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.
The health of a synovial joint is relied on normal function and coordination of a group of tissues such as articular cartilage (AC), ligaments, tendons and muscles. Osteoarthritis (OA), which is the most common joint disease, is clinically characterised by lesion of AC. Despite this, injury of a ligament or tendon or muscle generates a joint instability, which accelerates deterioration of AC and progression of OA. Traditional histology is often used to study the pathology of biological tissues. It requires tissue biopsy, which traumatises the donor tissues. Therefore, it is not an idea method for assessing AC, ligaments and tendons as the tissues have a poor healing capability. There is a worldwide demand of an imaging technique that diagnoses the microstructural changes of chondral and connective tissues without biopsy. Confocal arthroscopy (Optiscan Pty Ltd, Australia) possesses a Ø 6.3 mm probe and offers a 0.7 µm lateral imaging resolution and 7 µm axial resolution. It has been successfully used for examining the internal microstructural disorders in rotator cuff tendons of human cadavers without tissue biopsy (WU Four sheep keen joints were freshly donated by other research unrelated to this study. After 5 ml clinical grade fluorescein solution at 0.05 g/L was injected into the joint cavity of a knee joint, the joint was passively exercising for about 10 minutes. The joint was then open collaterally and washed thoroughly using PBS for acquiring the microstructure of AC, ligaments, tendons and muscles using the confocal arthroscopy. Results: without biopsy, confocal arthroscopy offers an imaging resolution for onsite distinguishing the subtle microstructural difference of AC in the weight-bearing and non-weight bearing region. It also permitted visualising the hierarchical collagen structure in ligaments and tendons at a fibre level, and characterising the muscle nuclei, motor-neurons, moto-neuron synapse and striates of myofibres. Confocal arthroscopy showed the early promise to act as optical histology for studying the microstructure of chondral and a range of connective tissues, which allows understand better the health status of a knee joint. Since a sheep knee joint is very small for operating a normal procedure of an arthroscopic examination, an open knee joint surgery was performed in this study to allow imaging the microstructure of AC and a range of connective tissues. This is accounted as a limitation in the study. Nevertheless, this study demonstrated the development of confocal arthroscopy may lead to optical histology of the internal microstructure of AC and a group of connective tissues, which offers understanding more comprehensively the healthy status of a knee joint.
A tendon is a fibrous connective tissue that acts to transmit tensile forces between muscles and bones. It mainly consists of soluble substance, collagen and small volume of elastic fibres, which are produced by tenoblasts and tenocytes. The Achilles tendon is the thickest tendon in the human body that subjects to some of the highest tensile force, thus disorders and ruptures commonly happen. As the insoluble fibrous components in Achilles tendons, the collagen fibrils and elastic fibres have unique spatial structure that plays important functional roles. Despite this, the understanding of relationship between them is still limited due to the lack of imaging evidence. Using confocal and second harmonic generation microscopy, this study aims to comprehensively investigate the spatial relationship of collagen, elastic fibres and tenocytes in hydrated tendons. Longitudinal sections of 50 µm thick and transverse sections of 20 µm thick were cryo-sectioned respectively from the mid-portion of ten rabbit Achilles tendons. Sections were stained with 0.03g/L Acridine Orange (AO) and 1mg/ml Sulforhodamine B (SRB) solution respectively for labelling the nucleus and elastic fibres. The Leica TCS SP2 multiphoton microscopy containing second harmonic generation microscopy can image collagen without labelling. The sections were scanned by the multiphoton microscopy, and images were processed and reconstructed into 3D images to study the spatial structure of collagen, elastic fibres and cells in Achilles tendons A rabbit Achilles tendon consists of three sub-tendons named flexor digitorum superficialis tendon, medial gastrocnemius tendon and lateral gastrocnemius tendon. Loose connective tissue connects the three sub-tendons and ensures efficient sliding between sub-tendons. The 3D network shows that the mid-portion of Achilles tendons is composed of longitudinal collagen and elastic fibres, while spindle tenocytes rest along the collagen and elastic fibres. Tenocytes appear to have a closer microstructural relationship with the elastic fibres. In comparison with the collagen, tenocytes and elastic fibres only occupy a very small volume in the 3D network. The elastic fibres exist in both tendon proper and endotenons. The tendon sheath and loose connective tissue have a higher cell density, and the cells are large and round while compared with tenocytes. As a component of the extracellular matrix (ECM) in Achilles tendons that closely mediates with the tenocytes, the elastin may participate in the force transition and interaction between tenocytes and the ECM. The elastic fibres may also endow Achilles tendons with unique mechanical properties to stand for tensile force.
