Experimental Research Of Treatment Of Rabbit Intervertebral Disc Degeneration By Transplantation Of Bone Mesenchymal Stem Cells And Functionalized Self-assembling Peptide Scaffold

Part 1 Biosynthesis of functionalized peptide and self-assembly of functionalized peptideObjective:To design and synthesize of functionalized peptide RLN containing core sequence (link N) of link protein and investigate its microstructure.Methods:The RLN and RADA16 peptides were dissolved in 10% sterile sucrose at a final concentration of 1%(lOmg/ml, m/v). Peptides solution of RLN and RADA16 (at a ratio 1:1) were mixed and treated with equal volume of Dulbecco's, Modified Eagle Medium F-12(DMEM-F12) solutions. The microstructures of peptide scaffolds were observed using atomic force microscopy (AFM).Results:The RADA16 and RLN peptides were successfully synthesized. A hydrogel was formed when the RLN was mixed with RADA16 at a ratio 1:1. The AFM images demonstrated that the nanofibers was observed in RLN/RADA16 peptide sample (D=31.9±3.8 nm), and the length of these fibers could be ranging from several hundreds nanometers to a few microns.Conclusions:A link N nanofiber scaffold (LN-NS) was self-assembled by mixing peptide solution of RLN and RADA16, which could be useful in the nucleus pulposus regeneration. Part 2The effect of functionalized self-assembling peptide nanofiber hydrogel scaffold on the biological behavior of rabbit nucleus pulposus cellsObjective:To investigate the effect of a novel designer functionalized self-assembling peptide hydrogel scaffold (LN-NS) on the biological behaviors of rabbit nucleus pulposus cells (NPCs).Methods:NPCs were obtained from immature nucleus pulposus (NP) of Japanese white rabbits (3 week-old) and identified by Transmission electron microscopy (TEM). The RLN/RADA16 mixture solution or pure RADA16 peptide solution was treated with equivalent standard cell culture medium DMEM-F12 to induce peptide self-assenbly (experimental group: functionalized LN-NS hydrogel; control group:RADA16 hydrogel). The rabbit NPCs were co-cultured with hydrogel scaffolds to observe the cellular toxicity of these scaffolds and the effect of these gel scaffolds on the biological behavior of NPCs. The biocompatibility of peptide hydrogel scaffold were observed by LIVE/DEAD cell viability assay (Calcein-AM/PI); the effect of the functionalized LN-NS hydrogel scaffold on cell adhesion and 3-D migration were examined by cell counting kit-8 (CCK-8) and real-time PCR.Results:The NPCs showed a cluster-like growth and were divided into two types (chondrocytes-like cells and notochordal cells). The notochordal cells were round or oval and filled with vesicles, and the chondrocytes-like cells were spindle or polygon. A large number of glycogen was gathered in the both type of cells. This designer functionalized LN-NS hydrogel scaffold exhibited little cytotoxicity and promoted NPCs adhesion obviously.In three-dimensional cell culture experiments, confocal reconstructed images testified that the functionalized LN-NS guided NPCs migration from the surface into the hydrogel considerably, in which the RADA16 scaffold did not. Moreover, the functionalized LN-NS significantly stimulated the biosynthesis of extracelluar matrices (ECM) by NPCs.Conclusions:The rabbit nucleus pulposus cells were cultured successfully. The designer functionalized LN-NS hydrogel containing link N exhibited excellent biocompatibility and bioactivity with rabbit NPCs and could be useful in the nucleus pulposus regeneration. Part 3The effect of functionalized self-assembling peptide nanofiber hydrogel scaffold on the biological behavior of rabbit bone marrow mesenchymal stem cellsObjective:To investigate the effect of a novel designer functionalized self-assembling peptide hydrogel scaffold (LN-NS) on the biological behaviors of rabbit bone marrow mesenchymal stem cells (BMSCs).Methods:Rabbit BMSCs were harvested by density gradient centrifugation and adherent screening in vitro. The rabbit BMSCs were identified by examining the expression of CD29, CD34, CD44, CD45 surface antigen. The rabbit BMSCs were co-cultured with hydrogel scaffolds (experimental group:functionalized LN-NS hydrogel; control group:RADA16 hydrogel) to observe the cellular toxicity of these scaffolds and the effect of these gel scaffolds on the biological behaviors of BMSCs. The biocompatibility of peptide hydrogel scaffold were observed by LIVE/DEAD cell viability assay (Calcein-AM/PI); the effect of the functionalized LN-NS hydrogel on cell adhesion and 3-D migration were examined by cell counting kit-8 (CCK-8) and immunofluorescence.Results:Purified cells exhibited fusiform and spindle-shaped growth after attaching to the culture flask. The flow cytometry showed that these cells were positive for CD44 (96.2%) and CD29 (88.1%), but were negative for CD45 (4.4%) and CD34 (3.9%). In 3-D cell culture experiments, this designer functionalized LN-NS hydrogel scaffold exhibited little cytotoxicity and promoted BMSCs adhesion obviously. Moreover, the immunofluorescence examination showed that the functionalized LN-NS hydrogel significantly stimulated the biosynthesis of type II collagen when compared with the RADA16 hydrogel.Conclusions:The rabbit BMSCs were cultured successfully. The novel functionalized self-assembling peptide nanofiber scaffold LN-NS, which exhibited little cytotoxicity and excellent biocompatibility, is a biological scaffold, it could be very useful for intervertebral disc (IVD) tissue engineering. Part 4 Experimental research of treatment of rabbit intervertebral disc degene-ration by conjunction of bone mesenchymal stem cells and functionalized self-assembling peptide scaffoldObjective:To investigate the treatment effect of transplantation of functionalized LN-NS hydrogel and rabbit BMSCs to repair the degenerative intervertebral disc.Methods:The rabbit intervertebral disc degeneration (IDD) model was made by annulus fibrosus aspiration method.12 Japanese white rabbits (2.0-2.5kg) were registered in this experimentation. The treatment of each intervertebral disc segment is as follows:L2-3, only aspiration of nucleus pulposus tissue; L3-4, RADA16 hydrogel+BMSCs were injected after aspiration of nucleus pulposus tissue; L4-5, functionalized LN-NS hydrogel+BMSCs were injected after aspiration of nucleus pulposus tissue and L1-2 segment was as blank control. MRI images was obtained preoperatively and 3 weeks postoperatively. These rabbits were sacrificed, and the intervertebral disc tissues were used for histological staining and immunohistochemical staining.Results:The intervention segment of intervertebral disc had a different degree of changes in comparison to the blank control group (L1-2 segment). The DHI and standardized T2WI (ST2WI) were measured using Image-proplus 6.0 and Mergee Film Workstation. The T2WI of L2-3 segment obtained 3 weeks postoperatively significantly decreased compared with that of pre-operation, the L3-4 segment slightly decreased and L4-5 segment had no obvious changes. HE staining showed that the number of NPCs-like significantly increased in the nucleus pulposus tissue of L3-4 and L4-5 segment. The nucleus pulposus tissue of L1-2 and L4-5 segment demonstrated significantly positive by stained with immunocytochemistry (typeⅡcollagen) when compared with the L2-3 segment.Conclusions:The method of annulus fibrosus aspiration could cause rabbit intervertebral disc degeneration successfully. Implantation of functionalized LN-NS hydrogel and rabbit BMSCs could effectively inhabit the process of intervertebral disc degeneration.