Exploring the Mechanism of CircRNA-vgll3 in Osteogenically Differentiated Human Bone Marrow Mesenchymal Stem Cells

Objective:To explore the mechanism of circRNA-vgll3 in osteogenic differentiation of human bone marrow mesenchymal stem cells.Methods:BMSCs cells were transfected with circRNA-vgll3,and divided into circRNA-vgll3 high-level group,circRNA-vgll3 low-level group,and negative control group(circRNA-vgll3 not transfected)according to the amount of transfection.The proliferation and apoptosis of BMSCs osteoblasts in each group were analyzed,and the alkaline phosphatase(ALP)activity,type I collagen gray value,bone morphogenetic protein 2(BMP-2),Runx2 protein,and mRNA expression levels were detected.Results:The circRNA-vgll3 low-level group had a significant inhibitory effect on the proliferation of BMSCs osteoblasts,and the apoptosis rate of the circRNA-vgll3 low-level group was significantly higher than that of the circRNA-vgll3 high-level group(P<0.05);ALP activity,type I collagen gray value,BMP-2,Runx2 protein,and mRNA expression levels in the high-level circRNA-vgll3 group were significantly higher than those in the low-level circRNA-vgll3 group,and the difference was statistically significant(P<0.05).Conclusion:Overexpression of circRNA-vgll3 can promote the osteogenic differentiation ability of BMSCs,while low expression of circRNA-vgll3 can inhibit the osteogenic differentiation ability of BMSCs.The main mechanism of action is that circRNA-vgll3 can affect osteogenic differentiation by regulating the Runx2 protein.

Microwave/Thermal Analyses for Human Bone Characterization

A novel imaging approach utilizing microwave scattering was proposed in order to analyze various properties of bone. Microwave frequencies of 900 MHz, 1 GHz, and 2.4 GHz were used during this study. This investigation’s objectives were to emphasize characteristics of abnormalities in human bones and to detect fine fractures through contrasts in bone density. The finite element method (FEM) presented here is generated from COMSOL software at different frequencies. The study identified the optimum transmission directed at the interface layers from an external microwave source. It was found that approximately 900 MHz microwave power was ideal for this application. This can be attributed to the penetration depth where the power dissipation is analyzed based on bone condition. The microwave energy was generated from an exterior antenna that was interfaced, via catheter, to skeletal bone. The power transmitted to bone was converted into thermal energy, and has led to a visible temperature distribution pattern, which reflects the bone density level, and accordingly, the type of bone under investigation. The electrical and thermal properties, including the dielectric permittivity, thermal conductivity, and heat flux absorption through the bone substance, have great implications on the FEM distribution. The boundary conditions using tangential matching of field components at the tissue-bone interface were incorporated into the finite element method. The average power from the electromagnetic fields (estimated from the Poynting’s vector, P = E*H), was assumed to be fully absorbed as heat due to the conductivity of the bone material. Furthermore, microwave energy was applied as a delta function and the thermal distributions have been analyzed in order to distinguish between normal healthy bone and bones with structural or metabolic abnormalities. The latter was emulated by different bone density to contrast normal bone anatomy. The FEM simulation suggests that thermography microwave imaging could be a good tool for bone characterization in order to detect skeletal abnormalities. This approach could be advantageous over other existing methods such as X-ray imaging.

The genomic landscapes of histone H3-Lys9 modifications of gene promoter regions and expression profiles in human bone marrow mesenchymal stem cells

Mesenchymal stem cells （MSCs） of nonembryonic origins possess the proliferation and multi-lineage differentiation potentials. It has been established that epigenetic mechanisms could be critical for determining the fate of stem cells, and MSCs derived from different origins exhibited different expression profiles individually to a certain extent. In this study, ChiP-on-chip was used to generate genome-wide histone H3-Lys9 acetylation and dimethylation profiles at gene promoters in human bone marrow MSCs. We showed that modifications of histone H3-Lys9 at gene promoters correlated well with mRNA expression in human bone marrow MSCs. Functional analysis revealed that many key cellular pathways in human bone marrow MSC self-renewal, such as the canonical signaling pathways, cell cycle pathways and cytokine related pathways may be regulated by H3-Lys9 modifications. These data suggest that gene activation and silencing affected by H3-Lys9 acetylation and dimethylation, respectively, may be essential to the maintenance of human bone marrow MSC self-renewal and multi-potency.

Human bone marrow mesenchymal stem cell transplantation attenuates axonal injury in stroke rats

Previous studies have shown that transplantation of human bone marrow mesenchymal stem cells promotes neural functional recovery after stroke, but the neurorestorative mechanisms remain largely unknown. We hypothesized that functional recovery of myelinated axons may be one of underlying mechanisms. In this study, an ischemia/reperfusion rat model was established using the middle cerebral artery occlusion method. Rats were used to test the hypothesis that intravenous transplantation of human bone marrow mesenchyrnal stem cells through the femoral vein could exert neuroprotective effects against cerebral ischemia via a mechanism associated with the ability to attenuate axonal injury. The results of behavioral tests, infarction volume analysis and immunohistochemistry showed that cerebral ischemia caused severe damage to the myelin sheath and axons. After rats were intravenously transplanted with human bone marrow mesenchymal stem cells, the levels of axon and myelin sheath-related proteins, including microtubule-associated protein 2, myelin basic protein, and growth-associated protein 43, were elevated, infarct volume was decreased and neural function was improved in cerebral ischemic rats. These findings suggest that intravenously transplanted human bone marrow mesenchymal stem cells promote neural function. Possible mechanisms underlying these beneficial effects include resistance to demyelination after cerebral ischemia, prevention of axonal degeneration, and promotion of axonal regeneration.

Vitamin D receptor expression in human bone tissue and dose-dependent activation in resorbing osteoclasts

The effects of vitamin D on osteoblast mineralization are well documented. Reports of the effects of vitamin D on osteoclasts, however, are conflicting, showing both inhibition and stimulation. Finding that resorbing osteoclasts in human bone express vitamin D receptor （VDR）, we examined their response to different concentrations of 25-hydroxy vitamin D3 [25（OH）D3] （100 or 500 nmol·L^-1） and 1,25-dihydroxy vitamin D3 [1,25（OH）2D3] （0.1 or 0.5 nmol·L^-1） metabolites in cell cultures. Specifically, CD14＋ monocytes were cultured in charcoal-stripped serum in the presence of receptor activator of nuclear factor kappa-B ligand （RANKL） and macrophage colony-stimulating factor （M-CSF）. Tartrate-resistant acid phosphatase （TRAP） histochemical staining assays and dentine resorption analysis were used to identify the size and number of osteoclast cells, number of nuclei per cell and resorption activity. The expression of VDR was detected in human bone tissue （ex vivo） by immunohistochemistry and in vitro cell cultures by western blotting. Quantitative reverse transcription-PCR （qRT-PCR） was used to determine the level of expression of vitamin D-related genes in response to vitamin D metabolites. VDR-related genes during osteoclastogenesis, shown by qRT-PCR, was stimulated in response to 500 nmol·L^-1 of 25（OH）D3 and 0.1-0.5 nmol·L^-1 of 1,25（OH）2D3, upregulating cytochrome P450 family 27 subfamily B member I （CYP27B1） and cytochrome P450 family 24 subfamily A member I （CYP24A1）. Osteoclast fusion transcripts transmembrane 7 subfamily member 4 （tm7sf4） and nuclear factor of activated T-cell cytoplasmic 1 （nfatcl） where downregulated in response to vitamin D metabolites. Osteoclast number and resorption activity were also increased. Both 25（OH）D3 and 1,25（OH）2D3 reduced osteoclast size and number when co-treated with RANKL and M-CSF. The evidence for VDR expression in resorbing osteoclasts in vivo and low-dose effects of 1,25（OH）2D3 on osteoclasts in vitro may therefore provide insight into the effects of clinical vitamin D treatments, further providing a counterpoint to the high-dose effects reported from in vitro experiments.

Construction of Adeno-associated Virus System for Human Bone Morphogenetic Protein 7 Gene

To construct the recombinant adeno-associated virus （rAAV） vector with human bone morphogenetic protein 7 （BMP7） and observe the BMP7 mRNA expression in vitro, BMP7 CDS sequence was cloned into expression plasmid pAAV-MCS of AAV Helper Free System. The recombinant plasmid was identified with enzyme digestion and sequencing. The recombinant plasmid, pAAV-RC, pHelper were co-transfected into AAV-293 cells according to the calcium phosphate-based protocol. The viral stock was collected by 4 rounds of freeze/thaw. After purified and concentrated, the recombinant virus titer was determined by dot-blot assay. HEK293 cells were transfected with the recombinant virus at different MOI, and the expression of BMP7 mRNA was detected by RT-PCR. The results showed rAAV-BMP7 was constructed and packaged successfully. The physical particle titer was 2.5×10^11 vector genomes/mL. There was different expression level of BMP7 mRNA after transfecton. These data suggested that recombinant AAV mediated a stable expression of hBMP7 mRNA in 293 cells. The AAV production method may pave the way of an effective strategy for the jaw bone defection around dental implants.

CORAL AS A CARRIER FOR RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN-2

By combining coral with recombinant human bone morphogenetic protein-2 (rhBMP-2), rhBMP-2/coral composite was obtained in this study. Following implantation of the composite into the muscle pouches of mice, cartilage growth was induced in the pores or on the surface of the implants at one week, woven bone at three week and lamellar bone with bone marrow at six week, and coral was absorbed partially. The induced formation of endochondral bone was time-related and rhBMP-2 dose-related. The results of this study indicate that the composite possesses a superior ability of osteogenesis, and coral acts as one of the most suitable rhBMP-2 slowrelease carriers currently available. The composite will be a new type of bone substitute to be used in orthopaedics and maxillofacial surgery.

Targeted Induction of Differentiation of Human Bone Mesenchymal Stem Cells into Neuron-like Cells

A systematic method of isolating and culturing human bone mesenchymal stem cells （hMSCs）, and inducing them to differentiate into neuron-like cells in vitro was established. The hMSCs were isolated from bone marrow with the lymphocyte-separating medium, cultured and expanded in vitro, and induced after addition of compound neuro-revulsants. The morphological changes of hMSCs were observed, and the expression of surface markers in induced hMSCs was immunocytochemically identified during induction period. The hMSCs could be separated, cultured and expanded in vitro. After induction by compound neuro-revulsants for 48 h, the changes of neuron-like cells, such as cellular shrinkage and neurite growth, were observed in some cells. The immunochemical staining revealed nestin （＋） or NF （＋）, and GFAP （-）. It was concluded that hMSCs were successfully cultured and induced to differentiate into neuron-like cells.

SPECIFIC BINDING OF HUMAN BONEMORPHOGENETIC PROTEIN （2A） WITH MOUSEOSTEOBLASTIC CELLS

Human bone morphogenetic protein 2A (hBMP2A) cDNA terminal 567 nucleotides were cloned and expressed in a phage display vector pCSM21. Human BMP2A C-terminal peptide displayed on the surface of the phage can bind specifically to the surface of mouse osteoblastic cell (MC3T3) membrane. ELISA assay showed a positive signal of the binding by using antibody against M13 phage gene 8 protein. After labeling with 3HTdR,the counts of the binding groups were 3 to 10 times higher than the control groups. It suggests that the surface of MC3T3 cells exist the receptor for hBMP2A.

CLONING AND SEQUENCING OF MATURE FRAGMENT OF HUMAN BMP4 GENE

Objective To study the cloning and sequencing of mature fragment of human bone morphogenetic protein 4 gene. Methods The template DNA was obtained from the human osteosarcoma cell line U2OS. By using RT PCR method, the cDNA coding for the mature fragment of BMP 4 was amplified, cloned into the vector pUC19, and sequenced by Sanger Dideoxy mediated Chain Termination method. Results The mature fragment of BMP4 cDNA was obtained by RT PCR and determined by sequencing. Through the computer search on Genebank, the analysis showed that the homology of nucleotides and amino acids between cDNA of rhBMP4 mature fragment of this study and the published sequence was 99%. Sequence analysis showed that there were two differences, one was at base 1154(201): G→C, which had no influence on the corresponding amino acids(Val). Another was at base1222(269):C→T, the mutation at the base 1222 had the change of Ala to Val. Conclusion The mature fragment of BMP4 gene has been cloned. The results will be of great significance in treatment of skeletal injuries and diseases.

EFFECTS OF TRANSFORMING GROWTH FACTOR β AND RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2 ON HUMAN PERIODONTAL LIGAMENT FIBROBLASTS

Objective To evaluate the effects of transforming growth factor β(TGF-β) and recombinant human bone morphogenetic protein 2 (rhBMP2) on human periodontal ligament fibroblasts (HPDLFs). Methods HPDLFs were done primary culture to detect the distinct concentrations of TGF-P and rhBMF2 on its proliferation, alkaline phosphatase (ALP) activity, osteocalcin (OC) synthesis and formation of the minerali-zed nodules, respectively. Results TGF-β (5～100ng/ml) significantly stimulated the proliferation of HPDLFs. The ALP activity of HPDLFs was evaluated evidently by 5ng/ml TGF-β. TGF-β( 0. 5 ～ 100ng/ml) had no effects on OC synthesis and formation of the mineralized nodules of HPDLFs. rhBMP2 (0. 25～2mg/ ml) had no remarkable effect on the proliferation of HPDLFs. The ALP activity, OC synthesis and forma-tion of the mineralized nodules of HPDLFs were significantly stimulated by 0. 5～ 2mg /ml rhBMP2. Conclusion The effects of TGF-β and rhBMP2 on HPDLFs are dose-dependent. TGF-P can stimulate HPDLFs to express the early marker of osteoblastic phenotype, and it lacks the ability to promote maturation of the osteogenic phenotype. rhBMP2 can not only stimulate the expression but also promote the maturation of osteoblas-tic phenotype of HPDLFs.

Construction of bicistronic green fluorescent protein labeled pSELECT GFPzeo human bone morphogenetic protein 2 eukaryotic expression vector

Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase

Chondrogenic differentiation of human bone mesenchymal stem cells treated with growth differentiation factor 5 under hypoxia

Objective To explore the feasibility and effectiveness of the self-assembly cartilage tissue engineered with chondrogenically differentiated human bone mesenchymal stem cells (hBMCs) induced by growth differentiation factor-5 (GDF-5)

Influence of sodium fluoride on alkaline phosphatase activity and bone gla protein synthesis in human yellow ligament cells in vitro

Objective To investigate the influence of sodium fluoride(NaF)on alkaline phosphatase(ALP)activity and bone gla protein(BGP)synthesis in yellow ligament cells from different surgical simples in vitro.Methods The human ligament cells

Influence of bone morphogenetic proteins-2 and strontium chloride on the human umbilical cord mesenchymal stem cells proliferation and osteogenic differentiation

Objective To study the effects of combination of bone morphogenetic protein-2 (BMP-2) and strontium chloride on proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells(hUCM-SCs)in vitro culture.

Bioactive materials from berberine-treated human bone marrow mesenchymal stem cells promote alveolar bone regeneration by regulating macrophage polarization

Alveolar bone regeneration has been strongly linked to macrophage polarization.M1 macrophages aggravate alveolar bone loss,whereas M2 macrophages reverse this process.Berberine(BBR),a natural alkaloid isolated and refined from Chinese medicinal plants,has shown therapeutic effects in treating metabolic disorders.In this study,we first discovered that culture supernatant(CS)collected from BBR-treated human bone marrow mesenchymal stem cells(HBMSCs)ameliorated periodontal alveolar bone loss.CS from the BBR-treated HBMSCs contained bioactive materials that suppressed the M1 polarization and induced the M2 polarization of macrophages in vivo and in vitro.To clarify the underlying mechanism,the bioactive materials were applied to different animal models.We discovered macrophage colony-stimulating factor(M-CSF),which regulates macrophage polarization and promotes bone formation,a key macromolecule in the CS.Injection of pure M-CSF attenuated experimental periodontal alveolar bone loss in rats.Colony-stimulating factor 1 receptor(CSF1R)inhibitor or anti-human M-CSF(M-CSF neutralizing antibody,Nab)abolished the therapeutic effects of the CS of BBR-treated HBMSCs.Moreover,AKT phosphorylation in macrophages was activated by the CS,and the AKT activator reversed the negative effect of the CSF1R inhibitor or Nab.These results suggest that the CS of BBR-treated HBMSCs modulates macrophage polarization via the M-CSF/AKT axis.Further studies also showed that CS of BBR-treated HBMSCs accelerated bone formation and M2 polarization in rat teeth extraction sockets.Overall,our findings established an essential role of BBR-treated HBMSCs CS and this might be the first report to show that the products of BBR-treated HBMSCs have active effects on alveolar bone regeneration.

Regulating the bioactivity of non-glycosylated recombinant human bone morphogenetic protein-2 to enhance bone regeneration

Recombinant human bone morphogenetic protein-2(rhBMP-2)is the predominant growth factor that effectively induces osteogenic differentiation in orthopedic procedures.However,the bioactivity and stability of rhBMP-2 are intrinsically associated with its sequence,structure,and storage conditions.In this study,we successfully determined the amino acid sequence and protein secondary structure model of non-glycosylated rhBMP-2 expressed by an E.coli expression system through X-ray crystal structure analysis.Furthermore,we observed that acidic storage conditions enhanced the proliferative and osteoinductive activity of rhBMP-2.Although the osteogenic activity of non-glycosylated rhBMP-2 is relatively weaker compared to glycosylated rhBMP-2;however,this discrepancy can be mitigated by incorporating exogenous chaperone molecules.Overall,such information is crucial for rationalizing the design of stabilization methods and enhancing the bioactivity of rhBMP-2,which may also be applicable to other growth factors.