OBJECTIVETo explore whether ultrasound-mediated microbubble destruction can enhance the expression efficiency of plasmid pIRES-rhBMP2-EGFP for bone morphogenetic protein-2(BMP-2) in mice skeletal muscle.

METHODSTwenty four male BALB/c mice were divided into four groups. The naked plasmid was injected into the pretibial muscle or the quadriceps muscle (group A and group C) without ultrasound-mediated microbubble destruction method. Micobubbles with plasmid were injected into the pretibial muscle or the quadriceps muscle (group B and group D) with destructing microbubbles by ultrasound immediately. Twelve mice (group A and group B, 30 microg plasmid injected) were killed after 7 days and the tissue samples of the pretibial muscle were obtained to observe the expression of enhanced green fluorescence protein (EGFP) by inverted fluorescence microscope, gene transfection efficiencies were quantified by counting EGFP positive fibers on mice skeletal muscle. After 14 days, the other twelve mice (group C and group D, 100 microg plasmid injected) were killed and immunnohistochemical technique was applied to detect the rhBMP-2 gene expression.

RESULTSThe percentage of GFP-positive fibers was significantly lower in the group A than that in the group B. After 14 days, expression of rhBMP-2 was detected in cells and interstitial spaces in the group C and group D, and expression efficiency of rhBMP-2 in the group D was significantly higher than that in the group C.

CONCLUSIONUltrasound-mediated microbubble destruction could enhance the transfection and expression efficiency of rhBMP-2 gene in skeletal muscle of mouse in vivo. It is a new gene therapy method for periodontal regeneration.

Animals ; Bone Morphogenetic Proteins ; Genetic Therapy ; Green Fluorescent Proteins ; Male ; Mice ; Mice, Inbred BALB C ; Microbubbles ; Muscle, Skeletal ; Plasmids ; Transfection ; Ultrasonics

OBJECTIVETo study the expression and interaction of core binding factor a1 (Cbfa1), bone morphogenetic proteins (BMPs) and osteopontin (OPN) in the developing periodontal tissues of mice.

METHODSA mice developing periodontal tissues study model was created histologically by 5-27 day postnatal BALB/c mice, then the immunohistochemical localization of Cbfa1, BMPs and OPN in different developing stages were undertaken.

RESULTSIn early stage of postnatal mice periodontal tissues development, only BMPs expressed in dental follicle cells, though the signal was weak. When root was forming, all of them were expressed in periodontal ligament cells and cementoblasts, while only OPN in acellular cementum, cellular cementum and the surface of alveolar bone, Cbfa1 only in cellular cementum and BMPs was seen in neither acellular cementum nor cellular cementum.

CONCLUSIONCbfa1, BMPs and OPN all involve in the development of periodontal tissues, while OPN is crucial for cementum.

Animals ; Bone Morphogenetic Proteins ; Bone and Bones ; Core Binding Factors ; Dental Cementum ; Mice ; Mice, Inbred BALB C ; Osteopontin ; Periodontal Ligament ; Tooth Root

OBJECTIVETo study the biological effects of phenytoin (PHT) on cultured human periodontal ligament fibroblasts (hPDLF), and explore the possibility of its accelerating periodontal regeneration.

METHODSIncreasing concentrations of PHT (1, 5, 20, 100, 500, 2 500 mg/L) were added into the medium of the fourth passage of cultured hPDLF, respectively. After co-incubated for 3 days, cell proliferation activity, the total amount of protein and alkaline phosphatase (ALP) activity were detected. Mineralized sodium and PHT (20, 100, 500 mg/L) were added into the medium of the fourth passage hPDLF. After co-incubated, the mineralized nodules formation were detected by Von Kossa staining. The third passage hPDLF were stimulated by PHT (20, 100 mg/L), bone morphogenetic protein-2 (BMP-2) concentration was analyzed by enzyme linked immunosorbent sandwich assay (ELISA).

RESULTSAt the concentration of 20 or 100 mg/L, PHT significantly enhanced the proliferating activity and ALP activity of hPDLF (P<0.01). PHT at 100 mg/L could increase protein synthesis of hPDLF (P<0.05). The capability of mineralization and BMP-2 expression of hPDLF were increased significantly (P<0.01) in 100 mg/L group when compared with that in the control group. However, higher concentration (2 500 mg/L) not only changed cell morphology, but also significantly inhibited cell activity.

CONCLUSIONThe results suggested that proper doses of PHT could promote proliferation and biosynthesis and also enhance osteogenesis by increasing the differentiation, mineralization and BMP-2 expression of hPDLF while higher concentrations of PHT had cytotoxic effect.

Cell Differentiation ; Cells, Cultured ; Fibroblasts ; Humans ; In Vitro Techniques ; Osteogenesis ; Periodontal Ligament ; Phenytoin

OBJECTIVETo investigate the transfection efficiency of the recombinant human bone morphogenetic protein-2 (hBMP-2) gene in targeted cells by ultrasound-mediated microbubble destruction.

METHODSNIH3T3 cells' anabiosis was completed and went down to the 3rd or 4th generation, and cultured in 6 well plates. The cells were divided into 2 groups: Plasmid DNA and Lipofectamine 2000 group (liposome group), plasmid DNA and ultrasound and microbubble group (ultrasound-mediated microbubble destruction group). Plasmid DNA was transfected into cells with liposome or ultrasound and microbubble. 24-48 hours later, the transfection efficiency and the concentrations of hBMP-2 were measured with fluoresence microscope and enzyme-linked immunosorbent assay (ELISA) respectively. The data were analyzed by curve fitting and t-test of SPSS 11.5.

RESULTSThe transfection efficiency rate was (7.30 +/- 1.58)% in liposome group, compared with (11.77 +/- 3.16)% in ultrasound-mediated microbubble destruction group (P< 0.05). The concentration of hBMP-2 after transfection was (1164.35 +/- 724.67) pg/mL in liposome group, versus (2932.70 +/- 656.27) pg/mL in ultrasound-mediated microbubble destruction group (P<0.05).

CONCLUSIONUltrasound-mediated microbubble destruction could significantly improve the transfection efficiency and expression of hBMP-2 gene in NIH3T3 cells. It may provide a new and effective gene delivery system for gene therapy in periodontal regeneration.

Animals ; Bone Morphogenetic Protein 2 ; Genetic Therapy ; Humans ; In Vitro Techniques ; Mice ; Microbubbles ; NIH 3T3 Cells ; Plasmids ; Recombinant Proteins ; Transfection ; Transforming Growth Factor beta ; Ultrasonics

OBJECTIVETo study role of dental follicle in tooth root development.

METHODSSixteen mandibular first molar dental germs from eight five-day postnatal Balb/c mice were divided into two groups randomly. Dental follicle of germs in one group was undetached and that of another group was removed. Subsequently, each of the germs was separately transplanted to back-muscles of adult nude mice. At seventh and fourteenth day after transplanting, the germs were collected, fixed, demineralized, dehydrated, and embedded in wax in sequence. Serial sections of 5 microm thick were made following the routine methods, stained with haematoxylin-eosin dying solution, and observed under a light microscope.

RESULTSAll implantations were located in the back-muscles with abundant capillary vasculature. Under microscope, although all tooth germs could further develop after grafting, tooth germs without dental follicle developed slowly with small size and low calcification compared to those with dental follicle. Although position of Hertwig's epithelial root sheath of all germs seemed no changing, roots of the group with dental follicle could further develop and the roots develop toward the apical direction; this tendency couldn't be seen in the germs of another group. Inflammatory cells could be seen in and out of the pulp cavity of the two groups at 7th day after grafting, while no obvious inflammatory cell was observed at 14th day after grafting.

CONCLUSIONDental follicle play an important role in tooth root development. It probably can lead tooth root to develop in normal direction.

Animals ; Dental Pulp Cavity ; Dental Sac ; Enamel Organ ; Mice ; Mice, Nude ; Molar ; Odontogenesis ; Tooth ; Tooth Germ ; Tooth Root

OBJECTIVETo establish a method for isolating and culturing mouse dental follicle cells and to study the phenotype characteristics of dental follicle cells.

METHODSMandibular first molars from 9 day old Balb/c mice were digested with 1% trypsin, subsequently, and the dental follicle was enucleated from the tooth germ and cultured. The shape and ultrastructural appearance of dental follicle cells were observed under phase-contrast microscope and scanning electron microscope (SEM). Immunocytochemistry was used to detect the expression of alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteopontin (OPN).

RESULTSThree types of cells were isolated: some were cuboidal/polygonal; some were elongated, spindle-shaped, fibroblast-like cells; and a minor, third cell type was very thin and elongated. The cytoplasm of the first two cell types was filled with abundant granules. The cells were pleomorphism under SEM and had many filipodia and microvilli. According to whether there were filaments overlying the surface, the cells could be divided into two subtypes. Some but not all follicle cells expressed ALP, BSP and OPN.

CONCLUSIONThe cultured dental follicle cells consisted of several cell phenotypes and had the potential of differentiating into cementoblasts, periodontal ligament fibroblasts and osteoblasts.

Alkaline Phosphatase ; Animals ; Cell Culture Techniques ; Cell Line ; Cells, Cultured ; Dental Cementum ; Dental Sac ; Fibroblasts ; Integrin-Binding Sialoprotein ; Mice ; Mice, Inbred BALB C ; Molar ; Osteoblasts ; Osteopontin ; Periodontal Ligament ; Phenotype ; Tooth Germ

OBJECTIVETo study the expression of Runx2/Cbfa1 in the developing dentin and differentiating odontoblasts.

METHODSA postnatal mice teeth developing model was built histologically. Immunohistochemical technique was adopted to determine the expression of Runx2/Cbfa1 in the developing pulpo-dentinal complex in mice.

RESULTSRunx2/Cbfa1 was merely present in predentin in the exact and before the 11th day's postnatal stages. Meanwhile, it was positively located in odontoblasts and dental pulp cells in root region, but negatively in coral part after the 11th day's stages.

CONCLUSIONRunx2/Cbfa1 may play an important role in the deposing of tooth dentin and in the differentiating of odontoblasts and pulp cells.

Animals ; Cell Differentiation ; Core Binding Factor Alpha 1 Subunit ; Dental Pulp ; Dentin ; Mice ; Odontoblasts ; Tooth

OBJECTIVETo examine the expression of periodontal ligament-associated protein-1 (PLAP-1) in the periodontal tissues and periodontal ligament cells (PDLC).

METHODSThe PLAP-1 expression in normal periodontal tissue was examined by immunohistochemistry. The protein expression and mRNA transcription of PLAP-1 in PDLC were investigated by immunocytochemistry and reverse transcription-polymerase chain reaction.

RESULTSPLAP-1 was expressed in periodontium but not in cementum, alveolar bone and gingival tissues. PLAP-1 expression was observed in cell plasma, but not in nuclei. There was a 350 bp electrophoresis band representing PLAP-1 mRNA.

CONCLUSIONSPLAP-1 may play a role in physiology of periodontal tissues and cells in normal adult rats.

Animals ; Extracellular Matrix Proteins ; genetics ; metabolism ; Immunohistochemistry ; Male ; Periodontal Ligament ; cytology ; metabolism ; Periodontium ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the effect of retinoic acid on differentiation of periodontal ligament cells (PDLCs) and gingival fibroblasts (GFs).

METHODSThe periodontal ligament cells and gingival fibroblasts were cultured, challenged with different concentrations of retinoic acid in medium and detected for the alkaline phosphatase (ALP) activity and its mRNA by biochemical technique, in situ hybridization and RT-PCR.

RESULTSALP activity in normal PDLCs was higher than that in GFs. The mRNA signals were positive in PDLCs, and negative in GFs. After treated with different concentrations of retinoic acid, ALP activity of PDLCs was increased than that of the control, and its mRNA signals were enhanced, especially in 5 x 10(-6) mol/L. The treated GFs showed a slight increase of ALP activity and a weak band of mRNA signals only in 5 x 10(-6) molUL concentration.

CONCLUSIONThere were differences between PDLCs and GFs in differentiating into osteoblast-like cells.

Cell Differentiation ; Fibroblasts ; Gingiva ; Osteoblasts ; Periodontal Ligament ; RNA, Messenger ; Tretinoin