Ji Bao

Construction of a portal implantable functional tissue-engineered liver using perfusion-decellularized matrix and hepatocytes in rats.

Innovative cell-based therapies, including hepatic tissue engineering following hepatocyte transplantation, are considered as theoretical alternatives to liver transplant or for partial replacement of liver function in patients. However, recent progress in hepatic tissue engineering has been hampered by low initial hepatocyte engraftment and insufficient blood supply in vivo. We developed an intact 3D scaffold of an extracellular matrix (ECM) derived from a decellularized liver lobe, with layer-by-layer (LbL) heparin deposition to avoid thrombosis, which we repopulated with hepatocytes and successfully implanted as a tissue-engineered liver (TEL) into the portal system. The TEL provided sufficient volume for transplantation of cell numbers representing up to 10% of whole-liver equivalents and was perfused by portal vein blood. Treatment of extended hepatectomized rats with a TEL improved liver function and prolonged survival; mean lifespan was extended from 16 to 72 h. At 72 h postoperation, the TEL sustained functional and viable hepatocytes. In conclusion, we propose the TEL as a state-of-the-art substitute for whole-liver transplantation and as a proof of concept for the technology that will eventually allow for the transplantation of a reconstituted liver.

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

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.

The Upregulation of Osteoblast Marker Genes in Mesenchymal Stem Cells Prove the Osteoinductivity of Hydroxyapatite/Tricalcium Phosphate Biomaterial

BACKGROUND Calcium phosphate (Ca-P), mainly concerning hydroxyapatite (HA), is the main inorganic component of the bodys hard tissue. It is acknowledged that Ca-P biomaterial not only has osteoconduction but also can form bone bonding to host bone, making an ideal tissue-engineering scaffold. However, whether Ca-P biomaterial possesses osteoinductivity is still debated. The present study was performed to explore the expression level of osteoblast maker genes in human mesenchymal stem cells (hMSCs) grown on a Ca-P biomaterial. MATERIALS AND METHODS hMSCs were cultured on the HA/tricalcium phosphate-(TCP) double-phase ceramic. After coculture for 5, 10, 15, or 20 days, the cells were digested for isolation of total RNA. Fluorescence quantitative polymerase chain reaction was used to detect the relative mRNA levels of Runx2, collagen type I, osteopontin, and osteocalcin, all of which are the marker genes for osteoblasts. The mg63 cell was recruited as the reference and un-cocultured hMSCs as the negative controls. Alkaline phosphatase (ALP) activity in the cells was also examined after culture for 10 or 20 days. RESULTS Our results showed that the expression levels of all four genes continued to rise during the first 10 days. Then, both collagen type I and Runx2 decreased. In contrast, osteocalcin mRNA reached its maximum at day 15 and osteopontin mRNA kept increasing throughout the whole experimental period. Additionally, ALP activity increased in a time-dependent manner. CONCLUSION The up-regulation of all four osteoblast marker genes in hMSCs grown on Ca-P biomaterial suggested that HA/TCP biomaterials possess osteoinductivity on hMSCs, cells a mechanism that requires further investigation.

Three-dimensional spheroid culture of human umbilical cord mesenchymal stem cells promotes cell yield and stemness maintenance

Mesenchymal stem cell (MSC) transplantation is a promising treatment of many diseases. However, conventional techniques with cells being cultured as a monolayer result in slow cell proliferation and insufficient yield to meet clinical demands. Three-dimensional (3D) culture systems are gaining attention with regard to recreating a complex microenvironment and to understanding the conditions experienced by cells. Our aim is to establish a novel 3D system for the culture of human umbilical cord MSCs (hUC-MSCs) within a real 3D microenvironment but with no digestion or passaging. Primary hUC-MSCs were isolated and grown in serum-free medium (SFM) on a suspension Rocker system. Cell characteristics including proliferation, phenotype and multipotency were recorded. The therapeutic effects of 3D-cultured hUC-MSCs on carbon tetrachloride (CCl4)-induced acute liver failure in mouse models were examined. In the 3D Rocker system, hUC-MSCs formed spheroids in SFM and maintained high viability and active proliferation. Compared with monolayer culture, the 3D-culture system yielded more hUC-MSCs cells within the same volume. The spheroids expressed higher levels of stem cell markers and displayed stronger multipotency. After transplantation into mouse, 3D hUC-MSCs significantly promoted the secretion of interferon-γ and interleukin-6 but inhibited that of tumor necrosis factor-α, thereby alleviating liver necrosis and promoting regeneration following CCl4 injury. The 3D culture of hUC-MSCs thus promotes cell yield and stemness maintenance and represents a promising strategy for hUC-MSCs expansion on an industrial scale with great potential for cell therapy and biotechnology.

Hemocompatibility improvement of perfusion-decellularized clinical-scale liver scaffold through heparin immobilization.

Whole-liver perfusion-decellularization is an attractive scaffold–preparation technique for producing clinical transplantable liver tissue. However, the scaffold’s poor hemocompatibility poses a major obstacle. This study was intended to improve the hemocompatibility of perfusion-decellularized porcine liver scaffold via immobilization of heparin. Heparin was immobilized on decellularized liver scaffolds (DLSs) by electrostatic binding using a layer-by-layer self-assembly technique (/h-LBL scaffold), covalent binding via multi-point attachment (/h-MPA scaffold), or end-point attachment (/h-EPA scaffold). The effect of heparinization on anticoagulant ability and cytocompatibility were investigated. The result of heparin content and release tests revealed EPA technique performed higher efficiency of heparin immobilization than other two methods. Then, systematic in vitro investigation of prothrombin time (PT), thrombin time (TT), activated partial thromboplastin time (APTT), platelet adhesion and human platelet factor 4 (PF4, indicates platelet activation) confirmed the heparinized scaffolds, especially the /h-EPA counterparts, exhibited ultralow blood component activations and excellent hemocompatibility. Furthermore, heparin treatments prevented thrombosis successfully in DLSs with blood perfusion after implanted in vivo. Meanwhile, after heparin processes, both primary hepatocyte and endothelial cell viability were also well-maintained, which indicated that heparin treatments with improved biocompatibility might extend to various hemoperfusable whole-organ scaffolds’ preparation.

Osteogenic differentiation of mesenchymal stem cells promoted by overexpression of connective tissue growth factor

ObjectiveLarge segmental bone defect repair remains a clinical and scientific challenge with increasing interest focusing on combining gene transfection with tissue engineering techniques. The aim of this study is to investigate the effect of connective tissue growth factor (CTGF) on the proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells (MSCs).MethodsA CTGF-expressing plasmid (pCTGF) was constructed and transfected into MSCs. Then expressions of bone morphogenesis-related genes, proliferation rate, alkaline phosphatase activity, and mineralization were examined to evaluate the osteogenic potential of the CTGF gene-modified MSCs.ResultsOverexpression of CTGF was confirmed in pCTGF-MSCs. pCTGF transfection significantly enhanced the proliferation rates of pCTGF-MSCs (P<0.05). CTGF induced a 7.5-fold increase in cell migration over control (P<0.05). pCTGF transfection enhanced the expression of bone matrix proteins, such as bone sialoprotein, osteocalcin, and collagen type I in MSCs. The levels of alkaline phosphatase (ALP) activities of pCTGF-MSCs at the 1st and 2nd weeks were 4.0- and 3.0-fold higher than those of MSCs cultured in OS-medium, significantly higher than those of mock-MSCs and normal control MSCs (P<0.05). Overexpression of CTGF in MSCs enhanced the capability to form mineralized nodules.ConclusionOverexpression of CTGF could improve the osteogenic differentiation ability of MSCs, and the CTGF gene-modified MSCs are potential as novel cell resources of bone tissue engineering.

Optimizing perfusion-decellularization methods of porcine livers for clinical-scale whole-organ bioengineering.

Aim. To refine the decellularization protocol of whole porcine liver, which holds great promise for liver tissue engineering. Methods. Three decellularization methods for porcine livers (1% sodium dodecyl sulfate (SDS), 1% Triton X-100 + 1% sodium dodecyl sulfate, and 1% sodium deoxycholate + 1% sodium dodecyl sulfate) were studied. The obtained liver scaffolds were processed for histology, residual cellular content analysis, and extracellular matrix (ECM) components evaluation to investigate decellularization efficiency and ECM preservation. Rat primary hepatocytes were seeded into three kinds of scaffold to detect the biocompatibility. Results. The whole liver decellularization was successfully achieved following all three kinds of treatment. SDS combined with Triton had a high efficacy of cellular removal and caused minimal disruption of essential ECM components; it was also the most biocompatible procedure for primary hepatocytes. Conclusion. We have refined a novel, standardized, time-efficient, and reproducible protocol for the decellularization of whole liver which can be further adapted to liver tissue engineering.

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.

Urinary connective tissue growth factor increases far earlier than histopathological damage and functional deterioration in early chronic renal allograft injury

Objective. To date, serum biochemistry examination and routine biopsy are the most commonly used methods to assess renal function after allogenic kidney transplantation. Connective tissue growth factor (CTGF) has been considered as a biomarker of chronic renal allograft injury characterized by tubular atrophy and interstitial fibrosis (TA/IF). This study explored the potential value of urinary CTGF as an early predictor of TA/IF using a rat model. Material and methods. A Fisher to Lewis allogenic rat kidney transplant model was established and the recipients were killed at weeks 4, 8 and 12 post-transplantation. TA/IF was graded based on Banff Schema 1997. The location and expression of CTGF mRNA were detected by oligonucleotide-primed in situ DNA synthesis and quantitative polymerase chain reaction. CTGF protein expression was examined with immunohistochemistry and immunoblotting. Urinary CTGF concentration was measured by enzyme-linked immunosorbent assay. The correlation between urinary CTGF concentration and serum creatinine (SCr) and Banff score was analysed statistically. Results. Typical morphological changes including TA/IF in allograft appeared at week 8 and became very severe at week 12 post-transplantation. CTGF expression in epithelium was up-regulated early and urinary CTGF was markedly elevated from week 4. SCr in recipients was stable before week 8 but increased tremendously at week 12. Urinary CTGF concentration was positively correlated with SCr and degree of interstitial fibrosis. Conclusion. Urinary CTGF increases earlier than the appearance of biochemical abnormalities and pathological changes. Measurement of urinary CTGF may offer a potential non-invasive strategy to predict the early onset of chronic renal allograft injury.

Loss of histone deacetylases 1 and 2 in hepatocytes impairs murine liver regeneration through Ki67 depletion

Histone deacetylases 1 and 2 (HDAC1 and HDAC2) are ubiquitously expressed in tissues, including the liver, and play critical roles in numerous physiopathological processes. Little is known regarding the role of HDAC1 and HDAC2 in liver regeneration. In this study we generated mice in which Hdac1, Hdac2 or both genes were selectively knocked out in hepatocytes to investigate the role of these genes in liver regeneration following hepatic injury induced by partial hepatectomy or carbon tetrachloride administration. The loss of HDAC1 and/or HDAC2 (HDAC1/2) protein resulted in impaired liver regeneration. HDAC1/2 inactivation did not decrease hepatocytic 5‐bromo‐2‐deoxyuridine uptake or the expression of proliferating cell nuclear antigen, cyclins, or cyclin‐dependent kinases. However, the levels of Ki67, a mitotic marker that is expressed from the mid‐G1 phase to the end of mitosis and is closely involved in the regulation of mitotic progression, were greatly decreased, and abnormal mitosis lacking Ki67 expression was frequently observed in HDAC1/2‐deficient livers. The down‐regulation of either HDAC1/2 or Ki67 in the mouse liver cancer cell line Hepa1‐6 resulted in similar mitotic defects. Finally, both HDAC1 and HDAC2 proteins were associated with the Ki67 gene mediated by CCAAT/enhancer‐binding protein β. Conclusion: Both HDAC1 and HDAC2 play crucial roles in the regulation of liver regeneration. The loss of HDAC1/2 inhibits Ki67 expression and results in defective hepatocyte mitosis and impaired liver regeneration. (Hepatology 2013; 58:2089–2098)