Keynote Lecture:
Developing the role of scientists in the Health Service

Professor Stephen Holgate

Southampton

We are living in times where application of science and technology to human health has never been greater. However, the workforce capacity to take on this challenge in our health services (of which the NHS is an example) is under great pressure, raising concerns that the scientists themselves will become frustrated and burdened by change, not supported by capacity and career development. Much of the emphasis on health service reform has focused on targets in specific disease areas, close to patient delivery of care, increased speed to diagnosis and treatment, rapid mobilisation after interventions such as surgery and evidenced- based practice. Much of the workforce emphasis has been around medical and nursing staffing in primary and secondary care. However, the health scientist has largely been ignored in this transformation agenda. Indeed, health scientists are increasingly being asked to meet service delivery targets but are not as engaged as they wish to be in the evaluation and uptake of new technologies. There is also an issue over career development and postgraduate training which has to be de-emphasised in the NHS to drive greater ‘productivity’! Scientists frequently feel disconnected from the other professionals linked to delivery of healthcare. Deskilling and reducing costs for providing pathology, physiology and imaging services are intimately related and require urgent action to revitalise the field and create the level of ownership that will secure a productive and rewarding future. Central to this is investment by the health scientists themselves to take greater ownership of their specialty fields and to work with others to enhance multidisciplinary and team working for the benefit of patients.

Plenary Lecture, Wednesday 26 September:
Skeletal Tissue Engineering - Harnessing Interdisciplinary Interactions from the laboratory to clinical translation

Richard OC Oreffo

Bone and Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Institute of Developmental Sciences, University of Southampton, Southampton, SO16 6YD, UK

Overview: Given the demographic challenges of an ageing population combined with rising patient expectation and the growing emphasis placed on cost containment by healthcare providers, economic regenerative medicine approaches for skeletal regeneration is a major clinical and socio-economic need. Mesenchymal stem cells or human bone marrow stromal stem cells are defined as multipotent progenitor cells with the ability to generate cartilage, bone, muscle, tendon, ligament and fat. These primitive progenitors exist postnatally and exhibit stem cell characteristics, namely low incidence and extensive renewal potential. These properties in combination with their developmental plasticity have generated tremendous interest in the potential use of mesenchymal stem cells to replace damaged tissues.

To date, relatively little is known concerning the phenotypic characteristics, whether from a morphological or biochemical standpoint whilst direct in vivo confirmation of the lineage potential and plasticity or interconversion potential that exists of mesenchymal stem cells and osteogenic progenitor cells remains. Nevertheless, strategies harnessing tissue engineering approaches offer much promise for skeletal regeneration using mesenchymal populations. Upon isolation of an appropriate progenitor population, repair and reconstruction of bone defects present additional challenges to the orthopaedic, reconstructive and maxillofacial surgeon including an ability to generate a functional microvascular network within engineered constructs to provide oxygen and nutrients that facilitates growth, differentiation, and tissue. The development of a functional vasculature is critical in respect to bone defects which can be extensive. To achieve this goal of skeletal regeneration, it will be necessary to harness the skills set from a variety of disciplines and there will be a need for close interactions between modelers, physical scientists, tissue engineers and clinicians.

Our work is centered on isolation, expansion and translational studies of humans fetal and adult mesenchymal populations for skeletal repair. Three areas of work will be reviewed using data from the group including:

1. isolation, expansion and characterisation of the plasticity of fetal and adult mesenchymal populations,
2. combination of progenitor cells with tailored growth factor containing biomimetic polymer scaffolds in an attempt to modulate the phenotype of the mesenchymal populations to generate mineralised bone tissue, and
3. translational studies to examine the efficacy of mesenchymal populations using impaction bone grafting as an exemplar.

The development of protocols, tools and, above all, multidisciplinary approaches for de novo bone formation that utilise marrow stromal cells and enriched mesenchymal populations containing mesenchymal stem cells may improve the quality of life for many as a result of strategies to augment skeletal regeneration.

Acknowledgements: The author gratefully acknowledges the BBSRC and EPSRC for support. The work presented and many useful discussions are derived from past and current members of the Bone and Joint Research Group as well as fruitful collaborations with Professors Shakesheff & Howdle (University of Nottingham), Professor Stephen Mann (University of Bristol), Professor Julian Chaudhuri (University of Bath), Professor Bruce Caterson (University of Cardiff) and Professor Walter Sebald (University of Wurzburg).

Plenary Lecture, Thursday 27 September:
The Matrix Metalloproteinases, from then to now

Charles M. Lapière

Presented by Professor Alain Colige
Laboratory of Connective Tissues Biology, University of Liège, Belgium

Then in 1962: the concept of Matrix Metalloproteinase was already established by the observation of a proteolytic (protease) activity able to degrade collagen (matrix) in its native triple-helical configuration. This enzyme was produced by resorbing tissues and required divalent ions (metallo). From 1962 to 1980 many other matrix degrading metalloproteinases were described. They were classified on the basis of a signature made of a typical zinc-containing catalytic site and a nearby methionine to form the family of the metzincins. The range of functions also extended upon the discovery of different lineages of proteases in the family (MMPs, ADAMS, ADAMTS, …). The biological function of several of the MMPs and other metalloproteinases was rapidly recognized in wound repair and regeneration, cancer, inflammation and development.

Now in 2007: the full description of the human genome has completed the list of the existing metzincins that contains 194 members. It appears that the range of activity of several of these enzymes outpasses the limits of their initial functions by recognizing additional substrates. As observed in many proteins, sequences of their gene encode for domains that participate in the control of important biological processes. The example of the ADAMTS 2, 3 and 14 in different aspects of wound healing will illustrate the concept.

Plenary Lecture, Friday 28 September:
Studying wound healing and inflammation in genetically tractable organisms

Prof Paul Martin

Departments of Physiology and Biochemistry,
School of Medical Sciences, University of Bristol

Embryos heal wounds very rapidly and efficiently and without leaving a scar. Studying how they do this can tell us much about the natural morphogenetic movements of embryogenesis as well as suggesting ways in which we might make adult tissues repair more efficiently. We observe a very reduced recruitment of inflammatory cells to sites of tissue damage during embryonic repair which hints that maybe inflammation is causal of fibrosis. Indeed, our studies of tissue repair in the neonatal PU.1 null mouse, which is genetically missing all leukocytic lineages, supports this speculation since PU.1 null wounds heal efficiently without significant fibrosis. Consequently, we have used a microarray approach with this mouse in order to identify a portfolio of candidate inflammation/fibrosis genes. In vitro co-culture experiments have suggested that, of the leukocytic lineages missing in the PU.1 null mouse, macrophages are the most likely source of the ‘fibrosis’ signal. We have begun a series of antisense ODN ‘knockdown’ experiments in wild-type wounds to test which of the candidate ‘fibrosis’ genes induced in wound fibroblasts after signals from inflammatory cells may indeed be causal of wound fibrosis. Finally, we have established models of inflammation in the Drosophila embryo and in the translucent zebra fish larval tail, which allow us to make DIC movies of the inflammatory response and to dissect the genetics of inflammation, in particular the signaling and cytoskeletal elements required to guide migration of macrophages towards wound sites; these data provide precise role for the various small GTPases and for their effectors, e.g., WASP, during in vivo inflammatory cell migrations.

Invited Speaker:
Measuring Wound Outcomes: Is this a recipe for growth?

David J. Margolis MD PhD

Departments of Biostatistics and Epidemiology and Dermatology, University of Pennsylvania School of Medicine, Philadelphia PA 19104, USA

Clinical trials of chronic wounds are limited by the one universally accepted outcome, a healed wound, and the prolonged period of time required reaching this outcome. This solitary outcome is often measured 12 to 24 weeks after the initiation of therapy. It is expensive to achieve this outcome in a clinical trial setting and disregards other important outcomes including improvements in the patient’s quality of life or of their wound related pain. A ‘healed’ outcome also prevents discovering new therapies that augment or prepare a wound for other therapies such that a second modality might become more effective. A ‘healed’ outcome certainly diminishes the impact that an improved wound has on the ease of self-administered care. One approach to discovering new outcomes is to better understand the natural history of chronic wounds and the association of surrogate markers and/or intermediate outcomes to a ‘healed’ outcome. We have studied more than 40,000 individuals with diabetic neuropathic wounds or venous leg ulcers in many different clinical settings. We have demonstrated that improvements in these wounds during the first four weeks of care are predictive of whether they will ultimately heal. These wound parameters include the percent change, log rate of change, and the ratio of log areas estimated by comparing the wound at baseline and four weeks later. These parameters can correctly classify wounds 70% of the time. Others have shown that changes in the first four weeks of care can be predictive of clinically whether a wound is ready for a skin graft and that changes in the size of a wound can also be important for predicting the success of treating pressure ulcers. Recent work has also demonstrated genomic differences between cells in the wound and the pen-wound tissue that may be indicative of a ‘genomic’ fingerprint of a healing wound. As a result, it is very likely that in the near future many outcomes and surrogate markers will be used to help guide healthcare providers as they help a patient care for their chronic wound.

Young Investigator Awards - Overall Winner (oral):
Hyaluronan facilitates TGFß1 mediated myofibroblastic differentiation

S. Meran¹, R. Steadman¹, A.O. Phillips¹, D.W. Thomas² and P. Stephens².

1. Institute of Nephrology, School of Medicine, and
2. Wound Biology Group, School of Dentistry

Cardiff University, Cardiff, CF14 4XN

Introduction:

The oral mucosa is distinct in that it demonstrates preferential healing with little/no scarring. In skin/internal organs, repair by scarring can lead to fibrous tissue accumulation, resulting in disability and organ dysfunction. Fibroblast to myofibroblast differentiation plays an important role in scarring and is associated with an accumulation of the matrix polysaccharide hyaluronan (HA). This study aims to understand the role of HA in influencing scarring by comparing its metabolism in fibroblasts with a non-scarring phenotype (oral mucosal fibroblasts; OMF) to patient-matched dermal fibroblasts (DF).

Methods:

OMF and DF (n = 4) differentiation to myofibroblasts was induced by TGFß1-stimulation and assessed by alpha-smooth muscle actin expression (QPCR/immunocytochemistry). A range of techniques was used to assess HA generation, assembly, HA Synthase (HAS) expression, Smad-signalling and proliferation including metabolic radio-labelling, column chromatography, western blots, 3H-thymidine incorporation and microarray analysis. HA synthesis was inhibited using 4-methyl-umbelliferone depletion of UDP-glucuronic acid and siRNA technology was used to down-regulate Smad3 expression.

Results:

The fibroblast populations demonstrated distinct differences in their TGFß1-responses. DF readily differentiated to myofibroblasts and demonstrated a proliferative response to TGFß1, whereas OMF were resistant to differentiation and demonstrated an anti-proliferative response to TGFß1. In DF, differentiation was associated with increased HA generation, HA pericellular coat assembly and increased HAS1&2 transcription. In contrast, resistance to differentiation in OMF was associated with reduced HA generation and inability to induce pericellular coat assembly or HAS1&2 transcription. Inhibiting HA synthesis in DF significantly attenuated differentiation, induced an anti-proliferative response to TGFß1 and attenuated Smad3-signalling. IL-1ß stimulation in DF induced HA synthesis and HAS1&2 transcription, however, it did not induce pericellular coat assembly or differentiation, suggesting that HA coat assembly is specifically required for differentiation. Both fibroblast populations demonstrated Smad3 phosphorylation; however, down-regulation of Smad3 (siRNA) led to loss of the TGFß1-mediated proliferative response in DF whereas it led to loss of the TGFß1- mediated anti-proliferative response in OMF.

Discussion:

Both fibroblast phenotypes respond to TGFß1. However, the levels of HA generated by the cells influences the outcome of this response such that increased HA facilitates TGFß1-mediated proliferation and differentiation. This represents an important target for future anti-scarring research.

Young Investigator Awards - Finalist (oral):
Myofibroblast Contraction Activates TGF-beta1 from the Extracellular Matrix in a Stress-Controlled Autocrine Loop

Pierre-Jean Wipff, Jean-Jacques Meister, and Boris Hinz

Laboratory of Cell Biophysics, Ecole Polytechnique Federale de Lausanne, Switzerland
This work was supported by Swiss National Science Foundation grants #3100A0-102150/1 and #3100A0-113733/1.

Introduction:

Mechanical stress and active TGFß1 are essential to convert fibroblasts into highly contractile myofibroblasts but a direct link between both factors was not yet established. Myofibroblasts deposit TGFß1 in the extracellular matrix (ECM) as large latent complex (LLC), further consisting of the latency associated protein and the latent TGFß binding protein-1. Different proteolytic and non-proteolytic mechanisms have been described to activate TGFß1 from the LLC. We here report myofibroblast contraction as a novel mechanism of TGFß1 activation that is controlled by ECM stiffness.

Material and methods:

Myofibroblasts were cultured for 7d, leading to enrichment of LLC in the ECM. To assess whether intracellular tension promotes TGFß1 activation, cells were treated with agonists and antagonists of cell contraction and of integrins. The minimal requirements for TGFß1 stress-activation were determined with Triton X-100-cytoskeletons and desoxycholate-ECM. Such cultures were subjected to 3-10% external stretch and contracted with ATP. To test whether ECM stiffness modulates TGFß1 activation, myofibroblasts were grown on silicone substrates with tunable compliance. Active TGFß1 was assessed using mink lung epithelial reporter cells, producing luciferase in response to TGFß1 (gift of D.B. Rifkin). In vivo, phosphorylation of TGFß1 downstream targets Smad2/3 was compared between stretched and relaxed myofibroblast-rich tissues.

Results:

Inducing contraction of myofibroblasts and of isolated cytoskeletons releases active TGFß1 from the ECM; this is inhibited by antagonizing integrins, by cell-relaxing drugs and by reducing ECM compliance. Stretching latent TGFß1-conditioned ECM in the presence of mechanically apposing stress fibers induces immediate activation of TGFß1. Moreover, activation of TGFß1 downstream targets Smad2/3 is higher in stressed compared to relaxed myofibroblast-rich tissues despite similar levels of total TGFß1 and its receptor.

Discussion:

We propose mechanical activation of TGFß1 as a checkpoint in the progression of fibrosis by restricting autocrine maintenance of myofibroblasts to a sufficiently remodeled and stiffened ECM.

Young Investigator Awards - Finalist (oral):
The ELR-Negative CXC Chemokine CXCL11 (IP-9 or I-TAC) facilitates Dermal and Epidermal Maturation during wound repair

Cecelia C. Yates, Diana Whaley, Priya Kulasekaran, Joseph Newsome, Patricia Hebda and Alan Wells.

Department of Pathology and Otolaryngology, University of Pittsburgh, and Pittsburgh VAMC.

In skin wounds, the regeneration of the ontogenically distinct mesenchymal and epithelial compartments must proceed coordinately to restore functionality. Orchestration of repair is conducted by signals including growth factors, chemokines, and matrix components. We recently postulated a novel model in which ELR-negative CXC chemokines (PF4/CXCL4, IP-10/CXCL10, MIG/CXCL9, and IP-9/CXCL11) that bind the common CXCR3 receptor limits or modulates fibroblast and keratinocyte responses to other signals. The results obtained in the absence of the CXCR3 signaling network in vivo during wound repair revealed a significant delay in dermal healing and the reepithelialization process. However, the key communicating protein involved has yet to be determined as several ELRnegatives CXC chemokines bind to the CXCR3 receptor.

We hypothesized that IP-9 produced by the redifferentiating keratinocytes mediates matrix maturation. We generated an antisense to IP-9(IP-9AS) mouse model. Full and partial thickness excisional wounds were created on both IP-9AS and WT mice and were histologically analyzed at days 7, 14, 21, 30, and 60. The wounds in the IP-9AS transgenes failed to present detectable IP-9 protein during the wound healing process, in distinction to robust IP-9 production during the regenerative phases of wound healing in wild type mice. Wound healing was impaired in the IP-9AS mice, with hypercellular and immature dermis being noted even as late as 60 days after wounding; there was delayed remodeling of the collagen in the dermis. Re-epithelialization was delayed by about seven days in comparison to the wild type mice. Even after epithelial coverage of the wound, the delineating basement membrane was ill-constructed. Provisional matrix components persisted in the dermis, and the mature basement membrane components laminin V and collagen IV were severely diminished. We conclude that IP-9 is the key ligand in the CXCR3 signaling system that promotes re-epithelialization, modulate dermal maturation and production of a mature matrix.

Young Investigator Awards:
Role of bacterial biofilms in chronic wounds

A. K. Deva, Q. Ngo and K. Vickery

University of New South Wales, Sydney, Australia

Purpose:

Bacterial biofilms are highly organized microbial communities living within a protective extracellular matrix. They are difficult to detect and highly resistant to immune or antibiotic killing. It has been suggested that biofilm presence may contribute to the intractable inflammatory processes seen in chronic wounds.

Method:

Twelve chronic wound samples from eight different patients were collected as part of routine debridement. These were examined for biofilm presence using light microscopy, confocal laser scanning microscopy, electron microscopy, and Fluorescent In Situ Hybridisation (FISH) technique. Microbiological profiles were established based on wound swabs.

Results:

Histological and microscopic evidence of bacterial biofilms were observed in seven out of twelve wounds. These were mostly found within the necrotic upper layer of the wound. A range of organisms were identified from swabs including the commonly isolated wound bacteria S.aureus, E.Coli, P.aeruginosa, and mixed anaerobes.

Conclusion:

Bacterial biofilms were found in almost twothird of chronic wounds examined. Given the patchy nature of its occurrence and the small area sampled in each biopsy, the true incidence could be much higher. The necrotic surface layer of wounds appeared to be more conducive to biofilm formation than deeper viable tissues. Such biofilms may then serve as niduses for continual bacterial seeding that perpetuate the inflammatory process seen in these wounds.

Poster Winner (A. K. Deva):
Effects of combined topical negative pressure (TNP) and antiseptic instillation on pseudomonas biofilm

A. K. Deva, Q. Ngo and K. Vickery

University of New South Wales, Sydney, Australia

Purpose:

TNP in the form of V.A.C. dressings has been successfully used to enhance chronic wound healing. An in vitro model of the chronic wound biofilm was created to test the effects of TNP with and without concurrent antimicrobial drug instillation on biofilms.

Method:

A P.aeruginosa biofilm model was constructed in vitro. Continuous TNP and TNP combined with varying frequencies of betadine instillation were tested on the biofilm model for 24 hours. Outcome parameters measured included viable bacterial count, fluorescent microscopy, confocal laser scanning microscopy, and electron microscopy. Statistical analysis was performed using ANOVA and MANOVA in SPSS 13.0.

Results:

Bacterial biofilms, when exposed to TNP alone, showed a 42% decrease in viable count; while exposure to betadine alone showed a reduction of up to 84%. Combined TNP and antiseptic instillation increased the bacteriocidal effect to 99.7% (p < 0.001). Exposure to TNP also resulted in a decrease in biofilm thickness, and diffusion distance, with an increase in surface area-to-volume ratio (p = 0.005, 0.002, and 0.009 respectively).

Conclusion:

TNP alone showed a modest effect on the reduction of bacterial biofilm. More importantly it appeared to compress and fragment the overall biofilm architecture. This physical deformity in turn led to more effective drug penetration into the highly resistant biofilm community. The result was an approximate 100-fold enhancement of bacteriocidal effect when betadine instillation was combined with TNP as compared to betadine alone.