Non-invasive imaging of hypoxia in tissue engineering

In tissue engineering, cells are grown on biomaterials in vitro and subsequently implanted. A critical parameter in effective proliferation and differentiation is the availability of nutrients. Few tools are currently available to monitor the nutritional status of cells. In this study, we have employed A4-4 cells [1], a Chinese hamster ovary cell line stably transfected with a luciferase gene driven by the hypoxia responsive element (HRE) from the promoter region of the VEGF gene [2, 3]. HRE activity, and thus luciferase activity, directly correlates with decreasing cellular O2 levels.The aim of this study is to investigate whether the HREluciferase construct can be used for non-invasive imaging of hypoxia in tissue engineering

Mesenchymal Stromal/Stem Cell- or Chondrocyte-Seeded Microcarriers as Building Blocks for Cartilage Tissue Engineering

In this study we have tested the use of mesenchymal stromal/stem cell (MSC)- or chondrocyte (hch)-laden microcarriers as building blocks for engineered cartilage tissue. MSCs and hchs expanded on microcarriers were used in chondrogenic coculture and compared with monoculture of MSCs or hchs. The use of cell-laden microcarriers as building blocks for cartilage tissue engineering led to a compact tissue formation with significant volume increase compared to the biomaterial-free approach. After 28 days of differentiation culture, formation of cartilaginous matrix in cocultures and chondrocyte monoculture approaches was observed. Coculture resulted in beneficial glycosaminoglycan deposition compared with monoculture of MSCs or chondrocytes attached to microcarriers. Further, the microcarrier-adhered coculture displayed increased levels of the differentiation marker ACAN and reduced levels of the dedifferentiation marker COL1A1. To our knowledge, this is the first article that successfully combines an innovative combination of cell expansion on microcarriers and the direct use of MSC- or hch-cell-laden microcarriers as building blocks in cartilage tissue engineering

Preparation of a Resorbable Osteoinductive Tricalcium Phosphate Ceramic

Over the past decade we have demonstrated numerous times that calcium phosphates can be rendered with osteoinductive properties by introducing specific surface microstructures1. Since most of these calcium phosphates contained hydroxyapatite, they are either slowly or not resorbable2. Resorbability is an often sought after characteristic of calcium phosphates so that they can be gradually replaced by newly formed bone. The objective of this study was to prepare a resorbable surface microstructured tricalcium phosphate (TCP) ceramic and evaluate its osteoinductive property and resorption rate after intramuscular implantation in dogs. This material was then compared to the established and slowly resorbable osteoinductive biphasic calcium phosphate ceramic (BCP)

Engineering vascularised tissues in vitro

Tissue engineering aims at replacing or regenerating tissues lost due to diseases or traumas (Langer and Vacanti, 1993). However, mimicking in vitro the physiological complexity of vascularized tissue is a major obstacle, which possibly contributes to impaired healing in vivo. In higher organisms, native features including the vascular network, the lymphatic networks and interstitial flow promote both mass transport and organ development. Attempts to mimic those features in engineered tissues will lead to more clinically relevant cell-based therapies. Aside from current strategies promoting angiogenesis from the host, an alternative concept termed prevascularization is emerging. It aims at creating a biological vasculature inside an engineered tissue prior to implantation. This vasculature can rapidly anastamose with the host and enhances tissue survival and differentiation. Interestingly, growing evidence supports a role of the vasculature in regulating pattern formation and tissue differentiation. Thus, prevascularized tissues also benefit from an intrinsic contribution of their vascular system to their development. From those early attempts are emerging a body of principles and strategies to grow and maintain, in vitro, those self-assembled biological vascular networks. This could lead to the generation of engineered tissues of more physiologically relevant complexity and improved regenerative potential

A Wnt/ß-catenin negative feedback loop inhibits IL-1-induced MMP expression in human articular chondrocytes.

Objective: Recent animal studies suggest that activation of Wnt/β-catenin signaling in articular chondrocytes might be a driving factor in the pathogenesis of osteoarthritis (OA) by stimulating amongst others the expression of matrix metalloproteinases (MMPs). This study aimed to investigate the role of Wnt/β-catenin signaling in IL-1β-induced MMP expression in human chondrocytes. Methods: Primary cultures of human, mouse and bovine articular chondrocytes as well as human mesenchymal stem cells (hMSCs) and mouse embryonic fibroblasts (MEFs) were used. Multiple strategies for activation and inhibition of signaling pathways were used. Reporter assays and co-immunoprecipitations were used to study the interaction between β-catenin and NF-κB. Results: In contrast to animal chondrocytes, in human chondrocytes Wnt/β-catenin is a potent inhibitor of MMP1, -3 and -13 expression and generic MMP activity both in basal conditions and after IL-1β stimulation. This effect is independent of TCF/LEF transcription factors but is due to an inhibitory protein-protein interaction between β-catenin and NF-κB. Furthermore we show that IL-1β indirectly activates β-catenin signaling by inducing canonical Wnt7B expression and by inhibiting the expression of canonical Wnt antagonists. Conclusion: Our data reveal an unexpected anti-catabolic role of Wnt/β-catenin signaling in human chondrocytes by counteracting NF-κB-mediated MMP expression induced by IL-1β in a negative feedback loop

Clinical Application of Human Mesenchymal Stromal Cells for Bone Tissue Engineering

The gold standard in the repair of bony defects is autologous bone grafting, even though it has drawbacks in terms of availability and morbidity at the harvesting site. Bone-tissue engineering, in which osteogenic cells and scaffolds are combined, is considered as a potential bone graft substitute strategy. Proof-of-principle for bone tissue engineering using mesenchymal stromal cells (MSCs) has been demonstrated in various animal models. In addition, 7 human clinical studies have so far been conducted. Because the experimental design and evaluation parameters of the studies are rather heterogeneous, it is difficult to draw conclusive evidence on the performance of one approach over the other. However, it seems that bone apposition by the grafted MSCs in these studies is observed but not sufficient to bridge large bone defects. In this paper, we discuss the published human clinical studies performed so far for bone-tissue regeneration, using culture-expanded, nongenetically modified MSCs from various sources and extract from it points of consideration for future clinical studies

Overlooked? Underestimated? Effects of Substrate Curvature on Cell Behavior

In biological systems, form and function are inherently correlated. Despite this strong interdependence, the biological effect of curvature has been largely overlooked or underestimated, and consequently it has rarely been considered in the design of new cell–material interfaces. This review summarizes current understanding of the interplay between the curvature of a cell substrate and the related morphological and functional cellular response. In this context, we also discuss what is currently known about how, in the process of such a response, cells recognize curvature and accordingly reshape their membrane. Beyond this, we highlight state-of-the-art microtechnologies for engineering curved biomaterials at cell-scale, and describe aspects that impair or improve readouts of the pure effect of curvature on cells

Distribution and viability of fetal and adult human bone marrow stromal cells in a biaxial rotating vessel bioreactor after seeding on polymeric 3D additive manufactured scaffolds

One of the conventional approaches in tissue engineering is the use of scaffolds in combination with cells to obtain mechanically stable tissue constructs in vitro prior to implantation. Additive manufacturing by fused deposition modeling is a widely used technique to produce porous scaffolds with defined pore network, geometry, and therewith defined mechanical properties. Bone marrow-derived mesenchymal stromal cells (MSCs) are promising candidates for tissue engineering-based cell therapies due to their multipotent character. One of the hurdles to overcome when combining additive manufactured scaffolds with MSCs is the resulting heterogeneous cell distribution and limited cell proliferation capacity. In this study, we show that the use of a biaxial rotating bioreactor, after static culture of human fetal MSCs (hfMSCs) seeded on synthetic polymeric scaffolds, improved the homogeneity of cell and extracellular matrix distribution and increased the total cell number. Furthermore, we show that the relative mRNA expression levels of indicators for stemness and differentiation are not significantly changed upon this bioreactor culture, whereas static culture shows variations of several indicators for stemness and differentiation. The biaxial rotating bioreactor presented here offers a homogeneous distribution of hfMSCs, enabling studies on MSCs fate in additive manufactured scaffolds without inducing undesired differentiatio

The effect of scaffold-cell entrapment capacity and physico-chemical properties on cartilage regeneration

An important tenet in designing scaffolds for regenerative medicine consists in mimicking the dynamic mechanical properties of the tissues to be replaced to facilitate patient rehabilitation and restore daily activities. In addition, it is important to determine the contribution of the forming tissue to the mechanical properties of the scaffold during culture to optimize the pore network architecture. Depending on the biomaterial and scaffold fabrication technology, matching the scaffolds mechanical properties to articular cartilage can compromise the porosity, which hampers tissue formation. Here, we show that scaffolds with controlled and interconnected pore volume and matching articular cartilage dynamic mechanical properties, are indeed effective to support tissue regeneration by co-cultured primary and expanded chondrocyte (1:4). Cells were cultured on scaffolds in vitro for 4 weeks. A higher amount of cartilage specific matrix (ECM) was formed on mechanically matching (M) scaffolds after 28 days. A less protein adhesive composition supported chondrocytes rounded morphology, which contributed to cartilaginous differentiation. Interestingly, the dynamic stiffness of matching constructs remained approximately at the same value after culture, suggesting a comparable kinetics of tissue formation and scaffold degradation. Cartilage regeneration in matching scaffolds was confirmed subcutaneously in vivo. These results imply that mechanically matching scaffolds with appropriate physico-chemical properties support chondrocyte differentiation