EUROPEAN  TISSUE  REPAIR  SOCIETY

Some of the Young Investigators Award and Poster Prize winners receiving their prizes
from Mark Ferguson and Esther Middelkoop, at the 13th Annual ETRS Meeting, in Amsterdam

STIMULATION OF SKIN REPAIR DEPENDS OF FIBROBLAST SOURCE

E N Lamme1, H J Wang2, J Pieper3, R Schotel3 and C A van Bliterswijk3
1UMC St Radboud, Dermatology, Nijmegen; 2Twente University, Institute of Biomedical Technology, Enschede, The Netherlands; 3Isotis SA, TE, Bilthoven, The Netherlands

Objectives: In this study, in vitro and in vivo function was compared of cultured dermal equivalent produced with fibroblasts either isolated from papillary dermis or adipose tissue.

Methods: Papillary and adipose fibroblast populations were seeding in synthetic PEGT/PBT scaffolds and cultured for 14 days. Cultured constructs were analysed biochemically for total collagen and GAG content and histologically. Simultaneously, these constructs + acellular scaffolds were implanted in athymic mouse full thickness wounds to evaluate angiogenesis (von Willebrand staining) and re-epithe-lialization after 10 and 21 days.

Results: Cell numbers in papillary and adipose fibroblast cultured constructs were comparable, but tissue morphology was different. Papillary-fibroblast had deposited significantly more GAG (214± 15 versus 159& plusmn; 1mg) and lower amounts of collagen (49& plusmn; 14 versus 111± 25 mg hydroxyproline) than adipose fibroblasts. Moreover, the latter constructs were significantly more contracted (78&Plusmn; 6 versus 96± 3%). The transplantation of acellular scaffolds, papillary and adipose-fibroblast-cultured constructs showed differences in angiogenesis and tissue growth after 10 days and in re-epithelialisation after 21 days. The level of vascular ingrowth was: adipose-fibroblast-cultured papillary-cultured acellular carrier. Complete wound re-epithelialisation (92± 12%) was only observed in the adipose fibroblast group. Wound contraction was not observed. Staining for HLA-ABC and a-smooth muscle active showed that human fibroblast had survived and only adipose fibroblasts expressed the actin isoform.

Conclusions: Fibroblast tissue source (or phenotype) and presence of ECM play a crucial role in the stimulation of (impaired) healing and engineering of dermal equivalents.

COLLAGENASE-3 AND TISSUE INHIBITOR OF MATRIX METALLOPROTEINASES-2 IN FIBROBLAST-MEDIATED COLLAGEN MATRIX REMODELLING

M Toriseva1, R Ala-aho2, A H Baker3, V Marjomaki4, J Heino5 and V-M Kahari6
1University of Turku and Abo Akademi, Turku Centre for Biotechnology, Turku, Finland, +358 (0)2333 8024, +358 (0)2333 8000, mervi.toriseva@btk.utu.fi; 2University of Turku and Abo Akademi, Turku Centre for Biotechnology, Turku, Finland, +358 (0)2333 8025, +358 (0)2333 8000, risto.ala-aho@btk.utu.fi; 3,University of Glasgow, Dept of Medicine and Therapeutics, Glasgow, UK, +44 (0)141 211 2100, +44 (0)141 211 1763, ab11f@clinmed.gla.ac.uk; 4,University of Jyvaskyla, Dept of Biological and Environmental Science, Jyvaskyla, Finland, +358 (0)14 260 2273, +358 (0)14 260 2271, vmarjoma@cc.jyu.fi; 5,University of Jyvaskyla, Dept of Biological and Environmental Science, Jyvaskyla, Finland, +358 (0)14 260 2240, +358 (0)14 260 2271, jyrki.heino@utu.fi; 6,University of Turku and Abo Akademi, Turku Centre for Biotechnology, Turku, Finland, +358 (0)2 333 8029, +358 (0)2 333 8000, veli-matti.kahari@btk.utu.fi

Background: Collagenase-3 (MMP-13) is a member of matrix metalloproteinase (MMP) family. It is not present in normal adult skin wounds but it is expressed by fibro-blasts in non-scarring adult gingival and fetal skin wounds.

Objective: We have studied the role of MMP-13 and tissue inhibitors of MMPs (TIMPs) in fibroblast-mediated contraction and remodelling of collagen matrix, a process that occurs during acute wound healing.

Methods: WE have used adenovirus-mediated gene delivery of MMP-13 and TIMP-2 to human adult skin fibro-blasts and studied their contraction capacity in a floating 3D collagen lattice.

Results: Adenoviral expression of human MMP-13 by dermal fibroblasts increased their collagen contraction capacity by 60% when compared to fibroblasts infected with a control virus. Addition of human recombinant MMP-13 to the medium of uninfected cells had a similar effect in a concentration dependent manner. The migration of fibroblasts on collagen was not affected by MMP-13 expression. Adenoviral expression of TIMP-2 reduced contraction by 50% and potently inhibited the activation of gelatinise-A (MMP-2). The morphology of TIMP-s expressing cells was rounded suggesting defects in cytoskeleton function or interaction between cells and surrounding matrix. The viability of MMP-13 and TIMP-2-expressing fibroblasts was not affected.

Conclusions: The results suggest that MMP-13 induces the reorganisation of collagen matrix by fibroblasts. Thus, it may enhance the wound closure or even promote scarless healing. The inhibitory effect of TIMP-2 expression on collagen lattice contraction suggests a role for MTI-MMP or MMP-2 in the process. That is, they may also be involved in regulation of collagen contraction by dermal fibroblasts in vivo during cutaneous wound healing.

DYNAMIC CHARACTERISATION OF THE MOLECULAR EVENTS CONTROLLING DERMAL FIBROBLASTS PROLIFERATION DURING WOUND HEALING
A A Chasson1, L Turchi2, J P Ortonne3, V Dulic4 and G Ponzio1
1INSERM, U385, Nice, France, +33 49 337 7790, +33 49 381 1404, chassot@unice.fr ; 2INSERM, U385, Nice, France, +33 49 337 7743, +33 49 938 11404, turcg@unice.fr ; 4INSERM, U385, Nice, France, CNRS-CRBM, UPR 1086, Montpellier, France, +33 046 761 3337, +33 046 752 1559, dulic@crbm,.cnrs-mop.fr ; 5INSERM, U358, Nice, France, + 33 049 337 7790, +33 048 11404, ponzio@unice.fr

Background: Skin wound healing is a complex process allowing the reconstruction of epidermal and dermal lesions caused by mechanical, chemical or thermal agents. Many genetic or acquired defects can perturb the normal wound healing leading to pathologies like hypertrophic scars or keloids, in which fibroblasts undergo an abnormal proliferation.

Objectives and methods: However, the molecular abnormalities at the origin of these pathologies are still unknown notably because the experimental systems used to study wound healing do not allow an exhaustive molecular analysis. To circumvent this hurdle, we have developed an original device that performs calibrated injuries of great length within confluent cell cultures or reconstructed dermis and skins, and thus enables the detection of a wide range of molecular events activated during wound healing.

Results: We demonstrate that mechanical lesions performed within quiescent dermal fibroblasts stimulate the cell proliferation. This stimulation correlates with 1) the up-regulation of cyclins D1, E, A and B1, 2) the inhibition of p27 Kip1, and consequently the stimulation of CDK4 and CDK2 kinase activities leading to the phosphorylation of pocket proteins family members. We show that the stimulation of cyclin D1 expression results from a transcriptional mechanism, whereas p275Kip1 decrease is regulated by two different and specific processes. In early G1 phase, p27Kip1 decrease results from a down-regulation of its mRNA. In G1/S phase, p27Kip1 is degraded via a protea-some-dependent pathway after being phosphorylated by cyclin A/CDK2 complexes. In conclusion, we propose a coherent scheme describing, for the first time, the cascade of events that initiate and regulate the fibroblasts proliferation during the dermal repair in vitro.

ALTERED CROSSLINKING AND THERMAL STABILITY OF COLLAGEN IN CHRONICALLY ISCHAEMIC SKIN

S J Dalton1, C V Whiting1, D C Mitchell2, J R Tarlton1
1 Bristol University, Matrix Building, Bristol, UK,
2 Southmead Hospital, Surgery, Bristol, UK.

Introduction: Dermal breakdown and impaired healing occurs in the ischaemic skin of patients with peripheral vascular disease (PVD), however, no mechanism for such failure has been established. Collagen metabolism and homeostasis is sensitive to changes in oxygen tension manifest in ischaemia. We hypothesise that in such skin, changes in collagen will occur which may predispose to weakening of the extracellular matrix and subsequent dermal breakdown.

Methods: Following quantification of ischaemia, paired biopsies of uninjured nonischaemic and ischaemic skin were harvested at view knee amputation from 16 patients with PVD. Age and site matched controls were taken at knee replacement and varicose vein operations. Collagen type I synthesis was assess by immunoassay and collagen content by determining hydroxyproline levels. Crosslinks were measured amino acid analyser. Differential scanning calorimetry was utilised to examine the thermal properties of the collagen and distribution of type I and III by immunohistochemistry.

Results: Collagen synthesis was increased in ischaemia (p<0.001) however no net change in total collagen was identified. Crosslinking profile revealed a decrease (p<0.05) in the intermediate crosslink hydroxylysinonorleucine and an increase in hydroxypyridinolione (p<0.01) when compared to non-ischaemic skin and controls. DSC revealed significant differences in the thermal stability of collagen molecules within the ischaemic population.

Discussion: Chronic ischaemia is associated with increased collagen turnover, altered crosslinking and a change in the thermal stability of collagen which may predispose to tissue breakdown and subsequent impaired healing, particularly when compounded with other problems of matrix homeostasis in patients with PVD.