Wound monitoring of pH and oxygen in patients after radiation therapy

Squamous cell carcinoma continues to be the most common of the head and neck malignant tumors. Depending on the progress of the disease, surgery as well as primary radiotherapy with or without concomitant chemotherapy have become established as treatment modalities. Salvage surgery is used for recurrences, but re-irradiation is also considered on a case-by-case basis [1, 2]. However, it is known that radiotherapy leads to short and long-term changes in the irradiated tissue and is therefore associated with an increased risk of wound healing disorders [3‐5]. Complications include infection [6], skin atrophy, tissue fibrosis [7], ulcerations, fistula formation [8] and in rare cases vascular rupture. The healing rate after flap transplantation also appears to be reduced [9]. A review by Lee, Chin et al. reports a rate of flap losses of 9.8% [10], while other studies report wound healing disorders after free flap reconstruction in irradiated areas in up to 40% of cases [11].

The management of these complications requires extensive efforts [12] and is even further complicated by a lack of assessment criteria to objectively measure treatment success. Thus, the current follow-up strategy for these complications is the independent observation and subjective assessment of the wounds by several physicians.

Previous studies on chronic wounds have shown that adequate tissue oxygenation and pH regulation are essential for cell proliferation and protein synthesis [13, 14]. Moreover, cell turnover, migration and enzymatic activity are affected by changes in pH und subsequent activation of various proton-sensing G protein-coupled receptors (GPCRs) [15].

The processes underlying wound healing are dormant in intact skin and are activated to restore the barrier integrity upon injury. This healing processes consist of three phases, inflammation, proliferation and remodeling [16, 17]. If the wound healing is functional, the pH of the wound decreases steadily, from pH values in the alkaline range (around pH 8–8.5) to the pH of intact skin upon completion of the healing process (5.5–6) [18]. However, if the pH remains at a high level, wound healing slows down or stops, partially by reduced cell proliferation and migration as seen in chronic wounds [13, 19]. In addition, chronic wounds exhibit a centripetal increase in pH, which is due to a centrifugally upregulated sodium / hydrogen exchanger isoform 1 (NHE1) expression [20]. This leads to an increased hydrogen-ion extrusion and thus acidification of the wound in the periphery [21, 22]. The low pH was shown to inhibit keratinocyte migration, proliferation and viability. Similarly, microvascular endothelial cells exhibit increased adhesion and lower migration rates in low pH conditions. In addition, there is a reduction in the release of mediators such as IL-6 and IL-8 [23, 24] and interferon-γ from keratinocytes in the acidic wound periphery compared to the wound center. The reduced IL-6 and IL-8 secretion potentially inhibits keratinocyte recruitment from the wound periphery to the center. Interferon-γ stimulates VEGF production, which is a key molecule for neoangiogenesis, implying that this process could be inhibited by the low pH in the wound periphery [25]. In conclusion, a low pH in the periphery of the wound seems to inhibit processes that are essential for functional wound healing. Similarly, the oxygen partial pressure (pO₂) differs in acute versus chronic wounds, with a pO₂ of about 60 mmHg in acute wounds without epithelialization and [26] an average of 35 mmHg in chronic wounds. Based on these findings, a monitoring of the pH and pO₂ suitable for everyday clinical practice could facilitate the objective assessment of wound healing disorders so that wound management could be adapted and improved. The aim of this study was to collect first in vivo data in irradiated tissues of patients with wound healing disorders after primary or adjuvant radiochemotherapy. To determine these parameters, a newly developed camera system was used allowing the direct measurement on a cell layer.

Wound healing complications can be side effects of radiation therapy [12].To address wound healing disorders in clinical practice, several approaches are pursued. An important progress was the intensity modulated radiotherapy (IMRT) by careful adaptation of the radiation field which should improve the therapeutic benefit and reduce side effects [30]. Computer-assisted inverse planning of IMRT and application with modern high-precision accelerators enables optimal sparing of surrounding normal tissues [31, 32]. As radiation techniques get better and treatment fields get smaller, re-irradation might occur more often resulting in a potential higher number of patients with wound healing disorders in previously irradiated areas. The resulting toxicity may lead to further wound healing disorders.

Various therapeutic approaches to manage wound healing disorders have already found their way into clinical routine and here complement the conventional wound management. Objective measurements analyzing the wound bed and the effects of intervention are still missing. A clinical review of our group summarizes the current treatment strategies in wound healing complications after radiotherapy [12], including hyperbaric oxygen, hydrogel membranes [33] and pluripotent stem cells [34].

Wound healing disorders in otorhinolaryngology are sometimes sustained not only by the tissue damage resulting from the previous irradiation. In the case of pharyngocutaneous fistula, the additional contamination by saliva and the associated bacterial colonization also has a negative effect on the treatment [6]. In recent years, negative-pressure wound therapy has established as an efficient treatment method [35‐37]. The wound is separated from the environment by means of a pressure-tight dressing. Using a portable pump negative pressure is built up to − 200 mmHg continuously or intermittently [38]. As a result, the wound base is cleared of secretions, granulation tissue is built up, thus promoting wound healing. Numerous clinical studies have now indicated that thus reducing the treatment time and a better outcome can be achieved.

In addition to the methods mentioned in the management of postradiogenic wound healing disorders, numerous others are discussed [12]. Common to all, however, is the problem of objectively assessing treatment success. Schreml et al. studied the pH [26] and oxygen [14] of intact skin as well as in acute and chronic wounds in detail. In addition, they established a method of 2D visualization of these parameters [13]. We adopted this method to develop a better understanding of the conditions in postradiogenic wound healing disorders.

While his group describe a pHe of about 6.5 in the periphery of the wound, we found values of 7.53 ± 0.26 (mean ± s.e.m.) in our experiments. Taking into account the molecular biological relationships described above, the conditions for wound closure in postradiogenic wounds appear to be more favorable with regard to the environment at the wound edge. However, pH-gradients play a crucial role in recruiting keratinocytes inwards from the wound edges.

Significantly less favorable are the conditions with regard to the oxygenation of the wound base in irradiated wounds. The oxygen partial pressure in acute wounds without epithelialization is about 60 mmHg [26] while chronic wounds have an average of 35 mmHg. A centripetal distribution pattern as in the case of the pH value is not found here [13]. While the oxygen barrier is regenerated with re-epithelialization, the measured values usually fall continuously [26]. Our measurements showed very pronounced hypoxia at 9.4 ± 2.2 (mean ± s.e.m.) mmHg and without any epidermal barrier. Since sufficient tissue oxygenation is crucial for keratinocyte proliferation and migration in healing [14, 39], extremely unfavorable conditions seem to exist for efficient wound healing. Should these results be confirmed in studies with larger numbers of cases, hyperbaric oxygen therapy as a therapeutic approach could address this problem. As described above, it is increasingly being used in the treatment of osteoradionecrosis. Thus, by improving the oxygen partial pressure, it could prove to be an effective therapy for wound healing disorders after radiotherapy in HNSCC patients. A Cochrane analysis by Bennett et al. in 2016 [40] found only two studies from 1999, in which hyperbaric oxygen therapy, applied pre- and postoperatively, described a significant advantage in the case of head-neck salvage surgery. However, these studies are only of limited significance due to methodological deficits.

The cause of the extremely hypoxic conditions in irradiated non-healing wounds could be damage to small vessels and fibrosis due to the radiation [41]. However, this will have to be investigated in future works.

On unirradiated skin we found a pH of 6.46 ± 0.18 (mean ± s.e.m.) and an oxygen saturation of 6.19 ± 0.83 (mean ± s.e.m.) mmHg. Low oxygen saturation on intact skin has already been described with this measurement method [26]. Due to the intact stratum corneum, the sensor foil is continuously deprived of oxygen without being refilled, which results in a reading of approximately 0 mmHg. Striking, however, is the high pH compared to previous work [42]. Using a similar measurement method, the authors described pH levels of about 5.1 in a patient population of a similar age. However, in contrast to our study, the skin areas on which the measurements were performed were standardized in that no cleaning or local application of care products was carried out 24 h before the pH was measured. Furtjes et al. describe an increase in pH after soap and water cleaning of the skin to up to 6.47 [43]. Our pH measurements were made in everyday clinical practice, so standardization in unirradiated skin was not part of our protocol. Thus, the difference in pH measured in this study compared to the aforementioned study could be due to personal hygiene or disinfecting measures.

In this study, we have for the first time measured the pH and pO₂ on irradiated skin. Here we found an average pH of 6.96 ± 0.26 (mean ± s.e.m.) and an oxygen saturation of 28.4 ± 2.4 (mean ± s.e.m.) mmHg. Within the stratum corneum, the formation of free fatty acids [44], the production of urocanic acid [45] as well as the expression of NHE1 [20] among others are responsible for the acidic pH of human skin. Deeper layers of the stratum corneum have a more alkaline pH, reaching pH neutral levels [18, 46, 47]. Together with the observation that measured oxygen levels on intact skin are close to 0 mmHg [26], the results indicate that the skin surface was affected due to irradiation and the values measured are a reflection of this side effect. The question arises whether the extent of damage to the skin could be deduced from the determination of these values in individual cases. Thus, before a salvage operation, the risk of a wound healing disorder could be estimated and discussed with the patient. Also, prophylactic measures could be taken.

In summary, there are unfavorable conditions in terms of pH and oxygen saturation in postradiogenic wound healing disorders. However, hypoxia appears to be more pronounced and thus potentially more influential in the course of healing. In addition, we were able to objectively measure the existing damage to irradiated skin by means of 2D luminescense imaging. Whether a prognosis of wound healing can be derived from this or whether prophylactic measures can reduce the development of postradiogenic wound healing disorders should be shown in further work.