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Evaluation of Permacol™ as a cultured skin equivalent
burns 34 (2008) 1169–1175
available at www.sciencedirect.com
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Evaluation of PermacolTM as a cultured skin equivalent T.M. MacLeod a,*, A. Cambrey a,1, G. Williams b,2, R. Sanders a,1, C.J. Green b,2 a b
Restoration of Appearance and Function Trust, Mount Vernon Hospital, Northwood, Middlesex HA6 2RN, UK Northwick Park Institute for Medical Research, Northwick Park Hospital, Harrow, Middlesex HA1 3UJ, UK
article info
abstract
Article history:
Skin loss following severe burn requires prompt wound closure to avoid such complications
Accepted 21 January 2008
as fluid and electrolyte imbalance, infection, immune suppression, and pain. In clinical situations in which insufficient donor skin is available, the development of cultured skin
Keywords:
equivalents (dermal matrices seeded with keratinocytes and fibroblasts) may provide a
PermacolTM
useful alternative. The aim of this study was to assess the suitability of a porcine-derived
Cultured skin equivalent
dermal collagen matrix (PermacolTM) to function as a cultured skin equivalent in supporting
Keratinocytes
the growth of keratinocytes in vitro and providing cover to full thickness wounds in the BALB
Fibroblasts
C/nude mouse model. A histological comparison was against Glycerol treated-Ethylene
Epidermis
Oxide Sterilised Porcine Dermis (Gly-EO Dermis) which has successfully been used as a cultured skin equivalent in previous studies. Both Gly-EO Dermis and to a lesser extent PermacolTM were able to support the growth of cultured keratinocytes following a 16-day period of cell culture, however, this study was only able to demonstrate the presence of an epidermal layer on Gly-EO dermis 2 weeks after grafting onto full-thickness wounds in the BALB C/nude mouse model. # 2008 Elsevier Ltd and ISBI. All rights reserved.
1.
Introduction
Skin loss following severe burn requires prompt wound closure to avoid such complications as fluid and electrolyte imbalance, infection, immune suppression, and pain. The ideal treatment for such wounds would be an unlimited supply of autologous skin graft that could be applied in a single procedure. In clinical situations in which insufficient donor skin is available, the development of cultured skin equivalents may provide a useful alternative. The combination of a dermal matrix with added keratinocytes and fibroblasts to resemble a conventional skin graft has been referred to as a ‘‘living skin equivalent’’, ‘‘bilayered skin equivalent’’, ‘‘composite’’ and
‘‘dermo/epidermal culture’’ but for the purposes of this study will be referred to as a ‘‘cultured skin equivalent’’ (CSE). The dermal component of a CSE provides stability, improves handling, promotes epithelial–mesenchymal interactions in vitro leading to greater keratinocyte attachment and differentiation [1]. When applied to a full-thickness wound the dermal component of the CSE acts as a dermal template or scaffold for vascularisation and remodelling into neo-dermis. The first type of dermal matrix used as a cultured skin equivalent incorporated fibroblasts into a gel of acid-extracted collagen overlayed with a suspension of keratinocytes [2]. Other synthetic dermal analogues studied include the collagen sponge [3], polyglycolic acid mesh [4], hyaluronic acid-derived
* Corresponding author. Tel.: +44 1923 835 815; fax: +44 1923 844 031. E-mail addresses: [email protected] (T.M. MacLeod), [email protected] (G. Williams), [email protected] (C.J. Green). 1
Tel.: +44 1923 835 815; fax: +44 1923 844 031. Tel.: +44 208 969 3265; fax: +44 208 869 3270. 0305-4179/$34.00 # 2008 Elsevier Ltd and ISBI. All rights reserved. doi:10.1016/j.burns.2008.01.013 2
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biomaterial [5], fibrin gels [6] and a collagen/glycosaminoglycan (C-GAG) mixture [7–10]. Most of the acceptable dermal substitutes produced thus far appear to be those which employ an acellular dermal matrix (ADM) derived from full or split-thickness skin then treated to remove epithelial (keratinocytes, sweat glands and sebaceous glands) and dermal cellular components (fibroblasts, vascular endothelium and smooth muscle cells). Most studies have focused on ADM derived from human cadaveric skin by various techniques [11,12] for use as a dermal substitute both in animal models [11–13] and in the clinical management of burn wounds [14,15]. The limited availablility of cadaver skin and risk of disease transmission limit the use of human cadaveric de-epidermalised dermis (DED) in dermal replacement. The aim of this study was to assess the suitability of PermacolTM for use as a cultured skin equivalent.
2.
Materials and methods
2.1.
PermacolTM
temperature-controlled (37 8C), humidified, CO2 (5%) incubators. Centrifugation was performed in a Heraeus Labofuge. Microscopic examination was achieved using an Olympus CK2 inverted phase contrast microscope. Cells were counted in a modified Neubauer haemocytometer (Weber Scientific International). Dulbecco’s phosphate-buffered saline (PBS), Dulbecco’s modified Eagles’ medium (DMEM), DMEM/Hams F12 medium and fetal calf serum were obtained from Gibco Ltd. The additives to the culture media: cholera toxin, epidermal growth factor, transferrin and insulin were obtained from Sigma Ltd.; and lyothyronine was obtained from Koch-Light Ltd. Antibiotics, trypsin, 0.05% ethylenediaminetetra-acetic acid (versene), di-methyl sulfoxide (DMSO), collagen type I and 0.4% trypan blue solution were also obtained from Sigma Ltd. The neutral protease Dispase1 and bacterial collagenase D were both purchased from Roche Diagnostics Ltd. Glycerol was obtained from BDH. Griener Labortechnik provided plastic ware including culture flasks and universals. Stainless steel rings and grids were manufactured in the Department of Bioengineering, Mount Vernon Hospital.
2.4. PermacolTM is a porcine-derived acellular dermal matrix whose manufacture involves trypsinisation (to remove all living cells and non-collagenous debris), solvent extraction (to remove all lipid and fat deposits), g-irradiation and isocyanate cross-linkage. It is manufactured by Tissue Science Laboratories as 5 cm 5 cm sterile sheets which are then double vacuum packed, heat sealed in sachets of aluminium foil (inner) and polyester/polythene (outer) sachets and stored at room temperature. This study used PermacolTM sheets 0.4 mm in thickness, which were kept moist in sterile saline.
2.2. Glycerol treated-Ethylene Oxide Sterilised Porcine Dermis (Gly-EO Dermis) Split-thickness skin grafts were obtained from a single large white pig (Fulmer Place Farm) using a modified Watson skin graft knife during euthanasia. The harvested skin was placed in 50:50 glycerol in phosphate-buffered saline for 4 h, 85:50 glycerol in phosphate-buffered saline for 4 h and pure glycerol for 40 h. After removing excess glycerol, the sample was placed in a bag and subjected to a standard ethylene oxide sterilisation process. The sterilised sample was rehydrated in PBS at 37 8C for 48 h and then placed in 1 mol/l sodium chloride for 16 h to produce a dermal/epidermal split. The epidermis was gently removed using a fine pair of forceps. Once the deepithelialised dermis (DED) was confirmed as sterile and acellular, it was stored in sterile universal containers in phosphate-buffered saline (Oxoid, Unipath, Hants, U.K.) with 625 mg/ml amphotericin B, 1000 mg/ml streptomycin (Gibco BRL, Life Technologies, Paisley, U.K.) at 20 8C.
2.3.
Fibroblast isolation and culture
Split-thickness skin was obtained from routine plastic surgery specimens of either breast reductions or abdominoplasties. Only specimens free from scars and striae were used. Fibroblasts were extracted from split-thickness skin grafts via collagenase digestion. Following incubation in trypsin and removal of the epidermis for keratinocyte isolation, the remaining dermal elements were separated out. The dermis was finely minced using a scalpel and placed in a 60 mm Petri dish. The tissue was then incubated in 50 ml of collagenase D solution (0.05% in DMEM) for 12 h at 37 8C. After filtration through a fine mesh tea strainer into another tube, this fibroblast suspension was spun down in a centrifuge at 1000 rpm for 5 min. The pellet was then re-suspended in FCM and counted. The fibroblasts were then inoculated into 75 cm2 culture flasks and the cultures continued to confluence. Media was changed twice a week and cultures split as required to a maximum of passage nine.
2.5.
Preparation of 3T3 feeders
Feeder cells were produced from Swiss mouse 3T3 fibroblast cell lines (passages 8–30). Frozen vials of cells were used to generate fresh stocks and discarded after 30 passages. These cells were grown in fibroblast culture medium (FCM) in 150 cm2 flasks (4 106 cells per flask) and routinely split using 0.05% trypsin in versene, two or three times per week. They were then spun down in FCM for 5 min at 1000 rpm and resuspended in keratinocyte culture medium (KCM) after counting. Using a c-irradiator they were lethally g-irradiated with 6000 rad (cobalt 60) and plated in a fresh flask at the appropriate seeding density.
Tissue culture 2.6.
Cell culture procedures were performed in sterile Class II laminar airflow hoods (LaminAir), purchased from Heraeus Instruments. Tissue culture was undertaken in Heraeus
Keratinocyte isolation and culture
Split-thickness skin graft samples were cut into 0.5 cm2 pieces and incubated overnight (12–18 h) at 4 8C in 0.1% (w/v) trypsin
burns 34 (2008) 1169–1175
solution. Epidermal and dermal layers were then peeled apart, and the keratinocytes collected in fetal calf serum by scraping the upper layer of the dermis and lower layer of the epidermis. The freshly isolated (P0) keratinocyte preparation was then centrifuged at 1000 rpm for 5 min and the pellet re-suspended in KCM before counting. P0 cells were then used to produce cultured skin equivalents grafts or expand their number by culture on plastic and produce passaged keratinocyte (P1) cell suspensions. Irradiated Swiss 3T3 mouse fibroblasts were seeded at a density of 2 106 cells per 75 cm2 tissue culture flask as a feeder layer. The flasks were then inoculated with keratinocytes at a density of 2 106 cells per flask. The first medium change was at 48 h. Thereafter, the medium was changed twice a week. Colonies of keratinocytes were seen at 3–4 days and reached 75–85% confluence by 1 week. These cells were detached from the plastic using 0.05% trypsin in versene and re-suspended at a known density in KCM. P1 cells were then used to produce CSE grafts.
2.7.
Seeding of fibroblasts: a sterilised stainless steel ring of 1 cm internal diameter was placed in the centre of the upper surface of the matrix and gently pressed down to ensure a watertight seal. One milliliter of fibroblast cell suspension (1 105 ml 1) was seeded into the steel ring and FCM added, both within and outside the ring, to a height just below the upper edge. This combination was incubated for 24 h, at which point the ring was removed. After 48 h incubation the seeded dermal matrix was turned over. Seeding of keratinocytes: a sterilised ring was placed in the centre of the upper surface of the matrix and gently pressed to ensure a watertight seal. One milliliter of keratinocyte suspension (1 106 ml 1) was seeded into the steel ring and keratinocyte culture medium then added, both within and outside the ring, to a height just below the upper edge. After 24 h the ring was removed and the media changed for fresh KCM. After 4 days the CSE was raised to the air–liquid interface on the stainless steel grids and maintained for a further 10 days changing the media twice a week.
Cryopreservation of cells 2.11.
Cell suspensions were centrifuged, counted and the pellet then re-suspended in cryopreservation media (90% fetal calf serum and 10% DMSO). Cells were frozen in cryovials, wrapped in four layers of tissue paper and then transferred to a 80 8C freezer. This allowed the cells to gradually freeze at approximately 1 8C per minute. Vials were later transferred to liquid nitrogen for long-term storage. Cells were recovered by thawing rapidly in a 37 8C water bath, spinning down in the centrifuge at 1000 rpm for 5 min, re-suspending in media and counting.
2.8.
Cell numbers
Where specific cell numbers were required, the standard trypan blue exclusion method was used. While viable cells are able to exclude the blue dye, non-viable cells are not and appear blue under microscopic examination. Single cell suspensions in a known volume of media were used, mixed in a 1:1 ratio with 0.4% solution of trypan blue. Cells were counted in a modified Neubauer haemocytometer and the cell concentration calculated accordingly.
2.9.
Preparation of matrices
All three biomaterials were divided into 1.5 cm 1.5 cm pieces and placed in phosphate-buffered saline prior to use. Six pieces of each type of matrix were used for the initial in vitro assessment and six pieces were used for the in vivo assessment.
2.10.
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Preparation of cultured skin equivalents
Pieces of the PermacolTM and Gly-EO Dermis (1.5 cm 1.5 cm) were placed in the wells of cell culture plates and incubated with fibroblast culture medium for 24 h at 37 8C as a sterility check. If the medium became turbid, the matrix was discarded. Sterile pieces were then placed in the wells of a six-well plate and seeded with cells as described below.
Transport of cultured skin equivalents
CSE grafts were transported between sites on their stainless steel grids at the air–liquid interface in fresh KCM. Six-well plates were put in a plastic container lined with wet paper towels (to maintain humidity) with an open tap on one side and the airtight lid replaced snugly. The container and lid were allowed to equilibrate with the temperature of the incubator and the CO2 concentration for 1 h. After this time the tap was closed as soon as the incubator door was opened and the container placed in a cool box for transportation. The container was left sealed until the skin equivalents were ready for transfer to the athymic mice 2 or 3 h later.
2.12.
Nude mice
All experimental surgical procedures were performed under License from the Home Office (Animals Scientific Procedures Act 1986). A maximum of 10 nude mice were kept in each large sterilised box, respectively and allowed to acclimatise to their surroundings for 1 week prior to any experimental procedure. After the first surgical procedure the animals were housed in individual cages to prevent them from disturbing each other’s dressings. Water and feed were supplied ad libitum. The room was kept at 22–24 8C with 45– 55% humidity. Lighting was adjusted to provide 12 h light and darkness per day. A Taconic Farms Mouse Scale (Pelouze Scale Company) was used to weigh the athymic mice for calculation of anaesthetic dosages. Hypnorm1 (fentanyl citrate and fluanisone) was purchased from Janssen Animal Health, Hypnoval1 (midazolam) from Roche Diagnostics Ltd. and diazepam from Phoenix Pharmaceuticals Ltd. Athymic mice were anaesthetised using an intra-peritoneal injection of ‘‘CRC cocktail’’ given (0.01 ml/g) consisting of one part Hypnorm1, two parts water for injection and one part Hypnoval1. The duration of anaesthesia was short (approximately 50 min) and animals recovered spontaneously. Six anaesthetic procedures were
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done in this way. Euthanasia was performed by cervical dislocation.
prior to their en bloc excision for processing of paraffin sections.
2.13.
2.17.
Histological analysis
2.17.1.
Epidermal attachment
Grafting of CSEs onto wounds in nude mouse model
Mice were anaesthetised and their skin disinfected with 0.5% (w/v) chlorhexidine gluconate in 70% (v/v) IMS. The mice were placed on sterile drapes and covered with surgical swabs to keep them warm. Each mouse had a 1 cm diameter silicone chambers inserted onto each flank. The skin at the site of the chamber was lifted up with fine forceps and amputated with sharp scissors, leaving a circular full-thickness defect with panniculus carnosus muscle fascia as the wound bed. The edge of the wound was undermined by 5 mm using scissors and the chamber inserted. The silicone chambers are malleable and were compressed then inserted and allowed to expand with the flange under the wound edge. The chambers did not require fixation to the skin. The grafts were trimmed to size and placed with the epidermal surface uppermost within the chamber. The acellular dermis was cut to leave a small rim around the epidermal culture, which sat underneath the flange of the chamber, effecting better graft immobilisation. A layer of non-adherent dressing gauze was placed directly over the graft, followed by two layers of swab soaked in normal saline solution and finally several dry layers of swab, all cut to size. The upper dressings were lightly compacted such that a gentle amount of pressure was exerted on the graft to aid immobilisation. Elastoplast bandage was then applied in a circumferential manner around the trunk of the animals. The bandage was applied loosely over the abdomen to permit free respiratory movements, but firmly on the dorsum to apply pressure on the wounds.
Epithelial attachment of cultured skin equivalents was viewed over the length of a section and expressed as a percentage (to the nearest 5%).
2.17.2.
2.17.3.
2.18.
In vitro assessment
After a total of 16 days in culture, six cultured skin equivalents based on PermacolTM and Gly-EO Dermis were fixed and processed for paraffin sections. A further six CSEs in each group were then used for in vivo experiments.
2.16.
Fibroblast penetration
Fibroblast penetration was graded as being: Grade 1, partial penetration of the matrix with fibroblasts; Grade 2, fibroblasts present across the matrix but sparse in the superficial part; and Grade 3, abundant fibroblasts throughout the matrix.
2.19.
Vascular penetration
Matrix vascularity was graded as being: Grade 1, partial penetration of the matrix; Grade 2, vessels present across the matrix but sparse in its superficial part; and Grade 3, abundant vessels present throughout the matrix.
Epidermal thickness
Histological examination
Tissue specimens were fixed in 4% paraformaldehyde in phosphate-buffered saline. After fixation, tissues were embedded in paraffin and 5 mm thick sections were cut and stained with haematoxylin and eosin or Masson’s trichrome.
2.15.
Dermal quality
Dermal quality was graded as being: Grade 1, large fractures of the dermis; Grade 2, small fissures of the dermis: and Grade 3, no obvious distortion of the dermis.
2.20. 2.14.
Dermal–epidermal junction
The dermal–epidermal junction was graded from: Grade 1, completely flat; to Grade 5, well-formed rete ridges throughout.
In vivo assessment
A total of six BALB C/nude mice were used for the in vivo assessment of cultured skin equivalents. Each mouse had two full-thickness wounds created (one on each flank) giving (n = 6) wounds for analysis in each of the two groups (PermacolTM and Gly-EO Dermis) Animals were euthanased 14 days after the grafting by cervical dislocation. All CSEs were found to be fully adherent to the underlying full-thickness wound bed
Epithelial thickness of cultured skin equivalents was calculated using Seescan image analysis. Histological sections of the matrices stained with Masson’s trichrome were viewed under the fluorescent microscope green filter. This clearly displayed the non-cornified epidermal layer in each section. The average height of epithelium within one microscopic field was then assessed by measuring the area of epidermis and dividing by the length of the dermo-epidermal junction (DEJ) present.
2.21.
Matrix thickness
Dermal thickness of cultured skin equivalents was calculated using Seescan image analysis. Histological sections of the matrices stained with Masson’s trichrome and viewed using the fluorescent microscope green filter clearly displayed the dermal layer in each section.
2.22.
Statistical analysis
It was not possible to analyse epidermal height, epidermal attachment or dermal–epidermal junction since we were only able to demonstrate the presence of a viable epidermal layer on Gly-EO Dermis. Dermal thickness was measured on a contin-
burns 34 (2008) 1169–1175
1173
uous scale and compared using analysis of variance with a contrast coefficient. Dermal quality, fibroblast penetration and vascular penetration were measured on ordered categorical scales and compared using the Mann–Whitney test.
3.
Results
3.1.
In vitro assessment
Gly-EO Dermis and to a lesser extent PermacolTM were both able to support the growth of human keratinocytes and fibroblasts in vitro.
3.2.
In vivo assessment
From the 12 grafts examined in vivo, two wounds were obviously infected (one Gly-EO Dermis and one PermacolTM graft) and these were omitted from further study.
3.3.
Graft stability
Both Gly-EO Dermis and PermacolTM were fully adherent to the underlying full-thickness wound bed after a period of 14 days in vivo.
3.4.
Epidermal assessment
This study was unable to demonstrate the presence of a viable epidermal cell layer on the surface of PermacolTM following a 2-week period in vivo in the BALB C/nude mouse model. Gly-EO Dermis showed an overlying epidermis that was a median of 95% adherent (range 30–100), 45.9 mm in thickness (range 27.3–78.2) and demonstrated a DEJ median score of 2 (range 0–4). As a result of this a statistically significant difference (P < 0.05) occurred between Gly-EO Dermis and PermacolTM with respect to epidermal attachment (P < 0.05).
3.5.
Dermal assessment (Fig. 1)
3.5.1.
Dermal thickness
Fig. 1 – Comparison of matrix thickness, matrix quality, fibroblast penetration and vascular penetration for Gly-EO Dermis and PermacolTM. Results shown for dermal thickness are means W standard deviation. Matrix quality, fibroblast penetration and vascular penetration are represented by median (and range). There was no significant difference in thickness between PermacolTM and Gly-EO Dermis (P = 0.67). Gly-EO Dermis showed a significantly inferior dermal quality compared to PermacolTM (P = 0.006). A significantly greater degree of fibroblast penetration was seen in Gly-EO Dermis compared to PermacolTM (P = 0.005). A significantly greater degree of vascular penetration was seen in Gly-EO Dermis compared to PermacolTM (P = 0.005).
remnants with only partial penetration across the surrounding matrix.
3.5.4. There was no significant difference in the thickness of 0.4 mm PermacolTM and Glycerol treated-Ethylene Oxide Sterilised Porcine Dermis (P = 0.67).
3.5.2.
Dermal quality
The quality of the dermis in PermacolTM was significantly better than that of the Glycerol treated-Ethylene Oxide Sterilised Porcine Dermis (Grades 1–2) (P = 0.006) with GlyEO dermis demonstrating either fissures or large fractures present in all specimens studied.
3.5.3.
Fibroblast penetration
A significantly greater degree of fibroblast penetration was seen in Gly-EO Dermis when compared to PermacolTM (P = 0.005). Fibroblast penetration into Gly-EO Dermis ranged from partial penetration across the CSE to abundant fibroblasts throughout the CSE whereas fibroblasts within PermacolTM were present mainly within the hair follicle
Vascular penetration
A significantly greater vascular penetration was seen in Gly-EO Dermis compared to PermacolTM (P = 0.005). Blood vessels were present throughout the full thickness of the Gly-EO dermis whereas PermacolTM showed vascular penetration into hair follicle remnants, with only sparse vessels seen in the surrounding matrix.
4.
Discussion
4.1.
In vitro studies
This study showed that both Gly-EO dermis and to a lesser extent PermacolTM were able to support the growth of keratinocytes and fibroblasts for an in vitro period of 16 days in cell culture. The study represented a simple morphological assessment and did not assess cell proliferation and differentiation. The ability of PermacolTM to support the growth of fibroblasts was in keeping with the work of Oliver
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et al. who demonstrated that normal human skin fibroblasts cultured on sheets of intact porcine acellular dermal matrix were able to proliferate and migrate into the collagen and survived for a period of up to 7 weeks [16]. The appearance of the keratinocytes grown on PermacolTM were in keeping with the typical features of skin composites lacking an initial basement membrane (BM), with most of the keratinocytes appearing disorganised and anucleate [17].
4.2.
In vivo studies in the nude mouse model
This study was unable to demonstrate the presence of an epidermal layer on the surface of PermacolTM following a 2week period of grafting in the nude mouse model. PermacolTM was instead invested in a layer of loose connective tissue. Again, this result is not that surprising considering the key role of basement membrane proteins in both keratinocyte adhesion and the formation of a stratified epithelium. Previous immunocytochemistry has shown that PermacolTM is composed primarily of collagen with complete absence of the basement membrane proteins—collagen type IV, laminin and fibronectin [18]. In contrast Glycerol treatedEthylene Oxide Sterilised Porcine Dermis is known to preserve these basement membrane proteins during processing [19]. In addition, the lack of fibroblast penetration into PermacolTM may have also been an inhibitory factor in preventing the normal keratinocyte–fibroblast interactions known to be important in enhancing basement membrane formation both in vitro [20] and in vivo [21]. Glycerol treatedEthylene Oxide Sterilised Porcine Dermis demonstrated a well-formed, mature epidermis with a stratum spinosum, granulosum and corneum in the majority of specimens after 14 days in vivo. PermacolTM demonstrated a significantly superior dermal quality to Gly-EO with the majority of Gly-EO Dermis specimens showing both fractures and small fissures. This difference in dermal quality may reflect the different methods of processing and storage of the two dermal matrices. Gly-EO Dermis is stored frozen in PBS which allows the formation of ice crystals whereas PermacolTM is g-irradiated and stored moist in normal saline. In conclusion, the results from this study suggest that PermacolTM is not suitable for use as a cultured skin equivalent due to its limited ability to support cultured keratinocytes in vitro and to generate a viable epidermal layer in vivo. It has been postulated that this may be due to the lack of substrate attachment sites and basement membrane proteins in this material which are necessary for the growth, differentiation and replication of many cell types in culture [22].
Acknowledgment This work was funded and supported by the Restoration of Appearance and Function Trust (RAFT).
Conflict of interest None.
reference
[1] Bouvard V, Germain L, Rompre P, Roy B, Auger FA. Influence of dermal equivalent maturation on the development of a cultured skin equivalent. Biochem Cell Biol 1992;70: 34–42. [2] Bell E, Ehrlich HP, Buttle DJ, Nakatsuji T. Living tissue formed in vitro and accepted as skin-equivalent tissue of full thickness. Science 1981;211:1052–4. [3] Maruguchi T, Maruguchi Y, Suzuki S, Matsuda K, Toda K, Isshiki N. A new skin equivalent: keratinocytes proliferated and differentiated on collagen sponge containing fibroblasts. Plast Reconstr Surg 1994;93:537–44. [4] Hansbrough JF, Morgan JL, Greenleaf GE, Bartel R. Composite grafts of human keratinocytes grown on a polyglactin mesh—cultured fibroblast dermal substitute function as a bilayer skin replacement in full-thickness wounds on athymic mice. J Burn Care Rehabil 1993;14: 485–94. [5] Zacchi V, Soranzo C, Cortivo R, Radice M, Brun P, Abatangelo G. In vitro engineering of human skin-like tissue. J Biomed Mater Res 1998;40:187–94. [6] Meana A, Iglesias J, Del Rio M, Larcher F, Madrigal B, Fresno MF, et al. Large surface of cultured human epithelium obtained on a dermal matrix based on live fibroblast-containing fibrin gels. Burns 1998;24: 621–30. [7] Hansbrough JF, Boyce ST, Cooper ML, Foreman TJ. Burn wound closure with cultured autologous keratinocytes and fibroblasts attached to a collagen–glycosaminoglycan substrate. JAMA 1989;262:2125–30. [8] Cooper ML, Hansbrough JF. Use of a composite skin graft composed of cultured human keratinocytes and fibroblasts and a collagen-GAG matrix to cover full-thickness wounds on athymic mice. Surgery 1991;109:198–207. [9] Boyce ST, Greenhalgh DG, Kagan RJ, Housinger T, Sorrell JM, Childress CP, et al. Skin anatomy and antigen expression after burn wound closure with composite grafts of cultured skin cells and biopolymers. Plast Reconstr Surg 1993;91:632–41. [10] Harriger MD, Warden GD, Greenhalgh DG, Kagan RJ, Boyce ST. Pigmentation and microanatomy of skin regenerated from composite grafts of cultured cells and biopolymers applied to full-thickness burn wounds. Transplantation 1995;59:702–7. [11] Takami Y, Matsuda T, Yoshitake M, Hanumadass M, Walter RJ. Dispase/detergent treated dermal matrix as a dermal substitute. Burns 1996;22:182–90. [12] Livesey SA, Herndon DN, Hollyoak MA, Atkinson YH, Nag A. Transplanted acellular allograft dermal matrix. Potential as a template for the reconstruction of viable dermis. Transplantation 1995;60:1–9. [13] Walter RJ, Jennings LJ, Matsuda T, Reyes HM, Hanumadass M. Dispase/triton treated acellular dermal matrix as a dermal substitute in rats. Curr Surg 1997;54:371–4. [14] Wainwright DJ. Use of an acellular allograft dermal matrix (AlloDerm) in the management of full-thickness burns. Burns 1995;21:243–8. [15] Wainwright D, Madden M, Luterman A, Hunt J, Monafo W, Heimbach D, et al. Clinical evaluation of an acellular allograft dermal matrix in full-thickness burns. J Burn Care Rehabil 1996;17:124–36. [16] Oliver RF, Barker H, Cooke A. In vitro growth of adult human skin fibroblasts on intact, trypsin-purified rat and pig dermal collagen; unpublished work. [17] Ralston DR, Layton C, Dalley AJ, Boyce SG, Freedlander E, Mac NS. The requirement for basement membrane
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antigens in the production of human epidermal/dermal composites in vitro. Br J Dermatol 1999;140:605–15. [18] Tissue Science Laboratories. The Implantation of Biomaterials. Hampshire, UK: Tissue Science Laboratories; 1998. [19] Chakrabarty KH, Dawson RA, Harris P, Layton C, Babu M, Gould L, et al. Development of autologous human dermal– epidermal composites based on sterilized human allodermis for clinical use. Br J Dermatol 1999;141(November (5)):811–23.
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[20] Konig A, Bruckner-Tuderman L. Epithelial–mesenchymal interactions enhance expression of collagen VII in vitro. J Invest Dermatol 1991;96:803–8. [21] Coulomb B, Friteau L, Baruch J, Guilbaud J, Chretien-Marquet B, Glicenstein J, et al. Advantage of the presence of living dermal fibroblasts within in vitro reconstructed skin for grafting in humans. Plast Reconstr Surg 1998;101:1891–903. [22] Kleinman HK, Klebe RJ, Martin GR. The role of collagen matrices in the adhesion and growth of cells. J Cell Biol 1981;88:473–85.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/burns
Evaluation of PermacolTM as a cultured skin equivalent T.M. MacLeod a,*, A. Cambrey a,1, G. Williams b,2, R. Sanders a,1, C.J. Green b,2 a b
Restoration of Appearance and Function Trust, Mount Vernon Hospital, Northwood, Middlesex HA6 2RN, UK Northwick Park Institute for Medical Research, Northwick Park Hospital, Harrow, Middlesex HA1 3UJ, UK
article info
abstract
Article history:
Skin loss following severe burn requires prompt wound closure to avoid such complications
Accepted 21 January 2008
as fluid and electrolyte imbalance, infection, immune suppression, and pain. In clinical situations in which insufficient donor skin is available, the development of cultured skin
Keywords:
equivalents (dermal matrices seeded with keratinocytes and fibroblasts) may provide a
PermacolTM
useful alternative. The aim of this study was to assess the suitability of a porcine-derived
Cultured skin equivalent
dermal collagen matrix (PermacolTM) to function as a cultured skin equivalent in supporting
Keratinocytes
the growth of keratinocytes in vitro and providing cover to full thickness wounds in the BALB
Fibroblasts
C/nude mouse model. A histological comparison was against Glycerol treated-Ethylene
Epidermis
Oxide Sterilised Porcine Dermis (Gly-EO Dermis) which has successfully been used as a cultured skin equivalent in previous studies. Both Gly-EO Dermis and to a lesser extent PermacolTM were able to support the growth of cultured keratinocytes following a 16-day period of cell culture, however, this study was only able to demonstrate the presence of an epidermal layer on Gly-EO dermis 2 weeks after grafting onto full-thickness wounds in the BALB C/nude mouse model. # 2008 Elsevier Ltd and ISBI. All rights reserved.
1.
Introduction
Skin loss following severe burn requires prompt wound closure to avoid such complications as fluid and electrolyte imbalance, infection, immune suppression, and pain. The ideal treatment for such wounds would be an unlimited supply of autologous skin graft that could be applied in a single procedure. In clinical situations in which insufficient donor skin is available, the development of cultured skin equivalents may provide a useful alternative. The combination of a dermal matrix with added keratinocytes and fibroblasts to resemble a conventional skin graft has been referred to as a ‘‘living skin equivalent’’, ‘‘bilayered skin equivalent’’, ‘‘composite’’ and
‘‘dermo/epidermal culture’’ but for the purposes of this study will be referred to as a ‘‘cultured skin equivalent’’ (CSE). The dermal component of a CSE provides stability, improves handling, promotes epithelial–mesenchymal interactions in vitro leading to greater keratinocyte attachment and differentiation [1]. When applied to a full-thickness wound the dermal component of the CSE acts as a dermal template or scaffold for vascularisation and remodelling into neo-dermis. The first type of dermal matrix used as a cultured skin equivalent incorporated fibroblasts into a gel of acid-extracted collagen overlayed with a suspension of keratinocytes [2]. Other synthetic dermal analogues studied include the collagen sponge [3], polyglycolic acid mesh [4], hyaluronic acid-derived
* Corresponding author. Tel.: +44 1923 835 815; fax: +44 1923 844 031. E-mail addresses: [email protected] (T.M. MacLeod), [email protected] (G. Williams), [email protected] (C.J. Green). 1
Tel.: +44 1923 835 815; fax: +44 1923 844 031. Tel.: +44 208 969 3265; fax: +44 208 869 3270. 0305-4179/$34.00 # 2008 Elsevier Ltd and ISBI. All rights reserved. doi:10.1016/j.burns.2008.01.013 2
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biomaterial [5], fibrin gels [6] and a collagen/glycosaminoglycan (C-GAG) mixture [7–10]. Most of the acceptable dermal substitutes produced thus far appear to be those which employ an acellular dermal matrix (ADM) derived from full or split-thickness skin then treated to remove epithelial (keratinocytes, sweat glands and sebaceous glands) and dermal cellular components (fibroblasts, vascular endothelium and smooth muscle cells). Most studies have focused on ADM derived from human cadaveric skin by various techniques [11,12] for use as a dermal substitute both in animal models [11–13] and in the clinical management of burn wounds [14,15]. The limited availablility of cadaver skin and risk of disease transmission limit the use of human cadaveric de-epidermalised dermis (DED) in dermal replacement. The aim of this study was to assess the suitability of PermacolTM for use as a cultured skin equivalent.
2.
Materials and methods
2.1.
PermacolTM
temperature-controlled (37 8C), humidified, CO2 (5%) incubators. Centrifugation was performed in a Heraeus Labofuge. Microscopic examination was achieved using an Olympus CK2 inverted phase contrast microscope. Cells were counted in a modified Neubauer haemocytometer (Weber Scientific International). Dulbecco’s phosphate-buffered saline (PBS), Dulbecco’s modified Eagles’ medium (DMEM), DMEM/Hams F12 medium and fetal calf serum were obtained from Gibco Ltd. The additives to the culture media: cholera toxin, epidermal growth factor, transferrin and insulin were obtained from Sigma Ltd.; and lyothyronine was obtained from Koch-Light Ltd. Antibiotics, trypsin, 0.05% ethylenediaminetetra-acetic acid (versene), di-methyl sulfoxide (DMSO), collagen type I and 0.4% trypan blue solution were also obtained from Sigma Ltd. The neutral protease Dispase1 and bacterial collagenase D were both purchased from Roche Diagnostics Ltd. Glycerol was obtained from BDH. Griener Labortechnik provided plastic ware including culture flasks and universals. Stainless steel rings and grids were manufactured in the Department of Bioengineering, Mount Vernon Hospital.
2.4. PermacolTM is a porcine-derived acellular dermal matrix whose manufacture involves trypsinisation (to remove all living cells and non-collagenous debris), solvent extraction (to remove all lipid and fat deposits), g-irradiation and isocyanate cross-linkage. It is manufactured by Tissue Science Laboratories as 5 cm 5 cm sterile sheets which are then double vacuum packed, heat sealed in sachets of aluminium foil (inner) and polyester/polythene (outer) sachets and stored at room temperature. This study used PermacolTM sheets 0.4 mm in thickness, which were kept moist in sterile saline.
2.2. Glycerol treated-Ethylene Oxide Sterilised Porcine Dermis (Gly-EO Dermis) Split-thickness skin grafts were obtained from a single large white pig (Fulmer Place Farm) using a modified Watson skin graft knife during euthanasia. The harvested skin was placed in 50:50 glycerol in phosphate-buffered saline for 4 h, 85:50 glycerol in phosphate-buffered saline for 4 h and pure glycerol for 40 h. After removing excess glycerol, the sample was placed in a bag and subjected to a standard ethylene oxide sterilisation process. The sterilised sample was rehydrated in PBS at 37 8C for 48 h and then placed in 1 mol/l sodium chloride for 16 h to produce a dermal/epidermal split. The epidermis was gently removed using a fine pair of forceps. Once the deepithelialised dermis (DED) was confirmed as sterile and acellular, it was stored in sterile universal containers in phosphate-buffered saline (Oxoid, Unipath, Hants, U.K.) with 625 mg/ml amphotericin B, 1000 mg/ml streptomycin (Gibco BRL, Life Technologies, Paisley, U.K.) at 20 8C.
2.3.
Fibroblast isolation and culture
Split-thickness skin was obtained from routine plastic surgery specimens of either breast reductions or abdominoplasties. Only specimens free from scars and striae were used. Fibroblasts were extracted from split-thickness skin grafts via collagenase digestion. Following incubation in trypsin and removal of the epidermis for keratinocyte isolation, the remaining dermal elements were separated out. The dermis was finely minced using a scalpel and placed in a 60 mm Petri dish. The tissue was then incubated in 50 ml of collagenase D solution (0.05% in DMEM) for 12 h at 37 8C. After filtration through a fine mesh tea strainer into another tube, this fibroblast suspension was spun down in a centrifuge at 1000 rpm for 5 min. The pellet was then re-suspended in FCM and counted. The fibroblasts were then inoculated into 75 cm2 culture flasks and the cultures continued to confluence. Media was changed twice a week and cultures split as required to a maximum of passage nine.
2.5.
Preparation of 3T3 feeders
Feeder cells were produced from Swiss mouse 3T3 fibroblast cell lines (passages 8–30). Frozen vials of cells were used to generate fresh stocks and discarded after 30 passages. These cells were grown in fibroblast culture medium (FCM) in 150 cm2 flasks (4 106 cells per flask) and routinely split using 0.05% trypsin in versene, two or three times per week. They were then spun down in FCM for 5 min at 1000 rpm and resuspended in keratinocyte culture medium (KCM) after counting. Using a c-irradiator they were lethally g-irradiated with 6000 rad (cobalt 60) and plated in a fresh flask at the appropriate seeding density.
Tissue culture 2.6.
Cell culture procedures were performed in sterile Class II laminar airflow hoods (LaminAir), purchased from Heraeus Instruments. Tissue culture was undertaken in Heraeus
Keratinocyte isolation and culture
Split-thickness skin graft samples were cut into 0.5 cm2 pieces and incubated overnight (12–18 h) at 4 8C in 0.1% (w/v) trypsin
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solution. Epidermal and dermal layers were then peeled apart, and the keratinocytes collected in fetal calf serum by scraping the upper layer of the dermis and lower layer of the epidermis. The freshly isolated (P0) keratinocyte preparation was then centrifuged at 1000 rpm for 5 min and the pellet re-suspended in KCM before counting. P0 cells were then used to produce cultured skin equivalents grafts or expand their number by culture on plastic and produce passaged keratinocyte (P1) cell suspensions. Irradiated Swiss 3T3 mouse fibroblasts were seeded at a density of 2 106 cells per 75 cm2 tissue culture flask as a feeder layer. The flasks were then inoculated with keratinocytes at a density of 2 106 cells per flask. The first medium change was at 48 h. Thereafter, the medium was changed twice a week. Colonies of keratinocytes were seen at 3–4 days and reached 75–85% confluence by 1 week. These cells were detached from the plastic using 0.05% trypsin in versene and re-suspended at a known density in KCM. P1 cells were then used to produce CSE grafts.
2.7.
Seeding of fibroblasts: a sterilised stainless steel ring of 1 cm internal diameter was placed in the centre of the upper surface of the matrix and gently pressed down to ensure a watertight seal. One milliliter of fibroblast cell suspension (1 105 ml 1) was seeded into the steel ring and FCM added, both within and outside the ring, to a height just below the upper edge. This combination was incubated for 24 h, at which point the ring was removed. After 48 h incubation the seeded dermal matrix was turned over. Seeding of keratinocytes: a sterilised ring was placed in the centre of the upper surface of the matrix and gently pressed to ensure a watertight seal. One milliliter of keratinocyte suspension (1 106 ml 1) was seeded into the steel ring and keratinocyte culture medium then added, both within and outside the ring, to a height just below the upper edge. After 24 h the ring was removed and the media changed for fresh KCM. After 4 days the CSE was raised to the air–liquid interface on the stainless steel grids and maintained for a further 10 days changing the media twice a week.
Cryopreservation of cells 2.11.
Cell suspensions were centrifuged, counted and the pellet then re-suspended in cryopreservation media (90% fetal calf serum and 10% DMSO). Cells were frozen in cryovials, wrapped in four layers of tissue paper and then transferred to a 80 8C freezer. This allowed the cells to gradually freeze at approximately 1 8C per minute. Vials were later transferred to liquid nitrogen for long-term storage. Cells were recovered by thawing rapidly in a 37 8C water bath, spinning down in the centrifuge at 1000 rpm for 5 min, re-suspending in media and counting.
2.8.
Cell numbers
Where specific cell numbers were required, the standard trypan blue exclusion method was used. While viable cells are able to exclude the blue dye, non-viable cells are not and appear blue under microscopic examination. Single cell suspensions in a known volume of media were used, mixed in a 1:1 ratio with 0.4% solution of trypan blue. Cells were counted in a modified Neubauer haemocytometer and the cell concentration calculated accordingly.
2.9.
Preparation of matrices
All three biomaterials were divided into 1.5 cm 1.5 cm pieces and placed in phosphate-buffered saline prior to use. Six pieces of each type of matrix were used for the initial in vitro assessment and six pieces were used for the in vivo assessment.
2.10.
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Preparation of cultured skin equivalents
Pieces of the PermacolTM and Gly-EO Dermis (1.5 cm 1.5 cm) were placed in the wells of cell culture plates and incubated with fibroblast culture medium for 24 h at 37 8C as a sterility check. If the medium became turbid, the matrix was discarded. Sterile pieces were then placed in the wells of a six-well plate and seeded with cells as described below.
Transport of cultured skin equivalents
CSE grafts were transported between sites on their stainless steel grids at the air–liquid interface in fresh KCM. Six-well plates were put in a plastic container lined with wet paper towels (to maintain humidity) with an open tap on one side and the airtight lid replaced snugly. The container and lid were allowed to equilibrate with the temperature of the incubator and the CO2 concentration for 1 h. After this time the tap was closed as soon as the incubator door was opened and the container placed in a cool box for transportation. The container was left sealed until the skin equivalents were ready for transfer to the athymic mice 2 or 3 h later.
2.12.
Nude mice
All experimental surgical procedures were performed under License from the Home Office (Animals Scientific Procedures Act 1986). A maximum of 10 nude mice were kept in each large sterilised box, respectively and allowed to acclimatise to their surroundings for 1 week prior to any experimental procedure. After the first surgical procedure the animals were housed in individual cages to prevent them from disturbing each other’s dressings. Water and feed were supplied ad libitum. The room was kept at 22–24 8C with 45– 55% humidity. Lighting was adjusted to provide 12 h light and darkness per day. A Taconic Farms Mouse Scale (Pelouze Scale Company) was used to weigh the athymic mice for calculation of anaesthetic dosages. Hypnorm1 (fentanyl citrate and fluanisone) was purchased from Janssen Animal Health, Hypnoval1 (midazolam) from Roche Diagnostics Ltd. and diazepam from Phoenix Pharmaceuticals Ltd. Athymic mice were anaesthetised using an intra-peritoneal injection of ‘‘CRC cocktail’’ given (0.01 ml/g) consisting of one part Hypnorm1, two parts water for injection and one part Hypnoval1. The duration of anaesthesia was short (approximately 50 min) and animals recovered spontaneously. Six anaesthetic procedures were
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done in this way. Euthanasia was performed by cervical dislocation.
prior to their en bloc excision for processing of paraffin sections.
2.13.
2.17.
Histological analysis
2.17.1.
Epidermal attachment
Grafting of CSEs onto wounds in nude mouse model
Mice were anaesthetised and their skin disinfected with 0.5% (w/v) chlorhexidine gluconate in 70% (v/v) IMS. The mice were placed on sterile drapes and covered with surgical swabs to keep them warm. Each mouse had a 1 cm diameter silicone chambers inserted onto each flank. The skin at the site of the chamber was lifted up with fine forceps and amputated with sharp scissors, leaving a circular full-thickness defect with panniculus carnosus muscle fascia as the wound bed. The edge of the wound was undermined by 5 mm using scissors and the chamber inserted. The silicone chambers are malleable and were compressed then inserted and allowed to expand with the flange under the wound edge. The chambers did not require fixation to the skin. The grafts were trimmed to size and placed with the epidermal surface uppermost within the chamber. The acellular dermis was cut to leave a small rim around the epidermal culture, which sat underneath the flange of the chamber, effecting better graft immobilisation. A layer of non-adherent dressing gauze was placed directly over the graft, followed by two layers of swab soaked in normal saline solution and finally several dry layers of swab, all cut to size. The upper dressings were lightly compacted such that a gentle amount of pressure was exerted on the graft to aid immobilisation. Elastoplast bandage was then applied in a circumferential manner around the trunk of the animals. The bandage was applied loosely over the abdomen to permit free respiratory movements, but firmly on the dorsum to apply pressure on the wounds.
Epithelial attachment of cultured skin equivalents was viewed over the length of a section and expressed as a percentage (to the nearest 5%).
2.17.2.
2.17.3.
2.18.
In vitro assessment
After a total of 16 days in culture, six cultured skin equivalents based on PermacolTM and Gly-EO Dermis were fixed and processed for paraffin sections. A further six CSEs in each group were then used for in vivo experiments.
2.16.
Fibroblast penetration
Fibroblast penetration was graded as being: Grade 1, partial penetration of the matrix with fibroblasts; Grade 2, fibroblasts present across the matrix but sparse in the superficial part; and Grade 3, abundant fibroblasts throughout the matrix.
2.19.
Vascular penetration
Matrix vascularity was graded as being: Grade 1, partial penetration of the matrix; Grade 2, vessels present across the matrix but sparse in its superficial part; and Grade 3, abundant vessels present throughout the matrix.
Epidermal thickness
Histological examination
Tissue specimens were fixed in 4% paraformaldehyde in phosphate-buffered saline. After fixation, tissues were embedded in paraffin and 5 mm thick sections were cut and stained with haematoxylin and eosin or Masson’s trichrome.
2.15.
Dermal quality
Dermal quality was graded as being: Grade 1, large fractures of the dermis; Grade 2, small fissures of the dermis: and Grade 3, no obvious distortion of the dermis.
2.20. 2.14.
Dermal–epidermal junction
The dermal–epidermal junction was graded from: Grade 1, completely flat; to Grade 5, well-formed rete ridges throughout.
In vivo assessment
A total of six BALB C/nude mice were used for the in vivo assessment of cultured skin equivalents. Each mouse had two full-thickness wounds created (one on each flank) giving (n = 6) wounds for analysis in each of the two groups (PermacolTM and Gly-EO Dermis) Animals were euthanased 14 days after the grafting by cervical dislocation. All CSEs were found to be fully adherent to the underlying full-thickness wound bed
Epithelial thickness of cultured skin equivalents was calculated using Seescan image analysis. Histological sections of the matrices stained with Masson’s trichrome were viewed under the fluorescent microscope green filter. This clearly displayed the non-cornified epidermal layer in each section. The average height of epithelium within one microscopic field was then assessed by measuring the area of epidermis and dividing by the length of the dermo-epidermal junction (DEJ) present.
2.21.
Matrix thickness
Dermal thickness of cultured skin equivalents was calculated using Seescan image analysis. Histological sections of the matrices stained with Masson’s trichrome and viewed using the fluorescent microscope green filter clearly displayed the dermal layer in each section.
2.22.
Statistical analysis
It was not possible to analyse epidermal height, epidermal attachment or dermal–epidermal junction since we were only able to demonstrate the presence of a viable epidermal layer on Gly-EO Dermis. Dermal thickness was measured on a contin-
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uous scale and compared using analysis of variance with a contrast coefficient. Dermal quality, fibroblast penetration and vascular penetration were measured on ordered categorical scales and compared using the Mann–Whitney test.
3.
Results
3.1.
In vitro assessment
Gly-EO Dermis and to a lesser extent PermacolTM were both able to support the growth of human keratinocytes and fibroblasts in vitro.
3.2.
In vivo assessment
From the 12 grafts examined in vivo, two wounds were obviously infected (one Gly-EO Dermis and one PermacolTM graft) and these were omitted from further study.
3.3.
Graft stability
Both Gly-EO Dermis and PermacolTM were fully adherent to the underlying full-thickness wound bed after a period of 14 days in vivo.
3.4.
Epidermal assessment
This study was unable to demonstrate the presence of a viable epidermal cell layer on the surface of PermacolTM following a 2-week period in vivo in the BALB C/nude mouse model. Gly-EO Dermis showed an overlying epidermis that was a median of 95% adherent (range 30–100), 45.9 mm in thickness (range 27.3–78.2) and demonstrated a DEJ median score of 2 (range 0–4). As a result of this a statistically significant difference (P < 0.05) occurred between Gly-EO Dermis and PermacolTM with respect to epidermal attachment (P < 0.05).
3.5.
Dermal assessment (Fig. 1)
3.5.1.
Dermal thickness
Fig. 1 – Comparison of matrix thickness, matrix quality, fibroblast penetration and vascular penetration for Gly-EO Dermis and PermacolTM. Results shown for dermal thickness are means W standard deviation. Matrix quality, fibroblast penetration and vascular penetration are represented by median (and range). There was no significant difference in thickness between PermacolTM and Gly-EO Dermis (P = 0.67). Gly-EO Dermis showed a significantly inferior dermal quality compared to PermacolTM (P = 0.006). A significantly greater degree of fibroblast penetration was seen in Gly-EO Dermis compared to PermacolTM (P = 0.005). A significantly greater degree of vascular penetration was seen in Gly-EO Dermis compared to PermacolTM (P = 0.005).
remnants with only partial penetration across the surrounding matrix.
3.5.4. There was no significant difference in the thickness of 0.4 mm PermacolTM and Glycerol treated-Ethylene Oxide Sterilised Porcine Dermis (P = 0.67).
3.5.2.
Dermal quality
The quality of the dermis in PermacolTM was significantly better than that of the Glycerol treated-Ethylene Oxide Sterilised Porcine Dermis (Grades 1–2) (P = 0.006) with GlyEO dermis demonstrating either fissures or large fractures present in all specimens studied.
3.5.3.
Fibroblast penetration
A significantly greater degree of fibroblast penetration was seen in Gly-EO Dermis when compared to PermacolTM (P = 0.005). Fibroblast penetration into Gly-EO Dermis ranged from partial penetration across the CSE to abundant fibroblasts throughout the CSE whereas fibroblasts within PermacolTM were present mainly within the hair follicle
Vascular penetration
A significantly greater vascular penetration was seen in Gly-EO Dermis compared to PermacolTM (P = 0.005). Blood vessels were present throughout the full thickness of the Gly-EO dermis whereas PermacolTM showed vascular penetration into hair follicle remnants, with only sparse vessels seen in the surrounding matrix.
4.
Discussion
4.1.
In vitro studies
This study showed that both Gly-EO dermis and to a lesser extent PermacolTM were able to support the growth of keratinocytes and fibroblasts for an in vitro period of 16 days in cell culture. The study represented a simple morphological assessment and did not assess cell proliferation and differentiation. The ability of PermacolTM to support the growth of fibroblasts was in keeping with the work of Oliver
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et al. who demonstrated that normal human skin fibroblasts cultured on sheets of intact porcine acellular dermal matrix were able to proliferate and migrate into the collagen and survived for a period of up to 7 weeks [16]. The appearance of the keratinocytes grown on PermacolTM were in keeping with the typical features of skin composites lacking an initial basement membrane (BM), with most of the keratinocytes appearing disorganised and anucleate [17].
4.2.
In vivo studies in the nude mouse model
This study was unable to demonstrate the presence of an epidermal layer on the surface of PermacolTM following a 2week period of grafting in the nude mouse model. PermacolTM was instead invested in a layer of loose connective tissue. Again, this result is not that surprising considering the key role of basement membrane proteins in both keratinocyte adhesion and the formation of a stratified epithelium. Previous immunocytochemistry has shown that PermacolTM is composed primarily of collagen with complete absence of the basement membrane proteins—collagen type IV, laminin and fibronectin [18]. In contrast Glycerol treatedEthylene Oxide Sterilised Porcine Dermis is known to preserve these basement membrane proteins during processing [19]. In addition, the lack of fibroblast penetration into PermacolTM may have also been an inhibitory factor in preventing the normal keratinocyte–fibroblast interactions known to be important in enhancing basement membrane formation both in vitro [20] and in vivo [21]. Glycerol treatedEthylene Oxide Sterilised Porcine Dermis demonstrated a well-formed, mature epidermis with a stratum spinosum, granulosum and corneum in the majority of specimens after 14 days in vivo. PermacolTM demonstrated a significantly superior dermal quality to Gly-EO with the majority of Gly-EO Dermis specimens showing both fractures and small fissures. This difference in dermal quality may reflect the different methods of processing and storage of the two dermal matrices. Gly-EO Dermis is stored frozen in PBS which allows the formation of ice crystals whereas PermacolTM is g-irradiated and stored moist in normal saline. In conclusion, the results from this study suggest that PermacolTM is not suitable for use as a cultured skin equivalent due to its limited ability to support cultured keratinocytes in vitro and to generate a viable epidermal layer in vivo. It has been postulated that this may be due to the lack of substrate attachment sites and basement membrane proteins in this material which are necessary for the growth, differentiation and replication of many cell types in culture [22].
Acknowledgment This work was funded and supported by the Restoration of Appearance and Function Trust (RAFT).
Conflict of interest None.
reference
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