Daniela Steffens

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.

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.

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.

Transplantation of mononuclear cells from human umbilical cord blood promotes functional recovery after traumatic spinal cord injury in Wistar rats

Cell transplantation is a promising experimental treatment for spinal cord injury. The aim of the present study was to evaluate the efficacy of mononuclear cells from human umbilical cord blood in promoting functional recovery when transplanted after a contusion spinal cord injury. Female Wistar rats (12 weeks old) were submitted to spinal injury with a MASCIS impactor and divided into 4 groups: control, surgical control, spinal cord injury, and one cell-treated lesion group. Mononuclear cells from umbilical cord blood of human male neonates were transplanted in two experiments: a) 1 h after surgery, into the injury site at a concentration of 5 x 106 cells diluted in 10 µL 0.9% NaCl (N = 8-10 per group); b) into the cisterna magna, 9 days after lesion at a concentration of 5 x 106 cells diluted in 150 µL 0.9% NaCl (N = 12-14 per group). The transplanted animals were immunosuppressed with cyclosporin-A (10 mg/kg per day). The BBB scale was used to evaluate motor behavior and the injury site was analyzed with immunofluorescent markers to label human transplanted cells, oligodendrocytes, neurons, and astrocytes. Spinal cord injury rats had 25% loss of cord tissue and cell treatment did not affect lesion extension. Transplanted cells survived in the injured area for 6 weeks after the procedure and both transplanted groups showed better motor recovery than the untreated ones (P < 0.05). The transplantation of mononuclear cells from human umbilical cord blood promoted functional recovery with no evidence of cell differentiation.

Development of a biomaterial associated with mesenchymal stem cells and keratinocytes for use as a skin substitute

AIM The present study has aimed to produce a cutaneous substitute, bringing together stem cells (mesenchymal stem cells) and keratinocytes, and an electrospun biomaterial. MATERIALS & METHODS Three groups of scaffolds were studied: group 1, poly-dl-lactic acid (PDLLA); group 2, hydrolyzed PDLLA (PDLLA/NaOH) and group 3, PDLLA/Lam - a PDLLA/NaOH scaffold linked to laminin protein. They were characterized by physicochemical and biological parameters. RESULTS As a result, the scaffolds presented well-formed and randomly distributed fibers. Group 3 showed the greatest hydrophilic characteristics. Group 1 showed a greater degradation rate after 14 days. Groups 2 and 3 presented molecular weight of about 40-50 Da. In general, group 3 showed the best results concerning cell adhesion and viability. CONCLUSION This study associated two revolutionary fields, stem cells and nanotechnology, for use in regenerative medicine.

Wettability and cell spreading enhancement in poly(sulfone) and polyurethane surfaces by UV-assisted treatment for tissue engineering purposes

The surface of Poly(sulfone) (PSU) and Polyurethane (PU) films were treated with ultraviolet (UV) light in the presence of oxygen to improve their wettability, adhesion and cell spreading properties. XPS and WCA results illustrated the effective conversion of the PSU and PU surfaces from hydrophobic to hydrophilic with grafting of new oxidized functional groups during the photochemical treatments. Treated films showed a larger number of adhered cells compared to the untreated films and that number of adhered cells was comparable to the number of adhered cells in the control group. The results showed that the cell response does not only depend on the hydrophilicity but on the chemical surface alterations which occur as a result of UV-assisted treatment in the presence of oxygen. Better cell adhesion, spreading and growing on the PSU and PU substrates modified by the present UV methodology confirmed the biocompatibility of the treated surfaces.

3D-printed PCL scaffolds for the cultivation of mesenchymal stem cells

Introduction Tissue engineering is a field which is currently under a great deal of investigation for the development and/or restoration of tissue and organs, through the combination of cell therapy with biomaterials. Rapid prototyping or additive manufacturing is a versatile technology which makes possible the fabrication of three dimensional (3D) structures from a wide range of materials with complex geometry and accuracy, such as scaffolds. Aim The aim of this study has been to investigate the interaction between mesenchymal stem cells with poly (ε-caprolactone) (PCL) biomaterials used for obtaining scaffolds through additive manufacturing. Materials and Methods Scanning electron microscopy, confocal microscopy and biological assays were performed to analyse the successful interaction between the cells and the biomaterials. Results As a result, the number of viable cells attached to the scaffolds was lower when compared to the control group; however, it was possible to observe cells in the scaffolds since day 1 of analysis, with regions of confluence after 21 days of seeding. Conclusions To conclude, these biomaterials are interesting if used as medical artifacts, principally in tissue with prolonged regeneration time and which requires 3D supports with good mechanical properties.

Update on the main use of biomaterials and techniques associated with tissue engineering

Regenerative medicine involves the study of cells, signaling cues and biomatrices to restore normal function of tissues and organs. To develop the matrices for use in tissue engineering there are three main groups of biomaterials: (i) naturally derived materials; (ii) synthetic polymers; and (iii) decellularized organ or tissue scaffolds. These biomaterials, in various forms such as hydrogels, nanofibers and 3D scaffolds, among others, have been employed for different tissue regeneration purposes, with several techniques involved in their production, including rapid prototyping, tissue decellularization and electrospinning. In this review, the main topics of hydrogels, 3D printing and electrospun scaffolds, other biomaterials and decellularization and recellularization will be discussed.