| Objective: To construct a cell-laden and orthogonal-multilayer tissue-engineered corneal stroma and to test the characteristics and activity.To explore the way of compressing and stretching the collagen hydrogel contain corneal stromal cells and multi-layer stacking methods.To construct the bionic tissue-engineered corneal stroma close to the native corneal stroma,and to explore the morphological and molecular biology changes of the tissue engineering corneal stroma.Methods: The primary rabbit corneal stromal cells(CSCs)cultured to passage 5 in vitro were as seed cells.Four-layer cell-laden compressed collagen(CC)and stretched compressed collagen(SCC)were constructed by plastic compression and stretching.They were crosslinked by transglutaminase.CC and SCC were then cultured in vitro for 7 days,and the transmittance and mechanical strength tests were performed.The surface and internal ultrastructure of CC and SCC were detected by scanning electron microscopy(SEM)and transmission electron microscopy(TEM).The viability of cells in CC and SCC was detected by the use of live-dead staining.The arrangement and length of cells were measured by fluorescent staining.The spatial arrangement of cells was examined by confocal microscopy.WB and q PCR were used to discovery the changes of gene expression in cells of CC,SCC and tissue culture plate(TCP).RNAseq was used to find out differential expression in the whole transcriptome.Based on differentially expressed genes(DEGs),molecular and pathway changes were analyzed.Finally,the biocompatibility and activity of the tissue-engineered corneal stroma were tested by rabbit corneal stromal transplantation experiments in vivo.Results: The average transmittances of hydrated CC,hydrated SCC and native corneal stroma were 63.7%,73.8% and 76.5%,respectively.The average transmittances of dehydrated CC,dehydrated SCC and native corneal stroma were 87.7%,89.7% and 98.5%,respectively.The tensile strength of dehydrated CC and SCC were 12.86±1.77 MPa and 17.38±1.06 MPa,respectively.The percentage of live CSCs was(83.5 ± 3.7)% in CC and(80.3 ± 2.6)% in SCC.The lengths of cells in CC and SCC were 108.0 ±28.4 ?m and 133.2 ± 36.1 ?m,respectively.The direction of cells in CC was random,without a specific direction.However,the arrangement of cells in SCC was concentrated within ± 30° of the stretching direction,with a distinct orientation.SEM and TEM results showed that the collagen fibers in CC were randomly arranged,while the collagen fibers in SCC were arranged in an orderly manner.The result of confocal microscopy showed that CC and SCC were composed of four cell layer sheets.The cells in CC were arranged randomly.However,each layer of cells in SCC was arranged in an orderly direction,orthogonal to its neighbours.WB and q PCR results showed that compared with the TCP group,CD34 and lumican were up-regulated in CC and SCC,and type I collagen,?-SMA and ?-actin were down-regulated.The expression of ACTA2,COL1A1,FN1,VIM,MKI67,SNAI1 and G0S2 is significantly increased,while LUM and DCN were down-regulated.Flow cytometry results showed that the ratios of cells in G0/G1 phase in CC,SCC and TCP were 56.0 ± 2.9%,55.7 ± 1.9% and 49.9 ± 1.4%,respectively,and they were 24.9 ± 0.3% and 24.5 ± 1.4% and 34.8 ± 2.3% in S phase respectively,in the G2/M phase were 18.6 ± 2.8%,19.5 ± 0.5%,and 15.2 ± 0.9%,respectively.The proportion of CC and SCC in G0/G1 and G2/M phases increased significantly,and the proportion in S phase significantly decreased.RNA-seq result showed that compared with the TCP group,there were 2440 genes down-regulated and 2324 genes up-regulated in CC.In addition,there were 2445 genes down-regulated and 2346 genes up-regulated in SCC.Compared with CC,there were 58 genes downregulated and 55 genes up-regulated in SCC.Cell division and DNA replication-related signaling pathways were down-regulated,and pathways such as Fox O,PI3K-Akt,axon guidance,and MAPK signaling pathways were up-regulated.Related genes such as cytoskeleton,collagen,activation,and proliferation were downregulated,and matrix metalloproteinases and gap junction related genes were up-regulated.In vivo study showed that CC and SCC were more transparent after being transplanted into rabbit corneal stroma for 6 weeks.There were cells in CC and SCC,and no cells in the control group.And cells in CC and SCC can normally expressed CD34 and vimentin,did not express ?-SMA,and CC and SCC have good biocompatibility.Conclusion: A bionic cell-laden tissue-engineered corneal stromal layer was fabricated by mechanical compression and stretching with orthogonal-multilayer orderly arrangement,quiescent stromal cells and good biocompatibility.It not only provides the basis for the design of future corneal and other tissue engineering organs,but also provides valuable new insights into the development,growth,remodeling,regeneration,and fibrosis of the cornea and other connective tissue. |
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