Role of Autologous Platelet Derived Growth Factor and Fibrin Rich Plasma in Management of Chronic Non-healing Ulcer–A Pilot Study

Background and Objectives : Chronic non-healing ulcer (CNHU) develops due to infections, trauma or underlying any medical and surgical conditions. Ulcer that have failed to response all available mode of treatment for long duration are more likely to develop gangrene and infection prone to limb amputation. This is a major public health problem. None of the conventional treatments are anticipated to stimulate active wound healing. The objectives of this study is to test the efficacy of topically applied autologous platelet derived growth factors and fibrin rich plasma in active repair of chronic non healing ulcer. Original Research Article Patel et al.; BJMMR, 12(12): 1-8, 2016; Article no.BJMMR.22635 2 Methods : Patients having one or more ulcers who have been receiving conventional treatment for their at least for more than 6 months duration but showed no evidence of healing till date were included in this study. Total of 30 skin ulcers were registered, of which 15 patients did not return for follow up. Rest 15 patients were included as the study group. All ulcers were treated with autologous platelet derived growth factors and fibrin rich plasma enriched antibiotic ointment. We observed that 73.33% ulcers were complete healed & rest ulcers had signs of improvement. Results: The study group showed complete healing in 73.33% ulcers and average 80% improvement observed in each ulcer, after applying autologous platelet derived growth factors and fibrin rich plasma. Significant ulcer healing was observed in patients who were less than 40 years of age, had no history of addiction for any toxic substance, and had ulcer size less than 30 sq cm. Ulcer healing rate was also found to be higher in cases whose duration of ulcer was within one year and those who did not have any history of systemic diseases. Conclusion: This study clearly shows the efficacy of topically applied autologous platelet derived growth factors and fibrin rich plasma in management of chronic non-healing ulcer.


INTRODUCTION
Ulcers that have failed to proceed through an orderly and timely reparative process over a period of 3 months are known as chronic nonhealing ulcer (CNHU) [1]. These ulcers were unable to produce satisfactory anatomic and functional integrity even after treatment with all available modalities.
Chronic wounds are common and ingenerate significant burden on health care services.
Nearly 6 million people suffer from chronic wounds in United States (US) with a rough prevalence of 2% of the general population [2,3]. The estimated prevalence of chronic ulcers in Indian population as depicted by Shukla et al. [4] in their study, was about 4.5 per 1000 population. They also found the incidence of acute wound to be more than double at 10.5 per 1000 population.
A recent study had revealed that in a course of life time, almost 10% of the population will develop a chronic wound, with a wound related mortality rate of 2.5% [5,6].
Vowden K et al. [7] in his study depicted a prevalence of 3.55per1000 population in United Kingdom (UK) with a cost burden of around 2.03 million pounds per 100,000 population.
As per the Wound healing Society, chronic wounds are classified into four categories: venous ulcers, pressure ulcers, diabetic ulcer and arterial insufficiency ulcer [8]. They are not limited to these aetiological factors. Most of the wound share common features of excessive inflammation, inability of dermal cells to respond to the reparative stimuli, cell-cycle dysfunction, changes in enzyme activities. But yet, the underlying patho-physiological derangements varies from ulcer to ulcer [9]. Aetiological factors like venous ulcers, vasculitis, trauma, communicable and non-communicable diseases have attributed to the aetiology of chronic wounds in Indian scenario. The principal systemic conditions that favors chronicity of wounds are like diabetes mellitus, atherosclerosis, tuberculosis, leprosy and even filariasis [4,5,10].
In US also diabetic ulcers and pressure ulcers are growing at double digit rates and 14-24% of diabetic foot ulcers end up with amputation [2,11]. A longer life expectancy along with the increasing trend of non-communicable diseases, the prevalence of chronic wounds is also likely to rise.
An interdisciplinary approach to systemic assessment and innovation of an optimal treatment strategy, are essential requisite to reverse the rising trend in wound chronicity and morbidity associated with it.
Wound healing practices has started since the time of Smith Papyrus in 1700 BC. But the revolutionary era of wound biology has embarked in 1962 when the first growth factor-epidermal growth factor was discovered. Since then, role of many growth factors in wound repair and regeneration have been studied. Focusing on such novel growth factors in wound healing will not only improvise human health but also reduce the healthcare cost.
Platelet derived growth factor is currently the only growth factor approved for treatment of chronic non healing ulcers [12,13]. Platelet derived healing factors are polypeptides produced in the platelets and are stored in the intracellular αgranules. These factors are released when the platelets are activated by thrombin and act in a paracrine fashion. They are potent mitogens for smooth muscle cells, capillary endothelial cells and fibroblasts, and thus promote neovascularization. Being a chemo-attractant for neutrophils and fibroblasts, these factors enable wound repair through fibroblast proliferation and collagen synthesis. These in vivo properties suggest that PDGF, derived from platelets at the site of injury, may play an important role in the initiation of repair process of wounds like deposition of neo-vascularised collagen mesh, granulation tissue formation and epithelialization. [9,13,14] Knighton et al. [15] in 1986, showed accelerated epithelialization and 100% healing of chronic non-healing ulcers by the use of autologous platelet derived wound healing factors (PDWHF). This was the first ever clinical demonstration of locally acting growth factors derived from autologous platelets that promote the healing of chronic cutaneous ulcers.
Steed et al had also reported significantly improved ulcer healing rates in patients receiving topical platelet derived growth factor versus a placebo group [16].
Saad Setta et al studied the healing effect of platelet releasate on the healing of chronic diabetic ulcers and compared it to that with platelet-poor plasma. They concluded that platelet-rich plasma (PRP) significantly enhanced the healing rate in of chronic diabetic ulcers [17].
In this study we tested the efficacy of topically applied autologous platelet derived growth factors and fibrin rich plasma in the repair of CNHU. Our aim was to establish certain guidelines on the use of platelet derived growth factors and fibrin rich plasma in management of CNHU. Our treatment protocol utilizes the fundamental treatment principles for the management of non-healing ulcers and can prove to be highly relevant in day to day practice as it is quite risk-free and easy on the pocket.

MATERIALS AND METHODS
This was a pilot study on a minimum of 15 patients/ulcers attending OPD of Dr. BRAM Hospital Raipur, Chhattisgarh. The study was conducted in the Department of Biochemistry, Pt J.N.M. Medical College Raipur, Chhattisgarh.
The Local Ethical Committee and Institutional Stem Cell Committee had provided clearance for this study on 15 patients. All patients were insured during this study.
13 patients were registered for the study. Of them, two patients had two ulcers each. So the number of chronic non-healing ulcers enrolled for the study was 15.

Selection Criteria
Ulcers formed due to any cause that fulfills the following criteria were selected for the study.

Methods
After taking informed consent from patients, 20 ml of patient's venous blood was drawn in plain sterile tube under aseptic condition. It was centrifuged at 2500 rpm at room temperature for 12 minutes without any interval. Fibrin clot was removed from the junction of packed red cells and plasma. The fibrin clot was mixed with 30 grams antibiotic ointment (Soframycin). This ointment was dispensed in a sterile container. The patients were advised to apply the ointment locally three times per day after cleaning the wound with normal saline. Patients were advised not to clean the wound with hydrogen peroxide. Of course, unhealthy wounds were debribed and cleaned once or twice with hydrogen peroxide in the hospital itself.
The study population was divided into different groups to study the effect of the following factors on ulcer healing: The patients were followed up every 15 days till healing is complete or at least for 6 months.
The clinical photographs have been taken during each visit for each of the patients compare the healing of the ulcers.
Data analysis was done using Chi Square to find the duration of healing and the percentage of conversion of non-healing ulcer to healing ulcer in the different groups. A 'p' value <0.05 was regarded as significant. Table 1 depicts the effects of different parameters on healing of chronic ulcers. Significant healing was documented in patients with ulcer size less than 30 sq.cm, and those without any type of addiction.

RESULTS
In Table 2, the effect of different parameters on the rate of healing has been shown. It reveals that the number of patient who could achieve complete healing within the first follow-up, are significantly more when compared to their respective counter-group.

DISCUSSION
Our study comprised of a total of 15 ulcers from 13 different patients diagnosed to have CNHU.  Two of these patients had two ulcers at two different sites and hence a total of 15 ulcers were included for the study. 73% of the total ulcers healed completely while the rest healed partially. The effects of different factors affecting ulcer healing have been represented in Table 1.
Of the ulcers those accomplished complete healing, 55 % of them achieved the same within the first follow-up period of one week. The effects of various factors on the rate of ulcer healing have been depicted in Table 2.
Increased age is a major risk factor for impaired wound healing. Many clinical and animal studies at the cellular & molecular level have depicted age related changes in microcirculation and delay in wound healing [18][19][20]. We observed that ulcers of 75% patients of more than 40 years of age, could not accomplish complete healing whereas 83% of younger study group displayed a significantly faster healing rate (p<0.05). Their ulcers got completely healed within the first follow-up period when compared to older group who required more than one follow-up in 80% cases. Aging is associated with altered inflammatory response such as delayed T cell infiltration in to the wound area with alteration in chemokine production and reduced macrophage phagocytic capacity. These entail in delayed reepithelialization, angiogenesis & collagen deposition, thus chronic non-healing ulcers. Reduced collagen turn over, remodeling & decreased wound strength have been observed in aged mice as compared with young mice. Exercise has been reported to improve cutaneous wound healing in older adults by increasing the blood flow to the site [21,22].
Retarded healing is strongly associated with exposure to cigarette smoke & tobacco. Nicotine has been identified as a potent vasoconstrictor that hampers blood flow reaching the areas undergoing regeneration [23]. In concurrence to this fact, our study population also revealed incomplete healing in 75% cases that were exposed to some sort of addiction when compared to patients without any addiction (p<0.05). Besides altered wound healing, smokers also evidenced an increase in a variety of complications such as infection, wound rupture, anostomotic leakage, wound and flap necrosis, epidermolysis and reduced tensile strength of wounds [24,25].
The healing rate was again found to be significantly high in non-addicted cases. About 83% of them accomplished complete healing by the first follow-up in comparison to the addicted cases who achieved the same in only 17% cases (p<0.05). Clinical evidence and animal experiments have also suggested findings relevant to alcohol induced immune-modulation and its consequences on host innate and adaptive immune responses. This resulted in diminished host resistance against microbial pathogens and host defense [23,26,27]. Connective tissue restoration is also influenced by alcohol intake, and results in decreased collagen content, and alterations in the protease balance at the wound site [28]. Even acute ethanol-intoxication remarkably impairs wound vascularity and precipitate increased wound hypoxia and oxidative stress [29].
Wound duration is a recognized indicator for potentially slow healing in a variety of wound types. When the effect of duration of ulcer (since the time it has appeared at that site) on its healing was analyzed, 75% of the partially healed group consisted of ulcer of longer duration. The study group revealed faster healing rate in those with short duration of ulcer history. 67% subjects accomplished healing within first follow-up (Table 2). Margolis et al. [30] had also correlated delayed healing with specific wound characteristics like large wound area, an ulcer of long duration and a reduced ankle-brachial pressure index. This could be due to decrease protease activity, development of senescent cell population that precede to release pro-inflammatory cytokines, impairs vascular supply and accelerate telomere degradation [31,32].
Several classification systems for diabetic foot ulceration have been devised to allow risk stratification. One such validated system is the S(AD)SAD classification system-Size (Area & Depth), Sepsis, Arteriopathy and Denervationwhich identifies cross-sectional area and depth of the ulcer as important factors associated with ulcer healing [33,34]. In this system lower grading is associated with rapid healing.
Similarly, our study group also depicted a significant link between the size of ulcer on its wound healing. Of the completely healed ulcers, around 91 % cases had wound size of less than 30 sq.cm (p<0.001). It was also noteworthy that of the patients with lesser surface area of ulcer, 100 % of them were healed completely within the first follow up (Fig. 1) when compared to those ulcers with a greater surface area (Fig. 2). The ulcers with larger surface area required more than one follow-up for healing. Kramer and Kearney have also indicated the size and depth of ulcers as good predictors for healing [35]. It has been suggested that large size ulcers provide greater surface area for various infections and thus there is a tendency for exaggerated inflammatory response that impede tissue repair, epithelialization and angiogenesis at the site [30,31]. Associated systemic disease is considered as a significant contributor for prolonged ulcer healing. 55% of ulcer patients that revealed complete healing were not associated with any evident systemic diseases. Of them 67% ulcers healed up completely within first follow-up. Treatment of chronic diseases like diabetes mellitus, obesity, malnutrition, peripheral vascular disease, malignancy, organ failure, sepsis plays a central role in wound management. Failure to attain optimum treatment of associated conditions will end up in chronic non-healing ulcers [23,31,36].

CONCLUSION
Autologous platelet derived growth factors and fibrin rich plasma promotes healing of chronic non-healing ulcers and leads to complete healing in about 74% ulcers in our study and others have also showed improvement. In the total ulcers, 40% ulcers were healed within 15 days. The duration of healing process depends on the age of patients, duration of ulcers, size of ulcer, addiction for alcohol and tobacco and presence of systemic disease or past history of systemic disease in the patients. There is no evidence of over healing, such as hypertrophy or keloid formation. The patient can apply medication at home without any difficulty. Patients feel benefitted as they don't have to undergo any surgery, only periodic out-patient examination is required and healing is achieved with little penny expenditure. However, to establish the validity of this hypothesis, a larger study group need to be recruited and necessitate more such studies.