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Abstract
Context: Chronic non-healing ulcer causes significant morbidity, high cost and reduced quality of life. Aims: To compare autologous platelet-rich fibrin matrix and transplantation of autologous non-cultured epidermal cell suspension in the treatment of chronic non-healing ulcers. Methods: The study was single-centre, prospective, randomised comparative study conducted in a tertiary care center in North India. Patients with chronic non-healing ulcer were included and randomly divided into two treatment groups- Group 1: Platelet-rich fibrin matrix (PRFM) procedure was done every 2 weeks with maximum three sittings and in Group 2: Transplantation of autologous noncultured epidermal cell suspension (NCES) procedure was done once. Follow-up was done every 2 weeks for 8 weeks then monthly for up to 5 months to evaluate the healing of the ulcer. The data were analysed by statistical package for social science (SPSS) trial version 22. To find out a significant difference in mean value between groups, the Chi-square test, student's t-test, and Mann–Whitney U test were used. Results: A total of 41 patients were included in the study. Complete healing of ulcers occurred in 89.5% of the patients in the PRFM group and 93.8% of the patients in the NCES group at the end of 5 months (P = 0.33). The mean duration of complete healing in PRFM was 1.7 months and in NCES was 2.13 months (P = 0.20). Conclusions: Both procedures were effective, and there was no significant difference between the two procedures.
Keywords: Chronic non-healing ulcer, leprotic ulcer, non-cultured epidermal cell suspension, platelet-rich fibrin
Introduction 
Chronic non-healing ulcer adversely affects activities of daily life and leads to loss of productivity and reduced quality of life. It increases the financial burden on the patient due to long-term management. The goal of the treatment of ulcers is to obtain faster-wound closure. In previous studies, platelet-rich fibrin matrix (PRFM) and noncultured epidermal cell suspension (NCES) were found to be effective for the faster healing of ulcers. We were unable to find any head-to-head trial comparing both procedures. Hence, we planned a randomised clinical trial between PRFM and NCES in the treatment of chronic ulcers.
Subjects and Methods A single-centred, prospective randomised comparative study. Ethical approval was taken from the institute's ethics committee, after which the study was registered with the Clinical Trials Registry of India (CTRI/2019/02/017788).
Patients suffering from chronic non-healing ulcers coming to the skin outpatient department, fulfilling the inclusion and exclusion criteria of the study were included in the period from February 2019 to March 2020. All the patients were explained the method, expected outcome, variable results, relapses, and informed consent was taken before participating in the study.
Inclusion criteria: Chronic non-healing ulcer for >6 weeks duration.
Exclusion criteria:
Age below 18 yearsPregnant or lactating womenUnderlying osteomyelitisSepsisMalignancyImmunocompromisedBleeding disorder, platelet count (<1 lakh), patient on anticoagulant medicationUncontrolled diabetes mellitus.The patients' demographic and wound characteristics are given in [Table 1]. Baseline investigations like complete blood count, total protein, blood sugar, and blood urea were done. Baseline swab culture results are given in [Table 2]. As per the clinical scenario, radiological examination and biopsy were done to rule out malignancy. The patients were admitted, proper antibiotics were prescribed, and wound care was done until the swab culture became sterile. Surgical debridement was done wherever necessary. Healthy granulation tissue at the base of the ulcer was required for the patients to proceed in the study. The study period for each patient was 5 months, which included procedures and follow-up. The patients were randomly divided into two treatment groups by a computer-generated random number sequence using the MS Excel Software.
Group 1: Platelet-Rich Fibrin Matrix (PRFM): -
Under strict aseptic precautions, 10 mL venous blood was drawn and added to a sterile centrifugation tube devoid of anticoagulant. Centrifugation was done at 3000 revolutions per minute (rpm) for 10 min. Three layers were obtained: upper straw-colored platelet-poor plasma (PPP), a red-coloured lower fraction containing red blood cells and a middle fraction containing PRFM. PPP was discarded, and PRFM was separated from red corpuscles at the base, preserving a small RBC layer measuring around one mm in length, which was transferred onto a sterile gauze. The middle membrane so obtained was compressed between two gauze pieces gently and was applied to wound bed followed by the application of a secondary non-absorbable dressing. The patients were advised to protect the area from trauma and keep it dry for 7 days. The secondary dressing and PRFM were removed from the wound bed after 1 week. The procedure was repeated every 2 weeks for a maximum of three sittings or healing of an ulcer. The patients were followed up weekly for 6 weeks then at eighth week, monthly till 5 months.
Group 2: Transplantation of autologous noncultured epidermal cell suspension:
After part preparation and maintaining an aseptic and sterile environment, we obtained a thin split-thickness skin graft with even thickness, depending upon the size of the ulcer from the anterolateral aspect of the thigh under local anesthesia (2% lignocaine), using a Humby knife. The donor to recipient size ratio of 1:4. The graft was incubated at 37°C for 50 min in a sterile bottle containing 0.25% trypsin- ethylenediamine tetraacetic acid (EDTA) solution. The action of trypsin was neutralized by washing the graft with phosphate buffer solution (PBS) in a sterile petri dish. The donor site was covered with chlorhexidine dressing. The graft was placed in a fresh sterile petri dish containing 3-4 mL PBS. The dermis was separated from the epidermis mechanically, and the dermis was discarded. The basal surface of the epidermis was rubbed gently leaving behind a transparent layer, the stratum corneum. The solution was centrifuged at 3000 rpm for 10 min. The supernatant was discarded, and the cell pellets thus obtained were suspended in PBS. The solution was vigorously pipetted out repeatedly to get a homogenous solution and was mixed with an equal amount of hydroxypropyl methylcellulose. An adequate amount of NCES was transplanted to the wound bed using a disposable syringe and evenly distributed followed by the application of a secondary nonabsorbable dressing. The patients were hospitalized for 2 nights, and advised to protect the area from trauma and keep it dry for 7 days. Dressing from the donor area was removed on the 3rd day of the procedure and from the wound bed after 1 week, and a topical antibiotic was applied. The patients were followed up every 2 weeks for the first 8 weeks and then monthly till 5 months. The tracing of the ulcer was recorded on a graph paper, and the area was calculated at the baseline and every visit, and the healing of the ulcer was assessed. Furthermore, the patients were advised to avoid pressure over the ulcer area by wearing cushioned footwear and walking with the help of stick. They were followed up to evaluate the healing of the ulcer and recurrences at the same site.
Statistical analysis
The data obtained were analysed by statistical package for social science (SPSS) trial version 22 for statistical analysis. Categorial data were presented in the form of numbers and percentages, and a Chi-square test had been used. Numerical data were presented in the form of mean and standard deviation, and the student's t-test was applied to find out a significant difference in the mean value between the study groups. If the data were found non-normal distribution, then Mann–Whitney U test was used. To find out the relationship between study variables, Karl Pearson's correlation coefficient and Spearman's rank correlation coefficient were calculated. P value less than 0.05 was taken as statistically significant.
Results A total of 90 patients were screened and finally, 41 patients were included and randomly divided into two groups: 23 patients in the PRFM group and 18 patients in the NCES group [Flow chart 1].
The baseline characteristics were similar between the two groups.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8] shows chronic non healing ulcer before and after intervention.
Figure 1: 12 months old leprotic ulcer after 3 sittings of PRFM (6 weeks)
Figure 6: Six weeks old leprotic ulcer after 3 sittings of PRFM (1.5 months)
Figure 8: 4 months old leprotic ulcer after 2 sittings of PRFM (4 weeks)Four patients in the PRFM group and two patients in the NCES group were lost to follow-up.
Antibiotic was started according to culture and sensitivity report. Most common antibiotic used was Levofloxacin. The most common organism isolated was Staphylococcus aureus in both groups (42.1% of the patients in the PRFM group and 43.8% in the NCES group). Multiple organisms were isolated from ulcers in both the groups: 21.05% of the patients in the PRFM group and 31.3% in the NCES group.
The majority of the ulcers healed in 3 months after the procedure. At the end of 5 months, there was complete healing of ulcers in the majority of the patients – 89.5% in the PRFM group and 93.8% % in the NCES group (P < 0.33) and incomplete healing of ulcers in 10.5% of the patients in PRFM group and 6.3% in NCES group [Table 4].
The mean duration of complete healing was 1.7 months in the PRFM group and 2.13 months in the NCES group [Table 5].
Recurrence of ulcer occurred at the same site in two (10.5%) patients in the PRFM group after 4 and 6 weeks and in one (6.3%) patient in the NCES group after 6 weeks.
No significant correlation was observed in the two procedures in the complete healing of ulcers at 3 months and age, sex of the patient, duration, baseline area of ulcer in both groups and baseline platelet count in the PRFM group [Table 6] and [Table 7].
Discussion Chronic ulceration of the lower leg is not uncommon and is mainly caused by venous insufficiency, diabetes, leprosy and vasculitis. In our specialty, leprosy is the leading cause of chronic non-healing ulcers. Chronic wound comes with a significant cost burden for the patients as well as the society.[1] Various treatment modalities are available for trophic ulcers such as moist wound dressings, vacuum-assisted closure, hyperbaric oxygen therapy, reconstructive surgeries, topical application of growth factors,[2] platelet-rich plasma (PRP) and PRFM therapy. PRFM acts as a biodegradable scaffold for cell migration, proliferation, differentiation and delivers growth factors.[3] Platelets trapped within the three-dimensional mesh ensure slow and continuous release of growth factors over time.[4] Various growth factors such as platelet-derived growth factors help in angiogenesis, neutrophilic chemotaxis and collagen synthesis; transforming growth factor-beta helps in regulating fibroblast proliferation and deposition of the bone matrix; insulin-like growth factor-1 and fibroblast growth factor enhance proliferation and differentiation of osteoblast and activate protein synthesis; vascular endothelial growth factor aids angiogenesis and endothelial cell migration; and IL- 1,6 stimulates T-cells and IL- 4 supports proliferation of activated B-cells and increases collagen synthesis.[5],[6],[7] Autologous cell transplantation is one of the ways to restore lost or injured tissue without the trepidation of rejection. Transplantation of cultured keratinocytes was a turning point in the novel technique of skin grafting. In vitro cultured epithelial autografts require a minimum of 2–3 weeks for clinical application, and cultured cells contain predominantly keratinocytes as the melanocytes get depleted with serial subcultivation.[8] To overcome the delay and cost, the use of non-cultured autologous cells has been explored. The autologous epidermal cell is an important cell in the reepithelization of cutaneous wounds. Many methods to prepare epidermal cell suspensions exist, and the application methods also differ from one another. Various application strategies have been attempted for delivering keratinocytes onto wounds. They are thought to work by stimulating proliferation and migration of the host epithelium from wound edges through the production of growth factors and other cytokines.[9],[10],[11],[12] Various studies have shown good results of autologous platelet-rich fibrin and non-cultured epidermal cell suspension in the treatment of chronic non-healing ulcers, but there is no study comparing the two procedures.[7],[13],[14],[15],[16],[17],[18]
To the best of our knowledge, this is the first randomised study comparing the two procedures in the treatment of chronic non-healing ulcers. In this study, the majority of chronic ulcers were due to leprosy as leprosy is still endemic in this part of our country. The mean age of the patients' was 40.14 years. The incidence of leprosy peaks in two age groups: 10–15 years and 30–60 years of age.[19] In an Indian study, the majority of the patients were 17–40 years of age. This indicates that the economically active population is the segment most affected by leprosy.[20]
The most common organism isolated was Staphylococcus aureus in 42.8% of the patients and multiple organisms were isolated from 25.7% of the ulcers in this study. In a bacteriological study on plantar ulcers of paucibacillary leprosy, the common infective agents isolated from the ulcer were Staphylococci (53%), Escherichia coli, Proteus sp. and Pseudomonas sp.[21] Polymicrobial growth was seen in 36% of the leprotic ulcers.[21] Swartz and Weinberg reported Staphylococcus aureus and Group A Streptococcus as the most common pathogens isolated from the chronic wound.[22]
In previous studies, PRFM was done weekly,[7],[13],[23],[24] and NCES was performed once[16],[17] in the treatment of non-healing ulcers. PRFM was reported to release growth factors continually for more than 7 to 14 days.[25],[26] Before starting this study, a pilot study was done on five patients in which PRFM was performed every 2 weeks because most of our patients come from distant places, and it is difficult for them to come weekly for follow-up. The results were encouraging then we decided to do the procedure at 2-weeks intervals. In this part of the country, there is a hot and humid climate, so the dressings were kept for a week. The procedure was repeated every 2 weeks, and it showed healing in the majority of the ulcers.
In previous studies on non-healing ulcers, the maximum duration of a study was 1–2 months,[7],[13],[23],[24] and there was partial healing. The duration of this study was 5 months in which we wanted to see the healing of the ulcer and also the recurrence of the ulcer at the same site after complete healing. The area of epithelization and percent healing of ulcers decreased at week 16 as compared to week 12 in the PRFM group because there was an increase in the area of ulcer in one patient [Table 3].
In a prior study, Agubata et al.[27] showed that hydrogels based on ofloxacin, hydroxypropyl methylcellulose and biodegradable microfibers are potential wound dressings with excellent physicochemical qualities and improved healing in wounds. In this study, hydroxy propyl methyl cellulose was used as a vehicle to increase the viscosity of the epidermal cell suspension to prevent runoff, increase contact time, and maintain the right moisture balance in the wound bed and absorb the wound exudates. It is non-adherent and can be easily removed without any damage to the wound bed. It is easily available and less expensive than other vehicles – fibrin and hyaluronic acid matrix.
In a randomised, open-labeled comparative study by Pravin et al.,[23] 30 patients with non-healing ulcers of various causes were treated with PRP versus leucocyte-PRF (L-PRF) at weekly intervals for a maximum of six treatments. The mean durations of healing of the ulcers were 5.7 weeks in L-PRF and 6.5 weeks in PRP (P = 0.034). About 100% resolution was seen in 73.3% of the ulcers in the L-PRF and 53.3% in the PRP at the end of the 6th treatment. In this study, complete healing at the 6th week occurred in 57.9% in the PRFM group and 50% in the NCES group. In a pilot study by Shukla et al.[16] using autologous epidermal cell suspension transplantation in chronic non-healing wounds, 60% of the patients healed completely within 20 weeks, 40% had incomplete but 50%–68% of the healing occurred by week 20. In our study, the majority of ulcers (81.3%) in the NCES group healed completely within 12 weeks, and complete healing occurred in 93.75% of the patients by the end of 5 months.
Most wounds in the elderly can heal but have a slower healing process, and all phases of wound healing are affected.[28] Studies indicate that oestrogen can improve the age-related impairment in healing in both men and women, whereas androgens regulate cutaneous wound healing negatively.[29],[30] In the present study, the rate of healing after the procedure did not vary significantly in relation to age and sex.
Two patients in the NCES group were lost to follow up, one after 1 month of the procedure, but the improvement was present and another after 1 week of procedure. Four patients in the PRFM group discontinued the study, first after 6 weeks, second after 3 months, third after 2 months and fourth after 1 week. Three out of these four patients discontinued the study because of no improvement or incomplete healing, and two of these had a venous ulcer. There was incomplete healing of ulcers in 10.5% of the patients in the PRFM group and 6.3% in the NCES group, and the reasons were mainly repeated trauma and pressure over the ulcer, improper care of anaesthetic foot and secondary infection.
In the present study, there was no significant difference between the two procedures in the treatment of chronic non-healing ulcers. The technique of transplanting non-cultured epidermal cell suspension is complex, requires the expertise of harvesting a thin split-thickness skin graft and epidermal cell separation, is time-consuming, more expensive and requires more equipment as compared to PRFM procedure. PRFM is a feasible, safe, simple, minimally invasive and inexpensive method. It can be done single-handedly, without any downtime, without much training and assistance, with minimum equipment even by beginners. All these advantages make it an affordable option for chronic non-healing wounds of varied causes. On an average, 10 mL of whole blood yields about 2.5 mL of PRFM.[7] In NCES, donor to recipient size ratio of 1:4,[17] which enabled a small single donor site to treat large wound areas. So, NCES may be preferred over PRFM in large ulcers.
Conclusion PRFM and NCES are affordable options for chronic ulcers of varied causes. These cause faster-wound closure. No significant difference was observed between the two procedures in the treatment of chronic non-healing ulcers.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References 
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