Modular protein matrices for cartilage repair
Abstract
Articular cartilage degradation (osteoarthritis) is a prevalent disease in the United States. Some disadvantages of current treatments for damaged cartilage include poor mechanical integrity, degradation with time, and donor site morbidity. One tissue engineering approach combines artificial proteins, with the use of human mesenchymal stem cells (MSCs). This approach offers a promising alternative to cartilage repair that has potential to be minimally invasive, low-cost and long-lasting. Modular proteins were designed as matrices for cartilage tissue engineering. The proteins feature mechanical domains derived from the rubber-like protein resilin and attempt to mimic the mechanical properties of natural cartilage. Bioactive domains are incorporated to interact with the MSCs and direct the cells to produce cartilage matrix. Bioactive domains derived from fibronectin are included into the resilin-based proteins, and other promising bioactive sequences are identified. Lysine residues serve as crosslinking sites. We manufactured long, repetitive resilin-based proteins using autoinduction. Changes to the standard protocols such as eliminating leaky expression, lowering the culture temperature and adjusting the harvest time produced the protein size of interest. Proteins manufactured (50 kDa) are larger than any protein consisting of repetitive resilin motifs made to date. Access to longer proteins allowed investigation of the effect of polypeptide length on thermal aggregation temperature. Oscillatory rheology experiments demonstrated that the proteins exhibit dual-phase behavior and form weak gels in response to temperature. This may be useful in tissue engineering and drug-delivery applications, so the thermal aggregation temperature was characterized via dynamic light scattering and ultraviolet-visible spectroscopy. Our findings suggest that cloud point and aggregation temperature can be modulated based on concentration and molecular weight but that molecular weight did not result in a predictable relationship. Crosslinking the modular proteins with tris(hydroxymethyl)phosphine resulted in viscoelastic hydrogels with a complex modulus of 22 ± 1 kPa and yield strain of 63%. The hydrogels had an unconfined compressive modulus of 2400 ± 200 kPa, which is on the same order of human cartilage. A LIVE/DEAD assay demonstrated that human MSCs cultured on the resilin-based protein had a viability of 95% after three days. A cell-spreading assay revealed that the cells interacted with the fibronectin-derived domain in a sequence-specific manner. A full-factorial experiment investigating the effect of four promising peptides on human MSCs in pellet culture identified a peptide derived from bone morphogenetic-2 (BMP-2) that had a significant effect on glycosaminoglycan (GAG) production. Upon further investigation, cells cultured with 100 µ/mL of the peptide produced significantly more GAG than the negative control. Over the course of four weeks of culture, comparable levels of GAG production were promoted by the peptide and BMP-2. Treatment with BMP-2 resulted in a large increase in hypertrophic markers such as AP activity and gene expression of type X collagen whereas treatment with the peptide resulted in little to no increase in these markers. These results suggest the BMP peptide sequence is a good candidate for incorporation into the resilin-based modular proteins for cartilage tissue repair. A laboratory module was developed featuring GAG quantification methods to educate female high school students about the chemical engineering profession. Survey data was taken to assess the impact of the activity.
Degree
Ph.D.
Advisors
Liu, Purdue University.
Subject Area
Biomedical engineering|Chemical engineering
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