Abstract: The authors describe three years of clinical experience in chronic wound management. The authors treated 315 patients (age 65–75 years) with chronic recurrent leg ulcers by allograft of cryopreserved dermis. Patients were divided into two groups (A and B) on the basis of patient preference and underwent two different reepithelization techniques. Patients in group A received autologous thin mesh grafts. Patients in group B received cultured autologous epidermal grafts. Proportions were compared by univariate analysis (c2 test with the Yates correction; EpiInfo version 5). P values <0.05 were considered significant. The cultured autologous epidermal graft (group B) was significantly more effective at one-month follow up (p=0.023, odd ratio=1.85), but no differences were seen at 6 and 12 months. Dermal allografts were obtained from Siena skin bank (Tuscan Region Tissue Bank). Autologous keratinocytes were obtained by skin biopsy from the buttocks of patients. Patients were treated as outpatients. At one-year follow up, complete wound closure was observed in 75 percent of the patients; recurrences occurred in six percent of patients.

Introduction

Chronic ulcers of the lower limbs are a major cause of morbidity in European countries, affecting 0.5 to 1.5 percent of the population. Lower-extremity chronic ulcers affect 3 to 5 percent of persons over 60 years of age, with a male:female ratio of 1:2. The most frequent causes are venous failure (70–90% of cases), arterial failure, and metabolic diseases, especially diabetes.[1]

Treatment is usually problematical, requiring a multidisciplinary approach to obtain healing and reduce morbidity. Even with a correct diagnostic-therapeutic approach, about 30 percent of leg ulcers suffer relapse.[2]

Besides treating the underlying pathology and vascular failure, medication of the ulcer to prepare the wound bed for grafting is necessary. Autologous skin grafts are the elective surgical therapy for skin repair; however, frequent recurrences and problems connected with repair of donor areas make it necessary to consider other surgical methods for permanent wound coverage. Reconstruction of both the epidermal and dermal compartments is another essential condition for stable functional repair of full-thickness ulcers. The utility of homologous dermis for this purpose has long been known.[3,4] The low immunogenicity of the fibrous dermal component ensures good taking and rapid colonization by host cells, at the same time preventing contraction of the graft and scarring.[5] Unlike bioengineered skin replacements, which are absorbed into the wound bed, homologous dermis has delayed absorption (more than 4 weeks),[6] and its collagen and elastic fibers remain integral forming scaffolding, which acts as a permanent template for infiltrating host cells.[7] Compared to bioenginereed skin equivalents, which are costly and suitable only for small wounds/grafts, sandwich techniques based on use of homologous de-epidermized dermis (~1/cm2) and autologous epithelial components are valid and less expensive alternatives for recalcitrant leg ulcers.

Materials and Methods

Patients. In 2000 through 2002, 315 patients (65–81 years of age) with chronic, recurrent leg ulcers were treated by allograft of cryopreserved de-epidermized dermis followed by mesh graft of autologous skin or graft of cultured autologous epidermis. In the authors’ study population, 91 percent of patients suffered from venous leg ulcers, and nine percent of patients had ulcers of combined etiology.[8] All patients had extensive skin loss (>100cm2). Patients were divided into two groups (A and B) on the basis of patient preference. Group A consisted of 150/315 patients (47.6%) who underwent dermal allograft and autologous mesh graft. Group B consisted of 165/315 patients (52.3%) who underwent dermal allograft and cultured autologous epidermis in order to reduce total donor site area. The latter had received autologous grafts in the past and denied consent to the extensive sampling necessary for mesh grafting. Informed consent was required.

All patients had standard therapy for venous ulcers consisting of saline wound irrigation, surgical debridement, appropriate dressing selected on the basis of clinical evidence, and elastic compression. Associated diseases (such as diabetes, infections, hematological disorders) were treated specifically. Ischemic arterial ulcers were excluded from the protocol. The procedure before grafting involved surgical debridement of the ulcers and topical and/or systemic antibiotic treatment in the case of concomitant super infection to obtain granulation tissue suitable to receive a graft. An allograft of cryopreserved homologous dermis, immobilized by sterile strips, was then placed on the wound, held firmly in place, and kept at ideal humidity and temperature with suitable absorbing dressing. In patients with full-thickness ulcers, multiple dermal allografts were performed. Five to seven days after the last allograft, an autologous thin mesh graft (6:1) or a cultured autologous epidermis graft was applied if donor areas were suitable. Patients could then be treated as outpatients with weekly medication, which consisted of petrolatum gauze held in place with slight compression. Follow up was performed after 1, 6, and 12 months.

Methods. Keratinocyte cultures. A superficial skin biopsy (400µm) of about 4cm2 was obtained from the buttock with an electric dermatome (Colibrì Intramatic 4000, 100 Watt). The biopsies were sent to the authors’ laboratory where they were used to grow primary epidermal cultures by the classical method.[9]

Briefly, the tissue was washed in phosphate buffered saline (PBS; Sigma) supplemented with penicillin/streptomycin (Sigma) and cut into thin strips, which were placed in Petri dishes containing a solution of trypsin (0.5g/L) and ethylenediaminotetracetic acid (EDTA 0.2g/L; Sigma). The dishes were incubated for at least three hours at 37 degrees C under five-percent CO2. A cell suspension was obtained by scraping the tissue on the dermal side. The cells were washed in Dulbecco-Modified Eagle’s Medium (D-MEM; Sigma) supplemented with antibiotics, 10-percent patient’s serum, and L-glutamine (Sigma) and then resuspended in 15mL culture epithelial cell medium (CEC) consisting of D-MEM/Ham’s F12 (3:1) with 10-percent patient’s serum, 0.584mg/mL L-glutamine (Sigma), 100 IU/mL penicillin (Sigma), 100mg/mL streptomycin (Sigma), 0.4µg/mL hydrocortisone succinate (Sigma), 5µg/mL insulin (Sigma), 5µg/mL transferrin (Sigma), 2 x 10-9 M triiodothyronine (Sigma), 10-10M cholera toxin (Sigma), and 1.8x104M adenine (Sigma) without epidermal growth factor (EGF). The cell suspension was seeded at a density of 30,000 cells/cm2 in 75cm2 culture flasks (Falcon) prepared with lethally irradiated 3T3 fibroblasts (density 20,000 cells/cm2). Subconfluent primary cultures were detached from the flasks with trypsin/EDTA. Secondary cultures were obtained from the resulting cell suspension, seeded at a density of 30,000 to 50,000 cells/cm2 on 10x10cm carrier sheets of hyaluronic acid ester polymer (Laserskin®, Fidia Advanced Biopolymers, Italy) in square Petri dishes prepared the day before with lethally irradiated 3T3 fibroblasts. The medium (CEC with EGF) was changed every two days until subconfluence. The sheets were placed in CEC medium without cholera toxin for 48 hours before grafting. Two weeks later the autologous keratinocytes on the carrier sheets were grafted.

Homologous de-epidermized dermis. De-epidermized dermis was obtained from Siena Skin Bank (Tuscan Region Tissue Bank). The skin was harvested by electric dermatome (0.5–0.8mm). Allografts were processed according to European Association of Tissue Banking (EATB) standards. Safety controls of allografts included tests for human immunodeficiency virus, hepatitis viruses (serological screening and polymerase chain reaction), human T-lymphotropic virus I/II, cytomegalovirus, and syphilis, as well as microbial and fungal contamination. De-epidermization was performed with one molar sodium chloride supplemented with antibiotics for three to eight hours at 37 degrees C, and the epidermis was removed using sterile forceps. The allografts were cryopreserved according to Stephen Kirby Skin Bank (Queen Mary’s Hospital, London) protocols, using 15-percent glycerol, amphotericin B, L-glutamine, gentamycin, pen/strept, and D-MEM.

Results

The area of the lesions was calculated by placing sterile film (OpSite™, Smith & Nephew Inc., Largo, Florida) over the ulcer and tracing the perimeter, which was then transferred to millimeter graph paper. This was done before treatment and at each follow up. Statistical analysis was performed by univariate analysis using the c2 test with Yates correction (EpiInfo version 5).

Wound closure (100% ulcer area reduction) was achieved in 71/315 cases (22.5%) at one-month follow up, 265/315 (84.1%) at six-month follow up, and 236/315 (74.9%) at 12-month follow up (Figures 1 and 2). Patients healed at one-month follow up included 26 in group A and 45 in group B; at 12-month follow up, they included 125 patients of group A and 111 patients of group B (Figures 3–6). The two methods did not show statistically significant differences except in the one-month follow-up period, when patients treated with cultured autologous epidermis showed significantly faster re-epithelization (p=0.023, odds ratio=1.85). Dermal reconstruction in vast ulcers required several allografts to obtain viable granulation tissue. Recurrences were observed at 12-month follow up in 18/315 patients (11 patients of group A and 7 of group B).

Figure 1

At the 12-month follow up, wound closure was achieved in 236 (75%) out of 315 patients, recurrence in 18 (6%) out of 315 patients, nonhealing in 61 (19%) out of 315 patients.

Figure 2

This graph shows the percentage of patients healed after 1, 6, and 12 months

Figure 3A

Figure 3B

Figure 3C

Figure 3. These photographs are of a 77-year-old patient treated with dermal allograft and autologous mesh graft: (A) before treatment; (B) one month after grafting; and (C) six months after grafting.

Figure 4A

Figure 4B

Figure 4. These photographs are of a 70-year-old patient treated with dermal allograft and cultured epidermis: (A) before grafting and (B) six months after grafting.

Figure 5A

Figure 5B

Figure 5. These photographs are of a 68-year-old patient treated with dermal allograft and cultured epidermis: (A) before grafting and (B) six months after grafting.

Figure 6A

Figure 6B

Figure 6. These photographs are of an 80-year-old woman treated with homologous dermis and autologous mesh graft: (A) before grafting and (B) 12 months after grafting.

Sixty-one patients (19%) did not respond to therapy. Fifty-four out of 61 patients had associated arterial involvement or connective tissue disease. Six patients dropped out due to side effects: in one case the authors observed immediate allergic reaction, probably to the antibiotics used for skin processing, and in five cases staph or streptococcal super-infection resistant to antibiotics used in the protocol. A 73-year-old patient died of heart failure.

Discussion

Autologous skin transplants are still the gold standard for accelerating healing in cases of extensive skin loss. However, when donor sites are lacking, combined transplant of autografts and allografts can be a successful surgical method for full-thickness wounds. The aim of the present study was to treat large, chronic, nonhealing leg ulcers by a composite skin graft technique employing cryopreserved allodermis and autologous epidermal cultures. Allodermis was used to guide and facilitate formation of granulation tissue and wound healing by host cell colonization of the dermal graft. Epithelization was achieved by autologous grafting. The two methods of reepithelization did not show statistically significant differences in efficacy at follow up after six and 12 months, but cultured autologous keratinocytes were more effective than autograft at one-month follow up, showing faster reepithelization. This may be due to the fact that cultured layers have a higher cell density than mesh grafts, which require migration of epidermal cells into the holes of the mesh. However, tissue arising from reepithelization with cultured cells is more fragile than autologous skin grafts.

Homologous skin obtained from cadavers has been used to treat severe burn patients since the second half of the 19th century.[10,11] Use of allografts to treat these patients has recently become accepted in clinical practice and has been extended to conditions characterized by extensive skin loss, such as ulcers, post-traumatic and post-surgical wounds, pressure ulcers, Lyell syndrome, congenital epidermolytic diseases, and other autoimmune diseases.[12,13]

In ulcer therapy, donor skin has offered new ways of using cultured epidermis in composite grafting, consisting of homologous skin/DED and cultured autologous epidermis.[14] Indeed, to be successful, grafts of autologous laminas of epidermis reconstructed in vitro require an integral dermis: the dermis protects the basal layer of the epidermis and is important for biological remodelling after the graft.[15] It has been observed in various studies that epidermal autografts are of limited utility and have a very low success rate if not associated with a dermal component.[15]

In cases of full-thickness ulcers or those in which autologous grafting cannot be done, it is important first to restore the dermal component by application of human DED or dermal equivalents, then to bring about reepithelization by thin autologous grafts or keratinocyte lamina.[16] The advantage of DED over homologous skin is that it avoids the need to remove the allograft epidermis five days after grafting, enabling direct use of a dermal component on which the cultured autologous epidermal component is then placed. Allodermis is also long lasting (more than 2.5 years) and accelerates skin
reconstruction.[7]

The authors’ complete wound closure rate (75%) was high, and the recurrence rate was lower than in other reports.[2] The authors’ mean reconstruction times (4 to 6 months) were also excellent for relapsing full-thickness ulcers already treated by other methods, especially considering that homologous dermis takes at least four weeks to vascularize.[6]

This technique makes it possible to reconstruct a viable, vascularized, nonscar dermis for autografting associated with a low rate of recurrence.[7] The two techniques of epithelization tested did not show substantial functional differences (p<0.10). Cultured autologous epidermal grafts may therefore be useful in cases of extensive ulcers when suitable donor areas are lacking, since with a biopsy of 3 to 4cm2, it is possibile to obtain more than 100cm2 of cultured epidermis, sparing skin graft donor sites. Side effects seldom occur. The authors’ results confirm the concept that allografts are contraindicated in cases of ischemic ulcers, which were excluded from this protocol; indeed, most nonhealing cases involved mixed ulcers with arteriovenous involvement.
Finally, use of the present techniques in various combinations can certainly be said to offer economic and social advantages. Medication is carried out once a week on an outpatient basis, whereas previously patients remained in hospital for long
periods.