Two of protein-protein interaction regulations on scaffold surface to direct stem cell differentiation

摘要

Background: Three elements involved in tissue engineering are cells, scaffolds and signals. Among these, signals and surface characters of scaffolds play important roles in facilitating cell adhesion, migration and differentiation etc. BMP-2 and EGF, bone morphogenetic protein 2 and epidermal growth factor, are two of important signaling proteins. In the beginning of signaling, both of them will bind with their receptor, BMP receptor and EGF receptor, to initiate following signaling transduction. Purpose: In this study, we try (1.) to prolong BMP and EGF signals through tethering them on surface; (2.) to enhance signaling strength through increasing their binding affinity by specific small molecular binders. Methods: In the study, BMP-2 and EGF are immobilized respectively on surface with or without TCM synergizing. Molecular binding, signal transduction, cell behaviors and osteo-induction are performed respectively by using SPR, over-expression cell line, bone marrow stromal cells, BMSC and rabbit calvarias defect. Results: In molecular binding, the result showed that in spite of immobilization, BMP-2 and EGF also can maintain the binding activity to BMPR and EGFR. Some of binding enhancer and inhibitor are found from 60 kinds of Traditional Chinese Medicines, TCM. Among 60 of TCMs, Gusuibu and Cortex Moutan Radicis are found to increase the affinity of BMP-2 and EGF binding to receptor, which are through increasing the quantity and association rate, ka, and decrease the dissociation rate, kd, of EGF bind to EGFR. From the aspect of signal transduction, tethered BMP-2 and EGF can stimulate BMPR and EGFR phosphorylation and down-stream signaling in C2C12 and A431 cell line. In the synergizing of TCM, Gusuibu and Cortex Moutan Radicis can enhance phosphorylation in C2C12 and A431. Gusuibu also are found to increase the proliferation in BMSC and osteoblastic differentiation in C2C12. From the aspect of tissue regeneration, both of tethered BMP-2 and TCM synergizing induce more osteoinduction comparing with BMSC control group in large bone defect rabbit model. Conclusion: Our results support us to give a summary that changing PPI binding are a potential approach to control cellular signaling. It will be applied on vascularization, integration of tissue-engineered bones.