Date Published: April 01, 2017
Author(s): Alan R.F. Godwin, Tobias Starborg, Michael J. Sherratt, Alan M. Roseman, Clair Baldock.
Extracellular matrix microfibrils are critical components of connective tissues with a wide range of mechanical and cellular signalling functions. Collagen VI is a heteromeric network-forming collagen which is expressed in tissues such as skin, lung, blood vessels and articular cartilage where it anchors cells into the matrix allowing for transduction of biochemical and mechanical signals. It is not understood how collagen VI is arranged into microfibrils or how these microfibrils are arranged into tissues. Therefore we have characterised the hierarchical organisation of collagen VI across multiple length scales. The frozen hydrated nanostructure of purified collagen VI microfibrils was reconstructed using cryo-TEM. The bead region has a compact hollow head and flexible tail regions linked by the collagenous interbead region. Serial block face SEM imaging coupled with electron tomography of the pericellular matrix (PCM) of murine articular cartilage revealed that the PCM has a meshwork-like organisation formed from globular densities ∼30 nm in diameter. These approaches can characterise structures spanning nanometer to millimeter length scales to define the nanostructure of individual collagen VI microfibrils and the micro-structural organisation of these fibrils within tissues to help in the future design of better mimetics for tissue engineering.
Cartilage is a connective tissue rich in extracellular matrix molecules and is tough and compressive to cushion the bones of joints. However, in adults cartilage is poorly repaired after injury and so this is an important target for tissue engineering. Many connective tissues contain collagen VI, which forms microfibrils and networks but we understand very little about these assemblies or the tissue structures they form. Therefore, we have use complementary imaging techniques to image collagen VI microfibrils from the nano-scale to the micro-scale in order to understand the structure and the assemblies it forms. These findings will help to inform the future design of scaffolds to mimic connective tissues in regenerative medicine applications.
Articular cartilage protects the articulating joints during movement; cartilage is hypocellular and is mainly composed of extracellular matrix . In adults, cartilage is poor at repairing itself after trauma which can lead to degenerative cartilage diseases such as Osteoarthritis (OA). One method of repairing this damage would be to use tissue engineering to regenerate damaged cartilage by differentiating stem cells, such as adipose-derived adult stem cells or mesenchymal stem cells, into chondrocytes using scaffolds which mimic the biomechanical properties of cartilage , . This goal however, has proven difficult to achieve, with scaffolds not supporting chondrocyte maintenance often forming fibrous or fibrocartilaginous tissue instead of hyaline cartilage . It is therefore important to understand the structural and mechanical properties of the matrix surrounding chondrocytes to create better biomaterials to stimulate cartilage regeneration. A key component of the PCM surrounding chondrocytes in articular cartilage is collagen VI which is important for maintaining the biochemical and mechanical properties of cartilage , , and has been shown to enhance cartilage tissue regeneration .
Here we present the first 3D reconstruction of collagen VI microfibrils using cryo-TEM. The model has a compact hollow head region composed of four lobe-like structures which likely contain the ten C-terminal VWA domains from the three α-chains. The intermediate region connects the head region to the two tail regions which have a compact C-shape which could accommodate the N1 VWA domains from the three α-chains. The additional N-terminal domains from the long α3 chain are absent from the structure but present in the microfibrils as shown by SDS-PAGE. Therefore the loss of density is likely due to heterogeneity in this region caused by flexibility of these domains. Indeed, SAXS studies on recombinant N-terminal VWA domain arrays have shown them to be flexible . The observed heterogeneity is less likely to be caused by different splice variants of the α3 chain as SDS-PAGE and AFM volume analysis of the bead region suggested that it was made up of a single species. Previous studies of bovine corneal collagen identified that collagen VI microfibrils were composed of VWA C1-N6+N8 from the α3 chain  and mass spectroscopy analysis did not identify α4, 5 or 6 chains. Therefore due to flexibility in this region it is still not clear how the N-terminal VWA domains are arranged in the bead region of the microfibrils. Reconstructions presented here could resolve part of the collagenous region which connects the two half-beads and which was poorly defined in previous negative stain studies . It is likely that the collagenous regions go around the outside of the hollow head region but increasing the resolution of this structure, for example by utilising the more sensitive direct detection devices , may in future allow resolution of these regions.
In this study, we have imaged collagen VI at different levels of hierarchy and at different length scales from the 3D nanoscale structure of purified microfibrils to the microscale networks of collagen VI in situ in the PCM of articular cartilage. Understanding the tissue structure of collagen VI will give greater insights into the role of collagen VI in health and diseases such as OA as well as providing insights into the role of collagen VI in organising PCM structure. A greater understanding of these structures will also be useful in engineering better replacements for regenerative medicine applications.