Xiaojiang Sun

In Vitro Proliferation and Differentiation of Human Mesenchymal Stem Cells Cultured in Autologous Plasma Derived from Bone Marrow

Expansion of human mesenchymal stem cells (hMSCs) in medium supplemented with fetal bovine serum (FBS) has a potential risk of transmitting viral and prion diseases and causing immunological rejection. The aim of our present study was to find a substitute for the traditional FBS in culture of hMSCs to facilitate the clinical application of hMSCs. We used autologous plasma derived from bone marrow (APM) as a substitute for FBS and found that, when cultured with APM, the cell surface markers and the proportion of hMSCs in the G(0)/G(1) phase and the S+G(2)/M phase resembled those cultured with FBS. However, there were fewer early apoptotic cells in APM-supplemented medium than in FBS (p < 0.01). APM resulted in much greater thymidine incorporation than FBS (p < 0.001). There were significantly more alkaline phosphatase (ALP)-positive fibroblast colony-forming units (CFU-Fs) covering larger areas in APM than in FBS (p < 0.01). Also, APM induced greater ALP activity, more mineralized nodules, and greater expression of osteogenic genes than did FBS. In addition, when cultured in adipogenic medium, there were fewer oil-red O-positive cells and lower expression of adipogenic gene with APM than with FBS. In conclusion, expansion of hMSCs in APM-supplemented medium instead of traditional FBS is more advantageous. It could promote cell proliferation, enhance osteogenic differentiation, and suppress adipogenic differentiation of hMSCs and is therefore a safer and more effective substitute for FBS in clinical cytotherapy processes.

Sox9 gene transfer enhanced regenerative effect of bone marrow mesenchymal stem cells on the degenerated intervertebral disc in a rabbit model.

Objective The effect of Sox9 on the differentiation of bone marrow mesenchymal stem cells (BMSCs) to nucleus pulposus (NP)-like (chondrocyte-like) cells in vitro has been demonstrated. The objective of this study is to investigate the efficacy and feasibility of Sox9-transduced BMSCs to repair the degenerated intervertebral disc in a rabbit model. Materials and Methods Fifty skeletally mature New Zealand white rabbits were used. In the treatment groups, NP tissue was aspirated from the L2-L3, L3-L4, and L4-L5 discs in accordance with a previously validated rabbit model of intervertebral disc degeneration and then treated with thermogelling chitosan (C/Gp), GFP-transduced autologous BMSCs with C/Gp or Sox9-transduced autologous BMSCs with C/Gp. The role of Sox9 in the chondrogenic differentiation of BMSCs embedded in C/Gp gels in vitro and the repair effect of Sox9-transduced BMSCs on degenerated discs were evaluated by real-time PCR, conventional and quantitative MRI, macroscopic appearance, histology and immunohistochemistry. Results Sox9 could induce the chondrogenic differentiation of BMSCs in C/Gp gels and BMSCs could survive in vivo for at least 12 weeks. A higher T2-weighted signal intensity and T2 value, better preserved NP structure and greater amount of extracellular matrix were observed in discs treated with Sox9-transduced BMSCs compared with those without transduction. Conclusion Sox9 gene transfer could significantly enhance the repair effect of BMSCs on the degenerated discs.

Mesenchymal stem cells deliver exogenous miR-21 via exosomes to inhibit nucleus pulposus cell apoptosis and reduce intervertebral disc degeneration

Although mesenchymal stem cells (MSCs) transplantation into the IVD (intervertebral disc) may be beneficial in inhibiting apoptosis of nucleus pulposus cells (NPCs) and alleviating IVD degeneration, the underlying mechanism of this therapeutic process has not been fully explained. The purpose of this study was to explore the protective effect of MSC‐derived exosomes (MSC‐exosomes) on NPC apoptosis and IVD degeneration and investigate the regulatory effect of miRNAs in MSC‐exosomes and associated mechanisms for NPC apoptosis. MSC‐exosomes were isolated from MSC medium, and its anti‐apoptotic effect was assessed in a cell and rat model. The down‐regulated miRNAs in apoptotic NPCs were identified, and their contents in MSC‐exosomes were detected. The target genes of eligible miRNAs and possible downstream pathway were investigated. Purified MSC‐exosomes were taken up by NPCs and suppressed NPC apoptosis. The levels of miR‐21 were down‐regulated in apoptotic NPCs while MSC‐exosomes were enriched in miR‐21. The exosomal miR‐21 could be transferred into NPCs and alleviated TNF‐α induced NPC apoptosis by targeting phosphatase and tensin homolog (PTEN) through phosphatidylinositol 3‐kinase (PI3K)‐Akt pathway. Intradiscal injection of MSC‐exosomes alleviated the NPC apoptosis and IVD degeneration in the rat model. In conclusion, MSC‐derived exosomes prevent NPCs from apoptotic process and alleviate IVD degeneration, at least partly, via miR‐21 contained in exosomes. Exosomal miR‐21 restrains PTEN and thus activates PI3K/Akt pathway in apoptotic NPCs. Our work confers a promising therapeutic strategy for IVD degeneration.

Ectopic Osteogenesis by Ex Vivo Gene Therapy Using Beta Tricalcium Phosphate as a Carrier

Injuries and other damage to large bone can result in defects that do not heal spontaneously and lead to severe functional impairment. Better therapies are greatly needed to address this worldwide problem. The objective of the present study was to determine whether adenoviral delivery of modified human BMP2 gene (AdBMP2) using beta tricalcium phosphate (ß-TCP) as a carrier could promote osteoblastic differentiation of bone marrow mesenchymal stem cells (BMSCs) and ectopic bone formation. Rabbit BMSCs were separated from tibia aspirates and expanded in vitro. The BMSCs were then infected with AdBMP-2. Expression of BMP2, alkaline phosphatase, type I collagen, osteonectin, osteopontin, and mineralization of the cells confirmed secretion of active BMP2. Cells were observed to differentiate and maintain the osteoblast phenotype. For additional in vivo experiments, subcutaneous pockets were created on the backs of nude mice, which were then implanted with AdBMP2-BMSCs/ß-TCP, Adβgal-BMSCs/ß-TCP, BMSCs/ß-TCP, or ß-TCP alone. The nude mice were sacrificed after 4 weeks for histological evaluation. Adβgal-BMSCs/ß-TCP, BMSCs/ß-TCP, and ß-TCP did not show bone formation, although extensive fibrous tissue formed in the subcutaneous space in the rats implanted with ß-TCP. However, new bone tissue formation was observed on the inner walls of the pores of the ß-TCP-treated animals, and ectopic bone formation (mainly ‘‘cartilage-bone inducing’’) was observed in the AdBMP2-BMSCs/ß-TCP composite. These results confirmed the osteogenic potential of BMSCs after AdBMP2 transduction and revealed that AdBMP2-BMSC/ß-TCP composites could provide the capacity for bone formation and maturation during the more advanced stages of healing.

Beta1 integrin inhibits apoptosis induced by cyclic stretch in annulus fibrosus cells via ERK1/2 MAPK pathway

Low back pain is associated with intervertebral disc degeneration (IVDD) due to cellular loss through apoptosis. Mechanical factors play an important role in maintaining the survival of the annulus fibrosus (AF) cells and the deposition of extracellular matrix. However, the mechanisms that excessive mechanical forces lead to AF cell apoptosis are not clear. The present study was to look for how AF cells sense mechanical changes. In vivo experiments, the involvement of mechanoreceptors in apoptosis was examined by RT-PCR and/or immunoblotting in the lumbar spine of rats subjected to unbalanced dynamic and static forces. In vitro experiments, we investigated apoptotic signaling pathways in untransfected and transfected AF cells with the lentivirus vector for rat β1 integrin overexpression after cyclic stretch. Apoptosis in AF cells was assessed using flow cytometry, Hoechst 33258 nuclear staining. Western blotting was used to analyze expression of β1 integrin and caspase-3 and ERK1/2 MAPK signaling molecules. In the rat IVDD model, unbalanced dynamic and static forces induced apoptosis of disc cells, which corresponded to decreased expression of β1 integrin. Cyclic stretch-induced apoptosis in rat AF cells correlated with the activation of caspase-3 and with decreased levels of β1 integrin and the phosphorylation levels of ERK1/2 activation level. However, the overexpression of β1 integrin in AF cells ameliorated cyclic stretch-induced apoptosis and decreased caspase-3 activation. Furthermore, ERK1/2-specific inhibitor promotes apoptosis in vector β1-infected AF cells. These results suggest that the disruption of β1 integrin signaling may underlie disc cell apoptosis induced by mechanical stress. Further work is necessary to fully elucidate the pathophysiological mechanisms that underlie IVDD caused by unbalanced dynamic and static forces.

Quantitative T2 mapping to characterize the process of intervertebral disc degeneration in a rabbit model

BackgroundTo investigate the potential of T2 mapping for characterizing the process of intervertebral disc degeneration (IDD) in a rabbit model.MethodsThirty-five rabbits underwent an annular stab to the L4/5 discs (L5/6 discs served as internal normal controls). Degenerative changes were graded according to the modified Thompson classification and quantified in T2 respectively at pre-operation, 1, 3, 6, 12 and 24xa0weeks postoperatively. After MRI analysis, expression analysis of aggrecan and type II collagen gene in nucleus pulposus (NP) was performed using real time polymerase chain reaction (real-time PCR). The longitudinal changes in NP T2 and gene expressions were studied by repeated measures and ANOVA, linear regression was performed for their correlations through the process of IDD. The reliability analysis of method of measurement of NP T2 was also performed.ResultsThere was a strong inverse correlation between NP T2 and Thompson grades (ru2009=u2009-0.85). The decline of L4/5 NP T2 through 24xa0weeks was nonlinear, the most significant decrease was observed in 3xa0weeks postoperatively (P<0.05). The tendency was confirmed at gene expression levels. NP T2 correlated strongly with aggrecan (R2u2009=u20090.85, P<0.01) and type II collagen (R2u2009=u20090.78, P<0.01) gene expressions. The intraclass correlation coefficients for interobserver and intraobserver reliability were 0.963 and 0.977 respectively.ConclusionsNP T2 correlates well with aggrecan and type II collagen gene expressions. T2 mapping could act as a sensitive, noninvasive tool for quantitatively characterizing the process of IDD in longitudinal study, help better understanding of the pathophysiology of IDD, assist us to detect the degenerative cascade, and develop a T2-based quantification scale for evaluation of IDD and efficacy of therapeutic interventions.

Bilateral decompression using a unilateral pedicle construct for lumbar stenosis

PurposeTo determine the effectiveness of bilateral decompression via a unilateral approach using unilateral pedicle screw fixation for two-level lumbar stenosis with instability.MethodsBetween October 2006 and October 2010, 98 patients (61 men and 37 women) who had reached the three-year follow-up interval were treated with unilateral pedicle screw fixation at the authors’ institution. All patients underwent two-level transforaminal lumbar interbody fusion (TLIF), and the mean age was 59.6xa0years (range, 40–72). Visual analog scale (VAS) scores and Oswestry Disability Index (ODI) were used to assess the pre-operative and postoperative clinical results. Fusion status, the disc space height, and the whole lumbar lordotic angle were analysed for the radiological evaluation.ResultsThe ODI scores decreased significantly in both early and late follow-up evaluations and the visual analog scale (VAS) score demonstrated significant improvement in late follow-up (Pu2009<u20090.01). The disc space height (Pu2009<u20090.05) and the whole lumbar lordotic angle (Pu2009<u20090.05) were increased at the final follow-up. Successful fusion was achieved in all patients.ConclusionBilateral decompression via a unilateral approach using unilateral pedicle screw fixation for two-level lumbar stenosis with instability, which can maintain the lumbar lordosis and the disc space height, is an effective and less invasive method than with bilateral constructs.

Clinical and radiographic outcomes of bilateral decompression via a unilateral approach with transforaminal lumbar interbody fusion for degenerative lumbar spondylolisthesis with stenosis

BACKGROUND CONTEXTnLaminectomy with posterior lumbar interbody fusion (PLIF) has been shown to achieve satisfactory clinical outcomes, but it leads to potential adverse consequences associated with extensive disruption of posterior bony and soft tissue structures.nnnPURPOSEnThis study aimed to compare the clinical and radiographic outcomes of bilateral decompression via a unilateral approach (BDUA) with transforaminal lumbar interbody fusion (TLIF) and laminectomy with PLIF in the treatment of degenerative lumbar spondylolisthesis (DLS) with stenosis.nnnSTUDY DESIGNnThis is a prospective cohort study.nnnPATIENT SAMPLEnThis study compared 43 patients undergoing BDUA+TLIF and 40 patients undergoing laminectomy+PLIF.nnnOUTCOME MEASURESnVisual analog scale (VAS) for low back pain and leg pain, Oswestry Disability Index (ODI), and Zurich Claudication Questionnaire (ZCQ) score.nnnMETHODSnThe clinical outcomes were assessed, and intraoperative data and complications were collected. Radiographic outcomes included slippage of the vertebra, disc space height, segmental lordosis, and final fusion rate. This study was supported by a grant from The National Natural Science Foundation of China (81572168).nnnRESULTSnThere were significant improvements in clinical and radiographic outcomes from before surgery to 3 months and 2 years after surgery within each group. Analysis of leg pain VAS and ZCQ scores showed no significant differences in improvement between groups at either follow-up. The mean improvements in low back pain VAS and ODI scores were significantly greater in the BDUA+TLIF group than in the laminectomy+PLIF group. No significant difference was found in the final fusion rate at 2-year follow-up. The BDUA+TLIF group had significantly less blood loss, shorter length of postoperative hospital stay, and lower complication rate compared with the laminectomy+PLIF group.nnnCONCLUSIONSnWhen compared with the conventional laminectomy+PLIF procedure, the BDUA+TLIF procedure achieves similar and satisfactory effects of decompression and fusion for DLS with stenosis. The BDUA+TLIF procedure appears to be associated with less postoperative low back discomfort and quicker recovery.

Hypertrophy and Fibrosis of the Ligamentum Flavum in Lumbar Spinal Stenosis is Associated with Increased Expression of LPA and LPAR1.

Study Design: Histologic, immunohistochemical (IHC), and enzyme-linked immunosorbent assay analysis of the human ligamentum flavum (LF). Objective: The purpose of this study was to determine the level of expression of lysophosphatidic acid (LPA) in the LF from lumbar spinal stenosis (LSS) patients and to analyze the relationship among LPA, LPA receptors (LPARs), and LF hypertrophy. Summary of Background Data: LF hypertrophy and fibrosis are important causes of LSS. LPA is involved in the fibrotic process in multiple organ systems. Therefore, we hypothesized that LPA and its receptors might also play a role in degeneration of the LF in LSS patients. Methods: Forty-one LF samples were enrolled in this study. The thickness of the LF was measured by magnetic resonance imaging. Histologic analysis using hematoxylin and eosin and Masson trichrome stain was performed for each LF to evaluate the architecture of the extracellular matrix. The content of LPA and connective tissue growth factor (CTGF) in LF samples was analyzed using enzyme-linked immunosorbent assay. The expression of LPAR1 was determined by IHC. Results: Degeneration of the LF was characterized by an increase in disorganized elastic fibers and fibrotic transformation by extracellular collagen deposition. The thickness of the LF and the concentration of LPA and CTGF in the hypertrophic LF group were significantly higher than the control group. Furthermore, the LPA and CTGF concentrations had a positive correlation with the LF thickness (r=0.91, P<0.001 and r=0.943, P<0.001, respectively). On the basis of IHC analysis, the expression of LPAR1 was increased in the hypertrophy group. Conclusions: The increased expression of LPA and LPAR1 is associated with the fibrosis and hypertrophy of the LF in patients with LSS. Further study on the mechanism underlying LF fibrosis may lead to new therapies for LF hypertrophy and fibrosis.

The role of cage height on the flexibility and load sharing of lumbar spine after lumbar interbody fusion with unilateral and bilateral instrumentation: a biomechanical study

BackgroundOne- and two-level lumbar interbody fusion with unilateral instrumentation is as effective as that with bilateral instrumentation. The height of the interbody cage influences the operated segment stability and the fusion technique success. The purpose of this research was to determine the effect of the fusion cage height (i.e. long and short) on both the stability (based on flexibility measures) and load sharing of the unilateral and bilateral instrumented transforaminal lumbar interbody fusion (TLIF) technique.MethodsThe flexibility and load sharing tests were performed on seven human lumbar spines. Different configurations combining a long or short cage with a unilateral, bilateral, or no posterior fixation were used to stabilize the operated segment. Two sets of modular cages were designed for each type of test to simulate the long and short cages. During the flexibility test, a pure-moment load of 7.5xa0Nm was applied. The range of motion (ROM) was recorded for flexion–extension, lateral bending, and axial rotation. During the load sharing test, an axial-compression load of 400xa0N was applied. The load bearing of the cages was recorded using a cage-embedded load cell.ResultsWhen the fusion cage height decreased 2xa0mm, the segment flexibility with unilateral fixation showed a significant increase in the ROM for flexion–extension, lateral bending, and axial rotation of 74.9, 83.8, and 175.2% (Pu2009<u20090.01), respectively. In contrast, for bilateral fixation, the height decrease resulted in no significant change in ROM for flexion–extension (Pu2009=u20090.686), lateral bending (Pu2009=u20090.698), and axial rotation (Pu2009=u20090.133). Using a short fusion cage, the load bearing decreased in 17.1, 21.5, and 54.1% (Pu2009<u20090.05) for the cage alone, unilateral, and bilateral fixation, respectively.ConclusionsA cage longer than the intervertebral space should be chosen to increase the stability and intervertebral graft load borne when performing TLIF with unilateral instrumentation.