Two-Photon Direct Laser Writing of 3D Scaffolds through C, H-Insertion Crosslinking in a One-Component Material System.

The popularity of two-photon direct laser writing in biological research is remarkable as this technique is capable of 3D fabrication of microstructures with unprecedented control, flexibility and precision. Nevertheless, potential impurities such as residual monomers and photoinitiators remaining unnoticed from the photopolymerization in the structures pose strong challenges for biological applications. Here, the first use of high-precision 3D microstructures fabricated from a one-component material system (without monomers and photoinitiators) as a 3D cell culture platform is demonstrated. The material system consists of prepolymers with built- in crosslinker motieties, requiring only aliphatic C, H units as reaction partners following two-photon excitation. The material is written by direct laser writing using two-photon processes in a solvent-free state, which enables the generation of structures at a rapid scan speed of up to 500 mm s-1 with feature sizes scaling down to few micrometers. The generated structures possess stiffnesses close to those of common tissue and demonstrate excellent biocompatibility and cellular adhesion without any additional modification. The demonstrated approach holds great promise for fabricating high-precision complex 3D cell culture scaffolds that are safe in biological environments.

On the Value of In Vitro Cell Systems for Mechanobiology from the Perspective of Yes-Associated Protein/Transcriptional Co-Activator with a PDZ-Binding Motif and Focal Adhesion Kinase and Their Involvement in Wound Healing, Cancer, Aging, and Senescence.

Mechanobiology comprises how cells perceive different mechanical stimuli and integrate them into a process called mechanotransduction; therefore, the related mechanosignaling cascades are generally important for biomedical research. The ongoing discovery of key molecules and the subsequent elucidation of their roles in mechanobiology are fundamental to understanding cell responses and tissue conditions, such as homeostasis, aging, senescence, wound healing, and cancer. Regarding the available literature on these topics, it becomes abundantly clear that in vitro cell systems from different species and tissues have been and are extremely valuable tools for enabling the discovery and functional elucidation of key mechanobiological players. Therefore, this review aims to discuss the significant contributions of in vitro cell systems to the identification and characterization of three such key players using the selected examples of yes-associated protein (YAP), its paralog transcriptional co-activator with a PDZ-binding motif (TAZ), and focal adhesion kinase (FAK) and their involvement in wound healing, cancer, aging, and senescence. In addition, the reader is given suggestions as to which future prospects emerge from the in vitro studies discussed herein and which research questions still remain open.

Molecular Research on Oral Diseases and Related Biomaterials: A Journey from Oral Cell Models to Advanced Regenerative Perspectives.

Oral diseases such as gingivitis, periodontitis, and oral cancer affect millions of people worldwide. Much research has been conducted to understand the pathogenetic mechanisms of these diseases and translate this knowledge into therapeutics. This review aims to take the reader on a journey from the initial molecular discoveries to complex regenerative issues in oral medicine. For this, a semi-systematic literature search was carried out in Medline and Web of Science databases to retrieve the primary literature describing oral cell models and biomaterial applications in oral regenerative medicine. First, an in vitro cell model of gingival keratinocytes is discussed, which illustrates patho- and physiologic principles in the context of oral epithelial homeostasis and carcinogenesis and represents a cellular tool to understand biomaterial-based approaches for periodontal tissue regeneration. Consequently, a layered gradient nonwoven (LGN) is described, which demonstrates that the key features of biomaterials serve as candidates for oral tissue regeneration. LGN supports proper tissue formation and obeys the important principles for molecular mechanotransduction. Furthermore, current biomaterial-based tissue regeneration trends, including polymer modifications, cell-based treatments, antimicrobial peptides and optogenetics, are introduced to represent the full spectrum of current approaches to oral disease mitigation and prevention. Altogether, this review is a foray through established and new concepts in oral regenerative medicine and illustrates the process of knowledge translation from basic molecular and cell biological research to future clinical applications.

Integrins, cadherins and channels in cartilage mechanotransduction: perspectives for future regeneration strategies.

Articular cartilage consists of hyaline cartilage, is a major constituent of the human musculoskeletal system and has critical functions in frictionless joint movement and articular homoeostasis. Osteoarthritis (OA) is an inflammatory disease of articular cartilage, which promotes joint degeneration. Although it affects millions of people, there are no satisfying therapies that address this disease at the molecular level. Therefore, tissue regeneration approaches aim at modifying chondrocyte biology to mitigate the consequences of OA. This requires appropriate biochemical and biophysical stimulation of cells. Regarding the latter, mechanotransduction of chondrocytes and their precursor cells has become increasingly important over the last few decades. Mechanotransduction is the transformation of external biophysical stimuli into intracellular biochemical signals, involving sensor molecules at the cell surface and intracellular signalling molecules, so-called mechano-sensors and -transducers. These signalling events determine cell behaviour. Mechanotransducing ion channels and gap junctions additionally govern chondrocyte physiology. It is of great scientific and medical interest to induce a specific cell behaviour by controlling these mechanotransduction pathways and to translate this knowledge into regenerative clinical therapies. This review therefore focuses on the mechanotransduction properties of integrins, cadherins and ion channels in cartilaginous tissues to provide perspectives for cartilage regeneration.

FAK Shutdown: Consequences on Epithelial Morphogenesis and Biomarker Expression Involving an Innovative Biomaterial for Tissue Regeneration.

By employing an innovative biohybrid membrane, the present study aimed at elucidating the mechanistic role of the focal adhesion kinase (FAK) in epithelial morphogenesis in vitro over 4, 7, and 10 days. The consequences of siRNA-mediated FAK knockdown on epithelial morphogenesis were monitored by quantifying cell layers and detecting the expression of biomarkers of epithelial differentiation and homeostasis. Histologic examination of FAK-depleted samples showed a significant increase in cell layers resembling epithelial hyperplasia. Semiquantitative fluorescence imaging (SQFI) revealed tissue homeostatic disturbances by significantly increased involucrin expression over time, persistence of yes-associated protein (YAP) and an increase of keratin (K) 1 at day 4. The dysbalanced involucrin pattern was underscored by ROCK-IISer1366 activity at day 7 and 10. SQFI data were confirmed by quantitative PCR and Western blot analysis, thereby corroborating the FAK shutdown-related expression changes. The artificial FAK shutdown was also associated with a significantly higher expression of filaggrin at day 10, sustained keratinocyte proliferation, and the dysregulated expression of K19 and vimentin. These siRNA-induced consequences indicate the mechanistic role of FAK in epithelial morphogenesis by simultaneously considering prospective biomaterial-based epithelial regenerative approaches.

Disruption of adherens junction and alterations in YAP-related proliferation behavior as part of the underlying cell transformation process of alcohol-induced oral carcinogenesis.

Accumulating evidences indicate that alcohol might play a causative in oral cancer. Unfortunately, in vitro cell systems, uncovering the molecular background of the underlying cell transformation process, are rare. Therefore, this study was conducted, to identify molecular changes and characterize their putative cell behavioral consequences in epitheloid (EPI) and fibroblastoid (FIB) oral keratinocyte phenotypes, arising from chronical alcohol treatment. Concerning adherens junctions (AJs), both EPI and FIB showed membrane-bound ß-catenin, but exhibited differences for E-cadherin and zyxin. While EPI revealed E-cadherin/ß-catenin membrane co-localization, which in parts also applied for zyxin, FIB membranes were devoid of E-cadherin and exhibited marginal zyxin expression. Fetal calf serum (FCS) administration in starved cells promoted proliferation in both keratinocyte phenotypes, whereat EPI and FIB yielded a strikingly modified FCS sensitivity on the temporal scale. Impedance measurement-based cell index detection yielded proliferation stimulation occurring much earlier in FIB (<20h) compared to EPI (>45h). Nuclear preference of the proliferation-associated YAP co-transcription factor in FIB was FCS independent, while it required FCS in EPI. Taken together, the lack of membrane-inherent E-cadherin/ß-catenin co-localization together with low zyxin - reveals perturbation of AJ integrity in FIB. Regarding cell behavior, perturbed AJs in FIB correlate with temporal proliferation sensitivity towards FCS. CYF of 5.6 strongly suggests involvement of chromatin-bound YAP in FIB's proliferation temperosensitivity. These molecular differences detected for EPI and FIB are part of the underlying cell transformation process of alcohol-induced oral carcinogenesis, and indicate FIB being in a more advanced transformation stage.

Biomechanical strain-induced modulation of proliferation coincides with an ERK1/2-independent nuclear YAP localization.

Biomechanical strain induces activation of the transcriptional co-activator yes-associated protein (YAP) by nuclear re-distribution. Recent findings indicate that the mechanically responsive mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) 1/2 is involved in the amount of nuclear YAP, reflecting its activation. In this context, we conducted experiments to detect how biomechanical strain acts on the subcellular localization of YAP in periodontal cells. To this end, cells were subjected to 2.5% static equiaxial strain for different time periods. Western blot and fluorescence imaging-based analyses revealed a clear modulation of nuclear YAP localization. This modulation fairly coincided with the altered course of the KI-67 protein amount in conjunction with the percentage of KI-67-positive and thus proliferating cells. The inhibition of the ERK1/2 activity via U0126 yielded an unchanged strain-related modulation of nuclear YAP localization, while YAP amount in whole cell extracts of strained cells was decreased. Administration of the YAP-inhibiting drug Verteporfin evoked a clear reduction of KI-67-positive and thus proliferating cells by approximately 65%, irrespective of strain. Our data reveal YAP as a regulator of strain-modulated proliferation which occurs in a MAPK-independent fashion.

Orthodontic strain affects the Hippo-pathway effector YAP concomitant with proliferation in human periodontal ligament fibroblasts.

Objectives: During orthodontic tooth movement (OTM), human periodontal ligament fibroblasts (hPDLFs) sense, and respond to mechanical forces. Since the molecular constituents involved in these processes are not fully elucidated, the objective of the present study was to identify further key molecules of the cellular strain response. Materials and Methods: Primary hPDLFs were strained with a static equiaxial strain of 2.5 per cent for 15 minutes, 1 hour, 6 hours, and 24 hours. Western blot (WB) and indirect immunofluorescence (IIF) analyses were performed to investigate the quantity and activation state of proteins involved in mechanotransduction, namely extracellular signal-regulated kinase (ERK) 1/2 and yes-associated protein (YAP). On the cell behavioural level, proliferation was assessed by the marker of proliferation KI-67. Results: In response to the applied strain, an early decline of phosphorylated and thus activated ERK1/2 was observed, followed by a mild recovery. Furthermore, both WB and IIF analyses revealed a modulation of nuclear YAP localisation. Concomitant with the modulation of YAP, the applied strain evoked an early increase in nuclear KI-67 amount, followed by a continuous decrease. Limitations: Consecutive studies will focus on scrutinising the suggested relationship between YAP and proliferation in response to static strain. Conclusions: Our findings provide evidence of ERK1/2 and YAP being biomechanically responsive molecular players in the context of OTM, among which YAP rather than ERK1/2 seems to be mechanistically interrelated with proliferation. Furthermore, the molecular and cell behavioural strain-induced early modulations may point to an involvement of the investigated molecules in the initial and the following lag phase of OTM.

Modulation of focal adhesion constituents and their down-stream events by EGF: On the cross-talk of integrins and growth factor receptors.

Within the concept of integrin growth factor receptor (GFR) cross-talk, little is known about the effects of GFRs on focal adhesions (FAs). Therefore, we tested the hypothesis whether EGF can modulate constituents of FAs and subsequent down-stream events. To this end, EGF-treated keratinocytes were subjected to combined fluorescence imaging and western blotting, to quantify expression and/or activation of molecules, involved in integrin GFR cross-talk, and receptor proximal and distal signaling events. Generally, EGF response revealed an amplified redistribution or activation of molecules under study, which will be explained in detail from the plasma membrane to the cell interior. In addition to significant activation of EGF receptor (EGFR) at tyrosine Tyr845, a remarkable redistribution was detectable for the focal adhesion constituents, integrin ß1 and ß3, and zyxin. Increased activation also applied to focal adhesion kinase (FAK) by phosphorylation at Tyr397, Tyr576, and Src at Tyr418, while total FAK remained unchanged. Risen activity was seen as well for the analyzed distal down-stream events, p190RhoGAP and MAP kinases p42/44. Intriguingly, Src-specific inhibitor Herbimycin A abrogated the entire EGF response except FAK Tyr397 phosphorylation, independent of EGF presence. Mechanistically, our results show that EGF modulates adhesion in a dual fashion, by firstly redistributing focal adhesion constituents to adhesion sites, but also by amplifying levels of activated RhoA antagonist p190RhoGAP, important for cell motility. Further, the findings suggest that the observed EGF response underlies an EGFR integrin cross-talk under recruitment of receptor proximal FAK and Src, and MAP kinase and p190RhoGAP as receptor distal events.

hMSC-Derived VEGF Release Triggers the Chemoattraction of Alveolar Osteoblasts.

Human mesenchymal stem cells (hMSCs) are promising candidates for regenerative periodontal strategies, due to the broad spectrum of supportive effects on cells and tissues at the site of application. Although positive effects are visible, the understanding of their underlying mechanisms still requires further elucidation. Recently, we have shown that hMSCs are capable to prompt osteogenic differentiation of alveolar osteoblasts, thereby presumably contributing to alveolar bone regeneration. Another issue that is critical in this context is the attraction of hard tissue-forming cells to regeneration sites, but it is an open question whether hMSCs can afford this. In the present manuscript, we show by life cell imaging that in interactive cocultures, hMSCs successfully trigger osteoblast chemotaxis. Gene expression analysis for hMSC-innate chemoattractive biomolecules, orchestrating this process, revealed vascular endothelial growth factor (VEGF), PgE synthase, osteoprotegerin (OPG), monocyte colony-stimulating factor, and transforming growth factor ß1, which was confirmed for VEGF and OPG on the protein level. Noteworthy, we showed that only corresponding levels of VEGF but not OPG attracted alveolar osteoblasts similar to hMSC coculture, while VEGF inhibitor abolished both the VEGF and the hMSC-triggered chemoattraction. In summary, we have identified secreted OPG and VEGF proteins as potential chemoattractants, of which further characterization yielded VEGF as a causative for hMSC-directed osteoblast chemotaxis. With respect to the better understanding of potential hMSC-based periodontal regeneration strategies, we propose hMSC-derived VEGF release as a mechanism in the recruitment of hard tissue-forming cells to alveolar bone sites in need of regeneration.

Multi-chromatic control of mammalian gene expression and signaling.

The emergence and future of mammalian synthetic biology depends on technologies for orchestrating and custom tailoring complementary gene expression and signaling processes in a predictable manner. Here, we demonstrate for the first time multi-chromatic expression control in mammalian cells by differentially inducing up to three genes in a single cell culture in response to light of different wavelengths. To this end, we developed an ultraviolet B (UVB)-inducible expression system by designing a UVB-responsive split transcription factor based on the Arabidopsis thaliana UVB receptor UVR8 and the WD40 domain of COP1. The system allowed high (up to 800-fold) UVB-induced gene expression in human, monkey, hamster and mouse cells. Based on a quantitative model, we determined critical system parameters. By combining this UVB-responsive system with blue and red light-inducible gene control technology, we demonstrate multi-chromatic multi-gene control by differentially expressing three genes in a single cell culture in mammalian cells, and we apply this system for the multi-chromatic control of angiogenic signaling processes. This portfolio of optogenetic tools enables the design and implementation of synthetic biological networks showing unmatched spatiotemporal precision for future research and biomedical applications.

A red/far-red light-responsive bi-stable toggle switch to control gene expression in mammalian cells.

Growth and differentiation of multicellular systems is orchestrated by spatially restricted gene expression programs in specialized subpopulations. The targeted manipulation of such processes by synthetic tools with high-spatiotemporal resolution could, therefore, enable a deepened understanding of developmental processes and open new opportunities in tissue engineering. Here, we describe the first red/far-red light-triggered gene switch for mammalian cells for achieving gene expression control in time and space. We show that the system can reversibly be toggled between stable on- and off-states using short light pulses at 660 or 740 nm. Red light-induced gene expression was shown to correlate with the applied photon number and was compatible with different mammalian cell lines, including human primary cells. The light-induced expression kinetics were quantitatively analyzed by a mathematical model. We apply the system for the spatially controlled engineering of angiogenesis in chicken embryos. The system's performance combined with cell- and tissue-compatible regulating red light will enable unprecedented spatiotemporally controlled molecular interventions in mammalian cells, tissues and organisms.

Shaping oral cell plasticity to osteogenic differentiation by human mesenchymal stem cell coculture.

In the context of cell-based oral hard tissue regeneration, especially assumed plasticity of oral host tissue cells in response to human mesenchymal stem cells (hMSCs), is poorly understood. To investigate this area, we assess osteogenic features in various oral cell types during hMSC coculture, including human alveolar osteoblasts (hOAs), periodontal ligament cells (hPDLs) and gingival fibroblasts (hGFs). Interactive hMSC coculture globally enhanced the transcription of osteogenic genes, in all oral cell types under study, as revealed by qRT-PCR and did not affect oral cell proliferation compared with controls in a transwell coculture system as evaluated by 5-bromo-2'-deoxyuridine proliferation assay. 3D gel-derived hMSC cocultures exhibited an abundance of bone-related key molecules in oral cells, which followed the ranking hOAs > hGFs > hPDLs. Compared to matched controls, this hierarchy also applied for the presence of higher amounts of extracellular matrix deposits and mineralization nodules in interactive hMSC coculture. Our results show for the first time that in the context of prospective periodontal tissue regeneration strategies, hMSCs influence oral cells by gradually shaping their plasticity, particularly features associated with an osteogenic phenotype. These novel findings contribute another piece to the conceptual hMSC action puzzle and valuably support the notion that hMSCs trigger osteogenesis in the oral cell context.

Focal adhesion kinase (FAK) perspectives in mechanobiology: implications for cell behaviour.

Mechanobiology is a scientific interface discipline emerging from engineering and biology. With regard to tissue-regenerative cell-based strategies, mechanobiological concepts, including biomechanics as a target for cell and human mesenchymal stem cell behaviour, are on the march. Based on the periodontium as a paradigm, this mini-review discusses the key role of focal-adhesion kinase (FAK) in mechanobiology, since it is involved in mediating the transformation of environmental biomechanical signals into cell behavioural responses via mechanotransducing signalling cascades. These processes enable cells to adjust quickly to environmental cues, whereas adjustment itself relies on the specific intramolecular phosphorylation of FAK tyrosine residues and the multiple interactions of FAK with distinct partners. Furthermore, interaction-triggered mechanotransducing pathways govern the dynamics of focal adhesion sites and cell behaviour. Facets of behaviour not only include cell spreading and motility, but also proliferation, differentiation and apoptosis. In translational terms, identified and characterized biomechanical parameters can be incorporated into innovative concepts of cell- and tissue-tailored clinically applied biomaterials controlling cell behaviour as desired.

Substrate elasticity as biomechanical modulator of tissue homeostatic parameters in corneal keratinocytes.

This study aimed at identifying putative modulations of tissue homeostatic parameters in corneal keratinocytes in response to biomechanical cues as basis for innovative cornea biomechanical-tailored biomaterials. Since cornea epithelial biomechanics is already described for contacts on nanostructures, we herein analyzed cell response to mechanical substrate elasticity. Therefore, corneal keratinocytes were established on physiologically-relevant elastic substrates of 40kPa, 130kPa but also on non-physiological stiff substrates of 1.74MPa for 3 days. qPCR revealed that changes in gene expression were only marginal between 40kPa and 130kPa, while significant modulations were seen on 1.74MPa substrates for most tissue-innate biomarkers under study. Gene expression fairly coincided with the protein, with differentiation progression biomarkers involucrin and fillagrin being already significantly increased between elasticities of 40kPa and 130kPa. Regarding focal adhesions, reinforcement was seen for ß1 integrin and phospho- p(125FAK) between 40kPa and 130kPa, while from 130kPa to 1.74MPa actin redistributed and phospho-p(125FAK) was strikingly up-regulated. These findings suggest elasticity dependence for differentiation progression and focal adhesion dynamics of corneal keratinocytes, supporting the concept of biomechanics governed regulation of tissue homeostasis. Moreover, this concept in turn can be translated into prospective cornea-tailored biomaterials for therapeutic approaches in ophthalmology.

Generation and evaluation of a human corneal model cell system for ophthalmologic issues using the HPV16 E6/E7 oncogenes as uniform immortalization platform.

The present study aimed at employing the human papillomavirus type 16 (HPV16) E6/E7 gene platform, to create a uniform authentic in vitro model cell system of the human cornea for ophthalmologic issues and here especially for prospective biomaterial evaluations for therapeutic regenerative approaches. Therefore, HPV16 E6/E7 genes were employed as uniform platform to immortalize primary human corneal keratinocytes (IHCK), fibroblasts (IHCF), and endothelial (IHCE) cells. qPCR revealed that E6/E7 mRNA transcription persisted at rising passages and FISH detection of the chromosome portfolio 1, 8, 10 and 18 showed fairly the disomic cytogenetic status. Hot spot passages proved oscillation of aneuploidies in the entire passage spectrum under study, while hot spot aneuploidies annotated prevalence for distinct chromosomes. Though IIF revealed general endurance, tissue-innate corneal biomarkers were modulated, i.e. expressed in a temporal-confluence, temporal-spatial or passage-dependent manner. In summary, by the fairly normal chromosomal status, and expression of tissue-innate biomarkers, we created for the first time a uniform authentic in vitro model cell system of the human cornea, by application of the HPV16 E6/E7 immortalization platform only. This system renders a precious tool for prospective iterative in vitro studies on issues such as corneal tissue homeostasis, pharmaceutical generics, and/or evaluation of new biomaterials for clinical corneal applications.

Analysis of soft tissue integration-supportive cell functions in gingival fibroblasts cultured on 3D printed biomaterials for oral implant-supported prostheses.

To date, it is unknown whether 3D printed fixed oral implant-supported prostheses can achieve comparable soft tissue integration (STI) to clinically established subtractively manufactured counterparts. STI is mediated among others by gingival fibroblasts (GFs) and is modulated by biomaterial surface characteristics. Therefore, the aim of the present work was to investigate the GF response of a 3D printed methacrylate photopolymer and a hybrid ceramic-filled methacrylate photopolymer for fixed implant-supported prostheses in the sense of supporting an STI. Subtractively manufactured samples made from methacrylate polymer and hybrid ceramic were evaluated for comparison and samples from yttria-stabilized tetragonal zirconia polycrystal (3Y-TZP), comprising well documented biocompatibility, served as control. Surface topography was analyzed by scanning electron microscopy and interferometry, elemental composition by energy-dispersive x-ray spectroscopy, and wettability by contact angle measurement. The response of GFs obtained from five donors was examined in terms of membrane integrity, adhesion, morphogenesis, metabolic activity, and proliferation behavior by a lactate-dehydrogenase assay, fluorescent staining, a resazurin-based assay, and DNA quantification. The results revealed all surfaces were smooth and hydrophilic. GF adhesion, metabolic activity and proliferation were impaired by 3D printed biomaterials compared to subtractively manufactured comparison surfaces and the 3Y-TZP control, whereas membrane integrity was comparable. Within the limits of the present investigation, it was concluded that subtractively manufactured surfaces are superior compared to 3D printed surfaces to support STI. For the development of biologically optimized 3D printable biomaterials, consecutive studies will focus on the improvement of cytocompatibility and the synthesis of STI-relevant extracellular matrix constituents.

Effect of dental composite dust on human gingival keratinocytes.

OBJECTIVE: The aim was to investigate the effect of particles released during grinding of dental composites on human gingival keratinocytes (HGK). METHODS: Specimens from Filtek™ Supreme XTE and ceram.x® universal were prepared and ground to dust. The dust was filtered (≤ 5 µm) and the particle size distribution was examined using NANO-flex®-180° dynamic light scattering (DLS). Suspensions at five concentrations (3, 10, 30, 100 and 300 µg/mL) were prepared using keratinocyte growth medium (KGM). These suspensions, as well as a positive (CuO) and a negative control (KGM) were added to HGK. The cells treated with Filtek™ Supreme XTE suspensions were analyzed by real-time monitoring using RTCA iCELLigence™. In addition, light and scanning electron microscopic images of the exposed cells were taken. Indirect immunofluorescence staining was performed to detect the extracellular matrix protein fibronectin. RESULTS: In distilled water, DLS showed similar particles' range (171.9 nm- 2.7 µm) for both composites. In saliva, larger particles were detected (Filtek™ Supreme XTE: 243 nm-6,5 µm; ceram.x® universal: 204 nm- 4,6 µm). iCELLigence™ revealed similar results of cell growth parameters for HGK incubated with composite dust (≤ 5 µm) at different concentrations. The microscopic images indicated unaltered cell structures and formation of large agglomerates with high particle concentration (> 100 µg/mL). Exposure to composite dust resulted in upregulation of fibronectin expression. SIGNIFICANCE: Grinding of dental composite materials generates dust particles of different sizes. The particle size distribution seems to be more influenced by the suspending medium than the material itself. While cell growth of HGK seem not to be affected by the particles, an upregulation of fibronectin in the intercellular space concomitant by increasing particle concentration may indicate an increase of cell migration/mobility.

Diffuse, Adult-Onset Nesidioblastosis/Non-Insulinoma Pancreatogenous Hypoglycemia Syndrome (NIPHS): Review of the Literature of a Rare Cause of Hyperinsulinemic Hypoglycemia.

Differential diagnosis of hypoglycemia in the non-diabetic adult patient is complex and comprises various diseases, including endogenous hyperinsulinism caused by functional ß-cell disorders. The latter is also designated as nesidioblastosis or non-insulinoma pancreatogenous hypoglycemia syndrome (NIPHS). Clinically, this rare disease presents with unspecific adrenergic and neuroglycopenic symptoms and is, therefore, often overlooked. A combination of careful clinical assessment, oral glucose tolerance testing, 72 h fasting, sectional and functional imaging, and invasive insulin measurements can lead to the correct diagnosis. Due to a lack of a pathophysiological understanding of the condition, conservative treatment options are limited and mostly ineffective. Therefore, nearly all patients currently undergo surgical resection of parts or the entire pancreas. Consequently, apart from faster diagnosis, more elaborate and less invasive treatment options are needed to relieve the patients from the dangerous and devastating symptoms. Based on a case of a 23-year-old man presenting with this disease in our department, we performed an extensive review of the medical literature dealing with this condition and herein presented a comprehensive discussion of this interesting disease, including all aspects from epidemiology to therapy.

An Uncommon Cause of Recurrent Presyncope, Dizziness, and Tachycardia: A Case Report of Diffuse, Adult-Onset Nesidioblastosis/Non-Insulinoma Pancreatogenous Hypoglycemia Syndrome (NIPHS).

Neurovegetative and autonomic symptoms are common presentations of various diseases, ranging from psychosomatic to severe organic disorders. A 23-year-old man presented with a history of recurrent presyncope, dizziness, and tachycardia. Repeated diagnostic work-up in various clinical settings could not identify any definite cause for approximately eight years. However, the incidental detection of postprandial and exercise-induced hypoglycemia was suggestive of an insulin-related disorder. A 72 h plasma glucose fasting test revealed endogenous hyperinsulinism. Upon imaging studies, no tumor mass potentially indicating insulinoma could be detected. 68Ga-DOTA-Exendin-4 PET/CT showed diffuse tracer enrichment throughout the whole pancreas. A subtotal pancreatectomy was performed, and the diagnosis of diffuse, adult-onset nesidioblastosis was established histopathologically. This corresponds to the clinical findings of a functional ß-cell disorder, also known as non-insulinoma pancreatogenous hypoglycemia syndrome (NIPHS). After nine months, the symptoms recurred, making complete pancreatectomy necessary. Postoperative laboratory evaluation exhibited no residual endogenous C-peptide production. This case illustrates the diagnostic challenges in patients presenting with unspecific, neurovegetative and autonomic symptoms with a severe and rare underlying cause.