New Endeavors of (Micro)Tissue Engineering: Cells Tissues Organs on-Chip and Communication Thereof.

The development of new therapies is tremendously hampered by the insufficient availability of human model systems suitable for preclinical research on disease target identification, drug efficacy, and toxicity. Thus, drug failures in clinical trials are too common and too costly. Animal models or standard 2D in vitro tissue cultures, regardless of whether they are human based, are regularly not representative of specific human responses. Approaching near human tissues and organs test systems is the key goal of organs-on-chips (OoC) technology. This technology is currently showing its potential to reduce both drug development costs and time-to-market, while critically lessening animal testing. OoC are based on human (stem) cells, potentially derived from healthy or disease-affected patients, thereby amenable to personalized therapy development. It is noteworthy that the OoC market potential goes beyond pharma, with the possibility to test cosmetics, food additives, or environmental contaminants. This (micro)tissue engineering-based technology is highly multidisciplinary, combining fields such as (developmental) biology, (bio)materials, microfluidics, sensors, and imaging. The enormous potential of OoC is currently facing an exciting new challenge: emulating cross-communication between tissues and organs, to simulate more complex systemic responses, such as in cancer, or restricted to confined environments, as occurs in osteoarthritis. This review describes key examples of multiorgan/tissue-on-chip approaches, or linked organs/tissues-on-chip, focusing on challenges and promising new avenues of this advanced model system. Additionally, major emphasis is given to the translation of established tissue engineering approaches, bottom up and top down, towards the development of more complex, robust, and representative (multi)organ/tissue-on-chip approaches.

Advancing Tumor Microenvironment Research by Combining Organs-on-Chips and Biosensors.

Organs-on-chips are microfluidic tissue-engineered models that offer unprecedented dynamic control over cellular microenvironments, emulating key functional features of organs or tissues. Sensing technologies are increasingly becoming an essential part of such advanced model systems for real-time detection of cellular behavior and systemic-like events. The fast-developing field of organs-on-chips is accelerating the development of biosensors toward easier integration, thus smaller and less invasive, leading to enhanced access and detection of (patho-) physiological biomarkers. The outstanding combination of organs-on-chips and biosensors holds the promise to contribute to more effective treatments, and, importantly, improve the ability to detect and monitor several diseases at an earlier stage, which is particularly relevant for complex diseases such as cancer. Biosensors coupled with organs-on-chips are currently being devised not only to determine therapy effectiveness but also to identify emerging cancer biomarkers and targets. The ever-expanding use of imaging modalities for optical biosensors oriented toward on-chip applications is leading to less intrusive and more reliable detection of events both at the cellular and microenvironment levels. This chapter comprises an overview of hybrid approaches combining organs-on-chips and biosensors, focused on modeling and investigating solid tumors, and, in particular, the tumor microenvironment. Optical imaging modalities, specifically fluorescence and bioluminescence, will be also described, addressing the current limitations and future directions toward an even more seamless integration of these advanced technologies.

Metabolic programming of mesenchymal stromal cells by oxygen tension directs chondrogenic cell fate.

Actively steering the chondrogenic differentiation of mesenchymal stromal cells (MSCs) into either permanent cartilage or hypertrophic cartilage destined to be replaced by bone has not yet been possible. During limb development, the developing long bone is exposed to a concentration gradient of oxygen, with lower oxygen tension in the region destined to become articular cartilage and higher oxygen tension in transient hypertrophic cartilage. Here, we prove that metabolic programming of MSCs by oxygen tension directs chondrogenesis into either permanent or transient hyaline cartilage. Human MSCs chondrogenically differentiated in vitro under hypoxia (2.5% O2) produced more hyaline cartilage, which expressed typical articular cartilage biomarkers, including established inhibitors of hypertrophic differentiation. In contrast, normoxia (21% O2) prevented the expression of these inhibitors and was associated with increased hypertrophic differentiation. Interestingly, gene network analysis revealed that oxygen tension resulted in metabolic programming of the MSCs directing chondrogenesis into articular- or epiphyseal cartilage-like tissue. This differentiation program resembled the embryological development of these distinct types of hyaline cartilage. Remarkably, the distinct cartilage phenotypes were preserved upon implantation in mice. Hypoxia-preconditioned implants remained cartilaginous, whereas normoxia-preconditioned implants readily underwent calcification, vascular invasion, and subsequent endochondral ossification. In conclusion, metabolic programming of MSCs by oxygen tension provides a simple yet effective mechanism by which to direct the chondrogenic differentiation program into either permanent articular-like cartilage or hypertrophic cartilage that is destined to become endochondral bone.

Skeletal tissue regeneration: where can hydrogels play a role?

The emerging field of tissue engineering reveals promising approaches for the repair and regeneration of skeletal tissues including the articular cartilage, bone, and the entire joint. Amongst the myriad of biomaterials available to support this strategy, hydrogels are highly tissue mimicking substitutes and thus of great potential for the regeneration of functional tissues. This review comprises an overview of the novel and most promising hydrogels for articular cartilage, osteochondral and bone defect repair. Chondro- and osteo-conductive and -instructive hydrogels are presented, highlighting successful combinations with inductive signals and cell sources. Moreover, advantages, drawbacks, and future perspectives of the role of hydrogels in skeletal regeneration are addressed, pointing out the current state of this rising approach.

Towards single-cell bioprinting: micropatterning tools for organ-on-chip development.

Organs-on-chips (OoCs) hold promise to engineer progressively more human-relevant in vitro models for pharmaceutical purposes. Recent developments have delivered increasingly sophisticated designs, yet OoCs still lack in reproducing the inner tissue physiology required to fully resemble the native human body. This review emphasizes the need to include microarchitectural and microstructural features, and discusses promising avenues to incorporate well-defined microarchitectures down to the single-cell level. We highlight how their integration will significantly contribute to the advancement of the field towards highly organized structural and hierarchical tissues-on-chip. We discuss the combination of state-of-the-art micropatterning technologies to achieve OoCs resembling human-intrinsic complexity. It is anticipated that these innovations will yield significant advances in realization of the next generation of OoC models.

3D Lung-on-Chip Model Based on Biomimetically Microcurved Culture Membranes.

A comparatively straightforward approach to accomplish more physiological realism in organ-on-a-chip (OoC) models is through substrate geometry. There is increasing evidence that the strongly, microscale curved surfaces that epithelial or endothelial cells experience when lining small body lumens, such as the alveoli or blood vessels, impact their behavior. However, the most commonly used cell culture substrates for modeling of these human tissue barriers in OoCs, ion track-etched porous membranes, provide only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically microcurved track-etched membranes. They recreate the mainly spherical geometry of the cells' native microenvironment. In this feasibility study, the membranes were given the shape of hexagonally arrayed hemispherical microwells by an innovative combination of three-dimensional (3D) microfilm (thermo)forming and ion track technology. Integrated in microfluidic chips, they separated a top from a bottom cell culture chamber. The microcurved membranes were seeded by infusion with primary human alveolar epithelial cells. Despite the pronounced topology, the cells fully lined the alveoli-like microwell structures on the membranes' top side. The confluent curved epithelial cell monolayers could be cultured successfully at the air-liquid interface for 14 days. Similarly, the top and bottom sides of the microcurved membranes were seeded with cells from the Calu-3 lung epithelial cell line and human lung microvascular endothelial cells, respectively. Thereby, the latter lined the interalveolar septum-like interspace between the microwells in a network-type fashion, as in the natural counterpart. The coculture was maintained for 11 days. The presented 3D lung-on-a-chip model might set the stage for other (micro)anatomically inspired membrane-based OoCs in the future.

Author Correction: On-chip recapitulation of clinical bone marrow toxicities and patient-specific pathophysiology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

On-chip recapitulation of clinical bone marrow toxicities and patient-specific pathophysiology.

The inaccessibility of living bone marrow (BM) hampers the study of its pathophysiology under myelotoxic stress induced by drugs, radiation or genetic mutations. Here, we show that a vascularized human BM-on-a-chip (BM chip) supports the differentiation and maturation of multiple blood cell lineages over 4 weeks while improving CD34+ cell maintenance, and that it recapitulates aspects of BM injury, including myeloerythroid toxicity after clinically relevant exposures to chemotherapeutic drugs and ionizing radiation, as well as BM recovery after drug-induced myelosuppression. The chip comprises a fluidic channel filled with a fibrin gel in which CD34+ cells and BM-derived stromal cells are co-cultured, a parallel channel lined by human vascular endothelium and perfused with culture medium, and a porous membrane separating the two channels. We also show that BM chips containing cells from patients with the rare genetic disorder Shwachman-Diamond syndrome reproduced key haematopoietic defects and led to the discovery of a neutrophil maturation abnormality. As an in vitro model of haematopoietic dysfunction, the BM chip may serve as a human-specific alternative to animal testing for the study of BM pathophysiology.

Mimicking the Articular Joint with In Vitro Models.

Treating joint diseases remains a significant clinical challenge. Conventional in vitro cultures and animal models have been helpful, but suffer from limited predictive power for the human response. Advanced models are therefore required to mimic the complex biological interactions within the human joint. However, the intricate structure of the joint microenvironment and the complex nature of joint diseases have challenged the development of in vitro models that can faithfully mimic the in vivo physiological and pathological environments. In this review, we discuss the current in vitro models of the joint and the progress achieved in the development of novel and potentially more predictive models, and highlight the application of new technologies to accurately emulate the articular joint.

In vivo screening of extracellular matrix components produced under multiple experimental conditions implanted in one animal.

Animal experiments help to progress and ensure safety of an increasing number of novel therapies, drug development and chemicals. Unfortunately, these also lead to major ethical concerns, costs and limited experimental capacity. We foresee a coercion of all these issues by implantation of well systems directly into vertebrate animals. Here, we used rapid prototyping to create wells with biomaterials to create a three-dimensional (3D) well-system that can be used in vitro and in vivo. First, the well sizes and numbers were adjusted for 3D cell culture and in vitro screening of molecules. Then, the functionality of the wells was evaluated in vivo under 36 conditions for tissue regeneration involving human mesenchymal stem cells (hMSCs) and bovine primary chondrocytes (bPCs) screened in one animal. Each biocompatible well was controlled to contain µl-size volumes of tissue, which led to tissue penetration from the host and tissue formation under implanted conditions. We quantified both physically and biologically the amounts of extracellular matrix (ECM) components found in each well. Using this new concept the co-culture of hMSCs and bPCs was identified as a positive hit for cartilage tissue repair, which was a comparable result using conventional methods. The in vivo screening of candidate conditions opens an entirely new range of experimental possibilities, which significantly abates experimental animal use and increases the pace of discovery of medical treatments.

Hypoxia inhibits hypertrophic differentiation and endochondral ossification in explanted tibiae.

PURPOSE: Hypertrophic differentiation of growth plate chondrocytes induces angiogenesis which alleviates hypoxia normally present in cartilage. In the current study, we aim to determine whether alleviation of hypoxia is merely a downstream effect of hypertrophic differentiation as previously described or whether alleviation of hypoxia and consequent changes in oxygen tension mediated signaling events also plays an active role in regulating the hypertrophic differentiation process itself. MATERIALS AND METHODS: Fetal mouse tibiae (E17.5) explants were cultured up to 21 days under normoxic or hypoxic conditions (21% and 2.5% oxygen respectively). Tibiae were analyzed on growth kinetics, histology, gene expression and protein secretion. RESULTS: The oxygen level had a strong influence on the development of explanted fetal tibiae. Compared to hypoxia, normoxia increased the length of the tibiae, length of the hypertrophic zone, calcification of the cartilage and mRNA levels of hypertrophic differentiation-related genes e.g. MMP9, MMP13, RUNX2, COL10A1 and ALPL. Compared to normoxia, hypoxia increased the size of the cartilaginous epiphysis, length of the resting zone, calcification of the bone and mRNA levels of hyaline cartilage-related genes e.g. ACAN, COL2A1 and SOX9. Additionally, hypoxia enhanced the mRNA and protein expression of the secreted articular cartilage markers GREM1, FRZB and DKK1, which are able to inhibit hypertrophic differentiation. CONCLUSIONS: Collectively our data suggests that oxygen levels play an active role in the regulation of hypertrophic differentiation of hyaline chondrocytes. Normoxia stimulates hypertrophic differentiation evidenced by the expression of hypertrophic differentiation related genes. In contrast, hypoxia suppresses hypertrophic differentiation of chondrocytes, which might be at least partially explained by the induction of GREM1, FRZB and DKK1 expression.

The effect of platelet lysate supplementation of a dextran-based hydrogel on cartilage formation.

In situ gelating dextran-tyramine (Dex-TA) injectable hydrogels have previously shown promising features for cartilage repair. Yet, despite suitable mechanical properties, this system lacks intrinsic biological signals. In contrast, platelet lysate-derived hydrogels are rich in growth factors and anti-inflammatory cytokines, but mechanically unstable. We hypothesized that the advantages of these systems may be combined in one hydrogel, which can be easily translated into clinical settings. Platelet lysate was successfully incorporated into Dex-TA polymer solution prior to gelation. After enzymatic crosslinking, rheological and morphological evaluations were performed. Subsequently, the effect of platelet lysate on cell migration, adhesion, proliferation and multi-lineage differentiation was determined. Finally, we evaluated the integration potential of this gel onto osteoarthritis-affected cartilage. The mechanical properties and covalent attachment of Dex-TA to cartilage tissue during in situ gel formation were successfully combined with the advantages of platelet lysate, revealing the potential of this enhanced hydrogel as a cell-free approach. The addition of platelet lysate did not affect the mechanical properties and porosity of Dex-TA hydrogels. Furthermore, platelet lysate derived anabolic growth factors promoted proliferation and triggered chondrogenic differentiation of mesenchymal stromal cells.

Self-attaching and cell-attracting in-situ forming dextran-tyramine conjugates hydrogels for arthroscopic cartilage repair.

Small cartilage defects are frequently treated with debridement or left untreated, predisposing to early onset osteoarthritis. We propose to fill these defects with a cell-free injectable hydrogel comprising dextran-tyramine conjugates (Dex-TA) that can be applied during arthroscopic procedures. In this study, we report on the adhesion mechanism between cartilage and Dex-TA hydrogels and enhancement of cell ingrowth by incorporation of Heparin-tyramine (Hep-TA) conjugates. The enzyme-catalyzed crosslinking reaction of Dex-TA and Hep-TA hydrogels is based on covalent bonding of hydroxyphenyl residues. We hypothesized that this reaction results in covalent bonding of the hydroxyphenyl residues in Dex-TA and Hep-TA to tyrosine residues in cartilage matrix proteins. The involvement of TA residues was confirmed by modelling the enzymatic reaction occurring during gelation. The mechanical analysis indicated that higher tyramine content led to stronger binding. Interfacial cartilage-hydrogel morphology and Raman spectroscopy demonstrated collagens' reorganization and evidenced the coupling of TA to tyrosine residues in collagen. Moreover, the addition of Hep-TA induced cell recruitment. Collectively, in vitro and ex vivo functional studies evidenced the covalent bonding of TA-containing hydrogels to tyrosine residues in cartilaginous matrix proteins. Moreover, the cell-attracting ability of these hydrogels could be explored to guide tissue repair in focal cartilage defects, preventing or delaying the onset osteoarthritis.

Cartilage tissue engineering.

Cartilage tissue engineering is the art aimed at repairing defects in the articular cartilage which covers the bony ends in the joints. Since its introduction in the early 1990s of the past century, cartilage tissue engineering using ACI has been used in thousands of patients to repair articular cartilage defects. This review focuses on emerging strategies to improve cartilage repair by incorporating fundamental knowledge of developmental and cell biology in the design of optimized strategies for cell delivery at the defect site and to locally stimulate cartilage repair responses.

Chondrogenesis in injectable enzymatically crosslinked heparin/dextran hydrogels.

In this study, injectable hydrogels were prepared by horseradish peroxidase-mediated co-crosslinking of dextran-tyramine (Dex-TA) and heparin-tyramine (Hep-TA) conjugates and used as scaffolds for cartilage tissue engineering. The swelling and mechanical properties of these hydrogels can be easily controlled by the Dex-TA/Hep-TA weight ratio. When chondrocytes were incorporated in these gels, cell viability and proliferation were highest for gels with a 50/50 weight ratio of Dex-TA/Hep-TA. Moreover, these hydrogels induced an enhanced production of chondroitin sulfate and a more abundant presence of collagen as compared to Dex-TA hydrogels. The results indicate that injectable Dex-TA/Hep-TA hydrogels are promising scaffolds for cartilage regeneration.

Chitosan scaffolds containing hyaluronic acid for cartilage tissue engineering.

Scaffolds derived from natural polysaccharides are very promising in tissue engineering applications and regenerative medicine, as they resemble glycosaminoglycans in the extracellular matrix (ECM). In this study, we have prepared freeze-dried composite scaffolds of chitosan (CHT) and hyaluronic acid (HA) in different weight ratios containing either no HA (control) or 1%, 5%, or 10% of HA. We hypothesized that HA could enhance structural and biological properties of CHT scaffolds. To test this hypothesis, physicochemical and biological properties of CHT/HA scaffolds were evaluated. Scanning electron microscopy micrographs, mechanical properties, swelling tests, enzymatic degradation, and Fourier transform infrared (FTIR) chemical maps were performed. To test the ability of the CHT/HA scaffolds to support chondrocyte adhesion and proliferation, live-dead and MTT assays were performed. Results showed that CHT/HA composite scaffolds are noncytotoxic and promote cell adhesion. ECM formation was further evaluated with safranin-O and alcian blue staining methods, and glycosaminoglycan and DNA quantifications were performed. The incorporation of HA enhanced cartilage ECM production. CHT/5HA had a better pore network configuration and exhibited enhanced ECM cartilage formation. On the basis of our results, we believe that CHT/HA composite matrixes have potential use in cartilage repair.

Chitosan/poly(epsilon-caprolactone) blend scaffolds for cartilage repair.

Chitosan (CHT)/poly(ɛ-caprolactone) (PCL) blend 3D fiber-mesh scaffolds were studied as possible support structures for articular cartilage tissue (ACT) repair. Micro-fibers were obtained by wet-spinning of three different polymeric solutions: 100:0 (100CHT), 75:25 (75CHT) and 50:50 (50CHT) wt.% CHT/PCL, using a common solvent solution of 100 vol.% of formic acid. Scanning electron microscopy (SEM) analysis showed a homogeneous surface distribution of PCL. PCL was well dispersed throughout the CHT phase as analyzed by differential scanning calorimetry and Fourier transform infrared spectroscopy. The fibers were folded into cylindrical moulds and underwent a thermal treatment to obtain the scaffolds. µCT analysis revealed an adequate porosity, pore size and interconnectivity for tissue engineering applications. The PCL component led to a higher fiber surface roughness, decreased the scaffolds swelling ratio and increased their compressive mechanical properties. Biological assays were performed after culturing bovine articular chondrocytes up to 21 days. SEM analysis, live-dead and metabolic activity assays showed that cells attached, proliferated, and were metabolically active over all scaffolds formulations. Cartilaginous extracellular matrix (ECM) formation was observed in all formulations. The 75CHT scaffolds supported the most neo-cartilage formation, as demonstrated by an increase in glycosaminoglycan production. In contrast to 100CHT scaffolds, ECM was homogenously deposited on the 75CHT and 50CHT scaffolds. Although mechanical properties of the 50CHT scaffold were better, the 75CHT scaffold facilitated better neo-cartilage formation.

Enzymatically crosslinked dextran-tyramine hydrogels as injectable scaffolds for cartilage tissue engineering.

Enzymatic crosslinking of dextran-tyramine (Dex-TA) conjugates in the presence of horseradish peroxidase and hydrogen peroxide was successively applied in the preparation of hydrogels. Depending on the molecular weight of the dextran (M(n,) (GPC) of 14000 or 31000 g/mol) and the degree of substitution (of 5, 10, or 15) with TA groups, the gelation times ranged from 20 s to 1 min. Hydrogels prepared from Dex31k-TA with a degree of substitution of 10 had storage moduli up to 60 kPa. Similar values were found when chondrocytes were incorporated into the hydrogels. Chondrocyte-seeded Dex-TA hydrogels were prepared at a molar ratio of hydrogen peroxide/TA of 0.2 and cultured in a chondrocyte medium. A live-dead assay and a methylthiazol tetrazolium assay revealed that almost all chondrocytes retained their viability after 2 weeks. Scanning electron microscopy analysis showed that the encapsulated chondrocytes were capable of maintaining their round shape. Histology and immunofluorescent staining demonstrated the production of glycosaminoglycans (GAGs) and collagen type II after culturing for 14 and 21 days. Biochemical analysis showed that GAG accumulation increased with time inside Dex-TA hydrogels. Besides, GAG/DNA for Dex-TA hydrogels was higher than that for agarose at day 28. These results indicate that Dex-TA hydrogels are promising 3D scaffolds for cartilage tissue engineering applications.