Angiopoietin-like 4 is upregulated by amphiregulin and activates cell proliferation and migration through p38 kinase in head and neck squamous cell carcinoma.

BACKGROUND: Angiopoietin-like 4 is a molecular hallmark that correlates with the growth and metastasis of head and neck squamous cell carcinoma, one of the most prevalent cancers worldwide. However, the molecular mechanisms by which angiopoietin-like 4 promotes head and neck squamous cell carcinoma tumorigenesis are unclear. METHODS: Using well-characterized cell lines of head and neck squamous cell carcinoma development, including human normal oral keratinocytes, dysplastic oral keratinocytes, oral leukoplakia-derived oral keratinocytes, and head and neck squamous cell carcinoma cell lines, HN13, HN6, HN4, HN12, and CAL27, we investigated the signaling pathways upstream and downstream of angiopoietin-like 4-induced head and neck squamous cell carcinoma tumorigenesis. RESULTS: We found that both epidermal growth factor receptor ligands, epithelial growth factor, and amphiregulin led to angiopoietin-like 4 upregulation in normal oral keratinocytes and dysplastic oral keratinocytes and cooperated with the activation of hypoxia-inducible factor-1 in this effect. Interestingly, amphiregulin and angiopoietin-like 4 were increased in dysplastic oral keratinocytes and head and neck squamous cell carcinoma cell lines, and amphiregulin-induced activation of cell proliferation was dependent on angiopoietin-like 4. Although both p38 mitogen-activated protein kinases (p38 MAPK) and protein kinase B (AKT) were activated by angiopoietin-like 4, only pharmacological inhibition of p38 MAPK was sufficient to prevent head and neck squamous cell carcinoma cell proliferation and migration. We further observed that angiopoietin-like 4 promoted the secretion of interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin-1 alpha (IL-1α), vascular endothelial growth factor, platelet-derived growth factor (PDGF), and tumour necrosis factor alpha (TNF-α), cytokines and chemokines previously implicated in head and neck squamous cell carcinoma pathogenesis. CONCLUSION: Our results demonstrate that angiopoietin-like 4 is a downstream effector of amphiregulin and promotes head and neck squamous cell carcinoma development both through direct activation of p38 kinase as well as paracrine mechanisms.

Effects of Innervation on Angiogenesis and Osteogenesis in Bone and Dental Tissue Engineering.

The repair and regeneration of critical-sized bone defects remain an urgent challenge. Bone tissue engineering represents an exciting solution for regeneration of large bone defects. Recently, the importance of innervation in tissue-engineered bone regeneration has been increasingly recognized. The cross talk between nerve and bone provides important clues for bone repair and regeneration. Furthermore, the promotion of angiogenesis by innervation can accelerate new bone formation. However, the mechanisms involved in the promotion of vascular and bone regeneration by the nervous system have not yet been established. In addition, simultaneous neurogenesis and vascularization in bone tissue engineering have not been fully investigated. This article represents the first review on the effects of innervation in enhancing angiogenesis and osteogenesis in bone and dental tissue engineering. Cutting-edge research on the effects of innervation through biomaterials on bone and dental tissue repairs is reviewed. The effects of various nerve-related factors and cells on bone regeneration are discussed. Finally, novel clinical applications of innervation for bone, dental, and craniofacial tissue regeneration are also examined.

Novel Remineralizing and Antibiofilm Low-Shrinkage-Stress Nanocomposites to Inhibit Salivary Biofilms and Protect Tooth Structures.

Recurrent caries remain a persistent concern, often linked to microleakage and a lack of bioactivity in contemporary dental composites. Our study aims to address this issue by developing a low-shrinkage-stress nanocomposite with antibiofilm and remineralization capabilities, thus countering the progression of recurrent caries. In the present study, we formulated low-shrinkage-stress nanocomposites by combining triethylene glycol divinylbenzyl ether and urethane dimethacrylate, incorporating dimethylaminododecyl methacrylate (DMADDM), along with nanoparticles of calcium fluoride (nCaF2) and nanoparticles of amorphous calcium phosphate (NACP). The biofilm viability, biofilm metabolic activity, lactic acid production, and ion release were evaluated. The novel formulations containing 3% DMADDM exhibited a potent antibiofilm activity, exhibiting a 4-log reduction in the human salivary biofilm CFUs compared to controls (p < 0.001). Additionally, significant reductions were observed in biofilm biomass and lactic acid (p < 0.05). By integrating both 10% NACP and 10% nCaF2 into one formulation, efficient ion release was achieved, yielding concentrations of 3.02 ± 0.21 mmol/L for Ca, 0.5 ± 0.05 mmol/L for P, and 0.37 ± 0.01 mmol/L for F ions. The innovative mixture of DMADDM, NACP, and nCaF2 displayed strong antibiofilm effects on salivary biofilm while concomitantly releasing a significant amount of remineralizing ions. This nanocomposite is a promising dental material with antibiofilm and remineralization capacities, with the potential to reduce polymerization-related microleakage and recurrent caries.

Glucose upregulates amphiregulin in oral dysplastic keratinocytes: A potential role in diabetes-associated oral carcinogenesis.

BACKGROUND: Compelling evidence implicates diabetes-associated hyperglycemia as a promoter of tumor progression in oral potentially malignant disorders (OPMD). Yet, information on hyperglycemia-induced cell signaling networks in oral oncology remains limited. Our group recently reported that glucose-rich conditions significantly enhance oral dysplastic keratinocyte viability and migration through epidermal growth factor receptor (EGFR) activation, a pathway strongly linked to oral carcinogenesis. Here, we investigated the basal metabolic phenotype in these cells and whether specific glucose-responsive EGFR ligands mediate these responses. METHODS: Cell energy phenotype and lactate concentration were evaluated via commercially available assays. EGFR ligands in response to normal (5 mM) or high (20 mM) glucose were analyzed by quantitative real-time PCR, ELISA, and western blotting. Cell viability and migration assays were performed in the presence of pharmacological inhibitors or RNA interference. RESULTS: When compared to normal keratinocytes, basal glycolysis in oral dysplastic keratinocytes was significantly elevated. In highly glycolytic cells, high glucose-activated EGFR increasing viability and migration. Notably, we identified amphiregulin (AREG) as the predominant glucose-induced EGFR ligand. Indeed, enhanced cell migration in response to high glucose was blunted by EGFR inhibitor cetuximab and AREG siRNA. Conversely, AREG treatment under normal glucose conditions significantly increased cell viability, migration, lactate levels, and expression of glycolytic marker pyruvate kinase M2. CONCLUSION: These novel findings point to AREG as a potential high glucose-induced EGFR activating ligand in highly glycolytic oral dysplastic keratinocytes. Future studies are warranted to gain more insight into the role of AREG in hyperglycemia-associated OPMD tumor progression.

Novel Bioactive Nanocomposites Containing Calcium Fluoride and Calcium Phosphate with Antibacterial and Low-Shrinkage-Stress Capabilities to Inhibit Dental Caries.

OBJECTIVES: Composites are commonly used for tooth restorations, but recurrent caries often lead to restoration failures due to polymerization shrinkage-stress-induced marginal leakage. The aims of this research were to: (1) develop novel low-shrinkage-stress (L.S.S.) nanocomposites containing dimethylaminododecyl methacrylate (DMADDM) with nanoparticles of calcium fluoride (nCaF2) or amorphous calcium phosphate (NACP) for remineralization; (2) investigate antibacterial and cytocompatibility properties. METHODS: Nanocomposites were made by mixing triethylene glycol divinylbenzyl ether with urethane dimethacrylate containing 3% DMADDM, 20% nCaF2, and 20% NACP. Flexural strength, elastic modulus, antibacterial properties against Streptococcus mutans biofilms, and cytotoxicity against human gingival fibroblasts and dental pulp stem cells were tested. RESULTS: Nanocomposites with DMADDM and nCaF2 or NACP had flexural strengths matching commercial composite control without bioactivity. The new nanocomposite provided potent antibacterial properties, reducing biofilm CFU by 6 logs, and reducing lactic acid synthesis and metabolic function of biofilms by 90%, compared to controls (p < 0.05). The new nanocomposites produced excellent cell viability matching commercial control (p > 0.05). CONCLUSIONS: Bioactive L.S.S. antibacterial nanocomposites with nCaF2 and NACP had excellent bioactivity without compromising mechanical and cytocompatible properties. The new nanocomposites are promising for a wide range of dental restorations by improving marginal integrity by reducing shrinkage stress, defending tooth structures, and minimizing cariogenic biofilms.

Calvaria defect regeneration via human periodontal ligament stem cells and prevascularized scaffolds in athymic rats.

BACKGROUND: Vascularization plays an important role in dental and craniofacial regenerations. Human periodontal ligament stem cells (hPDLSCs) are a promising cell source and, when co-cultured with human umbilical vein endothelial cells (hUVECs), could promote vascularization. The objectives of this study were to develop a novel prevascularized hPDLSC-hUVEC-calcium phosphate construct, and investigate the osteogenic and angiogenic efficacy of this construct with human platelet lysate (hPL) in cranial defects in rats for the first time. METHODS: hPDLSCs and hUVECs were co-cultured on calcium phosphate cement (CPC) scaffolds with hPL. Cell proliferation, angiogenic gene expression, angiogenesis, alkaline phosphatase activity, and cell-synthesized minerals were determined. Bone and vascular regenerations were investigated in rat critical-sized cranial defects in vivo. RESULTS: hPDLSC-hUVEC-CPC-hPL group had 2-fold greater angiogenic expressions and cell-synthesized mineral synthesis than hPDLSC-hUVEC-CPC group (p < 0.05). Microcapillary-like structures were formed on scaffolds in vitro. hPDLSC-hUVEC-CPC-hPL group had more vessels than hPDLSC-hUVEC-CPC group (p < 0.05). In cranial defects in rats, hPDLSC-hUVEC-CPC-hPL group regenerated new bone amount that was 2.1 folds and 4.0 folds, respectively, that of hPDLSC-hUVEC-CPC group and CPC control (p < 0.05). New blood vessel density of hPDLSC-hUVEC-CPC-hPL group was 2 folds and 7.9 folds, respectively, that of hPDLSC-hUVEC-CPC group and CPC control (p < 0.05). CONCLUSION: The hPL pre-culture method is promising to enhance bone regeneration via prevascularized CPC. Novel hPDLSC-hUVEC-CPC-hPL prevascularized construct increased new bone formation and blood vessel density by 4-8 folds over CPC control. CLINICAL SIGNIFICANCE: Novel hPDLSC-hUVEC-hPL-CPC prevascularized construct greatly increased bone and vascular regeneration in vivo and hence is promising for a wide range of craniofacial applications.

Injectable periodontal ligament stem cell-metformin-calcium phosphate scaffold for bone regeneration and vascularization in rats.

OBJECTIVES: Injectable and self-setting calcium phosphate cement scaffold (CPC) capable of encapsulating and delivering stem cells and bioactive agents would be highly beneficial for dental and craniofacial repairs. The objectives of this study were to: (1) develop a novel injectable CPC scaffold encapsulating human periodontal ligament stem cells (hPDLSCs) and metformin (Met) for bone engineering; (2) test bone regeneration efficacy in vitro and in vivo. METHODS: hPDLSCs were encapsulated in degradable alginate fibers, which were then mixed into CPC paste. Five groups were tested: (1) CPC control; (2) CPC + hPDLSC-fibers + 0% Met (CPC + hPDLSCs + 0%Met); (3) CPC + hPDLSC-fibers + 0.1% Met (CPC + hPDLSCs + 0.1%Met); (4) CPC + hPDLSC-fibers + 0.2% Met (CPC + hPDLSCs + 0.2%Met); (5) CPC + hPDLSC-fibers + 0.4% Met (CPC + hPDLSCs + 0.4%Met). The injectability, mechanical properties, metformin release, and hPDLSC osteogenic differentiation and bone mineral were determined in vitro. A rat cranial defect model was used to evaluate new bone formation. RESULTS: The novel construct had good injectability and physical properties. Alginate fibers degraded in 7 days and released hPDLSCs, with 5-fold increase of proliferation (p＜0.05). The ALP activity and mineral synthesis of hPDLSCs were increased by Met delivery (p＜0.05). Among all groups, CPC+hPDLSCs+ 0.1%Met showed the greatest cell mineralization and osteogenesis, which were 1.5-10 folds those without Met (p＜0.05). Compared to CPC control, CPC+hPDLSCs+ 0.1%Met enhanced bone regeneration in rats by 9 folds, and increased vascularization by 3 folds (p＜0.05). CONCLUSIONS: The novel injectable construct with hPDLSC and Met encapsulation demonstrated excellent efficacy for bone regeneration and vascularization in vivo in an animal model. CPC+hPDLSCs+ 0.1%Met is highly promising for dental and craniofacial applications.

Angiopoietin-like 4 induces head and neck squamous cell carcinoma cell migration through the NRP1/ABL1/PXN pathway.

OBJECTIVES: The molecular mechanisms whereby angiopoietin-like 4 (ANGPTL4), a pluripotent protein implicated in cancer development, contributes to head and neck squamous cell carcinoma (HNSCC) growth and dissemination are unclear. MATERIALS AND METHODS: We investigated ANGPTL4 expression in human normal oral keratinocytes (NOKs), dysplastic oral keratinocytes (DOKs), oral leukoplakia cells (LEUK1), and HNSCC cell lines, as well as in tissue biopsies from patients with oral dysplasia, and primary and metastatic HNSCC. We further examined the contribution of ANGPTL4 cancer progression in an HNSCC orthotopic floor-of mouth tumor model and the signaling pathways linking ANGPTL4 to cancer cell migration. RESULTS: ANGPTL4 expression was upregulated in premalignant DOKs and HNSCC cell lines compared to NOKs and was increased in tissue biopsies from patients with oral dysplasia, as well as in primary and metastatic HNSCC. We also observed that downregulation of ANGPTL4 expression inhibited primary and metastatic cancer growth in an HNSCC orthotopic tumor model. Interestingly, ANGPTL4 binding to the neuropilin1 (NRP1) receptor led to phosphorylation of the focal adhesion protein, paxillin (PXN), and tumor cell migration; this was dependent on the tyrosine kinase ABL1. Treatment with the ABL1 inhibitor, dasatinib and small interfering RNA silencing of NRP1 or ABL1 expression blocked PXN phosphorylation and tumor cell migration. CONCLUSION: Our findings suggest an early, sustained, and angiogenesis-independent autocrine role for ANGPTL4 in HNSCC progression and expose ANGPTL4/NRP1/ABL1/PXN as an early molecular marker and vulnerable target for the prevention of HNSCC growth and metastasis.

Degradable hydrogel fibers encapsulate and deliver metformin and periodontal ligament stem cells for dental and periodontal regeneration.

Human periodontal ligament stem cells (hPDLSCs) are promising cells for dental and periodontal regeneration. This study aimed to develop novel alginate-fibrin fibers that encapsulates hPDLSCs and metformin, to investigate the effect of metformin on the osteogenic differentiation of hPDLSCs, and to determine the regulatory role of the Shh/Gli1 signaling pathway in the metformin-induced osteogenic differentiation of hPDLSCs for the first time. CCK8 assay was used to evaluate hPDLSCs. Alkaline phosphatase (ALP) staining, alizarin red S staining, and the expression of osteogenic genes were evaluated. Metformin and hPDLSCs were encapsulated in alginate-fibrinogen solutions, which were injected to form alginate-fibrin fibers. The activation of Shh/Gli1 signaling pathway was examined using qRT-PCR and western blot. A mechanistic study was conducted by inhibiting the Shh/Gli1 pathway using GANT61. The administration of 50 µM metformin resulted in a significant upregulation of osteogenic gene expression in hPDLSCs by 1.4-fold compared to the osteogenic induction group (P < 0.01), including ALP and runt-related transcription factor-2 (RUNX2). Furthermore, metformin increased ALP activity by 1.7-fold and bone mineral nodule formation by 2.6-fold (P<0.001). We observed that hPDLSCs proliferated with the degradation of alginate-fibrin fibers, and metformin induced their differentiation into the osteogenic lineage. Metformin also promoted the osteogenic differentiation of hPDLSCs by upregulating the Shh/Gli1 signaling pathway by 3- to 6- fold compared to the osteogenic induction group (P<0.001). The osteogenic differentiation ability of hPDLSCs were decreased 1.3- to 1.6-fold when the Shh/Gli1 pathway was inhibited, according to ALP staining and alizarin red S staining (P<0.01). Metformin enhanced the osteogenic differentiation of hPDLSCs via the Shh/Gli1 signaling pathway. Degradable alginate-fibrin hydrogel fibers encapsulating hPDLSCs and metformin have significant potential for use in dental and periodontal tissue engineering applications. Alginate-fibrin fibers encapsulating hPDLSCs and metformin have a great potential for use in the treatment of maxillofacial bone defects caused by trauma, tumors, and tooth extraction. Additionally, they may facilitate the regeneration of periodontal tissue in patients with periodontitis.

Degradable hydrogel fibers encapsulate and deliver metformin and periodontal ligament stem cells for dental and periodontal regeneration

Abstract Human periodontal ligament stem cells (hPDLSCs) are promising cells for dental and periodontal regeneration. Objective This study aimed to develop novel alginate-fibrin fibers that encapsulates hPDLSCs and metformin, to investigate the effect of metformin on the osteogenic differentiation of hPDLSCs, and to determine the regulatory role of the Shh/Gli1 signaling pathway in the metformin-induced osteogenic differentiation of hPDLSCs for the first time. Methodology CCK8 assay was used to evaluate hPDLSCs. Alkaline phosphatase (ALP) staining, alizarin red S staining, and the expression of osteogenic genes were evaluated. Metformin and hPDLSCs were encapsulated in alginate-fibrinogen solutions, which were injected to form alginate-fibrin fibers. The activation of Shh/Gli1 signaling pathway was examined using qRT-PCR and western blot. A mechanistic study was conducted by inhibiting the Shh/Gli1 pathway using GANT61. Results The administration of 50 μM metformin resulted in a significant upregulation of osteogenic gene expression in hPDLSCs by 1.4-fold compared to the osteogenic induction group (P < 0.01), including ALP and runt-related transcription factor-2 (RUNX2). Furthermore, metformin increased ALP activity by 1.7-fold and bone mineral nodule formation by 2.6-fold (P<0.001). We observed that hPDLSCs proliferated with the degradation of alginate-fibrin fibers, and metformin induced their differentiation into the osteogenic lineage. Metformin also promoted the osteogenic differentiation of hPDLSCs by upregulating the Shh/Gli1 signaling pathway by 3- to 6- fold compared to the osteogenic induction group (P<0.001). The osteogenic differentiation ability of hPDLSCs were decreased 1.3- to 1.6-fold when the Shh/Gli1 pathway was inhibited, according to ALP staining and alizarin red S staining (P<0.01). Conclusions Metformin enhanced the osteogenic differentiation of hPDLSCs via the Shh/Gli1 signaling pathway. Degradable alginate-fibrin hydrogel fibers encapsulating hPDLSCs and metformin have significant potential for use in dental and periodontal tissue engineering applications. Clinical Significance Alginate-fibrin fibers encapsulating hPDLSCs and metformin have a great potential for use in the treatment of maxillofacial bone defects caused by trauma, tumors, and tooth extraction. Additionally, they may facilitate the regeneration of periodontal tissue in patients with periodontitis.

Effects of Metformin Delivery via Biomaterials on Bone and Dental Tissue Engineering.

Bone tissue engineering is a promising approach that uses seed-cell-scaffold drug delivery systems to reconstruct bone defects caused by trauma, tumors, or other diseases (e.g., periodontitis). Metformin, a widely used medication for type II diabetes, has the ability to enhance osteogenesis and angiogenesis by promoting cell migration and differentiation. Metformin promotes osteogenic differentiation, mineralization, and bone defect regeneration via activation of the AMP-activated kinase (AMPK) signaling pathway. Bone tissue engineering depends highly on vascular networks for adequate oxygen and nutrition supply. Metformin also enhances vascular differentiation via the AMPK/mechanistic target of the rapamycin kinase (mTOR)/NLR family pyrin domain containing the 3 (NLRP3) inflammasome signaling axis. This is the first review article on the effects of metformin on stem cells and bone tissue engineering. In this paper, we review the cutting-edge research on the effects of metformin on bone tissue engineering. This includes metformin delivery via tissue engineering scaffolds, metformin-induced enhancement of various types of stem cells, and metformin-induced promotion of osteogenesis, angiogenesis, and its regulatory pathways. In addition, the dental, craniofacial, and orthopedic applications of metformin in bone repair and regeneration are also discussed.

Periodontal ligament stem cell-based bioactive constructs for bone tissue engineering.

Objectives: Stem cell-based tissue engineering approaches are promising for bone repair and regeneration. Periodontal ligament stem cells (PDLSCs) are a promising cell source for tissue engineering, especially for maxillofacial bone and periodontal regeneration. Many studies have shown potent results via PDLSCs in bone regeneration. In this review, we describe recent cutting-edge researches on PDLSC-based bone regeneration and periodontal tissue regeneration. Data and sources: An extensive search of the literature for papers related to PDLSCs-based bioactive constructs for bone tissue engineering was made on the databases of PubMed, Medline and Google Scholar. The papers were selected by three independent calibrated reviewers. Results: Multiple types of materials and scaffolds have been combined with PDLSCs, involving xeno genic bone graft, calcium phosphate materials and polymers. These PDLSC-based constructs exhibit the potential for bone and periodontal tissue regeneration. In addition, various osteo inductive agents and strategies have been applied with PDLSCs, including drugs, biologics, gene therapy, physical stimulation, scaffold modification, cell sheets and co-culture. Conclusoin: This review article demonstrates the great potential of PDLSCs-based bioactive constructs as a promising approach for bone and periodontal tissue regeneration.

Human periodontal ligament stem cell encapsulation in alginate-fibrin-platelet lysate microbeads for dental and craniofacial regeneration.

OBJECTIVE: Tissue engineering is promising for dental and craniofacial regeneration. The objectives of this study were to develop a novel xeno-free alginate-fibrin-platelet lysate hydrogel with human periodontal ligament stem cells (hPDLSCs) for dental regeneration, and to investigate the proliferation and osteogenic differentiation of hPDLSCs using hPL as a cell culture nutrient supplement. METHODS: hPDLSCs were cultured with Dulbecco's modified eagle medium (DMEM), DMEM + 10% fetal bovine serum (FBS), and DMEM + hPL (1%, 2.5%, and 5%). hPDLSCs were encapsulated in alginate-fibrin microbeads (Alg+Fib), alginate-hPL microbeads (Alg+hPL), or alginate-fibrin-hPL microbeads (Alg+Fib+hPL). hPDLSCs encapsulated in alginate microbeads were induced with an osteogenic medium containing hPL or FBS. Quantitative real-time polymerase chain reaction (qRT-PCR), alkaline phosphatase (ALP) activity, ALP staining, and alizarin red (ARS) staining was investigated. RESULTS: hPDLSCs were released faster from Alg+Fib+hPL than from Alg+hPL. At 14 days, ALP activity was 44.1 ± 7.61 mU/mg for Alg+Fib+hPL group, higher than 28.07 ± 5.15 mU/mg of Alg+Fib (p<0.05) and 0.95 ± 0.2 mU/mg of control (p<0.01). At 7 days, osteogenic genes (ALP, RUNX2, COL1, and OPN) in Alg+Fib+hPL and Alg+Fib were 3-10 folds those of control. At 21 days, the hPDLSC-synthesized bone mineral amount in Alg+Fib+hPL and Alg+Fib was 7.5 folds and 4.3 folds that of control group, respectively. CONCLUSIONS: The 2.5% hPL was determined to be optimal for hPDLSCs. Adding hPL into alginate hydrogel improved the viability of the hPDLSCs encapsulated in the microbeads. The hPL-based medium enhanced the osteogenic differentiation of hPDLSCs in Alg+Fib+hPL construct, showing a promising xeno-free approach for delivering hPDLSCs to enhance dental, craniofacial and orthopedic regenerations.

Human Periodontal Ligament Stem Cell and Umbilical Vein Endothelial Cell Co-Culture to Prevascularize Scaffolds for Angiogenic and Osteogenic Tissue Engineering.

(1) Background: Vascularization remains a critical challenge in bone tissue engineering. The objective of this study was to prevascularize calcium phosphate cement (CPC) scaffold by co-culturing human periodontal ligament stem cells (hPDLSCs) and human umbilical vein endothelial cells (hUVECs) for the first time; (2) Methods: hPDLSCs and/or hUVECs were seeded on CPC scaffolds. Three groups were tested: (i) hUVEC group (hUVECs on CPC); (ii) hPDLSC group (hPDLSCs on CPC); (iii) co-culture group (hPDLSCs + hUVECs on CPC). Osteogenic differentiation, bone mineral synthesis, and microcapillary-like structures were evaluated; (3) Results: Angiogenic gene expressions of co-culture group were 6-9 fold those of monoculture. vWF expression of co-culture group was 3 times lower than hUVEC-monoculture group. Osteogenic expressions of co-culture group were 2-3 folds those of the hPDLSC-monoculture group. ALP activity and bone mineral synthesis of co-culture were much higher than hPDLSC-monoculture group. Co-culture group formed capillary-like structures at 14-21 days. Vessel length and junction numbers increased with time; (4) Conclusions: The hUVECs + hPDLSCs co-culture on CPC scaffold achieved excellent osteogenic and angiogenic capability in vitro for the first time, generating prevascularized networks. The hPDLSCs + hUVECs co-culture had much better osteogenesis and angiogenesis than monoculture. CPC scaffolds prevacularized via hPDLSCs + hUVECs are promising for dental, craniofacial, and orthopedic applications.

Novel calcium phosphate cement with biofilm-inhibition and platelet lysate delivery to enhance osteogenesis of encapsulated human periodontal ligament stem cells.

Osteomyelitis is caused by Staphylococcus aureus (S. aureus), with associated progressive bone loss. This study developed for the first time a calcium phosphate cement (CPC) for delivery of doxycycline (DOX) and human platelet lysate (hPL) to fight against S. aureus infection and enhance the osteogenesis of human periodontal ligament stem cells (hPDLSCs). Chitosan-containing CPC scaffolds were fabricated in the absence (CPCC) or presence of DOX (CPCC+DOX). In addition, hPL was encapsulated in alginate microbeads and incorporated into CPCC+DOX (CPCC+DOX+ hPL). Flexural strength of CPCC+DOX + hPL was (5.56 ± 0.55) MPa, lower than (8.26 ± 1.6) MPa of CPCC+DOX (p < 0.05), but exceeding the reported strength of cancellous bone. CPCC+DOX and CPCC+DOX + hPL exhibited strong antibacterial activity against S. aureus, reducing biofilm CFU by 4 orders of magnitude. The hPDLSCs encapsulated in microbeads were co-cultured with the CPCs. The hPDLSCs were able to be released from the microbeads and showed a high proliferation rate, increasing by about 8 folds at 14 days for all groups. The hPL was released from the scaffold and promoted the osteogenic differentiation of hPDLSCs. ALP activity was 28.07 ± 5.15 mU/mg for CPCC+DOX + hPL, higher than 17.36 ± 2.37 mU/mg and 1.34 ± 0.37 mU/mg of CPCC+DOX and CPCC, respectively (p < 0.05). At 7 days, osteogenic genes (ALP, RUNX2, COL-1, and OPN) in CPCC+DOX + hPL were 3-10 folds those of control. The amount of hPDLSC-synthesized bone mineral with CPCC+DOX + hPL was 3.8 folds that of CPCC (p < 0.05). In summary, the novel CPC + DOX + hPL-hPDLSCs scaffold exhibited strong antibacterial activity, excellent cytocompatibility and hPDLSC osteogenic differentiation, showing a promising approach for treatment and prevention of bone infection and enhancement of bone regeneration.

Fatty acid synthase mediates high glucose-induced EGFR activation in oral dysplastic keratinocytes.

BACKGROUND: Recent studies point to the epidermal growth factor receptor (EGFR) as a critical mediator of type 2 diabetes mellitus (T2DM)-induced renal, cardiac, and ocular complications. T2DM is considered a systemic contributing factor in oral carcinogenesis. Similarly, increased EGFR gene copy number and protein expression strongly predict tumor progression. Yet, the impact of hyperglycemia on EGFR activity in oral potentially malignant disorders remains unclear. We recently reported that fatty acid synthase (FASN), a key de novo lipogenic enzyme, mediates EGFR activation in nicotine-treated oral dysplastic keratinocytes. While in non-malignant tissues FASN expression is extremely low, it is frequently upregulated in several cancers, including oral squamous cell carcinoma. The present study was carried out to investigate whether high glucose conditions trigger pro-oncogenic responses in oral dysplastic keratinocytes via FASN-mediated EGFR activation. METHODS: Cell viability and migration of oral dysplastic keratinocytes were evaluated when exposed to normal (5 mM) or high (20 mM) glucose conditions in the presence of FASN and EGFR inhibitors. Western blotting was also performed to assess changes in FASN protein expression and EGFR activation. RESULTS: Oral dysplastic keratinocytes exposed to high glucose led to EGFR activation in a FASN-dependent manner. Likewise, high glucose significantly enhanced cell viability and migration in a FASN/EGFR-mediated fashion. Notably, EGFR inhibition by the anti-EGFR monoclonal antibody cetuximab significantly reduced the proliferation of FASN-overexpressing oral dysplastic keratinocytes. CONCLUSION: These novel findings suggest that FASN may act as a key targetable metabolic regulator of glucose-induced EGFR oncogenic signaling in oral potentially malignant disorders.

Bioactive small molecules in calcium phosphate scaffold enhanced osteogenic differentiation of human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) are exciting for regenerative medicine due to their multi-potent differentiation. SB431542 bioactive molecule can activate bone morphogenetic protein-signalling in osteoblasts. The objectives were to: (1) develop a novel injectable calcium phosphate cement (CPC)-SB431542 scaffold for dental/craniofacial bone engineering; and (2) investigate cell proliferation and osteo-differentiation of hiPSC-derived mesenchymal stem cells (hiPSC-MSCs) on CPC-SB431542 scaffold. Three groups were tested: CPC control; CPC with SB431542 inside CPC (CPCSM); CPC with SB431542 in osteogenic medium (CPC+SMM). SB431542 in CPC promoted stem cell proliferation and viability. hiPSC-MSCs differentiated into osteogenic lineage and synthesized bone minerals. CPC with SB431542 showed much greater osteo-expressions and more bone minerals than those without SB431542. In conclusion, hiPSC-MSCs on CPC scaffold containing SB431542 showed excellent osteo-differentiation and bone mineral synthesis for the first time. CPC was a suitable scaffold for delivering stem cells and SB431542 to promote bone regeneration in dental/craniofacial applications.

An injectable and antibacterial calcium phosphate scaffold inhibiting Staphylococcus aureus and supporting stem cells for bone regeneration.

Staphylococcus aureus (S. aureus) is the major pathogen for osteomyelitis, which can lead to bone necrosis and destruction. There has been no report on antibacterial calcium phosphate cement (CPC) against S. aureus. The aims of this study were to: (1) develop novel antibacterial CPC-chitosan-alginate microbead scaffold; (2) investigate mechanical and antibacterial properties of CPC-chitosan-penicillin-alginate scaffold; (3) evaluate the encapsulation and delivery of human umbilical cord mesenchymal stem cells (hUCMSCs). Flexural strength, elastic modulus and work-of-fracture of the CPC-chitosan-penicillin-alginate microbeads scaffold and CPC-chitosan scaffold were evaluated. Penicillin release profile and antibacterial effects on S. aureus were determined. The hUCMSC delivery and release from penicillin-alginate microbeads were investigated. Injectable CPC-chitosan-penicillin-alginate microbeads scaffold was developed for the first time. CPC-chitosan-penicillin-alginate microbeads scaffold had a flexural strength of 3.16 ± 0.55 MPa, matching that of cancellous bone. With sustained penicillin release, the new scaffold had strong antibacterial effects on S. aureus, with an inhibition zone diameter of 32.2 ± 2.5 mm, greater than that of penicillin disk control (15.1 ± 2.0 mm) (p < 0.05). Furthermore, this injectable and antibacterial scaffold had no toxic effects, yielding excellent hUCMSC viability, which was similar to that of CPC control without antibacterial activity (p > 0.05). CPC-chitosan-penicillin-microbeads scaffold had injectability, good strength, strong antibacterial effects, and good biocompatibility to support stem cell viability for osteogenesis. CPC-chitosan-penicillin-microbeads scaffold is promising for dental, craniofacial and orthopedic applications to combat infections and promote bone regeneration.

Antibacterial calcium phosphate cement with human periodontal ligament stem cell-microbeads to enhance bone regeneration and combat infection.

Infectious bone defects remain a significant challenge in orthopedics and dentistry. Calcium phosphate cement (CPC) have attracted significant interest in use as local drug delivery system, which with great potential to control release of antibiotics for the treatment of infectious bone defects. Within the current study, a novel antibacterial scaffold of chitosan-reinforced calcium phosphate cement delivering doxycycline hyclate (CPCC + DOX) was developed. Furthermore, the capacity of CPCC + DOX scaffolds for bone regeneration was enhanced by the human periodontal ligament stem cells (hPDLSCs) encapsulated in alginate beads. CPCC + DOX scaffolds were fabricated to contain different concentrations of DOX. Flexural strength of CPCC + DOX ranged from 5.56 ± 0.70 to 6.2 ± 0.72 MPa, which exceeded the reported strength of cancellous bone. Scaffolds exhibited continual DOX release, reaching 80% at 21 days. Scaffold with 5 mg/ml DOX (CPCC + DOX5mg) had a strong antibacterial effect, with a 4-log colony forming unit reduction against S. aureus and P. gingivalis. The proliferation and osteogenic differentiation of hPDLSCs encapsulated in alginate hydrogel microbeads were investigated in culture with CPCC + DOX scaffolds. CPCC + DOX5mg had no negative effect on proliferation of hPDLSCs. Alkaline phosphatase activity, mineral synthesis, and osteogenic gene expressions for CPCC + DOX5mg group were much higher than control group. DOX did not compromise the osteogenic induction. In summary, the novel CPCC + DOX scaffold exhibited excellent mechanical properties and strong antibacterial activity, while supporting the proliferation and osteogenic differentiation of hPDLSCs. The CPCC + DOX + hPDLSCs construct is promising to enhance bone regeneration and combat bone infections in dental, craniofacial, and orthopedic applications.

Biocompatible Nanocomposite Enhanced Osteogenic and Cementogenic Differentiation of Periodontal Ligament Stem Cells In Vitro for Periodontal Regeneration.

Decays in the roots of teeth is prevalent in seniors as people live longer and retain more of their teeth to an old age, especially in patients with periodontal disease and gingival recession. The objectives of this study were to develop a biocompatible nanocomposite with nano-sized calcium fluoride particles (Nano-CaF2), and to investigate for the first time the effects on osteogenic and cementogenic induction of periodontal ligament stem cells (hPDLSCs) from human donors.Nano-CaF2 particles with a mean particle size of 53 nm were produced via a spray-drying machine.Nano-CaF2 was mingled into the composite at 0%, 10%, 15% and 20% by mass. Flexural strength (160 ± 10) MPa, elastic modulus (11.0 ± 0.5) GPa, and hardness (0.58 ± 0.03) GPa for Nano-CaF2 composite exceeded those of a commercial dental composite (p < 0.05). Calcium (Ca) and fluoride (F) ions were released steadily from the composite. Osteogenic genes were elevated for hPDLSCs growing on 20% Nano-CaF2. Alkaline phosphatase (ALP) peaked at 14 days. Collagen type 1 (COL1), runt-related transcription factor 2 (RUNX2) and osteopontin (OPN) peaked at 21 days. Cementogenic genes were also enhanced on 20% Nano-CaF2 composite, promoting cementum adherence protein (CAP), cementum protein 1 (CEMP1) and bone sialoprotein (BSP) expressions (p < 0.05). At 7, 14 and 21 days, the ALP activity of hPDLSCs on 20% Nano-CaF2 composite was 57-fold, 78-fold, and 55-fold greater than those of control, respectively (p < 0.05). Bone mineral secretion by hPDLSCs on 20% Nano-CaF2 composite was 2-fold that of control (p < 0.05). In conclusion, the novel Nano-CaF2 composite was biocompatible and supported hPDLSCs. Nano-CaF2 composite is promising to fill tooth root cavities and release Ca and F ions to enhance osteogenic and cementogenic induction of hPDLSCs and promote periodontium regeneration.