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DEVELOPMENT
OF TECHNOLOGY FOR POINT-OF-CARE DIAGNOSTICS
THROUGH TO CELL-BASED THERAPY FOR CHRONIC WOUNDS
Stephen Britland1,2, Annie Thomas1, Abbas Din1, Annie
Smith1, Nick Crowther2, Donald Eagland2.
1School of Pharmacy, University of Bradford, Bradford, BD7 1DP, UK.
2AGT Sciences Ltd., Unit 25, Listerhills Science Park, Campus Road, Bradford.
MANY
of the emergent scientific strategies for correcting tissue dysfunction
after disease or trauma are increasingly referred to using the umbrella
term of tissue engineering. A literal interpretation of this somewhat
imprecise term has been suggested as ‘the persuasion of the
body to heal itself, through delivery to the appropriate sites of molecular
signals, cells and supporting structures’ (Williams, 1999).
One crucial factor affecting the likely success of even the most promising
tissue engineering therapy is whether or not the recipient’s tissue
will accept it. Even the most elegant treatments may ultimately fail if
subtle yet still contraindicating features of the underlying pathology
persist but are not recognised. One key to reducing this element of risk
could be rapid and cost-effective point-of-care diagnostics technology
capable of informing the clinical judgement of healthcare professionals.
We have adopted this strategy over the last ten years in developing technology
that spans the scope of treatment for intractable skin lesions from point-of-care
diagnosis, through anti-microbial intervention to cell-based therapy.
From left:
Mr Abbas Din, Dr Annie Smith, and
Dr Stephen Britland, School of Pharmacy, University of Bradford.
It has
been suggested that one person in every hundred in the United Kingdom
will at some point in their lifetime develop a long-lasting skin wound.
The financial cost to society of treating these patients is enormous.
Figures for 2000/1 show that approximately 4% of the entire NHS budget
was allocated to chronic wound care (Bennett et al. 2004). Although commonly
associated with poor peripheral circulation and complicated by diabetes,
rheumatoid arthritis, oedema or obesity, chronic wounds can be managed
and healed although usually over a protracted period. Treatment of leg
ulcers typically involves the application of various types of dressing
to promote healing and alleviate any predominant symptoms. Most often
the choice of dressing is selected on the basis of a practitioner’s
previous experience and personal preference. Unlike other diseases where
diagnosis is assisted by taking quantitative measurements of some sort,
chronic wounds are mostly judged from their appearance alone. Management
of chronic wounds, more than most other areas of medicine, therefore lacks
a comprehensive but also rapid diagnostic capability. Consider a situation
in which clinical ex examination was assisted by a diagnostic dressing
which, when applied to a wound in the same manner as a conventional dressing,
would reveal the status of that wound in terms of recognised prognostic
biochemical indicators. This would enable a more accurate assessment of
the wound and optimisation of treatment, so allowing wounds to heal more
effectively. Development of diagnostic devices for healthcare purposes
is a highly regulated process but is greatly assisted if there is a precedent
for use of elements of the technology, there is an overwhelming clinical
need, and it has a good chance of success. It is for this reason that
we have sought to develop a novel POC diagnostics
technology around a hydrogel material as they are currently deployed in
healthcare applications and are ideally suited to incorporation of colorimetric
detection systems. One prognostic indicator, the pH of the wound bed milieu,
has been the subject of research reports for many decades (Wilson et al.
1979), but the reported deviation from physiological pH and fluctuation
in chronic wounds (Dissemond et al. 2003) could be both symptomatic of,
and also an exacerbating factor in, abnormal wound biochemistry.
For example microbiological colonisation and infection could be detrimental
to wound pH and it is known that the activity of enzymes with identified
roles in wound healing is pH-dependent (Greener et al. 2005). Also, several
studies have, both directly (Westaby, 1986) and indi-rectly (Kaufman and
Berger, 1988, Leveen et al. 1973), found that the mitogenic activity of
cells with roles in wound healing is influenced by the pH of what, presumably,
is extracellular fluid. Despite its potential clinical relevance measurement
of wound pH is not routine, perhaps because to do so accurately requires
the use of calibrated meters which is not practical within the constraints
of a traditional wound management setting. We have developed a form of
hydrogel wound dressing that incorporates a colorimetric pH indicator
that provides a rapid mapping capability across the wound and which is
presently undergoing clinical trial. Moreover the indicator used in one
form of pH-sensitive dressing is a natural product with known antioxidant
properties and capable of controlled release.
This could be highly topical, in two senses, given recent observations
that conditions of localized oxidative stress, possibly related to neutrophil-associated
production of reactive oxygen species, are a feature of chronic leg ulcers
(James et al. 2003). Any effect that abnormal wound pH may have on endogenous
enzymatic activity would be superimposed on an already dysregulated system
of catalysts and their inhibitors (Trengove et al. 1999). The consensus
of published opinion on the implications of enzymatic dysregulation, notably
matrix metalloproteinases and neutrophil proteases, in chronic wounds
is that excessive activity (Rogers et al. 1995) and/or ineffective inhibition
(Bullen et al. 1995) is detrimental to healing. A similar aetiological
role for the aberrant expression of inflammatory mediators and growth
factors in the intractability of chronic wounds has been suggested (Trengove
et al. 2000), but to base a therapeutic strategy on correcting abnormal
levels of enzymatic activity (Greener et al. 2005) or growth factor expression
(Falanga, 1992), would first require technology capable of accurately
assessing their actual levels on a patient-bypatient basis within a meaningful
time frame in the wound care clinic. To address this technology vacuum
we have developed two hydrogel-based diagnostic systems called Gelisa™
and Zymogel™. Gelisa, which is intended primarily as a research
tool, is a refinement of the traditional ELISA based immunoassay but would
offer a mapping capability for the expression of molecules of interest
across a wound. The Zymogel system would offer a mapping capability together
with a semi-quantitative measure of enzymatic activity across a wound
and could be incorporated as a component part of a dressing designed principally
for other therapeutic purposes such as exudate absorbtion. Any treatment
which excessively diminished proteolytic activity in chronic wounds would
clearly be inappropriate, therefore re-establishing the normal levels
of activity could benefit from the use of technology such as Zymogel or
similar.
Smart dressings –
diagram of micro-engineered
hydrogel for cell-grafting.
From a purely functional
viewpoint a chronic wound represents a perpetual conduit for invasion
of body tissues by opportunistic micro-organisms that are otherwise held
in abeyance by the functional protective barrier that is the skin. Having
established a toehold, a bacterial presence may take several forms conditioned
in part by the host response from low-risk contamination to outright invasive
wound infection. Accurate identification and classification of abnormal
microbiological colonisation of broken skin is time consuming (Hill et
al. 2003) but is crucial given that inappropriate bacterial presence slows
wound healing (Dowsett, 2004). Incorrect diagnosis may lead to the inappropriate
use of potentially harmful antibacterial therapy (Howell-Jones et al.
2005) whereas excessive delay in confirming the type of bacteria may make
irradication more problematic. However, it has long been recognised that,
similar to the role of commensal bacteria on intact skin, a degree of
bacterial presence in wounds can be beneficial to healing although the
mechanisms underpinning this remain unclear (Edwards and Harding, 2004).
Ostensibly, as a foreign body it would seem logical to exclude bacteria
from the wound environment but the explanation for the paradoxical beneficial
effect of bacterial colonisation may lie in the nature of the subsequent
bacteria- host tissue interaction. Bacteria synthesise and release factors
capable of modulating the wound milieu (Teufel and, Gotz 1993) which invariably
frustrate the healing process but this, perhaps, is not the major problem.
Bacterial breakdown causes endotoxins to be released which, if present
at high enough concentration, can induce apoptotic cell death (Moazzam
et al. 2002) and cause tissue necrosis (Ovington, 2003). In the context
of wound healing bacterial endotoxin have also been found to inhibit fibroblast
migration and attachment within collagen gels (Pitaru et al. 1987) and
reduce the tensile strength of model surgical skin wounds in mice (Metzger
et al. 2002). However, recent findings have suggested that endotoxins
may play an important role in healing by indirectly stimulating upregulation
of cytokines such as VEGF, enzymes such as MMP- 3 and –9 (Warner
et al. 2004) and other transcriptional factors (Perfetto et al. 2003)
by fibroblastic cells. Moreover it is known that endotoxins can induce
cell division in fibroblasts and keratinocytes (Yang et al. 2002), possibly
through a toll-like receptor signalling pathway stimulating cell-cycle
entry (Hasan et al. 2005).
Excessive endotoxin is clearly damaging to tissues but limited quantities
may have a paradoxical facilitatory action on tissue regeneration. For
this reason we are developing a diagnostic hydrogel dressing capable of
rapid detection of endotoxin levels in wound exudate. The emergence of
such a capability would provide an objective measure of the consequence
of bacterial colonisation, that of hostile intent. Treatments options
could then be considered ranging from antibacterial therapy to dressings
capable of extracting and sequestrating bacteria from the wound milieu,
both of which are properties of the hydrogel used in the proposed diagnostic
applications. The separate phases of wound healing often described in
the literature must overlap, for example cell migration and connective
tissue remodelling. It is interesting therefore that reports have suggested
that the use of biosurgery (Beasley and Hirst, 2004), maggot therapy that
is, to debride wounds may also have a direct effect on cellular activity
within wounds (Horobin et al. 2003). Studies in our
own laboratory have shown that maggot secretions do indeed accelerate
the closure of ‘wounded’ epithelial and fibroblastic cell
monolayers in vitro and the mechanism underpinning this appears to be
a motogenic not a mitogenic effect. Despite its obvious clinical value
there are issues relating to patient acceptance of maggot therapy (Thomas
et al. 1988). This raises the question of whether it may be possible to
deliver maggot secretions topically onto a wound. Further experiments
carried out in our laboratory have confirmed that maggot secretions can
be released from hydrogel and other materials in a controlled fashion
whilst retaining its effectiveness on cell motility. Cellular migration
normally commences at an early stage in wound healing and, in the case
of keratinocytes, is oriented away from intact tissue in the direction
of the wound by population pressure. Contact guidance remains a theory
that has not yet been proven in vivo, but many reports have indicated
that the motility of human skin fibroblasts and keratinocytes can be accelerated
and oriented in vitro by motogenic substrata derived from extracellular
matrix (Sutherland et al. 2005). It is possible that this could be achieved
in acute and possibly chronic wounds by using the present hydrogel material
which can be microengineered with the necessary substratum-derived signals
to orientate cell division and migration whilst simultaneously acting
as a bridge across the wound bed. Experiments we have carried out using
micro-dissected rat vibrissae follicles in vitro have shown that putative
transit-amplifying cells emanating from a possible stem cell niche in
the follicular bulge (Morris et al. 2004), identified by expression of
stem cell integrins and keratin isoforms, are guided
by substratum topography (Thomas, 2005). This raises the possibility that
cells arising from stem cell niches in the skin could be encouraged to
re-seed damaged skin by an overlaid ‘smart hydrogel dressing’.
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| Above:
Fibroblasts growing on topographic substrate. |
Above:
Fluorescence image of cytoskeletal interaction with topographic
substrate. |
Above:
Keratinocytes growing on microengineered hydrogel. |
After severe burn injury, and also for chronic wounds, tissue-grafting
may be considered as a therapeutic option (Atiyeh et al. 2005). Most of
the reports describing grafting of cultured cells onto injury sites have
indicated that re-grafting may be necessary to establish viable colonies.
Shear and metabolic stress could explain why grafted cells often require
repeat application, which in turn infers that grafting devices which offer
some shielding against shear stress and buffering against metabolic stress
could improve the efficiency of the technique. We have found that human
keratinocytes and fibroblasts grafted onto burned (deepithelialised) human
dermis in vitro from media-hydrated hydrogel sheets incorporating protective
niches into the gel surfaces transfer cells very effectively (Britland
and Smith, 2005). There have been reports based on wound healing models
that re-epithelialization across meshed skin grafts is increased with
exposure to silver, suggesting that this also is underpinned by improved
cell viability. Our experience indicates that skin cells can remain viable
when grown in the proximity of hydrogel sheets containing antiseptics
and antibiotics.
To summarise, many useful strategies and products for healthcare applications
have and will emerge from the science of tissue engineering. However,
future deployment of tissue engineering technology, after scrutiny in
terms of cost-benefit analysis, may experience inertia unless it is certain
to be highly effective. Appropriate use of POC diagnostics technology
to inform the clinical decision to deploy a given tissue-engineered construct
should enhance its effectiveness. One thing those involved do not want
to see is tissue engineering joining the list of other technologies that
will forevermore remain full of potential.
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