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Practice of split-thickness skin graft storage andhistological assessment of tissue quality

Alicia Knapik a,d, K ai Kornmann a,d, K atrin Kerl b, Maurizio Calcagni a,Claudio Contaldo a, Brigitte Vollmar c, Pietro Giovanoli a,Nicole Lindenblatt a,*

a Division of Plastic and Reconstructive Surgery, Department of Surgery, University Hospital Zurich, Raemistrasse 100, 8091

Zü rich, Switzerland b Division of Dermatology, University Hospital Zurich, Raemistrasse 100, 8091 Zü rich, Switzerland c Institute for Experimental Surgery, University of Rostock, Schillingallee 70, 18055 Rostock, Germany

Received 22 August 2012; accepted 11 February 2013

KEYWORDSSkin graft;

Storage;Cell viability;Survey;Burn

Summary Storage of split-thickness skin grafts (STSGs) represents a standard procedure in

burn surgery. The purpose of this study was to evaluate clinical routine of STSG preservation.

Further, we aimed at investigating the effect of storage on tissue integrity and cell viability,proliferation, apoptosis and vascularization. A survey was performed among plastic surgery

centres in Europe. STSGs were harvested from healthy patients and analysed by histology

(HE, Verhoeff’s, Masson’s Trichrome, Sirius Red) and immunohistochemistry (Ki67, TUNEL,

CD31). Cell viability was determined by MTT assay. The survey revealed that storage of STSGs

up to 10 days is common practice. STSGs mostly were stored at 4 C in saline-moisturized

gauze. Histology showed no disintegration of the tissue or a decrease of collagen and elastic

fibres. Proliferation increased to 22.5% of total cells after 3 days. On day 7 of STSG storage

apoptotic cells amounted for 25% of total cells. Cell viability decreased by 50% after day 3

of storage. Even though reportedly superior methods for skin grafts storage exist, most study

participants applied the simplest method of storage. Our data underscore this practice. How-

ever, a reduced cell viability after 3 days of storage may have an influence on graft healing.

ª 2013 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by

Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: þ41 44 255 1111; fax: þ41 44 255 8977.E-mail address: [email protected] (N. Lindenblatt).

d Equal contribution as first authors.

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Please cite this article in press as: Knapik A, et al., Practice of split-thickness skin graft storage and histological assessment of tissuequality, Journal of Plastic, Reconstructive & Aesthetic Surgery (2013), http://dx.doi.org/10.1016/j.bjps.2013.02.003

1748-6815/$- seefront matter ª2013BritishAssociation ofPlastic,ReconstructiveandAestheticSurgeons.Publishedby ElsevierLtd.All rightsreserved.http://dx.doi.org/10.1016/j.bjps.2013.02.003

Journal of Plastic, Reconstructive & Aesthetic Surgery (2013) xx, 1e8

mailto:[email protected]://dx.doi.org/10.1016/j.bjps.2013.02.003http://dx.doi.org/10.1016/j.bjps.2013.02.003http://dx.doi.org/10.1016/j.bjps.2013.02.003http://dx.doi.org/10.1016/j.bjps.2013.02.003http://dx.doi.org/10.1016/j.bjps.2013.02.003http://dx.doi.org/10.1016/j.bjps.2013.02.003http://dx.doi.org/10.1016/j.bjps.2013.02.003http://dx.doi.org/10.1016/j.bjps.2013.02.003mailto:[email protected]


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Introduction

Storage of split-thickness skin grafts (STSGs) represents acommon practice in burn surgery to make use of surplusharvested skin. Preservation in the refrigerator at 4e6 C inmoist saline gauze is the standard method. Other media,e.g. Roswell Park Memorial Institute solution, University ofWisconsin solution and Histidine-tryptophan-ketoglutarate

solution are rarely used,1,2 even though several authors havereported their advantages over saline solution.3e6 Severalstudies have focused on skin viability after storage indifferent media,1,7 at different temperatures8 and for different time periods.9 A reduction of tissue viabilitygenerally was noted over time under all storage conditions.The viability changes were found to be related to multiplefactors including storage solution, temperature, and graftformat.6 Most of these studies analysed the viability of thepreserved skin by histology and concentrated on the evalu-ation of skin integrity and cellular changes in the epidermisand the epidermaledermal junction. Their focus was either to enhance the storage conditions or just to understand in

general the changes which occur during storage. Despitethese facts, no study up to date has focused on the routineof STSG short-term storage in a clinical setting.

Therefore, the present work has been designed to gaininsights into clinical routine concerning the storage andapplication of human STSGs. Further, it was the goal of thispaper to evaluate the effect of storage on STSG integrity andcell viability, cell proliferation, apoptosis and vascularity.

Materials and methods

On-line survey

An anonymous online survey was conducted among resi-dents, senior surgeons and chief plastic surgeons inSwitzerland, Germany, Austria, Great Britain and France.The questionnaire was created using survey monkey(http://www.surveymonkey.com). It included 19 questionsconcerning STSG storage conditions and clinical use. Thedetailed questionnaire can be viewed as an online content.

Patients and STSG harvest

The study was approved by the local ethics committee.Written and informed consent was given by every skin

donor (n Z 17). Healthy patients were between 28 and 71years old. Patients underwent different procedures ofdermolipectomy (abdominoplasty, belt lipectomy andthigh lift). STSGs of 300 mm thickness were taken with anair driven dermatome (Zimmer, Münsingen, Switzerland)and directly evaluated or stored for 3, 5 or 7 days in saline-moisturized gauze at 4 C in a refrigerator. In total skinfrom 17 patients was analysed. The following groups were

established depending on the storage time: fresh skin(n Z 5); 3 day storage (n Z 5), 5 day storage (n Z 5) 7 daystorage (n Z 5) and 14 day storage (n Z 4). Some skinsamples from the same patient were used after differentstorage periods.

Histopathological evaluation of tissue integrity

Skin tissues were fixed in 4% formalin and subsequentlyembedded in paraffin, according to standard procedures.Sections (4 mm) were stained with Sirius Red for collagenquantification, with a combination of Verhoeff’s elastic andMasson’s Trichrome for collagen and elastin fibers qualita-tive examination and with haematoxylin-eosin (HE) stainingfor general morphological aspect.10 Samples were scoredconcerning microscopic and macroscopic appearance by adermatopathologist in a blinded fashion. The score wasadapted from Ben-Bassat et al., Basaran et al. and Turhan-Haktanir et al. (Table 1)1,7,11 Collagen quantification wasbased on five random pictures taken with an AxioScope HRC(Zeiss, Feldbach, Switzerland) and a 40 dry objective.Pictures were further quantified with the analysis D soft-ware (Olympus Biosystems, Münster, Germany) based on aphase-contrast analysis and normalized to the totalsurface.

Histological assessment of capillary density

Paraffin sections (4 mm) were stained with mouse anti-human CD31 (Dako, Glostrup, Denmark) and visualizedusing diamino-benzidine (Dako, Glostrup, Denmark) as achromogen. Slides were scanned with the Mirax Midi SlideScanner (Zeiss, Feldbach, Switzerland) under a 20 dryobjective. Pictures were taken with a 3CCD colour camera(1360 1024 pixels). Capillaries were counted in 5 randomdermal areas of a cross section of the skin and normalizedto the total dermal surface.

Table 1 Parameters for microscopic and macroscopic assessment of graft quality after storage.

Microscopic parameter Score

0 1 2

Epidermal integrity Destroyed Partial Normal

Epidermaledermal junction Destroyed Partial Normal

Collagen organization Amorph Disturbed Normal

Apoptotic keratinocytes

& vacualisation

Complete Present Normal

Macroscopic parameter 0 1 2

Colour Blackened Mat grey Normal

Pliability Very rough Minimally hardened Pliable & smooth

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Immunohistochemical assessment of cellproliferation

Paraffin embedded sections (4 mm) were single-labelledwith rabbit anti-human Ki67 (Abcam, Cambridge, UK) after antigen-retrieval in boiling citrate buffer, followed byantibody incubation and detection using ABC-Vectastain(Reactolab, Servion, Switzerland). Quantification of stained

cells was performed by counting 10 representative fields for each skin section using a 20 objective. The proliferationrate is expressed as the ratio of the number of proliferatingcells divided by the total cell number.

Evaluation of cell viability by MTT assay

A 3-(4,5)-dimethylthiazol-2,5-diphenyl tetrasolium bromide(MTT) salt assay (Sigma, Buchs, Switzerland) was performedin skin biopsies taken from fresh and stored skin as sug-gested by Ge et al.8 (n Z 6). Cell viability was expressed asthe ratio of its OD570 to its weight (mg) and normalized tothe 100% viability of fresh skin. The test was performed intriplicates.

Determination of cell apoptosis rate

Apoptosis was assessed by in-situ Tdt-mediated dUTP-biotin nick-end labelling (TUNEL) using a commerciallyavailable kit (Qbiogene, Illkirch, France). Quantification ofstained cells was performed by counting 10 representativefields for each skin section using a 20 objective; theapoptotic cell rate is given as proportion of the number ofpositive cells/total number of cells.

Statistical analysis

Differences between values were assessed using 1-way-ANOVA or KruskalleWallis tests followed by the appropriate

post-hoc comparison tests (Holm-Sidak and Dunn’s test,respectively). All data were expressed as means SE andoverall statistical significance was set at p


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rate of stored STSGs, 50% estimated the graft take rategenerally as diminished after storage. The reported storagetime in over 50% of the hospitals was more than 7 days(Figure 2C), despite the fact that 95% of the surgeonsconsider the transplantation of fresh skin as the best

method (Figure 2D). Nonetheless, only 20% of the re-spondents refused to store skin and 40% would store it for amaximum of 10 days.

Skin graft proliferation, apoptosis and cell viability

Results for Ki67- and TUNEL-labelling are shown inFigure 3A. The proliferation rate about doubled (22.5%)

after 3 days of storage in comparison to fresh skin (10.6%)and decreased significantly after 7 days of storage( p 0.05). Cell viability of the graft dropped

within the first three days of storage significantly toapproximately 52% reaching finally a value of 44.1% incomparison with fresh skin until 14 days of storage(Figure 3B).

Macro - and microscopic graft evaluation according toBen-Bassat et al. revealed no atrophy of the tissues.11 Nodestruction of collagen and elastic fibres was detectedafter skin graft storage over a time period of 7 days

Figure 2 Results generated from the online survey concerning the storage of split-thickness skin grafts in the clinical setting. (A)

Storage conditions: Most participants stored STSGs at 4e6 C in the refrigerator is a moist piece of gauze. (B) Preservation media:

The most common medium was saline. (C) Average storage time: The majority of clinicians stored STSGs for more than 7 days. (D)Preferred period of storage before transplantation: Despite longer storage time most surgeons prefer to use fresh STSGs.

Figure 3 Effect of human STSG storage at 4 C for up to 7 days on cellular apoptosis and proliferation. (A) Proliferation rate

reached 22.5% on day 3 of storage and decreased significantly after 7 days of storage. On the other hand, apoptosis decreased by

10% within the first three days but afterwards increased to a level of 25%. (B) Viability change of human STSG stored at 4 C. Cell

viability decreased significantly after 3 days of storage to 44.1% and remained at this level during the subsequent days. Values are

means SE. * p


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(Figure 4A and B). Collagen mass ratio in the skin did notchange over the time of storage (Figure 4C). Tissue evalu-ation revealed no changes in total graft score over the timeperiod of 7 days (Figure 4D).

Effect of storage on graft vasculature

HE and CD31 staining showed no abnormalities of vascular

morphology and vessel density. Capillaries did not showsigns of degeneration. Neither thrombosis nor vascular obliterations were observed (Figure 5). Moreover, capillaryquantification revealed no significant changes of capillarydensity in fresh skin (15.6 7.6 capillaries/mm2) and skin,that had been stored for 7 days (15.0 7.8 capillaries/mm2).

Discussion

Skin graft storage and delayed application is a frequentpractice in plastic and burn surgery. The method for storagethat appears to be most cost effective and simple isrefrigeration at 4e6 C in moist saline gauze. Skin graftingto cover superficial acute and chronic wounds traditionallyprovides good results for patients with a variety of defects

and low donor site morbidity.12 However, little clinical dataconcerning actual practice in preservation of skin graftsexist. Despite the fact that many different electrolyte andnutrient rich solutions (e.g. Roswell Park Memorial Institutesolution, University of Wisconsin solution, Hartmann’s so-lution, Dulbecco’s Modified Eagle Medium, keratinocytenutrient MCDB 153, Ready Mix tissue culture medium,

Figure 4 Macro- and microscopical characterization of preserved human STSGs. HE (A) and Verhoeff’s elastic/Masson’s Trichrome

(B) staining for collagen and elastic fibers (magnification 200) after 0, 3, 5 and 7 days of storage. Samples showed no significant

difference. (C) Relative collagen content in STSGs (Sirius Red stain), which remained stable over a period of 7 days. (D) Total graft

score of STSGs stored at 4 C in a saline-soaked gauze showing no deterioration of tissue quality. Values are means SE. * p < 0.05.

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Marshall’s solution, McCoy’s 5A medium etc.) exist,6,8,13 itcan be concluded from the presented study that almost allof the respondents used the simple technique of storage insaline gauze at 4e6 C for preservation. Previous studiesreported that skin graft storage at 4 C in saline for over 4weeks preserved over 50% of keratinocyte viability.1,14 In

other studies, skin grafts stored under similar conditionslost about 90% of their viability after 10 days and no graftsurvival after 5 days of storage was observed.4,15

The application of fresh skin for transplantation remainsthe gold standard.16 Our data indicate that in Europepreservation of excess skin grafts for a possible use at alater date is routinely applied. Next to the widely usedmethod of storage in saline solution at 4 C other possibil-ities of storage were previously investigated e.g.: at thedonor site as described by Shepard or Ashbell, cryopreser-vation or the utilization of different storage media andtemperatures.8,17e19 Li et al. recommends a storage ofmeshed split-thickness skin grafts at 4 C in a culture media

(Dulbecco’s Modified Eagle Medium) instead of a saline so-lution and grafting within seven days because of a signifi-cant loss of keratinocyte growth potency.6 Ge et al. alsoinvestigated different storage temperatures (4 C/25 C)and storage media (saline solution/DMEM) with the result ofrecommended storage at 4 C in DMEM for maximum 96 h.8

In our survey, the time of storage is more than 7 days inover 50% of the institutions. 40% of participants felt thatstorage up to 10 days is feasible. The only alternative so-lutions used in two cases were Ready Mix solution andHartmann’s solution, all participants stored the graft at4 C in a refrigerator. Sterne et al. investigated also thedifference of storing the graft as a meshed or sheet graftand the difference of a rolled or flat graft at 4 C wrapped

in a saline dampened gauze. They recommend storage at4 C as a rolled sheet to achieve the best viability. However,this group observed severe changes of skin graftmorphology and integrity after 7 days of storage. In our split-thickness skin samples none of these changes wereseen as judged by an experienced clinical dermal patholo-gist. Since it is not stated in the paper how thick theinvestigated split-thickness skin grafts were, and yetchanges all over the dermis are described, an explanationmay be that these samples simply were thicker than our grafts with 300 mm thickness.20

As shown in an Australian survey by Lyall and Sinclair,and also in our case, the surgeons seem in general to be

satisfied with their preferred method without the need for alternatives.21 In addition it may preferably be used due toits practicality and inexpensiveness.3,20 However, 50% ofrespondents felt that the graft take rate is somewhat lessideal after storage. Interestingly, no specific conclusion wasdrawn from this and despite this assumption most surgeons

continue to store skin grafts and reuse them. One expla-nation may be that only 8 surgeons were from a hospitalwith a specialised burns unit and have extensive experiencewith skin grafting of large wounds. However, many surgeonsin Europe are involved in the coverage of smaller burns upto 15% TBS, which often are treated in the smaller pe-ripheral hospitals and not referred to a specialised burnsunit.

Since the simple storage in saline gauze proved to be thepreferred method of storage among the respondents in our survey, we further investigated the effect of storage on theskin graft tissue in vitro. Histological evaluation by a der-matopathologist revealed no macroscopic and only minor

microscopic changes within the graft over the time periodof 7 days. The overall total graft score showed no deteri-oration of colour and pliability as well as epidermal integ-rity, no detachment of the epidermaledermal junction, noabnormalities of collagen organization and no keratinocytevacuolization. Therefore it does not appear surprising thatin practice grafts are stored up to and even longer than 7days. These results are conform with previously publishedobservations of Sterne et al., who did not observe macro-scopic and only minor epidermal microscopic changeswithin the first seven days of storage at 4 C.20 Therefore itis striking that more than the half of the respondentsobserved macroscopic differences and diminished quality ofstored skin. However, despite their view, most clinicians

continue to use saline-stored stored skin grafts. It has to bespeculated that this is the case due to still sufficient - evenif slightly diminished - clinical take rates of STSGs. In lightof this it also has to be questioned, whether the reportedsuperiority of many other more expensive storage media isof any clinical interest.3e6

Rosenquist et al. compared graft viability of meshedand unmeshed skin and reported that meshing does notaffect the viability of the graft, demonstrating that evenmechanical stress does not affect the cell viability in along-lasting manner.22 In our study the MTT assay showed adecrease of cell viability to a level of w50% within the first3 days of storage and stayed on this level with only minor

Figure 5 Representative endothelial-cell staining (CD31) on paraffin sections of fresh and 7 days old skin (magnification 200).Vessel appearance on day 0 and 7 was identical without thrombosis or changes of cell morphology.

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changes until the final storage day (14 days). This r esult issimilar to previous studies of Castagnoli et al.23 andslightly lower than the viability reported by Ge et al.8

Additional tests for collagen or elastic fiber changes aswell as traditional scoring of the graft quality did notreveal noticeable changes within different time points ofstorage, which is congruent with the results of previousstudies. However, proliferation was found to be slightly

increased within the stored graft within the first 3 days,most likely due to the acute hypoxic condition. This is inline with previous studies showing increased proliferationand differentiation of cells under hypoxic conditions andinhibition of tumor cell proliferation by HIF1-alphasilencing.24,25 In parallel apoptosis within the graftincreased after day 5 to 25% of cells as a consequence oftissue storage. Cell viability was found be significantlydecreased to 50% already after 3 days of storage. In linewith this a recent study6 showed a progressive decrease ofcell viability over time during storage at 4e8 C. Cellculture media were better than saline and Hartmann’ssolution in this study in maintaining the viability andgrowth capability of skin cells.

Even though STSG storage is performed frequently, it isof interest, that the respondents indicated fresh skin as thebest graft as well as they observed diminished quality andgraft take after storage. Therefore, the diminished viabilityafter storage may have an impact on the graft take and theproliferative phase during wound healing.

In conclusion our study reveals that skin graft storage is awidely used practice in burn surgery and that most surgeonsprefer the method of preservation in saline-moistenedgauze at 4e6 C. Even though they have the impressionthat skin graft take rate may be diminished after storagethey continue to apply this method. Histological evaluationshowed no visible deterioration of tissue integrity and

vascularity. However, the skin graft exhibited a slight in-crease of apoptotic cells after 5 days and a decreasedproliferation rate. Cell viability was reduced to half after day 3 until day 14. Whether these observations haveimplication for the take rate, vascularization and woundhealing of STSGs will have to be studied further in an in vivosetting. Even though skin represents a complex organ withmultiple functions it can easily be preserved for up to 10days under certain conditions without major changes in itsfunctionality and appearance. This insight may be of rele-vance for researchers working on the development of the-shelf skin substitutes. Further, our findings could be trans-lated into new skin graft material designs, basing onreversible hypometabolism leading to an extended cell

survival prior to revascularisation and establishment ofnutrient and oxygen supply.

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Acknowledgements

This study was financially supported by the Swiss NationalScience Foundation (SNF-Grant 310030_127366).

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