Autologous Platelet Lysate Product Versus Placebo in Patients with Chronic Leg Ulcerations: A Pilot Study Using a Randomized, Do

P latelet growth factors are intimately involved with wound healing.^1–6 Whether they are indispensable to wound healing is unclear, because naturally occurring growth factor deficiency states have not been identified. Platelets contain at least five growth factors that may contribute to tissue formation and epithelialization: platelet-derived growth factor (PDGF), platelet factor 4, transforming growth factor-b, platelet-derived angiogenesis factor, and platelet-derived epidermal growth factor. Each growth factor has several biological activities. The combination of PDGF, platelet-derived angiogenesis factor, transforming growth factor-b, and possibly platelet-derived epidermal growth factor enhances granulation tissue growth while stimulating angiogenesis, fibroplasia, and extracellular matrix synthesis. Platelet factor 4 and PDGF attract neutrophils for host defense, and the combination of PDGF and platelet-derived epidermal growth factor stimulates epithelialization. PDGF is an extraordinarily potent mitogen for most mesenchymally derived connective tissue-forming cells, such as fibroblasts, smooth muscle cells, and glial cells. It is also chemotactic for the same cells for which it is mitogenic, including neutrophils and monocytes. After monocytes enter the wound, they convert to macrophages, which in turn serve as additional growth factor sources. Exposure to PDGF brings about numerous intracellular events, including calcium mobilization, increased DNA synthesis leading to cell proliferation, increased expression of certain genes, and increased RNA synthesis leading to increased production of collagen, proteoglycans, and elastic fiber proteins by connective tissue-forming cells. Platelet factor 4 is released from the alpha granule locally in injured tissue during platelet activation; it binds heparin and is chemotactic for polymorphonuclear leukocytes and monocytes. Transforming growth factor-b stimulates cell proliferation, protein synthesis, and collagen synthesis. It also inhibits growth of many epithelial tumor cells and fibroblastic cell lines. Platelet-derived angiogenesis factor is a polypeptide capable of stimulating new capillary growth by inducing migration of endothelial cells. Platelet-derived epithelial cell growth factor is partially responsible for the initial influx of neutrophils into the wound space; it is also a mitogen for many cells, including epithelial cells and fibroblasts. More recently, Herouy, et al.,⁷ demonstrated that autologous platelet releasate containing these growth factors significantly increased protein expression of alphavbeta3 integrin in leg ulcers. They suggested this as the mechanism by which platelet factors influence the process of angiogenesis and revascularization, thus promoting granulation tissue formation. These platelet growth factors can be collected either by activating the platelets with thrombin, inducing them to release the factors (releasate), or by lysing the platelets (lysate). Previous studies have found that platelet product containing growth factors may accelerate wound healing;^8–14 however, some studies have not found efficacy on comparison with placebo.^15,16 Of four prospective, randomized, placebo-controlled, blinded studies, two showed efficacy,^8,9 and two did not^15,16 (Tables 1 and 2). To assess the ability of autologous platelet lysate factors to facilitate the healing of chronic cutaneous ulcers, the authors conducted a prospective, randomized, double-blind, placebo-controlled trial in which the authors compared healing times for ulcers treated using autologous platelet lysate factors added to collagen (treatment group) with the healing times for ulcers treated with platelet-poor plasma plus collagen (placebo group). Methods Study design. Patients with chronic leg ulcers referred to the authors’ wound care center were eligible for entry into this study (inclusion and exclusion criteria are defined below). The patients were randomly assigned to receive either platelet lysate product mixed with collagen (the treatment group) or platelet-poor plasma mixed with collagen (the placebo group) for the first 12 weeks of therapy. After 12 weeks, there was a washout period of two weeks. During this washout period, patients applied only normal saline-moistened gauze twice daily to their ulcerations. Patients whose ulcers had not healed were then assigned to receive whichever treatment they had not received in the previous 12 weeks. For the duration of the trial after appropriate training and instruction, each group of patients applied their respective treatment twice daily after cleansing the ulcer. Patients were instructed to apply the product in a thin layer over the entire surface of the wound. Xeroform gauze was applied in a double layer over the platelet product, and a sterile 4in x 4in gauze dressing was placed over this. The entire wound site was covered with a gauze wrap. If signs of an infection developed in the wound bed of the ulcer, it was cultured and treated with an appropriate systemic antibiotic. Patients presented to the wound care center every week for two weeks and then every two weeks for the remainder of the study for measurement of the wound, photography, and general assessment of wound healing until healing occurred. All ulcers were surgically debrided (gentle debridement using forceps and scalpel following the application of topical lidocaine solution) if appropriate at each two-week visit. The principal end point was the occurrence of complete healing. Complete healing was defined as 100-percent epithelialization of the entire target ulcer. Therapy was initiated in either the inpatient hospital setting or the outpatient setting, with continued follow up in the outpatient setting until ulcer healing was complete. This study was approved by the Institutional Review Board of the authors’ institution. Informed consent was obtained in writing from study participants after the study procedures had been fully explained to them. Patient selection. Inclusion criteria. Patients who had nonhealing ulcers of the lower extremities for more than eight weeks were eligible to participate in the study, as were patients with multiple ulcers. A target ulcer was selected. Nonhealing ulcers of arterial, venous, neuropathic, or vasculitic (small-vessel) origin were considered. A transcutaneous partial pressure of oxygen (T_CPO₂) greater than 30mmHg in the vicinity of the ulcer was required. Additionally, a hemoglobin value greater than 9.0g/dL (normal, 12–15.5g/dL) and a platelet count greater than 100 x 10⁹/L (normal, 150–450 x 109/L) were required. Exclusion criteria. Because of the hemodynamic changes associated with preparations of platelet product, patients with the following conditions were excluded: unstable angina pectoris or angina at rest, persistent or recurrent symptomatic hypotension, myocardial infarction within four weeks, class III or IV congestive heart failure, clotting function disorders, or a platelet count less than 100 x 10,sup>9/L. Other conditions resulting in exclusion from the study were underlying osteomyelitis as determined by radiograph, bone scan, or direct biopsy/culture; wounds caused by burns or irradiation; wounds exceeding 100cm² in area; and pregnancy or lactation. Women of childbearing potential were also excluded unless they were using appropriate contraception and had a negative quantitative result for human chorionic gonadotropin from a blood sample drawn before entry. Positive blood culture results or clinical or laboratory evidence of active systemic infection were also reasons for exclusion. Active infection was demonstrated by the persistence of fever, increased leukocyte count greater than 15 x 10⁹/L (normal, 3.5–10.5 x 10⁹/L), or cellulitis. A complete history from the patient was reviewed, and all screening evaluations as outlined were performed. On obtaining informed consent and verifying adherence to all enrollment criteria, patients were randomly assigned into the study. Three patients were excluded postrandomization when it was discovered that these patients did not meet study criteria (two because osteomyelitis developed shortly after beginning the study and one because of location of the ulcer in the abdomen). Preparation of autologous platelet lysate product and placebo. An average of 240mL of leukocyte-reduced platelets and a similar amount of plasma were collected using the Spectra (Gambro Healthcare, Lakewood, Colorado) blood cell separator. Approximately 2.5 x 10¹¹ platelets were collected per patient. Aliquots of platelets and plasma were placed into 12 vials each and frozen at less than or equal to -18^oC within 12 hours of collection. Sterile technique was used throughout the preparation and application of the autologous platelet lysate product or plasma. Plasma and platelet lysate products were indistinguishable in physical appearance (yellow straw-colored material). Aliquots of the platelet lysate product were released for use in patients randomly assigned to the study group. Aliquots of plasma were released for use in patients randomly assigned to the placebo group. To obtain the platelet product (the material contained in the platelet that was released on activation plus the platelet membranes), the authors chose not to activate platelets with thrombin to extract the factors, as others have.^6,8 Instead, the authors lysed the platelets by freezing (a physical extraction as opposed to activation-release). The technique was validated by using enzyme-linked immunosorbent assay to measure the level of PDGF, which was found to be in a high concentration in the platelet-rich material and a low concentration in the platelet-poor material.¹⁷ A quantitative immunoassay (Quantikine®, R&D Systems Inc., Minneapolis, Minnesota) that detects platelet-derived growth factor-AB showed an average of 30,667pg/mL (range, 4,000–100,000) in the platelet product compared with an average of 743pg/mL (range, 190–1,800) in the corresponding platelet-poor plasma (p17 The authors’ finding was comparable to previously published levels.¹⁶ The placebo product was composed of platelet-poor plasma added to collagen. This placebo product looked, smelled, and behaved like the autologous platelet lysate product. Both products were packaged and prepared identically (i.e., freezing technique). To initiate the application of autologous platelet lysate product or the placebo (platelet-poor plasma), two vials of the substance were thawed and added to a 1-g jar of microcrystalline collagen, either Avitene (Alcon Puerto Rico, Humacao, Puerto Rico) or Medifil (BioCore Medical Technologies, Topeka, Kansas), to produce a sterile topical salve. The jar was used for one week and then discarded. Autologous platelet lysate product is usable for one year from the date of collection; however, each vial, once thawed, must be used within one week and during its use must be stored in a refrigerator at 1^oC to 6^oC. Statistical analysis. The primary end point for this study was whether the ulcer healed during the treatment period. Healing was defined as complete epithelialization of the wound bed and was assessed by ulcer photography and clinical examination. The authors tested the hypothesis that wounds treated with autologous platelet lysate product were more likely to heal than those treated with placebo. Patients were randomly assigned to receive active treatment or placebo; patients and caregivers were blinded to the treatment assignment. If the wound did not heal in the first 12 weeks, the patient was assigned to the other treatment for the second phase of the study. The study was designed this way to encourage enrollment by having each patient eligible to receive the active treatment during the course of the study. The Fisher exact test was used to test for a significant difference in the proportion of wounds healed in the first and second phases of the study. To use the information from both time periods, three possible outcomes were tabulated: 1) healed and did not receive the other treatment, 2) did not heal in either arm of the study, and 3) did not heal in the first arm of the study, received the other treatment, and healed in the second arm of the study. The authors assigned the scores of +1, 0, and -1 to each of the three outcomes, respectively. The Wilcoxon rank sum test was used with the patients retaining their initial group assignment to test for significant differences between the groups. A secondary end point was the rate at which the wound healed. To address the secondary end point of an increased rate of healing when the wound was treated with autologous platelet lysate product, the authors fit linear regression lines for each patient over time. Using the area of the ulceration (longest diameter of the ulceration [length] multiplied by measurement of the perpendicular length [width]) as the dependent variable and the time from the start of treatment as the independent variable, the authors fit regression lines to each patient in each group. The authors obtained the slope from each of these regression equations and used the rank sum test to look for a difference in the rate of healing between patients treated with autologous platelet lysate product mixed with collagen and those receiving collagen alone. Differences in baseline variables were compared using the Pearson x² test for discrete variables and one-way analysis of variance for continuous variables. Results Table 3 shows the baseline characteristics of the treatment and placebo groups. The mean ± SD age for patients randomly assigned to receive PDGF was 67.6±11.9 years; for those randomly assigned to receive placebo, it was 57.8±18.2 years. Fifty-three percent of treatment group patients and 63 percent of placebo patients were male. There were no statistically significant differences in these characteristics between patients randomly assigned to receive platelet lysate and those randomly assigned to receive placebo. No side effects of treatment were noted. These wounds had been present for an average of 372.3±327.5 days before patients entered the study. In the treatment group, 9 of 15 patients healed during the first 12 weeks of treatment with autologous platelet lysate. In the group receiving placebo, 4 of 11 healed during the first 12 weeks. There was not a statistically significant difference between the proportion healed in these two groups at 12 weeks (p=0.68). After the two-week washout period, the unhealed patients in the treatment group received placebo, and two additional patients healed during the second 12 weeks. Likewise, the unhealed patients in the placebo group received active autologous platelet lysate, and two more patients healed during the second 12 weeks. Comparing the eligible patients in the two groups over the second 12 weeks failed to show statistical significance (p=0.99). Throughout the study, 11 patients (42%) healed while being treated with active platelet product, 6 (23%) healed while receiving placebo, and 9 (35%) failed to heal. In the analysis using both time periods, there was not a significant difference between the treatment groups in the proportion healed (p=0.31). Figure 1 displays the area of the wounds by the randomization strategy at baseline, Week 12 (end of the first treatment period), and Week 28 (end of the study period). The rate of healing was not significantly different between the treatment group and the placebo group (p=0.56). The median slope estimating the rate of healing over time for patients receiving PDGF was -0.79; for those receiving placebo, it was -0.84. Table 4 includes the mean areas at baseline and after the first treatment phase for each group. Discussion Platelets contain products that have been shown to help initiate and promote wound healing. Topical application of both autologous and homologous platelet products to wounds has been shown in most previous studies to encourage wound healing (Table 2).^8–14 In this study, however, platelet product did not accelerate wound healing or lead to a statistically significant increase in rate of healing (although there was a trend toward healing in the treated group in the first 12 weeks [Table 4]). This finding is disappointing but consistent with the findings of two other studies (Table 1).^15,16 On the basis of studies using multiple growth factors and studies using single growth factors, the authors had anticipated that the platelet product would accelerate wound healing. Such studies of PDGF have led the US Food and Drug Administration (FDA) to approve the use of becaplermin for the management of venous and neuropathic ulcerations. Why did use of the authors’ platelet product not lead to statistically significant accelerated wound healing? There are several possibilities. First, the high proportion of the ulcers that healed overall may have been a result of the intensive wound care administered at a specialist wound care center. Additionally, multiple papers have documented the beneficial effects of topical collagen preparations in accelerating wound healing; topical collagen with plasma was the placebo in this study. Overall, 11 of 15 ulcers in the treatment group and 6 of 11 in the placebo group healed during the 26-week course of the study. These wounds had been present for an average of 372.3±327.5 days before patients entered the study. Thus, combining treatment and placebo groups, a total of 17 of 26 patients (65%) healed their wounds during the course of the study. Each of these figures is a high rate of healing for the period involved. Attendance at a wound care center, with intensive patient education and regular wound care, has been shown to result in a higher rate of healing of chronic wounds.¹⁸ Second, study design and recruitment contributed to the authors’ results. This study was originally designed to accrue 40 patients in each group; the actual number of patients enrolled in the study was small and not powered to detect significance. The study had to be terminated prematurely because of the difficulty of enrolling patients. There were two important reasons for this difficulty: 1) many patients did not want to participate in the preparatory protocol to obtain autologous platelet lysate product, because this involved returning for phlebotomy; and 2) becaplermin was given FDA approval and patients preferred to receive this drug after it became available. It must be remembered that failure to reject the null hypothesis (in the authors’ study, that there is no difference between autologous platelet lysate product and placebo) does not prove the hypothesis is true; it proves only that insufficient evidence exists to conclude that it is false. It could be argued that the first phase of this study was too short. In most wound care centers, chronic wounds heal within 10 to 12 weeks.^10,17 This was the authors’ rationale for using 12 weeks as the length of the first treatment phase. There are also differences between this and previous studies with regard to the method of the platelet product preparation. Two basic approaches are available for collecting the growth factors from platelets. The first and most common method in the literature involves activating platelets with thrombin, which induces the release of growth factors (releasate).⁶ The second, and the one the authors used, involves actual lysis of the platelets (lysate). Stacey, et al.,¹⁶ applied this method and sonicated the platelets to obtain lysis. The present study differs in that a physical lysis of platelets was obtained through freezing. The presence of growth factor was verified by using immunoassay to detect the presence of PDGF. The two studies that have used lysis instead of releasate have failed to demonstrate accelerated wound healing with the use of platelet-derived factors. The lack of a statistically significant effect of the authors’ platelet lysate could theoretically be due to either the level of growth factor in the lysate or the lysate activity. In common with many recent studies, the amount of lysate factors (growth factors in each product) was determined (and was as high as in other studies),¹⁶ but the activity of the lysate products was not specifically measured. Most previous studies have used a mitogenic assay to test the potency of their platelet product, because the ability to quantify the levels of growth factors was not available. In the present study, the authors did not perform mitogenic assays and, thus, did not specifically know the activity of the platelet product. Instead, the authors performed quantitative immunoassay (Quantikine) to detect the levels of platelet-derived growth factor-AB. The authors’ levels proved to be comparable to the platelet lysate levels obtained by Stacey, et al.¹⁶ However, more recent studies with recombinant PDGF have demonstrated that levels of growth factor approximately 1,000 to 10,000 times greater than were present in the authors’ lysate are needed to demonstrate a significant impact on wound healing.^19,20 Both efficacy (summarized in Table 2) and lack of efficacy (Table 1) have been demonstrated in previous reports for platelet lysate/releasate for various types of ulcers. Most studies included ulcerations that are multifactorial, as in the present study. The varying results may be due to several factors: study design, ulcer types, and platelet releasate/lysate preparation. Another possible reason for the failure of the present study to show a significant difference in healing between autologous platelet lysate and placebo is the effects of proteases and cytokines and the presence of biofilms found in chronic wounds. In recent years, elevated levels of matrix metalloproteinases (MMPs) (a family of zinc-dependent endopeptidases) and reduced expression of tissue inhibitors of MMPs have been demonstrated in chronic wounds.^21–24 It is possible that these biofilms and MMPs degraded the growth factors applied.²⁴ In summary, the results of the present study indicate that autologous platelet lysate plus collagen prepared and applied in the manner described did not accelerate the rate of wound healing or significantly decrease wound size compared with platelet-poor plasma with collagen. However, the authors recognize that the present study has many shortcomings, and, most important, it is underpowered because of the practical limitations outlined. The authors report this study because it is important and timely to revisit the issue of whether platelet releasate/lysate is effective for wounds. The authors designed a prospective, randomized, placebo-controlled, crossover, blinded trial to answer this question but could not adequately power the study. Although the study was underpowered, the authors did not notice a statistically significant improvement in wounds to which the platelet releasate/lysate was added to the collagen, and the authors believe it is important to present these data while recognizing that the study is underpowered. The authors’ pilot study adds to the growing body of evidence that questions the usefulness of these agents in the topical treatment of chronic wounds.