Angela Cristina Malheiros Luzo

Relevant Aspects of Centrifugation Step in the Preparation of Platelet-Rich Plasma

Introduction. Platelet-Rich Plasma (PRP) is rich in growth factors, playing important role in tissue healing. The wide variation of reported protocols for preparation of PRP leads to variable compositions, which induce different biological responses and prevent results comparison. This study aims to highlight relevant aspects of the centrifugation step to obtain reproducible results and overall quality. Material and Methods. Samples of blood were collected from 20 healthy donors that have signed free informed consent. Two centrifugation steps (spins) were analyzed for the influence of centrifugal acceleration, time, processed volume, and platelet gradient. The Pure Platelet-Rich Plasma (P-PRP) was characterized as platelet concentration, integrity, and viability (sP-selectin measurement). Results. Lower centrifugal accelerations favour platelet separation. The processing of 3.5 mL of blood at 100 ×g for 10 min (1st spin), 400 ×g for 10 min (2nd spin), withdrawing 2/3 of remnant plasma, promoted high platelet recovery (70–80%) and concentration (5x) maintaining platelet integrity and viability. The recovery of platelets was reduced for a larger WB volume (8.5 mL) processed. Conclusion. Centrifugal acceleration, time, WB processed volume, and minimization of the platelet gradient before sampling are relevant aspects to ensure reproducible compositions within the autologous nature of PRP.

Both Interleukin-3 and Interleukin-6 Are Necessary for Better Ex Vivo Expansion of CD133+ Cells From Umbilical Cord Blood

Umbilical cord blood (UCB), an ideal source for transplantable hematopoietic stem cells (HSC), is readily available and is rich in progenitor cells. Identification of conditions favoring UCB-HSC ex vivo expansion and of repopulating potential remains a major challenge in hematology. CD133+ cells constitute an earlier, less-differentiated HSC group with a potentially higher engraftment capacity. The presence of SCF, Flt3-L, and TPO are essential for CD133+ and/or CD34+ cells ex vivo expansion; however, IL-3 and IL-6 influence has not yet been clearly established. We investigated this influence on CD133+ cells from UCB ex vivo expansion and the effect of these cytokines upon cell phenotype. Immediately after isolation an 85% of CD133+ cell purity was obtained, diminishing after 4 and 8 days of ex vivo expansion. CD133+ fold-increase was higher using IMDM with SCF, Flt3-L, and TPO (BM)+IL-3 or BM+IL-3+IL-6 on day 8 (13.83- and 17.47-fold increase, respectively). BM+IL-6 presented no significant difference from BM alone. We demonstrated that 5.1% of the CD133+ cells expressed IL-6 receptor (IL-6R) after isolation. After 4 and 8 days in culture, the percentage of CD133+ cells that expressed IL-6R was as follows: BM alone (9.8% and 22.02%, respectively); BM+IL-3 (8.33% and 16.74%); BM+IL-6 (9.2% and 17.67%); and BM+IL-3+IL-6 (12.5% and 61.20%). Cell cycle analysis revealed quiescent cells after isolation, 95.5% CD133+ cells in the G0/G1 phase. Regardless of culture period or cytokine incubation, CD133+ cell cycle altered to 70% of CD133+ in the G0/G1 phase. Colony-forming unit (CFU) doubled in BM+IL-3+IL-6 after 8 days of incubation compared with BM group. SOX-2 and NANOG-relative gene expression was detected on day 0 after isolation. BM+IL-6 prevented the decrease in NANOG and SOX-2 gene expression level compared to BM+IL-3 or BM+IL-3+IL-6 incubated cells. Our results indicated that UCB-isolated CD133+ cells were better ex vivo expanded in the presence of SCF, Flt3-L, TPO, IL-3+IL-6. IL-3 probably promotes higher CD133+ cell expansion and IL-6 maintains immature phenotype.

Regulation of Chondrogenesis by Transforming Growth Factor-ss3 and Insulin-like Growth Factor-1 from Human Mesenchymal Umbilical Cord Blood Cells

Objective. Mature articular cartilage is vulnerable to injuries and disease processes that cause irreversible tissue damage because of its limited capacity for self-repair. Umbilical cord blood is a source of mesenchymal stem cells, which can give rise to cells of different lineages, including cartilage, bone, and fat. Cellular condensation is a required step in the initiation of mesenchymal chondrogenesis. We attempted to differentiate cells from umbilical cord blood into chondrocytes with insulin-like growth factor 1 (IGF-1) and transforming growth factor-ß3 (TGF-ß3). Methods. Cells were grown in high density micromass and monolayer culture systems and then evaluated for expression of type II collagen, aggrecan, and Sox9. Umbilical cord blood from 130 patients was harvested. Results. Expression of type II collagen, aggrecan, and Sox9 was detected after 14 days in TGF-ß3- and IGF-1-stimulated cells in both types of culture (monolayer and micromass). On Day 21 in the micromass culture, expression levels were greater than they were at 14 days for all genes. TGF-ß3 was found to be more efficient at promoting chondrogenesis than IGF-1. By western blot, we also found that after 3 weeks, the expression of type II collagen was greater in micromass culture with TGF-ß3. Conclusion. TGF-ß3 used in micromass culture is the best growth factor for promoting the proliferation and differentiation of mesenchymal cells from umbilical cord blood during chondrogenesis. This approach may provide an alternative to autologous grafting.

Neuroprotection and immunomodulation by xenografted human mesenchymal stem cells following spinal cord ventral root avulsion

The present study investigates the effects of xenotransplantation of Adipose Tissue Mesenchymal Stem Cells (AT-MSCs) in animals after ventral root avulsion. AT-MSC has similar characteristics to bone marrow mesenchymal stem cells (BM-MSCs), such as immunomodulatory properties and expression of neurotrophic factors. In this study, Lewis rats were submitted to surgery for unilateral avulsion of the lumbar ventral roots and received 5 × 105 AT-MSCs via the lateral funiculus. Two weeks after cell administration, the animals were sacrificed and the moto neurons, T lymphocytes and cell defense nervous system were analyzed. An increased neuronal survival and partial preservation of synaptophysin-positive nerve terminals, related to GDNF and BDNF expression of AT-MSCs, and reduction of pro-inflammatory reaction were observed. In conclusion, AT-MSCs prevent second phase neuronal injury, since they suppressed lymphocyte, astroglia and microglia effects, which finally contributed to rat motor-neuron survival and synaptic stability of the lesioned motor-neuron. Moreover, the survival of the injected AT- MSCs lasted for at least 14 days. These results indicate that neuronal survival after lesion, followed by mesenchymal stem cell (MSC) administration, might occur through cytokine release and immunomodulation, thus suggesting that AT-MSCs are promising cells for the therapy of neuronal lesions.

Viability of umbilical cord blood mononuclear cell subsets until 96 hours after collection

Umbilical cord blood (UCB) is a good source of hematopoietic stem cells for transplantation and cell therapy. In 2006, the Brazilian Public Network of Cord Blood Banks was founded; however, because our country is large, logistic problems could hamper the collection of numerous samples. Our aim was to evaluate the viability of several UCB cell subsets until 96 hours after collection, to examine whether this delay would be acceptable for processing and freezing the samples.

Fibrin network architectures in pure platelet-rich plasma as characterized by fiber radius and correlated with clotting time.

Abstract Fibrin networks are obtained through activation of platelet-rich plasma (PRP) for use in tissue regeneration. The importance of fibrin networks relies on mediation of release of growth factors, proliferation of tissue cells and rheological properties of the fibrin gels. Activation of PRP usually involves the decomposition of fibrinogen by agonists, in a wide range of concentrations. Therefore fibrin networks with a large structural diversity are formed, making comparative evaluations difficult. In order to standardize the fibrin networks, we used the statistical techniques central composite rotatable design and response-surface analysis, to correlate the radius of the fibers with the ratios between the agonists (autologous serum/calcium chloride) and agonist/PRP. From an individual and interactive analysis of the variables, architectures characterized by thick, medium and thin fibers were delineated on the response-surface. Furthermore, the architectures were correlated with coagulation time. This approach is valuable for standardizing the PRP preparation for clinical applications.

Effect of Human Adipose Tissue Mesenchymal Stem Cells on the Regeneration of Ovine Articular Cartilage

Cell therapy is a promising approach to improve cartilage healing. Adipose tissue is an abundant and readily accessible cell source. Previous studies have demonstrated good cartilage repair results with adipose tissue mesenchymal stem cells in small animal experiments. This study aimed to examine these cells in a large animal model. Thirty knees of adult sheep were randomly allocated to three treatment groups: CELLS (scaffold seeded with human adipose tissue mesenchymal stem cells), SCAFFOLD (scaffold without cells), or EMPTY (untreated lesions). A partial thickness defect was created in the medial femoral condyle. After six months, the knees were examined according to an adaptation of the International Cartilage Repair Society (ICRS 1) score, in addition to a new Partial Thickness Model scale and the ICRS macroscopic score. All of the animals completed the follow-up period. The CELLS group presented with the highest ICRS 1 score (8.3 ± 3.1), followed by the SCAFFOLD group (5.6 ± 2.2) and the EMPTY group (5.2 ± 2.4) (p = 0.033). Other scores were not significantly different. These results suggest that human adipose tissue mesenchymal stem cells promoted satisfactory cartilage repair in the ovine model.

Early proliferation of umbilical cord blood cells from premature neonates.

Background and Objective  Human umbilical cord blood (UCB) is an important source of haematopoietic stem cells; however, the behaviour of progenitor cells obtained from premature and full‐term neonates is still a controversy subject. Thus, the aim of this study was to evaluate cell cycle parameters and the proliferative capacity of UCB progenitor cells from premature and full‐term neonates.

Platelet-Rich Plasma

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.

Use of the second harmonic generation microscopy to evaluate chondrogenic differentiation of mesenchymal stem cells for cartilage repair

Articular cartilage injury remains one of the major concerns in orthopedic surgery. Mesenchymal stem cell (MSC) transplantation has been introduced to avoid some of the side effects and complications of current techniques.. With the aim to evaluate chondrogenic differentiation of mesenchymal stem cells, we used Second Harmonic Generation (SHG) microscopy to analyze the aggregation and orientation of collagen fibrils in the hyaline cartilage of rabbit knees. The experiment was performed using implants with type II collagen hydrogel (a biomaterial that mimics the microenvironment of the cartilage), one implant containing MSC and one other without MSC (control). After 10 weeks, the rabbit knees were dissected and fibril collagen distribution and spatial organization in the extracellular matrix of the lesions were verified by SHG. The result showed significant differences, whereas in histological sections of the cartilaginous lesions with MSC the collagen fibers are organized and regular; in the control sections the collagen fibers are more irregular, with absence of cells. A macroscopic analysis of the lesions confirmed this difference, showing a greater percentage of lesions filling in knees treated with MSC than in the knees used as controls. This study demonstrates that SHG microscopy will be an excellent tool to help in the evaluation of the effectiveness of MSC-based cell therapy for cartilage repair.