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Gelatin-enhanced porous titanium loaded with gentamicin sulphate and in vitro release behavior
Materials and Design 99 (2016) 459–466
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Gelatin-enhanced porous titanium loaded with gentamicin sulphate and in vitro release behavior Qiuyan Li a, Guo He a,b,⁎ a b
Shanghai Key Laboratory of Materials Laser Processing and Modification, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, China Collaborative Innovation Center for Advanced Ship and Deep-Sea Exploration, Shanghai 200240, China
a r t i c l e
i n f o
Article history: Received 26 January 2016 Received in revised form 15 March 2016 Accepted 18 March 2016 Available online 19 March 2016 Keywords: Titanium-gelatin composite Elastic modulus Drug release Gentamicin sulphate Bone repair
a b s t r a c t Porous titanium with entangled wire structure was enhanced by filling gelatin into the porous structure to form a titanium-gelatin (Ti-G) composite. The density of the composites was 1.6–2.5 g/cm3, similar to that of bone. The strength and elastic modulus of the composites reached 12.9–39.4 MPa and 2.5–7.4 GPa, respectively, which were comparable to that of the cortical bone. Gelatin in Ti-G composite acted as the carrier of gentamicin sulphate (GS). Antibacterial efficacy of GS-loaded Ti-G against Staphylococcus aureus strain was tested. The lack of growth of Staphylococcus aureus strains was found in Luria–Bertani (LB) medium containing GS-loaded Ti-G. GS release behavior from the GS-loaded Ti-G composites in phosphate buffer saline (PBS) was investigated. The effective GS release times from Ti30-G and Ti50-G were 120 h and 168 h, respectively. The gelatin in Ti-G was just degraded 3.2% at the immersion time of 168 h and 9.0% at 336 h. The degradation rate of gelatin was much slower than release rate of GS, indicated that the GS release from gelatin was less affected by the gelatin degradation. These Ti-G composites loaded with drug(s) are expected to be good candidates as functional bone repair materials with drug-delivery capabilities. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction As materials for bone repair, titanium and its alloy have attracted increasing attentions for its low density, good corrosion resistance and excellent biocompatibility [1–5]. However, many failures of titanium implants were attributed to the stress shielding effects caused by the mismatch between the elastic modulus of titanium and bone [6]. Research and development of porous materials is a way to solve this problem [7–9]. Porous titanium with entangled wire structure (p-Ti) has the advantages of easy to control porosity and easy for production [10,11]. However, its strength is too low for bone implants. When the porosity ranges from 50% to 70%, the yield strength and elastic modulus of p-Ti are 2.6–31.9 MPa and 0.44–0.82 GPa, respectively [11]. So it is necessary to enhance the strength of p-Ti. One of the methods is to infiltrate other biomaterial into the entangled structure, forming composite porous material. For this purpose, gelatin is a good choice for reinforcement of p-Ti.
⁎ Corresponding author at: Shanghai Key Laboratory of Materials Laser Processing and Modification, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, China. E-mail address: [email protected] (G. He).
http://dx.doi.org/10.1016/j.matdes.2016.03.103 0264-1275/© 2016 Elsevier Ltd. All rights reserved.
Gelatin is a natural polymer material derived from the hydrolysis of collagen. It has a long history of applying in antibacterial agents [12,13], anti-inflammatory drugs [14,15] and so on [16–18]. Gelatin has the advantages of good biocompatibility, good biodegradability, non-toxic and low antigenicity [19–23]. What's more important, the porous gelatin can be used as the carrier of antibiotics [24]. The utilization of antibiotic delivery systems is an effective measure to prevent wound infections. This method has the advantages of high local concentration, less dosage, low toxic and side effect [12,25,26]. Gentamicin sulphate (GS) is an aminoglycoside broad-spectrum antibiotic against Escherichia coli, Proteusbacillus vulgaris, Pseudomonas aeruginosa, Pneumobacillus, Salmonella bacteria, Shigella dysenteriae, Staphylococcus aureus and so on [27–29]. The bacterial resistance of gentamicin sulphate is lower than that of other aminoglycosides. So the gentamicin sulphate is widely used in the treatment of sepsis, respiratory infections, suppurative peritonitis, biliary infection, intracranial infection, urinary tract infections and infected lacerations [30–33]. In this paper, we used gelatin to reinforce p-Ti by forming p-Tigelatin (Ti-G) composite that was used as the carrier of GS. The antibacterial test showed that the GS-loaded Ti-G has obvious antibacterial efficacy. GS release behavior from the GS-loaded Ti-G materials in phosphate buffer saline (PBS) was investigated. Experimental results show that gelatin obviously enhanced the yield strength and elastic
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modulus of p-Ti. The elastic modulus of Ti30-G is up to 2.5 GPa, close to that of cortical bone (3–30 GPa) [34,35]. The elastic modulus of Ti50-G is up to 7.4 GPa, reaching the level of that of cortical bone. Its effective GS release time reached 168 h. 2. Materials and methods 2.1. Materials A commercial pure Ti wire (99.9% purity) with the diameter of 0.27 mm was used as raw material. The preparation of p-Ti was described in the previous paper [11]. In the process of sample preparation, the volume fractions of titanium were controlled by controlling the weight of titanium wire. It was calculated by the following formula: mTi ¼ V V Ti ρTi
ð1Þ
where VTi indicates the volume fraction of titanium wire; mTi indicates the weight of titanium wire; ρTi indicates the density of titanium wire; and V indicates the volume of sample. In this study, p-Ti has three kinds of porosity (70%, 60% and 50%). Sizes of compression samples and drug-loaded samples are Ø10 × 20 mm2 and Ø5 × 10 mm2, respectively. The gelatin and GS were bought from Aladdin Industrial Corporation. The gelatin water solution (25%) was prepared in the water bath at 100 °C. When the gelatin water solution was cooled to 60 °C, the pTi samples were immersed in it. A sustained ultrasonic oscillation was applied until the gelatin water solution cooled to 30 °C. Then the p-Ti samples filled with frozen gelatin was put in a refrigerator for 12 h at − 20 °C, and then put in a lyophilizer for 24 h at − 40 °C and 1.8 × 10− 2 mbar. After lyophilization, a 2% glutaraldehyde solution was used to crosslink the Ti-G for 6 h. After crosslinked, the Ti-G samples were washed in flowing water, and then cleared away the residues of glutaraldehyde by using firstly a 5% sodium borohydride solution and then pure water. Finally, the Ti-G samples were lyophilized again to dispel the residual water in the composite. After that, the Ti-G samples were fully immersed in a 50 mg/ml GS solution for 24 h at 4 °C for drug loading. The drug-loaded samples were then lyophilized again to obtain the gelatin-enhanced p-Ti materials with the gentamicin sulphate delivery system. The amount of GS loaded in Ti-G sample could be roughly estimated by the following formula: m ¼ ðm2 −m1 Þ C
ð2Þ
where m1 indicates the weight of the Ti-G sample. m2 indicates the weight of the Ti-G sample after immersion in the GS solution for 24 h. C indicates the concentration of the GS solution. Since the gelatin is absorbent to water, the porosity in GS-loaded TiG sample could be treated as the amount of water evaporation during lyophilization. It could be calculated by the following relation: P ¼ ðm2 −m3 Þ=V 100%
ð3Þ
where m3 indicates the weight of the GS-loaded Ti-G sample after lyophilized. P indicates the porosity of GS-loaded Ti-G sample. 2.2. Morphology and phase analysis The morphologies of the as-prepared Ti-G materials were observed by optical microscopy and scanning electron microscope (SEM). Information about the gelatin, GS, and GS-loaded gelatin was obtained by Fourier transform infrared spectrometer (FTIR, Nicolet 6700, Thermo Fisher, USA) analysis. The tests were performed at room temperature with wave number ranging from 4000 cm−1 to 500 cm−1.
2.3. Compression tests The compressive properties of Ti-G materials were evaluated by Zwick AG-100KN testing machine. The tests were conducted under displacement control with a cross-head speed of 1 mm/min. The average of three measurements was taken as the value. The yielding strength was determined according to the stress–strain curves, as described in the previous paper [36]. The elastic modulus of the Ti-G materials was calculated according to the slope of elastic stage of the stress–strain curve. 2.4. Test of antibacterial efficacy of GS-loaded Ti-G Antibacterial efficacy of GS-loaded Ti-G was tested using the Staphylococcus aureus strain. Inocula of bacterium were grown in Luria–Bertani (LB) medium. The Ti-G and GS-loaded Ti-G were put on the LB medium. During a 24 h incubation period (shaking: 35 rpm at 37 °C), LB medium samples from each experiment were tested for bacterial growth by measuring the bacteria inhibiting loop. 2.5. In vitro study of the release of gentamicin sulphate An in vitro method was employed to investigate the GS release behavior from the drug-loaded Ti-G materials. The sample was put in 10 ml PBS at pH 7.4 in a test tube for 14 days at 37 °C. The dissolution medium was collected at the time of 0.5 h, 1 h, 2 h, 4 h, 12 h, 24 h, 3 days, 7 days, 10 days, and 14 days. At the scheduled time, 3 ml of the dissolution medium were taken out from the test tube with pipette. The same volume of fresh PBS was added to keep the total volume of the release medium at 10 ml. GS release measurements were carried out using UV spectrophotometer (EV300, US Thermo Fisher) by Zhang's method [37]. Briefly, 1 ml GS solution, 1 ml isopropanol and 1 ml ophthaldialdehyde reagents reacted for 30 min at room temperature. The absorbance, which corresponds to the GS concentration, was then measured at 332 nm, at which the GS-phthaldialdehyde complex shows a maximum absorbance. These experiments were carried out in triplicate. Furthermore, calibration curve was made for each set of measurements and determined by taking absorbance versus GS concentration between 20 μg/ml and 400 μg/ml as parameters. 2.6. Test of gelatin degradation in PBS The gelatin degradation was tested in vitro. Ti-G samples were put in 10 ml PBS at pH 7.4 in a test tube for 14 days at 37 °C. At the time of 1 day, 3 days, 7 days, 10 days, and 14 days, three Ti-G samples were took out, respectively. The loss of gelatin was estimated by weighing after the Ti-G lyophilized. The average of three values was taken as the final value. 3. Results and discussion 3.1. Morphological characterization of the gelatin-enhanced p-Ti Morphology of as-prepared p-Ti was shown in Fig. 1(a), which was formed through titanium wires interweaving [10]. The pores distributed between titanium wires. In the case of the p-Ti with entangled wire structure, the pore size distribution and the average pore size mainly depend on the porosity. In this study, p-Ti materials with porosities of 70%, 60% and 50%, respectively, were prepared, which correspond to the Ti wire volume fractions of 30%, 40% and 50%, respectively. Thus, the three p-Ti materials were named as p-Ti30, p-Ti40 and p-Ti50, respectively. In the case of p-Ti30, the maximum pore diameter was about 500 μm and the average pore diameter was about 150 μm [11]. As the Ti wire volume fraction increases (the porosity decreases), the maximum pore diameter and the average pore size decrease. In other words, as the Ti wire volume fraction increased from 30% to 50%, the density of the p-Ti materials increased from 1.41 g/cm3 to 2.32 g/cm3.
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Fig. 1. As-prepared p-Ti (a) and gelatin-enhanced p-Ti (b).
After the p-Ti was filled with gelatin water solution, the porosity disappeared. When the water was evaporated during lyophilization, the porosity appeared again. But its value became smaller because the solid gelatin was left and embedded between the Ti wires as shown in Fig. 1(b) and Fig. 2(a). On the sections the embedded gelatin among the Ti wires was evident as shown in Fig. 2(b). Morphology of gelatin after lyophilization process is shown in Fig. 3. After lyophilization, there are many pores on the surface of gelatin. The pore size ranges from 2 μm to 30 μm. This porous gelatin can be used as the carrier of antibiotics. At the same Ti wire volume fraction, the density of Ti-G composites was reasonably larger than that of p-Ti materials. As the Ti wire volume fraction increased from 30% to 50%, the density of the Ti-G composites increased from 1.62 g/cm3 to 2.50 g/cm3. Since the density of natural bone is in the range from 1.8 to 2.1 g/cm3 [38], the Ti-G composites with about 34–41% Ti wire volume fraction would exhibit the density just in the same range as that of the bone as shown in Fig. 4. Compared
Fig. 2. Morphology of the Ti-G (a) and the section morphology of the Ti-G (b).
with the current mainstream bone implant materials such as Ti alloy (4.4–4.5 g/cm3), stainless steel (7.9–8.1 g/cm3) and Co-Cr alloy (8.3– 9.2 g/cm3) [38], the Ti-G composites have more close values in density with that of the bone. The low density of implant material can reduce the load of bone, and then decrease the risk of the implant failure. 3.2. Mechanical evaluation of the gelatin-enhanced p-Ti The compressive stress-strain curves of Ti-G composites exhibited typical three-stage deformation pattern, i.e., initial elastic deformation, then pseudo-plateau stress after yielding, and followed by a final densification region as shown in Fig. 5(a). It was evident that the strength of p-Ti materials was significantly enhanced after filled with gelatin. For example, when the titanium wire volume fraction was 30%, the yield strength of the p-Ti material was 5.2 MPa. The gelatin-enhanced p-Ti
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Fig. 3. Morphology of gelatin after lyophilization process.
material (i.e., Ti30-G composite) exhibited 12.9 MPa yield strength, that exceeded two times of the value of the non–enhanced p-Ti30 material. When the titanium wire volume fraction was 50%, the yield strength of the Ti50-G composite reached 39.4 MPa, that was about two times of the value of the non–enhanced p-Ti50 material (its yield strength was 21.8 MPa). The elastic modulus of the gelatin-enhanced p-Ti materials were evaluated directly from the stress-strain curves, which were 2.5 GPa for Ti30-G, 3.9 GPa for Ti40-G, and 7.4 GPa for Ti50-G. Compared with other porous entangled Ti materials that were enhanced by various methods [11,39,40], the gelatin-enhanced p-Ti materials had demonstrated their superiority in stiffness as shown in Fig. 5(b). It is known that the elastic modulus of cancellous bone is in the range of 0.01– 3 GPa [41,42], and that of cortical bone is in the range of 3–30 GPa [34, 35]. The Ti30-G composite investigated was close to the cortical bone in stiffness. The Ti40-G and Ti50-G composites exhibited the elastic modulus reached that of the cortical bone. The matched stiffness of the implanted materials would avoid stress shielding effect, and reduce the risk of implant failure.
Fig. 5. Compression stress-strain curves of the p-Ti materials and the Ti-G composites (a) and the elastic moduli of the Ti-G composites (b). Some data for the p-Ti enhanced by other methods were plotted for comparison.
3.3. Characterization of the GS-loaded Ti-G composites Since the gelatin is absorbent to GS solution, the drug-loading can be achieved by simple immersion in the GS solution. The amount of GS loaded can be directly calculated from the sample weights as shown in Fig. 6(a). When the dimension of the samples was Ø5 × 10 mm2, the amounts of the GS loaded were 3.65 mg for Ti30-G, 3.21 mg for Ti40-G, and 2.61 mg for Ti50-G. The real porosities of the Ti30-G, Ti40G and Ti50-G samples became 48.8%, 42.0% and 34.1%, respectively. With these data, two linear relationships between the real porosity (P) and Ti wire volume fraction (VTi), or, between the amounts of the GS loaded (mGS) and Ti wire volume fraction (VTi) could be roughly established (Fig. 6(b)): P ¼ 71:4−74:0V Ti
R ¼ 0:9956
mGS ¼ 5:33−5:40V Ti
Fig. 4. Densities of the p-Ti materials and the Ti-G composites with various titanium wire volume fractions.
R ¼ 0:9918
ð4Þ ð5Þ
where R is the correlation coefficient. Although the real porosities of the p-Ti materials became smaller after filled with gelatin, they were physiologically changeable. As the gelatin is degradable in the human body environment [12,13], it would gradually free extra space after implantation. The larger porosity was beneficial to the bone cell ingrowth and prosthesis fixation [38,43,44].
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and the amide group types III (1238 cm−1). The characteristic peaks of GS were the strong absorption peak of SO2− (1124 cm−1) and the 4 −1 2− medium absorption peak of SO4 (618 cm ). The GS-loaded Ti-G composite exhibited two peaks at 1107 cm−1 and 618 cm−1, which were the characteristic peaks of the SO42− in the GS. 3.4. Antibacterial efficacy of GS-loaded Ti-G The antibacterial efficacy of GS-loaded Ti-G in Luria–Bertani (LB) medium was shown in Fig. 8. After 24 h there was no bacteria inhibiting loop around Ti-G sample, but a distinct bacteria inhibiting loop around GS-loaded Ti-G sample. This GS-loaded Ti-G material seems to be sufficient to avoid the bacteria growth and thus reduce the frequency of infections after this biomaterial implanted. 3.5. GS release behavior from the GS-loaded Ti-G composites The absorbance of GS was scanned over the range of 190–400 nm, by a UV spectrophotometer (EV300, US Thermo Fisher). The maximum peak-absorption wave length of GS was observed at 332 nm. The linear relationships were established as standard curves between absorbance (A) and GS concentration (C) at the maximum peak-absorption wave length. The established standard curve was expressed as: A ¼ 0:01023 þ 0:00115C
Fig. 6. Weights of the Ti-G samples before and after immersion in GS solution 24 h, and weights after lyophilization (a) and the amounts of the loaded GS and the porosities of GS-loaded Ti-G composites (b).
To validate the GS loaded in the gelatin, a piece of GS-loaded gelatin was taken from the center of the GS-loaded Ti-G composite as the specimen for the FTIR analysis. The gelatin and GS were also analyzed by FTIR for comparison. The recorded patterns of three materials were plotted in Fig. 7. The characteristic peaks of gelatin were the amide group types I (1645 cm− 1), the amide group types II (1542 cm−1)
Fig. 7. FTIR analysis results of the gelatin, GS and GS-loaded gelatin.
R ¼ 0:9987 n ¼ 8:
ð6Þ
Based upon the obtained standard curves, the GS release profile was obtained from the absorbance of the sampling taken at the prefixed interval of time, as shown in Fig. 9. The curves could be divided into three stages: the burst release stage, the slow release stage and the final stage. The initial burst release was observed within the first 12 h, during which the GS release from Ti30-G composite was about 2370 μg, about 65% of the total amount loaded onto the composite. The GS release from Ti50-G composite was about 1350 μg, about 52% of the total amount loaded. In this initial stage, the average GS release rates of Ti30-G and Ti50-G were about 197.5 μg/h and 112.5 μg/h, respectively. The overall GS release from the Ti30-G was estimated to be about 82% in just 120 h, while about 75% was released from the Ti50-G during the same time period. In the time interval of 13 h to 120 h, the average GS release rates of Ti30-G and Ti50-G were about 5.6 μg/h and 5.8 μg/h, respectively. The former was about 1/35 of the release rate at the burst release stage, while the latter was about 1/20 of the release rate at its initial stage. After 120 h, there was no more GS release from the Ti30-G composite. After 168 h, there was no more GS release from the Ti50-G composites. At the initial burst release stage and the slower release stage, the concentration of GS released in PBS is higher than the minimum effective concentration for GS against most bacteria (1–4 mg/ml) [45]. It indicated that this GS-loaded Ti-G has 168 h antibacterial effect at least. The relationship between GS release rates of GS-loaded Ti-G and the immersion time in the PBS was shown in Fig. 10. At the initial burst release stage, the maximum GS release rates of Ti30-G and Ti50-G were 911 μg/h and 591 μg/h, respectively. The drug release from gelatin has
Fig. 8. Antibacterial test of GS-loaded Ti-G after 24 h.
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three kinds of mechanism: diffusion, gelatin dissolution and gelatin degradation [46]. The GS release occurred mainly following a diffusion mechanism. At the initial stage GS released from the surface and the accessible regions. The Ti-G composites with larger porosity would have more accessible surface area, thus provided a higher GS release rate. In addition, the higher GS concentration loaded in the Ti-G composite would lead to a larger GS release rate, because of a larger diffusion driving force at higher GS content. So that, the initial GS release rate was remarkably higher, and the GS release rate from Ti30-G was reasonably higher than that from Ti50-G (Fig. 10) because the GS-loaded Ti30-G had larger porosity and higher GS amount than the GS-loaded Ti50-G (Fig. 6(b)). At the time point of 12 h, the GS release rates of Ti30-G and Ti50-G were 63 μg/h and 38 μg/h, respectively. It was found that the GS release rate of Ti30-G tended to be lower than that from the Ti50-G after about 48 h (Fig. 10). For example, at the time point of 72 h, the GS release rates of Ti30-G and Ti50-G were 3.0 μg/h and 4.4 μg/h, respectively. At the time point of 120 h, the GS release rates became about 0.1 μg/h and 2.7 μg/h, respectively. At the slower release stage, GS mainly released from the interior regions. According to Higuchi model: Q =A ¼ 2C 0 ðDt=πÞ1=2
ð7Þ
Fig. 10. GS release rate profiles of the GS-loaded Ti-G composites.
where Q/A indicates the release of drug into solution unit area (mg/cm2), C0 indicates the initial concentration of drug (mg/ml), D indicates the diffusion coefficient of drug (cm2/min). t indicates the diffusion time of drug (min).
The Ti-G with higher porosity can load more GS. So the initial concentration (C0) of GS is higher than that of Ti-G with lower porosity. Thus, at the slower release stage, Ti30-G has higher released rate than Ti50-G. In this study, the GS release rate of Ti30-G approached zero after about 120 h, but that of Ti50-G approached zero after about 168 h. It is because the GS release was significantly influenced by the penetration of dissolution fluids into the gelatin matrix [47,48]. The Ti30-G with
Fig. 9. Cumulative GS release profiles from the GS-loaded Ti-G composites.
Fig. 11. Degradation of gelatin in PBS versus immersion time.
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larger porosity had more accessible surface area. Its GS release became faster and the sustained release time became shorter. So the effective GS release time of Ti50-G was longer than that of Ti30-G. 3.6. Degradation behavior of gelatin in PBS Fig. 11(a) shows the degradation of gelatin versus immersion time in PBS. In 24 h, the gelatin was almost no degradation. As time went on, the gelatin begin degrade. After 168 h, there was just 3.2% gelatin degrade. After 336 h, the degradation of gelatin reached 9.0%. Fig. 11(b) shows the relationship of GS release and gelatin degradation versus immersion time. There was not a linear relationship between the GS release and gelatin degradation. The results proved that the GS release from gelatin is less affected by the gelatin degradation. The morphologies of Ti-G after immersion in PBS phosphate buffer for 1 day (a) and 14 days (b) were shown in Fig. 12. The gelatin in TiG has almost no change after immersion in PBS phosphate buffer for 1 day. The morphology of Ti-G shows some changes after immersion in PBS phosphate buffer for 14 days. The gelatin in Ti-G was partly degraded. 4. Conclusions The p-Ti with entangled structure could be strengthened by filling gelatin into the porous structure to form titanium-gelatin composites. The density of the composites was 1.6–2.5 g/cm3, similar to that of bone. The strength and elastic modulus of the composites reached 12.9–39.4 MPa and 2.5–7.4 GPa, respectively, which were comparable
Fig. 12. Morphologies of Ti-G after immersion in PBS phosphate buffer for 1 day (a) and 14 days (b).
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to that of the cortical bone. The gelatin filled in p-Ti provided a carrier for drug loading purposes. The GS was successfully loaded into the gelatin-enhanced p-Ti composites. This GS-loaded Ti-G has obvious antibacterial efficacy. The GS sustained release time in the PBS reached about 120 h for the GS-loaded Ti30-G composite, and about 168 h for the GS-loaded Ti50-G composite. The effective GS release time from Ti50-G was longer than that from the Ti30-G. But, they all revealed that more than 50% GS released in the first 12 h. During the initial burst release, the GS release rate from Ti30-G was higher than from Ti50-G because the former with larger porosity had more accessible surface area. Their average GS release rates were approximately 197.5 μg/h and 112.5 μg/h, respectively. During the slow release stage, the GS release rate from Ti30-G tended to be lower than that from the Ti50-G. The latter was sustained longer release time.
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Gelatin-enhanced porous titanium loaded with gentamicin sulphate and in vitro release behavior Qiuyan Li a, Guo He a,b,⁎ a b
Shanghai Key Laboratory of Materials Laser Processing and Modification, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, China Collaborative Innovation Center for Advanced Ship and Deep-Sea Exploration, Shanghai 200240, China
a r t i c l e
i n f o
Article history: Received 26 January 2016 Received in revised form 15 March 2016 Accepted 18 March 2016 Available online 19 March 2016 Keywords: Titanium-gelatin composite Elastic modulus Drug release Gentamicin sulphate Bone repair
a b s t r a c t Porous titanium with entangled wire structure was enhanced by filling gelatin into the porous structure to form a titanium-gelatin (Ti-G) composite. The density of the composites was 1.6–2.5 g/cm3, similar to that of bone. The strength and elastic modulus of the composites reached 12.9–39.4 MPa and 2.5–7.4 GPa, respectively, which were comparable to that of the cortical bone. Gelatin in Ti-G composite acted as the carrier of gentamicin sulphate (GS). Antibacterial efficacy of GS-loaded Ti-G against Staphylococcus aureus strain was tested. The lack of growth of Staphylococcus aureus strains was found in Luria–Bertani (LB) medium containing GS-loaded Ti-G. GS release behavior from the GS-loaded Ti-G composites in phosphate buffer saline (PBS) was investigated. The effective GS release times from Ti30-G and Ti50-G were 120 h and 168 h, respectively. The gelatin in Ti-G was just degraded 3.2% at the immersion time of 168 h and 9.0% at 336 h. The degradation rate of gelatin was much slower than release rate of GS, indicated that the GS release from gelatin was less affected by the gelatin degradation. These Ti-G composites loaded with drug(s) are expected to be good candidates as functional bone repair materials with drug-delivery capabilities. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction As materials for bone repair, titanium and its alloy have attracted increasing attentions for its low density, good corrosion resistance and excellent biocompatibility [1–5]. However, many failures of titanium implants were attributed to the stress shielding effects caused by the mismatch between the elastic modulus of titanium and bone [6]. Research and development of porous materials is a way to solve this problem [7–9]. Porous titanium with entangled wire structure (p-Ti) has the advantages of easy to control porosity and easy for production [10,11]. However, its strength is too low for bone implants. When the porosity ranges from 50% to 70%, the yield strength and elastic modulus of p-Ti are 2.6–31.9 MPa and 0.44–0.82 GPa, respectively [11]. So it is necessary to enhance the strength of p-Ti. One of the methods is to infiltrate other biomaterial into the entangled structure, forming composite porous material. For this purpose, gelatin is a good choice for reinforcement of p-Ti.
⁎ Corresponding author at: Shanghai Key Laboratory of Materials Laser Processing and Modification, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, China. E-mail address: [email protected] (G. He).
http://dx.doi.org/10.1016/j.matdes.2016.03.103 0264-1275/© 2016 Elsevier Ltd. All rights reserved.
Gelatin is a natural polymer material derived from the hydrolysis of collagen. It has a long history of applying in antibacterial agents [12,13], anti-inflammatory drugs [14,15] and so on [16–18]. Gelatin has the advantages of good biocompatibility, good biodegradability, non-toxic and low antigenicity [19–23]. What's more important, the porous gelatin can be used as the carrier of antibiotics [24]. The utilization of antibiotic delivery systems is an effective measure to prevent wound infections. This method has the advantages of high local concentration, less dosage, low toxic and side effect [12,25,26]. Gentamicin sulphate (GS) is an aminoglycoside broad-spectrum antibiotic against Escherichia coli, Proteusbacillus vulgaris, Pseudomonas aeruginosa, Pneumobacillus, Salmonella bacteria, Shigella dysenteriae, Staphylococcus aureus and so on [27–29]. The bacterial resistance of gentamicin sulphate is lower than that of other aminoglycosides. So the gentamicin sulphate is widely used in the treatment of sepsis, respiratory infections, suppurative peritonitis, biliary infection, intracranial infection, urinary tract infections and infected lacerations [30–33]. In this paper, we used gelatin to reinforce p-Ti by forming p-Tigelatin (Ti-G) composite that was used as the carrier of GS. The antibacterial test showed that the GS-loaded Ti-G has obvious antibacterial efficacy. GS release behavior from the GS-loaded Ti-G materials in phosphate buffer saline (PBS) was investigated. Experimental results show that gelatin obviously enhanced the yield strength and elastic
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modulus of p-Ti. The elastic modulus of Ti30-G is up to 2.5 GPa, close to that of cortical bone (3–30 GPa) [34,35]. The elastic modulus of Ti50-G is up to 7.4 GPa, reaching the level of that of cortical bone. Its effective GS release time reached 168 h. 2. Materials and methods 2.1. Materials A commercial pure Ti wire (99.9% purity) with the diameter of 0.27 mm was used as raw material. The preparation of p-Ti was described in the previous paper [11]. In the process of sample preparation, the volume fractions of titanium were controlled by controlling the weight of titanium wire. It was calculated by the following formula: mTi ¼ V V Ti ρTi
ð1Þ
where VTi indicates the volume fraction of titanium wire; mTi indicates the weight of titanium wire; ρTi indicates the density of titanium wire; and V indicates the volume of sample. In this study, p-Ti has three kinds of porosity (70%, 60% and 50%). Sizes of compression samples and drug-loaded samples are Ø10 × 20 mm2 and Ø5 × 10 mm2, respectively. The gelatin and GS were bought from Aladdin Industrial Corporation. The gelatin water solution (25%) was prepared in the water bath at 100 °C. When the gelatin water solution was cooled to 60 °C, the pTi samples were immersed in it. A sustained ultrasonic oscillation was applied until the gelatin water solution cooled to 30 °C. Then the p-Ti samples filled with frozen gelatin was put in a refrigerator for 12 h at − 20 °C, and then put in a lyophilizer for 24 h at − 40 °C and 1.8 × 10− 2 mbar. After lyophilization, a 2% glutaraldehyde solution was used to crosslink the Ti-G for 6 h. After crosslinked, the Ti-G samples were washed in flowing water, and then cleared away the residues of glutaraldehyde by using firstly a 5% sodium borohydride solution and then pure water. Finally, the Ti-G samples were lyophilized again to dispel the residual water in the composite. After that, the Ti-G samples were fully immersed in a 50 mg/ml GS solution for 24 h at 4 °C for drug loading. The drug-loaded samples were then lyophilized again to obtain the gelatin-enhanced p-Ti materials with the gentamicin sulphate delivery system. The amount of GS loaded in Ti-G sample could be roughly estimated by the following formula: m ¼ ðm2 −m1 Þ C
ð2Þ
where m1 indicates the weight of the Ti-G sample. m2 indicates the weight of the Ti-G sample after immersion in the GS solution for 24 h. C indicates the concentration of the GS solution. Since the gelatin is absorbent to water, the porosity in GS-loaded TiG sample could be treated as the amount of water evaporation during lyophilization. It could be calculated by the following relation: P ¼ ðm2 −m3 Þ=V 100%
ð3Þ
where m3 indicates the weight of the GS-loaded Ti-G sample after lyophilized. P indicates the porosity of GS-loaded Ti-G sample. 2.2. Morphology and phase analysis The morphologies of the as-prepared Ti-G materials were observed by optical microscopy and scanning electron microscope (SEM). Information about the gelatin, GS, and GS-loaded gelatin was obtained by Fourier transform infrared spectrometer (FTIR, Nicolet 6700, Thermo Fisher, USA) analysis. The tests were performed at room temperature with wave number ranging from 4000 cm−1 to 500 cm−1.
2.3. Compression tests The compressive properties of Ti-G materials were evaluated by Zwick AG-100KN testing machine. The tests were conducted under displacement control with a cross-head speed of 1 mm/min. The average of three measurements was taken as the value. The yielding strength was determined according to the stress–strain curves, as described in the previous paper [36]. The elastic modulus of the Ti-G materials was calculated according to the slope of elastic stage of the stress–strain curve. 2.4. Test of antibacterial efficacy of GS-loaded Ti-G Antibacterial efficacy of GS-loaded Ti-G was tested using the Staphylococcus aureus strain. Inocula of bacterium were grown in Luria–Bertani (LB) medium. The Ti-G and GS-loaded Ti-G were put on the LB medium. During a 24 h incubation period (shaking: 35 rpm at 37 °C), LB medium samples from each experiment were tested for bacterial growth by measuring the bacteria inhibiting loop. 2.5. In vitro study of the release of gentamicin sulphate An in vitro method was employed to investigate the GS release behavior from the drug-loaded Ti-G materials. The sample was put in 10 ml PBS at pH 7.4 in a test tube for 14 days at 37 °C. The dissolution medium was collected at the time of 0.5 h, 1 h, 2 h, 4 h, 12 h, 24 h, 3 days, 7 days, 10 days, and 14 days. At the scheduled time, 3 ml of the dissolution medium were taken out from the test tube with pipette. The same volume of fresh PBS was added to keep the total volume of the release medium at 10 ml. GS release measurements were carried out using UV spectrophotometer (EV300, US Thermo Fisher) by Zhang's method [37]. Briefly, 1 ml GS solution, 1 ml isopropanol and 1 ml ophthaldialdehyde reagents reacted for 30 min at room temperature. The absorbance, which corresponds to the GS concentration, was then measured at 332 nm, at which the GS-phthaldialdehyde complex shows a maximum absorbance. These experiments were carried out in triplicate. Furthermore, calibration curve was made for each set of measurements and determined by taking absorbance versus GS concentration between 20 μg/ml and 400 μg/ml as parameters. 2.6. Test of gelatin degradation in PBS The gelatin degradation was tested in vitro. Ti-G samples were put in 10 ml PBS at pH 7.4 in a test tube for 14 days at 37 °C. At the time of 1 day, 3 days, 7 days, 10 days, and 14 days, three Ti-G samples were took out, respectively. The loss of gelatin was estimated by weighing after the Ti-G lyophilized. The average of three values was taken as the final value. 3. Results and discussion 3.1. Morphological characterization of the gelatin-enhanced p-Ti Morphology of as-prepared p-Ti was shown in Fig. 1(a), which was formed through titanium wires interweaving [10]. The pores distributed between titanium wires. In the case of the p-Ti with entangled wire structure, the pore size distribution and the average pore size mainly depend on the porosity. In this study, p-Ti materials with porosities of 70%, 60% and 50%, respectively, were prepared, which correspond to the Ti wire volume fractions of 30%, 40% and 50%, respectively. Thus, the three p-Ti materials were named as p-Ti30, p-Ti40 and p-Ti50, respectively. In the case of p-Ti30, the maximum pore diameter was about 500 μm and the average pore diameter was about 150 μm [11]. As the Ti wire volume fraction increases (the porosity decreases), the maximum pore diameter and the average pore size decrease. In other words, as the Ti wire volume fraction increased from 30% to 50%, the density of the p-Ti materials increased from 1.41 g/cm3 to 2.32 g/cm3.
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Fig. 1. As-prepared p-Ti (a) and gelatin-enhanced p-Ti (b).
After the p-Ti was filled with gelatin water solution, the porosity disappeared. When the water was evaporated during lyophilization, the porosity appeared again. But its value became smaller because the solid gelatin was left and embedded between the Ti wires as shown in Fig. 1(b) and Fig. 2(a). On the sections the embedded gelatin among the Ti wires was evident as shown in Fig. 2(b). Morphology of gelatin after lyophilization process is shown in Fig. 3. After lyophilization, there are many pores on the surface of gelatin. The pore size ranges from 2 μm to 30 μm. This porous gelatin can be used as the carrier of antibiotics. At the same Ti wire volume fraction, the density of Ti-G composites was reasonably larger than that of p-Ti materials. As the Ti wire volume fraction increased from 30% to 50%, the density of the Ti-G composites increased from 1.62 g/cm3 to 2.50 g/cm3. Since the density of natural bone is in the range from 1.8 to 2.1 g/cm3 [38], the Ti-G composites with about 34–41% Ti wire volume fraction would exhibit the density just in the same range as that of the bone as shown in Fig. 4. Compared
Fig. 2. Morphology of the Ti-G (a) and the section morphology of the Ti-G (b).
with the current mainstream bone implant materials such as Ti alloy (4.4–4.5 g/cm3), stainless steel (7.9–8.1 g/cm3) and Co-Cr alloy (8.3– 9.2 g/cm3) [38], the Ti-G composites have more close values in density with that of the bone. The low density of implant material can reduce the load of bone, and then decrease the risk of the implant failure. 3.2. Mechanical evaluation of the gelatin-enhanced p-Ti The compressive stress-strain curves of Ti-G composites exhibited typical three-stage deformation pattern, i.e., initial elastic deformation, then pseudo-plateau stress after yielding, and followed by a final densification region as shown in Fig. 5(a). It was evident that the strength of p-Ti materials was significantly enhanced after filled with gelatin. For example, when the titanium wire volume fraction was 30%, the yield strength of the p-Ti material was 5.2 MPa. The gelatin-enhanced p-Ti
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Fig. 3. Morphology of gelatin after lyophilization process.
material (i.e., Ti30-G composite) exhibited 12.9 MPa yield strength, that exceeded two times of the value of the non–enhanced p-Ti30 material. When the titanium wire volume fraction was 50%, the yield strength of the Ti50-G composite reached 39.4 MPa, that was about two times of the value of the non–enhanced p-Ti50 material (its yield strength was 21.8 MPa). The elastic modulus of the gelatin-enhanced p-Ti materials were evaluated directly from the stress-strain curves, which were 2.5 GPa for Ti30-G, 3.9 GPa for Ti40-G, and 7.4 GPa for Ti50-G. Compared with other porous entangled Ti materials that were enhanced by various methods [11,39,40], the gelatin-enhanced p-Ti materials had demonstrated their superiority in stiffness as shown in Fig. 5(b). It is known that the elastic modulus of cancellous bone is in the range of 0.01– 3 GPa [41,42], and that of cortical bone is in the range of 3–30 GPa [34, 35]. The Ti30-G composite investigated was close to the cortical bone in stiffness. The Ti40-G and Ti50-G composites exhibited the elastic modulus reached that of the cortical bone. The matched stiffness of the implanted materials would avoid stress shielding effect, and reduce the risk of implant failure.
Fig. 5. Compression stress-strain curves of the p-Ti materials and the Ti-G composites (a) and the elastic moduli of the Ti-G composites (b). Some data for the p-Ti enhanced by other methods were plotted for comparison.
3.3. Characterization of the GS-loaded Ti-G composites Since the gelatin is absorbent to GS solution, the drug-loading can be achieved by simple immersion in the GS solution. The amount of GS loaded can be directly calculated from the sample weights as shown in Fig. 6(a). When the dimension of the samples was Ø5 × 10 mm2, the amounts of the GS loaded were 3.65 mg for Ti30-G, 3.21 mg for Ti40-G, and 2.61 mg for Ti50-G. The real porosities of the Ti30-G, Ti40G and Ti50-G samples became 48.8%, 42.0% and 34.1%, respectively. With these data, two linear relationships between the real porosity (P) and Ti wire volume fraction (VTi), or, between the amounts of the GS loaded (mGS) and Ti wire volume fraction (VTi) could be roughly established (Fig. 6(b)): P ¼ 71:4−74:0V Ti
R ¼ 0:9956
mGS ¼ 5:33−5:40V Ti
Fig. 4. Densities of the p-Ti materials and the Ti-G composites with various titanium wire volume fractions.
R ¼ 0:9918
ð4Þ ð5Þ
where R is the correlation coefficient. Although the real porosities of the p-Ti materials became smaller after filled with gelatin, they were physiologically changeable. As the gelatin is degradable in the human body environment [12,13], it would gradually free extra space after implantation. The larger porosity was beneficial to the bone cell ingrowth and prosthesis fixation [38,43,44].
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and the amide group types III (1238 cm−1). The characteristic peaks of GS were the strong absorption peak of SO2− (1124 cm−1) and the 4 −1 2− medium absorption peak of SO4 (618 cm ). The GS-loaded Ti-G composite exhibited two peaks at 1107 cm−1 and 618 cm−1, which were the characteristic peaks of the SO42− in the GS. 3.4. Antibacterial efficacy of GS-loaded Ti-G The antibacterial efficacy of GS-loaded Ti-G in Luria–Bertani (LB) medium was shown in Fig. 8. After 24 h there was no bacteria inhibiting loop around Ti-G sample, but a distinct bacteria inhibiting loop around GS-loaded Ti-G sample. This GS-loaded Ti-G material seems to be sufficient to avoid the bacteria growth and thus reduce the frequency of infections after this biomaterial implanted. 3.5. GS release behavior from the GS-loaded Ti-G composites The absorbance of GS was scanned over the range of 190–400 nm, by a UV spectrophotometer (EV300, US Thermo Fisher). The maximum peak-absorption wave length of GS was observed at 332 nm. The linear relationships were established as standard curves between absorbance (A) and GS concentration (C) at the maximum peak-absorption wave length. The established standard curve was expressed as: A ¼ 0:01023 þ 0:00115C
Fig. 6. Weights of the Ti-G samples before and after immersion in GS solution 24 h, and weights after lyophilization (a) and the amounts of the loaded GS and the porosities of GS-loaded Ti-G composites (b).
To validate the GS loaded in the gelatin, a piece of GS-loaded gelatin was taken from the center of the GS-loaded Ti-G composite as the specimen for the FTIR analysis. The gelatin and GS were also analyzed by FTIR for comparison. The recorded patterns of three materials were plotted in Fig. 7. The characteristic peaks of gelatin were the amide group types I (1645 cm− 1), the amide group types II (1542 cm−1)
Fig. 7. FTIR analysis results of the gelatin, GS and GS-loaded gelatin.
R ¼ 0:9987 n ¼ 8:
ð6Þ
Based upon the obtained standard curves, the GS release profile was obtained from the absorbance of the sampling taken at the prefixed interval of time, as shown in Fig. 9. The curves could be divided into three stages: the burst release stage, the slow release stage and the final stage. The initial burst release was observed within the first 12 h, during which the GS release from Ti30-G composite was about 2370 μg, about 65% of the total amount loaded onto the composite. The GS release from Ti50-G composite was about 1350 μg, about 52% of the total amount loaded. In this initial stage, the average GS release rates of Ti30-G and Ti50-G were about 197.5 μg/h and 112.5 μg/h, respectively. The overall GS release from the Ti30-G was estimated to be about 82% in just 120 h, while about 75% was released from the Ti50-G during the same time period. In the time interval of 13 h to 120 h, the average GS release rates of Ti30-G and Ti50-G were about 5.6 μg/h and 5.8 μg/h, respectively. The former was about 1/35 of the release rate at the burst release stage, while the latter was about 1/20 of the release rate at its initial stage. After 120 h, there was no more GS release from the Ti30-G composite. After 168 h, there was no more GS release from the Ti50-G composites. At the initial burst release stage and the slower release stage, the concentration of GS released in PBS is higher than the minimum effective concentration for GS against most bacteria (1–4 mg/ml) [45]. It indicated that this GS-loaded Ti-G has 168 h antibacterial effect at least. The relationship between GS release rates of GS-loaded Ti-G and the immersion time in the PBS was shown in Fig. 10. At the initial burst release stage, the maximum GS release rates of Ti30-G and Ti50-G were 911 μg/h and 591 μg/h, respectively. The drug release from gelatin has
Fig. 8. Antibacterial test of GS-loaded Ti-G after 24 h.
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three kinds of mechanism: diffusion, gelatin dissolution and gelatin degradation [46]. The GS release occurred mainly following a diffusion mechanism. At the initial stage GS released from the surface and the accessible regions. The Ti-G composites with larger porosity would have more accessible surface area, thus provided a higher GS release rate. In addition, the higher GS concentration loaded in the Ti-G composite would lead to a larger GS release rate, because of a larger diffusion driving force at higher GS content. So that, the initial GS release rate was remarkably higher, and the GS release rate from Ti30-G was reasonably higher than that from Ti50-G (Fig. 10) because the GS-loaded Ti30-G had larger porosity and higher GS amount than the GS-loaded Ti50-G (Fig. 6(b)). At the time point of 12 h, the GS release rates of Ti30-G and Ti50-G were 63 μg/h and 38 μg/h, respectively. It was found that the GS release rate of Ti30-G tended to be lower than that from the Ti50-G after about 48 h (Fig. 10). For example, at the time point of 72 h, the GS release rates of Ti30-G and Ti50-G were 3.0 μg/h and 4.4 μg/h, respectively. At the time point of 120 h, the GS release rates became about 0.1 μg/h and 2.7 μg/h, respectively. At the slower release stage, GS mainly released from the interior regions. According to Higuchi model: Q =A ¼ 2C 0 ðDt=πÞ1=2
ð7Þ
Fig. 10. GS release rate profiles of the GS-loaded Ti-G composites.
where Q/A indicates the release of drug into solution unit area (mg/cm2), C0 indicates the initial concentration of drug (mg/ml), D indicates the diffusion coefficient of drug (cm2/min). t indicates the diffusion time of drug (min).
The Ti-G with higher porosity can load more GS. So the initial concentration (C0) of GS is higher than that of Ti-G with lower porosity. Thus, at the slower release stage, Ti30-G has higher released rate than Ti50-G. In this study, the GS release rate of Ti30-G approached zero after about 120 h, but that of Ti50-G approached zero after about 168 h. It is because the GS release was significantly influenced by the penetration of dissolution fluids into the gelatin matrix [47,48]. The Ti30-G with
Fig. 9. Cumulative GS release profiles from the GS-loaded Ti-G composites.
Fig. 11. Degradation of gelatin in PBS versus immersion time.
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larger porosity had more accessible surface area. Its GS release became faster and the sustained release time became shorter. So the effective GS release time of Ti50-G was longer than that of Ti30-G. 3.6. Degradation behavior of gelatin in PBS Fig. 11(a) shows the degradation of gelatin versus immersion time in PBS. In 24 h, the gelatin was almost no degradation. As time went on, the gelatin begin degrade. After 168 h, there was just 3.2% gelatin degrade. After 336 h, the degradation of gelatin reached 9.0%. Fig. 11(b) shows the relationship of GS release and gelatin degradation versus immersion time. There was not a linear relationship between the GS release and gelatin degradation. The results proved that the GS release from gelatin is less affected by the gelatin degradation. The morphologies of Ti-G after immersion in PBS phosphate buffer for 1 day (a) and 14 days (b) were shown in Fig. 12. The gelatin in TiG has almost no change after immersion in PBS phosphate buffer for 1 day. The morphology of Ti-G shows some changes after immersion in PBS phosphate buffer for 14 days. The gelatin in Ti-G was partly degraded. 4. Conclusions The p-Ti with entangled structure could be strengthened by filling gelatin into the porous structure to form titanium-gelatin composites. The density of the composites was 1.6–2.5 g/cm3, similar to that of bone. The strength and elastic modulus of the composites reached 12.9–39.4 MPa and 2.5–7.4 GPa, respectively, which were comparable
Fig. 12. Morphologies of Ti-G after immersion in PBS phosphate buffer for 1 day (a) and 14 days (b).
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to that of the cortical bone. The gelatin filled in p-Ti provided a carrier for drug loading purposes. The GS was successfully loaded into the gelatin-enhanced p-Ti composites. This GS-loaded Ti-G has obvious antibacterial efficacy. The GS sustained release time in the PBS reached about 120 h for the GS-loaded Ti30-G composite, and about 168 h for the GS-loaded Ti50-G composite. The effective GS release time from Ti50-G was longer than that from the Ti30-G. But, they all revealed that more than 50% GS released in the first 12 h. During the initial burst release, the GS release rate from Ti30-G was higher than from Ti50-G because the former with larger porosity had more accessible surface area. Their average GS release rates were approximately 197.5 μg/h and 112.5 μg/h, respectively. During the slow release stage, the GS release rate from Ti30-G tended to be lower than that from the Ti50-G. The latter was sustained longer release time.
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