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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 26  |  Issue : 1  |  Page : 66-71

Can removal of epidermis delay rejection of cadaveric dermis-only allograft? A proof of concept study


1 Department of Surgery, Plastic Surgery Unit, NSCB Government Medical College, Jabalpur, Madhya Pradesh, India
2 Department of Surgery, NSCB Government Medical College, Jabalpur, Madhya Pradesh, India
3 Department of Pathology, NSCB Government Medical College, Jabalpur, Madhya Pradesh, India

Date of Web Publication11-Mar-2019

Correspondence Address:
Prof. Pawan Agarwal
292/293, Napier Town, Jabalpur - 482 003, Madhya Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijb.ijb_14_18

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  Abstract 


Background: Skin allograft, used as a biological dressing, is usually rejected after 2 weeks. Epidermis contains immunogenic cells and its removal reduces antigenicity of allograft and delays its rejection.
Materials and Methods: This proof of concept observational study was conducted in 50 patients of deep burns and posttraumatic wounds to evaluate the biological response of cadaveric glycerol-preserved dermis-only allograft. The epidermis was removed surgically, and the endpoints of healing or graft rejection were compared with historical controls of full-thickness allografts.
Results: Dermis-only allograft take was seen in 82% patients at 20 days; in 34% patients at 4 weeks and in 16% patients at 8 weeks.
Conclusion: Surgical removal of epidermis from cadaver allograft reduces its antigenicity and enables its longer survival.

Keywords: Burn, cadaveric, epidermis depleted allograft, wound


How to cite this article:
Agarwal P, Sharma D, Agarwal A, Patel L K. Can removal of epidermis delay rejection of cadaveric dermis-only allograft? A proof of concept study. Indian J Burns 2018;26:66-71

How to cite this URL:
Agarwal P, Sharma D, Agarwal A, Patel L K. Can removal of epidermis delay rejection of cadaveric dermis-only allograft? A proof of concept study. Indian J Burns [serial online] 2018 [cited 2019 May 25];26:66-71. Available from: http://www.ijburns.com/text.asp?2018/26/1/66/253843




  Introduction Top


Autologus skin graft is the gold standard for coverage of burn injury wounds. However, if patient's own skin is not available for any reason, use of skin allograft is the best alternative as a temporary cover for the burn wounds.[1],[2],[3],[4] The use of allograft skin – as a permanent skin transplant – is limited by its immunogenic properties; which lead to its rejection around 2 weeks. This immunogenic response is directed primarily against the Langerhans cells (LC) of the epidermis and dendritic cells (DC) of the dermis.[5],[6] Medawar showed that long-term survival of allograft is possible if antigenic components of allograft are reduced by removing epidermis as it renders the remaining dermal component less immunogenic.[7] This hypothesis was investigated in the present proof of concept study by assessing the take/survival of epidermis-depleted glycerol-preserved dermis-only allograft in deep burns and posttraumatic wounds.


  Materials and Methods Top


This prospective “proof of concept observational” study was conducted in burn and plastic surgery unit in a tertiary referral center in Central India over a period of 1 year. Institutional Ethical Committee Approval and written/informed consent from all the patients were obtained. We certify that we 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.

A total of 50 consecutive patients with deep second-/third-degree thermal burn raw area and posttraumatic full thickness raw area were included in this study. Posttraumatic patients with compound fracture and those need flap were excluded from the study. Patients with scalds, electrical, and chemical burns were also excluded from the study. All the patients who died with malignancy, jaundice, sepsis, and poisoning were excluded as cadaveric donors.

Skin graft was harvested from cadavers within 24 h after death with usual operating room technique. First epidermis was removed by Humby's knife ensuring complete removal till glistening white dermis was exposed. And then, medium thickness (0. 012”–0. 018” or 0. 3–0. 45 mm) split thickness dermal graft was harvested.[8] The sheet of epidermis was discarded. Immediately after harvesting, the grafts were immersed in 85% glycerol solution for a period of 2 h at 4°C; then graft sheets were laid on sterile wet gauze piece and packed in presterilized polythene bags. These polythene bags were stored in deep freezer at −4°C for maximum of 3 weeks. Detailed records of all the allograft beginning from harvesting to application on recipient were kept. A blood sample was taken by femoral puncture from all the cadavers and sent for HIV, hepatitis B surface antigen, and Venereal Disease Research Laboratory. In case of positive sample, allograft from that cadaver was discarded. A small piece of allograft was sent for qualitative bacterial culture before its application and if culture was found positive, this allograft was discarded. Before applying, allograft was washed thoroughly with normal saline to remove glycerol.

All patients with deep second-/third-degree thermal burn were analyzed regarding total body surface area (TBSA) and depth of burn and treated with standard burn treatment protocol by initial resuscitation, antibiotics; wound care, and nutritional support according to the American Burn Association Practice Guidelines.[9]

Burn wounds were treated with daily dressing using silver sulfadiazine cream till healthy granulation tissue appeared on the bed. Posttraumatic wounds were treated by debridement, antibiotics, and wound care by daily dressing using normal saline till healthy granulation tissue appeared on the bed.

Once the wounds get granulated glycerol treated cryopreserved dermis-only allo-graft sheet were applied under full aseptic precautions under general/regional anesthesia using standard skin grafting technique. All patients were given a combination of cephalosporin and amikacin postoperatively. The assessment of biological response of allograft was done clinically, histologically, and bacteriologically. Clinical assessment for graft take/loss was done on 5th, 7th, 10th, 20th postoperative days and up to 2 months by two independent observers (consultant plastic surgeons) by simple clinical observation and photographs was taken. Percentage of allograft take/loss, presence of infection, and duration of wound healing were noted. Complete wound healing or detachment of allograft was considered as endpoint of the study.

Punch biopsy was taken from the allograft edge on 14th day, fixed and stained in hematoxylin and eosin and examine under microscope with ×400 magnification. Pus was sent for culture and sensitivity to detect micro-organisms on 5th day, no quantitative culture was done. The endpoint was healing or graft rejection. The outcome was compared with historical controls.


  Results Top


A total of 50 patients (25 patients of deep second-/third-degree burn and 25 patients of posttraumatic wounds) were included in the study. The youngest patient was 8 years old and oldest was 65 years old (mean age 27.02 years). Thirty-two (64%) patients were females and 18 (36%) were males. The mean TBSA of burn was 33.65% (range 10–50%). The size of the wound ranges from 4 cm × 3 cm to 20 cm × 15 cm. Glycerol-treated cryopreserved cadaver dermis-only allografts [Figure 1] were applied to the granulating wounds [Figure 2] and [Figure 3] in all 50 patients. It was applied on the chest in 3 (6%) patients, back in 1 (2%) patient, abdomen in 1 (2%) patient, arm in 1 (2%) patients, forearm in 6 (12%) patients, thigh in 15 (30%) patients, leg in 9 (18%) patients, and foot in 14 (28%) patients. On 5th postoperative day, all grafts were pale without swelling or exudates [Figure 4]. On 7th postoperative day, 46 (92%) dermal allograft turned to pink suggesting neovascularization [Figure 5]. On 10th postoperative day, dermal allograft remained pink in 44 (88%) patients. In 6 patients (12%), allograft became hard, leathery, and was rejected on 10th day. On 20th postoperative day, totally 9 patients (18%) had their graft rejected while in 41 (82%) patients skin graft stayed on the wound bed. At the end of 4 weeks, the grafts were healthy and stuck to bed in 17 (34%) [Figure 6] patients while in 33 patients grafts were necrosed and rejected. At the end of 8 weeks in 8 (16%) patients graft was still stuck to bed while in 42 patients, graft was detached from their bed and wound was completely healed [Table 1].
Figure 1: Dermis-only allograft

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Figure 2: Posttraumatic raw area leg

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Figure 3: Dermis-only allograft applied day-0

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Figure 4: Dermis-only allograft- day-5

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Figure 5: Dermis-only allograft- day 7

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Figure 6: Dermis-only allograft- 4-week postoperative

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Table 1: Number of patients showing the duration of allograft take and rejection

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Histopathological evaluation on 14th postoperative day showed infiltration of mononuclear cells and fibroblasts with raised level of collagen in all survived grafts. Angiogenesis was seen at the junction of graft and bed [Figure 7]. Rejected graft showed apoptosis, foreign body giant cell reaction, and irregular collagen formation and degenerative changes in vessel wall [Figure 8].
Figure 7: Histopathology showing take of allograft- neovascularization and collagen formation

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Figure 8: Histopathology showing allograft rejection -apoptosis, foreign body giant cell reaction, and irregular collagen formation

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Pus culture examination on 5th day showed bacterial growth in 18 patients (36%) while in 32 (64%) patient's pus culture was sterile. Staphylococcus and Pseudomonas were present in the majority of the cases.


  Discussion Top


Allografts and skin banks are increasingly playing a critical role in the management of burns injury.[2],[10],[11],[12] When viable allograft is transplanted on a healthy recipient, rejection of the graft occurs within 2 weeks.[1],[13],[14],[15],[16]

This graft rejection is mediated by the activation of T cells which is directed primarily against the LC of epidermis and DC of dermis. The LCs and DCs are both potent antigenic and initiate the rejection by migrating from the skin graft to the draining lymph node of the recipient where they activate the T cells.[5],[17] Treatment of allograft with glycerol results in cell death; therefore, after transplantation, the migration of LC and DC cannot occur to draining lymph nodes preventing its rejection. The survival of glycerol treated allograft is longer but not permanent. There are two mechanisms which initiate rejection of dead allograft. First, the host T cells which infiltrate the graft recognize the antigenic cells which are present in allograft and initiate rejection. Second, allograft skin cells deliver immunogenic molecules after transplantation which is taken by antigen processing cells of host and initiate immune response leading to its rejection. Therefore, even the dead allograft may initiate rejection. Efforts to prevent rejection have included methods that might reduce antigen expression by controlling LC activity in the allograft skin. Treatment of the allografts with ultraviolet light irradiation, incubation of the skin in glucocorticoids and using cultured epidermal allografts – which lack LCs and DCs – have been used.[18],[19]

Better understanding of immunology of skin transplantation has led to the realization that reducing the immunogenicity of allograft by removing the epidermis delays the rejection; which then takes place through indirect route mediated by macrophages.[6],[20] First attempt at removal of epidermis to reduce antigenicity of an allograft – and thereby delay its rejection - was made in 1986.[21],[22]

Four methods have been described for the separation of epidermis from underlying dermis: Heating, enzymatic digestion, chemicals, and surgical separation. Heat (50°C) causes some denaturation of protein of cells.[23] Enzymatic digestion can be done with trypsin/dispase which causes detachment of the epidermis from dermis. Epidermis can be separated from the underlying dermis by treatment of the skin with chemicals such as iso-osmotic ammonium hydroxide, acetic acid (1.5%; volume per volume), or sodium thiocyanate. These solutions disturb the electrolyte equilibrium of the cells which cannot be restored even with washing with saline.[24],[25],[26] All these methods have their own limitations and are complicated, therefore, we have used surgical separation of epidermis from dermis. It is a simple and quick way of removing epidermis and lowering the immunogenic potential of the allograft. This is the first study using surgical removal of the epidermis and using dermis-only allografts in humans.

Nearly 34% patients showed allograft adherence at 4 weeks and 16% had allograft adherence at 8 weeks. Overall, the duration of dermal allograft adherence to the wound bed in our study was on average 24.8 days which is better than other studies using full thickness skin allografts [Table 2]. Allografts when applied serve as a temporary cover on freshly excised/granulating wounds; as an overlay on widely expanded autografts; promote reepithelialization, provide dermal template for epidermal grafts, and improve the quality of the wound bed before autografting.[27],[28],[29],[30],[31] Extending these advantages by an additional 10–14 days is a profitable trade-off.
Table 2: Comparison with historical studies showing duration of allografts adherence

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We have used the allograft in small wounds which can be treated by autografts, as the biological response of allograft can be assessed easily on these wounds and small wounds get healed completely even when allograft got rejected. Only 9 patients (18%) whose allograft rejected within 20 days required secondary surgery in the form of autografting. Our results are much better than those obtained by Peeters et al.[32] (31% patients required secondary surgery) and Brans et al.[33] (53% required secondary surgery out of 45 patients).

None of the preserved allografts showed positive bacterial culture when tested before application as preservation and storage of cadaver skin in 85% glycerol has very strong antimicrobial and virucidal effects. Our study showed absence of postoperative infection in 32 (64%) patients because glycerol served as virucidal and antibacterial agent.[34],[35] We also confirmed the clinical observation of take by histological evaluation. Dermis-only allograft finally detached from the bed when the underlying wound had reepithelialized or because of superimposed infection which accelerated the detachment.[36]


  Conclusion Top


This proof of concept observational study shows that surgical removal of epidermis from cadaver allograft enables its long-term survival.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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7.
Medawar PB. A second study of the behaviour and fate of skin homografts in rabbits: A report to the war wounds committee of the medical research council. J Anat 1945;79:157-74.  Back to cited text no. 7
    
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Gupta DK. Thin and Ultra Thin Split Thickness Skin Grafts (STSG-UT, STSG-T). In: Microskin Grafting for Vitiligo. London: Springer; 2009.  Back to cited text no. 8
    
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Blome-Eberwein S, Jester A, Kuentscher M, Raff T, Germann G, Pelzer M, et al. Clinical practice of glycerol preserved allograft skin coverage. Burns 2002;28 Suppl 1:S10-12.  Back to cited text no. 10
    
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Gore MA, De AS. Deceased donor skin allograft banking: Response and utilization. Indian J Plast Surg 2010;43:S114-20.  Back to cited text no. 12
    
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Khoo TL, Halim AS, Saad AZ, Dorai AA. The application of glycerol-preserved skin allograft in the treatment of burn injuries: An analysis based on indications. Burns 2010;36:897-904.  Back to cited text no. 13
    
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Burd A, Lam PK, Lau H. Allogenic skin: Transplant or dressing? Burns 2002;28:358-66.  Back to cited text no. 14
    
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Eldad A, Din A, Weinberg A, Neuman A, Lipton H, Ben-Bassat H, et al. Cryopreserved cadaveric allografts for treatment of unexcised partial thickness flame burns: Clinical experience with 12 patients. Burns 1997;23:608-14.  Back to cited text no. 15
    
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Mat Saad AZ, Khoo TL, Dorai AA, Halim AS. The versatility of a glycerol-preserved skin allograft as an adjunctive treatment to free flap reconstruction. Indian J Plast Surg 2009;42:94-9.  Back to cited text no. 16
    
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Larsen CP, Steinman RM, Witmer-Pack M, Hankins DF, Morris PJ, Austyn JM, et al. Migration and maturation of Langerhans cells in skin transplants and explants. J Exp Med 1990;172:1483-93.  Back to cited text no. 17
    
18.
Odling KA, Halliday GM, Muller HK. Effects of low or high doses of short wavelength ultraviolet light (UVB) on Langerhans cells and skin allograft survival. Immunol Cell Biol 1987;65:337-43.  Back to cited text no. 18
    
19.
Calota DR, Nitescu C, Florescu IP, Lascar I. Surgical management of extensive burns treatment using allografts. J Med Life 2012;5:486-90.  Back to cited text no. 19
    
20.
Richters CD, Hoekstra MJ, van Baare J, du Pont JS, Kamperdijk EW. Morphology of glycerol-preserved human cadaver skin. Burns 1996;22:113-6.  Back to cited text no. 20
    
21.
Cuono C, Langdon R, McGuire J. Use of cultured epidermal autografts and dermal allografts as skin replacement after burn injury. Lancet 1986;1:1123-4.  Back to cited text no. 21
    
22.
Cuono CB, Langdon R, Birchall N, Barttelbort S, McGuire J. Composite autologous-allogeneic skin replacement: Development and clinical application. Plast Reconstr Surg 1987;80:626-37.  Back to cited text no. 22
    
23.
Baumberger JP, Suntzeff V, Cowdry EV. Methods for the separation of epidermis from dermis and some physiologic and chemical properties of isolated epidermis. J Natl Cancer Inst 1942;2:413-23.  Back to cited text no. 23
    
24.
Macdiarmid J., Wilson JB. Separation of epidermal tissue from underlying dermis and primary keratinocyte culture. In: Wilson JB, May GH, editors. Epstein-Barr Virus Protocols. Methods in Molecular Biology™. Vol. 174. Totowa, NJ: Humana Press; 2001.  Back to cited text no. 24
    
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Lawson TA, Pound AW. A new method for separation of the epidermis and dermis. Biochem Med 1975;12:162-5.  Back to cited text no. 25
    
26.
Diaz LA, Heaphy MR, Calvanico NJ, Tomasi TB, Jordon RE. Separation of epidermis from dermis with sodium thiocyanate. J Invest Dermatol 1977;68:36-8.  Back to cited text no. 26
    
27.
Wong L. The many uses of allograft skin. Ostomy Wound Manage 1995;41:36-8, 40-2.  Back to cited text no. 27
    
28.
Mackie D. Postal survey on the use of glycerol-preserved allografts in clinical practice. Burns 2002;28 Suppl 1:S40-4.  Back to cited text no. 28
    
29.
Moerman E, Middelkoop E, Mackie D, Groenevelt F. The temporary use of allograft for complicated wounds in plastic surgery. Burns 2002;28 Suppl 1:S13-5.  Back to cited text no. 29
    
30.
Tzeng YS, Chen SG, Dai NT, Fu JP, Chang SC, Deng SC, et al. Clinical experience using cadaveric skin for wound closure in Taiwan. Wounds 2012;24:293-8.  Back to cited text no. 30
    
31.
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32.
Peeters R, De Caluwe D, Neetens C, Hubens A. Use of glycerolized cadaver skin for the treatment of scalds in children. Burns 1994;20 Suppl 1:S32-3.  Back to cited text no. 32
    
33.
Brans TA, Hoekstra MJ, Vloemans AF, Kreis RW. Long-term results of treatment of scalds in children with glycerol-preserved allografts. Burns 1994;20 Suppl 1:S10-3.  Back to cited text no. 33
    
34.
Marshall L, Ghosh MM, Boyce SG, MacNeil S, Freedlander E, Kudesia G, et al. Effect of glycerol on intracellular virus survival: Implications for the clinical use of glycerol-preserved cadaver skin. Burns 1995;21:356-61.  Back to cited text no. 34
    
35.
Saegeman VS, Ectors NL, Lismont D, Verduyckt B, Verhaegen J. Short- and long-term bacterial inhibiting effect of high concentrations of glycerol used in the preservation of skin allografts. Burns 2008;34:205-11.  Back to cited text no. 35
    
36.
Blome-Eberwein S, Jester A, Kuentscher M, Raff T, Germann G, Pelzer M, et al. Clinical practice of glycerol preserved allograft skin coverage. Burns 2002;28 Suppl 1:S10-2.  Back to cited text no. 36
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

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