| Nanofibrous scaffold can be developed as biomimetic natural extracellular matrix, thus it has become a research hotspot in tissue engineering. Collagen(Col) fibrous scaffold can be prepared by self-assembly method, however, it cannot satisfy the requirements of ideal bionic scaffold due to its poor mechanical properties and rapid degradation behavior. A polyelectrolyte brush(CH) was synthesized by grafting Low-molecular-weight heparin(LMWH-CHO) onto carboxymethylcellulose(CMC) backbone, combine the high mechanical strength and hydrature properties of carboxymethyl cellulose with the property of protecting collagen from degradation, regulating biological signal transduction, protecting the growth factor activity of heparin(HEP). This work aimed to prepare Col/CH composite fibrous scaffolds through self-assembly technology, study their physic-chemical properties and evaluated their potential application as tissue engineering scaffold.Firstly, heparin was degraded by nitrous acid to prepare LMWH with a aldehyde group at their reduce ends(LMWH-CHO), the MW of LMWH-CHO was 7.007×103 g/mol, APTT and PT value was 117 s and 16.8 s, respectively, suggesting LMWH-CHO reserves a certain anticoagulant activity. While CMC was reacted with ethylene diamine(EDA) by EDC catalyzed amide bond formation between amino group of EDA and carboxylic groups of CMC to introduce primary amino groups to its backbone(CMC-NH2). Finally LMWH-CHO was grafted onto CMC-NH2 by Schiff-base reaction. The chemical structure and molecular weight of the synthesized hyperbranched copolymer were characterized by FTIR, NMR and GPC-MALLS, respectively. The results showed the grafted ratio of LMWH-CHO was 23.7%.Secondly, use CH polyelectrolyte brush to induce collagen self-assemble, thus to prepare Col/CH composite scaffolds. The results of SEM showed a electrostatic-spinning-like high fibrosis porous network structure in the inner space of composite scaffolds. Besides, the composite scaffold showed a high porosity(up to 95%), good equilibrium-swelling ratio(maximum 30) and novel mechanical properties(highest strain was 3.90 MPa, the stress was 10.91%), lower degradation ratio in vitro(reduced from 100% to 11.26±0.95% in 24 h). In addition, the composite scaffolds exhibit excellent biocompatibility, which not only showed no poisonous to the mouse embryonic fibroblast, but also can promote cell proliferation. The mouse embryonic fibroblast was grown on scaffolds and cultivated together, which were observed with inverted fluorescence microscope. The results revealed that the composite scaffolds can not only promote cell adhesion and proliferation on the surface, can also lead internal migration into the scaffolds, suggesting the composite scaffolds can temporarily replace organization and play a role of supporting cell adhesion, proliferation and migration.Finally, the composite scaffold had great inhibition on inflammatory cytokines, i.e., NO secretion was reduced by 51.25%, TNF-α secretion was reduced by 23.52%, IL-1β secretion was reduced by 26.51% and IL-6 secretion was reduced by 44.56%.In conclusion, the stable three-dimensional nanofibrous Col/CH composite scaffolds were successfully prepared without any crosslinker agent, which not only mimic extracellular matrix in the composition and structure, but also showed good result in biological performance. The scaffolds combined the advantages of Col, CMC, Hep and the structure characteristic of the polyelectrolyte brushes, which can not only preserve good mechanical strength to support tissue regeneration, but also provide a good environment for cell adhesion and migration. The self-assembly collagen-based composite scaffolds were simply induced by CH polysaccharide and bebaves like electrostatic spinning scaffolds, thus the Col/CH composite scaffolds were expected to be developed as a new tissue engineering scaffold used in wound healing and tissue regeneration. |
PDF Full Text Request