OBJECTIVE: Stem cells from human exfoliated teeth (SHED) were sorted by magnetically activated cell sorting (MACS) technique to obtain the CD146 positive and negative cell subpopulation. Then the biological characteristics of these subpopulations were compared to explore their specific application potential in tissue engineering. METHODS: In this study, freshly extracted deciduous teeth without any caries or dental pulp disease were obtained. SHED was isolated using enzyme digestion method and then sorted by MACS, CD146 positive cells and CD146 negative cells were obtained after cell sorting. The biological characteristics of the unsorted mixed cells, CD146 positive subpopulation and CD146 negative subpopulation were compared. The proliferation ability was detected through cell counting kit-8 (CCK-8) and colony-forming unit (CFU). After osteogenic induction, alizarin red staining was performed and the gene expression of osteogenic related markers was detected by quantitative real-time polymerase chain reaction(qPCR). After adipogenic induction, oil-red O staining was performed and the gene expression of adipogenic related markers was detected. After neurogenic differentiation induction, the expression of neural markers was detected by immunofluorescence and the gene expression of neural markers was detected by qPCR. RESULTS: SHED of the fifth passage was sorted by MACS. And the CD146 positive cell subpopulation and CD146 negative cell subpopulation were obtained. CCK8 assay showed that the proliferative tendency of the three cell groups was consistent, but the proliferation potential of CD146 positive and negative cell subpopulations was significantly lower than that of the unsorted cells. The colony forming rates of the unsorted mixed cell group, CD146 positive and negative populations were 28.6%±3%,17.1%±2.3% and 27.5%±2.5%, respectively. After 21 days of osteogenic induction, alizarin red staining and qPCR showed that the CD146 positive cell population had more mineralized nodule formation and expressed higher level of osteogenic related genes compared with the other two groups. After 21 days of adipogenic induction, oil red O staining and qPCR results showed that the CD146 negative subpopulation produced more lipid droplets and the expression of lipid related genes increased more significantly. After 14 days of neural induction, cell immunofluorescence and qPCR results showed that the unsorted mixed cell group and CD146 positive subpopulation expressed glial cell marker, and the expressions of neural precursor cells and neuronal marker increased significantly in negative subpopulation. CONCLUSION The unsorted mixed cells showed better proliferative potential than CD146 positive and negative subpopulations. The CD146 positive subpopulation was most potent in osteogenic differentiation; it was more suitable for bone tissue engineering. The CD146 negative cells had stronger adipogenic differentiation potential than the other two cell groups; different subpopulations differed in neural differentiation.
Bone and Bones ; CD146 Antigen/analysis* ; Cell Differentiation ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Humans ; Mesenchymal Stem Cells ; Neural Stem Cells ; Neurons ; Osteogenesis ; Staining and Labeling ; Tissue Engineering ; Tooth, Deciduous/cytology*

OBJECTIVEWe explored the expressions of the Notch and Wnt signaling pathways and their significance in the repair process of alveolar bone defects by establishing animal models with a composite of autologous bone marrow mesenchymal stem cells (BMSCs) and platelet-rich fibrin (PRF) to repair bone defects in the extraction sockets of rabbits.

METHODSA total of 36 two-month-old male New Zealand white rabbits were randomly divided into four groups, and the left mandibular incisors of all the rabbits were subjected to minimally invasive removalunder general anesthesia. BMSC-PRF compounds, single PRF, and single BMSC were implanted in Groups A, B, and C. No material was implanted in Group D (blank control). The animals were sacrificed at 4, 8 and 12 weeks after surgery, the bone defect was immediately drawn, and the bone specimens underwent surgery after four, eight, and twelve weeks, with three rabbits per time point. The expressions of Notch1 and Wnt3a in the repair process of the bone defect were measured via immunohistochemical and immunofluorescence detection.

RESULTSImmunohistochemistry showed that the expressions of Notch1 and Wnt3a in Groups A, B, and C were higher than that in Group D at the fourth and eighth week after operation (P<0.05). By contrast, the expressions of Notch1 and Wnt3a in Group D were higher than those in Groups A, B, and C at the twelfth week (P<0.05). Immunofluorescence showed that the expressions of both Notch1 and Wnt3a reached their peaks in the new bone cells of the bone defect after four weeks following surgery and gradually disappeared when the bone was repaired completely.

CONCLUSIONNotch1 and Wnt3a signaling molecules are expressed in the process of repairing bone defects using BMSC-PRF composites and can accelerate the healing by regulating the proliferation and differentiation of BMSCs. Moreover, the expressions of Notch and Wnt are similar, and a crosstalk between them may exist it.

Alveolar Bone Grafting ; methods ; Animals ; Blood Platelets ; Bone Marrow Cells ; cytology ; Bone Transplantation ; methods ; Bone and Bones ; abnormalities ; Cell Differentiation ; Fibrin ; administration & dosage ; Male ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; Platelet-Rich Plasma ; Rabbits ; Random Allocation ; Receptor, Notch1 ; metabolism ; Tissue Engineering ; Wnt Signaling Pathway ; Wnt3A Protein ; metabolism ; Wound Healing

The research of bone tissue engineering bases on three basic directions of seed cells, scaffold materials and growth information. Stem cells have been widely studied as seed cells. Human urine-derived stem cell (hUSC) is extracted from urine and described to be adhesion growth, cloning, expression of the majority of mesenchymal stem cell markers and peripheral cell markers, multi-potential and no tumor but stable karyotype with passaging many times. Some researches proposed that hUSC might be a new source of seed cells in tissue engineering because of their invasive and convenient obtention, stable culture and multiple differentiation potential.
Bone and Bones ; Cell Differentiation ; Humans ; Mesenchymal Stromal Cells ; Stem Cells ; cytology ; Tissue Engineering ; Urine ; cytology

OBJECTIVETo investigate a method that constructing a tissue-engineered tendon with a continuous and heterogeneous transition region.

METHODSFibroblasts derived from rabbit epithelial tissue were cultured in vitro and collagen gel was prepared. The experimental groups were scaffold only group, fibroblasts+ chondrocytes group (Fb+ CC group), fibroblasts+ osteoblasts group (Fb+ OB group), fibroblasts+ chondrocytes+ osteoblasts group (Fb+ CC+ OB group). Heterogeneous cell populations(fibroblasts, chondrocytes and osteoblasts) with collagen gel were seeded within three predesigned specific regions (fibrogenesis, chondrogenesis, and osteogenesis) of decellularized rabbit achilles tendons to fabricate a stratified scaffold containing three biofunctional regions supporting fibrogenesis, chondrogenesis, and osteogenesis. The tests of morphology, architecture and cytocompatibility of the scaffolds were performed. Gradient tissue-specific matrix formation was analysed within the predesignated regions via histological staining and immunofluorescence assays.

RESULTSThe HE staining and scanning electron microscopy analysis demonstrated that no major cell fragments or nuclear material was evident, and increased intra-fascicular and inter-fascicular spaces were found, the cytocompatibility of the scaffolds showed that the numbers of viable cells on the scaffold surfaces increase steadily, no significant differences were found between the scaffold only containing ordinary culture medium and scaffold containing gel groups. Histological staining and immunofluorescence assays demonstrated that the cartilage-related markers (GAG, COL2A1) were found only in the chondrogenesis region, but bone-related proteins only in the osteogenesis region of bone tunnel, and fibrosis was remarkable for the fibrogenesis region in the joint cavity. The transitional architecture with ligament-fibrocartilage-bone was constructed in the ligament-bone tunnel interface.

CONCLUSIONSA transitional interface (fiber-fiberocartilage-bone) could be replicated in a decellularized tendon through stratified tissue integration in vitro. The cell-tendon complex offers the advantages of a multi-tissue transition involving controlled cellular interactions and matrix heterogeneity.

Animals ; Bone and Bones ; Cells, Cultured ; Chondrocytes ; cytology ; Collagen ; Fibroblasts ; cytology ; Ligaments ; Osteoblasts ; cytology ; Rabbits ; Tendons ; Tissue Engineering ; methods

The regulation of epigenetics on bone marrow mesenchymal stem cells (BMSCs) has been a research hot spot in medical area. This paper mainly summarizes the progress of the regulation of DNA methylation, histone acetylation, small interfering RNA (siRNA) induced gene silence and microRNA (miRNA) on BMSCs. Our analysis shows that the regulation of epigenetics on BMSCs plays a significant role in the repair of bone tissue, nervous tissue and cardiac muscle.
Acetylation ; Bone and Bones ; DNA Methylation ; Epigenesis, Genetic ; Hematopoietic Stem Cells ; Histones ; Humans ; Mesenchymal Stromal Cells ; cytology ; MicroRNAs ; Myocardium ; RNA, Small Interfering ; Wound Healing

Notch signaling is highly conserved in evolution and regarded as a key factor in cell fate determination. It mediates cell-to-cell interactions that are critical for embryonic development and tissue renewal, and is involved in the occurrence and metastasis of neoplasm. Recent researches have found that such signaling plays an important role in modulating the differentiation of chondrocytes, osteoblasts and osteoclasts. Dysfunction of Notch signaling can result in many skeletal diseases such as bone tumor, disorders of bone development or bone metabolism.
Animals ; Bone Development ; Bone Diseases ; genetics ; metabolism ; Bone and Bones ; metabolism ; Humans ; Osteoblasts ; cytology ; metabolism ; Osteogenesis ; Receptors, Notch ; genetics ; metabolism ; Signal Transduction

OBJECTIVEThe purpose of this study was to review the current status of calcium phosphate (CaP) scaffolds combined with bone morphogenetic proteins (BMPs) or mesenchymal stem cells (MSCs) in the field of bone tissue engineering (BTE).

DATE SOURCESData cited in this review were obtained primarily from PubMed and Medline in publications from 1979 to 2014, with highly regarded older publications also included. The terms BTE, CaP, BMPs, and MSC were used for the literature search.

STUDY SELECTIONReviews focused on relevant aspects and original articles reporting in vitro and/or in vivo results concerning the efficiency of CaP/BMPs or CaP/MSCs composites were retrieved, reviewed, analyzed, and summarized.

RESULTSAn ideal BTE product contains three elements: Scaffold, growth factors, and stem cells. CaP-based scaffolds are popular because of their outstanding biocompatibility, bioactivity, and osteoconductivity. However, they lack stiffness and osteoinductivity. To solve this problem, composite scaffolds of CaP with BMPs have been developed. New bone formation by CaP/BMP composites can reach levels similar to those of autografts. CaP scaffolds are compatible with MSCs and CaP/MSC composites exhibit excellent osteogenesis and stiffness. In addition, a CaP/MSC/BMP scaffold can repair bone defects more effectively than an autograft.

CONCLUSIONSNovel BTE products possess remarkable osteoconduction and osteoinduction capacities, and exhibit balanced degradation with osteogenesis. Further work should yield safe, viable, and efficient materials for the repair of bone lesions.

Animals ; Bone Morphogenetic Proteins ; chemistry ; Bone and Bones ; cytology ; Calcium Phosphates ; chemistry ; Humans ; Mesenchymal Stromal Cells ; cytology ; Tissue Engineering ; methods ; Tissue Scaffolds ; chemistry

This study was aimed to investigate the distribution of compact bone mesenchymal stem cells(MSC) marked with lentiviral plasmid pGC FU-RFP-LV in lung tissue and bone marrow of mouse. The MSC were infected by lentivirus with infection efficiency 78%, the infected MSC were injected into BALB/c mice via tail veins in concentration of 1×10(6) /mouse. The mice were randomly divided into 4 group according to 4 time points as 1, 2, 5 and 7 days. The lung tissue and bone marrow were taken and made of frozen sections and smears respectively in order to observed the distributions of MSC. The results indicated that the lentiviral infected MSC displayed phenotypes and biological characteristics which conformed to MSC by immunophenotyping analysis and induction differentiation detection. After the MSC were infected with optimal viral titer MOI = 50, the cell growth no significantly changed; the fluorescent microscopy revealed that the distributions of MSC in bone marrow on day 1, 2, 5 and 7 were 0.50 ± 0.20, 0.67 ± 0.23, 0.53 ± 0.14, 0.33 ± 0.16; those in lung tissue were 0.55 ± 0.15, 0.47 ± 0.13, 0.29 ± 0.13, 0.26 ± 0.08. It is concluded that the distribution of MSC in lung tissue reaches a peak on day 1, while distribution of MSC in bone marrow reaches a peak on day 2. The distribution of mouse MSC relates with RFP gene expression and implantation of MSC in lung tissue and bone marrow.
Animals ; Bone Marrow Cells ; cytology ; Bone and Bones ; cytology ; Female ; Lung ; cytology ; Male ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL

In the present research, the effects of sintered bone modified with surface mineralization/P24 peptide composite biomaterials on the adhesion, proliferation and osteodifferentiation of MC3T3-E1 cells were investigated. The experiments were divided into three groups due to biomaterials used: Group A (composite materials of sintered bone modified with surface mineralization and P24, a peptide of bone morphogenetic protein-2); Group B (sintered bone modified with surface mineralization) and Group C (sintered bone only). The three groups were observed by scanning electron microscopy (SEM) before the experiments, respectively. Then MC3T3-E1 cells were cultured on the surfaces of the three kinds of material, respectively. The cell adhesion rate was assessed by precipitation method. The proliferative ability of MC3T3-E1 cells were measured with MTT assay. And the ALP staining and measurement of alkaline phosphatase (ALP) activity were performed to assess the differentiation of cells into osteoblasts. The SEM results showed that the materials in the three groups retained the natural pore structure and the pore sizes were in the range between 200-850 μm. The adhesive ratio measurements and MTT assay suggested that adhesion and proliferation of MC3T3-E1 cells in Group A were much higher than those in Group B and Group C (P < 0.05). The ALP staining and ALP activity of MC3T3-E1 cells in Group A were significantly higher than those in Group B and Group C (P < 0.05). The sintered bone modified with surface mineralization/P24 composite material was confirmed to improve the adhesion rate and proliferation and osteodifferentiation of MC3T3-E1 cells, and maintained their morphology.
3T3 Cells ; Animals ; Biocompatible Materials ; Bone Morphogenetic Protein 2 ; Bone and Bones ; Cell Adhesion ; Cell Differentiation ; Mice ; Microscopy, Electron, Scanning ; Osteoblasts ; cytology ; Peptides ; Staining and Labeling

OBJECTIVEPolyethylene glycol/bone morphogenetic protein-2 (PEG/BMP-2) nanoparticles were transfected into Rabbit bone mesenchymal stem cells (rBMSCs) and the expression of BMP-2 was detected.

METHODSDissociated rBMSCs were primarily cultured in vitro and BMP-2 gene was transfected into rBMSCs by PEG/BMP-2 nanoparticals and lipofectamine, respectively. The efficiency of transfection was detected by flow cytometry and the expression of BMP-2 was detected by Western Blot and real time RT-PCR.

RESULTSPEG/BMP-2 nanoparticals were successfully synthesized and transfected into rBMSCs. Compared with the lipofectamine transfection group, PEG/BMP-2 transfection group had higher efficiency and higher BMP-2 expression.

CONCLUSIONPEG/BMP-2 nanoparticals transfected rBMSCs highly expressed BMP-2,which provided novel strategies for the treatment of bone defect.

Animals ; Bone Diseases ; genetics ; therapy ; Bone Morphogenetic Protein 2 ; chemistry ; genetics ; metabolism ; Bone and Bones ; cytology ; Gene Expression Regulation ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Nanoparticles ; chemistry ; Polyethylene Glycols ; chemistry ; RNA, Messenger ; genetics ; metabolism ; Rabbits ; Transfection ; methods