Narrow Results

The most exciting discoveries in the last two decades have been the mapping, sequencing, and understanding of genes in our body. In this chapter, we will discuss how to specifically map expression profiles of gene products — mRNA, protein, and reporter gene (X-gal) — using in situ hybridization, immunohistochemistry, and X-gal staining, respectively.

Cartilage damage is irreversible due to its properties of avascularity and lack of undifferentiated cells. Studying the biology of chondrocytes and cartilage is therefore critical to understand the underlying mechanism and to explore the potential repair approaches. It has been reported that three-dimensional (3D) chondrocyte culture behaves very differently from two-dimensional (2D) monolayer culture. Therefore, a native 3D culture of chondrocytes is valuable for such a research purpose, and may also be an option for repairing cartilage defects. This chapter describes a detailed protocol for high-yield chondrocyte isolation and the modified pellet culture technique (using a 3D chondrocyte culture), which can synthesize a bioengineered tissue up to 8 mm in diameter. The authors aim at providing a platform for researchers to study chondrocyte behavior in a 3D environment and to explore the application of scaffold-free tissue for transplantation into a large cartilage defect model.

Perhaps the most difficult task in bone histology work is the preparation of a good specimen. It is impossible to obtain a good image of bone or cartilage without careful preparation and processing of specimens. This chapter will describe very basic skills in the preparation of specimens. These techniques include the paraffin method, plastic method, and frozen method.

Bone or cartilage cells are essentially colorless, and it is difficult to distinguish their morphologies under a light microscope. To better visualize these structures, various staining techniques have been developed; due to page constraints, we will only describe the most common methods for readers. These staining assays include hematoxylin and eosin (H&E) stain, used for general histology of the cell; Safranin O stain, used for the cartilage; and von Kossa and van Gieson stains as well as modified Goldner's trichrome stain, used for nondecalcified tissues.

Attrition and eventual loss of articular cartilage are crucial elements in the pathophysiology of osteoarthritis. Preventing the breakdown of cartilage is believed to be critical in order to preserve the functional integrity of a joint. Magnetic resonance imaging (MRI) and advanced digital postprocessing techniques have opened novel possibilities for in vivo quantitative analysis of cartilage morphology, structure, and function in health and disease. Techniques of semiquantitative scoring of human knee cartilage pathology and quantitative assessment of human cartilage have recently been developed. Though cartilage represents a thin layer of material relative to the size of voxels typically used for MRI, cartilage thickness and volume have been quantified in human and in small animals. MRI-detected cartilage loss has been shown to be more sensitive than radiography-detected joint space narrowing. Progress made in MRI technology in the last few years allows longitudinal studies of knee cartilage with an accuracy good enough to follow disease-caused changes and to evaluate the therapeutic effects of chondroprotective drugs.

Articular cartilage is hyaline cartilage which has very limited self-repairingcapacity after its degeneration or injury. Recently, three-dimensional (3D) bioprinting provides a promising method for repair and regeneration of articular cartilage. Significant progress has been made in 3D bioprinting for cartilage regeneration, particularly in printing hydrogels in combination of cells and growth factors. In this chapter, we reviewed recent progress in cartilage 3D bioprinting, including the use of various cell sources and growth factors for cartilage formation. We also discussed the challenges and the future research directions of cartilage regeneration.