It is well established that the piezoelectric effect plays an important physiological role in bone growth, remodeling and fracture healing. Barium titanate, as a well-known piezoelectric ceramic, is especially an attractive material as a scaffold for bone tissue engineering applications. In this regard, we tried to fabricate a highly porous barium titanate based scaffolds by foam replication method and polarize them by applying an external electric field. In order to enhance the mechanical and biological properties, polarized/non-polarized scaffolds were coated with gelatin and nanostructured HA and characterized for their morphologies, porosities, piezoelectric and mechanical properties. The results showed that the compressive strength and piezoelectric coefficient of porous scaffolds increased with the increase of sintering temperature. After being coated with Gel/HA nanocomposite, the interconnected porous structure and pore size of the scaffolds almost remain unchanged while the Gel/nHA-coated scaffolds exhibited enhanced compressive strength and elastic modulus compared with the uncoated samples. Also, the effect of polarizing and coating of optimal scaffolds on adhesion, viability, and proliferation of the MG63 osteoblast-like cell line was evaluated by scanning electron microscope (SEM) and MTT assay. The cell culture experiments revealed that developed scaffolds had good biocompatibility and cells were able to adhere, proliferate and migrate into pores of the scaffolds. Furthermore, cell density was significantly higher in the coated scaffolds at all tested time-points. These results indicated that highly porous barium titanate scaffolds coated with Gel/HA nanocomposite have great potential in tissue engineering applications for bone tissue repair and regeneration.
KEYWORDS: bone tissue engineering, foam replication method, piezoelectric ceramics, Barium titanate, BaTiO3,d33
Tissue engineering is a promising therapeutic strategy that utilizes specific biocompatible scaffolds as well as different cells types and growth factors in order to restore or replace injured tissues [1, 2]. Scaffolds, as the main component of the bone tissue engineering, provide a supporting structure for cell colonization, proliferation, and subsequent new bone formation. An ideal scaffold should possess several criteria including highly porous structure, appropriate mechanical properties, excellent bioactivity and good biodegradability [3, 4]. Moreover, it is known that natural bone exhibits the electrical potential in response to mechanical stress due to its inherent piezoelectric property. The piezoelectricity generated in bone is found to be applicable in sensing the biologic forces, which promotes the bone regeneration at the repair site [5-7]. Therefore, efforts have been made to develop the biomaterials with inherent piezoelectricity such as Barium titanate (BaTiO3; BT) to resemble self-healing of the natural bone [8-10].
Barium titanate belongs to a class of ferroelectric materials which display high piezoelectricity, and with regard to biomedical applications, show excellent biocompatibility and ability to form a strong interfacial bond with surrounding bone [11, 12]. The piezoelectric ceramic materials consist of a large number of the unit structures with the same polarization direction which is called domain. Each domain has a specific polarization direction and they are randomly oriented so that the net polarization of the material remains zero in the absence of an external electric field. Through a poling process, a strong external voltage at a proper elevated temperature aligns the domains parallel to the direction of the applied electric field, as the positive side of the electric dipoles facing the negative electrode and the negative side facing the positive electrode. Thus, one side of the scaffold derives a net positive charge and the opposite side derives a net negative charge [13, 14]. The presence of negative surface charge as a result of polarization have a significant effect on the crystallization of inorganic ions dissolved in vitro or in vivo and influence the behaviors and activity of cells deposited on it[15-17].
Different fabrication techniques have been developed to fabricate biomedical porous scaffolds [3, 18]. Among them, foam replication method has the capacity to produce highly porous structure through the impregnation of a polymeric sponge with a bioceramic slurry of proper viscosity. Then, the sponge is burned out during a careful heat treatment, which also sinters the ceramic powder. The high porosity and large pore size of resulted scaffolds are favorable for osteogenesis and vascularization throughout the entire implanted construct. However, such highly porous structure will exhibit high brittleness and very low mechanical strength for bone tissue engineering applications [19, 20].
One approach being investigated to enhance the mechanical properties and bioactivity of highly porous ceramic scaffolds is coating the struts of the implant with biocompatible materials such as polymers, ceramics, and composite materials. Composites have proven to be more effective at increasing the both mechanical strength and bioactivity, compared to monolithic ceramics and polymers [21, 22]. Gelatin, one of the derivations of collagen, has been shown to be able to significantly improve the mechanical properties of ceramic scaffolds. This is due to its strengthening and toughening effects which can be linked to a micro-scale crack-bridging mechanism, leading to an enhancement of the scaffold toughness in a similar behavior of collagen fibers in bone [23-25]. In comparison with collagen, gelatin is completely resorbable in vivo, does not express antigenicity in physiological conditions and its physicochemical properties can be suitably modulated. As a water-soluble natural polymer, gelatin degrades rapidly in aqueous environments. In order to decrease the degradation rate, gelatin can be cross-link by chemical agents such as glutaraldehyde or genipin [26-28].
In recent years, nanotechnology has emerged to be one of the most promising technologies in applied biomedical sciences . Surface chemistry and microstructure are critical factors in bioactivity and early osseointegration of a bioimplant. It is well known that the inorganic phase of human bone is composed of nanosized hydroxyapatite (HA) crystals. A nHA surface, significantly improves the biocompatibility, biological activity, and osteoconductivity of the underlying biomaterial. In addition, compared to conventional ceramic scaffolds, those with a nanostructured surface have greater osteoinductive potential. According to above explanations, the incorporation of Nano-structural HA in a polymeric matrix, as a nanocomposite for surface coating, can efficiently improve both mechanical and biological properties of the scaffold [30, 31].
Therefore, the development of the porous piezoelectric bioceramic scaffolds has attracted attention to achieve the desirable combination of electrical and mechanical properties. The integration of above consequences suggests that the porous polarized BT scaffolds coated with a polymer-ceramic nanocomposite can actively promote the osseointegration and mechanical properties of the construct.
To this perspective, the present study attempted to fabricate highly porous BT based scaffolds by foam replication method through a tailored sintering schedule. The prepared samples were then polarized by applying external electric field. In order to enhance the bioactivities, mechanical and biological properties, polarized and non-polarized scaffolds were coated with gelatin and nanostructured HA and characterized for their morphologies, porosities, piezoelectric and mechanical properties. Finally, the effects of polarizing and coating of optimal scaffolds on adhesion, viability, and proliferation of the human osteosarcoma cells (MG63) were studied by SEM and MTT assay.
2. Materials and methods
2.1 Scaffold fabrication
2.1.1. Preparation of BaTiO3 scaffolds
Barium titanate-based scaffolds were prepared by using the foam replication method, involves preparation of green bodies of ceramic foams by coating a polyurethane (PU) sponge with the ceramic slurry. The slurry was prepared by dissolving 6 % w/v polyvinyl alcohol (PVA) in deionized water at 80 °C, in which BT powder was added up to a concentration of 50 wt%. The whole procedure was carried out under vigorous magnetic stirring. In the following, PU foams with the desired size were immersed and rotated in the prepared slurry to ensure homogeneous slurry infiltration. The foams were taken out and the extra slurry was completely squeezed out from the foams. The samples were then dried in an oven at 60 oC for overnight and subsequently were placed in a heat treatment furnace. The temperature profile of the sintering process was programmed, as shown in Fig.1. The burning condition of the PU foams was the same for all samples: 350 oC/30 min, with a heating rate of 1 °C/min.
2.1.2. Polarization of porous scaffolds:
Following sample preparation, porous scaffolds which were sintered at 1300 and 1400 oC were electrically polarized. The polarization was carried out in a silicon oil bath at the temperature and an electric field of 120 oC (near the Curie temperature of BaTiO3) and 1 kV/mm, respectively, for 30 min. The piezoelectric charge coefficient, d33, was measured by a d33 meter (YE2730A d33 METER, Amirkabir University of Technology). The negatively charged surface of the porous scaffolds was labeled prior to gelatin/CaP coating.
2.1.3. Surface coating with gelatin and HA:
In this experiment, dip-coating and in situ precipitation process were used to form gelatin and HA coating on the surface of porous scaffolds. First, a solution containing gelatin (1 % w/v) and Ca with a pH value of about 7.4 was prepared by dissolving gelatin (type A-Merck) and Calcium nitrate tetrahydrate (0.1 M Ca(NO3)2⋅4H2O-Merck) together in distilled water under magnetic stirring at 50 °C. In order to get a better infiltration of the solution, scaffolds were placed and fixed in the front end of the cylindrical tube of a syringe and the piston of syringe was pulled along the inside of the tube, allowing to take in the Gelatin/Ca solution as it passes through the porous scaffold, then it was pushed and expelled the solution. This process of taking in and expelling the solution was repeated 3-4 times. After that, the scaffolds were taken out and dried in a freeze-drier system for 24 h. Then, coated gelatin were cross-linked with glutaraldehyde vapor to improve its water-resistant ability. Crosslinking process was carried out by placing the coated scaffolds in a sealed desiccator containing 5 ml aqueous glutaraldehyde solution (%25) in a petri dish, for 2 days at room temperature. After cross-linking, the samples were exposed in a fume hood for 2 h followed by circular vacuumization to remove residual GTA. In order to complete precipitation reaction, the scaffolds were exposed to a phosphate-rich solution (0.6 M (NH4) H2PO4, pH= 11) using the method explained above. Finally, the samples were rinsed thoroughly with deionized water and freeze-dried.
2.2 Characterization of scaffolds
2.2.1. X-ray diffraction (XRD)
X-ray diffraction analysis (Siemens- Brucker D5000 diffractometer, 40 kV/40 mA, Cu-Ka) were applied to gain information about the crystalline phases and probable structural changes of the initial BT powder and uncoated/coated scaffolds sintered at 1400 oC. For qualitative analysis, XRD diagrams were recorded in the interval 10280 at the scan speed of 28/min, being 0.028 step size and 1 s step time. The crystalline phases were identified on the basis of the JCPDS reference data.
2.2.2. Fourier transform infrared spectroscopy (FTIR)
FTIR spectroscopy was used to characterize the presence of specific chemical groups in uncoated and Gel/nHA coated BT scaffolds sintered at 1400 oC using a FTIR spectrometer (ABB Bomem, Quebec, Canada). For this analysis, 1 mg of the powder sample was mixed with 300 mg of potassium bromide (KBr) (infrared grade) and pelletized under vacuum. Then, the pellet was analyzed in the range of 500-4,000 cm-1 at the scan speed of 23 scans/min with 4 cm-1 resolution.
2.2.3. Scanning electron microscopy (SEM)
The surface morphology and elemental map, microstructure, and pore size measurement of uncoated and coated scaffolds fabricated at 1400 oC were evaluated using scanning electron microscopy (SEM). The samples were coated with a thin layer of Gold (Au) and observed on a scanning electron microscope (SEM-Philips XL30) at an accelerating voltage of 15 kV.
2.3. Mechanical Properties.
Mechanical behavior of the coated and uncoated scaffoldssintered at different temperatures (1100-1400 oC) was investigated by a Universal Testing Machine (SANTAM, Iran) with a crosshead velocity of 0.5 mmmin−1 and a 50N load cell. For compressive testing, the samples ( = 5) were cylinders of approximately 6mm in diameter and 10mm in height in accordance with the compression mechanical test guidelines set in American Standard Test and Measurement (ASTM F 451-95). Specimens were compressed to ∼40% of their original height and the values were expressed as the means ± standard error.
2.4. Analytical methodology
Dimensions of the test specimens were measured and weighed to an accuracy of 0.01 mm and 0.01 g, respectively, before and after heat treatment and compression of the samples.
2.4.1. Porosity measurement
The porosity of the scaffolds was measured according to the below formula:
Pore volume (Vp) was calculated according to Eq. (1):
VTis the volume of the scaffold, which is calculated using its outer dimension (cm3),
MBTis weight of the scaffold and
ρBTis the density of barium titanate (6.02 g/cm³).
Thus the calculation formula of porosity (%) is defined as follows Eq (2):
2.4.2. Measurement of volume shrinkage:
The percentage of volume shrinkage of sintered samples was obtained by the following equation:
(%) Volume shrinkage:
V1 and V2 are the volume of samples before and after sintering, respectively.
The density of the sintered samples was calculated using the mass and bulk volume data obtained by weighing and measuring, respectively.
2.5. Cell experiments
2.5.1. Cell culture and cell seeding
Cell studies were carried out using MG-63 cell line (Human osteosarcoma cell line-National Cell Bank of Iran, Pasture Institute). Cell lines were cultured in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS), 100U/mL (1%) penicillin–streptomycin and incubated at 37°C in a humidified incubator with 5% CO2 and 95% humidity. The culture media was refreshed every 3 days and when cells reached to 80–90 % confluency, they were washed with phosphate buffered saline (PBS) and passaged with 0.25% trypsin/EDTA (Gibco).
All cell culture studies were carried out on the porous scaffolds sintered at 1400oC. Prior to cell seeding, scaffolds were sterilized by UV radiation for 40min and incubated in culture medium for 1 h at 37 oC. Cells were seeded on the sterilized scaffolds at the concentrations of 4×104 cells per sample. The cell-loaded scaffolds were then placed at 37°C in a humidified incubator under standard conditions.
2.5.2. Cytotoxicity evaluation
The viability and proliferation of cells grown on the uncoated and coated scaffolds were evaluated by 3-(4, 5 Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test. 1, 3 and 7 days after cell seeding, 150 L of MTT solution (5mg/mL MTT in PBS 0.1 M) was added to750 mL of sample medium (RPMI). Following 4h incubation at 37oC, the medium was removed and 600µL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals. 100 L of the resulting supernatant was transferred into a 96-well plate and the absorbance (optical densities) was measured by an ELISA reader at 570 nm.
2.5.3. Cell adhesion and morphology on the coated and uncoated scaffolds
The cellular adhesion and morphology of MG-63 cells on theuncoated and Gel/nHA-coated scaffolds were evaluated using scanning electron microscopy (SEM). Following culture for 72h, cell-loaded scaffolds were washed twice with phosphate-buffered saline (PBS) and the cells were fixed with 2.5% glutaraldehyde for 1h. Post fixation for each sample was performed with 1% osmium tetroxide followed by dehydration in a graded series of alcohols (70%, 80%, 96%, and two changes of 100% ethanol). Samples were then freeze-dried and kept dry using silica gel. Dried samples were coated with a thin layer of Gold (Au) and then the morphology of the cells was observed using a scanning electron microscope (Philips XL30, Netherland) that operated at the acceleration voltage of 25 kV.
2.6. Statistical analysis
The results of cell experiment were expressed as the mean ± standard deviation (n=3) and statistical analysis of data was performed by one-way analysis of variance (ANOVA) and paired Student t-test. A value of p <0.05 was considered to be statistically significant.
3. Results and discussion:
3.1. Microstructure characterization of BT and Gel/nHA-coated BT scaffolds
Figure 2 displays the XRD patterns of the as-received BT powder and sintered scaffolds at 1400 oC with and without Gel/nHA-coating. XRD analysis of the raw powder is performed to detect the initial phase of the material and compare it with sintered phases. Fig. 2a confirms phase purity and polycrystalline nature of the initial BT powder (PDF#01-072-0138). The XRD patterns of fabricated scaffolds (Fig. 2b-c) show that there is no phase transformation or decomposition after sintering at 1400 oC. Moreover, in Fig. 2c, the diffraction peak corresponding to the coating was consistent with the HA standard card (PDF#00-025-0166) and suggests that our method of rapid mineralization was effective in producing HA phase.
Fig. 3 shows the FTIR spectra, in the 500–4000cm–1 spectral range, of uncoated and coatedscaffolds. The FTIR spectrum of uncoated BT scaffold is shown in Fig. 3a. The band observed at 521 cm-1 is due to the Ti-O stretching of BT and the band around 858 cm-1 is due to the C-C stretching vibrations. The bands observed at 1384 and 1637 cm-1 is due to C=O and C=C stretching respectively. The characteristic absorption band at 3419 cm–1 can be assigned to O-H stretching vibrations of the adsorbed water. FTIR analysis was performed on coated scaffolds to confirm the presence of the gelatin and precipitated HA. The result (Fig. 3b) exhibited a number of characteristic spectral bands related to gelatin and HA, while the characteristic spectral bands for the BT was present in the spectrum as well (listed in Table. 1). The most characteristics of them were protein spectrums such as: N-H bending vibration at 1245cm–1 for the amide III, C=O stretching vibration at 1742cm–1 for the amide I and C-H bending vibration at 2970cm–1 for the amide B. The precipitated HA is characterized by the absorption bands of 1111 and 612cm-1 correspond to the P-O stretching vibration and O-P-O bending mode respectively, whereas the band at 780 cm–1 is assigned to the carbonate group. The band at about 1430cm–1 indicates the formation of the chemical bond between carboxyl groups from Gel and Ca2+ ions from the HA that has been mentioned in former studies too [32, 33]. Thus, from the FTIR spectra, the successful formation of Gel/nHA coating can be confirmed.
Typical morphologies of the uncoated and Gel/nHA coated BT scaffolds were observed by SEM (Fig. 4a-f). The microstructure of the uncoated scaffolds illustrates the highly porous structure with interconnected macroporous networks with the pore size in the range of 200 – 500 μm (Fig.4 a-b). High porosity and interconnected pore structure are suitable for cell migration and proliferation, vascularization and new bone formation throughout the entire 3D scaffold. Higher magnification image of struts after sintering at1400 °C is shown in Fig. 4c. The sintered structure demonstrates the high degree of densification which consequently leads to higher mechanical properties. Although, microstructural image clearly show the presence of micro pores dispersed on the scaffold walls/struts. Also, as it is well known from the literatures, scaffolds fabricated by the foam replication method usually shows surface defects such as microcracks and a remaining central hole inside the struts which is the result of the removal of the polyurethane foam [34, 35]. Clearly, such highly porous and microcracked structure limit the mechanical properties of the fabricated scaffolds which are the main reason why polymer coatings are considered, as will be discussed in more detail in the next section. After coating with Gel/nHA, the interconnected pore structure and pore sizes of the scaffolds were maintained as confirmed by Fig. 4(d). The thin layer of gelatin coating filled the existing open micropores and sealed micro cracks on the surface of the scaffolds to form an interlaced structure. The scaffold surfaces appear to be rougher and crystal-like materials are embedded in the polymer matrix in very close proximity to each other. In the micrographs with higher magnification (Fig. 4e), the morphology of the mineral nanocrystals precipitated on the scaffold surfaces is also visible. As the figure depicts, the HA coating has a nanostructure with crystallite size in the range of 70–300 nm and crystal growth occurred in the form of needle-shaped crystals in the aligned parallel directions on the scaffold surface. The observed appearance can be attributed to the applied method for surface coating. In fact, HA crystals grow in the same direction of the coating solution while taking in and expelling it from the syringe tube. The occurrence of nanoscale needle-shaped and parallel organized crystals is predominantly a feature of hard tissues biominerals such as bone and dentin. This nano-rough surface can improve cell adhesion and growth for tissue regeneration. Additionally, elemental mapping was performed to identify the distribution of the HA nanoparticles present in the coated scaffold surfaces. Figure. 4f shows that calcium was dispersed throughout the surface, which confirms the presence of nHA crystals coating on the scaffold substrate.
3.2. Mechanical characterization of the scaffolds:
Mechanical properties of uncoated and Gel/nHA-coated porous BT scaffolds fabricated at different sintering temperature were evaluated by the uniaxial compression test. Typical compressive stress–strain curves of uncoated and Gel/nHA coated scaffolds are shown in Fig. 5. The effect of sintering temperature and Gel/nHA-coating on the porosity, shrinkage and compressive strength and elastic modulus of the macroporous scaffolds is shown in Table 2. As it can be seen, the compressive strength and elastic modulus increases with increasing the sintering temperature from 1100 °C (0.02 ± 0.01 MPa, 0.28 0.07 MPa) to 1400 oC (0.64± 0.09 MPa, 2.34 0.57 MPa). It can be explained by the increasing densification via shrinkage of scaffold material due to the expanding small contact areas between the particles and decreasing the total void volume which leads to decreasing porosity and finally improved mechanical properties. Moreover, the mechanical strength of Gel/nHA-coated scaffolds fabricated at different sintering temperature was significantly higher than that of uncoated scaffolds. The improved mechanical properties of the polymer coated scaffolds can be explained by the micro-scale crack-bridging mechanism. The polymeric coating fills and covers the micropores and microcracks situated on the strut surfaces, forming continuous bridges which increase the structural integrity of the scaffolds. This lead to an enhancement of the scaffold toughness, in a similar manner as collagen fibers increase the fracture toughness of bone [21, 36]. Since the porosity of the scaffolds slightly reduced after coating, it is obvious that the presence of the gelatin coating has such notable effect on the mechanical properties of the scaffolds. In overall, the highest value of compressive strength and elastic modulus (1.39 0.03 MPa, 3.25 0.26 MPa) was obtained from the coated scaffolds fabricated at sintering temperature of 1400 oC. (See Table 2).
3.3. Piezoelectric properties:
Figure 5(a-b) shows the effects of polarization time and electric field intensity on the piezoelectric coefficient (d33) of porous scaffolds sintered at 1300 and 1400 oC. The polarization of the samples was performed at 120 oC, the Curie point temperature of BaTiO3 in which the ferroelectric-paraelectric transition occurs . As it is shown in Fig. 4, the d33 value of piezoelectric ceramic scaffolds initially increased with the increasing of polarization time and applied electric field, reached a maximum value of 4.5 pC/N and then decreased. As explained in Section 1. Introduction, through a poling process, domains are urged to move when the material is heated up to the required temperature and therewith array into a single direction under the application of high-intensity electric field (~1 kV/mm). The extent of alignment depends on the parameters such as the magnitude of the applied external electric field, poling duration and temperature. An applied external electric field causes any of the domains which are favorably oriented with respect to this field grow at the expense of unfavorably oriented domains. As the intensity of the electric field is increased, these parallel domains become larger and eventually all of the dipole moments become aligned parallel to the field. However, excessive poling electric field tends to over-pole the samples which leads to physical flaws and dielectric breakdown of the porous scaffolds. On the other hand, with the increase of the polarization time, the electric domain gradually overcame resistance to orientation, and the d33 value increase. However, when the polarization time is more than 30 min, the d33 decreased due to the burn-out of the scaffold material following long exposure to the high-intensity electric field. Therefore, the optimal polarization parameters which were obtained for the polarization electric field intensity was 1 kV/mm, with polarization time of 30 min, and polarization temperature of 120 °C were chosen based on the previously published researches. Porous scaffolds sintered at 1300 oC showed the same variation but as it can be seen, with the reduction of sintering temperature, the d33 decreased from 4.5 to 3.9 pC/N. As mentioned earlier, increasing the sintering temperature to 1400 oC, increase the grain size, densify the material and reduce the number and size of the air voids which affect the dielectric and piezoelectric properties of the piezoelectric barium titanate scaffolds. Moreover, the increase in the degree of orientation during electrical polarization, induced by the development of large grains. It is notable that, because of the low compressive and dielectric strength, electrical polarization of the porous scaffolds fabricated at 1100 and 1200 oC was not possible.
3.4. Biocompatibility evaluation:
The viability and proliferation of MG-63 cells on the uncoated and Gel/nHA-coated scaffolds were determined by MTT test. MTT is a quantitative colorimetric assay to evaluate cell viability based on the reduction of tetrazolium salt by mitochondrial enzymes into a colored formazan. As shown in Figure 6, the cell density was increased with the culture time in both tested groups, which confirm that both samples have good biocompatibility and scaffold materials and coating process had no cytotoxic effects on viability and proliferation of the cells. However, the cell density on the Gel/nHA-coated scaffolds were significantly higher than that on the uncoated BT scaffolds. Therefore, although both samples could support the adhesion and proliferation of cells over time in culture, the Gel/nHA-coated BT scaffolds supported better cell proliferation, indicating that the Gel/nHA coating on the scaffold surface enhance the attachment and subsequent proliferation and viability of seeded cells, as reported by previous studies.
3.5. Cell adhesion
SEM micrographs were used to study the adhesion and morphology of cells on the polarized and non-polarized porous scaffolds with and without gelatin/nHA coating. SEM images of seeded cells incubated for 72 h showed that cells attached to the pore walls of the scaffolds. However, there was a significant difference in cell morphology and spreading in a surface dependent manner. As shown in Fig. 7, on day 3, a few cells were distributed on the BT scaffolds and exhibited elongated, flattened morphology with few filopodia extensions (Fig. 7a). More cells attached on the surface of polarized scaffolds compared with non-polarized samples. Cells produced much filopodia in multiple directions, adhered on the negatively-charged surfaces. In the physiological environment or culture medium, cations adhere strongly to the negatively charged surface through electrostatic interactions and attract proteins such as integrin and fibronectin that mediate cell adhesion and proliferation [38, 39]. On the other hand, cell adhesion and proliferation on polarized and non-polarized scaffolds with Gel/nHA-coating were significantly greater than that of the uncoated scaffolds. A large number of cells attached on the surfaces with numerous lamellipodia and filopodia extensions, and grew to a confluent layer, as it can be seen in Fig. 7c and d. There is no obvious distinction between polarized and nonpolarized scaffolds. In fact, Bone mimicking coatings provide an appropriate complex microenvironment for cell adhesion and growth in the early stage of cell-surface interactions. The microstructure of gelatin, as a natural polymer, contain the extracellular substance which acts as a temporary extracellular matrix for successful cell migration and colonization. Moreover, the CaP coating provides adhesion sites for proteins and other macromolecules, leading to a biological layer that facilitates the adhesion and migration of cells at the surface and osteogenic activity of cultured cells at an early stage, as it was shown in our previous studies . The combination of the polarization effect and surface coating can efficiently improve the cell adhesion and proliferation, and made the scaffold favorable for bone regeneration.
In summary, highly porous barium titanate based scaffolds were successfully fabricated by the foam replication method. Fabricated scaffolds were electrically polarized and coated with gelatin/HA nonocomposite in order to improve their mechanical and biological properties. The mechanical strength and piezoelectric coefficient were observed to increase with increasing sintering temperature being, the highest at 1400 oC. After coating, the porosity and pore interconnectivity of the scaffolds almost remain unchanged while the Gel/nHA-coated scaffolds exhibited enhanced compressive strength and elastic modulus compared with the uncoated samples. In vitro tests indicated that developed scaffolds were biocompatible with MG-63 cells and osteoblasts were able to adhere, proliferate and migrate into pores of scaffolds. However, it is well-known that polarized piezoelectric materials produce an electric charge when a mechanical stress is applied. Surface-charged piezoelectric ceramic and the electric field has been proven to influence proliferation, differentiation, and extracellular matrix deposition of osteogenic cells. Therefore, further investigations such as in-vitro and in-vivo tests are in progress to study the effects of applied mechanical stress on the porous piezoelectric BT scaffolds in regulating cellular behaviors.
Fig. 1. Heat treatment program designed for burning-out the polyurethane templates and sintering the barium titanate green bodies.
Fig. 2. XRD patterns of (a) barium titante powder, (b) uncoated scaffolds, and (c) coated scaffolds fabricated at sintering temperature of 1400 oC.
Fig. 3. FTIR spectra of (a) uncoated scaffolds and (b) coated scaffolds fabricated at sintering temperature of 1400 oC.
Fig. 4. SEM micrographs of the morphologies and microstructures of the BT scaffolds before and after coating with Gel/HA nanocomposites at different magnifications: (a-c) without coating, (d-e) coated with Gel/nHA, and (f) map scanning of Ca element.
Fig. 5. Compressive stress–strain curves of uncoated and Gel/nHa coated scaffolds.
Fig. 6. Effects of polarization process on the d33 of BT piezoelectric bioceramic. (A) Polarization time, (B) Polarized electric field intensity.
Fig. 7. Cytotoxicity assay of MG-663 cell cultured on the uncoated and Gel/nHA coated scaffolds at 1, 3 and 7 days (p < 0.05).
Fig. 8. SEM morphology of MG-63 cells on the scaffolds for 3 days. (a) non-polarized BT scaffolds, (b) polarized BT scaffolds, (c) non-polarized BT scaffolds with Gel/nHA surface coating, and (d) polarized BT scaffolds with Gel/nHA surface coating.
Table. 1. Infrared assigned and chemical relevant bonds in prepared GEL/nHA coated scaffolds.
Table. 2. Compared mechanical properties of uncoated and Gel/nHA coated scaffolds fabricated at different sintering temperature.