Augmentation and repair tissue formation of the nucleus pulposus after partial nucleotomy in a rabbit model.

Disc degeneration alters disc height and mechanics of the spinal column and is associated with lower back pain. In preclinical studies gel-like materials or resorbable polymer-based implants are frequently used to rebuild the nucleus pulposus, aiming at tissue regeneration and restoration of tissue function. To compare the outcome of tissue repair, freeze-dried resorbable polyglycolic acid-hyaluronan (PGA/HA) implants without any bioactive components or bioactivated fibrin (fibrin-serum) was used in a degenerated disc disease model in New Zealand white rabbits. Animals with partial nucleotomy only served as controls. The T2-weighted/fat suppression sequence signal intensity in the nuclear region of operated discs as assessed by magnet resonance imaging was reduced in operated compared to healthy discs, indicating loss of water and did not change from week 1 to month 6 after surgery. Quantification of histological and immunohistochemical staining indicated that the implantation of PGA/HA leads to significantly more repair tissue compared to nucleotomy only. Type II collagen content of the repair tissue formed after PGA/HA or fibrin-serum treatment is significantly increased compared to controls with nucleotomy only. The data indicate that intervertebral disc augmentation after nucleotomy has a positive effect on repair tissue formation and type II collagen deposition as shown in the rabbit model.

Effect of degeneration on gene expression of chondrogenic and inflammatory marker genes of intervertebral disc cells: a preliminary study.

AIM: New techniques for biological repair in the treatment of degenerative disc disease (DDD) have been developed recently. The question arises whether it is possible to find a predictive marker to identify a patient population which could benefit from this new treatment option. Standard magnetic resonance imaging (MRI) fails to differentiate between pathologic painful and asymptomatic aging discs. Neurological symptoms contribute to identifying the pathological level. In this preliminary translational research study we analysed the gene expression of structure proteins and inflammatory mediators as well as histological features of lumbar intervertebral discs in symptomatic patients with various signs of degeneration in the MRI. METHODS: Specimens of intervertebral disc tissue were obtained from 20 patients undergoing lumbar nucleotomy. Preoperatively, a group selection based on four pre-defined MRI-criteria was performed: Group 1 (mild signs of degeneration), group 2 (moderate), group 3 (moderate-severe), group 4 (severe). RESULTS: An increase of the expression of structural proteins and inflammatory markers could be observed in MRI-groups 2 and 3. Gene expression of collagen type I and II and aggrecan went along with levels of cyclooxygenase-2 (COX-2) and (fibroblast growth factor-2) FGF-2 expression. Histological examination showed signs of granulation tissue in only 35% of cases, but no differences between the groups. CONCLUSION: Our findings implicate that the gene expression of structural proteins might correlate with the appearance of inflammatory mediators in symptomatic patients with moderate disc changes in the MRI in this preliminary clinical subset. The assessment of cell activity and protein expression in a larger number of patients could be next step to support and supplement the present data.

Human intervertebral disc-derived cells are recruited by human serum and form nucleus pulposus-like tissue upon stimulation with TGF-beta3 or hyaluronan in vitro.

The aims of this work were to test whether human intervertebral disc-derived nucleus pulposus cells (hNP-cells) are attracted by human serum and to analyze if matrix generation from hNP-cells is promoted under the influence of transforming growth factor-beta3 (TGF-beta3) or hyaluronan (HA) in vitro. Using the multi-well chemotaxis assay to determine cell migration under the influence of different concentrations of human serum, it was demonstrated that dedifferentiated hNP-cells are able to migrate towards a serum fraction gradient in a concentration-dependent manner. Re-differentiation capacity of hNP-cells in 3D micro-masses under the influence of TGF-beta3 or hyaluronan was also tested. Gene expression analysis of types I, II, III and IX collagen, as well as aggrecan, COMP and LINK of hNP-cells in 3D-micro-mass cell-culture revealed a strong increase of these markers in TGF-beta3 treated cells. Furthermore, histochemical and immuno-histochemical staining after 28d showed proteoglycan and type II collagen-rich matrix for both, the TGF-beta3 and the hyaluronan treated cells. These findings show that TGF-beta3 or hyaluronan are able to induce the differentiation and that human serum stimulates the migration of hNP-cells in vitro. Therefore, hyaluronan and serum are suited for cell-free biomaterials as cell migration and differentiation inducing factors intended for biological treatment strategies of the intervertebral disc.

Murine pre-eclampsia induced by unspecific activation of the immune system correlates with alterations in the eNOS and AT1 receptor expression in the kidneys and placenta.

It remains arguable if an animal model can be of use in pre-eclampsia (PE) studies, as it is clearly a human disease not observed spontaneously in other species. The aim of this study was to investigate whether PE-like signs in mice inoculated with activated Th1 cells were accompanied by abnormal expression of molecules related to the regulation of blood pressure, viz. nitric oxide synthase enzymes (eNOS and iNOS) and angiotensin (Ang) II receptors (AT1R and AT2R), in order to analyse the relevance of this model for human disease. In this model, C57/BL6-mated BALB/c females received lymphocytes crosslined with anti-CD3 and cultured with interleukin (IL)-2 and IL-12 to mimic PE pathology. Control mice received PBS. eNOS, iNOS and AT1R but not AT2R expression was augmented in the kidneys of PE-mice compared with control pregnant mice. The expression of eNOS but not of iNOS was augmented at the fetal-maternal interface of PE-mice as compared with the controls. NOSs regulate the synthesis of NO, a blood pressure and parturition mediator. As its expression is increased in PE patients, our data suggest that the Th1 cells-induced signs in this model are due to similar mechanisms as in humans. AT1R and AT2R mediate the effect of Ang II, and particularly the AT1R appears to be involved in the pathogenesis of human PE. The increased AT1R expression in the kidneys of PE-mice reinforces the theory that Th1 cells elicit a pathological situation closely resembling the human PE. All together, our data support the use of this animal model to study mechanisms underlying clinically overt PE.

Upregulation of Bcl-2 at the foetal-maternal interface from mice undergoing abortion.

Several burning questions remain unanswered in pregnancy-related research. Pro- and anti-inflammatory cytokines orchestrate an intriguing interaction leading either to the development of a normal individual or to its rejection. Augmented Th1 cytokines' production is involved in immunological rejection of the foetus. Excessive production of Th1 cytokines, particularly of tumour necrosis factor (TNF)-alpha, also triggers apoptosis. Thus, in the present work we investigated the incidence of apoptosis in a well-known experimental model of Th1-induced abortion, characterized by increased local TNF-alpha levels. Apoptosis of lymphocytes as well as their Th1 and Th2 cytokine production were analysed by flow cytometry. TNF-alpha mRNA levels were additionally analysed by real time reverse transcription-polymerase chain reaction (RT-PCR) in placental and decidual samples. Total placental apoptosis activity was investigated by measuring caspase-3 activity and by TdT-mediated dUTP nick end label staining. Immunohistochemistry, Western blot and real time RT-PCR were used to localize and quantify several anti- and pro-apoptotic molecules at the foetal-maternal interface. Despite elevated Th1 levels at the foetal-maternal interface, mice undergoing abortion presented comparable apoptotic rates. Interestingly, we found a significant upregulation of the anti-apoptotic Bcl-2 protein at the foetal-maternal interface from abortion-prone mice, while no changes could be observed for pro-apoptotic molecules. In the light of our results, we conclude that there is no evidence of increased apoptosis in mice undergoing immunological abortion in spite of elevated TNF-alpha levels. This is probably due to a selective upregulation of anti-apoptotic pathways (i.e. Bcl-2) at the foetal-maternal interface as a compensatory and/or protective mechanism.