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