Crosslinking methacrylated porcine pericardium by radical

Subscriber access provided by UNIV OF TEXAS DALLAS

Bio-interactions and Biocompatibility

Crosslinking methacrylated porcine pericardium by radical polymerization confers bioprosthetic heart valves enhanced extracellular matrix stability, reduced calcification, and mitigated immune response Linhe Jin, Gaoyang Guo, Wanyu Jin, Yang Lei, and Yunbing Wang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00091 • Publication Date (Web): 21 Feb 2019 Downloaded from http://pubs.acs.org on February 26, 2019

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 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 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.

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 36 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 Biomaterials Science & Engineering

Crosslinking methacrylated porcine pericardium by radical polymerization confers bioprosthetic heart valves enhanced extracellular matrix stability, reduced calcification, and mitigated immune response Linhe Jin‡, Gaoyang Guo‡, Wanyu Jin, Yang Lei*, Yunbing Wang* National Engineering Research Center for Biomaterials, Sichuan University, No.24 South Section 1 Yihuan Road, Chengdu 610064, P.R.China ‡ Linhe Jin and Gaoyang Guo contributed equally to this work.

*Correspondence

authors at: National Engineering Research Center for Biomaterials, Sichuan

University, No.24 South Section 1 Yihuan Road, Chengdu 610064, P.R.China. Tel: +86 28 85415280;

Fax:

+86

28

85410246;

E-mail:

[email protected]

(Dr.

Yang

Lei);

[email protected] (Prof. Yunbing Wang, Director of National Engineering Research Center for Biomaterials)

KEYWORDS: bioprosthetic heart valves, radical polymerization, elastin stabilization, immune response, anti-calcification

ACS Paragon Plus Environment

1

ACS Biomaterials Science & Engineering 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 2 of 36

ABSTRACT: The aging population and the development of transcatheter aortic valve replacement (TAVR) technology largely expand the usage of bioprosthetic heart valves (BHVs) in patients. Almost all of the commercial BHVs are treated with glutaraldehyde (GA). However, the GA treated BHVs display the drawbacks such as extracellular matrix (ECM) degradation, cytotoxicity, immune response, and calcification. In this study, radical polymerization reaction, a powerful tool commonly used in preparing polymers and hydrogels, has been developed to fix decellularized ECM instead of GA treatment. Porcine pericardium (PP) is taken as an example of ECM for BHVs fabrication to investigate the impact of radical polymerization on the tissue properties. The radical polymerization method better stabilizes collagen and elastin of PP than GA treatment and produces a soft biomaterial more like the native heart valve. Furthermore, radical polymerization crosslinked PP exhibits excellent cytocompatibility. After implanted subcutaneously in rats for 30 days, radical polymerization crosslinked PP shows better elastin stability, mitigated immune response, and reduced calcification than GA-PP. All these results suggest that radical polymerization is an ideal crosslinking method for BHVs or tissue engineering heart valve scaffolds and it also has the potential for creating a variety of ECM-polymer hybrid biomaterials in the future.


2

Page 3 of 36 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


Introduction

Valve replacement is the optimal choice to treat severe valvular heart diseases such as valvular stenosis, regurgitation, and congenital defects.1-2 The currently available heart valves prostheses to replace the native one are mechanical heart valves (MHVs) and bioprosthetic heart valves (BHVs). BHVs have the advantages in hydrodynamic performance without the need of taking long-term anti-coagulation drug and are increasingly used in patients. The aging population is expected to further expand the usage of BHVs. Since the 2000s, transcatheter aortic valve replacement (TAVR), as an implantation method without the need of thoracotomy, has become a preferred alternative to surgical aortic valve replacement.3-4 This new type of BHVs decreases the risk of morbidity of reoperation through “valve in valve” procedure, which extends the use of BHVs to younger patients. Based on these facts, the implanted BHVs may increase from 300,0005 in 2015 to 850,000 in the year 2050.6 Currently, all commercially available BHVs are crosslinked by glutaraldehyde (GA). There are several shortcomings of GA-treated valves including inefficient extracellular matrix (ECM) stability, toxicity effect, inflammatory response, and accelerated calcification.7 The lack of protecting elastin in GA-treated valve leads to elasticity deficiency and elastin-degradation related calcification. Cytotoxicity reduces the adhesion and proliferation of host endothelial cells and further hurdle endothelialization.8 Smooth muscle cells tend to migrate and proliferate on the nonendothelialized BHVs’ surface. The smooth muscle cells induce the aggregation of platelets and leucocytes to cause the thrombus and inflammation, even some of them have the potential to osteogenesis.9 Besides, the development of ectopic calcification and less adaptability to the host tissue, which may be caused by the toxicity of GA, have been implicated in the use of GA as a crosslinking agent.10 The residual inherent immunogenicity in xenogeneic tissues and GA


3


Page 4 of 36

crosslinker are the main contributors to the inflammatory response causing ECM degradation and leaflet fibrosis.11 Calcification stiffens the BHVs and increases localized mechanical stresses, then the tear of leaflets may occur.8, 12 Many studies have reported some alternative fixatives of GA, however, their clinical use has not been reported. It is well known that radical polymerization is one powerful tool to prepare functional biomaterials such as polymers and hydrogels because of the diversified monomers, gentle reaction conditions, and simple process.13-16 However, few reports about their applications in crosslinking decellularized ECM are published. We have previously studied the methacrylic anhydride as the crosslinker to crosslink the porcine pericardium. Herein, we have tried another method of free radical polymerization crosslinking and it shows ideal biocompatibility. In this study, the porcine pericardium as a model of ECM for BHVs fabrication was modified by glycidyl methacrylate (GMA) firstly and then crosslinked by radical polymerization. The chemical structure, ECM stability, biomechanics, cytotoxicity, immune response, and calcification were further examined in vitro or in vivo.

Experimental Section

Materials The fresh PP was kindly provided by VENUS MEDTECH Inc (Hangzhou, China). Collagenase Ⅱ, Rosewell Park Memorial Institute 1640 (RPMI 1640) Medium, Fetal Bovine Serum (FBS), and phosphate buffered saline (PBS) were purchased from Invitrogen (NY, USA). Elastase and ninhydrin were purchased from Macklin Biochemical Co., Ltd (Shanghai, China).


4

Page 5 of 36 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


Glycidyl methacrylate (GMA) was purchased from Aladdin biotechnology Co., Ltd (Shanghai, China). Glutaraldehyde (GA) was purchased from Best-Reagent (Chengdu, China). Crosslinking of Porcine Pericardium Fresh PP was decellularized according to a protocol which was described previously.17 Briefly, fresh PP was incubated with 10 mM Tris buffer (pH = 8) containing 0.1% EDTA and 10 KIU mL-1 aprotinin for 1 h under vigorous stirring. The supernatant was discarded and the PP was incubated with 10 mM Tris buffer (pH = 8) containing 0.1% sodium dodecyl sulfate (SDS), 20 μg mL-1 RNase A, and 200 μg mL-1 DNase for another 48 h. After rinsed 3 times with distilled water for one hour, the decellularized PP (D-PP) was reacted to GMA with three different concentrations (1%, 3%, 5% v v-1) in PBS buffer under constant stirring for 7 days at 37 ℃ (Figure 1). The GMA modified PP was abbreviated to GMA-PP, and the prefix of “1%-”, “3%-”, and “5%-” represented their treated concentration of GMA solutions. The resulting GMA-PP was subsequently crosslinked by incubating with PBS buffer containing 5 mM ammonium persulfate (APS) and 5 mM sodium hydrogen sulfite (SHS) as radical polymerization initiator with shaking at 120 rpm at 37 ℃ for 24 hours (Figure 1). The polymerized GMA-PP (PGMA-PP) was rinsed with aqueous 50% alcohol twice and then with distilled water 3 times in an ultrasonic cleaner.


5


Page 6 of 36

Figure 1. Schematic illustration for presumable mechanism of crosslinking methacrylated PP. Methacrylated PP was synthesized through the reaction of reactive groups within PP to glycidyl methacrylate (GMA). Methacrylated PP was then crosslinked in the presence of redox-initiator ammonium persulfate/sodium hydrogen sulfite (APS/SHS) to initiate radical polymerization.


6

Page 7 of 36 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


Chemical Characterization Free Amine Content Measurement by Ninhydrin Assay The ninhydrin assay was commonly used to quantify free amine group present in the xenogeneic tissue.18 Briefly, ninhydrin was dissolved in citric acid aqueous solution (0.1 M, pH = 5) with a concentration of 1% (w v-1). D-PP, GA-PP, and GMA-PP (5*5 mm2, n=6 from each group) were immersed in 2 mL ninhydrin solution and incubated at 95℃ for 45 min. Then the solutions were cooled down to room temperature and the supernatant was aspirated from the tube. The absorbance at 567 nm of the supernatant was immediately measured using a UV-Vis spectrophotometer (UV-3200 spectrophotometry, MAPADA Instruments). The tissue samples were freeze-dried and weighed (W) for the normalization of free amine group content. The amine group content was calculated with the equation below.

Amine Content =

𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒𝑆𝑎𝑚𝑝𝑙𝑒/𝑊𝑆𝑎𝑚𝑝𝑙𝑒 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒𝐷 ― 𝑃𝑃/𝑊𝐷 ― 𝑃𝑃

× 100%

（1）

NMR Spectroscopy D-PP, GMA-PP, and PGMA-PP (1*1 cm2) were hydrolyzed in 35% deuterium chloride in 15 mL centrifuge tubes at 37 ℃ for 24 h. The digestive solution was centrifuged to remove the insoluble and the supernatant was collected. About 0.5 mL aliquot of the supernatant diluted by a factor of 5 with deuteroxide was analyzed by 1H NMR spectroscopy (Avance II-400 MHz, Bruker).


7


Page 8 of 36

Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy PPs (0.5*0.5 cm2) were flattened on a glass slide and covered with a coverslip. The glass slide and coverslip were immobilized by tape to ensure the PPs were flat during the whole procedure. Then, the PPs were freeze-dried and characterized by ATR-FTIR spectrometer (NEXUS 670, Nicolet). Enzymatic Degradation The collagen and elastin stability of the crosslinked PPs was measured by the weight loss percentage after collagenase Ⅱ and elastase treatment.5 PPs (about 1 * 1 cm2, n=6 from each group) were freeze-dried and weighed (W0). The dried pieces were incubated in Tris buffer (Tris 0.1 M, CaCl2 0.05 M, pH = 7.4) containing 125 U mL-1 collagenase Ⅱ or Tris buffer (Tris 0.1 M, CaCl2 0.001 M, pH = 7.8) containing 30 U mL-1 elastase for 24 h, respectively. The enzyme-treated PPs were rinsed with distilled water 3 times before freeze-dried and weighted (Wt). The fraction of weight-loss was calculated as:

Weight Loss Ratio =

𝑊0 ― 𝑊𝑡 𝑊0

× 100%

（2）

Uniaxial Tensile Test The mechanical property of PP was tested by a universal testing machine (Instron 5967). PPs were cut into rectangles (3*0.6 cm2, n=6 from each group) and the thickness was measured at three random locations by thickness gauge and the average value of thickness was taken for the stress


8

Page 9 of 36 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


calculation. The PPs were mounted into the fixture of the universal testing machine and preloaded with 0.1 N force. PPs were uniaxial stretched under a constant velocity of 12 mm min-1 until fractured. Cytotoxicity The cytotoxicity of tissue samples was assessed according to ISO 10993-5 standard. More specifically, the crosslinked PPs (0.3g) were washed with distilled water thoroughly and sterilized with 75% ethanol overnight before rinsed in sterile PBS buffer. The leach liquor was prepared by soaking the PPs into 1.5 mL complete cell culture medium (RPMI 1640 Medium containing 100 μg mL-1 streptomycin, 100 U mL-1 penicillin and supplemented with 10% FBS) at 37℃ for 3 days. Untreated RPMI 1640 Medium was set as a control. L929 fibroblasts were seeded in 96-well plate in triplicates 24 h prior to replacing the old medium with the extract liquid and cultured in humid 5% CO2 atmosphere at 37 ℃. After 2, 4, and 6 days culture, the medium was discarded and the cells in each well were incubated with 100 µL fresh culture medium supplemented with 10% Cell Counting Kit-8 (CCK-8) reagent for 90 min at 37℃. The absorbance at 450 nm was measured by a microplate reader. Cell viability was defined as the ratio between the absorbance of PPs and untreated RPMI 1640 Medium. Implantation and Explantation The conducted animal experiments have been approved by an institutional review from Medical Ethics Committee of Sichuan University. Pieces of PP with a size of 1*1 cm2 were implanted subcutaneously in 200 g-weighted male Sprague-Dawley (SD) rats for 3 days, 7 days, 14 days, and 30 days. The 2 pieces of PPs (1 piece of 5%-PGMA-PP and 1 piece of GA-PP) were implanted subcutaneously in each SD rat. There were 6 replicates of each type of PPs implanted


9


Page 10 of 36

in 6 different SD rats for each implanted time. Altogether 24 SD rats were utilized to study histology in order to provide statistical significance. Briefly, PPs were sterilized with 70% ethanol for 24 h, then thoroughly rinsed with sterile PBS buffer, stored in the same buffer until use. SD rats were anesthetized using pentobarbital sodium (at the dose of 1mL/kg) anesthesia and the dorsal hair of SD rats was shaved. Two longitudinal surgical incisions (no longer than 2 cm) were made on either side of the back of each rat using surgical scissors to create pockets for the subcutaneous space. One PP was placed in each pocket. After implantation, the incisions were closed using surgical sutures. After implantation at 3, 7, 14, and 30 days, rats were euthanized and PPs together with the newly formed surrounding tissues were excised. Explanted PPs at 3, 7, and 14 days were promptly fixed in formaldehyde fixative overnight and embedded in paraffin wax according to a standard process. The explanted PPs at 30 days were cut in the middle. Half of the PP was embedded in paraffin as above and the other half of the PP was used for quantitative calcification study. Histological Analysis For each PP, 6 μm sections were cut and stained for light microscopy analysis. Picrosirius red was used to stain collagen with red. Victoria blue stain was used to visualize elastin. Alizarin red stains were used to stain calcium deposition. For the immunohistochemical analysis, 6μm sections were cut following by deparaffinized and rehydrated. When the antigen was recovery, the primary antibodies were dropped on the section and incubated at 4 ℃ overnight. Mouse antirat CD68 antibody (Bioss) and rabbit antirat CD3 (Servicebio) antibody were utilized to label macrophage cells and T lymphocytes. Both of the antibodies were diluted 100 times. The bound antibodies were visualized by DAB kit (Servicebio). Images were captured by slide scanner (MIRAX Desk,


10

Page 11 of 36 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


Carl Zeiss Microimaging GmbH, Germany) and the immunopositive cells were Semi-determined using Image-Pro Plus software. Quantitative Calcium Analysis Inductively Coupled Plasma Optical Emission Spectrometer （ ICP-OES ） was utilized to study the calcium content of subcutaneously implanted PPs. Briefly, 6 pieces of explanted PPs for each group were utilized to study the quantitative calcium analysis. Explanted PPs were washed with PBS 3 times before digested with 1ml 6 M HCl solution at 96 ℃ for 24 h. Then, the hydrolysate was diluted 10 times and detected the calcium content with ICP-OES (PQ ExCell). Statistical Analysis All the results were expressed as the mean ± standard error of the mean. All statistical analysis was performed using single factor analysis of variance (ANOVA). Differences between the means were determined using least-significant difference with a significance level of α=0.05.

Results

Chemical characterization of PPs In order to verify the conjugation of GMA to D-PP, amine content of GMA-PP was measured assuming that amine group was one of the main reaction sites to the epoxy group. The residual amine content of 1%-GMA-PP, 3%-GMA-PP, and 5%-GMA-PP was 42.06%, 26.63%, and 13.28%, respectively (Figure 2A). The increasing GMA concentration resulted in decreasing


11


Page 12 of 36

content of amine groups, indicating the participation of amine group in reaction to the epoxy group. Meanwhile, the amount of consumed amine group represented the degree of substitution (DS) of the methacrylate group to some extent. The more the amine group was consumed, the more the methacrylate groups were introduced. This result showed that the DS of the methacrylate group could be controlled by the amount of added GMA. Besides the indirect evidence from amine conversion, the introduction of methacrylate group into PP and their polymerization were directly monitored by 1H-NMR technique. The emergence of characteristic peaks of the double bond (5.67 ppm and 6.09 ppm) assigned to methacrylic acid hydrolyzed from GMA-PP confirmed the introduction of the methacrylate groups (Figure 2B).19 After polymerization, the disappearance of the double bond signal in PGMA-PP demonstrated that the polymerization reaction of GMA-PP was complete.

Figure 2. The alteration of functional groups within PPs. (A) The content of amine groups (n = 6, mean values ± s.d.) in the metharcylated PPs. D-PP and GA-PP were used as references. 1%GMA-PP, 3%-GMA-PP, and 5%-GMA-PP refer to the methacrylated PPs obtained by incubating


12

Page 13 of 36 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


with 1%, 3%, 5% (v v-1) GMA in PBS, respectively. * p < 0.05. (B) Representative 1H NMR spectrum of the hydrolyzed production of D-PP (blue), GMA-PP (green), and 5%-PGMA-PP (red).

Componential Stability The weight-loss ratio of PPs after collagenase treatment was used to measure the protection of collagen by different stabilizing methods. 1%-PGMA-PP, 3%-PGMA-PP, 5%-PGMA-PP, and GA-PP showed the weight-loss percentages of 12.37%, 6.69%, 4.19%, and 5.72%, respectively (Figure 3A). All of them were significantly lower than D-PP with a weight loss of 92.87%. This result demonstrated that both radical polymerization and GA crosslinking could stabilize collagen. The collagen stability increased with the DS of methacrylate group in an order of 5%-PGMA-PP > 3%-PGMA-PP > 1%-PGMA-PP. These results indicated that the ability to protect the collagen of PGMA-PP correlated with the crosslinking density. The optimized option among the PGMAPPs with the lowest weight-loss ratio (4.19%) was 5%-PGMA-PP and 5%-PGMA-PP didn’t show a significant difference in the weight-loss ratio of collagen compared with GA-PP (5.21%). This result indicated that 5%-PGMA-PP exhibited ideal collagen preservation as well as GA-PP. When PGMA-PPs, GA-PP, and D-PP were challenged with elastase, all the PGMA-PPs lost lower mass (1%-PGMA-PP: 5.61%, 3%-PGMA-PP: 4.98%, and 5%-PGMA-PP: 2.60%) than GA-PP (11.74%) and D-PP (24.17%), indicating that PGMA-PPs could protect elastin more effectively than GA-PP and D-PP (Figure 3B). Similar to the pattern of collagenase degradation assays, 5%PGMA-PP with the highest crosslinking density showed a slightly less weight-loss ratio of elastin than 1%-PGMA-PP and 3%-PGMA-PP. According to the results of in vitro collagenase and


13


Page 14 of 36

elastase digestion assays, 5%-PGMA-PP exhibited the best ability to protect collagen and elastin and was selected for further study. In order to further verify the collagen and elastin stability of 5%-PGMA-PP in vivo, we implanted the PPs subcutaneously in SD rats for 30 days to simulate the degradation in vivo enzyme level. Histologic sections of 5%-PGMA-PP and GA-PP explants were stained by Picrosirius red to visualize collagen and Victoria blue to visualize elastin. According to the Picrosirius red sections (Figure 3C), both 5%-PGMA-PP and GA-PP showed protection of collagen. The collagenous fiber was intact and coherent without fracture. This result indicated that both PPs didn’t show significant degradation of collagen in 30 days subcutaneously implantation. 5%-PMGA-PP showed a dense distribution of fibrous elastin within the implanted PP (Figure 3D). In contrast, no visible elastin was observed within the implanted GA-PP (Figure 3D). This result indicated that radical polymerization crosslinking showed a better ability to protect elastin than GA crosslinking.


14

Page 15 of 36 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


Figure 3. The ability of PPs to resist enzymatic degradation. (A-B) Weight-loss ratio (n = 6, mean values ± s.d.) of 1%-PGMA-PP, 3%-PGMA-PP, 5%-PGMA-PP, GA-PP, and D-PP after incubating with collagenase (A) and elastase (B) for 24 h at 37℃. ** p < 0.01. (C) In vivo collagen stability of 5%-PGMA-PP and GA-PP. Picrosirius red staining on sections of 5%-PGMA-PP (top) and GA-PP (bottom) after 30 days implantation in male juvenile rats. The black bar indicates 50 µm. (D) In vivo elastin stability of 5%-PGMA-PP and GA-PP. Victoria blue staining on sections of 5%-PGMA-PP (top) and GA-PP (bottom) after 30 days implantation in male juvenile rats. Fibrous elastin was stained blue (red arrow). The black bar indicates 50 µm.


15


Page 16 of 36

Mechanical Property The secant modulus and extensibility

20

were calculated based on the stress-strain curve as

shown in Figure 4A. Briefly, secant modulus at 10% and 15% strain was expressed as the slope of secant at 10% and 15% strain. Extensibility was defined as the intercept of a linear fit to the high-modulus region against the strain. As shown in Figure 4B, the stress-strain curve of GA-PP was gentle within the initial 8% strain and had a precipitous rise after 10% strain. Differently, the stress-strain curve of 5%-PGMAPP exhibited a gentle trend before 25% strain and then showed a sharp rise. As described in the previous study, the initial lower slope in stress-strain curves corresponded to elastin as the major load-bearing component and the follow-up high slope region was allotted to collagen component in tissue.21 Elastin could preserve the original structure under cyclic loading, however, while the strain reached beyond the elastin-bearing window to the collagen-bearing window, the shape and geometry of leaflet would gradually change and more stress would be transferred to the collagen structure leading to its damage and tearing.20 Therefore, the wide elastin-bearing window of 5%PGMA-PP provided more opportunity for elastin to share stress, which preferably mitigated the cyclic loading on collagen. Additionally, the stress-strain curve of 5%-PGMA-PP was similar to that of D-PP indicating that radical polymerization did not significantly alter the mechanical behavior of native tissue. BHV as a switch of blood flowing has a lower strain and higher frequency movements during the opening and closing stages. A soft heart valve more like the native heart valve facilitates the immediate response to the blood flowing and reduces the burden of heart. The secant modulus at low strain such as 10% and 15% strain was usually used to evaluate the stiffness of BHVs (Figure


16

Page 17 of 36 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


4C).22-23 The secant modulus of GA-PP was significantly higher than 5%-PMGA-PP and D-PP at both 10% strain and 15% strain. The secant modulus at 10% and 15% strain was 1.01 MPa and 3.02 MPa for 5%-PGMA-PP, 4.70 MPa and 10.29 MPa for GA-PP, and 1.51 MPa and 2.46 MPa for D-PP. This result showed that 5%-PGMA-PP was softer than GA-PP and might show a lower pressure gradient and better property of hydromechanics. Extensibility was an integrated parameter of the elastin-bearing window and elasticity modulus.20 Routinely, the damage of elastin led to a reduction of extensibility. In BHVs, the initial mechanical response was due to the elastin.20 Therefore, higher extensibility might contribute to maintaining the mechanical properties of BHVs. Also, extensibility was wildly used to evaluate the ductility. The extensibility of 5%PGMA-PP (28.01%) was significantly higher than GA-PP (15.00%) and D-PP (14.96%), indicating that 5%-PGMA-PP was more ductile than GA-PP and D-PP (Figure 4D). All of these results showed that 5%-PGMA-PP, as soft as the native tissue, was softer than GA-PP and had better ductility than GA-PP and native tissue.


17


Page 18 of 36

Figure 4. Uniaxial tensile testing. (A) Definition of secant modulus and extensibility. (B) Representative strain-stress curve of 5%-PGMA-PP, GA-PP, and D-PP. Secant modulus (C) and extensibility (D) of 5%-PGMA-PP, GA-PP, and D-PP (n = 6, mean values ± s.d.). * p < 0.05; ** p

Crosslinking methacrylated porcine pericardium by radical

Recommend Documents