Improvement of physico-chemical properties of dextran-chitosan composite scaffolds by addition of nano-hydroxyapatite

Nano-hydroxyapatite was incorporated into polymer matrix of Dextran/Chitosan to achieve a novel composite scaffold by freeze drying technique. The synthesized composite scaffolds were recognized by different performances such as: X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) and Scanning electron microscope (SEM). The results revealed the complex formation between dextran and chitosan with an excellent dispersion of nHA inside the polymer matrix. The SEM images showed the presence of interconnected pore structure inside the scaffolds. The porosity of the composites was found to decrease from 82% to 67% by adding nanohydroxyapatite to the polymer matrix of Dextran/Chitosan. The mechanical properties of the scaffolds were measured by compression test. The obtained results verified that the presence of nHA can noticeably enhance young’s modulus and compressive strength of the composite scaffolds. All the obtained results essentially recommend that these composites can be a good candidate for bone tissue engineering applications.

geometries. However, up to date, there is still no research on the preparation of a Dex-CS/nHA composite scaffold. Hence, here we first combined nano-hydroxyapatite (nHA) into Dex-CS to achieve a novel composite scaffold of Dex-CS/nHA by freeze-drying method.
In this paper, we investigate the preparation of Dex-CS/nHA composite scaffolds with different weight percentage of nHA of (0, 20, 30 and 40 Wt%). Also, the influence of nHA on the morphological and mechanical properties of the scaffolds is studied and compared.
A suspension of 1 M Ca(NO 3 ) 2 ·4H 2 O was prepared. The Ca:P ratio for stoichiometric HA = 1.67. Keeping that ratio constant, a suspension of 0.6 M (NH 4 ) 2 HPO 4 was organized by dissolving (NH 4 ) 2 HPO 4 in distilled water. A 1 M calcium salt solution was dropped slowly from a burette into a stirred solution of 0.6 M aqueous phosphate solution with heating up to 60 °C on a hot plate. Then NaOH solution was added dropwise to control the pH of the solution mixture at ≈11 during the preparation process keeping the solution to stirred for 3 hours. The obtained suspension was aged at room temperature for 5 days. The HA precipitates were filtered from the solution, washed with distilled water and dried in a furnace at 60 °C. Finally, the obtained powders were sintered at 1050 °C for 1 hr then grounded to fine particles 10 .
Preparation of the Composite Scaffold. Dextran-Chitosan composite scaffold was synthesized via blending method. A dextran-chitosan aqueous solution of 8 Wt% was prepared by dissolving dextran and chitosan powders into acidified distilled water (3 Wt% acetic acid) with constant stirring overnight at room temperature to get a homogeneous mixture. Then the mixture was moulded, frozen to freeze the solvent and lyophilized in a freeze-dryer at −90 °C for 48 h and a porous scaffold was achieved. This sample was denoted by A. Then, HA nanoparticles (nHA) powder was added with different proportions into a dextran-chitosan aqueous solution of 3 Wt% acetic acid (20,30 and 40 Wt%) with constant stirring overnight at room temperature to get a homogeneous mixture. After that, the mixture was moulded, frozen to freeze the solvent and lyophilized in a freeze-dryer (SCANVAC, Denmark) at −90 °C for 48 h to get porous scaffolds. These samples were symbolized by B, C and D, respectively.
Characterization of the scaffolds. X-ray diffraction (XRD). X-ray diffraction (XRD) measurements were aquired using a diffractometer model (Type PHILIPS X' Pert Diffractometer) having a Cukα (λ = 0.154056 nm) at room temperature. The diffraction patterns were recorded automatically with scanning speed (4°/min) and chart speed (20 mm/min). The X-ray diffraction patterns were investigated in the range of 2θ between 4° and 60°.
Fourier transform infrared spectroscopy (FTIR). FT-IR spectra of dextran, chitosan and all composite scaffolds (A, B, C and D) were obtained using an FT-IR spectrometer model (FT/IR-6100 type A).The scaffolds were grounded into fine powder. Then, the obtained powder was mixed with KBr powder and compacted into pellets for FT-IR examination. The spectra were investigated in the wave number range of 400-4000 cm −1 .
Scanning electron microscopy (SEM). The morphologies of the synthesized scaffolds (A, B, C and D) were studied by scanning electron microscope (SEM). Scaffolds were cut into thin sections with a razor. After being coated with gold in a sputtering device, the samples were examined with a scanning electron microscope (QANTA FEG 250).
Porosity and density. The total porosity (P) and density of the prepared scaffolds were measured using a liquid displacement method 11 . Scaffolds with a given dry weight were putted in absolute ethanol for 24 h. Ethanol was used because it can penetrate through the scaffolds without bulging or shrinking the matrix and the scaffolds would not be dissolved in ethanol under room temperature 12 . The total amount of ethanol that the scaffolds were capable to absorb during 24 h was estimated by utilizing the following relationship: where W 1 and W 2 indicate the weight of the scaffolds before and after submerging in ethanol, respectively, V is the volume of the scaffold before submerging and ρ is a constant density of ethanol. When nHA particles added to Dex-CS composite with different proportions (20, 30 and 40 Wt%), the crystalline features of the constituents HA and Dex-CS still exists, as verified from the existence of their respective characteristic crystalline peaks. Moreover, XRD pattern of the composite B shows the two characteristics peaks of Dex-CS composite at 2θ of 20.4° and 39.37° and the high intensity diffracted peak of nHA at 32.21°, which suggests that the obtained composite has low crystallinity. As the amount of nHA percentage increases (composites C and D), an increasable change in both crystallinity and the intensity of the reflection planes has been observed in the respective XRD pattern. It is worthy to note that the crystallinity of the Dex-CS/nHA composites scaffolds is lower than pure nHA, which can be attributed to the occurrence of the polymer matrix 13 .
The broadening of the diffraction peaks verified the nanocrystalline characteristics of the samples. The peak broadening of XRD pattern can be used to determine the crystallite size (L) in the perpendicular direction of the crystallographic plane based on Scherrer's formula 14,15 .
where K is a constant related to the crystallite shape (K ≈ 1), λ is the wavelength of X-ray radiation (0.154056 nm), β is the full width of the high intensity peak at half of its maximum intensity (rad). The average values of crystallite sizes of all composites lie in the range of 3-33 nm as tabulated in Table 1. It could be observed that the estimated crystallite size of composite with high concentration of nHA (composite D) is ~23 which is analogous to the crystallite size of bioapatite in bone tissue. The average size of bone apatite is normally ~20 nm, which was evidenced by different experimental techniques 16 . Lattice parameters (a and c) have been calculated from the diffraction planes (300) and (002), respectively, using the typical HCP unit cell plane spacing relationship 17 :  Table 1. The obtained results reveal that there is a variation in the lattice parameters values of hydroxyapatite present in biopolymer/hydroxyapatite nanocomposites in comparison with pure hydroxyapatite. The biomimetic hydroxyapatite displayed analogous behavior in all nanocomposites, i.e., positive shift (elongation) was found along c-axis and a-axis (a = b). The ratio (R) of peak intensity of the planes (002) and (300), R 002/300 = I (002) /I (300) , for the Dex-CS/nHA composites, is larger than that of the pure nHA as shown in Table 1. This means that the nHA crystals in the Dex-CS/nHA composites grow along the c-axis.  signifying chemical interaction between the amino groups of CS and the Ca 2+ in the nHA or between the amino groups and PO 4 3-23 . The stretching vibrations of C-H band of Dex-CS shifted from 2928 cm −1 to 2923 cm −1 in Dex-CS/nHA with decreasing in its intensity by increasing nHA percentage. Figure 2 also shows that by enlarging HA content, the typical band of PO 4 at 500-700 cm −1 became stronger 24 . Meanwhile, the wavelength and intensity of the other bands of Dex-CS changed slightly with no significant trend. The absorption bands are given in Table 2.

Scanning electron microscopy (SEM). The SEM images of the composite scaffolds (A, B, C and D)
are shown in Fig. 3. It could be observed that all scaffolds exhibit porous structures with good interconnection which directly influence the biological performance of materials. The cause of the porous morphology is that in the frozen process ceramic particles are extruded into gaps among ice crystals 25 . The high magnification SEM Then by adding nHA, the surface becomes rougher. All composite scaffolds are similar in their macroscopic morphology, which indicate that the addition of HA to system is not affect the porous structure; however, the microscopic morphology on pore wall surfaces is quite different because of different crystals created. It is clearly seen that the surface morphology of the scaffolds significantly altered by adding nHA, where the surface of the pure Dex-CS composite scaffold appeared smooth.
Comparing the four scaffolds, it could be noticed that the pore diameter of the scaffold decreases with increasing HA content as shown in Fig. 4. The average pore diameters have been estimated by analyzing scaffolds SEM images and were tabulated in Table 3. Also, it was observed that the incorporation of HA nanoparticles into the Dex-CS composite scaffolds decreased the porosity which is confirmed by the porosity assessment results.    Moreover, the number of SEM images on scaffolds implied that HA nanoparticles collapsed the pore structure which is in convenient with that earlier discussed by Jin et al. 26 .
From the above results of SEM, it could be found that the achieved composite scaffolds have good structural integrity and porosity. So, they are perfect candidates for tissue engineering and bone regeneration.
Porosity and density. Density and porosity are important characteristics of the scaffolds which are related to each other. The apparent density of the scaffold can effect its mechanical strength, permeability, and existence of structural defects due to its porous nature 11 . Also, the porosity is a vital factor for a perfect scaffold to be used in tissue engineering applications 27 . Cells can mobile through the pores and become attached at appropriate positions in the scaffold for additional proliferation. Since a higher density of a scaffold generally leads to higher mechanical strength while a high porosity offers a promising biological environment, a balance between the porosity and density of a scaffold must be recognized for the specific application. The density of the composite scaffolds (A, B, C and D) has been measured, the obtained results ranges from 0.809 to 0.838 g/cm 3 as shown in Table 3. These values are in good accordance with the apparent density of trabecular bone which ranges from 0.14 to 1.10 g/cm 3 . It is observed that, adding nHA to Dex-CS matrix increases the scaffold density. The increased density of the composite scaffold is ascribed to the greater material content in scaffold, as the water volume during the preparation of the scaffolds has been fixed. The enlarged density of the composite scaffold contributes to the better compressive performance.
The composites have a porosity range from 67.5 to 82.9% (Table 3 and Fig. 5), which is appropriate for bone regeneration, according to what was previously designated by Kruyt et al. 28 . The porosity of the composite decreased by increasing the nHA content due to the deposition of the nHA over the scaffold matrix and the intermolecular hydrogen bonding between -NH 2 groups of chitosan and -OH groups of nHA, these results are similar  to previous studies 29,30 . Other earlier researchers have found that the porosity decreased with adding ceramic particle content, since the increment in the density of the scaffold is confirmed 31 . The present nanocomposite scaffolds have sufficient porosity which helps in the passage of nutrients and oxygen to the inner regions of the composite scaffolds.
Mechanical Properties. The scaffolds for bone tissue engineering should have sufficient mechanical properties for maintaining their integrity after implantation till new bone tissue regenerates and to transfer the load properly. The mechanical properties of freeze-dried scaffolds are usually tested by compression mode 32 . The influence of nHA on the mechanical properties of the Dex-CS scaffolds in dry state has been explored. The obtained values of Young's modulus and compressive strength have been summarized in Table 3.
The linear variation of stress versus strain (up to the strain value of about 15%) was detected in the first phase of the compression investigation. This is the stage of elastic distortion and this linear stage of the curve was used to estimate the Young's modulus of the composite. Within this stage of strain, the scaffolds distorted elastically and returned to their initial dimensions when the load was removed. After the linear stage of the curve, the scaffolds did not return to their initial dimensions when the load was removed and the starting of the destruction process was detected.
As previously designated in literature, the polymers used for scaffold fabrication, such as chitosan and dextran, existing weak mechanical properties which limit their applicability in bone tissue engineering 33 . To overcome this handicap, the adding of calcium phosphate ceramics to chitosan has been described to enhance the mechanical properties of the scaffolds 33,34 . Therefore, as can be observed from Figs 6 and 7, addition of nHA particles to the Dex-CS matrix improves both Young's modulus and compression strength.
The Young's moduli of the prepared composites were moderately low and different from those of natural spongy bone (0.18-0.33 GPa) 35 and compact bone (17)(18)(19) 36 . It could be proposed that the strain was taken by Dex/CS matrix while the ceramic granules were only slightly deformed through compression testing.
The increased compressive strength provides evidence that nHA interacted well within pore wall of CS matrix. Furthermore, these results confirm the excellent dispersion of nHA inside the polymer matrix has a remarkable influence on the improvement of the mechanical properties of the porous composite scaffolds. Also, the enlarged density and reduced porosity of the composite scaffold by totaling nHA contribute to the enhancement in the mechanical properties.

Conclusions
In this study, a novel Dex-CS/nHA composite scaffolds with different HA contents (0, 20, 30 and 40 Wt%) were synthesized by freeze-drying method. The crystallite size of composite scaffold with HA content of 40 Wt% is ~23 nm, which is close to the crystallite size of bioapatite in bone tissue. The SEM results revealed that the scaffolds had an extremely interconnected porous structure which directly influences the biological performance of materials. As the HA content was regularly increased, the surface of the scaffold became rougher. The prepared nanocomposite scaffolds have sufficient porosity which helps in the passage of nutrients and oxygen to the internal regions of the composite scaffolds. The compressive strength of the composite scaffolds increases by increasing HA content. It can be established that the fabricated scaffolds had desirable physico-chemical properties to meet the essential requirement for bone tissue engineer materials.