Artificial extracellular matrix proteins for substrates in skin substitutes

• Main Author: Tjin, Monica Suryana
• Other Authors: Fong Wen Mei Eileen
• Format: Theses and Dissertations
• Language: English
• Published: 2015
• Subjects: DRNTU::Engineering::Materials::Biomaterials
• Online Access: https://hdl.handle.net/10356/65237
• Institution: Nanyang Technological University

For decades, there has been intense research to develop novel substrates for skin repair. Several tissue-engineered skin products are available currently, however, autograft remains the gold standard in skin repair. Recently, protein-based biomaterials have recently received great attention for use in biomedical applications as they can be genetically engineered to mimic the biological properties of the native tissue. This research aims to develop novel protein-based biomaterials for use as tissue engineered skin. It was hypothesized that the incorporation of integrin-specific cell binding domains on aECM proteins will enhance keratinocytes adhesion and viability; promote faster re-epithelialization and in-situ human dermal fibroblast infiltration during wound healing. For this purpose, recombinant DNA technologies were used to create artificial extracellular matrix (aECM) proteins that promote integrin-specific interactions between human epidermal keratinocytes (HEKs) and the underlying substratum. Artificial ECM proteins were developed as elastin-like polypeptide fusion proteins containing different types of cell binding domains: FN910 (FN910 aECM), PPFLMLLKGSTR (LN-5 aECM), and GEFYFDLRLKGDK (Col-IV aECM). In this work, we found that keratinocyte attachment and proliferation were mediated through specific interactions between the integrins and cell-binding domain present in aECM proteins. Specifically, keratinocyte attachment on FN910 aECM protein was mediated by alpha5 beta1 integrin, while their attachment on LN-5 aECM protein was mediated by alpha3 beta1 integrin. We also showed that aECM proteins were able to support proliferation and colony-forming abilities of keratinocytes stem cell. The cell-aECM proteins interactions were also examined in the context of cell migration. Similar to the native FN, we found that FN910 aECM protein promoted faster wound closure compared to BSA control. In contrast, slower wound closure rate was observed on LN-5 aECM compared to native LN. This could be attributed to the lower cell speed and less directional movement observed on keratinocytes migrating on this aECM. Likewise, cell migrating individually on LN-5 aECM substrates also moved with slower speeds and little persistence. Furthermore, addition of anti-integrin antibodies led to severe inhibition of cell migration and loss of directed motility. Taken together, our results suggest that keratinocytes primarily utilized alpha3beta1 integrin to migrate on LN-5 aECM, while alpha5beta1 is engaged by keratinocytes to migrate on FN910 aECM. Finally, we evaluated the feasibility of developing the artificial ECM proteins into a mechanically intact free-standing scaffold to be used as keratinocytes cell delivery system. We showed that aECM protein scaffold produced by freeze-drying method was able to support keratinocytes attachment and viability. Further, there was sufficient porosity within the aECM protein scaffolds to allow human dermal fibroblasts infiltration. Hence, we demonstrate that aECM protein scaffolds are suitable for use as a cell carrier, potentially serving as a substrate to aid wound repair in the clinic.