Two-step modification process to improve mechanical properties and bioactivity of hydroxyfluorapatite scaffolds

Author’s Accepted Manuscript Two-Step Modification Process to Improve Mechanical Properties and Bioactivity of Hydroxyfluorapatite Scaffolds Sorour Sadeghzade, Rahmatollah Emadi, Batol Soleimani, Fariborz Tavangarian www.elsevier.com/locate/ceri

PII: DOI: Reference:

S0272-8842(18)31973-4 https://doi.org/10.1016/j.ceramint.2018.07.231 CERI18953

To appear in: Ceramics International Received date: 3 July 2018 Revised date: 25 July 2018 Accepted date: 25 July 2018 Cite this article as: Sorour Sadeghzade, Rahmatollah Emadi, Batol Soleimani and Fariborz Tavangarian, Two-Step Modification Process to Improve Mechanical Properties and Bioactivity of Hydroxyfluorapatite Scaffolds, Ceramics International, https://doi.org/10.1016/j.ceramint.2018.07.231 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Two-Step Modification Process to Improve Mechanical Properties and Bioactivity of Hydroxyfluorapatite Scaffolds

Sorour Sadeghzadea, Rahmatollah Emadia, Batol Soleimania, Fariborz Tavangarianb* a

Biomaterials research Group, Department of Materials engineering, Isfahan University of Technology, Isfahan

84156-83111, Iran b

Mechanical Engineering Program, School of Science, Engineering and Technology, Pennsylvania State

University, Harrisburg, Middletown, PA 17057, USA *

Corresponding author. Tel.: +(717) 948 6125; fax: +(717) 948 6401. [email protected]

Abstract Porous ceramic scaffolds are synthetic implants, which support cell migration and establish sufficient extracellular matrix (ECM) and cell-cell interactions to heal bone defects. Hydroxyapatite (HA) scaffolds is one of the most suitable synthetic scaffolds for hard tissue replacement due to their bioactivity, biocompatibility and biomimetic features. However, the major disadvantages of HA is poor mechanical properties as well as low degradability rate and apatite formation ability. In this study, we developed a new method to improve the bioactivity, biodegradability and mechanical properties of natural hydroxyfluorapatite (HFA) by applying twostep coating process including ceramic and polymer coats. The structure, morphology and bioactivity potential of the modified and unmodified nanocomposite scaffolds were evaluated using transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy 1

(FTIR) and energy dispersive spectroscopy (EDS). The scaffold with optimized mechanical properties was HFA30 wt.%HT (HT stands for hardystonite) with a total porosity and pore size of 89 ± 1 and 900-1000 µm, respectively. The compressive modulus and strength of HFA (porosity ~ 93 ± 1) were improved from 108.81 ± 11.12 to 251.45 ± 12.2 MPa and 0.46 ± 0.1 to 1.7 ± 0.3 MPa in HFA-30 wt.%HT sample, respectively. After applying poly(ɛ-caprolactone fumarate (PCLF) polymer coating, the compressive strength and modules increased to 2.8 ± 0.15 and 426.1 ± 15.14 MPa, respectively. The apatite formation ability of scaffolds was investigated using simulated body fluid (SBF). The results showed that applying the hardystonite coating improve the apatite formation ability; however, the release of ions increased the pH. Whereas, modified scaffolds with PCLF could control the release of ions and improve the apatite formation ability as well.

Keywords A. Sol-Gel; B. Composite; C. Mechanical properties; E. Biomedical applications

1. Introduction Tissue engineering is one of the useful methods for the reconstruction of massive bones [1, 2]. Nowadays, biological replacement such as biofunctional prostheses, injectable substrates and hydrogels are being used for bone reconstruction purposes [3-5]. One of the biological replacements in bone injuries is ceramic scaffolds [6, 7]. Scaffolds with a 3D porous structure play an effective role in bone regeneration through providing a suitable environment for cell migration, adhesion, vascularization, cell differentiation and diffusion nutrients. An ideal bone scaffold must have close mechanical properties similar to bone to prevent stress shielding [6-8]. Also pore 2

size, and interconnectivity of the scaffolds should mimic the bone structure for vascularization and bone ingrowth [6-9]. It is worth to mention that, pore size has an inverse relationship with mechanical properties, as increasing the pores size, decrease the mechanical properties of scaffolds [6, 7]. Among bioceramics, hydroxyapatite is the first ceramic that used as a bioceramic in body due to the close chemical component to that of bone [10-12]. This ceramic has been frequently used in clinical application because of its high biocompatibility, bioactivity, osteoconductivity and osteoinductivity [13, 14]. But its low biodegradability and mechanical properties limited its application as a bone replacement scaffold [10, 13, 14]. Various methods can be used to improve the mechanical properties and degradability of this bioceramic scaffold such as applying the ceramic and polymer coating [15-18]. During the last decades, silica-based ceramics are recognized as ceramics with excellent biological and mechanical properties close to natural bone. Hardystonite (Ca2ZnSi2O6) is a member of zinc-silicate system with a tetragonal structure [19-21]. High bioactivity, biocompatibility and mechanical properties of hardystonite compared to natural HA increased its clinical applications as a reinforcement and coating on the surface of scaffolds [20, 21]. An alternative approach to improve the mechanical properties of scaffolds is applying a polymer coating on the surface of the scaffolds. The polymer can make a bridge between the crack walls and fill them and thus improve the toughness of bio-ceramic scaffolds [22-24]. Various studies have reported the use of polymers with HA for using in hard and soft tissue engineering application [25-27]. Polycaprolactone (PCL) is hydrophobic and biodegradable polyester that can be used for hard tissue engineering scaffolds. It is also a semi-crystalline polymer that is approved by the Food and Drug Administration (FDA) agency of the USA [28, 29]. PCLF is a cross linkable derivative of PCL that had been shown to be suitable for tissue engineering such as bone replacement. The mechanical properties of this polymer 3

depend on the design of PCLF polymer (its tensile modulus is in the range of 0.87-138 MPa) [29-32]. These values only depend on the molecular weight of PCL precursor chosen [31, 32]. The broad range of mechanical properties allows PCLF to be used in many applications such as bone regeneration [30-32]. Various studies have been done to improve the mechanical and physical properties of HA [33, 34]. Emadi et al. [33] improved the compressive strength of porous hydroxyapatite scaffolds by applying a forsterite coating. The HA scaffold with a forsterite coating was successfully synthesized with 83% porosity and mean pore size of about 740 µm. The compressive strength of natural HA scaffold in that study was increased from 0.12 to 1.61 MPa. The aim of this study was to modify the mechanical properties of HFA scaffolds with applying the two-step coating processes. For this purpose, natural HFA was coated with different percentage of hardystonite. The optimum percentage of HT to improve the mechanical and physical properties of HFA was determined. Then the optimum ceramic composite scaffold was coated with PCLF as the secondary modifying process. The mechanical and physical properties of new scaffolds were evaluated. Also, the apatite formation ability of produced scaffolds was investigated as well.

2. Materials and methods 2.1. Preparation of nanostructured hardystonite powder In this research, combustion synthesis method was used to produce hardystonite (Ca 2ZnSi2O6) powder [21]. Raw materials were calcium nitrate tetrahydrate (Ca(NO3)2. 4H2O, 99% purity, Merck), zinc nitrate hexahydrate (Zn(NO3)2.6H2O, 99% purity, Merck) and tetraethyl orthosilicate (TEOS, (C2H5O)4Si, 99% purity, Merck), 4

(Zn:Ca:Si ratio was 1:2:2). First TEOS and deionized water were mixed together for 30 min with a molar ratio of 1:8. Then zinc nitrate and calcium nitrate powders were added to the aqueous solution with the molar ratio of 1:2. Sucrose (C12H22O11, 99% purity, Aldrich) with molar ratio to metal of 2:1 and polyvinyl alcohol polymer (PVA, molecular weight 66,000) with molar ratio to metal of 0.4:1 were gently added to alkoxide solution and stirred for 1 h. At the end, pH was adjusted to 1 utilizing nitric acid (1 M). The solution was dried in an oven at 120 °C for 48 h. Finally, the dry gel was sintered at 1050 °C for 3 h.

2.2. Preparation of natural HFA scaffold and coating procedure The spongy part of bovine bone was cut to rectangular specimens with 6 mm × 6 mm × 12 mm in size. The sintering temperature was set to 800 °C for 3 h [35]. Then the scaffolds were coated with 0, 10, 20, 30 and 50 wt.% nanostructured hardystonite slurry. The schematic of the scaffold preparation process with two-step coating is shown in Fig. 1. Briefly, polyvinyl alcohol polymer (PVA) (10 wt.%) was dissolved in 50 cc water at 70 °C. Then the various content of hardystonite was added to the solution and stirred for 3 h. The prepared scaffold was immersed in the slurry for 20 min. The coated scaffold was dried in an oven for 12 h at 40 °C. Then the scaffolds were sintered at 1000 °C for 3 h. To infiltrate the scaffolds with PCLF, first PCLF (6 wt.%) was dissolved in dichloromethane (DCM). Benzoyl peroxide (BPO, 2 wt.%) and N-vinyl pyrrolidone (NVP, 2wt%) were added to the polymer solution which cause crosslinking the polymer. In order to coat the HFA-HT scaffolds, the samples were soaked in polymer solution twice (30 min each time). During the heating process, ceramic scaffolds with polymer coating were first heated at 70 °C for 30 min and then at 90 °C for 30 min in an oven.

5

2.3. Sample characterization 2.3.1. XRD analysis Phase transformation of the produced powder and scaffolds was characterized by X-ray diffraction (XRD, Philips Xpert) with Cu Kα radiation (λ = 0.154 nm , 20 kV and 30 mA) in the 2θ range of 20-70 ° (step size 0.05 ° and time per step 1 s).

2.3.2. SEM analysis Scanning electron microscopy (Philips XL30, acceleration voltage of 30V) and energy- dispersive spectroscopy (EDS) were used to evaluate particles morphology as well as micro/macropores. ImageJ software was utilized to assess the size of powder particles and pores.

2.3.3. TEM analysis The powder morphology was investigated by transmission electron microscopy (TEM, Philips, EM208S). The applied voltage was 100 kV. In addition, the particle size of prepared hardystonite nanopowder was measured by ImageJ software.

6

2.3.4. Mechanical Testing To evaluate the mechanical properties of scaffolds the compressive strength and modulus were measured by Hounsfield (H25KS) with the speed of crosshead set to 0.5 mm/min. The sample were prepared with dimension of 6mm × 6mm ×12mm in size.

2.3.5. Porosity Measurement The total porosity of scaffolds was assessed using Archimedes principle .The total porosity of the scaffolds including both interconnected and closed pores can be measured according to the following equation [21]:

(1)

Where

is the true density of the scaffolds,

is the dry weight,

is the wet weight, and

is the wet weight

suspended in water.

2.3.6. In vitro characterization In order to evaluate the bioactivity of scaffolds, the modified and unmodified samples were soaked in a simulated body fluid (SBF) [36] with pH=7.38 for 1, 7, 14, and 21 days. All samples were kept in Ben Marry bath at the 7

temperature of 37 °C. After soaking, the scaffolds were dried at 70 °C for 5 h. Scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS) and Fourier transform infrared spectroscopy (FTIR, JASCO 680 PLUS, in the range of 400-4000 cm-1) were utilized to evaluate the formation of bone-like apatite. pH changes of SBF was examined by a pH meter every 2 days.

2.4. Statistical Analysis In order to evaluate the pH data statistically significant differences, the Graph Pad Prism was used with Two-way ANOVA, P-value < 0.05 (Table S1 in the supplementary section). In addition, all data were examined as average values ± standard deviation for n = 3.

3. Results and Discussion The TEM micrograph of prepared nanostructured hardystonite powder is shown in Fig. 2a. In addition, the average crystallite size of nanostructured hardystonite is presented in Fig. 2b. The crystallite size of pure nanostructured hardystonite with spherical morphology was about 22.22 ± 1 nm. Based on the results of TEM micrograph the hardystonite powder has a high degree of agglomeration. Fig. 2c shows the XRD pattern of sintered powder after 3 h annealing at 1050 ºC. The XRD results showed only the characteristic peaks of hardystonite (JCPDS data file 01-075-0916) phase. In our previous study, the effect of mechanical activation time and subsequent annealing temperature on the formation of hardystonite were studied [37]. The results showed that nanostructure hardystonite powder can be 8

synthesized after 20 h of milling with subsequent annealing at 900 °C for 3 h. The measured crystallite size in that study was 28 ± 2 nm. Comparison of the results showed that producing hardystonite using combustion synthesis leads to the finer powder particles with higher purity and more uniform morphology [21]. In another study conducted by Wu et al. [38], hardystonite were prepared by sol-gel process. The calcined temperature was 1100 and 1200 ºC. The produced hardystonite was not pure and had willemite as a secondary phase for those samples annealed at 1100 ºC. Although willemite was vanished after increasing the annealing temperature to 1200 °C, an increase in crystallite size was observed which can reduce the mechanical and biological properties of hardystonite. The size of particles was in the range of 5-40 µm, while the size of particles in our study was measured to be 22.22 ± 1 nm. These results showed that combustion synthesis method is more effective than sol-gel technique which is a common procedure to synthesize bioceramic powders. Fig. 3 shows the XRD patterns of all coated scaffolds with different percentage of HT after sintering the scaffolds at 1000 °C for 3 h. The first pattern is related to the HFA which is in a good agreement with the standard card of hydroxyfluorapatite (XRD JCPDS data file No. 9-432). As can be seen in the XRD patterns of the coated scaffolds, some peaks related to hardystonite were detected besides of hydroxyfluorapatite peaks. With increasing the percentage of hardystonite powder, the intensity of its main peaks increased which confirmed the presence of hardystonite on the surface of the scaffolds. Also, no additional phase was observed during the sintering process. In a previous study, Gheisari et al. [17] developed hydroxyapatite - hardystonite (HT) bulk nanocomposite ceramics with 5, 10 and 15 wt.% HT. In that study, they could improve the mechanical properties, density, bioactivity and degradability of HA by adding 10 wt.% HT as a result of the formation of glassy bonds between the particles and matrix.

9

The mechanical and physical properties of HFA with various percentages of HT as well as the composite scaffold with polymer coating are shown in Table. 1. The amount of porosity was measured using Archimedes principle method [20]. The porosity of HFA scaffold was 93 ± 1%. After coating the HFA scaffolds with HT, the porosity of scaffold decreased to 86 ± 1.1 % for HFA-50wt.%. In addition, compressive strength and modulus of HFA was measured to be 0.46 ± 0.1 and 108 ± 11.12 MPa, respectively. Based on the mechanical properties results, the highest compressive strength and modulus of composite scaffolds were achieved by addition of 30 wt.% hardystonite. As can be seen the compressive strength and modulus of HFA-30wt.%HT was 1.7 ± 0.3 and 251.45 ± 12.2 MPa, respectively. In order to prevent stress shielding phenomena [39], using biomaterials with similar mechanical properties to human bone is suggested and our produced composite has mechanical properties close to natural spongy bone with compressive strength and modulus in the range of 0.2-4 and 120-1100 MPa, respectively [6]. In other study [20], HT- 15wt.% diopside nanocomposite was produced by the space holder method and the compressive strength was reported to be 1.655 MPa. The compressive modulus and crystallite size was about 45.45 MPa and 41 nm, respectively, which is much lower than the scaffolds fabricated in this study. With increasing the amount of hardystonite to 50 wt.%, a 3% reduction in porosity as well as 41% reduction in compressive strength (due to the weak bonding of hardystonite particles to HFA surface) was observed. Also, with increasing the fraction of hardystonite, the amount of glassy phase increased between hardystonite coating and surface of HFA which reduced the mechanical properties [17]. According to the mechanical and physical properties, the coated scaffold with 30wt.% HT was chosen as the best scaffold in this study to be coated with 6wt.% PCLF polymer solution. As mentioned before the major disadvantages of porous ceramic scaffolds is their inherent brittleness which limited their application in hard tissue engineering. Applying a polymer coating can

10

increase the toughness and mechanical properties of ceramic scaffolds. In fact, polymer coatings can fill and bridge the cracks which improve the fracture toughness (similar to the function of collagen in natural bone) [2224]. As can be seen, compressive strength, compressive modulus and porosity of HFA-30wt.%HT-6wt.% PCLF scaffold were 2.8 ± 0.15 MPa, 426.1 ± 15.14 MPa, and 82 ± 0.8%, respectively. The unsaturated PCLFs macromere cannot enhance the mechanical properties of HA by itself due to its weak nature, which just acts as a paste [24]. To reduce the brittleness of HFA and enhance the toughness of coating, a small amount of Nvinylpyrrolidone (NVP) monomer was added. Although PCLF is a self-cross-linked polymer, adding NVP monomer enhance crosslinking efficiency [24, 30, 40, 41]. NVP polymer is a small molecule so it possesses more mobility than PCLF chains and bridged two adjacent unsaturated fumarate groups so improve the adhesion between coating layer and substrate[24, 30, 40, 41]. On the other hand, the composite strength of scaffolds depends on macro porosity, its distribution and pore size. When the scaffold was coated with polymer, the macro porous areas were infiltrated by polymer and any possible defect of the scaffolds was reduced or eliminated, resulted in a polymer ceramic composite with increased strength compared to ceramics composite [6, 7]. The morphology of scaffolds before and after the modifying process with polymer coating was evaluated by SEM and is shown in Fig. 4. As can be seen in Fig. 4a and b the pore size of HFA-30wt.%HT scaffold was about 900-1000 µm. After coating the aforementioned scaffold with PCLF polymer (Fig. 4c and d, HFA-30wt.%HT6wt.%PCLF sample), the pore size decreased to 500 - 800 µm. As seen on the SEM micrographs, a smooth surface without any crack was observed in HFA-30wt.%HT-6wt.%PCLF scaffold. Whereas, the SEM micrograph of scaffold before polymer coating, confirmed the presence of cracks on the wall of macropores (Fig. 4a-b). These results are in a good agreement with the results obtained from mechanical properties tests. Roohani et al. [16] developed a route to enhance compressive strength of HA. They coated HA with bioactive glass.

11

Compressive strength of highly porous HA improved from 0.22 to 1.49 MPa. While, in our study applying the two steps modifying procedure cause to enhance compressive strength and modulus of HFA from 0.46 ± 0.1 and 108.81 ± 11.12 MPa to 2.8 and 426.1 ± 15.14 MPa, respectively. The EDS spectrum of HFA-30wt.%HT was shown on Fig. 4e which confirms the presence of hardystonite coating on the surface of HFA ceramic. To confirm the presence of PCLF polymer, FTIR test was carried out (Fig. 5). The presence of ester carbonyl functional group (C=O) at 1725 cm-1 confirmed the construction of polyester material. The stretching bands of functional fumarate group (C=C), is detected at around 1645 cm-1 [22, 32]. In fact, the main peak which proved the formation of PCLF appeared in this range, indicating the entry of the fumarate unit into the base polymeric chain. Stretching characteristic peak of 1161 cm-1 and 1104 cm-1 are related to the asymmetric stretching motion of C-O-C and C-C (= O)-O bands [22, 32]. Specific peaks for adsorption, asymmetric and symmetric bending of methylene (C-H) bands was observed at 2945, 1460, 1419, and 1367 cm-1, respectively [22, 32]. The peaks of hydroxyl group were detected at around 3400 and 3500 cm-1 [22]. The presence of these peaks confirms the conversion of PCL-diol to PCLF. Kim et al. [9] coated hydroxyapatite porous scaffold with HA and PCL composite. The HA scaffold obtained by a polymeric reticule method. This method could only enhance compressive strength of pure HA from 0.16 to 0.45MPa and elastic modulus from 0.79 to 1.43 MPa with 87% porosity. However, the pore size was about 150-200 µm which cannot provide an appropriate environment encourage the cell migration, vascularization and proliferation [6]. In order to evaluate the bioactivity of scaffolds before and after modification, the samples were immersed in SBF for up to 21 days. Fig. 6 shows SEM micrographs of various samples. Some fine spherical white particles was observed on the surface of HFA (Fig. 6a-c), HFA-30wt%HT (Fig. 6d-f) and HFA-30wt%HT-6wt%PCLF (Fig. 12

6g-i). The EDS results of the particles on the surface of scaffolds confirmed the formation of apatite after soaking the scaffolds in SBF for 21 days (Fig. 6j-K). Applying the hardystonite coating on the surface of HFA led to an increase in the formation and deposition of apatite. Based on the previous studies [42-44], the major disadvantages of hydroxyapatite ceramic in comparison to bioactive glasses and other bioceramics is its low reactivity with bone. The bioactivity index (IB) of HA is 3.1 while the bioactivity of 45S5 and 55S4 bio-glasses is 12.5 and 33.7, respectively [41-43]. Therefore, improving the bioactivity of HA can increase its demand in the biomedical applications. Previous studies [42, 44] reported that substitution of Si and carbonate ions increased rate of bone apposition around the Si-HA and carbonate substitute in hydroxyapatite (CHA) compared to pure HA as a result of higher solubility. In this study, we showed another way to improve the bioactivity of HFA. In fact, by applying the hardystonite ceramic coating on the surface of HFA, higher exchange rate of ions with surrounding environment improved the apatite formation ability of HFA-30wt.%HT scaffolds. The size of apatite particles was measured to be in the range of 1-3 µm. applying the polymer coating on the surface of HFA30wt.%HT, on the other hand decreased apatite formation ability (Fig. 6g-i). The FTIR spectra of modified and unmodified samples after immersing for 7 days in SBF are shown in Fig. 7(ac). Also, the changes in pH of solutions as a function of time is illustrated in Fig. 7(d). The results of statistical analysis of this experiment is shown in the supplementary materials section (Table S1). All the comparisons were performed between the samples which were significantly different. Solubility of scaffolds was the main reason of pH changes. As seen, the pH curve of HFA-30wt.%HT showed a noticeable increase to 7.91 after soaking in SBF for 5 days (Fig. 7d). After immersing the scaffolds in the SBF solution, Zn, Ca and Si ions of the scaffolds were exchanged with H+ or H3O+ to form silanol groups (Si-OH) on the surface. With the migration of Ca2+ and PO43- to the surface of the scaffolds, a calcium phosphate rich layer 13

was formed on the surface of the scaffolds which lead to a reduction in pH to 7.74 (after 9 days). Subsequently, crystallization of the Ca-phosphate amorphous layer was occurred with incorporation of OH- or CO32- from SBF solution [21, 45]. This hydroxyl carbonate apatite layer can join implants to bone tissue. As seen in Fig. 7d, the solubility of HFA samples is really low, so that up to 9 days soaking in SBF the pH increased to 7.63 and then decreased (after 13 days). Therefore, it is observed that there is delay to form of apatite on the surface of HFA. Whereas, after applying the hardystonite coating the required time for apatite formation reduced to 5 days. Applying the polymer coating controlled the biodegradability of HFA-30wt.%HT scaffolds. It is worth to mention that the extreme increase of the ions concentration around the tissue as a result of the release of ions from implants can lead to tissue necrosis (the pH around the surrounding tissue affects the cell growth and proliferation) [37, 39, 45]. Therefore, controlling the release of ions and concentration is a key element on designing the bio-ceramic scaffolds. It was found that the apatite formation was delayed in HFA-30wt.%HT6wt.%PCLF. This phenomenon was observed in FTIR spectra as well. The characteristic peaks of PO 43- group was observed in FTIR spectrum of HFA at 1090, 1054, 962, 602 and 571 cm-1 (Fig. 7a). The vibrational and stretching OH- bands of HFA scaffold is observed at 635 cm-1 [15, 16, 34]. After soakings the HFA scaffold in SBF solution for 7 days, the new adsorption peaks of phosphate group was appeared at 874 cm-1 [34]. Also, the intensity of phosphate group (in the range of 1100 - 960 and 500-600 cm-1) increased by increasing the soaking time in SBF. This phenomenon can be ascribed to the nucleation of apatite on the surface of the scaffolds after 79 days which is in a good agreement with pH changes curve. The FTIR spectrum of HFA-30wt.%HT before and after soaking was shown in Fig. 7b. The bending and stretching vibration bands of Si-O-Si was detected in the range of 1100-900 and 500-600 cm-1 which had an overlap with phosphate group peaks in HFA [46]. However, the presence of peaks related to Ca-O and Zn-O at 568 and 512 cm-1 showed the existence of hardystonite on the 14

surface of scaffolds which was confirm previously by the XRD results [19, 20, 47]. After soaking HFA30wt.%HT for 7 days, the new adsorption peaks related to P-O bands was also observed at 1030, 970, 917, 679 and 604 cm-1 [15-16, 19]. Detecting the characteristic peaks of carbonate groups at 1406 and 837 cm-1 in hydroxyl apatite structure, proved the formation of crystalline apatite on the surface of the scaffold with 30% hardystonite, just after 7 days soaking in SBF [47]. Also, the scaffold with PCLF coating did not show the nucleation of apatite even after 7 days soaking in SBF. Considering the pH changes, it can be expected that the nucleation of apatite started after 15 days. The SEM micrographs of scaffold with PCLF coating proved the presence of apatite particles after 21 days soaking.

4. Conclusion In this study, we successfully synthesized the modified and unmodified HFA-HT-PCLF nanocomposite scaffolds by two-step coating process and characterized the samples to evaluate their potential for biomedical applications. An improvement in mechanical properties of HFA scaffolds was observed with applying the two-step coating process. Based on the results, the compressive strength, modulus, porosity and pore size of HFA-30wt.%HT and HFA-30wt.%HT-6wt.%PCLF were measured to be 1.7 ± 0.3 MPa, 251.45 ± 12.2 MPa, 89 % ± 1, 900-1000 µm and 2.8 ± 0.15 MPa, 426.1 ± 15.14 MPa, 82 % ± 0.8, 500-800 µm, respectively, compared to HFA scaffold with compressive strength and modulus of 0.46±0.1 and 108.81 ± 11.12 (porosity ~ 93± 1), respectively. The biological ability, mechanical and physical properties of these modified scaffolds showed a great potential to be used as biological substitutes in hard tissue engineering applications.

15

References ]1[A. Yazdanpanah, R. Kamalian, F. Moztarzadeh, M. Mozafari, R. Ravarian, L. Tayebi, Enhancement of fracture toughness in bioactive glassbased nanocomposites with nanocrystalline forsterite as advanced biomaterials for bone tissue engineering applications, Ceramics International, 38 (2012) 5007-5014. ]2[S.-I. Roohani-Esfahani, Y. Chen, J. Shi, H. Zreiqat, Fabrication and characterization of a new, strong and bioactive ceramic scaffold for bone regeneration, Materials Letters, 107 (2013) 378-381. ]3[M .Razavi, M. Fathi, O. Savabi, D. Vashaee, L. Tayebi, In vitro study of nanostructured diopside coating on Mg alloy orthopedic implants, Materials Science and Engineering: C, 41 (2014) 168-177. ]4[W. Zhang, G. Wang, Y. Liu, X. Zhao, D. Zou, C. Zhu, Y. Jin ,Q. Huang, J. Sun, X. Liu, The synergistic effect of hierarchical micro/nanotopography and bioactive ions for enhanced osseointegration, Biomaterials, 34 (2013) 3184-3195. ]5[H. Zreiqat, Y. Ramaswamy, C. Wu, A. Paschalidis, Z. Lu, B. James, O. Birke, M .McDonald, D. Little, C.R. Dunstan, The incorporation of strontium and zinc into a calcium–silicon ceramic for bone tissue engineering, Biomaterials, 31 (2010) 3175-3184. ]6[L.-C. Gerhardt, A.R. Boccaccini, Bioactive glass and glass-ceramic scaffolds for bone tissue engineering, Materials, 3 (2010) 3867-3910. ]7[S. Sadeghzade, F. Shamoradi, R. Emadi, F. Tavangarian, Fabrication and characterization of baghdadite nanostructured scaffolds by space holder method, Journal of the mechanical behavior of biomedical materials, 68 (2017) 1-7. ]8[G. Wang, Z. Lu, D. Dwarte, H. Zreiqat, Porous scaffolds with tailored reactivity modulate in-vitro osteoblast responses, Materials Science and Engineering: C, 32 (2012) 1818-1826. ]9[H.-W. Kim, J.C. Knowles, H.-E. Kim ,Hydroxyapatite/poly (ε-caprolactone) composite coatings on hydroxyapatite porous bone scaffold for drug delivery, Biomaterials, 25 (2004) 1279-1287. ]11[H.-H. Jin, S.-H. Min, W.-K. Lee, H.-C. Park, S.-Y. Yoon, The Effect of MgO Content on the Preparation of Porous Hydroxyapaite Scaffolds by Polymer Sponge Method, Korean Journal of Materials Research, 16 (2006) 715-718. ]11[S.K. Padmanabhan, F. Gervaso, M. Carrozzo, F. Scalera, A. Sannino, A. Licciulli, Wollastonite/hydroxyapatite scaffolds with improved mechanical, bioactive and biodegradable properties for bone tissue engineering, Ceramics International, 39 (2013) 619-627. ]12[M. Zhang, C. Liu, J. Sun, X. Zhang, Hydroxyapatite/diopside ceramic composites and their behaviour in simulated body fluid ,Ceramics International, 37 (2011) 2025-2029. ]13[M. Eilbagi, R. Emadi, K. Raeissi, M. Kharaziha, A. Valiani, Mechanical and cytotoxicity evaluation of nanostructured hydroxyapatitebredigite scaffolds for bone regeneration, Materials Science and Engineering: C, 68 (2016) 603-612. ]14[H.-W. Kim, J.C. Knowles, H.-E. Kim, Hydroxyapatite porous scaffold engineered with biological polymer hybrid coating for antibiotic Vancomycin release, Journal of materials science: materials in medicine, 16 (2005) 189-195. ]15[R. Emadi, S.R. Esfahani, F. Tavangarian, A novel, low temperature method for the preparation of ß-TCP/HAP biphasic nanostructured ceramic scaffold from natural cancellous bone, Materials Letters, 64 (2010) 993-996. 16

]16[S.R. Esfahani, F. Tavangarian ,R. Emadi, Nanostructured bioactive glass coating on porous hydroxyapatite scaffold for strength enhancement, Materials Letters, 62 (2008) 3428-3430. ]17[H. Gheisari, E. Karamian, M. Abdellahi, A novel hydroxyapatite–hardystonite nanocomposite ceramic ,Ceramics International, 41 (2015) 5967-5975. ]18[Z. Xihua, L. Changxia, L. Musen, B. Yunqiang, S. Junlong, Fabrication of hydroxyapatite/diopside/alumina composites by hot-press sintering process, Ceramics International, 35 (2009) 1969-1973. ]19[H. Mohammadi, M. Hafezi, S. Hesaraki, M.M. Sepantafar, Preparation and characterization of Sr-Ti-hardystonite (Sr-Ti-HT) nanocomposite for bone repair application, Nanomedicine journal, 2 (2015) 203-210. ]21[S. Sadeghzade, R. Emadi, S. Labbaf, Hardystonite-diopside nanocomposite scaffolds for bone tissue engineering applications, Materials Chemistry and Physics, 202 (2017) 95-103. ]21[S. Sadeghzade, R. Emadi, F. Tavangarian, Combustion assisted synthesis of hardystonite nanopowder, Ceramics International, 42 )2116( .14661-14656 ]22[S. Joughehdoust, A. Behnamghader, M. Imani, M. Daliri, A.H. Doulabi, E. Jabbari, A novel foam-like silane modified alumina scaffold coated with nano-hydroxyapatite–poly (ε-caprolactone fumarate) composite layer, Ceramics International, 39 (2013) 209-218. ]23[S. Roohani-Esfahani, C. Dunstan, B. Davies, S. Pearce, R. Williams, H. Zreiqat, Repairing a critical-sized bone defect with highly porous modified and unmodified baghdadite scaffolds, Acta biomaterialia, 8 (2012) 4162-4172. ]24[S. Sharifi, Y. Shafieyan, H. Mirzadeh, S. Bagheri‐Khoulenjani, S.M. Rabiee, M. Imani, M. Atai, M.A. Shokrgozar, A. Hatampoor, Hydroxyapatite scaffolds infiltrated with thermally crosslinked polycaprolactone fumarate and polycaprolactone itaconate, Journal of Biomedical Materials Research Part A, 98 (2011) 257-267. ]25[L. Lao, Y. Wang, Y. Zhu, Y. Zhang, C. Gao, Poly (lactide-co-glycolide)/hydroxyapatite nanofibrous scaffolds fabricated by electrospinning for bone tissue engineering, Journal of Materials Science: Materials in Medicine, 22 (2011) 1873-1884. ]26[A. Rainer, C. Spadaccio, P. Sedati, F. De Marco, S. Carotti, M. Lusini, G. Vadalà, A. Di Martino, S. Morini, M. Chello, Electrospun hydroxyapatite-functionalized PLLA scaffold: potential applications in sternal bone healing, Annals of biomedical engineering, 39 (2011) 18821890. ]27[S. Zadegan, M. Hosainalipour, H. Rezaie, H. Ghassai, M. Shokrgozar, Synthesis and biocompatibility evaluation of cellulose/hydroxyapatite nanocomposite scaffold in 1-n-allyl-3-methylimidazolium chloride, Materials Science and Engineering: C, 31 (2011) 954-961. ]28[B.K. Chen, A.M. Knight, N.N. Madigan, L. Gross, M. Dadsetan, J.J. Nesbitt, G.E. Rooney, B.L. Currier, M.J. Yaszemski, R.J. Spinner, Comparison of polymer scaffolds in rat spinal cord: a step toward quantitative assessment of combinatorial approaches to spinal cord repair, Biomaterials, 32 (2011) 8077-8086. ]29[N. Shokri, H.A. Javar, S. Fouladdel, A. Khalaj, R. Dinarv, E. Azizi, Preparation and characterization of crosslinked and non-crosslinked polycaprolactone fumarate (PCLF) NPs as carriers for doxorubicin HCl, African Journal of Pharmacy and Pharmacology, 5 (2011) 797-805. ]31[M.B. Runge, H. Wang, R.J. Spinner, A.J. Windebank, M.J. Yaszemski, Reformulating polycaprolactone fumarate to eliminate toxic diethylene glycol: effects of polymeric branching and autoclave sterilization on material properties, Acta biomaterialia, 8 (2012) 133-143. ]31[M. Todo, S.-D. Park, T. Takayama, K. Arakawa, Fracture micromechanisms of bioabsorbable PLLA/PCL polymer blends, Engineering Fracture Mechanics, 74 (2007) 1872-1883. 17

]32[S. Wang, M.J. Yaszemski, J.A. Gruetzmacher, L. Lu, Photo-crosslinked poly (ɛ-caprolactone fumarate) networks: roles of crystallinity and crosslinking density in determining mechanical properties, Polymer, 49 (2008) 5692-5699. ]33[R. Emadi, F. Tavangarian, S.I.R. Esfahani, A. Sheikhhosseini, M. Kharaziha, Nanostructured forsterite coating strengthens porous hydroxyapatite for bone tissue engineering, Journal of the American Ceramic Society, 93 (2010) 2679-2683. ]34[R. Emadi, F. Tavangarian, S.R. Esfahani, Biodegradable and bioactive properties of a novel bone scaffold coated with nanocrystalline bioactive glass for bone tissue engineering, Materials Letters, 64 (2010) 1528-1531. ]35[F. Tavangarian, R. Emadi, S.I.R. Esfahani, A novel method to synthesis of β-TCP/HA biphasic nanocrystalline powder by using bovine bone, International Journal of Modern Physics B, 24 (2010) 3365-3372. ]36[M. Bohner, J. Lemaitre, Can bioactivity be tested in vitro with SBF solution?, Biomaterials, 30 (2009) 2175-2179. ]37[S. Sadeghzade, R. Emadi, S. Labbaf, Formation mechanism of nano-hardystonite powder prepared by mechanochemical synthesis, Advanced Powder Technology, 27 (2016) 2238-2244. ]38[C. Wu, J. Chang, W. Zhai, A novel hardystonite bioceramic: preparation and characteristics, Ceramics international, 31 (2005) 27-31. ]39[S. Sadeghzade, R. Emadi, F. Tavangarian, M. Naderi, Fabrication and evaluation of silica-based ceramic scaffolds for hard tissue engineering applications, Materials Science and Engineering: C, 71 (2017) 431-438. ]41[E. Jabbari, S. Wang, L. Lu, J.A. Gruetzmacher, S. Ameenuddin, T.E. Hefferan, B.L. Currier, A.J. Windebank, M.J. Yaszemski ,Synthesis, material properties, and biocompatibility of a novel self-cross-linkable poly (caprolactone fumarate) as an injectable tissue engineering scaffold, Biomacromolecules, 6 (2005) 2503-2511. ]41[S. Sharifi, H. Mirzadeh, M. Imani, F. Ziaee, M. Tajabadi, A. Jamshidi, M. Atai, Synthesis, photocrosslinking characteristics, and biocompatibility evaluation of N‐vinyl pyrrolidone/polycaprolactone fumarate biomaterials using a new proton scavenger, Polymers for Advanced Technologies, 19 (2008) 1828-1838. ]42[I.R. Gibson, W. Bonfield, Novel synthesis and characterization of an AB‐type carbonate‐substituted hydroxyapatite, Journal of Biomedical Materials Research Part A, 59 (2002) 697-708. ]43[L.L. Hench, Ö. Andersson, Bioactive glasses, An introduction to bioceramics, World Scientific, 1993, pp. 41-62. ]44[A. Porter, N. Patel, J. Skepper, S. Best, W. Bonfield, Comparison of in vivo dissolution processes in hydroxyapatite and silicon-substituted hydroxyapatite bioceramics, Biomaterials, 24 (2003) 4609.4621]45[M. Diba, O.-M. Goudouri, F. Tapia, A.R. Boccaccini, Magnesium-containing bioactive polycrystalline silicate-based ceramics and glassceramics for biomedical applications, Current opinion in solid state and materials science, 18 (2014) 147-167. ]46[H. Ghomi, R. Emadi, S.H. Javanmard, Fabrication and characterization of nanostructure diopside scaffolds using the space holder method: effect of different space holders and compaction pressures, Materials & Design, 91 (2016) 193-200. ]47[S. Mirhadi ,F. Tavangarian, R. Emadi, Synthesis, characterization and formation mechanism of single-phase nanostructure bredigite powder, Materials Science and Engineering: C, 32 (2012) 133-139.

18

Figure Caption Fig. 1. Fabrication procedure of composite scaffolds.

19

Fig. 2. a) TEM images, b) frequency distribution as a function of crystallite size and c) XRD patterns of hardystonite powder after sintering at 1050°C for 3 h.

20

Fig. 3. XRD patterns of nanocomposite scaffolds in different percentage of hardystonite after sintering at 1000 °C for 3 h.

21

Fig. 4. SEM micrographs of , (a-b) HFA-30wt.%HT, (c-d) HFA-30wt.%HT-6wt.%PCLF, e) EDS spectrum of HFA-30wt.%HT.

Fig. 5. FTIR spectrum of PCLF polymers, HFA scaffolds and modified scaffolds with polymer and hardystonite.

22

Fig. 6. The SEM micrographs of, a-c) HFA, d-f) HFA-30wt.%HT and g-i)HFA-30wt.%HT-6wt.%PCLF, j) EDS spectra of HFA-30wt.%HT and k) HFA , scaffolds after 21 days soaking in SBF solution

23

Fig. 7. FTIR spectra of, a) HFA, b) HFA-30wt.%HT and c) HFA-30wt%HT-6wt%PCLF scaffolds before and after 7 days soaking in SBF, d) pH changing curve of different samples as a function of time.

24

Tables Table. 1. Mechanical and physical properties of composite scaffolds. Samples

Porosity (%)

Compressive strength

Compressive modulus

(MPa)

(MPa)

HFA

0.46 ± 0.1

108.81 ± 11.12

93 ± 1

HFA-

0.88 ± 0.12

141.21 ± 8

93 ± 1

1.14 ± 0.1

191.22 ± 8.5

92 ± 0.5

1.7 ± 0.3

251.45 ± 12.2

89 ± 1

1.01 ± 0.1

152.2 ± 5.65

86 ± 1.1

2.8 ± 0.15

426.1 ± 15.14

82 ± 0.8

10wt.%HT HFA20wt.%HT HFA30wt.%HT HFA50wt.%HT HFA30wt.%HT6wt.%PCLF

25