The cause of Dupuytren’s disease (DD) is believed to be multifactorial with a genetic disposition and concerns predominantly middle-aged Caucasian men [1]. It’s a benign fibromatosis affecting the palmar fascia of the hand, characterized by an increased subcutaneous nodule, followed by cord formation dependent on the progression of this disease. Responsible for cord or nodule formation is an excessive proliferation and differentiation of fibroblasts to myofibroblasts, so that the myofibroblasts becomes the predominant cell type in Dupuytren’s tissue [2]. And the benign but undesired growth of Dupuytren’s myofibroblasts is supported by their decreased susceptibility to undergo apoptosis [3–5]. In general, differentiated myofibroblasts show characteristics of fibroblasts and smooth muscle cells, and express α-smooth muscle actin (α-SMA), which allows the cells to contract. Therefore myofibroblasts assume mandatory functions in wound healing enabling wound closure. Hence, the common method to identify (Dupuytren’s) myofibroblasts is proving the intracellular α-SMA-amount. The pathological staging of DD is based on the definition given by Luck et al. [6], who suggested to differentiate between proliferative, involutional, and residual stages. In the proliferative phase, α-SMA-expression increases due to the uncontrolled proliferation of myofibroblasts, leading to nodule formation influenced by local mediators such as transforming growth factor-β1 (TGF-β1). During the progression of Dupuytren’s disease myofibroblasts synthesize an elevated level of α-SMA, collagen III and fibronectin [7]. So, the involutional stage is marked by an elevated proportion of collagen III, resulting in the formation of the characteristic cords and an increased extracellular matrix deposition. In the residual phase, nodules and myofibroblasts decrease and α-SMA and collagen III were diminished [8], and replaced by scar tissue.

Furthermore, TGF-β1 seems to play a superordinate role in the formation and maintenance of DD, especially because TGF-β1 induces the expression of α-SMA, collagen III and fibronectin, and has been shown to increase the contraction of myofibroblasts [9, 10]. So, TGF-β is localized in myofibroblasts in all phases of DD [1]. This is not surprising, since TGF-β1 is an abundant pro-inflammatory cytokine, involved in pathological scarring and fibrosis [11].

One therapy option for advanced DD is the partial fasciectomy, which is unfortunately accompanied by significant recurrence rates [12]. This surgical technique removes involved tissue, and contains extensive dissection of diseased longitudinal cords or nodules, which are removed from the surrounding fascia [13]. Needle fasciotomy or enzyme injection as collagenase [14] were performed as alternative methods, and at least 88% of the collagenase-treated patients were observed to have their movement improved by up to 48° movement in a one-year follow-up study [15].

The use of photobiomodulation-based therapies as a treatment option for DD is a promising alternative, which has not been applied to this day. Visible light could mediate its therapeutic effect via the interaction with endogenous photoreceptors, chromophores such as porphyrins, cytochrome c oxidase, nitrosated and flavo- proteins, opsins, and ion-gate channels [16, 17], dependent on the specific characteristics of the irradiated tissue.

It has already been shown, that blue light, dependent on the wavelength, intensity (irradiance) and radiant exposure, has anti-inflammatory effects in keratinocytes [18] and suppresses dendritic cell activation [19]. In rodents blue light inhibits skin tumors [20] and improves wound healing [21]. But, in the context of DD, blue light is particularly interesting, because mitosis and proliferation of cultured cells could be inhibited [22]. Furthermore, the proliferation and induced α-SMA-expression in human dermal myofibroblasts could be diminished [23]. These preconditions could render the irradiance with blue light (λ = 453 nm, intensity 38 mW/cm²) an interesting tool for the treatment of Dupuytren’s fibroblasts. Therefore, the therapeutic benefit of blue light (with these parameters) on Dupuytren’s disease was evaluated in the present work.

Partial fasciectomy, enzyme injection, and needle fasciotomy are todays treatment options for Dupuytren’s disease (DD) [24]. Due to the limitations of these treatment methods, researchers and clinicians are forced to search for better therapies. The usage of photobiomodulation strategies may provide a solution to this problem, because the combination of intensity, radiant exposure, wavelength, and their specific interaction with the absorption properties of the target tissue can have a therapeutic effect [25]. So, blue light (50 mW/cm²; 15, 30 Joule/cm²; λ = 420 nm) has already been proven to inhibit TGF-β-induced α-SMA expression in differentiated human dermal fibroblasts [23]. Especially in Dupuytren’s fibroblasts, the increased and pathological differentiation of fibroblasts to myofibroblasts, is associated with an elevated α-SMA-expression. Hence, in this study the therapeutic benefit of blue light irradiation on Dupuytren’s fibroblasts was analyzed for the first time.

Dupuytren’s fibroblasts and normal palmar fascia (NPF) fibroblasts were irradiated (with a wavelength of λ = 453 nm and an intensity of 38 W/cm²), and as the most promising radiant exposure 40 Joule/cm² was used for the subsequent experimental procedures (Fig 1). Since the irradiation with 40 Joule/cm² hardly inhibited the cell viability/metabolic activity of Dupuytren’s fibroblasts and especially of NPF, which is required for a therapeutic application of blue light in DD.

Up to 60 Joule/cm² irradiation, metabolic activity of Dupuytren’s fibroblasts was increased compared to NPF (Fig 1). This result is supported by several research groups, demonstrating that the proliferation of Dupuytren’s fibroblasts is elevated compared to control cells by the activation of the TGF-β/SMAD- and extracellular signal-regulated kinase (ERK) signaling [26], and an autocrine regulation through epidermal growth factor (ERBB)-2 and insulin growth factor (IGF)-1 receptors, as well as Akt-signaling [27].

Furthermore, it could be demonstrated that after 24 h cell viability of Dupuytren’s fibroblasts appeared to be considerably higher than NPF fibroblasts viability (Fig 1, 0 Joule), although in both groups the same cell number was seeded. This is a remarkable result, because “cell viability” was indirectly determined by measuring metabolic activity (CellTiter-Blue Assay). These results and the elevated ROS amount in Dupuytren’s fibroblasts compared with NPF fibroblasts shown in Fig 4 indicate, that Dupuytren’s fibroblasts have, by nature, a higher metabolism than the respective controls from patients suffering from carpal tunnel syndrome. And this may be an explanation why DD fibroblasts were more affected by the blue light irradiation, than the NPF fibroblasts. This assumption is solidified by the findings, that the expression of the housekeeping gene Glyerinaldehyd-3-phosphat-Dehydrogenase (GAPDH) is increased in Dupuytren’s fibroblasts (data not shown) treated with TGF-β1 compared to NPF.

It could be ascertained that the exposure with λ = 453 nm does not change α-SMA expression in neither resting NPF nor in Dupuytren’s fibroblasts after 72 h and 120 h (Fig 2A and 2C and Fig 3). (It has to be considered, that only one donor causes those high standard deviations of irradiated Dupuytren’s fibroblasts.) Furthermore, to tighten standard deviation is difficult in Dupuytren’s research, because it is a multifactorial disease, and tissue specimen were not classified in regard to secondary diseases like diabetes, alcohol abuse, smoking, or just relapse. And to exacerbate these issues, the amount of tissue specimen (quantity of Dupuytren’s tissue specimen is low, but quantity of NPF tissue specimen is even lower) is not sufficient for technical replicates. But interestingly, if NPF were pretreated with TGF-β1 and irradiated with blue light, α-SMA protein expression was significantly diminished after 72 h, and this effect could be further enhanced after 120 h (Fig 2B and 2D and Fig 3). And special attention should be paid to the observation, that in TGF-β1 pretreated Dupuytren’s fibroblasts, α-SMA protein expression is significantly decreased by the blue light irradiation (Fig 2B and 2D and Fig 3).

It was assumed that the effect of the blue light irradiation is mediated by the generation of reactive oxygen species (ROS), such as singlet oxygen [28] or hydrogen peroxide (H₂O₂) [23]. And it was presumed, that photons interact with endogenous photoreceptor molecules, such as lipofuscin [29], cytochrome c oxidase [30], or flavin-based photo sensors [31]. In fact, Taflinski et al. determined that an irradiation with blue light (420 nm) led to an increased amount of ROS as well as α-SMA reduction, and the induction of intracellular oxidative stress was thought to be the cause for that [23]. These findings intrigued the present work to examine ROS in Dupuytren’s fibroblasts after the exposure to blue light. It could be assured, that Dupuytren’s fibroblasts tended to produce more ROS, which was detected (after 24 h seeding the same cell number) with DHR123, an unspecific ROS indicator (Fig 4). As mentioned before, this might be due to a higher metabolic activity of Duypuytren’s fibroblasts. And the irradiation in general induced increased ROS formation in DD and NPF fibroblasts in tendency (Fig 4). As postulated by Taflinski et al., we assume that repeated smaller doses of blue light irradiation induce subtoxic levels of intracellular oxidative stress (as shown here in Fig 4), which may induce energy-consuming cellular responses against oxidative stress, which may in turn result in a proliferation stop and interfere with myofibroblasts differentiation [23].

The excessive proliferation of myofibroblasts [32], combined with the diminished capability to undergo apoptosis [3, 4] in the proliferative and involutional stages are further characteristics determining the phenotype of DD. In order to proof, if the beneficial effect of blue light is mediated via apoptosis, and to investigate, if photobiomodulation makes Dupuytren’s fibroblasts more sensitive to apoptotic signals, the apoptotic capacity of (Dupuytren’s) fibroblasts was analysed after five days of irradiation and TGF-β1 treatment. To induce cell death staurosporine was used. Interestingly, it was measured that in Dupuytren’s fibroblasts incubated for five days with TGF-β1, apoptosis was significantly higher compared to NPF (Fig 5). But even more important is, that blue light irradiation with a wavelength of λ = 453 nm, intensity of 38 mW/cm², and a radiant exposure of 40 Joule/cm², does not reinforce apoptosis in neither Dupuytren’s fibroblasts nor in NPF fibroblasts.

The present study highlights, that the application of blue light could inhibit the differentiation of Dupuytren's fibroblasts into myofibroblasts and the accompanied α-SMA expression. Our data suggests that, especially in the proliferative and involutional stages, or after surgery when TGF-β concentration is increased, an irradiation with blue light could be beneficial. Consequently, blue light therapy could be a promising therapy option for DD, and especially for relapse prevention. Our results suggest that the usage of blue light is a promising tool for therapeutic treatment for further fibrotic diseases such as keloids, hypertrophic scarring, and scleroderma.