Periodontitis is a chronic infective disease characterized as the destruction of the supporting tissues of the teeth. Bone marrow mesenchymal stem cells, which are ideal adult stem cells for the regeneration of supporting tissues, may play important roles in restoring the structure and function of the periodontium and in promoting the treatment of periodontal disease. As a consequence, the characteristics, especially osteogenic differentiation mechanism, of these stem cells have been extensively investigated. The regulation of the physiological behavior of these stem cells is associated with BMAL1 gene. This gene is a potential treatment target for periodontal disease, although the specific mechanism remains inconclusive. This study aimed to describe the characteristics of BMAL1 gene and its ability to regulate the osteogenic differentiation of stem cells.
ARNTL Transcription Factors ; genetics ; Adult ; Adult Stem Cells ; Bone Marrow Cells ; physiology ; Cell Differentiation ; Hematopoietic Stem Cells ; Humans ; Mesenchymal Stromal Cells ; physiology ; Osteogenesis ; physiology ; Periodontal Ligament ; Periodontitis ; Periodontium ; Regeneration ; Tooth

OBJECTIVEThis work investigated mesenchymal stem cells (MSCs) modified with Runt-related transcription factor 2 (Runx2) therapy for bone regeneration in rabbit mandibular distraction osteogenesis.

METHODSForty-eight New Zealand mature white rabbits were randomly divided into three groups after the rabbit model of mandibular distraction osteogenesis was established: reconstruction plasmid modified with Runx2 (group A), plasmid without Runx2 (group B), and the same dose of saline as control (group C). At the fifth day of distraction phase, MSCs with reconstruction plasmid modified with adv-hRunx2-gfp were injected into the distraction gap of group A. MSCs with reconstruction plasmid modified with adv-gfp was injected into the distraction gap of group B, whereas group C was injected with the same dose of saline. At 8 weeks after injection, all animals were sacrificed, and the distracted mandibles were harvested. The general imaging histological observation and three-point bending test were used for evaluation.

RESULTSCT plain scan and histological analysis confirmed that the amount of new bone forming in the distraction gap of group A was significantly higher than those in groups B and C. Dual-energy X ray and three-point bending test results also showed that the bone mineral density, bone mineral content, and maximum load of the distraction gap of group A were significantly higher than those of groups B and C (P<0.01).

CONCLUSIONRunx2-ex vivo gene therapy based on MSCs can effectively promote the bone regeneration in rabbit mandibular distraction osteogenesis and shorten the stationary phase. Therefore, reconstruction of craniofacial fracture would be a valuable strategy

Absorptiometry, Photon ; Animals ; Bone Density ; Bone Regeneration ; physiology ; Core Binding Factor Alpha 1 Subunit ; genetics ; pharmacology ; Genetic Therapy ; Mandible ; physiology ; surgery ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; Osteogenesis ; genetics ; Osteogenesis, Distraction ; methods ; Plasmids ; Rabbits ; Random Allocation ; Transcription Factors ; genetics ; physiology ; Treatment Outcome

This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCl) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which was also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel.
Ameloblasts ; physiology ; Amelogenesis ; genetics ; Amelogenin ; analysis ; Bone Morphogenetic Protein 4 ; pharmacology ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cell Line ; Cell Lineage ; Embryonic Stem Cells ; drug effects ; physiology ; Epithelial Cells ; drug effects ; physiology ; Fibroblast Growth Factor 8 ; analysis ; Hedgehog Proteins ; analysis ; Homeodomain Proteins ; analysis ; Humans ; Keratins ; analysis ; classification ; Lithium Chloride ; pharmacology ; MSX1 Transcription Factor ; analysis ; Mouth Mucosa ; cytology ; Phenotype ; Regeneration ; physiology ; Skin ; cytology ; Transcription Factors ; analysis ; Tretinoin ; pharmacology

Bone morphogenetic protein-2 (BMP-2) is used to promote bone regeneration. However, the bone regeneration ability of BMP-2 relies heavily on the delivery vehicle. Previously, we have developed heparin-conjugated fibrin (HCF), a vehicle for long-term delivery of BMP-2 and demonstrated that long-term delivery of BMP-2 enhanced its osteogenic efficacy as compared to short-term delivery at an equivalent dose. The aim of this study was to compare the bone-forming ability of the BMP-2 delivered by HCF to that delivered by clinically utilized BMP-2 delivery vehicle collagen sponge. An in vitro release profile of BMP-2 showed that HCF released 80% of the loaded BMP-2 within 20 days, whereas collagen sponge released the same amount within the first 6 days. Moreover, the BMP-2 released from the HCF showed significantly higher alkaline phosphatase activity than the BMP-2 released from collagen sponge at 2 weeks in vitro. Various doses of BMP-2 were delivered with HCF or collagen sponge to mouse calvarial defects. Eight weeks after the treatment, bone regeneration was evaluated by computed tomography, histology, and histomorphometric analysis. The dose of BMP-2 delivered by HCF to achieve 100% bone formation in the defects was less than half of the BMP-2 dose delivered by collagen sponge to achieve a similar level of bone formation. Additionally, bone regenerated by the HCF-BMP-2 had higher bone density than bone regenerated by the collagen sponge-BMP-2. These data demonstrate that HCF as a BMP-2 delivery vehicle exerts better osteogenic ability of BMP-2 than collagen sponge, a clinically utilized delivery vehicle.
Alkaline Phosphatase/metabolism ; Animals ; Bone Density ; *Bone Morphogenetic Protein 2/administration & dosage/genetics ; Bone Regeneration/*genetics ; Cells, Cultured ; Collagen Type I/chemistry/metabolism ; *Fibrin/chemistry/metabolism ; *Gene Transfer Techniques ; *Heparin/chemistry/metabolism ; Mice ; Osteogenesis/genetics ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the mechanism of Huogu I formula (I) in treating osteonecrosis of femoral head.

METHODSForty-eight healthy female Leghorn chickens were randomly divided into control group, model group and Huogu I group, and each group consisted of 16 chickens. At the meantime of model establishment, chickens of the Huogu I group were administrated with decoction, while the model and control group with distilled water by gavage. At the 8th and 16th week after medication, blood samples were obtained for blood lipid detection while both sides of femoral head were harvested for the rest of examinations. Specifically, expressions of bone morphogenetic protein-2 (BMP2), transforming growth factor beta1 (TGFβ(1)), Smad4 and Smad7 were evaluated by immunohistochemistry, while expression of osteoprotegerin/receptor activator of nuclear factor kappaB ligand (OPG/RANKL) mRNA was detected by in situ hybridization.

RESULTSCompared with the control group, serum levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in the model group rose significantly. Positive cell counting of BMP2, TGFβ(1), Smad4 and OPG in femoral head of the model group dropped prominently. Positive cell counting of Smad7 and RANKL increased dramatically. In contrast with the model group, levels of TC, TG and LDL-C in Huogu I group reduced significantly. Positive cell counting of BMP2, TGFβ(1), Smad4 and OPG in femoral head of the Huogu I group increased prominently. Indices of Smad7 and RANKL both decreased significantly. Especially at the 8th week, these variations were more significant.

CONCLUSIONHuogu I formula is effective in promoting repair of necrotic femoral head by regulating the expressions of BMP2, TGFβ(1), Smads and OPG/RANKL of osteoclast in femoral head.

Animals ; Bone Morphogenetic Protein 2 ; metabolism ; Bone Regeneration ; drug effects ; physiology ; Chickens ; Chondrocytes ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; Female ; Femur Head Necrosis ; chemically induced ; drug therapy ; metabolism ; Lipid Metabolism ; physiology ; Osteocytes ; metabolism ; Osteoprotegerin ; genetics ; metabolism ; Receptor Activator of Nuclear Factor-kappa B ; genetics ; metabolism ; Smad4 Protein ; metabolism ; Smad7 Protein ; metabolism ; Steroids ; pharmacology ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo explore effect of interleukin 10 (IL-10) gene-modified bone marrow-derived liver stem cells (BDLSCs) transplantation on hepatic inflammatory response and liver regeneration in rats with liver fibrosis.

METHODS50 female Wistar rats were randomly divided into 4 groups: (1) control group: 10 rats were subcutaneously injected with olive oil for 8 weeks; (2) fibrosis groups: 16 rats were subcutaneously injected with carbon tetrachloride (CCl4) for 8 weeks to induce liver fibrosis; (3) BDLSC group: 12 rats were subcutaneously injected with CCl4 for 8 weeks, and were transplanted with 2 x 10(5) BDLSC at week 4; (4) BDLSC/IL-10 group: 12 rats were subcutaneously injected with CCl4 for 8 weeks, and were transplanted with 2 x 10(5) IL-10 gene-modified BDLSC at week 4. IL-10 and tumor necrosis factor alpha (TNFa) in liver tissues were detected by ELISA. HE stained liver tissues were observed under light microscope. The expression of hepatocyte growth factor (HGF) was quantified by real-time RT-PCR, and the expression of proliferating cell nuclear antigen (PCNA) was determined by immunohistochemistry.

RESULTSThe ratio of IL-10/TNFa in fibrosis group (0.05+/-0.01) was lower than that in control group (0.26+/-0.04) (P < 0.01). Transplantation of untreated BDLSCs did not improve the ratio (P > 0.05), however, transplantation of IL-10 modified BDLSCs improved the ratio significantly (P < 0.01). Severe inflammatory response and fibrosis were observed in fibrosis group. Inflammatory response was alleviated to some extent in the BDLSC group, and the histopathology of BDLSC/IL-10 group was not significantly different from that of the control group. Compared to the control group, the expression of HGF mRNA and PCNA protein was increased in the fibrosis group (P < 0.01). The expression of HGF and PCNA was further increased by BDLSCs or IL-10 modified BDLSCs transplantation. Compared to BDLSCs, IL-10 gene-modified BDLSCs were more potent to induce the expression of HGF and PCNA.

CONCLUSIONTransplantation of IL-10 gene-modified BDLSCs can alleviate hepatic inflammatory response and promote liver regeneration in hepatic fibrosis rats.

Adenoviridae ; genetics ; Animals ; Bone Marrow Cells ; cytology ; Cell Proliferation ; Disease Models, Animal ; Female ; Genetic Therapy ; methods ; Hepatocyte Growth Factor ; genetics ; metabolism ; Immunohistochemistry ; Interleukin-10 ; genetics ; Liver ; metabolism ; pathology ; Liver Cirrhosis ; genetics ; pathology ; therapy ; Liver Regeneration ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cell Transplantation ; methods ; Transduction, Genetic ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo evaluate the efficacy of hBMP-4 gene modified tissue engineered bone graft in the enhancement of rabbit spinal fusion and find an ideal kind of substitute for the autograft bone.

METHODSRabbit BMSCs were cultured and transfected with AAV-hBMP-4 using different MOI value. The optimal MOI value were determined by observing cell's morphology change. BMSCs were then transfected with AAV-hBMP4 and AAV-EGFP respectively, following which the transfected cells were evenly suspended in a collagen sponge I, and implanted to either side of the L5,6 intertransverse spaces posterolateral in the New Zealand rabbits to induce spinal fusion. Fourteen rabbits were randomly divided into 2 groups. Group 1: AAV-hBMP-4 transfected BMSCs in the right side (hBMP-4 side) and autograft bone in the left side. Group 2: AAV-hBMP-4 transfected BMSCs in the right side (hBMP-4 side) and AAV-EGFP transfected BMSCs in the left side (EGFP side). Radiographs and three-dimensional CT of the spine, manual palpation, gross and histological examination of the fusion masses for all the animals were performed subsequent to animals having been sacrificed at 12 weeks after surgery.

RESULTSEvaluation has been taken in 12 New Zealand rabbits delivered into 2 groups which meet the criterion after operation. Eleven in 12 implemented sides involved hBMP-4 achieved bony fusion, to which 5 in 6 autografted sides was similar. But only 2 in 6 sides in EGFP-group achieved bony fusion meanwhile. Three-dimensional CT scan and palpation also evidenced the results. Bone formation was observed obviously on specimen both in hBMP4 sides and autografted ones. EGFP-group also got bony integration, but the quantity was small.

CONCLUSIONTissue-engineered bone graft constructed from application of hBMP4 is a fine substitute for autograft. Effective enhancement of bony integration in spinal fusion surgery has been evidenced in vivo.

Animals ; Bone Morphogenetic Protein 4 ; genetics ; Bone Regeneration ; Bone Substitutes ; Bone Transplantation ; methods ; Genetic Vectors ; Lentivirus ; genetics ; Male ; Myeloid Progenitor Cells ; Rabbits ; Random Allocation ; Spinal Fusion ; methods ; Stromal Cells ; Tissue Engineering ; Transfection

Planarian is among the simplest animals that possess a centralized nervous system (CNS), and its neural regeneration involves the replacement of cells lost to normal 'wear and tear' (cell turnover), and/or injury. In this review, we state and discuss the recent studies on molecular control of neural regeneration in planarians. The spatial and temporal expression patterns of genes in intact and regenerating planarian CNS have already been described relatively clearly. The bone morphogenetic protein (BMP) and Wnt signaling pathways are identified to regulate neural regeneration. During neural regeneration, conserved axon guidance mechanisms are necessary for proper wiring of the nervous system. In addition, apoptosis may play an important role in controlling cell numbers, eliminating unnecessary tissues or cells and remodeling the old tissues for regenerating CNS. The bilateral symmetry is established by determination of anterior-posterior (A-P) and dorsal-ventral (D-V) patterns. Moreover, neurons positive to dopamine, serotonin (5-HT), and gamma-aminobutyric acid (GABA) have been detected in planarians. Therefore, planarians present us with new, experimentally accessible contexts to study the molecular actions guiding neural regeneration.
Animals ; Apoptosis ; Bone Morphogenetic Proteins ; metabolism ; Central Nervous System ; cytology ; Fibroblast Growth Factors ; Gene Expression ; physiology ; Nerve Regeneration ; genetics ; Neurotransmitter Agents ; metabolism ; Planarians ; genetics ; physiology ; Signal Transduction ; physiology ; Wnt Proteins ; physiology

OBJECTIVETo evaluate the therapeutic effect of bone morphogenetic protein 2 (BMP-2) gene modified tissue engineering bone (GMB) combined with vascularized periosteum in the reconstruction of segmental bone defect.

METHODSAdenovirus carrying BMP-2 gene (Ad-BMP-2) was transfected into the isolated and cultured rabbit bone marrow stromal cells (MSCs). The transfected MSCs were seeded on bovine cancellous bone scaffolds (BCB) to construct gene modified tissue engineering bone (GMB). The bilateral rabbits radial defects (2.5 cm long) were created as animal model. The rabbits were divided into five groups to reconstruct the defects with CMB combined with vascularized periosteum (group A); or GMB combined with vascular bundle implantation (group B); or GMB combined with free periosteum (group C); or GMB only (group D); or BCB scaffolds only (group E). Angiogenesis and osteogenesis were observed by X-ray, histological examination, biomechanical analysis and capillary ink infusion.

RESULTSIn group A, the grafted GMB was revascularized rapidly. The defect was completely reconstructed at 8 weeks. The mechanism included both intramemerbrane and endochondral ossification. In group B, the vascular bundle generated new blood vessels into the grafted GMB, but the osteogenesis process was slow in the central zone, which healed completely at 12 weeks. In group C, the free graft of periosteum took at 4 weeks with angiogenesis. The thin extremal callus was formed at 8 weeks and the repairing process almost finished at 12 weeks. Better osteogenesis was found in group D than in group E, due to the present of BMP2 gene-transfected MSCs. The defects in group D were partial repaired at 12 weeks with remaining central malunion zone. The defects in group E should nonunion at 12 weeks with only fibre tissue.

CONCLUSIONSBMP-2 gene modified tissue engineering bone combined with vascularized periosteum which provides periosteum osteoblasts as well as blood supply, has favorable ability of osteogenesis, osteoinduction and osteoconduction. It is an ideal method for the treatment of segmental bone defect.

Animals ; Bone Marrow Cells ; cytology ; Bone Morphogenetic Protein 2 ; genetics ; Bone Regeneration ; Bone Substitutes ; Bone Transplantation ; methods ; Bone and Bones ; pathology ; Cattle ; Mesenchymal Stromal Cells ; cytology ; Periosteum ; blood supply ; transplantation ; Rabbits ; Surgical Flaps ; blood supply ; Tissue Engineering ; methods ; Tissue Scaffolds ; Transfection

OBJECTIVETo investigate bone defect healing by true bone ceramic complex carrying core binding factor a1 (Cbfa1) gene modified rabbit skin fibroblasts.

METHODSTransfect rabbit skin fibroblasts (RSF) with both eukaryotic expression vector pSG5 which could express Cbfa1 gene and pSG5. After being cultured for 48 h, the transfected RSF were seeded into true bone ceramic (TBC) of 2 cm in length and 4 mm in diameter to construct pSG5-Cbfa1/RSF/TBC complex and pSG5/RSF/TBC complex. Forty-eight bone defect model rabbits were randomized into four groups, each has 6 rabbits (12 radius), due to different treatment. group I: with pSG5-Cbfa1/RSF/TBC complex, group II: with pSG5/RSF/TBC complex, group III: with TBC, Group IV: empty control. After being seeded and cultured for about 24 h the complexes were implanted into 2 cm long bone defects in the middle of bilateral radius of rabbits. The radius were inspected by X-ray and then the specimens were collected at the end of the fourth and twelfth weeks after operation. Then, the specimens were decalcified and histologically investigated with Hematoxylin eosin staining and Masson staining methods. Newly synthesized trabecular bone was inspected by image analysis system and the strength of bone defect area treated with graft-implantation was tested with biomechanical method-three point bending test.

RESULTSIn group I, trabecular bone was actively synthesized to generate a great amount of trabecular bone and osteon. Preliminary union and bone defect healing were completed with good biomechanical characteristics. There were no newly synthesized trabecular in the other three groups, and bone defect healing were not discovered. The amount of newly synthesized trabecular bone and the results of biomechanical testing differed significantly between group I and the other three (P < 0.01). The efficacy of group I was significantly better than that of the other three groups.

CONCLUSIONTrue bone ceramic complex composed with Cbfa1 gene modified rabbit skin fibroblasts can effectively heal bone defect in rabbits.

Animals ; Bone Regeneration ; Bone Substitutes ; Bone Transplantation ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; genetics ; Disease Models, Animal ; Fibroblasts ; cytology ; metabolism ; Plasmids ; genetics ; Rabbits ; Radius ; injuries ; physiopathology ; surgery ; Random Allocation ; Skin ; cytology ; Tissue Engineering ; methods ; Transfection