Local release of metalloproteinases and their inhibitors after a successful revascularisation procedure

Abstract

An altered balance between metalloproteinases (MMPs) and their inhibitor tissue inhibitor of metalloproteinases (TIMPs) may influence the healing process of a minor amputation following a successful vein graft. To speed up this process, negative pressure wound therapy (NPWT) and advanced moist wound dressing have been proposed. We determined the systemic and local release of MMP‐1, ‐2, ‐3, ‐9, TIMP‐1, and TIMP‐2 by enzyme linked immunosorbent assay (ELISA) technique and their influences in the healing process in 26 patients who underwent minor amputation after a successful revascularisation procedure. Twelve patients (group 1) were medicated with NPWT and 14 (group 2) with advanced moist wound dressing. Plasma samples were collected on the morning of surgery and thereafter at 1, 3, and 5 months; exudates were collected 3 days after surgery when amputation was performed and thereafter at 1, 3, and 5 months. Fifteen age‐matched healthy male volunteers served as controls. All wounds healed in 5 ± 0.5 months. Follow‐up plasma and local release of MMP‐1, ‐2, ‐3, and ‐9 were overall significantly lower when compared with the preoperative levels, while those of TIMP‐1 and ‐2 were significantly higher with no differences among the groups. Despite no differences in the healing process being observed among the two types of medications, at 1 month the local release of MMP‐2 and ‐9 was significantly lower (P = .013 and .047, respectively) and that of TIMP‐1 was significantly higher (P = .042) in group 1 as compared to group 2. A correct and aggressive local approach to the wound is able to promote the healing of the lesion stimulating the extracellular matrix turnover with local MMP/TIMP adequate balance and favouring the creation of granulation tissue. However, a successful restoration of an adequate blood flow remains the key point of a durable and rapid wound healing.

1. INTRODUCTION

Endovascular or open revascularisation procedures often in association with minor amputations are the current therapeutic option to treat critical limb ischaemia (CLI). Because of the disease severity at presentation, the failure rate of minor amputations, even after a successful revascularisation procedure, is approximately 30% to 45%,1, 2 and a major amputation may be required in 25% to 30% of these cases.3 In non‐healing wounds, the antibiotic therapy for infected lesions,4 repeated local debridement, bioengineered tissue or skin substitutes,5 growth factors,6 and spinal cord stimulation7, 8 can accelerate the healing process. Conversely, advanced moist wound dressing associated with different drugs and appropriated surgical treatment can guarantee excellent results in the management of chronic vascular ulcers.9, 10, 11 Negative pressure wound therapy (NPWT) represents a new emerging non‐invasive system based on localised delivery of continuous negative sub‐atmospheric pressure through a pump, which is connected to the resilient, foam‐surface dressing that collects wound exudates.12 Evidence in patients with CLI showed that it could be helpful to promote and accelerate wound healing of foot lesions after restoration of an adequate distal blood flow through surgical revascularisation.13

The pivotal role of the inflammatory process in the natural history of CLI and wound healing is well known14, 15; elevated circulating levels of matrix metalloproteinases (MMPs) and cytokines are shown to be associated with the worst outcomes after revascularisation in terms of wound healing and limb salvage.16, 17 Moreover, a number of research studies revealed that tissue MMPs may follow all stages of the wound healing process in different vascular settings, and the variation in their concentration seems to be directly proportional to the extent of healing.18, 19 The activity of MMPs is also controlled by tissue inhibitors tissue inhibitor of metalloproteinases (TIMPs) and an imbalance between MMPs and TIMPs may contribute to the development of vascular diseases such as advanced atherosclerosis, aneurysms, and varicose veins.20, 21, 22

The purpose of this study was to evaluate the systemic and local exudative levels of different MMPs and their natural inhibitors in patients treated with a successful revascularisation procedure followed by minor amputation with the application of either NPWT or advanced moist wound dressing to accelerate wound healing.

2. METHODS

2.1. Study design

Between January 2015 and December 2018, a total of 26 patients of both sexes affected with Rutherford Grade III Category 5‐6 of CLI23 and a moderate‐to‐high risk of limb amputation (clinical stage 3‐4 according to Wound Ischemia Foot Infection—WIfI classification),24 with occlusion of the superficial femoral and popliteal arteries with one or two tibial vessels runoff who underwent successful infrapopliteal reversed vein bypass graft, and minor amputations left intentionally open, were enrolled, inconsecutively, in the present retrospective study. The study was approved by the Ethical Committee of “Sapienza” University of Rome in accordance with the Declaration of Helsinki and the Guidelines for Good Clinical Practice. Before the beginning of the study, all participants provided written informed consent for intervention. This report is in accordance with the STROBE statement checklist of reporting on cohort studies.25

The patients were divided into two groups. Group 1 consisted of 12 patients affected with peripheral arterial disease (PAD) who underwent infrapopliteal reversed vein bypass graft, minor amputations, and NPWT; and group 2 consisted of 14 patients affected with PAD who underwent infrapopliteal reversed vein bypass graft, minor amputations, and advanced moist wound dressing.15, 26 Group 3 consisted of 15 patients who are male age‐matched healthy volunteers (mean age, 69 ± 10 years; median age, 65 years; age range, 52‐84 years), non‐smokers, without atherosclerotic lesions (excluded by carotid and aortic ultrasonography, and ankle brachial index (ABI) measurements) and with normal glycaemic profile, low density lipoprotein‐cholesterol values, served as a reference for biological parameters.

All wounds had the application of either NPWT or advanced moist wound dressing to promote healing. No attempt to endorse an advance medication over the other was the purpose of the study, and the choice was done according to the surgeons' preference (Paolo Sapienza and Elvira Tartaglia).

Exclusion criteria were the presence of chronic venous insufficiency; arterial aneurysms; insulin dependent or independent diabetes; acute ischaemia; connective tissue disorders including rheumatoid arthritis; and the use of medications impairing wound healing (ie, cytotoxic antineoplastic, immunosuppressive agents, and corticosteroids), blood disorders (clotting factor deficiencies and prothrombotic state due to cancer), alcohol use abuse, non‐healing wounds related to vein graft failure, and revision of the graft or patients' death occurred before wound healing.

2.2. Collection of baseline data

Demographic data including age, gender, risk factors, and the associated diseases were retrospectively collected. The risk factors included active tobacco use, cardiac disease (prior myocardial infarction, stable or unstable angina, or ST segment alteration on electrocardiogram), hypertension (diastolic blood pressure, ≥85 mmHg), renal disease (blood urea nitrogen, >7.1 mmol/L; creatinine level, >266 μmol/L; creatinine clearance, <50 mL/min), pulmonary disease (PO₂, <60 mmHg; PCO₂, >50 mmHg; pulmonary function tests, <80% of predicted), total cholesterol level ≥5 mmol/L, low‐density lipoprotein level ≥4 mmol/L, high‐density lipoprotein level ≤1 mmol/L, and non‐fasting triglyceride level ≥1.7 mmol/L. Associated disease was represented by obesity (body mass index—kg/m² >20% of ideal). Diagnostic studies consisting of B‐mode ultrasonography and colour imaging and computed tomography angiography (CTA), inflow and run‐off status, surgical details, minor and major complications, length of stay, and early‐ and mid‐term outcomes were recorded.

2.3. Operative procedures and clinical follow‐up

The surgical risk was determined according to the standard (ASA Physical Status Classification System) proposed by the American Society of Anesthesiologists,27 and all patients belonged from class II to class IV. CTA images were used to plan the surgical treatment. Intraoperative angiography was performed in all patients via the ipsilateral or contralateral femoral artery to confirm preoperative exams and to assess the outcome of the revascularisation.

A reversed autologous vein graft, femorotibial bypass was then performed using standard techniques. At the end of the procedure, the vein graft was studied with angiography to identify flow disturbances or intrinsic vein disease. Preferentially, all bypass grafts were performed to revascularise the ischaemic angiosomes.28 Three days after the revascularisation, the midfoot (transmetatarsal, Lisfranc, or Chopart amputations) was removed. All wounds were left open and no attempts to direct closure were made. A broad‐spectrum short course (2‐4 days) iv antibiotic therapy was started. Vacuum Assisted Closure (VAC Therapy System) (KCI Medical S.r.l. via Meucci, 1 Assago, Italy) or advanced dressing using collagen alginate, hydrogel and antimicrobial/cytolytic dressing, and gauze packaged according to the characteristics of the wound, were used to accelerate wound healing.

Postoperative medical therapies consisted of aspirin 100 mg/d and atorvastatin 10 or 20 mg/d. All patients were followed up with physical examination, ABI measurement, and B‐mode ultrasonography and colour imaging every 4 months. Suspected graft occlusions were confirmed by means of B‐mode ultrasonography and colour imaging and CTA.

2.4. Wound healing

Wound healing was assessed through direct ulcer tracing into clear plastic sheet and subsequent computerised planimetry. Healing was calculated by subtracting the final ulcer area expressed in centimetres from the initial ulcer area expressed in centimetres and dividing by the number of follow‐up months to obtain the total area healed per month (cm²/mo).

2.5. Sample collection

Blood samples were collected at the morning of surgery and thereafter at 1, 3 and 5 months by a direct venipuncture on an antecubital vein of the arm. The samples were placed into tubes containing K2‐EDTA (Terumo Europe NV, Leuven, Belgium) or sodium citrate (BD, Plymouth, UK) and immediately transported to our laboratory under controlled conditions of temperature and humidity.29

Three days after vein graft at the end of the amputation procedures and, thereafter at 1, 3, and 5 months, the wounds were covered with a transparent polyurethane occlusive film dressing (Tegaderm 3 M, Milano, Italy) under which exudates accumulated for 2‐3 hours.19 The exudates were then aspirated through the film dressing using a 30‐gauge hypodermic needle mounted on a 1‐cc tuberculin syringe.

2.6. Enzyme linked immunosorbent assay

Plasma and exudates were centrifuged at 3000 rpm for 10 minutes or 14 000 rpm for 15 minutes, respectively and, then, stored at 70°C for further analysis. Plasma and exudate levels of 72‐kDa MMP‐2 and 92‐kDa MMP‐9 were determined using the enzyme linked immunosorbent assay (ELISA) technique (Quantikine human MMP‐2 and ‐9, R&D Systems Europe Ltd, Abingdon, UK). Plasma and exudate levels of 52‐kDa MMP‐1, 57‐kDa MMP‐3, 28.5‐kDa TIMP‐1, and 21‐kDa TIMP‐2 were determined by the ELISA technique with Biotrak assay systems (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK).

2.6.1. Statistical analysis

Data were analysed with a computer software program (SPSS Ver. 25.0.0.1; SPSS, Chicago, Illinois for macOS High Sierra version 10.13.6, Apple Inc., Cupertino, California). All results are expressed as the mean ± SD. Continuous variables were analysed with either the Mann‐Whitney U test or Kruskal‐Wallis H test (one‐way analysis of variance), followed by the Bonferroni post‐hoc test calculated dividing the P value (.05) by the number of paired comparisons made. Categorical variables were analysed using the chi‐square test or Fisher's exact test. Survival, primary patency, and limb salvage rates were assessed by the Kaplan‐Meier method. SE values of survival, primary patency, and limb salvage rates were estimated at each censored case. Patency and limb salvage rates were obtained by analysing the number of limbs at risk and survival rates of patients were included. Differences with an α level of <.05 were considered statistically significant.

3. RESULTS

3.1. Clinical features

All patients had similar risk factors, associated diseases, and clinical presentation and are depicted in Table 1.

Table 1.

Patients' demographics and preoperative characteristics of patients affected with peripheral arterial disease

	Group 1 (n = 12)	Group 2 (n = 14)	Significance
Sex (male/female)	9/3	12/2	.635
Age (year ± SD)	71 ± 8	74 ± 6	.657
Active tobacco use	7 (58)	10 (71)	.683
Cardiac disease	5 (42)	3 (21)	.401
Hypertension	7 (58)	6 (43)	.695
Renal disease	2 (17)	—	.203
Pulmonary disease	1 (8)	2 (14)	1.000
Total cholesterol (mmol/L ± SD)	5.4 ± 0.6	5.7 ± 0.5	.916
Low‐density lipoprotein (mmol/L ± SD)	3.3 ± 0.7	3 ± 0.6	.545
High‐density lipoprotein (mmol/L ± SD)	0.6 ± 0.4	0.8 ± 0.3	.977
Non‐fasting triglyceride (mmol/L ± SD)	1.9 ± .3	1.9 ± 0.3	.977
Obesity	1 (8)	2 (14)	1.000
Preoperative ABI (mmHg ± SD)	0.36 ± 0.7	0.35 ± 0.8	.590
ASA (II/III/IV)	5/6/1	8/5/1	.729
Vessel runoff (1/2)	9/3	10/4	1.000
Distal anastomosis
AT/PT/P	5/4/3	5/4/5	.840
Minor complications
Lymphorrhea/haematoma	3/1	3/1	.966
Amputation
Transmetatarsal/lisfranc/chopart	6/4/2	5/6/3	.763

Note: Percentages are reported within brackets.

Abbreviations: ABI, ankle brachial index; ASA, American Society of Anesthesiologists; AT, anterior tibial artery; P, peroneal artery; PT, posterior tibial artery.

3.2. Early and mid‐term revascularisation outcomes until and after wound healing

Table 1 describes the level of the distal anastomosis and the level of amputations. Minor complications consisted of lymphorrhea at the level of the groin in six cases and haematoma in two cases. These complications were resolved without operative treatment. No deaths, revision, or failure of the vein graft occurred until the healing of the wound. Complete follow‐up information was available for all patients and mean follow‐up was 17 ± 6 months (range minimum 7‐maximum 28 months; median 17.5 months). After wound healing, we observed 4 (15%) deaths (due to myocardial infarction 2, stroke 1, and colon cancer 1 at 15, 19, 21, and 26 months) and 4 (15%) graft failures at 14, 14, 15, and 16 months. Four (15%) limbs because of the graft failure had major amputations at 15, 15, 15, and 18 months (3 above and 1 below the knee). Twenty‐four survival rates, primary patency, and limb salvage rates were 52% (SE = 0.23), 76% (SE = 0.11), and 75% (SE = 0.11), respectively.

3.3. Wound healing

Table 2 describes the healing process among the groups. After midfoot amputations, mean wound diameter was 21 ± 4 cm² (range minimum 15‐maximum 31 cm²; median 21 cm²). No statistical difference was observed among the groups. All wound healed within 6 months (mean 5 ± 0.5 months; range minimum 5‐maximum 6 months; median 6 months).

Table 2.

Wound healing process

	Group 1 (n = 12)	Group 2 (n = 14)	Significance
1‐mo	3.1 ± 0.4 cm2/mo	3.0 ± 0.2 cm2/mo	.987
3‐mo	4.9 ± 0.3 cm2/mo	4.8 ± 0.2 cm2/mo	.756
5‐mo	4.4 ± 0.6 cm2/mo	4.3 ± 0.5 cm2/mo	.542

3.4. Plasma levels of MMPs and TIMPs release

Preoperative MMP‐1, MMP‐2, MMP‐3, and MMP‐9 plasma levels of group 1 (32 ± 3 ng/mL, 429 ± 82 ng/mL, 65 ± 8 ng/mL, and 49 ± 6 ng/mL) and group 2 (31 ± 3 ng/mL, 426 ± 63 ng/mL, 65 ± 13 ng/mL, and 46 ± 10 ng/mL) were significantly higher when compared with controls (15 ± 3 ng/mL, 175 ± 12 ng/mL, 38 ± 5 ng/mL, and 8 ± 3 ng/mL, respectively) (P = .001). Preoperative TIMP‐1 and TIMP‐2 plasma levels of group 1 (7 ± 1 ng/mL and 24 ± 4 ng/mL, respectively) and group 2 (7 ± 1 ng/mL and 22 ± 3 ng/mL, respectively) were significantly lower when compared with controls (21 ± 6 ng/mL and 82 ± 10 ng/mL, respectively) (P = .001). At 1‐, 3‐, and 5‐months, the plasma levels of MMP‐1, ‐2, ‐3, and ‐9 were overall slightly lower with respect to the preoperative levels, while the plasma levels of TIMP‐1 and TIMP‐2 were slightly higher. However, no statistical differences with the preoperative levels and among the groups were observed at each interval (Figure 1A).

Figure 1.

A, Plasma level of metalloproteinases (MMPs) and TIMPs among the two groups; no statistically significant differences were recorded. B, Exudate levels of MMP‐2 and MMP‐9 were significantly lower (P = .013 and .047, respectively), whereas those of TIMP‐1 were significantly higher (P = .042) in group 1 as compared to group 2 at 1 month (asterisks indicate significances)

3.5. Exudate release of MMPs and TIMPs

The local release of MMP‐1 (35 ± 3 ng/mL), MMP‐2 (465 ± 62 ng/mL), MMP‐3 (72 ± 10 ng/mL), and MMP‐9 (57 ± 7 ng/mL) immediately after the amputation procedure were overall significantly higher when compared with the preoperative plasma levels (P = .001, .001, .001, and .001, respectively), whereas the release of TIMP‐1 (8 ± 1 ng/mL) and TIMP‐2 (24 ± 4 ng/mL) was significantly lower with respect to the preoperative plasma levels (P = .001 and .011, respectively).

During the follow‐up, the overall local release of MMP‐1 (1 month = 29 ± 3 ng/mL; 3 months = 28 ± 2 ng/mL; 5 months = 25 ± 3 ng/mL), MMP‐2 (1 month = 374 ± 69 ng/mL; 3 months = 339 ± 51 ng/mL; 5 months = 317 ± 53 ng/mL), MMP‐3 (1 month = 68 ± 9 ng/mL; 3 months = 65 ± 8 ng/mL; 5 months = 61 ± 8 ng/mL), and MMP‐9 (1 month = 50 ± 7 ng/mL; 3 months = 48 ± 5 ng/mL; 5 months = 39 ± 5 ng/mL) decreased significantly (P = .001, .001, .001, and .001, respectively) when compared with the immediate postoperative levels. On the other hand, the local release of TIMP‐1 (1 month = 10 ± 1 ng/mL; 3 months = 11 ± 1 ng/mL; 5 months = 13 ± 1 ng/mL) and TIMP‐2 (1 month = 21 ± 4 ng/mL; 3 months = 20 ± 4 ng/mL; 5 months = 18 ± 3 ng/mL) increased significantly at each follow‐up interval (P = .001, and .001, respectively).

Figure 1B describes the local release of MMPs and TIMPs from the exudate at 1, 3, and 5 months among the two groups. Interestingly, at 1 month, the release of MMP‐2 and ‐9 was significantly lower (P = .013 and .047, respectively), whereas those of TIMP‐1 were significantly higher (P = .042) in group 1 as compared to group 2.

4. DISCUSSION

Several authors showed that graft patency, restenosis, limb salvage, amputation‐free survival, and overall survival are the main endpoints to evaluate the effectiveness of the revascularisation procedures.30 Hoffman et al31 pointed out that in clinical studies, ulcer healing is rarely reported as an effective parameter to evaluate the revascularisation therapies in patients affected with CLI; less than 1% of all studies in fact provided data on complete healing and healing times and no randomised controlled studies were present. Furthermore, the role of the wound‐free period after revascularisation is not well elucidated because clinical limb outcome is considered successful when limb is rescued from major amputation without any consideration for minor amputations.32 In our opinion, this is an extremely important aspect of the infrapopliteal arteries reconstructive surgery.

CLI and the wound healing process are strictly associated with the release of numerous inflammatory cytokines: the recruitment, adhesion, and subsequent transendothelial migration of leucocytes are necessary to start and maintain the inflammatory process. Different cytokines and MMPs characterise the entire inflammation process with local and systemic implications, and therefore, it is the link between a successful revascularisation procedure and wound healing.17 Extracellular matrix, a hard network of interlacing macromolecules, that forms a supporting structure for vascular wall and skin integrity, is dynamically maintained by the action of MMPs and their inhibitors.33, 34, 35 Under normal conditions, MMPs are expressed at low levels, but these increase rapidly in the presence of inflammation; the physiological activity of MMPs is essential for many events in wound healing, particularly autologous wound debridement, provisional matrix turnover, and long‐term tissue remodelling, facilitating inflammation, fibroplasia, epithelialisation, and angiogenesis.19, 36 Chronic inflammatory condition results in polynuclear and mononuclear cells' infiltration at the site of injury, resulting in continuous secretion of collagenases, gelatinases, and elastase into the wound.9, 22, 37, 38 TIMPs are secreted by many cells and TIMP‐1 inhibits the activity of MMP‐1, ‐2, ‐3, and ‐9, while TIMP‐2 seems to limit MMP‐2 activity.39

We recently demonstrated that elevated plasma levels of VCAM‐1, ICAM‐1, IL‐6, TNF‐α, and MMP‐2 and ‐9 are strongly related to impaired wound healing and revascularisation failure.16, 17 In the present study, we first evaluated the release and production of MMPs and TIMPs from exudate at the amputation sites treated with either advanced dressings or NPWT. An extensive pattern of protease activity was identified, and the results of the study traced the pathophysiological pathway of postamputation wound heal after revascularisation. We also evaluated the efficacy of two medications on the healing process in patients with CLI and the correlation between the healing process and the release and production of MMPs and TIMPs.

In our study, all patients presented, before revascularisation, high plasma levels of MMP‐1,‐2,‐3, and ‐9 and low levels of TIMP‐1 and ‐2 when compared with the control, thus, at least theoretically, demonstrating the direct relationship with the aggressiveness of the atherosclerotic process and the severity of the clinical presentation. This finding is also supported by the fact that in the postoperative period these markers remained increased and similar to those measured preoperatively. On the other hand, MMP levels evaluated from the exudates at the moment of amputation were significantly higher with a consistently lower level of TIMPs when compared with the plasma level; this finding demonstrates the local action and increased release of these enzymes and their inhibitors.

The revascularisation procedure itself did not modify, if not partially, the triggering of the inflammatory process. In both groups, after the revascularisation procedures, the plasma levels of MMP‐1, ‐2, ‐3, and ‐9 remained elevated with a slight decrease with respect to the preoperative levels.18, 40 Similarly, no significant modifications of the plasma level of TIMP‐1 and ‐2 were observed.

After the revascularisation procedure, the local release of MMP‐2 and ‐9 significantly decreased, at each interval, more effectively when compared with plasma levels. Furthermore, a significant increase of TIMP‐1 and ‐2 release was also detected. This particular trend might be, at least theoretically, the expression of both a successful revascularisation procedure and the healing process because it continued along the follow‐up and these events are strictly related.

It is also extremely interesting that NPWT determined a rapid and persistent local reduction of MMP‐2 and ‐9 as much as the increase of TIMP‐1 at the 1‐month follow‐up. We may also suppose that patients with CLI and non‐healing ulcers have an immune dysfunction caused by chronic and prolonged inflammation, which also may favour progressive critical colonisation and disruptive effects of bacteria on the extracellular matrix. NPWT exerts beneficial effect in applying mechanical forces on the wound bed,41 it shows positive effects on both the proliferation of the granulation tissue and the stimulation of extracellular matrix turnover, and it may increase the local concentration of drugs in the treated tissue enhancing their systemic effects beginning from the initial phases of treatment.42 These hypotheses may explain the increased release of TIMP‐1 at 1 month. However, no differences among the two groups in the wound healing process were observed. Subsequently, the two medications exerted a similar action in the local release of MMPs and TIMPs.

After the wound healed, the plasma levels of MMPs and TIMPs decreased to values significantly lower than those recorded preoperatively. Our data suggest that the positive or negative predictive value of these markers is quite high. This experience confirms the central role of wound healing as the indicator of outcomes after limbs revascularisation; an adequate and aggressive wound management strategy is needed and recommended in patient with CLI, and the use of NPWT can help to debride, hydrate, or granulate and to speed up the healing process.41, 42, 43, 44 Furthermore, our findings also raise the important question whether a more aggressive anti‐inflammatory therapy associated with peripheral arterial reconstruction may improve the postoperative outcomes of these patients.45, 46 We prescribed the systematic use of aspirin and statin, but the eventual modulation in the release and production of MMPs and their natural inhibitors was not elucidated.

We are aware that our study has some limitations: first, the small sample size. Second, the measurements were performed at 2‐month intervals to reduce the costs of biological kits and follow‐up. Therefore, we could not exclude the variability of plasma levels during the time course. Third, wound bed can be hypoxic even when the reconstructions were patent due to local pathological changes in the vascular bed, peri‐wound fibrosis, and oedema, which increases the distance between capillaries, simply because in the phase of healing peripheral collaterals will dilate in an attempt to supply the wound with the required nutrients, yet the base of the wound is lacking, particularly in oxygen. This in turn might also alter the release of the local biomarkers. Fourth, since the atherosclerotic process affects several arterial districts, the plasma level of MMP and their inhibitors represent the ubiquitous involvement of this disease.

In conclusion, our study allowed drawing a complete and detailed map of the biomolecular pathophysiology of wound healing after a successful revascularisation procedure and the role of MMPs and their natural inhibitors in the various phases of this process. An aggressive therapy of the wound either with NPWT or with an advanced moist wound dressing is able to promote the healing of the lesions after restoration of an adequate distal blood flow through surgical revascularisation.

Grande R, Brachini G, Sterpetti AV, et al. Local release of metalloproteinases and their inhibitors after a successful revascularisation procedure. Int Wound J. 2020;17:149–157. 10.1111/iwj.13249