Nucleation Of Phosphate Research Articles

Redefined ion association constants have consequences for calcium phosphate nucleation and biomineralization

Calcium orthophosphates (CaPs), as hydroxyapatite (HAP) in bones and teeth are the most important biomineral for humankind. While clusters in CaP nucleation have long been known, their speciation and mechanistic pathways to HAP remain debated. Evidently, mineral nucleation begins with two ions interacting in solution, fundamentally underlying solute clustering. Here, we explore CaP ion association using potentiometric methods and computer simulations. Our results agree with literature association constants for Ca2+ and H2PO4−, and Ca2+ and HPO42-, but not for Ca2+ and PO43− ions, which previously has been strongly overestimated by two orders of magnitude. Our data suggests that the discrepancy is due to a subtle, premature phase separation that can occur at low ion activity products, especially at higher pH. We provide an important revision of long used literature constants, where association of Ca2+ and PO43− actually becomes negligible below pH 9.0, in contrast to previous values. Instead, [CaHPO4]0 dominates the aqueous CaP speciation between pH ~6–10. Consequently, calcium hydrogen phosphate association is critical in cluster-based precipitation in the near-neutral pH regime, e.g., in biomineralization. The revised thermodynamics reveal significant and thus far unexplored multi-anion association in computer simulations, constituting a kinetic trap that further complicates aqueous calcium phosphate speciation.

Characterization of aTiO2 surfaces functionalized with CAP-p15 peptide.

Functionalization of Titanium implants using adequate organic molecules is a proposed method to accelerate the osteointegration process, which relates to topographical, chemical, mechanical, and physical features. This study aimed to assess thepotential of a peptide derived from cementum attachment protein (CAP-p15) adsorbed onto aTiO2 surfaces to promote the deposition of calcium phosphate (CaP) minerals and its impact on the adhesion and viability of human periodontal ligament cells (hPDLCs). aTiO2 surfaces were synthesized by magnetron sputtering technique. The CAP-p15 peptide was physically attached to aTiO2 surfaces and characterized by atomic force microscopy, fluorescence microscopy, and water contact angle measurement. We performed in vitro calcium phosphate nucleation assays using an artificial saliva solution (pH 7.4) to simulate the oral environment. morphological and chemical characterization of the deposits were evaluated by scanning electronic microscopy (SEM) and spectroscopy molecular techniques (Raman Spectroscopy, ATR-FTIR). The aTiO2 surfaces biofunctionalized with CAP-p15 were also analyzed for hPDLCs attachment, proliferation, and in vitro scratch-healing assay. The results let us see that the homogeneousamorphous titanium oxide coating was 70 nanometers thick. The CAP-p15 (1 μg/mL) displayed the ability to adsorb onto the aTiO2 surface, increasing the roughness and maintaining the hydrophilicity of the aTiO2 surfaces. The physical adsorption of CAP-p15 onto the aTiO2 surfaces promoted the precipitation of a uniform layer of crystals with a flake-like morphology and a Ca/P ratio of 1.79. According to spectroscopy molecular analysis, these crystalline deposits correspond to carbonated hydroxyapatite. Regarding cell behavior, the biofunctionalized aTiO2 surfaces improved the adhesion of hPDLCs after 24 h of cell culture, achieving 3.4-fold when compared to pristine surfaces. Moreover, there was an increase in cell proliferation and cell migration processes. Physical adsorption of CAP-p15 onto aTiO2 surfaces enhanced the formation of carbonate hydroxyapatite crystals and promoted the proliferation and migration of human periodontal ligament-derived cells in in vitro studies. This experimental model using the novel bioactive peptide CAP-p15 could be used as an alternative to increasing the osseointegration process of implants.

Nucleation Domains in Biomineralization: Biomolecular Sequence and Conformational Features.

Biomolecules play a vital role in the regulation of biomineralization. However, the characteristics of practical nucleation domains are still sketchy. Herein, the effects of the representative biomolecular sequence and conformations on calcium phosphate (Ca-P) nucleation and mineralization are investigated. The results of computer simulations and experiments prove that the line in the arrangement of dual acidic/essential amino acids with a single interval (Bc (Basic) -N (Neutral) -Bc-N-Ac (Acidic)- NN-Ac-N) is most conducive to the nucleation. 2α-helix conformation can best induce Ca-P ion cluster formation and nucleation. "Ac- × × × -Bc" sequences with α-helix are found to be the features of efficient nucleation domains, in which process, molecular recognition plays a non-negligible role. It further indicates that the sequence determines the potential of nucleation/mineralization of biomolecules, and conformation determines the ability of that during functional execution. The findings will guide the synthesis of biomimetic mineralized materials with improved performance for bone repair.

Counterintuitive Crystallization: Rate Effects in Calcium Phosphate Nucleation at Near-Physiological pH

Counterintuitive Crystallization: Rate Effects in Calcium Phosphate Nucleation at Near-Physiological pH

Effects of carbonate species and chloride ions on calcium phosphate nucleation of biological apatite

Apatite is the principal inorganic component of biological hard tissues such as bone and teeth, and thus is essential to the existence of vertebrate life. Because carbonate species and chloride ions are ubiquitous in the fluids of vertebrate life forms, and these species are known to compete with phosphate species, it is critical to understand the influence of these anions on the nucleation of biological apatite. In this study, the important roles of carbonate and chloride ions in mediating inorganic calcium phosphate (CaP) phase nucleation are revealed both theoretically and experimentally. Theoretical investigations suggest that the Ca-O coordination number is an important descriptor for the nucleation of CaP and the interface interaction of the CaP-additive. Positive surface charges on the outermost Ca2+ ions of CaP clusters could attract carbonate species and Cl− ions, promoting the formation of CaP phase. In the presence of carbonate species and Cl− ions, the crystallization of hydroxyapatite (HA) from its precursor brushite (DCPD) has been observed in our experimental studies. The Cl− ion-doped B-type HA is proposed to be the most likely biological apatite based on equilibrium oxygen isotope fractionation and vibrational spectroscopic analysis. The insight gained here may help rationalize the control of biomineralization, and will guide the synthesis of anion-doped biological functional apatite materials.

Biomimetic Mineral Synthesis by Nanopatterned Supramolecular-Block Copolymer Templates.

Supramolecular structures of matrix proteins in mineralizing tissues are known to direct the crystallization of inorganic materials. Here we demonstrate how such structures can be synthetically directed into predetermined patterns for which functionality is maintained. The study employs block copolymer lamellar patterns with alternating hydrophilic and hydrophobic regions to direct the assembly of amelogenin-derived peptide nanoribbons that template calcium phosphate nucleation by creating a low-energy interface. Results show that the patterned nanoribbons retain their β-sheet structure and function and direct the formation of filamentous and plate-shaped calcium phosphate with high fidelity, where the phase, amorphous or crystalline, depends on the choice of mineral precursor and the fidelity depends on peptide sequence. The common ability of supramolecular systems to assemble on surfaces with appropriate chemistry combined with the tendency of many templates to mineralize multiple inorganic materials implies this approach defines a general platform for bottom-up-patterning of hybrid organic-inorganic materials.

Calcification of Various Bioprosthetic Materials in Rats: Is It Really Different?

The causes of heart valve bioprosthetic calcification are still not clear. In this paper, we compared the calcification in the porcine aorta (Ao) and the bovine jugular vein (Ve) walls, as well as the bovine pericardium (Pe). Biomaterials were crosslinked with glutaraldehyde (GA) and diepoxide (DE), after which they were implanted subcutaneously in young rats for 10, 20, and 30 days. Collagen, elastin, and fibrillin were visualized in non-implanted samples. Atomic absorption spectroscopy, histological methods, scanning electron microscopy, and Fourier-transform infrared spectroscopy were used to study the dynamics of calcification. By the 30th day, calcium accumulated most intensively in the collagen fibers of the GA-Pe. In elastin-rich materials, calcium deposits were associated with elastin fibers and localized differences in the walls of Ao and Ve. The DE-Pe did not calcify at all for 30 days. Alkaline phosphatase does not affect calcification since it was not found in the implant tissue. Fibrillin surrounds elastin fibers in the Ao and Ve, but its involvement in calcification is questionable. In the subcutaneous space of young rats, which are used to model the implants' calcification, the content of phosphorus was five times higher than in aging animals. We hypothesize that the centers of calcium phosphate nucleation are the positively charged nitrogen of the pyridinium rings, which is the main one in fresh elastin and appears in collagen as a result of GA preservation. Nucleation can be significantly accelerated at high concentrations of phosphorus in biological fluids. The hypothesis needs further experimental confirmation.

Vibrational Study on the Structure, Bioactivity, and Silver Adsorption of Silk Fibroin Fibers Grafted with Methacrylonitrile

Natural fibers have received increasing attention as starting materials for innovative applications in many research fields, from biomedicine to engineering. Bombyx mori silk fibroin has become a material of choice in the development of many biomedical devices. Grafting represents a good strategy to improve the material properties according to the desired function. In the present study, Bombyx mori silk fibroin fibers were grafted with methacrylonitrile (MAN) with different weight gains. The potential interest in biomedical applications of MAN functionalization relies on the presence of the nitrile group, which is an acceptor of H bonds and can bind metals. IR and Raman spectroscopy were used to characterize the grafted samples and the possible structural changes induced by grafting. Afterward, the same techniques were used to study the bioactivity (i.e., the calcium phosphate nucleation ability) of MAN-grafted silk fibroins after ageing in simulated body fluid (SBF) for possible application in bone tissue engineering, and their interaction with Ag+ ions, for the development of biomaterials with enhanced anti-microbial properties. MAN was found to efficiently polymerize on silk fibroin through polar amino acids (i.e., serine and tryptophan), inducing an enrichment in silk fibroin-ordered domains. IR spectroscopy allowed us to detect the nucleation of a thin calcium phosphate layer and the uptake of Ag+ ions through the nitrile group, which may foster the application of these grafted materials in biomedical applications.

Water-Mediated attraction between Like-charged species involved in calcium phosphate nucleation

There is overwhelming evidence that the biomineralization pathway involves prenucleation clusters (PNCs). Studies of calcium phosphate precipitation have shown that the thermodynamically favorable pathway to form the PNC identified as calcium triphosphate (Ca(HPO4)34-) includes two stable ion species (CaHPO4, Ca(HPO4)22–). However, the underlying mechanism remains unclear. Here, we reveal that the formation of these three species exhibits extremely distinct thermodynamic mechanisms. In sharp contrast to an entropy-driven Ca2+-HPO42- (CaP1) pairing compensated by unfavorable energy variation, free energy calculations display an energy-dominated association for CaHPO4-HPO42- (CaP2) and Ca(HPO4)22–-HPO42- (CaP3) with a slightly favorable entropy change. Further free energy decompositions demonstrate that the ion-induced favorable entropic and energetic variations are responsible for CaP1 and CaP2 associations, while the solvent-induced energy change accounts for the CaP3 pairing between two species carrying the same charges. In the complete prenucleation process starting from aqueous solution, the solvent-induced energy change initially hinders the CaP1 pairing but eventually makes a favorable contribution to the CaP3 association. Specifically, the enhanced stability and alignment of the hydrated water molecules around the ion species with charges of the same sign lead to the substantial favorable water-induced energy change and thus promote the CaP3 association even without any counterions.

Novel Utilization of Therapeutic Coatings Based on Infiltrated Encapsulated Rose Bengal Microspheres in Porous Titanium for Implant Applications.

Despite the increasing progress achieved in the last 20 years in both the fabrication of porous dental implants and the development of new biopolymers for targeting drug therapy, there are important issues such as bone resorption, poor osseointegration, and bacterial infections that remain as critical challenges to avoid clinical failure problems. In this work, we present a novel microtechnology based on polycaprolactone microspheres that can adhere to porous titanium implant models obtained by the spacer holder technique to allow a custom biomechanical and biofunctional balance. For this purpose, a double emulsion solvent evaporation technique was successfully employed for the fabrication of the microparticles properly loaded with the antibacterial therapeutic agent, rose bengal. The resulting microspheres were infiltrated into porous titanium substrate and sintered at 60 °C for 1 h, obtaining a convenient prophylactic network. In fact, the sintered polymeric microparticles were demonstrated to be key to controlling the drug dissolution rate and favoring the early healing process as consequence of a better wettability of the porous titanium substrate to promote calcium phosphate nucleation. Thus, this joint technology proposes a suitable prophylactic tool to prevent both early-stage infection and late-stage osseointegration problems.

Amyloid-like amelogenin nanoribbons template mineralization via a low-energy interface of ion binding sites

Protein scaffolds direct the organization of amorphous precursors that transform into mineralized tissues, but the templating mechanism remains elusive. Motivated by models for the biomineralization of tooth enamel, wherein amyloid-like amelogenin nanoribbons guide the mineralization of apatite filaments, we investigated the impact of nanoribbon structure, sequence, and chemistry on amorphous calcium phosphate (ACP) nucleation. Using full-length human amelogenin and peptide analogs with an amyloid-like domain, films of β-sheet nanoribbons were self-assembled on graphite and characterized by in situ atomic force microscopy and molecular dynamics simulations. All sequences substantially reduce nucleation barriers for ACP by creating low-energy interfaces, while phosphoserines along the length of the nanoribbons dramatically enhance kinetic factors associated with ion binding. Furthermore, the distribution of negatively charged residues along the nanoribbons presents a potential match to the Ca–Ca distances of the multi-ion complexes that constitute ACP. These findings show that amyloid-like amelogenin nanoribbons provide potent scaffolds for ACP mineralization by presenting energetically and stereochemically favorable templates of calcium phosphate ion binding and suggest enhanced surface wetting toward calcium phosphates in general.

Effect of Al2O3 content on amorphous phase-separation and self-limited crystallization of phosphosilcate glasses

Amorphous phase-separation and crystallization behavior of phosphosilicate glasses with different Al2O3 contents were systematically investigated. We demonstrate that nanophase-separation spontaneously occurs during cooling of all the glass melts. The two separated phases are found to be the phosphate-rich droplet phase with high ion movability and the rigid silicate-rich matrix. With increasing Al2O3 content, nucleation rate decreases whereas growth rate is enhanced in terms of nucleation-growth type phase-separation. In addition, for the compositions with 0 and 2 mol% Al2O3, sodium phosphate nanocrystals simultaneously form in the droplets in the cooling process, this is further confirmed from the micro-morphologies the samples heat-treated at 1.2 Tg for various durations. In this case, the growth of sodium phosphate is self-limited by the surrounding silicate-rich phase. Our results show that the addition of Al2O3 tends to inhibit nucleation of sodium phosphate rather than crystal growth. This work indicates that phase-separation can be used as a route to the preparation of amorphous/nanocrystal composites by adjusting Al2O3 content in phosphosilcate glass system.

Biomineralization from the Perspective of Ion Aggregation: Calcium Phosphate Nucleation in the Physiological Environment.

Biomineralization is an important process of bone tissue generation. Calcium (Ca) and phosphate (P) ions aggregate and nucleate under the regulation of biomolecules at the initial mineralization stage. Due to the complexity of the physiological environment, the movement behavior and mineralization mechanism of calcium and phosphate ions, as well as the effect of biomolecules on them, are not clear. In this study, computer simulations and experimental verification were applied to investigate the characteristics of the initial biomineralization from the view of ion aggregation and nucleation. The results prove that P ions play a more important role in mineralization than Ca ions. The guanidyl group and surrounding carboxyl terminal groups are a potential excellent nucleation domain on proteins. The interval distribution of acidic/basic residues on protein is more conductive to the formation of large Ca and P ions clusters. The involvement of protein could increase the probability of hydroxyapatite phase precipitation, especially in the presence of a helical conformation. The detailed information on Ca and P ions behavior provided by the computer simulations is helpful for further understanding the mechanism of biomineralization, which will promote the development of bone repair materials to the biomimetic mineralized materials.

Improvement of the corrosion resistance of mild steel in sulfuric acid by new organic-inorganic hybrids of Benzimidazole-Pyrophosphate: Facile synthesis, characterization, experimental and theoretical calculations (DFT and MC)

In this work, a new generation of corrosion inhibitors composed of xMCl2-yNa4P2O7 (MCl2 = 2,2’-dibenzimidazolyl butane dichlorhydrates) hybrids materials have been synthesized successfully with five molar ratios (R1, R2, R3, R4, and R5) by soft chemistry route and characterized. The synthesis was performed in aqueous solutions by the ionic exchange between the pyrophosphate anions and the 2,2′-dibenzimidazolyl butane dichlorhydrates cations. The structure and morphology of the synthesized hybrids are studied by infrared spectroscopy (FT-IR), UV–Visible analysis (UV-vis), X-ray diffraction (XRD), and scanning electron microscopy (SEM). The infrared results show the formation of a new band N-O-P confirming the hybrid structure. The crystalline structure was improved after adding pyrophosphate, confirming the phosphate nucleation and a better settlement of the crystals. These crystals were clearly shown in the SEM images. The corrosion inhibition performance of the prepared compounds were studied on mild steel in 0.5 M sulfuric acid by electrochemical impedance spectroscopy (EIS) and polarization studies. The inhibition efficiency increased with increasing inhibitor concentration, while the corrosion current density decreased. Indeed, the best inhibitor of these hybrids is R1 with an inhibition power of 97.1 % at 300 ppm. The effect of temperature on inhibitory efficiency in a range of 298 to 328K is discussed. The SEM images are also confirmed that the R1 retards the corrosion on the mild steel surface. The EDX analysis presented that this surface is mainly composed of N and P elements. The theoretical calculation results show that the benzimidazole cycle and the heteroatoms of N, O and P are the active sites of inhibitor R1.

Investigation of the effect of some plant aqueous extracts on calcium phosphate precipitation as a simulation of initial dental calculus formation in vitro

Background and Aims: Calcium phosphate is most of the inorganic content of dental calculus. Therefore, knowing or controlling the precipitation mechanism of calcium phosphate is very important for the inhibition of dental calculus formation at the beginning. Plants have been known to be excellent sources of many nutritional and phytochemical content. The aim of this study is to investigate the effects of Petroselinum crispum, Eruca vesicaria ssp. sativa, Beta vulgaris L.var.cicla, Rumex cristatus DC. and Cotinus coggygria Scop. aqueous extracts on calcium phosphate precipitation, which is thought to reflect the onset of dental calculus formation in vitro. Methods: The optical density (OD) increases first with the calcium phosphate nucleation and when the balance is reached, the optical density decreases gradually when the nuclei begin to aggregate and precipitate. The OD change was monitored by recording the absorbance at 620 nm. Results: The effect on the calcium phosphate precipitation varied differently among the 5 types of aqueous extracts. The smoke tree (Cotinus coggygria Scop.) extract activated calcium phosphate precipitation while all others inhibited precipitation. Conclusion: These results suggest that some types of plant aqueous extracts may have protective potential against dental calculus initially and, therefore they may be used in toothpastes or in mouthwashes.

Phosphatidylserine controls calcium phosphate nucleation and growth on lipid monolayers: A physicochemical understanding of matrix vesicle-driven biomineralization

Bone biomineralization is an exquisite process by which a hierarchically organized mineral matrix is formed. Growing evidence has uncovered the involvement of one class of extracellular vesicles, named matrix vesicles (MVs), in the formation and delivery of the first mineral nuclei to direct collagen mineralization. MVs are released by mineralization-competent cells equipped with a specific biochemical machinery to initiate mineral formation. However, little is known about the mechanisms by which MVs can trigger this process. Here, we present a combination of in situ investigations and ex vivo analysis of MVs extracted from growing-femurs of chicken embryos to investigate the role played by phosphatidylserine (PS) in the formation of mineral nuclei. By using self-assembled Langmuir monolayers, we reconstructed the nucleation core - a PS-enriched motif thought to trigger mineral formation in the lumen of MVs. In situ infrared spectroscopy of Langmuir monolayers and ex situ analysis by transmission electron microscopy evidenced that mineralization was achieved in supersaturated solutions only when PS was present. PS nucleated amorphous calcium phosphate that converted into biomimetic apatite. By using monolayers containing lipids extracted from native MVs, mineral formation was also evidenced in a manner that resembles the artificial PS-enriched monolayers. PS-enrichment in lipid monolayers creates nanodomains for local increase of supersaturation, leading to the nucleation of ACP at the interface through a multistep process. We posited that PS-mediated nucleation could be a predominant mechanism to produce the very first mineral nuclei during MV-driven bone/cartilage biomineralization.

Assessing the Onset of Calcium Phosphate Nucleation by Hyperpolarized Real-Time NMR.

We report an experimental approach for high-resolution real-time monitoring of transiently formed species occurring during the onset of precipitation of ionic solids from solution. This is made possible by real-time nuclear magnetic resonance (NMR) monitoring using dissolution dynamic nuclear polarization (D-DNP) to amplify signals of functional intermediates and is supported by turbidimetry, cryogenic electron microscopy, and solid-state NMR measurements. D-DNP can provide drastic signal improvements in NMR signal amplitudes, permitting dramatic reductions in acquisition times and thereby enabling us to probe fast interaction kinetics such as those underlying formation of prenucleation species (PNS) that precede solid–liquid phase separation. This experimental strategy allows for investigation of the formation of calcium phosphate (CaP)-based minerals by 31P NMR—a process of substantial industrial, geological, and biological interest. Thus far, many aspects of the mechanisms of CaP nucleation remain unclear due to the absence of experimental methods capable of accessing such processes on sufficiently short time scales. The approach reported here aims to address this by an improved characterization of the initial steps of CaP precipitation, permitting detection of PNS by NMR and determination of their formation rates, exchange dynamics, and sizes. Using D-DNP monitoring, we find that under our conditions (i) in the first 2 s after preparation of oversaturated calcium phosphate solutions, PNS with a hydrodynamic radius of Rh ≈ 1 nm is formed and (ii) following this rapid initial formation, the entire crystallization processes proceed on considerably longer time scales, requiring >20 s to form the final crystal phase.

Ion redistributions at interfaces facilitate nucleation and growth of branched Ag3PO4 polypods

Interfaces of liquid-solid and air-liquid are known to change the chemical and physical properties of the liquid solution and thus utilized for multiple applications. However, their role in crystal growth, especially of branched structures, is seldom investigated. Here we synthesized branched silver phosphate polypods in a thin layer of aqueous solution (thickness of 810 µm or below) taking advantage of the unique properties of both the air-liquid and a hydrophilic liquid-solid interfaces. Our results indicate that the synergism of long-range electric fields (ion distributions) at the interfaces promotes not only the nucleation and growth of silver phosphate but also the formation of branched structures. This work can be a reference for facilitating crystal nucleation and growth and controlling structures by understanding and mimicking the properties at the interfaces.

Structure–Activity Relationships of Hydroxyapatite-Binding Peptides

Elucidating the structure-activity relationships between biomolecules and hydroxyapatite (HAP) is essential to understand bone mineralization mechanisms, develop HAP-based implants, and design drug delivery vectors. Here, four peptides identified by phage display were selected as model HAP-binding peptides (HBPs) to examine the effects of primary amino acid sequence, phosphorylation of serine, presence of charged amino acid residues, and net charge of the peptide on (1) HAP-binding affinity, (2) secondary conformation, and (3) HAP nucleation and crystal growth. Binding affinities were determined by obtaining adsorption isotherms by mass depletion, and the conformations of the peptides in solution and bound states were observed by circular dichroism. Results showed that the magnitude of the net charge primarily controlled binding affinity, with little dependence on the other HBP features. The binding affinity and conformation results were in good agreement with our previous molecular dynamics simulation results, thus providing an excellent benchmark for the simulations. Transmission electron microscopy was used to explore the effect of these HBPs on calcium phosphate (Ca-PO4) nucleation and growth. Results indicated that HBPs may inhibit nucleation of Ca-PO4 nanoparticles and their phase transition to crystalline HAP, as well as control crystal growth rates in specific crystallographic directions, thus changing the classical needle-like morphology of inorganically grown HAP crystals to a biomimetic plate-like morphology.

Promotion effect of immobilized chondroitin sulfate on intrafibrillar mineralization of collagen

Chondroitin sulfate (CS) is widespread in mineralized tissues and is considered to play crucial roles during the mineralization process. However, its role in biomineralization remains controversial. In the present study, CS is immobilized to collagen fibrils to mimic its state in biomineralization. The results demonstrate that immobilized CS on collagen fibrils accelerates calcium phosphate nucleation and significantly promotes collagen mineralization by accumulating calcium ions in collagen fibrils. The stochastic optical reconstruction microscopy results confirm that CS gives the specific nucleation sites for calcium phosphate to preferentially form, the improved intrafibrillar heterogeneous nucleation of calcium phosphate facilitates intrafibrillar mineralization. It is found remarkably accelerated remineralization of CS immobilized demineralized dentin is achieved. This study offers insight on the understanding of the function of the biomacromolecule CS on the biomineralization front. In addition, CS effectively promotes intrafibrillar mineralization, which highlights fine prospect for CS to reconstruct collagen-mineralized tissues as a natural material.