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International Journal of Biological Macromolecules 120 (2018) 876–885

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Development of 3D scaffolds using nanochitosan/silk-fibroin/hyaluronic acid biomaterials for tissue engineering applications S. Gokila a, T. Gomathi a, K. Vijayalakshmi a, Faleh A. Alsharani b, Sukumaran Anil c, P.N. Sudha a,⁎ a b c

Biomaterials Research Lab and Department of Chemistry, D.K.M. College for Women, Vellore, Tamil Nadu, India Department of Oral and Maxillofacial Surgery, King Fahad Medical City, Riyadh, Saudi Arabia Department of Periodontics, Saveetha Dental College and Hospitals, Saveetha University, Poonamallee High Road, Chennai 600077, India

a r t i c l e

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Article history: Received 7 July 2018 Accepted 26 August 2018 Available online 29 August 2018 Keywords: Nanochitosan Silk fibroin Hyaluronic acid Blend Scaffolds Antibacterial activity Tissue regeneration

a b s t r a c t Bone tissue engineering put emphasis on fabrication three-dimensional biodegradable porous scaffolds that supporting bone regeneration and functional bone tissue formation. In the present work, we prepared novel 3D tripolymeric scaffolds of nanochitosan (NCS)/silk fibroin (SF)/hyaluronic acid (HA) ternary blends and demonstrating the synergistic effect of scaffolds and its use in tissue engineering applications. The physico-chemical characterization of the prepared scaffold was evaluated by FTIR, XRD and SEM studies. The FT-IR and XRD results confirmed the interfacial bonding interaction existing between polymers. SEM images showed good interconnected porous structure with rough surface morphology. The in vitro cytocompatibility tests carried out with osteoblast cells by the MTT assay demonstrated that the blended scaffold favors the early adhesion, growth and proliferation of preosteoblast MC3T3-E1 cells. The alizarin red assay indicated that the prepared scaffold can promote the osteogenic differentiation and facilitate the calcium mineralization of MC3T3-E1 cells. The alkaline phosphatase assay confirmed that the NCS/SF/HA scaffold provide conducive environment for osteoblast proliferation and mineral deposition. The bactericidal action of NCS/SF/HA scaffold reveals that the prepared sample has the potential to kill the microorganisms to a greater extent. Hence the overall findings concluded that the NCS/SF/HA scaffolds have better applications in tissue engineering. © 2018 Elsevier B.V. All rights reserved.

1. Introduction Bone plays an important role in a number of ways in our day today activities. As the mortality rate is reducing and the elderly population is increasing the degeneration of bone is in the increasing side. Also the rate of accidents is increasing with the increase in the transport facilities. This also demands easy and fast regeneration of body parts particularly bone. Recent approach to solve this problem is based on regeneration of new tissues by growing cells in a biologically compatible vessel called scaffolds. The process of regeneration of the required tissues through their growth within extracellular matrix mimicking materials is called tissue engineering. Tissue engineering is an interdisciplinary field that applies the principles of engineering and the life sciences towards the development of biological substitutes that restore, maintain or improve tissue function [1]. Demand is growing towards tissue and organ transplantations. In order to facilitate the growth of the damaged tissues use of three main factors are necessary such as biomaterials, scaffolds and cell combinations [2].

⁎ Corresponding author. E-mail address: [email protected] (S. P.N.).

https://doi.org/10.1016/j.ijbiomac.2018.08.149 0141-8130/© 2018 Elsevier B.V. All rights reserved.

Scaffold fabrications are the most important factor in tissue engineering and it is the central main matrix by supporting cell adhesion, proliferation, cell to cell contact and cell migrations [3]. Scaffold with highly porous well interconnected pore structure provides desired volume, shape and mechanical support. Moreover, the production of scaffolds can be made with different materials such as the biopolymers [4]. Biopolymers are useful in the biomedical field because of their structure and unique properties such as bioactivity, biodegradability, and non-toxicity for humans and the ability to form complexes [5]. Scaffolds with appropriate morphological structure usually aid the adhesion of the cells and proliferation. A good scaffold also facilitates the differentiation of the cells into a particular type. Also the scaffoldic materials are expected to be mechanically strong and also biodegradable and are replaced once the tissues are formed [6]. Natural polymers such as Collagen, gelatin, silk, alginate, chitosan, hyaluronic acid and peptides are ideal for the preparation of scaffolds due to their biocompatibility and biodegradability. Due to the presence of several components at nano scale in ECM, scaffolds with nanofibrous structure would mimic the ECM aiding the beneficial effects of bone regeneration. Chitosan biopolymer is an aminopolysaccharide composed of poly (1–4)-2-amino-2-deoxy-D-glucopyranose units consisting of amino group in its structure. Chitosan has been widely used as an important and promising biopolymeric material in tissue engineering due to its

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anti-microbial activity, biodegradability, biocompatibility and non-toxic Nature [7]. However, it is not the ideal material for tissue engineering as its bioactivity need to be improved for specific tissue. One of the methods used to modify chitosan is to convert it into its nanoform. In this work nanochitosan has been prepared by ionic crosslinking method using sodium tripolyphosphate [8]. Nanochitosan has some unique properties such as high surface reactivity, non-toxic, small size and ecofriendly material [9]. The nanosized particles are soft and brittle in nature. To overcome these limitations and to fulfill the requirements for bone tissue engineering scaffolds, it is blended with another natural polymer silk fibroin to form extracellular matrix. Silk biomaterials have, repetitive protein sequences with different amino acids in its backbone, received increasing attention as promising scaffolds for tissue engineering, although they can be processed into various forms like sponge, film, gel, fibers and particles, their biological suitability remains to be established [10–13]. In particular, the silk fibroin (SF) secreted by the silkworm (Bombyx mori) has certain desirable properties, including biocompatibility, biodegradability, optical performance and thus the development and application of silk fibroin in the field of biomedical materials has gained increasing attention [14–17]. Silk fibroin film forming property is highly desirable in tissue engineering and also it triggers the conformational transition of the SF molecule from random coil to β-sheet structure via hydrogen-bonding interactions with the compounded polymers [18–20]. Silk-based scaffolds in combination with components like chitosan, Hyaluronic acid and collagen indicates that it enhances cell adhesion, proliferations and increases the mineralization [21]. Hence, this provides an impressive toolbox which allows silk fibroin scaffolds to be blended with another natural

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polymeric material Hyaluronic acid is hydrophilic polysaccharides and hence it is utilized in many biomedical applications. Hyaluronic acid (HA), is a uniform, linear and unbranched nonsulfated glycosaminoglycan made up of glucuronic acid and N-acetyl glucosamine disaccharide units with highly variable length and molecular weight (up to 106 Da) present in all connective tissue [22]. It displays several properties like excellent viscoelasticity, water solubility, biocompatibility, consistency, hydrophilicity and non-immunogenicity have made it an excellent products as well as a potential biomaterial in tissue engineering [23]. However, HA without modification tends to be absorbed rapidly in human body, which makes it unqualified in tissue engineering. To overcome this defect, chemical modification is indispensable [24]. A new type of hydrogen bonding was obtained by its hydroxyl and carboxylic group with amino group of nanochitosan [25]. The histocompatibility, chemical modification and biodegradability of HA make it an ideal scaffold for tissue engineering. In current research work, it was intended to use the three novel polymeric biomaterials namely nanochitosan (NCS), silk fibroin (SF) and hyaluronic acid (HA) to produce a new ternary blends (NCS/SF/ HA). Hence these (NCS/SF/HA) plays an important role as the bonding material of the cell interactions and to utilize the same in wound healing or in the fields of tissue engineering. 1.1. Materials Chitosan was purchased from India Sea Foods, Cochin, Kerala 10 × 10 5 Da. Degummed silk in which sericin is removed were purchased from the sericulture farm in Vaniyambadi, Vellore

Fig. 1. FT-IR spectrum of (a) nanochitosan and (b) 3D-porous scaffold of NCS/SF/HA.

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District and hyaluronic acid was purchased from Sisco chemicals Pvt., Ltd., Maharastra. MC3T3-E1 cell line was purchased from National Cell Science Centre Pune. The crosslinking agent sodium tripolyphosphate and the solvent formic acid and glacial acetic acid were procured from Finar chemicals, Ahmedabad and Thomas Bakers chemicals Pvt. Ltd., Mumbai respectively. All the chemicals utilized in this present study were of analytical grade.

1.2. Methods 1.2.1. Preparation of nanochitosan As per the ionic gelation method, the nanochitosan was synthesized by the interaction of negatively charged sodium tripolyphosphate (TPP) with positively charged chitosan biopolymer. In order to prepare it, initially a homogeneous viscous chitosan gel was prepared by completely stirring known amount of chitosan (1 g) dissolved in 200 mL of 2% acetic acid for a period of 20 min. Sodium tripolyphosphate (0.8 g of sodium tripolyphosphate dissolved in 107 mL of deionized water) was then added dropwise to the above prepared homogeneous chitosan solution with rapid stirring for over a period of 30 min. A milky emulsion like appearance of nanochitosan obtained was then allowed to stand overnight to settle as suspension. The supernatant solution was decanted and finally the thick suspension of nanochitosan settled at the bottom of the beaker was washed several times with deionized water and preserved in the refrigerator for further use.

1.2.2. Preparation of silk fibroin Silk fibers of 3 mm length were cut and 0.5 g of it was dissolved in 100 mL of 10% LiCl in formic acid. This silk fibroin solution was then stirred well under magnetic stirrer for a period of 2 h. After this process is over, finally the thick emulsion of silk was preserved in the refrigerator.

1.2.3. Preparation of hyaluronic acid Hyaluronic acid (HA) (80–150 kDa, 0.5 mg) was dissolved in deionized water (20 mL) and this solution was stirred well for 2 h at room temperature to activate the HA carboxylic group. The solution was then stored in the refrigerator and it is utilized for further use. 1.2.4. Preparation of nanochitosan/silk fibroin/hyaluronic acid scaffold The above three prepared nanochitosan, silk fibroin and hyaluronic acid solutions were mixed, neutralized and stirred well for 2 h to remove the air bubbles completely. This prepared solution mixture was then freeze- dried to −80 °C for overnight and then lyophilized for 1 day and after this process, the scaffold was subjected to further studies. 1.3. Physico-chemical characterization FT-IR spectrum of the prepared sample was measured in the wavenumber range from 4000 to 650 cm−1 using a 100 FT-IR Perkin Elmer spectrophotometer. The powder X-ray diffractogram (XRD) of ternary blended nanochitosan/silk fibroin/hyaluronic acid scaffold was measured in a SHIMADZU XRD 6000 (Japan) diffractometer using CuKα radiation (λ = 1.5406 Ǻ) with 30 mA, 40 kV and scanning rate of 3°/min. The morphology, cell spreading and the microstructure of scaffolds was observed by JEOL JSM 6460-LV scanning electron microscope, Japan. 1.4. Cell culture Cell types were mainly used to assess the effect of the scaffold composition onto the different stages of cell differentiation within the osteoblast lineage. MC3T3-E1 cells were cultured in Iscove's Medium (Sigma, USA) and MC3T3-E1 cells were added in the culture media to induce osteoblastic differentiation. The cell-line was then washed, hydrated for 2 h with PBS prior to cell seeding and thereafter the scaffolds were placed in a 24-well cell culture plate. After this process,

Fig. 2. X-ray diffractogram (a) nanochitosan and (b) 3D-porous scaffold of NCS/SF/HA.

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Fig. 3. Scanning Electron microscopy images of 3D-porous scaffolds NCS/SF/HA.

then 2 × 104 cells/scaffold was seeded in a volume that soaked the scaffold and were incubated for 3 h. Five hundred μL of culture medium was added into each well. After 24 h, the scaffolds were changed to new culture wells in order to analyze only the cells growing into the scaffolds. Empty scaffolds (without cells added) were treated in the same manner and used as controls, to obtain proliferation and differentiation data. 1.5. MTT assay Cell adhesion and proliferation rates were estimated by MTT assay. For cell adhesion, the cells were loaded onto the scaffold and left for 24 h. After 24 h, the scaffolds were removed and the attached biomass was measured at 570 nm. The cytotoxicity properties of the fabricated pure scaffolds were evaluated. Briefly, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St Louis, MO, USA) assay was used which was prepared in phosphate buffered saline

(PBS) at a final concentration of 5 mg/mL. For cell adhesion, the cells were loaded onto the scaffold and left for 24 h. After 24 h, the scaffolds were removed and the attached biomass was measured at 570 nm. Then, the supernatant was withdrawn and centrifuged to prepare the conditioned extracts before the cytotoxicity test. Cell viability and proliferation were measured by MTT assay [55] from which 50% cytotoxic concentration (IC50) was calculated (American Type Culture Collection (ATCC), Manassas, VA, USA). 1.6. Alkaline phosphatase assay (ALP) Cell differentiation was evaluated by alkaline phosphatase (ALP) activity. The samples were permeabilized with 0.5% Triton X-100 and incubated with a 20 nM p-nitrophenyl phosphate (Sigma, USA) solution. According to the manufacturer's instructions, the ALP activity of the MC3T3-E1 cells was then evaluated by a standard procedure. Briefly,

Scheme 1. Proposed mechanism for the formation of NCS/SF/HA 3D scaffold.

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The whole solution was transferred to a tube and centrifuged at 13,000 rpm for 10 min at 4 °C. ALP activity was measured by mixing 50 μL of supernatant with 50 μL p-nitrophenyl phosphate (5 mM) in 150 mM 2-amino-2-methyl-1-propanol buffer solution. After 30 min incubation at 37 °C, the reaction was stopped by the addition of 50 μL of 0.2 N NaOH and the OD was measured at 520 nm using an ELISA reader (Bio-Rad Model 550, USA). 1.7. Alizarin Red S assay (ARS)

Fig. 4. Antibacterial Activity of 3D-porous scaffolds NCS/SF/HA against E. coli and Pseudomonas aureus.

samples and control group were cultured 1, 4, 7 and 10 days, irrigated with PBS three times to remove as much residual serum as possible and followed by this 1 mL of 0.1% Triton X-100 were placed on the samples and control group at 4 °C through overnight to break up the cells.

1000µg/ml

10µg/ml

0.3µg/ml

By utilizing alizarin red S (ARS) staining of the MC3T3-E1 cells, the calcium deposition can be determine. Osteoblasts and osteocytes are involved in the formation and mineralization of bone. Modified (flattened) osteoblasts become the lining cells that form a protective layer on the bone surface. The mineralized matrix of bone tissue has an organic component of mainly collagen called ossein and an inorganic component of bone mineral made up of various salts. This study describes a sensitive method for the recovery and semiquantification of Alizarin Red S in a stained monolayer by acetic acid extraction and neutralization with ammonium hydroxide followed by colorimetric detection at

100µg/ml

3µg/ml

control

Fig. 5. In vitro cytotoxicity and cell proliferation of NCS/SF/HA scaffolds by MTT assay.

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405 nm in a 96-well format. Cells were cultured in different osteogenic differentiation medium for 7 days, fixed for ARS staining and quantified for mineral deposit using the kit. 1.8. Antibacterial studies The antimicrobial properties of NCS/SF/HA ternary blended scaffolds were determined by the agar diffusion method with Muller Hinton Agar as medium (MHA) as the medium. The gram positive bacterial strain namely Pseudomonas aureus and gram negative bacterial strain namely E. coli were used for determining the antimicrobial properties. The microorganisms Escherichia coli and Pseudomonas aureus were inoculated initially on Muller Hinton Agar (MHA) medium and spread uniformly using sterile spreader in Petri plates. After solidification of the MHA medium, a small amount of the NCS/SF/HA ternary blended scaffold was then placed on different cultured agar plates and the plates were incubated on individual racks for 24 h at 37 °C. By utilizing the ruler, the antibacterial activities of the prepared samples was evaluated by measuring the diameter of zone of inhibition grown around the samples against the test microorganisms. 2. Results and discussion 2.1. FT-IR spectroscopy The FT-IR spectrum of the prepared nanochitosan and NCS/SF/HA 3D scaffold was given is the Fig. 1 and a. Compared with the nanochitosan, the prepared blend showed the characteristic bands of the blended moieties. The FTIR spectrum of nanochitosan showed strong absorption bands at 3385 and 1635 cm−1 indicating the presence of OH hydroxyl group, NH stretching vibrations and C_O stretching vibrations respectively. The wider band indicates the participation of functional groups in ionic crosslinking with the polyanion sodium tripolyphosphate. The FT-IR spectrum of NCS/SF/HA scaffold shows a strong broad absorption band at around 3442 cm−1 shows the intermolecular hydrogen bonds stretching vibrations of OH and NH groups [26]. On comparing the FTIR spectrum of nanochitosan with NCS/SF/HA scaffold, the enhancement of hydrogen bonding is seen. This indicates the involvement

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of functional groups of SF and HA in hydrogen bond interaction with NCS [27]. Absorption bands present at 2945 and 1456 cm−1 are assigned to the CH stretching and bending modes of vibration respectively. Generally, Silk fibroin (SF) shows an absorption band in four specific areas such as 1645, 1531, 1240 and 588 cm−1 for amide II (N\\H bending), amide III (C\\N stretching) [28], β-sheet [31] and amide V [29]. These bands while mixing with NCS and HA, the wavenumbers of the bands were shifted and some of them gets overlapped with the bands of nanochitosan, TPP and HA. Certain absorption bands observed at 1386 cm−1, 1159 cm−1 and 449 cm−1 were indicative of OH in plane bending, P_O stretching and C\\O bending respectively [30]. Thus the observed FTIR spectrum proves the effective blending of the three polymers during scaffold formation. The addition of SF and HA in nanochitosan was well pronounced in the FTIR spectrum of scaffold compared with pure nanochitosan.

2.2. X-ray diffraction studies X-ray diffraction study deals about the crystallinity and crystalline structure of the prepared scaffold material. The XRD pattern of the nanochitosan/silk fibroin/hyaluronic acid ternary blended scaffold was represented in Fig. 2a. Generally, in the XRD pattern when the reflection gets broadened with high noise and low peak intensities, it showed that the prepared material had poorly crystalline or amorphous nature [31]. Similarly, in the present work, the novel 3D scaffold showed only a broad hump in the diffraction pattern over a large range from 2θ = 20 to 40° due to noncrystalline form/amorphous state of the scaffold. The XRD pattern supports the FTIR spectrum results. XRD pattern of nanochitosan showed one broad peak at 18.86° (Fig. 2), whereas while forming the 3D scaffold with SF and HA the peak gets broadened. This denoted the presence of strong interaction which was formed between the functional groups of NCS, SF and HA and this phenomenon reduce the crystallinity of the individual polymers [32,33]. In other words, it confirms the co-existence of three substances in the scaffolds [34,35]. The higher amorphous structure with a highly functionalized surface area is expected to be a favorable material for good cell attachment and proliferation.

Fig. 5a. Percentage of cell viability of MTT Assay using NCS/SF/HA scaffold.

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Nanochitosan exhibited higher antibacterial activity than chitosan itself, due to the interaction between positively charged nanochitosan and negatively charged microbial cell wall [41]. The antibacterial Activity of 3D-porous scaffolds NCS/SF/HA against E. coli and Pseudomonas aureus are represented in Fig. 4. Generally, when the zone of inhibition value was less than 10 mm, then the material is considered as resistant towards the corresponding microorganism [42]. But the antibacterial results indicate that the zone of inhibition values exhibited are 16 mm and 14 mm by the NCS/ SF/HA scaffold against the growth of the selected E. coli and Pseudomonas aureus bacteria respectively. From the zone of inhibition values it was evident that the prepared NCS/SF/HA scaffold exhibits good antibacterial activity against E. coli and Pseudomonas aureus. Fig. 6. Alkaline phosphatase (ALP) activity in MC3T3-E1 cells culture assessed by 3D scaffold of NCS/SF/HA ternary blend.

2.3. Scanning electron microscopic (SEM) studies The scanning electron microscopic (SEM) technique helps in determining the microstructure, morphology and chemical composition of material. The scanning electron micrograph of NCS/SF/HA ternary blended scaffold was represented in Fig. 3. The magnified SEM images of NCS/SF/HA scaffold illustrate the open pore structure with distinct pore walls and high degree of pore interconnectivity [36], embedded with small grooves looking like a birds nest [37], that allow to visualize pores in its interior which indicates the interconnectivity that these pores have in the matrix. The observed results also showed that the prepared scaffold is highly porous with an even pore size distribution ranging from 0.5 μm to 1 μm, which will be a favorable size range for cell growth [38]. In addition to this macroporosity requirement for cell interaction, microstructural features in terms of microporosity or surface roughness are also preferable which may facilitate cell-scaffold interactions [39]. Therefore on close observation, it has been found that NCS/SF/HA scaffold has highly interconnected pores with the typical reticulated structure rather than irregular sheets. Thus the characterization results reveals the extraordinary interactions formed between NCS, HA and SF, which makes it highly suitable for a cell attachment and proliferation. The proposed mechanism for the formation of NCS/SF/HA scaffold was given in Scheme 1. 2.4. Antimicrobial studies Being a cationic polymer, Chitosan has a good antimicrobial property. When chitosan was converted into nanochitosan, due to the increased surface area the microbes are expected to be adhered to the surface significantly in a short time. This interaction leads to a slight shock for and there by exhibit very good antimicrobial activity [40].

2.5. MTT assay The cell viability of the prepared scaffolds was investigated through the MTT assay by seeding the known concentration of MC3T3-E1cells onto NCS/SF/HA scaffolds (Figs. 5 and 5a). The MC3T3-E1 cells were incubated with NCS/SF/HA 3D scaffold test solutions of different concentration to determine their effect on cell viability. The dehydrogenase enzyme secreted from the mitochondria is mainly responsible for formation of purple formazan crystals and the amount of purple formazan reflect the level of cell viability on the material [43]. The percentage cell viability by NCS/SF/HA scaffold was represented in Fig. 5a. The results confirm that the response of the MTT cytotoxicity assay was strongly dependent on the cell concentration [44]. Also, the cell growth experiments carried out using NCS/SF/HA scaffold demonstrated the interaction of MC3T3-E1 cells with the scaffolds. Generally, a strong positive charge at the biomaterial surface can induce unnaturally strong focal adhesion and integrin binding; however, from a chemical point of view, the cell membrane is negatively charged, thus favoring electrostatic interactions with positively charged surfaces [45], so there is a formation of a new ionic bond that leads to the growth of the viability of proliferated cells increased with increasing concentrations from 0.1 to 1000 μg/mL. The data presented are the means ± SD of results from three independent experiments and the IC-50 concentration is observed to be 48 μg/mL for GTI which was calculated by Graph pad Prism Software 5.0. [63], reported that cell viability by chitosan nanoparticles was clearly observed in a dose- and time-dependent manner. Similarly, cell viability results revealed that the cell viability percentage increases and toxicity decreases as the concentration and time of incubation of the 3D scaffold increases on MC3T3 –E1 cells during the in vitro culture period. Therefore, suggesting that the 3D scaffold did not retarded the cell proliferation, which shows the it is nontoxic and cell compatible.

ALIZARIN RED ASSAY

Control

25mcg/ml

50mcg/ml

Fig. 7. Alizarin Red S (ARS) images of MC3T3-E1 cell cultured on NCS/SF/HA scaffold.

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Fig. 7a. Alizarin Red S (ARS) activity of MC3T3-E1 cell culture assessed by 3D scaffold of NCS/SF/HA ternary blend.

2.6. Alkaline phosphatase (ALP) assay Cell differentiation has been evaluated by measuring ALP activity of MC3T3-E1 cells cultured on NCS/SF/HA scaffolds (Fig. 6) as this enzyme play an important role in the initiation of the mineralization process [46]. ALP activity is one of the characteristic parameter of osteoblast cells differentiation [47]. The ALP activity assessment indicated that cells on the scaffolds showed a higher differentiation level than cultured in conditioned medium. The addition of silk Fibroin-Hyaluronic Acid could develop the properties of Nanochitosan to fit bone tissue engineering, because it can promote the important growing factor bone matrix deposition and providing sufficient cell adhesion [48]. From Fig. 6, it was observed that the NCS/SF/HA scaffolds exhibited the higher ALP activity than the control, which is widely known to accelerate the mineralization process [49]. In a study by [50], CS grafted PDLLA nanofibers were developed by a two-step process, and these nanofibers enhanced biomineralizing ability in SBF due to the incorporation of bioactive amino groups on their surfaces. Also, the grafted amino groups promoted the proliferation of mouse pre-osteoblastic cells (MC3T3-E1) and increased their ALP activity [50]. The ALP activity was significantly increased when the concentration of NCS/SF/HA scaffold increases, which is known to encourage mineralization process of precursor cells and as well as it develops bone formation. 2.7. Alizarin Red S (ARS) assay Alizarin Red S (ARS), an anthraquinone dye, has been widely used to evaluate calcium deposits in cell culture. The mineralization is mainly

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assessed by extraction of calcified mineral at low pH, neutralization with ammonium hydroxide and colorimetric detection at 405 nm in a 96-well format. The ARS staining is quite versatile because the dye can be extracted from the stained monolayer of cells and readily assayed. The bioactivity of the bone implants was evaluated and the capacity of minerals deposition is a late stage marker of osteogenic differentiation that can be used to conform that MC3T3-E1 cells entered into the mineralization phase to deposit mineralize ECM [51]. Figs. 7 and 7a show the Alizarin Red S (ARS) images of MC3T3-E1 cell cultured on NCS/SF/HA scaffold and the ARS activity of the of MC3T3-E1 cell culture assessed by 3D scaffold of NCS/SF/HA. The Alizarin Red S staining showed reddish dots on the NCS/SF/HA scaffold and this indicate that the prepared sample can facilitate the calcium mineralization of MC3T3-E1 cells [52–54]. The higher porous structure and larger surface area of the scaffold are the important parameters to facilitate the binding of cells and calcium, which improved the cells growth on the surface of the scaffold had increased mineralized nodule formation [55]. Hence it was concluded that the prepared 3D NCS/SF/HA scaffold can promote the osteogenic differentiation and facilitate the calcium mineralization of MC3T3-E1 cells. 2.8. Cell colonization and proliferation In this study, the in vitro cell attachments, cell colonization and cell proliferation of the NCS/SF/HA scaffold was analyzed using fluorescence microscopic technique (Fig. 8). This technique is mainly used to enhance the imaging capabilities in bone tissue research that is not possible with other traditional optical microscopes [55]. The visualization of cell adhesion and colonization of the scaffolds, cell/scaffold constructs were evaluated using fluorescence microscopy after performing live/ dead staining. Based on the simultaneous staining of live (green labeled) and dead (red labeled) cells, the cell behavior in terms of viability, proliferation, morphology and distribution was qualitatively investigated. The in vitro cell attachments, cell colonization and cell proliferation of the NCS/SF/HA scaffold on MC3T3-E1 Cell culture assessed was analyzed at three different concentrations [56,57]. The cells on the scaffolds staining indicate that on one day after seeding, MC3T3-E1 cells were viable (stained green) and the amount of cells mineralized scaffold appeared similar and fewer cells were detected on the control scaffolds. The amount of dead cells (stained red) was limited on the scaffolds and also the NCS/SF/HA scaffolds demonstrated high background fluorescence after staining [58]. High cellular viability was revealed the good surface interactions of all studied compositions as well as on the control scaffold during one week of culture [59] and this observation was confirmed by the low amount of dead cells found in all cell-material systems. However, cell

FLUORESCENT ASSAY

CONTROL

25µg/ml

50µg/ml

Fig. 8. Fluorescence microscopic images of MC3T3-E1 Cell culture assessed by living(green-labeled) and dead (red-labeled) 3D-porous scaffolds, NCS/SF/HA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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density on the surface of sample NCS/SF/HA was visibly higher than the control, suggesting a higher proliferation rate [60]. Additionally, cell distribution on the surface of the sample NCS/SF/HA was observed to be homogenous, suggesting a uniform and ordered microporous structure which was able to allow the cells to adhere [61]. The complex biomechanical bone system which requires an ideal scaffold possessing good biocompatibility to osteoconductivity where the scaffold lets the bone cells to adhere, proliferate and form the extracellular matrix on its surface and pores [62]. These properties can be fulfilled by this prepared NCS/SF/HA scaffold and therefore suitable for bone tissue engineering applications.

3. Conclusion In the present study, the NCS/SF/HA scaffold was successfully prepared and evaluated for its suitability in bone tissue engineering applications. FTIR and XRD results reveal the presence of specific functional groups and its interaction for scaffold formation. SEM images of NCS/ SF/HA scaffold showed the porous and rough surface with a net like morphology. The anti-bacterial activity showed that the scaffold is having good bactericidal property. In vitro cell viability studies such as MTT and ALP assay reveals that the prepared scaffold support cell adhesion/ attachment and proliferation and also the noncytotoxic nature. Alizarin Red dye and cell staining assay proved the enhancement of biomineralization and suggesting the NCS/SF/HA scaffold for higher proliferation rate and cell colonization. Thus, in the near future, it is most likely that the NCS/SF/HA scaffold based systems would help to reconcile the clinical and commercial demands in tissue engineering.

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