Effects of Serial Expansion of Septal Chondrocytes on Tissue-Engineered Neocartilage Composition

Abstract

OBJECTIVES: Cartilage grafts for reconstructive surgery may someday be created from harvested autologous chondrocytes that are expanded and seeded onto biodegradable scaffolds in vitro. This study sought to quantify the biochemical composition of neocartilage engineered from human septal chondrocytes and to examine the effects of cell multiplication in monolayer culture on the ultimate composition of the neocartilage.METHODS: Human septal chondrocytes from 10 donors were either seeded immediately after harvest (passage 0 (P0)) onto polyglycolic acid (PGA) scaffolds or underwent multiplication in monolayer culture before scaffold seeding at passage 1 (P1) and passage 2 (P2). Cell/scaffold constructs were grown in vitro for 7, 14, and 28 days. Neocartilage constructs underwent histologic analysis for matrix sulfated glycosaminoglycan (S-GAG) and type II collagen as well as quantitative assessment of cellularity (Hoescht 33258 assay), S-GAG content (dimethylmethylene blue assay), and collagen content (hydroxyproline assay).RESULTS: Histologic sections of constructs seeded with P0cells stained strongly for S-GAG and type II collagen, whereas decreased staining for both matrix components was observed in constructs derived from P1and P2cells. Cellularity, S-GAG content, and total collagen content of constructs increased significantly from day 7 to day 28. S-GAG accumulation in P0constructs was higher than in either P1( P < 0.05) or P2( P < 0.01) constructs, whereas cellularity and total collagen content showed no difference between passages.CONCLUSION: Neocartilage created from chondrocytes that have undergone serial passages in monolayer culture exhibited decreased matrix S-GAG and type II collagen, indicative of cellular dedifferentiation.SIGNIFICANCE: The alterations of matrix composition produced by dedifferentiated chondrocytes may limit the mechanical stability of neocartilage constructs.The process of tissue engineering of cartilage was introduced by Vacanti et al1,2more than a decade ago as a potential solution to the limited supply of cartilage autografts available for reconstructive surgery. One strategy of tissue engineering is initiated by cartilage harvest from a donor site such as the nasal septum or the auricle. After digestion of the cartilage extracellular matrix, the chondrocytes can be isolated and grown in vitro using standard cell culture methods. Cell populations are expanded in culture to yield large numbers of chondrocytes. These cells can then be seeded onto biodegradable scaffolds and induced to deposit an extracellular matrix, thus forming new cartilage. Such neocartilage could potentially be implanted for structural support during reconstructive surgical procedures.There are several potential advantages of using tissue-engineered cartilage over native cartilage for autografting. First, the ability of chondrocytes to replicate in vitro allows for the expansion of cell numbers to produce theoretically limitless supplies of cartilage autografts. Furthermore, by varying the geometric configurations of the scaffolds, neocartilage autografts could potentially be designed in any desired size and shape. Finally, neocartilage constructs derived from autologous chondrocytes have a lower risk of immune rejection and infection transmission than that encountered with cartilage allografts or xenografts.Although a variety of scaffold materials exist, much research in tissue engineering has focused on scaffolds consisting of a nonwoven mesh of bioresorbable fibers such as polyglycolic acid (PGA), polylactic acid, or their copolymer.3The interlacing scaffold fibers provide a 3-dimensional structure to which cells can adhere. Chondrocytes grown in these scaffolds deposit extracellular matrix around themselves that is remodeled as the synthetic fibers degrade, theoretically creating cartilaginous tissue in the shape of the original scaffold.Chondrocytes for tissue engineering research have been obtained from a variety of sources, including articular, costal, nasal, and auricular cartilage from both animals and humans.4 – 9Septal cartilage can be harvested with less morbidity than articular or costal cartilage and has superior mechanical stability compared with elastic cartilage from the ear. Septal chondrocytes might therefore be used to form neocartilage that replicates the mechanical properties of native septal cartilage. Despite these advantages, limited studies have used septal chondrocytes for neocartilage formation on biodegradable scaffolds.8 – 10These studies have used histologic architecture as an outcome measure of successful neocartilage creation on scaffolds. Indeed, these authors have demonstrated the production of neocartilage that histologically resembles native cartilage.8 – 10When reimplanted into animal models, however, neocartilage constructs have uniformly lacked long-term structural integrity, demonstrating invasion by fibroblasts and loss of shape over time.6,11Unknown differences in the composition of neocartilage compared with native cartilage may be responsible for these phenomena.Native cartilage is composed of nests of cells embedded in an extensive extracellular matrix consisting of large proteoglycan molecules interwoven with collagen fibrils. Proteoglycans are macromolecules consisting of a protein core with hundreds of sulfated glycosaminoglycan side chains. These glycosaminoglycan chains consist of repeating disaccharide units whose highly negative charge attracts osmotically active cations. The osmotic ingression of water confers turgor to the tissue, which allows the cartilage to resist compressive forces. Collagen fibrils in the extracellular matrix serve a complementary mechanical function by resisting tensile forces. In hyaline cartilage, type II collagen predominates over other collagen subtypes, whereas the matrix of other connective tissues (skin, tendon, bone, ligaments) is primarily composed of type I collagen.Because the mechanical properties of cartilage are largely due to the composition and structure of its extracellular matrix, it is possible that a deficient matrix composition plays a role in the lack of neocartilage integrity to date. In support of this hypothesis is the observation that neocartilage engineered on scaffolds from calf articular or costal chondrocytes demonstrated less matrix glycosaminoglycan than native articular cartilage.4,5Thus far, no published reports have quantified the biochemical constituents of neocartilage constructed from human septal chondrocytes on biodegradable scaffolds.The distinction between data obtained from calf articular cells and adult human septal chondrocytes is important. It is unclear whether cells from different species will behave similarly under equivalent culture conditions. Furthermore, chondrocytes obtained from different anatomic locations may have different properties owing to the vastly different function served by cartilage in different areas (eg, load bearing in articular cartilage, rigid structural support for septal cartilage, and the deformation with elastic recoil characteristic of auricular cartilage). Finally, cells would be expected to grow and produce matrix in a manner that is dependent on donor maturity and age. Results from previous experimental work12,13have demonstrated an age-dependent decline in the synthesis of extracellular matrix components by cultured chondrocytes, suggesting that cells from juvenile subjects may have superior capacity for regenerating cartilage. Cells from younger subjects also are likely to expand in number faster and retain their chondrocyte phenotype for longer periods ex vivo. Thus, the translation of experimental data from fetal or juvenile animal chondrocytes to adult human septal cells is not straightforward.In addition, previous work has not routinely addressed another important issue relating to the practical application of cartilage engineering. As stated previously, one major advantage of tissue engineering is the potential ability to produce greater amounts of cartilage than are available from harvest during traditional autografting. This requires the expansion of cell numbers in culture, which potentially induces the chondrocytes to dedifferentiate. It is well established that with increasing passage, cultured chondrocytes exhibit a progressive transformation toward a more fibroblastic phenotype.14Thus, the effect on matrix formation of the necessary and potentially detrimental step of chondrocyte expansion remains to be established.The objective of the current study was to quantify the accumulation of the major cartilage matrix constituents in neocartilage engineered from adult human nasal septal chondrocytes. Additionally, the variation of the composition of neocartilage constructs from cell populations after expansion of cell numbers through multiple passages in culture was examined.