Cardiovascular Stent Materials Research Articles

Albumin Protein Impact on Early-Stage In Vitro Biodegradation of Magnesium Alloy (WE43).

Mg and its alloys are promising biodegradable materials for orthopedic implants and cardiovascular stents. The first interactions of protein molecules with Mg alloy surfaces have a substantial impact on their biocompatibility and biodegradation. We investigate the early-stage electrochemical, chemical, morphological, and electrical surface potential changes of alloy WE43 in either 154 mM NaCl or Hanks' simulated physiological solutions in the absence or presence of bovine serum albumin (BSA) protein. WE43 had the lowest electrochemical current noise (ECN) fluctuations, the highest noise resistance (Zn = 1774 Ω·cm2), and the highest total impedance (|Z| = 332 Ω·cm2) when immersed for 30 min in Hanks' solution. The highest ECN, lowest Zn (1430 Ω·cm2), and |Z| (49 Ω·cm2) were observed in the NaCl solution. In the solutions containing BSA, a unique dual-mode biodegradation was observed. Adding BSA to a NaCl solution increased |Z| from 49 to 97 Ω·cm2 and decreased the ECN signal of the alloy, i.e., the BSA inhibited corrosion. On the other hand, the presence of BSA in Hanks' solution increased the rate of biodegradation by decreasing both Zn and |Z| while increasing ECN. Finally, using scanning Kelvin probe force microscopy (SKPFM), we observed an adsorbed nanolayer of BSA with aggregated and fibrillar morphology only in Hanks' solution, where the electrical surface potential was 52 mV lower than that of the Mg oxide layer.

Effect of Mn addition on the microstructure, mechanical, and corrosion properties of an extruded biodegradable Zn-0.2Mg alloy

In this study, the effect of Mn addition on microstructural evolution, mechanical properties, and biocorrosion behaviors of as-extruded Zn-0.2 Mg alloy as candidates for degradable biomaterials were investigated. The microstructural observations indicated that the addition of Mn element significantly refined α-Zn grains, and the average size decreased from 17.23 μm to 7.14 μm. Moreover, Mn-rich intermetallic precipitated alongside eutectic Mg2Zn11 phases. A result of electron backscattered diffraction indicated that manganese has caused grains to rotate 19°, facilitating the activation of non-basal slip as the Schmid factor increases. Mechanical test results showed that the yield strength (YS), ultimate tensile strength (UTS), and elongation (El) of Zn-0.2Mg-0.5Mn alloy is improved by 35.92%, 31.51%, and 6.65%, respectively, compared with Zn-0.2 Mg alloy. The main mechanisms responsible for the improvement of mechanical properties were higher volume fraction of the particles, grain refinement, texture modification, and deformation twining formed by Mn addition. The electrochemical test in SBF solution at human body temperature revealed that the corrosion rate of Mn-containing alloy increased from 0.664 mm/year to 0.87 mm/year compared to the base alloy. It is evident from these results that the Zn-0.2Mg-0.5Mn alloy has a great deal of potential as a cardiovascular stent material.

Blending with transition metals improves bioresorbable zinc as better medical implants.

Zinc (Zn) is a new class of bioresorbable metal that has potential for cardiovascular stent material, orthopedic implants, wound closure devices, etc. However, pure Zn is not ideal for these applications due to its low mechanical strength and localized degradation behavior. Alloying is the most common/effective way to overcome this limitation. Still, the choice of alloying element is crucial to ensure the resulting alloy possesses sufficient mechanical strength, suitable degradation rate, and acceptable biocompatibility. Hereby, we proposed to blend selective transition metals (i.e., vanadium-V, chromium-Cr, and zirconium-Zr) to improve Zn's properties. These selected transition metals have similar properties to Zn and thus are beneficial for the metallurgy process and mechanical property. Furthermore, the biosafety of these elements is of less concern as they all have been used as regulatory approved medical implants or a component of an implant such as Ti6Al4V, CoCr, or Zr-based dental implants. Our study showed the first evidence that blending with transition metals V, Cr, or Zr can improve Zn's properties as bioresorbable medical implants. In addition, three in vivo implantation models were explored in rats: subcutaneous, aorta, and femoral implantations, to target the potential clinical applications of bioresorbable Zn implants.

Influence of bovine serum albumin on corrosion behaviour of pure Zn in phosphate buffered saline

Zinc (Zn) and its alloys have received increasing attention as new alternative biodegradable metals. However, consensus has not been reached on the corrosion behaviour of Zn. As cardiovascular artery stent material, Zn is supposed to contact with plasma that contains inorganic salts and organic components. Protein is one of the most important constitute in the plasma and could adsorb on the material surface. In this paper, bovine serum albumin (BSA) was used as a typical protein. Influences of BSA on pure Zn corrosion in phosphate buffered saline is investigated as a function of BSA concentrations and immersion durations by electrochemical techniques and surface analysis. Results showed that pure Zn corrosion was progressively accelerated with BSA concentrations (ranging from 0.05 to 5 g L−1) at 0.5 h. With time evolves, formation of phosphates as corrosion product was delayed by BSA adsorption, especially at concentration of 2 g L−1. Within 48 h, the corrosion of pure Zn was alleviated by BSA at concentration of 0.1 g L−1, whereas the corrosion was enhanced after 168 h. Addition of 2 g L−1 BSA has opposite influence on the pure Zn corrosion. Furthermore, schematic corrosion behaviour at protein/Zn interfaces was proposed. This work encourages us to think more about the influence of protein on the material corrosion and helps us to better understand the corrosion behaviour of pure Zn.

Designing HA/PEI nanoparticle composite coating on biodegradable Mg–Zn–Y-Nd alloy to direct cardiovascular cells fate

Biodegradable magnesium (Mg) alloy as a new generation of cardiovascular stent material has attracted great attention. However, the problems of excessive degradability and lack of corresponding biological functions of Mg alloys still need to be solved urgently. In the present study, series of hyaluronic acid (HA)/polyethyleneimine (PEI) nanoparticles (NP) with optimized parameters were designed and fixed on the fluorination/poly-dopamine (PDA) treated Mg–Zn–Y-Nd alloy to prepare a multi-functional composite coating. Our data indicated that the HA/PEI NPs composite coatings greatly improved the corrosion resistance and biological functions of the Mg–Zn–Y-Nd alloy. We hope this technology contribute to better feasibility of the new generation of biodegradable Mg alloy stents.

Biocompatibility analysis of PLA based candidate materials for cardiovascular stents in a rat subcutaneous implant model

Modification of Polylactic acid (PLA), a biopolymer, is a strategy still to be fully explored for the next generation of bioresorbable vascular stent (BVS) biomaterials. With this focus, inclusions upto 5% of Polycaprolactone (PCL) and Magnesium in PLA were tested in the rat subcutaneous model and their cellular and tissue interactions characterized, specifically with respect to inflammatory response, angiogenesis and capsularization. The cytokines IL6, TNF Alpha and IL-1Beta were estimated in the peri-implant tissue, all of which showed a non-significant difference between the non-implanted animals and those containing PLA by 8 weeks, speaking to the benign nature of PLA as an implant biomaterial. Both modified materials, had increased macrophage counts and cytokine levels, except IL6 at 8 weeks. Vascularization only at 8 weeks in PLA PCL containing tissue was significantly higher than pure PLA, which may be more carefully controlled along with the material hydrophobicity for possible efforts towards therapeutic angiogenesis. Capsule thickness, measured by staining with both Hematoxylin & Eosin and Masson’s Trichome did not show any differences between materials, including PLA.

Conjugating heparin, Arg-Glu-Asp-Val peptide, and anti-CD34 to the silanic Mg-Zn-Y-Nd alloy for better endothelialization.

Magnesium alloy is generally accepted as a potential cardiovascular stent material due to its good mechanical properties, biocompatibility, and biodegradability, and has become one of the research hotspots in this field. However, too fast degradation rate and delayed surface endothelialization have been the bottleneck of further application of magnesium alloy stent. In this study, we selected Mg-Zn-Y-Nd, a kind of biodegradable magnesium alloy for cardiovascular stent, and passivated its surface by alkali heat treatment and silane treatment to improve the corrosion resistance, subsequently conjugated Arg-Glu-Asp-Val (REDV) peptide and anti-CD34 to promote endothelial cells adhesion and capture endothelial progenitor cells respectively, further improving surface endothelialization. In addition, the heparin was also immobilized to the Mg-Zn-Y-Nd surface for the consideration of anti-coagulation and anti-inflammation. Systematic material characterization and biological evaluation show that we have successfully developed this composite surface on Mg-Zn-Y-Nd alloy, and achieved multiple functions such as corrosion resistance, promoting endothelialization, and inhibiting platelet/macrophage adhesion.

Layer-by-layer deposition of bioactive layers on magnesium alloy stent materials to improve corrosion resistance and biocompatibility

Magnesium alloy is considered as one of the ideal cardiovascular stent materials owing to its good mechanical properties and biodegradability. However, the in vivo rapid degradation rate and the insufficient biocompatibility restrict its clinical applications. In this study, the magnesium alloy (AZ31B) was modified by combining the surface chemical treatment and in-situ self-assembly of 16-phosphonyl-hexadecanoic acid, followed by the immobilization of chitosan-functionalized graphene oxide (GOCS). Heparin (Hep) and GOCS were alternatively immobilized on the GOCS-modified surface through layer by layer (LBL) to construct the GOCS/Hep bioactive multilayer coating, and the corrosion resistance and biocompatibility were extensively explored. The results showed that the GOCS/Hep bioactive multilayer coating can endow magnesium alloys with an excellent in vitro corrosion resistance. The GOCS/Hep multilayer coating can significantly reduce the hemolysis rate and the platelet adhesion and activation, resulting in an excellent blood compatibility. In addition, the multilayer coating can not only enhance the adhesion and proliferation of the endothelial cells, but also promote the vascular endothelial growth factor (VEGF) and nitric oxide (NO) expression of the attached endothelial cells on the surfaces. Therefore, the method of the present study can be used to simultaneously control the corrosion resistance and improve the biocompatibility of the magnesium alloys, which is expected to promote the application of magnesium alloys in biomaterials or medical devices, especially cardiovascular stent.

Tailoring of cardiovascular stent material surface by immobilizing exosomes for better pro-endothelialization function

Stent intervention as available method in clinic has been widely applied for cardiovascular disease treatment for decades. However, the restenosis caused by late thrombosis and hyperplasia still limits the stents long-term application, and the essential cause is usually recognized as endothelial functionalization insufficiency of the stent material surface. Here, we address this limitation by developing a pro-endothelial-functionalization surface that immobilized a natural factors-loaded nanoparticle, exosome, onto the poly-dopamine (PDA) coated materials via electrostatic binding. This PDA/Exosome surface not only increased the endothelial cells number on the materials, but also improved their endothelial function, including platelet endothelial cell adhesion molecule-1 (CD31) expression, cell migration and nitric oxide release. The pro-inflammation macrophage (M1 phenotype) attachment and synthetic smooth muscle cell proliferation as the interference factors for the endothelialization were not only inhibited by the PDA/Exosome coating, while the cells were also regulated to anti-inflammation macrophage (M2 phenotype) and contractile smooth muscle cell, which may contribute to endothelialization. Thus, it can be summarized this method has potential application on surface modification of cardiovascular biomaterials.

Towards revealing key factors in mechanical instability of bioabsorbable Zn-based alloys for intended vascular stenting

Zn-based alloys are recognized as promising bioabsorbable materials for cardiovascular stents, due to their biocompatibility and favorable degradability as compared to Mg. However, both low strength and intrinsic mechanical instability arising from a strong strain rate sensitivity and strain softening behavior make development of Zn alloys challenging for stent applications. In this study, we developed binary Zn-4.0Ag and ternary Zn-4.0Ag-xMn (where x = 0.2–0.6wt%) alloys. An experimental methodology was designed by cold working followed by a thermal treatment on extruded alloys, through which the effects of the grain size and precipitates could be thoroughly investigated. Microstructural observations revealed a significant grain refinement during wire drawing, leading to an ultrafine-grained (UFG) structure with a size of 700 nm and 200 nm for the Zn-4.0Ag and Zn-4.0Ag-0.6Mn, respectively. Mn showed a powerful grain refining effect, as it promoted the dynamic recrystallization. Furthermore, cold working resulted in dynamic precipitation of AgZn3 particles, distributing throughout the Zn matrix. Such precipitates triggered mechanical degradation through an activation of Zn/AgZn3 boundary sliding, reducing the tensile strength by 74% and 57% for Zn-4.0Ag and Zn-4.0Ag-0.6Mn, respectively. The observed precipitation softening caused a strong strain rate sensitivity in cold drawn alloys. Short-time annealing significantly mitigated the mechanical instability by reducing the AgZn3 fraction. The ternary alloy wire showed superior microstructural stability relative to its Mn-free counterpart due to the pinning effect of Mn-rich particles on the grain boundaries. Eventually, a shift of the corrosion regime from localized to more uniform was observed after the heat treatment, mainly due to the dissolution of AgZn3 precipitates. Statement of SignificanceOwing to its promising biodegradability, zinc has been recognized as a potential biodegradable material for stenting applications. However, Zn's poor strength alongside intrinsic mechanical instability have propelled researchers to search for Zn alloys with improved mechanical properties. Although extensive researches have been conducted to satisfy the mentioned concerns, no Zn-based alloys with stabilized mechanical properties have yet been reported. In this work, the mechanical properties and stability of the Zn-Ag-based alloys were systematically evaluated as a function of microstructural features. We found that the microstructure design in Zn alloys can be used to find an effective strategy to not only improve the strength and suppress the mechanical instability but also to minimize any damage by augmenting the corrosion uniformity.

Layered hydroxide/polydopamine/hyaluronic acid functionalized magnesium alloys for enhanced anticorrosion, biocompatibility and antithrombogenicity in vascular stents.

Magnesium alloys are promising cardiovascular stent materials due to the favourable physical properties and complete biodegradability in vivo. However, the rapid degradation, poor cytocompatibility and tendency of thrombogenesis hinder practical clinical applications. In order to solve these problems, a facile and highly efficient strategy of alkali treatment combined with subsequent layer-by-layer assembly was used to fabricate a multifunctional coating. A bottom layer hydroxyl (–OH) with negative charge after alkali treatment first formed a solid bond with magnesium matrix to provide a rough outer surface for the further immobilization of functional biomolecules. Afterwards, polydopamine and hyaluronic acid were successively immobilized on alkali-treated magnesium surface via strong electrostatic adsorption and covalent bonding between carboxyl group of hyaluronic acid and amine or hydroxyl of polydopamine to form magnesium/OH/polydopamine/hyaluronic acid. Hydroxyl significantly improves the corrosion resistance while polydopamine and hyaluronic acid layers act as a further barrier to provide better anticorrosion. A balance between biocompatibility and antithrombogenicity has been achieved by adjusting the content of hyaluronic acid on polydopamine surface. The multifunctional magnesium/OH/polydopamine/hyaluronic acid coating with lower hyaluronic acid concentrations expose more active sites of polydopamine molecules to promote endothelial cell proliferation while retaining the intrinsic antithrombogenic function of hyaluronic acid to offer a potential application for vascular stents.

Evolution of metallic cardiovascular stent materials: A comparative study among stainless steel, magnesium and zinc

A cardiovascular stent is a small mesh tube that expands a narrowed or blocked coronary artery. Unfortunately, current stents, regardless metallic or polymeric, still largely fall short to the ideal clinical needs due to late restenosis, thrombosis and other clinical complications. Nonetheless, metallic stents are preferred clinically thanks to their superior mechanical property and radiopacity to their polymeric counterparts. The emergence of bioresorbable metals opens a window for better stent materials as they may have the potential to reduce or eliminate late restenosis and thrombosis. In fact, some bioresorbable magnesium (Mg)-based stents have obtained regulatory approval or under trials with mixed clinical outcomes. Some major issues with Mg include the too rapid degradation rate and late restenosis. To mitigate these problems, bioresorbable zinc (Zn)-based stent materials are being developed lately with the more suitable degradation rate and better biocompatibility. The past decades have witnessed the unprecedented evolution of metallic stent materials from first generation represented by stainless steel (SS), to second generation represented by Mg, and to third generation represented by Zn. To further elucidate their pros and cons as metallic stent materials, we systematically evaluated their performances in vitro and in vivo through direct side-by-side comparisons. Our results demonstrated that tailored Zn-based material with proper configurations could be a promising candidate for a better stent material in the future.

Towards Revealing Key Factors in Mechanical Instability of Bioabsorbable Zn-Based Alloys for Vascular Stenting

Zn-based alloys are recognized as promising bioabsorbable materials for cardiovascular stents, due to their biocompatibility and favorable degradability as compared to Mg. However, both low strength and intrinsic mechanical instability arisen from strong strain rate sensitivity and strain softening behavior make development of Zn alloys challenging for stent applications. In this study, we developed binary Zn-4.0Ag and ternary Zn-4.0Ag-xMn (where x= 0.2-0.6wt%) alloys. An experimental methodology was designed by cold working followed by a thermal treatment on extruded alloys, through which the effects of the grain size and precipitates could be thoroughly investigated. Microstructural observations revealed a significant grain refinement during wire drawing, leading to an ultrafine-grained (UFG) structure with a size of 670nm and 240nm for the Zn-4.0Ag and Zn-4.0Ag-0.6Mn, respectively. Mn showed a powerful grain refining effect as it promoted the dynamic recrystallization. Furthermore, cold working resulted in dynamic precipitation of AgZn3 particles, distributing throughout the Zn matrix. Such precipitates triggered mechanical degradation through an activation of Zn/AgZn3 boundary sliding, reducing the tensile strength by 74% and 57% for Zn-4.0Ag and Zn-4.0Ag-0.6Mn, respectively. The observed precipitation softening caused strong strain rate sensitivity in cold drawn alloys. Short-time annealing significantly mitigated the mechanical instability by reducing the AgZn3 fraction. The ternary alloy wire showed superior microstructural stability to its Mn-free counterpart due to the pinning effect of Mn particles on the grain boundaries. Eventually, a shift of the corrosion regime from localized to more uniform was observed after the heat treatment, mainly due to the dissolution of AgZn3 precipitates.

Effects of extrusion temperature on microstructure, mechanical properties and in vitro degradation behavior of biodegradable Zn-3Cu-0.5Fe alloy.

Zn-based alloys as biodegradable materials for cardiovascular stents have drawn more and more attention in recent years. However, the hot plastic deformation of Zn-based alloys does not get enough attention. In this study, Zn-3Cu-0.5Fe alloy was prepared and then hot extruded at 140, 180, 220 and 260 °C. Effects of extrusion temperature on the microstructure, mechanical properties and degradation behavior were investigated. Nano-scaled particles precipitated during extrusion and the quantity decreased with the increasing of extrusion temperature. As the extrusion temperature increased from 140 to 260 °C, the grain size increased from 1.15 to 4.38 μm, the yield strength and ultimate tensile strength increased from 203 ± 3.23 and 221 ± 1.56 MPa to 269 ± 2.65 and 302 ± 8.01 MPa, increased by 32.5% and 45%, while the degradation rate was decreased gradually from 62.8 ± 0.72 μm/year to 49.9 ± 3.35 μm/year, decreased by 20.5%. In addition, the degradation behavior changes from a relatively uniform degradation mode to a localized degradation mode with the increase of the grain size. Dislocation gliding is replaced by grain-boundary movement and dynamic recrystallization (DRX) in the room temperature tensile deformation process. Lower extrusion temperature is beneficial for higher elongation and degradation rate as well as relatively uniform degradation mode, which is better suitable for the clinical application of cardiovascular stents.

Micromechanical Fatigue Modelling of the Size Effect in Micro-Scale 316L Stainless Steel Specimens

For many years, computational modelling and simulation studies have been used by developers to advance device design and have been reported in regulatory medical device submissions. However, cardiovascular stent materials in such computational models are typically assumed to behave as a continuum. This approach assumes that bulk material properties apply to the micro-sized structure, i.e. material behavior is scale independent. However, as size is reduced, mechanical size effects arise as the grain size to specimen width ratio drops below a critical value. These size effects cause material behavior to deviate significantly from bulk material behavior. If such a deviation in material behavior is to be captured within computational models, it is necessary to represent the crystalline structure of a metal and to capture the anisotropic behavior of individual grains within these models. This paper describes the development of such a modelling methodology to investigate the phenomenon of strain localization within grains of a 316L stainless steel specimen under fatigue loading conditions.

In Vitro and in Vivo Studies on Two-Step Alkali-Fluoride-Treated Mg-Zn-Y-Nd Alloy for Vascular Stent Application: Enhancement in Corrosion Resistance and Biocompatibility.

Bioabsorbable magnesium alloys are becoming prominent materials for cardiovascular stents, as their desirable mechanical properties and favorable biosafety. However, the rapid corrosion of magnesium alloys under physiological conditions hinders their wider application as medical implant materials. Fluoride chemical conversion treatment is an effective and simple technique to improve the corrosion resistance for magnesium alloys. Despite previous literature reporting on fluoride chemical conversion treatment with hydrofluoric acid (HF) in different conditions, some defects are still present on the surface of the coating. In this study, we report on a two-step alkali-fluoride treatment of magnesium alloy by effectively removing the second phase in the substrate surface and form a dense and flawless magnesium fluoride (MgF2) coating to endow the magnesium alloy greater corrosion resistance. The results showed that the serious pitting corrosion caused by galvanic corrosion could be effectively prevented after removing of the second phase of the surface. In vivo tests in a rat subcutaneous implantation model showed that two-step alkali-fluoride-treated MgZnYNd alloy (MgZnYNd-A-F) uniformly corroded with a low corrosion rate. No subcutaneous gas cavities or significant inflammatory cell infiltration were observed for MgZnYNd-A-F in in vivo tests. The two-step alkali-fluoride treatment can significantly improve the corrosion resistance and biocompatibility of magnesium alloy, which has great potential in the application of vascular stents because of its simplicity and effectiveness.

Fretting-corrosion of cardiovascular stent materials: The role of electrochemical polarisation on debris generation mechanisms

Fretting-corrosion of cardiovascular stent materials: The role of electrochemical polarisation on debris generation mechanisms

Serum albumin can influence magnesium alloy degradation in simulated blood plasma for cardiovascular stenting

Magnesium (Mg) alloys are promising materials for biodegradable cardiovascular stents due to their good biocompatibility and low thrombogenicity. Since dissolved Mg is unlikely to cause any adverse effects, magnesium materials become safe choice in humans. Herein, we report on the in vitro degradable properties of a new bioresorbable implant for conceivable cardiovascular stent application, namely AZ80 magnesium alloy. The degradation behavior in simulated blood plasma (SBP) without and with 5_ 40 g/L bovine serum albumin (BSA) was assessed using open circuit potential, electrochemical impedance spectroscopy and potentiodynamic polarization measurements. The results show that degradation susceptibility of the alloy at any exposure period depends very much on the surrounding electrolyte composition. Additions of 10_20 g/L BSA doses in SBP fluid lead to decrease the susceptibility of the alloy degradation due to forming an integrated protective adsorbed layer on the surface. Plausible explanations were given for the stimulating effect induced by BSA at concentrations lower than 10 g/L or higher than 20 g/L. The synergistic influence of the negatively charged adsorbed albumin molecules at the physiological concentration (40 g/L) and Cl− ions in the contacting medium was also investigated. The electrochemical results are very consistent with SEM and EDX surface analyses, as well as the antibacterial efficiency of the alloy.

Surface Modification of Cardiovascular Stent Material 316L SS with Estradiol-Loaded Poly (trimethylene carbonate) Film for Better Biocompatibility.

A delay in the endothelialization process represents a bottleneck in the application of a drug-eluting stent (DES) during cardiovascular interventional therapy, which may lead to a high risk of late restenosis. In this study, we used a novel active drug, estradiol, which may contribute to surface endothelialization of a DES, and prepared an estradiol-loaded poly (trimethylene carbonate) film (PTMC-E5) on the surface of the DES material, 316L stainless steel (316L SS), in order to evaluate its function in improving surface endothelialization. All the in vitro and in vivo experiments indicated that the PTMC-E5 film significantly improved surface hemocompatibility and anti-hyperplasia, anti-inflammation and pro-endothelialization properties. This novel drug-delivery system may provide a breakthrough for the surface endothelialization of cardiovascular DES.

Study on the Mg-Li-Zn ternary alloy system with improved mechanical properties, good degradation performance and different responses to cells.

Mg-Li alloys are promising as absorbable metallic biomaterials, which however have not received significant attention since the low strength, controversial corrosion performance and the doubts in Li toxicity. The Mg-Li-Zn alloy in the present study revealed much improved mechanical properties higher than most reported binary Mg-Li and ternary Mg-Li-X alloys, with superior corrosion resistance in cell culture media. Surprisingly, the addition of Li and Zn showed increased nitric oxide release. The present study indicates good potential of Mg-Li-Zn alloy as absorbable cardiovascular stent material.