Organ and tissue shortage are known as a crucially important public health problem as unfortunately a small percentage of patients receive transplants. In the context of emerging regenerative medicine, researchers are trying to regenerate and replace different organs and tissues such as the liver, heart, skin, and kidney. Liver tissue engineering (TE) enables us to reproduce and restore liver functions, fully or partially, which could be used in the treatment of acute or chronic liver disorders and/or generate an appropriate functional organ which can be transplanted or employed as an extracorporeal device. In this regard, a variety of techniques (e.g., fabrication technologies, cell-based technologies, microfluidic systems and, extracorporeal liver devices) could be applied in tissue engineering in liver regenerative medicine. Common TE techniques are based on allocating stem cell-derived hepatocyte-like cells or primary hepatocytes within a three-dimensional structure which leads to the improvement of their survival rate and functional phenotype. Taken together, new findings indicated that developing liver tissue engineering-based techniques could pave the way for better treatment of liver-related disorders. Herein, we summarized novel technologies used in liver regenerative medicine and their future applications in clinical settings.

Almost all patients with chronic liver diseases (CLD) show altered bone metabolism. Depending on the etiology, this manifests in a severe osteoporosis in up to 75% of the affected patients. Due to high prevalence, the generic term hepatic osteodystrophy (HOD) evolved, describing altered bone metabolism, decreased bone mineral density, and deterioration of bone structure in patients with CLD. Once developed, HOD is difficult to treat and increases the risk of fragility fractures. Existing fractures affect the quality of life and, more importantly, long-term prognosis of these patients, which presents with increased mortality. Thus, special care is required to support the healing process. However, for early diagnosis (reduce fracture risk) and development of adequate treatment strategies (support healing of existing fractures), it is essential to understand the underlying mechanisms that link disturbed liver function with this bone phenotype. In the present review, we summarize proposed molecular mechanisms favoring the development of HOD and compromising the healing of associated fractures, including alterations in vitamin D metabolism and action, disbalances in transforming growth factor beta (TGF-β) and bone morphogenetic protein (BMP) signaling with histone deacetylases (HDACs) as secondary regulators, as well as alterations in the receptor activator of nuclear factor kappa B ligand (RANKL)–osteoprotegerin (OPG) system mediated by sclerostin. Based on these mechanisms, we give an overview on the limitations of early diagnosis of HOD with established serum markers.

5-Azacytidine (5-AZA), a DNA methyl transferase inhibitor, is a clinically used epigenetic drug for cancer therapy. Recently, we have shown that 5-AZA upregulates ten-eleven translocation (TET) protein expression in hepatocellular carcinoma (HCC) cells, which induce active demethylation. Vitamin C facilitates TET activity and enhances active demethylation. The aim of this study is to investigate whether vitamin C is able to enhance the effect of 5-AZA on active demethylation and to evaluate its consequence in HCC cell lines. HCC cell lines (Huh7 and HLE) were treated with 5-AZA and vitamin C. After 48 h of treatment, viability (resazurin conversion), toxicity (lactose dehydrogenase (LDH) release), and proliferation ((proliferating cell nuclear antigen (PCNA)) of single- and combined-treated cells were assessed. The effect of the treatment on 5-hydroxymethylcytosine (5hmC) intensity (immunofluorescence (IF) staining), TET, Snail, GADD45B, and P21 mRNA (real-time PCR) and protein expression (Western blot) were investigated. Our results indicated that vitamin C enhances the anti-proliferative and apoptotic effect of 5-AZA in HCC cell lines. By further analyzing the events leading to cell cycle arrest, we have shown for the first time in HCC that the combination of 5-AZA and vitamin C leads to an enhanced downregulation of Snail expression, a key transcription factor governing epithelial-mesenchymal transition (EMT) process, and cell cycle arrest. We conclude that when combined with 5-AZA, vitamin C enhances TET activity in HCC cells, leading to induction of active demethylation. An increase in P21 expression as a consequence of downregulation of Snail accompanied by the induction of GADD45B expression is the main mechanism leading to cell cycle arrest in HCCs.

Bone tissue undergoes constant remodeling and healing when fracture happens, in order to ensure its structural integrity. In order to better understand open biological and clinical questions linked to various bone diseases, bone cell co-culture technology is believed to shed some light into the dark. Osteoblasts/osteocytes and osteoclasts dominate the metabolism of bone by a multitude of connections. Therefore, it is widely accepted that a constant improvement of co-culture models with both cell types cultured on a 3D scaffold, is aimed to mimic an in vivo environment as closely as possible. Although in recent years a considerable knowledge of bone co-culture models has been accumulated, there are still many open questions. We here try to summarize the actual knowledge and address open questions.

Liver organoids (LOs) are receiving considerable attention for their potential use in drug screening, disease modeling, and transplantable constructs. Hepatocytes, as the key component of LOs, are isolated from the liver or differentiated from pluripotent stem cells (PSCs). PSC-derived hepatocytes are preferable because of their availability and scalability. However, efficient maturation of the PSC-derived hepatocytes towards functional units in LOs remains a challenging subject. The incorporation of cell-sized microparticles (MPs) derived from liver extracellular matrix (ECM), could provide an enriched tissue-specific microenvironment for further maturation of hepatocytes inside the LOs. In the present study, the MPs were fabricated by chemical cross-linking of a water-in-oil dispersion of digested decellularized sheep liver. These MPs were mixed with human PSC-derived hepatic endoderm, human umbilical vein endothelial cells, and mesenchymal stromal cells to produce homogenous bioengineered LOs (BLOs). This approach led to the improvement of hepatocyte-like cells in terms of gene expression and function, CYP activities, albumin secretion, and metabolism of xenobiotics. The intraperitoneal transplantation of BLOs in an acute liver injury mouse model led to an enhancement in survival rate. Furthermore, efficient hepatic maturation was demonstrated after ex ovo transplantation. In conclusion, the incorporation of cell-sized tissue-specific MPs in BLOs improved the maturation of human PSC-derived hepatocyte-like cells compared to LOs. This approach provides a versatile strategy to produce functional organoids from different tissues and offers a novel tool for biomedical applications.