- Email: [email protected]
Viability of frozen allografts
Viability of Frozen Allografts Thomas L. Wachtel, MD, San Diego, California John Ninnemann, PkD,” San Diego, California Jack C. Fisher, MD, San Diego, California Hugh A. Frank, MD, San Diego, California Wayne Inancsi, PA-C, San Diego, California
Closure of the burn wound is the ultimate goal in the care of the burned patient. Often the need for tissue does not correspond with the availability of autograft; therefore, alternative methods must be employed. Allograft cadaver skin was applied first to burn wounds in 1881 [1,2]. Grafting techniques were refined greatly in subsequent years until skin grafting became commonplace [3-51. The importance of human allotypic skin as a biologic dressing in the treatment of burns is well established [5-71. It is now generally recognized that no alternative is available that can approximate the biologic properties of human skin. During the past 2 decades allograft skin has proved invaluable in the temporary closure of open granulating burn wounds [g-13]. With the acceptance of this indication has come an increased demand for tissue, leading to the establishment of tissue procurement centers and skin banks at several institutions [7,13,14]. Research subsequent to the establishment of skin banks has resulted in improved methods for harvesting, treating, transporting, storing, and evaluating preserved tissue [6,15-221. Fresh human cadaver allograft is considered the best biologic membrane for temporary closure of the burned wound. There is a “take” in which vascularization and actual bonding of the graft to the recipient site are present. The allograft will remain for a finite period until the host rejects the allograft as a foreign body. This rejection phenomenon is postponed in the endogenously immunosuppressed burned patient and, therefore, the fresh allograft can From the Department of Surgery, University of California San Diego, San Dieoo. California. This work was suocorted in part by Grant GM-23126 from theNational Institute of General Medical Science of the National Institutes of Health, Bethesda, Maryland. a Recipient of Research’Career Award GM-00264 from the National Institute of General Medical Sciences, Bethesda, Maryland. Reprint requests shoukl be addressed to Thomas L. Wachtel, MD, Regional Rum Treatment Center, 229 Dickinson Street, San Diego, California 92103. Presented at the 31st Annual Meeting of the Southwestern Surgical Congress, Las Vegas, Nevada, April 23-26, 1979.
Volume 136, December
1979
be used as an intermediate allograft (long-term allograft) as well as a biologic membrane. Unfortunately, the demand for fresh allograft is not always matched by the supply of available cadavers [16,23]. The shelf life of fresh allograft at normal suggested refrigeration temperature (4OC) is limited [16,24]. To extend the shelf life of this biologic product, it is frozen in liquid nitrogen (-lSO’C), thus permitting a more adequate balance of supply and demand [23]. Nitrogen-frozen skin is commonly accepted as a viable product [16] that will adhere by vascularization and otherwise participate in normal healing mechanisms such as epithelial migration. However, there is considerable controversy over the viability of such frozen, stored tissue [23-251, partly because of the cellular trauma of the freezing and thawing process [23,24]. Techniques are available for determining the number of remaining viable cells by organ culture, radioautography [16], and assays of enzyme activity [24,26]; however, the ultimate criteria are whether the frozen allograft will “take” on the recipient wound and indicate viability by demonstrating self-proliferation in the form of growth. Our study was conducted to determine the viability of frozen allografts harvested and preserved by standard skin banking techniques using well-known clinical methods. Emphasis was placed on assessing the viability ‘of the frozen biologic membrane for long-term allografting (intermediate allografting). Furthermore, we compared the effect of rapid thawing of the allograft in a microwave oven with the more common and standard water bath thawing techniques. Material and Methods
Fresh and thawed frozen allografts were compared at 17 burned patients. Patients were selected who would require a biologic membrane for wound closure using sites on eight
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the usual clinical criteria [16,23]. Allografts for each comparison were taken from the same donor. All fresh allografts were placed within 7 days of procurement. The techniques for harvesting, preserving, and thawing human cadaver allograft have been detailed [23]. Briefly, skin donations were accepted from cadaver donors aged 12 to 60 years with no history of cancer or hepatitis and no present evidence of systemic infection, jaundice, skin disease, or venereal disease. Skin surfaces were shaved, prepared with iodophor solution, and draped with sterile sheets. Skin (0.015 inch in thickness) was harvested from one region at a time and placed in 50 ml sterile Falcon@ 2070 tubes (Falcon Division, Becton Dickinson, Oxnard, CA) with reference numbers corresponding to bacteriologic controls. Each roll contained approximately 12 to 15 in2 of allograft. The fresh allograft was stored at 4°C until used. The allograft to be frozen was cryoprotected in 15 per cent glycerol in lactated Ringer’s solution for 2 hours at 4’C. At the end of 2 hours, excess cryoprotective solution was poured off and the tubes were resealed. Tubes containing cryoprotected allograf;t were placed directly into the vapor phase liquid nitrogen (-180°C) in a Linde Star@‘30 liquid nitrogen freezer (Union Carbide, Linde Division, Indianapolis, IN) [23]. Just before the clinical experiment, the appropriate number of tubes of frozen skin were removed from the liquid nitrogen freezer. Half of these tubes were thawed in a standard water bath at 37OC [16] while the other half were thawed in a microwave oven as previously described [23]. After thawing, the tubes were placed in an ice-filled transfer container and the allograft was delivered to the operating theater. All allografts stored in glycerol were rinsed in warm Physiosole (Abbott Laboratories, North Chicago, IL) before use. Allografts were meshed at a ratio of 1:1.5 with a Tanner Meshgraft II@(Zimmer-USA, Warsaw, IN). Fresh allograft, frozen allograft thawed in the microwave oven, and frozen allograft thawed in the water bath were expanded to maximal extent and placed side by side on similar recipient
beds (granulation tissue, fat, or fascia) (Figure 1). When possible, autograft meshed at a 1:1.5 ratio was placed on the recipient bed as a control. The order of application was chosen by random methods. The test area was dressed with Furacin@ (nitrofurazone, Eaton Laboratories, Norwich, NY) gauze occlusive dressings. Serial clinical evaluations were performed on postoperative days 3,5,7,10,14,21, and 28. Special attention was directed to the rate of skin growth as shown by closure of the interstices of the meshed skin by epithelial migration. Color, adherence (resistance to removal of membrane in the absence of exudate), percentage of take (a pink appearance at 3 to 5 days that blanches on pressure and returns after release), and complications were also evaluated. Photographic documentation was performed to coincide with clinical evaluation. Quantitative bacterial cultures were taken under each test site before application of the allografts. Similar cultures were obtained during the postgrafting period, particularly when nonadherence occurred or the experiment terminated. Biopsy specimens were obtained through each of the allografts for histologic section. Results
In each instance meshed autograft became ‘adherent and the interstices closed in 12 f 2 days (mean f standard error of the mean) (Table I). The fresh allograft also adhered, but closure of the graft interstices occurred more slowly (16 f 4 days). The frozen allograft adhered, but the interstices failed to close in all but three case,s (that is, 82 per cent failed to grow). Nevertheless, meshed frozen allograft often remained adherent for 30 days, serving as a reliable biologic membrane (Figure 2). Mechanical dislodgment and hematoma formation accounted for graft loss from three sites. All preoperative quantitative cultures contained less than lo5 organisms and were thereby compatible with potential graft take. Quantitative cultures contained less than lo5 organisms beneath adherent grafts and usually greater than lo5 organisms when graft rejection occurred. Histologic sections showed normal epithelial architecture and keratin development of the autograft.
TABLE
I
Clinical Evaluation of Grafts
Graft
Figure 7. Test site with grafts meshed at 7: 7.5, expanded, placed and skte by side on a gramdating b&L Aufografi (A), fresh aNogratV(F), fmzen aliograft thawed the in water bath ( W), and frozen allograft thawed In the microwave oven (M) were used.
784
Autograft Fresh allograft Frozen allograftwater bath thaw Frozen allograftmicrowave thaw
No.
Good Adherence at 10 Days
Interstices Closed No. of No. of Sites Days*
15 17 17
15 16 15
15 16 3
12f 2 16f4 18 f 4
17
15
3
18f4
’ Mean f standard error of the mean.
The American Journal of Surgery
Viability of Frozen Allografts
The dermis was healthy with normal vascularization. The inflammatory response was minimal. Fresh cadaver allograft was viable with normal-appearing epithelium that became flat along the basal layer as it advanced to close the interstices (Figure 3). The dermis was healthy with good vascularity. Mild chronic inflammation cells were present in many of the sect.ions, particularly toward the end of the study period. The frozen cadaver allograft showed viable epithelium initially, but at studies at 3 and 4 weeks most of the epithelium was dead (Figure 4). Sometimes the deep papillary aspects of the epithelium were viable, but ghosts of the epithelial cells were present throughout most of this layer. The dermis became more edematous, usually had chronic inflammatory cells and hemorrhage, and often had acute inflammatory cells and foreign body giant cells as well. Vascularization was not apparent. There was remarkably little difference in the histologic appearance of frozen allograft thawed by microwave and t.hat thawed in a water bath. Roth underwent slow progressive degeneration with preservation of the t.issue layers and the cornified epithelium.
here best to beds ready for autografting and thus have been used as a test of autograft acceptance [23]. Nonfrozen allograft, harvested, processed as described, and refrigerated at 4’C, has consistently vascularized on recipient wounds with less than lo” organisms per gram of tissue. Allograft skin refrigerated at 4°C for longer than 1 week should not be used because Brown and Kemble [24] reported that the longest period a graft could be stored at - 10°C and remain viable was 12 days; a few were nonviable before 10 days. Using these strict crit.eria, we have kept fresh allograft skin in place as a wound covering for as long as 2.5 months without exogenous immunosuppression. Moreover, we have consist.ently had
Comments
The use of skin allografts as a biologic dressing offers many distinct advantages over conventional dressing techniques [27-291. Frozen, thawed allograft has helped to prepare recipient granulation beds for autografting, to protect wounds from bacterial contamination, to reduce protein and water loss from the wound surface, and to increase the comfort and well-being of the patient. In addition, allografts ad-
Figure 2. Test site 28 days after application the of meshed atlograft. frozen The allograft thawed by water bath ( Hz 0) and microwave energy ( M W) remain adherent with open interstices. The fresh a&graft (Fresh) and the autograft (Auto) are adherent ad the interstices are closed. Stainless steel staples were used to attach these grafts.
Figure 3. flIstoh?gic section of fresh atlograft 30 days atier applkation showing viable’ eplthefi&m an@ heatthy dermfs attaclied to the granu/at/on ttssue. There is mtnlmat Irlftammatory reactkn and Qood vasculartty. (MagnifkiMoq X 100, reduced 26 pqr cent. ) Volume 138. December IBTB
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Figure 4. Histologic section of frozen allograft 10 days after application showing viabiltty the of papillary deep aspects of the eptthelium and ghosts of epitheliat cells on the surface. The dermis is edematous. There are chronic inflammatory ceL in the both dermis and the granulating tis&e. Vascularizattoh of the gratt is not appa’rent, ( Magnlftcatton X 100, reduced 27 per cent. )
success with meshed fresh allograft over extensive open burn wounds with closure of inter&ices. Liquid nitrogen frozen and microu&e-thawed allograft consistently adhered to wounds ready for biologic membranes and, when applied to healthy granulation tissue, adhered in a manner comparable to fresh tissue. The use of microwave energy to thaw rapidly the frozen allograft reduces the risk of ice crystal formation during the thawing p’rocedure and also solves some additional problems. Skin is available more quickly than with other thawing techniques, and because allograft never leaves the sterile Falcon tube container, the procedure does not risk product contamination [23]. The basic problem encountered in storing any viable tissue or organ is to control the destructive effects of anaerobic metabolism. Freezing provides the most complete metabolic inhibition and is the only method to date that has provided ., indefinite storage of viable tissue. However, because freezing introduces physical and physiologic“cel1 damage, precautions must be taken if the tissue is to survive. One source of damage during freezing Is related to freezing out of water, leaving behind a lethal concentration of salts greater than five times normal. Cryoprotective agents that act through their ability to partially bind water are used to protect against this type of damage. Another source of damage during freezing is the formation of intracellularice crystals. Control of the rate of freezing reduces this type of damage [22]. Several investigators have reported techniques for freezing human skin, preserving it at low temperatures with eventual thawing and transplantation 786
[15,30,31]. Some studies have demonstrated that frozen skin grafts subjected to slow freezing and storage at liquid nitrogen temperatures possess the same biologic and clinical properties as freshly harvested skin grafts [ 161. The results of our study failed to confirm this universal enthusiasm. Fresh cadaver allograft was definitely a superior product for 'longer term allografting. The viability of preserved skin is determined only with difficulty by processes such as radioautography and metabolic studies. Although it was not one of the criteria in our current studyi the radioautographic experiments using tritiated thymidine provided evidence for epithelial ceil multiplication in both the conventionally used unfrozen split-thickness graft and the frozen preserved split-thickness graft with no detectable difference between epidermal cell migration in either of these two groups [16]. Our studies failed to show equal epithelial migration when closure of interstices of the fresh and frozen allograft was the criterion. The single most important reason for failure of split-thickness skin grafts on burn patients was nonviability of the graft at the time of application. Brown and Kemble [24] determined the viability of the graft by measuring relative tetrazolium reductase activity. Repeated freezing and thawing of stored skin grafts reduced the length of viability by 40 per cent compared with that of grafts that were frozen and thawed only once [24], indicating that freezing and thawing did, in fact, cause significant cellular injury. D&sing and S@rensen [25] demonstrated that the functional integrity of freeze-dried skin’ is partly The American Journal of Surgery
Viability
impaired and is preserved only about 3 weeks after transplantation. Freeze-dried allografts are useful even though they are dead tissue. Histologic study showed that mitosis did not occur in the freeze-dried allograft that underwent progressive degeneration although the tissue layers and cornified epithelium were preserved. Summary
The use of meshed skin grafts allows an evaluation of the biologic properties of stored tissues such as skin. Frozen allograft may serve as an excellent biologic membrane, but it is not a satisfactory product for longer term allografting (intermediate allografting). The method of thawing (microwave oven versus water bath) appeared to have little effect on the ultimate viability and outcome of the frozen allograft. Acknowledgment: Viviano, Joan knowledged.
The assistance of Paul Wolf, MD, Pat Riley, and Debbie Brown is gratefully ac-
References 1. Girdner JH: Skin gafting with grafts taken from ths deed subject. MedRec 20: 119. 1881. 2. Rogers 80: Guide and bibliography for research into the skin homograft problem. flasf Reconsh Surg 7: 169, 195 1. 3. Brown JB, McDowell F: Massive repairs of burns with thick split-skin grafts: emergency “dressings” with homografts. Ann Surg 115: 656. 1942. 4. Moore FD: Transplant: The Give and Take of Tissue Transplantation. New York, Simon 8. Schuster, 1972. 5. Sell KW, Friedlander GE (ed): Proceedings of the tissue bank symposium. Transplant hoc 6: Suppl I: 2, 1976. 6. Robson MC. Krizek TJ: Predicting skin gaft survival. J Trauma 13: 213 1973. 7. Trier WC, Sell KW: ulited States Navy skin bank. fbsf Reconsfr Surg41: 543, 1968. a. Burke JF, Bondoc CC: Combined burn therapy utilizing immediate skin allografts and 0.5% AgN03. Arch Surg 97: 716, 1968. 9. Jackson D. Topley E. Cason JS. Lowburg EJ: Primary excision and grafting of large burns. Ann Surg 152: 167. 1960. 10. Artz CP, Becker JM, Sakq Y, Bronwell AW: Postmortem skin hornografts in the treatment of extensive burns. Arch Surg 71: 682, 1955. 11. Haynes BW Jr: Skin homografts: a life-saving measure in severely burned children. J Trauma 3: 217, 1963. 12. MacMillian BG: Homoyaft skin: a valuable adjunct to the treatment of thermal burns. J Trauma 2: 130, 1962. 13. Malinin TI: University of Miami tissue bank: collection of postmortem tissues for clinical use and laboratory investigation. Transplant Proc 6 (suppl I): 53. 1976. 14. Roberts M: The role of the skin bank. Ann R Co// Surg Eng/ 56: 70, 1976. 15. Athreya BH, Grimes EL, Lehr HB. Greene AE, Corlell LL: Differential susceptibi!ity of epithelial cells and fibroblasts of human skin to freeze injury. Cryobiology 5: 262. 1969. 16. Bondoc CC, Burke JF: Clinical experience with viable frozen human skin and a frozen skin bank. Ann Surg 174: 371, 1971.
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Allografts
17. Ccchrane T: The low temperature storage of skin: a preliminary report. f3r J flast Surg 21: 1 ia,1966. 18. Donati L. Klinaer _ A. Montorsi W: Comparison of clinical activity of allo- and xeno-grafts of fresh and cryopreserved skin. Minh Med 65: 3654, 1974. 19. Graham WP. Hamilton RW. Lehr HB: Versatility of skin allografts: desirability of a viable frozen tissue bank. J Trauma 11: 494. 1971. 20. Neifeld JP, Chretien PB: An improved technique of excision and skin grafting for primary malignant melanomas. Surg Gynecol Obstet 142: 564, 1976. 21. Ostrowski K: Current problems of tissue banking. Transplant Proc 1: 126. 1969. 22. Perry VP: A review of skin preservation. Cryobiology 3: 109, 1966. 23. Ninnemann JL, Fisher JC, Frank HA: Clinical skin banking: a simplified system for processing, storage, and retrieval of human allografts. J Trauma 16: 723, 1976. 24. Brawn RFR, Kemble JVH: Tetrazolium reductase as an index of viability of stored skin. Burns 1: 179, 1975. 25. wssing M, S@rensen. B: Freeze-c&d and non-freeze-dried-skin allografts on excised burns. Burns 2: 36, 1976. 26. Prows J. Nathan P: Ths viability of epidermal cells from stored refrigerated skin. Presented at the 1 lth annual meeting of the American Burn Association, March 15. 1979. necessary? 27. Gillman T, Hathorn M, Penn J: Is skin howrafting A reexamination of the rationale for auto- or homo-grafting of cutaneous injuries and a preliminary report on the action of plastic dressings. Plast Reconstr Skg 16: 260, 1956. 28. Jackson D: A clinical study of the use of skin homografts for burns. Br J Plast Surg 7: 26, 1954. 29. Zaroff L. Mills W Jr, Duckett J Jr, Switzer WE, Moncrief JA: Multiple uses of viable cutaneous homografts in the burned patient. Surgery 59: 366. 1966. 30. Berggren RB, L&r HB: Clinical use of viable frozen human skin. JAMA 194: 149. 1965. 31. Billingham RE. Medawar PB: The freezing. drying, and &rage of mammalian skin. J Exp Viol 29: 454, 1952.
Discussion Jerry M. Shuck (Albuquerque, NM): This report is excellent for the following reasons: (1) The purpose was clearly stated. (2) The experimental design was simple and well controlled and the end point was easily recognizable. (3) The results were both biologically and clinically significant and useful. (4) It confirms my own biases. Two questions come to mind. First. does now knowing that fresh homograft skin is viable and will generate epithelium really make a difference in the temporary biologic dressing as we currently use it? Second. would you consider repeating the study of fresh homograft at 2 weeks or longer to establish the temporal parameters of viability? Thomas L. Wachtel (closing): U’e currently apply fresh allograft for temporary dressings if we have it because we still think it best. Sometimes we can let the allograft remain in place 30 LO4.5 days, close the wound, and see the patient through a number of problems. We do use the frozen allograft as a biologic dressing and often remove it within 2 weeks to replace it with fresh allograft if we are trying to get a patient with a huge burn over the long haul. Perhaps we have become a little nonchalant in our use of fresh allograft, but when available we use it for both temporary dressings and Ilmg-term allografting.
787
Closure of the burn wound is the ultimate goal in the care of the burned patient. Often the need for tissue does not correspond with the availability of autograft; therefore, alternative methods must be employed. Allograft cadaver skin was applied first to burn wounds in 1881 [1,2]. Grafting techniques were refined greatly in subsequent years until skin grafting became commonplace [3-51. The importance of human allotypic skin as a biologic dressing in the treatment of burns is well established [5-71. It is now generally recognized that no alternative is available that can approximate the biologic properties of human skin. During the past 2 decades allograft skin has proved invaluable in the temporary closure of open granulating burn wounds [g-13]. With the acceptance of this indication has come an increased demand for tissue, leading to the establishment of tissue procurement centers and skin banks at several institutions [7,13,14]. Research subsequent to the establishment of skin banks has resulted in improved methods for harvesting, treating, transporting, storing, and evaluating preserved tissue [6,15-221. Fresh human cadaver allograft is considered the best biologic membrane for temporary closure of the burned wound. There is a “take” in which vascularization and actual bonding of the graft to the recipient site are present. The allograft will remain for a finite period until the host rejects the allograft as a foreign body. This rejection phenomenon is postponed in the endogenously immunosuppressed burned patient and, therefore, the fresh allograft can From the Department of Surgery, University of California San Diego, San Dieoo. California. This work was suocorted in part by Grant GM-23126 from theNational Institute of General Medical Science of the National Institutes of Health, Bethesda, Maryland. a Recipient of Research’Career Award GM-00264 from the National Institute of General Medical Sciences, Bethesda, Maryland. Reprint requests shoukl be addressed to Thomas L. Wachtel, MD, Regional Rum Treatment Center, 229 Dickinson Street, San Diego, California 92103. Presented at the 31st Annual Meeting of the Southwestern Surgical Congress, Las Vegas, Nevada, April 23-26, 1979.
Volume 136, December
1979
be used as an intermediate allograft (long-term allograft) as well as a biologic membrane. Unfortunately, the demand for fresh allograft is not always matched by the supply of available cadavers [16,23]. The shelf life of fresh allograft at normal suggested refrigeration temperature (4OC) is limited [16,24]. To extend the shelf life of this biologic product, it is frozen in liquid nitrogen (-lSO’C), thus permitting a more adequate balance of supply and demand [23]. Nitrogen-frozen skin is commonly accepted as a viable product [16] that will adhere by vascularization and otherwise participate in normal healing mechanisms such as epithelial migration. However, there is considerable controversy over the viability of such frozen, stored tissue [23-251, partly because of the cellular trauma of the freezing and thawing process [23,24]. Techniques are available for determining the number of remaining viable cells by organ culture, radioautography [16], and assays of enzyme activity [24,26]; however, the ultimate criteria are whether the frozen allograft will “take” on the recipient wound and indicate viability by demonstrating self-proliferation in the form of growth. Our study was conducted to determine the viability of frozen allografts harvested and preserved by standard skin banking techniques using well-known clinical methods. Emphasis was placed on assessing the viability ‘of the frozen biologic membrane for long-term allografting (intermediate allografting). Furthermore, we compared the effect of rapid thawing of the allograft in a microwave oven with the more common and standard water bath thawing techniques. Material and Methods
Fresh and thawed frozen allografts were compared at 17 burned patients. Patients were selected who would require a biologic membrane for wound closure using sites on eight
783
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et
al
the usual clinical criteria [16,23]. Allografts for each comparison were taken from the same donor. All fresh allografts were placed within 7 days of procurement. The techniques for harvesting, preserving, and thawing human cadaver allograft have been detailed [23]. Briefly, skin donations were accepted from cadaver donors aged 12 to 60 years with no history of cancer or hepatitis and no present evidence of systemic infection, jaundice, skin disease, or venereal disease. Skin surfaces were shaved, prepared with iodophor solution, and draped with sterile sheets. Skin (0.015 inch in thickness) was harvested from one region at a time and placed in 50 ml sterile Falcon@ 2070 tubes (Falcon Division, Becton Dickinson, Oxnard, CA) with reference numbers corresponding to bacteriologic controls. Each roll contained approximately 12 to 15 in2 of allograft. The fresh allograft was stored at 4°C until used. The allograft to be frozen was cryoprotected in 15 per cent glycerol in lactated Ringer’s solution for 2 hours at 4’C. At the end of 2 hours, excess cryoprotective solution was poured off and the tubes were resealed. Tubes containing cryoprotected allograf;t were placed directly into the vapor phase liquid nitrogen (-180°C) in a Linde Star@‘30 liquid nitrogen freezer (Union Carbide, Linde Division, Indianapolis, IN) [23]. Just before the clinical experiment, the appropriate number of tubes of frozen skin were removed from the liquid nitrogen freezer. Half of these tubes were thawed in a standard water bath at 37OC [16] while the other half were thawed in a microwave oven as previously described [23]. After thawing, the tubes were placed in an ice-filled transfer container and the allograft was delivered to the operating theater. All allografts stored in glycerol were rinsed in warm Physiosole (Abbott Laboratories, North Chicago, IL) before use. Allografts were meshed at a ratio of 1:1.5 with a Tanner Meshgraft II@(Zimmer-USA, Warsaw, IN). Fresh allograft, frozen allograft thawed in the microwave oven, and frozen allograft thawed in the water bath were expanded to maximal extent and placed side by side on similar recipient
beds (granulation tissue, fat, or fascia) (Figure 1). When possible, autograft meshed at a 1:1.5 ratio was placed on the recipient bed as a control. The order of application was chosen by random methods. The test area was dressed with Furacin@ (nitrofurazone, Eaton Laboratories, Norwich, NY) gauze occlusive dressings. Serial clinical evaluations were performed on postoperative days 3,5,7,10,14,21, and 28. Special attention was directed to the rate of skin growth as shown by closure of the interstices of the meshed skin by epithelial migration. Color, adherence (resistance to removal of membrane in the absence of exudate), percentage of take (a pink appearance at 3 to 5 days that blanches on pressure and returns after release), and complications were also evaluated. Photographic documentation was performed to coincide with clinical evaluation. Quantitative bacterial cultures were taken under each test site before application of the allografts. Similar cultures were obtained during the postgrafting period, particularly when nonadherence occurred or the experiment terminated. Biopsy specimens were obtained through each of the allografts for histologic section. Results
In each instance meshed autograft became ‘adherent and the interstices closed in 12 f 2 days (mean f standard error of the mean) (Table I). The fresh allograft also adhered, but closure of the graft interstices occurred more slowly (16 f 4 days). The frozen allograft adhered, but the interstices failed to close in all but three case,s (that is, 82 per cent failed to grow). Nevertheless, meshed frozen allograft often remained adherent for 30 days, serving as a reliable biologic membrane (Figure 2). Mechanical dislodgment and hematoma formation accounted for graft loss from three sites. All preoperative quantitative cultures contained less than lo5 organisms and were thereby compatible with potential graft take. Quantitative cultures contained less than lo5 organisms beneath adherent grafts and usually greater than lo5 organisms when graft rejection occurred. Histologic sections showed normal epithelial architecture and keratin development of the autograft.
TABLE
I
Clinical Evaluation of Grafts
Graft
Figure 7. Test site with grafts meshed at 7: 7.5, expanded, placed and skte by side on a gramdating b&L Aufografi (A), fresh aNogratV(F), fmzen aliograft thawed the in water bath ( W), and frozen allograft thawed In the microwave oven (M) were used.
784
Autograft Fresh allograft Frozen allograftwater bath thaw Frozen allograftmicrowave thaw
No.
Good Adherence at 10 Days
Interstices Closed No. of No. of Sites Days*
15 17 17
15 16 15
15 16 3
12f 2 16f4 18 f 4
17
15
3
18f4
’ Mean f standard error of the mean.
The American Journal of Surgery
Viability of Frozen Allografts
The dermis was healthy with normal vascularization. The inflammatory response was minimal. Fresh cadaver allograft was viable with normal-appearing epithelium that became flat along the basal layer as it advanced to close the interstices (Figure 3). The dermis was healthy with good vascularity. Mild chronic inflammation cells were present in many of the sect.ions, particularly toward the end of the study period. The frozen cadaver allograft showed viable epithelium initially, but at studies at 3 and 4 weeks most of the epithelium was dead (Figure 4). Sometimes the deep papillary aspects of the epithelium were viable, but ghosts of the epithelial cells were present throughout most of this layer. The dermis became more edematous, usually had chronic inflammatory cells and hemorrhage, and often had acute inflammatory cells and foreign body giant cells as well. Vascularization was not apparent. There was remarkably little difference in the histologic appearance of frozen allograft thawed by microwave and t.hat thawed in a water bath. Roth underwent slow progressive degeneration with preservation of the t.issue layers and the cornified epithelium.
here best to beds ready for autografting and thus have been used as a test of autograft acceptance [23]. Nonfrozen allograft, harvested, processed as described, and refrigerated at 4’C, has consistently vascularized on recipient wounds with less than lo” organisms per gram of tissue. Allograft skin refrigerated at 4°C for longer than 1 week should not be used because Brown and Kemble [24] reported that the longest period a graft could be stored at - 10°C and remain viable was 12 days; a few were nonviable before 10 days. Using these strict crit.eria, we have kept fresh allograft skin in place as a wound covering for as long as 2.5 months without exogenous immunosuppression. Moreover, we have consist.ently had
Comments
The use of skin allografts as a biologic dressing offers many distinct advantages over conventional dressing techniques [27-291. Frozen, thawed allograft has helped to prepare recipient granulation beds for autografting, to protect wounds from bacterial contamination, to reduce protein and water loss from the wound surface, and to increase the comfort and well-being of the patient. In addition, allografts ad-
Figure 2. Test site 28 days after application the of meshed atlograft. frozen The allograft thawed by water bath ( Hz 0) and microwave energy ( M W) remain adherent with open interstices. The fresh a&graft (Fresh) and the autograft (Auto) are adherent ad the interstices are closed. Stainless steel staples were used to attach these grafts.
Figure 3. flIstoh?gic section of fresh atlograft 30 days atier applkation showing viable’ eplthefi&m an@ heatthy dermfs attaclied to the granu/at/on ttssue. There is mtnlmat Irlftammatory reactkn and Qood vasculartty. (MagnifkiMoq X 100, reduced 26 pqr cent. ) Volume 138. December IBTB
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Wachtel et al
Figure 4. Histologic section of frozen allograft 10 days after application showing viabiltty the of papillary deep aspects of the eptthelium and ghosts of epitheliat cells on the surface. The dermis is edematous. There are chronic inflammatory ceL in the both dermis and the granulating tis&e. Vascularizattoh of the gratt is not appa’rent, ( Magnlftcatton X 100, reduced 27 per cent. )
success with meshed fresh allograft over extensive open burn wounds with closure of inter&ices. Liquid nitrogen frozen and microu&e-thawed allograft consistently adhered to wounds ready for biologic membranes and, when applied to healthy granulation tissue, adhered in a manner comparable to fresh tissue. The use of microwave energy to thaw rapidly the frozen allograft reduces the risk of ice crystal formation during the thawing p’rocedure and also solves some additional problems. Skin is available more quickly than with other thawing techniques, and because allograft never leaves the sterile Falcon tube container, the procedure does not risk product contamination [23]. The basic problem encountered in storing any viable tissue or organ is to control the destructive effects of anaerobic metabolism. Freezing provides the most complete metabolic inhibition and is the only method to date that has provided ., indefinite storage of viable tissue. However, because freezing introduces physical and physiologic“cel1 damage, precautions must be taken if the tissue is to survive. One source of damage during freezing Is related to freezing out of water, leaving behind a lethal concentration of salts greater than five times normal. Cryoprotective agents that act through their ability to partially bind water are used to protect against this type of damage. Another source of damage during freezing is the formation of intracellularice crystals. Control of the rate of freezing reduces this type of damage [22]. Several investigators have reported techniques for freezing human skin, preserving it at low temperatures with eventual thawing and transplantation 786
[15,30,31]. Some studies have demonstrated that frozen skin grafts subjected to slow freezing and storage at liquid nitrogen temperatures possess the same biologic and clinical properties as freshly harvested skin grafts [ 161. The results of our study failed to confirm this universal enthusiasm. Fresh cadaver allograft was definitely a superior product for 'longer term allografting. The viability of preserved skin is determined only with difficulty by processes such as radioautography and metabolic studies. Although it was not one of the criteria in our current studyi the radioautographic experiments using tritiated thymidine provided evidence for epithelial ceil multiplication in both the conventionally used unfrozen split-thickness graft and the frozen preserved split-thickness graft with no detectable difference between epidermal cell migration in either of these two groups [16]. Our studies failed to show equal epithelial migration when closure of interstices of the fresh and frozen allograft was the criterion. The single most important reason for failure of split-thickness skin grafts on burn patients was nonviability of the graft at the time of application. Brown and Kemble [24] determined the viability of the graft by measuring relative tetrazolium reductase activity. Repeated freezing and thawing of stored skin grafts reduced the length of viability by 40 per cent compared with that of grafts that were frozen and thawed only once [24], indicating that freezing and thawing did, in fact, cause significant cellular injury. D&sing and S@rensen [25] demonstrated that the functional integrity of freeze-dried skin’ is partly The American Journal of Surgery
Viability
impaired and is preserved only about 3 weeks after transplantation. Freeze-dried allografts are useful even though they are dead tissue. Histologic study showed that mitosis did not occur in the freeze-dried allograft that underwent progressive degeneration although the tissue layers and cornified epithelium were preserved. Summary
The use of meshed skin grafts allows an evaluation of the biologic properties of stored tissues such as skin. Frozen allograft may serve as an excellent biologic membrane, but it is not a satisfactory product for longer term allografting (intermediate allografting). The method of thawing (microwave oven versus water bath) appeared to have little effect on the ultimate viability and outcome of the frozen allograft. Acknowledgment: Viviano, Joan knowledged.
The assistance of Paul Wolf, MD, Pat Riley, and Debbie Brown is gratefully ac-
References 1. Girdner JH: Skin gafting with grafts taken from ths deed subject. MedRec 20: 119. 1881. 2. Rogers 80: Guide and bibliography for research into the skin homograft problem. flasf Reconsh Surg 7: 169, 195 1. 3. Brown JB, McDowell F: Massive repairs of burns with thick split-skin grafts: emergency “dressings” with homografts. Ann Surg 115: 656. 1942. 4. Moore FD: Transplant: The Give and Take of Tissue Transplantation. New York, Simon 8. Schuster, 1972. 5. Sell KW, Friedlander GE (ed): Proceedings of the tissue bank symposium. Transplant hoc 6: Suppl I: 2, 1976. 6. Robson MC. Krizek TJ: Predicting skin gaft survival. J Trauma 13: 213 1973. 7. Trier WC, Sell KW: ulited States Navy skin bank. fbsf Reconsfr Surg41: 543, 1968. a. Burke JF, Bondoc CC: Combined burn therapy utilizing immediate skin allografts and 0.5% AgN03. Arch Surg 97: 716, 1968. 9. Jackson D. Topley E. Cason JS. Lowburg EJ: Primary excision and grafting of large burns. Ann Surg 152: 167. 1960. 10. Artz CP, Becker JM, Sakq Y, Bronwell AW: Postmortem skin hornografts in the treatment of extensive burns. Arch Surg 71: 682, 1955. 11. Haynes BW Jr: Skin homografts: a life-saving measure in severely burned children. J Trauma 3: 217, 1963. 12. MacMillian BG: Homoyaft skin: a valuable adjunct to the treatment of thermal burns. J Trauma 2: 130, 1962. 13. Malinin TI: University of Miami tissue bank: collection of postmortem tissues for clinical use and laboratory investigation. Transplant Proc 6 (suppl I): 53. 1976. 14. Roberts M: The role of the skin bank. Ann R Co// Surg Eng/ 56: 70, 1976. 15. Athreya BH, Grimes EL, Lehr HB. Greene AE, Corlell LL: Differential susceptibi!ity of epithelial cells and fibroblasts of human skin to freeze injury. Cryobiology 5: 262. 1969. 16. Bondoc CC, Burke JF: Clinical experience with viable frozen human skin and a frozen skin bank. Ann Surg 174: 371, 1971.
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17. Ccchrane T: The low temperature storage of skin: a preliminary report. f3r J flast Surg 21: 1 ia,1966. 18. Donati L. Klinaer _ A. Montorsi W: Comparison of clinical activity of allo- and xeno-grafts of fresh and cryopreserved skin. Minh Med 65: 3654, 1974. 19. Graham WP. Hamilton RW. Lehr HB: Versatility of skin allografts: desirability of a viable frozen tissue bank. J Trauma 11: 494. 1971. 20. Neifeld JP, Chretien PB: An improved technique of excision and skin grafting for primary malignant melanomas. Surg Gynecol Obstet 142: 564, 1976. 21. Ostrowski K: Current problems of tissue banking. Transplant Proc 1: 126. 1969. 22. Perry VP: A review of skin preservation. Cryobiology 3: 109, 1966. 23. Ninnemann JL, Fisher JC, Frank HA: Clinical skin banking: a simplified system for processing, storage, and retrieval of human allografts. J Trauma 16: 723, 1976. 24. Brawn RFR, Kemble JVH: Tetrazolium reductase as an index of viability of stored skin. Burns 1: 179, 1975. 25. wssing M, S@rensen. B: Freeze-c&d and non-freeze-dried-skin allografts on excised burns. Burns 2: 36, 1976. 26. Prows J. Nathan P: Ths viability of epidermal cells from stored refrigerated skin. Presented at the 1 lth annual meeting of the American Burn Association, March 15. 1979. necessary? 27. Gillman T, Hathorn M, Penn J: Is skin howrafting A reexamination of the rationale for auto- or homo-grafting of cutaneous injuries and a preliminary report on the action of plastic dressings. Plast Reconstr Skg 16: 260, 1956. 28. Jackson D: A clinical study of the use of skin homografts for burns. Br J Plast Surg 7: 26, 1954. 29. Zaroff L. Mills W Jr, Duckett J Jr, Switzer WE, Moncrief JA: Multiple uses of viable cutaneous homografts in the burned patient. Surgery 59: 366. 1966. 30. Berggren RB, L&r HB: Clinical use of viable frozen human skin. JAMA 194: 149. 1965. 31. Billingham RE. Medawar PB: The freezing. drying, and &rage of mammalian skin. J Exp Viol 29: 454, 1952.
Discussion Jerry M. Shuck (Albuquerque, NM): This report is excellent for the following reasons: (1) The purpose was clearly stated. (2) The experimental design was simple and well controlled and the end point was easily recognizable. (3) The results were both biologically and clinically significant and useful. (4) It confirms my own biases. Two questions come to mind. First. does now knowing that fresh homograft skin is viable and will generate epithelium really make a difference in the temporary biologic dressing as we currently use it? Second. would you consider repeating the study of fresh homograft at 2 weeks or longer to establish the temporal parameters of viability? Thomas L. Wachtel (closing): U’e currently apply fresh allograft for temporary dressings if we have it because we still think it best. Sometimes we can let the allograft remain in place 30 LO4.5 days, close the wound, and see the patient through a number of problems. We do use the frozen allograft as a biologic dressing and often remove it within 2 weeks to replace it with fresh allograft if we are trying to get a patient with a huge burn over the long haul. Perhaps we have become a little nonchalant in our use of fresh allograft, but when available we use it for both temporary dressings and Ilmg-term allografting.
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