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The Evolution of Platelet-Based Wound Therapy
Chronic wounds of the lower extremity are common complications of diabetic peripheral neuropathy and diabetes-associated peripheral vascular disease. Patients with diabetes frequently present with wounds that remain open for several weeks due to repetitive trauma related to a loss of protective sensation, glycosylation of the cells involved in the healing cascade, and macro- and micro-vascular insufficiency, which place the patients at a higher risk for infection and tissue necrosis.1 Not only do chronic wounds take a toll on patients’ physical and emotional health but they also affect patients’ financial well-being. Standard of care procedures alone frequently fail to close wounds in an expeditious manner, resulting in infection and/or amputations.
Sheehan and colleagues determined that the change in wound surface area over a four-week period is a strong predictor of healing at 12 weeks.2 If a 50 percent or more reduction of wound surface area does not occur by week four, the patient is defined as a non-healer and will most likely need additional treatment as standard of care is not adequate.2 This standard has been endorsed by payors and physicians alike to justify intervention using advanced, and often more expensive, therapies such as cellular and acellular tissue grafts and modalities such as non-contact ultrasound, shockwave therapy and negative pressure therapy.
How PRP Works
Since its original introduction in the 1970s, platelet-rich plasma (PRP) therapy has grown to be a common addition to standard care practices in the treatment of wounds.3 PRP imparts its beneficial physiologic effects in upregulating wound healing by utilizing the natural mechanisms of platelets and platelet-derived cytokines. The autologous growth factors derived from concentrated, activated platelets aid in evoking cell differentiation, proliferation, and neo-angiogenesis, which are all processes involved in the natural course of wound healing and tissue regeneration.4
Autologous PRP is formulated from the patient’s own peripheral blood through a centrifugation process separating red blood cells from the plasma and concentrating platelets in the solution created. The blood is centrifuged with a low RPM or “soft” spin with anticoagulant, citrate dextrose solution (ACD-A) to prevent coagulation during the production of PRP. The plasma-rich supernatant then undergoes a “hard spin” at higher speed to obtain the platelet concentrate.5 Autologous PRP avoids adverse effects, such as autoimmune reactions and disease transmission, seen with tissues produced from allogenic sources.6 However, rare reactions attributed to additives have been recorded.7,8 PRP is delivered in its liquid form via a syringe or coagulated with calcium and bovine thrombin before being applied as a gel directly to the wound bed; this process is often conducted multiple times on a weekly basis.9 Research has shown repetitive treatment application to increase the effectiveness of PRP but requires weekly clinic visits and in some situations, hospital admittance for access to the technology.5
Despite its seemingly long list of advantageous effects, PRP treatments are not always what they seem. As outlined by Gentile and colleagues, there are many different PRP products on the market, each obtained via various methods, using different additives, centrifugation times, and centrifugal force. Each of these factors affects the bioavailability of the growth factors and chemokines in the final PRP product, contributing to the varying degrees of efficacy seen in various studies on wound healing.4
What You Should Know About Platelet-Rich Fibrin
PRP has evolved, since its original introduction, to another form of treatment: Platelet-rich fibrin (PRF) therapy. PRF is derived from autologous blood, centrifuged without ACD-A, allowing platelets to activate, release various cytokines and begin to form a clot matrix that concentrates the patient’s own inflammatory cells and growth factors in a soft, handleable tissue matrix. The increased density of this medium allows continual release of growth factors and cells and shows promising wound healing potential.
Similar to PRP, many factors are at play in the formation of this matrix. One such integral factor is the relative centrifugation force. Herrera and coworkers found that using different RPMs in centrifugation produces different relative centrifugal force (RCF) values and produces different cellular responses in the tissue created. A low-speed centrifugation showed an increase in the amounts of growth factor released, and researchers observed an enhancement of the tissue’s angiogenic properties.10 Finding the ideal RCF value has been difficult, and delayed its implementation. Another problem encountered with PRF is its period of viability. According to a systematic review conducted by Al-Maawi and coworkers, the benefits of PRF therapy may be limited to the acute wound stage. Despite PRF’s efficacy in soft tissue regeneration through the concentration and activation of fibroblasts and endothelial cells, it only exerts its benefits in the first two or three months of a wound’s lifetime.11
These limitations of PRF have been addressed by combining it with leukocytes, collectively known as leukocyte- and platelet-rich fibrin (L-PRF). L-PRF is an autologous platelet concentrate that includes leukocytes and a higher fibrin density. Pure PRP preparations are composed primarily of platelets since a cell separator is used to filter out all leukocytes. Depending on the technology used, L-PRF can contain up to 100 percent of platelets and 50 percent of the leukocytes of the original blood sample.12 The increased leukocyte and fibrin concentration of L-PRF, in addition to platelet concentration, allows it to promote and enhance wound healing processes including cell migration as well as cytokine, growth factor, and matrix protein release for an extended period of time.13,14 The high fibrin concentration also extends protection of growth factors from proteolysis and rapid hydrolyzation.1,12
Leukocytes, specifically, have been shown to have antimicrobial and immune properties.15 A study conducted by Pravin and colleagues directly compared the effects of PRP and L-PRF on chronic, non-healing wounds. Their results showed that L-PRF healed the ulcer faster (5.7 weeks in L-PRF and 6.5 weeks in PRP (P = .034) and allowed complete healing in a greater number of wounds than those treated with PRP (73.3 percent in L-PRF vs 53.3 percent in PRP). They concluded that the added anti-inflammatory and antimicrobial benefits of leukocytes were most likely responsible for imparting the greater efficacy of L-PRF.16
Treatment with L-PRF exhibited favorable results in treating previously chronic, non-healing wounds, as further explored by Pinto and colleagues in a prospective cohort study. The application of L-PRF to diabetic foot ulcers (DFUs), venous leg ulcers, and pressure ulcers led to increased healing tendencies in all patients and culminated in complete wound closure in all patients who did not prematurely discontinue therapy due to financial reasons or switching hospitals. The DFUs, for example, had a mean baseline size of 6.7 cm2 ± 8.2 cm2; with 8 wounds ≤ 10 cm2, and all attained full closure with a mean of 6.8 L-PRF applications over nine weeks or fewer. The study showed L-PRF to be a cost-effective and efficient treatment of a financially and physically devastating pathology.14
In another study, Wang and colleagues retrospectively evaluated 42 patients with DFUs. They determined that L-PRF allowed a significantly faster wound healing rate and significantly higher overall wound cure rate than the control group treatment course with mupirocin ointment and recombinant human epidermal growth factor gel. By the end of the fifth week, 20 of the 21 DFUs treated with L-PRF were completely healed while 12 of the 21 DFUs in the control group were healed. Ultimately, treatment with L-PRF was proven to positively impact wound healing in DFUs.1
The Lancet study by Game and coworkers consisted of a larger 269-patient, multicenter, observer-masked randomized controlled trial.17 Researchers showed that the application of a multi-layered L-PRF, in addition to standard of care practices, showed a statistically significant increase in wound healing ability and a decrease in wound surface area and healing time when compared to exclusive standard of care, such as offloading, antibiotics, and/or debridement.17
Insights on an Innovative L-PRF Disc
The multi-layered L-PRF disc is fabricated at bedside using 18mL of the patient’s venous blood, which is drawn during the appointment. One would transfer this blood into a patented centrifuge-specific device, without any additives. The blood then undergoes a process of centrifugation, coagulation, and compaction. In about 20 minutes, this forms a L-PRF disc combining autologous fibrin, leukocytes, and platelets, referred to as the 3C Patch® (Reapplix), formerly known as the LeucoPatch®.12,18 This automated centrifugation process at a predetermined RPM ensured the maximum effectiveness of the L-PRF tissue. This process results in a soft, handleable, patented collection mechanism of 2.5 cm in diameter, which remains in the wound bed until the low-density fibrin matrix starts to dissolve after application.13,14
Direct application of the L-PRF patch to a clean and debrided wound continuously releases growth factors and active immune cells into the wound for the next four to five days. This confers beneficial biological healing processes relating to inflammation and tissue repair at the wound site.17 The patch can be replaced weekly for up to 20 weeks or until wound closure occurs.
Lundquist, a co-inventor of the original LeucoPatch® technology, performed a study to evaluate its clinical efficacy on 15 patients with 16 wounds of various etiologies including venous ulcers, diabetic foot ulcers and post-surgical wounds.19 The wound duration ranged from 2 to 104 months prior to treatment. Application of the L-PRF product led to reduction in wound area and increased granulation tissue in all the included study participants. The mean percentage of wound area reduction at 6 weeks was 64.7% (95% CI = 45.6% to 83.8%).
The proportion of granulation tissue at the wound bed showed a statistically significant increase thefrom 33% (95% CI = 9% to 57%) at baseline to 72% (95% CI = 55% to 90%) after the six-week treatment period. In fact, four of the chronic wounds healed completely with L-PRF treatment. This pilot study shows evidence that “the LeucoPatch® is well tolerated, safe, and has potential to adequately treat difficult-to-heal chronic wounds.”19
Another study performed by Crisci and colleagues was the first to use L-PRF to treat diabetic foot ulcers with a risk for osteomyelitis.20 Prior to treatment, all three study participants’ wounds had been present for more than six months, showed a positive probe-to-bone test and displayed evidence of osteomyelitis on magnetic resonance imaging (MRI) and bone cell culture. Treatment with L-PRF combined with culture specific antibiotics led to complete wound closure and abatement of all further clinical signs of infection. At the two-year follow-up, all wound closures had persisted. The authors deemed L-PRF–based treatment of osteomyelitis in a diabetic foot ulcer to be effective and worthy of further study.20
In Conclusion
One should consider L-PRF as a viable addition to the collection of treatments for chronic wounds. Platelets, leukocytes, and fibrin each impart unique advantages to the process of wound healing through what is essentially a blood clot over the wound surface.14 By upregulating multiple cell lines, especially fibroblasts and pre-keratinocytes, and increasing the potential for neo-angiogenesis, the application of L-PRF helps cut down on both the time and cost of wound healing without adverse impacts to the patient.21 The International Working Group on the Diabetic Foot in 2019 accepted autologous combined leukocyte, platelet, and fibrin L-PRF therapy as a significant adjuvant therapy used in the treatment of diabetic foot ulcers but this warrants further larger studies in other chronic wound types.17,22,23
Shivani J. Patel, BS, is a fourth-year podiatric medical student at Temple University School of Podiatric Medicine in Philadelphia.
Dr. Khan is a Clinical Associate Professor in the Department of Podiatric Medicine at the Temple University School of Podiatric Medicine in Philadelphia.
Dr. McGuire is a Professor in the Department of Medicine at the Temple University School of Podiatric Medicine in Philadelphia. He is the Director of the Leonard Abrams Center.
References
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