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Tissue Engineering and Regenerative Medicine

Non-glutaraldehyde fixation for off#the#shelf decellularized bovine pericardium in anti-calcification cardiac valve applications Jing Liu, Huimin Jing, Yibo Qin, Binhan Li, Zhiting Sun, Deling Kong, Xigang Leng, and Zhihong Wang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01311 • Publication Date (Web): 30 Jan 2019 Downloaded from http://pubs.acs.org on February 2, 2019

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ACS Biomaterials Science & Engineering

Non-glutaraldehyde fixation for off‐the‐shelf decellularized bovine pericardium in anti-calcification cardiac valve applications Jing Liu1, Huimin Jing1, Yibo Qin1, Binhan Li1, Zhiting Sun1, Deling Kong1,2, Xigang Leng1*, Zhihong Wang1*

1Tianjin

Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering, Chinese

Academy of Medical Sciences and Peking Union Medical College, Tianjin 300192, China 2Key

Laboratory of Bioactive Materials of Ministry of Education; State Key Laboratory of

Medicinal Chemical Biology, College of Life Science, Nankai University, Tianjin 300071, China

* Corresponding author Zhihong Wang, PhD E-mail: [email protected] Xigang Leng, MD E-mail: [email protected] Mailing address: Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Baidi Road 236, Nankai District, Tianjin 300192, China

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Abstract In valvular replacement surgery, especially in the construction of bioprosthetic valves with decellularized pericardial xenograft, glutaraldehyde (GA) is routinely utilized as the golden standard reagent to fix bovine or porcine pericardial tissues. However, the apparent defects of GA, including cytotoxicity and calcification, increase the probability of leaflet failure and motivate the exploration for alternatives. Thus, the aim of this study is to develop non-glutaraldehyde combined-crosslinking reagents composed of alginate-EDC/NHS (Alg) or oxidized alginate-EDC/NHS (Alg-CHO) as substitute for GA, which is confirmed to be less toxic and more biocompatible. Evaluations of the fixed acellular bovine pericardial tissues included mechanical performance, thermodynamics/enzymatic/in vivo stability tests, blood compatibility assay, cytocompatibility assay, in vitro anti-calcification, and in vivo anti-calcification assay by subcutaneous implantation in juvenile Wistar rats. The data revealed that the tissues fixed with the combined crosslinking reagents were superior to GA control and commercially available Sino product in terms of better in vitro hemocompatibility

&

cytocompatibility,

lower

calcification

levels,

better

thermodynamics stability and better regenerative capacity in subcutaneous implants, while the mechanical strength and in vivo stability were comparable. Considering all above performances, it indicated that both Alg and Alg-CHO are appropriate to replace GA as the crosslinkers for biological tissue, particularly as a non-glutaraldehyde fixation for off‐the‐shelf decellularized bovine pericardial tissue in the anti-calcification cardiac valve applications. Nevertheless, studies on the long-term durability and calcification-resistance capacity in large animal model are further needed.

Keywords: bioprosthetic heart valve; tissue crosslinking; extracellular matrix stability; anti-calcification; biocompatibility


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Introduction Valvular heart diseases, particularly progressive structural and functional deteriorations of bioprosthetic grafts, have become more popular and led to serious mortality and morbidity in recent years.1 The total heart valve replacement cases are predicted to increase from 290,000 in 2003 to 850,000 by 2050.2 The most frequent clinical symptoms reflected in valve leaflet are calcification and chronic inflammation, which will further result in valvular tissue degradation and deterioration.3 At present, the golden standard therapeutic strategy for valvular heart disease is valvular replacement.4, 5 These valve replacement operations usually adopt mechanical prosthetic valves or bioprosthetic valves such as decellularized pericardial xenograft,1, 6 decellularized aortic valve xenograft,7-8 and cryopreserved homografts.9 These decellularized tissue engineering products have shown the potential capacity to become the commercial “off-the-shelf” products, which benefit the patients with longer storage life and operation window.10 However, bioprosthetic valves usually end with ultima valve failure attributed to mechanical failure or leaflet mineralization,11 only a few prosthetic heart valves are commercially available with limited durability of 15 - 20 years. Thus, the tendency of valvular material study mainly focuses on the prevention of leaflet calcification.12 These studies concentrate on optimizing material composition, host-related factors, dynamic mechanics environment and chemical processing.13 Meanwhile, the anti-calcification strategies include employing material modification and local drug delivery14 to mimic the native tissue15 and reduce



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antigenicity. While the most widely-used and effective strategy to increase chemical stability and mechanical strength is chemical crosslinking,16 which can form solid covalent bonds among amino, hydroxy or carboxyl groups within various polypeptide chains in the extracellular matrix (ECM).16 These crosslinkers largely include glutaraldehyde

(GA),17

epoxy

1-ethyl-3-(3-dimethylaminopropyl)

compounds,18

tannic

carbodiimide

acid,19

genipin,20

hydrochloride

(EDC)/N-hydroxysuccinimide (NHS),21 and polyphenols.7 The crosslinked ECM exhibited remarkably enhanced toughness, mechanical strength, and anti-degradation capacity after treating with GA.22 However, the application of GA crosslinking is limited as it is frequently reported to induce serious toxicity and calcification,23 which are caused by residual free aldehyde groups or phospholipids.13 Besides, the effect of the targeted in vitro detoxification strategies by amino acids and citric acid is also limited.24 While EDC-fixed ECM mainly involves the activation of carboxyl group in the peptide chain to be nucleophilic attacked by free amino group and formation of amido bond.20 However, it was reported that the EDC-crosslinking is not that stable as the GA-crosslinking, because EDC cannot bond distant collagen molecules. Nevertheless, a recent study reported that utilizing carbodiimide, neomycin trisulfate, and pentagalloyl glucose as combined crosslinkers can get valve leaflets with decreased calcification in addition to better stabilization and biocompatibility.6 Alginate (Alg) has been widely applied in biomedical field because of its non-thrombogenicity, mild immune response and superb biocompatibility.25 It derives from natural polysaccharide biosynthesized by certain bacteria or brown seaweed,25


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and constitutes of acidic components including 1, 4-linked β-d-mannuronic acid (M) and α-l-guluronic acid (G).26 Abundant carboxyl groups in alginate give it great potential to be used as component in combined crosslinkers together with EDC/NHS to bond free amino groups in ECM substrates; while both the aldehyde groups and carboxyl groups in partially oxidized alginate (Alg-CHO) can react with free amino groups with the assistance of EDC/NHS to form solid chemical crosslinking. To our knowledge, there is no reported study so far. Thus, in this study, we combined carboxyl group and aldehyde group in Alg and Alg-CHO as well as EDC/NHS to constitute the combined crosslinkers to react with free amino groups in acellular bovine pericardium (ABP) derived ECM. Further, we characterize the decellularization process and the surface properties, as well as mechanical performance, thermodynamics/enzymatic stability and in vivo degradation after treating with the combined crosslinkers. In addition, blood/cellular compatibility were routinely tested before the in vitro anti-calcification assay and the in vivo anti-calcification assay with subcutaneous implantation in juvenile Wistar rats, corresponding histological analysis and tissue calcium levels were analyzed to compare among different groups. Materials and Methods Decellularization method and ECM evaluation. Fresh bovine pericardia obtained from Fuhua meat co. LTD (Hebei, China) were cleaned, rinsed in sterile saline, cut into strips, and decellularized. Briefly, the pericardia were incubated on an orbital shaker with 1% (w/v) sodium dodecyl sulfate (SDS, Thermo Fisher) at room



temperature for 6 h, which was followed by 1% (v/v) Triton X-100 (Sigma) incubation for 30 min. After thoroughly washed with phosphate buffered solution (PBS, Thermo Fisher) for 7 days at 4 ℃, the samples were then treated with 2 U·mL-1 deoxyribonuclease (DNase grade Ⅱ, Sigma‐Aldrich) and 20 mg/mL ribonuclease (RNase, Sigma‐Aldrich) overnight at 37 ℃ with gentle shaking. Acellular pericardia were sterilely stored in 4 ℃ PBS until use. Histological analyses were performed with fresh and acellular bovine pericardial tissue. Samples were snap-frozen in liquid nitrogen and cut into 6 μm thick sections after embedding in optimal cutting temperature compound (OCT, SAKURA, USA). Standard histological staining, including hematoxylin & eosin (H&E), Masson's trichrome, Sirius red and Safranin O (Zhongshan Golden Bridge Biotechnology, China) were carried out to analyze the distribution of collagen, glycosaminoglycan (GAG) and elastin. Images were observed under a light microscope (Leica DM3000, German). Besides, DNA contents in both fresh and acellular samples were quantified using DNeasy Blood & Tissue Kit (Qiagen, Milan, Italy) following the manufacturer’s instructions. Quantifications of collagen and GAG were carried out according to the protocol of Insoluble Collagen Assay (S1000, Biocolor, UK) and Glycosaminoglycan Assay Blyscan™ (B1000, Biocolor, UK), respectively. Fresh and acellular samples were sputter-coated with gold and then imaged by scanning electron micrograph (SEM, Hitachi s-4800). Crosslinking of acellular bovine pericardium (ABP). Sodium alginate (viscosity of 1% (w/v) solution at 20 ℃ was 160 mPa·s) was purchased from Qingdao


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Jingyan co. LTD (China), and 30% oxidized sodium alginate was self-prepared by adding 0.65 g sodium periodate into 200 mL PBS containing 4.00 g sodium alginate before reacting for 24 h in dark. Extra sodium periodate was removed by equimolar ethylene glycol and 60 h dialysis (molecular weight cut off is 3000), oxidized sodium alginate was freeze-dried and kept in dryer until use. Crosslinking solutions were prepared by dissolving 0.4 g sodium alginate or oxidized sodium alginate together with 0.4 g EDC and 0.8 g NHS in 40 mL PBS. Pericardia were fixed with the crosslinking solutions for 48 h at 37 ℃ with gentle shaking before being washed with PBS for extra 48 h and stored at 4 ℃. The sodium alginate or oxidized sodium alginate crosslinked samples were respectively named as “Alg” and “Alg-CHO” for short. Similarly, GA-crosslinked sample was prepared by treating pericardia with 0.625% (v/v) GA. Both the GA-crosslinked sample (named as “GA”) and commercially available valve leaflet product (named as “Sino product”) from Sino Medical Sciences Technology Inc. (Tianjin, China) were used as control groups. Crosslinked samples were sputter-coated with gold and then imaged by SEM. To detect functional group on the surfaces of crosslinked samples, attenuated total reflectance-Fourier transform infrared spectrometry (ATR-FTIR, NICOLET 750, America) was scanned at 1.0 cm-1 resolution in 675 - 4,000 cm-1 frequency range. Mechanical performance and thermodynamics/enzymatic/in vivo stability. The mechanical performance of both crosslinked and uncrosslinked samples (n = 6) was assessed by a tensile-testing machine with a load capacity of 1 kN at the rate of



10 mm/min (Instron). Tensile strength and elongation at break were directly read from the stress-strain curve, while elastic modulus was calculated from the slope of the second linear region of the curve. To test the thermodynamic stability of the crosslinked species, differential scanning calorimetry (DSC) was performed based on calorimetric heat flow (W/g) against temperature (℃). Briefly, 5 - 10 mg pericardia were cut and sealed hermetically in a DSC pan, and was heated at an increasing heating rate of 10 ℃/min from 0 ℃ to 140 ℃ in a N2 gas environment with Thermal Analysis Instrument (DSC 800, Perkin Elmer). The denaturation temperature (DT) defined at the endo-thermic peak indicated the crosslinking degree of the collagen. For the enzymatic stability study, crosslinked and uncrosslinked samples underwent collagenase hydrolysis as follows: samples were lyophilized and dry weights were recorded, about 10 mg dry bovine pericardia (n = 18) was incubated in 1 mL collagenase solution (5 units/mL, in Tris with 1 mM CaCl2 and 0.02% (w/v) NaN3, pH 7.8), after 3, 6 and 12 days (n = 6 for each time point), samples were thoroughly rinsed with deionized water before finally lyophilized and weighed. Weight loss ratio (%) was calculated as follows: (weight before hydrolysis - weight after hydrolysis)/weight before hydrolysis × 100%. The in vivo experiment was authorized by the Center of Tianjin Animal Experiment Ethics Committee and Authority for Animal Protection (Approval No. SYXK (Jin) 2011-0008). The in vivo degradation assay was conducted by subcutaneous implantation with adult Sprague Dawley (SD) rat (200 g, n = 12). After


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the anesthesia with 10% (w/v) chloral hydrate (300 mg/kg), rats were fixed and shaved. After scrubbing with iodine, two 1.0 cm dorsal midline incisions were made over the back dorsum. Sterile pericardia (1.0 × 1.0 mm, n = 12) were wiped, weighed and inserted between skin and fascia at both sides for each incision. Finally, the incisions were sutured with polypropylene 3-0 and incubated for 3, 6, 12 days (n = 4 for each time point) before taken out, decellularized and weighted. Weight loss ratio (%) was calculated as follows: (weight before implantation - weight after implantation)/weight before implantation × 100%. Blood compatibility. Hemolytic test was primarily conducted to evaluate the blood compatibility of both crosslinked and uncrosslinked species. Initially, the sodium citrate anticoagulated whole blood from New Zealand white rabbit was diluted with normal saline (4:5, v/v). The Alg, Alg-CHO and GA crosslinked bovine pericardium as well as Sino product were cut into 1 × 1 cm slice and pre-incubated with 10 mL normal saline for 30 min at 37 ℃. After adding 200 μL diluted blood into each incubation media, the mixtures were further incubated for another 60 min and centrifuged at 1000 G for 5 min. 100 μL supernatants were read at 540 nm by spectrophotometer (Model 680, BioRad) and recorded as ODs. 100 μL normal saline without any sample was used as negative control (ODn), while D.I. water as positive control (ODp). The hemolysis ratio was calculated with the following equation (n = 5): Hemolysis ratio (%) = (ODs - ODn)/(ODp - ODn) × 100%. Furthermore, platelet adhesion/activation assay was conducted to evaluate the platelets behaviors including adhesion and activation after incubating on the surfaces



of each pericardium sample. In brief, 400 μL platelet-rich plasma (PRP) was evenly deposited onto sample surfaces and kept in 37 °C incubator for 60 min which was followed by fixation with 2.5% (v/v) GA solution and dehydration with 70% - 100% (v/v) gradient alcohol. Finally, samples were dried by hexamethyldisilazane (HMDS) and imaged by SEM. Besides, the adhered platelet number per 5000× magnification field was calculated randomly (n = 5). In vitro cytocompatibility. Primary human umbilical vein endothelial cells (HUVEC) were utilized for in vitro cytocompatibility (between passages 3 - 8). Cells were cultured with Endothelial Cell Medium Kit (ECM, Sciencell) containing 1% (v/v) penicillin/streptomycin antibiotics (P/S, HyClone) at 37 ℃ with 5% CO2 and 95% relative humidity. Cell viability assay was conducted via the indirect assay according to ISO10993-12: 2012 to assess the cytotoxicity of Alg, Alg-CHO, GA crosslinked bovine pericardium and Sino product. 1 × 1 cm samples (n = 18) were sterilized with UV radiation (254 nm) overnight before being immersed in 1.6 mL ECM media for each sample. HUVEC were harvested at confluence and reseeded in 96-well plates at a concentration of 3,000 cells per well. 24 h later, the media was replaced by 100 μL extract supernatant and incubated for another 1, 3, and 5 days. Upon each time point, 10 μL cell counting kit-8 (CCK-8, Neuronbc) was added into each well before incubating for 4 h. ECM media with serum worked as blank control. Optical density (OD) was read by spectrophotometer (Model 680, BioRad) at 450 nm, with the reference wavelength at 630 nm. Data were recorded as ODs and ODb for tested


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samples and blank control, respectively. Cell viability was calculated with the following equation (n = 6): Cell viability (%) = ODs/ODb × 100%. For the direct assay, HUVECs were digested and replanted on the surface of sterile samples at the density of 30,000 cells per well and further stained with Calcein-AM/PI Kit (CA1630, Solarbio) to identify the live and dead cells after 24 h incubation with samples. In addition, cellular adhesion behaviors were observed by actin and nucleus staining. 30,000 cells were similarly implanted on surface of samples and cultured for 24 h and 72 h in 24-well plates. Cells were orderly fixed with 4% (w/v) paraformaldehyde and permeabilized with 0.1% (v/v) Triton X-100 (Sigma). Cellular actin and nuclei were stained with 1.0% (v/v) FITC-phalloidin (Sigma) and 1 µg/mL DAPI (CST) respectively. Adhered HUVECs for both live/dead staining and actin/nuclei staining were imaged by confocal microscopy (A1R-si, Nikon, Japan), cell numbers were counted randomly (n = 5) from 10× magnification field. Moreover, HUVECs incubated on sample surfaces for 14 days were further stained with rabbit anti-vWF antibody (ab6994, Abcam) to identify endothelium layer formation. Briefly, samples were firstly embedded into OCT, cut into 6 μm thick sections and then fixed by cold acetone. The sections were washed with PBS, followed by blocking with 20% (v/v) goat serum (Thermo Fisher) for 30 min at room temperature. The sections were then treated with rabbit anti-vWF antibody (dilution 1:100, Invitrogen) overnight at 4 °C, Alexa Fluor 594 goat anti-rabbit IgG (dilution 1:200, Invitrogen) was used as the secondary antibody. Finally, these sections were counterstained with DAPI and observed under immunofluorescence microscope



(Zeiss Axio Imager Z1, Germany). In vitro anti-calcification assay. In vitro anti-calcification assay of the crosslinked and uncrosslinked samples were conducted according to the authorized patent using artificial plasma solution.27 1 L artificial plasma was prepared by mixing 12 mL 10% (w/v) CaCl2 solution with 988 mL 6% (w/v) hydroxyethyl starch sodium chloride solution. Each 1 × 1 cm sample (n = 30) including Alg, Alg-CHO, GA crosslinked bovine pericardium and Sino product was incubated in 10 mL artificial plasma at 37 ℃ for 7, 14, 21, 30, 60 days under 120 rpm shaking. Upon each time point (n = 6), samples were collected and washed with deionized water thoroughly and dried to constant weight. Dried species were digested with Microwave Digestion System (Metash, MWD-620) before testing calcium level with inductively coupled plasma-mass spectrometry (ICP-MS, PerkinElmer Optima 8300, German) and the data were normalized by the dry weight of each sample. In vivo anti-calcification assay-Surgical implantation. The golden standard screening model for calcification-prevention assay is the subdermal implantation,13 thus the in vivo assay was conducted by subcutaneous implantation in juvenile Wistar rat (45 - 65 g, n = 16) similarly as above in vivo degradation assay. Histological analysis. After 2 and 4 weeks, tissue samples were explanted and snap-frozen in liquid nitrogen and then cut into 6 μm thick sections after embedding in OCT. The samples underwent H&E and Von Kossa staining under manufacturer’s protocol (Zhongshan Golden Bridge Biotechnology, China). DNA content and calcium level. DNA contents in harvested samples were


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quantified using DNeasy Blood & Tissue Kit (Qiagen, Milan, Italy) following the manufacturer’s instructions. Furthermore, calcium levels were obtained by digesting harvested samples with Microwave Digestion System and measuring the media with ICP-MS, finally the data were normalized by the dry weight of each sample. Statistical analysis. All data were presented as the mean ± standard deviation. The two-tailed unpaired t-test was taken to compare between two groups, one-way ANOVA was performed to compare among three or more groups. p value lower than 0.05 was considered statistically significant, i.e., * represented p < 0.05, ** represented p < 0.01 and *** represented p < 0.001. Results Decellularization and ECM evaluation. Bovine pericardium tissue was decellularized and the ECM was evaluated in terms of general tissue morphology, distribution of collagen, GAG and elastin, and the quantification of DNA, collagen, and GAG, fresh pericardium was analyzed for comparison (Figure 1). Qualitatively, as shown in Fig. 1f, most of the original cells were removed as compared to Fig. 1a, and the DNA content was significantly decreased from 0.89 ± 0.13 μg/mg to 0.11 ± 0.03 μg/mg, whose DNA level was even much lower than Sino product control (0.28 ± 0.04 μg/mg). Additionally, the general fiber structure was maintained without any structural destruction, this could also be confirmed with SEM images of fiber structure in Fig. 1j. Moreover, both collagen fiber and muscle fiber (stained blue and red in Masson’s trichrome staining respectively) were slightly decreased after the decellularization procedure as compared between Fig. 1g and Fig. 1b, and this was



also evidenced by the collagen quantification assay (Fig. 1l), in which collagen level in acellular pericardium group was slightly lower without significant difference. Sirius red staining (Fig. 1c & Fig. 1h) indicated that the collagen contained in bovine pericardium tissue and its acellular ECM were both type I collagen fiber, which was in accordance with human aortic valve cusps,11 and the content of type I collagen almost stayed the same after acellular procedure. The extracellular cartilage matrix and chondrocytoplasm (stained red in Safranine O staining, Fig. 1d) were almost removed after decellularization, while collagen fiber (stained celadon in Safranine O staining, Fig. 1d & Fig. 1i) remained almost the same as before. GAG content in acellular pericardium (1.0 ± 0.2 μg/mg) was significantly lower (p < 0.01) compared with fresh pericardium (2.7 ± 0.3 μg/mg). Thus, this decellularization procedure could remove the cell component thoroughly and maintain the ECM structure without losing integrity.

Figure 1 Decellularization of bovine pericardium tissue and ECM evaluation. Standard H&E, Masson's trichrome, Sirius red, Safranin O and SEM images are shown in (a)-(j), scale bar is 20


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μm; (a)-(e) are fresh pericardium, (f)-(j) are acellular pericardium; DNA content, collagen content, and GAG content are shown in (k)-(m), respectively. * is versus fresh pericardium, ** represents p < 0.01 and *** represents p < 0.001.

Surface characterization. Surface characterization of the crosslinked ABP included the FTIR spectra and surface morphology by SEM images (Figure 2). The characteristic absorption at 1725 cm-1 detected on GA-crosslinked sample was attributed to the C=O group in GA, while the obvious absorption at 2853 - 2926 cm-1 as labelled in Fig. 2a indicated -CH2 symmetric and anti-symmetric stretching vibrations. Moreover, the peak at 1050 cm-1 belonged to ether bond in both alginate and oxidized alginate. However, some peaks including 1600 cm-1, 1690 cm-1, 3200 cm-1 absorption ascribed to the N-H in -NH2 group, C=O in carboxyl group, and O-H in hydroxy group, respectively, were all basal groups of collagen existed in ECM substrates which was similar with published FIIR spectra.28 Furthermore, the surface morphologies of various groups were observed (Fig. 2b-e), which revealed no obvious difference in fiber configuration or pore size between the uncrosslinked group and the GA, Alg, and Alg-CHO crosslinked groups.

Figure 2 Surface characterization of uncrosslinked and GA, Alg, Alg-CHO crosslinked ABP. (a) ATR-FTIR spectra indicated the characteristic chemical groups and bonds; (b)-(e) SEM images of sample surfaces, scale bar is 20 μm.



Mechanical performance and thermodynamics/enzymatic/in vivo stability. The mechanical performance for the uncrosslinked and GA, Alg, Alg-CHO crosslinked ABP was measured by a tensile-testing machine, and the data were shown in Figure 3. The stress-strain curve (Fig. 3a) of the four crosslinked ECM exhibited a regular curve like other pericardial tissue,1 showing a mild increasing at the initial stage, which was followed by a second linear region with sharp increased,8 then reached the peak tensile stress and finally ruptured after a high elongation. As calculated with the slope of the second linear region, for all the crosslinked groups, including the GA (34.4 ± 8.2 MPa), Sino product (38.6 ± 7.9 MPa), Alg (33.7 ± 4.1 MPa), and Alg-CHO (36.9 ± 13.4 MPa), the elastic modulus (Fig. 3b) did not exhibit big difference among each group, while showed a significant improve (p < 0.05) compared to the uncrosslinked group (20.7 ± 6.7 MPa), indicated the reliability of our Alg and Alg-CHO crosslinking strategy in the enhancement of mechanical strength. As for the tensile strength (Fig. 3c), which is the maximum stress before rupture, the data for Alg group (8.9 ± 0.7 MPa) and Alg-CHO (7.5 ± 1.1 MPa) were higher than GA group (7.4 ± 1.3 MPa) and Sino product (6.9 ± 1.8 MPa) without significant difference, while were markedly (p < 0.05) higher than the uncrosslinked group (5.0 ± 1.2 MPa). In addition, the elongation at break in the Alg-crosslinked group and Alg-CHO-crosslinked group were 59.6 ± 3.3% and 52.5 ± 5.2%, respectively (Fig. 3d), both of which were higher than that of uncrosslinked group (39.3 ± 7.8%).


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Figure 3 Mechanical performance and thermodynamics/enzymatic/in vivo stability of uncrosslinked, Sino product and GA, Alg, Alg-CHO crosslinked ABP. (a) Stress-strain curve; (b)-(d) Elastic modulus, tensile strength and elongation at break calculated from stress-strain curve, * is versus uncrosslinked group, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001; (e)-(f) DSC curve and DT; (g) In vitro enzymatic hydrolysis was tested in collagenase solution; (h) In vivo degradation data over 12-day implantation.

The thermodynamics, enzymatic denaturation and in vivo degradation were conducted to evaluate the stability and crosslinking degree of samples facing heating, enzyme, and in vivo environment. As can be calculated from the DSC curve (Fig. 3e), the DT (Fig. 3f) at which collagen denatured were 97.1 ± 1.5 ℃ and 96.5 ± 2.9 ℃ for Alg and Alg-CHO, which were even slightly higher than the 93.1 ± 11.2 ℃ of GA group. Furthermore, the enzymatic degradation reflected by weight loss in collagenase solution could also characterize the stability of the crosslinked materials (Fig. 3g), every group except the GA group exhibited a sharp rise in weight loss ratio during the first 3 days, while slowed down till the end of 12 days, and ended up with the weight loss ratio of 70.1 ± 0.6% and 76.6 ± 9.7% for Alg and Alg-CHO crosslinked pericardium, respectively, which were much lower than the uncrosslinked group (94.3 ± 2.5%), and was comparable to the commercially available Sino product (66.3 ± 3.7%), while much lower than the GA control group (7.2 ± 1.1%). However, the in



vivo degradation data indicted a comparable degradation ratio between Alg (1.59 ± 0.65% after 12-day implantation), Alg-CHO group (1.61 ± 0.46 %) and GA group (1.62 ± 0.28%), which were much lower than the uncrosslinked group (22.50 ± 1.70 %). Blood compatibility. Blood compatibility measurements were carried out with hemolysis ratio and platelet adhesion/activation assay, the data are shown in Figure 4. Both the Alg (1.0 ± 0.1%) and Alg-CHO (2.0 ± 0.1%) groups as well as the Sino product (0.9 ± 0.1%) showed a distinct reduction in hemolysis ratio than the traditional GA-crosslinked pericardium (3.6 ± 0.2%), while the data of all the testing groups were below 5.0%, which is the standard requirement for medical apparatus and instruments. Moreover, the hemolysis ratio for both Alg and Alg-CHO groups were comparable with the commercial Sino product without significant difference.

Figure 4 Blood compatibility include hemolytic test and platelet adhesion/activation assay of uncrosslinked, Sino product and GA, Alg, Alg-CHO crosslinked ABP. (a) Hemolysis ratio (%) (n = 5), *** represents p < 0.001; (b)-(f) SEM images of adhered platelet adhesion/activation, (g) the calculated adhered platelet number per 5000× magnification field was calculated randomly (n = 5). Scale bar is 10 μm, * is versus GA group, *** represents p < 0.001.

The morphology and calculated number of the adhered platelets on surface of various crosslinked ABP after incubating with PRP for 1 h are shown in Fig. 4b-g. Generally, the adhered platelets on the surfaces of Alg group (Fig. 4e) and Alg-CHO


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group (Fig. 4f) revealed a round shape with rarely any pseudopodia stretching out, whose morphology was similar with those on uncrosslinked group (Fig. 4b) as well as control groups of GA group (Fig. 4c) and Sino product (Fig. 4d), which were scarcely activated. Additionally, Fig. 4g shows the adhered platelets numbers on sample surfaces. Generally, the assembled platelet numbers on Sino product (25 ± 4 per field), Alg group (23 ± 3 per field) and Alg-CHO group (18 ± 6 per field) were markedly lower (p < 0.001) than the GA control group (72 ± 8 per field). In short, after overall considering of the hemolysis ratio and platelets adhesion/activation assay data, applying Alg and Alg-CHO as crosslinking reagents of ABP not only avoided platelets assembling and activation, but also owned the merits of lowering the hemolysis ratio compared to the traditional GA crosslinking. In vitro cytocompatibility. In order to evaluate the in vitro cytocompatibility of the crosslinked ABP, the cellular responses, including cell proliferation and adhesion behaviors were studied29 and shown in Figure 5. These behaviors were largely influenced by material toxicity, surface chemical bond, surface topography, charge, hydrophilicity and so on.30



Figure 5 In vitro cytocompatibility of uncrosslinked, Sino product and GA, Alg, Alg-CHO crosslinked ABP. (a) Cell viability (%) of blank control (n = 6); (b)-(f) HUVEC incubated on sample surfaces for 14 days were stained with rabbit anti-vWF antibody to identify endothelium layer formation, scale bar is 100 μm; (g)-(k) Live (green) and dead (red) cells were stained with Calcein-AM/PI Kit after 24 h incubation with samples, (l) cell numbers were counted randomly (n = 5) from 10× magnification field; (m)-(v) Cellular actin (green) and nuclei (blue) were stained with FITC-phalloidin and DAPI, (w) cell numbers were counted randomly (n = 5) from 10× magnification field. Scale bar is 20 μm; * is versus GA group, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001.

For the cytotoxicity tests, HUVEC (Fig. 5a) was incubated with the 24-h extract liquid of Alg and Alg-CHO crosslinked ABP over 5 days, tissue culture plate acted as blank control, the resulting cell viabilities were normalized against blank control. For Alg and Alg-CHO crosslinked ABP, cell viabilities exhibited the continuous growth over the whole period, and were initially in par and then even higher than the blank control and ranged between 109.7% - 223.1% (Alg) and 114.4% - 258.6% (Alg-CHO), which accorded with the adhesion assay in Fig. 5m-v. Specifically, for the Alg and Alg-CHO group, the HUVEC viability data were notably higher than the GA group (72.8% - 99.1%) and Sino product (102.9% - 204.1%). The cells incubated on sample surface for 24 h were further stained with


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Calcein-AM/PI Kit to identify their live (green) or dead (red) state (Fig. 5g-k). In correspondence with the cell viability data, the surface of Alg and Alg-CHO group showed both the highest adhered number (170 ± 18 - 238 ± 24 per field) and the highest viability rates of 92.4% - 96.5%, whose viability rates were even higher than the traditional GA group (78.3%) and the commercial Sino product (81.1%). For the cellular actin and nuclei staining (Fig. 5m-v), most of cells in GA group became contracted without any spreading filopodia, and hardly any actin filament was stained. Contrarily, the HUVECs on Alg and Alg-CHO crosslinked ABP fully spread out with the characteristic filopodia and identifiable cytoskeleton, which were quite similar as the blank control.31 Quantitatively (Fig. 5w), after 24 h incubation, there were significant advantage (p < 0.001) in adhered cell number on the surface of Alg (189 ± 23 cells per field) and Alg-CHO crosslinked ABP (169 ± 18 cells per field) over the GA group (28 ± 9 cells per field) and the Sino product (78 ± 12 cells per field), and the data were even slightly higher than the uncrosslinked group (158 ± 15 cells per field). Moreover, the 72-h data showed the same trend as 24-h, which further confirmed the priority in better cytocompatibility of our crosslinking method. Additionally, after incubation for 14 days under the same condition (Fig. 5b-f), an obvious endothelium layer (green line in Fig. 5e-f) formed on the surface of Alg and Alg-CHO crosslinked ABP, encouraging the survival and proliferation of HUVEC for long period with signs of widespread cell coverage. While the GA-crosslinked pericardium and Sino product only exhibited some nonspecific dyeing, as they could not support adequate HUVEC proliferation to form the endothelialization.



In vitro anti-calcification assay. In vitro anti-calcification assay of uncrosslinked, Sino product and GA, Alg, Alg-CHO crosslinked ABP were investigated using artificial plasma solution, accumulated Ca2+ contents over 60 days are shown in Figure 6.

Figure 6 In vitro anti-calcification assay of uncrosslinked, Sino product and GA, Alg, Alg-CHO crosslinked ABP were investigated using artificial plasma solution, accumulated Ca2+ contents over 60 days were shown in the graph.

GA group revealed a continuous rise from the beginning to the end of the testing period and ended up with 6.7 ± 0.1 μg/mg, which was considerably higher than the other groups. Particularly, the Alg group (1.4 ± 0.2 μg/mg) and Alg-CHO group (1.7 ± 0.1 μg/mg) showed the lowest data with only slight increase over 60 days, which were even lower than Sino product (1.8 ± 0.1 μg/mg). In vivo anti-calcification assay. In vivo anti-calcification assay was conducted by subcutaneous implantation with juvenile Wistar rat for 2 & 4 weeks, and corresponding data are displayed in Figure 7. As indicated by the H&E staining (Fig. 7a-h), there was no big difference among the four groups in general tissue structure, while the infiltration cell differed a lot among the Sino product and GA, Alg, Alg-CHO crosslinked ABP, i.e., much more cell grew into the Alg and Alg-CHO


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crosslinked materials and distributed more homogeneous, which would be beneficial for valve regeneration, these were in contrast to the GA treated samples and Sino product. This was further convinced in terms of the DNA quantification in Fig. 7q, as the DNA contents detected in Alg group were 0.80 ± 0.23 μg/mg at 2 weeks and 1.56 ± 0.31 μg/mg at 4 weeks, and data for Alg-CHO group were 0.94 ± 0.23 μg/mg at 2 weeks and 1.34 ± 0.09 μg/mg at 4 weeks, which were more than two times higher than the GA group (0.12 ± 0.01 - 0.56 ± 0.03 μg/mg). Repopulating and revitalizing of the acellular pericardium endowed its potential to recreate a living tissue that had the proper function and durability.15

Figure 7 In vivo anti-calcification assay of Sino product and GA, Alg, Alg-CHO crosslinked ABP. (a)-(h) H&E staining; (i)-(p) Von Kossa staining, scale bar is 20 μm; (q) DNA concentration in the implantation; (r) Ca2+ concentration in the implantation. * is versus GA group, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001.

As for the anti-calcification ability assay, Von Kossa staining (calcium phosphates were stained black) was adopted to determine the calcium distribution qualitatively (Fig. 7i-p). Distinctly, more detective calcium element was discovered in



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GA group and Sino product for both 2 weeks and 4 weeks, which was in accordance with the Ca2+ quantifications (Fig. 7r), the Ca2+ concentration in Sino product (0.7 ± 0.1 - 1.2 ± 0.1 μg/mg), Alg group (0.1 ± 0.1 - 0.7 ± 0.3 μg/mg) and Alg-CHO group (1.0 ± 0.6 - 1.1 ± 1.4 μg/mg) were all significantly lower than the GA group (21.8 ± 0.1 - 32.6 ± 12.8 μg/mg). Discussion The excellent properties make the BHV become more popular in the valve replacement operations. In the United States, more than 80% of patients choose biological valves due to the merits of not taking anticoagulants, but the life span is limited to 10 - 15 years, which is the biggest weakness. In addition, the emerging transcatheter aortic valve replacement (TAVR) has become a prospective treatment for valvular diseases, which avoids thoracotomy but gives rise to expansive need for biological tissues.32-33 Moreover, calcification has been found to be one of the main causes that result in biological heart valves failure.34-35 After investigating the mechanism of biological valve calcification, it is widely accepted by the public that the calcification is caused by traditional crosslinking agent GA to a large extent. All these factors put forward an urgent demand for developing more robust biomaterials with longer durability and anti-calcification over the current commercially available BHV.

Many

modification

methods

non-glutaraldehyde fixation chemistry6,

36, 4

have

been

developed,

including

and detoxification strategies targeted at

GA.13 Furthermore, some strategies to remove phospholipid are also reported to prevent calcification.35, 37 However, the improvement is so limited that no clinical data


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is available till now. Due to the good cytocompatibility, low cost, non-thrombogenic feature, slight immune response and mild reaction condition, alginate has been used in a wide range of biomedical applications, such as wound repair, drug delivery and especially cardiovascular tissue engineering scaffold.25 Trials in clinic and animal models have demonstrated its safety and therapeutic effect. For example, Randall Lee et al injected the alginate into the ventricle wall of heart failure patients undergoing open-heart surgery and demonstrated that the heart function was dramatically improved and the left ventricle dilatation was significantly retarded, indicating alginate itself could be therapeutic agent.38 Moreover, alginate is used as growth factors and cells delivery vehicle for angiogenesis treatment after myocardial ischemia (MI).39-41 Additionally, alginate hydrogel has been comprehensively investigated in the field of tissue engineered heart valves by Jonathan Butcher group. They developed tissue engineered heart valves using 3D bio-printing technique with alginate hydrogel and valve interstitial cells (VIC).42-43 However, alginate was used as different forms of scaffolds in most of these studies, few have reported using alginate as crosslinker component. In this study, we first used it as crosslinker to enhance the mechanical properties and stability of bovine pericardium tissue. The results showed satisfactory results compared to the GA crosslinker with dramatically reduced toxicity and calcification level, solving the biggest weakness of GA. More importantly, our data gave an evidence that alginate could accelerate endothelial cells’ proliferation, leading to complete re-endothelialization on the surface of the bovine pericardium within 14



days, which was dramatically faster than GA and Sino product. Meanwhile, cell infiltrations into the in vivo implantation materials were dramatically improved in the alginate fixed group compared to GA and Sino group. Re-endothelialization and fast cell infiltration are two crucial factors that determine fast tissue regeneration after implantation in situ, which are positively associated with the long durability. Taken together, alginate was demonstrated to promote the regeneration process of bovine pericardium derived heart valves. In addition, it is unavoidable to concern the probability of the calcification while using alginate. As we known, the most common crosslinking method for alginate is through calcium ion. Thus, we consider about this issue deeply and measured the calcification level in the present work by in vitro assay and in vivo assay. Fortunately, we found that calcium level was dramatically reduced in the alginate modified group in comparison with regular GA crosslinked group, as several groups have also reported similar results of reducing calcification of bovine BHV.44 In addition, literature reported that alginate could inhibit 40% mineralization in the rat model.45 Of course, converse results were also reported in several works that alginate could promote tissue calcification.46 But it should be considered that in these studies, they usually used alginate in the crosslinked form with large amounts of Ca2+, which is sharply higher than the Ca2+ concentration in the fabrication process and application environment of cardiac valves. These distinctions may lead to totally different results. Additionally, since extra aldehyde group is considered to be related to high calcification, we supposed that the oxidized alginate may be beneficial for


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anti-calcification, as the lower aldehyde group concentration compared to GA could be fully consumed by -NH2 in ECM.17 Moreover, slightly higher calcification level was still found in the oxidized alginate group compared to the alginate group while much lower than GA group. Of course, it is reasonable that multiple factors except the residual aldehyde group can regulate calcification, such as the microenvironment,47 the stiffness of the materials.48-49 Therefore, much more investigations are needed before its clinical application. The thermodynamics stability of alginate treated group was better than GA group. But the in vitro degradation of the alginate treated group was faster than the GA crosslinked group as demonstrated by the enzymatic hydrolysis test, as the enzyme concentration in this accelerated degradation model is dramatically higher than the in vivo atmosphere, and the Alg-EDC/NHS or Alg-CHO-EDC/NHS treated bovine pericardium was comparable with the commercially available Sino product. The in vivo degradation assay closer to the application atmosphere was also conducted by subcutaneous implantation in adult SD rats to give more accurate degradation information, and properly concluded that stability of the alginate-fixed system is comparable to glutaraldehyde fixation. Moreover, considering the hemocompatibility, cytocompatibility

and

resistance

to

calcification,

the

Alg-EDC/NHS

and

Alg-CHO-EDC/NHS were superb than GA as the crosslinkers for ABP. Of course, advances to slow down the degradation are deserved to develop further, such as combining alginate and GA as combined crosslinkers. Furthermore, the durability and calcification resistance of the alginate-EDC/NHS or oxidized alginate-EDC/NHS



treated bovine pericardium as BHV remain to be investigated in the large animal model before clinical application. Conclusions In this study, we explored the feasibility of Alg-EDC/NHS or Alg-CHO -EDC/NHS as a combined non-glutaraldehyde fixation for ECM-derived scaffolds, especially decellularized bovine pericardial tissue in the anti-calcification cardiac valve applications. Both Alg and Alg-CHO group exhibited improved in vitro hemocompatibility & cytocompatibility, anti-calcification property, thermodynamics stability and regenerative capacity compared to the GA group and commercially available Sino product. Furthermore, the in vitro enzymatic stability of Alg and Alg-CHO group were worse than GA group, but comparable with Sino product, while the more reliable in vivo stability and mechanical properties were comparable with GA fixation. The subcutaneous implantation in juvenile Wistar rats confirmed the tissue regeneration and reduction in calcification in the in vivo environment. This strategy provides a non-glutaraldehyde fixation alternative for fabricating off‐the‐shelf anti-calcification cardiac valve. Author information Corresponding Author *E-mail: [email protected] (Z.W.) *E-mail: [email protected] (X.L.) ORCID Zhihong Wang: 0000-0001-7341-0201


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Xigang Leng: 0000-0002-5149-7630 Notes The authors declare no competing financial interest. Acknowledgments This work was financially supported by Natural Science Foundation of China (no. 81530059, no. 31800815, no. 31771059), Chinese Academy of Medical Sciences, CAMS Initiative for Innovative Medicine (2017-12M-3-002), Science & Technology Projects of Tianjin of China (no. 18JCQNJC14200 and no. 16JCQNJC14100), the Fundamental Research Funds for the Central Universities (3332018119), Specific Program for High-Tech Leader &Team of Tianjin Government. Reference 1.

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