Introduction: Vertebroplasty (VP) is a new prophylactic treatment for preventing osteoporotic compression fractures of vertebral bodies. During this procedure polymethylmethacrylate (PMMA) is injected into several vertebral bodies. It has been shown that fat embolism (FE) with acute cardiopulmonary deterioration occurs during VP as in a variety of other orthopaedic procedures (e.g. knee and hip replacements). The aim of the study is to investigate cardiovascular changes during FE caused by multiple VP using an animal model. Method: In six sheep, PMMA was injected unilaterally, into L1 – L6, with ten minutes in-between injections. Arterial, venous and pulmonary artery pressure, cardiac output and blood gas values were recorded pre injection and 1, 3, 5 and 10 minutes post injection. Post mortem lungs were harvested and the histopathologic score (percentage of lung fields occupied by intravascular fat globules as seen in the microscope) was calculated. Results: The sequential injection of bone cement into six vertebral bodies from values pre injection of L1 to 10 minutes post injection of L6 resulted in: significant falls in arterial blood pressure (P 2 and pH (P significant rises in pulmonary arterial pressure (P=0.0005) and pCO 2 (P Conclusion: This study clearly shows that multiple VP in sheep leads to FE with major cardiovascular reactions. Arterial blood pressure showed a stepwise, cumulative fall and was clearly the best parameter to demonstrate these reactions. This suggests, in human patients, particular attention should be paid to falls in arterial blood pressure during multiple VP.

Human bone marrow-derived mesenchymal stem cells (MSCs) have limited growth potential in vitro and cease to divide due to replicative senescence, which from a tissue-engineering perspective has practical implications, such as defining the correct starting points for differentiation and transplantation. Time spent in culture before the loss of required differentiation potential is different and reflects patient variability, which is a problem for cell expansion. This study aimed to develop a score set which can be used to quantify the senescent state of MSCs and predict whether cells preserve their ability to differentiate to osteogenic, adipogenic and chondrogenic phenotypes, based on colony-forming unit (CFU) assay, population doubling time (PDT), senescence-associated -galactosidase (SA--Gal) activity, cell size, telomere length and gene expression of MSCs cultured in vitro over 11 passages. This set of morphological, physiological and genetic senescence markers was correlated to the ability of MSCs to differentiate. Differentiation efficiency was assessed by marker genes and protein expression. CFUs decreased with increasing passage number, whereas SA--Gal activity and PDT increased; however, the correlation with MSCs' differentiation potential was sometimes unexpected. The expression of genes related to senescence was higher in late-passage cells than in early-passage cells. Early-passage cells underwent efficient osteogenic differentiation, with mid-passage cells performing best in chondrogenic differentiation. Late-passage cells preserve only adipogenic differentiation potential. Based on this marker set, we propose a senescence score in which combined markers give a reliable quality control of MSCs, not depending only on mechanistic passage number. Copyright (c) 2013 John Wiley & Sons, Ltd.

OBJECT The authors investigated the relevance of 2D MRI measurements for the diagnosis of critical cervical spinal canal stenosis. Among patients who had sustained a minor cervical spine trauma, they compared MRI measurements of the cervical spine between those with acute cervical spinal cord injury (CSCI) and those without. They also investigated the correlation between the MRI measurements and the severity of CSCI as well as classification accuracy concerning the diagnosis of critical spinal canal stenosis for acute CSCI after a minor trauma.METHODS The authors conducted a single-center retrospective radiological case-control study of patients who had sustained CSCI after a minor trauma to the cervical spine from January 2000 to August 2010. The controls were patients who had sustained a cervical trauma without clinical or radiological signs of cervical spinal cord pathology. On axial T2-weighted MR images, the following were measured: the transverse spinal canal and cord area, the transverse and sagittal cord diameter, and the sagittal canal diameter of the cervical spine (C3-7). Using these measurements, the authors calculated the cord-canal-area ratio by dividing the transverse cord area by the transverse canal area, the space available for the cord by subtracting the sagittal canal diameter from the sagittal cad diameter, and the compression ratio by dividing the transverse cord diameter by its sagittal diameter. The severity of CSCI was assessed by using the motor index scores of 10 key muscles at different time points (initially, 1, 3, and 12 months after injury) obtained from the clinical records. The intra- and interobserver reliability of the MRI measurements, intra- and intergroup differences, and correlations between variables were also investigated. Receiver operating characteristic curves were calculated for evaluating the classification accuracy of the imaging variables for CSCI.RESULTS Data for 52 CSCI patients and 77 control patients were analyzed. The intra- and interobserver reliability regarding the MRI measurements ranged from good (0.72) to perfect (0.99). Differences between the CSCI group and the control group were significant (p < 0.001) for all parameters, except for the cord area. The most prominent differences between the groups existed for the spinal canal area, cord-canal-area ratio, and space available for the cord. The classification accuracy was best for the cord-canal-area ratio and the space available for the cord; areas under the curve were 0.99 (95% CI 0.97-1.0) and 0.98 (95% CI 0.95-0.99), respectively. There was no significant (p > 0.05) correlation between any of the imaging parameters and the motor index score at any time point.CONCLUSIONS The cord-Canal-area ratio (> 0.8) or the space available for the cord (< 1.2 mm) measured on MR images can be used to reliably identify patients at risk for acute CSCI after a minor trauma to the cervical spine. However, there does not seem to be any association between spinal canal imaging characteristics and the severity of or recovery from CSCI after a minor trauma.

BACKGROUND CONTEXT: Neuropathic (Charcot) spinal arthropathy (CSA) is a rare but progressive and severe degenerative disease that develops in the absence of deep sensation, for example, after spinal cord injury. The diagnosis of CSA is often delayed as a result of the late onset or slow progression of the disease and the nonspecific nature of the reported clinical signs. Considering risk factors of CSA in combination with the common clinical signs may facilitate timely diagnosis and prevent severe presentation of the disease. However, there is a lack of data concerning the early signs and risk factors of CSA. Furthermore, the complications and outcomes after surgical treatment are documented insufficiently.PURPOSE: To investigate the early signs and risk factors of CSA after spinal cord injury, as well as the complications and outcome after surgical treatment.STUDY DESIGN: Retrospective case series from a single center.PATIENT SAMPLE: Twenty-eight patients with 39 Charcot joints of the spine.OUTCOME MEASURES: Clinical signs, radiological signs, risk factors, and complications.METHODS: The case histories and radiological images of patients suffering from CSA were investigated.RESULTS: The first clinical symptoms included spinal deformity, sitting imbalance, and localized back pain. Long-segment stabilization, laminectomy, scoliosis, and excessive loading of the spine were identified as risk factors for the development of the disease. Postoperative complications included implant loosening, wound healing disturbance, and development of additional Charcot joints. All patients were able to return to their previous levels of activities.CONCLUSIONS: Radiological follow-up of the entire thoracic and lumbar spine should be performed in paraplegic patients. Risk factors in combination with typical symptoms should be considered to facilitate early detection. Functional restoration can be achieved with appropriate surgical techniques. (C) 2014 Elsevier Inc. All rights reserved.

In der Sportmedizin bestehen zum Herzratenvariabilität (HRV) – Schwellenkonzept keine vergleichenden experimentellen Grundlagen mit Standardtestverfahren im Wasser. Bei 12 anamnestisch gesunden und trainierten Probanden wurde während Ergometerbelastungen (Arm-/Beindrehkurbel) im Wasser und an Land spirografisch der Gasstoffwechsel gemessen. Zeitsynchron mit der Erfassung respiratorischer Parameter wurde die hochfrequente Komponente der HRV (logarhithmierte HF-Leistung, lnHF) mit einem EKG auf jeder Belastungsstufe zeitkontinuierlich aufgezeichnet. Aus dem Produkt lnHF und der Zentralherzfrequenz wurde verlaufskinetisch die Leistung (Watt) an der respiratorischen Schwelle 2 quantitativ bestimmt und mit der reliablen Messmethodik der Gasanalyse verglichen. Die Differenzen der mittleren Leistungskennwerte unterscheiden sich nicht (p>0,05). Der mittlere Bias weicht nur wenig von Null ab (−0,6 Watt). Die Übereinstimmungsgrenzen liegen zwischen −20,1 und 18,8 Watt. Auf der Basis eines quantitativen Vergleichs der Leistungskennwerte stimmt das HRV-Schwellenkonzept mit dem respiratorischen Referenzverfahren auch bei Arm-/Bein-Drehkurbelarbeit im Wasser gut überein.

Background and purpose — There are very few data concerning the outcome after short-segment posterior stabilization and anterior spondylodesis with rib grafts in patients suffering from unstable thoracolumbar burst fractures. We have therefore investigated the clinical and radiographic outcome after posterior bisegmental instrumentation and monosegmental anterior spondylodesis using an autologous rib graft for unstable thoracolumbar burst fractures. Patients and methods — This was a retrospective analysis of 32 consecutive patients at a single center. The monosegmental Cobb angle was measured preoperatively, postoperatively, then 6 and 12 months postoperatively, and also after implant removal. Anterior vertebral fusion was graded on conventional radiographs according to the criteria proposed by Molinari. Results — Segmental kyphosis at the fracture site was corrected from a median of -20° (95% CI: -21.2 to -18.8) to -1.0° (95% CI: -2.7 to 0.7) postoperatively. 1 year after surgery, the segmental angle had decreased by a median of 2.0° (95% CI: 0.2 to 2.8). The spondylodesis fused in all patients, which was evident from incorporation and remodeling of the rib grafts. The median correction loss after implant removal was 0.0° (95% CI: -0.5 to 0.5). 26 of the 32 patients reported having no back complaints at the last follow-up (2 years postoperatively). 1 patient suffered from intercostal neuralgia, and 5 patients reported mild to moderate back pain. Interpretation — Short-segment posterior instrumentation and anterior spondylodesis using an autologous rib graft resulted in sufficient correction of posttraumatic segmental kyphosis. There was no clinically relevant correction loss, and the majority of patients had no back complaints at the 2-year follow-up.

BACKGROUND CONTEXT:Acute spinal cord injury (SCI) after a minor trauma to the cervical spine has been reported in patients without preceding neurologic symptoms. Spinal canal stenosis may be the reason for the discrepancy between the severity of the injury and that of the trauma. The spinal canal to vertebral body ratio is often used to assess canal stenosis on conventional radiographs. However, the ratio does not appraise soft-tissue stenosis and canal narrowing at the level of the intervertebral disc. Parameters measured on magnetic resonance (MR) images may thus be more meaningful. The relevance of MR image parameters for predicting the risk and severity of acute SCI in patients after a minor trauma to the cervical spine has not yet been established. PURPOSE:To investigate MR image parameters of the cervical spine in patients suffering from acute SCI after a minor trauma to the cervical spine. To investigate the use of these parameters for predicting the risk and severity of acute cervical SCI after a minor trauma to the cervical spine. STUDY DESIGN/SETTING:Retrospective radiological study of consecutive patients. PATIENT SAMPLE:Fifty-two patients suffering from acute cervical SCI and 131 patients showing no neurologic deficits after a minor trauma to the cervical spine. OUTCOME MEASURES:On sagittal MR images: vertebral body diameter, midvertebral canal diameter, disc-level canal diameter, and spinal cord diameter. On lateral conventional radiographs: vertebral body diameter and midvertebral canal diameter. METHODS:Conventional lateral radiographs and sagittal T2-weighted MR images of the cervical spine (C3-C7) were analyzed. The following calculations were performed using measurements from MR images: the spinal canal to vertebral body ratio, the space available for the cord, and the canal-to-cord ratio. Using measurements from conventional radiographs, the spinal canal to vertebral body ratio was determined. Receiver-operating curves were calculated for evaluating the classification accuracy of the different parameters for predicting the risk, severity, and course of SCI. RESULTS:All investigated MR image parameters in the SCI group were significantly (p<.001) smaller compared with the control group. However, there was no significant (p>.9) difference in any parameter among the different American Spinal Injury Association impairment score groups. A cutoff value of 8.0 mm for the minimal sagittal disc-level canal diameter yielded the largest positive predictive value and likelihood ratio for predicting SCI. CONCLUSIONS:Patients at risk of acute SCI after a minor trauma to the cervical spine can be identified by applying a disc-level canal diameter cutoff value (measured on MR images) of 8 mm. Additional factors to the radiological characteristics of the spinal canal affect the severity of acute SCI after a minor trauma to the cervical spine.

Exercise in water compared to land-based exercise (LE) results in a higher release of natriuretic peptides, which are involved in the regulation of exercise-induced adipose tissue lipolysis. The present study was performed to compare the release of atrial natriuretic peptide (ANP) and free fatty acids (FFA) during prolonged aerobic water-based exercise (WE) with the release after an identical LE. 14 untrained overweight subjects performed 2 steady state workload tests on the same ergometer in water and on land. Before and after exercise, venous blood samples were collected for measuring ANP, FFA, epinephrine, norepinephrine, insulin and glucose. The respiratory exchange ratio (RER) was determined for fat oxidation. The exercises resulted in a significant increase in ANP in LE (61%) and in WE (177%), and FFA increased about 3-fold in LE and WE with no significant difference between the groups. Epinephrine increased, while insulin decreased similarly in both groups. The RER values decreased during the exercises, but there was no significant difference between LE and WE. In conclusion, the higher ANP concentrations in WE had no additional effect on lipid mobilization, FFA release and fat oxidation. Moderate-intensity exercises in water offer no benefit regarding adipose tissue lipolysis in comparison to LE.

BACKGROUND CONTEXT:Testosterone (T) is a hormone and regulator involved in the processes of development of the organism (ie, promoting development of bone and muscle mass). Although T effects on the mesenchyme-derived muscle, bone, and adipose tissues are well studied, T effects on intervertebral disc (IVD) have not been reported. PURPOSE:The aim was to test the following hypothesis: if a physiological concentration of T (∼30 nM) can improve in vitro chondrogenesis of human IVD cells and mesenchymal stem cells (MSCs). STUDY DESIGN/SETTING:Human IVD cells and MSCs were differentiated to chondrogenic lineage on gelatin scaffolds for 4 weeks, in the presence or absence of T. METHODS:Chondrogenesis was assessed by cell viability, by measuring gene expression with quantitative polymerase chain reaction and extracellular matrix (ECM) accumulation with immunoblotting, immunohistochemical, and biochemical methods. RESULTS:Supplementation of T to chondrogenic culture did not affect viability. In male IVD cells, T had a beneficial impact on chondrogenesis, particularly in nucleus pulposus cells, demonstrated by an increased expression of aggrecan, collagen type I, and especially collagen type II. Conversely, T had no effects on chondrogenesis of female IVD cells or MSCs from both genders. A gene expression array of transforming growth factor β/bone morphogenetic protein signaling cascade showed that in male IVD cells, T promoted a stable general but nonsignificant increase in gene expression. Furthermore, aromatase inhibitor anastrazole repressed the effect of T on ECM expression by IVD cells. The results suggest that T increased ECM accumulation in male IVD cells in combination with its conversion to estradiol by the enzyme aromatase. CONCLUSIONS:We demonstrated that T effectively enhances in vitro chondrogenesis in male IVD cells, rising the interest in the possible role of sex hormones in IVD degeneration. Nevertheless, T does not affect chondrogenic differentiation of female IVD cells and MSCs from both genders.

Objectives: Heart rate variability (hrv) is an established parameter for analyzing performance in sports medicine. However, no valid data are available for standard test situations in water. Methods: 12 trained healthy persons performed maximal graded exercise tests on a whole body cycling ergometer. Gas exchange (VO2, VCO2) and the vagally modulated short time variability parameter lnHF were monitored with a Holter device during each step. The expired air and lnHF were analyzed for exceeding anaerobic energy expenditure (ventilatory threshold 2, VT2) and compared with each other. Results: There was no significant difference between the means of the VT2 determined by the 2 different methods (p>0.05), and the bias was close to zero (-0.6 Watt). The confidence interval (1.96 SD) of the mean differences has been found to be between -20.1 and 18.8 Watts. Discussion: The quantitative comparison of gas exchange measurements with hrv showed a strong correlation between both parameters on a whole body ergometer in water. Both methods can be used interchangeably.

Ziel der Studie war es, die Reliabilität der Einstellwerte eines neu entwickelten Arm-/Beindrehkurbelergometer (Reha-Aquabike) bei gestufter Belastungsführung im Wasser zu überprüfen. In wiederholten Messungen wurde bei Arm-/Beinarbeit (A+B) und Beinarbeit (B) in Wasser (W) und an Land (L) mit demselben Ergometer das Belastungsverhalten 12 gesunder männlicher Probanden anhand der Sauerstoffaufnahme (VO2) bestimmt. Die Streuung der Wiederholungsmessungen wurde mit der Bland-Altman-Methode quantifiziert. Die Mittelwerte der Testwiederholungen unterschieden sich nicht (p>0,05). ­Innerhalb der Übereinstimmungsgrenzen 68,9±423 ml*min − 1 (A+B) und 69,7±354 ml*min − 1 (B) lagen 95% der Differenzen. Das Wiederholungsverfahren lieferte gleiche VO2-Ergebnisreihen. Innerhalb der Übereinstimmungsgrenzen sind die Einstellwerte des Wasser­ergometers präzise und das Bremsverhalten ­beständig.

Purpose: Our objective was to investigate the reliability of a newly developed whole body water ergometer (Reha-Aquabike). Materials and methods: 12 healthy subjects performed repeated exercise tests. There were 4 tests which included combined arm-leg-ergometry (A+B) and cycling alone (B) in water (W) and on land (L). A revolution and a step protocol were followed. Oxygen and carbon dioxide were measured to calculate gas exchange. The scatter of the oxygen consumption (VO2) values was analyzed with the Bland-Altman method and ANOVA.. Results: The results of the repeated tests showed no difference in the means (p > 0.05). Limits of agreement were 68.9 +/- 423 ml*min(-1) (A+B) and 69.7+/-354 ml*min(-1) (B) and included 95% of the values. Discussion: The repeated measures of VO2 were comparable and the step protocol was reproducible. Conclusions: The cut-off-points of the Reha-Aquabike are adequate and the control system is accurate.

INTRODUCTION:Cell-based therapies for regeneration of the degenerated intervertebral disc (IVD) are an alternative to current surgical intervention. Mesenchymal stem cells (MSCs), in combination with a scaffold, might be ideal candidates for regenerating nucleus pulposus (NP), the pressure-distributing part of the IVD. While the use of growth factors for MSCs differentiation currently receives major attention, in this study we compare the performance of sponge-like matrixes in supporting cell differentiation into NP-like cells.MATERIALS AND METHODS:Four types matrixes approved as medical devices for other applications were tested as scaffolds for MSCs: two made of equine or porcine collagen, one of gelatin and one of chitosan. Bone marrow-derived human MSCs were seeded in these scaffolds or embedded in alginate, as a three-dimensional control. After five weeks in culture, NP-like differentiation of the cell-scaffold constructs was analyzed by qRT-PCR, histology, total DNA quantification, proteoglycan accumulation and immunohistochemistry.RESULTS:MSCs in collagen matrixes and gelatin produced more mRNA and proteins of the chondrogenic markers collagen type I, collagen type II (COL2) and aggrecan (ACAN), when compared with cells embedded in alginate or chitosan. Proteoglycan accumulation and cell survival were also higher in collagen and gelatin matrixes. Gene expression results were also confirmed by histological and immunohistochemical staining. In contrast to alginate control, the gene expression of the undesired bone marker osteopontin was lower in all tested groups. In porcine collagen supports, MSC expression ratio between COL2/ACAN closely resembled the expression of nucleus pulposus cells, but gene expression of recently described NP markers keratin19, PAX1 and FOXF1 was lower.CONCLUSIONS:Collagen supports provide a readily available, medically approved and effective scaffold for chondrogenic differentiation in vitro, but the phenotype of differentiated MSCs is not yet completely equivalent to that of NP cells.

Vertebral cement augmentation is reported to be a safe and effective technique for providing stabilization and pain relief. However, adjacent intervertebral discs may be at risk of accelerated degeneration as a result of aggravated nutritional constraints. Therefore, we investigated the effects of injecting polymethylmethacrylate (PMMA) into three adjacent lumbar vertebrae on intervertebral disc and vertebral bone tissue in 12 skeletally mature sheep. After 6 and 12 months of augmentation, the sheep were euthanized and their spines were processes for histological evaluation. Semiquantitative histomorphological analysis of discs and endplates was conducted using published criteria. Histomorphological changes in the augmented bone were assessed qualitatively. Approximately 80% of the length of the endplates was in contact with PMMA. However, there was no significant difference between the histopathological score of the discs adjacent to augmented vertebrae and the score of the control discs. Bone tissue reaction to PMMA was characterized by a thin fibrous tissue layer and occasional foreign-body reactions. New bone formation was present in all augmented vertebrae. Concerns about aggravation of disc degeneration as a result of vertebral cement augmentation seem to be unsubstantiated. Furthermore, adverse effects of PMMA cement on bone biology do not seem to be a relevant issue.

Anamnese Ein 57-jahriger Patient wurde nach einem Sturz einen steilen Berghang hinunter (ca. 70 m) wegen einer Lungenkontusion und einer Rissquetschwunde an der Stirn extern versorgt. Es wurde damals auf den externen konventionellen Rontgenbildern der Halswirbelsaule (HWS) in zwei Ebenen (antero-posterior und lateral inkl. Schwimmeraufnahme) keine Fraktur diagnostiziert (Abb. 1 x). Der Patient wurde nach neun Tagen Hospitalisation nach Hause entlassen. Ungefahr acht Wochen nach dem Unfall wurde der Patient wegen zunehmender Nackenschmerzen beim Hausarzt vorstellig. Dieser veranlasste eine radiologische Abklarung (MRT) der HWS (Abb. 2 x) und wies uns den Patienten aufgrund der Befunde (Luxationsfraktur der HWS) notfallmassig zu.

There is evidence that mesenchymal stem cells (MSCs) can differentiate towards an intervertebral disc (IVD)-like phenotype. We compared the standard chondrogenic protocol using transforming growth factor beta-1 (TGFß) to the effects of hypoxia, growth and differentiation factor-5 (GDF5), and coculture with bovine nucleus pulposus cells (bNPC). The efficacy of molecules recently discovered as possible nucleus pulposus (NP) markers to differentiate between chondrogenic and IVD-like differentiation was evaluated. MSCs were isolated from human bone marrow and encapsulated in alginate beads. Beads were cultured in DMEM (control) supplemented with TGFß or GDF5 or under indirect coculture with bNPC. All groups were incubated at low (2 %) or normal (20 %) oxygen tension for 28 days. Hypoxia increased aggrecan and collagen II gene expression in all groups. The hypoxic GDF5 and TGFß groups demonstrated most increased aggrecan and collagen II mRNA levels and glycosaminoglycan accumulation. Collagen I and X were most up-regulated in the TGFß groups. From the NP markers, cytokeratin-19 was expressed to highest extent in the hypoxic GDF5 groups; lowest expression was observed in the TGFß group. Levels of forkhead box F1 were down-regulated by TGFß and up-regulated by coculture with bNPC. Carbonic anhydrase 12 was also down-regulated in the TGFß group and showed highest expression in the GDF5 group cocultured with bNPC under hypoxia. Trends in gene expression regulation were confirmed on the protein level using immunohistochemistry. We conclude that hypoxia and GDF5 may be suitable for directing MSCs towards the IVD-like phenotype.

BACKGROUND:MMP28 (epilysin) is a recently discovered member of the MMP (matrix metalloproteinase) family that is, amongst others, expressed in osteoarthritic cartilage and intervertebral disc (IVD) tissue. In this study the hypothesis that increased expression of MMP28 correlates with higher grades of degeneration and is stimulated by the presence of proinflammatory molecules was tested. Gene expression levels of MMP28 were investigated in traumatic and degenerative human IVD tissue and correlated to the type of disease and the degree of degeneration (Thompson grade). Quantification of MMP28 gene expression in human IVD tissue or in isolated cells after stimulation with the inflammatory mediators lipopolysaccharide (LPS), interleukin (IL)-1β, tumor necrosis factor (TNF)-α or the histondeacetylase inhibitor trichostatin A was performed by real-time RT PCR.RESULTS:While MMP28 expression was increased in individual cases with trauma or disc degeneration, there was no significant correlation between the grade of disease and MMP28 expression. Stimulation with LPS, IL-1β, TNF-α or trichostatin A did not alter MMP28 gene expression at any investigated time point or any concentration.CONCLUSIONS:Our results demonstrate that gene expression of MMP28 in the IVD is not regulated by inflammatory mechanisms, is donor-dependent and cannot be positively or negatively linked to the grade of degeneration and only weakly to the occurrence of trauma. New hypotheses and future studies are needed to find the role of MMP28 in the intervertebral disc.

Degeneration of intervertebral discs (IVD) is one of the main causes of back pain and tissue engineering has been proposed as a treatment. Tissue engineering requires the use of highly expensive growth factors, which might, in addition, lack regulatory approval for human use. In an effort to find readily available differentiation factors, we tested three molecules--dexamethasone, triiodothyronine (T3) and insulin--on human IVD cells isolated after surgery, expanded in vitro and transferred into alginate beads. Triplicates containing 40 ng/ml dexamethasone, 10 nM T3 and 10 µg/ml insulin, together with a positive control (10 ng/mL transforming growth factor (TGF)-beta 1), were sampled weekly over six weeks and compared to a negative control. Furthermore, we compared the results to cultures with optimized chondrogenic media and under hypoxic condition (2% O2). Glycosaminoglycan (GAG) determination by Alcian Blue assay and histological staining showed dexamethasone to be more effective than T3 and insulin, but less than TGF-beta1. DNA quantification showed that only dexamethasone stimulated cell proliferation. qPCR demonstrated that TGF-beta1 and the optimized chondrogenic groups increased the expression of collagen type II, while aggrecan was stimulated in cultures containing dexamethasone. Hypoxia increased GAG accumulation, collagen type II and aggrecan expression, but had no effect on or even lowered cell number. In conclusion, dexamethasone is a valuable and cost-effective molecule for chondrogenic and viability induction of IVD cells under normoxic and hypoxic conditions, while insulin and T3 did not show significant differences.