Rational Design of Coordination Polymers Composited Hollow Multishelled Structures for Drug Delivery.

Multifunctional drug delivery systems (DDS) are in high demand for effectively targeting specific cells, necessitating excellent biocompatibility, precise release mechanisms, and sustained release capabilities. The hollow multishelled structure (HoMS) presents a promising solution, integrating structural and compositional design for efficient DDS development amidst complex cellular environments. Herein, starting from a Fe-based metal-organic framework (MOF), amorphous coordination polymers (CP) composited HoMS with controlled shell numbers are fabricated by balancing the rate of MOF decomposition and shell formation. Fe-CP HoMS loaded with DOX is utilized for synergistic chemotherapy and chemodynamic therapy, offering excellent responsive drug release capability (excellent pH-triggered drug release 82% within 72 h at pH 5.0 solution with doxorubicin (DOX) loading capacity of 284 mg g-1). In addition to its potent chemotherapy attributes, Fe-CP-HoMS possesses chemodynamic therapy potential by continuously catalyzing H2O2 to generate ·OH species within cancer cells, thus effectively inhibiting cancer cell proliferation. DOX@3S-Fe-CP-HoMS, at a concentration of 12.5 µg mL-1, demonstrates significant inhibitory effects on cancer cells while maintaining minimal cytotoxicity toward normal cells. It is envisioned that CP-HoMS could serve as an effective and biocompatible platform for the advancement of intelligent drug delivery systems in the realm of cancer therapy.

Hydro-Sensitive, In Situ Ultrafast Physical Self-Gelatinizing, and Red Blood Cells Strengthened Hemostatic Adhesive Powder with Antibiosis and Immunoregulation for Wound Repair.

Immediate and effective hemostatic treatments for complex bleeding wounds are an urgent clinical demand. Hemostatic materials with characteristics of adhesion, sealing, anti-infection, and concrescence promotion have drawn growing concerns. However, pure natural multifunctional hemostatic materials with in situ ultrafast self-gelation are rarely reported. In this study, a hydro-sensitive collagen/tannic acid (ColTA) natural hemostatic powder is developed that can in situ self-gel to form adhesive by the non-covalent crosslinking between tannic acid (TA) and collagen (Col) in liquids. The physical interactions endow ColTA adhesive with the characteristics of instantaneous formation and high adhesion at various substrate surfaces. Crucially, ColTA powder adhesive shows an enhanced adhesion performance in the presence of blood due to the electrostatic interactions between ColTA adhesive and red blood cells, conducive to effective in situ sealing and rapid hemostasis. The biocompatible and hemocompatible ColTA adhesive can effectively control bleeding and seal the wounds of the caudal vein, liver, heart, and femoral arteries in rats. Furthermore, the low-cost and ready-to-use ColTA adhesive powder also possesses good antibacterial and inhibiting biofilm formation ability, and can efficiently regulate immune response by the NF-κB pathway to promote wound repair, making it a highly promising hemostatic material with great potential for biomedical applications.

Immunomodulatory Properties: The Accelerant of Hydroxyapatite-Based Materials for Bone Regeneration.

The immunoinflammatory response is the prerequisite step for wound healing and tissue regeneration, and the immunomodulatory effects of biomaterials have attracted increasing attention. Hydroxyapatite [Ca10(PO4)6(OH)2] (HAp), a common calcium phosphate ceramic, due to its structural and functional similarity to the inorganic constituent of natural bones, has been developed for different application purposes such as bone substitutes, tissue engineering scaffolds, and implant coatings. Recently, the interaction between HAp-based materials and the immune system (various immune cells), and the immunomodulatory effects of HAp-based materials on bone tissue regeneration have been explored extensively. Macrophages-mediated regenerative effect by HAp stimulation occupies the mainstream status of immunomodulatory strategies. The immunomodulation of HAp can be manipulated by tuning the physical, chemical, and biological cues such as surface functionalization (physical or chemical modifications), structural and textural characteristics (size, shape, and surface topography), and the incorporation of bioactive substances (cytokines, rare-earth elements, and bioactive ions). Therefore, HAp ceramic materials can contribute to bone regeneration by creating a favorable osteoimmune microenvironment, which would provide a more comprehensive theoretical basis for their further clinical applications. Considering the rapidly developed HAp-based materials as well as their excellent biological performances in the field of regenerative medicine, this review discusses the recent advances concerning the immunomodulatory methods for HAp-based biomaterials and their roles in bone tissue regeneration. Impact statement This review summarized the immunomodulatory methods for hydroxyapatite-based biomaterials in bone tissue regeneration, and further discussed the affecting factors of immunomodulation as well as the challenges for the immunomodulatory strategies. The comprehensive understanding of immunomodulatory strategies for tissue regeneration would provide more guidance for the development of novel hydroxyapatite composite biomaterials.

Neuregulin-1 promotes the proliferation, migration, and angiogenesis of human periodontal ligament stem cells in vitro.

Neuregulin-1 (NRG-1) can promote the proliferation, migration, and angiogenesis of multiple stem cells, as well as prohibit cell apoptosis. In the present study, we aimed to explore the effects of NRG-1 on the proliferation, migration, apoptosis, angiogenic, and osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in vitro. The expression of erythroblastic leukemia viral oncogene homolog 2 (ERBB2), ERBB3, and ERBB4 on PDLSCs were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence. The effects of NRG-1 on the proliferation, migration, apoptosis, angiogenic and osteogenic differentiation of PDLSCs were assessed by cell proliferation and viability assays, transwell migration assay, flow cytometry assay, tubule formation assay, alkaline phosphatase (ALP) activity, and Alizarin Red S staining, respectively. Gene expression of angiogenesis and osteogenesis-related markers were detected by qRT-PCR. Among the ERBB family members, ERBB2 had the highest expression level in PDLSCs. Further, 10 ng/ml NRG-1 exhibited the maximal effect on proliferation, migration and remarkably inhibited the apoptosis of PDLSCs (p < .05). Moreover, NRG-1 upregulated the expression of vascular endothelial growth factor (VEGF), platelet/endothelial cell adhesion molecule-1 (CD31), hypoxia-inducible factor (HIF), kinase insert domain-containing receptor (KDR) in a dose-dependent manner as well as induced more tube formation. However, NRG-1 did not affect osteogenesis (p > .05). In summary, our study demonstrated that NRG-1 promotes the proliferation, migration, and angiogenesis and inhibits the apoptosis of PDLSCs in vitro and can potentially be used in tissue engineering for periodontal regeneration.

Red fluorescent nanoprobe based on Ag@Au nanoparticles and graphene quantum dots for H2O2 determination and living cell imaging.

A sensitive and turn-on fluorescence nanoprobe based on core-shell Ag@Au nanoparticles (Ag@AuNPs) as a fluorescence receptor and red emissive graphene quantum dots (GQDs) as a donor was fabricated. They were conjugated together through π-π stacking between the GQDs and single-strand DNA modified at the Ag@AuNPs surface. The absorption spectrum of the receptor significantly overlapped with the donor emission spectrum, leading to a strong Förster resonance energy transfer (FRET) and thus a dramatic quenching. The sensing mechanism relies on fluorescence recovery following DNA cleavage by •OH produced from Fenton-like reaction between the peroxidase-like Ag nanocore and H2O2. The red emissive feature (Ex/Em, 520 nm/560 nm) provides low background in physiological samples. The •OH production, great spectrum overlapping, and red emission together contributes to good sensitivity and living cell imaging capability. The fluorescence assay (intensity at 560 nm) achieves a low detection limit of 0.49 µM H2O2 and a wide linear range from 5 to 200 µM, superior to most of the reported fluorescent probes. The RSD value for 100 µM H2O2 was 1.4%. The nanoprobe exhibits excellent anti-interferences and shows low cytotoxicity. The recovery of 100 µM standard H2O2 in a cancer cell lysate was 85.8%. Most satisfactorily, it can realize monitoring and imaging H2O2 in living cells. This study not only presents a sensitive H2O2 probe but also provides a platform for detecting other types of reactive oxygen species.

Immunomodulatory functions of oral mesenchymal stem cells: Novel force for tissue regeneration and disease therapy.

Mesenchymal stem cells (MSCs)-based therapeutic strategies have achieved remarkable efficacies. Oral tissue-derived MSCs, with powerful self-renewal and multilineage differentiation abilities, possess the features of abundant sources and easy accessibility and hold great potential in tissue regeneration and disease therapies. Oral MSCs mainly consist of periodontal ligament stem cells, gingival mesenchymal stem cells, dental pulp stem cells, stem cells from human exfoliated deciduous teeth, stem cells from the apical papilla, dental follicle stem cells, and alveolar bone-derived mesenchymal stem. Early immunoinflammatory response stage is the prerequisite phase of healing process. Besides the potent capacities of differentiation and regeneration, oral MSCs are capable of interacting with various immune cells and function as immunomodulatory regulators. Consequently, the immunomodulatory effects of oral MSCs during damage repair seem to be crucial for exploring novel immunomodulatory strategies to achieve disease recovery and tissue regeneration. Herein, we reviewed various oral MSCs with their immunomodulatory properties and the potential mechanism, as well as their effects on immunomodulation-mediated disease therapies and tissue regeneration.

Immunomodulatory effect of dimethyloxallyl glycine/nanosilicates-loaded fibrous structure on periodontal bone remodeling.

BACKGROUND/PURPOSE: Relieving immuno-inflammatory responses is the prerequisite step for treating periodontitis. The angiogenic small molecule, dimethyloxalylglycine (DMOG), and osteoinductive inorganic nanomaterial, nanosilicate (nSi) have a powerful effect on bone regeneration, whereas the roles in osteoimmunomodulation have not been totally uncovered. Our study aimed to explore the immunomodulatory effect of DMOG/nSi-loaded fibrous membranes on periodontal bone remodeling. MATERIALS AND METHODS: The fibrous membranes were prepared by incorporating DMOG and nSi into poly (lactic-co-glycolic acid) (PLGA) with electrospinning. The morphology features, surface chemical property and biocompatibility of DMOG/nSi-PLGA fibrous membranes were characterized. Thereafter, the fibrous membranes were implanted into rat periodontal defects, bone remodeling potential and immunomodulatory effect were evaluated by micro-computed tomography (micro-CT), histological evaluation and immunohistochemical analysis. RESULTS: DMOG/nSi-PLGA membranes possessed favorable physicochemical properties and biocompatibility. After the fibrous membranes implanted into periodontal defects, DMOG/nSi-PLGA membranes could relieve immuno-inflammatory responses of the defects (reduction of inflammatory cell infiltration, CD40L and CD11b-positive cells), increased CD206-positive M2 macrophages, and eventually facilitated periodontal bone regeneration. CONCLUSION: DMOG/nSi-PLGA fibrous membranes exert protective effects during periodontal bone defect repairing, and steer immune response towards bone regeneration. Consequently, DMOG/nSi-PLGA fibrous membranes may serve as a promising scaffold in periodontal tissue engineering.

[Sitagliptin inhibits lipopolysaccharide-induced inflammatory response in human gingival fibroblasts by blocking nuclear factor-κB signaling pathway].

OBJECTIVES: This study was performed to clarify the effects of sitagliptin on Porphyromonas gingivalis-lipopolysaccharide (LPS)-induced inflammatory response in human gingival fibroblasts (HGFs), explore the molecular mechanism of its roles, and provide a foundation for clinical therapeutics in periodontitis. METHODS: Healthy gingival samples were collected from the donors. HGFs were isolated with enzymic digestion method and identified. The effects of LPS and sitagliptin on cell viability were detected by cell-counting kit-8 (CCK8). The mRNA levels of inflammatory cytokines, namely, interleukin (IL)-6, IL-8, C-C motif ligand 2 (CCL2), and superoxide dismutase 2 (SOD2), were evaluated by quantity real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immune sorbent assay (ELISA) was used to measure the secretion protein levels of IL-6, IL-8, and CCL2. Western blot analysis was used to further investigate the activation of nuclear factor (NF)-κB signaling pathway. The effect of NF-κB pathway inhibitor BAY11-7082 on LPS-induced HGF inflammatory cytokines at the gene level was verified by qRT-PCR. RESULTS: Low concentrations of sitagliptin (0.1, 0.25, and 0.5 µmol·L-1) did not affect HGF growth in 24 and 48 h, whereas high concentrations of sitagliptin (5-1 000 µmol·L-1) significantly inhibited cell proliferation. Sitagliptin suppressed 5 µg·mL-1 of LPS-induced IL-6, IL-8, CCL2, and SOD2 gene expression levels in HGF in a concentration-dependent manner. Furthermore, sitagliptin significantly decreased the elevated secretion of IL-6, IL-8, and CCL2 protein induced by LPS. Western blot analysis showed that 0.5 µmol·L-1 of sitagliptin significantly inhibited LPS-induced NF-κB signaling pathway activation. Results of qRT-PCR analysis indicated that 0.5 µmol·L-1 of sitagliptin and 5 µmol·L-1 of BAY11-7082 significantly inhibited LPS-induced IL-6, IL-8, CCL2, and SOD2 gene expressions. CONCLUSIONS: Sitagliptin could significantly inhibit LPS-induced HGF inflammatory response by blocking the NF-κB signaling pathway activation.

6-Bromoindirubin-3'-oxime Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells and Facilitates Bone Regeneration in a Mouse Periodontitis Model.

Effective bone tissue engineering is important to overcome the unmet clinical challenges of periodontal tissue regeneration. Successful bone tissue engineering comprises three key factors: stem cells, growth factors, and scaffolds. 6-Bromoindirubin-3'-oxime (BIO) is an inhibitor of glycogen synthase kinase-3 (GSK-3) that can activate the Wnt signaling pathway by enhancing ß-catenin activity. In this study, the effects of BIO on the proliferation, migration, and osteogenic differentiation of periodontal ligament stem cells (PDLSCs) were investigated. Poly(lactic-co-glycolic acid) (PLGA) and hyaluronic acid (HA) emerged as promising biomaterials; thus, we developed a novel HA hydrogel embedded with BIO-encapsulated PLGA microspheres and injected the formulation into the gingival sulcus of mice with experimental periodontitis. The release speed of this system was fast in the first week and followed a sustained release phase until week 4. In vivo experiments showed that this PLGA-BIO-HA hydrogel system can inhibit periodontal inflammation, promote bone regeneration, and induce the expression of bone-forming markers alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteocalcin (OCN) in a mouse periodontitis model. Therefore, this PLGA-BIO-HA hydrogel system provides a promising therapeutic strategy for periodontal bone regeneration.

Dimethyloxallyl glycine/nanosilicates-loaded osteogenic/angiogenic difunctional fibrous structure for functional periodontal tissue regeneration.

The coupled process of osteogenesis-angiogenesis plays a crucial role in periodontal tissue regeneration. Although various cytokines or chemokines have been widely applied in periodontal in situ tissue engineering, most of them are macromolecular proteins with the drawbacks of short effective half-life, poor stability and high cost, which constrain their clinical translation. Our study aimed to develop a difunctional structure for periodontal tissue regeneration by incorporating an angiogenic small molecule, dimethyloxalylglycine (DMOG), and an osteoinductive inorganic nanomaterial, nanosilicate (nSi) into poly (lactic-co-glycolic acid) (PLGA) fibers by electrospinning. The physiochemical properties of DMOG/nSi-PLGA fibrous membranes were characterized. Thereafter, the effect of DMOG/nSi-PLGA membranes on periodontal tissue regeneration was evaluated by detecting osteogenic and angiogenic differentiation potential of periodontal ligament stem cells (PDLSCs) in vitro. Additionally, the fibrous membranes were transplanted into rat periodontal defects, and tissue regeneration was assessed with histological evaluation, micro-computed tomography (micro-CT), and immunohistochemical analysis. DMOG/nSi-PLGA membranes possessed preferable mechanical property and biocompatibility. PDLSCs seeded on the DMOG/nSi-PLGA membranes showed up-regulated expression of osteogenic and angiogenic markers, higher alkaline phosphatase (ALP) activity, and more tube formation in comparison with single application. Further, in vivo study showed that the DMOG/nSi-PLGA membranes promoted recruitment of CD90+/CD34- stromal cells, induced angiogenesis and osteogenesis, and regenerated cementum-ligament-bone complex in periodontal defects. Consequently, the combination of DMOG and nSi exerted admirable effects on periodontal tissue regeneration. DMOG/nSi-PLGA fibrous membranes could enhance and orchestrate osteogenesis-angiogenesis, and may have the potential to be translated as an effective scaffold in periodontal tissue engineering.

AGGF1 inhibits the expression of inflammatory mediators and promotes angiogenesis in dental pulp cells.

OBJECTIVES: To determine the role of angiogenic factor with G-patch and FHA domain 1 (AGGF1) in inflammatory response of human dental pulp cells (DPCs) and the underneath mechanism and to explore its role in angiogenesis. MATERIALS AND METHODS: The expression of AGGF-1 in human healthy and inflammatory pulp tissues was detected by immunohistochemistry. RT-qPCR and Western blot were used to evaluate the expression of AGGF1 in DPCs stimulated by lipopolysaccharide (LPS). After AGGF1 was knocked down, the expression of LPS-induced inflammatory cytokines in DPCs was quantified by RT-qPCR and ELISA. Immunofluorescence and Western blot were used to assess the activation of NF-κB signaling. Inflammatory cytokines were detected by RT-qPCR and ELISA in DPCs pretreated with NF-κB pathway inhibitors before LPS stimulation, and then the effect of AGGF1 on angiogenesis was also evaluated. RESULTS: AGGF1 expression increased in inflammatory dental pulp tissues. In DPCs stimulated by LPS, AGGF1 was upregulated in a dose-dependent manner (P < 0.05). In AGGF1 knockdown cells, the expression of IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1/CCL-2) increased by LPS stimulation (P < 0.001). Nuclear translocation of p65 was promoted, and the addition of NF-κB inhibitors inhibited the expression of inflammatory factors. Meanwhile, knockdown of AGGF1 inhibited vascularization. CONCLUSIONS: AGGF1 inhibited the synthesis of inflammatory cytokines through NF-κB signaling pathway and promoted the angiogenesis of DPCs. CLINICAL RELEVANCE: This study might shed light in the treatment of pulpitis and regeneration of dental pulp tissues; however, more clinical trials are required to validate these findings.

Nanotextured silk fibroin/hydroxyapatite biomimetic bilayer tough structure regulated osteogenic/chondrogenic differentiation of mesenchymal stem cells for osteochondral repair.

OBJECTIVES: Articular cartilage plays a vital role in bearing and buffering. Injured cartilage and subchondral bone repair is a crucial challenge in cartilage tissue engineering due to the peculiar structure of osteochondral unit and the requirement of osteogenic/chondrogenic bi-directional differentiation. Based on the bionics principle, a nanotextured silk fibroin (SF)-chondroitin sulphate (CS)/hydroxyapatite (HAp) nanowire tough bilayer structure was prepared for osteochondral repair. METHODS: The SF-CS/HAp membrane was constructed by alcohol-induced ß-sheet formation serving as the physical crosslink. Its osteochondral repairing capacity was evaluated by culturing bone marrow mesenchymal stem cells (BMSCs) in vitro and constructing a rat osteochondral defect model in vivo. RESULTS: The bilayer SF-CS/HAp membrane with satisfactory mechanical properties similar to natural cartilage imitated the natural osteochondral unit structural layers and exerted the function of bearing and buffering timely after in vivo implantation. SF-CS layer upregulated the expression of chondrogenesis-related genes of BMSCs by surface nanotopography and sustained release CS. Meanwhile, nanotextured HAp layer assembled with nanowire endowed the membrane with an osteogenic differentiation tendency for BMSCs. In vivo results proved that the biomimetic bilayer structure dramatically promoted new cartilage formation and subchondral bone remodelling for osteochondral defect model after implantation. CONCLUSIONS: The SF-CS/HAp biomimetic bilayer membrane provides a promising strategy for precise osteochondral repair.

N-WASP knockdown upregulates inflammatory cytokines expression in human gingival fibroblasts.

OBJECTIVE: The neuronal wiskott-aldrich syndrome protein (N-WASP) is a member of the wiskott-aldrich syndrome protein (WASP) family. N-WASP plays a vital role in promoting cell migration, receptor signaling and immune inflammatory responses. This study aimed to observe the changes in the expression of inflammatory factors and involving pathways after N-WASP knockdown in human gingival fibroblasts (HGFs). DESIGN: Gingival inflammatory condition of N-WASP knockout mice was evaluated by H&E staining. N-WASP in HGFs was knockdown by siRNA and the best knockdown efficiency was determined by qRT-PCR and immunofluorescence. The mRNA levels of interleukin (IL)-6, IL-8, C-C motif ligand 2 (CCL2), superoxide dismutase 2 (SOD2) and prostaglandin endoperoxide synthase 2 (PTGS2) were evaluated by qRT-PCR after N-WASP knockdown with or without mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) inhibitors. The protein levels of IL-6, IL-8 and CCL2 were assessed by ELISA. Western blotting was used to detect the activation of NF-κB and MAPK signaling pathways. RESULTS: Gingival tissue from N-WASP knockout mice exhibited an inflammatory reaction. The expression of IL-6, IL-8, CCL2, SOD2 and PTGS2 was significantly upregulated after N-WASP knockdown in HGFs for 6, 24 and 48 h, except for the SOD2 at 6 h. N-WASP knockdown significantly activated the signaling pathways of NF-κB and MAPK. The inhibitors of p65, p38, ERK and JNK clearly decreased IL-6, IL-8, CCL2, SOD2 and PTGS2 expression after N-WASP knockdown. CONCLUSION: These data indicated that N-WASP deficiency in HGFs increases the production of inflammatory cytokine and is regulated via NF-κB and MAPK signaling pathways.

A method to visually observe the degradation-diffusion-reconstruction behavior of hydroxyapatite in the bone repair process.

Nanostructured hydroxyapatite (HAp) has been applied widely as a scaffold material for bone tissue engineering for its good osteoinduction and biodegradability. However, the degradation process and the distribution of degraded HAp within the bone-defect cavity is still not clear. To visually study the behavior of HAp in bone repair process, a membrane of HAp/terbium (Tb)-HAp nanowires (NWs) was prepared with a concentric circle structure (CCS), of which the inner circle and the outer ring were constructed with Tb-HAp and HAp NWs, respectively. HAp/Tb-HAp CCS membrane possessed good osteogenic capacity and efficient fluorescence in the center for visualization. The in vitro experimental results proved that the Tb-HAp and HAp NWs membranes both presented high cytocompatibility and adequate efficiency to induce osteogenic differentiation of bone marrow stem cells (BMSCs). HAp/Tb-HAp CCS membranes were then implanted into a rat calvarial bone-defect model to study the behavior of HAp in bone repair process in vivo by tracking the fluorescence distribution. The results showed that the fluorescence of Tb-HAp diffused gradually from the inner circle to the outer ring, which suggested that the HAp was first degraded, and then the degraded product was diffused and finally reconstructed. Further, the histological results proved that the doping of Tb did not impair the promotive effect of HAp on bone repair process. Therefore, this study provided a visual method to observe the degradation-diffusion-reconstruction behavior of HAp nanomaterials in bone repair process. STATEMENT OF SIGNIFICANCE: The study of dynamic degradation process of implanted hydroxyapatite (HAp) materials in bone-defect cavity is of great significance to bone tissue engineering applications. Here, we designed a HAp/Tb-HAp nanowires (NWs) membrane with concentric circle structure (CCS) to visibly observe the behavior of HAp during bone repair process. HAp/Tb-HAp CCS membrane possessed both osteoinduction ability and fluorescence property. Calvarial bone-defect repair experiments in vivo showed that the fluorescence of Tb-HAp diffused gradually from inner circle to outer ring, which suggested that HAp was first degraded, then diffused and finally reconstructed. Therefore, this invention provides not only a visible method to observe the degradation-diffusion-reconstruction behavior of HAp-based biomaterials, but also a basic understanding of the dynamic change of HAp-based biomaterials.

Sequential application of bFGF and BMP-2 facilitates osteogenic differentiation of human periodontal ligament stem cells.

BACKGROUND AND OBJECTIVES: Basic fibroblast growth factor (bFGF) promotes cells proliferation and chemotaxis and maintains stemness while inhibits mineralized nodule formation. Bone morphogenetic protein 2 (BMP-2) shows great potential in promoting bone formation. However, sequential application of these two growth factors on periodontal ligament stem cells (PDLSCs) has not been explored. In this study, we aimed to identify the optimal concentration and time of bFGF on PDLSCs proliferation, migration and then investigate the sequential delivery of bFGF and BMP-2 on osteogenic differentiation of PDLSCs in vitro. MATERIALS AND METHODS: Periodontal ligament stem cells were isolated by limiting dilution method. Dose-dependent additive effects of bFGF and BMP-2 on PDLSCs were detected. Cell counting assay, cell migration assay, alkaline phosphatase (ALP) activity assay, Alizarin red staining, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis were used to determine different application modalities of bFGF and BMP-2 on proliferation, migration, and osteogenic differentiation of PDLSCs. RESULTS: 50 ng/mL bFGF significantly promoted PDLSCs proliferation and chemotaxis while time-dependently inhibited BMP-2 induced ALP activity. Sequential application of 25 ng/mL bFGF for first 3 days and followed with 50 ng/mL BMP-2 for another 9, 18, and 25 days significantly promoted PDLSCs osteogenic differentiation. Compared with bFGF and BMP-2 simultaneous group, sequential application of bFGF and BMP-2 group significantly enhanced ALP activity, osteogenesis-related genes and proteins expression and mineral deposition. CONCLUSION: Sequential application of bFGF and BMP-2 synergistically promoted osteogenic differentiation of PDLSCs, and this sequential application modality of growth factors would provide a new strategy for periodontal regeneration.

Oxytocin facilitates the proliferation, migration and osteogenic differentiation of human periodontal stem cells in vitro.

OBJECTIVE: To explore the effect of oxytocin (OT) on the proliferation, migration, and osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in vitro. DESIGN: PDLSCs were obtained by limiting dilution method. Immunofluorescence (IF), cell-counting kit-8 (CCK8), cell migration assay, Alizarin Red S staining, cetylpyridinium chloride (CPC) colorimetry, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis were used to examine the effect of OT on oxytocin receptor (OTR) expression, cell proliferation, migration and osteogenic differentiation of PDLSCs. RESULTS: Our study showed that PDLSCs expressed OTR. One hundred nanomolar OT exhibited the maximal effect on migration, while only 50 nM OT significantly promoted proliferation of PDLSCs, as well as mineralized nodule formation and calcium deposition (p < 0.05). Furthermore, 50 nM OT significantly up-regulated expression of osteogenesis-related genes-alkaline phosphatase (ALP), Collagen I (Col I), runt related transcription factor 2 (Runx 2), osteopontin (OPN) and osteocalcin (OCN)-at specific time points compared with osteogenic inductive medium (p < 0.05). In addition, western blot analysis demonstrated that 50 nM OT enhanced protein levels of ALP, Col I, and Runx 2 at day 7 and day 14 (p < 0.01), as well as activating the phosphorylation of extracellular-signal-regulated kinase (ERK) and protein kinase B (AKT) pathway; notably, 50 nM OT inhibited phosphorylation of the phosphatidylinositol 3-kinase (PI3K) pathway (p < 0.05). CONCLUSIONS: OT promoted proliferation, migration, and osteogenic differentiation of PDLSCs in vitro. Furthermore, the effect of OT on osteogenic differentiation was mediated through ERK and AKT pathway. Thus, OT may have potential for use in periodontal regeneration.

Prolyl hydroxylase inhibitor DMOG suppressed inflammatory cytokine production in human gingival fibroblasts stimulated with Fusobacterium nucleatum.

OBJECTIVE: Fusobacterium nucleatum (F. nucleatum) is one of the most common bacteria involved in the initiation and progression of periodontal diseases. Pharmacological inhibitor of prolyl hydroxylases (PHDs), dimethyloxallyl glycine (DMOG), has been reported to exert anti-inflammatory effects. The aim of this investigation was to evaluate the role of DMOG in inflammatory cytokine production of human gingival fibroblasts (HGFs) stimulated with F. nucleatum. MATERIAL AND METHODS: HGFs were pretreated with 10, 50, and 100 µM DMOG for 24 h before infected with F. nucleatum (MOI = 100). Cell morphology and survival after infection with F. nucleatum were determined by crystal violet staining assay. The mRNA levels of interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α, and IL-1ß were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The production of IL-6, IL-8, TNF-α, and IL-1ß was assessed by enzyme-linked immunosorbent assay (ELISA). RESULTS: F. nucleatum did not affect the morphology and survival of HGFs by the concentrations of MOI (multiplicity of infection) = 10, 50, and 100. The mRNA levels of IL-6, IL-8, TNF-α, and IL-1ß were significantly enhanced with the stimulation of F. nucleatum, and the maximal effect reached at 6 h. The secretion of IL-6, IL-8, and TNF-α was significantly upregulated by the infection of F. nucleatum while the production of IL-1ß was nearly unchanged. Above all, DMOG suppressed F. nucleatum-stimulated IL-6, IL-8, TNF-α, and IL-1ß expressions. CONCLUSIONS: These data indicate that prolyl hydroxylase inhibitor DMOG partly downregulates inflammatory cytokine expression in F. nucleatum-infected HGFs. CLINICAL RELEVANCE: DMOG may provide a novel strategy for the therapy of periodontitis.

Prolyl hydroxylases positively regulated LPS-induced inflammation in human gingival fibroblasts via TLR4/MyD88-mediated AKT/NF-κB and MAPK pathways.

OBJECTIVES: Prolyl hydroxylases (PHDs) play essential roles in oxygen-sensing system, whereas the effects of PHDs on inflammation have not been totally uncovered. Our study aimed to investigate the role of PHDs in lipopolysaccharide (LPS)-induced inflammation of human gingival fibroblasts (HGFs) and clarify the potential mechanisms. MATERIALS AND METHODS: A pan hydroxylase inhibitor, dimethyloxallyl glycine (DMOG), and RNA interference were used to explore the role of PHDs in inflammation. Cytotoxic effect of DMOG was determined by cell-counting kit-8 and flow cytometry respectively. The secretion levels of IL-6 and IL-8 were assessed by ELISA. The mRNA levels of inflammatory cytokines, Toll-like receptor (TLR) 4 and MyD88 were evaluated by quantitative real-time PCR. The activation of NF-κB, mitogen-activated protein kinase (MAPK) and PI3K/AKT pathways were detected by western blot and the nuclear translocation of NF-κB p65 was examined by immunofluorescence. Downregulation of PHD1 and PHD2 was performed with siRNA transfection. RESULTS: Dimethyloxallyl glycine inhibited LPS-induced inflammatory cytokine, TLR4 and MyD88 expression in gene level and the elevated secretion of IL-6 and IL-8 was also downregulated. Additionally, LPS-induced activation of NF-κB, MAPK and AKT pathways was abolished by DMOG treatment. Importantly, LPS-induced inflammatory cytokine expression was merely suppressed by PHD2 knockdown. CONCLUSIONS: Prolyl hydroxylases acted as a positive regulator in LPS-induced inflammation of HGFs via TLR4/MyD88-mediated NF-κB, MAPK and AKT signalling pathways and PHD2 among three isoforms was principally responsible for the effects.

Rho-kinase inhibitor Y-27632 downregulates LPS-induced IL-6 and IL-8 production via blocking p38 MAPK and NF-κB pathways in human gingival fibroblasts.

BACKGROUND: Porphyromonas gingivalis lipopolysaccharide (LPS) plays a major role in the initiation and progression of chronic periodontitis. Human gingival fibroblasts (HGFs) interact with bacteria or bacterial products and trigger inflammatory signaling pathways that destroy periodontal tissues. RhoA regulates cytokine production in various cell types. This study investigated the role of Rho-kinase inhibitor Y-27632 in LPS-induced nuclear factor-kappa B (NF-κB) and p-38 mitogen-activated protein kinase (MAPK) activation, and inflammatory cytokine production in HGFs. METHODS: Effects of Y-27632, SB203580 (p38 MAPK inhibitor), and BAY11-7082 (NF-κB inhibitor) were assessed in lipopolysaccharide (LPS)-treated HGFs. Cytotoxicity assays were used to determine the effect of the drugs on HGF viability. Enzyme-linked immunosorbent assays and quantitative real-time polymerase chain reaction were applied to evaluate the levels of interleukin (IL)-6, IL-8, and Toll-like receptors (TLRs). NF-κB and p38 MAPK pathway activation was detected by western blot and immunocytochemistry. RESULTS: P. gingivalis LPS at 5 µg/mL, 10 µM Y-27632, 10 µM SB203580, and 5 µM BAY11-7082 exhibited no toxicity in HGFs. LPS activated NF-κB and p38 MAPK by increasing degradation of IκBα and phosphorylation of IκBα, p65, and p38, and facilitating p65 translocation from the cytoplasm to nuclei. The activation of NF-κB and p38 MAPK induced overproduction of IL-6 and IL-8 at both mRNA and protein levels. However, Y-27632 attenuated LPS-induced NF-κB and p38 MAPK activation and inflammatory cytokine production. CONCLUSIONS: Rho-kinase inhibitor Y-27632 downregulates LPS-induced IL-6 and IL-8 production by blocking NF-κB and p38 MAPK activation in HGFs.

[Preparation of porous ceramics based on waste ceramics and its Ni2+ adsorption characteristics].

The preparation conditions of porous ceramics were determined by SEM, XRD and FT-IR characterizations as well as the nickel removal ability of porous ceramics to be: the mass fraction w of sesbania powder doped was 4%, and the calcination temperature was 800 degrees C. SEM and pore structure characterization illustrated that calcination caused changes in the structure and morphology of waste ceramics. With the increase of calcination temperature, the specific surface area and pore volume decreased, while the aperture increased. EDS analyses showed that the main elements of both the original waste porcelain powder and the porous ceramics were Si, Al and O. The SEM, XRD and FT-IR characterization of porous ceramics illustrated that the structure of porous ceramics was stable before and after adsorption. The series of experiments of Ni2+ adsorption using these porous ceramics showed that when the dosage of porous ceramics was 10 g x L(-1), the adsorption time was 60 min, the pH value was 6.32, and the concentration of nickel-containing wastewater was below 100 mg x L(-1), the Ni2+ removal of wastewater reached 89.7%. Besides, the porous ceramics showed higher removal efficiency on nickel in the wastewater. The Ni(2+)-containing wastewater was processed by the porous ceramics prepared, and the adsorption dynamics and adsorption isotherms of Ni2+ in wastewater by porous ceramics were investigated. The research results showed that the Ni2+ adsorption process of porous ceramics was in accordance with the quasi second-order kinetic model (R2 = 0.999 9), with Q(e) of 9.09 mg x g(-1). The adsorption process can be described by the Freundlich equation and Langmuir equation, and when the temperature increased from 20 degrees C to 40 degrees C, the maximum adsorption capacity Q(m) increased from 14.49 mg x g(-1) to 15.38 mg x g(-1).