Matrix metalloproteinase controlled degradation of an ECM analogue hydrogel based on a bespoken elastin-like recombinamer for tissue engineering

摘要

Introduction: Current studies in tissue engineering reveal a lack of scaffolds with adequate degradation dynamics that would support cell proliferation but also ensure degradation of the platform to permit the substitution of the material by newly formed tissue. Elastin-like recombinamers are emerging biomaterials with tunable biomechanical characteristics, suitable for different biomedical applications. This work presents an ELR-based hydrogel that sustains cell colonization and long-term proliferation by being sensitive to degradation by various cell-secreted enzymes, the matrix metalloproteinases (MMPs). Materials and Methods: The two recombinamers used were obtained by iterative-recursive recombinant technology and expressed in an E.coli strain. The proteins were purified by inverse transition cycling and characterized by NMR, DSC, MALDI-TOF, FT-IR and HPLC. Subsequently, a chemical modification was performed by adding on[Cr1] the MMP sensitive recombinamer an activated alkyne, and an azide group on the component carrying an RGD cell adhesion sequence. The products of modification were crosslinked at cold aqueous conditions for about 15min, by catalyst free click chemistry. The attained hydrogel was characterized mechanically. The degradation by the MMPs was studied for the recombinamer in solution and the hydrogel as well. Cytocompatibility, cell proliferation and scaffold degradation were tested with HUVECs, HASMCs, HFF1s and macrophages. Figure 1. Scheme of the MMP-sensitive recombinamer structure Results and Discussion: The purity and structure of the two synthesized recombinamers have been confirmed, as well as the chemical modifications involved in the catalyst free click reaction that brings to a crosslinking ensuring adequate mechanical stability of the scaffold. Rheology showed the possibility to adjust the stiffness of the scaffold to a specific application as the modulus increases with recombinamer concentration, covering a range of 1-5 kPa. SEM evaluation showed high porosity of the structure. SDS-page analysis highlighted the formation of recombinamer fragments in time, as the recombinamer and the hydrogel in solution with MMPs are being degraded. Cytocompatibility, cell adhesion and proliferation of HUVECs, HFF1s and HASMCs have been studied and proved the degradable platform supports not only cell growth, but also favors the substitution of the scaffold by a newly formed natural one and is consequently a promising material for future in vivo studies. Figure 2. Mechanical versatility of the material for various applications in dependence of the stiffness Conclusions: The initial aim to design a mechanically stable and enzymatically cleavable ELR-based analogue extracellular matrix, obtained by highly compatible techniques with scaling up potential, was attained. As MMPs are well known for being released by numerous cell lines, colonizing cells encounter a favourable environment for proliferation and degrade themselves the artificial support, forming simultaneously their own new ECM. Therefore, the material is taught to be suitable for numerous tissue engineering and regenerative medicine applications.