Liver regeneration using decellularized splenic scaffold: a novel approach in tissue engineering

BACKGROUND： The potential application of decellularized liver scaffold for liver regeneration is limited by severe shortage of donor organs. Attempt of using heterograft scaffold is accompanied with high risks of zoonosis and immunological rejection. We proposed that the spleen, which procured more extensively than the liver, could be an ideal source of decellularized scaffold for liver regeneration. METHODS： After harvested from donor rat, the spleen was processed by 12-hour freezing/thawing ×2 cycles, then circulation perfusion of 0.02% trypsin and 3% Triton X-100 sequentially through the splenic artery for 32 hours in total to prepare decellularized scaffold. The structure and component characteristics of the scaffold were determined by hematoxylin and eosin and immumohistochemical staining, scanning electron microscope, DNA detection, porosity measurement, biocompatibility and cytocompatibility test. Recellularization of scaffold by 5×106 bone marrow mesenchymal stem cells（BMSCs） was carried out to preliminarily evaluate the feasibility of liver regeneration by BMSCs reseeding and differentiation in decellularized splenic scaffold.RESULTS： After decellularization, a translucent scaffold, which retained the gross shape of the spleen, was generated. Histological evaluation and residual DNA quantitation revealed the remaining of extracellular matrix without nucleus and cytoplasm residue. Immunohistochemical study proved the existence of collagens I, IV, fibronectin, laminin and elastin in decellularized splenic scaffold, which showed a similarity with decellularized liver. A scanning electron microscope presented the remaining three-dimensional porous structure of extracellular matrix and small blood vessels. The poros-ity of scaffold, aperture of 45.36±4.87 μm and pore rate of 80.14%±2.99% was suitable for cell engraftment. Subcutaneous implantation of decellularized scaffold presented good histocompatibility, and recellularization of the splenic scaffold demonstrated that BMSCs could locate and survive in the decellularized matrix. CONCLUSION： Considering the more extensive organ source and satisfying biocompatibility, the present study indicated that the three-dimensional decellularized splenic scaffold might have considerable potential for liver regeneration when combined with BMSCs reseeding and differentiation.

Green Electrospun Silk Fibroin/Galactose Chitosan Composite Nanofibrous Scaffolds for Hepatic Tissue Engineering

The electrospun nanofibrous scaffolds made of proteins and polysaccharides were thought to be able to simulate the structure of natural extracellular matrix well.Silk fibroin(SF)and chitosan(CS)are probably the most widely used natural materials in biomedical fields including liver tissue engineering for their good properties and wide variety of sources.The asialoglycoprotein receptors of hepatocyte were reported to specifically recognize and interact with galactose.In this work,a green electrospun SF/galactosylated chitosan(GC)composite nanofibrous scaffold was fabricated and characterized.The data indicated that the addition of GC greatly influenced the spinning effect of SF aqueous solution,and the average diameter of the composite nanofibers was about 520nm.Moreover,the green electrospun SF/GC nanofibrous scaffolds were demonstrated significantly enhancing the adhesion and proliferation of hepatocyte(RH35)according to our data.The present study did a useful exploration on constructing scaffolds for liver regeneration by green electrospinning,and also laid a good foundation for the further applicative research of this green electrospun scaffolds in liver tissue engineering.

Polyethylene glycol crosslinked decellularized single liver lobe scaffolds with vascular endothelial growth factor promotes angiogenesis in vivo

Background: Improving the mechanical properties and angiogenesis of acellular scaffolds before transplantation is an important challenge facing the development of acellular liver grafts. The present study aimed to evaluate the cytotoxicity and angiogenesis of polyethylene glycol(PEG) crosslinked decellularized single liver lobe scaffolds(DLSs), and establish its suitability as a graft for long-term liver tissue engineering. Methods: Using mercaptoacrylate produced by the Michael addition reaction, DLSs were first modified using N-succinimidyl S-acetylthioacetate(SATA), followed by cross-linking with PEG as well as vascular endothelial growth factor(VEGF). The optimal concentration of agents and time of the individual steps were identified in this procedure through biomechanical testing and morphological analysis. Subsequently, human umbilical vein endothelial cells(HUVECs) were seeded on the PEG crosslinked scaffolds to detect the proliferation and viability of cells. The scaffolds were then transplanted into the subcutaneous tissue of Sprague-Dawley rats to evaluate angiogenesis. In addition, the average number of blood vessels was evaluated in the grafts with or without PEG at days 7, 14, and 21 after implantation. Results: The PEG crosslinked DLS maintained their three-dimensional structure and were more translucent after decellularization than native DLS, which presented a denser and more porous network structure. The results for Young’s modulus proved that the mechanical properties of 0.5 PEG crosslinked DLS were the best and close to that of native livers. The PEG-VEGF-DLS could better promote cell proliferation and differentiation of HUVECs compared with the groups without PEG cross-linking. Importantly, the average density of blood vessels was higher in the PEG-VEGF-DLS than that in other groups at days 7, 14, and 21 after implantation in vivo. Conclusions: The PEG crosslinked DLS with VEGF could improve the biomechanical properties of native DLS, and most importantly, their lack of cytotoxicity provides a new route to promote the proliferation of cells in vitro and angiogenesis in vivo in liver tissue engineering.

Long-term functional maintenance of primary hepatocytes in vitro using macroporous hydrogels engineered through liquid-liquid phase separation

Preserving the functionality of hepatocytes in vitro poses a significant challenge in liver tissue engineering and bioartificial liver,as these cells rapidly lose their metabolic and functional characteristics after isolation.Inspired by the macroporous structures found in native liver tissues,here we develop synthetic hydrogel scaffolds that closely mimic the liver’s structural organization through the phase separation between polyethylene glycol(PEG)and polysaccharides.Our hydrogels exhibit interconnected macroporous structures and appropriate mechanical properties,providing an optimal microenvironment conducive to hepatocyte adhesion and the formation of sizable aggregates.Compared to two-dimensional hepatocyte cultures,enhanced functionalities of hepatocytes cultured in our macroporous hydrogels were observed for 14 days,as evidenced by quantitative reverse-transcription–polymerase chain reactions(qRT-PCR),immunofluorescence,and enzyme linked immunosorbent assay(ELISA)analyses.Protein sequencing data further confirmed the establishment of cell–cell interactions among hepatocytes when cultured in our hydrogels.Notably,these hepatocytes maintained a protein expression lineage that closely resembled freshly isolated hepatocytes,particularly in the Notch and tumor necrosis factor(TNF)signaling pathways.These results suggest that the macroporous hydrogels are attractive scaffolds for liver tissue engineering.