1.Introduction

     Wound healing is a complex biological repair procedure commonly developing in four concurring stages, including hemostasis, inflflammation, proliferation and remodeling [1]. This elaborate and precarious procedure is moderately effective but may degenerate, causing chronic wound (CW) conditions, which are unresponsive to a natural biological healing mechanism. Such CW may remain at a proliferative or inflflammatory state of wound healing [2] and typically revert to an inflflammatory state [3] due to specifific biochemical, microbial, and cellular abnormalities that interfere with healing progression (Figure 1).

         The mechanism concurring to CW varies considerably but involves metabolic diseases(such as diabetes) [4], factors inflfluencing blood supply (peripheral vascular disease) [5],elderly age [6], peripheral neuropathy [7], infection [8], malnutrition [9], altering of immune function, medications, or previous local tissue injury (trauma or radiation therapy). Moreover, external factors such as sustained pressure, temperature, and moisture also play an important role in enabling a wound to heal [10]. Although the etiology differs, CW are characterized by excessive levels of proinflflammatory cytokines, proteases, reactive oxygen species, senescent cells, and a defificiency of functional stem cells [11,12].

        A  retrospective study conducted in 2018 reported that approximately 8.2 million people  worldwide were affected by infected or non-infected wounds,which lead to hard-to-heal ulcers.Furthermore,significant  interest and demand has emerged regaiding wound care dressings,above  all in the USA and Europe,currently  representing the two largest markets in the world.Indeed,in 2014,the annual cost of wound care was approximately  USD 2.8 billion;in 2021,spending increased to USD 3.5 billion[13].The spending forecast is estimated at around USD 15 billion for 2022 and USD 22 billion for 2024[14].These costs highlight the importance of designing appropriate outpatient instruments to enhance healing.For this reason,Falanga and colleagues developed the concept of wound bed preparation[15,16].Wound bed preparation is crucial foe CW  due to the difficuit management of such atypical wounds[17].This preparation aims to  remove healing resistance as  well as enhancing healing  mechanisms with the formation of a stable wound,containing appropriate granulation tissue and a well-vascularized wound bed,providing the oxygen required  to improve the ulcer [18].The fifirst and most important intervention is the debridement process, which is normally considered a simple removal of necrotic and infected tissue [19]. Additionally, debridement is applied to maintain an optimal uninfected wound bed, essential for the cutaneous regeneration process [20]. Two methods of debridement are herein described: surgical and enzymatic. Surgical debridement is commonly used for large surface wounds that are severely infected and often require the removal of exposed infected noble structures (bones, cartilage, and tendons). This debridement is more rapid and effective compared to other complementary methods, such as VAC and OTI therapy [21,22],even though it is more invasive.

         Enzymatic debridement is often conducted in smaller wounds not requiring surgical intervention or wounds that necessitate such a selective microenvironment [23], able to identify devitalized tissues only. It consists of the topical use of natural products, such as proteolytic enzymes or proteinases, whose activity is useful to eliminate necrotic tissue through a digestive mechanism of the not-vital tissue, but also to promote cell migration responsible for skin regeneration at wound bed level [24]. Several enzymatic debridement agents have been proposed such as trypsin [25], streptokinase–streptodornase combination [26], and subtilisin [27]. Bromelain is under investigation as an enzymatic debriding agent consisting of endopeptidases and other enzymes [28] that derive from the pineapple. However, the two most used proteolytic agents are papain and collagenase [29]. The application of both ointments (Accuzyme®(Healthpoint, Fort Worth, TX, USA) and Noruxol® (Smith&Nephew, Agrate Brianza (MB), Italy), respectively) has been incorporated into the treatment regimen of burn injuries and CW with benefificial effect. Papain, derived from Carica papaya, is part of nonselective preparations [30], while collagenase, derived from Clostridium histolyticum, is a selective preparation [31]. Collagenase has been successfully adopted for the degradation of collagen and elastin but was reported irrelevant in fifibrin. Papain–urea is mainly used to solubilize fifibrin. Investigations have revealed its safety and effectiveness in the debridement of cutaneous ulcers, and it is favorably applicable in long-term care and home care contexts.

        Although the most widely used collagenase preparation is derived from Clostridium histolyticum [31], in 2011, researchers characterized a new collagenase, derived from highpurity Vibrio alginolitycus [32], with ability to hydrolyze collagen molecules and to preserve periwound skin. It seems to be more selective and less aggressive compared to the other one [33]. Moreover, its association with hyaluronic acid (HA) is apparently able to enhance the proliferative effects of the collagen peptides [34,35].

       The purpose of our retrospective study is to assess the clinical effects concerning Bionect Start® (Fidia Farmaceutici, Abano Terme (PD), Italy) ointment when treating chronic hard-to-heal wounds.

2、Materials and Methods

       A single-center observational and retrospective study was conducted to assess the effificacy and safety of HA/collagenase (Bionect Start®, Hyalo4 Start®, Fidia Farmaceutici, Abano Terme (PD), Italy) ointment in relation to the recovery rate of chronic hard-toheal wounds in patients treated between January 2017 and December 2020. The study was performed in compliance with the Declaration of Helsinki and the Guidelines for Good Clinical Practice. The study protocol was approved by the local Ethics Committee (396/2021). Demographic data, medical history and wound measurements were collected (Table 1).

2.1 Patient Selection

       This study included 70 patients (45 male and 25 female) with an average age of 59.2 years (32–85 years) affected by multifactorial hard-to-heal ulcers, i.e., diabetes mellitus (n = 20), post-traumatic ulcers (n = 35), chronic burns degrees I and II (not healed in 12 weeks) (n = 10), and pressure ulcers (n = 5). Patients with hard-to-heal wounds and presence of necrotic tissue were included. All patients underwent the same therapeutic protocol, including wound disinfection with Amukine Med, saline solution cleansing, and application of HA/collagenase (hyaluronic acid sodium salt 0.2% + collagenase) ointment to the wounds in a 2 mm thick layer. A sterile tongue blade was employed to spread the ointment preparation over the whole wound area. A barrier ointment was applied to the surrounding periwound area. The wound was covered with a non-adherent gauze and then with a secondary dressing. We applied dressings to the ulcers twice a week at our clinical center, and performed follow-up visits (photographs, wound characteristics, exudates, edges, size and periwound skin evaluation, and infections by bacterial swab) at V0 (pre-treatment), V1 (two weeks), V2 (four weeks), V3 (two months), and V4 (one year). Treatment was continued until the wound was completely clean and covered by granulating tissue. No advanced wound dressing was applied so as to avoid interference with healing time. Wounds were diagnosed as fully healed when the normal reepithelization process was complete.

2.2 Assessment Methods

     During each visit, wound size, wound bed, and wound quality were evaluated.Changes in wound size are diffificult to measure by length and width due to the irregular shape of the wounds; hence, we measured the surface area in cm2 using squared and transparent paper on which we traced the wound area with indelible marker pens (Oxford Health NHS Foundation Trust guidelines). On wound closure, a second assessment was performed after 2 weeks to confifirm complete healing of the wound, according to AHRQ recommendations. the wound bed score [36] was adopted to evaluate black eschar, eczema/dermatitis, depth, scarring (fifibrosis), colour of wound bed, oedema, resurfacing epithelium, exudate amount (Table 2).

       Each parameter was assigned a score of 0, 1, or 2. The scores were determined considering 0 as a poor healing outcome and 14 as the best healing outcome. The scores were divided into 4 quartiles [16]. The percentage of necrotic area was calculated by dividing the necrotic area, expressed in cm2 , by the total ulcer area, expressed in cm2 , and the result was reported as a percentage [16]. The scores were computed, considering 76–100% as poor and 0–25% as the best healing score. Patients with successful debridement were considered among those with a percentage of 0–25% (necrotic area out of the total area). A global assessment of each patient (PTGA) was conducted with the participant required to express aesthetic satisfaction using a fifive-point Likert scale: “much worse than expected”, “worse than expected”, “as expected”, “better than expected”, or “much better than expected”. A clinical observer global assessment (COGA) was performed, and the investigator was required to evaluate wound status using a fifive-scale set: 1 = deterioration, 2 = no change, 3 = slight improvement, 4 = moderate improvement, or 5 = signifificant improvement. Furthermore, pain during dressing change and any undesired systemic or local effects were also evaluated and recorded. The pain was scored using the Visual Analogic Scale (VAS 0-10). Side effects were documented from study initiation to study termination. All adverse events were reported as separate events, including signs, symptoms, or morbidities, and were monitored until complete recovery or conclusive physical examination and assessment.

2.3 Statistical Analysis

      Measurements were conducted in relation to the groups. Statistically signifificant differences were measured by the Student t-test. The threshold for statistical difference was based on the p-value, signifificant at <0.05. The statistical analysis software (SPSS) was used to conduct the two-tailed Student t-test along with the Kolmogorov–Smirnov test.

3、Results

      Between January 2017 and December 2020, 70 patients were enrolled in the study. Of the total, 45 (64.3%) were male and 25 (35.7%) were female, with a mean age of 59.2 (range 32–85 years). Demographic data, medical history, and wound measurements were collected (Table 3). Wound healing was completed at V3 (over a two-month period) in 62 patients (87%) out of 70 subjects, and 100% complete wound healing was observed, with 0% re currence after one year. A signifificantly higher wound recovery rate was observed at each evaluation compared to baseline: at V1 (100% vs. 74% (26% reduction); 95% confifidence interval (CI) = 20.7–31.1%; p = 0.000), at V2 (100% vs. 6%; (94% reduction) 95% CI = 90–99.8; p = 0.000), at V3 (100% vs. 5.4%; (95% reduction) 95% CI = 91–98%; p = 0.000), and at V4 (100% vs. 0%; (100% reduction) 95% CI = 100–100%; p = 0.000).

      The rate of debridement effificacy considering cleansing of the wound bed signifificantly increased during the treatment (p < 0.001). The total ulcer area signifificantly decreased at V2 (after week 4 of application) compared to V0 (baseline pre-treatment) (p = 0.000), while the necrotic area signifificantly decreased at V1 (after week 2)（p<0.000). The necrotic component of the ulcer decreased at a higher rate compared to the total area (Figure 2).

Figure 2. The rate of debridement effificacy. Trends of the percentage of necrotic area (orange line) and total ulcer area (blue line) during the treatment. Boxes represent the 95% confifidence intervals.

        A signifificant improvement was observed regarding qualitative parameters of the ulcer, such as black eschar, eczema/dermatitis, depth of scarring (fifibrosis), color of wound bed, oedema, resurfacing epithelium, and exudate amount (Table 4).

       A signifificant shift in wound healing progression was observed during treatment compared to baseline (Figure 3). Pain reduction after two months was remarkable in all patients, with a statistically signifificant reduction (p < 0.001) in pain experience. Moreover, a bacterial wound culture was performed with resulting positive V0 swabs in 60% of patients. Exudate was observed in 70% of patients. During the treatment, a progressive reduction in contaminated swabs and exudate was found compared to baseline (p < 0.001). At V0,analysis of wound exudate gave a positive result for Staphylococcus aureus (6 patients),Pseudomonas aeruginosa (10 patients), Enterococcus faecalis (4 patients), Bacillus cereus (2 patients), and for Klebsiella pneumoniae (1 patient). Infected wounds were treated after antibiogram results with specifific oral antibiotics administration. At the end of the treatment, the swab culture analysis revealed the absence of bacterial pathogens. However, two lesions were positive for Enterococcus faecalis, and three lesions were positive for Pseudomonas aeruginosa.

       Further investigations were conducted to evaluate secondary objectives, specifically patient satisfaction regarding wound outcomes (PTGA) and clinician’s global assessment of the wound (COGA) over time (Figure 4).

       No signifificant differences were observed in patient satisfaction and investigator evaluation. No side effects were reported in relation to the procedures or to the experimental product. Figures 5–7 show examples of clinical cases.

4、Discussion

      CW are indeed an arduous clinical and social issue, with 6.5 million people affected in the United States and 2 million people in Europe [37]. In the world’s largest wound-dressing regions, the United States and Europe, a significant demand for wound care products is evident. Globally, the annual cost for wound care was an average of USD 3.5 billion in 2021, which is expected to rise to USD 22 billion in 2024. Wound bed preparation plays a fundamental role in CW management with numerous debridement procedures being applied in clinical contexts [38]. Mechanical debridement is often a restrictive procedure due to the patient experiencing significant pain in order to achieve adequate wound cleansing, which must be performed in an operating room under anesthesia. For this reason, proteolytic enzymes have been successfully used in difficult ulcers[39], and enzymatic debridement is one current, non-invasive, painless method to remove necrotic tissue in long-term care. The most widely used enzyme in Europe is collagenase [40]. Collagenase is an ideal enzymatic debriding agent. Collagenase ointment has been favorably proposed as a selective enzymatic debriding agent, with current commercial use derived from the bacterial strain Clostridium histolyticum and from the bacterial strain Vibrio alginolyticus [33,41]. Reports from in vitro investigations have demonstrated that the Vibrio alginolyticus enzyme is as effective as Clostridium histolyticum digestion of collagen type I. Conversely, Vibrio alginolyticus collagenase degrades fifibronectin and decorin to a lesser extent than Clostridium histolyticum collagenase. This is an important insight, as decorin and fifibronectin possess structurally important components of dermal extracellular matrix (ECM), and fifibronectin is essential to dermal fifibroblast migration within the wound-healing process [42]. The difference may be attributable to a higher selectivity of Vibrio alginolyticus collagenase, in which a completely active enzyme is observed on the collagen fifilaments binding the necrotic tissue to the wound bed. Conversely, the same collagenase remains inactive against crucial elements of the dermal ECM [33]. This response may explain the moderate effect of collagenase ointment from Vibrio alginolyticus on perilesional, healthy skin [16,41,43,44]. Several in vivo studies have demonstrated the effectiveness of Vibrio alginolyticus collagenase with a lower rate of perilesional skin complications compared with collagenase-based products derived from Clostridum histolyticum.Moreover, collagenase from Vibrio alginolyticus achieved an average inferior healing time compared to Clostridium histolyticum. Many authors have speculated that the combination of the fifibrinolytic enzyme with a product able to stimulate tissue regeneration, such as HA, is a decisive factor in promoting rapid and effective healing of CW. Accordingly, in association with the specifific action of the Vibrio alginolyticus enzyme, HA facilitates the recall of cells in the injured area and guides the deposition of ECM fifibrous components, protecting the newly formed granulation tissue from oxygen-free radicals that impair wound healing [45].

       This retrospective observational study has highlighted the importance and effectiveness of advanced bioactive dressings, such as Bionect Start® ointment, in CW treatment. The application of Bionect Start® promotes vascularized tissue and reduces formation of fifibrin and exudate. This reduction is most likely due to a more specifific presence of collagenase, accountable for fifibrin and necrotic tissue lysis, and for macrophage engagement at the wound site and the consequent digestion of eschar and devitalized material at the wound bed.

       We observed complete wound bed healing in 87% of patients in 2 months. On the other hand, some authors reported complete healing at the same endpoint in 27% of patients [33].

      Total ulcer area signifificantly decreased after 4 weeks compared to baseline. This fifinding is in agreement with those found in the literature, compared to placebo [37].

      Moreover, necrotic area signifificantly decreased after 2 weeks, as reported in the literature [45,46] and compared to mechanical debridement and Clostridium hystoliticum collagenase [33], with a statistically signifificant wound reduction over time.

      The improved healing conditions may be in relation to hyaluronan in the ointment composition. This molecule maintains an optimal moist environment and promotes the healing process, as can be seen from our results, which show a clear reduction in discomfort, erythema, and swelling; the presence of hyaluronan might inhibit scar formation and yield more satisfactory outcomes [47]. Hyaluronan acid also exerts a protective action on wound edges through ECM matrix formation, modulation of the inflflammatory reaction, and collagen deposition.

      Moreover, pain reduction was signifificantly higher after 2 months compared to baseline, and no differences were found between patient satisfaction and clinics’ evaluation. These fifindings confifirm the safety and effificacy of this ointment.

      Despite the great results obtained, this retrospective, observational, and longitudinal study had some limitations. Firstly is the lack of a comparison arm, as previously performed [41,48]. A further limitation was the lack of in vitro analysis, such as histological and immune-histochemical evaluation, which enables the analysis of an authentic regenerative potential regarding Bionect Start®. In this regard, the authors are currently performing studies to actualize these points. Nevertheless, this study had a very long-term follow-up period, with a large sample of participants, allowing the observation of the absence of long-term recurrences.

5、Conclusions

       In conclusion, based on this retrospective observational study, Bionect Start® ointment shows a higher wound recovery rate over time and a signifificative reduction in total ulcer area and necrotic area compared to baseline. It displayed chemical, physical, and clinical potential to confifirm easy operability in CW treatment and its role in protecting periwound skin. This is probably mainly due to collagenase association with HA, which allows better control of inflflammation and modulates collagen deposition.

       At the end of the study, all patients reached complete wound debridement. Furthermore, all other parameters associated with the ulcer, such as pain and patient satisfaction, showed signifificant improvement over time. No adverse events or hypersensitivity reactions were reported.

      Further studies are necessary to prove the regenerative effect of Bionect Start® ointment through an in vitro specifific analysis and compared to in vivo outcomes. Moreover, perspective and randomized in vivo clinical trials are preferred.

Author Contributions: Conceptualization, F.D.F.; methodology, F.D.F. and M.D.F.; formal analysis, F.D.F. and M.D.F.; investigation, F.D.F.; resources, M.R.; data curation, M.D.F.; writing—original draft preparation, F.D.F.; writing—review and editing, F.D.F. and M.R.; visualization, M.R.; supervision, M.R.; project administration, M.R. All authors have read and agreed to the published version of the manuscript.

Funding: This research received no external funding.

Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of AOU Ospedali Riuniti di Ancona (protocol code 396/2021 in date 23 September 2021).

Informed Consent Statement: Written informed consent has been obtained from the patients to publish this paper.

Data Availability Statement: The clinical data used to support the fifindings of this study are included within the article.

Acknowledgments: The authors are grateful to Giuseppina Caraglia, mother tongue expert, University of Naples “Vanvitelli” for performing English revision.

Conflflicts of Interest: The authors declare no conflflict of interest.

Abbreviations

COGA clinician’s global assessment of the wound

CW chronic wound

ECM extracellular matrix

HA hyaluronic acid

PTGA patient satisfaction regarding wound outcomes

VAS visual analogic scale

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