Samantha Chan

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.

Outcome assessment of bracing in adolescent idiopathic scoliosis by the use of the SRS-22 questionnaire.

The SRS-22 questionnaire is specifically designed for the assessment of quality of life in spinal deformity patients. This study is the first to use it to assess the quality of life of adolescent idiopathic scoliosis patients under brace treatment and compares the results with an observational group matched by age and curve magnitude. Forty-six patients were enrolled into each group. Overall, it was found that patients under observation had a significantly better quality of life than braced patients. Specifically, the domains for function/activity and self-image were most affected. This effect was most apparent in those with a curve magnitude of under 20°. The scores did not improve significantly with the duration of brace wear, suggesting little adaptation. This study has implications for treatment, and more attention will need to be given to those with mild but progressive curves to help improve patients’ understanding of their treatment and hence their compliance and satisfaction.RésuméLe questionnaire SRS-22 est spécifiquement dévolu à l’évaluation de la qualité de vie chez les patients présentant une déformation rachidienne. Cette étude est la première qui utilise l’information relative à la qualité de vie des adolescents présentant une scoliose idiopathique, traitée par plâtre, en comparant ces résultats avec un groupe identique sur le plan de l’âge et de l’importance de la déformation. Quarante-six patients ont été étudiés dans chaque groupe. Il est évident que les patients sous simple surveillance ont une qualité de vie bien supérieure à ceux traités par plâtre. De façon spécifique, ce sont surtout les critères d’activité et d’image de soi-même qui sont les plus affectés. Cet effet est surtout apparent chez les patients dont la courbe est inférieure à 20°. Les scores ne s’améliorent pas avec le temps, ce qui aurait pu suggérer une petite adaptation. Cette étude a des implications sur le traitement et une attention particulière doit être apportée à tous les patients présentant une courbe d’aggravation progressive de façon à améliorer leur compréhension du traitement et améliorer leur compréhension, leur compliance et leur satisfaction vis-à-vis du traitement.

Biological Response of the Intervertebral Disc to Repetitive Short-Term Cyclic Torsion

Study Design. In vitro study of the biological response of the intervertebral disc (IVD) to cyclic torsion by using bovine caudal IVDs. Objective. To evaluate the biological response of the IVD to repetitive cyclic torsion of varying magnitudes at a physiological frequency. Summary of Background Data. Mechanical loading is known to be a risk factor for disc degeneration (DD) but the role of torsion in DD is controversial. It has been suggested that a small magnitude of spinal rotation decreases spinal pressure, increases spinal length, and enhances nutrition exchange in the IVD. However, athletes who participate actively in sports involving torsional movement of the spine are frequently diagnosed with DD and/or disc prolapse. Methods. Bovine caudal discs with end plates were harvested and kept in custom-made chambers for in vitro culture and mechanical stimulation. Torsion was applied to the explants for 1 hour/day over four consecutive days by using a servohydraulic testing machine. The biological response was evaluated by cell viability, metabolic activity, gene expression, glycosaminoglycan content, and histological evaluation. Results. A significantly higher cell viability was found in the inner annulus of the 2˚ torsion group than in the static control group. A trend of decreasing metabolic activity in the nucleus pulposus with increasing torsion magnitude was observed. Apoptotic activity in the nucleus pulposus significantly increased with 5˚ torsion. No statistical significant difference in gene expression was found between the three torsion angles. No visible change in matrix organization could be observed by histological evaluation. Conclusion. The IVD can tolerate short-term repetitive cyclic torsion, as tested in this study. A small angle of cyclic torsion can be beneficial to the IVD in organ culture, possibly by improving nutrition and waste exchange, whereas large torsion angle may cause damage to disc in the long term.

Papain-induced in vitro disc degeneration model for the study of injectable nucleus pulposus therapy

BACKGROUND CONTEXT Proteolytic enzyme digestion of the intervertebral disc (IVD) offers a method to simulate a condition of disc degeneration for the study of cell-scaffold constructs in the degenerated disc. PURPOSE To characterize an in vitro disc degeneration model (DDM) of different severities of glycosaminoglycans (GAG) and water loss by using papain, and to determine the initial response of the human mesenchymal stem cells (MSCs) introduced into this DDM. STUDY DESIGN Disc degeneration model of a bovine disc explant with an end plate was induced by the injection of papain at various concentrations. Labeled MSCs were later introduced in this model. METHODS Phosphate-buffered saline (PBS control) or papain in various concentrations (3, 15, 30, 60, and 150 U/mL) were injected into the bovine caudal IVD explants. Ten days after the injection, GAG content of the discs was evaluated by dimethylmethylene blue assay and cell viability was determined by live/dead staining together with confocal microscopy. Overall matrix composition was evaluated by histology, and water content was visualized by magnetic resonance imaging. Compressive and torsional stiffness of the DDM were also recorded. In the second part, MSCs were labeled with a fluorescence cell membrane tracker and injected into the nucleus of the DDM or a PBS control. Mesenchymal stem cell viability and distribution were evaluated by confocal microscopy. RESULTS A large drop of GAG and water content of the bovine disc were obtained by injecting >30 U/mL papain. Magnetic resonance imaging showed Grade II, III, and IV disc degeneration by injecting 30, 60, and 150 U/mL papain. A cavity in the center of the disc could facilitate later injection of the nucleus pulposus tissue engineering construct while retaining an intact annulus fibrosus. The remaining disc cell viability was not affected. Mesenchymal stem cells injected into the protease-treated DDM disc showed significantly higher cell viability than when injected into the PBS-injected control disc. CONCLUSIONS By varying the concentration of papain for injection, an increasing amount of GAG and water loss could be induced to simulate the different severities of disc degeneration. MSC suspension introduced into the disc has a very low short-term survival. However, it should be clear that this bovine IVD DDM does not reflect a clinical situation but offers exciting possibilities to test novel tissue engineering protocols.

Region Specific Response of Intervertebral Disc Cells to Complex Dynamic Loading: An Organ Culture Study Using a Dynamic Torsion-Compression Bioreactor

The spine is routinely subjected to repetitive complex loading consisting of axial compression, torsion, flexion and extension. Mechanical loading is one of the important causes of spinal diseases, including disc herniation and disc degeneration. It is known that static and dynamic compression can lead to progressive disc degeneration, but little is known about the mechanobiology of the disc subjected to combined dynamic compression and torsion. Therefore, the purpose of this study was to compare the mechanobiology of the intervertebral disc when subjected to combined dynamic compression and axial torsion or pure dynamic compression or axial torsion using organ culture. We applied four different loading modalities [1. control: no loading (NL), 2. cyclic compression (CC), 3. cyclic torsion (CT), and 4. combined cyclic compression and torsion (CCT)] on bovine caudal disc explants using our custom made dynamic loading bioreactor for disc organ culture. Loads were applied for 8 h/day and continued for 14 days, all at a physiological magnitude and frequency. Our results provided strong evidence that complex loading induced a stronger degree of disc degeneration compared to one degree of freedom loading. In the CCT group, less than 10% nucleus pulposus (NP) cells survived the 14 days of loading, while cell viabilities were maintained above 70% in the NP of all the other three groups and in the annulus fibrosus (AF) of all the groups. Gene expression analysis revealed a strong up-regulation in matrix genes and matrix remodeling genes in the AF of the CCT group. Cell apoptotic activity and glycosaminoglycan content were also quantified but there were no statistically significant differences found. Cell morphology in the NP of the CCT was changed, as shown by histological evaluation. Our results stress the importance of complex loading on the initiation and progression of disc degeneration.

Cryopreserved intervertebral disc with injected bone marrow-derived stromal cells: a feasibility study using organ culture

BACKGROUND CONTEXT 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. PURPOSE This study examined the viability over time of endogenous and injected BMSCs in cryopreserved disc under simulated-physiological loading conditions. STUDY DESIGN/ SETTING: An in vitro study of BMSCs injected into cryopreserved bovine caudal discs. METHODS Bovine caudal discs were harvested and cryopreserved at -196 degrees C. After thawing, PKH-26-labeled BMSCs embedded in peptide hydrogel carrier were injected into the nucleus pulposus. Two BMSC injection quantities, that is, 1x10(5) and 2.5x10(5) were examined. Discs with injected cells were maintained in a bioreactor for 7 days under simulated-physiological loading. Cell viability (staining), gene expression (reverse transcription-polymerase chain reaction) profile, and proteoglycan content (histologically) were evaluated. RESULTS Forty percent of endogenous cell viability was maintained after freeze thawing. Over the 7-day culture, this did not change further. However, there was upregulation of Col1a2 and Mmp-13 and downregulation of Col2a1gene expression. Sixty percent of BMSCs survived the initial injection procedure, and only 20% remained alive after 7 days of culture. Bone marrow-derived stromal cell implantation did not alter the viability of the endogenous cells, but discs injected with 1x105 BMSCs showed significantly higher ACAN expression than sham discs. CONCLUSIONS Although only 40% of cells survived cryopreservation, these endogeneous cells continued to survive over 7 days if maintained under simulated-physiological loading conditions. Although only a small portion of injected BMSCs survived, they did have some effect on the matrix protein gene expression profile. Their influence on native cells requires long-term evaluation.

Activation of intervertebral disc cells by co-culture with notochordal cells, conditioned medium and hypoxia

BackgroundNotochordal cells (NC) remain in the focus of research for regenerative therapy for the degenerated intervertebral disc (IVD) due to their progenitor status. Recent findings suggested their regenerative action on more mature disc cells, presumably by the secretion of specific factors, which has been described as notochordal cell conditioned medium (NCCM). The aim of this study was to determine NC culture conditions (2D/3D, fetal calf serum, oxygen level) that lead to significant IVD cell activation in an indirect co-culture system under normoxia and hypoxia (2% oxygen).MethodsPorcine NC was kept in 2D monolayer and in 3D alginate bead culture to identify a suitable culture system for these cells. To test stimulating effects of NC, co-cultures of NC and bovine derived coccygeal IVD cells were conducted in a 1:1 ratio with no direct cell contact between NC and bovine nucleus pulposus cell (NPC) or annulus fibrosus cells (AFC) in 3D alginate beads under normoxia and hypoxia (2%) for 7 and 14 days. As a positive control, NPC and AFC were stimulated with NC-derived conditioned medium (NCCM). Cell activity, glycosaminoglycan (GAG) content, DNA content and relative gene expression was measured. Mass spectrometry analysis of the NCCM was conducted.ResultsWe provide evidence by flow cytometry that monolayer culture is not favorable for NC culture with respect to maintaining NC phenotype. In 3D alginate culture, NC activated NPC either in indirect co-culture or by addition of NCCM as indicated by the gene expression ratio of aggrecan/collagen type 2. This effect was strongest with 10% fetal calf serum and under hypoxia. Conversely, AFC seemed unresponsive to co-culture with pNC or to the NCCM. Further, the results showed that hypoxia led to decelerated metabolic activity, but did not lead to a significant change in the GAG/DNA ratio. Mass spectrometry identified connective tissue growth factor (CTGF, syn. CCN2) in the NCCM.ConclusionsOur results confirm the requirement to culture NC in 3D to best maintain their phenotype, preferentially in hypoxia and with the supplementation of FCS in the culture media. Despite these advancements, the ideal culture condition remains to be identified.

Assessment of the matrix degenerative effects of MMP-3, ADAMTS-4, and HTRA1, injected into a bovine intervertebral disc organ culture model.

Study Design. In vitro study to develop an intervertebral disc degeneration organ culture model, using coccygeal bovine intervertebral discs (IVDs) and injection of proteolytic enzymes MMP-3, ADAMTS-4, and HTRA1. Objective. This study aimed to develop an in vitro model of enzyme-mediated intervertebral disc degeneration to mimic the clinical outcome in humans for investigation of therapeutic treatment options. Summary of Background Data. Bovine IVDs are comparable with human IVDs in terms of cell composition and biomechanical behavior. Researchers injected papain and trypsin into them to create an intervertebral disc degeneration model with a degenerated nucleus pulposus (NP) area. They achieved macroscopic cavities as well as a loss of glycosaminoglycans (GAGs). However, none of these enzymes are clinically relevant. Methods. Bovine IVDs were harvested maintaining the endplates. Active forms of MMP-3, ADAMTS-4, and HTRA1 were injected at a dose of 10 &mgr;g/mL each. Phosphate-buffered saline was injected as a control. Discs were cultured for 8 days and loaded diurnally (days 1–4 with ≈0.4 MPa for 16 hr) and left under free swelling condition from days 4 to 8 to avoid expected artifacts because of dehydration of the NP. Outcome parameters included disc height, metabolic cell activity, DNA content, GAG content, total collagen content, relative gene expression, and histological investigation. Results. The mean metabolic cell activity was significantly lower in the NP area of discs injected with ADAMTS-4 than the day 0 control discs. Disc height was decreased after injection with HTRA1 and was significantly correlated with changes in GAG/DNA of the NP tissue. Total collagen content tended to be lower in groups injected with ADAMTS4 and MMP-3. Conclusion. MMP-3, ADAMTS-4, and HTRA1 provoked neither visible matrix degradation nor major shifts in gene expression. However, cell activity was significantly reduced and HTRA1 induced loss of disc height that positively correlated with changes in GAG/DNA content. The use of higher doses of these enzymes or a combination thereof may, therefore, be necessary to induce disc degeneration.

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.

Minimally invasive photopolymerization in intervertebral disc tissue cavities

Photopolymerized hydrogels are commonly used for a broad range of biomedical applications. As long as the polymer volume is accessible, gels can easily be hardened using light illumination. However, in clinics, especially for minimally invasive surgery, it becomes highly challenging to control photopolymerization. The ratios between polymerizationvolume and radiating-surface-area are several orders of magnitude higher than for ex-vivo settings. Also tissue scattering occurs and influences the reaction. We developed a Monte Carlo model for photopolymerization, which takes into account the solid/liquid phase changes, moving solid/liquid-boundaries and refraction on these boundaries as well as tissue scattering in arbitrarily designable tissue cavities. The model provides a tool to tailor both the light probe and the scattering/absorption properties of the photopolymer for applications such as medical implants or tissue replacements. Based on the simulations, we have previously shown that by adding scattering additives to the liquid monomer, the photopolymerized volume was considerably increased. In this study, we have used bovine intervertebral disc cavities, as a model for spinal degeneration, to study photopolymerization in-vitro. The cavity is created by enzyme digestion. Using a custom designed probe, hydrogels were injected and photopolymerized. Magnetic resonance imaging (MRI) and visual inspection tools were employed to investigate the successful photopolymerization outcomes. The results provide insights for the development of novel endoscopic light-scattering polymerization probes paving the way for a new generation of implantable hydrogels.