Insulin-like growth factor-I and growth differentiation factor-5 promote the formation of tissue-engineered human nasal septal cartilage

Abstract

Introduction: Tissue engineering of human nasal septal chondrocytes offers the potential to create large quantities of autologous material for use in reconstructive surgery of the head and neck. Culture with recombinant human growth factors may improve the biochemical and biomechanical properties of engineered tissue. The objectives of this study were to (1) perform a high-throughput screen to assess multiple combinations of growth factors and (2) perform more detailed testing of candidates identified in part I.

Methods: In part I, human nasal septal chondrocytes from three donors were expanded in monolayer with pooled human serum (HS). Cells were then embedded in alginate beads for 2 weeks of culture in medium supplemented with 2% or 10% HS and 1 of 90 different growth factor combinations. Combinations of insulin-like growth factor-I (IGF-1), bone morphogenetic protein (BMP)-2, BMP-7, BMP-13, growth differentiation factor-5 (GDF-5), transforming growth factor β (TGFβ)-2, insulin, and dexamethasone were evaluated. Glycosaminoglycan (GAG) accumulation was measured. A combination of IGF-1 and GDF-5 was selected for further testing based on the results of part I. Chondrocytes from four donors underwent expansion followed by three-dimensional alginate culture for 2 weeks in medium supplemented with 2% or 10% HS with or without IGF-1 and GDF-5. Chondrocytes and their associated matrix were then recovered and cultured for 4 weeks in 12 mm transwells in medium supplemented with 2% or 10% HS with or without IGF-1 and GDF-5 (the same medium used for alginate culture). Biochemical and biomechanical properties of the neocartilage were measured.

Results: In part I, GAG accumulation was highest for growth factor combinations including both IGF-1 and GDF-5. In part II, the addition of IGF-1 and GDF-5 to 2% HS resulted in a 12-fold increase in construct thickness compared with 2% HS alone (p < 0.0001). GAG and type II collagen accumulation was significantly higher with IGF-1 and GDF-5. Confined compression modulus was greatest with 2% HS, IGF-1, and GDF-5.

Conclusion: Supplementation of medium with IGF-1 and GDF-5 during creation of neocartilage constructs results in increased accumulation of GAG and type II collagen and improved biomechanical properties compared with constructs created without the growth factors.

FIG. 1.

Weight (A) and thickness (B) for alginate-recovered chondrocyte constructs in each of the five medium conditions in part II. Constructs created with IGF-1 and GDF-5 were significantly thicker and heavier than those created without growth factors (see Table 3 for p-values). For each condition, four constructs from each of four donors were measured. GDF-5, growth differentiation factor-5; IGF-1, insulin-like growth factor-I.

FIG. 2.

Biochemical properties of the alginate-recovered chondrocyte constructs for each of the five conditions in part II. DNA per construct (A), DNA per mg of tissue wet weight (B), GAG per wet weight (C), and type II collagen per wet weight (D) are shown. Insufficient material was available for analysis of type II collagen in the 2% HS condition. There was significantly greater GAG and type II collagen per mg of tissue in constructs created with 2% HS + IGF-1 + GDF-5 compared with each of the other conditions (see Table 3 for p-values). For each condition, measurements were made from two constructs from each of four donors. GAG, glycosaminoglycan; HS, human serum.

FIG. 3.

Immunohistochemistry staining of neocartilage constructs created with 2% HS + IGF-1 + GDF-5. There was abundant staining for type II collage with minimal staining for type I collagen and nonspecific IgG. Alcian blue staining reveals abundant GAG. Color images available online at www.liebertonline.com/ten.

FIG. 4.

Photographs of representative constructs. The 2% HS alone constructs had insufficient rigidity to handle. Color images available online at www.liebertonline.com/ten.

FIG. 5.

Confined compression modulus for neocartilage constructs created with IGF-1 and GDF-5. The confined compression modulus was significantly greater in the 2% HS + IGF-1 + GDF-5 condition compared with the 10% HS + IGF-1 + GDF-5 condition (p < 0.0001).