(336b) Bioactive Peptides for Mesenchymal Stem Cell Differentiation In Cartilage Tissue Engineering

Osteoarthritis is characterized by degradation of articular cartilage, and there is strong demand for long-lasting and minimally invasive cartilage replacements. One promising approach for cartilage regeneration is to use the patient's own bone marrow-derived stem cells (MSCs) to seed an artificial protein matrix embedded with bioactive differentiation cues. This method could allow MSCs embedded in the matrix to be directly implanted, which would reduce ex vivo culturing time. In addition, this technique could lead to the development of materials with spatially patterned differentiation cues, which would aid in the development of complex tissues such as osteochondral grafts.

We are developing an artificial protein matrix that directs human MSC (hMSC) differentiation and maintains the desired chondrogenic phenotype based on embedded biochemical cues. Short peptide sequence candidates were screened and characterized for their effect on hMSC differentiation to cartilage. To evaluate the efficacy of agonist peptides derived from growth factors, cells were cultured utilizing a modified pellet culture in a 96-well plate.

The most promising of the screened peptides is derived from the knuckle epitope of bone morphogenetic protein-2 (BMP-2). To study the effect of concentration, the BMP-2 peptide was added to medium at a range from 0.02 to 200 µg/mL. After two weeks of culture, we found that glycosaminoglycan (GAG) production of hMSCs cultured with >100 ug/mL of the peptide was 74% of the positive control (cells cultured with 200 ng/mL BMP-2 growth factor). Further characterizations were performed with the peptide at 100 µg/mL and included histology, measurements of matrix production over time, and gene expression. From one to four weeks, the peptide promoted GAG production at levels comparable to the BMP-2 positive control. The peptide had little effect on the production of collagen but did not increase the alkaline phosphatase (AP) activity over time, which suggests that it does not promote hypertrophy. In addition, the BMP peptide did not enhance GAG production in TGF-β3 mediated chondrogenesis, whereas BMP-2 does, suggesting a different mechanism for the bioactivity. Finally, the peptide promoted more homogenous distribution of extracellular matrix in the pellets compared to BMP-2, making the peptide an interesting new tool in hMSC differentiation to cartilage.