Patricia Pranke

Electrospinning for regenerative medicine: a review of the main topics

Electrospun fibers are promising tissue engineering scaffolds that offer the cells an environment that mimics the native extracellular matrix. Fibers with different characteristics can be produced by the electrospinning technique according to the needs of the tissue to be repaired. In this review, the process of electrospinning was examined, providing a description of the common techniques used for the physicochemical and biological characterization of electrospun fibers. The review also discusses the potential applications of electrospun scaffolds for tissue engineering, based on scientific literature.

Hematologic and immunophenotypic characterization of human umbilical cord blood

In this work, cord blood cells from 30 healthy term newborns were analyzed for complete blood counts with an automated cytometer and, in part of the sample, for surface molecules in cord blood monocytes, lymphocytes and CD34+ cells by two-color flow cytometry. Hematological parameters were as follows: WBC = 12.85 (5.24–15.10) ×109/l; platelets = 304.33 (156.00–469.00) × 109/l; Hb = 14.45 (11.90–17.82) g/dl; RBC = 3.99 (3.14–5.12) × 1012/l; MCV 107.25 (99.60–115.00) fl; reticulocytes = 157.80 (101.00–124.00) × 109/l or 3.99 (2.45–6.01)%; erythroblasts = 0.88 (0.15–2.58) per 100 WBC or 6.63 (2.86–16.80) × 109/l. The mononuclear population, as evaluated by flow cytometry, was composed of 22.9 ± 7.2% monocytes and 77.05 ± 7.24% lymphocytes, among which 46.59 ± 15.62% were T lymphocytes (43.94 ± 16.94% CD3+/CD4+ and 13.45 ± 7.46% CD3+/CD8+). CD34+ cells were on average 0.54 ± 0.24% of the mononuclear fraction. CD11c, CD49e and HLA-DR were found mainly on monocytes, and CD31 and CD62L occurred in similar levels on monocytes and lymphocytes. CD117+ cells were less than 5% of these populations. Among CD34+ cells, CD31 and HLA-DR were the molecules with higher frequencies (79.7 ± 19.9 and 65.7 ± 23.0%, respectively), followed by CD62L (41.8 ± 31.9%) and CD117 (20.1 ± 15.8%). The presence of CD11c and CD49e on CD34+ cells was low (below 10%). The results stress the phenotypic heterogeneity of cord blood CD34+ cells, and the different behavior of the cells when manipulated in vitro in different degrees of isolation.

Points to consider in the development of seed stocks of pluripotent stem cells for clinical applications: International Stem Cell Banking Initiative (ISCBI)

In 2009 the International Stem Cell Banking Initiative (ISCBI) contributors and the Ethics Working Party of the International Stem Cell Forum published a consensus on principles of best practice for the procurement, cell banking, testing and distribution of human embryonic stem cell (hESC) lines for research purposes [1], which was broadly also applicable to human induced pluripotent stem cell (hiPSC) lines. Here, we revisit this guidance to consider what the requirements would be for delivery of the early seed stocks of stem cell lines intended for clinical applications. The term ‘seed stock’ is used here to describe those cryopreserved stocks of cells established early in the passage history of a pluripotent stem cell line in the lab that derived the line or a stem cell bank, hereafter called the ‘repository’.

Preparation of nanofibers containing the microalga Spirulina (Arthrospira)

Spirulina is a microalga which offers biological functions highly favorable for tissue engineering. Highly porous scaffolds can be produced by electrospinning containing biomass of Spirulina. The goal of this contribution was therefore to establish spinning conditions allowing to produce well defined nanofibers with diameters down to about 100 nm and to produce nanofibers with various concentration of the biomass for subsequent studies in tissue engineering applications. The experimental results reveal that the blend system PEO/biomass is behaved surprisingly well in electrospinning. Very thin bead-free nanofibers with diameters of about 110 nm can be produced for different biomass contents of up to 67 wt.% of the nanofibers and for PEO concentrations in the spinning solution well below 4 wt.%. These results suggest to us the use of the biomass containing nanofibers as extracellular matrices for stem cell culture and future treatment of spinal chord injury.

Effects of cryopreservation on the characteristics of dental pulp stem cells of intact deciduous teeth

OBJECTIVES The aim of this study was to isolate and cultivate cells from the pulp of 7-day-cryopreserved intact deciduous human teeth and evaluate the effect of cryopreservation on dental pulp stem cell (DPSC) characteristics. DESIGN Twenty-six deciduous teeth were collected and allocated in two groups: immediate cell isolation (non-cryopreserved group) and intact cryopreserved (cryopreserved group). The teeth were cryopreserved in dimethylsulfoxide solution and recovered after 7 days. The success rate of isolation, proliferation, surface markers (CD14, CD29, CD34, CD45, CD73, CD90, and HLA-DR), differentiation capacity, and morphology were evaluated. RESULTS Isolation success rate was 61% and 30% for the non-cryopreserved and cryopreserved groups, respectively. There were no statistical differences between the groups for the tested surface markers. The cells in both groups were capable of differentiating into three mesenchymal lineages. No statistical differences between the groups were observed through the time course proliferation assay (0, 1, 3, 5, and 7 days); however, the mean time between isolation and the fifth passage was shorter for the non-cryopreserved group (p=0.035). The morphology of the cells was considered altered in the cryopreserved group. CONCLUSION DPSCs were obtained from cryopreserved intact deciduous teeth without changes in the immunophenotypical characteristics and differentiation ability; however, lower culture rates, proliferation potential, and morphological alterations were observed in relation to the control group.

Advantages and challenges offered by biofunctional core–shell fiber systems for tissue engineering and drug delivery

Whereas highly porous scaffolds composed of electrospun nanofibers can mimick major features of the extracellular matrix in tissue engineering, they lack the ability to incorporate and release biocompounds (drugs, growth factors) safely in a controlled way. Here, electrospun core-shell fibers (core made from water and aqueous solutions of hydrophilic polymers and the shell from materials with well-defined release mechanisms) offer unique advantages in comparison with those that have helped make porous nanofibrillar scaffolds highly successful in tissue engineering. This review considers the preparation and biofunctionalization of such core-shell fibers as well as applications in various areas, including neural, vascular, cardiac, cartilage and bone tissue engineering, and touches on the topic of clinical trials.

The use of stem cells for the treatment of autoimmune diseases

Autoimmune diseases constitute a heterogeneous group of conditions commonly treated with anti-inflammatory, immunosuppressant and immunomodulating drugs, with satisfactory results in most cases. Nevertheless, some patients become resistant to conventional therapy. The use of high doses of drugs in such cases results in the need for bone marrow reconstitution, a situation which has stimulated research into the use of hematopoietic stem cells in autoimmune disease therapy. Stem cell transplantation in such diseases aims to destroy the self-reacting immune cells and produce a new functional immune system, as well as substitute cells for tissue damaged in the course of the disease. Significant results, such as the reestablishment of tolerance and a decrease in the recurrence of autoimmune disease, have been reported following stem cell transplantation in patients with autoimmune disease in Brazil and throughout the world. These results suggest that stem cell transplantation has the potential to become an important therapeutic approach to the treatment of various autoimmune diseases including rheumatoid arthritis, juvenile idiopathic arthritis, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, Crohns disease, autoimmune blood cytopenias, and type I diabetes mellitus.

Development of a new nanofiber scaffold for use with stem cells in a third degree burn animal model.

The combination of mesenchymal stem cells (MSCs) and nanotechnology to promote tissue engineering presents a strategy for the creation of new substitutes for tissues. Aiming at the utilization of the scaffolds of poly-d,l-lactic acid (PDLLA) associated or not with Spirulina biomass (PDLLA/Sp) in skin wounds, MSCs were seeded onto nanofibers produced by electrospinning. These matrices were evaluated for morphology and fiber diameter by scanning electron microscopy and their interaction with the MSCs by confocal microscopy analysis. The biomaterials were implanted in mice with burn imitating skin defects for up to 7 days and five groups were studied for healing characteristics. The scaffolds demonstrated fibrous and porous structures and, when implanted in the animals, they tolerated mechanical stress for up to two weeks. Seven days after the induction of lesions, a similar presence of ulceration, inflammation and fibrosis among all the treatments was observed. No group showed signs of re-epithelization, keratinization or presence of hair follicles on the lesion site. In conclusion, although there was no microscopical difference among all the groups, it is possible that more prolonged analysis would show different results. Moreover, the macroscopic analysis of the groups with the scaffolds showed better cicatrization in comparison with the control group.

The effect of sterilization methods on electronspun poly(lactide‐co‐glycolide) and subsequent adhesion efficiency of mesenchymal stem cells

The sterilization of scaffolds is an essential step for tissue engineering in vitro and, mainly, clinical biomaterial use. However, this process can cause changes in the structure and surface of the scaffolds. Therefore, the objective of this study was to investigate the effect of sterilization by ethanol, ultraviolet radiation (UVR) or antimicrobial solution (AMS) on poly(lactide-co-glycolide) (PLGA) scaffolds produced by the electrospinning technique. The properties of nanofibers and the cellular adhesion of mesenchymal stem cells to the scaffolds were analyzed after the treatments. All methods generated sterile scaffolds but showed some kind of damage to the scaffolds. Ethanol and AMS caused changes in the morphology and scaffold dimensions, which were not observed when using the UVR method. However, UVR caused a greater reduction in polymeric molecular weight, which increased proportionally with exposure time of treatment. Nanofibers sterilized with AMS for 1 h and 2 h showed greater cellular adhesion than the other methods, demonstrating their potential as a method for sterilizing PLGA nanofibers.

Viability of mesenchymal stem cells during electrospinning

Tissue engineering is a technique by which a live tissue can be re-constructed and one of its main goals is to associate cells with biomaterials. Electrospinning is a technique that facilitates the production of nanofibers and is commonly used to develop fibrous scaffolds to be used in tissue engineering. In the present study, a different approach for cell incorporation into fibrous scaffolds was tested. Mesenchymal stem cells were extracted from the wall of the umbilical cord and mononuclear cells from umbilical cord blood. Cells were re-suspended in a 10% polyvinyl alcohol solution and subjected to electrospinning for 30 min under a voltage of 21 kV. Cell viability was assessed before and after the procedure by exclusion of dead cells using trypan blue staining. Fiber diameter was observed by scanning electron microscopy and the presence of cells within the scaffolds was analyzed by confocal laser scanning microscopy. After electrospinning, the viability of mesenchymal stem cells was reduced from 88 to 19.6% and the viability of mononuclear cells from 99 to 8.38%. The loss of viability was possibly due to the high viscosity of the polymer solution, which reduced the access to nutrients associated with electric and mechanical stress during electrospinning. These results suggest that the incorporation of cells during fiber formation by electrospinning is a viable process that needs more investigation in order to find ways to protect cells from damage.