Speaker
Description
Purpose
The primary role of articular cartilage (AC) is to provide frictionless joint movement while transferring loads to the underlying bone [1]. Thanks to its unique hierarchical arcade-like collagen type II fiber organization, AC withstands extreme mechanical forces. While numerous regenerative medicine approaches strive to replicate the native architecture of AC, none have achieved reproduced this characteristic structure. This study explores collagen type II orientation in engineered microtissues to further understand driving forces for collagen type II alignment.
Methods and Materials
Negative molds of different geometrical shapes (circular, triangular, square) were fabricated in different diameters (diameter =150, 200, and 300 µm) using the stereolithography 3D printing technique (SLA, Formlabs). Subsequently, microwells were prepared using 3D printed molds and agarose. Articular cartilage progenitor cells (ACPCs) were seeded into the microwells and cultured for 28 days. Cells were observed by light (Olympus BX-43) and fluorescence microscopy (Leica Thunder Live Cell Imager). Moreover, deposition of extracellular matrix proteins such as collagen type II and glycosaminoglycans were evaluated by (immuno)histochemistry and biochemistry. Also, samples were stained with picrosirius red and collagen fiber organization was observed with polarized light microscopy (Olympus BX-50, U-POT filter).
Results and Discussion
ACPCs self-assembled into cellular aggregates in geometries reflecting predefined shapes during 28- days cell culture period. Fluorescence images show the formation of the cellular aggregates in triangular, square and circular shapes with cytoskeleton morphology and collagen type II deposition on days 3 and 28 (Figure 1A). Histological evaluations confirmed the production of cartilaginous extracellular matrix, indicating collagen type II deposition (Figure 1B). Moreover, polarized light imaging revealed an initial circular alignment of collagen fibers, regardless of the predefined outer geometries. During maturation of the microtissues, collagen alignment became concentrated specifically at the outer edges of the predefined shapes (Figure 1B). Furthermore, upward tissue growth was observed in the vertical sections of histology slides (Figure 1C).
Consclusion
This study demonstrates a proof-of-principle of guided collagen type II deposition through ACPCs differentiation within predefined shapes, providing insights into how this shape-driven tissue formation can be adopted in biofabrication strategies to engineer functional and long-lasting AC grafts.
Acknowledgements
The authors would like to acknowledge the financial support from the Gravitation Program “Materials Driven Regeneration”, funded by the Netherlands Organization for Scientific Research (024.003.013) and the LS-NeoCare project (project number NWA.1389.20.192).
References
[1] Benninghoff, A. Form und Bau der Gelenkknorpel in ihren Beziehungen zur Funktion. Z.Zellforsch 2, 783–862 (1925).
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