Robocast Scaffolds Research Articles

Preparation of trivalent rare-earth element-substituted bioactive glass robocast scaffolds for osteosarcoma treatment: structural, morphological, mechanical, drug delivery, and biological properties

In this study, trivalent rare-earth ion (Eu3+, Gd3+, and Yb3+)-substituted silicate-based bioactive glass scaffolds were prepared by robocasting method using sol–gel-derived bioactive glass powders for tissue engineering applications and cancer therapy. The structural, morphological, and mechanical properties of the prepared scaffolds as well as their in vitro bioactivity in simulated body fluid (SBF) were investigated in detail. In addition, an anticancer drug (5-FU) adsorption and release behavior of the scaffolds was studied as a function of time. In vitro, cytotoxicity and alkaline phosphatase activity were investigated using human skin fibroblast BJ and osteosarcoma SaOS-2 cells. Results showed that using lanthanide ion-containing (0.5, 1, 3, and 5 wt%) sol–gel-derived bioactive glass powders it was possible to successfully fabricate periodic, mesh-like patterned robocast glass scaffolds. All of the scaffolds prepared in the study sintered at 675 °C showed an amorphous structure. The compressive strength of scaffolds was in the range of 8.8 MPa to 13.6 MPa and the highest strength values were obtained in the Yb3+-containing scaffolds. Hydroxyapatite formation was obtained for the scaffolds immersed in SBF for 28 days. The fluorouracil adsorption amount was calculated to be ~ 25% for all types of scaffolds and the cumulative drug release was in the range of 20–25% depending on the dopant concentration. Results of the in vitro cell culture experiments revealed that all of the scaffolds fabricated in the study were not cytotoxic to fibroblast and osteosarcoma cells for up to 7 days under in vitro conditions. An increase was obtained for the ALP activities for both types of cells as the incubation time was increased.

Robocasting of multicomponent sol-gel–derived silicate bioactive glass scaffolds for bone tissue engineering

Robocasting is universally recognized as an affordable and reproducible manufacturing strategy to process glass and glass-ceramic materials in the form of highly ordered porous scaffolds for bone tissue engineering (BTE) applications. Nevertheless, while being widely applied to melt-derived bioactive glasses, this technique was seldom implemented with sol-gel materials due to the intrinsic difficulties in producing suitable inks for extrusion and thus, good printing outcomes. The present experimental work describes a new and relatively easy method to manufacture multicomponent sol-gel bioactive silicate scaffolds (oxide system: 47.5SiO2–20CaO–10MgO–10Na2O-10 K2O-2.5P2O5, mol.%) using dried gels as basic material within the ink composition, which allows by-passing intermediate heat treatments that are usually detrimental to the bioactive potential of the material in physiological environment. The scaffolds, exhibiting a total porosity of 81 vol%, were characterized in terms of morphological-compositional features and bioactivity in simulated body fluid (SBF), paying special attention to ion release and surface modifications occurring upon soaking (apatite-forming ability). The compressive strength of the scaffolds (around 5 MPa) was comparable to that of human cancellous bone. Collectively, the results supported the possibility of using robocast sol-gel–derived scaffolds in BTE approaches. Furthermore, a comparison with robocast scaffolds based on a melt-derived glass with the same composition was reported in order to investigate the effect of the synthesis route on the dissolution behavior and morphological features of the final biomaterials.

Robocasting of Single and Multi-Functional Calcium Phosphate Scaffolds and Its Hybridization with Conventional Techniques: Design, Fabrication and Characterization

In this work, dense, porous, and, for the first time, functionally-graded bi-layer scaffolds with a cylindrical geometry were produced from a commercially available hydroxyapatite powder using the robocasting technique. The bi-layer scaffolds were made of a dense core part attached to a surrounding porous part. Subsequently, these bi-layer robocast scaffolds were joined with an outer shell of an antibacterial porous polymer layer fabricated by solvent casting/salt leaching techniques, leading to hybrid ceramic-polymer scaffolds. The antibacterial functionality was achieved through the addition of silver ions to the polymer layer. All the robocast samples, including the bi-layer ones, were first characterized through scanning electron microscopy observations, mechanical characterization in compression and preliminary bioactivity tests. Then, the hybrid bi-layer ceramic-polymer scaffolds were characterized through antimicrobial tests. After sintering at 1300 °C for 3 h, the compressive strengths of the structures were found to be equal to 29 ± 4 MPa for dense samples and 7 ± 4 MPa for lattice structures with a porosity of 34.1%. Bioactivity tests performed at 37 °C for 4 weeks showed that the precipitated layer on the robocast samples contained octacalcium phosphate. Finally, it was evidenced that the hybrid structure was effective in releasing antibacterial Ag+ ions to the surrounding medium showing its potential efficiency in limiting Staphylococcus aureus proliferation during surgery.

Robocasting of SiO2-Based Bioactive Glass Scaffolds with Porosity Gradient for Bone Regeneration and Potential Load-Bearing Applications.

Additive manufacturing of bioactive glasses has recently attracted high interest in the field of regenerative medicine as a versatile class of fabrication methods to process bone substitute materials. In this study, melt-derived glass particles from the SiO2-P2O5-CaO-MgO-Na2O-K2O system were used to fabricate bioactive scaffolds with graded porosity by robocasting. A printable ink made of glass powder and Pluronic F-127 (binder) was extruded into a grid-like three-dimensional structure with bimodal porosity, i.e., the inner part of the scaffold had macropores with smaller size compared to the periphery. The crystallization behavior of the glass powder was studied by hot-stage microscopy, differential thermal analysis, and X-ray diffraction; the scaffolds were sintered at a temperature below the onset of crystallization so that amorphous structures could be obtained. Scaffold architecture was investigated by scanning electron microscopy and microtomographic analysis that allowed quantifying the microstructural parameters. In vitro tests in Kokubo’s simulated body fluid (SBF) confirmed the apatite-forming ability (i.e., bioactivity) of the scaffolds. The compressive strength was found to slightly decrease during immersion in SBF up to 4 weeks but still remained comparable to that of human cancellous bone. The pH and concentration of released ions in SBF were also measured at each time point. Taken together, these results (favorable porosity, mechanical strength, and in vitro bioactivity) show great promise for the potential application of these robocast scaffolds in bone defect repair.

Effect of pore architecture on the mesenchymal stem cell responses to graphene/polycaprolactone scaffolds prepared by solvent casting and robocasting

In the study graphene-containing porous, three dimensional polycaprolactone (PCL) scaffolds were prepared by solvent casting-salt leaching and robocasting methods for tissue engineering applications. Graphene nanopowders in the form of nanoflakes were incorporated into the polymer matrix at different concentrations namely 1, 3, 5 and 10 wt%. The dichloromethane was used as the solvent and sodium chloride crystals were utilized as the water-soluble porogen for the formation of an interconnected porous network (with non-oriented pores) inside the composite scaffolds in solvent casting-salt leaching method. On the other hand, acetone was utilized as solvent and PCL solutions were prepared at 20 wt% in robocasting method to construct scaffolds (with oriented pores) having a grid-like structure. The biological response of bone marrow mesenchymal stem cells seeded on these composite constructs having different architecture were tested using MTT method, live-dead cell viability assay and Alcian blue stanining. Cytotoxicity experiments revealed that mesenchymal stem cells did not show toxic response to composite robocast scaffolds. Cells proliferate and differentiate well on the surface of the robocast scaffolds compared to solvent-cast scaffolds under the same conditions. Results showed that scaffolds prepared in the study have potential to be used in cartilage tissue engineering in the presence of electric stimulation.

Bioceramic scaffolds fabrication: Indirect 3D printing combined with ice-templating vs. robocasting

Ice-templating combined with indirect 3D printing is proposed as a promising method for preparation of scaffolds with multiscale porosity, including a well-defined interconnected macro-channel network. Robocasting was used as a comparative technique to produce scaffolds with comparable porosity at the introduced macroporosity and the inter-grain microporosity levels. Porosity, phase composition and mechanical stability were measured and compared for bio-scaffolds prepared by both techniques. Comparable total porosities could only be achieved in robocasting by choosing a significantly lower sintering temperature (950 °C vs. 1200 °C). The compressive strength of robocast scaffolds was significantly greater (6.5 ± 1.19 MPa vs. 2.3 ± 1.00 MPa, respectively). However, the increased level of interconnected multiscale porosity coupled to a finer grain size of ice-templated samples sintered at 1200 °C (∼ 500 nm vs. 2.5 μm for robocast parts) could prove to be beneficial for the development of highly porous bioactive scaffolds with enhanced biological performance.

Optimisation and biological activities of bioceramic robocast scaffolds provided with an oxygen-releasing coating for bone tissue engineering applications

Hypoxia is one of the major challenges after scaffold implantation which can lead to cell necrosis and bacterial infection. Using of supplemental oxygen can increase the cell proliferation, encourage the cell differentiation and prevent the infections. Developing an engineered scaffold with a sustained oxygen release is an outstanding way for addressing the challenges of oxygen deficiency. In this study, the bioceramic scaffolds were fabricated from biphasic calcium phosphate (BCP) powder with the composition of 60% hydroxyapatite (HA) and 40% beta-tricalcium phosphate (β-TCP). The Robocasting technique was utilised for producing a porous structure comprising interpenetrated ceramic rods in a 3-dimensional tetragonal mesh. The scaffold was modelled by the finite element method (FEM) for computing the stress fields and predicting their mechanical performance. Calcium peroxide (CPO), as an oxygen-producing and antimicrobial biomaterial, was mixed with a polycaprolactone (PCL) solution and was coated on the scaffolds by the dip-coating method. The coating layer possessed three different percentages of CPO (1, 3 and 5 wt%). The oxygen-releasing profile proved that this design of coating-scaffold could be effective as a system of oxygen delivery. According to the antibacterial investigations, releasing of CPO from the scaffolds could inhibit the growth of E. coli and S. aureus. SBF tests confirmed that the coated scaffolds because of CPO particles on their surface presented superior apatite precipitation in comparison with the uncoated one. The differentiated osteoblastic function was monitored by measuring the alkaline phosphatase (ALP) activity. The coated BCP scaffolds with 3% and 5% CPO exhibited higher ALP activity compared to the other samples. The results demonstrated that the proposed bioceramic-based scaffolds containing oxygen-generating coating could be optimised to supply an antibacterial performance, ideal mechanical properties, improved ALP activity and higher apatite formation ability. Therefore, these scaffolds can be a promising candidate for applying in bone tissue engineering.

Enhancing the mechanical and in vitro performance of robocast bioglass scaffolds by polymeric coatings: Effect of polymer composition.

The effect of different polymeric coatings, including natural and synthetic compositions, on the mechanical performance of 45S5 bioglass robocast scaffolds is systematically analyzed in this work. Fully amorphous 45S5 bioglass robocast scaffolds sintered at 550 °C were impregnated with natural (gelatin, alginate, and chitosan) and synthetic (polycaprolactone, PCL and poly-lactic acid, PLA) polymers through a dip-coating process. Mechanical enhancement provided by these coatings in terms of both compressive strength and strain energy density was evaluated. Natural polymers, in general, and chitosan, in particular, were found to produce the greater reinforcement. The effect of these coatings on the in vitro bioactivity and degradation behavior of 45S5 bioglass robocast scaffolds was also investigated through immersion tests in simulated body fluid (SBF). Coatings from natural polymers, especially chitosan, are shown to have a positive effect on the bioactivity of 45S5 bioglass, accelerating the formation of an apatite-like layer. Besides, most coating compositions reduced the degradation (weight loss) rate of the scaffold, which has a positive impact on the evolution of their mechanical properties.

Custom Repair of Mandibular Bone Defects with 3D Printed Bioceramic Scaffolds

Implanting artificial biomaterial implants into alveolar bone defects with individual shape and appropriate mechanical strength is still a challenge. In this study, bioceramic scaffolds, which can precisely match the mandibular defects in macro and micro, were manufactured by the 3-dimensional (3D) printing technique according to the computed tomography (CT) image. To evaluate the stimulatory effect of the material substrate on bone tissue regeneration in situ in a rabbit mandibular alveolar bone defect model, implants made with the newly developed, mechanically strong ~10% Mg-substituted wollastonite (Ca90%Mg10%SiO3; CSi-Mg10) were fabricated, implanted into the bone defects, and compared with implants made with the typical Ca-phosphate and Ca-silicate porous bioceramics, such as β-tricalcium phosphate (TCP), wollastonite (CaSiO3; CSi), and bredigite (Bred). The initial physicochemical tests indicated that although the CSi-Mg10 scaffolds had the largest pore dimension, they had the lowest porosity mainly due to the significant linear shrinkage of the scaffolds during sintering. Compared with the sparingly dissolvable TCP scaffolds (~2% weight loss) and superfast dissolvable (in Tris buffer within 6 wk) pure CSi and Bred scaffolds (~12% and ~14% weight loss, respectively), the CSi-Mg10 exhibited a mild in vitro biodissolution and moderate weight loss of ~7%. In addition, the CSi-Mg10 scaffolds showed a considerable initial flexural strength (31 MPa) and maintained very high flexural resistance during soaking in Tris buffer. The in vivo results revealed that the CSi-Mg10 scaffolds have markedly higher osteogenic capability than those on the TCP, CSi, and Bred scaffolds after 16 wk. These results suggest a promising potential application of customized CSi-Mg10 3D robocast scaffolds in the clinic, especially for repair of alveolar bone defects.

Direct ink writing of highly bioactive glasses

Direct ink writing (DIW), or Robocasting, is an additive manufacturing technique that offers the opportunity to create patient specific bioactive glass scaffolds and high strength scaffolds for bone repair. The original 45S5 Bioglass® composition crystallises during sintering and until now, robocast glass scaffolds contained at least 51.9mol% SiO2 or B2O3 to maintain their amorphous structure. Here, ICIE16 and PSrBG compositions, containing <50mol% SiO2, giving silicate network connectivity close to that of 45S5, were robocast and compared to 13–93 composition. Results showed Pluronic F-127 can be used as a universal binder regardless of glass reactivity and that particle size distribution affected the ink “printability”. Scaffolds with interconnects of 150μm (41–43% porosity) had compressive strengths of 32–48MPa, depending on the glass composition. Robocast scaffolds from these highly reactive bioactive glasses promise greatly improved bone regeneration rates compared with existing bioactive glass scaffolds.

Effect of the drying process on the compressive strength and cell proliferation of hydroxyapatite‐derived scaffolds

AbstractThe compressive strength and MC3T3 cell proliferation response of robocast hydroxyapatite‐derived scaffolds was evaluated for samples fabricated by conventional and freeze‐drying methods followed by sintering at 1100 or 1300°C. Both the sintering temperature and, especially, the drying method affected significantly the size and morphology of the residual microporosity within the robocast scaffold's struts. The freeze‐drying method generated a persistent large (1‐10 μm) microporosity of dendritic morphology that was found to improve the biological response of hydroxyapatite‐derived scaffolds. Conversely, conventional drying enhances the compressive strength of the structures. Strength was also increased at the higher sintering temperature, although at the expense of a poorer cell proliferation behavior. The results of this study suggest that the use of a freeze drying process after printing by robocasting provides a very appropriate method for enhancing the biological performance and reliability of bioceramic robocast scaffolds without severely reducing their compressive strength. And, thus, shows promise as an effective method to optimize the performance of robocast scaffolds for bone tissue regeneration.

Case study: Reinforcement of 45S5 bioglass robocast scaffolds by HA/PCL nanocomposite coatings.

The purpose of this study is to analyze the mechanical enhancement provided by nanocomposite coatings deposited on robocast 45S5 bioglass (BG) scaffolds for bone tissue regeneration. In particular, a nanocomposite layer consisting of hydroxyapatite (HA) nanoparticles, as reinforcing phase, in a polycaprolactone (PCL) matrix was deposited onto the surface of the BG struts conforming the scaffold. Three different HA nanopowders were used in this study. The effect of particle size and morphology of these HA nanopowders on the mechanical performance of 45S5 BG scaffolds is evaluated.

Robocast 45S5 bioglass scaffolds: in vitro behavior

The in vitro bioactivity and degradation behavior of 45S5 bioglass robocast scaffolds is investigated by immersion tests in simulated body fluid (SBF). A comparative study is conducted between fully amorphous scaffolds, sintered at 550 °C, and mostly crystalline structures, sintered at 1000 °C. The degradation behavior is evaluated through measurements of the weight loss and solution pH variations upon immersion in SBF. The concurrent formation of calcium-rich precipitates on the scaffold surface during immersion in SBF solution was also monitored. The effect of the degradation on the scaffolds’ mechanical performance in compression is subsequently analyzed. Finally, in vitro cell culture experiments were performed to evaluate the proliferation behavior of osteoblast-like cells onto both types of robocast scaffolds. Amorphous scaffolds exhibited an enhanced in vitro degradation and faster calcium carbonate precipitation. However, cell proliferation was not enhanced in the glassy structures compared to the crystalline scaffolds, which demonstrated also a better mechanical performance, as expected.

Understanding the role of dip-coating process parameters in the mechanical performance of polymer-coated bioglass robocast scaffolds

The effect of different dip-coating variables—solvent, deposition temperature and polymer concentration—on the mechanical performance of polycaprolactone-coated 45S5 bioglass robocast scaffolds is systematically analyzed in this work. The reproducible geometry of the scaffolds produced by this additive manufacturing technique makes them an optimal model system and facilitates the analysis. The results suggest that the mechanical performance of the hybrid scaffolds is improved monotonically with polymer concentration, but this concentration cannot be increased indefinitely if the macroporosity interconnectivity, and thus the scaffold׳s capacity to promote tissue ingrowth, are to be preserved. An optimal concentration, and therefore viscosity (~1–4Pas in the present case), exists for any given set of process variables (scaffold geometry and material, polymer, solvent and process temperature) that yields coatings with optimal reinforcement and minimal reduction of scaffold functionality. Solvent and process temperature do not directly affect the strengthening provided by the polymeric coating. However they can determine the maximum concentration at the critical viscosity, and thereby the maximum achievable mechanical performance of the resulting hybrid scaffold.

Electrically functional 3D-architectured graphene/SiC composites

Lightweight, three-dimensional (3D) cellular structures of graphene/silicon carbide (SiC) showing very low densities (<1.6 g cm−3) as compared to the dense skeleton (<3.2 g cm−3) have been fabricated by a filament-based printing technique known as Robocasting. These scaffolds have been assembled from pseudoplastic inks containing homogeneous mixtures of SiC ceramic powders and up to 20 vol.% of graphene nanoplatelets (GNPs), and densified by pressureless spark plasma sintering. The electrical conductivity (σ) of the scaffolds shows certain anisotropy with the structure orientation and increases with the GNPs volume fraction. In this way, σ values of up to 611 S m−1 for the longitudinal and 273 S m−1 for the transverse orientations of the structures relative to the extruded rods are achieved. First attempts to model the electrical behavior of robocast scaffolds using both analytical and finite-element methods are described. The models provide a correct description of general trends in the conductivity and anisotropy, and are expected to be useful as a first approach to anticipate the trends of other properties, as the thermal conductivity, of such complex multifunctional cellular materials, thus narrowing the experimental workload. Scaffolds tested under compression show crushing strengths in the range of 10–50 MPa, which increase with the relative density of the skeletons.

Enhanced osteogenic differentiation and biomineralization in mouse mesenchymal stromal cells on a β-TCP robocast scaffold modified with collagen nanofibers

B-TCP robocast scaffolds modified with collagen nanofibers promote osteoblastic differentiation and biomineralization of mMSCs.

Poly-(lactic acid) infiltration of 45S5 Bioglass® robocast scaffolds: Chemical interaction and its deleterious effect in mechanical enhancement

Strengthening and toughening of robocast 45S5 Bioglass®-derived scaffolds sintered at different temperatures (550–1000°C) is attempted by PLA infiltration following two different approaches: immersion of sintered scaffolds in molten PLA, to obtain fully-impregnated structures, or in a solution of PLA to produce polymeric coatings. PLA-coated scaffolds showed significantly higher strength and toughness than bare scaffolds both in compression and, especially, in bending. However, molten PLA degraded chemically in the presence of the alkaline ions in 45S5 Bioglass®, an interaction never before reported in the literature, which resulted in color change and cracking of PLA, and which prevented fully-impregnated structures from exhibiting a significant mechanical enhancement over simply coated structures.

Influence of sintering temperature on the mechanical properties of ϵ-PCL-impregnated 45S5 bioglass-derived scaffolds fabricated by robocasting

The effect of sintering temperature on the mechanical performance of 45S5 Bioglass® scaffolds fabricated by robocasting and infiltrated by ϵ-polycaprolactone (ϵ-PCL) has been investigated in this work. Infiltration was made by immersion of the robocast scaffolds in a polymer melt to produce fully impregnated, dense co-continuous composites, or in a polymeric solution to produce a polymer-coated bioceramic structure. Polymer infiltration produced a significant increase in the strength and toughness of the scaffolds, both under compression and bending. The strengthening and toughening provided by the polymer was larger in fully impregnated structures than in coated scaffolds, and the toughening was especially dramatic (up to more than two orders of magnitude) under flexural stresses. The strengthening and toughening produced by polymer infiltration increased monotonically with the level of microporosity in the ceramic struts, i.e., they decreased with the sintering temperature. In spite of that, denser ceramic struts yield stronger and tougher hybrid structures after polymer infiltration.

Improving mechanical properties of 13–93 bioactive glass robocast scaffold by poly (lactic acid) and poly (ε-caprolactone) melt infiltration

The effect of polymer infiltration on the mechanical properties of 13–93 bioactive glass scaffolds fabricated by robocasting has been investigated. Biodegradable polymers—poly (lactic acid) (PLA) and poly (ε-caprolactone) (PCL)—were infiltrated into selected robocast scaffolds by immersion of the structure in a polymer melt. The mechanical enhancement achieved upon impregnation on robocast 13-93 structures with fully-dense struts was found to be lower than in the currently existing literature due to the absence of microporosity. The modulus increase is negligible and the strengthening achieved is limited (<40% in the most favorable case of PLA impregnation under bending). However, the toughening produced by the polymer infiltrate, while lower than previously reported values in microporous structures, is still large (over one order of magnitude); especially when a ductile polymer infiltrate such as PCL is used. Indeed, the 13-93/PCL co-continuous composites were able to survive large strains with significant load-bearing capacity, even under bending stresses, or a rough, manual milling. On the other hand, the dense 13–93 frames fabricated by robocasting acted as good mechanical reinforcements for both polymers, with the exception of PLA under bending stresses.

Effect of Polymer Infiltration on the Flexural Behavior of β-Tricalcium Phosphate Robocast Scaffolds

The influence of polymer infiltration on the flexural strength and toughness of β-tricalcium phosphate (β-TCP) scaffolds fabricated by robocasting (direct-write assembly) is analyzed. Porous structures consisting of a tetragonal three-dimensional lattice of interpenetrating rods were impregnated with biodegradable polymers (poly(lactic acid) (PLA) and poly(ε-caprolactone) (PCL)) by immersion of the structure in a polymer melt. Infiltration increased the flexural strength of these model scaffolds by a factor of 5 (PCL) or 22 (PLA), an enhancement considerably greater than that reported for compression strength of analogue materials. The greater strength improvement in bending was attributed to a more effective transfer of stress to the polymer under this solicitation since the degree of strengthening associated to the sealing of precursor flaws in the ceramic rod surfaces should remain unaltered. Impregnation with either polymer also improved further than in compression the fracture energy of the scaffolds (by more than two orders of magnitude). This increase is associated to the extraordinary strengthening provided by impregnation and to a crack bridging toughening mechanism produced by polymer fibrils.