In vitro biocompatibility study of biodegradable polyester scaffolds constructed using Fused Deposition Modeling (FDM) Sabino Marcos*,***, Moret Josnell**, Fermín Zulielfre*,***, Rodríguez Dubravska**, Rodrigo A. Rezende***, Paulo I. Neto***, Frederico D. Sena***, Jorge V. L. Silva***, Alvarez José** *Departamento de Química, Grupo B5IDA, USB, AP89000, Caracas 1080-A, Venezuela (Tel: +58-212-9063990; e-mail:
[email protected]) **Laboratorio de Ingeniería de Tejidos Humanos, Fundación IDEA, Caracas, Venezuela ***Divisão de Tecnologias Tridimensionais–Centro de Tecnologia da Informação Renato Archer-Campinas, SP-Brasil Abstract: The growing interest in tissue engineering has stimulated the research of biomaterials that can be used as cellular supports and/or scaffolds to subsequently stimulate and/or regenerate tissues. Based on this premise, biodegradable polyesters: amorphous Poly (Lactic-acid) (PLA) and semi-crystalline Poly(caprolactone) (PCL), were used for manufacturing 3D scaffolds. These structures were designed using a a free software called Rhinoceros ® version 4.0. The software parameters considered for the design of these structures were: the distance between adjacent filaments, number of layers and the filaments orientation between layers. Through this information, and using PLA and PCL filaments (with diameters 2mm ≤ ø ≤ 3 mm, obtained by extrusion), scaffolds were fabricated using Fused Deposition Modeling (FDM), a rapid prototyping technology. The Morphology of all structures was observed by Scanning Electron Microscopy (SEM). To assess biocompatibility, human fibroblasts were seeded on these scaffolds, and cultured for 4 and 8 days. The biocompatibility was assessed by a metabolic activity assay based on MTT, where an increase in metabolic activity is interpreted as cell proliferation. The results led to appreciate the interaction of fibroblast cultures with these materials, with a noticeable increase in the cellular metabolism indicative of the material´s cytocompatibility and its capacity to support proliferation, making them strong candidates for tissue engineering.
1. INTRODUCTION For tissue engineering (an important area in the field of regenerative medicine), supports or scaffolds are temporary 3D structures, in which cells can grow, proliferate, differentiate and induce tissue formation or regeneration. The design and manufacturing of these structures must take into account considerations such as the material used, the manufacturing technique, control of topography and surface roughness, porosity, pore size and shape, as all of them are important to ensure the cell recognition processes, and increase cell migration on and from its surface [1, 2].
monolayer cultures of human fibroblasts in their third passage were used. Additionally, the following reagents were used: Trypsin-EDTA (Gibco, USA), DMEM culture medium (Dulbecco's modified essential medium) low in glucose (Gibco, USA), fetal bovine serum (FBS, Gibco, Brazil), antibiotic and antimycotic (anti-anti, Gibco - USA), tetrazolium methyl 3 - (4,5-Dimethyl-2-thiazolyl) -2,5diphenyl-2H-tetrazolium bromide (Sigma, USA) (also known as reactive MTT) and Dimethyl sulfoxide (Thermo Scientific).
There is a variety of techniques and computational tools for scaffold design and fabrication [4]. Among these, the free software called Rhinoceros ® version 4.0 is found, and it was chosen for this study. Fused Deposition Modeling (FDM) is an additive manufacturing technology commonly used for modeling, prototyping, and production applications. FDM works on an "additive" principle by laying down material in layers. A plastic filament or metal wire is unwound from a coil and supplies material to an extrusion nozzle which can turn the flow on and off. The nozzle is heated to melt the material and can be moved in both horizontal and vertical directions by a numerically controlled mechanism, directly controlled by a computer aided manufacturing software. The model or part is produced by extruding a thermoplastic material to form layers as the material hardens immediately after extrusion from the nozzle. Stepper motors or servo motors are typically employed to move the extrusion head. For the design of these 3D structures, there are importants parameters to consider, such as the distance between adjacent filaments, number of layers and the orientation of the filaments between layers.
The first stage of the development of the scaffolding was to obtain by extrusion, the PLA and PCL filaments with uniform diameter, 2mm ≤ ø ≤ 3mm, which were used as raw materials to fuel the rapid prototyping machine FAB@CTI (designed, built and evaluated by the CTI in Campinas, Brazil). For long filaments, a co-rotating twin screw extruder, BERSTORFF model ECS20 was used.
For scaffold fabrication, the materials used must be biocompatible and bioabsorbable, capable of undergoing reactions of hydrolytic degradation under physiological conditions within the body and removed by metabolic pathways [3]. These properties are presented by biodegradable polyesters, among which are the poly(lactic acid) (PLA) and poly(ε-caprolactone) (PCL), the first of them more susceptible to hydrolysis than the latter. Based on this information, the aim of the present study was to fabricate several scaffolds through the rapid prototyping technique, Fused Deposition Modelling (FDM), using PLA and PCL filaments, previously extruded with uniform diameter, 2mm ≤ ø ≤ 3mm; and to assess their cytocompatibility.
2.2 Scaffold Construction by FDM The three dimensional structures or scaffolds were designed using a free program called Rhinoceros ® version 4.0. The parameters considered in the design of these structures in the software are summarized in Table 1: Table 1. Parameters for PLA and PCL scaffold fabrication
Parameters
PLA
PCL
Temperature (°C) Diameter of scaffold filament (mm) Rate deposition of filaments (mm/s)
170
80
0,55
0,69
0,125
0,160
0,5; 0,7; 1 6, 8, 9, 10 36°, 40°, 56° , 90° and 120°
0,10; 0,20; 0,30; 0,50; 0,70 6, 8, 9, 10 40°, 56°, 90° and 120°
Distance between filaments (mm) Number of layers Filaments orientation between layers
The equipment used for modeling the scaffold was a FAB@CTI machine, as shown in the diagram in Figure 1.
2. EXPERIMENTAL PROCEDURE 2.1 Materials Polymers used were: pellets of amorphous PLA Biopolymer from NatureWorks 3001D, with a glass transition temperature around 60-65°C (experimentally determined by thermal analysis DSC) and viscosimetric molecular weight (Mv) of ~1x105 g/mol. Semicrystalline PCL from Solvay®, 6505 CAPA-6505 in powder presentation, with melting temperature 60 °C and viscosimetric molecular weight (Mv) of ~ 5x104 g/mol. For biocompatibility testing, primary
Fig 1. Rapid prototyping process using FDM [6] 2.3 Scaffold characterization.
Each scaffold was coated with gold in a sputtering equipment, BALZERS, under vacuum, at 200V, for 5 minutes. After this, sample morphology was observed using a scanning electron microscope (SEM) of high resolution, JEOL JSM6390 (at 25 kV).
obtained at 570 nm, using a TECAN-Infinite M200 plate reader.
2.4. Biocompatibility assays
As the PLA and PCL are polymers of different nature, but with different molecular weights, one amorphous and one semi-crystalline, it was necessary to vary the processing conditions for scaffold fabrication. It was noted that the PLA can be significantly affected by the process, given the marked dependence of rheological properties with temperature [7]. Accordingly, it was decided to design the PLA only homogeneous porous scaffolds, considering a single pore size, 0.7 mm.
For the development of bioassays, scaffolds were washed with sterile distilled water under stirring for 15 min. and subsequently dried with air flow. The sterilization was performed using UV light by exposing each side of the scaffold in 30 min cycles. Primary cultures of human fibroblasts were maintained at 37° C, 95% relative humidity and 5% CO2, making periodic changes of DMEM culture medium supplemented with 10% FBS and 1% antibiotic and antifungal. For cell seeding onto the scaffolds, cells were detached from the monolayer culture plate by the enzymatic action of trypsin, to subsequently count and seed on scaffolds of PLA and PCL, at a density of 150,000 and 250,000 cells/scaffold, respectively. The cell-scaffold systems were cultured for 4 and 8 days, after which cytocompatibility tests were conducted using the methyl-thiazol-tetrazolium (MTT) technique. MTT will be reduced by mitochondrial succinate dehydrogenase in viable cells, forming blue-violet formazan crystals whose colorimetric change is proportional to the number of metabolically active cells [5]. For this, the scaffolds were incubated with the cells in a fresh solution of MTT at 0.4 mg/mL for 2 hours. This reaction was stopped by removing the MTT, and placing an equal volume of DMSO, yielding the formazan coloration. Spectrophotometric measures were
Software Rhinoceros®
SEM PLA scaffold
3. RESULTS AND DISCUSSION
On the contrary, in the case of the PCL, which exhibits greater ease of processing, it allowed for the fabrication of scaffolds with different pore sizes: 0.3; 0.5 and 0.7 mm. Furthermore, for both polyesters, it was possible to modify the filament orientation in four angles (36 °, 40 °, 56 ° and 120 °), which in turn changed the number of layers placed. In order to verify the structure and orientation estimated by the software and the real morphology obtained during bioextrusión, SEM was performed, and the comparison is made in Figure 2. First, the morphology set designed in 3D software and built scaffolds was observed. Secondly, the figure shows the morphology and porous architecture achieved by the different conditions (pore size, orientation of the filaments, see Table 1). Also, SEM allowed verifying proper sintering between filaments (which ensures good dimensional stability), porous interconnectivity, and thereby good mechanical properties expected in these structures (as is show in figure 3).
Software Rhinoceros®
SEM PCL scaffold
0,7mm /36°/36°/10 layers
0,5 mm /40°/40°/9 layers
0,7mm/40°/40°/9 layers
0,7mm /56°/56°/6 layers
0,7mm/56°/56°/6 layers
0,5mm/120°/120°/6 layers
0,7mm/120°/120°/6 layers
0,3mm-0,2mm-0,5mm/90°/9 layers
0,7mm-0,5mm-1mm/90°/9 layers
0,3mm-0,2mm-0,1mm/90°/9 layers
Fig. 2. 3D Model and SEM correspondence for PLA and PCL prototyping scaffolds design with Rhinoceros ® v.4.0
Fig. 3. SEM micrographs for 3D scaffolds, is show porous interconnectivity between layers and sintering (with arrows).
As for the in vitro biocompatibility tests, it is seen that the sterilization method was efficient and does not deteriorate the scaffolding, allowing the removal of contaminants, which is needed to avoid contaminating of cell culture. The scaffolding, after incubation with MTT reagent, had a purple coloration on the surface. This is due to formation of formazan crystals coming from adherent cells grown on the surface of the filaments of the scaffold, thus showing good interaction between the cells and structures of PLA and PCL in study. The crystals were detached from the scaffold
surfaces by washes with DMSO and a buffered saline solution, and the supernatant could be used for measurements. Additionally, for both types of scaffolds, a significant increase in cellular metabolism from day 4 to day 8 was highlighted, as shown in Figures 4 and 5, respectively, indicating that cell proliferation took place on the surfaces of the scaffolds. These results demonstrate the cytocompatibility of the
scaffolds, allowing not only the adhesion but also the proliferation of the cells on their surfaces, which are key cellular events to validate a structure to be considered suitable for tissue engineering applications. The results presented in the present work demonstrate the in vitro
biocompatibility of the scaffolds in the conditions tested, consistent with the results reported in the literature[8,9], concerning the biocompatibility of PCL and PLA scaffolds with structural differences between them.
Fig 4. Cytocompatibility test (proliferation) on PLA scaffolds.
Fig 5. Cytocompatibility test (proliferation) on PCL scaffolds.
3. CONCLUSIONS Different scaffolding structures made out of PLA and PCL were obtained by rapid prototyping, through the FDM
technology. For both polymers, a correspondence was evident when comparing the structures designed with the Rhinoceros® software to those rapid prototyped, as observed by SEM. The orientation of the filaments that form the scaffolds was verified.
In terms of the in vitro biocompatibility tests, the obtained results showed the cell-material interactions that are evidenced by cellular adhesion and proliferation, making them strong candidates for tissue engineering applications. 5. REFERENCES [1] Hench, L y Jones, J. (2005). Biomaterials, Artificial Organs and Tissue Engineering. Woodhead Publishing Limited. Cambrigde, Inglaterra. p.p. 202. [2] Cheung, H-Y., Lau, K-T., Lu T-P., Hui, D. A critical review on polymer-based bio-engineered materials for scaffold development. Composites: Part B. 38 (2007); 291–300. [3] Khang, W, Kim, M, Lee, H. A Manual For Biomaterials/Scaffold Fabrication Technology. World Scientific Publishing. U.S.A. 2007. p.p. 199. [4] LIU, C, XIA, Z, CZEMUSZKA, T. Design And Development Of Three-Dimensional Scaffolds For Tissue Engineering. Chemical Engineering Research and Design. 2007. Vol. 85. p.p. 1051-1064.
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