1. Tissue engineering: autologous full- thickness skin substitute for healing chronic wounds A. Bellomi, G. Calabrese, A. Cassisa, F. Colpani, R. Fante, L. Gaetti, G. Granchelli, S. Negri Servizio Anatomia Patologica Ospedale C. Poma – Mantova

2. Anatomia Patologica, Mantova. Az. Ospedaliera C.Poma Chronic wounds, inclouding venous and arteriosclerotic leg ulcers, diabetic foot ulcers, decubitus and trauma induced wounds, represent a major problem in our society. These wounds occur with high incidence and exist for prolonged periods of time and therefore have a great socioeconomic impact. The problem increases as the average age of the population increases and therefore new therapies in wound healing are continously being sought. The aim of this study is to develop an autologous, full-thickness skin substitute and to evaluate its efficiency and applicability in closing long-standing ulcers that have proven non-responsive to the currently available wound-healing therapies (topical therapy, antibiotic treatment, surgical debridment, external compression). Introduction

3. Anatomia Patologica, Mantova. Az. Ospedaliera C.Poma Method We included 20 patients with long-standing ulcers of which 13 venous and arteriosclerotic (65%), 3 diabetic (15%), 3 trauma-induced and 1 burn wounds (5%). Age of patients varies from 57 to 91 (average 75). The lesions were present since at least two years. A single punch biopsy (diameter cm 0,6) or a surgical biopsy (cm 1,5x1) obtained from the patients upper leg were required. After 3-4 weeks we obtaneid three autologous products on collagen support (Antema soft, Opocrin S.P.A): fibroblasts (fig.1 ), fibroblasts and keratinocytes (fig.2 ) and keratinocytes (fig.3 ). Sheets of keratinocytes present basal melanocytes (fig.4 ); between keratinocytes and fibroblasts we observe basement membrane ( fig.5 ). Depending on ulcers depth and dimensions our patients underwent multiple applications (at least two). All procedures were performed with the Ethics Committee approval and patient consent.

4. Anatomia Patologica, Mantova. Az. Ospedaliera C.Poma Fig.1: haematoxylin-eosin stain (200x)

5. Anatomia Patologica, Mantova. Az. Ospedaliera C.Poma Fig.2: haematoxylin-eosin stain (400x)

6. Anatomia Patologica, Mantova. Az. Ospedaliera C.Poma Fig.3: haematoxylin-eosin stain (400x)

7. Anatomia Patologica, Mantova. Az. Ospedaliera C.Poma Fig. 4: immunohistochemical detection of basement membrane - collagen IV- 400 x

8. Anatomia Patologica, Mantova. Az. Ospedaliera C.Poma Fig. 5: immunohistochemical detection of melanocytes - HMB45 - 200 x

9. Anatomia Patologica, Mantova. Az. Ospedaliera C.Poma The success rate in culturing biopsies was 100%. The skin substitute visibly resembled an autograft. Ten of the 13 (77%) chronic venous ulcers (size 6-300 cmq) healed between 8 and 48 weeks (case 1 and 2). One of the 3 (33%) diabetic ulcers (size 3-28 cmq) healed within 12 weeks. Three (100%) trauma induced ulcers (size 4-6 cmq) healed between 6 and 12 weeks. One (100%) burn ulcer (size 12 cmq) healed within 4 weeks. Skin substitutes were very well tollerated and pain relief was immediate after application. Results

10. Anatomia Patologica, Mantova. Az. Ospedaliera C.Poma Female 80 years old, with vascular ulcer since three years non responsive to the currently available therapies... Case n° 1 Female 80 years old, with vascular ulcer since three years non responsive to the currently available therapies...

11. Anatomia Patologica, Mantova. Az. Ospedaliera C.Poma The same woman after autologous full-thicness skin substitute application Case n° 1 The same woman after autologous full-thicness skin substitute application

12. Anatomia Patologica, Mantova. Az. Ospedaliera C.Poma Female 73 years old, with vascular ulcer since two years non responsive to the currently available therapies... Case n° 2 Female 73 years old, with vascular ulcer since two years non responsive to the currently available therapies...

13. Anatomia Patologica, Mantova. Az. Ospedaliera C.Poma The same woman after autologous full-thicness skin substitute application Case n° 2 The same woman after autologous full-thicness skin substitute application

14. Anatomia Patologica, Mantova. Az. Ospedaliera C.Poma The application of this noval skin sobstitute provides a promising new therapy for healing chronic wounds resistant to conventional therapies. It is also necessary to point out the importance of suitable cyto-histological and immunohistochemical studies for evaluating the correct cell morphology and phenotype. Conclusion