Diabetic ulcer regeneration: stem cells, biomaterials, growth factors

Abstract

The impairment of ulcer wound healing in diabetic patients is a vital clinical problem affecting millions of patients. Several clinical and basic science studies have demonstrated that stem cell therapy, to be effective in healing diabetic ulcer. Furthermore, these ulcer wounds may be healed from molecular maneuvering of growth factors to improve microcirculation within the ulcer wound. In addition, ulcer wound dressings may be employed as medicated systems, through the delivery of drugs, growth factors, peptides and stem cells. These dressing materials can include natural, modified and synthetic polymers, as well as their mixtures or combinations. This review paper will give a summary of some of the recent advances on the application of stem cells, biomaterials and growth factors in the treatment of diabetic ulcer wound.

Keywords:

• Diabetic ulcer
• growth factors
• wound dressings
• stem cells
• biomaterials

Introduction

It was reported that about 36 million adults live with diabetic as at 2011. Worldwide global projections indicate that there will be an increase to about 552 million adults by 2030. In North America and Europe, the number of adults with diabetes is expected to increase by 42.4% and 20%, respectively [1,2]. The diabetic ulcer, a major complication of diabetes mellitus, has remained an important clinical challenge. Presently, the standard for the diagnosis and classification of diabetic ulcer is the Wagner classification system and the University of Texas Diabetic Wound Classification system [3]. Local ulcer wound care with dressing and repeated debridement of necrotic tissue is typical clinical treatment of diabetic ulcer. However, the results are not encouraging, and about 14–20% of the patients with diabetic ulcer still get amputated. Several methods have been developed for diabetic ulcer wound healing, based on inflammation or growth factors [4,5]. However, stem cell therapy has been proven to be promising as clinical and non-clinical science studies have demonstrated that these therapies can be useful in providing solution by tackling multiple factors during diabetic wound healing, including cell proliferation, extracellular matrix (ECM) synthesis, growth factor release, and vascularization.

Galkowska and colleagues reported that the analysis of human diabetic ulcers shown the differential expression of growth factors, chemokines, cytokines, and their receptors, which are important to different phases of the normal wound-healing process [6]. In addition, matrix-metallo-proteinases has been identified to be involve in tissue remodelling are also differentially expressed in chronic wounds, thereby leading to the dysfunctional breakdown of the extracellular matrix [7]. Khanna et al. stated that macrophages isolated from diabetic ulcerative mice demonstrated a reduced ability to phagocyte dead cells, thereby leading to a prolonged inflammatory response [8]. Tumour necrosis factor-α (TNF-α), a potent pro-inflammatory cytokine, are responsible for the stimulation of fibroblasts and keratinocytes, the expression of growth factors and up-regulation of antimicrobial defences [9], however, in diabetic ulcer wound healing, increase in TNF-α causes an impairment in the proliferation of fibroblast [10]. Xu and co-workers also reported that inhibition of angiogenesis; proliferation, differentiation, migration and an increase in apoptosis are all as a result of high levels of TNF-α. In addition, they reported that TNF-α inhibition mitigates the effect of diabetes-enhanced TNF-α which provides potentially new therapeutic option for treatment of chronic diabetic foot ulcers [11]. In this review paper, we will give a summary of some of the available recent advances on the application of stem cells, biomaterials and growth factors in the treatment of diabetic ulcer wound.

Growth factors and diabetic ulcer wound healing

Growth factors, or cytokines, are biologically active polypeptides that are responsible for modifying the growth, differentiation and metabolism of target cells [12]. Growth factors can act by both paracrine and autocrine mechanisms, and can bring about cellular communication via binding to specific cell surface receptors with the resultant induction of a complex cascade of signal transduction pathways (Figure 1). Increased expression of various growth factors has also been reported in numerous studies, including EGF, KGF, TGF-beta1, VEGF and PDGF [13,14]. These growth factors contribute to the repair, the regeneration, and the neovascularization in the diabetic wound (Table 1).

Figure 1. Showing schematizes the phases and growth factors involved in diabetic wound healing processes in comparison with a regular wound. The cells involved include blood platelets, red blood cells, epithelial cells (blue coloured), fibroblast (green coloured), macrophages, fibrin, neutrophils and blood vessels (red) [78].

Table 1. Showing principal growth factors involved in wound healing [79].

Growth factors	Repair process	Source	Target cells/effects in wound repair	Clinical uses
Platelet-derived growth factor (PDGF)	Inflammation, tissue formation and tissue remodelling	Platelets, macrophages,endothelial cells	Chemo-attractant forneutrophils and fibroblasts, smooth muscle cells.	Neuropathic diabetic foot ulcers treatment
Fibroblast growth factor (FGF2)	Tissue formation and tissue remodelling	Fibroblasts, endothelial cells	Mitogenic for endothelial cells, fibroblasts and keratinocytes.	Have been demonstrated in pressure ulcers but no known effect on diabetic foot ulcers
Keratinocyte growth factor (KGF)	Tissue formation	Fibroblasts	Mitogenic and chemotactic for keratinocytes	No reported clinical trials
Epidermal growth factor (EGF)	Tissue formation	Platelets, macrophages and keratinocytes	Mitogenic for fibroblasts,endothelial cells and keratinocytes	There have been effects on venous ulcer but they are insignificant. No reported clinical trials on diabetic ulcer.
Transforming growth factor (TGF-β)	Inflammation, tissue formation and tissue remodelling	Platelets, macrophages,fibroblasts, keratinocytes, neutrophils	Stimulates fibroplasia and angiogenesis.	No reported clinical trials
Vascular endothelial growthfactor (VEGF)	Tissue formation	Neutrophils, platelets	Stimulates the process of angiogenesis	It promotes collateral blood vessel formation in peripheral arterial disease
Granulocyte colony-stimulating factor (G-CSF)	Inflammation	Monocytes, fibroblasts and lymphocytes	Formation of neutrophils	There has been report of clinical studies on diabetic foot ulcer.

Tables 1 and 2 show several growth factors involved in diabetic ulcer wound healing and randomized clinical trials of healing of diabetic foot ulcers respectively.

Table 2. Showing randomized clinical trials of growth factors in diabetic foot ulcers [79].

Growth factor	Trial	Results	References
Platelet-derived growth factor (PDGF)	A double blinded and multicentre clinical trial on 118 patients	There was evidence of ulcer healing at 20 weeks: p = .01 There was reduction in ulcer volume at 20 weeks: p = .09	[15]
Platelet-derived growth factor (PDGF)	A double blinded and multicentre clinical trial on 379 patients	There was evidence of ulcer healing at 36 weeks: p = .007 The time to healing was 127 days; p = .013	[16]
Platelet-derived growth factor (PDGF)	A double blinded and multicentre clinical trial on 250 patients	There was evidence of ulcer healing at 36 weeks	[17]
Fibroblast growth factor (FGF)	A double blinded clinical trial on 17 patients	There was evidence ulcer healing at 33 weeks; p = .23	[18]
Granulocyte colony-stimulating factor(G-CSF)	A double blinded clinical trial on 40 patients	There was ulcer healing at 7 days: p = .09 The time to resolution of cellulitis was after 12 days; p = .03	[19]
Granulocyte colony-stimulating factor (G-CSF)	A double blinded clinical trial on 40 patients	There was amputation by 9 weeks: p = .038 The duration of antibiotic therapy was two months and 2 days	[20]
Platelet-derived wound healing factors (PDWHFs)	A double blinded clinical trial on 13 patients	There was evidence of ulcer healing: five of seven vs. one of six There was reduction in ulcer area; p < .02	[21]

Stem cell therapy for diabetic ulcer

Stem cells are also referred to master cells, capable of both self-renewal and multi-lineage differentiation [22]. Stem cell therapy, an interventional approach that involves the treatment of a disease or injury by the administration of adult stem cells into the damaged tissue, has also been studied as a treatment option for diabetic ulcers [22]. There have been several research reports and results in preclinical models of diabetic ulcer wound healing. Mesenchymal stem cells (MSCs) have been demonstrated to suppress the local immune response, decrease inflammation, and via secretion of growth factors MSCs are able to stimulate the differentiation and proliferation of local progenitor cells [23]. Aggarwal et al. demonstrated in their report that MSCs are capable of reducing inflammation by reducing the amount of pro-inflammatory cytokines; TNF-α and interferon-γ [24]. Nakamura and co-workers were able to develop new granulation tissue and form new blood vessel in their preclinical models of diabetic wound healing via MSCs [25,26], and increased wound closure. Wu and colleagues were able to demonstrate in their animal studies that when MSCs are injected into a diabetic ulcer wound of a model mice, there is an increased rate of wound closure, re-epithelialization, and angiogenesis [27].

In another small clinical study by Yoshikawa et al., they demonstrated the effectiveness of MSCs in the treatment of chronic diabetic ulcer wounds [28]. Zou et al. also reported that angiogenesis is vital for diabetic ulcer wound healing and they have demonstrated that the pro-angiogenic nature of MSCs and their ability to secrete platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF) [29]. In another large level I RCT study by Dash et al., where they used bone marrow-derived MSCs in wounds of the limbs [30]. They concluded that their study trial demonstrated that MSC therapy significantly reduced ulcer wound size. In addition, another study in China showed improved ulcer healing rates with bone marrow mesenchymal stem cells (BMMSC) [31].

Mesenchymal stem cells (MSCs) and diabetic ulcer wound healing

Mesenchymal stem cells are the most widely and common stem cell used in clinical and non-clinical science studies and also known as stromal cells, fibroblast-like self-renewing stem cells in the bone marrow. Several studies have demonstrated that MSC transplantation can have several positive effects on diabetic wound healing, like promoting cell proliferation, collagen synthesis, growth factor release, wound contraction, neovascularization, and cellular recruitment to diabetic ulcer wounds [32].

Kwon et al. reported in a rat diabetic ulcer wound healing model, that systemic and local treatment with BM-MSCs on diabetic wounds enhanced the breaking strength through increased type I–V collagen in the ulcer wound bed [14]. In another recent study Mizuno and Lee, they showed that CD34 + and CD34 − adipose-derived stromal cells (ADSCs) have a better proliferative capacity and a greater differentiating capacity respectively [33,34]. Kim and colleagues also reported their findings in diabetic nude mice where they studied the effect of human adipose-derived stem cells on ischemic wounds. They concluded that ADSC-treated animals showed signs of early formation of new blood vessels and better tissue remodelling when compared to the control group. In addition, they reported that there was a lower rate of auto-amputation and a higher survival rate in the ADSC-treated group [35]. Furthermore, Lee and co-workers used a human ADSC culture medium to treat human keratinocytes and dermal fibroblasts in an in-vitro study. They concluded in their study that there was an increase in cell proliferation in both cell types and in addition, there was an increase in the expression of type I collagen [33].

Bone-marrow-derived mononuclear cells (BM-MNCs) and diabetic ulcer wound healing

BM-MNCs are a group cells comprised of different kinds of stem cells and differentiated cells [36]. There have been several reports of application of BM-MNCs in the treatment of diabetics ulcer wound. Sivan-Loukianova and colleagues reported that in a diabetic mouse wound healing model, BM-MNCs treatment could improve and accelerate epidermal healing and promptly and radically fast-track revascularization of the ulcer wounds [37]. In another clinical study, Ruiz-Salmeron et al. performed an autologous MNC transplantation in diabetic patients with peripheral artery disease. They reported after 3 to 12 months, that all patients showed signs of clinical improvement with a significant proliferation in vascular network [38].

Furthermore, Wu et al. suggested that MNC transplantation can lessen local inflammation [39]. Pedroso and colleagues also confirmed the ability of MNCs to differentiate into endothelial cells, thereby speeding up neovascularization [36,40]. Table 3 below shows different application and efficacy of several stem cells especially BM-MNCs in the treatment of diabetic ulcer.

Table 3. Showing clinical trials of stem cell therapy on the treatment of diabetic ulcer [80].

Clinical trials	Diabetic ulcer cases	Stem cell therapy	Efficacy
[41]	24	BM-MNCs/BM-MSCs	80% of the 24 cases significantly improved.
[42]	8	BM-MNCs	There was total wound healing in 3 cases and the other 5 cases improved significantly.
[43]	25	BM-MNCs	There was an increase in the rate of chronic lower extremity wound healing.
[30]	12	BM-MSCs	There was improvement evidence in pain-free walking distance and decrease in ulcer size.
[44]	3	BM-MNCs	There was evidence of wound healing.
[28]	20	MSCs	There was evidence of wound healing in 18 of the 20 cases.
[45]	1	BM-MNCs	There was evidence of total wound healing.
[46]	4	BM-MNCs	Total wound healing was achieved.
[44]	8	MSCs	It took an average of 7 weeks after surgery for all wounds to be healed totally.
[47]	1	BM-MNCs and bFGF	There was evidence of total wound healing.
[48]	2	BM-MNCs	Decrease in wound size was achieved and evidences of increased vascularization were noted.
[49]	1	Fibroblast and MSCs	There was evidence of both closing and healing of the non-healing diabetic ulcer.
[50]	3	BM-MNCs	There was dermal renewal and evidence of closure of non-healing chronic wounds.

There have been extensive reports on the application of stem cells and growth factors on specifically on the treatment of diabetic foot ulcer DFU. Table 4 gives a summary of different treatment options for DFU.

Table 4. Showing application of stem cell and growth factors for diabetic foot ulcer (DFU) treatment [78].

Bioactive substances	Models used	Results	References
VEGF	db/db diabetic mice	There was an improved neovascularization, mobilization of bone marrow-derived cells into the wound site to fast-track the wound healing process.	[51]
PDGF	Streptozotocin (STZ) diabetic rats	The process of angiogenesis was enhanced as well as cell proliferation and epithelialization.	[52]
bFGF	Human patients with DFUs	There was about 75% decrease of the wound area in treated patients.	[53]
SDF-1α	STZ diabetic mice	There was an increase in EPC mobilization, homing process and as well as wound healing process.	[54]
BMSCS	STZ diabetic rats	The process of wound healing was accelerated and growth factors like TGF-β, KGF, EGF and VEGF were expressed.	[14]
CD133+ cells	STZ diabetic rats	Wound closure was fast tracked and promoted angiogenesis	[55]
Human adipose-derived stromal cells	db/db diabetic mice	Wound closure was fast tracked and that stimulated production of extracellular matrix proteins.	[56]
Embryonic stem cells	STZ diabetic rats	There was evidence of decreased in wound size significantly and as well as increased expression of EGF and VEGF	[57]
EPCs	db/db diabetic mice	Accelerated wound healing and induced expression of VEGF and bFGF	[58]

Biomaterials and diabetic ulcer therapy

Several component polymeric materials, demonstrating diverse chemical, physical and biological properties has been reported to be used in the treatment of diabetic ulcer [59]. In addition, modified polymeric materials of diverse polymers and copolymers have also been reported. [60]. Polymers-based biomaterials have been reported to be used in DFU treatment and are presented in the Table 5 below.

Table 5. Showing natural and synthetic poly based biomaterials used in DFUs treatment [78].

Polymers	Bioactive substance	Models used	Results	References
Chitosan-crosslinked collagen	Recombinant human aFGF	STZ diabetic rats	There were evidences of fast tracked wound healing process	[61]
Chitosan with different degrees of deacetylation	Acetylglucosamine oligomers	Human diabetic	There was reduction in the wound size and the process of angiogenesis was stimulated.	[62]
Thiolated chitosan-oxidized dextran hydrogel		STZ diabetic mice	There were non-cytotoxicity and resistant to degradation.	[63]
Chitosan, alginate, and poly(γ-glutamic acid) hydrogel		STZ diabetic rats	There was improvement in wound healing and increased deposition of collagen and hydroxyproline levels.	[64]
Microbial-derived cellulose hydrogel	Polyhexamethylasebiguanide (PHMB)	Human patients with non- healing ulcers	There was an inhibition of bacteria proliferation, provided an optimal moist healing micro-environment. There was also removal of necrotic tissue
Carboxymethyl cellulose hydrogel	Chestnut honey	db/db mice	There was reduction of wound area significantly and an increase of tissue granulation.	[65]
Alginate hydrogel	Phenytoin	Human patients with DFUs	Infections were removed and there was reduction in pain level.	[66]
Alginate gel	Honey	Human patients with DFUs	The rate of healing was fast tracked and increased tissue reepithelization
Gelatine microspheres	bFGF	db/db mice	There was decreased rate of infection, accelerated fibroblast proliferation.	[67]
Collagen matrix, Integra^® (Integra LifeSciences Corp., USA)		Human patients with DFUs at high risk of amputation	Saved 46% of the limbs and increased the rate of bacterial clearance.	[68]
Collagen–gelatine foam	bFGF	db/db mice	Fast tracked dermis tissue formation and stimulated increase in number of new capillaries.
Dextran-allyl isocyanate-ethylamine/polyethylene glycol diacrylate hydrogel		Human patients with chronic wounds	There was evidence of dermal regeneration and accelerated the early inflammatory cell infiltration	[69]
PVA aminated hydrogel	NO	db/db mice	There were evidences of increased collagen production	[70]
PCL-PEG block copolymer	rhEGF	STZ diabetic mice	Induced accelerated wound healing process.
PEG-PCL nanofibres	EGF and bFGF	STZ diabetic mice	There were evidences of increased accumulation of collagen and keratin.	[71]
PEGylated fibres	rhaFGF	STZ diabetic rats	Fast tracked rate of wound closure and tissue collagen formation as well as TGF expression.	[72]
Poly(ethylene imine) and PEG	Plasmid bFGF	STZ diabetic rats	There was increase in wound recovery rate significantly and improved collagen deposition.	[73]
Poly (vinyl methyl ether co-maleic anhydride) and PVP	NO	STZ diabetic rats	Accelerated wound closure was evident.	[74]
PHEMA and by PEG hydrogel	Matrix metalloproteinase(MMP) inhibitors	Human patients with DFUs	MMPs in the chronic wound fluid are inhibited.
PLGA microspheres	rhEGF	STZ diabetic rats	The growth rate of fibroblasts was significantly improved.	[75]
PCL nanofibres	Curcumin	STZ diabetic mice	These nanofibres improved the rate of wound closure.	[76]
PLA fibres	Curcumin	STZ diabetic mice	These nanofibres augmented the rate of wound closure.	[77]

Conclusion

Diabetic ulcer remains a critical clinical challenge over this past few years and much research work has been directed towards the development of an efficient novel therapeutic approach for its treatment. Stem cell transplantation can provide systemic enhancement to the wound site, including improved cell proliferation, extracellular matrix synthesis, growth factor release, and neovascularization. However, there is still concern that these stem cells may not be oncologically safe as reported by some studies that stem cells may promote tumour growth. Effects of growth factors on wound healing has also been promising from in vitro and in vivo studies, however, this has not been translated into practical clinical treatments. Furthermore, development of more efficient and less expensive biocompatible and biodegradable medicated dressings that can deliver important DFU healing factors to the wound site in order to improve patient care and quality of life.

Disclosure statement

The authors declare that they have no conflict of interest.