Cross-linking porcine peritoneum by oxidized konjac glucomannan: a novel method to improve the properties of cardiovascular substitute material

The use of natural polysaccharide crosslinkers for decellularized matrices is an effective approach to prepare cardiovascular substitute materials. In this research, NaIO4 was applied to oxidize konjac glucomannan to prepare the polysaccharide crosslinker oxidized konjac glucomannan (OKGM). The as-prepared crosslinker was then used to stabilize collagen-rich decellularized porcine peritoneum (DPP) to construct a cardiovascular substitute material (OKGM-fixed DPP). The results demonstrated that compared with GA-fixed DPP and GNP-fixed DPP, 3.75% OKGM [1:1.5 (KGM: NaIO4)]-fixed DPP demonstrated suitable mechanical properties, as well as good hemocompatibility, excellent anti-calcification capability, and anti-enzymolysis in vitro. Furthermore, 3.75% OKGM [1:1.5 (KGM: NaIO4)]-fixed DPP was suitable for vascular endothelial cell adhesion and rapid proliferation, and a single layer of endothelial cells was formed on the fifth day of culture. The in vivo experimental results also showed excellent histocompatibility. The current results demonstrted that OKGM was a novel polysaccharide cross-linking reagent for crosslinking natural tissues featured with rich collagen content, and 3.75% OKGM [1:1.5 (KGM: NaIO4)]-fixed DPP was a potential cardiovascular substitute material.


Introduction
Heterogeneous decellularized tissues have been widely used in the preparation of vascular grafts because their construction is suitable for cell adhesion, proliferation, and migration. The products currently developed include the Artegraft bovine common carotid artery [1], ProCol bovine mesenteric vein [2,3], and Synergraft bovine ureter [4]. However, some decellularized tissues have not been used clinically, including the human umbilical vein, porcine carotid artery, porcine saphenous artery, and other acellular matrices. All the decellularized scaffolds were rich in collagen and elastin. Elastin is the main extracellular matrix component of vascular tissues. Lack of elastin can lead to failure of blood vessels, including aneurysms, thrombosis, and intimal hyperplasia [5][6][7][8]. The peritoneum of the Bama pig is also a tissue rich in elastin and is easier to obtain than other tissues. The peritoneum, rich in blood capillaries, lymphatic vessels, and nerves, exhibits flexibility and a smooth surface. The peritoneum has the same embryologic origin as blood vessels and a low risk of thrombosis. Additionally, the Bama pig peritoneum demonstrates desired permeability along with a certain degree of regenerative properties, adequate mechanical properties, and a thickness similar to that of the 2 mm diameter-natural vessels (300-500 μm). Therefore, it is suitable for use in preparing artificial vessels with diameters of 2 mm.
However, the direct application of porcine peritoneum onto the construction of replacement grafts can cause immune rejection. DPP can greatly reduce its immunogenicity, but its poor mechanical properties and stability in physiological environments prevent its direct use or preservation. Therefore, the pre-fixation of these animal-derived decellularized tissues is required to address these problems [9]. Glutaraldehyde (GA), the most common crosslinking agent used for pre-fixation, can enhance the biomechanical properties of acellular tissues and improve their stability. However, as many researchers have confirmed, the application of Glutaric dialdehyde (GA) is limited by calcification and cytotoxicity as well as the inability to crosslink elastin [10]. Genipin (GNP) is an excellent, naturally occurring crosslinking agent that has been developed in recent years. Its cytotoxicity is lower than that of GA alone. However, the color of tissues crosslinked by GNP is dark blue, and the thickness of GNP-fixed tissues increases excessively [11]. These shortcomings restrict the further application of GNP. There is an urgent need to find a suitable crosslinking agent to pre-fix acellular tissues to improve their mechanical properties, further reduce their antigenicity, and enhance their resistance to enzymatic degradation while maintaining their original ultrastructure.
Konjac glucomannan (KGM) is a high-molecularweight, water-soluble, non-ionic natural polysaccharide. It exhibits good biocompatibility, biodegradability, and excellent gel-forming properties and is used in cosmetics, drug-controlled release materials, and plasma substitutes. KGM is linked by β-1,4 glycosidic bonds and is rich in hydroxyl groups, providing opportunities for this polysaccharide to be modified for other biomedical applications. The o-hydroxyl groups of KGM can be selectively oxidized by sodium periodate (NaIO 4 ) into aldehyde groups, yielding oxidized KGM (OKGM) with multiple functional aldehyde groups. OKGM can bridge amine groups in heterogeneously derived acellular tissues through reactive formyl-aldehyde groups to form a stable cross-linking structure [12][13][14].
This research evaluated various physical and chemical properties of OKGM-fixed DPP, including its ultrastructure, mechanical properties, stability, cytocompatibility, hemocompatibility, in vitro calcification, and in vivo inflammation. This research first provides an experimental basis for OKGM as a new cross-linking reagent for fixing natural tissues featured with rich collagen content.

Preparation and characterization of OKGM
After 5 g of KGM (K8170, Solarbio) was dissolved in 500 mL distilled water, 7.5 g of NaIO 4 (S817518, Solarbio) was added to the KGM solution. The oxidation reaction proceeded in the dark at room temperature for 48 h. Subsequently, 20 mL ethylene glycol was added to the mixture to remove excess NaIO 4 . Crude OKGM was obtained via precipitation with excess ethanol and suction filtration. Finally, the obtained OKGM was immediately dialyzed (with a molecular weight cutoff of 3000) with deionized water for 3 days and subsequently lyophilized using a vacuum freeze dryer to obtain purified OKGM.
The aldehyde content of OKGM was determined by the potentiometric titration of hydroxylamine hydrochloride. The weight-average molecular weight (Mw), numberaverage molecular weight (Mn), and polydispersity (Mw/ Mn) of OKGM were determined using aqueous gel permeation chromatography (Waters 1515). Zeta potential (ζ) measurements of products were analyzed in a Malvern Zeta sizer (Nano Model ZS90) at a constant temperature of 25 °C. For the FTIR measurement of OKGM, 4 mg of dry samples and 40 mg of KBr were pressed into a disc. A Nicolet 560 infrared spectrometer was used to measure the FTIR spectra at a resolution of 4 cm −1 in the wavenumber range of 400-4000 cm −1 .

Preparation of DPP and fixation index of OKGM-fixed DPP
The fresh peritoneum was immersed in 1% Triton X-100/ deionized water and 0.5% SDS/deionized water and shaken (60 rpm) for 3 h. Subsequently, the samples were soaked while undergoing continuous magnetic mechanical stirring for 3 h. Finally, the samples were washed several times with PBS to remove cellular remnants. DNase I (200 U/mL) was used to treat samples at 37 °C for 4 h. DPP was obtained using the decellularization process described above. DPPs were immersed in a series of OKGM/PBS solutions with different OKGM concentrations (3.75, 7.5, and 15% m/v) to fix the samples. The DPPs were all fixed at 37 °C for 24 h in a constant temperature oscillator.
At predetermined times, all the OKGM-fixed DPPs were lyophilized for at least 12 h to a constant weight. After heating the NHN solution to boiling for 20 min, lyophilized tissues were removed, and the optical absorbance of the solution was measured using an enzymelabeled instrument (Multiskan FC, Thermo Fisher) at 570 nm. Glycine at various known concentrations was used as the standard, and the fixation index (FI) was calculated using the following formula:

Surface characterization of samples
Field-emission scanning electron microscopy (FE-SEM, Oberkochen) was used to evaluate the surface topographies of the samples. The hydrophilicity of the surfaces of the samples (lyophilized 2.5 cm × 2.5 cm) was characterized by a water contact angle goniometer (WCA, SL200KS).

Hemocompatibility measurement 2.5.1 Hemolytic test
Red blood cell (RBCs) separation 10 mL of rabbit blood anticoagulated with sodium citrate were added to 20 mL DPBS to ensure uniform dispersion. The RBCs were separated by centrifugation at 500 × g for 10 min. Thereafter, the RBCs were rinsed with DPBS several times and re-suspended in 100 mL DPBS solution. RBCs dispersed in DPBS were used as negative controls, while those dispersed in distilled water were used as positive controls. Gradient concentration hemolysis test All OKGM-fixed DPP samples were ground to powders and dispersed in PBS at gradient concentrations to form a suspension. 0.4 mL of RBC solution was then added to 1.6 mL suspensions at each concentration soon afterward, and all mixtures were incubated for 3 h at 37 °C.
Direct contact hemolysis test The sample was cut into 2 cm × 2 cm pieces and placed flat in a 6-well plate. Then, 2 mL of RBC suspension was added to each well, and the samples were incubated for 3 h at 37 °C.
The suspension was centrifuged, and the absorbance of the supernatant was measured at 540 nm. The hemolysis rate (%) was calculated as follows:

Static platelet adhesion test
Whole blood was centrifuged (1000 r/min for 10 min) to separate platelet-rich plasma (PRP). 400 μL of PRP was dropwise added on the surface of DPP-samples, which have been pre-immersed and equilibrated in PBS at 37 °C for at least 1 h. Then, they were incubated at 37 °C for 1 h. These DPP-samples were mildly washed with PBS to remove non-adherent platelets before being fixed with a 2.5% GA/PBS solution for 8 h at 4 °C. Finally, the DPPsamples were dehydrated using gradient ethanol (50, 70, 85, 90, 95, and 100%) for 10 min per concentration, and the morphology and aggregation of the attached platelets were observed using SEM.

Ex vivo blood contact test
The DPP-samples were cut into square patches (4 mm × 9 mm) and pre-immersed in PBS for 1 h. The patches were then attached to the internal walls of silicone tubes (ID = 3.1 mm) pre-soaked with heparin. General anesthesia of experimental rabbits weighing 3 kg each was induced through the intravenous administration of 10 mg/kg pentobarbital sodium salt (Sigma, 57-33-0). 40 μg/kg of the sedative dexmedetomidine hydrochloride (Zoetis, 1852761) was also administered intravenously. 0.3 mg/kg Butorphanol (MSD, A141A04) for preemptive analgesia was administered intramuscularly. After opening the abdominal cavity from the midline, 2 cm of the abdominal aorta was separated and exposed. The vessel was clamped using arterial clamps at the distal and proximal ends. Next, the artery was cut from the middle, and the vascular stump was flushed with heparinized saline. A cannula (0.8 mm × 1.6 mm) was inserted into the vascular stump and bound using sutures to prevent cannula dislodgement. Blood was collected from the proximal arterial drainage tube and infused into degassed sample tubes to interact with the materials using peristaltic pumps (YZ1515x, Longer). Blood was then injected into the distal arterial drainage tube to complete circulation. The infusion lasted for 2 h. After completing the perfusion procedure, the DPP-samples were removed, fixed with 2.5% GA solution, and observed using SEM.

Enzymatic hydrolytic resistance
After being lyophilized to constant weight, all samples were immersed into 1.5 mL collagenase I/PBS (S10053, Yuanye Shanghai) (25 U/mL), collagenase IV/ PBS (S10056, Yuanye Shanghai) solutions (25 U/mL), Elastase/PBS (S10165, Yuanye Shanghai) solutions (25 U/ mL), respectively, and incubated at 37 °C with constant shaking. The samples were then transferred to a 10 mM EDTA solution to terminate the enzymatic hydrolysis reaction at predetermined time points (0.5, 1, 3, 6, 12, 24, 48, and 72 h). The enzymatically hydrolyzed samples were lyophilized and weighed. The percentage of weight loss (ΔW%) was calculated using the following formula: W 0 represents the initial weight before degradation, and W t represents the weight of the corresponding sample after degradation.

In vitro calcification
The lyophilized and sterilized 1 cm × 1 cm samples were immersed in 2 mL of simulated body fluid (SBF, JISS-KANG) and incubated in a constant temperature incubator at 37 °C. The SBF was changed weekly and the entire process remained aseptic. The samples were collected and washed with distilled water at pre-determined times (after 30, 60, and 90 d). After dehydration by gradient alcohol and drying at the critical point of CO 2 , the surfaces of the samples were observed by SEM, and the surface calcium content of the samples was detected using EDS.

Mechanical properties 2.8.1 Uniaxial tensile test
The samples were investigated before and after fixation to determine if any improvement in the biomechanical properties could be detected. The samples were cut into rectangular strips of 40 mm × 10 mm, and their thicknesses were measured by a micrometer. Uniaxial tensile tests were performed using an Instron material testing machine (Instron Co., USA) at an extension rate of 7 mm/min. The ultimate tensile strain and the ultimate tensile stress were recorded when the sample was torn. The ultimate elastic modulus was determined from the ultimate tensile stress-strain curve.

Suture retention strength
The suture retention test was performed according to the American National Standard Institute /Association for the Advancement of Medical Instruments (ANSI/AAMI) VP20 standard. Each sample was cut into rectangular strips (40 mm × 10 mm). A single 5-0 absorbable suture loop was created 2 mm away from the sample edge and secured to a hook connected to the clamp of the testing device. The suture retention strength was defined as the maximum force recorded at an extension rate of 7 mm/ min when the sample was torn. Suture retention tension was obtained by normalizing the suture retention strength to the thickness of each sample.

Subcutaneous implantation
Subcutaneous implantation in rats is the standard FDAaccepted technique for evaluating cardiovascular materials. SD rats were purchased from Chengdu Dossy Experimental Animals CO, Ltd. Rats were inhalation anesthetized with of 1-2% isoflurane gas, and sterilized DPP-samples were implanted subcutaneously on both sides of their backs. After recovery from anesthesia, all rats were fed a standard diet. Rats were euthanized with excess isoflurane gas after 90 days, and each collected implantation sample was fixed in 4% formaldehyde for HE staining analysis.

Statistical analysis
Data are expressed as mean ± standard deviation and were analyzed by one-way ANOVA using SPSS (version 20.0), p < 0.05.

Oxidation degree, molecular weight, and polydispersity of OKGMs prepared with different feeding ratios of KGM/NaIO 4
With the increase in NaIO 4 , the Mn and Mw of OKGM showed a downward trend, and the aldehyde group content within OKGM increased because of the breaking of the polysaccharide chain ( Table 1). The molecular weights and physiological activities of the crosslinking agents are different. OKGM with a large molecular weight has both low cytotoxicity and low crosslinking properties owing to its better diffusion and ability to reach more reaction sites to react with the amino groups within the tissue. In addition, the molecular weight of the crosslinking agent has an important relationship with its metabolic time in vivo. The larger the molecular weight of OKGM, the more complex its structure and the more difficult it is for OKGM in vivo [15,16]. Therefore, appropriate NaIO 4 consumption is key to the preparation of OKGM. According to the pre-experiment, we selected three OKGM samples prepared with different feeding ratios of KGM/NaIO 4 (mass ratios of KGM/NaIO 4 were 1:1, 1:1.5, and 1:2) to further investigate their crosslinking properties and the physicochemical and biological properties of OKGM-fixed samples to ascertain the best matching of KGM and NaIO 4 for preparing OKGM used as a crosslinking agent.

FTIR analysis of OKGM
OKGM was prepared by the NaIO 4 oxidation method (Fig. 1a). The presence of dialdehyde groups was verified by FTIR analysis. As shown in Fig. 1b, the spectrum of OKGM shows a new characteristic peak at 1727.5 cm −1 , which is the most characteristic band of the C=O vibrations of the aldehyde group. This peak clearly indicates the formation of active aldehyde groups in the OKGM molecular chain. A new infrared band at 892 cm −1 suggests the formation of hemiacetals between the aldehyde groups and non-oxidized hydroxyl groups. Therefore, the aldehyde group was successfully introduced through periodate oxidation.

Zeta potential of OKGM
The OKGMs with different degrees of oxidation presented different zeta potential values. This may be owing to the different types and numbers of functional groups on the surface of OKGMs. The higher the feeding ratio of NaIO 4 , the higher thetable degree of oxidation of an OKGM (Table 2), and more aldehyde groups are formed in the OKGM molecular chain. Owing to the electronegativity of oxygen atoms in aldehyde groups, the electron cloud of the carbon-oxygen double bond will shift toward the oxygen atoms, eventually leading to an increase in the electronegativity of the overall system. This increase in polarity is manifested as an increase in the absolute value of the potential.

Decellularization of peritoneum and characterization of DPP
The fresh porcine peritoneum (FPP) is a semi-transparent film with a thickness of approximately 0.2-0.5 mm. After decellularization, the tissue thickness significantly increased to about 0.8 mm (Additional file 1: Fig. S1).
Lyophilized DPP appeared as a white sheet with good flexibility, as shown in Fig. 2a.

Residual DNA of DPP
Residual cellular components in ECM can cause adverse host reactions in vivo and increase the risk of heterogeneous antigen transmission. Double-stranded DNA (dsDNA) can be quantitatively assessed as an indicator of cell residue in the ECM. Previous studies have suggested that the minimal standard concentration of dsDNA in the ECM is < 50 ng/mg. Our research demonstrates that the total DNA content of DPP is significantly decreased by 91.5% (44 ± 12 ng/mg) after decellularization when compared to that of FPP (519 ± 80 ng/mg) (Fig. 2c), which meets the standard of clinical application of ECM. DAPI staining of fresh and decellularized porcine peritoneum was also performed to determine the extent of DNA removal. As shown in Fig. 2b, the number of intact nuclei in the FPP is much higher than that in the DPP. Almost no nuclei are observed in the DPP. HE staining is a simple qualitative indicator used to observe whether tissues contain nuclei to confirm the removal of nucleic acids from tissues [17]. In this experiment, there are no residual cells with intact structures in the DPP fiber voids after decellularization, based on the HE staining results (Fig. 2e).

Ultrastructure of DPP and FPP
The SEM results (Fig. 2d) show that after decellularization, the fibers in the peritoneum became loose but less broken, and the directions of the fibers are similar.
The results of HE, Masson, and EVG staining (Fig. 2f ) show that collagen and elastic fibers remain intact after decellularization.

The expression level of α-Gal, α-SMA, collagen I and collagen IV in DPP
α-Gal is widely expressed in most mammalian tissues (including humans and higher primates), and α-Gal remaining in the cytomembrane can cause hyperacute immune rejection reactions after xenotransplantation [18,19]. The absence of α-Gal could be considered a sign of successful decellularization. α-SMA is a marker protein of vascular smooth muscle, and its expression level can be used as an indicator of the degree of clearance of smooth muscle cells [20]. The weak expression of α-Gal and α-SMA (Fig. 2f ) in DPP indicates the successful decellularization of FPP and the safety of this animal-derived substitutional material.
Collagen accounts for approximately one-third of the total proteins in all vertebrates. It is biodegradable with weak antigenicity and good biocompatibility [21]. However, after decellularization, its natural assembly structure is destroyed, resulting in decreased mechanical properties and stability. The results of collagen immunohistochemical staining (Fig. 2f ) shows that the expression of type I and type IV collagen both in FPP or DPP is abundant and mainly concentrated in the basal layer [22,23].

Preparation and characterization of OKGM-fixed DPP
After crosslinking with OKGM, GNP, and GA, it was found that GNP-fixed DPP becomes dark bluish, while the color of both GA-fixed and OKGM-fixed DPP turn yellow. The freeze-dried DPP-samples are shown in Fig. 3b.

The fixation of DPP by OKGM
The FI was used to quantitatively evaluate the degree of crosslinking. The results demonstrated that the FI of OKGM-fixed DPP was correlated with the feed ratio of KGM/NaIO 4 for preparing OKGM and the concentration of OKGM. As shown in Fig. 3a, the fixation of samples by 7.5 and 3.75% OKGM prepared with different feeding ratios of KGM/NaIO 4 (the mass ratio of KGM/ NaIO 4 is 1:1, 1:1.5, 1:2) is stable, and after 24 h of fixation, their FI reaches a maximum (> 60%). There is no significant difference in FI during the entire course of the test between the 3.75 and 7.5% OKGM-fixed DPP.
Studies have reported that the degradation rate of decellularized tissue affects tissue regeneration after implantation [24]. The in vivo degradation rate of animalderived scaffolds could be controlled by their FI. The 95%-FI bovine pericardium was more resistant to enzymatic degradation than its 60%-FI counterpart in research of GNP-fixed bovine pericardium [25]. Consequently, tissue regeneration was limited in the 95%-FI acellular tissue throughout the entire research (1 year post-operatively), whereas tissue regeneration was observed in the 60%-FI acellular tissue [26]. In conclusion, FI determines the degradation rate of acellular tissue and its regeneration pattern. Therefore an appropriate FI (≥ 60%) should be taken into account the ability to resist enzymolysis and tissue regeneration.

Water contact angle
Appropriate surface hydrophilicity is a prerequisite and a basis for good cytocompatibility of biomaterials. Superhydrophilic or superhydrophobic surfaces are not conducive to cell spreading and adhesion. The water contact angle (WCA) of the DPP was approximately 77° (Fig. 3c). After crosslinking with GA and OKGM, the WCA of the fixed samples increased to 87.6° and 91.1°, respectively. This may be because of the massive consumption of amino groups on the surface of DPP by GA or OKGM, leading to a weakened interaction force between the fixed sample and the water droplet. Therefore, the surfaces of DPP fixed with GA or OKGM exhibited slight hydrophobicity compared to that of DPP alone. After crosslinking with GNP, the WCA of the fixed samples decreased significantly to 14.1°. This might be because a large number of amino groups remained on the DPP surface due to the different crosslinking mechanisms of GNP. Moreover, the carboxyl and hydroxyl groups on the GNP were exposed. All these events resulted in a reduction in WCA. The surfaces of GA-fixed DPP and OKGM-fixed DPP are neither too hydrophilic nor too hydrophobic, which could effectively promote cell adhesion and spreading.

The fiber structure of fixed DPP
After crosslinking with GA and GNP, the DPP fibers exhibited anisotropic characteristics (Fig. 3d). The fibers in the GA-fixed DPP were tighter than those in the GNPfixed DPP. For OKGM-fixed DPP, with the increase in NaIO 4 concentration, the aldehyde group content within OKGM (the oxidation of OKGM) increased, the structures of the fibers were more compact, and the fiber orientation was basically the same. This indicated that the cross-linking by OKGM did not destroy the original structure of the fiber but rather made the fiber structure denser.

Cytocompatibility and in vitro endothelialization of OKGM-fixed DPP
The lumen surface of a vascular graft can trigger thrombosis owing to the deposition of platelets. The absence or  [27]. Re-endothelialization is the first key point in inhibiting intimal hyperplasia and thrombosis. A completely spread monolayer of endothelial cells can ensure normal blood flow and the functional expression of EC [28,29]. After seeding HUEVCs on fixed DPP and culturing for 1, 3, and 5 days, the proliferation of HUEVCs was detected by CCK8 (Fig. 4a). The adhesion of HUEVCs on the second and third days was detected by live/dead cell fluorescence staining (Fig. 4b) and SEM (Fig. 4c), respectively. The proliferation and endothelialization of HUEVCs on samples on the fifth day tested by HE staining (Fig. 4d). The results showed that the samples in the 1:1 OKGM-fixed and 1:1.5 OKGM-fixed groups had similar relative generation rates (RGR) of HUEVCs, basically maintaining around 70-90% within 5 days. This was significantly higher than that of the GA-, GNP-, and 1:2 OKGM-fixed DPP. Live/Dead cell fluorescence staining and SEM results showed that HUEVCs had good adhesion on the surface of 1:1 OKGM-fixed and 1:1.5 OKGM-fixed DPP, spreading fully with their characteristic fusiform morphology on sample surfaces. In contrast, the GA-and 1:2 OKGM-fixed samples showed a certain degree of cytotoxicity, with no obvious normal adhesion of HUEVCs on 1:2 OKGM-fixed DPP. As shown in Fig. 5d, compared with counterparts in other groups, significantly confluent monolayers are found on the surfaces of the 1:1 OKGM-and 1:1.5 OKGM-fixed DPP. The above results indicated that 1:1 OKGM-and 1:1.5 OKGM-fixed DPP presented low cytotoxicity and high HEVC cytocompatibility. This was partly because of the good biocompatibility of OKGM. Furthermore, OKGM could also crosslink collagen and elastin, which enhances the ability of OKGM-fixed DPP to resist enzymatic hydrolysis in vitro, avoiding the cytotoxicity resulting from the small molecule toxic substances produced by rapid degradation of samples in vitro. Conversely, the excessive aldehyde groups in the 1:2 OKGM-fixed DPP could result in some cytotoxicity.

Hemocompatibility of OKGM-fixed DPP
Hemocompatibility is the most important criterion for the successful application of cardiovascular substitute materials in vivo [30]. Figure 5a shows the hemolysis rate of various DPP-samples as a function of suspension concentration of the samples after incubation with RBCs at 37 °C for 3 h. As shown in the figure, the hemolysis rate of OKGM-fixed samples is lower than that of GA-fixed and GNP-fixed samples. In addition, the hemolysis rate is less than 5% for 1:1 and 1:1.5 OKGM-fixed samples at a concentration of 25 μg/mL, which was much higher than the concentration value that was possibly reached in vivo. The complete DPP-sample direct contact hemolysis test results (Fig. 5b) show that the hemolysis rate of the OKGM-fixed samples is significantly lower than that of the GA-fixed and GNP-fixed samples, and the hemolysis rates of all samples are less than 5%. The hemolysis rate tested by the two methods met the clinical requirements for implants according to the international standard ISO 10993-4:2017(E).
Anticoagulation is an essential factor for the longterm patency of blood vessels [31]. Platelet adhesion and deposition are the leading causes of thrombosis and intimal hyperplasia. Therefore, inhibition of platelet adhesion on cardiovascular substitute materials is very important for small-diameter vascular grafts. Owing to the participation of GP lb, vWF, and integrin α II β 3d , the adhesion of platelets onto the surface of cardiovascular substitute materials undergoes five processes (flowingdisc-shaped platelets, rolling ball-shaped platelets, hemisphere-shaped platelets, firm reversible adhesion, and spreading irreversible adhesion), and the platelet morphology undergoes corresponding changes during these processes. Among these processes, only the last step (spreading irreversible adhesion) is irreversible, which is conducive to capturing more proteins and platelets to adhere to the surface of the cardiovascular substitute materials, leading to platelet aggregation and thrombus formation of thrombi [32]. As observed by SEM (Fig. 5c), more flat platelets are spread irreversibly and aggregated on the DPP surface. There were many hemispherical platelets on the GA-fixed DPP, which were basically in the rolling ball-shape stage, on the GNP-fixed DPP, the platelets were slightly flat and their pseudopodia were more stretched, and most of them were in the hemisphere shape stage. The number of platelets adhering to OKGM-fixed DPP was significantly lower than that of the GA-fixed and GNP-fixed samples. Among these OKGMfixed DPP, no platelets spread irreversibly on the surface of the 1:1.5 OKGM-fixed DPP. The results showed that OKGM-fixed DPP, especially 1:1.5 OKGM-fixed DPP, could significantly inhibit platelet adhesion.
The carotid artery in rabbits is thin and prone to thrombosis after puncturing owing to hemodynamic changes at the site of the puncture needle and cannula. In view of this, a semi-ex vivo circulation (Fig. 6a, b) was constructed by ligating the abdominal aorta and cannulating the upper and lower ends of the vessel, and using a peristaltic pump to set the blood flow rate in vivo. The sheet sample was fixed to the inner surface of the blood circulation line, and arterial blood was extracted from rabbits for semi-ex vivo circulation to assess the anticoagulation properties and leukocyte adhesion of the sample. After circulation for 2 h, no thrombus is observed on the surface of the samples in each group (Fig. 6c). As shown in the SEM image (Fig. 6d), there are variable amounts of adherent platelets without a fibrin network on the sample surface. Among them, more platelets are on the GA-and GNP-fixed DPP. In addition, a certain amount of leukocytes is also observed to adhere to their surfaces. The number of platelets adhering to the OKGM-fixed DPP was lower than that of the DPP, GA-, and GNP-fixed samples, and no leukocytes were found on their surfaces. This indicated a lower risk of thrombosis for OKGMfixed DPP and also that GA-and GNP-fixed DPP might trigger a more severe inflammatory response. An important feature of the inflammatory response process in vessels is that leukocytes adhere to and infiltrate the vascular endothelium and engulf pathogens or tissue debris from exudation. Therefore, the amount of leukocyte adhesion and exudation could reflect the degree of inflammation [33]. At the site of vascular injury, excessive leukocyte adhesion needs to be avoided as excessive leukocyte adhesion and accumulation could lead to atherosclerosis [34]. Conversely, excessive leukocyte adhesion and infiltration is an important manifestations of vascular endothelial dysfunction [35]. Based on these results, the OKGM-fixed DPP, especially the 1:1.5 OKGM-fixed DPP, had better hemocompatibility.

In vitro anti-enzymatic hydrolysis of OKGM-fixed DPP
The main structural proteins in mammals are collagen and elastin, which provide mechanical support, strength, and elasticity in various tissues and organs. Collagen and elastin are readily available, biodegradable, biocompatible, and can stimulate cell growth [36]. In the anti-enzymatic hydrolysis experiments (collagenase I and collagenase IV) of fixed samples in vitro, the ability of GNP-fixed DPP to resist collagenase digestion is better than that of GA-fixed DPP at 72 h, and OKGM-fixed DPP is equivalent to GA-fixed DPP (Fig. 7). This indicates that GNP presents a better crosslinking effect on collagen than OKGM and GA, while OKGM and GA present the same crosslinking effect on collagen. Masson staining results (Additional file 1: Figs. S2-S3) show that the structure of collagen fiber in GNP-fixed DPP is well preserved. The same result was also observed for GA-fixed and 1:1.5 OKGM-fixed DPP. Elastin keeps ECM in its original shape under stressful conditions. Elastin is mainly composed of hydrophobic amino acids [37]. Due to the lack of lysine residues as reactive groups, it is difficult to fix and stabilize natural elastin through covalent interactions. Therefore, the commercial crosslinker GA cannot effectively crosslink elastin. This conclusion was confirmed in the present research. The ability of GNP-fixed and OKGM-fixed DPP to resist elastase digestion was better than that of GAfixed DPP and DPP alone. EVG staining results showed no obvious elastic fibers were observed in the GA-fixed DPP after 72 h of elastase-enzymolysis in vitro, and its microstructure was seriously damaged. The elastic fiber microstructure (Additional file 1: Fig. S4) in the 1:1.5 and 1:2 OKGM-fixed DPP samples was preserved as well as that of the GNP-fixed DPP. The results indicated that GA had a poor crosslinking effect on elastin and was unsuitable for crosslinking DPP. However, GNP had a good crosslinking effect on both collagen and elastin, and their resistance to enzymatic hydrolysis (collagenase and elastase) in vitro was the best. The crosslinking effect of OKGM on collagen and elastin was second only to that of GNP. It also demonstrated a good crosslinking effect on DPP.

In vitro calcification of OKGM-fixed DPP
The calcification process of the vessel and valve is similar to bone mineralization, and there are two main stages: calcium phosphate nucleation and further growth of crystals [38][39][40]. The initiation and progression of calcification depend on the microstructure of the biomaterials (including collagen fibers and elastic fibers). The low crosslinked elastin protein has large open spaces between molecules because of the lack of functional groups that will react with aldehydes and epoxy compounds. It has many calcium-binding sites that provide a central site for crystal nucleus formation. Therefore, the more elastin the biomaterials contain, the faster their calcification rates. As representative small-molecule crosslinking agents, GA and GNP are usually used to crosslink bovine pericardium to construct artificial heart valves. However, this artificial heart valve presents a serious risk of long-term calcification, which limits its long-term implantation. Grafts fixed by other crosslinking agents (including EDC, ethylene oxide, proanthocyanidins, sodium alginate, etc.) also demonstrated serious instances of calcification either in vitro or in vivo, causing implantation failure [41]. We developed a novel OKGM to crosslink DPP and detected the calcification of OKGM-fixed DPP in vitro through EDS (Fig. 8a) and SEM (Fig. 8b). DPP exhibited the maximum degree of calcification, and its calcium content reached more than 30% after incubation for 60 and 90 days. The calcium content of GA-fixed and GNPfixed DPP was approximately 1% after incubation for 60 and 90 days. The OKGM-fixed DPP presented the lowest degree of calcification because of its smooth surface with almost no calcium deposits. The results suggested that OKGM-fixed DPP had significantly better anti-calcification capability in vitro than GA-fixed and GNP-fixed DPP. Although OKGM does not wholly prevent the first stage of calcification in the peritoneum, it can delay the mineralization of elastin and reduce the second stage of calcification. However, the underlying mechanism remains unclear. We speculate that one of the reasons for this is that OKGM can simultaneously crosslink collagen and elastin.

The mechanical properties of OKGM-fixed DPP
Adequate mechanical properties are essential for cardiovascular materials, particularly for artificial heart valves and small-diameter vascular grafts [42]. According to the literature, the axial E-modulus of a 6 mm aorta is 9.13 MPa, and the ultimate tensile stress is 4.03 MPa [43][44][45]. Uniaxial tensile testing (Fig. 9) is one of the most commonly used methods for measuring the mechanical properties of cardiovascular materials. The ultimate tensile stress (Fig. 9a, b) of OKGM-fixed DPP is 3.43 MPa, which is slightly lower than that of the artery, GA-fixed DPP and GNP-fixed DPP, but there is no significant difference in the ultimate tensile stress among them. Conversely, the E-modulus of OKGM-fixed DPP (Fig. 9c) is 12 MPa, which is slightly higher than that of the artery, GA-fixed DPP and GNP-fixed DPP. However, there was still no significant difference in the E modulus among them. Vascular grafts require a certain suture retention strength to ensure clinical use, and the demand for suture retention strength of vascular grafts is at least 1 N. The suture retention strength results for various samples are shown in Fig. 9d. The suture retention strength of GAfixed DPP and GNP-fixed DPP was significantly higher than that of the 6 mm artery and OKGM-fixed DPP. Furthermore, OKGM-fixed DPP had an equivalent suture retention strength to that of the 6 mm artery, which was approximately 7.9-9.2 N. In fact, suture retention strength that is too high is not conducive to anastomosis. The above results confirm that the mechanical properties of the OKGM-fixed DPP is the nearest to the human artery, meeting the mechanical performance requirements for vascular grafts.

Degradation and inflammation of OKGM-fixed DPP in vivo
A subcutaneous implantation model of the SD rat subjects for 90 days was used to investigate the biocompatibility of the samples. As shown in Fig. 10a, compared with OKGM-fixed DPP, there are more blood vessels around the DPP and GA-fixed DPP/GNP-fixed DPP 12 weeks after subcutaneous implantation. This demonstrated that the inflammation caused by DPP, GAfixed DPP, and GNP-fixed DPP was still obvious, and the sample morphologies showed noticeable changes due to degradation in vivo. The morphology of the GNP-fixed DPP and OKGM-fixed DPP remained relatively complete. No apparent morphological changes due to degradation and no obvious inflammation were observed in the OKGM-fixed samples, indicating their good histocompatibility. Samples were retrieved from the subdermal sites for histological analysis. HE staining results showed (Fig. 10b) that DPP presented more fiber breakage in vivo and more inflammatory cell infiltration compared with samples in other groups. GAfixed DPP and GNP-fixed DPP maintained better fiber morphology because of their ability to resist enzymatic digestion, but inflammatory cell infiltration in these samples was also high, indicating serious inflammation. OKGM-fixed DPP presented fewer broken fibers, and its shape retention was second only to GNP-fixed DPP. It is to be noted that there was no inflammatory cell infiltration observed in the sample, while massive neovascularization around the sample was found. This indicated that OKGM-fixed DPP had good histocompatibility in vivo, which was beneficial to the migration of endothelial cells, and thus could promote angiogenesis in implants in vivo.

Conclusion
The peritoneum is a thin elastic membrane. After decellularization, DPP is a promising animal-derived biomaterial that remains intact ECM and suitable mechanical properties. In this research, NaIO 4 was applied to oxidize KGM to prepare a polysaccharide crosslinker, OKGM, and then used OKGM to stablize DPP to prepare a novel cardiovascular substitute material. For the preparation of OKGM, the results showed that OKGM obtained using a feeding ratio of 1:1.5 (KGM:NaIO 4 ) possessed an appropriate molecular weight and degree of oxidation, with almost no cytotoxicity. Furthermore, 3.75% OKGM [1:1.5 (KGM:NaIO 4 )] was used as a cross-linking agent to fix DPP to prepare a cardiovascular substitute material and the prepared material exhibited good cross-linking characteristics. Compared to GA-fixed DPP and GNPfixed DPP, this novel 3.75% OKGM-fixed DPP exhibited suitable mechanical strength, excellent HEVC-cytocompatibility, hemocompatibility, anti-calcification capability, and resistance to enzymatic degradation in vitro, as a cardiovascular substitute material. It also exhibits good histocompatibility in vivo. This research provides an experimental basis for OKGM as a new cross-linking reagent for fixing natural tissues featured with rich collagen content and 3.75% OKGM-fixed DPP as a potential cardiovascular substitute material.