Characterization of human nasal septal chondrocytes cultured in alginate.

BACKGROUND: After serial passages in monolayer, chondrocytes dedifferentiate into a fibroblast-like phenotype. Our objective was to determine if culture in alginate affects the phenotype of dedifferentiated human nasal septal chondrocytes. STUDY DESIGN: Human nasal septal chondrocytes were seeded at low density and passaged in monolayer culture. At passages (P) 1, 2, and 3 a portion of cells were cultured in alginate. Collagen, glycosaminoglycan (GAG), and DNA production were assessed. RESULTS: Chondrocytes in alginate proliferated less yet produced higher levels of GAG and collagen than those in monolayer culture. Alginate encapsulated P1 chondrocytes stained strongly for GAG and collagen type II, and minimally for collagen type I. Monolayer cells at P0 and P1 stained positively for collagen type II. All monolayer passages stained positive for collagen type I with minimal GAG staining. CONCLUSIONS: Compared with monolayer culture, alginate stimulates deposition of GAG and collagen type II, and supports the chondrocyte phenotype through P1, but does not promote redifferentiation.

Review of injectable materials for soft tissue augmentation.

Although a variety of injectable agents are available for soft tissue augmentation, no perfect filler material exists. The advantages and disadvantages of commonly used materials such as bovine collagen have been well documented. However, many newer injectables are still undergoing experimentation to determine their clinical efficacy and long-term safety. This article details recent scientific work to compare injectable materials from the following categories: xenografts (bovine collagen and hyaluronic acid derivatives), autografts (autologous fat, Isolagen, and Autologen), homografts (Dermalogen and Cymetra), and synthetic materials (fluid silicone and Artecoll).

A compositional analysis of human nasal septal cartilage.

BACKGROUND: Nasal septal cartilage is well established as an autograft material. Tissue engineering methods are now being developed to synthesize cartilage constructs with the properties of this type of cartilage. However, important baseline data on the composition of native septal cartilage is sparse. OBJECTIVES: To characterize quantitatively the major biochemical constituents of native adult human septal cartilage and determine age- or sex-related variation in composition. METHODS: Cartilage was harvested from the inferior region of the nasal septum in 33 patients (mean +/- SD age, 47.0 +/- 13.5 years; range, 24-80 years) during routine septoplasty or septorhinoplasty. Biochemical assays were used to determine the quantities, relative to wet weight, of the major constituents of cartilage: water, collagen (from hydroxyproline), sulfated glycosaminoglycan (sGAG), and chondrocytes (from DNA). RESULTS: On average, each gram of wet cartilage contained 77.7% water, 7.7% collagen, 2.9% sGAG, and 24.9 million cells. Hydration and collagen content showed no significant age variation. Advancing age was associated with a reduction in sGAG content (7.7% per decade, P =.02) and cellularity (7.4% per decade, P =.05). No significant sex differences were found in any of these cartilage constituents. CONCLUSIONS: This study represents the first biochemical characterization of the composition of native human septal cartilage. The data serve as a baseline for future comparison of the properties of tissue-engineered neocartilage constructs. Furthermore, the age-associated variations in cartilage composition have implications for patient selection for reconstructive procedures.

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. STUDY DESIGN: Laboratory research. METHODS: Chondrocytes were isolated from human nasal septal cartilage of five donor patients (age, 35.8 +/- 9.3 y). Cell populations were seeded at low density (30,000 cells/cm2) into monolayer culture and expanded for 4 to 6 days. Following trypsin release, chondrocytes were placed into three different systems for neocartilage formation: alginate beads, polyglycolic acid scaffolds, and monolayer. After 7 and 14 days of growth, neocartilage was analyzed using histological and quantitative biochemical assessment of cellularity (Hoechst 33258 assay) and sulfated glycosaminoglycan content (dimethyl methylene blue assay). RESULTS: Histologically, alginate beads contained spherical chondrocytes surrounded by dense extracellular matrix, an appearance similar to that of native cartilage. In contrast, polyglycolic acid scaffolds and monolayer cultures contained elongated cells with scant staining for matrix sulfated glycosaminoglycans, which are features that are characteristic of dedifferentiated chondrocytes. Biochemical analysis demonstrated a lower level of cell proliferation (P <.001) in scaffolds (+52% over baseline) and alginate (+96% over baseline) than in monolayer (+366% over baseline), as well as a higher content of sulfated glycosaminoglycans per cell (P <.001), after 14 days of growth in alginate culture than in either polyglycolic acid scaffolds (19-fold difference) or monolayer (98-fold difference). CONCLUSIONS: Of the systems compared, monolayer-expanded human septal chondrocytes demonstrated the greatest accumulation of sulfated glycosaminoglycans per cell when grown in alginate beads. Future research on cartilage tissue engineering may use alginate culture for reverting dedifferentiated cells back to the chondrocytic phenotype.

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 [P(0)]) onto polyglycolic acid (PGA) scaffolds or underwent multiplication in monolayer culture before scaffold seeding at passage 1 (P(1)) and passage 2 (P(2)). 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 P(0) cells stained strongly for S-GAG and type II collagen, whereas decreased staining for both matrix components was observed in constructs derived from P(1) and P(2) cells. Cellularity, S-GAG content, and total collagen content of constructs increased significantly from day 7 to day 28. S-GAG accumulation in P(0) constructs was higher than in either P(1) (P < 0.05) or P(2) (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.