It is known that low-frequency pulsed electromagnetic fields (PEMFs) can promote the differentiation and maturation of rat calvarial osteoblasts (ROBs) cultured in vitro. However, the mechanism that how ROBs perceive the physical signals of PEMFs and initiate osteogenic differentiation remains unknown. In this study, we investigated the relationship between the promotion of osteogenic differentiation of ROBs by 0.6 mT 50 Hz PEMFs and the presence of polycystin2 (PC2) located on the primary cilia on the surface of ROBs. First, immunofluorescence staining was used to study whether PC2 is located in the primary cilia of ROBs, and then the changes of PC2 protein expression in ROBs upon treatment with PEMFs for different time were detected by Western blotting. Subsequently, we detected the expression of PC2 protein by Western blotting and the effect of PEMFs on the activity of alkaline phosphatase (ALP), as well as the expression of Runx-2, Bmp-2, Col-1 and Osx proteins and genes related to bone formation after pretreating ROBs with amiloride HCl (AMI), a PC2 blocker. Moreover, we detected the expression of genes related to bone formation after inhibiting the expression of PC2 in ROBs using RNA interference. The results showed that PC2 was localized on the primary cilia of ROBs, and PEMFs treatment increased the expression of PC2 protein. When PC2 was blocked by AMI, PEMFs could no longer increase PC2 protein expression and ALP activity, and the promotion effect of PEMFs on osteogenic related protein and gene expression was also offset. After inhibiting the expression of PC2 using RNA interference, PEMFs can no longer increase the expression of genes related to bone formation. The results showed that PC2, located on the surface of primary cilia of osteoblasts, plays an indispensable role in perceiving and transmitting the physical signals from PEMFs, and the promotion of osteogenic differentiation of ROBs by PEMFs depends on the existence of PC2. This study may help to elucidate the mechanism underlying the promotion of bone formation and osteoporosis treatment in low-frequency PEMFs.
Alkaline Phosphatase/metabolism* ; Animals ; Electromagnetic Fields ; Osteoblasts/metabolism* ; Osteogenesis/genetics* ; Rats ; TRPP Cation Channels/physiology*

Mesenchymal stem cells (MSCs) are characterized by their self-renewing capacity and differentiation potential into multiple tissues. Thus, management of the differentiation capacities of MSCs is important for MSC-based regenerative medicine, such as craniofacial bone regeneration, and in new treatments for metabolic bone diseases, such as osteoporosis. In recent years, histone modification has been a growing topic in the field of MSC lineage specification, in which the Su(var)3-9, enhancer-of-zeste, trithorax (SET) domain-containing family and the Jumonji C (JmjC) domain-containing family represent the major histone lysine methyltransferases (KMTs) and histone lysine demethylases (KDMs), respectively. In this review, we summarize the current understanding of the epigenetic mechanisms by which SET domain-containing KMTs and JmjC domain-containing KDMs balance the osteogenic and adipogenic differentiation of MSCs.
Adipogenesis ; genetics ; physiology ; Cell Differentiation ; genetics ; physiology ; Cell Lineage ; genetics ; Epigenesis, Genetic ; genetics ; F-Box Proteins ; genetics ; physiology ; Histone Demethylases ; genetics ; physiology ; Histone-Lysine N-Methyltransferase ; genetics ; physiology ; Humans ; Jumonji Domain-Containing Histone Demethylases ; genetics ; physiology ; Mesenchymal Stromal Cells ; enzymology ; physiology ; Methyltransferases ; genetics ; physiology ; Osteogenesis ; genetics ; physiology

OBJECTIVETo investigate the effect of gene transfection at different time on bone mineral density and strength of newly formed bone in mandibular distraction gap in rabbit, so as to explore the optimal time for gene therapy and enhance the therapeutic effect.

METHODS48 New-Zealand rabbits were employed to receive mandibular osteotomy and implantation of distraction devices bilaterly. Then the rabbits were randomly divided into 4 groups as group A, B and C and D. The animals in group A, B, and C were transfected with recombinant plasmids pIRES-hBMP2-hVEGF165 via electroporation-mediated approach at latency period, distraction period, consolidation period respectively. Group D was used as control group without gene transfection. After 3 days of latency period, the distraction devices were activated at the rate of 0.8 mm per day for 10 days. Three rabbits in each group were sacrificed at 1 wk, 2 wk, 4 wk and 8 wk of consolidation respectively. The mandibles were harvested and the left mandible received X-ray examination for bone healing, and quantitative computed tomography (QCT) dectection for the bone mineral density (BMD) of newly formed bone in the distraction gap. The biomechanical properties of the new generation bone at 4 th and 8 th week of consolidation in each group were detected by three point bending test.

RESULTSThe bone mineral density and the biomechanical strength of newly formed bone increased along the length of consolidation in each group. After 1 week of consolidation, there was no significant difference in BMD among group A (83.43 +/- 9.96), group B (92.29 +/- 11.25), group C (89.93 +/- 14.15), P > 0.05. But the BMD of group A, B and C was higher than that of group D (70. 31 +/- 3.30), P < 0.05. After 2wk, 4 wk and 8 wk of consolidation, the BMD of group B (137.54 +/- 7.20,492.93 +/- 17.57, 790.48 +/- 12.19) was significantly higher than those of group A (121.44 +/- 9.27, 396.15 +/- 15.70, 603.39 +/- 16.46), C (125.06 +/- 7.24, 464.15 +/- 15.45, 764.15 +/- 17.28), and D (98.86 +/- 8.13, 336.45 +/- 11.95, 577.89 +/- 18.43), P < 0.05. The biomechanical parameters were also higher in group B than those of group A, C and D after four and eight weeks of consolidation (P < 0.05).

CONCLUSIONIt is better to transfect gene at the beginning of distraction (distraction period) than at other stages of DO. In this way, more remarkable effect could be obtained on new bone formation. It suggests that the distraction stage is the optimal time for gene therapy.

Animals ; Bone Density ; genetics ; physiology ; Electroporation ; Genetic Therapy ; Mandible ; physiology ; surgery ; Osteogenesis, Distraction ; Osteotomy ; Rabbits ; Time Factors ; Transfection

Spine fusion stabilize adjacent vertebral segments by achieving bone union. The preferred method of spine fusion involves decortication of the host bed and transplantation of autologous bone graft from the iliac crest. Although autologous bone grafting is the gold standard, this procedure has significant complication. Gene therapy represents the new frontier of medical science that holds much promise for the improvement of spinal arthrodesis. Studies have shown the efficacy in using liposome-mediated and adenovirus-mediated gene transfer of bone morphogenetic protein (BMP) and related gene in animal models. Several studies in which gene transfer has been used specifically to enchance spine fusion in animal models are reviewed. Current main areas of research, including the elucidation of gene expression profiles during bone formation and the development of new gene transfer vehicles, promises the development of clinically applicable techniques in the near future.
Animals ; Bone Morphogenetic Proteins ; genetics ; physiology ; Genetic Therapy ; methods ; Genetic Vectors ; Osteogenesis ; genetics ; Spinal Fusion ; methods ; Transfection

Mesenchymal stem cells (MSCs) are progenitors of connective tissues, which have emerged as important tools for tissue engineering due to their differentiation potential along various cell types. In recent years, accumulating evidence has suggested that the regulation of mitochondria dynamics and function is essential for successful differentiation of MSCs. In this paper, we review and provide an integrated view on the role of mitochondria in MSC differentiation. The mitochondria are maintained at a relatively low activity level in MSCs, and upon induction, mtDNA copy number, protein levels of respiratory enzymes, the oxygen consumption rate, mRNA levels of mitochondrial biogenesis-associated genes, and intracellular ATP content are increased. The regulated level of mitochondrial ROS is found not only to influence differentiation but also to contribute to the direction determination of differentiation. Understanding the roles of mitochondrial dynamics during MSC differentiation will facilitate the optimization of differentiation protocols by adjusting biochemical properties, such as energy production or the redox status of stem cells, and ultimately, benefit the development of new pharmacologic strategies in regenerative medicine.
Adipogenesis ; physiology ; Animals ; Cell Differentiation ; physiology ; Chondrogenesis ; physiology ; Humans ; Mesenchymal Stem Cells ; cytology ; metabolism ; Mitochondria ; genetics ; metabolism ; Mitochondrial Proteins ; genetics ; metabolism ; Osteogenesis ; physiology ; RNA ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; RNA, Mitochondrial ; Reactive Oxygen Species ; metabolism

OBJECTIVETo investigate effects of intermittent negative pressure on osteogenesis in human bone marrow-derived stroma cells (BMSCs) cultured in vitro.

METHODSThe third passage cells were divided into negative pressure treatment group and control group. The cells in the treatment group were induced by negative pressure intermittently (pressure: 17 kPa, 30 min per time, and four times of each day). The cells in the control group were cultured in conventional condition. The osteogenesis of BMSCs was examined by phase-contrast microscopy. The alkaline phosphatase (ALP) activities were determined. The expression of collagen type I was detected by immunohistochemistry method. The mRNA expressions of osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL) in BMSCs were analyzed by real-time polymerase chain reaction (PCR).

RESULTSBMSCs showed a typical appearance of osteoblast after 2 weeks of induction by intermittent negative pressure. The activity of ALP increased significantly, and the expression of collagen type I was positive. In the treatment group, the mRNA expression of OPG increased significantly (P < 0.05) and the mRNA expression of OPGL decreased significantly (P < 0.05) after 2 weeks, compared with the control. However, 3 days after the exposure to 2-week negative pressure, these were no significantly different from that of the control group (P > 0.05).

CONCLUSIONIntermittent negative pressure could promote osteogenesis in BMSCs in vitro.

Bone Marrow Cells ; physiology ; Cell Culture Techniques ; Collagen Type I ; analysis ; Humans ; Osteogenesis ; Osteoprotegerin ; genetics ; Pressure ; RANK Ligand ; genetics ; RNA, Messenger ; analysis ; Stromal Cells ; physiology

BACKGROUND: Osteopenia has been well documented in adolescent idiopathic scoliosis (AIS). Bone marrow stem cells (BMSCs) are a crucial regulator of bone homeostasis. Our previous study revealed a decreased osteogenic ability of BMSCs in AIS-related osteopenia, but the underlying mechanism of this phenomenon remains unclear. METHODS: A total of 22 AIS patients and 18 age-matched controls were recruited for this study. Anthropometry and bone mass were measured in all participants. Bone marrow blood was collected for BMSC isolation and culture. Osteogenic and adipogenic induction were performed to observe the differences in the differentiation of BMSCs between the AIS-related osteopenia group and the control group. Furthermore, a total RNA was extracted from isolated BMSCs to perform RNA sequencing and subsequent analysis. RESULTS: A lower osteogenic capacity and increased adipogenic capacity of BMSCs in AIS-related osteopenia were revealed. Differences in mRNA expression levels between the AIS-related osteopenia group and the control group were identified, including differences in the expression of LRRC17 , DCLK1 , PCDH7 , TSPAN5 , NHSL2 , and CPT1B . Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed several biological processes involved in the regulation of autophagy and mitophagy. The Western blotting results of autophagy markers in BMSCs suggested impaired autophagic activity in BMSCs in the AIS-related osteopenia group. CONCLUSION Our study revealed that BMSCs from AIS-related osteopenia patients have lower autophagic activity, which may be related to the lower osteogenic capacity and higher adipogenic capacity of BMSCs and consequently lead to the lower bone mass in AIS patients.
Humans ; Adolescent ; Scoliosis/genetics* ; Cell Differentiation/physiology* ; Osteogenesis/genetics* ; Bone Diseases, Metabolic/genetics* ; Kyphosis ; Autophagy/genetics* ; Bone Marrow Cells ; Cells, Cultured ; Doublecortin-Like Kinases

Aging of craniofacial skeleton significantly impairs the repair and regeneration of trauma-induced bony defects, and complicates dental treatment outcomes. Age-related alveolar bone loss could be attributed to decreased progenitor pool through senescence, imbalance in bone metabolism and bone-fat ratio. Mesenchymal stem cells isolated from oral bones (OMSCs) have distinct lineage propensities and characteristics compared to MSCs from long bones, and are more suited for craniofacial regeneration. However, the effect of epigenetic modifications regulating OMSC differentiation and senescence in aging has not yet been investigated. In this study, we found that the histone demethylase KDM4B plays an essential role in regulating the osteogenesis of OMSCs and oral bone aging. Loss of KDM4B in OMSCs leads to inhibition of osteogenesis. Moreover, KDM4B loss promoted adipogenesis and OMSC senescence which further impairs bone-fat balance in the mandible. Together, our data suggest that KDM4B may underpin the molecular mechanisms of OMSC fate determination and alveolar bone homeostasis in skeletal aging, and present as a promising therapeutic target for addressing craniofacial skeletal defects associated with age-related deteriorations.
Aging ; Cell Differentiation ; Facial Bones/physiology* ; Humans ; Jumonji Domain-Containing Histone Demethylases/genetics* ; Mesenchymal Stem Cells/cytology* ; Osteogenesis ; Osteoporosis

BACKGROUNDBone morphogenetic protein 9 (BMP9) and Wnt/β-catenin signaling pathways are able to induce osteogenic differentiation of mesenchymal stem cells (MSCs), but the role of Wnt/β-catenin signaling pathway in BMP9-induced osteogenic differentiation is not well understood. Thus, our experiment was undertaken to investigate the interaction between BMP9 and Wnt/β-catenin pathway in inducing osteogenic differentiation of MSCs.

METHODSC3H10T1/2 cells were infected with recombinant adenovirus expressing BMP9, Wnt3a, and BMP9+Wnt3a. ALP, the early osteogenic marker, was detected by quantitative and staining assay. Later osteogenic marker, mineral calcium deposition, was determined by Alizarin Red S staining. The expression of osteopotin (OPN), osteocalcin (OC), and Runx2 was analyzed by Real time PCR and Western blotting. In vivo animal experiment was carried out to further confirm the role of Wnt3a in ectopic bone formation induced by BMP9.

RESULTSThe results showed that Wnt3a enhanced the ALP activity induced by BMP9 and increased the expressions of OC and OPN, with increase of mineral calcium deposition in vitro and ectopic bone formation in vivo. Furthermore, we also found that Wnt3a increased the level of Runx2, an important nuclear transcription factor of BMP9.

CONCLUSIONCanonical Wnt/β-catenin signal pathway may play an important role in BMP9-induced osteogenic differentiation of MSCs, and Runx2 may be a linkage between the two signal pathways.

Blotting, Western ; Cell Differentiation ; genetics ; physiology ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Growth Differentiation Factor 2 ; genetics ; metabolism ; Humans ; Osteocalcin ; genetics ; metabolism ; Osteogenesis ; genetics ; physiology ; Wnt3A Protein ; genetics ; metabolism

BACKGROUNDMost of the basic and clinical studies of osteonecrosis of the femoral head (ONFH) are restricted to bone tissues only, whereas various systems are involved in the onset and development of ONFH, including nervous system. Peptidergic nerve participates in the neuronal regulation of bone metabolism and anabolism, and plays key roles in the growth, repair and reconstruction of bone. Calcitonin gene-related peptide (CGRP), which is secreted by peptidergic nerve, is the main mediator of bone metabolism. It dramatically promotes the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Additionally, it enhances the osteoblast mass and the rate of osteoblast formation, and reduces the bone resorption by acting on osteoblasts and osteoclasts. Hence, we aimed to construct recombinant retrovirus vector pLNCX(2)-hCGRPα and to investigate the proliferation and osteogenic potential of hCGRPα-producing BMSCs (BMSCs/pLNCX(2)-hCGRPα) after virus infection.

METHODSThe constructed recombinant retrovirus vector pLNCX(2)-hCGRPα was transfected into PT67 packaging cells by lipofectamine 2000. Virus was collected for BMSCs infection. The mRNA and protein expression of hCGRPα was examined by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively. The cell proliferation was determined by methyl thiazoleterazolium (MTT) assay. The osteogenic potential of BMSCs was evaluated by alkaline phosphatase (ALP) activity.

RESULTSBoth mRNA and protein expression of hCGRPα was detected in BMSCs/pLNCX(2)-hCGRPα cells. These cells exhibited significantly elevated proliferation and ALP value as compared with control BMSCs (P < 0.05).

CONCLUSIONBMSCs/pLNCX(2)-hCGRPα cells could stably express hCGRPα and showed promoted proliferation ability and osteogenic potential as compared with control BMSCs.

Alkaline Phosphatase ; genetics ; metabolism ; Animals ; Blotting, Western ; Bone Marrow Cells ; cytology ; Calcitonin Gene-Related Peptide ; genetics ; metabolism ; Cell Differentiation ; genetics ; physiology ; Cell Proliferation ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; Osteogenesis ; genetics ; physiology ; Rabbits ; Reverse Transcriptase Polymerase Chain Reaction