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Electrospun Polyhydroxybutyrate /Poly (#-caprolactone) /58S Sol-gel Bioactive Glass Hybrid Scaffolds with Highly Improved Osteogenic Potential for Bone Tissue Engineering Yaping Ding, Wei Li, Teresa Müller, Dirk W. Schubert, Aldo R. Boccaccini, Qingqing Yao, and Judith Anna Roether ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03997 • Publication Date (Web): 13 Jun 2016 Downloaded from http://pubs.acs.org on June 19, 2016

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ACS Applied Materials & Interfaces

1. Introduction Organic/inorganic composites and hybrids are attracting increasing attention developing into a cutting-edge research field with various applications, especially in bone tissue engineering. In this context diverse properties can be integrated and balanced, such as mechanical strength, hydrophilicity, bioactivity, and osteoconductivity.1 Electrospinning is a cost-effective technique to obtain submicron or nano-fibrous scaffolds with high porosity, high surface area, which can mimic the extracellular matrix structure and act as attractive temporary templates during tissue formation in bone tissue engineering.2 Inorganic components can be incorporated into the polymer matrix through electrospinning to prepare functional nano- or micro fibers with superior mechanical and biological properties.3 Polyhydroxybutyrate (PHB) and poly (ε-caprolactone) (PCL) are both biocompatible and biodegradable polymers, which attract great interest for tissue engineering applications.4 However, the intrinsic brittleness of PHB as well as its relatively high cost limits its application. Great efforts are being devoted to developing modifications of PHB. For instance, PCL possesses relatively low stiffness and high flexibility but is less expensive and can be blended with PHB thus integrating the high strength and modulus of PHB with the flexibility of PCL.5, 6 In the last 10 years, inorganic nanoparticles such as hydroxyapatite, silica, calcium carbonate, tri-calcium phosphate, bioactive glass, Ag, Au, and multi-walled carbon nanotubes have been mixed into PHB or PCL matrices and were then processed using electrospinning to impart bioactivity, long term antibacterial functions and biological stimulation.7-11 The conventional way to incorporate particles in a polymer matrix involves distributing them in the matrix by simple mechanical mixing, or with the assistance of ultrasonication. However, these methods normally lead to particle agglomeration due to the high surface energy of nanoparticles. It is well known that the sol-gel technique enables the formation of glasses or ceramics from precursor solutions mixed at a molecular level at low temperatures, and multicomponent materials can be created based on wet chemistry;12, 13 therefore, the sol-gel method is considered to be a versatile approach to homogeneously distribute inorganic substances in polymer matrices.14 Through the combination of electrospinning and sol-gel method, organicinorganic fibrous networks may be blended on a molecular level with a strong or weak interaction force, leading to hybrid structures.15, 16 Sol-gel derived silica is one of the oldest and most popular inorganic materials prepared by the solgel technique,12 and studies have shown that sol-gel derived silica is able to promote osteoblastic differentiation in the context of bone regeneration approaches.17, 18 In our previous research, up to

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50 wt% of sol-gel derived silica was successfully incorporated into the PHB/PCL blend system through a combined electrospinning and sol-gel method.19 To further enhance the bioactivity of the hybrid scaffolds, calcium and phosphorus were considered as necessary ingredients since they are the main elements in the bone mineral phase.20 In the literature, polymer/binary or ternary bioactive glass hybrid systems like PVA/70SiO2-30CaO, gelatin/70SiO2-25CaO-5P2O5 and PCL/70SiO226CaO-4P2O5 have been prepared via the sol-gel technique and processed using different methods including electrospinning.21-25 The in vitro cell cultivation of the above systems has indicated the increased cell viability and osteogenic differentiation in comparison to the plain polymers.24, 25 As one of the well-known ternary glasses, sol-gel derived 58S (60SiO2−36CaO−4P2O5, mol%) bioactive glass has been shown to be able to release ions upon dissolution, which can upregulate osteoblast-specific gene expression.26, 27 To the best of our knowledge, hybrid scaffolds fabricated through electrospinning of PHB/PCL blend and 58S sol-gel derived bioactive glass have not been reported so far. In the present work, PHB/PCL/58S hybrid fibermats were fabricated through electrospinning and sol-gel method, aiming to integrate the high strength of PHB, the suitable flexibility of PCL and the favorable bioactivity of sol-gel derived 58S glass. The cell adhesion, viability, proliferation and ALP activity of MG-63 osteoblast-like cells on contact with the new hybrid scaffolds were investigated. 2. Materials and methods 2.1 Materials Polyhydroxybutyrate (PHB, Mw = 437 kDa) and poly (ε-caprolactone) (PCL, Mw = 48–90 kDa) were used. Tetraethyl orthosilicate (TEOS) and triethyl phosphate (TEP) were used as the precursor for SiO2 and P2O5. CaCl2·2H2O was used as calcium source. Ethanol (EtOH), chloroform (CF) and N, N-Dimethylformamide (DMF) were used as solvents for sol-gel preparation and electrospinning. All chemicals and polymers were purchased from Sigma-Aldrich and were of analytical grade. 2.2 Preparation of electrospun PHB/PCL/sol-gel derived 58S bioactive glass hybrid scaffolds PHB/PCL (7:3 w/w) was dissolved in CF/DMF (8:2 v/v) solvent mixture at a concentration of 5% w/v at 70 ˚C under vigorous magnetic stirring until a clear solution was obtained for further use. TEOS, TEP, EtOH, H2O and CaCl2·2H2O with the molar ratio of 1: 0.133: 2: 2: 0.6 were mixed and stirred to prepare the 58S bioactive glass sol under acid catalyst. The sol was aged at 40 ˚C for 2 3 ACS Paragon Plus Environment

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hours and subsequently kept at room temperature for 24 hours before being added into the polymer solution. Before electrospinning, predetermined amounts of the sol were added into the above mentioned blended polymer solution to obtain hybrid solutions with varying polymer/inorganic component ratios. The flow chart of the preparation process is shown in Fig. 1. The obtained solutions were then transferred into 10 ml syringe for electrospinning, which were carried out using the following the parameters: flow rate at 2 ml/h, applied voltage of 8 kV, and the collecting distance of 15 cm. Based on the designed organic/inorganic weight ratios of 1:0, 20:1, 10:1 and 5:1, the samples were labeled as P1B0, P20B1, P10B1, and P5B1. The electrospinning process was carried out at room temperature with humidity around 30% − 40%. The prepared samples were dried in the vacuum oven at 40 ˚C overnight for 24 hours before further characterization.

Fig. 1. Schematic flowchart of the hybrid scaffolds fabrication. 2.3 Morphologies The morphologies of the obtained fibrous scaffolds were observed using scanning electron microscopy (SEM, LEO 435VP, Zeiss Leica). All samples were sputter-coated with Au before observation. Fiber diameters and distributions were analyzed through SEM images by Image J, measuring more than 100 fibers randomly for each sample. 2.4 Mechanical properties To quantitatively evaluate the physical strength of fibrous structures, standard tensile tests (Frank, Karl Frank GmbH, Germany) were performed on strips with dimensions of 40 mm × 5 mm, which were cut from obtained samples. The width and length were measured by calipers (Mitutoyo, UK) and the thickness was measured by a thickness gauge (TESA DIGICO 1). The stress-strain curves were recorded when samples were stretched at a speed of 10 mm/min with an initial span length of 4 ACS Paragon Plus Environment


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20 mm and the capacity of the load cell was 50 N. The ultimate strength, elongation at break and Young’s modulus were determined from the stress-strain curves. For each composition, at least 5 specimens were tested. 2.5 Wettability Wettability of the samples was measured by contact angle measurements (DSA30, Kruess GmbH, Germany). Three specimens were tested for each composition and five measurements were carried on each specimen. 2.6 Fourier transform infrared spectroscopy Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR, Nicolet 6700, Thermo Scientific) was utilized to investigate the chemical structure and the possible organicinorganic interactions in the samples. For each measurement, 32 spectral scans were repeated in the wavenumber range of 4000 cm-1 – 525 cm-1. 2.7 Thermogravimetric analysis and differential scanning calorimetry To determine the actual weight ratios and thermal stability of scaffolds, thermogravimetric analysis (TGA, Q5000, TA Instruments) was conducted from room temperature to 600 ˚C under nitrogen atmosphere at a heating rate of 10 ˚C /min. The weight loss curves were recorded. Indeed the intended application of the present scaffolds is at human body temperature, these thermal tests are however relevant as they can reveal the intrinsic interactions among different components, and disclose the actual organic/inorganic ratios involved, which are important for confirming the initial design of the materials. Furthermore, to study the component interaction, melting temperature and crystallinity were investigated through differential scanning calorimetry (DSC, Q2000, TA Instruments). In the DSC measurement, 5 mg of each sample went through a heating- cooling-heating cycle from 0 ˚C to 200 ˚C at a rate of 10˚C/min under nitrogen atmosphere. The crystallinity Xc of the polymer was calculated using the following equation from the melting enthalpy of the DSC endothermic peak.28 Xc = ∆Hm / (f ×∆Hmo)

(1)

where ∆Hm is the melting enthalpy of PHB or PCL during the heating cycles and ∆Hmo is the fusion enthalpy of 100% crystalline PHB or PCL, which equals to 146.6 J/g

29

or 136.1 J/g

28

. f is the

weight fraction of PHB or PCL in the hybrid mixture. 5 ACS Paragon Plus Environment

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2.8 X-ray diffraction analysis X-ray diffraction (XRD) analysis (Philips X'Pert PRO, 30 kV/30 mA, Cu Kα) was performed to further characterize the obtained fibrous structures. Scans were carried out from 10° to 50° (2θ) with the speed of 2 °/min. 2.9 In vitro cell cultivation and cell adhesion MG-63 osteoblast-like cells were seeded onto the electrospun scaffolds P1B0 and P5B1 for biological studies. Blank tissue culture plates (TCPs) were used as control samples. After the UV light sterilization, cells were seeded on the samples in 24 well plates at a density of 4 × 104 cells /well and cultivated in culture medium at 37 ˚C in 5% CO2 for 8 h. When the samples were taken out, 4% paraformaldehyde was used to fix the cells for 10 min, and then PBS was used to wash the samples three times. Before the cell adhesion behavior was observed by confocal laser scanning microscopy (CLSM, LSM 710, Zeiss, Germany), the cells were permeabilized with 0.1% Trion X100 for 5 min, rinsed with PBS and blocked with 1% BSA solution for 20 min. After that, cell cytoskeletons were fluorescently stained with phalloidin for 20 min and nuclei were stained using Hoechst 33258 for 5 min. 2.10 Cell viability To investigate the cell viability on the prepared scaffolds, MG-63 cells were seeded onto P1B0 and P5B1 in a 24 well plate at a density of 3 × 103 cells/well, and then analyzed by a cell counting kit-8 (CCK-8, Dojindo, Japan). After 1 day and 3 days of cell culture, the culture medium was removed and samples were incubated in a fresh cell culture medium containing 10 vol% CCK8 solutions at 37 ˚C in 5% CO2 for another 2 hours. Then 100 µl of the reacted medium were measured at 450 nm using a Microreader (Model 680, Bio-Rad Laboratories) to determine the amount of formazan dye, which proportionally indicated the live cells number. The tests were repeated 3 times and 6 parallel repeats were carried out for each sample. 2.11 Cell proliferation morphologies To observe the cell proliferation morphologies, MG-63 cells were cultured on P1B0 and P5B1 at a density of 2 × 104 cells/well for 3 and 7 days. To stabilize the cells, cell-seeded samples were fixed in 2.5 % glutaraldehyde for 30 minutes, rinsed with PBS several times, and then dehydrated through concentration graded ethanol at 30%, 50%, 70%, 80%, 90%, and 100% for 15 min each. The dehydrated samples were sputter-coated with gold before SEM observation.



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2.12 Alkaline phosphate activity The Alkaline phosphate activity (ALP) is regarded as an initial indicator of the osteoblast phenotype and can be the enzyme to converse para-nitrophenyl phosphate (pNPP) to yellow p-nitrophenol (pNP).30 An ALP assay kit (Beyondtime Bio-Tech, China) was utilized to measure the ALP activity. In brief, after 7 days and 14 days of cell cultivation, 50 µl of cell lysate was collected to react with 50 µl pNPP solution at 37 ˚C for 30 min to determine the production of pNP from the light absorbance at 405 nm through a microreader. To analyze the experimental values, pNPP solutions with varying concentrations from 10 µM to 100 µM were prepared to create a standard curve. All results were normalized by protein content. (Unit definition: 1 DEA unit is defined as the amount of alkaline phosphatase needed to hydrolyze para-nitrophenyl phosphate to get 1 µmol p-nitrophenol per min at 37 ˚C). 2.13 Quantitative assessment of alizarin red S staining for mineralization Mineralization of cells is another sign of osteogenic differentiation of relevance to bone tissue engineering. Alizarin red S (ARS) staining was used to detect the presence of calcified calcium on MG-63 cells cultured on P1B0 and P5B1 scaffolds at a density of 5 × 103 cells / well for 4 weeks. After cell culture, both samples were rinsed with PBS and fixed with 95% ethanol for 10 min. Afterwards, they were immersed in ARS staining solution (pH = 4) for 10 min to stain the calcium followed by rinsing in distilled and deionized water to remove excess dye. Finally, mineralization was quantified by extraction and measurement of ARS uptake considering the following steps: 1) to extract the ARS dye from the cells by using a 10% cetylpiridinium chloride solution for 1h; 2) to determine the optical density by using the microplate reader at a wavelength of 570 nm. 2.14 Statistical analysis All data regarding cell viability, ALP activity and ARS staining are presented as mean ± standard deviation. The values were analyzed by the single-factor analysis of variance. P < 0.05 was considered to be statistically significant. 3. Results and discussion 3.1 Morphology To closely mimic the extra cellular matrix (ECM), three dimensional porous structures with interconnected pore channels for cell growth and nutrient diffusion throughout the whole architecture are desired.31 Fig. 2 shows the surface topography of the electrospun hybrid scaffolds 7 ACS Paragon Plus Environment

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P1B0, P20B1, P10B1, and P5B1 with designed inorganic component ranging from 0 to 16.7 wt.%. All samples exhibit highly porous fibrous architecture constructed by uniform and smooth fibers with diameters in the range of 0.7 – 1.2 µm. From the values in Table 1, the average fiber diameter shows an increasing trend as the sol content increased, which may be caused by the decreased volatility of glass sol due to the increasing amount of water. It is worth noting that the addition of the sol greatly changed the solution electro-spinnability. In preliminary exploratory experiments, pure PHB/PCL blend could be electrospun into various fibrous structures for a wide range of parameter combinations.32 However, in the case of fresh P5B1 mixture, for instance, no regular fibrous membrane could be obtained in any parameter combinations because of the ionization effect of salts.33 After the aging process, which was expected to confine calcium inside the 3D sol-gel network, the hybrid fibermats could still only be prepared when the voltage was equal or below 8 kV, otherwise, a non-stable porous cotton-like structure was formed.19 If the sol was aged for a longer time, a phase separation occurred in the solution owing to the immiscibility of high viscosity sol and polymer solution. Moreover, the hybrid solution would be immediately phase-separate if the sol content exceeded 16.7 wt.%, due to the fact that H2O is immiscible with organic solvents.



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Fig. 2. SEM images and fiber diameter distributions of (a) P1B0, (b) P20B1, (c) P10B1, and (d) P5B1 fibrous scaffolds. 3.2 Mechanical properties Scaffolds are also required to mechanically support cell expansion and new tissue regeneration in addition to withstanding manipulation.34 Mechanical properties of the scaffolds, such as stiffness and flexibility, play a crucial role in cell behavior regulation.35, 36 Fig.3 presents the typical stressstrain curves of the prepared samples. As the curves show, all specimens exhibit ductile behavior. Although PHB is characterized by a completely brittle fracture behavior,5 30 wt% PCL addition to PHB through electrospinning can effectively enhance the fiber flexibility. Table 1 summarizes the average values of tensile strength, strain and Young’s modulus of all samples and Fig.3 (b) displays the variation of the properties as a function of 58S glass content. In contrast to the reinforcement using inorganic nanoparticles, the incorporation of varying amount of glass sol actually does not strengthen the matrix within the experimental error, whereas it weakens the PHB/PCL blend slightly. One reason for this could be the fact that the glass sol incorporated in the fibers by electrospinning may still contain H2O molecules and residue of the precursor which cannot be removed by vacuum heating thus generating weak points in the matrix. Moreover, the crystallinity of the polymer could be reduced by the sol addition, leading to the decrease of strength and strain simultaneously. Similar phenomena were observed in our previous research19 and in the study of Gowsihan et al. in the case of electrospun silica/PLLA fibers with calcium18. The crystallinity change was thoroughly studied by DSC (See the following section). Although the Young’s modulus decreased by 34% and the strain decreased by 25% with around 16.7 wt.% sol addition, the modulus value was still nearly 10 times higher than that reported for pure PCL electrospun fibermats.37 The strain values were almost 40 – 50 times higher than those reported for pure PHB electrospun fibermats,38 which means that the hybridization approach leads to notable improvement of the properties of the single components. Table1. Physical properties of electrospun fibrous scaffolds. (The values are mean ± standard deviation (n = 5)) Diameter / µm

Tensile strength / MPa

Strain / %

Young’s modulus / MPa

P1B0

0.7 ± 0.2

2.8 ± 0.3

158 ± 23

101 ± 11

P20B1

0.9 ± 0.3

2.2 ± 0.2

179 ± 56

87 ± 20

P10B1

1.0 ± 0.2

2.9 ± 0.3

139 ± 27

85 ± 13


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P5B1

1.2 ± 0.2

1.9 ± 0.2

119 ± 43

67 ± 9

Fig. 3. Representative stress-strain curves (a) and trend of average values (b) of P1B0, P20B1, P10B1, and P5B1 samples. 3.3 FTIR analysis and wettability In order to confirm the incorporation of silicate sol into the electrospun fibermats, and to further investigate the structure variation, FTIR analysis was performed on different samples and the results are displayed in Fig. 4 (a). PHB and PCL spectra were studied in detail in our previous investigation,19 thus only the silicate sol related information was analyzed. Additionally, the main vibration bands of 58S sol-gel are summarized in Table 2. Compared to P1B0, a broad band at around 3400 cm-1 corresponding to hydroxyl groups appeared in sol containing samples, i.e., P20B1, P10B1 and P5B1, which is responsible for the hydrophilic character of the fibers as discussed below. In the pattern of sol containing samples, typical peaks at around 1167, 1075, 953, 797, 548 cm-1 corresponding to Si-O-Si, PO43-, Si-O-Ca, Si-O-Si, PO43- stretching, bending and vibration modes were overlapped with typical polymer peaks. Moreover, the whole transmission intensity decreases at around 500 cm-1, which is ascribed to Si-O-Si bending mode and P-O bending vibration.39 These pattern changes verified the incorporation of 58S sol-gel in the polymer fibermats. No other bands appeared or shifted, which implies that no strong interaction occurred between the organic and inorganic components; however, weak interactions such as hydrogen bonding may form between the –OH groups of 58S sol-gel and C=O bonds of PHB and PCL.40 As is well-known, surface wettability can influence not only protein adsorption, but also cell attachment and proliferation. Studies have shown that cell attachment on hydrophilic surfaces is 10 ACS Paragon Plus Environment


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usually more efficient than on hydrophobic materials.41 The wettability of all samples is presented in Fig.4 (b). Due to the hydrophobic nature of PHB and PCL, pure polymer fibermats P1B0 were also highly hydrophobic. However, the wettability of P20B1, P10B1 and P5B1 switched to completely hydrophilic owing to the silicate sol addition (Fig. 4 (b)). It was observed that during the measurement, the contact angles decreased with time and finally the H2O drop was absorbed into the samples within 30 seconds indicating a significant water uptake by the scaffold itself and therefore turning the web to an apparent hydrophilic character. Fig. 4 (b), inset (1), presents the typical image of hydrophobicity for pure polymer fibermats P1B0 and inset (2) shows the typical image of hydrophilicity for sol containing samples P20B1, P10B1 and P5B1. On these samples, the droplet would disappear in a few seconds. The results indicate that the silicate sol addition can reverse the wettability behavior of pure PHB/PCL blend fibermats.

Fig. 4. FTIR patterns (a) and contact angle values (b) of P1B0, P20B1, P10B1, and P5B1. Table 2. Main vibration bands and assignments in 58S sol-gel39, 42, 43 Wave numbers/ cm-1

Vibrational mode assignments

3400

Hydroxyl group –OH

1632

C-O band

1167

Si-O-Si asymmetric stretching

1075

PO43- asymmetric stretching

953

Si-O-Ca vibration

797

Si-O-Si bending

548

PO43- bending

460

Si-O-Si rock


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3.4 Thermal properties and crystalline structure The results of TGA analysis in Fig.5 were used to determine the actual organic/inorganic ratio through residue weight calculation. All samples seemed to undergo a multiphase degradation due to their multicomponent composition. The derivative curves of the TGA measurements in Fig.5 (b) illustrate the different phases more clearly.

Fig.5. (a) TGA curves of samples heated from room temperature to 600°C; (b) derivative curves of TGA curves in (a). Table 3. Thermal degradation results from TGA analysis. #

T1 / °C

1st loss

T2 / °C

T3 / °C

T4 / °C

/% 58S

−

P1B0

−

P20B1

2nd, 3rd and

Residue

Organic/inor

4th loss / %

weight %

ganic ratio

−

−

−

55.0

−

204−286

304−426

−

99.8

0.2

99.8/0.2

RT − 68

2

208−275

275−376

403−506

95.2

2.8

19.8

P10B1

RT − 87

2.7

209−278

278−359

406−500

91.7

5.6

11.0

P5B1

RT − 90

5.3

209−274

296−360

405−520

83

11.7

4.9

Notes: T1, T2, T3 and T4 represent the temperature range of the four degradation phases; Estimated organic/inorganic ratio = (weight loss in stage 2, 3 and 4)/ (weight loss in stage 1 + residue weight)



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When heating to 600°C, two separate decomposition periods corresponding to PHB and PCL appeared in sample P1B0 as described in our previous study;19 however, four stages occurred in P20B1, P10B1 and P5B1 owing to 58S addition. Using 58S gel as reference, evaporation of H2O molecules and residual precursor (which could not be removed by the vacuum oven) are thought to cause the 1st stage weight loss until 100°C;44 then PHB and PCL decomposition mainly causes the 2nd and 3rd stage weight loss.19 The slight weight loss in the 4th stage could be the last phase of the densification process of the 58S gel.44 It is worth noting that the oxidation of organics and the densification process in 58S gel took place throughout the whole heating period, not only in the final weight loss period, thus it is difficult to accurately calculate the exact organic/inorganic ratio. Table 3 lists the temperature range of the four periods and the organic/inorganic ratio was approximated from the weight loss. DSC curves of all samples after a heating-cooling-heating cycle from 0 °C to 200 °C are displayed in Fig. 6 (a) and (b). The first heating curves of the samples could reflect the influence of the solution properties and the electrospinning process on the melting behavior of the obtained structures, while the second heating run is useful for investigating the material interactions since the first heating run has already eliminated the thermal history. In the first heating, P1B0 and P20B1 exhibited two endothermic peaks at 56°C and 175°C corresponding to the melting point of PCL and PHB crystalline, respectively. As 58S content increased for P10B1 and P5B1, more endothermic peaks appeared at 41°C and around 100°C, which was possibly caused by H2O molecules and residual precursor. In the second heating, the PHB in P1B0 exhibits dual melting peaks, which indicate a recrystallization process in PHB. The recrystallization phenomenon occurred as a meltcrystallize-remelt process, which corresponds to the melting of crystalline phases from the previous cooling run and the subsequent heating run, respectively.38 This phenomenon disappeared in P20B1, P10B1 and P5B1, which implies that the addition of the inorganic substance suppresses the thermal crystallization during the second heating run. Additionally, the crystallinity of PHB and PCL were also calculated using equation (1) and labeled in Fig.6 (b). As the results show, the crystallinity of PHB and PCL decreased from 54.4% to 41.1% and from 39.8% to 27.5%, respectively. The disappearance of the recrystallization process and the significant reduction of crystallinity suggest an inhibition effect of the inorganic component on the crystalline process of PHB and PCL. In other words, in contrast to acting as a nucleation agent for crystallization as reported commonly in nanomaterial reinforced polymer systems,45 the homogeneously distributed 58S here did not promote crystalline formation, but inhibit the macromolecule movement leading to the decrease of overall crystallinity. The possible reason could be the weak interaction, for instance, hydrogen


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bonding between polymer chains and the inorganic component, which restrains the crystal formation. W. Yu et al. drew a similar conclusion in a study of electrospun PHBV with 1wt% ZnO addition,40 proposing that intermolecular forces between PHBV and ZnO were the main cause of the crystallinity decrease.

Fig.6. DSC curves of prepared samples heated to 200 °C: (a) first heating run; (b) second heating run.

Fig. 7. X-ray diffraction (XRD) patterns of P1B0 and P5B1 showing characteristic peaks of PHB and PCL.46, 47 Two representatives of hybrid scaffolds, i.e. P5B1 and P1B0, were chosen for X-ray diffraction analysis to investigate the crystalline structure, and results are presented in Fig.7. PHB and PCL are both semicrystalline biopolymers, and the characteristic peaks of PHB and PCL are labeled on the graph. The results indicate that the incorporated 58S glass sol did not significantly change the



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crystalline structure of both polymers in the system. However, the peaks of P5B1 pattern become weaker compared to P1B0, which also implies a crystallinity reduction.48 3.5 In vitro cell-culture evaluation Unlike conventional nanoparticles, sol-gel derived components can rapidly release ions into the local culture environment.49 Although PHB and PCL have been proved to be biocompatible in many reports, a detailed examination on the effect of the sol-gel derived component and the unreacted precursor on cell-material interaction is necessary. Since P5B1 contains the highest amount of inorganic component in this system, we choose P5B1 and pure polymer membrane P1B0 to investigate the influence of added silicate sol on the in vitro biological properties. MG-63 osteoblast-like cells were cultivated up to 28 days to primarily investigate the cell behavior.

Fig.8. CLSM images of MG-63 osteoblast-like cells cultured for 8 hours on (a1) (a2) P1B0, (b1) (b2) P5B1 fibrous scaffolds. Favorable cell adhesion on a material surface is considered as the prerequisite to the subsequent cell activity.50 Firstly, after 8 hours of culture, the cell attachment was evaluated through CLSM as shown in Fig. 8. Elongated cells were all spindle-shaped, flat and adhered well on the substrate regardless of its composition, which means the cells can effectively attach to the matrix. The only 15 ACS Paragon Plus Environment

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subtle distinction is that cells cultured on P5B1 sample possess longer guided filopodia than those cultured on P1B0, which may be attributed to the effect of calcium released from 58S. It has been demonstrated that filopodia length and numbers can be affected by altering calcium concentration.51 In S. Cheng et al’ s research,52 either a global or local increase in calcium concentration in the short term was reported to cause the filopodia elongation and increase in length. The well-developed guided filopodia of cells on P5B1 were expected to facilitate cell migration since filopodia are not only the initiation sites for adhesion, but also act as a sensing organelle to produce guidance cues and traction force to move the cell body.53, 54 Furthermore, in vitro cytotoxicity and proliferation of cells were examined by CCK8 assay. As shown in Fig. 9, both P1B0 and P5B1 showed much higher cell viability than the control (P < 0.001) after 3 days of culture, although no significant differences appeared after 1 day, which means that both fibrous structures are non-toxic to MG-63 cells. Additionally, there is no significant difference in cell viability between the two samples.

Fig.9. Cell viability after 1 day and 3 days for samples P1B0 and P5B1 (** P < 0.01, *** P < 0.001, control sample is TCP). Cell morphologies on P1B0 and P5B1 scaffolds after 3 days and 7 days of cell culture are shown in Fig.10. Cells proliferated well on both P1B0 and P5B1 scaffolds and showed well spread morphologies after 3 days of culture. However, on day 7, more cells were found on P5B1 scaffold and the majority of cells on P5B1 presented multipolar polygonal morphologies (stellate) with short filopodia, while cells on P1B0 still showed bipolar spindle morphologies with relatively long filopodia. Stellate cells can firmly attach, spread and eventually proliferate well on the matrix, while bipolar spindle cells are still migrating to locate sites for subsequent spreading and proliferation.55 Thus, it is reasonable to conclude that the addition of silicate sol could promote the proliferation of MG-63 cells. The cell morphology changes and improved proliferation capacity can be mainly 16 ACS Paragon Plus Environment


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ascribed to two reasons: the calcium-dependent regulation and the highly enhanced hydrophilicity provided by the presence of the inorganic phase. Filopodia consist of cross-linked actin filament bundles which can be affected by a calcium sensitive regulator factor -fragmin. It was reported that fragmin could increase the number of actin filaments, decreasing the length of the filaments only when free calcium concentration was above 1×10-6 M,56 which may lead to stellate cell formation. In other words, the local concentration change of free calcium causes a change in cell from bipolar spindle to multipolar stellate. Additionally, studies have shown that hydrophilic surfaces can enhance cellular adhesion and proliferation to a greater extent than hydrophobic surface since the former exhibit higher vitronectin and fibronectin absorption, which are essential proteins to bind with cell surface receptors.57, 58

Fig.10. Morphologies of MG-63 cells on P1B0 (a, b) and P5B1 (c, d) after 3 and 7 days of cultivation. Osteogenicity represents the potential capacity of scaffolds to stimulate and support cell differentiation and tissue mineralization.59 As an important enzyme and the most widely recognized marker of osteoblast differentiation, ALP activity is considered an essential factor to assess osteogenicity.60 Fig.11 (a) displays the ALP activity of MG-63 cells cultured on P1B0 and P5B1 for 2 weeks. The ALP activity of cells on P1B0 and P5B1 scaffolds was enhanced significantly 17 ACS Paragon Plus Environment

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compared to control (P < 0.001) after 1 week of culture. Meanwhile, the P5B1 sample showed a much higher ALP activity than P1B0 after 1 week (P < 0.001), indicating greatly enhanced osteogenicity. After 2 weeks in culture, the ALP activities were further increased on both samples. All the above results demonstrated that P1B0 and P5B1 scaffolds could promote the osteoinduction of MG-63 cells, and the existence of 58S glass sol will further accelerate osteoinduction.

Fig.11. (a) ALP activity after 7 days and 14 days of culturing MG-63 cells on P1B0 and P5B1 fibrous scaffolds; (b) Mineral calcium nodules content measurements of MG-63 cells cultured on P1B0 and P5B1 samples for 4 weeks (* P < 0.05, ** P < 0.01, *** P < 0.001, control sample is TCP). Other than ALP activity, the cell differentiation on scaffolds can be further characterized by evaluating mineralization ability thorough measurement of calcification.50,

61

In this study the

mineralization capacity of MG-63 cells on P1B0 and P5B1 scaffolds was quantitatively determined by detecting the mineral calcium nodules after a period of 28 days in culture, which is proportionally related to the optical density of ARS staining extraction (Fig. 11(b)). As Fig. 11(b) illustrates, cells on both P1B0 and P5B1 scaffolds showed higher mineral calcium nodule content compared to the control (P < 0.001), and significantly higher amount of calcified calcium content was revealed in the cells cultured on P5B1 scaffolds than on P1B0 scaffolds (P < 0.01). The highly promoted mineralization of calcium nodules on MG-63 cells cultured on P5S1 further demonstrated the significantly improved osteogenicity. Thorough investigations have been conducted by Danoux et al.62 and Kim et al.63 to separately study the influence of silicon, calcium and inorganic phosphate ions on stem cell and osteoblast cell behavior, and results showed increased ALP activity and mineral deposition for all three ions. In our study, the significant enhancement of ALP activity and mineralization deposition on silicate containing samples (P5B1) confirm that the inorganic 18 ACS Paragon Plus Environment


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component addition in this system can effectively improve the osteogenic properties owing to the release of the inorganic ions, for instance Si, Ca and P, into the cell culture environment.50 Thus the obtained hybrid PHB/PCL/58S scaffolds are considered favorable candidates for bone tissue engineering applications on the basis of their biological features. In the future, efforts will be mainly made on expanding these fibrous structures from simple 2D scaffolds to hierarchical 3D constructs to match the pore dimension requirements in bone tissue engineering, adapting techniques that have been already suggested in this context 64,65. 4. Conclusions PHB/PCL/58S hybrid fibrous scaffolds were successfully prepared through the combination of electrospinning and sol-gel methods. These hybrid systems combined the advantageous properties of the single components in terms of both physical and biological behavior. The obtained hybrid structures exhibit high stiffness of PHB, flexibility of PCL and the high bioactivity of sol-gel 58S bioactive glass. The FTIR and wettability measurements verified the incorporation of 58S bioactive glass in hybrid scaffolds. In vitro MG-63 cell cultivation on pure polymer scaffolds and silicate containing scaffolds demonstrated that the addition of the inorganic component did not lead to toxicity whilst it enabled improved cell adhesion, proliferation, leading to significantly enhanced osteogenicity. The present bioactive, biodegradable fibrous scaffolds are thus attractive in bone regeneration applications and should be characterized in vivo in further investigations. 5. Acknowledgements Yaping Ding and Wei Li would like to acknowledge the China Scholarship Council (CSC) for financial support. The authors are grateful to Alfred Frey and Heinz Mahler for technical support, Inge Herzer for DSC operation, and Jennifer Reiser for TG analysis. Financial support for this work from Zhejiang National Nature Science Foundation (LQ15H18003) is acknowledged. 6. References 1.

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