Human Umbilical Cord-derived Mesenchymal Stem Cells Differentiation To NP-like Cells In Chitosan-glycerophosphate Hydrogels

ChapterⅠ In vitro isolation、culture、identification and differentiation ofhuman umbilical cord-derived mesenchymal stem cellsObjective: To explore the methods of ilsolating、culturing、identification and differentiation ofmesenchymal stem cells from human umbilical cord. Methods: Human umbilical cord from8neonatuswith cesarean section surgery were collected to extract human umbilical cord-derived mesenchymalstem cells (hUC-MSCs) using digestion by0.075%collagenase I at37℃temperature. During the wholeculturing and passaging period, morphologic characteristics of hUC-MSCs were observed underinverted microscope. Flow cytometry assaying was used to detect the expression of cell surface markerslike CD90、CD105、CD44、CD34、CD45and HLA-DR. After identification, hUC-MSCs were inducedby specific inducing medium to detect their differentiating potentials towards osteocyte、adipose andchondrocyte. After induction for21days, Alizarin red S staining、Oil red staining、Alcian blue stainingand immunocytochemistry staining of collagen Ⅱ were used to evaluate their differentiation. Results:hUC-MSCs from umbilical cord of neonatus could be extracted by digestion of0.075%collagenase Ⅰ.It took an average of3days for the polygonal primary hUC-MSCs to achieve adherence and about14days to reach a confluence of80%. After3passaging, the polygonal cells gradually changed intospindle-like cells, which demonstrated a swirling clustering. They had positive expression of CD90、CD105、CD44and negative expression of CD34、CD45、HLA-DR surface markers. The hUC-MSCs hadpositive results of Alizarin red S、Oil red、Alcian blue staining and immunocytochemistry staining ofcollagen Ⅱ after an induction period of21days. Conclusions: hUC-MSCs can be isolated fromumbilical cord by use of0.075%collagenase Ⅰ. They had the multilineage differentiation potentialssuch as osteocyte、adipose and chondrocyte. Chapter Ⅱ Preparation and biocompatibility of thermo-sensitivechitosan-glycerophosphate hydrogelsObjective: To study the influence of different concentrations of chitosan hydrochloride andβ-glycerophosphate on the gelation properties of the hydrogel systems. To explore the optimisedformulation of chitosan-based injectable hydrogel which can be used as intervertebral disc tissueengineering scaffolds, and to evaluate it’s biocompatibility. Methods: Chitosan hydrochloride andβ-glycerophosphate were combined to give a final concentration of3%,2%,1%chitosan in0.25M,0.5M,0.75M glycerophosphate respectively. The pH values were detected by a pH acidimeter at4℃，room temperature and37℃. The gelation time was detected in water bath at37℃. Hydrogelmorphology score was made by naked eye view with reference to the hydrogel morphology score sheet.The morphologies of the chitosan hydrogel with2%chitosan hydrochloride in0.5Mβ-glycerophosphate were observed by HE staining, Masson staining and scanning electron microscopy.The cytotoxicity of the extracts of the2%chitosan hydrogel with different glycerophosphateconcentration was detected by CCK-8assay, and growth curves of hUC-MSCs were made. Invertedmicroscope and DAPI staining were adopted to evaluate the performance of cell adhesion on thehydrogel membrane. Cells in the chitosan-based hydrogel were observed by scanning electronmicroscopy. The hydrogel was subcutaneously implanted in mices, and in vivo compatibility of thehydrogel was observed by naked eye view and HE staining. Results: Concentrations of chitosanhydrochloride and β-glycerophosphate have great effect on the pH value, gelation time and morphologyof the hydrogel, but environment temperature and autoclave treatment had no significant effect on them.The chitosan hydrogel with2%chitosan hydrochloride in0.5M β-glycerophosphate had pH value of7.04, and the gelation time at37℃was8.5min. HE staining and scanning electron microscopy showedthe hydrogel had porous network structure which was irregularly shaped, and the cells were located inthe network structure. The cytotoxicity test of the extracts showed that0.5M glycerophosphate wasconductive to the growth of the cells. Microscopy combined with DAPI staining showed hUC-MSCscould well adhere to the gel membrane. The in vivo experiments showed gelation in situ after injection,and there was no significant inflammatory response in the injection site.4weeks after inoculation, thescaffolds were partially absorbed, with no obvious inflammatory cells infiltration or foreign bodyreaction. Conclusions: The concentrations of Chitosan hydrochloride and β-glycerophosphate had greatinfluence on the gelation properties and biocompatibility of the thermo-sensitive hydrogel systems, andthe hydrogel with2%chitosan hydrochloride in0.5M β-glycerophosphate had potential use inintervertebral disc tissue engineering scaffold. Chapter Ⅲ hUC-MSCs differentiation to NP-like cells inchitosan-glycerophosphate hydrogelsObjective: To study the biological changes of hUC-MSCs differentiation to NP-like cells in thethermo-sensitive chitosan-glycerophosphate hydrogel. Methods: Hydrogels with2%chitosanhydrochloride in0.5M β-glycerophosphate were seeded with hUC-MSCs and cultured in24-well platesfor a period of2-4weeks in standard medium. The morphological features of the cells/scaffoldscomplex were observed by HE staining and Masson staining. Gene expression analysis of type Icollagen, type II collagen, Aggrecan and SOX-9was done by quantitative real-time PCR. Cell matrixsynthesis was qualitatively observed by immunofluorescence staining of type II collagen and Aggrecan.Results: HE and Masson staining showed the hydrogel had porous network structure which wasirregularly shaped, the cells were located in the nest-like structure of the scaffold, and cell aggregationwas observed. Cultured for2weeks, quantitative real-time PCR demonstrated significantly highexpression of type II collagen, Aggrecan and SOX-9（P<0.05）, and demonstrated higher expressionafter4weeks of culture (P<0.05). But there was no significant change of gene expression of type Icollagen. Immunofluorescence also found increased of proteoglycan and type II collagen synthesis after2-4weeks in culture. Conclusions: hUC-MSCs can differentiate to NP-like cells in thermo-sensitivechitosan scaffolds; hUC-MSCs combined with thermo-sensitive chitosan scaffolds has potential use intissue engineering nucleus in the treatment of intervertebral disc degeneration.