Leo De Ridder

Isolation, proliferation and differentiation of osteoblastic cells to study cell/biomaterial interactions: comparison of different isolation techniques and source

A sufficient amount of easily obtained and well-characterized osteoblastic cells is a useful tool to study biomaterial/cell interactions essential for bone tissue engineering. Osteoblastic cells were derived from adult and fetal rat via different isolation techniques. The isolation and in vitro proliferation of primary cultures were compared. The osteogenic potential of subcultures was studied by culturing them in osteogenic medium and compared with respect to alkaline phosphatase activity, nodule formation and mineralization potential. Calvaria cells were easier to obtain and the amount of cells released by enzymatic isolation was higher than for the long bone cells. The expansion of the cells in primary culture was highest for fetal calvaria cells compared to fetal and adult long bone cells. All cultures expressed high alkaline phosphatase activity except for calvaria cells obtained by spontaneous outgrowth. Enzymatic isolation of fetal calvaria and long bone cells favoured the osteogenic differentiation. Enzymatically isolated calvaria cells formed well-defined three-dimensional nodules which mineralized restricted to this area. On the contrary, cultures derived from fetal as well as adult long bones mineralized in ill-defined deposits throughout the culture and only formed occasionally nodular-like structures. The mineral phase of all osteoblastic cultures was identified as a carbonate-containing apatite. The present study demonstrates that considering the isolation method, proliferation capacity and the osteogenic potential, the enzymatically released fetal calvaria cells are most satisfactory to study cell/biomaterial interactions.

Is the root entry/exit zone important in microvascular compression syndromes?

OBJECTIVE Microvascular compression syndromes such as trigeminal neuralgia, hemifacial spasm, and disabling positional vertigo involve an artery or vein compressing a cranial nerve. A cranial nerve is composed of a central nervous system (CNS) segment and a peripheral nervous system (PNS) segment separated by the root entry/exit zone (REZ). Although vascular compression can occur at any point along the cranial nerve, it has been generally assumed that only vascular contact at the REZ of the affected cranial nerve can cause symptoms. On the basis of personal surgical experience, we propose that vascular compression of the CNS segment alone causes symptoms. This has important repercussions for the future diagnosis and treatment of microvascular compression syndromes, especially the cochleovestibular compression syndrome. METHODS For the anatomic study, four autopsy specimens and one surgical biopsy specimen of the vestibulocochlear nerve were microscopically and ultramicroscopically analyzed for structural differences between the CNS and PNS segments. For the clinical study, five patients with the clinical picture of cochleovestibular compression syndrome were treated by microsurgical decompression at the level of the CNS segment and not the REZ. One patient underwent reoperation for recurrent symptoms 4 years later, and a 4-mm vestibular neurectomy was performed at that stage. We performed an epidemiological analysis to demonstrate that the known incidences of trigeminal neuralgia, hemifacial spasm, and glossopharyngeal neuralgia are related to the length of their respective CNS segments. RESULTS Histological differences between the PNS and CNS segments suggest that the PNS segment is more resistant to compression. This was confirmed by neurophysiological data from intraoperative monitoring in posterior fossa surgery and experimental studies. We found a clear epidemiological correlation between the length of the CNS segment, which differed among cranial nerves, and the incidence of the microvascular compression syndrome. Successful decompression of the CNS segment in patients without compression at the REZ of the vestibulocochlear nerve for disabling positional vertigo provides clinical support for this hypothesis. CONCLUSION The evidence we present supports the hypothesis that vascular compression syndromes arise from vascular contact along the CNS segment of the cranial nerves.

Quantification of apoptosis in lymphocyte subsets and effect of apoptosis on apparent expression of membrane antigens.

Annexin V binding to phosphatidylserine was evaluated by flow cytometry to examine apoptosis in different lymphocyte subsets of peripheral blood mononuclear cells after a 24 h in vitro culture period. We also applied a 2 Gy dose gamma-irradiation prior to incubation to evaluate the additional apoptogenic effect of radiation on the lymphocyte subsets. Overall, B lymphocytes showed the highest number of apoptotic cells, followed by T lymphocytes. Within the T lymphocytes, CD4-positive and CD45RA-negative cells were more prone to apoptosis than the CD8-positive and CD45RA-positive cells. Natural killer cells turned out to be most apoptosis-resistant. In the irradiated samples about twice as many apoptotic cells were found and the differences between lymphocyte subpopulations remained. Backgating of the annexin V-positive cells showed that these cells had a clearly decreased forward scatter signal. The antibody binding capacity (ABC) of lymphocyte membrane antigens was determined with CD3-fluorescein isothiocyanate (FITC), CD45RA-FITC, CD4-phycoerythrin (PE), CD8-PE, CD56-PE, and CD20-PE in viable and apoptotic cells. In the apoptotic cells a decrease of ABC was found for all antigens, except for CD20. There was no significant cell loss in the cultures. We conclude that the change in scatter and in ABC must be considered in immunophenotyping experiments on cells kept in culture for 24 h. If these changes are taken into account, percentages of subpopulations or the numbers of cells that stain positive for the studied markers do not significantly change.

Invasiveness of primary brain tumors

SummaryPrimary malignant neoplasms of the nervous system differ from other types of malignancy in several ways. Clinical progression is due to local invasive growth, while metastases outside the skull are rare. The tumors show no sharp delimitation from the surrounding normal tissue. At the edge, an ill-defined area of invasive tumor cells, reacting glial cells and inflammatory cells is present. At the same time the primary brain tumors are biologically heterogeneous.In this review, a short survey of markers for malignancy in primary brain tumors is given, and some properties of importance for invasive behavior, are listed. These include different cellular enzymes, phagocytotic property, locomotive and proliferative characteristics.Studies of primary brain tumors in situ show invasive growth into the surrounding brain tissue, often tollowed by hemorrhage and necrosis. In addition spread of tumor cells takes place along preexisting intracranial structures. Recently, several systems for the study of brain tumor invasiveness in culture have been elaborated. Both experimental and human gliomas have been tested. The target tissues include organ culture of embryonic chick heart muscle, chorioallantoic membrane, fetal rat brain tissue and reconstructed vessel walls. It has been shown that glioma cells are able to split junctions between normal cells. They destroy and phagocytose the normal cells and penetrate the normal tissue. The use of brain tissue and reaggregated brain cell cultures as target for glioma cells in culture opens the possibility for an elucidation of invasiveness as one of the most important properties of malignancy in the nervous system.

Telomerase activity and expression of telomerase reverse transcriptase correlated with cell proliferation in meningiomas and malignant brain tumors in vivo

Abstract. Telomerase activity was examined in intracranial tumors and compared with gene expression of the two core components of telomerase – the reverse transcriptase subunit (hTERT) and the RNA subunit (hTR) – and the proliferative index. We investigated 32 tumors across three to five sampled regions (20 meningiomas, 1 acoustic schwannoma, 1 pituitary adenoma, 8 gliomas, and 2 medulloblastomas). Telomerase activity was demonstrated by the telomeric repeat amplification protocol (TRAP) assay in seven (22%) intracranial tumors (four malignant brain tumors, two atypical meningiomas and one ependymoma) but could not be detected in the 18 (100%) benign meningiomas. hTERT and hTR mRNA were detected using reverse-transcription polymerase chain reaction (RT-PCR). hTERT mRNA was present in 20 (63%) intracranial tumors. Whereas hTERT mRNA transcripts were consistently low or absent in meningiomas, malignant brain tumors exhibited elevated hTERT mRNAs. Multiple regions of glioblastomas showed differences in telomerase activity and in the presence of hTERT mRNA. RT-PCR analysis revealed, for the first time in intracranial tumors, the presence of hTERT mRNA spliced products, corresponding to full-length mRNA as well as spliced mRNAs with critical reverse transcriptase motifs deleted. Only tumors with marked telomerase activity showed all hTERT spliced messages simultaneously. The absence of a positive correlation between telomerase activity and hTERT mRNA could not be attributed to the presence of hTERT spliced variants. We found a significant correlation between telomerase activity scores and Ki-67 proliferation index. A positive association is also seen between Ki-67 staining and the degree of hTERT mRNA expression. This shows that there seems to be a relationship between telomerase activity or the degree of hTERT expression and proliferation rate in intracranial tumors.

Pulsatile tinnitus and the intrameatal vascular loop: why do we not hear our carotids?

OBJECTIVE: Pulsatile tinnitus is characterized by hearing the heart beat or respiration in one or both ears. In 15% of patients with pulsatile tinnitus, no cause can be found. Other investigators have suggested that a vascular loop entering the internal auditory meatus can be another cause of arterial, pulse synchronous tinnitus. If so, we should constantly hear the arterial pulsations of the carotid arteries passing through the petrous bone. METHODS: Using magnetic resonance imaging, 17 patients with unilateral pulsatile tinnitus and 46 with non-pulsatile tinnitus were analyzed for the presence of a vascular loop entering into the internal acoustic meatus. Four temporal bones were sectioned to find structural differences between the internal acoustic meatus and the pericarotid area. Four patients with intrameatal vascular loops and ipsilateral pulsatile tinnitus underwent surgery by Teflon interpositioning between the loop and the cochlea. RESULTS: In unilateral pulsatile tinnitus, a statistically highly significant amount of intrameatal vascular loops was noted in comparison to non-pulsatile tinnitus. A well-developed pericarotid venous plexus was found histologically. Three of the four patients who underwent surgery were initially tinnitus free, but pulsations recurred after 3 months in one patient. CONCLUSION: Vascular loops in the internal auditory canal may generate pulsatile tinnitus. It may be treated by placing Teflon between the cochlea and the intrameatal vascular loop. One then does not hear the pulsation of the carotids due to a dampening effect of a pericarotid venous plexus.

Failure of nimodipine to prevent brain damage in a global brain ischemia model in the rat

In view of our negative results with the calcium antagonist nimodipine as a cerebroprotective agent in a cardiopulmonary resuscitation model in the rat, we examined the protective effects of nimodipine in the four-vessel (carotid and vertebral) occlusion model, a model of global brain ischemia without important cardiovascular depression. Survival and neurological status were monitored and after 72 h the hippocampus was resected and examined for histological evaluation. The animals were treated blindly and randomly with either nimodipine, its solvent or saline given subcutaneously. In two separate studies, high doses (total dose: 5 mg/kg) or low doses of nimodipine (total dose: 1.6 mg/kg) were administered. In the high dose series, the survival rates at 72 h in the nimodipine group, the saline group and the solvent group were 4% (2/44), 19% (7/37) and 20% (8/41), respectively; in the low dose series, the figures were 26% (13/50), 34% (15/44) and 39% (18/46), respectively. The differences between nimodipine, solvent and saline were not statistically significant. Likewise, no differences in neurological status or histological brain damage were found. These data suggest that nimodipine offers no cerebral protection in global brain ischemia and may even be toxic, especially when given in high doses.

Invasiveness of primary and secondary brain tumors in vitro correlated with clinical results

One hundred fifty-one tumor fragments were collected in the neurosurgical operating amphitheater immediately after removal. Small tumor fragments were transferred into culture flasks and cultured until a confluent monolayer was formed by the outgrowing cells. Flaps of these cell monolayers were mechanically scraped from the culture flasks and confronted with embryonic chick heart tissue in vitro. The evolution of the confrontations was followed for a week. Histological analysis of the confrontations demonstrated three different morphological patterns of interaction between the heart tissue and the tumor-derived cells: 1) progressive engulfment of the tumor-derived cells by the heart tissue (Type I), 2) survival of both the heart tissue and the tumor-derived cells (Type II), and 3) progressive replacement of the heart tissue by tumor-derived cells (Type III). The replacement of the heart tissue by tumor-derived cells was only observed in cells originating from malignant tumors that were invasive and metastatic in vivo. Thus, invasiveness in confrontation culture is correlated with malignancy in vivo.

Effects of Ionizing Radiation on the Metabolism and Longitudinal Growth of Cartilaginous Embryonic Chick Tibiae in Vitro

The effect of ionizing radiation on the metabolism and longitudinal growth of cartilaginous tibiae of 6.5-day-old chick embryos was studied in vitro over a 3-day period. Before being cultured, tibiae received absorbed doses of 2 to 200 Gy. Of each pair, the counterpart served as control. Compared to the strong inhibition of [3H]thymidine incorporation, already 50 percent at 10 Gy, the effects of ionizing radiation on [3H]uridine and [3H]proline incorporation were limited: 20 and 40 percent respectively at 150 Gy. Metabolism of the cartilage cells in our organ culture was almost completely arrested at 200 Gy. Light and electron microscopy showed no morphological differences between irradiated and sham-irradiated tibiae until 150 Gy. At 200 Gy necrosis of most of the cells was observed. No differences in form and arrangement of extracellular fibers were noticed. The results of the metabolic studies and the morphological observations were correlated with the effects of ionizing radiation on the longitudinal growth. In contrast to DNA synthesis, RNA transcription and synthesis of collagen fibres were radioresistant processes.

Micronucleus assay reveals no radiation effects among nuclear power plant workers.

The micronucleus assay was applied as biomarker for exposure effect and radiosensitivity in a group of 99 radiation workers of the Nuclear Power Plant Doel (Belgium). The difference in micronucleus frequency between the group of radiation workers with annual dose exceeding 2 mSv and a non-exposed control population was statistically not significant. With respect to the micronucleus frequency after an in vitro challenge dose of 2 Gy, which can be considered as biomarker for radiosensitivity, the data of present study can be represented by a normal distribution without a high frequency tail. This means that a subpopulation of workers with elevated radiosensitivity could not be identified in the population under study.