Three-Dimensional Porous Sponges from Collagen Biowastes

Subscriber access provided by UNIV OF NEBRASKA - LINCOLN

Article

Three-Dimensional Porous Sponges from Collagen Bio-Wastes Ashokkumar Meiyazhagan, Alin Cristian Chipara, Narayanan Tharangattu Narayanan, Anumary Ayyappan, Sruthi Radhakrishnan, Thanikaivelan Palanisamy, Robert Vajtai, Mani A. Sendurai, and Pulickel M Ajayan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04582 • Publication Date (Web): 24 May 2016 Downloaded from http://pubs.acs.org on May 31, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Three-Dimensional Porous Sponges from Collagen Bio-Wastes Meiyazhagan Ashokkumar,†* Alin Cristian Chipara,† Narayanan Tharangattu Narayanan,§ Ayyappan Anumary,† ‡ Radhakrishnan Sruthi,† Palanisamy Thanikaivelan,‡ Robert Vajtai,† Sendurai A Mani¥ and Pulickel M Ajayan†* †

Department of Materials Science & NanoEngineering, Rice University, Houston, Texas, USA, Tata Institute of Fundamental Research-Centre for Interdisciplinary Sciences, Hyderabad, India, 500075 ‡ Advanced Materials Laboratory, Centre for Leather Apparels & Accessories Development (CLAD), Central Leather Research Institute (Council of Scientific and Industrial Research), Adyar, Chennai, India, 600020 §

¥

Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030 KEYWORDS: Collagen; Skin wastes; Porous sponge; Environmental Applications; Cell Viability ABSTRACT: Three dimensional, functional, porous scaffolds can find applications in a variety of fields. Here we report the synthesis of hierarchical, interconnected porous sponges using a simple freeze-drying technique, employing collagen extracted from animal skin wastes and superparamagnetic iron oxide nanoparticles. The ultra-lightweight, high surface area sponges exhibit excellent mechanical stability and enhanced functional properties such as high absorption capacity for oils and organic dye molecules. Additionally these bio-composite sponges display significant cellular biocompatibility, which opens new prospects in bio-medical uses. The approach highlight innovative ways of transforming bio-wastes into advanced hybrid materials using simple and scalable synthesis techniques.

sponge or scaffold is expected to possess eco-friendliness, high porosity, mechanical stiffness, and flexibility to accomplish wide range of applications.10, 11 Collagen based scaffolds were widely synthesized by hybridization with synthetic or natural polymers. Different polymers such as chitin,12 poly(DL-lactic-co-glycolic acid),13 etc. were mixed with collagen to form scaffolds with improved mechanical and degradation properties. The formed sponges or scaffolds were widely utilized in biomedical applications such as wound dressing agents, drug and gene carriers, and tissue growth due to their excellent biocompatibility and biodegradability.14-17 Although collagen possesses several fascinating characteristics and applications, they degrade easily under mild acidic and hot conditions. To improve their stability and degradation property, collagen is chemically cross-linked using cross-linking agents such as aldehydes,18 chromium,19 acyl azide,20 etc. or other physical treatments namely UV21 and gamma irradiation.22 These cross-linking agents form an exacerbating effect on the calcification of prosthesis materials and are also cytotoxic.23 To overcome these limitations, the collagen fibers were stabilized by blending them with iron oxide nanoparticles in aqueous or non-aqueous medium.24, 25 Porous 3D scaffolds developed using interconnected collagen fibrils will be highly intriguing for various environmental and bio-related applications. Here, collagen extracted from animal skin waste was mixed with super paramagnetic iron

1. INTRODUCTION The demand for the synthesis of distinct multifunctional composite materials continues to grow every day because of their diversified properties such as mechanical, thermal, biocompatible, biodegradable, electrical, magnetic, etc., which suits a myriad of attractive applications.1 Most of these composites were developed through the combination of hybrid organic-inorganic materials that are arranged into different hierarchies or structures and thus can perform a multitude of various functions. Although nature has inspired humans in developing new and advanced materials, precise assembling of nanoscopic modules into multifunctional macroscopic structures still remains challenging. Nevertheless, efforts are being made by researchers to develop biomimetic, hybrid and engineered composites with complex nanoscale features and interactions. This is an emerging and interdisciplinary area at the crossroads between life sciences, materials science, and nanotechnology. Numerous reports are available for the synthesis of different polymer-based sponges/scaffolds for applications such as tissue engineering,2-4 catalytic supports,5 water treatment,6 heavy metal removal,7 and oil spill remediation.8, 9 Collagen, a biodegradable natural polymer, is well suited for the preparation of hierarchical porous scaffolds. It is a biological byproduct from the leather industry, which can be used for novel material synthesis thereby reducing environmental waste. An ideal

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 15

at 110°C under vacuum to remove moisture. The mechanical data was acquired using a DMA Q800 in compression mode. The pure collagen and the composite sponge were exposed to different loading and unloading at 0.1, 0.5, 1, 1.5 and 2 N respectively (at a rate of 0.5 N/min). Both the pure collagen and composite sponge underwent a thermal sweep from -25 to 125°C with isothermal steps at -25, 25, 50, 75, 100 and 125°C. The isothermal steps each lasted 5 min and the material was heated and cooled at a constant rate of 2°C/min. Room temperature magnetic measurements of the formed sponges were studied using a vibrating sample magnetometer (VSM, Lakeshore Model 7410). Cell Viability Studies: For the study, 293T cells were cultured in a Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin. 1000 cells were seeded in a 96 well plate. About 0.1 mg of the sponge was added to the culture and the samples with cells was incubated for 24 and 48 h. The experiments were conducted in triplicates and average values were plotted. The MTS reagent was added to the cells and incubated for an hour before reading the optical density at 490nm using a microplate reader. The images were taken using the Zeiss Axio Observer A1 microscope with Axiovision software). The statistical significance between the control and the treated cells were analyzed by paired students t test. Oil and Dye Absorption Studies: The separation of oil from a mixture of oil and water was carried out by immersing a known weight of the SPION incorporated collagen sponge to a mixture of distilled water and engine oil. The magnetic field (~2000 Oe) of the permanent magnet was used to track the sponge and the samples were weighed at regular intervals from 5 to 60 min. The amount of oil absorbed by the sponge was calculated by subtracting the initial weight from final oil absorbed weight. The experiment was repeated and their average values were considered. The UV-Visible spectrophotometric dye absorption studies using the developed sponge were carried out using a UV-1800 Shimadzu UV-VIS-NIR spectrophotometer. The dye, Violet 90, used for this experiment was kindly provided by DyStar, India. A stock solution of about 50 ppm was prepared by dissolving Violet 90 in deionized water. Adsorption studies were carried out by immersing about 12 mg of the Col-SPION sponge into 10 ml of the prepared dye solution. The spectrum was scanned from 200-800 nm with 0.5 nm of scanning intervals. The change in concentration of dye was estimated spectrophotometrically at regular intervals (1 to 30 min) by monitoring the absorbance peak, which occurs at around λmax 554 nm.

oxide nanoparticles (SPION) for the stabilization of its triple helical structure and lyophilized to prepare highly interconnected magnetically responsive 3D sponges with very low bulk density (Figure. 1). The incorporation of ferro fluids into the collagen matrix would result in strong reinforcement and improvement in their properties and performance. The integration of magnetic functionality into the scaffolds without compromising its bulk density and open pore microstructure are expected to bring a paradigm shift in collagen-based applications. Due to the presence of an interconnected porous structure and multifunctional characteristics, we employed the developed sponge as an efficient candidate for biological and environmental applications. Insert Figure. 1 here

2.

EXPERIMENTAL SECTION Preparation of the Sponge: Collagen wastes in the form of bovine hide trimming pieces were collected from a local tannery at Chennai, India. The hide trimmings were processed using conventional technique to remove the unwanted non-proteinaceous materials, flesh and hair as reported elsewhere.26, 27 The processed hide pieces were soaked in 35 and 70% acetone followed by 100% methanol to completely remove the moisture. Finally, the hide pieces were thoroughly dried in a vacuum drier and grounded finely into powder using a Willy mill of mesh size 2 mm. The obtained dry hide powders were used as the source for further experiments. About 1 L stock solution of collagen (10 mg/ml) was prepared by soaking hide powder in 0.5 N acetic acid followed by blending sporadically to obtain a homogenous viscous collagen solution. To 10mL of collagen solution, 300 µL of SPION was added drop wise which was prepared as reported elsewhere.24, 28 and the whole contents were gently heated (40±3°C) and stirred for 12 h at 300 rpm. The homogenously mixed solutions were finally poured into glass templates and freeze dried at 4°C for 18 h. The developed Col-SPION sponges were removed from templates and stored for further studies. Characterization of the Sponge: The presence of functional groups corresponding to both the collagen and ColSPION sponges was analyzed using a Nicolet Fourier Transform-Infrared (FT-IR) spectrometer. Both samples were pressed into small pellets and analyzed in the range of 400-4000 cm-1. Each spectrum was obtained at an average of 50 scans with a resolution of 2 cm-1. Differential scanning calorimetric (DSC) analysis of both the sponges were carried out using TA instruments (Q600 simultaneous TGA/DSC) under nitrogen atmosphere at a flow rate of 50 ml/min. The heating was carried out from 25 to 130°C at a rate of 2°C/min. High resolution field emission scanning electron microscopic (SEM) analysis of the developed sponges was carried out using a FEI Quanta 400F ESEM FEG operated at 20 kV. The porosity and specific surface area of the sponges were determined using BrunauerEmmett-Teller (BET) method. The device Quantachrome Autosorb-3b BET Surface Analyzer was used for the analysis. Before analysis all the samples were evacuated for 24 h

3.

RESULTS AND DISCUSSION The method of preparation of porous SPION complexed collagen (Col-SPION) sponge with appreciable surface area using freeze-drying technique is illustrated in Figure.1. We observed that freeze-drying helps in preventing collapse of the interconnected network and also helps give rise to a hierarchical porous structure for multifunctional applications. The porous sponges with different shapes formed using freeze-drying are shown in Figure. 1. The formed SPION incorporated collagen sponges are lightweight, highly magnetic and get easily attracted towards permanent magnets

2


Page 3 of 15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces cannot act as an efficient material to hold foreign matter into their structure due to the absence of porosity. On other hand, the Col-SPION sponge shows a honeycomb-like porous structure with excellent interconnected linkages and an even distribution of pores (Figure. 3b and c). The presence of a minor amount of inorganic nanoparticles (SPION) had a noticeable influence on the microstructure of the developed sponge, indicating that the collagen fibrils were cross-linked with the inorganic nanoparticles forming greater networks. As a result, highly porous interconnected three-dimensional sponge with more pores was obtained (see Figure. S2 for additional electron microscopic images). These pores could anchor micro and nanoparticles and provide channels for facile migration of foreign matters and their by-products.

(~2000 Oe) (Figure. 1 inset and Supp. Movie SV1). The nature of magnetization was further confirmed using vibrating sample magnetometer (VSM) where the pure collagen sponge shows a diamagnetic behavior (Supp. Info. Figure. S1a), while Col-SPION sponge displays superparamagnetic behavior predominantly due to the presence of superparamagnetic iron oxide nanoparticles (Supp. Info. Figure. S1b). The infrared spectrum (Figure. 2a) corresponding to both native and SPION complexed collagen sponges exhibit similar characteristic peaks at 3307, 1636, 1548, 1450, and 1235cm-1. This indicates that the inclusion of SPION into the collagen matrix did not alter or modify the triple helical structure of the collagen in the composite sponge. The stretching corresponding to C=O of amide I occurs at 1636 cm-1, the sharp band at 1548 cm-1 corresponds to N-H bending of amide II, and the small peak around 1235 cm-1 is due to the presence of C-N stretching and N-H in plane bending of amide III. The presence of hydroxyl (O-H) groups is confirmed by the existence of a broad band at 3307 cm-1. The symmetric and asymmetric band corresponding to CH2 stretch is seen at 2920 cm-1. These observations are in agreement with previous reports.29

Insert Figure. 3 here To get better insight into porosity, pore sizes and surface area of the developed sponges, nitrogen adsorption-desorption isotherm measurements were carried out using BrunauerEmmett-Teller (BET) technique (Figure. 4). The Col-SPION sponge exhibited a BET surface area of 47.73 m2g-1, which is significantly higher than the pristine collagen sponge (see Supp. Info. Figure. S3). The pore size distribution of the composite sponge demonstrates the presence of a large number of macro pores with pore sizes around 20 to 60 µm (Figure. 4b). The analysis elucidates that the composite sponge consists of a mixture of both macropores (>50 nm) and mesopores (2-50 nm), which are considered most ideal for tissue engineering and several other applications.35

Insert Figure 2 here Thermal stability of the native collagen and Col-SPION sponges (Figure. 2b) was analyzed using differential scanning calorimetry (DSC). From previous literature, it is known that the native collagen exhibits shrinkage around 64±2°C, which primarily corresponds to the loss of the triple helical structure of collagen.24, 29, 30 Here, we observed a similar shrinkage value for the pristine collagen sponge. To improve the shrinkage temperature of native collagen, tanners and researchers employ metal salts such as chromium (III) sulfate, aluminum (III) sulfate and other salts based on titanium, zirconium, etc.31-33 These metal salts are anticipated to form multiple bonding with the collagen subunits thereby interacting strongly with carboxyl groups of amino acid side chains or through weak interactions within the peptide linkage. Similarly here, incorporation of SPION into collagen leads to fairly strong interaction of iron oxide nanoparticles with the triple helical structure of collagen thereby exhibiting remarkable improvement in the shrinkage temperature up to 92°C, as also observed in our previous study.24 Slight improvement in the shrinkage of the structure of Col-SPION sponge could be due to the freeze-drying technique adopted in the present study, which helps in creating a porous structure of the collagen macromolecules consequently paving way for the iron oxide nanoparticles to easily penetrate and interact with the fibrillar structure. The morphology of both sponges was analyzed using scanning electron microscopy (SEM) as shown in Figure. 3. The presence of porosity coupled with an interconnected pore network is observed only in Col-SPION composite sponge (Figure. 3b and c). Whereas, the pristine collagen sponge displays a sheath like appearance with a dense surface and fewer pores (Figure. 3a). In general, sponges or scaffolds with dense surfaces and reduced pores cannot act as a good absorbent or help in cell adhesion/proliferation due to the difficulty of interpenetration by foreign matter into the deeper structure.34 Hence, the pristine collagen sponge that we formed

Insert Figure. 4 here The incorporation of Fe3O4 nanoparticles into the collagen is anticipated to assist in strong interactions of nanoparticles with the collagen matrix paving way for the formation of a porous interconnected honeycomb structure, which eventually results in the enhancement of multifunctional properties of the developed composite sponge. However, presence of excess porosity can also reduce the mechanical properties of the sponge, leading to very slow structural recovery or permanent destruction. Appropriate mechanical responses are required for any material to be considered for a range of applications, specifically where mechanical stability is an essential prerequisite. Ideally, the material should temporarily deform upon loading with little structural collapse and recover to its original geometry upon unloading. In the case of structural collapse with a high degree of incomplete recovery or permanent deformation upon loading, the developed engineered materials are considered inappropriate for most applications. Dynamic mechanical analysis (DMA) for the pure collagen and Col-SPION composite was done using controlled forces under compressive mode. The load-unload data for both sponge materials exhibit moderate progressive cyclic stress softening (Figure. 5a and d). Although this is similar to the Mullins effect, this behavior differs since the softening is much lower than that would be expected from the Mullins effect.36 Additionally, the Col-SPION sponge (Figure. 5d) shows lower stiffness than the pure collagen sponge, which can be attributed to the

3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 15

supports cell adhesion, growth and migration by facilitating the transport of nutrients and regulatory factors.39 Also Fe3O4 nanoparticles are investigated with great interest for both in vitro and in vivo applications.40 These interesting characteristics along with the mechanical properties motivated us to look into the biocompatibility of both the sponges. The study was done using 293T cells on both the sponges and evaluated using (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay at 490 nm after 24 and 48 h of cell proliferation and the results are displayed in Figure. 6. Interestingly, the Col-SPION sponge did not inhibit cell proliferation relative to the untreated control cells. In fact, the 293T cells grow better in the presence of the Col-SPION sponge than the pristine collagen sponge after 24 and 48 h of cell seeding (Figure. 6e). This significant improvement in cell viability of the ColSPION sponge even after 48 h of cell seeding might be due to the presence of enhanced interconnected porosity, owing to the non-covalent crosslinking between collagen and Fe3O4 nanoparticles, which has considerable influence on the cell attachment, proliferation and migration. The Fe3O4 nanoparticles and collagen macromolecules are bound through electrostatic interactions between the positively charged side chain amino groups of collagen and the citric acid coated on the Fe3O4 nanoparticles.24 Due to this interaction, the Fe3O4 nanoparticles are anchored tightly to the collagen fibrils leading to improved mechanical performance and thus preventing swelling of the porous structure. Images acquired using a phase contrast microscope are shown in Figure. 6a-d, and they corroborate the observation in the MTS assay. Thus, the composite sponge developed in this study exhibited excellent biocompatibility and can be facilely used for various biomedical applications.

difference in density, structure (honeycomb vs. free layers) and porosity originating from non-covalent interactions and the freeze-drying protocol.37, 38 The dynamic mechanical data for the pure collagen and ColSPION composite shows trends originating from the honeycomb structure as seen through SEM (Figure. 3) and the BET data, which indicates differences in pore sizes. Strain data shows irreversible deformations in both pure collagen and the Col-SPION sponge; however, the Col-SPION sponge (Figure. 5d) exhibits a higher overall strain than pure collagen and is consistent with the low stiffness of the material. A nominal increase in residual strain (~52% for Col-SPION sponge as seen in Figure. 5d vs. ~48% for pure collagen as seen in Figure. 5a) in the Col-SPION sponge is also observed. The enhanced strain can be attributed to the inclination of the hexagonal walls of the composite due to loading, which lead to an increase in the moment of the system thus lowering the force necessary to increase strain. Both materials did not exhibit failure even up to 60% strain. However, the addition of iron oxide nanoparticles should increase the lifetime of the material as the softening effect continues with repeated cycles. Additional data on softening of pure collagen and Col-SPION sponge are given in Figure. S4. The addition of iron oxide nanoparticles into the collagen matrix dampened the system, as seen from high tan δ value (tan δ for pure collagen sponge ≈ 0.07, Figure. 5c; tan δ for Col-SPION sponge ≈ 0.09, Figure. 5f). This is due to the honeycomb structure created by the local immobile points and free space in the system. This implies that the engineered collagen sponge material has potential for high energy absorption and mechanical damping. Insert Figure. 5 here To further analyze the effects of iron oxide nanoparticles on collagen, we used a temperature sweep to identify the glass transition and shrinking of the material. The material was tested in compression using thermal sweeps with isothermal steps inserted in the procedure to prevent any thermal lag within the material (Figure. 5 b-c and e-f). The temperature sweep of the Col-SPION sponge revealed several key inflections in the material as well as shifts in behavior compared to pure collagen. The Col-SPION sponge displayed a shift in the shrinking temperature of collagen from 69°C (as seen in Figure. 5c and literature) to approximately 100°C (Figure. 5f). This data is in agreement with the DSC results (Figure. 2). Further mechanical studies of the sponge based on application of a constant static force over a time period are shown in Figure. S5. The obtained strain and stiffness data of both the sponges are in agreement with the above results. Overall, the honeycomb structure of the Col-SPION sponge elicits a low stiffness, fairly higher strain and higher damping than pure collagen. From the above observations, it is evident that the developed SPION incorporated collagen sponge exhibits a remarkable improvement in thermal stability, porosity, pore interconnectivity and mechanical properties. Hence, we further investigated the usability of this material for biological and environmental applications. Interconnected macroporous sponges/scaffolds are studied with great interest for tissue engineering applications, since it

Insert Figure. 6 here On the other hand, presence of an open interconnected porous structure and excellent thermo-mechanical properties are expected to assist in trapping oil contaminants6 or toxic organic dyes easily from water without absorption of water. We used the developed hybrid Col-SPION sponge as an effective absorbent to selectively remove motor oil from an oil-water mixture (Figure. 7a-c). A few milligrams of the formed hybrid sponge were added to the oil-water mixture and tracked throughout the petri dish using a permanent magnet (approx. 2000 Oe) to effectively trap the oil from the water all over their porous surfaces (Supp. Movie SV2). The hybrid sponge instantaneously absorbed the oil (Figure. 7c); such fast absorption kinetics could be attributed to the strong oleophilic nature of the hybrid sponge. Due to its low density and excellent interconnected porous structure, the hybrid sponge is found to float on the surface of water before and after absorbing the oil. Thus, the oil can be easily removed from water, which is of great significance for practical uses especially for spill remediation. The extraordinary performance of the hybrid sponge was predominantly due to critical point drying, where the surface area and pore volume of the formed sponge were increased tremendously, thus allowing the oil to easily penetrate into their porous structure exhibiting superoleophilic characteristics. From the graph (Figure. 7c), it can be observed that the oil absorption capability increases gradually

4


Page 5 of 15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


from 10 g/g to a maximum of 14 g/g as the time progresses. This experiment was repeated three times and the average values are plotted (Figure. 7c). These values are higher than those reported earlier for magnetic biocomposite materials.24 These results demonstrate that the as-prepared Col-SPION sponge can act as superior oleophilic materials to remove oil contaminants from water bodies. This developed biological composite material can also be utilized as facial oil-plotting material due to their excellent biocompatibility and for their efficient oil absorption properties. Another interesting application of the prepared Col-SPION sponges can be absorption of organic dyes from wastewater (Figure.7d-f). Here we tested the dye absorbing capability of the developed sponge using Violet 90. It is seen that the intensity of the absorption band with λmax at 554 nm corresponding to Violet 90 decreases as time progresses from 1 to 30 min (Figure. 7d). Moreover, the concentration of Violet 90 dye solution becomes negligible after 30 min (Figure. 7d and 7f). This result reveals that the absorption capability of the formed sponge increases with contact time. The presence of high porosity, low density and high absorption capabilities makes the Col-SPION sponge a remarkable absorbent for toxic dyes. The mechanism of absorption of toxic organic molecules is mainly physical, where the molecules are stored in the interconnected porous networks.

AUTHOR INFORMATION Corresponding Author * (Meiyazhagan Ashokkumar) Email: [email protected] ( Pulickel M Ajayan) Email: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT MA thanks USIEF, India for the Fulbright-Doctoral and Professional Research (DPR) Fellowship, Air Force Office of Scientific Research, (Grant No. AFOSR FA9550-13-1-0084) and Fred L. Hartley Family Foundation for financial assistance.

REFERENCES (1) Faivre, D., Multifunctional Materials Dry but flexible magnetic materials. Nature Nanotechnology 2010, 5, (8), 562-563. (2) Hirata, E.; Uo, M.; Takita, H.; Akasaka, T.; Watari, F.; Yokoyama, A., Development of a 3D Collagen Scaffold Coated With Multiwalled Carbon Nanotubes. Journal of Biomedical Materials Research Part B-Applied Biomaterials 2009, 90B, (2), 629-634. (3) Lee, C. K.; Shin, S. R.; Mun, J. Y.; Han, S. S.; So, I.; Jeon, J. H.; Kang, T. M.; Kim, S. L.; Whitten, P. G.; Wallace, G. G.; Spinks, G. M.; Kim, S. J., Tough Supersoft Sponge Fibers with Tunable Stiffness from a DNA Self-Assembly Technique. Angewandte Chemie-International Edition 2009, 48, (28), 51165120. (4) Svagan, A. J.; Samir, M. A. S. A.; Berglund, L. A., Biomimetic foams of high mechanical performance based on nanostructured cell walls reinforced by native cellulose nanofibrils. Advanced Materials 2008, 20, (7), 1263-+. (5) Anderson, M. L.; Stroud, R. M.; Rolison, D. R., Enhancing the activity of fuel-cell reactions by designing three-dimensional nanostructured architectures: Catalyst-modified carbon-silica composite aerogels (vol 2, pg 239, 2002). Nano Letters 2003, 3, (9), 1321-1321. (6) Choi, S. J.; Kwon, T. H.; Im, H.; Moon, D. I.; Baek, D. J.; Seol, M. L.; Duarte, J. P.; Choi, Y. K., A Polydimethylsiloxane (PDMS) Sponge for the Selective Absorption of Oil from Water. Acs Applied Materials & Interfaces 2011, 3, (12), 4552-4556. (7) Munoz, J. A.; Gonzalo, A.; Valiente, M., Arsenic adsorption by Fe(III)-loaded open-celled cellulose sponge. Thermodynamic and selectivity aspects. Environmental Science & Technology 2002, 36, (15), 3405-3411. (8) Moura, F. C. C.; Lago, R. M., Catalytic growth of carbon nanotubes and nanofibers on vermiculite to produce floatable hydrophobic "nanosponges" for oil spill remediation. Applied Catalysis B-Environmental 2009, 90, (3-4), 436-440. (9) Gui, X. C.; Wei, J. Q.; Wang, K. L.; Cao, A. Y.; Zhu, H. W.; Jia, Y.; Shu, Q. K.; Wu, D. H., Carbon Nanotube Sponges. Advanced Materials 2010, 22, (5), 617-+. (10) Wang, S.; Falk, M. M.; Rashad, A.; Saad, M. M.; Marques, A. C.; Almeida, R. M.; Marei, M. K.; Jain, H., Evaluation of 3D nano-macro porous bioactive glass scaffold for hard tissue engineering. Journal of Materials Science-Materials in Medicine 2011, 22, (5), 1195-1203.

4.

CONCLUSION In summary, we prepared a hierarchically arranged magnetic biocomposite sponge by freeze-drying technique using collagen extracted from biological waste source and superparamagnetic Fe3O4 nanoparticles. The nature of magnetization of both the sponges was confirmed using VSM. The addition of SPION to collagen and freeze drying stabilized its macromolecular structure thereby displaying appreciable improvement in structure and properties. Thermal stability of the Col-SPION sponge as analyzed through DSC shows improvement in shrinkage temperature due to non-covalent interaction of SPION with the triple helical structure of collagen. The Col-SPION sponge as observed through SEM and BET shows remarkable improvement in surface area and porosity when compared to native collagen sponge. Additionally, the hybrid sponge displays robust improvement in mechanical stability even at higher temperatures. We also demonstrate that the developed hybrid sponges can be utilized as an efficient material for biological and environmental applications suggesting a sustainable approach for the management of bio-waste. The results of this study hint that this new material has immense implications in several other areas apart from biological and environmental fields.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Magnetization results of the sponge material, BET studies, Additional SEM and DMA of the sponge materials

5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(11) Ramay, H. R.; Zhang, M. Q., Preparation of porous hydroxyapatite scaffolds by combination of the gel-casting and polymer sponge methods. Biomaterials 2003, 24, (19), 3293-3302. (12) Lee, S. B.; Kim, Y. H.; Chong, M. S.; Lee, Y. M., Preparation and characteristics of hybrid scaffolds composed of betachitin and collagen. Biomaterials 2004, 25, (12), 2309-2317. (13) Chen, G. P.; Ushida, T.; Tateishi, T., Hybrid biomaterials for tissue engineering: A preparative method for PLA or PLGAcollagen hybrid sponges. Advanced Materials 2000, 12, (6), 455+. (14) Friess, W., Collagen - biomaterial for drug delivery. European Journal of Pharmaceutics and Biopharmaceutics 1998, 45, (2), 113-136. (15) Shakespeare, P., Burn wound healing and skin substitutes. Burns 2001, 27, (5), 517-522. (16) Nimni, M. E.; Cheung, D.; Strates, B.; Kodama, M.; Sheikh, K., Chemically Modified Collagen - a Natural Biomaterial for Tissue Replacement. Journal of Biomedical Materials Research 1987, 21, (6), 741-771. (17) Yannas, I., Regeneration templates. The biomedical engineering handbook 1995, 1619-1633. (18) Sheu, M. T.; Huang, J. C.; Yeh, G. C.; Ho, H. O., Characterization of collagen gel solutions and collagen matrices for cell culture. Biomaterials 2001, 22, (13), 1713-1719. (19) Usha, R.; Ramasami, T., Effect of crosslinking agents (basic chromium sulfate and formaldehyde) on the thermal and thermomechanical stability of rat tail tendon collagen fibre. Thermochimica Acta 2000, 356, (1-2), 59-66. (20) Petite, H.; Rault, I.; Huc, A.; Menasche, P.; Herbage, D., Use of the Acyl Azide Method for Cross-Linking Collagen-Rich Tissues Such as Pericardium. Journal of Biomedical Materials Research 1990, 24, (2), 179-187. (21) Weadock, K. S.; Miller, E. J.; Bellincampi, L. D.; Zawadsky, J. P.; Dunn, M. G., Physical Cross-Linking of Collagen-Fibers Comparison of Ultraviolet-Irradiation and Dehydrothermal Treatment. Journal of Biomedical Materials Research 1995, 29, (11), 1373-1379. (22) Damink, L. H. H. O.; Dijkstra, P. J.; Vanluyn, M. J. A.; Vanwachem, P. B.; Nieuwenhuis, P.; Feijen, J., Influence of Ethylene-Oxide Gas Treatment on the in-Vitro Degradation Behavior of Dermal Sheep Collagen. Journal of Biomedical Materials Research 1995, 29, (2), 149-155. (23) Gough, J. E.; Scotchford, C. A.; Downes, S., Cytotoxicity of glutaraldehyde crosslinked collagen/poly(vinyl alcohol) films is by the mechanism of apoptosis. Journal of Biomedical Materials Research 2002, 61, (1), 121-130. (24) Thanikaivelan, P.; Narayanan, N. T.; Pradhan, B. K.; Ajayan, P. M., Collagen based magnetic nanocomposites for oil removal applications. Scientific Reports 2012, 2. (25) Alliraja, C.; Rao, J. R.; Thanikaivelan, P., Magnetic collagen fibers stabilized using functional iron oxide nanoparticles in nonaqueous medium. Rsc Advances 2015, 5, (27), 20939-20944. (26) Ashokkumar, M.; Sumukh, K. M.; Murali, R.; Narayanan, N. T.; Ajayan, P. M.; Thanikaivelan, P., Collagen-chitosan biocomposites produced using nanocarbons derived from goatskin waste. Carbon 2012, 50, (15), 5574-5582.

Page 6 of 15

(27) Meiyazhagan, A.; Thangavel, S.; Daniel, P. H.; Pulickel, M. A.; Palanisamy, T., Electrically conducting nanobiocomposites using carbon nanotubes and collagen waste fibers. Materials Chemistry and Physics 2015, 157, 8-15. (28) Narayanan, T. N.; Mary, A. P. R.; Shaijumon, M. M.; Ci, L. J.; Ajayan, P. M.; Anantharaman, M. R., On the synthesis and magnetic properties of multiwall carbon nanotubesuperparamagnetic iron oxide nanoparticle nanocomposites. Nanotechnology 2009, 20, (5). (29) Anumary, A.; Thanikaivelan, P.; Ashokkumar, M.; Kumar, R.; Sehgal, P. K.; Chandrasekaran, B., Synthesis and Characterization of Hybrid Biodegradable Films From Bovine Hide Collagen and Cellulose Derivatives for Biomedical Applications. Soft Materials 2013, 11, (2), 181-194. (30) Covington, A. D., Modern tanning chemistry. Chemical Society Reviews 1997, 26, (2), 111-126. (31) Sreeram, K. J.; Ramasami, T., Sustaining tanning process through conservation, recovery and better utilization of chromium. Resources Conservation and Recycling 2003, 38, (3), 185212. (32) Deng, D. H.; Tang, R.; Liao, X. P.; Shi, B., Using collagen fiber as a template to synthesize hierarchical mesoporous alumina fiber. Langmuir 2008, 24, (2), 368-370. (33) B. Shi, Y. D., Plant polyphenols. (1st ed.)Science Press, Beijing 2000, 73-91. (34) O'Brien, F. J.; Harley, B. A.; Yannas, I. V.; Gibson, L. J., The effect of pore size on cell adhesion in collagen-GAG scaffolds. Biomaterials 2005, 26, (4), 433-441. (35) Hofmann, S.; Hagenmuller, H.; Koch, A. M.; Muller, R.; Vunjak-Novakovic, G.; Kaplan, D. L.; Merkle, H. P.; Meinel, L., Control of in vitro tissue-engineered bone-like structures using human mesenchymal stem cells and porous silk scaffolds. Biomaterials 2007, 28, (6), 1152-1162. (36) Munster, S.; Jawerth, L. M.; Leslie, B. A.; Weitz, J. I.; Fabry, B.; Weitz, D. A., Strain history dependence of the nonlinear stress response of fibrin and collagen networks. Proceedings of the National Academy of Sciences of the United States of America 2013, 110, (30), 12197-12202. (37) Kinneberg, K. R. C., Tissue Engineering Strategies to Improve Tendon Healing and Insertion Site Integration. 2011, 159 3469920. (38) Lakes, R., Materials with Structural Hierarchy. Nature 1993, 361, (6412), 511-515. (39) Gelinsky, M.; Welzel, P. B.; Simon, P.; Bernhardt, A.; Konig, U., Porous three-dimensional scaffolds made of mineralised collagen: Preparation and properties of a biomimetic nanocomposite material for tissue engineering of bone. Chemical Engineering Journal 2008, 137, (1), 84-96. (40) Laurent, S.; Saei, A. A.; Behzadi, S.; Panahifar, A.; Mahmoudi, M., Superparamagnetic iron oxide nanoparticles for delivery of therapeutic agents: opportunities and challenges. Expert Opinion on Drug Delivery 2014, 11, (9), 1449-1470.

6


Page 7 of 15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure Captions Figure 1. Schematic showing the formation of hybrid composite sponge. Insets show different shapes of sponges, a sponge attracted towards a permanent magnet (~2000 Oe) and electron microscopic view of the composite sponge. Figure 2. (a) FTIR spectra of pristine collagen sponge and Col-SPION sponge; (b) DSC traces of pristine collagen sponge and Col-SPION sponge. Figure 3. Scanning electron microscopic images of (a) pristine collagen sponge and Col-SPION hybrid sponge at (b) lower and (c) higher magnifications showing hexagonal honeycomb like structure (marked in red). Figure 4. BET observation of the developed Col-SPION hybrid sponge. (a) Nitrogen adsorption-desorption isotherms and (b) Pore size distribution curve. Figure 5. DMA Analysis of pure collagen and Col-SPION composite sponge. (a-c) Show the DMA data of the pure collagen sponge. a) Load-unload cycles and deformation behavior for collagen sponge with a maximum strain over 55%. b) Change in stiffness of collagen sponge as temperature is decreasing (green region) and increasing (red region). The glass transition of collagen is highlighted in the figure. c) Changes in tan delta as the temperature decreases (green area) and increases (red area). (d-f) Show the DMA data for the Col-SPION composite sponge and demonstrate the load-unload cycle and deformation behavior (exhibiting a maximum strain over 60%) of the composite sponge (d), the change in stiffness with temperature (e), and the tan delta transitions with temperature (f). This more clearly highlights the transitions in the Col-SPION composite. Figure 6. Cell viability of developed sponges as determined by phase contrast microscopy and MTS assay; (a and c) Representative phase contrast images of 293T cells exposed to pristine collagen sponge for 24 and 48 h, respectively. (b and d) Representative phase contrast image of 293T cells exposed to Col-SPION sponge for 24 and 48 h, respectively. (e) Viability of 293T cells measured by MTS assay cultured in the presence of collagen, Col-SPION compared to untreated cells for 24 and 48 h at 0.1 mg concentrations as determined by MTS assay. Figure 7. Oil and dye absorption of the Col-SPION sponge. (a) Oil in water medium placed in petri dish. Inset shows small pieces of Col-SPION sponge used for the study. (b) Col-SPION sponge added to the oil in water medium placed in petri dish. Inset shows oil absorbed Col-SPION sponge and squeezed out oil. (c) Oil absorption ability of the Col-SPION sponge as a function of time using used motor oil. (d) UV-Visible absorption spectra displaying dye absorption ability of Col-SPION sponge as a function of time. (e) Concentrated dye solution before immersing Col-SPION sponge. (f) Image of dye solution taken after soaking Col-SPION sponge for 30 min.

7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 15

Figure 1. Schematic showing the formation of hybrid composite sponge. Insets show different shapes of sponges, a sponge attracted towards a permanent magnet (~2000 Oe) and electron microscopic view of the composite sponge.

8


Page 9 of 15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. (a) FTIR spectra of pristine collagen sponge and Col-SPION sponge; (b) DSC traces of pristine collagen sponge and ColSPION sponge.

9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 15

Figure 3. Scanning electron microscopic images of (a) pristine collagen sponge and Col-SPION hybrid sponge at (b) lower and (c) higher magnifications showing hexagonal honeycomb like structure (marked in red).

10


Page 11 of 15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. BET observation of the developed Col-SPION hybrid sponge. (a) Nitrogen adsorption-desorption isotherms and (b) Pore size distribution curve.

11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 15

Figure 5. DMA Analysis of pure collagen and Col-SPION composite sponge. (a-c) Show the DMA data of the pure collagen sponge. a) Load-unload cycles and deformation behavior for collagen sponge with a maximum strain over 55%. b) Change in stiffness of collagen sponge as temperature is decreasing (green region) and increasing (red region). The glass transition of collagen is highlighted in the figure. c) Changes in tan delta as the temperature decreases (green area) and increases (red area). (d-f) Show the DMA data for the Col-SPION composite sponge and demonstrate the load-unload cycle and deformation behavior (exhibiting a maximum strain over 60%) of the composite sponge (d), the change in stiffness with temperature (e), and the tan delta transitions with temperature (f). This more clearly highlights the transitions in the Col-SPION composite.

12


Page 13 of 15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Cell viability of developed sponges as determined by phase contrast microscopy and MTS assay; (a and c) Representative phase contrast images of 293T cells exposed to pristine collagen sponge for 24 and 48 h, respectively. (b and d) Representative phase contrast image of 293T cells exposed to Col-SPION sponge for 24 and 48 h, respectively. (e) Viability of 293T cells measured by MTS assay cultured in the presence of collagen, Col-SPION compared to untreated cells for 24 and 48 h at 0.1 mg concentrations as determined by MTS assay.

13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 15

Figure 7. Oil and dye absorption of the Col-SPION sponge. (a) Oil in water medium placed in petri dish. Inset shows small pieces of Col-SPION sponge used for the study. (b) Col-SPION sponge added to the oil in water medium placed in petri dish. Inset shows oil absorbed Col-SPION sponge and squeezed out oil. (c) Oil absorption ability of the Col-SPION sponge as a function of time using used motor oil. (d) UV-Visible absorption spectra displaying dye absorption ability of Col-SPION sponge as a function of time. (e) Concentrated dye solution before immersing Col-SPION sponge. (f) Image of dye solution taken after soaking Col-SPION sponge for 30 min.

14


Page 15 of 15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Porous sponge prepared using collagen bio-wastes and SPION 180x159mm (300 x 300 DPI)


Three-Dimensional Porous Sponges from Collagen Biowastes

Recommend Documents