Search

To quantify and visualize both bone formation and resorption within osteochondral explants cultured ex vivo is challenging with the current analysis techniques. An approach that enables monitoring of bone remodeling dynamics is longitudinal microcomputed tomography (µCT), a non‐destructive technique that relies on repeated µCT scanning and subsequent registration of consecutive scans. In this study, a two‐compartment culture system suitable for osteochondral explants that allowed for µCT scanning during ex vivo culture was established. Explants were scanned repeatedly in a fixed orientation, which allowed assessment of bone remodeling due to adequate image registration. Using this method, bone formation was found to be restricted to the outer surfaces when cultured statically. To demonstrate that the culture system could capture differences in bone remodeling, explants were cultured statically and under dynamic compression as loading promotes osteogenesis. No quantitative differences between static and dynamic culture were revealed. Still, only in dynamic conditions, bone formation was visualized on trabecular surfaces located within the inner cores, suggesting enhanced bone formation towards the center of the explants upon mechanical loading. Taken together, the ex vivo culture system in combination with longitudinal µCT scanning and subsequent registration of images demonstrated potential for evaluating bone remodeling within explants. [ABSTRACT FROM AUTHOR] more...

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. [ABSTRACT FROM AUTHOR] more...

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. [ABSTRACT FROM AUTHOR] more...

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. [ABSTRACT FROM AUTHOR] more...

An ex vivo aneurysm model that closely resembles the in vivo situation can provide an important tool to test therapeutic approaches while avoiding animal experimentation. The model should mimic a variety of conditions, such as in vivo hemodynamics and native arterial structure and characteristics. The aim of this study was to develop an ex vivo aneurysm model by stiffening vessel walls 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 vessel distensibility. Rose Bengal is a sono-sensitive compound that is activated by several ultrasound frequencies, resulting in stiffening of the treated arteries; the most effective combination of concentration and frequency was determined. In a pulsatile ex vivo vascular bioreactor, treated and control porcine carotid arteries were subjected to physiological conditions for 10 days. Treated arteries showed increased mean pressure and decreased pulsatility compared to controls. Changes in vessel morphology and a significant increase of distal diameter were observed in the treated arteries but not in the controls. Histology revealed distal dissection-like lesions and proximal aneurysm-like structure in the treated arteries. Finally, a stent graft was deployed and cultured in one treated artery, 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 represents a promising tool for early stage device testing, thereby reducing the need for animal studies. [ABSTRACT FROM AUTHOR] more...

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. more...

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-β (TGF-β) 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-β3 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-β3 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-β3 is favorable for cartilage tissue-engineering studies, because both stimulate the formation of a more homogeneous ECM and consequently result in improved mechanical properties. more...