Febe Bruwer, Clinical Nurse Specialist in Wound Care, Woundclinic, Nianwi Healthcare, Johannesburg, 
And Vice President of the Wound Healing Association of Southern Africa

Introduction

Wound healing is a dynamic and complex cascade of cellular, molecular, and biochemical events. Individual patient factors such as age, weight, nutrition and hydration status, co-morbidities and medication, together with factors that influence the microenvironment of the wound, such as inflammation, oxidative stress, and protease levels, determine healing outcomes (Diegelmann and Evans, 2004; Wild et al, 2010; McCarty and Percival, 2013).

Proteases, such as matrix metalloproteinases (MMPs) and elastases, are enzymes that break down proteins, and they have a role in all phases of normal wound healing. In the inflammatory phase, proteases aid in autolytic debridement and the breakdown of damaged extracellular matrix (ECM) and foreign material, to aid wound repair (McCarty and Percival, 2013). During the proliferative phase, proteases are responsible for the degradation of the capillary basement membrane around blood vessels, aiding detachment of pericytes and endothelial cells and the subsequent migration of endothelial cells to enable angiogenesis (Rundhaug, 2005; Eming et al, 2014). Finally, during the remodelling phase, proteases are responsible for contraction and remodeling of the ECM (Eming et al, 2014). Moreover, proteases detach the connection between intergrins and collagen and allow the keratinocytes at the wound edge and epidermis to advance over the wound substratum to close the deficit (Eming et al, 2014).

In acute wounds, the production and activation of proteases is tightly regulated by cellular and molecular mechanisms in the different phases of wound healing. In chronic wounds with unresolved inflammation and microbial contamination, proteases are constantly produced and activated, keeping the wounds in a chronic state (Eming et al, 2014). It is therefore expected that in wounds where there is a lot of debris, such as with sloughy tissue, there is more inflammation and thus a higher volume of proteases (Atkin 2014; Erfurt-Berge and Renner, 2014).
In chronic wounds, protease activity is increased by the presence of damaged tissue, foreign material, micro-organisms or biofilms, which subsequently prevents wound healing from progressing normally. When excessive levels of proteases are present, it can lead to the degradation of newly formed ECM and other proteins needed for healing, such as growth factors, making it impossible to form new granulation tissue (Eming et al, 2014; Westby et al, 2016). However, in chronic wounds, proteases may also be elevated as a consequence of chronic inflammation, even in the absence of tissue debris (International Consensus, 2011). An imbalance in the proteases present can subsequently keeps the wounds stagnated by constantly disintegrating newly formed ECM (Eming et al, 2014). Therefore, it is important to control protease activity present so that debris and inflammation are removed/resolved, without affecting the formation of new tissue. Interventions that reduce harmful proteases or modulate the wound environment may aid healing (Smeets et al, 2008; Erfurt-Berge and Renner, 2014). An expert consensus report (International Consensus, 2011) stated that an elevated protease level is associated with delayed healing especially in chronic wounds and monitoring protease activity could guide treatment choices.

Honey has been used for centuries as a wound treatment and a wealth of evidence exists to demonstrate its positive influence on the wound healing process, including in burns and chronic non-healing wounds (Gulati et al, 2014; Martinotti et al, 2018; Yilmaz and Aygin, 2020). Medical Grade Honey (MGH) follows strict guidelines to guarantee the safety and efficacy of honey for medical purposes (Hermanns et al, 2020).
MGH stimulates autolytic debridement via multiple mechanisms. The osmotic activity encourages lymphatic outflow as occurs with negative pressure therapy, creating moist conditions and cleansing of the wound bed. MGH increases plasmin activity (an enzyme that specifically digests fibrin that attaches slough to the wound surface), while the low pH of MGH (pH 3.2–4.5) makes the wound environment less favourable for destructive proteases by increasing the release of oxygen from haemoglobin (Molan and Rhodes, 2015). The antimicrobial activity of MGH is also orchestrated via a wide range of properties, including its hygroscopic activity that dehydrates microorganisms, the acidic pH that is inhospitable to pathogens, and the steady release of low amounts of hydrogen peroxide and molecules that have direct antimicrobial activity (Cremers et al, 2020; Smaropoulos et al, 2020a,b). Moreover, MGH possesses anti-inflammatory and anti-oxidative activity, creating a favourable wound environment (Yaghoobi et al, 2013).

Thus MGH has multiple mechanisms by which a beneficial switch in the microenvironment of the wound can be made, including the possible reduction of protease levels to promote healing in chronic wounds. This study therefore evaluates the effect of MGH on protease levels in chronic wounds during the early phase of therapy.

Methodology

Patient inclusion and wound care

Patients presenting with wounds of different aetiologies to the Nianwi Healthcare Wound Clinic were evaluated for inclusion in the evaluation. Seven patients were included based on the inclusion and exclusion criteria described in Table 1.

Convenience sampling was used to select patients as they presented to the clinic until inclusion of the study group was completed. Patient demographics are presented in Table 2.

Table 1: Inclusion and exclusion criteria for the study.

Inclusion criteria
Exclusion criteria
Chronic wound present for at least 4 weeks
Acute wound
Size not more than 10x10cm (dressing is 10cmx10cm)
Wound more than 10x10cm
Elevated protease activity as indicated by WOUNDCHEK™ Protease Status
Negative for raised protease level as indicated on WOUNDCHEK™ Protease Status

Table 2: Patient characteristics and previous treatments.

Case# Patient gender and (years of age) Wound type Wound present for Previous treatment Protease analysis (day of MGH treatment) Adjuviant treatment/Standard of care
1 Female (47) Trauma 6 weeks Parrafin Gauze and topical antibiotic 25 Compression
2 Female (79) Venous ulcer 6 weeks Non adherent dressing 2 Compression
3 Male (50) Venous ulcer 6 months Foam dressing 21 Modified compression
4 Male (96) Venous ulcer 6 months Parrafin Gauze 45 Compression
5 Male (70) Ulcer 4 months Toppical Antibiotic cream 6 Compression
6 Male (58) Ulcer 8 months Silver foam dressing 14 Compression
7 Male (66) Venous ulcer 2 years Foam dressing 28 Compression
The same clinician (the author) treated all patients. Before the application of MGH, wounds were cleaned using normal saline solution and protease activity was checked after cleansing.  Patients were treated with the MGH-based wound care product L-Mesitran® Hydro (Triticum Exploitatie BV, Maastricht, the Netherlands), a honey hydrogel dressing. In the case of deeper wounds, L-Mesitran® Soft (Triticum Exploitatie BV, Maastricht, the Netherlands), a MGH gel, was first applied to make sure the contours of the wound bed where in contact with MGH (cases 6 and 7), then L-Mesitran® Hydro applied. Dressings were changed every 2–5 days, depending on the stage and size of the wound and the amount of exudate produced. Compression therapy was also applied where indicated (Table 2).
 

Protease activity measurement

Protease levels were measured using the WOUNDCHEK™ Protease Status test (WOUNDCHEK ™ Laboratories, North Yorkshire, United Kingdom). WOUNDCHEK™ Protease Status is a point of care diagnostic test for the qualitative assessment of human neutrophil-derived inflammatory protease activity in chronic wounds, including MMPs and human neutrophil-derived elastase. 

Protease status was measured following manufacturer’s instructions, at the start of the treatment (Day 0), and subsequently during dressing change. The timing of the protease activity measurement was dependent on the wound type and determined ad hoc based on the size and visual progression of the wound. The wounds differed in size and origin, with the smaller wounds rapidly progressing and therefore requiring a relative early measurement (e.g. cases 2 and 6) when compared to the larger wounds.
 

Results

Protease activity

Following treatment with MGH, all wounds showed a reduction in protease levels as indicated by WOUNDCHEK™ Protease Status testing (Figure 1). Upon use, a control line (C) appeared to confirm the test was valid. High levels of protease activity were indicated when the colour of the test line (T) was less intense than the line on the reference strip (R), or the test line was not visible at all. Low levels of protease activity were indicated when the intensity of the test line was more than or equal to the reference strip line (Figure 1).
A).
B).
Figure 1. Example of readout of WOUNDCHEK™ Protease Status assay. A). Positive readout for elevated protease levels before L-Mesitran application. B). Reduced level of proteases as indicated by lighter test strip after application of 2–3 L-Mesitran dressings.
 
 

Individual case results will now be presented.

Case 1

A 47-year-old female presented with a traumatic injury to the shin that developed into a chronic ulcer (Figure 2a). The wound was present for about 6 weeks before starting treatment with L-Mesitran. Previous treatment included the topical application of antibiotics. After 25 days of MGH treatment (Figure 2b), protease activity was decreased.


Case 1

Figure 2. a. The wound on Day 0 of MGH treatment; b. Day 25 of MGH treatment.

Case 2

A 79-year-old female with venous disease presented with a recurrent ulcer (CEAP C6 classification) (Figure 3a). The wound was present for about 6 weeks before L-mesitran treatment, and had previously treated with a topical antibiotic. After one treatment with MGH, the inflammation subsided (Figure 3b) and wound protease levels decreased.

Treatment with topical antibiotics, although not advisable, is still a standard treatment in South Africa as the patient’s first contact after injury is the general practitioner or pharmacy. Within the wound care fraternity, topical antibiotic treatment is not the standard of care, but the patients are usually referred to the clinic only after seeing the general practitioner/pharmacy.


Case 2

Figure 3. a. The wound on Day 0 of MGH treatment; b. Day 2 of MGH treatment.



 

Case 3

A 50-year-old male presented with a recurrent venous lower leg ulcer (CEAP C6 classification) (Figure 4a). The wound was present for about 6 months prior to L-Mesitran treatment and was previously treated with paraffin gauze. After 3 weeks L-Mesitran treatment, the wound showed improvement (Figure 4b) and the protease levels were decreased. At our facility compression therapy for the treatment of venous lower leg ulcers is standard care.


Case 3

Figure 4. a. The wound on Day 0 of MGH treatment; b. Day 21 of MGH treatment.

Case 4

A 96-year-old male presented with a chronic venous ulcer to the lower leg (Figure 5a). The wound was present for about 6 months and was previously treated with parrafin gauze before starting L-Mesitran treatment. After 45 days, the wound tissue was debrided, more vital and became smaller by reepithelialisation (Figure 5b), and protease activity decreased.


Case 4

Figure 5. a. The wound on Day 0 of MGH treatment; b. Day 45 of MGH treatment.



 

Case 5

A 70-year-old male presented with a recurrent ulceration (CEAP C6 classification) (Figure 6a) which was present for about 4 months and previously treated with topical antibiotics. After 6 days, the inflammation subsided, the wound epithelialized and protease activity decreased (Figure 6b).


Case 5

Figure 6. a. The wound on Day 0 of MGH treatment; b. Day 6 of MGH treatment.

Case 6

A 58-year-old male with diabetes had a chronic ulcer on the achilles due to surgery (Figure 7a). The wound was present for 8 months and previously treated with polyurethane foam before starting L-Mesitran treatment. L-Mesitran was applied every second day, until the patient unfortunately discontinued care himself after 14 days. At this timepoint, reepithelialisation was ongoing (Figure 7b) and protease levels were decreased.


Case 6

Figure 7. a. The wound on Day 0 of MGH treatment; b. Day 14 of MGH treatment.



 

Case 7

A 66-year-old male with chronic venous insufficiency had a venous ulcer (CEAP C6 classification) on the lower leg present for more than 2 years (Figure 8a). At day 28, the wound was debrided, granulating, more vital and became smaller by reepithelialisation (Figure 8b), while protease activity was decreased.


Case 7

Figure 8. a. The wound on Day 0 of MGH treatment; b. Day 28 of MGH treatment.



 

Discussion

This case series investigated the effect of MGH on elevated protease levels in chronic wounds. In these wounds, excessive protease activity results in the continued breakdown of new ECM, preventing the generation of new granulation tissue and thus healing. 

MGH has proven antimicrobial and wound healing properties and this evaluation measured the effect of MGH on the activity of proteases in chronic wounds. The results demonstrated that in all seven chronic wound cases, the level of proteases decreased after treatment with the L-Mesitran MGH dressings. It has previously been suggested that honey can suppress protease activity in wounds because of its acidity which is not favourable to protease activity, however, clinical studies that substantiate this are lacking (Simon et al, 2009; Yaghoobi et al, 2013). 

In addition, all wounds showed signs of progressed healing. All wounds were chronic having been present for at least 4 weeks and a history in which other therapies such as topical antibiotics, paraffin gauze, and (silver) foam dressings, had proven ineffective (Table 2). Thus, therapy with MGH may have played a role in progressing the wounds towards healing. Compression therapy was applied in most cases, and the effect of this on wound progress must also be considered. However, while compression therapy may stimulate blood and lymph flow, the activity on autolyic debridement and wound healing is likely limited when compared to the well-described mechanisms of MGH. As demonstrated by the results, MGH stimulated autolytic debridement as shown by the resolution of nectrotic and sloughy tissue and new granulation tissue was clearly formed. The wounds became more vital, indicated by the presence of healthy red coloured tissue, also demonstrating that angiogenesis was enhanced. In addition, wound dimensions decreased with reepithelialisation clearly visible around the edges of the wounds. The anti-inflammatory and anti-oxidative activity of MGH together with the change in protease activity changed the microenvironment of the wound bed, creating a switch from stagnating towards healing. 

Further research is needed to  investigate the exact molecular mechanisms behind the change in the wound microenvironment created by MGH. It is unclear whether honey modulates protease activity directly, or indirectly, e.g. by its anti-inflamamtory and anti-oxidative effects. Larger randomized controlled trials are advised to further substantiate the findings and prove causality. 

No pain or negative side effects of MGH were experienced by any of the patients during the treatment of these wounds. Furthermore, previous publications confirmed MGH to be a safe and cost-effective therapy, even in severe wounds in paediatric and neonatal patients (Smaropoulos et al, 2020a; Smaropoulos and Cremers, 2021; Yilmaz et al, 2020).
 

Conclusion

This case series supports the hypothesis that the application of MGH decreases excessive protease levels that are often observed in chronic wounds. MGH is indicated in the treatment of multiple wound types, offering the opportunity to modulate protease activity in addition to its well documented effects on the wound microenvironment, in order to promote healing. 

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