Osteoarthritis, just one example of a joint impairment, involves the wearing down of articular cartilage, the tissue found at the ends of bones. Millions of people worldwide suffer from this joint disease, and it is estimated that last year, more than half a million Americans received bone defect repairs, the cost of which exceeded $5 billion [1]. However, the healing of relevant tissue in this case is extremely difficult, as cells must regrow in very precise patterns for the joints to regain original functionality. Our team concluded that we must target the positioning and growth of cells with our project to solve this problem.
The extracellular matrix (ECM) of the cell is extremely important in determining its attachment properties to surfaces and other cells. Common proteins found in the ECM include collagen, fibronectin, integrin, heparin, etc [2]. Individual cells interact with each other via these proteins, influencing their relative positioning and growth patterns. Because the ECM of each cell type features a unique composition of the aforementioned proteins, its attachment properties are similarly unique. Thus, the growth patterns of each cell type can be selectively influenced by the protein content of the binding surface via these ECM interactions.
Taking advantage of the phenomenon above, a base framework with a customizable surface can be made to bring about the desired growth patterns of each cell type. For our project, this framework takes the form of a hydrogel, a watery gel-like scaffold on which cells can grow. The surface of our hydrogel features a prokaryotic protein called Streptococcal collagen-like protein (Scl2), which was originally discovered in gram-positive Streptococcus pyogenes.
The Scl2 protein has demonstrated production and technical advantages. Scl2 is easily expressed in E. coli and lacks the requirement of post-translational modifications for functionality [3]. Perhaps most importantly, customizable binding motifs for common ECM proteins can be easily inserted into the Scl2 protein. The significance of this customization lies in the incorporation of these Scl2 proteins into the hydrogel. By creating a spatial distribution of these various Scl2 proteins and their associated binding motifs, we can deliberately control which combination of binding motifs are found in which area of the hydrogel, thus indirectly controlling which cell type is best suited for growth in that precise area.
For these reasons, we chose Scl2 as the backbone for Collatrix—a collagen-mimetic gradient hydrogel for bone-soft tissue interfaces. By customizing our hydrogel with these fibronectin and integrin binding sites, our project can be put toward applications at the bone-cartilage interface in the knee and elbow joints.
Before work can begin with hydrogels made of Scl2 proteins, we must first characterize the general interactions between collagen and other molecules of a mammalian cell’s ECM. Because rat tail collagen is readily available and does not need to be produced via recombinant protein expression, our initial hydrogels were created using regular eukaryotic collagen as opposed to Scl2, which has similar properties minus the presence of binding domains.
Our system uses an E. coli chassis to constitutively express Scl2 with a coding sequence modified in specific locations to include fibronectin and integrin binding sites. The protein will include polyhistidine tags in order to perform purification and confirm protein production. After the collagen is produced, we tested cross-linking both chemically (with the use of glutaraldehyde) and with UV light in order to create hydrogels. After production of the hydrogels, Mouse Fibroblast L929 and Mouse Myoblast C2C12 will be introduced in order to analyze how different cells will interact with our collagen gel matrix product, Collatrix.
Figure 1: This is an overview of our genetic circuit, which constitutively expresses Scl2 with variable binding sites
The customizability of Scl2, as demonstrated in our project, along with its low cytotoxicity, low likelihood of immune response, and ability to be produced in high amounts [4, 5] makes it an appealing material for biomedical applications. The use of Scl2 in such applications has been studied in existing literature [4]. To build off of this, Collatrix serves as an in vitro representation of the ability of customized Scl2 contained in hydrogels to allow aggregation of certain cell types. In the future, these hydrogels could be used as scaffolds. Scaffolds like these are especially useful in tissue engineering, as they are completely customizable, biocompatible, and degrade naturally over time, allowing for the body to interface with and eventually fully incorporate implanted materials. Further, the experimental results and conclusions from testing customized Scl2 hydrogels could realistically be extended to human collagen, modifying the native collagen structure to create similar biomedical scaffolds. This can be done by aligning Scl2 with human collagen proteins to identify key structural regions, inserting binding domains, and incorporating customized human collagen into a hydrogel.
- [1] Maji, K. (2018). Biomaterials for Bone Tissue Engineering: Recent Advances and Challenges. In B. Li & T. Webster (Eds.), Orthopedic Biomaterials: Progress in Biology, Manufacturing, and Industry Perspectives (pp. 429–452). Springer International Publishing. https://doi.org/10.1007/978-3-319-89542-0_17
- [2] Frantz, C., Stewart, K. M., & Weaver, V. M. (2010). The extracellular matrix at a glance. Journal of Cell Science, 123(24), 4195–4200. https://doi.org/10.1242/jcs.023820
- [3] An, B., Kaplan, D. L., & Brodsky, B. (2014). Engineered recombinant bacterial collagen as an alternative collagen-based biomaterial for tissue engineering. Frontiers in Chemistry, 2, 40. https://doi.org/10.3389/fchem.2014.00040
- [4] Walker, M., Luo, J., Pringle, E. W., & Cantini, M. (2021). ChondroGELesis: Hydrogels to harness the chondrogenic potential of stem cells. Materials Science and Engineering: C, 121, 111822. https://doi.org/10.1016/j.msec.2020.111822
- [5] Peng, Y. Y., Yoshizumi, A., Danon, S. J., Glattauer, V., Prokopenko, O., Mirochnitchenko, O., Yu, Z., Inouye, M., Werkmeister, J. A., Brodsky, B., & Ramshaw, J. A. M. (2010). A Streptococcus pyogenes derived collagen-like protein as a non-cytotoxic and non-immunogenic cross-linkable biomaterial. Biomaterials, 31(10), 2755–2761. https://doi.org/10.1016/j.biomaterials.2009.12.040
- [6] O’Brien, F. J. (2011). Biomaterials & scaffolds for tissue engineering. Materials Today, 14(3), 88–95. https://doi.org/10.1016/S1369-7021(11)70058-X