Bone Gamma-carboxyglutamic Protein Research Articles

Neural Dynamics of the Primate Attention Network

The objective of this study was to investigate the effects of maternal nutritional restriction on fetal growth and bone metabolism in early pregnancy. Twelve twin-bearing goats (Xiangdong black goats) in second pregnancy, with similar weight (31.2 ± 8.1 kg) and age (2.0 ± 0.3 year) were assigned to two dietary treatments in early gestation (26–65 days): control group (CON, 100% feed) and intake restriction group (IR, 60% of CON intake). The umbilical blood, right femur and liver of the fetuses were sampled on day 65 to determine the bone metabolism indicators and expression of genes involved in bone development. Maternal intake restriction did not affect the growth performance of pregnancies (P > 0.05), increased (P < 0.05) the body weight, body length, thoracic circumference and umbilical circumference of fetal goats, but decreased (P < 0.05) the concentrations of cross-linked N-terminal telopeptides of type I collagen and cross-linked C-terminal telopeptides of type I collagen, which involved in bone metabolic activity, in the fetal umbilical blood of the IR. In fetal femurs, the mRNA expression of bone gamma-carboxyglutamate protein, osteoprotegerin and mothers against decapentaplegic homolog 1 both participating in osteoclast differentiation were increased (P < 0.05), whereas transcript of the low density lipoprotein receptor related protein 5 involved in osteoblast differentiation was decreased (P < 0.05), compared with the fetuses from the CON. These results indicate that nutrition restriction in early pregnancy regulated the fetal growth and promoted formative activity of femur. The molecular mechanism of maternal intake restriction on bone metabolism needs further investigation.

Calcium silicate-based cements cause environmental stiffness and show diverse potential to induce osteogenesis in human osteoblastic cells

Calcium silicate-based cements differ markedly in their radiopacifiers and the presence of calcium sulfate, aluminates, carbonates and other components that can affect their biological properties. This study aimed to compare the biological properties of six calcium silicate cements in human osteoblastic cell culture (Saos-2 cells): Bio-C Repair (Bio-C), PBS HP (PBS-HP), Biodentine (Biodentine), MTA Repair HP (MTA-HP), NeoMTA Plus (NeoMTA-P), and ProRoot MTA (ProRoot). After exposure to these materials, the cells were analyzed by MTT, wound healing, cell migration, and alkaline phosphatase activity (ALP) assays, real-time PCR (qPCR) analysis of the osteogenesis markers (osteocalcin or bone gamma-carboxyglutamate protein, BGLAP; alkaline phosphatase, ALPL; bone sialoprotein or secreted phosphoprotein 1, BNSP), and alizarin red staining (ARS). Curiously, the migration rates were low 24–48 h after exposure to the materials, despite the cells showing ideal rates of viability. The advanced and intermediate cell differentiation markers BGLAP and BNSP were overexpressed in the Bio-C, MTA-HP, and ProRoot groups. Only the Biodentine group showed ALPL overexpression, a marker of initial differentiation. However, the enzymatic activity was high in all groups except Biodentine. The mineralization area was significantly large in the NeoMTA-P, ProRoot, PBS-HP, MTA-HP, and Bio-C groups. The results showed that cellular environmental stiffness, which impairs cell mobility and diverse patterns of osteogenesis marker expression, is a consequence of cement exposure. Environmental stiffness indicates chemical and physical stimuli in the microenvironment; for instance, the release of cement compounds contributes to calcium phosphate matrix formation with diverse stiffnesses, which could be essential or detrimental for the migration and differentiation of osteoblastic cells. Cells exposed to Bio-C, PBS-HP, ProRoot, NeoMTA-P, and MTA-HP seemed to enter the advanced or intermediate differentiation phases early, which is indicative of the diverse potential of cements to induce osteogenesis. Cements that quickly stimulate osteoblast differentiation may be ideal for reparative and regenerative purposes since they promptly lead to dentin or bone deposition.

Cold Atmospheric Plasma Promotes Regeneration-Associated Cell Functions of Murine Cementoblasts In Vitro.

The aim of the study was to examine the efficacy of cold atmospheric plasma (CAP) on the mineralization and cell proliferation of murine dental cementoblasts. Cells were treated with CAP and enamel matrix derivates (EMD). Gene expression of alkaline phosphatase (ALP), bone gamma-carboxyglutamate protein (BGLAP), periostin (POSTN), osteopontin (OPN), osterix (OSX), collagen type I alpha 1 chain (COL1A1), dentin matrix acidic phosphoprotein (DMP)1, RUNX family transcription factor (RUNX)2, and marker of proliferation Ki-67 (KI67) was quantified by real-time PCR. Protein expression was analyzed by immunocytochemistry and ELISA. ALP activity was determined by ALP assay. Von Kossa and alizarin red staining were used to display mineralization. Cell viability was analyzed by XTT assay, and morphological characterization was performed by DAPI/phalloidin staining. Cell migration was quantified with an established scratch assay. CAP and EMD upregulated both mRNA and protein synthesis of ALP, POSTN, and OPN. Additionally, DMP1 and COL1A1 were upregulated at both gene and protein levels. In addition to upregulated RUNX2 mRNA levels, treated cells mineralized more intensively. Moreover, CAP treatment resulted in an upregulation of KI67, higher cell viability, and improved cell migration. Our study shows that CAP appears to have stimulatory effects on regeneration-associated cell functions in cementoblasts.

Role of 1,25-Dihydroxyvitamin D3 on Osteogenic Differentiation and Mineralization of Chicken Mesenchymal Stem Cells.

1,25-dihydroxyvitamin D3 (1,25OHD) has been suggested to play an important role in osteogenic differentiation and mineralization. However, limited data have been reported in avian species. In the present study, the direct role of 1,25OHD on osteogenic differentiation and mineralization in chicken mesenchymal stem cells (cMSCs) derived from day-old broiler bones was investigated. cMSCs were treated with control media (C), osteogenesis media (OM), OM with 1, 5, 10, and 50 nM 1,25OHD, respectively. The messenger RNA (mRNA) samples were obtained at 24 and 48 h and 3 and 7 days to examine mRNA expression of key osteogenic genes [runt related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2), collagen type I alpha 2 chain (COL1A2), bone gamma-carboxyglutamate protein (BGLAP), secreted phosphoprotein 1 (SPP1), and alkaline phosphatase (ALP)]. Cells were stained at 7, 14, and 21 days using Von Kossa (mineralization), Alizarin Red (AR; mineralization), and Alkaline Phosphatase (early marker) staining methods. From the mRNA expression results, we found a time-dependent manner of 1,25OHD on osteoblast differentiation and mineralization. In general, it showed an inhibitory effect on differentiation and mineralization during the early stage (24 and 48 h), and a stimulatory effect during the late cell stage (3 and 7 days). The staining showed 1,25OHD had an inhibitory effect on ALP enzyme activities and mineralization in a dosage-dependent manner up to 14 days. However, at 21 days, there was no difference between the treatments. This study provides a novel understanding of the effects of 1,25OHD on osteogenic differentiation and mineralization of cMSCs depending on cell stage and maturity.

Role of 25-Hydroxyvitamin D3 and 1,25-Dihydroxyvitamin D3 in Chicken Embryo Osteogenesis, Adipogenesis, Myogenesis, and Vitamin D3 Metabolism.

A study was conducted to understand the effects of 25-hydroxyvitamin D3 (25OHD) and 1,25-dihydroxyvitamin D3 (1,25OHD) administration on the expression of key genes related to osteogenesis, adipogenesis, myogenesis, and vitamin D3 metabolism in the chicken embryo. A total of 120 fertilized Cobb 500 eggs were used in the current study and were reared under standard incubation conditions. On embryonic day 3 (ED 3), PBS (C), PBS with 40ng 1,25OHD (1,25D-L), 200ng 1,25OHD (1,25D-H), 40ng 25OHD (25D-L), or 200ng 25OHD (25D-H) were injected into the dorsal vein of developing embryos. Whole embryos were harvested at 1, 3, and 6h post-injection for gene expression analyses (n=8). Gene expression for key osteogenesis markers (RUNX2: runt-related transcription factor 2; BMP2: bone morphogenetic protein 2; COL1A2: collagen type I alpha 2 chain; BGLAP: bone gamma-carboxyglutamate protein; SPP1: secreted phosphoprotein 1; and ALP: alkaline phosphatese), adipogenesis markers (PPAR-γ: peroxisome proliferator-activated receptor gamma; FASN: fatty acid synthase; and FABP4: fatty acid binding protein 4), myogenesis markers (MYOG: myogenin; MYOD1: myogenic differentiation 1; and MYF5: myogenic factor 5), and the enzyme responsible for vitamin D3 inactivation (CYP24A1: cytochrome P450 family 24 subfamily A member 1) were measured using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Data were normalized by the ΔΔCT method and analyzed using a one-way ANOVA. Results indicated that at 1h post-injection, no differences were found among treatments. At 3h, the early osteogenesis differentiation marker, ALP, was increased by 1,25D-H and 25D-H, and 25D-H also stimulated the expression of adipogenesis markers (FAPB4 and FASN). In contrast, the expression of myogenesis markers (MYOD1 and MYF5) was suppressed by 25OHD or 1,25OHD treatments, respectively. At 6h, a late osteogenic differentiation marker, SPP1, was increased by 25D-H. MYOD1 and MYF5 were continuously suppressed by 25OHD treatments or 1,25D-H. The evidence of vitamin D3 metabolite retention was assessed by measuring CYP24A1 expression. At 1h, there were no differences in CYP24A1 expression. At 3h, all treatments upregulated CYP24A1 expression relative to control (PBS) embryos. However, at 6h, only the 25D-H group retained higher CYP24A1 expression compared to the other treatments. In conclusion, the results suggested both 1,25OHD and 25OHD induced chicken embryo osteogenesis and adipogenesis, but inhibited myogenesis during early chicken embryo development. The higher dosage of 25OHD showed a possibility of a longer retention time in the embryos.

1α,25-dihydroxyvitamin D3 promotes early osteogenic differentiation of PDLSCs and a 12-year follow-up case of early-onset vitamin D deficiency periodontitis

Periodontitis is a chronic periodontal disease that contributes to tooth loss. In recent years, many animal studies have reported that vitamin D (VitD) deficiency results in chronic periodontitis. However, no studies have reported cases of early-onset periodontitis with VitD deficiency. This study reports a 5-year-old male patient with early-onset periodontitis, VitD deficiency and VitD receptor (VDR) mutation. The patient was treated with VitD and calcium, and received systematic periodontal treatment. During the 12-year treatment, the periodontal conditions of this patient were stable. Our in vitro study found that VitD could promote the expression of alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), bone morphogenetic protein 2 (BMP2), bone gamma-carboxyglutamate protein (BGLAP), and VDR in the early osteogenic differentiation of periodontal ligament stem cells (PDLSCs). Meanwhile, VitD could downregulate mRNA expression levels of Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-1β (IL-1β) and protein levels of IL-6 in the tumor necrosis factor-α (TNF-α) -induced inflammation of PDLSCs. Therefore, sufficient VitD supply can be a potential treatment for VitD deficiency induced early-onset periodontitis.

Autophagy receptor OPTN (optineurin) regulates mesenchymal stem cell fate and bone-fat balance during aging by clearing FABP3

ABSTRACT Senile osteoporosis (OP) is often concomitant with decreased autophagic activity. OPTN (optineurin), a macroautophagy/autophagy (hereinafter referred to as autophagy) receptor, is found to play a pivotal role in selective autophagy, coupling autophagy with bone metabolism. However, its role in osteogenesis is still mysterious. Herein, we identified Optn as a critical molecule of cell fate decision for bone marrow mesenchymal stem cells (MSCs), whose expression decreased in aged mice. Aged mice revealed osteoporotic bone loss, elevated senescence of MSCs, decreased osteogenesis, and enhanced adipogenesis, as well as optn– / – mice. Importantly, restoring Optn by transplanting wild-type MSCs to optn– / – mice or infecting optn– / – mice with Optn-containing lentivirus rescued bone loss. The introduction of a loss-of-function mutant of OptnK193R failed to reestablish a bone-fat balance. We further identified FABP3 (fatty acid binding protein 3, muscle and heart) as a novel selective autophagy substrate of OPTN. FABP3 promoted adipogenesis and inhibited osteogenesis of MSCs. Knockdown of FABP3 alleviated bone loss in optn– / – mice and aged mice. Our study revealed that reduced OPTN expression during aging might lead to OP due to a lack of FABP3 degradation via selective autophagy. FABP3 accumulation impaired osteogenesis of MSCs, leading to the occurrence of OP. Thus, reactivating OPTN or inhibiting FABP3 would open a new avenue to treat senile OP. Abbreviations: ADIPOQ: adiponectin, C1Q and collagen domain containing; ALPL: alkaline phosphatase, liver/bone/kidney; BGLAP/OC/osteocalcin: bone gamma carboxyglutamate protein; BFR/BS: bone formation rate/bone surface; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CDKN1A/p21: cyclin-dependent kinase inhibitor 1A; CDKN2A/p16: cyclin dependent kinase inhibitor 2A; CDKN2B/p15: cyclin dependent kinase inhibitor 2B; CEBPA: CCAAT/enhancer binding protein (C/EBP), alpha; COL1A1: collagen, type I, alpha 1; Ct. BV/TV: cortical bone volume fraction; Ct. Th: cortical thickness; Es. Pm: endocortical perimeter; FABP4/Ap2: fatty acid binding protein 4, adipocyte; H2AX: H2A.X variant histone; HE: hematoxylin and eosin; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MAR: mineral apposition rate; MSCs: bone marrow mesenchymal stem cells; NBR1: NBR1, autophagy cargo receptor; OP: osteoporosis; OPTN: optineurin; PDB: Paget disease of bone; PPARG: peroxisome proliferator activated receptor gamma; Ps. Pm: periosteal perimeter; qRT-PCR: quantitative real-time PCR; γH2AX: Phosphorylation of the Serine residue of H2AX; ROS: reactive oxygen species; RUNX2: runt related transcription factor 2; SA-GLB1: senescence-associated (SA)-GLB1 (galactosidase, beta 1); SP7/Osx/Osterix: Sp7 transcription factor 7; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 (human T cell leukemia virus type I) binding protein 1; Tb. BV/TV: trabecular bone volume fraction; Tb. N: trabecular number; Tb. Sp: trabecular separation; Tb. Th: trabecular thickness; μCT: micro computed tomography.

Curcumin promotes osteogenic differentiation of human periodontal ligament stem cells by inducting EGR1 expression

ObjectiveHuman periodontal ligament stem cells (hPDLSCs) attract attention for the periodontal regeneration therapy. Curcumin may promote osteogenic differentiation of hPDLSCs. This research aims to elucidate whether Curcumin displays promoting osteogenic differentiation and its mechanism. MethodsThe hPDLSCs were isolated from human periodontal ligament by immunomagnetic beads, identified with immumofluorescence. hPDLSCs were treated with 0, 5, 10, 20, 50, 100 μmol/L Curcumin. The early growth response gene 1 (EGR1) siRNA or plasmind were tranfected into the hPDLSCs. The viability, Alkaline Phosphatase (ALP) activity and mineralizaiton level of hPDLSCs were measured with 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, ALP Assay Kit or Alizarin Red staining. The expression of EGR1, RUNX family transcription factor 2 (Runx2), bone gamma-carboxyglutamate protein (OC), secreted phosphoprotein 1 (OPN) and collagen type I alpha 1 chain (Collagen I), in hPDLSC were determined by Western blotting and quantitative reverse transcription-polymerase chain reaction. ResultsThe isolated hPDLSCs were spindle or irregular, arranged in radial shape and shown positive expression of STRO-1, CD146 and Vimentin. Curcumin 10 μmol/L treatment maximal promoting the cells viability, ALP activities, mineralization, and levels of Runx2, OC, OPN, Collagen I and EGR-1 in hPDLSCs. EGR-1 siRNA transfection inversed Curcumin’s promoting effect on ALP activities, mineralization, and levels of Runx2, OC, OPN, Collagen I and EGR-1 in hPDLSCs. While the EGR-1 plasmid transfection enhanced Curcumin’s promoting effect on these parameters of hPDLSCs. ConclusionCurcumin promotes the osteogenic differentiation of hPDLSCs, which may work through the EGR1. Curcumin may be a promising medicine for periodontitis treatment and periodontal regeneration.

Establishment of human immortalized mesenchymal stem cells lines for the monitoring and analysis of osteogenic differentiation in living cells

Mesenchymal stem cells (MSCs) are expected to be useful in bone regeneration treatment for various diseases and conditions, including cleft lip and palate, fracture, and bone absorption. However, to date, MSCs have failed to produce satisfactory results in clinical settings. This is primarily due to the low rate of induced osteogenic differentiation. To realize MSC potential, it is necessary to establish methods for the isolation of MSC-derived living osteoblasts. However, no osteoblast markers have been reported to date. In an attempt to develop a method for the assessment of osteoblast differentiation, we established reporter human immortalized MSC (hiMSC) lines for in vitro monitoring of bone gamma-carboxyglutamate protein (BGLAP, osteocalcin) expression. To this end, we successfully knocked-in an enhanced green fluorescent protein (EGFP) gene cassette immediately downstream of the first ATG of BGLAP via CRISPR-Cas9, and established hiMSC lines expressing EGFP to monitor osteogenic differentiation. On differentiation day 7, EGFP-positive cells were collected by flow cytometric cell sorting, and the expression of EGFP and endogenous BGLAP was analyzed. During osteogenic differentiation, EGFP upregulation was found to correlate with expression of endogenous BGLAP. Moreover, mineralization was confirmed using Alizarin red-S staining after two weeks of osteogenic differentiation of the modified hiMSC lines. The modified hiMSC lines, as well as the derived differentiated osteoblasts obtained herein, are valuable tools for the monitoring osteoblast gene and protein expression, and can be used to develop novel methods for isolating living osteoblasts.

Kaempferol promotes proliferation and osteogenic differentiation of periodontal ligament stem cells via Wnt/β-catenin signaling pathway

AimsKaempferol, a type of flavonoid, is widely present in fruits, vegetables and medicinal herbs. This study was designed to investigate the effects of kaempferol on proliferation and osteogenesis of periodontal ligament stem cells (PDLSCs) and to identify the related pathway. Materials and methodsPDLSCs were isolated from extracted premolars and cultured in vitro. Cell-counting kit-8 (CCK-8) and colony formation assays were performed to determine the effect of kaempferol, at various concentrations, on the proliferation of PDLSCs. Alkaline phosphatase (ALP) activity was analyzed both quantitatively and qualitatively, and extracellular matrix mineralization was examined by alizarin red-S staining. In addition, the mRNA and protein expression levels of ALP, RUNX Family Transcription Factor 2 (RUNX2), Sp7 Transcription Factor (SP7; Osterix), Bone Gamma-Carboxyglutamate Protein (BGLAP; osteocalcin) and catenin beta 1 (CTNNB1; β-catenin) were monitored by qPCR and Western blot analysis. Additionally, the tankyrase inhibitor, XAV939, was used to determine the role of the Wnt/β-catenin pathway. Key findingsThe results illustrated that 10−6 M kaempferol markedly promoted the proliferation, ALP activity and mineral deposition of PDLSCs (P < 0.05). The expression levels of ALP, RUNX2, SP7, BGLAP and β-catenin were all upregulated (P < 0.05). After blocking the Wnt/β-catenin pathway with XAV939, the effects of kaempferol were apparently reversed. Significancekaempferol enhanced proliferation and osteogenesis of PDLSCs by activating the Wnt/β-catenin signaling, which suggests the potential application of kaempferol for periodontal tissue regeneration.

Effects of miR-103 by negatively regulating SATB2 on proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells.

The proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (HBMScs) are modulated by a variety of microRNAs (miRNAs). SATB homeobox 2 (SATB2) is a critical transcription factor that contributes to maintain the balance of bone metabolism. However, it remains unclear how the regulatory relationship between miR-103 and SATB2 on HBMScs proliferation and osteogenic differentiation. HBMScs were obtained from Cyagen Biosciences and successful induced osteogenic differentiation. The proliferation abilities of HBMScs after treatment with agomiR-103 and antagomiR-103 were assessed using a cell counting Kit-8 (CCK-8) assay, and osteogenic differentiation was determined using alizarin red S staining and alkaline phosphatase (ALP) activity assay. The expression levels of miR-103, SATB2, and associated osteogenic differentiation biomarkers, including RUNX family transcription factor 2 (RUNX2), bone gamma-carboxyglutamate protein (BGLAP), and secreted phosphoprotein 1 (SPP1), were evaluated using real-time qPCR and Western blot. The regulatory sites of miR-103 on SATB2 were predicted using bioinformatics software and validated using a dual luciferase reporter assay. The underlying mechanism of miR-103 on SATB2-medicated HBMScs proliferation and osteogenic differentiation were confirmed by co-transfection of antagomiR-103 and SATB2 siRNA. The expression of miR-103 in HBMScs after induction of osteogenic differentiation was reduced in a time-dependent way. Overexpression of miR-103 by transfection of agomiR-103 suppressed HBMScs proliferation and osteogenic differentiation, while silencing of miR-103 by antagomiR-103 abolished these inhibitory effects. Consistently, RUNX2, BGLAP and SPP1 mRNA and protein expression were decreased in agomiR-103 treated HBMScs compared with those in agomiR-NC group. Meanwhile, antagomiR-103 upregulated the mRNA and protein expression levels of RUNX2, BGLAP and SPP1 in HBMScs. Further studies revealed that SATB2 was a direct target gene of miR-103. BMSCs transfected with agomiR-103 exhibited significantly downregulated protein expression level of SATB2, whereas knockdown of miR-103 promoted it. Additionally, rescue assays confirmed that silencing of SATB2 partially reversed the effects of antagomiR-103 induced HBMScs proliferation and osteogenic differentiation. The present results suggested that miR-103 negatively regulates SATB2 to serve an inhibitory role in the proliferation and osteogenic differentiation of HBMScs, which sheds light upon a potential therapeutic target for treating bone-related diseases.

Differentiation Potential of Synovial Mesenchymal Stem Cells Isolated From Hip Joints Affected by Femoroacetabular Impingement Syndrome Versus Osteoarthritis

To establish the characteristics of synovium-derived mesenchymal stem cells (MSCs) from the hip joints of patients with femoroacetabular impingement syndrome (FAIS) and osteoarthritis (OA), particularly their proliferation and differentiation potentials. We further investigated their functional differences. Synovium samples were harvested from 21 patients with FAIS who underwent hip arthroscopic surgery and from 14 patients with OA who underwent total hip arthroplasty. The MSC number, colony-forming units, cell viability, and differentiation potential were compared. Real-time polymerase chain reaction assessed the differentiation potential into adipose, bone, and cartilage tissues. The number of colonies at a density of 104 at passage 0 from OA synovium was significantly greater than that from FAIS synovium (P < .01). However, their proliferation and viability were significantly lower than those of FAIS synovium cells (P= .0495). The expression of lipoprotein lipase mRNA in OA synovium cells was greater than that in FAIS synovium cells (P < .01). Meanwhile, the fraction of colonies positive for von Kossa and alkaline phosphatase staining, as well as the level of bone gamma-carboxyglutamate protein expression in OA synovium cells, were greater than those in FAIS synovium cells (P < .01). In chondrogenic pellet culture experiments, the expression of COL10A1 mRNA was lower in OA synovium than in FAIS synovium (P < .01). Synovial MSCs from patients with OA had greater colony numbers but less viability and proliferative potential. They also showed greater osteogenic and adipogenic potentials, whereas those from patients with FAIS showed greater chondrogenic potential. MSCs from patients with FAIS exhibited good potential as cell sources for stem cell therapy in case of cartilage damage in the hip joint.

Evaluation of Osteoblastic Gene Expression in Human Mesenchymal Stem Cells

BackgroundOsteoblastic differentiation is a primary pathway to stimulate bone remodeling within regenerative medicine. Human mesenchymal stem cells (hMSCs) are an attractive model for bone regeneration due to their ability to differentiate into osteoblasts, thus promoting an osteogenic environment. With the development of bone biomaterials, it is important to establish a solid baseline of the genes involved in osteoblast differentiation. Once established, the genes regulating osteoblastic differentiation can be compared to any biomaterial product that may potentially speed up the process of bone regeneration. Therefore, the purpose of this study is to examine gene expression as hMSCs undergo osteoblastic differentiation.MethodshMSCs were extracted from human adipose tissue and cultured in DMEM‐F12 medium, 10% FBS. For experimental conditions, cells were cultured in either a growth medium (GM) or an osteogenic induced medium (OIM), supplemented with β‐glycerophosphate, ascorbic‐2‐phosphate, and dexamethasone. Cells were stained with Alizarin Red S staining to confirm calcium deposits of cells cultured in GM or OIM. hMSCs cultured in OIM were extracted for total RNA at days 3, 7, and 21, followed by cDNA synthesis for RT2 Profiler PCR Human Osteogenesis Array (Qiagen, Hilden, Germany). Gene expressions were analyzed using Qiagen Gene Globe software to determine the relative fold change.ResultshMSCs appear to express alkaline phosphatase (ALPL) and bone gammacarboxyglutamate protein (BGLAP) early throughout OIM culture. In contrast, Sp7 expression increases over time, while Sox9 peaks at Day 7 in comparison to the control at Day 3.ConclusionOur data collectively show a change in gene expressions associated with osteoblastic differentiation such as ALPL, BGLAP, Sp7, and SOX9. Sp7, a master osteoblastic transcription factor, increases over time suggesting hMSCs favoring into osteoblastic transformation. Interestingly, Sox9 is another transcription regulator known to commit hMSCs into osteo‐chondrogenic progenitors, and our data show its expression to increase before diminishing near control levels. However, further studies are needed to confirm the genetic pattern of hMSCS undergoing osteoblastic differentiation as well as the upstream mechanisms controlling their activation. This information will be useful in the application of bone biomaterials as a therapeutic treatment for bone regeneration.

The effect of adrenocorticotropic hormone on alpha-2-macroglobulin in osteoblasts derived from human mesenchymal stem cells.

Nowadays, alpha‐2‐macroglobulin (A2M) gene has allocated escalating interest among several genes involved in the pathogenesis of avascular necrosis of the femoral head (ANFH). This molecule could interact with several osteogenic‐related proteins. It was reported that adrenocorticotropic hormone (ACTH) affects bones through its receptor located on osteoblasts, suggesting it as a potential target in ANFH treatment. In this study, the effect of ACTH on A2M expression was investigated in osteoblasts as well as during the differentiation of human mesenchymal stem cells (MSCs) into osteoblasts. In this study, MSCs derived from bone marrow were isolated and purified using Ficoll gradient and several passaging. MSCs were characterized by induction with osteogenic and adipogenic medium followed by Oil Red O, Alizarin Red and alkaline phosphatase staining. Besides, MSCs were exposed to various concentrations of ACTH to evaluate the cell variability by MTT assay. MSCs and differentiated osteoblasts were treated with 10−8 molar ACTH for 16 and 26 days, respectively. Then, the total RNA was extracted and A2M expression was quantified by real‐time qPCR. The protein expression levels of osteoblast markers including alkaline phosphatase (ALPL) and bone gamma‐carboxyglutamate protein (BGLAP) were also measured. The results showed that A2M expression in cells treated with ACTH was up‐regulated significantly compared to the control group. Similarly, the expression of osteoblast gene markers including ALPL and BGLAP was significantly increased. ACTH, as an osteoblastic differentiation enhancer, up‐regulates A2M, which promotes osteoblastic differentiation probably through TGF‐β induction.

The synergistic effect of low oxygen tension and macromolecular crowding in the development of extracellular matrix-rich tendon equivalents

Cellular therapies play an important role in tendon tissue engineering, with tenocytes being the most prominent and potent cell population available. However, for the development of a rich extracellular matrix tenocyte-assembled tendon equivalent, prolonged in vitro culture is required, which is associated with phenotypic drift. Recapitulation of tendon tissue microenvironment in vitro with cues that enhance and accelerate extracellular matrix synthesis and deposition, whilst maintaining tenocyte phenotype, may lead to functional cell therapies. Herein, we assessed the synergistic effect of low oxygen tension (enhances extracellular matrix synthesis) and macromolecular crowding (enhances extracellular matrix deposition) in human tenocyte culture. Protein analysis demonstrated that human tenocytes at 2% oxygen tension and with 50 μg ml−1 carrageenan (macromolecular crowder used) significantly increased synthesis and deposition of collagen types I, III, V and VI. Gene analysis at day 7 illustrated that human tenocytes at 2% oxygen tension and with 50 μg ml−1 carrageenan significantly increased the expression of prolyl 4-hydroxylase subunit alpha 1, procollagen-lysine 2- oxoglutarate 5-dioxygenase 2, scleraxis, tenomodulin and elastin, whilst chondrogenic (e.g. runt-related transcription factor 2, cartilage oligomeric matrix protein, aggrecan) and osteogenic (e.g. secreted phosphoprotein 1, bone gamma-carboxyglutamate protein) trans-differentiation markers were significantly down-regulated or remained unchanged. Collectively, our data clearly illustrates the beneficial synergistic effect of low oxygen tension and macromolecular crowding in the accelerated development of tissue equivalents.

Osteogenic and odontogenic differentiation potential of dental pulp stem cells isolated from inflamed dental pulp tissues (I-DPSCs) by two different methods

Objective: The objective of the present study is to isolate stem cells from inflamed dental pulp tissues (I-DPSCs) and study the characteristic such as surface markers, osteo/odontogenic differentiation potential between the outgrowth (OG) and enzymatic digestion (COL) methods.Materials and methods: I-DPSCs harvested by both methods were analysed for Mesenchymal Stem Cell marker expression by flow cytometry. The metabolic activity of the isolated cells was assessed by MTT assay. The Alkaline Phosphatase (ALP) and Alizarin red staining was done to analyse the osteogenic potential of isolated cells. The osteo/odontogenic differentiation was done by checking the expression of Dentine Matrix Protein 1 (DMP1), Dentine Sialophosphoprotein (DSPP), ALP and Bone Gamma-Carboxyglutamate Protein (BGLAP) by Real time PCR.Results: The isolated cells were positive for MSC markers such as CD-90, CD-105 and CD-73 and negative for CD-14, CD-45 and STRO-1. MTT assay indicated that the I-DPSCs from OG method showed higher metabolic activity than cells from COL. However, the osteo/odontogenic differentiation was in favour of cells isolated by COL method.Conclusion: Although the cell metabolic rate was more in OG, the osteo/odontogenic differentiation was higher in COL, suggesting that the isolation method and culture conditions do affect the differentiation capacity of isolated cells.

Effects of maternal intake restriction during early pregnancy on fetal growth and bone metabolism in goats

• Effect of maternal intake restriction on bone metabolism of goats was investigated. • Maternal intake restriction during early gestation decreased the INTP and CTX-1 in fetal umbilical blood. • Nutritional restriction during early gestion regulated the femur metabolic activity of fetal goats. The objective of this study was to investigate the effects of maternal nutritional restriction on fetal growth and bone metabolism in early pregnancy. Twelve twin-bearing goats (Xiangdong black goats) in second pregnancy, with similar weight (31.2 ± 8.1 kg) and age (2.0 ± 0.3 year) were assigned to two dietary treatments in early gestation (26–65 days): control group (CON, 100% feed) and intake restriction group (IR, 60% of CON intake). The umbilical blood, right femur and liver of the fetuses were sampled on day 65 to determine the bone metabolism indicators and expression of genes involved in bone development. Maternal intake restriction did not affect the growth performance of pregnancies ( P > 0.05), increased ( P < 0.05) the body weight, body length, thoracic circumference and umbilical circumference of fetal goats, but decreased ( P < 0.05) the concentrations of cross-linked N-terminal telopeptides of type I collagen and cross-linked C-terminal telopeptides of type I collagen, which involved in bone metabolic activity, in the fetal umbilical blood of the IR. In fetal femurs, the mRNA expression of bone gamma-carboxyglutamate protein, osteoprotegerin and mothers against decapentaplegic homolog 1 both participating in osteoclast differentiation were increased ( P < 0.05), whereas transcript of the low density lipoprotein receptor related protein 5 involved in osteoblast differentiation was decreased ( P < 0.05), compared with the fetuses from the CON. These results indicate that nutrition restriction in early pregnancy regulated the fetal growth and promoted formative activity of femur. The molecular mechanism of maternal intake restriction on bone metabolism needs further investigation.

1977-P: Methylglyoxal May Contribute to Bone Loss via Differential Effects on Osteoblast and Osteoclasts

Purpose of the Study: Diabetes mellitus is characterized by chronic hyperglycemia and diabetic complications. Methylglyoxal (MG) is a reactive advanced glycation end-product precursor in which abnormal accumulation is found in the serum of diabetic patients. In clinical studies, they are found to have higher risk of bone fracture. Yet, the underlying mechanism has not been elucidated. Bone remodeling, depending on the interaction between osteoblast (OB) and osteoclast (OC), is to maintain bone structural integrity, such to resorb aged cells while replacing with newly formed cells and to support the metabolic function of bones. Both OB and OC were found to be severely affected by MG. Hence the effect of MG leading to subsequent bone loss was investigated. Methods: In this study, the effects of MG on bone in both animal and cell models were investigated. SD rats were treated with MG (50mM, 10mL/kg) for 8 weeks. Proximal end of tibia were collected for analysis. The bone mineral density (BMD) and mRNA expression of bone biomarkers were measured using micro-CT and RT-PCR respectively. Results: Using micro-CT scanning, MG-treated group has lower trabecular BMD (318.1 ± 10.9 mg HA/ccm) compared with control group (383.2 ± 17.7 mg HA/ccm). Using RT-PCR, it was found there were decreased expressions in OB biomarkers including Bone Gamma-Carboxyglutamate Protein (0.718 fold), Collagen I (0.25 fold); while increasing RANKL/OPG (9.46 fold) and Sclerostin (3.51 fold) after MG treatment, indicating the inhibition of bone formation. Besides, increased expressions in OC biomarkers Cathepsin K (1.51 fold), Osteoclast-associated receptor (1.90 fold), Receptor activator of nuclear factor kappa B (1.34 fold) and Tartrate-resistant acid phosphatase (2.57 fold) were observed indicating activation of OC. Conclusions: MG activates OC and inhibits OB, leading to lower BMD and more porous bone architecture. Therefore, MG may contribute to the bone loss through imbalanced bone remodeling in both OC and OB. Disclosure K. Yue: None. F. Lee: None.

The effect of embryonic origin on the osteoinductive potential of bone allografts

Statement of problemAllografts with osteoinduction potential are widely used to augment bone in surgical and prosthetic rehabilitations. However, osteoinduction potential varies among commercially available allografts. Donor bones are derived from different embryonic origins, either the neural crest or mesoderm. Whether the origin of the bones affects the osteoinductivity of allograftsis is unclear. PurposeThe purpose of this ex vivo study was to investigate the osteoinduction potential of allografts derived from bones with distinct embryonic origins. Material and methodsAllografts were obtained from human frontal and parietal bones at 2 different ages (fetal and adult). The specimens were divided into 4 groups: adult frontal (n=5), adult parietal (n=5), fetal frontal (n=10), and fetal parietal (n=10). Two investigations were conducted to assess the osteoinductive potential of these allografts. First, the osteogenesis of human osteoblasts exposed to these allografts were evaluated by analyzing the expression of runt-related transcription factor 2 (RUNX2), collagen type 1 alpha 2 chain (COL1A2), and bone gamma-carboxyglutamate protein (BGLAP) genes using quantitative real-time polymerase chain reaction (qRT-PCR). Second, the protein content of the adult frontal and parietal bone matrices was analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). One-way ANOVA and the t test were used for statistical analyses of the gene and protein expression of the groups (α=.05). ResultsNo difference was found in the gene expression of the cells exposed to frontal or parietal bones. However, all 3 genes were significantly overexpressed in cells treated with fetal bones compared with adult bones. No difference was found in protein expression between adult frontal and adult parietal bones. ConclusionsNo difference was found in the osteoinductive capacity of frontal and parietal bones used as allografts. However, the osteoinductivity of fetal bones can be higher than that of adult bones. Further microanalyses are needed to determine the protein content of fetal bones.

Activation of local bone RAS by maternal excessive glucocorticoid participated in the fetal programing of adult osteopenia induced by prenatal caffeine exposure

This study was aimed to investigate whether and how prenatal caffeine exposure (PCE) could induce osteopenia in the adult offspring. Pregnant rats were treated with prenatal caffeine 12 mg/100 g body weight per day from pregnant day 9 to 20, while rat bone marrow mesenchymal stem cells (BMSCs) were treated with exogenous corticosterone during osteogenic induction. Shorter femur and primary ossification center was observed in the PCE offspring, as well as less bone trabecular and poor biomechanical intensity. Local gene expression of glucocorticoid receptor (GR) and angiotensin converting enzyme (ACE), as well as angiotensin 2 content, was found to be stimulated, while the expression of bone gamma-carboxyglutamate protein (BGLAP), alkaline phosphatase (ALP) and bone sialoprotein (BSP) was found to be suppressed, with hypomethylation of ACE promoter. Corticosterone (1250 nM) suppressed osteogenic differentiation of BMSCs and gene expression of BGLAP, ALP and BSP, which was attenuated by enalapril, while it stimulated ACE mRNA expression and induced hypomethylation of ACE promoter, which was attenuated by mifepristone. It indicated that PCE caused bone growth retardation and adult osteopenia in offspring, which might be triggered by the activation of local RAS induced by excessive maternal glucocorticoid, while the hypomethylation of ACE gene might be the key point of the sustained activation of the local RAS.