Ângela Cristina Malheiros Luzo

Prediction and Modulation of Platelet Recovery by Discontinuous Centrifugation of Whole Blood for the Preparation of Pure Platelet-Rich Plasma

Abstract The aim of this study was to describe the behavior of the separation of red blood cells (RBCs) by discontinuous centrifugation (DC) of whole blood to modulate and control the platelet recovery in the preparation of pure platelet-rich plasma (P-PRP). P-PRP is a platelet-rich plasma (PRP) in which the white blood cell layer is not included. To achieve this goal, an analytical model was derived that takes into account the packing of RBCs and predicts the behavior of platelet and plasma recovery efficiencies (PtPlRE) based on the volume of whole blood, the hematocrit, and the volume of supernatant, as a function of the operating variables, centrifugal acceleration, and time. The model was derived from the basic equation of DC, which originates from the equilibrium balance of forces on a particle, and included the addition of one factor that corrected the terminal velocity of RBCs and was also correlated to the PtPlRE in the supernatant. This factor was the ratio between the fractional volume concentrations of plasma and RBCs in the centrifugation pellet after centrifugation. The model was validated and the variability of the data was determined using experimental data from 10 healthy donors in the age range of 25–35 years. The predicted behavior for the packing of RBCs and the PtPlRE was consistent with the behavior seen in the experimental data. Thus, the PtPlRE could be modulated and controlled through centrifugal acceleration, time, and hematocrit. Use of this model based on a physical description of events is the first step of a reliable standardization of PRP preparations.

In vitro performance of injectable chitosan-tripolyphosphate scaffolds combined with platelet-rich plasma

This study aimed to evaluate the in vitro biological effectiveness of chitosan microparticles crosslinked with sodium tripolyphosphate (TPP) in combination with activated pure platelet-rich plasma (aP-PRP) as an injectable composite scaffold for growth factors release, cell proliferation and osteogenic differentiation. Two main novelties were addressed in the field of scaffolds in regenerative medicine: the first is the approach including simultaneously the three vertices of the proliferation triangle formed by the capabilities genic progenitor cells, conductive scaffolds and inductive growth factors, which are provided by platelet rich plasma (PRP); secondly, the approach of an injectable composite scaffolds formed by the fibrin network from aP-PRP and the chitosan microparticles crosslinked with TPP. The microparticles were prepared by vortexing the chitosan and TPP solutions. The ionic crosslinking of chitosan with TPP was made at mass ratios of 2:1, 5:1, and 10:1 at pH 4.0. P-PRP was obtained via the controlled centrifugation of whole blood. The composite scaffolds were prepared by adding the microparticles to immediately activated P-PRP. The results showed that the microparticles had adequate physicochemical and mechanical properties for injection. Furthermore, the microparticles controlled the release of growth factors from P-PRP. The proliferation of human adipose-derived mesenchymal stem cells was lower than in aP-PRP alone but significant at a 2:1 chitosan-TPP mass ratio. Osteogenic differentiation was stimulated at all studied mass ratios, as indicated by the alkaline phosphatase activity. These results offer perspectives for optimizing the composite scaffold, and to prove its potential as an injectable scaffold in regenerative medicine.

Useful properties of undifferentiated mesenchymal stromal cells and adipose tissue as the source in liver-regenerative therapy studied in an animal model of severe acute fulminant hepatitis

BACKGROUND AIMS End-stage liver diseases frequently require liver transplantation. Cell therapy could be an alternative. This study aimed to analyze whether undifferentiated mesenchymal stromal cells (U-MSCs) or MSC-derived hepatocyte-like cells (DHLCs) from adipose tissue (AT), umbilical cord blood (UCB) and bone marrow (BM) would better restore damaged liver. METHODS AT was obtained from lipo-aspiration, UCB from an Umbilical Cord Blood Bank and BM from a BM Transplantation Unit. AT (collagenase digestion), UCB and BM (Ficoll gradient) were cultured (Dulbeccos modified Eagles medium, low glucose, FBS) for 3 days. Detached adherent cells, at passage 4, were characterized as MSCs. Genetic stability was investigated by means of telomerase enzyme activity and karyotype. Hepatocyte differentiation protocol was performed with the use of Dulbeccos modified Eagles medium, hepatocyte growth factor, basic fibroblast growth factor and nicotinamide (7 days); maturation medium (oncostatin, dexamethasone, insulin, transferrin and selenium) was added at 36 days. Hepatogenesis analyses were performed by use of morphology and albumin, AF, tyrosine-aminotransferase and glutamine synthetase gene expression and quantitative reverse transcription-polymerase chain reaction on days 9, 18, 25 and 36. Functionality was assessed through glycogen storage detection, indocyanine green absorption and transplantation procedure. U-MSCs and DHLCs were injected 48 h after induced fulminant hepatitis (intraperitoneal injection of carbon tetrachloride) in SCID/BALB-c mice. Histopathologic analyses were performed on days 7 and 15. Human origin included albumin and CK19 human markers. RESULTS All MSCs differentiated into functional hepatocyte-like cells, stored glycogen and absorbed indocyanine green. AT-MSC DHLC gene expression was more consistent with a normal hepatogenic-differentiation profile. UCB-MSCs expanded weakly, impairing their use for the transplantation procedure. AT and BM U-MSCs and DHLCs regenerated liver injury equally. Regenerated hepatocytes exhibited human origin. CONCLUSIONS AT might be the source and U-MSCS the stem cells useful for liver-regenerative therapy.

Platelet Rich Plasma and Its Growth Factors: The State of the Art

This study aims to offer a general idea of the current progress and discussions about the aspects of technical preparation and biological foundation of PRP for clinical application. We seek to gather the best therapeutic indications that have a scientific foundation on the use of this new tool of Regenerative Medicine. The articles of this study were acquired from the leading data bases of medical literature.

Mesenchymal stromal cells from adipose tissue attached to suture material enhance the closure of enterocutaneous fistulas in a rat model

BACKGROUND AIMS Surgical treatment for enterocutaneous fistulas (EF) frequently fails. Cell therapy may represent a new approach to treatment. Mesenchymal stromal cells (MSCs) have high proliferative and differentiation capacity. This study aimed to investigate whether MSCs could adhere to suture filament (SF), promoting better EF healing. METHODS MSCs, 1 × 10(6), from adipose tissue (ATMSCs) were adhered to a Polyvicryl SF by adding a specific fibrin glue formulation. Adhesion was confirmed by confocal and scanning electron microscopy (SEM). A cecal fistula was created in 22 Wistar rats by incising the cecum and suturing the opening to the surgical wound subcutaneously with four separate stitches. The animals were randomly allocated to three groups: control (CG)-five animals, EF performed; injection (IG)-eight animals 1 × 10(6) ATMSCs injected around EF borders; and suture filament (SG): nine animals, sutured with 1 × 10(6) ATMSCs attached to the filaments with fibrin glue. Fistulas were photographed on the operation day and every 3 days until the 21st day and analyzed by two observers using ImageJ Software. RESULTS Confocal and SEM results demonstrated ATMSCs adhered to SF (ATMSCs-SF). The average reduction size of the fistula area at 21st day was greater for the SG group (90.34%, P < 0.05) than the IG (71.80%) and CG (46.54%) groups. CONCLUSIONS ATMSCs adhered to SF maintain viability and proliferative capacity. EF submitted to ATMSCs-SF procedure showed greater recovery and healing. This approach might be a new and effective tool for EF treatment.

Red Blood Cell Antigen Alloimmunization in Liver Transplant Recipients

Orthotopic liver transplantation (OLT) is a life-saving procedure for patients with end-stage liver disease. Transfusion support is an important part of OLT. Intraoperative transfusion of large volumes of blood products is recognized to be a poor prognostic factor, probably due to the negative effects of blood transfusions, such as transfusion reactions, infectious contamination of blood products, or immune modulation of the transfused patient. The aim of this study was to evaluate the frequency of alloimmunization and its specificity to red blood cell (RBC) antigens among patients undergoing OLT. We identified 74 RBC alloantibodies in 70 (23%) patients when the indirect antiglobulin test (IAT) was performed. The most common RBC alloantibodies were against Rh system antigens. The majority (41.9%) were directed against the E antigen. Despite the ethnic heterogeneity of our population there were no cases of intravascular hemolysis. The incidence of alloimmunization (23%) was slightly higher among patients than in the literature, most probably as a consequence of our ethnic heterogeneity.

Contributions for classification of platelet rich plasma – proposal of a new classification: MARSPILL

Platelet-rich plasma (PRP) has emerged as a significant therapy used in medical conditions with heterogeneous results. There are some important classifications to try to standardize the PRP procedure. The aim of this report is to describe PRP contents studying celular and molecular components, and also propose a new classification for PRP. The main focus is on mononuclear cells, which comprise progenitor cells and monocytes. In addition, there are important variables related to PRP application incorporated in this study, which are the harvest method, activation, red blood cells, number of spins, image guidance, leukocytes number and light activation. The other focus is the discussion about progenitor cells presence on peripherial blood which are interesting due to neovasculogenesis and proliferation. The function of monocytes (in tissue-macrophages) are discussed here and also its plasticity, a potential property for regenerative medicine treatments.

Natural Type II Collagen Hydrogel, Fibrin Sealant, and Adipose-Derived Stem Cells as a Promising Combination for Articular Cartilage Repair

Objective Articular cartilage is an avascular tissue with limited ability of self-regeneration and the current clinical treatments have restricted capacity to restore damages induced by trauma or diseases. Therefore, new techniques are being tested for cartilage repair, using scaffolds and/or stem cells. Although type II collagen hydrogel, fibrin sealant, and adipose-derived stem cells (ASCs) represent suitable alternatives for cartilage formation, their combination has not yet been investigated in vivo for focal articular cartilage defects. We performed a simple experimental procedure using the combination of these 3 compounds on cartilage lesions of rabbit knees. Design The hydrogel was developed in house and was first tested in vitro for chondrogenic differentiation. Next, implants were performed in chondral defects with or without ASCs and the degree of regeneration was macroscopically and microscopically evaluated. Results Production of proteoglycans and the increased expression of collagen type II (COL2α1), aggrecan (ACAN), and sex-determining region Y-box 9 (SOX9) confirmed the chondrogenic character of ASCs in the hydrogel in vitro. Importantly, the addition of ASC induced a higher overall repair of the chondral lesions and a better cellular organization and collagen fiber alignment compared with the same treatment without ASCs. This regenerating tissue also presented the expression of cartilage glycosaminoglycan and type II collagen. Conclusions Our results indicate that the combination of the 3 compounds is effective for articular cartilage repair and may be of future clinical interest.

Umbilical cord blood CD34(+) stem cells and other mononuclear cell subtypes processed up to 96 h from collection and stored at room temperature maintain a satisfactory functionality for cell therapy.

Umbilical cord blood (UCB) is a good stem cell source for cell therapy. We recently demonstrated that cord blood mononuclear cell (MNCs) subtypes were viable and functional until 96 h after collection, even stored at room temperature. Now, we analyzed the viability and functionality of the cells before and after cryopreservation.

Use of Platelet-Rich Plasma (PRP) in Treating Chronic Wounds

The healing process is dynamic and involves complex events that include hemostasis, inflammation, granulation tissue formation, epithelialization, neovascularization, collagen synthesis, and wound contraction. Several experimental clinical studies have demonstrated the reduction of growth factors of chronic wounds. Platelet aggregation has the leading role in the process of skin healing since it is responsible for releasing growth factors, adhesion molecules and lipids, which regulate migration, proliferation and function of keratinocytes, fibroblasts and endothelial cells. The platelet-leukocyte gel (L-PRP), besides releasing the growth factors that start tissue regeneration, can also strengthen the antimicrobial activity, which shows its potential as an infection prevention and treatment agent. PRP is a powerful weapon for treating chronic ulcers, providing healing, reducing infection rates, besides its preventive action, which reduces amputation rates.