Objective: To investigate the effects of long non-coding RNA (lncRNA) LINC01133 on the cementogenic differentiation of human periodontal ligament stem cells (hPDLSC) and the underlying mechanism. Methods: A total of 12 teeth were harvested from 10 patients aged 17-30 years in the Department of Oral and Maxillofacial Surgery, School of Stomatology, The Fourth Military Medical University for impacted or orthodontic reasons from September 2021 to January 2022. The hPDLSCs were isolated from the teeth and transfected with small interfering RNA-LINC01133 (si-LINC01133) or small interfering RNA-negative control (si-NC). The si-LINC01133 was regarded as the experimental group, and the si-NC was regarded as the control one. The silencing efficiency of LINC01133 in the hPDLSCs was evaluated by real-time quantitative PCR (RT-qPCR). Western blotting was used to detect the protein expression levels of cementogenic differentiation-related factors including bone sialoprotein (BSP), cementum attachment protein (CAP), and cementum protein-1 (CEMP-1). Mitochondrial reactive oxygen species (mtROS) production was assessed using the MitoSox by flow cytometry. Mitochondrial membrane potential (MMP) was detected by JC-1 fluorescence staining. Mitochondrial respiratory chain complexes proteins including NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8 (NDUFB8), succinate dehydrogenase complex flavoprotein subunit A (SDHA), ubiquinol-cytochrome c reductase core protein 1 (UQCR1), cytochrome c oxidase subunit 4 isoform 1 (COXⅣ), and ATP synthase F1 subunit alpha (ATP5A) were evaluated by Western blotting. Results: The expression levels of LINC01133 could be suppressed by more than 60% with si-LINC01133 (control group: 1.000±0.000, experimental group: 0.385±0.128) (t=10.72, P<0.01). Suppression of LINC01133 in hPDLSCs decreased the levels of cementogenic differentiation-related proteins including BSP (control group: 1.000±0.000, experimental group: 0.664±0.179) (t=4.62, P<0.01) and CAP (control group: 1.000±0.000, experimental group: 0.736±0.229) (t=2.83, P<0.05). Suppression of LINC01133 in hPDLSCs increased the production of mtROS (control group: 1.000±0.000, experimental group: 1.458±0.185) (t=4.96, P<0.05) and the expression of NDUFB8 (control group: 1.000±0.000, experimental group: 1.683±0.397) (t=3.45, P<0.05), as well as decreased MMP levels (control group: 1.000±0.000, experimental group: 0.209±0.029) (t=53.99, P<0.01) and the expression of SDHA (control group: 1.000±0.000, experimental group: 0.428±0.228) (t=5.02, P<0.05). No significant changes in the UQCR1, COXⅣ, and ATP5A expression levels were found between the control group and exprimental group (P>0.05). Conclusions: LINC01133 regulates the cementogenic differentiation of hPDLSCs possibly via modulating the mitochondrial functions.
Humans ; Periodontal Ligament ; RNA, Long Noncoding/metabolism* ; Cells, Cultured ; Stem Cells ; Cell Differentiation ; Integrin-Binding Sialoprotein/metabolism* ; Mitochondrial Proteins/metabolism* ; Mitochondria/genetics* ; RNA, Small Interfering/metabolism* ; Osteogenesis

Amelogenin (AMG) is a cell adhesion molecule that has an important role in the mineralization of enamel and regulates events during dental development and root formation. The purpose of the present study was to investigate the effects of recombinant human AMG (rhAMG) on mineralized tissue-associated genes in cementoblasts. Immortalized mouse cementoblasts (OCCM-30) were treated with different concentrations (0.1, 1, 10, 100, 1000, 10,000, 100,000 ng · mL) of recombinant human AMG (rhAMG) and analyzed for proliferation, mineralization and mRNA expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen type I (COL I), osteopontin (OPN), runt-related transcription factor 2 (Runx2), cementum attachment protein (CAP), and alkaline phosphatase (ALP) genes using quantitative RT-PCR. The dose response of rhAMG was evaluated using a real-time cell analyzer. Total RNA was isolated on day 3, and cell mineralization was assessed using von Kossa staining on day 8. COL I, OPN and lysosomal-associated membrane protein-1 (LAMP-1), which is a cell surface binding site for amelogenin, were evaluated using immunocytochemistry. F-actin bundles were imaged using confocal microscopy. rhAMG at a concentration of 100,000 ng · mL increased cell proliferation after 72 h compared to the other concentrations and the untreated control group. rhAMG (100,000 ng · mL) upregulated BSP and OCN mRNA expression levels eightfold and fivefold, respectively. rhAMG at a concentration of 100,000 ng · mL remarkably enhanced LAMP-1 staining in cementoblasts. Increased numbers of mineralized nodules were observed at concentrations of 10,000 and 100,000 ng · mL rhAMG. The present data suggest that rhAMG is a potent regulator of gene expression in cementoblasts and support the potential application of rhAMG in therapies aimed at fast regeneration of damaged periodontal tissue.
Alkaline Phosphatase ; metabolism ; Amelogenin ; physiology ; Animals ; Biomarkers ; metabolism ; Calcification, Physiologic ; Cell Adhesion Molecules ; metabolism ; Cell Proliferation ; Cementogenesis ; physiology ; Collagen Type I ; metabolism ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Gene Expression Regulation ; In Vitro Techniques ; Integrin-Binding Sialoprotein ; metabolism ; Mice ; Microscopy, Confocal ; Osteocalcin ; metabolism ; Osteopontin ; metabolism ; Real-Time Polymerase Chain Reaction

OBJECTIVES: The effects of bone morphogenetic protein-2 (BMP-2) and enamel matrix derivative (EMD) respectively with mineral trioxide aggregate (MTA) on hard tissue regeneration have been investigated in previous studies. This study aimed to compare the osteogenic effects of MTA/BMP-2 and MTA/EMD treatment in MC3T3-E1 cells. MATERIALS AND METHODS: MC3T3-E1 cells were treated with MTA (ProRoot, Dentsply), BMP-2 (R&D Systems), EMD (Emdogain, Straumann) separately and MTA/BMP-2 or MTA/EMD combination. Mineralization was evaluated by staining the calcium deposits with alkaline phosphatase (ALP, Sigma-Aldrich) and Alizarin red (Sigma-Aldrich). The effects on the osteoblast differentiation were evaluated by the expressions of osteogenic markers, including ALP, bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN) and osteonectin (OSN), as determined by reverse-transcription polymerase chain reaction analysis (RT-PCR, AccuPower PCR, Bioneer). RESULTS: Mineralization increased in the BMP-2 and MTA/BMP-2 groups and increased to a lesser extent in the MTA/EMD group but appeared to decrease in the MTA-only group based on Alizarin red staining. ALP expression largely decreased in the EMD and MTA/EMD groups based on ALP staining. In the MTA/BMP-2 group, mRNA expression of OPN on day 3 and BSP and OCN on day 7 significantly increased. In the MTA/EMD group, OSN and OCN gene expression significantly increased on day 7, whereas ALP expression decreased on days 3 and 7 (p < 0.05). CONCLUSIONS: These results suggest the MTA/BMP-2 combination promoted more rapid differentiation in MC3T3-E1 cells than did MTA/EMD during the early mineralization period.
Alkaline Phosphatase ; Calcium ; Dental Enamel* ; Gene Expression ; Integrin-Binding Sialoprotein ; Osteoblasts ; Osteocalcin ; Osteonectin ; Osteopontin ; Polymerase Chain Reaction ; Regeneration ; RNA, Messenger ; Pemetrexed

PURPOSE: This study focused on in vitro cell differentiation and surface characteristics in a magnesium coated titanium surface implanted on using a plasma ion source. MATERIALS AND METHODS: 40 commercially made pure titanium discs were prepared to produce Ti oxide machined surface (M) and Mg-incorporated Ti oxide machined surface (MM). Surface properties were analyzed using a scanning electron microscopy (SEM). On each surface, alkaline phosphatase (ALP) activity, alizarin red S staining for mineralization of MC3T3-E1 cells, and quantitative analysis of osteoblastic gene expression, were evaluated. Actin ring formation assay and gene expression analysis of TRAP and GAPDH performing RT-PCR were performed to characterize osteoclast differentiation on mouse bone marrow-derived macrophages (BMMs). RESULTS: MM showed similar surface morphology and surface roughness with M, but was slightly smoother after ion implantation at the micron scale. M was more hydrophobic than MM. No significant difference between surfaces on ALP activity at 7 and 14 days were observed. Real-time PCR analyses showed similar levels of mRNA expression of the osteoblast phenotype genes; osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), and collagen 1 (Col 1) in cell grown on MM at 7, 14 and 21 days. Alizarin red S staining at 21 days showed no significant difference. BMMs differentiation increased in M and MM. Actin ring formation assay and gene expression analysis of TRAP showed osteoclast differentiation to be more active on MM. CONCLUSION: Both M and MM have a good effect on osteoblastic cell differentiation, but MM may speed the bone remodeling process by activating on osteoclast differentiation.
Actins ; Alkaline Phosphatase ; Animals ; Bone Remodeling ; Cell Differentiation ; Collagen ; Gene Expression ; Integrin-Binding Sialoprotein ; Macrophages ; Magnesium* ; Mice ; Microscopy, Electron, Scanning ; Osteoblasts* ; Osteocalcin ; Osteoclasts* ; Osteopontin ; Phenotype ; Plasma ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; Surface Properties ; Titanium*

OBJECTIVETo investigate the roll of bone sialoprotein (BSP), a secreted glycoprotein, found in mineralized tissues in the development and progression of human esophageal squamous cell carcimoma (ESCC), and explore its association with clinicopathological characteristics and five-year survival of the patients.

METHODSThe expression of BSP was determined in 211 primary ESCC tumors and their paired nontumorous tissues using tissue-array, RT-PCR and immunohistochemistry.

RESULTSPrimary ESCC tissues showed a significantly higher expression rate of BSP mRNA than their paired nontumorous tissues (93.8% vs. 16.6%, P < 0.001), the same with BSP protein (56.9% vs. 31.3%, P < 0.001). The expression rate of BSP protein was correlated to lymph node metastasis and TNM stage (P < 0.05). The 5-year survival rate of BSP protein-positive ESCC patients was significantly lower than that of BSP protein-negative ESCC patients (P < 0.05). Multivariate analysis showed that tumor differentiation, TNM staging and BSP protein expression were independent factors affecting the prognosis of ESCC patients (P < 0.05).

CONCLUSIONSThe abnormal expression of BSP may play a significant role in the malignant progression and prognosis of ESCC, and BSP might be a marker reflecting the biologial behavior of ESCC.

Blotting, Western ; Carcinoma, Squamous Cell ; diagnosis ; metabolism ; Esophageal Neoplasms ; diagnosis ; metabolism ; Humans ; Immunohistochemistry ; Integrin-Binding Sialoprotein ; genetics ; metabolism ; Lymphatic Metastasis ; Neoplasm Staging ; Prognosis ; RNA, Messenger ; Survival Rate

PURPOSE: The purpose of this study was to assess the surface characteristics and the biocompatibility of zirconium (Zr) coating on Ti-6Al-4V alloy surface by radio frequency (RF) magnetron sputtering method. MATERIALS AND METHODS: The zirconium films were developed on Ti-6Al-4V discs using RF magnetron sputtering method. Surface profile, surface composition, surface roughness and surface energy were evaluated. Electrochemical test was performed to evaluate the corrosion behavior. Cell proliferation, alkaline phosphatase (ALP) activity and gene expression of mineralized matrix markers were measured. RESULTS: SEM and EDS analysis showed that zirconium deposition was performed successfully on Ti-6Al-4V alloy substrate. Ti-6Al-4V group and Zr-coating group showed no significant difference in surface roughness (P>.05). Surface energy was significantly higher in Zr-coating group than in Ti-6Al-4V group (P<.05). No difference in cell morphology was observed between Ti-6Al-4V group and Zr-coating group. Cell proliferation was higher in Zr-coating group than Ti-6Al-4V group at 1, 3 and 5 days (P<.05). Zr-coating group showed higher ALP activity level than Ti-6Al-4V group (P<.05). The mRNA expressions of bone sialoprotein (BSP) and osteocalcin (OCN) on Zr-coating group increased approximately 1.2-fold and 2.1-fold respectively, compared to that of Ti-6Al-4V group. CONCLUSION: These results suggest that zirconium coating on Ti-6Al-4V alloy could enhance the early osteoblast responses. This property could make non-toxic metal coatings on Ti-6Al-4V alloy suitable for orthopedic and dental implants.
Alkaline Phosphatase ; Alloys* ; Biocompatible Materials ; Cell Proliferation ; Coated Materials, Biocompatible ; Corrosion ; Dental Implants ; Gene Expression ; Integrin-Binding Sialoprotein ; Orthopedics ; Osteoblasts* ; Osteocalcin ; RNA, Messenger ; Surface Properties ; Titanium ; Zirconium*

OBJECTIVETo regenerate dentin-pulp complex by tissue engineering with human stem cells from apical papilla cells (SCAP) as the seed cells.

METHODSSCAP was separated from from normal human impacted third molars with immature roots by outgrowth culture. The cells were then cultured in the differentiation medium for 3 weeks or in normal medium for 60 days, and analyzed for mineralization potential by Alizarin red staining. The osteo/odontogenic markers including alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OC) and dentin sialoprotein (DSP) were investigated by immunofluorescence staining and reverse transcription-polymerase chain reaction. The co-cultured mixture of SCAP and HA/TCP, or HA/TCP alone was implanted subcutaneously on the back of nude mice for 8 weeks, and the implants were collected and examined by HE and immunohistochemical staining.

RESULTSRound alizarin red-positive nodules formed in the isolated cells after cell culture in the differentiation medium for 3 weeks or in normal medium for 60 days with positive staining for osteo/odontogenic markers. SCAP with HA/TCP could regenerate pulp-dentin complex-like tissue in nude mice. The cells near the dentin-like tissue were positive for DSP. No mineral tissue was found in mice receiving HA/TCP implantation.

CONCLUSIONSSCAP may serve as a promising seed cell for dentin-pulp complex tissue engineering.

Adolescent ; Adult ; Alkaline Phosphatase ; analysis ; Animals ; Cell Culture Techniques ; Cell Differentiation ; Coculture Techniques ; Dental Papilla ; cytology ; Dental Pulp ; cytology ; Extracellular Matrix Proteins ; analysis ; Female ; Humans ; Integrin-Binding Sialoprotein ; analysis ; Mice ; Mice, Nude ; Odontogenesis ; physiology ; Osteocalcin ; analysis ; Phosphoproteins ; analysis ; Sialoglycoproteins ; analysis ; Stem Cells ; chemistry ; physiology ; Tissue Engineering ; methods ; Young Adult

PURPOSE: The aim of this study was to evaluate the surface properties and in vitro bioactivity to osteoblasts of magnesium and magnesium-hydroxyapatite coated titanium. MATERIALS AND METHODS: Themagnesium (Mg) and magnesium-hydroxyapatite (Mg-HA) coatings on titanium (Ti) substrates were prepared by radio frequency (RF) and direct current (DC) magnetron sputtering.The samples were divided into non-coated smooth Ti (Ti-S group), Mg coatinggroup (Ti-Mg group), and Mg-HA coating group (Ti-MgHA group).The surface properties were evaluated using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The surface roughness was evaluated by atomic force microscopy (AFM). Cell adhesion, cell proliferation and alkaline phosphatase (ALP) activity were evaluated using MC3T3-E1 cells. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed. RESULTS: Cross-sectional SEM images showed that Mg and Mg-HA depositionson titanium substrates were performed successfully. The surface roughness appeared to be similaramong the three groups. Ti-MgHA and Ti-Mg group had improved cellular responses with regard to the proliferation, alkaline phosphatase (ALP) activity, and bone-associated markers, such as bone sialoprotein (BSP) and osteocalcin (OCN) mRNA compared to those of Ti-S group. However, the differences between Ti-Mg group and Ti-MgHA group were not significant, in spite of the tendency of higher proliferation, ALP activity and BSP expression in Ti-MgHA group. CONCLUSION: Mg and Mg-HAcoatings could stimulate the differentiation into osteoblastic MC3T3-E1 cells, potentially contributing to rapid osseointegration.
Alkaline Phosphatase ; Biocompatible Materials ; Calcium Phosphates ; Calcium* ; Cell Adhesion ; Cell Proliferation ; Coated Materials, Biocompatible ; Integrin-Binding Sialoprotein ; Magnesium* ; Microscopy, Atomic Force ; Microscopy, Electron, Scanning ; Osseointegration ; Osteoblasts* ; Osteocalcin ; Photoelectron Spectroscopy ; Polymerase Chain Reaction ; Reverse Transcription ; RNA, Messenger ; Surface Properties ; Titanium*

OBJECTIVE: To investigate the effects of the hypoxia inducible factor-1 (HIF-1) activation-mimicking agent cobalt chloride (CoCl2) on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and elucidate the underlying molecular mechanisms. STUDY DESIGN: The dose and exposure periods for CoCl2 in hMSCs were optimized by cell viability assays. After confirmation of CoCl2-induced HIF-1alpha and vascular endothelial growth factor expression in these cells by RT-PCR, the effects of temporary preconditioning with CoCl2 on hMSC osteogenic differentiation were evaluated by RT-PCR analysis of osteogenic gene expression, an alkaline phosphatase (ALP) activity assay and by alizarin red S staining. RESULTS: Variable CoCl2 dosages (up to 500 microM) and exposure times (up to 7 days) on hMSC had little effect on hMSC survival. After CoCl2 treatment of hMSCs at 100 microM for 24 or 48 hours, followed by culture in osteogenic differentiating media, several osteogenic markers such as Runx-2, osteocalcin and osteopontin, bone sialoprotein mRNA expression level were found to be up-regulated. Moreover, ALP activity was increased in these treated cells in which an accelerated osteogenic capacity was also verified by alizarin red S staining. CONCLUSIONS: The osteogenic differentiation potential of hMSCs could be preserved and even enhanced by CoCl2 treatment.
Alkaline Phosphatase ; Anoxia ; Anthraquinones ; Cell Survival ; Cobalt ; Durapatite ; Gene Expression ; Humans ; Integrin-Binding Sialoprotein ; Mesenchymal Stromal Cells ; Osteocalcin ; Osteopontin ; RNA, Messenger ; Vascular Endothelial Growth Factor A

OBJECTIVETo investigate the effects of Osterix (Osx) overexpression on the osteogenic differentiation of human periodontal ligament cells in response to mechanical force.

METHODSHuman periodontal ligament cells were isolated and cultured in vitro with explant method. Cells were transfected with either an Osx expression vector pcDNA3.1 flag-Osx or the mock control vector pcDNA3.1 flag. Then, cells were centrifuged for 6 h. After transfection and centrification, the expression of Osx mRNA and protein in untransfected cells, mock-transfected cells and Osx-transfected cells were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively. Furthermore, the changes of mRNA expressions of core-binding factor cal (Cbfal), alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OC), bone sialoprotein(BSP) and collagen protein al (Col I ) genes were measured to assess the differentiation of human periodontal ligament cells.

RESULTSAt 24 h after transfection, Osx mRNA and protein level increased significantly in Osx-transfected cells (P < 0.01), while there were no significant difference in Osx mRNA and protein levels between mock-transfected cells and untransfected cells(P > 0.05). Simultaneously, the upregulated mRNA expressions of all the five osteogenic genes were observed (P < 0.05, P < 0.01). After 6 h of mechanical stimulation, a significant increase in Osx expression was shown in all three groups. However, compared to mock-transfected and untransfected cells, Osx-transfected cells further showed the highest Osx mRNA and protein expression level. Furthermore, the mRNA expressions of all five osteogenic markers in Osx-transfected cells also exhibited the greater increase and showed the highest levels.

CONCLUSIONThe overexpression of Osx promotes the mechanical stress-induced osteogenic differentiation of human periodontal ligament cells. Osx may be essential for mechanical stress-induced differentiation of human periodontal ligament cells to osteoblas tic-like cells and be involved in orthodontic osteogenic remodeling.

Alkaline Phosphatase ; Cell Differentiation ; Cells, Cultured ; Humans ; Integrin-Binding Sialoprotein ; Osteocalcin ; Osteogenesis ; Osteopontin ; Periodontal Ligament ; RNA, Messenger ; Stress, Mechanical ; Transfection