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.

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.

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.

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.

Characterization of a Stemness-Optimized Purification Method for Human Dental-Pulp Stem Cells: An Approach to Standardization.

Human dental pulp stem cells (hDPSCs) are promising for oral/craniofacial regeneration, but their purification and characterization is not yet standardized. hDPSCs from three donors were purified by magnetic activated cell sorting (MACS)-assisted STRO-1-positive cell enrichment (+), colony derivation (c), or a combination of both (c/+). Immunophenotype, clonogenicity, stemness marker expression, senescence, and proliferation were analyzed. Multilineage differentiation was assessed by qPCR, immunohistochemistry, and extracellular matrix mineralization. To confirm the credibility of the results, repeated measures analysis and post hoc p-value adjustment were applied. All hDPSC fractions expressed STRO-1 and were similar for several surface markers, while their clonogenicity and expression of CD10/44/105/146, and 166 varied with the purification method. (+) cells proliferated significantly faster than (c/+), while (c) showed the highest increase in metabolic activity. Colony formation was most efficient in (+) cells, which also exhibited the lowest cellular senescence. All hDPSCs produced mineralized extracellular matrix. Regarding osteogenic induction, (c/+) revealed a significant increase in mRNA expression of COL5A1 and COL6A1, while osteogenic marker genes were detected at varying levels. (c/+) were the only population missing BDNF gene transcription increase during neurogenic induction. All hDPSCs were able to differentiate into chondrocytes. In summary, the three hDPSCs populations showed differences in phenotype, stemness, proliferation, and differentiation capacity. The data suggest that STRO-1-positive cell enrichment is the optimal choice for hDPSCs purification to maintain hDPSCs stemness. Furthermore, an (immuno) phenotypic characterization is the minimum requirement for quality control in hDPSCs studies.

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.

Novel In Situ-Cross-Linked Electrospun Gelatin/Hydroxyapatite Nonwoven Scaffolds Prove Suitable for Periodontal Tissue Engineering.

Periodontal diseases affect millions of people worldwide and can result in tooth loss. Regenerative treatment options for clinical use are thus needed. We aimed at developing new nonwoven-based scaffolds for periodontal tissue engineering. Nonwovens of 16% gelatin/5% hydroxyapatite were produced by electrospinning and in situ glyoxal cross-linking. In a subset of scaffolds, additional porosity was incorporated via extractable polyethylene glycol fibers. Cell colonization and penetration by human mesenchymal stem cells (hMSCs), periodontal ligament fibroblasts (PDLFs), or cocultures of both were visualized by scanning electron microscopy and 4',6-diamidin-2-phenylindole (DAPI) staining. Metabolic activity was assessed via Alamar Blue® staining. Cell type and differentiation were analyzed by immunocytochemical staining of Oct4, osteopontin, and periostin. The electrospun nonwovens were efficiently populated by both hMSCs and PDLFs, while scaffolds with additional porosity harbored significantly more cells. The metabolic activity was higher for cocultures of hMSCs and PDLFs, or for PDLF-seeded scaffolds. Periostin and osteopontin expression was more pronounced in cocultures of hMSCs and PDLFs, whereas Oct4 staining was limited to hMSCs. These novel in situ-cross-linked electrospun nonwoven scaffolds allow for efficient adhesion and survival of hMSCs and PDLFs. Coordinated expression of differentiation markers was observed, which rendered this platform an interesting candidate for periodontal tissue engineering.

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.

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.

Low-dose Bisphenol A and its analogues Bisphenol F and S activate estrogen receptor ß and slightly modulate genes in human gingival keratinocytes.

OBJECTIVES: This study investigated the putative activation of estrogen receptor ß (ERß) and possible effects related on gene expression in oral mucosal cells in response to the endocrine disruptor Bisphenol A (BPA) and its analogues Bisphenol F (BPF) and Bisphenol S (BPS). METHODS: Human gingival keratinocytes (HGK) were exposed to BPA-, BPF-, and BPS-solutions in concentrations of 1.3 µM, 0.16 µM and 11.4 nM as well as 200 pM and 100 nM estradiol (E2) for 6 h, 24 h and 4 d. Indirect immunofluorescence (IIF) was performed to detect a possible ERß activation. Additionally, transcription of keratinocyte-relevant biomarkers was analyzed by quantitative real-time PCR (qRT-PCR). A linear mixed model and pairwise comparisons were applied for statistical analyses. RESULTS: The tested concentrations of BPA, BPF, BPS and E2 revealed distinct activation of ERß at all time periods, whereat 100 nM E2 induced the most pronounced activation. Despite the detected ERß activation, the concentrations of BPA and its analogues induced only moderate modulation of the tested keratinocyte-relevant biomarker genes at all time periods. This also applied to 200 pM E2, while in case of 100 nM E2 significant changes (p < 0.05) were detected for almost all analyzed genes. SIGNIFICANCE: Though BPA and its analogues induce activation of ERß irrespective from the chosen concentrations and incubation periods, they lack significant modulation of gene expression of keratinocyte-relevant biomarkers. Although limited to a selected number of genes, the sparse modulation of gene expression may give a hint that the substances do slightly affect transcription of gingival-keratinocyte-innate genes, since the concentrations applied to HGK were of physiological importance.

Controlling osteoblast morphology and proliferation via surface micro-topographies of implant biomaterials.

Current research on surface modifications has yielded advanced implant biomaterials. Various implant surface modifications have been shown to be promising in improving bone target cell response, but more comprehensive studies whether certain implant surface modifications can directly target cell behavioural features such as morphogenesis and proliferation are needed. Here, we studied the response of primary alveolar bone cells on various implant surface modifications in terms of osteoblast morphology and proliferation in vitro. Analyses of surface modifications led to surface-related test parameters including the topographical parameters micro-roughness, texture aspect and surface enlargement as well as the physicochemical parameter surface wettability. We compared osteoblast morphology and proliferation towards the above-mentioned parameters and found that texture aspect and surface enlargement but not surface roughness or wettability exhibited significant impact on osteoblast morphology and proliferation. Detailed analysis revealed osteoblast proliferation as a function of cell morphology, substantiated by an osteoblast size- and morphology-dependent increase in mitotic activity. These findings show that implant surface topography controls cell behavioural morphology and subsequently cell proliferation, thereby opening the road for cell instructive biomaterials.

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.

In vivo evaluation of an electrospun gelatin nonwoven mat for regeneration of epithelial tissues.

One major objective in epithelial tissue engineering is to identify a suitable biomaterial that supports epithelial tissue formation. Therefore, the purpose of this study is to elucidate a novel electrospun gelatin nonwoven mat (NWM) for epithelial tissue engineering purposes in vivo. This NWM was seeded with either human gingival keratinocytes (GK, in coculture with gingival fibroblast) or human skin epithelial keratinocytes (EK, in coculture with skin dermal fibroblasts). These constructs were ex vivo cultured for 4 days before subcutaneous implantation into athymic nude mice. After 7 days, the constructs were explanted and investigated by immunohistology. Our results show that GK form a stratified epithelium on the surface of the NWM, mostly independent of a fibroblastic counterpart. Like native mucosa, the regenerated epithelium showed expression of epidermal growth factor receptor, cytokeratin-14 and -1, and involucrin. Only the expression of the basement membrane constituent laminin 5 was more pronounced in cocultures. Comparing GK and skin EK, we found that skin EK form a less developed epithelial tissue. Furthermore, the NWM allows not only for epithelial tissue formation by GK, but also for infiltration of human fibroblasts and mouse immune cells, thus representing a biomaterial with potential regenerative capacity for oral mucosa tissue engineering applications. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1605-1614, 2019.

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.

Nanofibered Gelatin-Based Nonwoven Elasticity Promotes Epithelial Histogenesis.

Regarding tissue regeneration, mechanics of biomaterials gains progressive importance. Therefore, this study reports on in situ crosslinked electrospun gelatin nonwoven mats (NWMs) whose distinct modulus of elasticity (ME) promotes epithelial tissue formation in a graded manner. NWMs, comprising fiber diameters in various distributions, yield an ME of about 2.1, 3.2, and 10.9 kPa. A two-step approach of preclinical in vitro validation identifies the elasticity of 3.2 kPa as superior to the other, regarding the histogenetic epithelial outcome. Hence, this 3.2 kPa candidate NWM is colonized with oral mucosal epithelial keratinocytes in the absence or presence of mesenchymal fibroblasts and/or endothelial cells. Evaluation of epithelial histogenesis at days 1 to 10 occurs by colorimetric and fluorescence-based immunohistochemistry (IHCH) of specific biomarkers. These include cytokeratins (CK) 14, CK1, and involucrin that indicate different stages of epithelial differentiation, as well as the basement membrane constituent collagen type IV and Ki-67 as a proliferation marker. Intriguingly, histogenesis and IHCH reveal the best resemblance of the native epithelium by the NWM alone, irrespective of other cell counterparts. These findings prove the gelatin NWM a convenient cell matrix, and evidence that NWM mechanics is important to promote epithelial histogenesis in view of prospective clinical applications.