Experimental Study On Angiogenesis Of Tissue Engineered Nerves Repairing Peripheral Nerve Defects

Objective: To observe and analyze the natural biological process of angiogenesis of tissue engineered nerve grafts(TENG) repairing peripheral nerve defects in vivo, and to explore key regulatory genes and internal molecular mechanisms during angiogenesis by morphological observations and microarray-based big data analysis.Methods: 1. Tissue engineered nerve graft construction. Tissue engineered nerve grafts were constructed in vitro using Schwann cells differentiated from rat skin-derived precursors(SKPs)(SKP-SCs) as supporting cells and chitosan nerve conduits combined with silk fibroin fibers as scaffolds in a rotary perfusion cell culture bioreactor following the successful preparation of freeze-dried chitosan nerve conduits. Tissue engineered nerve grafts were rinsed twice with normal saline(NS) and stored in NS for use. 2. Sciatic nerve defect model establishment. Female SPF Sprague-Dawley(SD) rats were divided into four groups randomly: TENG(tissue engineered nerve graft) group, Autograft(autologous nerve graft) group, Scaffold(chitosan nerve conduit) group and Sham(sham surgery) group. Eight different time points, 1d, 4d, 1w, 2w, 3w, 4w, 8w and 12 w, were included. Models of rat sciatic nerve 10 mm defect were established conventionally. 3. Stereoscopic microscope observation. The tissue engineered nerve or autologous nerve grafts were fully dissociated and exposed after anesthesia of animals(n=3 in TENG group and n=3 in Autograft group at each time point). The whole grafts were placed in the vision of stereoscopic microscope. The surface blood vessels of tissue engineered nerve or autologous nerve grafts were focused and photographed. 4. HE staining. The tissue engineered nerve or autologous nerve grafts were harvested following stereoscopic microscope photograph(n=3 in TENG group and n=3 in Autograft group at each time point). The animals specimens were fixed, sliced and HE stained. The inner blood vessels of tissue engineered nerve or autologous nerve grafts were observed and photographed in light microscope. 5. Micro-CT scanning, blood vessel reconstruction and parameter analysis. The perfusion of MICROFIL contrast agents was conducted after anesthesia of animals at 4w post surgery(n=3 in TENG group and n=3 in Autograft group). After the contrast agents had solidified, the tissue engineered nerve or autologous nerve grafts were dissociated and collected. The cleared specimens were observed in stereoscopic microscope. Micro-CT scanning, blood vessel reconstruction and parameter analysis were conducted for the specimens with well blood vessel perfusion of contrast agents. 6. Microarray detection and big data analysis. The graft tissues were dissociated and collected rapidly(n=9 in TENG group, n=9 in Autograft group, n=9 in Scaffold group and n=9 in Sham group)(same section of sciatic nerves in sham group) at each time point; a mixing tube included three specimens at each time point of each group) after anesthesia of animals. RNA extraction and microarray detection were performed. Genes related to blood vessel were analyzed. 7. Real-time RT-PCR detection and data analysis. The graft tissues were dissociated and collected rapidly(same section of sciatic nerves in sham group)(n=3 in TENG group, n=3 in Autograft group, n=3 in Scaffold group and n=3 in Sham group at each time point; three specimens became to a mixing tube at each time point of each group) after anesthesia of animals. RNA extraction, reverse transcription and RT-q PCR were performed to detect key genes. 8. Immunofluorescence staining. Key regulatory genes at time points of significant process of sprouting angiogenesis were selected(n=3 in TENG group and n=3 in Autograft group). The tissue slices were immunofluorescence stained to locate the spatial relationship between the key proteins and vascular endothelial cells.Results: 1. Stereoscopic microscope observation of the surface blood vessels. Blood vessels grown into tissue engineered nerve grafts from the two ends at 4d post surgery. The surface of tissue engineered nerve grafts were filled with blood vessels at 2w post surgery. Blood vessels of autologous nerve grafts increased significantly at 1w and 2w post surgery. 2. Light microscope observation of the inner blood vessels by HE staining. The mature lumen of blood vessels in the wall of nerve conduits appeared at 1w post surgery, and blood vessels in the wall increased significantly at 2w post surgery in TENG group.Meanwhile a few of blood vessels were seen in the regenerated nerve tissues at 1w post surgery in TENG group. A large number of blood vessels were observed in the regenerated nerve tissues at 2w-12 w post surgery in TENG group. Less blood vessels had been seen in the nerve tissues at 1d and 4d post surgery, and the blood vessels increased with no significant changes at following time points in Autograft group. 3. Blood vessel reconstruction by Micro-CT scanning. A large number of microvessels, also vessels connecting the proximal and distal ends, were seen in both TENG and Autograft group. New blood vessels grown into tissue engineered nerve grafts from three main directions: the proximal end, the distal end, and the middle. 4. Parameter analysis of blood vessel reconstruction. The average blood vessel diameter in TENG group was significantly smaller than that in Autograft group(p<0.05). The number, connection, and spatial distribution of the blood vessels had no significant difference between two groups(p>0.05). There were mainly microvessels and capillaries in both two groups. The diameters of blood vessels in TENG group were relatively smaller. However, blood vessels in TENG group were distributal broader. 5. Clustering analysis of microarray data. The gene expression values of three samples displayed well reproducibility at each time point respectively in TENG, Autograft and Scaffold groups. The gene expression values at 1d post surgery were relatively independent to those at other time points in three groups. 6. Principal component analysis of microarray data. The gene expression values at 1d post surgery were a stage, which were distant from those at other time points in TENG, Autograft and Scaffold groups respectively. The gene expression values at 4d, 1w, 2w, 3w, 4w, 8w and 12 w were another stage, which were close to each other in three groups respectively. 7. The number of differentially expressed genes analysis of microarray data. The number of both up-regulated and down-regulated differentially expressed genes increased significantly at 1w post surgery in TENG and Scaffold groups. However, the number of up-regulated and down-regulated differentially expressed genes was stable relatively with little change at each time points in Autograft group. The dynamic trend of the number of differentially expressed genes was similar between TENG and Scaffold groups, which was slightly different from that in Autograft group. 8. Biological process analysis of microarray data. Angiogenesis during nerve regeneration included “hypoxia onset”(1d post surgery), “sprouting angiogenesis”(4d-3w post surgery) and “blood vessel remodeling”(4w-12 w post surgery) in all TENG, Autograft and Scaffold groups. The local microenvironment was more conducive to sprouting angiogenesis(sooner appearance) and blood vessel remodeling(longer duration) in TENG group than that in Autograft and Scaffold groups. 9. Venn diagram analysis of angiogenesis genes. In the situation of genes involved, the process of cellular response to hypoxia was more significant in Scaffold group compared with that in other two groups; the process of sprouting angiogenesis was more similar in TENG and Autograft groups; the process of blood vessel remodeling was more significant in TENG and Scaffold groups than that in Autograft group. 10. Analysis of key angiogenesis genes. UCN2 plays a key role in process of cellular response to hypoxia in TENG and Autograft groups; PTGS2 is critical in Scaffold group. VEGFA and E2F8 play a central role in process of sprouting angiogenesis in TENG group; SEMA3 E is important in Autograft group. DBH has significant effect on process of blood vessel remodeling in TENG and Scaffold groups; CSRP3 is pivotal in Autograft group. 11. Dynamic regulation analysis of angiogenesis genes. Compared with that in Autograft group, the number of differentially expressed genes related to both sprouting angiogenesis and blood vessel remodeling was larger in TENG and Scaffold groups. Futhermore, the number of differentially expressed genes of sprouting angiogenesis in TENG and Autograft groups and blood vessel remodeling in Autograft group began to reduce at late time points, which showed a trend of decline following increase; but the number of differentially expressed genes of sprouting angiogenesis in Scaffold group and blood vessel remodeling in TENG and Scaffold group did not display a trend of decline at later time points. 12. Real-time RT-PCR detection. The key genes of cellular response to hypoxia, sprouting angiogenesis and blood vessel remodeling during the process of angiogenesis had similar trends of expression and good correlations with time changes between the detection results by RT-q PCR and the detection results by microarray. 13. Immunofluorescence staining. At the time points of siganificant process of sprouting angiogenesis, the key genes VEGFA(1d post surgery in TENG group), E2F8(1w post surgery in TENG group) and SEMA3E(2w post surgery in Autograft group) were all translated expression and co-localized with vascular endothelial cells, indicating that they play a major role in the process of sprouting angiogenesis.Conclusions: 1. Angiogenesis during peripheral nerve regeneration includes “hypoxia onset”, “sprouting angiogenesis” and “blood vessel remodeling” three main phases. 2. There is no essential difference of the angiogenesis network between tissue engineered nerve and autologous nerve repairing peripheral nerve defect; however, the phases and major regulatory molecules of different stages of angiogenesis vary. 3. Schwann cells differentiated from skin-derived precursors as supporting cells of tissue engineered nerve are in favor of “sprouting angiogenesis” and “blood vessel remodeling” during peripheral nerve regeneration.