Gene expression profiling of Jagged1-treated human periodontal ligament cells.

OBJECTIVE: Jagged1 regulates several biological functions in human periodontal ligament cells (hPDLs). The present study aimed to evaluate mRNA expression profiling of Jagged1-treated hPDLs using microarray technique. METHODS: Notch ligands, Jagged1, were indirectly immobilized on tissue culture surface. Subsequently, hPDLs were seeded on Jagged1 immobilized surface and maintained in growth medium for 48 hr. Total RNA was collected and processed. Gene expression profiling was examined using microarray technique. Real-time polymerase chain reaction and immunofluorescence staining were employed to determine mRNA and protein expression levels, respectively. Cell proliferation and colony-forming unit assay were performed. Cell cycle was evaluated using propidium iodide staining and flow cytometry analysis. RESULTS: The isolated cells demonstrated fibroblast-like morphology and exhibited the co-expression of CD44, CD90, and CD105 surface markers. After stimulated with Jagged1, the total of 411 genes was differentially expressed, consisting both coding and non-coding genes. For coding genes, 165 and 160 coding genes were upregulated and downregulated, respectively. Pathway analysis revealed that the upregulated genes were mainly involved in cellular interactions, signal transduction, and collagen formation and degradation while the downregulated genes were in the events and phases in cell cycle. Jagged1 significantly decreased cell proliferation, reduced colony-forming unit ability, and induced G0/G1 cell cycle arrest in hPDLs. CONCLUSION: Jagged1 regulates various biological pathways in hPDLs. This gene expression profiling could help to understand the mechanisms potentially involved in the Notch signaling regulation in periodontal homeostasis.

Kit W-sh Mutation Prevents Cancellous Bone Loss during Calcium Deprivation.

Calcium is essential for normal bone growth and development. Inadequate calcium intake increases the risk of osteoporosis and fractures. Kit ligand/c-Kit signaling plays an important role in regulating bone homeostasis. Mice with c-Kit mutations are osteopenic. The present study aimed to investigate whether impairment of or reduction in c-Kit signaling affects bone turnover during calcium deprivation. Three-week-old male WBB6F1/J-Kit W /Kit W-v /J (W/W v ) mice with c-Kit point mutation, Kit W-sh /HNihrJaeBsmJ (W sh /W sh ) mice with an inversion mutation in the regulatory elements upstream of the c-Kit promoter region, and their wild-type controls (WT) were fed either a normal (0.6% calcium) or a low calcium diet (0.02% calcium) for 3 weeks. µCT analysis indicated that both mutants fed normal calcium diet had significantly decreased cortical thickness and cancellous bone volume compared to WT. The low calcium diet resulted in a comparable reduction in cortical bone volume and cortical thickness in the W/W v and W sh /W sh mice, and their corresponding controls. As expected, the low calcium diet induced cancellous bone loss in the W/W v mice. In contrast, W sh /W sh cancellous bone did not respond to this diet. This c-Kit mutation prevented cancellous bone loss by antagonizing the low calcium diet-induced increase in osteoblast and osteoclast numbers in the W sh /W sh mice. Gene expression profiling showed that calcium deficiency increased Osx, Ocn, Alp, type I collagen, c-Fms, M-CSF, and RANKL/OPG mRNA expression in controls; however, the W sh mutation suppressed these effects. Our findings indicate that although calcium restriction increased bone turnover, leading to osteopenia, the decreased c-Kit expression levels in the W sh /W sh mice prevented the low calcium diet-induced increase in cancellous bone turnover and bone loss but not the cortical bone loss.

Mouse genome-wide association and systems genetics identify Asxl2 as a regulator of bone mineral density and osteoclastogenesis.

Significant advances have been made in the discovery of genes affecting bone mineral density (BMD); however, our understanding of its genetic basis remains incomplete. In the current study, genome-wide association (GWA) and co-expression network analysis were used in the recently described Hybrid Mouse Diversity Panel (HMDP) to identify and functionally characterize novel BMD genes. In the HMDP, a GWA of total body, spinal, and femoral BMD revealed four significant associations (-log10P>5.39) affecting at least one BMD trait on chromosomes (Chrs.) 7, 11, 12, and 17. The associations implicated a total of 163 genes with each association harboring between 14 and 112 genes. This list was reduced to 26 functional candidates by identifying those genes that were regulated by local eQTL in bone or harbored potentially functional non-synonymous (NS) SNPs. This analysis revealed that the most significant BMD SNP on Chr. 12 was a NS SNP in the additional sex combs like-2 (Asxl2) gene that was predicted to be functional. The involvement of Asxl2 in the regulation of bone mass was confirmed by the observation that Asxl2 knockout mice had reduced BMD. To begin to unravel the mechanism through which Asxl2 influenced BMD, a gene co-expression network was created using cortical bone gene expression microarray data from the HMDP strains. Asxl2 was identified as a member of a co-expression module enriched for genes involved in the differentiation of myeloid cells. In bone, osteoclasts are bone-resorbing cells of myeloid origin, suggesting that Asxl2 may play a role in osteoclast differentiation. In agreement, the knockdown of Asxl2 in bone marrow macrophages impaired their ability to form osteoclasts. This study identifies a new regulator of BMD and osteoclastogenesis and highlights the power of GWA and systems genetics in the mouse for dissecting complex genetic traits.

Deproteinized bovine bone and freeze-dried bone allograft in sinus floor augmentation: A randomized controlled trial.

PURPOSE: The aim of this study was to investigate the effects of deproteinized bovine bone (DBB, Bio-Oss®) and freeze-dried bone allograft (FDBA, SureOss®) on bone healing during maxillary sinus floor augmentation (MSFA) using histology, immunohistochemistry, and gene expressions of the marker genes including Runx2, Opn, Ocn, Col1a1, Rankl, and Tnf-α. MATERIALS AND METHODS: Fourteen participants who required two-stage maxillary sinus augmentation were randomly assigned to DBB and FDBA bone grafting groups. Six months after the sinus augmentation procedure, bone samples were collected before implant placement with a trephine bur. Gene expression of Runx2, Opn, Ocn, Col1a1, Rankl, and Tnf-α of the bone samples was assessed by real-time polymerase chain reaction as a primary outcome. Histological analysis of H&E-stained sections, immunohistochemistry for OPN quantification, and CBCT-based bone tissue examination were performed to investigate the bone healing effects of DBB and FDBA substitutes. RESULTS: The FDBA treated group showed higher gene expression when compared with the DBB treated group in Opn (2.83 ± 1.23 vs. 1.40 ± 0.69; p = 0.04), Runx2 (1.49 ± 0.44 vs. 0.67 ± 0.14; p = 0.01), and Rankl (2.34 ± 0.85 vs. 0.69 ± 0.39; p = 0.03). In the DBB treated group a downregulated expression was found of Ocn relative to maxillary edentulous bone (1.18 ± 0.40 vs. 2.51 ± 0.78; p = 0.02). CONCLUSION: Two-stage maxillary sinus augmentation with FDBA upregulated specific bone remodeling genes when compared to DBB. The outcome of gene expression matched with the ones for OPN immunoreactivity, being higher in the FDBA group. FDBA had an expression pattern similar to native bone and showed stronger expression of bone forming related-genes suggesting it may be clinically preferable over DBB. This clinical trial was not registered prior to participant recruitment and randomization (clinical registration number TCTR20221217002).

Inflammation-Related Gene Profile Histology and Immunohistochemistry of Soft Connective Tissue Covering Bone Grafted with Deproteinized Bovine Bone Mineral and Demineralized Freeze-Dried Bone Allograft.

PURPOSE: To determine the profile of gene expression of soft connective tissue covering bone grafted with deproteinized bovine bone mineral (DBBM) or demineralized freeze-dried bone allograft (DFDBA) in comparison to that without grafting. MATERIALS AND METHODS: Calvaria defects of mice were created and treated as follows: (1) defect without a graft as a control, (2) grafted with DBBM, or (3) grafted with DFDBA. After 1 month, the animals were sacrificed. Soft connective tissue covering the defect area was collected by a punch technique. RNA was isolated and processed to cDNA. Gene expression was evaluated with the microarray technique. Pathway analyses were performed via the PANTHER Overrepresentation test and WikiPathway analysis. Inflammatory marker genes were chosen for mRNA expression using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Tissue sections were used for the histologic and immunohistologic evaluation. RESULTS: The numbers of genes that were significantly differently expressed were 312 (DBBM vs control), 82 (DFDBA vs control), and 113 (DBBM vs DFDBA). Inflammation-related genes were upregulated in DBBM vs control (16 genes), DFDBA vs control (3 genes), and DBBM vs DFDBA (15 genes). Two of these genes, Bcl2a1 and Cxcl9, were significantly upregulated in both the DBBM and DFDBA groups compared with the control. Pathway analysis indicated that Bcl2a1 and Cxcl9 are dominantly expressed in inflammation-related pathways. RT-qPCR and immunohistochemistry showed upregulation of Bcl2a1 and Cxcl9 in DBBM compared with the control and DFDBA groups. Cxcl9 showed a significantly higher expression in the DBBM group. Bcl2a1 at the protein level was equally expressed in all groups. Any sign of inflammation, however, was not seen by histology in any of the groups. CONCLUSION: After a 1-month healing period, soft tissue covering bone grafted with DBBM expressed a higher number of inflammation-related genes compared with those non-grafted or grafted with DFDBA. DFDBA resulted in a decreased expression of inflammation-related genes.

Whole Genome Gene Expression: Histologic and Immunohistologic Study of Grafted Bone Using Allograft and Xenograft.

PURPOSE: The aim of this study was to explore the influence of different bone grafts, demineralized freeze-dried bone allograft (DFDBA, OraGraft), and deproteinized bovine bone mineral (DBBM, Bio-Oss) implanted in mouse calvaria defects on gene expression. MATERIALS AND METHODS: Male C57BL/6MLac mice were separated into three groups as follows: group 1-defect without graft as control, group 2-DFDBA, and group 3-DBBM. Affymetrix DNA microarrays were used to characterize gene expression in bone after 3 months of graft healing. Differential expression of designated genes discovered by microarray analysis was confirmed using real-time polymerase chain reaction (PCR) and immunohistochemistry. RESULTS: Compared with normal bone healing, 355 and 1,108 coding genes of bone grafted with DFDBA were upregulated and downregulated, respectively. The upregulated genes were mainly involved in chemokine signaling, macrophage activity, osteoclast activity, cytokine expression, T-cell receptor signaling, apoptosis, and MAPK signaling. The downregulated genes were predominantly involved in calcium regulation in cardiac cells, chemokine signaling, MAPK signaling, and adipogenesis. A total of 306 and 817 coding genes of bone grafted with DBBM were upregulated and downregulated, respectively. The upregulated genes were mainly involved in osteoclast activity, chemokine signaling, B cell receptor signaling, macrophage activity, and signaling of T-cell receptor, MAPK, IL-5, and IL-1. The downregulated genes were predominantly involved in calcium regulation in the cardiac cell and osteoclast activity. Real-time PCR revealed that the DFDBA and DBBM groups showed a higher mRNA level of MMP12, Bcl2A1, S100A4, and Postn compared with the control (P < .05). Histology showed that, compared with the control, the volume of new bone was higher in both types of bone grafts. Immunohistochemistry using an MMP12 antibody confirmed the microarray results because the MMP12 immunoreactivity intensified, and a positive expression of MMP12 increased significantly in the DFDBA and DBBM groups. CONCLUSION: Both DFDBA and DBBM had a gene expression network involved in new bone formation, which coincided with an increased expression of MMP-12 and osteoclast activity. Both types of graft materials appeared to connect with genes that stimulate bone remodeling at 3 months of bone grafting.

Effect of Jagged1 on the expression of genes in regulation of osteoblast differentiation and bone mineralization ontology in human dental pulp and periodontal ligament cells.

OBJECTIVE: To define the effect of Jagged1 on the gene expression in osteoblast differentiation, regulation of osteoblast differentiation and regulation of bone mineralization ontology. METHODS: Human dental pulp and periodontal ligament cells were isolated using explant method. Cells were seeded on Jagged1 immobilized surface. The mRNA expression was determined using real-time polymerase chain reaction. Mineral deposition was evaluated using alizarin red s staining. Publicly available database of gene expression profiles (GSE126249 and GSE94989) were downloaded and performed bioinformatic analysis to identify gene expression in osteoblast differentiation and regulation of osteoblast differentiation and mineralization ontology. RESULTS: Both human dental pulp cells and human periodontal ligament cells expressed higher Notch target gene (HES1 and HEY1) when cells were seeded on Jagged1 immobilization surface. Jagged1 significantly increased an in vitro mineral deposition in both cell types after maintaining in osteogenic induction medium for 14 days. Correspondingly, the significant increase of ALPL mRNA expression was observed, while there was no significant change in ANKH and ENPP1 mRNA expression in Jagged1 treated condition. From bioinformatic analysis of genes in osteoblast differentiation ontology, IARS, COL1A1, ALPL, COL6A1, CREB3L1, and SNAI2 mRNA levels were upregulated while GJA1 mRNA levels were decreased upon Jagged1 exposure. In the regulation of bone mineralization ontology, evidences supported that TFGB1 and TGFB3 were upregulated in Jagged1 treated condition. CONCLUSION: Jagged1 promote the mRNA expression of several genes in osteoblast differentiation related gene ontology. This could be further employed to identify the mechanism by which Jagged1 promoted osteogenic differentiation in human dental pulp and periodontal ligament cells.

Jagged1 promotes mineralization in human bone-derived cells.

OBJECTIVES: The present study aimed to investigate the expression of Notch signaling components during osteogenic differentiation in vitro and bone healing in vivo. In addition, the influence of Notch signaling on osteogenic differentiation of human bone-derived cells was examined. METHODS: Gene expression profiling of osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells in vitro (GSE80614) and bone healing period of murine tibial fracture in vivo (GSE99388) was downloaded from Gene Expression Omnibus database. The expression of Notch signaling components was obtained from bioinformatic tools. Human bone-derived cells were isolated from alveolar and iliac bone. Cells were seeded on Jagged1 immobilized surface. Osteogenic marker gene expression and mineralization were examined using real-time polymerase chain reaction and alizarin red s staining, respectively. RESULTS: From bioinformatic analysis of gene expression profiling, various Notch signaling components were differentially expressed during osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells in vitro and bone healing period of murine tibial fracture in vivo. The common genes differentially regulated of these two datasets were Hes1, Aph1a, Nsctn, Furin, Adam17, Hey1, Pcsk5, Nedd4, Jag1, Heyl, Notch3, Dlk1, and Hey2. For an in vitro analysis, the mineral deposition markedly increased after seeding human bone-derived cells on Jagged1 immobilized surface, correspondingly with the increase of ALP mRNA expression. Jagged1 treatment downregulated TWIST2 mRNA expression in both human alveolar and iliac bone-derived cells. CONCLUSION: Notch signaling is regulated during osteogenic differentiation and bone healing. In addition, the activation of Notch signaling promotes osteogenic differentiation in human alveolar and iliac bone-derived cells. Therefore, Notch signaling manipulation could be a useful approach for enhancing bone regeneration.

Gene Expression and Microcomputed Tomography Analysis of Grafted Bone Using Deproteinized Bovine Bone and Freeze-Dried Human Bone.

PURPOSE: Bio-Oss and demineralized freeze-dried bone allograft (DFDBA) are two commercial bone grafts that have been associated with clinical success for many years. However, there are few in vivo studies regarding their healing mechanism. The purpose of this study was to investigate the level of bone formation using microcomputed tomography (micro-CT) and gene expression in mouse calvaria at 1 and 3 months after bone grafting with deproteinized bovine bone and freeze-dried human bone, and compare them to natural bone healing. MATERIALS AND METHODS: Thirty-six mice were divided into three groups (n = 6 per group) according to the type of bone graft used: group 1 (control)-an empty defect without bone graft; group 2-treatment with deproteinized bovine xenograft (Bio-Oss); group 3-treatment with DFDBA. The bone graft was inserted into two 3-mm calvarial defects created on both sides of the parietal bone. At 1 and 3 months, the mice were sacrificed and bone volume was evaluated using micro-CT and gene expression analysis using reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Micro-CT analysis demonstrated that the parietal bone of mice grafted with Bio-Oss had significantly greater bone volume than both the DFDBA and control groups at both 1 and 3 months. The expression of bone marker genes (Runx2, Osterix [Osx], alkaline phosphatase [ALP], osteopontin [OPN], and osteocalcin [OCN]) were significantly increased from 1 month in both Bio-Oss and DFDBA groups at 3 months. Runx2 and Osx had significantly higher expression in the Bio-Oss and DFDBA groups compared to the control group at 3 months. No statistically significant difference was observed among groups after 1 month. CONCLUSION: These results showed that both bone graft materials promoted bone regeneration. Bio-Oss demonstrated high osteoconductive properties.

Inflammation related genes are upregulated in surgical margins of advanced stage oral squamous cell carcinoma.

AIMS: Advanced stage of oral squamous cell carcinoma (OSCC) exhibits different properties compared with the early stage for example an invasion ability. The present study investigated a differential gene expression of surgical margin between advanced and early stage of OSCC. METHODS: Gene Expression Omnibus dataset (GSE31056) was downloaded and re-analyzed. Surgical margin samples were categorized into 2 groups; early stage and late stage. Differential gene expression analysis was performed. Dysregulated genes were further analyzed for gene ontology, enriched pathway, and disease association using a network-based analysis tools. RESULTS: Eighty-five dysregulated genes were identified in margin of late stage OSCC. Metabolic process and biological regulation were the main gene ontology of dysregulated genes. Genes involved in Jak-STAT signaling pathway were upregulated in late stage of surgical margin samples. In addition, seven upregulated genes in late stage group, namely CEBPB, S1PR1, IL6, CEBPD, CHI3L1, PTX3, and SOCS3, were categorized in acute phase reaction and inflammation categories of disease association analysis. CONCLUSION: The differential expressed genes in surgical margin of late stage OSCC could be further employed to understand cancer's behavior and to identify target pathway to prevent OSCC invasion.

Maxillary Sinus Floor Augmentation Using Xenograft: Gene Expression and Histologic Analysis.

PURPOSE: Many histologic and histomorphometric studies as well as systematic reviews have shown the clinical success of the use of anorganic bovine bone (ABB, Bio-Oss) in maxillary sinus floor augmentation (MSFA). The molecular processes involved in bone healing are, however, still unknown. The aims of this study were to explore gene expression associated with bone remodeling and inflammation in MSFA sites. MATERIALS AND METHODS: The mRNA expression levels of runt related transcription factor 2 (RUNX2), receptor activator of NF-kB ligand (RANKL), osteoprotegerin (OPG), matrix metallopeptidase 9 (MMP-9), tartrate-resistance acid phosphatase (TRAP), and interleukin-1beta (IL-1ß), as well as the ratio of RANKL/OPG were compared between alveolar bone of a group after MSFA with ABB and a maxillary posterior edentulous bone group. Twenty-one bone samples were collected at the time of implant placement after 6 months of MSFA or tooth extraction. Fourteen bone samples from the MSFA group and from the maxillary posterior edentulous bone without MSFA group were taken to analyze gene expression by real-time reverse transcription polymerase chain reaction (RT-PCR). Seven bone samples from the MSFA group were used for histologic analysis. RESULTS: Real time RT-PCR revealed no statistically significant difference in gene expression level of RUNX2, RANKL, OPG, MMP-9, TRAP, and IL-1ß, or in the ratio of RANKL/OPG. Histology showed bone-lining cells at the edge and osteocyte inside newly formed bone. Residual grafted particles were in close contact with new bone. CONCLUSION: After a healing period of 6 months, ABB particles did not have an effect on the expression of genes associated with bone remodeling and inflammation. In addition, histologic evidence supports that ABB particles are replaced by new bone formation and do not affect bone healing.

Systems genetics analysis of mouse chondrocyte differentiation.

One of the goals of systems genetics is the reconstruction of gene networks that underlie key processes in development and disease. To identify cartilage gene networks that play an important role in bone development, we used a systems genetics approach that integrated microarray gene expression profiles from cartilage and bone phenotypic data from two sets of recombinant inbred strains. Microarray profiles generated from isolated chondrocytes were used to generate weighted gene coexpression networks. This analysis resulted in the identification of subnetworks (modules) of coexpressed genes that then were examined for relationships with bone geometry and density. One module exhibited significant correlation with femur length (r = 0.416), anteroposterior diameter (r = 0.418), mediolateral diameter (r = 0.576), and bone mineral density (r = 0.475). Highly connected genes (n = 28) from this and other modules were tested in vitro using prechondrocyte ATDC5 cells and RNA interference. Five of the 28 genes were found to play a role in chondrocyte differentiation. Two of these, Hspd1 and Cdkn1a, were known previously to function in chondrocyte development, whereas the other three, Bhlhb9, Cugbp1, and Spcs3, are novel genes. Our integrative analysis provided a systems-level view of cartilage development and identified genes that may be involved in bone development.

A genetic association study of single nucleotide polymorphisms in FGFR1OP2/wit3.0 and long-term atrophy of edentulous mandible.

BACKGROUND: After dental extraction, the external surface of alveolar bone undergoes resorption at various rates, and a group of patients develop excessive jawbone atrophy. Oral mucosa overlying the atrophied jawbone is unusually thin; therefore, we have hypothesized that excessive jawbone atrophy may be associated with abnormal oral mucosa contraction. FGFR1OP2/wit3.0, a cytoskeleton molecule initially identified in oral wound fibroblasts, has been shown to induce oral mucosa contraction after dental extraction. This study examined the genetic association between single nucleotide polymorphisms (SNPs) of FGFR1OP2/wit3.0 and excessive atrophy of edentulous mandible. METHODS AND FINDINGS: First, the expression of FGFR1OP2/wit3.0 was determined in gingival tissues of 8 subjects before and after dental extraction. In situ hybridization revealed that all subject increased FGFR1OP2/wit3.0 expression in the post-operative oral mucosa tissues; however, significantly high levels of FGFR1OP2/wit3.0 were observed in 3 out of 8 subjects. In a separate study, 20 long-term edentulous subjects (66.4 ± 9.4 years) were recruited. Tag-SNPs in the FGFR1OP2/wit3.0 allele were determined by Taqman-based polymerase chain reaction. The mandibular bone height was determined following the American College of Prosthodontists (ACP) protocol. Subjects with minor allele of rs840869 or rs859024 were found in the highly atrophied group by the ACP classification (Chi square test, p = 0.0384 and p = 0.0565, respectively; Fisher's Exact, p= 0.0515 and p = 0.2604, respectively). The linear regression analysis indicated a suggestive association between rs859024 and the decreased bone heights (Mann-Whitney, p = 0.06). The average bone height of the subjects with rs840869 or rs859024 minor alleles (10.6 ± 3.2 mm and 9.6 ± 3.2 mm, respectively) was significantly smaller than that of those subjects with the major alleles (14.2 ± 4.5 mm, p<0.05). CONCLUSIONS: The patients with the minor allele of rs840869 or rs859024 were associated with excessive atrophy of edentulous mandible. This study may provide the basis for a genetic marker identifying susceptible individuals to develop jawbone atrophy after dental extraction.