Human dental pulp stem cell adhesion and detachment in polycaprolactone electrospun scaffolds under direct perfusion

Brazilian Journal of Medical and Biological Research (2018) 51(5): e6754, http://dx.doi.org/10.1590/1414-431X20186754 ISSN 1414-431X Research Article 1/10 Human dental pulp stem cell adhesion and detachment in polycaprolactone electrospun scaffolds under direct perfusion A. Paim1,2,3, D.I. Braghirolli3, N.S.M. Cardozo2, P. Pranke3,4 and I.C. Tessaro1 1 Laboratório de Separac¸ão por Membranas, Departamento de Engenharia Química, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil 2 Laboratório de Simulac¸ão, Departamento de Engenharia Química, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil 3 Laboratório de Hematologia e Células-Tronco, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil 4 Instituto de Pesquisa com Células-Tronco, Porto Alegre, RS, Brasil Abstract Cell adhesion in three-dimensional scaffolds plays a key role in tissue development. However, stem cell behavior in electrospun scaffolds under perfusion is not fully understood. Thus, an investigation was made on the effect of flow rate and shear stress, adhesion time, and seeding density under direct perfusion in polycaprolactone electrospun scaffolds on human dental pulp stem cell detachment. Polycaprolactone scaffolds were electrospun using a solvent mixture of chloroform and methanol. The viable cell number was determined at each tested condition. Cell morphology was analyzed by confocal microscopy after various incubation times for static cell adhesion with a high seeding density. Scanning electron microscopy images were obtained before and after perfusion for the highest flow rate tested. The wall pore shear stress was calculated for all tested flow rates (0.005–3 mL/min). An inversely proportional relationship between adhesion time with cell detachment under perfusion was observed. Lower flow rates and lower seeding densities reduced the drag of cells by shear stress. However, there was an operational limit for the lowest flow rate that can be used without compromising cell viability, indicating that a flow rate of 0.05 mL/min might be more suitable for the tested cell culture in electrospun scaffolds under direct perfusion. Key words: Cell adhesion; Perfusion; Shear stress; Stem cell; Electrospun scaffolds Introduction In tissue engineering, scaffolds are used as substitutes for damaged tissue and act as a support for cell proliferation, differentiation, and migration. In order to promote the formation of natural extracellular-matrix, a scaffold must be designed with appropriate biocompatibility, biodegradability, architecture, and mechanical properties (1). An important class of scaffolds for tissue engineering is based on electrospun polymer-based structures comprising solid microfibers or nanofibers, which can present high packing density and interconnected pore network (2). Nanofiber scaffolds favor higher mesenchymal stem cell viability than smooth surfaces (3). However, nanofiber scaffolds usually present small pores (4) that can hinder cell infiltration through three-dimensional structures (2). On the other hand, microfiber scaffolds can provide structures with bigger pores, allowing the cell migration and colonization inside the matrix (5). Correspondence: A. Paim: <> Received September 12, 2017 | Accepted January 11, 2018 Braz J Med Biol Res | doi: 10.1590/1414-431X20186754 Perfusion culture systems enhance mass transfer in scaffold-containing bioreactors and provide increased nutrient transport and cell viability (6), migration (7), growth, and differentiation (8). In addition, perfusion bioreactors can reduce the accumulation of toxic metabolites and degradation byproducts and the polymer degradation rate (9). Nevertheless, high shear stress can provoke cell detachment followed by cell death (10). Consequently, the cell number in three-dimensional (3D) scaffolds under perfusion is influenced by the cell detachment provoked by shear stress (11) and the capability of the cells to remain adhered to the scaffold and to proliferate, differentiate, and migrate is strongly dependent on the flow rate and the pore size employed. This is important because in order to obtain a homogeneous and effective regeneration of damaged tissue, it is essential to produce a biomaterial with an adequate cell number for implantation. Cell detachment in scaffolds under perfusion Therefore, it is necessary to quantify the cell drag and the final cell number in perfusion bioreactors to produce tissue substitutes that fit the quality standard required in a medical environment. Despite this, many studies on perfusion systems based on 3D scaffolds focus on the flow rate and shear stress effect on nutrient transport and stem cell proliferation and differentiation (12–14), without evaluating the cell detachment from the scaffold. This work addressed the reduction of the shear stress effects inside the scaffold pores under perfusion to produce cellularized electrospun structures for clinical application. An investigation was made of flow rate and shear stress under direct perfusion in polycaprolactone electrospun scaffolds on human dental pulp stem cell detachment. The influence of the adhesion time on cell adhesion and detachment under static conditions was also evaluated. Different seeding densities were tested under perfusion to evaluate the detachment. Material and Methods Scaffold production The scaffolds were produced in an electrospinning apparatus with temperature and humidity control (EC-CLI, IME Technologies, Netherlands). A 16% w/w solution of polycaprolactone (Sigma-Aldrich, USA; Mw 70-90 kDa) in a chloroform:methanol 9:1 vol% mixture was electrospun at 38% humidity, 19°C, 35 cm distance between the needle and the collector, flow rate of 0.1 mL/min, and voltage of 17 kV. The scaffolds were cut into 16 mm diameter disks and sterilized by ultraviolet radiation (UV) for 1 h. Cell isolation and expansion The pulp of human deciduous teeth was used to obtain dental pulp stem cells with the approval of the Research Committee and the Ethics Committee of the Universidade Federal do Rio Grande do Sul (project No. 33177214.1. 3001.5330), according to the methodology described by Werle et al. (15). Human deciduous teeth with physiologic root resorption were extracted and immersed in DMEM (Dulbecco’s modified Eagle’s culture medium)/Hepes (Sigma-Aldrich), supplemented with 10% fetal bovine serum (FBS; Gibco, USA), 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco), for transportation. The dental pulp tissue was removed with the use of endodontic instruments and the cells were isolated from the pulp by a mechanic and enzymatic process. The isolated cells were incubated for 24 h at 37°C and 5% CO2. The primary cultures and further passages were subcultured when a confluence of 90% was reached, with medium exchange every 3 or 4 days. Five primary culture cells (between the third and eighth passages) were used in this work. The cells were characterized as mesenc (...truncated)