Interconnecting Bone Nanoparticles by Ovalbumin Molecules to Build

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Surfaces, Interfaces, and Applications

Interconnecting Bone Nanoparticles by Ovalbumin Molecules to Build a Three-Dimensional Low-density and Tough Material Peter Samora Owuor, Thierry Tsafack, Hye Yoon Hwang, Mohamad Sajadi, Seohui Jung, Tong Li, Sandhya Susarla, Bingqing Wei, Robert Vajtai, Jun Lou, Sanjit Bhowmick, Chandra Sekhar Tiwary, and Pulickel M. Ajayan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b13681 • Publication Date (Web): 31 Oct 2018 Downloaded from http://pubs.acs.org on November 1, 2018

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ACS Applied Materials & Interfaces

Interconnecting Bone Nanoparticles by Ovalbumin Molecules to Build a Three-Dimensional Low-density and Tough Material Peter Samora Owuor1, Thierry Tsafack1, HyeYoonHwang1, Mohamed Sajadi1, Seohui Jung 1,Tong Li2, Sandhya Susarla1, Bingqing Wei2, Robert Vajtai1,Jun Lou1,Sanjit Bhowmick3 , Chandra Sekhar Tiwary*1,4,Pulickel M. Ajayan*1 1Department

of Materials Science and NanoEngineering, Rice University, Houston, Texas

77005, USA 2University of Delaware, Delaware, 19716, USA 3Bruker Nano Surfaces, Minneapolis, MN, USA 55344. 4 Metallurgical and materials engineering, Indian Institute of Technology, Kharagpur, WestBengal, India. 721302.

Abstract Natural building blocks like proteins and hydroxyapatite (HA) are found in abundance. However, their effective utilization to fabricate environmentally friendly strong, stiff and tough materials remains a challenge. This work reports on the synthetic of a layered material from entirely natural building blocks. A simple process to extract HA from bones, while keeping collagen intact, is presented. These HA nanocrystals have a high aspect ratio as a result of the extraction method that largely retains the pristine nature of the HA. To fabricate the materials, polymerized egg white is used to induce toughness to the crystals where it acts like load transfer entity between the crystals. As shown by atomic force microscope modulus mapping, the result is a layered material with a modulus that ranges from 3GPa to 180GPa. Furthermore, the material exhibits self-stiffening behavior. Hydrogen and ionic bonds are likely to regulate the chemical interactions at the egg white/HA interface and are likely to be responsible for the observed high toughness and stiffness, respectively. The use of the HA/egg white composite as printed scaffolds is also demonstrated together with their biocompatibility. Keywords: Hydroxyapatite, egg white, polymerization, 3D printing, primary amine Corresponding Authors: [email protected], [email protected]

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Introduction The efficiency of materials design achieved by nature is still unmatched by their synthetic counterparts.1–3 However, great strides have been achieved in mimicking natural designs to create multifunctional materials.2,4–7 What makes natural designs so remarkable is their ability to utilize simple molecules, through a combination of hierarchy and geometry,8–10 to make very strong and tough materials.11–18 As recently demonstrated by Kotov lab,19 the parallel micro/nanoscale ceramics columns in a soft polymer of the tooth enamel is responsible for the extreme vibration resistance observed. The same design principles are exhibited by the wellstudied nacre structure.2,3,12,15,20,21 Efforts to mimic such elegant natural designs still rely heavily on the use of synthetic building blocks which themselves are still limited. For instance, the inorganic synthetic reinforcing ‘bricks’ in nacre need surface modification to induce high affinity with the ductile polymer matrix.1,15,16,22 On the other hand, natural building blocks are in abundance but difficult to use effectively. If judicious processing methods can be developed to take advantage of them, advanced environment-friendly materials with improved multifunctionality can be designed. Such environment-friendly materials can be prime candidates for various biomedical applications like bone healing.15,23–27 For example, an autologous bone graft is the traditional method for healing bone damage28,29 but it falls short as a safer material, the patient has to endure pain during the process, and other consequences like contamination may occur. Creating functional and effective bone scaffolds has been attempted with synthetic and biomaterials or a combination of the two with varying success.28,29 For the organic components, collagen and polylactic acid have been used while ceramic and calcium phosphate derivatives have been selected for the inorganic phase.30 The majority of scaffolds are made from pure hydroxyapatite tri-calcium


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phosphate ceramic or other calcium phosphate derivatives which are inherently limited by the lack of interconnected pores resulting in frequent fractures.29 Another method, three-dimensional (3D) powder printing is also limited by poor degradability31 of final products. In addition, 3D powder printing by-products, mainly brushite and tricalcium phosphate, are insoluble in the human body.31 Other rapid prototyping methods may be used but the materials, polylactic acid (PLA) and collagen derivatives, lack structural integrity and are not bio-compatible with the body.30 In the current work, a method to design a layered structure of hydroxyapatite (HA) and polymerized egg white is presented. Hydroxyapatite (HA) is extracted from bones without degrading the collagenous structure using an acid (citric acid) in a simple etching method. Egg white is polymerized by addition of primary amine compounds such as urea, diethylenetriamine (DETA) and phenylethylamine. We have recently shown that amine-based molecules introduce strong bonds with carboxylic ends of albumen amino acids in condensation reaction.32 The introduction of amine compounds into a stiff albumen results into a ductile interconnected network. The interconnected networks comprise interfaces of hard (albumen) and soft part (amine) leading to a tough cross-linked egg white material. The resulting complete biodegradable matrix of cross-linked egg white is then used to provide ductility to HA crystals. The composite exhibits high modulus and stiffening response in dynamic loads. Incorporating polymerized egg white into hydroxyapatite complements the structural fragility of hydroxyapatite. Due to their natural occurring, egg white and hydroxyapatite prevent the necessity for removing the bone scaffolds from the body during malfunctioning. The procedure is also relatively simple and cost-effective. To make the scaffolds, predetermined structures like bone or ear mold is printed on 3D printer using polyvinly alcohol (PVA) filament. A major



advantage of 3D printed scaffolds is the high degree of precision and complexity that can be achieved at micron level details. This means complex scaffolds that were not possible or timeconsuming with conventional methods are now possible. Furthermore, 3D printing tends to produce scaffolds faster than traditional methods thus being more cost-effective in the long term. Utilizing 3D printing, HA/egg white is cured in a 3D printed mold by PVA filament. Removing the PVA mold is a quick procedure due to the high solubility of PVA in water. This work opens new possibilities for efficient use of bio-derived building blocks to provide solutions to some of the bottlenecks faced by material scientists. Result and Discussion A simple one-step chemical process protocol is adopted to synthesize hydroxyapatatite (HA) from bones. The bones are first dried in an oven, dipped in hydrogen peroxide to remove any remaining soft tissue33 and placed in solution of citric acid. The citric acid opens the collagenous matrix to release the ‘brick’ like HA as shown in Fig. S1. Unlike other methods of extracting HA bone, this method retains the collagenous matrix morphology with no degradation (Fig. S2). Transmission electron microscope (TEM) shows the HA crystals are a combination of regular and irregular shapes (Figs. S3a and b). The bricks are polycrystalline as shown by diffraction pattern from the TEM (inset Fig. S3c). Also, x-ray diffraction of the bricks exhibits a typical pattern associated with polycrystalline materials (Fig. S4). The peaks are observed at 32°, 40°, 47° and 51° with the highest peak at 32°. These peaks are similar to what is normally observed for HA. To ascertain this, FTIR test is done to the powder and signatures for –PO4, CO3 and O2 are clearly visible (Fig. S5). The XPS plots show the constituents elements present in hydroxyapatite. The spin orbit splitting of Ca 2p shows that it exists as Ca2+. The detailed fitting of C 1s however shows three peaks: C-C; C-N/C-O; O-C=O confirming the presence of some organic component in addition to the inorganic one. This is further confirmed by detailed fitting


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of N 1s and O 2p peaks which again shows the presence of C=N; C-NH2 and C-O; C-OH respectively. This method of producing HA in a single procedure in large quantity is important for wide applications of these nano-bricks (Fig. S6). Following the typical procedure to design nacre-like structure, we use liquid egg white ‘polymerized’ by primary amine compounds (diethylenetriamine in this case) as a biopolymer to provide flexibility to the inorganic HA. Fig. 1a shows an artistic representation for the adopted methodology to make the material. Primary amine compounds such as urea, phenylethylamine, diethylenetriamine are effective crosslinkers for liquid egg white and here we use diethylenetriamine to cross-link the egg white. The mixture is later added to the inorganic bricks from bones and cured as shown by SEM images (Figs. 1b and c) resulting in a three-dimensional materials (Fig. 1d). The material is inherently layered, similarly to nacre (Figs. 1e, f, and g). High magnification SEM images depict a material where the polymerized egg white has formed the junctions between the inorganic HA. Unlike other nacre-like synthetic materials that have to rely on the surface modification of the inorganic bricks, this work shows that bio-derived building blocks do not require such sophisticated methods to achieve structural stability. On the other hand, the inorganic bricks assembled in the absence of polymerized egg white does not possess the mechanical stability shown by the HA/egg white as clearly exhibited by cracks development on the structure (Fig. S7). The polymerized egg white acts like an effective glue to the bricks resulting in the observed high mechanical stability. Mechanical characterization is done on an Instron instrument to understand the addition of polymerized egg white in the inorganic bricks of HA under compression. There is an increase of load with strain for HA/egg white material (Fig. 2a). The curve is characterized by the zigzag form, an indication of stepwise failure. The ultimate tensile strength of the HA/egg white



materials is 1.86MPa compared to only 0.03MPa for the pristine HA powder as shown in Fig. 2b. Modulus mapping on the atomic force microscope (AFM) shows a ranging modulus of 0.6 – 8.9 GPa (Figs. 2c, S8a) for the pristine HA while the HA/egg white exhibits a modulus of 0.030.18TPa (Figs. 2d, S8b). The addition of cross-linked egg white greatly improves on the modulus of the composite. Egg white by itself is an extremely brittle material with high stiffness, but once cross-linked by primary amine compounds, elasticity is induced. It is therefore believed that the high stiffness of egg white combines with the HA crystals to result in a strong and tough material. Layered materials such as nacre34,35 are thought as next frontier in designing synthetic materials with improved mechanical properties. It has been suggested that reinforcements within the nanometer scale are much stronger than those in micrometer due to their high aspect ratio in the matrix.36 Many failure mechanisms of such nanocomposites have been proposed. For instance it has been shown that discrete element model can reliably model the random staggered37 reinforcement such as aragonite ‘nanobrick’. Indeed, interfaces with inherent large shear strain along with high hardening influences strength, toughness and stiffness of the nanocomposite. In the present study, we envision HA to provide the load carrying capacity while the elastic cross-linked egg white provides the load or stress transfer required to achieve the above mechanical properties. In addition, the non-uniformity of the HA may impede smooth or efficiency load carrying capacity of the nanocomposite. However, when a crack develops, it does travel in a straight line rather in a staggered manner which is the ideal situation. Local deformation is important to understand the failure mechanism of nanocomposite. The layered material deformation test was conducted using an in-situ nano-indentation test equipped with an SEM.38,39 Load-displacement curves are shown in Fig. 2e. For consistency, the test is done under load control at the same peak force. High elastic modulus was observed at the


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composite of HA/egg region. The unloading curve exhibits a complete elastic recovery at HA/egg sample unlike only HA that has shown higher plastic strain due to lack of ‘glue’ between the bricks. In-situ SEM indentation shows large plastic deformation creating a crater after unloading on the pristine HA (Fig. 2f) unlike HA/egg white which does not show any large scale observable deformation (Fig. 2g). With such desirable mechanical properties and the easy of making, HA/egg white material can find application in biomedical field where biodegradable scaffolds are in high demand. To overcome some of the complexity designs encountered in such scaffolds, 3D printing can be an important tool to solve those issues. The noticeable increase in toughness induced by the addition of Egg white (EW) and diethylenetriamine (DETA) to calcium hydroxyapatite (HA) can be attributed to the nature of the chemical interactions between HA surface ions on one hand and EW amino acid terminals and water molecules on the other hand. Calcium hydroxyapatite, Ca10(PO4)6(OH)2, is a highly porous hexagonal crystal40 whose unit cell can be mapped into a illustrative oval in Fig. 3. It is a network of six phosphate groups (PO43-) held together by coulomb interactions between the double-bonded oxygen in the phosphate group and one calcium cation (Ca++) located at the center of the structure (see dash-dot black lines in Fig. 3). Additional Ca++ ions electrostatically connect two O- ions belonging to each phosphate group (see dotted black lines in Fig. 3). Other Ca++ ions connect the remaining O- ions belonging to two adjacent phosphate groups (see dash black lines in Fig. 3). Hydroxyl ions OH- located in opposite sides of the circle add to the ionic nature of HA’s structure. This high porosity along with strong and localized electrostatic interactions hold PO43-, Ca++, and OH- ions together thereby giving HA not only its unique combination of lightweight and stiffness but also its brittleness. Almora-Barrios at al.,41 showed that HA surfaces show different patterns of PO43-, Ca++, O- and OH- ions that are chemically



favorable to interfacing with highly polar and/or ionic molecules. The water molecules (88%) and the amino acids32 (11%) comprising egg white as well as DETA, do indeed match the chemical environment at HA surfaces in two fundamental respects: (1) Hydrogen bonds form between water molecules in EW and OH-/O- ions at the surface of HA. They also form between amine terminals (NH2/NH3+) in normal/zwitterionic glutamic acid and OH-/O- ions at the surface of HA (see dash blue lines in Fig. 3). Additionally, hydrogen bonds form between DETA’s amine ends (NH2) and OH-/O- ions at the surface of HA. (2) Ionic bonds form between O/O- ends of normal/zwitterionic glutamic acid in EW and Ca++ ions on the surface of HA (see dash-dot blue lines in Fig. 3). While ionic bonds at the HA/egg white interface are likely to contribute to the stiffness, hydrogen bonds, because of their reformable nature, tend to prevent crack propagation and increase the composite plastic regime, thus significantly increasing the composite’s toughness. The combination of hydrogen and ionic bonds at the interface between EW and HA is believed to be responsible for the remarkable increase in ductility without loss in stiffness. Three-dimensional (3D) printing has received a lot of attention in recent years. The possibility of printing human tissues and organs is being pursued by many research groups. However, there still exists plethora of technological and biological challenges such biocompatibility, high thoughput etc. that require a significant amount of research to overcome. Typically bio-printing still follows three methodologies; pre-processing, processing and postprocessing. The pre-processing stage is where the ‘blue print’ for the organ is created by utilizing bio-imaging techniques. The processing stage involves the actual printing process by bioprinters. Finally, the post-processing will entail important procedures in transforming printed


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structures in functional engineered organs. In our 3D printed materials from HA and egg white, we first design molds (bone and ear). Then the bone- and ear-like molds are printed using polyvinyl alcohol (PVA) filament. Once finished, mixed HA/egg white are poured in and cured. Polyvinyl alcohol is finally dissolved in water to result in the predetermined structures (Fig. 4a). The ‘artificial bone’ printed has a diameter of 11mm and weigh 9.6g and can carry a weight of 500g (Fig. S9) The same procedure is used to make an ear-like structure (Fig. 4b). The stiffness measured on a section of the ‘artificial bone’ show a self-stiffening tendency (Fig. 4c) with high stiffness. Damping or loss factor under temperature was also done show a constant value of 0.5 from room temperature to around 78°C and decreases above this temperature. For typical application, this is an acceptable temperature range for the material to retain its stiffness (Fig. S10). The above biological applications necessitate the need to study biocompatibility of the HA/egg white. The biocompatibility of HA/egg white was assessed by evaluation of in-vitro cell cultures utilizing chondrocyte cells. The sample was put in cell media and toxicity of the material measured via MTT test. The test is done over a period of 7 days where the cells shows better cell growth over the said period (Fig. 4d). Conclusion This work has shown that natural building blocks, proteins and hydroxyapatite from readily available sources, can be used to design layered material exhibiting high strength and toughness. Hydroxyapatite (HA) nanocrystals were extracted from waste bones in a simple method that leaves the collagen matrix intact. These hydroxyapatite nanocrystals were then reinforced by polymerized liquid egg white responsible for the toughness required. The liquid egg white was polymerized by addition of primary compounds like diethylenetriamine (DETA) where amine ends from these molecules reacted with carboxylic ends in amino acids in a condensation process



to form peptide bonds, a process identical to the polymerization procedurre performed by Owuor et al.,32 Experimental tests reveal high modulus, stiffness and toughness of the HA with polymerized egg white. The high toughness likely occurs as a result of the nature of the chemical interaction between the polymerized egg white amino acids and surface ions on the HA. Polymerized egg white provides ductility while ionic bonds at the HA/egg white interface contributes to the high stiffness. In addition, hydrogen bonds because of their reformable nature serve as an obstacle to crack propagation within the composite. The use of the HA/egg white composite as printed scaffolds is also demonstrated together with their biocompatibility. This work opens new avenues of utilizing naturally available building blocks to fabricate multifunctionality materials. Experimental Section Hydroxyapatite crystals and composite synthesis To extract the hydroxyapatite (HA) from bone, chicken bones are first washed to remove any impurities. Next, the bones are dried at 90C for 24 hours. The bones are later immersed in a prepared 30 wt.% H2O2 to remove soft tissues and fats. Citric acid (20- 4 wt.0%) is then used to remove HA from bone leaving the collagen matrix intact. The powder are oven dried to remove any remnant solvent. The composite is made by first adding the polymerization initiator to the egg white liquid which are primary amine compounds such urea, phenylethylamine, diethylenetriamine (DETA) (Sigma Aldrich, USA). This discussion is based on DETA. A 10% DETA is added to egg white liquid and this mixture is later added to the HA crystals. Egg white/DETA makes 5-10% of HA. The whole mixture is later cured at 55˚C for an hour (Across International, China) to result in material (θ = 10mm, t = 2mm) with a density of 0.11gcm-3. As a comparison, HA powders without polymerized egg white are also made as the baseline samples.


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Morphology characterization The morphology characterizations are done on a FEI Quanta 400 ESEM and transmission electron microscopy (TEM). Mechanical Characterization Mechanical response was done on an Instron (USA) in a compression mode at a strain rate of 2mm/min on circular samples (θ = 10mm, t = 2mm). Nano-indentation tests were done on a quasi-static uniaxial compression loading inside a scanning electron microscope (SEM) utilizing a SEM PicoIndenter, xrPI85 (Hysitron, Inc., USA). Local mapping were done on an atomic force microscope (AFM) with contact mode cantilever (FESP) equipped with a spring constant of about 5N/m is used. Stiffness of the 'artificial bone' section is done on a dynamical mechanical analysis (DMA) Q800 (TA Instrument, USA). The test is conducted in a compression mode at ambient conditions with a 1Hz cycle in a controlled strain rate mode. For all mechanical tests, at least three samples are tested for each test. 3D models of bone and ear The 3D models of 'artificial bone and ear' are first designed on Solidworks. The files are then changed into STL format and printed on a 3D printer with a resolution of 100µm by 100µm using poly lactic acid (PVA) filament. The HA/egg white is then filled in these molds, cured and water used to dissolve PVA from the structure. A major concern with any scaffold is the ability to completely biodegrade without causing any side effects. A cell viability test is also conducted to show that cells can grow on the HA/cross-linked material showing the possibility that microorganisms may degrade the material.



Supporting Information Fig. S1 Procedure to remove nano-ceramics from bones. Fig. S2 Transparent collagen structure after nano-ceramics removal. Fig. S3 TEM images of the nano-ceramics. Fig. S4 XRD pattern. Fig. S5 FTIR spectra. Fig. S6 XPS fitting of the nano-ceramics. Fig. S7 Cracks formation in pristine nano-ceramics material without polymerized egg white. Fig. S8 Three dimensional modulus mapping. Fig. S9 Strength of artificial bone. Fig S10 Tan delta values. Notes: The authors declare no competing financial interest. Acknowledgements The authors thank the Air Force Office of Scientific Research (Grant FA9550-13-1-0084) for funding this research, and Air Force Office of Scientific Research MURI Grant FA9550-12-10035 financial support of this research.

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Fig. 1 Synthesis of hydroxyapatite. (a) An artistic visualization of the process adopted to synthesize HA/egg white, DETA/egg white is added to HA cured at 50˚C. The polymerized egg



white with diethylenetriamine (DETA) acts like glue providing load carrying property to the composite. (b) SEM of hydroxyapatite nanocrystals from bones showing ‘brick’ like morphology with high surface area (b,c). (d) Three dimensional composite of HA/polymerized egg white after curing. Hydroxyapatite/egg white composite with its layered structure as observed from the top (e), side view (f) and inner (g). Clear interconnections cab be seen between the inorganic crystals and egg white/DETA polymer.


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Fig. 2 Mechanical characterization (a) Compression test showing high load carrying capability of HA/egg white with a characteristic step-wise failure. (b) High ultimate tensile strength of polymerized egg white/HA. (c) AFM modulus mapping of HA with no polymer with a modulus of 0.6 to 8.9 GPa. (d) Extremely high modulus (30-180GPa) of reinforced HA. (e) Local in-situ loading exhibiting a high modulus of the sample. (f) In-situ SEM showing the failure of HA with no polymer unlike reinforced HA (g) which does not exhibit major failure (scale bar 50µm).


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Fig. 3 Chemical reactions likely to occur at the interface between HA and eggwhite. Illustrative structure of hydroxyapatite (light-blue oval) interfacing with water molecules, DETA and normal/zwitterionic glutamic acid. Glutamic acid is taken here as an example of amino acid present in eggwhite. Coulomb (dash-dot blue lines) and hydrogen interactions (dash blue lines) at the interface play key roles in the composite’s toughness.



Fig. 4 Fabrication of model ‘artificial bone and ear’ (a,b) Bone and ear are modeled in solid works and its corresponding mold printed in a 3D printer with polyvinyl alcohol (PVA) filament. HA/egg white is then filled into the mold, cured and later PVA dissolved in water. (c) Stiffening behavior of a bone section. (d) Biocompatibility test of HA/egg white composite


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Interconnecting Bone Nanoparticles by Ovalbumin Molecules to Build

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