Normothermic Ex Vivo Liver Platform Using Porcine Slaughterhouse Livers for Disease Modeling.

Metabolic and toxic liver disorders, such as fatty liver disease (steatosis) and drug-induced liver injury, are highly prevalent and potentially life-threatening. To allow for the study of these disorders from the early stages onward, without using experimental animals, we collected porcine livers in a slaughterhouse and perfused these livers normothermically. With our simplified protocol, the perfused slaughterhouse livers remained viable and functional over five hours of perfusion, as shown by hemodynamics, bile production, indocyanine green clearance, ammonia metabolism, gene expression and histology. As a proof-of-concept to study liver disorders, we show that an infusion of free fatty acids and acetaminophen results in early biochemical signs of liver damage, including reduced functionality. In conclusion, the present platform offers an accessible system to perform research in a functional, relevant large animal model while avoiding using experimental animals. With further improvements to the model, prolonged exposure could make this model a versatile tool for studying liver diseases and potential treatments.

High level of polarized engraftment of porcine intrahepatic cholangiocyte organoids in decellularized liver scaffolds.

In Europe alone, each year 5500 people require a life-saving liver transplantation, but 18% die before receiving one due to the shortage of donor organs. Whole organ engineering, utilizing decellularized liver scaffolds repopulated with autologous cells, is an attractive alternative to increase the pool of available organs for transplantation. The development of this technology is hampered by a lack of a suitable large-animal model representative of the human physiology and a reliable and continuous cell source. We have generated porcine intrahepatic cholangiocyte organoids from adult stem cells and demonstrate that these cultures remained stable over multiple passages whilst retaining the ability to differentiate into hepatocyte- and cholangiocyte-like cells. Recellularization onto porcine scaffolds was efficient and the organoids homogeneously differentiated, even showing polarization. Our porcine intrahepatic cholangiocyte system, combined with porcine liver scaffold paves the way for developing whole liver engineering in a relevant large-animal model.

An ex vivo human osteochondral culture model.

To reduce animal experimentation and to overcome translational issues in cartilage tissue engineering, there is a need to develop an ex vivo human tissue-based approach. This study aims to demonstrate that a human osteochondral explant at different stages of osteoarthritis (OA) can be kept in long-term culture while preserving its viability and composition. Osteochondral explants with either a smooth or fibrillated cartilage surface, representing different OA stages, were harvested from fresh human tibial plateaus. Explants were cultured for 2 or 4 weeks in a double-chamber culture platform. The biochemical content of the cartilage of cultured explants did not significantly change over a period of 4 weeks and these findings were supported by histology. Chondrocytes mostly preserved their metabolic activity during culture and active bone and marrow were found in the periphery of the explants, while metabolic activity was decreased in the bone core in cultured explants compared to fresh explants. In fibrillated explants, chondrocyte viability decreased in the periphery of the sample in cultured groups compared to fresh explants (fresh, 94 ± 6%; cultured, 64% ± 17%, 2 weeks, and 69% ± 17%, 4 weeks; P < .05). Although biochemical and histological results did not show changes within the cartilage tissue, the viability of the explants should be carefully controlled for each specific use. This system provides an alternative to explore drug treatment and implant performance under more controlled experimental conditions than possible in vivo, in combination with clinically relevant human osteochondral tissue.

Cellulose Nanofibril Hydrogel Promotes Hepatic Differentiation of Human Liver Organoids.

To replicate functional liver tissue in vitro for drug testing or transplantation, 3D tissue engineering requires representative cell models as well as scaffolds that not only promote tissue production but also are applicable in a clinical setting. Recently, adult liver-derived liver organoids are found to be of much interest due to their genetic stability, expansion potential, and ability to differentiate toward a hepatocyte-like fate. The current standard for culturing these organoids is a basement membrane hydrogel like Matrigel (MG), which is derived from murine tumor material and apart from its variability and high costs, possesses an undefined composition and is therefore not clinically applicable. Here, a cellulose nanofibril (CNF) hydrogel is investigated with regard to its potential to serve as an alternative clinical grade scaffold to differentiate liver organoids. The results show that its mechanical properties are suitable for differentiation with overall, either equal or improved, functionality of the hepatocyte-like cells compared to MG. Therefore, and because of its defined and tunable chemical definition, the CNF hydrogel presents a viable alternative to MG for liver tissue engineering with the option for clinical use.

Development of an ex vivo aneurysm model for vascular device testing.

An ex vivo aneurysm model that closely resembles the in vivo situation can provide an important tool for testing therapies. The model should mimic a variety of conditions, such as in vivo hemodynamics and native arterial structure and characteristics, avoiding animal experimentation.  Therefore, the aim of this study is to develop an ex vivo aneurysm model by vessel wall stiffening to be used to assess treatment strategies. Porcine carotid arteries from slaughterhouse animals were used to evaluate the acute effect of different concentrations of Rose Bengal on distensibility. This sono-sensitive compound was activated by several ultrasound frequencies, resulting in stiffening of the treated arteries of which the most effective combination was selected. In a pulsatile ex vivo vascular bioreactor treated and control porcine carotid arteries were subjected to physiological conditions for 10 days. During culture, hemodynamics showed increased mean pressure and decreased pulsatility in treated arteries compared to controls. Change in vessel morphology and significant increase of distal diameter was observed in the treated arteries but not in the controls. Histology of treated arteries revealed dissection-like lesions distally and aneurysm-like structure proximally. Finally, a stent graft was deployed in one treated artery and cultured demonstrating the feasibility of testing endovascular devices in the model. In conclusion, we developed an ex vivo model reproducing the onset of aneurysm formation. This could represent a promising tool for early stage device testing thereby reducing the need for animal studies.

Biomedical Applications of Photo- and Sono-Activated Rose Bengal: A Review.

Objective: The aim of this review is to discuss and compare the extensive range of biomedical applications of photo- and sono-activated Rose Bengal (RB). Background data: RB is a xanthene dye that due to its interesting photo- and sono-sensitive properties is gaining attention in the scientific field. Methods: This study is a literature review using the database PubMed. Results: As a photosensitizer, RB converts the triplet oxygen molecule into reactive oxygen species after irradiation with green light (532 nm). This mechanism allows for the use of photo-activated RB in photochemical tissue bonding, photodynamic therapy, antimicrobial therapy and cancer treatment, photothrombotic animal models, and other applications, including tissue engineering and treatment of tauopathies. As a sono-sensitive compound, RB is applied for sonodynamic therapy, cancer treatment, and antimicrobial therapy. Conclusions: This review outlines the versatility and effectiveness of photo- and sono-activated RB in numerous biomedical applications.

Flow-perfusion interferes with chondrogenic and hypertrophic matrix production by mesenchymal stem cells.

Flow-perfusion is being promoted as a way to grow tissue-engineered cartilage in vitro. Yet, there is a concern that flow-perfusion may induce unwanted mechanical effects on chondrogenesis and terminal differentiation. Therefore, the aim of this study is to evaluate the effect of fluid flow on chondrogenesis and chondrocyte hypertrophy of MSCs in a well-established pellet culture model. Human MSC pellets were mounted into 3D-printed porous scaffolds in basic chondrogenic differentiation medium, containing TGF-ß2. Constructs were then allowed to form cartilaginous matrix for 18 days, before they were transferred to a custom-built flow-perfusion system. A continuous flow of 1.22ml min(-1) was applied to the constructs for 10 days. Controls were maintained under static culture conditions. To evaluate chondrogenic and hypertrophic differentiation, RNA was isolated at day 20 and 28 and histology, immunohistochemistry and western blot analyses were performed after 28 days of culture. Abundant matrix was formed in the constructs, but production of chondrogenic and hypertrophic matrix components was affected by flow-perfusion. Although gene expression levels of the (late) hypertrophic and osteogenic marker osteocalcin increased by flow-perfusion, this did not result in more collagen type X protein deposition. Decreased GAG release, in combination with diminished collagen II staining, indicates reduced chondrogenesis in response to flow-perfusion. Caution should thus be taken when applying flow-perfusion to cultures to improve nutrient diffusion. Although we show that it is possible to influence the differentiation of chondrogenic differentiated MSCs by flow-perfusion, effects are inconsistent and strongly donor-dependent.

Sliding indentation enhances collagen content and depth-dependent matrix distribution in tissue-engineered cartilage constructs.

BACKGROUND: Current tissue-engineered cartilage constructs contain insufficient amounts of collagen, whose function is to resist tension. We postulate that dynamic tension is necessary to stimulate collagen formation. Another shortcoming is that tissue-engineered cartilage does not possess native zonal variations. We hypothesize that applying depth-varying mechanical cues would stimulate extracellular matrix (ECM) synthesis depth dependently. We developed a dedicated loading regime called sliding indentation, which enables us to apply dynamic tension as well as depth-varying strain fields to the chondrocyte-seeded agarose constructs. OBJECTIVE: In 2 study designs, we explored whether sliding indentation would increase collagen content and induce depth-varying ECM distribution. METHODS: In the first study, we developed an agarose-sandwich model that involves embedding of a thin chondrocyte-seeded 0.5% agarose layer between two cell-free 3% agarose layers. In the second study, 3-mm-thick chondrocyte-seeded agarose constructs were created. Sliding indentation at 10% depth and 1 Hz was applied to constructs in both studies for 4 h/day during 28 days, and unloaded constructs served as control. RESULTS: Sliding indentation resulted in an increased amount of collagen in the produced cartilage layer. Further, sliding indentation for 7 days resulted in a depth-dependent response at gene expression levels, with the highest response in the regions that received highest strains. Analysis of protein expression after 28 days showed a similar depth-dependent distribution in all constructs, which further enhanced by sliding indentation. CONCLUSIONS: Sliding indentation can increase collagen content and enhances depth-dependent ECM distribution, and is therefore a promising strategy for culturing cartilage with improved properties.

Low agarose concentration and TGF-ß3 distribute extracellular matrix in tissue-engineered cartilage.

BACKGROUND: The mechanical properties of articular cartilage are dominated by the interterritorial matrix, as the matrix in this region is stiffer, greater in volume, and more interconnected compared to that in the pericellular and territorial region. Hence, tissue-engineered constructs in which a newly synthesized matrix accumulates in the pericellular and territorial regions may be of a lower mechanical quality compared to constructs in which the interterritorial region contains abundant matrix. OBJECTIVE: In this study, we explored the extent to which matrix distribution may be modulated by altering the agarose concentration and the presence of the transforming growth factor-ß (TGF-ß) and how this affects the mechanical properties of cultured cartilage constructs. METHODS: Cartilage development in constructs with agarose concentrations varying from 1%, 2%, and 3% (study 1) and in constructs with no or very low agarose concentrations of 0.25%, 0.5%, and 1% (study 2) were compared. In both studies, the effect of TGF-ß3 was compared to fetal bovine serum. After 21 and 42 days of culture, the matrix content and distribution were analyzed and mechanical properties were assessed at day 42. RESULTS: Culture in lower agarose concentrations did not significantly influence the matrix content per wet weight, but did result in a more homogeneous distribution. Constructs cultured with less agarose also showed a higher equilibrium modulus. The presence of TGF-ß3 resulted in an increased extracellular matrix (ECM) deposition, a more homogeneous matrix distribution, and an equilibrium modulus. CONCLUSIONS: Culturing with no or low agarose concentrations and TGF-ß3 is favorable for cartilage tissue-engineering studies, because both stimulate the formation of a more homogeneous ECM and consequently result in improved mechanical properties.

RGD-dependent integrins are mechanotransducers in dynamically compressed tissue-engineered cartilage constructs.

Recent studies have shown that integrins act as mechanoreceptors in articular cartilage. In this study, we examined the effect of blocking RGD-dependent integrins on both ECM gene expression and ECM protein synthesis. Chondrocytes were isolated from full-depth porcine articular cartilage and seeded in 3% agarose constructs. These constructs were loaded in compression with 15% strain at 0.33 and 1 Hz for 12h, in the presence or absence of GRGDSP, which blocks RGD-dependent integrin receptors. The levels of mRNA for aggrecan, collagen II and MMP-3 were determined by semi-quantitative PCR at several time points up to 24h post-stimulation. DNA and sGAG content were determined at several time points up to 28 days post-stimulation. At 0.33 Hz, the mRNA levels for aggrecan and MMP-3 were increased after loading, but the mRNA levels for collagen II remained unchanged. Incubation with GRGDSP counteracted these effects. Loading at 1 Hz led to increased mRNA levels for all three molecules directly after loading and these effects were counteracted by incubation with GRGDSP. The constructs that were loaded at 0.33 Hz showed a lower amount of sGAG, compared to the unstrained control. In contrast, loading at 1 Hz caused an increase in sGAG deposition over the culture period. Blocking integrins had only a counteracting effect on the long-term biosynthetic response of constructs that were compressed at 1 Hz. The results confirmed the role of RGD-dependent integrins as mechanotransducers in the regulation of both ECM gene expression and matrix biosynthesis for chondrocytes seeded in agarose under the applied loading regime. Interestingly, this role seems to be dependent on the applied loading frequency.