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.

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.

Response of human gingival keratinocytes to hybrid CAD/CAM material eluates.

OBJECTIVES: The aim of this study was to investigate the influence of hybrid CAD/CAM-blocks on immortalized human gingival keratinocytes (HGK). METHODS: Samples of two different hybrid CAD/CAM materials [Lava™ Ultimate (3 M); VITA Enamic® (VITA Zahnfabrik)], a composite material [ceram.x® universal (Dentsply Sirona)] and a CAD/CAM ceramic [VITABLOCS® (VITA Zahnfabrik)] were stored in cell culture medium for 72 h to prepare eluates according to ISO-10993-12:2012. HGK were exposed to eluates for 6, 24 and 48 h. Cell monitoring was performed by RTCA iCELLigence™ system. The morphological changes were evaluated using phase contrast imaging. Specific biomarkers of apoptosis and terminal differentiation (Caspase-3, Involucrin) were analyzed semi quantitatively by indirect immunofluorescence (IIF). Protein levels and activation of MAP kinases ERK1/2 (p44/42) were quantified by Western blot. Data were statistically analyzed by unpaired t-test (p < 0.05). RESULTS: Regarding Vita Enamic® and Lava™ Ultimate, results of RTCA iCELLigence™ and Western blots showed no statistically significant differences (p > 0.05) compared to the negative control (HGK in native keratinocyte growth medium). No aberrant expression of Caspase-3 and Involucrin was detected in cells incubated with Vita® Enamic eluates Cells incubated with Lava™ Ultimate showed a higher expression of Involucrin after 24 h of incubation compared to the negative control. Statistically significant differences (p < 0.01) were found between cells incubated with ceram.x® universal and the negative control in RTCA iCELLigence™ assay and in quantitative measurements of Western blots after 6 h against phospho-p44/42 (p = 0.044). Increased expression of Caspase-3 and Involucrin were detected by IIF in cells after incubation with eluates of ceram.x® universal. SIGNIFICANCE: The present data show no significant effect of hybrid materials on analyzed functions of cell behavior. A cytotoxic influence of ceram.x® universal eluates was observed in HGK in terms of a strong modulation of proliferation, morphology and protein expression.

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.

From the Matrix to the Nucleus and Back: Mechanobiology in the Light of Health, Pathologies, and Regeneration of Oral Periodontal Tissues.

Among oral tissues, the periodontium is permanently subjected to mechanical forces resulting from chewing, mastication, or orthodontic appliances. Molecularly, these movements induce a series of subsequent signaling processes, which are embedded in the biological concept of cellular mechanotransduction (MT). Cell and tissue structures, ranging from the extracellular matrix (ECM) to the plasma membrane, the cytosol and the nucleus, are involved in MT. Dysregulation of the diverse, fine-tuned interaction of molecular players responsible for transmitting biophysical environmental information into the cell's inner milieu can lead to and promote serious diseases, such as periodontitis or oral squamous cell carcinoma (OSCC). Therefore, periodontal integrity and regeneration is highly dependent on the proper integration and regulation of mechanobiological signals in the context of cell behavior. Recent experimental findings have increased the understanding of classical cellular mechanosensing mechanisms by both integrating exogenic factors such as bacterial gingipain proteases and newly discovered cell-inherent functions of mechanoresponsive co-transcriptional regulators such as the Yes-associated protein 1 (YAP1) or the nuclear cytoskeleton. Regarding periodontal MT research, this review offers insights into the current trends and open aspects. Concerning oral regenerative medicine or weakening of periodontal tissue diseases, perspectives on future applications of mechanobiological principles are discussed.

Enhancing the soft-tissue integration of dental implant abutments-in vitro study to reveal an optimized microgroove surface design to maximize spreading and alignment of human gingival fibroblasts.

Within this work, we demonstrate the influences of different microgrooved surface topographies on the alignment and spreading of human gingival fibroblast (HGF) cells and present the optimal parameters for an improved soft-tissue integration design for dental implant abutments for the first time. Microgrooves with lateral widths from 2.5 to 75 µm were fabricated by UV-lithography and wet etching on bulk Ti6Al4V ELI material. The microstructured surfaces were compared to polished and ground surfaces as current state of the art. The resulting microtopographies were analyzed using vertical scanning interferometry and scanning electron microscopy. Samples loaded with HGF cells were incubated for 8 and 72 hr and cell orientation, spreading, resulting area, and relative gene expression were analyzed. The effect of contact guidance occurred on all microstructured surfaces yet there is a clear preferable range for the lateral widths of the microgrooves between approx. 11.5 and 13.9 µm and depths between 1.6 and 2.4 µm for an abutment surface design, where cell orientation and spreading maximizes. For structures larger than 30 µm, cell orientation, spreading and even gene expression of intercellular adhesion molecule-1 and yes-associated protein decrease.

Force-responsive Zyxin modulation in periodontal ligament cells is regulated by YAP rather than TAZ.

In the context of mechanically induced force transmission, the modification of the actin cytoskeleton through involvement of zyxin is an established concept. However, in cells of the periodontal ligament (PDL), which is physiologically subjected to intermittent mechanical forces, the force-responsive modulation of zyxin and the molecular key players, which orchestrate its cellular regulation, have not yet been elucidated. By employing indirect immunofluorescence and western blotting with different subcellular fractions, we show here in stretch force-exposed human PDL fibroblasts (hPDLFs) that (i) the zyxin protein is modulated, and (ii) its subcellular localization is altered. More importantly, using a pharmacological intervention approach, to inhibit the nuclear presence of the co-transcriptional activator yes-associated protein (YAP), we evidence for the first time that on the molecular level, the cellular abundance of zyxin, among the Thyrotrophic Embryonic Factor (TEF)-binding proteins, is regulated by YAP rather than TAZ. Our findings provide novel insights into the topic how cells of the periodontium and the periodontal ligament in particular respond and may adapt to mechanical forces, and first time identify YAP as the key player of the intracellular regulation of the mechano-sensor and mechano-transducer zyxin in hPDLFs. Moreover, the findings broaden the current knowledge on YAP, since so far, currently only very few YAP-regulated genes have been identified.

On the relationship of YAP and FAK in hMSCs and osteosarcoma cells: Discrimination of FAK modulation by nuclear YAP depletion or YAP silencing.

The HIPPO pathway effector YAP has been shown to be regulated by FAK-signaling. However, the existence of an inverse relationship between YAP and FAK is unknown. Here we demonstrate in hMSCs and in the human osteosarcoma derived cell line Saos that Verteporfin- or RNAi-dependent YAP depletion has opposing influence on FAK. While Verteporfin strikingly reduced cellular FAK protein and phosphorylation, RNAi led to an increase of both molecules and point on a generalizable aspect of the YAP/FAK interrelationship. YAP depletion also caused down-regulation of osteogenic genes in hMSCs, irrespective from the YAP intervention mode. Verteporfin induced topological changes in conjunction with reduced protein levels of ß1 integrin, paxillin, and zyxin of focal adhesions (FAs) in hMSCs, suggesting FAK-decrease-related alterations in FAs, which seems to be a FAK-dependent mechanism. On the cell behavioral level, YAP-FAK-interrelation involves proliferation and senescence, as indicated by proliferation inhibition and increase of ß-Galactosidase-activity in hMSCs. Our findings, derived from this dual strategy of YAP intervention, reveal a YAP-FAK relationship in conjunction with molecular and cell behavioral consequences. Moreover, they deepen the current scientific knowledge on YAP from a different scientific point of view, since this inverse YAP/FAK-relationship seems to be transferrable to other cell types, including cell entities with pathological background.

Gelatin nonwovens-based epithelial morphogenesis involves a signaling axis comprising EGF-receptor, MAP kinases ERK 1/2, and ß1 integrin.

In biomaterials research, biomechanics which support tissue regeneration steadily gains of importance. Hence, we have previously shown that gelatin-based electrospun nonwoven mats (NWMs) with a distinct modulus of elasticity (3.2 kPa) promotes epithelial morphogenesis. Since molecular mechanisms of this morphogenesis are still unknown, the present study aims at identifying molecules, involved herein. Epithelia established on the NMWs showed persistence of the activated state of the epidermal growth factor receptor (EGF-R), phosphorylated at the src-specific tyrosine 845 (EGF-RT845 ) throughout the observation period of 10 days. To elucidate whether the observed morphogenesis mechanistically involves EGF-R signaling, we inhibited EGF-R, by employing the EGF-RT845 specific inhibitor Gefitinib (IRESSA®). Gefitinib administration yielded a reduced expression of the ß1 integrin subunit, a well-known cell-matrix interaction receptor, concomitant with downregulation of p42/44 ERK1/2 MAP-kinase activity. To elucidate whether the observed downregulation of ß1 is EGF-RT845 -dependent or emerging from ERK1/2 signaling, we exposed epithelia, grown on the NWMs, with the ERK1/2-directed inhibitor U0126. In the absence of Gefitinib, inhibition of p42/44 MAP-kinase activity resulted in decreased ß1 integrin protein levels, thus indicating that ß1 expression is dependent on ERK1/2 and not EGF-RT845 . Our results showed the first time that an EGF-R-ß1 integrin-signaling axis, including ERK1/2, promotes NWM-elasticity-based epithelial morphogenesis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 663-677, 2019.

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.

Antibiotic-free nanotherapeutics: ultra-small, mucus-penetrating solid lipid nanoparticles enhance the pulmonary delivery and anti-virulence efficacy of novel quorum sensing inhibitors.

Cystic fibrosis (CF) is a genetic disease mainly manifested in the respiratory tract. Pseudomonas aeruginosa (P. aeruginosa) is the most common pathogen identified in cultures of the CF airways, however, its eradication with antibiotics remains challenging as it grows in biofilms that counterwork human immune response and dramatically decrease susceptibility to antibiotics. P. aeruginosa regulates pathogenicity via a cell-to-cell communication system known as quorum sensing (QS) involving the virulence factor (pyocyanin), thus representing an attractive target for coping with bacterial pathogenicity. The first in vivo potent QS inhibitor (QSI) was recently developed. Nevertheless, its lipophilic nature might hamper its penetration of non-cellular barriers such as mucus and bacterial biofilms, which limits its biomedical application. Successful anti-infective inhalation therapy necessitates proper design of a biodegradable nanocarrier allowing: 1) high loading and prolonged release, 2) mucus penetration, 3) effective pulmonary delivery, and 4) maintenance of the anti-virulence activity of the QSI. In this context, various pharmaceutical lipids were used to prepare ultra-small solid lipid nanoparticles (us-SLNs) by hot melt homogenization. Plain and QSI-loaded SLNs were characterized in terms of colloidal properties, drug loading, in vitro release and acute toxicity on Calu-3 cells. Mucus penetration was studied using a newly-developed confocal microscopy technique based on 3D-time-lapse imaging. For pulmonary application, nebulization efficiency of SLNs and lung deposition using next generation impactor (NGI) were performed. The anti-virulence efficacy was investigated by pyocyanin formation in P. aeruginosa cultures. Ultra-small SLNs (<100nm diameter) provided high encapsulation efficiency (68-95%) according to SLN composition, high burst in phosphate buffer saline compared to prolonged release of the payload over >8h in simulated lung fluid with minor burst. All types and concentrations of plain and QSI-loaded SLNs maintained the viability of Calu-3 cells. 3D time-lapse confocal imaging proved the ability of SLNs to penetrate into artificial sputum model. SLNs were efficiently nebulized; NGI experiments revealed their deposition in the bronchial region. Overall, nanoencapsulated QSI showed up to sevenfold superior anti-virulence activity to the free compound. Most interestingly, the plain SLNs exhibited anti-virulence properties themselves, which was shown to be related to anti-virulence effects of the emulsifiers used. These startling findings represent a new perspective of ultimate significance in the area of nano-based delivery of novel anti-infectives.