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 et al., 2015). This study investigates the capability of confocal arthroscopy as optical histology for assessing the internal microstructure of AC, ligaments, tendons and muscles in a knee joint.

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.

Introduction and Aims: Autologous chondrocyte implantation (ACI) is emerging as a leading technique for the treatment of articular cartilage defects. However, there exists some debate regarding which ACI technique is best able to regenerate hyaline cartilage. To this end, the development of a non-invasive technique enabling the examination of microstructure after ACI is essential.

Method: In this study, we have developed a novel 2D Laser Scanning Confocal Arthroscope (LSCA) in the assessment of articular cartilage and examined the microstructure of knee articular cartilage from rabbits and patients with total knee arthroplasty. The LSCA system consists of the LSA handheld probe, a Launch and Detection Unit (LDU) with a built in 488nm–514nm Krypton Argon Laser and Master Control unit (MCU). Human and rabbit knee articular cartilage stained with Fluoroscein (5g/L) and Acriflavine (0.5g/L) were used to examine the microstructure of cartilage by LSCA.

Results: By LSCA we have generated optical histology images of normal human and rabbit articular cartilage from the femoral condyle. Optical histology of normal articular cartilage tissue reveals typically smooth surface texture with relatively homogenous sub-surface distribution of viable chondrocyte cells. The general orientation of collagen fibres is occasionally visible in surface images. Optical histology of arthritic cartilage of humans showed clusters of round-shaped chondrocytes mixed with spindle-shaped cells. Surface cracking typically indicative of tissue damage is also evident by LSCA observation. Examination of rabbit knee six weeks after ACI showed high density of chondrocytes and homogeneous matrix on the site of the defect.

Conclusion: In short, we have shown the efficacy of LSCA in the non-destructive assessment of articular cartilage in vivo. Further study is required to evaluate the clinical significance of optical histology of LSCA.