14.1 General Structure
and Mechanism of Action
The term ‘human skin equivalents’ (HSE) refers to living composite grafts composed of a der- mal component containing human living fibro- blasts and an epidermal component containing living keratinocytes. HSE is also referred to as
‘graftskin’, ‘bio-engineered tissue’, ‘bilayered cellular matrix’ or ‘bilayered skin substitute’.
The manufacturing of HSE is based on an original idea mooted by Bell [1] in 1981. Two available commercial HSE products are FDA approved: Apligraf® and OrCel®. Apligraf® is the product with which the most experience
Human Skin Equivalents:
When and How to Use 14
Contents
14.1 General Structure and Mechanism of Action 177
14.2 Product Description 178 14.2.1 Apligraf® 178
14.2.2 OrCel® 178 14.3 Indications 178 14.4 Instructions for Use 179 14.4.1 Preparing the Ulcer Bed 179 14.4.2 Steps to Take Prior to Applying
the Product to the Ulcer Bed 179 14.4.3 Grafting Procedure 180 14.4.4 Dressing the HSE Layer 180 14.4.5 Following Grafting 180 14.5 Contraindications 181 14.6 Efficacy 181
14.7 Concluding Remark 181 References 183
has been gathered in the treatment of cutane- ous ulcers. Therefore, most of the following dis- cussion is based on research studies investigat- ing Apligraf®. Further types of commercial composite grafts are expected to be available in forthcoming years. As described in Chap. 13, some studies have been carried out with HSE products not in commercial use and intended for research purposes only.
HSE products manufactured in vitro from neonatal foreskins represent a modification of allogeneic grafting. They have an internal der- mal layer containing living human fibroblasts and an external epidermal layer containing liv- ing keratinocytes. Histologically, the currently available HSE products do not contain Langer- hans’ cells, macrophages, melanocytes, lym- phocytes, blood vessels, or hair follicles [2, 3].
Having the above-mentioned qualities, an
HSE product functions as an effective biolog-
ical dressing, providing an optimal environ-
ment for wound repair. Its dermal layer may
serve as a structural template for dermal gener-
ation. Fibroblasts incorporated in the dermal
layer, as well as the epidermal cells, induce se-
cretion of growth factors, by which HSE are
considered to exert their healing effect [4–7]. In
fact, positive immunostaining has been shown
within the dermal component of Apligraf® for
platelet-derived growth factor (PDGF), trans-
forming growth factor-beta (TGF-β), and basic
fibroblast growth-factor (FGF-2) [4]. OrCel®,
according to the manufacturer, contains several
growth factors including fibroblast growth fac-
tor (FGF), granulocyte-macrophage colony
stimulating factor (GM-CSF), and keratinocyte
growth factor (KGF). It has been suggested that
the new allogeneic cells incorporated in HSE
may initiate a cascade of growth factors pro-
duced within the treated ulcer [5, 8].
Apligraf® has also been shown to produce certain antibacterial peptides, such as human β-defensin 2, a substance active against gram- negative bacteria, yeasts, and fungi [4]. In any case, the allogeneic cells do not survive on the transplanted ulcer bed, although their rejection may be delayed in conditions of abnormal im- mune status [9].
14.2 Product Description
14.2.1 Apligraf®
The supporting internal dermal layer of Apli- graf® consists of living human fibroblasts em- bedded in a bovine type-1 collagen. The exter- nal epidermal layer contains living keratinocy- tes. The behavior of Apligraf® is similar to that of human skin, and its epidermal layer is able to produce differentiated stratum corneum. In vi- tro, it demonstrates self-healing capacities fol- lowing injury.
Each unit of Apligraf® is kept in a sealed polyethylene bag in a plastic dish, over a layer of an agar-like material. The product should be kept in the sealed bag until used, within a tem- perature range of 20°–31°C, in order to ensure its viability. Each sheet is manufactured as a cir- cular disc measuring 7.5 cm in diameter and is 0.75 mm thick.
According to the manufacturer’s informa- tion, the agar-based Apligraf® medium con- tains agarose,
L-glutamine, hydrocortisone, bo- vine serum albumin, bovine insulin, human transferrin, triiodothyronine, ethanolamine, O- phosphoryletanolamine, adenine, selenious ac- id, DMEM powder, HAM’s F-12 powder, sodium bicarbonate, calcium chloride, and water for in- jection. All components of the product undergo thorough screening for various potential infec- tious agents, including viruses (HIV and hepa- titis A, B, and C), bacteria (such as syphilis), and fungi [3]. The cells are also screened for iden- tification of chromosomal abnormalities and biochemical defects. The product is packed so that its dermal, glossy layer is placed face down on the agar substrate, while its epidermal (dull) layer faces up.
14.2.2 OrCel®
The product OrCel® is made of two separate compartments of a porous, type-I bovine colla- gen sponge. It contains cultured allogeneic ke- ratinocytes and fibroblasts, both originating from human neonatal foreskin [10]. It measures approximately 6 × 6 cm (36 cm
2).
The donor’s cells are screened for viruses (HTLV I and II, Hepatitis B, HIV I and II, EBV and HHV-6), bacteria, fungi, yeast, mycoplas- ma, and tumorigenicity. The final product should fulfill the required criteria as to mor- phology, cell density, cell viability, sterility, and physical container integrity.
14.3 Indications
HSE products induce repair in ulcers that have been considered recalcitrant to conventional therapy.
Apligraf® is FDA approved for the condi- tions listed below, i.e., for venous ulcers and diabetic ulcers:
5 Venous ulcers that fulfill the follow- ing criteria:
– Of partial or full thickness – Non-infected
– Not showing significant improvement following one month of conventional therapy
5 Diabetic ulcers that fulfill the fol- lowing criteria:
– Of full thickness – Non-infected
– With neuropathic etiology – Not showing significant improve-
ment following three weeks of conventional therapy
– Not extending into tendons, mus- cles or bones
– Located on the plantar, medial, or lateral aspects of the foot (ex- cluding the heel)
Chapter 14 Human Skin Equivalents: When and How to Use 178
14
t
However, Apligraf® has also been reported as beneficial for other types of cutaneous ulcers, such as those caused by polyarteritis nodosa, those due to sarcoidosis, and epidermolysis bullosa lesions [11–13]. Apligraf® has also been used for excised burn wounds [14].
Currently, OrCel® is FDA approved for:
5 Split-thickness donor sites of burn patients
5 Surgical wounds and donor sites as- sociated with mitten-hand defor- mities secondary to recessive dys- trophic epidermolysis bullosa
Note that OrCel® has recently been reported to be beneficial in the treatment of diabetic neu- ropathic foot ulcers [15] (see Table 14.1).
14.4 Instructions for Use
The main principles described below are based on our experience with Apligraf®. However, sim- ilar techniques should be implemented when dealing with other HSE products. The instruc- tions also apply, at least in their main principles, to dermal grafting products (such as Derma- graft®). There are unique guidelines for the grafting of each product, and manufacturers’ in- structions should be followed precisely and thoroughly.
14.4.1 Preparing the Ulcer Bed
The product should be applied onto a clean, vi- able granulation tissue. Debridement is re- quired in order to obtain an optimal substrate for grafting.
The ulcer bed should be prepared as fol- lows:
5 Prior to debridement, the ulcer should be cleansed thoroughly with a saline solution.
5 Any necrotic tissue present should be removed by scalpel and forceps.
Shaving the ulcer’s surface until a minor degree of bleeding can be seen is required to create an opti- mal, vascular substrate for the HSE treatment [8, 16]. The principles that apply here are the same as those for preparing the ulcer bed for the ap- plication of preparations containing growth factors [17].
5 Shaving the upper layer of the ulcer bed, superficially and horizontally, may be performed with a scalpel or with a fine curette. A fine curette can be used for the removal of layers near the ulcer’s margin – its shape enabling an accurate outlin- ing. Debridement of the ulcer’s mar- gin is extremely important, since peripheral epithelialization can be stimulated by this procedure. This issue is detailed in Chap. 9.
5 Bleeding following debridement may be dealt with by applying gen- tle pressure with a sterile gauze. Fol- lowing debridement, the ulcer should be cleansed with saline or Ringer’s lactate solution.
14.4.2 Steps to Take Prior to Applying the Product to the Ulcer Bed
Check the expiry date of the product to be used. Assure that the product has been kept under appropriate conditions prior to grafting (temperature range according to manufactu- rer’s instructions). Note: Products should be used within a limited period following removal from their packaging (i.e., 15 min for Apligraf®).
t
t
14.4.3 Grafting Procedure
Grafting should be done as follows:
5 The technique demands attention to absolute sterility. Sterile gloves should be worn. The plastic tray, af- ter its removal from its protective polyethylene cover, should be placed on a sterile surface (Fig. 14.1).
5 The HSE sheet should be fenestrat- ed, with its epidermal side facing down, to enable drainage of secre- tions from the ulcer bed. The HSE should be trimmed to conform to ulcer size.
5 Ascertain that the right side of the graft (its dermal layer) is placed in direct contact with the wound bed (Fig. 14.2).
5 After the HSE sheet has been placed on the wound bed, it should be straightened and smoothed using a saline-moistened cotton applicator (Fig. 14.3), in order to achieve con- tinuous contact between the HSE layer and the wound bed, without wrinkles or folds. Alternatively, Apli- graf® can be meshed, thereby allow- ing the product to expand and to cover a larger ulcer area.
14.4.4 Dressing the HSE Layer
Once the graft has been correctly applied, it should be covered with a non-adherent pri- mary dressing, followed by a cotton gauze dressing (Fig. 14.4).
The treated area is now bandaged. The use of sutures or staplers to attach the graft sheet to the ulcer bed is not recommended, since these cause unnecessary tissue damage and increase the risk of local infection in the treated area.
For the same reasons, it is not recommended to use an occluding substance such as vaseline over the HSE layer.
Chapter 14 Human Skin Equivalents: When and How to Use 180
14
Fig. 14.1.Lifting the HSE sheet prior to its application onto the ulcer
Fig. 14.2.The HSE sheet is placed onto the ulcer bed
14.4.5 Following Grafting
Frequency of dressing replacement will depend on the amount of secretions from the ulcer. If, based on a swab culture, there is evidence of col- onization by ‘notorious’ bacteria (Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus), frequent examination of the treated ul-
t
cer is recommended. Additional applications of HSE may be used according to the progress of healing.
14.5 Contraindications
HSE products are contraindicated under the following conditions:
5 For infected wounds/ulcers
5 For patients known to be hypersen- sitive to bovine collagen
5 For patients known to be hypersen- sitive to substances contained in the preserving/shipping medium
OrCel® may contain traces of penicillin, strep- tomycin, gentamicin, and amphotericin B. It should therefore be avoided in patients with known allergies to these drugs.
In any case, current indications and contra- indications of each product should be verified and updated according to manufacturers’
guidelines and regulations as determined by medical/legal authorities of each country.
14.6 Efficacy
The efficacy of HSE products which have been applied to chronic cutaneous ulcers, as exam- ined in several clinical trials, is detailed in Ta- ble 14.1. Figures 14.5–14.7 illustrate the progress of an ulcer following the application of Apli- graf®.
14.7 Concluding Remark
Human skin equivalents represent an advanced mode of therapy that may be used for cutane- ous ulcers. Their use is intended for relatively clean ulcers. HSE has been found to be effective in difficult-to-heal ulcers of long duration
. Fig. 14.3.Straightening and smoothing the ApligrafFig. 14.4.Dressing the treated area
Fig. 14.5.One day after the application of Apligraf®
t
Chapter 14 Human Skin Equivalents: When and How to Use 182
14
Table 14.1.Efficacy ofHSE products applied to chronic ulcers
S tud y Y ear D esi g n P ar ticip an ts Dur at io n In ter ven tio n Ou tc o me A PLI GRAF ®
Falanga 1998쐌randomized쐌309 patients suffering 쐌8 weeks oftreatment쐌146 patients:Apligraf® 쐌63% ofulcers treated et al.[2]쐌controlledfrom chronic venous 쐌6 months follow-up,with compression with Apligraf® healed, 쐌open-labelulcersplustherapyvs 쐌multi-centered쐌293 patients were 쐌6 months safety 쐌129 patients:external 쐌49% in the control treated,from whichevaluationdressing containing group 쐌275 patients were zinc-oxide paste with evaluatedcompression therapy Falanga 1999쐌randomized쐌120 patients with 쐌8 weeks treatment쐌72 patients:Apligraf® 쐌47% ofulcers treated et al.[18]쐌controlleddifficult-to-heal 쐌6 months follow-upwith compressionwith Apligraf® 쐌open-labelvenous ulcerstherapyachieved complete 쐌multi-centeredoflong duration쐌48 patients:external wound closure (more than 1 year)dressing containingvs zinc-oxide paste with 쐌19% in the control compression therapygroup Pham 1999쐌randomized쐌33 with diabetic 쐌12 weeks (treatment 쐌16 patients:Apligraf®쐌75% ofulcers treat- et al.[19]쐌controlledfoot ulcerswith Apligraf® within 쐌17 patients:woven ed with Apligraf® 쐌open-labelfirst four weeks) gauze kept moist byachieved complete salinewound closure (12 patients),vs 쐌41% in the control group (7 patients) 쐌The median time for ulcer closure was 38.5 days for Apligraf® vs 91 days in the control groupO R CEL ®
Lipkin2003쐌randomized쐌40 patients with 쐌12 weeks쐌20 patients:쐌35% oftreated ulcers et al.[15]쐌controlledchronic,neuropathic,OrCel® with standard achieved complete heal- 쐌open-labeldiabetic foot ulcerscare (moist saline ing (7 of20 wounds),vs 쐌multi-centeredgauze cover)쐌20% in the control 쐌20 patients:standardgroup (4 out of20 care alonewounds)References
1. Bell E, Ehrlich HP, Buttle DJ, et al: Living tissue formed in vitro and accepted as skin-equivalent tis- sue of full thickness. Science 1981; 211 : 1052–1054 2. Falanga V, Margolis D, Alvarez O, et al: Rapid healing
of venous ulcers and lack of clinical rejection with an allogeneic cultured human skin equivalent. Arch Dermatol 1998; 134 : 293–300
3. Wilkins LM, Watson SR, Prosky SJ, et al: Develop- ment of a bilayered living skin construct for clinical applications. Biotechnol Bioeng 1994; 43 : 747–756 4. Schmid P: Apligraf – phenotypic characteristics and
their potential implications for the treatment of dia-
betic foot ulcers. A satellite symposium at the 36th annual meeting of the European association for the study of diabetes (EASD). Jerusalem, September 2000
5. Phillips TJ, Manzoor J, Rojas A, et al: The longevity of a bilayered skin substitute after application to ve- nous ulcers. Arch Dermatol 2002; 138 : 1079–1081 6. Navsaria HA, Myers SR, Leigh IM, et al: Culturing
skin in vitro for wound therapy. Trends Biotechnol 1995; 13 : 91–100
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10. Still J, Glat P, Silverstein P, et al: The use of a collagen sponge/living cell composite material to treat donor sites in burn patients. Burns 2003; 29: 837–841 11. Pennoyer JW, Susser WS, Chapman MS, et al: Ulcers
associated with polyarteritis nodosa treated with bi- oengineered human skin equivalent (Apligraf). J Am Acad Dermatol 2002; 46 : 145
12. Streit M, Bohlen LM, Braathen LR: Ulcerative sarcoi- dosis successfully treated with apligraf. Dermatolo- gy 2001; 202 : 367–370
13. Falabella AF,Valencia IC, Eaglstein WH, et al: Tissue- engineered skin (Apligraf) in the healing of patients with epidermolysis bullosa wounds. Arch Dermatol 2000; 136 : 1225–1230
14. Waymack P, Duff RG, Sabolinski M: The effect of a tissue engineered bilayered living skin analog, over meshed split-thickness autografts on the healing of excised burn wounds. Burns 2000; 26 : 609–619 15. Lipkin S, Chaikof E, Isseroff Z, et al: Effectiveness of
bilayered cellular matrix in healing of neuropathic diabetic foot ulcers: results of a multicenter pilot trial. Wounds 2003; 15 : 230–236
16. Brem H, Balledux J, Sukkarieh T, et al: Healing of ve- nous ulcers of long duration with a bilayered living skin substitute: results from a general surgery and dermatology department. Dermatol Surg 2001; 27 : 915–919
17. Steed DL, Donohoe D, Webster MW, et al: Effect of extensive debridement and treatment on the healing of diabetic foot ulcers. J Am Coll Surg 1996; 183 : 61–64
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19. Pham HT, Rosenblum BI, Lyons TE, et al: Evaluation of a human skin equivalent for the treatment of dia- betic foot ulcers in a prospective, randomized, clini- cal trial. Wounds 1999; 11 : 79–86
Fig. 14.6.After eight days
Fig. 14.7.After 12 days
