Towards Reaching the Target: Clinical Application of Mesenchymal Stem Cells for Diabetic Foot Ulcers

Abstract

Mesenchymal stem cells (MSCs) hold great promise for therapeutic application in non-healing ulcers and tissue regeneration because of their multi-lineage differentiation potential. MSCs delivered may migrate to the sites of injury and improve wound healing by stimulating angiogenesis and promoting revascularization. The incidence of type 2 diabetes mellitus (T2DM) is increasing worldwide. It is associated with peripheral neuropathy and peripheral arterial occlusive disease (PAOD), which predispose patients to develop non-healing foot ulcers following minor trauma. A high rate of amputation exists among diabetic patients due to non-healing foot ulcers, which are a significant burden for the society despite new therapeutic protocols developed. In recent years, stem cell transplantation has been considered as a new therapeutic option for diabetic foot ulcers (DFUs). The regeneration potential of MSCs has been demonstrated in the experimental and clinical trials. Here we review the potential efficacy and systematic use of MSCs for the treatment of non-healing DFUs, current advances, MSC delivery systems, and possible options to enhance the therapeutic potential of stem cell for wound healing.

Introduction

Diabetic foot ulcers (DFUs) are a variety of chronic non-healing ulcers that pose a serious challenge to global health. Because the number of people with type 2 diabetes mellitus (T2DM) will double by 2030,¹ the burden of diabetic foot disease is also likely to increase.² When a patient with diabetes develops an injury, closure of the foot wound is hampered by both physiological impairments in wound healing and an increased susceptibility to infection. Lower limb ischemia due to narrowing of arteries is the most significant complication in individuals with diabetes,³ and the amputation rates are high despite current advances in clinical trials. Translating the results of mesenchymal stem cell (MSC) application in in vitro and in vivo experiments from animal models to human clinical trials is still in its infancy. Current advances in stem cells research suggests MSCs as a novel treatment strategy for ulcer healing and prevention of amputation.⁴

Adult stem cells have become an important source for cell-based therapy and tissue repair. Stem cells (embryonic, adult, or induced pluripotent cells) have the ability for self-renewal and differentiation into many cell types. Bone marrow contains many distinguished types of MSCs, which contribute to the regeneration of mesenchymal tissues.⁵ MSCs are the preferred candidates for therapeutic approaches because there is no ethical controversy and the cells can be isolated in appropriate amounts from several sources. Autologous bone marrow–derived MSCs (BM-MSCs) have yielded encouraging results not only in the treatment of peripheral arterial disease but also in DFUs.⁶ Studies have investigated the therapeutic use of MSCs to treat DFUs, but a suitable medium for MSCs, systematic delivery systems, and long-term follow-up after transplantation have not yet been well discussed.

Here we bring together a number of research studies that share some ideas to elucidate the mechanisms by which MSCs could be an ideal candidate for DFUs. In this review, we focus our attention on recent advances in clinical trials and the challenges that exist before MSC therapy in DFUs. In addition, we will also discuss available stem cell delivery systems based on published reports and the useful information that holds the key for safe use of cell-based therapy.

Pathogenesis of DFUs

Diabetic patients face serious complications that may affect the eyes, kidneys, feet, skin, and heart. Foot ulceration is one of the most common complications in patients with DM. Diabetic neuropathy and peripheral arterial occlusive disease (PAOD) are the two major events of diabetes and contribute to DFUs. Most commonly, DFUs are caused by peripheral neuropathy, and up to 50% of people with T2DM have neuropathy and are at risk for complications of deformity, callus formation, and trauma.^2,7,8 DFUs are also linked to secondary medical complications like osteomyelitis and amputation. Major lower limb amputations in patients with diabetes arise from ulcers in 85% of cases,⁹ and 50% of these ulcerations become infected, resulting in lower extremity amputation. Moreover, 69% of diabetic amputees will not survive more than 5 years.¹⁰ Foot ulceration occurs due to many overlapping factors—they put the tissue of the foot at risk, precipitate a break in the skin, and impair healing. The most common risk factors for ulcer formation are diabetic neuropathy and PAOD.

Diabetic neuropathy

In diabetes, hyperglycemia causes metabolic disorders and excess oxidative stress, leading to loss of neurovascular support. Prolonged hyperglycemic episodes and oxidative stress result in a series of metabolic and vascular damage. Excess glucose is converted into sorbitol and fructose, which results in decrease the synthesis of myoinositol, leading to defects in myelin synthesis and nerve conduction. Decresed sodium-potassium adenosoine triphosphatase (ATPase) activity results in decreased production of nitric oxide (NO), which causes vasoconstriction and nerve injury.

Diabetic neuropathy has three major components, namely sensory neuropathy, motor neuropathy, and autonomic neuropathy. Sensory neuropathy in a diabetic patient causes either partial or complete loss of sensation in the lower limb, as a result of which minor foot trauma may go unnoticed¹¹ and the protective sensation is lost, resulting in ulcer formation. The patient continues to walk because there is no sensation of pain present, which in turn delays healing of traumatized wound.

Motor neuropathy causes muscle weakness and intrinsic muscle imbalance leading to digital deformities, resulting in abnormal bony prominences prone to trauma. Damage to innervations of interosseous and small intrinsic muscles of the foot leads to imbalance between flexion and extension of the affected foot, resulting in hammertoe and clawed toe deformities. Alteration in morphology results in appearance of pressure points susceptible to trauma and subsequent ulceration. The tips of the toes and the area under the first metatarsal head are very vulnerable to ulceration.

Autonomic dysfunction (and denervation of dermal structures) also results in loss of skin integrity, which provides an ideal site for microbial invasion.¹² There is decreased autonomic innervation of sweat and oil glands, and the loss of function makes the diabetic foot dry and keratinized, predisposing to cracks. Autonomic neuropathy may also result in arteriovenous shunting, resulting in the dilation of small arteries and consequent edema; swelling and erythema can result in the skin to break down.

Peripheral arterial occlusive disease

PAOD results from atherosclerosis, an embolism, or thrombus formation, leading to stenosis. It causes either acute or chronic ischemia (lack of blood supply). PAOD is a major contributor to diabetic foot problems in T2DM patients, causing endothelial and smooth muscle cell dysfunction in peripheral arteries. Identification of PAOD is essential because appropriate treatment at right time can prevent the amputation of a diabetic foot. Macroangiopathy in form of atherosclerotic blockage of large vessels typically involves infrapopliteal arteries with compromised collateral circulation. Hyperglycemia, dyslipoproteinemia, and smoking along with genetic factors predispose to atherosclerotic blockage. The decrease of blood flow through stenosed vessels impairs the flow of oxygen to cells, leading to necrosis and gangrene, which in turn may require amputation. Microangiopathy is also a major problem in diabetes patients. It affects small blood vessels, resulting in thickened basement membrane, altered microcirculation, and endothelial function. Besides genetic, immunologic, and behavioral factors, long-standing hyperglycemia contributes to microangiopathy. Oxidative stress, advanced glycation end products (AGEs), alteration in the polyol pathway and sorbitol metabolism, and impaired sodium-potassium ATPase activity all contribute to microangiopathy. The lack of healthy blood flow may lead to ulceration and impaired wound healing.¹¹

Several causative factors for pathogenesis of DFUs and the therapeutic options are outlined in Table 1. Stem cell application may be an option for patients suffering from DFUs or critical limb ischemia. But once an ulcer develops, aggressive management is required to prevent amputation.

Table 1.

Pathogenesis of Diabetic Foot Ulcers: Underlying Factors and Therapeutic Approaches

Underlying factor	Mechanism	Treatment modality	Comprehensive treatment for DFUs
Diabetic neuropathy	1. Sensory neuropathy: Sensory loss resulting in breaks in skin tissue, even in minor painless trauma	• Continuous patient education on prevention is the best treatment. • Offloading high-pressure areas of foot with special shoes, heel protectors, leg braces, wheelchairs, etc.	The essential components of DFU management are: 1. Treating underlying pathology 2. Restoring adequate vascularity 3. Pressure offloading 4. Local wound care
	2. Motor neuropathy:Loss of structural alignment in anatomy of foot and joints, causing gait change and appearance of abnormal bony prominences vulnerable to trauma	• Prophylactic surgery to address foot deformities (metatarsal osteotomy, resection of metatarsal heads, percutaneous tenotomy)• Well-equipped protective shoes if surgery is not advisable• Regular removal of callus	1. Treating underlying pathology:• Patient education• Lifestyle modification; stop smoking and alcohol• Control blood sugar and hyperlipidemia• Treating neuropathic pain with tricyclic antidepressants• Addressing foot deformities
	3. Autonomic neuropathy:Impaired autonomic nerve roots leads to reduced sweating resulting in dry stiff skin, which is prone to cracks and infection.Arterio-venous shunting causes defects in microcirculation with reduced blood flow resulting in edema.	• Proper skin care.• Compression stocking to reduce edema• Hyperbaric oxygen	2. Restoring adequate vascularity:• Arterial reconstruction• Vascular surgery (femorodistal bypass with autologous tissue or prosthetic grafts, subintimal angioplasty)
Peripheral arterial occlusive disease (PAOD)	1. Macroangiopathy involving infrapopliteal arteries obstructs blood flow resulting in gangrene.2. Microangiopathy results in basement membrane thickening decrease oxygen and nutrient delivery causing impaired wound healing.	• Options for revascularization include angioplasty, thrombolysis, and bypass surgery.• Hyperbaric oxygen as adjunct to treatment modalities• Broad-spectrum empirical antimicrobial therapy in adequate dosage, duration, and route• G-CSF and PDGF have been effective in ulcer healing.• Autologous stem cells are under clinical trial and are effective in ulcer healing and angiogenesis.	3. Pressure offloading:• Adequate offloading of high-pressure areas• Use of removable cast, total contact cast, healing sandals, pneumatic walker, crutches, and wheelchairs4. Local wound care:• Adequate microbiological coverage with broad-spectrum antibiotics• Tissue debridement; surgical, larval, autolytic.Adequate tissue dressing (hydrogels, hydrocolloids, alginates, foams)• Products for advanced wound care: G-CSF, bioengineered skin substitutes, PDGF (becaplermin)• Autologous cultured stem cellsAmputation if all options fail

DFUs, diabetic foot ulcers; G-CSF, granulocyte colony-stimulating factor; PDGF, platelet-derived growth factor.

For appropriate treatment, careful examination of the diabetic foot is essential. DFU examination must include comprehensive foot and ulcer evaluation with laboratory screening, nutritional evaluation, and a neurological and vascular assessment. The patient's history is taken and a physical assessment is made of dermatologic changes in the surrounding skin and documentation of ulcer characteristics, including location, shape, and size of the wound (measurement of length, width, and depth); determination of the condition of the wound edges, wound bed, wound base, surrounding skin, and exudates; and determination of the presence of necrosis along with assessment of wound-associated pain.^12,13 Most of the options for treatment of DFU are in the different phase of clinical trials; a recent popular option is stem cell therapy. Early adoption of advanced therapies is advocated by many reports to speed up wound healing and decrease complications. Autologous stem cell therapy is an effective therapy, whereby stem cells can be preferentially located at damaged tissue sites to induce angiogenesis and regenerate the epidermis.

MSCs and Their Therapeutic Application

Cellular therapies are rapidly evolving and paving the way for novel therapeutic targets. Stem cells from diverse sources, including bone marrow, umbilical cord, amniotic fluid, placenta, and adipose tissue, have propelled researchers to evaluate their potential in treatment of diabetes and other complicated diseases.^5,14 MSCs are multi-potent stromal cells that were discovered in bone marrow by Friedenstein et al.¹⁵ They exhibit plasticity, i.e., they are capable of differentiating into many cell types (Table 2) in the appropriate microenvironment.⁵ Characterization of MSCs relies on expression of specific cell-surface markers. They differentiate into fat, bone, and cartilage when exposed to appropriate culture conditions.

Table 2.

Major Sources of Mesenchymal Stem Cells and Their Differentiation into Different Cell Types

Bone marrow	Muscle	Adipose tissue	Synovial membrane	Blood	Pericyte	Trabecular Bone	Periosteum
AdipocyteAstrocyteCardiomyocyteChondrocyteHepatocyteMesangial cellMyocyteNeuronOsteoblastStromal cell	AdipocyteEndothelial cellNeuronChondrocyteOsteocyte	ChondrocyteMyocyteOsteoblastStromal cell	AdipocyteChondrocyteMyocyteOsteoblast	AdipocyteFibroblastOsteoblastOsteoclast	Chondrocyte	AdipocyteChondrocyteOsteoblastMyocyte	ChondrocyteOsteoblast

In vivo and in vitro studies have shown that MSCs are an effective therapeutic agent in various experimental models. Exogenous application of MSCs has been successful in animal and human trials in myocardial infarction, liver diseases, Parkinsonism, connective tissue disorders, spinal cord injury, chronic non-healing ulcers, critical leg ischemia, and musculoskeletal disorders. Studies have shown that BM-MSCs promote regeneration of infarcted myocardium by promoting neovascularization.^16,17 Al-Khalid et al.¹⁸ demonstrated blood flow improvement in a chronic limb ischemia model with therapeutic application of autologous bone marrow stromal cells. BM-MSCs have been applied to promote cutaneous wound healing in human and murine models.^19,20

Muscle-derived stem cells (MDSCs) have been evaluated for use with fibrin and collagen gels to fill bone defects in the craniofacial skeleton.²¹ Chondrogenesis in a murine model of osteoarthritis was achieved by intra-articular injection of MDSCs expressing bone morphogenetic protein-4 (BMP-4) and vascular endothelial growth factor (VEGF).²² MDSCs may also be a suitable therapeutic option for Duchenne muscular dystrophy (DMD) patients, as demonstrated in dystrophic mdx mice.²³ Umbilical cord MSCs are gaining importance in the field of liver diseases.²⁴ Studies on the possibility of using MSCs in regenerative and reparative therapies for neurological disorders, particularly Parkinsonism, are underway.²⁵ BM-MSCs and other sources of MSCs have been explored actively in recent years, but their biology still remains poorly understood. BM-MSCs are easy to isolate, expand, characterize, and differentiate into many cell lineages. Hence most of the current research focus on MSC application is centered on BM-MSCs. BM-MSCs constitute nearly 10% of the hematopoietic stem cells (HSCs), and they are always regarded as a component of the HSC niche.¹⁵ Autologous BM-MSCs have shown efficacy in the treatment of the non-healing diabetic ulcers.^4
–6 BM-MSCs participate in all phases of wound healing and help to reconstitute the dermal, vascular, and other components required for optimal healing.¹⁸

Wound Healing and Role of BM-MSCs

Normal wound healing

Normal wound healing is a biological phenomenon involving a wide variety of cells, the extracellular matrix (ECM), and an array of cytokines and growth factors playing a pivotal role in angiogenesis and subsequent events. This dynamic process spreads over the four interrelated phases of hemostasis, inflammation, proliferation, and remodeling.²⁶ The hemostasis phase is initiated by loss of structural integrity and provides the much needed stimulus for the inflammatory phase. Following the coagulation cascade, platelets release cytokines essential for recruitment of cells that play a key role in inflammatory phase. The inflammatory phase is marked by increased vascular permeability, chemotactic migration of cells to the site, secretion of cytokines and growth factors, and proliferation of active migrating cells. The proliferative stage is characterized by the formation of granulation tissue, consisting of a capillary bed, fibroblasts, macrophages, and a loose arrangement of collagen, fibronectin, and hyaluronic acid. Remodeling or wound contraction appears to occur by a complex interaction of the extracellular materials and fibroblasts, which is not completely understood. The entire process may last from few weeks to years to complete.²⁷

Defects in DFU

The normal healing process entails a complex interplay between growth factor activation, cellular activity, and formation of connective tissue. All of the three physiologic processes are altered in non-healing ulcers and contribute to poor healing. In a non-healing DFU, the following abnormalities are seen: Decreased or impaired growth factor production,^19,28 angiogenic response,²⁰ macrophage function,²⁹ collagen accumulation, epidermal barrier function, abnormal quantity of granulation tissue, keratinocyte and fibroblast migration and proliferation, along with a reduction in the number of epidermal nerves.³⁰

The balance between the accumulation of ECM components and their remodeling by matrix metalloproteinases (MMPs) is also altered. The final result of the above changes keeps the diabetic ulcer stuck in the inflammatory phase of the wound healing process. Under normal circumstances, this phase lasts only 2–3 days, followed by the proliferative phase characterized by angiogenesis, expression of numerous growth factors, cell migration, collagen production, all of which results in wound closure. However, a DFU fails to progress to the proliferative phase and remains in a chronic inflammatory phase.

Role of BM-MSCs in wound healing

Earlier, researchers postulated that MSCs bring about repair through direct participation and eventual incorporation into damaged sites.³¹ Due to lack of evidence, it is now proposed that grafted MSCs modulate the host environment through indirect mechanisms, leading to enhanced healing, rather than through direct participation and incorporation into tissue.³² Therefore, engrafted MSCs may directly initiate or facilitate the host repair response and also act as a vector to deliver the appropriate signal.³³ Direct action of MSCs can occur under the appropriate microenvironment. MSCs have been shown to differentiate into cardiomyocytes, adipocytes, chondrocytes, neuronal cells, and dermal epithelial cells. In vivo and in vitro, it has been shown that MSCs can differentiate into epidermal keratinocytes and other skin appendages. In a wound environment, MSC differentiate into keratinocytes and express keratinocyte-specific keratin protein.³¹ Direct engraftment of exogenous MSCs as mature keratinocytes has been shown in green fluorescent protein (GFP)-positive MSCs in murine models.³⁴ Although direct incorporation and differentiation of transplanted MSCs into keratinocytes and vascular cells were demonstrated in a murine model, ³¹ the efficacy of engraftment of transplanted MSCs is still debated, with some studies declining incorporation of MSCs into newly formed blood vessels.³⁵ MSCs in vivo can migrate to sites of injury in response to chemotactic signals modulating inflammation, repairing damaged tissue, and facilitating tissue regeneration.

BM-MSCs improve wound healing, and the humoral and secretory factors play a vital role in repairing damaged tissue. The humoral factors play an important role because they recognize and combat the newly presented antigens at the site of injury and participate in the debridement of the wound area contributing to the healing. In chronic wound inflammation and infection, immunoglobulin M (IgM) and IgG in particular dominate the immune profile and serve as principal opsonins against pathogens. Growth factors, in addition to their role in immunomodulation and vascularization in situ, also recruit stem cells from the circulation or even from bone marrow to participate in angiogenesis.³⁶ Now, evidence shows that paracrine secretion of growth factors is the important therapeutic mechanism of these stem cells.

The paracrine action includes secretion of cytokines, growth factors, and chemokines that regulate a number of cells, such as epithelial cells, endothelial cells, keratinocytes, and fibroblasts, and has productive effects on immunoregulation, angiogenesis, epithelialization, and fibro-proliferation during wound repair. Analysis of paracrine factors released by BM-MSCs shows that BM-MSCs secreted distinctively increased amounts of VEGF-α, insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), keratinocyte growth factor, angiopoietin-1, stromal cell–derived factor-1 (SDF-1), macrophage inflammatory protein-1α and -β, and erythropoietin. These molecules are known to be important in normal wound healing.³⁶

MSCs influence the wound's ability to progress beyond the inflammatory phase and not regress to a chronic wound state. MSCs inhibit macrophage pro-inflammatory cytokine production, such as tumor necrosis factor-α (TNF-α), IL- 6, and interferon-α (IFN-α³⁷ and stimulate anti-inflammatory cytokine IL-10 and IL-12 production.³⁸ By doing so, immune cell activation and local inflammatory processes are limited, reducing tissue damage. Cytokine secretion by MSCs has an influence on immune system and other cells responsible for wound healing. Post-injury T cells secrete pro-inflammatory cytokines, which may stall the wound in inflammatory phase. MSCs can suppress several T lymphocyte activities both in vitro and in vivo by secreting mediators such as IL-10, transforming growth factor-β (TGF-β), indoleamine 2,3-dioxygenase (IDO), nitric oxide (NO), and prostaglandin E2 (PGE2).³⁹ IDO induces the depletion of tryptophan from the local environment, which is essential for lymphocyte proliferation. NO mediates its effect partly through phosphorylation of signal transducer and activator of transcription-5 (STAT-5), which results in suppression of T cell proliferation, by inhibiting either NO synthase or prostaglandin synthesis. In addition to its effect on the Janus kinase (JAK)/STAT pathway, NO may also influence mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB), which reduces the gene expression of pro-inflammatory cytokines. MSCs can suppress the activation of M1 macrophages (pro-inflammatory form) and induce proliferation of M2 macrophages (anti-inflammatory form), thereby favoring secretion of anti-inflammatory cytokines, and enhance resolution of chronic inflammation.

MSCs can also influence neutrophil recruitment and their action. Prolonged presence of neutrophils after phagocyting microbes can lead to respiratory burst and further tissue damage by reactive oxygen species (ROS). MSCs inhibit neutrophil migration and decrease their ROS production, but at the same time do not decrease their phagocytic capacity. MSCs also assist in clearing the wound site by promoting apoptotic cell phagocytosis. These anti-inflammatory properties of MSCs make them beneficial to the healing process of chronic ulcers because they force the stalled wound to advance past a chronic inflammatory state into the next stage of healing. MSCs play a central role in tissue regeneration during the proliferation phase. Angiogenic support provided by MSCs is crucial for recovery of damaged tissues. MSCs secrete SDF-1, VEGF, and other cytokines important for angiogenesis like basic fibroblast growth factor (bFGF) and MMPs. SDF-1 activity is essential for endothelial cell survival, vascular branching, and pericyte recruitment.⁴⁰ SDF-1 recruits CXCR4-positive progenitor cells into hypoxic tissues and retains angio-competent stem cells. It also assists pericytes and smooth muscle cell recruitment for stabilizing and maturing newly formed blood vessels.^41,42 Thus, SDF-1 has been shown to assist neovascularization by accelerating endothelial progenitor cell (EPC) recruitment into the ischemic site.⁴³ VEGF, apart from being a potent angiogenic cytokine, not only mobilizes EPCs from bone marrow but also inhibits EPC apoptosis. VEGF binds to Flt-1 (VEGFR-1) and KDR (VEGFR-2) receptors, which are members of the type III tyrosine kinase family. These receptors are present on endothelial surface of developing and mature blood vessels. VEGF binding to the kinase domain receptor (KDR) stimulates NO synthase (NOS) and cyclo-oxygenase activities promoting vasodilation and vascular permeability. The proliferative and anti-apoptotic properties of VEGF have been shown to be partially mediated by either MAP2K1/2/MAPK3/1 or phosphoinositide 3-kinase (PI3K)/AKT1 pathways⁴⁴ with subsequent inhibition of pro-apoptotic signaling.⁴⁵ BM-MSCs increase expression of pro-angiopoietic factor angiopoietin-1 (Ang-1), which serves as a ligand for the Tie2 receptor tyrosine kinase present on endothelial cells and endothelial progenitor cells. Ang-1–Tie2 interactions facilitate newly formed vessels to mature and maintain vessel integrity through the recruitment of peri-endothelial cells.⁴⁶ In the proliferative stage, MSCs reinforce vascular supply by secreting a number of angiogenic factors and facilitate production of ECM. In the rat diabetic wound-healing model, Kwon et al.⁴⁷ found that systemic and local treatment with BM-MSCs on diabetic wounds improved collagen levels (types I–V) in the wound bed.⁴⁷ MSCs secrete a variety of cytokines and growth factors that have anti-fibrotic properties, including hepatic growth factor (HGF), IL-10, adrenomedullin,⁴⁸ and MMP-9,⁴⁹ that promote turnover of the ECM,⁵⁰ keratinocyte proliferation,⁵¹ and inhibition of myofibroblast differentiation.⁵²

In a nutshell, the action of stem cells includes differentiation into specialized cells of the dermis and epidermis by paracrine or autocrine effects through the secretion of trophic factors, such as production of soluble mediators for neo-angiogenesis and immunomodulation.

Clinical Perspective of MSCs and Delivery Systems

Clinical perspectives

For an effective therapeutic application of MSCs in DFU, a complementary delivery vehicle is essential to optimize the delivery of cells to the wound. Studies so far predominantly have used either direct systemic administration or local injection of MSCs. The major disadvantages are optimal retention of the stem cells at the wound site and widespread distribution of MSCs, cellular trafficking, and malignancy. Some MSCs that do reach their intended location after infusion face a proteolytic wound environment that is chronically inflamed and inherently hostile to the proliferation of unprotected cells.⁵³ Cells often fail to anchor onto the bed of the ulcer, resulting in cell death and diminished therapeutic effect.⁵⁴ Thus, a protective vehicle is necessary to promote MSC engraftment and enable these transplanted cells to exert their regenerative or paracrine effects.⁵⁵

Table 3 presents published results that have been proved effective in clinical practice for non-healing ulcers, including our previous experiment. The positive outcome after stem cell therapy showed either enhanced healing rate of the wound or improved pain-free walking distance in the patients. Few reports have shown the safety and efficacy of ex vivo–expanded BM-MSCs or have been compared with BM-MNCs in the treatment of diabetic critical limb ischemia (CLI) and foot ulcers. BM-MSCs from diabetic patients were found to secrete more VEGF, FGF-2, and Ang-1 than BM-MNCs under normoxic and hypoxic conditions.⁵⁶ Interestingly, rat BM-MSCs release more VEGF and bFGF than BM-MNCs in serum-free medium.⁵⁷ When BM-MSCs are transplanted locally into the hind limbs, regional blood flow during ischemia improves much better than when BM-MNCs are transplanted systemically.⁵⁷ These effects may partly contribute to the difference in the clinical outcomes.

Table 3.

Clinical Trials on Stem Cell–Based Therapy and Their Efficacy Assessment

References	Patient condition	Source of stem cells for therapy	Stem cell administration	Outcomes
Li et al. (2013)⁹²	Type 2 diabetic patients with foot disease	Human umbilical cord blood mesenchymal stem cell (hUCB-MSCs)	Injected into the quadriceps thigh muscles (cell quantity at 2×10⁶ per point)	Post- transplantation reduction in blood glucose level with an decrease in insulin requirement
Kirana et al. (2012)⁹³	Diabetic patients with critical limb ischemia	Bone marrow mononuclear cells (BM-MNCs) and expanded bone marrow cells enriched in CD90⁺ cells;Tissue repair cells (TRCs)	Intramuscular administration of 30×10⁶ TRCs and 50×10⁶ BM-MNCs	Improved microcirculation and complete wound healing
Ravari et al. (2011)⁹⁴	Chronic lower extremity wounds	Bone marrow mononuclear cells (BM-MNCs)	Injected topically (15–22×10⁸ cells) into the wound along with platelets, fibrin glue, and bone marrow-impregnated collagen matrix	Complete wound healing in few patients and some others with improvements
Lu et al. (2011)⁵⁶	Diabetic critical limb ischemia (CLI) and foot ulcer	BM-MNCs and BM-MSCs	Intramuscular into the lower limb (20 sites, 3 cm in intervals, 1–1.5 cm in depth, and 0.5–1 mL BM-MSCs or BM-MNCs per site)	BM-MSCs therapy is more effective than BM-MNCs for increasing lower limb perfusion and promoting foot ulcer healing
Jain et al. (2011)⁹⁵	Chronic lower limb wounds in patients with diabetes	BM-MNCs	Injected and over sprayed with a total of 5 cc of autologous bone marrow–derived cells and autologous peripheral blood cells	Complete wound healing in 20 weeks; improvement in perfusion status and angiogenesis in the forefoot stump was demonstrated
Dash et al. (2009)⁴	Chronic lower extremity wounds	Bone marrow mesenchymal stem cells (BM-MSCs)	MSCs injected in and around ulcer, covered by dressing	Reduced ulcer size at 12 weeks; significant improvement in pain-free walking distance and reduction in ulcer size
Rogers et al. (2008)⁹⁶	Chronic lower extremity wounds	BM-MNCs	20 mL of bone marrow from the distal tibial metaphysis and placed directly on the wound	Complete wound healing in 47–60 days

Despite encouraging results from numerous preclinical studies, ongoing clinical trials of stem cell therapy have demonstrated moderate and inconsistent benefits, indicating an urgent need to optimize the cellular therapeutic platform. Human (h) MSCs have great potential for tissue engineering, but the challenge remains to generate functional cell types that are useful for transplantation. MSCs from different tissue sources can be cultured for differentiation to a specific cell type in precise conditions on scaffolds provided with a biomimetic microenvironment to mimic the features of natural ECM. Scaffolds made of polymeric biomaterials may interact with the cellular components and provide structural support for stem cell attachment, proliferation, and differentiation. Investigators have used a three-dimensional culture system for MSCs, and proved it to be a better option as compared to standard two-dimensional culture systems for reproducing the tissue environment and delivering cells for in vivo applications.

Pre-clinical evidence suggests that use of scaffolds combined with MSCs promotes osteogenesis. MSCs from rat bone marrow were cultured with collagen gel and a macro-channeled polycaprolactone scaffold with flow perfusion culture for osteogenic development of MSCs. This approach resulted in significant alteration of the transcription of bone-related genes.⁵⁸ Several biomaterials are currently available. Fibrin-based constructs are used for guiding the cells during tissue repair or regeneration due to their biocompatibility and biodegradability. Trombi et al.⁵⁹ used hMSCs and a fibrin scaffold, which differentiated into osteoblasts in vitro. Neuss et al.⁶⁰ cultured hMSCs on three-dimensional collagen scaffolds, and after a 50-day culture period, large numbers of mature adipocytes and osteoblasts were identified within the scaffolds. Nanofibrous scaffolds have been widely used to mimic the native ECM. Zhong et al.⁶¹ investigated MSC morphology on a biomimetic microenvironment, in this case an integrated microfluidic platform embedded with biomimetic nanofibrous scaffolds. Their report suggested that the crucial role of mechanotransduction in regulating fibrochondrogenic differentiation of MSCs may be mediated by the RhoA/Rho-associated protein kinase (ROCK) pathway and Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) pathway to play a key role in regulating cytoskeletal dynamics and stem cell differentiation.

Further profound understanding of stem cell niches and their effects on novel biomaterials will be useful for modeling stem cell therapeutic application technologies. Detailed knowledge on the role played by the scaffold design for enhancing stem cell differentiation needs further study.

Stem cell delivery system

Delivering stem cells to the wound remains a technical challenge for developing an optimized therapy. An ideal delivery medium is essential for cell adhesion, proliferation, migration, and differentiation of MSCs⁶² in the non-healing wound environment, characterized by increased proteolytic activity and chronic inflammation.⁵³ It should also be a simple applications procedure and less costly to the patients. Published results on the delivery systems that are effective in animal models may serve as a good vehicle for human DFUs patients (Table 4). One of the most common delivery systems used in DFUs is hydrogels. Hydrogels are three-dimensional insoluble polymer networks capable of absorbing and maintaining large amounts of water or biological fluids.⁶³ Hydrogels formed from polyethylene glycol (PEG) are useful as scaffolds for promoting stem cell growth and differentiation toward the formation of tissues.⁶⁴ Cellular functions that are important in determining and maintaining stem cell phenotype can be controlled in PEG hydrogels.⁶⁵ A biomimetic hydrogel system based on the polymer pullulan, a carbohydrate glucan known to exhibit potent anti-oxidant capabilities, can act as an effective cell delivery system and it can improve MSC survival and engraftment in high oxidative stress environments.⁶⁶ Rustad et al.⁶⁷ examined the capacity of a biomimetic pullulan–collagen hydrogel to create a functional biomaterial-based stem cell niche for the delivery of MSCs into wounds.

Table 4.

Stem Cell Delivery Systems Used in Preclinical Studies

References	Stem cell delivery system	Disease model	Stem cell type	Outcomes
Ballos et al. (2010)⁹⁷	Hyaluronan and methylcellulose (HAMC)	Retinal degenerative disease in mice model	Retinal stem-progenitor cell (RSPC)	RSPCs delivered in HAMC were more evenly distributed in the sub-retinal space than control.
Lü et al. (2010)⁹⁸	Scaffold with chitosan hydrogel was co-injected with nuclear-transferred embryonic stem (NTES) cells.	Rat myocardialInfarcted hearts	NTES cells	The NTES cells with chitosan hydrogel have possessed therapeutic potential to improve the function of infarcted heart.
Lu et al. (2009)⁹⁹	Temperature-responsive chitosan hydrogel alone or together with mouse embryonic stem cells (ESCs)	Rat myocardial infarcted hearts	Mouse ESCs	After 4 weeks of implantation, heart function, wall thickness, and micro-vessel densities within the infarct area improved in the chitosan+ESC injected areas.
Tang et al. (2010)¹⁰⁰	Gene and stem-cell therapies combined stem cell, chemokine, and angiogenic growth factor SDF-1-MSC, Ad-VEGF-MSC, or Ad-SDF-VEGF-MSC (Ad-SDF-1-VEGF-MSC)	Rat myocardial infarcted hearts	MSCs	Combination of chemokine, angiogenic factor gene, and stem cells could enhance angiogenesis and improves cardiac function after acute myocardial infarction.
Zhang et al. (2008)¹⁰¹	Collagen-based matrices with circulating progenitor cells (CPCs)	Ischemic hind limb of rats	CPCs	A collagen matrix enhances progenitor cell retention and limits distribution to nonspecific tissues.
Xiong et al. (2011)¹⁰²	Human embryonic stem cells (hESCs)-derived endothelial cells (ECs) and hESC-derived smooth muscle cells (SMCs) in a fibrin three-dimensional porous scaffold biomatrix	Post-infarction porcine model	hESCs-derived ECs and SMCs	The transplantation of hESC-derived vascular cells resulted in significant improvement in LV function.
Falanga et al. (2007)¹⁹	Mesenchymal stem cells (MSCs) using a fibrin polymer spray system with a double-barreled syringe	Non-healing lower extremity wounds in human and experimental wounds in mice	MSCs	Generation of new elastic fibers stimulated the wound repair process.
Piao et al. (2007)¹⁰³	Bone marrow-derived mononuclear cells (BM-MNCs) seeded on biodegradable poly-glycolide-co-caprolactone (PGCL) scaffold	Rat myocardial infarcted hearts	BM-MNCs	BM-MNC-seeded PGCL scaffolding influences BM-MNC migration, differentiation to cardiomyocytes, and induction of neovascularization.
Barandon et al. (2004)¹⁰⁴	Abdominal muscle patches BM-MNCs embedded in a collagen-rich three-dimensional matrix	Rat myocardial infarcted hearts	BM-MNC	Angiogenesis and cell survival in the scar were improved by patch+BMC treatment. Left ventricular geometry and cardiac function were improved.

SDF-1, stromal cell derived factor-1; Ad-VEGF, adenovirus expressing vascular endothelial growth factor.

Commercially available fibrin sealants have been the most widely used hydrogel technology⁶⁸ in wound healing because fibrin is biocompatible, bioresorbable, and essential in normal wound healing. There have been numerous studies to develop fibrin-based injectable cell delivery systems. Ho et al.⁶⁹ defined that the outcome of fibrin cell delivery depends on concentrations of fibrinogen and thrombin solutions. Furthermore, these investigators suggested that the concentrations of fibrinogen and thrombin solutions must be carefully considered for cell delivery because they affect cell proliferation. Bensaid et al.⁷⁰ reported that activity of fibrinogen and thrombin was optimal for producing fibrin scaffolds that would allow good hMSCs spreading and proliferation. Fibrinogen–thrombin formulation of fibrin sealant combined with fibroblasts and platelet-derived growth factor-BB (PDGF-BB) enhance cutaneous wound healing and suggest that the repair process can be enhanced by ex vivo cell delivery in fibrin sealant.⁷¹ Cultured autologous MSCs were applied to the wounds using a fibrin polymer spray system, and application of BM-MSCs in a fibrin hydrogel spray improves wound healing in mice and humans.¹⁹ Autologous biografts composed of autologous skin fibroblasts on biodegradable collagen membrane (Coladerm) in combination with autologous BM-MSCs⁷² resulted in an overall decrease in wound size and an increase in the vascularity of the dermis and in the dermal thickness of the wound.

Jiang et al.⁷³ developed a surface carrier of medical-grade silicone coated by plasma polymerization with a thin layer of acrylic acid (PPAAc) and used as a carrier to deliver human adipose tissue (AT)-derived MSCs to murine full-thickness excisional skin wounds in vivo. Carrier-delivered AT-MSCs were demonstrated to down-modulate TNF-α–dependent inflammation, increase anti-inflammatory M2 macrophage numbers, and induce TGF-β–dependent angiogenesis, myofibroblast differentiation, and granulation tissue formation, thereby enhancing overall tissue repair.⁷³

BM-MSCs delivered by an epidermal growth factor (EGF) microspheres–based engineered skin model may be a promising strategy to repair sweat glands and improve cutaneous wound healing after injury. It might be beneficial for BM-MSCs administration clinically.⁷⁴ From these results, it is clear that suitable delivery methods and formulation have a large impact on the in vitro and in vivo study.

Role of extracellular matrix

The ECM is the largest component of the dermal skin layer and a key feature of wound healing. The effects of ECM components vary with wound stage and are influenced by their interactions with cells and growth factors in a dynamic, reciprocal manner.⁷⁵ Microenvironments appear to be of prime importance in stem cell lineage specification. MSCs determine the specificity and lineage and commit themselves to phenotypes with extreme sensitivity to tissue-level elasticity.⁷⁶

An important factor is how the synthetic biomaterials mimic the natural ECM. Kim et. al.⁴⁹ suggested that MSCs in a three-dimensional collagen gel scaffold accelerated wound healing by early activation of MMP-9 and VEGF. Collagen is an attractive vehicle for MSC delivery because it is the predominant ECM component.⁷⁷ MSC adhesion, proliferation, and multipotent differentiation in vitro show that collagen-containing hydrogels are a good substrate for MSC culture.⁷⁸ Tay et al.⁷⁹ used fibronectin printed on poly(lactic-co-glycolic) acid (PLGA) thin film forming spatially defined geometries as a means to control the morphology of bone marrow–derived hMSCs.

With advances in the understanding the stem cell niche, it may be possible to develop advanced biomaterials that direct stem cell fate in novel ways. MSC encapsulation with PEG has been considered to be a good tool because it is porous, immunogenically nonreactive, and able to absorb large amounts of water, and its cross-linking density can be easily controlled. ^62,65 A major limitation of MSC encapsulation within synthetic materials is the cells' tendency to undergo apoptosis, resulting from lack of cell–ECM interactions.^80
–82 Cell–ECM interactions initiate many of the biochemical events responsible for cell proliferation, differentiation, and migration. Although these commercially available materials have been used to deliver other human cells,⁸³ they are less successful with MSCs because they lack a domain to which the adherence-dependent MSCs can attach. Cell attachment to ECM adhesive components is principally mediated by integrin (transmembrane heterodimeric surface receptors), which play a major role in cell survival and migration.⁸² Degradation of the synthetic polymers is another challenge.⁶² To address this challenge, investigators are attempting to incorporate adhesive components into the network, particularly those from the ECM that can improve therapeutic efficacy in chronic wounds.

Challenges for MSC Therapy

Adult stem cells could resolve the some of the problems that embryonic stem cells (ESCs) potentially face. Treatment of patients with their own cells avoids ethical issues and tissue rejections. The priority for stem cell therapy in recent times is patient safety and clinical efficacy. The majority of clinical studies on MSC therapy for DFUs are short-term analysis. A major difficulty encountered is the identification of stem cells from the population of cells isolated or cultured. Another important challenge is compatibility with the immune system of the patient, which is not a problem in the case of patients receiving autologous stem cells. Immunological rejection is a major barrier for heterologous stem cell transplantation, but it is not associated with autologous stem cell transplantation. MSCs home to tumors and contribute to the formation of tumor-associated stroma.⁸⁴ MSCs have the capacity to differentiate into fibroblastic-like cells known as tumor-associated (myo) fibroblasts (TAF), carcinoma-associated fibroblasts (CAF), fibrocytes, or pericytes within the tumor microenvironment.⁸⁵ In a suitable environment, production of growth factors, cytokines, chemokines, matrix-degrading enzymes, and immunomodulatory mechanisms by MSCs augments tumor progression, and the subsequent TAF phenotype arises from the MSCs, ultimately promotes tumor growth.⁸⁶ Interestingly, Hall et al. in 2007⁸⁶ suggested that MSCs can be genetically modified to produce high levels of anti-cancer agents that inhibit the tumor growth. MSCs within the tumor microenvironment may be useful as delivery vehicles for anti-tumor proteins. In situ administration of human autologous stem cell therapy in a patient with lupus nephritis has induced angiomyeloproliferative lesions at the sites of injection and hematuria.⁸⁷ Again, when MSCs are injected endogenously, immunosuppression is achieved. MSC therapy may compromise the host's defense against infections.⁸⁸

Treatment with MSCs in DFU patients is full of challenges. Failures may be due to different reasons, like, methodological flaws in the clinical trials. Lack of information on safety, method of recruitment, randomization methods, and blinding strategy for outcome assessments has also been reported. Development of techniques for cell identification within a mixed cell culture population and follow-up of transplanted cells will also be essential to ascertaining the potential of in vivo invasive therapeutic processes. The standard set for conventional drug should apply to MSCs in terms of purity, safety, and functionality. Simultaneously, the total cost of MSCs as a new therapeutic product should not challenge the total cost of patient care.

Future Directions and Conclusion

Improving methods of isolation, culture, and efficacy of treatment will firmly place stem cells as a better therapeutic option in the future. Preclinical studies suggest that BM-MSCs represent an effective and safe therapeutic strategy in the treatment of non-healing wounds. However, before considering MSCs for DFUs, certain conditions need to be taken care of.

Clinical use of cell therapy should focus on the important factors, like determination of medium in which stem cells should be delivered, culture conditions, and purity of cell population. More clinical studies on human subjects, including better data management of the patients and long-term follow-up of the patient's conditions, are necessary. We suggest certain procedures that may be useful for future use of MSCs in DFUs. First, bone marrow–derived EPCs carrying prospective candidate genes like VEGF ⁸⁹ serve better than bone marrow–derived EPCs alone in experimental therapeutic angiogenesis, as suggested by Iwaguro et al. ⁸⁹ In our view, more research work is required for establishment of this method, and most importantly development of virus-free medium will be a better option for therapeutic applications. Second, important aspect is improved culture systems that enable a purified version of cell therapy with specific cell marker expression. MSCs must be able to differentiate into osteoblasts, adipocytes, and chondroblasts in vitro ⁹⁰ and promote cell proliferation, collagen synthesis, growth factor release, neovascularization, and cellular recruitment to wounds and wound contraction. Third, improved stem cell delivery vehicles in large-scale human clinical trials may be promising for DFUs. For example, hydrogels interact with ECM components and appear to be an optimal approach for devising a stem cell delivery vehicle that can be adapted for clinical applications.⁹¹ Investigations to improve and optimize the function of synthetic delivery systems by incorporating ECM-adherent components to improve cell survival and migration may be a better option.

Stem cell transplantation to treat the diabetic foot is a promising treatment, and some patients have benefited from this technology. There are no serious complications or side effects, but its therapeutic mechanisms, effects, and standardization still require further research. In conclusion, cell-based therapy for diabetes foot disease has emerged as a highly promising therapeutic approach that will expand the benefits provided by current pharmacologic and revascularization approaches. It is difficult to completely cure diabetes or DFUs, but effective therapies can reduce their incidence through appropriate management and prevention programs.

Footnotes

Acknowledgments

The authors are highly thankful to SCB Medical College Cuttack Odisha, India, and Utkal University of Odisha, India, for providing support and internal funding to this work.

Author Disclosure Statement

No competing financial interests exist.

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