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. 2023 Nov 10:24:602-616.
doi: 10.1016/j.reth.2023.11.003. eCollection 2023 Dec.

Enhancing osteogenic differentiation in adipose-derived mesenchymal stem cells with Near Infra-Red and Green Photobiomodulation

Daniella Da Silva  1 , Anine Crous  1 , Heidi Abrahamse  1
Affiliations

Enhancing osteogenic differentiation in adipose-derived mesenchymal stem cells with Near Infra-Red and Green Photobiomodulation

Daniella Da Silva et al. Regen Ther. .

Abstract

Worldwide, osteoporosis is the utmost predominant degenerative bone condition. Stem cell regenerative therapy using adipose-derived mesenchymal stem cells (ADMSCs) is a promising therapeutic route for osteoporosis. Photobiomodulation (PBM) has sparked considerable international appeal due to its' ability to augment stem cell proliferation and differentiation properties. Furthermore, the differentiation of ADMSCs into osteoblast cells and cellular proliferation effects have been established using a combination of osteogenic differentiation inducers and PBM. This in vitro study applied dexamethasone, β-glycerophosphate disodium, and ascorbic acid as differentiation inducers for osteogenic induction differentiation media. In addition, PBM at a near-infrared (NIR) wavelength of 825 nm, a green (G) wavelength of 525 nm, and the novel combination of both these wavelengths using a single fluence of 5 J/cm2 had been applied to stimulate proliferation and differentiation effectivity of immortalised ADMSCs into early osteoblasts. Flow cytometry and ELISA were used to identify osteoblast antigens using early and late osteoblast protein markers. Alizarin red Stain was employed to identify calcium-rich deposits by cells within culture. The morphology of the cells was examined, and biochemical assays such as an EdU proliferation assay, MTT proliferation and viability assay, Mitochondrial Membrane Potential assay, and Reactive Oxygen Species assay were performed. The Central Scratch Test determined the cells' motility potential. The investigative outcomes revealed that a combination of PBM treatment and osteogenic differentiation inducers stimulated promising early osteogenic differentiation of immortalised ADMSCs. The NIR-Green PBM combination did appear to offer great potential for immortalised ADMSC differentiation into early osteoblasts amongst selected assays, however, further investigations will be required to establish the effectivity of this novel wavelength combination. This research contributes to the body of knowledge and assists in the establishment of a standard for osteogenic differentiation in vitro utilising PBM.

Keywords: Adipose-derived mesenchymal stem cells (ADMSC); Differentiation inducers; Osteogenic induction; Photobiomodulation (PBM); Stem cell therapy.

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Conflict of interest statement

All authors declare that they have no conflicts of interest that could be perceived as influencing the research, data interpretation, or publication of this work. The funders have no involvement in the design, execution, or reporting of the research.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
The proposed mechanisms of Near-Infrared and Green light. Photobiomodulation using a Near-Infrared light of 825 nm wavelength penetrates through the cell membrane reaching the mitochondria of the cell. The enzymatic chromophore, CcO, is found within the mitochondria and absorbs the infrared light. The enzyme plays a role in the electron transport chain during ATP production. Increased amounts of ATP bring about an increased gene transcription within the nucleus resulting in increased DNA and RNA synthesis causing the cells to proliferate. Photobiomodulation using a Green light of 525 nm wavelength penetrates the cell membrane and excites the downstream target opsin receptor, the transient receptor potential (TRP) channel. After the application of a stimulus, TRP channels will open and allow an overflow of calcium (Ca2+) ions into the cell cytoplasm. The Ca2+ influx enables calcium/calmodulin dependent kinase II (CAMKII) activity triggering cAMP response element-binding protein (CREB) phosphorylation in the nucleus. The gene transcription changes initiated by CREB are believed to produce the favourable effects of PBM such as cell differentiation.
Fig. 2
Fig. 2
Theoretical clinical application. The isolation of adipose-derived mesenchymal stem cells from the host via a biopsy, from which cells are then stimulated to proliferate and differentiate using various osteogenic differentiation inducers and PBM as a physical stimulant. The post-differentiated osteogenic cells are transplanted back into the host for therapeutic application.
Fig. 3
Fig. 3
Experimental cell culture model. Cells in Group A received neither osteogenic differentiation inducers nor photobiomodulation treatment. Cells in Group B received osteogenic differentiation inducers but did not receive photobiomodulation treatment. Cells in Group C received both osteogenic differentiation inducers and Near Infra-red photobiomodulation treatment at an 825 nm wavelength at a 5 J/cm2 dose. Cells in Group D received both osteogenic differentiation inducers and Green photobiomodulation treatment at a 525 nm wavelength at a 5 J/cm2 dose. Cells in Group E received both osteogenic differentiation inducers and Near Infra-Red-Green combined photobiomodulation treatment at 825 nm and 525 nm wavelengths at a 5 J/cm2 dose.
Fig. 4
Fig. 4
Experimental methodology. Immortalised adipose-derived mesenchymal stem cells were resuscitated and sub-cultured until desirable confluency was attained. Dexamethasone, β-glycerol phosphate disodium, and ascorbic acid differentiation inducers were used to initiate and direct osteogenic differentiation during a several days incubation. The cells were irradiated with a single fluence of 5 J/cm2 at wavelengths of Near-Infrared 825 nm, Green 525 nm, and their combination wavelengths for enhanced cellular differentiation into osteoblasts and cellular proliferation. An experimental standard in which cells received neither osteogenic differentiation inducers nor photobiomodulation treatment, as well as an experimental control in which cells received only osteogenic differentiation inducers and not photobiomodulation treatment, were included. The cell samples were collected at 24 h, 48 h and 7 days post-irradiation. Cell characterisation, via the use of flow cytometry as a quantitative assay, identified early protein markers (FGFR-3 and Col-1) and late osteogenic protein markers (Osteocalcin, Biglycan, DMP-1 and FGF-23). Calcium deposit identification was recognised by Alizarin red Stain morphology. Morphological analysis was seen by inverted light microscopy. Cell migration was investigated via the ‘Central Scratch Test’ method. Biochemical analysis such as cell viability, cell proliferation, membrane permeability, mitochondrial membrane potential and reactive oxygen species were determined. The protein expression of osteogenic early (RUNX2) and late (Osteocalcin and Osterix) transcription factors were identified using ELISA.
Fig. 5
Fig. 5
Characterisation of early osteogenic surface markers, FGFR-3, and Col-1, investigated at 24 h and 7 days post-photobiomodulation irradiation. A statistically significant increase at 24 h post-photobiomodulation treatment in FGFR-3 marker expression by the control, Near-Infrared and Green photobiomodulation groups compared to the Near Infrared-Green experimental photobiomodulation group occurred. A statistically significant increase in Col-1 marker expression by the control, Green and Near Infrared-Green photobiomodulation was identified at 24 h post-irradiation. At 7 days post-photobiomodulation treatment, a statistically significant increase in FGFR-3 and Col-1 marker expression by Near Infrared-Green photobiomodulation compared to the control group and amongst the experimental photobiomodulation groups was observed.
Fig. 6
Fig. 6
Characterisation of late osteogenic surface markers, FGF-23, BGLAP, BGN and DMP-1, investigated at 24 h and 7 days post-photobiomodulation irradiation. A statistically significant increase at 24 h post-photobiomodulation treatment in FGF-23, BGN and DMP-1 marker expression by Near Infrared-Green photobiomodulation compared to the control group and amongst the experimental photobiomodulation groups was identified. A statistically significant increase in BGLAP marker expression by Near Infra-red photobiomodulation was identified at 24 h post-irradiation compared to the control and the other experimental photobiomodulation groups occurred. At 7 days post-photobiomodulation treatment, a statistically significant increase in FGFR-3 and DMP-1 marker expression by Near Infra-red photobiomodulation compared to the control group and amongst the experimental photobiomodulation groups was observed. A statistically significant increase in BGLAP marker expression by the control group and Green photobiomodulation at 7 days post-irradiation treatment was identified. A statistically significant increase was observed in BGN marker expression at 7 days post-photobiomodulation by the Near Infrared-Green photobiomodulation group compared to the control and other photobiomodulation treatment groups.
Fig. 7
Fig. 7
Protein expression of i) RUNX2, ii) Osteocalcin and iii) Osterix using ELISA analysis. A statistically significant increase at 7 days post-photobiomodulation treatment in RUNX2 and Osteocalcin protein expression by Near Infrared-Green photobiomodulation compared to the control group and amongst the experimental photobiomodulation groups.
Fig. 8
Fig. 8
i) Morphological characterisation of cells using an Alizarin red Stain. ii) Quantification of colour intensity of Alizarin red Stain. i) The identification of bright orange deposits at 24 h and 7 days post-photobiomodulation treatment indicative of calcium deposition presence, which is suggestive of osteogenic differentiation (C, D, E, H, I and J). ii) A statistically significant increase occurred at 7 days post-photobiomodulation treatment in Alizarin red Stain colour intensity by Green photobiomodulation and Near Infrared-Green photobiomodulation compared to the control group.
Fig. 9
Fig. 9
Morphology of immortalised adipose-derived mesenchymal stem cell differentiation 24 h, 48 h, and 7 days post-photobiomodulation treatment using inverted light microscopy. The cell morphology had become rounded and/or shorter spindle shaped at 24 h post-photobiomodulation treatment (D and E), at 48 h post-photobiomodulation treatment (H, I and J) and at 7 days post-photobiomodulation treatment (L, M, N and O). The noticeable changes in cell morphology are similar in appearance to the characteristic cell shape of osteoblast-like cells, suggestive of cell differentiation.
Fig. 10
Fig. 10
i) Morphological representation of cellular motility of differentiated immortalised adipose-derived mesenchymal stem cells. Cellular migration was visibly apparent by artificial wound closure at 24 h post-photobiomodulation treatment (H and I), at 48 h post-photobiomodulation treatment (K-M) and at 7 days post-photobiomodulation treatment (P-T). ii) Motility analysis of differentiated immortalised adipose-derived mesenchymal stem cells. A statistically significant enhancement of cell motility occurred at 24 h and 48 h post-photobiomodulation amongst the experimental photobiomodulation groups compared to the control. This is suggestive of an enhanced cell homing capacity. At 7 days post-photobiomodulation treatment, all the experimental photobiomodulation groups including the standard and the control presented with statistically significant increases in artificial wound closure. The wound closure identified in the standard group at 7 days post-photobiomodulation may be explained by cell overcrowding.
Fig. 11
Fig. 11
EdU Proliferation of osteogenic differentiated immortalised adipose-derived mesenchymal stem cells at 24 h, 48 h, and 7 days post-photobiomodulation treatment. A right shift occurred at 24 h and 48 h post-photobiomodulation treatment in EdU expression compared to the control group (C, D and J). A left shift occurred at 7 days post-photobiomodulation treatment in EdU expression compared to the control group (O). The left shift may be an outcome of cell culture contact competition due to cell over-crowding and a lack of available nutrients and/or the redirection of cellular energy for cellular differentiation.
Fig. 12
Fig. 12
i) Cellular proliferation/viability analysis using the MTT assay demonstrated a statistically significant decline in cell proliferation at 24 h post-photobiomodulation treatment amongst the Green and Near Infrared-Green photobiomodulation groups compared to the control and Near-infrared photobiomodulation groups. The premature decline is suggestive of ATP redistribution from cellular proliferation to cellular differentiation. ii) Cellular viability using the trypan blue assay provided a reassurance of using photobiomodulation to differentiate immortalised adipose-derived mesenchymal stem cells whilst maintaining cell health as cell viability remained consistent at 24 h, 48 h, and 7 days post-photobiomodulation treatment.
Fig. 13
Fig. 13
Morphological analysis of immortalised adipose-derived mesenchymal stem cell mitochondrial membrane potential at 24 h, 48 h, and 7 days post-photobiomodulation treatment. The cell morphology demonstrated an increase in fluorescence amongst the experimental photobiomodulation groups at 48 h post-photobiomodulation treatment (H-J). A further increase in fluorescence were identified in the control group and experimental photobiomodulation groups at 7 days post-photobiomodulation treatment (L-O), whereby, the Near-infrared-Green experimental group displayed fluorescence to a greater extent (O). The noticeable bright mitochondrial fluorescence is suggestive of cellular differentiation.
Fig. 14
Fig. 14
Reactive oxygen species production had displayed a significant increase amongst the experimental photobiomodulation groups at 24 h and 48 h post-photobiomodulation compared to the standard group and at 7 days post-photobiomodulation treatment in comparison to the control group. Due to the reassurance of consistent and sustainable cell viability despite photobiomodulation treatment (Fig. 12 ii), the significant increases in reactive oxygen species production are not suggestive of cell harm but instead, implies stem cell fate direction of differentiated immortalised adipose-derived mesenchymal stem cells.
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