| Summary: | 博士 === 國立陽明大學 === 醫學工程研究所 === 100 === Cartilage is a tissue without vessels, nerves and lymph, and thus difficult to regenerates adequately after injury without the supplement of cells and matrix. The goal of this study is to develop a hydrogel capable of self-assembly as a cell carrier. We combine the gelling capacity of Fibrinogen (Fgn) and high hydration and physiological activity of Hyaluronic acid (HA) to synthesize a series of biohybrid molecules designated as SXF (S, short chain HA, abbreviated as sHA; F, Fgn; X, graft ratio of S to F) with gelling capability. This SXF was obtained by reducing-end amination of the terminal aldehyde of HA with the free amine of Fgn. The SXF copolymers were characterized, and the physical properties and structures of SXF gels were investigated. The effects of SXF gels on growth and chondrogenic differentiation of ATDC5 (a chondrogenic cell line) and human umbilical cord blood mesenchymal stem cells (hcbMSCs) were also investigated in this study.
We found that SXF copolymer had higher graft ratio (number of sHA molecules coupled per fibrinogen) as feed ratio of HA to Fgn increased. The size of the SXF hybrid molecules increased as graft ratio increased (particle size analysis). The results of zeta potential and native gel electrophoresis confirm that sHA is bound to fibrinogen through covalent bonding. Kinetic data based on the change of solution turbidity and microstructural studies of and the resulted gel demonstrated that clotting time of SXF copolymers was prolonged, and the microstructure of fibrin fiber became thinner and branched, accompanied with better swelling property. However, the gelling capacity dropped dramatically when graft ratio was above 6, and the fiber structure changed with disappeared band pattern. Therefore, SXF with graft ratio higher than 6 are not suitable for carrying cells alone.
ATDC5 cells were cultivated without induction on SXF surface and in 3D SXF fibrin gels. The proliferation of ATDC5 cells was not promoted, but cells in 3D gel exhibited round cell morphology, seemed to be favorable for chondrogenesis. When ATDC5 cells were induced in 3D SXF fibrin gels for 14 days, cells remained round cell morphology, similar to chondrocyte phenotype. In addition, cells in SXF gels expressed Col II, Sox9 and Agc at day3, superior to those in FC (without grafted sHA) and SXFmix gels. According the results of histological analysis, cells in SXF gel expressed the most lacunae and abundant cartilage extracellular matrix: GAGs and type II collagen, and exhibited higher GAG/DNA content, better than FC and SXFmix gels. Chondrogenesis of hcbMSCs revealed similar results. It suggests that grafted-sHA not only facilitates signaling transduction with cells, but also provides fibrin gel with a better 3-D microenvironment. In conclusion, we have demonstrated that sHA- modified fibrin gels with suitable graft ratio could be a potential injectable material to carry cells for tissue engineering application, such as cartilage regeneration.
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