Susanne Proksch

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.

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.

Environmental Biomechanics Substantiated by Defined Pillar Micropatterns Govern Behavior of Human Mesenchymal Stem Cells

While evidence on the impact of the biomechanical environment elasticity on human mesenchymal stem cell (hMSC) behavior is growing, the aspect of micropatterning is still poorly understood. Thus, the present study aimed at investigating the influence of defined environmental micropatterning on hMSC behavior. Following characterization, hMSCs were grown on defined pillar micropatterns of 5, 7, 9, and 11 μm. With respect to cell behavior, primary hMSC adhesion was detected by indirect immunofluorescence (iIF) for paxillin, vinculin, integrin αV, and actin, while proliferation was visualized by histone H3. Morphogenesis was monitored by scanning electron microscopy and the expression of stem cell-specific biomarkers by real-time PCR. Favoritism of primary adhesion of hMSCs on pillar tops occurred at smaller pillar micropatterns, concomitant with cell flattening. While vinculin, integrin αV, and paxillin appeared initially more cytoplasmic, high pillar micropatterns favored a progressive redistribution with polarization to cell tension sites and at cell borders. Accomplishment of morphogenesis at day 3 revealed establishment of fully rotund cell somata at 5 μm, while hMSCs appeared progressively elongated at rising micropatterns. The hMSC proliferation capacity was influenced by pillar micropatterns and gene expression analysis of stem cell- and differentiation-associated biomarkers disclosed clear modulation by distinct pillar micropatterns. In response to environmental biomechanics, our results show that hMSC behavior is governed by pillar micropatterning. In turn, these findings may form the basis to prospectively direct lineage specificity of hMSCs in a customized fashion.

Melatonin as a Candidate Therapeutic Drug for Protecting Bone Cells From Chlorhexidine-Induced Damage

BACKGROUND Melatonin was proposed for use in periodontitis and peri-implantitis therapy due to its bone-supportive effects. This issue is of interest because standard adjuvant antiseptics, namely chlorhexidine (CHX), prove damaging for osteoblasts. Thus, the aim of this study is to investigate if melatonin is suitable as an auxiliary agent for protecting osteoblasts from CHX damage. METHODS MC3T3 osteoblast response was determined following administration of various CHX concentrations in the absence or presence of melatonin. Osteoblast morphology was evaluated, total reactive oxygen species (ROS) and superoxide levels were quantified, ratios of apoptotic and necrotic cells were identified by flow cytometry, metabolic activity of remaining cells was assessed, and effects were calculated with repeated measures analysis and post hoc P value adjustment. RESULTS CHX led to poor morphology, increased total ROS and superoxide levels, and rigorously diminished the number of vital and metabolic active osteoblasts in a concentration-dependent manner. However, simultaneous melatonin supply supported cell morphogenesis and growth, reduced ROS and superoxide generation, shifted the percentage of CHX-damaged cells from necrotic/late to early apoptotic events, and modulated metabolic activity in osteoblasts. CONCLUSION These data reveal that melatonin protects osteoblasts in the CHX context, thereby implicating melatonin as a promising drug in periodontitis and peri-implantitis treatment.

Strain response in fibroblasts indicates a possible role of the Ca2+-dependent nuclear transcription factor NM1 in RNA synthesis

On the mechanistic level, response of periodontal fibroblasts permanently exposed to mechanical strain forces in vivo still lacks in clarity. Therefore, we first investigated putative strain modulation of proteins by combined 1D gel electrophoresis-based protein profiling and electrospray tandem mass spectrometry (ESI-MS). Thereafter, the exponential-modified protein abundance index (emPAI) identified strain modulation of cytoskeleton-associated molecules, including decrease in talin and microtubule-associated protein 4 (MAP4), and significant increase in myosin IC (Myo IC), the latter ones regulated by Ca(2+). These findings were corroborated by western blotting (WB) and indirect immunofluorescence (IIF). Regarding the dual function of Myo IC as actin-based cytoplasmic motor protein and nuclear transcription factor NM1, WB and IIF revealed inverse correlation for Myo IC and NM1. During strain application, cytoplasmic increase of Myo IC was counteracted by nuclear NM1 deprivation, the latter coinciding with a decline in RNA quantity. Independent on strain, cytoplasmic Myo IC and nuclear NM1 abundance could be abrogated by the Ca(2+) channel blocker nifedipine, suggesting Ca(2+) dependency of cytoplasmic and/or nuclear Myo IC/NM1 expression. Mechanistically, we conclude that, application of strain appears as causative for the decline in RNA by impacting NM1, thereby indicating the possible role of NM1 in RNA synthesis.

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

Abstract 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. Graphical abstract Figure. No Caption available. HighlightsStrain‐induced modulation of nuclear YAP amount coincides with proliferation.YAP‐inhibiting drug Verteporfin yields distinct reduction of KI‐67‐positive cells.This demonstrates regulatory involvement of nuclear YAP in proliferation.Strain‐induced YAP modulation is mechanistically independent from ERK1/2 activity.ERK1/2 activity influences total YAP amount, exclusively in strained cells.

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 β1, 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. Stem Cells 2015;33:3114—3124

Human saliva exposure modulates bone cell performance in vitro

Various situations encountered by a clinician during the daily routine including surgical periodontitis therapy, dental implant insertion, or tooth extraction involve the contact of saliva with the jaw bone. However, there are only sparse data concerning the influence of saliva on bone cells. Saliva specimens were incorporated within culture medium and administered to murine MC3T3 osteoblasts, of which the morphology (REM), proliferation (EZ4U), and differentiation (qRT-PCR, alkaline phosphatase activity, extracellular matrix calcification) were assessed. Simultaneously, the composition of saliva media was analyzed with respect to the content of lactoferrin, activities of classical salivary enzymes, and the ability to provoke inflammatory cytokine production (enzyme-linked immunosorbent assay) in MC3T3 osteoblasts. The morphology, proliferation, and expression of differentiation-associated genes were seriously handicapped by saliva contact. Saliva-touched cells exhibited less alkaline phosphatase but normal levels of extracellular matrix mineralization. Saliva-containing culture media featured physiological activities of salivary enzymes and considerable amounts of lactoferrin but almost completely lacked salivary alkaline phosphatase and unspecific proteases. Upon saliva incubation, MC3T3 osteoblasts did not release noteworthy levels of interleukin-1beta or tumor necrosis factor alpha. Although saliva is generally considered to vitalize oral tissues, this study reveals that it harms osteoblast-like cells more due to the presence of salivary enzymes than by triggering of inflammation. This issue is clinically relevant because it broadens the understanding of the bone cell fate within the rather complex cosmos of the oral cavity thereby providing a basis for clinical decision making and treatment guidelines. It seems to be reasonable to restrict the contact period between saliva and bone.

Effect of gabapentin-lactam and gamma-aminobutyric acid/lactam analogs on proliferation and phenotype of ovine mesenchymal stem cells.

PURPOSE Classic tissue engineering consists of three components: scaffold, cells, and growth or differentiation factors. Currently, expensive bone morphogenetic proteins are the most common substance used for hard tissue regeneration. An alternative could be gamma-aminobutyric acid/lactam (GABA-lactam) analogs. MATERIALS AND METHODS The effects of gabapentin-lactam, cis- and trans-8-tertbutyl-GABA-pentinlactam (trans-TB-GBP-L), and phenyl-GABA-lactam were tested in this study on ovine mesenchymal stem cell (MSC) proliferation. MSCs were selected from bone marrow aspirate concentrate by plastic adherence and amplified. Aliquots of the cells were incubated in medium, with four different concentrations of the GABA-lactam analogs dissolved in dimethyl sulfoxide. Cells in medium with and without dimethyl sulfoxide served as controls. Cell proliferation was tested with a nonradioactive assay. Before and after GABA-lactam analog influence, the MSC character was evaluated by the ability of the cells to differentiate into osteoblasts, chondrocytes, and adipocytes. RESULTS Proliferation was significantly increased under the influence of the analogs, depending on their concentration. MSCs cultured in 1 nmol/L trans-TB-GBP-L showed the highest proliferation rate. The MSC character was not altered. CONCLUSIONS GABA-lactam analogs could be suited to stimulate MSC proliferation for tissue engineering applications. Further in vivo studies are necessary to evaluate the possible clinical potential of GABA-lactam analogs for hard tissue regeneration.

Comparative differentiation analysis of distinct oral tissue-derived cells in response to osteogenic stimulation

BackgroundMixed cell populations from oral tissues may be superior to pure stem cells for regenerative approaches. Therefore, the aim of the present study was to explore the osteogenic potential of mixed cells derived from oral connective tissues compared to alveolar osteoblasts.Materials and methodsPrimary cells were isolated from the alveolar bone, periodontal ligament and gingiva. Following characterization by colony formation, growth capacity and flow cytometry, all cells were subjected to osteogenic differentiation induction and screened for a large panel of osteogenic markers using western blots, qPCR arrays, and matrix mineralization and alkaline phosphatase quantification.ResultsNon-induced mixed cells from gingiva showed higher colony formation efficiency but decreased proliferation compared to non-induced periodontal mixed cells, while both entities revealed similar surface markers tested in this setup. Following osteogenic induction, all cell populations individually expressed receptors with distinctively activated downstream effectors. Gene expression of induced periodontal mixed cells was similar to alveolar osteoblasts, but was differently modulated in gingival mixed cells. The latter failed to achieve osteogenic differentiation in terms of matrix mineralization and alkaline phosphatase activity, which was well observed in periodontal mixed cells and osteoblasts.ConclusionMixed cells from periodontal ligament but not from gingiva feature an inherent osteogenic capacity in vitro. From these results, it can be concluded that periodontal cells do not require further stem cell enrichment in order to qualify for bone regeneration.Clinical relevanceOur data contribute to the development of novel cell-based therapies using mixed cells from the periodontal ligament in regenerative periodontics.