Piezo1 and BKCa channels in human atrial fibroblasts: Interplay and remodelling in atrial fibrillation.

AIMS: Atrial Fibrillation (AF) is an arrhythmia of increasing prevalence in the aging populations of developed countries. One of the important indicators of AF is sustained atrial dilatation, highlighting the importance of mechanical overload in the pathophysiology of AF. The mechanisms by which atrial cells, including fibroblasts, sense and react to changing mechanical forces, are not fully elucidated. Here, we characterise stretch-activated ion channels (SAC) in human atrial fibroblasts and changes in SAC- presence and activity associated with AF. METHODS AND RESULTS: Using primary cultures of human atrial fibroblasts, isolated from patients in sinus rhythm or sustained AF, we combine electrophysiological, molecular and pharmacological tools to identify SAC. Two electrophysiological SAC- signatures were detected, indicative of cation-nonselective and potassium-selective channels. Using siRNA-mediated knockdown, we identified the cation-nonselective SAC as Piezo1. Biophysical properties of the potassium-selective channel, its sensitivity to calcium, paxilline or iberiotoxin (blockers), and NS11021 (activator), indicated presence of calcium-dependent 'big potassium channels' (BKCa). In cells from AF patients, Piezo1 activity and mRNA expression levels were higher than in cells from sinus rhythm patients, while BKCa activity (but not expression) was downregulated. Both Piezo1-knockdown and removal of extracellular calcium from the patch pipette resulted in a significant reduction of BKCa current during stretch. No co-immunoprecipitation of Piezo1 and BKCa was detected. CONCLUSIONS: Human atrial fibroblasts contain at least two types of ion channels that are activated during stretch: Piezo1 and BKCa. While Piezo1 is directly stretch-activated, the increase in BKCa activity during mechanical stimulation appears to be mainly secondary to calcium influx via SAC such as Piezo1. During sustained AF, Piezo1 is increased, while BKCa activity is reduced, highlighting differential regulation of both channels. Our data support the presence and interplay of Piezo1 and BKCa in human atrial fibroblasts in the absence of physical links between the two channel proteins.

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

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

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

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

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

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

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

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

Biomechanical Modulation of Dental Pulp Stem Cell (DPSC) Properties for Soft Tissue Engineering.

Dental pulp regeneration strategies frequently result in hard tissue formation and pulp obliteration. The aim of this study was to investigate whether dental pulp stem cells (DPSCs) can be directed toward soft tissue differentiation by extracellular elasticity. STRO-1-positive human dental pulp cells were magnetically enriched and cultured on substrates with elasticities of 1.5, 15, and 28 kPa. The morphology of DPSCs was assessed visually. Proteins relevant in mechanobiology ACTB, ITGB1, FAK, p-FAK, TALIN, VINCULIN, PAXILLIN, ERK 1/2, and p-ERK 1/2 were detected by immunofluorescence imaging. Transcription of the pulp marker genes BMP2, BMP4, MMP2, MMP3, MMP13, FN1, and IGF2 as well as the cytokines ANGPT1, VEGF, CCL2, TGFB1, IL2, ANG, and CSF1 was determined using qPCR. A low stiffness, i.e., 1.5 kPa, resulted in a soft tissue-like phenotype and gene expression, whereas DPSCs on 28 kPa substrates exhibited a differentiation signature resembling hard tissues with a low cytokine expression. Conversely, the highest cytokine expression was observed in cells cultured on intermediate elasticity, i.e., 15 kPa, substrates possibly allowing the cells to act as "trophic mediators". Our observations highlight the impact of biophysical cues for DPSC fate and enable the design of scaffold materials for clinical pulp regeneration that prevent hard tissue formation.

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-1 beta 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.

On the biocompatibility of endodontic sealers.

Periapical tissue may be exposed to root canal filling materials in consequence of root canal therapy. There is scant scientific data about the biocompatibility of root canal filling materials of various chemistry on the periapical area. This study aimed to investigate the effects of different root canal sealers and their eluates on human alveolar osteoblasts in terms of cell proliferation, adhesion, morphology and gene expression in vitro. Five endodontic sealers (AH Plus®, Apexit®, Tubli-Seal®, Real Seal SE®, EndoRez®) and one gutta-percha obturation material (BeeFill®) were tested. Human alveolar osteoblasts derived from 3 different donors following incubation with sealer eluates after 24 h and 72 h were investigated by means of qPCR (gene expression). Morphological reactions of the alveolar osteoblasts were measured by culturing the cells for 3 d, and 7 d and 14 d, respectively, followed by scanning electron microscopy (morphology, adhesion) and fluorescence imaging of the actin cytoskeleton (morphology, proliferation). A repeated measures analysis was performed and p-values were adjusted by Tukey. While all sealers influenced the cell morphology and the expression of genes associated with apoptosis (Casp3), proliferation (histone H3), and inflammation (interleukin-6 and matrix metalloproteinases 1 and 3), mainly AH Plus® and Apexit® yielded a regular actin cytoskeleton and beneficial gene expression patterns. Regarding cell adhesion, only AH Plus® supported proper anchorage for alveolar osteoblasts. Our results provide evidence for the biocompatibility of epoxy resin-based endodontic sealers, i.e. AH Plus®, while other sealers proved cytotoxic for alveolar osteoblasts. Further studies are needed for understanding the bone cell reactions after endodontic treatment and the clinical decision-making regarding the sealer of choice for root canal fillings.

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.

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.

Does the human skeletal muscle harbor the murine equivalents of cardiac precursor cells?

The limited plasticity of adult muscle- or bone marrow- derived stem cells intended for cardiac regeneration impedes their conversion into cardiomyocytes. Since murine skeletal muscle was reported to harbor cardiac precursor cells, we assessed whether similar cells exist in man. Skeletal muscle biopsies obtained from 39 patients were sorted by flow cytometry which generated three populations (CD90+/CD34(-), CD34+/CD90(-), CD90(-)/CD34(-)) expressing similar levels of cardiac (Nkx2.5, cTn-T, cTn-I, Cx43) and skeletal muscle (Myf-5, MyoD, myogenin) mRNAs, as assessed by quantitative reverse transcriptase-PCR. However, compared to unpurified myoblasts, CD34+/CD90(-) cells expressed greater amounts of endothelium-specific mRNAs and were, therefore, selected for transplantation experiments. Thirty immunosuppressed rats then underwent coronary artery ligation and, 4 weeks later, were intramyocardially injected with culture medium, myoblasts, or CD34+/CD90(-) cells. After 1 month, left ventricular ejection fraction was significantly higher in the CD34+/CD90(-) group than in the control and myoblast-injected hearts, which was associated with smaller fibrosis and greater angiogenesis. The low engraftment rate suggested a paracrine mechanism supported by the greater release of growth factors by CD34+/CD90(-) cells than by unsorted myoblasts. In conclusion, the human skeletal muscle does not harbor cardiac-specified cells but contains a CD34+ fraction endowed with an angiogenic potential providing superior functional and structural benefits.

Effect of high glucose levels and lipopolysaccharides-induced inflammation on osteoblast mineralization over sandblasted/acid-etched titanium surface.

BACKGROUND AND PURPOSE: Poorly controlled diabetes mellitus has been related to higher risk of implant treatment complications due to increased susceptibility to infection and delayed wound healing. Lipopolysaccharides (LPS) stimulate cytokine production leading to chronic inflammation and immunological host response that accentuates the destruction of periodontal tissues. This study aimed to evaluate the effect of different glycemic conditions on secretion and mineralization of bone matrix under sterile inflammation induced by LPS on osteoblasts seeded over sandblasted/acid-etched (SLA) titanium surface. MATERIALS AND METHODS: Osteoblast cell viability was performed to determine the influence of different glucose concentrations (5.5, 8, 12, and 24 mM), which were chosen to reflect normal, postprandial, and high glucose values, similar to those typically seen in Diabetes mellitus under clinical conditions. Cells were seeded on titanium SLA discs (Straumann AG, Waldenburg, Switzerland) and exposed to glucose concentrations and LPS (1µg/mL) in order to test inflammatory response (qPCR) and mineralization (Alizarin Red staining). RESULTS: Osteoblast viability was severely decreased when exposed to higher glucose levels (≥12 mM) and LPS (P < .05) compared to control. When the osteoblasts were exposed to LPS and glucose at ≥8 mM, the gene transcripts of inflammatory cytokines were ≈2.5-fold upregulated, while ≤8 mM glucose elicited no significant change compared to control without glucose treatment (P > .05). Osteoblasts exposed to LPS produced sparse extracellular matrix mineralization, especially combined with higher glucose values (≥12 mM), together with decreased calcium deposition compared to control (P < .05). CONCLUSIONS: High glucose levels combined with LPS inflammatory stimulation elicited an adverse effect on the volume and quality of mineralized hard tissue formation on SLA titanium surfaces in vitro. Hence, both normal glucose levels and infection control including low levels of circulating LPS during initial osseointegration period may be required to increase the success rate of dental implants.

Cellular transcriptional response to zirconia-based implant materials.

OBJECTIVE: To adequately address clinically important issues such as osseointegration and soft tissue integration, we screened for the direct biological cell response by culturing human osteoblasts and gingival fibroblasts on novel zirconia-based dental implant biomaterials and subjecting them to transcriptional analysis. METHODS: Biomaterials used for osteoblasts involved micro-roughened surfaces made of a new type of ceria-stabilized zirconia composite with two different topographies, zirconium dioxide, and yttria-stabilized zirconia (control). For fibroblasts smooth ceria- and yttria-stabilized zirconia surface were used. The expression of 90 issue-relevant genes was determined on mRNA transcription level by real-time PCR Array technology after growth periods of 1 and 7 days. RESULTS: Generally, modulation of gene transcription exhibited a dual dependence, first by time and second by the biomaterial, whereas biomaterial-triggered changes were predominantly caused by the biomaterials' chemistry rather than surface topography. Per se, modulated genes assigned to regenerative tissue processes such as fracture healing and wound healing and in detail included colony stimulating factors (CSF2 and CSF3), growth factors, which regulate bone matrix properties (e.g. BMP3 and TGFB1), osteogenic BMPs (BMP2/4/6/7) and transcription factors (RUNX2 and SP7), matrix collagens and osteocalcin, laminins as well as integrin ß1 and MMP-2. SIGNIFICANCE: With respect to the biomaterials under study, the screening showed that a new zirconia-based composite stabilized with ceria may be promising to provide clinically desired periodontal tissue integration. Moreover, by detecting biomarkers modulated in a time- and/or biomaterial-dependent manner, we identified candidate genes for the targeted analysis of cell-implant bioresponse during biomaterial research and development.

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.

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.

Crosstalk on cell behavior in interactive cocultures of hMSCs with various oral cell types.

When prospectively applied for regenerative therapies, human bone-marrow-derived mesenchymal stem cells (hMSCs) interact with the locally residing host cells. With respect to the developmentally particular origin of oral cells, little is known about the putatively discriminative behavioral responses of hMSCs in interaction with various oral cell types, including human alveolar bone osteoblasts (hOAs), periodontal ligament fibroblasts (hPDLs), and gingival fibroblasts (hGFs). To assess the crosstalk between hMSCs and oral cells, interactive cocultures were established by combining well-characterized hMSCs with hOAs, hPDLs, or hGFs, and the behavioral hMSC aspects, that is, proliferation and gene expression, were measured by employing a 5-bromo-2'-deoxyuridine assay and real-time polymerase chain reaction, while apoptosis was quantified by in situ cell death detection kit. hMSCs expressed the typical antigen spectrum lacking CD34, CD45, CD14, CD19, and HLA-DR, while expressing CD73, CD90, and CD105, and could successfully be transformed into adipocytes, osteocytes, and chondrocytes. Monocultured control hMSCs proliferated readily, whereas a general reduction of BrdU-labeled cells was observed in cocultures. Globally, upon extending time periods, interactive coculture combinations of hMSCs with hOAs reduced both osteogenic gene and stem cell marker transcription in hMSCs, a phenomenon appearing less pronounced by combining hMSCs with hPDLs, such that the observed effects in terms of proliferation and gene expression followed the same ranking: hOAs>hGFs>hPDLs. Vice versa, in interactive hMSC cocultures, the cell survival rate was significantly increased, irrespective from the combined coculture cell counterpart. Our results show for the first time that behavior of hMSCs reflected by proliferation and gene expression was governed by interaction with various oral cells in a cell-type-discriminative manner. In addition, hMSC coculture restrains apoptosis, such that influences on cell behavior appear as a crosstalk. In summary, interactive cocultures render the basis for a prospective prediction of mutual cell behavior in hMSC-based oral tissue regeneration disclosing that oral cells shift hMSC behavior from proliferation to differentiation and apoptosis-repressing features.

Strain response in fibroblasts indicates a possible role of the Ca(2+)-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.