Synergistic Effect of Porous Hydroxyapatite Scaffolds Combined with

Jan 30, 2019 - Jeong-Hyun Ryu†‡ , Jae-Sung Kwon† , Kwang-Mahn Kim†‡ , Hye Jin ... Hyo-Jung Lee∥⊥ , Heon-Jin Choi∥ , Seong Yi⊥ , Hyun...
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Article Cite This: ACS Omega 2019, 4, 2302−2310

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Synergistic Effect of Porous Hydroxyapatite Scaffolds Combined with Bioactive Glass/Poly(lactic-co-glycolic acid) Composite Fibers Promotes Osteogenic Activity and Bioactivity Jeong-Hyun Ryu,†,‡ Jae-Sung Kwon,† Kwang-Mahn Kim,†,‡ Hye Jin Hong,§ Won-Gun Koh,§ Jaejun Lee,∥ Hyo-Jung Lee,∥,⊥ Heon-Jin Choi,∥ Seong Yi,⊥ Hyunjung Shin,# and Min-Ho Hong*,#

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†

Department and Research Institute for Dental Biomaterials and Bioengineering and ‡BK21 PLUS Project, Yonsei University College of Dentistry, Seoul 03722, Republic of Korea § Departm.ent of Chemical and Biomolecular Engineering and ∥Department of Materials Science and Engineering, Yonsei University, Seoul 03722, Republic of Korea ⊥ Department of Neurosurgery, Spine and Spinal Cord Institute, Yonsei University College of Medicine, Seoul 03722, Republic of Korea # Nature Inspired Materials Processing Research Center, Department of Energy Science, Sungkyunkwan University, Suwon 16419, Republic of Korea S Supporting Information *

ABSTRACT: Porous hydroxyapatite (HAp) scaffolds are commonly used for hard tissue regeneration because of their biocompatibility and osteoconduction properties, but they are limited in terms of bioactivity and osteoinduction. This study investigated the fabrication of HAp scaffolds coated with poly(lactic-co-glycolic acid)/ 45S5 bioactive glass (PLGA/BG) composite microfibers using the sponge replica method and electrospinning process for improved bioactivity and osteoinductivity during osteogenesis. Characterization of the HAp/PLGA/BG (HPB) scaffold was carried out by examining morphology and ion release. The biological evaluation of HPB scaffolds was carried out by assessing cytotoxicity, cell proliferation, and cell differentiation using MC3T3-E1 preosteoblasts. The results showed that all HPB scaffolds exhibited a high porosity of 89.2% and had porous structures coated with a layer of composite containing BG ions (Si−Ca−Na−P). These scaffolds enabled controlled release of Si, Ca, Na, and P ions for up to 28 days. There was no significant difference in cytotoxicity between the scaffolds. Cell proliferation on HPB scaffolds was increased from day 1 to 3. In addition, cell viability on the HPB scaffolds was confirmed with LIVE/DEAD assay. Cell differentiation, as shown by alkaline phosphatase activity and western blot analyses, indicated that HPB scaffolds with 20 and 30 min of coating induced higher levels of osteogenesis-related markers compared to other scaffolds. Furthermore, immunocytochemistry indicated osteopontin expression. Alizarin red staining indicated that HPB scaffolds with 20 min of coating were more effective than the HAp scaffold in terms of mineralization. In conclusion, HAp scaffolds coated with PLGA/BG for 20 min are promising materials for osteogenic activity and may be a potential bone substitute for tissue engineering.

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INTRODUCTION Tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences to develop biological substitutes that restore, maintain, or improve tissue function.1 Hard-tissue engineering may be required following injury, trauma, or tumor of the bone, which may consequently lead to the formation of large defects.2 The reconstruction of such large or critical-sized bone defects remains the main challenge in medical fields such as oral, craniofacial, and orthopedic surgery.3,4 Large bone defects are classically treated by replacement with autologous bone, which is considered to be the gold standard in reconstructing bone defects because of its strong osteogenic potential.5 However, supply of autologous © 2019 American Chemical Society

bones is limited in the case of patients with a damaged bone, and therefore, bone-substitute materials are developed as an alternative choice. A wide range of biomaterials and synthetic bone substitutes are currently being used as scaffolds, including hydroxyapatite (HAp), β-tricalcium phosphate, other calcium phosphate-based materials, and glass ceramics. HAp is a type of calcium phosphate-based bioceramics that constitutes the majority of the inorganic component in bones and teeth.6,7 HAp is widely used for various bone regeneration Received: October 22, 2018 Accepted: January 14, 2019 Published: January 30, 2019 2302

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Figure 1. Morphology of the HAp and HPB scaffolds. (A) Representative SEM images of porous HAp scaffolds. (B) Representative SEM image of HPB scaffolds with the thickness of BG-containing PLGA microfibers. (C) SEM−Energy dispersive spectroscopy (EDS) analyses of the HPB10 scaffold. Ca and P ions were detected in HAp, and Si, Ca, Na, and P ions were detected in BG-containing PLGA microfibers.

together with biodegradable PLGA to compensate for the disadvantage of HAp. HAp is generally known to have low electrical conductivity (∼10−12 S cm−1).23 However, the fabricated HAp scaffold in this study has macrosized pores and interconnective pore structure. The structural properties of the porous HAp scaffold have helped to apply the electrospinning method, even though it has low conductivity. The aim of this study was to develop porous HAp scaffolds that are selectively covered with a BG-containing PLGA microfiber layer [named HAp/PLGA/BG (HPB) scaffold from here on] and to investigate the proliferation and differentiation of peripheral osteoblasts cultured on the developed scaffolds.

applications as it has excellent biological properties such as biocompatibility, bioactivity, cell migration, and osteoconduction.8,9 Nevertheless, its reactivity with existing bone tissue in defected bones is low. In fact, the minimal degradability of HAp in physiological circumstances often results in the material not being absorbed or replaced by new bone formation.7,10 45S5 bioactive glass (BG) is a well-known inorganic biomaterial characterized by its higher bioactivity index compared to that of HAp. The multistage mechanism begins as BG comes in contact with physiological fluids.11,12 BG is able to bond to soft tissues and hard ones, and reactions on the material surface induce the release and exchange of critical concentrations of soluble Si, Ca, Na, and P ions.13,14 Out of these ions, it is well known that Si-ion concentration affects the osteoblast growth cycle and promotes proliferation and differentiation.15,16 Hence, BG is known to be highly bioactive, osteoconductive, and osteoinductive, whereas it promotes bone formation from beneficial intracellular and extracellular responses.17,18 In the past years, HAp has been sintered to be combined with BG to overcome the disadvantages of HAp.19,20 Nevertheless, the phases of HAp and BG are changed by high temperature after sintering, and the combination of BG with HAp via such a method was unable to maintain the bioactivity of BG previously mentioned. Thus, in this study, fibers consisting of biopolymers were applied to maintain the properties of both HAp and BG. Poly(lactic-co-glycolic) acid (PLGA) is an FDA-approved biodegradable biopolymer that is well known for its properties including good ductility and biocompatibility.21 PLGA can control the rate of degradation by the ratio of lactide to glycolide. Also, the fibrous structure of PLGA obtained using electrospinning has been previously adopted for the controlled release of growth factors or drugs.22 Therefore, BG, which has the advantage of bioactivity and osteoinductivity, was applied

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RESULTS Surface Morphology and Structural Analysis. The surface morphology and structure of HAp and HPB scaffolds were observed using scanning electron microscopy (SEM) (Figure 1) and microcomputed tomography (μ-CT) (Figure S1). The SEM images of HAp scaffolds show macroporous structures (Figure 1A), which were due to irregular sintering at 1250 °C, whereas the highly porous structure of HAp scaffolds is evident by μ-CT (Figure S1). The SEM images show composite fibers in HPB scaffolds (Figure 1B), where the PLGA/BG composite fibers in the HPB30 scaffold were the thickest compared to all other groups. Figure 1C shows the surface chemistry composition of the HPB10 scaffold representatively (results of other groups are shown in Figure S2). Spot 1 indicates the presence of Si, Ca, Na, and P. Spot 2 indicates the presence of Ca and P only (without the presence of Si or Na). These results show that the HPB scaffolds generally maintained the components of HAp and BG. In Vitro Ion-Release Profile. In general, the accumulation of Ca and P ions increased in all scaffolds during the 28 days, 2303

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Figure 2. Ion release of HAp and HPB scaffolds into distilled water, represented by cumulative release of Ca, P, Si, and Na ions (n = 3, Si and Na ions are not contained in the HAp scaffold).

Figure 3. (A) Cell proliferation on HAp and HPB scaffolds, revealing that cells had good viability within all groups (n = 5). (B) Alkaline phosphatase (ALP) expression (normalized by DNA in the sample) of MC3T3-E1 within HAp and HPB scaffolds after 3 and 7 days. Osteoinductivity of HAp and HPB scaffolds in vitro (n = 3). (C) Osteogenic expression levels of osteopontin (OPN) normalized to GADPH (n = 3, *; p < 0.05 and **; p < 0.001).

7 until day 28 for HPB10 and HPB20. However, the HPB30 scaffold released Na ions until day 14, and the release then plateaued until day 28. Therefore, the durations of Si, Ca, Na, and P ion release were the longest in the HPB30 scaffold, and the accumulated ions were also the highest in the HPB30 scaffold. Biocompatibility of HPB Scaffolds. Figures 3A and 4 show the proliferation and viability of MC3T3-E1 cells on HAp and HPB scaffolds. On day 1, cell proliferation on the HPB30 scaffold was significantly lower than that on HAp, HPB10, and HPB20, whereas on day 3, cell proliferation on the HPB30 scaffold was significantly lower than that on HAp and HPB10. Only HPB10 in the HPB groups showed higher cell proliferation

whereas additional accumulation of Si and Na ions was evident on all scaffolds except HAp (Figure 2 and Table S1). The release of Ca and P ions into distilled water from each scaffold continually increased during the 28 days for all scaffolds. For Si ions, sharp release of ions from each HPB scaffold into distilled water was evident during the first day and the seventh day of immersion. However, for HPB10 and HPB20, the release of Si plateaued from day 14 until day 28, whereas constant release of the HPB30 scaffold into distilled water was evident during the same period. For Na ions, a burst release from each HPB scaffold into distilled water was noticed during the first day and the seventh day of immersion. The release then plateaued from day 2304

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Figure 4. LIVE/DEAD staining of the HAp and HPB scaffolds after 7 days showing cells on the HAp strut and BG-containing PLGA microfiber layer (scale bar = 200 μm).

BG continuously releases a greater amount of ions than HAp in physiological conditions. However, HAp is mainly composed of calcium and phosphate, whereas BG mainly consists of Si, meaning that these two bone-substitute materials are different in crystal or amorphous structure. In this sense, it is reasonable to conclude that these two materials might have separate roles in maintaining osteoblasts in bone defects. Thus, we raised the question whether HAp and BG coexist in human bone tissue within a bone defect area, and we hypothesized that the scaffold consisting of BG-loaded PLGA fibers coated around HAp might play a significant role in promoting bone growth and regeneration. In this study, we confirmed that HPB scaffolds maintained the porous structure of HAp scaffolds, whereas the other component is composed of BG/PLGA fibers (Figure 1A,B). The pore size and porosity of porous scaffolds ranged from 200 to 500 μm and 80−90%, respectively, which were similar to those of cancellous bone and therefore would provide effective cell proliferation and differentiation.26 On the contrary, the composite electrospun fibers of HPB scaffolds had pore sizes that were smaller than those of the porous HAp scaffold. However, the electrospun fibers of the HPB scaffolds provided the specific surface area for cell adhesion. In previous studies, electrospun fibers were indicated to be effective for cell attachment.22 We performed analysis to confirm the components of HAp and BG in the HPB scaffolds (Figures 1C and S2). We observed that BG was in a pure state in the composite fibers, which were combined with the HAp scaffold. Previously, BG and HAp were detected in pure states in fibrous composite scaffolds.27,28 In addition, we performed quantitative analysis of PLGA composite fibers (Figure S3). We confirmed that the electrospun composite fibers of HPB20 and HPB30 had the same ratio of PLGA to BG before making electrospun fibers. However, the electrospun composite fibers of HPB10 were not efficient as they had a lower ratio of PLGA to BG than those of HPB20 and HPB30. Thermogravimetric analysis conducted on HPB scaffolds by comparing the ratio of raw materials demonstrated that short electrospinning time is not efficient for the electrospun BG-containing PLGA fibers. HAp and HPB scaffolds were compared in terms of their cytotoxicity (Figure S4) and cell proliferation rate (Figure 3A). The evaluation showed an absence of cytotoxicity in the HPB scaffolds. Notably, lactic acid caused toxicity by biodegradation of PLGA. However, the components of the HPB scaffolds, HAp and BG ions, neutralized the lactic acid. According to Vergnol et

than HAp. These results were also confirmed with LIVE/DEAD staining (Figure 4), where green fluorescence indicated live cells and red fluorescence indicated dead cells. There was no obvious difference in the number of red cells on each scaffold, whereas most cells were alive with green fluorescence. Cells were present on the HAp scaffolds but for HPB scaffolds, they were also attached to the PLGA/BG composite fibers. Osteogenic Differentiation of MC3T3-E1 Cells on HPB Scaffolds. We assessed ALP activity and OPN expression in MC3T3-E1 cells on HPB scaffolds (Figure 3B,C). The ALP activity of the HPB30 scaffold was significantly lower than those of HAp, HPB10, and HPB20 on day 3, but on day 7, the ALP activity of the HPB30 scaffold was higher than those of HAp and HPB10. In terms of OPN expression, there was no significant difference between HAp and HPB scaffolds at 1 week (Figure 3C). However, OPN expression was higher on HPB30 than on the other groups at 2 weeks. To further consider the osteogenic properties of the materials, immunocytochemical (ICC) analyses were carried out for collagen I (COL-I), runt-related transcription factor 2 (RUNX2), and OPN in MC3T3-E1 cells cultured on HAp and HPB scaffolds for 7 and 14 days (Figure 5). First, osteogenic activity was detected in MC3T3-E1 on both day 7 and 14. HAp and HPB scaffolds showed similar levels of COL-I and RUNX2 expression. However, the expression of OPN was only evident in MC3T3-E1 cells on HPB scaffolds and was absent on the HAp scaffold, on both day 7 and 14. Mineralization of Osteoblastic MC3T3-E1 via Released Ions from Scaffolds. We investigated the effect of released ions from HAp and HPB scaffolds on the mineralization of MC3T3-E1 cells during 14 and 21 days (Figure 6). There was no evidence of bone mineralization in all groups after 14 days of culture and in both the control (only MC3T3-E1 cells) and HAp group after 21 days of culture. On day 21, the HPB groups showed mineralization. In particular, HPB20 and HPB30 showed high levels of mineralization.

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DISCUSSION HAp is a widely used material for osteoconduction because of its high biocompatibility and ability to obtain a porous bonelike structure.24 However, the limitation of the material lies in the lack of characteristics such as osteoinduction, which is required for filling in the defect sites of bone that would assist osteoblasts in the repair process. On the other hand, BG promotes a higher level of osteoinduction and osteoconduction in bone defects.25 The difference is due to the release of ions from each material. 2305

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al., the released ions from BG enable cells to dwell in the poly(lactic acid) (PLA). The degradation of PLA may, in turn, easily cause cell death because of changes in pH. Both polymeric and ceramic composite materials demonstrated an absence of cytotoxicity.29 When cells were seeded on the surface of the HAp and HPB scaffolds, we observed that the cells adhered and proliferated. The HAp structure and the PLGA/BG composite fibers in the HPB scaffolds affected the attachment of the cells. According to Hong et al., MC3T3-E1 cells were attached to the structure of HAp scaffolds.30 In addition, PLGA composite fibers at the side of the HPB scaffolds showed increased cell attachment compared to those on the HAp scaffold. We confirmed that the specific surface area of the HPB scaffolds increased more than that of the HAp scaffold with the SEM images in this study. It was previously reported that electrospun PLGA fibers enhanced the attachment of osteoblasts and imaging of three-dimensional cell growths.22 According to the results of inductively coupled plasma optical emission spectroscopy (ICP-OES) (Figure 2), Si and Na ions were quickly released in comparison to Ca and P ions, possibly because of the accelerated degradation of BG. The release kinetics of BG in PLGA fibers could be easily controlled by regulating the Si−Ca−Na−P ions (Table S1). BG has shown improved osteogenesis since first reported by Hench.31 In the present study, we demonstrated that the released Si ions from PLGA/BG composite fibers on the HPB scaffolds were important for stimulating osteogenesis. Some studies have reported that Si ions released from BG enhanced osteogenesis.32−34 Also, BG released Ca and P ions persistently, which improved osteoblastic differentiation.35 We showed that HAp and BG could enhance the osteogenic activity of MC3T3-E1, as indicated by cells on the HPB scaffolds compared to those on the HAp scaffold. The HPB30 scaffold showed that ALP activity, which is a marker of early osteogenic differentiation, has the tendency to dramatically increase (Figure 3B). Tsigkou et al. reported that culture medium with BG ions had higher ALP activity than that of culture medium without BG ions.36 In addition, Rath et al. reported that BG stimulated high amounts of ALP at an earlier time in adipose stem cells and mesenchymal stem cells.37 The HPB30 scaffold significantly promoted osteogenic differentiation by enhancing the expression of bone-related genes (Figure 3C). We demonstrated that the HPB30 scaffold induced the protein expression of OPN in MC3T3-E1. BG significantly enhanced the osteoinductive capacity of the HPB scaffolds, which is critical for bone repair and regeneration.38 In our experiment, the ICC analysis suggested that the BG in HPB scaffolds stimulated the upregulation of three genes (COL-I, RUNX2, and OPN) associated with the process of osteogenic differentiation (Figure 5). RUNX2 has been identified as the

Figure 5. Osteogenic activity of MC3T3-E1 cells cultured on HAp and HPB scaffolds as indicated by ICC analyses of the osteogenic protein expression level (COL-I, RUNX2, and OPN) following culture for (A) 7 and (B) 14 days (scale bar = 20 μm).

Figure 6. Mineralization of MC3T3-E1 cells by ions released around HPB scaffolds during days 14 and 21 (scale bar = 1 mm). 2306

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Figure 7. Schematic representation of the fabrication of porous HAp scaffolds covered with a BG-containing PLGA fiber layer.

lum bromide, and dimethyl sulfoxide were purchased from Sigma-Aldrich (St. Louis, MO, United States). Osteoblastic MC3T3-E1 cells (CRL-2593, subclone 4) from mouse calvariae were obtained from the American Type Culture Collection (Manassas, VA, United States), and L929 fibroblasts were obtained from the Korean Cell Line Bank (Seoul, Korea). Alpha minimum essential medium (α-MEM), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), trypsin−ethylenediaminetetraacetate, penicillin, phosphate-buffered saline (PBS), and distilled PBS were purchased from Gibco (Grand Island, NY, United States). A protein extraction solution for Bradford protein assay was purchased from PROPREP, iNtRON Biotechnology (Seongnam, Korea). Polyvinylidene difluoride membranes were purchased from Millipore (Schwalbach, Germany). Antibodies against OPN (ab11503), COL-I (ab34710), RUNX2 (ab76956), and glyceraldehyde dehydrogenase (GAPDH, ab8245) were purchased from Abcam (Cambridge, UK). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). All secondary antibodies used in immunocytochemistry were purchased from Jackson ImmunoResearch (West Grove, PA, United States). All reagents and chemicals were used in this study without further purification. Fabrication of HAp Scaffold. The porous HAp scaffolds were fabricated with polyurethane (PU) foam by the replica method. As a binder, 1 g of PVB was stirred vigorously in 20 mL of absolute ethanol for 2 h, and 6 g of HAp nanopowders was added in the stirring PVB solution. The mixture was stirred for an additional 24 h. PU foam templates were punched to form a three-dimensional cylindrical shape with a volume of 7d × 9h mm3 and immersed in the HAp slurry. After blowing with an air gun to disperse the slurry uniformly throughout the porous scaffolds without blocking the pores, the sponges were dried at 90 °C for 30 min. These dipping and drying steps were repeated twice. The sponge-coated HAp slurry was heat-treated to burn out the sponge and binder at 600 °C for 1 h in air at a heating rate of 5 °C/min. A Lindberg furnace (Lindberg/MPH, Riverside, MI, United States) was then used to sinter the scaffolds at 1250 °C for 3 h (heating rate of 5 °C/min). Fabrication of HPB Scaffolds. BG was loaded into the microfiber layer, which was made with PLGA with a lactide/ glycolide ratio of 65:35. The PLGA 6535 containing drugs or ions displayed the longest acting effects compared 50:50 PLGA.45 Considering the period of in vitro tests, PLGA having a ratio exceeding 65:35 was not selected. To prepare the electrospinning dope, PLGA was dissolved in TFE at a

major transcription factor controlling osteogenic differentiation from the preosteoblastic to osteoblastic stage. The expression levels of late differentiation marker genes COL-I and OPN were upregulated by RUNX2.39 Hence, RUNX2 can activate osteoblast-related gene expression. OPN is a substantial component of the bone matrix and is thought to be responsible for cell attachment to the electrospun fibers.40 Earlier research indicated that osteogenic differentiation marker genes were stimulated and increased in response to BG.41,42 Osteogenic differentiation was further confirmed by evaluating Ca deposition via alizarin red staining in response to HPB30 scaffold treatment with preosteoblasts after 21 days (Figure 6). Increased Ca deposition was observed in response to HPB scaffolds along with an environment that stimulated osteogenesis. Notably, Si ions released by BG ions played a role in osteoblast function.43 The ability of Si ions to produce cell mineralization and nodules is important for the development of appropriate materials for bone repair.44 Some studies have found that bone nodules were formed in the presence of BG32 or extracted medium from BG ions.36 Hence, it is suggested that HPB scaffolds induce the promotion of osteoblastic differentiation from preosteoblasts in an osteogenic environment.

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CONCLUSIONS Overall, HPB scaffolds prepared by the two-step fabrication method as described in this research are promising materials for rapid critical bone regeneration. We expect that our HPB scaffolds will be useful for the treatment of bone defects as HAp and BG are suitable as biomaterials for the human bone. HPB scaffolds not only mimic human cancellous bone but are also able to support cell migration, allowing the growth of MC3T3E1. Most importantly, HPB scaffolds controlled the release of ions from BG, which was shown to be effective in osteogenesis and bone mineralization compared to the HAp scaffold. Based on our study, BG ions are released to control the HPB scaffolds where among the 45S5 ions, Si ions were indicated to stimulate bone repair. Taken together, HPB scaffolds can act as a bone substitute to support the restoration of bone structure and efficient function of damaged bone.

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EXPERIMENTAL SECTION Materials. Commercially available HAp powder (Ossgen, Daegu, Korea) and BG microparticles (bioactive glass 45S5, SCHOTT, Landshut, Germany) were used. Poly(vinyl butyral) (PVB), poly(D,L-lactide-co-glycolide) 6535 (PLGA 6535; MW 24 000−38 000), 2,2,2-trifluoroethanol (TFE), Tween 20, Triton X-100, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo2307

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concentration of 0.5 g/mL at 50 °C, and the BG was dispersed (BG/PLGA = 1:10) by vortex agitation and sonication for 10 min. When the temperature of the PLGA solution decreased to room temperature, the viscosity increased and the BG could be kept in a dispersed state in the PLGA solution. The prepared PLGA solution with BG was coated around the HAp scaffolds through the electrospinning (NanoNC, Seoul, Korea) process. The HAp scaffolds were mounted on a metallic stick that would replace the rotating drum of the electrospinning apparatus (Figure 7). In detail, the PLGA solution with BG was loaded in a syringe pump, and the solution was pumped toward the rotating stick at a rate of 1 mL/h through a 23-gauge metallic needle. The stick with mounted HAp scaffolds was rotated at a speed of 230 rpm with an applied voltage of 13 kV. The overall spinning time differed depending on the experimental groups, including 10, 20, and 30 min (Table 1). Morphology and Composition Analysis. The surface morphology of the scaffolds was observed by SEM (JSM-7001F, JEOL, Tokyo, Japan). In brief, the scaffolds were coated with Pt using a sputter coater at a gas pressure of 45 mTorr and current of 40 mA for 150 s. The coated scaffolds were analyzed at an accelerating voltage of 20 kV. EDS (Octane Plus, EDAX,

was added to the cell pellets, and the cells were resuspended by brief vortexing. The cells were transferred to a black 96-well plate and analyzed using a FlexStation III microplate reader (Molecular Devices) with the fluorescence mode at Ex/Em = 480/520 nm. The viability of the cells on the scaffold was analyzed using the LIVE/DEAD viability/cytotoxicity kit for mammalian cells (Invitrogen, Carlsbad, CA, United States). In brief, the scaffolds seeded with cells were incubated for 7 days and washed with PBS. The cells on the scaffold were then stained in LIVE/DEAD reagents for 30 min. The HPB scaffolds were then observed by confocal laser scanning microscopy (LSM 700, Zeiss, Oberkochen, Germany) to assess the viability of the cells on the scaffold. ALP Activity Assay. To confirm the initial stage of osteogenic differentiation, ALP activity was evaluated on days 3 and 7. The cultured cells in the scaffolds were homogenized, and the obtained cell lysates were tested for ALP activity using a SensoLyte pNPP alkaline phosphatase assay kit (Anaspec, An Jose, CA, United States) according to the manufacturer’s protocol. The ALP activity was normalized using the total protein amounts by using the Bradford method (Pierce, Rockford, IL, United States). Western Blot Analysis. The cells in the scaffold were harvested in lysis buffer. After sonication, the samples were centrifuged for 5 min at 13 000 rpm. Protein contents were quantified by using the Bradford method. The prepared proteins in each group were separated using sodium dodecyl sulfate− polyacrylamide gel (4 and 15%) electrophoresis. After electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes, and the blots were subsequently probed with OPN antibodies. For detection, HRP-conjugated secondary antibodies were used. Normalization of results was ensured by running parallel Western blots with GAPDH antibodies. Immunocytochemistry. ICC staining was performed to detect specific expression patterns exhibited by proteins. Primary antibodies against COL-I, RUNX2, and OPN were used to identify osteogenic differentiation. In detail, cells cultured on the scaffolds were fixed in 4% paraformaldehyde on days 7 and 14, permeabilized with 0.3% Triton X-100 in PBS, and blocked in 10% normal donkey serum. After blocking, the cells were incubated overnight at 4 °C with primary antibodies targeting the osteoblast-specific marker proteins. After washing with PBS, secondary antibodies conjugated to fluorescein isothiocyanate (711-095-152), Cy3 (715-165-151), and Alexa Fluor 647 (705-606-147) were added to the cells for 1 h, and DAPI was used for nuclei staining. Representative fluorescence images of stained cells on the scaffold were obtained using a confocal laser scanning microscope. Alizarin Red S Staining. Alizarin red staining was performed to observe mineralized bonelike cells on days 14 and 21. In brief, the cells on the plate with the scaffolds were washed with PBS and fixed with 70% EtOH for 1 h. The fixed cells were stained for 10 min with 40 mM alizarin red S solution, pH 4.2, at 25 °C. After washing out the remaining dye with deionized water, the cells were observed under a light microscope and representative views were photographed. Following rinsing with distilled water, the bound stain was eluted with 10% cetylpyridinium chloride and absorbance of the solution was measured at 562 nm. Statistical Analysis. Experimental data were processed with one-way analysis of variance followed by Tukey’s post hoc analysis (SPSS, Chicago, IL, United States) to establish

Table 1. Representative Groups in This Study HPB groups HAp group coating time of BG-containing PLGA microfibers (min)

HPB10

HPB20

HPB30

10

20

30

Mahwah, NJ, United States) was used to provide qualitative information on the elemental composition of the scaffolds. Glass Ion-Release Test. The ion concentrations of released Si, Ca, Na, and P after immersion of the scaffold in distilled water (1 g of scaffold in 50 mL of distilled water) were measured after 1, 7, 14, 21, and 28 days at 37 °C. The concentrations of each ion released from the scaffold were measured by ICP-OES (ICP Optima 8300, PerkinElmer, Waltham, MA, USA). Cell Culture. Calvaria-derived MC3T3-E1 subclone 4 preosteoblasts from newborn mice and L929 fibroblasts (only for the cytotoxicity test) from mice were cultured in α-MEM (for MC3T3-E1) and RPMI 1640 (for L929) supplemented with 10% FBS and 100 μg/mL antibiotic/antimycotic mix at 37 °C in an atmosphere with 95% humidity and 5% CO2. The complete medium was replaced every two to three days, and confluent cells were subcultured through trypsinization. The cultured cells were seeded in the scaffolds at a density of 1 × 105 cells/scaffold for in vitro tests. Differentiation was then induced through the addition of 50 μg/mL ascorbic acid, 10 mM βglycerophosphate, and 100 nM dexamethasone to the growth medium. The differentiation medium was subsequently replaced every two days. Cell Proliferation and Cell Viability Tests. Cell proliferation was analyzed using a CyQUANT cell proliferation assay kit (Molecular Probes, Eugene, OR, United States) according to the manufacturer’s protocol.46 In brief, the cells were cultured for one and three days. The scaffolds were washed with PBS, and the adherent cells were detached and suspended by treatment with trypsin. The prepared cell suspension with cell-lysis buffer was centrifuged for 5 min at 2000 rpm, and the supernatant was carefully removed. The CyQUANT GR dye 2308

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statistical significance. Error bars represent the mean ± standard deviation of the measurements.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02898. Calculated pore properties of scaffolds by μ-CT; SEM− EDS results of HPB20 and HPB30 scaffolds; results of thermogravimetric analysis of PLGA/BG compositions in the HPB scaffolds; cytotoxicity of scaffolds; MC3T3-E1 cell attachment on HAp and HPB10 scaffolds; and raw data of the released ion profile (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Won-Gun Koh: 0000-0002-5191-2531 Hyunjung Shin: 0000-0003-1284-9098 Min-Ho Hong: 0000-0001-9268-9906 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF2016R1A6A3A11932752) and the Ministry of Science and ICT for the Bioinspired Innovation Technology Development Project (NRF-2018M3C1B7021994).

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