INTRODUCTION

Diabetes mellitus is one of the leading causes of morbidity and mortality, and it is a significant risk factor for the early onset of various dysfunctions¹. In 2019, it was estimated that 463 million adults aged 20 to 79 were living with the disease. Projections for 2045 indicate that this number will reach 700 million people². This condition can lead to complications such as peripheral neuropathy, microangiopathy, local immunodeficiency, and vascular disease. Additionally, genetic variations related to the tissue repair process can contribute as major factors in the development of chronic ulcers, specifically diabetic foot³,⁴.

Lower limb ulcers have a high prevalence, affecting more than 30% of the diabetic population over 40 years of age⁵. If these lesions are not treated appropriately, there is a risk of progression to chronic ulcers, leading to the development of severe complications such as limb amputation⁴.

Following tissue injury, it is expected that ulcers will regenerate physiologically. However, if this process is interrupted or delayed, extending beyond six weeks, the lesions are classified as chronic. A notable characteristic of these ulcers is the presence of a prolonged inflammatory phase, which impairs the ability of dermal and epidermal cells to respond adequately to the healing process. This repair process in diabetic individuals can be complicated⁶,⁷,⁸ and involves a complex interaction of regenerative properties and mechanisms.

The effective restoration of these lesions requires the coordination of multiple cell types and the interaction of various biochemical molecules. The skin regeneration process can be divided into four distinct and sequential phases: hemostasis, inflammation, proliferation, and remodeling⁶, each phase contributes to the complete restoration of tissue integrity⁹,¹⁰. The complete development of all phases of the healing process is crucial to restore the skin’s protective function. Irregularities or interruptions in this process can lead to the formation of chronic wounds. This scenario has a substantial impact on healthcare systems, increasing costs and significantly affecting the quality of life of affected individuals⁸,⁹.

Therapies for infected chronic ulcers have been challenging due to the presence of biofilms that hinder antibiotic penetration. Various systemic and local approaches have been proposed, but studies indicate that the isolated use of high-dose antibiotics is ineffective in treating these ulcers¹¹,¹². In addition to antimicrobials, other systemic treatments such as antibodies, peptides, hormones, amino acids, or derivatives are utilized. Although they have shown positive impact, systemic administration is limited by challenges in selectively targeting affected tissue, adverse effects, high cost, and application complexity⁶,¹³,¹⁴,¹⁵.

Skin lesions can be locally addressed with ointments, creams, solutions, and/or topical medications aimed at cleansing, anti-inflammatory, and anti-infective agents. Various types of dressings can also be used; however, a significant number of them exhibit low to moderate efficacy and generally entail high costs. Therefore, developing more effective and cost-efficient strategies is crucial for promoting wound healing, such as in diabetic foot ulcers.

Hemoderivative therapy is a therapeutic practice used in medicine for over five decades. Over time, this approach has been continuously refined, especially in the treatment of skin wounds⁴,¹⁵,¹⁶ and is recognized as one of the most traditional techniques in the field of regenerative medicine.

Platelet-Rich Plasma (PRP) is a method used in the treatment of skin wounds. It is a safe and effective technique that accelerates the wound healing process, considered effective for refractory ulcers and complex wounds¹⁷,¹⁸. Despite its benefits, the process of obtaining PRP is complex, and the high cost per session makes the technology inaccessible for many patients¹⁷,¹⁹. Platelets play a crucial role in wound healing, extending beyond blood clotting and hemostatic activities. They promote the formation of new blood vessels, regulate inflammation, contribute to immunity, and facilitate wound healing, underscoring their importance in various biological processes¹¹.

However, PRP lacks the size and structure necessary for the treatment of external and complex wounds, which poses a complication, especially when used in isolation¹⁹,²⁰. This issue can be mitigated by combining fibrin or another form of matrix¹⁸, which, in turn, increases costs and complicates method reproducibility.

This method provides reproducible, low-cost, and highly effective therapeutic options for the treatment of chronic ulcers that do not respond to conventional treatment¹⁷,²¹. Currently, second-generation blood concentrates have various protocols with variations in centrifugation time²²,²³, aiming to improve cellularity²⁴ and promote both histogenesis and angiogenesis²⁵. These can be applied as membranes or in their injectable liquid form, both promoting tissue bio-stimulation²⁶,²⁷,²⁸ and modulation of the inflammatory response²⁹. However, membranes produced by this method have been limited in size, which hinders their application in extensive ulcers³⁰,³¹,³².

With the aim of improving strength, cellular distribution, and membrane dimensions, the Tissue Regeneration Research Laboratory (LAPERT) at the Federal University of Uberlândia (FUU), Brazil has developed a third-generation matrix called Progressive Platelet-Rich Fibrin (PRO-PRF). Initially used only for gingival lesions or small skin injuries, this matrix has now been adapted for larger wounds such as diabetic foot ulcers while maintaining a low-cost method. Therefore, this study aims to describe a case series of diabetic ulcers treated with PRO-PRF.

METHODS

This is a prospective experimental case series study conducted at the vascular surgery outpatient clinic of the Santo André Municipal Hospital Center (CHMSA).

All patients included in this case series have provided informed consent for the publication of their respective cases. This consent includes permission to publish their medical history, clinical findings, diagnostic images, and any other relevant information.

The study was planned according to the Declarations of Helsinki, and was approved by the Research Ethics Committee of the FMABC University Center (protocol nº. 5,346,616).

The full protocol description of this study has been published in protocols.io at https://dx.doi. org/10.17504/protocols.io.n2bvj8wqpgk5/v1. The method has been published in: https://doi.org/10.1371/journal.pone.0284701.

The absence of a control group in this study is justified by the aim to explore a new intervention and treatment in a specific clinical context, focusing on detailed case descriptions and evaluation of response to the intervention.

Patients with chronic lower limb ulcers of diabetic origin, without active infection after local care with debridement and antimicrobials, were included. Patients diagnosed with ischemic macroangiopathic disease were excluded.

RESULTS

Twenty-nine patients were evaluated with a mean age of 60.54 ± 9.75 years. 82.8% of the sample was male, with a mean body mass index (BMI) of 29.29 ± 8.04 kg/m². 72.4% of the patients had never smoked. Only 1 patient had type 1 diabetes mellitus. This sample consisted of 72.4% of patients with systemic arterial hypertension and 55.2% of them had dyslipidemia. The majority of the lesions (41.4%) were located on the hallux (table 1). The median treatment duration was 14 days (25th percentile of 7 and 75th percentile of 63 days) (table 1).

In table 2, we can observe the mean, median, standard deviation, 25th and 75th percentiles of wound area, and the percentage of healed wound from the lesion at the beginning of the study, after the first intervention, and at the final assessment. The median initial area decreased from 15 cm² to 4 cm² (p<0.001), demonstrating a significant percentage reduction (73.55%, p<0.001) at the last assessment (figures 1, 2).

Legend: *Friedman test.

Figure 1 Comparison of the areas in square centimeters (cm2) of the ulcers at baseline, after the first intervention with a giant platelet-rich fibrin membrane with progressive centrifugation (P-PRF) and at the end (discharge from treatment) 

Legend: *Friedman test.

Figure 2 Comparison of the median percentage (%) of ulcer reduction at the following moments: initial (percentage 1), after the first intervention with a giant platelet-rich fibrin membrane with progressive centrifugation (percentage 2) and final (discharge from treatment - percentage 3) 

In table 3, we observe a direct correlation between the initial area of the lesion and the area after the first intervention with the number of times the Giant PRO- PRF membrane was used (Rho=0.436, p=0.018 and Rho=0.461, p=0.012 respectively).

DISCUSSION

Tissue engineering is at the forefront in the quest for efficient solutions for tissue regeneration. By combining stem cells, protein matrices, and morphogenic signals, this promising field aims to create structures that facilitate regeneration³³. Despite advances in biomaterials and understanding of wound healing⁶, current therapies are often expensive, complex, and not always effective, limiting their widespread application. Tissue engineering emerges as a promising alternative to overcome these challenges, offering the potential for accessible and effective regeneration of damaged tissues, and promising improved therapeutic options for various medical conditions³³.

The use of PRO-PRF in the treatment of diabetic ulcers lies in its rich and complex composition, which provides a conducive environment for accelerated tissue healing. Components within the matrix, such as growth factors, cytokines, and other bioactive molecules, have been capable of stimulating cell proliferation and angiogenesis, thereby promoting accelerated tissue healing.

For over three decades, PRP has stood out as an innovative resource in regenerative medicine and tissue engineering. It acts as a catalyst in wound healing and shows significant potential in bone regeneration by stimulating the formation of new bone tissue and aiding in fracture consolidation³⁴.

Individuals with chronic wounds face changes in body image, reduced mobility, deficits in self-care, inability to perform daily activities, pain, and discomfort, which diminishes their quality of life³⁵. The non-healing of these ulcers is associated with high morbidity and loss of function¹⁷ and, in some cases, limb amputation. The mortality rate five years after limb amputation is approximately 50%, underscoring the severity of this condition⁴.

The treatment of diabetic ulcers involves several general principles such as metabolic control and management of comorbidities, lifestyle changes, cholesterol control, and regulation of blood pressure and glucose levels. However, surgical orthopedic interventions are often necessary to correct excessive focal pressure in the area. Additionally, relief from abnormal pressure can be achieved with orthoses, devices that offload pressure from the ulcer region. It is also crucial to emphasize proper cleaning of the ulcer area, preferably using antiseptic dressings³⁶,³⁷.

Medication use is initiated solely for the management of neuropathic pain in the lower limbs. Typically, treatment starts with non-opioid analgesics, and in cases of therapeutic failure or intense pain, tricyclic antidepressants or anticonvulsants may be employed³⁸.

The therapeutic approach to diabetic foot infections may depend on the type of infection, whether bacterial or fungal. For fungal infections, topical treatment typically involves 2% miconazole or 2% ketoconazole. In cases of recurrent infections, an alternative is the use of 150 mg fluconazole³⁸. When diagnosed with onychomycosis, therapy is conducted using 100 mg of itraconazole³⁹.

The recommended treatment for bacterial infections in diabetic foot depends on the severity of the infection. In mild cases, outpatient treatment with oral or intramuscular antibiotics is recommended. For patients with moderate infections, hospitalization may be necessary for intravenous antibiotic administration. In severe cases, hospitalization is required for intravenous antibiotic therapy³⁹.

However, it is known that approximately 85% of lower limb amputations in people diagnosed with Diabetes Mellitus are preceded by ulcerations³⁹. In light of this, there is a pressing need to implement a comprehensive and effective therapeutic approach for the treatment of ulcers in diabetic patients, aiming to prevent amputation.

Among the various tissue engineering-based therapies for chronic wound healing, the local application of blood concentrates such as Leucocyte-and platelet- rich fibrin (L-PRF) stands out. The study by Pinto et al. (2018)⁴ demonstrated a promising potential of L-PRF in wound healing without adverse events, positioning it as a low-cost and highly effective strategy in the treatment of chronic wounds. Furthermore, L-PRF also contributes to obtaining regenerated tissue of better quality, with no recurrence in the first year of follow-up¹⁷. However, the reduced dimensions of the membranes limit their use in extensive wounds, necessitating improvements to expand their clinical application.

The extracellular matrix (ECM) plays a fundamental role in wound healing by providing a bioactive environment that directs cellular behavior through chemical and mechanical stimuli. Its components directly influence cell proliferation, adhesion, migration, differentiation, and cell death, while also regulating growth factors, receptors, tissue hydration, and pH⁴⁰.

In ulcers and tissue injuries, the blood clot acts as a temporary immunological defense and a repairing tissue. Its cellular and molecular elements assume the role of cells and the extracellular matrix of the injured tissue until the proliferative phase¹⁹,²¹,²²,³⁴,⁴¹. Considering their structure and biological properties, blood concentrates can be seen as optimized clots¹⁷, accelerating the tissue repair process, enhancing the microenvironment, and creating favorable conditions for tissue restructuring.

In this scenario, for over a decade, our research group has been applying second-generation membranes as temporary fillers and natural autologous dressings¹², despite their reduced dimensions. Due to the need for larger membrane sizes, in 2019, the third generation, PRO- PRF, was introduced, which stands out for its applicability in extensive lesions and acceleration in the healing of chronic skin wounds.

However, this pioneering study marks the unprecedented application of PRO-PRF in a standardized protocol for treating chronic wounds in diabetic feet. The systematic implementation of this innovative technology has the potential to enhance treatment outcomes for patients with lower limb diabetic ulcers.

The application of PRO-PRF membrane, fixed with cyanoacrylate adhesive, promotes the stabilization of the growth cell reservoir in the ideal location, favoring traction balance and the healing of extensive and complex wounds²¹,²²,²³,²⁴,⁴¹.

The greater tensile strength of the PRO-PRF membrane results from a dense and flexible fibrin network. This characteristic is attributed to the high concentration of fibrin molecules and the formation of equilateral junctions during progressive centrifugation, a process that allows slow and orderly polymerization of molecules, resulting in thicker and more robust fibers⁴².

Furthermore, through the utilization of this protocol, the membranes modulate the inflammatory response, confer immune protection to the wound²², and exhibit antimicrobial potential²⁶,²⁷,²⁸. The findings of Snyder et al. (2020) demonstrated a low cost of PRO-PRF, both in Brazilian standards and international standards, particularly when compared to other regenerative matrices and industrialized dressings³².

PRO-PRF stands out as a promising therapeutic option for the treatment of chronic diabetic ulcers. By accelerating the repair process, angiogenesis and histogenesis, this new approach corrects cellular and molecular deficiencies present in chronic wounds. Its simplicity, safety, low cost and production from the patient’s own blood make it an advantageous alternative. Future studies are crucial to elucidate the mechanisms of tissue repair induced by PRO-PRF and optimise its therapeutic potential.

CONCLUSION

The use of PRO-PRF proved to be an easy-to- use, low-cost and effective therapeutic option for treating chronic skin wounds in diabetic feet.