Bio-inspired mineralization of hydroxyapatite in 3D silk fibroin hydrogel for bone tissue engineering

Accepted Manuscript Title: Bio-inspired Mineralization of Hydroxyapatite in 3D Silk Fibroin Hydrogel for Bone Tissue Engineering Author: Yashi Jin Banani Kundu Yurong Cai Subhas C. Kundu Juming Yao PII: DOI: Reference:

S0927-7765(15)30046-1 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.07.015 COLSUB 7216

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

18-3-2015 28-5-2015 7-7-2015

Please cite this article as: Yashi Jin, Banani Kundu, Yurong Cai, Subhas C.Kundu, Juming Yao, Bio-inspired Mineralization of Hydroxyapatite in 3D Silk Fibroin Hydrogel for Bone Tissue Engineering, Colloids and Surfaces B: Biointerfaces http://dx.doi.org/10.1016/j.colsurfb.2015.07.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Bio-inspired Mineralization of Hydroxyapatite in 3D Silk Fibroin Hydrogel for Bone Tissue Engineering Yashi Jin1, Banani Kundu2,3, Yurong Cai1, Subhas C. Kundu2, Juming Yao1*

1

The Key Laboratory of Advanced Textile Materials and Manufacturing Technology of

Ministry of Education, College of Materials and Textiles, Zhejiang Sci-Tech University, Hangzhou 310018, China 2

Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal -

721302, India 3

Institute of Tissue Regeneration Engineering (ITREN) & Department of Nanobiomedical Science BK21 Plus NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 330-714, South Korea

Graphical abstract

Highlights: 

Silk fibroin hydrogel fabricated in a self-assembled way without any additives.

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Hydroxyapatite formed in silk fibroin hydrogel scaffold via a biomimetic method.

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Ca2+ inserted hydrogel provides the oriented sites for the nucleation of crystals.

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Organic-inorganic hybrid composition improves the compressive strength of hydrogel.

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The mineralized hydrogel is the promising candidate material for bone repair.

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* Corresponding author:

Email: yaoj@ zstu.edu.cn (Juming Yao) Tel.: (86-571)8684-3618 Fax: (86-571)8684-3619 Address: 5 Second Avenue, Xiasha Higher Education Zone, Hangzhou, China, 310018 1

Bio-inspired Mineralization of Hydroxyapatite in 3D Silk Fibroin Hydrogel for Bone Tissue Engineering

Abstract To fabricate hard tissue implants with bone-like structure using a biomimetic mineralization method is drawing much more attentions in bone tissue engineering. The present work focuses in designing 3D silk fibroin hydrogel to modulate the nucleation and growth of hydroxyapatite crystals via a simple ion diffusion method. The study indicates that Ca2+ incorporation within the hydrogel provides the nucleation sites for hydroxyapatite crystals and subsequently regulates their oriented growth. The mineralization process is regulated in a Ca2+ concentration- and minerlization timedependent way. Further, the compressive strength of the mineralized hydrogels is directly proportional with the mineral content in hydrogel. The orchestrated organic/inorganic composite supports well the viability and proliferation of human osteoblast cells; improved cyto-compatibility with increased mineral content. Together, the present investigation reports a simple and biomimetic process to fabricate 3D bonelike biomaterial with desired efficacy to repair bone defects.

Keywords: Silk fibroin; hydrogel; crystal growth; hydroxyapatite; biomaterials; bone tissue engineering

1.Introduction Biominerals exist widely in nature, often with precise architectural order over several length scales. The orchestrated structure endows biominerals with excellent characteristics including unique optical properties, high strength and fracture toughness over other synthetic materials [1,2]. The detail insight of biomineralization progression is still elusive due to its complex nature. The efforts are made to figure out including the phase transitions during the mineral formation [3] and the role of organic matrices [4]. The biomineralization process in living system is critically influenced by the organic 2

components, which construct a hydrogel environment to regulate the crystal nucleation and growth, confine the crystal size and shape and finally assemble the crystals into complex structures through molecular recognition in mild environments [5]. Bone is a typical biomineral composed of 10-20 % collagen, 60-70 % minerals (approximately defined as hydroxyapatite) and 9–20 % water by weight. In addition, it contains small quantities of other organic compounds, such as proteins, polysaccharides and lipids [6]. The deformation and degeneration of bone with age, trauma, congenital defects, and tumor cause not repairable damage to bone, result in increasing requirements for bone implants. Reproducing natural mineralization in the laboratory to fabricate biominerals with tailored physiochemical property is an attractive prospect for bone tissue regenerative therapy. Recently, hydrogels gained popularity due to their intrinsic elasticity, water retention ability and structural homology with 3D collagen matrix of natural bone [7, 8] and considered to serve as ideal candidates for bonelike biomaterials from structure and composition point view of [9]. Silk is natural proteinaceous biopolymer, chiefly consist of glue like protein sericin and core protein fibroin, which possess outstanding biocompatibility, biodegradability, and low inflammatory response [10-13]. High-technology applications such as ligament tissue repair patches [14-16], guided bone regeneration [17, 18] and conduits for nerve [19] or blood vessel [20, 21] based on silk biomaterials are already reported. To mimetic the organic–inorganic hybrid tissue, researchers develop silk-based HAps composites materials such as co-solution of SF and SBF (SF/1.5SBF) is used to find out the SF mediated nucleation of HAps and self-assembly of SF in SBF [22]. Pandaa et al. fabricates a blended eri-tasar silk fibroin nanofibrous scaffold with nHAp deposition on the surface; results in improved osteogenic differentiation [23]. Low crystalline hydroxyapatite (LHA) modified silk fibroin scaffold is also employed for successful repair of bone/ligament defects [24]. However, mineralization in silk fibroin framework more closely mimics the biomineralization under physiological condition. In the present report, silk fibroin hydrogel system is designed, which serves as a 3D niche to regulate the nucleation and growth of hydroxyapatite crystals via a simple 3

ion diffusion method. The aim is to understand further the growth process of biomineral via a biomimetic method for subsequent preparation of biomedical materials. The cytocompatibility of this mineralized hydrogel is also investigated using human osteosarcoma cells (MG-63 cells).

2.Materials and Methods 2.1. Materials Na2CO3, LiBr, NaOH, CaCl2 and Na2HPO4 were analytical grade (Mike Chemical Agents Company, Hangzhou, China), MTT (Sigma, USA), tissue culture grade polystyrene plastic flasks and plates (Tarsons, India), Dulbecco’s modified eagle medium (DMEM), fetal calf serum, trypsin, penicillin-streptomycin antibiotics (Gibco BRL, USA) and alamar blue (Invitrogen, USA) were purchased for this study. B. mori silkworm cocoons were obtained from Huzhou Academy of Agricultural Science, China. All solutions were prepared with picopure water (Synergy Millipore, Billerica, MA; resistivity 18 MΩ·cm at 25 °C) Human osteoblast-like cells (MG-63) were obtained from National Centre for Cell Science (NCCS), Pune, India. 2.2. Preparation of silk fibroin solution and hydrogel Bombyx mori 6 wt% regenerated silk fibroin solution was prepared following the established protocol [25]. The concentration of the silk fibroin solution was diluted to 5 wt% using CaCl2 solutions with different Ca2+ concentrations (0, 10, 30 and 80 mM). The mixture was dwelled at 60 ℃ for 24 h to induce gelation [26]. The hydrogel thus obtained was cut into 10×10×10 mm3 cubes and kept in refrigerator for further use. 2.3. In vitro mineralization within silk fibroin hydrogel Gels with different Ca2+ concentrations (10, 30 and 80 mM) were immersed in 50 ml Na2HPO4 solution (pH 8.5) for mineralization. Further, individual investigation was carried out to evaluate the effect of phosphate concentration and time on mineralization. Concentration-dependent mineralization using various phosphate concentrations (6, 18 and 48 mM respectively) was carried out at 37℃ for 24 h; while time-dependent 4

mineralization (at time points like 0.5, 2, 6, 12, 24 and 72 h) was carried out at 37℃ using the concentrations of Ca2+ and phosphate of 30 and 18 mM respectively. All the mineralization conditions of the silk fibroin hydrogels are summarized in Table 1. The mineralized hydrogels were lyophilized for further biophysical characterization. 2.4. Biophysical characterizations The morphology of the hydrogels was observed using a field emission scanning electron microscope (FE-SEM, S4800, Hitachi) with an accelerating voltage of 1 kV. The samples were dispersed in distilled water under the assistance of ultrasonic treatment, followed by dropping onto the carbon-coated copper grids for the observation of TEM (JEM 1230, JEOL) at 80 KV and HRTEM (JEM 2010, JEOL) at 200 kV. The chemical composition and crystallinity of samples were analyzed with X-ray diffraction instrument (XRD, ARL X'TRA, Thermo Electron) using a monochromatic CuKα radiation (λ=1.54056 Å) in the range of 2θ=10-60° with a step of 0.04° and a scanning rate of 3.0°/min. Fourier transform infrared spectra (Nicolet 5700, Thermo Electron) of the samples were acquired by using the KBr pellet method in the range of 400-2000 cm1

with a resolution of 4 cm-1. Mineralized samples were also evaluated using a

thermogravimetric analyzer (METTLER TOLEDO) under a nitrogen atmosphere and heated up to 800℃ at a heating rate of 20℃/min. For the compressive strength test, samples under wet conditions with dimensions of approximately 10×10×10 mm3 were loaded at a crosshead speed of 2 mm/min using a screw driven load frame (Instron). The stress and strain responses of the samples were monitored during the compressive strength tests. The experiment was repeated for three times to obtain the average value along with its standard deviation. 2.5. Cell culture and assay Osteoblast-like cells (MG-63) were maintained in DMEM supplemented with 10% fetal calf serum, 1% penicillin/streptomycin at 37℃ in 5% CO2 atmosphere. The cells were trypsinized and seeded on hydrogels in a seeding density of 1×105 cells/ hydrogel. 2.5.1. Cell seeding and maintenance of constructs Prior to cell culture, the hydrogels were cut into small pieces of 5 mm (diameter) ×5 5

mm (thickness). These hydrogel pieces were UV sterilized while immersing in 70% ethanol for 30 min then rinsed thoroughly with PBS (pH 7.4). The hydrogels were further incubated with cell culture media (DMEM) for 4 h to home the cells and partially dehydrated for 2 h before cell seeding for better cell infiltration. The cell seeding was carried out by adding the suspension of cells in drop wise manner on both side of the hydrogel disc. The cell-laden hydrogels were maintained in DMEM (mentioned above) for 10 days with regular medium replenishment on every other day. 2.5.2. Cell survivability assay The toxic effect of mineralization was determined by MTT assay. The hydrogels were incubated after cell culture within MTT solution [1 mg/ml stock solution diluted in PBS (pH 7.4) in a ratio of 1:10]. The formed formazan crystal was solubilized in dimethyl sulfoxide and the optical density was examined spectrophotometrically (Thermo Scientific Multiskan Spectrum, Japan) at 570 nm. 2.5.3. Alkaline phosphatase assay Neo-matrix synthesis and secretion by the cells within the construct was measured spectrophotometrically by alkaline phosphatase assay kit following manufacture’s protocol (SKU#75DP, Span Diagnostics, India) [27]. The constructs were taken out at each time point (day 1, 5 and 10), sonicated and homogenized within Tris-buffer (1M, pH 8.0). The obtained tissue lysate was then incubated with p-nitrophenyl phosphate solution (16 mM) for 5 mins followed by spectrophotometric analysis of p-nitrophenol production at 405 nm. 2.5.4. Cell morphology and distribution assay For Hematoxylin–Eosin (H–E) staining, the fixed constructs were treated with methanol, followed by treatment with 99% ethanol for 2 mins. The constructs were subsequently washed with PBS (pH 7.4) and incubated in hematoxylin solution for 1 min. Next, constructs were again rinsed (PBS, pH 7.4) several times;stained with Eosin for 30 seconds and imaged using bright field microscope (Nikon Eclipse TS 100, Japan). Hydrogels without cells were served as control to normalize the background illumination obtained from mineralized matrices. 6

2.5.5 Alizarin red S staining The calcification by the constructs was investigated using Alizarin Red S staining. Briefly, the constructs were fixed in 4% paraformaldehyde for 1 h, stained using 1 wt% alizarin red S (Sigma Aldrich, St Louis, USA) solution and counter stained with Hematoxylin. Images were obtained using ECLIPSE TS100 (Nikon, Japan). 2.6. Statistical analysis All data were expressed as mean ± standard deviation (SD) of samples in triplicate (n = 3). Statistical evaluation of data from different compositions was carried out using one way analysis of variance (ANOVA). * p < 0.05 between the groups was considered as statistical significant.

3. Results and Discussion Suitable organic matrix is necessary to template the mineral formation and following material application. The choice of silk fibroin protein as hydrogel matrix is due to its natural origin, abundance in nature, low cost and notable biocompatibility [28]. Mineralization process of gels containing different Ca2+ concentration was characterized using FE-SEM (Fig.1a-d). The hydrogel containing 0 mM Ca2+ was not mineralized, exhibiting uniform porous network (Fig.1a). A few crystals were observed in 10 mM Ca2+ containing hydrogels with thicker pore wall (as shown in Fig.1b). The poor mineralization was due to the low Ca2+ concentration, which corroborated with the observation of Kino et al. [29]. Flake-like crystals homogenously distributed along the pore wall of hydrogels with 30 mM Ca2+ concentration (Fig.1c). As Ca2+ concentration rasied to 80 mM, a white fog emerged quickly once the gel was immersed in Na2HPO4 solution. The phenomenon is possibly due to the immediate formation of the crystals, when the dissociative Ca2+ in gel directly encountered with PO43- in the reaction system. Fig.1d showed the formation of flower-like aggregates filling the whole porous structure of the gel. The results concluded that the presence of Ca2+ is necessary to modulate mineralization of fibroin hydrogel. An appropriate content of Ca2+ in gel directs the oriented nucleation and growth of the calcium phosphate crystals along the pore wall of 7

the hydrogel, which in turn significantly affect the overall topography of fibroin hydrogel. The effect of reaction time on biomineralized hydrogel is evaluated using gel of 30 mM Ca2+ concentration. The chemical composition of fibroin hydrogels after mineralization at different time intervals (0, 2, 6, 12, 24 and 72 h) was detected using XRD (Fig.2a). Diffraction peaks of all the samples were broadening due to the poor crystallinity of samples and high fibroin content (Fig.2a). A broad peak at about 20° is attributed to the silk β form of fibroin. The prominent peak at 2 of 31.8° is assigned to the (211) planes of hydroxyapatite (HAP) according to the standard card of HAP (JCPDS09-0432) [30]. The peak became shaper with increasing mineralization time, indicating enhanced crystallinity of HAP with time and corroborated with the findings of Yang et al., [31, 32]. They reported the similar nucleation behavior of HAP in presence of another silk protein sericin. FTIR bands at about 1095 and 1033 cm−1(Fig.2b) are ascribed to the P-O stretching vibration modes of HAP; while the band at 603 cm−1 is attributed to the O-P-O bending mode [33]. The results indicate the formation of HAP after mineralization and are consistent with the XRD results. Band at 875 cm−1 corresponds to C–O stretching vibration mode of the CO32- group and attributed by dissolved CO2 from atmosphere during mineralization. The characteristic absorption bands of silk fibroin at 1627 (amide I), 1524 (amide II) and 1232 cm−1 (amide III) are also observed, which are ascribed to β-sheet structure of silk fibroin [22, 34]. The morphologies of biomineralized hydrogels obtained at different time interval were investigated using SEM (Fig.3). Some sparse particles were appeared after 2h of mineralization (Fig.3a), which increased in size, quantity, and homogeneity into the pore wall of fibroin gel after 6h and correspondingly expected to increase the roughness of hydrogels. When mineralization time reached 12h, pore wall of hydrogel was almost covered with nanoplates. The quantity of crystals further increased when the time rose to 24 and 72h. A lot of flake-like crystals existing along the pore wall of hydrogel were observed. The phenomenon implies that the nucleation and growth of the crystals are 8

modulated by fibroin hydrogel and is hypothesized that the Ca2+ ions in hydrogels attract PO43- to induce the HAPs nucleation along with the β-sheet assembly of silk fibroin chain by H-bond interaction [35]. The fine structured crystals formed after 24h mineralization treatment were detected using HRTEM (Fig.4). The crystals were uniform flake-like with predominant lattice spacing of 0.282 nm, corresponding to (211) planes of HAP cryatal. The result indicates oriented growth of HAP crystals along [211] a direction. This phenomenon is significantly different from our previous work [4], in which thin rod-like HAP particles with various crystal lattice spaces existed in absence of structural modifier. Consequently, we presume that the fibroin gels have a confinement effect on the crystal growth. The crystal quantities within the mineralized hydrogels were further evaluated using thermogravimetric analysis. All weight loss curves were divided into three stages (Fig.5a): first stage was below 120℃, which corresponds to the evaporation of adsorbed water; the second stage at 270-310 ℃ is attributed to the decomposition of organic components (fibroin) [36]; and subsequent stage is ascribed to burning of decomposed organic molecules. The weight residue at 800 ℃ is attributed to crystals obtained and 33.8, 36.6, 37.4, 37.8 and 38.8 % are corresponding to mineralization time of 0.5, 2, 12, 24 and 72 h respectively. The results suggest that the amount of HAP is directly proportional to the mineralization time and consistent with the results of FE-SEM. Compressive strength of mineralized hydrogels was measured using universal testing machine (Fig.5b). All the mineralized samples exhibited improved mechanical properties than non-mineralized fibroin hydrogel. It is directly related to the extent of mineralization and mineralization time. The increased mineral content and the combination of organic and inorganic phases in orchestrated structure imparts the mechanical robustness into the hydrogel [37]. The probable effect of silk fibroin hydrogel on HAP crystal growth is proposed in Fig.6. Firstly, the disperse Ca2+ ions in fibroin hydrogel chelate the functional groups of fibroin protein results in the concentration of Ca ions on the surface of silk fibroin. The 9

encountering with phosphate cause heterogeneous nucleation of HAP crystals at these sites to reduce the surface energy and the continuous growth of crystal takes place as a follow-up process. As the chelating sites on fibroin protein are limited, so only appropriate Ca2+ ion quantity in gel helps to modulate the growth of crystal along the fibroin hydrogel contributing to fabricate orchestrated organic/inorganic composite and interconnected pore structure. Therefore, fibroin hydrogel not only plays an important role in guidance nucleation of crystal, but also support the crystal growth as a 3D template. Proteins are reported to critically regulate the physical characteristics of minerals, which in turn affects its biological responses [38]. For this reason the cellular response towards the mineralized samples was evaluated. The osteoblast-like cell laden hydrogels turned opaque during culture over time. Metabolic activity evaluated by MTT analysis confirms that higher activity of osteoblasts seeded within 72 h constructs compared to 24 h mineralized constructs (p < 0.05) (Fig.7a) throughout the culture period. The metabolic activity of cells correlates with the viability of cells within the constructs and the toxic effect of the seeded matrix on cells. The MG-63 cells cultured within 72 h mineralized constructs revealed the highest viability and lowest toxicity during all days of measurement. No adverse effect of matrix mineralization on cellular proliferation was observed, which corroborated with the findings of MTT assay indicating enhanced mineralization promote cellular proliferation. It is further interesting to note that the rate of proliferation of cells within D1 and D5, and D5-D10-72 h mineralized constructs was slightly different, perhaps due to time require by cells to adjust within 3D environment; therefore, slower down the proliferation rate [39]. The osteogenic activity was measured in terms of alkaline phosphate activity, the early marker of osteoblast differentiation [40] and mineralization. The MG-63 cells within the both constructs (24 and 72 h) revealed that the alkaline phosphate activity was increased over the culture period, greater in 72 h construct over 24 h. Mineralization by the osteoblast cells was evaluated by the calcium deposition, which stains in Alzarin red stain. Intense staining is indicative of high calcium content 10

and observed in 72 h construct (Fig.8). Furthermore, histological analysis (H & E) showed the distribution of cells within the hydrogels (Fig.8). MG-63 cells were relatively homogeneously distributed and packed into multicellular aggregates in 72 h constructs in comparison to the segregated individual cells in 24 h constructs. Based on the observations, it can be said that the prolong mineralization tailors the matrix composition. This in turn effects the initial cellular interaction contributing to materialtissue compatibility.

4. Conclusions Silk protein fibroin hydrogel containing calcium ion can be used as 3D architecture template for hydroxyapatite formation via a simple ion diffusion method. The process is Ca2+ concentration and time - dependent. Fibroin hydrogel plays regulatory role in oriented nucleation and growth of hydroxyapatite crystals. The obtained 3D hydroxyapatite-fibroin composites exhibit improved compressive strength due to the combination of organic and inorganic phases in the orchestrated structure. The mineralized hydrogel promotes the viability, proliferation and differentiation of MG-63. This implies its excellent biocompatibility. In summary, silk fibroin template driven mineralization technique can serve as efficient approach towards the designing and fabrication of 3D bone-like biomaterial for bone tissue engineering.

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Figure 1: FE-SEM images of fibroin hydrogels containing different Ca2+ concentration 14

of (a) 0 mM; (b) 10 mM; (c) 30 mM and (d) 80 mM after 24 h mineralization Figure 2: The characteritics of the mineralized fibroin hydrogels: (a) XRD patterns; (b) FTIR spectra Figure 3: FE-SEM images of hydrogels after (a)0h; (b)2h; (c)6h; (d)12h; (e)24h; (f)72h mineralization treatment. Figure 4: HRTEM image of 24h mineralization sample. Figure 5: (a) Thermogravimetric analysis curves and (b) compressive modules of hydrogels mineralized for different times, data are presented as mean ± SD, n = 4. Figure 6: Illustration of the formation of fibroin directed hydroxyapatite with different Ca ion concentrations: (a) low concentration of calcium ion, (b) high concentration of calcium ion. Figure 7: Human osteoblast like cells (MG-63) response towards the mineralized matrix with different calcium contents: (a) The metabolic activity and viability of the constructs determined by MTT assay; (b) cell proliferation and osteoblast differentiation marker synthesis by the cells during 10 days culture period indicates greater osteogenic response of 72 h constructs compare to 24 h. * p < 0.05, n=3 at each time point (One way ANOVA followed). Figure 8: Alzarin red S staining: Calcification by human osteoblast like cells reveals concentrated alizarin red S staining; more intense in 72 h constructs compared to few patches in 24 h. Scale bar = 50 μm. H & E staining: The cellular organization and distribution within after 10 days. Greater cell-cell contact exhibits in 72 h constructs with well stretched cellular morphology. Scale bar = 50 μm. Acellular (hydrogels without cells) constructs serve as reference templates for comparison.

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Table 1: Mineralization conditions of silk fibroin hydrogels, kept T=37℃, pH=8.5 as constant. Concentration of Ca 2+ (mM) 10 30 80

Concentration of phosphate (mM) 6 18 48

20

Mineralization time (h) 24 0.5, 2, 6, 12, 24 and 72 24