Bioengineering/Nanomedicine
Therapy with autologous adipose-derived regenerative cells for the care of chronic ulcer of lower limbs in patients with peripheral arterial disease

https://doi.org/10.1016/j.jss.2013.05.024Get rights and content

Abstract

Background

An ulcer is a trophic lesion with loss of tissue that often has a multifactorial genesis. It typically diverges from the physiologic processes of regeneration because it rarely tends to heal spontaneously. In this study, we used purified adipose-derived stem and regenerative cells (ADRCs) extracted from autologous fat, for the care of chronic ulcers of the lower limbs of arteriopathic patients. The primary objective of this study was complete re-epithelization of chronic ulcers; the secondary objective was a decrease in diameter and depth.

Methods

From January 2010 to January 2012, 20 patients with peripheral arterial disease, with an ankle-brachial index between 0.30–0.40, in the age range 60–70 y (14 men and six women), with chronic ulcers of the lower limb, were involved in the study. Only 10 arteriopathic patients (seven men and three women) with chronic ulcers of the lower limb were surgically treated. Using the Celution system, we isolated a solution of ADRCs in about 150 min. The isolated cells were injected through a 10-mL syringe into the edges of the ulcer, taking care to spread it in all directions. Using a small amount of Celution extract, we performed cell characterization by flow cytometry analysis and cell viability assay.

Results

We monitored patients treated with ADRC or untreated at 4, 10, 20, 60, and 90 d. In all cases treated with ADRC, we found a reduction in both diameter and depth of the ulcer, which led to a decrease in pain associated with the ulcer process. In six of 10 cases there was complete healing of the ulcer. Characterization of the cells by FACS clearly showed that the ADRC cells contained adipose-derived stem cells. Viability assays demonstrated that partial or total closure of the ulcer was attributable exclusively to ADRC cells present in the Celution extract, and not to growth factors extracted during the process of purification of the Celution and injected together with the cells.

Conclusions

For the first time, the Celution method has been applied for the care of chronic ulcers in the lower extremity of patients with peripheral arterial disease. Our results demonstrate that the technique is feasible for autologous cell application and is not associated with adverse events. Moreover, the transplantation of autologous stem cells extracted with Celution may represent a valuable method for the treatment of chronic ulcers in lower limbs of arteriopathic patients.

Introduction

An ulcer results from a breakdown at the epidermal layer and may extend through the subcutaneous tissue to the bone, severely compromising the viability of the limb. The moment during genesis of an ulcerative lesion in patients with peripheral arterial disease (PAD) is the development of a tissue hypoxia condition in the site of lesion, which alters the physiologic processes of tissue viability and then contrasts with the system of regeneration post-lesion. Atherosclerosis is the most common cause of PAD of the lower limbs; shrinkage or obstruction of the arteries caused by atherosclerosis reduces blood flow in the legs. Arterial ulcers are related to arterial insufficiency of one or both limbs, which causes tissue hypoperfusion. There are two kinds of ulcers in patients with PAD: ones leading to rapid resolution after recanalization for reactivation of blood flow, and ones that do not respond to this treatment. These are called chronic ulcers. Therapeutic approaches based on cells have recently been introduced to treat chronic ulcers, with good results [1], [2]. Stem cells isolated from adipose tissue, defined as adipose-derived stem cells (ADSCs), have been used to treat chronic skin ulcers [3]. The mechanism of ulcer healing by ADSCs is based on direct differentiation toward lineage-committed cells or on the production of angiogenic growth factors, such as basic fibroblast growth factor, platelet-derived growth factor, and vascular endothelial growth factor [4]. The protocols are not standardized, which makes them challenging to apply under certain clinical conditions. Using automated cell isolation such as the Celution System (Cytori Therapeutics, Inc), we significantly reduced the possibility of making mistakes during the critical steps of cell isolation and enrichment that could be caused by human subjectivity. Furthermore, this method provides in a short time (about 2 h) a cellular array consisting of adipose cell stem cells, endothelial cells, endothelial progenitor cells, vascular smooth muscle cells, and pericytes, defined as adipose-derived stem and regenerative cells (ADRCs), which can be promptly injected into the chronic ulcer site [5], [6]. In this study, for the care of lower limb chronic ulcers of arteriopathic patients, we used ADRCs (after extraction from autologous fat and then purification). The primary objective of this study was the complete re-epithelization of chronic ulcers; the secondary objective was a decrease in diameter and depth.

Section snippets

Selection of patients

From January 2010 to January 2012, 20 patients (Tables 1 and 2) with PAD, with an ankle-brachial index (ABI) between 0.30–0.40, in the age range of 60–70 y (14 men and six women), with chronic ulcers of the lower limbs, were involved in the study. Of the 20 patients, 18 had diabetes mellitus type 2 for about 20 y. In addition, of the 20 patients, five had heart disease and 6 had chronic obstructive pulmonary disease. All patients underwent revascularization procedures for the reactivation of

Characterization of ADSC cells by flow cytometry

We cultured an aliquot of Celution extract (ADRC) for 20 d (nine passages) for immunophenotype stabilization [9] and observed it daily to assess morphologic homogeneity. The population was analyzed by expression of CD13, CD34, CD44, CD45, CD73, CD105, CD117, and CD133 markers. Immunologic characterization revealed that the cells were negative for CD34, CD45, CD117, and CD133 markers, whereas they were positive for CD13, CD44, CD73, and CD105 (Fig. 4). These data demonstrate that ADRC cells

Discussion

Application of stem cells to repair and regenerate tissues has recently been investigated at a clinical level in plastic surgery and in chronic radiation injury [10]. Some authors showed that bone marrow–derived mesenchymal stem cells and ADSCs had the same ability to differentiate when grown in differentiation media [11], [12]. Adipose-derived stem cells can be harvested through minimally invasive liposuction. In radiation injury after ADRC treatment, a neovascular increase was found to avoid

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