Subscriber access provided by UNIV LAVAL
Article
Highly compliant vascular grafts with gelatinsheathed coaxially-structured nanofibers Naveen Nagiah, Richard Johnson, Roy Anderson, Winston Howard Elliott, and Wei Tan Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b03177 • Publication Date (Web): 03 Nov 2015 Downloaded from http://pubs.acs.org on November 5, 2015
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.
Langmuir 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 32
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
Langmuir
Highly compliant vascular grafts with gelatin-sheathed coaxiallystructured nanofibers Naveen Nagiah, Richard Johnson, Roy Anderson, Winston Elliott, Wei Tan Department of Mechanical Engineering, University of Colorado at Boulder, Boulder, CO
Corresponding author: Dr. Wei Tan Associate Professor ECME 265 Department of Mechanical Engineering University of Colorado Boulder Boulder Colorado, USA 80302 Email :
[email protected] ACS Paragon Plus Environment
Langmuir
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 We have developed three types of materials composed of polyurethane-gelatin, polycaprolactonegelatin or polylactic acid-gelatin nanofibers by coaxially electrospinning the hydrophobic core and gelatin sheath with a ratio of 1:5 at fixed concentrations. Results from attenuated total reflection-fourier transformed infrared spectroscopy demonstrated the gelatin coating around nanofibers in all the materials. Transmission electron microscopy images further displayed the core-sheath structures showing the coreto-sheath thickness ratio varied greatly with the highest ratio found in polyurethane-gelatin nanofibers .Scanning electron microscopy images revealed similar, uniform fibrous structures in all the materials, which changed with genipin crosslinking due to inter-fiber interactions. Thermal analyses revealed varied interactions between the hydrophilic sheath and hydrophobic core among the three materials, which likely caused different core-sheath structures, and thus physico-mechanical properties. The addition of gelatin around the hydrophobic polymer and their interactions led to the formation of graft scaffolds with tissuelike viscoelasticity, high compliance, excellent swelling capability, and absence of water permeability while maintaining competent tensile modulus, burst pressure and suture retention. The hydrogel-like characteristics are advantageous for vascular grafting use, because of the capability of bypassing preclotting prior to implantation, retaining vascular fluid volume, and facilitating molecular transport across the graft wall as shown by co-culturing vascular cells sandwiched over a thick-wall scaffold. Varied core-sheath interactions within scaffolding nanofibers led to differences in graft functional properties such as water swelling ratio, compliance, and supporting growth of co-cultured vascular cells. The PCL-gelatin scaffold with thick gelatin-sheathed nanofibers demonstrated more compliant structure, elastic mechanics and high water swelling property. Our results demonstrate a feasible approach to produce new hybrid, biodegradable nanofibrous scaffold biomaterials with interactive core-sheath structure, good biocompatibility and tissue-like viscoelasticity, which may reduce potential problems with the use of individual polymers for vascular grafts.
Keywords: Coaxial electrospinning, gelatin, nanofibers, vascular tissue, engineered scaffolds
ACS Paragon Plus Environment
Page 2 of 32
Page 3 of 32
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
Langmuir
1. Introduction Vascular disease is one of the leading causes of morbidity and mortality in developed countries, and thereby necessitates the replacement of diseased artery or vein through surgical intervention [1, 2]. Tissue engineering offers an alternative approach of designing biomaterials for small diameter artery regeneration [1]. A typical tissue-engineering scaffolds for artery grafting must enable the adhesion and proliferation of endothelial cells and smooth muscle cells leading to the deposition of extracellular matrix (ECM) proteins and withstand physiological hemodynamic pressures [3]. Of the various methods used for mimicking the structure and function of the tissue ECM, electrospinning has proven to be a facile method for generating micro- and nano- fibers from a variety of materials that include biodegradable polymers. In electrospinning, polymer fibers are generated when an electrified jet of polymer solutions is continuously stretched due to the electrostatic repulsions between the surface charges and the evaporation of solvent [4]. The high ratio of the surface area to volume or to mass of these fibers is highly advantageous in biomedical applications such as tissue engineering and drug delivery [5-8]. The use of a mixture of hydrophobic and hydrophilic polymers or a mixture of natural and synthetic polymers has been attempted in the past for synthesis of tissue-engineering scaffolds [9, 10]. However, using conventional methods such as blending fibers or layering materials, the adhesion and interaction between the blended systems were often too low to obtain a mechanically stable scaffold while providing desired pore size and porosity. Coaxial electrospinning provides a unique approach to construct structured polymer blend, wherein, the polymers are highly interactive at the nanoscale to bring about novel properties. Coaxially electrospun fibers in comparison with coated or blended fibers have shown enhanced biocompatible and mechanical properties for tissue engineering and regenerative applications [11, 12]. Few attempts have been made using coaxial electrospinning to develop small diameter artery grafts [13-17]. In coaxial electrospinning, two compartments containing either different polymer solutions or a mixture of a polymer solution for the formation of shell and a non-polymeric Newtonian liquid or even a powder for the formation of core, are used to initiate a core–shell jet. With the core–shell jet solidifying, core–shell fibers are deposited on a grounded electrode [5]. In most fabrication conditions,
ACS Paragon Plus Environment
Langmuir
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
the shell fluid is able to be processed with electrospinning, while the core fluid is not electrospinnable [18]. Biocompatible and biodegradable hydrophobic polymers like polycaprolactone (PCL), polyurethane (PU) and polylactic acid (PLA) have been used in myriad tissue engineering applications including vascular regeneration [19, 20, 21]. In spite of the high mechanical stability of these polymers, they lack the innate reactive sites for cell adhesion. The hydrophobic nature of PCL, PU and PLA also tends to attract platelet and plasma protein adhesion, leading to the aggregation and intimal hyperplasia of the artificial blood vessels [22]. Collagen is one of the most predominant ECM proteins found in the vasculature. However, electrospinning with the use of strong organic solvents always causes the denaturation of collagen into gelatin [23]. Gelatin possesses competent biodegradable properties along with excellent biocompatibility and non-antigenicity comparable to collagen [24, 25]. The combination of hydrophobic polymer and gelatin may provide appropriate graft strength, compliance and viscoelasticity. This study attempts to coaxially electrospin 3 different types of vascular grafts composed of non-antigenic hydrophilic gelatin sheathing with the hydrophobic core made of PU, PCL or PLA, which forms fibrous scaffolds for potential small diameter (< 6mm in diameter) vascular grafts. The hydrophobic core provides the mechanical strength and stability of the scaffold, while the gelatin sheath ensures its enhanced biocompatibility and mimetic viscoelasticity. The physical, mechanical, and biological performances of the hybrid nanofibrous scaffolds are evaluated here.
ACS Paragon Plus Environment
Page 4 of 32
Page 5 of 32
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
Langmuir
2. Experimental: Materials and Methods 2.1 Materials All polymers and chemicals were purchased from Sigma Aldrich Inc. (St Louis, MO) unless specified otherwise.
2.2 Electrospinning of polymer fibers The apparatus used for obtaining coaxial fibers was developed in house. Core hydrophobic polymer solution was passed through the inner needle of 22 G (0.71 mm in internal diameter) and the sheath gelatin solution was passed through the outer needle of 16 G (1.65 mm in internal diameter). A dual syringe holder was used to place the syringes loaded with polymer solutions. This design allows the solutions to be extruded simultaneously. Polymer solutions of 1wt% concentration of PU, PCL, or PLA and 5 wt% gelatin were prepared by dissolving a predetermined amount of PU, PLA or PCL and gelatin in 1,1,1,3,3,3 hexafluoro-2-propanaol (HFP). The solutions obtained after stirring for 6 to 8 hours were loaded in 5 mL syringes connected to the positive terminal of a high voltage ES30P 10 W power supply (Gamma High Voltage Research, Ormond Beach, FL). The polymer solutions were extruded at 1 mL/h using syringe pumps (Pump 11 Plus, Harvard Apparatus, Boston, MA) and was subjected to an electric potential of 1kV/cm. The fibers were deposited onto a grounded static aluminum substrate or cylindrical aluminum rod of 5mm in diameter rotating at 150rpm and placed at a distance of 10 cm perpendicular to the needle. The aluminum rod was coupled to a BMU230AP-A-3 brushless DC motor (Oriental Motor Corp, USA). The obtained samples were stored at room temperature until further use.
2.3 Crosslinking of electrospun fibers The scaffolds composed of coaxially electrospun fibers were subjected to crosslinking for 48 hr by immersion in solution containing 0.25 % (w/v) genipin dissolved in 100% ethanol. Immediately after the crosslinking process, fibrous scaffolds were rinsed with phosphate buffered saline (pH 7.4) and used as such or air dried overnight.
ACS Paragon Plus Environment
Langmuir
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
2.4 Material Characterizations 2.4.1 Scanning Electron Microscopy The aluminum substrates coated with the electrospun fibers were mounted on brass stubs and observed under a scanning electron microscope (JEOL JSM 6480 LV, USA) operating at an accelerating voltage of 5–20 kV. The diameters of about 50 different fibers were measured in each of the cases using the UTHSCA Image tool to obtain their average diameter.
2.4.2 Transmission electron spectroscopy The nanostructure of the coaxial fibers was observed using H7650 transmission electron microscope (Hitachi Ltd, Tokyo, Japan) operated at 80 kV. The electrospun nanofiber samples for TEM observation were prepared by directly depositing the as-spun fibers on carbon-coated TEM grids.
2.4.3 ATR-FTIR Spectroscopy Attenuated total reflection - fourier transform infrared spectroscopy (ATR-FTIR) measurements were carried out on peeled fibrous membranes using Nicolet 6700 FTIR spectrometer (Thermo Fisher Scientific, USA) equipped with a diamond ATR crystal. Typically, 30 scans were signal-averaged to reduce spectral noise. The spectrum of the samples was recorded from 600 to 4000 cm-1.
2.4.4 Thermal Analysis – Thermogravimetric analysis, differential thermal analysis and differential scanning calorimetry analysis Thermogravimetric analysis (TGA) of the fibers was performed using universal Netzsch 204 F1 (Phoenix, AZ). About 5 mg of the samples were heated at 10ºC min–1 in a temperature range of 0 – 600ºC using platinum crucibles. Differential thermal curves were obtained from the TGA curves by plotting a graph between derivative weight % and temperature.
ACS Paragon Plus Environment
Page 6 of 32
Page 7 of 32
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
Langmuir
Differential scanning calorimetry (DSC) analysis of the fibers was performed from 0ºC to 300 ºC at 20ºC min-1 using Netzsch 204 F1 (Phoenix, AZ). The instrument was calibrated using an indium standard and the calorimeter cell was flushed with liquid nitrogen at 20 mL min-1.
2.4.5 Tensile strength measurement Samples of electrospun nanofibrous membranes with dimensions of 100mm x 5 mm were tested for their tensile strength. Load-elongation measurement was carried out at a crosshead speed of 5 mm/min, 25⁰C temperature and relative humidity of 65%. Percentage of elongation at break (%) was measured using a universal testing machine (INSTRON model 1405, Shakopee, MN), at an extension rate of 5 mm/min.
2.4.6 Rheometry testing The viscoelastic properties of coaxial scaffolds were investigated under shear deformation using the ARES or ARG2 rheometer (TA Instrument, New Castle, DE). The ARES is designed to measure the expended torque. Samples were analyzed on ARES using the plate geometry (diameter = 8-10 mm; gap set between 0.25-0.5mm). Strain sweep measurements spanning from 0.1 % to 50 % strain were conducted at 10 points each decade of strain (e.g. 0.1% - 1%). Frequency sweeps were conducted at a stress strain range of constant oscillation frequency. Oscillatory frequency sweep tests were conducted with logarithmic step increases between 0.1 and 50 Hz to identify the linear region for all scaffolds. The storage modulus (G′) and loss modulus (G″) of scaffolds as well as tan δ (tan δ = G′/ G") were determined in the linear viscoelastic region (LVR). Using G′ and G" the complex storage modulus (G*) can be calculated using: G* = G′ + iG". Shear stress was determined in response to constant strain application, reflecting the difference in material viscosity. In order to avoid sample slipping during rheometry measurement, the glass slides were treated for sample attachment. Briefly, slides are immersed in 20% sulfuric acid for ~30 minutes, and then washed in deionized water three times changing the water at 5minute intervals. Subsequently, the slides were immersed in acetone for 30 minutes and then in
ACS Paragon Plus Environment
Langmuir
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
aminopropyltriethoxysilane overnight. Then, the slides were washed in acetone three times, followed by three washes in deionized water. Lastly, the slides were immersed in 2.5% glutaraldehyde for 1 hour followed by two washes in deionized water, before they were used to collect nanofibers for rheometry measurements.
2.4.7. Swelling and water retention capacity of coaxially electrospun fibers The electrospun samples were placed in de-ionized water for a period of 72 hr. The samples were then removed, centrifuged for 15 min at 1500 rpm (Eppendorf Centrifuge 5415D, Hauppauge, NY), and weighed. Water retention capacity was determined with the increase in the weight of the fibers through the following equation.
Water retention (%) = (w- wd) X 100% wd where w is the weight of each specimen after submersion for a certain period of time and wd is the weight of the specimen in its dry state before submersion.
2.5. Vascular graft functional characterizations The water permeability, burst strength, compliance, and suture retention strength of vascular grafts were determined according to the methods prescribed in the industrial standard—ANSI/AAMI/ISO 7198:1998/2001/(R) 2004 (Cardiovascular Implants: Tubular Vascular Prostheses). A custom-made apparatus designed for determination of water permeability, burst pressure and compliance was used.
2.5.1. Water permeability Tube adapters were inserted onto both ends of the multilayer graft. Paraffin films were used to secure the graft on the adapters. Then, the graft was cannulated by connecting one end of the graft to a water reservoir with a flow valve and the other end was connected to the other end of the water reservoir. Water pressurized at 16 kPa (120 mmHg), was imposed using a pulsatile flow pump. Once the flow became
ACS Paragon Plus Environment
Page 8 of 32
Page 9 of 32
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
Langmuir
steady, the permeated fluid through the graft due to the pressure was collected in a beaker and the water volume collected within 1 min was measured. The graft permeability (units: mL/cm2/min-1) was determined using the equation: S=Q/A where S is the graft permeability, Q is the fluid volume passing through the graft, and A is the crosssectional area of the aperture in the sample holder.
2.5.2. Burst strength Tube adapters were inserted on both ends of the vascular grafts. Paraffin films were used to secure the graft onto the latex tube near the adapters. The graft was then loaded onto a custom-made fixture and connected to a flow network via pressure gauges on both ends of the graft. Water was pressurized through the graft at increments of 10mmHg. The increasing pressure led to an expansion of the graft diameter and surface area. The expansion was allowed until graft failure occurred. The pressure at which this failure of the vascular graft occurred was recorded as the burst strength or burst pressure of the graft.
2.5.3. Compliance Compliance was measured by similar method adopted for burst pressure. Tubular graft was cannulated with a latex tube inserted in a way similar to the burst strength test. The graft was then loaded onto a custom-made fixture and connected to a flow network via pressure gauges on both ends of the graft. The pressure in the graft was controlled using varying speed of a water pump, to obtain a steady pressure from 10 mmHg to 200 mmHg, with an increment of 10 mmHg. At each pressure, images of the graft dilatation were taken using a camera (Canon EOS 450D, Japan). These images were analyzed using a customized script in MatlabVR (MathWorks, USA) to obtain the graft diameter at each pressure point. Compliance was measured using the equation C= [(R2-R1)/R1]/ (P2-P1) * 104
ACS Paragon Plus Environment
Langmuir
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 10 of 32
Where C is % of compliance, R1 is the original graft diameter, R2 is the changed graft diameter, P1 is the inlet pressure and P2 is the outlet pressure.
2.5.4. Suture retention Suture retention was measured by cutting the 3D vascular graft into rectangular strips with dimensions of 10 mm X 5 mm. A suture was inserted 2 mm from the end of the stretched strip of the graft through the graft wall to form a half loop and the other end of the graft was attached to the lower clamp of the tensile testing system. The other end of the suture was attached to a 2 kN load cell. The suture was pulled at the rate of 10 mm/min. The force required to rupture the graft wall was recorded as suture retention.
2.6 Biocompatibility of the electrospun fibers In vitro biocompatibility by MTT assay Human pulmonary artery endothelial cells (hECs) from Lonza Inc (Basel, Switzerland) were cultured on the luminal (top) surface of coaxial electrospun fibrous mat at a seeding density of 1X 105 cells/cm2 at 37 °C with 5% CO2, 95% air and complete humidity for 72 hr. Human pulmonary artery smooth muscle cells (hSMCs) were then seeded on the back (bottom) side of the scaffolds with hECs at a seeding density of 5X104 cells/cm2 for 72 hr. The cell culture medium was composed of DMEM with 10% FBS, 4mL of 1mM VEGF and supplemented with an antibiotic cocktail of penicillin (120 units per mL), streptomycin (75mg/mL), gentamycin (160mg/mL), and amphotericin B (3mg/mL). After 6 days, the culture medium was replaced with a serum-free medium containing thioazolyl blue (MTT) and incubated for 4 hr at 37ºC in a humidified atmosphere of 5% CO2. The medium was aspirated and the formazan needles were dissolved in 500 µL of dimethyl sulfoxide. The absorbances of the solutions were read at 570 nm and the results were expressed as means of three experiments. Data were statistically analyzed using ANOVA with a sample size of at least 3 (n≥3) for each set with p
