| Backgrounds:Low back pain (LBP) is an extremely common disorder in modern society, theincidence of which is second only to upper respiratory infections in the USA. Degenerativedisc disease (DDD) is generally thought to be the main cause for discogenic LBP. As theaging process of the population in our country, DDD will impact the people in living andworking more and more severely.Treatment of DDD remains still a great challenge to both clinical physicians and basicresearchers. Conservative treatment modalities involve physical therapy and medications.For those patients who fail extensive conservative treatment, surgical treatment may beconsidered and usually involves discectomy or fusion of the involved intervertebral disk(IVD) levels. Although discectomy and spine fusion have relieved the suffering of manypatients, these lead to an irreversible loss of function of the treated segment with theresulting risks of adjacent segment degeneration and segmental instability. On the otherhand, all of those disc prostheses under investigations are composed of variable syntheticmaterials, which could hardly mimic the biological and biomechanical properties of thenative IVD. Thus, the ideal treatment of DDD is not only relieving the symptom of pain,but also reconstruct the structure and biological function of IVD.In recent years, with the rapid development of tissue engineering technique, surgeryhas entered a new era of regenerative medicine. Tissue engineered IVD and cell therapicshave also been an ideal strategy to treat DDD, and this may open a new way to treat DDDbiologically. The nutrition supply of implanted tissues and/or cells is a problem that allthese therapical methods faced. The cartilage endplate is the main structure for the nucleuspulposus nutrition. Many authors regard cartilage endplate degeneration as an initial anddevelopmental factor for DDD. Therefor, keeping the normal function and structure ofcartilage endplate and preventing cartilage endplate from degeneration is a key factor for the success of above treatments. The decreased cells in the cartilage endplate will lead tothe cartilage endplate degeneration. Thus, stimulating the stem cells in situ of cartilageendplate to proliferate, differentiate and synthesis extracellular matrix (ECM) is a keyfactor to solve the above problems.ObjectiveThe degenerated cartilage endplate was obtained, cleaned, then isolated cartilageendplate cells were cultured in the agarose culture medium. The cell clones were obtainedand expanded in vitro for the in vitro and in vivo experiment. The experiments were used toconfirm the existence of stem cells in the degenerated cartilage endplate, which could beinduced into chondrogenesis, osteogenesis and adipogenesis. The cartilage endplate derivedstem cells (CESCs) were compared with bone marrow mesenchyme stem cells (BM-MSCs)from the morphology, cell proliferation, cell cycle, stem cell markers, and mutilineagedifferentiation. And the results were to confirm the characteristics of CESCs, which wouldpull the way for the research of biological function.MethodThe CEP used in this study was obtained from seven patients who underwent posteriordiscectomy and fusion for lumbar degenerative disease. Surgically explanted CEP wascleaned of any adherent extraneous tissue under a sterilized dissecting microscope. Thenucleus pulposus, annulus fibrosus, and subchondral bone tissues around blocks of CEPwas removed. The cells were subcultured in the agarose culture and select the cell clonesand expanded invitro for the assays of stem cell markers, cell clone, mutilineagedifferentiation and related assays which are to confirm the existence of stem cells in thecartilage endplate. The CESCs were loaded into the hydroxyapatite ceramic and implantedinto the naked mouse. The immunohistochemisty was used to confirm the osteogenesispotential of CESCs in vivo. To further clarify the characteristics of CESCs, the BM-MSCsfrom the same patient were compared from the cell proliferation, stem cell markers, cellcycle, mutilineage differentiation and related assays. Software SPSS10.0was used instatistical treatment.Staining positive and strong positive were included in positive group,compared with the negative group. The difference was statistically significant whenP<0.05. Results1. There are cells in the degenerated cartilage endplate which are positive for stem cellmarkers such as STRO-1,CD105,CD73,CD90by the confocal scan microscope. Afterculture in agarose for6weeks, a portion of the cells formed cell clusters. Expaned cells invitro exhibited a homogeneous fibroblast-like morphology with a spindle shapedappearance, formed clone unit, and were positive for the stem cell markers(CD90, CD73,STRO-1, CD105)and negative for CD14, CD19, CD34, CD45and HLA-DR. The results of in vitromultileanage differentiation also confirmed the existence of stem cells in the endplate. The result ofreverse transcription polymerase chain reaction (RT-PCR) before and after induction for osteogenesis andadipogenesis indicated that the CESCs were derived from mesenchymal tissues but not embryo tissues.2. In vivo experiment showed that there is homogeneous ECM staining red in the pores by HE.Toluidine blue staining also indicated the heterophile ECM which secreted by chondrocytes. Theimmunohistochemistry results also showed the collagen â… , â…© were expressed in the tissues, andcollagen â…¡ was minimally stained in the tissues loaded in the pores.3. CESCs and BM-MSCs shared the similar characteristics in the cell morphology, cellproliferation, cell cycle, cell immunophenotype, and trilineage indifferentiation.4. The expression of CD105and CD166in the CESCs were lower than that ofBM-MSCs in the cell immunophenotype. Regarding the osteogenesis, the results of mineraldeposit, alkaline phosphatase activities (ALP), osteogenesis specific genes indicated thatthe osteogenesis of CESCs was superior to that of BM-MSCs in the early and late stage ofosteogenesis. Regarding the adipogenesis, there was no significance between CESCs andBM-MSCs. However, a greater variability was detected in the adipogenic potential ofBM-MSCs. As to the chondrogenesis, the results of wet weigh of cell sphere, alsinlandensity,chondrogenic specific genes, and western blot indicated that the chodrogenicpotential of CESCs was higher than that of BM-MSCs.Conclusion1. All the results in this study indicated that the CESCs were existed in the degeneratedcartilage endplate and they were derived from mesenchymal tissues but not embryo tissues.2. The CESCs can be induced into the chondrogenesis and/or osteogenesis in thenaked mouse.3. CESCs and BM-MSCs shared the similar characteristics in the cell morphology, cell proliferation, cell cycle, cell immunophenotype, and trilineage indifferentiation.However, the expression of CD105and CD166in the CESCs were lower than that ofBM-MSCs. Regarding the osteogenesis and chondrogenesis, the CESCs was superior toBM-MSCs. |
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