Freeze-cast Scaffolds Research Articles

Control over the mechanical properties of surface-magnetized alumina magnetically freeze-cast scaffolds

Control over the mechanical properties of surface-magnetized alumina magnetically freeze-cast scaffolds

Complex architectural control of ice-templated collagen scaffolds using a predictive model

The architectural and physiomechanical properties of regenerative scaffolds have been shown to improve engineered tissue function at both a cellular and tissue level. The fabrication of regenerative three-dimensional scaffolds that precisely replicate the complex hierarchical structure of native tissue, however, remains a challenge. The aim of this work is therefore two-fold: i) demonstrate an innovative multidirectional freeze-casting system to afford precise architectural control of ice-templated collagen scaffolds; and ii) present a predictive simulation as an experimental design tool for bespoke scaffold architecture. We used embedded heat sources within the freeze-casting mold to manipulate the local thermal environment during solidification of ice-templated collagen scaffolds. The resultant scaffolds comprised complex and spatially varied lamellar orientations that correlated with the imposed thermal environment and could be readily controlled by varying the geometry and power of the heat sources. The complex macro-architecture did not interrupt the hierarchical features characteristic of ice-templated scaffolds, but pore orientation had a significant impact on the stiffness of resultant structures under compression. Furthermore, our finite element model (FEM) accurately predicted the thermal environment and illustrated the freezing front topography within the mold during solidification. The lamellar orientation of freeze-cast scaffolds was also predicted using thermal gradient vector direction immediately prior to phase change. In combination our FEM and bespoke freeze-casting system present an exciting opportunity for tailored architectural design of ice-templated regenerative scaffolds that mimic the complex hierarchical environment of the native extracellular matrix. Statement of significanceBiomimetic scaffold structure improves engineered tissue function, but the fabrication of three-dimensional scaffolds that precisely replicate the complex hierarchical structure of native tissue remains a challenge. Here, we leverage the robust relationship between thermal gradients and lamellar orientation of ice-templated collagen scaffolds to develop a multidirectional freeze-casting system with precise control of the thermal environment and consequently the complex lamellar structure of resultant scaffolds. Demonstrating the diversity of our approach, we identify heat source geometry and power as control parameters for complex lamellar orientations. We simultaneously present a finite element model (FEM) that describes the three-dimensional thermal environment during solidification and accurately predicts lamellar structure of resultant scaffolds. The model serves as a design tool for bespoke regenerative scaffolds.

Structural, mechanical, and in vitro characterization of freeze-cast scaffolds prepared using a sol-gel-derived bioactive glass from the SiO2–CaO–Na2O–P2O5–K2O–MgO system

This work deals with the preparation of freeze-cast scaffolds using a bioactive glass from the SiO2–CaO–Na2O–P2O5–K2O–MgO system. This material could be sintered at lower temperatures (650 °C) than other variations of bioactive glasses, which is an important advantage in terms of energy and cost savings. This behavior represents a great advantage in terms of energy and cost savings. The freeze-casting step was conducted using water as a solvent and liquid nitrogen as a coolant. The prepared samples were examined according to their pore structure, thermal behavior, mechanical stability, and bioactivity. The glass transition temperature (Tg), crystallization onset temperature (Tx), and maximum crystallization temperature (Tc) evaluated for this bioactive glass were about 660 °C, 690 °C, and 705 °C. Consequently, the freeze-cast scaffolds could be sintered at 650 °C for 2–8 h, which favored viscous flow sintering without crystallization. Bioactivity assays were conducted by soaking the scaffolds in simulated body fluid for up to 21 days, showing that these materials present a bioactive behavior, inducing hydroxyapatite formation. These materials' mechanical properties and biocompatibility make them promising candidates for use in trabecular bone repair.

Poly(d,l-lactide)-Grafted Bioactive Glass Nanoparticles: From Nanobricks to Freeze-Cast Scaffolds for Bone Substitution

This paper focuses on an integrative “bricks-and-mortar” approach involving bioactive glass nanoparticles (bricks) covalently functionalized with a customized polymer (mortar) combined with the freeze-casting process. With the aim of obtaining a macroporous composite for bone substitution, composed of a spatially homogeneous assembly of nanoscale objects, we establish a method for the systematic elaboration of nanocomposite scaffolds. It was implemented through several steps from the synthesis of functionalized poly(d,l-lactide) (PDLLA) and SiO2–CaO binary bioactive glass nanoparticles (diameter around 164 nm) to the unidirectional freeze-casting process. The different stages include the first description of controlled PDLLA (Mn 8400 g·mol–1) grafting onto bioactive glass nanoparticle surfaces, their fine characterization and grafting quantification, and their mixing with free PDLLA chains (83 400 g·mol–1) during suspension formulation. This paper emphasizes the effect of the working temperature during the freeze-casting process on the multiscale spatial organization of resulting scaffolds such as the porosity morphology (lamellar and tubular), size (from 30 to 380 μm), anisotropy, and orientation. In addition to porosity, our results demonstrate a rosary-like organization of PDLLA-grafted nanoparticles in pore walls. The higher homogeneity in the spatial distribution of grafted nanoparticles over the height of scaffolds and at a micron scale confirms the validity of the “bricks-and-mortar” concept to prevent or limit aggregation. In particular, this study highlights the correlation between nanoparticle functionalization and mechanical properties, especially the recovery rate after compression tests. These results lay the foundation for the development of tunable materials for bone substitution, via potential enhancement of bioactivity and cell colonization.

Yttrium Oxide Freeze-Casts: Target Materials for Radioactive Ion Beams.

Highly porous yttrium oxide is fabricated as ion beam target material in order to produce radioactive ion beams via the Isotope Separation On Line (ISOL) method. Freeze casting allows the formation of an aligned pore structure in these target materials to improve the isotope release. Aqueous suspensions containing a solid loading of 10, 15, and 20 vol% were solidified with a unidirectional freeze-casting setup. The pore size and pore structure of the yttrium oxide freeze-casts are highly affected by the amount of solid loading. The porosity ranges from 72 to 84% and the crosslinking between the aligned channels increases with increasing solid loading. Thermal aging of the final target materials shows that an operation temperature of 1400 °C for 96 h has no significant effect on the microstructure. Thermo-mechanical calculation results, based on a FLUKA simulation, are compared to measured compressive strength and forecast the mechanical integrity of the target materials during operation. Even though they were developed for the particular purpose of the production of short-lived radioactive isotopes, the yttria freeze-cast scaffolds can serve multiple other purposes, such as catalyst support frameworks or high-temperature fume filters.

Bamboo-inspired tubular scaffolds with functional gradients

Bamboo achieves its mechanical efficiency in bending and compression, meaning mechanical performance per unit mass, due to its hierarchical structure. As an orthotropic tube with a higher strength and stiffness parallel to the tube axis and with a density and property gradient across the tube wall, in which fiber bundles are embedded in a porous matrix, the bamboo culm is both stiffer and stronger in bending and less prone to ovalization and catastrophic failure than an orthotropic tube without property gradients would be. Few engineered materials exist that emulate bamboo's mechanical efficiency. The results of the study presented here demonstrate that freeze casting (ice templating) is a manufacturing process with which bamboo-inspired tubular scaffolds with property gradients across the tube wall can be custom-made. A highly aligned, honeycomb-like porosity is generated by ice crystal growth opposite to the direction of heat flow. Using a core-shell mold, the microstructure of the tube wall material, such as the pore size, geometry, and alignment, is defined by the mold materials' properties and applied cooling conditions. These also allow to custom-design the desired property gradient across the section. Further customization of the tube gradient structure and properties is possible through the deposition of additional layers on the freeze-cast scaffolds. Characterizing the pore structures of the tubes using X-ray microtomography, pore morphology and property gradients can be analyzed and correlated to both the processing conditions and the resulting mechanical properties determined in three-point bending, longitudinal and radial compression. The resulting fundamental structure-property-processing correlations support the custom design of tubular scaffolds that are ideally suited for applications that range from conduits for peripheral nerve repair to ureteral stents.

Freeze-FRESH: A 3D Printing Technique to Produce Biomaterial Scaffolds with Hierarchical Porosity.

Tissues are organized in hierarchical structures comprised of nanoscale, microscale, and macroscale features. Incorporating hierarchical structures into biomaterial scaffolds may enable better resemblance of native tissue structures and improve cell interaction, but it is challenging to produce such scaffolds using a single conventional scaffold production technique. We developed the Freeze-FRESH (FF) technique that combines FRESH 3D printing (3DP) and freeze-casting to produce 3D printed scaffolds with microscale pores in the struts. FF scaffolds were produced by extrusion 3DP using a support bath at room temperature, followed by freezing and lyophilization, then the FF scaffolds were recovered from the bath and crosslinked. The FF scaffolds had a hierarchical pore structure from the combination of microscale pores throughout the scaffold struts and macroscale pores in the printed design, while control scaffolds had only macroscale pores. FF scaffolds frozen at −20 °C and −80 °C had similar pore sizes, due to freezing in the support bath. The −20 °C and −80 °C FF scaffolds had porous struts with 63.55% ± 2.59% and 56.72% ± 13.17% strut porosity, respectively, while control scaffolds had a strut porosity of 3.15% ± 2.20%. The −20 °C and −80 °C FF scaffolds were softer than control scaffolds: they had pore wall stiffness of 0.17 ± 0.06 MPa and 0.23 ± 0.05 MPa, respectively, compared to 1.31 ± 0.39 MPa for the control. The FF scaffolds had increased resilience in bending compared with control. FF scaffolds supported MDA-MB-231 cell growth and had significantly greater cell numbers than control scaffolds. Cells formed clusters on the porous struts of FF scaffolds and had similar morphologies as the freeze cast scaffolds. The FF technique successfully introduced microscale porosity into the 3DP scaffold struts to produce hierarchical pore structures that enhanced MDA-MB-231 growth.

Freeze-cast composite scaffolds prepared from sol-gel derived 58S bioactive glass and polycaprolactone

This work deals with the preparation of freeze-cast scaffolds using sol-gel derived 58S bioactive glass and a hypoeutectic naphthalene-camphor mixture as the starting powder and freezing vehicle, respectively. After the freeze-casting step, samples were air sintered at 1250 °C for 2 h, which led to the crystallization of 58S. The obtained scaffolds were subsequently infiltrated with poly(ε-caprolactone) (PCL), a biodegradable polymer with potential application for bone tissue repair. The prepared materials were examined by helium pycnometry, laser granulometry, scanning electron microscopy (SEM), Archimedes tests, X-ray microtomography (micro-CT), Fourier transform infrared spectroscopy (FTIR), N2 adsorption, X-ray diffraction (XRD), and uniaxial compression tests. Samples cytotoxicity was evaluated by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction (MTT) and LIVE/DEAD assays. Their biocompatibility was also examined after soaking in a simulated body fluid (SBF) solution at 37 °C for up to 14 days. It was observed that the infiltration of PCL into the 58S scaffolds greatly increased their mechanical stability. Moreover, it was shown that these composites displayed a high cell viability (above 70%), which reveals that they did not interfere in the production of osteoblast cells. A hydroxyapatite coating was observed on the samples surface upon soaking in SBF, reinforcing that they are biocompatible materials. As far as we know, this is the first time that freeze-cast scaffolds were obtained using sol-gel derived 58S particles and a naphthalene-camphor mixture. Besides, as the infiltration of PCL into freeze-cast bioactive glass scaffolds improved their mechanical stability without impairing their bioactivity, this is a promising approach to prepare samples for load-bearing applications in bone tissue engineering.

Effects of pore orientation on in-vitro properties of retinoic acid-loaded PLGA/gelatin scaffolds for artificial peripheral nerve application

Different manufacturing processes of scaffolds affect their main properties by altering the microstructure of pores. In this study, poly (lactic-co-glycolic acid) (PLGA) scaffolds were synthesized by both freeze casting and freeze drying methods. Scanning electron microscopy (SEM) micrographs demonstrated a unidirectional microstructure in the freeze-cast and a number of random pores in the freeze-dried scaffolds. According to the results, the formation of a lamellar microstructure increased the mechanical strength, swelling ratio, biodegradation behavior and drug release level. Addition of gelatin to the PLGA freeze-cast constructs led to an increase in the average diameter of pores, hydrophilicity and cellular spreading. The gelatin-containing scaffolds showed a decreased mechanical strength, but at the same time, an enhanced swelling ratio, biodegradation rate and drug release level. Differentiation of P19 cells and expression of β-tubulin III, Pax-6 and Nestin were improved by incorporating retinoic acid in PLGA-Gelatin freeze-cast scaffolds. This is a good option with initial necessary features for regenerating peripheral nervous system (PNS).

Bioinspired composites from freeze casting with clathrate hydrates

Freeze casting with isopropanol (IPA)-H2O as a freezing agent has shown the potential to create porous scaffolds with enlarged pores. Though not experimentally proven, this effect has been suggested to be the result of non-stoichiometric structures called clathrate hydrates forming during the freezing process. In this manuscript, we build upon these results to provide experimental evidence of the formation of clathrate hydrates during the freeze casting process when using IPA-H2O as a freezing agent and explain the observed maximum in pore area through observations of the enthalpy of the transitions. Additionally, these enlarged pores are harnessed in order to create two-phase bioinspired composites. These compos- ites exhibit an increase in mechanical strength over both constituents, a phenomena that is common to complex biological composites. Previous reports have shown that the characteristic method of failure for many freeze cast scaffolds is buckling of the lamellar walls within the porous microstructure. It is proposed that this increase in mechanical strength is due to the support of the second phase, which resists this characteristic buckling failure mode.

Morphological Comparison of PLGA/Gelatin Scaffolds Produced by Freeze Casting and Freeze Drying Methods

In this research, PLGA/Gelatin scaffolds were prepared by both freeze drying and freeze casting methods and their physical, mechanical and morphological observations were evaluated and compared to each other. The pore size and percent of porosity was measure for scaffolds fabricated by both freeze drying and freeze casting techniques. These values were more than 200μm and 95%, respectively. The results of scanning electron microscope (SEM) showed that freeze-cast scaffolds had aligned structures in comparison with freeze drying scaffolds whereas these kinds of microstructure were not seen for samples produced by freeze drying method. The compressive strength for freeze-cast scaffolds (3.2 MPa) was higher than freeze drying samples (2.1 MPa). Although adsorption percentage of freeze cast scaffolds was 650%, this parameter was 450% for freeze drying samples. Based on the obtained results, it seems that the freeze-cast scaffolds are able to support cell attachment, to maintain the required structural integrity and to prevent the pores of the scaffolds from collapsing during neo-tissue formation.

Directionally solidified biopolymer scaffolds: Mechanical properties and endothelial cell responses

Vascularization is a primary challenge in tissue engineering. To achieve it in a tissue scaffold, an environment with the appropriate structural, mechanical, and biochemical cues must be provided enabling endothelial cells to direct blood vessel growth. While biochemical stimuli such as growth factors can be added through the scaffold material, the culture medium, or both, a well-designed tissue engineering scaffold is required to provide the necessary local structural and mechanical cues. As chitosan is a well-known carrier for biochemical stimuli, the focus of this study was on structure-property correlations, to evaluate the effects of composition and processing conditions on the three-dimensional architecture and properties of freeze-cast scaffolds; to establish whether freeze-cast scaffolds are promising candidates as constructs promoting vascularization; and to conduct initial tissue culture studies with endothelial cells on flat substrates of identical compositions as those of the scaffolds to test whether these are biocompatible and promote cell attachment and proliferation.