Potential effect of platelet rich fibrin prepared under differen centrifugation protocols on stem cells from the apical papila / O potencial efeito da fibrina rica em plaquetas preparada sob diferentes protocolos de centrifugação nas células tronco da papila apical

Objetivo: Este estudo objetivou avaliar o potencial proliferativo e de diferenciação das células tronco da papila cultivadas conjuntamente com fibrina rica em plaquetas (PRF) preparados sob dois protocolos de centrifugação distintos. Material e Métodos: Protocolos padrão e avançado de PRF foram utilizados. As células foram divididas em 4 grupos: controle negativo, controle positivo, padrão (L-PRF) e avançado (A-PRF). A contagem de células e ensaio de viabilidade foram realizados para verificar a capacidade proliferativa. Coloração vermelho de alizarina S, atividade de fosfatase alcalina e imunofluorescência para o receptor ativador do fator nuclear kappa-B (RANKL) foram utilizados para avaliar o potencial osteogênico e de diferenciação celular. Resultados: Ambos os tipos de PRF aumentaram o número de células, viabilidade celular sem toxicidade o que refletiu no aumento da proliferação e diferenciação de acordo com os testes realizados. Conclusão: O grupo A-PRF aumentou significativamente a proliferação e diferenciação comparado com o grupo L-PRF.(AU)

Objectives: The present work was designed to evaluate the proliferation and differentiation potential of stem cells from the apical papilla (SCAP) seeded along with platelet rich fibrin (PRF) scaffolds prepared under two different centrifugation protocols. Materials and Methods: Standard and advanced PRF protocols were used. Cells were divided into 4 groups: negative control, positive control, standard (L-PRF) and advanced (A-PRF) groups. Cell count and cell viability assays were carried out to assess the proliferation capacity. Alizarin red S (ARS) stain, Alkaline phosphatase (ALP) activity and Receptor activator of nuclear factor-kappa B ligand (RANKL) immunofluorescence staining were used to evaluate the osteogenic potential in the differentiated cells. Results:Both types of platelet rich fibrin increased the cell count, cell viability with no cytotoxicity that was reflected on increased proliferation and differentiation in terms of the performed tests. Conclusion: A-PRF group showed significant increase in proliferation and differentiation potentials compared to L-PRF group

Preliminary study on centrifugation time of liquid plasmatrix for soft tissue regeneration / 中华口腔医学杂志

Objective: To provide reference for clinical application of liquid plasmatrix, and to investigate the optimal centrifugation time of liquid plasmatrix prepared by horizontal centrifugation for soft tissue regeneration from the aspects of mechanical properties, biological properties, and the effect of promoting soft tissue regeneration. Methods: Venous blood was collected from 6 healthy volunteers [3 males and 3 females, aged (26±2) years, with informed consent] who volunteered to donate blood at School of Stomatology, Wuhan University from September to November 2021. The collected venous blood was centrifuged at 500 ×g for 3, 5, 8 and 12 min to obtain liquid plasmatrix. The volume, weight, solidification time, and mechanical properties of liquid plasmatrix prepared at different centrifugation time were measured and recorded (the sample size at each time point was 3). The microstructure of different groups of liquid plasmatrix clot was observed by scanning electron microscope (SEM). The rheological properties of each group of liquid plasmatrix clot were measured by rheological test. The number and concentration of cells in the whole blood group and in each liquid plasmatrix group were measured using complete blood count test. The distribution of cells in the liquid plasmatrix clots was observed by hematoxylin-eosin staining. The effect of control group (Dulbecco's modified Eagle's medium containing 20% fetal bovine serum) and liquid plasmatrix clot exudates in 3, 5, 8, 12 min group (the sample size at each time point was 3) on gingival fibroblast migration was detected by cell migration method. Finally, the effects of control group and liquid plasmatrix clot exudates on the morphology of gingival fibroblasts were observed by fluorescence microscope. Results: The volume of liquid plasmatrix in 3, 5, 8 and 12 min group were approximately (2.47±0.12), (2.67±0.12), (3.53±0.12) and (3.73±0.12) ml, respectively. The weight of liquid plasmatrix in 3, 5, 8 and 12 min group were approximately (0.35±0.01), (0.46±0.02), (0.88±0.06) and (1.03±0.01) g, respectively. The maximum tensile force of liquid plasmatrix clots in 3, 5, 8 and 12 min group were (0.55±0.03), (0.56±0.03), (1.31±0.05) and (1.38±0.02) N, respectively. SEM results showed that the fibers inside the liquid plasmatrix clot became denser with increased centrifugation time. Compared with other groups, the concentrations of leukocytes, neutrophils, monocytes and lymphocytes in 8 min group were the highest, and the distribution of cell was more even. Compared with other groups, the efficiency of stimulating gingival fibroblast migration in 8 min group was the best (1.60±0.01). Fluorescence staining test showed that the liquid plasmatrix clot exudates could make gingival fibroblasts more stretched compared with control group. Conclusions: The present study shows that liquid plasmatrix prepared by centrifugation with 500 ×g centrifugal force for 8 min has higher concentration of viable cells and the ability to promote the migration of gingival fibroblasts.

Alcance de las células madre derivadas de tejido adiposo / Scope of stem cells derived from adipose tissue

Introducción: En el tejido adiposo se han identificado células madre mesenquimales con capacidad autorrenovadora y multipotencial. Mediante digestión enzimática y centrifugado del lipoaspirado se libera una población heterogénea de células denominada fracción vascular estromal, con innumerables potencialidades terapéuticas en el campo de la medicina regenerativa. Objetivo: Actualizar el alcance de las células madre derivadas de tejido adiposo en la terapia regenerativa. Método: Se revisaron 38 artículos entre los años 2000 y 2019 en las bases de datos Scielo, ScienceDirect, Medline y Pubmed. Desarrollo: Las células de la fracción vascular estromal se caracterizan por su capacidad de generar tejido adiposo y vasos sanguíneos, y por la producción de factores de crecimiento que ayudan en la supervivencia de los adipocitos y la formación de una red vascular. El principal mecanismo de acción de las células madre derivadas de tejido adiposo parece deberse a su acción paracrina y a la sinergia con células endoteliales. En el campo de la medicina regenerativa se han utilizado en el tratamiento de cicatrices patológicas y de fibrosis deformantes con impotencia funcional, en las reconstrucciones de secuelas por cáncer y en el cierre precoz de zonas cruentas. Conclusiones: La lipotransferencia es un procedimiento con un mínimo de complicaciones que constituye una de las opciones terapéuticas más empleadas para corregir defectos en los tejidos, debido a que no solo es un medio de relleno, sino que también permite la regeneración y restauración tisular. La presencia de células madre en el tejido adiposo, unido a su accesibilidad, disponibilidad e histocompatibilidad, ha motivado su aplicación cada vez más expandida en la medicina estética, reconstructiva y regenerativa(AU)

Introduction: Mesenchymal stem cells with self-renewing and multipotential capacity have been identified in adipose tissue. By means of enzymatic digestion and centrifugation of the lipoaspirate a heterogeneous population of cells called vascular stromal fraction is released. It has innumerable therapeutic potentialities in the field of regenerative medicine. Objective: To update the scope of stem cells derived from adipose tissue in regenerative therapy. Method: 38 articles published between 2000 and 2019 in the Scielo, ScienceDirect, Medline and Pubmed databases were reviewed. Development: The cells of the vascular stromal fraction are characterized by generating adipose tissue and blood vessels and by the production of growth factors that help in the survival of adipocytes and the formation of a vascular network. The main mechanism of action of stem cells derived from adipose tissue appears to be due to their paracrine action and synergy with endothelial cells. Stem cells derived from adipose tissue have been used in regenerative medicine for the treatment of pathological scars and deforming fibrosis with functional impotence, in the reconstruction of cancer sequelae and in the early closure of bloody areas. Conclusions: Lipotransfer is a procedure with a minimum of complications that constitutes one of the most widely used therapeutic options to correct tissue defects, since it is not only a filling medium, but also allows tissue regeneration and restoration. The presence of stem cells in adipose tissue, together with their accessibility, availability and histocompatibility, has motivated their increasingly widespread application in aesthetic, reconstructive and regenerative medicine(AU)

Effect of plasma separation techniques and sperm selection on sperm recovery and viability of cooled pony stallion semen for 48h at 5°C / [Efeito das técnicas de separação de plasma e de seleção de esperma na recuperação e viabilidade do sêmen de garanhão pônei resfriado por 48h a 5°C]

Separation techniques of seminal plasma [centrifugation (SC) and Sperm Filter® (SF)] and sperm selection [Androcoll-E (SCA) and filtration glass wool (GW)] were used in 24 ejaculates from 6 stallions. In experiment 1, the ejaculates were allocated into control (no spin), centrifugation at 600 g x 10min, SF and GW. In experiment 2, semen was submitted to SC, SGA and filtered through GW. Following the treatments in both experiments, samples were kept chilled at 5°C to 50 x 106 sperm/ml for 48h. The variables measured on fresh and cooling semen were pH, motility, membrane viability function by 6-carboxyfluorescein diacetate and propidium iodide (CFDA / PI), viability or vitality (eosin / nigrosine) and mitochondrial activity. In experiment 1, centrifugation to remove seminal plasma resulted in greater damage to sperm than separation by sperm filter, and selection by glass wool was more efficient in separating viable cells and maintaining viability during cooling. In experiment 2 Androcoll-E and glass wool treatments resulted in higher (P <0.0001) motility, membrane function, mitochondrial activity, and viability than centrifuged semen. Both selection by Androcoll- E and glass wool improved the quality of semen pony stallions for preservation for up to 48h to 5ºC.(AU)

As técnicas de separação do plasma seminal (centrifugação, SpermFilter) e de seleção espermática (Androcoll-E e filtração por lã de vidro) foram aplicadas em 24 ejaculados de seis garanhões da raça Pônei Brasileiro. Após coleta e separação da fração gel, os ejaculados foram diluídos 1:1 com diluente à base de leite em pó. No experimento 1, os ejaculados foram distribuídos em controle (sem centrifugação), centrifugação a 600g x 10min, SpermFilter e filtração por lã de vidro. No experimento 2, o sêmen foi submetido aos procedimentos: centrifugado (SC), centrifugado com Androcoll-E e filtrado por lã de vidro. Após os procedimentos de ambos os experimentos, as amostras foram mantidas refrigeradas a 5ºC, com 50 x 106 espermatozoides/mL, por 48h. As variáveis mensuradas a fresco, 24h e 48h foram: pH, motilidade, funcionalidade de membrana, viabilidade por diacetato de carboxifluoresceína e iodeto de propídio (CFDA/PI, vitalidade (eosina/nigrosina) e atividade mitocondrial. Já osmolaridade e morfologia espermática foram avaliadas somente imediatamente após a coleta. No experimento 1, a centrifugação para retirada do plasma seminal resultou em maiores danos aos espermatozoides do que a separação por SpermFilter. A filtração por lã de vidro mostrou-se mais eficiente em separar células viáveis e manter a viabilidade durante o resfriamento. No experimento 2, os tratamentos com Androcoll-E e filtrado por lã de vidro foram superiores (P<0,0001) ao sêmen centrifugado quanto à motilidade, à funcionalidade de membrana, à atividade mitocondrial e à viabilidade, tanto nas amostras de sêmen fresco como de sêmen refrigerado. O Androcoll-E e a lã de vidro permitiram manter por 48h, a 5ºC, o sêmen de garanhões pôneis utilizando-se diluente à base de leite.(AU)

Supervivencia del injerto graso. Factores pronósticos / Survival of the fatty graft. Prognostic factors

Utilizar la grasa del propio paciente como material de elección para restaurar volúmenes permite describir distintas formas de obtención de la misma; con o sin infiltración de la zona donante, distintas presiones negativas de succión; diferentes tipos de procesamiento (lavado, filtrado, decantación o centrifugación) y varias formas de implantarla según el plano utilizado, el grosor de los depósitos, la separación de los mismos entre sí, con resultados muy dispares. Para aumentar el volumen de una región, basta con obtener grasa y colocarla en el sitio deseado, siempre sobrevivirá una parte del injerto y siempre existirá una célula madre propia dirigiendo el proceso regenerativo. Pero, ¿cómo lograr que sobreviva todo el tejido trasplantado? La diversidad de criterios aún discutibles, unido a la falta de métodos objetivos no invasivos de evaluación de la sobrevida de los adipocitos, hacen que surja la necesidad de establecer las variables predictoras de supervivencia del lipoinjerto.

Using the patient's own fat as the material of choice to restore volumes has led to the description of different ways of obtaining it; with or without infiltration of the donor area, different negative suction pressures; different types of processing (washing, filtering, decanting or centrifuging) and various ways of implanting it according to the plane used, the thickness of the deposits, their separation from each other, with very different results. To increase the volume of a region, it is enough to obtain fat and place it in the desired site, a part of the graft will always survive and there will always be its own stem cell directing the regenerative process. But how to make all the transplanted tissue survive? The diversity of still debatable criteria, together with the lack of objective non - invasive methods to assess adipocyte survival, lead to the need to establish the predictive variables for lipograft survival.

Antibacterial effects of platelet-rich fibrin produced by horizontal centrifugation / 国际口腔科学杂志·英文版

Platelet-rich fibrin (PRF) has been widely used owing to its ability to stimulate tissue regeneration. To date, few studies have described the antibacterial properties of PRF. Previously, PRF prepared by horizontal centrifugation (H-PRF) was shown to contain more immune cells than leukocyte- and platelet-rich fibrin (L-PRF). This study aimed to compare the antimicrobial effects of PRFs against Staphylococcus aureus and Escherichia coli in vitro and to determine whether the antibacterial effects correlated with the number of immune cells. Blood samples were obtained from eight healthy donors to prepare L-PRF and H-PRF. The sizes and weights of L-PRF and H-PRF were first evaluated, and their antibacterial effects against S. aureus and E. coli were then tested in vitro using the inhibition ring and plate-counting test methods. Flow-cytometric analysis of the cell components of L-PRF and H-PRF was also performed. No significant differences in size or weight were observed between the L-PRF and H-PRF groups. The H-PRF group contained more leukocytes than the L-PRF group. While both PRFs had notable antimicrobial activity against S. aureus and E. coli, H-PRF demonstrated a significantly better antibacterial effect than L-PRF. Furthermore, the antimicrobial ability of the PRF solid was less efficient than that of wet PRF. In conclusion, H-PRF exhibited better antibacterial activity than L-PRF, which might have been attributed to having more immune cells.

L-PRF y Ciclo Celular. Revisión Narrativa / L-PRF and cell cycle. Narrative Review

RESUMEN: Los concentrados plaquetarios han emergido como un potencial material regenerativo, utilizado de forma aislada o como andamiaje para otros materiales de injerto. Son extractos de sangre, obtenidos después de procesar una muestra de sangre completa, mediante centrifugación. El primer reporte data de 1970, con un CP utilizado como pegamento para mejorar cicatrización de heridas de piel. En 1998, se usaron en cirugía oral y maxilofacial. Desde entonces, se han desarrollado diferentes técnicas y una variedad de preparaciones. Entre ellas, cabe destacar el plasma rico en plaquetas, fibrina rica en plaquetas y leucocitos (L-PRF) y plasma rico en factores de crecimiento (PRGF). El desarrollo de estos biomateriales, se debe en parte, a la posibilidad de alterar la concentración de mediadores químicos liberados en una lesión que provoque la formación de un coágulo, que pueda madurar conforme transcurran las fases del proceso inflamatorio y concluya con la regeneración íntegra del tejido dañado. El objetivo de este manuscrito fue describir las principales vías de señalización intracelular que se activan en presencia del L-PRF en cirugía oral, y sus efectos en la regulación del ciclo celular.

ABSTRACT: Platelet concentrates (PC) have emerged as a potential regenerative material, used in isolation or as scaffolding for other graft materials. They are blood extracts, obtained after processing a sample of whole blood, by centrifugation. The first report dates from 1970, with a PC used glue to improve the healing of skin wounds. In 1998, they were used in oral and maxillofacial surgery. Since then, different techniques and a variety of preparations have been developed. These include platelet-rich plasma, fibrin rich in platelets and leukocytes (L-PRF) and plasma rich in growth factors (PRGF). The development of these biomaterials, is due in part to the possibility of altering the concentration of chemical mediators released in a lesion that causes the formation of a clot, which can mature as the phases of the inflammatory process pass and conclude with the complete regeneration of the damaged tissue. The aim of this manuscript was to describe the main intracellular signaling pathways that are activated in the presence of LPRF in oral surgery, and its effects on the regulation of the cell cycle.

Efeito da bactofugação nas contagens e diversidade microbiana de psicrotróficos do leite cru / Effect of bactofugation of raw milk on counts and microbial diversity of psychrotrophs

Esse estudo teve por objetivo verificar o efeito da bactofugação do leite na quantidade e diversidade de psicrotróficos. Como o leite cru era pré-aquecido (≈55ºC) para ser bactofugado, foram coletadas amostras de três lotes de leite cru refrigerado, pré-aquecido e bactofugado a 10.000g. O pré-aquecimento foi suficiente para eliminação de 99,99% dos psicrotróficos do leite cru. A bactofugação reduziu 89,66% das contagens de psicrotróficos do leite pré-aquecido. Lysinibacillus fusiformis predominou (45, 7%) no leite cru, pré -aquecido (37,5%) e bactofugado (60%). Bacillus invictae (20%), Enterococcus faecalis (10%) e Kurthia gibsonii (10%) também foram remanescentes à bactofugação, que aparentemente não tem efeito sobre uma população específica de micro-organismos, mas reduz proporcionalmente toda a microbiota psicrotrófica do leite cru.

Influência do tempo de centrifugação em testes de coagulação: tempo de protrombina e tempo de tromboplastina parcial / Influence of the centrifuge time in coagulation tests: prothrombin time and partial thromboplastin time

Objetivo: Analisar os testes de coagulação: tempo de protrombina (TP) e tempo de tromboplastina parcial (TTP) em diferentes tempos de centrifugação da amostra da biológica, com relação ao protocolo padrão do Clinical Laboratory Standards Institute (CLSI). Métodos: As amostras foram divididas em cinco alíquotas de 1 mL. Foi realizada a centrifugação em 15, 10, 5, 2 e 1 minuto, com velocidade de 1500 g. O TP e TTP foram imediatamente analisados em aparelho automatizado. Os plasmas foram analisados para presença de elementos residuais: eritrócitos, leucócitos e plaquetas. Resultados: Observou-se alteração dos valores do TP nos tempos de centrifugação 10, 5, 2 e 1 minuto e do TTP em 5, 2 e 1 minuto, com relação ao protocolo padrão. Na interpretação de Bland Altman, observou-se um viés significativo do limite clínico aceitável para o TP e para o TTP em todos os tempos de centrifugação, com relação ao protocolo padrão. Apenas no tempo de centrifugação de 15 minutos não foram encontradas células residuais nas amostras analisadas. Conclusão: O tempo de centrifugação de 15 minutos é o ideal para remoção completa das células sanguíneas residuais e para garantia da confiabilidade dos resultados dos testes de coagulação TP e TTP.

Objective: To analyze the coagulation tests: prothrombin test (PT) and partial thromboplastin time (PTT) in different centrifugation times of the sample, in relation to the standard protocol of the Clinical Laboratory Standards Institute (CLSI). Methods: The selected samples were splitted up into five aliquots of 1 mL. Centrifugation of these aliquots was carried out at 15, 10, 5, 2 and 1 minute at 1500 g. The PT and PTT were analyzed in an automated apparatus. The plasmas were analyzed for presence of residual elements: erythrocytes, leukocytes and platelets. Results: The results showed a change in the values of PT at the 10, 5, 2 and 1 minute centrifugation times and the PTT at 5, 2 and 1 minutes, relative to the standard protocol. In the interpretation of Bland Altman, a significant bias of the acceptable clinical limit for TP and TTP at all centrifugation times was observed, relative to the standard protocol. Only in the 15 minute centrifugation time no residual cells were found in the analyzed samples. Conclusion: The present study demonstrated that the 15-minute centrifugation time is ideal for complete removal of residual blood cells and to ensure the reliability of the results of the PT and PTT coagulation

Turn down - turn up: a simple and low-cost protocol for preparing platelet-rich plasma

OBJECTIVE: To describe and analyze a new protocol for the extraction of platelet-rich plasma (PRP) for use in clinical practice and compare this technique with methods that have been previously described in the medical literature. METHODS: We extracted PRP from 20 volunteers using four different protocols (single spin at 1600 ×g, single spin at 600 ×g, double spin at 300 and 700 ×g, and double spin at 600 and 900 ×g). In another group of 12 individuals, we extracted PRP with our new technique (named 'turn down-turn up') consisting of a double spin (200 ×g and 1600 ×g) closed system using standard laboratory equipment (including an ordinary benchtop centrifuge), where the blood remained in the same tube during all processes, reducing the risk of contamination. Platelet counts adjusted to baseline values were compared using analysis of covariance (ANCOVA). RESULTS: Using the four previously described protocols (mentioned above), we obtained concentrations of platelets that were 1.15-, 2.07-, 2.18-, and 3.19-fold greater than the baseline concentration, respectively. With the turn down-turn up technique, we obtained a platelet count that was 4.17-fold (95% confidence interval (CI): 3.09 to 5.25) greater than the baseline platelet count (p=0.063 compared with the double spin at 600 and 900 ×g method). The total cost of the disposable materials used in the extraction process was less than US$10.00 per individual. CONCLUSION: In the present study, we described a simple and safe method for obtaining PRP using low-cost devices.

Comparison of methodologies for detecting Trypanosoma cruzi parasites by microscopic observation of microhematocrit capillary tubes

Abstract INTRODUCTION: The microscopic examination of microhematocrit tubes (mHCT) has been proposed as the gold standard for acute and congenital Chagas disease diagnosis. We compared different mHCT methodologies detecting T. cruzi parasites in the blood. METHODS: The rotating method, water mount, and immersion oil methods were compared for their suitability, sensitivity, and specificity. RESULTS: The rotating method was easier, faster, and more sensitive than the others with 100% specificity. CONCLUSIONS: The rotating method is feasible for laboratory technicians with standard training in microscopic techniques and is recommended for the diagnosis of acute Chagas disease in primary health care facilities.

Evaluation of BacT/Alert FAN Plus Bottles for the Culture of Peritoneal Dialysate / 대한임상미생물학회지

BACKGROUND: A major complication of peritoneal dialysis (PD) is peritonitis, and bacterial culture of PD effluent in a blood culture bottle is the preferred technique for diagnosis of peritonitis. In this study, we compared dialysate inoculation and culture using the BacT/AlerT® Fastidious Antimicrobial Neutralization Plus blood culture bottles (FAN Plus; bioMérieux, France) to the conventional centrifugation culture method.METHODS: A total of 170 PD effluents were simultaneously processed by the conventional centrifugation culture method and by culture using FAN Plus media with two different inoculation procedures: inoculation after centrifugation and direct bedside inoculation.RESULTS: Of the 52 cultures that were positive on at least one of the culture methods, 27 samples were positive on conventional centrifugation. However, 46 samples showed growth following inoculation into the FAN Plus media after centrifugation, and 47 samples were positive on the direct FAN Plus inoculation method. Using the case definition for PD peritonitis to classify samples, sensitivity of the conventional method was 50.0% (95% CI, 33.7–66.3%), whereas the sensitivity of the FAN Plus media was 78.9% (95% CI, 62.2–89.9%) by inoculation after centrifugation and 86.8% (95% CI, 71.1–95.1%) by direct inoculation. Use of both inoculation methods with FAN Plus media resulted in 92.1% sensitivity (95% CI, 89.2–99.9%).CONCLUSION: Culture using FAN Plus media demonstrated a superior bacterial recovery rate to the conventional centrifugation culture method. A combination of the two inoculation methods with FAN Plus media is recommended for the best diagnostic yield, while direct inoculation alone can be useful due to its simplicity and cost-effectiveness.

Effects of Pre-analytical Variables on Cell-free DNA Extraction for Liquid Biopsy

BACKGROUND: Extraction of cell-free DNA (cfDNA) is a key step for determining the quality of cfDNA-related molecular diagnostics. We evaluated the effect of sample containers and sample storage conditions on cfDNA extraction. METHODS: The cfDNA extraction using the MagMAX Cell-Free DNA Isolation Kit from five healthy controls and five lung cancer patients was evaluated according to the type of sample container and storage conditions: K2-EDTA container, <1, 6, 24, and 48 hr storage at 4℃ after immediate plasma separation; and Cell-Free DNA BCT container, <1, 3, 7, and 14 days stored at room temperature. Mutation analysis of EGFR exons 18–21 was performed. To assess the effect of a delay in centrifugation, EDTA whole blood samples from five healthy individuals were stored at 4℃ for 6, 12, and 24 hr before plasma separation. RESULTS: There was no significant difference in the amount and nucleic acid size of cfDNA in both controls and patients with cancer when EDTA plasma was stored at 4℃ up to 48 hr. The amount and size of cfDNA in the BCT container were not different up to 7 days; however, the 14-day sample showed an increase in cfDNA concentration due to genomic DNA contamination. EGFR mutations were detected on EDTA containers up to 48 hr and with BCT containers up to 14 days. When EDTA whole blood was stored at 4℃ and plasma separation was delayed, the cfDNA concentration increased from 24 hr. CONCLUSIONS: The cfDNA extraction was affected by the sample containers and storage conditions.

Reduction in post extraction waiting period for dental implant patients using plasma rich in growth factors: an in vivo study using cone-beam computed tomography

OBJECTIVES: This study examined the effects of plasma-rich growth factors (PRGF) on accelerating bone regeneration/repair in fresh extraction sockets, and determined the quality and quantity of bone by assessing the bone density using cone-beam computed tomography (CBCT). MATERIALS AND METHODS: Twenty patients, who had undergone bilateral extractions, were included in this study. In one extraction socket, PRGF was used and covered with an autologous fibrin plug. Nothing was used in the opposite side extraction socket. Thirteen weeks post extraction, the level of bone regeneration was evaluated on both sides with CBCT. RESULTS: At the end of the study, the mean bone density according to the Hounsfield units (HU) in the control group and PRGF group was 500.05 HU (type III bone type) and 647.95 HU (type II bone type), respectively. CONCLUSION: This study recommends the use of PRGF in post extraction sites to accelerate the rate of bone regeneration and improve the quality of regenerated bone. The technique to process PRGF was simple compared to previously mentioned techniques used for platelet-rich plasma (PRP) preparation. PRP preparation requires a two-cycle centrifugation procedure, leading to a longer processing time.

An experimental investigation of the effects of chronic stress on bone-to-implant contact

OBJECTIVES: This study aimed to investigate the effects of chronic restraint stress on the osseointegration of titanium implants. MATERIALS AND METHODS: Twenty adult male Wistar albino rats were used in the study. After surgical insertion of titanium implants into the metaphyseal part of the tibial bone, rats were randomly divided into two groups: a control group (CNT group) and an experimental restraint stress group (RS group). In the CNT group, titanium implants were inserted surgically, and rats received no further treatment during the 47-day experimental period. In the RS group, restraint stress was applied for 3 hours per day for 45 days, beginning 2 days after implant surgery. Weight of the rats was measured prior to surgery and at the end of the study to analyze the effects of stress. At the end of the experimental period, rats were euthanized, and implants and surrounding bone tissues were used for undecalcified histological analysis. Serum cortisol levels were assessed in cardiac blood samples from the rats following centrifugation. RESULTS: Average weight of rats in the RS group was lower than that of rats in the CNT group after the experimental protocol had been completed (P0.05). CONCLUSION: The data analyzed in this study suggest that chronic restraint stress did not adversely affect rats during a 45-day osseointegration period.

Facial aesthetic fat graft retention rates after filtration, centrifugation, or sedimentation processing techniques measured using three-dimensional surface imaging devices / 中华医学杂志(英文版)

OBJECTIVE@#How to increase the long-term retention rate of autologous fat grafting has been widely discussed. This study aimed to evaluate long-term fat graft retention rates for the most widely used fat processing methods in the area of facial esthetic surgery, including centrifugation, filtration, and sedimentation, using three-dimensional (3D) imaging.@*DATA SOURCES@#PubMed, Embase, Wiley/Cochrane Library, and Web of Science databases were comprehensively searched from inception to July 2018 according to the guidelines of the American Society of Plastic Surgeons Fat Graft Task Force Assessment Methodology.@*STUDY SELECTION@#Articles were screened using predetermined inclusion and exclusion criteria. Data collected included patient characteristics, follow-up devices, fat grafting techniques, and clinical outcomes. Patient cohorts were pooled, and fat graft retention rates were calculated. Complications were summarized according to different clinical characteristics.@*RESULTS@#Of 77 articles, 10 clinical studies met the inclusion criteria and reported quantified measurement outcomes with 3D imaging which provide precise volumetric data with approximately 2% standard deviation compared to real volumes. Data of 515 patients were included. Fat grafting retention varied from 21% to 82%. We found filtration and centrifugation techniques could result in better retention outcomes. However, retention varied within each processing technique, with no significant difference among the 3 techniques. Twenty-two complications were reported among 515 patients, including donor-site hematoma (1 case), mild post-operative erythema (2 cases), mild volumetric asymmetries (2 cases), chronic edema (2 cases), overcorrection (2 cases), skin irregularity (6 cases), and headache or dysesthesia (7 cases).@*CONCLUSIONS@#Filtration and centrifugation techniques may result in better fat grafting retention outcomes than gravity sedimentation; however, more accurate statistical evidence is needed. Controversies continue to exist with respect to the performance of the different fat-processing techniques in fat graft retention.

Bone healing of dental alveoli in smokers with platelet-rich plasma obtained using single or double centrifugation

Introduction: the habit of smoking alters the bone healing process, a problem to consider in oral surgery. objective: to evaluate the bone healing of dental alveoli with PRP obtained using single or double centrifugation in smokers. methodology: extraction of mandibular third molars was performed in a study population divided into smoking group (A), which had PRP applied with the protocol using a single centrifugation step (P1C) in the alveolus of tooth 38 and the protocol of double centrifugation (P2C) in alveolus of tooth 48; a smoking group(B), to whom no PRP was applied; and a non-smokers group (C) to whom PRP was applied obtained using P1C and P2C protocol. radiographic examination was performed at 8, 30 and 60 days post procedure. results: thirty patients met the criteria, 57 percent were female. when evaluating bone healing between the group of smokers and non-smokers, statistically significant differences were observed in the non-smoking group at 30 and 60 days, showing better results with the P2C protocol (p<0.005). statistically significant differences were found at 30 and 60 days (p<0.005), both with the P1C and P2C when comparing bone healing of group A and B. conclusions: bone healing in the alveoli of mandibular third molars that which PRP applied was higher in non-smoking patients, compared with the group of smokers. bone healing was better in patients smokers to whom PRP was applied than those without PRP treatment. regarding the method of obtaining PRP, bone healing was better when a double centrifugation protocol (P2C) was applied.

Non-cultured Epidermal Cell Suspension Transplantation Using Suction Blisters to Treat Refractory Vitiligo: A Retrospective Study of 20 Cases / 대한피부과학회지

BACKGROUND: As nonsurgical interventions for vitiligo are not always successful, various surgical modalities have been used in patients with refractory vitiligo. Of these, non-cultured epidermal suspension transplantation (NCES) was recently introduced to treat large recipient sites using cells from small donor tissue. OBJECTIVE: We assessed the effectiveness and safety of NCES as a surgical treatment for patients with refractory vitiligo. METHODS: We retrospectively reviewed 20 cases in 17 patients (11 females; median age 25 years) who underwent NCES from July 2015 through March 2018. Suction blisters (20 mm in diameter) were collected from the patient's inner thigh at a donor-to-recipient area ratio of 1:5. After the addition of 5 mL recombinant trypsin solution to the suction blisters, followed by incubation at 37℃ for 60 min, epidermal cells were manually scraped off the blister surface, and epidermal cell suspension was obtained by centrifugation at 1,500 RPM for 5 min. The suspension was applied to the vitiligo regions after epidermal ablation of those regions. Phototherapy resumed 1 month later. Treatment success was defined as ≥75% repigmentation of the surgical site, and all adverse events were noted. RESULTS: Overall, 85.0% of cases (17/20) exhibited treatment success. Adverse events included hyperpigmentation (20%) and surgical site infection (5%), but the treatment was tolerable in all cases. CONCLUSION: NCES is a reliable surgical option for patients with vitiligo refractory to nonsurgical treatment. Large areas of vitiligo can be treated by NCES, and use of this technique should be encouraged in Korea.

Removing Lipemia in Serum/Plasma Samples: A Multicenter Study

BACKGROUND: Lipemia, a significant source of analytical errors in clinical laboratory settings, should be removed prior to measuring biochemical parameters. We investigated whether lipemia in serum/plasma samples can be removed using a method that is easier and more practicable than ultracentrifugation, the current reference method. METHODS: Seven hospital laboratories in Spain participated in this study. We first compared the effectiveness of ultracentrifugation (108,200×g) and high-speed centrifugation (10,000×g for 15 minutes) in removing lipemia. Second, we compared high-speed centrifugation with two liquid-liquid extraction methods—LipoClear (StatSpin, Norwood, USA), and 1,1,2-trichlorotrifluoroethane (Merck, Darmstadt, Germany). We assessed 14 biochemical parameters: serum/plasma concentrations of sodium ion, potassium ion, chloride ion, glucose, total protein, albumin, creatinine, urea, alkaline phosphatase, gamma-glutamyl transferase, alanine aminotransferase, aspartate-aminotransferase, calcium, and bilirubin. We analyzed whether the differences between lipemia removal methods exceeded the limit for clinically significant interference (LCSI). RESULTS: When ultracentrifugation and high-speed centrifugation were compared, no parameter had a difference that exceeded the LCSI. When high-speed centrifugation was compared with the two liquid-liquid extraction methods, we found differences exceeding the LCSI in protein, calcium, and aspartate aminotransferase in the comparison with 1,1,2-trichlorotrifluoroethane, and in protein, albumin, and calcium in the comparison with LipoClear. Differences in other parameters did not exceed the LCSI. CONCLUSIONS: High-speed centrifugation (10,000×g for 15 minutes) can be used instead of ultracentrifugation to remove lipemia in serum/plasma samples. LipoClear and 1,1,2-trichlorotrifluoroethane are unsuitable as they interfere with the measurement of certain parameters.

Clostridium botulinum spores in Polish honey samples

The aim of this study was an examination of 240 multifloral honey samples collected from Polish apiaries to determine Clostridium botulinum occurrence. Honey was collected from apiaries directly after the extraction process. Samples were inoculated by using the dilution and centrifugation method. Suspected isolates were examined by using mouse bioassay, polymerase chain reaction (PCR), and real-time PCR methods. C. botulinum type A and B strains were detected in 5 of 240 examined honey samples (2.1%). Bacterial strains were also detected that were phenotypically similar to C. botulinum but that did not exhibit the ability to produce botulinum toxins and did not show the presence of the botulinum cluster (ntnh and bont genes) or expression of the ntnh gene. The methods used in the examination, especially the expression analysis of ntnh gene, enabled specific analysis of suspected strains and could be used routinely in environmental isolate analyses of C. botulinum occurrence.