Ghép Da: Chỉ Định, Ứng Dụng Và Các Nghiên Cứu Hiện Tại (2011)

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Fig. 1. Typical scar formation results after sandwich grafting, the mesh of the autologous
skin graft pattern is still visible.
2. Development of dermal substitute
Dermal substitutes can be obtained from animal or human tissue as well as synthetic
materials. The advantage of human skin is the presence of the natural collagen- elastin fibre
network, providing the optimal three dimensional fibre structure for ingrowing fibroblast
and blood vessels. Antigenic structures must be absent from the dermal substitute to avoid
the induction of inflammatory processes that can worsen final scar quality. Human
allogeneic skin has been applied to the wound as dermal substitute in the past as described
by Cuono et al., 1987. In this method, the more immunogenic epidermis was removed
several days after grafting the human donor skin and replaced by autogous cultured
epithelial sheets. Technically, this method is difficult and in the allogeneic dermis cells of the
donor remain present that can induce inflammation after wound closure. Therefore, several
methods have been developed to remove the donor cells and hairs from the donor skin. This
resulted in products known as Alloderm, Surederm, Graft jacket using enzymes and freeze
drying techniques.
We have developed a cost-effective method to eliminate the antigenic structures by using
low concentrations of sodium hydroxide (Richters et al. 2008). De-cellularization of glycerol
85% preserved skin starts by rinsing out the glycerol, thereafter the skin is incubated in
0.06M NaOH for 6 weeks. The NaOH solution is refreshed every week and finally
neutralized. The acellular dermis is again preserved in glycerol 85%. This method avoids
freezing which may damage the collagen and elastin fibers and, at the same time, it is very
effective in washing out the donor cells and hairs. The prototypes, consisting of an intact,
native collagen-elastin matrix obtained using the NaOH method were tested in a porcine
model. Full thickness wounds of 4x4 cm were transplanted with the prototype and an
autologous split skin graft meshed 1:3. Contraction of the wounds treated with this

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prototype was significantly lower compared to the control wounds, transplanted with only
autologous skin (Richters et al., 2008, Pirayesh et al., 2007 and 2008). The six weeks
treatment with NaOH showed the best results. Further experiments showed a two stage
procedure was more optimal with the take rate of the autologous skin > 90% if the interval
between application of the prototype and the autologous skin was 7 days. Final result with
respect to contraction was comparable to Integra in this porcine model but the scar was
more smooth (Pirayesh et al., 2011, manuscript in preparation). This encouraging results let
to the first pilot of clinical experiments on burn patients.
3. Clinical application of Glyaderm
This novel dermal substitute is called Glyaderm, which is an abbreviation for Glycerol-
preserved Acellular Dermis. Before application of Glyaderm, the wound bed must show
viable granulation tissue. In most cases, woundbed preparation with allogeneic donor skin
is needed; in burns this takes about 7 days after debridement. Glyaderm is preserved in




Fig. 2. Example of Glyaderm

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85% glycerol and must be rinsed in sterile 0.9% NaCl before use on the patient. The
Glyaderm is meshed 1:1 to allow drainage of wound fluid. Glyaderm was used on the
patients also in a two stage procedure in clinical studies, approved by the local ethics
committee of the University of Ghent, Belgium. A pilot study and a comparative study were
performed sofar.
4. Pilot study
In the first non-randomized pilot experiments 15 patients with full thickness burns (TBSA <
40%) were selected to study the optimal wound dressing, to avoid dehydration and
bacterial contamination of the Glyaderm during the first days before the autologous skin
was applied. Then a group of 10 patients with full thickness wounds was selected (5 with
burns and 5 with reconstructive wounds) and was treated to define the optimal time
between the two operations using Laser Doppler Imaging. The depth of the burn wounds
was first assessed using laser Doppler imaging (Monstrey et al., 2007) to assure the wounds
were deep dermal. The patients were recruited and treated with Glyaderm and split
thickness skin graft after wound bed preparation with glycerol preserved allografts. Patient
demographics were recorded and Laser Doppler imaging was used to monitor the
vascularization of glyaderm. We also discovered that Laser Doppler Imaging allows us to
measure wound tissue perfusion and monitor daily ingrowth of blood vessels into the
dermal substitute. This allows us to know the exact timeframe in which the dermal bed is
vascularised enough and skin grafting can be carried out. This is typically after 5-7 days,
much shorter than the period necessary for ingrowth of the currently most widely used
commercial dermal substitute Integra (Dantzer et al., 2001)
4.1 Randomised, controlled, paired, intra-individual Comparative study in 30 patients
After completing the pilot studies, a comparative study was performed. Patients with full
thickness burns or full thickness lower arm defects after free flap harvesting were recruited.
Part of the wounds were treated with Glyadem and split thickness skin and the other part
with split thickness skin only. This was done in anatomically related areas to allow paired,
intra-individual comparison of the wound healing. Preferably, a left/right comparison was
made and the experimental treatment was randomized. Patient demographics were
recorded and at regular follow up time points of 1, 3, 6 and 12 months after wound closure
biopsies were taken and objective and subjective scar assessment was performed. The
dermatospectrometer was used to measure scar colour, Dermalab cutometer for pliability
and the modified Vancouver scar scale as well as a contour scale (Brusselaers et al 2010)
were used. All data were recorded in a purpose designed database and subjected to
statistical analysis with SPSS software package.
5. Results of the pilot study
All recruited patients responded well to a dressing regimen of surfasoft, a semipermeable
membrane, with betadine gel and paraffin gauze in terms of bacterial control and
prevention from desiccation of Glyaderm with capital. Laser Doppler Imaging proved to
show enhanced vascularization from day 1 to day 5 allowing to exactly delineate the
optimal engraftment interval. This was found to be 6 days with a 1 day standard deviation.

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The increase in blood vessel growth can be observed clearly in Figure 3, the areas with a red
color represent blood vessel flow and these are larger on day 7 in this patient. The take of
the Glyaderm was > 90% and the take of the autologous skin > 95%. The results with respect
to scar formation in this pilot study were encouraging. Figure 4 shows pictures of the
wound and final scar of the same patient as in Figure 3. The color of the scar is not red and
the patient is able to move the head normally, indicating good elasticity.














Fig. 3. Shows the growth of blood vessels in a deep burn wound treated with Glyaderm
6. Results of the comparative study
The take rates in the STSG on Glyaderm were comparable to the take rates in the wounds
with a STSG alone (Wilcoxon Singed Ranks Test Z = -0.823, p = .41). Pliability and Visual
Scores and contour scale assessment indicate a trend of improvement with Glyaderm
treatments; however, follow-up times are currently too short with small numbers to be able
to draw conclusions, we expect a necessary follow-up interval of two years. Comparing the
Dermalab measurements for elasticity “Glyaderm + STSG” versus “STSG alone” statistics
indicate that: Glyaderm + STSG” has significantly more elasticity when compared to “STSG
alone” 1 month and 12 months after wound closure.

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A: Burn wound before treatment


B: Two years follow-up
Fig. 4. Final results of this wound with respect to scar formation show a cosmetic good
quality of the scar.
7. Conclusion
The results of the comparative, controlled, intra-individual clinical study show beneficial
effects of a dermal substitute on the elasticity of the scar. The natural structure of the
collagen and elastin fibers in glyaderm may have contributed to this effect. Glyaderm is the
first non-commercial dermal substitute that can compete with all currently available dermal

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equivalents. Laser Doppler imaging allows monitoring of vascular ingrowth in dermal
substitutes such as Glyaderm. Although most burn experts advocate the use of dermal
substitutes, the challenge remains to objectively show the perceived benefit over split
thickness skin grafting alone. The evolving evaluation with objective scar assessment tools
within these studies may help to demonstrate this benefit in the near future.
Our next study with Glyaderm has started recently; a multi-centre clinical study in
major European Burn centers. To allow also quality of life and cost-effectiveness
studies, 60 patients will be treated with glyaderm + STSG and 60 patients with STSG
alone.
Researchers continue their quest for the ideal skin substitute, and in the future it should be
possible to create such an advanced skin substitute, containing melanocytes, hair follicles
and sebaceous glands. The available products remain rather expensive, due to commercial
incentives, high manufacturing costs, special shipment and storage conditions.
Nevertheless, accelerated healing and closure of the wound will reduce the labour intensive
dressing changes, hospital stay and the need for reconstructive surgery. Until the optimal
off the shelve skin substitute becomes available, the burn surgeon can improve aesthetic and
functional outcome by choosing from the gamut of currently available scaffolds for
bilayered skin restoration. Glyaderm may well be an optimal cost-effective solution to
bridge this gap in the near future.
8. References
Brusselaers N, Pirayesh A, Hoeksema H, Richters CD, Verbelen J, Beele H, Blot SI, Monstrey
S. J Trauma 2010; 68: 490-501.
Brusselaers N, Pirayesh A, Hoeksema H, Verbelen J, Blot S, Monstrey S. Burn scar
assessment: a systematic review of objective scar assessment tools. Burns 2010,
36:1157-1164.
Cuono CB, Langdon R, Birchall N, Barttelbart S, McGuire J. Composite autologous-
allogeneic skin replacement: development and clinical application. Plast Recon
Surg 1987, 80: 626-637.
Dantzer E, Braye FM. Reconstructive surgery using an artificial dermis (Integra): results
with 39 grafts. Br J Plast Surg. 2001, 54: 659-664.
Hermans MHE. Clinical experience with glycerol preserved donor skin treatment in partial
thickness burns. Burns 1989; 15: 57-60.
Jones I., Currie L., Martin P. A guide to biological skin substitutes. Br J Plast Surg 2002, 55:
185-193.
Kreis RW, Vloemans AFPM., Hoekstra MJ, Mackie DP, Hermans RP. The use of non-
viable glycerol-preserved cadaver skin combined with widely expanded
autografts in the treatment of extensive third-degree burns. J Trauma 1989;
29:51-55
Monstrey S, Hoeksema H, Verbelen J, Pirayesh A, Blondeel P. Assessment of burn depth
and burn wound healing potential. Burns 2008; 34:761-769.
Pirayesh A, Monstrey S, Blondeel P, Hoekstra H, Vanoorbeek J, Hoeksema H, Richters CD.
Development of a novel dermal substitute based on glycerinized allograft: Clinical
(Phase I) and experimental evaluation. Burns 2007; 33:S13

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Pirayesh A., Dur A.F.M., Paauw N.J., Monstrey S., Kreis R.W., Hoekstra M.J., Richters C.D.
Evaluation of acellular dermis for closure of abdominal wall defects in a rat model.
Eur Surg res 2008; 41 :345-352.
Pirayesh A., Hoeksema H, Richters CD, Monstrey S. Determination of the optimal time
interval between application of a dermal substitute and grafting with autologous
skin using Laser Doppler Imaging. Manuscript in preparation.
Richters C.D., Pirayesh A., Hoeksema H., Kamperdijk E.W.A., Kreis R.W., Dutrieux R.P.,
Monstrey S., Hoekstra M.J. Development of a dermal matrix from glycerol
preserved allogeneic skin. Cell and Tissue Banking 2008; 9 : 309-315.
Van der Veen V, Van der Wal MB, Van Leeuwen MC, Ulrich MM. Midelkoop E. Biological
background of dermal substitutes. Burns 2010; 36: 305-321.

21
External Wire Frame Fixation
for Skin Grafts
Rei Ogawa and Hiko Hyakusoku
Department of Plastic, Reconstructive and Aesthetic
Surgery, Nippon Medical School, Tokyo
Japan
1. Introduction
Skin grafting has been used widely for various reconstructions. The complete survival of the
skin graft is necessary for functional and aesthetic restoration, as incomplete survival delays
wound healing and results in severe primary and secondary contraction of the graft and
scar contractures. To promote successful skin grafting, bleeding of the recipient wound bed
must be arrested during surgery and the graft must be appropriately compressed and
completely fixed to prevent shearing against the wound bed. After the operation, it is
necessary to maintain the compression force and fixation of the graft for at least 5–7 days.
One of the difficulties associated with skin grafting is differences in recipient site conditions.
Areas that are difficult to graft are mobile areas such as the joints and the neck, and areas
that have a free border, such as the eyelids and the perioral area. The authors currently use
the external wire-frame fixation method1-5 for reliable skin grafting, in particular when these
areas are involved. This technique is useful for securing the graft to the wound bed and it
prevents the graft edges from lifting. In addition, it can sometimes be used to fix joints at the
same time. This technique is introduced and discussed in this chapter.
2. Technique and methods
During surgery, the skin graft is fixed with sutures as usual (Fig. 1-1). At the same time, a wire
frame of 0.7–1.0 mm-diameter Kirschner wire that has the shape of the graft is created. In the
case of digital skin grafts, the wire frame can be slightly larger than the recipient site so that the
digital joint(s) can be fixed at the same time and results in a three-dimensional wire frame. To
ensure that the frame edges do not stick out, the ends of the Kirschner wire meet each other in
a thin plastic tube, such as the outer sheath of an indwelling needle. The wire frame shape
should be adjusted finely to ensure that it does not induce a pressure ulcer.
The wire frame is placed onto the graft and attached with the same sutures that were used
to stitch the graft (Figs. 1-2 – 1-7). Finally, tie-over fixation is performed in the usual manner
with appropriate pressure (Figs. 1-8 – 1-11).
The tie-over and the wire frame are removed about seven days later in the case of full-
thickness skin grafts, and five days later in the case of split-thickness skin grafts, prior to
removing the grafted skin sutures. The patient can then begin rehabilitating the affected
sites.

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Fig. 1-1. The skin graft is sutured to the recipient site by 4-0 or 5-0 nylon or polypropylene
sutures.


Fig. 1-2. One end of each suture is lifted and bound to the others, thus forming a hank.


Fig. 1-3. The external wire frame is placed over the hank formed by the sutures.


Fig. 1-4. The external wire frame is fitted onto the grafted skin.

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Fig. 1-5. The sutures are released from the hank.


Fig. 1-6. To tie up and fix the external wire frame, the sutures are knotted.


Fig. 1-7. The external wire frame is fixed tightly, with care taken to ensure that a pressure
ulcer does not arise.


Fig. 1-8. Tie-over gauze is placed onto the grafted skin.

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Fig. 1-9. The tie-over gauze is arranged so that its volume is distributed equally over the
graft.


Fig. 1-10. The sutures are divided into 4–6 sets and bound into hanks.


Fig. 1-11. The suture hanks that oppose each other diametrically are tied up, thus
completing the tie-over fixation.
3. Discussion
With regard to eyelid skin grafts, the wire frame can eliminate the need for tarsorrhaphy, as
an external wire frame allows the patient to open his or her eyes immediately after surgery
without causing the skin grafts to move2,5. With regard to the perioral area, an external wire

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frame allows the patient to start eating immediately after surgery (Fig. 2-1 and 2-2).
Moreover, the three-dimensional external wire frames3 used with digits are useful for fixing
the digital joint(s) as well as the skin graft (Fig. 3-1 and 3-2). If external fixation method is
used for digital skin grafting, the digital joints do not need to be fixed by pinning the digit
with Kirschner wire. Thus, external wire frames are particularly useful for the grafting of the
palmar surfaces of the fingers.
In summary, external wire frame fixation can secure skin grafts to the wound bed with
homogeneous pressure and reduce shear force at the periphery, thereby promoting skin
graft take. It provides reliable but not excessive fixation that permits the early movement
and rehabilitation that promotes functional and aesthetic restoration. It is a simple, cheap
and individualized technique that is associated with easy perioperative management and
nursing.


Fig. 2-1. Skin graft with external wire frame on the upper lip (intra-operative view)
Patient started to eatimmediately after surgery.


Fig. 2-2. Skin graft with external wire frame on the upper lip (7 days post-operative view)

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Grafted skin survived completely.


Fig. 3-1. Three-dimensional external wire frames used with digits (intra-operative view)
The digital joints did not need to be fixed by pinning using Kirschner wire.


Fig. 3-2. Three-dimensional external wire frames used with digits (7 days post-operative
view)

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The grafted skin survived completely.


Fig. 3-3. Three-dimensional external wire frames used with digits (6 months post-operative
view)
There were no functional limitation.


Fig. 3-4. Three-dimensional external wire frames used with digits (6 months post-operative
view)
There were no functional limitation.

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4. References
[1] Hirai T, Hyakusoku H, Fumiiri M. The use of a wire frame to fix grafts externally. Br J
Plast Surg 1991; 44: 69-70.
[2] Murakami M, Hyakusoku H, Ishimaru S. External wire frame fixation of eyelid graft. Br
J Plast Surg 2003; 56: 312-13.
[3] Ogawa R, Aoki S, Aoki M, Oki K, Hyakusoku H. Three-dimensional external skin graft
fixation of digital skin graft. Plast Reconstr Surg. 2007 Jan;119(1):440-2.
[4] Ogawa R, Hyakusoku H, Ono S. Useful tips for successful skin grafting. J Nippon Med
Sch 2007; 74: 386-92.
[5] Murakami M, Ogawa R, Hyakusoku H. Efficacy of 'rugby-ball shape' skin grafting using
external wire frame fixation without tarsorrhaphy for lower eyelid reconstruction. J
Plast Reconstr Aesthet Surg 2010; 63: e220-1.

22
Efficacy of Autogenous Dermis
Graft for Wound Coverage
Seung-Kyu Han
Department of Plastic Surgery
Korea University College of Medicine Seoul,
Korea
1. Introduction
Skin grafting is the easiest and the most reliable way to obtain proper wound coverage.
However, the two major concerns of skin grafting are the poor color match in the recipient
sites and donor site morbidity including pain, discomfort, and hypertrophic scarring. Major
pigment mismatches are common in split-thickness grafts particularly in darker skinned
patients including Asians (Fig. 1). Several factors may play a role in the color mismatch of a
regular skin graft. These include the amount of melanin, the degree of transfer of
melanosomes to keratinocytes, and the number of melanocytes (Carlson et al., 2002; Velangi
& Rees, 2001). Because the melanocytes are localized to the basal cell layer of the epidermis,
the origin of the color mismatch has been assumed to be in the epidermal melanin (Tyack et
al., 1997; Swope et al., 2002). Because regular skin grafts transferred to new locations
maintain their epidermal specificity, a pigmentation difference with the surrounding skin is
unavoidable.
To minimize the limitations of the classic skin graft, the author has developed a dermis
graft, which is a deepithelialized split thickness skin graft, and have reported promising
results of the method for coverage of small to medium sized wounds on the body (Han et
al., 2007). The important aspects of this method involve the immediate return of the
epidermis to the donor site in order to overcome the donor site morbidity and minimize the
pigment mismatch between graft and surrounding skin by restoring the epidermal portion
of the recipient site through inducing epithelization from the adjacent skin.
The purpose of this chapter is to present usefulness of the dermis graft in a wound coverage.
2. Surgical technique
After sharp debridement of the recipient site, skin and underlying subcutaneous tissue of
the wound margin is undermined approximately 5mm in length along the periphery and
meticulous hemostasis is obtained. The defect size of the recipient site measures. A thin
epidermal flap is elevated on the gluteal area or the lateral thigh using a Zimmer
dermatome, in which the thickness is set to 0.010 inches. The blade is then reset to 0.012 to
0.020 inches and the dermis to be grafted is cut from the same area. The previously elevated
epidermal flap is replaced on the donor site and sutured with prolene. The harvested dermis
is tailored to the size and shape of the defect including the undermined area and transferred

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Fig. 1. Two major concerns of skin grafting. (Above) Poor color match in the recipient sites.
(Below) Hypertrophic scar formation on the donor sites.
to the recipient site. The edge of the dermis graft is inserted into the undermined wound
margin and fixed to the wound bed along the circumference of the defect using PDS sutures.
The undermined marginal skin over the grafted dermis is then fixed to the underlying
dermis using prolene sutures (Fig. 2). The skin sutures are removed after approximately 5 - 7
days in order to reduce the stitch marks. Sunshine is avoided for the first three to six months
using sun-blocking agents.

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3. Indication
The dermis graft is indicated in any cases with small to medium sized skin defect (smaller
than 42.0 cm2 based on the author’s experience). Especially, a dermis graft is likely to have
its greatest reconstructive role on the exposed areas such as the face, neck, forearms, and
hands. In fairly large wounds (larger than 42.0 cm2 based on the author’s experience), the
return of the patients to their normal daily activities is likely to be delayed because the
epithelization of the graft takes a longer time. There is no data on the critical size or
thickness of the dermis that can produce scar-free wound healing. Therefore, further study
will be needed.



Fig. 2. (Above) The thin epidermal flap is elevated on the gluteal area, the dermis to be
grafted is then cut on the same area. (Below, left) The edge of the dermis graft is inserted into
the undermined wound margin and fixed to the wound bed along the circumference of the
defect. (Below, right) The previously elevated epidermal flap is replaced on the donor site.

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It was found that a full thickness small sized wound, which was left to heal by secondary
intention, sometimes resulted in acceptable scarring in a concave area of the skin. In some
wounds, however, a period of several weeks or months is required for complete healing,
particularly in actinically damaged, fragile skin. Wounds on convex areas of the skin heal
rather poorly (depressed scar) via secondary intention. The resultant wound contraction,
which is an important clinical component, is not an uncommon problem (Fig. 3). Skin or
dermal substitutes, which are currently produced by advanced technology, may replace the
regular skin graft (Kuroyanagi et al., 2001; Terino et al., 2001). However, these procedures
usually heal with a significantly conspicuous and unfavorable scar (Figs. 4 and 5). Further
improvements are required in such technology to obtain a favorably aesthetic result as
obtained with the dermis graft method.








Fig. 3. An open wound on nasal dorsum, which was left to heal by secondary intention,
resulted in a conspicuous wound contraction.

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Fig. 4. A full thickness skin defect on a lower eyelid healed by grafting of an artificial
dermis. The resultant scar was prominent with contraction.



Fig. 5. A wound on cheek healed by grafting of an allogenic dermis. The resultant scar was
unfavorable.

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4. Author’s experience
4.1 Clinical research
The author has evaluated the efficacy of the dermis graft used for wound coverage by
comparing it with that of a regular skin graft (Han et al., 2007). The patients included in the
study provided informed written consent for the study, but has not been registered in an
official database of the institutional review board.
4.1.1 Patients and methods
From April of 2001 to March of 2004, the dermis graft was applied to 53 patients (17 male, 36
female), ranging in age from 6 to 61 years (average, 36.5 years). The size of the wound
ranged from 2.7 to 42.0 cm2, with a mean and a median size of 12.0 and 10.5 cm2,
respectively. The recipient sites were located as follows: 8 on the face, 6 on the forearm, 34
on the hand, 4 on the lower extremities, and 1 on the back. Simultaneously, a regular split
thickness skin graft procedure was performed on the wounds of approximately the same
size and location as the dermis graft in 33 patients. The size of the regular skin graft wound
ranged from 2.3 to 42.0 cm2, with a mean and a median size of 14.0 and 12.0 cm2,
respectively. The locations of the wound were as follows: 3 on the face, 6 on the forearm, 21
on the hand, and 3 on the lower extremities. This provided an opportunity to compare the
results of the dermis graft with those of the regular skin graft. Prior to classifying the
patients into the two treatment groups, detailed information of the new procedure (dermis
graft), which included the surgical techniques, advantages, and disadvantages, was
provided to each patient. The patients were classified according to their compliance.
Compliant patients who fully understood and agreed to the new procedure underwent a
dermis graft and noncompliant patients who did not want the new procedure underwent a
regular skin graft.
The healing time for both groups was compared. The mean follow-up time was 12.4 months
ranging from 3 months to 2 years. Twenty-six dermis graft patients and 20 regular skin graft
patients had more than a 12-month follow-up period. The scar condition was evaluated in
these long-term follow-up patients by two blind observers (plastic surgery residents in
training at the author’s institution) after a 12 to 24 month follow-up (average, 15.7 months
for the dermis graft group and 15.0 months for the regular skin graft group) according to the
Vancouver Scar Scale (VSS), which included pigmentation, scar height, pliability, and
vascularity (Vloemans et al., 2003; Sullival et al., 1990). The averaged scores of the two
observers were used as data for the analysis.
The patients’ satisfaction for the donor and recipient site was also surveyed in the long-term
follow-up patients. The patients’ satisfaction was compared using a Visual Analogue Scale
(VAS) with a score of 0 being the worst and 5 being the best. Statistical comparisons for the
data of the VSS and the VAS were performed using a Mann-Whitney U-test. P values < 0.05
were considered significant.
4.1.2 Results
The graft take was complete in all patients in both groups. Refilling of the blood vessels and
multiple pin point bleeding was observed on the grafted dermis within the first 2 to 3 days.
Reepithelization occurred progressively from the periphery to the center of the grafted
dermis.

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The whole wound of the dermis graft had reepithelialized after grafting within 11 to 20 days
(average 15.5+1.9 days). The skin grafted wounds had healed by 7 to 16 days (average
11.8+1.6 days). In the long-term follow-up, good quality skin characteristics were achieved
in the dermis graft.
A comparison of the scars at the recipient site, as assessed by the VSS, showed that the
dermis graft was superior to the regular skin graft in terms of pigmentation, height, and
vascularity (p<0.05). No significant difference in pliability was detected. The overall
average VSS scores of the dermis and skin graft were 1.0+1.1 and 1.6+1.1, respectively
(p<0.05). The patients’ satisfaction in the dermis graft group, as assessed by the VAS, was
also better than that in skin graft group (4.2+0.9 and 3.4+1.1 each).
Regarding the donor sites, the results of the dermis graft were also satisfactory. All the
donor sites of dermis graft healed within 9 days (average 7.5+0.8 days). In contrast, those of
the regular skin graft required 11 to 16 days to heal (average 12.8+1.1 days). The donor sites
of the dermis graft were also superior to those of the skin graft in terms of scar quality and
patient satisfaction. The average of overall VSS scores for the donor sites of the dermis graft
and regular skin graft were 0.7+0.9 and 1.3+0.7 each (p<0.05). The average VAS scores for
the dermis and skin graft donors were 4.1+1.3 and 2.8+0.8 each (p<0.05). There were no
significant complications and no functionally relevant scar in either group (Fig. 6).


Fig. 6. A woman received a venous island flap coverage to reconstruct an amputated finger
tip. (Above, left) The donor site was covered by a dermis graft harvested from her buttock.
(Above, right) Two-month postoperative view. (Below, left) Eight-month postoperative view.
(Below, right) Fourteen-month postoperative view, which demonstrated excellent color
match with the adjacent skin.

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4.2 Dermis graft on face after removal of skin cancer
The author has evaluated the reliability of the dermis graft for covering defects after
removal of non-melanoma skin cancer on the face. Surgical excision of skin cancer leaves a
soft tissue defect which requires reconstruction. Proper selection of reconstruction method is
the key point to get a successful result on the face. In planning reconstruction for facial
defects, reconstructive efforts should start from the safest and least invasive methods while
achieving optimal functional and cosmetic outcomes. A variety of methods can be used to
cover the defects; healing by secondary intention, a primary closure, a skin graft, and a local
flap. Healing by secondary intention usually leaves a significantly conspicuous and
unfavorable scar. Darker skin is more prone to hypertrophic scarring or keloids. The role of
primary closure for reconstruction after complete eradication of skin cancer on the face is
very limited because of defect size and shape. Only small defects with elliptical shapes yield
satisfactory results after primary closure. A local flap may provide several advantages,
including decreased scar contracture and satisfactory contour, color, and texture match. In
some cases, however, a local flap is not feasible, mainly due to the limitation of size and arc
of rotation, particularly in young patients. Poor flap design and inappropriate incisions may
contribute to unacceptable scars, violation of aesthetic subunits, and resultant disfigurement
on difficult-to-treat areas. In addition, compromise of flap vascularity and inadequate
wound closure created by insufficient undermining, lack of deep closure, excessive tension,
or inadequate approximation of wound edges predispose to unfavorable results.
4.2.1 Patients and methods
Thirty-eight patients (20 male, 18 female) were treated for the facial defects created by the
resection of the non-melanoma skin cancer on the face from January 2006 and December
2008. The patients had more than 12-months of follow-up and the clinical records of all
patients were reviewed. The patients’ age ranged from 57 to 83 years (average, 68.7±6.3
years). The defect size ranged from 3.3 to 6.5 cm2 with a mean of 5.1±0.9 cm2. The location of
the defect was as follows: 17 cases on the nose, 14 in the orbital area, 4 on the cheek, 2 on the
temple area, and 1 on the forehead.
Functional and cosmetic outcomes, focusing on scar contracture, pain, itching, scar height,
color, texture, and postoperative complications were assessed. The patients’ satisfaction was
also evaluated by using a VAS with a score of 0 being the worst and 10 being the best. The
patients were followed up for 12 to 36 months (mean, 20.5±6.2 months).
4.2.2 Results
The graft was well taken by all patients. Reepithelization occurred progressively from the
periphery to the center of the grafted dermis. The entire dermis graft reepithelialized after
grafting within 17 to 27 days (mean, 25.1±2.4 days).
All patients had satisfactory results in both functional and cosmetic matters with high
quality skin characteristics. No significant scar contracture was observed and none of the
patients complained of pain or itching. In the scar height, 20 scars were flat and 18 scars
were slightly depressed. Neither hypertrophic nor keloid scar was observed. In the color
parameter, most of the scars (30 scars) showed favorable color match to the surrounding
skin (Figs. 7 and 8) and 8 scars were hypopigmented (Fig. 9). Hyperpigmentation or redness
was not observed. In terms of the skin texture, favorable smooth texture was obtained in 25
cases and shiny texture in 13 cases. Obviously conspicuous scar was not identified in all

Efficacy of Autogenous Dermis Graft for Wound Coverage

315
cases and no revision procedure was needed to improve scar quality. There were no
significant complications and no recurrences were observed during the follow-up. The
patients’ satisfaction with the dermis graft was also excellent (mean score 8.3±1.3).
Regarding the donor sites, the results of the dermis graft were also satisfactory. All the
donor sites of dermis graft healed within 9 days (average 7.2±1.1 days). There were no
significant complications and no functionally relevant scar (Fig. 10).
5. Advantages and disadvantages
The primary advantage of this method is to provide a graft that is similar to the surrounding
skin, leaving minimal scars and color mismatch. Since the epidermal portion can be
restored by epithelization, induced by the migration and proliferation of adjacent epidermal
cells including melanocytes, the density and activity of the melanocytes as well as the
precursor melanocytes of the epidermis of the graft become similar to those observed in the
adjacent skin.


Fig. 7. A skin defect created by removal of basal cell carcinoma treated with the dermis graft.
(Above, left) Immediate postoperative view. (Above, right) One-month postoperative view.
(Below) Two-year postoperative view, which showed excellent results with minimal scar
contraction.

Skin Grafts – Indications, Applications and Current Research

316

Fig. 8. A skin defect seen on a lower eyelid created by removal of basal cell carcinoma
treated with the dermis graft. (Above, left) Preoperative view. (Above, right) After removal of
the lesion. (Below, left) One-month postoperative view. (Below, right) One-year postoperative
view. The resultant scar was acceptable.
Regarding wound contraction of the recipient site after dermis grafting, the contraction of
myofibroblasts can be inhibited by grafting with a more dermal portion than a regular skin
graft. In previous studies, the inhibition of scar contraction was attributed to the amount of
dermal collagen rather than to the amount of epidermis (Chou et al., 2001; Rudolph, 1979;
Brown et al., 1990). Another advantage of the dermis graft is the quick healing of the donor
site without an obvious scar appearance because the epidermis can be replaced immediately
to ensure the closure of the donor bed. Hypertrophic scarring is rare, and the level of
pigmentation is less than that of the donor site in a regular skin graft. The patient has less
donor site pain and discomfort, which is a very important component from the patient’s
point of view. Care of the donor site is also very simple. Dermis grafts are feasible for all
wounds that are candidates for regular skin grafts including acute facial burns or post facial
cancer excisions.
The only possible demerit with dermis grafting is the delay in complete healing of the
recipient site. As previously described, there was a 3.7 day delay in graft healing of the
recipient site (15.5 and 11.8 days in the dermis graft and the regular skin graft groups,
respectively). However, it is believed that the 3.7 day delay would not cause significant
trouble, and what ever problems there are can be overcome by selecting suitable sized
wounds.

Efficacy of Autogenous Dermis Graft for Wound Coverage

317

Fig. 9. A skin defect on a nose created by removal of basal cell carcinoma treated with the
dermis graft. (Above, left) Preoperative view. (Above, right) Immediate postoperative view.
(Below, left) One-month postoperative view. (Below, right) One-year postoperative view.
Hypopigmentation of the grafted site was noted in this case.


Fig. 10. Healed donor sites of the dermis grafts demonstrate acceptable cosmetic results.
6. Conclusion
The dermis graft technique for wound coverage is superior to a regular skin graft technique
in terms of the aesthetic results of both the recipient and donor sites. The donor site healing

Skin Grafts – Indications, Applications and Current Research

318
process is faster, and the patient’s pain and discomfort can also be reduced significantly. The
dermis graft can be used reliably for covering small to medium sized wounds on exposed
areas and may be considered as the first choice.
7. References
Brown D, Garner W, Young L. Skin grafting: Dermal components in inhibition of wound
contraction. South Med J. 1990;83:789-793.
Carlson JA., Grabowski R., Mu XC., Del Rosario A. Possible mechanisms of
hypopigmentation in lichen sclerosus. Am J Dermatopathol. 2002;24:97-101
Chou TD., Chen SL., Lee TW., Chen SG. Reconstruction of burn scar of the upper extremities
with artificial skin. Plast Reconstr Surg. 2001;108:378-384.
Han SK, Yoon TH, Kim JB, Kim WK. Dermis graft for wound coverage. Plast Reconstr Surg.
2007 ;120:166-172.
Kuroyanagi Y, Yamada N, Yamashita R, Uchinuma E. Tissue-engineered product: allogenic
cultured dermal substitute composed of spongy collagen with fibroblasts. Artificial
Organs. 2001;25:180-186.
Rudolph R. Inhibition of myofibroblasts by skin grafts. Plast Reconstr Surg. 1979;63:473-478.
Sullival, T., Smith, J., Kermode, J., McIver, E., and Courtermanche, D. J. Rating the burn scar.
J Burn Care Rehabil. 1990;11:256-261.
Swope VB., Supp AP., Boyce ST. Regulation of cutaneous pigmentation by titration of
human melanocytes in cultured skin substitutes grafted to athymic mice. Wound
Repair Regen. 2002;10:378-385.
Terino EO. AlloDerm acellular dermal graft: applications in aesthetic soft tissue
augmentation. Clin Plast Surg. 2001;28:83-90.
Tyack ZF., Pegg S., Ziviani J. Postburn dyspigmentation : Its assessment, management, and
relationship to scarring. J Burn Care Rehabil. 1997;18:435-440.
Velangi SS., Rees JL. Why are scars pale? An immunohistochemical study indicating
preservation of melanocyte number and function in surgical scars. Acta Derm
Venereol. 2001;81:326-332.
Vloemans AF., Soesman AM., Suijker M., Kreis RW. Arandomised clinical trial comparing a
hydrocolloid-derived dressing and glycerol preserved allograft skin in the
management of partial thickness burns. Burns. 2003;29-34.

23
Cryopreservation of Skin Tissues
for Skin Grafts
Suong-Hyu Hyon
Institute for Frontier Medical Sciences,
Kyoto University 53 Kawahara-cho,
Shogoin, Sakyo-ku, Kyoto
Japan
1. Introduction
Preservation of living tissue and organs are very important to ensure successful
transplantation. The storage of living tissue, from the time of removal of the donor tissue
until transplantation, is one of the most important factors for successful tissue
transplantation. The purpose of living tissue storage is to maintain the viability of the
mammalian cells, and various methods have been developed to lengthen the time donor
tissue can be stored without loss of cell integrity.
At present, low temperature is used as the major organ preservation method. Generally,
cells are preserved in a frozen state at -196°C (1-3). The survival rate after such storage has
been enhanced by controlled temperature freezing. However, cell survival after freezing
can be low (namely, 20～40%), as with ES cells (embryonic stem cells),EG cells (embryonic
genital cells) and induced pluripotent stem (iPS) cells. It would be of great advantage to
researchers in the field of stem cell research to be able to preserve these cells more
successfully. It would also be of benefit to be able to preserve other cells, such as platelets,
over the long term without freezing. Likewise, research continues in our attempts to
prolong the time for transplantation of solid organs, and in the development of optimal
perfusion fluids that protect against ischemia remains an active subject of investigation.
Various storage solutions for organ preservation, such as the UW solution developed at the
University of Wisconsin (USA) are also in current clinical use. However, it is necessary to
develop storage solutions that can maintain the viability of tissues and organs for longer
periods because of the limitations of storage in UW solution.
After transplantation, many organs suffer from the generation of free radicals following
reperfusion. The restoration of blood flow becomes a trigger for injury, with subsequent
lipid peroxidation of the biomembrane leading to membrane failure and as a result, the
transplanted organ fails. A logical goal would then be the development of a preservation
fluid that would limit cell damage by preventing peroxylipid generation. Such a
preservation fluid should limit cell division and multiplication. A room temperature
storage state could also potentially prevent the injury to the small vessel endothelium seen
with freezing and to delicate tissues, such as the cornea, which do not survive freezing well.
Although these tissues can be held from 4 to 24 hours at 4oC, large organs impose a severe

Skin Grafts – Indications, Applications and Current Research
320
time limitation on the medical team (4-6). In addition, through advances in tissue
engineering, cultured skin and cultured cartilage have reached the level of clinical
application and demanded long term storage techniques for optimum utilization.
Transplantation of xenogeneic organs from genetically prepared animal donors would
likewise benefit from the possibility of longer periods of organ storage.
It has now been found that the polyphenols in green tea promote the preservation of
tissues, such as blood vessels, cornea, nerves, islet tissues, articular cartilage and
myocardium, at room temperature.(24) Furthermroe, in the case of hematopoetic stem
cells, the polyphenols suppress the differentiation of the cells into erythrocytes, T cells
and B cells. These findings suggest the possibility of a new method for tissue banking
that does not require freezing.
We have been conducting the research on the applications of green tea polyphenol
(epigallocatechin-3-0-gallate, EGCG) to the regenerative medicine (7-14). In the field of
transplantation medicine, which is a subfield of regenerative medicine, we have faced
challenging problems associated with graft tissues and organs, including loss of viability
and function (15-18), hyperplasia and immunological rejection of grafted tissues after
transplantation (19,20). To overcome these problems, we propose the use of a preservation
medium containing EGCG, which has been shown to have anti-oxidative (22-34), anti-
proliferative (8)and immunosuppresive properties (19).
Polyphenols have recently attracted attention as components of functional foods, and have
been shown to have various bioactivities such as anticancer activity, antimicrobal and anti-
virus activity since the 1980’s.(7-11) Therefore, there are many papers and patents on the use
of polyphenols for various applications. However, there has been no research which applies
to the use of polyphenols for the preservation of various tissues and organs. We believe that
such applications are possible, and we recently found an interesting phenomena related to
the effects of polyphenols on mammalian cells and living tissues. We herein describe the
effects of polyphenols on living cells and tissues, and present possible applications of
polyphenols for their preservation.
2. Preservation solutions for organ and tissue transplantation
Polyphenols have a hydroxyl group attached to the 2nd carbon, and have properties
completely different from other phenolic chemicals such as hydroxybenzene. The
chemical structure of green tea polyphenol is shown in Figure 1. It is possible to classify
these polyphenols into flavonoid hydrolysis type tannins and other polyphenols. Various
chemical compounds are known within the polyphenol group. Representative members
include catechin, which is mainly found in green tea and oolong tea, and anthocyanin,
which is the red pigment in red wine The antioxidative effects of green tea polyphenol
and catechin, as well as proanthocyanidin, are especially potent, and these agents are
known to be associated with a lower morbidity from heart disease (35-37). It has also
recently been reported in Nature that the proliferation of cancer cells was suppressed by
polyphenols (38-39).
The success rate of organ transplantation has increased due to the improvements in surgical
techniques and the development of new immunosuppressive agents in recent ten years. For
instance, in the USA where transplantation is frequently practiced with organs or tissues
from brain-dead donors, there were 4 million transplants performed in 2002.

Cryopreservation of Skin Tissues for Skin Grafts
321
EC
EGC
ECG
EGCG

chemical structures of various catechins

Fig. 1. The chemical structure of green tea polyphenol.
The donor organs or tissues are routinely transported in various preservation solutions. The
UW (University of Wisconsin) solution and Euro-Collins solution are the first and the
second most frequently used preservation solutions. These preservation solutions are
mainly used for the transport of kidneys, liver, or pancreas. However, they cannot preserve
the organs for longer than 24 hours at 4℃ (40-44). In light of the increasing demand for
donor organs and tissues, preservation solutions with better preserving abilities and the
research and development of such solutions are urgently needed.
When organs or tissues are isolated from a donor, their blood circulation stops, and
physiological activities rapidly decrease. Varying degrees of ischemia are commonly
observed for these organs, and free radicals are generated upon the restart of blood flow,
which leads to lipid peroxidation of the cell membrane, causing membrane damage and
various dysfunctions of the transplanted organs. The development of a preservation
solution that can minimize the oxidation and cell damage can solve this problem.
One of the traditional methods used to solve this problem has been freeze preservation (45-
47). Although living tissues and cells are routinely preserved at -196℃, freezing and
subsequent thawing cause some structural damage. For example, frozen blood vessels have
damage that often make their transplantation difficult, and corneas cannot be preserved at
4℃ for longer than one week without a significant degree of damage. Studies indicate that
the cell damage is primarily caused by activated oxygen molecules emerging from the
freezing and thawing processes, but also occurs even after exposing the living tissues and
cells to ordinary temperatures after removal from the donor.
Recent advances in tissue engineering are now on the cusp of providing clinically useful
cultured skin, cartilage and cornea specimens. However, these cultured tissues also require

Skin Grafts – Indications, Applications and Current Research
322
preservation, and better methods for long term preservation would be beneficial for
ensuring that they can be successfully applied in the clinic field. To this end, we introduce
the anti-oxidant polyphenol, EGCG, as a means to prevent the cell damage associated with
organ and tissue preservation. Using EGCG, we can preserve living tissues and organs
allowing for longer storage and more successful transplantation (13-34).
3. Control of mammalian cell proliferation
To study the effects of EGCG on cell proliferation, the rat fibroblast cell line, L-929, was
cultivated in EMEM (with kanamycin 60mg/1) supplemented with 10% fetal bovine serum.
A cell proliferation test was carried out at a cell density of 1.76×105 cells/ml. The
polyphenol (250 μg/ml concentration) was added to another culture system as a control.
The effects of the polyphenol in the rat fibroblast culture are shown in Figure 2. In the
polyphenol system, the cells became round, although cell proliferation was still active, and
the cell population was increased to 1×106 cells/ml on the fourth day after cultivation, but
the proliferation decreased after 1 week of treatment, and resumed when the polyphenol
was removed from the culture media.

Input
polyphenol
Polyphenol non-treated cells
0 day
4 days
2 days
3days
4days
15 days
Removal of polyphenol
(a) Fibroblast cells
TIME (days)
0
10
20
30
40
50
Cell Number
0e+0
1e+6
2e+6
3e+6
4e+6
5e+6
6e+6
a
b
c
d
e
Polyphenol treatment point
Proliferation curve of fibroblast
treated by polyphenol

Fig. 2. The effect of polyphenols on the multiplication of the fibroblast.
( ) The polyphenol free. ( ) The polyphenol addition.
We also assessed the effects of the polyphenol on the cell cycle. The results of treatment of
the fibroblasts with the polyphenol as determined by flow cytometry are shown in Table 1.
For the polyphenol-treated cells, the number of cells in the G0, G1 and G2/M-phases
increased, although after 9 hours in culture, the number of cells in the S phase reached 0. A
similar phenomenon was observed in porcine hepatocytes. In addition, the viable cell
population did not decrease. Good results were also obtained for the protective effects of
the green tea polyphenol against reactive oxygen species that induce oxidative stress in
cultured rat calvarial osteoblasts.

Cryopreservation of Skin Tissues for Skin Grafts
323
Cycle(%)
Time(hr)
Polyphenol
0
2
4
9
48*
Non-treated
cells
Treated
cells
G0G1
G2M
S
G0G1
G2M
S
22.18
2.58
75.24
22.18
2.58
75.24
15.69
1.12
83.19
33.05
11.00
56.95
22.86
11.93
65.22
52.96
13.83
33.21
18.99
20.85
60.16
70.07
29.93
0
71.33
20.54
8.13
・
・
・
*Cell cycle fibroblasts cultured in medium only during 48hr after removal of p
olyphenol from medium

Table 1. Time changes in cell cycle of untreated and polyphenol treated (250 mcg/ml)
fibroblasts.
4. Green tea polyphenol affects the preservation of rats skin and improves
the success rate of skin grafts
Although skin allografts are used in the treatment of serious burn injuries, skin disorders,
and skin defects their use is limited because of the limited amount of normal skin that can be
removed for such grafts (48). The best graft for the functional closure of wounds is the
autograft. At the end of an autograft procedure, the remaining donor skin is routinely stored
in a saline solution and applied to the open wound when graft loss or superficial wound
breakdown occurs postoperatively. (49) However, long-time storage in saline leads to poor
engraftment, and we were prompted to study other preservation media which would
extend the storage time of skin grafts. Recently, it has been found that the polyphenols in
green tea promote the preservation of tissues, such as the blood vessel, cornea, nerve, islet
cells, articular cartilage and myocardium, at room temperature (12-34). These findings
suggest the possibility of a new method for tissue banking without freezing.
As previously stated the dysfunction of transplants occurs as a result of free radicals due to
ischemia, which triggers lipid peroxidation of the cell membrane when blood flow is
restarted. It is reported that EGCG prevents peroxylipid generation. (50)
To determine whether the addition of EGCG to conventional cell culture medium could
enhance the viability of stored skin grafts and also extend storage time, we performed a
study using the skin from rats. The storage solution chosen for this study was Dulbecco's
Modified Eagle Medium (Sigma, St. Louis, MO, USA) supplemented with 10% Fetal Calf
Serum (FCS; Sigma), add and 1% antibiotic solution (including 10000U penicillin and 10 mg
streptomycin, Sigma). EGCG was dissolved in the storage solution at a final concentration of

Skin Grafts – Indications, Applications and Current Research
324
1 mg/ml. Transgenic Sprague-Dawley rats (8 weeks old, male) expressing the green
fluorescent protein (GFP) (green 2 Kim et al., produced with the constructs used for the
green mouse (51)), were a kind gift from Dr. Masaru Okabe (Genomic Information Research
Center, Osaka, Japan).
For anesthesia, pentobarbital (50 mg/Kg) was administered intraperitoneally to the GFP
transgenic rats. After shaving and depilating the backs of the rats, the back skins were
biopsied. The muscular layers were immediately stripped from the skin biopsies. Skin
samples measuring 1×1 cm were kept in sterile containers with 50ml preservation solution
at 4℃ and 37℃ for up to 8 weeks. Periodically, some of preserved skin (30 min, 1, 2, 4, 6, 7, 8
weeks) were used for grafts in nude mice or were directly examined histologically.
From histological examinations of the 4℃ preservation skin, it was noted that there was a
decrease in the GFP value in the non-EGCG skin noted during the second week (Figure 3),
as was a slight degeneration of the epidermal layer. The degeneration of the epidermal and
dermal layers were noted beginning at 5 weeks in all 4℃ groups (Figure 4).


Preservation
Time and
Temperature

Fig. 3. Photographs showing cryosection of the preserved GFP rat skins. GFP value was
slightly decreased according to preservation time.
In the 37℃ preserved groups, degeneration and flakiness of the epidermal layer were noted
beginning after one week of preservation, both with or without EGCG, and a decrease in the
GFP value was observed, regardless of the presence of EGCG, at 2 weeks. Moreover, the
epidermis was damaged in the tissue samples preserved without EGCG, although not in the
EGCG group.

Cryopreservation of Skin Tissues for Skin Grafts
325
Preservation
Time and
Temperature

Fig. 4. Photographs showing H.E. stain of preservation GFP rat’s skins. Degeneration of the
epidermis and dermis was observed, according to preservation time. The degeneration is
improved in EGCG groups compared to EGCG(-) groups.

Preservation
Time and
Temperature

Fig. 5. Appearances 4 weeks after grafting the preserved skins to the nude mice. Thirty
minutes preserved skin with EGCG, holds hair (a). On the other hand, skin without EGCG
(b) has no hair and the graft size is smaller than that with EGCG groups (a). After 2 weeks
preservation with EGCG at 4℃, grafts color were dark like necrosis, but it only remain
EGCG. Grafts size were smaller or only scar were observed at all groups, except
preservation under 4℃ with EGCG, after 2weeks preservation.

Skin Grafts – Indications, Applications and Current Research
326
The rat skin grafted onto the nude mice after 30 minute preservation with EGCG showed a
better condition than those implanted without EGCG, which showed loss of hair and
shrinkage. In the EGCG-preserved groups, the skin color was dark and looked like there
was necrosis superficially, however, the color was found to be due to the presence of the
EGCG (Figure 5). After 2 to 7 weeks of preservation in EGCG at 4℃, there was good graft
survival. In the other groups, the grafts shrunk or were only scar-like because of contracture
or rejection.
In the histological analysis, grafts preserved at 4℃ with EGCG were found to have been
completely accepted, with both epidermal and dermal layers remaining. On the other hand,
grafts preserved at 4℃ without EGCG had no GFP positive keratinocytes or fibroblasts
observed, and only phagocytes were observed in the dermal layer (likely targeting the GFP).
In the 37℃ preserved groups, no GFP positive rat cells were observed, with or without
EGCG (Figure 6). No grafts were successful from the 37℃ preserved groups (with or
without EGCG). However, the 4℃ preserved grafts were accepted, and the success rate for
the EGCG-treated grafts was 100% even after 4 weeks of preservation (Figure 7).
Wide skin defects caused by burns or trauma should be covered with skin grafts, but
sometimes skin grafts fail due to various causes such as hematoma. When skin grafts are
partially unsuccessful, small skin defects can be repaired, using the skin leftover from the
primary grafting, if it is preserved. Moreover, injuries leading to complicated tears or small
flap-like. In such cases, if the skin can be temporarily preserved, it can later be used as part
of the graft after the wound condition is improved. Freeze-drying porcine skin, xenografts,

30min
4W,37℃
6W,4℃
2W,4℃
2W,37℃
4W,4℃
7W,4℃
8W,4℃
EGCG(+) EGCG(-)
EGCG(+) EGCG(-)

Fig. 6. Photographs showing cryosection of the GFP rat skins 28 days after grafting to nude
mice (n=4-6). EGCG positive keratinocytes were observed only in the grafts preserved in 30
minutes preservation groups and 4℃ preservation groups with EGCG, 2weeks to 6 weeks
(a, c, g, k). EGCG positive fibroblasts were seen only in grafts preserved at 4℃ with EGCG,
30 minutes to 7 weeks (a, c, g, k, m). In other groups, only phagocytes which ate the GFP
were observed.

Cryopreservation of Skin Tissues for Skin Grafts
327
and frozen allografts are also used for wound coverage, but they are not usually
permanently incorporated. Therefore, it is very useful to develop better technologies for the
preservation of autografts.


Fig. 7. The take rate of preserved skin grafts to nude mice, judged 28 days after surgery. No
grafts took in 37℃ preserved groups with or without EGCG. Take rates were improved by
addition of EGCG, at 4℃ preservation.
There have been a few reports on the preservation of the cornea, blood vessels, nerves,
pancreatic islet cells, saphenous veins and peripheral nerves using EGCG. In the first
reported experiment, histological examination revealed that EGCG improved the length of
preservation time a skin sample. With time, skin samples start to degenerate from the
epidermal layer to the dermal layer. Skins samples preserved at 4℃ seem to undergo less
degeneration than the ones at 37℃. However, it was difficult to judge the cell viabilities
based on only histological examinations, because GFP was still present in all of the skin
samples. Therefore, we investigated the viability of the skin specimens by examining
whether the samples could lead to successful grafts. It has been reported that the dead tissue
or skin is rejected by around two weeks after implantation. (52, 53)
We used immunodeficient mice as recipient animals and judged the success of skin grafts
from GFP-Tg rats. The results indicated that the preservation of the skin with 37℃ is not
possible even if EGCG is added. Similarly, AE CRAM et al. reported that only 1/3 of their
grafts were successful at ten days after transplanting skin samples preserved at 4℃ for two
weeks. In our study, at 4 weeks after transplantation of skins preserved for 4 weeks at 4℃
with EGCG were successful, and one-third were still successful even after preservation for 7
weeks at 4℃ with EGCG. We therefore concluded that EGCG was beneficial for the
preservation of skin samples by decreasing the degeneration of the epidermal layer and
suppressing the postoperative graft contraction.
There might be several reasons why EGCG improved the preservation of the skin. EGCG
has strong anti-oxidative activities. The strong anti-oxidative activity of EGCG might inhibit
the lipid peroxidation of cell membrane of the preserved skin. M. Kapoor et al. reported that

Skin Grafts – Indications, Applications and Current Research
328
EGCG had anti-inflammatory and free radical scavenging effects in vitro. (54) EGCG was
also reported to control cell division. We believe that a combination of these effects
improved the condition of the preserved skin. In addition, the actions of EGCG, such as was
strengthening the scaffold structure, and its antibacterial activity also seem to have
contributed to its preserving effects. EGCG also improved the quality of scars after full
thickness incisions in rats, likely because EGCG increases iNOS (inducible nitric oxide
synthase), COX-2 (cyclooxygenase-2), and VEGF (vascular endothelial growth factor)
expression, leading to the formation of new blood vessels. EGCG also has been shown to
decrease arginase-I activity and protein levels. (54)
Our results suggest the possibility of the future clinical use of EGCG for skin preservation
without freezing, although the mechanism underlying how EGCG exerts its beneficial
effects on skin preservation still remains unclear.
5. Long term preservation of rat skin tissue by EGCG
EGCG enhances the viability of stored skin grafts and also extends the storage time for up to
7 week at 4°C. The addition of EGCG to conventional freezing medium, “Cell Banker” (CB),
could enhance the viability of skin grafts stored at −196°C and also extend their storage
time. Metabolic assays have been used as a surrogate measure of overall viability in the
grafts. Skin tissue was transplanted from GFP transgenic rats into immunodeficient mice,
and cell migration in graft tolerance of GFP positive cells was investigated after
transplantation (55).
For anesthesia, pentobarbital (50 mg/kg) was administered intraperitoneally. After shaving
and depilating the back of the rats, the back skin was elevated. After procurement, the
muscular layer was immediately stripped from the skin biopsies. Skin samples from GFP
rats measuring 1 × 1 cm were kept under sterile containers and refrigerated with PBS
solution with or without EGCG for 1 night. Skin samples were then transferred to CB
solution with or without EGCG and were stored in liquid nitrogen (-196°C) for up to about
24 weeks. Periodically, the preserved skin grafts of GFT rats were transplanted into nude
mice (2, 8, and 24 weeks). Circular skin biopsies (8 mm in diameter) were also made using a
sterile dermal biopsy punch (Kai Industries Co., Ltd. Gifu, Japan) and preserved in the same
way for in vitro analysis.
A full-thickness excisional square wound (1 × 1 cm) was created on the dorsum of each
nude mouse, and the GFP rat skin specimens were sutured to the adjacent normal skin
with 6-0 ProleneTM thread. After surgery, the mice were housed in separate cages. Skin
grafts were excised with 2–3 mm of the surrounding tissue, bisected, and processed for
histology 14 days after transplantation (n =4–6).
The in vitro results showed that 1 mg/ml EGCG had the highest protective effect on skin
samples when it was used at −196℃ for 2 weeks. At concentrations higher than 1.5 mg/ml,
EGCG delayed the recovery of the metabolic activity of skin samples after thawing (Figure
8). Figure 9 shows that glucose consumption was gradually increased with time and the skin
samples preserved with EGCG showed higher glucose consumption in all groups after
preservation. The in vitro data showed decreased glucose consumption in the 8-week-
preserved group in comparison to the 2-week-preserved group. By comparing Figures 8 and
9, We concluded that the presence of DMSO and EGCG in the freezing medium led to more
effective maintenance of glucose consumption than freezing medium alone. Glucose

Cryopreservation of Skin Tissues for Skin Grafts
329

Fig. 8. The optimum concentration of EGCG for rat skin preservation was determined by
measuring the metabolic activity. Skin biopsy samples were preserved in cell culture
medium with various concentrations of EGCG and preserved at −196C for 2 weeks.
Glucose consumption was measured after 6 days of 37C incubation after thawin.


Fig. 9. Glucose consumption of the preserved skin biopsies after thawing. Skin biopsy
samples were preserved in freezing medium (CB) with or without EGCG at −196C for 4
and 8 weeks. There was no significant difference; however, the amount of glucose
consumption in the EGCG groups showed faster metabolic recovery in comparison to the
CB groups.
consumption by the 2-week-preserved skin samples treated with only EGCG after 6 days
was about 220 mg/dl while the 4-week skin samples preserved with EGCG and DMSO was
almost 300 mg/dl. After day 7 the possibility of bacterial contamination increased and false-
positive data were obtained. All skin samples were washed with PBS containing antibiotics
every 2 days. The GFP fluorescence appeared to be similar between the 2- and 8-week-
preserved groups (Figure 10). The degradation of the epidermal layer to the dermal layer
was observed in all groups, regardless of preservation. However, while the Skin grafts of
GFP rats preserved in CB showed only green florescence around the dermis of the
transplanted area in the 24-week-preserved group, the fluorescence of the epidermal layer

Skin Grafts – Indications, Applications and Current Research
330
and some viable cells were confirmed in the EGCG- preserved skin grafts. The degradation
was therefore improved by EGCG in comparison to the CB group. An evaluation of the
viability of the transplanted skin grafts was difficult with GFP confirmation only. The
histology of the transplanted skin grafts of the GFP rats and nude mice was also therefore
evaluated using H&E-stained sections (Figure 11). The grafts preserved with EGCG
showed a dense dermal matrix and an intact epidermal layer, as shown in the GFP pictures.
Micrographs of CB preserved skin grafts were not fully rejected; however, separation
between the transplanted skin of the GFP rat and immunodeficient mouse was observed
near the location of the original epidermal layer. The histological score was marked with
micrographs of H&E-stained specimens based on the attached area of the transplanted skin
graft and the condition of the surviving skin grafts (Figure 12). Dead and completely
rejected skin grafts scored 0. The surviving skin grafts with a widely attached area, intact
epidermis, and dermis with hair follicles and a dense matrix scored 5. Neither the survival
rate of the skin grafts nor the histological scores indicated a clear difference between the 2-,
8-,and 24-week-preserved groups. However, both the survival rate of the skin grafts and the
histological scores were increased by EGCG in all groups.


Fig. 10. Cryosections of GFP rat skin graft to a nude mouse (left: 2-week-preserved group,
middle: 8-week-preserved group, fight: 24-week-preserved group). These micrographs are
representative of four to six independent experiments, and showed similar results.
EGCG therefore enhances the viability of stored skin grafts and extends the storage time up
to 7 weeks at 4℃. In this study, the storage time of the skin grafts was extended to 24 weeks
by cryopreservation with both DMSO and EGCG. The survival rate of the transplanted skin
grafts reached almost 100% in the 24-week-preserved group, indicating that long term
storage is possible.(62)
Cryopreserved cells and tissues are increasingly being used for stem cell transplantation and
tissue engineering. However, they are highly sensitive to freezing, storage, and thawing,
which suggests the need for improved cryopreservation methods. When storing any living
tissue, the destructive effects of hypoxic metabolism must be controlled. A tissue removed
from its blood supply will die unless cellular metabolic activity is decreased or nutrients are
provided. By reducing metabolism and providing nutrients, viability can be improved (56).
EGCG was reported to control cell division, which causes the energy metabolism to decrease

Cryopreservation of Skin Tissues for Skin Grafts
331

Fig. 11. Histological observation in skin grafts with H&E-stained micrographs (top: original
magnification 5, bottom: original magnification 20). Degradation of the epidermal layer to
the dermal layer was observed in the CB groups. The degradation was improved by EGCG
in comparison to the CB groups. These photographs are representative of four to six
independent experiments, and showed similar results.


Fig. 12. (A) The survival rate of grafted GFP rat skin after 2 weeks. (B) Considering the
epidermal morphology and dermal integrity of the transplanted grafts, a score was given
from 0 to 5. A higher score indicates better condition of the skin grafts. The results are
reported as the mean SE (n 4–6) and analyzed by Fisher’s PLSD test. The value marked
with an asterisk is significantly (p 0.05) different from the nontreatment groups.

Skin Grafts – Indications, Applications and Current Research
332
by inhibiting the cell cycle. Therefore, the combination of such effects improves the
condition of the preserved skin. In addition, EGCG reduces ischemia/reperfusion injury by
attenuating nitric oxide synthase expression and activity, and improving hypoxic
metabolism in preserved skin (57). Furthermore, other activities of EGCG, such as
strengthening of the scaffold structure, immunomodulatory effects, and antibacterial
activity, may also contribute to its preservative effects.
In the present study, the protection afforded by EGCG was similar for all three groups.
EGCG and DMSO in the freezing medium were more effective for maintaining glucose
consumption. These results suggest that oxygen radicals scavenged by EGCG may be
responsible for damage at the membrane level, and thus, exogenous EGCG can protect cell
membranes. These phenomena may be related to the intrinsic characteristics of polyphenol
compounds, which readily penetrate cell membranes due to their amphipathic properties.
These compounds are easily absorbed by lipid bilayers, extracellular matrices (e.g., collagen,
fibronectin), and various cell membrane receptors. The absorption of polyphenolic
compounds by such proteins is rapid, while the adsorption rates are slow. Consequently,
the skin graft could be protected from freeze–thaw injuries due to absorption of EGCG by
various membrane proteins and lipids (28). The glucose consumption of thawed tissues was
assessed after 2, 4, and 6 days of incubation under tissue culture conditions. This post-thaw
incubation period was selected to allow for recovery of the metabolic activity in damaged
cells. These delayed measurements may be more characteristic of the actual viability of the
skin allografts than measurements taken immediately after thawing and dilution. The cells
may sustain lethal damage during the thawing and dilution process but have not yet
completely disintegrated; thus, there may be residual metabolic activity in the lethally
damaged cells immediately after thawing.
The viability of the preserved skin biopsy samples was investigated by examining whether
skin specimens could be successfully grafted after preservation. It is reported that dead
tissue or skin is rejected in around 2 weeks (58,59). Therefore, immunodeficient mice were
used as recipient animals to judge the success of skin grafts from GFP rats (61). GFP
fluorescent protein does not need any chemical substrate for visualization. By using a GFP
transgenic animal, transplanted cells or tissues having GFP can be detected by direct
visualization. Therefore, the GFP transgenic rat is thought to be a suitable skin donor for
skin storage and transplantation studies.
A histological analysis demonstrated that the preserved skin began to degenerate from
the epidermis to the dermis. The epidermis is easily degenerated during the preservation
processes. Considering the histological data presented in previous studies, the results
obtained after 2 weeks of preservation seem to be better for samples stored at 4℃ than
those with cryopreservation. However, the success of the 4℃ preserved skin grafts was
limited to a 7-week or shorter period, as the samples at 8 weeks no longer led to
successful grafts.
EGCG therefore enhances the viability of stored skin grafts and extends the storage time up
to 7 weeks at 4℃ (61). In this study, the storage time of skin grafts was extended to 24 weeks
by cryopreservation using EGCG, and the survival rate was almost 100% in the samples
preserved for 24 weeks. These findings suggest that there may be future clinical applications
for EGCG as an agent for skin preservation without freezing, although the mechanism by
which EGCG promotes skin preservation remains to be elucidated (62).

Cryopreservation of Skin Tissues for Skin Grafts
333
6. Induction of hibernation of the cells and long term storage of living tissues
The control of cellular proliferation may be important for the prolonged storage of tissues in
polyphenol solutions without freezing. Polyphenol is a kind of antioxidant that has similar
antioxidant potential as vitamins C and E and superoxide dismutase (SOD). Because of its
amphiphilic properties, it also dissolves well in both water and oil. It also has a very high
affinity for protein, which allows it to combine with the protein, and yet dissociate with the
progression of time, which is characteristic of a reversible adsorption. For the control of cell
proliferation and tissue preservation, the latter characteristic is very important. Due to the
high affinity between the polyphenol and proteins, the polyphenol can adsorb to a receptor
on the cell surface that is integral to the process of cell division when cells are cultured, as
shown in Figure 13. The signaling between cells is then blocked when the polyphenol bonds
to this receptor, and the cell cycle is inhibited, resulting in no cells entering the S phase,
inducing a type of hybernation. As the polyphenol reversibly leaves the cell membrane
with the progression of time, the S phase of the cell cycle, and cell proliferation, can resume.
The successful long-term storage in the unfrozen state is probably also due to the adsorption
of the polyphenol to the collagen and proteoglycan of the extracellular matrix, where it may
easily generate temporary cross-linking reactions. Basic research on the interaction between
polyphenols
and
proteins
have
demonstrated
interesting
reversible
absorption
phenomena.(63)

The hybernation mechanism of the
various mamamian cells
necleus
DNA
polyphenol
mitochondoria
cytokine
cytodifferentiation
Cell prorification
Protein production
mRNA
Control protein
Signal
interception
LO・
LOO・
LOOH
O2
-
H2O2
-OH
1O2
cell mambrane
protein
membrane damage by oxidation!
Fe・O2
Red blood cells
O2

Fig. 13. The cell hibernation mechanism induced by the polyphenol.
7. Potential mechanisms for non-frozen preservation of mammalian tissues
and organs by polyphenols
All of these studies were started based on the author’s observation that polyphenols
appeared to be useful for the physiological preservation of tissues or organs, particularly rat
pancreatic islet cells [13]. Since then, evidence has been accumulating showing the beneficial
effects of polyphenols on cell preservation. The extension of that observation to the
preservation of tissues or even organs for transplantation will make it possible to store them
for longer periods by regulating the concentration of polyphenols present in the storage
solutions. Recently, it was reported that polyphenols can reduce the ischemia/reperfusion
injury in rat lungs and preserve various types of tissues or organs including canine lungs,

Skin Grafts – Indications, Applications and Current Research
334
rat aorta, rat peripheral nerves and mammalian pancreatic islet cells (12-34), supporting the
author’s hypothesis.
As shown in Figure 13, this non-frozen preservation of mammalian cells, tissues or organs
might be mediated by hibernation, a reversible regulation of cell proliferation and survival
through modulation of cell cycle-related genes by the polyphenols at the cellular level. With
regard to this hypothesis, it has already been reported that the hibernation phenomenon
triggered by polyphenols might be related to their intrinsic characteristics, including their
bind with and penetration into the cell membrane or tissue matrix due to their amphipathic
properties. The absorption of polyphenolic compounds to the proteins is generated early,
but the desorption rate is very slow. Consequently, mammalian cells, tissues or organs
could be physiologically preserved through adsorption of the compounds to membranous
proteins and extracellular matrices, leading to both the reduction in structural deterioration
and the prevention of oxidative damage (12-34).
8. Conclusion
As described above, the polyphenol was able to control the proliferation of various cell
types, and was demonstrated to be very useful for the long term storage of various tissues,
including islet cells, blood vessels, cartilage, corneas, nerves, and skin. Currently in the
U.S.A., approximately 850,000 tissue allografts are transplanted into patients annually.
Much of this tissue is stored frozen. The method of cryopreservation at -196℃ was adopted
at the University of Tokyo Hospital and the Osaka National Cardiovascular Center in April
1999. Cryopreservation methods are being used for the long term storage of blood vessel,
cartilage and skin. However, current research is still being directed at improving the
effectiveness of cryopreservation and of preservation fluids, such as the University of
Wisconsin solution, for tissue transplantation. We have herein demonstrated that the
preservation of various tissues for up to three months without freezing is now possible, with
excellent maintenance of the histological and biomechanical characteristics of the tissue.
The authors have also succeeded in preserving the rat sciatic nerve, guinea pig periodontal
ligament and rat myocardium in a non-frozen condition for long term storage prior to
preservation. This has been made possible by the development of a new preservation fluid
with polyphenol as an additive antioxidant. Therefore, the cryopreservation of many tissues
may eventually be superseded by their storage in the newly developed polyphenol tissue
banking fluid. This method of cell and tissue preservation would be of great benefit, not
only in Japan, but throughout the world, especially in developing countries where
cryopreservation is often not possible.
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Sentinel Skin Allograft for Monitoring of
Composite Tissue Transplants Rejection
Zamfirescu DG1, Lascar I1 and Lanzetta M2
1Carol Davila Bucharest Medical University
2University of Canberra
1Romania
2Australia
1. Introduction
In contrast to visceral solid-organ transplants, a vascularized composite tissue allograft
(CTA) is not a single tissue, and is histologically heterogeneous. It is a neurovascularized
module of nonvital tissues which include structural, functional and aesthetic units. CTAs
are composed of a large spectrum of ectodermal tissues: epidermis and epidermal
derivatives such as nails and hair, nerves and mesodermal tissues such as dermis, muscles,
bones, articular cartilage, ligaments, tendons and paratenon and other supportive and
connective tissues, adipose tissue, vessels. In addition there are hematopoietic tissues and
cells from bone marrow and lymph nodes and these latter elements are immunocompetent.
Each tissue has differing antigenicity, displays different antigen expression and presentation
mechanisms. As a result, CTAs elicit nonsynchronized immune responses, of differing
intensity, among their tissue components.
So far only 135 CTA transplantations were carried out on human patients: 68 hand
transplantations (49 patients), 12 abdominal wall transplantations, 9 bone and vascularized
articulations transplantations, 7 peripheral nerves, 2 tendons, 23 larynxes, 1 right abdominal
muscle, 1 tongue, a lobe made of the cephalic cervical skin and 2 ears, and 11 faces.
The transplantation of a composite tissue allograft is only justified when excellent functional
outcome can be achieved. This aim is more important for CTAs then for organ allografts as
the procedure is not lifesaving and lifelong immunosuppressive therapy, with its associated
risks and side effects, is difficult to justify. Even mild and reversible rejection episodes can
decrease the chance of a favorable outcome.
Skin is an important component of a CTA and is the most immunogenic of the component
tissues. The skin is more sensitive to rejection than any other tissues or visceral organ, and
the primary reason is probably its unique immunologic defense function, with its special
intrinsec antigenic and immunologic properties. Boss et al. identified these properties as "the
skin immune system” and demonstrate, that the skin is not only the largest organ in the
body but is itself an immunologic effector organ, with almost half its cells belonging or
related to the immune system (especially the large quantity of dendritic cells within epider-
mis and dermis). For CTAs the best marker of rejection is by visual and histopathological
examination of the skin, as it is the skin that is first involved in the rejection process.
Skin is an important component of a CTA and is the most immunogenic of the component
tissues. The skin is more sensitive to rejection than any other tissues or visceral organ, and

Skin Grafts – Indications, Applications and Current Research

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the primary reason is probably its unique immunologic defense function, with its special
intrinsec antigenic and immunologic properties. Boss et al. identified these properties as "the
skin immune system” and demonstrate, that the skin is not only the largest organ in the
body but is itself an immunologic effector organ, with almost half its cells belonging or
related to the immune system (especially the large quantity of dendritic cells within epider-
mis and dermis). For CTAs the best marker of rejection is by visual and histopathological
examination of the skin, as it is the skin that is first involved in the rejection process.
Monitoring of CTA rejection is facilitated because the first tissue to be rejected is the skin,
which is the outermost tissue of a CTA. Skin biopsy provides a more specific diagnosis to
confirm the visual assessment but delays diagnosis. Repeated skin biopsies also lead to
scarring that represents a considerable aesthetic problem in the case of a hand or face
transplants. The ideal marker of CTA rejection should therefore obviate the need for biopsy
of the transplant and provide easily warning of rejection of the graft itself. Early detection of
the first stages of rejection enables advanced administration of salvage therapy, resulting in
a rapid and efficient reversal of rejection.
2. Distant sentinel skin allograft (DSSG) - experimental study
Some studies report a more rapid rejection of isolated skin grafts compared to rejection of
skin that is part of a CTA. This has led us to examine the use of distant sentinel skin allograft
(DSSG) of donor origin transplanted simultaneously with a limb, as a marker of rejection.
In one of our studies, using a hind limb transplantation model in rats we demonstrated that
the DSSG serves as a predictive marker for visual assessment, as well as a site for repeated
biopsies that does not damage the CTA itself.
Sixty rats with hind limb transplants were included in this study. They were divided into
two groups. Group 1 (n=15) received limb transplants and immunosuppressive therapy
(control group). Group 2 (n=45) received a limb transplant and a simultaneous sentinel skin
graft, plus immunosuppressive therapy.
Surgical procedure
Brown Norway (BN) rats served as donors and Lewis rats as recipients of orthotopic hind
limb transplants and free skin flap allografts. The previously described surgical technique
for limb replantation and transplantation was used.7-11 A free full thickness skin graft 4 cm x
5 cm was obtain from the abdominal region of BN donors and transplanted to the inter


Fig. 1. Full thickness sentinel skin allograft and the hind limb transplantation