Associations of parental age at pregnancy with adolescent cognitive development and emotional and behavioural problems: a birth cohort in rural Western China.

BACKGROUND: The relationship between parental age at pregnancy and offspring development in low- and middle-income countries remains unclear. We aimed to examine the associations of parental age at pregnancy with adolescent development in rural China. METHODS: We conducted a prospective birth cohort study of offspring born to pregnant women who participated in an antenatal micronutrient supplementation trial in rural Western China. Adolescent cognitive development and emotional and behavioural problems were assessed by using the Wechsler Intelligence Scale for Children-IV and the Youth Self-Report-2001, respectively. After accounting for the possible nonlinear relationships, we examined the linear associations between parental age (in years) at pregnancy and scores of adolescent cognitive development and emotional and behavioural problems by performing generalized estimating equations. RESULTS: Among 1897 adolescents followed from birth to early adolescence, 59.5% were male with a mean age of 11.8 (standard deviation (SD): 0.8) years. The mean ages of mothers and fathers at pregnancy were 24.6 (SD: 4.4) and 27.9 (SD: 4.1) years old, respectively. All the P values of the nonlinear terms between parental age and adolescent development in all domains were greater than 0.05. Each one-year increase in maternal age at pregnancy was associated with a 0.29-point (95% confidence interval (CI) 0.06, 0.52) increase in the full-scale intelligence quotient in early adolescence. After parental age was categorized into quartiles, the total behavioural problem scores of adolescents with fathers with an age in the fourth quartile (Q4) were 6.71 (95% CI 0.86, 12.57) points higher than those of adolescents with fathers with an age in the first quartile (Q1), with a linear trend P value of 0.01. Similarly, higher scores (worse behavioural problems) were observed for internalizing behavioural problems and other emotional and behavioural symptoms related to anxiety, withdrawal, social problems, thought problems and aggressive behaviour. CONCLUSIONS: At conception, older maternal age was independently linked to better adolescent cognitive development, whereas advanced paternal age was independently associated with a greater risk of adolescent emotional and behavioral problems. These findings suggest that public health policies targeting an optimal parental age at pregnancy should be developed in the context of offspring developmental consequences.

Predicting YAP/TAZ nuclear translocation in response to ECM mechanosensing.

Cells translate mechanical cues from the extracellular matrix (ECM) into signaling that can affect the nucleus. One pathway by which such nuclear mechanotransduction occurs is a signaling axis that begins with integrin-ECM bonds and continues through a cascade of chemical reactions and structural changes that lead to nuclear translocation of YAP/TAZ. This signaling axis is self-reinforcing, with stiff ECM promoting integrin binding and thus facilitating polymerization and tension in the cytoskeletal contractile apparatus, which can compress nuclei, open nuclear pore channels, and enhance nuclear accumulation of YAP/TAZ. We previously developed a computational model of this mechanosensing axis for the linear elastic ECM by assuming that there is a linear relationship between the nucleocytoplasmic ratio of YAP/TAZ and nuclear flattening. Here, we extended our previous model to more general ECM behaviors (e.g., viscosity, viscoelasticity, and viscoplasticity) and included detailed YAP/TAZ translocation dynamics based on nuclear deformation. This model was predictive of diverse mechanosensing responses in a broad range of cells. Results support the hypothesis that diverse mechanosensing phenomena across many cell types arise from a simple, unified set of mechanosensing pathways.

Effect of ILK on small-molecule metabolism of human periodontal ligament fibroblasts with mechanical stretching.

BACKGROUND: Mechanical stimuli can cause periodontal tissue reconstruction. Studies have found that changes in metabolites can be the terminal effect of integrin-mediated mechanical signaling. As a key kinase in integrin regulation, integrin-linked kinase (ILK) mediates mechanical signal transduction, which may contribute to metabolite changes. Defining the components of small-molecule metabolites can optimize mechanical stimuli and periodontal tissue reconstruction. Our purpose is to detect the effect of ILK-mediated mechanical signaling on intracellular small-molecule metabolites (amino acids and organic acids) in human periodontal ligament fibroblasts (HPDLFs). METHODS: Primary HPDLFs were isolated by enzyme digestion method. Tensile stresses were applied on HPDLFs in vitro using a Flexcell system. ILK gene in HPDLFs was knocked down by RNA interference (RNAi). Twenty common amino acids and seven organic acids in HPDLFs were analyzed by gas chromatography/mass spectrometry technique. RESULTS: Five amino acids (ie, alanine, glutamine, glutamate, glycine, and threonine) and three organic acids (ie, pyruvate, lactate, and citric acid) were found to be changed remarkably after mechanical stretching. In addition, baseline levels of four amino acids (ie, glutamate, glutamine, threonine, and glycine) and two organic acids (ie, lactate and citric acid) were significantly different in ILK knockdown compared with wild-type HPDLFs. CONCLUSION: This study suggests that five amino acids (ie, alanine, glutamine, glutamate, glycine, and threonine) and three organic acids (ie, pyruvate, lactate, and citric acid) may act as cellular mediators for mechanical signals in HPDLFs. Among them, four amino acids (ie, glutamate, glutamine, threonine, and glycine) and two organic acids (ie, lactate and citric acid) may be closely linked to ILK.

shRNA knockdown of integrin-linked kinase on hPDLCs migration, proliferation, and apoptosis under cyclic tensile stress.

OBJECTIVE: To investigate the roles of integrin-linked kinase (ILK) in mediating the cell migration, proliferation, and apoptosis of human periodontal ligament cells (hPDLCs) in response to cyclic tensile stress. METHODS: Primary hPDLCs were obtained through the enzyme digestion and tissue culture method. Short hairpin ILK-expressing hPDLCs were constructed using a recombinant lentiviral vector that specifically targeted ILK gene expression. The silencing of the ILK gene was identified by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The hPDLCs were seeded on a flexible substrate and loaded with cyclic tensile stress at 0.5 Hz for 0, 2, 4, and 8 hr, consecutively, with the Flexcell Tension System. The response of cell migration was tested by the scratch assay. Cell proliferation was characterized by optical density (OD) value of cell counting kit-8 (CCK-8) test and Ki67 mRNA expression of qRT-PCR. Cell apoptosis was determined by flow cytometry and Caspase-3 mRNA expression of qRT-PCR. RESULTS: Knocking down ILK substantially reduces migration and proliferation as well as regulates the sensitivity of hPDLCs to apoptosis under cyclic tensile stress. CONCLUSIONS: ILK can promote the proliferation and migration as well as inhibit apoptosis of hPDLCs under cyclic tensile stress.

Synergistic Effect of Matrix Stiffness and Inflammatory Factors on Osteogenic Differentiation of MSC.

Mesenchymal stem cells (MSCs) in vivo reside in a complex microenvironment. Changes of both biochemical and biophysical cues in the microenvironment caused by inflammation affect the differentiation behaviors of MSCs. Most studies, however, only focus on either biochemical or biophysical cues, although the synergistic effect of matrix stiffness and inflammatory factors on osteogenic differentiation of MSCs has not been explored yet. Here, we showed that there was a matrix stiffness-dependent modulation in the osteogenic differentiation of human MSCs (hMSCs) with higher matrix stiffness favoring osteogenesis bias. However, when interleukin-1 ß (IL-1ß) was added, the osteogenic differentiation of hMSCs was suppressed, which was independent of increasing matrix stiffness. Both experimental observations and mathematical modeling confirmed that matrix stiffness and IL-1ß could activate the ERK1/2 signaling and contribute to osteogenic differentiation. The p38 signaling activated by IL-1ß has a strong role in inhibiting osteoblastic differentiation, thus diminishing the vital effect of ERK1/2 signaling. In addition, sensitivity analysis of the model parameters revealed that activation/deactivation dynamics of sensitive factors (e.g., FAK, ERK, and p38) also played a key role in the synergistic effect of matrix stiffness and IL-1ß on the osteogenic differentiation of hMSCs. The outcomes of this study provide new insights into the synergistic effect of biochemical and biophysical microenvironments on regulating MSC differentiation.

TGF-ß1 promotes osteogenesis of mesenchymal stem cells via integrin mediated mechanical positive autoregulation.

Positive autoregulation (PAR), one type of network motifs, provides a high phenotypic heterogeneity for cells to better adapt to their microenvironments. Typical mechanosensitive proteins can also form PAR, e.g., integrin mediated PAR, but the role of such mechanical PAR in physiological development and pathological process remains elusive. In this study, we found that transforming growth factor ß1 (TGF-ß1) and integrin levels decrease with tissue softening after the development of paradentium in vivo in rat model of periodontitis (an inflammatory disease with bone defect). Interestingly, TGF-ß1 could induce the formation of mechanical PAR involving the integrin-FAK-YAP axis in mesenchymal stem cells (MSCs) by both in vitro experiments and in silico computational model. The computational model predicted a mechanical PAR involving the bimodal distribution of focus adhesions, which enables cells to accurately perceive extracellular mechanical cues. Thus, our analysis of TGF-ß1 mediated mechanosensing mechanism on MSCs may help to better understand the molecular process underlying bone regeneration.

Substrate Stiffness Regulates the Proliferation and Apoptosis of Periodontal Ligament Cells through Integrin-Linked Kinase ILK.

The hallmark of orthodontic tooth movement (OTM) is time-consuming during clinical treatments. The acceleration of OTM through modulating proliferation and apoptosis of periodontal ligament cells (PDLCs) possesses the potential application in clinical treatments. Here, we established an in vitro model with a graded increase in substrate stiffness to investigate the underlying mechanism of proliferation and apoptosis of PDLCs. The role of the integrin-linked kinase (ILK) in response to substrate stiffness was investigated by the depletion model of PDLCs. We found that the proliferation and apoptosis of PDLCs show a stiffness-dependent property with stiffer substrates favoring increased bias at the transcript level. Depleting integrin-linked kinase diluted the correlation between PDLCs behaviors and substrate stiffness. Our results suggest that ILK plays a significant role in modulating PDLC proliferation and apoptosis and can serve as a potential target for accelerating OTM.

Nanoscale integrin cluster dynamics controls cellular mechanosensing via FAKY397 phosphorylation.

Transduction of extracellular matrix mechanics affects cell migration, proliferation, and differentiation. While this mechanotransduction is known to depend on the regulation of focal adhesion kinase phosphorylation on Y397 (FAKpY397), the mechanism remains elusive. To address this, we developed a mathematical model to test the hypothesis that FAKpY397-based mechanosensing arises from the dynamics of nanoscale integrin clustering, stiffness-dependent disassembly of integrin clusters, and FAKY397 phosphorylation within integrin clusters. Modeling results predicted that integrin clustering dynamics governs how cells convert substrate stiffness to FAKpY397, and hence governs how different cell types transduce mechanical signals. Existing experiments on MDCK cells and HT1080 cells, as well as our new experiments on 3T3 fibroblasts, confirmed our predictions and supported our model. Our results suggest a new pathway by which integrin clusters enable cells to calibrate responses to their mechanical microenvironment.

Point-of-Care Periodontitis Testing: Biomarkers, Current Technologies, and Perspectives.

Periodontitis has become one of the most universal chronic inflammatory diseases worldwide. Subclinical symptom progression, ultimately leading to permanent damage, calls for early diagnosis and long-term monitoring. However, traditional clinical diagnostic methods are complex and expensive, and cannot meet these requirements. Recently, with more biomarkers and the development of new technologies, various point-of-care testing (POCT) platforms have been developed for periodontitis diagnosis and monitoring. These are easy to perform, rapid, low-cost, and are perfectly suited for high-frequency diagnosis of periodontitis at the point-of-care (POC). We summarize existing biomarkers of different periodontitis stages and recent developed POCT platforms (including lab-on-a-chip, paper-based platforms, and chairside tests), discuss their existing challenges and future potential, and provide some inspiration and guidelines for future POC periodontitis testing.

Combining enamel matrix proteins with mechanical stimuli potentiates human periodontal ligament fibroblasts proliferation and periodontium remodeling.

BACKGROUND: Collagen I (Col-I) and matrix metalloproteinase-1 (MMP-1) have been implicated in the regeneration and remodeling of the periodontium. Studies have shown that enamel matrix proteins (EMPs) and mechanical stimuli can promote the synthesis and degradation, respectively, of Col-I and MMP-1. However, the effects of the combination of EMPs and mechanical stimuli on human periodontal ligament are not known. OBJECTIVE: Our aim was to test the combined effects of EMPs and mechanical stimuli on the proliferation of human periodontal ligament fibroblasts (HPDLFs) and Col-I and MMP-1 mRNA expression. METHODS: Primary HPDLFs were isolated using an enzyme digestion method. To select the optimum EMP concentration and the optimum magnitude and loading time of mechanical stimuli, HPDLFs were stimulated with gradient concentration of EMPs (0 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL and 200 µg/mL) and mechanical stimuli (0 kPa, 25 kPa, 50 kPa, 100 kPa, and 200 kPa for 0 h, 3 h, 6 h, 12 h, and 24 h), respectively. The cell proliferative response was tested by the MTT assay. The impact of EMPs combined with mechanical stimuli on Col-I and MMP-1 mRNA expression were measured by reverse transcription polymerase chain reaction. RESULTS: 100 µg/mL of EMPs and a 50 kPa mechanical stimulus were chosen as the optimum parameters due to the higher proliferation rates than other doses. The combination of 100 µg/mL of EMPs and a 50 kPa mechanical stimulus significantly stimulated HPDLFs proliferation and increased Col-I and MMP-1 expression levels compared with incubation with two factors alone. CONCLUSIONS: We concluded that the combination of EMPs and mechanical stimulus have synergistic effects on cell growth, cell number, collagen turnover, and periodontium remodeling.

Bioprinting-Based PDLSC-ECM Screening for in Vivo Repair of Alveolar Bone Defect Using Cell-Laden, Injectable and Photocrosslinkable Hydrogels.

Periodontitis is an inflammatory disease worldwide that may result in periodontal defect (especially alveolar bone defect) and even tooth loss. Stem-cell-based approach combined with injectable hydrogels has been proposed as a promising strategy in periodontal treatments. Stem cells fate closely depends on their extracellular matrix (ECM) characteristics. Hence, it is necessary to engineer an appropriate injectable hydrogel to deliver stem cells into the defect while serving as the ECM during healing. Therefore, stem cell-ECM interaction should be studied for better stem cell transplantation. In this study, we developed a bioprinting-based strategy to study stem cell-ECM interaction and thus screen an appropriate ECM for in vivo repair of alveolar bone defect. Periodontal ligament stem cells (PDLSCs) were encapsulated in injectable, photocrosslinkable composite hydrogels composed of gelatin methacrylate (GelMA) and poly(ethylene glycol) dimethacrylate (PEGDA). PDLSC-laden GelMA/PEGDA hydrogels with varying composition were efficiently fabricated via a 3D bioprinting platform by controlling the volume ratio of GelMA-to-PEGDA. PDLSC behavior and fate were found to be closely related to the engineered ECM composition. The 4/1 GelMA/PEGDA composite hydrogel was selected since the best performance in osteogenic differentiation in vitro. Finally, in vivo study indicated a maximal and robust new bone formation in the defects treated with the PDLSC-laden hydrogel with optimized composition as compared to the hydrogel alone and the saline ones. The developed approach would be useful for studying cell-ECM interaction in 3D and paving the way for regeneration of functional tissue.