Antibodies against cell cycle regulatory proteins (CDK2, CDK4, cyclin A, cyclin D1 and cyclin E) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-RB antibody was purchased from Pharmingen (BD Biosciences, San Jose, CA, USA).

Participants with acute and chronic ulcers were recruited through the dermatology clinic. The recruitment period was between January 2010 and December 2010 and the study was completed in March 2011. Exclusion criteria included the presence of active infection, compromised immune function and coagulation disorders. These processes were approved by the institutional review board of Keimyung University and DongSan Hospital. Written informed patient consent was obtained from the patient.

In total, 16 patients (means ± SD age, 60±13, 39–83 years) with various ulcers, including traumatic ulcer, livedoid vasculitis, stasis ulcer, venous leg ulcer, burn ulcer and pressure ulcer, were included. All patients had ulcers involving different body parts: lower leg (7 patients), toe (4), sole (2), heel (1), hand dorsum (1) and finger (1). Ulcer grade was grade 2 (14 patients) or grade 3 (2 patients).

PRP was prepared from the patient’s own blood. A small volume of blood (12 ml) was collected using a commercial kit (MyCells Autologous Preparation kit^®, Holon, Israel) according to the manufacturer’s instructions.

Activated PRP was applied to the wound as a liquid or gel form according to wound size, location and condition. Prior to the application of PRP, necrotic tissues or eschars were debrided using a scalpel and images were captured with a digital camera (450D; Canon, Tokyo, Japan). Following PRP application, a foam dressing (Allevin^®, Smith and Nephew, Huntingdon, UK) was placed on the ulcer for 48 h. Time intervals between treatments varied from twice a week to once a week. The number of treatments differed depending on the wound state. After each treatment, the same dermatologist assessed clinical improvement and epithelization rate. Overall degree of ulcer improvement was assessed every week until complete epithelization occurred.

HaCaT keratinocyte cell lines were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂ in DMEM supplemented with 10% heat inactivated fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 100 g/ml streptomycin. For the experiments, cells (5×10⁴ cells/ml) were seeded in a culture dish and maintained in a tissue culture incubator.

HaCaT cells were seeded at a density of 15×10⁴ cells/well in 6-well culture plates. The cells were cultured using serum-free DMEM supplemented with 0 (control), 0.05, 0.5, 1 or 2% activated PRP for 3 days. The cultured cells were assayed for proliferation using the trypan blue exclusion method. Briefly, cells were washed with phosphate-buffered saline and stained with trypan blue dye. Viable cells were observed as light reflecting cells, but dead cells were observed as dark cells. The numbers of viable cells were measured under an inverted phase contrast microscope.

For the measurement of cell migration, confluent keratinocytes, which were kept in serum-free medium for 24 h, were wounded with a plastic micropipette tip. After washing, the medium was replaced by serum-free medium or serum-free medium supplemented with 0.01, 0.05, 0.5, 1 or 2% activated PRP for 24 h. Images of the wounded area were taken every 24 h by phase-contrast microscopy under crystal violet staining.

Whole cell extracts were prepared in lysis buffer [10 mM Tris (pH 7.4), 5 mM EDTA, 130 mM NaCl, 1% Triton X-100, phenylmethylsulphonyl fluoride (PMSF, 10 g/ml), aprotinin (10 g/ml), leupeptin (10 g/ml), 5 mM phenanthroline and 28 mM benzamidine-HCl]. The protein concentration of extracts was estimated with Bradford reagent (Bio-Rad Laboratories, Hercules, CA, USA) using bovine serum albumin as the standard. Equal amounts of protein (40 μg/lane) were resolved by 6.5–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. The membrane was then washed with Tris-buffered saline (10 mM Tris, 150 mM NaCl) containing 0.05% Tween-20 (TBST) and blocked in TBST containing 5% non-fat dried milk. The membrane was further incubated with respective specific antibodies. The membrane was continuously incubated with appropriate secondary antibodies coupled to horseradish peroxidase and developed in the ECL Western detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

We analyzed the effect of PRP on migration and proliferation rates of the HaCaT keratinocyte cell line. As shown in Fig. 1A, PRP treatment resulted in a dose-dependent increase of the proliferation rates of HaCaT cells. In addition, PRP treatment resulted in markedly increased rates of migration of HaCaT cells, even with a low concentration of PRP (0.005–0.5%; Fig. 1B).

Cell cycle regulatory proteins are important in proliferation and migration of HaCaT keratinocyte cell lines. We have investigated the question of whether PRP treatment promotes the proliferative and migratory activities of HaCaT cells through upregulation of G1/S or G2/M transition regulatory proteins. In order to determine the basal expression levels of cell cycle regulatory proteins in HaCaT cells, cells were cultured without fetal bovine serum (FBS) for 3 days. After serum starvation for 3 days, cells were cultured under 5% FBS or 2% (v/v) PRP with DMEM (Fig. 2). Expression levels of G1/S transition regulatory proteins, namely, ppRb, CDK4 and cyclin A, were decreased in serum-starved HaCaT cells. Notably, 2% PRP induced increased expression of cyclin A and CDK4 in HaCaT cells, resulting in increased expression of ppRb. Markedly increased expression of cyclin D1, cyclin E and CDK4 was not observed.

Based on the laboratory data that support the enhancing effect of PRP on wound healing, we performed a clinical application of PRP to acute and chronic wounds and evaluated its clinical wound healing effect. The clinical characteristics and outcomes of patients are summarized in Table I.

Eleven patients with chronic ulcers (cases 1–11) presented with stasis ulcer, diabetic ulcer, venous leg ulcer, livedoid vasculitis, claw foot and traumatic ulcer. The mean period of the disease was 8.54 months. However, in 15.18 days, 9 patients showed 90–100% epithelization. Case 2 presented with large-sized stasis ulcers and had been refractory to standard treatment for 2 months. However, almost a complete epithelization was shown in 24 days after 3 PRP treatments at 3-day intervals (Fig. 3A). In case 4, the patient had been suffering from a penetrating non-adhesion deep ulcer on an amputated foot for 18 months (Fig. 3B). The wound had not been healed by sutures. Thus, we applied PRP gel (6 times) on the ulcer and observed a complete epithelization 20 days after treatment. In case 11, a round-shaped ulcer on the middle interphalangeal joint of the left 3rd toe following corn treatment using cryotherapy (Fig. 3C) also showed epithelization in 23 days after treatment 3 times with PRP gel solution.

In 5 patients with acute ulcers (cases 12–16), an 80–100% epithelization rate was achieved in 20 days. These wounds had occurred for an average of 2 months, including special indications such as dehiscence (case 12, Fig. 3D), open wound, pressure ulcer (case 14, Fig. 3E) and burn wound (case 16, Fig. 3F). Three months after the treatment, the majority of the patients (90%), including acute and chronic patients, evaluated the ulcer appearance as having good to excellent improvement and the remaining patients (10%) rated the improvement as moderate.