Synthesis and characterization of Bioglass-based bone grafts with Gelatine substitution for biomedical applications

Abstract

Background:

Syntesizing alternative bone graft materials are important in biomedical applications. Their morphology, mechanical properties and cell viability plays an important role in tissue engineering.

Objective:

Bioglass (B) based bone grafts with Gelatin (G) substitution were syntesized via the sol-gel method and were compared with various Gelatin and Bioglass concentrations (wt%).

Methods:

Syntesized bone grafts were characterized by Scanning Electron Microscopy (SEM) and X-ray diffraction (XRD) to show the structural and morphological changes of the fabricated B-based bone grafts.

Results:

It was demonstrated that the concentration of pore size increased with increasing amounts of Gelatin in wt%. The biograft-B40G20 produced the highest flexture strength and hardness. Increasing the pore size caused a decrease in hardness and flexture stress of B-based biografts.

Conclusions:

Cell viability tests were conducted on the fabricated biografts and it was shown that the cell viability increased in fabricated B-based bone grafts.

Keywords

Bioglass sol gel mechanical properties cell viability

1. Introduction

The surface reactivity of a biograft with a direct bone contact can improve bonding strength between the biograft and surrounding bone tissue. The Bioglass 45S5 was first developed by Hench exhibiting a pronounced bioactivity and more importance in the field of reconstructive surgery [1–4]. Bioactive glasses would be suitable especially to fill bone defects, but can also be used as coating material for implants in direct bone contact in order to enhance the osseointegration between bone and metallic implant [5–8]. It was reported that the crystallized variant of the bioglass 45S5 reveals a good osseointegration characteristics via some in vivo studies [9,10]. In a contact of bioglass to bone, a bone-like apatite showed good bone-bonding characteristics on the coating surface [11].

The preparation of uniform porous materials and their degradation behavior plays an important role in tissue engineering processes [12]. Once implanted in a body, porous bone graft should maintain mechanical properties and structural integrity until the loaded cells adapt to the environment. Degradation of porous scaffolds for tissue engineering [13–19] and enzymatic degradation behaviors were also examined [20]. From the engineering viewpoint, the mechanical forces experienced by the cell culture in a scaffold are likely to be influenced by the mechanical behaviors of the scaffold and microstress environment [13,21]. However, the structural changes related to cell viability, B-based bone graft was not reported or eluciated in literature.

In the current study, B-based porous bone grafts were syntesized by using Gelatin as a template for pore formation. The structures and properties of the fabricated B-based biografts at various wt% B and G were compared to each other via structural, mechanical and cell viability tests. Furthermore, the effects of Gelatin addition at different proportions (wt%) on the porosity, mechanical properties and cell viability of fabricated biografts were ivestigated.

Table 1
Per cent concentrations of the bioglass based biografts

Biografts KH₂PO₄ (%) Na₂CO₃ (%) P₂O₅ (%) Gelatin (%) Bioglass (%)

B40G20 10 15 15 20 40

B20G50 2.5 5.0 2.5 50 40

B10G75 5.0 5.0 5.0 75 10

Biografts	KH₂PO₄ (%)	Na₂CO₃ (%)	P₂O₅ (%)	Gelatin (%)	Bioglass (%)
B40G20	10	15	15	20	40
B20G50	2.5	5.0	2.5	50	40
B10G75	5.0	5.0	5.0	75	10

2. Experimental work

2.1. Materials and sol gel process

In order to syntesize a porous bone graft structure via sol gel process, commercially provided Bioglass, 45S5 was chemically mixed with KH₂PO₄ (2.5–10 wt%), Na₂CO₃ (5–15 wt%), and partially with P₂O₅ (5–10 wt%). As given in Table 1, Gelatin was used at various concentrations (20, 50, 75 wt%) to obtain gel. The chemical, physical and mechanical performance of the syntesized porous biografts were compared with each other. The mechanical properties of bioglass, 45S5 (10 up to 40 wt%) supplemented with KH₂PO₄ (2.5–10 wt%), Na₂CO₃ (5–15 wt%) and partially with P₂O₅ (5–10 wt%) were tested and compared to each other. During the preparation of the sol, the ingredients were stirred up at 800 rpm rotor speed for 30 minutes in the ethanol/distilled water (at ratio $1 / 3$ ) mixture using a magnetic stirrer and a gel-like structure was obtained. After finding a homogeneous gel structure using an homogenizer (750 W, 35 Hz) for 10 mins, the obtained gel was poured into the cylindrical molds (5 × 2 mm) and let to dry at room temperature. Five samples from each group (B40G20, B20G50 and B10G75) were prepared at three different compositions. The produced cylindrical bone graft disc specimens were calcined in an a furnace at 120°C for 24 hours at normal atmospheric conditions. They were later heated at 350°C for 2 hours and the Gelatin was evaporated from the gel structure and pores were formed in the cylindrical discs. Finally, the sintering was carried out for those fabricated bone graft discs at 1200°C for 2.5 h.

2.2. Characterization

The structure and properties of the fabricated bone graft samples at different B and G wt% rates (B40G20, B20G50 and B10G75) were characterized by FTIR, XRD (Brucker) and SEM (JEOL, JSM-7001F). XRD analysis was conducted using the specimen holder and ( $λ = 1.5406$ Å) marked via fully automated diffractometer. The diagrams were produced by conducting the measurements recorded in the range of $2 θ = 3 – 70$ ° at a scanning rate of 2°/min and 1 s at a constant time gap. The spectroscopy was employed in a wavelength range of 4000–400 cm⁻¹. Mechanical properties of sintered B based biograft structures with the sintering additives KH₂PO₄ was evaluated under compressive loads. The ultimate stress of these biograft structures were evaluated by using a Shimadzu (5 kN) universal testing machine with a constant crosshead speed of 5 mm/min. The Vickers (Leica) microhardness of these biograft structures was evaluated under 20 N preload for 5 seconds.

The fabricated biografts were finally subjected to viability tests in a immature osteoblast culture environments by diluting in a 1 mol of KCl and HCl mixture in order to decrease concentration of the mixture down to 1 mg/ml. Cell viability evaluation were executed via the MTS (cell proliferation assay) (3-(4.5-dimetiltiazol-2-yl)-5-(3-karboksimetoksifenil)-2-(4-sülfofenil) tetrazolium, (CellTiter 96 Aqueous One Solution Assay) test in 96 well culture plates. First of all, the osteoblast cells were planted into 96 blank plates with 100 μl culture environment as each plate having 5000 cells and left to grow for 24 hours at 37°C incubation temperature. Next day, the culture environment was withdrawn and the cells were added into the cells of a culture environment (DMEM). The culture environment (DMEM) was used as negative and 20% DMSO was used as positive control. When the incubation time ended, the culture environment in wells was withdrawn and added to the blanks as to obtain 10 μl MTS + 100 μl culture environment. The cells subjected to MTS were left into incubation at 37°C and 5% CO₂ for 2–3 hours and after incubation, the cell viability was read by the Petri dish reader (Elisa plate reader). The grafts were investigated by counting cells in bakers at different concentrations (0.1–0.5 μm/ml) from 24 to 72 hours time intervals.

3. Results

3.1. FTIR

Figure 1 and Fig. 2 display the FTIR and XRD spectras, respectively for the B-based bone grafts at varying Gelatin contents (20, 50 and 75 wt% forming the biografts B40G20, B20G50 and B10G75. The FTIR spectras of the three different types of biografts were plotted in Fig. 1. All biografts displayed similar characteristic peaks in the range of 1200–400 cm⁻¹ depending on the vibrations. In the peaks, of PO₄ ⁻³ compounds were observed in the range of 1023–570 cm⁻¹ and sharp peaks were observed in the 590–560 cm⁻¹ range. The less sharp and broad peaks were observed in the structure of post sintering. Results of XRD analysis of the syntesized Biografts (B40G20, B20G50 and B10G75) were given in Fig. 2. The all syntesized B-based biografts yielded similar peaks in which increasing Gelatin content did not yield any considerable difference in peak widths.

Fig. 1.

FTIR spectrums of the syntesized biografts B40G20, B40G50 and B10G75.

Fig. 2.

XRD patterns of the syntesized biografts B40G20, B40G50 and B10G75.

Fig. 3.

SEM views of the syntesized biografts (a) B40G20, (b) B40G50 and (c) B10G75 biografts.

3.2. SEM

The structural images of the biografts by dopping Gelatin into Bioglass were analyzed through SEM analysis. The analyses were executed in comparison of the syntesized samples at various B and G combinations. Figure 3(a)–(c) displays SEM images of the B40G20, B20G50 and B10G75 biografts and compared with each other in terms of structure and porosity. Figure 3(a) displays the SEM image of the biograft B40G20 with 40% bioglass and 20% Gelatin contents. The macro and micropores were observed to be homogeneously dispersed as displayed in the image. SEM image of the B40G50 with 50% Gelatin content was shown in Fig. 3(c). The SEM image of the biograft (B20G75) with 75% Gelatin revealed the presence of the hill-like ridges among micro-porous structures. Along with the micro-pores that were formed on the surface, the bonds linking the pores were observed to be well connected (Fig. 3(c)).

3.3. Mechanical tests

Mechanical resistance of the syntesized B was evaluated via Compression and Hardness tests. The Stress-% deformation plots of the B-based biografts supplemented with 20, 50 and 75% Gelatin (B40G20, B20G50, and B10G75, respectively) were presented in Fig. 4. There was no significant statistical difference between B20G50 and B10G75 in terms of cell viability rates. As seen from the graphical representation, the results were similar for the biografts B40G20, B20G50, and B10G75. Hardness measurements were carried out for the syntesized B-based biografts that were supplemented with Gelatin at concentration ratios of 20, 50 and 75% (Fig. 5). Vickers hardness test (Emcotest) was conducted for 5 s under 20 N on the bioglass based biografts (B40G20, B25G50 and B10G75). The highest hardness value among the syntesized biografts was measured for the sample having the B40G20 sample with 0.75 GPa and the lowest hardness value among the porous biografts was measured for the sample B10G75 having 0.43 GPa.

Fig. 4.

Maximum stress (a) and strains (b) after compression tests of the syntesized biografts B40G20, B40G50 and B10G75.

Fig. 5.

Hardness results for the biografts B40G20, B40G50 and B10G75.

Fig. 6.

Cell viability changes for (a) B40G20 and (b) B10G75 biografts from 1 to 3 days of incubation periods.

3.4. Cell viability

Cell viability tests for B-based biografts (20 and 75% Gelatin) were executed into the osteoblast culture at (0.1–0.3 μg/ml) concentrations (B40G20, B10G75). From the plotted results given in Fig. 6(a)–(b), it was observed that the viability rates for B40G20 and B10G75 biografts increased proportionally with incubation time at both concentrations.

4. Discussion

FTIR analysis shown in Fig. 1 indicate that all biografts displayed such similar characteristic peaks associated with PO₄ ⁻³ in the range of 1200–400 cm⁻¹ depending on the vibrations. Similar peak ranges were identified for the fabricated B-based biografts. Therefore, the (PO₄ ⁻³ peaks are observed to be broad and in between 1136.16–570.21 cm⁻¹ range. FTIR spectras confirm such multiple bands obtained in the symmetric and asymmetric stretching mode region of P–O group in the crystal [14]. However, the FTIR spectrums of B40G20 and B20G50 samples showed no peaks around 789.64 cm⁻¹. Besides, the intensity of peaks were decreased for B40G20 and B20G50 samples due to the short P–O bond lengths. The characteristics bands due to PO₄ ⁻³ in the B-based biografts were the present at 1023.2 cm⁻¹ and 574.31 cm⁻¹. It is believed that the basic structure of B-based biografts were affected by the compositon of biografts. Such dense and sharp peaks were observed in the 590–560 cm⁻¹ range indicating the metallurgical bonds (M–O) occurred in the post sintering structure.

The results of XRD analysis of the syntesized biografts B40G20, B20G50 and B10G75 are displayed in Fig. 2. As shown, the peaks indicated the formation of both HA and β-TCP phases. All biografts were observed to yield similar peaks at similar angles and phases of the HA and β-TCP phases with increasing Gelatin content [13,14]. A comparison of the results of FTIR and XRD analyses indicated that the identification of both HA and β-TCP phases in XRD analysis as well as the presence of PO₄ ⁻³ peaks in the FTIR analysis. HA spectrum revealed the bands belonging to PO₄ ⁻³ group at 470 cm⁻¹, 560–610 cm⁻¹, 960 cm⁻¹ and 1000–1100 cm⁻¹ bands [15,16].

A cross-comparison of the SEM images for the syntesized biografts indicated that the biograft morphology and chemical structure varied with respect to the chemical content. The formation of pores was facilitated in the B-based biografts upon the supplementation and removal of polymer (gelatin) at various weight ratios (20, 50, and 75%) followed by the removal of the polymer from the structure at elevation temperatures e.g. 200°C (Fig. 3(a)–(c)). The analysis indicated that the porous structure was achieved with G addition and the number of pores increased with increasing gelatin content (Fig. 3(a)–(c)). SEM images of the samples showed that highest porosity was determined for 20% Bioglass with 50 and 75% Gelatin addition (B20G50–B10G75). However, with increasing concentration of Gelatin, the pore size decreased, which is in accordance with a previous report [14]. A comparison in the images between the B20G50 and B40G20 indicated that as the Gelatin content was increased, the porosity also increased and the macro-pores were being replaced with micro-pores. In addition, the micro-pores in the B20G50 were of different sizes and were better bonded although the grain boundaries could not be clearly identified. Furthermore, the biograft B10G75 was observed to constitute of spherical-like pores of varying sizes with an homogeneous pore distribution (Fig. 3(c)).

Compression and Hardness tests were conducted on the syntesized biografts and the related values were plotted in Figs 4 and 5, respectively. As seen from the figures that the compression stress of HA was found to be 294 MPa (Fig. 4) and Hardness value (HV) of that graft was measured as 3.43 GPa (Fig. 5), as similar results was reported in [15]. The results of the Compression test for the B-based biografts indicated a lower compression resistance with increasing Gelatin content (Fig. 4). Additionally, per cent deformation value was observed to decrease upon increasing the gelatin content of the B based biografts (from 20% to 50%), whereas a further G increase up to 75% did not yield any further changes in the % deformation. Furthermore, the stress value was observed to decrease with increasing Gelatin content and so the porosity while the highest stress was measured for the biograft B40G20 (177 MPa) and the lowest stress value was found to have 130 MPa for the biograft B10G75. In comparison with non-porous grafts, the highest hardness value among the porous biografts was measured 0.75 GPa for the sample B40G20. The lowest hardness value was measured as 0.43 GPa for the sample B10G75 among the synthesized porous biografts. The compression stress and hardness of the porous B-based biografts having 20, 50, and 75% Gelatin supplement were compared to each other. The biograft B10G75 delivered the lowest hardness value (HV = 0.43 GPa) and the hardness value was observed to decrease with increasing Gelatin concentration ratio for the all syntesized biografts (Fig. 5). As displayed in Fig. 4, the porous biografts indicated that the compression stress and the strain (%) values were observed to be decreased with increasing G addition (wt%) into the B-based biografts.

Osteoblasts are immature life cells that are proliferated and transformed to bone cell [22]. Figure 6(a)–(b) shows the changes in cell viabilities for B40G20 and B10G75 biografts by detecting the osteoblast cells in between 0.1–0.3 μg/ml concentrations for various incubation periods from 1 to 3 days. From the cell viability tests, it was observed that the biografts B40G20 and B10G75 have produced the higher cell viability rates at especially 0.3 μm/ml concentration. Also in B40G20 biograft at different concentrations and incubation times, the cell viability ratios were found to be over the control (n.c). B10G75 biograft also exhibited an increase in the density of the live cells at 0.1 μm/ml at the end of the third day of incubation time. Figure 6(b) shows the cell viability results of B10G75 biograft at the end of the third day shows the cell viability ratio was also above the control (n.c). The live cell density was a little below from the control n.c level of cell viability rate at 0.3 μg/ml concentration in which indicating a kind of the risk of toxic effect. However, B10G75 biograft at both concentrations show no toxicity as keeping the cell viability ratio over n.c (Fig. 6(b)). It can be concluded throughout the current experimental work that the cell viability increased as high as 40% and 60% for 20 and 75% G contents, respectively (Fig. 6(a)–(b)).

5. Conclusions

In the present study, B-based biografts were synthesized and the following conclusions are summarized;

Increasing the G content from 20 to 50% also increased the number of pores however, decreased the pore size, whereas further increasing the Gelatin content up to 75% was observed to decrease the number of pores while increasing the pore size.

The compression strength and Hardness values were observed to decrease with increasing G content and the porosity, the highest strength value was obtained from the biograft B40G20.

The cell viability rate in the fabricated B-based grafts was increased.

Addition of the G into the B-based biografts had a significant effect on the cell viability, e.g. B10G75 showed about 60% higher life cells compared to control in the 3rd day of incubation time.

Conflict of interest

The authors have no conflict of interest to report.

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Synthesis and characterization of Bioglass-based bone grafts with Gelatine substitution for biomedical applications

Abstract

Background:

Objective:

Methods:

Results:

Conclusions:

Keywords

1. Introduction

Table 1 Per cent concentrations of the bioglass based biografts Biografts KH2PO4 (%) Na2CO3 (%) P2O5 (%) Gelatin (%) Bioglass (%) B40G20 10 15 15 20 40 B20G50 2.5 5.0 2.5 50 40 B10G75 5.0 5.0 5.0 75 10

2.1. Materials and sol gel process

2.2. Characterization

3. Results

3.1. FTIR

3.3. Mechanical tests

4. Discussion

5. Conclusions

Conflict of interest

References

Table 1
Per cent concentrations of the bioglass based biografts

Biografts KH₂PO₄ (%) Na₂CO₃ (%) P₂O₅ (%) Gelatin (%) Bioglass (%)

B40G20 10 15 15 20 40

B20G50 2.5 5.0 2.5 50 40

B10G75 5.0 5.0 5.0 75 10