The healing of the tendon-bone interface is a complex dynamic process involving the interaction of multiple cellular and molecular signaling pathways. Bone mesenchymal stem cells (BMSCs) have the potential to differentiate into various types of cells, including osteoblasts, chondrocytes and adipocytes, etc., and have the potential to regenerate damaged tissues. They are potential seed cells for promoting tendon-bone healing. How to precisely regulate the proliferation and differentiation of BMSCs to accelerate the process of tendon-bone healing is a current research hotspot. Monomers of traditional Chinese medicine can promote tendon-bone healing by regulating signaling pathways such as Wnt/β-catenin and BMP/Smad to induce osteogenic and chondrogenic differentiation of BMSCs. This article reviews from several aspects such as the regulatory role of related signaling pathways on tendine-bone healing, traditional Chinese medicine monomers and their mechanism of regulating BMSCs to promote tendine-bone healing in order to providing new ideas for promoting tendine-bone healing.
Mesenchymal Stem Cells/cytology* ; Humans ; Animals ; Bone Marrow Cells/cytology* ; Bone and Bones/drug effects* ; Wound Healing/drug effects* ; Medicine, Chinese Traditional ; Tendons/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Signal Transduction/drug effects* ; Cell Differentiation/drug effects*

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*

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

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

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

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

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

OBJECTIVE: To prepare nHA/gelatin porous scaffold and to evaluate its physical and chemical properties and biocompatibility. METHODS: We used nano-powders of HA and gelatin to prepare 3D porous composite scaffold by freeze-drying technique, and used scanning electron microscope, fourier transform infrared spectroscopy and universal testing machine to characterize the composite material. Osteoblasts were primarily cultured, and the third-passage osteoblasts were co-cultured with the composite material. The cell adhesion and morphology were examined under scanning electron microscope. The cell viability analysis was performed by MTT assay, and the alkaline phosphatase activity was measured with alkaline phosphatase kit. RESULTS: Scanning electron microscope showed that the scaffold possessed a 3-dimensional interconnected homogenous porous structure with pore sizes ranging from 150 to 400 μm. Fourier transform infrared spectroscopy showed that the composite material had a strong chemical bond between the inorganic phase and organic phase. The scaffold presented the compressive strength of (3.28 ± 0.51) MPa and porosities of (80.6 ± 4.1)%. Composite materials showed features of had good biocompatibility. Mouse osteoblasts were well adhered and spread on the materials. The grade of the cell toxicity ranged from I to II. On the 5th and 7th day the proliferative rate of osteoblasts on scaffolds in the composite materials was significantly higher than that in the control group. The activity of alkaline phosphatase was obviously higher than that in the control group on Day 1 and 3. CONCLUSION Nano-hydroxyapatite and gelatin in certain proportions and under certain conditions can be prepared into a composite biomimetic porous scaffolds with high porosity and three-dimensional structure using freeze-drying method. The scaffold shows good biocompatibility with mouse osteoblasts and may be a novel scaffolds for bone tissue engineering.
Animals ; Biocompatible Materials ; Biomimetic Materials ; Bone and Bones ; Cell Adhesion ; Coculture Techniques ; Durapatite ; Gelatin ; Mice ; Nanostructures ; Osteoblasts ; cytology ; Porosity ; Spectroscopy, Fourier Transform Infrared ; Tissue Engineering ; Tissue Scaffolds

AIM: Osteoblasts are key functional cells in the process of bone metabolic balance. Phytoestrogens have an important influence on the proliferation and differentiation of osteoblasts. Puerarin, a plant estrogen, has a wide range concentration in vitro on the function of osteoblasts. The current study investigates the effect of the phytoestrogen puerarin on the proliferation, differentiation, and mineralization of osteoblasts in vitro. METHODS: The calvaria bone of eight-ten Wistar rats which were born within 24 h were obtained in aseptic condition. After enzyme digestion, isolation, purified osteoblasts of rats were cultured for further study. The cells of the first to third generation were divided into a control group and a puerarin-treated group with 10(-3)-10(-10) mol·L(-1) puerarin. The cells were exposed to the medium containing a low level of carbohydrates, 10% (V/V) FBS for 24 h. After 1 to 4 days of culture, the OD values on the proliferation of osteoblasts in each group were determined by microplate reader. The cells were cultured in the medium containing 50 μg·mL(-1) vitamin C, 10(-2) mol·L(-1) sodium glycerophosphate, 10% FBS and the medium was changed every 3 to 4 days. After 2 to 8 days of culture, expression of alkaline phosphatase were tested and compared by microplate reader. The mineral nodes of osteoblasts were dyed using alizarin red or improved Von Kossa way after four weeks. RESULTS: Compared with those in the 10(-5)-10(-9) mol·L(-1) puerarin, the proliferation of osteoblasts, the expression of alkaline phosphatase, and the number of mineral nodes of osteoblasts were significantly decreased in the control group. The increase was the fastest in the third day, while on the fourth day it was decreased, and arrived at statistical significance compared with the alkaline phosphatase activities and control group. The 10(-6) mol·L(-1) group was the most distinct, and formed the most mineralized nodule. Compared with the 10(-3) mol·L(-1) puerarin group, those changes were markedly increased in the control group. CONCLUSIONS Puerarin has proliferation, differentiation, and mineralization effects on osteoblasts in a dose-dependent manner, and has a double-way effect on the osteoblasts in vitro. A low-dose showed positive effects on the development of osteoblasts, and high-dose puerarin could inhibit the formation of bone.
Alkaline Phosphatase ; metabolism ; Animals ; Bone Density ; Bone and Bones ; cytology ; drug effects ; Calcification, Physiologic ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Female ; In Vitro Techniques ; Isoflavones ; pharmacology ; Osteoblasts ; drug effects ; Phytoestrogens ; pharmacology ; Rats, Wistar