As an alternative to conventional bottom-up assembly approach for creating synthetic tissue scaffolds, this research project aims not to recreate, but improve on nature’s tissue scaffold by developing non-covalent collagen functionalization methods which allow controlled presentation of cellular signals in biologically derived scaffolds. Due to the inherent complexity associated with tissue development, I believe that immediate clinical success in tissue engineering (TE) will mostly likely come from systematic discovery-driven research that employs natural or natural-synthetic hybrid scaffold systems as reflected by the TE products recently approved by the FDA. However natural scaffolds have been employed in such research almost in ad hoc formulation with little systematic and hypothesis driven efforts because their delicate structures and heterogeneous composition prevent the use chemical conjugation commonly used for changing the physico-chemical properties of synthetic scaffolds.

Study of the hybridization mechanism:

To overcome the limitations of conventional “chemical” modification which is non-selective and non-reversible, we developed a novel “physical” collagen modification technique that is based on collagen’s native ability to associate into triple-helical molecular architecture (Fig. 1) (17). This research was inspired by structural similarity between collagen and DNA which are helical multiplex stabilized by inter-chain hydrogen bonds. We discovered that, collagen mimetic peptide (CMP), a biochemically inert synthetic peptide composed of collagen-like repetitive amino acid sequence, exhibits structure-dependent binding affinity to natural collagen. Our binding experiments have indicated that collagen films and gels attract only single stranded CMPs, (ProHypGly)x and not triple helical form of CMP nor control peptides comprising scrambled peptide sequence. Initially, we speculated that the CMP binding could take place on the thermally unstable domains of the native collagen or parts of the collagen that are partially denatured during the purification and fiber regeneration (17, 16, 14). The study of templated heterotrimeric CMP that model such binding interactions confirmed the dramatic stabilization effect when the synthetic CMP hybridize with the unstable domain of natural collagen (6). Recently, we developed caged CMPs which can be photo-triggered to fold into triple helix and hybridize with collagen strands (1). This work addressed the major problem of using heat to control CMP folding and collagen binding: CMPs with high collagen binding affinity typically need to be heated to temperatures above 70°C before application to collagen which results in collagen denaturation complicating the collagen binding study as well as in vivo and ex vivo tissue imaging experiments. Therefore we explored non-thermal means to control triple helix folding and identified photo-caging as an ideal alternative. With the new caged-CMPs and control CMP with opposite helical twist, we were able to confirm the stereo-selective, triple-helix hybridization between CMP and natural collagen strands as well as image tumor micro-environment in vivo and photo-pattern collagen-based tissue scaffolds (1).

Functionalization of collagen scaffolds:

We have developed various CMP derivatives that can be used to functionalize collagen matrix and control cellular behaviors. PEG-CMP can reduce the cell adhesiveness of a collagen substrates which can be useful for treating pathological conditions caused by uncontrolled migration and proliferation of cells through collagen scaffolds (17, 12). Anionically charged CMP can bind to collagen matrix and signal endothelial cells by attracting vascular endothelial cell growth factor (VEGF) within a 3D collagen gel (10). Recently, a pro-angiogenic peptide was conjugated to the CMP resulting in a new peptide with both the collagen binding and angiogenic capacities (3). This peptide simulates the morphogenic activity of a matrix bound vascular endothelial growth factor (VEGF), a key factor in the production of organized microvasculature which is considered to be a main challenge in engineering artificial tissues with long-term viability. Using this peptide, we were able to control areas of peptide immobilization and induce cell morphogenesis in pre-defined areas within wider cell culture (Fig. 2). The ability to encode spatially defined morphogenic signals in natural scaffolds is expected to provide new pathways for engineering complex tissues for regenerative medicine.

Targeting collagens in pathologic tissues:

 Since many pathologic conditions are associated with abnormal metabolic collagen activity, collagens in diseased tissue and circulation are potential diagnostic and therapeutic targets. For example, tumor progression involves proteolytic remodeling of the ECM that results in the accumulation of stromal collagen with a unique structural and biochemical signature. We reasoned that our CMPs would represent a new and effective class of collagen targeting agents because they are small peptides that can bind to not only healthy collagen fibers but also to unstructured collagens that are common in tissues undergoing rapid remodeling. Fig 3 shows the in vivo imaging of prostate cancer tissue by CMP hybridization (1). Fluorescence images of mice with internal organs exposed at 4 days post injection showed significant CMP uptake at the tumor sites with residual uptake in salivary glands, liver, and the gastrointestinal tract, while the scrambled peptide had largely been cleared. Considering the abundance of collagens in various organs, it is remarkable to see such localized and apparently stable accumulation of CMPs in the tumors. More work is underway to determine the exact state of the collagens targeted by CMPs (e.g. intact collagens, digested collagens, or collagen fragments); however we believe that CMP mediated in vivo collagen targeting offers an entirely new direction in clinical imaging and treatment of cancers and possibly of other diseases associated with ECM remodeling and fibrous tissue formation.

PEG-CMP hydrogels:

 We have been also working on PEG-based hydrogels that carry CMP side chains which function as non-covalent and digestible cross-links as well as sites for scaffold functionalization. This hydrogels can retain cell secreted collagen and promote maintenance of chondrocytes in a three-dimensional culture system (15) and can direct differentiation of stem cells into predefined pathway (9). In addition, recent study demonstrated that the triple helical crosslinks of this hydrogels can be disrupted by addition of single stranded CMPs that compete for triple helix formation (8). By injecting free CF-CMP directly into the preformed hydrogel, we were able to create a gradient of CMPs immobilized to the PEG-CMP hydrogel evidenced by the reconstructed fluorescence microscopy (Fig. 4). When analyzed by particle tracking microrheology, the gel showed gradual change in stiffness along the CMP gradient. Although these experiments are preliminary, they demonstrate that triple helical association of CMP can produce spatially-defined signals in PEG-hydrogels which will be useful for studying cellular behaviors.

 

References:

1. Y. Li, C. A. Foss, D. D. Summerfield, M. G. Pomper and S. M. Yu (2011) Targeting Collagen Strands by Photo-Triggered Triple Helix Hybridization. submitted to Nature Materials.
2. C. Kelliher, S. Chakravarti, N. Vij, S. Mazur; P.  J. Stahl, C. Engler, M. Matthaei, S. M. Yu, A. S. Jun (2011) A Cellular Model for the Investigation of Fuchs' Endothelial Dystophy. Exp. Eye. Res., in press.
3. T. R. Chan, P. J. Stahl, and S. M. Yu (2011)  Matrix-Bound VEGF Mimetic Peptides: Design and Endothelial Cell Activation in Collagen Scaffolds. Adv. Funct. Mater., in press. DOI: 10.1002/adfm.201101163
4. D. Farrar, K. Ren, D. Cheng, S. Kim, W. Moon, W. L. Wilson, J. E. West, and S. M. Yu (2011) Permanent Polarity and Piezoelectricity of Electrospun α-Helical Poly(α-amino acid) Fibers. Adv. Mater. 23, 3954.
5. S. M. Yu, Y. Li, and D. Kim (2011) Collagen Mimetic Peptides: Progress Towards Functional Applications. Soft Matter 7, 7927.
6. Y, Li, X. Mo, D, Kim, and S. M. Yu, (2011) Template-Tethered Collagen Mimetic Peptides for Studying Heterotrimeric Triple-Helical Interactions Biopolymers 95, 94.
7. Y. Hwang, D. Farrar, J. E. West, S. M. Yu and W. Moon (2011) Piezoelectric Properties of Polypeptide-PMMA Molecular Composites Fabricated by Contact Charging. Polymer 52, 2723.
8. P. Stahl, N. Romano, D. Wirtz, and S. M. Yu, (2010) “PEG-Based Hydrogels with Collagen Mimetic Peptide-Mediated and Tunable Physical Crosslinks” Biomacromolecules 11, 2336.
9. Lee, J. H., Yu, C., Chansakul, T., Hwang, N. S., Varghese, S., Yu, S. M., and Elisseeff J. H. (Enhanced Chondrogenesis of Mesenchymal Stem Cells in Collagen Mimetic Peptide-Mediated Microenvironment. Advanced Tissue Engineering (2010), Chapt. 15, Eds P.C. Johnson and A.G. Mikos.
10. Wang, A. Y., Leong, S., Liang Y.-C., Huang, R. C., Chen, C. S.,  and Yu, S. M. (2008) Immobilization of growth factors on collagen scaffolds mediated by polyanionic collagen mimetic peptides and its effect on endothelial cell morphology. Biomacromoleulces 9, 2929.
11. Farrar, D., West, J. W., Busch-Vishniac, I. J., and Yu, S. M. (2008) Fabrication of Polypeptide-Based Piezoelectric Composite Polymer Film. Scripta Materialia 59, 1051.
12. Wang, A. Y., Foss, C. A., Leong, S., Mo, X., Pomper, M. G., and Yu, S. M. (2008) Spatio-temporal modification of collagen scaffolds mediated by triple helical propensity. Biomacromoleulces 9, 1755.
13. Lee, J. H., Yu, C., Chansakul, T., Hwang, N. S., Varghese, S., Yu, S. M., and Elisseeff J. H. (2008) Enhanced Chondrogenesis of Mesenchymal Stem Cells in Collagen Mimetic Peptide-Mediated Microenvironment. Tissue Engineering 14, 1843.
14. Mo, X., An, Y. J., Yun, C. S., and Yu, S. M. (2006) Nanoparticle-assisted visualization of binding interactions between collagen mimetic peptide and collagen fibers. Angewandte Chemie-International Edition 45, 2267-2270.
15. Lee, J. H., Lee, J.-S., Chansakul, T., Yu, C., Elisseeff, J. H., and Yu, S. M. (2006) Collagen mimetic peptide-conjugated photopolymerizable PEG hydrogel. Biomaterials 27, 5268-5276.
16. Mo, X., Krebs, M. P., and Yu, S. M. (2006) Directed synthesis and assembly of nanoparticles using purple membrane. Small 2, 526-529.
17. Wang, A. Y., Mo, X., Chen, C. S., and Yu, S. M. (2005) Facile modification of collagen directed by collagen mimetic peptides. Journal of the American Chemical Society 127, 4130-4131.
18. Fukuto, M., Heilmann, R. K., Pershan, P. S., Yu, S. J. M., Soto, C. M., and Tirrell, D. A. (2003) Internal segregation and side chain ordering in hairy-rod polypeptide monolayers at the gas/water interface: An x-ray scattering study. Journal of Chemical Physics 119, 6253-6270.
19. Fukuto, M., Heilmann, R. K., Pershan, P. S., Yu, S. M., Soto, C. M., and Tirrell, D. A. (2002) Confinement-induced order of tethered alkyl chains at the water/vapor interface. Physical Review E 66.
20. Yu, S. M., McQuade, D. T., Quinn, M. A., Hackenberger, C. P. R., Krebs, M. P., Polans, A. S., and Gellman, S. H. (2001) An improved tripod amphiphile for membrane protein solubilization.  Protein Science 10, 1089-1089.
21. Yu, S. J. M., and Tirrell, D. A. (2000) Thermal and structural properties of biologically derived monodisperse hairy-rod polymers. Biomacromolecules 1, 310-312.
22. Yu, S. J. M., Soto, C. M., and Tirrell, D. A. (2000) Nanometer-scale smectic ordering of genetically engineered rodlike polymers: Synthesis and characterization of monodisperse derivatives of poly(gamma-benzyl alpha,L-glutamate). Journal of the American Chemical Society 122, 6552-6559.
23. McQuade, D. T., Quinn, M. A., Yu, S. M., Polans, A. S., Krebs, M. P., and Gellman, S. H. (2000) Rigid amphiphiles for membrane protein manipulation. Angewandte Chemie-International Edition 39, 758.
24. Fukuto, M., Heilmann, R. K., Pershan, P. S., Yu, S. J. M., Griffiths, J. A., and Tirrell, D. A. (1999) Structure of poly(gamma-benzyl-L-glutamate) monolayers at the gas-water interface: A Brewster angle microscopy and x-ray scattering study. Journal of Chemical Physics 111, 9761-9777.
25. Song, J.-J., Yoon, S.-C., Yu, M. S., and Lenz, R. W. (1998) Differential Scanning Calorimetric Study of Poly(3-hydroxyoctanoate) Inclusions in Bacterial Cells. International Journal of Biological Macromolecules 23, 165.
26. He, S. J., Lee, C., Gido, S. P., Yu, S. J. M., and Tirrell, D. A. (1998) A twist grain boundary-like twisted smectic phase in monodisperse poly(gamma-benzyl alpha,L-glutamate) produced by recombinant DNA techniques. Macromolecules 31, 9387-9389.
27. Fukuto, M., Heilmann, R. K., Pershan, P. S., Griffiths, J. A., Yu, S. J. M., and Tirrell, D. A. (1998) X-ray measurements of noncapillary spatial fluctuations from a liquid surface. Physical Review Letters 81, 3455-3458.
28. Yu, S. J. M., Conticello, V. P., Zhang, G. H., Kayser, C., Fournier, M. J., Mason, T. L., and Tirrell, D. A. (1997) Smectic ordering in solutions and films of a rod-like polymer owing to monodispersity of chain length. Nature 389, 167-170.
29. Jin, J.-I., and Yu, S. M. (1995) New Polyarylates Prepared from 2,5-Bis(α-phenyl-isopropyl)hydroquinone, Terephthalic Acid and Isophthalic Acid. Bull. Korean Chem.  Soc. 16, 17.