Osteogenic Experiments Research Articles

Application of gelatin methacryloyl/minocycline-chitosan-nanoparticles composite hydrogel for the treatment of periodontitis.

This study aimed to investigate the feasibility of gelatin methacryloyl (GelMA) hydrogel loa-ded with minocycline-chitosan-nanoparticles (MCN) for the treatment of periodontitis in vitro and vivo. MCN were synthesized by ionic gel method. GelMA/MCN composite hydrogels were prepared by compounding MCN with GelMA hydrogel. The materials were characterized by transmission electron microscope, scanning electron microscopy, and Fourier transform infrared spectroscopy. The degradation behavior and drug release rates of hydrogels were evaluated. The antibacterial activity of GelMA/MCN hydrogel against Porphyromonas gingivalis was detected, and the minimum antibacterial concentration was determined. Biocompatibility and osteogenic experiments were conducted under a simulated periodontitis environment. A rat model of periodontitis was constructed to observe the therapeutic effects of GelMA/MCN hydrogel. MCN was successfully synthesized with a particle size of about 80 nm, while the structures of GelMA/MCN had no significant differences from GelMA. MCN and GelMA/MCN released minocycline slowly and steadily. Bacterial growth was completely inhibited when the MCN concentration was higher than or equal to 0.2 mg·mL-1. GelMA/MCN hydrogels exhibited good biocompatibility at effective antimicrobial concentrations under the simulated periodontitis environment with the enzyme. The in vivo results showed that GelMA/MCN prevented the progression of periodontitis and promoted the repair of bone defects. GelMA/MCN composite hydrogel can release minocycline slowly and steadily and has good antibacterial activity and biocompatibility to promote the repair of periodontitis bone defects.

MicroRNA-124-3p affects myogenic differentiation of adipose-derived stem cells by targeting Caveolin-1 during pelvic floor dysfunction in Sprague Dawley rats.

BackgroundThe aim of this study was to investigate using myogenic differentiation of adipose stem cells for the treatment of female pelvic floor dysfunction (PFD) and aimed to further study the influences of microRNA-124-3p (miR-124-3p) in the process of myogenic differentiation of adipose-derived stem cells (ADSCs) through targeting Caveolin-1 (Cav1) during PFD in Sprague Dawley (SD) rats.MethodsThe ADSCs were separated from 6–8-week-old female SD rats (n=25) and were cultivated. Then, we observed the cell status and conducted fat and osteogenic experiments. We then constructed an ADSC-green fluorescent protein (GFP) stable transfer strain. Flow cytometry was used to identify the positive rates of CD44, CD90, and CD45 in ADSCs and ADSC-GFP. Real-time quantitative polymerase chain reaction (qRT-PCR) and western blotting were used to mRNA and protein expression levels. Myogenic differentiation of ADSCs was measured with immunofluorescence methods. A dual-luciferase reporter assay was executed to affirm whether Cav1 was a target of miR-124-3p.ResultsThe isolated ADSCs cells were in good condition under the microscope. The results of flow cytometry showed that the positive rate of CD44 and CD90 was high, and the positive rate of CD45 was low in ADSCs and ADSC-GFP. Under normal culture conditions, ADSCs-GFP cells can be massively adipated and osteogenic. After 5-Aza induced ADSC-GFP myogenic differentiation, the level of miR-124-3p was significantly increased. We found that MiR-124-3p mimics promoted the myogenic differentiation of ADSCs. Moreover, we discovered that Cav1 was a target gene of miR-124-3p and was negatively regulated by miR-124-3p. The results of leak point pressure (LPP), hematoxylin and eosin (HE), and Masson showed that the collagen fiber content of the PFD group was lower than that of the control group; the collagen fiber content of ADSC-GFP, 5-Aza, or miR-124-3p mimics were increased after intervention. Furthermore, the outcomes qRT-PCR, western blotting, and immunofluorescence suggested that miR-124-3p facilitated the survival ADSC-GFP fat transplantation by regulating many key factors in vivo.ConclusionsThese results proofed that miR-124-3p could accelerate myogenic differentiation of ADSCs by down-regulating Cav1 to improve PFD in SD rats, which will pave the way for therapeutic delivery of miRNA targeting PFD disease.

In vitro study of the biological interface of Bio‐Oss: implications of the experimental setup

To systematically investigate the biological interface of Bio-Oss by analysing dissolution-precipitation behaviour and osteogenic responses using in vitro experimental systems. Different concentrations (1-100mg/ml) of Bio-Oss were incubated in cell culture medium for 24h before elemental concentrations for calcium, phosphorus and silicon in the medium were analysed with inductive coupled plasma-optical emission spectroscopy. Radioactive calcium-45 isotope labelling technique was used to study possible precipitation of calcium on the Bio-Oss particle. Biological interface of Bio-Oss was studied in osteogenic experiments using mineralization medium and three different sources of cells (primary mouse bone marrow stromal cells, primary rat calvarial cells and MC3T3-E1 mouse pre-osteoblast cell line). Cells were fixed and stained with Toulidine blue, von Kossa or Alizarin Red staining for confirmation of extracellular matrix mineralization. Elemental analysis of the cell culture medium demonstrated a significant decrease of calcium and phosphorus and a dose-dependent release of silicon to the medium after incubation with Bio-Oss. A significant decrease of calcium and phosphorus in the medium occurred even at low concentrations of Bio-Oss. Uptake of calcium on the Bio-Oss particle was confirmed with radioactive calcium-45 isotope labelling technique. In osteogenic experiments with Bio-Oss (<1mg/ml), matrix mineralization around the Bio-Oss particles were demonstrated in all three cell types with von Kossa and Alizarin Red staining. Dissolution-precipitation reactions occur at the surface of Bio-Oss, and osteogenic responses are seen at the biological interface. The concentration of Bio-Oss is a key factor for the experimental in vitro results, and may also have implications for the clinic.