Effect of platelet rich plasma in diabetic foot healing

PRP (platelet rich plasma) is a natural source of growth factors obtained from the patient's blood which are involved in the wound healing process. PRP therapy is successfully applied in the medical field, being used nowadays in diabetic patients who develop as complication the diabetic foot. The purpose of this review is to present the composition of PRP growth factors, PRP preparation stages and the results obtained in patients diagnosed with diabetic foot.


IntroductIon
PRP is an autologous biological product that is obtained from the patient's blood following a centrifugation process. This endogenous therapeutic produs is increasingly used in regenerative medicine due to its potential to stimulate and accelerate the healing process. PRP growth factors are polypeptide substances that regulates cell growth, proliferation, differentiation and metabolism for different cell types (1,2). PRP promotes endothelial and epithelial regeneration, stimulates angiogenesis, collagen synthesis, homeostasis and soft tissue healing (3).
In a healthy person the normal number of platelets varies between 150,000 and 350,000 cells/µl of blood. Platelets are small, discoid, nucleus-free cells that are formed in the bone mar-row (from megakaryocytes), cannot reproduce and have a lifespan of about 7-10 days. Platelets are involved in hemostasis and coagulation. When bleeding occurs, platelets are activated and release growth factors that stimulate the inflammatory cascade and healing process. Proteins and other biomolecules are secreted by three types of granules (alpha, delta and lambda) located inside the platelets. α granules are the most abundant, representing 10% of platelet volume (50-80 α granules per platelet) and secretes over 300 soluble proteins (4,5).

StageS of wound healIng
The wound healing process is a dynamic one and includes the following phases: • hemostasis • inflammatory response • cell proliferation, reepithelialization, granulation and angiogenesis • matrix remodeling and scar formation Immediately after skin injury, platelets are stimulated to aggregate themselves to form the fibrin clot and recruitment of different cell types. Growth factors, cytokines and other hemostasis-specific factors are activated during the first phage. The inflammatory stage is characterized by the release of histamine, serotonin, bioactive factors that lead to the arrival of inflammatory cells at the site of injury such as neutrophils, leukocytes and macrophages. Neutrophils with the help of macrophages are involved in the prevention of bacterial infections and activates keratinocytes, fibroblasts and immune cells. At the end of the inflammatory phase, the macrophages release growth factors that will be involved in the third stage of wound healing. Extracellular matrix remodeling is a complex process that includes the participation of cytokines, growth factors, which promote cell migration, epithelialization, adhesion and wound contraction (11).

the role of growth factorS In the wound healIng proceSS
Currently, PDGF is approved in USA by the FDA (Food and Drug Adminstration) and European Authority for clinical applications in patients. Is one of the first growth factors that are secreted after injury, promoting many metabolic processes such as collagen and protein synthesis, chemotaxis of fibroblasts and smooth muscle cells, as well as collagenase activity (9,12). PDGF present angiogenic effects promoting the proliferation and migration of endothelial cells.
It can stimulate the production of TGF-β which initiates the synthesis of coalgen, but also the production of IGF (9,12). PDGF present strong chemotactic effects on neutrophils, monocytes, smooth muscle cells, promoting mitosis of fibrobalts, smooth muscle cells and endothelial cells. This growth factor stimulates the process of angiogenesis, wound contraction, granulation tissue formation and finally wound remodeling. PDGF is secreted in the proliferative stage of wound healing. In addition to platelets, macrophages, keratinocytes, endothelial cells and muscle cells can secrete PDGF (9)(10)(11)(12).
EGF is involved in the epithelialization phase of wound healing, being synthesized by platelets, fibroblasts, macrophages and mesenchymal stem cells. Plays a vital role in proliferation, differentiation, growth and migration of epithelial and keratinocyte cells (13,14).
Macrophages, endothelial cells, fibroblasts, T lymphocytes and mast cells secrete FGF, being involved in the proliferative phase of wound healing ( in re-epithelialization, angiogenesis) (15). It stimulates fibroblast proliferation, collagen accumulation and accelerates the formation of the granulation tissue (16). IGF is implicated in the proliferative and inflammatory phase of wound healing, being secreted by fibroblasts on which it exerts autocrine effects. IGF in combination with PDGF promotes keratinocyte maturation, enhancing tissue repair (17,18).
VEGF is secreted by platelets, fibroblasts, macrophages and keratinocytes, is active in the proliferative phase of wound healing. VEGF present paracrine effects on endothelial cells, is the main growth factor promoting granulation tissue angiogenesis (19,20).
TGF-β growth factors family, includes 3 isoforms: TGF-β1,TGF-β2 and TGF-β3 which are in-volved in different stages of wound healing. Are secreted by platelets, macrophages, T lymphocytes, endothelial cells, fibroblasts, muscle cells and keratinocytes. TGF-β1 and TGF-β2 are present in the inflammatory phase, TGF-β3 is active in the remodeling phase of the wound healing process (21,22). TGF-β3 is involved in the angiogenesis process, and the first 2 isoforms in fibrosis and scarring. These growth factors promote differentiation of myoblasts and fibroblasts, extracellular matrix formation, wound contraction and finally wound healing (23). KGF is present in the epithelialization of the healing process, inducing keratinocyte proliferation and migration (16,17).

prp productIon
Currently, there are many different commercial products, capable of obtaining PRP from the patient's blood usually through two centrifugation processes. After the first centrifuge, the red blood cells will be separated from the plasma and will be found in the bottom of the centrifuge container. Above the erythrocytes layer, buffy coat of white blood cells is formed, platelets will have the highest concentration in plasma just above the buffy coat (24,25).

prp preparatIon StageS
• Blood collection: around 30 ml of venous blood is collected into tubes containing sodium citrate anticoagulant to prevent platelet activation • First stage of centrifugation: the blood is first centrifuged with reduced forces (soft rotation from 200 g to 600 g): three layers are separated: the upper layer containing platelets and white blood cells called platelet-poor plasma (PPP); an intermediate thin layer rich in white blood cells; and the bottom layer containing most of the red blood cells • Second stage of centrifugation: takes place at a higher speed (hard rotation from 700 g to 2,300 g) to concentrate platelets: soft pellets (erythrocytes-platelets) are obtained at the bottom of the tube; the lower one-third of the tube which contains around 5 mL of plasma is homogenised by gently shaking to create PRP (26).

prp therapy and dIabetIc foot treatment
Diabetes is a global health problem, both in developing and developed countries, considering that by 2030 there will be 366 million diabetic patients. One of the most serious complications of diabetes is the diabetic foot, most often leading to foot amputation (27,28).
To test PRP effects on diabetic subjects, many studies were performed on laboratory diabetic animals. Clinical and experimental studies in animals or humans have reported that VEGF accelerates wound healing by increasing epithelialization, angiogenesis and granulation tissue formation (29)(30)(31).
Nashwa Fathy El-Tahawy et al. conducted a study on diabetic male albino rats. The purpose of the study was to test PRP effects on the endocrine pancreas. PRP injections were administrated twice a week for 3 weeks in a dose of 0.5 mg / kg. The results of the study showed the regeneration of pancreatic islets of the albino rats in response to PRP therapy (32). Navid Hosseini Mansoub et al. tested the effect of PRP therapy on burn diabetic rats. The results of the study confirm the beneficial effects of PRP therapy by healing burn injuries (33).
Babei V et al. conducted a clinical trial between 2011 and 2014, which included 150 patients with diabetic foot, who received PRP therapy. Diabetic foot was completely healed after 8.8 weeks and after 8 months of PRP treatment, none of the wounds reopened (34).
A prospective study conducted by Singh SP et al. between 2013 and 2015, which included 29 patients with diabetic foot, who received PRP therapy, reported complete healing of diabetic patients after 36.7 ± 3 days compared with 60.6 ± 3.7 days in the control group. No side effects have been reported due to PRP therapy (35).
By 2018, around eleven clinical trials have been performed in patients with diabetic foot to test PRP therapy. All eleven studies reported diabetic foot healing without side effects after PRP administration. The healing period reported by these clinical trials was between 7.8 ± 2.7 and 8.3 ± 3.7 weeks (36). concluSIonS PPR can be regarded as a cocktail of growth factors that are involved in different stages of the healing process, with positive results in the medical field. The incidence of diabetes has increased alarmingly in the population worldwide, which present many complications. PRP therapy for diabetes mellitus started first on laboratory animals, and then extended to diabetic patients.
All clinical studies performed still new on diabetic foot patients following PRP therapy reported complete healing between 7 to 8 weeks. The clinical studies performed did not report any side effects following PRP administration.