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Functional Biocompatible Matrices from Mussel Byssus Waste Devis Montroni, Francesco Valle, Stefania Rapino, Simona Fermani, Matteo Calvaresi, Matthew James Harrington, and Giuseppe Falini ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00743 • Publication Date (Web): 27 Nov 2017 Downloaded from http://pubs.acs.org on December 4, 2017

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ACS Biomaterials Science & Engineering

Functional Biocompatible Matrices from Mussel Byssus Waste Devis Montroni1, Francesco Valle2, Stefania Rapino1, Simona Fermani1, Matteo Calvaresi1, Matthew J. Harrington*, §,3, Giuseppe Falini*,1 1

Dipartimento di Chimica “Giacomo Ciamician”, Alma Mater Studiorum Università di Bologna,

via Selmi 2, 40126 Bologna, Italy. 2

National Research Council (CNR), Institute for Nanostructured Materials (ISMN), Via P.

Gobetti 101, 40129 Bologna, Italy. 3

Department. of Biomaterials, Max-Planck Institute for Colloids and Interfaces, Research

Campus Golm, Am Mühlenberg 1, Potsdam 14424, Germany. Corresponding Authors * Prof. Giuseppe Falini, email: [email protected] * Prof. Matthew J. Harrington, email: [email protected]

KEYWORDS: Byssus; Waste materials; Matrix; Biocompatible; Biorenewable; Metalation; Dyes.

ACS Paragon Plus Environment

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ABSTRACT The mussel byssus is a biorenewable, protein-based material produced by marine mussels, which has attracted the interest of material scientists due to its remarkable mechanical and self-healing properties. Large quantities of byssus waste material from mussel mariculture are produced every year, which have great potential as a raw starting material for producing sustainable advanced materials. In this work, we developed a facile and scalable method to synthesize whole byssus-based porous matrices that retain part of the hierarchical organization of the pristine material at the nano-scale. The resulting material is biocompatible and maintains important native byssus features - metal ion chelation (≥ 12 mg/g), collagen domains and hierarchical organization, with tunable properties controlled via metal ion content. Furthermore, these biocompatible matrices showed a dye absorbing efficiency (up to 64 mg/g for anionic dyes) that was similar to or higher than that of the pristine byssus, a proof of preservation of structural motifs. These findings indicate that biorenewable matrices originating from byssus waste could have potential use in biomedical engineering and applied material science.

Introduction It is widely recognized that animal byproducts from the mariculture industry offer a largely untapped and potentially useful source of biorenewable starting material for generating advanced materials with potential biomedical and industrial applications.1-7 This includes the mussel byssus, which is a proteinaceous fibrous material produced by mussels to anchor to hard substrates and resist lift and drag forces from waves.8 The byssus, which is a biorenewable waste material from mussel farming, has attracted the attention of researchers based on its impressive mechanical/adhesive properties, its high resistance to chemical degradation and its rather unique biochemical composition. Notably, byssal threads exhibit toughness comparable to Kevlar when pulled to break,9,

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as well as a distinctive self-healing behaviour during cyclic tensile


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deformation, both of which arise from the presence of sacrificial protein-metal coordination bonds mediated by specific amino acidic residues in the thread proteins.11 In the present study, we processed natural byssus waste material acquired from mussel mariculture to create multifunctional porous matrices that retain certain distinctive chemical and structural features of the native material. The byssus (Figure 1) is comprised of four different regions:12 i) the fibrous core which acts as a shock absorbing tether to the surface, ii) the cuticle, a hard protective sheath, which surrounds the thread core and is enriched in the aromatic amino acid 3,4-dihydroxyphenylalanine (DOPA) iii) the plaque, responsible for the adhesion of the byssus to a substrate and also composed of DOPA-rich proteins, and iv) the stem, which connects the byssus threads to the organism. The byssus is comprised of an assortment of more than ten different protein building blocks.13, 14 The fibrous core of the byssus is mainly composed of a family of modified collagens called preCols,15 each consisting of a central collagen domain, two adjacent flanking domains at both ends of the collagen domain and two terminal histidine (His)–rich domains, which bind transition metal ions (mainly Cu2+ and Zn2+).11, 16, 17 The cuticle is only known to be composed of a single protein, mussel foot protein-1 (mfp-1),18 characterized by minimal secondary structure and 10-15 mol% of DOPA content. DOPA residues in the cuticle are involved in coordination bonds with Fe3+ that contribute to the high hardness.19 Finally, plaques are composed of at least five different mfps having different percentages of DOPA content.14, 20 The high content of DOPA and His residues in the byssus are utilized to form diverse cross-link that stabilize the fibers, with both metal coordination crosslinking between DOPA and His residues, as well as DOPA-based covalent oxidative cross-linking.21,

22, 23

This high level of

sclerotization makes the byssus resistant to solubilization, as well as chemical, physical and


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biological degradation.24 However, previously, research groups were able to extract solubilized byssus proteins from the bysuss24 and mussel foot organ,17, 25 which could be used for in vitro formation of films and fibers, respectively, with properties reminiscent of native byssus, demonstrating the potential of byssus as a starting material for generating new materials. Currently, mussel mariculture produces an enormous amount of byssus waste, which could otherwise be utilized and applied in material production. Mussel cultivation and processing for human consumption generates significant amounts of solid waste (20–50 wt%), equivalent to about 0.5 Gtons per year.26 This waste material is mainly composed of shells and a smaller fraction of byssus, which can be easily collected.27 Previously, byssal threads from mariculture were utilized to extract a soluble byssus protein hydrolysate by means of a basic treatment.24 These solubilized proteins were then precipitated to produce water-insoluble self-standing films for soft tissue engineering and drug delivery applications, which showed pH- and metalmodulated mechanical properties.24, 28 It was also previously reported that byssus thread material is able to remove aromatic dyes from polluted water according to their metalation state, suggesting that this material may have further potential applications.30 Here, the production of porous free-standing matrices using the whole byssus from mussel cultivation was achieved utilizing insoluble byssus material following a short acid treatment. Matrices were tested for various functionalities including, biocompatibility, mechanical performance and dye removal. The scalable process for producing the matrices required the development of a new methodology for breaking byssal threads down into collagen-rich microfibrils and DOPA-enriched matrix material, which could then be cast into thick matrices. The processed material consisted of a high yield of the insoluble portion of the byssus and retained a large amount of the native structure and chemical functionality. The properties of the


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synthetic byssus-derived matrices could be tuned by controlling the content of metal ions (Zn2+, Cu2+ and Fe3+), which are believed to interact with amino acid functional groups (i.e. DOPA and His) in the byssus proteins. Our findings indicate that byssus-derived matrices exhibit a higher surface area to volume ratio compared to the denser and more highly organized source material, while still maintaining part of the byssus supramolecular structure, including local organization of protein building blocks and metal dependent cross-linking. Thus, we have a demonstrated and scalable route for a producing a new biocompatible material with tunable performance that has potential application in biomedical engineering, material science and water remediation.

Materials and methods Materials All the reagents and solvents were purchased from Sigma Aldrich and utilized without any further purification. Methylene blue (MB) and Eosin Y (EosY) fresh solutions were prepared for each experiment in PremilliQ water. Byssus preparation The byssus, from Mytilus galloprovincialis, was collected from a musselbreeding farm close to Fano (Italy). After collection the byssus was washed with tap water and soap until washing water was completely clear. Then it was rinsed with distilled water until all the soap was eliminated and stirred in ethanol two times for 30 minutes each and washed again with distilled water for 15 minutes to restore the hydration sphere of the material. The clean byssus was conserved dry in a desiccator under vacuum. Matrix synthesis The byssus used for matrices synthesis was ground with a mortar and pestle following liquid nitrogen freezing, and dried again. The porous materials were synthesized using 150 mg of grinded byssus adding 4 mL of a water/HCl solution at pH 1. The dispersion was stirred vigorously for 6 hours at 80 °C, checking the pH after 30 and 60 minutes of treatment


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resetting it to pH 1 every time. After 6 h the suspension obtained was filtered on a filter paper, washed with distilled water and the precipitate collected was formed into films via filtration. Finally, the partially de-metalized material (hereafter native matrix) obtained was dried in a desiccator under vacuum (Figure 2B). Matrix remetalation In order to reintegrate metals in the matrix after the synthesis we put the casted material in a mixture of 0.01 M FeCl3, 0.01 M ZnSO4 and 0.01 M CuSO4 in a 0.1 M TRIS buffer solution at pH 8.4 under mechanical stirring for 24 hours. The matrix was then stirred 2 times with PremilliQ water for 1 hour each time to wash away the metal unbound residues. Finally, the matrix (hereafter metal treated matrix) was dried in a desiccator (Figure 2A). Matrix demetalation Demetalation of the byssus matrices was performed according to the experimental procedure reported in Schmitt et al.,30 utilizing ethylenediaminetetraacetic acid (EDTA) in acidic conditions as a metal chelation agent. However, in the current study the Tris concentration was increased to 0.1 M and two more 1 hour washing in milliQ water were added at the end of the procedure. Samples (hereafter EDTA-treated matrix) were stored dry in a desiccator until further testing (Figure 2C). NBT/Glycinate assay30 Sample sections of 20 µm were cut using a Cryo-microtom, Cryostat Microm HM560. The sections were put on a glass slide and placed in a solution of 98 vol% of potassium glycinate solution and 2 vol% of Nitro Blue Tetrazolium (NBT) stain (10 mg/mL in water) for 40 minutes in a dark room, then was washed with distilled water. The stained sample was fixed using: ethanol 95 vol% for 2-5 minutes, absolute ethanol for 2-5 minutes and RotiHistol or xylene for 2-5 minutes. Finally, it was covered with a cover glass fixed with Canadian balsam.


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Picrosirius Red Stain assay31 Sample sections on a glass slide were placed in a solution of Picrosirius Red Stain ( Direct Red 1 g/L in Picric Acid 1.3 vol% in water) for 1 h, washed 2 times with acetic acid 0.5 vol% in water and then with distilled water. The sample was fixed as in the NBT/Glycinate assay. Trichrome stain assay This staining assay was done using a kit bought from Sigma Aldrich. First, the sample was put in a Bierbrich Scarlet-Acid Fuchsine solution for 5 minutes. Second, we put the sample in a solution made of one part of phosphotungstic acid, one of phosphomolybdic acid and two of water for 5 minutes. Third, we put it in an Aniline blue solution for 5 minutes. Finally, we put the glass in a solution of acetic acid (1 vol% in water) for 2 minutes and then we rinsed it with distilled water. The sample was fixed as in the NBT/Glycinate assay. Scanning electron Microscopy (SEM) images SEM images of uncoated samples were collected by using a Phenom G2 Pure and applying a tension of 3 kV. Different dry samples were glued on a carbon tape in different geometries allowing to observe the matrix sections and outer surfaces. Atomic Force Microscopy (AFM) images Samples for AFM observations were prepared depositing some reaction solutions on a mica surface for 10 minutes, washing away the excess sample with milliQ water and gently drying it with a nitrogen flow. An AFM Multimode 8 controlled by Nanoscope 5 electronic was used. The microscope was set on ScanAsyst mode and the cantilever were ScanAsyst with an elastic constant of 0.40 N/m. Amino acid analysis Samples for amino acid analysis were hydrolyzed for 24 h in 6 M HCl with 5% phenol at 110°C under vacuum. The amino acid composition of the samples was analyzed using a post-column ninhydrin-based amino acid analyzer (Sykam S433, Fürstenfeldbruck,


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Germany). Resolution of the phenylthiohydantoin (PTH) derivatives of DOPA and alanine was effected by a modified gradient program.32 Spectroscopic Analysis In the Raman micro-spectroscopy investigations a continuous laser beam was focused down to a micron-sized spot on the sample through a confocal Raman microscope (CRM200, WITec, Ulm, Germany) equipped with a piezo- scanner (P-500, Physik Instrumente, Karlsruhe, Germany). A diode-pumped 785 nm near infrared (NIR) laser excitation (Toptica Photonics 34 AG, Graefelﬁng, Germany) was used in combination with a water immersed 60X (Nikon, NA ¼ 1.0) microscope objective. The linearly polarized laser light was rotated using a half-wave plate, and scattered light was ﬁltered by introducing a further polarizer before the confocal microscope pinhole. The spectra were acquired using a CCD (PI-MAX, Princeton Instruments Inc., Trenton, NJ, USA) behind a grating (300 g mm 21) spectrograph (Acton, Princeton Instruments Inc., Trenton, NJ, USA) with a spectral resolution of approximately 6 cm1

. For our measurements, we utilized an integration time of 1 second and 40 accumulations. Data

were processed with OPUS software (Bruker) by background subtracting a polynomial baseline, averaging three spectra and smoothing with a Savitzky-Golay filter. Induced Coupling Plasma coupled with Optical Emission Spectroscopy (ICP-OES) measurements The metal content in the matrices was measured by degrading them in 5 mL of a hypochlorite solution (10-15 vol%), adjusting the volume with water and acidifying with HNO3 (atomic absorption grade) till a concentration of 0.5 M. All the samples were measured in duplicate and each sample was measured three times, 12 seconds each and 50 seconds of prerunning, using an ICP-OES, Spectro Arcos-Ametek, Inductive Coupled Plasma Optical Emission Spectroscopy with axial torch and high salinity kit. The iron signal was measured at 238.2 nm,


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the zinc signal at 213.9 nm and the copper signal at 324.8 nm. The calibrating curve was done using certified standards in a solution of nitric acid and hypochlorite. Swelling properties The measures of the matrix swelling was carried out putting a sample of dryweighed material in water and measuring its weight every 5 minutes, cleaning it from water drops before every measurement. Mass measurements were performed using a Radweg AS82/220.X2 balance. Uniaxial compression tests Each uniaxial compression experiment was performed on five different samples using an INSTRON Testing Machine 4465 and the Series IX software package. Each sample was cylindrical, about 10 mm of diameter and 4 mm of height (determined by micrometer) and was hydrated for 2 hours before being measured. The compression tests were performed with a velocity of 1 mm/min. The measures did not exhibit an elastic region. The measure of the stress-strain slope was performed interpolating the data between 0 and 5% of strain for metal-treated matrices and between 0 and 10% for the other matrices. The densification region of the three matrices was identified as the point where the slope reached a value of 0.2. Biocompatibility tests In order to test the matrix biocompatibility NIH cells were cultured on it. A cell growth curve analysis in the presence of a byssus matrix was performed. Before the cell plating, the samples were sterilized using ethanol and then incubated in the cell growth environment solution to eliminate all the acid residues from the treatment. 50,000 cells were plated per dish in a six-well, after 24 h of incubation to permit the cells to seed, the byssus-based material samples were placed in the cell cultures. The cells were counted, using erythrosin coloration as marker of vitality, 24, 48 and 72 hours after introducing the samples in order to define the influence of the material on the cell growth.


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Water remediation experiments Water remediation experiments were carried out putting half of a casted matrix (≈ 30-35 mg) in 1 mL of stain solution at different concentrations for different times (depending on the adsorption kinetic) and measuring the stain concentration, using UV-Vis spectroscopy, before and after the matrix insertion. The concentrations studied were 0.01, 0.1, 0.5, 1 and 2 mM both for MB and EosY. Each experiment was carried out in double and the spectra were collected using a Cary60 from Agilent Technologies. Adsorption and desorption kinetics Adsorption kinetics were carried out using half of a casted matrix in 1 mL of 0.01 mM solution of stain and recording spectra every 10 minutes for 300 minutes and then every 15 minutes till 72 h. The desorption kinetics were done with the same parameters, using a loaded matrix in 1 mL of PremilliQ water. All the kinetics studies were carried out using a Cary60 equipped with an 18 cell holder.

Results Matrix synthesis In order to produce matrices from the byssus waste material, we aimed first to develop a protocol for breaking the byssus down into microfibers that could be cast into matrices. As an initial step, a wide screening of solvents and degradation conditions (Table SI1) was performed with the primary aim to retain the characteristic microfibrillar morphology of the thread core.33 The degradation products resulting from the treatments were screened using SEM and AFM imaging, as well as polarized light microscopy, due to the birefringence of the ordered core region.34 Based on this initial screening, the optimal treatment was determined to be degradation in HCl at pH 1, stirring at 80 °C under reflux. Additional pH values were tested as well, but the degradation process was slower, although leading to the same yield and material.


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To determine the optimal time of the HCl degradation treatment to achieve a reasonably homogenous population of minimally degraded microfibers, we again utilized AFM imaging and polarized light microscopy to observe the insoluble portion of the hydrolysate at various time points (Figures 3 and 4). These measurements revealed the presence of micron scale birefringent fibers and nanoscale fibers observed with AFM, as well as a more amorphous matrix material. Hydrolysis of byssal threads with the plaque removed showed only fibrous structures in light microscopy, indicating that the matrix is likely the product of the plaque degradation. Analysis of degradation products at various time points revealed that the microfiber population remained stable in size and density until around 6 hours; however, between 6–18 hours the microfibers appear to be degraded further. On the other hand, the non-fibrous matrix treated for 3 hours appeared less homogeneous than the 6-hour treated byssus, leading to the selection of 6 hours as the optimal treatment time. Using the 6-hour treated byssus as a starting material, we next aimed to cast freestanding films using a range of different methodologies (Table SI1). In the end, filtration exhibited the best results because it allowed the removal of the soluble component of the suspension, leading to a stable and water insoluble matrix. Using the methodologies above, it was possible to synthesized fibrous rich homogenous matrix stable in water with a yield of 50 ± 4 wt%. Matrix characterization Considering the relatively harsh conditions utilized for breaking down the byssus material, amino acid analysis was utilized in order to evaluate whether the byssus proteins were degraded during the chemical treatment and or if specific components were lost in the soluble fraction during the filtration process. In comparing the composition of the native byssus and the casted films (Table SI2), they are largely similar. However, a concomitant drop in the glycine (Gly), hydroxyproline (Hyp) and proline (Pro) composition are likely associated with


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preferential loss of preCol proteins comprising the thread core relative to the other byssus proteins associated with the cuticle and plaque.35 The composition of the material was further investigated using histological stains on 20 µm thick matrix

cross-sections

produced

with

a

cryo-microtome

(Figure

5).

Nitro

blue

tetrazolium/Glycinate (NBT) assay is a stain that can identify quinone groups (e.g. oxidized DOPA residues), which stain blue in spite of the relatively low signal of DOPA in the amino acid analysis measurements, which can arise from hydrolysis issues. Picrosirius Red stain (PRS) selectively binds collagen triple helices, staining them in red. Moreover, the samples were observed using cross polarized light to detect the presence of birefringent and crystalline fibers in the matrices. The results of the staining experiments showed that the fibers were rich in collagen, but poor in DOPA residues, whereas the non-fibrous material of the matrix was rich in DOPA residues, but poor in collagen domains, suggesting that it might arise from the degradation of plaques and/or cuticles. To address the source of the non-fibrous component, cuticle or plaques, hydrolysates using only threads without plaques and one enriched in plaques were synthesized. These were then stained using Trichrome stain (TC) assay, which stains collagen blue and other proteinaceous materials red. The optical images of the TC stained precipitates (Figure SI1) showed only collagen-rich material for the precipitate obtained from the thread, confirming that the non-fibrous components were largely due to the plaque and stem material, while cuticles were probably mostly degraded during the treatment. Confocal Raman spectroscopy (Figure SI2) was used to investigate whether plaque-derived amorphous material in the films still retains the ability to coordinate metal ions via DOPA.14, 36 Using a near infrared laser (λ = 785 nm), Raman spectra of films was measured before and after treatment in a solution 0.1 M of vanadium chloride. Vanadium was chosen because DOPA-V


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coordination was shown to exhibit a much higher resonance intensity enhancement at this laser wavelength than DOPA-Fe in the byssus cuticle.14, 38 Spectra collected from the untreated films were highly fluorescent and did not show the typical peaks indicative of DOPA-metal coordination. However, following treatment with VCl3, very clear peaks corresponding to DOPA-V coordination including three distinct peaks between 500–700 cm-1 and four strong peaks between 1200–1500 cm-1 appear throughout the film (Figure SI1), clearly indicating that DOPA residues are still present in the material and able to bind metal ions, even following the harsh processing. The observation of a transversal section of the native matrix by optical microscopy (Figure 6A) shows an open structure, with millimeter size interconnected pores and channels. This open structure remains also at a micron scale, as shown in Figure 6B, where the stratification of the fibrous and non-fibrous matrix components is visible. The SEM images of the casted material surface (Figure 6C), instead, show a heterogeneous structure in which dispersed fibers are observed. The biocompatibility of the native matrices was evaluated using in vitro cell cultures. The cell growth curve analysis in Figure 7, shows that this material does not affect the cell number and their vitality. Only a small reduction of overall cell number was observed in respect to the control substrate (six-well dish in absence of the byssus matrix). This difference is due to the physical encumbrance of the samples, which, occupying part of the dish plate, limit the dish area for cells growth. This result is confirmed by the fact that the density of cells in the control dishes and in the culture dishes with the matrix is the same. Furthermore, the cell adhesion and growth directly on the native matrix was tested: NIH cells were plated on the matrix and after 48 hours of incubation, they were fixed and marked with 4',6-diamidino-2-phenylindole (DAPI) fluorophore. The sample was imaged using fluorescence microscopy (Figure 7), and the recorded


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image showed that the cells could adhere and proliferate on the matrix without any visible sign of toxicity. The role of metals in influencing the chemical and physical features of the byssus films was further investigated by analyzing the materials just after synthesis, after a de-metalation treatment using EDTA at acidic pH, and after reinserting metals using a solution of Zn, Cu and Fe at pH 8.4. The metal content in the films was evaluated using ICP-OES (Table 1). Measurements revealed that for Cu and Zn, the metal-treated film contains significantly higher amount of metal than the native and EDTA-treated films, while the Zn content was not significantly different between the native and EDTA treated films and the Cu was only slightly higher for the native films (all data were compared using a t-test with p=0.05 and ν=10). In contrast to the other two metals, Fe gave completely different results, showing an approximately 20-fold higher concentration than either Zn or Cu in the native films. This concentration was reduced to about two-thirds the native value during EDTA treatment, but surprisingly, the Fe content did not increase during subsequent treatment in the metal solution. This could be due to a precipitation of iron as iron hydroxide at pH 8.4 (sea water pH) during the treatment. This compound, compared to the other two potential precipitating hydroxides from Cu2+ and Zn2+ shows a much lower solubility (Ksp (Cu(OH)2 = 4.8×10-20, Ksp Zn(OH)2 = 3.0×10-17, Ksp Fe(OH)3 = 2.8×10-39).38 At the sea water pH of 8.4 ([OH-] = 2.5 10-6 mol/dm3) the maximum concentration of Fe3+, Zn2+ and Cu2+ in solution can be 1.7 10-22, 7.6 10-9 and 4.8 10-6 mol/dm3, respectively. That low concentration of Fe3+ in solution favors its removal from the coordination sites into the byssus in favor of its precipitation. This is consistent with the hypothesis that the iron present in the byssus is first concentrated in the mussel soft tissue through filter feeding and actively added during thread formation.34, 36


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The three different matrices were also characterized using uniaxial compression tests, as reported in Figure SI3 and Table 2. The stress strain curves showed that the metal-treated matrix was the one that went in densification region (steep region of the curve) at the lowest strain, followed by the EDTA-treated higher matrix and native one. The first one appeared significantly different from the other two matrices, which appeared not significantly different between them (t-test performed with p=0.05, ν=8). Considering the stress-strain slope between 0-10 % of strain, we also observed a value for the metal-treated matrix which was significantly higher compare to the other two matrices which, as before, did not appear to be significantly different (t-test performed with p=0.05, ν=8). The matrices capability in absorbing water was measured by swelling experiments that showed an increase in the matrix weight around 240 wt% (Figure SI4) in less than one hour for the native matrix, 170 wt% for the EDTA-treated matrices and 80 wt% for the metal-treated matrices. Dye water removal experiments The dye absorption kinetics of the matrix was tested using 0.01 mM EosY and 0.01 mM MB solutions. Absorption profiles using native, EDTA-treated and metal-treated matrices reported in Figure SI5 showed that all the matrices reached a plateau after around 72 h, except for EosY in the native films, which plateaued in less than 24 h. The results of the loading are shown in Figure 8 and Table SI4. The data of EosY removal from a 2 mM solution were not significantly different (t-test performed with p=0.05, ν=2) for native and metaltreated films, while the EDTA-treated films absorbed significantly more. The MB absorption from a 2 mM solution was significantly different for the three different matrix typologies, with the EDTA-treated films being the most efficient, followed by the native and metal-treated matrices. After the loading experiments, some of the dye containing matrices were used for the desorption kinetics experiments, the results are reported in Table SI4. In agreement with the


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loading results, the binding between EosY and the byssus matrices appeared stronger and no detectable amount of dye was lost after 72 h for native and EDTA-treated matrices, while metaltreated matrix lost less than 1 wt% of its dye content. On the other hand, the binding with MB appear weaker, exhibiting desorption of a detectable amount of dye from the 0.1 mM treated samples of native and EDTA-treated matrices, which showed a lower affinity for the dye, and less than 1 wt% of its dye content form the metal-treated matrix treated with the 1 mM solution.

Discussion Natural waste products from industrial processing of animal and plant-based materials have emerged as an important biorenewable source for the sustainable production of new materials for a large range of applications.1-7 In the present study, a new method for processing mussel byssus mariculture waste to form open-cell matrices is reported. In the preparation of these free-standing open matrices, the insoluble byssus material obtained from the entire byssus from the industrial mussel processing was used. Previous studies have produced fibers17 and films24 from byssus, but in these studies, the water-soluble components of the byssus extracted either from the mussel foot tissue or via degradation of the byssus material, were used. The use of the whole waste material provides a fast and simple way of processing in which tedious manual separation of the different byssus regions are avoided. The byssus is a highly cross-linked that is chemically resistant to solubilization (Table SI1); thus, the use of its insoluble fraction has the double advantage to increase yield of recovery and to keep the majority of the chemical and structural features of the byssus threads. Indeed, the structural analysis of the byssal matrix revealed that fibrils of tens of microns in diameter and up


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to hundreds of microns in length are retained during the process; however, biochemical analysis showed that the collagen proteins were selectively lost during the process, leaving behind a network of fibrils embedded in an amorphous non-collagenous material. The strong birefringence observed under cross-polarizers suggests that the collagenous core fibrils retain a high degree of their characteristic hierarchical structure, which was found to be critical to the mechanical performance of the byssus thread.33 Histological staining for quinones with a NBTglycinate stain and Raman spectroscopic analysis of the matrix confirmed that the matrices maintain the characteristic ability of byssus proteins to coordinate with trivalent metal ions via DOPA residues. Furthermore, ICP measurements demonstrated that the matrices kept the byssus capability to bind strongly to Cu2+ and Zn2+ following soaking in metal-rich solutions, which is likely mediated via histidine residues in the preCol proteins. However, at this point there is no direct evidence for this interaction. Thus, in spite of the harsh treatment, the matrices cast from the insoluble material remaining from byssus hydrolysis preserve many of the structural and biochemical features that contribute to the impressive properties of the mussel byssus. As a consequence, the obtained open matrices show a mechanical resistance to compression that depends on the content of metal ions. Previous studies utilizing the byssus as a raw starting material for making new materials were focused mainly on their mechanical performance and function, demonstrating nicely the maintenance of characteristic pH- and metal-dependent properties, such as high toughness and self-healing capacity.24, 28 As proof of chemical similarity between the byssus matrices and their source, the pristine byssus thread, we observed that they could also function as an adsorbent material for capturing aromatic dye molecules in contaminated water, with an improvement compared to byssus threads.29 The


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capability of the byssal matrices to uptake dyes was dependent on the degree of metalation and of the same order of magnitude or even higher when compared with other absorbents obtained from natural fibrous biomaterials38-41 or waste materials.1-7 As for byssal threads,29 also the matrices’ capability for retaining dyes without desorbing them could be a starting point to produce future functional materials. The metal-treated films exhibited much lower water absorption (~ 80 wt%) compared to the native and EDTA-treated films (~ 200 wt%). Moreover, in the compression tests the metaltreated matrices reach the densification region at a lower strain and with a higher stress-strain slope. It is tempting to speculate that these physicochemical and functional differences might arise from the degree metal ion coordination present within the films. In fact, many (bio)polymeric materials containing DOPA or His functional groups, or collagenous molecules have properties that are modified in the presence of metal ions.29, 6, 42-45

Conclusions In this study, byssus-based functional biocompatible matrices were synthesized starting from byssus thread, which is a waste material from fishery industries, developing an easy and cheap synthetic method. These matrices preserved part of the supramolecular fibrous organization of the pristine material and were biocompatible and tunable in their chemical and physical properties by controlling their metal ions concentration. They also showed an ability in dye sorption, as demonstrated for the pristine threads. The application of these biorenewable matrices to other fields, such as substrates for regenerative medicine, is envisioned due to their mechanical resistance, porosity and biocompatibility.


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ASSOCIATED CONTENT Supporting Information. Tables SI1-4; Fig. SI1 (Raman spectrum); Fig. SI2 (hydration kinetics); Fig. SI3 (mechanical tests); Fig. SI4 (dye absorption kinetics); Fig. SI5 (dye loading). AUTHOR INFORMATION Present Addresses §

Prof. Matthew J. Harrington: Dept. of Chemistry, McGill University, 801 Sherbrooke St. W,

Montreal, QC, H3A 0B8 Canada Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT GF thanks Prof. Corrado Piccinetti for providing the raw byssus threads. GF and SF thank the Consorzio Interuniversitario per la Chimica dei Metalli nei Sistemi Biologici for the support. MJH acknowledges support of the Max Planck Society.

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Table 1. Metal content in the different matrices (mg/g), measured using ICP-OES.

EDTA-treated matrix Native matrix2

1

3

Metal-treated matrix

Fe

Zn

Cu

0.830 ± 0.004

0.070 ± 0.004

0.039 ± 0.002

1.270 ± 0.005

0.064 ± 0.005

0.059 ± 0.002

0.666 ± 0.007

5. 699 ± 0.007

6.299 ± 0.003

1

The casted matrix was treated using an acidic EDTA treatment to remove metal ions.

2

The sample correspond to the pristine dried casted matrix.

3

The casted matrix was treated with Fe3+, Cu2+ and Zn2+ salts at pH 8.4.

Table 2. Stress-strain slope (kPa / %) for the different matrices, measured between 0-5% of strain for the metal-treated matrix and between 0-10% for the others. The strain % indicates the starting point of the densification region, which was arbitrarily identified as the point where the slope started to be higher than 0.2 (Figure SI3).

3

Metal-treated matrix Native matrix2

EDTA-treated matrix

1

Stress-strain slope

Strain (dens.)

(kPa/%)

(%)

1.3 ± 0.4

72 ± 8

0.5 ± 0.1

97 ± 2

0.3 ± 0.1

92 ± 6

1

The casted matrix was treated using an acidic EDTA treatment to remove metal ions.

2

The sample correspond to the pristine dried casted matrix.

3

The casted matrix was treated with Fe3+, Cu2+ and Zn2+ salts at pH 8.4.


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Figure 1. (A) A photo of a byssus removed from a mussel, indicating (1) the plaque, (2) the stem and (3) the thread. (B) A SEM image of a byssal thread that has been ripped open revealing (4) the cuticle and (5) the fibrous core. (C) A schematic representation of a PreCol, the main molecular protein component of the fibrous core.13, 15

Figure 2. The three different hydrated matrices obtained: (A) the metal-treated matrix, (B) the matrix after the synthesis and (C) the EDTA-treated matrix. The matrix becomes more dark increasing the metal content.


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Figure 3. AFM images of precipitate obtained from degraded byssal threads. top A lower magnification with two different population of fibers. bottom (A) a higher magnification of the smaller fibers. (B) a magnification of the bigger fiber population.

Figure 4. Polarized light microscopy images of the suspension obtained from the reaction at different times: 3 h (A), 6 h (B) and 18 h (C). The samples where stained with Picrosirius Red stain to enhance the birefringence of the fibers.


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Figure 5. Matrix sections stained with NBT (on the left) and Sirius (on the right). A and B are optical images of stained sections of the matrix. C and D are the same image observed using polarized light, to enhance the birefringent fibers. E and F are higher magnificence on the fibers.

Figure 6. (A) Optical image of a byssus matrix transversal section. (B) SEM image of the byssus matrix section. (C) SEM image of the byssus matrix observed from the top. The insets show higher magnifications. These images are representative of the whole sample.


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