- Email: [email protected]
Combined use of a collagen-based dermal substitute and a fibrin-based cultured epithelium: a step toward a total skin replacement for acute wounds
Burns 30 (2004) 713–719
Combined use of a collagen-based dermal substitute and a fibrin-based cultured epithelium: a step toward a total skin replacement for acute wounds Béatrice Mis∗ , Eric Rolland, Vincent Ronfard Isotis SA, 18-20 Avenue de Sévelin, 1004 Lausanne, Switzerland Accepted 26 April 2004
Abstract Integra® , a dermal replacement, is used as an immediate and temporary coverage for acute wounds, after which, autograft is used to reconstitute permanently the epidermal coverage. The fibrin sheet-cultured epithelium autograft (FS-CEA) could provide an effective alternative to the surgical procedure. To evaluate this hypothesis, we compared the association of Integra® /FS-CE to Integra® /control-cultured epithelium (control-CE). Their respective abilities: (1) to produce dermal–epidermal construct in vitro; (2) to generate skin replacement when grafted onto athymic mice were studied. We have shown that: (1) 83% of the FS-CE attached to the artificial dermis in vitro compared to only 33% for control-CE; (2) retraction of the grafted area was significantly lower 2 weeks after grafted with FS-CE than with the control-CE (P < 0.05); (3) 83% of the mice grafted with FS-CE showed the presence of a differentiated human epidermis 21 days after grafting, while such an epidermis was absent in all the animals of the control-CE group. We found that the use of FS-CE greatly improved adhesion, development of the epithelium and graft take onto the artificial dermis. We believe this technology should significantly improve the performance of dermal–epidermal skin replacement for acute wounds. © 2004 Elsevier Ltd and ISBI. All rights reserved.
1. Introduction Today, patients with burns encompassing 90% of their body surface can survive because of improvements in burn management and the development of new technologies [1,2]. Full-thickness burns, which extend to the skin’s deepest layers, require immediate care to prevent infection and fluid loss. The first step of treatment is to debride all of the tissue that has been burned. Once this is done, allografts are often used to cover the wounds, temporarily protecting the body until autologous skin is available from donor sites on the unburned body area. If the patient does not have enough donor skin to cover the wound, the physician may need to consider autologous cell culture [3,4] and tissue engineering [5,6] in order to save the patient’s life. Despite many advantages, allografts from cadavers are not always available to surgeons, their quality can vary from donor to donor, and, they may carry the risk of potential adventitious viral
∗
Corresponding author. Fax: +41 21 620 6060. E-mail address: [email protected] (B. Mis).
0305-4179/$30.00 © 2004 Elsevier Ltd and ISBI. All rights reserved. doi:10.1016/j.burns.2004.04.007
transmission. Therefore, a skin substitute to replace allografts is needed. We recently evaluated the performance of a combined utilization of two classes of biomaterials to cover the wound: (1) an artificial skin made of silicone, collagen and glycosaminoglycan and (2) an autologous epithelium grown on a fibrin sheet. [7,8] These products have been used separately in clinics with good clinical outcomes [9–12]. Integra® artificial skin can be applied immediately and provides a temporary coverage for 2–4 weeks, during which time the patient’s own cells make their way into the scaffold. This allows time to produce fibrin sheet-cultured epithelium autograft over about 2–3 weeks. FS-CEA has considerable advantages [10]. One of them is that the fibrin sheet provides a biodegradable matrix that plays an important role for epithelial attachment, survival and development onto the neodermis. To validate the combined utilization of the two biomaterials, we evaluated the performance of human keratinocytes grown on a fibrin sheet when applied to the artificial dermis. We evaluated its ability to generate: (1) a coherent dermal–epidermal construct in vitro, and (2) its ability to generate a skin replacement when grafted onto athymic mice.
714
B. Mis et al. / Burns 30 (2004) 713–719
2. Materials and methods
2.5. Combined in vivo grafting procedures
2.1. Culture
Twenty-three athymic mice (NIH Swiss Nude) approximately 6–8 weeks in age (24–32 g in weight) were used for experiments according to Swiss regulations for the care and use of laboratory animals. They were anesthetized by 2.5% isoflurane inhalation and a full thickness skin wound of approximately 1.7 cm2 was made on the central dorsum of each animal and covered with Integra® . Then, Vaseline gauze and adhesive bandages were applied onto the graft. Dressings were changed after 7 days. The silicone membrane was removed from the artificial dermis and cultured epithelia were grafted 3 weeks later (six mice for control-CE and six mice for FS-CE). A silicone membrane was used to transfer control-CE and FS-CE onto the wound bed. Four mice were not grafted with any CE and used as open wound controls. Two weeks after cultured epithelia grafting, dressings were removed and the grafts were left uncovered. Biopsies were harvested from 15 and 21 days after grafting. The size of the graft was measured on each animal with a caliper (TESA digit-cal CAPA RS 150 mm).
Keratinocytes were cultured according to the Rheinwald and Green method as modified by Limat and colleagues [3,13]. Briefly, keratinocytes were cultured in the presence of growth arrested irradiated human fibroblasts, in F-12/DME medium, added in a 1:3 ratio, containing adenine at 1.8 × 10−4 M, 10% fetal bovine serum (FBS, Gibco, Life Technologies), hydrocortisone 0.5 ng/ml, insulin 5 mg/ml, triiodothyronin 2 × 10−9 M, choleratoxin 10 ng/ml and epidermal growth factor 10 ng/ml (Sigma). Human keratinocytes were obtained from a skin biopsy of an adult undergoing plastic surgery. They were amplified and used in the third passage. 2.2. Control-CE When the cultures reached confluence, they were treated with Dispase as previously described [14] in order to detach the epithelial sheet from the bottom of the dish. Then, with the help of a silicone membrane (Perthese® , Perouse-Plastie, France), the epithelial sheet was lifted off the plate and applied onto the artificial dermis. 2.3. FS-CE The fibrin substrate was made from freeze-dried surgical fibrinogen prepared from human plasma obtained from blood donors (Tissucol® kit, Baxter). The freeze-dried fibrinogen from a 2 ml Tissucol® kit was reconstituted with 5 ml of a sodium chloride (2%) solution containing 1 mM of calcium chloride. It was then mixed (v/v) with sodium chloride 2.2% solution containing 2 mM calcium chloride human thrombin (2.5 IU/ml) and distributed into culture dishes before clotting occurred. Human keratinocytes were cultivated on the top of the fibrin matrix in the presence of aprotinin (Trasylol® , Bayer), as previously described [10]. When the culture reached confluence, the epithelial sheet along with the fibrin was applied to the artificial dermis. 2.4. Combined in vitro cultures Integra® pieces (0.8 cm × 0.8 cm) were seeded with 2 × human dermal fibroblasts and cultured for 5 days. They were fed every other day with the medium as described for the keratinocytes except that the medium did not contain any choleratoxin. Vitamin C (ascorbic acid, Sigma, 50 g/ml) was added at each medium change. Then, the artificial dermis was placed on inserts (0.78 cm2 of culture surface and 0.4 m of membrane pore size, Corning), so that they were fed from underneath. Control epithelium and FS-CE were placed on the top of the artificial dermis and covered with a silicone membrane.
105
2.6. Integra® and FS-CEA grafting procedures Athymic mice (NIH Swiss Nude) approximately 6–8 weeks in age (24–32 g in weight) were used for experiments according to the Swiss regulations for the care and use of laboratory animals and were anesthetized as previously described. Twenty-three animals were grafted with Integra® alone applied to an open wound (1.7 cm2 ) and left in place for 3 weeks. Then, biopsies were harvested and processed for histological evaluation. Independently, ten other animals were grafted with FS-CE alone transplanted on the inner part of a dorsal skin flap of athymic mice as previously described [15]. 2.7. Histology In vitro samples: samples were fixed in Lang solution (10% acetic acid in sublimated formol). After dehydration, samples were embedded in resin (Basic Historesin and Hardener, Leica) and stained with haematoxylin and eosin. In vivo samples: biopsies from grafts were fixed in buffered formalin (5%) and embedded in paraffin wax. Five micrograms sections were stained with haematoxylin and eosin. Immunochemical staining was performed on deparaffinized and rehydrated sections. Human involucrin was detected using an immuno-kit consisting of highly specific human involucrin rabbit antibody and the necessary reagents to peroxidase staining (BT-600, Biomedical Technology Inc. Stoughton, USA). Hematoxylin was used for counterstaining. This kit was used to identify the human origin of the regenerated epidermis from the mouse tissue.
B. Mis et al. / Burns 30 (2004) 713–719
3. Results 3.1. Combined in vitro cultures Behavior of the cultured epithelium combined with the artificial dermis was examined in vitro. Pieces of artificial dermis (0.8 cm × 0.8 cm) without cells or pre-seeded with human dermal fibroblasts were placed on inserts and covered with cultured epithelia grown in the presence or in the absence of fibrin (FS-CE or Control-CE respectively). After 2 and 5 days of culture, histological cross-sections of the cultured epithelia showed that the presence of fibroblasts into the dermal matrix improved the attachment of the epithelium onto the artificial dermis. The control-CE did attach more firmly when the dermal matrix was populated with cells compared to unpopulated matrix. In all cases, the edges of the epithelium were unattached and were often folded. Numerous dying cells with pyknotic nuclei and differentiating cells were seen all along the dermal–epidermal junction. This indicates that the “in vitro take” of the control-CE onto the dermal matrix was poor (Fig. 1(A) and (B)). In
715
comparison, FS-CE did attach well onto the matrix (83%, n = 12, of attached FS-CE versus 33% for control-CE, n = 12); keratinocytes migrated from the fibrin layer to the matrix. FS-CE adheres uniformly on both fibroblast populated and unpopulated dermis matrix. Close junctions were seen between the fibrin layer and fibroblasts in the fibroblast populated matrices. The epithelial layer was made up of normal proliferating cells: small cells with round nucleus, no intercellular spaces or intracellular vacuoles as seen in the control epithelia. A fibrin layer provides a suitable environment for the keratinocytes to adhere to and to grow on the top of the artificial dermis (Fig. 1(C) and (D)). 3.2. Grafting of Integra® and FS-CE independently on Nude mice To evaluate the graft take of Integra® and FS-CE separately, both were grafted onto the back of athymic mice. The artificial dermis was grafted on an open wound and left in place for a period of 3 weeks. At the end of this period, the dermis was well integrated with the host tissue; cells from
Fig. 1. Appearance of the cultured epithelium applied on the top of Integra® artificial dermis after 2 days of culture. (A) Control-CE on Integra® without fibroblasts; (B) control-CE on Integra® with fibroblasts; (C) FS-CE on Integra® without fibroblasts; (D) FS-CE on Integra® with fibroblasts. Note the well-organized epithelium obtained with FS-CE (C, D) compared to control-CE (A, B) (90×).
716
B. Mis et al. / Burns 30 (2004) 713–719
Fig. 2. Histology cross-sections of Integra® and FS-CE before and after grafting. (A) Integra® artificial dermis; (B) Integra® 3 weeks after grafting on Nude mouse; (C) FS-CE; (D) FS-CE 11 days after grafting on Nude mice (90×).
the wound colonized the matrix as compared to the original material (Fig. 2(A) and (B)). FS-CE was grafted on the inner part of a dorsal skin flap of each mouse. The formation of a stratified epithelium resembling human epidermis was observed 9–11 days after grafting. The different epidermal layers were present and the epidermis was well attached to the wound bed (Fig. 2 (C) and (D)).
3.3. Combined grafts on Nude mice Three weeks after grafting, the artificial dermis was well integrated with the host tissue (Fig. 2(B)). Some neovessels were observed at the edges of the matrix. Host cells were far more numerous at the edge of the matrix (coming from the surrounding dermis) than in the matrix itself at the cen-
Fig. 3. Evaluation of the size of the wound on Nude mice up to 21 days after cultured epithelium grafting. Integra® was applied on all mice on day 0 and left undisturbed for 19 days. Then, no epithelium (open wound control), epithelium cultivated without fibrin (control-CE) or with fibrin (FS-CE) were applied onto Integra® artificial dermis.
B. Mis et al. / Burns 30 (2004) 713–719
717
Fig. 4. Histological appearance of the regenerated epidermis 21 days after grafting on Nude mice. (A) Integra® alone; (B) Integra® with control-CE; (C) Integra® with FS-CE (47×). Grafts take occurred only when FS-CE was applied onto Integra® as shown by the presence of the human keratinocyte marker, involucrin (white arrowheads).
718
B. Mis et al. / Burns 30 (2004) 713–719
ter of the wound (sparse colonization from underneath the matrix). Microbial infection has been observed in 7 mice out of 23 within 19 days after Integra® grafting. These mice were discarded and not grafted with cultured epithelia. Cultured epithelia (control and FS) were grafted 3 weeks after the implantation of dermal matrix. Biopsies were harvested from 15 and 21 days after grafting. The contraction rate of the initial wound size was significantly lower 2 weeks after grafting for the animals grafted with FS-CE compared to those grafted with control-CE or the open wound control (P < 0.05) (Fig. 3). Human involucrin antibody was used to identify human keratinocytes from mouse tissue. Human epidermis was identified at the wound site only in animals grafted with FS-CE (five out of six animals), no human epidermis was found with control-CE grafts (six out of six animals) (Fig. 4(A)–(C)). In FS-CE and control-CE grafts, some desquamation as well as edema could be observed macroscopically at 17 days post grafting, 2 days after bandage removal.
4. Discussion In case of third-degree burns, all damaged tissues need to be removed. The common procedure is to cover the wound with split thickness skin grafts for which donor sites are limited in severely burned patients. Usually, these grafts are overlaid with available meshed skin allografts [16]. Another procedure is to prepare the wound bed with meshed skin allografts before replacing the alloepidermis with cultured or meshed autologous skin within 3 weeks [17]. The aim of the experiments reported here was to demonstrate that the combined use of an artificial dermis and a FS-CEA could provide an efficient procedure to treat full-thickness wounds. Since 1984, Gallico and colleagues [3] have shown that third-degree burn patients can be saved by using cultured epithelium autografts (CEAs), and, the use of CEAs has saved thousands of patients around the world. Despite these results, one of the problems associated with the use of CEAs, is the difficulty of transferring the epithelial sheet onto the wound bed. This can be solved by the use of FS-CEAs [7]. FS-CEAs offer many complementarities to the artificial dermis. Fibrin is the product of an activated coagulation system, and it plays a major role during cutaneous wound healing [18]. It has been shown that the provisional fibrin matrix induces keratinocytes migration to cover the wound [19,20]. Fibrin glue has been widely used as an adhesive or delivery vehicle in plastic and reconstructive surgery [21]. The physical properties of fibrin greatly facilitate the preparation and transplantation of the CEAs, which had a high rate of take on humans [9,10]. The physical properties of the fibrin substrate greatly improve the transplantation and the manipulation of the CEAs and make this product “user friendly” in burn treatment and reconstructive and plastic surgery. On the other hand, Integra® provides a safe artificial skin. After removing the damaged skin, surgeons cover the
excised wound with it [21]. The temporary epidermal substitute layer is removed after 2–4 weeks when the host cells have colonized the dermal matrix. Then, an autograft must be applied to close the wound and restore the barrier function of the skin permanently. Yannas and Burke were the first to develop the use of this bilayered membrane on humans [6]. Other clinical studies have been performed and published elsewhere [22]. Numerous studies have been performed to evaluate the use of CEAs to epidermalize this dermal matrix [2]. Recently, Steven Boyce and colleagues [6] have shown in a human clinical trial that an autologous cultured skin (CCS-Cincinnati) grafted onto Integra® successfully regenerated the skin with excellent aesthetic and functional outcomes. In our study, we have shown that 83% of the FS-CE adheres onto the artificial dermis cultured in vitro whether if it was populated or not with human fibroblasts; this result compares to only 33% adherence for the control-CE populated with fibroblasts. Only sparse areas of the control-CE adhesion onto fibroblast unpopulated dermal matrix were observed. On one hand, this could be explained by the quality of the culture system. The fibrin sheet provides a natural substrate to the keratinocytes. The interaction of the cells with fibrin or some other component of the matrix such as fibronectin would create a microenvironment preserving cell growth. On the other hand, Dispase treatment used to detach the epithelium from the culture dish results in a significant loss of clonogenic cells [10]. Microbial infection has been observed in 7 mice out of 23 within 19 days after Integra® grafting. It has been reported elsewhere that artificial skin is prone to infection under the silicone layer [2,6]. All CEAs were grafted on non-infected mice. After grafting on Nude mice, retraction of the grafting area was two times less when the epithelium was grown in the presence of the fibrin sheet compare to the control epithelium. This result is related to the fact that no control epithelium was able to survive on the top of the artificial dermis. Therefore, the rate of contraction was high and identical to the control without epithelium. Overall, 83% of the human epithelium grown on fibrin was able to develop a normal skin on the top of the artificial dermis, while the epithelium control was absent 21 days after grafting. The quality of the FS-CE, the basement membrane molecules synthesized during the culture and co-grafted with the fibrin substrate allows rapid adhesion of the FS-CE onto the wound bed. These molecules are absent with the control epithelium. It may be possible that the fibronectin contained in the substrate and the fibrinopeptides resulting from the degradation of the fibrin substrate would induce angiogenesis and improve the take rate of the FS-CE. Another advantage of the FS-CE is its shelf-life compare to the control CE. Control cultured epithelium is very fragile, and its shelf-life is 48 h, while the shelf-life of the epithelium produced on fibrin is 5–7 days (data not shown) with an excellent recovery of keratinocyte colony-forming cells [9,10]. Sheridan and Tompkins said earlier [1] “A promising approach is to combine CEAs with one of the currently avail-
B. Mis et al. / Burns 30 (2004) 713–719
able dermal analogues”. We found that human keratinocytes cultured on a fibrin matrix greatly improve the adhesion and the development of an epidermis on the top of an artificial dermis. This new treatment approach should significantly improve the performance of total skin replacement for burns in the near future. Acknowledgements We would like to thank our colleagues at Isotis SA, Lausanne: Jacques Essinger for the continuous support of this project, Stephane Germain for the skillful animal work, and Nathaliane Burger and Marie Vauthey for technical assistance. This work was supported in full by Isotis SA. References [1] Sheridan RL, Tompkins RG. Skin substitutes in burns. Burns 1999;25(2):97–103. [2] Loss M, Wedler V, Kunzi W, Meuli-Simmen C, Meyer VE. Artificial skin, split-thickness autograft and cultured autologous keratinocytes combined to treat a severe burn injury of 93% of TBSA. Burns 2000;26(7):644–52. [3] Gallico 3rd GG, O’Connor NE, Compton CC, Kehinde O, Green H. Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med 1984;311(7):448–51. [4] De Luca M, Albanese E, Bondanza S, Megna M, Ugozzoli L, Molina F, et al. Multicentre experience in the treatment of burns with autologous and allogenic cultured epithelium fresh or preserved in a frozen state. Burns 1989;15(5):303–9. [5] Carsin H, Ainaud P, Le Bever H, Rives J, Lakhel A, Stephanazzi J, et al. Cultured epithelial autografts in extensive burn coverage of severely traumatized patients: a five year single-center experience with 30 patients. Burns 2000;26(4):379–87. [6] Burke JF, Yannas IV, Quinby Jr WC, Bondoc CC, Jung WK. Successful use of a physiologically acceptable artificial skin in the treatment of extensive burn injury. Ann Surg 1981;194(4):413– 28. [7] Boyce ST, Kagan RJ, Meyer NA, Yakuboff KP, Warden GD. The 1999 clinical research award. Cultured skin substitutes combined with Integra Artificial Skin to replace native skin autograft and allograft for the closure of excised full-thickness burns. J Burn Care Rehabil 1999;20(6):453–61.
719
[8] Ronfard V, Broly H, Mitchell V, Galizia JP, Hochart D, Chambon E, et al. Use of human keratinocytes cultured on fibrin glue in the treatment of burn wounds. Burns 1991;17:181. [9] Ronfard V, Barrandon Y. Keratinocytes colony-forming cells as determinants of the transplantability of human squamous epithelium cultivated on a fibrin substrate. Geaorg Thieme Verlag, Horch, Munster, Achauer, editors. Cultures human keratinocytes and tissue engineered skin substitutes, 2001. p. 52–59. [10] Pellegrini G, Ranno R, Stracuzzi G, Bondanza S, Guerra L, Zambruno G, et al. The control of epidermal stem cells (holoclones) in the treatment of massive full-thickness burns with autologous keratinocytes cultured on fibrin. Transplantation 1999;68:868. [11] Ronfard V, Rives JM, Neveux Y, Carsin H, Barrandon Y. Long term regeneration of human epidermis on third-degree burns transplanted with autologous cultured epithelium grown on a fibrin matrix. Transplantation 2000;70:1588. [12] Heimbach DM, Warden GD, Luterman A, Jordan MH, Ozobia N, Ryan CM, et al. Multicenter postapproval clinical trial of Integra dermal regeneration template for burn treatment. J Burn Care Rehabil 2003;24(1):42–8. [13] Wisser D, Steffes J. Skin replacement with a collagen-based dermal substitute, autologous keratinocytes and fibroblasts in burn trauma. Burns 2003;29(4):375–780. [14] Limat A, Hunziker T, Boillat C, Bayreuther K, Noser F. Post-mitotic human dermal fibroblasts efficiently support the growth of human follicular keratinocytes. J Invest Dermatol 1989;92(5):758–62. [15] Green H, Kehinde O, Thomas J. Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc Natl Acad Sci USA 1979;76(11):5665–8. [16] Alexander JW, MacMillan BG, Law E, Kittur DS. Treatment of severe burns with widely meshed skin autograft and meshed skin allograft overlay. J Trauma 1981;21(6):433–8. [17] Cuono CB, Langdon R, Birchall N, Barttelbort S, Mc Guire J. Composite autologous-allogeneic skin replacement: development and clinical application. Plast Reconstr Surg 1987;80(4):626–37. [18] Barrandon Y, Li V, Green H. New techniques for the grafting of cultured human epidermal cells onto athymic animals. J Invest Dermatol 1988;91(4):315–8. [19] Martin P. Wound healing—aiming for perfect skin regeneration. Science 1997;276(5309):75–81. [20] Odland G, Ross R. Human wound repair. I. Epidermal regeneration. J Cell Biol 1968;39(1):135–51. [21] Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med 1999;341(10):738–46. [22] Yannas IV, Lee E, Orgill DP, Skrabut EM, Murphy GF. Synthesis and characterization of a model extracellular matrix that induces partial regeneration of adult mammalian skin. Proc Natl Acad Sci USA 1989;86(3):933–7.
Combined use of a collagen-based dermal substitute and a fibrin-based cultured epithelium: a step toward a total skin replacement for acute wounds Béatrice Mis∗ , Eric Rolland, Vincent Ronfard Isotis SA, 18-20 Avenue de Sévelin, 1004 Lausanne, Switzerland Accepted 26 April 2004
Abstract Integra® , a dermal replacement, is used as an immediate and temporary coverage for acute wounds, after which, autograft is used to reconstitute permanently the epidermal coverage. The fibrin sheet-cultured epithelium autograft (FS-CEA) could provide an effective alternative to the surgical procedure. To evaluate this hypothesis, we compared the association of Integra® /FS-CE to Integra® /control-cultured epithelium (control-CE). Their respective abilities: (1) to produce dermal–epidermal construct in vitro; (2) to generate skin replacement when grafted onto athymic mice were studied. We have shown that: (1) 83% of the FS-CE attached to the artificial dermis in vitro compared to only 33% for control-CE; (2) retraction of the grafted area was significantly lower 2 weeks after grafted with FS-CE than with the control-CE (P < 0.05); (3) 83% of the mice grafted with FS-CE showed the presence of a differentiated human epidermis 21 days after grafting, while such an epidermis was absent in all the animals of the control-CE group. We found that the use of FS-CE greatly improved adhesion, development of the epithelium and graft take onto the artificial dermis. We believe this technology should significantly improve the performance of dermal–epidermal skin replacement for acute wounds. © 2004 Elsevier Ltd and ISBI. All rights reserved.
1. Introduction Today, patients with burns encompassing 90% of their body surface can survive because of improvements in burn management and the development of new technologies [1,2]. Full-thickness burns, which extend to the skin’s deepest layers, require immediate care to prevent infection and fluid loss. The first step of treatment is to debride all of the tissue that has been burned. Once this is done, allografts are often used to cover the wounds, temporarily protecting the body until autologous skin is available from donor sites on the unburned body area. If the patient does not have enough donor skin to cover the wound, the physician may need to consider autologous cell culture [3,4] and tissue engineering [5,6] in order to save the patient’s life. Despite many advantages, allografts from cadavers are not always available to surgeons, their quality can vary from donor to donor, and, they may carry the risk of potential adventitious viral
∗
Corresponding author. Fax: +41 21 620 6060. E-mail address: [email protected] (B. Mis).
0305-4179/$30.00 © 2004 Elsevier Ltd and ISBI. All rights reserved. doi:10.1016/j.burns.2004.04.007
transmission. Therefore, a skin substitute to replace allografts is needed. We recently evaluated the performance of a combined utilization of two classes of biomaterials to cover the wound: (1) an artificial skin made of silicone, collagen and glycosaminoglycan and (2) an autologous epithelium grown on a fibrin sheet. [7,8] These products have been used separately in clinics with good clinical outcomes [9–12]. Integra® artificial skin can be applied immediately and provides a temporary coverage for 2–4 weeks, during which time the patient’s own cells make their way into the scaffold. This allows time to produce fibrin sheet-cultured epithelium autograft over about 2–3 weeks. FS-CEA has considerable advantages [10]. One of them is that the fibrin sheet provides a biodegradable matrix that plays an important role for epithelial attachment, survival and development onto the neodermis. To validate the combined utilization of the two biomaterials, we evaluated the performance of human keratinocytes grown on a fibrin sheet when applied to the artificial dermis. We evaluated its ability to generate: (1) a coherent dermal–epidermal construct in vitro, and (2) its ability to generate a skin replacement when grafted onto athymic mice.
714
B. Mis et al. / Burns 30 (2004) 713–719
2. Materials and methods
2.5. Combined in vivo grafting procedures
2.1. Culture
Twenty-three athymic mice (NIH Swiss Nude) approximately 6–8 weeks in age (24–32 g in weight) were used for experiments according to Swiss regulations for the care and use of laboratory animals. They were anesthetized by 2.5% isoflurane inhalation and a full thickness skin wound of approximately 1.7 cm2 was made on the central dorsum of each animal and covered with Integra® . Then, Vaseline gauze and adhesive bandages were applied onto the graft. Dressings were changed after 7 days. The silicone membrane was removed from the artificial dermis and cultured epithelia were grafted 3 weeks later (six mice for control-CE and six mice for FS-CE). A silicone membrane was used to transfer control-CE and FS-CE onto the wound bed. Four mice were not grafted with any CE and used as open wound controls. Two weeks after cultured epithelia grafting, dressings were removed and the grafts were left uncovered. Biopsies were harvested from 15 and 21 days after grafting. The size of the graft was measured on each animal with a caliper (TESA digit-cal CAPA RS 150 mm).
Keratinocytes were cultured according to the Rheinwald and Green method as modified by Limat and colleagues [3,13]. Briefly, keratinocytes were cultured in the presence of growth arrested irradiated human fibroblasts, in F-12/DME medium, added in a 1:3 ratio, containing adenine at 1.8 × 10−4 M, 10% fetal bovine serum (FBS, Gibco, Life Technologies), hydrocortisone 0.5 ng/ml, insulin 5 mg/ml, triiodothyronin 2 × 10−9 M, choleratoxin 10 ng/ml and epidermal growth factor 10 ng/ml (Sigma). Human keratinocytes were obtained from a skin biopsy of an adult undergoing plastic surgery. They were amplified and used in the third passage. 2.2. Control-CE When the cultures reached confluence, they were treated with Dispase as previously described [14] in order to detach the epithelial sheet from the bottom of the dish. Then, with the help of a silicone membrane (Perthese® , Perouse-Plastie, France), the epithelial sheet was lifted off the plate and applied onto the artificial dermis. 2.3. FS-CE The fibrin substrate was made from freeze-dried surgical fibrinogen prepared from human plasma obtained from blood donors (Tissucol® kit, Baxter). The freeze-dried fibrinogen from a 2 ml Tissucol® kit was reconstituted with 5 ml of a sodium chloride (2%) solution containing 1 mM of calcium chloride. It was then mixed (v/v) with sodium chloride 2.2% solution containing 2 mM calcium chloride human thrombin (2.5 IU/ml) and distributed into culture dishes before clotting occurred. Human keratinocytes were cultivated on the top of the fibrin matrix in the presence of aprotinin (Trasylol® , Bayer), as previously described [10]. When the culture reached confluence, the epithelial sheet along with the fibrin was applied to the artificial dermis. 2.4. Combined in vitro cultures Integra® pieces (0.8 cm × 0.8 cm) were seeded with 2 × human dermal fibroblasts and cultured for 5 days. They were fed every other day with the medium as described for the keratinocytes except that the medium did not contain any choleratoxin. Vitamin C (ascorbic acid, Sigma, 50 g/ml) was added at each medium change. Then, the artificial dermis was placed on inserts (0.78 cm2 of culture surface and 0.4 m of membrane pore size, Corning), so that they were fed from underneath. Control epithelium and FS-CE were placed on the top of the artificial dermis and covered with a silicone membrane.
105
2.6. Integra® and FS-CEA grafting procedures Athymic mice (NIH Swiss Nude) approximately 6–8 weeks in age (24–32 g in weight) were used for experiments according to the Swiss regulations for the care and use of laboratory animals and were anesthetized as previously described. Twenty-three animals were grafted with Integra® alone applied to an open wound (1.7 cm2 ) and left in place for 3 weeks. Then, biopsies were harvested and processed for histological evaluation. Independently, ten other animals were grafted with FS-CE alone transplanted on the inner part of a dorsal skin flap of athymic mice as previously described [15]. 2.7. Histology In vitro samples: samples were fixed in Lang solution (10% acetic acid in sublimated formol). After dehydration, samples were embedded in resin (Basic Historesin and Hardener, Leica) and stained with haematoxylin and eosin. In vivo samples: biopsies from grafts were fixed in buffered formalin (5%) and embedded in paraffin wax. Five micrograms sections were stained with haematoxylin and eosin. Immunochemical staining was performed on deparaffinized and rehydrated sections. Human involucrin was detected using an immuno-kit consisting of highly specific human involucrin rabbit antibody and the necessary reagents to peroxidase staining (BT-600, Biomedical Technology Inc. Stoughton, USA). Hematoxylin was used for counterstaining. This kit was used to identify the human origin of the regenerated epidermis from the mouse tissue.
B. Mis et al. / Burns 30 (2004) 713–719
3. Results 3.1. Combined in vitro cultures Behavior of the cultured epithelium combined with the artificial dermis was examined in vitro. Pieces of artificial dermis (0.8 cm × 0.8 cm) without cells or pre-seeded with human dermal fibroblasts were placed on inserts and covered with cultured epithelia grown in the presence or in the absence of fibrin (FS-CE or Control-CE respectively). After 2 and 5 days of culture, histological cross-sections of the cultured epithelia showed that the presence of fibroblasts into the dermal matrix improved the attachment of the epithelium onto the artificial dermis. The control-CE did attach more firmly when the dermal matrix was populated with cells compared to unpopulated matrix. In all cases, the edges of the epithelium were unattached and were often folded. Numerous dying cells with pyknotic nuclei and differentiating cells were seen all along the dermal–epidermal junction. This indicates that the “in vitro take” of the control-CE onto the dermal matrix was poor (Fig. 1(A) and (B)). In
715
comparison, FS-CE did attach well onto the matrix (83%, n = 12, of attached FS-CE versus 33% for control-CE, n = 12); keratinocytes migrated from the fibrin layer to the matrix. FS-CE adheres uniformly on both fibroblast populated and unpopulated dermis matrix. Close junctions were seen between the fibrin layer and fibroblasts in the fibroblast populated matrices. The epithelial layer was made up of normal proliferating cells: small cells with round nucleus, no intercellular spaces or intracellular vacuoles as seen in the control epithelia. A fibrin layer provides a suitable environment for the keratinocytes to adhere to and to grow on the top of the artificial dermis (Fig. 1(C) and (D)). 3.2. Grafting of Integra® and FS-CE independently on Nude mice To evaluate the graft take of Integra® and FS-CE separately, both were grafted onto the back of athymic mice. The artificial dermis was grafted on an open wound and left in place for a period of 3 weeks. At the end of this period, the dermis was well integrated with the host tissue; cells from
Fig. 1. Appearance of the cultured epithelium applied on the top of Integra® artificial dermis after 2 days of culture. (A) Control-CE on Integra® without fibroblasts; (B) control-CE on Integra® with fibroblasts; (C) FS-CE on Integra® without fibroblasts; (D) FS-CE on Integra® with fibroblasts. Note the well-organized epithelium obtained with FS-CE (C, D) compared to control-CE (A, B) (90×).
716
B. Mis et al. / Burns 30 (2004) 713–719
Fig. 2. Histology cross-sections of Integra® and FS-CE before and after grafting. (A) Integra® artificial dermis; (B) Integra® 3 weeks after grafting on Nude mouse; (C) FS-CE; (D) FS-CE 11 days after grafting on Nude mice (90×).
the wound colonized the matrix as compared to the original material (Fig. 2(A) and (B)). FS-CE was grafted on the inner part of a dorsal skin flap of each mouse. The formation of a stratified epithelium resembling human epidermis was observed 9–11 days after grafting. The different epidermal layers were present and the epidermis was well attached to the wound bed (Fig. 2 (C) and (D)).
3.3. Combined grafts on Nude mice Three weeks after grafting, the artificial dermis was well integrated with the host tissue (Fig. 2(B)). Some neovessels were observed at the edges of the matrix. Host cells were far more numerous at the edge of the matrix (coming from the surrounding dermis) than in the matrix itself at the cen-
Fig. 3. Evaluation of the size of the wound on Nude mice up to 21 days after cultured epithelium grafting. Integra® was applied on all mice on day 0 and left undisturbed for 19 days. Then, no epithelium (open wound control), epithelium cultivated without fibrin (control-CE) or with fibrin (FS-CE) were applied onto Integra® artificial dermis.
B. Mis et al. / Burns 30 (2004) 713–719
717
Fig. 4. Histological appearance of the regenerated epidermis 21 days after grafting on Nude mice. (A) Integra® alone; (B) Integra® with control-CE; (C) Integra® with FS-CE (47×). Grafts take occurred only when FS-CE was applied onto Integra® as shown by the presence of the human keratinocyte marker, involucrin (white arrowheads).
718
B. Mis et al. / Burns 30 (2004) 713–719
ter of the wound (sparse colonization from underneath the matrix). Microbial infection has been observed in 7 mice out of 23 within 19 days after Integra® grafting. These mice were discarded and not grafted with cultured epithelia. Cultured epithelia (control and FS) were grafted 3 weeks after the implantation of dermal matrix. Biopsies were harvested from 15 and 21 days after grafting. The contraction rate of the initial wound size was significantly lower 2 weeks after grafting for the animals grafted with FS-CE compared to those grafted with control-CE or the open wound control (P < 0.05) (Fig. 3). Human involucrin antibody was used to identify human keratinocytes from mouse tissue. Human epidermis was identified at the wound site only in animals grafted with FS-CE (five out of six animals), no human epidermis was found with control-CE grafts (six out of six animals) (Fig. 4(A)–(C)). In FS-CE and control-CE grafts, some desquamation as well as edema could be observed macroscopically at 17 days post grafting, 2 days after bandage removal.
4. Discussion In case of third-degree burns, all damaged tissues need to be removed. The common procedure is to cover the wound with split thickness skin grafts for which donor sites are limited in severely burned patients. Usually, these grafts are overlaid with available meshed skin allografts [16]. Another procedure is to prepare the wound bed with meshed skin allografts before replacing the alloepidermis with cultured or meshed autologous skin within 3 weeks [17]. The aim of the experiments reported here was to demonstrate that the combined use of an artificial dermis and a FS-CEA could provide an efficient procedure to treat full-thickness wounds. Since 1984, Gallico and colleagues [3] have shown that third-degree burn patients can be saved by using cultured epithelium autografts (CEAs), and, the use of CEAs has saved thousands of patients around the world. Despite these results, one of the problems associated with the use of CEAs, is the difficulty of transferring the epithelial sheet onto the wound bed. This can be solved by the use of FS-CEAs [7]. FS-CEAs offer many complementarities to the artificial dermis. Fibrin is the product of an activated coagulation system, and it plays a major role during cutaneous wound healing [18]. It has been shown that the provisional fibrin matrix induces keratinocytes migration to cover the wound [19,20]. Fibrin glue has been widely used as an adhesive or delivery vehicle in plastic and reconstructive surgery [21]. The physical properties of fibrin greatly facilitate the preparation and transplantation of the CEAs, which had a high rate of take on humans [9,10]. The physical properties of the fibrin substrate greatly improve the transplantation and the manipulation of the CEAs and make this product “user friendly” in burn treatment and reconstructive and plastic surgery. On the other hand, Integra® provides a safe artificial skin. After removing the damaged skin, surgeons cover the
excised wound with it [21]. The temporary epidermal substitute layer is removed after 2–4 weeks when the host cells have colonized the dermal matrix. Then, an autograft must be applied to close the wound and restore the barrier function of the skin permanently. Yannas and Burke were the first to develop the use of this bilayered membrane on humans [6]. Other clinical studies have been performed and published elsewhere [22]. Numerous studies have been performed to evaluate the use of CEAs to epidermalize this dermal matrix [2]. Recently, Steven Boyce and colleagues [6] have shown in a human clinical trial that an autologous cultured skin (CCS-Cincinnati) grafted onto Integra® successfully regenerated the skin with excellent aesthetic and functional outcomes. In our study, we have shown that 83% of the FS-CE adheres onto the artificial dermis cultured in vitro whether if it was populated or not with human fibroblasts; this result compares to only 33% adherence for the control-CE populated with fibroblasts. Only sparse areas of the control-CE adhesion onto fibroblast unpopulated dermal matrix were observed. On one hand, this could be explained by the quality of the culture system. The fibrin sheet provides a natural substrate to the keratinocytes. The interaction of the cells with fibrin or some other component of the matrix such as fibronectin would create a microenvironment preserving cell growth. On the other hand, Dispase treatment used to detach the epithelium from the culture dish results in a significant loss of clonogenic cells [10]. Microbial infection has been observed in 7 mice out of 23 within 19 days after Integra® grafting. It has been reported elsewhere that artificial skin is prone to infection under the silicone layer [2,6]. All CEAs were grafted on non-infected mice. After grafting on Nude mice, retraction of the grafting area was two times less when the epithelium was grown in the presence of the fibrin sheet compare to the control epithelium. This result is related to the fact that no control epithelium was able to survive on the top of the artificial dermis. Therefore, the rate of contraction was high and identical to the control without epithelium. Overall, 83% of the human epithelium grown on fibrin was able to develop a normal skin on the top of the artificial dermis, while the epithelium control was absent 21 days after grafting. The quality of the FS-CE, the basement membrane molecules synthesized during the culture and co-grafted with the fibrin substrate allows rapid adhesion of the FS-CE onto the wound bed. These molecules are absent with the control epithelium. It may be possible that the fibronectin contained in the substrate and the fibrinopeptides resulting from the degradation of the fibrin substrate would induce angiogenesis and improve the take rate of the FS-CE. Another advantage of the FS-CE is its shelf-life compare to the control CE. Control cultured epithelium is very fragile, and its shelf-life is 48 h, while the shelf-life of the epithelium produced on fibrin is 5–7 days (data not shown) with an excellent recovery of keratinocyte colony-forming cells [9,10]. Sheridan and Tompkins said earlier [1] “A promising approach is to combine CEAs with one of the currently avail-
B. Mis et al. / Burns 30 (2004) 713–719
able dermal analogues”. We found that human keratinocytes cultured on a fibrin matrix greatly improve the adhesion and the development of an epidermis on the top of an artificial dermis. This new treatment approach should significantly improve the performance of total skin replacement for burns in the near future. Acknowledgements We would like to thank our colleagues at Isotis SA, Lausanne: Jacques Essinger for the continuous support of this project, Stephane Germain for the skillful animal work, and Nathaliane Burger and Marie Vauthey for technical assistance. This work was supported in full by Isotis SA. References [1] Sheridan RL, Tompkins RG. Skin substitutes in burns. Burns 1999;25(2):97–103. [2] Loss M, Wedler V, Kunzi W, Meuli-Simmen C, Meyer VE. Artificial skin, split-thickness autograft and cultured autologous keratinocytes combined to treat a severe burn injury of 93% of TBSA. Burns 2000;26(7):644–52. [3] Gallico 3rd GG, O’Connor NE, Compton CC, Kehinde O, Green H. Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med 1984;311(7):448–51. [4] De Luca M, Albanese E, Bondanza S, Megna M, Ugozzoli L, Molina F, et al. Multicentre experience in the treatment of burns with autologous and allogenic cultured epithelium fresh or preserved in a frozen state. Burns 1989;15(5):303–9. [5] Carsin H, Ainaud P, Le Bever H, Rives J, Lakhel A, Stephanazzi J, et al. Cultured epithelial autografts in extensive burn coverage of severely traumatized patients: a five year single-center experience with 30 patients. Burns 2000;26(4):379–87. [6] Burke JF, Yannas IV, Quinby Jr WC, Bondoc CC, Jung WK. Successful use of a physiologically acceptable artificial skin in the treatment of extensive burn injury. Ann Surg 1981;194(4):413– 28. [7] Boyce ST, Kagan RJ, Meyer NA, Yakuboff KP, Warden GD. The 1999 clinical research award. Cultured skin substitutes combined with Integra Artificial Skin to replace native skin autograft and allograft for the closure of excised full-thickness burns. J Burn Care Rehabil 1999;20(6):453–61.
719
[8] Ronfard V, Broly H, Mitchell V, Galizia JP, Hochart D, Chambon E, et al. Use of human keratinocytes cultured on fibrin glue in the treatment of burn wounds. Burns 1991;17:181. [9] Ronfard V, Barrandon Y. Keratinocytes colony-forming cells as determinants of the transplantability of human squamous epithelium cultivated on a fibrin substrate. Geaorg Thieme Verlag, Horch, Munster, Achauer, editors. Cultures human keratinocytes and tissue engineered skin substitutes, 2001. p. 52–59. [10] Pellegrini G, Ranno R, Stracuzzi G, Bondanza S, Guerra L, Zambruno G, et al. The control of epidermal stem cells (holoclones) in the treatment of massive full-thickness burns with autologous keratinocytes cultured on fibrin. Transplantation 1999;68:868. [11] Ronfard V, Rives JM, Neveux Y, Carsin H, Barrandon Y. Long term regeneration of human epidermis on third-degree burns transplanted with autologous cultured epithelium grown on a fibrin matrix. Transplantation 2000;70:1588. [12] Heimbach DM, Warden GD, Luterman A, Jordan MH, Ozobia N, Ryan CM, et al. Multicenter postapproval clinical trial of Integra dermal regeneration template for burn treatment. J Burn Care Rehabil 2003;24(1):42–8. [13] Wisser D, Steffes J. Skin replacement with a collagen-based dermal substitute, autologous keratinocytes and fibroblasts in burn trauma. Burns 2003;29(4):375–780. [14] Limat A, Hunziker T, Boillat C, Bayreuther K, Noser F. Post-mitotic human dermal fibroblasts efficiently support the growth of human follicular keratinocytes. J Invest Dermatol 1989;92(5):758–62. [15] Green H, Kehinde O, Thomas J. Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc Natl Acad Sci USA 1979;76(11):5665–8. [16] Alexander JW, MacMillan BG, Law E, Kittur DS. Treatment of severe burns with widely meshed skin autograft and meshed skin allograft overlay. J Trauma 1981;21(6):433–8. [17] Cuono CB, Langdon R, Birchall N, Barttelbort S, Mc Guire J. Composite autologous-allogeneic skin replacement: development and clinical application. Plast Reconstr Surg 1987;80(4):626–37. [18] Barrandon Y, Li V, Green H. New techniques for the grafting of cultured human epidermal cells onto athymic animals. J Invest Dermatol 1988;91(4):315–8. [19] Martin P. Wound healing—aiming for perfect skin regeneration. Science 1997;276(5309):75–81. [20] Odland G, Ross R. Human wound repair. I. Epidermal regeneration. J Cell Biol 1968;39(1):135–51. [21] Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med 1999;341(10):738–46. [22] Yannas IV, Lee E, Orgill DP, Skrabut EM, Murphy GF. Synthesis and characterization of a model extracellular matrix that induces partial regeneration of adult mammalian skin. Proc Natl Acad Sci USA 1989;86(3):933–7.