Development of a one-step approach for the reconstruction of full thickness skin defects using...

JBUR-4174; No. of Pages 9

Development of a one-step approach for thereconstruction of full thickness skin defects usingminced split thickness skin grafts andbiodegradable synthetic scaffolds as a dermalsubstitute

Kavita Sharma a, Anthony Bullock b, David Ralston a, Sheila MacNeil b,*aDepartment of Plastic and Reconstructive Surgery, Sheffield Teaching Hospitals, NHS Foundation Trust, United

KingdombDepartment of Materials Science and Engineering, Kroto Research Institute, University of Sheffield, Sheffield S3 7HQ,

United Kingdom

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a r t i c l e i n f o

Article history:

Accepted 30 September 2013

Keywords:

Skin substitute

Burn reconstruction

Electrospun scaffold

Minced human skin

Wound reconstruction

a b s t r a c t

Introduction: Tissue engineering has progressed in delivering laboratory-expanded kerati-

nocytes to the clinic; however the production of a suitable alternative to a skin graft,

containing both epidermis and dermis still remains a challenge.

Aim: To develop a one-step approach to wound reconstruction using finely minced split

thickness skin and a biodegradable synthetic dermal substitute.

Methods: This was explored in vitro using scalpel diced pieces of split thickness human skin

combined with synthetic electrospun polylactide (PLA) scaffolds. To aid the spreading of

tissue, 1% methylcellulose was used and platelet releasate was examined for its effect on

cellular outgrowth from tissue explants. The outcome parameters included the metabolic

activity of the migrating cells and their ability to produce collagen. Cell presence and

migration on the scaffolds were assessed using fluorescence microscopy and SEM. Cells

were identified as keratinocytes by immunostaining for pan-cytokeratin. Collagen deposi-

tion was quantified by using Sirius red.

Results: Skin cells migrated along the fibers of the scaffold and formed new collagen. 1%

methylcellulose improved the tissue handling properties of the minced skin. Platelet

releasate did not stimulate the migration of skin cells along scaffold fibers. Immunohis-

tochemistry and SEM confirmed the presence of both epithelial and stromal cells in the new

tissue.

Conclusion: We describe the first key steps in the production of a skin substitute to be

assembled in theatre eliminating the need for cell culture. Whilst further experiments are

needed to develop this technique it can be a useful addition to armamentarium of the

reconstructive surgeon.

# 2013 Elsevier Ltd and ISBI. All rights reserved.

* Corresponding author at: The Kroto Research Institute North Campus, University of Sheffield Broad Lane, Sheffield S3 7HQ, UK.Tel.: +44 0114 222 5995; fax: +44 0114 222 5943.

Available online at www.sciencedirect.com

ScienceDirect

journal homepage: www.elsevier.com/locate/burns

E-mail address: [email protected] (S. MacNeil).

Please cite this article in press as: Sharma K, et al. Development of a one-step approach for the reconstruction of full thickness skin defects usingminced split thickness skin grafts and biodegradable synthetic scaffolds as a dermal substitute. Burns (2013), http://dx.doi.org/10.1016/j.burns.2013.09.026

0305-4179/$36.00 # 2013 Elsevier Ltd and ISBI. All rights reserved.http://dx.doi.org/10.1016/j.burns.2013.09.026

http://dx.doi.org/10.1016/j.burns.2013.09.026

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http://dx.doi.org/www.elsevier.com/locate/burns



b u r n s x x x ( 2 0 1 3 ) x x x – x x x2

1. Introduction

Split thickness skin grafts (STSGs) remain the gold standard

treatment for resurfacing full thickness burns in the acute

setting and for subsequent release of contractures. In larger

burns (>30% total body surface area) donor sites become a

limiting factor. Meshing and re-using donor site are employed

to overcome this, however meshing is associated with a less

aesthetically pleasing outcome and continued use of donor

sites results in unpleasant scarring, delayed healing and

pigmentation problems [1]. Over the last thirty years advances

in the development of tissue-engineered products have

revolutionized our ability to manage wounds [2,3]. Dermal

substitutes can be used to replace contractures resulting from

full thickness burns or poorly healed split thickness grafts and

can facilitate the removal and reconstruction of large benign

and malignant skin lesions [4]. There is strong evidence that

small amounts of cells or minced skin tissue can improve the

healing of donor sites [5,6] and that inclusion of a cellulose

based gel can help reduce trans-epithelial water loss (TEWL)

[7] which also aids re-epithelialization.

The ‘‘ideal skin substitute’’ is inexpensive, has a long shelf

life, is usable off the shelf, non-antigenic, durable, flexible,

prevents water loss, is a barrier against microorganisms,

drapes well, is easy to secure, and can be applied in a one-stage

procedure [8]. A material possessing all of these properties is

yet to be produced. Our aim is to develop a skin substitute for

small-scale reconstructive surgery by combining knowledge of

tissue engineering of skin with recent progress in developing a

synthetic biodegradable poly-L-lactide (PLA) scaffold. We seek

Fig. 1 – Cartoon of our proposed approach from biopsy to appli

skin, (B) minced skin placed in 1% methylcellulose, (C) suspens

electrospun scaffold and (D) application onto patient.

Please cite this article in press as: Sharma K, et al. Development of a one-stminced split thickness skin grafts and biodegradable synthetic scaffoj.burns.2013.09.026

to develop a one-step approach to reconstruction of full

thickness skin defects.

In this study we examined the ability of an electrospun

scaffold to support outgrowth of keratinocytes and fibroblasts

from minced pieces of split thickness skin. We examined how

to introduce skin pieces to scaffolds and the potential benefits

of adding methylcellulose as a spreading agent and platelet

releasate as a potential stimulator of cell outgrowth from skin

pieces.

2. Materials and methods

Fig. 1 shows a cartoon of our proposed approach. A biopsy with

the thickness of a conventional split thickness skin graft (10/

1000 in.) would be harvested and finely minced using standard

scalpel blades. The skin would then be taken up into a solution

(for clinical use we anticipate this to be sterile saline to which a

thickening agent (1% methylcellulose) would have been added).

This suspension would then be spread onto a layer of scaffold,

followed by second layer of scaffold on top. The construct would

then be placed in the wound site and secured using fibrin glue.

2.1. Preparation of split thickness human skin

STSGs were obtained after gaining fully informed written

consent from patients following elective abdominoplasty or

breast reduction surgery for the use of excess skin under a

Human Tissue Authority research tissue bank licence 08/

H1308/39. All tissue was used on an anonymous basis. STSGs

were stored in phosphate buffered saline (PBS) supplemented

cation in the clinical setting. (A) Mincing biopsy of patient

ion of minced skin placed in between two layers of

ep approach for the reconstruction of full thickness skin defects usinglds as a dermal substitute. Burns (2013), http://dx.doi.org/10.1016/



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with 0.625 mg/ml amphotericin B, 100 IU/ml penicillin and

100 mg/ml streptomycin at 4 8C until used – normally within

24 h of receipt.

2.2. Preparation of poly-L-lactide (PLA) Scaffolds

Scaffolds were electrospun aseptically in a clean room

environment, using reagents and equipment which were

either procured sterile or rendered so before use by autoclav-

ing. A 10% (w:w) solution of medical grade PLA (Purac, the

Netherlands) in dichloromethane (Sigma–Aldrich, Poole,

United Kingdom) was electrospun from 4 syringes fitted with

11 gauge blunt tipped needles at a flow rate of 30 ml/min/

syringe (giving a total of 120 ml/min). An accelerating voltage of

�17,000 V DC from a power supply (Genvolt, United Kingdom)

connected to the needles induced a Taylor cone from which

fibers were extruded toward an earthed rotating drum (the

collector) wrapped in aluminum foil. The collector was rotated

at 200RPM – a rotational speed previously been shown to allow

for a random fiber arrangement (Fig. 2). Each 15 � 20 cm sheet

was coated with fibers derived from 6 ml of PLA solution

equating to 0.6 g PLA. Sheets of scaffold were dried at room

temperature overnight in a laminar flow cabinet. Sheets were

then sealed and stored at 4 8C until use. Pore size of scaffold

was calculated to be 20 mm. For experiments, sheets were cut

into 2 � 2 cm squares under sterile conditions (Fig. 2).

2.3. Preparation of 1% methylcellulose

Methylcellulose powder was obtained from Sigma–Aldrich, UK

and sterilized by autoclaving. A 1% w/v solution of sterile

methylcellulose in Green’s media supplemented with 10% v/v

fetal calf serum (FCS) was used in these experiments.

Fig. 2 – The technique of electrospinning. (A) Electrospinning de

syringes, rotating collector and power supply, (B) sample of elec

lactide (PLA) scaffolds showing fiber morphology and random a


2.4. Preparation of platelet releasate

Whole blood (60 ml) from a single donor was collected and

added to 0.9 ml of anticoagulant (ACD) (38 mM anhydrous

citric acid, 75 nM sodium citrate and 124 mM D-Glucose in

dH20). Platelet rich plasma (PRP) was created by centrifuga-

tion of the blood at 150 g for 10 min, the resulting PRP above

the packed suspension of red cells was retained. Prosta-

glandin E1 (PGE1) was added to the PRP (1 mM) and gently

mixed. After centrifugation at 720 g (2500RPM) for 10 min,

the resulting platelets were resuspended in 10% Green’s

media at a concentration of 2 � 108 cells/ml. Thrombin

(0.1 U/ml) was added, gently mixed, and incubated at

37 8C for 20 min. The resulting solution was separated

from the clotted material, and filtered through a 0.2 mm

pore filter to create 10 ml of sterile platelet releasate in

Green’s media.

2.5. Application of skin pieces to PLA scaffolds

Small pieces of split thickness skin (approximately 2 � 2 cm)

were cut into 5 � 5 mm squares which were each further

minced into 16 pieces each using a 22 blade scalpel. To this

1 ml of Green’s media or Green’s media containing varying

concentrations of methylcellulose was added. The suspen-

sion of minced skin (from one 5 mm � 5 mm – (0.25 cm2))

was placed over a 1 cm diameter circle (0.79 cm2) in the

center of each PLA scaffold representing a 3-fold increase in

area. A second layer of PLA scaffold was added on top of

some samples and held in place with a 1 cm internal

diameter sterile medical grade stainless steel ring (Medical

Engineering Department, Royal Hallamshire Hospital,

Sheffield).

vice in clean room comprising syringe pump, polymer filled

trospun sheet, (C and D) SEM image of electrospun Poly-L-

rrangement at 60 mm (C) and at 20 mm (D).




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2.6. Assessment of use of methylcellulose as a spreadingagent

Initial experiments used a range of concentrations of

methylcellulose (from 0.1 to 2%) to determine a concentration,

which increased the viscosity of media such that the pieces of

skin could be evenly spread on the scaffold. It was found that

1% methylcellulose added to media produced a slightly

viscous suspension of skin pieces in media, which could then

be spread on surfaces.

2.7. Assessment of effect of platelet releasate on themigration of skin cells on the scaffold

Platelet releasate (0.5 ml releasate from 2 � 108 platelets/ml

PRP) in Green’s media was added to constructs either with or

without methylcellulose in the center of each metal ring.

2.8. Assessment of the performance of skin pieces on thescaffolds

All skin/scaffold constructs were incubated at 37 8C, 5% CO2

atmosphere with media changes every 48–72 h for 7, 14, and 21

days respectively. The performance of the cells growing out

from the skin pieces and the impact of including methylcellu-

lose or platelet releasate or a combination of both on the cells

was assessed by looking at their metabolic activity and their

ability to produce collagen. Cell presence and migration on the

scaffolds were seen by staining for cell nuclei with DAPI and

the relationship of cells to the scaffold fibers were visualized

using SEM. Cells were also identified as keratinocytes by

immunostaining for pan-cytokeratin.

2.9. Fixation of cells

Samples were washed three times with PBS followed by the

addition of 3mls of 3.7% formaldehyde in PBS. These were

incubated at 37 8C, 5% CO2 atmosphere for 20 min.

2.10. Detection of cellular migration onto scaffold fibersusing fluorescence microscopy

DAPI (40, 6-diamidino-2-phenylindole) 1 mg/ml in PBS was

added to each fixed sample and incubated at 37 8C in the dark

for 30 min. Samples were washed three times with PBS then

imaged using an Axon ImageXpress microscope (AxonCorp,

USA) at an excitation wavelength of 360 nm and emission

wavelength of 480 nm. DAPI stained cell nuclei appearing on

the scaffold fibers adjacent to minced tissue would indicate

migration.

2.11. Quantification of collagen deposition in scaffolds bySirius red staining

1 mg/ml of Sirius red F3B (C.I. 35780, Direct Red 80, Sigma–

Aldrich) in saturated picric acid was added to each of the

dissected constructs and subjected to mild shaking for 18 h.

The constructs were washed with PBS until no further red

color was eluted. Stained electrospun scaffold fibers

were observed under direct light microscopy and digital


photographs obtained. For a quantitative analysis, the stain

from scaffolds was eluted with 500 ml of de-stain solution

(0.2 M NaOH/methanol 1:1) for 30 min. 100 ml samples of the

resulting solution from each sample were diluted with 1:1 with

100 ml of distilled water, and placed into a 96 well plate. Optical

density was then measured at 490 nm with a plate reader

spectrophotometer (Bio-tek ELx800, USA). The dilution step

was necessary to obtain reading within the measurable range

of the plate reader.

2.12. Histology

Samples were fixed in 10% buffered formalin for 2 h prior to an

overnight incubation in OCT compound. Samples were then

frozen, mounted, and 10 mm cryosections were cut. Sections

were soaked in water for 2 min before being incubated in

Harris’s Hematoxylin for 2 min, washed with water for 1 min

followed by Eosin Y (water based) for 5 min. Samples were

rinsed and mounted with a small amount of glycerol and a

coverslip.

Samples were then incubated in 2.5% glutaraldehyde in

0.1 M phosphate buffer for 3 h at 4 8C followed by incubation in

aqueous 2% osmium tetraoxide for 1 h. Samples were then

dehydrated in ascending grades of alcohol, freeze dried,

bisected and mounted on 12.5 mm stubs. The samples were

then sputter coated with approximately 25 nm of gold and

then examined using a scanning electron microscope (Philips/

FEI XL-20 SEM) at an accelerating voltage of between 10 and

15 kV and a SPOT size between 2 and 3 [9].

2.13. Statistics

Values are expressed as the mean � standard error of the

mean (SEM). Student’s unpaired t-test was used to assess the

statistical significance of differences between sample groups.

3. Results

Initial work focused on determining that skin cells would grow

out from small pieces of skin irrespective of their orientation.

This was important as for ease of use it would be more

convenient to finely mince the skin pieces, immerse them into

a thickening agent and then apply the suspension onto

scaffold without having to consider the orientation of the skin

pieces. This is illustrated in Fig. 3A and B. These constructs

cultured for 14 days, show cells emerging and colonizing

scaffold fibers as evidenced by collagen deposition. This is

seen as a visible stained margin of Sirius red around each

explant irrespective of orientation. H&E staining of finely

minced STSG confirmed outgrowth of cells onto scaffold

(Fig. 3C). It should be noted that the scaffold material was

degraded by the solvents in the staining process leaving the

tissue and cell material behind.

Additionally we conducted a series of experiments using a

range of concentrations of methylcellulose from 0.1 to 2% to

determine the optimal concentration that would be required

for its use in aiding the spread of minced skin onto scaffold

fibers. We found that a concentration of 1% methylcellulose

allowed minced skin to be in suspension and at the same time




Fig. 3 – (A) Split thickness skin graft (STSG) placed on electrospun scaffold for 14 days then stained with Sirius red in different

orientations from epidermal side up to epidermal side down and then finally minced into small pieces and randomly

orientated. The red stain denotes collagen deposition using Sirius red. (B) Sirius red stain of the edge of a minced piece of

STSG showing collagen deposition by cells migrating out of the STSG. (C) Hematoxylin & Eosin staining of a cryosection of

finely minced STSG in scaffold illustrating the arrangement of minced skin in between scaffold. (D) Examples of the even

distribution of suspension stained with Sirius red (For interpretation of the references to color in this figure legend, the

reader is referred to the web version of the article.).

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be viscous enough to allow an even spread onto scaffold fibers.

This is illustrated in Fig. 3D, where constructs cultured for 14

days, showed an even distribution of collagen deposition

highlighted with Sirius red staining.

We then compared two methodologies for applying tissue

to scaffolds as illustrated in Fig. 4. In the first one minced

tissue in media with 1% methylcellulose was placed on a

monolayer of scaffold, in the second it was sandwiched

between two layers of scaffold. The latter was found to be very

convenient to prevent loss of the tissue and provided for a

longer contact time between fibers of the scaffold and the

minced skin to facilitate the migration of cells from tissue

pieces. Fig. 4D–F shows H&E sections of the outward migration

to be broadly comparable for cells in media and for cells in

media with 1% methylcellulose. In both cases cells are growing

out onto the scaffold. Methylcellulose neither inhibited nor

enhanced outgrowth (this is quantified further on). Where the

tissue was placed between two sheets of scaffold, outgrowth

was visible in all directions around the scaffold. Fibroblasts

were seen on scaffolds fibers in all cases, as shown in

examples in Fig. 4G–I. In contrast where keratinocytes

migrated out and proliferated in sufficient numbers at the

surface of the scaffolds they were seen to form a monolayer

within 14–21 days. Confocal microscopy of such layers

confirmed the presence of keratinocytes by immunostaining

for keratins with pancytokeratin staining. This revealed areas

where layers of keratinocytes were 1–2 cell layers thick (Fig. 5).

We then assessed the extent of cell outgrowth over 21 days

and production of collagen using Sirius red. This showed that


migration occurred in all cases. Examples of outgrowth after 7

and 21 days are shown in Fig. 6A and B respectively. Sirius red

staining revealed that cells deposited collagen during the

colonization of the scaffold indicated by the red margin, which

increased over time during incubation. Elution of the stain

showed an increase in total collagen over time (Fig. 7). Sirius

red stain indicating collagen deposition was always seen in a

halo around each piece of skin tissue embedded in the scaffold

(Fig. 6C and D).

Fig. 7 demonstrates the quantitative analysis of collagen

deposited over time. Collagen deposition increased with time

for all experiments. There was no significant effect on collagen

production with the addition of methylcellulose and there was

a reduction in collagen deposition at 21 days in samples

containing platelet releasate, but this was not statistically

significant. Samples which contained both methylcellulose

and platelet releasate showed identical collagen production to

control samples over 21 days (Table 1).

4. Discussion

The aim of this study was to commence the development of a

one-stage approach for reconstruction of full thickness skin

defects by combining minced pieces of human skin with a

synthetic dermal substitute.

The principle of using small pieces of split thickness skin

graft and spreading them over a wide area to regenerate skin

barrier function has been long established with the technique




Fig. 4 – Images depicting the various arrangements scaffold (one (A + B) versus two (C) layers) minced split thickness skin and

methylcellulose. Hematoxylin & Eosin staining of cryosections of each arrangement of scaffold after 14 days of culture (D–F),

SEM images of each arrangement (G–I). Scale bar for figures D–F = 400 mm; scale bar for figure G = 50 mm and scale bar for

figures H–I = 20 mm.

Fig. 5 – Confocal microscopy of keratinocytes after 4 weeks, cells are immunostained with pancytokeratin antibody (red) and

DAPI (blue). (A) Plan view, the dark branches are the shadows left by the scaffold. (B) Confocal projection of a layer of

keratinocytes allowing us to see a section through the layer of keratinocytes. (For interpretation of the references to color in

this figure legend, the reader is referred to the web version of the article.)

b u r n s x x x ( 2 0 1 3 ) x x x – x x x6


Please cite this article in press as: Sharma K, et al. Development of a one-step approach for the reconstruction of full thickness skin defects usingminced split thickness skin grafts and biodegradable synthetic scaffolds as a dermal substitute. Burns (2013), http://dx.doi.org/10.1016/j.burns.2013.09.026



Fig. 6 – Microscopy of scaffolds after 7 (A and C), and 21-days (B and D) cultures showing DAPI stained nuclei (A and B) and

Sirius red staining (C and D), scale bar = 200 mm.

b u r n s x x x ( 2 0 1 3 ) x x x – x x x 7


of MEEK grafting. In this technique, considerable care is taken

to create precise skin squares and maintain their orientation

with the epidermal surface uppermost on the underlying

wound bed. This technique although clinically successful is no

longer in widespread use as it is time consuming and has been

0 7 14 210

10

20

30

40

50

60

70

80

90

100

Siri

us re

d st

ain

(abs

) / g

sca

ffold

Time (days)

Control Methyl cellulose Platelet releaseate Methyl cellulose + Platelet releasate

Fig. 7 – Graph illustrating collagen deposition on fibers of

scaffold at 7, 14 and 21-day time intervals in constructs

with no treatment, 1% methylcellulose, platelet releasate

and a combination of 1% methylcellulose and platelet

releasate.


superseded by the meshed split skin graft. Meanwhile

advances in tissue engineering have led to methodologies

for rapid expansion of keratinocytes and for the production of

skin equivalents containing both an epidermis and a dermis

[3]. However the latter take time to produce and can fail to

survive on the wound bed because of delayed neovasculariza-

tion [10].

Surgically one of the most challenging problems lies in the

treatment of full thickness skin defects avoiding additional

donor sites, unpleasant scarring and contractures. Hence in

this study we have combined minced split thickness skin with

layers of synthetic biodegradable scaffold to evaluate whether

skin cells (both fibroblasts and keratinocytes) will migrate

from the skin pieces onto the scaffold, produce new matrix

and form a new epidermal barrier. The study confirms that

finely cut up small pieces of skin can be placed within layers of

biodegradable scaffold without concern for the orientation of

the skin pieces and that cells, both fibroblasts and keratino-

cytes, will migrate onto the scaffold producing a new matrix

and an epidermal barrier.

Minced skin contains elevated levels of tumor necrosis

factor alpha, platelet derived growth factor and basic fibroblast

growth factor, all of which favor re-epithelialization, neo-

angiogenesis and extracellular matrix deposition [11]. The

principle of using fine minced skin is already in clinical use.

Minced skin covered with polyurethane foam has been found

to be beneficial in elderly or debilitated patients with thin poor

quality skin where it has been reported to decrease the




Table 1 – Collagen deposition after 7, 14 and 21-day of culture measured by Sirius red staining.

Time interval (days) Control 1% methylcellulose (MC) Platelet releasate (PR) 1% MC + PR

7 50.26 � 8.05 55.69 � 8.128 51.52 � 8.33 57.67 � 6.75

14 66.49 � 7.10 70.53 � 9.084 59.12 � 6.23 75.32 � 4.28

21 66.61 � 11.22 82.61 � 2.96 37.69 � 16.10 82.92 � 5.10

Values are average � SEM (n = 3).

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incidence of hypopigmentation, hyperpigmentation and red-

ness [6].

Previous work from this laboratory has developed synthetic

biodegradable electrospun scaffolds for a wide range of

purposes [9]. These have been designed to act as replacements

for human donor dermis in the production of tissue

engineered skin. There are many advantages to working with

PLA scaffolds. These materials are already used in resorbable

sutures, and are FDA approved. As they are synthetic they can

be sterilized using g-irradiation [12] hence their use would

remove any risk of disease transmission as currently exists in

the use of allo- or xenografts. Most importantly they remove

the need for additional donor sites and their associated co-

morbidities. The rate of degradation of the scaffold in vivo

(study in experiments in rats) can be predicted by varying the

ratio of glycolic acid to lactic acid. Thus scaffolds that are 100%

PLA persist in the body for at least 12 months while those with

25% PGA and 75% polyglycolic acid disappear within three

months. More recently scaffolds composed of 50% PLA and

50% PGA have been shown to degrade in vitro within

approximately six weeks and are under development as an

alternative to the amniotic membrane for the delivery of human

limbal stem cells to the cornea [12]. Thus one can design a

scaffold to degrade over a predictable period of time. We

envisage that degradation over a year would allow sufficient

time for dermal remodeling, but if necessary this can be fine-

tuned by modifying the polymer or polymer blend used. The

design of the scaffold is such that one would expect it to degrade

fully without incident – scaffold degradation is associated with

vigorous macrophage activity but little or no lymphocyte

response. When these are designed for tissue engineering

purposes the intention is that the new cells transplanted to the

body produce a neo-tissue. Hence in this study it was important

to determine that the cells moving out from the skin explants

would produce new tissue rapidly and this was assessed by

looking at the production of total collagen.

Having established that minced skin results in outward

migration of skin cells onto scaffold fibers, it was challenging to

obtain an even distribution of the minced skin on the scaffolds.

Adding methylcellulose to Green’s media resulted in a fluid of a

thicker viscosity, which improved the efficiency of the proce-

dure by keeping the minced skin in continuous contact with the

scaffold. Methylcellulose is a cheap, non-toxic, inert substance,

which is part of the cellulose family. It is used in many

applications including lubricant eye drops for patients with

keratoconjunctivitis sicca, and laxatives to aid bowel move-

ment. In our experiments constructs containing methylcellu-

lose did not appear to have more outward migration of cells

compared to the control, but more importantly it did not hinder

migration of cells but allowed for better tissue handling.

Activated platelets release platelet-derived growth factor,

transforming growth factor; vascular endothelial growth


factor and epidermal growth factor. These substances have

roles in cell-to-cell communication, vascular remodeling,

coagulation, and vessel growth [13,14]. Clinically this has

been used to accelerate wound healing and rejuvenate aging

skin. Patients undergoing laser rejuvenation who have topical

platelet releasate applied had objective improvements in skin

elasticity and decreased erythema while subjectively patients

were more satisfied with their results.

Platelet releasate is widely available in the clinical

environment and is easily processed in the hospital laborato-

ry. It has been shown to have promise in promotion of normal

wound healing responses [15]. However, it did not appear to

have an effect on the outward migration of skin cells onto

scaffold fibers. This may be due to the dosage or treatment

regime of one initial dose, which would have eventually

dissipated and fallen prey to metabolism. It is possible that the

minced skin does not have the properties of an acute or

chronic wound hence the benefit of the platelet releasate was

not experienced or that regular doses would be of benefit.

The use of single stage tissue engineered procedures has

already been implemented into the clinical arena in the

treatment of damaged corneas. Limbal biopsies obtained from

the contralateral healthy eye or a donor are minced into fine

pieces and spread onto human amniotic membrane secured

with fibrin glue (TISSEEL Kit from Baxter AG, Vienna, Austria).

This is placed onto surgically prepared corneas in a one-step

approach. At six week follow up stable epithelialized corneas

were noted [16]. This study neatly highlights the advantages of a

one-step approach. Firstly less donor tissue is required as it is

minced into very tiny pieces and no cell expansion is required.

The need for specialist laboratory equipment, trained personnel

and consumables is abolished, and the expense of a clean room

based cell expansion is avoided. Current composite substitutes

require 2–3 weeks of cell culture in the laboratory. In our

technique this step is eliminated hence the process is

expedited. Moreover the formation of the substitute and the

planned operation should coincide, as any delays in either leg

would result in wastage of the tissue engineered product which

is expensive and time consuming. This further supports the

need for a simplified one-stage approach, which can be flexible

to suit the needs of the ever-changing clinical environment.

In summary we have achieved the first steps towards the

establishment of a one-stage in theatre skin substitute for the

reconstruction of full thickness skin defects. Future studies

will now require investigation of the ability of these scaffolds

to become rapidly vascularized so that they will survive on the

wound bed.

Conflict of interest

None.




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Funding

Burns Research Fund from the Sheffield Burns Unit, Northern

General Hospital, Sheffield.

Acknowledgment

Mr John Williamson – Department of Medical Illustration,

Northern General Hospital, Sheffield.

r e f e r e n c e s

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[3] MacNeil S. Progress and opportunities for tissue-engineered skin. Nature 2007;445:874–80.

[4] MacNeil S, Shepherd J, Smith L. Production of tissue-engineered skin and oral mucosa for clinical andexperimental use. Methods Mol Biol 2011;695:129–53.

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Development of a one-step approach for the reconstruction of full thickness skin defects using...

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