Properties of PLDLA/bioglass scaffolds produced by selective laser sintering

Transcription

1 DOI /s ORIGINAL PAPER Properties of PLDLA/bioglass scaffolds produced by selective laser sintering Gean V. Salmoria 1 Rafaela V. Pereira 1 Márcio C. Fredel 1 Ana P. M. Casadei 2 Received: 31 July 2016 / Revised: 29 May 2017 / Accepted: 10 June 2017 Ó Springer-Verlag GmbH Germany 2017 Abstract This paper reports an evaluation of the structure, the flexural and viscoelastic properties, and the cytotoxicity of scaffolds produced from PLDLA and bioglass (BG) by selective laser sintering. The process conditions used in the scaffold preparation resulted in microstructures with a high sintering degree. The range of porosity values obtained for the scaffolds was 26 30%, which is appropriate for scaffold applications, and the average pore size in the PLDLA/BG scaffolds was 200 lm. The flexural modulus, ultimate strength, and elongation values for the PLDLA/BG scaffold specimens decreased for scaffolds with 20 and 30% of BG, probably due to the low chemical affinity between the polymeric and the ceramic phase. The PLDLA/BG scaffolds exhibited flexural modulus values between 10 and 79 MPa, depending on the scaffold composition, which lie within the range reported for human bone. In the direct cytotoxicity tests, cells seeded on PLDLA/BG scaffolds showed a high viability, indicating that the PLDLA/BG scaffolds prepared by selective laser sintering can be applied in bone repair. Keywords PLDLA/bioglass Scaffold properties Selective laser sintering & Gean V. Salmoria gean.salmoria@ufsc.br 1 2 Nimma, Departamento de Engenharia Mecânica, Universidade Federal de Santa Catarina, UFSC, Florianópolis, SC , Brazil Departamento de Morfologia, Universidade Federal de Santa Catarina, UFSC, Florianópolis, SC , Brazil

2 Introduction Polymeric scaffolds can be used to regenerate osseous tissue failure, restoring the shape and physiological functions of the damaged tissue. The scaffolds employed must fit into the anatomical defect, possess mechanical properties that will bear in vivo loads, enhance tissue in-growth, and not produce toxic degradation byproducts [1 4]. The success of tissue engineering will rely on the ability to generate complex 3D structures. Therefore, methods that can be used for the precise engineering of the architecture and topography of scaffolding materials will represent a critical aspect of functional tissue engineering [5]. Due to the lack of control over the architecture and porosity, the production of complex scaffold structures can involve both bottom-up and top-down approaches involving microand nano-manufacturing [6]. Selective laser sintering (SLS) is a solid free-form fabrication method that can be used to create scaffolds and biomedical devices to match a complex anatomical geometry [7 9]. SLS enables the design and fabrication of desirable microstructures and properties, allowing control over the pore size, porosity, permeability, mechanical properties, and material composition [9 12]. The control of these characteristics can enhance cell infiltration and the mass transport of nutrients and metabolites throughout the scaffold. Poly(L-co-D,L)lactic acid (PLDLA) is usually employed as the matrix in the production of bioabsorbable scaffolds, avoiding the need for a second surgery to remove the device after complete bone regeneration. However, PLDLA degradation in the body can result in undesirable side effects. The acid nature of monomers and oligomers produced from PLDLA hydrolysis can promote an inflammatory process. To minimize this type of body response, bioactive ceramic particles can be used as a dispersed phase in the polymeric matrix. Bioglass 58S (BG58S) particles, which show osteoconductive and basic behavior, can be used to reduce the concentration of acid species in the region of the PLDLA scaffold implantation [13 15]. This paper reports an evaluation of the structure, the flexural and viscoelastic properties, and the cytotoxicity of PLDLA/bioglass scaffolds produced by selective laser sintering. Experimental Scaffold fabrication PLDLA particles with diameters of lm were synthetized in the Biomaterials Laboratory of PUC/Sorocaba [16], SP and have been previously characterized. Bioglass 58S [60% SiO 2, 36% CaO, 4% P 2 O 5 (% mol)] was supplied as a powder (d 50 = lm) by Cermat. PLDLA and the bioglass (BG) powder were mixed in a Y-mixer for 45 min, in compositions with 10, 20, and 30% of BG content. Rectangular scaffolds ( ) were built by SLS using the previously determined processing parameters. The specimens were manufactured using a CO 2 laser with a power of 5 W, beam focus diameter of 250 lm and scan

3 speed of 40 mm/s. The chamber temperature was 40 C. The building layer thickness used was 700 lm and the spacing between the laser scans was 125 lm. Microscopy analysis and density measurements A JEOL JSM-6390LV scanning electronic microscope was used in the microstructural analysis. Prior to the analysis by SEM, all samples were coated with a thin layer of gold in a sputter diode D2 sputtering system. The apparent density was obtained in a pycnometer (50 cm 3 ). The average value was obtained from four samples of mm. The volumetric density was calculated considering the size and mass of the specimens. Mechanical tests A DMA Q800 analyzer (TA instruments) with a single cantilever clamp was used for the flexural tests of four specimens for each scaffold type. Stress strain curves were obtained at a loading rate of 2 N/min and 30 C. Dynamic mechanic analysis (DMA) was conducted for three specimens for each scaffold type to obtain the values for the storage modulus (E 0 ), loss modulus (E 00 ), and tan delta (d) ata frequency of 1 Hz within the temperature range of C using a heating rate of 3 C/min and strain of 0.5%. Cytotoxicity analysis The scaffolds were packed in self-seal sterilization pouches for gamma irradiation. Gamma-irradiation sterilization was achieved at a dose of 20 kgy at the CBE Embrarad Co. at room temperature. In vitro test of the cytotoxicity was performed using PLDLA and PLDLA scaffold samples according to ISO Part 5, by the neutral red uptake methodology [17]. Samples gamma sterilized were added to Eagle s minimum medium (MEM) in a proportion of 1 cm 2 /ml and incubated for 48 h at 37 C. Serial dilutions were made of extracts from the samples. The cell line NCTC clone L929 used was acquired from the American Type Culture Collection (ATCC) bank and maintained in MEM supplemented with 10% fetal calf serum, 20 mm glutamine, and 1% non-essential amino acids (complete MEM) in a humidified incubator with 5% CO 2 at 37 C. The cells were detached by trypsin, washed twice with calcium and magnesium-free phosphate buffer solution and the cell suspension was adjusted to about cell/ml. 0.2 ml of the cell suspension was seeded in flat-bottomed 96-well microplate (Costar, Cambridge, MA, USA). The microplate was incubated for 24 h at 37 C inaco 2 humidified incubator. After this period, the medium of the plate was discarded and replaced with 0.2 ml of serially diluted extract of each sample (100, 50, 25, 12.5, and 6.25%). Control of cell culture medium was replaced with complete MEM. In the same microplate was ran a positive control (0.02% phenol solution) and a negative control (atoxic tin-stabilized polyvinyl chloride). Samples and controls were tested in triplicate. The plate was incubated again for 24 h under the same conditions. After 24 h, the culture medium and extracts were discarded and replaced with 0.2 ml of

4 0.005% neutral red diluted in MEM. After 3 h of incubation at 37 C, the dye medium was discarded and the microplate was washed twice with phosphate saline buffer. The cells were washed with a solution of 1% CaCl 2 in 0.5% formaldehyde. The rupture of cells and neutral red release was obtained by the addition of 0.2 ml/ well of extractant solution containing 50% ethanol in 1% acetic acid. The absorbances were read in a 540-nm filter on an RC Sunrise model-tecan spectrophotometer for ELISA. With the average of the optical density of each extract dilution of samples, negative, and positive controls, the cell viability percentages were calculated in relation to the cell control (100%) and plotted in a graph against the extract concentrations. The cytotoxicity potential of the investigated materials was expressed as a cytotoxicity index [IC50(%)] and can be obtained from this graph. IC50(%) is the concentration of the extract which injures or kills 50% of the cell population in the assay due to toxic elements extracted from the tested sample. Results and discussion The microstructure of the pure PLDLA scaffold fabricated by SLS showed a higher sintering degree than the composite specimens, with particles joined by extensive neck formations, as can be observed in the micrograph shown in Fig. 1a. The Fig. 1 Micrographs of PLDLA (a) and PLDLA/BG composites with BG content of 10% (b), 20% (c), and 30% (d). Amplification 930

5 microstructure of the composite scaffolds fabricated by SLS showed the presence of different quantities of BG powder with an average particle size of 10 lm, the size varying according to the composite composition (Fig. 1b d). The BG particles tend to agglomerate at higher concentrations. The porosity, which refers to the percentage of voids in the volume of the scaffold specimen, and the internal pore architecture, in terms of the pore diameter, was analyzed by density measurements and SEM images, to evaluate the manufactured scaffold morphologies. The scaffolds were designed to have a volumetric porosity ranging from 35 to 45%. The manufactured scaffolds had porosity values of 26 38% (Table 1), which can be considered as consistent with the scaffold design. The process conditions used in the scaffold preparation resulted in microstructures with a high sintering degree, as shown in Fig. 2. The microstructure of the sintered scaffold specimens had irregular distributions of interconnected pores, the Table 1 Values for the porosity of the PLDLA scaffolds prepared with different BG contents Scaffold Porosity (%) Pure PLDLA 38 ± 2 PLDLA/BG 10% 26 ± 2 PLDLA/BG 20% 27 ± 3 PLDLA/BG 30% 30 ± 4 Fig. 2 Micrographs of PLDLA (a) and PLDLA/BG composites with BG content of 10% (b), 20% (c), and 30% (d). Amplification 9500

6 Table 2 Flexural mechanical properties of PLDLA and PLDLA/BG scaffolds Scaffold Flexural modulus (MPa) Ultimate strength (MPa) Elongation (%) PLDLA 68 ± ± ± 6.1 PLDLA/BG 10% 79 ± ± ± 3.9 PLDLA/BG 20% 21 ± ± ± 2.6 PLDLA/BG 30% 10 ± ± ± 5.1 average size being related to the particle size and shape of the original PLDLA powder (Fig. 2). The sizes of the pores observed in the scaffold microstructures varied as a function of the particle size used in the preparation and the sintering degree. The PLDLA and PLDLA/BG scaffolds had average pore sizes of 200 lm and in the PLDLA/BG scaffolds there were pores within the BG particles. Table 2 shows the average values for the flexural modulus, ultimate strength, and elongation for the pure PLDLA and PLDLA/BG scaffolds. The majority of sintered PLDLA/BG composites had a lower value for the flexural modulus and ultimate strength than the pure PLDLA scaffold in the flexural tests. The low values for the ultimate strength and elastic modulus of the sintered composites with 20 and 30% of BG indicate the low chemical affinity between the PLDLA and BG phases. High BG content means less polymer matrix and consequently less material to fuse and facilitate composite particle coalescence. On the other hand, the higher elastic modulus of composites with 10% of BG is related to the fact of the quantity of BG is still low cause this dilution effect in flexural modulus, and PLDLA matrix combined with 10% of BG reach an optimum flexural behavior on this property combining Fig. 3 Storage modulus (E 0 ) as a function of temperature for PLDLA (A) and PLDLA/BG with 10% (B), 20% (C), and 30% (D) of BG content

7 tensile and compressive stiffness. The dilution effect was observed on the reduction of the ultimate strength and the elongation at failure for composites with 10% of BG (Table 2). These polymer properties are more sensible to the presence of heterogeneous phases even on flexural tests [7, 11, 12]. The complex relationship between the effects of the applied laser energy, laser absorption by the materials, the chemical interactions, and material composition on the microstructure formation (sintering degree, co-continuous phases and porosity) plays an important role in the specimen properties, and can lead to high variation on mechanical properties of binary materials sintered by laser as related in the literature [18 20]. The mechanical modulus values of human bone range from 1.0 to 5000 MPa, with mean values of approximately 194 and 4 MPa for flexural modulus and ultimate strength, respectively [21]. The flexural modulus values for the PLDLA/ BG scaffolds varied between 10 and 79 MPa, depending on the scaffold composition, which lies within the range reported for human bone. To evaluate the viscoelastic properties of the PLDLA and PLDLA/BG scaffold specimens, DMA analysis was carried out. The variations in the E 0 and tan d values as a function of temperature for PLDLA and the PLDLA/BG composites measured at a frequency of 1 Hz are shown in Figs. 3 and 4. At a temperature of 30 C, E 0 Fig. 4 Loss tangent (tan d) as a function of temperature for PLDLA (A) and PLDLA/BG with 10% (B), 20% (C), and 30% (D) of BG content Table 3 Glass transition temperatures of PCL/PG blend specimens from DMA and DSC Scaffold T g ( C) by DMA T g ( C) by DSC PLDLA 54 ± 1 59 ± 1 PLDLA/BG 10% 54 ± 2 59 ± 1 PLDLA/BG 20% 53 ± 2 57 ± 2 PLDLA/BG 30% 52 ± 2 58 ± 1

8 Fig. 5 DSC thermograms for PLDLA (A) and PLDLA/BG with 10% (B), 20% (C), and 30% (D) ofbg content values for the pure PLDLA and PLDLA/BG scaffolds showed the same characteristic observed in quasi-static flexural tests (Table 2). The storage modulus E 0 to PLDLA/BG scaffold with 10% of BG presented higher value than the pure PLDLA and other PLDLA/BG scaffolds with 20 and 30% of BG due to the dilution effect of the ceramic phase into the polymeric matrix.

9 The loss tangent (tan d) for the PLDLA decreased after the addition of BG, suggesting that there is no significant interaction between the two components. The maximum loss tangent was obtained between 52 and 54 C. The maximum tan d peak showed a trend toward lower temperatures for the PLDLA/BG composites with 20 and 30% of BG content (Fig. 4), suggesting that the chain rotation at the glass transition temperature (T g ) in PLDLA was slightly affected in these cases. The glass transition temperatures, T g (at the maximum tan d), for the PLDLA and PLDLA/BG scaffolds are reported in Table 3. DSC curves (Fig. 5) show the transitions for pure PLDLA scaffold and for the PLDLA/BG scaffolds. The scaffold curves present a clear glass transition between 51 and 64 C. The glass transition temperatures (T g ) obtained by DSC for the PLDLA and PLDLA/BG scaffolds are reported in Table 4. The T g values similar (around 58 C) for the pure PLDLA and PLDLA/BG scaffolds confirm the inexistence of significant chemical interaction between PLDLA and the ceramic phase. In vitro tests of cytotoxicity were performed to evaluate the toxicity of pure PLDLA and PLDLA/BG scaffolds. Figure 6 shows the viability curves of the pure PLDLA and PLDLA/BG scaffold compositions and the positive and negative controls in the cytotoxicity assay by the neutral red uptake methodology. From these curves, it is possible to observe that the extracts of pure PLDLA and PLDLA/BG scaffolds even with high extract concentrations do not cause death or injury of the cell population, indicating that these materials present no cytotoxicity. All studied scaffold showed the same behavior in the negative control. Only the positive control showed cytotoxicity presenting a cytotoxicity index (IC50%) of about 55% indicating that the extract of the positive control at a concentration of 55% injured or killed 50% of the cell population in the assay. The test showed that there is no contamination by the processing in significant amounts to compromise the Fig. 6 Cell viability in the cytotoxicity test for the scaffolds produced

10 experiment. The results for the direct cytotoxicity presented a high viability of the cells seeded on both the PLDLA and PLDLA/BG scaffolds. Conclusions The process conditions used in the scaffold preparation resulted in microstructures with a high sintering degree. The PLDLA/BG scaffolds showed sintered porous structures with BG particles distributed over the polymeric matrix, and their size varied according to the composite composition. The range of porosity values obtained for the scaffolds was 26 30%, which is appropriate for scaffold applications, and the average pore size in the PLDLA/BG scaffolds was 200 lm. The flexural modulus, ultimate strength, and elongation values for the PLDLA/BG scaffold specimens decreased for scaffolds with 20 and 30% of BG, probably due to the low chemical affinity between the polymeric and the ceramic phase. Storage modulus E 0 decreased for scaffolds with 20 and 30% of BG at 30 C, which is consistent with the results obtained in the quasi-static flexural test. The PLDLA/BG scaffolds exhibited flexural modulus values between 10 and 79 MPa, depending on the scaffold composition, which lie within the range reported for human bone. In the direct cytotoxicity tests, cells seeded on PLDLA/BG scaffolds showed a high viability, indicating that the PLDLA/BG scaffolds prepared by selective laser sintering can be applied in bone repair by grafting. Acknowledgements The authors would like to thank PRONEX-FAPESC, CNPq, and CAPES for financial support and also LCME-UFSC for the SEM micrographs. References 1. Mercuri LG (1998) Alloplastic temporomandibular joint reconstruction. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 85(6): Langer R, Tirrell DA (2004) Designing materials for biology and medicine. Nature 428(6982): Agrawal CM, Ray RB (2001) Biodegradable polymeric scaffolds for musculoskeletal tissue engineering. J Biomed Mater Res 55(2): Das S, Hollister SJ (2003) Tissue engineering scaffolds. In: Buschow KHJ, Cahn RW, Flemings MC, Ilschner B, Kramer EJ, Mahajan S (eds) Encyclopedia of materials: science and technology. Elsevier, Amsterdam 5. Gauvin R, Chen Y, Lee JW, Soman P, Zorlutuna P, Nichol JW, Bae H, Chen S, Khademhosseini AK (2012) Microfabrication of complex porous tissue engineering scaffolds using 3D projection stereolithography. Biomaterials 33(15): doi: /j.biomaterials Chryssolouris G, Stavropoulos P, Tsoukantas G, Salonitis K, Stournaras A (2004) Nanomanufacturing processes: a critical review. Int J Mater Prod Technol 21(4): Salmoria GV, Fancello EA, Roesler CRM, Dabbas F (2013) Functional graded scaffold of HDPE/HA prepared by selective laser sintering: microstructure and mechanical properties. Int J Adv Manuf Technol 65: Salmoria GV, Klauss P, Zepon K, Kanis LA, Roesler CRM, Vieira LF (2012) Development of functionally-graded reservoir of PCL/PG by selective laser sintering for drug delivery devices. Virtual Phys Prototyp 7(2): Salmoria GV, Klauss P, Zepon KM, Kanis LA (2013) The effects of laser energy density and particle size in the selective laser sintering of polycaprolactone/progesterone specimens: morphology and drug release. Int J Adv Manuf Technol 66:

11 10. Salmoria GV, Hotza D, Klauss P, Kanis LA, Roesler CRM (2014) Manufacturing of porous polycaprolactone prepared with different particle sizes and infrared laser sintering conditions: microstructure and mechanical properties. Adv Mech Eng 2014:art. no doi: /2014/ Salmoria GV, Leite JL, Paggi RA (2009) The microstructural characterization of PA6/PA12 blend specimens fabricated by selective laser sintering. Polym Test 28(7): Salmoria GV, Leite JL, Vieira LF, Pires ATN, Roesler CRM (2012) Mechanical properties of PA6/ PA12 blend specimens prepared by selective laser sintering. Polym Test 31(3): Hutmacher DW (2000) Scaffolds in tissue engineering bone and cartilage. Biomaterials 21(24): Chen Q, Roether JA, Boccaccini AR (2008) Tissue engineering scaffolds from bioactive glass and composite materials. In: Ashammakhi N, Reis R, Cjielini F (eds) Topics in tissue engineering. TBE Group, Illinois (e-book) 15. Hoppe A, Güldal NS, Boccaccini AR (2011) A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics. Biomaterials 32(11): Motta AC, Duek EAR (2007) Synthesis and characterization of the copolymer poly(l-co-d,l lactic acid). Polímeros 17: ISO document , 1992 Biological evaluation of medical devices: part 5. Tests for cytotoxicity: in vitro methods 18. Leite JL, Salmoria GV, Paggi RA, Ahrens CH, Pouzada AS (2012) Microstructural characterization and mechanical properties of functionally graded PA12/HDPE parts by selective laser sintering. Int J Adv Manuf Technol 59: Salmoria GV, Paggi RA, Beal VE (2017) Graded composites of polyamide/carbon nanotubes prepared by laser sintering. Lasers Manuf Mater Process 4:36. doi: /s Leite JL, Salmoria GV, Paggi RA, Ahrens CH, Pouzada AS (2010) A study on morphological properties of laser sintered functionally graded blends of amorphous thermoplastics. Int J Mater Prod Technol 39: Goulet RW, Goldstein SA, Ciarelli MJ, Kuhn JL, Brown MB, Feldkamp LA (1994) The relationship between the structural and orthogonal compressive properties of trabecular bone. J Biomech 27(4):