Solid Phase Synthesis of a Functionalized Bis-Peptide Using …
Published 5/15/2012
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In JoVE (1)
Other Publications (9)
- Tissue Engineering. Part C, Methods
- Spine
- Spine
- The Spine Journal : Official Journal of the North American Spine Society
- European Spine Journal : Official Publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the Cervical Spine Research Society
- Methods in Molecular Biology (Clifton, N.J.)
- European Spine Journal : Official Publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the Cervical Spine Research Society
- European Spine Journal : Official Publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the Cervical Spine Research Society
- Spine
Articles by Benjamin Gantenbein-Ritter in JoVE
Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture
ARTORG Center for Biomedical Engineering, University of Bern
This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.
Other articles by Benjamin Gantenbein-Ritter on PubMed
Accuracy of Three Techniques to Determine Cell Viability in 3D Tissues or Scaffolds
Several different assays are commonly used to evaluate survival of cells inside tissues or three-dimensional carriers, but their accuracy and reliability have not been evaluated. Here, we compare three methods for cell viability (CV) determination: (i) lactate dehydrogenase (LDH) staining on cryosections, (ii) calcein AM/ethidium homodimer-1 (CaAM/EthH) staining, and (iii) carrier digestion and trypan blue (TB) assay. Living and dead cell populations were generated from bovine chondrocytes and combined to produce approximately 0%, 25%, 50%, 75%, and 100% CV mixtures. CV ratios were measured with TB assay (MIX) before seeding cells into fibrin carriers. CV was then determined using the three methods (n = 5/method). Custom-written macros were used to process LDH- and CaAM/EthH-stained images, and hand counting with hemocytometer was used for the TB method. Absolute error and intraclass correlation (ICC) were used for accuracy and reliability evaluation. All methods estimated CV values close to MIX values. TB method was the most accurate (ICC = 0.99) followed by CaAM/EthH (ICC = 0.98) and LDH (ICC = 0.97). As for absolute quantification of living and dead cells, TB and LDH methods performed well (ICC = 0.75-0.96), whereas CaAM/EthH largely overestimated cell numbers (living, ICC = 0.30; dead, ICC = 0.30). Although TB was the most accurate, LDH and CaAM/EthH provide valuable information on cell shape and spatial distribution of cells in tissue or a scaffold.
Effect of Limited Nutrition on in Situ Intervertebral Disc Cells Under Simulated-physiological Loading
Whole ovine caudal intervertebral discs (IVD) were cultured in sufficient and limited nutrition under simulated-physiologic loading for 7 and 21 days.
The Combined Effects of Limited Nutrition and High-frequency Loading on Intervertebral Discs with Endplates
Whole ovine caudal intervertebral discs were cultured under simulated-physiologic or high-frequency loading and either sufficient or limited nutrition for 7 days.
Cryopreserved Intervertebral Disc with Injected Bone Marrow-derived Stromal Cells: a Feasibility Study Using Organ Culture
A recent clinical study demonstrated that cryopreserved allogeneic intervertebral disc transplantation relieved pain and preserved motion, thus opening up a new treatment option for degenerative disc disease. However, these transplanted discs continued to degenerate, possibly due to a lack of viable cells. Bone marrow-derived stromal cell (BMSC) implantation has been shown to delay disc degeneration.
Differential Response of Human Bone Marrow Stromal Cells to Either TGF-β(1) or RhGDF-5
Cell therapy along with growth factor injection is currently widely investigated to restore the intervertebral disc. However, there is increasing evidence that transplanted unconditioned bone marrow-derived stromal cells (BMSCs) cannot thrive in the intervertebral disc "niche". Moreover, uncertainty exists with respect to the cell phenotype that would be suitable to inject. The intervertebral disc cell phenotype only recently has been started to be characterised using transcriptomics profiling. Recent findings suggest that cytokeratin 19 (KRT-19) could be used as a potential candidate marker for the intervertebral disc, or more specifically the nucleus pulposus cell (NPC) phenotype. We present in vitro cell culture data using alginate bead culture of primary human BMSCs exposed to the standard chondrogenic stimulus, transforming growth factor beta-1 (TGF-β), the growth and differentiation factor 5 and/or bovine NPCs to induce a potential "discogenic" pathway. Chondrogenic induction via TGF-β pathway provoked down-regulation of KRT-19 gene expression in four out of five donors after 18 days of culture, whereas KRT-19 expression remained unchanged in the "discogenic" groups. In addition, the ratio of aggrecan/collagen II gene expression showed a remarkable difference (of at least 3 magnitudes) between the chondrogenic stimulus (low ratio) and the discogenic stimulus (high ratio). Therefore, KRT-19 and aggrecan/collagen II ratio may be potential markers to distinguish chondrogenic from "discogenic" differentiation.
Confocal Imaging Protocols for Live/dead Staining in Three-dimensional Carriers
In tissue engineering, a variety of methods are commonly used to evaluate survival of cells inside tissues or three-dimensional (3D) carriers. Among these methods confocal laser scanning microscopy opened accessibility of 3D tissue using live cell imaging into the tissue or 3D scaffolds. However, although this technique is ideally applied to 3D tissue or scaffolds with thickness up to several millimetres, this application is surprisingly rare and scans are often done on slices with thickness <20 μm. Here, we present novel protocols for the staining of 3D tissue (e.g. intervertebral disc tissue) and scaffolds, such as fibrin gels or alginate beads.
The Effects of Dynamic Loading on the Intervertebral Disc
Loading is important to maintain the balance of matrix turnover in the intervertebral disc (IVD). Daily cyclic diurnal assists in the transport of large soluble factors across the IVD and its surrounding circulation and applies direct and indirect stimulus to disc cells. Acute mechanical injury and accumulated overloading, however, could induce disc degeneration. Recently, there is more information available on how cyclic loading, especially axial compression and hydrostatic pressure, affects IVD cell biology. This review summarises recent studies on the response of the IVD and stem cells to applied cyclic compression and hydrostatic pressure. These studies investigate the possible role of loading in the initiation and progression of disc degeneration as well as quantifying a physiological loading condition for the study of disc degeneration biological therapy. Subsequently, a possible physiological/beneficial loading range is proposed. This physiological/beneficial loading could provide insight into how to design loading regimes in specific system for the testing of various biological therapies such as cell therapy, chemical therapy or tissue engineering constructs to achieve a better final outcome. In addition, the parameter space of 'physiological' loading may also be an important factor for the differentiation of stem cells towards most ideally 'discogenic' cells for tissue engineering purpose.
The Evolutionary Importance of Cell Ratio Between Notochordal and Nucleus Pulposus Cells: an Experimental 3-D Co-culture Study
INTRODUCTION: Notochordal cells and nucleus pulposus cells are co-existing in the intervertebral disc at various ratios among different mammalians. This fact rises the question about the interactions and the evolutionary relevance of this phenomenon. It has been described that these relatively large notochordal cells are mainly dominant in early lifetime of all vertebrates and then differences occur with ageing. Human, cattle, sheep, and goat lose the cells with age, whereas rodents and lagomorphs maintain these throughout their lifetime. MATERIALS AND METHODS: Here, we addressed the importance of cell ratio using alginate bead 3-D co-culture of bovine nucleus pulposus cells (bNPC) and porcine notochordal cells (pNCs) for 14 days using culture inserts. RESULT: We found a significant stimulation of bNPC in the presence of pNC in terms of cell activity and glycosaminoglycan production, but not for proliferation (DNA content). Relative gene expression was significantly stimulated for collagen type 2 and aggrecan. CONCLUSION: The stimulating effect of NC was confirmed and the ideal ratio of NPC: NC was found to be ~50:50. This has direct implications for tissue-engineering approaches, which aim to repopulate discs with NP-like precursor cells.
Biological Response of the Intervertebral Disc to Repetitive Short-term Cyclic Torsion
In vitro study of the biological response of the intervertebral disc (IVD) to cyclic torsion by using bovine caudal IVDs.