Jian Ping Wu

Study of the collagen structure in the superficial zone and physiological state of articular cartilage using a 3D confocal imaging technique

IntroductionThe collagen structure in the superficial zone of articular cartilage is critical to the tissues durability. Early osteoarthritis is often characterized with fissures on the articular surface. This is closely related to the disruption of the collagen network. However, the traditional histology can not offer visualization of the collagen structure in articular cartilage because it uses conventional optical microscopy that does not have insufficient imaging resolution to resolve collagen from proteoglycans in hyaline articular cartilage. This study examines the 3D collagen network of articular cartilage scored from 0 to 2 in the scoring system of International Cartilage Repair Society, and aims to develop a 3D histology for assessing early osteoarthritis.MethodsArticular cartilage was visually classified into five physiological groups: normal cartilage, aged cartilage, cartilage with artificial and natural surface disruption, and fibrillated. The 3D collagen matrix of the cartilage was acquired using a 3D imaging technique developed previously. Traditional histology was followed to grade the physiological status of the cartilage in the scoring system of International Cartilage Repair Society.ResultsNormal articular cartilage contains interwoven collagen bundles near the articular surface, approximately within the lamina splendens. However, its collagen fibres in the superficial zone orient predominantly in a direction spatially oblique to the articular surface. With age and disruption of the articular surface, the interwoven collagen bundles are gradually disappeared, and obliquely oriented collagen fibres change to align predominantly in a direction spatially perpendicular to the articular surface. Disruption of the articular surface is well related to the disappearance of the interwoven collagen bundles.ConclusionA 3D histology has been developed to supplement the traditional histology and study the subtle changes in the collagen network in the superficial zone during early physiological alteration of articular cartilage. The fibre confocal imaging technology used in this study has allowed developing confocal arthroscopy for in vivo studying the chondrocytes in different depth of articular cartilage. Therefore, the current study has potential to develop an in vivo 3D histology for diagnosis of early osteoarthritis.

High-resolution measurements of the multilayer ultra-structure of articular cartilage and their translational potential.

Current musculoskeletal imaging techniques usually target the macro-morphology of articular cartilage or use histological analysis. These techniques are able to reveal advanced osteoarthritic changes in articular cartilage but fail to give detailed information to distinguish early osteoarthritis from healthy cartilage, and this necessitates high-resolution imaging techniques measuring cells and the extracellular matrix within the multilayer structure of articular cartilage. This review provides a comprehensive exploration of the cellular components and extracellular matrix of articular cartilage as well as high-resolution imaging techniques, including magnetic resonance image, electron microscopy, confocal laser scanning microscopy, second harmonic generation microscopy, and laser scanning confocal arthroscopy, in the measurement of multilayer ultra-structures of articular cartilage. This review also provides an overview for micro-structural analysis of the main components of normal or osteoarthritic cartilage and discusses the potential and challenges associated with developing non-invasive high-resolution imaging techniques for both research and clinical diagnosis of early to late osteoarthritis.

Microstructural analysis of collagen and elastin fibres in the kangaroo articular cartilage reveals a structural divergence depending on its local mechanical environment

OBJECTIVEnTo assess the microstructure of the collagen and elastin fibres in articular cartilage under different natural mechanical loading conditions and determine the relationship between the microstructure of collagen and its mechanical environment.nnnMETHODnArticular cartilage specimens were collected from the load bearing regions of the medial femoral condyle and the medial distal humerus of adult kangaroos. The microstructure of collagen and elastin fibres of these specimens was studied using laser scanning confocal microscopy (LSCM) and the orientation and texture features of the collagen were analysed using ImageJ.nnnRESULTSnA zonal arrangement of collagen was found in kangaroo articular cartilage: the collagen fibres aligned parallel to the surface in the superficial zone and ran perpendicular in the deep zone. Compared with the distal humerus, the collagen in the femoral condyle was less isotropic and more clearly oriented, especially in the superficial and deep zones. The collagen in the femoral condyle was highly heterogeneous, less linear and more complex. Elastin fibres were found mainly in the superficial zone of the articular cartilage of both femoral condyle and distal humerus.nnnCONCLUSIONSnThe present study demonstrates that the collagen structure and texture of kangaroo articular cartilage is joint-dependent. This finding emphasizes the effects of loading on collagen development and suggests that articular cartilage with high biochemical and biomechanical qualities could be achieved by optimizing joint loading, which may benefit cartilage tissue engineering and prevention of joint injury. The existence of elastin fibres in articular cartilage could have important functional implications.

Elastin fibers display a versatile microfibril network in articular cartilage depending on the mechanical microenvironments

Elastin fibers are major extracellular matrix macromolecules that are critical in maintaining the elasticity and resilience of tissues such as blood vessels, lungs and skins. However, the role of elastin in articular cartilage is poorly defined. The present study investigated the organization of elastin fiber in articular cartilage, its relationship to collagen fibers and the architecture of elastin fibers from different mechanical environments by using a kangaroo model. Five morphologies of elastin fibers were identified: Straight fiber, straight fiber with branches, branching fibers directly associated with chondrocyte, wave fiber and fine elastin. The architecture of the elastin network varied significantly with cartilage depth. In the most superficial layer of tibial plateau articular cartilage, dense elastin fibers formed a distinctive cobweb‐like meshwork which was parallel to the cartilage surface. In the superficial zone, elastin fibers were well organized in a preferred orientation which was parallel to collagen fibers. In the deep zone, no detectable elastin fiber was found. Moreover, differences in the organization of elastin fibers were also observed between articular cartilage from the tibial plateau, femoral condyle, and distal humerus. This study unravels the detailed microarchitecture of elastin fibers which display a well‐organized three‐dimensional versatile network in articular cartilage. Our findings imply that elastin fibers may play a crucial role in maintaining the integrity, elasticity, and the mechanical properties of articular cartilage, and that the local mechanical environment affects the architectural development of elastin fibers.

Confocal arthroscopic assessment of osteoarthritis in situ.

PURPOSEnThis study aimed to assess the ability of the laser scanning confocal arthroscope (LSCA) to evaluate cartilage microstructure, particularly in differentiating stages of human osteoarthritis (OA) as classified by the International Cartilage Repair Society (ICRS) OA grade definitions.nnnMETHODSnTen tibial plateaus from total knee arthroplasty patients were obtained at the time of surgery. Cartilage areas were visually graded based on the ICRS classification, imaged by use of a 7-mm-diameter LSCA (488-nm excitation with 0.5% [wt/vol] fluorescein, 20-minute staining period), and then removed with underlying bone for histologic examination with H&E staining. The 2 imaging techniques were then compared for each ICRS grade to ascertain similarity between the methods and thus gauge the techniques diagnostic resolution. Cartilage surface degeneration was readily imaged and OA severity accurately gauged by the LSCA and confirmed by histology.nnnRESULTSnLSCA and histologic images of specimens in the late stages of OA were seen to be mutually related even though they were imaged in planes that were orthogonal to each other. Useful and comparable diagnostic resolution was obtained in all imaged specimens from subjects with various stages of OA.nnnCONCLUSIONSnThis study showed the LSCAs ability to image detailed cartilage surface morphologic features that identify grade 1 through 4 of the ICRS OA grading system. The LSCAs imaging potential was best shown by its ability to resolve the fine collagen network present under the lamina splendens. The incorporation of high-magnification confocal technology within the confines of an arthroscopic probe has proved to provide the imaging requirements necessary to perform detailed cartilage condition assessment.nnnCLINICAL RELEVANCEnIn comparison to video arthroscopy, LSCA provides increased magnification along with improved contrast and resolution.

The development of confocal arthroscopy as optical histology for rotator cuff tendinopathy

MRI, ultrasound and video arthroscopy are traditional imaging technologies for noninvasive or minimal invasive assessment of the rotator cuff tendon pathology. However, these imaging modalities do not have sufficient resolution to demonstrate the pathology of rotator cuff tendons at a microstructural level. Therefore, they are insensitive to low‐level tendon diseases. Although traditional histology can be used to analyze the physiology of rotator cuff tendons, it requires biopsy that traumatizes the rotator cuff, thus, potentially comprising the mechanical properties of tendons. Besides, it cannot offer real‐time histological information. Confocal endoscopy offers a way to assess the microstructural disorder in tissues without biopsy. However, the application of this useful technique for detecting low‐level tendon diseases has been restricted by using clinical grade fluorescent contrast agent to acquire high‐resolution microstructural images of tendons. In this study, using a clinical grade sodium fluorescein contrast agent, we have reported the development of confocal arthroscopy for optical histological assessment without biopsy. The confocal arthroscopic technique was able to demonstrate rotator cuff tendinopathy in human cadavers, which appeared macroscopically normal under video arthroscopic examinations. The tendinopathy status of the rotator cuff tendons was confirmed by corresponding traditional histology. The development of confocal arthroscopy may provide a minimally invasive imaging technique for real‐time histology of rotator cuff without the need for tissue biopsy. This technique has the potential for surgeons to gain in real time the histological information of rotator cuff tendons, which may assist planning repair strategies and potentially improve intervention outcomes.

Microstructural and compositional features of the fibrous and hyaline cartilage on the medial tibial plateau imply a unique role for the hopping locomotion of kangaroo.

Hopping provides efficient and energy saving locomotion for kangaroos, but it results in great forces in the knee joints. A previous study has suggested that a unique fibrous cartilage in the central region of the tibial cartilage could serve to decrease the peak stresses generated within kangaroo tibiofemoral joints. However, the influences of the microstructure, composition and mechanical properties of the central fibrous and peripheral hyaline cartilage on the function of the knee joints are still to be defined. The present study showed that the fibrous cartilage was thicker and had a lower chondrocyte density than the hyaline cartilage. Despite having a higher PG content in the middle and deep zones, the fibrous cartilage had an inferior compressive strength compared to the peripheral hyaline cartilage. The fibrous cartilage had a complex three dimensional collagen meshwork with collagen bundles parallel to the surface in the superficial zone, and with collagen bundles both parallel and perpendicular to the surface in the middle and deep zones. The collagen in the hyaline cartilage displayed a typical Benninghoff structure, with collagen fibres parallel to the surface in the superficial zone and collagen fibres perpendicular to the surface in the deep zone. Elastin fibres were found throughout the entire tissue depth of the fibrous cartilage and displayed a similar alignment to the adjacent collagen bundles. In comparison, the elastin fibres in the hyaline cartilage were confined within the superficial zone. This study examined for the first time the fibrillary structure, PG content and compressive properties of the central fibrous cartilage pad and peripheral hyaline cartilage within the kangaroo medial tibial plateau. It provided insights into the microstructure and composition of the fibrous and peripheral hyaline cartilage in relation to the unique mechanical properties of the tissues to provide for the normal activities of kangaroos.

The three dimensional microstructural network of elastin, collagen and cells in Achilles tendons

Similar to most biological tissues, the biomechanical, and functional characteristics of the Achilles tendon are closely related to its composition and microstructure. It is commonly reported that type I collagen is the predominant component of tendons and is mainly responsible for the tissues function. Although elastin has been found in varying proportions in other connective tissues, previous studies report that tendons contain very small quantities of elastin. However, the morphology and the microstructural relationship among the elastic fibres, collagen, and cells in tendon tissue have not been well examined. We hypothesize the elastic fibres, as another fibrillar component in the extracellular matrix, have a unique role in mechanical function and microstructural arrangement in Achilles tendons. It has been shown that elastic fibres present a close connection with the tenocytes. The close relationship of the three components has been revealed as a distinct, integrated and complex microstructural network. Notably, a “spiral” structure within fibril bundles in Achilles tendons was observed in some samples in specialized regions. This study substantiates the hierarchical system of the spatial microstructure of tendon, including the mapping of collagen, elastin and tenocytes, with 3‐dimensional confocal images.

Study of Altered Mechanical Properties of Articular Cartilage in Relation to the Collagen Network.

Articular cartilage is a semitransparent elastic material that covers on the two articulating bones in synovial joints. It acts as a cushion between the bones that transfers loads from one to another while attenuating dynamic stresses and providing almost frictionless contact surfaces for normal use of synovial joints without pains. Osteoarthritis causes a chronic joint pain and it is mainly due to malfunction of articular cartilage. The mechanical function of articular cartilage is derived from its unique microstructure. Therefore, study of the relationship between the mechanical function and microstructure of articular cartilage comprehends the aetiology and pathology of osteoarthritis. Confocal microscopy permits studying the internal microstructure of buck biological tissues without tissue sectioning and dehydration. This provides a way to study the relationship between the mechanical function and microstructure of articular cartilage. Using a fibre optic laser scanning confocal microscope, this study examines the pathological status of articular cartilage in relation to the mechanical function and 3D collagen network of articular cartilage. The results show that the 3D collagen structure and the mechanical function are different between normal and arthritic cartilage. Loss of the integrity of the 3D collagen network is closely related to cartilage softening.

Three-dimensional printing of shape memory hydrogels with internal structure for drug delivery

Hydrogels with shape memory behavior and internal structure have wide applications in fields ranging from tissue engineering and medical instruments to drug delivery; however, creating the hydrogels has proven to be extremely challenging. This study presents a three-dimensional (3D) printing technology to fabricate the shape memory hydrogels with internal structure (SMHs) by combining sodium alginate (alginate) and pluronic F127 diacrylate macromer (F127DA). SMHs were constituted by a dual network structure. One is a stable network which is formed by F127DA photo-crosslinking; the other one is a reversible network which is formed by Ca2+ cross-linked alginate. SMHs recovery ratio was 98.15% in 10min after Ca2+ was removed in the Na2CO3 solution, and the elastic modulus remains essentially stable after the shape memory cycle. It showed that the drug releasing rate is more rapid compared with traditional drug-loaded hydrogels in in vitro experiments. The viability of 3T3 fibroblasts remained intact which revealed its excellent biocompatibility. Therefore, SMHs have a huge prospect for application in drug carriers and tissue engineering scaffold.