When healthy skin gets wounded, the proteins and growth factors in the skin stimulate the body to regenerate new skin.
This is the normal wound healing process.
However with certain diseases (like diabetes and circulatory problems), the skin is missing these biological substances, and the healing cycle is broken.
This leads to the development of non-healing ulcers and wounds. (Chen et al., 2008; Walter et al., 2010)

Foot ulcers account for over 84% of all non-traumatic lower limb amputations and the 3-year mortality after a first amputation has been estimated as 20-50%.
Foot ulcers are costly, more so depending on the seriousness of the condition.
The average cost to healing a single ulcer is $8000, an infected ulcers $17,000 and an amputation $45,000. A $27,987 excess cost is attributed to diabetic foot ulcers and their sequelae (in the 2 years following first presentation).

Diabetic people or individuals at risk can cryopreserve their cells for a prompt use for future ulcer treatment.
The immediate cure of a diabetic ulcer, within 12 week of onset, reduces the risk of unfavorable disease evolutions such as chronic forms or amputations.

The cell therapies based on skin cells to date have not yielded important results due to the lack of extracellular matrix in the wound bed that prevented the engraftment of new implanted cells.
The Adipose Derived Cells have their ability to:
• reconstitute the extracellular matrix that represents the scaffolding on which to organize the cells from the wound edges to rebuild tissues and play
• active role by delivering to the wound living cells, proteins produced by the cells and collagen, which are important for healing.

In studies, Adipose Derived Cells has been proven to heal chronic wounds significantly faster than standard wound care such as moist dressing, compression therapy, debridement, off-loading and compression therapy.
Clinical studies have also shown that Adipose Derived Cells can help close many severe wounds that have been resistant to standard therapy. LIPOSKILL is safe, hi-end and well tolerated. (Dash et al., 2009).

LIPOSKILL is an advanced treatment for wound healing that uses autologous ADC. It is an innovative approach for healing ulcers such as diabetic foot and venous leg ulcers that are not responding despite treatment with conventional therapies. Briefly described characteristics of the ADC explain why this type of cells are one of the most promising types in advanced therapies currently being applied in wound healing.
This is due to their potentials and also because they are very accessible within the body. ADC can be isolated through mini liposuction technique from fat tissue and then processed in a series of isolation and purification steps in the laboratory.

Skin wounds can happen for a myriad of reasons during the course of one’s life. Injuries, cuts, burns, poor circulation, ulcers from pressure sores, and illnesses such as diabetes can all cause wounds that temporarily compromise the normal function and structure of the skin. If the body is unable to heal these wounds, they become chronic and fester over time. Surprisingly, this happens more often than one would expect, and at any given time 1% of the population is living with a chronic skin wound.
Half of these wounds never heal. As well known, when healthy skin gets wounded, the proteins and growth factors in the skin stimulate the body to regenerate new skin. This is the normal wound healing process. However when the skin is compromised with such factors due to a certain diseases (diabetes and circulatory problems), the healing cycle is damaged resulting in non healing. This leads to non-healing of ulcers and wounds. (Chen et al., 2008; Walter et al., 2010)

About 12 days after the small FAT collection (10 ml), almost five vials of ADC are produced, each containing about ten million ADC. One vial can be used for an immediate Wound Care treatment and remaining vials are frozen for the future treatments.

Cell-to-cell direct contact and also paracrine activation through secretory factors ensures effectivenes of the treatment. ADC enhanced the secretion of type I collagen in HDFs by regulating the mRNA levels of extracellular matrix (ECM) proteins: up-regulation of collagen type I, III and fibronectin and down-regulation of MMP-1.
ADC showed stimulatory effect on migration of HDFs and significantly reduced the wound size and acceleration of re-epithelialization from the edge. Moreover, huge benefit of these cells is that they are immune response modulators, helping transplanted cells to fly ‘under the radar’ without provoking the same type of vigorous immune response which often leads to graft rejection.

The cell therapies based on skin cells up to date have not yielded important results due to the lack of extracellular matrix in the wound bed that prevented the engraftment of new implanted cells. The Adipose Derived Cells have their ability to:
• Reconstitute the extracellular matrix that play an important role of scaffolding on which the cells organize to rebuild the tissues from the wound
• Delivering proteins produced by the cells is important for healing of the wounded living cells
• Modulate the immune response, reducing the local inflammation
• Participate in new vessels formation secreting the angiogenic cytokines

It has been proven in various studies that Adipose Derived Cells heal chronic wounds significantly faster than standard wound care such as moist dressing, compression therapy, debridement, off-loading and compression therapy. Clinical studies have also shown that Adipose Derived Cells can help close many severe wounds that have been resistant to standard therapy.

Adipose-derived stem cells (ADC) are multipotential stem cells capable of differentiation into various cell lineages and secretion of angiogenic growth factors. The gross and histological results showed that ADC significantly accelerated wound closure in normal and diabetic patients, including increased epithelialization and granulation tissue deposition indicating spontaneous site- specific differentiation into epithelial and endothelial lineages.
These data suggest that ADC not only contribute to cutaneous regeneration, but also participate in new vessels formation. Moreover, ADC were found to secret angiogenic cytokines in vitro and in vivo, including VEGF, HGF, and FGF2, which increase neovascularization and enhance wound healing in injured tissues.

It is important to know that diabetes impairs the angiogenic potential of adipose-derived stem cells by selectively depleting cellular subpopulations. Diabetes alters the ADC niche in situ and that diabetic ADC are compromised in their ability to establish a vascular network both in vitro and in vivo. Moreover, these diabetic cells were less effective in promoting soft tissue neovascularization and wound healing. Perturbations in specific cellular subpopulations, visible only on a single-cell level, represent a previously unreported mechanism for the dysfunction of diabetic ADC. These data suggest that the banking of autologous ADC for cell-based therapies in patients with diabetes is a must.
Diabetic people or individuals at risk can cryopreserve their cells for a prompt use for future ulcer treatment. The immediate cure of a diabetic ulcer, within 12 week of onset, reduces the risk of unfavorable disease evolutions such as chronic forms or amputations.

Use of Adipose Derived Stem Cells for the treatment of diabetic wounds

Mesenchymal stem cells (MSC) are multipotent adult cells capable of self-renewal and differentiation into various cell lineages. Bone marrow (BM) MSC are currently considered the gold standard, by which newly discovered sources of MSC are compared on the basis of renewal and multipotency.
Recent studies have shown that subcutaneous adipose tissue provides a clear advantage over other MSC sources due to the ease with which adipose tissue can be accessed (under local anaesthesia and with minimum of patient discomfort) as well as to the ease of isolating stem cells from the harvested tissue [Casteilla et al., 2005].
Stem cell frequency is significantly higher in adipose tissue than in BM and maintenance of the proliferating ability in culture seems to be superior in ADSC compared with BM-MSC [Puissant et al., 2005].
ADSC are isolated through an initial enzymatic digestion of harvested adipose tissue, that yields a mixture of stromal and vascular cells (preadipocytes, fibroblasts, vascular smooth muscle cells, endothelial cells, resident monocytes/ macrophages, lymphocytes and ADSC [16427974]) referred to as stromal-vascular fraction (SVF) [Zuk et al., 2001]. SVF is a rich source of pluripotent ADSC [8, 9], which were first identified by Zuk and named processed lipoaspirate (PLA) cells [8, 10]. Morphologically, ADSC are fibroblast-like cells and preserve their shape after in vitro expansion [8, 12, 13]. Minimal criteria have been proposed to define MSC -and by similarity, ADSC- by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy [18]. These are: plastic adherence ability; tripotential mesodermal differentiation potency into osteoblasts, chondrocytes, and adipocytes; immunomodulatory capability [6]. Several groups demonstrated ADSC multipotency showing differentiation toward various cells derived from ectoderm (epithelial cells and neurons), mesoderm (connective stroma, cartilage, fat and bone cells) and endoderm (muscle cells, gut epithelial cells and lung cells) [10, 15, 19-21].
Based on beta-galactosidase activity, ADSC have been shown to exhibit telomerase activity similar to BM-MSC that, although lower than that in cancer cell lines, indicates maintenance of the capacity for self-renewal and proliferation even after transplantation. A recent study investigated the fundamental changes of ADSC in long-term culture by studying the morphological feature, growth kinetic, surface marker expressions, expression level of the senescence- associated genes, cell cycle distribution and ß-galactosidase activity.The morphology of ADSC in long-term culture showed the manifestation of senescent feature at P15 and P20 and all the results showed that culture till 10 in vitro passages is safe (22391697).

ADSC, immunomodulation and clinical implications
An emerging body of evidence shows that MSC possess intrinsic immunomodulatory properties and immunoprivileged characteristics and can modulate a wide range of target cells within the innate and adaptive arms of the immune system (19172693).
The mechanism underlying MSC inhibitory effect is not fully elucidated and is likely to be multifactorial and to result from the communication between various immune cells and cytokine generation. Therefore, a combination of the paracrine activity of MSC and direct cell-cell contact is at the basis of their immunomodulatory effects.
Besides being immunosuppressive, ADSC are also immunoprivileged: they have low immunogenicity due to low expression of human leukocyte antigen (HLA) class I and no expression of class II molecules, as demonstrated by flow cytometry. The lack of HLA-DR expression and the above described immunosuppressive properties of ADSC make them suitable even for allogenic transplantation procedures, irrespective of MHC incompatibility [25]. Because of the great immunomodulatory effects of MSC in vivo and in vitro and since the first report by Le Blanc et al. [15121408], MSC have been mainly used for treating or preventing acute graft versus host disease (GVHD) during allogeneic hematopoietic stem cell (HSC) transplantation. The efficiency of such therapy is greatest in liver and gut GVHD and in children (17910665).
On the whole, their immunomodulatory function as well as the approved clinical trials, suggest that these cells are efficient for treatment for several classes of autoimmune diseases and that their application is safe [48].

ADSC differentiation and clinical applications
The broad range of clinical applications for ADSC largely depends on their potential for differentiation and on their ability to migrate and to recruit endogenous stem cells from the niches.
There are numerous scientific publications demonstrating that ADSC possess the potential to differentiate towards a variety of cell lineages both in vitro and in vivo:
• ADSC capability to differentiate towards adipogenic lineages has implications for breast soft tissue reconstruction after tumor surgery, for breast cancer, breast asymmetry and soft tissue and subdermal defects, after trauma, surgery or burn injury.
As the differentiation of ADSC into adipocytes is not in any doubt and as there is a strong demand in reconstructive and cosmetic surgery, progression of related clinical treatments and trials is further advanced than for other differentiation lineages.
• ADSC can be used for skeletal regeneration of inherited and tumor- or trauma-induced bone defects, thanks to their osteogenic differentiation.
Following osteogenic differentiation, ADSC can acquire even bone cell-like functional properties, such as responsiveness to fluid shear stress [16411823) and increase their expression of both alkaline phosphatase and mechanosensitive genes, such as osteopontin, collagen type I1, and COX-2 after mechanical loading.
• ADSC chondrogenic commitment is relevant for joint and disc defects repair and for plastic reconstruction of ear and nose defects.
• Given their myogenic and cardiomyogenic properties, ADSC are useful for muscle reconstruction after trauma and surgery, dystrophic muscle disorders, heart muscle regeneration, functional improvement after myocardial infarction or heart failure. Cultured ADSC have the potential for differentiation into a cardiomyocyte-like phenotype with specific cardiac marker gene expression and pacemaker activity [14656930]. However, the myogenic potential of ADSC may be harvested in the treatment of Duchenne muscular dystrophy (DMD), an inherited genetic disorder characterized by progressive degeneration of skeletal muscle. In vivo murine studies have shown that the implantation of ADSC into dystrophin-deficient, immunocompetent mice resulted in restoration of dystrophin expression, both in the muscle at the site of injection and in adjacent muscles over the long-term [21874281].
• ADSC have been shown as useful for neovascularization and, therefore, for ischemic diseases.
• As far as the differentiation into cells of the mesodermal lineages and regeneration of mesodermal tissues is concerned, ADSC can differentiate as well as into tenogenic and periodontogenic lineages [21547120].
• Given their ability to differentiate both morphologically and functionally into neurons, they are currently under investigation for neurological diseases, for brain injury, stroke, neuronal protection and peripheral nerve injury.
• Finally, it has been shown that ADSC can differentiate into endoderm lineage cells. Several reports have shown that ADSC have the potential to differentiate into hepatocytes as indicated by the presence of HGF and FGF-1 and 4 [22224071].
ADSC can be induced to become not only hepatic cells, critical for chronic liver failure, hepatic regeneration or even hepatocyte transplantation, but also pancreatic/endocrine insulin-secreting cells, relevant for type 1 diabetes mellitus. Timper et al. [100] were successful in differentiating human ADSC into cells with a pancreatic endocrine ph

ADSC mechanism of action
The potential benefits of ADSC are promising for regenerative medicine because they have several advantages: ability to continue proliferating after transplantation [37], to differentiate into endothelial cells and to induce neovascularization [38] and to release angiogenic and anti-apoptotic growth factors [39].
In terms of the proposed mechanisms for regeneration of ADSC, because they are known to differentiate into various cell types, it was thought initially that the mechanism involved engraftment and differentiation into injured tissue. Interestingly, a robust engraftment into the target tissue is not a requisite for ADSC to mediate their clinical effects. Only a minority (often < 1%) of intravenously infused MSC reaches the damaged tissue and disappears after few days (19172693).
Despite this low level of engraftment, ADSC mediate their therapeutic effects through paracrine actions rather than transdifferentiation, stimulating proliferation and differentiation of resident progenitor cells (10102814- 17700588).
A number of papers have described the secretory profiles of pre-adipocytes, ADSC, or adipose tissue, which were determined using Enzyme Linked Immunoabsorbent Assays (ELISA) or related techniques. ADSC secrete high levels of epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), transforming growth factor-beta (TGF-β), insulin-like growth factor (IGF), and brain-derived neurotrophic factor (BDNF) [40-45].
They also secrete cytokines such as Flt-3 ligand, granulocyte colony stimulating factor (G-CSF), granulocyte/ macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-11 (IL-11), interleukin-12 (IL-12), leukemia inhibitory factor (LIF), and tumor necrosis factor-alpha (TNF-α) [43, 46]. These angiogenic and anti-apoptotic growth factors are secreted in bioactive levels by ADSC.
Therefore, the capacity of ADSC to exert a potent tissue protective effect by several mechanisms (i.e., immunoprotection and paracrine stimulation) suggest that they might trigger endogenous mechanisms of regeneration that self-sustain, accordingly to the input they receive from the transplanted tissue, and that survive after cells disappearance.

ADSC safety
To date, any studies suggesting that hADSC (human ADSC) have a tumorigenic potential are either inconclusive or have been retracted [49, 50] (+20631079), due to cross-contamination of the examined hADSC cultures with immortalized tumor cell lines.
In a recent study culture-expanded hADSC were applied to immunosuppressed mice. At one year, animals were no different in weight nor life span from controls and showed no signs of tumorigenesis [51].
The same result on the safety of ADSC systemic administration had been obtained by Ra and coworkers: to test the toxicity of hADSC, different doses of hADSC were injected intravenously into immunodeficient mice, and the mice were observed for 13 weeks. Even at the highest cell dose (2.5×108 cells/kg body weight), the SCID mice were viable and had no side effects. A tumorigenicity test was performed also in Balb/c-nu nude mice for 26 weeks and again no evidence of tumor development was found (21303266).
To conclude, it must be recognized that the risk of neoplasia from stem cells, particularly MSC, has long been managed in clinical trial development.
Ongoing studies will add substantially to the database of safety information (sustained ventricular arrhythmias, ectopic tissue formation, or sudden unexpected death).
Very importantly, phase I clinical trials have already specifically monitored for unwanted tissue formation. It has to be noted, however, that patients with increased oncological risk due to underlying comorbidities or to ongoing immunosoppression treatments have been excluded by most trial designs and that, for the moment, most trial have a short follow up (19958962, 21415390).
The evidence that so far in a quite large cohort of patients (more than 700) with hematologic tumors or non-neoplastic disease, exposed to MSC therapy, none has developed new neoplasias even on long-term follow-up, can be taken as a reasonable proof of the safety of the procedure.

Scientific Background
In this frame, Bioscience developed LIPOSKILL, a technology that allows isolation, characterization and in vitro expansion of autologous ADSC, starting from a fat tissue sample. Once expanded, cells will be released for treatment as a suspension in a hyaluronic acid containing solution. Briefly, LIPOSKILL is obtained from about 10 ml of fat harvested using surgical liposuction or in one outpatient session.
After collection, the sample is placed in an isothermal container which guarantees the best security, temperature and pressure conditions for the shipment of biological material.
The kit is collected by a specialized delivery service and delivered to Bioscience Cell Factory in a very short time, respecting the necessary procedures required by biological material.
Upon arrival, biologists at Bioscience process the sample to isolate and expand a homogeneous population of ADSC according to the maximum biological security conditions.
After expansion, cells can either undergo programmed freezing in order to interrupt senescence and be ready in case of future need or be released for treatment.
Briefly, diabetic patients with a clinical diagnosis of diabetic wounds and without gangrene on any part of the affected foot will be enrolled after receiving all relevant information on the trial and after signature of informed consent. Patients will undergo an abdominal liposuction for the harvest of at least 20 ml of fat. The lipoaspirate will be sent to Bioscience Clinic laboratories for ADSC isolation and in-vitro expansion, according to Good Manufacturing Practice (GMP).
After about two weeks (depending on the individual cell culture) or when an appropriate cells number is achieved through cell expansion, cells will be administered to the patients. Cells will be injected into the periphery and debrided surfaces of chronic wounds by means of a syringe and the number of injected cells (and the number of injections, as well) will be determined by the surgeon, as a function of total area to be treated.
At follow up visits, percent change in wound size and improvement in life quality from baseline will be assessed. Major adverse events, i.e. death, target limb amputation, will be also monitored.
The pathophysiology of diabetic wound healing is characterized by microcirculatory ischemia and an abnormal wound healing cascade due to glycosylated cells and proteins. In the skin, the normal wound healing cellular cascade mechanism acts to restore epithelial components and ends in collagen deposition and scar formation. Imperative to this process is angiogenesis, cellular signaling, and cellular mitosis.
Various approaches have been developed for diabetic wound healing, but most of these approaches have centered on one facet of wound healing, such as inflammation or growth factors (Yang et al., 2013). With a multifactorial etiology, therapy that focuses on one facet has limited therapeutic efficacy (Yang et al, 2011; Menke et al., 2007). On the contrary, wound healing processes can be promoted by stem cell transplantation.
Stem cell therapy, which refers to an interventional strategy that introduces adult stem cells into damaged tissue in order to treat disease or injury, has been studied largely as a treatment for diabetic ulcers (Ren et al., 2012) and has shown promise. Clinical and basic science studies show that this therapy can provide a comprehensive solution by addressing multiple factors during diabetic wound healing, including cell proliferation, extracellular matrix (ECM) synthesis, growth factor release, and vascularization.
We will now focus on stem cell therapies for diabetic foot, mainly focusing on MSC and, in particular, on ADSC.

In 2002, Tateishi-Yuyama et al. were the first to publish a pilot randomized, controlled trial to test autologous non- cultured bone marrow derived mononuclear cells (BMMNCs), which were intra-muscularly reimplanted in Critical Limb Ischemia (CLI) patients (Tateishi-Yuyama et al., 2002). They reported a significant increase in transcutaneous oxygen pressure (TcPO2) and pain-free walking time.
Since then, and in the context of ischemia, several cell types have been tested and reviewed for their therapeutic potential. Most of these are derived from bone marrow or peripheral blood (Lu et al., 2011; Losordo et al., 2004). Kirana and coworkers, for example, used bone marrow mononuclear cells in comparison with expanded bone marrow cells enriched in CD90+ cells in the treatment of diabetic ulcers to induce revascularisation.
The transplantation of both cell populations proved to be safe and feasible and improvements of microcirculation and complete wound healing were observed in the transplant groups (Kirana et al., 2012).
Prochàzka and coworkers demonstrated the prevention of major limb amputation in a group of diabetic patients using a local application of autologous bone marrow stem cells concentrate (Prochàzka et al., 2010).
In a recent meta-analysis including all types of bone marrow derived cell populations, Teraa et al. concluded that there is a true and significant benefit of bone marrow derived cell therapy (Tera et al., 2013).
The use of BMMNC displays the advantage of preparing the therapeutic cell product at bench side.
Unfortunately, this is counterbalanced by the need for large amounts of bone marrow aspirate with systemic or epidural anesthesia and the great heterogeneity of the therapeutic product that cannot be controlled in the time of intervention.
Adipose Derived Stem Cells (ADSC) may overcome these limits because they can:
• be easily harvested with minimal donor site morbidity and higher yields compared to BMC;
• be in vitro expanded;
• be characterized as a homogeneous population;
• have a differentiation potential similar to other MSCs and
• display pleiotropic regenerative effects, mainly through their marked paracrine activity and their strong angiogenic potential.

ADSC displayed strong angiogenic properties in vitro as well as in vivo in an experimental model of CLI (Planat-Benard et al., 2004).
Kim et al. studied the effect of human ADSC on healing of ischemic wounds in diabetic nude mice.
The authors found that ADSC-treated animals had an earlier and abundant neovessel formation and better tissue remodelling than the control group without any treatment.
Lower rates of amputation and a survival rate comparable to group II were also observed in the ADSC-treated group (Kim et al., 2011).
Maharlooe et al. evaluates the effect of ADSC on wound healing in a diabetic rat model and concluded that ADSC enhances diabetic wound healing rate probably by other mechanisms rather than enhancing angiogenesis or accumulating collagen fibers (Maharlooe et al., 2011).
These results were confirmed by numerous reports, highlighting complementary mechanisms for ADSC regenerative capacities (Kondo et al., 2009; Moon et al., 2006; Nakagami et al., 2005; Cao et al. 2005) and showing the lack of biological toxicity for ADSC infusions (Toupet et al., 2013).
On these basis, the use of ADSC for wound treatment has been translated from bench to bedside.
ADSC reparative capabilities are illustrated in a study by Rigotti et al., which examines the role of ADSCs in treating severe and irreversible radiation-induced lesions with atrophy, fibrosis, ulceration and retraction.
Repeated transplants of purified autologous lipoaspirates into irradiated areas resulted in improvement of ultrastructural tissue characteristics with neovessel formation as well as significant clinical improvements (Rigotti et al., 2007).
Bura et al. describes the intramuscular injection of 108 expanded ADSC to treat no-option patients with CLI and assesses feasibility and safety:
no karyotype abnormality is reported, trans-cutaneous oxygen pressure tended to ncrease in most patients and ulcer evolution and wound healing showed improvement (Bura et al., 2014).
Lee et al. utilizes intramuscular injections of ADSCs to treat patients with thromboangiitis obliterans and diabetic feet with improvement in pain rating scores in the majority of patients as well as improved walking distances measured in a subset of patients.
Digital subtraction angiography before and 6 months after implantation showed formation of numerous vascular collateral networks across affected arteries (Lee et al., 2012).

On the basis of the wealth of published preclinical and clinical results on safety and efficacy of stem cells administration for wound healing, several clinical trials have been granted by FDA using mesenchymal stem cells (either from bone marrow, adipose tissue or umbilical cord blood) for the treatment of diabetic wounds. Here is a selection:
• Adipose Derived Regenerative Cellular Therapy of Chronic Wounds (NCT02092870, Phase II) explores the effects of ADSC on chronic wounds. Patients receive a single treatment with ADSC in the form of multiple injections of cells within and immediately surrounding the wound. Cells are delivered using a 1 cc syringe with an appropriate gauge and length needle.
Each injection has a volume less than 250 microliters. The number of injections is determined by the surgeon as a function of total wound volume. Percent change in wound size from baseline at 12 weeks is measured as primary outcome.
• Human Adipose Derived Mesenchymal Stem Cells for Critical Limb Ischemia (CLI) in Diabetic Patients (NCT01257776, Phase 1/2) recruits 30 non-diabetic patients with critical chronic ischemia in at least one of the lower limbs (CLI) and without possibility of revascularization. Experimental group includes two dose levels: 0,5 x 106 cells/kg of body weight and 1 x 106 cells/kg of body weight, administered by intra-arterial administration through a selective cannulation of target common femoral artery. At baseline and 6 months follow up, neovasculogenesis is assessed angiographically by a dedicated software.
• ACELLDream for Adipose CELL Derived Regenerative Endothelial Angiogenic Medicine (ACELLDREAM) (NCT01211028, Phase I/II) evaluates safety and feasibility of regenerative therapy with expanded adipose derived stroma/stem cells (dose 100 million of expanded cells), administered intramuscularly in patients with critical leg ischemia. It assesses number and nature of adverse events at 15 days, 1, 2, 3, 4, 5 and 6 months). The Role of Lipoaspirate Injection in the Treatment of Diabetic Lower Extremity Wounds and Venous Stasis Ulcers (NCT00815217) is a prospective, single blinded randomized clinical study to determine if the injection of lipoaspirate into diabetic or venous stasis wounds promotes wound healing or wound closure at a faster rate than conventional treatment.
• Induced Wound Healing by Application of Expanded Bone Marrow Stem Cells in Diabetic Patients With Critical Limb Ischemia (NCT01065337, Phase II) recruits diabetic foot patients with chronic limb ischemia and without the option for surgical or interventional revascularization. Patients are randomized to control or intervention group, whereas the intervention is divided into bone marrow cells administered intramuscular or intraarterial or expanded bone marrow cells administered intramuscular or intraarterial resulting in five distinct groups.To measure the therapeutic effects of the various treatment arms patients are evaluated for ankle brachial index (ABI), transcutaneous oxygen partial pressure (TcPO2), and reactive hyperemia (Blood Oxygen Level Dependent [BOLD]). Patients also undergo imaging with angiographic methods. At 1 year follow up, wound areas and healing of wounds are evaluated.
• Endogenous Progenitors Cell Therapy for Diabetic Foot Ulcers (AMD3100) (NCT0133937, phase I) proposes a novel combination of two drugs (Mozobil® and Regranex®Gel) to mobilize a specific sub-type of stem cells (endothelial progenitor cells) from the bone marrow and traffic them toward the wound. At 1 year follow up, rate of wound closure is evaluated.
• Stem Cell Therapy for Patients With Vascular Occlusive Diseases Such as Diabetic Foot (NCT02304588, Phase I) uses MSC injections (10-20 X106 cells (up to volume of 20 ml, depending on wound size and patient weight) for treatment of diabetic foot and lower limb ischemia. It evaluates frequency of adverse events and relative wound area regression (time frame: more than 6 weeks).
• Safety Study of Stem Cells Treatment in Diabetic Foot Ulcers (NCT01686139, Phase I/II) is a study whose aim is the determination of safety and efficacy of cultured Bone Marrow Mesenchymal Stromal Cells (BM-MSCs) from allogeneic donors for treatment of chronic leg wound of diabetic patients. Enrolled patient receive multiple injections in one session during the study. The injections take place in the chronic wound bed and in the third distal part of the treated shin (in the form of a ring). Maximal amount of ABMD-MSCs injected id 10-20x106 (up to volume of 20 ml, depending on wound size and patient weight).
• Autologous Bone Marrow Stem Cell Transplantation for Critical, Limb-threatening Ischemia (BONMOT) (NCT00434616, Phase II/III) compares the efficiency of concentrated bone marrow cells injected into the critically ischemic limb compared to a placebo procedure where only saline is injected. Cells are injected directly into the muscle of the diseased leg. After the follow-up of three months, the rate of death and amputations and the wound healing process are compared between groups. Adverse and serious adverse events are recorded during this time period. Diagnostic studies are obtained to measure blood flow in the treated leg during the follow up period and include skin oxygen measurements, pressure recordings in the leg and arteriography. Also, quality of life, pain and wound healing are assessed.
• Autologous Bone Marrow Stem Cell Transfer in Patients With Chronical Critical Limb Ischemia and Diabetic Foot (NCT01232673, Phase II) evaluates the efficacy-safety profile of autologous bone marrow stem cells transplantated into chronically and critically ischemic limb and into diabetic foot in stage IV Fontaine, Rutherfod 4-6 classification. The efficacy/safety of this therapy is assessed using several endpoints such as (a) prevention of amputation, (b) wound healing and (c) degree of angiogenesis.
• Safety and Efficacy of Autologous Bone Marrow Stem Cells for Lower Extremity Ischemia Treating (NCT01903044, Phase I/II) determines whether autologous bone marrow-derived stem cells are effective in the treatment of lower extremity ischemia. BM-MNCs are injected in aliquots of 1 ml at multiple regions of the leg muscles and wound healing (wound size, wound stage) is assessed at 3 months follow up.
• Umbilical Cord Mesenchymal Stem Cells Injection for Diabetic Foot (NCT01216865, Phase I/II) aims at determining whether treatment with umbilical cord Mesenchymal Stem Cells is safe and effective in the management of diabetic foot ischemia. Expanded MSC from human umbilical cord blood are administered by multiple injections at 5x107 cells/ischemic limb. Follow-up index includes efficacy (pain, ulcer healing rate, lower limb amputation rate and ankle-brachial index, magnetic resonance angiography, electromyogram) and safety (infection of the injection site, fever, and tumor generation).

• Abetz et al., The diabetic foot ulcer scale: a quality of life instrument for use in clinical trials. Pract Diab Int 2002.
• Almoutaz Alkhier Ahmed et al., The Diabetic Foot in the Arab World. The Journal of Diabetic Foot Complications, 2011.
• Armstrong et al., Guest editorial: are diabetes related wounds and amputations worse than cancer? Int Wound J 2007.
• Brownrigg et al., The association of ulceration of the foot with cardiovascular and all-cause mortality in patients with diabetes: a meta-analysis. Diabetology 2012.
• Bura et al., Phase I trial: the use of autologous cultured adipose-derived stroma/stem cells to treat patients with non-revascularizable critical limb ischemia. Cytotherapy, 2014.
• Cao et al., Human adipose tissue-derived stem cells differentiate into endothelial cells in vitro and improve postnatal neovascularization in vivo. Biochem Biophys Res Commun. 2005.
• Diabetes UK. State of the nation 2012 – England. London: Diabetes UK, 2012.
• Kim et al., “The effect of human adipose derived stem cells on healing of ischemic wounds in a diabetic nude mouse model,” Plastic and Reconstructive Surgery, 2011.
• Kirana et al.,, Autologous stem cell therapy in the treatment of limb ischaemia induced chronic tissue ulcers of diabetic foot patients. Int J Clin Pract. 2012.
• Kondo et al., Implantation of adipose-derived regenerative cells enhances ischemia-induced angiogenesis. Arterioscler Thromb Vasc Biol. 2009.
• Lee et al., Safety and effect of adipose tissue-derived stem cell implantation in patients with critical limb ischemia: a pilot study. Circ J. 2012.
• Losordo et al., Therapeutic angiogenesis and vasculogenesis for ischemic disease, part ii: cell-based therapies. Circulation. 2004.
• Lu D et al., Comparison of bone marrow mesenchymal stem cells with bone marrow-derived mononuclear cells for treatment of diabetic critical limb ischemia and foot ulcer: a double-blind, randomized, controlled trial. Diabetes Res Clin Pract. 2011.
• Maharlooei et al.,, Adipose tissue derived mesenchymal stem cell (AD-MSC) promotes skin wound healing in diabetic rats. Diabetes Res Clin Pract. 2011.
• Menke et al., “Impaired wound healing,” Clinics in Dermatology, 2007.
• Moon et al., Human adipose tissue-derived mesenchymal stem cells improve postnatal neovascularization in a mouse model of hindlimb ischemia. Cell Physiol Biochem. 2006.
• Nakagami et al., Novel autologous cell therapy in ischemic limb disease through growth factor secretion by cultured adipose tissue-derived stromal cells. Arterioscler Thromb Vasc Biol. 2005.
• Planat-Benard et al., Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives. Circulation. 2004.
• Procházka et al., Cell therapy, a new standard in management of chronic critical limb ischemia and foot ulcer. Cell Transplant. 2010.
• Rigotti et al., Clinical treatment of radiotherapy tissue damage by lipoaspirate transplant: a healing process mediated by adipose-derived adult stem cells. Plast Reconstr Surg. 2007.
• Ren et al., “Concise review:mesenchymal stem cells and translational medicine: emerging issues,” Stem Cells Translational Medicine, 2012.
• Teraa et al., Autologous bone marrow-derived cell therapy in patients with critical limb ischemia: a meta-analysis of randomized controlled clinical trials. Ann Surg. 2013.
• Toupet et al. Long-term detection of human adipose derived mesenchymal stem cells after intra-articular injection. Arthritis Rheum. 2013.
• Yang, et al., “Bone marrow derived mesenchymal stem cells transplantation accelerates tissue expansion by promoting skin regeneration during expansion,” Annals of Surgery, 2011.
• Yang et al., Stem Cell Therapy for Lower Extremity Diabetic Ulcers: Where Do We Stand?BioMed Research International, 2013.