Papers by Thorsten Steinberg
Tissue Engineering Part C-methods, Nov 1, 2013
Though recent studies report decisive positive effects on cells, elicited by ultraviolet (UV)-ind... more Though recent studies report decisive positive effects on cells, elicited by ultraviolet (UV)-induced bioactivation of biomaterial implant surfaces, they frequently employ cells other than of human origin or cells not representing oral implant targets. Therefore, the present study aims at exploring distinct cell functions of primary human alveolar bone osteoblasts (PHABO) in response to bioactivated microstructured titanium and zirconia implant surfaces with matched controls. UV-treatment significantly reduced surface carbon, while concomitantly increasing wettability. In case of titanium or zirconia biomaterial source of equal roughness, bioactivation did not significantly improve cell functions, including initial cell attachment, morphogenesis, proliferation, and gene expression of osteogenic biomarkers osteocalcin, alkaline phosphatase and collagen type I. However, cell functions discriminated surface roughness by either comparing titanium and zirconia or interindividual zirconia surfaces. While rough surfaces primarily favored primary adhesion, proliferation appeared improved on smooth surfaces, and gene expression seemed to be stronger modulated on the smoothest biomaterial. Our results show for the first time that bioactivation appears to be not the main causative for the observed modulation of the distinct cell functions analyzed in PHABO, but add to the body of evidence that they were more governed by surface architecture rather than by bioactivation.
Biochimica et biophysica acta. Molecular cell research, 2018
Disruption of adherens junction and alterations in YAP-related proliferation behavior as part of ... more Disruption of adherens junction and alterations in YAP-related proliferation behavior as part of the underlying cell transformation process of alcohol-induced oral carcinogenesis. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Bbamcr(2017),
Biomedicines, Jun 17, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Biomedicines, Jun 16, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Tissue Engineering Part A, Jul 1, 2014
Standard preclinical assessments in vitro often have limitations regarding their transferability ... more Standard preclinical assessments in vitro often have limitations regarding their transferability to human beings, mainly evoked by their nonhuman and tissue-different/nontissue-specific source. Here, we aimed at employing tissue-authentic simple and complex interactive fibroblast-epithelial cell systems and their in vivo-relevant biomarkers for preclinical in vitro assessment of nonwoven-based gelatin/polycaprolactone membranes (NBMs) for treatment of soft tissue defects. NBMs were composed of electrospun gelatin and polycaprolactone nanofiber nonwovens. Scanning electron microscopy in conjunction with actin/focal contact integrin fluorescence revealed successful adhesion and proper morphogenesis of keratinocytes and fibroblasts, along with cells' derived extracellular matrix deposits. The ''feel-good factor'' of cells under study on the NBM was substantiated by forming a confluent connective tissue entity, which was concomitant with a stratified epithelial equivalent. Immunohistochemistry proved tissue authenticity over time by abundance of the biomarker vimentin in the connective tissue entity, and chronological increase of keratins KRT1/10 and involucrin expression in epithelial equivalents. Suitability of the novel NBM as wound dressing was evidenced by an almost completion of epithelial wound closure in a pilot mini-pig study, after a surgical intervention-caused gingival dehiscence. In summary, preclinical assessment by tissue-authentic cell systems and the animal pilot study revealed the NBM as an encouraging therapeutic medical device for prospective clinical applications.
Ultraviolet (UV) light treatment of implant surfaces has been demonstrated to enhance their bioac... more Ultraviolet (UV) light treatment of implant surfaces has been demonstrated to enhance their bioactivity significantly. This study examined the effect of UV treatment of different zirconia surfaces on the response of primary human alveolar bone-derived osteoblasts (PhABO). Disks of two zirconia-based materials with two different surface topographies (smooth, roughened) were exposed to UV light. Qualitative and quantitative assessment of PhABO on zirconia surfaces, by means of immunofluorescence, scanning electron microscopy and DNA quantification at 4 and 24 h revealed a higher number of initially attached osteoblasts on UV-treated surfaces. Cell area and perimeter were significantly larger on all UV-treated surfaces (p < 0.05). The proliferation activity was significantly higher on both roughened UV-treated surfaces than on untreated samples at day 3 of culture (p < 0.05). The expression levels of collagen I, osteopontin and osteocalcin at day 14 and alkaline phosphatase activity at day 7 and 14 of culture period were similar among UV-treated and untreated surfaces. Alizarin-Red-Staining at day 21 demonstrated significantly more mineralised nodules on UV-treated samples than on untreated samples. Contact angle measurements and X-ray photoelectron spectroscopy showed that UV light transformed zirconia surfaces from hydrophobic to (super-) hydrophilic (p < 0.05) and significantly reduced the atomic percentage of surface carbon. The results showed that UV light pre-treatment of zirconia surfaces changes their physicochemical properties and improves their attractiveness against PhABO, primarily demonstrated by an augmented cell attachment and spreading. This may result in faster healing and better bone-to-implant contact of zirconia implants in vivo following such a pre-treatment.
Journal of Biomedical Materials Research Part A, Dec 10, 2018
Thi s article has been accepted for publication and undergone full peer review but has not been t... more Thi s article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as
Journal of Biomedical Materials Research Part A, Apr 9, 2019
This article has been accepted for publication and undergone full peer review but has not been th... more This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as
Journal of Dental Research, Apr 17, 2021
Novel findings broaden the concept of mechanotransduction (MT) in biophysically stimulated tissue... more Novel findings broaden the concept of mechanotransduction (MT) in biophysically stimulated tissues such as the periodontium by considering nuclear MT, convergence of intracellular MT pathways, and mechanoresponsive cotranscription factors such as Yes-associated protein 1 (YAP1). Regarding periodontal disease, recent studies have elucidated the role of bacterial gingipain proteases in disturbing the barrier function of cadherins, thereby promoting periodontal inflammation. This leads to dysregulation of extracellular matrix homeostasis via proteases and changes the cell’s biophysical environment, which leads to alterations in MT-induced cell behavior and loss of periodontal integrity. Newest experimental evidence from periodontal ligament cells suggests that the Hippo signaling protein YAP1, in addition to integrin-FAK (focal adhesion kinase) mechanosignaling, also regulates cell stemness. By addressing mechanosignaling-dependent transcription factors, YAP1 is involved in osteogenic and myofibroblast differentiation and influences core steps of autophagy. Recent in vivo evidence elucidates the decisive role of YAP1 in epithelial homeostasis and underlines its impact on oral pathologies, such as periodontitis-linked oral squamous cell carcinogenesis. Here, new insights reveal that YAP1 contributes to carcinogenesis via overexpression rather than mutation; promotes processes such as apoptosis resistance, epithelial-mesenchymal transition, or metastasis; and correlates with poor prognosis in oral squamous cell carcinoma. Furthermore, YAP1 has been shown to contribute to periodontitis-induced bone loss. Mechanistically, molecules identified to regulate YAP1-related periodontal homeostasis and disease include cellular key players such as MAPK (mitogen-activated protein kinase), JNK (c-Jun N-terminal kinase), Rho (Ras homologue) and ROCK (Rho kinase), Bcl-2 (B-cell lymphoma 2), AP-1 (activator protein 1), and c-myc (cellular myelocytomatosis). These findings qualify YAP1 as a master regulator of mechanobiology and cell behavior in human periodontal tissues. This review summarizes the most recent developments in MT-related periodontal research, thereby offering insights into outstanding research questions and potential applications of molecular or biophysical strategies aiming at periodontal disease mitigation or prevention.
Cellular Signalling, Nov 1, 2019
The HIPPO pathway effector YAP has been shown to be regulated by FAK-signaling. However, the exis... more 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-Galactosidaseactivity 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.
Advanced Functional Materials, Oct 9, 2008
European Journal of Orthodontics, Mar 17, 2017
Objectives: During orthodontic tooth movement (OTM), human periodontal ligament fibroblasts (hPDL... more 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 yesassociated 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.
Macromolecular Materials and Engineering, Nov 12, 2020
Vaccine, 2005
Immunization with a codon-optimized HPV 16 E7 gene was shown to yield higher levels of E7-specifi... more Immunization with a codon-optimized HPV 16 E7 gene was shown to yield higher levels of E7-specific cytotoxic T cells [Liu WJ, Gao F, Zhao KN, Zhao W, Fernando GJ, Thomas R, et al. Codon modified human papillomavirus type 16 E7 DNA vaccine enhances cytotoxic T-lymphocyte induction and anti-tumour activity. Virology 2002;301:43]. Here, we sought to verify the hypothesis that there is a direct correlation between the level of protein expression and immunogenicity in mice. We generated HPV 16 E7 expression plasmids where the genes were inserted either as authentic sequence (wt) or after optimizing the codons for use in mammalian cells (opt). For enhancement of translation of the E7 gene a 5 Kozak sequence (K) was added. Transfection experiments revealed the strength of expression in the order of E7opt + K, E7opt − K, E7wt + K and E7wt − K. After immunization of C57/B6 mice we observed an equally strong CD8 + T-cell response with the E7opt plasmids (+ or −K), followed by the E7wt + K and E7wt − K DNAs. The same difference in efficiency was obtained in tumor protection experiments. Regression of pre-existing tumors and CTL activity was observed only with the E7opt + K plasmid. From these data, we conclude that the level of protein expression correlates with the efficiency of CTL response and hence testing by transfection of cells in culture may allow a pre-selection of expression plasmids prior to DNA immunization.
PLOS ONE, Oct 31, 2014
Bacterial infection of biomaterials is a major concern in medicine, and different kinds of antimi... more Bacterial infection of biomaterials is a major concern in medicine, and different kinds of antimicrobial biomaterial have been developed to deal with this problem. To test the antimicrobial performance of these biomaterials, the airborne bacterial assay is used, which involves the formation of biohazardous bacterial aerosols. We here describe a new experimental setup which allows safe handling of such pathogenic aerosols, and standardizes critical parameters of this otherwise intractable and strongly user-dependent assay. With this new method, reproducible, thorough antimicrobial data (number of colony forming units and live-dead-stain) was obtained. Poly(oxonorbornene)-based Synthetic Mimics of Antimicrobial Peptides (SMAMPs) were used as antimicrobial test samples. The assay was able to differentiate even between subtle sample differences, such as different sample thicknesses. With this new setup , the airborne bacterial assay was thus established as a useful, reliable, and realistic experimental method to simulate the contamination of biomaterials with bacteria, for example in an intraoperative setting.
Macromolecular Chemistry and Physics, Aug 1, 2020
Common sugar alcohols used as artificial sweeteners and components of polymer networks represent ... more Common sugar alcohols used as artificial sweeteners and components of polymer networks represent low molecular weight polyhydroxymethylenes (PHMs) with the general formula [CH(OH)] n H 2 but very low degree of polymerization (n = 2-6). Herein high molecular weight PHM (n >> 100) unparalleled in nature is tailored for 3D printing and medical applications by free radical polymerization of 1,3-dioxol-2-one vinylene carbonate to produce polyvinylene carbonate (PVCA) which yields PHM by hydrolysis. Furthermore, PVCA is solution processable and enables PHM functionalization, membrane formation, and extrusion-based 3D printing. Opposite to cellulose, amorphous PHM is plasticized by water and is readily functionalized via PVCA aminolysis/hydrolysis to produce polyhydroxymethylene urethane (PHMU), enable PHM crosslinking and coupling of PHM with amine-functional components like gelatin. After hydrolysis/aminolysis the original PVCA shapes are retained. PVCA solution casting yields PVCA and PHM which exhibits uniform and hierarchic pore architectures. Asymmetric membranes, hydrogels, PHM/collagen blends, and electrospun nonwovens of PVCA, PHM, and PHMU are readily tailored for medical applications. 3D printing of PVCA dispersions containing hydroxyapatite affords porous PVCA, PHMU, and PHM scaffolds useful in regenerative medicine. PHM and functionalized PHMs as carbohydrate-inspired multifunctional materials indicate in vitro biocompatibility and hold great promise for applications in medicine and health care.
Biomaterials, Dec 1, 2011
Due to the architecture of solid body tissues including bone, three-dimensional (3D) in vitro mic... more Due to the architecture of solid body tissues including bone, three-dimensional (3D) in vitro microenvironments appear favorable, since herein cell growth proceeds under more physiological conditions compared to conventional 2D systems. In the present study we show that a 3D microenvironment comprising a fibronectin-coated PMMA/PC-based micro-chip promotes differentiation of primary human osteoblasts as reflected by the densely-packed 3D bone cell aggregates and expression of biomarkers indicating osteoblast differentiation. Morphogenesis and fluorescence dye-based live/dead staining revealed homogenous cell coverage of the microcavities of the chip array, whereat cells showed high viability up to 14 days. Moreover, Azur II staining proved formation of uniform sized multilayered aggregates, exhibiting progressive intracellular deposition of extracellular bone matrix constituents comprising fibronectin, osteocalcin and osteonectin from day 7 on. Compared to 2D monolayers, osteoblasts grown in the 3D chip environment displayed differential mostly higher gene expression for osteocalcin, osteonectin, and alkaline phosphatase, while collagen type I remained fairly constant in both culture environments. Our results indicate that the 3D microenvironment, based on the PMMA biomaterial chip array promotes osteoblast differentiation, and hereby renders a promising tool for tissue-specific in vitro preconditioning of osteoblasts designated for clinically-oriented bone augmentation or regeneration.
Biochimica et biophysica acta. Molecular cell research, Oct 1, 2015
Within the concept of integrin growth factor receptor (GFR) cross-talk, little is known about the... more 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 downstream 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 downstream 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.
Nucleic Acids Research, Apr 26, 2013
The emergence and future of mammalian synthetic biology depends on technologies for orchestrating... more 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 UVBresponsive system with blue and red light-inducible gene control technology, we demonstrate multichromatic 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.
This review is aimed at providing a depiction of molecules and their topography which characteriz... more This review is aimed at providing a depiction of molecules and their topography which characterize native gingiva and PDL fibroblasts, to describe their function in tissue maintenance, and to discuss their possible modulation due to orthodontic tooth movement. Maintenance of the human periodontium requires the balance of proliferation and differentiation in the respective tissues’ cells. Moreover, the cells must synthesize the extracellular matrix molecules and receptors that facilitate adhesion. To describe the molecules that contribute to periodontal tissue maintenance, we illustrate the localization of their expression and topography on frozen sections from native gingival tissue and primary cell cultures derived from periodontal ligament. In native gingival epithelium, proliferation is confined to basal and parabasal cells. Keratin K14, when used as structural marker, is visible in the entire epithelium, while K13, an indicator of early differentiation, is restricted to the suprabasal cell compartment. Vimentin indicates mesenchymal cells in the subgingival connective tissue. Concerning the matrix molecules, collagen type-IV is abundant at the epithelium-lamina propria interface, and fibronectin is apparent throughout the mesenchyme. The matrix receptor integrin � 1 reveals a pericellular localization in basal and parabasal cells, while focal adhesion kinase p125 FAK is seen pericellularly in all epithelial layers. Cultures of primary periodontal ligament (PDL) fibroblasts (PDL-F) reveal expression of vimentin, strong proliferation, synthesis and extracellular deposition of collagen type-I and fibronectin. The integrin subunits � 1 and p125 FAK are largely detectable at the cell periphery.
Uploads
Papers by Thorsten Steinberg