Bioactive glass combined with zein as composite

Biomed. Glasses 2018; 4:72–81

Research Article Jasmin Hum*, Shiva Naseri, and Aldo R. Boccaccini

Bioactive glass combined with zein as composite material for the application in bone tissue engineering https://doi.org/10.1515/bglass-2018-0007 Received Feb 20, 2018; revised Jun 07, 2018; accepted Jun 13, 2018

Abstract: The present study has focused on the development of new composite scaffolds based on the combination of zein with bioactive glass for the application in bone tissue engineering. Porous polymeric matrices were produced by the salt leaching technique. By incorporating 45S5 bioactive glass particles the lack of bioactivity can be remedied. However, the addition of bioactive glass is influencing the plasticization behavior of the zein matrix during the salt leaching which negatively affects the compression strength as well as the degradation behavior. This paper describes the process during leaching and explains the different behavior of zein with and without the presence of bioactive glass. Keywords: Bioactive glass, zein, bone tissue engineering, composite material, plasticization, scaffold

1 Introduction The demographic evolution shows a worldwide progressive population aging over the past centuries. This phenomenon can be assigned to the improved medical care and high standards of hygiene, achieved especially in the developed countries. However, this development not only influences the economic situation but also entails a high demand for the healthcare system, in particular

*Corresponding Author: Jasmin Hum: Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Cauerstrasse 6, 91058 Erlangen, Germany; Email: [email protected] Shiva Naseri: Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Cauerstrasse 6, 91058 Erlangen, Germany; Department of Mining and Materials Engineering, McGill University, Montreal, QC, Canada Aldo R. Boccaccini: Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Cauerstrasse 6, 91058 Erlangen, Germany Open Access. © 2018 J. Hum et al., published by De Gruyter. NonCommercial-NoDerivatives 4.0 License

the availability of “body parts” to replace damaged and non-functional tissue and organs. The replacement or repair of tissue damaged by disease or trauma is a growing challenge faced by thousands of surgeons every day. Nowadays, autografts are the gold standard [1–3] to replace lost tissue but the availability is strongly limited. Allografts as well as xenografts carry the risk of infection, rejection and incompatibility. Next to this, also ethical concerns are a significant issue when applying xenografts. Therefore, a solution approach is based on the development and application of alloplastic grafts which consist of non-biological engineered materials like metals, ceramics or polymers. A main field for the application of alloplastic grafts is bone tissue engineering. In general, tissue engineering combines approaches from natural and engineering sciences to develop artificial and three-dimensional constructs which support or replace damaged tissues or organs [4, 5]. Bone tissue engineering (BTE) focuses on the regeneration process of native bone tissue [6, 7]. Therefore, research efforts are being devoted to find suitable materials for bone tissue engineering. One promising material is zein as it is a plant-derived protein from corn [8] which offers biodegradability and biocompatibility. It is widely used as coating material in pharmaceutical and food industries [8, 9]. Extracted from renewable resources, it is increasingly considered for biomedical applications [10]. Another material highly attractive for bone tissue engineering is bioactive glass. This material offers many advantages like bioactivity, osteoconductivity and the released ions are known to stimulate angiogenesis and promote cell proliferation and differentiation of osteoblastic cells [11]. Composite scaffolds with zein and bioactive glasses are rarely reported in the literature. Therefore, this paper reports the production of porous zein and zein- bioactive glass composite scaffolds. The samples are investigated in terms of mechanical properties, bioactivity and biodegradability for the application in bone tissue engineering.

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Bioactive glass combined with zein in bone tissue engineering | 73

2 Materials and methods 2.1 Materials For the experiments, only chemicals of analytic grade were used. Zein was received from Sigma-Aldrich (Germany). Commercially available bioactive glass powder (mean particle size ~ 2 µm) with the 45S5 composition (45% SiO2 , 24.5% CaO, 24.5% Na2 O and 6% P2 O5 in wt.%) was purchased from Schott (Vitryxxr, Germany). Sodium chloride (NaCl) was obtained from VWR Chemicals (Germany). Ultrapure water (UPW) with electrical conductivity of ≤ 0.067 µS/cm was received from Purelab R 7 (Veolia, Germany).

2.2 Preparation of porous zein scaffolds without bioactive glass particles (pure zein) Three-dimensional porous zein scaffolds without bioactive glass particles were produced by the leaching technique using sodium chloride (particle size < 125 µm) as porogen [12]. Zein was homogeneously mixed with NaCl particles (ratio 1:2) and compressed applying a load of 104 N in an electrohydraulic pressing device (Mauthe Maschinenbau, Germany). The received cylindrical specimens (Ø = 10 mm, h = 6.5 mm) were leached at 80∘ C in a water bath for 2 hours to receive a porous structure. After leaching, samples were washed in UPW and lyophilized for further use.

2.3 Preparation of reinforced porous zein scaffolds with bioactive glass particles (zein-BG) For the preparation of three-dimensional porous zein scaffolds with bioactive glass reinforcement, a concentration of 70:30 (in wt.%) was chosen for zein and bioactive glass particles, respectively, based on optimized parameters of previous studies [13]. According to the preparation of pure zein scaffolds, a homogenous mixture of zein and bioactive glass particles was prepared. The zein-bioactive glass mixture was then mixed with NaCl particles (ratio 1:2) and pressed into cylindrical shape, as described above. Samples were immersed in a water bath (UPW) and leached at 80∘ C for 2 h. Received samples were washed in UPW and lyophilized for further use.

2.4 Porosity The porosity P of pure zein scaffolds and zein-bioactive glass composites was determined according to the following equation: (︂ )︂ ρ sample P = 1− · 100 (1) ρ material where ρ sample is the volumetric mass density of the investigated sample and ρ material is the density of material (in case of pure zein ρ material = 1.22 g/cm3 ) [14]. For the ascertainment of ρ material of the composite scaffold, the different ratios of zein and bioactive glass have to be considered. As dissolution of bioactive glass particles during the leaching process is assumed, the correct ratio of zein and bioactive glass was determined by thermogravimetric analysis (TGA, STA 449 F3 Jupiter, Netzsch, Germany). TGA was carried out in air with a heating rate of 5 K/min. Based on the results of the TGA measurement, the density ρ composite was calculated as ρ composite =

m composite m + m BG = zein V composite V zein + V BG

(2)

where m zein and V zein represent the mass and volume of zein and m BG and V BG represent the mass and volume of 45S5 bioactive glass (2.7 g/cm3 ) [15], respectively.

3 Swelling properties For the evaluation of the swelling properties of porous zein and zein-bioactive glass composite scaffolds, sorption experiments were carried out. Samples were immersed in phosphate buffered saline (PBS) at room temperature for up to 24 h. After different time points, samples were removed and the excess solution was dried with a filter paper (qualitative filter paper 410, VWR Chemicals, Germany). The water uptake of each sample was calculated according to (︂ )︂ mw − md Water uptake [%] = · 100 (3) md where m w and m d represent the mass of the wet and dry samples, respectively.

3.1 Enzymatic degradation The degradation behavior of zein and zein-bioactive glass composite scaffolds was investigated in vitro in the presence of enzymes. Bacterial collagenase from Clostridium


74 | J. Hum et al. histolyticum with a collagenase activity of 125 CDU/mg was used (Sigma-Aldrich, Germany). A buffer solution (pH = 7.4) was prepared with 0.1 M TRIS-HCl (Sigma-Aldrich, Germany), 0.005 M CaCl2 (VWR Chemicals, Germany) and 0.05 mg/ml NaN3 (Merck Millipore, Germany). Samples of zein and zein-bioactive glass were soaked in 1 ml of TRIS-HCl buffer solution. After one hour of immersion, 1 ml of Tris-HCl buffer with 200 CDU/ml was added to get the desired concentration of 100 CDU/ml. The enzymatic degradation was stopped after 1 and 24 h by addition of 0.25 M EDTA buffer solution (Sigma-Aldrich, Germany). Scaffolds were washed in UPW and lyophilized. The degradation rate was calculated as (︂ )︂ mi − ma Degradation [%] = · 100 (4) mi

3.4 Fourier transform infrared spectroscopy (FTIR)

where m i is the initial weight of each scaffold and m a is the dry weight after degradation, respectively. Scaffolds, which were immersed in pure Tris-HCl buffer solution without enzymes, were used as reference.

3.5 Mechanical characterization

3.2 Evaluation of bioactivity in vitro The bioactive behavior of zein and zein-BG composite scaffolds was assessed in vitro by immersion in simulated body fluid (SBF) using the standard protocol introduced by Kokubo et al. [16]. Samples were put in closable containers of polypropylene, immersed in 50 ml of SBF and placed in an orbital shaker with 90 rpm at 37∘ C. After different time points, samples were removed, washed in UPW and dried at 60∘ C. The acellular mineralization of the samples was evaluated by the formation of hydroxyapatite, which was investigated by scanning electron microscopy (SEM) and analyzed by Fourier transform infrared spectroscopy (FTIR).

3.3 Scanning electron microscopy (SEM) A high resolution scanning electron microscope (Auriga, Zeiss, Germany) was used to investigate the microstructure and morphology of the different scaffolds. Samples were fixed with conductive silver, sputtered with gold (Quorum Q150T S, United Kingdom) and examined with 2 kV at a working distance of ~ 5 mm.

For the analysis of the chemical composition of the different scaffolds, Fourier transform infrared spectroscopy (FTIR) was used. 1 wt.% of the sample to be analyzed was mixed with KBr (Potassium bromide for IR spectroscopy Uvasolr, Merck Millipore, Germany) and pressed into a pellet (Ø 12 mm) applying a load of 8·104 N in an electrohydraulic pressing device (Mauthe Maschinenbau, Germany). The spectrum was recorded in absorbance mode (Nicolet 6700 FTIR spectrometer, Thermo Scientific, USA) and collected between 4000 and 400 cm−1 . Pure KBr was used to correct the background noise.

The mechanical properties of the obtained scaffolds were investigated by compression strength tests. Ten specimens of each type were uniaxially compressed until fracture with a preload of 0.1 N, a crosshead velocity of 1 mm/min and a maximum applied force of 1 kN (Zwick Z050, Germany).

4 Results 4.1 Morphological and microstructural characterization 4.1.1 Pure zein scaffolds The microstructure of the pure zein scaffolds after the leaching process was observed by SEM. In Figure 1, the top surface as well as the cross-section of the samples are presented. In the SEM images, the porous structure of the zeinbased scaffolds can be observed. The top of the scaffold shows many concavities with macropores between 100 and 400 µm and struts with diameter in the range of 75 to 400 µm. At higher magnification, micropores < 1 µm can be clearly identified. The cross-section shows a continuous porosity inside the scaffold. Macro- and micropores form a cave-like structure with interconnected porosity confirming the successful leaching of sodium chloride particles not only at the outside but also inside the scaffold structure. The porosity of the zein-based scaffolds (n = 4) was calculated according to equation (1) and determined to be 76 ± 2%.



Figure 1: SEM images showing the top surface and cross-section of porous zein-based scaffolds after the leaching process (at different magnifications).

After the leaching process, the scaffolds were investigated by FTIR to analyze the structural change occurring during leaching. Figure 2 shows the spectra of zein asreceived and zein after leaching, recorded in absorbance mode. Most characteristic peaks are labeled.

Figure 2: FTIR spectra of zein scaffolds before and after the leaching process. Relevant peaks are labeled and discussed in the text.

Pure zein shows the typical amide A band in the region between 3500 and 2800 cm−1 , which is described in the literature [10, 17, 18] and can be associated with the stretching of N-H and O-H bonds of the amino acids. In addition, amide I, II and III can be identified at 1662 cm−1 , 1535 cm−1 and 1240 cm−1 , respectively, and can be attributed to the C=O stretching of the peptide (amide I), the C-N stretching and N-H deformation (amide II) and the N-H and C-N deformation (amide III) [10, 17, 18]. After the leaching of sodium chloride particles, the FTIR spectrum showed reduced intensity of the peaks. As received, zein shows a glass transition temperature Tg at 165∘ C [8]. Due to the high content of water during the leaching process, the Tg of zein is reduced. Gillgren et al. [19] reports a Tg of zein below 60∘ C with 10 wt.% of water. It is assumed that the penetrating water interacts with hydrogen bonds of the amide-amide bonding in the structure of zein, visible by the reduced intensity of the FTIR peaks. This interaction weakens the zein structure as these hydrogen bonds are essential for the secondary and tertiary structure of zein [20]. As a result, also the glass transition temperature is reduced. As the leaching takes places at 80∘ C, zein becomes viscous and is plasticized [21]. Therefore, the SEM images (Figure 1) show a smooth surface of the zein scaffold caused by the plasticization process.


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Figure 3: SEM images showing porous zein-based scaffolds reinforced with 30 wt.% of 45S5 bioactive glass particles after the leaching process (at different magnifications).

4.1.2 Zein scaffolds with bioactive glass reinforcement Zein scaffolds reinforced with 30 wt.% of 45S5 bioactive glass particles are represented in Figure 3. SEM images show macro- and micropores with interconnected porosity. Small agglomerations of bioactive glass particles (< 10 µm) are homogenously distributed inside the scaffold structure. However, bioactive glass particles are not embedded inside the zein matrix which can be explained by the hydrophilicity of the bioactive glass and the hydrophobicity of zein. The FTIR spectra of zein after leaching with and without 45S5 bioactive glass reinforcement are shown in Figure 4. The spectra were recorded in the range between 1800 and 650 cm−1 in absorbance mode. Amide I, II and III can be identified at 1662 cm−1 , 1535 −1 cm and 1240 cm−1 , respectively, and belong to the zein structure as already explained above. At 1040 cm−1 and

940 cm−1 the presence of bioactive glass can be clearly shown. The great peak at 1040 cm−1 can be attributed to the Si-O-Si stretching vibrations in the glass network [22] whereas the smaller peak at 940 cm−1 is assigned to the vibrational mode of the Si-O-2NBO bond [22]. However, after the addition of bioactive glass particles, the intensity of the peaks is seen to increase again. It is assumed that water has a higher affinity to interact with bioactive glass than with the hydrogen bonds of the amide-amide linkage. As a consequence, also the plasticization process is affected. This hypothesis can be supported by observations during handling of the different samples. Pure zein scaffolds showed a rubber-like structure after the salt leaching whereas zein-bioactive glass composite scaffolds exhibited a more brittle and fragile behavior which can be confirmed by the mechanical properties of the different kind of scaffolds. The porosity of the zein-bioactive glass composite scaffolds was determined according to equation (1). Based on the TGA measurements, which revealed a ratio of 74:26 (in wt.%) for zein and bioactive glass particles after salt leaching, respectively, ρ composite was calculated as 1.42 g/cm3 (using equation 2). Therefore, porosity of the composite scaffolds was determined as 84 ± 3%. The difference compared to pure zein (~ 75%) is likely caused by the additional leaching of bioactive glass particles.

4.2 Swelling properties and degradation behavior 4.2.1 Swelling properties Figure 4: FTIR spectra of zein after the leaching process with and without 45S5 bioactive glass reinforcement. Relevant peaks are labeled and discussed in the text.

The water uptake of pure zein and zein-bioactive glass composite scaffolds was determined after 1, 3, 24 and 48 h. The results are plotted in Figure 5.



Figure 5: Swelling properties of zein and zein-bioactive glass composite scaffolds after 1, 3, 24 and 48 h.

Results show that the equilibrium for pure zein was reached after 24 h. As zein contains leucine, proline and alanine, which are hydrophobic amino acid residues, it shows only a relatively low water uptake (~ 400%.) However, for zein-bioactive glass composite scaffolds equilibrium was already reached after 3 h of immersion in PBS. In addition, a faster swelling behavior can be observed in the initial stage attributed to the presence of the bioactive glass particles which additionally create channels for the ingress of fluid into the polymer matrix.

4.2.2 Degradation behavior The structural stability of zein and zein-bioactive glass composite scaffolds was investigated in presence of enzymes for up to 14 days. The degradation rate was determined according to equation (4). Results are presented in Figure 6. After 14 days of immersion in TRIS-HCl solution + collagenase, pure zein scaffolds revealed a high stability against enzymatic degradation. Calculated values show a degradation rate of 36 ± 1%. Similar results have been reported in the literature before [12]. However, zein-bioactive glass composite scaffolds were completely destroyed after 14 days of incubation. As composite scaffolds also show a higher swelling behavior compared to pure zein scaffolds, the higher degradation rate is a natural consequence. On the other hand, reference samples which were incubated without enzymes exhibit a very low rate of degradation. Pure zein did not show any weight loss after 14 days which suggest a high stability of zein in water-based solutions. Composite scaffolds with bioactive glass reinforce-

Figure 6: Enzymatic degradation behavior of zein and zein-bioactive glass composite scaffolds after 1, 24, 168 and 336 h.

ment showed a degradation rate of 13 ± 1% which is not connected to degradation of the zein matrix but to the dissolution of the bioactive glass particles.

4.3 Evaluation of in vitro bioactivity Pure zein and zein-bioactive glass composite scaffolds were immersed in SBF for different time periods. Samples were investigated in terms of hydroxyapatite formation by SEM observation and FTIR measurement. Figure 7 and 8 show SEM images and FTIR spectra of pure zein, respectively, after 7 and 14 days of immersion. Pure zein did not exhibit any bioactive behavior after immersion in SBF for 14 days. SEM images (Figure 7) did not show any indication of formation of hydroxyapatite on the surface of the porous scaffolds. In addition, FTIR spectra (Figure 8) did not indicate the mineralization of the pure zein matrix. Only amide I, II and III can be identified which belong to the zein structure, as already discussed above. After the reinforcement of the zein matrix with bioactive glass particles, a clear change in bioactivity can be observed. After 3 days in SBF, SEM images (Figure 9) show small round-shaped crystals randomly distributed in the inner structure of the composite scaffolds. With increasing immersion time, a denser hydroxyapatite layer is formed on the surface. After 14 days, the surface is homogenously covered with the typical cauliflower structure of hydroxyapatite [23, 24]. The formation of hydroxyapatite can also be confirmed by the FTIR spectra represented in Figure 10. Next


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Figure 7: SEM images of pure zein scaffolds after 7 and 14 days of immersion in SBF showing no hydroxyapatite formation.

Figure 8: FTIR spectra of pure zein scaffolds after 0, 1, 3, 7 and 14 days of immersion in SBF. Relevant peaks are labeled and discussed in the text.

to the typical peaks of the zein structure (amide I, II and III), growing peaks in the 1469–1499 cm−1 and 1100-1000 cm−1 regions can be assigned to the C-O stretching [25, 26] and P-O-stretching [25] in hydroxyapatite crystals, respectively. In addition, the broad peak at 1235 cm−1 and the small peak at 960 cm−1 are attributed to the P-O stretching in the calcium phosphate layer [25, 27]. Finally, the peak at 875 cm−1 indicates the formation of carbonated hydroxyapatite on the surface of the zein-bioactive glass composite scaffolds which can be attributed to the C-O bending [25, 26]. The peak at 798 cm−1 is characteristic for the formation of a silica rich layer [28].

The strength was measured by the maximum stress immediately before the fracture point. Results revealed high compressive strength for pure zein scaffolds (4.1 ± 0.8 MPa) compared to zein-bioactive glass composite scaffolds which reached values of 2.2 ± 0.9 MPa. In the literature, similar scaffolds have been reported showing values between 2.5 ± 1.2 MPa and 11.8 ±1.7 MPa for compressive strength [12]. Gong et al. [12], for example, describe the mechanical properties of salt-leached pure zein matrices depending on the mass fraction of NaCl/zein and the particle size of sodium chloride. Mass fractions of 1:1.4 and 1:2.5 for zein:NaCl (w/w), respectively, were used with particle sizes between 38.5 and 220 µm. The authors report relatively high compressive strength (11.8 ±1.7 MPa) for low particle sizes (38.5 to 75 µm) and mass fraction of 1:1.4 (zein:NaCl). On the other hand, samples reveal lower compressive strength (2.5 ± 1.2 MPa) when higher mass fraction (1:2.5 for zein:NaCl) and larger particle sizes (150 – 220 µm) were used. During this study, sodium chloride with particle size < 125 µm was applied as porogen. Zein and NaCl were homogenously mixed at a ratio of 1:2. Accordingly, results for the compressive strength reported in this paper show comparable values for pure zein-based scaffolds. As composite scaffolds exhibit a higher porosity than pure zein, also the compressive strength is reduced. In addition, it is assumed that the addition of bioactive glass hampers the plasticization process during leaching, which also explains the reduced mechanical properties. Nevertheless, compressive strength of natural cancellous bone ranges between 2 and 12 MPa. Thus, received values for pure zein and zein composite scaffolds match these requirements which makes them of great interest for the potential application in bone tissue engineering.

4.4 Mechanical characterization To characterize the mechanical properties of the different scaffolds, uniaxial compression tests were carried out.



Figure 9: SEM images of zein-bioactive glass composite scaffolds after 3, 7 and 14 days of immersion in SBF showing the formation of hydroxyapatite.

ence of the porogen on the mechanical properties and the degradation behavior. However, the described scaffolds exhibit no bioactive behavior which is a big drawback for the application in bone tissue engineering. To continue and further improve the previous work, this study was aimed at developing similar scaffold with enhanced bioactivity by the reinforcement with 45S5 bioactive glass particles. In addition, the processes during salt leaching of the zein structure are explained which are not mentioned in the literature yet. As the presence of bioactive glass influences the plasticization of zein, this paper for the first time provides an explanation of zein scaffold processing, which can be helpful for further investigations in this field.

Figure 10: FTIR spectra of zein-bioactive glass composite scaffolds after 0, 1, 3, 7 and 14 days of immersion in SBF. Relevant peaks are labeled and discussed in the text.

5 Discussion Previous studies are already available describing the fabrication of porous zein scaffolds based on the salt leaching method [12]. Porous scaffolds were produced with porosity between 75 and 79% and compressive strength in the range of 2 to 12 MPa. The paper mainly investigated the influ-

6 Conclusion The present paper described the fabrication of porous zein and zein-bioactive glass composite scaffolds. By the socalled salt leaching technique novel bioactive composites with compressive strength of 2.2 ± 0.9 MPa, interconnected porosity of around 84% and high stability against enzymatic degradation were developed. In the literature, the fabrication of stable and porous zein scaffolds is already described [12, 29]. However, the significant drawback of pure zein for tissue engineering applications is the lack of bioactivity of this vegetal protein. By the combination


80 | J. Hum et al. with bioactive glass this problem was overcome, as reported in this work. Nevertheless, it has to be considered that the presence of 45S5 bioactive glass particles influences the plasticization process of zein, which results in lower compressive strength and reduced stability against enzymatic degradation compared to pure zein. An interesting approach for further studies is the development of zein composites with enhanced bioactivity and improved mechanical properties at the same time. This may be realized by the optimization of the bioactive glass content. Also the size of the bioactive glass particles should be considered. Furthermore, zein can be combined with different polymers to increase the mechanical performance, e.g. PCL. Other bioactive materials next to 45S5 bioactive glass can be incorporated to enhance the biomineralization process without affecting the plasticization process of zein. Acknowledgement: The authors want to thank Dr. Judith Roether, Dr. Yaping Ding and Dirk Dippold (Institute of Polymer Materials, University Erlangen-Nuremberg) as well as Sabine Fiedler (Institute of Glass and Ceramics, University Erlangen-Nuremberg) and Giulia Rella (Institute of Biomaterials, University Erlangen-Nuremberg) for the support during all relevant measurements. Conflict of interest: The authors state no conflict of interest. Ethical approval: The conducted research is not related to either human or animals use.

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