Construction Of Self-assembling KLD-12 Peptide As Cell Scaffold In Tissue Engineering And Regenerative Effects Of Transplanting MSCs Embedded In KLD-12 Peptide Hydrogel To The Degenerated Intervertebral Disc

Objective:To synthesize self-assembling KLD-12 peptide with a sequence of AcN-KLDLKLDLKLDL-CNH2 and investigate its self-assembly and biocompatibility.Methods:KLD-12 peptide was synthesized by using a peptide synthesizer, which was purified and tested with high performance liquid chromatography (HPLC) and mass spectroscopy (MS). KLD-12 peptide solution with concentration of 0.5%,0.25% and 0.1% were induced to self-assembly with PBS in vitro, and the self-assembled peptide hydrogel was morphologically observed. Atomic Force Microscope (AFM) was employed to examine the self-assembled peptide nano-fibers. The fresh bone marrow was taken out and Rabbit mesenchymal stem cells (MSCs) were isolated by means of Percoll density gradient centrifugation, and then those cells were cultured in vitro. The cells (P3) were characterized by the expression of CD34, CD90, CD44 and CD45 with a flow cytometry. The peptide solution was diluted with complete culture medium to obstain a final peptide concentration of 0.01%,0.03%, and 0.05%, and MSCs (P3) were cultured with those peptide solutions, the complete medium was considered as control group.24h and 48h after the culture, cell viability was evaluated by using MTT test. Hemolysis rate of KLD-12 peptide was detected by using Hemolysis Test. Skin Irritation Test and Skin Implantation Test was used to evaluate the respone of the local skin for KLD-12 peptide.Results:The relative molecular mass of KLD-12 peptide was 1467.83, with a purity quotient of 95.36%. The KLD-12 peptide solution (0.5%) could be self-assembled to produce a hydrogel when it was triggered with PBS, which was structurally integral and homogeneous had sufficient cohesion to retain the shape of hydrogel. But the self-assembled hydrogel of KLD-12 with concentration of 0.1% and 0.25% was heterogeneous, with only few hydrogel granules formed in some areas. AFM demonstrated that the KLD-12 peptide could self-assemble to produce a kind of nano-fibers with a diameter of 30-40 nm (mean:13.7±4.7 nm) and a length of hundreds of nm. These nanofibers interwove to form a porous network structure. Isolated cells were adherent to culture flask and presented spindle-shape. Flow cytometry demonstrated that the expression of CD90 and CD44 by cells were positive, however, the expression of CD34 and CD45 was negative. The MSCs cultured with peptide solutions grew well and presented spindle-like or fusiform shape. At the 24 th h and 48 th h, the A values of the experimental groups were not significantly different from those of the control group (P>0.05). Hemolysis rate demonstrated that the hemolysis rate of KLD-12 peptide was 0.112%. Skin Irritation Test showed that the skin injected with KLD-12 solution remained normal without edema, erythema, and eschar, and the score of skin irritation was 0. The Histological Examination with HE staining exhibited that the skin layers were clear and there was no infiltration with neutrophilic granulocytes and lymphocytes.Conclusions:KLD-12 peptide was synthesized successfully. KLD-12 peptide could self-assemble to produce hydrogel and form nano-fibers. The self-assembling KLD-12 peptide had a good biocompatibility with host rabbit and MSCs. Objective:To culture Nucleus pulposus (NP) cells in 3-D self-assembling KLD-12 peptide hydrogel in vitro and evaluate the biological viability and phenotype of NP cell, the main objective was to investigate the feasibility of KLD-12 peptide as cell scaffold in tissue engineering of intervertebral disc.Methods:The NP cells were cultured in self-assembling KLD-12 peptide hydrogel for 2 weeks in vitro. The cells were observed under inverted microscope.1,3,7, and 14 days after the culture, peptide hydrogel/cells were taken out for cell proliferation evaluation by CCK-8.7 and 14 days after incubation, Calcein-AM (CAM) and Propidium Iodide (PI) Staining were used to detect the live and dead cells and calculate the cell survival rate in the hydrogel.14 days after incubation, Reverse Transcription Polymerase Chain Reaction (RT-PCR) was employed to detect the expression of type II collagen mRNA and Aggrecan mRNA of NP cells. The expression of type I/type II collagen were determinated by using Immunofluorescence Staining. The medium were collected for glycosaminoglycan (GAG) assay 1,4,7,10 and 13 days after the incubation.Results:Isolated NP cells were adherent to culture flask and presented spindle-shape. The NP cells distributed uniformly in hydrogel and maintained round shape. However, the cells at the bottom of hydrogel presented spindle-like shape. Cell Proliferation Test exhibited that the A value increased gradually over the culture time. A great number of live cells grew well in the hydrogel as revealed by CAM/PI fluorescence staining and maintained round shape, but the cells at the bottom of hydrogel presented spindle-like or fusiform shape, Some NP cells proliferated and were of dumb-bell shape. At the 14th day, the cells in peptide hydrogel still grew well. Compared with the cells at the 7th day, cell morphology remained unchanged, but cell proliferation was more apparent. The cell survival rate was 92.6% at the 7th day and 14th day after the incubation. RT-PCR results showed that the NP cells expressed mRNA of collagen II and Aggrecan. Immunofluorescence Staining showed that expression of collagen typeⅡwas high and most of the NP cells in hydrogel were positive, in addition, extracellular matrix was moderately stained for type II collagen. However, only a few of cells expressed collagen typeⅠ. GAG assay proved that GAG content in medium increased gradually over the culture time, though the increase was not linear.Conclusion:The peptide hydrogel could provide a conducive microenvironment for NP cells to survive and division in vitro. And the NP cells in hydrogel maintained phenotypically stability and normal disc function at gene and molecular level. It was conclude that KLD-12 self-assembling peptide could serve as a good alternative of biological scaffold for tissue engineering of intervertebral disc.Objective:To culture MSCs in 3-D self-assembling KLD-12 peptide hydrogel and construct tissue-engineered disc grafts with self-assembling KLD-12 peptide as scaffold material containing MSC in vitro. Tissue-engineered disc grafts of KLD-12 peptide/autologous MSC were transplanted into a rabbit model of disc degeneration, the main objective was to evaluate regenerative effects of the tissue-engineered grafts to the degenerated intervertebral disc.Methods:In vitro:The MSCs (P3) were cultured in the 3-D KLD-12 peptide hydrogel for 2 weeks in vitro. The cells were observed under inverted microscope.1, 3,7, and 14 days after the culture, CCK-8 Test was used to detect the cells proliferation. At the 7 th and 14 th day after the culture, Calcein-AM/PI Fluorescence Staining was performed to determine the Live/dead cell and calculate the cells survival rate. In vivo:To induce the rabbit disc degeneration. Peptide hydrogel/MSCs were transplanted into degenerated disc.12 weeks after transplantation, Lateral plain radiographs were taken to measure the vertebral body heights and disc heights. The disc height index (DHI) was calculated and changes in the DHI were expressed as %DHI (%DHI=postoperative DHI/preoperative DHI×100%). MRI images were used to measure the signal intensity of each disc on T2-weighted image. At the 12 weeks after transplantation, the spines were harvested for Macroscopic Evaluation and HE-staining to observe the disc degeneration. Toluidine Blue Staining was employed to assess the proteoglycan in discs. Reverse Transcription Polymerase Chain Reaction (RT-PCR) was used for the determination of the expression of typeⅡcollagen mRNA and Aggrecan mRNA in the disc. Some MSCs infected with Hochest fluorescence stain were encapsulated within the KLD-12 peptide hydrogel, and then the peptide/MSCs were transplanted into rabbit models of disc degeneration.4 weeks after transplantation, the spines were harvested to observe the MSCs distribution in disc.Results:In vitro:The MSCs distributed uniformly in KLD-12 peptide hydrogel and maintained round shape, but the cells at the bottom of hydrogel presented spindle-like shape. Cell Proliferation Test exhibited that the A value of peptide/MSCs increased gradually over the culture time. A great number of live cells grew well in the hydrogel as revealed by CAM/PI Fluorescence Staining and maintained round shape, but the cells at the bottom of the hydrogel presented spindle-like or fusiform shape, Some MSCs proliferated and were of dumb-bell shape. At the 14th day, the cells in peptide hydrogel still grew well. Compared with the cells at the 7th day, cell morphology remained unchanged, but cell proliferation was more apparent. The cell survival rate was 92.15% at the 7 th day and 91.18% at the 14 th day, there existed no difference in cell survival rates between two groups (P>0.05). In vivo:The transplanted MSCs were located in the nucleus pulposus (NP) and distributed uniformly. A few of MSCs migrated into inner annular fibrosus (AF). Radiographic Analysis demonstrated significant disc space narrowing in sham and light narrowing in transplantation groups, the % DHI was 90.21% in the transplantation group and 60.56% in the sham. There were statistically significant differences in % DHI between two groups (P<0.05). MRI T2-weighted signal intensities decreased significantly in the sham and decreased lightly in the transplantation group, there were statistically significant differences in signal intensities between two groups (P<0.05). Macroscopic Evaluation showed significant degenerative discs without NP regeneration in the sham. However, in the transplantation group, the basic structure of AF was normal, the discs demonstrated NP regeneration, but the structure of NP was incomplete. Histological Analysis demonstrated that Sham-operated discs showed significantly disordered AF without NP and NP cells. The basic structure of normal discs was observed in the transplantation group, the transplantation-operated discs showed slightly disordered AF with NP and NP cells regeneration. The discs were graded as 1-2 in the transplantation group and as 4-5 in the sham. Toluidine Blue Staining revealed the NP was moderately stained in the transplantation group, the sham-operated discs lacked of NP and the inner AF was stained poorly. RT-PCR confirmed that all of the discs expressed mRNA of typeⅡcollagen and Aggrecan. There were not statistically significant differences in expression of theⅡcollagen mRNA among three groups (P>0.05). However, the expression of Aggrecan mRNA in the transplantation group, which similar to the normal disc, was higher significantly than that in the sham (P<0.05)Conclusion:The peptide hydrogel could provide a conducive microenvironment for MSC to survive and proliferate in vitro. Tissue-engineered disc grafts of peptide/ MSC could restore the discs space height and increase the water content and proteoglycan content in the degenerated discs, which illustrated that the peptide/ MSC effectively led to regeneration of the degenerated intervertebral discs.