The Effects of Platelet-Rich Fibrin in the Behavior of Mineralizing Cells Related to Bone Tissue Regeneration-A Scoping Review of In Vitro Evidence.

Platelet-rich fibrin (PRF) is a second-generation blood concentrate that serves as an autologous approach for both soft and hard tissue regeneration. It provides a scaffold for cell interaction and promotes the local release of growth factors. PRF has been investigated as an alternative to bone tissue therapy, with the potential to expedite wound healing and bone regeneration, though the mechanisms involved are not yet fully understood. This review aims to explore the in vitro evidence of PRF's effects on the behavior of mineralizing cells related to bone tissue regeneration. A systematic electronic search was conducted up to August 2023, utilizing three databases: PubMed, Web of Science, and Scopus. A total of 76 studies were selected, which presented in vitro evidence of PRF's usefulness, either alone or in conjunction with other biomaterials, for bone tissue treatment. PRF membranes' influence on the proliferation, differentiation, and mineralization of bone cells is linked to the constant release of growth factors, resulting in changes in crucial markers of bone cell metabolism and behavior. This further reinforces their therapeutic potential in wound healing and bone regeneration. While there are some notable differences among the studies, the overall results suggest a positive effect of PRF on cell proliferation, differentiation, mineralization, and a reduction in inflammation. This points to its therapeutic potential in the field of regenerative medicine. Collectively, these findings may help enhance our understanding of how PRF impacts basic physiological processes in bone and mineralized tissue.

Antimicrobial effect of platelet-rich fibrin: A systematic review of in vitro evidence-based studies.

This systematic review (SR) aimed to evaluate the antimicrobial potential of different types of platelet-rich fibrin (PRF) often used in regenerative treatments. An electronic search was performed in four databases and in Gray literature for articles published until January, 2023. The eligibility criteria comprised in vitro studies that evaluated the antimicrobial effect of different types of PRF. For the analysis of the risk of bias within studies, the modified OHAT (Office of Health Assessment and Translation) tool was used. For the evaluation of the results, a qualitative critical analysis was carried out in the synthesis of the results of the primary studies. Sixteen studies published between 2013 and 2021 were included in this SR. The antimicrobial effects of PRF variations (PRF, injectable PRF [I-PRF], PRF with silver nanoparticles [agNP-PRF], and horizontal PRF [H-PRF]), were analyzed against 16 types of bacteria from the oral, periodontal, and endodontic environments. All types of PRF showed significant antimicrobial action, with the antibacterial efficacy being more expressive than the fungal one. The I-PRF, H-PRF, and agNP-PRF subtypes improve antimicrobial activity. According to the OHAT analysis, no study was classified as having a high risk of bias. Evidence suggests that PRF variations have significant antimicrobial activity, with bacterial action being greater than fungal. Evolutions such as I-PRF, H-PRF, and agNP-PRF improve antimicrobial activity. Future studies analyzing the clinical effect of these platelets are fundamental. This SR was registered in INPLASY under number INPLASY202340016.

Biological and Cellular Properties of Advanced Platelet-Rich Fibrin (A-PRF) Compared to Other Platelet Concentrates: Systematic Review and Meta-Analysis.

Platelet concentrates are used for cell induction and stimulation in tissue repair processes. The aim of the present systematic review and meta-analysis was to compare the biological and cellular properties of advanced platelet-rich fibrin (A-PRF) to those of other platelet concentrates. Searches were conducted on the PubMed/Medline, Scopus, Web of Science, Embase and LILACS databases using a search strategy oriented by the guiding question. A total of 589 records were retrieved. Seven articles of in vitro experimental studies were selected for qualitative data analysis and four were selected for meta-analysis. The release of growth factors, distribution of cells in the fibrin membrane, and cell viability, the fibrin network, and fibroblast migration were investigated. In the final analysis, statistically significant differences were found for the A-PRF group with regard to platelet-derived growth factor, transforming growth factor, epidermal growth factor and vascular endothelial growth factor at all assessment times. A difference was found with regard to bone morphogenetic protein only in the later assessment, and no differences among groups were found with regard to platelet-derived growth factor or insulin-like growth factor. The results of this systematic review and meta-analysis suggest that A-PRF has superior cellular properties and better release of growth factors compared to other platelet concentrates.

The impact of gender and peripheral blood parameters on the characteristics of L-PRF membranes.

Aim: The purpose of this study was to evaluate the impact of gender and peripheral blood parameters on the characteristics of Leucocyte-and Platelet-Rich Fibrin (L-PRF) membranes and to describe histologically three different zones of L-PRF membranes. Methods: Blood was collected from twenty healthy donors (10 men and 10 women). Peripheral blood parameters including leucocyte and platelet counts, and fibrinogen levels were recorded. L-PRF membranes were prepared to quantify the release of growth factors (PDGF, VEGF, BMP-2, and BMP-9) at 1, 2, 3 and 7 days and for histological examination. Three zones within each L-PRF membrane (face, body, and tail) were analysed separately, quantifying the area of leucocytes, platelets, and fibrin in percentage. The Young's modulus of the membranes was also considered (during tensile and compression tests). Results: Women had significantly higher fibrinogen levels in their peripheral blood, and a higher release of BMP-9, whereas men showed a significantly higher Young's modulus in compression tests. The histology revealed significant differences in cellular content and fibrin concentration between the 3 areas, with the face being biologically the richest. Conclusion: Several factors influenced the final characteristics of L-PRF membranes. These need to be taken into consideration when interpreting the results of research, but especially in clinical practice.

Autologous platelet concentrates for facial rejuvenation

Abstract Autologous platelet concentrates (APCs) are promising therapeutic agents in facial rejuvenation since they are a great source of cytokines, growth factors and other biologically active substances. Obtained from the patient's blood, they have the advantages of reducing immunological reactions, making the procedure safer, well tolerated, with minimal adverse effects and lower cost. Currently, they are used for facial rejuvenation both in combination with microneedling and in mesotherapy techniques, as well as to treat facial acne scars, melasma and wounds after laser ablative treatments. This review summarizes current knowledge on the use of APCs, ranging from basic concepts related to their composition and mechanisms of action to up-to-date information on their clinical efficacy. Methodology MEDLINE (PubMed) was searched from inception through 2021 for English language publications on APCs for facial rejuvenation. Results A total of 100 files were found. Based on the available literature, APCs for skin rejuvenation are safe and well tolerated. The most studied product is the first-generation material, platelet-rich plasma (PRP). Conclusions The results are in general favorable, but the quality of the studies is low. The second and third generation products, platelet-rich fibrin (PRF) and injectable platelet-rich fibrin (i-PRF), respectively, are easier to be obtained and, at least in vitro , seem to induce greater collagen production than PRP, especially under lower relative centrifugation forces, but to date only a few clinical trials evaluating these products exist. More high-quality trials with appropriate follow-up are necessary to provide adequate evidence that may help to improve the treatment regimens with APCs. Many aspects should be considered when designing clinical trials to evaluate APCs, such as the patients' characteristics that best predict a favorable response, the optimal number of sessions and the interval between them, the characteristics of the studies and the development of better instruments to evaluate skin aging.

Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates / Otimização de um método baseado em PCR em tempo real para rastreamento de bactérias em concentrados de plaquetas

Bacterial contamination of blood components remains a major challenge in transfusion medicine, particularly, platelet concentrates (PCs) due to the storage conditions that support bacterial proliferation. In this study, we develop a rapid, sensitive and specific real-time PCR protocol for bacterial screening of PCs. An internally controlled real-time PCR-based method was optimized and validated with our proprietary 16S Universal PCR Master Mix (IBMP/Fiocruz), which targets a conserved region of the bacterial 16S rRNA gene. Nonspecific background DNA was completely eliminated by treating the PCR Master Mix with ethidium monoazide (EMA). A lower limit of detection was observed for 10 genome equivalents with an observed Ct value of 34±1.07 in calibration curve generated with 10-fold serial dilutions of E. coli DNA. The turnaround time for processing, including microbial DNA purification, was approximately 4 hours. The developed method showed a high sensitivity with no non-specific amplification and a lower time-to-detection than traditional microbiological methods, demonstrating it to be an efficient means of screening pre-transfusion PCs.(AU)

A contaminação bacteriana dos componentes sanguíneos é um grande desafio na medicina transfusional, principalmente nos concentrados de plaquetas (PCs) devido às condições de armazenamento que favorecem a proliferação bacteriana. Neste estudo, desenvolvemos um protocolo de PCR em tempo real rápido, sensível e específico para a triagem bacteriana de PCs. Um método baseado em PCR em tempo real, controlado internamente, foi otimizado e validado com um Master Mix Universal PCR 16S (IBMP / Fiocruz), que detecta uma região conservada do gene 16S rRNA bacteriano. O background de DNA não específico foi completamente eliminado tratando a PCR Master Mix com monoazida de etídio (EMA). O limite de detecção inferior observado foi de 10 cópias equivalentes do genoma com um valor de Ct 34 ± 1,07, a curva de calibração foi gerada com diluições seriada de 10 vezes do DNA de E. coli. O tempo de processamento, incluindo a purificação microbiana do DNA, foi de aproximadamente 4 horas. O método desenvolvido mostrou alta sensibilidade sem amplificação inespecífica e menor tempo de detecção do que os métodos microbiológicos tradicionais, demonstrando ser um meio eficiente de triagem de PCs pré-transfusionais.(AU)

Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates / Otimização de um método baseado em PCR em tempo real para rastreamento de bactérias em concentrados de plaquetas

Abstract Bacterial contamination of blood components remains a major challenge in transfusion medicine, particularly, platelet concentrates (PCs) due to the storage conditions that support bacterial proliferation. In this study, we develop a rapid, sensitive and specific real-time PCR protocol for bacterial screening of PCs. An internally controlled real-time PCR-based method was optimized and validated with our proprietary 16S Universal PCR Master Mix (IBMP/Fiocruz), which targets a conserved region of the bacterial 16S rRNA gene. Nonspecific background DNA was completely eliminated by treating the PCR Master Mix with ethidium monoazide (EMA). A lower limit of detection was observed for 10 genome equivalents with an observed Ct value of 34±1.07 in calibration curve generated with 10-fold serial dilutions of E. coli DNA. The turnaround time for processing, including microbial DNA purification, was approximately 4 hours. The developed method showed a high sensitivity with no non-specific amplification and a lower time-to-detection than traditional microbiological methods, demonstrating it to be an efficient means of screening pre-transfusion PCs.

Resumo A contaminação bacteriana dos componentes sanguíneos é um grande desafio na medicina transfusional, principalmente nos concentrados de plaquetas (PCs) devido às condições de armazenamento que favorecem a proliferação bacteriana. Neste estudo, desenvolvemos um protocolo de PCR em tempo real rápido, sensível e específico para a triagem bacteriana de PCs. Um método baseado em PCR em tempo real, controlado internamente, foi otimizado e validado com um Master Mix Universal PCR 16S (IBMP / Fiocruz), que detecta uma região conservada do gene 16S rRNA bacteriano. O background de DNA não específico foi completamente eliminado tratando a PCR Master Mix com monoazida de etídio (EMA). O limite de detecção inferior observado foi de 10 cópias equivalentes do genoma com um valor de Ct 34 ± 1,07, a curva de calibração foi gerada com diluições seriada de 10 vezes do DNA de E. coli. O tempo de processamento, incluindo a purificação microbiana do DNA, foi de aproximadamente 4 horas. O método desenvolvido mostrou alta sensibilidade sem amplificação inespecífica e menor tempo de detecção do que os métodos microbiológicos tradicionais, demonstrando ser um meio eficiente de triagem de PCs pré-transfusionais.

Efectividad de los concentrados plaquetarios en el tratamiento de los desórdenes temporomandibulares / Effectiveness of platelet concentrates in the treatment of temporomandibular disorders

Introducción: Los desórdenes temporomandibulares son un grupo de trastornos que afectan la articulación temporomandibular y/o los tejidos musculoesqueléticos asociados. Objetivo: Determinar la efectividad de los concentrados de plaquetas en el tratamiento de los desórdenes temporomandibulares. Métodos: La búsqueda de la literatura fue realizada desde enero del 2014 hasta abril del 2019, en las bases de datos biomédicas: PubMed, Embase, SciELO, Scopus, Science Direct, Sistema de información sobre literatura gris en Europa, Literatura Latinoamericana y del Caribe en Ciencias de la Salud, Google Académico y el Registro Central de Ensayos Clínicos Cochrane. Se definieron como criterios de selección de los estudios que fueran ensayos clínicos aleatorizados, con una antigüedad máxima de cinco años, que reportarann la efectividad (reducción del dolor y aumento de apertura máxima) de los concentrados plaquetarios en el tratamiento de los desórdenes temporomandibulares. El riesgo de sesgo de los estudios fue analizado por medio del Manual Cochrane de revisiones sistemáticas de intervenciones. Resultados: La estrategia de búsqueda resultó en nueve artículos, de los cuales el 100 por ciento reportó que no había diferencia en la reducción del dolor y el aumento de apertura máxima de los concentrados plaquetarios en el tratamiento de los desórdenes temporomandibulares. Conclusiones: La literatura revisada sugiere que existe una ligera evidencia de los beneficios potenciales de las inyecciones intraarticulares de los concentrados plaquetarios en pacientes con desórdenes temporomandibulares. Sin embargo, es necesario establecer un protocolo estandarizado para la preparación y aplicación de estos concentrados(AU)

Introduction: Temporomandibular disorders are a group of dysfunctions which affect the temporomandibular joint and/or associated musculoskeletal tissues. Objective: Determine the effectiveness of platelet concentrates in the treatment of temporomandibular disorders. Methods: A bibliographic search was conducted from January 2014 to April 2019 in the biomedical databases PubMed, Embase, SciELO, Scopus, Science Direct, System for Information on Gray Literature in Europe, Latin American and Caribbean Health Sciences Literature, Google Scholar, and Cochrane Central Register of Clinical Trials. The following selection criteria were defined for the studies: randomized clinical trials published in the last five years and reporting on the effectiveness (pain reduction and maximum opening increase) of platelet concentrates in the treatment of temporomandibular disorders. Bias risk analysis was based on the Cochrane manual of systematic reviews of interventions. Results: Nine papers were retrieved, of which 100 percent reported no differences in pain reduction or maximum opening increase resulting from the use of platelet concentrates in the treatment of temporomandibular disorders. Conclusions: The literature review conducted suggests that there is slight evidence of the potential benefits of intra-articular injections of platelet concentrates in patients with temporomandibular disorders. However, a standardized protocol should be established for the preparation and application of these concentrates(AU)

Technical Procedures for Preparation and Administration of Platelet-Rich Plasma and Related Products: A Scoping Review.

INTRODUCTION: Platelet-rich plasma is widely used for different types of clinical situations, but universal standardization of procedures for its preparation is still lacking. METHODS: Scoping review of comparative studies that have assessed at least two alternatives in one or more stages of preparation, storage and/or administration of PRP or its related products. A systematic search was conducted in MEDLINE, Embase, and LILACS. Two authors screened references independently. Data extraction was performed iteratively, and results were presented for each included comparison. RESULTS: Thirty-nine studies were included after assessing full texts, focusing on the comparison of PRP to a related product, types of anticoagulants, centrifugation protocols, commercial kits, processing time, methods for activation, and application concomitantly to other substances. Only laboratory outcomes were assessed, as platelet, leukocyte and growth factor concentrations. CONCLUSION: Results showed great variability related to methods employed in different stages of PRP processing, which may explain the variability observed in clinical trials assessing the efficacy of PRP for different clinical situations.

Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates

Abstract Bacterial contamination of blood components remains a major challenge in transfusion medicine, particularly, platelet concentrates (PCs) due to the storage conditions that support bacterial proliferation. In this study, we develop a rapid, sensitive and specific real-time PCR protocol for bacterial screening of PCs. An internally controlled real-time PCR-based method was optimized and validated with our proprietary 16S Universal PCR Master Mix (IBMP/Fiocruz), which targets a conserved region of the bacterial 16S rRNA gene. Nonspecific background DNA was completely eliminated by treating the PCR Master Mix with ethidium monoazide (EMA). A lower limit of detection was observed for 10 genome equivalents with an observed Ct value of 34±1.07 in calibration curve generated with 10-fold serial dilutions of E. coli DNA. The turnaround time for processing, including microbial DNA purification, was approximately 4 hours. The developed method showed a high sensitivity with no non-specific amplification and a lower time-to-detection than traditional microbiological methods, demonstrating it to be an efficient means of screening pre-transfusion PCs.

Resumo A contaminação bacteriana dos componentes sanguíneos é um grande desafio na medicina transfusional, principalmente nos concentrados de plaquetas (PCs) devido às condições de armazenamento que favorecem a proliferação bacteriana. Neste estudo, desenvolvemos um protocolo de PCR em tempo real rápido, sensível e específico para a triagem bacteriana de PCs. Um método baseado em PCR em tempo real, controlado internamente, foi otimizado e validado com um Master Mix Universal PCR 16S (IBMP / Fiocruz), que detecta uma região conservada do gene 16S rRNA bacteriano. O background de DNA não específico foi completamente eliminado tratando a PCR Master Mix com monoazida de etídio (EMA). O limite de detecção inferior observado foi de 10 cópias equivalentes do genoma com um valor de Ct 34 ± 1,07, a curva de calibração foi gerada com diluições seriada de 10 vezes do DNA de E. coli. O tempo de processamento, incluindo a purificação microbiana do DNA, foi de aproximadamente 4 horas. O método desenvolvido mostrou alta sensibilidade sem amplificação inespecífica e menor tempo de detecção do que os métodos microbiológicos tradicionais, demonstrando ser um meio eficiente de triagem de PCs pré-transfusionais.

Detection of bacterial contamination in platelet concentrates from Brazilian donors by molecular amplification of the ribosomal 16S gene.

OBJECTIVE: The aim of our work was to establish a semi-automated high-throughput DNA amplification method for the universal screening of bacteria in platelet concentrates (PCs). BACKGROUND: Among cases of transfusion transmission of infectious agents, bacterial contamination ranks first in the number of events, morbidity and mortality. Transmission occurs mainly by transfused PCs. Automated culture is adopted by some blood banks for screening of bacterial contamination, but this procedure is expensive and has a relatively long turnaround time. METHODS: PCs were spiked with suspensions of five different bacterial species in a final concentration of 1 and 10 colony-forming units (CFU) per millilitre. After incubation, the presence of bacteria was investigated by real-time polymerase chain reaction (PCR) and by the Enhanced Bacterial Detection System (eBDS, Pall) assay as a reference method. Real-time PCR amplification was performed with a set of universal primers and probes targeting the 16S rRNA gene. Co-amplification of human mitochondrial DNA served as an internal control. RESULTS: Using the real-time PCR method, it was possible to detect the presence of all bacterial species tested with an initial concentration of 10 CFU mL-1 24 h after contamination, except for Staphylococcus hominis. The PCR assay also detected, at 24 h, the presence of Serratia marcescens and Enterobacter cloacae with an initial concentration of 1 CFU mL-1 . CONCLUSIONS: The real-time PCR assay may be a reliable alternative to conventional culture methods in the screening of bacterial contamination of PCs, enabling bacterial detection even with a low initial concentration of microorganisms.

Differential Expression Profile of MicroRNAs During Prolonged Storage of Platelet Concentrates As a Quality Measurement Tool in Blood Banks.

Platelet concentrate (PC) is a key blood component, which even in good storage conditions, susceptible to cellular damage over time. Hence, blood banks discard unused PC bags after 5 days of storage. Biomarkers of PC quality are therefore highly sought after in blood bank governance. We used the data (Gene Expression Omnibus: GSE61856) generated with next-generation sequencing to examine the expression profiles of microRNAs (miRNAs) from PCs that were stored for 6 days in a blood bank, that is, 1 day longer than is normally stored PC. We identified the 14 most differentially expressed miRNAs by comparing a control PC on the first day of storage with the PCs on each of the subsequent 5 days of storage from day 1 to 6. In all, we identified nine miRNAs with the downregulated profile (miR-145-5p, miR-150-5p, miR-183-5p, miR-26a-5p, miR-331-3p, miR-338-5p, miR-451a, miR-501-3p, and miR-99b-5p) and five upregulated miRNAs (miR-1304-3p, miR-411-5p, miR-432-5p, miR-668-3p, and miR-939-5p). These miRNAs were validated by real-time quantitative PCR in 100 PC units. As each PC unit is composed of platelets of five individuals, the validation was thus performed in 500 individuals (250 men and 250 women, comprised 18-40 years old adults). The data were analyzed with hierarchical clustering and principal component analysis, which revealed the variation of mean relative expression and the instability of miRNAs half-life on the fourth day of PC storage, which coincides with time of onset of platelet storage lesions. These new observations can usefully inform future decision-making and governance in blood banks concerning PC quality.

Análisis estructural de la fibrina rica en plaquetas y sus aplicaciones en odontología regenerativa / Structural Analysis of Platelet-Rich Fibrin and its Applications in Regenerative Dentistry

Antecedentes: La fibrina rica en plaquetas (PRF) es un concentrado plaquetario que se está usando con mayor frecuencia en medicina y odontología. Los resultados clínicos son variables posiblemente porque hay diferentes protocolos de obtención, equipos de centrifugado y técnicas de colocación. El desconocimiento de los aspectos estructurales puede afectar el resultado clínico. Objetivo: Describir las características estructurales de la PRF en las diferentes zonas de la membrana. Métodos: Se realizó un estudio experimental in vitro con 15 muestras de sangre periférica tomada de cinco voluntarios adultos, sanos, asistentes a la clínica odontológica de la Universidad Antonio, Popayán. Se hizo hemograma inicial, se recolectó sangre y se centrifugó (10 min x 3000 rpm). Las muestras se analizaron histológicamente y con microscopía electrónica de barrido (SEM). Se describió la estructura de la fibrina, las plaquetas y los leucocitos. Resultados: El promedio de recuento de plaquetas en sangre total fue de 251±31,74 x103 x mm3 y en PRF fue de 832±123,43 x103 x mm3. Macroscópicamente, se identificaron tres zonas del PRF: una superior con pocas plaquetas, una zona leucocitaria (BC) y una zona corpuscular roja. En el análisis de microscopía óptica muestra que en la zona BC hay mayor concentración plaquetaria. El análisis por SEM comprueba que la estructura de la red de fibrina y el contenido celular son diferenciales en cada zona. Conclusión: A partir del conocimiento estructural del PRF se pueden proponer aplicaciones que mejoren el rendimiento del material y por tanto los resultados clínicos.

Background: Platelet rich fibrin (PRF) is a platelet concentrate that is used most frequently in medicine and dentistry. Clinical results are variables because there are different protocols, centrifugation equipment and placement techniques. Ignorance of structural elements can affect the clinical outcome. Purpose: To describe the structural characteristics of Platelet Rich Fibrin in the different zones of the membrane. Methods: Experimental in vitro study with 15 blood samples taken from five healthy adult volunteers attending the dental clinic of the University Antonio Nariño, Popayán. The basal blood count was made, then blood was collected and centrifuged (10 minutes x 3000 rpm). The samples were analyzed histologically and with scanning electron microscopy (SEM). The structure of fibrin, platelets and leukocytes was described. Results: The average platelet count in whole blood was 251 ± 31.74 x103 x mm3 and in PRF it was 832±123.43 x103 x mm3. Macroscopically, three areas of the PRF were identified: an upper one with few platelets (FPP), a buffy coat (BC) and the corpuscular network zone (RBC). In the analysis of optical microscopy shows that in the BC area there is a higher platelet concentration. The SEM analysis proves that the structure of the fibrin network and cellular content is differential in each zone. Conclusion: From the structural knowledge of the PRF, applications that may be improve the performance of the biomaterial and therefore the clinical results can be proposed.

Autologous Platelet Concentrates as Treatment for Avascular Necrosis of Femoral Head in a Dog.

Avascular necrosis of the femoral head is a developmental disturbance that generally affects young dogs of small breeds and produces ischemic necrosis of the femoral head resulting in an incongruous and malformed joint. The most common treatment is the excisional arthroplasty of the head and femoral neck. The aim of this study is to describe the treatment of avascular necrosis in a Yorkshire dog using intra-articular injections of autologous platelet concentrate. Evaluations were made at 0, 15, 30, 60, and 120 days of treatment, describing the following parameters: clinical gait analysis, perimetry, goniometry, and radiographic evaluations. The results obtained in this case suggest that the autologous platelet concentrate may be an alternative for the treatment of avascular necrosis of the femoral head in dogs.

Macroscopic and histomorphometric evaluation of different healing stages of skin wounds in horses treated with leukocyte-poor plateletrich plasma

Skin injuries are frequent in horses, and one of the treatments used for such injuries is the platelet-rich plasma (PRP). The objective of this experiment was to macroscopically and microscopically investigate the process of healing by second intention in skin wounds of eight healthy gelding crossbred horses treated (T) or untreated (UT) with a single dose of leukocyte-poor PRP (LP-PRP). Three squareshaped wounds were induced in both gluteal regions. After 12 h of wound induction, 0.5 mL LP-PRP was administered in each border of the lesion in one of gluteal region. Contralateral wounds were UT. Six skin biopsies were obtained with a 6-mm Punch. Macroscopic variables of one of the non-biopsied wounds were evaluated. Samples were processed for histomorphometric evaluation. No difference was observed between the time required for wound closure in the two groups. Histomorphometric analysis performed 14 days after wound induction revealed higher (p = 0.034) angiogenesis and number (p = 0.0179) of total leukocytes in T wounds. Fibrocytes numbers increased significantly (p = 0.023) on day 7 after injury in the UT group. General microscopic evaluation performed independently of scores and morphometric analysis revealed that the majority of the T wounds showed better healing variables in the sections analyzed after complete macroscopic closure of the wound. A single injected dose of LP-PRP 12 h after wound induction in horses does not interfere with the wound healing process but reveals that majority of the T wounds exhibit better healing histomorphometric characteristics.(AU)

Macroscopic and histomorphometric evaluation of different healing stages of skin wounds in horses treated with leukocyte-poor plateletrich plasma

Skin injuries are frequent in horses, and one of the treatments used for such injuries is the platelet-rich plasma (PRP). The objective of this experiment was to macroscopically and microscopically investigate the process of healing by second intention in skin wounds of eight healthy gelding crossbred horses treated (T) or untreated (UT) with a single dose of leukocyte-poor PRP (LP-PRP). Three squareshaped wounds were induced in both gluteal regions. After 12 h of wound induction, 0.5 mL LP-PRP was administered in each border of the lesion in one of gluteal region. Contralateral wounds were UT. Six skin biopsies were obtained with a 6-mm Punch. Macroscopic variables of one of the non-biopsied wounds were evaluated. Samples were processed for histomorphometric evaluation. No difference was observed between the time required for wound closure in the two groups. Histomorphometric analysis performed 14 days after wound induction revealed higher (p = 0.034) angiogenesis and number (p = 0.0179) of total leukocytes in T wounds. Fibrocytes numbers increased significantly (p = 0.023) on day 7 after injury in the UT group. General microscopic evaluation performed independently of scores and morphometric analysis revealed that the majority of the T wounds showed better healing variables in the sections analyzed after complete macroscopic closure of the wound. A single injected dose of LP-PRP 12 h after wound induction in horses does not interfere with the wound healing process but reveals that majority of the T wounds exhibit better healing histomorphometric characteristics.

Prevalence of bacterial contamination in platelet concentrates at the National Center of Blood Transfusion (Mexico).

OBJECTIVES: Most common bacterial sepsis associated with transfusion is caused by contaminated Platelet Concentrates (PC). The screening of PC to detect bacterial contamination is obligatory in Mexico, and it is carried out in quality control programs. In Mexico, the identification and molecular characterization of bacterial contaminants to detect contamination sources have not been implemented due to high costs; however, it is an actual current need. MATERIAL AND METHODS: One hundred PC were randomly selected and microbiologically analyzed. This sample size corresponds to 1% of the PC obtained by the National Center of Blood Transfusion (NCBT) in Mexico City according to the Official Mexican Standard NOM-253-SSA1-2012. Additionally, molecular biology tests were implemented in order to identify the possible contamination sources. RESULTS: Nine of the 100 PC analyzed (9%) showed bacterial contamination; analysis of the nucleotide sequences revealed the presence of characteristic microbiota from donor skin and soil. Diverse clonal relationship between the strains was identified in Staphylococcus epidermidis. CONCLUSION: Detection of contaminants associated with environmental and skin flora, shows the need to implement measures in the process of disinfecting skin at the site of phlebotomy and cleaning each of the areas involved in blood collection.

Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates

Abstract Bacterial contamination of blood components remains a major challenge in transfusion medicine, particularly, platelet concentrates (PCs) due to the storage conditions that support bacterial proliferation. In this study, we develop a rapid, sensitive and specific real-time PCR protocol for bacterial screening of PCs. An internally controlled real-time PCR-based method was optimized and validated with our proprietary 16S Universal PCR Master Mix (IBMP/Fiocruz), which targets a conserved region of the bacterial 16S rRNA gene. Nonspecific background DNA was completely eliminated by treating the PCR Master Mix with ethidium monoazide (EMA). A lower limit of detection was observed for 10 genome equivalents with an observed Ct value of 34±1.07 in calibration curve generated with 10-fold serial dilutions of E. coli DNA. The turnaround time for processing, including microbial DNA purification, was approximately 4 hours. The developed method showed a high sensitivity with no non-specific amplification and a lower time-to-detection than traditional microbiological methods, demonstrating it to be an efficient means of screening pre-transfusion PCs.

Resumo A contaminação bacteriana dos componentes sanguíneos é um grande desafio na medicina transfusional, principalmente nos concentrados de plaquetas (PCs) devido às condições de armazenamento que favorecem a proliferação bacteriana. Neste estudo, desenvolvemos um protocolo de PCR em tempo real rápido, sensível e específico para a triagem bacteriana de PCs. Um método baseado em PCR em tempo real, controlado internamente, foi otimizado e validado com um Master Mix Universal PCR 16S (IBMP / Fiocruz), que detecta uma região conservada do gene 16S rRNA bacteriano. O background de DNA não específico foi completamente eliminado tratando a PCR Master Mix com monoazida de etídio (EMA). O limite de detecção inferior observado foi de 10 cópias equivalentes do genoma com um valor de Ct 34 ± 1,07, a curva de calibração foi gerada com diluições seriada de 10 vezes do DNA de E. coli. O tempo de processamento, incluindo a purificação microbiana do DNA, foi de aproximadamente 4 horas. O método desenvolvido mostrou alta sensibilidade sem amplificação inespecífica e menor tempo de detecção do que os métodos microbiológicos tradicionais, demonstrando ser um meio eficiente de triagem de PCs pré-transfusionais.

Antimicrobial susceptibility profile and research of mec A and erm genes in coagulase-negative staphylococci isolated from platelet concentrates bags

Abstract In recent years, several studies have described the clinical impact of bacterial infection associated with transfusion of platelet concentrates (PCs). Among the blood components, PCs are responsible for the highest rates of bacterial contamination as well as septic transfusion reactions. We assessed antimicrobial susceptibility profile, resistance to methicillin (MRCoNS), and resistance to macrolides, lincosamides and streptogramins of group B (MLSB) of 16 coagulase-negative staphylococci (CoNS) isolates from an investigation in 691 PCs bags. We then compared conventional and automated phenotypic methods, disc diffusion test (DD) and VITEK(r) 2, respectively as well as phenotypic and genotypic methods (Polymerase Chain Reaction - PCR). All CoNS were susceptible to vancomycin. The disc diffusion test characterized 18.75% as MRCoNS and 37.5% with inducible resistance to MLSB (iMLSB), and with VITEK(r) 2, 6.3% and 31.25%, respectively. The mecA gene was detected in 18.75% and the erm gene in 31.25% of the isolates. In this study, we found equal percentage values between presence of the mecA gene by PCR and resistance to methicillin using cefoxitin by DD test, evidence of the erm gene by PCR, and iMLSB resistance by automation (VITEK(r) 2). Moreover, we identified three strains with beta-lactamase overproduction, and the occurrence of a bigger mistake was verified when automation was compared with DD test. And we observed that D-test was the most reliable for the detection of iMLSB resistance in Staphylococcus sp.

Fibrina rica en plaquetas (FRP): Una alternativa terapéutica en odontología

El objetivo de esta revisión, fue describir de una manera sencilla las principales características de la Fibrina Rica en Plaquetas (FRP), su composición, propiedades y aplicación clínica. La FRP, biomaterial autógeno y concentrado plaquetario de segunda generación, es una matriz de fibrina que contiene leucocitos, plaquetas y factores de crecimiento, que son necesarios para los procesos de cicatrización, lo que brinda a este biomaterial, una gran utilidad en diversas áreas de la salud, incluyendo la odontología. Con esta revisión concluimos que la FRP es una alternativa real para mejorar la cicatrización de procedimientos quirúrgicos y potenciar otros biomateriales regenerativos en diversas áreas de la odontología, además de su accesibilidad y bajo costo.

The aim of this review was to describe in a simple way the main characteristics of platelet rich Fibrin (PRF), its composition, properties and clinical application. PRF, biomaterial autologous and platelet concentrate of second generation, is a fibrin matrix containing leukocytes, platelets and growth factors, which are necessary for the healing process, giving to this biomaterial, very useful in various areas of health, including dentistry. With this review, we conclude that FRP is a real alternative to improve healing of surgical procedures and enhance other regenerative biomaterials in several areas of dentistry, in addition to accessibility and low cost.