Co-culture of Adult Mesenchymal Stem Cells and Nucleus Pulposus Cells in Bilaminar Pellets for Intervertebral Disc Regeneration

Background: Our goal is to optimize stem cell-based tissue engineering strategies in the context of the intervertebral disc environment. We explored the benefits of co-culturing nucleus pulposus cells (NPC) and adult mesenchymal stem cells (MSC) using a novel spherical bilaminar pellet culture system where one cell type is enclosed in a sphere of the other cell type. Our 3D system provides a structure that exploits embryonic processes such as tissue induction and condensation. We observed a unique phenomenon: the budding of co-culture pellets and the formation of satellite pellets that separate from the main pellet. Methods: MSC and NPC co-culture pellets were formed with three different structural organizations. The first had random organization. The other two had bilaminar organization with either MSC inside and NPC outside or NPC inside and MSC outside. Results: By 14 days, all co-culture pellets exhibited budding and spontaneously generated satellite pellets. The satellite pellets were composed of both cell types and, surprisingly, all had the same bilaminar organization with MSC on the inside and NPC on the outside. This organization was independent of the structure of the main pellet that the satellites stemmed from. Conclusion: The main pellets generated satellite pellets that spontaneously organized into a bilaminar structure. This implies that structural organization occurs naturally in this cell culture system and may be inherently favorable for cell-based tissue engineering strategies. The occurrence of budding and the organization of satellite pellets may have important implications for the use of co-culture pellets in cell-based therapies for disc regeneration. Clinical Relevance: From a therapeutic point of view, the generation of satellite pellets may be a beneficial feature that would serve to spread donor cells throughout the host matrix and restore normal matrix composition in a sustainable way, ultimately renewing tissue function. © 2009 The Spine Arthroplasy Society

Bovine explant model of degeneration of the intervertebral disc

BACKGROUND: Many new treatments for degeneration of the intervertebral disc are being developed which can be delivered through a needle. These require testing in model systems before being used in human patients. Unfortunately, because of differences in anatomy, there are no ideal animal models of disc degeneration. Bovine explant model systems have many advantages but it is not possible to inject any significant volume into an intact disc. Therefore we have attempted to mimic disc degeneration in an explant bovine model via enzymatic digestion. METHODS: Bovine coccygeal discs were incubated with different concentrations of the proteolytic enzymes, trypsin and papain, and maintained in culture for up to 3 weeks. A radio-opaque solution was injected to visualise cavities generated. Degenerative features were monitored histologically and biochemically (water and glycosaminoglycan content, via dimethylmethylene blue). RESULTS AND CONCLUSION: The central region of both papain and trypsin treated discs was macro- and microscopically fragmented, with severe loss of metachromasia. The integrity of the surrounding tissue was mostly in tact with cells in the outer annulus appearing viable. Biochemical analysis demonstrated greatly reduced glycosaminoglycan content in these compared to untreated discs. We have shown that bovine coccygeal discs, treated with proteolytic enzymes can provide a useful in vitro model system for developing and testing potential new treatments of disc degeneration, such as injectable implants or biological therapies

Pedicle Screw Surgery in the UK and Ireland: A Questionnaire Study

Pedicle screw (PS) malpositioning rates are high in spine surgery. This has resulted in the use of computed navigational aids to reduce the rate of malposition; but these are often expensive and limited in availability. A simple mechanical device to aid PS insertion might overcome some of these disadvantages. The purpose of this study was to determine the demand and design criteria for a simple device to aid PS placement, as well as to collect opinions and experiences on PS surgery in the UK and Ireland. A postal questionnaire was sent to 422 spinal surgeons in the UK and Ireland. 101 questionnaires were received; 67 of these (16% of total sent) contained useful information. 78% of surgeons experienced problems with PS placement. The need for a simple mechanical device to aid PS placement was expressed by 59% of respondent surgeons. The proportion of respondents that inserted PSs in the cervical spine was 14%; PSs are mainly inserted in the thoracic, lumbar and sacral spine, but potential exists for a PS placement aid for the cervical and thoracic spine. From the experiences of these 67 surgeons, there is evidence to suggest that surgeons would prefer a pedicle aid that is multiple use, one-piece, hand-held, radiolucent, unilateral and uses the line of sight principle in traditional open surgery. Based on the experiences of 67 surgeons, there is evidence to suggest that computed navigational aids are not readily used in PS surgery and that a simple mechanical device could be a better option. This paper provides useful data for improving the outcomes of spinal surgery

Finite element analysis of the effect of cementing concepts on implant stability and cement fatigue failure

Background and purpose Two contradictory cementing techniques (using an undersized stem versus a canal-filling stem) can both lead to excellent survival rates, a phenomenon known as the “French paradox”. Furthermore, previous studies have indicated that the type of bone supporting the cement mantle may affect implant survival. To further evaluate the mechanical consequences of variations in cementing technique, we studied the effect of implant size and type of bone supporting the cement mantle on the mechanical performance of cemented total hip arthroplasty, using finite element analysis

Simulated-Physiological Loading Conditions Preserve Biological and Mechanical Properties of Caprine Lumbar Intervertebral Discs in Ex Vivo Culture

Low-back pain (LBP) is a common medical complaint and associated with high societal costs. Degeneration of the intervertebral disc (IVD) is assumed to be an important causal factor of LBP. IVDs are continuously mechanically loaded and both positive and negative effects have been attributed to different loading conditions

Trauma induces apoptosis in human thoracolumbar intervertebral discs

BACKGROUND: Vertebral fractures resulting from high energy trauma often comprise the risk of posttraumatic degenerative changes in the affected intervertebral discs (IVD). Particularly in conservatively treated patients, or in cases after implant removal of an exclusively posterior stabilization, consecutive disc degeneration and the associated functional losing of the spinal segment clearly represent detrimental treatment results. In this regard, apoptosis of IVD cells has been suggested to be involved in the critical changes of the extracellular matrix. METHODS: To investigate whether fractures of the vertebrae induce apoptosis in the affected IVD, disc tissue from patients (n = 17) undergoing open reduction and internal fixation of thoracolumbar spine fractures were analysed in regards to caspase activity, apoptosis-receptor expression levels and gene expression of apoptosis-regulating proteins such as Bax and Bcl-2. Healthy IVD tissue (n = 3) obtained from patients undergoing surgical resection of adjacent vertebrae were used as control samples. RESULTS: In contrast to healthy control IVD tissues, samples from traumatic thoracolumbar IVD showed positive TUNEL staining and a significant increase of caspase-3/7 activity. Interestingly, analyses of the initiator caspase-8 and -9 revealed significantly increased activation levels compared to control values, suggesting the coexistent activation of both the extrinsic (receptor-mediated) and intrinsic (mitochondria-mediated) apoptosis pathway. Accordingly, expression levels of the Fas receptor (FasR) mRNA were significantly increased. Although the TNF receptor I (TNFR I) was only slightly upregulated, corresponding TNFα from trauma IVD presented significantly increased mRNA expression values. Furthermore, traumatic IVD cells demonstrated significantly reduced expression of the mitochondria-bound anti-apoptotic Bcl-2, thereby maintaining baseline transcriptional levels of the pro-apoptotic Bax protein when compared to control IVD cells. CONCLUSION: Our data suggest that thoracolumbar fractures induce early caspase-dependent apoptosis in IVD cells of the affected intervertebral disc, in part, by downregulation of the anti-apoptotic protein Bcl-2 (intrinsic apoptosis pathway), as well as signalling via the death receptor complex (TNFR I and FasR)

Urocortin protects chondrocytes from NO-induced apoptosis: a future therapy for osteoarthritis?

Osteoarthritis (OA) is characterized by a loss of joint mobility and pain resulting from progressive destruction and loss of articular cartilage secondary to chondrocyte death and/ or senescence. Certain stimuli including nitric oxide (NO) and the pro-inflammatory cytokine tumor necrosis factor α (TNF-α have been implicated in this chondrocyte death and the subsequent accelerated damage to cartilage. In this study, we demonstrate that a corticotrophin releasing factor (CRF) family peptide, urocortin (Ucn), is produced by a human chondrocyte cell line, C-20/A4, and acts both as an endogenous survival signal and as a cytoprotective agent reducing the induction of apoptosis by NO but not TNF-α when added exogenously. Furthermore, treatment with the NO donor S-nitroso-N-acetyl-D-L-penicillamine upregulates chondrocyte Ucn expression, whereas treatment with TNF-α does not. The chondroprotective effects of Ucn are abolished by both specific ligand depletion (with an anti-Ucn antibody) and by CRF receptor blockade with the pan-CRFR antagonist α-helical CRH(9-41). CRFR expression was confirmed by reverse transcription-PCR with subsequent amplicon sequence analysis and demonstrates that C-20/A4 cells express both CRFR1 and CRFR2, specifically CRFR1α and CRFR2β. Protein expression of these receptors was confirmed by western blotting. The presence of both Ucn and its receptors in these cells, coupled with the induction of Ucn by NO, suggests the existence of an endogenous autocrine/paracrine chondroprotective mechanism against stimuli inducing chondrocyte apoptosis via the intrinsic/mitochondrial pathway

The Effect of Sustained Compression on Oxygen Metabolic Transport in the Intervertebral Disc Decreases with Degenerative Changes

Intervertebral disc metabolic transport is essential to the functional spine and provides the cells with the nutrients necessary to tissue maintenance. Disc degenerative changes alter the tissue mechanics, but interactions between mechanical loading and disc transport are still an open issue. A poromechanical finite element model of the human disc was coupled with oxygen and lactate transport models. Deformations and fluid flow were linked to transport predictions by including strain-dependent diffusion and advection. The two solute transport models were also coupled to account for cell metabolism. With this approach, the relevance of metabolic and mechano-transport couplings were assessed in the healthy disc under loading-recovery daily compression. Disc height, cell density and material degenerative changes were parametrically simulated to study their influence on the calculated solute concentrations. The effects of load frequency and amplitude were also studied in the healthy disc by considering short periods of cyclic compression. Results indicate that external loads influence the oxygen and lactate regional distributions within the disc when large volume changes modify diffusion distances and diffusivities, especially when healthy disc properties are simulated. Advection was negligible under both sustained and cyclic compression. Simulating degeneration, mechanical changes inhibited the mechanical effect on transport while disc height, fluid content, nucleus pressure and overall cell density reductions affected significantly transport predictions. For the healthy disc, nutrient concentration patterns depended mostly on the time of sustained compression and recovery. The relevant effect of cell density on the metabolic transport indicates the disturbance of cell number as a possible onset for disc degeneration via alteration of the metabolic balance. Results also suggest that healthy disc properties have a positive effect of loading on metabolic transport. Such relation, relevant to the maintenance of the tissue functional composition, would therefore link disc function with disc nutrition