Abstract
High resolution imaging techniques such as atomic force microscopy, provide a platform to study the fibrillary architecture of biological tissues, but are not capable of imaging the internal microstructure of tissues in 3D. Conversely, multiphoton microscopes facilitate 3D imaging to study the spatial relationship of micro-components within tissues, but without the resolution of atomic force microscopy. The lamina splendens is the most superficial layer of articular cartilage. It is believed to play a crucial role in the health of the tissue. However, the precise form of this layer is uncertain as it has never been independently studied. Here, we use multiphoton microscopy and atomic force microscopy to demonstrate the anatomic form of the lamina splendens. The lamina splendens were peeled from the femoral condyles of healthy, adult sheep (n=20). Using atomic force microscopy, we show that the collagen and elastin form an interweaving fibrillary network at the surface of the lamina splendens and at the interface of the lamina splendens with the underlying cartilage. Moreover, using fluorescent stains; sulforhodamine B and acridine orange, multiphoton microscopy shows the heterogeneous distribution of collagen, elastin and chondrocytes throughout the depth of the lamina splendens. Our results demonstrate the fibrillary and component level architecture of the lamina splendens. We believe our findings provide the backbone of knowledge to advance tissue engineering techniques that will lead to more promising strategies to treat cartilage pathologies, including osteoarthritis. Furthermore, our results provide a starting point to determine the role of the lamina splendens in cartilage pathology.
