INTRODUCTION

Nonhealing leg ulcer is defined as a chronic defect in the skin below the level of knee persisting for more than 6 weeks and shows no tendency to heal after 3 or more months.[1] The incidence of ulceration is rising due to increasing risk factors for atherosclerotic occlusion such as smoking, obesity, and diabetes.[1] Prevalence of nonhealing ulcers in India is found to be 4.5/1000 population.[2] Regardless of the underlying etiology, nonhealing ulcers tend to have chronic pain, discharge, sleep impairment, and subsequent adverse repercussions in quality of life; posing many therapeutic challenges. Principles of therapy are addressal to the underlying cause, symptom management, interventions to improve circulation, and promotion of wound healing.

Autologous platelet-rich plasma (PRP) provides anti-inflammatory action on the wound bed as well as contents of the alpha granules (platelet-derived growth factors and vascular endothelial growth factors) released locally to stimulate the initiation of healing.[3] Autologous platelet-rich fibrin matrix (PRFM) provides growth factors, especially transforming growth factor beta (TGF)-beta which is profibrotic, increases biomechanical strength, and helps in epithelial resurfacing and differentiation.[4] Epidermal growth factor (EGF) is quintessential for wound healing. By acting on epithelial cells and fibroblasts, recombinant human epidermal growth factors (rhEGF) promote faster re-epithelization.[5] Collagen dressings act as a scaffold as well as spare the healthy collagen by acting as a decoy for the wound matrix metalloproteinases, thereby accelerating wound healing.[6]

The aim of this study was to compare the therapeutic efficacy of PRP, PRFM, rhEGF, and collagen particles: four relatively newer modalities in the management of nonhealing ulcers.

SUBJECTS AND METHODS

An open-labeled, randomized prospective interventional study was conducted in the Department of Dermatology in a single tertiary care center after informed patient consent and approval by Institutional Ethical Committee from July 2018 to August 2020. The inclusion criteria of the study were patients aged 18–65 years and either sex having nonhealing ulcers on the lower extremity of any etiology, defined as ulcer ≥1 year duration and/or showing no/inadequate signs of healing after 12 weeks of appropriate treatment. The exclusion criteria of the study were pregnant and lactating females, bleeding disorders, and/or oral anticoagulant therapy, uncontrolled diabetes, proven malignancy, actively infected ulcers, ulcers >10 cm² area, ulcers with exposed bone with no underlying granulation tissue, HIV/AIDS and those with unrealistic expectations and unwilling to give consent for treatment or photography. The detailed case history was elicited as per a predesigned proforma. General, systemic and local examination was performed noting the attributes of the ulcer, surrounding skin, and regional lymph nodes. All patients were subjected to the following baseline investigations: complete blood count, liver and renal function tests, coagulation profile PT/INR, APTT, random blood sugar, HbA1c, viral markers––HIV, HBsAg, HCV, and ultrasonography with Doppler of limbs. Ulcers cleansing, slough debridement, and treatment of the underlying condition(s) were done. Fifty consecutive patients fulfilling the study criteria were randomly allocated into one of the four treatment arms [Figure 1]:

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Figure 1

Therapeutic modalities employed for nonhealing ulcer in this study are depicted. (A) Injection of autologous PRP into the base and sides of the ulcer in Group A. (B and C) Separation of PRFM from adherent blood clot and placement over the cleansed wound bed, in Group B. (D) Application of rhEGF cream to the wound in Group C. (E) Collagen particles in Group D to be sprinkled adequately over the wound. (F) Paraffin-impregnated non-adherent gauze dressing applied after the procedure

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• Group A: Autologous PRP

• Group B: Autologous PRFM

• Group C: rhEGF

• Group D: Collagen particles

RESULTS

From a total of 52 patients fulfilling the study criteria, 48 completed the study: 12 patients in each of the four groups and were included in the final analysis, whereas four were lost to follow-up (one in Groups A and B each due to lack of feasibility to return for the procedure, and two from Group C due to lack of response). The mean age of patients was 42.37 ± 4.56 years with a range of 32–56 years. The male-to-female ratio was 2.6:1 with 72.9% (n = 35) males and 27.1% (n = 13) females. The most common occupation was manual labor in 35.4% (n = 17) patients and 52.1% (n = 25) belonged to lower socio-economic status. The most common presenting complaints were pain and discharge from the ulcer in all groups. Underlying comorbidities eventuating into ulcers were varicosities in 43.75% (n = 21), followed by traumatic in 25% (n = 12), diabetes in 22.91% (n = 11) and leprosy in 8.34% (n = 4). Addiction in the form of alcohol intake was seen in 31.3% (n = 15), smoking in 27.1% (n = 13), and both in 31.3% (n = 15) patients. The majority of patients 52% (n = 25) had ulcers for the past 8 to 10 months duration, 25% (n = 12) patients had an ulcer for more than 10 months, and 23% (n = 11) patients had an ulcer for less than 8 months. At baseline, all groups were comparable in terms of baseline characteristics as well as ulcer size and duration (P > 0.05).

Table 1 shows the decline in the area of the ulcer from baseline at the end of 12 weeks. Figure 2 shows the differences between groups in terms of ulcer healing at 8 and 12 weeks.

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Figure 2

Excellent improvement in Physicians’ Global Assessment scores. At the end of 8 weeks, excellent ulcer improvement* was seen in majority of the Group B patients (PRFM arm). This difference between groups was statistically highly significant (P = 0.0001). At the end of 12 weeks, difference between excellent ulcer healing between groups was statistically insignificant (P = 0.05)

(*≥75% reduction in the size of the ulcer from baseline was defined as excellent improvement)

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Early endpoint (8 weeks)

On applying the Physician Global Assessment score, excellent improvement (>75%) was achieved in a total 18 out of 48 patients, that is, 37.5% of patients at the end of 8 weeks, with improvement in maximum patients of Group B, 83.33%, that is, 10 out of 12 patients, followed by Group A (33.33%, n = 4), Group C (25%, n = 3), and Group D (8.33%, n = 1) [Figures 3,4,5–6]. The difference in ulcer healing at 8 weeks was statistically highly significant (P = 0.0001).

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Figure 3

(A) Baseline: Posttraumatic ulcer in a 34-year-old man. (B) Week 8: After six sessions of autologous platelet-rich plasma therapy (Group A)

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Figure 4

(A) Baseline: Varicose ulcer in a 54-year-old man. (B) Week 8: After four sessions of autologous platelet-rich fibrin therapy (Group B)

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Figure 5

(A) Baseline: Trophic ulcers in a 56-year-old diabetic woman. (B) Week 8: After recombinant human epidermal growth factor application (Group C)

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Figure 6

(A) Baseline: Trophic ulcers in a 30-year-old man with leprosy. (B) Week 8: After collagen particle dressing (Group D)

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DISCUSSION

In the Indian context, nonhealing leg ulcers may stem from systemic conditions such as diabetes, atherosclerosis, leprosy, venous ulcers, pressure ulcers, vasculitis, and inappropriate management of acute trauma.[2] Despite adequate preparation of wound bed, infection control, and a moist environment, chronic leg wounds may fail to respond, necessitating the use of techniques that deliver and enhance dermal matrix components, that is, growth factors, fibrin preparations, and/or scaffold proteins.[7] In our study, the majority of patients achieved complete ulcer healing in all four modalities with excellent patient acceptability, but PRFM showed slightly better efficacy along with faster wound healing [Figure 7]. Comparison between studies in the literature is difficult, due to differences in the underlying etiologies, procedural and protocol variations, and lack of similar head-on comparative studies between the four modalities in this cohort.

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Figure 7

Mean decrease in ulcer size. Comparison of time needed for decline in the mean ulcer size at 4, 8, and 12 weeks of therapy across all groups is depicted. Faster healing times were seen with Group B (PRFM arm)

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In a landmark study by Margolis et al.[8] which included 26,599 patients, it was found that ulcers treated with “platelet releasate” tended to heal faster than patients who were treated without them. PRFM due to its fibrinous matrix acts as a novel natural carrier to deliver three-fold higher growth factor concentrates to the wound bed with a controlled release, versus PRP despite the latter having higher platelet concentrates.[910] This theoretically translates into superior efficacy of PRFM over PRP. In our study, both PRFM and PRP showed ulcer resolution in 91.67% of patients albeit faster with the PRFM group, versus a recent randomized controlled trial by Pravin et al.,[11] where 73.3% and 53.3% of patients achieved ulcer clearance with PRFM and PRP, respectively. Mean time to complete healing in our study was 4.73 weeks (corresponding to approximately four to five sittings), which is comparable with Sarvajnamurthy et al.[12] and Dorjay et al.,[13] where complete healing took approximately 5 weeks (corresponding to 5 sittings). Apart from providing a plethora of growth factors, PRFM behaves like a biologically active tissue, akin to tissue graft for cutaneous wounds.[14] It contains leucocytes which help combat and prevent secondary infections, enabling its use across leg ulcers of multiple causes.

EGF family comprises four proteins: EGF, TGF-alpha, heparin-binding EGF, and amphiregulin.[15] Acting as a mitogen, EGF stimulates the proliferation and migration of keratinocytes, granulation tissue formation, and fibroblast motility.[5] Recombinant human EGF has the best evidence for use in diabetic ulcers. Recent meta-analysis involving 530 patients showed that intralesional injection and/or topical application of rhEGF in diabetic foot ulcers had better rates of healing than conventional modalities (combined odd’s ratio of 4.005, P < 0.001).[16] The indications have now expanded to nonhealing ulcers of other etiologies. Sachez et al.[17] studied rhEFG in chronic venous ulcers and found that 100% of ulcers re-epithelialized; 71% achieved wound closure in 8 weeks or less, and the remaining within 9 and 12 weeks. This was comparable with our subset of five patients in Group C with venous ulcers of which four, that is, 80% of patients healed completely in approximately 8–10 weeks. There is a paucity of studies that compare the efficacy of this modality to platelet-derived modalities/ collagen dressings. A recent study on microbiome colonization showed that patients treated with EGF had no infections during the follow-up period, and there was a significant difference versus patients treated with PRP who had Staphylococcus aureus and Pseudomonas aeruginosa colonization (P = 0.0078).[18] However, no patient in this study had infections either in PRP or rhEGF arm.

Collagen is fundamental to wound healing. Chalimidi et al.[6] found a significant decrease in wound size by using collagen particles versus conventional dressings. Collagen particle dressings belong to protease-modulating matrix (PMM) treatments that improve healing by physically removing proteases from the wound fluid. Evidence regarding the superiority of PMM over non-PMM concept dressings is unclear in literature as per a recent review of 784 patients.[19] Qureshi et al.[20] conducted a study on 102 patients with nonhealing ulcer and found PRP to be safer and more effective with faster healing rates and time compared with collagen dressing (P < 0.05); a similar finding was seen in our study. Durvasula et al.[21] did a comparative study on 30 patients with trophic ulcers secondary to leprosy>diabetes>venous insufficiency, comparing the efficacy of collagen particles impregnated with antibiotics (similar to our cohort) with PRFM. Complete healing was seen in 80% and 53% of patients in collagen and PRFM arms, respectively, in a mean time of 6 weeks in both arms, versus 66.67% and 91.67% of patients, respectively, in our study in the mean time of 7–8 weeks for collagen arm and 4–5 weeks for PRFM arm. Impregnation with antibiotics potentially limits the rate of secondary infections.

This study broadly compares the efficacy of treatment options in nonhealing leg ulcers. Limitations of this study include non-homogeneity in patient comorbidities and ulcer profiles limiting a perfect comparison with other studies or drawing inferences about merits/demerits of a particular modality in a specific clinical scenario. A relatively small sample size leading to bias and lack of long-term follow-up are some other limitations.

CONCLUSION

Autologous PRFM resulted in faster healing of chronic leg ulcers when compared with PRP, rhEGF, and collagen particles at 8 weeks, with a shorter mean time to healing of approximately 4–5 weeks with excellent patient acceptability. It is a feasible, relatively simple, and efficacious treatment option for nonhealing leg ulcers.