Sustained Release of BMP-2 from Porous Particles with Leaf-Stacked

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Biological and Medical Applications of Materials and Interfaces

Sustained Release of BMP-2 from Porous Particles with Leaf-Stacked Structure for Bone Regeneration Ho Yong Kim, Jin Ho Lee, Han A Reum Lee, Ji-Sung Park, Dong Kyun Woo, Hee-Chun Lee, Gyu-Jin Rho, June-Ho Byun, and Se Heang Oh ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02141 • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 4, 2018

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ACS Applied Materials & Interfaces 1

Sustained Release of BMP-2 from Porous Particles with Leaf-Stacked Structure for Bone Regeneration

Ho Yong Kim,1, † Jin Ho Lee,1, ‡ Han A Reum Lee,† Ji-Sung Park,§ Dong Kyun Woo,∥ Hee-Chun Lee,⊥ Gyu-Jin Rho,§ June-Ho Byun,**,# and Se Heang Oh*,†,○

†

Department of Nanobiomedical Science, Dankook University, Cheonan 31116, Republic of

Korea ‡

Department of Advanced Materials, Hannam University, Daejeon 34054, Republic of Korea,

§

Department of Theriogenology and Biotechnology, College of Veterinary Medicine,

Gyeongsang National University, Jinju 52828, Republic of Korea ∥

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Gyeongsang

National University, Jinju 52828, Republic of Korea ⊥

Department of Veterinary Medical Imaging, College of Veterinary Medicine, Gyeongsang

National University, Jinju 52828, Republic of Korea #

Department of Oral and Maxillofacial Surgery, Gyeongsang National University School of

Medicine, Gyeongsang National University Hospital, Institute of Health Sciences, Gyeongsang National University, Jinju, 52727, Republic of Korea

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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○

Department of Pharmaceutical Engineering Dankook University, Cheonan 31116, Republic

of Korea

KEYWORDS: growth factors, polycaprolactone, delivery system, tissue engineering, miniature pig


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ABSTRACT: Sustained release of bioactive molecules from delivery systems is a common strategy for ensuring their prolonged bioactivity and for minimizing safety issues. However, residual toxic reagents, the use of harsh organic solvents, and complex fabrication procedures in conventional delivery systems are considered enormous impediments towards clinical use. Herein,

we

describe

bone

morphogenetic

protein-2

(BMP-2)-immobilized

porous

polycaprolactone (PCL) particles with unique leaf-stacked structures (LSS particles) prepared using clinically feasible materials and procedures. The BMP-2 immobilized in these LSS particles is continuously released up to 36 days to provide an appropriate environment for osteogenic differentiation of human periosteum-derived cells (hPDCs) and new bone formation. Thus, the leaf-stacked structures of these LSS particles provide a simple but clinically applicable platform for effectively delivering a variety of bioactive molecules, such as growth factors, hormones, cytokines, peptides, etc.



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■

INTRODUCTION

Growth factors synthesized by cells are fundamental signaling molecules for precise orchestration of biological processes and maintenance of a healthy body.1-3 The growth factors that regulate cell behaviors (e.g., growth, migration, differentiation, maturation, etc.) are also key components for tissue regeneration. These bioactive molecules can be secreted by cells neighboring small-sized tissue defects to enable spontaneous tissue restoration. However, large defects are not restored as easily because of insufficient quantities and concentrations of these factors as well as a lack of a scaffolding matrix for the generation of new tissues. To compensate for such limitations, techniques in tissue engineering that integrate cells, signal molecules (e.g., growth factors), and scaffolds have been introduced and spotlighted in research and clinical fields.4,5 More recently, such techniques applied for the endogenous regeneration of damaged tissues have gained interest.6 These techniques for in situ tissue engineering involve recruitment and accommodation of host cells to damaged sites to stimulate new tissue growth.7-9 This approach minimizes the risk of pathogen transmission and reduces the cost/time for cell cultivation compared to cell-based approaches.7,10-12 However, growth factors are inherently unstable (via degradation or inactivation), and their practical application is further challenged by their high sensitivity to temperature, pH, and proteolytic degradation.13,14 Therefore, in initial strategies, large amounts of growth factors


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were applied at sites of tissue defects, risking potential cell/tissue damage and cancer formation, with a heavy economic burden for patients.3 A variety of delivery systems enabling sustained release of growth factors from appropriate matrices have been developed to provide long-term stability/bioactivity and minimize adverse outcomes.15 These systems incorporate growth factors on the surfaces or within matrices by chemical or physical means. The chemical means involve noncovalent hydrophobic interactions,16 hydrogen bonding,17 electrostatic attraction,18 and heparinmediated19,20 or enzyme-mediated21 binding, as well as covalent interactions from carbodiimide-mediated22 and photoinitiated3,22,23 reactions, click chemistry,24 maleimide thiol chemistry,25 and plasma treatments.26 Physical methods can also be used, in which growth factors are encapsulated in hydrogels, solids, and liposomes.27-29 Despite

benefits

from

matrix-delivered

growth

factors

(e.g.,

accelerated

nerve/cartilage/bone regeneration), their clinical use is plagued by residual toxic reagents in the

matrices,

the

use

of

harsh

organic

solvents

(leading

to

growth

factor

denaturation/inactivation), and complex fabrication procedures.30 In recent years, bone morphogenetic proteins (BMPs) with osteogenic activity have gained steady attention as a growth factor for enhanced bone reconstruction.31 BMP-2, with the most effective osteogenic activity among BMPs, has been adapted in clinical fields for bone diseases, such as nonunions, open fractures, and spinal fusions.32 The BMP-2 containing bone graft (Infuse;



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Medtronic, Inc.) was approved as a bone substitute by the U.S. Food and Drug Administration (FDA) in 2002.33 However, potential safety issues have been reported regarding the excessive use of BMP-2 and its burst release from collagen sponge.34 To the best of our knowledge, reports on clinically adaptable delivery systems for the sustained release of growth factors are few. Our main goals of this study were (i) to fabricate a delivery matrix that facilitates continuous release of growth factor using clinically acceptable materials and procedures for human use; and (ii) to confirm that the growth factor remains bioactive after it is released from the delivery matrix in vitro and in vivo. This report describes the development of a unique porous matrix with a leaf-stacked structure (LSS particles). The leaf-stacked structure is expected to provide a large surface area and complex three-dimensional maze-like release path for growth factors, thus enabling sustained release and long-term bioactivity without further chemical/physical modifications (Figure 1). BMP-2 with osteogenic activity was chosen as a model growth factor to confirm whether the LSS particles could allow sustained release of growth factors. PCL is used in clinical practices for biodegradable sutures and staples for wound closure, and tetraglycol is a nontoxic solvent used as a pharmaceutical excipient for intravenous/intramuscular injections.35-37 In particular, PCL is one of the most frequently utilized synthetic polymers to prepare 3D printed tissue engineering scaffold for bone regeneration.38 Morphology and BMP-2 release profile of the LSS particles, as well as


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the induction of osteogenic diﬀerentiation, were compared with those of PCL-based microporous (MP) particles and dense (DS) particles.39 We also evaluated each of these particles for promoting bone regeneration in a miniature pig (mandibular defect model).

■

EXPERIMENTAL SECTION Materials. PCL (MW, 81,000 Da; Lakeshore Biomaterials) and tetraglycol (Sigma-

Aldrich) were used to prepare LSS particles. Pluronic F127 (EG99PG65EG99, MW, 12,500 Da; Sigma-Aldrich) and NaCl particles (sieved to sizes of 25–53 µm; Daejung) were used to fabricate DS and MP particles. Recombinant human BMP-2 [Source, Chinese Hamster Ovary (CHO) cell line; R&D Systems] was used as a growth factor for osteogenic cell differentiation and bone reconstruction. Water was purified to ultrapure grade using an EXL®3 pure & ultrapure water system (Vivagen). For in vitro cell culture and in vivo animal studies, PCL particles were sterilized by ethylene oxide. Preparation and characterization of PCL particles. Preparation of porous particles with leaf-stacked structure (LSS particles). The LSS particles were fabricated by a sprayprecipitation method.40 In brief, a hot (90°C) PCL solution (15 wt% in tetraglycol) was placed in a 10-mL syringe, and the hot solution was spattered through a double-nozzle spray (outer nozzle, N2 purging of 1 L/min) into 50% ethanol (17°C; precipitation solution) (Figure 2A). The speed of which the PCL solution was controlled at 60 mL/h using a syringe pump (inner nozzle). The syringe and spray were warmed (90°C) using a heating tape (HT2510; Misung



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Scientific) to preclude PCL from solidifying during the process. The distance between the nozzle tip and the precipitation solution was 20 cm. Precipitated PCL particles were immersed in precipitation solution for 6 h, and the PCL particles were washed by water for 24 h. The PCL particles were then separated by size in the range of 1.0–1.4 mm using standard testing sieves (Chunggye Industrial Co.) with aperture size 1.0 mm and 1.4 mm, and freeze-dried. To demonstrate structural change in PCL solution droplets during precipitation, the hot PCL solution (300 µL) was dropped on slide glass at 17°C (same temperature as the coagulation solution). And the precipitation process was observed over time under a light microscope (BX51; Olympus). Preparation of DS and MP particles. DS and MP PCL particles (1.0–1.4 mm in size) were prepared as controls using isolated particle-melting method and melt-molding particulateleaching method with DS particles, respectively.39 For DS particles, PCL pellets were crushed using a freezer mill (SPEX 6750; SPEX SamplePrep) and separated for a size range of 1.0– 1.4 mm using standard testing sieves with aperture size 1.0 mm and 1.4 mm. The crushed PCL particles were evenly mixed with Pluronic F127 solution (4 oC, 17.5 wt%, solid state, 1:50 [w/v]), and the mixture was positioned in a prewarmed (65°C) water for 30 min. Pluronic F127 changed into a gel state (gelation), and the random-shaped PCL pellets were melted into sphere shapes in the Pluronic F127 gel matrix. DS particles were collected after cooling at room temperature (RT), washing with excess water for 24 h, and vacuum drying. For MP particles, NaCl particles (25–53 µm in size) and DS particles (500–710 µm, 1/40


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[w/w]) were put in a brass mold (diameter, 18 mm; thickness, 2.5 mm). The mold was compressed under 10 MPa (80°C, 3 min) and sequentially compressed under 15 MPa (1.5 min) using a compression molding equipment. The salt-infiltrated particles were removed from the particles by washing in excess water for 24 h. MP particles of 1.0–1.4 mm in size were collected after vacuum drying and microsieving using standard testing sieves with aperture size 1.0 mm and 1.4 mm. Characterization of PCL particles. Morphologies of DS, MP, and LSS particles were observed under a scanning electron microscope (SEM; S-4300; Hitachi). Cross-sectional sections of samples frozen in liquid nitrogen were cut with a cold blade. Surface areas of DS, MP, and LSS particles were analyzed with the Brunauer–Emmett–Teller (BET) technique. For this, each 0.5-g particle underwent nitrogen adsorption and desorption using a BET surface area analyzer (AsiQwin; Quantachrome) at 77 K. The specific surface area was calculated by using BET equation.41 Porosities of DS, MP, and LSS particles were also analyzed with a porosimeter using mercury (PoreMaster 33GT, Quantachrome). Fourier Transform Infrared Spectroscopy (FTIR; 640-IR; Varian) was used to confirm any changes of chemical structures (functional groups) of the PCL particles during each fabrication procedure. BMP-2 immobilization and release behavior. To immobilize BMP-2 on the DS, MP, and LSS particles, 10-mL syringes were filled with each particle type (2 mL) and BMP-2 solution (1 µg/mL in phosphate-buffered saline [PBS] containing 1% bovine serum albumin [BSA]).



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The syringes containing BMP-2 solution and particles were stored at 4°C for 3 h under positive pressure to permeate BMP-2 solution into porous MP and LSS particles when the plunger was pushed in. Then, the BMP-2 solution was aspirated and freeze-dried. The amount of BMP-2 immobilized on each particle was quantified directly via an enzyme-linked immunosorbent assay (ELISA),42 in which the standard ELISA processes of the kit (antibody source, monoclonal mouse IgG1; Duoset®, R & D systems) was followed but the particle was used as the primary substance. Each BMP-2-immobilized particle type was blocked by reagent diluent (1% BSA) in 96-well plates (Corning Inc.) for 1 h. After washing with Tween 20 solution (0.05% in PBS) three times, the particles were immersed in a detection antibody solution for 2 h. After washing, the particles were immersed in Strept Avidin-HRP solution for 20 min. These wet particles were put in a cryostat, frozen at - 20 oC, and cryosectioned into 200 µm. These sections were laid on the bottom of wells (96-well plate). Finally, color reagent A (H2O2)/color reagent B (tetramethylbenzidine) (1/1, v/v) substrate solution was added to each well and color development was monitored with a series of BMP-2 standard solutions. After 20 min, stop solution (2N H2SO4) was added to each well, and the absorbance was measured at 450 nm using a plate reader. The BMP-2 loading efficiency in each particle (occupied volume of particle, 2 mL) was calculated by the following formula: Loading amount of BMP-2 immobilized in the particle / 100% loading amount of BMP-2 in the particle [total volume of BMP-2 solution (mL) wetted in the particle x initial concentration of BMP-2 solution (1 µg/mL)] x 100. To compare the release patterns of PCL particles with different morphologies, BMP-2loaded particles (5 mg) were incubated in PBS (1 mL; containing 1% BSA) at 37°C for 36


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days with shaking at 50 rpm. The incubated medium was collected every day and changed with new PBS. The amount of BMP-2 released from each particle group was measured using ELISA kit. To visualize the release pattern of BMP-2 from each particle, BMP-2 was conjugated to rhodamine using a rhodamine fast conjugation kit (Abcam). The rhodamine-conjugated BMP2 was then immobilized on the DS, MP, and LSS particles and incubated in PBS using the same procedures described above. At 0, 1, 3, 7, 14, 21, and 28 days, particle cross-sections (central region) were observed under a confocal laser scanning microscope (LSM 700; Carl Zeiss). Cell culture. Human periosteum-derived cells (hPDCs) which can be differentiated into osteoblasts were used to estimate bioactivity of BMP-2 released from the DS, MP, and LSS particles. The hPDCs were collected from periosteal tissues of patients after obtaining informed consent. This process was approved by the Ethics Committee of Gyeongsang National University Hospital (GNUH 2014-05-012). To harvest the hPDCs, alveolar periosteal specimens were put in culture dishes (100-mm) and incubated in growth medium.43 To examine cell behaviors, the hPDCs (passage 3) were seeded on well (24-well plates, Corning Inc.; cell density, 3 × 104 cells/well) and incubated in the growth medium for 24 h. Then, the medium was changed with osteogenic medium44 and plate inserts (polycarbonate membrane; pore size, 8.0-µm; Corning Inc.) were put in each well. BMP-2-loaded PCL particles [16 mg; to provide effective concentration of BMP-2 for osteogenic differentiation



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(0.1 - 800 ng/mL)45 in culture medium by the LSS particles] were put in the insert wells and incubated for 4 wk. A cell only group without particles or BMP-2 was used as a control. At 0, 1, 2, and 4 wk, cell proliferation was observed by using a CCK-8 assay (Dojindo, Japan). In vitro osteogenic differentiation of hPDCs. Alkaline phosphatase (ALP) activity. ALP staining was performed for hPDCs stimulated by BMP-2 released from each particle type. After 1, 2, and 4 wk in culture, cells were washed with PBS supplemented with 0.05% Tween 20 (Sigma-Aldrich) and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 5 min (at RT). The fixation solution was aspirated, and cells were incubated with 5-bromo-4-chloro-3indolyl phosphate/nitro blue tetrazolium solution (SigmaFast BCIP/NBT tablet; SigmaAldrich) for 10 min. The stained cells were examined under an inverted microscope (CKX41; Olympus). For quantitative analyses, the hPDCs from each group were harvested after 0, 1, 2, and 4 wk in culture, and ALP activity was determined using an ALP enzyme kit (Abcam), and was quantified on an ELISA plate reader (Spark 10M; Tecan) at 405 nm. ALP activity was normalized by DNA contents as determined by a PicoGreen dsDNA quantification kit (Molecular Probes). Calcium deposition. Mineralization of the hPDCs was estimated by alizarin red S (ARS) staining. After 1, 2, and 4 wk in culture, the cells in each group were fixed with 4% paraformaldehyde for 10 min (at RT) and treated with 40 mM ARS (Sigma-Aldrich) for 45 min. The mineralization of cells was detected under an inverted microscope (CKX41), and the concentrations of ARS in each well were determined by a colorimetric technique. Ten percent


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acetic acid (Daejung) was added in each well then incubated at RT for 30 min under shaking at 50 rpm. Resultant solutions were incubated at 85°C for 10 min, cooled on ice, then centrifuged. Supernatants (200 µL) were neutralized with ammonium hydroxide (75 µL), and ARS concentrations were determined with a plate reader (Spark 10M; Tecan) at 405 nm. After 0, 1, 2, and 4 wk in culture, the hPDCs were washed by Tyrode’s balanced salt solution then decalcified by 0.6 N HCl (300 µL) at RT for 24 h. Calcium contents in the resultant solutions were measured using a calcium colorimetric assay kit (calcium C-test Wako; Wako Pure Chemical Industries). Quantitative real-time PCR. After 0, 1, 2, and 4 wk in culture with PCL particles, RNA was extracted from cells using TRIzol reagent (Molecular Research Center) and converted into cDNA using a First Strand cDNA synthesis kit (Applied Biosystems Inc.). Primers and probes (Runx2, catalog no. Hs00231692-m1; type 1 collagen, catalog no. Hs0016004-m1; osteocalcin, catalog no. Hs00609452-g1; osteopontin, catalog no. Hs00959010_m1; glyceraldehyde 3-phosphate dehydrogenase [GAPDH], catalog no. Hs02758991-g1; TaqMan gene expression assay kit [Applied Biosystems Inc.]) were used for amplification on a ViiA 7 real-time PCR system (Applied Biosystems Inc.). The data were normalized to GAPDH expression levels. Immunocytochemical analysis. After 1, 2, and 4 wk in culture, osteogenic differentiation of hPDCs stimulated by BMP-2 released from each particle type was visualized via immunocytochemical staining. Cells in each group were rinsed with PBS, fixed with 4%



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paraformaldehyde (20 min), permeabilized with PBS containing 0.1% Triton X-100 (5 min), and blocked with PBS supplemented with 10% BSA (1 h). The cells were incubated first with antibodies against Runx2 (1:100 dilution; Abcam) and osteocalcin (1:100 dilution; R&D Systems) for 12 h (at 4°C), and then with DyLight 488 (1:200 dilution; Abcam) and NorthernLights 557 (1:200 dilution; R&D Systems) secondary antibodies, respectively, for 1 h (at RT). Cell nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole, Vector Laboratories), and the cells were examined under a confocal laser microscope (LSM 700). Animal study. To demonstrate in vivo bioactivity of BMP-2 released from the DS, MP, and LSS particles, miniature pigs (~25 kg) with mandibular defects were chosen as an animal model. Animal studies were approved by the Animal Center of Biomedical Experimentation at Gyeongsang National University (GNU-160913-P0047). Animals were anesthetized with tiletamine-zolazepam (10 mg/kg) and azaperone (4 mg/kg). Mandibular bodies and rami were exposed through submandibular incisions. Three bone defects (4 mm in depth and 12 mm in diameter) were created on each mandible. The BMP-2-loaded PCL particles (DS, MP, and LSS particles; 50 mg) were implanted in the bony defects, and the incisions were closed in two layers using sutures. To prevent infections at the surgery lesions, the pigs received intramuscular injections of a first-generation cephalosporin twice daily for 7 days. Radiographic images and computed tomography (CT) scans were collected 0, 4, 8, and 12 wk after implantation from pigs anesthetized with tiletamine-zolazapam. Open mouth radiographs of the mandibles were made from pigs in right lateral recumbent positions. CT


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scans were acquired with a CT scanner (Somatom Emotion Duo; Siemens AG). Images were obtained on a Lucion image post processing system (Infinitt Technology). Three-dimensional (3D) reconstructions of mandible regions of interest (ROIs) were made to examine bone healing process. Bone regeneration in the target region was quantified by Hounsfield units (HUs) bone density measurement of CT scans. Briefly, HU values were calculated from pixel data on DICOM CT images of circular ROIs (0.5 cm2). The miniature pigs were sacrificed at 12 wk after implantation and the mandible specimens were harvested for histological observation (hematoxylin and eosin [H&E] staining) under a light microscope (CKX41). Statistical analysis. The data obtained each experiment are expressed as means ± standard deviations (SDs). Statistical analyses were performed using GraphPad Prism software (GraphPad Software). Data were evaluated by one-way analysis of variance (ANOVA). After verifying that the value was significant by ANOVA, Tukey’s multiple comparison test was conducted. Comparisons with p values of

Sustained Release of BMP-2 from Porous Particles with Leaf-Stacked

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