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Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
1
In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds Bo-ram Kim1, B.S, Nguyen Thuy Ba Linh1, 3, Ph.D, Young-Ki Min2,3, Ph.D, Byong-Taek Lee1, 3*, Ph.D 1
Department of Regenerative medicine, College of Medicine, Soonchunhyang University, 366-1, Ssangyong dong,
Cheonan, Chungnam, 330-090, Republic of Korea 2
Department of Physiology, College of Medicine, Soonchunhyang University, 366-1, Ssangyong dong, Cheonan, Chungnam,
330-090, Republic of Korea 3
Institute of Tissue Regeneration, College of Medicine, Soonchunhyang University, 366-1, Ssangyong dong, Cheonan,
Chungnam, 330-090, Republic of Korea
*Corresponding author: Byong-Taek Lee Telephone: +82-41-570-2427; Fax: +82-41-577-2415 Email: [email protected] 1
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Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
2 Abstract To confirm the effect of recombinant human bone morphogenetic protein-2 (BMP-2) for bone regeneration, BMP-2 loaded polycaprolactone (PCL)-gelatin (Gel)-biphasic calcium phosphate (BCP) fibrous scaffolds were fabricated using the electrospinning method. The electrospinning process to incorporate BCP nanoparticles into the PCL-Gel scaffolds yielded an extracellular matrix-like microstructure that was a hybrid system composed of nano and micro sized fibers. BMP-2 was homogeneously loaded on the PCL-Gel-BCP scaffolds for enhanced induction of bone growth. BMP-2 was initially released at high levels, and then showed sustained release behavior for 31 days. Compared with the PCL-Gel-BCP scaffold, the BMP-2 loaded PCL-Gel-BCP scaffold showed improved cell proliferation and cell adhesion behavior. Both scaffold types were implanted in rat skull defects for 4 and 8 weeks to evaluate the biological response under physiological conditions. Remarkable bone regeneration was observed in the BMP-2/PCL-Gel-BCP group. These results suggest that BMP-2 loaded PCL-Gel-BCP scaffolds should be considered for potential bone tissue engineering applications. Keywords: electrospinning, polycaprolactone, gelatin, BCP, BMP-2, bone tissue engineering, bone regeneration
2
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Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
3 1. Introduction Large sized bone injuries and defects are often repaired using alternative materials such as bone substitutes. Scaffolds play a vital role by serving as templates for host cells and supporting the regeneration of bone defects 1,2. An ideal scaffold should be biocompatible and biodegradable and function in a similar manner to the extracellular matrix (ECM) 3. Fibrous scaffolds fabricated by electrospinning have been used recently for biomedical applications because they can easily create a three-dimensional porous structure resembling the morphology of natural ECM
4-6
. The microstructure of electrospun scaffolds such as a fiber
diameter and porosity can be controlled by adjusting solution properties and operating parameters. Due to the large surface area to volume ratio of electrospun scaffolds, cell adhesion, migration, and proliferation improve 7-9. Various types of biomaterials such as natural polymers, synthetic polymers, ceramics, and their composites can be applied to the electrospinning process. The combination of natural and synthetic polymers improves mechanical properties as well as biocompatibility. Polycarprolactone (PCL) is a typical hydrophobic biodegradable synthetic polymer that degrades slowly inside the human body. Due to its slow degradation, it has been investigated mainly as long-term implant material for drug release and support of bone formation
10-12
.
Gelatin (Gel), which is natural polymer derived from collagen, has been widely used for biomaterial applications such as wound dressings, bone and cartilage regeneration, and drug delivery systems
13-16
because of its excellent biocompatibility and biodegradability. Several
studies have investigated PCL-Gel electrospun scaffolds
17-20
. This approach of using a
synthetic/natural polymer blend overcomes the drawback of individual synthetic and natural polymers and improves mechanical properties and biocompatibility.
3
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Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
4 However, a PCL-Gel polymer system alone cannot provide support for an ideal scaffold because it is not an osteoconductive or osteoinductive material 21. Natural ECM is composed of fibrous collagen organized in a three-dimensional porous network, and hydroxyapatite (HAp) crystals are dispersed within the collagen fibers
22
. In this study, biphasic calcium
phosphate (BCP) nanoparticles were dispersed into a PCL-Gel blend matrix to synthesize a bone mimicking ECM. BCP is a biodegradable and osteoconductive ceramic, which consists of HAp and tricalcium phosphate (TCP) phase compositions. It forms tight bonds with host bone tissues and allows osteogenesis. BCP is a more effective ceramic for bone repair and regeneration than pure HAp or pure TCP alone because it controls scaffold degradation rate 23,24
. Combining polymers with inorganic materials such as BCP particles can improve the
bioactivity of electrospun scaffolds and enhance cell attachment, proliferation, and osteoblastic differentiation 25-27. Bone regeneration is regulated by various bioactive agents such as growth factors (bone morphogenetic proteins [BMPs], transforming growth factor-beta, fibroblast growth factor, platelet-derived growth factor, and insulin-like growth factor), which are important signals in the osteogenic microenvironment
28,29
. Using osteoinductive growth factors to enhance bone
reconstruction and formation on this type of scaffold is the most efficient way to perform protein therapy
30,31
. Among the various types of growth factors, BMPs promote
differentiation by inducing mesenchymal stem cells to transform into chondrocytes and osteoblasts and cartilage and bone to form
32,33
. In this study, recombinant human BMP-2-
loaded PCL-Gel-BCP scaffolds were fabricated using a blend electrospinning process to encapsulate BMP-2 directly inside the fibers. We investigated the effects of BMP-2 on cell proliferation and differentiation of MC3T3-E1 cells and focused on osteogenesis in a rat calvarial skull model. 4
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Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
5 2. Materials and methods 2.1. Preparation of the BMP2-loaded PCL-Gel-BCP solution PCL (Mn 70,000–90,000) and Gel type A (300 Bloom) from porcine skin were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2,2,2-Trifluoroethanol (TFE, purity > 99.0%, Alfa Aesar, Cambridge, UK) was used as the solvent. BCP nanopowder was synthesized by a microwave-assisted process 34. The concentration and ratio of each solution were prepared according to a method reported previously
17
. To fabricate the electrospun
scaffolds, 10% w/v PCL and 10% w/v Gel solutions were prepared by dissolving 1 g of each polymer in TEF separately followed by stirring overnight at room temperature. The 10% w/v Gel solution was added to the 10% w/v PCL solution until it was mixed completely at a volume 50:50 ratio of PCL: Gel. BCP particles (size, 45–50 μm) were dispersed in TFE by ultrasound and added to the PCL-Gel solution at a ratio of BCP: PCL: Gel = 1:1:1 (w/w) and stirred for 2 hr to make the scaffold blended BCP. In addition, 0.5 μg/ml recombinant BMP-2 (R&D Systems Minneapolis, MN, USA; 10 µg, > 95% purity) was mixed with the PCL-GelBCP solution before starting the electrospinning process 35. 2.2. Fabrication of the electrospun scaffolds The PCL-Gel-BCP scaffolds and BMP2 loaded PCL-Gel-BCP scaffolds were fabricated by an electrospinning process. Five mL of the solution was loaded into a 12 mL plastic syringe fitted with an 18-gauge needle and injected using a syringe-pump at a flow rate of 0.5 mL/hr. The collector was covered with a piece of aluminum foil. The distance between the tip of the needle and the collector was about 10 cm, and 20 kV voltage was applied. The
fabricated
electrospun
scaffolds
were
cross-linked
using
1-ethyl-3
(3-
dimethylaminopropyl) carbodiimide (EDAC) and N-hydroxysuccinimide (Sigma-Aldrich) at 5
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Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
6 a 5:2 molar ratio in 80% ethanol for 1 hr at 4 ºC, rinsed three times with distilled water for 5 min to remove residual EDAC, frozen at −20ºC for 4 hr, and then freeze-dried at −80ºC. 2.3. Scaffold characterization 2.3.1. Morphological analysis The surfaces of the PCL-Gel-BCP and BMP2/PCL-Gel-BCP scaffolds were sputtercoated with platinum (Cressington 108 Auto, Cambridge, UK) and observed by scanning electron microscopy (SEM, JSM-6701F, JEOL, Tokyo, Japan) at an accelerating voltage of 10 kV. The average fiber diameter of the electrospun fibers was measured from SEM images. At least 10 positions on the fiber mat were tested to measuring electrospun fiber diameter. Energy dispersive X-ray spectrometer (EDS, JSM-7401F) was used to analyze the elemental composition of the electrospun fibers. The elemental composition of the crystal structures and the composition of the scaffolds were analyzed by X-ray diffraction (XRD, Rigaku, D/MAX2500V). The diffraction angle was varied from 10° to 60° 2θ. 2.3.2. BMP-2 release study To determine whether BMP-2 loaded successfully, BMP-2 loaded PCL-Gel-BCP scaffolds were observed by immunofluorescent staining and compared with PCL-Gel-BCP scaffolds (negative control). The scaffolds were washed with phosphate buffered saline (PBS) and treated with 5% bovine serum albumin (BSA) for 30 min. The scaffolds were blocked with BMP-2 primary antibody (BMP-2/4 [H-1]; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:50) for 4 hr and rinsed with PBS three times. BMP-2 secondary antibody (goat antimouse IgG-FITC, Santa Cruz Biotechnology) (1:100) was applied for 1 hr and washed again with PBS. After staining, confocal microscopy (FV10i-W) was performed to measure BMP-2 loading on the scaffolds. 6
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Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
7 The in vitro release rate of BMP-2 from the PCL-Gel-BCP scaffolds was measured with a BMP-2 enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems). To collect the supernatant, the BMP-2 loaded PCL-Gel-BCP scaffolds were immersed in 1 ml PBS and incubated at 37ºC. At various times, all of the supernatant was collected, and replaced with fresh PBS. The amount of BMP-2 in the supernatants was determined according to the manufacturer’s protocol. Absorbance was read with an ELISA reader (Infinite F50, Tecan, Zurich, Switzerland) at a wavelength of 450 nm. 2.4. In vitro study 2.4.1. Cell culture Pre-osteoblast MC3T3-E1 cells, derived from mice, were cultured in MEM alpha (MEM-α, Gibco, Grand Island, NY USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (P/S). The MC3T3-E1 cells were maintained in a humidified incubator at 37ºC in a 5% CO2 atmosphere. 2.4.2. Cell adhesion Electrospun nanofibers were initially sterilized under UV light, sterilized further with 70% ethanol, washed with PBS, and finally soaked in MEM-α. MC3T3-E1 cells were seeded on 1 cm2 of scaffolds at a density of 104 cells/cm2 in a 24-well plate with culture medium. To observe cell adhesion, the cells were cultured for 60 and 90 min. After culturing, the cells were fixed in 2% glutaraldehyde (DeaJung Co., Seoul, Korea) and dehydrated in an ethanol series (50, 70, 90, and 100%) (Merck, Darmstadt, Germany). The dehydrated cells were dried in hexamethyldisilazane (DeaJung Co) and observed by SEM. The cells were immunostained using fluorescein isothiocyanate (FITC) conjugated phalloidin (25 μg/ml, Sigma-Aldrich) and vinculin (Millipore, Billerica, MA, USA) for confocal microscopy. The cells on the scaffolds 7
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Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
8 were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature, permeabilized in 0.25% TritonX-100 (Sigma-Aldrich) for 10 min, and blocked in 2.5% BSA for 1 hr. The cells were immunostained using vinculin antibody at 4ºC overnight. After washing with PBS, the cells were stained with FITC conjugated phalloidin for 30 min. Nuclei were stained with Hoechst. Finally, the scaffolds were mounted on glass slides and visualized under a confocal fluorescent microscope (FV10i-W) using the accompanying FV10i-ASW 3.0 viewer software. 2.4.3. Cell viability To evaluate cell proliferation, the PCL-Gel-BCP and BMP-2/PCL-Gel-BCP scaffolds were
analyzed using the MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyltetrazolium
bromide) assay (Sigma-Aldrich)36. After 1, 3, and 7 days of culture, 100 μl of MTT solution was added to each well filled with PCL-Gel-BCP and BMP-2/PCL-Gel-BCP scaffolds and incubated for 4 hr. After removing the media, 1 ml dimethyl sulfoxide (Samchun Pure Chemical Co.) was added to each well to extract the formazan crystals under gentle shaking. The extract of each scaffold was transferred to a 96-well plate and the absorbance intensities were measured at 595 nm using an ELISA reader. 2.4.4. Cell proliferation and attachment Confocal microscopy was performed to observe growth of MC3T3-E1 cells (1 × 104 cells/well) seeded on the PCL-Gel-BCP and BMP-2/PCL-Gel-BCP scaffolds. Briefly, after 1, 3, and 7 days of culture, the scaffolds were rinsed with PBS twice and fixed in 4% paraformaldehyde for 10 min at room temperature, permeabilized in 0.25% TritonX-100 for 10 min, and blocked in 2.5% BSA for 1 hr. The cells were immunostained using FITC
8
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Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
9 conjugated phalloidin overnight at 4ºC. Nuclei were stained with Hoechst. Cell behavior was observed under a confocal microscope at 10× and 60× magnifications. 2.4.5. Cell differentiation The
osteogenic
differentiation
of
MC3T3-E1
cells
was
analyzed
using
immunocytochemical analysis of alkaline phosphate (ALP) and osteopontin (OPN). MC3T3E1 cells were cultured on PCL-Gel-BCP and BMP-2/PCL-Gel-BCP scaffolds using osteogenic medium consisting of MEM supplemented with 10% FBS, 1% P/S, 10nM dexamethasone (Sigma), 50 µg/ml L-ascorbic acid (Sigma), and 10 mM β-glycerophosphate disodium salt hydrate (Sigma). After 7 and 14 days, cells on the scaffolds were stained with rabbit ALP antibody (1:50, Santa Cruz Biotechnology) and mouse OPN antibody (1:50, Santa Cruz Biotechnology) overnight at 4ºC. Then, the scaffolds were incubated in secondary antibody (Alexa Fluor 488, 1:1000, Invitrogen, Carlsbad, CA, USA). The images were visualized under a confocal microscope using 60× magnification. 2.5. In vivo study 2.5.1. Implantation of the BMP-2/PCL-Gel-BCP scaffolds in the rat skull A total of 16 male Sprague–Dawley rats, weighing 300 g, were used for the in vivo study of PCL-Gel-BCP and BMP-2/PCL-Gel-BCP scaffolds (four rats each), after the protocol was approved by the Animal Ethical Committee of Soonchunhyang University. The healing process was observed 4 and 8 weeks after implantation. The PCL-Gel-BCP and BMP-2/PCLGel-BCP scaffolds were sterilized with 70% ethanol, washed with PBS, and finally freezedried at −80ºC for 3 hr before use. The rats were anesthetized with diethyl ether, and the hair on the skull was shaved and sterilized with 70% ethanol and povidone iodine. After exposure of the parietal skull, two defects were made on the left and right side of the skull (5 mm 9
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Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
10 diameter, and 1 mm thickness) using a trephine drill. One defect was grafted with PCL-GelBCP scaffolds and the other defect was sutured without a sample as a negative control. The BMP-2/PCL-Gel-BCP scaffolds were implanted in the same way. The subcutaneous tissue was closed and the skin was re-sutured. The rats were sacrificed 4 and 8 weeks after implantation. 2.5.2. Micro-computed tomography (micro-CT) and histological analysis Four and eight weeks of after implantation, the rats were sacrificed and the entire portion of the defected skull was removed. The samples were fixed in 10% formalin solution at room temperature. Micro-CT (Skyscan 1076, Antwerp, Belguim) was performed to observe new bone formation at the defect sites. Each sample was fixed on the object stage, and imaging was performed on the sample for 360°of rotation with an exposure rate of 20 min per frame. Micro-CT images were reconstructed using CTAn (Skyscan) and CTVol (Skyscan) to make three-dimensional images. The bone volume (BV) and percent bone volume (bone volume (BV)/tissue volume (TV) (%) were calculated to evaluate the new bone quantity. The rat skulls fixed in 10% formalin solution were decalcified using 5% HNO3. The tissues were embedded in a paraffin block and serially cut using a microtome (HM 325, Thermo Scientific, Rockford, IL, USA). The 4 ± 2 μm thick sections were mounted on microscope slides. The slides were deparaffinized and hydrated with xylene and an alcohol series. The slides were then stained with hematoxylin/eosin (H&E) and Masson’s trichrome (M-T). The tissue sections were viewed with an Olympus BX53 microscope and photographed with an Olympus DP72 camera. Images were analyzed using Cellsens software.
10
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Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
11 2.5.3. Immunohistochemistry Immunohistochemical staining of the BMP-2/PCL-Gel-BCP implanted group at 8 weeks was performed using the EnVisionTM kit (K5007, Dako, Carpentaria, CA, USA) to determine differentiation simulation in vivo. The tissue sections were heated with citrate buffer for antigen retrieval, blocked with normal blocking serum for 15 min, and then incubated with anti-OPN (1:100, Santa Cruz Biotechnology) and anti-osteocalcin (OCN; 10 µg/ml, Abcam, Cambridge, UK) for 1 hr. After washing, the sections were incubated with secondary antibody (Envision/HRP) for 30 min and finally were stained with substrate-chromogen solution (DAB) and hematoxylin. The tissue sections were viewed and examined using an Olympus BX53 light microscope equipped with CellSens software. 2.6. Statistical analysis All statistical analyses were performed using SPSS (Statistical Package for the Social Science, version 16, SPSS Inc., USA). Results are expressed as mean ± standard deviation37. Student’s t-test was used to compare different treatment groups, with significance assigned at P
Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
1
In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds Bo-ram Kim1, B.S, Nguyen Thuy Ba Linh1, 3, Ph.D, Young-Ki Min2,3, Ph.D, Byong-Taek Lee1, 3*, Ph.D 1
Department of Regenerative medicine, College of Medicine, Soonchunhyang University, 366-1, Ssangyong dong,
Cheonan, Chungnam, 330-090, Republic of Korea 2
Department of Physiology, College of Medicine, Soonchunhyang University, 366-1, Ssangyong dong, Cheonan, Chungnam,
330-090, Republic of Korea 3
Institute of Tissue Regeneration, College of Medicine, Soonchunhyang University, 366-1, Ssangyong dong, Cheonan,
Chungnam, 330-090, Republic of Korea
*Corresponding author: Byong-Taek Lee Telephone: +82-41-570-2427; Fax: +82-41-577-2415 Email: [email protected] 1
Page 2 of 36
Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
2 Abstract To confirm the effect of recombinant human bone morphogenetic protein-2 (BMP-2) for bone regeneration, BMP-2 loaded polycaprolactone (PCL)-gelatin (Gel)-biphasic calcium phosphate (BCP) fibrous scaffolds were fabricated using the electrospinning method. The electrospinning process to incorporate BCP nanoparticles into the PCL-Gel scaffolds yielded an extracellular matrix-like microstructure that was a hybrid system composed of nano and micro sized fibers. BMP-2 was homogeneously loaded on the PCL-Gel-BCP scaffolds for enhanced induction of bone growth. BMP-2 was initially released at high levels, and then showed sustained release behavior for 31 days. Compared with the PCL-Gel-BCP scaffold, the BMP-2 loaded PCL-Gel-BCP scaffold showed improved cell proliferation and cell adhesion behavior. Both scaffold types were implanted in rat skull defects for 4 and 8 weeks to evaluate the biological response under physiological conditions. Remarkable bone regeneration was observed in the BMP-2/PCL-Gel-BCP group. These results suggest that BMP-2 loaded PCL-Gel-BCP scaffolds should be considered for potential bone tissue engineering applications. Keywords: electrospinning, polycaprolactone, gelatin, BCP, BMP-2, bone tissue engineering, bone regeneration
2
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Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
3 1. Introduction Large sized bone injuries and defects are often repaired using alternative materials such as bone substitutes. Scaffolds play a vital role by serving as templates for host cells and supporting the regeneration of bone defects 1,2. An ideal scaffold should be biocompatible and biodegradable and function in a similar manner to the extracellular matrix (ECM) 3. Fibrous scaffolds fabricated by electrospinning have been used recently for biomedical applications because they can easily create a three-dimensional porous structure resembling the morphology of natural ECM
4-6
. The microstructure of electrospun scaffolds such as a fiber
diameter and porosity can be controlled by adjusting solution properties and operating parameters. Due to the large surface area to volume ratio of electrospun scaffolds, cell adhesion, migration, and proliferation improve 7-9. Various types of biomaterials such as natural polymers, synthetic polymers, ceramics, and their composites can be applied to the electrospinning process. The combination of natural and synthetic polymers improves mechanical properties as well as biocompatibility. Polycarprolactone (PCL) is a typical hydrophobic biodegradable synthetic polymer that degrades slowly inside the human body. Due to its slow degradation, it has been investigated mainly as long-term implant material for drug release and support of bone formation
10-12
.
Gelatin (Gel), which is natural polymer derived from collagen, has been widely used for biomaterial applications such as wound dressings, bone and cartilage regeneration, and drug delivery systems
13-16
because of its excellent biocompatibility and biodegradability. Several
studies have investigated PCL-Gel electrospun scaffolds
17-20
. This approach of using a
synthetic/natural polymer blend overcomes the drawback of individual synthetic and natural polymers and improves mechanical properties and biocompatibility.
3
Page 4 of 36
Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
4 However, a PCL-Gel polymer system alone cannot provide support for an ideal scaffold because it is not an osteoconductive or osteoinductive material 21. Natural ECM is composed of fibrous collagen organized in a three-dimensional porous network, and hydroxyapatite (HAp) crystals are dispersed within the collagen fibers
22
. In this study, biphasic calcium
phosphate (BCP) nanoparticles were dispersed into a PCL-Gel blend matrix to synthesize a bone mimicking ECM. BCP is a biodegradable and osteoconductive ceramic, which consists of HAp and tricalcium phosphate (TCP) phase compositions. It forms tight bonds with host bone tissues and allows osteogenesis. BCP is a more effective ceramic for bone repair and regeneration than pure HAp or pure TCP alone because it controls scaffold degradation rate 23,24
. Combining polymers with inorganic materials such as BCP particles can improve the
bioactivity of electrospun scaffolds and enhance cell attachment, proliferation, and osteoblastic differentiation 25-27. Bone regeneration is regulated by various bioactive agents such as growth factors (bone morphogenetic proteins [BMPs], transforming growth factor-beta, fibroblast growth factor, platelet-derived growth factor, and insulin-like growth factor), which are important signals in the osteogenic microenvironment
28,29
. Using osteoinductive growth factors to enhance bone
reconstruction and formation on this type of scaffold is the most efficient way to perform protein therapy
30,31
. Among the various types of growth factors, BMPs promote
differentiation by inducing mesenchymal stem cells to transform into chondrocytes and osteoblasts and cartilage and bone to form
32,33
. In this study, recombinant human BMP-2-
loaded PCL-Gel-BCP scaffolds were fabricated using a blend electrospinning process to encapsulate BMP-2 directly inside the fibers. We investigated the effects of BMP-2 on cell proliferation and differentiation of MC3T3-E1 cells and focused on osteogenesis in a rat calvarial skull model. 4
Page 5 of 36
Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
5 2. Materials and methods 2.1. Preparation of the BMP2-loaded PCL-Gel-BCP solution PCL (Mn 70,000–90,000) and Gel type A (300 Bloom) from porcine skin were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2,2,2-Trifluoroethanol (TFE, purity > 99.0%, Alfa Aesar, Cambridge, UK) was used as the solvent. BCP nanopowder was synthesized by a microwave-assisted process 34. The concentration and ratio of each solution were prepared according to a method reported previously
17
. To fabricate the electrospun
scaffolds, 10% w/v PCL and 10% w/v Gel solutions were prepared by dissolving 1 g of each polymer in TEF separately followed by stirring overnight at room temperature. The 10% w/v Gel solution was added to the 10% w/v PCL solution until it was mixed completely at a volume 50:50 ratio of PCL: Gel. BCP particles (size, 45–50 μm) were dispersed in TFE by ultrasound and added to the PCL-Gel solution at a ratio of BCP: PCL: Gel = 1:1:1 (w/w) and stirred for 2 hr to make the scaffold blended BCP. In addition, 0.5 μg/ml recombinant BMP-2 (R&D Systems Minneapolis, MN, USA; 10 µg, > 95% purity) was mixed with the PCL-GelBCP solution before starting the electrospinning process 35. 2.2. Fabrication of the electrospun scaffolds The PCL-Gel-BCP scaffolds and BMP2 loaded PCL-Gel-BCP scaffolds were fabricated by an electrospinning process. Five mL of the solution was loaded into a 12 mL plastic syringe fitted with an 18-gauge needle and injected using a syringe-pump at a flow rate of 0.5 mL/hr. The collector was covered with a piece of aluminum foil. The distance between the tip of the needle and the collector was about 10 cm, and 20 kV voltage was applied. The
fabricated
electrospun
scaffolds
were
cross-linked
using
1-ethyl-3
(3-
dimethylaminopropyl) carbodiimide (EDAC) and N-hydroxysuccinimide (Sigma-Aldrich) at 5
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Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
6 a 5:2 molar ratio in 80% ethanol for 1 hr at 4 ºC, rinsed three times with distilled water for 5 min to remove residual EDAC, frozen at −20ºC for 4 hr, and then freeze-dried at −80ºC. 2.3. Scaffold characterization 2.3.1. Morphological analysis The surfaces of the PCL-Gel-BCP and BMP2/PCL-Gel-BCP scaffolds were sputtercoated with platinum (Cressington 108 Auto, Cambridge, UK) and observed by scanning electron microscopy (SEM, JSM-6701F, JEOL, Tokyo, Japan) at an accelerating voltage of 10 kV. The average fiber diameter of the electrospun fibers was measured from SEM images. At least 10 positions on the fiber mat were tested to measuring electrospun fiber diameter. Energy dispersive X-ray spectrometer (EDS, JSM-7401F) was used to analyze the elemental composition of the electrospun fibers. The elemental composition of the crystal structures and the composition of the scaffolds were analyzed by X-ray diffraction (XRD, Rigaku, D/MAX2500V). The diffraction angle was varied from 10° to 60° 2θ. 2.3.2. BMP-2 release study To determine whether BMP-2 loaded successfully, BMP-2 loaded PCL-Gel-BCP scaffolds were observed by immunofluorescent staining and compared with PCL-Gel-BCP scaffolds (negative control). The scaffolds were washed with phosphate buffered saline (PBS) and treated with 5% bovine serum albumin (BSA) for 30 min. The scaffolds were blocked with BMP-2 primary antibody (BMP-2/4 [H-1]; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:50) for 4 hr and rinsed with PBS three times. BMP-2 secondary antibody (goat antimouse IgG-FITC, Santa Cruz Biotechnology) (1:100) was applied for 1 hr and washed again with PBS. After staining, confocal microscopy (FV10i-W) was performed to measure BMP-2 loading on the scaffolds. 6
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Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
7 The in vitro release rate of BMP-2 from the PCL-Gel-BCP scaffolds was measured with a BMP-2 enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems). To collect the supernatant, the BMP-2 loaded PCL-Gel-BCP scaffolds were immersed in 1 ml PBS and incubated at 37ºC. At various times, all of the supernatant was collected, and replaced with fresh PBS. The amount of BMP-2 in the supernatants was determined according to the manufacturer’s protocol. Absorbance was read with an ELISA reader (Infinite F50, Tecan, Zurich, Switzerland) at a wavelength of 450 nm. 2.4. In vitro study 2.4.1. Cell culture Pre-osteoblast MC3T3-E1 cells, derived from mice, were cultured in MEM alpha (MEM-α, Gibco, Grand Island, NY USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (P/S). The MC3T3-E1 cells were maintained in a humidified incubator at 37ºC in a 5% CO2 atmosphere. 2.4.2. Cell adhesion Electrospun nanofibers were initially sterilized under UV light, sterilized further with 70% ethanol, washed with PBS, and finally soaked in MEM-α. MC3T3-E1 cells were seeded on 1 cm2 of scaffolds at a density of 104 cells/cm2 in a 24-well plate with culture medium. To observe cell adhesion, the cells were cultured for 60 and 90 min. After culturing, the cells were fixed in 2% glutaraldehyde (DeaJung Co., Seoul, Korea) and dehydrated in an ethanol series (50, 70, 90, and 100%) (Merck, Darmstadt, Germany). The dehydrated cells were dried in hexamethyldisilazane (DeaJung Co) and observed by SEM. The cells were immunostained using fluorescein isothiocyanate (FITC) conjugated phalloidin (25 μg/ml, Sigma-Aldrich) and vinculin (Millipore, Billerica, MA, USA) for confocal microscopy. The cells on the scaffolds 7
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Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
8 were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature, permeabilized in 0.25% TritonX-100 (Sigma-Aldrich) for 10 min, and blocked in 2.5% BSA for 1 hr. The cells were immunostained using vinculin antibody at 4ºC overnight. After washing with PBS, the cells were stained with FITC conjugated phalloidin for 30 min. Nuclei were stained with Hoechst. Finally, the scaffolds were mounted on glass slides and visualized under a confocal fluorescent microscope (FV10i-W) using the accompanying FV10i-ASW 3.0 viewer software. 2.4.3. Cell viability To evaluate cell proliferation, the PCL-Gel-BCP and BMP-2/PCL-Gel-BCP scaffolds were
analyzed using the MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyltetrazolium
bromide) assay (Sigma-Aldrich)36. After 1, 3, and 7 days of culture, 100 μl of MTT solution was added to each well filled with PCL-Gel-BCP and BMP-2/PCL-Gel-BCP scaffolds and incubated for 4 hr. After removing the media, 1 ml dimethyl sulfoxide (Samchun Pure Chemical Co.) was added to each well to extract the formazan crystals under gentle shaking. The extract of each scaffold was transferred to a 96-well plate and the absorbance intensities were measured at 595 nm using an ELISA reader. 2.4.4. Cell proliferation and attachment Confocal microscopy was performed to observe growth of MC3T3-E1 cells (1 × 104 cells/well) seeded on the PCL-Gel-BCP and BMP-2/PCL-Gel-BCP scaffolds. Briefly, after 1, 3, and 7 days of culture, the scaffolds were rinsed with PBS twice and fixed in 4% paraformaldehyde for 10 min at room temperature, permeabilized in 0.25% TritonX-100 for 10 min, and blocked in 2.5% BSA for 1 hr. The cells were immunostained using FITC
8
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Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
9 conjugated phalloidin overnight at 4ºC. Nuclei were stained with Hoechst. Cell behavior was observed under a confocal microscope at 10× and 60× magnifications. 2.4.5. Cell differentiation The
osteogenic
differentiation
of
MC3T3-E1
cells
was
analyzed
using
immunocytochemical analysis of alkaline phosphate (ALP) and osteopontin (OPN). MC3T3E1 cells were cultured on PCL-Gel-BCP and BMP-2/PCL-Gel-BCP scaffolds using osteogenic medium consisting of MEM supplemented with 10% FBS, 1% P/S, 10nM dexamethasone (Sigma), 50 µg/ml L-ascorbic acid (Sigma), and 10 mM β-glycerophosphate disodium salt hydrate (Sigma). After 7 and 14 days, cells on the scaffolds were stained with rabbit ALP antibody (1:50, Santa Cruz Biotechnology) and mouse OPN antibody (1:50, Santa Cruz Biotechnology) overnight at 4ºC. Then, the scaffolds were incubated in secondary antibody (Alexa Fluor 488, 1:1000, Invitrogen, Carlsbad, CA, USA). The images were visualized under a confocal microscope using 60× magnification. 2.5. In vivo study 2.5.1. Implantation of the BMP-2/PCL-Gel-BCP scaffolds in the rat skull A total of 16 male Sprague–Dawley rats, weighing 300 g, were used for the in vivo study of PCL-Gel-BCP and BMP-2/PCL-Gel-BCP scaffolds (four rats each), after the protocol was approved by the Animal Ethical Committee of Soonchunhyang University. The healing process was observed 4 and 8 weeks after implantation. The PCL-Gel-BCP and BMP-2/PCLGel-BCP scaffolds were sterilized with 70% ethanol, washed with PBS, and finally freezedried at −80ºC for 3 hr before use. The rats were anesthetized with diethyl ether, and the hair on the skull was shaved and sterilized with 70% ethanol and povidone iodine. After exposure of the parietal skull, two defects were made on the left and right side of the skull (5 mm 9
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Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
10 diameter, and 1 mm thickness) using a trephine drill. One defect was grafted with PCL-GelBCP scaffolds and the other defect was sutured without a sample as a negative control. The BMP-2/PCL-Gel-BCP scaffolds were implanted in the same way. The subcutaneous tissue was closed and the skin was re-sutured. The rats were sacrificed 4 and 8 weeks after implantation. 2.5.2. Micro-computed tomography (micro-CT) and histological analysis Four and eight weeks of after implantation, the rats were sacrificed and the entire portion of the defected skull was removed. The samples were fixed in 10% formalin solution at room temperature. Micro-CT (Skyscan 1076, Antwerp, Belguim) was performed to observe new bone formation at the defect sites. Each sample was fixed on the object stage, and imaging was performed on the sample for 360°of rotation with an exposure rate of 20 min per frame. Micro-CT images were reconstructed using CTAn (Skyscan) and CTVol (Skyscan) to make three-dimensional images. The bone volume (BV) and percent bone volume (bone volume (BV)/tissue volume (TV) (%) were calculated to evaluate the new bone quantity. The rat skulls fixed in 10% formalin solution were decalcified using 5% HNO3. The tissues were embedded in a paraffin block and serially cut using a microtome (HM 325, Thermo Scientific, Rockford, IL, USA). The 4 ± 2 μm thick sections were mounted on microscope slides. The slides were deparaffinized and hydrated with xylene and an alcohol series. The slides were then stained with hematoxylin/eosin (H&E) and Masson’s trichrome (M-T). The tissue sections were viewed with an Olympus BX53 microscope and photographed with an Olympus DP72 camera. Images were analyzed using Cellsens software.
10
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Tissue Engineering Part A In vitro and in vivo studies of BMP-2 loaded PCL-Gelatin-BCP electrospun scaffolds (doi: 10.1089/ten.TEA.2014.0081) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
11 2.5.3. Immunohistochemistry Immunohistochemical staining of the BMP-2/PCL-Gel-BCP implanted group at 8 weeks was performed using the EnVisionTM kit (K5007, Dako, Carpentaria, CA, USA) to determine differentiation simulation in vivo. The tissue sections were heated with citrate buffer for antigen retrieval, blocked with normal blocking serum for 15 min, and then incubated with anti-OPN (1:100, Santa Cruz Biotechnology) and anti-osteocalcin (OCN; 10 µg/ml, Abcam, Cambridge, UK) for 1 hr. After washing, the sections were incubated with secondary antibody (Envision/HRP) for 30 min and finally were stained with substrate-chromogen solution (DAB) and hematoxylin. The tissue sections were viewed and examined using an Olympus BX53 light microscope equipped with CellSens software. 2.6. Statistical analysis All statistical analyses were performed using SPSS (Statistical Package for the Social Science, version 16, SPSS Inc., USA). Results are expressed as mean ± standard deviation37. Student’s t-test was used to compare different treatment groups, with significance assigned at P
