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

Abstract

Introduction: The collagen structure in the superficial zone of articular cartilage is critical to the tissue's 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.

Methods: Articular 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.

Results: Normal 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.

Conclusion: A 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.

Figures

Figure 1

(A). A 3D image of normal cartilage (ICRS grade 0, shown in Fig. 1(E) from a cow femoral head at a lower magnification view using digital zooming shows more clearly that the structure of the interwoven collagen bundles (ICB) near the articular surface (the arrows in Fig 1(A)).(B). A 3D image of normal cartilage from a cow femoral condyle at a higher magnification view using digital zooming shows more clearly the orientation of the collagen fibres in the superficial zone, which is align predominantly in a spatial direction oblique the AC surface. (C): MBI reconstructed from the top eight of 2D images in Fig 1(A) shows clearly the interwoven collagen bundles near the articular surface. (D): The MBI reconstructed from the image stack used to reconstruct the 3D image in Fig 1(B) is analogous to an en face 2D observation, by which the collagen fibres in the superficial zone appear to align predominantly in a direction parallel to the AC surface in 2D images. (E): The corresponding traditional histology of a cow femoral condyle used for ICRS grading shows proteoglycans (blue) are highly deposited in normal cartilage.

Figure 2

(A). A semitransparent membrane corresponding to the lamina splendens (LS) was physically peeled off from normal articular cartilage (N) of a cow femoral head (unloading region).(B). A 3D image of the lamina splendens shows the collagen network within it is compromised of unique interwoven collagen bundles (ICB). (C). The corresponding MBI of the collagen network in LS in Fig (B). (D). Traditional histology shows the site where the lamina splendens was separated from the normal (cow) cartilage. (E). Traditional histology of early arthritic cartilage from a human femoral head shows disrupting the articular surface in early OA is a process similar to physically peeling off the lamina splendens. (F). Traditional histology of normal cartilage physically peeled the lamina splendens (indicated as CP (cartilage peeled lamina splendens) in Fig 2(A)) shows loss of the most superficial layer of articular cartilage can expose some chondrocytes near the surface to the joint cavity.

Figure 3

(A). In approximately half of aged cartilage specimens (from cadaver femoral heads) with little surface lesion (ICRS Grade 0, shown in Fig 3(C)), the collagen fibres in the superficial zone are oriented predominantly in a direction spatially oblique to the AC surface. However, the fibres are rarely integrated the interwoven collagen bundles on the surface. (B). The corresponding MBI of the collagen network is analogous to an en face 2D image. (C). Traditional histology shows the cartilage is almost at ICRS grade 0 but it contains less proteoglycans than the normal cartilage.

Figure 4

Approximately another fifty percent of the aged specimens (Fig 4(C)) from human femoral heads display a similar physiological condition (approximate ICRS Grade 1) to a small proportion of arthritic cartilage specimens (Figs 4(C1)) from human femoral heads and the cartilage (from cow femoral heads) physically peeled off the lamina splendens (Fig 4(C2)). These cartilage specimens, as shown in Figs (A), (A1) and (A2), also have a 3D collagen structure similar to each other and contain the collagen fibres that oriented predominantly in a spatial direction perpendicular to the AC surface. Figs 4(B), (B1) and (B2) are the corresponding MBI images, which are analogous to enface 2D images. Figs 4(C), (C1) and (C2) are the corresponding histology used for ICRS grading. The field of the 3D collagen network in images

Figure 5

(A). The 3D collagen network (33 μm × 33 um ×) of the cartilage with a matte surface (ICRS Grade 1–2 in Fig 5(C)) obtained from human femoral heads is disrupted and compromised of the collagen fibres aligning predominantly in a direction spatially perpendicular to the AC surface. (A1). The 3D collagen network (33 μm × 33 um) of fibrillated cartilage (ICRS grade 3 in Fig 5(C1)) has an abnormal microstructure and collagen orientation. Images (B)-(B1) are the corresponding MBIs of images (A) and (A1), which are analogous to en face 2D images. Images (C)-(C1) are the corresponding histological images used for ICRS grading.

Figure 6

A schematic structure of the collagen network in AC shows that the interwoven collagen bundles in the lamina splendens integrate the obliquely oriented collagen fibres and those in the deeper region to form a 3D collagen scaffold, which anchors to the subchondral bone. It is well accepted that the 3D collagen scaffold arched on the subchondral bone of AC. It reinforces the swelling pressure of proteoglycan (PG) gel to provide the AC with loading capacities and considerable tensile strength to withstand for wear and shear stresses. Peeling off the lamina splendens where the interwoven collagen bundles reside reduces the wear and shearing resistance of the AC. It also leads to change of the osmotic pressure in AC and gradually release of PGs to the joint cavity. The tensile strength and lateral integrity of the interwoven collagen bundles permitted peeling off the most superficial layer from AC. This explains why torn articular surface occurs during excessive sports and exercises.