The present study, in conjunction with our previous study13, has found interesting differences between these four wavelengths of PBM. Broadly speaking there appears to be distinct differences between blue and green light on the one hand, and red and NIR light on the other hand. The two studies also highlight interesting differences between differentiation and proliferation programs in hASC. We previously found13 that blue/green wavelengths were more effective in triggering osteogenic differentiation (when the hASC were cultured in osteogenic medium, compared with same dose of red/NIR. The involvement of TRP ion channels in this mechanism was implicated by the finding that ion channel inhibitors CPZ and SKF96365 (SKF) abrogated the osteogenic differentiation produced by blue/green light.

The literature reports of the effects of blue light on various cellular processes are highly confusing. Most of the studies however do agree that the overall effect of blue light PBM is to increase the level of cellular ROS. This ROS increase is likely to inhibit proliferation in many cases14, 15, and can even produce apoptosis and outrightcytotoxicity if the doses are high enough16, 17. However matters are complicated by the fact that the term “blue light” has been used to refer to wavelengths spanning a range of over 100 nm. Violet light (395–425 nm) is known to be antibacterial and the chromophore responsible for this action is thought to be generation of ROS via photoexcitation of metal free porphyrins that have a large Soret band at about 405 nm18. Royal blue light (430–470 nm) is also known to generate ROS and in this case the chromophores are thought to be flavins, flavoproteins or cryptochromes19. However, it is not clear to what extent cryptochromes are expressed in mammalian cells compared to insects, plants and marine organisms20. Blue-green light (475–510 nm) is thought to be primarily absorbed by opsins (OPNs). Opsins are a group of cis-retinal dependent G-protein coupled receptors (the best known of which are the visual pigments (present in the retinal photoreceptors (OPN1 in cones and OPN2 in rods)21. However, there are now 5 known members of the mammalian opsin family, all of which have different absorption maxima22. OPN3 is called encephalopsin or panopsin and absorbs blue-green light23, OPN4 is called melanopsin and has a maximum absorption at 480 nm24 while OPN5 is called neuropsin and absorbs 380 nm25. Opsins signal via two main pathways depending on the type of G-protein they are coupled with26, 27. Those opsins (OPN1, OPN2, OPN3, OPN5) that are coupled with G o , G i, G t , G s proteins signal via a pathway involving cyclic nucleotides (cAMP and cGMP). On the other hand OPN4 is coupled to Gq and signals via the phospholipase C pathway leading to production of inositol triphosphate and di-acylglycerol. It is known that activation of retinal opsins by blue light can generate ROS, which is responsible for ocular phototoxicity caused by violet and blue light28. One of the main downstream signaling pathways activated by opsins, is related to members of the family of transient receptor potential (TRP) cation channels such as TRPV1 (vanilloid sub-family). TRP channels are a superfamily of 28 members identified by amino acid sequence similarities and sub-divided into organized into six subfamilies29. TRPV1 (capsaicin receptor) has been shown to be activated by various wavelengths of light, including green, red and NIR30,31,32,33. The first law of photobiology states that there must be a specific photoacceptor molecule (or chromophore) for any biological effect to be caused by light34. At present the leading hypothesis to explain the light activation of TRP channels, is that the photons (especially blue and green photons) are absorbed by light-sensitive proteins in the family of mammalian opsins (comprising OPN1-5)35, 36 and that the opsins are linked to TRP channels. OPN4 (known as melanopsin) has been shown to be involved in photoentraiment or regulation of the circadian rhythm37. TRP channels have been called “a missing bond in the photoentrainment mechanism of peripheral clocks throughout evolution”26.

Our results with red and NIR light are in broad agreement with previously published data from many other investigators9. It has been shown that NIR light could produce a biphasic increase in ATP, and a biphasic increase in MMP with a peak response at 3 J/cm2 in cultured cortical neurons38. One important mechanistic issue in this regard, is the realization that ROS can be produced in mitochondria when the MMP is lowered, but also ROS are produced when the MMP is increased above the baseline. If the MMP is lowered by mitochondrial dysfunction, then the action of PBM in raising the MMP will lead to a drop in the levels of ROS generated in the mitochondria39. On the other hand if the mitochondria are normal then the action of PBM will be to raise MMP above baseline, which will lead to a brief and relatively modest burst of ROS40.

We were able to distinguish some subtle differences between the effects of blue light and green light, although overall they were broadly similar, especially when compared to the stark differences produced by red and NIR light. While the effects of blue and green to inhibit proliferation of hASCs and to reduce ATP levels, were almost identical, it was clear that blue light produced higher quantities of ROS and a more pronounced drop in MMP compared to green light. On the other hand green light produced a bigger change in intracellular calcium than did blue light, and a slightly more pronounced drop in intracellular pH. This could be explained if blue light had a more damaging effect against mitochondrial chromophores (CCO and other cytochromes) than did green light, as might be expected by the fact that heme prosthetic groups have a much higher absorption in the blue than the green. The more pronounced action of green light in elevating intracellular calcium concentrations could be explained by the action of green light to trigger opening of TRP ion channels by absorption of photons by a member of the opsin family. By contrast, it was striking how similar the responses of the hASCs to red light and NIR light actually were. This similarity underlines the often proposed theory that both wavelength ranges are absorbed by CCO and stimulate mitochondrial respiration, increase ATP, and only produce modest levels of ROS. It was interesting that the CPZ ion channel inhibitor, partly (but not completely) blocked the generation of ROS by green light, and to a lesser extent by blue light. This implies that blue light generates ROS by for instance (an adverse effect in mitochondria) and also by triggering TRPV channel opening. On the other hand, green light produced ROS more by activating TRPV ion channels and less by an adverse effect in mitochondria. It should be noted that the TRPV1 agonist has been shown to signal by (among other pathways) the generation of ROS41.

We believe that the most likely explanation for these observations is that there are (at least two) two mechanisms and two different chromophores at play here. The effects of red and NIR light are consistent with a mitochondrial chromophore (giving more ATP, increasing MMP, and producing only a low level of ROS). The different effects of blue and green light may also be mediated by two different chromophores, one for green and another for blue. However there also clearly is considerable overlap between these two wavelength ranges, which might be expected considering that the bandwidth of biological chromophores can be quite wide. For blue light the effects seem to be more likely to adversely affect mitochondria and cause ROS production, while for green light the effects appear to more concern ion channel activation and reduction in intracellular pH.

Evidence is emerging concerning the role of light and heat-activated ion channels in various PBM experiments. There is evidence that green light in particular, can activate TRPV1. Gu et al.30 studied activation of the TRPV1 channel that had been exogenously expressed in Xenopus oocytes by red (637 nm) and green (532 nm) laser light. They found that green laser produced more activation TRPV1 than red laser as measured by patch-clamp experiments. TRPV channels are involved in mast cell activation leading to degranulation42. Laser activation of TRPV1 in mast cells was abrogated by SKF and ruthenium red (broad-spectrum inhibitors of mammalian ion channels). Wu et al.32 compared histamine release from mast cells with different wavelengths, and found that blue laser was better than red and green had little effect. Yang et al.33 found that the mast cell degranulation and intracellular calcium increased equally after irradiation with blue or green laser.

We recently published43 another study, which examined whether there was a difference in the response of these identical hASCs to two different wavelengths of near-infrared light. We used a 810 nm laser and a 980 nm laser. Interestingly both wavelengths displayed a biphasic dose response with the cells being responsive to much lower energy densities from a 980 nm laser (0.3 J/cm2 maximum response), than they did to 810 nm laser (3 J/cm2 maximum response). Again we were surprised to discover that the mechanisms of action showed distinct and remarkable differences. Similar to the data reported in the present study, we found that 810 increased MMP and ATP. However 980 nm appeared to activate TRP ion channels, as shown by a pronounced increase in intracellular calcium, and the ability of ion channel blockers (CPZ and SKF) to inhibit the hASC responses.

Our studies on PBM of hASCs taken together (present work and13, 43, have highlighted some interesting facts concerning the relative abilities of PBM to stimulate proliferation or to trigger differentiation in stem cells. Admittedly the culture medium is the most important single factor in deciding whether the hASC will proliferate or alternatively differentiate. Various additional factors added to different kinds of medium will specify the exact differentiation pathway that the cells progress down, i.e. to form osteoblasts, neurons, chondrocytes, adipocytes, hepatocytes, etc. Our particular focus was on differentiation into osteoblasts. We found that PBM wavelengths (415 nm/540 nm/980 nm) that activated TRPV ion channels, increased intracellular calcium, reduced ATP, and produced larger amounts of ROS, and all tended to encourage osteogenic differentiation, while at the same time inhibiting proliferation (415 nm/540 nm). On the other hand, wavelengths (660 nm/810 nm) that increased ATP, raised MMP, and only produced modest amounts of ROS, stimulated proliferation, while having no particular effect on differentiation. There are some studies that suggest that treatments that cause some degree of cell stress, can promote hASC differentiation. These treatments may include ROS44, heat shock45 and gamma irradiation46.

In conclusion, blue and green light inhibits proliferation of hASCs, while red and NIR (810 nm) light stimulate proliferation all at the same dose (3 J/cm2). Considering the pronounced biphasic nature of the dose response curve in PBM, it would be necessary to test other doses of light at different wavelengths before drawing any firm conclusions, especially considering the differences we found in the maximum dose between 810 nm and 980 nm43. We found differences between the mechanisms of action of blue/green as compared to red/NIR, and even subtle differences between the mechanisms of blue and green. To answer those who point out that blue light does not penetrate tissue, we might point out that a blue light LED device (Philips BlueTouch) has recently received regulatory approval for relief of back pain, suggesting that the clinical application of blue light for PBM applications may not be completely impracticable.