Structural and morphological investigation of polycaprolactone multi-component scaffold containing collagen microsphere/bioglass nanoparticles

INTRODUCTION
Bone tissue engineering is the most important topics in tissue engineering. Successful bone tissue replacement is highly dependent on the used scaffold properties such as biocompatibility, bioactivity, porosity, mechanical properties, and three-dimensional architecture for migration and attachment of bone cells [1-4]. Medical advances have led to increase the life expectancy of people. Bone fractures cost 17 billion $ a year in Europe and 20 billion $ in the US. Bone tissue is one of the vital and multifunctional tissues of the body known as a major component for the adult skeleton [5-8]. Bone tissue is a source of calcium, phosphate and other ions that can be stored to be effective in stabilizing the concentration of these elements in biological environment. One of the major clinical challenges in bone tissue is to repair after disease or physical injury [9-14].
The goal of tissue engineering as a field of high-tech and interdisciplinary knowledge is to repair damaged tissues and organs with modern medical tools. Therefore, one of the important goals of porous bone scaffold design is to obtain an appropriate physical, biological and mechanical features [15-24]. In bone tissue engineering, three key factors are including stem cells (precursors), signals or morphogens, and scaffolds [25-29]. A porous substrate can create a suitable place to allow cell growth and proliferation. In this study, it was observed that addition of Ce may not have a negative effect on biocompatibility and formation of hydroxyapatite (HA). These elements can increase the mechanical properties by forming CeO2 in the synthetic compound [30-39]. Therefore, PCL scaffold as a support and 3D cell culture medium, Ce and BG as an amorphous base material used for bone tissue approaches which has multiple effects on better cell attachment and proliferation. The collagen microspheres also can be useful for bone tissue approaches that using this materials confine Ce and BG as well as a substrate for the production of HA.

MATERIALS AND METHODS
In this article the synthesis of bioglass was done by sol-gel method according to the previous work [32]. Then, compounds with 1% cerium were able to form a more uniform apatite layer over a 30-day period in SBF solution according to the Kokubo protocol. Also, in Fourier transform infrared (FTIR) test indicated that the formation of these calcium phosphates (CaPs) accelerates from day 15 onwards. The purpose of this study was to prepare a scaffold with high flexibility for biological imitation of extracellular matrix (ECM) to simulate bone tissue.  In this article, the aim was to prepare a 3D scaffold using PCL polymer with high elastic modulus that can create a porous substrate to allow cell growth and proliferation. The effect of Ce in BG on the repair process was also evaluated. The presence of BG mineral phase in the composition and microstructure of the scaffold can lead to the formation of CaPs and eventually HA in the scaffold. Also, the collagen layer on the surface of BG makes it possible to prevent its initial degradation in a liquid environment. Preparation of PCL scaffolds containing surface collagen microspheres can craete bone-like properties. Finally, the adhesion and growth of osteoblast cells on the scaffold were studied to evaluate the biocompatibility properties of the fabricated scaffold. Tetraethyl orthosilicate, type 1 collagen, glutaraldehyde and PCL (PCL, MW = 80 kDa) were purchased from Sigma Aldrich (USA). Calcium nitrate tetrahydrate (Ca(NO3) · 4H2O), triethyl phosphate (C6H15O4P,), cerium nitrate hexahydrate (Ce(NO3)3·6H2O), dimethyl sulfoxide (DMSO), RPMI-1640 culture medium, phosphate buffer saline, chloroform, ethanol and methanol were purchased from Merck (Germany). Streptomycin, penicillin, bovine fetal serum and trypsin were purchased from Initrogen (Gibco, Germany). Human MG63 cell line was obtained from Pasteur Institute of Iran (Tehran, Iran). Bioglass nanopowder containing different amounts of cerium based on SiO2–36CaO-4P2O5 (60-x) system was prepared by sol-gel method according to the previous study. In this article, for the synthesis of bioglass by sol-gel method, from tetraethyl ortho-silicate as a precursor of SiO2, from triethyl phosphate (TEP) as a precursor of P2O5, from cerium nitrate hexahydrate for the supply of alkaline oxides. The required feed is used in the composition of the glass as well as the supply of cerium, deionized water twice distilled as solvent and 2M nitric acid as catalyst. Then, specific amount of water and acid were mixed with a magnetic stirrer at 400 rpm at 25°C for 20 minutes to obtain a homogeneous solvent. Then, a certain amount of tetraethyl orthosilicate was weighed and gradually added to a solution of nitric acid and distilled water and mixed on a magnetic stirrer at 400 rpm for 15 minutes at room temperature. Tetraethyl orthosilicate was initially insoluble in acid and water solvents and its droplets appeared to be suspended in the liquid, but after hydrolysis began. During this process, the reaction vessel was heated, indicating a chemical reaction. After hydrolysis of tetraethyl orthosilicate, an appropriate amount of triethyl phosphate, calcium nitrate tetrahydrate and cerium nitrate hexahydrate were added to the solution. The resulting solution was stirred for 30 minutes with a magnetic stirrer at ambient temperature to obtain a clear and homogeneous tuberculosis solution. These calculations were performed to obtain 25 g of bioglass powder containing cerium. In this synthesis method, the volume ratio of water to nitric acid is 1:6 and the molar ratio of tetraethyl ortho-silicate-triethyl phosphate solution to water is 12:1. In this study, bioglass nanoparticles containing 1, 3 and 10% cerium (mass-mass) were prepared. Cerium-free bioglass nanoparticles were also synthesized as a control sample. The resulting tuber solution was incubated at 37°C for 7 days to give a gel. For this purpose, the gel was poured into humans and completely sealed with paraffin paper to prevent the penetration of dust by opening and closing the incubator and also to prevent the evaporation of the gel water. In the next step, the gel was dried in two steps. First, the gel was exposed to 70°C for 24 hours and the low temperature allowed the gel to dry slowly over 24 hours. The gel was then incubated at 120°C for 48 hours and to obtain a smooth and uniform powder from bioglass nanoparticles containing stabilized cerium, it was pulverized into a very soft powder by satellite milling. After complete milling and obtaining fine powder, the bioglass nanoparticle powder containing stabilized cerium was sieved to uniform size and remove large particles. For this purpose, an 8-inch steel sieve with a particle size of 37 microns. Collagen microspheres containing bioglass nanopowder with different cerium content were prepared using water-in-oil emulsion system. To do this, 1% by volume of the collagen solution was prepared by dissolving 100 mg of collagen in 10 ml of a water acetic acid solvent mixture. The volume ratio of water/acetic acid solvent mixture is 19:1 (9.5 ml of water and 0.5 ml of acetic acid), respectively. Then bioglass nanopowders containing cerium were added to the solution. The weight ratio of bioglass nanoparticles containing cerium to collagen solution is 1 to 19. Collagen solution/bioglass/cerium using ultrasonic homogenizer (80% power, 50% -50% on-off cycle) for 5 minutes Spread evenly on the ice bath. To prepare collagen microspheres containing bioglass/cerium, 50 ml of olive oil was heated to 45°C on a magnetic stirrer at 800 RPM and 10 ml of bioglass/cerium/collagen solution was quickly sprayed into it with a syringe and the mixture was stirred for 3 hours to form an oil-in-water emulsion. To further reduce the size of the prepared microspheres, the emulsion prepared by the ultrasonic homogenizer (100% power, 70% -30% on-off cycle) was homogenized twice for 10 minutes (each time) on an ice bath. Next, to precipitate the collagen microspheres containing bioglass/cerium, the resulting solutions were placed on an ice bath at 4°C for 30 minutes to cool. Then, 40 ml of pre-cooled acetone was added to the solutions at 4°C to dehydrate and precipitate the collagen microspheres. Toluene-saturated glutaraldehyde was used to crosslink the collagen microspheres containing bioglass/cerium. For this purpose, 0.25% (w/v) of saturated glutaraldehyde in toluene was added to the solution at room temperature and incubated under laboratory hood for 18 hours. To prepare toluene-saturated glutaraldehyde, first glutaraldehyde was diluted to about 1%, then equal amounts of 1% glutaraldehyde and toluene were removed and poured into a decanter funnel and shaken gently for 10 minutes to obtain a homogeneous mixture. The above mixture was given enough time for the two phases to separate completely. The supernatant was isolated and used as a saturated glutaraldehyde in toluene. A centrifuge at 2000 rpm was used for 10 minutes to separate the cross-linked microspheres. Crosslinked microspheres were deposited on the bottom of the Falcon and collected. To remove unwanted and excess residues from the surface of the formed microspheres (separation of oil phase and glutaraldehyde residue), they were mixed twice with acetone and then centrifuged at 2000 rpm for 5 minutes. Then it was washed 3 times with distilled water and gently to remove the remaining acetone. The obtained microspheres were dried at laboratory temperature for 24 hours. Then, 400 mg of polycaprolactone (PCL) 8% (mass-volume) in 5 ml (2.5 ml of chloroform and 2.5 ml of methanol, 50:50) was added and placed on a magnetic stirrer for 24 hours to obtain a one-handed spinning solution. Collagen microspheres containing bioglass/cerium prepared in man were poured and stirred with a magnetic stirrer to evaporate the solvent (acetone). In the last step, the PCL solution was added to the microsphere collagen mixture containing bioglass/cerium and stirred on a magnetic stirrer at 100 rpm for 4 hours. Wet electrospinning of PCL scaffold containing microcirculation/cerium/bioglass nanoparticles the PCL solution containing microcirculation/bioglass nanoparticles was poured into a 2.5 cc syringe after 24 hours of steering and placed on the injection site in a syringe pump (KDS-200, Nanoscale Technologists, Iran). Syringe pump device (uniform injection of injection solution) and voltage devices (potential difference suppliers) used for the electrospinning column (water surface supplier as collector). In this design, a single-syringe (or single nozzle) injection pump was used. Because the collector in this design was water surface and the type of electrospinning was vertical, the injection needle had to be placed on top of the electrospinning column. Therefore, a holder was placed on the spinning column to hold the injection needle, and one of the poles of the high-voltage device was attached to the tip of the needle. Due to the distance of the injection syringe inside the syringe pump to the injection needle, the injection serum hose was used to transfer the injection solution. Also, to create the required potential difference, a metal ring was installed in the bottom of the container, which was connected to the negative pole of voltage devices. The device was a constant current potential difference (DC) with a potential difference of 0-30 kV (nanoscale technologists, Iran). This device had a potential difference resolution of 0.1 kV (100 V). To create the required potential difference, the opposite pole had to be placed in the same direction in the solution inside the electrospinning column. For this purpose, a metal ring was used which was placed in the bottom of the column and the opposite pole was connected to it through the floor hole of the device. In this design, the distance from the nozzle to the collector (in this design, the solution surface inside the electrospinning column) the syringe is placed vertically on the water/ethanol bath.  

Investigation of bioactivity of prepared scaffold
After spinning the scaffold on a chloroform/methanol bath, the prepared scaffold is carefully collected and placed under a laboratory hood for 24 hours to dry. Then, for 24 hours before freezing, it was placed in the -80 freezer. Next, place the PCL/microspheric collagen/bioglass/cerium scaffold in the freezer for 72 hours to dry completely. In this study, gour types of PCL scaffolds/collagen microsphere/bioglass/containing 1, 3 and 10% cerium as experimental groups and without cerium as negative control in body simulated solution (SBF) with solid/liquid ratio of 20 mg.ml according to protocol was reviewed. The samples were soaked in SBF plastic bottles and stored at 37°C in a water bath for 7, 14 and 28 days. 

Investigation of MG63 cell line binding
First, to start the study of survival coefficient and cell binding, cut the scaffold of PCL/collagen microspheres/bioglass/containing cerium with concentrations of 1, 3 and 10% (mass-mass) in the dimensions of a 24-well plate and with a sterile steel ring. It was placed at the bottom of the well. PCL/collagen microsphere/bioglass fiber scaffolds were used as negative control. According to previous research to sterilize the scaffolds, each scaffold was placed in a clean room under the UV light of a cell culture hood for 30 minutes.

Evaluation of survival coefficient on MG63 cell line
MTT colorimetric method was used to evaluate cell viability. For this purpose, MG63 cells were cultured on PCL/microfiber collagen/bioglass containing/without cerium as prepared by the above method. First, as in the mentioned protocol, the scaffolds were cut to the size of the 48-well plate and then sterilized. Sterile rims were used to hold the scaffold in the bottom of the well. After counting the cells with a neobar slide, 10-4 cells per well were added to the scaffold. After loading the cells, the plates were incubated in a cell culture incubator for 1, 3 and 7 days at 37°C and 5% CO2. After 7-day of treatment, the cell culture medium was changed on the third day. At the end of the treatment period, the supernatant was discarded and the scaffold surface was gently washed with PBS to remove all excess culture medium and dead cells. Then, 20 μl of MTT solution (5 mg/ml) in PBS (pH 7.4) was added to each well and incubated for 4 hours in an incubator at 37°C and 5% CO2. 

RESULT AND DISCUSSION
The single observed peak of Bioglass 58 S sample can be related to the partial crystallization of stabilized calcium silicate under a temperature treatment of 600°C.  Direct sweeping OH (Process ① in Fig. 1), and OONO− were investigated by cerium nanoparticles. In addition, previous studies have shown that cerium nanoparticles have similar effects to catalase and superoxide dismutase (SOD) (processes 1 and 2 in Fig. 1). Both effects are closely related to surface concentrations of Ce3+ and Ce4+, pH, H2O2 and chelating ligand concentrations. The level of hydrophilicity of the scaffold prepared by the contact angle analysis was evaluated. As shown in Fig. 2, the hydrophilicity of the surface increases slightly with increasing cerium ion, but this increase is not significant. As shown in Fig. 2, the contact angle of the droplet with the surface in the initial second is above 100°. In the 2th second, this angle dropped below 100° (except for the control group). In the third second, the contact angle decreased again until in the fourth second, the angle decreased below 50° and the drop was absorbed by the scaffold. The MG-63 cell line is relatively immature osteoblast cells that are well-characterized and widely used for biomaterial testing, with numerous similarities to isolated human bone-derived cells. Previous research has shown that the size of the MG-63 cell line reaches more than 50 microns after 24 hours when connected to the surface. Fig. 2 indicate the cell size at 24 h after culture, which is similar to our finding, and the cell attachment to the surface of the PCL fiber scaffold containing collagen microspheres/bioglass 58S nanoparticles/cerium 1, 3 and 10% (mass). The obtained results presented after 7 days, the rate of cell proliferation and growth increased compared to the initial days (except for PCL fiber scaffold containing collagen microspheres/bioglass 58S nanoparticles/10% cerium (mass-mass). One of the strongest probabilities about these results can be related to the high rate of cell proliferation in this sample. As can be seen it is possible that the rate of cell proliferation has reached more than 95% due to dietary and spatial limitations and possibly media acidosis. Also, the cells are exposed to food stress and pH, resulting in cell necrosis which leads to the cell density decreased. The reason can be the dark spots on the surface of the PCL fiber scaffold containing collagen microspheres/bioglass 58S nanoparticles/10% cerium on the day 7, which have light margins. Other results confirm the results of cytotoxicity test in which PCL fiber scaffold containing collagen microspheres/bioglass 58S nanoparticles/10% cerium (mass-mass) has a significantly higher survival rate than other samples. However, it should be noted that the number of sided cells as well as the size of the wells in the cell binding test as well as the cytotoxicity are different and the probability of cell density in the MTT test is not even close to 95%. The tensile strength of the prepared scaffolds is in accordance with Figs. 5 (a-e). Fig. 5 (a-e) shows the control sample (PCL nanofiber scaffold/collagen microsphere/BG nanoparticles) with a thickness of 0.5 mm and a scaffold ribbon width of 11.8, the maximum force tolerance was 2.7 N and the scaffold is stretched to 41.23 mm against tension. In the bioglass control sample (PCL nanofiber scaffold/bioglass nanoparticles), the scaffold with a thickness of 0.5 mm and a width of 10 mm had the highest compressive strength up to 1.5 N. The scaffold has increased in length up to 24.52 mm before tearing under pressure and it has the highest tensile strength of 0.12 N. The scaffold has increased in length up to 57.62 mm before tearing under pressure. It has the highest tensile strength of 3.1 N. The scaffold length increased up to 40.10 mm before tearing under pressure. Wet electrospinning equipment was used to create three-dimensional structures and chloroform/methanol solvent with a ratio of 1: 9 in PCL to produce thin and elongated fibers. Also, water and ethanol baths and a combination of the two were used in the collector and the formation of the resulting foam was investigated according to the porosity. It was found that the ratio of 9.1 water and ethanol can create the best porosity in the scaffold and more uniform fibers were spun [33-44]. Researchers indicated that each of the effective parameters such as polymer concentration, solvent type, device voltage, spraying speed, needle tip to collector distance can be variable factors that can affect the size and diameter of spun fibers [45-62]. Liu et al. studied the effect of Ce on the ossification process of cells. Cerium has been shown to play an important role in osteogenesis factors and can improve ossification activity. Also, the concentration of Ce in the composition may have a direct effect on cell proliferation and alkaline phosphatase activity [3]. Calcium phosphate deposition with the presence of bioglass in the collagen scaffold was observed from the third day in the SBF test, which also precipitated hydroxyapatite particles until the 14 days. The average diameter of PCL fibers was measured at 260 nm and by loading the bioglass 58S encapsulating microspheres containing cerium inside the PCL fibers, due to the physical loading of the collagen microspheres inside the fibers, the average diameter of the PCL fibers increased to 320 ± 17 nm. Measurement of PCL scaffold fibers containing bioglass 58S encapsulating microspheres containing different concentrations of 1, 3 and 10% cerium (mass-mass) does not show any significant size difference. The average diameter of PCL fibers containing bioglass 58S encapsulated microspheres containing 3% (mass-mass) Ce concentration increased to 370 nm and also some collagen microspheres were detected on the surface of PCL nanofibers. In PCL fiber scaffolds containing bioglass 58S encapsulated microspheres containing different concentrations of 10% (mass-mass) cerium, microspheres are more detectable on the surface of the fibers and visible defects in the formation of continuous fibers due to the accumulation of microfibers in the microspheres. For example, PCL fiber scaffolds containing collagen microspheres/bioglass 58S nanoparticles/10% cerium (mass-mass), wider band in 956 cm-1 compared to the sharp band of PCL fiber scaffold specimens containing microspheres of 58S 956 cm-1. It has been observed that the effect of Ce nanoparticles on the crystal lattice disruption of silica groups is consistent with the SEM observations. Overlap of non-poly oxygen groups with Si-O-Si groups after seria incorporation into 58S bioglass results in the formation of a wider band in the FTIR of PCL fiber scaffold specimens containing collagen microspheres/bioglass 58S/cerium 10% in the range of 900 cm-1 and 1000 cm-1. In the bioglass control sample, the scaffold with a thickness of 0.5 mm and a width of 10 mm had the highest compressive strength up to 1.5 N. This sample is also stretched to 24.11 mm before tearing under tensile pressure. As shown in Fig. 5, the sample is a PCL nanofiber/collagen/bioglass/different concentrations of 1% cerium with a scaffold diameter of 5.0 mm and width 13 mm had the highest tensile strength of 2 N. The length of scaffold increased upto 24.52 mm before tearing under pressure. In the sample of PCL/microsphere collagen/bioglass nanofibers, different concentrations of 3% cerium with a scaffold diameter of 0.45 mm and a width of 13.5 mm had the highest tensile strength of 0.12 N. 
Biological activity plays an important role in the development of materials for bone tissue engineering applications. To evaluate the effect of collagen microspheres/bioglass 58S nanoparticles/10% cerium enclosed in PCL fiber scaffolds, a variety of samples were prepared (PCL fiber scaffolds containing collagen microspheres/PCL nanoparticles collagen/bioglass nanoparticles 58S/cerium 1, 3 and 10% (mass-mass)) were soaked in SBF solution for 7, 14 and 28 days. The SEM images of the samples at each time shown in Fig. 4. After 7 days of immersion of the sample in the SBF, no visible apatite formation was observed on the surface of PCL scaffold fibers containing collagen microspheres/bioglass 58S nanoparticles or PCL fiber scaffolds containing collagen microspheres/Ce bioglass nanoparticles 58%. As can be seen, with increasing the concentration of cerium encapsulated in the microsphere within the fibers of PCL scaffolds, the crystallization rate of the phosphate group increased by about 588 cm-1. It is noteworthy that the sharpest peak in the FTIR of the PCL fiber scaffold sample containing collagen microspheres/bioglass 58S nanoparticles/10% cerium (mass-mass) in the 1038 cm-1 region could be related to PO4-3 over-formation. The porosity of the final scaffold was checked by Image-J software. The porosity was observed around 70.4%, which was a good amount for nanofiber scaffold. As shown in Fig. 2, the level of surface hydrophilicity increases slightly with increasing cerium ion, but this increase is not significant. As shown in Fig. 2, the contact angle of the droplet with the surface in the initial second above 100°. In the second second, this angle dropped below 100° (except for the control group). In the third second, in all groups, the contact angle decreased again until in the fourth second, the angle decreased below 50° and the drop was absorbed by the scaffold. The results of this study showed that the PCL fiber scaffold sample containing collagen microspheres/Bioglass 58S nanoparticles/10% cerium has higher biocompatibility due to its cell proliferation and higher viability at different time points. In the last decade, sodium dioxide nanoparticles have attracted much attention due to their redox properties and potential therapeutic applications. Attempts have been made to discover the potential uses of sodium dioxide as a drug. The ability of Ce-NPs to reverse the oxidation states of Ce3+ and Ce4+ makes them a suitable mediator for reactive oxygen species. Researchers worked on different nanoparticles, including bioglass and bioceramic nanoparticles, which have the ability to improve the mechanical and chemical properties of scaffolds. Also, these scaffolds were evaluated by many researchers by simulation software and suggested for dental and orthopedic applications [63-83]. The functional group OH, NO, and OONO− have been investigated by Ce-NPs. In addition, previous studies have shown that cerium nanoparticles have similar effects to catalase and superoxide dismutase (SOD). Both effects are closely related to surface concentrations of Ce3+ and Ce4+, pH, H2O2 and chelating ligand concentrations. The MG-63 cell line is relatively immature osteoblast cells that are well-characterized and widely used for biomaterial testing which have many similarities to isolated human bone-derived cells. 

CONCLUSION
The designed of porous scaffold is important to create a three-dimensional structure with sufficient porosity. Some parameters such as high temperature, high pH and the use of magnets in the bath can reduce the surface tension of the scaffold on water and create microstructure. In the current study, PCL fiber containing collagen microspheres-bioglass 58S nanoparticles with 1 wt%, 3 wt% and 10 wt% were successfully fabricated. The particle size of bioglass-S58 containing Ce decreases compared to bioglass, which indicates the effect of cerium on reducing the size of bioglass. The mean diameters of collagen microspheres/bioglass 58S nanoparticles and collagen microspheres/bioglass 58S/10% cerium (mass-mass) was 340 nm and 280 nm, respectively. Biocompatibility evaluation of the prepared scaffolds indicated high cell binding and proliferation in the sample of PCL fiber scaffolds containing collagen microspheres-Bioglass 58S/3 nanoparticles (directly) and 10% (indirectly). The obtained results showed that PCL fiber scaffolds containing collagen microspheres/Bioglass 58S nanoparticles/3% and 10% (mass-mass) can be suitable choice for repairing bone defects. Further studies should be performed in the future to evaluate the in vivo performance of PCL fiber scaffolds containing collagen microspheres/Bioglass 58S nanoparticles containing 3 wt% and 10 wt% for regeneration of bone defects.