hydroxyapatite

International Journal of Biological Macromolecules 112 (2018) 448–460

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International Journal of Biological Macromolecules journal homepage: https://www.journals.elsevier.com/ijbiomac

Novel alginate/hydroxyethyl cellulose/hydroxyapatite composite scaffold for bone regeneration: In vitro cell viability and proliferation of human mesenchymal stem cells Khairy M. Tohamy a, Mostafa Mabrouk b,⁎, Islam E. Soliman a, Hanan H. Beherei b, Mohamed A. Aboelnasr a a b

Biophysics Branch, Faculty of Science, Al-Azhar University, Nasr City, 11884 Cairo, Egypt Refractories, Ceramics and Building Materials Department, (Biomaterials Group), National Research Centre, 33El Bohouth st.(former EL Tahrir st.), Dokki, Giza P.O. 12622, Egypt

a r t i c l e

i n f o

Article history: Received 22 September 2017 Received in revised form 21 January 2018 Accepted 28 January 2018 Available online 31 January 2018 Keywords: Hydroxyethylcellulose Bovine serum albumin Human mesenchymal stem cells

a b s t r a c t Sodium alginate (SA)/hydroxyethylcellulose (HEC)/hydroxyapatite (HA) composite scaffolds were explored for enhanced in vitro bone regeneration. The SA/HEC/HA composites were synthesized using the lyophilization technique and further cross-linked in the presence of calcium ions to form composite hydrogel networks. The physicochemical, thermal behavior and morphology properties of the prepared scaffolds were characterized through XRD, DSC/TGA, FTIR and SEM. Furthermore, the mechanical behavior of the under investigated scaffolds was determined using texture analyzer. The in vitro bioactivity in SBF and adsorption of bovine serum albumin as well as cell viability for all the prepared scaffolds were also tested. The results indicated that the higher HA concentration (40 wt%) enhanced the mechanical properties (23.9 MPa), bioactivity and protein adsorption. Cell viability of the tested scaffolds confirmed the non-toxicity of the fabricated systems on the human mesenchymal stem cells (hMSCs). Proliferation capability was also confirmed for the tested scaffolds after 3 and 7 days, but the higher HA-containing scaffold showed increased cell populations specially after 7 days compared to HA-free scaffolds. This novel composite material could be used in bone tissue engineering as a scaffold material to deliver cells and biologically active molecules. © 2018 Elsevier B.V. All rights reserved.

1. Introduction In recent years, one of the significant difficulties in tissue engineering field is the manufacturing of acceptable biomaterials that could be utilized as substrate for cell bonding, multiplication, development and reproduction [1]. Tissue engineering is a field in which biology, medicine and engineering are integrated. A standout among the most basic issues is to develop appropriate three-dimensional (3D) biomechanical scaffolds as tissue replacement [2–4]. To accomplish this task, the perfect scaffold should meet some particular criteria, including biocompatibility, convenient porous structure, adequate mechanical quality, and controllable biodegradation [5–7]. Polymeric scaffolds gained a magnificent attention in the last 20 years due to their biocompatibility, durability and simplicity of fabrication. A lot of strategies have been used for fabrication of biopolymer scaffolds [8–10]. Natural polysaccharides- especially alginate- had been attractive due to their impressive properties. They are renewable, cheap, biodegradable, non-toxic upon in vivo administration and abundant available natural polymers. In addition to that, they are tunable for

⁎ Corresponding author. E-mail address: [email protected] (M. Mabrouk).

https://doi.org/10.1016/j.ijbiomac.2018.01.181 0141-8130/© 2018 Elsevier B.V. All rights reserved.

the continuous development of advanced biomaterials with new modification behavior [11–15]. Sodium alginate is an anionic linear polysaccharide consisting of αL-guluronic acid (G units) and (1-4)-linked β-D-mannuronic acid (M units), which hold together by covalent bond in various site and series distributions along network polymer chain, depending on the alginate source [16]. The functional properties of alginate depend on its monomer composition (M/G) ratio and sequence [17]. For example, MG blocks (MGMGMGM) form the most flexible chains and G blocks (GGGGGGG) form stiff chains. Moreover, alginate can be prepared with a wide range of molecular weights (typically 10–103 kDa) [17,18]. It is well known that alginate can formed stabilized scaffolds through crosslinked ionotropic in the existence of divalent cations with little concentrations, such as Ca2+ ions, due to its anionic property that enables alginate to form complex with the Ca cations [19,20]. In this manner, alginate is effectively manufactured into porous three-dimension scaffolds or scaffold networks by lyophilization technique. Nevertheless, alginate (without Ca2+ ions crosslinking) showed weak mechanical strength, responses in biological activity and controlled biodegradability [21,22], which seriously confine their reasonable applications in tissue engineering. When alginate is utilized as a porous scaffolds as a part of in vivo experiment, ionically crosslinked alginate degradation starts when Ca2+ substitution takes place by another

K.M. Tohamy et al. / International Journal of Biological Macromolecules 112 (2018) 448–460

ions from extracellular fluid such as Na+ or K+ [23,24]. This fact would cause a very long degradation time (slow degradation rate) depending on the rate of ions exchange process, thus -in turn- it would delay the scaffold replacement with new tissue. The addition of another polymer (non-crosslinked with Ca2+ ions) to the alginate would alter the degradation mechanism of the crosslinked alginate scaffold and make it more controllable. Controlling the scaffolds degradation is one of the biggest challenges facing the tissue engineering scientists, thus it would certainly ensure simultaneous tissue replacement upon scaffold degradation. Hydroxyethylcellulose (HEC), is a non-ionic carbohydrate polymer with surface active properties that can function as a “protective” colloid, which is good with an extensive variety of other water-dissolvable polymer [25]. HEC has many applications such as a thickener in latex paint and paper finishing; although HEC showed the higher lysosomal degradation rate than SA, it can't support in vitro cells growth for tissue regeneration systems. One promising process that deals with adequately defects in the above restrictions is the blending of inorganic materials within the polymer scaffold as acceptable filler. Among different biomaterials, hydroxyapatite (HA) possessed greater advantages for evolution of organic/ inorganic composite scaffold; it is widely used in many applications for bone tissue engineering, because of its biocompatibility, bioresorbability, perfect cell adhesiveness, and excellent bioactivity with good mechanical strength [26–29]. This study focused on understanding the effect of blending HEC with Na alginate incorporated with and without HA on degradation, mechanical properties and bioactivity of the prepared scaffolds. The 3D scaffolds of various blend ratios were formed using lyophilization technique. Simulated Body Fluid (SBF) was utilized in order to investigate the scaffolds bioactivity and biodegradation. In vitro bovine serum albumin (BSA) adsorption was undertaken in phosphate buffer saline (PBS). Cells viability and cell proliferation were conducted on human mesenchymal stem cells (hMSCs). 2. Experimental details 2.1. Material Sodium alginate (SA) was obtained from Kibun Food Chemipha, Japan Aldrich chemical Co. (MW = 500,000 g/mol). Hydroxyethylcellulose (HEC), (MW = 90,000 g/mol), was acquired from Aldrich Sigma (St. Louise, MO, USA). The cross linkers employed were the chloride salts of calcium (Ca2+), (CaCl2·2H2O) (MW = 147.02 g/mol) from Sigma–Aldrich and bovine serum albumin (BSA) were purchased from Sigma–Aldrich (Chem. Lab, Germany). HA starting materials: Calcite (Anala R, specified minimum assay 99% CaCO3, 14 μm average particular size, BDH Laboratories Ltd.) and CaHPO4 (with particle size in rang of 30–50 μm, Lumifax Ltd.). 2.2. Preparation of monoclinic hydroxyapatite Monoclinic hydroxyapatite (HA) was prepared using solid state preparation method as reported in Morgan et al. [30] with little modifications. In details, 81.666 g of DCPA and 39.930 g of calcite was utilized to give the stoichiometric amounts of CaCO3 and CaHPO4 according to the following chemical reaction: Final actual 100 g of HA were yielded. The constituents were well mixed using ball mill. Afterwards, the mixed powder was fired at 1000 °C with air atmosphere for 3 h.

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Table 1 Different composite scaffolds. Sample code

SA

HEC

HA

S1 S2 S3 S4 S5

100 50 50 50 50

0 50 50 50 50

0 0 10 20 40

for overnight, thereafter; the freezed samples were lyophilized for 24 h at −56 °C. Finally, the scaffolds were immersed in 5% CaCl2 for 3 h for crosslinking. Furthermore, the crosslinked scaffolds were removed, washed three times by distilled water, dried at room temperature and kept in the desiccator for further analysis. 2.4. Scaffold characterization 2.4.1. Crystallization behavior of the prepared scaffolds 2.4.1.1. Thermal analysis. In order to figure out the effect of HEC and HA on the crystallization behavior of the prepared scaffolds, Differential Scanning Calorimetry (DSC) and Thermo gravimetrical Analysis (TGA) were done utilizing a modernized SETARAM labsys™. DSC tests were conducted by heating 25 mg of HA powder and all scaffolds at a fixed rate flow of 10 °C/min, using the same amount of Al2O3 as the reference material in range of (25–1000 °C). The TGA analysis was performed by Shimadzu TGA-50 Hz instrument at heating rate of 10 °C/min under a constant flowing atmosphere. The precise mass of all samples before heat treatment was 1.131 mg and then they were placed in a platinum crucible. 2.4.1.2. XRD analysis. XRD is a useful method to confirm the amorphous or crystalline nature of the samples; the selected samples which were visually tested were milled in an agate mortar before measuring. XRD analysis were assessed for the prepared scaffolds to determine the effect of HEC and HA on their crystallization behavior. XRD patterns of the specimens were examined by utilizing [Axs D8 ADVANCE], [Cukα = 1.54056 A°] radiation. The applied current and voltage were 40 mA and 40 kV. XRD was taken at 2ϴ angle range of 10–70 and the operation condition was scan move size 0.02 (2ϴ) and scan move time 0.05 s. 2.4.2. Chemical integrity of the prepared scaffolds In order to estimate the effect of HEC and HA on the chemical integrity of the composite samples, Infra-red (FTIR) spectroscopy estimations were registered at room temperature in wavenumber range of 400– 4000 cm−1, by Demonstrate 1600, Perkin-Elmer USA. The scaffolds were completely dried, then well grinded and blended completely with KBr at a proportion of 1:10 (Specimen: KBr), which was subsequently pressed into pellet in an evacuated die.

2.3. Fabrication of SA/HEC/HA composite scaffolds

2.4.3. Microstructural properties of scaffolds The microstructural properties of the scaffold samples before immersion in SBF were determined by Scanning Electron Microscope (SEM) (Demonstrate XL30, Philips) connected with component investigation of an X-ray detector (EDX) unit, with 20–25 KV quickening voltage, amplification image up to 400,000× and its determination is (3.5 nm). SEM micrographs were acquired subsequently via covering the samples with gold for excellent observation, utilizing Edwareds 5150 sputter coating (England).

Scaffolds were prepared employing freeze drying Technique. Firstly, SA and HEC (20% W/V) solution was prepared by dissolving SA and HEC powders in distilled water and were kept for 1 h stirring at 60 °C. HA powder was blended with the achieved polymer mixture according to Table 1. The mixtures were casted in Petri dishes and kept at −18 °C

2.4.4. Swelling studies The scaffolds capacity for swelling was carried out by utilizing distilled water at room temperature [31]. Initially, three equal pieces of each sample were weighed and noted as Wd. The scaffolds were immersed in distilled water for different time durations (3, 6, 9, 15 and

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30 days). After each time period, the specimens were collected, gently put on a filter paper to eliminate the adsorbed water, then was weighed, recorded as Ww and the ratio of swelling was calculated by Eq. (2). Swelling % ¼

Ww Wd Wd

ð2Þ

Swelling % was registered as mean ± SD (n = 3). 2.4.5. Scaffold porosity The porosity of the prepared scaffolds was estimated by liquid displacement technique as early reported [31]. Three specimens of each scaffold were submersed in glycerol for 1 h and then centrifugated at 5000 rpm for 15 min, afterwards the samples were left submersed at room temperature for 48 h and the porosity of the samples was determined using Eq. (3). P% ¼

W1 W3 W2 W3

100

ð3Þ

Where W1: weight of the scaffold before immersion, W2: weight of the scaffold after immersion and W3: weight after drying and (P%) is the porosity percentage. 2.4.6. Mechanical properties of composite scaffolds In this study, the mechanical properties of the composites scaffolds were evaluated by measuring the compression strength as well as the resilience (%). Resilience (%) of the scaffold could be defined as the efficiency of the scaffold to release located energy under flexible disfigurement, and returned vitality subsequent after unloading [32,33], Practically, the specimens were evenly cut from the scaffolds to form blocks with dimensions of 10 × 5 × 5 mm3. The scaffold samples were situated between parallel plates utilizing a component EMIC DL3000 and compressed with a crosshead speed of 0.5 mm/min and a 1.0 kN load cell. Three pieces (n = 3) of each composite scaffold specimens were investigated to calculate the average values. Furthermore, the resilience (%) was indirectly calculated by the Texture analyzer (TA.XT plus Stable Microsystems, Surrey, UK). 2.5. In vitro studies 2.5.1. Scaffolds bioactivity in SBF Simulated Body Fluid (SBF) was used to determine the bioactivity of samples. The scaffolds were immersed in SBF at 37 °C as early reported by Kokubo et al. [34], it is worth to highlight that the bioactivity of the scaffolds assesses their ability for deposition of hydroxyapatite layer on their surface [35]. Scaffolds (10 × 10 × 5 mm3) were immersed in SBF (60 mL, pH = 7.4), and they were left in a thermodynamic incubator at 37 °C up to 28 days [36,37]. After each immersion period, the specimens were filtered, washed three times by distilled water, and air dried at room temperature. The changes happened on scaffolds surface were, then, examined through XRD, SEM, EDX and FTIR techniques. The crystallinity degree of the precipitated phase on the scaffolds surfaces was determined by calculating the crystallinity index. The crystallinity index XC, relating to the portion of crystalline HA phase was detected in the examined volume of surface scaffold samples. The crystallinity (XC) for the scaffolds could be assessed by Eq. (4) [38].

Xc ¼

0:24 3 β

ð4Þ

Where XC is the crystallinity index, β is full width at half maximum peak (FWHMP).

2.5.2. Scaffolds biodegradation in SBF The degradation (%) of the scaffolds was determined in SBF (pH 7.4) at 37 °C in thermostatic incubator [39]. Three equal pieces in volume and area from all composite scaffolds were immersed in SBF and kept at 37 °C for 1, 2, 3 and 4 weeks, respectively, each scaffold was weighted prior immersion, recorded as Wi. Afterwards, the specimens were removed from SBF, washed three times with distilled water to stop the ions adsorption on the scaffolds surface and air dried. Then, all scaffolds were weighed and noted (Wt); the degradation ratio was estimated by Eq. (5). Degradation ð%Þ ¼

Wi Wt 100 Wi

ð5Þ

Degradation (%) was registered as mean ± SD (n = 3). 2.5.3. Protein adsorption analysis Scaffolds were soaked in 2 mg/ml Bovine Serum Albumin (BSA) solution prepared in PBS, and aged in static thermo incubator for predetermined time (30 min, 1, 3, 6, 9 and 12 h). After each duration time, the scaffolds were well rinsed thrice with PBS, washed with distilled water three times to remove the non-adsorbed protein. The rinsed scaffolds were, then, incubated for 1 h at 37 °C. The protein adsorption was investigated using FTIR and gradual decrease in concentration of BSA was measured by UV spectrophotometer (Thermo Scientific Orion aquamate 8000 UV spectra, USA), at a wavelength of 595 nm, standard calibration curve of different concentration of BSA (0.4–2 mg/ml) was plotted. Based on this curve, the amount of the adsorbed protein could be calculated by comparing the absorption values of the aliquot solution as in Eq. (6). q¼

ðCi Cf ÞV m

ð6Þ

Where Ci and Cf are the initial concentration of protein and the concentration of protein remaining in solution after scaffolds removal from solution, respectively; V is the total volume of the solution; and m is the total surface area of scaffolds embedded into the solution. 2.5.4. In vitro cell culture studies In this study, the newly synthesized scaffolds were subject to cytotoxic evaluation on human normal cell line. Doxorubicin was used in this experiment as a positive control. The scaffolds were dissolved in 20% DMSO in concentration 1 mg/mL. Serial dilutions were made reaching final concentration of the prepared sample to 6.25, 12.5, 25, 50 and 100 μg/mL; all chemicals used in this study are of high analytical grade. They were obtained from (either Sigma-Aldrich or Biorad). The human mesenchymal stem cells (hMSCs) were obtained frozen in liquid nitrogen (−180 °C) from the American Type Culture Collection (ATCC) and was maintained at the National Cancer Institute, Cairo, Egypt, by serial subculturing. The cytotoxic activity was measured in vitro on human normal cell line (hMSCs) using Sulforhodamine-B stain (SRB) assay applying the method of Skehan et al. [40], Seeding density used in this assessment was 1.5 × 104 cells per mL. Cells were plated in 96 multi well plates for 24 h before treatment with prepared scaffold samples to allow attachment of the cells to the wall of the plate, after that, different concentrations of the scaffolds under test (0, 6.25, 12.5, 25, 50 and 100 μg/mL) were added to the cell monolayer. Triplicate wells were prepared for each individual dose. Monolayer cells were incubated with the samples for 3 and 7 days at 37 °C and in atmosphere of 5% CO2. After determined periods cell was fixed, washed and stained with Sulforhodamine B stain. Excess stain was washed with acetic acid and attached stain was recovered with Tris EDTA buffer. Colour intensity was measured in an ELISA reader with 570 nm wavelength. The relation between surviving fraction and sample concentration was plotted and IC50 (the concentration required for


50% inhibition of cell viability) was calculated for each sample by Sigma plot software. In addition to that, hMSCs were seeded on the scaffold surfaces in 12 multi well plates for 21 days under the above mentioned conditions. In details, the scaffolds were washed 3 times with 70% ethanol, exposed to U.V light for 1 h and then washed twice with the used medium. HMSCs were seeded on the scaffold surfaces at density of 10 × 106, and incubated at 37 °C in DMEM containing 20% FBS, 100 μg/mL penicillin & streptomycin and 1% glutamax, Lascorbic acid 2-phosphate, β-glycerol phosphate and dexamethasone for 21 days. Furthermore, scaffolds were fixed using Glutaraldehyde. Finally, the hMSCs were fixed on the scaffold surfaces and were characterized by SEM after gold coating. 3. Results and discussion 3.1. Crystallization behavior of scaffolds 3.1.1. Thermal analysis DSC/TGA analysis was used to investigate the thermal behavior of the prepared scaffold samples, through this analysis both Tg and Tc could be determined for all the prepared scaffolds to figure out the effect of HA concentrations. According to DSC curves (Fig. 1), four regions of interest were observed: the first region, exhibited endothermic peak around 100 °C (initiated ∼25 °C and continued till 180 °C) corresponded to ∼17.71% weight loss (Fig. 1(a)). This loss was attributed to disposal of greater part of the water and the amorphous transition temperature [37]. A great number of scientists have reported that there are three sorts of assimilated water within the hydrophilic polymers network; free, solidifying bound and non-solidifying water or restricted bonded water [41,42]. For pure sodium alginate scaffold (S1), the region from 20 °C to 180 °C could be divided into two sub regions, the losses of weight in the principal sub locale stage can be assigned to the disposal of free water as illustrated in Fig. 1(a). These particles of water are the initial atoms leaving the specimen (weight loss approximately 11.59%). Above 100 °C the specimens still containing water. Actually, on expanding the temperature flow, the specimen's weight keeps on continuous diminishing, in spite of the fact that the decay processes have not been included yet. In this region, most solidifying and non-solidifying water were removed (weight loss ≈6.12%). Furthermore, Fig. 1(b) showed a remarkable change for scaffold S2, at 180 °C mass loss increased to ≈27.29% especially sub region under 100 °C ≈ 17.54%, due to the presence of HEC. The decomposed water was attributed to the independent water, while S1 possessed both free and bound water. This observable contrast was related to the different chemical nature of both SA and HEC. The addition of HA to the blend of SA/HEC had affected water interaction. In detail, Fig. 1(c) confirmed that the addition of HA considerably influences the quantity of retained water, especially the quantity of bound water absorbed was decreased in HA-loaded scaffolds, which perhaps ascribed to the increased bonding between scaffolds component, resulted from chemical the interaction of HEC and SA grouping with HA structure. This resulted in decreasing of polymeric network free rotation in composite [43,44]. S1 scaffold exhibited an exothermic peaks at 270 °C (initiated ∼180 °C

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and continued till 340 °C) were attributed to the degradation of polysaccharide network [44], corresponded to the major weight loss (31%) due to the breaking up of C\\H bonds (i.e. degradation of the biopolymer) which was initiated from 240 °C and continued to 340 °C, this exothermic peak was shifted to 300 °C upon the addition of HEC as observed for S2 scaffold [33]. All the prepared scaffolds exhibited exothermic peaks at 460 °C corresponding to 9.23% weight loss; this could be ascribed to the pyrolysis of calcium alginate. The HA-loaded scaffold (S5) showed an exothermic peak at 820 °C, associated to crystallization temperature (Tc) of CaO, and corresponding to weight loss (about 10%) also an exothermic peak was observed at 680 °C, which was ascribed to the dehydroxylation behavior of HA [45,46]. The obtained results suggested two effects: the first effect was the reduction of Tg and Tc upon HEC addition (Tg from 240 to 210 °C; Tc from 770 to 650 °C). This result was in the same line with early reported by Russo et al. [37]. The second effect was the reduction of weight loss and water absorption due to the presence of HA. This result was due to the potential chemical reaction between SA/HEC modular groups with HA, thus, it increased both Tg, Tc again (Tg from 210 to 250 °C; Tc from 650 to 655 °C). 3.1.2. X-ray diffraction analysis In this study, the XRD was also used to investigate the crystallinity and to determine the obtained phases for the prepared scaffolds. According to XRD curves presented in Fig. 2(a), the diffraction peaks of SA exhibited completely amorphous nature with a broad peak between 20 and 40 (2ϴ), which could be attributed to the polymer network [24]. The XRD spectrum of S2 showed the amorphous nature with two broad peaks, first broadening between 20 and 35 (2ϴ) that proved the existence of sodium alginate, second broadening between 35 and 50 (2ϴ) owing to the presence of HEC [24,47] The HA containing scaffolds (S3, S4 and S5) exhibited predominant peaks of XRD pattern in Fig. 2(a) that was referred to HA matched with standard card of JCPDS 760694. The higher HA concentration in the scaffolds (S5) demonstrated higher peaks intensity. 3.1.3. FTIR analysis FTIR spectroscopic technique was used to determine the interaction and bonding occurring between the scaffold components. IR spectra of pure alginate scaffold (S1), is represented in Fig. 2(b). Major common bands, especially those above 3000 cm−1 (3000:3700) assigned to hydroxyl group (O\\H) stretching vibration mode; the bands represented at 2923 cm−1 assigned to methylene stretching vibrational group (C\\H); and band at 1108 cm−1 assigned to C\\O group in case of stretching vibration. The S1 spectra showed two distinguished bands found at 1617 and 1421 cm−1 may be attributed to asymmetric stretching of –COO– and symmetric –COO–, respectively. The Ca2+ crosslinking process was confirmed by obvious shift to higher wavenumbers for the –COO− group stretching bands, resulting from the ionic bond formation between Ca2 + ions and the –COO– group of alginate scaffold [48–50]. The appearance of band at 460 cm−1 in S1 spectrum indicated the presence of Ca\\O vibration, suggesting the presence of (Ca (OH)2 ion complex. In

Fig. 1. DSC/TGA curves of S1, S3 and S5 scaffolds.

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Fig. 2. Illustrates a) XRD; b) FTIR patterns of all scaffold samples before immersed in SBF.

addition, alginate characteristic bands were also observed, small two bands which are located at 817 and 877 cm−1 assigned to C\\C\\H, skeletal C\\C, C\\O\\H and C\\O\\C symmetric vibration of 1,4-glycosidic link [51], as well as 1036 cm−1 (C\\O\\C stretching).

HEC addition to SA (S2) scaffold showed no remarkable features when it is compared with the spectrum of S1. However, the characteristic O\\H deformation band in both SA and HEC at 616 cm−1, showed a slight shift to lower wavenumber (from 619 in S1 to 611 cm−1 in S2.

Fig. 3. SEM images of scaffolds sample a) S2, b) S3, c) S4 and D) S5 before immersion in SBF.


This shift may be related to the bonding between SA and HEC groups. The intensity of this band was increased through Ca-cross-linking [24], suggesting a good distribution of HEC particles in SA matrix [52]. The higher intensity and shifting of –OH absorption band that was observed for S2 (at 3409 cm−1) compared with S1 (at 3416 cm−1), proved also the well blending of HEC with SA and suggested also that the developed hybrid scaffold possesses higher hydrophilicity than pure SA scaffold S1, the FTIR results are in the same line with thermal analysis results. Remarkable changes were imposed by presence of HA as shown in Fig. 2(b). particularly, the bands located at, 1037, 609 and 566 cm−1 atwere observed for scaffolds S3, S4 tributed to phosphate group PO3− 4 and S5. The higher intensities of these bands, especially at 603 and 566 cm−1 confirmed the presence of well crystallized HA. This sharpness was increased with the increase of HA content [53]. Kikuchi et al. reported that a weak ion-dipole bond could be formed among oxygen anion in carbohydrate group of SA (C_O) and the Ca cations of hydroxyapatite [54,55]. From the FTIR results it could be confirmed that some chemical interactions are taking place between the scaffold components. These interactions would in turn enhance the overall physicochemical and mechanical properties as well as the in vitro bioactivity and the protein adsorption ability of the prepared scaffolds. 3.1.4. SEM analysis Pore size is a very important factor for the cells growth and diffusion of nutrients, thus would facilitate the cell attachment as well as the vascularization [39,56]. Fig. 3. showed the SEM images of S2, S3, S4 and S5 composite scaffolds (a, b, c and d), respectively. Generally, all the prepared scaffolds showed a lamellated microstructure, also it is worthy noted that the HA-free scaffold (S2) exhibited a smooth surface compared with the HA-loaded composite scaffold (S3, S4 and S5). This may be attributed to the addition of HA that essentially increased the outer surface area of their scaffolds. Undoubtedly, the rough surface

453

and the higher surface area are more favorable for cell attachment and proliferation [33]. The pore size was observed to be in the range of 100–300 μm, this pore size is suitable for tissue engineering applications as previously reported [57].

3.1.5. Porosity (%) The porosity percentages of the prepared composite scaffolds are shown in Fig. 4(a) with reference to pure alginate (S1) scaffold. The percentage porosity was increased from ≈82 ± 4.3% (S1) to 87 ± 5.1% upon the addition of HEC. Moreover, the porosity (%) was decreased gradually to 79.25 ± 5.4, 73.17 ± 6.3, 66.7 ± 3.2% for S3, S4, S5 respectively due to the presence of HA. Pores are fundamental parameter for the transport of oxygen and nutrients from the extracellular matrix to the interior of the scaffolds. A porosity decrease of the composite samples was noted with a controlled manner due to the presence of HA, but still sufficiently favorable for bone replacement applications. However, it is well known that the presence of HA in the composite scaffold would support the cells in growth through the scaffolds [39].

3.1.6. Swelling behavior Swelling is the capability of scaffolds to uptake and preserves the water within their structure. It is an important feature for developing suitable tissue engineering constructs for regeneration of bone defect [58]. It is worthy noted that the blend SA/HES (S2) scaffold possessed higher swelling (%) compared with SA (S1) scaffold as in Fig. 4(b). This difference could be attributed to the addition of HEC, which had increased the OH– groups of S2, which in turn increased the absorbed water during the immersion time. On the other side, HA incorporated into the scaffolds decreased the swelling (%) compared with the native polymers. This might be because of the diminishment in pore volume with the increment of HA powder content.

Fig. 4. Demonstrates a) Porosity (%) by Liquid displacement method b) Swelling (%), c) Compressive strength and d) Resilience (%) for all the scaffolds.

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Overall, the swelling ratio was observed to be increased with time until 4 weeks. Swelling with pores volume under certain ratio helps in the diffusion of nutrients into the scaffolds [39,59]. In addition, porosity (%) increased also the exposed surface area for cells to ensure better cell adhering. The cells attachment is very essential for tissue regeneration. However, increased swelling could affect the mechanical properties of the implanted material; in this case a controlled swelling is recommended for hard tissue regeneration [56].

and created mechanical interlocking within the polymer matrix. The mechanical stability of composite scaffolds samples relies upon the kind of polymer, porosity, and crosslinking chemistry. These results demonstrated a reasonable mechanical stability for all the prepared scaffolds. However, better mechanical properties were observed for scaffold containing the higher concentration of HA.

3.1.7. Mechanical analysis Scaffolds for bone tissue engineering should possess an appropriate mechanical strength to help bone tissue recover at the implantation site and ensure suitable integrity through both in vitro and in vivo cell proliferation [60–61]. One of the real difficulties in the creation of porous scaffolds is the tradeoff between the needed porosity and mechanical strength [61]. From the mechanical test, it was very obvious, that the addition of HEC to the SA showed a negative effect on the mechanical properties (1.31 + 0.09%) resilience (%) and (8.83 ± 0.34 MPa) compressive strength of (S2) compared with (S1) resilience (%) (3.54 + 0.08) and (10.13 ± 0.51) compressive strength as illustrated at Fig. 4 (c). This could be due to the negative effect of high porosity (%) recorded for S2, thus it resulted in lower mechanical properties for S2. This case was consistent with the early reported results [62]. It was observed that the compressive strength of the scaffolds was gradually increased from 18.56 ± 0.76 to 21.22 ± 0.71 and 23.96 ± 0.82 MPa for S3, S4 and S5 respectively, contrariwise resilience decreased from 2.97 ± 0.12, 2.73 ± 0.13 and 2.61 ± 0.15% as shown in Fig. 4(c and d). These were attributed to HA powder addition into the scaffold polymer matrix, which reduced the number of open pores

3.2.1. XRD analysis after soaking in SBF Fig. 5(a) showed the XRD patterns of the prepared scaffolds after being immersed for 28 days in SBF. In general, four sharp peaks located at 2θ 22.8°, 25.6°, 31.76°, 32.16° and 33° were observed after immersion in SBF, these diffraction peaks were allocated to (111), (002), (211), (300) planes and attributed to HA layer formed on the scaffold surface as confirmed by the standard JCPDS document no. (82-1943), the broad reflections peak at (2θ) running from 31.2°: 34.3° is related to the interference of (211), (112), (300) and (202) which refers to a good crystallization for hydroxyapatite phase [63–65]. The intensity of apatite peaks increased with the increased concentration of HA in scaffolds. The mechanism of hydroxyapatite (HA) phase formation is related to an interaction that takes place between the Ca2+ of samples and the H3O+ in aqueous solution, and then the polymer network breakdown takes place (de-polymerization). Afterwards, cross-linked (calcium alginate), forming carbonyl bonds that repolymerize to form a hydrated stimulate apatite nucleation site. The formed latest nuclei was then developed, starting the apatite formation [66]. The formation of the HA on the scaffolds surfaces confirmed the scaffolds bioactivity.

3.2. In vitro bioactivity analysis

Fig. 5. (a) Showed the XRD patterns of the prepared scaffolds after being immersed for 28 days in SBF.


XRD results suggested that, the HA was the precipitated phase on the scaffolds surfaces after ion exchange in SBF, the addition of HA favors to the crystalline calcium phosphate hydroxide phase is formed on scaffold surface. Moreover, the intensity of the observed peaks is a fingerprint for crystallinity HA phases [67]. For S4 and S5 the HA crystalline phase is more pronounced. This result could be explained due to higher concentration of HA in S4 and S5 samples compared with the non-containing HA scaffolds (S1 and S2). 3.2.2. Crystallinity estimation of HA by XRD The crystallinity index (XC), is related to the quantity of crystalline HA in the examined area of surface scaffolds. From Fig. 5(b), The Xc gradual increase was found to be dependent on the HA concentration. Particularly, the sample S5 exhibited higher XC than those of HA low concentrations as well as the native polymers. This result was consistent with the results reported previously [68]. It is notable that the HA may be able to contact with the network of the native polymer, has the ability to change easily its coordination between oxygen and calcium ions and hence can form variable structural units. 3.2.3. FTIR after SBF (bioactivity) The important and conclusive role of adding hydroxyapatite on the improvement of scaffold bioactivity was confirmed by the FTIR spectra as demonstrated in Fig. 5(c). Generally, two bands at 564 cm−1 and 603 cm−1 were observed for all the prepared scaffolds after immersion in SBF [15] as well as broad band is observed at 1024 cm−1 and 1108 cm−1 confirming the presence of HA phase [20,69,70]. The precipitation of HA was found to be dependent on the HA concentration in the scaffolds as confirmed by the characteristic O\\H bands located at 1637 and 3419 cm−1, that increased gradually from S1 to S5. This result could be due to the hydrogen bonds and electrostatic forces presented between ions and groups in the composite scaffolds. Particularly, chemical interaction between –C_O groups in SA biopolymer and charged groups such as Ca2+ and PO3− 4 of the HA filler. Additionally, the bonds that were initiated between Ca2+ group of HA as a filler and –COO– group of SA as a network polymer were early reported [71–75]. Thus, the high concentrations and uniform distribution of HA particles in the SA/HEC matrix enhanced the bioactivity of the scaffolds [72]. It is worth to highlight that the higher content of

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HA in the composite, the better bioactivity could be achieved [76,77]. The obtained FTIR results refer to both HEC and HA important role on enhancing the bioactivity of SA polymer, this is in the same line with the XRD results after immersion in SBF. 3.2.4. SEM and EDX after immersion in SBF Fig. 6. shows the SEM images after immersion in SBF for 28 days. The SEM images confirmed the formation of new layer on the scaffolds surface. SBF is a metastable calcium phosphate solution supersaturated with apatite [78]. While a biomaterial scaffolds was being soaked in simulated body fluid, the layer of appetite was formed on scaffold surfacethrough series of biochemical interactions-these biochemical interactions include minerals deposition from the SBF onto the scaffold surface and nucleation of the precipitated initial hydroxyapatite, that lead to the development of bonding with calcium ions on scaffolds surface, which plays a dominant role in the formation of hydroxyapatite. During the immersion time, the previously mentioned negative particles on the HA surface pulled in the positive Ca2+ particles from surrounding SBF solution, which in turn added positive charges to the specimen surface. Furthermore, negative ions groups (OH– and PO3− 4 ) were attracted from SBF, thus, they induced the development of the apatite layered crystals [79]. It is worth to highlight that the apatite layer could be shaped on the composite with consistent pores, which gave available areas to the deposition of HA on scaffolds surface. In this manner, in a similar interim of time, more apatite was growing on S5 surface than on the other scaffolds [80]. The EDX results are shown in Fig. 6(c and d), for S1 and S5 scaffolds after in immersion in SBF for 28 days. The spectrum of both scaffolds was distinguished by appearance of multi peaks, for the following elements P, O, Cl, Ca, k, and Na. A reasonable improvement of the Ca and P peaks was recorded. Essential examination of the precipitated minerals on the scaffold surfaces by means of EDX investigation demonstrated the presence of calcium and phosphorous elements, with Ca/P proportion of 2.45 and 2.58 for S1 and S5, respectively. The highly Ca/ P ratio result could be explained by overlapped identification of both the Ca2+ from deposited mineral and the Ca2+ from alginate crosslinking on the scaffolds surfaces. Generally, mineral precipitation on the surface is often interrupted when pores are filled [40]. The combined FTIR, SEM and EDX results suggest that the scaffolds degradation

Fig. 6. Demonstrates a) SEM images of S1, b) SEM images of S5, c) EDX spectra of S1 and d) EDX spectra of S5 after soaking in SBF for 28 days.

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Fig. 7. Demonstrates a) In vitro degradation profiles of the prepared scaffolds in SBF, b) Bradford standard calibration curve of different protein concentration 0.4–2 mg/ml and c) Protein adsorption studies for all scaffolds in BSA at 37 °C for different time intervals.

Fig. 8. Morphological observation on fibroblast cells cultured on pure SA scaffold (S1); hybrid SA/HEC (S2) and composite SA/HEC-HA (S5).


and deposition of a Ca–P layer takes place spontaneously which is more reliable for tissue engineering, specifically bone tissue replacement [31,78,81–83]. 3.2.5. In vitro degradation studies Due to the fundamental significance of knowing the degradation rate of any scaffold materials that could be implanted in the human body, degradation was conducted in the SBF at different time intervals (1, 2, 3 and 4 weeks), under the same conditions of biomineralization test. The in vitro degradation profiles of scaffolds are shown in Fig. 7 (a), revealed that the degradation (%) was increased with the immersion time for all scaffolds compared with the pure SA (S1) scaffold. This was due to the higher crosslinking/polymer ratio in sample S1 scaffold compared with other scaffolds according to the scaffolds compositions [84]. The presence of HEC caused more degradation to take place, because of the weak bonding interaction between the two polymers with Ca2+ ions (cross-linker) due to reduction of carboxylic group, thus, it resulted in higher degradation rate for S2, S3, S4 and S5 scaffold compared with S1, this was more articulated for extended immersion time in SBF. The actuality that illustrates this phenomenon is that the hydrogel SA/ HEC/HA composite scaffolds could bolster a biochemical reaction between HEC and SA and SBF solution via Ca2+ ions. Accordingly, the ions exchanging phenomenon (the calcium divalent cations diffuse from samples into SBF due to the presence of monovalent cations such as phosphate ions and potassium ions located in SBF) has a higher chance in the presence of both HEC and HA. The degradation result confirm the fulfillment of the main objective of this study, alerting and controlling the crosslinked alginate degradation in the SBF [85]. All the conducted characterizations after soaking the scaffolds in SBF had revealed the impressive role of HEC in the present formulations in enhancing the deposition of Ca-P layers and control its degradation, thus in turn makes the under investigated scaffolds more suitable for bone tissue regeneration. 3.2.6. Protein adsorption studies According to the performed Bradford standard calibration curve for different protein concentration (0.4–2 mg/mL) as showed in Fig. 7(b), the amount of the adsorbed protein could be calculated by comparing the absorption values of the aliquot solution. It is worthy to mention that there is a significant increase in the protein adsorption for the SA/ HEC (S2) composite scaffold compared with the pure SA (S1) scaffold with the increment of immersion time in BSA solution. The existence of functional groups on scaffold surface, such as (hydroxyl, carbonyl and oxygen), would increase the protein adsorption [86,87], addition of HEC to SA caused formation of excess hydroxyl group on the scaffold surface. Thus, it imparted higher hydrophilicity which- in turn- increased the protein adsorbing affinity of scaffolds. In details, more hydrophilicity would increase the electrostatic forces, Vander Waals force, thus, would result in an induced protein adsorption onto the surface of SA/HEC (S2) scaffold. It is worth to highlight that the increment of surface area of the composite scaffolds would increases the number of protein attracting sites on the scaffold surface [88].

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It is worth to highlight that the BSA is the significant calciumrestricting protein existing in the human blood and consists of 19 albumin molecule calcium binding sites destinations on its imidazole groups, Just 10% of these destinations are possessed by Ca2+ ions in physiological medium. It is also important to mention that the calcium-binding property of albumin is not steady on drug pharmacokinetics [89]. In general, there are many factors affecting on protein adsorption onto the materials surface, such as (a) functional groups found in their surface, (b) ability to absorbed water (swelling) and (c) surface topography [87]. In our case, addition of HEC and HA to SA, has modified surface functional groups as confirmed by FTIR, like hydroxyl and carbonyl groups, Evidently, from both porosity and swelling studies, the amount of HA when blend to SA/HEC could control wettability and surface topography by interaction happened between HA and Ca ions (crosslinker), all these changes increase the number of protein attracting sites on the scaffold surface. In addition to that, the protein adsorption was increased with the higher concentration of HA and adsorption rate changed with the prolongation of immersing time. This could be explained due to the fact that the higher concentrations of HA possessed a higher affinity for Ca2+ ions and COOH– groups of BSA, which caused an increase in the BSA adsorption on scaffolds surfaces. This could be due to that the bonding with BSA acts as a bridge between BSA and HA surface [90]. It is known that the increases of the adsorbed amount of protein would enhance the cells attachment through biomolecule adhesion keys, such as BSA. 3.2.7. In vitro cell culture analysis Fig. 8 shows the morphological observation on the cells after scaffold samples exposure to normal human MSCs. No dead cells were observed in the well plates for all the prepared scaffolds which means that all the tested samples had no negative effect on normal environment. After 3 days of incubation, normal cell growth was recorded and continued to increase up to 7 days. The cell viability assay revealed that the IC50 (minimum concentration needed to induce 50% of cell death following exposure to the samples) was not obtainable; in detail, the toxicity values were below 50% of cell viability percentage for different concentrations of scaffolds. This result could be due to the non-toxic properties of the individual components of the scaffolds (SA, HEC and HA) as early reported [91–93]. Moreover, SEM images of hMSCs Fig. 9(a) showed cells anchoring on a alginate pure scaffold and the cells attachment to the scaffold surface was noted. The cells are indicated as “C”, cell–cell junctions, indicated as “J”, open pores, indicated as “P” were also detected. Extracellular matrix (EX), attached to scaffold surface, of alginate and HEC/alginate (Fig. 9 (b)) scaffolds was also detected. The differentiation of hMSCs was accompanied by the production of clusters of bone like apatite mineralization surrounded by extracellular matrix. The observed mineralization is in the same line with the Ca-P layer deposited on the scaffold surfaces after soaking in SBF without cells, indicating the self-mineralization property for the prepared scaffolds. Several junctions that were expanded between the cells within and above the scaffold matrix are very obvious. The joints and the extracellular matrix were highly

Fig. 9. The SEM images of a) pure SA scaffold (S1), b) hybrid SA/HEC (S2) and c) composite SA/HEC-HA (S5) after seeding of hMSCs on the scaffold surfaces for 21 days.

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Fig. 10. Illustrates a) Cells viability assay and b) Cell proliferation assay.

pronounced for the scaffold containing HA Fig. 9(c), and the secretion of hMSCs increased also in the presence of HA. These results proved the osteogenic functions and rapid bone formation of novel scaffolds after 21 days. Furthermore, hMSCs seeded on tissue culture exhibited significant differences in cell number after 7 days of incubation as shown in Fig. 10(a, b). In details, the number of recorded cells after 3 days for samples S1, S2 and S5, respectively was increased to 8 × 104 ± 8 × 103, 8.32 × 104 ± 5.26 × 103, 7.7 × 105 ± 1.1 × 103 compared with control 8.25 × 103 ± 1.25 × 103. After 7 days the proliferation effect for the prepared scaffolds was confirmed by increased cells population recorded for samples S1, S2 and S5, respectively 2.62 × 105 ± 1.3 × 104, 2.96 × 105 ± 2.0 × 104 and 3.9 × 105 ± 1.91 × 104 compared with control 2.45 × 105 ± 1.47 × 104. These results suggested that the human MSCs attachment and proliferation were governed by the presence of both HEC and HA. In addition, the observed increase in human MSCs attachment is an important advantage of the HA inside scaffold, since cell attachment is important for the survival, proliferation, and differentiation of multiple cell types including bone-forming cells. Recent studies suggest that HA based materials may intrinsically promote ectopic bone formation in vivo, even without inclusion of exogenous cells or growth factors. Indeed, previous studies have demonstrated that HA substrates may have a positive influence on osteogenic differentiation of human MSCs in vitro [94] and in vivo [80,95]. Furthermore, the presence of SA and HEC will be useful as scaffold matrix for bone replacement that can also be used as a carrier for bone growth factors. Many scientists, recently, have used the calcium phosphate biomaterials affinity for proteins to deliver proteins in sustained manure [80]. 4. Conclusion The present work illustrated the feasibility to prepare SA and SA/ HEC/HA scaffolds by freeze drying technique. The new 3D scaffolds showed great physico-chemical and mechanical behavior that make them suitable matrix for cells attachment and growth. The prepared scaffolds are promising compounds in bone tissue regeneration, Furthermore, the addition of HEC to the fabricated scaffolds elevated the porosity (%) and swelling (%) but decreased the compressive strength, therefore, selecting HA to be incorporated in the SA/HEC has clearly enhanced the bioactivity, mechanical properties and supported the ability of scaffold to increase adsorbed protein such as bovine serum albumin as well as cells viability and proliferation. In terms of our study, the HEC alerted and controlled the degradation rate, cells attachment and proliferation of prepared scaffolds, thus increased the feasibility of fabricated scaffolds to be suitable for bone tissue engineering. Therefore, SA/HEC/HA scaffolds are recommended for multi-application in bone tissue engineering.

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