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It is well-accepted that articular (ART) cartilage composition and tissue architecture are intimately related to mechanical properties. On the other hand, very little information about other cartilage tissues is available, such as elastin-rich auricular (AUR) cartilage. While thorough investigation of ART cartilage has enhanced osteoarthritis research, ear cartilage reconstruction and tissue engineering (TE) could benefit in a similar way from in-depth analysis of AUR cartilage properties. This study aims to explore the constituent-function relationships of AUR cartilage, and how elastin influences mechanical behavior. Stress-relaxation indentation and tensile tests were performed on bovine ART and AUR cartilage. Elastase incubation was performed to simultaneously deplete elastin and sulfated glycosaminoglycans (sGAG), while hyaluronidase incubation was used to deplete sGAG-only, in order to systematically investigate matrix components in material behavior. ART and AUR cartilages showed different viscoelastic behaviors, with AUR cartilage exhibiting a more elastic behavior. Higher equilibrium properties and limited viscous dissipation of strain energy were observed in AUR cartilage, while ART cartilage exhibited a rapid viscous response and high resistance to instantaneous loading. In conclusion, loss of sGAG had no effect on auricular mechanics in contrast to articular cartilage where GAG loss clearly correlated with mechanical properties. Auricular cartilage without elastin lost all compressive mechanical integrity, whereas in articular cartilage this was provided by collagen. This work shows for the first time the involvement of elastin in the mechanical behavior of ear cartilage. In future, this data can be used in AUR cartilage TE efforts to support reproduction of tissue-specific mechanical properties. (C) 2015 Elsevier Ltd. All rights reserved.

Tissue engineering provides a promising alternative therapy to the complex surgical reconstruction of auricular cartilage by using ear-shaped autologous costal cartilage. Bacterial nanocellulose (BNC) is proposed as a promising scaffold material for auricular cartilage reconstruction, as it exhibits excellent biocompatibility and secures tissue integration. Thus, this study evaluates a novel bilayer BNC scaffold for auricular cartilage tissue engineering. Bilayer BNC scaffolds, composed of a dense nanocellulose layer joined with a macroporous composite layer of nanocellulose and alginate, were seeded with human nasoseptaLchondrocytes (NC) and cultured in vitro for up to 6 weeks. To scale up for clinical translation, bilayer BNC scaffolds were seeded with a low number of freshly isolated (uncultured) human NCs combined with freshly isolated human mononuclear cells (MNC) from bone marrow in alginate and subcutaneously implanted in nude mice for 8 weeks. 3D morphometric analysis showed that bilayer BNC scaffolds have a porosity of 75% and mean pore size of 50 +/- 25 pm. Furthermore, endotoxin analysis and in vitro cytotoxicity testing revealed that the produced bilayer BNC scaffolds were non-pyrogenic (0.15 +/- 0.09 EU/ml) and non-cytotoxic (cell viability: 97.8 +/- 4.7%). This study demonstrates that bilayer BNC scaffolds offer a good mechanical stability and maintain a structural integrity while providing a porous architecture that supports cell ingrowth. Moreover, bilayer BNC scaffolds provide a suitable environment for culture-expanded NCs as well as a combination of freshly isolated NCs and MNCs to form cartilage in vitro and in vivo as demonstrated by immunohistochemistry, biochemical and biomechanical analyses. (C) 2014 Elsevier Ltd. All rights reserved.

Journal of the Mechanical Behavior of Biomedical Materials, 22, ISSN:1751-6161, ISSN:1878-0180

It is key for successful auricular (AUR) cartilage tissue-engineering (TE) to ensure that the engineered cartilage mimics the mechanics of the native tissue. This study provides a spatial map of the mechanical and biochemical properties of human auricular cartilage, thus establishing a benchmark for the evaluation of functional competency in AUR cartilage TE. Stress-relaxation indentation (instantaneous modulus, Ein; maximum stress, (sigma(max), equilibrium modulus, E-eq; relaxation half-life time, rip; thickness, h) and biochemical parameters (content of DNA; sulfated-glycosaminoglycan, sGAG; hydroxyproline, HYP; elastin, ELN) of fresh human AUR cartilage were evaluated. Samples were categorized into age groups and according to, their harvesting region in the human auricle (for AUR cartilage only). AUR cartilage displayed significantly lower E-in, sigma(max), E-eq, sGAG content; and significantly higher t(1/2), and DNA content than NAS cartilage. Large amounts of ELN were measured in AUR cartilage ( > 15% ELN content per sample wet mass). No effect of gender was observed for either auricular or nasoseptal samples. For auricular samples, significant differences between age groups for h, sGAG and HYP and significant regional variations for E-in, sigma(max). E-eq, t(1/2), h, DNA and sGAG were measured. However, only low correlations between mechanical and biochemical parameters were seen (R< 0.44). In conclusion, this study established the first comprehensive mechanical and biochemical map of human auricular cartilage. Regional variations in mechanical and biochemical properties were demonstrated in the auricle. This finding highlights the importance of focusing future research on efforts to produce cartilage grafts with spatially tunable mechanics. (C) 2015 Elsevier Ltd. All rights reserved.

Background: Cartilage tissue engineering can offer promising solutions for restoring cartilage defects in the head and neck area and has the potential to overcome limitations of current treatments. However, to generate a construct of reasonable size, large numbers of chondrocytes are required, which limits its current applicability. Therefore, the authors evaluate the suitability of a combination of cells for cartilage regeneration: bone marrow-derived mesenchymal stem cells and ear or nasal chondrocytes. Methods: Human bone marrow-derived mesenchymal stem cells were encapsulated in alginate hydrogel as single-cell-type populations or in combination with bovine ear chondrocytes or nasal chondrocytes at an 80:20 ratio. Constructs were either cultured in vitro or implanted directly subcutaneously into mice. Cartilage formation was evaluated with biochemical and biomechanical analyses. The use of a xenogeneic coculture system enabled the analyses of the contribution of the individual cell types using species-specific gene-expression analyses. Results: In vivo, human bone marrow-derived mesenchymal stem cells/bovine ear chondrocytes or human bone marrow-derived mesenchymal stem cells/bovine nasal chondrocytes contained amounts of cartilage components similar to those of constructs containing chondrocytes only (i.e., bovine ear and nasal chondrocytes). In vitro, species-specific gene-expression analyses demonstrated that aggrecan was expressed by the chondrocytes only, which suggests a more trophic role for human bone marrow-derived mesenchymal stem cells. Furthermore, the additional effect of human bone marrow-derived mesenchymal stem cells was more pronounced in combination with bovine nasal chondrocytes. Conclusions: By supplementing low numbers of bovine ear or nasal chondrocytes with human bone marrow-derived mesenchymal stem cells, the authors were able to engineer cartilage constructs with properties similar to those of constructs containing chondrocytes only. This makes the procedure more feasible for future applicability in the reconstruction of cartilage defects in the head and neck area because fewer chondrocytes are required. CLINICAL QUESTIONS/LEVEL OF EVIDENCE: Therapeutic, V.

Tissue Engineering. Part B, Reviews, 21 (2), ISSN:1937-3368, ISSN:1937-3376

Tissue Engineering. Part B, Reviews, 20 (1), ISSN:1937-3368, ISSN:1937-3376

Scaffolds are widely used to reconstruct cartilage. Yet, the fabrication of a scaffold with a highly organized microenvironment that closely resembles native cartilage remains a major challenge. Scaffolds derived from acellular extracellular matrices are able to provide such a microenvironment. Currently, no report specifically on decellularization of full thickness ear cartilage has been published. In this study, decellularized ear cartilage scaffolds were prepared and extensively characterized. Cartilage decellularization was optimized to remove cells and cell remnants from elastic cartilage. Following removal of nuclear material, the obtained scaffolds retained their native collagen and elastin contents as well as their architecture and shape. High magnification scanning electron microscopy showed no obvious difference in matrix density after decellularization. However, glycosaminoglycan content was significantly reduced, resulting in a loss of viscoelastic properties. Additionally, in contact with the scaffolds, human bone-marrow-derived mesenchymal stem cells remained viable and are able to differentiate toward the chondrogenic lineage when cultured in vitro. These results, including the ability to decellularize whole human ears, highlight the clinical potential of decellularization as an improved cartilage reconstruction strategy.