Indian Journal of Burns

ORIGINAL ARTICLE
Year
: 2016  |  Volume : 24  |  Issue : 1  |  Page : 13--17

Assessment of biological response of lyophilized cadaveric skin allograft in post burn raw area and nonhealing ulcers


Pawan Agarwal, Anoop Jain, Dhananjaya Sharma 
 Department of Surgery, Plastic Surgery Unit, Netaji Subhash Chandra Bose Government Medical College, Jabalpur, Madhya Pradesh, India

Correspondence Address:
Pawan Agarwal
292/293 Napier Town, Jabalpur - 482 001, Madhya Pradesh
India

Abstract

Introduction: Extensive full-thickness burns and major injuries resulting in extensive damage to the skin make spontaneous regeneration difficult and compromise patient survival. When a donor area is available, it is always preferable to proceed with autograft, which is considered to be the best option. However, if the affected region is extensive, donor area is not enough, or when medical condition of the patient does not permit immediate grafting with autologous skin; in such conditions lyophilized skin allograft can be used for temporary coverage of the wounds. Materials and Methods: In this prospective study, evaluation of biological response of lyophilized cadaveric skin allografting was done in 100 cases of thermal burn and nohealing ulcers. Results: In this study, 19% cases lyophilized skin grafts got rejected in 3 weeks, 65% in 1 month, and in 16% patients graft got rejected at the end of 2 months. Rejection is defined as when skin graft becomes hard and starts separating from wound edges and is finally totally detached from the bed. Out of 100 patients, wounds healed completely in 81 patients, while 19 patients required autografting. On pus culture examination out of 100 patients, 26% patients showed the presence of infection. Histopathological study was done on the 21st postoperative day and all those who survived skin graft showed infiltration of mononuclear cells, fibroblasts, keratinization, uniformly arranged collagen bundles, and angiogenesis at the junction of graft and graft bed. All grafts that were rejected, showed acanthosis, spongiosis, and degenerative changes in vascular walls. Neovascularization was not observed in these grafts. Conclusion: Lyophilized cadaveric allograft acts as mechanical and physiological barrier and the process of lyophilization helps in minimizing the immunoreactivity of the graft, therefore, rejection is not rapid. Lyophilized cadaveric allografts can be lifesaving in cases of severe burn.



How to cite this article:
Agarwal P, Jain A, Sharma D. Assessment of biological response of lyophilized cadaveric skin allograft in post burn raw area and nonhealing ulcers.Indian J Burns 2016;24:13-17


How to cite this URL:
Agarwal P, Jain A, Sharma D. Assessment of biological response of lyophilized cadaveric skin allograft in post burn raw area and nonhealing ulcers. Indian J Burns [serial online] 2016 [cited 2017 Sep 21 ];24:13-17
Available from: http://www.ijburns.com/text.asp?2016/24/1/13/195526


Full Text

 Introduction



Extensive full-thickness burns and major injuries resulting in extensive damage to the skin make spontaneous regeneration difficult and compromise patient survival. It additionally necessitates immediate coverage to restore normal skin function. When a donor area is available, it is always preferable to proceed with autograft, which is considered to be the best option. However, if the affected region is extensive, donor area is not enough or when the medical condition of the patient does not permit immediate grafting with autologous skin, in such conditions lyophilized skin allograft can be used for temporary coverage of the wounds and it is considered as mainstay in the treatment of burns.[1]

Human split-thickness skin allograft can be prepared and stored by various methods. Processing methods used for banking of skin for subsequent therapeutic use depend on whether the skin is to retain viability or not. For viable skin grafts, sterilization techniques cannot be applied; however, antibiotics and antimycotics may be used to disinfect the tissue with respect to bacteria and fungi. Cryopreservation is the preferred method for long-term storage of viable skin grafts. If viability is not required, then additional long-term preservation methods may be used including deep-freezing, freeze-drying, or high concentration solute preservation. All three methods work by reducing water activity.[2] The “nonviable” skin allograft can be incorporated into the wound and make an appropriate dermal bed for further application of autologous skin grafts.[3]

The use of allograft skin as a permanent skin transplant is limited by immunogenic properties. Although, allograft skin initially takes on full thickness wound but ultimately it gets rejected. This immunogenic response from the skin is directed primarily against the Langerhans cells of the epidermis but studies shows that lyophilized allogenic cadaveric skin does not induce the detectable host sensitivity and appears to be immunologically inert.[4]

Lyophilization (freeze-drying) is considered as a logical choice for long-term storage of nonviable tissues. This process yields strict preservation of tissue structure. This is accomplished by first freezing, which rigidly holds internal structures in constant relationship with one another, followed by evacuation of almost all the free water by the physiochemical process of sublimation. Sublimation, which is enhanced in a vacuum, is the transfer of water from the frozen state into vapor without passing through a liquid phase. This phenomenon of water transfer allows the retention of tissue integrity. After freeze-drying, the solid structures of the tissue remain in an undisturbed relationship with one another, resembling a honeycomb, because the water has been removed.[5] If 95% or more of the tissue water is removed, sufficient arrest of biochemical process occurs which allows for safe, long-term storage at room temperature.[6],[7]

This study was conducted to identify the clinical success rate, infection, and rejection of cadaveric lyophilized skin allograft in thermal burns and nonhealing ulcers.

 Materials and Methods



This study was conducted in the plastic surgery unit, Department of Surgery, Netaji Subhash Chandra Bose (NSCB) Medical College, Jabalpur, Madhya Pradesh, India, over a period of 2 years. In this prospective study, evaluation of biological response of lyophilized cadaveric skin allografting was done in cases of thermal burn and nonhealing ulcers. Before commencing the study, approval of the institutional (NSCB Government Medical College, Jabalpur, Madhya Pradesh, India) ethics committee was taken. Skin grafts were harvested from cadavers within 24 h after death. All the patients who died with malignancy, jaundice, sepsis, and poisoning were excluded. Skin grafts were harvested with usual sterile operating room technique. Skin grafts were taken from the thigh, arm, and forearm. Strict records of all the allograft beginning from harvesting to application on recipient were kept. Blood samples were taken by femoral puncture from all the cadavers and sent for human immunodeficiency virus (HIV), HbSAg, and venereal disease research laboratory (VDRL). In case of positive sample, all the allografts from that cadaver were discarded.

After harvesting the graft, it was kept in normal saline, ciprofloxacin, and metronidazole solution. The grafts were lyophilized within 6 h of allograft harvest. Lyophilization of skin graft was done in FD-3 lyophilizer system. (Shanghai Bilon Instrument Company, Limited. located in Shanghai, China) This mobile small lyophilizer is most widely used because of its low cost, self-contained portable unit. This unit consists of a two-tiered lab cart with a pump and a trap on the lower tier. A rubber vacuum tubing then connects the lower trap to a manifold that is mounted on the upper level. The larger manifold suggested by the manufacturer has 12 ports with maximum capacity of 100 mL in each port. On the side of the unit is McLeod Gauge for checking vacuum in the whole system. The inner trap and the lower trap both have a capacity of 2.8 L, which will maintain temperature for up to 12 h. In this system, initial pre-lyophilization freezing was done at −10°C for 24 h, then rapid freezing was done at up to −65°C for 8-12 h. After lyophilization, grafts were stored at room temperature in a sterile container. Just before grafting, the lyophilized skin was soaked in normal saline solution for 20 min at 37°C.

Recipient sites were prepared by cleaning with normal saline and dressed using silver sulphadiazine cream every day till healthy granulation tissues appeared on the bed. Depth of wound was mixed partial thickness, and full thickness in majority of cases. All patients were given broad spectrum antibiotics (combination of cephalosporin and Amikacin) preoperatively and grafting was done under full aseptic precautions. Wound margin were infiltrated with 2% Xylocaine and 10% Xylocaine was sprayed over the wound gave good anesthesia. After removal of remaining necrotic tissue, grafts were applied on the granulating bed and fixed with the help of staplers. The wounds were dressed with medicated paraffin gauge and betadine soaked gauze pieces. Postoperative adjacent joints were immobilized using a plaster of Paris slab. Postoperatively, broad spectrum antibiotics were continued. The assessment of biological response of lyophilized allograft was done clinically on the basis of color, swelling, exudation, texture, and take of graft on the 5th, 10th, 14th, and 21st postoperative days. Assessment of graft take or rejection was done by two qualified plastic surgeons working in the same unit. Pus culture was taken on the 5th postoperative day and biopsy from the edge of the grafted area was taken on the 21st day. End point of the study was complete wound healing or when their allograft got rejected from the wound bed.

 Results



100 patients of post burn raw area and nonhealing ulcers were included in this study. The evaluation of biological response of lyophilized cadaveric allograft in the form of clinical response of allograft, take of the graft and final healing of wounds was done. Out of 100 patients, 62 were male and 38 were female. According to etiology there were 58 patients having post thermal burn raw area and 42 patients of nonhealing ulcers. Out of 42 nonhealing ulcer patients, 30 were posttraumatic and 12 were following infection [Figure 1]. The youngest patient in the study was a 5-year-old female and the oldest patient was a 60-year-old male. Cadaveric skin allografts were applied on the different sites of body such as the chest in six cases, the back in two cases, the abdomen in two cases, the arm in six cases, the forearm in eight cases, the thigh in 30 cases, the legs in 18 cases, and foot in 28 cases. Size of the raw area ranges from 4 cm × 5 cm to 20 cm × 14 cm with the average size being 53.38 cm 2.{Figure 1}

On clinical examination, grafts were pale in color till the 10th day in all cases, suggesting ischemia and avascularity and there was no swelling and exudation. On the 14th postoperative day, the color of skin allograft turned to pink and there was some swelling in all patients suggesting neovascularization [Figure 2]. On the 21st postoperative day, the color of skin allograft remained pink in 81 (81%) [Figure 3] patients, while in 19 patients the graft was totally detached from the bed leading to raw area [Graph 1]. Graft take on the 21st day was more than 90% in 32 cases, 80-90% in 30 cases, 70-80% in 19 cases, and total loss was present in 19 cases. In our study, epidermis was started to be separated on the 5th postoperative day and on the 10th day 28% of the patient's epidermis was separated from the dermis. In the remaining 72 patients, the epidermis got separated on the 14th day. The consistency of applied skin graft was assessed by palpation. On the 10th postoperative day, all the allografts were soft in consistency. On the 14th postoperative day the consistency was still soft in 81 patients whereas in 19 patients the consistency changed to hard and on the 21st postoperative day, in 19 (19%) patients the grafts that had hard consistency got rejected while in 81 (81%) patients, the graft was taken. At the end of 1 month the graft was lost in 65 patients and their wound was healed. At 2 months remaining in 16 patients the majority of graft got separated, resulting in complete wound healing but in some areas the part of the graft was still in place and seems to be permanently taken up [Table 1]. 19 patients in whom allograft was rejected within 2-3 weeks were retreated with autograft. {Figure 2}{Figure 3}[INLINE:1]{Table 1}

On pus culture examination, out of 100 patients only 26 (26%) patients showed bacterial growth, while in 74 (74%) patients pus culture was sterile. Staphylococcus was found in 11 (11%) patients. Escherichia coli was found in two (2%) patients. Pseudomonas was found in nine (9%) patients and others (Kliebsiella, Streptococcus) were found in four (4%) patients.

Histopathological study was done on the 21st postoperative day. All survived grafts showed infiltration of mononuclear cells, fibroblasts, keratinization, uniformly arranged collagen bundles, and angiogenesis at the junction of graft and graft bed. All grafts that were rejected showed acanthosis, spongiosis, and degenerative changes in vascular walls. Neovascularization was not observed in these grafts.

 Discussion



Lyophilization of the cadaveric skin graft proved to be a physiologically ideal method by which the skin can be stored for years together. Use of lyophilized skin graft as a temporary biological dressing in severely burned patients has been lifesaving in many instances.[8],[9] Lyophilization of the skin leads to reduction or even loss of antigenicity and lyophilized grafts failed to provide initial sensitization. Lyophilization of the skin maintains most of the structural details of cells and leaves many of proteins and enzymes unchanged, leading to most satisfactory initial take as compared to refrigerated nutrients media and glycerol frozen grafts.

In contrast with autograft, the allograft skin capillaries collapsed and necrotic immediately after transplantation. No endothelial cell reactivity was seen until 2 weeks. At 2 weeks, the graft became positive for endothelial cells as endothelial cells migrated along the damaged capillaries. Dermis of allograft was gradually replaced by new collagen fiber that extended from the borders of the graft. In our study as well, histopathological evaluation of allograft on the 21st postoperative day showed that there was infiltration of mononuclear cells and fibroblasts, keratinization, angiogenesis, acanthosis, spongiosis with raised level of collagen in all well survived grafts. The graft incorporated into the recipient bed as epidermal cells cover it.[10]

These grafts are reported to stay on the wound bed up to 2 months. Finally, all these graft underwent a rejection characterized by vitrification followed by separation of the eschar and lifting of the margin of the graft from the edges of the wound.[6] The allograft dermis felicitates the proliferation and migration of various cellular components such as epithelial cells, endothelial cells, and fibroblasts and thus helps in epithelialization of wounds. In cases of small wounds, the wounds healed and allograft peeled off from the bed and in larger wounds the wound decreased in size and remaining wound was ready to accept autograft.

The ideal wound dressing should have good adherence, water transport, elasticity, durability, create barrier for bacteria, absence of antigenicity, and a longer shelf life. Lyophilized cadaveric skin graft meets all these requirements. Lyophilized graft has all the advantages of biological membrane such as the reduction of bacterial contamination, pain, heat, and protein loss from the wound. It promotes neovascularization and development of healthy granulation tissues, thus preparing the wound for autografting.[11] Cadaveric skin allografts are used in burns surgery both as a temporary bio-dressing and occasionally as definitive management of partial thickness burns. Lyophilized cadaveric allograft has a positive and definite role as an adjunct to conventional dressing and grafting where available, particularly in patients with large total body surface area (TBSA) burns.[12],[13] The major indications for lyophilized cadaveric skin graft includes early physiologic closure of small wounds, testing of wounds for autograft take, rapid preparation of wound for autograft, and temporary closure of major life-threatening wounds.[11]

It is assumed that a higher level of viability of cells in allograft is an important advantage and contributes to better healing. There is no evidence that viability of the graft influences healing outcomes. Literature review does not support this assumption. Thus, rather than viability, antimicrobial safety and cost should be the primary driver for the use of allograft in the treatment of partial thickness burns.[14]

In this study in 19% cases, the graft got rejected in 3 weeks' time leading to a raw area. Lyophilized skin graft stays on wounds till 1 month in 65% patients and their wounds healed. While in 16 patients after 2 months, the majority of graft got separated and the wound healed completely but in some areas, the part of the graft was still in place and seems to be permanently taken up. Therefore, in our study, in 81% patients wounds healed completely while 19 patients required autografting. Snyder [15] as well reported healing of 70% of their patients after the application of allograft.

Priutt [16] observed staphylococcus aureus and pseudomonas to be the most common organism found in burn wounds. In our study as well, these two were the most common organisms. On pus culture examination out of 100 patients 26 (26%) patients showed the presence of infection. Eade [17] verified the widely held opinion that bacterial colony count decreases rapidly after a skin graft is placed over a burn wound. It is because of trapping of bacteria in fibrin matrix between the graft and the bed and then phagocytosed without antibody production or opsonins.[18]

 Conclusion



Lyophilized cadaveric allograft acts as mechanical and physiological barrier when applied on patients of burn and nonhealing ulcers. The process of lyophilization helps in minimizing the immunoreactivity of the graft, therefore, rejection is not rapid and it seems to be not due to immunological mechanism. Lyophilization process preserves the cellular integrity, natural protein structures and certain enzymes, which helps in long-term preservation of allograft. The lyophilized cadaveric allografts are ultimately going to be rejected but part of it may get permanently revascularized. Lyophilized cadaveric allografts can be lifesaving in cases of severe burn. We have used the lyophilized graft on post burn raw area and nonhealing ulcers but the assessment of the behavior of lyophilized graft on wounds of different etiologies needs to be assessed as a separate study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Pruitt BA Jr. The evolutionary development of biologic dressings and skin substitutes. J Burn Care Rehabil 1997;18:S2-5.
2Kearney JN. Guidelines on processing and clinical use of skin allografts. Clin Dermatol 2005;23:357-64.
3Mahdavi-Mazdeh M, Nozary Heshmati B, Tavakoli SA, Ayaz M, Azmoudeh Ardalan F, Momeni M. Human split-thickness skin allograft: Skin substitute in the treatment of burn. Int J Organ Transplant Med 2013;4:96-101.
4Abbott WM, Dupree EL Jr. The procurement, storage, and transplantation of lyophilized human cadaver dura mater. Surg Gynecol Obstet 1970;130:112-8.
5Perry VP. A review of skin preservation. Cryobiology 1966;3:109-30.
6Flosdorf EW, Hyatt GW. The preservation of bone grafts by freeze-drying. Surgery 1952;31:716-9.
7Wolf DL, Capozzi A, Pennisi VR. Evaluation of biological dressings. Ann Plast Surg 1980;5:186-90.
8Hyatt GW. Storage of human tissues for surgical application. In: Parks AS, Smith AU, editors. Recent Research in Freeze and Drying. Oxford: Blackwell Scientific Publication; 1960. p. 251-80.
9Abbott WM, Hembree JS. Absence of antigenicity in freeze-dried skin allografts. Cryobiology 1970;6:416-8.
10Omi T, Kawanami O, Matsuda K, Tsujii A, Kawai M, Henmi H, et al. Histological characteristics of the healing process of frozen skin allograft used in the treatment of burns. Burns 1996;22:206-11.
11Spence RJ, Wong L. The enhancement of wound healing with human skin allograft. Surg Clin North Am 1997;77:731-45.
12Sarkar A, Rakshit P, Majumdar BK, Bain J, Adhya A, Saha JK, et al. Use of cadaveric skin allograft in management of deep burn wounds: Our experience. Int J Basic Appl Med Sci 2013;3:186-8.
13Rose JK, Desai MH, Mlakar JM, Herndon DN. Allograft is superior to topical antimicrobial therapy in the treatment of partial-thickness scald burns in children. J Burn Care Rehabil 1997;18:338-41.
14Hermans MH. Preservation methods of allografts and their (lack of) influence on clinical results in partial thickness burns. Burns 2011;37:873-81.
15Snyder RJ, Simonson DA. Cadaveric allograft as adjunct therapy for nonhealing ulcers. J Foot Ankle Surg 1999;38:93-101.
16Pruitt BA Jr, Levine NS. Characteristics and use of biologic dressings and skin substitutes. Arch Surg 1984;119:312-22.
17Eade GG. The relationship between granulation tissue, bacteria, and skin grafts in burned patients. Plast Reconstr Surg Transplant Bull 1958;22: 42-55.
18Burleson R, Eiseman B. Mechanisms of antibacterial effect of biologic dressings. Ann Surg 1973;171:181-6.