Strategies for neurotrophin-3 and chondroitinase ABC release from freeze-cast chitosan-alginate nerve-guidance scaffolds.

Freeze casting, or controlled unidirectional solidification, can be used to fabricate chitosan-alginate (C-A) scaffolds with highly aligned porosity that are suitable for use as nerve-guidance channels. To augment the guidance of growth across a spinal cord injury lesion, these scaffolds are now evaluated in vitro to assess their ability to release neurotrophin-3 (NT-3) and chondroitinase ABC (chABC) in a controlled manner. Protein-loaded microcapsules were incorporated into C-A scaffolds prior to freeze casting without affecting the original scaffold architecture. In vitro protein release was not significantly different when comparing protein loaded directly into the scaffolds with release from scaffolds containing incorporated microcapsules. NT-3 was released from the C-A scaffolds for 8 weeks in vitro, while chABC was released for up to 7 weeks. Low total percentages of protein released from the scaffolds over this time period were attributed to limitation of diffusion by the interpenetrating polymer network matrix of the scaffold walls. NT-3 and chABC released from the scaffolds retained bioactivity, as determined by a neurite outgrowth assay, and the promotion of neurite growth across an inhibitory barrier of chondroitin sulphate proteoglycans. This demonstrates the potential of these multifunctional scaffolds for enhancing axonal regeneration through growth-inhibiting glial scars via the sustained release of chABC and NT-3. Copyright © 2014 John Wiley & Sons, Ltd.

Nacre-like hybrid films: Structure, properties, and the effect of relative humidity.

Functional materials often are hybrids composed of biopolymers and mineral constituents. The arrangement and interactions of the constituents frequently lead to hierarchical structures with exceptional mechanical properties and multifunctionality. In this study, hybrid thin films with a nacre-like brick-and-mortar microstructure were fabricated in a straightforward and reproducible manner through manual shear casting using the biopolymer chitosan as the matrix material (mortar) and alumina platelets as the reinforcing particles (bricks). The ratio of inorganic to organic content was varied from 0% to 15% and the relative humidities from 36% to 75% to determine their effects on the mechanical properties. It was found that increasing the volume fraction of alumina from 0% to 15% results in a twofold increase in the modulus of the film, but decreases the tensile strength by up to 30%, when the volume fraction of alumina is higher than 5%. Additionally, this study quantifies and illustrates the critical role of the relative humidity on the mechanical properties of the hybrid film. Increasing the relative humidity from 36% to 75% decreases the modulus and strength by about 45% and triples the strain at failure. These results suggest that complex hybrid materials can be manufactured and tailor made for specific applications or environmental conditions.

Combined multi-nozzle deposition and freeze casting process to superimpose two porous networks for hierarchical three-dimensional microenvironment.

An engineered three-dimensional scaffold with hierarchical porosity and multiple niche microenvironments is produced using a combined multi-nozzle deposition-freeze casting technique. In this paper we present a process to fabricate a scaffold with improved interconnectivity and hierarchical porosity. The scaffold is produced using a two-stage manufacturing process which superimposes a printed porous alginate (Alg) network and a directionally frozen ceramic-polymer matrix. The combination of two processes, multi-nozzle deposition and freeze casting, provides engineering control of the microenvironment of the scaffolds over several length scales; including the addition of lateral porosity and the ratio of polymer to ceramic microstructures. The printed polymer scaffold is submerged in a ceramic-polymer slurry and subsequently, both structures are directionally frozen (freeze cast), superimposing and patterning both microenvironments into a single hierarchical architecture. An optional additional sintering step removes the organic material and densifies the ceramic phase to produce a well-defined network of open pores and a homogenous cell wall material composition. The techniques presented in this contribution address processing challenges, such as structure definition, reproducibility and fine adjustments of unique length scales, which one typically encounters when fabricating topological channels between longitudinal and transverse porous networks.

Platelets self-assemble into porous nacre during freeze casting.

Nacre possesses a remarkable combination of mechanical properties. Its high stiffness, strength and toughness are attributed to a highly aligned structure of aragonite platelets "glued" together by a small fraction (∼5vol%) of polymer; theoretically it can be described by a shear-lag model of staggered tensile elements between which loads are transferred via shear. Despite extensive research, it has not been possible yet to manufacture this aligned structure as a bulk material of considerable volume with a fast and easy production process. Particularly porous materials would benefit from enhanced wall material properties to compensate for performance loss due to their high porosity. An important application for such porous materials are tissue scaffolds for bone substitution. Bone, like nacre, exhibits excellent mechanical properties, particularly an exceptionally high toughness, because of its composite structure of hydroxyapatite platelets aligned in a ∼35vol% polymer matrix. Through the freeze casting process, which results in a fast and straightforward self-assembly of platelet-shaped particles during directional solidification, highly porous bulk materials with nacre-like cell walls can now be created. This porous nacre outperforms by a factor of 1.5-4 in terms of stiffness, strength and toughness materials that have the same amount of porosity but do not exhibit the nacre-like microarchitecture. The self-assembly process presented in this study thus has tremendous potential for the creation of highly porous, yet mechanically strong tissue scaffolds for low or medium load bearing bone substitute materials. Due to the versatility of the freeze casting process, materials with a self-assembled cell wall structure can be created from high-aspect ratio particles of all material classes. This enables material optimization for a great variety of applications such as impact protection, filtration, catalysis, energy generation and storage, in addition to those with excellent mechanical properties at high porosity.

Structure-property-processing correlations in freeze-cast composite scaffolds.

Surprisingly few reports have been published, to date, on the structure-property-processing correlations observed in freeze-cast materials directionally solidified from polymer solutions, or ceramic or metal slurries. The studies that exist focus on properties of sintered ceramics, that is materials whose structure was altered by further processing. In this contribution, we report first results on correlations observed in alumina-chitosan-gelatin composites, which were chosen as a model system to test and compare the effect of particle size and processing parameters on their mechanical properties at a specific composition. Our study reveals that highly porous (>90%) hybrid materials can be manufactured by freeze casting, through the self-assembly of a polymer and a ceramic phase that occurs during directional solidification, without the need of additional processing steps such as sintering or infiltration. It further illustrates that the properties of freeze-cast hybrid materials can independently be tailored at two levels of their structural hierarchy, allowing for the simultaneous optimization of both mechanical and structural requirements. An increase in freezing rate resulted in decreases in lamellar spacing, cell wall thickness, pore aspect ratio and cross-sectional area, as well as increases in both Young's modulus and compressive yield strength. The mechanical properties of the composite scaffolds increased with an increasing particle size. The results show that both structure and mechanical properties of the freeze-cast composites can be custom-designed and that they are thus ideally suited for a large variety of applications that require high porosity at low or medium load-bearing capacity.

An ice-templated, linearly aligned chitosan-alginate scaffold for neural tissue engineering.

Several strategies have been investigated to enhance axonal regeneration after spinal cord injury, however, the resulting growth can be random and disorganized. Bioengineered scaffolds provide a physical substrate for guidance of regenerating axons towards their targets, and can be produced by freeze casting. This technique involves the controlled directional solidification of an aqueous solution or suspension, resulting in a linearly aligned porous structure caused by ice templating. In this study, freeze casting was used to fabricate porous chitosan-alginate (C/A) scaffolds with longitudinally oriented channels. Chick dorsal root ganglia explants adhered to and extended neurites through the scaffold in parallel alignment with the channel direction. Surface adsorption of a polycation and laminin promoted significantly longer neurite growth than the uncoated scaffold (poly-L-ornithine + Laminin = 793.2 ± 187.2 µm; poly-L-lysine + Laminin = 768.7 ± 241.2 µm; uncoated scaffold = 22.52 ± 50.14 µm) (P < 0.001). The elastic modulus of the hydrated scaffold was determined to be 5.08 ± 0.61 kPa, comparable to reported spinal cord values. The present data suggested that this C/A scaffold is a promising candidate for use as a nerve guidance scaffold, because of its ability to support neuronal attachment and the linearly aligned growth of DRG neurites.

Biomaterials by freeze casting.

The functional requirements for synthetic tissue substitutes appear deceptively simple: they should provide a porous matrix with interconnecting porosity and surface properties that promote rapid tissue ingrowth; at the same time, they should possess sufficient stiffness, strength and toughness to prevent crushing under physiological loads until full integration and healing are reached. Despite extensive efforts and first encouraging results, current biomaterials for tissue regeneration tend to suffer common limitations: insufficient tissue-material interaction and an inherent lack of strength and toughness associated with porosity. The challenge persists to synthesize materials that mimic both structure and mechanical performance of the natural tissue and permit strong tissue-implant interfaces to be formed. In the case of bone substitute materials, for example, the goal is to engineer high-performance composites with effective properties that, similar to natural mineralized tissue, exceed by orders of magnitude the properties of its constituents. It is still difficult with current technology to emulate in synthetic biomaterials multi-level hierarchical composite structures that are thought to be the origin of the observed mechanical property amplification in biological materials. Freeze casting permits to manufacture such complex, hybrid materials through excellent control of structural and mechanical properties. As a processing technique for the manufacture of biomaterials, freeze casting therefore has great promise.