| Background and Objective:At present,in the process of repair and regeneration of the degenerative intervertebral discs?IVD?,stem cells are still the most important candidate cell type for the reconstruction of IVD biological functions.In the exogenous treatment of IVD degeneration,supplementation of exogenous mesenchymal stem cells?MSCs?was the common method.When the degenerative bovine IVDs were injected with MSCs alone,the glycosaminoglycan?GAG?content in the IVD can be restored[1].A follow-up study of autologous mesenchymal bone marrow cells in patients with low-back pain revealed that,after stem cell transplantation,the clinical symptoms of these patients improved significantly[2].Although the clinical effect can be determined,there are still various factors in which this technology can be affected,such as age,cell type and cell number,cell leakage?induce osteophyte formation?,the carrier or scaffold of cell transplantation,the method of directional differentiation and culture of stem cells before transplantation,and the degree of degeneration of the transplanted IVD are taken into account.All bioactive reagents involve safety concerns on the localization,appropriate dose,time and side effects.Considering these,in recent years,more ideal endogenous repair has attracted more and more attention in the treatment of IVD diseases.Recent studies have shown that the progenitor cells in the IVDs may play a potential role in the regeneration process[3].Besides the progenitor cells,there are some stem cell niches within the IVDs?ISN?,which have been identified in the outer AF near the perichondrium region,it may be the source of some stem cells of the IVDs and is considered to play an important role in the intrinsic repair of the IVD[4].The latest study showed that there are some differences between ISN-SCs and BMSCs in biological functions,and interestingly,ISN-SCs seems to have better performance in osteogenic and chondrogenic differentiations[5].The homing ability of MSCs in a variety of tissues and animals has gradually reached a consensus.After the tissue injury/inflammation,stem cells can perceive the decrease of the number of surviving functional cells and the structural injuries,and then migrated selectively into the damaged areas.ISN-SCs are endogenous stem cells with similar biological characteristics with BMSCs,and its homing mechanism may be very similar to that of MSCs,and regulated by chemokines and their receptors.However,there are still few studies on the potential mechanism of ISN-SCs migration and repair,which limits the application of the therapeutic strategies for endogenous repair of IVDs.In this study,the purpose is to detect ISN-SCs migration in degenerated IVDs and evaluate the role of chemokines in these cell recruitment process.Methods:We established a rabbit model of disc degeneration by using a 20-gauge hypodermic needle to stab the anterolateral anulus fibrosus.The identification of intervertebral disc degeneration were performed by magnetic resonance imaging,picrosirius red and hematoxylin-eosin staining,separately.Quantitative real-time PCR was used to detect the mRNA expression of CD29 and CD90 in anulus fibrosus?AF?at 1,2,3,4,5 weeks after acupuncture treatment.At 2 weeks after surgery,the number of BrdU positive cells in the IVD tissue was detected by immunofluorescence.The ISN tissues were separared under an anatomical microscope.Bone marrow mesenchymal stem cells?BMSCs?and ISN-SCs from IVD tissues were cultured and amplified in vitro.Then,the passage 3 of BMSCs and ISN-SCs were compared regarding the morphology and immunophenotypic expression.Quantitative real-time PCR was also performed to detect the mRNA expression of chemokines CCL3\CCL4\CCL5\CCL7\CXCL8 at 2 weeks after acupuncture treatment.And then QPCR and immunohistochemistry were used to detect the expression of CCL5 at different time points after acupuncture.Meanwhile,we performed cell migration assays to study the effects of the degenerative IVD cells supernatant and rhCCL5 on migration of these stem cells.Results:The MRI results showed that the discs were rapidly degenerated at 3 weeks after apuncture treatment,Correspondingly,the picrosirius red staining demonstrated that compared with the normal group?non-puncture?,the progressively increasing type I collagen fibres and gradually reduced collagen type II fibres.These changes began to occur in the first week after operation,and showed a progression in the 2 and 3 weeks,but in the4 and 5 weeks,almost all the normal structures in the nucleus pulposus were completely disintegrated,and the disc degeneration was severed.Accordingly,the results of HE staining showed same trend,the acupuncture group exhibited more severe degeneration.To summarize,these results indicated that the degenerative changes of the IVDs occurred after acupuncture,and this change was aggravated with the prolongation of postoperative time.The expression of CD29,CD90 mRNA in the second week after acupuncture expressed a higher level.The immunofluorescence showed that only a small number of BrdU+cells existed in the control group,but the number in the the outer AF near the perichondriuml region was significantly increased in the acupuncture group after 2 weeks of the model establishment.The mRNA expressions of CCL3,CCL4,CCL5,CCL7 and CXCL8 after acupuncture were all significantly higher than that of the control groups.Among these changes,CCL5increased with the most multiplier.Next,immunohistochemistry showed that the expression of CCL5 was strongest at second weeks and then decreased gradually.ISN-SCs and BMSCs cultured from IVD tissues gradually formed colonies about 7days after growing,and IVD cells gradually formed colonies only for 4 days after growing in culture.The expression of CD45 and CD19 in ISN-SCs and BMSCs was lower than that of CD29,CD90 and CD44.Meanwhile,the expression of CD44 in ISN-SCs was significantly higher than that in BMSCs.Cell migration assay suggested that the culture supernatants of degenerated IVD cells could significantly promote the migration of BMSCs and ISN-SCs which compared with the normal IVD cells culture supernatants.When the neutralizing CCL5-antibody?2?g/ml?was added,the migration of BMSCs and ISN-SCs could be reversed.Finally,the rhCCL5?1 and 10 ng/ml?was added,the migration of ISN-SCs could be promoted significantly.Conclusions:1.The chemokines in degenerative intervertebral discs can promote the migration of ISN-SCs.2.CCL5 is the key to ISN-SCs recruitment in degenerative intervertebral discs. |
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