Scaffold-free tracheal cartilage engineering using roller bottle culture

Resection with primary anastomosis can only repair up to 50% of the adult trachea and up to 30% of the pediatric trachea when damaged. There is a strong clinical need for long-segment tracheal replacements. The goal of this research was to create a seamless, scaffold-free cartilage cylinder for tracheal tissue engineering in vitro. Primary bovine articular chondrocytes were seeded onto tracheal moulds for roller bottle culture and the effect of rotational speed, growth factor supplementation, and chondrocyte layering were investigated. After the 4-week culture period, samples were evaluated biochemically, histologically, and biomechanically. The results indicated that rotation was necessary for full tissue coverage, with slower rotational speeds generating thicker tissue with an improved extracellular matrix, IGF-1 supplementation generating thicker tissue rich in glycosaminoglycans with inferior mechanical properties, and chondrocyte layering producing thinner tissue with increased mechanical properties. Overall, scaffold-free tissue engineering can generate seamless cylindrical cartilage constructs using roller bottle culture for future applications in long-segment tracheal replacement.

Scaffold-free tracheal cartilage engineering using roller bottle culture

Resection with primary anastomosis can only repair up to 50% of the adult trachea and up to 30% of the pediatric trachea when damaged. There is a strong clinical need for long-segment tracheal replacements. The goal of this research was to create a seamless, scaffold-free cartilage cylinder for tracheal tissue engineering in vitro. Primary bovine articular chondrocytes were seeded onto tracheal moulds for roller bottle culture and the effect of rotational speed, growth factor supplementation, and chondrocyte layering were investigated. After the 4-week culture period, samples were evaluated biochemically, histologically, and biomechanically. The results indicated that rotation was necessary for full tissue coverage, with slower rotational speeds generating thicker tissue with an improved extracellular matrix, IGF-1 supplementation generating thicker tissue rich in glycosaminoglycans with inferior mechanical properties, and chondrocyte layering producing thinner tissue with increased mechanical properties. Overall, scaffold-free tissue engineering can generate seamless cylindrical cartilage constructs using roller bottle culture for future applications in long-segment tracheal replacement.

Inhibition of CD44 intracellular domain production suppresses bovine articular chondrocyte de-differentiation induced by excessive mechanical stress loading

CD44 fragmentation is enhanced in chondrocytes of osteoarthritis (OA) patients. We hypothesized that mechanical stress-induced enhancement of CD44-intracellular domain (CD44-ICD) production plays an important role in the de-differentiation of chondrocytes and OA. This study aimed to assess the relationship between CD44-ICD and chondrocyte gene expression. Monolayer cultured primary bovine articular chondrocytes (BACs) were subjected to cyclic tensile strain (CTS) loading. ADAM10 inhibitor (GI254023X) and γ-secretase inhibitor (DAPT) were used to inhibit CD44 cleavage. In overexpression experiments, BACs were electroporated with a plasmid encoding CD44-ICD. CTS loading increased the expression of ADAM10 and subsequent CD44 cleavage, while decreasing the expression of SOX9, aggrecan, and type 2 collagen (COL2). Overexpression of CD44-ICD also resulted in decreased expression of these chondrocyte genes. Both GI254023X and DAPT reduced the production of CD44-ICD upon CTS loading, and significantly rescued the reduction of SOX9 expression by CTS loading. Chemical inhibition of CD44-ICD production also rescued aggrecan and COL2 expression following CTS loading. Our findings suggest that CD44-ICD is closely associated with the de-differentiation of chondrocytes. Excessive mechanical stress loading promoted the de-differentiation of BACs by enhancing CD44 cleavage and CD44-ICD production. Suppression of CD44 cleavage has potential as a novel treatment strategy for OA.

Evaluation of Surface Properties and In Vitro Characterization of Surface Modified In Situ TiO2 Nanofibers

In situ TiO2 nanofiber arrays have been successfully produced directly on a Ti-6Al-4V substrate by using thermal oxidation under a limited supply of oxygen. Their morphology, elemental composition, crystal structure, surface roughness and surface wettability were characterized by field-emission scanning electron microscope (FESEM), energy-dispersive X-ray spectroscopy (EDX), X-ray diffractometer (XRD), atomic force microscope (AFM) and contact angle goniometer, respectively. The results of material characterization studies revealed that TiO2 nanofibers possessed greater surface roughness and wettability, as well as the degree of crystallinity. In vitro characterization have also been evaluated by using bovine articular chondrocytes on the resulting TiO2 nanofibrous surface at different time points. Cell adhesion was observed qualitatively by using FESEM and cell proliferation was determined quantitatively by using AlamarBlue reduction assay. The results showed that the TiO2 nanofibrous substrate triggers enhanced chondrocytes adhesion, proliferation, and production of extracellular matrix (ECM) fibrils compared to untreated substrate. These results suggest that the oxidation process produces a surface structure to which chondrocytes affinity, and thus this surface would has potential use in implants designed for cartilaginous applications.

Determination of Mechanical Properties of Chondrocytes in Articular Cartilage Using Atomic Force Microscopy

Atomic Force Microscopy (AFM) based nanoindentation is a widely used technique for measuring mechanical properties of living cells, providing information for understanding their mechanobiological behavior. However, very local properties of cell surfaces have not been characterized earlier. The goal of this study was to develop an AFM-based technique to determine local elastic properties of bovine articular chondrocytes. The Youngs modulus of chondrocytes was 19.3 ± 5.6 kPa for spread cells and 10 ± 4.1 kPa for the round cells. The results were compared to previous studies in which different techniques were used to obtain more global properties of chondrocytes. Our findings suggest that using nanosized AFM tips, the very local cell properties can be measured.