Method for perfusion decellularization of porcine whole liver and kidney for use as a scaffold for clinical-scale bioengineering engrafts.

BACKGROUND: Whole-organ engineering provides a new alternative source of donor organs for xenotransplantation. Utilization of decellularized whole-organ scaffolds, which can be created by detergent perfusion, is a strategy for tissue engineering. In this article, our aim is to scale up the decellularization process to human-sized liver and kidney to generate a decellularized matrix with optimal and stable characteristics on a clinically relevant scale. METHODS: Whole porcine liver and kidney were decellularized by perfusion using different detergents (1% SDS, 1% Triton X-100, 1% peracetic acid (PAA), and 1% NaDOC) via the portal vein and renal artery of the liver and kidney, respectively. After rinsing with PBS to remove the detergents, the obtained liver and kidney extracellular matrix (ECM) were processed for histology, residual cellular content analysis, and ECM components evaluation to investigate decellularization efficiency, xenoantigens removal, and ECM preservation. RESULTS: The resulting liver and kidney scaffolds in the SDS-treated group showed the most efficient clearance of cellular components and xenoantigens, including DNA and protein, and preservation of the extracellular matrix composition. In comparison, cell debris was observed in the other decellularized groups that were generated using Triton X-100, PAA, and NaDOC. Special staining and immunochemistry of the porcine liver and kidney ECMs further confirmed the disrupted three-dimension ultrastructure of the ECM in the Triton X-100 and NaDOC groups. Additionally, Triton X-100 effectively eliminated the residual SDS in the SDS-treated group, which ensured the scaffolds were not cytotoxic to cells. Thus, we have developed an optimal method that can be scaled up for use with other solid whole organs. CONCLUSIONS: Our SDS-perfusion protocol can be used for porcine liver and kidney decellularization to obtain organ scaffolds cleared of cellular material, xenoimmunogens, and preserved vital ECM components.

[Anticoagulant Ability and Heparinization of Decellularized Biomaterial Scaffolds].

In order to enhance the anticoagulant properties of decellularized biological materials as scaffolds for tissue engineering research via heparinized process, the decellularized porcine liver scaffolds were respectively immobilized with heparin through layer-by-layer self-assembly technique (LBL), multi-point attachment (MPA) or end-point attachment (EPA). The effects of heparinization and anticoagulant ability were tested. The results showed that the three different scaffolds had different contents of heparin. All the three kinds of heparinized scaffolds gained better performance of anticoagulant than that of the control scaffold. The thrombin time (TT), prothrombin time (PT) and activated partial thromboplastin time (APTT) of EPA scaffold group were longest in all the groups, and all the three times exceeded the measurement limit of the instrument. In addition, EPA scaffolds group showed the shortest prepared time, the slowest speed for heparin release and the longest recalcification time among all the groups. The decellularized biological materials for tissue engineering acquire the best effect of anticoagulant ability in vitro via EPA heparinized technique.

PTFE-covered TIPS is an effective treatment for secondary preventing variceal rebleeding in cirrhotic patients with high risks.

AIM: To compare the effectiveness and safety of transjugular intrahepatic portosystemic shunt (TIPS) with endoscopic therapy plus non-selective ß-blockers (NSBBs) for secondary prevention of gasroesophageal variceal bleeding (GEVB) in cirrhotic patients with high-risk factors of treatment failure. METHODS AND MATERIAL: A total of 122 cirrhotic patients with history of gasroesophageal variceal bleeding and high factors including hepatic vein pressure gradient (HVPG) ≥ 20 mmHg, portal vein thrombosis (PVT), gastrorenal shunt (GRS), or extraluminal para-gastric veins (ep-GVs) detected by endoscopic ultrasound, were analyzed retrospectively. Seventy-seven patients underwent TIPS with PTFE-covered stent (group A) and 102 patients received endoscopic therapy combined with nonselective ß-blockers (NSBBs) (group B). According to above high-risk factors, both groups were stratified into four paired subgroups (A1-A4 and B1-B4). Two-year rebleeding rate, overt hepatic encephalopathy, overall survival, and procedure-related adverse events were compared between the two groups and paired subgroups. RESULTS: The 2-year cumulative probability of free of variceal rebleeding was higher in group A than group B (93 vs. 62%, P < 0.001). Similarly, the 2-year cumulative probability of free of variceal rebleeding was also higher in the subgroups A1-A4 than the subgroups B1-B4 (91 vs. 67%, P = 0.022, 90 vs. 67%, P = 0.021, 94 vs. 59%, P = 0.029, and 90 vs. 58%, P = 0.016, respectively). There was no significant difference between the two groups and corresponding subgroups in overt hepatic encephalopathy and survival. CONCLUSION: Compared to secondary prophylaxis with endoscopic therapy plus NSBBs, polytetrafluoroethylene-covered TIPS could significantly reduce the variceal rebleeding rate in cirrhotic patients with HVPG ≥ 20 mmHg, PVT, GRS, or ep-GVs, without increasing the incidence of hepatic encephalopathy.

Microplastics in the surface water of small-scale estuaries in Shanghai.

This study involved an assessment of the levels of microplastic pollution in seven small-scale estuaries in Shanghai for the first time. The abundance of microplastics ranged from 13.53 ±â€¯4.6 to 44.93 ±â€¯9.41 particles L-1, with a mean abundance of 27.84 ±â€¯11.81 particles L-1. Microplastics collected from samples were classified into four types (fiber, film, granule, and fragment), and granules were the most abundant type. Up to 99.5% of microplastics were <2 mm in diameter. The microplastics had a variety of colors, with black being the dominant color. Polypropylene (37.5%) and polyethylene (50%) were the main types of microplastic component validated. Our study showed severe microplastic pollution in small-scale estuaries, and the associated rivers need urgent attention for microplastic pollution prevention.

Genipin crosslinking reduced the immunogenicity of xenogeneic decellularized porcine whole-liver matrices through regulation of immune cell proliferation and polarization.

Decellularized xenogeneic whole-liver matrices are plausible biomedical materials for the bioengineering of liver transplantation. A common method to reduce the inflammatory potential of xenogeneic matrices is crosslinking. Nevertheless, a comprehensive analysis of the immunogenic features of cross-linked decellularized tissue is still lacking. We aimed to reduce the immunogenicity of decellularized porcine whole-liver matrix through crosslinking with glutaraldehyde or genipin, a new natural agent, and investigated the mechanism of the immune-mediated responses. The histologic assessment of the host's immune reaction activated in response to these scaffolds, as well as the M1/M2 phenotypic polarization profile of macrophages, was studied in vivo. The genipin-fixed scaffold elicited a predominantly M2 phenotype response, while the glutaraldehyde-fixed scaffold resulted in disrupted host tissue remodeling and a mixed macrophage polarization profile. The specific subsets of immune cells involved in the responses to the scaffolds were identified in vitro. Crosslinking alleviated the host response by reducing the proliferation of lymphocytes and their subsets, accompanied by a decreased release of both Th1 and Th2 cytokines. Therefore, we conclude that the natural genipin crosslinking could lower the immunogenic potential of xenogeneic decellularized whole-liver scaffolds.