| Recent years have brought about the rise of regenerative medicine,and in particular the application of tissue engineering scaffolds with stem cells to repair malfunctional tissues/organs becomes a hotspot of research.Supercritical fluid technology,denoted as a set of novel ecofriendly techniques,is attractive and superior to other scaffold fabrication methods taking advantages of mild operation condition,avoidance of organic solvents and easy tunability.Diabetes is one of the four non-communicational diseases.The number of patients suffering from diabetes keeps expanding through the decades,making it urgent to find a cure for the disease.Tissue engineering uses scaffolds to transplant pancreatic cells or the pluripotent stem cell-derived pancreatic cells.It is a promising solution to cure diabetes,an alternative to insulin administration treatment.However,the methodology and mechanism of either well-designed scaffold or high efficient in vitro differentiation protocol remains poorly understood.In this thesis,the research of supercritical CO2(scCO2)foaming technique focused on the influence of main operation parameters on pore formation during simple foaming and two-step depressurization process,and the mechanism of pore growth was detailedly depicted.Meanwhile,the protocol of pancreatic progenitor differentiation from human embryonic stem cells/induced pluripotent stem cells in vitro was optimized,and a novel mechanism/protocol of generating definitive endoderm was developed,as well as an Activin A/FGF-free protocol of pancreatic progenitor differentiation.To begin with,mono-modal poly(ε-caprolactone)(PCL)scaffolds were fabricated via scCO2 simple foaming.The effects of soaking time(ST),temperature,pressure and depressurization rate(DPR)on pore morphology and structure were investigated.Results revealed that pore size decreased and pore size distribution was narrowed when ST extended from 0.5-2 h.Obviously,pore size and size distribution remained stable for ST longer than 2 h due to saturation of CO2 in the sample.Temperature showed a profound impact on pore formation,characterized by the facts that foaming failed at temperature lower than 35℃ and beyond 60℃,and that pore size increased from 17.5±7.7μm to 150.8±37.2μm when elevating temperature from 35 to 60℃.Interestingly,the reduction of PCL melting point by scCO2 altered the pattern of foaming,denoted as "solid-state foaming" for temperature lower than 40℃ and "melt-state foaming" for temperature over 40℃.Influence of pressure relied greatly on temperature,exhibiting a narrow size window between 20-30μm for the range of 10-25 MPa at 40℃ and an increase from 25.2±4.3μm to 238±32μm at 60℃.Pore size increased from 23.8±5.0μmto 345±54μm/s with lowering from 5 to 0.005 MPa/s.Also,temperature showed a strong influence on porosity,characterized by 60.8±3.2%at 35℃ and 80%for higher temperature.Interconnectivity was mostly affected by DPR,showing an increase from 41%to 71%as DPR increased from 0.03 MPa/s to 5 MPa/s.Based on the study of scCO2 simple foaming,the procedure was modified into two-step depressurization foaming to fabricate bi-modal porous PCL scaffolds.It was discovered that large and small pores expanded and size distribution widened when raising temperature,characterized by shift of peaks at 35 μm and 140 μm for small and large pores at 40℃respectively,to 70 μm and 160 μm at 50℃.Mono-modal porous scaffold with pore size 31.9±2.9 μm was fabricated when ST=0.5 h;Cell density increased from(186±15)×105/cm3 to(215±81)×105/cm3 when extending ST from 1 to 2 h,and the coalescence of cells rendered increase of large pore size while small pores remained unchanged.The pore density of large pores decreased from(3.17±0.48)×105/cm3 to(1.75±0.41)×105/cm3 when holding time(HT)was elongated from 0.5 to 1 h.Pressure exhibited a distinctive influence on pore structure.When intermediate pressure(Pinter)was in the range of 7-12 MPa,size of both large and small pores increased with decrease of Pinter;whilePinter was in the range of 5-7 MPa,size of both large and small pores decreased with decrease of Pinter.With the aid of foaming break experiment,pore growth profile during scCO2 two-step depressurization foaming was depicted:(a)in soaking stage,CO2 was dissolved into PCL matrix;(b)in the first depressurization stage,the precursors of large pore nucleated and grew in a certain extent;(c)during holding stage,the generated bubbles/cells coalescenced to form large bubbles/cells;(d)in the second depressurization stage.venting of CO2 from the matrix either expanded the existing cells or formed small pores in the unfoamed region.Depletion of CO2 caused vitrification of foaming matrix,thus bi-modal porous scaffold was generated.The unfolded mechanism offered a guidance to the fabrication of bi-/multi-modal porous scaffolds.On the basis of scCO2 foaming experiments,physical model for bubble growth profile during supercritical CO2 foaming was established by resorting to cell model.Mathematical modeling of the bubble growth was deducted by calculating mass and momentum balance using Zaremba-Dewitt model as a constitutive equation.For the purpose of solving this model,PC-SAFT EOS was introduced to simulate the CO2 solubility in PCL matrix.By extracting the solubility data,initial conditions for cell model were obtained.By introducing the experimental data and calculated initial conditions,the bubble growth curves in varying pressure and DPR were profiled.The calculated data were consistent with experimental data,evidencing the reliability of the model in describing and predicting pore growth during scCO2 simple foaming.On another aspect,the protocol of differentiating hESC/hiPSC into pancreatic progenitors in vitro was studied in detail.Interestingly,a novel method using GSK3β inhibitor,chir99021(CH)to induce SOX 17 expression was developed.It was found that in the CH concentration range of 2-6μM,44.7%SOX17 expressing H1 cells at 4 μM and 82.5%SOX17 expressing 6-9-9 cells at 3μM were detected respectively.Higher or lower CH concentration would decrease the SOX 17+ population,which was then validated by a peak mcherry expression at 3 μM in a knock-in H9-SOX17-mcherry line.Meanwhile,treatment of BMP inhibitor,dorsomorphin(DM),together with CH was found to significantly increase SOX17+population,characterized by an elevated percentage of 78.1%with DM over 38.4%without DM on D5.The fact that TGF-βinhibitor repressed the SOX17 expression induced by CH indicated that TGF-β signaling was involved in the induction.RT-qPCR result showed downregulation of pluripotent markers Nanog and Oct4 on D1,transient expression of primitive streak markers T,Mixl1,GSC on D1-D2,and the sequential upregulation of definitive endoderm markers Foxa2 and SOX 17 from D1 to D4,demonstrating the formation of definitive endoderm.The endoderm potential of these CH induced D4 cells was later evidencd by successful differentiation into pancreatic progenitor and liver bud cells.Finally,RNA-seq analysis confirmed that induction of definitive endoderm bypassing Activin pathway was mediated by TGF-β signaling pathway through the activation of Nodal signaling pathway by CH.Using CH+DM derived definitive endoderm cells,the differentiation protocol of pancreatic progenitor was optimized.It was found that NKX6.1+population was upregulated by either prolonging S3 duration from 2 to 4 days,or addition of PKC activator PDBu in S3,or addition of 50μM ROCK inhibitor Y27632.Moreover,diluting and replating D4 cells with a ratio of 1:6 promoted PDX1 expressing population to over 90%,yet suppressing the expression of NKX6.1,evidencing the profound impact of cell density on pancreatic differentiation.Furthermore,efforts were made to substitute FGF10 with FGF2 during the protocol,only to find no significant variation of PDX1 expression;it was also discovered that the expression of both PDX1 and NKX6.1 was not affected by removal of FGF content in the protocol,suggesting the independent regulation of pancreatic progenitor differentiation on FGF signaling pathway.To conclude with,scaffolds with tunable pore size and structure could be fabricated by means of scCO2 foaming technique,and a closer look into the profile and mechanism of pore formation during scCO2 foaming was displayed.Meanwhile,pancreatic progenitor cells could be efficiently generated in vitro using the novel differentiation protocol.Combination of scaffolding and stem cell differentiation,the thesis proposed a promising insight into the application of tissue engineering to repair tissue/organs. |
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