- Email: [email protected]
Controlled-release naringin nanoscaffold for osteoporotic bone healing
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/dema
Controlled-release naringin nanoscaffold for osteoporotic bone healing Yan Ji a,b , Lu Wang a,b , David C. Watts e,f , Hongmei Qiu d , Tao You a,b , Feng Deng b,c , Xiaohong Wu a,b,∗ a
Department of Prosthodontics, Stomatological Hospital of Chongqing Medical University, No. 426 Songshibei Road, Yubei District, Chongqing 401147, China b Chongqing key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing 401147, China c Department of Orthodontics, Stomatological Hospital of Chongqing Medical University, No. 426 Songshibei Road, Yubei District, Chongqing 401147, China d Key Laboratory of Biochemistry and Molecular Pharmacology, Department of Pharmacology, School of Pharmacy, Chongqing Medical University, Yixueyuan Road, Yuzhong District, Chongqing 400016, China e School of Dentistry and Photon Science Institute, The University of Manchester, Oxford Road, Manchester M13 9PL, UK f Institute of Material Science and Technology, Friedrich-Schiller-University, Jena, Löbdergraben 32, 07743, Germany
a r t i c l e
i n f o
a b s t r a c t Objectives. Osteoporosis is one of the most common bone diseases in the world and results from an imbalance of bone cell functions. In the process of guided bone regeneration, osteo-
Keywords:
porosis weakens the bonding strength between scaffold and bone. Naringin is evidenced to
Bone tissue regeneration
be effective for the treatment of osteoporosis and bone resorption and the aim was to explore
Electrospinning
methods and benefits of its incorporation.
Nanoscaffold
Methods. In this study, naringin was incorporated in the electrospun nanoscaffold containing
Naringin
poly(-caprolactone) (PCL) and poly(ethylene glycol)-block-poly(-caprolactone) (PEG-b-PCL).
Osteoporosis
Results. The nanoscaffold demonstrated unchanged chemical structure, improved hydrophilicity, thinner and more uniform nanofibers by Fourier-transform infrared spectroscopy, contact angle measurement and scanning electron microscopy. The nanoscaffold also showed faster degradation rate and controlled-release of naringin. Osteoblastnanoscaffold interactions were studied by the evaluation of adhesion, proliferation, differentiation of MC3T3-E1 osteoblasts and mineralization of ECM on the nanoscaffolds. Meanwhile, the response of osteoclasts to nanoscaffolds was evaluated in a mouse calvarial critical size defect organ culture model. The osteoclasts around the bone defect were shown by tartrate resistant acid phosphatase staining.
∗ Corresponding author at: Department of Prosthodontics, Stomatological Hospital of Chongqing Medical University, No. 426 Songshibei Road, Yubei District, Chongqing 401147, China. Tel.: +86 23 88860086; fax: +86 23 88860086. E-mail address: [email protected] (X. Wu). http://dx.doi.org/10.1016/j.dental.2014.08.381 0109-5641/© 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1264
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
Significance. The results demonstrated that controlled-release naringin nanoscaffolds supported greater osteoblast adhesion, proliferation, differentiation, and mineralization and suppressed osteoclast formation. © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Extensive craniomaxillofacial bone defects caused by tumor, infection, trauma and dysplasia are regularly repaired by bone substitution materials. However, the restorative effect depends not only on the implant properties but also on the characteristics of the host bone. Researchers pay increasing attention to the patients’ own diseases. Among them, osteoporosis is the most common systemic skeletal disorder that weakens the bonding strength between the implants and bone, delays bone defect repair, lowers the quality of new bone formation and even causes prosthesis failure [1,2]. With the aging of populations, osteoporosis is a still increasing prevalence. As a part of the whole body bone, craniomaxillofacial bone is inevitably influenced by osteoporosis, showing reduced bone mass, bone mineral content and bone density, deterioration of the microstructure of the trabecular bone and lower osteogenesis ability. Although osteoporosis is not an absolute contraindication for bone repair, it increases implant failure and the risk of complications. Therefore, the treatment of bone defects in osteoporosis patients remains a challenge in dental medicine. Osteoporosis occurs when bone resorption is greater than bone formation. Thus, osteoclasts and osteoblasts imbalance correction may be a therapeutic modality for bone repair of the patients with osteoporosis. The most widely used drugs to treat osteoporosis are bisphosphonates. Bisphosphonates maintain bone mass by inhibiting the function of osteoclasts [3]. However, bisphosphates have been reported to cause osteoclast dysfunction and even osteonecrosis [4,5]. Researchers showed that naringin plays a dual role in regulation of osteoblasts and osteoclasts. Naringin, a polymethoxylated flavonoid, is an active constituent of Rhizoma Drynariae (Traditional Chinese Medicine). Naringin has been reported to stimulate proliferation and differentiation of osteoblastic cell lines [6,7] and inhibit osteoclast formation [8–10]. Naringin has been shown to inhibit HMG-CoA reductase, activate the BMP-2 promotor, increase bone formation [11] and induce the expression of osteogenic markers of osteoblasts [12]. Naringin can also dosedependently suppress the number of osteoclasts formed by the treatment with interleukin-1 (IL-1) [9]. Chen et al. [10] has reported that naringin increased alkaline phosphatase activity, osteocalcin level, bone mineral density and decreased the number of osteoclasts. In addition, naringin has been shown to improve bone quality of rats with osteoporosis induced by retinoic acid [13] and orchidectomized rats [14]. Guided bone regeneration (GBR) is a method to repair bone defects, which emerged with the development of biology and bioengineering. GBR scaffolds are used as barriers to prevent soft tissue ingrowth and create space for slowly regenerating periodontal and bony tissues. For the past few years, the elec-
trospinning technique has been used widely in fabrication of scaffolds for bone [15,16], tendon [17], vasculature [18] and neural [19] tissue regeneration. Electrospun scaffolds with a large surface area-to-volume ratio, high porosity and morphology similar to the extracellular matrix (ECM) of natural tissue can serve as an ideal GBR scaffold [20]. In this study, the advantages of electrospinning technology and an active constituent of a Traditional Chinese Medicine (Rhizoma Drynariae) were combined for the first time. Naringin was incorporated into the electrospun nanofibers, which served as a controlled-release GBR scaffold. The properties of the controlled-release naringin scaffold (such as chemical, hydrophilic and morphological characteristics, drug release, material degradation, osteoblast-scaffold interactions, and osteoclast-scaffold interactions) were studied to evaluate its potential for the treatment of bone defects in osteoporosis patients.
2.
Materials and methods
2.1.
Materials
Poly--caprolactone (PCL) (Mw = 80,000), poly(ethylene glycol)block-poly(-caprolactone) (PEG-b-PCL) (PEG Mw = 5000; PCL Mw = 5000) and naringin were obtained from Sigma–Aldrich (St. Louis, MO). MC3T3-E1 osteoblast line and 4-day-old CD1 mice were obtained from the life science Institute of Chongqing Medical University. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), antibiotics and trypsin–EDTA from GIBCO Invitrogen (USA) were used.
2.2.
Electrospinning
PCL was dissolved in acetone to obtain a 15% (w/v) solution. PCL and PEG-b-PCL were mixed at a weight ratio of (1:1) to obtain PCL/PEG-b-PCL solution. Naringin (3.33 mg/ml) was added to PCL and PCL/PEG-b-PCL solution to obtain PCL/naringin and PCL/PEG-b-PCL/naringin solution. The solutions were separately loaded in 5 ml syringes, delivered at a constant flow rate (Q = 3 ml/h, Harvard Apparatus PHD 2000 syringe pump, Holliston, MA). A voltage of 15 kV was applied using a high voltage power supply (High Voltage System, Gamma High Voltage Research, FL, USA). A positively charged jet was formed from the Taylor Cone. Nanofibers were sprayed onto a grounded aluminum plate kept 12 cm away from the needle tip. Nanofibers were collected on the coverslips (10 mm × 10 mm) for cell and organ culture experiments, while those collected on the aluminum foil were used for Fourier-transform infrared spectroscopy (FT-IR), contact angle measurement, scanning electron microscopy (SEM), drug release and in vitro degradation studies. The
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
1265
electrospun nanofibers were stored in desiccators for several days to remove the residual acetone.
for 24 h. The weight loss of the scaffolds was calculated according to the following equations:
2.3.
W% =
FT-IR
FT-IR (Nicolet 5DXC spectrometer, USA) was used to qualitatively analyze and the materials before and after electrospinning. The naringin and PEG-b-PCL samples were pressed into pellets with KBr. The PCL samples were hot pressed into membranes. The controlled-release naringin nanoscaffolds were observed directly by FT-IR.
2.4.
Contact angle measurement
For determination of wettability of nanoscaffolds, the contact angles of electrospun nonwoven materials were measured by a video contact angle system (DSA100, Siber Hegner, China) mounted with a CCD camera at an ambient temperature. Distilled water was used as the test liquid. Each experiment was repeated six times. Means were shown as the results.
2.5.
Morphologies of nanoscaffolds
The morphologies of electrospun nanoscaffolds were observed by SEM (JSM-5600LV, JEOL, Japan) at an accelerating voltage of 30 kV after gold coating. The average fiber diameters of the electrospun nanofibers were measured using Image-ProPlus 5.02 (Media Cybernetics, USA).
2.6.
Drug release assay in vitro
A specimen of nanoscaffold (PCL/PEG-b-PCL/naringin and PCL/naringin) (20 mm × 20 mm) was first placed in a vial filled with 2 ml phosphate buffer solution (PBS). Naringin release studies were carried out at 37 ◦ C and 100 rotation/min (rpm) in a thermostatic shaking incubator (THZ-92A, GuangTe Instrument Co., Ltd., China). In this case, 0.5 ml samples were taken from the medium after 0, 1, 2, 6, 10, 14, 21, 30, 60, 90 days, and then the same volume of fresh PBS was added as replacement. Naringin in the medium was determined by a UV–vis spectrophotometer (Nanodrop2000, Thermo, USA) at the wavelength of 284 nm. The results were presented in terms of cumulative release as a function of release time: Cumulative amount of release (%) =
Mt × 100 M∞
where Mt was the amount of naringin released from a sample at time t. The total amount of naringin in a sample was calculated and regarded as M∞ in this study. Three samples were tested for each electrospun nanoscaffold and the results were reported as average values.
2.7.
Nanoscaffold degradation assay in vitro
PCL and PCL/PEG-b-PCL/naringin nanoscaffolds (20 mm × 20 mm) were placed in 24-well plates containing 1 ml PBS in each well and were incubated at 37 ◦ C for different periods of time (0, 10, 20, 30, 40, 50, 60 days). After each degradation period, the scaffolds were washed and subsequently dried in a vacuum oven at room temperature
Wi − Wt × 100 Wi
W% is the percentage of weight loss, Wi is the initial weight of the scaffold and Wt is the weight of the scaffold at the time t of the experiment after drying to constant weight.
2.8.
Osteoblast-nanoscaffold interactions
2.8.1.
Cell seeding and culture
Both sides of the nanoscaffolds were exposed to UV radiation for 2 h, washed three times with PBS for 20 min and incubated with DMEM for 24 h before cell seeding. MC3T3-E1 osteoblasts were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin. After reaching 80–90% confluence, the cells were detached by trypsin–EDTA. Each of the nanoscaffolds on coverslips (10 mm × 10 mm) was placed in a 24-well plate and pressed with cell crowns (Sigma–Aldrich, USA) to avoid possible floating during cell culture. Cells were further seeded on nanoscaffolds at a density of 2 × 104 cells/cm2 and cultured with DMEM at 37 ◦ C, 5% CO2 and 95% humidity. The cell-seeded nanoscaffolds were replenished with fresh medium every 3 days.
2.8.2.
Cell adhesion and proliferation by MTS
In order to observe the cell adhesion and proliferation at different times, viable cells were determined by colorimetric MTS assay (CellTiter 96® AQueous Assay). After 1, 2, 4 h and 2, 6, 10, 14 days of cell seeding in the 24-well plates, nanoscaffolds with MC3T3-E1 osteoblasts (constructs) were washed with PBS and incubated with 20% of MTS reagent containing serum free media. After 4 h of incubation at 37 ◦ C in 5% CO2 and 95% humidity, aliquots were pipetted into a 96-well plate and the absorbance was read at 490 nm using an ELISA microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA).
2.8.3.
Cell differentiation by ALP
The differentiation ability of MC3T3-E1 seeded on different nanoscaffolds was analyzed by the expression of alkaline phosphatase (ALP) activity of the cells. In this study, cells were cultured on the nanoscaffolds for 2, 6, 10 and 14 days, and then assessed for ALP activity. The cell-scaffold constructs were washed twice with PBS prior to analysis. To determine ALP activity, the adhered cells were lysed with 1% TritonX100 (Huamei Biological Engineering Co., China) overnight. A commercially available ALP Colorimetric Assay Kit (Jiancheng Biological Engineering Institute, Nanjing, China) was used to quantify ALP concentration in the lysate. The absorbance was measured at 405 nm using an ELISA microplate reader (BioTek Instruments Inc., Winooski, VT, USA). And converted to the units of ALP per liter.
2.8.4.
SEM observations
In order to observe cell morphologies on different nanoscaffolds, cells were fixed after 7 days of culture. A cellular construct was rinsed twice with PBS and subsequently fixed in 2% glutaraldehyde for 1.5 h at room temperature. Then each cellular construct was rinsed with distilled water, dehydrated
1266
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
with graded concentrations of ethanol solution and finally dried in hexamethyldisilazane overnight. Dried cellular constructs were sputter coated with gold and observed under SEM at an accelerating voltage of 20 kV.
2.8.5.
Alizarin Red-S (ARS) staining
Cellular constructs were washed five times in PBS and fixed in ice cold 70% ethanol for 1 h. Then these constructs were washed three times with distilled water and stained with 40 mM ARS for 30 min at room temperature. Microscopic images of the stain showing calcium deposition were taken.
2.9.
Osteoclast-scaffold interactions
2.9.1.
Mouse calvarial organ culture
The heads of 4-day-old CD-1 mice were provided by the life science Institute of Chongqing Medical University. The calvarial of mouse heads were exposed. The parietal bones were dissected out aseptically. During dissection, periosteum and dura mater were left intact on the bone surfaces. Full-thickness, 1.5 mm in diameter, circular defects were created through the parietal bones. The paired bones were divided into two groups. The critical size defect (CSD) model of mouse calvarial bone was based on our previous research [21]. In the present study, the CSD models were used for the evaluation of the responses of osteoclasts to PCL/PEG-b-PCL/naringin and PCL nanoscaffolds. These two types of scaffolds were cut into small disks (1.5 mm diameter) by a hollow steel tube, and then grafted into the bone defect. The bones with scaffolds were cultured concave side down on the tissue culture plate in DMEM with 10% FBS and 1% penicillin/streptomycin. Incubations were carried out at 37 ◦ C, 5% CO2 and 95% humidity. Media were changed every 3 days.
2.9.2. Osteoclast observing by tartrate resistant acid phosphatase (TRAP) staining After 14 days’ culture, the bones with scaffolds were stained for TRAP (Acid Phosphatase Kit 387-A; Sigma–Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. TRAP-positive cells appeared red or purple. Images were taken by transmitted light microscopy (Olympus IX71, Japan) with a digital camera (Canon 500D, Japan).
2.10.
Statistical analysis
All data presented are expressed as mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) and significance was considered at p < 0.05.
3.
Results
naringin (1644 cm−1 ) as referenced from the pure PCL, PEG-b-PCL and naringin. The result indicated that the chemical structures of PCL, PEG-b-PCL and naringin remained unchanged in the electrospinning process. Water contact angles of the nanoscaffolds are shown in Table 1. The contact angles of PCL and PCL/naringin scaffolds were about 141 ± 2.8◦ and 137 ± 2.1◦ , which implied that these scaffolds were hydrophobic. By contrast, PCL/PEGb-PCL and PCL/PEG-b-PCL/naringin scaffolds tended to be hydrophilic with their contact angles 26 ± 0.6◦ and 27 ± 0.5◦ , respectively. Their improved wettability was due to the presence of hydrophilic PEG segments on the surface of the nanofibers. All types of the nanoscaffolds showed nonwoven, bead free, porous and interconnected fibrous structures (Fig. 2). The diameters of PCL, PCL/naringin, PCL/PEG-b-PCL and PCL/PEGb-PCL/naringin nanofibers were 356 ± 32 nm, 367 ± 38 nm, 242 ± 21 nm and 248 ± 27 nm, respectively (Table 1). The fiber diameters of PCL/PEG-b-PCL and PCL/PEG-b-PCL/naringin nanofiber were similar. The nanofibers (Fig. 2c and d) containing PEG-b-PCL were found to be significantly (p < 0.05) thinner and more uniform than PCL and PCL/naringin nanofibers (Fig. 2a and b).
3.2. Naringin release and nanoscaffold degradation assay in vitro Cumulative naringin release profiles of PCL/naringin and PCL/PEG-b-PCL/naringin nanoscaffolds are shown in Fig. 3. Naringin was released rapidly as much as 63% from PCL/naringin nanoscaffolds on the first day, followed by a slow rise from the 2nd to 6th day, and was barely detected after 30 days. The cumulative release at the end of the experiment was 78%. Compared with the abrupt initial burst in the PCL/naringin scaffold, a much lower initial burst was detected in PCL/PEG-b-PCL/naringin scaffold (less than 20% within a day). The naringin embedded within the PCL/PEG-b-PCL/naringin was released gradually over a 90-day period, and the final cumulative release was 93%. The weight loss vs. time of the nanoscaffolds is presented in Fig. 4. Weight loss of the pure PCL scaffold was less than 10% during the period of 60 days, while the PCL/PEGb-PCL/naringin scaffold degraded much faster, losing 55% weight. This result showed that adding PEG-b-PCL into the scaffold could accelerate the rate of degradation.
Table 1 – Fiber diameter and contact angles of different electrospun nanoscaffolds (n = 6).
3.1. Chemical, hydrophilic and morphological characteristics of nanoscaffolds
Samples
Fig. 1 shows the FT-IR spectra of the electrospun PCL/PEGb-PCL/naringin nanoscaffolds and their composition. The PCL/PEG-b-PCL/naringin nanoscaffold showed the mixed bands of PCL (2943, 1243 cm−1 ), PCL-b-PEG (1724 cm−1 ) and
PCL PCL/naringin PCL/PEG-b-PCL PCL/PEG-bPCL/naringin
Fiber diameter 356 367 242 248
± ± ± ±
32 nm 38 nm 21 nm 27 nm
Contact angle in degree 141 137 26 27
± ± ± ±
2.8◦ 2.1◦ 0.6◦ 0.5◦
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
1267
Fig. 1 – Fourier-transform infrared spectra of (a) PCL, (b) PEG-b-PCL, (c) naringin and (d) electrospun PCL/PEG-b-PCL/naringin nanoscaffolds.
Fig. 2 – SEM images of electrospun: (a) PCL, (b) PCL/naringin, (c) PCL/PEG-b-PCL and (d) PCL/PEG-b-PCL/naringin nanoscaffolds.
1268
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
Fig. 3 – The naringin release profiles from the PCL/naringin and PCL/PEG-b-PCL/naringin nanoscaffolds.
Fig. 5 – The MC3T3-E1 adhesion on PCL, PCL/PEG-b-PCL, PCL/naringin and PCL/PEG-b-PCL/naringin nanoscaffolds (n = 3). Bar represent means + SD. a: p < 0.05 vs. PCL nanoscaffolds.
Fig. 4 – Weight loss vs. immersion time for PCL and PCL/PEG-b-PCL/naringin nanoscaffolds.
3.3.
Osteoblast-nanoscaffold interactions
The osteoblast attachment and proliferation behavior on the nanoscaffolds were evaluated by MTS assay after a short-term (4 h) and a long-term period (14 days). Fig. 5 shows the measure of osteoblast attachment on different nanoscaffolds. Data on all nanoscaffolds increased with time. However, the data of PCL/PEG-b-PCL and PCL/PEG-b-PCL/naringin groups were significantly higher than PCL groups at 1, 2, 4 h (p < 0.05). The proliferation of osteoblasts was evaluated after 2, 6, 10 and 14 days by MTS assay (Fig. 6). The data of PCL/naringin and PCL/PEG-b-PCL/naringin groups were higher than those for the PCL group at all time points (p < 0.05). The proliferation of osteoblasts on the PCL/PEG-b-PCL/naringin nanoscaffolds was significantly higher than all the other groups on the 14th day. The ALP activity of MC3T3-E1 osteoblasts cultured on the nanoscaffolds after 2, 6, 10 and 14 days of cell seeding are shown in Fig. 7. After 6 days of culture, the ALP activities of osteoblasts cultured on the PCL/PEG-b-PCL/naringin and PCL/naringin nanoscaffolds were higher than that on
Fig. 6 – The MC3T3-E1 proliferation on PCL, PCL/PEG-b-PCL, PCL/naringin and PCL/PEG-b-PCL/naringin nanoscaffolds (n = 3). Bar represent means + SD. a: p < 0.05 vs. PCL nanoscaffolds; b: p < 0.05 vs. PCL/naringin nanoscaffolds.
PCL nanoscaffolds (p < 0.05). Moreover, the ALP activity of osteoblasts on PCL/PEG-b-PCL/naringin nanoscaffolds was significantly higher than all the other groups on the 14th day. SEM images of the PCL/PEG-b-PCL/naringin nanoscaffolds with osteoblasts were taken after 7 days’ culture. Mineralization nodules were observed on the basal surfaces of nanoscaffolds (Fig. 8a). Moreover, multilayers of mineral deposits were detected on the osteoblasts (Fig. 8c) of PCL/PEGb-PCL/naringin nanoscaffolds, while few mineral deposits were detected on the osteoblasts of PCL nanoscaffolds (Fig. 8d). Mineralization nodules on the 7th day are shown in Fig. 8a and b at different magnifications. These nodules were further evidenced by ARS staining, which was used to detect mineralization [22]. Fig. 9 shows images of the stained nanoscaffolds under the optical microscope after 10 days of cell culture. ARS
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
1269
were observed on the PCL/PEG-b-PCL/naringin nanoscaffolds, which showed the presence of calcium (Fig. 9a). However, similar stains were not observed on the PCL nanoscaffolds (Fig. 9b).
3.4.
Osteoclast-nanoscaffold interactions
TRAP, an isoenzyme of acid phosphatase, is the specific enzyme of the osteoclast [23]. As shown in Fig. 10, the bone defect treated with PCL/PEG-b-PCL/naringin nanoscaffold (left one) stained redder than that with the PCL nanoscaffold (right one). The microscopic images showed that the density of osteoclasts in bone with a PCL/PEG-b-PCL/naringin nanoscaffold was much lower than that in bone with a PCL nanoscaffold (Fig. 11). The results suggested that PCL/PEG-b-PCL/naringin nanoscaffold was effective for suppressing osteoclast.
Fig. 7 – ALP activity of MC3T3-E1 cultured on PCL, PCL/PEG-b-PCL, PCL/naringin and PCL/PEG-b-PCL/naringin nanoscaffolds. Bar represent means + SD. a: p < 0.05 vs. PCL nanoscaffolds; b: p < 0.05 vs. PCL/naringin nanoscaffolds.
4.
Discussion
Degradable biopolymer with nanofibrous structures have attracted great interest as promising drug delivery systems and GBR scaffolds [24]. Due to its lack of toxicity, low cost and slow degradation, PCL was first suggested to be a degradable
Fig. 8 – SEM images of MC3T3-E1 osteoblasts on PCL/PEG-b-PCL/naringin and PCL nanofscaffolds after 7 days’ culture. (a) Mineralization nodules on PCL/PEG-b-PCL/naringin nanoscaffolds (2000×), (b) mineralization nodules on PCL/PEG-b-PCL/naringin nanoscaffolds (1200×), (c) multilayers of mineralization on the surfaces of osteoblasts on PCL/PEG-b-PCL/naringin nanoscaffolds (1000×), and (d) few mineral deposits on the surfaces of osteoblasts on PCL nanoscaffolds (1200×).
1270
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
Fig. 9 – Alizarin Red-S staining for calcium mineralization in MC3T3-E1 osteoblasts on (a) PCL/PEG-b-PCL/naringin (200×) and (b) PCL nanoscaffolds after 10 days’ culture (200×).
nanofiber matrix for bone regeneration in vitro [25]. However, osteoporosis weakens the bonding strength between the scaffold and bone, delays bone defect repair and lowers the quality of new bone formation [1,2]. Naringin, isoflavonoids isolated from plants, has been shown to reduce bone resorption and had an osteogenic effect on osteoblasts [10]. However, the hydrophilic naringin is difficult to dissolve in the hydrophobic PCL/acetone solvent systems and is not compatible with the hydrophobic polymers, which leads to phase separation during electrospinning. So successful incorporation and sustained release of hydrophilic naringin is a challenge. PEGb-PCL, like other types of amphiphilic block copolymers, has been reported to incorporate hydrophilic drugs into hydrophobic polymers by electrospinning to produce controlled-release nanofiberous scaffolds [26]. In this study, PEG-b-PCL was added into the PCL/naringin/acetone system to form a transparent and homogenous mixed solution. When the solution was electrospun, naringin could be embedded into the hydrophobic polymer fibers.
Fig. 10 – Macro image of TRAP staining for osteoclasts on mouse calvarial bone after 14 days’ culture. (left one) The critical size defects of mouse calvarial bone grafted with PCL nanoscaffold; (right one) the critical size defects of mouse calvarial bone grafted with PCL/PEG-b-PCL/naringin nanoscaffold.
During the electrospinning process, random nanofibers were formed. PEG-b-PCL could assist in achieving thinner and more uniform medicated fibers in this study, probably due to improved solubilization of the drug and polymer in a solvent. The solution with improved solubilization could achieve strong combinations through the process of electrospinning and avoid phase separation [27]. The reduced diameters of the fibers also suggested that the drug was dispersed homogeneously in the electrospun fibers [28]. The greatly (p < 0.05) reduced diameters have greater surface areas, which are beneficial to cell adhesion and drug distribution. Besides a desired range of fiber diameters, surface wettability is also an important physicochemical property that could affect the cell behavior. Some studies indicated that cells tended to attach and spread much more widely on hydrophilic surfaces than on hydrophobic surfaces [29–32], but there was no significant difference in cell proliferation among these surfaces [32]. Table 1 shows that PEG-b-PCL remarkably changed the wettability of both PCL/PEG-b-PCL and PCL/PEGb-PCL/naringin nanoscaffolds. FT-IR results of PCL/PEG-b-PCL/naringin nanoscaffolds showed the mixed vibrational absorbance bands of pure PCL, PCL-b-PEG and naringin as referenced, and indicated that the chemical structures of PCL, PEG-b-PCL and naringin remained unchanged even though they had been subjected to a high electrical voltage. The initial (first day) drug release was as high as 63% from the PCL/naringin nanoscaffolds due to the drug not being encapsulated efficiently. Like other polyester biopolymers, PCL is quite hydrophobic. Hydrophilic naringin could not be encapsulated effectively by conventional electrospinning techniques. Naringin covered on the surfaces of the fibers directly diffused in the medium, which caused the initial burst release followed by a plateau after 2 days. By contrast, PCL/PEG-b-PCL/naringin nanoscaffolds showed a better naringin release behavior. It released low concentrations with a slow-release effect. The release period was prolonged to 90 days and the total release was more than 90%. Naringin was embedded into the PCL/PEG-b-PCL/naringin nanofibers. Thus,
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
1271
Fig. 11 – Microscopic images of TRAP staining for osteoclast on mouse calvarial bone after 14 days’ culture. (a) The critical size defects of mouse calvarial bone grafted with PCL/PEG-b-PCL/naringin nanoscaffold (200×). (b) The critical size defects of mouse calvarial bone grafted with PCL nanoscaffold (200×). B: bone and S: scaffold.
naringin released steadily and slowly. The polymer fibers gradually degraded, rather than directly diffused into the medium. Moreover, the cumulative release of PCL/PEG-b-PCL/naringin nanoscaffolds was 93%, which was greater than the control group (78%). The narrower the fiber diameters, the larger the total surface areas of the scaffolds for the same mass were. Thus, more naringin could be loaded within the PCL/PEG-bPCL/naringin nanoscaffolds. The addition of amphiphilic block copolymers PEG-bPCL not only showed controlled-release behavior but also increased the scaffold degradation rate [26]. PCL is a semicrystalline aliphatic polymer and has a slower degradation rate than most biopolymers [33]. PCL is characterized by a degradation time in excess of 1 year [34]. However, reconstruction of human bone tissue usually takes about 17 weeks [35]. Therefore, it is necessary to speed up the scaffold’s degradation rate. The high water uptake of PEG-b-PCL may increase PCL degradation by providing water channels since PCL polyester is degraded mainly via a hydrolytic process. Also, according to Bolgen et al. [36], PCL nanofibers with thinner diameter size degraded faster than thicker ones. Although PCL is relatively stable against in vitro hydrolysis, it is known that microorganisms can degrade it [37]. So further studies in vivo are needed to adjust the degradation rate to a suitable range. The MC3T3-E1 pre-osteoblastic cell line is a well-accepted model for osteogenesis study in vitro, as it differentiates into post-mitotic osteoblasts capable of expressing the osteogenic phenotype [38]. Traditional osteoblast culture medium is alpha-MEM which is a nutrient-rich medium and can promote osteogenesis. However, in order to observe the osteogenic effect of naringin, DMEM was used [39]. Based on the shortterm MTS assay, cells attached rapidly on the nanoscaffolds containing PEG-b-PCL (1, 2, 4 h, p < 0.05) (Fig. 5). This could be attributed to the hydrophilic segments PEG on the surfaces of PCL/PEG-b-PCL and PCL/PEG-b-PCL/naringin nanoscaffolds. The hydrophilic surface provides more suitable conditions for initial cell adhesion. Some studies showed that the adhesive protein fibronectin known to promote cell adhesion tended
to adhere onto a hydrophilic surface [40,41]. However, no significant difference was observed in osteoblast proliferation between the hydrophilic and hydrophilic surfaces. This result was in accordance with that reported by Wei et al. [32]. Previously, naringin has been reported to improve the proliferation of osteoblasts [42]. In this study, PCL/naringin nanoscaffolds showed improved osteoblast proliferation on the 2nd day, which was consistent with the burst release of naringin in PCL/naringin nanoscaffolds for the first 2 days. However, naringin release from PCL/naringin nanoscaffolds gradually declined after 6 days, meanwhile, naringin continued to be released from PCL/PEG-b-PCL/naringin nanoscaffolds. Thus, the osteoblast proliferation on PCL/PEG-b-PCL/naringin nanoscaffolds was significantly (14 d, p < 0.05) higher than that on PCL/naringin nanoscaffolds (Fig. 6). ALP is a key component of bone matrix vesicles because of its role in the formation of apatitic calcium phosphate and it is an early indicator of immature osteoblast activity [43]. All ALP levels increased with culture time, but ALP levels of the nanoscaffolds with naringin were significantly higher than those of the PCL nanoscaffold on the 6th, 10th and 14th day. In agreement with a previous report [42], a certain concentration of naringin can affect cell differentiation. There are significant differences of ALP activity between the PCL/naringin and PCL/PEG-b-PCL/naringin nanoscaffold on the 14th day. Up to 63% naringin released from the PCL/naringin nanoscaffolds was lost on the first day because the culture medium was replenished every 3 days. Then, the released naringin continued to fall. So the significant difference between PCL/naringin and PCL/PEG-b-PCL/naringin nanoscaffold was not apparent until the 14th day. These results indicated that PCL/PEG-bPCL/naringin nanoscaffolds were superior in supporting cell attachment, proliferation and differentiation. The naringin controlled released from PCL/PEG-b-PCL/naringin nanoscaffolds could promote long-term bone formation. SEM images and the subsequent ARS staining results indicated that osteoblasts seeded on PCL/PEG-b-PCL/naringin nanoscaffolds displayed accelerated mineralization compared
1272
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
to PCL nanoscaffolds. This might be explained in two ways. First, the addition of PEG-b-PCL changed the nanoscaffold surface morphology to a hydrophilic condition, which helped initial osteoblast adhesion. Consequently, a large number of osteoblasts synthesized and secreted ECM to form nodules for bone formation. Second, naringin has been shown to have an HMG-CoA reductase inhibiting effect [11]. Therefore, it is possible that naringin may also activate the BMP-2 promotor and increase bone formation [11]. BMP-2 has been extensively studied and demonstrated to play a crucial role in inducing osteoblast differentiation and bone matrix calcification during embryonic skeletal development and postnatal bone remodeling [44–47]. On the other hand, naringin decreases bone resorption by suppressing osteoclast formation [8–10]. Ang et al. [48] showed that naringin perturbed osteoclast formation and bone resorption by inhibiting RANK-mediated NF-B and ERK signaling. Naringin suppressed gene expression of key osteoclast marker genes. Naringin was found to inhibit RANKL-induced activation of NF-B by suppressing RANKLmediated IB-␣ degradation. In addition, naringin inhibited RANKL-induced phosphorylation of ERK. The CSD in vitro model was used [49] to evaluate the responses of osteoclasts to scaffolds. PCL/PEG-b-PCL/naringin nanoscaffolds inhibited bone resorption by decreasing osteoclast formation.
5.
Conclusion
In this study, an active constituent of the Traditional Chinese Medicine, naringin, regarded as an effective anti-osteoporosis drug, was encapsulated in PCL/PEG-b-PCL nanofibers using electrospinning for the first time. The introduction of PEGb-PCL significantly improved the hydrophilic properties, drug release behavior and the material degradation rate of the scaffolds. The controlled-release naringin nanoscaffolds, which were shown to enhance functions of osteoblasts and suppress formation of osteoclasts, showed the potential to be used as GBR scaffolds for the repair of osteoporotic bone defects.
Acknowledgements X. Wu is grateful to the financial support from the national Natural Science Foundation of China (grant number 81200767/H1402), Chongqing Science & Technology Commission, China (grant number CSTC 2010BB5102) and Chongqing Municipal Health Bureau, China (grant number 2012-2-8).
references
[1] Erdogan O, Shafer DM, Taxel P, Freilich MA. A review of the association between osteoporosis and alveolar ridge augmentation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;104:738 e1–413e. [2] Qi M, Hu J, Li J, Dong W, Feng X, Yu J. Effect of zoledronate acid treatment on osseointegration and fixation of implants in autologous iliac bone grafts in ovariectomized rabbits. Bone 2012;50:119–27. [3] Rodan GA, Martin TJ. Therapeutic approaches to bone diseases. Science 2000;289:1508–14.
[4] Salesi N, Pistilli R, Marcelli V, Govoni FA, Bozza F, Bossone G, et al. Bisphosphonates and oral cavity avascular bone necrosis: a review of twelve cases. Anticancer Res 2006;26:3111–5. [5] Merigo E, Manfredi M, Meleti M, Guidotti R, Ripasarti A, Zanzucchi E, et al. Bone necrosis of the jaws associated with bisphosphonate treatment: a report of twenty-nine cases. Acta Biomed 2006;77:109–17. [6] Wang XL, Wang NL, Zhang Y, Gao H, Pang WY, Wong MS, et al. Effects of eleven flavonoids from the osteoprotective fraction of Drynaria fortunei (KUNZE) J. SM. on osteoblastic proliferation using an osteoblast-like cell line. Chem Pharm Bull (Tokyo) 2008;56:46–51. [7] Jeong JC, Lee JW, Yoon CH, Lee YC, Chung KH, Kim MG, et al. Stimulative effects of Drynariae Rhizoma extracts on the proliferation and differentiation of osteoblastic MC3T3-E1 cells. J Ethnopharmacol 2005;96:489–95. [8] Jeong JC, Kang SK, Youn CH, Jeong CW, Kim HM, Lee YC, et al. Inhibition of Drynariae Rhizoma extracts on bone resorption mediated by processing of cathepsin K in cultured mouse osteoclasts. Int Immunopharmacol 2003;3:1685–97. [9] Hirata M. Naringin suppresses osteoclast formation and enhances bone mass in mice. J Health Sci 2009;55: 463–7. [10] Chen LL, Lei LH, Ding PH, Tang Q, Wu YM. Osteogenic effect of Drynariae rhizoma extracts and Naringin on MC3T3-E1 cells and an induced rat alveolar bone resorption model. Arch Oral Biol 2011;56:1655–62. [11] Wong RW, Rabie AB. Effect of naringin on bone cells. J Orthop Res 2006;24:2045–50. [12] Wu JB, Fong YC, Tsai HY, Chen YF, Tsuzuki M, Tang CH. Naringin-induced bone morphogenetic protein-2 expression via PI3K, Akt, c-Fos/c-Jun and AP-1 pathway in osteoblasts. Eur J Pharmacol 2008;588:333–41. [13] Wei M, Yang Z, Li P, Zhang Y, Sse WC. Anti-osteoporosis activity of naringin in the retinoic acid-induced osteoporosis model. Am J Chin Med 2007;35:663–7. [14] Mandadi K, Ramirez M, Jayaprakasha GK, Faraji B, Lihono M, Deyhim F, et al. Citrus bioactive compounds improve bone quality and plasma antioxidant activity in orchidectomized rats. Phytomedicine 2009;16:513–20. [15] Sui G, Yang X, Mei F, Hu X, Chen G, Deng X, et al. Poly-l-lactic acid/hydroxyapatite hybrid membrane for bone tissue regeneration. J Biomed Mater Res A 2007;82: 445–54. [16] Nie H, Soh BW, Fu YC, Wang CH. Three-dimensional fibrous PLGA/HAp composite scaffold for BMP-2 delivery. Biotechnol Bioeng 2008;99:223–34. [17] Sahoo S, Ouyang H, Goh JC, Tay TE, Toh SL. Characterization of a novel polymeric scaffold for potential application in tendon/ligament tissue engineering. Tissue Eng 2006;12:91–9. [18] Lee SJ, Yoo JJ, Lim GJ, Atala A, Stitzel J. In vitro evaluation of electrospun nanofiber scaffolds for vascular graft application. J Biomed Mater Res A 2007;83:999–1008. [19] Schnell E, Klinkhammer K, Balzer S, Brook G, Klee D, Dalton P, et al. Guidance of glial cell migration and axonal growth on electrospun nanofibers of poly-epsilon-caprolactone and a collagen/poly-epsilon-caprolactone blend. Biomaterials 2007;28:3012–25. [20] Venugopal J, Low S, Choon AT, Kumar AB, Ramakrishna S. Electrospun-modified nanofibrous scaffolds for the mineralization of osteoblast cells. J Biomed Mater Res A 2008;85:408–17. [21] Wu X, Downes S, Watts DC. Evaluation of critical size defects of mouse calvarial bone: an organ culture study. Microsc Res Tech 2010;73:540–7. [22] Gregory CA, Gunn WG, Peister A, Prockop DJ. An Alizarin red-based assay of mineralization by adherent cells in
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
culture: comparison with cetylpyridinium chloride extraction. Anal Biochem 2004;329:77–84. Janckila AJ, Yam LT. Biology and clinical significance of tartrate-resistant acid phosphatases: new perspectives on an old enzyme. Calcif Tissue Int 2009;85:465–83. Sill TJ, von Recum HA. Electrospinning: applications in drug delivery and tissue engineering. Biomaterials 2008;29:1989–2006. Yoshimoto H, Shin YM, Terai H, Vacanti JP. A biodegradable nanofiber scaffold by electrospinning and its potential for bone tissue engineering. Biomaterials 2003;24: 2077–82. Kim K, Luu YK, Chang C, Fang D, Hsiao BS, Chu B, et al. Incorporation and controlled release of a hydrophilic antibiotic using poly(lactide-co-glycolide)-based electrospun nanofibrous scaffolds. J Control Release 2004;98:47–56. Yan J, Ye Z, Chen M, Liu Z, Xiao Y, Zhang Y, et al. Fine tuning micellar core-forming block of poly(ethylene glycol)-block-poly(epsilon-caprolactone) amphiphilic copolymers based on chemical modification for the solubilization and delivery of doxorubicin. Biomacromolecules 2011;12:2562–72. Xu X, Chen X, Ma P, Wang X, Jing X. The release behavior of doxorubicin hydrochloride from medicated fibers prepared by emulsion-electrospinning. Eur J Pharm Biopharm 2008;70:165–70. Chen M, Zamora PO, Som P, Pena LA, Osaki S. Cell attachment and biocompatibility of polytetrafluoroethylene (PTFE) treated with glow-discharge plasma of mixed ammonia and oxygen. J Biomater Sci Polym Ed 2003;14:917–35. Ozdemir Y, Hasirci N, Serbetci K. Oxygen plasma modification of polyurethane membranes. J Mater Sci Mater Med 2002;13:1147–51. Altankov G, Grinnell F, Groth T. Studies on the biocompatibility of materials: fibroblast reorganization of substratum-bound fibronectin on surfaces varying in wettability. J Biomed Mater Res 1996;30:385–91. Wei J, Yoshinari M, Takemoto S, Hattori M, Kawada E, Liu B, et al. Adhesion of mouse fibroblasts on hexamethyldisiloxane surfaces with wide range of wettability. J Biomed Mater Res B: Appl Biomater 2007;81:66–75. Shor L, Guceri S, Wen X, Gandhi M, Sun W. Fabrication of three-dimensional polycaprolactone/hydroxyapatite tissue scaffolds and osteoblast-scaffold interactions in vitro. Biomaterials 2007;28:5291–7. Pitt CG, Gratzl MM, Kimmel GL, Surles J, Schindler A. Aliphatic polyesters II. The degradation of poly (dl-lactide), poly (epsilon-caprolactone), and their copolymers in vivo. Biomaterials 1981;2:215–20. Weng B. Development of resorbable potentials for bone regeneration. In: Symposium on: difficult treatment option for bone regeneration. 1995. Bolgen N, Menceloglu YZ, Acatay K, Vargel I, Piskin E. In vitro and in vivo degradation of non-woven materials made of
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
1273
poly(epsilon-caprolactone) nanofibers prepared by electrospinning under different conditions. J Biomater Sci Polym Ed 2005;16:1537–55. Singh NK, Das Purkayastha B, Roy JK, Banik RM, Yashpal M, Singh G, et al. Nanoparticle-induced controlled biodegradation and its mechanism in poly(epsilon-caprolactone). ACS Appl Mater Interfaces 2010;2:69–81. Pereira CT, Huang W, Sayer G, Jarrahy R, Rudkin G, Miller TA. Osteogenic potential of polymer-bound bone morphogenetic protein-2 on MC3T3-E1 two-dimensional cell cultures. Plast Reconstr Surg 2009;124:2199–200 [author reply 200]. Coelho MJ, Fernandes MH. Human bone cell cultures in biocompatibility testing. Part II: effect of ascorbic acid, beta-glycerophosphate and dexamethasone on osteoblastic differentiation. Biomaterials 2000;21:1095–102. Howlett CR, Evans MD, Wildish KL, Kelly JC, Fisher LR, Francis GW, et al. The effect of ion implantation on cellular adhesion. Clin Mater 1993;14:57–64. Hao L, Lawrence J. On the role of CO2 laser treatment in the human serum albumin and human plasma fibronectin adsorption on zirconia (MGO-PSZ) bioceramic surface. J Biomed Mater Res A 2004;69:748–56. Zhang P, Dai KR, Yan SG, Yan WQ, Zhang C, Chen DQ, et al. Effects of naringin on the proliferation and osteogenic differentiation of human bone mesenchymal stem cell. Eur J Pharmacol 2009;607:1–5. Luben RA, Wong GL, Cohn DV. Biochemical characterization with parathormone and calcitonin of isolated bone cells: provisional identification of osteoclasts and osteoblasts. Endocrinology 1976;99:526–34. Suzuki A, Ghayor C, Guicheux J, Magne D, Quillard S, Kakita A, et al. Enhanced expression of the inorganic phosphate transporter Pit-1 is involved in BMP-2-induced matrix mineralization in osteoblast-like cells. J Bone Miner Res 2006;21:674–83. Suzuki D, Yamada A, Aizawa R, Funato S, Matsumoto T, Suzuki W, et al. BMP2 differentially regulates the expression of Gremlin1 and Gremlin2, the negative regulators of BMP function, during osteoblast differentiation. Calcif Tissue Int 2012;91:88–96. Miron RJ, Saulacic N, Buser D, Iizuka T, Sculean A. Osteoblast proliferation and differentiation on a barrier membrane in combination with BMP2 and TGFbeta1. Clin Oral Investig 2013;17(3):981–8. Rawadi G, Vayssiere B, Dunn F, Baron R, Roman-Roman S. BMP-2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop. J Bone Miner Res 2003;18:1842–53. Ang ES, Yang X, Chen H, Liu Q, Zheng MH, Xu J. Naringin abrogates osteoclastogenesis and bone resorption via the inhibition of RANKL-induced NF-kappaB and ERK activation. FEBS Lett 2011;585:2755–62. Schmitz JP, Hollinger JO. The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop Relat Res 1986;29:9–308.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/dema
Controlled-release naringin nanoscaffold for osteoporotic bone healing Yan Ji a,b , Lu Wang a,b , David C. Watts e,f , Hongmei Qiu d , Tao You a,b , Feng Deng b,c , Xiaohong Wu a,b,∗ a
Department of Prosthodontics, Stomatological Hospital of Chongqing Medical University, No. 426 Songshibei Road, Yubei District, Chongqing 401147, China b Chongqing key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing 401147, China c Department of Orthodontics, Stomatological Hospital of Chongqing Medical University, No. 426 Songshibei Road, Yubei District, Chongqing 401147, China d Key Laboratory of Biochemistry and Molecular Pharmacology, Department of Pharmacology, School of Pharmacy, Chongqing Medical University, Yixueyuan Road, Yuzhong District, Chongqing 400016, China e School of Dentistry and Photon Science Institute, The University of Manchester, Oxford Road, Manchester M13 9PL, UK f Institute of Material Science and Technology, Friedrich-Schiller-University, Jena, Löbdergraben 32, 07743, Germany
a r t i c l e
i n f o
a b s t r a c t Objectives. Osteoporosis is one of the most common bone diseases in the world and results from an imbalance of bone cell functions. In the process of guided bone regeneration, osteo-
Keywords:
porosis weakens the bonding strength between scaffold and bone. Naringin is evidenced to
Bone tissue regeneration
be effective for the treatment of osteoporosis and bone resorption and the aim was to explore
Electrospinning
methods and benefits of its incorporation.
Nanoscaffold
Methods. In this study, naringin was incorporated in the electrospun nanoscaffold containing
Naringin
poly(-caprolactone) (PCL) and poly(ethylene glycol)-block-poly(-caprolactone) (PEG-b-PCL).
Osteoporosis
Results. The nanoscaffold demonstrated unchanged chemical structure, improved hydrophilicity, thinner and more uniform nanofibers by Fourier-transform infrared spectroscopy, contact angle measurement and scanning electron microscopy. The nanoscaffold also showed faster degradation rate and controlled-release of naringin. Osteoblastnanoscaffold interactions were studied by the evaluation of adhesion, proliferation, differentiation of MC3T3-E1 osteoblasts and mineralization of ECM on the nanoscaffolds. Meanwhile, the response of osteoclasts to nanoscaffolds was evaluated in a mouse calvarial critical size defect organ culture model. The osteoclasts around the bone defect were shown by tartrate resistant acid phosphatase staining.
∗ Corresponding author at: Department of Prosthodontics, Stomatological Hospital of Chongqing Medical University, No. 426 Songshibei Road, Yubei District, Chongqing 401147, China. Tel.: +86 23 88860086; fax: +86 23 88860086. E-mail address: [email protected] (X. Wu). http://dx.doi.org/10.1016/j.dental.2014.08.381 0109-5641/© 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1264
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
Significance. The results demonstrated that controlled-release naringin nanoscaffolds supported greater osteoblast adhesion, proliferation, differentiation, and mineralization and suppressed osteoclast formation. © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Extensive craniomaxillofacial bone defects caused by tumor, infection, trauma and dysplasia are regularly repaired by bone substitution materials. However, the restorative effect depends not only on the implant properties but also on the characteristics of the host bone. Researchers pay increasing attention to the patients’ own diseases. Among them, osteoporosis is the most common systemic skeletal disorder that weakens the bonding strength between the implants and bone, delays bone defect repair, lowers the quality of new bone formation and even causes prosthesis failure [1,2]. With the aging of populations, osteoporosis is a still increasing prevalence. As a part of the whole body bone, craniomaxillofacial bone is inevitably influenced by osteoporosis, showing reduced bone mass, bone mineral content and bone density, deterioration of the microstructure of the trabecular bone and lower osteogenesis ability. Although osteoporosis is not an absolute contraindication for bone repair, it increases implant failure and the risk of complications. Therefore, the treatment of bone defects in osteoporosis patients remains a challenge in dental medicine. Osteoporosis occurs when bone resorption is greater than bone formation. Thus, osteoclasts and osteoblasts imbalance correction may be a therapeutic modality for bone repair of the patients with osteoporosis. The most widely used drugs to treat osteoporosis are bisphosphonates. Bisphosphonates maintain bone mass by inhibiting the function of osteoclasts [3]. However, bisphosphates have been reported to cause osteoclast dysfunction and even osteonecrosis [4,5]. Researchers showed that naringin plays a dual role in regulation of osteoblasts and osteoclasts. Naringin, a polymethoxylated flavonoid, is an active constituent of Rhizoma Drynariae (Traditional Chinese Medicine). Naringin has been reported to stimulate proliferation and differentiation of osteoblastic cell lines [6,7] and inhibit osteoclast formation [8–10]. Naringin has been shown to inhibit HMG-CoA reductase, activate the BMP-2 promotor, increase bone formation [11] and induce the expression of osteogenic markers of osteoblasts [12]. Naringin can also dosedependently suppress the number of osteoclasts formed by the treatment with interleukin-1 (IL-1) [9]. Chen et al. [10] has reported that naringin increased alkaline phosphatase activity, osteocalcin level, bone mineral density and decreased the number of osteoclasts. In addition, naringin has been shown to improve bone quality of rats with osteoporosis induced by retinoic acid [13] and orchidectomized rats [14]. Guided bone regeneration (GBR) is a method to repair bone defects, which emerged with the development of biology and bioengineering. GBR scaffolds are used as barriers to prevent soft tissue ingrowth and create space for slowly regenerating periodontal and bony tissues. For the past few years, the elec-
trospinning technique has been used widely in fabrication of scaffolds for bone [15,16], tendon [17], vasculature [18] and neural [19] tissue regeneration. Electrospun scaffolds with a large surface area-to-volume ratio, high porosity and morphology similar to the extracellular matrix (ECM) of natural tissue can serve as an ideal GBR scaffold [20]. In this study, the advantages of electrospinning technology and an active constituent of a Traditional Chinese Medicine (Rhizoma Drynariae) were combined for the first time. Naringin was incorporated into the electrospun nanofibers, which served as a controlled-release GBR scaffold. The properties of the controlled-release naringin scaffold (such as chemical, hydrophilic and morphological characteristics, drug release, material degradation, osteoblast-scaffold interactions, and osteoclast-scaffold interactions) were studied to evaluate its potential for the treatment of bone defects in osteoporosis patients.
2.
Materials and methods
2.1.
Materials
Poly--caprolactone (PCL) (Mw = 80,000), poly(ethylene glycol)block-poly(-caprolactone) (PEG-b-PCL) (PEG Mw = 5000; PCL Mw = 5000) and naringin were obtained from Sigma–Aldrich (St. Louis, MO). MC3T3-E1 osteoblast line and 4-day-old CD1 mice were obtained from the life science Institute of Chongqing Medical University. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), antibiotics and trypsin–EDTA from GIBCO Invitrogen (USA) were used.
2.2.
Electrospinning
PCL was dissolved in acetone to obtain a 15% (w/v) solution. PCL and PEG-b-PCL were mixed at a weight ratio of (1:1) to obtain PCL/PEG-b-PCL solution. Naringin (3.33 mg/ml) was added to PCL and PCL/PEG-b-PCL solution to obtain PCL/naringin and PCL/PEG-b-PCL/naringin solution. The solutions were separately loaded in 5 ml syringes, delivered at a constant flow rate (Q = 3 ml/h, Harvard Apparatus PHD 2000 syringe pump, Holliston, MA). A voltage of 15 kV was applied using a high voltage power supply (High Voltage System, Gamma High Voltage Research, FL, USA). A positively charged jet was formed from the Taylor Cone. Nanofibers were sprayed onto a grounded aluminum plate kept 12 cm away from the needle tip. Nanofibers were collected on the coverslips (10 mm × 10 mm) for cell and organ culture experiments, while those collected on the aluminum foil were used for Fourier-transform infrared spectroscopy (FT-IR), contact angle measurement, scanning electron microscopy (SEM), drug release and in vitro degradation studies. The
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
1265
electrospun nanofibers were stored in desiccators for several days to remove the residual acetone.
for 24 h. The weight loss of the scaffolds was calculated according to the following equations:
2.3.
W% =
FT-IR
FT-IR (Nicolet 5DXC spectrometer, USA) was used to qualitatively analyze and the materials before and after electrospinning. The naringin and PEG-b-PCL samples were pressed into pellets with KBr. The PCL samples were hot pressed into membranes. The controlled-release naringin nanoscaffolds were observed directly by FT-IR.
2.4.
Contact angle measurement
For determination of wettability of nanoscaffolds, the contact angles of electrospun nonwoven materials were measured by a video contact angle system (DSA100, Siber Hegner, China) mounted with a CCD camera at an ambient temperature. Distilled water was used as the test liquid. Each experiment was repeated six times. Means were shown as the results.
2.5.
Morphologies of nanoscaffolds
The morphologies of electrospun nanoscaffolds were observed by SEM (JSM-5600LV, JEOL, Japan) at an accelerating voltage of 30 kV after gold coating. The average fiber diameters of the electrospun nanofibers were measured using Image-ProPlus 5.02 (Media Cybernetics, USA).
2.6.
Drug release assay in vitro
A specimen of nanoscaffold (PCL/PEG-b-PCL/naringin and PCL/naringin) (20 mm × 20 mm) was first placed in a vial filled with 2 ml phosphate buffer solution (PBS). Naringin release studies were carried out at 37 ◦ C and 100 rotation/min (rpm) in a thermostatic shaking incubator (THZ-92A, GuangTe Instrument Co., Ltd., China). In this case, 0.5 ml samples were taken from the medium after 0, 1, 2, 6, 10, 14, 21, 30, 60, 90 days, and then the same volume of fresh PBS was added as replacement. Naringin in the medium was determined by a UV–vis spectrophotometer (Nanodrop2000, Thermo, USA) at the wavelength of 284 nm. The results were presented in terms of cumulative release as a function of release time: Cumulative amount of release (%) =
Mt × 100 M∞
where Mt was the amount of naringin released from a sample at time t. The total amount of naringin in a sample was calculated and regarded as M∞ in this study. Three samples were tested for each electrospun nanoscaffold and the results were reported as average values.
2.7.
Nanoscaffold degradation assay in vitro
PCL and PCL/PEG-b-PCL/naringin nanoscaffolds (20 mm × 20 mm) were placed in 24-well plates containing 1 ml PBS in each well and were incubated at 37 ◦ C for different periods of time (0, 10, 20, 30, 40, 50, 60 days). After each degradation period, the scaffolds were washed and subsequently dried in a vacuum oven at room temperature
Wi − Wt × 100 Wi
W% is the percentage of weight loss, Wi is the initial weight of the scaffold and Wt is the weight of the scaffold at the time t of the experiment after drying to constant weight.
2.8.
Osteoblast-nanoscaffold interactions
2.8.1.
Cell seeding and culture
Both sides of the nanoscaffolds were exposed to UV radiation for 2 h, washed three times with PBS for 20 min and incubated with DMEM for 24 h before cell seeding. MC3T3-E1 osteoblasts were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin. After reaching 80–90% confluence, the cells were detached by trypsin–EDTA. Each of the nanoscaffolds on coverslips (10 mm × 10 mm) was placed in a 24-well plate and pressed with cell crowns (Sigma–Aldrich, USA) to avoid possible floating during cell culture. Cells were further seeded on nanoscaffolds at a density of 2 × 104 cells/cm2 and cultured with DMEM at 37 ◦ C, 5% CO2 and 95% humidity. The cell-seeded nanoscaffolds were replenished with fresh medium every 3 days.
2.8.2.
Cell adhesion and proliferation by MTS
In order to observe the cell adhesion and proliferation at different times, viable cells were determined by colorimetric MTS assay (CellTiter 96® AQueous Assay). After 1, 2, 4 h and 2, 6, 10, 14 days of cell seeding in the 24-well plates, nanoscaffolds with MC3T3-E1 osteoblasts (constructs) were washed with PBS and incubated with 20% of MTS reagent containing serum free media. After 4 h of incubation at 37 ◦ C in 5% CO2 and 95% humidity, aliquots were pipetted into a 96-well plate and the absorbance was read at 490 nm using an ELISA microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA).
2.8.3.
Cell differentiation by ALP
The differentiation ability of MC3T3-E1 seeded on different nanoscaffolds was analyzed by the expression of alkaline phosphatase (ALP) activity of the cells. In this study, cells were cultured on the nanoscaffolds for 2, 6, 10 and 14 days, and then assessed for ALP activity. The cell-scaffold constructs were washed twice with PBS prior to analysis. To determine ALP activity, the adhered cells were lysed with 1% TritonX100 (Huamei Biological Engineering Co., China) overnight. A commercially available ALP Colorimetric Assay Kit (Jiancheng Biological Engineering Institute, Nanjing, China) was used to quantify ALP concentration in the lysate. The absorbance was measured at 405 nm using an ELISA microplate reader (BioTek Instruments Inc., Winooski, VT, USA). And converted to the units of ALP per liter.
2.8.4.
SEM observations
In order to observe cell morphologies on different nanoscaffolds, cells were fixed after 7 days of culture. A cellular construct was rinsed twice with PBS and subsequently fixed in 2% glutaraldehyde for 1.5 h at room temperature. Then each cellular construct was rinsed with distilled water, dehydrated
1266
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
with graded concentrations of ethanol solution and finally dried in hexamethyldisilazane overnight. Dried cellular constructs were sputter coated with gold and observed under SEM at an accelerating voltage of 20 kV.
2.8.5.
Alizarin Red-S (ARS) staining
Cellular constructs were washed five times in PBS and fixed in ice cold 70% ethanol for 1 h. Then these constructs were washed three times with distilled water and stained with 40 mM ARS for 30 min at room temperature. Microscopic images of the stain showing calcium deposition were taken.
2.9.
Osteoclast-scaffold interactions
2.9.1.
Mouse calvarial organ culture
The heads of 4-day-old CD-1 mice were provided by the life science Institute of Chongqing Medical University. The calvarial of mouse heads were exposed. The parietal bones were dissected out aseptically. During dissection, periosteum and dura mater were left intact on the bone surfaces. Full-thickness, 1.5 mm in diameter, circular defects were created through the parietal bones. The paired bones were divided into two groups. The critical size defect (CSD) model of mouse calvarial bone was based on our previous research [21]. In the present study, the CSD models were used for the evaluation of the responses of osteoclasts to PCL/PEG-b-PCL/naringin and PCL nanoscaffolds. These two types of scaffolds were cut into small disks (1.5 mm diameter) by a hollow steel tube, and then grafted into the bone defect. The bones with scaffolds were cultured concave side down on the tissue culture plate in DMEM with 10% FBS and 1% penicillin/streptomycin. Incubations were carried out at 37 ◦ C, 5% CO2 and 95% humidity. Media were changed every 3 days.
2.9.2. Osteoclast observing by tartrate resistant acid phosphatase (TRAP) staining After 14 days’ culture, the bones with scaffolds were stained for TRAP (Acid Phosphatase Kit 387-A; Sigma–Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. TRAP-positive cells appeared red or purple. Images were taken by transmitted light microscopy (Olympus IX71, Japan) with a digital camera (Canon 500D, Japan).
2.10.
Statistical analysis
All data presented are expressed as mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) and significance was considered at p < 0.05.
3.
Results
naringin (1644 cm−1 ) as referenced from the pure PCL, PEG-b-PCL and naringin. The result indicated that the chemical structures of PCL, PEG-b-PCL and naringin remained unchanged in the electrospinning process. Water contact angles of the nanoscaffolds are shown in Table 1. The contact angles of PCL and PCL/naringin scaffolds were about 141 ± 2.8◦ and 137 ± 2.1◦ , which implied that these scaffolds were hydrophobic. By contrast, PCL/PEGb-PCL and PCL/PEG-b-PCL/naringin scaffolds tended to be hydrophilic with their contact angles 26 ± 0.6◦ and 27 ± 0.5◦ , respectively. Their improved wettability was due to the presence of hydrophilic PEG segments on the surface of the nanofibers. All types of the nanoscaffolds showed nonwoven, bead free, porous and interconnected fibrous structures (Fig. 2). The diameters of PCL, PCL/naringin, PCL/PEG-b-PCL and PCL/PEGb-PCL/naringin nanofibers were 356 ± 32 nm, 367 ± 38 nm, 242 ± 21 nm and 248 ± 27 nm, respectively (Table 1). The fiber diameters of PCL/PEG-b-PCL and PCL/PEG-b-PCL/naringin nanofiber were similar. The nanofibers (Fig. 2c and d) containing PEG-b-PCL were found to be significantly (p < 0.05) thinner and more uniform than PCL and PCL/naringin nanofibers (Fig. 2a and b).
3.2. Naringin release and nanoscaffold degradation assay in vitro Cumulative naringin release profiles of PCL/naringin and PCL/PEG-b-PCL/naringin nanoscaffolds are shown in Fig. 3. Naringin was released rapidly as much as 63% from PCL/naringin nanoscaffolds on the first day, followed by a slow rise from the 2nd to 6th day, and was barely detected after 30 days. The cumulative release at the end of the experiment was 78%. Compared with the abrupt initial burst in the PCL/naringin scaffold, a much lower initial burst was detected in PCL/PEG-b-PCL/naringin scaffold (less than 20% within a day). The naringin embedded within the PCL/PEG-b-PCL/naringin was released gradually over a 90-day period, and the final cumulative release was 93%. The weight loss vs. time of the nanoscaffolds is presented in Fig. 4. Weight loss of the pure PCL scaffold was less than 10% during the period of 60 days, while the PCL/PEGb-PCL/naringin scaffold degraded much faster, losing 55% weight. This result showed that adding PEG-b-PCL into the scaffold could accelerate the rate of degradation.
Table 1 – Fiber diameter and contact angles of different electrospun nanoscaffolds (n = 6).
3.1. Chemical, hydrophilic and morphological characteristics of nanoscaffolds
Samples
Fig. 1 shows the FT-IR spectra of the electrospun PCL/PEGb-PCL/naringin nanoscaffolds and their composition. The PCL/PEG-b-PCL/naringin nanoscaffold showed the mixed bands of PCL (2943, 1243 cm−1 ), PCL-b-PEG (1724 cm−1 ) and
PCL PCL/naringin PCL/PEG-b-PCL PCL/PEG-bPCL/naringin
Fiber diameter 356 367 242 248
± ± ± ±
32 nm 38 nm 21 nm 27 nm
Contact angle in degree 141 137 26 27
± ± ± ±
2.8◦ 2.1◦ 0.6◦ 0.5◦
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
1267
Fig. 1 – Fourier-transform infrared spectra of (a) PCL, (b) PEG-b-PCL, (c) naringin and (d) electrospun PCL/PEG-b-PCL/naringin nanoscaffolds.
Fig. 2 – SEM images of electrospun: (a) PCL, (b) PCL/naringin, (c) PCL/PEG-b-PCL and (d) PCL/PEG-b-PCL/naringin nanoscaffolds.
1268
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
Fig. 3 – The naringin release profiles from the PCL/naringin and PCL/PEG-b-PCL/naringin nanoscaffolds.
Fig. 5 – The MC3T3-E1 adhesion on PCL, PCL/PEG-b-PCL, PCL/naringin and PCL/PEG-b-PCL/naringin nanoscaffolds (n = 3). Bar represent means + SD. a: p < 0.05 vs. PCL nanoscaffolds.
Fig. 4 – Weight loss vs. immersion time for PCL and PCL/PEG-b-PCL/naringin nanoscaffolds.
3.3.
Osteoblast-nanoscaffold interactions
The osteoblast attachment and proliferation behavior on the nanoscaffolds were evaluated by MTS assay after a short-term (4 h) and a long-term period (14 days). Fig. 5 shows the measure of osteoblast attachment on different nanoscaffolds. Data on all nanoscaffolds increased with time. However, the data of PCL/PEG-b-PCL and PCL/PEG-b-PCL/naringin groups were significantly higher than PCL groups at 1, 2, 4 h (p < 0.05). The proliferation of osteoblasts was evaluated after 2, 6, 10 and 14 days by MTS assay (Fig. 6). The data of PCL/naringin and PCL/PEG-b-PCL/naringin groups were higher than those for the PCL group at all time points (p < 0.05). The proliferation of osteoblasts on the PCL/PEG-b-PCL/naringin nanoscaffolds was significantly higher than all the other groups on the 14th day. The ALP activity of MC3T3-E1 osteoblasts cultured on the nanoscaffolds after 2, 6, 10 and 14 days of cell seeding are shown in Fig. 7. After 6 days of culture, the ALP activities of osteoblasts cultured on the PCL/PEG-b-PCL/naringin and PCL/naringin nanoscaffolds were higher than that on
Fig. 6 – The MC3T3-E1 proliferation on PCL, PCL/PEG-b-PCL, PCL/naringin and PCL/PEG-b-PCL/naringin nanoscaffolds (n = 3). Bar represent means + SD. a: p < 0.05 vs. PCL nanoscaffolds; b: p < 0.05 vs. PCL/naringin nanoscaffolds.
PCL nanoscaffolds (p < 0.05). Moreover, the ALP activity of osteoblasts on PCL/PEG-b-PCL/naringin nanoscaffolds was significantly higher than all the other groups on the 14th day. SEM images of the PCL/PEG-b-PCL/naringin nanoscaffolds with osteoblasts were taken after 7 days’ culture. Mineralization nodules were observed on the basal surfaces of nanoscaffolds (Fig. 8a). Moreover, multilayers of mineral deposits were detected on the osteoblasts (Fig. 8c) of PCL/PEGb-PCL/naringin nanoscaffolds, while few mineral deposits were detected on the osteoblasts of PCL nanoscaffolds (Fig. 8d). Mineralization nodules on the 7th day are shown in Fig. 8a and b at different magnifications. These nodules were further evidenced by ARS staining, which was used to detect mineralization [22]. Fig. 9 shows images of the stained nanoscaffolds under the optical microscope after 10 days of cell culture. ARS
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
1269
were observed on the PCL/PEG-b-PCL/naringin nanoscaffolds, which showed the presence of calcium (Fig. 9a). However, similar stains were not observed on the PCL nanoscaffolds (Fig. 9b).
3.4.
Osteoclast-nanoscaffold interactions
TRAP, an isoenzyme of acid phosphatase, is the specific enzyme of the osteoclast [23]. As shown in Fig. 10, the bone defect treated with PCL/PEG-b-PCL/naringin nanoscaffold (left one) stained redder than that with the PCL nanoscaffold (right one). The microscopic images showed that the density of osteoclasts in bone with a PCL/PEG-b-PCL/naringin nanoscaffold was much lower than that in bone with a PCL nanoscaffold (Fig. 11). The results suggested that PCL/PEG-b-PCL/naringin nanoscaffold was effective for suppressing osteoclast.
Fig. 7 – ALP activity of MC3T3-E1 cultured on PCL, PCL/PEG-b-PCL, PCL/naringin and PCL/PEG-b-PCL/naringin nanoscaffolds. Bar represent means + SD. a: p < 0.05 vs. PCL nanoscaffolds; b: p < 0.05 vs. PCL/naringin nanoscaffolds.
4.
Discussion
Degradable biopolymer with nanofibrous structures have attracted great interest as promising drug delivery systems and GBR scaffolds [24]. Due to its lack of toxicity, low cost and slow degradation, PCL was first suggested to be a degradable
Fig. 8 – SEM images of MC3T3-E1 osteoblasts on PCL/PEG-b-PCL/naringin and PCL nanofscaffolds after 7 days’ culture. (a) Mineralization nodules on PCL/PEG-b-PCL/naringin nanoscaffolds (2000×), (b) mineralization nodules on PCL/PEG-b-PCL/naringin nanoscaffolds (1200×), (c) multilayers of mineralization on the surfaces of osteoblasts on PCL/PEG-b-PCL/naringin nanoscaffolds (1000×), and (d) few mineral deposits on the surfaces of osteoblasts on PCL nanoscaffolds (1200×).
1270
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
Fig. 9 – Alizarin Red-S staining for calcium mineralization in MC3T3-E1 osteoblasts on (a) PCL/PEG-b-PCL/naringin (200×) and (b) PCL nanoscaffolds after 10 days’ culture (200×).
nanofiber matrix for bone regeneration in vitro [25]. However, osteoporosis weakens the bonding strength between the scaffold and bone, delays bone defect repair and lowers the quality of new bone formation [1,2]. Naringin, isoflavonoids isolated from plants, has been shown to reduce bone resorption and had an osteogenic effect on osteoblasts [10]. However, the hydrophilic naringin is difficult to dissolve in the hydrophobic PCL/acetone solvent systems and is not compatible with the hydrophobic polymers, which leads to phase separation during electrospinning. So successful incorporation and sustained release of hydrophilic naringin is a challenge. PEGb-PCL, like other types of amphiphilic block copolymers, has been reported to incorporate hydrophilic drugs into hydrophobic polymers by electrospinning to produce controlled-release nanofiberous scaffolds [26]. In this study, PEG-b-PCL was added into the PCL/naringin/acetone system to form a transparent and homogenous mixed solution. When the solution was electrospun, naringin could be embedded into the hydrophobic polymer fibers.
Fig. 10 – Macro image of TRAP staining for osteoclasts on mouse calvarial bone after 14 days’ culture. (left one) The critical size defects of mouse calvarial bone grafted with PCL nanoscaffold; (right one) the critical size defects of mouse calvarial bone grafted with PCL/PEG-b-PCL/naringin nanoscaffold.
During the electrospinning process, random nanofibers were formed. PEG-b-PCL could assist in achieving thinner and more uniform medicated fibers in this study, probably due to improved solubilization of the drug and polymer in a solvent. The solution with improved solubilization could achieve strong combinations through the process of electrospinning and avoid phase separation [27]. The reduced diameters of the fibers also suggested that the drug was dispersed homogeneously in the electrospun fibers [28]. The greatly (p < 0.05) reduced diameters have greater surface areas, which are beneficial to cell adhesion and drug distribution. Besides a desired range of fiber diameters, surface wettability is also an important physicochemical property that could affect the cell behavior. Some studies indicated that cells tended to attach and spread much more widely on hydrophilic surfaces than on hydrophobic surfaces [29–32], but there was no significant difference in cell proliferation among these surfaces [32]. Table 1 shows that PEG-b-PCL remarkably changed the wettability of both PCL/PEG-b-PCL and PCL/PEGb-PCL/naringin nanoscaffolds. FT-IR results of PCL/PEG-b-PCL/naringin nanoscaffolds showed the mixed vibrational absorbance bands of pure PCL, PCL-b-PEG and naringin as referenced, and indicated that the chemical structures of PCL, PEG-b-PCL and naringin remained unchanged even though they had been subjected to a high electrical voltage. The initial (first day) drug release was as high as 63% from the PCL/naringin nanoscaffolds due to the drug not being encapsulated efficiently. Like other polyester biopolymers, PCL is quite hydrophobic. Hydrophilic naringin could not be encapsulated effectively by conventional electrospinning techniques. Naringin covered on the surfaces of the fibers directly diffused in the medium, which caused the initial burst release followed by a plateau after 2 days. By contrast, PCL/PEG-b-PCL/naringin nanoscaffolds showed a better naringin release behavior. It released low concentrations with a slow-release effect. The release period was prolonged to 90 days and the total release was more than 90%. Naringin was embedded into the PCL/PEG-b-PCL/naringin nanofibers. Thus,
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
1271
Fig. 11 – Microscopic images of TRAP staining for osteoclast on mouse calvarial bone after 14 days’ culture. (a) The critical size defects of mouse calvarial bone grafted with PCL/PEG-b-PCL/naringin nanoscaffold (200×). (b) The critical size defects of mouse calvarial bone grafted with PCL nanoscaffold (200×). B: bone and S: scaffold.
naringin released steadily and slowly. The polymer fibers gradually degraded, rather than directly diffused into the medium. Moreover, the cumulative release of PCL/PEG-b-PCL/naringin nanoscaffolds was 93%, which was greater than the control group (78%). The narrower the fiber diameters, the larger the total surface areas of the scaffolds for the same mass were. Thus, more naringin could be loaded within the PCL/PEG-bPCL/naringin nanoscaffolds. The addition of amphiphilic block copolymers PEG-bPCL not only showed controlled-release behavior but also increased the scaffold degradation rate [26]. PCL is a semicrystalline aliphatic polymer and has a slower degradation rate than most biopolymers [33]. PCL is characterized by a degradation time in excess of 1 year [34]. However, reconstruction of human bone tissue usually takes about 17 weeks [35]. Therefore, it is necessary to speed up the scaffold’s degradation rate. The high water uptake of PEG-b-PCL may increase PCL degradation by providing water channels since PCL polyester is degraded mainly via a hydrolytic process. Also, according to Bolgen et al. [36], PCL nanofibers with thinner diameter size degraded faster than thicker ones. Although PCL is relatively stable against in vitro hydrolysis, it is known that microorganisms can degrade it [37]. So further studies in vivo are needed to adjust the degradation rate to a suitable range. The MC3T3-E1 pre-osteoblastic cell line is a well-accepted model for osteogenesis study in vitro, as it differentiates into post-mitotic osteoblasts capable of expressing the osteogenic phenotype [38]. Traditional osteoblast culture medium is alpha-MEM which is a nutrient-rich medium and can promote osteogenesis. However, in order to observe the osteogenic effect of naringin, DMEM was used [39]. Based on the shortterm MTS assay, cells attached rapidly on the nanoscaffolds containing PEG-b-PCL (1, 2, 4 h, p < 0.05) (Fig. 5). This could be attributed to the hydrophilic segments PEG on the surfaces of PCL/PEG-b-PCL and PCL/PEG-b-PCL/naringin nanoscaffolds. The hydrophilic surface provides more suitable conditions for initial cell adhesion. Some studies showed that the adhesive protein fibronectin known to promote cell adhesion tended
to adhere onto a hydrophilic surface [40,41]. However, no significant difference was observed in osteoblast proliferation between the hydrophilic and hydrophilic surfaces. This result was in accordance with that reported by Wei et al. [32]. Previously, naringin has been reported to improve the proliferation of osteoblasts [42]. In this study, PCL/naringin nanoscaffolds showed improved osteoblast proliferation on the 2nd day, which was consistent with the burst release of naringin in PCL/naringin nanoscaffolds for the first 2 days. However, naringin release from PCL/naringin nanoscaffolds gradually declined after 6 days, meanwhile, naringin continued to be released from PCL/PEG-b-PCL/naringin nanoscaffolds. Thus, the osteoblast proliferation on PCL/PEG-b-PCL/naringin nanoscaffolds was significantly (14 d, p < 0.05) higher than that on PCL/naringin nanoscaffolds (Fig. 6). ALP is a key component of bone matrix vesicles because of its role in the formation of apatitic calcium phosphate and it is an early indicator of immature osteoblast activity [43]. All ALP levels increased with culture time, but ALP levels of the nanoscaffolds with naringin were significantly higher than those of the PCL nanoscaffold on the 6th, 10th and 14th day. In agreement with a previous report [42], a certain concentration of naringin can affect cell differentiation. There are significant differences of ALP activity between the PCL/naringin and PCL/PEG-b-PCL/naringin nanoscaffold on the 14th day. Up to 63% naringin released from the PCL/naringin nanoscaffolds was lost on the first day because the culture medium was replenished every 3 days. Then, the released naringin continued to fall. So the significant difference between PCL/naringin and PCL/PEG-b-PCL/naringin nanoscaffold was not apparent until the 14th day. These results indicated that PCL/PEG-bPCL/naringin nanoscaffolds were superior in supporting cell attachment, proliferation and differentiation. The naringin controlled released from PCL/PEG-b-PCL/naringin nanoscaffolds could promote long-term bone formation. SEM images and the subsequent ARS staining results indicated that osteoblasts seeded on PCL/PEG-b-PCL/naringin nanoscaffolds displayed accelerated mineralization compared
1272
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
to PCL nanoscaffolds. This might be explained in two ways. First, the addition of PEG-b-PCL changed the nanoscaffold surface morphology to a hydrophilic condition, which helped initial osteoblast adhesion. Consequently, a large number of osteoblasts synthesized and secreted ECM to form nodules for bone formation. Second, naringin has been shown to have an HMG-CoA reductase inhibiting effect [11]. Therefore, it is possible that naringin may also activate the BMP-2 promotor and increase bone formation [11]. BMP-2 has been extensively studied and demonstrated to play a crucial role in inducing osteoblast differentiation and bone matrix calcification during embryonic skeletal development and postnatal bone remodeling [44–47]. On the other hand, naringin decreases bone resorption by suppressing osteoclast formation [8–10]. Ang et al. [48] showed that naringin perturbed osteoclast formation and bone resorption by inhibiting RANK-mediated NF-B and ERK signaling. Naringin suppressed gene expression of key osteoclast marker genes. Naringin was found to inhibit RANKL-induced activation of NF-B by suppressing RANKLmediated IB-␣ degradation. In addition, naringin inhibited RANKL-induced phosphorylation of ERK. The CSD in vitro model was used [49] to evaluate the responses of osteoclasts to scaffolds. PCL/PEG-b-PCL/naringin nanoscaffolds inhibited bone resorption by decreasing osteoclast formation.
5.
Conclusion
In this study, an active constituent of the Traditional Chinese Medicine, naringin, regarded as an effective anti-osteoporosis drug, was encapsulated in PCL/PEG-b-PCL nanofibers using electrospinning for the first time. The introduction of PEGb-PCL significantly improved the hydrophilic properties, drug release behavior and the material degradation rate of the scaffolds. The controlled-release naringin nanoscaffolds, which were shown to enhance functions of osteoblasts and suppress formation of osteoclasts, showed the potential to be used as GBR scaffolds for the repair of osteoporotic bone defects.
Acknowledgements X. Wu is grateful to the financial support from the national Natural Science Foundation of China (grant number 81200767/H1402), Chongqing Science & Technology Commission, China (grant number CSTC 2010BB5102) and Chongqing Municipal Health Bureau, China (grant number 2012-2-8).
references
[1] Erdogan O, Shafer DM, Taxel P, Freilich MA. A review of the association between osteoporosis and alveolar ridge augmentation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;104:738 e1–413e. [2] Qi M, Hu J, Li J, Dong W, Feng X, Yu J. Effect of zoledronate acid treatment on osseointegration and fixation of implants in autologous iliac bone grafts in ovariectomized rabbits. Bone 2012;50:119–27. [3] Rodan GA, Martin TJ. Therapeutic approaches to bone diseases. Science 2000;289:1508–14.
[4] Salesi N, Pistilli R, Marcelli V, Govoni FA, Bozza F, Bossone G, et al. Bisphosphonates and oral cavity avascular bone necrosis: a review of twelve cases. Anticancer Res 2006;26:3111–5. [5] Merigo E, Manfredi M, Meleti M, Guidotti R, Ripasarti A, Zanzucchi E, et al. Bone necrosis of the jaws associated with bisphosphonate treatment: a report of twenty-nine cases. Acta Biomed 2006;77:109–17. [6] Wang XL, Wang NL, Zhang Y, Gao H, Pang WY, Wong MS, et al. Effects of eleven flavonoids from the osteoprotective fraction of Drynaria fortunei (KUNZE) J. SM. on osteoblastic proliferation using an osteoblast-like cell line. Chem Pharm Bull (Tokyo) 2008;56:46–51. [7] Jeong JC, Lee JW, Yoon CH, Lee YC, Chung KH, Kim MG, et al. Stimulative effects of Drynariae Rhizoma extracts on the proliferation and differentiation of osteoblastic MC3T3-E1 cells. J Ethnopharmacol 2005;96:489–95. [8] Jeong JC, Kang SK, Youn CH, Jeong CW, Kim HM, Lee YC, et al. Inhibition of Drynariae Rhizoma extracts on bone resorption mediated by processing of cathepsin K in cultured mouse osteoclasts. Int Immunopharmacol 2003;3:1685–97. [9] Hirata M. Naringin suppresses osteoclast formation and enhances bone mass in mice. J Health Sci 2009;55: 463–7. [10] Chen LL, Lei LH, Ding PH, Tang Q, Wu YM. Osteogenic effect of Drynariae rhizoma extracts and Naringin on MC3T3-E1 cells and an induced rat alveolar bone resorption model. Arch Oral Biol 2011;56:1655–62. [11] Wong RW, Rabie AB. Effect of naringin on bone cells. J Orthop Res 2006;24:2045–50. [12] Wu JB, Fong YC, Tsai HY, Chen YF, Tsuzuki M, Tang CH. Naringin-induced bone morphogenetic protein-2 expression via PI3K, Akt, c-Fos/c-Jun and AP-1 pathway in osteoblasts. Eur J Pharmacol 2008;588:333–41. [13] Wei M, Yang Z, Li P, Zhang Y, Sse WC. Anti-osteoporosis activity of naringin in the retinoic acid-induced osteoporosis model. Am J Chin Med 2007;35:663–7. [14] Mandadi K, Ramirez M, Jayaprakasha GK, Faraji B, Lihono M, Deyhim F, et al. Citrus bioactive compounds improve bone quality and plasma antioxidant activity in orchidectomized rats. Phytomedicine 2009;16:513–20. [15] Sui G, Yang X, Mei F, Hu X, Chen G, Deng X, et al. Poly-l-lactic acid/hydroxyapatite hybrid membrane for bone tissue regeneration. J Biomed Mater Res A 2007;82: 445–54. [16] Nie H, Soh BW, Fu YC, Wang CH. Three-dimensional fibrous PLGA/HAp composite scaffold for BMP-2 delivery. Biotechnol Bioeng 2008;99:223–34. [17] Sahoo S, Ouyang H, Goh JC, Tay TE, Toh SL. Characterization of a novel polymeric scaffold for potential application in tendon/ligament tissue engineering. Tissue Eng 2006;12:91–9. [18] Lee SJ, Yoo JJ, Lim GJ, Atala A, Stitzel J. In vitro evaluation of electrospun nanofiber scaffolds for vascular graft application. J Biomed Mater Res A 2007;83:999–1008. [19] Schnell E, Klinkhammer K, Balzer S, Brook G, Klee D, Dalton P, et al. Guidance of glial cell migration and axonal growth on electrospun nanofibers of poly-epsilon-caprolactone and a collagen/poly-epsilon-caprolactone blend. Biomaterials 2007;28:3012–25. [20] Venugopal J, Low S, Choon AT, Kumar AB, Ramakrishna S. Electrospun-modified nanofibrous scaffolds for the mineralization of osteoblast cells. J Biomed Mater Res A 2008;85:408–17. [21] Wu X, Downes S, Watts DC. Evaluation of critical size defects of mouse calvarial bone: an organ culture study. Microsc Res Tech 2010;73:540–7. [22] Gregory CA, Gunn WG, Peister A, Prockop DJ. An Alizarin red-based assay of mineralization by adherent cells in
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1263–1273
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
culture: comparison with cetylpyridinium chloride extraction. Anal Biochem 2004;329:77–84. Janckila AJ, Yam LT. Biology and clinical significance of tartrate-resistant acid phosphatases: new perspectives on an old enzyme. Calcif Tissue Int 2009;85:465–83. Sill TJ, von Recum HA. Electrospinning: applications in drug delivery and tissue engineering. Biomaterials 2008;29:1989–2006. Yoshimoto H, Shin YM, Terai H, Vacanti JP. A biodegradable nanofiber scaffold by electrospinning and its potential for bone tissue engineering. Biomaterials 2003;24: 2077–82. Kim K, Luu YK, Chang C, Fang D, Hsiao BS, Chu B, et al. Incorporation and controlled release of a hydrophilic antibiotic using poly(lactide-co-glycolide)-based electrospun nanofibrous scaffolds. J Control Release 2004;98:47–56. Yan J, Ye Z, Chen M, Liu Z, Xiao Y, Zhang Y, et al. Fine tuning micellar core-forming block of poly(ethylene glycol)-block-poly(epsilon-caprolactone) amphiphilic copolymers based on chemical modification for the solubilization and delivery of doxorubicin. Biomacromolecules 2011;12:2562–72. Xu X, Chen X, Ma P, Wang X, Jing X. The release behavior of doxorubicin hydrochloride from medicated fibers prepared by emulsion-electrospinning. Eur J Pharm Biopharm 2008;70:165–70. Chen M, Zamora PO, Som P, Pena LA, Osaki S. Cell attachment and biocompatibility of polytetrafluoroethylene (PTFE) treated with glow-discharge plasma of mixed ammonia and oxygen. J Biomater Sci Polym Ed 2003;14:917–35. Ozdemir Y, Hasirci N, Serbetci K. Oxygen plasma modification of polyurethane membranes. J Mater Sci Mater Med 2002;13:1147–51. Altankov G, Grinnell F, Groth T. Studies on the biocompatibility of materials: fibroblast reorganization of substratum-bound fibronectin on surfaces varying in wettability. J Biomed Mater Res 1996;30:385–91. Wei J, Yoshinari M, Takemoto S, Hattori M, Kawada E, Liu B, et al. Adhesion of mouse fibroblasts on hexamethyldisiloxane surfaces with wide range of wettability. J Biomed Mater Res B: Appl Biomater 2007;81:66–75. Shor L, Guceri S, Wen X, Gandhi M, Sun W. Fabrication of three-dimensional polycaprolactone/hydroxyapatite tissue scaffolds and osteoblast-scaffold interactions in vitro. Biomaterials 2007;28:5291–7. Pitt CG, Gratzl MM, Kimmel GL, Surles J, Schindler A. Aliphatic polyesters II. The degradation of poly (dl-lactide), poly (epsilon-caprolactone), and their copolymers in vivo. Biomaterials 1981;2:215–20. Weng B. Development of resorbable potentials for bone regeneration. In: Symposium on: difficult treatment option for bone regeneration. 1995. Bolgen N, Menceloglu YZ, Acatay K, Vargel I, Piskin E. In vitro and in vivo degradation of non-woven materials made of
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
1273
poly(epsilon-caprolactone) nanofibers prepared by electrospinning under different conditions. J Biomater Sci Polym Ed 2005;16:1537–55. Singh NK, Das Purkayastha B, Roy JK, Banik RM, Yashpal M, Singh G, et al. Nanoparticle-induced controlled biodegradation and its mechanism in poly(epsilon-caprolactone). ACS Appl Mater Interfaces 2010;2:69–81. Pereira CT, Huang W, Sayer G, Jarrahy R, Rudkin G, Miller TA. Osteogenic potential of polymer-bound bone morphogenetic protein-2 on MC3T3-E1 two-dimensional cell cultures. Plast Reconstr Surg 2009;124:2199–200 [author reply 200]. Coelho MJ, Fernandes MH. Human bone cell cultures in biocompatibility testing. Part II: effect of ascorbic acid, beta-glycerophosphate and dexamethasone on osteoblastic differentiation. Biomaterials 2000;21:1095–102. Howlett CR, Evans MD, Wildish KL, Kelly JC, Fisher LR, Francis GW, et al. The effect of ion implantation on cellular adhesion. Clin Mater 1993;14:57–64. Hao L, Lawrence J. On the role of CO2 laser treatment in the human serum albumin and human plasma fibronectin adsorption on zirconia (MGO-PSZ) bioceramic surface. J Biomed Mater Res A 2004;69:748–56. Zhang P, Dai KR, Yan SG, Yan WQ, Zhang C, Chen DQ, et al. Effects of naringin on the proliferation and osteogenic differentiation of human bone mesenchymal stem cell. Eur J Pharmacol 2009;607:1–5. Luben RA, Wong GL, Cohn DV. Biochemical characterization with parathormone and calcitonin of isolated bone cells: provisional identification of osteoclasts and osteoblasts. Endocrinology 1976;99:526–34. Suzuki A, Ghayor C, Guicheux J, Magne D, Quillard S, Kakita A, et al. Enhanced expression of the inorganic phosphate transporter Pit-1 is involved in BMP-2-induced matrix mineralization in osteoblast-like cells. J Bone Miner Res 2006;21:674–83. Suzuki D, Yamada A, Aizawa R, Funato S, Matsumoto T, Suzuki W, et al. BMP2 differentially regulates the expression of Gremlin1 and Gremlin2, the negative regulators of BMP function, during osteoblast differentiation. Calcif Tissue Int 2012;91:88–96. Miron RJ, Saulacic N, Buser D, Iizuka T, Sculean A. Osteoblast proliferation and differentiation on a barrier membrane in combination with BMP2 and TGFbeta1. Clin Oral Investig 2013;17(3):981–8. Rawadi G, Vayssiere B, Dunn F, Baron R, Roman-Roman S. BMP-2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop. J Bone Miner Res 2003;18:1842–53. Ang ES, Yang X, Chen H, Liu Q, Zheng MH, Xu J. Naringin abrogates osteoclastogenesis and bone resorption via the inhibition of RANKL-induced NF-kappaB and ERK activation. FEBS Lett 2011;585:2755–62. Schmitz JP, Hollinger JO. The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop Relat Res 1986;29:9–308.