Maximizing the safety of glycerol preserved human amniotic membrane as a biological dressing

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Maximizing the safety of glycerol preserved human amniotic membrane as a biological dressing Serag M. Zidan *, Samy A. Eleowa, Mahmoud A. Nasef, Magdy A. Abd-Almoktader, Amr M. Elbatawy, Ahmad G. Borhamy, Mohammad A. Aboliela, Ahmad M. Ali, Mahmoud R. Algamal Department of Plastic Surgery and Burn, Faculty of Medicine, Al-Azhar University, Almokhayyam Aldaem Street, Nasr City, Cairo, Egypt

article info

abstract

Article history:

Introduction: Preservation of human amniotic membrane (HAM) in glycerol 85% has been

Accepted 7 March 2015

used clinically but the use of glycerol 98% can give the maximum virucidal activity and increases the safety of HAM.

Keywords:

Objective: To determine the degree of clinical efficacy of HAM preserved in glycerol 98% as a

Human amniotic membrane

biological dressing in management of donor site of split thickness skin graft (STSG).

Glycerol preservation

Patients and methods: 40 subjects were enrolled in this randomized, controlled study con-

Skin graft donor site

ducted in Al-Azhar University Hospitals from August 2013 to June 2014. We compared HAM preserved in glycerol 98% to vaseline gauze. Patients were randomly allocated to STSG donor site dressing with one of these materials. Outcome measures included pain scores at postoperative days 2, 6 and 10, time to re-epithelialization, and incidence of infection. Results: Both groups were homogenous regarding age, gender, cause of burn and size. The HAM group showed significantly less pain on postoperative days 2 and 6 (4 and 2.7 vs. 5.6 and 4.2 respectively with p value <0.05). Shorter time to re-epithelialization was also found in the HAM group (11.7 vs. 15.4 with p value <0.05). No significant difference was found between both groups in the incidence of infection. Conclusion: HAM preserved in glycerol 98% is clinically effective as a biological dressing. The higher glycerol concentration increases the safety of HAM with retaining the clinical effect at the same time. # 2015 Elsevier Ltd and ISBI. All rights reserved.

1.

Introduction

The use of human amniotic membrane (HAM) in burn and wound management was reported in the literature approximately hundred years ago [1–3]. The object of these early attempts was to achieve durable wound coverage and, although the body ultimately rejected the

membrane, lack of infections and alleviation of pain were noted [4]. In 1952, Duglas reported the use of amniotic membranes to temporarily cover burn wounds [5]. In the following decades, the advantages of amnion as a temporary dressing became more evident [6–10]. Since the early 1990s, there has been an increasing body of literature addressing the use of amnion in chronic wounds

* Corresponding author. Tel.: +20 1020107311. E-mail addresses: [email protected], [email protected] (S.M. Zidan). http://dx.doi.org/10.1016/j.burns.2015.03.009 0305-4179/# 2015 Elsevier Ltd and ISBI. All rights reserved.

Please cite this article in press as: Zidan SM, et al. Maximizing the safety of glycerol preserved human amniotic membrane as a biological dressing. Burns (2015), http://dx.doi.org/10.1016/j.burns.2015.03.009

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and burns. Moreover, a review by Kesting et al. found 31 articles dealing with the use of amnion in burns published in the main international burn journals in the period 1987–2007 [11]. The proven properties of HAM as a biological temporary dressing include promotion of epithelialization and wound healing due its content of growth factors [12,13] along with analgesic effects [14–16]. Dressing of burns with HAM limits fluid and protein loss which reflects on general patient’s outcome [17,18]. When compared with many other dressing materials, HAM had significantly stronger effect in suppression of bacterial proliferation [18–20]. Furthermore, the antigenicity of HAM is very low and was found of no sequence [21]. HAM can also help to reduce scar formation. In experimental study on merino lambs, Fraser et al. found that HAM resulted in reduced scar tissue as assessed histopathologically [22]. There are multiple methods of HAM preservation including; cryopreservation [23], glycerol preservation [24] and freeze–drying (lyophilization) and gamma-irradiation [18,25]. Of these methods, glycerol preservation has the advantage of simplicity and low cost. Glycerol also has antibacterial and antiviral activity giving it additional advantage [26]. The efficacy of antibacterial and antiviral properties of glycerol is directly proportionate to the concentration of glycerol and the initial storage temperature [27,28]. We conducted this study to determine the degree of clinical efficacy of HAM preserved in glycerol 98% as a biological dressing. We compared its effect as to that of the chlorhexidine impregnated vaseline gauze in management of donor site of split thickness skin graft (STSG). The parameters used were rapidity of healing, degree of pain and incidence of infection.

2.

Patients and methods

2.1.

Preparation of HAM

Amniotic membranes were harvested only from placentas delivered by caesarian sections to ensure sterile harvesting conditions. An informed consent is provided by all donors and a blood sample is simultaneously taken so that the donor is screened for human immuno-deficiency virus, hepatitis B and hepatitis C viruses. The membrane was separated from the placenta, washed thoroughly from blood and then kept in povidone iodine 10% for half an hour. The membrane was then washed again with saline and transferred to a sterile sealed container filled with glycerol 98%. The containers were labeled with donation data including the donor’s name, medical recording number, date of harvest and the name of harvesting resident. The HAM containers were kept in room temperature for 1 month to gain the maximum antibacterial and antiviral effects of glycerol. During this month the membranes from donors with positive serology for viral diseases were excluded. After this period, the HAM containers were stored in refrigerator and were ready for use. At the time of dressing, the glycerolized HAM is taken from its container, washed thoroughly with saline. It is then kept in saline for 20 minutes before use to get rid from any glycerol remnants (Fig. 1).

2.2.

Study design

Forty subjects were enrolled in this randomized, controlled clinical trial conducted in Al-Azhar University Hospitals in the period from August 2013 to June 2014. We included patients indicated for split thickness skin grafting due to thermal burns or trauma. We excluded cases with raw areas due to other causes as postinflammatory raw area. We limited the study to patients between ages of 10 and 50 years. We excluded those who had other comorbidities affecting wound healing as well as patients with burns of more than 40% of total body surface area (TBSA) or associated inhalation injuries. Subjects were randomly allocated to one of the two groups according to the method of donor site dressing. Each group is formed of 20 patients. The first group was called the HAM group. Dressing of patients in this group was achieved by covering the donor site with the HAM as a primary dressing. The membrane was then covered with paraffin gauze wrapped with cotton gauze dressing and held by bandage. The second group of patients is called the control group. Dressing of patients in this group was achieved by covering the donor site with chlorhexidine-impregnated paraffin gauze as a primary dressing then covered with cotton gauze dressing and held by bandage. For both groups dressing change was done after 48 hours. We opened the external dressing and then the primary dressing was checked. If adherent, the primary dressing was left undisturbed and if not, it was changed. Afterwards, wound care was done day by day in the same way. Outcome measures included pain scores at postoperative days 2, 6 and 10, time to re-epithelialization, and incidence of infection. Pain score was measured by asking the patient to grade the pain on a scale from 1 to 10. This pain scoring system is valid for children above 9 years old as well as for adults [29]. Donor site was considered infected when there are local signs of infections as sever intolerable pain, surrounding erythema, induration, purulent discharge or bad odor. Results of the study were assessed for significance using the independent t-test and the Chi-square test. The independent t-test was used to compare continuous variables among groups of patients. The Chi-square test was used to compare nominal variables. All p values were considered significant if less than 0.05.

3.

Results

Both groups were homogenous regarding age (27  13 in HAM group and 23  10 in control group) with p value >0.05 using independent t-test. Gender distribution and cause of burn had no statistically significant difference between the groups with p value >0.05 using Chi-square test. The difference in the size of the open area, presented as percent of total body surface area, was not statistically significant too (7.4  2.6% TBSA in HAM group and 6.1  2.6% TBSA in control group) with p value >0.05 using independent t-test.

Please cite this article in press as: Zidan SM, et al. Maximizing the safety of glycerol preserved human amniotic membrane as a biological dressing. Burns (2015), http://dx.doi.org/10.1016/j.burns.2015.03.009

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Fig. 1 – Amniotic membrane while being separated from the placenta (upper right). A 5 months preserved amniotic membrane after washing with saline showing easy handling (upper left). A large sheet of amnion easily spread on the wound without being torn or fragmented (lower).

The HAM group shows significantly lower pain scores in postoperative days 2 and 6 (4  0.8 and 2.7  0.9 vs. 5.6  1 and 4.2  1.2 respectively) with p values <0.05 using independent ttest. Although the pain score was less in the HAM group in the 10th postoperative day (Fig. 2A), this was not statistically significant (1.8  0.9 vs. 2.3  0.3) with p value >0.05 using independent t-test. Time to re-epithelialization was found significantly shorter in the HAM group (11.7  2.4 days) than in control group (15.4  3.7 days) with p value <0.05 using independent t-test (Figs. 2B and 3). Donor site infection occurred in 2 patients in HAM group and 3 patients in control group (Fig. 2C). This difference was found statistically insignificant with p value >0.05 using chisquare test. All infected patients were treated conservatively and eventually healing occurred. The results are summarized in Table 1.

4.

Discussion

HAM lies in the middle of the spectrum of wound dressings. It was proved better than antimicrobial dressings and some

synthetic dressings in many aspects of wound care [16,18,30]. On the other hand, Biobrane witch is a synthetic biological dressing, had comparable effects with HAM; however, preserved human skin was the most effective [11]. Moreover, the most appealing feature of HAM as a biological dressing is its abundance, low cost and ease of harvesting [31]. As fresh amnion has a short shelf life and it may not always be available when needed, various preservation methods have been introduced [32]. Of these preservation methods, we chose glycerol preservation. The concentration of glycerol described in literature for HAM preservation was 85% [11]. HAM preserved that way has been tested experimentally [4] and in management of partial thickness burns with good results [24]. However, we used a higher concentration, glycerol 98%, that was not used before in literature for preservation of HAM. Marshal et al. tested the antiviral activity of different concentrations of glycerol on intracellular viral survival. They found that glycerol 98% inactivated both enveloped and nonenveloped intracellular viruses in the same circumstances at which glycerol 85% did not inactivate the non-enveloped intracellular virus tested [27]. The risk of disease transmission is a possible drawback of using human-derived biological dressings. All measures

Please cite this article in press as: Zidan SM, et al. Maximizing the safety of glycerol preserved human amniotic membrane as a biological dressing. Burns (2015), http://dx.doi.org/10.1016/j.burns.2015.03.009

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Fig. 2 – Summary of the study outcome (A) pain scores in postoperative days 2, 6 and 10 in both groups. (B) Days to reepithelialization in both groups. (C) Infection rate in both groups.

should be taken to minimize that risk. The fact that there is a risk of disease transmission with glycerol 85% preservation, even very low, can make any one change to other safer methods of preservation (e.g. irradiation). Glycerolization, however, is

simpler and more economic and the presence of a concentration of glycerol that can eliminate this risk is very fortunate. We chose the donor site management as the test field for this technique in preservation for more than one reason. First, the donor site wound is surgically created, more standardized and is considered very homogenous wound healing model

Table 1 – Demographics, clinical characteristics, and outcomes of the HAM and control groups. HAM group

P value NS NS

Age in years Sex (male/female)

27  12.5 11/9

23  9.9 14/6

Cause of raw area Burn Other trauma

16 4

17 3

7.4  2.6

6.1  2.6

NS

4  0.8 2.7  0.9 1.8  0.9 11.7  2.4 10%

5.6  1 4.2  1.2 2.3  0.3 15.4  3.7 15%

<0.05 <0.05 NS <0.05 NS

Size of raw area (percent of TBSA)

Fig. 3 – Wound healing after 12 days (upper). The lower photo shows another case with amniotic membrane that was still adherent to the wound after healing of most of it after 10 days.

Control group

Pain score Day 2 postoperatively Day 6 postoperatively Day 10 postoperatively Days to re-epithelialization Infection rate

NS

NS, not significant; TBSA, total body surface area.

Please cite this article in press as: Zidan SM, et al. Maximizing the safety of glycerol preserved human amniotic membrane as a biological dressing. Burns (2015), http://dx.doi.org/10.1016/j.burns.2015.03.009

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[33]. Second reason was that several kinds of dressings are being used in donor site care, but which is superior has not been determined [34]. Moreover, pain control is a well approved feature in using HAM as a dressing. This advantage is very important in dressing of a donor site due to its highly painful nature [35]. During harvesting and preparation of HAM we strictly followed the standard protocols of using such human allograft. We harvested HAM only from caesarian sections of screened donors who provided written consent for donation. All steps of preparation and use were carried out under sterile conditions and only by assigned plastic surgery residents. This is together with strict recording and reporting system. Pain relief in donor sites treated by HAM processed by other techniques has been proved in multiple studies [36–38]. In contrary, Bari et al. observed sever pain in five donor sites covered by oven dried gamma irradiated HAM. The results of our study support the assumption of analgesic effect of HAM on donor site. We found HAM preserved in glycerol 98% to be more effective in pain relief than conventional dressing using paraffin gauze. We used pain scoring system on a scale from 1 to 10. The score was significantly lower in the study group in the 2nd and 6th postoperative days. Although the difference in 10th day was no significant, the mean pain score in the HAM group remained lower. Multiple studies have shown that HAM is accompanied by rapid re-epithelialisation and promotion of wound healing by inhibition of leukocyte protease activity and angiogenesis stimulation [39]. The promotion of epithelialisation is also due to high concentration of growth factors in stromal and epithelial amnion regions [12]. HAM improved healing rates of burn wounds, donor site wounds and even non-healing ulcers of different etiologies [40]. In our study we found significantly higher rate of donor site healing treated by HAM than conventional dressing. This finding helps proving the clinical advantage of HAM even with preservation in a very high concentration of glycerol. Rapid healing can be primarily attributed to the epithelialisation promoting effect of HAM rather than its antimicrobial activity as the incidence of infection was not significantly different between both groups. There are multiple dressing techniques for donor site management discussed in literature. The main concerns are usually the rapidity of healing and pain relief. However, the cost of the dressing is of matter in developing countries and should be put in consideration when choosing a dressing technique. Demirtas et al. compared five types of dressing materials used in donor site management. The cost of treatment ranged from $0.014 to $0.155 for each square cm2 treated [35]. The amniotic membrane from one placenta measuring 400–500 cm2 costs only about $3. Thus, the cost of treatment with HAM is less than the cheapest one in spite of its advantages of healing promotion and pain relief. Contrary to that reported by Bujang-Safawi et al. [32], we found HAM preserved in glycerol 98% easier in use than dried irradiated HAM in our experience. It has the same ease of handling and use as the fresh amnion (Fig. 1). It is washed easily with saline; once it is free from glycerol remnants, it can be spread easily on the wound without being torn or fragmented.

5

This study has shown that HAM preserved in glycerol 98% was better than conventional method in donor site dressing as regards the rate of wound healing and pain control. Its use can be extended to other indications specially dressing of partial thickness burns. Based on these results, we recommend using glycerol 98% as a preservative medium for HAM banking particularly that based on hospital level.

5.

Conclusion

Human amniotic membrane is an excellent natural temporary skin substitute. Preservation of amniotic membrane in glycerol 98% minimizes the risk of disease transmission by utilizing the strong antiviral and antibacterial activity of such concentration. Its clinical efficacy is maintained as regards healing promotion and pain relief effects. The economic value and ease of preparation remains highly appealing advantages especially in developing countries.

Conflict of interest The authors deny any financial or personal relationships with other people or organizations that could inappropriately influence that work.

Acknowledgments The authors acknowledge all the staff members of Al-Azhar university hospitals who had an active share in this work. Our gratitude for Dr. Maged Almofty, professor of plastic surgery; Al-Azhar University, for his valuable help and advice.

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