| Ex vivo expansion of hematopoietic stem cells(HSCs)was an effective method to address the scarce amount of transplantable HSCs from umbilical cord blood.Existing ex vivo expansion of HSCs technologies had not yet provided a culture microenvironment similar to the bone marrow microenvironment in vivo,which was difficult to rapidly expand HSCs in a short period of time,and easy to make HSCs lose their original biological functions after expansion.HSCs existed in the three-dimensional bone marrow microenvironment,which mainly consisted of stromal cells,extracellular matrix and a multiplicity of soluble factors.These specific structures and components provided complex biochemical and physical signals that jointly regulated HSCs.Therefore,how to construct biomimetic hematopoietic bone marrow microenvironment was the key to the success of HSCs expansion in vitro.In recent years,it has been recognized that osteoblasts(OBs)were stromal cells in a niche area.Besides,OBs played a key role in the controlling of the migration,proliferation,self-renewal and maintenance of the multi-direction differentiation potential of HSCs.This thesis focused on the theme of the ex vivo expansion of HSCs.Starting from the design and preparation of biomaterials,chitosan polyphenol derivative scaffolds with antioxidant activity were designed and prepared.The strategy of inducing MSCs into OBs to fabricate biomimetic microenvironment was used to construct biomimetic hematopoietic niche(COB).The cell permeable trehalose glycopolymers were synthesized by epichlorohydrin modified trehalose,which realized the DMSO-free cryopreservation of COB.In addition,the function of COB after cryopreservation was similar to that of fresh COB.A three-dimensional co-culture system of HSCs based on COB was constructed,which could effectively support ex vivo expansion of functional HSCs.As follows:Firstly,given that the increase in the level of intracellular reactive oxygen species(ROS)during the differentiation of MSCs into OBs is a limiting factor for their successful differentiation.Seven kinds of chitosan-polyphenol derivative scaffolds 2a～2g with antioxidant activity were obtained by chemically modifying chitosan(CS)using polyphenol derivatives.The effects of different phenolic hydroxyl groups,double bonds and chain lengths of the 2a～2g scaffolds on the differentiation of MSCs into OBs were investigated.Based on alkaline phosphatase(ALP)activity and calcium content,phenolic hydroxyl groups of the 2a～2g scaffolds promoted the differentiation of MSCs into OBs.Among the 2a-2g scaffolds,the 2c scaffold had the strongest antioxidant capacity.Owing to the strongest antioxidant capacity of 2c scaffold,the lower the intracellular ROS of MSCs on the 2c scaffold,the stronger the ability of 2c scaffold to promote the differentiation of MSCs into OBs.Therefore,2c scaffold promoted differentiation of MSCs into OBs by reducing intracellular ROS.Based on the above experimental results,the engineering artificial HSCs niche based on 2c scaffold and OBs differentiated from MSCs(COB)was fabricated to support ex vivo expansion of HSCs.The expansion of CD34+cells,CD34+CD38-cells and CD34+CD38-CD45R-CD90+CD49f+ cells in COB cultured strategy on day 14 were 39.5±4.7 folds,25.5±1.6 folds,and 6.05±0.4 folds,which was significantly higher than conventional two-dimensional CD34+culture group(14.1±0.6 folds,9.8±0.7 folds,and 1.5±0.1 folds).In addition,the secondary amplification ability and colony formation ability of CD34+cells in COB cultured strategy after 14 days of expansion were higher than those of conventional two-dimensional CD34±cell culture group.These results indicated that the COB established in this paper could effectively support ex vivo expansion of functional HSCs.Secondly,an effective cryopreservation method to achieve off-the-shelf biomimetic hematopoietic niche was developed in this paper.Epichlorohydrin was used to modify trehalose to improve its permeability in order to obtain anon-toxic trehalose glycopolymers S2 with good biocompatibility and cell low temperature protection effect,and to establish the cryopreservation method of biomimetic hematopoietic niche based on trehalose glycopolymers S2.When COB was cryopreserved in liquid nitrogen with cryopreservation solution containing 5.0 wt%S2 for 30 days,the cell recovery rate and survival rate of COB reached 63.14±2.85%and 80.29±4.72%,respectively,which was no significant difference with conventional cell cryopreservation solution containing 10%DMSO group(61.46±3.28%and 86.29±2.95%).Moreover,COB could maintain normal morphology and function.COB resuscitated with liquid nitrogen cryopreservation solution containing 5.0 wt%S2 for 30 days was used for culture of CD34+cells.The expansion of CD34+cells,CD34+CD38-cells and CD34+CD38-CD45RACD90+CD49f+ cells from COB cryopreserved with 5.0 wt%S2 group on day 14 were 27.7±2.6 folds,19.3±1.9 folds,and 9.2±1.3 folds,which was no significant difference between the freshly prepared COB cultured cell group(32.2±2.3,20.4±2.5 and 11.7±2.1 folds).In addition,the secondary amplification ability and colony formation ability of CD34+cells from COB cryopreserved with 5.0 wt%S2 group were similar to those of CD34+cells cultured with freshly prepared COB system.In conclusion,the cryopreservation method based on trehalose glycopolymers S2 developed in this paper could effectively maintain the function of COB,and the cryopreservation COB was able to effectively support ex vivo expansion of functional HSCs,realizing the off-the-shelf biomimetic hematopoietic niche.Finally,since Wnt/β-catenin signaling pathway played a pivotal role in the differentiation of MSCs into OBs,Epigallocatechin-3-gallate(EGCG)has been selected to promote the osteogenic differentiation of MSCs via the activation of Wnt/β-catenin signaling.However,EGCG is easy to be metabolized by cells,so it is difficult to promote the differentiation of MSCs into OBs in vitro.In this paper,EGCG was encapsulated in chitosan to prepare EGCGloaded chitosan nanoparticles(ECN).Owing to the properties of CS and alginate(Alg)were similar to those of bone extracellular matrix,ECN was entrapped into CS/Alg scaffolds to form CS/Alg-ECN scaffolds for improving the bioavailability of EGCG.The results showed that the CS/Alg-ECN scaffolds continuously released EGCG for up to 16 days,and the EGCG release rate of the CS/Alg-ECN scaffolds reached 0.18±0.04～2.82±0.36 m/mL.Compared with the group without EGCG,the expression level of ALP and calcium content of MSC on CS/AlgECN scaffold were significantly increased.Moreover,osteogenic genes such as ALP,Runx2,OPN,OCN and Col-I of MSC on CS/Alg-ECN scaffold were significantly elevated than the group without EGCG.Wnt/β-catenin signaling pathway related proteins of MSCs on CS/AlgECN scaffolds were further detected.The protein expression levels of Wnt 3A,β-catenin,PGSK3 β,TCF1/TCF7 and Cycin D1 of MSC on CS/Alg-ECN scaffolds were significantly higher than those without EGCG group.These results suggested that CS/Alg-ECN scaffolds activated Wnt/β-catenin signaling pathway by releasing EGCG to promote differentiation of MSCs into OBs.Taken together,in this paper,CS/Alg system was used to construct sustainedrelease CS/Alg-ECN scaffolds to promote the differentiation of MSCs into OBs,providing a new idea for the construction of biomimetic microenvironment components for ex vivo expansion of HSCs.In this paper,starting from the perspective of biomaterials and cells,the strategy of inducing MSCs into OBs to fabricate biomimetic microenvironment was used to construct a three-dimensional co-culture system.In addition,the off-the-shelf COB application could be realized through cryopreservation,which improved ex vivo expansion of HSCs,and provided a technical support for the construction of bionic bone marrow hematopoietic microenvironment. |
