| BackgroundWith the change of lifestyle and coming of ageing population, neck shoulder pain or lumbocrural pain (NSCP) has been become the main threat to human health and the main cause of disability and labour loss. It is estimated that about 80% of the population may experience NSCP at least one time in their lifetime, and NSCP bring heavy social and economic burden. Therefore, treatment of NSCP diseases is one of the most important issues to be solved urgently.Currently, the intervertebral disc degeneration(IVDD) was recognized as the main cause of NSCP. However, the mechanism underlying IVDD is still unclear. The commonly accepted risk factors include heredity, age, biomechanics, trauma, occupation and others. As the main organ to share load of the upper part of the body, intervertebral disc (IVD) is susceptible to the damage caused by the continuously enormous compressive stress and shear stress. Meanwhile, the loss of notochord cells and avascular character of IVD in adults greatly influence the regenerative ability, which further aggravate the irreversible process of IVDD.Generally, conservative treatment and surgical treatment are two major strategies for IVDD. Conservative treatment include medication therapy, acupuncture, physiotherapy which are aimed at temporary symptomatic relief in the earlier stage of IVDD instead of prevention of IVDD. As the final option following the conservative treatment, surgical treatment including discectomy, spinal fusion, artifical disc replacement can maintain clinical effectiveness for longer period, however, complications result from surgical treatment, such as adjacent disc degeneration, have greatly disturbed doctors and patients. Therefore, seeking a new therapeutic strategy is an important task for treatment of IVDD, and biological therapeutics of IVD have provide a new option for renovation of IVDD.The fate of IVD depends on the balance between the degenerative process and endogenously self-regenerative potential. Aforementioned risk factors are involved in the degenerative process. However, the endogenous regneration of IVD involves two manners. On the one hand, inherent cells in IVD including IVD terminal cells (like nucleus pulposus cells, annulus fibrosus cells, endplate cartilage cells) and IVD progenitor (like nucleus pulposus mesenchymal stem cells, annulus fibrosus mesenchymal stem cells, endplate cartilage mesenchymal stem cells) function through secreting excellular matrix and differentiating toward terminal IVD cells respectively. On the other hand, bone mesenchymal stem cells(BMSCs) function through migrating into IVD with the help of growth factors produced in the process of degeneration. Therefore, enhancement of endogenous repairement through activating endougenous IVD cells and chemotactic migrating BMSCs is expected to became a potential therapeutic strategy for IVD degeneration.As an important member of transforming growth factor-β(TGF-β) family, bone morphogenetic protein-7(BMP-7) plays a similar role in activating chondrocytes and nucleus pulposus cells. Many ex-vivo studies have demonstrated that BMP-7 can greatly enhance the expression of aggrecan (AGG), type II collagen (COLII) and sex determining region Y-box 9 (SOX-9) at both gene and protein levels, and promote cell proliferation and inhibit cell apoptosis. Besides, BMP-7 can faciliate the migration and chondrogenic differentiation of BMSCs. Based on above requirements, BMP-7 can be selected as a ideal growth factor for enhancement of endogenous regeneration of IVD. However, BMP-7 has disadvantages like short half-life, multiple injection and risk of ossification which retard the process of clinic application.Studies revealed that BMP-7 contains three bioactive peptide (KPSS/SNVI/KAIS) and KPSS behaved best in promoting cell proliferation and poorest in ossification. In our previous study, we conjugated these three peptides onto the C terminal of self-assembly peptide nanofiber material RAD A16-1 respectively to produce three types of functional self-assembly peptide nanofiber materials RADA-SNVI, RADA-KPSS, RADA-KAIS. Then, we respectively combined these three functional self-assembly peptides with RADA16-I at the volume ratio of 1:1 to produce another three mixed peptides like RADKPS、RADSNV、RADKAI. We have compared these functional self-assembly peptide by biological methods and demonstrated that RADKPS behaved better in promoting prodution of AGG and type COLII and maintained longer effectiveness. However, there remain no studies on avtivation of human BMSCs and prevention of IVDD by RADKPS. Therefore, based on our previous studies, we are to further investigate the biological effectiveness of RADKPS in vitro and in vivo. There were three objectives in this study. Firstly, we aim to study the role RADKPS on the chemotactic migration of human BMSCs in vitro and the process of human BMSCs migrating into nucleus pulposus through cartilage endplate using ex-vivo bovine coccyx IVD organ model. Secondly, we aim to detect the effect of RADKPS on the proliferation and differentiation toward nucleus pulposus cells of human BMSCs. Thirdly, we aim to detect the effect of RADKPS on the regenerative effectiveness of IVDD and the chemotactic migration of PKH26 labeled human BMSCs toward IVDs in rabbits.Part 1 Effect of RADKPS on the chemotactic migration of human BMSCs in vitroObjective:To observe effect of RADKPS on the chemotactic migration of human BMSCs with Transwell model and bovine coccyx IVD organ culture model.Methods:1.Self-assembly peptide RADA16-I and functional self-assembly peptide RADA16-KPSS were manufactured by Hangzhou Chinese Peptide Company. Peptide powders (RADA16-I/RADA16-KPSS) were dissolved with 10% sucrose solution at the concentration of lmg/mL(m/v), and then these two types of solution were mixed at the volume ratio of 1:1 to produce the mixed functional self-assembly peptide solution RADKPS. Then the gelling properties and histological structure were evaluated. About 400 uL serum-free Dulbecco’s modified Eagle’s medium (SF-DMEM) was added into each well of 24 wells plate with Transwells inserted inside, and then 100 uL of RADA16-I and RADKPS solutions were added into the each insert. After culturing in incubator for 30 mins, the gelling properties and the structure of gelatin was observed through HE staining.2.Primary human BMSCs were isolated by gradient centrifugation method and cells P3 generation were obtained by ex-vivo expansion. Multiple differentiation potential (Osteogenic, Adipogenic, Chondrogenic) were induced by conditioned medium and results were tested by Alizarin red staining, Oil red staining, Immunocytochemical staining of COLII. Human BMSCs in P3 generation were made into cells suspension for further studies.3.The ability of chemotactic migration of human BMSCs influcenced by RADKPS was evaluated by in vitro Transwell migration model. Experiment was divided into three groups:RADKPS, RADA16-I, SF-DMEM. Human BMSCs in P3 generation were suspended with SF-DMEM at the concentration of 1×105/mL, and 100 uL of cells suspension(1×104 cells in total) was added into each Transwell insert inside 24 wells plate. After incubating in incubator for 48 h, crystal violet staining was performed to observe the migrated cells on the lower surface of filter membrane inside Transwell insert. Then the number of migrated cells was counted under each view of × 100.4. Under aseptic condition, bovine coccyx spinal motion units were isolated. Bony endplate was removed by abrasive drilling and annulus fibrosus was incised longitudinally followed by removel of small amount of nucelus pulposus tissue. About 50 uL of RADKPS、RADA16-Ⅰ and SF-DMEM was injected into the defect inside the IVD and the incision was sutured layer by layer. All the discs were pre-cultured in incubator for 24 h. Human BMSCs in P3 generation were labeled with PKH26 fluorescent dye and seeded onto the upper surface of the IVD followed by incubation in incubator untill cells were adherented. Then, complete medium was added untill the entire IVD was immersed and cultured for 48 h in incubator. Samples were fixed with formaldehyde, embeded in paraffin, sectioned coronally at the center and observed under fluorescence microscope.Results:Both RAD A16-Ⅰ and RADKPS solutions can swiftly form into transparent hydrogel when serum-free medium was added. Meanwhile, HE staining showed that both two types of hydrogel stain red evenly. After differentiation induction, Alizarin red staining, Oil red staining, Immunocytochemical staining of COLII for human BMSCs all stained positively. After migrating in vitro for 48 h, the number of migrated cells on the lower surface of filter membrane was 115.33±11.30 in RADKPS group,20.89±5.49 in RADA16-I group,8.44±2.65 in SF-DMEM group respectively, RADKPS>RADA16-I>SF-DMEM, and there were significant differences among groups(P<0.05). In the model of bovine coccyx IVD organ, the number of migrated cells into nucleus pulposus within central-coronal section in group RADKPS was 41.75±10.81, and that in group RADA16-I and group SF-DMEM were 16.25±6.13 and 10.00±4.16 respectively. There were signifcant difference between RADKPS group and other groups (P<0.05). The difference between RADA16-I group and SF-DMEM group was significant (P>0.05). Conclusion:Both RADKPS and RADA16-I can migrate human BMSCs in vitro, and RADKPS displayed superior ability than RADA16-I. However, only RADKPS displayed ability of enhancing chemotactic migration of human BMSCs in bovine coccyx IVD model.Part 2 Effect of RADKPS on the ability of differentiation toward nucleus pulposus cell of human BMSCsObjective:To study the effect of RADKPS on the viability, proliferation and ability of differentiation toward nucleus pulposus cell of human BMSCs in vitro.Methods:1.Human BMSCs in P3 generation was suspended with complete medium at specific concentration varied with different assays. Cells suspension was mixed with RADKPS or RADA16-I at the volume ratio of 1:5 to construct cells-hydrogel hybrids. FDA/PI staining was performed to determine the viability of hybrids after culturing for 7 days. And MTT test was performed at 12 h,3 days and 7 days to determine the proliferation of the cells on hydrogels.2.Cells-hydrogel hybrids were constructed according to procedure (1) and cultured in vitro for 21 days. The relative levels of mRNA expression for nucleus pulposus cell phenotype like SOX-9、COLⅡ、CA-12、KRT-19 and AGG were determined by Real-time PCR. Then contents of COLII and AGG in cells-hydrogel hybrids at 7 days and 21 days were determined by ELISA assay.Results:After cells-hydrogel hybrids were cultured in vitro for 7 days, cells survival rate remained about 90% in both RADKPS group and RADA16-I group. MTT test showed that there were no significant difference in cell number between RADKPS group and RADA16-I group at 12 h and 3 days (P>0.05). The cell number in RADKPS group was higher than that in RADA16-I at 7 days(P<0.05). Real-time PCR showed the relative level of mRNA expression for SOX-9、COLⅡ、CA-12. KRT-19 and AGG in RADKPS group was higher than that in RADA16-I group after culture in vitro for 21 days, the difference was significant(P<0.05). ELISA assay showed the content of COLII in RADKPS group was slightly higher than that in RADA16-I group at 7 days, but there was no statistically significant difference (p> 0.05). However, the content of COLII was higher in RADKPS group at 21 days (P<0.05). The content of AGG in RADKPS group was higher than RADA16-Ⅰ group both at 7 days and 21 days (P<0.05). Conclusion:Compared with RADA16-I, RADKPS conjugated with bioactive short functional peptide of BMP-7 can maintain the viability and enhance proliferation of human BMSCs, more importantly, promote the differentiation toward nucleus pulposus cells.Part 3 The study of retardation of degeneration process of rabbit IVD by RADKPSObjective:To study the effect of RADKPS on retardation of the process of IVDD in rabbit and the chemotactic migration of human BMSCs in vivo.Methods:1.Twenty four rabbits(genders unlimited, weight about 2.5-3.0 kg) without IVDD were selected by MRI scan test. After anaesthetized by intramuscular injection, FVDs between L2-5 were exposed aseptically through the retroperitoneal lateral (left) approach. Annulus fibrosus was punctured with 21G needle connected with 10 mL syringe and proper nucelus pulposus tissue was aspirated under the constant negative pressure to induce IVD degeneration.2. At 4 weeks after degeneration induction, animals with degenerative FVDs were evaluated by MRI scan and X ray test. Rabbits with degnerative IVDs at Pfirrmann Ⅱ-Ⅱ were assigned to subsequent animal trials refering to IVD regeneration. Three consecutive degenerated IVDs and L5-6 in the same rabbit were assigned to four treatment groups:PBS buffer solution(L2-3)、RADA16-I solution(L3-4)、RADKPS solution(L4-5)、Normal control(L5-6). After anaesthetized by intramuscular injection with ketamine and Sumianxin Ⅱ, IVDs between lumber 2 and lumber 5 were exposed aseptically through the retroperitoneal lateral (right) approach. Then, about 20 μL of solution(PBS solution, RADA16-I and RADKPS) was injected into corresponding IVDs with Hamilton microinjector (27 G needle). At 4 weeks after therapeutic operation,1 mL of PKH26-labeled cells suspension at the density of 1 × 106/mL was intravenously injected through ear vein.3. At 4 weeks、16 weeks after therapeutic operation, rabbits were anaesthetized by intramuscular injection of ketamine and Sumianxin Ⅱ, and relative signal intensity (RGI) and change in the relative disc height index(%DHI) were evaluated by MRI and X ray test. After performing MRI and X ray test at 16 weeks after therapeutic operation, animals were executed by air embolism. All the IVDs between L2-L6 were harvested with part of vertebral body. Parts of IVDs were fixed within 10% formalin solution for 48 h. After processes of decalcification, paraffin embedding and section, safranin O staining and HE staining were stained with performed to observe the histological structure of IVD. Then sections without staining were stained with DAPI and observated under fluorescence inversion microscope to identify the migrated cells in nucleus pulposus. transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) fluorescence staining was performed to evaluate the apoptosis in nucleus pulposus tissue. The rest of IVDs were incised to harvest nucleus pulposus and preserved in liquid nitrogen for ELIS A analysis of content of COLⅡ and AGG in nucleus pulposus.Results:After degeneration induced for 4 weeks, the results of MRI scan and X ray test revealed that target IVDs degenerated homogeneously. There were no significant difference among different levels of IVDs in RGI and %DHI (P>0.05). At 4 weeks and 16 weeks after therapeutic operation, X ray assay revealed that there were no significant difference among different treated groups in %DHI (P>0.05). However, the%DHI in three treated groups were all less than normal group (P<0.05). At 4 weeks and 16 weeks after therapeutic operation, results from MRI assay revealed that RGI of nucleus pulposus in RADKPS group was higher than that in both RADA16-I group and PBS group (P<0.05). The RGI in RADA16-I group was slightly higher than that in PBS group (P>0.05). The RGI in three treated groups were all less than that in normal control group (P<0.05).At 16 weeks after therapeutic operation, Safranin 0 staining showed that NP tissue stained red deeply in normal control group, RADKPS group stained stronger than RAD A16-1 group and PBS group. HE staining showed that the boundary between AF and NP was clear and clusters of huge cells was remained in normal control group.The boundary between AF and NP was exist and the cell shape was chondrocyte-like in RADAKPS group. However, in RADA16-I group and PBS group, the boundary was disappeared and the chondrocyte like cells were reduced and gradually replaced by fibroblast-like cells. After stained with DAPI, PKH26-labeled human BMSCs (red fluorescence) with complete nuclear(blue fluorescence) were observed in RADKPS group, there were no red fluorescence was observed in other groups. TUNEL fluorescence staining showed little apoptotic cells were observed in normal control group, and the rate of apoptotic cells to all nucleus pulposus cells was lower in RADKPS group than that in RADA16-I group and PBS group(P<0.05). ELISA assay test showed that content of COLII and AGG in RADKPS group was higher than that in RADA16-I group and PBS group(P<0.05), but lower than that in normal control group(P<0.05).Conclusion:Compared with RADA16-I, RADKPS can retard IVDD through decreasing apoptosis, promoting migration of BMSCs from bone marrow and production of COLII and AGG in rabbit IVDs. |
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