Abstract: BACKGROUND: With the in-depth research of hydrogel materials, the applicable fields of hydrogel have been gradually broadened, and carrying stem cells for disease treatment has become a new direction of research, but how to construct a hydrogel suitable for stem cell growth is the key problem that needs to be solved at present.
OBJECTIVE: To investigate the physicochemical properties of chitosan-chitosan biguanide hydrochloride-collagen composite hydrogels and to evaluate their ability to load mouse umbilical cord mesenchymal stem cells. 
METHODS: The hydrogels were prepared by physically cross-linking chitosan, chitosan biguanide hydrochloride and collagen with the cross-linking agents β-glycerophosphate sodium and sodium bicarbonate, and the suitable hydrogels were screened according to the gel formation time and gel formation effect (noted as Gel-1, Gel-2 and Gel-3 in this way). Morphology, porosity, swelling properties, and degradability of the three groups of hydrogels were observed by scanning electron microscopy. Hemolysis experiments were performed to examine the hemolysis of the three groups of hydrogels. The mouse umbilical cord mesenchymal stem cells were co-cultured with the hydrogel with the best comprehensive performance of characterization. The cytotoxicity, cell survival and adhesion effect of the composite hydrogel were determined to evaluate the performance of this hydrogel loaded with umbilical cord mesenchymal stem cells. 
RESULTS AND CONCLUSION: (1) Scanning electron microscopy characterization results showed that all three groups had porous mesh structures inside, and the internal structure of Gel-2 and Gel-3 with the addition of chitosan biguanide hydrochloride was more porous and three-dimensional. (2) The hydrogel porosity of the Gel-3 group was higher than the remaining two groups, with high porosity and uniform pore size distribution. (3) The swelling performance of all three groups of hydrogels was above 100%, and the swelling performance of hydrogels with chitosan biguanide hydrochloride component was better. (4) The degradation rate of the three groups of hydrogels could be degraded by more than 90% in a time scale of 15 days, with good degradation performance. (5) The results of the hemolytic properties showed that the absorbance values measured by each group of hydrogels carrying chicken erythrocytes were basically the same as those of saline  carrying chicken erythrocytes, and no hemolysis occurred. (6) The toxicity experiment and living and dead cell staining showed that the survival rate of umbilical cord mesenchymal stem cells in each group of hydrogels was above 100%, indicating that there was no obvious cytotoxicity. Umbilical cord mesenchymal stem cells could survive under the hydrogel package and the hydrogels had a positive effect on the survival rate of umbilical cord mesenchymal stem cells. (7) The cells in the umbilical cord mesenchymal stem cell adhesion assay can survive under the hydrogel package and can adhere to the surface of the hydrogel with normal morphology.

Key words: smart hydrogel, umbilical cord mesenchymal stem cell, physical crosslinking, chitosan, type II diabetes