Barbara L. Schumacher

Human Septal Chondrocyte Redifferentiation in Alginate, Polyglycolic Acid Scaffold, and Monolayer Culture†

Objectives/Hypothesis Tissue engineering laboratories are attempting to create neocartilage that could serve as an implant material for structural support during reconstructive surgery. One approach to forming such tissue is to proliferate chondrocytes in monolayer culture and then seed the expanded cell population onto biodegradable scaffolds. However, chondrocytes are known to dedifferentiate after this type of monolayer growth and, as a result, decrease their production of cartilaginous extracellular matrix components such as sulfated glycosaminoglycans. The resultant tissue lacks the biomechanical properties characteristic of cartilage. The objective of the study was to determine whether different culture systems could induce monolayer‐expanded human septal chondrocytes to redifferentiate and form extracellular matrix.

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

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 P0 cells stained strongly for S-GAG and type II collagen, whereas decreased staining for both matrix components was observed in constructs derived from P1 and P2 cells. Cellularity, S-GAG content, and total collagen content of constructs increased significantly from day 7 to day 28. S-GAG accumulation in P0 constructs 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 al 1,2 more 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. 3 The 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. 4–9 Septal 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. 8–10 These 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. 8–10 When reimplanted into animal models, however, neocartilage constructs have uniformly lacked long-term structural integrity, demonstrating invasion by fibroblasts and loss of shape over time. 6,11 Unknown 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. 4,5 Thus 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 work 12,13 have 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. 14 Thus, 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.

Detection of Superficial Zone Protein in Human and Animal Body Fluids by Cross-Species Monoclonal Antibodies Specific to Superficial Zone Protein

In this report we describe the purification of human superficial zone protein (SZP), the generation of cross-species monoclonal antibodies (MAbs) and the detection of this protein in human and animal body fluids. Human SZPs, used as immunizing antigens, were purified either from culture media of human cartilage organ cultures or from human synovial fluids. The immunizing antigens were mixed with RIBI adjuvant in one of three forms: nonmodified SZP, superficial zone protein-keyhole limpet hemocyanin conjugate (SZP-KLH), or a mixture of superficial zone protein and hyaluronic acid (SZP-HA). A panel of MAbs including GW4.23, S6.79, S13.52, S13.233, and S17.109 were generated and characterized. Monoclonal antibody (MAb) S6.79, an IgG2b with K(D) 3.14 x 10(-9) M from SZP-KLH immunization, is of particular interest. It reacts strongly to a large molecular weight form of SZP in both enzyme-linked immunosorbent assay (ELISA) and Western blotting. It stains the most superficial layer of articular cartilage in immunohistochemistry, whereas the middle and deep zones of cartilage are not stained. When MAb S6.79 was applied to Western blots of human body fluids, a strong 345-kDa band was detected in samples of synovial fluid and weaker bands of similar size were detected in samples of plasma and serum. MAb S6.79 also showed cross-species immunoreactivity with SZP in samples of synovial fluids harvested from bovine, dog, guinea pig, and rabbit, as demonstrated by Western blotting and antibody absorption experiments. This cross-species MAb will be a useful tool in human and animal model studies for monitoring SZP levels and tissue distribution. It may help define the roles of SZP in normal articular joints and may be of diagnostic or prognostic value for the measurement of SZP in pathological conditions such as osteoarthritis, rheumatoid arthritis, and camptodactyly-arthropathy-coxa vara-pericarditis.

Human knee and ankle cartilage explants: catabolic differences

The prevalence of osteoarthritis (OA) is lower in some joints, i.e., the ankle, than in the knee. We have compared the cartilages from these two joints of the same limb in adult donors (matched pairs). Our data to date suggest that there are metabolic, biochemical and biomechanical differences between the cartilages of the two joints. The current study has focused on extending the metabolic studies comparing the response of chondrocytes to Interleukin‐1β (IL‐1β) and osteogenic protein 1 (OP‐1) by analyzing changes in sulfate incorporation into glycosaminoglycans (GAGs) as a measure of proteoglycan (PG) synthesis. Human adult chondrocytes from normal knees (tibiofemoral) and ankles (talocrural) joints cultured as explants both responded to IL‐1β after 72 h by decreasing PG synthesis; however, the IC50 for the knee chondrocytes was 6.2 pg/ml, while that for the ankle was 35 pg/ml. When the explants were incubated for 72 h with IL‐1β and allowed to rebound without IL‐1β, synthesis of PG was significantly elevated by ankle chondrocytes within five days; knee chondrocytes were unable to significantly increase synthesis even after eight days. However, in both knee and ankle, application of OP‐1 enhanced PG synthesis in the rebound phase. In response to IL‐1, an upregulation of proteinase activity was detectable by an increase in the neoepitopes proteolytically‐generated by both aggrecanase and matrix metalloproteinases (MMPs), in the deep zone of the knee cartilage. Stromelysin and collagenase were upregulated as well. The data emerging from these studies confirm that the ankle is less responsive to catabolic stimulation and more responsive to anabolic stimulation following IL‐1 removal. These differences in metabolic activity between the cartilages of the two joints could in part help to explain their differences in susceptibility to OA.

Tissue-Engineered Human Nasal Septal Cartilage Using the Alginate-Recovered-Chondrocyte Method†

Objectives Tissue engineering of nasal septal cartilage has numerous potential applications in craniofacial reconstruction. Chondrocytes suspended in alginate gel have been shown to produce a substantial cell‐associated matrix. The objective of this study was to determine whether cartilage tissue could be generated using the alginate‐recovered‐chondrocyte (ARC) method, in which chondrocytes are cultured in alginate as an intermediate step in tissue fabrication.

Horizontally oriented clusters of multiple chondrons in the superficial zone of ankle, but not knee articular cartilage.

Osteoarthritis is a progressive disease that is initiated at the surface of articular cartilage and proceeds to destroy the entire depth of the cartilage. The prevalence of osteoarthritis varies in different joints; e.g., the ankle joint has a very low prevalence of the disease compared to the knee joint. To better understand any inherent differences between the articular cartilage of the ankle and that of the knee that would account for the difference in occurrence of osteoarthritis, studies were undertaken to examine differences between the superficial zones in these two joint cartilages obtained from human donors. Chondrocytes in the superficial zones of the normal ankle (talocrural) and the normal knee (tibiofemoral) joints were identified with a monoclonal antibody specific for the superficial zone protein (SZP). When the chondrocytes from both joints were compared in serial horizontal sections, the chondrocytes in the superficial zone of the knee cartilage were seen either as isolated single cells or as doublets. However, the chondrocytes within the superficial zone of normal ankle cartilage were arranged in planar clusters containing multiple chondrons composed of 2–13 cells. There were no detectable differences in the chondrocyte clusters in the superficial zone of the ankle with respect to age, gender, or site on the cartilage surface. Adjacent to a lesion in an ankle joint with degenerative changes, the clusters were larger, containing up to 22 chondrocytes. This is the first report documenting the presence of multiple chondrons in the superficial zone of normal human adult articular cartilage. Anat Rec 266:241–248, 2002.

Effect of growth factors on cell proliferation, matrix deposition, and morphology of human nasal septal chondrocytes cultured in monolayer.

Objectives: Tissue engineering of septal cartilage provides ex vivo growth of cartilage from a patients own septal chondrocytes for use in craniofacial reconstruction. To become clinically applicable, it is necessary to rapidly expand a limited population of donor chondrocytes and then stimulate the production of extracellular matrix on a biocompatible scaffold. The objective of this study was to determine favorable serum‐free culture conditions for proliferation of human septal chondrocytes using various concentrations and combinations of four growth factors.

ARTICULAR CARTILAGE TENSILE INTEGRITY: MODULATION BY MATRIX DEPLETION IS MATURATION-DEPENDENT

Articular cartilage function depends on the molecular composition and structure of its extracellular matrix (ECM). The collagen network (CN) provides cartilage with tensile integrity, but must also remodel during growth. Such remodeling may depend on matrix molecules interacting with the CN to modulate the tensile behavior of cartilage. The objective of this study was to determine the effects of increasingly selective matrix depletion on tensile properties of immature and mature articular cartilage, and thereby establish a framework for identifying molecules involved in CN remodeling. Depletion of immature cartilage with guanidine, chondroitinase ABC, chondroitinase AC, and Streptomyces hyaluronidase markedly increased tensile integrity, while the integrity of mature cartilage remained unaltered after depletion with guanidine. The enhanced tensile integrity after matrix depletion suggests that certain ECM components of immature matrix serve to inhibit CN interactions and may act as modulators of physiological alterations of cartilage geometry and tensile properties during growth/maturation.

Human serum for tissue engineering of human nasal septal cartilage

Objective To compare the chondrogenic and proliferative effects of pooled human serum (HS) and fetal bovine serum (FBS) on tissue-engineered human nasal septal chondrocytes. Study Design and Setting Human chondrocytes were expanded for one passage in monolayer in medium supplemented with 10% FBS, 2% HS, 10% HS, or 20% HS. Cells were then suspended in alginate beads for 3D culture for 2 weeks with 10% FBS, 2% HS, 10% HS, or 20% HS. Results Monolayer cell yields were greater with HS than FBS. In alginate, cellular proliferation, glycosaminoglycan production per cell, and type II collagen were significantly higher with 10% HS compared to 10% FBS controls. Conclusion HS results in increased proliferation and production of cartilaginous extracellular matrix by tissue-engineered human nasal septal chondrocytes, compared to FBS controls. Significance Culture with human serum may facilitate creation of neocartilage constructs that more closely resemble native tissue.

Effects of equine joint injury on boundary lubrication of articular cartilage by synovial fluid: Role of hyaluronan

OBJECTIVE To compare equine synovial fluid (SF) from injured and control joints for cartilage boundary lubrication function; concentrations of the putative boundary lubricant molecules hyaluronan (HA), proteoglycan 4 (PRG4), and surface-active phospholipids (SAPLs); relationships between lubrication function and composition; and lubrication restoration by addition of HA. METHODS Equine SF from normal joints, joints with acute injury, and joints with chronic injury were analyzed for boundary lubrication of normal articular cartilage (kinetic friction coefficient [μ(kinetic) ]). Equine SF samples were analyzed for HA, PRG4, and SAPL concentrations and HA molecular weight distribution. The effect of the addition of HA, of different concentrations and molecular weight, on the μ(kinetic) of equine SF samples from normal joints and joints with acute injury was determined. RESULTS The μ(kinetic) of equine SF from joints with acute injury (0.036) was higher (+39%) than that of equine SF from normal joints (0.026). Compared to normal equine SF, SF from joints with acute injury had a lower HA concentration (-30%) of lower molecular weight forms, higher PRG4 concentration (+83%), and higher SAPL concentration (+144%). Equine SF from joints with chronic injury had μ(kinetic) , PRG4, and SAPL characteristics intermediate to those of equine SF from joints with acute injury and normal equine SF. Regression analysis revealed that the μ(kinetic) value decreased with increasing HA concentration in equine SF. The friction-reducing properties of HA alone improved with increasing concentration and molecular weight. The addition of high molecular weight HA (4,000 kd) to equine SF from joints with acute injury reduced the μ(kinetic) to a value near that of normal equine SF. CONCLUSION In the acute postinjury stage, equine SF exhibits poor boundary lubrication properties, as indicated by a high μ(kinetic) . HA of diminished concentration and molecular weight may be the basis for this, and adding HA to deficient equine SF restored lubrication function.