Asymmetric Cyclodextrin-Dimer-Involved Nanoassemblies by Selective Host-Guest Interactions: Concentration-Dependent Morphology Evolution and Light-Regulated Biomedical Applications.

Supramolecular assembly has attracted significant attention and has been applied to various applications. Herein, a ß-γ-CD dimer was synthesized to complex different guest molecules, including single-strand polyethylene glycol (PEG)-modified C60 (PEG-C60), photothermal conversion reagent (IR780), and dexamethasone (Dexa), according to the complexation constant-dependent specific selectivity. Spherical or cylindrical nanoparticles, monolayer or bilayer vesicles, and bilayer fusion vesicles were discovered in succession if the concentration of PEG-C60 was varied. Moreover, if near-infrared light was employed to irradiate these nanoassemblies, the thermo-induced morphological evolution, subsequent cargo release, photothermal effect, and singlet oxygen (1O2) generation were successfully achieved. The in vitro cell experiments confirmed that these nanoparticles possessed excellent biocompatibility in a normal environment and achieved superior cytotoxicity by light regulation. Such proposed strategies for the construction of multilevel structures with different morphologies can open a new window to obtain various host-guest functional materials and achieve further use for disease treatment.

Analyses of key mRNAs and lncRNAs for different osteo-differentiation potentials of periodontal ligament stem cell and gingival mesenchymal stem cell.

Both human periodontal ligament stem cells (hPDLSCs) and human gingival mesenchymal stem cells (hGMSCs) are candidate seed cells for bone tissue engineering, but the osteo-differentiation ability of the latter is weaker than the former, and the mechanisms are unknown. To explore the potential regulation of mRNAs and long non-coding RNAs (lncRNAs), this study obtained the gene expression profiles of hPDLSCs and hGMSCs in both undifferentiated and osteo-differentiated conditions by microarray assay and then analysed the common and specific differentially expressed mRNAs and lncRNAs in hPDLSCs and hGMSCs through bioinformatics method. The results showed that 275 mRNAs and 126 lncRNAs displayed similar changing patterns in hPDLSCs and hGMSCs after osteogenic induction, which may regulate the osteo-differentiation in both types of cells. In addition, the expression of 223 mRNAs and 238 lncRNAs altered only in hPDLSCs after osteogenic induction, and 177 mRNAs and 170 lncRNAs changed only in hGMSCs. These cell-specific differentially expressed mRNAs and lncRNAs could underlie the different osteo-differentiation potentials of hPDLSCs and hGMSCs. Finally, dickkopf Wnt signalling pathway inhibitor 1 (DKK1) was proved to be one regulator for the weaker osteo-differentiation ability of hGMSCs through validation experiments. We hope these results help to reveal new mRNAs-lncRNAs-based molecular mechanism for osteo-differentiation of hPDLSCs and hGMSCs and provide clues on strategies for improving stem cell-mediated bone regeneration.

Downregulation of Prolactin-Induced Protein Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells.

BACKGROUND Periodontal ligament stem cells (PDLSCs) are promising seed cells for bone tissue engineering and periodontal regeneration applications. However, the mechanism underlying the osteogenic differentiation process remains largely unknown. Previous reports showed that prolactin-induced protein (PIP) was upregulated after PDLSCs osteogenic induction. However, few studies have reported on the function of PIP in osteogenic differentiation. The purpose of the present study was to investigate the effect of PIP on osteogenic differentiation of PDLSCs. MATERIAL AND METHODS The expression pattern of PIP during PDLSCs osteogenic differentiation was detected and the effect of each component in the osteogenic induction medium on PIP was also tested by qRT-PCR. Then, the PIP knockdown cells were established using lentivirus. The knockdown efficiency was measured and the proliferation, apoptosis, and osteogenic differentiation ability were examined to determine the functional role of PIP on PDLSCs. RESULTS QRT-PCR showed that PIP was sustainedly upregulated during the osteogenic induction process and the phenomenon was mainly caused by the stimulation of dexamethasone in the induction medium. CCK-8 and flow cytometer showed that knocking down PIP had no influence on proliferation and apoptosis of PDLSCs. ALP staining and activity, Alizarin Red staining, and western blot analysis demonstrated PIP knockdown enhanced the osteogenic differentiation and mineralization of PDLSCs. CONCLUSIONS PIP was upregulated after osteogenic induction; however, PIP knockdown promoted PDLSCs osteogenic differentiation. PIP might be a by-product of osteogenic induction, and downregulating of PIP might be a new target in bone tissue engineering applications.

A comprehensive study on donor-matched comparisons of three types of mesenchymal stem cells-containing cells from human dental tissue.

BACKGROUND AND OBJECTIVE: Mesenchymal stem cells (MSCs) have been widely used in tissue engineering, such as for regenerating the supporting structures of teeth destroyed by periodontal diseases. In recent decades, dental tissue-derived MSCs have drawn much attention owing to their accessibility, plasticity and applicability. Dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs) and gingival MSCs (GMSCs) are the most readily available MSCs among all types of dental MSCs. The purpose of this study was to comprehensively compare the characteristics of MSCs from dental pulp (DP), periodontal ligament (PDL) and gingiva (G) in vitro and thus provide insight into optimizing the performance of cells and seed cell selection strategies for tissue regeneration. MATERIALS AND METHODS: In this study, patient-matched (n = 5) cells derived from DP, PDL and G which, respectively, contained DPSCs, PDLSCs and GMSCs were evaluated using multiple methods in terms of their proliferation, senescence, apoptosis, multilineage differentiation and stemness maintenance after long-term passage. RESULTS: Mesenchymal stem cells-containing cells from G (MSCs/GCs) showed superior proliferation capability, whereas patient-matched MSCs-containing cells from PDL (MSCs/PDLCs) exhibited excellent osteogenic and adipogenic differentiation ability; MSCs-containing cells from DP (MSCs/DPCs) achieved mediocre results in both aspects. In addition, MSCs/GCs were the least susceptible to senescence, while MSCs/PDLCs were the most prone to ageing. Furthermore, the biological properties of these three types of cells were all affected after long-term in vitro culture. CONCLUSION: These three types of dental MSCs showed different biological characteristics. MSCs/PDLCs are the best candidate cells for bone regeneration, but the application of MSCs/PDLCs might be limited to certain number of passages. Improving the differentiation of MSCs/GCs remains the key issue regarding their application in tissue engineering.

The Crosstalk between HDPSCs and HUCMSCs on Proliferation and Osteogenic Genes Expression in Coculture System.

Objectives: The present study established a non-contact coculture system in vitro, aiming to investigate the crosstalk between human dental pulp stem cells (hDPSCs) and human umbilical cord mesenchymal stem cells (hUCMSCs) on proliferation activity and osteogenic genes expression through paracrine. Materials and methods: The stemness of hDPSCs and hUCMSCs were identified by flow cytometric analysis and multipotential differentiation assays. With the help of transwell inserts, the non-contact coculture system in vitro was established between hDPSCs and hUCMSCs. EdU labeling analysis and Western Blot were used to detect the proliferation activity. The mRNA and protein levels of osteogenic genes were evaluated by RT-PCR and Western Blot. The expression of elements in Akt/mTOR signaling pathway were detected by Western Blot. Results: Both hDPSCs and hUCMSCs were positive to MSCs specific surface markers and had multi-differentiation potential. The proportion of EdU-positive cells increased and the expression of CDK6 and CYCLIN A were up-regulated in cocultured hDPSCs. Both prior coculture and persistent coculture improved mRNA and protein levels of osteogenic genes in hDPSCs. While in cocultured hUCMSCs, no statistical differences were observed on proliferation and osteogenesis. The phosphorylation of Akt and mTOR was up-regulated in cocultured hDPSCs. Conclusions: The crosstalk between hDPSCs and hUCMSCs in coculture system increased the proliferation activity and enhanced osteogenic genes expression in hDPSCs. Akt/mTOR signaling pathway might take part in the enhancing effects in both cell proliferation and gene expression.

Nanoparticles-encapsulated doxorubicin alleviates drug resistance of osteosarcoma via inducing ferroptosis.

To determine the effects of polymeric nanoparticle for doxorubicin (Dox) delivery and treatment of drug-resistant Osteosarcoma (OS) cells. Methoxy-polyethylene glycol amino (mPEG-NH2) and platinum bio-mimetic polycaprolactone-cysteine (PtBMLC) were crosslinked to obtain glutathione (GSH)-responsive mPEG-NH2-PtBMLC polymer to encapsulate Dox (named as Nano-Dox). The particle size and zeta potential of the nanoparticles were measured, and internalization of Dox by OS cells was observed. After treatment with Nano-Dox, cell proliferation was determined by cell counting kit 8 (CCK-8) and colony formation assay. Cell migration and invasion were determined by Transwell assay. Cell cycle arrest was assessed by flow cytometry. The induction of ferroptosis was analyzed by abnormal accumulation of total iron, Fe2+. Nano-Dox exhibited a stronger localization in OS cells (p < 0.01). Nano-Dox induced more significant suppression of drug-resistant OS cell growth (p < 0.01), migration (p < 0.01), and invasion (p < 0.01), compared with the single Dox treatment group, along with decreased expression of N-cadherin, Snail, and Vimentin, suggesting impaired cancer migration and invasion. The treatment with Nano-Dox induced notable cell cycle arrest at G0/G1 phase (p < 0.01) and accumulation of iron, Fe2+, and MDA (p < 0.01), as well as suppressed the protein levels of glutathione peroxidase 4 (GPX4) and SLC7A11. Administration of ferroptosis inhibitor (Fer-1) reversed the anti-proliferation effects of Nano-Dox (p < 0.01). The Dox delivered by the polymeric nanoparticle system notably enhanced its effects on suppressing the growth, migration, and invasion of drug-resistant OS cells via inducing ferroptosis. The application of environment response polymer enhanced the delivery of Dox and the therapeutic effects on OS.

Effects of rutin on the oxidative stress, proliferation and osteogenic differentiation of periodontal ligament stem cells in LPS-induced inflammatory environment and the underlying mechanism.

Periodontitis can cause damage to dental support tissue and affect the function of periodontal ligament cells. Rutin, a common flavonoid, plays a key role in anti-inflammatory responses, tissue repair and bone development. The purpose of this study was to investigate the effects of rutin on the oxidative stress, proliferation, and osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) in an inflammatory environment and the underlying mechanism. Lipopolysaccharide (LPS) was used to stimulate PDLSCs to mimic an inflammatory environment model. Reactive oxygen species (ROS) levels were detected by the dichlorodihydrofluorescein diacetate (DCFH-DA) probe and the oxidative stress factors were tested by an oxidative stress factor detection kit. Moreover, the proliferation of PDLSCs was evaluated by cell counting kit-8 (CCK-8) assay. In addition, the osteogenic differentiation of PDLSCs was determined by alkaline phosphatase (ALP) staining, ALP activity test, alizarin red staining, and alizarin red semi-quantitative analysis. Furthermore, the protein levels of AKT and p-AKT were detected by Western blot. The results showed that rutin inhibited the release of ROS and increased the secretion of oxidative stress factors [superoxide dismutase (SOD) and glutathione (GSH)] and promoted the proliferation of PDLSCs in an inflammatory environment. Moreover, rutin upregulated ALP activity and enhanced the number of mineralized nodules. Conversely, the use of LY294002 (an inhibitor of PI3K) blocked the activation of the PI3K/AKT signaling pathway and prevented the beneficial effects of rutin. In conclusion, rutin promoted the antioxidative stress ability, proliferation and osteogenic differentiation of PDLSCs through PI3K/AKT signaling pathway in LPS-induced inflammatory environment.

1,25(OH)2D3 supports the osteogenic differentiation of hPDLSCs under inflammatory conditions through inhibiting PLAP-1 expression transcriptionally.

BACKGROUND: Periodontal ligament-associated protein-1 (PLAP-1) is a newly identified negative regulator which is the mineralization of human periodontal ligament stem cells (hPDLSCs). The aim of the present study is to determine whether 1α, 25-dihydroxyvitamin D3 (1,25(OH)2D3) could enhances the osteoblastic differentiation of hPDLSCs under inflammatory condition, and if PLAP-1 is involved in this process. MATERIALS AND METHODS: hPDLSCs were in combination or alone cultured with lipopolysaccharide (LPS) and 1,25(OH)2D3, in osteo-inductive medium. The expression levels of osteoblastic markers and PLAP-1 of hPDLCs during osteo-inductive culture were assessed by western blot and real-time quantitative PCR(qRT-PCR). The potential vitamin D receptor elements (VDREs) which were located in PLAP-1 promoter region were identified and confirmed. RESULTS: The data showed that LPS inhibited osteoblastic differentiation and induced the expression of PLAP-1 in hPDLSCs. The increasing addition of 1,25(OH)2D3 reversed the LPS-induced inhibition of osteoblastic differentiation of hPDLSCs through the suppression of PLAP-1 expression. Moreover, a potential VDRE within the PLAP-1 promoter region was identified and shown to bind with VDR by chromatin immunoprecipitation (ChIP) assays. This negative region was also found to mediate suppressor reporter gene activity. CONCLUSIONS: 1,25(OH)2D3 could enhances the osteogenic differentiation of hPDLSCs under inflammatory condition through inhibiting PLAP-1 expression transcriptionally.

Lipopolysaccharide from Escherichia coli stimulates osteogenic differentiation of human periodontal ligament stem cells through Wnt/ß-catenin-induced TAZ elevation.

Human periodontal ligament stem cells (PDLSCs), a type of dental tissue-derived mesenchymal stem cells (MSCs), can be clinically applied in periodontal tissue regeneration to treat periodontitis, which is initiated and sustained by bacteria. Lipopolysaccharide (LPS), the major component of the outer membrane of gram-negative bacteria, is a pertinent deleterious factor in the oral microenvironment. The aim of this study was to investigate the effect of LPS on the proliferation and osteogenic differentiation of PDLSCs, as well as the mechanisms involved. Proliferation and osteogenic differentiation of PDLSCs were detected under the stimulation of Escherichia coli-derived LPS. The data showed that E. coli-derived LPS did not affect the proliferation, viability, and cell cycle of PDLSCs. Furthermore, it promoted osteogenic differentiation with the activation of TAZ. Lentivirus-mediated depletion of TAZ (transcriptional activator with a PDZ motif) was used to determine the role of TAZ on LPS-induced enhancement of osteogenesis. PDLSCs cultured in osteogenic media with or without LPS and DKK1 (Wnt/ß-catenin pathway inhibitor) were used to determine the regulatory effect of Wnt signaling. We found that TAZ depletion offset LPS-induced enhancement of osteogenesis. Moreover, treatment with DKK1 offset LPS-induced TAZ elevation and osteogenic promotion. In conclusion, E. coli-derived LPS promoted osteogenic differentiation of PDLSCs by fortifying TAZ activity. The elevation and activation of TAZ were mostly mediated by the Wnt/ß-catenin pathway. PDLSC-governed alveolar bone tissue regeneration was not necessarily reduced under bacterial conditions and could be modulated by Wnt and TAZ.

High-fat and high-glucose microenvironment decreases Runx2 and TAZ expression and inhibits bone regeneration in the mouse.

BACKGROUND: Type 2 diabetes mellitus (T2DM) and hyperlipidemia are negatively related to bone regeneration. The aim of this study was to evaluate the effect of high-fat and high-glucose microenvironment on bone regeneration and to detect the expression of runt-related transcription factor 2 (Runx2) and transcriptional co-activator with PDZ-binding domain (TAZ) during this process. METHODS: After establishing a high-fat and high-glucose mouse model, a 1 mm × 1.5 mm bone defect was developed in the mandible. On days 7, 14, and 28 after operation, bone regeneration was evaluated by hematoxylin-eosin staining, Masson staining, TRAP staining, and immunohistochemistry, while Runx2 and TAZ expression were detected by immunohistochemistry, RT-PCR, and Western blot analysis. RESULTS: Our results showed that the inhibition of bone regeneration in high-fat and high-glucose group was the highest among the four groups. In addition, the expression of Runx2 in high-fat, high-glucose, and high-fat and high-glucose groups was weaker than that in the control group, but the expression of TAZ only showed a decreasing trend in the high-fat and high-glucose group during bone regeneration. CONCLUSIONS: In conclusion, these results suggest that high-fat and high-glucose microenvironment inhibits bone regeneration, which may be related to the inhibition of Runx2 and TAZ expression.

Comparative analysis of lncRNA and mRNA expression profiles between periodontal ligament stem cells and gingival mesenchymal stem cells.

Oral tissue-derived mesenchymal stem cells, such as periodontal ligament stem cells (PDLSCs) and gingival mesenchymal stem cells (GMSCs), possess different biological characteristics, but the molecular mechanism remains unclear, which restricts their application in tissue engineering. Long noncoding RNAs (lncRNAs) are known to be significant regulators of gene expression, but our knowledge about their roles in the regulation of stem cell biological properties is still limited. This study compared the lncRNA and mRNA expression profiles between PDLSCs and GMSCs through microarray analysis, and applied bioinformatics methods to analyze and predict the function and connection of differentially expressed genes, aiming to screen potential key regulators of diverse biological characteristics in PDLSCs and GMSCs. Microarray analysis showed that 2162 lncRNAs and 1347 mRNAs were significantly differentially expressed between PDLSCs and GMSCs. Gene ontology (GO) analysis and pathway analysis indicated that these differentially expressed genes were involved in diverse biological processes and signaling pathways. The gene signal network and pathway relation network predicted some potentially important regulators. The coding-noncoding gene coexpression network (CNC network) revealed many potential lncRNA-mRNA connection pairs that participated in the regulation of biological behaviors. These results stressed the roles of lncRNAs in controlling stem cell biological behaviors and provided guides for molecular mechanistic study of different biological characteristics in PDLSCs and GMSCs.

Expression pattern of YAP and TAZ during orthodontic tooth movement in rats.

Orthodontic tooth movement (OTM) is a periodontal tissue remodeling and regeneration process that is caused by bio-mechanical stimulation. This mechanical-chemical transduction process involves a variety of biological factors and signaling pathways. It has been shown that the Hippo-YAP/TAZ signaling pathway plays a pivotal role in the mechanical-chemical signal transduction process. Moreover, YAP and TAZ proteins interact with RUNX family proteins via different mechanisms. To explore the regulation of the Hippo signaling pathway during periodontal tissue remodeling, we examined the upper first molar OTM model in rats. We examined YAP, TAZ and RUNX2 expression at 12 hours, 24 hours, 2 days (2d), 4 days, 7 days (7d) and 14 days (14d) after force application. Haemotoxylin and eosin staining, immunohistochemical staining and western blot analysis were used to examine the expression level and localization of these proteins. We found that YAP, TAZ and RUNX2 expression started increasing at 2d, YAP and TAZ expression was proportional to the orthodontic force applied until peaking at 7d, and at 14d the expression started to decrease. YAP and TAZ were observed in osteocytes, bone matrix and periodontal ligament cells during OTM. Furthermore, using double labeling immunofluorescence staining, we found that the increase in TAZ expression was associated with RUNX2 expression, however, YAP and RUNX2 showed different expression patterns. These results suggest that the Hippo-YAP/TAZ signaling pathway participates in periodontal tissue remodeling through various mechanisms; TAZ may adjust bone tissue remodeling through RUNX2 during OTM, while YAP may regulate periodontal cell proliferation and differentiation.

An In Vitro Comparative Study of Multisource Derived Human Mesenchymal Stem Cells for Bone Tissue Engineering.

Mesenchymal stem cells (MSCs) have been considered promising tools for tissue engineering and regenerative medicine. However, the optimal cell source for bone regeneration remains controversial. To better identify seed cells for bone tissue engineering, we compared MSCs from seven different tissues, including four from dental origins, dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), gingival MSCs (GMSCs), and dental follicle stem cells (DFSCs); two from somatic origins, bone marrow-derived MSCs (BM-MSCs) and adipose-derived stem cells (ADSCs); and one from birth-associated perinatal tissue umbilical cord (UCMSCs). We cultured the cells under a standardized culture condition and studied their biological characteristics. According to our results, these cells exhibited similar immunophenotype and had potential for multilineage differentiation. MSCs from dental and perinatal tissues proliferated more rapidly than those from somatic origins. Simultaneously, DPSCs and PDLSCs owned stronger antiapoptotic ability under the microenvironment of oxidative stress combined with serum deprivation. In respect to osteogenic differentiation, the two somatic MSCs, BM-MSCs and ADSCs, demonstrated the strongest ability for osteogenesis compared to PDLSCs and DFSCs, which were just a little bit weaker than the formers. However, GMSCs and UCMSCs were the most pertinacious ones to differentiate to osteoblasts. We also revealed that the canonical intracellular protein kinase-based cascade signaling pathways, including PI3K/AKT, MAPK/ERK, and p38 MAPK, possessed different levels of activation in different MSCs after osteoblast induction. Our conclusions suggest that PDLSCs might be a good potential alternative to BM-MSCs for bone tissue engineering.

CTHRC1 promotes osteogenic differentiation of periodontal ligament stem cells by regulating TAZ.

Collagen triple helix repeat containing 1 (CTHRC1) is associated with bone metabolism. Alveolar bone has an ability to rapidly remodel itself to adapt its biomechanical environment and function. However, whether CTHRC1 is expressed in alveolar bone tissue and the role of CTHRC1 in alveolar bone remodeling remain unclear. We used orthodontic tooth movement (OTM) rat model to study the effects of CHTRC1 in alveolar bone remodeling in vivo. We found that CTHRC1 was expressed in normal physiological condition of osteocytes, bone matrix, and periodontal ligament cells in rat. During the OTM, the expression of CTHRC1, Runx2 and TAZ were increased. We further studied the effects of CTHRC1 on osteogenic differentiation of human periodontal ligament stem cells in vitro. CTHRC1 can positively regulate the expression of TAZ and osteogenic differentiation markers like Col1, ALP, Runx2 and OCN. Overexpression of CHTRC1 increased osteogenic differentiation of PDLSCs, which could be abolished by TAZ siRNA. Our results suggest that CTHRC1 plays an important role in alveolar bone remodeling and osteogenic differentiation of PDLSCs.