G 525 nm Diode Laser, and the combined NIR-G wavelengths at fluences of 3 J/cm², 5 J/cm², and 7 J/cm². Samples were collected for analysis at both 24 hours and 7 days following irradiation. Cell characterization was conducted using flow cytometry to identify the stem cell protein marker Thy-1, as well as markers for early osteoblasts (Alkaline phosphatase and Runt-related transcription factor-2), late osteoblasts (Biglycan), early osteocytes (Osteocalcin), and late osteocytes (Sclerostin). Calcium deposition was assessed with Alizarin red staining, and cell morphology was analysed using inverted light microscopy. Additional biochemical assessments included cell viability,
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proliferation, reactive oxygen species (ROS) levels, and membrane permeability. Gene expression of osteogenic transcription factors was measured using qPCR.
The results demonstrated that the combined effects of specific PBM wavelengths and fluencies significantly promoted osteogenic differentiation, as evidenced by increased expression of osteogenic markers and distinct morphological changes associated with osteoblast-like and osteocyte-like phenotypes. Analysis revealed that PBM, particularly at 5 J/cm2 and 7 J/cm2 fluences, markedly enhanced osteogenic marker expression at both early and late stages of differentiation. Protein level analyses demonstrated that PBM at 5 J/cm2 for G and 7 J/cm2 for NIR significantly enhanced the expression of critical osteogenic markers, including RUNX2, BGLAP, BGN, and SOST. This increase not only indicates PBM’s role in early osteogenic differentiation but also supports immortalised ADMSC maturation towards osteoblast-like and osteocyte-like phenotypes. The 5 J/cm2 fluence consistently resulted in high osteoblast marker expression across both the 24 hours and 7 days post-PBM treatment time points, suggesting PBM’s effectiveness in sustaining osteogenic commitment over time. Early osteogenic marker ALP activity was significantly elevated following PBM treatment, particularly at G 5 J/cm2 and NIR 7 J/cm2 across both 24 hour and 7-day time points. Morphological assessment supported PBM’s impact on cell shape, with inverted light microscopy revealing a shift from elongated, spindle-shaped ADMSC forms to compact, cuboidal osteoblast-like shapes. The addition of RGD peptides further enhanced these morphological adaptations, especially in the NIR 7 J/cm2 and G 5 J/cm2 groups, where cells displayed a stellated shape indicative of osteocyte-like morphology by day 7. Alizarin Red S staining confirmed significant calcium deposition, which is a marker of bone matrix mineralization, within the G 5 J/cm2 and NIR 5 J/cm2 groups as early as 24 hours post-treatment. This rapid mineralization persisted through day 7 post-PBM treatment, demonstrating PBM’s potential to accelerate bone-like matrix formation. Gene expression analysis via qPCR provided further insight into PBM’s effect on the osteogenic differentiation pathway, with specific gene expression patterns varying according to wavelength and fluence. The stem
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cell marker CD90 was downregulated in response to the combination treatment at 3 J/cm2 after 24 hours post-treatment, while osteogenic genes RUNX2 and BGLAP were consistently upregulated in the NIR 7 J/cm2 and G 3 J/cm2 and 5 J/cm2 groups. These patterns persisted at day 7 post-PBM exposure, highlighting RUNX2’s role in early differentiation and underscoring BGLAP and BGN’s importance in advancing osteogenesis. The increased expression of SOST in NIR 7 J/cm2 and G 5 J/cm2 groups indicates its involvement in regulating osteocyte maturation, supporting the conclusion that PBM influences immortalised ADMSC progression into mature osteogenic cell types.
The combined use of osteogenic DIs, dextran hydrogel matrices, and PBM proves to be a promising approach for advancing the osteogenic differentiation of immortalised ADMSCs. This study demonstrates that PBM, when precisely modulated in terms of wavelength and fluence, enhances differentiation processes at both protein and genetic levels, with clear implications for regenerative medicine applications. The dextran hydrogel provided a supportive 3D scaffold that aided PBM mediated differentiation, while varying hydrogel rigidities could be explored in future studies to more accurately mimic the bone extracellular matrix and promote late-stage differentiation of cells into osteocyte-like cells. This study contributes a valuable foundation for optimizing PBM protocols in bone tissue engineering, presenting as a potential precursor to osteo-degenerative therapies.