Bilayered Bioengineered Skin Substitute (Apligraf®)

Summary

Abstract

The bilayered bioengineered skin substitute (BBSS) [Apligraf®] is used for the treatment of venous leg ulcers and diabetic foot ulcers. It has an epidermal layer formed from human keratinocytes and a dermal layer composed of human fibroblasts in a bovine type I collagen matrix. BBSS does not contain any antigen-presenting cells such as Langerhans cells, dermal dendritic cells, endothelial cells or leucocytes. In clinical trials, there was no evidence of clinical rejection and immunological tests indicated no humoral or cellular response to the keratinocytes or fibroblasts of BBSS. Further clinical trials are required to identify the exact mechanism of action of BBSS in chronic wounds.

BBSS plus compression therapy was well tolerated and was superior in efficacy to compression therapy alone in a multicentre, randomised trial in patients with venous leg ulcers. At 6 months’ follow-up, complete wound healing occurred in 63 versus 49% of patients and the median time to wound closure was 61 versus 181 days. In a subgroup of patients with hard-to-heal ulcers (>1 year’s duration), wound healing was achieved in significantly more patients (47 vs 19%) and the median time to wound healing was significantly shorter (181 days vs not attained).

In a multicentre, randomised trial, BBSS was well tolerated and effective in patients with full-thickness neuropathic diabetic foot ulcers. Ulcer healing occurred in significantly more patients (56 vs 38%) and the median time to wound healing was shorter (65 vs 90 days) with BBSS than with saline-moistened gauze at 12 weeks’ follow-up. Patients in both groups also received standard diabetic foot care.

The cost effectiveness of BBSS in patients with chronic ulcers has yet to be examined in well designed, prospective clinical trials. However, according to a modelled analysis incorporating data from a multicentre randomised trial, BBSS was cost effective in patients with hard-to-heal venous leg ulcers. The average annual medical cost of managing patients with ulcers of >1 year’s duration was estimated to be $US20 041 per patient treated with BBSS plus compression therapy and $US27 493 per patient treated with compression therapy alone (1996 costs).

Conclusions: Clinical trials have shown that BBSS in conjunction with standard compression therapy was effective and well tolerated in patients with venous leg ulcers, especially patients with ulcers of s >6 months’ duration or that extended to the subcutaneous tissue. In addition, BBSS in conjunction with standard diabetic foot care was effective and well tolerated in patients with full-thickness neuropathic diabetic foot ulcers. BBSS represents a useful adjuvant to standard ulcer therapy in patients with venous leg ulcers or full-thickness neuropathic diabetic foot ulcers that do not respond to conventional ulcer therapy.

Structural and Functional Properties

BBSS consists of an epidermal layer (formed from human keratinocytes) and a dermal layer (composed of active human fibroblasts in a bovine type I collagen matrix). The epidermis has a well-differentiated stratum corneum and well-defined spinous and granular layers. The basal layer divides at a rate comparable to that of human skin. The fibroblasts in the dermal layer are mitotically and metabolically active, and secrete collagen and other matrix components.

BBSS lacks many of the cells normally present in skin including melanocytes and mast cells, cells involved in the immune response (Langerhans cells, lymphocytes, macrophages and endothelial cells) and various skin structures (e.g. nerves, hair follicles, sebaceous glands, rete ridges and blood vessels). BBSS lacks a basement membrane.

In vitro BBSS provides a significant barrier to water and responds to irritants in a similar manner to human skin.

Manufacture

BBSS is manufactured under aseptic conditions from human neonatal foreskins that were obtained under informed-consent guidelines. The foreskins are aseptically cleaned, the fibroblasts and keratinocytes are isolated and then serially cultured under separate tissue culture conditions to establish cell banks.

Fibroblasts are then seeded on a semipermeable membrane along with bovine type I collagen. The fibroblasts divide, multiply and then contract the collagen filaments to form a dermal matrix after approximately 6 days. The epidermal layer is formed by seeding the keratinocytes onto the surface of this contracted dermal matrix and allowing them to proliferate. After 4 days, the skin culture is incubated at the air-liquid interface for 7 to 10 days to promote keratinocyte differentiation and the formation of a stratum corneum.

The keratinocytes and fibroblasts cell banks and the blood of the mother of the foreskin donor undergo screening for pathogens. The cell banks are also screened for tumorigenicity, chromosomal abnormalities and biochemical defects. The bovine type I collagen is extracted from cattle herds bred and raised within the US that are free from bovine spongiform encephalopathy.

Mechanism of Action

In chronic wounds, it is proposed that BBSS may aid healing by stimulating the healing process by supplying matrix materials, cytokines and other regulatory materials in the correct sequence and amount.

In a multicentre, randomised, nonblind study in 275 evaluable patients with venous leg ulcers, 59% of BBSS recipients, with healed ulcers at 6 months, had healed by secondary intention. There was no obvious persistence of the BBSS in the wound. In the other 41% of the BBSS recipients with healed ulcers, the appearance of graft ‘take’ and at least temporary persistence of BBSS occurred, but remodelling of the graft and probable replacement with the patient’s own skin cells occurred in 63% of these patients.

Immunogenicity

BBSS does not contain any antigen-presenting cells such as Langerhans cells, dermal dendritic cells, endothelial cells and leucocytes. Keratinocytes and fibroblasts do not express human leucocyte antigens class II antigens and common costimulatory molecules and, consequently, allogenic T cells are not activated. The bovine type I collagen used in BBSS did not produce an immediate or delayed immune response in healthy volunteers.

In clinical trials in patients with venous leg ulcers, with diabetic ulcers, or with acute wounds, there was no clinical evidence of rejection of BBSS. Immunological tests indicated no humoral or cellular response to the keratinocytes or fibroblasts of BBSS.

Therapeutic Efficacy

In a multicentre, nonblind trial, BBSS in combination with compression therapy (nonadherent primary dressing, cotton gauze bolster and a self-adherent elastic wrap) was more effective than active control treatment (compression therapy plus zinc oxide-impregnated paste bandage) in patients with venous leg ulcers.

At 6 months’ follow-up, the incidence of complete healing (63 vs 49%; p = 0.02) was greater with BBSS plus compression therapy (n = 146) than with control treatment (n = 129). Complete ulcer healing took place at a significantly faster rate in patients treated with BBSS plus compression therapy than with control treatment (median 61 vs 181 days). Healing was faster with BBSS plus compression therapy than control treatment in patients with ulcers of >6 months’ duration and in patients with stage III (extending to subcutaneous tissue) ulcers. In a subgroup analysis of patients with ulcers of >1 year’s duration, wound healing at 6 months was achieved in more patients (47 vs 19%; p < 0.005) and the median time to complete healing was shorter (median 181 days vs not attained; p < 0.005) with BBSS plus compression therapy (n = 72) than control treatment (n = 48).

In a multicentre, nonblind comparative trial, BBSS was effective in patients with full-thickness neuropathic diabetic foot ulcers. Patients were randomised to BBSS (n = 112) or control treatment (saline-moistened gauze; n = 96). All patients received adjunctive diabetic foot care therapy including off-loading and surgical debridement. At 12 weeks’ follow-up, significantly more diabetic foot ulcers were completely healed (56 vs 38%) and the median time to complete wound healing was significantly lower (65 vs 90 days) in recipients of BBSS than in recipients of control treatment.

Tolerability

BBSS was well tolerated in patients with venous leg ulcers or diabetic foot ulcers in randomised, clinical trials.

The incidence of adverse events in patients with venous leg ulcers attributable to treatment was similar in recipients of BBSS plus compression therapy (nonadherent primary dressing, a gauze bolster and a self-adherent elastic wrap) and in recipients of control treatment (compression therapy plus zinc oxide-impregnated bandage) during 12 months’ follow-up. The three most commonly reported adverse events were wound infection, cellulitis, or pain, with no significant difference in incidence between treatment groups.

Similarly, in patients with diabetic foot ulcers receiving standard diabetic foot care, there was no significant difference in the incidence of wound infection and cellulitis in recipients of BBSS or control (saline-moistened gauze) treatment during the 6 months’ follow-up. However, the incidences of osteomyelitis and amputation of the study limb were both significantly lower in recipients of BBSS than control treatment.

Pharmacoeconomic and Quality-of-Life Studies

The cost effectiveness of BBSS in patients with chronic ulcers (venous or diabetic foot ulcers) has yet to be examined in well designed, prospective clinical trials.

However, BBSS was cost effective in patients with hard-to-heal venous leg ulcers, according to data from a modelled analysis incorporating results from a multicentre, randomised trial. The average annual direct medical cost of managing patients with hard-to-heal ulcers was estimated to be $US20 041 per patient treated with BBSS plus compression therapy and $US27 493 per patient treated with control therapy (1996 costs). The cost was based on a mean of 3.34 applications of BBSS.

Another economic model estimated the cost of 12 weeks of wound care with various treatment modalities in patients with venous ulcers for a hypothetical managed-care plan. Costs per patient healed were $US15 053 for BBSS, $US2939 for saline-, paraffin- or zinc oxide-impregnated dressings and $US1873 for hydrocolloid D dressings (year of costing 2000). However, this analysis had a number of limitations, including the short follow-up period.

Life was considered to be ‘much better’ after BBSS administration in 79% of patients with venous leg ulcers (n = 14) in a retrospective noncomparative study. Pain scores were significantly better after BBSS application.

Application and Administration

In the US, BBSS is indicated for use with standard therapeutic compression in the treatment of patients with noninfected partial and/or full-thickness skin ulcers (the result of venous insufficiency) of at least 1 month’s duration that have not adequately responded to conventional ulcer therapy.

BBSS is also indicated for use with standard diabetic foot care for the treatment of patients with full-thickness diabetic foot ulcers of neuropathic etiology of >3 weeks’ duration that have not adequately responded to conventional ulcer therapy and which extend through the dermis (but not with tendon, muscle, capsule or bone exposure).

The product should be kept at 20 to 31°C and placed on the wound within 15 minutes of opening the package. The date of expiry and the pH of the product should be checked.

BBSS should be applied under aseptic conditions to a clean, debrided wound. Venous leg ulcers should be dressed with a nonadherent primary dressing, a nonocclusive dressing such as mesh gauze (this may be folded or rolled as a bolster) and an elastic compression bandage. Diabetic ulcers should be dressed with a nonadherent, saline-moistened dressing, a layer of dry gauze, a layer of petrolatum gauze and a gauze wrap. The dressing in contact with BBSS needs to be changed once weekly until healing occurs. The tolerability and efficacy of BBSS has not been established for patients treated with >5 applications of the product.