fabrication-mechanically-tunable-bioactive-metal-scaffolds-for

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Seoul National University

Knowledge of the structure-property-function relationships of dermal scales of armoured fish could enable pathways to improved bioinspired human body armour, and may provide clues to the evolutionary origins of mineralized tissues. Here, we present a multiscale experimental and computational approach that reveals the materials design principles present within individual ganoid scales from the 'living fossil' Polypterus senegalus. This fish belongs to the ancient family Polypteridae, which first appeared 96 million years ago during the Cretaceous period and still retains many of their characteristics. The mechanistic origins of penetration resistance (approximating a biting attack) were investigated and found to include the juxtaposition of multiple distinct reinforcing composite layers that each undergo their own unique deformation mechanisms, a unique spatial functional form of mechanical properties with regions of differing levels of gradation within and between material layers, and layers with an undetectable gradation, load-dependent effective material properties, circumferential surface cracking, orthogonal microcracking in laminated sublayers and geometrically corrugated junctions between layers.

Materials design principles of ancient fish armour.

In this study, a quantitative investigation of the microstructure and composition of field-caught marine Gasterosteus aculeatus (threespine stickleback) armor is presented, which provides useful phylogenetic information and insights into biomechanical function. Micro-computed tomography (microCT) was employed to create full three-dimensional images of the dorsal spines and basal plate, lateral plates, pelvic girdle and spines and to assess structural and compositional properties such as the spatial distribution of thickness (approximately 100-300 microm), the heterogeneous cross-sectional geometry (centrally thickened), plate-to-plate juncture and overlap (approximately 50% of the plate width), and bone mineral density (634-748 HA/cm(3)). The convolution of plate geometry in conjunction with plate-to-plate overlap allows a relatively constant armor thickness to be maintained throughout the assembly, promoting spatially homogeneous protection and thereby avoiding weakness at the armor unit interconnections. Plate-to-plate junctures act to register and join the plates while permitting compliance in sliding and rotation in selected directions. Mercury porosimetry was used to determine the pore size distribution and volume percent porosity of the lateral plates (20-35 vol.%) and spines (10-15 vol.%). SEM and microCT revealed a porous, sandwich-like cross-section beneficial for bending stiffness and strength at minimum weight. Back-scattered electron microscopy and energy dispersive X-ray analysis were utilized to quantify the weight percent mineral content (58-68%). Scanning electron microscopy and surface profilometry were used to characterize the interior and exterior surface topography (tubercles) of the lateral plates. The results obtained in this study are discussed in the context of mechanical function, performance, fitness, and survivability.

Quantitative microstructural studies of the armor of the marine threespine stickleback (Gasterosteus aculeatus).

It has been hypothesized that predatory threats are a critical factor in the protective functional design of biological exoskeletons or "natural armor", having arisen through evolutionary processes. Here, the mechanical interaction between the ganoid armor of the predatory fish Polypterus senegalus and one of its current most aggressive threats, a toothed biting attack by a member of its own species (conspecific), is simulated and studied. Finite element analysis models of the quad-layered mineralized scale and representative teeth are constructed and virtual penetrating biting events simulated. Parametric studies reveal the effects of tooth geometry, microstructure and mechanical properties on its ability to effectively penetrate into the scale or to be defeated by the scale, in particular the deformation of the tooth versus that of the scale during a biting attack. Simultaneously, the role of the microstructure of the scale in defeating threats as well as providing avenues of energy dissipation to withstand biting attacks is identified. Microstructural length scale and material property length scale matching between the threat and armor is observed. Based on these results, a summary of advantageous and disadvantageous design strategies for the offensive threat and defensive protection is formulated. Studies of predator-prey threat-protection interactions may lead to insights into adaptive phenotypic plasticity of the tooth and scale microstructure and geometry, "adaptive stalemates" and the so-called evolutionary "arms race".

Threat-protection mechanics of an armored fish.

A common feature of the outer layer of protective biological exoskeletons is structural anisotropy. Here, we directly quantify the mechanical anisotropy and fracture of an individual material layer of a hydroxyapatite-based nanocomposite exoskeleton, the outmost ganoine of Polypterus senegalus scale. Uniaxial compression was conducted on cylindrical micropillars of ganoine fabricated via focused ion beam at different orientations relative to the hydroxyapatite rod long axis (θ = 0°, 45°, 90°). Engineering stress versus strain curves revealed significant elastic and plastic anisotropy, off-axial strain hardening, and noncatastrophic crack propagation within ganoine. Off-axial compression (θ = 45°) showed the lowest elastic modulus, E (36.2 ± 1.6 GPa, n ≥ 10, mean ± SEM), and yield stress, σ(Y) (0.81 ± 0.02 GPa), while compression at θ = 0° showed the highest E (51.8 ± 1.7 GPa) and σ(Y) (1.08 ± 0.05 GPa). A 3D elastic-plastic composite nanostructural finite element model revealed this anisotropy was correlated to the alignment of the HAP rods and could facilitate energy dissipation and damage localization, thus preventing catastrophic failure upon penetration attacks.

Direct quantification of the mechanical anisotropy and fracture of an individual exoskeleton layer via uniaxial compression of micropillars.

Caffeine is white crystalline xanthine alkaloid that is naturally found in some plants and can be produced synthetically. It has various biological effects, especially during pregnancy and lactation. We studied the effect of caffeine on heartbeat, survival and the expression of cell damage related genes, including oxidative stress (HSP70), mitochondrial metabolism (Cyclin G1) and apoptosis (Bax and Bcl2), at early developmental stages of zebrafish embryos. We used 100 µm concentration based on the absence of locomotor effects. Neither significant mortality nor morphological changes were detected. We monitored hatching at 48 h post-fertilization (hpf) to 96 hpf. At 60 and 72 hpf, hatching decreased significantly (P < 0.05); however, the overall hatching rate at 96 hpf was 94% in control and 93% in caffeine treatment with no significant difference (P > 0.05). Heartbeats per minute were 110, 110 and 112 in control at 48, 72 and 96 hpf, respectively. Caffeine significantly increased heartbeat - 122 and 136 at 72 and 96 hpf, respectively. Quantitative RT-PCR showed significant up-regulation after caffeine exposure in HSP70 at 72 hpf; in Cyclin G1 at 24, 48 and 72 hpf; and in Bax at 48 and 72 hpf. Significant down-regulation was found in Bcl2 at 48 and 72 hpf. The Bax/Bcl2 ratio increased significantly at 48 and 72 hpf. We conclude that increasing exposure time to caffeine stimulates oxidative stress and may trigger apoptosis via a mitochondrial-dependent pathway. Also caffeine increases heartbeat from early phases of development without affecting the morphology and survival but delays hatching. Use of caffeine during pregnancy and lactation may harm the fetus by affecting the expression of cell-damage related genes.

Exposure time to caffeine affects heartbeat and cell damage-related gene expression of zebrafish Danio rerio embryos at early developmental stages.

Prostate cancer is the most frequently diagnosed cancer in Western men, and more men have been diagnosed at younger ages in recent years. A high-fat Western-style diet is a known risk factor for prostate cancer and increases oxidative stress.

High animal fat intake enhances prostate cancer progression and reduces glutathione peroxidase 3 expression in early stages of TRAMP mice.

This study reports the first case of Capillaria hepatica infection in a nutria in Korea. Ten nutrias, captured near the Nakdong River, were submitted to our laboratory for necropsy. White-yellowish nodules were found in the liver of 1 of the nutrias at necropsy. Histologically, the lesions were granulomatous, and infiltrations of lipid-laden macrophages, eosinophils, and several multinucleated giant cells were observed. The lesions consisted of numerous eggs and necrotic hepatocytes. The eggs were lemon-shaped and had polar plugs at the ends of both long sides. The eggs were morphologically identified as those of C. hepatica. Worldwide, C. hepatica infection in nutrias is very rare. Nutrias are a kind of livestock, as well as wildlife; therefore, an epidemiological study for parasitic infections needs to be conducted.

The first case of Capillaria hepatica infection in a nutria (Myocastor coypus) in Korea.

Metals have been used as biostructural materials because of outstanding mechanical reliability. However, low bioactivity and high stiffness in biological environments have been major issues of metals, causing stress shielding effects or foreign body reactions after implantation. Therefore, in this study, densified porous titanium has been introduced to achieve comparable mechanical properties to hard tissues and bioactivity that promote a better interface between the implant and bone. Porous titanium scaffolds were successfully fabricated through dynamic freezing casting, and were densified, controlling the degree of densification by applied strain. During densification, structural integrity of porous titanium was well maintained without any mechanical deterioration, exhibiting good pore connectivity and large surface area. Densified porous titanium possesses two important features that have not been achieved by either dense titanium or porous titanium: 1) mechanical tunability of porous scaffolds through densification that allows scaffolds to be applied ranging from highly porous fillers to dense load-bearing implants and 2) improved bioactivity through bioactive coating that is capable of sustainable release through utilizing high surface area and pore connectivity with controllable tortuosity. This simple, but effective post-fabrication process of porous scaffolds has great potential to resolve unmet needs of biometals for biomedical applications.

Novel strategy for mechanically tunable and bioactive metal implants.

Many armored fish scale assemblies use geometric heterogeneity of subunits as a design parameter to provide tailored biomechanical flexibility while maintaining protection from external penetrative threats. This study analyzes the spatially varying shape of individual ganoid scales as a structural element in a biological system, the exoskeleton of the armored fish Polypterus senegalus (bichir). X-ray microcomputed tomography is used to generate digital 3D reconstructions of the mineralized scales. Landmark-based geometric morphometrics is used to measure the geometric variation among scales and to define a set of geometric parameters to describe shape variation. A formalism using continuum mechanical strain analysis is developed to quantify the spatial geometry change of the scales and illustrate the mechanisms of shape morphing between scales. Five scale geometry variants are defined (average, anterior, tail, ventral, and pectoral fin) and their functional implications are discussed in terms of the interscale mobility mechanisms that enable flexibility within the exoskeleton. The results suggest that shape variation in materials design, inspired by structural biological materials, can allow for tunable behavior in flexible composites made of segmented scale assemblies to achieve enhanced user mobility, custom fit, and flexibility around joints for a variety of protective applications.

Article	Year
Materials design principles of ancient fish armour. Nature materials\| PubMed ID: 18660814	2008
Quantitative microstructural studies of the armor of the marine threespine stickleback (Gasterosteus aculeatus). Journal of structural biology\| PubMed ID: 20433929	2010
Threat-protection mechanics of an armored fish. Journal of the mechanical behavior of biomedical materials\| PubMed ID: 21565718	2011
Direct quantification of the mechanical anisotropy and fracture of an individual exoskeleton layer via uniaxial compression of micropillars. Nano letters\| PubMed ID: 21755939	2011
Exposure time to caffeine affects heartbeat and cell damage-related gene expression of zebrafish Danio rerio embryos at early developmental stages. Journal of applied toxicology : JAT\| PubMed ID: 22886764	2013
High animal fat intake enhances prostate cancer progression and reduces glutathione peroxidase 3 expression in early stages of TRAMP mice. The Prostate\| PubMed ID: 25053105	2014
The first case of Capillaria hepatica infection in a nutria (Myocastor coypus) in Korea. The Korean journal of parasitology\| PubMed ID: 25352702	2014
Novel strategy for mechanically tunable and bioactive metal implants. Biomaterials\| PubMed ID: 25453937	2015
Morphometric structural diversity of a natural armor assembly investigated by 2D continuum strain analysis. Journal of structural biology\| PubMed ID: 26481418	2015

Juha Song

Biography

JoVE Journal Publications

Other Publications