Cartilage Biopsy and Handling

The technology of autologous chondrocyte transplantation is based on the use of autologous cartilage for cell processing. The theoretical background to the autologous harvest and not other cell sources (e.g., bone marrow) is the fact that the joint and the articular cartilage have a unique developmental pathway in comparison to other cells with chondrogenic potential. The articular chondrocytes will have the capacity to form de novo cartilage that is halted before the hypertrophic differentiation and subsequent mineralization.

Theoretical Background

The joint consists of different anatomical structures with different cell types (i.e., bone, ligament, synovium, fibrous capsule, and articular cartilage). The cartilage provides the initial mold of the skeleton in the embryo; in the adult, cartilage provides a mechanical and structural support for breathing, articulation, locomotion, and hearing.

Formation of the Synovial Joint

The understanding of embryonic tissue formation will enable us to understand and control the processes of repair and regeneration in adult tissue. The first sign of joint formation in the embryo is the appearance of an interzone where the cells gives rise to the articular layer of the future long bones. It has been unclear whether the interzone cells derive from transdifferentiation of local prechondrocytes into interzone cells or from migration of mesenchymal cells into the joint site, or a combination.¹ The prechondrocytes in the interzone differentiate to early chondroblasts, thereafter becoming and remaining articular chondrocytes. However, recently studies have shred new light of the role of this population of cells expressing GDF5 (growth and differentiation factor 5; also known as CDMP-1 [cartilage-derived morphogenic protein 1]), a member of the transforming growth factor-beta family. The role of GDF5 expressing cells was demonstrated in ROSA-LacZ-reporter mice where GDF5 reporter expressing cells from the interzone from E13.5 were followed postnatally and remain present in structures that appear to arise from the interzone, namely the articular cartilage, synovium, and other joint tissues.²

This strengthens the hypothesis that there are two populations of cells with distinct phenotypes: the transient chondrocytes forming the epiphysis and the GDF5 expressing cells forming the articular cartilage during interzone formation. These facts support the established method of autologous cartilage biopsy as a source for chondrocytes isolation and subsequent cartilage regeneration in treated patients.

The Adult Articular Hyaline Cartilage

Two questions are often asked: if full thickness biopsies or only superficial biopsies can be taken, and if osteoarthritic cartilage or free bodies can be used.

The articular cartilage tissue is an avascular, noninnervated, and alymphatic tissue where the nutrition of the chondrocytes comes from passive diffusion. The only cell type in cartilage is the chondrocyte, constituting about 2% to 5% of the tissue. The function of chondrocytes is to build, maintain, and remodel the extracellular matrix composed of collagens, proteoglycans, noncollagenous proteins, and water. The articular cartilage has specialized load-bearing properties and the ability to withstand compressive, tensile, and shear forces because of the composition and structural integrity of its extracellular matrix (ECM), which consists of fibrillar and nonfibrillar macromolecules. The principal fibrillar component is collagen type II, which accounts for 90% to 95% of the collagen in articular cartilage; it forms a three-dimensional cross-linked network (together with smaller amounts of other minor collagens such as collagen types IX and XI). The collagen fibrils on the surface are oriented tangentially to create maximal strength, whereas deeper in the cartilage the fibrils are more vertically oriented. Collagen type X is exclusively produced by prehypertrophic and hypertrophic chondrocytes in the calcified layer.

The main nonfibrillar component consists of sulphated aggrecan monomers attached to hyaluronic acid forming large polyanionic aggregates. The collagen network is constructed in a complex three-dimensional network that acts to resist the osmotic pressure induced by the hydrophilic aggrecan monomers. If the collagen network fails, the cartilage will swell and lose its resilient properties. The small proteoglycans include decorin, biglycan, and fibromodulin. They seem not to contribute directly to the mechanical behavior of the tissue. Instead they bind to other macromolecules and probably influence cell function.

Articular cartilage also consists of noncollagenous matrix proteins, which are important for the interaction and assembly of the various macromolecules. Cartilage oligomeric protein (COMP) is a glycoprotein belonging to the thrombospondin family, also named thrombospondin 5. COMP interacts with collagen types I, II and IX, and it has been used as a diagnostic marker in serum for the progress of matrix degradation.³

Depending on matrix composition and cellular appearance, the articular cartilage is divided into several zones with different functional roles: superficial, transitional, radial, and calcified zones. Facing the joint cavity is the tangential layer/superficial zone with small, flattened cells parallel to the surface and mainly collagen type I fibers arranged tangentially to the articular surface and small amounts of proteoglycans. The surface is covered with a thin sheet of fine fibrils and cells lubricated by a thin layer of synovial fluid, sometimes called the “lamina splendens.” The production of the lubricin protein providing the frictionless surface of articular cartilage is a specific property for the cells at the surface of cartilage.⁴ Below the superficial zone is the transitional zone with larger rounded chondrocytes arranged in groups and as single cells. The matrix is composed almost entirely of proteoglycan and aggrecan, and is rich in collagens. The collagen fibrils are more randomly arranged than in the superficial zone. This zone displays the typical morphological features of hyaline cartilage. The zone is followed by the radial zone of the articular cartilage, where the cell density is lowest and the large chondrocytes form radial columns and produce a matrix rich in proteoglycans, especially aggrecan. The content of collagen is low. The tidemark is the zone separating the cartilage from the underlying bone, and below the tidemark is the calcified cartilage layer, with chondrocytes demonstrating hypertrophic phenotype and a matrix rich in type X collagen but without proteoglycans.

The growth properties of chondrocytes from different layers are poorly studied. The superficial cells express markers of stem cells or progenitor cells (e.g., the notch receptor). Deeper layers are more differentiated but the cloning capacity of cells in the middle or deep layer is not negligible. Based on these uncertainties a full thickness cartilage biopsy is preferred. Several limiting factors are associated with the use of chondrocytes from osteoarthritis (OA) joints, including the number of cells that can be obtained from a diseased tissue, the capacity of cells to proliferate in vitro, and the responsiveness to growth factors necessary to trigger redifferentiation process. Chondrocyte numbers are decreased by 38% in OA cartilage as assessed histomorphometrically and via the number of isolated cells. Whereas the proliferative capacity of chondrocytes in OA cartilage is increased in vitro and may account for chondrocyte clustering observed in vivo, results in vitro are still inconclusive given that both lower and higher proliferative rates have been reported. Despite all the identified differences, recent data indicate that OA chondrocytes retain their differentiation potential upon isolation and proliferation in vitro.⁵ In the micromass pellet cultures, OA chondrocytes continued to proliferate for 14 days, thus increasing the pellet size in contrast to normal chondrocytes. The proteoglycan production was comparable to normal chondrocytes, and the collagen-rich matrix was present, although the total collagen was significantly lower. Additionally, in a 3D-scaffold based on hyaluronic acid, OA chondrocytes were also able to produce cartilage-specific matrix proteins. These results raise hope that despite their differences in comparison to normal chondrocytes, OA chondrocytes could be employed as a cell source for tissue engineering (TE) treatment providing that the disease can be controlled. Recent data using human chondrocytes from patients with the history of trauma demonstrated that cells exposed to a hyaluronan-based scaffold reduced apoptosis and decreased gene expression as well as secretion of degradation cytokines, namely, MMP-1, and MMP-13. At the same time, the expression of cartilage-specific genes SOX9, collagen type II, and aggrecan indicated differentiation toward chondrogenesis.⁶

In conclusion, osteoarthritic cartilage contains cells that differ significantly from normal cartilage especially when studying inflammatory response. However, in culture, especially in cultures with hyaluronan matrices, the difference between normal and OA cartilage is negligible. Theoretically, using hyaluronan scaffolds (e.g., Hyaff-11, Fidia Advanced Biopolymers, Abano Terme, Italy), cartilage harvests could be considered. In technologies using monolayer cultures only where treatment is based on singe-cell solutions OA cartilage specimens are not recommended.