Enhanced cellular migration and prolonged chondrogenic differentiation in decellularized cartilage scaffolds under dynamic culture conditions

Lesions of aural, nasal and tracheal cartilage are frequently reconstructed by complex surgeries which are based on harvesting autologous cartilage from other locations such as the rib. Cartilage tissue engineering (CTE) is regarded as a promising alternative to attain vital cartilage. Nevertheless, CTE with nearly natural properties poses a significant challenge to research due to the complex reciprocal interactions between cells and extracellular matrix which have to be imitated and which are still not fully understood. Thus, we used a custom-made glass bioreactor to enhance cell migration into decellularized porcine cartilage scaffolds (DECM) and mimic physiological conditions. The DECM seeded with human nasal chondrocytes (HPCH) were cultured in the glass reactor for 6 weeks and examined by histological and immunohistochemical staining, biochemical analyses and real time-PCR at 14, 28 and 42 days. The migration depth and the number of migrated cells were quantified by computational analysis. Compared to the static cultivation, the dynamic culture (DC) fostered migration of HPCH into deeper tissue layers. Furthermore, cultivation in the bioreactor enhanced differentiation of the cells during the first 14 days, but differentiation diminished in the course of further cultivation. We consider the DC in the presented bioreactor as a promising tool to facilitate CTE and to help to better understand the complex physiological processes during cartilage regeneration. Maintaining differentiation of chondrocytes and improving cellular migration by further optimizing culture conditions is an important prerequisite for future clinical application

Adenosine receptor 2B activity promotes autonomous growth, migration as well as vascularization of head and neck squamous cell carcinoma cells

Adenosine is a signaling molecule that exerts dual effects on tumor growth: while it inhibits immune cell function and thereby prevents surveillance by the immune system, it influences tumorigenesis directly via activation of adenosine receptors on tumor cells at the same time. However, the adenosine-mediated mechanisms affecting oncogenic processes particularly in head and neck squamous cell carcinomas (HNSCC) are not fully understood. Here, we investigated the role of adenosine receptor activity on HNSCC-derived cell lines. Targeting the adenosine receptor A2B (ADORA2B) on these cells with the inverse agonist/antagonist PSB-603 leads to inhibition of cell proliferation, transmigration as well as VEGFA secretion in vitro. At the molecular level, these effects were associated with cell cycle arrest as well as the induction of the apoptotic pathway. In addition, shRNA-mediated downmodulation of ADORA2B expression caused decreased proliferation. Moreover, in in vivo xenograft experiments, chemical and genetic abrogation of ADORA2B activity impaired tumor growth associated with decreased tumor vascularization. Together, our findings characterize ADORA2B as a crucial player in the maintenance of HNSCC and, therefore, as a potential therapeutic target for HNSCC treatment

Histological Image Processing for the Assessment of Tissue Engineered Cartilage

In a recently developed perfusion bioreactor, human nasal septum chondrocytes were seeded on decellularized porcine collagen scaffolds and cultivated for 6 weeks. Afterwards, the samples were stained with alcian blue and multiple histological microscope images were taken to assess the migration of the human cells and their redifferentiation. In this paper, we propose an image processing algorithm to objectively quantify the cultivation results. This algorithm performs a white balance, detects cell nuclei and their migration depth, and detects lacunae and their orientation. To evaluate performance, sensitivity and precision are calculated using manually labeled data over 50 images. The detection of cell nuclei has a reasonable precision (73%), but the level of sensitivity (42%) should be further improved. The proposed method demonstrates acceptable level of precision (83%) and sensitivity (86%) for the detection of lacunae

Neuer Knorpel aus dem Reaktor? Entwicklung eines Tissue-Engineering-Bioreaktors für Knorpel des Kopf-Hals-Bereichs

Knorpel in Ohren, Nase und Luftröhre bestimmt deren Form und biomechanische Eigenschaften. Kommt es aufgrund von Unfällen oder Erkrankungen zu einem Knorpelverlust, kann dies bei Ohr und Nase zu einer erheblichen funktionellen und psychischen Belastung des Patienten führen. Bei der Luftröhre sind sogar lebensbedrohliche Folgen möglich. Bisherige Behandlungsoptionen bestehen darin, entweder dem Patienten Knorpel an anderer Stelle zu entnehmen oder Knorpelersatzmaterialien einzusetzen. Bei der ersten Variante ist zu beachten, dass nur eine begrenzte Knorpelmenge verfügbar ist und der zusätzliche operative Eingriff zu Komplikationen führen kann. Körperfremde Ersatzmaterialien weisen oft nicht die gewünschten mechanischen Eigenschaften auf und können zu Immunreaktionen führen. Besser wäre es, aus eigenen Zellen des Patienten Knorpel zu züchten und dann zu replantieren. Im BMBF-Projekt BioopTiss wurde an der Hochschule Ulm in Kooperation mit der HNO-Klinik des Universitätsklinikums Ulm die Entwicklung eines dafür geeigneten Bioreaktors begonnen und soll auch zukünftig fortgesetzt werden

Mean, precision error (PE_SD), precision error expressed as a percentage of coefficient of variation of the repeated measurements (PE_%CV) and intraclass correlation coefficient (ICC) computed using a two-way model (absolute agreement for single measurements) for inter-rater reproducibility.

<p>Mean, precision error (PE_SD), precision error expressed as a percentage of coefficient of variation of the repeated measurements (PE_%CV) and intraclass correlation coefficient (ICC) computed using a two-way model (absolute agreement for single measurements) for inter-rater reproducibility.</p

Inter-rater (a, b c) and intra-rater (d, e, f) repeated measures for cartilage volume (Cg.V), cartilage surface (Cg.S) and mean cartilage thickness (Cg.Th) for all 14 volunteers.

<p>Good reproducibility is observed for Cg.V and Cg.S values. Additionally low patient-to-patient variations are observed for Cg.Th, i.e. Cg.Th for all 14 volunteers and all three raters is 1.15±0.10 mm, whereas the average Cg.V and Cg.S are 2295±415 mm³ and 5102±667 mm², respectively.</p

Correlation between values measured with contrast-enhanced micro-CT (after dissection) and values obtained by clinical MRI imaging combined with manual segmentation.

<p>Solid line represents y = x. (a) A correlation of r = 0.99 is observed for cartilage surface (Cg.S), (b) r = 0.99 for cartilage volume (Cg.V), (c) r = 0.96 for mean cartilage thickness (Cg.Th), dotted lines represented a 1-voxel error (0.45 mm) on the MRI datasets.</p

Mean, PE_SD, PE_%CV and ICC for intra-rater reproducibility.

<p>PE_%CV values are below 5% for Cg.S and below 10% for Cg.V and Cg.Th which demonstrates good precision. ICC values obtained in both inter-rater and intra-rater measurements are good for Cg.V and Cg.S, but very low for Cg.Th. This indicates that the proposed method is adequate to distinguish patient-specific variations for Cg.V and Cg.S only.</p

Pairwise comparisons of the three repeated masks obtained by rater 1 for a typical volunteer.

<p>The three masks obtained by segmentation are display in the diagonal. These masks are superimposed two-by-two, each overlapped pair of masks (left-hand side) and a corresponding cross-section (right-hand side) are displayed. Common regions are represented in yellow. The areas that belong to only one of the two masks are semi-transparent. Large common regions are observed in all three overlapped pairs of masks, i.e. the overall integrity of shape is maintained, with minor surface variations. This demonstrates that the inability to detect patient-specific variation for mean Cg.Th (low ICC) does not adversely affect the capacity of the proposed clinical method to detect local shape variations within a volunteer auricle. Scale bar: 1 cm.</p

Adenosine receptor 2B activity promotes autonomous growth, migration as well as vascularization of head and neck squamous cell carcinoma cells

Adenosine is a signaling molecule that exerts dual effects on tumor growth: while it inhibits immune cell function and thereby prevents surveillance by the immune system, it influences tumorigenesis directly via activation of adenosine receptors on tumor cells at the same time. However, the adenosine-mediated mechanisms affecting oncogenic processes particularly in head and neck squamous cell carcinomas (HNSCC) are not fully understood. Here, we investigated the role of adenosine receptor activity on HNSCC-derived cell lines. Targeting the adenosine receptor A2B (ADORA2B) on these cells with the inverse agonist/antagonist PSB-603 leads to inhibition of cell proliferation, transmigration as well as VEGFA secretion in vitro. At the molecular level, these effects were associated with cell cycle arrest as well as the induction of the apoptotic pathway. In addition, shRNA-mediated downmodulation of ADORA2B expression caused decreased proliferation. Moreover, in in vivo xenograft experiments, chemical and genetic abrogation of ADORA2B activity impaired tumor growth associated with decreased tumor vascularization. Together, our findings characterize ADORA2B as a crucial player in the maintenance of HNSCC and, therefore, as a potential therapeutic target for HNSCC treatment