| Objective:The objective of this study is to develop a novel chitosan-gelatin hydrogel system, which can be crosslinked by chemical and ionic agents, and further optimize the hydrogel 1) supporting stem cell 3D culture; 2) improving stem cell proliferation and osteogenic differentiation; 3) controlling long-term release of growth factors, and 4) delivering stem cells for bone tissue regeneration. Methods:1) The chitosan-gelatin hydrogels were prepared by both chemical crosslinker genipin and ionic crosslinker glycerol phosphate. 2) The volume ratios of chitosan to gelatin and the concentrations of genipin and glycerol phosphate were adjusted to optimize the hydrogels’gelation time and mechanical properties for 3D stem cell culture. 3)the biocompatibilities of hydrogels were evaluated based on the morphology, proliferation and differentiation of encapsulated stem cells inside hydrogels.4)The tailored hydrogels were used in the following in vitro and in vivo experiments.An oscillatory rheometer was used to evaluate the gelation process and the mechanical properties of hydrogels with different amounts of crosslinkers. 5)the microstructures of hydrogels were characterized by scanning electron microscopy (SEM).6)The rhBMP-2 release profiles from hydrogels were quantified by ELISA. 7)The migration of bone marrow derived mesenchymal stem cells (MSCs) response to the released BMP-2 from hydrogels was analyzed using Costar Transwell invasion chambers. Cell proliferation was evaluated by the MTT assay. 8)Cell differentiation was inspected by real-time PCR and immunohistochemistry.the radial bone critical defect model in rabbits was established in this study to investigate the transplanted hydrogels-mediated bone regeneration. 9)There were 4 groups applied to treat the bone defect, including blank, hydrogel, hydrogel+BMP-2 and hydrogel+BMP-2+bone marrow MSCs.To evaluate their biocompatibility, hydrogels were implanted subcutaneously in SD and nude rats, respectively. Macro-inspectionspecific bone tissue staining and immunohistochemistry were applied. There were 3 groups in this experiment: blank, hydrogel and hydrogel+BMP-2. Results:1)Hydrogel preparation:1.85% w/v chitosan solution, distilled water and 1.8% w/v gelatin solution were mixed with the ratio of 4:1:1 under stirring for 30 min and then heated to 35 ℃. The hydrogel precursor solutions were crosslinked by 8μL of genipin solution (50 mM) for 20 min and then further crosslinked by 0.8% w/v glycerol phosphate solution with the ratio of 160 to 1 under stirring for another 5 min and incubated at 37℃ for 20 min. The order of the proper ratios is as followed:4-1+ Nx (12x,16x,8x,4x,32x,44x,2x,52x).2) The morphologies of lyophilized hydrogels with different concentrations of genipin were analyzed by SEM. Hydrogels displayed a highly porous network structure with a rough surface. With the increasing of genipin, the pore size decreased. Compared to other ratios of hydrogels, the 12X hydrogel exhibited much more clearly and connected network structure with smoother surface, which may benefit cell attachment, growth and proliferation. Hydrogels with a large amount of water can facilitate oxygen exchange, drug release and the exchange of nutrients and wastes as a scaffold for 3D cell culture.3) Hydrogels were prepared with different concentrations of genipin. The rheological analysis was performed to investigate the gelation time and the shear storage modulus (G’) and loss modulus (G") of these hydrogels. It has been shown that the range of G" was from 1.80 ± 0.05 kPa to 1.05 ± 0.12 kPa, wherever the range of G’was from 22.42 ± 3.20 kPa to 2.57 ± 0.65 kPa. With the increasing of genipin, the hydrogel gelation time decreased. Average gelation time was between 14.44 min and 2.36 min. The gelation times of hydrogels with the ratios of 4-1+ Nx (12x,16x,8x) are long enough to do 3D cell culture.4) Confocal laser scanning microscope was used to observe the cell morphology in 3D hydrogels. Cells could attach, spread and proliferate inside the hydrogels with the ratios of 8x,12x and 16x.5) The ELISA was used to study the BMP-2 in vitro release kinetics from hydrogels loaded with different concentrations of BMP-2 (0.5,1, and 5 μg/mL). The hydrogels could provide a stable sustained release of active BMP-2. There was a burst release followed by a slow steady release. The release amount could reach to 6.40% ± 0.55 at day 28. With the increase of the loading amount of BMP-2, the release amount increased accordingly. 6) Osteoblasts were seeded on the surface of hydrogels, and their proliferation was studied by the MTT assay. Their proliferation rate was significantly faster than those seeded on the culture plate, leading to cells on the surface of hydrogels quickly reaching confluence.7) Cell migration: a transwell chamber was used to study the migration of cells responded to released BMP-2 from hydogels. All of the BMP-2 loaded hydrogels have shown to attract cells migrating through the transwell membrane. Different amounts of loaded rhBMP-2 resulted in cell response differently. Specifically, among all the groups, the 12x hydrogel with the 500 ng/mL BMP-2 displayed the highest capacity to recruit cells. 8) In order to investigate the effect of encapsulated BMP-2 on osteogenic differentiation of bone marrow stem cells, osteogenic gene expression was determined by quantitative real time-PCR analysis. Compared with the control group, BMP-2 at low concentration could significantly promote the expression of osteogenic genes. With the increase of BMP-2 amount, the gene expression increased until reached the highest at the BMP-2 concentration of 200 ng/mL. 9) For in vivo study, the radial bone critical defect model in rabbit was used to investigate the hydrogel-mediated bone regeneration. At 4,8 and 12 weeks, the group of hydrogel loaded with BMP-2 and osteoblast cells had more bone formation compared to other groups through radiographic analysis, micro-computed tomography and histology. Conclusion: We have successfully developed a new type of chitosan-gelatin hydrogel system based on relevant literature, rational design and a variety of tests. After optimizing the physical properties and in vitro biological characteristics of hydrogels, we have demonstrated that our hydrogels can be used as a scaffold for 3D stem cell culture. Our hydrogels can be also used to controlled long-term release of a variety of drugs and bioactive substances. The in vivo experiments confirmed that our hydrogels not only had good biocompatibility but also promoted bone regeneration of the radial critical defect in rabbit. Our novel hydrogels can be applied as a scaffold for bone tissue regeneration, which offers a new strategy of thinking and treatment for the clinical problem of the long bone defect. |
PDF Full Text Request