In vitro construction of cartilage organoids based on extracellular matrix microcarriers of cartilage
Objective To study the in vitro construction of functional and self-renewing cartilage organoids based on cartilage acellular extracellular matrix(ECM)microcarriers.Methods Fresh porcine articular cartilage was taken.The merely crushed cartilage particles were set as natural cartilage group and ECM microcarriers of appropriate particle size,which were prepared by the acellular method of combining physical centrifugation and chemical extraction,were set as microcarrier group.Cartilage organoids were constructed by loading human umbilical cord mesenchymal stem cells(hUCMSCs)and human chondrocytes(hCho)with a ratio of 3:1 with microcarriers through a rotating bioreactor.The organoids with different induction times were divided into 0-,7-,14-,and 21-day induction groups.The cell residues of the microcarrier group and natural cartilage group were evaluated by 4',6-diaminidine 2-phenylindole(DAPI)fluorescence staining and DNA quantitative analysis.The retention of microcarrier components was observed by Safranin O and toluidine blue stainnings,and the collagen and glycosaminoglycan(GAGs)levels in the microcarrier group and the natural cartilage group were determined by colorimetric method and dimethyl-methylene blue(DMMB)method.The microcarriers were further characterized by scanning electron microscopy and energy dispersive spectroscopy.The hUCMSCs cultured with Dulbecco's Modified Eagle's Medium(DMEM)supplemented with fetal bovine serum(FBS)in a volume fraction of 10％was used as the control group and the hUCMSCs cultured with the microcarrier extract was used as the experimental group.Subgroups of hUCMSCs cultured at 3 time points:1,3 and 5 days were set up in the two groups separately.Cell Counting Kit 8(CCK-8)was used to detect the biocompatibility of the two groups.The cellular activity of the organoids of the 0-,7-,14-,and 21-day induction groups was detected by live/dead staining and the self-renewal ability of the cartilage organoids of the 14-day induced group was identified by Ki67 fluorescence staining.The organoids of the 7-,14-,and 21-day induction groups were detected by RT-PCR in terms of the expression levels of chondrogenesis-related marker aggrecan(ACAN),type Ⅱcollagen(COL2A1),SRY-related high mobility group-box gene-9(SOX9),cartilage hypertrophy-and mineralization-related marker type Ⅰ collagen(COL1A1),Runt-related transcription factor-2(RUNX2),and osteocalcin(OCN).Colorimetric and DMMB assays were performed to determine the ability of organoids in the 0-,7-,14-,and 21-day induction groups to secrete collagen and GAGs.Results The results of DAPI fluorescent staining showed that the natural cartilage group had a large number of nuclei while the microcarrier group hardly had any nuclei.The DNA content of the microcarrier group was(7.8±1.8)ng/mg,which was significantly lower than that of the natural cartilage group[(526.7±14.7)ng/mg](P＜0.01).Saffranin O and toluidine blue staining showed that the microcarrier was dark-and uniform-colored and it kept a lot of cartilage ECM components.The collagen and GAGs contents of the microcarrier group were(252.9±1.4)μg/mg and(173.4±0.8)μg/mg,which were significantly lower than those of the natural cartilage group[(311.9±2.2)μg/mg and(241.3±0.7)μg/mg](P＜0.01).Scanning electron microscopy showed that the surface of the microcarriers had uneven and interleaved collagen fiber network.The results of energy spectrum analysis showed that elements C,O and N were evenly distributed in the microcarriers,indicating that the composition of the microcarrier was uniform.The microcarrier had good biocompatibility and there was no statistical significance in the results of CCK-8 test between the control group and the experimental group after 1 and 3 days of culture(P＞0.05).After 5 days of culture,the A value of the experimental group was 0.53±0.02,which was better than that of the control group(0.44±0.03)(P＜0.05).In the 0-,7-,14-,and 21-day induction groups,hUCMSCs and hCho were attached to the surface of the microcarriers,with good cellular activity,and the live/death rates were(70.6±1.1)％,(80.5±0.6)％,(94.5± 0.9)％,and(90.8±0.5)％respectively(P＜0.01).There were a large number of Ki67 positive cells in cartilage organoids.RT-PCR showed that the expression levels of ACAN,COL2A1,SOX9,COL1A1,RUNX2 and OCN were 1.00±0.09,1.00±0.24,1.00±0.18,1.00±0.03,1.00±0.06 and 1.00±0.13 respectively in the 7-day induction group;4.16±0.28,5.09±1.25,5.65±1.05,0.47±0.01,1.68±0.02 and 0.21± 0.06 respectively in the 14-day induction group;13.42±0.92,3.07±0.21,1.84±1.08,2.72±0.17,2.91± 0.18 and 3.32±1.20 respectively in the 21-day induction group.Compared with the 7-day induction group,the expression levels of ACAN,COL2A1,SOX9 and RUNX2 in the 14-day group were increased(P＜0.05),but COL1A1 expression level was decreased(P＜0.05),with no significant difference in OCN expression level(P＞0.05).Compared with the 7-day induction group,the expression levels of ACAN,COL1A1 and RUNX2 in the 21-day induction group were significantly increased(P＜0.01),with no significant differences in the expression levels of COL2A1,SOX9 and OCN(P＞0.05).Compared with the 14-day induction group,the expression levels of ACAN,COL1A1,RUNX2 and OCN in the 21-day group were increased(P＜0.05 or 0.01),with no significant difference in the expression level of COL2A1(P＞0.05),but the expression level of SOX9 was decreased(P＜0.05).The contents of collagen in 0-,7-,14-and 21-day induction groups were(219.15±0.48)μg/mg,(264.07±1.58)μg/mg,(270.83±0.84)μg/mg and(280.01±0.48)μg/mg respectively.The GAGs contents were(171.18±1.09)μg/mg,(184.06±1.37)μg/mg,(241.08±0.84)μg/mg and(201.14±0.17)μg/mg respectively.Compared with the 0-day induction group,the contents of collagen and GAGs in 7-,14-,and 21-day induction groups were significantly increased(P＜0.01),among which the content of collagen was the lowest in 7-day induction group(P＜0.01)but the highest in the 21-day induced group(P＜0.01);the content of GAGs was the lowest in the 7-day induced group(P＜0.01)but the highest in the 14-day induction group(P＜0.01).Conclusions The microcarriers prepared by combining physical and chemical methods are decellularized successfully,with more matrix retention,uniform composition and on cytotoxicity.By loading microcarriers with hUCMSCs and hCho,cartilage organoids are successfully constructed in vitro,which are characterized by good cell activity,self-renewal ability,strong expression of genes related to chondrogenesis and secretion of collagen and GAGs.The cartilage organoids constructed at 14 days of induction have the best chondrogenic activity.
万方数据
维普
