Chronic wound healing by fetal cell therapy may be explained by differential gene profiling observed in fetal versus old skin cells

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Abstract

Engineering of fetal tissue has a high potential for the treatment of acute and chronic wounds of the skin in humans as these cells have high expansion capacity under simple culture conditions and one organ donation can produce Master Cell Banks which can fabricate over 900 million biological bandages (9 × 12 cm). In a Phase 1 clinical safety study, cases are presented for the treatment of therapy resistant leg ulcers. All eight patients, representing 13 ulcers, tolerated multiple treatments with fetal biological bandages showing no negative secondary effects and repair processes similar to that seen in 3rd degree burns. Differential gene profiling using Affymetrix gene chips (analyzing 12,500 genes) were accomplished on these banked fetal dermal skin cells compared to banked dermal skin cells of an aged donor in order to point to potential indicators of wound healing. Families of genes involved in cell adhesion and extracellular matrix, cell cycle, cellular signaling, development and immune response show significant differences in regulation between banked fetal and those from banked old skin cells: with approximately 47.0% of genes over-expressed in fetal fibroblasts. It is perhaps these differences which contribute to efficient tissue repair seen in the clinic with fetal cell therapy.

Introduction

Considerable interest and research has been dedicated to the understanding of wound healing and the associated process. Whereas adult cutaneous wounds heal more slowly and with scar formation to restore tissue integrity, fetal skin, in utero, is observed to have rapid and scar-less tissue repair characterized by regeneration of an organized dermis with normal appendages and by a relative lack of inflammation. (Beanes et al., 2002, Bullard et al., 2003, Cass et al., 1997, Adzick and Lorenz, 1994, Armstrong and Ferguson, 1995, Dang et al., 2003, Lorenz et al., 1995). Fundamental differences between fetal and adult skin and the fetal and adult skin wound environment may be important in inducing efficient tissue repair. Chronic wounds, more specifically leg ulcers, are a major health concern. Patient’s suffering has long been underestimated and recent evaluations of quality-of-life reveal that patients with leg ulcers have complaints that are substantial and similar to those previously reported for patients with chronic obstructive pulmonary disease, osteoarthritis or angina (Kahn et al., 2004). Costs are considerable and were calculated to exceed 0.5 billion euros/year in Germany and the direct cost of leg ulcers in the UK were calculated at 400 million euros a year (Hafner et al., 1999). Similarly in the United States, estimations are as high as 3 billion dollars for treatment of all forms of leg ulcers. Important variables for cost differences between countries are the frequency of bandage changes and duration of time for each bandage change (Ragnarson and Hjelmgren, 2005). Etiological treatment of venous ulcers is mandatory in order to prevent relapse. Compression therapy is the cornerstone of venous ulcer treatment and high rates of healing (up to 83% in 6 months) may be obtained by different techniques (Booza et al., 2005). However, such results are not obtained by all teams, and considerable biases of recruitment do exist. In countries with a high standard of health care and a good prevention of venous insufficiency, resistant ulcers are more and more frequent and present as a difficult therapeutic challenge (Chaby et al., 2006, Marklund et al., 2000).

Local methods for accelerating healing are based on modern wound dressings. However, there is no evidence in the literature that their use really improves the healing rate of venous ulcers (Booza et al., 2005). Therefore, there is an urgent need for new techniques in the treatment of refractory leg ulcers.

New biological therapies for wound healing have significantly advanced including growth factors, skin substitutes, gene and stem cell therapies as well as tissue engineering. The origin of cell choice, their interaction with a biomaterial and the simplicity of preparation is extremely important for eventual therapeutic usage.

Autologous skin fibroblasts and substitutes of various nature have been used on leg ulcers to date (Limat et al., 1996, Uccioli, 2003, Caravaggi et al., 2003, Cavallini, 2007) with different degrees of efficiency. Major drawbacks are that production time is long since patient tissues are necessary for processing. Allogenic skin substitutes using foreskin tissue (Dermagraft®, Apligraf® and Orcel®) have shown decreased healing times for diabetic foot ulcers, other ulcers and non-weight bearing wounds (Falanga et al., 1998, Marston et al., 2003). The production time is long and we have recently shown high differences in gene expression of banked fetal and foreskin cells used in tissue engineering (Hirt-Burri et al., 2008) which could be, in part, responsible for differences of efficiency seen in the clinic to date.

As banked fetal skin fibroblasts have been previously shown to efficiently induce tissue repair in burns and in acute wounds (Hohlfeld et al., 2005, de buys Roessingh et al., 2006, Quintin et al., 2007), it was of interest to look at the effect on recalcitrant leg ulcers with different etiologies in the elderly to see if a full clinical trial would be of merit. In this Phase I safety clinical study, we particularly wanted to evaluate the tolerance of multiple treatments with fetal cell biological bandages and interaction with the chronic wound environment. With these specifically banked fetal skin cells used in tissue engineering, we performed microarray analysis to identify differences on the molecular level between specific gene clusters compared to old skin cells, banked in the same manner, which could give some insight into significant parameters involved in efficient wound healing.

Section snippets

Skin donations and cell bank synthesis

Cell lines established in the University Hospital of Lausanne from a fetal skin biopsy at 14 weeks of gestation (14 week male fetal skin;14 wFS) obtained after pregnancy termination with informed and written consent and approval from the local Medical School Ethics Committee. Normal skin from an old male donor (75 yr old skin; 75 yOS) was obtained in the Department of Dermatology in the University Hospital of Lausanne from a non-sun-exposed skin site (buttocks) also with informed consent and

Preliminary assessment of fetal skin constructs on chronic wounds

In a total of 9 patients treated with fetal cell therapy from 3 to 31 weeks concerning 13 ulcers, we were able to see 8 ulcers closed completely, 4 with significant amelioration in size but not complete closure and 1 which was lost to follow-up because the patients estimated that there was a substantial improvement. We present the detail of 4 patients herein as we have completed data including venous (Duplex) and arterial investigations (ABI and duplex) described according to CEAP

Discussion

In the clinical setting, we have seen practical advantages of fetal cell therapy applied to preliminary patients with chronic wounds (Ramelet et al., 2001). The cells are able to exert promoting effects on adhesion, proliferation and migration of existing cells as the repaired wounds tend to heal gradually and the skin is much less atrophic. Also, the pain alleviation following fetal construct application was universal. In other studies using other autologous and allogenous grafting techniques (

Acknowledgments

The authors wish to thank Baxter (Switzerland) for the donation of the matrix (TissueFleece®) for these studies. We also thank Drs. Zwahlen and Poussin for helpful discussions on micro-array analysis. These studies were supported by the Foundation for Orthopedics for Swiss Romande and a grant from Nestlé (Switzerland) for working on aging.

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      Considering the fact that fetal skin wounds heal rapidly and regenerate faster compared to adults, it is presumed that fetal cells hold the key for tissue regeneration [12,13]. Several studies have shown that compared to adult fibroblasts, fetal fibroblasts possess greater proliferative and migratory capacity, larger amounts of extracellular matrix deposition and different growth factors [14–16]. Given the importance of fetal fibroblasts in accelerating fetal wound healing, researchers have tried to mimic the fetal wound healing conditions by using fetal cell-based skin substitutes [12,15].

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    1

    Present address: Department of Pediatric Surgery, University Hospital, Lausanne.

    2

    These authors contributed equally in this study.

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