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.     

Temporal changes of mechanical signals and extracellular composition in human intervertebral disc during degenerative progression

In this study, a three-dimensional finite element model was used to investigate the changes in tissue composition and mechanical signals within human lumbar intervertebral disc during the degenerative progression. This model was developed based on the cell-activity coupled mechano-electrochemical mixture theory. The disc degeneration was simulated by lowering nutrition levels at disc boundaries, and the temporal and spatial distributions of the fixed charge density, water content, fluid pressure, Von Mises stress, and disc deformation were analyzed. Results showed that fixed charge density, fluid pressure, and water content decreased significantly in the nucleus pulposus (NP) and the inner to middle annulus fibrosus (AF) regions of the degenerative disc. It was found that, with degenerative progression, the Von Mises stress (relative to that at healthy state) increased within the disc, with a larger increase in the outer AF region. Both the disc volume and height decreased with the degenerative progression. The predicted results of fluid pressure change in the NP were consistent with experimental findings in the literature. The knowledge of the variations of temporal and spatial distributions of composition and mechanical signals within the human IVDs provide a better understanding of the progression of disc degeneration.     

Comparative immunolocalisation of fibrillin-1 and perlecan in the human foetal, and HS-deficient hspg2 exon 3 null mutant mouse intervertebral disc

The aim of this study was to examine the comparative localisations of fibrillin-1 and perlecan in the foetal human, wild-type C57BL/6 and HS-deficient hspg2Δ^3?/Δ^3? exon 3 null mouse intervertebral disc (IVD) using fluorescent laser scanning confocal microscopy. Fibrillin-1 fibrils were prominent components of the outer posterior and anterior annulus fibrosus (AF) of the foetal human IVD. Finer fibrillin-1 fibrils were evident in the inner AF where they displayed an arcade-type arrangement in the developing lamellae. Relatively short but distinct fibrillin-1 fibrils were evident in the central region of the IVD and presumptive cartilaginous endplate and defined the margins of the nuclear sheath in the developing nucleus pulposus (NP). Fibrillin-1 was also demonstrated in the AF of C57BL/6 wild-type mice but to a far lesser extent in the HS-deficient hspg2Δ^3?/Δ^3? exon 3 null mouse. This suggested that the HS chains of perlecan may have contributed to fibrillin-1 assembly or its deposition in the IVD. The cell–matrix interconnections provided by the fibrillin fibrils visualised in this study may facilitate communication between disc cells and their local biomechanical microenvironment in mechanosensory processes which regulate tissue homeostasis. The ability of fibrillin-1 to sequester TGF-β a well-known anabolic growth factor in the IVD also suggests potential roles in disc development and/or remodelling.     

TGFβ and BMP Dependent Cell Fate Changes Due to Loss of Filamin B Produces Disc Degeneration and Progressive Vertebral Fusions

Spondylocarpotarsal synostosis (SCT) is an autosomal recessive disorder characterized by progressive vertebral fusions and caused by loss of function mutations in Filamin B (FLNB). FLNB acts as a signaling scaffold by linking the actin cytoskleteon to signal transduction systems, yet the disease mechanisms for SCT remain unclear. Employing a Flnb knockout mouse, we found morphologic and molecular evidence that the intervertebral discs (IVDs) of Flnb^–/–mice undergo rapid and progressive degeneration during postnatal development as a result of abnormal cell fate changes in the IVD, particularly the annulus fibrosus (AF). In Flnb^–/–mice, the AF cells lose their typical fibroblast-like characteristics and acquire the molecular and phenotypic signature of hypertrophic chondrocytes. This change is characterized by hallmarks of endochondral-like ossification including alterations in collagen matrix, expression of Collagen X, increased apoptosis, and inappropriate ossification of the disc tissue. We show that conversion of the AF cells into chondrocytes is coincident with upregulated TGFβ signaling via Smad2/3 and BMP induced p38 signaling as well as sustained activation of canonical and noncanonical target genes p21 and Ctgf. These findings indicate that FLNB is involved in attenuation of TGFβ/BMP signaling and influences AF cell fate. Furthermore, we demonstrate that the IVD disruptions in Flnb^–/–mice resemble aging degenerative discs and reveal new insights into the molecular causes of vertebral fusions and disc degeneration.     

Sox9治疗兔椎间盘退变的效果及机制研究

目的:探究Sox9用于治疗椎间盘退变的效果及调控机制。方法:将Ad-sox9和Ad-GFP各20μL分别转染至椎间盘退变兔的髓核组织中,转染后3、7、30、60天取材,采用免疫组化、免疫荧光和MRI等研究方法检测椎间盘髓核组织中II型胶原、蛋白多糖的表达情况,并分析对椎间盘退变的改善情况。结果:免疫组化染色显示sox9组中椎间盘髓核组织中II型胶原、蛋白多糖的表达明显升高,MRI显示sox9组椎间盘T2像信号有明显改善(P<0.05)。结论:体内转染腺病毒介导的sox9基因能够增加椎间盘内II型胶原和蛋白多糖的表达,并抑制椎间盘的退变进程。     

Sox9‐expressing precursors are the cellular origin of the cruciate ligament of the knee joint and the limb tendons

Sox9 expression defines cell progenitors in a variety of tissues during mouse embryogenesis. To establish a genetic tool for cell‐lineage tracing and gene‐function analysis, we generated mice in which the CreERT2 gene was targeted to the endogenous mouse Sox9 locus. In Sox9^CreERT2/+;R26R embryos, tamoxifen activated Cre recombinase exclusively in Sox9‐expressing tissues. To determine the suitability of this mouse line for developmental stage‐specific gene recombination, we investigated the cellular origins of the cruciate ligaments of the knee joint and the limb tendons, in which precursor cells have not been defined. The cells in these tissues were labeled after tamoxifen treatment before or at the stage of chondrogenic mesenchymal condensation, indicating that ligament and tendon cells originated from Sox9‐expressing cells and that cell fate determination occurred at mesenchymal condensation. This mouse line is a valuable tool for the temporal genetic tracing of the progeny of, and inducible gene modification in Sox9‐expressing cells. genesis 48:635–644, 2010. © 2010 Wiley‐Liss, Inc.     

A quantitative and qualitative RNA expression profiling assay for cell culture with single cell resolution

Cells are often characterized by their gene expression profile. However, commonly used methods to detect mRNA require cell pooling and could therefore mask differences in gene expression within heterogeneous cell populations. q²PISH allows for the analysis of both qualitative and quantitative (q²) gene expression on cultured cells for quality control measures with single cell resolution. q²PISH was optimized for the subsequent use of two alkaline phosphatase substrates in combination with a cell nucleus count to allow for accurate quantification of gene expression per cell and simultaneously qualitative assessment of potential culture population drift or heterogeneity. As proof of principle the assay was applied to cell lines derived from different areas of the bovine intervertebral disc, showing significant difference in the expression of Col1a1, Col2a1, Acan and Sox9. Furthermore, the assay served to explore a potential impact on cultured cells when substituting a critical media component, fetal bovine serum (FBS), suggesting no significant difference in gene expression for the biomarkers analyzed. As a tool, q²PISH serves as an accurate quality control with single cell resolution for cultured cells.     

Characterization of Krt19 CreERT allele for targeting the nucleus pulposus cells in the postnatal mouse intervertebral disc

Intervertebral disc degeneration and associated back pain are relatively common but sparsely understood conditions, affecting over 70% of the population during some point of life. Disc degeneration is often associated with a loss of nucleus pulposus (NP) cells. Genetic mouse models offer convenient avenues to understand the cellular and molecular regulation of the disc during its formation, growth, maintenance, and aging. However, due to the lack of inducible driver lines to precisely target NP cells in the postnatal mouse disc, progress in this area of research has been moderate. NP cells are known to express cytokeratin 19 (Krt19), and tamoxifen (Tam)-inducible Krt19^CreERT allele is available. The current study describes the characterization of Krt19^CreERT allele to specifically and efficiently target NP cells in neonatal, skeletally mature, middle-aged, and aged mice using two independent fluorescent reporter lines. The efficiency of recombination at all ages was validated by immunostaining for KRT19. Results show that following Tam induction, Krt19^CreERT specifically drives recombination of NP cells in the spine of neonatal and aged mice, while no recombination was detected in the surrounding tissues. Knee joints from skeletally mature Tam-treated Krt19^CreERT/+; R26^tdTOM mouse show the absence of recombination in all tissues and cells of the knee joint. Thus, this study provides evidence for the use of Krt19^CreERT allele for genetic characterization of NP cells at different stages of the mouse life.     

Targeting the annulus fibrosus of the intervertebral disc: <Emphasis Type="Italic">Col1a2-Cre(ER)T</Emphasis> mice show specific activity of Cre recombinase in the outer annulus fibrosus

Degeneration of the intervertebral disc (IVD) is a major underlying contributor to back pain—the single leading cause of disability worldwide. However, we possess a limited understanding of the etiology underlying IVD degeneration. To date, there are a limited number of mouse models that have been used to target proteins in specific compartments of the IVD to explore their functions in disc development, homeostasis and disease. Furthermore, the majority of reports exploring the composition and function of the outer encapsulating annulus fibrosus (AF) of the IVD have considered it as one tissue, without considering the numerous structural and functional differences existing between the inner and outer AF. In addition, no mouse models have yet been reported that enable specific targeting of genes within the outer AF. In the current report, we discuss these issues and demonstrate the localized activity of Cre recombinase in the IVD of Col1a2-Cre(ER)T;ROSA26mTmG mice possessing a tamoxifen-dependent Cre recombinase driven by a Cola2 promoter and distal enhancer and the mTmG fluorescent reporter. Following tamoxifen injection of 3-week-old Col1a2-Cre(ER)T;ROSA26mTmG mice, we show Cre activity specifically in the outer AF of the IVD, as indicated by expression of the GFP reporter. Thus, Col1a2-Cre(ER)T;ROSA26mTmG mice may prove to be a valuable tool in delineating the function of proteins in this unique compartment of the IVD, and in further exploring the compositional differences between the inner and outer AF in disc homeostasis, aging and disease.