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 x 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 x 10(6) 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 porosity 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.

Using a decellularized splenic matrix as a 3D scaffold for hepatocyte cultivation in vitro: a preliminary trial.

Using a decellularized liver matrix (DLM) to reengineer liver tissue is a promising therapy for end-stage liver disease. However, the limited supply of donor organs still hampers its potential clinical application, while a xenogenic decellularized matrix may bring a risk of zoonosis and immunological rejection. Therefore, an appropriate alternative scaffold is needed. In this research, we established a decellularized splenic matrix (DSM) in a rodent model, which preserved the 3D ultrastructure, the components of the extracellular matrix (ECM) and the native vascular network. The DSM and DLM had similar components of ECM, and similar mechanical properties. Hepatocytes were seeded to the DSM and DLM for dynamic culturing up to 6 d, and distributed both in decellularized sinusoidal spaces and around the vessels. The TUNEL-positive cell percentage in a dynamic culturing decellularized splenic matrix (dDSM) was 10.7% ± 3.6% at 3d and 25.8% ± 5.6% at 5d, although 14.2% ± 4.5% and 24.8% ± 2.9%, respectively, in a dynamic culturing decellularized liver matrix (dDLM) at the same time point (p > 0.05). Primary hepatocytes in the dDSM and dDLM expressed albumin, G6pc and Ugt1a1. The gene expression of Cyp2b1, Cyp1a2 and HNF1α in the gene transcription level revealed hepatocytes had lower gene expression levels in the dDSM compared with the dDLM at 3d, but better than those in a sandwich culture. The cumulative albumin production at 6 d of culture was 80.7 ± 9.6 µg per million cells in the dDSM and 89.6 ± 4.6 µg per million cells in the dDLM (p > 0.05). In summary, the DSM is a promising 3D scaffold for hepatocyte cultivation in vitro.