Ex vivo intervertebral disc cultures: degeneration-induction methods and their implications for clinical translation

Because low back pain is frequently a result of intervertebral disc degeneration (IVDD), strategies to regenerate or repair the IVD are currently being investigated. Often, ex vivo disc cultures of non-human IVD organs or tissue explants are used that usually do not exhibit natural IVDD. Therefore, degenerative changes mimicking those reported in human IVDD need to be induced. To support researchers in selecting ex vivo disc cultures, a systematic search was performed for them and their potential use for studying human IVDD reviewed. Five degeneration induction categories (proinflammatory cytokines, injury/damage, degenerative loading, enzyme, and other) were identified in 129 studies across 7 species. Methods to induce degeneration are diverse and can induce mild to severe degenerative changes that progress over time, as described for human IVDD. The induced degenerative changes are model-specific and there is no "one-fits-all" IVDD induction method. Nevertheless, specific aspects of human IVDD can be well mimicked. Currently, spontaneously degenerated disc cultures from large animals capture human IVDD in most aspects. Combinatorial approaches of several induction methods using discs derived from large animals are promising to recapitulate pathological changes on several levels, such as cellular behaviour, extracellular matrix composition, and biomechanical function, and therefore better mimic human IVDD. Future disc culture setups might increase in complexity, and mimic human IVDD even better. As ex vivo disc cultures have the potential to reduce and even replace animal trials, especially during preclinical development, advancement of such models is highly relevant for more efficient and cost-effective clinical translation from bench-to-bedside

Ex vivo intervertebral disc cultures: degeneration-induction methods and their implications for clinical translation

Because low back pain is frequently a result of intervertebral disc degeneration (IVDD), strategies to regenerate or repair the IVD are currently being investigated. Often, ex vivo disc cultures of non-human IVD organs or tissue explants are used that usually do not exhibit natural IVDD. Therefore, degenerative changes mimicking those reported in human IVDD need to be induced. To support researchers in selecting ex vivo disc cultures, a systematic search was performed for them and their potential use for studying human IVDD reviewed. Five degeneration induction categories (proinflammatory cytokines, injury/damage, degenerative loading, enzyme, and other) were identified in 129 studies across 7 species. Methods to induce degeneration are diverse and can induce mild to severe degenerative changes that progress over time, as described for human IVDD. The induced degenerative changes are model-specific and there is no “one-fits-all” IVDD induction method. Nevertheless, specific aspects of human IVDD can be well mimicked. Currently, spontaneously degenerated disc cultures from large animals capture human IVDD in most aspects. Combinatorial approaches of several induction methods using discs derived from large animals are promising to recapitulate pathological changes on several levels, such as cellular behaviour, extracellular matrix composition, and biomechanical function, and therefore better mimic human IVDD. Future disc culture setups might increase in complexity, and mimic human IVDD even better. As ex vivo disc cultures have the potential to reduce and even replace animal trials, especially during preclinical development, advancement of such models is highly relevant for more efficient and cost-effective clinical translation from bench-to-bedside

In vivo biofunctional evaluation of hydrogels for disc regeneration

Purpose Regenerative strategies aim to restore the original biofunctionality of the intervertebral disc. Different biomaterials are available, which might support disc regeneration. In the present study, the prospects of success of two hydrogels functionalized with anti-angiogenic peptides and seeded with bone marrow derived mononuclear cells (BMC), respectively, were investigated in an ovine nucleotomy model. Methods In a one-step procedure iliac crest aspirates were harvested and, subsequently, separated BMC were seeded on hydrogels and implanted into the ovine disc. For the cell-seeded approach a hyaluronic acid-based hydrogel was used. The anti-angiogenic potential of newly developed VEGF-blockers was investigated on ionically crosslinked metacrylated gellan gum hydrogels. Untreated discs served as nucleotomy controls. 24 adult merino sheep were used. After 6 weeks histological, after 12 weeks histological and biomechanical analyses were conducted. Results Biomechanical tests revealed no differences between any of the implanted and nucleotomized discs. All implanted discs significantly degenerated compared to intact discs. In contrast, there was no marked difference between implanted and nucleotomized discs. In tendency, albeit not significant, degeneration score and disc height index deteriorated for all but not for the cell-seeded hydrogels from 6 to 12 weeks. Cell-seeded hydrogels slightly decelerated degeneration. Conclusions None of the hydrogel configurations was able to regenerate biofunctionality of the intervertebral disc. This might presumably be caused by hydrogel extrusion. Great importance should be given to the development of annulus sealants, which effectively exploit the potential of (cell-seeded) hydrogels for biological disc regeneration and restoration of intervertebral disc functioningThis work was supported by the EU-project Disc Regeneration (NMP3-LA-2008-213904). Technical assistance of Iris Baum and the whole animal surgery team of the Institute of Orthopaedic Research and Biomechanics, Ulm, are gratefully acknowledged. DDAHA hydrogels were kindly provided by Cristina Longinotti (DDAHA, Anika Therapeutics, Abano Therme, Italy)

Actin Fusion Proteins Alter the Dynamics of Mechanically Induced Cytoskeleton Rearrangement

Mechanical forces can regulate various functions in living cells. The cytoskeleton is a crucial element for the transduction of forces in cell-internal signals and subsequent biological responses. Accordingly, many studies in cellular biomechanics have been focused on the role of the contractile acto-myosin system in such processes. A widely used method to observe the dynamic actin network in living cells is the transgenic expression of fluorescent proteins fused to actin. However, adverse effects of GFP-actin fusion proteins on cell spreading, migration and cell adhesion strength have been reported. These shortcomings were shown to be partly overcome by fusions of actin binding peptides to fluorescent proteins. Nevertheless, it is not understood whether direct labeling by actin fusion proteins or indirect labeling via these chimaeras alters biomechanical responses of cells and the cytoskeleton to forces. We investigated the dynamic reorganization of actin stress fibers in cells under cyclic mechanical loading by transiently expressing either egfp-Lifeact or eyfp-actin in rat embryonic fibroblasts and observing them by means of live cell microscopy. Our results demonstrate that mechanically-induced actin stress fiber reorganization exhibits very different kinetics in EYFP-actin cells and EGFP-Lifeact cells, the latter showing a remarkable agreement with the reorganization kinetics of non-transfected cells under the same experimental conditions

Capacitative calcium influx and proliferation of human osteoblastic-like MG-63 cells

Adult bone tissue is continuously being remodelled and bone mass is maintained by a balance between osteoclastic bone resorption and osteoblastic bone formation. Alteration of osteoblastic cell proliferation may account in part for lack of balance between these two processes in bone loss of osteoporosis. There is calcium (Ca2+) control in numerous cellular functions; however, involvement of capacitative Ca2+ entry (CCE) in proliferation of bone cells is less well investigated. OBJECTIVES: The study described here was aimed to investigate roles of CCE in the proliferation of osteoblast-like MG-63 cells. MATERIALS AND METHODS: Pharmacological characterizations of CCE were undertaken in parallel, with evaluation of the expression of transient receptor potential canonical (TRPC) channels and of cell proliferation. RESULTS: Intracellular Ca2+ store depletion by thapsigargin induced CCE in MG-63 cells; this was characterized by a rapid transient increase of intracellular Ca2+ followed by significant CCE, induced by conditions that stimulated cell proliferation, namely serum and platelet-derived growth factor. Inhibitors of store-operated Ca2+ channels (2-APB and SKF-96365) prevented CCE, while voltage-dependent Ca2+ channel blockers had no effect. Expression of various TRPC channels was shown in the cells, some having been shown to be responsible for CCE. Voltage-dependent Ca2+ channel blockers had no effect on osteoblast proliferation while thapsigargin, 2-APB and SKF-96395, inhibited it. Cell cycle analysis showed that 2-APB and SKF-96395 lengthen the S and G2/M phases, which would account for the reduction in cell proliferation. CONCLUSIONS: Our results indicate that CCE, likely attributed to the activation of TRPCs, might be the main route for Ca2+ influx involved in osteoblast proliferation

Three-Dimensional Cell and Tissue Patterning in a Strained Fibrin Gel System

Techniques developed for the in vitro reproduction of three-dimensional (3D) biomimetic tissue will be valuable for investigating changes in cell function in tissues and for fabricating cell/matrix composites for applications in tissue engineering techniques. In this study, we show that the simple application of a continuous strain to a fibrin gel facilitates the development of fibril alignment and bundle-like structures in the fibrin gel in the direction of the applied strain. Myoblasts cultured in this gel also exhibited well-aligned cell patterning in a direction parallel to the direction of the strain. Interestingly, the direction of cell proliferation was identical to that of cell alignment. Finally, the oriented cells formed linear groups that were aligned parallel to the direction of the strain and replicated the native skeletal muscle cell patterning. In addition, vein endothelial cells formed a linear, aligned vessel-like structure in this system. Thus, the system enables the in vitro reproduction of 3D aligned cell sets replicating biological tissue patterns

Thermally Triggered Hydrogel Injection Into Bovine Intervertebral Disc Tissue Explants Induces Differentiation Of Mesenchymal Stem Cells And Restores Mechanical Function.

We previously reported a synthetic Laponite® crosslinked pNIPAM-co-DMAc (L-pNIPAM-co-DMAc) hydrogel which promotes differentiation of mesenchymal stem cells (MSCs) to nucleus pulposus (NP) cells without additional growth factors. The clinical success of this hydrogel is dependent on: integration with surrounding tissue; the capacity to restore mechanical function; as well as supporting the viability and differentiation of delivered MSCs. Bovine NP tissue explants were injected with media (control), human MSCs (hMSCs) alone, acellular L-pNIPAM-co-DMAc hydrogel or hMSCs incorporated within the L-pNIPAM-co-DMAc hydrogel and maintained at 5% O2 for 6 weeks. Viability of native NP cells and delivered MSCs was maintained. Furthermore hMSCs delivered via the L-pNIPAM-co-DMAc hydrogel differentiated and produced NP matrix components: aggrecan, collagen type II and chondroitin sulphate, with integration of the hydrogel with native NP tissue. In addition L-pNIPAM-co-DMAc hydrogel injected into collagenase digested bovine discs filled micro and macro fissures, were maintained within the disc during loading and restored IVD stiffness. The mechanical support of the L-pNIPAM-co-DMAc hydrogel, to restore disc height, could provide immediate symptomatic pain relief, whilst the delivery of MSCs over time regenerates the NP extracellular matrix; thus the L-pNIPAM-co-DMAc hydrogel could provide a combined cellular and mechanical repair approach