EUROPEAN  TISSUE  REPAIR  SOCIETY

SELECTED ABSTRACTS

Abstracts from 16 September (Pisa meeting)

LIVER REGENERATION AND REPAIR
Alexis Desmoulière, INSERM E0362, Université Victor
Segalen Bordeaux 2, Bordeaux, France

The process of regeneration in the liver is observed in living donor transplantation, after hepatic resection, portal branch embolisation, and after liver injury. After partial hepatectomy, in rats and mice, the original liver mass is restored to approximately 100% in 7–10 days. In humans, there is a very rapid increase in liver mass during the first seven days after partial liver transplantation, leading to complete restoration by 3–6 months. After injury, the processes of liver repair and of fibrogenesis resemble a wound healing process. When injury and the associated acute inflammation response result in moderate cell necrosis and extracellular matrix damage, tissue repair normally takes place. In this process, dead cells are replaced by normal tissue, with regeneration of specialised cells by proliferation of the surviving ones, formation of a granulation tissue, and tissue remodelling with scar formation. The specific regenerative capacities of the liver generally allow it to reconstitute itself entirely following acute, moderate lesions. However, chronic injuries to the liver do not always heal as effectively and fibrosis is the main complication of the many known chronic liver diseases. The endpoint of liver fibrosis, defined as the abnormal accumulation of extracellular matrix in the liver, is cirrhosis, which is responsible for a significant morbidity and mortality. Cirrhosis is an advanced stage of fibrosis, characterised by the formation of regenerative nodules of liver parenchyma separated by fibrotic septa. In all these situations, it is necessary to ascertain whether hepatocytes responsible for liver regeneration originated from the replication of existing hepatocytes, were generated by differentiation of oval cells (hepatic progenitor cells), or were produced from bone marrow cells. Bone marrow cells can generate hepatocytes in transplanted livers but so far, the frequency of hepatocytes produced by this route is very low (however, bone marrow cells are an important source of nonparenchymal cells such as endothelial cells). In contrast, normally quiescent, highly differentiated hepatocytes have an enormous proliferative potential, unique among the differentiated cells in mammalian tissues. Furthermore, oval cells which originate in the canals of Hering, function as a reserve compartment and are capable of differentiating into hepatocytes or cholangiocytes. It is thought that the canals of Hering which have a highly strategic distribution in the liver, and the surrounding stromal cells may constitute a niche for hepatic stem cells. All these data could generate in the future, new therapeutic strategies for liver repopulation.

GUIDED REGENERATION OF SMALL DIAMETER ARTERY USING HYALURONIC ACID BASED PROSTHESIS
G. Abatangelo1, S. Lepidi2, V. Vindigni3, B. Zavan1, C. Tonello1 and R. Cortivo1
1) Dpt. di Istologia, Microbiologia e Biotecnologie Mediche. 2) Dpt. di Scienze Cardiologiche, Toraciche e Vascolari – Clinica di Chirurgia Vascolare. 3) Dpt di Chirurgia Plastica, Università di Padova, Italy

Introduction:
The aim of present paper was to answer the lack of a prosthetic graft capable of performing adequately as a small diameter conduit (2 mm diameter) using an in vivo tissue engineering approach. We decided to reproduce and positively guide the remodeling process directly in vivo, using a 2 mm diameter and 1 cm in length hyaluronic acid based tubular scaffold (HYAFF-11) that functioned only as temporary absorbable guide, like an in vivo ‘artery-bioregeneration assist tube’ (ABAT). We chose this biomaterial for its documented capacity to promote adhesion and proliferation of endothelial and smooth muscle vascular cells. Its biodegradability and biocompatibility were also well documented.

Material and methods:
Thirty male Wistar rats weighing 250–350 g were used. The aorta, from the renal arteries to the aortic trifurcation, was exposed and isolated. A segment of aorta (1 cm) was incised and a HYAFF 11™ tube was anastomized first proximally then distally in an end-to-end fashion with interrupted stitches of 10.0 nylon suture. No anticoagulants were used either before or after the operation. Histological (haematoxylin-eosin and Weighert solution), immunohistochemical (antibodies to von Willebrand factor, CD34, vascular endothelial growth factor receptor-2 and to Myosin Light Chain Kinase) and ultra-structural analysis were used to evaluate the results at 5, 15, 30, 60, 120, and 180 days after surgery.

Results and conclusion
Three novel findings stood out from result evaluation: endothelialization of the tube luminal surface within 5 days; sequential regeneration of the other vascular components that leads to a complete vascular walls regeneration after 15 days from surgery (Figure 1); temporariness of the tube: biomaterial was entirely degraded after four months from implantation, and after that, a new artery remained to connect artery stumps. This study assesses the feasibility to create a completely biodegradable vascular regeneration guide in vivo. The most important novel finding is represented by the ability of proposed vascular prostheses to sequentially orchestrate vascular regeneration events needed for very small artery reconstruction that up to now, given the great difficulty to obtain their in vivo significant long term patency and good wall mechanical strength, is defined as the holy grail of vascular biology.

Figure 1

SKIN REGENERATION AFTER ACCIDENTAL GAMMA IRRADIATION USING MESENCHYMAL STEM CELL
THERAPY
Doucet Christelle, PhD, Centre de Transfusion Sanguine des Armées, Unité de Thérapie Cellulaire, BP410, 92141
Clamart, France

Local irradiation syndrome is marked by necrosis that may extend to the deep subcutaneous structures. Today, treatment is surgery, excision, graft and flap with sometimes bad results. It has been suggested that Mesenchymal Stem Cells (MSC) therapy could be used in order to treat numerous tissue lesions. We report here a novel therapeutic
approach of the local irradiation syndrome by using local MSC therapy in a clinical case of an accidental irradiation localized to the left buttock and the left hand. The patient was admitted at the Burn Department of the Percy Military Hospital two weeks after the accident. Due to the severity of the lesions (threatening necrosis, pain uncontrolled by opiates) and because of our preclinical data obtained in collaboration with Drs Chapel and Benderitter (Institut de Recherche et de Sureté Nucléaire: IRSN), MSC therapy was proposed. The IRSN scientists were in charge to estimate the irradiation dose received by the patient. The Cell Therapy Unit of the Military Blood Transfusion Center (CTSA) produced MSC in GMP conditions after an informed consent was obtained from the patient.

Autologous bone marrow cells were collected from the unexposed iliac crest. For GMP production, MSC were
expanded in a closed system (MacoPharma partnership) containing an innovative serum free medium supplemented with human platelet lysate as previously described (Doucet et al., J. Cell Physiol., 2005). Quality control assays evidenced that expanded cell population retained typical MSC characteristics.

Autologous skin graft was completed with local injection of MSC (76.106) in and around the irradiated tissues of the hand. Concerning the buttock lesion, first action was surgically with excision of all tissues exposed to 20 Gy followed by autologous skin graft. MSC injection was achieved as an adjuvant treatment of surgery, first injection of 168.106 MSC was followed a week later by a second one with 226.106 MSC.

The pain disappeared within days. Cicatrisation appeared to be quicker and better following the MSC injection
than that usually observed in irradiated lesions treated by graft alone.

This report relates the first evidence of human MSC therapy efficiency in the context of acute cutaneous and
subcutaneous damage repairs following irradiation. This approach might be also of high clinical importance for other skin lesions such as thermal burns.

OXIDATIVE STRESS HYPOTHESIS AND CHRONIC INFLAMMATION: MOLECULAR IMPLICATIONS AND PREVENTIVE THERAPEUTICAL STRATEGIES
R. Colognato, Dept. of Human and Environmental Sciences, Faculty of Medicine, University of Pisa, Via S. Giuseppe 22, 56100 Pisa, Italy

Oxidative stress-induced cell damage has been, since some years, implicated in a wide range of pathologies such as cancers, diabetes, cardiovascular dysfunctions and neurodegenerative disorders. This oxidative hypothesis has now been demonstrated (by us and other authors) to be a key factor in the onset and likely a cause in the progression of degenerative disorders in which chronic inflammation play the major rôle. Inflammation, cellular and redox signalling mechanisms play major rôles in the pathophysiology of numerous disease states. Although pharmacological therapies ensure stabilization of such pathological progression, for many chronic and degenerative diseases still the therapeutical strategy for a complete remission is missing. Besides this type of problem, increasing evidence is becoming available on the fact that many drugs are indeed themselves reactive species for oxidative stress induction. Moreover, strategies for any kind of intervention and prevention for these type of pathological conditions require an understanding of the basic molecular mechanism(s) of prophylactic agents (dietary antioxidant factors from food plants and medicinal plants in this context) that may potentially prevent or reverse the promotion or progression of such diseases. Considering the fact that vitamin antioxidant are still to be used although no clear effectiveness has been reported, since some years an increasing number of data are published on pilot study or clinical trials with non-vitamin antioxidant of natural origin (polyphenols, carotenoids, catechin, etc., etc.) Extracts from food plants and medicinal plants continue to be used in herbal medicine practice for the treatment of many chronic or acute diseases, viral pathologies and as immune modulators. Indeed, dietary antioxidants and components of fruit and vegetable extracts are increasingly suggested to have the capacity to significantly contribute to the modulation of the complex mechanisms of these diseases. The view being that they may be essential in optimizing in vivo antioxidant defenses. Defining if the presence of oxidative stress
triggering to more severe pathological condition, like chronic inflammation, in various diseases and how these could be attenuated by the administration of antioxidant compounds in food plants coupled with establishing the relationship to the presence of particular genetic polymorphism and modulation of the complex cell signalling cascades involving gene transcription remains a major future scientific challenge. Marchetti and Abbracchio have argued that ‘novel therapeutic approaches must rely on potentiation of endogenous anti-inflammatory pathways and a combination of treatment involving immune modulation and anti-inflammatory therapies’.

EPIDERMAL REGENERATION FROM CORNEAL EPITHELIUM
Danielle Dhouailly, Biologie de la Differenciation Epithéliale, UMR CNRS 5538, Grenoble, France

The epidermis and the corneal epithelium are two tissues that derive from the embryonic ectoderm and which express different keratins as markers of their differentiation state, as well as having different stem cell locations. We previously showed that the formation of a dense dermis is a crucial stage during skin morphogenesis. Different signals, including Wnt1, Shh and Noggin are required for this dense dermis formation, and are able to trigger experimentally the formation of a dense dermis from even the extra-embryonic mesoderm, i.e., from the amnion somatopleure. Once this dense dermis stage is reached during development, not only is the dermis endowed with the ability to induce cutaneous appendage formation, but also to induce the segregation of its associated stem cells. Likewise, an embryonic dense dermis is able to induce the formation of hairs from an adult central corneal epithelium, and subsequently the replacement of this epithelium by an epidermis. This transformation occurs via a multi-step process, which involves cell dedifferentiation of those epithelial cells in contact with the dermis, resulting from ß- catenin activation by Wnt signals from the embryonic dermis. This transdifferentiation includes the down-regulation of the corneal-specific keratin pair 3/12, the down regulation of Pax6 and the formation of hair placodes. Finally epidermal cells which express keratin pair K1, 2/ K10 emerge from the newly formed hair follicles and join to form a continuous epidermal sheet, which displaces the
corneal epithelium. Three main concepts arise from these results. First: transdifferentiation occurs via dedifferentiation to a stem cell state. Second: trans differentiation can result from submitting committed adult cells to a new embryonic environment. Third: as they are both of ectodermal origin, epidermal stem cells are similar to corneal epithelial stem cells, with the latter having, in addition preactivation of Pax6 and of still unknown other transcription factors.

THE ROLE OF PATHOLOGY IN CHRONIC WOUNDS DIAGNOSIS AND TREATMENT
Giovanni Borroni, MD, Olga Ciocca, MD and Giorgia Ronzi. Department of Dermatology, University of Pavia,
Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.

If wound healing is regarded today as a complex series of reactions and inter-reactions between wound round, cells and mediators, which encompass the classic three overlapping phases of inflammation, proliferation and remodelling, totally different pathogenetic mechanisms may lead to the formation of wounds, whose clinical presentation could be, from a diagnostic point of view, not univocal.

Most chronic leg ulcers are secondary to venous insufficiency, arterial disease, neuropathy or to a combination of these factors. However, chronic leg ulcers in collagen-vascular disease, in cryoglobulinemia, in arterial-venous insufficiency, in superficial pyoderma gangrenosum and in many other cases may be difficult to be correctly diagnosed, even in an adequate clinical and laboratory setting. Despite its reasonable approach to the diagnosis of chronic non-healing wound, biopsy is recommended only to exclude carcinoma. This presentation considers biopsies taken from the border of chronic wounds, mostly but not exclusively from leg ulcers, in order to delineate the histopathologic features of the different types of wounds, and to contribute to the definition of the diagnosis. Small vessel vasculitic ulcers were characterized by a neutrophilic infiltrate around the superficial plexus, and interstitially, with leukocytoclasis, fibrin in the vessel walls, and around them, and thrombi in the lumina. Sjögren syndrome associated with cryoglobulinemia was also characterized by leukocytoclastic vasculitis. Rheumatoid ulcers were characterized by leukocytoclastic vasculitis, with a diffuse neutrophilic infiltrate, usually associated with many plasma cells, with thrombi, involving also the subcutaneous fat. Fibrin cuff was a common feature, both in superficial and deep vessels. Cryoglobulinemic ulcers were characterized
by diffuse neutrophilic infiltrate, prominent leukocytoclasis, destruction of small blood vessels. Pyoderma gangrenosum ulcer was characterized by epidermal hyperplasia, and diffuse neutrophilic infiltrate, sometimes with a necrotizing vasculitis of small vessels. Post-traumatic ulcers were characterized by extravasation of erythrocytes with siderophages, diffuse neutrophilic infiltrates, with fibrin deposits in the vessel walls, without leukocytoclasis. Venous ulcers were recognisable for stasis changes, with frequent glomeruli-like grouping of small vessels, surrounded by fibrosing connective tissue. Inflammatory neutrophilic infiltrate, associated with plasma cells, greatly varied with bacterial presence. Fibrin deposits were also found around the vessels in proximity of the ulcer’s border, also at deeper levels. These findings, associated with neutrophilic infiltration of the vessel walls, makes very difficult to distinguish this pattern from that seen in true vasculitis. Chronic acquired lymphedema wounds were characterized, on the contrary, by a scant inflammatory infiltrate in the dermis, made of plasma cells, with striking epidermal hyperplasia and hyperkeratosis of the border of the ulcer, with papillomatosis. Dermal fibrosis and sclerosis, stasis changes and absence of lymphatic dilation characterized the advanced stages of the disease. A punch biopsy with histopathology may substantially contribute to
the definition of the diagnosis of chronic wounds. Even when, in many cases (30–40% in our series) histopathology is not per se decisive, it gives useful integration in addressing clinical, laboratory, instrumental and immunopathologic diagnosis.

CLINICAL AND HISTOLOGICAL CORRELATION IN CHRONIC WOUNDS: SERIES OF CASE REPORT
Paolo Romanelli, MD, Department of Dermatology and Cutaneous Surgery,
University of Miami, USA

Chronic wounds are a common clinical problem especially in the elderly and/or debilitated population, the laboratory evaluation of biopsy material with standard hematoxylin-eosin, special stains for infectious, vascular or degenerative etiologies or immunohistochemistry for malignancies, is a very important component of the diagnosis and management. Clinical histopathological correlation should be the gold standard of any chronic, non healing wound, especially when the clinical diagnosis is not confirmed. Herein we describe some cases where the histopathology assessment was crucial in establishing the correct diagnosis of non-healing chronic wounds and we also would like to introduce a few novel, pioneer and potentially extremely helpful histopathology diagnostic modalities to better assess the pathogenesis and prognosis of chronic wounds.

INFLAMMATION DURING WOUND REPAIR: FRIEND OR FOE?
Sabine A. Eming, Axel Roers and Thomas Krieg,
Department of Dermatology, University of Cologne, Cologne, Germany

In postnatal life the inflammatory response is an inevitable consequence to tissue injury. Experimental studies established the dogma that inflammation is essential to the establishment of cutaneous homeostasis following injury
and in recent years information about specific subsets of inflammatory cell lineages and the cytokine network orchestrating inflammation associated with tissue repair has increased. Recently, this dogma has been challenged and
reports have raised questions on the validity of the essential prerequisite of inflammation for efficient tissue repair. Indeed, in experimental models of repair inflammation has been shown to delay healing and to result in increased scarring. Further, chronic inflammation as e.g., in nonhealing wounds predisposes tissue to cancer development. Thus, a more detailed understanding in mechanisms controlling the inflammatory response during repair and how inflammation directs the outcome of the healing process will serve as a significant milestone in the therapy of pathological tissue repair. In the presented work we will delineate cellular and molecular mechanisms that control inflammation during healing and might provide a rationale target to modulate the outcome of the healing response.

FIBROBLAST-KERATINOCYTE-INTERACTIONS LEAD TO THE RECONSTITUTION OF AN EPITHELIAL PHENOTYPE BY IMMORTALIZED GINGIVAL KERATINOCYTES
Pascal Tomakidi

We report here that the organotypic co-culture (OCC) system allows for significant preservation of the tissue-specific
phenotype of human gingival keratinocytes (IHGK) immortalised with the E6/E7 gene of the human papilloma virus type 16 (HPV 16). The approach taken is based on the OCC system facilitating spatially separated cell growth and cell-to-cell interactions via diffusible growth factors. Generally, IHGK reveal transcription of the HPV 16 E6/ E7 gene at rising passages. Fluorescence in situ hybridisation performed for chromosomes 1, 8, 10 and 18 demonstrates that disomic fractions differ between the tested chromosomes but otherwise remain fairly constant.Monosomies of chromosome 18 are more prominent in late passages 81 and 83, while polysomies of chromosome 10 and 18 are detected in early passages 25 and 27.

In comparison with corresponding monolayer cultures (MCs), IHGK in OCCs form stratified epithelia, proliferate and express gingival-specific gene products in vitro. Moreover, mRNA gene transcription for growth factors IL-1ß, GM-CSF, FGF-7 and EGF in OCCs is different from that in MCs.

When grafted onto nude mice, IHGK develop hyperplastic, differentiated surface epithelia devoid of malignant growth. We are not aware of any other OCC system comprising of IHGK, which allows for site-specific expression of gingival epithelial markers. This substantiates reconstitution of a gingival epithelial phenotype in vitro.