Biomaterials (v.26, #30)

Calendar (I).

The mechanisms involved in the inhibition of protein adsorption by polyethylene oxide (PEO) are not completely understood, but it is believed that PEO chain length, chain density and chain conformation all play a role. In this work, surfaces formed by chemisorption of PEO-thiol to gold were investigated: the effects of PEO chain density, chain length (600, 750, 2000 and 5000 MW) and end-group (–OH, –OCH3) on protein adsorption from plasma are reported. Similar to previous single protein adsorption studies (L.D. Unsworth et al., Langmuir 2005;21:1036–41) it was found that, of the different surfaces investigated, PEO layers formed from solutions near the cloud point adsorbed the lowest amount of fibrinogen from plasma. Layers of hydroxyl-terminated PEO of MW 600 formed under these low solubility conditions showed almost complete suppression (versus controls) of the Vroman effect, with 20±1 ng/cm2 adsorbed fibrinogen at the Vroman peak and 6.7±0.6 ng/cm2 at higher plasma concentration. By comparison, Vroman peak adsorption was 70±20 and 50±3 ng/cm2, respectively, for 750-OCH3 and 2000-OCH3 layers formed under low solubility conditions; adsorption on these surfaces at higher plasma concentration was 16±9 and 12±3 ng/cm2. Thus in addition to the effect of solution conditions noted previously, the results of this study also suggest a chain end group effect which inhibits fibrinogen adsorption to, and/or facilitates displacement from, hydroxyl terminated PEO layers. Fibrinogen adsorption from plasma was not significantly different for surfaces prepared with PEO of molecular weight 750 and 2000 when the chain density was the same (∼0.5 chains/nm2) supporting the conclusion that chain density may be the key property for suppression of protein adsorption. The proteins eluted from the surfaces after contact with plasma were investigated by SDS-PAGE and immunoblotting. A number of proteins were detected on the various surfaces including fibrinogen, albumin, C3 and apolipoprotein A-I. The blot responses were zero or weak for all four proteins of the contact system; some complement activation was observed on all of the surfaces studied.
Keywords: Surface modification; Fibrinogen; Plasma protein; Protein adsorption; Protein resistant surface; Vroman effect; Western blots; Poly(ethylene oxide);

In this study, modifications of glass ionomer cements (GICs) were made by adding bioactive glass (BAG) to GIC to obtain bioactive restorative materials. This study used SEM, EDS and visual analysis to examine the bioactivity and the ability of the study materials to mineralize dentin. Conventional cure and resin-modified light-curing GIC were used. The materials consisted of powder and liquid. Three experimental materials were made by mixing 10–30 wt% of BAG powder with GIC powders. Commercially available GIC without BAG were used as controls. Class III restorations were made in altogether 62 intact beagle dog teeth, and the operation was performed under general anesthesia. The restorations were followed clinically for 1, 3 or 6 weeks. Resin-modified GIC containing BAG showed uniform CaP surface formation on the restorations. Mineral depositions in the close vicinity of the restoration–dentin interface and in deeper parts of dentin tubules were also noticed in resin-modified GIC containing BAG particles. It can be concluded that resin-modified GIC containing BAG have good potential in clinical applications where enhanced mineralization is expected.
Keywords: Bioactive glass; Glass ionomer; SEM; In vivo test;

Monocyte viability on titanium and copper coated titanium by Felicia Suska; Christina Gretzer; Marco Esposito; Pentti Tengvall; Peter Thomsen (5942-5950).
The role of apoptosis/cell death in the inflammatory response at the implanted materials is unexplored. Two surfaces with different cytotoxic potential and in vivo outcomes, titanium (Ti) and copper (Cu) were incubated in vitro with human monocytes and studied using a method to discriminate apoptotic and necrotic cells (Annexin V/PI staining). Further, staurosporine, a potent inducer of apoptosis, was added to the surface adherent monocytes. Lactate dehydrogenase (a marker of cell membrane injury) and TNF-α and IL-10, cytokines, previously suggested to play a major role in the monocyte apoptosis, were assayed in the culture medium.The results demonstrated that Ti surfaces displayed enhanced monocyte survival and production of IL-10 and TNF-α. Cu adherent cells exhibited apoptotic signs as early as 1 h after incubation. In contrast to Ti, after 48 h the predominance of apoptotic cells switched to apoptotic/necrotic cells on Cu surfaces. Staurosporine treatment of Ti adherent cells mediated similar type of cell death. LDH and cytokine contents were low around Cu surfaces, partly explained by interference between Cu ions and LDH and cytokines.This study suggests that material properties rapidly influence the onset of human monocyte apoptosis and progression to late apoptosis/necrosis. Early detection of apoptosis and cell death may be important for the understanding of the biological response to implanted materials.
Keywords: Titanium; Copper; Biocompatibility; Monocyte; Apoptosis; Cell viability; Cytokine;

The objective of this study was to characterize a series of anionic biodegradable polymer resins for their compatibility in a biological environment, comparing them with respect to the influence of ionic function on enzyme catalyzed biodegradation when the polymers were incorporated into a porous calcium polyphosphate (CPP) 3-D structure to form an interpenetrating phase composite (IPC). The swelling behavior of the polymers was investigated by immersing the cured polymer resins in growth media at 37 °C. In vitro cytotoxicity of the polymer resins was assessed using a HeLa cell line. Cell viability increased when the amount of low molecular weight monomer was minimized. Despite observing that the addition of carboxylic acid groups into the polymer resin chains contributed to an improvement of the chemical bonding between the polymer and the CPP, the addition of high ionic content into the resin led to the greatest loss of bending strength for the samples incubated in phosphate buffer and cholesterol esterase enzyme solutions, when compared to their as made state. The increased degradation for the higher ionic component materials and their loss of physical strength was attributed to enhanced hydrolysis within the materials and by water transport deep within the composites, via the anionic components of the resin. The findings indicated that the introduction of anionic content must be optimized to promote increased mechanical performance for the CPP, balancing the features of polymer CPP bonding versus polymer swelling and cytotoxicity.
Keywords: Enzymes; Biodegradation; Bending strength; Cytotoxicity; Interfacial shear strength; Interpenetrating phase composite;

Polysaccharide-protein surface modification of titanium via a layer-by-layer technique: Characterization and cell behaviour aspects by Kaiyong Cai; Annett Rechtenbach; Jianyuan Hao; Jörg Bossert; Klaus D. Jandt (5960-5971).
To improve the surface biocompatibility of titanium films, a layer-by-layer (LBL) self-assembly technique, based on the polyelectrolyte-mediated electrostatic adsorption of chitosan (Chi) and gelatin (Gel), was used leading to the formation of multilayers on the titanium thin film surfaces. The film growth was initialized by deposition of one layer of positively charged poly(ethylene imine) (PEI). Then the thin film was formed by the alternate deposition of negatively charged Gel and positively charged Chi utilizing electrostatic interactions. The LBL film growth was monitored by several techniques. The chemical composition, surface topography as well as wettability were investigated by using X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), confocal laser scanning microscopy (CLSM) and water contact angle measurement, respectively. Quantitative XPS analysis showed the alternative change of C/N ratio after four sequential cycles coating of Ti/PEI/Gel/Chi/Gel, which indicated the discrete layer structure of coatings. Uncoated titanium (control sample) displayed a smooth surface morphology (root mean square (RMS) roughness was around 2.5 nm). A full coverage of coating with Gel/Chi layers was achieved on the titanium surface only after the deposition layers of PEI/(Gel/Chi)2. The PEI/Gel/(Chi/Gel)3 layer displayed a rough surface morphology with a tree-like structure (RMS roughness is around 82 nm). These results showed that titanium films could be modified with Chi/Gel which may affect the biocompatibility of the modified titanium films. To confirm this hypothesis, cell proliferation and cell viability of osteoblasts on LBL-modified titanium films as well as control samples were investigated in vitro. The proliferation of osteoblasts on modified titanium films was found to be greater than that on control ( p < 0.05 ) after 1 and 7 days culture, respectively. Cell viability measurement showed that the Chi/Gel-modified films have higher cell viability ( p < 0.05 ) than the control. These data suggest that Chi/Gel were successfully employed to surface engineer titanium via LBL technique, and enhanced its cell biocompatibility. The approach presented here may be exploited for fabrication of titanium-based implant surfaces.
Keywords: Titanium; Layer-by-layer composite; Chitosan; Gelatin; In vitro assay; Surface engineering; AFM; XPS; Contact angle; Laser scanning confocal microscopy; Interfaces; Surfaces;

Protein binding properties of surface-modified porous polyethylene membranes by George Greene; Harish Radhakrishna; Rina Tannenbaum (5972-5982).
In this study, we quantified the adsorption of immunoglobulin G (IgG) protein onto several polyelectrolyte-modified sintered porous polyethylene (PPE) membranes. The polymer surfaces had both cationic and anionic charges obtained via the adsorption of polyethylenimine (PEI) and polyacrylic acid (PAA), respectively, onto plasma-activated PPE. The amount of IgG adsorption was determined by measuring the gamma radiation emitted by [125I]-IgG radio labeled protein. By studying the impact of pH and ionic strength on IgG adsorption, we attempted to characterize the role and nature of the electrostatic interactions involved in the adsorption process to better understand how these interactions were influenced by the charge and structure of immobilized polyelectrolyte complexes at modified membrane surfaces. We were able to show that surface modification of PPE membranes with adsorbed PEI monolayers and PEI–PAA bilayers can greatly improve the IgG binding ability of the membrane under optimized conditions. We also showed that the observed improvement in the IgG binding is derived from electrostatic interactions between IgG and the polyelectrolyte surface. In addition, we found that the greatest IgG adsorption occurred when the IgG and the surface possessed predominantly opposite charges, rather than when the surface possessed the greatest electrostatic charge. Finally, we have found that the molecular weight of the terminating polyelectrolyte has a noticeable effect upon the electrostatic interactions between IgG and the PEI–PAA bilayer-modified PPE surfaces.
Keywords: Protein adsorption; Specific interactions; Polyelectrolytes; Immunoglobulin;

Chitosan: A versatile biopolymer for orthopaedic tissue-engineering by Alberto Di Martino; Michael Sittinger; Makarand V. Risbud (5983-5990).
Current tissue engineering strategies are focused on the restoration of pathologically altered tissue architecture by transplantation of cells in combination with supportive scaffolds and biomolecules. In recent years, considerable attention has been given to chitosan (CS)-based materials and their applications in the field of orthopedic tissue engineering. Interesting characteristics that render chitosan suitable for this purpose are a minimal foreign body reaction, an intrinsic antibacterial nature, and the ability to be molded in various geometries and forms such as porous structures, suitable for cell ingrowth and osteoconduction. Due to its favorable gelling properties chitosan can deliver morphogenic factors and pharmaceutical agents in a controlled fashion. Its cationic nature allows it to complex DNA molecules making it an ideal candidate for gene delivery strategies. The ability to manipulate and reconstitute tissue structure and function using this material has tremendous clinical implications and is likely to play a key role in cell and gene therapies in coming years. In this paper we will review the current applications and future directions of CS in articular cartilage, intervertebral disk and bone tissue engineering.
Keywords: Chitosan; Tissue engineering; Orthopaedics; Cartilage; Intervertebral disc; Bone;

The effect of incorporating RGD adhesive peptide in polyethylene glycol diacrylate hydrogel on osteogenesis of bone marrow stromal cells by Fan Yang; Christopher G. Williams; Dong-an Wang; Hyukjin Lee; Paul N. Manson; Jennifer Elisseeff (5991-5998).
Advances in tissue engineering require biofunctional scaffolds that can not only provide cells with structural support, but also interact with cells in a biological manner. To achieve this goal, a frequently used cell adhesion peptide Arg–Gly–Asp (RGD) was covalently incorporated into poly(ethylene glycol) diacrylate (PEODA) hydrogel and its dosage effect (0.025, 1.25 and 2.5 mm) on osteogenesis of marrow stromal cells in a three-dimensional environment was examined. Expression of bone-related markers, osteocalcin (OCN) and Alkaline phosphatase (ALP), increased significantly as the RGD concentration increased. Compared with no RGD, 2.5 mm RGD group showed a 1344% increase in ALP production and a 277% increase in OCN accumulation in the medium. RGD helped MSCs maintain cbfa-1 expression when shifted from a two-dimensional environment to a three-dimensional environment. Soluble RGD was found to completely block the mineralization of marrow stromal cells, as manifested by quantitative calcium assay, phosphorus elemental analysis and Von Kossa staining. In conclusion, we have demonstrated that RGD-conjugated PEODA hydrogel promotes the osteogenesis of MSCs in a dosage-dependent manner, with 2.5 mm being optimal concentration.
Keywords: RGD; Marrow stromal cells; Osteogenesis; Photopolymerizing; Hydrogel;

Electrospun protein fibers as matrices for tissue engineering by Mengyan Li; Mark J. Mondrinos; Milind R. Gandhi; Frank K. Ko; Anthony S. Weiss; Peter I. Lelkes (5999-6008).
Keywords: Electrospinning; Nanofibers; Gelatin; Collagen; Elastin; Tropoelastin;

Mechanical properties of gamma irradiated morselized bone during compaction by Adam M. Butler; David A.F. Morgan; Richard Verheul; William Robert Walsh (6009-6013).
This study examined the effects of gamma irradiation on the compressive properties of morselized cancellous bone from human femoral heads. Twelve bone samples, mean age of 68 years (range 92–39), were divided into 3 groups ( N = 12 ) of varying irradiation level (0, 15 and 25 kGy). Each specimen was compacted in a controlled fashion in steps of 0.5 mm at 0.5 mm/min (up 12 mm). The load and stiffness increased with compaction, but this relationship was not linear. There was no statistical significant difference in the compacting load or stiffness between groups ( p > 0.05 ) until the last 1 mm of compaction, where the 25 kGy group were significantly stiffer compared to controls but not different to the 15 kGy group. This may be due to decreased interlocking of bone particles caused by higher irradiation levels resulting in a stiffer graft.
Keywords: Gamma irradiation; Bone graft; Mechanical properties; Compression;

Mechanical properties and osteoconductivity of porous bioactive titanium by Mitsuru Takemoto; Shunshuke Fujibayashi; Mashashi Neo; Jun Suzuki; Tadashi Kokubo; Takashi Nakamura (6014-6023).
Porous bioactive titanium implants (porosity of 40%) were produced by a plasma-spray method and subsequent chemical and thermal treatments of immersion in a 5 M aqueous NaOH solution at 60 °C for 24 h, immersion in distilled water at 40 °C for 48 h, and heating to 600 °C for 1 h. Compression strength and bending strength were 280 MPa (0.2% offset yield strength 85.2 MPa) and 101 MPa, respectively. For in vivo analysis, bioactive and nontreated porous titanium cylinders were implanted into 6 mm diameter holes in rabbit femoral condyles. The percentage of bone–implant contact (affinity index) of the bioactive implants (BGs) was significantly larger than for the nontreated implants (CGs) at all postimplantation times (13.5 versus 10.5, 16.7 versus 12.7, 17.7 versus 10.2, 19.1 versus 7.8 at 2, 4, 8 and 16 weeks, respectively). The percentage of bone area ingrowth showed a significant increase with the BGs, whereas with the CGs it appeared to decrease after 4 weeks (10.7 versus 9.9, 12.3 versus 13.1, 15.2 versus 9.8, 20.6 versus 8.7 at 2, 4, 8 and 16 weeks, respectively). These results suggest that porous bioactive titanium has sufficient mechanical properties and biocompatibility for clinical use under load-bearing conditions.
Keywords: Osteoconduction; Porous titanium; Surface treatment; Mechanical properties; Osteointegration;

Extent of iron pick-up in deforoxamine-coupled polyurethane materials for therapy of chronic wounds by Jennifer E. Taylor; Peter R. Laity; John Hicks; Steven S. Wong; Keith Norris; Peck Khunkamchoo; Anthony F. Johnson; Ruth E. Cameron (6024-6033).
Polyurethane net substrates (PNS) coupled with deferoxamine (DFO) have been studied to determine the extent of Fe2+ pick-up for use in chronic wound therapy. A m solution of ferrous sulphate (FeSO4) was used to generate ferrous ions similar to those found in chronic wounds. The concentration of Fe as a function of position through the dressings was evaluated using a variety of techniques. Atomic force microscopy (AFM) and energy-filtered transmission electron microscopy (EFTEM) revealed a rough precipitated layer at the surface of activated PNS exposed to FeSO4 solution. Optical microscopy (OM) and backscattered environmental scanning electron microscopy (ESEM) showed a clear layer of Fe3+-enriched material in the surface regions exposed to DFO. The penetration depth of DFO into activated dressings was found to be 20–30 μm. Energy-dispersive X-ray (EDX) analysis was used to approximate the distribution of bound- and unbound-Fe as a function of position within BPNS and DFO-activated dressings after immersing them in a FeSO4 solution for various times. These studies have shown the activity of iron with respect to ionic state in DFO-activated PNS for potential using as dressing for chronic wounds.
Keywords: Wound dressing; Inflammation; Polyurethane; Bioactivity;

Polyethylene wear particle generation in vivo in an alumina medial pivot total knee prosthesis by Yukihide Minoda; Akio Kobayashi; Hiroyoshi Iwaki; Masatsugu Miyaguchi; Yoshinori Kadoya; Hirotsugu Ohashi; Kunio Takaoka (6034-6040).
Polyethylene wear particle generation is one of the most important factors affecting mid- to long-term results of total knee arthroplasties. It has been reported that the medial pivot total knee prosthesis (MP) design and alumina ceramic femoral component reduce polyethylene wear. The aim of this study is to evaluate in vivo polyethylene wear particle generation in the newly introduced alumina MP, in comparison with a metal MP. Synovial fluid was obtained from 11 knees with alumina MP and 15 knees with metal MP at nine months after the operation. Polyethylene particles were isolated, and examined using scanning electron microscope and image analyzer. Total number of particles in each knee was 7.10±2.86×106 in alumina (mean±standard error), and 5.70±2.82×107 in metal MP ( p = 0.048 ). Particle size (equivalent circle diameter) was 0.78±0.04 μm in alumina, and 0.66±0.06 μm in metal MP ( p = 0.120 ). Particle shape (aspect ratio) was 1.52±0.05 in alumina, and 1.88±0.11 in metal MP ( p = 0.014 ). Apart from the femoral component, the material and manufacturing method of polyethylene insert differed between the two groups, although the sterilization method was the same. Alumina MP generated fewer and rounder polyethylene wear particles than metal MP in early clinical stage, and could potentially reduce prevalence of osteolysis and aseptic loosening.
Keywords: In vivo test; Polyethylene; Wear debris; Alumina; Knee replacement;

Chitosan (CS)/β-lactoglobulin (βlg) core–shell nanoparticles (CS-βlg nanoparticle) were successfully prepared with the aim of developing a biocompatible carrier for the oral administration of nutraceuticals. The effects of pH and initial concentrations (C β lg) of native and denatured βlg on the properties of the nanoparticles were investigated. Uniform nanoparticles were prepared by ionic gelation with sodium tripolyphosphate (TPP). The surface charge of the particles was positive, with a zeta potential of 20–60 mV. βlg loading efficiency (LE) spanned a broad range (1–60%); and was highly sensitive to formulation pH. This adsorption can be mainly attributed to electrostatic, hydrophobic interactions and hydrogen bonding between βlg and CS. Brilliant blue (BB) release experiments showed that the nanoparticles prepared with native βlg had favorable properties to resist acid and pepsin degradation in simulated gastric conditions unlike those prepared with denatured βlg or denatured βlg crosslinked with Ca2+. When transferred to simulated intestinal conditions, the βlg shells of the nanoparticles were degraded by pancreatin.
Keywords: Chitosan; β-lactoglobulin; Nanoparticle; In vitro test;

Injectable glycosaminoglycan hydrogels for controlled release of human basic fibroblast growth factor by Shenshen Cai; Yanchun Liu; Xiao Zheng Shu; Glenn D. Prestwich (6054-6067).
Synthetic hydrogel mimics of the extracellular matrix (ECM) were created by crosslinking a thiol-modified analog of heparin with thiol-modified hyaluronan (HA) or chondroitin sulfate (CS) with poly(ethylene glycol) diacrylate (PEGDA). The covalently bound heparin provided a crosslinkable analog of a heparan sulfate proteoglycan, thus providing a multivalent biomaterial capable of controlled release of basic fibroblast growth factor (bFGF). Hydrogels contained >97% water and formed rapidly in <10 min. With as little as 1% (w/w) covalently bound heparin (relative to total glycosaminoglycan content), the rate of release of bFGF in vitro was substantially reduced. Total bFGF released increased with lower percentages of heparin; essentially quantitative release of bFGF was observed from heparin-free hydrogels. Moreover, the hydrogel-released bFGF retained 55% of its biological activity for up to 28 days as determined by a cell proliferation assay. Finally, when these hydrogels were implanted into subcutaneous pockets in Balb/c mice, neovascularization increased dramatically with HA and CS hydrogels that contained both bFGF and crosslinked heparin. In contrast, hydrogels lacking bFGF or crosslinked heparin showed little increase in neovascularization. Thus, covalently linked, heparin-containing glycosaminoglycan hydrogels that can be injected and crosslinked in situ constitute highly promising new materials for controlled release of heparin-binding growth factors in vivo.
Keywords: Heparin; Hyaluronan; Chondroitin sulfate; Wound healing; Neovascularization; Synthetic extracellular matrix;

This research developed a novel bioadhesive drug delivery system, poly(d,l-lactide-co-glycolide)/montmorillonite (PLGA/MMT) nanoparticles, for oral delivery of paclitaxel. Paclitaxel-loaded PLGA/MMT nanoparticles were prepared by the emulsion/solvent evaporation method. MMT was incorporated in the formulation as a matrix material component, which also plays the role of a co-emulsifier in the nanoparticle preparation process. Paclitaxel-loaded PLGA/MMT nanoparticles were found to be of spherical shape with a mean size of around 310 nm and polydispersity of less than 0.150. Adding MMT component to the matrix material appears to have little influence on the particles size and the drug encapsulation efficiency. The drug release pattern was found biphasic with an initial burst followed by a slow, sustained release, which was not remarkably affected by the MMT component. Cellular uptake of the fluorescent coumarin 6-loaded PLGA/MMT nanoparticles showed that MMT enhanced the cellular uptake efficiency of the pure PLGA nanoparticles by 57–177% for Caco-2 cells and 11–55% for HT-29 cells, which was dependent on the amount of MMT and the particle concentration in incubation. Such a novel formulation is expected to possess extended residence time in the gastrointestinal (GI) tract, which promotes oral delivery of paclitaxel.
Keywords: Anticancer drugs; Biodegradable polymers; Chemotherapy; Controlled release; Drug formulation; Medical clay;

A combinatorial approach to the selective capture of circulating malignant epithelial cells by peptide ligands by Saurabh Aggarwal; Samuel Janssen; Randy M. Wadkins; James L. Harden; Samuel R. Denmeade (6077-6086).
Early detection is critical in the administration of definitive and curative therapy of cancer. However, current detection methods are ineffective at identifying the presence of circulating metastatic cancer cells in the blood because they typically sample only a relatively small volume of blood. One strategy for sampling larger blood volumes would be to capture circulating cells in vivo over an extended period of time. The development of such a method would be substantially facilitated by the identification of peptide ligands that bind selectively to metastatic cancer cells in the blood with high affinity. To identify such ligands a combinatorial peptide library was synthesized on polyethylene acrylamide (PEGA) resin and screened for binding to malignant epithelial cells. Using Biacore, cell binding assays were performed to demonstrate that peptides selected from PEGA bead screen can bind selectively to malignant epithelial cancer cells and not to circulating leukocytes under physiologic shear stress conditions. One peptide, with the sequence QMARIPKRLARH, was used to demonstrate selective labeling of malignant epithelial cells spiked in whole blood. When immobilized on appropriate surfaces, these peptides could be used in both in vivo and ex vivo cell separation devices to efficiently and selectively capture metastatic epithelial cancer cells from flowing blood.
Keywords: Peptide; Biosensor; Cell adhesion; Epithelial cell; Catheter;

This study investigates the fracture properties of nacre using a discrete lattice model based on continuous damage random threshold fuse network. The discrete lattice topology of the model is based on nacre's unique brick and mortar microarchitecture. The mechanical behavior of each of the bonds in the discrete lattice model is governed by the characteristic modular damage evolution of the organic matrix and the mineral bridges between the aragonite platelets. The numerical results obtained using this simple discrete lattice model are in very good agreement with the previously obtained experimental results, such as nacre's stiffness, tensile strength, and work of fracture. The analysis indicates that nacre's superior toughness is a direct consequence of ductility (maximum shear strain) of the organic matrix in terms of repeated unfolding of protein molecules, and its fracture strength is a result of its ordered brick and mortar architecture with significant overlap of the platelets, and shear strength of the organic matrix.
Keywords: Nacre; Fracture toughness; Biomimetic material; Fracture mechanism; Nanocomposite; Random fuse model;