Combined light treatment and chemical signals increase bone cell development from fat stem cells in 3D culture

Osteoporosis and other degenerative bone diseases are a major global health concern, affecting over 200 million people worldwide and contributing to millions of fractures annually, according to the World Health Organization [1,2]. These conditions significantly impact mobility, quality of life, and healthcare systems. Current treatments, including anti-resorptive drugs and hormone replacement therapy, aim to slow bone loss but often fail to restore full bone integrity [3]. Therefore, effective strategies that actively promote bone regeneration are essential [4].

Regenerative medicine has emerged as a promising avenue for addressing this challenge, particularly through stem cell-based therapies. Among these, adipose-derived stem cells (ADSCs) have gained attention due to their accessibility, minimal invasiveness in harvesting, and high differentiation potential [5,6]. Compared to bone marrow-derived mesenchymal stem cells, ADSCs can be obtained in larger quantities with less patient burden [7]. However, a limitation of ADSCs is their inherent tendency to differentiate into adipocytes rather than osteoblasts, necessitating strategies that more effectively direct osteogenic differentiation [8]. Osteogenic differentiation inducers (DIs), such as dexamethasone, β-glycerophosphate, and ascorbic acid, are commonly used to guide this process, but their efficacy alone is often insufficient to overcome the adipogenic bias [9].

Recent studies have highlighted the potential of integrating osteogenic DIs with three-dimensional (3D) hydrogels and photobiomodulation (PBM) to enhance osteogenic differentiation outcomes [10]. Photobiomodulation uses specific wavelengths of light to modulate cellular activity, promoting proliferation and differentiation under carefully calibrated conditions [11]. Simultaneously, 3D hydrogels mimic the extracellular matrix, providing a supportive scaffold for cell adhesion, migration, and tissue-like organisation essential for successful differentiation [12]. The combination of PBM and 3D hydrogels creates a microenvironment that closely resembles the natural bone niche, potentially improving the precision and robustness of osteogenic differentiation [13].

The choice of 825 nm and 525 nm wavelengths for PBM in this study was informed by their documented effects on cellular processes critical to osteogenesis. Near-infrared (NIR) light at 825 nm penetrates deeper into biological tissues and has been shown to enhance mitochondrial activity, ATP production, and cell proliferation, processes that are essential for initiating and sustaining osteogenic differentiation [14,15]. Green light at 525 nm, while less penetrative, has demonstrated efficacy in modulating oxidative stress and cellular signalling pathways that influence differentiation outcomes [16,17]. The combined application of these wavelengths seeks to leverage their complementary effects, with NIR supporting energy metabolism and proliferation, and green light enhancing differentiation-specific pathways. The fluencies of 3, 5, and 7 J/cm² were selected based on previous findings, indicating these energy levels effectively stimulate osteogenic differentiation without inducing phototoxicity [18,19]. By fine-tuning these parameters, this study aims to maximise the osteogenic potential of PBM within a physiologically relevant model.

This study investigates the synergistic effects of osteogenic DIs, a fast-dextran hydrogel matrix, and PBM at specific wavelengths and fluences on the osteogenic differentiation of immortalised ADSCs. The analysis focused on key markers, including CD90 (a stem cell surface marker), RUNX2 (a transcription factor indicative of early osteoblast differentiation), BGLAP (osteocalcin, an early osteocyte marker), BGN (biglycan, a late osteoblast marker), and SOST (sclerostin, a late osteocyte marker). These markers were evaluated to determine differentiation outcomes and to explore how PBM influences their regulation. These markers were chosen for their critical roles in the osteogenesis process, providing insights into the transition from stem cells to mature osteoblasts and osteocytes. By optimising the interplay between DIs, the hydrogel matrix, and PBM parameters, this study aims to develop a targeted and physiologically relevant model for enhancing osteogenic differentiation.

The findings of this study align with previous research demonstrating PBM’s efficacy in promoting osteogenic differentiation in ADSCs under varied parameters. This is consistent with the studies by Peng et al. and Oliveira et al., which showed significant increases in osteogenic markers following PBM. Our study highlights PBM’s role in stemness retention through CD90 expression shortly after treatment [38,39]. This retention, especially evident in the G 5 J/cm² group, suggests a phased progression toward differentiation, essential for controlled osteogenic signalling [20]. Likewise, the elevated CD90 levels in the NIR 7 J/cm² group at 7 days post-treatment emphasises PBM’s potential to balance proliferation and early differentiation, aligning with studies on matrix-interacting stem cells [21,22]. Runt-Related Transcription Factor-2 upregulation, notably in the NIR groups, corroborates Yu et al.’s findings on RUNX2’s critical role in early osteogenic signalling [40]. Specifically, NIR 3 J/cm² and 7 J/cm² fluencies in our study were effective in inducing RUNX2 expression, suggesting ADSC commitment to an osteogenic pathway, as seen in similar osteogenic differentiation studies [41]. The sustained expression in the NIR 7 J/cm² group further suggests an effective phased differentiation, supporting findings from research on NIR-induced osteogenic priming [23]. The early yet transient BGLAP expression at 24 h in the NIR 5 J/cm² group marks an initial osteogenic response that may drive subsequent differentiation processes, as observed in studies using hydrogel matrices and PBM with ADSCs. This expression pattern, reported in early studies on BGLAP, underlines PBM’s role in initiating osteogenic signalling [42]. Increased BGN expression, observed in the G 5 J/cm² and later in the NIR 5 J/cm² groups, indicates PBM’s contribution to advancing osteoblast maturation stages earlier than expected. Yaralı Çevik et al. similarly reported that PBM expedites osteoblast marker expression, with implications for scaffold integration in tissue engineering applications [24,25]. This outcome underscores PBM’s role in enhancing osteogenic maturation and matrix signalling, offering insights into PBM’s optimisation for cellular maturation. The consistent upregulation of SOST in the G 5 J/cm² group, aligning with Zaccara et al.’s findings, suggests PBM’s influence on osteoblast signalling for bone homeostasis, indicating PBM’s capacity to control osteogenic differentiation in regenerative bone therapies [26].

Morphological analysis revealed that PBM-treated ADSCs undergo changes reflective of osteogenic differentiation stages, transitioning from spindle-shaped progenitor cells to mature, matrix-secreting osteoblasts and osteocyte-like cells. Initially, the fibroblast-like shape of ADSCs, indicative of their mesenchymal origin as noted by Lu et al., progresses to a cuboidal morphology in the NIR 7 J/cm² and G 5 J/cm² groups within 24 h, signalling early osteoblast differentiation [43]. This cuboidal shape, which enhances matrix secretion capacity, was sustained in the NIR 5 J/cm², NIR 7 J/cm², and G 5 J/cm² groups at 7 days. The appearance of stellate extensions in these groups suggests differentiation toward an osteocyte-like state, supporting matrix maintenance and intercellular communication essential for bone stability. These morphological changes underscore PBM’s ability to drive ADSC adaptation toward bone-regenerative potential within a 3D matrix environment.

The observed ROS elevation following PBM treatment suggests its involvement in osteogenic signalling, aligning with its established role in differentiation pathways. ROS are essential modulators of redox-sensitive pathways for osteogenic differentiation, as reported by Yaralı Çevik et al. [44]. In the present study, significant ROS increases across all treatment groups at 24 h highlight PBM’s ability to initiate an oxidative response, potentially activating downstream osteogenesis-related pathways [45]. The enhanced ROS levels in the NIR-G 7 J/cm² group, surpassing those with NIR alone, indicate that specific wavelength combinations can optimise ROS-mediated signalling. At 7 days, sustained ROS levels in the NIR 5 J/cm², G 5 J/cm², G 7 J/cm², and NIR-G 7 J/cm² groups suggest continuous ROS involvement in differentiation-promoting pathways, highlighting ROS as active contributors to osteogenic lineage commitment [46]. This prolonged ROS response likely reflects ongoing pathway activation within the 3D matrix environment, underlining ROS’s role in promoting ADSC differentiation toward osteogenesis.

The qPCR results illustrate a dynamic response in osteogenic marker expression following PBM treatment, indicating distinct shifts from the early 24 h time point to the later 7-day time point differentiation stages. The CD90’s significant downregulation at 24 h suggests an early commitment to osteogenesis, supported by studies linking CD90 reduction to osteogenic initiation [27,28]. Early RUNX2 and BGLAP upregulation underscores PBM’s role in accelerating transcriptional and mineralisation readiness, with peak responses at 825 nm and higher fluencies [29,30]. The sustained expression of RUNX2, BGLAP, and BGN at 7 days highlights PBM’s lasting impact on matrix deposition and maturation processes [33,34,35]. Interestingly, CD90 re-upregulation at later stages suggests a balance between retaining stemness and advancing differentiation within the PBM-treated hydrogel matrix, indicating a complex regulatory effect induced by PBM [32]. Elevated SOST expression at 7 days suggests PBM’s influence on bone remodelling pathways and its potential role in promoting cellular maturation and transition to a more specialised osteocyte phenotype [47,48]. This modulation of SOST, which is critical for maintaining bone homeostasis [36,37], provides insights into the mechanisms of osteogenesis, highlighting how PBM may enhance cellular specialisation and contribute to a more refined understanding of treatment effects in bone regeneration. These findings highlight PBM’s potential as a tool for controlled osteogenic differentiation, with wavelength and fluency optimisation supporting applications in bone regeneration and repair.

This study evaluated the combined impact of DIs, dextran hydrogel matrices, and PBM on the osteogenic differentiation of immortalised ADSCs. Results show that specific PBM wavelengths and fluences, integrated with osteogenic DIs and a 3D hydrogel scaffold, significantly increase osteogenic marker expression and facilitate ADSC differentiation into osteoblast- and osteocyte-like cells. Through assessments of protein markers, morphological changes, cell stress responses, and gene expression, this research identifies optimised PBM settings for efficient osteogenic differentiation within controlled laboratory conditions.

Protein analysis revealed that PBM at G 5 J/cm² and NIR 7 J/cm² fluences increased essential osteogenic markers such as RUNX2, BGLAP, BGN, and SOST, suggesting that PBM promotes both early differentiation stages and continued maturation of ADSCs into osteoblast- and osteocyte-like cells. Notably, the 5 J/cm² fluence sustained high expression of osteoblast markers across both 24-h and 7-day time points, implying a role for PBM in maintaining osteogenic progression. The distinct responses of these markers to different PBM parameters emphasise the importance of wavelength and fluence selection for targeted stem cell differentiation, supporting the development of fully functional osteogenic cells.

Morphological analyses confirmed PBM’s role in influencing cell shape. Light microscopy revealed shifts from elongated, spindle-shaped ADSCs to cuboidal, osteoblast-like structures, consistent with prior studies linking morphology to osteogenic commitment [12]. These early morphological changes observed in the G and NIR groups at 24 h, and maintained through 7 days, indicate that PBM promotes structural transformations necessary for osteogenesis. Additionally, RGD peptides in combination with PBM enhanced these morphological adaptations, particularly in NIR-treated groups at 7 J/cm² and G-treated groups at 5 J/cm² by day 7, yielding stellate shapes associated with osteocyte-like phenotypes.

Gene expression analysis via qPCR further elucidated PBM’s effects, revealing that the expression of osteogenic markers varied with wavelength and fluence. At 24 h post-PBM treatment, a downregulation of the CD90 stem cell marker was observed in the combination group at 3 J/cm², while RUNX2 and BGLAP were upregulated with NIR at 7 J/cm² and G at 3 J/cm² and 5 J/cm². This pattern persisted through day 7, suggesting that RUNX2 supports early differentiation while BGLAP and BGN drive osteogenic progression. Sclerostin upregulation at 825 nm and 7 J/cm², as well as at 525 nm and 5 J/cm², implies its regulatory role in differentiation, with continued expression indicative of a mature osteocyte phenotype.

Overall, these findings highlight the potential of optimised PBM parameters to guide osteogenic differentiation of immortalised ADSCs, which could be highly beneficial for regenerative medicine and tissue engineering applications that aim to replicate bone structures. Dextran hydrogels also proved to be effective as 3D scaffolds, providing an environment conducive to PBM-induced differentiation. Future studies may explore hydrogels of varying stiffness to better mimic the bone extracellular matrix, potentially supporting later-stage differentiation into osteocyte-like cells. Furthermore, additional investigations should evaluate the long-term functionality of differentiated cells and examine the translational viability of this approach in preclinical models of bone injury.

The authors express their gratitude to the University of Johannesburg and the Laser Research Centre for providing access to their resources and facilities. They also thank the National Laser Centre for granting permission to use their laser equipment. Figures have been created by Biorender.com.

Conceptualization, D.D.S. and A.C.; methodology, D.D.S. and A.C.; validation, D.D.S. and A.C.; formal analysis, D.D.S.; investigation, D.D.S.; resources, A.C. and H.A.; data curation, D.D.S.; writing—original draft preparation, D.D.S.; writing—review and editing, D.D.S., A.C. and H.A.; visualisation, D.D.S.; supervision, A.C. and H.A.; project administration, D.D.S., A.C. and H.A.; funding acquisition, D.D.S., A.C. and H.A. All authors have read and agreed to the published version of the manuscript.

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of the University of Johannesburg Faculty of Health Sciences (REC-1466-2022). This study did not include any research involving humans or animals.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

This work is supported by the National Research Foundation of South Africa Thuthuka Instrument, grant number TTK2205035996; the Department of Science and Innovation (DSI) funded African Laser Centre (ALC), grant number HLHA23X task ALC-R007; the University Research Council, grant number 2022URC00513; the Department of Science and Innovation South African Research Chairs Initiative (DSI-NRF/SARChI), grant number 98337, and the APC was funded by the University of Johannesburg Faculty of Health Sciences.