| Hydrogels are currently receiving a great deal of interest in the filed of biology and iatrology because their physical properties are similar with the tissue of organism. The selection of hydrogel is a key factor to the success for their applications. Among biopolymers of interest, chitosan(CS), a natural polymer, stands out by a unique combination of favourable biological properties such as non-toxicity, biocompatiblility and biodegradablility. In this study, highly porous thermosensitive hydrogels are prepared with CS andα,β-glycerophosphate(α,β-GP), and the parameters which affected the hydrogel characterizations are studied. The CS-α,β-GP hydrogel that possess a physiological pH and can form monolithic gels at 25℃is prepared according to the influence of changing parameters to hydrogel characterization. The biocompatiblility, biodegradablility and the feasibility as a 3D culture systerm for penaeus chinensis lymphoid cells of the hydrogel are also investigated.In the current study, highly porous CS-α,β-GP hydrogels are prepared by changing the CS/α,β-GP ratio. These formulations possess a physiological pH and can be held liquid below room temperature and form monolithic gels at higher temperature. The gelation temperatures (Tgel) of these gels determined from rheological analysis indicated that the Tgel decreased by increasingα,β-GP content. The absorbency at 600 nm (OD600) of CS-α,β-GP hydrogel at Tgel was time dependent. The dynamics curve of turbidity decreased rapidly along with time extension till 50 s, and then it undergo a rapidly increased part following a slowly increased part. Results from FTIR, pNMR and TG confirm the CS-α,β-GP hydrogel is physically crosslinked hydrogel and is formed by various reversible links.The characterizations of the hydrogels are also studied during the changing of hydrogel preparation parameters. The influences of the dynamic viscosity of CS to the characterizations of the hydrogel are not obvious. With the increasing of deacetylation degree of CS or concentration of CS solution, the pH value of the hydrogel increase, the Tgel, gelation time and pore size of the hydrogel decrease. The characterizations are different when the CS is dissolved with different solvent or some salts are added in the CS solution. When the CS is dissolved with identical acid during the process of hydrogel preparation, the OD600 at Tgel and pH value increase and the OD600 at 4℃, Tgel and gelation time decrease with decreasing solvent strength. The OD600 at 4℃decrease and the OD600 at Tgel, Tgel and gelation time increase with the increasing of NaCl concentration.The equilibrium swelling ratios (ESR) of the dry hydrogels in water and PBS at 25℃and 37℃increase with increasing CS/α,β-GP ratio. The results form swelling kinetics indicate that the interconnecting and stable internal channel system of the hydrogel would enable fast convective water transport inwards or outwards; make possible fast swelling and deswelling by water convection. The swelling speed of the hydrogel at 37℃is faster than at 25℃. The effects of the pepsin, trypsin, bacteriolysin, cellulase and lipase to CS-α,β-GP hydrogel are not abvious at 25℃and physiological pH. But at 37℃and physiological pH, the weight loss reach 66.14% and 64.26% respectively when the hydrogels incubated in trypsin and bacteriolysin for 6 h. The results of the degradation kinetics and SEM images indicate that the degradation of highly porous CS-α,β-GP hydrogels in aqueous solutions belongs to the bulk-erosion process.The CS-α,β-GP hydrogel that possess a physiological pH and can form monolithic gels at 25℃is designed according to the beforehand results. The effects of steam sterilization and UV sterilization on chitosan and CS-α,β-GP hydrogel are investigated. The results show that UV sterilization for 2 h will be an appropriate method to sterilize chitosan powder.To study the biocompability of the hydrogel, the tests such as in vivo injection, hemocompatibility tests and cell toxicity experiment have been investigated. In this study, homogeneous hydrogel is injected and formed in situ in dorsal subcutaneously of mouse. The results of in vivo injection show that the CS-α,β-GP hydrogel has no obvious effect on hematopoiesis and has no toxicity to the body. Histological analysis over several weeks confirm that the CS-α,β-GP hydrogel is safety to the organism. The morphology of CS-α,β-GP hydrogel develop rough surfaces and become irregularly shaped after 45 d and the hydrogel is degraded completely after 60 d. The acute toxicity test making further proof that the hydrogel has no toxicity to the organism. The hemolysis rate of the hydrogel is less than 5% and is regard as non-toxic. Bovine serum albumin (BSA) adsorption tests show that the amount of BSA proteins adsorbed on CS-α,β-GP hydrogel is little and the adsorption between the hydrogel and BCS is physical adsorption, indicate the hydrogel had good hemocompatibility. The cell compatibility of CS-α,β-GP hydrogel is evaluated by measuring the relative growth rate of MEF cell, and the results show that the hydrogel has good cell compatibility.The feasibility of the CS-α,β-GP hydrogel as a 3D culture systerm for penaeus chinensis lymphoid cells is investigated. The results indicate that lymphoid cells cultured on the surface of the CS-α,β-GP hydrogel or cultured encapsulated inside the hydrogel can growth and reproduce. After 20 d culture on the surface of the hydrogel or encapsulated inside the hydrogel, the lymphoid cells are still have good activity, and could subculture on the surface of the hydrogel or encapsulated inside the hydrogel.Based on the above considerations, the CS-α,β-GP hydrogel is physically crosslinked hydrogel and is formed by various reversible links. And the CS-α,β-GP hydrogels can be better designed according to their applications. The CS-α,β-GP hydrogels have good biocompatibility and biodegradability, and could be as a 3D culture systerm of penaeus chinensis lymphoid cells for primary culture and subculture.
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