Self-Assembling Peptide Hydrogels Promote in Vitro Chondrogenesis of Bone Marrow-Derived Stromal Cells: Effects of Peptide Sequence, Cell Donor Age, and Method of Growth Factor Delivery

The inability of articular cartilage to heal after damage or disease has motivated investigation of novel cartilage tissue engineering technologies. The objective of this thesis was to advance the use of self-assembling peptide hydrogel scaffolds for cartilage repair by encapsulating bone-marrow-stromal cells (BMSCs) and incorporating chondrogenic cues to stimulate differentiation and neotissue production.

To test the hypothesis that self-assembling peptide hydrogels provide cues which enhance the chondrogenic differentiation of BMSCs, a technique for rapid, high-viability BMSC encapsulation was developed. BMSCs were cultured in two peptide hydrogel sequences and compared to agarose hydrogels. BMSCs in all three hydrogels underwent TGF-β1-mediated chondrogenesis as demonstrated by comparable gene expression and ECM biosynthesis. Cell proliferation occurred only in the peptide hydrogels, not in agarose, resulting in higher sulfatedglycosaminoglycan content and more spatially uniform proteoglycan and type II collagen deposition. These data showed that self-assembling peptide hydrogels enhance chondrogenesis compared to agarose.

To evaluate the capacity for BMSCs from young and adult equine donors to produce cartilage-like ECM, neotissue formation was compared to that for animal-matched primary chondrocytes. Young chondrocytes stimulated by TGF-β1 accumulated ECM with higher sulfated-glycosaminoglycan content than adult chondrocytes and BMSCs of either age. BMSCs produced neotissue with higher dynamic stiffness than young chondrocytes. Measurement of aggrecan core-protein and chondroitin-sulfate length by atomic-force microscopy revealed BMSCs produce longer core protein and chondroitin-sulfate, and fewer catabolic-cleavage products than chondrocytes. Therefore, BMSC-produced aggrecan appears to have a younger phenotype than chondrocyte-produced aggrecan. These advantages make BMSCs a potentially superior cell source for peptide-hydrogel-based cartilage repair.

To deliver TGF-β1 to BMSCs via a bioactive scaffold, BMSCs were encapsulated in peptide hydrogels with both tethered and adsorbed TGF-β1 and cultured in TGF-β1-free medium. Chondrogenesis was compared to that of unmodified peptide hydrogels with medium-delivered TGF-β1. Adsorbed-TGF-β1 peptide hydrogels stimulated chondrogenesis of BMSCs as demonstrated by cell proliferation and cartilage-like ECM accumulation, while tethered TGF-β1 was not different from TGF-β1-free controls. TGF-β1 adsorbed to self-assembling peptide hydrogels can stimulate BMSC chondrogenesis.

BMSC-seeded self-assembling peptide hydrogels, modified for controlled delivery of pro-chondrogenic factors, generate cartilage-like neotissue and are compatible with a singlesurgery, autologous therapy for cartilage repair.

Year	Entry
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Year

Entry

2003

Ng L, Grodzinsky AJ, Patwari P, Sandy J, Plaas A, Ortiz C. Individual cartilage aggrecan macromolecules and their constituent glycosaminoglycans visualized via atomic force microscopy. J Struct Biol. September 2003;143(3):242-257.

1992

Buschmann MD, Gluzband YA, Grodzinsky AJ, Kimura JH, Hunziker EB. Chondrocytes in agarose culture synthesize a mechanically functional extracellular matrix. J Orthop Res. November 1992;10(6):745-758.

2001

Williamson AK, Chen AC, Sah RL. Compressive properties and function—composition relationships of developing bovine articular cartilage. J Orthop Res. November 2001;19(6):1113-1121.

2004

Fitzgerald JB, Jin M, Dean D, Wood DJ, Zheng MH, Grodzinsky AJ. Mechanical compression of cartilage explants induces multiple time-dependent gene expression patterns and involves intracellular calcium and cyclic AMP. J Biol Chem. May 7, 2004;279(19):19502-19511.

1982

Benya PD, Shaffer JD. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell. August 1982;30(1):215-224.