b u r n s 3 3 ( 2 0 0 7 ) 2 8 2 – 2 9 1

Review

Stem cell strategies in burns care

A. Burd *, K. Ahmed, S. Lam, T. Ayyappan, L. Huang

Division of Plastic & Reconstructive Surgery, Department of Surgery, The Chinese University of Hong Kong,

Prince of Wales Hospital, Hong Kong, China

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282

2. Stem cells and skin tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283

3. Stem cells and wound healing modulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285

4. Clinical challenge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285

5. Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287

6. Cord blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287

7. Stem cell fractions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

8. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289

a r t i c l e i n f o

Article history:

Accepted 15 August 2006

Keywords:

Stem cells

Marrow suppression

Burns

a b s t r a c t

The prospect of being able to replace damaged tissue by the process of regeneration would

dramatically and irrevocably change the impact, management and outcome of burns. The

current understanding of stem cell-based modulation and therapy together with their

potential developments do bring this prospect ever closer to a clinical reality. This paper

gives a background to stem cell strategies in burns care and identifies actual or prospective

applications which, collectively, will forever change burns care throughout the world.

# 2006 Elsevier Ltd and ISBI. All rights reserved.

avai lable at www.sc iencedi rec t .com

journal homepage: www.e lsevier .com/ locate /burns

1. Introduction

The last 30 years has seen a tremendous evolution in

conceptual strategies in the field of burns care. The three

‘R’’s, replace, reconstruct, regenerate have their clinical

application in transplantation, tissue engineering and now,

stem cells therapies [1]. When Rheinwald and Green described

the serial cultivation of strains of human epidermal kerati-

nocytes with the formation of keratinizing colonies from

single cells over 30 years ago [2], naive claims were made by

* Corresponding author. Tel.: +852 2632 2639; fax: +852 2632 4675.E-mail address: andrewburd@surgery.cuhk.edu.hk (A. Burd).

0305-4179/$30.00 # 2006 Elsevier Ltd and ISBI. All rights reserved.doi:10.1016/j.burns.2006.08.031

some laboratory scientists that the burns care of the future

would be a simple matter of quick and easy cover after

the excision of burn wounds. Unfortunately this is far from

reality [3,4]. The next decade brought the concept of tissue

engineering.

When the term ‘tissue engineering’ was officially coined at

a National Science Foundation Workshop in the USA in 1988, it

was understood to mean ‘the application of principles and

methods of engineering and life sciences toward the funda-

mental understanding of structure–function relationships in

Table 1 – Potential plasticity of adult stem cells (adaptedfrom Rosenthal [22])

Location of stem cell Type of cells generated

Brain Neurons, oligodendrites, skeletal

muscle, blood cells

Bone marrow Endothelial cells, blood cells, cartilage,

bone, adipocytes, cardiac muscle,

skeletal muscle, neuronal cells, dermal

fibroblasts, oval cells, gastrointestinal

tract cells, thymus, pulmonary

epithelial cells

Skeletal muscle Skeletal muscle, bone, cartilage, fat,

smooth muscle

Myocardium Myocytes, endothelial cells

Skin Keratinocytes

Liver Liver cells

Tests and ovaries Gonads

Pancreatic ducts Islet cells

Adipose tissue Fat, muscle, cartilage, bone

b u r n s 3 3 ( 2 0 0 7 ) 2 8 2 – 2 9 1 283

normal and pathological mammalian tissues and the devel-

opment of biological substitutes to restore, maintain or

improve tissue function’. This concept has unfortunately led

to some serious misconceptions that have resulted in the early

promise of skin tissue engineering being slow to be realized in

clinical practice. The misconception was that skin is a tissue,

like cartilage, and would be relatively simple to address as a

tissue engineering challenge. Skin however is NOT a tissue but

an extremely complex organ that brings into conjunction cells

from three different embryological origins, ectoderm, meso-

derm and neural crest [5]. It serves multiple functions [6]. The

original futuristic claims of producing ‘off-the shelf’ skin

replacements have become far more restrained in their

expectation and now tissue engineering skin products are

being described as skin substitutes to aid healing and repair,

temporary skin replacements and occasionally aids to

regeneration [7–19].

How will the current era of stem cell exploration be

reviewed in retrospect? The ‘stem cell’ concept in burns care is

not new and it was Barrendon, working in Howard Green’s

Harvard Laboratory who introduced the concept of the clonal

differentiation of cultured keratinocytes identifying the

holoclone with the greatest proliferative capacity [20]. Can

holoclones be regarded as adult skin stem cells?

Stem cells can be variously classified. One way is to identify

the source and thus we can regard stem cells to be embryonic,

foetal or adult in nature. An alternative perspective is to look at

the stage of evolution of the stem cell line and describe the

cells as being totipotent, pluripotent and multipotent.

The totipotent cells contain all the complete genetic

information needed to manufacture all the cells of the body

as well as the placenta. These cells are present immediately

after fertilization of the egg and for three to four divisions

thereafter. As the cells become more specialized they are

described as pluripotent. The cells are extremely adaptable

and can develop into any cell type with the exception of

placenta. Further division of the pluripotent cells will give rise

to multipotent cells. These are far more specialized and can

only generate a limited number of cell types [21].

The true stem cell must satisfy certain criteria: it must be

clonogenic i.e. capable of unlimited self-renewal by symmetric

division; it must also be able to divide asymmetrically, with

one daughter cell resembling the mother (to perpetuate the

clone) but the other capable of giving rise to multiple types of

differentiated cells which indeed represent derivatives of all

three primitive embryonic germ layers.

It is this concept of ‘plasticity’ which makes the stem cell so

attractive to the tissue engineer and so one critical aspect of

stem cells will be the variable expression of plasticity related

to source. In Table 1 the potential plasticity of some adult stem

cells is detailed which does indicate the wide range of cells

that have thus far been generated from bone marrow cells [22].

It is evident, however, that this plasticity is limited in adult

stem cells, certainly compared to stem cells derived from the

inner cell mass of the early embryonic blastocyst (ES cells),

which can both proliferate indefinitely but also give rise to

virtually any type of cell [1].

Adult stem cells have been incorporated into tissue

engineered constructs used by reconstructive surgeons. A

recent review focused on the four components in developing

tissue substitutes: gene therapy, growth factors and pharma-

logical preparations, scaffolds and cells. It is this last

component, the cells, where increasing attention is focusing

on stem cells particularly in true tissue, as opposed to organ,

engineering. Bone, cartilage, tendon and muscle have all been

developed to some degree of success from adult stem cells.

The preferred source of adult stem cells, however, remains

uncertain and the functional plasticity of adult stem cells

exists more of interest in scientific experimentation than in

clinical application [23,24].

2. Stem cells and skin tissue engineering

The skin is indeed a complex structure incorporating a

fusion of multiple cell types, integrated within a three-

dimensional matrix containing both fibrillar and non-

fibrillar elements. To synthesize such a complex structure

by identifying the component parts and to put them together

is neither practical nor realistic. It must be observed,

however, that this integrative strategy has been the major

one used in skin tissue engineering during its less productive

phase [19]. The great attraction of stem cells in the

construction of a complex tissue or organ is that the

component parts can be simplified at the initial stage and

a significantly greater proportion of the neogenerative

process can be driven by the intrinsic bioengineering

capacity of the cells and tissues. It is important to realize

that cells by themselves cannot generate organs and the

matrix provides a critical element in defining appropriate

differentiation and three-dimensional organization. Stem

cells are going to play an increasingly important role in

tissue engineering and they will be derived from a range of

sources as described above. Of particular interest is human

umbilical cord blood (HUCB). Considerable attention is now

being addressed to expanding the number of stem cells at

differing stages of maturity. Expansion factors of 10 to more

than 1000 have been claimed and one aspect of considerable

significance is that undifferentiated stem cells can be

expanded. Cell process engineering for expansion does rely

in three-dimensional matrices in bioreactors [25–27].

Fig. 1 – Biological concepts of skin tissue engineering.

b u r n s 3 3 ( 2 0 0 7 ) 2 8 2 – 2 9 1284

Cord blood and cells derived from the umbilical cord

have been used in a variety of tissue engineering projects.

Living patches of tissue have been fabricated from a

combination of synthetic polymers (PGA/P4HB) seeded with

fibroblasts harvested from umbilical cord tissue and

endothelial progenitor cells which were cultured in a

perfusion bioreactor. These patches have the potential of

being used in congenital cardiac conditions [28]. Attempts to

engineer microvessels using similar endothelial progenitor

cells and poly-glycolic acid–poly-L-lactic acid (PGA–PLLA)

scaffolds demonstrated a lack of vessel formation. However,

when the same cells were co-cultured with human smooth

muscle cells, microvessel formation was observed on the

porous (PGA–PLLA) scaffolds [29]. Skeletal myogenic differ-

entiation was observed in mesenchymal cells isolated from

HUCB [30].

Fig. 2 – The conceptual pr

The 21st century tissue engineer faces the challenges of

both possibility and practicality. Complex and costly products

will not find commercial applications. In the meantime the

biological complexity of the skin has been appreciated and it is

no longer viewed as a bilaminar tissue but a multidimensional

organ. The tissue engineer must ask how much of this

complex biological tissue is going to be constructed. Fig. 1

shows the ‘biological’ as opposed to the ‘engineering’ concepts

of the skin structure. The skin began in the embryonic stage as

a conjunction of cells which integrated their specialized

functions. The key cell of the epidermis is the keratinocyte

which produces keratin. It is the outer layer of keratin filled,

dead cells, the stratum corneum that is responsible for the

principle protective functions of the skin. The mesodermal

fibroblasts are principally involved in the formation and

maintenance of the dermis. The problem for the tissue

ocess of construction.

b u r n s 3 3 ( 2 0 0 7 ) 2 8 2 – 2 9 1 285

engineer is that the time scale for cellular matrix production is

too slow in clinical application. A preformed matrix is required

either as a permanent or temporary scaffold. The problem

with the permanent scaffold is biocompatibility and the problem

with the temporary scaffold is stability.

For the permanent replacement of lost skin it is necessary

to use a dermal analogue which is slowly replaced by an

autologous dermis. Such a product might be formed by an

immune modulated allogeneic or xenogenic dermis modified

with mesenchymal stem cells, either of marrow or adipocyte

origin. This layer would need to be seeded with a stem cell-

enriched keratinocyte formulation possibly including mela-

nocytes. The attachment is critical and will require a medium

such as hyaluronic acid which can support the cells whilst

promoting proliferation and attachment. These ex vivo

derived components would be combined for in vivo culture.

Such a process can be simply conveyed diagrammatically

(Fig. 2) but the practicality of such a construct presents

considerable challenges. The history of the evolution of skin

tissue engineering does suggest, however, that for permanent

skin replacement the body’s own ‘tissue engineering’ capacity

must be harnessed using stem cell technology with or without

synthesized matrix support. For tissue-engineered skin sub-

stitutes to provide temporary wound cover materials, science

and technology already show promise but the challenge is to

reduce costs. Again for products which are aimed at

modulating wound healing, living cells can be genetically

engineered to restore molecular balance to chronic wounds.

Again, knowledge and technology are available but the

challenge is to develop products that are safe, effective, easy

to use but also affordable. Another fascinating prospect is the

clinical application of human umbilical cord-derived stem

cells. Preliminary results suggest that these can be expanded

in culture and applied to burn wounds where they differ-

entiate into non-antigenic keratinocytes. This truly would

have a dramatic impact on burns care but of course further

indepth studies will be needed to explore the real clinical

potential of such an approach [31].

3. Stem cells and wound healing modulation

In this situation cells will be placed into a pathological

environment for example a chronic or non-healing burn

wound. They will undertake an assessment of the physiolo-

gical deficiencies in terms of matrix composition and cytokine

milieu and correct these by producing the appropriate wound

healing modulators. In a sense this means that the cells are

acting as an intelligent, interperative biofeedback control

mechanism that can autoregulate biological systems. Bone

marrow, peripheral blood and umbilical cord blood have all

been used in chronic wounds to modulate the healing

response. Whilst the early experience is limited, the prospects

are promising and some of the concerns about incorporating

stem cell-derived tissue, into the body are unwarranted [32].

Of particular interest is the effect of topical applications of

bone marrow-derived cells on chronic wounds. Although the

reports are few, the consistent theme is that a chronic wound

changes its nature to become an acute wound that heals or

becomes healthy and can be closed with a skin graft [33–35].

Laboratory studies looking at cutaneous healing in a chimeric

mouse model indicate that when marrow is transplanted it

can contribute to the reconstitution of the dermal fibroblast

population in the wound although local cutaneous cells

reconstitute the epidermis [36]. An in vitro study indicated

that collagen synthesis and levels of bFGF and VEGF were

much higher in bone marrow stromal cells than those in

dermal fibroblasts. This suggests the potential of topically

applied bone marrow cells to accelerate wound healing [37]. A

further study examined the effects of a bone marrow-

impregnated collagen matrix and found a significantly

increased angiogenic effect in an experimental mouse model.

This same group applied an autogenous bone marrow-

impregnated collagen matrix to a patient with a chronic leg

ulcer and observed a dramatic healing response [38].

In our own experience we applied autologous bone marrow

to a chronic unhealed burn wound, a donor site that had

repeatedly failed to heal and a chronic wound at the extremity

of latissimus dorsi free muscle flap where the graft was being

traumatized by footwear as well as non-burns related leg

ulcers. The burn wound changed from being chronic and non-

healing to re-epithelializing and was closed with a graft. The

donor site that had failed to heal and also had repeatedly failed

to take a graft, healed with no grafting necessary. The chronic

heel wound and leg ulcers became more vascular and were

definitively closed with skin grafts. Another recent report

describes the use of allogenic bone marrow mesenchymal

stem cells for the treatment of a patient with deep skin burns

[39].

4. Clinical challenge

Bone marrow suppression has documented association with

major burns either as a result of toxicity by silver sulphadia-

zine [40] or as a result of sepsis [41,42]. The relation of

mesenchymal stem cell activity and burn wound healing is,

however, a newer concept that has received little attention.

The following case illustrates a number of dilemmas in

contemporary burn management.

A 53-year-old man presented with 94% BSA burns which

were of mixed depth. The proposed strategy was to treat the

wounds conservatively with the expectation that the majority

were capable of healing within 3–4 weeks as judged by the

depth of burn. By the end of the fourth week the face had

healed and other areas of the trunk and limbs were showing

signs of punctate re-epithelialization. At this stage the patient

began to become progressively anaemic with no evidence of

blood loss and the progress of healing appeared to have halted.

The patient was treated with blood transfusions and oxan-

drolone and the potential for restimulating wound healing by

topical preparations were considered. Our evolving hypoth-

esis is that mesenchymal stem cells (MSC) are biological

catalysts that catalyse a change in profile from a chronic to an

acute wound. We considered the increasing urgency of

intervention in our patient and also the availability of ethically

approved stem cell sources. We discounted allogenic marrow

in view of the donor supply and time needed for MSC

expansion and sought our institutional ethical committee

approval for the use of human umbilical cord blood. This has,

Fig. 3 – Cells of dipolar, spindle-shaped and fibroblastic

morphology derived from bone marrow after 48 days of

culture (40T magnification).

Fig. 5 – The tibial shaft has been opened and reveals a

complete absence of red marrow.

b u r n s 3 3 ( 2 0 0 7 ) 2 8 2 – 2 9 1286

as yet, been untried in acute burns management and required

due deliberation by the ethical committee. In the meantime

the patients condition continued to deteriorate and he was

returned to theatre on the 47th post-burn day. Bone marrow

aspiration was attempted from both iliac crests using aseptic

technique. Eight negative aspirations were performed during

which time X-ray control was used to confirm placement in

the marrow cavity. Finally, 12 ml of marrow was aspirated and

10 ml were spread onto porcine skin and applied to 4% BSA of

the right anterior thigh and 2% BSA of the chest. The

remaining 2 ml were anti-coagulated in preservative-free

heparin and sent for analysis. The bone marrow sample

was hypocellular; the nucleated cell count being

3.6 � 106 ml�1. The numbers of CD34+ cells and CD3+ cells

were 0.49% and 23.1% of CD45+ cells, respectively. Bone

marrow nucleated cells (1.2 � 107) in 5 ml culture medium,

(Advanced Dulbecco’s Modified Eagle’s Medium supplemen-

ted with 2% foetal bovine serum and 20 ng/ml each epidermal

growth factor and basic fibroblast growth factor) were seeded

Fig. 4 – Cells of dipolar, spindle-shaped and fibroblastic

morphology derived from bone marrow after 48 days of

culture (100T magnification).

onto a 25 cm2 flask and incubated at 37 8C in a humidified

atmosphere containing 5% carbon dioxide. After a week,

suspended cells were removed and adherent cells were

continuously cultured with a change of culture medium every

3–4 days. On day 17, the culture of 70% confluency was

segregated with 0.05% trypsin–EDTA and replated at a cell

density of 2 � 103 cm�2 in culture flasks. Subsequent passages

of cells were performed in every 10–12 days. Homogeneous

population of cells of dipolar, spindle-shaped and fibroblastic

morphology were noted having cultured for 48 days (Figs. 3

and 4).

Unfortunately whilst the culture of the MSC was being

established the patient’s condition continued to deteriorate

and the patient succumbed from lung associated sepsis. The

post-mortem examination confirmed complete absence of red

marrow in the tibia (Fig. 5) and scanty macroscopic red

marrow distribution in the sternum.

The management of the extensive partial thickness burns

is a challenging process in terms of deciding when and how

to intervene in the healing process. An interesting report in

the Lancet suggested using fetal skin constructs [43] but the

clinical application in larger partial thickness burns was

questioned in a discussion of this paper [44]. Our previous

experience with this type of burn led to our preference for

conservative treatment. Admittedly previous patients have

been younger but the good initial response of this patient

suggested that a correct decision had been made. It was not

until 4 weeks after burn that concern began to be focused on

the rate of healing. The patient described above was

receiving full metabolic support and was never overtly

septic. It was at this stage that the patient began to develop

progressive anaemia with no obvious blood loss which

would fit with marrow suppression. With the wounds

beginning to appear more ‘chronic’ in nature we considered

various options to re-stimulate the healing process. After

general support has been given in terms of nutrition

and metabolic stimulation attention focused on the wound.

A range of strategies have been described from the

single cytokine approach first described by Knighton with

b u r n s 3 3 ( 2 0 0 7 ) 2 8 2 – 2 9 1 287

autologous—platelet derived growth factor (PDGF) [45] to the

use of cells within a morphological construct that can

develop targeted cytokine responses [46]. Our previous

experience with chronic wound healing stimulation

involved autologous bone marrow application. Autologous

bone marrow is a rich source of adult mesenchymal stem

cells which are multipotent besides containing a host of

healing factors. By virtue of being an autologous transfer the

question of immune related problems and ethical issues are

non-existent (graft versus host reaction is a potential

concern if human umbilical cord blood is used). Another

potential advantage of applying the cells directly without

subjecting them to expansion in the laboratory is saving

time. There has been a report published describing the

beneficial effects of applying allogenic fibroblast-like bone

marrow mesenchymal stem cells onto the surface of a deep

thermal burn [39]. In that case the chronic non-healing burn

wound changed in appearance with more angiogenesis and

the patient was healed with autologous skin grafts.

5. Hypothesis

Our evolving hypothesis is that the burn wound has a

multifactorial effect on bone marrow activity with toxic

bacteriological products having a suppressive effect on

marrow proliferation. At the same time there are increased

demands for marrow-derived cells stimulated by inflamma-

tion and autolysis, invasive infection and cutaneous wound

healing. The combined effect of this in the very extensive,

partial thickness burn would be marrow exhaustion or failure

(Fig. 6). The clinical correlate of this hypothesis is that if bone

marrow stem cells are the key to healing of extensive burn

wounds, attempts to speed up healing should target main-

tenance of a healthy marrow pool. This hypothesis is

supported by observations of cutaneous wound healing in

Fig. 6 – Diagrammatic repres

rodent models which indicate the reconstitution of dermal

fibroblasts from marrow-derived cells [36,47].

The post-mortem examination supported the evidence of

bone marrow depletion as evidenced by absent marrow in the

tibia and scanty marrow in sternum. It is difficult to speculate

how the wounds would have behaved had the patient

survived, but the post-mortem examination showed that

the raw areas that were treated with stem cells were covered

with firmly adherent porcine skin with no evidence of

infection as compared to untreated areas.

6. Cord blood

Another aspect of stem cell management that has already

been alluded to is the potential for using human umbilical cord

blood (HUCB) and, indeed human umbilical cord-derived stem

cells. Each year the human race produces over 10 million litres

of HUCB. It is a free, natural resource. A recent report has

indicated the potential of HUCB stem cells to differentiate into

keratinocytes under in vitro conditions [48]. The potential of

systemic infusion of ABO compatible HUCB and/or the topical

application of such blood to burns and other wounds opens up

the possibility of providing cheap and accessible burns and

wound care to more of the world’s neediest patients. The

attraction of supplies of pluripotent stem cells remains as a

prized resource by scientist and clinicians. The annual global

100 million human birth rate underlines the possibility that

umbilical cord blood represents the world’s largest untouched

stem cell resource. The particular advantages of stem cells

from this source include ethical acceptability, a naive immune

status and relatively unshortened telomere length. Claims are

already being made that stem cells with embryonic char-

acteristics can be produced from human umbilical cord blood

[49]. Because of the many ethical and biological issues

involved it will be many years before true embryonic stem

entation of hypothesis.

b u r n s 3 3 ( 2 0 0 7 ) 2 8 2 – 2 9 1288

cells reach the clinic. Research in this field has not been helped

by the highly publicized claims of scientific fraud by some high

profile researchers [50]. Cord blood on the other hand, is being

used for an increasing number of clinical and experimental

applications which highlighted the reduced incidence of graft

versus host reaction when the haemopoietic fraction is used

as an allogenic transplant [51].

A point of note is the difference between transplantation

and transfusion. In the context of using the stem cell

component to be a permanent replacement for the host stem

cell source as in haematological malignancies, then the use of

the term transplant is appropriate. The concept is of a

procedure with a long term, if not life long, implication for

safety and efficacy. Transfusion is another matter where the

time scale of interaction is shorter. When considering HUCB as

an adjunct therapy for a critically ill patient it would be more

appropriate to regard it as a transfusion and not demand such

strict compatibility criteria.

The response and interaction of stem cells and target cell

differentiation is a focus of particular interest to the Burn

Surgeon. The review by Heng et al. discusses strategies for

directing keratinocyte stem cell lineage in vitro using selective

purification and proliferation [52]. A three-step process is

described with the first-step being to induce commitment of

the (non-epidermal) stem cell into keratinocyte progenitors.

These progenitors have to then be selected and purified and

finally this purified population of committed keratinocyte

progenitors has to be expanded by proliferation and allowed to

differentiate. A recent article in Burns has re-affirmed this

potential strategy in resurfacing burns [48].

Various strategies are discussed for committing stem cells

to the keratinocyte lineage including induction with exogen-

ous cytokines, growth factors, chemicals and extracellular

matrix. It is becoming obvious that stem cell biology is very

complex if such strategies are to be adopted to direct stem cell

differentiation and maintain it.

It is evident, however, that there are going to be consider-

able challenges in the development of reliable protocols that

can confidently preclude risks of teratoma formation and

other, as yet unforeseen complications.

Nevertheless we do see cord blood as a biological wound

healing modulator. It is of interest to note that amniotic

membranes have been used as biological dressings for wounds

but concerns about risks of disease transmission have

severely limited this practice in many parts of the world

[53]. Similarly the question of potential risk of disease

transmission when using cord blood may also be raised.

However, there are already well-defined screening processes

to reduce and/or elimate such risks as applied in routine blood

banking. Another consideration is that this use of cord blood is

temporary and the relative lack of immunogenicity will limit

adverse effects.

7. Stem cell fractions

As the range and nature of the stem cell composition from

various sources becomes more clear, it may be possible to

apply more selective fractions onto both chronic and acute

wounds to modulate the biological healing mechanisms.

Fractionation of cells is conventionally performed using

FicollTM gradient centrifugation, although more and more

advanced cell filtration devices are being developed. By the

conventional method, a cell fraction of mononuclear cells

(MNCs), which contains the significant number of stem/

progenitor cells, will be isolated. The reduction rates of RBCs,

platelets (75–85%) and granulocytes (80%) were significant,

which may be minimally effective for the preparation of stem

cells [54]. Stem cells, however, constitute only a small fraction

(less than 1%) of all MNCs in the BM, and an even smaller

percentage of cells in the peripheral blood and cord blood.

Both hematopoietic stem cells (HSCs) and mesenchymal stem

cells (MSCs) have been isolated from all the three sources.

Although the exact relationship between HSCs and MSCs is yet

unclear, was reported that purified HSC fraction is not limited

to forming blood cells, but may also function as multipotent

progenitors for non-hematopoietic cells, for example, to

generate liver, etc. [55]. Conversely, MSCs play very important

roles in the maintenance, proliferation and differentiation of

HSCs. There has been some debate about the ease of isolation

of MSC from the different fractions. Wexler identified adult

bone marrow as the richest source compared to umbilical cord

and adult peripheral blood [56]. Kern again observed better

rates of isolation of MSC from bone marrow (100%) compared

to cord blood (63%) but interestingly also reported 100%

success rate in isolated MSC’s from adipose tissue isolated

from cosmetic liposuction [57]. The isolation of MSC from

adult peripheral blood can be enhanced by prior treatment

with granulocyte colony stimulating factor (G-CSF) [58]. The

significance of the MSC fractions is already a major research

area issue. The heterogenicity of the fractions isolated from

human umbilical cord blood is demonstrated by differential

gene expression profiling [59,60]. Experimental studies in

animals also indicate a wide variety of conditions that can be

treated, delayed or otherwise modulated by IV administration

of human umbilical cord MSC fractions. These conditions

include prostate cancer [61], type 1 [62] and type 2 [63] diabetes,

Parkinson’s disease [64], Alzheimer’s disease [65] and amyo-

trophic lateral sclerosis [66].

It would certainly be a mistake to underestimate the

complexity of stem cell biology and the clinical applications.

Nevertheless research and developments in this field will

undoubtedly have far reaching effects on burns practice

throughout the world.

8. Summary

We are moving into a new and exciting era of burns care where

perhaps we are going to exercise more humility in the face of

the enormous complexity of biological processes than

acknowledged by our purely science oriented colleagues.

Would it be true to say that one stem cell is worth 10n

individually characterized and engineered proteins? Currently

the challenge is to define the optimum source, processing and

method of application of stem cells as well as defining their

role. The clinical application of embryonic stem cells is likely

to beset with numerous ethical but also safety concerns. Fetal

tissue, likewise, will be associated with ethical issues. The

reality of widespread applications of stem cells devoid of

Fig. 7 – Diagrammatic overview of stem cell strategies.

b u r n s 3 3 ( 2 0 0 7 ) 2 8 2 – 2 9 1 289

complex ethical dimensions really begins with human

umbilical cord blood. This has been used in a number of

clinical ‘haematopoetic’ applications as a ‘transplant’ which

underlines the safety and efficacy of this stem cell source [67–

73]. There have been an even wider range of application of

HUCB as a transfusion in conditions as diverse as malaria,

cancer, tuberculosis and autoimmune degenerative arthritis

[74–77]. So why not burns? Fig. 7 gives an overview of the

present ‘state of the art’ with regard to stem cell applications.

Concerning the case presented, it is somewhat salutary to

observe that several weeks after the demise of the patient the

mesenchymal stem cells were flourishing and proliferating

actively. This has caused us to reflect on new paradigms of

care in the extensive burn. At present part of our routine

protocol is to take a skin biopsy to establish keratinocyte

cultures. We are now considering the aspiration of 5–10 ml

bone marrow after admission and before starting silver-based

dressings. Marrow stem cells can be selected, expanded in

culture and banked. The route and mode of subsequent

clinical administration is an area wide open for research;

systemic infusion or topical administration. Similarly with the

topical application of autologous marrow on the smaller

wounds there is much to discover: is it better to apply whole

marrow aspirate, or selected fractions; clotted or unclotted;

carriers; adjunct dressings; repeat applications? Whatever the

outcome it is certain that there will be an exponential rise in

the research output in this exciting field and stem cell

strategies in burns care will play an ever increasing role in

our management options. If we truly are really beginning to

uncover the secrets of regeneration of skin then this may well

be the greatest and most significant phase in the evolution of

burns care.

Acknowledgment

We would like to acknowledge the help and advice regarding

bone marrow culture from Dr. Kent K.S. Tsang, Scientific

Officer, Blood and Marrow Transplantation, Department of

Anatomical & Cellular Pathology, The Chinese University of

Hong Kong, Prince of Wales Hospital, Hong Kong.

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