Accepted Manuscript

Title: Platelet rich plasma and growth factors cocktails for

diabetic foot ulcers treatment: state of art developments and
future prospects

Authors: I.V. Gonchar, A.R. Lipunov, I.M. Afanasov, V

Larina, A.P. Faller, A.V. Kibardin

PII: S1871-4021(17)30294-1
DOI: http://dx.doi.org/10.1016/j.dsx.2017.09.007
Reference: DSX 869

To appear in: Diabetes & Metabolic Syndrome: Clinical Research & Reviews

Received date: 22-8-2017

Accepted date: 20-9-2017

Please cite this article as: Gonchar IV, Lipunov AR, Afanasov IM, Larina V, Faller
AP, Kibardin A.V.Platelet rich plasma and growth factors cocktails for diabetic foot
ulcers treatment: state of art developments and future prospects.Diabetes and Metabolic
Syndrome: Clinical Research and Reviews http://dx.doi.org/10.1016/j.dsx.2017.09.007

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Platelet rich plasma and growth factors cocktails for dia-
betic foot ulcers treatment: state of art developments and
future prospects
Gonchar I.V.1, Lipunov A. R.2, Afanasov I.M3, Larina N.A.4, Faller A.P.4, Kibardin A. V.5,6, #
1 Center for Theoretical Problems of Physicochemical Pharmacology, RAS, Moscow, Russia
2 Moscow State University, Chemistry department, Moscow, Russia
3 GF Group LLC, Moscow, Russia
4 Pirogov Russian National Research Medical University, Moscow, Russia
5 Institute of Gene Biology of the Russian Academy of Sciences, Moscow, Russia
6 Dmitry Rogachev National Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia

Email address:
artem.lipunov112@gmail.com (Lipunov A.R.)
# Corresponding author: Institute of Gene Biology, Russian Academy of Sciences, Vavilova Str., 34/5, Moscow, 119334 Russia
e-mail: alexeyvk@igb.ac.ru

Abstract: Current advances in diabetic foot ulcers (DFU) treatment are discussed. Normal and pathological wound healing
process are observed and the role of growth factors (GFs) is elucidated. Current techniques involving GFs and platelet rich
plasma (PRP) are compared. Up-to-date research suggests that treatment with single growth factor (GF) could be insufficient
and not encompassing all pathological changes in DFU bed. Efficiency of PRP is rather controversial and lacks evidence. Thus
the use of cocktail of particular GFs is suggested. Pro et contra of each approach are discussed.

Keywords: growth factors, diabetic foot ulcer, platelet rich plasma, growth factors cocktail, diabetes

1. Introduction

Diabetes mellitus (also referred to as diabetes) is a highly-frequent and fast-spreading disease in the world. According to Inter-
national Diabetes Federation, 415 million adults (1 of 11) have diabetes. About 15 % of them suffer from diabetic foot ulcers
(DFU) which can lead to severe consequences if no appropriate treatment is provided. More than 88 % of lower limb amputa-
tions are caused by diabetic foot ulcers complicated by necrosis [1], gangrene and osteomyelitis [2, 3], because ulcers are high-
ly susceptible to infections [3]. These wounds are mostly chronic and are caused by several factors that result in different
pathways of DFU formation. Peripheral neuropathy hampers ability to feel pain and affect muscles involved in moving thus
callus is likely to appear. Patient’s awareness of wounding is decreased and ulcer can easily develop if no adequate treatment is
provided [4]. Diabetic angiopathy causes peripheral vascular disease and hampers perfusion on macrovascular level [5, 6].
Often ulcers with vascular insufficiency need revascularization by angiosurgery, thrombolysis and bypass surgery [4]. In con-
trast, neuropathic ulcers could be treated medically [7]. Also structural and functional changes of microcirculation occur that
lead to ischemia and changes in the metabolism of cells corrupting normal wound healing mechanism. Other common reasons
for ulcers are foot deformity, high plantar pressures, peripheral oedema, and external trauma, the latter one could cause DFU
[2].

Thus it is important to diagnose the disease at early stages and provide effective management to avoid dramatic consequences.
Current standard of care for DFUs starts with assessment for vascular disease, skin, soft tissue or bone infection and neuropa-
thy [8]. Conventional techniques for DFU treatment include debridement of the wound and offloading [9]. Debridement aims
at removing all devitalized tissues, including callus, necrotic, and infected tissue, and leaving only healthy one and effectively
converting a chronic wound into an acute one [10]. In experiments with DFU exudates placed in acute wounds significant in-
hibition of healing was observed [11]. Thus offloading is essential for eliminating further tissue destruction and is highly im-
portant for ulcers of neuropathic etiology [9]. A number of systematic reviews concluded that good-quality evidence for a ben-
eficial effect of this approach on ulcer healing is lacking and amputation often is still necessary [9].

Nowadays the outcome of existing standard methods is not sufficient, so that modern techniques should be widely introduced
to overcome existing limitations and improve wound healing process. These techniques include use of modern wound dress-
ings and bioengineered skin substitutes, negative pressure therapy, topical therapies, hyperbaric oxygen therapy, growth fac-
tors, electrophysical therapy and alternative therapy. Good-quality evidence is also lacking to justify the use of many of these
therapies [9].

It is reported that one of the potent origins of chronic pattern is an increased activity of metalloproteases [12] changing extra-
cellular matrix. However, there is strong evidence that changes in metabolism affects growth factor production in diabetic pa-
tients and this imbalance plays a major role in prolonged ulcer healing [13-15, 16].

Growth factors are biologically active polypeptide molecules that act in para- or autocrine manner and regulate chemotaxis,
cell migration, division, cellular proliferation, extracellular matrix formation and cellular interactions. It has been shown that
GFs play a central role in wound healing process [17]. The depletion of GFs is considered to be the main reason for chronic
pattern of DFU [18]. Thus GFs are considered to be perspective mechanism for DFU management. Efficacy of GFs in wound
management has been elucidated in several in vivo and in vitro studies on animal models and in clinical trials [19]. Nowadays
several products containing single GF such as REGRANEX, Hebermin, Easyef have been approved by regulatory authorities
of various countries, some others undergo laboratory tests and clinical trials.

It is known that platelets contain great variety of GFs that are released after platelet activation and initiate wound healing via
locally acting GFs [20]. Thus the usage of platelet-rich plasma was introduced and is used in clinics as adjunctive therapy. It is
prepared from autologous plasma by centrifugation and calcium activation and then PRP is injected in the wound. Variety of
GFs in PRP is considered to better enhance healing compared to single GF use [19].

However, the efficacy of all mentioned remedies is controversial and more detailed research is required [9]. In this paper we
encompass pro et contra’s of PRP, single GF and their cocktails in DFU treatment. We propose the use of GF cocktails as a
possible solution to boost the advantages of PRP and single GFs.

2. Wound healing and growth factors

Wound healing process is well studied and could be divided into three stages: inflammation, proliferation, and remodeling
[18]. At first, blood clot and fibrin-based matrix are formed; platelets are activated by collagen of cell matrix and platelets se-
cret mediators and cytokines from their granules [21]. Secondly, neutrophils and monocytes are attracted in the wound site.
Inflammatory cells exert GFs and other signal molecules that stimulate cells division and migration. Macrophages, exerting
GFs, control collagen deposition in the wound by fibroblasts [22]. Fibroblasts and other cells in tissue produce new GFs, epi-
thelial cells start to migrate and new blood vessels grow from pre-existing ones (angiogenesis). After that fibroblasts, keratino-
cytes and new blood vessels migrate into the wound site to produce granulation tissue [23]. Finally, reepithelialization and ma-
trix remodeling of the wound surface occurs, resulting in the formation of a relatively acellular mature scar. Epithelialization
occurs as cells migrate from the edge of the wound over a collagen-fibronectin surface [22].

However, in diabetic patients several deficiencies of wound healing are present. They include decreased or impaired GFs pro-
duction, impaired angiogenic response, altered macrophage function, collagen accumulation and faster remodeling of extracel-
lular matrix by metalloproteases [24]. These factors lead to impaired healing due to decreased migration of fibroblasts and
keratinocytes.

All stages of wound healing process are regulated by GFs, that are secreted by platelets and pro-inflammatory cells at first
stages of healing. These GFs alter activity and morphology of fibroblasts, keratinocytes and epithelial cells leading to changes
in extracellular matrix and production of GFs by cells in tissue. Both factors contribute to prolonged GFs presence in wound
site [23]. Also it should be noted that the mechanism of GFs influence is very complicated and involves great variety of signal
pathways [25]. In diabetic patients normal balance of different GFs and their concentration is corrupted, leading to abnormali-
ties of healing [18].

2.1. Platelet-derived growth factor

Platelet-derived growth factor (PDGF) is produced by platelets, macrophages, vascular endothelium, fibroblasts and keratino-
cytes [26]. In humans, PDGF is found in surgically created acute wounds, but in chronic non-healing wounds it is poorly pre-
sented [27]. Its concentration there is drastically decreased because of increased protease activity [28]. Platelets are the earliest
and largest source of PDGF that attracts monocytes into the wound that convert into macrophages and also secrete PDGF. It
also stimulates production of fibronectin and hyaluronic acid that are the main components of extracellular matrix. Also en-
hanced proliferation of fibroblasts results in increased collagen production [29]. PDGF is a chemoattractant and mitogen for
fibroblasts, smooth muscle cells and endothelial cells, and acts synergistically with transforming growth factor beta (TGF-β)
and epidermal growth factor (EGF). During epithelialization phase of wound healing, PDGF up-regulates the production of
insulin growth factor 1 (IGF-1) and thrombospondin-1 [29]. PDGF attracts inflammatory cells such as neutrophils, monocytes,
and fibroblasts. As a result, granulation tissue production is also increased. Although PDGF does not directly affect keratino-
cytes, wounds in animals have an increased rate of epithelialization [22]. It can be explained as follows: GFs from macrophag-
es and fibroblasts attracted into the wound site. Also it has been reported that diabetic patients have an increased plasma levels
of PDGF and EGF [30].

2.2. Transforming growth factors

Transforming growth factors (TGFs) are GFs that affect different types of cells and can reversibly stimulate or inhibit their
growth and transformations.

TGF-α in wound site is produced by macrophages and keratinocytes [22]. TGF-α as well as EGF is mitogen for keratinocytes
and fibroblasts, but TGF-α also stimulates angiogenesis.

TGF-β are a group of proteins that can reversibly inhibit growth of cells originating from ectoderm. Members of this protein
family are synthesized by platelets, macrophages, fibroblasts, keratinocytes and lymphocytes. These GFs are released at early
stages in a wound healing process and their deficiency increases time of wound healing. Broadly involved in wound healing
process, TGF-β1 serves as a chemoattractant for neutrophils, macrophages, and fibroblasts. It stimulates collagen synthesis,
granulation tissue formation and reepithelialization [31]. TGF-β1 alters growth or differentiation of many different cells. TGF-
β1 stimulates chemotaxis in inflammatory cells and trigger cells to produce extracellular matrix. TGF-β1 is a pleiotropic GF,
its action is dose-dependent. For instance, it enhances fibroblast proliferation at low concentrations, but induces differentiation
at high concentrations [23].
2.3. Epidermal growth factor

EGF is found in different tissues, including kidney, salivary glands and skin. EGF shares a receptor with TGF-α and increases
wound healing by stimulating fibroblasts, keratinocytes, vascular endothelium, smooth muscles and epithelial cells. EGF in-
creases the number of fibroblasts in the wound that produce collagen and improves the wound strength. Also this GF increases
motility of keratinocytes and promotes epidermal regeneration and corneal epithelialization as it has been shown on animal
models. EGF stimulates epithelial cells to grow across the wound, enhancing epithelialization in early human wound repair and
is reported to enhance healing in chronic wounds such as diabetic foot [23, 32].

2.4. Fibroblast growth factor

Fibroblast growth factor family (FGF) includes several GFs. In wound healing FGF2, FGF7 (also referred to as keratinocyte
growth factor — KGF) and FGF10 (or KGF2) are integrated. FGFs are synthesized by keratinocytes, fibroblasts, endothelial
cells, smooth muscle cells and chondrocytes [29]. FGF are mitogens for cells of mesodermal and neuroectodermal origin.
FGFs are potent mitogens for endothelial cells and function as angiogenesis factors by stimulating growth of new blood vessels
through proliferation of capillary endothelial cells. [22]. FGF2 or basic FGF (bFGF) is increased in acute wounds and plays a
role in granulation tissue formation, re-epithelialization and tissue remodeling. It enhances proliferation, differentiation and
migration of keratinocytes and fibroblasts. Also FGF2 increases collagenase production by fibroblasts. FGF2 is involved in
initiating granulation tissue formation, targeting angiogenesis in granulation tissue by stimulating the infiltration and prolifera-
tion of capillary endothelial cell resulting in shorter time of closure and increased strength of the wound [23]. The levels of
FGF2 are decreased in chronic wounds [33] hence they are less likely to close. Keratinocyte growth factor (KGF) is a member
of FGF family and is secreted only by fibroblasts and affects proliferation and migration of keratinocytes [23] supporting epi-
thelialization of the wound. Also KGF upregulates VEGF synthesis [29].

2.5. Insulin-like growth factor

Insulin-like growth factor (IGF) is a family of 2 proteins that have homology with insulin. In injured skin IGF1 is produced by
epithelial cells. IGF1 induces chemotaxis of endothelial cells and stimulates keratinocyte proliferation and re-epithelialization
of the wound [34]. IGF1 also activate synthesis of glycosaminoglycans that are the main compound of extracellular matrix
[22]. Both IFG1 and IGF2 enhance collagen synthesis in fibroblasts. IGF2 is important in fetal development. In mature skin
IGF2 is expressed in the epidermis. However, in diabetic skin IGF1 is impaired and such depletion could lead to delayed
wound closure.

2.6. Vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is similar to PDGF and is a potent angiogenic factor. It is released by keratinocytes
and epithelial cells during hypoxic conditions, although synthesis of VEGF is regulated by PDGF, FGF2 and TGF-β. VEGF
acts synergistically with FGF2 and placenta-derived growth factor [35], thus combination of these factors can be potent stimu-
lator of angiogenesis. VEGF causes increased vascular permeability and formation of blood vessels [23]. It activates mono-
cytes, but does not affect fibroblasts or vascular smooth muscle cells. Application of VEGF to the wound site improves granu-
lation tissue formation in ischemic rabbit wounds, although it has no effect on epithelium formation [37]. In diabetic patients
plasma concentration of VEGF is increased [38]. Disruption in microcirculation cause hypoxia and increased VEGF produc-
tion. It is reported that in diabetic patients depletion of VEGF in wound is a major cause of ulceration [39].

3. Platelet rich plasma

Platelets secrete vast variety of GFs after activation and degranulation. These GFs initiate wound healing process, therefore in
chronic wounds with reduced amount of native GFs platelet extract would be effective source of GFs. For the last 25 years
platelet-derived wound-healing formula (PDWHF) and PRP have been used. These are autologous preparations consisting of
the secretory granules of a patient’s own centrifugated platelets that are either lysated or activated [11, 31]. Preparation of PRP
involves obtaining several milliliters of blood and special systems for enrichment of the sample. The price of this procedure
strongly varies [40, 41]. Such plasma should contain not less than 1 million platelets per microliter and could serve as a defini-
tion of PRP [25]. Several protocols of PRP preparation are appreciated by FDA and variety of commercial devices are present
on the market. However, there is still no consistent research regarding PRP production process and its influence on the growth
factor levels in PRP.

It has been shown that there are differences in PDGF and TGF concentrations depending on the preparation protocol [42]. Not
every device is approved for human use and preserves viable platelets [20]. Often obtained PRP does not undergo any control
of early platelet activation, that leads to platelet poor plasma (PPP), or low platelet concentration which was demonstrated to
be inefficient in several studies.[21]. In this case we should note that usage of PPP coupled with PRP could have positive effect
as PPP contains nutrients viable for healing in ischemic conditions [43]. Moreover, legal constraints suppress wide use of PRP
systems in clinics. Thus further clinical trials on the basis of evidence-based medicine should be conducted.

PRP serves as drug delivery system, which transports GFs (PDGF, TGF-β, VEGF, EGF, IGF1), fibronectin and other molecules
to the wound site. It remains controversial [44] whether stimulatory effect of PRP on cells is caused by compounds from plate-
lets or high levels of GFs [25]. Nevertheless, a variety of active substances at high concentrations are released [21] and they
enhance healing in acute and chronic wounds through stimulation of cellular migration and proliferation [45]. PRP serves as a
growth factor agonist and has both mitogenic and chemotactic properties on various cell types, present in a wound, such as
fibroblasts, keratinocytes and epithelial cells [20]. It stimulates local production of GFs and enhances wound closure, improves
angiogenesis and re-epithelialization. Also it has been reported that PRP may suppress cytokine release and limit inflammation,
interacting with macrophages to improve tissue healing and regeneration [46].

As of 2017, no large statistically significant and evidence-based research was conducted [41]. Existing trials confirming posi-
tive effects of PRP [19] can be considered methodologically questionable or do not show significant enhancement of DFU
healing compared to control group or patients treated with standard techniques [9, 47]. However, significant enhancement of
DFU healing with PRP in combination with other therapies is shown [9].

4. Growth factors treatment

4.1. Single growth factors

As it has been already mentioned, according to corrupted wound healing process in DFU and major role of GFs in its activa-
tion and regulation, topical use of GFs is considered to be perspective method to treat DFU in order to replenish lack of GFs
[9]. Some researchers underline that efficacy of GFs could be reduced because of increased protease activity in wound bed or
deficient knowledge about signal pathways in wound healing [11]. To prolong GFs presence in wound, several matrices for
GFs preserving have been recently introduced, such as bioengineered skin substitutes [3, 22, 49] and liposomes [50].
Bennett et al studied healing of full-thickness skin wound ingenetically diabetic mice [51], and attributed non-optimal wound
healing rate to the imbalance in growth factors. Application of EGF, TGF-α, FGF, and the combination of PDGF and IGF-I
increased epithelialization rate by 20–30 %. It was also noted that prolonged exposure of cells to GFs via matrices significantly
increase the effectiveness of the treatment.

However, available data show controversial results of such approach for DFU treatment. One of the GF products, that has been
approved by FDA for use on neuropathic DFU, is recombinant PDGF in REGRANEX gel (Smith & Nephew) [52]. It decreas-
es wound healing time and increases proportion of healed ulcers only when it is used as adjuvant therapy. However, its effect is
neglected in 3 months perspective [22]. Meta-analysis also shows positive effect at short-time range but available data poorly
supports the efficacy of PDGF at long-term scale therefore further studies should be performed [41].

TGF-β was tested in diabetic ulcers [53] and showed better results compared with collagen sponge, however, patients treated
with standard care healed better than those patients treated with TGF-β. Efficiency of EGF on animal models was verified in
studies on pigs and rats [23]. EGF has been tested in various in vivo and clinical studies. [22] shows that positive effect was
observed but there is a lack of data. Review of valid clinical trials [9] revealed several trials with recombinant human EGF that
had superior effect in comparison to placebo. 41] Game F. et al report improvement in DFU healing under EGF application for
12 and 16 weeks in several clinical trials [41].

FGF2 in several studies did not induce any altered wound healing compared to placebo in 12 weeks [9, 41]. However, the use
of FGF2 leads to significant reduction (75 %) of ulcer area but methodological drawbacks weaken positive result [54]. Recom-
binant human FGF10 — repifermin — has been used in clinical trials and shoved controversial results: one group report about
75 % better closure over placebo [55], but in another reported no significant difference in time of closure after 20 weeks of
topical repifermin application was revealed [56].

Two clinical trials (phases I [57] and II [58]) have been conducted with recombinant human VEGF (Telbermin, Genentech,
Inc.) in patients with neuropathic DFUs. It showed positive effect of reduced closure time. Another group of researchers conju-
gated collagen-binding domain and VEGF. This construct, named CBD-VEGF, was put on collagen matrix and showed better
performance in wound healing, great cellularization and vascularization [59].

4.2. Growth factors cocktails — perspectives

All encompassed data indicate that neither PRP nor single GF could be regarded as reliable and sufficient therapy for DFU.
PRP needs laboratory equipment and medical personnel to conduct the procedure, it is invasive and the benefits of PRP therapy
are lacking scientific proof, however further investigation is required. Single GFs affect only particular types of cells and can-
not shift the imbalance that emerges in DFU due to pathological physiology of tissues. Current evidences are not sufficient for
clinical use of single GFs. Therefore new techniques and methods should be studied and introduced in clinical practice.

According to physiology of wound healing and conditions in DFU, normalization of GF concentrations and relative propor-
tions should have groundbreaking results. However, this aim could not be reached with single GFs applied onto a wound sur-
face. We propose to use a cocktails (mixtures) of GFs that would create and keep the “healing balance”, hence supplying cells
in DFU with insufficient GFs and avoiding inhibitory role of proinflammatory cytokines and proteases. Optimal ratio between
different GFs in that mixture remains to be investigated and may differ significantly from such found in tissue under normal
physiological conditions. Such an approach has several advantages:

1) Optimization and balancing the healing process.

Since wound healing is a complex process that consists of several stages, each one promoted or inhibited by different GFs and
at every stage of wound closure diverse GFs are required, a cocktail of GFs would precisely optimize the healing process and
influence each cell involved (for example: fibroblasts, keratinocytes, macrophages, dendritic cells, endothelial cells and oth-
ers).
As wound environment and activity of metalloproteases are changing over time in wound healing process, various GFs should
be delivered at wound site in different concentrations. To enhance stability of mixtures of GFs and to promote their gradual and
controlled release GFs can be physically/chemically conjugated with “storing and delivery vehicles” - polymeric matrices/gels
or liposomes.

2) Normalization of paracrine regulation.

GFs in the cocktail can influence various signal pathways inducing cell proliferation and GFs synthesis.

3) Modern technologies of recombinant protein biosynthesis allowed to obtain animal and human component free GFs, avoid-
ing possible contamination with pathogens (especial by viruses) which lead to high safety of the approach. Moreover artificial
mixture could be prepared in personal way, according to particular cytokine insufficiency of an individual patient.

5. Conclusions
Existing methods and techniques for DFU treatment exhibit limited efficacy. Novel approaches are currently implemented,
including the use of single GF and PRP - some of them show prominent results and several products are already on the market.
The use of GFs cocktails combines the benefits of PRP and single GF and could be suggested as an alternative approach to
treat DFU. The main problem for future investigation is an absence of proper animal models both for study DFU pathogenesis
and treatment efficiency of materials of human origin without species specificity. In this way further detailed clinical trials of
GFs cocktails with particular well characterized lists of recombinant proteins in compositions are promising and most required.

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