Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division: implications for cartilage tissue repair

Abstract

Emerging medical technologies for effective and lasting repair of articular cartilage include delivery of cells or cell-seeded scaffolds to a defect site to initiate de novo tissue regeneration. Biocompatible scaffolds assist in providing a template for cell distribution and extracellular matrix (ECM) accumulation in a three-dimensional geometry. A major challenge in choosing an appropriate scaffold for cartilage repair is the identification of a material that can simultaneously stimulate high rates of cell division and high rates of cell synthesis of phenotypically specific ECM macromolecules until repair evolves into steady-state tissue maintenance. We have devised a self-assembling peptide hydrogel scaffold for cartilage repair and developed a method to encapsulate chondrocytes within the peptide hydrogel. During 4 weeks of culture in vitro, chondrocytes seeded within the peptide hydrogel retained their morphology and developed a cartilage-like ECM rich in proteoglycans and type II collagen, indicative of a stable chondrocyte phenotype. Time-dependent accumulation of this ECM was paralleled by increases in material stiffness, indicative of deposition of mechanically functional neo-tissue. Taken together, these results demonstrate the potential of a self-assembling peptide hydrogel as a scaffold for the synthesis and accumulation of a true cartilage-like ECM within a three-dimensional cell culture for cartilage tissue repair.

Figure 1

(A) Molecular model of a single KLD-12 self-assembling peptide. The alternating hydrophobic and hydrophilic residues on the backbone promote β-sheet formation. The positively charged lysines (K) and negatively charged aspartic acids (D) are on the lower side of the β-sheet, and the hydrophobic leucines (L) are on the upper side. This molecular structure facilitates self-assembly through intermolecular interactions. (B) A 12-mm chondrocyte-seeded peptide hydrogel plug, punched from 1.6-mm-thick slabs. (C) Light microscope image of chondrocytes encapsulated in peptide hydrogel.

Figure 2

Radiolabel incorporation of ³H-proline and ³⁵S-sulfate as measures of the synthesis of proteins and sulfated proteoglycans, respectively, by chondrocytes within cell-seeded peptide and agarose hydrogels.

Figure 3

Matrix accumulation in chondrocyte-seeded peptide hydrogel. (A) Total GAG accumulation in cell-seeded peptide hydrogel cultured in FBS and ITS/FBS medium and in cell-seeded agarose. (B) Toluidine blue staining of chondrocyte-seeded peptide hydrogel cultured in 10% FBS, day 15. (C) Immunohistochemical staining for type II collagen in cell-seeded peptide hydrogel cultured in 10% FBS, day 15. Image width for B and C = 175 μm. (D) SDS/PAGE of collagens extracted from day 35 samples of chondrocyte-seeded peptide hydrogel cultured in 1% ITS with 0.2% FBS. Standards: Chick cartilage for collagen II and XI banding pattern. Mouse skin identifies collagen I α-helix 2, indicative of collagen expression of a dedifferentiated, fibroblastic phenotype.

Figure 4

Mechanical properties of chondrocyte-seeded peptide hydrogels. Equilibrium modulus and dynamic stiffness measured in uniaxial confined compression, evaluated in 6-mm diameter plugs initially seeded at a density of 15 × 10⁶ cells per ml and cultured in 10% FBS medium. * indicates 3-mm diameter plugs seeded at 30 × 10⁶ cells per ml and cultured in ITS/FBS medium, conditions that resulted in markedly increased stiffness values, as high as 20–33% that of human and animal cartilages (26).

Figure 5

Cell division of chondrocytes seeded in peptide hydrogel. (A) Calibration curve for the MTS assay in chondrocyte-seeded agarose hydrogel. (B) MTS measurement of the density of cells in chondrocyte-seeded peptide and agarose hydrogels. (C and D) BrdUrd incorporation in chondrocyte-seeded hydrogel 3 days (C) and 7 days (D) after seeding.