The effects of platelet gel-released supernatant on human fibroblasts.

In recent years, interest in the topical use of platelet gel (PG) to stimulate wound healing has rapidly extended into various clinical applications and specialized fields. Many recent in vitro and in vivo studies have attempted to explain the biological mechanisms involved in PG-induced tissue regeneration/reparation. However, it remains unclear which parameters should be used in clinical applications to obtain satisfactory results in the healing of wounds. Toward this end, the present study focused on understanding the relationship between platelet concentrations and the cellular parameters of the cell types, i.e., fibroblasts, involved in wound healing. Normal human dermal fibroblasts were treated with PG-released supernatant at various concentrations in different assays (proliferation, migration, invasion, and in vitro scratch wound closure) to identify the most effective concentration to promote the fibroblasts' activities. Different concentrations of platelets per microliter in PG have different levels of efficacy in inducing fibroblast activity. The most effective concentration was obtained from PG at a concentration of approximately 0.5-1.5 × 10(6) plt/µL; higher concentrations were less effective. This study shows that excessively high concentrations of platelets per microliter have an inhibitory effect on the wound healing processes and are, therefore, counterproductive.

Identification of an optimal concentration of platelet gel for promoting angiogenesis in human endothelial cells.

BACKGROUND: Numerous studies have supported the use of topical blood components to improve wound healing and tissue regeneration. Platelet gel (PG), a hemocomponent obtained from mix of activated platelets (PLTs) and cryoprecipitate, is currently being used clinically in an attempt to improve tissue healing. The present study sought to define the most effective PG concentration to promote angiogenesis in vitro. STUDY DESIGN AND METHODS: The effects of PG-released supernatant at different concentrations on human endothelial cells were studied using different in vitro assays (proliferation, migration, invasion, cord formation, and wound healing). RESULTS: The concentration of PG-released supernatant had a significant influence on the angiogenic potential of endothelial cells. The optimal concentration for the stimulation of angiogenesis was 1.5 x 10(6) PLTs per microL in most of the in vitro experiments used in this study. Lower or higher concentrations of PG displayed a lower angiogenic potential. CONCLUSION: An optimal concentration of PG to promote angiogenesis in human endothelial cells was identified. Excessively high PG concentrations may inhibit the angiogenic process, thereby being counterproductive for wound healing in a clinical setting.

Leukocyte depletion does not affect the in vitro healing ability of platelet rich plasma.

The clinical use of platelet-rich plasma (PRP) containing or deprived of leukocytes remains a subject of debate and a controversial issue. It is not yet clear whether leukocyte content has a positive or negative effect on tissue healing processes. Several studies, conducted mainly in the orthopedic field, support the use of leukocyte-poor (LP) PRP, whereas other studies have not identified any significant differences between the use of LP and leukocyte-rich PRP. In the present study, the role of leukocytes contained in PRP was assessed to verify their in vitro effect on fibroblasts and endothelial cells, which have a leading role in the biological processes associated with wound healing (including angiogenesis and matrix remodeling). The original sample of PRP was divided into two aliquots, one of which remained unaltered, while the other was deprived of leukocytes. The two aliquots were used in in vitro tests in order to verify the effects of leukocytes on proliferation, wound healing and tube formation, and in molecular analyses of growth factor and enzyme content. The present results highlighted a substantial overlap between the two formulations. This may be explained by similar levels of growth factors (vascular endothelial growth factor, thrombospondin-1, interferon-γ, platelet-derived growth factor-BB, -AA and -B, tumor growth factor-ß1, fibroblast growth factor 7 and tumor necrosis factor-α) and enzymes (gelatinases and plasminogen activators) in the two formulations. These results support the hypothesis that the ability of the PRP to affect the in vitro biological response of endothelial cells and fibroblasts does not rely on the presence of leukocytes.

Infected Nonhealing Wound in a Kidney Transplant Recipient: Successful Treatment With Topical Homologous Platelet-Rich Gel.

Impaired would healing is a known adverse effect of chronic immunosuppression. Solid-organ transplant recipients undergoing major abdominal surgery have an increased risk of wound-related complications compared with the general population. In this subset of patients, surgical site infections and wound dehiscence must be aggressively treated to avoid sepsis, graft loss, and death. Recently, topical application of platelet-rich plasma has been proposed as an alternative therapeutic option to enhance wound healing in difficult cases. Unfortunately, randomized controlled trials evaluating the efficacy of platelet-rich plasma compared with standard or advanced wound management are lacking, and the literature mostly refers to anecdotal reports in patients with no evidence of wound infection. This report documents a kidney transplant recipient who experienced spontaneous bladder rupture because of gangrenous cystitis. After an exploratory laparotomy and bladder repair, the patient developed a deep surgical site infection by multidrug resistant Acinetobacter baumannii and extensive wound dehiscence. Advanced wound management and vacuum-assisted closure therapy were ineffective. Topical homologous platelet-rich gel was used resulting in significant wound healing, without infections or immunologic complications.

Oral Platelet Gel Supernatant Plus Supportive Medical Treatment Versus Supportive Medical Treatment in the Management of Radiation-induced Oral Mucositis: A Matched Explorative Active Control Trial by Propensity Analysis.

OBJECTIVES: In this active control trial, the rate of radio-induced WHO grade 3/4 oral mucositis and the change in quality of life, assessed by OMWQ-HN, were measured in subjects with head and neck cancer treated by platelet gel supernatant (PGS) and supportive medical treatment versus subjects treated by supportive medical treatment alone. MATERIALS AND METHODS: Eighty patients with nonmetastatic head and neck cancer underwent curative or adjuvant radiotherapy. All patients underwent supportive medical treatment and/or PGS at the beginning and during radiotherapy. Sixteen patients received PGS in association with supportive medical treatment. To obtain 2 groups virtually randomized for important clinical characteristics subjects were matched, by propensity analysis, with a group of subjects (64 patients) treated with supportive medical treatment alone. RESULTS: Subjects treated with standard supportive treatment experienced significant higher WHO grade 3/4 toxicity (55%; 35/64) than subjects treated by PGS (13%; 3/16). The reduced toxicity found in PGS group paralleled with the evidence that they developed later symptoms with respect to controls. The Cox proportional hazard model indicated that patients treated with standard supportive medical treatment experienced 2.7-fold increase (hazard ratio=2.7; 95% confidence interval, 1.3-5.7) in the occurrence of WHO grade 3/4 toxicity. PGS group significantly experienced higher quality of life than control groups as measured by OMWQ-HN. A significant decrease in the opioid analgesics usage was found in the PGS group. CONCLUSIONS: These preliminary data should be interpreted with caution and could serve as a framework around which to design future trials.

Microfracture versus microfracture and platelet-rich plasma: arthroscopic treatment of knee chondral lesions. A two-year follow-up study.

PURPOSE: the aim of this study was to describe and compare the clinical results obtained in patients affected by chondral lesions of the knee submitted to an arthroscopic treatment with the microfracture technique or microfracture + intraoperative autologous platelet-rich plasma (PRP) injection. METHODS: a prospective observational study was performed in patients affected by chondral lesions of the knee (classed as grade III-IV according to Outer-bridge's classification) and early osteoarthritis (classed as grade 1-2 according to the Kellgren-Lawrence classification). Their mean age was 52.4 years. Thirteen patients were treated with the microfracture technique according to Steadman (Group A), while 14 were treated with microfracture + PRP injection (Group B). Both groups were assessed using series of measures (a visual analog scale for pain, the 36-Item Short Form Health Survey and the International Knee Documentation Committee Subjective Knee Form) to compare pre-operative and postoperative values at 3, 6, 12 and 24 months. Statistical analysis was conducted using a two-factor ANOVA for repeated measures. RESULTS: the VAS score decreased from a pre-operative value of 6.62±1.26 to 3.54 ±2.26 at 24 months in Group A (p<0.001), and from 6.43±1.91 to 3.36±2.84 in Group B (p<0.001). The IKDC subjective score increased from a pre-operative value of 37.02±12.00 to 62.13±19.00 at two years in Group A (p<0.001) and from 34.63±15.00 to 67.11±26.74 in Group B (p<0.001); the SF-36 scores showed a similar trend. Although an improvement was recorded over time in both groups, in the short term the IKDC subjective score improvement seemed to be better in Group B; a similar trend was shown by the SF-36 and VAS scores. At two years, the IKDC Subjective Scale, VAS and SF-36 scores seemed to be similar in the two groups. Over time, no significant differences were found between the two groups in any of the three outcomes. CONCLUSIONS: the use of autologous PRP in association with the microfracture technique seems to give better clinical and functional results in short-term follow-up, above all as regards pain. At two-year follow-up, however, the clinical results of the two groups were similar. LEVEL OF EVIDENCE: Level II, prospective cohort study.

Platelet concentration in platelet-rich plasma affects tenocyte behavior in vitro.

Since tendon injuries and tendinopathy are a growing problem, sometimes requiring surgery, new strategies that improve conservative therapies are needed. Platelet-rich plasma (PRP) seems to be a good candidate by virtue of its high content of growth factors, most of which are involved in tendon healing. This study aimed to evaluate if different concentrations of platelets in PRP have different effects on the biological features of normal human tenocytes that are usually required during tendon healing. The different platelet concentrations tested (up to 5 × 10(6) plt/µL) stimulated differently tenocytes behavior; intermediate concentrations (0.5 × 10(6), 1 × 10(6) plt/µL) strongly induced all tested processes (proliferation, migration, collagen, and MMPs production) if compared to untreated cells; on the contrary, the highest concentration had inhibitory effects on proliferation and strongly reduced migration abilities and overall collagen production but, at the same time, induced increasing MMP production, which could be counterproductive because excessive proteolysis could impair tendon mechanical stability. Thus, these in vitro data strongly suggest the need for a compromise between extremely high and low platelet concentrations to obtain an optimal global effect when inducing in vivo tendon healing.

Platelet gel-released supernatant modulates the angiogenic capability of human endothelial cells.

BACKGROUND: Platelet gel is used to facilitate wound healing in virtue of the growth factors released from activated platelets at the site of lesion, but little is known about the specific mechanisms underlying cellular repair. AIMS: To evaluate, in vitro, cellular effects of different concentrations of platelet gel -released supernatant on endothelial cells. MATERIAL AND METHODS: Platelet concentrate was produced at the Service of Immunohaematology and Transfusion of San Salvatore Hospital of L'Aquila, using multiple bags. Platelet gel was obtained by adding thrombin and calcium gluconate to the concentrates and then centrifuging to recover the supernatant. Human umbilical vein endothelial cells were isolated from umbilical cord veins and grown in appropriate conditions. To study their viability, cells were treated with different concentrations of supernatant and XTT assays were performed on the 3 days following treatment. Endothelial cell motility and invasiveness were assayed using modified Boyden chambers with filters coated with 0.1% gelatin (for the motility test) or with a thick layer of the reconstituted basement membrane Matrigel (for the invasion test). The supernatant, added at various concentrations to the lower compartment of the chamber, was used as an attractant. Umbilical cells were added to the upper compartment of the chamber. After 4 hours (for the motility test) or 6 hours (for the invasion test), filters were stained and the migrated cells in five high-power fields were counted. RESULTS: When used at specific concentrations, platelet gel-released supernatant is able to induce proliferation and to stimulate motility and invasiveness of endothelial human cells. Higher concentrations induce a reversion of the stimulatory processes. CONCLUSIONS: There is a large body of evidence indicating that platelets and their derivatives have the potential for a substantial therapeutic role in tissue regeneration. The results of this in vitro study highlight the need for an in-depth analysis of technical protocols for the most appropriate and effective use of platelet gel for in vivo applications.