Ceramic Implant Rehabilitation: Consensus Statements from Joint Congress for Ceramic Implantology.

The objectives of the study group focused on the following main topics related to the performance of one- and two-piece ceramic implants: defining bone-implant-contact percentages and its measurement methods, evaluating the pink esthetic score as an esthetic outcome parameter after immediate implantation, recognizing the different results of ceramic implant designs, as redefined by the German Association of Oral Implantology, incorporating the patient report outcome measure to include satisfaction and improvement in oral health-related quality of life, and conducting preclinical studies to address existing gaps in ceramic implants. During the Joint Congress for Ceramic Implantology (2022), the study group evaluated 17 clinical trials published between 2015 and 2021. After extensive discussions and multiple closed sessions, consensus statements and recommendations were developed, incorporating all approved modifications. A one-piece implant design features a coronal part that is fused to the implant body or interfaces with the post-abutment restoration platform, undergoing transmucosal healing. Long-term evaluations of this implant design have been supported by established favorable clinical evidence. Inaccuracies in the pink esthetic score and bone-implant-contact percentages were managed by establishing control groups for preclinical studies and randomizing clinical trials. The patient-reported outcome measures were adjusted to include an individual visual analog scale, collected from each clinical study, that quantified improved oral health and quality of life. Preclinical investigations should focus on examining the spread of ceramic debris and the impact of heat generation on tissue and cellular levels during drilling. Further technical advancements should prioritize wound management and developing safe drilling protocols.

Recombination hotspot in NF1 microdeletion patients.

Neurofibromatosis type 1 (NF1) patients that are heterozygous for an NF1 microdeletion are remarkable for an early age at onset and an excessive burden of dermal neurofibromas. Microdeletions are predominantly maternal in origin and arise by unequal crossover between misaligned NF1REP paralogous sequence blocks which flank the NF1 gene. We mapped and sequenced the breakpoints in several patients and designed primers within each paralog to specifically amplify a 3.4 kb deletion junction fragment. This assay amplified a deletion junction fragment from 25 of the 54 unrelated NF1 microdeletion patients screened. Sequence analysis demonstrated that each of the 25 recombination events occurred in a discrete 2 kb recombination hotspot within each of the flanking NF1REPs. Two recombination events were accompanied by apparent gene conversion. A search for recombination-prone motifs revealed a chi-like sequence; however, it is unknown whether this element stimulates recombination to occur at the hotspot. The deletion-junction assay will facilitate the prospective identification of patients with NF1 microdeletion at this hotspot for genotype-phenotype correlation studies and diagnostic evaluation.

Neuregulin signaling through a PI3K/Akt/Bad pathway in Schwann cell survival.

beta-Neuregulin (betaNRG) is a potent Schwann cell survival factor that binds to and activates a heterodimeric ErbB2/ErbB3 receptor complex. We found that NRG receptor signaling rapidly activated phosphoinositide 3-kinase (PI3K) in serum-starved Schwann cells, while PI3K inhibitors markedly exacerbated apoptosis and completely blocked NRG-mediated rescue. NRG also rapidly signaled the phosphorylation of mitogen-activated protein kinase (MAPK) and the serine/threonine kinase Akt. The activation of Akt and MAPK in parallel pathways downstream from PI3K resulted in the phosphorylation of Bad at different serine residues. PI3K inhibitors that blocked NRG-mediated rescue also blocked the phosphorylation of Akt, MAPK, and Bad. However, selective inhibition of MEK-dependent Bad phosphorylation downstream from PI3K had no effect on NRG-mediated survival. Conversely, ectopic expression of wild-type Akt not only enhanced Bad phosphorylation but also enhanced autocrine- and NRG-mediated Schwann cell survival. Taken together, these results demonstrate that NRG receptor signaling through a PI3K/Akt/Bad pathway functions in Schwann cell survival.

Schwann cell-conditioned medium inhibits angiogenesis in vitro and in vivo.

BACKGROUND: Neuroblastomas are biologically heterogeneous tumors that consist of two main cell populations: neuroblastic/ganglionic cells and Schwann cells. The amount of Schwannian stroma strongly impacts prognosis. Low tumor vascularity, localized stage, and favorable outcome are associated with tumors that are Schwannian stroma-rich/stroma-dominant. PROCEDURE: To investigate if Schwann cells play a role in inhibiting angiogenesis in neuroblastoma tumors, we examined the ability of human Schwann cell-conditioned medium to affect bFGF- and VEGF-induced endothelial cell proliferation and migration, and in vivo angiogenesis. RESULTS: Schwann cell-conditioned medium significantly inhibited bFGF- and VEGF-induced endothelial cell proliferation and migration. This effect appears to be specific for endothelial cells as smooth muscle cell and fibroblast proliferation were not inhibited by this medium. Schwann cell-conditioned medium also inhibited in vivo angiogenesis in rat corneal assays. CONCLUSIONS: Schwann cells produce a potent inhibitor(s) of angiogenesis that may be responsible for the low level of vascularity and more benign clinical behavior of Schwannian stroma-rich/stroma-dominant neuroblastoma tumors. Studies to identify the inhibitor(s) are ongoing.

Schwann cell-conditioned medium inhibits angiogenesis.

Neuroblastomas are biologically heterogeneous tumors that consist of two main cell populations: neuroblastic/ganglionic cells and Schwann cells. The amount of Schwannian stroma strongly impacts prognosis, and favorable outcome is associated with tumors that are Schwannian stroma rich/stroma dominant. At the present time, there is controversy regarding the origin of Schwann cells in neuroblastoma tumors. However, recent studies have suggested that the Schwann cells in mature neuroblastoma tumors may be normal cells that produce soluble substances that enhance the survival and differentiation of neuroblastoma cell lines. Previously, we reported that in neuroblastoma, high vascular index correlated with clinically aggressive disease. In contrast, tumors with favorable histology and abundant Schwannian stroma had low tumor vascularity. As a first step toward investigating whether Schwann cells also play a role in inhibiting angiogenesis in neuroblastoma tumors, we examined the ability of conditioned medium collected from normal human Schwann cells to affect basic fibroblast growth factor- and vascular endothelial growth factor-induced endothelial cell proliferation and migration and in vivo angiogenesis. In vitro angiogenesis assays were also performed with conditioned medium collected from Schwann cells derived from a Schwannian stroma-dominant neuroblastoma tumor. Our results indicate that Schwann cells derived from either adult nerve or tumor tissue produce a potent inhibitor(s) of angiogenesis. Expression studies revealed tissue inhibitor of metalloproteinase (TIMP)-2 in conditioned medium collected from both normal and tumor-derived Schwann cells. In addition, TIMP-2 was detected in the cytoplasm of Schwann cells and ganglion cells in stroma-rich/stroma-dominant neuroblastoma tumors by immunohistochemistry studies. We postulate that the low level of vascularity and more benign clinical behavior of Schwannian stroma-rich/stroma-dominant neuroblastoma tumors result from the Schwann cell production of TIMP-2 and/or other inhibitors of angiogenesis.

Genetic and cellular defects contributing to benign tumor formation in neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a common inherited cancer predisposition syndrome. The NF1 gene product, neurofibromin, is hypothesized to function as a tumor suppressor and nearly all NF1 patients develop benign peripheral nerve tumors. These neurofibromas presumably arise from NF1 inactivation in S100(+)Schwann cells, but there is no formal proof for this mechanism. We demonstrate that fibro-blasts isolated from neurofibromas carried at least one normal NF1 allele and expressed both NF1 mRNA and protein, whereas the S100(+)cells typically lacked the NF1 transcript. Our findings further indicate that additional molecular events aside from NF1 inactivation in Schwann cells and/or other neural crest derivatives contribute to neurofibroma formation.

Signals for proinflammatory cytokine secretion by human Schwann cells.

Wallerian degeneration is a post-traumatic process of the peripheral nervous system whereby damaged axons and their surrounding myelin sheaths are phagocytosed by infiltrating leukocytes. Our studies indicate that Schwann cells could initiate the process of Wallerian degeneration by releasing proinflammatory cytokines involved in leukocyte recruitment and differentiation including IL-1beta, MCP-1, IL-8 and IL-6. A comparison of the secretory pattern between nerve explants and cultured Schwann cells showed that each cytokine was differentially regulated by growth factor deprivation or axonal membrane fragments. Since Wallerian-like degeneration occurs in a wide variety of peripheral neuropathies, Schwann cell-mediated cytokine production may play an important role in many disease processes.

Germline mutations in NF1 patients with malignancies.

We have analyzed 98.5% of the coding region of the NF1 gene at the cDNA level in seven NF1 patients who developed malignant peripheral nerve sheath tumors. Seven germline mutations were detected in six individuals: a 6-bp in-frame deletion in exon 28, a splice acceptor mutation in intron 31 resulting in a premature stop of translation, a missense mutation in exon 38, and three total NF1 gene deletions. In one of the patients with a total NF1 gene deletion, a missense mutation in exon 16 on the other NF1 allele was detected. These data indicate that NF1 patients developing malignant neoplasms can have any type of NF1 germline mutation such as a total gene deletion, a frameshift mutation, an in-frame deletion, or a missense mutation. We conclude that in our series no specific type of NF1 germline mutation was found in NF1 individuals with malignancies, but that large NF1 gene deletions were more frequently found in this group than reported for the general population of NF1 individuals. Genes Chromosomes Cancer 26:376-380, 1999.

Molecular studies in 20 submicroscopic neurofibromatosis type 1 gene deletions.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder characterized by a marked variability in expression. A more severe phenotype is frequently observed in the group of patients carrying a large NF1 deletion. To study the extent of the microdeletion in these NF1 patients, we generated a partial physical map of the NF1 flanking region. We describe seven PACs and three new polymorphic dinucleotide repeats located outside the NF1 gene and analyzed 20 unrelated individuals with an NF1 microdeletion in a collaborative study. We detected one individual with a substantially smaller deletion including only the NF1 gene and its three embedded genes. In the other 19 patients, the deletion extended at least 1 Mb. The parental origin of the deletion was determined in 15 individuals and was maternal in 13 and paternal in two cases. The new molecular tools described here can be used to unequivocally diagnose a possible extragenic extension of an NF1 deletion.

Reciprocal Id expression and myelin gene regulation in Schwann cells.

Id proteins are thought to act as dominant negative antagonists of basic helix-loop-helix (bHLH) transcription factors that direct differentiation in various cell types. We found that Schwann cells express all four Id-family genes and that their transcript levels were reciprocally regulated in pairs during nerve maturation in vivo and cAMP-mediated differentiation in vitro. The rapid induction as part of the early response to axonal membranes and cytokines suggested that Id3 is involved in myelin gene repression. An inverse relationship between Id1/3 and myelin P0 expression was consistent with a role for these two Id proteins as inhibitors of differentiation, and Id1/3 proteins strongly repressed myelin gene promoter activity. Nuclear factors isolated from Schwann cells and intact sciatic nerves were found to bind three different HLH recognition sequences (E boxes) in the proximal region of the P0 promoter, and production of these DNA binding complexes was altered during differentiation. These data support the concept that Id proteins regulate myelin gene expression by controlling the formation of specific bHLH DNA binding complexes with different E-box preferences.

Schwann cell-conditioned medium promotes neuroblastoma survival and differentiation.

Neuroblastomas are histopathologically heterogeneous, ranging from immature malignant tumors to benign ganglioneuromas. The amount of Schwann cell stroma greatly increases with neuroblastoma differentiation, and these Schwann cells appear to be normal cells that infiltrate the tumor. To determine whether Schwann cells influence neuroblast differentiation, four human neuroblastoma cell lines were cultured in the presence or absence of human Schwann cell-conditioned medium for 7 days. Neuroblastoma cell survival, as determined by a colorimetric assay, more than doubled in three of the four neuroblastoma cell lines in the Schwann cell-conditioned medium. There was a corresponding reduction in apoptosis as measured by a nick-end labeling assay, with little change in mitotic rate. Schwann cell-conditioned medium induced extensive neurite outgrowth in all of the neuroblastoma cell lines, and these processes contained mature neurofilament in three of the cell lines. These results indicate that Schwann cells produce soluble substances capable of supporting survival and differentiation in neuroblastoma cell lines. This may represent a biological mechanism responsible for neuronal differentiation in stroma-rich neuroblastomas.

A role for Pak protein kinases in Schwann cell transformation.

Neurofibromatosis type 1 (NF1), a common autosomal dominant disorder caused by loss of the NF1 gene, is characterized clinically by neurofibromas and more rarely by neurofibrosarcomas. Neurofibromin, the protein encoded by NF1, possesses an intrinsic GTPase accelerating activity for the Ras proto-oncogene. Through this activity, it is a negative regulator of Ras. The Pak protein kinase is a candidate for a downstream signaling protein that may mediate Ras signals because it is activated by Rac and Cdc42, two small G proteins required for Ras signaling. Here, we use Pak mutants to explore the role of Pak in Ras signaling in Schwann cells, the cells affected in NF1. Whereas an activated Pak mutant does not transform cells, dominant negative Pak mutants are potent inhibitors of Ras transformation of rat Schwann cells and of a neurofibrosarcoma cell line from an NF1 patient. Although activated Pak stimulated jun-N-terminal kinase, inhibition of Ras transformation by dominant negative Pak did not require inhibition of jun-N-terminal kinase. Instead, the Pak mutants appeared to inhibit transformation by preventing Ras activation of the ERK/mitogen-activated protein kinase cascade. These results have implications for our understanding of NF1 because a neurofibrosarcoma cell line derived from a patient with NF1 was reverted by stable expression of the Pak dominant negative mutants.

RalGDS functions in Ras- and cAMP-mediated growth stimulation.

Thyroid-stimulating hormone stimulates proliferation through both the cAMP-dependent protein kinase and Ras but not through Raf-1 and mitogen-activated and extracellular signal-related kinase kinase. We now report that thyroid-stimulating hormone represses mitogen-activated protein kinase activity and that microinjection of an effector domain mutant Ha-Ras protein, Ras(12V,37G), defective in Raf-1 binding and mitogen-activated protein kinase activation, stimulates DNA synthesis in quiescent and thyroid-stimulating hormone-treated thyrocytes. A yeast two-hybrid screen identified RalGDS as a Ras(12V,37G) binding protein and therefore a potential effector of Ras in these cells. Associations between Ras and RalGDS were observed in extracts prepared from thyroid cells. Microinjection of a mutant RalA(28N) protein thought to sequester RalGDS family members reduced DNA synthesis stimulated by Ras as well as cAMP-mediated DNA synthesis in two cell lines which respond to cAMP with mitogenesis. These results support the idea that RalGDS may be an effector of Ras in cAMP-mediated growth stimulation.

Interleukin 12: a potential link between nerve cells and the immune response in inflammatory disorders.

BACKGROUND: The nervous system has been implicated in several inflammatory skin disorders based on evidence such as the role of stress in inducing lesions, symmetry of lesions, and sparing of denervated skin. Interleukin 12 (IL-12) is a cytokine recently shown to promote cellular immune responses characterized by delayed-type hypersensitivity and production of the TH1-lymphokine, interferon-gamma. MATERIALS AND METHODS: Using immunohistochemistry, IL-12 immunoreactivity was identified in cryostat sections of normal and diseased human skin samples, and in the peripheral and central nervous system of rodents and human tissue samples. IL-12 p35 and p40 mRNAs were detected using reverse transcriptase-polymerase chain reaction in tissue samples and cultured cells. IL-12 protein levels were also examined by ELISA and quantitative bioassay utilizing an IL-12-dependent cell line. RESULTS: By immunostaining IL-12 was detected in free nerve ending in the epidermis of normal and diseased skin samples, and also in the dermal nerve fibers. Strong reactivity was detected in axonal processes and in various glial cell types. In addition, IL-12 protein and mRNA were contained within cutaneous peripheral nerves and spinal cord tissues, and functional levels of IL-12 were produced by cultured Schwann cells. CONCLUSIONS: It is likely that IL-12 is important in initiating or propagating selected inflammatory skin lesions and in determining the pattern of disease that will develop. The presence of IL-12 in neural tissue suggests a mechanism whereby the nervous system can modify or amplify cutaneous and perhaps other immune responses.

The zinc finger transcription factor Zif268/Egr-1 is essential for Schwann cell expression of the p75 NGF receptor.

Nerve injury alters the function of Schwann cells from quiescent, myelin forming cells to proliferating cells that facilitate nerve repair. The transcription factor, Zif268, may be involved in transmitting injury-related signals since its expression is rapidly induced by nerve transection in vivo and without intervening protein synthesis by injury-related signals in vitro. Expression of the low-affinity p75 nerve growth factor receptor (NGFRp75) by Schwann cells after nerve injury closely correlated with the zif268 expression profile, and Zif268 transactivated the NGFRp75 promoter in transient transfection assays. Conversely, the NGFRp75 gene was not expressed when Zif268 protein was depleted by stable transfection of antisense cDNA. Moreover, nuclear proteins corresponding to Zif268 bound to the NGFRp75 promoter by Southwestern blotting, indicating that a direct interaction of Zif268 with the NGFR gene is required for its expression in Schwann cells.

Purification and expansion of human Schwann cells in vitro.

The ability to culture cells from the human nervous system provides new insight into the pathophysiology of neurological diseases and could be crucial to the development of gene replacement therapies and neural transplantation. We report that the proliferation of human Schwann cells isolated from paediatric and adult nerves is sustained in vitro by recombinant glial growth factor. Agents that increase intracellular cyclic cAMP were also mitogenic towards Schwann cells but suppress growth of contaminating fibroblasts. As the lifespan of highly enriched cultures can be extended for up to twelve population doublings, large numbers of cells can be generated from nerve biopsies.

Lack of NF1 expression in a sporadic schwannoma from a patient without neurofibromatosis.

The neurofibromatosis type 1 (NF1) gene encodes a tumor suppressor protein, neurofibromin, which is expressed at high levels in Schwann cells and other adult tissues. Loss of NF1 expression has been reported in Schwann cell tumors (neurofibrosarcomas) from patients with NF1 and its loss is associated with increased proliferation of these cells. In this report, we describe downregulation of NF1 expression in a single spinal schwannoma from an individual without clinical features of neurofibromatosis type 1 or 2. Barely detectable expression of NF1 RNA was found in this tumor by in situ hybridization using an NF1-specific riboprobe as well as by Northern blot and reverse-transcribed (RT)-PCR analysis. In Schwann cells cultured from this schwannoma, abundant expression of NF1 RNA could be detected by Northern blot and RT-PCR analysis. These results suggest that, in some tumors, expression of NF1 may be downregulated by factors produced within the tumor and may represent a novel mechanism for inactivating these growth suppressing genes and allowing for increased cell proliferation in tumors.

Sources of human Schwann cells and the influence of donor age.

We evaluated several tissues as possible sources for culturing human Schwann cells. The average cell yield (total cell number/mg of nerve fascicle) obtained from adult autopsy cases and transplant organ donors was similar (2 x 10(4) and 2.9 x 10(4), respectively), but significantly higher yields were obtained from dorsal roots of pediatric patients undergoing selective dorsal rhizotomy (6.1 x 10(4)). Fresh tissue was not essential since cells isolated from 0 to 20 h postmortem were equally viable. However, we found evidence that donor age affects the intrinsic growth rate of Schwann cells and perineurial fibroblasts in culture.

Modulation of the neurofibromatosis type 1 gene product, neurofibromin, during Schwann cell differentiation.

Neurofibromin, the product of the neurofibromatosis type 1 (NF1) gene, is a approximately 250 kDa protein expressed predominantly in cortical neurons and oligodendrocytes in the central nervous system (CNS) and sensory neurons and Schwann cells in the peripheral nervous system (PNS). To gain insight into the biological role of neurofibromin in Schwann cells, the modulation of NF1 gene expression in a Schwann cell line (MT4H1) stimulated to either proliferate or differentiate in response to agents that elevate intracellular cAMP was examined. Untreated cells and cells exposed to mitogenic doses of forskolin (1-10 microM) or 8-bromo-cAMP (0.1 mM) expressed low levels of NF1 mRNA and the protein was barely detectable. High doses of forskolin (100 microM) or 8-bromo-cAMP (1 mM) induced the expression of both myelin P0 protein and neurofibromin with an identical time course. Although NF1 mRNA levels peaked within 1-6 hr, the rise in neurofibromin was not apparent until 24-48 hr and peaked 72 hr after treatment. P0 and neurofibromin were also coinduced by cell-cell contact in high density, untreated cultures. Moreover, differentiation initiated by either cAMP stimulation or high density culture conditions was associated with predominant expression of the type 2 NF1 mRNA isoform. In contrast, type 1 NF1 mRNA isoform expression was observed in untreated Schwann cells or those stimulated with mitogenic doses of forskolin or 8-bromo-cAMP. A switch from the type 1 neurofibromin that can efficiently downregulate p21-ras to the type 2 isoform with reduced activity may facilitate a p21-ras signaling pathway associated with Schwann cell differentiation.