The use of acellular porcine dermis, hyaluronic acid and polynucleotides in the treatment of cutaneous ulcers: Single blind randomised clinical trial

Abstract

Reconstruction of chronic ulcers is often hampered by lack of local tissues and poor general conditions. Conservative approaches with debridement and advanced medications, such as polyurethane foam, stand as mainstays. However, the healing process is often slow, thus increasing the risk for infection or other complications. In such cases, porcine dermis (PD) and polynucleotides‐added hyaluronic acid (PAHA) were previously reported to accelerate healing. The aim of the study was to compare the efficacy of PD, PAHA and polyurethane foam in chronic ulcers. Thirty patients were randomly divided into 3 groups: group 1 was treated with advanced medications, group 2 with PD, group 3 with PAHA. Standardised photographs and biopsies were taken before treatment and at 30‐day follow‐up. Photographs were processed to calculate the wound area. Specimens were stained with Haematoxylin/Eosin, Masson trichrome, and immunohistochemically for CD34, alpha‐Smooth Muscle Actin (α‐SMA), Collagen types I and III, Ki67. The re‐epithelialized area was larger in patients treated with PD and PAHA compared with those treated with polyurethane foam (P < .05 and P < .01, respectively). Specimens from patients treated with PD and PAHA showed a higher number of myofibroblasts (α‐SMA+, P < .01), neo‐angiogenesis (CD34+, P < .01), proliferating dermal cells (Ki67+, P < .01), proliferating keratinocytes (Ki67+, P < .01) and collagen type 1 deposition (P < .05). No difference was found between PD and PAHA. PD and PAHA proved to be more effective than polyurethane foam in the treatment of chronic ulcers. These approaches are a versatile and reliable option to address such cases.

1. INTRODUCTION

A wound that failed to re‐epithelialize after 3 months is defined as chronic. More than 4 million patients in the United States and 1.5‐2 million people in Europe are affected by chronic wounds, and around 50 billion US dollars are spent every year on their treatments. ¹ , ² Such wounds are a major issue for patients and a challenge for clinicians and surgeons. They may complicate with infection, sepsis or cancerization, sometimes leading to amputation. Even with uncomplicated ulcers, the patient's quality of life is reduced by pain, need for frequent medications as well as by functional and social limitations. Surgical reconstruction is often unfeasible due to poor local tissues, comorbidities and local infection. As a consequence, surgical debridement followed by secondary healing stands as a mainstay. It would be paramount for these patients to speed up the re‐epithelization process. Advanced medications, such as polyurethane foam (PF), are effective in promoting healing and preventing contamination. However, they require every‐other‐day turnover, thus implying a significant logistic and economic burden for the patient. Moreover, their regenerative effect is limited and often slow or inadequate to achieve complete re‐epithelization. Porcine dermis (PD) and polynucleotides‐added hyaluronic acid (PAHA) were previously reported to reduce hospitalisation and to promote healing, as well as cell proliferation and matrix production in skin ulcers. ³ , ⁴ , ⁵ , ⁶ , ⁷ To date, there is still lack of comparative data about the clinical and biological effects of such medical devices. Moreover, the reduction in the overall monthly expense for treatment is still debated. The average monthly cost for 10 × 10 cm polyurethane foam is around 110‐150 $, considering the every‐other‐day turnover. On the other hand, a single 8 × 10 cm sheet of PD ranges from 90 to 140$, while a 2 mL syringe of PAHA is sold at about 30$. The aim of the study was to compare the clinical, biological and economical efficacy of polyurethane foam, porcine dermis and polynucleotides‐added hyaluronic acid in the treatment of chronic wounds.

2. MATERIALS AND METHODS

The study was designed as a single blind, randomised, prospective clinical trial and it was approved by the local ethical committee. Thirty patients were enrolled between January 2016 and June 2017, according to the following criteria:

• inclusion criteria: venous ulcer not improving after 3 months; age over 18 years old.

• exclusion criteria: systemic infection; connectivities or rheumatic diseases; cancer; ongoing chemotherapy or therapy with biologic drugs, corticoids or immunosuppressants; diabetes; neoplastic or Marjolin ulcer.

Thirty patients were enrolled and randomised by mean of block randomization to one of three treatment groups (10 patients for each group): polyurethane foam, porcine dermis, polynucleotides‐added hyaluronic acid. In the whole cohort, a digital photo of the wound was taken with the patient in supine or prone position (from a distance of 50 cm and including a centimetric ruler in the picture). A biopsy was performed at one of the wound edges including both damaged and healthy tissue (Figure 1).

FIGURE 1.

Haematoxylin–Eosin showing normal tissue (continuous line) adjacent to granulation tissue (dashed line) in a biopsy (merged from original magnification ×40)

All the patients underwent surgical debridement and were immediately treated according to the randomization group:

Group 1: polyurethane foam with silver complex (Biatain Ag non‐adhesive, Coloplast Italia, Bologna, Italy) was applied, followed by every other day change.

Group 2: porcine dermis (EzDERM, Mölnlycke Health Care AB, Gothenburg, Sweden) was applied once.

Group 3: polynucleotides‐added HA (NukliaSkin S, Mastelli SRL, Sanremo [IM], Italy) was applied once.

Standardised pictures were taken 15, 30, and 45 days post‐operatively. At 30 days follow‐up, a biopsy was performed harvesting tissue on the wound edge opposite to the previously biopsied one.

The digital photographs were manually processed with MOWA (Mobile wound Analyser, Healthpat) software. The ulcerated area was calculated in square centimetres. The wound area reduction was calculated in square centimetres and as a percentage.

2.1. Tissue samples

Tissue specimens were fixed in 4% neutral buffered formaldehyde, embedded in paraffin and cut into 3 μm slices to achieve a transversal view of the tissue layers. They were subsequently stained by haematoxylin/eosin and immunohistochemistry antibodies for alpha smooth muscle actin (α‐SMA, as a marker of myofibroblasts, Clone 1A4 Dako), CD34 (as a marker of neo‐angiogenesis, Clone QBEnd/10 Biocare Medical), Ki67 (in keratinocytes and dermal fibroblasts, as a marker of cell proliferation, Clone MIB‐1 Dako), collagen type 1 (COL I Polyclonal IgG, AbD Serotec) and collagen type 3 (COL III, Polyclonal IgG, AbD Serotec). Secondary antibodies and processing were performed as previously described. ⁸ The positivity for α‐SMA was evaluated at a 400x magnification in three randomly chosen fields, then averaged and expressed a percentage of positive cells. Neo‐angiogenesis was evaluated by CD34 positivity, with the Weidner method. ⁹ Collagen positivity was assessed as previously described, ⁸ Ki67 positivity of keratinocytes and dermal cells was evaluated as the average percentage of positive cells in three randomly chosen 400 x fields. Light microscopy images were captured by a videocam (SPOT Insight; Diagnostic Instrument, Inc., Sterling Heights, Michigan) connected to an Olympus BX‐51 light microscope (Olympus, Tokyo, Japan) and processed with an image analysis system (Delta Sistemi, Rome, Italy). The operator evaluating clinical and histological outcomes was blinded to treatment group and patient's data. Wound area reduction at postoperative day 30 was considered as the primary endpoint. The secondary endpoints were the increases in neo‐angiogenesis, cellular proliferation, collagen deposition and number of myofibroblasts. Data were expressed as mean (± Standard Deviation) or median with 95% confidence interval and analysed as both absolute value and percentage. Independent samples T test were performed to assess any statistically significant difference between groups. Paired T test was used to assess the eventual difference between pre‐operative and 30 days postoperative values. Mann–Whitney U test was performed where necessary. In case of categorical variables, the χ ‐square test was used. Correlations were evaluated by mean of Spearman correlation coefficient. All tests were two‐tailed and considered statistically significant for p values lower than 0.05.

3. RESULTS

Mean age of the patients(±SD) was 69 (±13) years. The average pre‐operative size of the wounds was 28.28 (±19.03), 10.14 (±14.94), and 30.9 (±21.45) cm² in the PU, PAHA and PD groups, respectively. Among groups, no statistically significant difference was found in the pre‐operative wound area. Neither complication nor loss to follow up were experienced in the cohort. Treatment outcomes for the primary endpoint at 30 days follow‐up (Figure 2) are summarised in Table 1.

FIGURE 2.

Above: patient from group 3; left, pre‐operative view; right, post‐operative view. Below: patient from group 2; left, pre‐operative view; right, post‐operative view

TABLE 1.

Mean wound area reduction

Treatment	Mean wound area reduction (± SD)
Porcine dermis	6.14 (±3.67) cm2	P<.05 a
	20.95 (±13.06) %	P<.01 a
Polynucleotides‐added hyaluronic acid	3.64 (±2.24) cm2	P<.01 a
	74.14 (±32.94) %	P<.01 a
Polyurethane foam	0.4 (±0.17) cm2
	1.6 (±0.58) %

^aCompared with the polyurethane foam group.

Both PD and PAHA groups had a statistically significant reduction in the wound area compared with the PU foam group. The percent reduction of the wound area was significantly higher in the PAHA group compared with the PD group (P < .05) (Figure 3).

FIGURE 3.

Mean wound area reduction

PAHA and PD groups showed, compared with PU group (Figure 3):

• higher Collagen type I deposition (P < .05 and P < .05, respectively);

• higher ki67 positivity in dermal cells (P < .01 and P < 0.01, respectively) and basal keratinocytes (P < .01 and P < .01, respectively);

• higher CD34 expression (P < .01 and P < .01, respectively)

• higher number of α‐SMA positive cells (P < .01 and P < .01, respectively).

In PAHA and PD groups, each of these secondary endpoints showed a statistically significant increase compared with the average value at time 0.

In the PU group, only the number of α‐SMA positive cells showed a statistically significant increase compared with the average value at time 0. There was no statistically significant difference in any of the secondary endpoint between PAHA and PD groups (Figure 4). No significant difference in Collagen type III expression was found among groups (Figure 5).

FIGURE 4.

Immunohistochemical results. Collagen I: from left to right, PU, PD and PAHA (×40); CD34: from left to right, PU, PD and PAHA (×40); KI 67: from left to right, PU(×40), PD (×100) and PAHA (×40); α‐sma: from left to right, PU, PD and PAHA (×40). PD, porcine dermis; PAHA, polynucleotides‐added hyaluronic acid

FIGURE 5.

Box‐plot representing immunohistochemical expression of the different markers analysed. * P < .05 vs PU; ** P < .01 vs PU. T0 = average pre‐operative value

4. DISCUSSION

Chronic ulcers are a major issue of modern medicine. They extremely jeopardise the patient's quality of life as well as increase the cost of hospitalisation and outpatient treatment. Surgical reconstruction is often unfeasible, thus making surgical debridement followed by secondary, slower, healing stand as a mainstay. In our current era of increasing health care costs, aging population, and increasing prevalence of obesity and diabetes, it is important to identify novel therapeutic options to better address the growing burden of chronic wounds. Polyurethane foam stands out as one of the most versatile and effective advanced medications. It is regarded among the gold standards for wounds with moderate secretion. Nevertheless, it is expensive and sometimes inadequate in achieving prompt tissue healing. The use of porcine dermis in cutaneous ulcers was previously investigated. Zhang et al reported its efficacy in promoting healing, reduce hospitalisation and complications in perineal and lower limb wounds. ³ , ⁴ Zajicek and colleagues demonstrated that such dermal substitute promoted the proliferation and differentiation of human keratinocytes. ⁵ Moreover, it was found to increase collagen synthesis, stem cells proliferation as well as PDGF, EGF and FGF expression. ¹⁰ Hyaluronic acid is a polysaccharide common in all species. Several studies found it to improve the healing of wounds resulting from burns, venous insufficiency, diabetes and surgery. ⁶ , ⁷ The combination of HA and polynucleotides was demonstrated to be more effective than HA alone in promoting re‐epithelialization of venous ulcers. ¹¹ In the present study, we found that both PD and PAHA allowed for larger healing than PU at 30 days follow‐up. Preoperatively the ulcers had different size, and the average wound size of the PD group was a little larger than that of PAHA; as a consequence we analysed the percent wound area reduction and found that PAHA performed better than PD. Tissue healing is a dynamic process, requiring cell proliferation, neo‐angiogenesis, and matrix deposition. Compared with PU group, PD and PAHA ones resulted in higher cellular proliferation, number of myofibroblasts, neo‐angiogenesis. From a biological standpoint, all these factors contribute to tissue healing and have inter‐plays, as demonstrated by the here‐found correlation. Thirty days post‐operatively, all the above‐cited histological parameters showed an increase in PD and PAHA groups compared pre‐treatment status. On the other hand, only the number of myofibroblasts was increased in the PU group. Such a finding means that, although debridement and PU application results in some tissue reaction, it may be not enough to activate all the factors involved in tissue healing and regeneration. From a clinical standpoint, it may be the biological reason underlying the slower or inadequate healing found at clinical examinations. On the other hand, the experimental devices upregulate the response to tissue damage by providing a stable extra‐cellular matrix as well as positive feedback interactions for cell proliferation. Moreover, they performed economically better at 30 days follow‐up. Given the above‐cited prices, the monthly expense was the lowest with PAHA, while similar between PD and PU. Nevertheless, PD treatment was still cheaper than PU as it needed a single application, thus reducing the secondary costs of transportation, medical personnel, working time loss etc. associated with an every‐other‐day medication change.

5. CONCLUSION

PD and PAHA proved to be more effective than PU in addressing chronic wounds, both clinically, histologically and economically. Although further studies, with a larger number of patients and more selective inclusion criteria, are needed to compare the two medical devices, our study provides a first evidence of the advantage in using PD and PAHA compared with PU.

CONFLICT OF INTEREST

The authors do not have any financial interest or commercial association with subject matter and/or products mentioned in the manuscript.

Segreto F, Carotti S, Marangi GF, et al. The use of acellular porcine dermis, hyaluronic acid and polynucleotides in the treatment of cutaneous ulcers: Single blind randomised clinical trial. Int Wound J. 2020;17:1702–1708. 10.1111/iwj.13454