Systematic assessment of growth factor treatment on biochemical and biomechanical properties of engineered articular cartilage constructs

Abstract

Objective: To determine the effects of bone morphogenetic protein-2 (BMP-2), insulin-like growth factor (IGF-I), and transforming growth factor-beta1 (TGF-beta1) on the biochemical and biomechanical properties of engineered articular cartilage constructs under serum-free conditions.

Methods: A scaffoldless approach for tissue engineering, the self-assembly process, was employed. The study consisted of two phases. In the first phase, the effects of BMP-2, IGF-I, and TGF-beta1, at two concentrations and two dosage frequencies each were assessed on construct biochemical and biomechanical properties. In phase II, the effects of growth factor combination treatments were determined. Compressive and tensile mechanical properties, glycosaminoglycan (GAG) and collagen content, histology for GAG and collagen, and immunohistochemistry (IHC) for collagen types I and II were assessed.

Results: In phase I, BMP-2 and IGF-I treatment resulted in significant, >1-fold increases in aggregate modulus, accompanied by increases in GAG production. Additionally, TGF-beta1 treatment resulted in significant, approximately 1-fold increases in both aggregate modulus and tensile modulus, with corresponding increases in GAG and collagen content. In phase II, combined treatment with BMP-2 and IGF-I increased aggregate modulus and GAG content further than either growth factor alone, while TGF-beta1 treatment alone remained the only treatment to also enhance tensile properties and collagen content.

Discussion: This study determined systematically the effects of multiple growth factor treatments under serum-free conditions, and is the first to demonstrate significant increases in both compressive and tensile biomechanical properties as a result of growth factor treatment. These findings are exciting as coupling growth factor application with the self-assembly process resulted in tissue engineered constructs with functional properties approaching native cartilage values.

Fig. 1

Schematic diagram indicating experimental designs of phases I and II. The experimental design depicted in phase I was carried out for each individual growth factor separately (blocked by growth factor). The best treatment for each growth factor was selected for phase II. Phase II assessed the effects of each growth factor individually and in all combinations of two and three.

Fig. 2

Photomicrographs of collagen and GAG staining for no growth factor control constructs, BMP-2 + IGF-I treated constructs, and TGF-β1 treated constructs. 10× original magnification.

Fig. 3

Biomechanical and biochemical properties of BMP-2 treated constructs in phase I. All BMP-2 treatments significantly increased (a) aggregate modulus with no effect on (b) Young’s modulus. Likewise, all BMP-2 treatments significantly increased (c) GAG/WW with no effect on (d) collagen/WW. Columns and error bars represent means and standard deviations. Groups denoted by different letters are significantly different (p<0.05).

Fig. 4

Biomechanical and biochemical properties of IGF-I treated constructs in phase I. All IGF-I treatments, except 10 ng/ml continuous, significantly increased (a) aggregate modulus with no effect on (b) Young’s modulus. Likewise, all IGF-I treatments, except 10 ng/ml continuous, significantly increased (c) GAG/WW with no effect on (d) collagen/WW. Columns and error bars represent means and standard deviations. Groups denoted by different letters are significantly different (p<0.05).

Fig. 5

Biomechanical and biochemical properties of TGF-β1 treated constructs in phase I. TGF-β1 treatment at 30 ng/ml and 2-wk continuous dosage significantly increased (a) aggregate modulus and (b) Young’s modulus, with corresponding increases in (c) GAG/WW and (d) collagen/WW. Columns and error bars represent means and standard deviations. Groups denoted by different letters are significantly different (p<0.05).

Fig. 6

Biomechanical and biochemical properties of constructs in phase II. Combined treatment with BMP-2 and IGF-I led to the greatest enhancement of aggregate modulus and GAG/WW, while TGF-β1 alone was the only treatment to enhance both compressive and tensile stiffness. (a) aggregate modulus, (b) Young’s modulus, (c) GAG/WW and (d) collagen/WW. Columns and error bars represent means and standard deviations. Groups denoted by different letters are significantly different (p<0.05).