Osteocalcin mRNA Expression Research Articles

Effect of the lipoxygenase inhibitor baicalein on bone tissue and bone healing in ovariectomized rats

BackgroundOsteoporosis is one of the world’s major medical burdens in the twenty-first century. Pharmaceutical intervention currently focusses on decelerating bone loss, but phytochemicals such as baicalein, which is a lipoxygenase inhibitor, may rescue bone loss. Studies evaluating the effect of baicalein in vivo are rare.MethodsWe administered baicalein to sixty-one three-month-old female Sprague-Dawley rats. They were divided into five groups, four of which were ovariectomized (OVX) and one non-ovariectomized (NON-OVX). Eight weeks after ovariectomy, bilateral tibial osteotomy with plate osteosynthesis was performed and bone formation quantified. Baicalein was administered subcutaneously using three doses (C1: 1 mg/kg BW; C2: 10 mg/kg BW; and C3: 100 mg/kg BW) eight weeks after ovariectomy for four weeks. Finally, femora and tibiae were collected. Biomechanical tests, micro-CT, ashing, histological and gene expression analyses were performed.ResultsBiomechanical properties were unchanged in tibiae and reduced in femora. In tibiae, C1 treatment enhanced callus density and cortical width and decreased callus area. In the C3 group, callus formation was reduced during the first 3 weeks after osteotomy, correlating to a higher mRNA expression of Osteocalcin, Tartrate-resistant acid phosphatase and Rankl. In femora, baicalein treatments did not alter bone parameters.ConclusionsBaicalein enhanced callus density and cortical width but impaired early callus formation in tibiae. In femora, it diminished the biomechanical properties and calcium-to-phosphate ratio. Thus, it is not advisable to apply baicalein to treat early bone fractures. To determine the exact effects on bone healing, further studies in which baicalein treatments are started at different stages of healing are needed.

Effect of psoralen on the expression of PPAR\u03b3, osteocalcin, and trabecular bone area in rabbits with steroid-induced avascular necrosis of the femoral head

ObjectivePsoralen is a natural plant toxin which has the function of protecting fungi, insects, and herbivores. In this study, we aim to investigate the effect and mechanism of psoralen on steroid-induced avascular necrosis of femoral head (SANFH).MethodsThirty rabbits were randomly divided into blank group (n = 10), model group (n = 10), and experimental group (n = 10). Rabbits in blank and model groups were treated with normal saline, and rabbits in experimental group were treated with psoralen. Total RNA of bone marrow was extracted by trizol, and the mRNA expression of PPARγ and osteocalcin were detected by q-PCR. Then, the mRNA expression of PPARγ and osteocalcin in the three groups were compared. Western blot was used to detect the PPARγ protein expression in the bone of rabbits. ELISA was used to measure the osteocalcin protein.ResultsThe mRNA expression of PPARγ in model group significantly increased compared with blank group. The mRNA expression of osteocalcin in model group decreased compared with blank group. However, the mRNA and protein expressions of PPARγ in experimental group were significantly decreased compared with the model group. The protein expressions of osteocalcin increased compared with the model group. There was no significant difference of trabecular bone area (TBA) between experimental and blank groups (P > 0.05). TBA in model group was lower than the experimental group (P < 0.05). There was no significant difference of TBA between experimental and blank groups (P > 0.05).ConclusionThis research confirms that psoralen plays a positive role in the rehabilitation of SANFH.

Heparin is biocompatible and can induce differentiation of human dental pulp cells.

To investigate the biocompatibility, osteogenic bioactivity and mRNA expression of the osteo/odontogenic markers bone morphogenetic protein 2 (BMP-2), osteocalcin (OC) and alkaline phosphatase (ALP), induced by heparin in human dental pulp cells (hDPCs). hDPCs were exposed to the heparin, and cell viability was assessed by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT), and cell death was evaluated by flow cytometry. Osteogenic bioactivity was evaluated by the alkaline phosphatase (ALP) assay, and the detection of calcium deposits by alizarin red staining (ARS). The gene expression of BMP-2, OC and ALP was quantified with real-time PCR. Statistical analysis was performed with ANOVA and Bonferroni or Tukey post-test and t-test (α=0.05). Heparin had no cytotoxic effect and did not induce apoptosis. After 3days, heparin had significantly higher ALP activity in comparison with the control (P<0.05). Heparin had a significant (P<0.05) stimulatory effect on the formation of mineralized nodules. BMP-2 and OC mRNA expressions were significantly higher in cells exposed to heparin than control group after 1day (P<0.05). Heparin was biocompatible in hDPCs, induced osteogenic bioactivity and enhanced mRNA expression of osteo/odontogenic markers BMP-2 and OC. These results suggest that heparin has potential to induce osteo/odontogenic cell differentiation of hDPCs.

MiR-146a regulates osteogenic differentiation and proliferation of bone marrow stromal cells in traumatic femoral head necrosis.

To investigate the regulatory mechanism of micro ribonucleic acid (miR)-146a in osteogenic differentiation and proliferation of bone marrow stromal cells (BMSCs) in traumatic femoral head necrosis. Femoral neck fracture patients undergoing surgery were divided into necrosis group and non-necrosis group. The expression level of miR-146a in BMSCs isolated from these patients was detected via quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The clinical correlation of miR-146a with BMSCs in traumatic femoral head necrosis was explored. The regulatory effects of miR-146a on osteogenic differentiation and proliferation of BMSCs in traumatic femoral head necrosis were detected. Moreover, the cell proliferation was analyzed via cell counting kit-8 (CCK-8) assay. The deposition of calcium on the cell surface was detected via alizarin red staining to evaluate the osteogenic differentiation. The messenger RNA (mRNA) expressions of osteogenesis-specific genes, alkaline phosphatase (ALP), and osteocalcin (Ocn) in BMSCs undergoing osteogenic differentiation were detected via qRT-PCR. Expression level of miR-146a in BMSCs of necrosis group was significantly lower than that in non-necrosis group, and the difference was statistically significant (p<0.01). CCK-8 assay revealed that the proliferation of BMSCs was significantly enhanced in miR-146a-mimic group compared with that in miR-NC group, while it significantly declined in miR-146a-inhibitor group compared with that in miR-NC group. The results of alizarin red staining showed that the deposition of calcium obviously increased in miR-146a-mimic group compared with that in miR-NC group, indicating that the osteogenic differentiation ability is significantly enhanced, while it markedly decreased in miR-146a-inhibitor group compared with that in miR-NC group. The detection of osteogenesis-specific genes via qRT-PCR manifested that the mRNA expressions of ALP and Ocn remarkably increased in miR-146a-mimic group compared with those in miR-NC group, and there were statistically significant differences (p<0.05). The mRNA expressions of ALP and Ocn remarkably decreased in miR-146a-inhibitor group compared with those in miR-NC group, and there were statistically significant differences (p<0.05), suggesting the inhibited osteogenic differentiation ability. We showed that miR-146a regulates the osteogenic differentiation and proliferation of BMSCs in traumatic femoral head necrosis.

MPMBP, a novel bisphosphonate with an antioxidant side chain, stimulates bone formation through inhibition of NF-κB nuclear translocation

Bisphosphonates (BPs) are chemically characterized by a P-C-P bond with two lateral side chains on the carbon atom, and have been widely used as anti-resorptive agents in various metabolic bone diseases. 4-[(methylthio) phenylthio] methanebisphosphonate (MPMBP) is a novel non-nitrogen-containing BP with an antioxidant side chain that possesses anti-inflammatory properties. Since inflammation is known to be a cause of the pathological bone resorption, we investigated the effects of MPMBP on bone metabolism both in vitro and in vivo. The results showed that: i) MPMBP dose-dependently increased alkaline-phosphatase activity in a culture of osteoblastic MC3T3-E1 cells, ii) MPMBP increased the synthesis of collagen (type-I) in an organ culture of mouse calvaria, iii) local injection of MPMBP to alveolar bone induced prominent increases in both the bone mass and thickness of alveolar bone at the local site of injection in rabbits, iv) MPMBP increased the mRNA expression of alkaline-phosphatase, type-I collagen, osteocalcin, and bone sialoprotein in MC3T3-E1 cells, v) MPMBP inhibited the translocation of NF-κB/p65 to the nuclei in osteoblasts of cultured mouse calvaria. Taken together, these findings suggest that MPMBP is a promising agent to prevent bone loss, or even accelerate new bone formation, through inducing an uncoupling between bone resorption and bone formation, which is preferable to maintain bone mass and quality.

(-)-Epigallocatechin-3-Gallate (EGCG) Enhances Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells.

Osteoporosis is the second most-prevalent epidemiologic disease in the aging population worldwide. Cross-sectional and retrospective evidence indicates that tea consumption can mitigate bone loss and reduce risk of osteoporotic fractures. Tea polyphenols enhance osteoblastogenesis and suppress osteoclastogenesis in vitro. Previously, we showed that (−)-epigallocatechin-3-gallate (EGCG), one of the green tea polyphenols, increased osteogenic differentiation of murine bone marrow mesenchymal stem cells (BMSCs) by increasing the mRNA expression of osteogenesis-related genes, alkaline phosphatase activity and, eventually, mineralization. We also found that EGCG could mitigate bone loss and improve bone microarchitecture in ovariectomy-induced osteopenic rats, as well as enhancing bone defect healing partially via bone morphogenetic protein 2 (BMP2). The present study investigated the effects of EGCG in human BMSCs. We found that EGCG, at concentrations of both 1 and 10 µmol/L, can increase mRNA expression of BMP2, Runx2, alkaline phosphatase (ALP), osteonectin and osteocalcin 48 h after treatment. EGCG increased ALP activity both 7 and 14 days after treatment. Furthermore, EGCG can also enhance mineralization two weeks after treatment. EGCG without antioxidants also can enhance mineralization. In conclusion, EGCG can increase mRNA expression of BMP2 and subsequent osteogenic-related genes including Runx2, ALP, osteonectin and osteocalcin. EGCG further increased ALP activity and mineralization. Loss of antioxidant activity can still enhance mineralization of human BMSCs (hBMSCs).

Leukemia Cells Remodel Adipocyte Niches and Their Progenitor Functions to Generate Leukemia Favoring Niche

Increasing evidence suggests that the cancer cells take shelter in different osteoblastic and adipocytic niches, where they hide from chemotherapy and continue to survive. As yet, how leukemia cells alter the bone marrow (BM) adipocytic niches to facilitate their expansion and assist them in evading chemotherapy is unclear. We have previously shown that the acute myeloid leukemia (AML) cells directly or through their exosomes, reprogram BM osteoblastic niche which facilitates their expansion and suppress normal hematopoiesis(Kumar B et al, Leukemia 2018,32(3):575-587). In this study , we provide further evidences that AML and Acute lymphoid leukemia (ALL) transformed the BM adipocytic niche to facilitate their expansion and suppress normal hematopoiesis.Using MLL-AF9 (AML) knock-in mouse, MLL-AF9 or BCR-ABL(p190, ALL) HSC transduction transplantation leukemia mice models, we performed flow cytometry analysis to show that the leukemia cells stimulate expansion of BM derived CD45-Ter119-CD31-CD166-Sca1+CD140a+(PaS)MSCs population compared to normal mice (p=0.04, p=0.001 and p=0.002 respectively).Further, the BM osteoblasts specific Osteocalcin mRNA expression in sorted stroma cells and flow cytometry based osteoblasts population (CD45-Ter119-CD31- CD166+Sca1-) numbers were also significantly reduced in AML (p=0.04) and ALL (p=0.02) mice models suggesting bone loss with the leukemia development. Similar to osteoblasts loss, mature adipocytes (Perilipin, PPARg mRNA) were also significantly reduced in the ALL/AML mice compared to control. The triglyceride content and white adipose tissue(WAT) mass was diminished in leukemic mice , suggesting leukemia may have utilized adipocyte for survival. Adipocyte loss in the leukemia mice was accompanied by long term hematopoietic stem cells(LT-HSC ) and erythroid megakaryocyte progenitor (MEP) populations reduction in the leukemic mice (p=0.01 and p=0.02 respectively).To dissect the mechanism of adipocytes reduction is either due to adipocyte loss or adipocytes maturation defect in leukemic mice, we analyzed different stromal progenitors in normal and leukemic mice and identified that the leukemia cells stimulate the growth of BM derived adipocytic committed progenitors (CD45-Ter119-CD31-CD166-Sca1+CD140a+CD29+CD24-) and blocked the chondrocyte/osteoblastic/adipocytic multipotent progenitors (CD45-Ter119-CD31- CD166-Sca1+CD140a+CD29+CD24+) population (p=0.02,p=0.01 respectively).Despite the increase in number of MSCs and adipocytic progenitors, the in-vitro adipocytic differentiation potential of sorted adipocyte committed progenitors was severely compromised in ALL and AML compared to control. The WAT western blot analysis showed significantly increased expression of ATGL and pHSL(Ser-563) expression involved in triglyceride lipolysis in the leukemic mice .The ALL leukemic adipocytic stroma had increased expression of IL-1β and IL-6 cytokine levels compared to normal stroma and provided more survival advantage to leukemia cells in in-vitro co-culture experiments in nutrient deprived conditions and during chemo-radiotherapy treatment. Further, the ATGL and HSL pharmacological inhibitors rescued leukemia induced lipolysis, reduced leukemia proliferation and increased chemotherapy induced apoptosis in leukemia cells.Overall, this data strongly suggests the notion of progressive decline in functional LT-HSCs & normal hematopoiesis, adipocytes and osteoblasts numbers with leukemia progression due to activation of lipolytic enzymes resulting in increased availability of fatty acids for leukemia expansion and is a common feature in both lymphoid and myeloid leukemias. DisclosuresNo relevant conflicts of interest to declare.

MicroRNA-146a reverses the inhibitory effects of Porphyromonas gingivalis lipopolysaccharide on osteogenesis of human periodontal ligament cells

Objective: To investigate the effects and mechanisms of microRNA-146a (miR-146a) on osteogenic differentiation of human periodontal ligament cells (hPDLC) stimulated by lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg). Methods: hPDLC were cultured in vitro and induced to the phase of osteogenic differentiation. These cells were divided into five groups: non-osteogenic differentiation cells, osteogenic differentiation cells, osteogenic differentiation cells treated with Pg LPS, osteogenic differentiation cells treated with Pg LPS and miR-146a mimic, osteogenic differentiation cells treated with Pg LPS and miR-146a negative control. Osteogenic markers and mineralization were detected via quantitative real-time PCR (qPCR) and alizarin red staining, respectively. Meanwhile, non-radioactive transcription factor assay was applied to explore the nuclear activity of nuclear factor kappa B (NF-κB) p65 in nuclear extracts of hPDLC. Results: Compared with cells of osteogenic differentiation in non-LPS-stimulated groups, Pg LPS could decrease the markers of osteogenic differentiation of hPDLC such as collagen Ⅰ (Col-Ⅰ), alkaline phosphatase (ALP), Runt-related transcription factor-2 (RUNX2) and osteocalcin (OCN) (P<0.05), inhibit mineralization, and stimulate NF-κB p65 nuclear activity expression (non-LPS stimulated group: 1.023±0.217, LPS stimulated group: 6.252±0.613, P=0.008). However, compared with cells in Pg LPS/miR-146a negative control group, miR-146a increased Col-Ⅰ (P=0.007) and OCN (P=0.049) mRNA expression, rather than ALP (P=0.167) and RUNX2 (P=0.580) at day 3; miR-146a also upregulated mRNA levels of Col-Ⅰ, ALP, RUNX2 and OCN (P<0.05) at day 7 and day 14, and enhance mineralization. Meanwhile, miR-146a mimic could decrease the nuclear activity of NF-κB p65 induced by Pg LPS in hPDLC (miR-146a: 2.427±0.354, negative control: 5.863±0.482, P=0.019). Conclusions: miR-146a could reverse the inhibitory effects of Pg LPS on osteogenic differentiation of hPDLC through enhancing the expression of osteogenic markers and decreasing inflammatory pathway in hPDLC.

Effect of hydroxyapatite nanoparticles and wedelolactone on osteoblastogenesis from bone marrow mesenchymal stem cells.

Regenerative medicine has a high demand for the development of scaffold materials combined with other osteogenic inducers to generate bioactive composite materials for bone replacement therapies. Previously, we reported that wedelolactone promoted osteoblastogenesis of bone marrow mesenchymal stem cells (BMSCs). In this study, the effect of hydroxyapatites (HAps), bone composite materials we prepared, and the combined effect of wedelolactone and HAps on osteoblastogenesis differentiation was first evaluated. Three kinds of HAps constructed by a rod-like shape with particle size of 25 nm (HAp-1), 37 nm (HAp-2), and 33 nm (HAp-3) did not affect BMSC survival, but induced activity of alkaline phosphatase(ALP), a marker enzyme for osteoblastogenesis. HAp-1 treatment resulted in a more significant increase in the number of ALP staining-positive BMSC, and maintained an extended time for the increased number of ALP staining-positive BMSC. Moreover, HAp-1 combined with wedelolactone induced a higher ALP activity for a longer time than HAp-2 and HAp-3, and also increased the bone mineralization level. Osteoblastogenesis-related marker genes expression including osteorix, osteocalcin, and runx2 were increased after BMSC were treated with HAp-1 for 6 days. Although three kinds of HAps treatment for 9 days increased osteorix mRNA expression, osteocalcin, and runx2 mRNA expression levels were upregulated only by HAp-1. Similarly, only HAp-1 enhanced wedelolactone-induced osteocalcin, osteorix, and runx2 mRNA expression after 9 days treatment. Together, these results suggested that HAps with different sizes generated different effect on osteoblastogenesis. HAp-1 combined with wedelolacone can exert an enhanced effect on osteoblastogenesis, which has potential for treating osteoporosis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 145-153, 2019.

Study on cytotoxicity of three-dimensional printed β-tricalcium phosphate loaded poly (lactide-co-glycolide) anti-tuberculosis drug sustained release microspheres and its effect on osteogenic differentiation of bone marrow mesenchymal stem cells

To study the effect of three-dimensional (3D) printed β-tricalcium phosphate (β-TCP) scaffold loaded poly (lactide-co-glycolide) (PLGA) anti-tuberculosis drug sustained release microspheres on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and its cytotoxicity. Isoniazid and rifampicin/PLGA sustained release microspheres were prepared by W/O/W multiple emulsion method. The β-TCP scaffolds were prepared by 3D printing technique. The microspheres were loaded on the scaffolds by centrifugal oscillation method to prepare composite materials. The BMSCs of Sprague Dawley rat were isolated and cultured by whole bone marrow adherent method, and the third generation cells were used for the following experiments. BMSCs were co-cultured with osteogenic induction medium (group A), PLGA anti-tuberculosis drug sustained release microsphere extract (group B), 3D printed β-TCP scaffold extract (group C), and 3D printed β-TCP scaffold loaded PLGA anti-tuberculosis drug sustained release microsphere composite extract (group D), respectively. Cytotoxicity was detected by cell counting kit 8 (CCK-8) method; the calcium deposition was observed by alizarin red staining; and the mRNA expressions of alkaline phosphatase (ALP), osteocalcin (OCN), and bone sialoprotein (BSP) were detected by real-time fluorescence quantitative PCR (RT-qPCR). CCK-8 assay showed that the absorbance ( A) value of groups A, B, C, and D increased gradually with the culture time prolonging. After cultured for 24, 48, and 72 hours, the A value decreased in the order of groups A, C, B, and D. There was no significant difference between groups B and D ( P>0.05), but there were significant differences between other groups ( P<0.05). The cytotoxicity was evaluated as grade 0-2, and the toxicity test was qualified. Alizarin red staining showed that red mineralized nodules were formed in all groups at 21 days after osteogenic induction, but the number of mineralized nodules decreased sequentially in groups C, D, A, and B. RT-qPCR test results showed that the relative expressions of OCN and BSP genes in groups A, B, C, and D increased gradually with the culture time prolonging. The relative expression of ALP gene increased at 7 and 14 days, and decreased at 21 days. After cultured for 7, 14, and 21 days, the relative expressions of ALP, OCN, and BSP genes decreased sequentially in groups C, D, A, and B; the differences were significant between groups at different time points ( P<0.05). 3D printed β-TCP loaded PLGA anti-tuberculosis drug sustained release microsphere composites have no obvious cytotoxicity to BMSCs, and can promote BMSCs to differentiate into osteoblasts to a certain extent.

Friday, September 28, 2018 10:30 AM–12:00 PM abstracts: innovation, surface technology and biomechanics: 175. The comparison of 3D porous titanium, nonporous titanium, and tantalum interbody cages: in vitro and in vivo study

BACKGROUND CONTEXT For interbody fusion implant material, autogenous bone graft is current gold standard, but it also carries several disadvantages. Tantalum and titanium are both ideal materials for interbody cages for their biocompatibility. New cage design utilizing three-dimensional (3D) printing technique, selective laser melting (SLM), to conjugate titanium powder into 3D-porous structure (porous titanium, PTi) may be an optimal choice for interbody fusion. PURPOSE To further investigate the cell properties and bone in-growth characteristics, the study aims to evaluate the PTi interbody device on the biocompatibility and the in-vivo bone ingrowth characteristics compared to nonporous titanium (NPTi), porous tantalum (PTa) interbody device. STUDY DESIGN/SETTING In vitro and in vivo study. PATIENT SAMPLE A total of six cages in six groups for in vitro study (total 36 cages). Twelve pigs for in vivo bone ingrowth study. Each pig had three cages implanted (total 36 cages). OUTCOME MEASURES Cell attachment and osteogenic markers were evaluated for in vitro study. Cage migration, subsidence, bone ingrowth were evaluated for in vivo study. METHODS For in vitro study, we made uniform PTi (20%, 40%, 60%, 80%), NPTi and PTa cages (n=6 in each group, total 36 cages) to investigate the cell attachment and osteogenic markers using MG63 cell line. SEM was used to observe the cell attachment on the implants. Osteogenic markers were evaluated with the mRNA expression and activity of alkaline phosphatase and mRNA expression of osteocalcin. In vivo study was performed using pig posterior instrumented anterior interbody fusion model comparing PTi (60%), NPTi, and PTa interbody device in twelve pigs.For evaluating the migration and subsidence of implants, plain radiographs were performed before and after surgery, and postoperative 1, 3, and 6 months. All implants (total 33 cages) were harvested the implants with neighboring vertebral bodies after sacrifice. The harvested implants were assessed by micro-CT, histologic section for their bone ingrowth and bone attachment. RESULTS The in vitro micrographics were showed that cells had similar attachment behavior on PTi, NPTi and PTa cages. More cellular migration into cage pores was noticedin 60% and 80% PTi cages. The osteogenic markersALP activity and mRNA expression were highest in 60% cages while the osteocalcin was highest in NPTi cages.In vivo study eleven pigs were completed the radiographic exams. One pig was expired early due to poor postoperative recovery after 1 month. All interbody cages had successful fusion without obvious migration or cage failure. However, 60% PTi cage had less subsidence compared to other cages. The micro-CT showed better pore bone-ingrowth percentage (18%, 8%, 0% in PTi, PTa, NPTi cages, respectively) and the bone histomorphometry demonstrated better new-bone mineralization and trabeculation in PTi cages. CONCLUSIONS In conclusion, our in vitro and in vivo results express that the innovative designed PTi device that manufacture by SLM, it is biocompatible and work well in lumbar interbody fusion surgery. The fusion character of PTi cages seems to be faster with less subsidence compared to PTa and NPTi cages.

The impact of photofunctionalized gold nanoparticles on osseointegration

ObjectivesThe aims of this study were to create a new surface topography using simulated body fluids (SBF) and Gold Nanoparticles (GNPs) and then to assess the influence of UV Photofunctionalization (PhF) on the osteogenic capacity of these surfaces. Materials and methodsTitanium plates were divided into six groups All were acid etched with 67% Sulfuric acid, 4 were immersed in SBF and 2 of these were treated with 10 nm GNPs. Half of the TiO2 plates were photofunctionalized to be compared with the non-PhF ones. Rat's bone marrow stem cells were seeded into the plates and then CCK8 assay, cell viability assay, immunofluorescence, and Scanning electron microscopy (SEM) were done after 24 hours. Gene expression analysis was done using real time quantitative PCR (qPCR) one week later to check for the mRNA expression of Collagen-1, Osteopontin and Osteocalcin. Alkaline phosphatase (ALP) activity was assessed after 2 weeks of cell seeding. ResultsOur new topography has shown remarkable osteogenic potential. The new surface was the most biocompatible, and the 10 nm GNPs did not show any cytotoxicity. There was a significant increase in bioactivity, enhanced gene expressions and ALP activity. ConclusionsGNPs enhances osteogenic differentiation of stem cells and Photofunctionalizing GNPs highly increases this. We have further created a novel highly efficient topography which highly enhances the speed and extent of osseointegration. This may have great potential for improving treatment outcomes for implant, maxillofacial as well as orthopedic patients.

Phenotype and Viability of MLO-Y4 Cells Is Maintained by TGFβ₃ in a Serum-Dependent Manner within a 3D-Co-Culture with MG-63 Cells.

The osteocyte network inside the bone matrix is of functional importance and osteocyte cell death is a characteristic feature of pathological bone diseases. Osteocytes have emerged as key regulators of bone tissue maintenance, yet maintaining their phenotype during in vitro culture remains challenging. A 3D co-culture system for osteocytes with osteoblasts was recently presented, enabling the determination of more physiological effects of growth factors on cells in vitro. MLO-Y4 cells were embedded within a type I collagen gel and cultured in the presence of surface MG-63 cells. Co-culture was performed in the presence or absence of TGFβ3. Gene expression by quantitative PCR, protein expression by fluorescent immunohistochemistry and cell viability tests were performed. The 3D co-culture induced cell differentiation of MG-63 cells seen by increased type I collagen and osteocalcin mRNA expression. TGFβ3 maintained osteocyte differentiation of MLO-Y4 cells during co-culture as determined by stable E11 and osteocalcin mRNA expression till day 4. Interestingly, most of the effects of TGFβ3 on co-cultured cells were serum-dependent. Also, TGFβ3 reduced cell death of 3D co-cultured MLO-Y4 cells in a serum-dependent manner. This study shows that 3D co-culture upregulates differentiation of MG-63 cells to a more mature osteoblast-like phenotype; while the addition of TGFβ3 maintained the characteristic MLO-Y4 osteocyte-like phenotype and viability in a serum-dependent manner.

Recombinant amelogenin regulates the bioactivity of mouse cementoblasts in vitro

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-1) 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-1 increased cell proliferation after 72 h compared to the other concentrations and the untreated control group. rhAMG (100,000 ng · mL-1) upregulated BSP and OCN mRNA expression levels eightfold and fivefold, respectively. rhAMG at a concentration of 100,000 ng · mL-1 remarkably enhanced LAMP-1 staining in cementoblasts. Increased numbers of mineralized nodules were observed at concentrations of 10,000 and 100,000 ng · mL-1 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.

NOX4 Deletion in Male Mice Exacerbates the Effect of Ethanol on Trabecular Bone and Osteoblastogenesis.

Chronic alcohol consumption increases bone resorption and decreases bone formation. A major component of ethanol (EtOH) pathology in bone is the generation of excess reactive oxygen species (ROS). The ROS-generating NADPH oxidase-4 (NOX4) is proposed to drive much of the EtOH-induced suppression of bone formation. Here, 13-week-old male wild-type (WT) and NOX4-/- mice were pair fed (PF) a high-fat (35%), Lieber-DeCarli liquid diet with or without EtOH at 30% of their total calories for 12 weeks. Micro-computed tomography analysis demonstrated significant decreases in trabecular bone volume/total volume (BV/TV) percentage and cortical thickness in WT, EtOH-fed mice compared with PF controls. EtOH-fed NOX4-/- mice also displayed decreased trabecular BV/TV and trabecular number compared with PF (P < 0.05). However, NOX4-/- mice were protected against EtOH-induced decreases in cortical thickness (P < 0.05) and decreases in collagen1 and osteocalcin mRNA expression in cortical bone (P < 0.05). In WT and NOX4-/- vertebral bone, EtOH suppressed expression of Wnt signaling components that promote osteoblast maturation. A role for NOX4 in EtOH inhibition of osteoblast differentiation was further demonstrated by protection against EtOH inhibition of osteoblastogenesis in ex vivo bone marrow cultures from NOX4-/-, but not p47phox-/- mice lacking active NADPH oxidase-2. However, bone marrow cultures from NOX4-/- mice formed fewer osteoblastic colonies compared with WT cultures (P < 0.05), suggesting a role for NOX4 in the maintenance of mesenchymal progenitor cell populations. These data suggest that NOX4 deletion is partially protective against EtOH effects on osteoblast differentiation, but may predispose bone to osteogenic impairments.

MicroRNA-214-5p/TGF-β/Smad2 signaling alters adipogenic differentiation of bone marrow stem cells in postmenopausal osteoporosis

Postmenopausal osteoporosis (OPM) is a common type of osteoporosis in females. It is a systemic, chronic bone disease that presents as microstructure degradation of osseous tissue, decreased bone mineral density and increased osteopsathyrosis caused by hypoovarianism and reduced estrogen levels in the body following menopause. In the present study, the role of microRNA (miR)-214-5p in the regulation of the expression of bone marrow stem cells (BMSCs) was investigated, and its molecular mechanism of osteogenic induction in vitro was assessed. When dexamethasone-induced adipogenic differentiation was performed, miR-214-5p expression was increased compared with the control group, as determined by RT-qPCR. Furthermore, oil red O staining, RT-qPCR and western blot analysis demonstrated that overexpression of miR-214-5p promoted adipogenic differentiation, inhibited alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteocalcin (OC) and collagen α-1 (I) chain (COL1A1) mRNA expression, and suppressed transforming growth factor (TGF)-β, phosphorylated (p)-Smad2 and collagen type IV α1 chain (COL4A1) protein expression in BMSCs. Additionally, downregulation of miR-214-5p increased the ALP, Runx2, OC and COL1 mRNA expression and increased TGF-β, Smad2 and COL4A1 protein expression in BMSCs. Furthermore, a TGF-β inhibitor was employed to inhibit TGF-β expression in BMSCs following miR-214-5p downregulation, which led to reduced Smad2, TGF-β and COL4A1 protein expression, and ALP, Runx2, OC and COL1 mRNA expression was also reduced, compared with the miR-214-5p downregulation only group. It was demonstrated that miR-214-5p may weaken osteogenic differentiation of BMSCs through regulating COL4A1. In conclusion, the results of the present study indicated that miR-214-5p may promote the adipogenic differentiation of BMSCs through regulation of the TGF-β/Smad2/COL4A1 signaling pathway, and potentially may be used to develop a novel drug for postmenopausal osteoporosis.

FGF23 Neutralizing Antibody Partially Improves Bone Mineralization Defect of HMWFGF2 Isoforms in Transgenic Female Mice.

Mice overexpressing high molecular weight FGF2 isoforms (HMWTg) in osteoblast lineage phenocopy human X-linked hypophosphatemic rickets (XLH) and a Hyp murine model of XLH demonstrating increased FGF23/FGF receptor signaling and hypophosphatemic rickets/osteomalacia. Because HMWFGF2 was upregulated in bones of Hyp mice and abnormal FGF23 signaling is important in XLH, HMWTg mice were used to examine the effect of the FGF23 neutralizing antibody (FGF23Ab). Eight-week-old female Vector control mice and HMWTg mice were treated with FGF23Ab or control IgG. A single injection of FGF23Ab rescued abnormal hypophosphatemia in HMWTg. The decreased type II sodium-dependent phosphate co-transporter (Npt2a) was rescued by FGF23Ab treatment. Inappropriately low serum 1,25(OH)2 D in HMWTg mice was normalized by FGF23Ab treatment, which is accompanied by increased anabolic vitamin D hydroxylase Cyp27b1 and decreased catabolic vitamin D hydroxylase Cyp24 mRNA in kidney. Long-term treatment with FGF23Ab normalized femur length and significantly increased vertebrae BMD and BMC, and femur BMC in HMWTg mice compared to IgG-treated HMWTg mice. Micro-computed tomography (μCT) revealed increased cortical porosity and decreased cortical apparent density in the HMWTg-IgG group compared with the Vector-IgG group; however, FGF23Ab treatment rescued defective cortical mineralization, decreased porosity, and increased apparent density in HMWTg mice. Bone histomorphometry analysis showed FGF23Ab treatment decreased osteoid volume, increased intra-label thickness, mineralization apposition rate, and bone formation rate in HMWTg mice. FGF23Ab improved disorganized double labeling in femurs from HMWTg mice. Quantitative real-time PCR analysis of tibia shafts showed FGF23Ab treatment normalized the osteocalcin (Ocn) mRNA expression in HMWTg mice, but further increased expression of SIBLING protein-related and pyrophosphate-related genes that are important in matrix mineralization, suggesting that HMWFGF2 modulates these genes independent of FGF23. We conclude that FGF23Ab partially rescued hypophosphatemic osteomalacia in HMWTg. However, long-term treatment with FGF23Ab further increased SIBLING protein-related genes and pyrophosphate-related genes in bone that could contribute to incomplete rescue of the mineralization defect. © 2018 American Society for Bone and Mineral Research.

Supplying osteogenesis to dead bone using an osteogenic matrix cell sheet

To evaluate whether osteogenic matrix cell sheets can supply osteogenesis to dead bone. Femur bone fragments (5mm in length) were obtained from Fisher 344 rats and irradiated by a single exposure of 60Gy to produce bones that were no longer viable. Osteogenic matrix cell sheets were created from rat bone marrow-derived stromal cells (BMSCs). After wrapping the dead bone with an osteogenic matrix cell sheet, it was subcutaneously transplanted into the back of a rat and harvested after 4 weeks. Bone formation around the dead bone was evaluated by X-ray imaging and histology. Alkaline phosphatase (ALP) and osteocalcin (OC) mRNA expression levels were measured to confirm osteogenesis of the transplanted bone. The contribution of donor cells to bone formation was assessed using the Sry gene and PKH26. After the cell sheet was transplanted together with dead bone, X-ray images showed abundant calcification around the dead bone. In contrast, no newly formed bone was seen in samples that were transplanted without the cell sheet. Histological sections also showed newly formed bone around dead bone in samples transplanted with the cell sheet, whereas many empty lacunae and no newly formed bone were observed in samples transplanted without the cell sheet. ALP and OC mRNA expression levels were significantly higher in dead bones transplanted with cell sheets than in those without a cell sheet (P<0.01). Sry gene expression and cells derived from cell sheets labeled with PKH26 were detected in samples transplanted with a cell sheet, indicating survival of donor cells after transplantation. Our study indicates that osteogenic matrix cell sheet transplantation can supply osteogenesis to dead bone.

Neurotrophin 3 promotes osteogenic differentiation of human dental follicle cells

This study aims to investigate the effect of neurotrophin 3 (NT-3) on the osteogenic differentiation of human dental follicle cells (hDFCs). hDFCs were isolated and cultured in vitro. Immunocytochemical staining was used to identify the origin of hDFCs. The effects of different NT-3 concentrations on hDFCs proliferation were detected by using CCK-8 assay. The alkaline phosphatase (ALP) activities and mRNA expression levels of bone morphogenetic protein-2 (BMP-2) and osteocalcin (OCN) were determined to investigate the effects of NT-3 on hDFCs osteogenesis. The difference in the number of mineralized nodules was detected using alizarin red staining. Vimentin and cytokeratin staining results showed that hDFCs originated from the mesenchymal cells. NT-3 exerted no evident effect on hDFCs proliferation. The ALP activity and the BMP-2 and OCN mRNA expression levels of hDFCs were significantly improved under treatment with different NT-3 concentrations (25, 50, and 100 ng·mL ⁻¹) compared with those in the control group. BMP-2 and OCN mRNA relative expression levels of hDFCs reached the highest when the NT-3 concentration was 100 ng·mL ⁻¹. The number of mineralized nodules reached the maximum when the hDFCs were treated with 50 and 100 ng·mL ⁻¹ NT-3. Appropriate mass concentration of NT-3 can promote the osteogenic differentiation of hDFCs.

Carbon monoxide releasing molecule‑3 promotes the osteogenic differentiation of rat bone marrow mesenchymal stem cells by releasing carbon monoxide.

Stem cell‑based therapies are promising strategies to stimulate bone regeneration.Carbon monoxide releasing molecule‑3 (CORM‑3) exhibits multiple regulatory effects in a number of cells by releasing carbon monoxide (CO). The present study aimed to investigate the influence of CORM‑3 on the osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs). BMSCs were divided into five groups: A CORM‑3‑osteogenic group, in which cells were pretreated with CORM‑3 and subjected to osteogenic differentiation induction using osteogenic medium; an osteogenic group, in which cells were cultured in osteogenic medium; a degassed CORM‑3‑osteogenic group, in which cells were pretreated with degassed CORM‑3 and subjected to osteogenic differentiation induction; a CORM‑3 group, in which cells were cultured in control medium containing CORM‑3; and a control group, in which cells were cultured in control medium alone. The osteo‑specific mRNA and protein expression of runt‑related transcription factor2 (Runx2), osteocalcin (OCN) and osteopontin (OPN) were assessed using reverse transcription‑quantitative polymerase chain reaction and western blot analysis. Alkaline phosphatase (ALP) activity was also examined and mineralization was detected using alizarin red staining. Levels of Runx2, OCN and OPN mRNA and protein in the CORM‑3‑osteogenic group were significantly increased compared with the osteogenic group (P<0.05), with the exception of OCN protein levels on day3. The mRNA and protein expression of Runx2, OCN and OPN in the degassed CORM‑3‑osteogenic and osteogenic groups were similar. In addition, the mRNA and protein expression of Runx2, OCN and OPN in the CORM‑3 and control group were similar. ALP activity in the CORM‑3‑osteogenic group was increased from day3 and remained significantly higher compared with all other groups on days3,5and7 (P<0.05). Additionally, the results indicated that the optical density value of alizarin red staining in the CORM‑3‑osteogenic group was significantly increased compared with the other groups (P<0.05). Therefore, the present study demonstrated that CORM‑3 may promote the osteogenic differentiation of BMSCs by releasing CO.