EUROPEAN  TISSUE  REPAIR  SOCIETY

MATRIX FORMATION
Phil Stephens, Department of Oral Surgery, Medicine & Pathology, Dental School, University of Wales College of Medicine, Cardiff, UK

The wound repair process is an extremely complex yet highly orchestrated process of which matrix formation represents a crucial phase. The principle cell type involved in this activity is the fibroblast which migrates into the wound space using the provisional extracellular matrix (ECM) as a scaffold over which to relocate. Interaction with the ECM is facilitated via cell surface integrin recep-tors and migration is stimulated by growth factors/cyto-kines, the local ECM environment and fragments of damaged matrix. Migration is facilitated through a fine balance between control of the cytoskeleton and attachment to and dissolution from the surrounding ECM environment. Dissolution is brought about through the action of matrix metalloproteinases and their inhibitors along with the urokinase plasminogen activator/plasmin/plasminogen activator inhibitor pathway. As cellular migration occurs, growth factor/cytokine stimulated cellular proliferation commences such that within days fibroblasts, rather than macrophages, predominate within the wound space. Fibro-blasts then undergo a phenotypic alteration with the commencement of the production of new ECM/granulation tissue. Production of this nascent dermal tissue (e.g., collagens, proteoglycans, glycosaminoglycans and glycopro-teins) is under the control of numerous growth factors/cytokines and is a temporary matrix which will eventually be remodelled over a considerable period of time to form the mature scar tissue.

STEM CELLS AND HEALING

CORNEAL STEM CELLS IN OCULAR SURFACE REPAIR AND RECONSTRUCTION
Julie Daniels, Moorfields Eye Hospital, London, UK

The cornea provides the eye with protection and refractile properties essential for visual acuity. The transparent epithelium is highly specialised with basal cells and stratified squamous cells that are renewed throughout life from a stem cell (SC) population. The SCs are thought to reside at the outer corneal limbus. SCs, characterised by a long cell cycle, have extensive potential for error-free proliferation. A number of proteins including -enolase, EGF receptor, cytochrome oxidase and keratin 19 preferentially localise to slow cycling limbal basal cells. Asymmetric SC division produces one SC and one daughter transient amplifying (TA) cell. TA cells continue to divide replacing cells lost from the epithelial surface during homestasis. Following injury this process is up-regulated to replace the cell deficit unless the SC population is depleted, e.g., by an alkali burn. Limbal SC deficiency results in re-epithe-lialisation of the cornea by the neighbouring conjunctival epithelium causing pain, poor vision and even blindness. Corneal SCs can be cultured and grafted with encouraging patient results. However, whilst SC deficiency is recognised clinically and purposed SC can be grafted, a definitive corneal epithelial SC marker remains elusive. This enigma may be explained by recent research suggesting that the term 'stem cells' most accurately describes a biological function rather than a distinct cell type.
JTDaniels is funded by the Royal National Institute for the Blind.

THE UTILITY OF VARIOUS CHONDROPROGENITOR CELLS AND THEIR CARRIER SYSTEMS IN CARTILAGE REPAIR
Ernst B. Hunziker, Müller Institute for Biomechanics, University of Bern, Switzerland

Adult human articular cartilage, being avascular, alym-phatic and aneural, has a very limited potential to repair spontaneously. Lesions confirmed to articular cartilage fail completely in this capacity, and even those that penetrate the vascularised layer of subchondral bone heal poorly, the repair cartilage formed being qualitatively inferior to native tissue and of limited durance.
Recent efforts to improve the quality and durability of repair cartilage have focused on tissue engineering approaches. Such strategies involve the introduction of a matrix into the lesion void. A number of materials have been employed for this purpose, including polylactic acid, polyglycolic acid, hyaluronan, fibrin and collagen. In most instances, the matrices are applied together with undifferentiated or differentiated chondroprogenitor periosteum, perichondrium, cartilage or synovium. In this presentation, the features desired of an ideal carrier system are delineated and the problems encountered with those currently available discussed. The usefulness of various chondroprogenitor cells, their mode of application and the conditions that need to be established for a successful outcome are also dealt with. Experimental data will be analysed in the light of its clinical applicability and the current status of clinical treatment strategies.

REPAIR IN THE RENAL TRACT

ROLE OF DECORIN IN RESPONSE TO KIDNEY INJURY
Liliana Schaefer, University of Munster, Germany

Glomerulosclerosis and interstitial fibrosis are uniform end-stage features of chronic kidney injury. Decorin, a small dermatan sulfate proteoglycan, has successfully been used to treat fibrotic disorders with TGF-b- overproduction. The underlying mechanisms, however, remain unclear since upon complex formation with TGF-b- the cytokine's activity may become increased, decreased or not influenced at all. Based on our studies in human diabetic nephropathy, we postulate two other mechanisms by which decorin modulates TGF-b- mediated injury:
1) Increased quantities of glomerular decorin are synthesized and form complexes with TGF-b-, which are then removed via glomerular capillaries or the urinary tract.
2) In the presence of type-I collagen decorin is able to sequester TGF-b- in the extracellular matrix, thereby withdrawing the cytokine from its cell surface receptors.
Following unilateral ureteral obstruction, a model of tubulointerstitial injury, in decorin-/- mice additional effects of decorin were discovered. In the absence of decorin the diseased kidneys showed an earlier and more pronounced induction of caspases and a higher degree of apoptosis of tubular epithelial cells together with a more pronounced accumulation of macrophages. Based on these findings, it appears that decorin may play a much more complex role in inflammation and tissue repair as previously thought.

HA IN THE KIDNEY: FRIEND OR FOE?
Aled Phillips, Institute of Nephrology, University of Wales College of Medicine, Cardiff, UK

Hyaluronan (HA) is a ubiquitous connective tissue polysaccharide which in vivo is present as a key component of the extracellular matrix. It has mow clear however that alterations in HA generation play an important role in wound healing. In the normal kidney HA and its major receptor CD44, are expressed predominantly in the interstitium of the renal papilla. Numerous studies have demonstrated that the expression of both HA and its receptor CD44 may be altered during renal disease, thus raising the question as to the source and the function of HA at this sites in these models of renal injury?
We have demonstrated increased HA synthesis by human renal proximal tubular cells in response to either IL-1b, to mimic the inflammatory state, or elevated 25 mM D-glucose, to mimic the diabetic state. This increased generation of HA was associated with a change in the molecular weight distribution of the HA generated. Stimulation with either IL-1b or 25 mM D-glucose also resulted in increased expression of the functional-HA binding form of the HA receptor CD44, the net result being increased binding of low-molecular weight HA to the cell surface.
An interstitial macrophage influx has been implicated in the pathogenesis of interstitial fibrosis related to progressive renal disease of diverse aetiology. HA induces monocyte chemoattractant protein-1 expression and up-regulation of ICAM-1 and VCAM-1 in renal tubular epithelial cells. Significantly both adhesion molecule up-regulation and induction of chemokine synthesis in PTC were stimulated by low molecular weight HA polysaccharies such as those stimulated by exposure to elevated D-glucose concentrations. This suggests that there may be a positive feedback loop by which inflammation stimulates HA generation which may then amplify the inflammatory response by increasing recruitment of inflammatory cells to the site of injury. In vitro ar least these responses to low molecular weight HA may be abrogated by addition of high molecular weight forms of HA therefore suggesting that 'HA, friend or foe', is dependent not only on the amount of the HA generated but also on its physical characteristics.

FIBROSIS

CONNECTIVE TISSUE GROWTH FACTOR IN RENAL INJURY
Roger Mason, Imperial College of Medicine, London,UK

Connective tissue growth factor (CTGF) is a secreted monomeric protein of molecular mass 36-38 kDa and belongs to the CTGF, Cyr61/Cef10, and NOV family of growth regulators (CCN). Members of this family have four conserved domains; an IGF-binding protein domain, a Von Willebrand factor type C domain, a TSP-1 type 1 repeat domain and a heparin binding domain at the C-terminus. CTGF expression is activated by serum growth factors, TGFb, BMP2, dexamethasone, thrombin, lysophosphatidic acid, 5-hydroxytryptamine, high glucose and mechanical strain. The physiological role of CTGF has not been fully elucidated but it may be involved in the pathogenesis of fibrosis, possibly acting as a mediator of TGFb. Increased CTGF expression has been reported in inflammatory bowel disease, scleroderma and systemic sclerosis, renal fibrosis, liver fibrosis, idiopathic pulmonary fibrosis and pulmonary sarcoidosis. We have investigated CTGF expression in diabetic nephropathy in man and in the non-obese diabetic (NOD) mouse. Marked increases in CTGF protein occur in the glomeruli compared to non-diabetic kidneys. Transfection of transformed human glomerular mesangial cells stimulated increased fibronectin and plasminogen activator inhibtior-1 expression. Transfection with a CTGF antisense construct reduced elevated synthesis of these proteins in cells maintained in high glucose. Likewise, an anti-CTGF neutralising antibody and CTGF antisense oligonucleotide reduced elevated fibronectin synthesis in normal mesangial cells cultured in high glucose. CTGF is likely to be a key mediator of glomerulosclerosis in the diabetic kidney.

PROTEOGLYCANS AND MYOFIBROBLAST TRANSFORMATIONS
Bob Steadman, Institute of Nephrology, University of Wales College of Medicine, Cardiff, UK

Myofibroblasts are the principal cells implicated as the source of the expanded matrix in fibrotic disease and wound healing. Matrix synthesis by these cells is under the control of a variety of growth factors, such as the family of fibroblast growth factors (FGF). The response of the cells to many of these, however, is mediated through a primary interaction with cell surface and matrix-associated proteoglycans, through their glycosaminoglycan (GAG) side chains. Specific sequences within heparan sulfate (HS) GAG chains mediate FGF interaction with its receptor. We have specifically focussed on differences in HS structure between fibroblasts and myofibroblasts which may affect the responses of the cells to FGF.
Myofibroblasts incorporated approximately twofold more label into secreted, and extracellular matrix proteo-glycans (PG) than did fibroblasts. In contrast, there was a decreased incorporation of label into cell surface HSPGs. This was associated, however, with an increase in the size of their HS GAG chains. Functionally this difference was associated with a loss of the proliferative response of myofibroblasts to FGF-2 but not to PDGF or FCS. The detailed structure of these chains, therefore, was investigated by selective digestion with specific heparinases. While sequences of low sulfation were longer in fibroblast HS chains, there was no difference, in the total disaccharide composition of the chains. HS chains from myofibroblasts, however, had two compared to one potential FGF-2 binding site. Nevertheless there was, no difference between the HS chains in the binding affinity for FGF-2. The level at which the differences in HS GAG structure affect the response to FGF-2 is currently under investigation.