Collagen Scaffolds Loading Insulin PLGA

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

Porous nano-hydroxyapatite/collagen scaffolds loading insulin PLGA particles for restoration of critical size bone defect Xing Wang, Xia Wu, Helin Xing, Guilan Zhang, Quan Shi, Lingling E, Na Liu, Tingyuan Yang, Dongsheng Wang, Feng Qi, Lianyan Wang, and Hongchen Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13566 • Publication Date (Web): 03 Mar 2017 Downloaded from http://pubs.acs.org on March 4, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

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

ACS Applied Materials & Interfaces

Porous nano-hydroxyapatite/collagen scaffolds loading insulin PLGA particles for restoration of critical size bone defect Xing Wang,1,a, bXia Wu, 1,aHelin Xing,1,a Guilan Zhang, aQuan Shi,aLingling E, aNa Liu,a Tingyuan Yang, cDongsheng Wang,aFeng Qi,c Lianyan Wang,c, * Hongchen Liu a, * a Institute of Stomatology, Chinese PLA General Hospital, Beijing 100853, China b Hospital of Stomatology, Shanxi Medical University, Taiyuan, 030001, China c National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, China. 1These authors contributed equally to this paper. * Corresponding author.

Address for correspondence and reprints: Hongchen Liu, Ph.D. Institute of Stomatology, Chinese PLA General Hospital, Fuxing Lu 28# Beijing 100853, China, E-mail addresses: [email protected]. Lianyan Wang, Ph.D. National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, China, E-mail addresses: [email protected]

ACS Paragon Plus Environment


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

ABSTRACT Insulin is considered as a classical central regulator of energy homeostasis. Recently, the effect of insulin on bone has gained a lot of attentions, but little has been paid to the application in bone tissue engineering. In this study, porous nano-hydroxyapatite/collagen (nHAC) scaffolds incorporating poly lactic-co-glycolic- acid (PLGA) particles were successfully developed as an insulin delivery platform for bone regeneration. Bioactive insulin was successfully released from the PLGA particles within scaffold, and the size of the particles as well as the release kinetics of the insulin could be efficiently controlled through Shirasu porous glass premix membrane emulsification technology. It was indicated that the nHAC/PLGA composite scaffolds possessed favorable mechanical and structural properties for cells adhesion and proliferation, as well as the differentiation into osteoblasts. It was also demonstrated that the nHAC/PLGA scaffolds implanted into a rabbit critical-size mandible defect possessed tissue compatibility and higher bone restoration capacity compared with the defects that were filled with or without nHAC scaffolds. Furthermore, the in vivo results showed that the nHAC/PLGA scaffolds which incorporated insulin-loaded microspheres with size of 1.61µm significantly accelerated bone healing compared with another two composite scaffolds. Our study indicated that the local insulin released at the optimal time could substantially and reproducibly improve bone repair. Keywords: Insulin, Composite Scaffold, Drug delivery system, Bone tissue engineering, PLGA, Hydroxyapatite


Page 2 of 40

Page 3 of 40

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


1. INTRODUCTION Non-union defects of bone are the major challenges in orthopedics, maxillofacial surgery and reconstructive medicine. In the 13 million fractures that occur yearly in the United States, about 10% of them fail to heal.1 In many cases, some synthetic implants can provide a temporary well-defined region of containment in the defects, but the limited osteoinductivity and inadequate cellular responses will the delay bone healing.2,3 As a traditional remedy, autogenous bone grafting is the most predictable procedure for the bone regeneration, but the donor site morbidity, limited tissue availability and additional surgical pain restrict its clinical applications.4 Therefore, development of advanced biomaterials combined with inducible factors that can support damaged tissue, promote osteogenesis and rapid healing in patients is highly desired. Insulin, a drug but also a hormone, is widely considered as a central regulator of energy homeostasis. Recently, it has been increasingly recognized as an inducible factor for osteogenesis and bone turnover. Bone marrow mesenchymal stromal cells (BMSCs) and osteoblasts express functional insulin receptor(IR)5and respond to exogenous insulin by increasing rates of proliferation,6 alkaline phosphatase production7 and collagen synthesis.8 Moreover, in vivo studies have shown that insulin induces increased bone formation to two or three times compared with the initial level when administered locally in bone defect models or around titanium implants.9 However, in these applications, the bioavailability of insulin is very low due to the leakage and/or loss of bioactivity in the short term.10 Therefore, a controlled, localized insulin delivery system is very important in the protection of insulin bioactivity and maintenance of drug concentration at the defect



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

site for effective bone regeneration. Poly lactic-co-glycolic-acid (PLGA) possesses excellent biocompatibility and biodegradability and has been widely applied to deliver cytokines andproteins.11The bioactivity of protein can be largely protected during the encapsulation and release. Meanwhile, the release behavior can be regulated by adjusting the molecular weight, preparation method, particle size and so on. However, in terms of bone regeneration applications, ideal material would serve not only as a drug delivery carrier, but also as a porous scaffold for cellular activities. Recently, more attentions have focused on nano-structural hydroxyapatite/collagen (nHAC) due to its three-dimensional porous structure and similar composition as natural bone, which favors cells adhesion, proliferation and deposition of calcium-containing minerals on its surface.12,13Although several techniques have been proposed to incorporate drug in nHAC, it is difficult to regulate the drug release behavior before the nHAC degradation. Therefore, scaffolds composed of a single above component are not ideal bone substitutes because of their shortcomings, and a composite scaffold of PLGA and nHAC may be a preferable strategy. Based on this, we encapsulated insulin into PLGA particles and further loaded the particles inside the nHAC scaffold to prepare composite scaffold. Given that the healing of bone defect is a complex, coordinated temporal and multistage process, we hypothesized that the beneficial effects of insulin might be limited to a specific stage of healing. Thus, to achieve the optimal biological function of this factor, composite scaffolds with different insulin release kinetics regulated by PLGA particle size were investigated in this study.


Page 4 of 40

Page 5 of 40

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


2. MATERIALS AND METHODS 2.1 Materials PLGA with a molar ratio of D, L-lactide/glycolide 75/25 (Mw 13 kDa) was purchased from Lakeshore Biomaterials (Birmingham, AL, USA). Human recombinant insulin was provided by Wako Pure Chemical Industries, Ltd (Osaka, Japan). Poly (vinyl alcohol) (PVA-217, degree of polymerization 1700, degree of hydrolysis 88.5%) was provided by Kuraray (Tokyo, Japan). Shirasu porous glass (SPG) membranes were purchased from SPG Technology Co. Ltd. (Miyazaki, Japan). The SPG premix membrane emulsification equipment (FMEM-500M) was designed by National Engineering Research Center for Biotechnology (NERCB, Beijing, PR China). The nano-hydroxyapatite/collagen (nHAC) was provided by Beijing Allgens Medical Science & Technology Co., Ltd. (Beijing, China).The nHAC scaffolds are similar to that of natural bone: hydroxyapatite content of 45%±5%, a porosity of 75-88% and a pore size of 80-150 µm.14,15All other reagents were analytical grade.

Figure 1 Preparation process of insulin nHAC/PLGA composite scaffolds



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

2.2 Preparation of insulin-loaded particles Microspheres were prepared using SPG premix membrane emulsification combined with the W 1/O/W 2 double emulsion solvent evaporation method (Figure 1). First, 1 mL of insulin solution (3%, w/v ) as W 1 was emulsified with 8 mL of organic solvent (methylene dichloride, O) containing PLGA (10%, w/v) via ultrasonication (S-450D Digital Sonifier, Branson, USA) at 120 W for 60 s to form primary emulsions of W 1/O. The W 1/O emulsions were mixed with an external aqueous phase (W 2) containing PVA (2%, w/v) and NaCl (0.5%, w/v) to form coarse double emulsions (W 1/O/W 2). Then, the coarse emulsions were poured into a premix reservoir. Uniform-sized droplets with diameters of 20µmand 2µmwere obtained by extruding the coarse double emulsions through 50.4µmand 2.8µmSPG membrane with 5kpa and 50kpaof N2 pressure, respectively. Then, the emulsion droplets were stirred for 12 h at room temperature to allow the solvent evaporation. Finally, the microspheres were collected via centrifugation at 300-3000 g, and washed with distilled water for 5 times and lyophilized. Blank PLGA microspheres were fabricated using distilled water as internal aqueous phase instead of insulin solution, and the procedures was same with as above. For the nanospheres, 1 mL of insulin solution (3%, w/v ) as W 1 was emulsified with 8 mL of methylene dichloride containing PLGA (10%, w/v) via ultrasonication at 200 W for 40 s to form primary emulsions of W 1 /O. The W 1 /O emulsions were mixed with an external aqueous phase containing sodium cholate hydrate (1%, w/v) to form double emulsions (W1/O/W2) by ultrasonication at 200 W for 160 s. Next, the W1/O/W2 emulsions were stirred at 600rpm for 5 min. Then, they were solidified at room


Page 6 of 40

Page 7 of 40

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


temperature at 250 rpm for 5 h. Finally, the nanospheres were collected and washed using a 100,000Da ultrafiltration membrane for 5 times. 2.3 Surface morphology observation and size distributions measurement The shape and surface morphology of particles were observed by a scanning electron microscope (SEM, JSM-6700 F, JEOL, Japan). The particle size and size distributions were measured by laser diffraction using Zetasizer Nano (Malvern, UK) and Mastersizer 2000 (Malvern, UK). The size uniformity of particles was referred as Span value and calculated as follows: Span =

D v , 90% − Dv ,10% D v , 50%

Where Dv,90% , Dv,50% and Dv,10% are volume size diameters of particles at 90%, 50% and 10% of the cumulative volume, respectively. 2.4 Measurement of insulin loading efficiency and encapsulation efficiency PLGA particles (5 mg) were dispersed in 10 mL of 0.04 M NaOH solution and kept in an orbital shaker at constant gentle shaking of 110 rpm at 37 °C for 12 hours. The quantitative insulin loaded in PLGA particles were then determined using a Micro BCA protein assay kit according to the manufacturer instructions. Blank PLGA particles were treated with the same process as the control. The Loading Efficiency (LE) and Encapsulation Efficiency (EE) of insulin were calculated by following equations: Mass of drug in particles × 100% Mass of particles Loading efficiency EE(%, w / w) = × 100% Theoretical loading efficiency LE (%, w / w) =

2.5 Drug distribution in the PLGA particles



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

Page 8 of 40

To observe the drug distribution in PLGA particles, insulin labeled with Super Fluor 488 SE (Fanbo Biochemicals, China) was encapsulated into particles. The PLGA particles were observed by confocal laser scanning microscopy (CLSM, Leica, Germany) at an excitation wavelength of 488 nm. 2.6 Insulin release from the PLGA particles Insulin-loaded PLGA particles (20mg) were placed in a 10,000 Da bag filter, and were incubated in 10 mL of 10 mM phosphate buffer saline medium (pH 7.4) under a constant vibration of 30 rpm at 37°C. At each time interval, liquid was pipetted from the bag filter and replaced with equal volume fresh buffer. The concentrations of insulin in the collected

liquid

were

determined

using

insulin

ELISA

Kit

(Mercodia,

Sweden). Approximately 10 mL of 100mU/L insulin solution was also incubated as control. The measurements were performed in triplicate, and the error was expressed as the standard deviation. 2.7 Fabrication of nHAC/ PLGA composite scaffolds The suspensions of insulin-loaded PLGA particles with different sizes (4 mL, 0.5%, w/v) were prepared at first. Then, 10×5×3 mm3 of the nHAC scaffold was immersed into the PLGA particles suspensions under a 0.1 MPa vacuum suction for 10 min, and was subsequently shacked by vortex for 10 min (Figure 1). The sample of nHAC scaffold immersed into 4 ml insulin solution (0.5%, w/v) under a 0.1 MPa vacuum suction was used as control (I-nHAC). After vacuum suctioning, the suspensions were centrifuged and the unloaded PLGA particles were collected to detect the residual insulin using ELISA Kit. The amount of insulin loaded into nHAC was calculated by subtracting the residual insulin


Page 9 of 40

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


from the total amount. The measurements were performed in triplicate, and the error was expressed as the standard deviation. 2.8 Physical characterizations of scaffolds The effects of particles incorporation on the mechanical properties of scaffolds were assessed and compared with blank nHAC scaffolds. The morphological characteristics of the surface and cross-section of the obtained nHAC/ PLGA composite scaffolds were observed using a scanning electron microscope (SEM). In order to make a quantitative analysis to the structure of nHAC/ PLGA composite scaffolds, scaffolds were embedded in glycolmethacrylate (Polysciences Inc., UK), sectioned and stained with toluidine blue. Using MATLAB® pore topology analyzer software, the stained images were subjected to uneven lighting removal, image enhancement, binarization, removing interference objectives to calculate pore diameters and porosity of the scaffolds .16 The compressive strengths of the scaffold specimens (size of 10×5×3 mm3) were measured by a universal testing machine (Z050, Zwick/Roell, Germany) at a constant loading rate of 0.5 mm/min.17 The compressive strength was determined by the maximum point of the stress-strain curve. The reported data was the average of three samples within each group. 2.9Insulin release kinetics of scaffolds To examine the insulin release profiles, 10×5×3 mm3 of nHAC/ PLGA and I-nHAC scaffolds were placed in a 10,000 Da bag filter. These were incubated in 10 mL of 10 mM phosphate buffer saline medium (pH 7.4) under a constant vibration of 30 rpm at 37°C. At each time interval, liquid was taken from the bag filter and replaced with equal volume



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

fresh buffer. The concentrations of insulin in the collected liquid were determined using insulin ELISA Kit. The measurements were performed in triplicate, and the error was expressed as the standard deviation. 2.10 In vitro bioactivity assessment of insulin delivered from scaffolds Human bone marrow mesenchymal stromal cells (BMSCs) were isolated from bone marrow specimens and cultured as described in a previously protocol.18 Bone marrow specimens were obtained as femoral tissue discarded during primary hip arthroplasty. These methods were approved by the Chinese PLA General Hospital Review Board. To minimize other possible impacts on BMSCs, the following exclusion criteria were as follows: age over 55 or less than 20, rheumatoid arthritis, cancer, osteoporosis and diabetes. A total of 23 subjects (14 women and 9 men) with ages from 26 to 51 years-old, were included in this study. P4 BMSCs (106/ml, 100µl) were seeded dropwise into nHAC, I-nHAC and nHAC/ PLGA composite scaffolds, respectively. Afterwards, minimum essential alpha medium (αMEM; Gibco, USA) with 15% fetal bovine serum (FBS, Gibco) was added to each well (24-well plates, 37 °C, 5% CO2). The medium was renewed twice each week. To analyze the influence of scaffolds on cell proliferation, DNA assay was quantified after 3, 7 and 14 days of culture. The BMSCs + nHAC/PLGA scaffolds were homogenized in lysis buffer (SensoLyte pNPP Alkaline Phosphatase Assay Kit, AnaSpec, USA) using a rotor-stator homogenizer (Omni International, Germany). Then the suspension was centrifuged at 2500 ×g for 10 min at 4 °C and analyzed for DNA content using the Quanti-iT TM PicoGreen DNA Molecular Probes (OR, USA). 14Fixed numbers of cells


Page 10 of 40

Page 11 of 40

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


were analyzed to convert DNA content into cell number per sample. Alkaline phosphatase (ALP) and osteocalcin (OCN) were applied to evaluate the osteogenic differentiation of BMSCs cultured in scaffolds. At 3, 7 and 14 days, the supernatants were collected and replaced with an equal amount of fresh αMEM (with 15% FBS). The ALP activity in the medium was detected using Roche Diagnostics ALP kits on the cobas e602 platform (Roche Diagnostics, Mannheim, Germany), and calcium content and phosphonium content were measured using Roche Diagnostics Ca/P kits. OCN activity was tested using N-MID Osteocalcin kits, based on electrochemiluminescence immunoassay techniques. The measurements were performed in triplicate. All data were measured and analyzed on the cobas 8000 platform (Roche Diagnostics). Similarly, 1 × 105 BMSCs were cultured on different scaffolds in αMEM (with 15% FBS). At Day 21, the cells were fixed with 2.5% glutaraldehyde and stained with Alizarin red. To quantify the amount of Alizarin Red, the deposition was extracted by 10% (w ⁄v) cetylpyridinium chloride in 10 mM sodium phosphate (pH 7.0) at room temperature for 2 h, and the amount of AlizarinRed stain in the extraction buffer was determined by measuring the optical density (OD) of the solution at 560 nm.19 2.11In vivo studies of restoration of critical-size bone defect The bone restoration capacity of the nHAC/PLGA composite scaffolds was evaluated in a critical-size mandible bone defect model of New Zealand white rabbits. Thirty five female rabbits with weight 2.50~3.00 kg were housed. All surgical procedures and care were performed with the approval of the Chinese PLA General Hospital Review Board. The rabbits were intravenously anesthetized using 2% pentobarbital sodium (30 mg/kg).



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

Under aseptic conditions, a 15 mm incision was made along the edge of the right mandible of each rabbit. A segmental critical-size defect (10×5×3 mm) was prepared in the mandible using a surgical oscillating saw.20 Eighteen defects were treated with nHAC/PLGA composite scaffolds. Untreated defect (Blank) and defects treated with nHAC scaffolds were used as the negative control. Four and eight weeks after the surgical procedure, rabbits were sacrificed successively to collect mandible specimens. Then, the specimens were surgically removed, fixed, dehydrated, and embedded undecalcified in methyl methacrylate. Tissue sections (20 µm thick) were cut, ground and stained with toluidine blue and methylene blue/acid fuchsin. All samples were observed by a Leica DMLB microscope (Leica Microsystems Inc., Wetzlar, Germany) and analyzed by BioQuant software (R&M Biometrics, Nashville, USA). Ten successive histological sections were prepared from each implant according to Cavalierie’s principle.21 The amount of bone formed was determined in each section as a percentage of bone volume per total volume (BV/TV) by point counting using a grid.22 The specimens of eight weeks after the surgical procedure were monitored using a Quantum FX micro computed tomography Imaging System (Micro CT, Caliper, USA). Field of view (FOV) scanning at 36 mm and a 4.5 µm voxel size resolution were selected to obtain 512 binary images. The calculated Bone mineral density (BMD) and BV/TV in this area was presented as the percentage of defect regeneration.23 2.12 Statistical analysis SPSS 16.0 software was applied to analyze the data. All data were expressed as mean ± standard deviation. One-way ANOVA and two-tailed Student t tests were used to


Page 12 of 40

Page 13 of 40

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


determine statistical significance, and p

Collagen Scaffolds Loading Insulin PLGA

Recommend Documents