Magnesium-zinc scaffold loaded with tetracycline for tissue engineering application: In vitro cell biology and antibacterial activity assessment
E. Dayaghi, H.R. Bakhsheshi-Rad, E. Hamzah, A. Akhavan- Farid, A.F. Ismail, M. Aziz, E. Abdolahi
Reference: MSC 9631
To appear in: Materials Science & Engineering C
Received date: 27 September 2017
Revised date: 25 May 2018
Accepted date: 3 April 2019
Please cite this article as: E. Dayaghi, H.R. Bakhsheshi-Rad, E. Hamzah, et al., Magnesium-zinc scaffold loaded with tetracycline for tissue engineering application: In vitro cell biology and antibacterial activity assessment, Materials Science & Engineering C, https://doi.org/10.1016/j.msec.2019.04.010
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Magnesium-zinc scaffold loaded with tetracycline for tissue engineering application: In vitro cell biology and antibacterial activity assessment
E. Dayaghi1, H.R. Bakhsheshi-Rad1,2,*, E. Hamzah2, A. Akhavan-Farid3, A.F. Ismail4, M. Aziz4, E. Abdolahi1
1Advanced Materials Research Center, Department of Materials Engineering, Najafabad Branch, Islamic Azad University, Najafabad, Iran
2Department of Materials, Manufacturing and Industrial Engineering, Faculty of Mechanical Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor Bahru, Johor, Malaysia
3Department of Mechanical, Materials and Manufacturing Engineering, University of Nottingham Malaysia Campus, 43500 Semenyih, Malaysia
4Advanced Membrane Technology Research Center (AMTEC), Universiti Teknologi Malaysia, 81310 Skudai, Johor Bahru, Johor, Malaysia
Recently, porous magnesium and its alloys are receiving great consideration as biocompatible and biodegradable scaffolds for bone tissue engineering application. However, they presented poor antibacterial performance and corrosion resistance which limited their clinical applications. In this study, Mg-Zn (MZ) scaffold containing different concentrations of tetracycline (MZ-xTC, x=1, 5 and 10%) were fabricated by space holder technique to meet the desirable antibacterial activity and corrosion resistance properties. The MZ-TC contains total porosity of 63–65% with pore sizes in the range of 600–800 µm in order to accommodate bone cells. The MZ scaffold presented higher compressive strength and corrosion resistance compared to pure Mg scaffold. However, tetracycline incorporation has less significant effect on the mechanical and corrosion properties of the scaffolds. Moreover, MZ-xTC scaffolds drug release profiles show an initial immediate release which is followed by more stable release patterns. The bioactivity test reveals that the MZ-xTC scaffolds are capable of developing the formation of HA layers in simulated body fluid (SBF). Next, Staphylococcus aureus and Escherichia coli bacteria were utilized to assess the antimicrobial activity of the MZ-xTC scaffolds. The findings indicate that those scaffolds that incorporate a high level concentration of tetracycline are tougher against bacterial organization than MZ scaffolds. However, the MTT assay demonstrates that the MZ scaffolds containing 1 to 5% tetracycline are more effective to sustain cell viability, whereas MZ-10TC shows some toxicity. The alkaline phosphatase (ALP) activity of the MZ-(1-5)TC wasconsiderably higher than that of MZ-10TC on the 3 and 7 days, implying higher osteoblastic differentiation. All the findings suggest that the MZ-xTC scaffolds containing 1 to 5% tetracycline is a promising candidate for bone tissue healing due to excellent antibacterial activity and biocompatibility.
Keywords: Mg composite scaffold; Drug delivery; Antibacterial activity; Biocompatibility; Bioactivity
Porous scaffold plays a cardinal role in tissue engineering through assisting osseointegration, body fluid transportation, drug delivery and cutting down on implant loosening [1-3]. Particularly, metallic scaffolds are widely surveyed for orthopedic applications for their desirable mechanical stability [4,5]. Foremost among the various elements used in scaffold fabrication, magnesium and its alloys have been largely used in orthopedics reconstruction due to their close density to the human cortical bone (1.75 g/cm3), while the density of the most common of Ti alloys, Ti6Al4V, is 4.47 g/cm3 . As for biocompatibility, magnesium ions that are abundant in human body take part in many of metabolic functions and biological mechanisms.
Magnesium consists nearly 35 g of the human body weighing 70 Kg and the daily need for the component is approximately 375 gr per day . In addition, Mg and its alloys potentially degrade in physiological environment, resulting in the elimination of a second surgery and the side effects of implant removal after bone healing . However, Mg alloys are suffer from low corrosion resistance and high degradation rate, which resulted in a loss of mechanical strength before completion of bone healing and thus limits its potential applications [9, 10]. Hence, to tackle the problem, mechanical alloying as a promising technique has been employed to enhance the biological corrosion resistance of magnesium . In the present study, mechanical strength and corrosion resistance were raised by addition of Zn, in order to fulfill the clinical requirements [11, 12]. As one of the most common nutritional elements, Zn is omnipresent in all human body tissues . Zn is a frequent alloying element to reinforce mechanical strength and corrosive resistance of magnesium alloys with the solubility rate of 6.2 wt.% . In this context, it has been proposed  that Mg-6wt.% Zn demonstrates suited mechanical stability with tensile strength and elongation of 279 MPa and 18.8% at room temperature, respectively. However, the insufficient antibacterial characteristic of Mg-based composites has restricted their applicationsand brought about some post-operation hurdles .
The number of bone infections resulted from multi-resistant gram-positive and gram-negative organisms was noticeable enough to draw great attention to antibacterial effect at the surgical site [17-19]. Typically, drug administration can warranty antibacterial effect to some extent, whereby drugs are absorbed in bloodstream [20, 21]. However, this delivery route underlies the appearance of some drawbacks including toxicity and insufficient drug delivery to the target tissue. Moreover, the distribution of an antibacterial agent directly to the surgical spot brings about an alternative treatment . Therefore, in the current study, the MZ scaffolds were doped with antibiotics such as tetracycline, which is a broad-spectrum antibiotic. Since tetracycline is an effective antibiotic against gram-positive and gram-negative microorganisms, it has been already used in some scaffold formulations [22, 23]. Until now, numerous researches [24, 25] have been conducted to cast light on the mechanical and biological of Mg-based scaffolds properties. However, up to now, no study has been reported to address the drug delivery system development which relies on antibacterial Mg-based bone scaffolds.
Accordingly, in the present study, Mg-Zn alloying scaffolds were fabricated to meet the desirable mechanical and corrosion resistance properties. Then they were loaded with different percentages of tetracycline in order to fulfill the antibacterial and drug delivery expectations.
2. Materials and methods
2.1. Materials and preparation
The process commences by combining pure magnesium powder (purity ≥99%, 5–20 µm particle size) with 6%.wt zinc powder (99.5% purity, particle size <80 μm), purchased from SIGMA- ALDRICH. The mixing process was followed by drying in which Mg-6.0 wt.%Zn composite powders, denoted as MZ, were dried in a vacuum dry oven at 220 °C for 10 h. To ensure well- proven mixing and refine reduction of particle size the dried mixture was mixed again in a planter ball-mill for 3 h in an argon atmosphere. The space holder method was implemented for creation of pore networks. For this purpose, spherical carbamide particles (CO(NH2)2 ; SIGMA- ALDRICH) as the spacer, in the range of 600-800 μm were mixed with MZ nano composite powders in the weight ratio of 65:35. The powder was uniaxially pressed in the form of a cylindrical green compact under 200 MPa at a crosshead speed rate of 2.0 mm/min with a size of 10mm (diameter) × 15mm (thickness). The heat treatment process consisted of two stages. First, the green MZ compacts were heated to 180°C for 2 h to burn out the spacer particles, and thenthe temperature rose to 580 °C for 2 h at a heating rate of 5 °C/min in a tube furnace under the protection of argon gas. Tetracycline as an antibiotic with different concentrations of 10, 50, and 100 mg was dissolved in 100 ml of distilled water to prepare three kinds of tetracycline hydrochloride solutions (TC; C22H24 N2O8·HCl; Sigma-Aldrich) labeled as MZ-1TC, MZ-5TC, and MZ-10TC. The loading process was carried out by immersing MZ scaffolds in each of the tetracycline hydrochloride solutions which had been formerly vacuumed for 5 min. The freeze drying method was employed to dry overnight the tetracycline doped MZ scaffolds according to the principles offered in Ref. . Schematic presentation of this method is shown in Scheme 1. The scaffolds porosity was accounted according to Archimedes principle and the procedures suggested in Ref. . In equation (1), the true density of MZ scaffold based on the percentage of Mg and Zn was calculated. In equation (2), the total porosity consisting of interconnected pore network was measured where Wd and Ws account for the weight of sample in dry and water saturated conditions, respectively, and ρ is the true density of MZ (1.987 g/cm3).
True density = wt.%Mg . ρMg + wt.%Zn . ρZn (1)
Total porosity = 1–Wd / ρ(Wd-Ws) × 100 (2)
2.2. Mechanical properties
To measure the compressive strength before and after 3 days of immersion in the SBF solution, the cylindrical green compact of scaffolds with 10 mm diameter and 15 mm height was pressed under the Instron-5569 universal testing machine at a crosshead speed of 0.5 mm/min and a load cell of 10 kN at room temperature. At least three scaffolds of each type were examined to assess the reproducibility of test results.
2.3. In vitro corrosion behavior
As for electrochemical measurement, the MZ-TC scaffolds with a surface area of 1 cm2 were prepared to be measured by PARSTAT 2263 potentiostat/galvanostat (Princeton Applied Research). They were submerged in a three-electrode cell filled with Kokubo SBF at 37 °C and with a pH value of 7.44 to meet the requirements of Ref. . The changes in pH of the SBF were monitored throughout the immersion test (24 h). In order to study the bioactivity behavior, each of the MZ-TC scaffolds was soaked in 100 ml simulated body fluid (SBF) under the
condition of 37 °C for 7 days. After the period, they were taken off from the SBF cups and rinsed with distilled water and dried in the open air.
2.4. In vitro antibacterial activity
Gram-positive Staphylococcus aureus (S. aureus, ATCC 12600) and gram-negative Escherichia coli (E. coli, ATCC 9637) bacteria were utilized to evaluate the antibacterial attitude of MZ-TC scaffolds in agreement with disc diffusion antibiotic sensitivity and liquid medium microdilution requirements. The antibiotic gentamicin (10 μg /disc) was employed as the positive control. The antibacterial effect of MZ scaffolds incorporating different amounts of tetracycline was evaluated by assisting inhibition zone (IZ) . An overnight culture of E. coli and S. aureus was diluted with broth medium to reach the absorbance value of 0.1-0.2 at 625 nm. After the completion of the bacterial culture, the specimens with the weight of 10mg were added to the suspension and incubated at 37° and they were shaken on a shaker platform at the rate of 100 rpm/min. UV-vis spectrophotometer (Lambda 25) was used to determine the absorbance value. For this purpose, at 4 and 24 h intervals, the absorbance value of all tested solutions was read at 625 nm to calculate the bacterial inhibition according to the following equation, where Ic indicates the bacterial suspension absorbance value and Is demonstrates the bacterial value of each suspension including one of the three different MZ-TC scaffolds.
Bacterial inhibition (%) = Ic–Is / Ic × 100 (3)
For studying the in-vitro TC release of MZ-TC scaffolds, the scaffold samples were soaked in a phosphate-buffered saline (PBS) and maintained at 37°C. At each regular interval, 1mL of the solution was substituted with a fresh one. Finally, the content of TC was measured by ultraviolet- visible spectrophotometry (UV- spec), in which the peak of 345 nm was contrasted to the standard calibration curve.
2.5. Osteoblastic cytocompatibility
The in vitro cytotoxicity of MZ-TC scaffolds was determined by indirect 3-(4,5-dimethylthiazol
-2-yl)-2,5-diphenyltetra-zolium-bromide (MTT, Sigma, Saint Louis, USA) assay based on the extraction method. Briefly, the culture medium was added to the scaffolds (5mg) and incubated at 37°C for 1 and 2 days. MG-63 cells (1× 104 cells/well) were seeded on the MZ-TC scaffolds. Afterward, the cell medium was refreshed with 1 and 2-day extracts. The medium was removed after another 24 h and 100 μL of MTT agents (0.5 mg/mL in PBS) was inoculated into each well and kept in the incubator for 4 h. After 4h, 100 µl of DMSO was inserted to the well to dissolve the formazan crystals. Finally, the absorbance was read at 545 nm by using an ELISA Reader (Stat Fax-2100, Miami, USA) and normalized by free scaffolds culture medium as a control group. Nuclear staining with DAPI (4΄, 6-diamidino-2-phenylindole, blue fluorescence in live cells) was performed in order to examine the MG-63 cell proliferation on the MZ-TC scaffolds under fluorescence microscopy.
Alizarin red (AR) staining was conducted to demonstrate the influence of the MZ-TC scaffolds on calcium deposition of MG-63 cells. After a 7 days osteogenic culture, the cells cultured with the scaffolds extracts were diluted at various ratios and the culture medium comprising the scaffolds extract and 50 μM were initially fixed in 4% paraformal-dehyde and subsequently stained in 1% AR solution for 30 min at 37 °C. The optical density (OD) was measured on a microplate reader (Thermo Scientific Multiskan GO, USA) at 612 nm to determine the cell viability. Cell attachment to various scaffolds was evaluated by acridine orange fluorescent staining for 72 h. The scaffolds were placed in 95% ethanol and stained in 4×10-4 mg/ml acridine orange for 1 min. After rinsing with PBS, the scaffolds were evaluated under a fluorescent microscope, and the number of attached MG-63 cells was measured in a 2 mm2 in random areas. The ALP activity assay was performed on the third and seventh days to assess the influence of tetracycline on the early osteogenic differentiation of M-G63 cells. The cells were seeded at a concentration of 104 cells/ml placed individually in a 24-well plate. The cells were left to grow for different days at 37 ºC in a humidified atmosphere of 5% CO2 according to Ref. .
2.6. Morphological characterization
Fourier Transform Infrared spectroscopy (FTIR), which was adjusted in the spectral range of 4000-450 cm-1, was used to demonstrate the surface functional group MZ-TC scaffolds. In addition, scanning electron microscopy (SEM, JEOL JSM-6380LA) and transmission electron microscopy (TEM, HT7700 Hitachi, Japan) were employed to study the MZ-TC scaffolds microstructures. An X-ray diffractometer (Siemens D5000) was employed to reveal the phase components through Cu-Kα radiation (45 kV, 40 mA) over the diffraction angles (2θ) of 20–75° at a scanning speed of 4°/min. Finally, the crystallite size was determined by the Williamson– Hall method  according to the following equation:
β cot θ= 0.45λ
+ ε (4)
2 Sin θD
In the above equation, β accounts for diffraction peak width at the mid-height, λ indicates X-ray wavelength, and D is the average crystallite size (nm). Microstrain and the Bragg diffraction angle are indicated in the formula by ε and θ, respectively.
3. Results and discussion
3.1 Structural and micrographic characterization
Fig. 1a illustrates the XRD patterns of MZ composite powders, in which the two observed peaks are ascribed to α‐ Mg matrix and Mg12Zn13. The crystallite size of MZ composite powders were around 72 nm, based on the Williamson-Hall equation . Since the crystallite size of composite powders influences mechanical properties and “in vitro bioactivity" , TEM analysis was performed to ensure the former calculated crystallite size and to depict the morphology of the MZ composite powders. According to Fig.1b, the particle size is in the range of 30-100 nm which is in good agreement with the calculated particle size relying on the XRD data and the Williamson-Hall equation. It was recommended  that the composite consisting of particle size below 100 nm fulfills the mechanical and biological expectations.
The FT-IR spectra (Fig. 2) showed the characteristic peaks of TC at 1669 cm–1 and 1634 cm–1 which are related to C=O vibration of Amide I and C=O vibration of A-ring, respectively. The bands at 1535 cm–1 and 1456 cm–1 are attributed to the NH2 deformation of Amide II and C=C vibration of aromatic ring, respectively . The MZ-TC composite scaffolds presented the characteristic peaks of MZ-TC composite scaffold including the peak at 1083 cm–1 and 3696 cm–1, which is attributed to the Mg–O stretching vibration besides TC peaks.
Porosity analyses and SEM images in Fig. 3 show a negligible change in the porosity of MZ and MZ-xTC (x=1, 5 and 10%) in the range of 63-65%. According to the listed porosity for the bare and tetracycline incorporated MZ scaffolds, addition of tetracycline has no significant influence on the porosity content of these scaffolds. As for the pore size; MZ, MZ-1TC, MZ-5TC, and MZ-10TC scaffolds pore size are 670 ± 25 μm, 690 ± 28 μm, 740 ± 32 μm and 720 ± 30 μm, respectively. This implies that carbamide as the space holder agent with the particle size of 600- 800 µm can potentially reserve their initial spherical particle shape through the fabrication process. According to the former studies [1, 5], those kinds of bone scaffolds possessing interconnected pore networks beside porosity size (>300µm) demonstrate more potential tendency for cell developing and tissue growing. Furthermore, the scaffold porosity consists ofmacro pores, shown by the arrow, in the range of 100 to 250 μm which were resulted from the revolution of carbon dioxide. In relevance to the findings of Roohani et al. , the presence of macro pores is essintially required, because they facilitate the capillary in-growth and cell-matrix interactions. The image in the frame obviously indicates the presence of micro pores at the surface of the scaffolds which is required for the absorption process of the drug. As the fracture cross-section SEM image images exhibit, the presence of some necks between the pores, involving half of the cross-section porosity and with 100-200 μm and interconnected porosities indicates a suited level of interconnectivity (Fig 3e-h).
3.2 Mechanical characterization
Mechanical strength as a trade-off between the porosity and mechanical strength is a big challenge in fabrication of porous bone scaffolds. Having appropriate mechanical strength emphasizes cell attachment, cell proliferation through pores, and osteogenic behaviors. It is evidently an issue when mesoporous calcium silicate is three dimensionally printed into the scaffolds due to its amorphous feature [35-37]. It was reported [38, 39] that bone scaffolds should possess a compressive strength very close to that of the neighboring bones. Furthermore, mechanical properties of synthetic scaffolds are in close relation with their porosity, constituents, density, and grain size. In order to avoid the failure of the whole database, the compressive strength of MZ scaffolds including different amounts of tetracycline was initially measured in order to assure the insignificant influence of tetracycline on the scaffolds mechanical strength. As it was previously mentioned, the mechanical strength changes are free of adding tetracycline, because tetracycline has no significant influence on the size and shape of porosity. In contrast, by adding 6 wt.% Zn to the magnesium scaffolds, the compressive strength raised noticeably from
3.2 to 4.8 MPa versus the pure Mg scaffold which exhibited comparable compressive strength to cancellous bone (2-12 MPa)  as can be seen in Fig. 4a. According to the previous studies, the refining of grain, solution strength as well as second phase strengthening account for the compressive strength enhancement. As it was discussed earlier, 6.2 wt.% is reported  for the maximum solubility of Zn in Mg-Zn system at ambient temperature at equilibrium . Moreover, the precipitation of Mg12Zn13 in grain boundaries and α-Mg matrix may put up a barrier which blocks the boundary sliding and dislocation motion, hence elevating mechanical strength. The compressive stress–strain curves of the MZ and MZ-TC scaffolds after 72 h in the
SBF solution are presented in Fig. 4b. The compressive strength of both pure Mg and MZ-TC scaffolds declined after soaking for 72 h in the SBF solution, but the mechanical stability of the MZ-TC scaffolds stood at higher level than that of the pure Mg demonstrating a much more decline trend. It can be seen that the compressive stress of the pure Mg scaffolds increases after addition of Zn into the Mg scaffolds, the compressive strength of the immersed Mg scaffold went up noticeably from 1.8 to 3.1 MPa, respectively after 72 h immersion. In fact, when 6 wt.% Zn is added to the scaffold many Mg-Zn phases precipitated form the matrix that causes an increase in the strength through dispersion mechanism. So, Zn can be used act as a component to refine the microstructure of the Mg matrix and to augment the mechanical stability. But, no significant differences were found in the compressive strength of the MZ and MZ-TC scaffolds after incubation in the SBF, indicating that the tetracycline has no effect on porosity, nor does it change pore size of the MZ scaffolds.
3.3 Electrochemical behavior
The polarization curves of the MZ and MZ-TC scaffolds in the simulated body fluid (SBF) are depicted in Fig. 5a. The presence of Zn as a reinforcement in Mg scaffolds has been reported to increase the corrosion potential (Ecorr) of pure Mg from -1811 mVSCE to a nobler direction (-1741 mVSCE). In the present study, the MZ samples possessed a lower corrosion current density (icorr; 47.4 µA∙cm2) than pure Mg scaffold specimen (213.2 µA∙cm-2). This dichotomy is ascribed to the existence of Zn in the MZ scaffold which could settle the formation of brucite, Mg(OH)2. In addition to brucite, the aforementioned phase (brucite) can operate as a protective layer which slows down the diffusion of SBF into the underneath layers. Hence, the direct contact with the scaffold surface is interrupted. It was also reported  that Zn forms a zinc oxide layer on the surface of the SBF immersed Mg-Zn alloyed which passivates the scaffold surface. It has also been proposed  that in the SBF solution, Zn takes the place of Mg2+ cations in Mg(OH)2 forming protective layer at the surface of the alloy. Regardless of the different concentrations of tetracycline, the MZ-TC scaffolds exhibit a similar corrosion behavior in SBF and present similar Ecorr and icorr very close to that of the MZ scaffold, indicating a less significant change observed in the corrosion rate with increasing TC from 1 to 10%.
3.4 In vitro drug release and antibacterial activity
In vitro release experiment was performed in phosphate buffer solution with a pH value of 7.4 for 48h in order to assess the drug release behavior of MZ-xTC (x=1, 5 and 10%) scaffolds. According to Fig. 5b, a two-stage (bimodal) of drug-release pattern is observed. An initial rapid step released about 30-40% of the drug within 6h, followed by a sustained phase through which nearly 100% of the tetracycline was released during 16h. The first fast rate release observed initially is attributed to the water-soluble low molecular character of tetracycline. Furthermore, the small MZ-TC particle size of 74 nm leads to a high ratio of surface area to volume which can account for the initial rapid rate of drug release . Then the drug release process progressed into a more controlled fashion. Generally, the gram-positive S. aureus and gram-negative E. coil were used in order to evaluate the antibacterial activity of MZ and MZ-TC scaffolds. Figs. 6a and b exhibit the antibacterial activity of MZ-TC scaffolds during the 24 h incubation period. Fig. 6a reveals the inadequacy of the MZ scaffold antibacterial activity indicating that it is not able to completely prevent the bacterial growth on E. coil, whereas the MZ scaffolds incorporating tetracycline inhibit the bacterial growth which is more pronounced in the MZ- 10TC scaffold containing higher concentration of tetracycline. As can be seen in Fig. 6b, the diameter of zone inhibition for MZ, MZ-1TC, MZ-5TC and MZ-10TC scaffolds against E. coli was 0.68±0.12, 1.83±0.23, 2.52±0.34 and 3.84±0.32 mm, respectively. The antibacterial test performed on S. aureus shows results that are identical with the former one: the MZ scaffold less significantly inhibit bacteria growth, while the MZ-TC scaffold interrupted the bacterial growth. The inhibition zone against S. aureus was also 0.83±0.15, 2.33±0.22, 3.48±0.35 and 4.37±0.27 mm for MZ, MZ-1TC, MZ-5TC and MZ-10TC scaffolds, respectively. In this context, Ren et al
 showed that pure Mg indicated higher antibacterial activity than Mg coated with porous silicon and fluorine owing to the raise of pH value throughout its degradation in the bacterial solution. However, the result of present study exhibited antibacterial performance is more pronounced in MZ-TC compared to the MZ scaffold due to drug release during degradation process of the scaffolds. It seems clear that the scaffold with higher concentrations of TC demonstrates more bactericidal effects on S. aureus, implying that the bacterial inhibition effect is solely connected to the concentration of tetracycline. Generally, the effect of tetracycline hydrochloride is tougher against S. aureus than E. coli bacteria. It is due to the dichotomy ofcellular wall structures between E. coli as a gram-negative bacterium and S. aureus as a gram- positive bacterium .
The percentage of bacterial inhibition of all MZ and MZ-TC scaffolds are contrasted with respect to the incubation times in Figs. 6c, d. As can be seen, the bacterial inhibition of MZ-TC scaffolds increased with increasing tetracycline concentration. Regarding the antibacterial inhibition, no significant difference between the MZ-10TC and pure gentamicin was observed, recommending the close competence between the MZ-10TC and pure gentamicin. In contrast, MZ scaffolds lacking drug presented poor antibacterial activity. Therefore, tetracycline as a drug inhibits bacterial growth and the intense of antibacterial activity is directly correlated with the drug concentration.
3.5 In vitro biocompatibility
Cell attachment is presumably the most critical stage of the cell interaction with a biomaterial because it underlies other cellular activities. Fig. 7a-d depicts the fluorescence images of the cells adhesion on magnesium based composite scaffolds for osteoblasts proliferation experiments. As can be seen, the cells could adhere well to the MZ and MZ-1TC scaffolds after 2 days of culture, and the cell nuclei were thus stained with DAPI as shown in Figs. 7a,b. This implies excellent osteoblasts proliferation accommodated to the scaffold maintaining a low concentration of TC. However, the concentration of tetracycline does not align with the cell attachment, and the number of cells adhered to the MZ-TC scaffolds falls significantly as TC concentration increases to 10% (Figs. 7c,d). The cytotoxicity assay measured the viability of the cell after contact with the scaffold samples through the MTT test. Since this assay relies on the enzymes activity, it shows both the cells number and metabolic activity. In the present work, the cells viability to attach to the fabricated scaffolds decreases in the MZ scaffold containing 1-5% TC and this decline is much greater for the MZ-10TC scaffold. MZ-1TC and MZ-3TC scaffolds decreased the number of viable cells while these scaffolds were not cytotoxic. But the MZ-10TC scaffold presented some toxicity (Fig. 7e). Thus, the more drug concentration means the lower cell attachment. The viability of MG-63 cell cultured on MZ-TC scaffolds slightly weakened as the incubation time increased to the 48 days. It is due to the more release of tetracycline from the MZ-TC scaffold which weakens the cells-scaffold attachment.
Acridine orange as a versatile fluorescence dye was used to visualize the cells under fluorescent microscope. It was revealed that after culturing the cells in the day 3, the MZ-TC scaffolds containing various concentration of tetracycline presented lower cell density than that of the MZ scaffold (Fig. 8). In comparison with the scaffolds containing higher tetracycline, the cells attached on the scaffolds with lower tetracycline presented higher cell numbers after 3-day cell culture. Tetracycline appears to depress cell proliferation and in this case MZ-1TC scaffold shows the most cell proliferation in the system of MZ-TC samples after 3 days, while MZ-10TC scaffolds exhibits the opposite response. In fact, as the tetracycline concentration increases, MG- 63 cells population declines and the cell state deteriorates slightly, indicating that the MZ-1TC is beneficial to initial adhesion and growth of MG-63 cells. This fact demonstrates the non- cytotoxic delivery system to the cell media in the low- tetracycline MZ-TC extracts, and that the amount of delivered drug in this extent does not exert cytotoxic effect on the cells and its surroundings.
Alizarin red (AR) staining has been carried out to visualize the calcific deposition of the cells cultured in the scaffold extracts. The content of calcific deposition on various MZ-TC scaffolds was investigated by Alizarin red staining which turns to bright red strains when free calcium ions form precipitates with alizarin. Fig. 9a-d reveals mineralized calcium nodules on all the scaffolds as more extracellular matrix (ECM) mineralization on MZ scaffolds is found than MZ-TC scaffolds containing various concentration of tetracycline. The ECM mineralization levels on MZ-10TC and MZ-5TC are less than those on the MZ-1TC and MZ scaffolds. Particularly, MZ- 10TC shows significantly suppressed ECM mineralization. Hence, it is assumed that MZ-TC scaffolds containing lower tetracycline induces more ECM mineralized nodule formation and so enhances collagen producing. Consistent with cell staining, highest OD absorbance was detected for MZ scaffold by quantitative test (Fig. 9e). The results indicate that the calcium nodule- formation of the cells on the MZ-1TC and MZ-5TC scaffolds was significantly more than on MZ-10TC scaffold.
The ALP activities of MG-63 cells after cultivating on the MZ and MZ-TC scaffolds for 3 and 7 days are shown in Fig. 9f. The ALP activity of MZ is higher than that of MZ-TC scaffolds throughout the experimental period. MZ-1TC shows slightly higher ALP activity than MZ-5TC on both days 3 and 7 and the lowest ALP activity is observed from MZ-10TC scaffold. ALP expression suggests that the MZ-TC scaffolds containing lower tetracycline concentrationbenefits osteoblastic differentiation. A minimal decrease in the ALP activity could be related to the existence of antibiotics in the media and it is a sign of the antibiotic effect on the differentiation process . It can be observed that ALP activity of all MZ-TC scaffolds is incubation time-dependent mode, which cell differentiation in the MZ-TC scaffold extracts increases with increasing incubation time. In this study, higher level of ALP activity of MZ-TC scaffolds containing lower concentration of tetracycline extracts suggests that they are able to promote the cell differentiation.
According to the above cell tests, MZ and MZ-1TC scaffolds exhibit better cell response including cell adhesion, cell growth and differentiation. To the best of our knowledge, several reasons account for it. The dissolution of Mg ions as a degradation product from Mg matrix at suitable concentration in a medium contributes to bone regeneration. Furthermore, magnesium can increase osteogenic differentiation and growth of peristeum-drived stem cells by production of calcitonin gene-related peptide (CGRP). Zn exists in various enzymes and also is involved in many important biological functions. Meanwhile, Zn has been shown to stimulate osteogenic differentiation and prohibit osteoclasts mature. Thus, MZ and MZ-1TC scaffolds with lower corrosion rates and pH values may offer superior biocompatibility and osteogenic differentiation compared with MZ-5TC and MZ-10TC scaffolds. Likewise, Liu et al  proposed that the release of Mg2+ and Zn2+ from Mg-Nd-Zn alloy scaffold contribute to enhance in vitro osteogenic differentiation and proliferation behaviors. Wong et al  showed that the Mg/PCL porous scaffold retains better cytocompatibility in comparison with the pure PCL scaffold owing to the release of Mg ions as degradation product was able to enhance the viability of pre- osteoblasts.
3.6 In vitro bioactivity
SEM micrographs represented in Fig. 10 show the MZ and MZ-xTC (x=1, 5 and 10%) scaffolds surface incubated in SBF for 7 days. As can be observed, the surfaces of all scaffold samples include the spherical aggregates of apatite which are about 0.8–1.4 μm in diameter. Despite the different compositions of formed HA layers, indicated with arrows, they share the same morphologies of spherical aggregates. The high resolution of HA demonstrates flower-like nano flakes including hierarchical structure. The more even distribution of apatite clusters is observed in MZ in contrast to the MZ-TC scaffolds. However, in all of the specimens the morphology the apatite layers was not affected by the tetracycline concentration; instead, it increased the number of small apatite particles (Figs. 10a-d). This effect of tetracycline on the apatite layer is attributed to the nucleation rate of apatite which is greater than its growth rate. It is more plausible to rely on the presence of zinc which is reported  to enhance the heterogeneous of apatite nucleation on the surface of nanocomposite samples. In this context, it was asserted  that the apatite nucleation and growth starts at the beginning of the immersion and progresses with the extension of the immersion. According to the obtained results, the formation of apatite on the surface of the scaffolds with different concentrations of tetracycline can imply the fact that tetracycline cannot hinder the bioactivity of the MZ scaffold.
The EDS results in Fig.11a reveal the presence of the O, P, C, Mg, and Ca elements determine as the corrosion products of the MZ scaffold. The atomic ratio of Ca to P (Ca/P) was found to be almost 1.47, indicating the deposition of the calcium deficit hydroxyapatite on the surfaces of all the four models of scaffolds. Similar products including Ca, Mg, C, P, and O were observed in the MZ scaffold containing TC, suggesting that the product contains HA (Fig.11b). The XRD pattern (Fig. 11c) further confirms the result of EDX analysis since MZ and MZ-1TC scaffolds presented the HA peaks, implying its apatite formation ability in the SBF solution. Since SBF as the in vitro testing solution consists of Cl–, H2PO4- and Ca2+ , the corrosion products are likely to be Mg(OH)2 and HA. Broad Mg(OH)2 diffraction peaks can be related to the poor crystallinity of these phases.
Fig. 12a shows the pH values of the SBF solution incubating the pure Mg and MZ-TC scaffolds at different immersion times. It can be seen that the pH values of the Kokubo solution containing MZ and MZ-TC scaffolds are evidently lower than that of the pure Mg during the immersion test. The graph also depicts that the pH values of the solution for all scaffolds increased remarkably in response to increasing immersion duration. All the scaffolds present a rapid growth in the pH value in the initial stage of immersion, and then the pH enhanced moderately after 1 day. After 3 days, the pH values reached 11.23, 10.45 and 10.39 for Mg, MZ and MZ-TC scaffolds, respectively. The degradation of Mg(OH)2 lead to the rapid rise in the initial pH due to the release of OH– ions based on the following reactions:
MgO + H2O → Mg(OH)2 (5)
Mg(OH)2 → Mg2+ + 2OH– (6)
However, the pH value of MZ and MZ-TC scaffolds becomes stable in longer time. The degradation products coating on the surface of scaffolds may be the central reason for pH stabilization. Likewise, it was reported that  the rapid increase of pH in the early immersion period is due to the fact that the initial noticeable cathodic reaction took place near the scaffolds surface. Once the reactions between all the ions reach equilibrium, the SBF pH values converged at a relative stable level. However, there is no observable difference in the pH values of various MZ-TC scaffolds. In this context, Shi et al  showed that Mg substrate samples at various pH exhibited similar drug loading system and rapamycin spread at a slow release rate after the initial sudden release, indicating the pH variation in the range of 7.4 ~9.7 hardly influenced the release rate.
The average mass loss of MZ and MZ-TC scaffolds is shown in Fig. 12b, the mass losses measured from MZ-TC scaffolds are significantly lower than that from pure Mg scaffold at three time points. The above results demonstrate that alloying Mg with Zn significantly declines the degradation rate of Mg scaffold. However, the weight loss seems have no big difference between MZ and MZ-TC scaffolds, the possible principal reason for this phenomenon was that an increase in tetracycline concentration has no effect on the scaffold porosity and pore size, thus similar contact area of the scaffolds was exposed to the corrosive solution and resulting in comparable degradation rate. It can be also observed than as corrosion goes ahead, more degradation products formed on the surface of the MZ-TC scaffolds to decelerate the corrosion rate and pH rise.
In the present research, magnesium-zinc (MZ) and MZ composite scaffolds containing different tetracycline concentrations (MZ-TC) were synthesized by using the space holder method. The fabricated scaffolds were homogenous containing spherical and interconnected porosities of 600- 800 μm pore size. The addition of zinc to the magnesium scaffold enhanced the compressive strength and corrosion resistance as well. The corrosion current density fell down from 213.2 to
47.4 µA∙cm-2 and the compressive strength increased by 3.2 MPa reaching to 4.8 MPa compared with the pure magnesium scaffold. However, the presence of tetracycline in the MZ scaffolds had minimal effect on both the mechanical strength and corrosion rate. It has also proved that the MZ scaffolds loading with tetracycline are more effective to drug delivery to secure antibacterialeffects and long-term hailing. Moreover, the bioactivity of the synthesized scaffolds was examined in the SBF solution. The presence of apatite layers formed on the surface scaffolds after 7 days was granted to support the bioactivity of MZ and MZ-TC scaffolds. The existence of tetracycline in the composition of the fabricated scaffolds guarantees the antimicrobial character, as the inhibition zone grows directly with the increase of tetracycline concentration. Based on the results of this study, MZ as well as the MZ-TC scaffolds incorporating 1% and 5% tetracycline are biocompatible, whereas the more tetracycline concentration in MZ-10TC demonstrates toxicity. The development of biodegradable, bioactive and drug delivery function of MZ-(1- 5)TC makes them potential candidates in bone tissue engineering scaffolds.
The authors would like to thank the Universiti Teknologi Malaysia and Islamic Azad University for providing the facilities for this research.
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