As the development of orthopaedics and tranmstology, treatment of bone defect is a major puzzle, and is so apparent that we need a more effective therapeusis urgently. The periosteum contains a lot of osteogenitor cells, which can self-duplicate and multi-directional differentiation. When the tissue is damaged, these cells will participate in tissue repair. In vitro cultured periosteal cells and periosteal cells injected into the small coloboma of bone have been succeeded. On the adequate biodegradable scaffolds, being regulated by some cell factors, periosteal cells can generate bone tissue that are at equal pace in tissue morphology and chemical composition of natural bone. The new bone can be juncture with vivo-bone and continue growing. It is of importance to seek a suitable scaffold materials and in vitro induction condition and to make bone formation in vitro from periosteal cells in bone tissue engineering research for clinical application.

BACKGROUND:The in vitro construction, maturation and differentiation of cellscaffold complexes into tissue-engineered bone is the necessary process of bone tissue engineering construction, but there are no uniform
methods and standards.
OBJECTIVE:To summarize the basic method and technology to build bone tissue engineering at present and to discuss the related development.
METHODS:A compute-based online search was conducted on the PubMed database and CNKI database for the articles related to the in vitro construction and culture of bone tissue engineering bone from January 1997 to
January 2013 with the key words of“bone tissue engineering, cellbiological scaffold, cellinoculation, seeding density, culture in vitro, bioreactor”in English and Chinese. Final y, 44 articles were included according to the inclusion and exclusion criteria.
RESULTS AND CONCLUSION:As the carrier of bone tissue engineering seed cells, the primary prerequisite of biological scaffold is sterile, because the sterile biological scaffold can be able to survive. Sterilization of biological scaffolds includes ultraviolet sterilization, 60Coγ-ray sterilization, soaking in ethanol with the volume fraction of 75%, autoclave method, and ethylene oxide sterilization. 60Coγ-ray sterilization is the common method in the biological scaffold sterilization. The inoculation density of seed cells is the key factors that influence the adhesion growth and proliferation of seed cells on the scaffolds. The adhesion between cells and scaffold materials wil be affected by the affinity of scaffolds, celladhesion and gravity, and other factors. The method for the inoculation of bone tissue engineering seed cells includes static inoculation and dynamic inoculation. Each construction method has its advantages and disadvantages. Overcomeing these disadvantages, forming a uniform construction method and ful y clinical application are the direction of future development.

BACKGROUND: Many factors are expressed during bone formation and osteonosus, which can induce cartilage to transformed into bone, or induce periosteal cells to transform into chondrocytes.OBJECTIVE: To summarize the osteogenic factors in recent years, and explore the important roles of these factors in bone formation and osteonosus.METHODS: A computer-based online search of Medline database (2008-01/2009-07) and CNKI (2008-01/2009-07) was performed using the key words of "osteogenic factors, bone disease, bone formation, Intercellular Signaling Peptides and Proteins,osteogenesis" in English and "osteogenic factors, bone formation, bone disease, cytokine, stimulating factor" in Chinese. All the articles were limited to the English and Chinese languages. The content about the bone factors, bone formation or osteonosus was included. Repeated or old studies were excluded.RESULTS AND CONCLUSION: According to the exclusion criteria, 158 literatures were collected and the roles of osteogenic factors in bone formation and osteonosus were analyzed. Bone formation and osteonosus are complex processes, influenced by many hormones and systemic or local growth factors. A variety of bone growth factors participate in process of bone healing and bone metabolism-related disease, which act on the target calls through local autocrine/paracrine mode and influence the distant target cells through blood circulation mode. A great number of factors are involved in the process of bone cell proliferation,differentiation, matrix synthesis and osteonosus metabolism, such as the fibroblast growth factor, bone morphogenetic protein,platelet-derived growth factor, transforming growth factor, interleukin-1, insulin-like growth factor, growth hormone etc.Osteogenic factors play an important role in the process of bone formation and bone-related diseases. More and more factors have been used in clinical therapy and bone tissue engineering research, and their effects have been clinically proven.

Objective To investigate the properties of cultured periosteal cells associated with bone morphogenetic protein(BMP) to find a new treatment for bone defects.Methods The periosteal cells were cultured,expanded and passage cultured in vitro to investigate the characteristics and functional changes of rabbit periosteal cells.Animal models of 1.0cm gaps with complete removal of the bilateral radius medium bone and periosteum were induced in 24 New Zealand rabbits,which were randomly divided into 4 groups to receive the defect repair with periosteal cells combined with BMP,periosteal cells,BMP and blank control respectively.X-ray and histological examination were made periodically after operation to evaluate the effectiveness of bone formation.Results Periosteal cell morphology can be divided into three stages: cell division phase,cell growth phase and cell functional phase.Cellular configuration had no significant difference with primary cells after anagenesis.The callus can be formed after the periosteal cells were transplanted into the bone defection.The osteogenesis ability of periosteal cells combined with BMP was better than that of the BMP,and the osteogenesis ability of the BMP was better than that of the periosteal cells.The mechanism of bone regeneration for periosteal cells was similar to intramembranous ossification,and that of BMP was similar to endochondral ossification.In the periosteal cells combined with BMP group,both intramembranous and endochondral ossification existed,and the former was obvious.Conclusion Both periosteal cells and BMP can improve the repair of bone defect in vivo.Cultured periosteal cells combined with BMP had substantial ability of synergistic osteogenesis,and it was an ideal treatment for bone defection.

BACKGROUND:The reports on bone morphogenetic protein-7 as a stimulating factor to induce osteogenic are relatively rare.
OBJECTIVE:To study the expression of alkaline phosphatase of periosteal cel s after induced by bone morphogenetic protein-7 in vitro.
METHODS:Periosteal cel s were obtained from adult tibial periosteum, and then the periosteal cel s were
cultured by routine method in vitro. The cel s were divided into experimental group and control group, and then cultured with bone morphogenetic protein-7 plus osteoblast culture adjuvants and simple osteoblast culture
adjuvants, respectively. The phase contrast microscope was used to observe the morphology and ultrastructure of periosteal cel s. Each group was observed at 7, 14 and 21 days, and three samples were observed at each time point. Alkaline phosphatase kit was used to detect the expression of osteoblast-specific markers alkaline phosphatase.
RESULTS AND CONCLUSION:After cultured for 7 days, the proliferation of periosteal cel s in the experimental group and the control group was increased obviously, and the expression of alkaline phosphatase was detected but less. The cel s were spindle in shape, while the expression of alkaline phosphatase in the experimental group was higher than that in the control group. After cultured for 14 days, the proliferation of periosteal cel s in the
experimental group and the control group was increased obviously, the cel morphology was changed from spindle-shaped to wide spindle-shaped, and the expression of alkaline phosphatase in the experimental group was
increased significantly when compared with the control group. After cultured for 21 days, the proliferation of periosteal cel s was detected in the experimental group and the control group, and the proliferation in the experimental group was more significant than that in the control group, the cel morphology was wide spindle-shaped, and the number of alkaline phosphatase in the experimental group was higher than that in the control group. Statistical analysis showed that the
positive rate of osteogenic markers alkaline phosphatase of bone morphogenetic protein-7 induced periosteal cel s in the experimental group was higher than that in the control group (P<0.01). It suggested that periosteal cel s had the
osteogenic and regeneration ability, the bone morphogenetic protein-7 could induce periosteal cel s, promote the expression of alkaline phosphatase, and could induce the periosteal cel s to transform into osteoblasts.

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Objective To discuss clinical effect of minimally invasive surgery, on herniated lumbar disc.Methods 50 patients of herniated lumbar disc were selected, and 25 patients were treated by micro endoscopy discectomy (MED).Results The treatment effect was better in treatment group, and the surgery time was shorter.1 case in treatment group had erector spinae hematoma, and 9 patients in control group had lumbago, while 2 cases had sciatica behavior.Conclusion The injury of MED was small,the surgery time was shorter,and the recovery was quicker,and it could be popularized in the clinic.

Objective To explore biological characteristics of chondrogenic differentiation of human periosteum-derived cells and the role of BMP4 in chondrogenic differentiation of these cells.Methods From October 2009 to September 2012,periosteum was obtained from tibia of patients undergoing leg amputation surgery,and isolated periosteum-derived cells by tissue culture method.Cells were cultured in DMEM/F12 containing 10％ fetal bovine serum,and morphology of cells were observed under inverted microscope.Periosteum-derived cells growth and the effect of BMP4 on cells growth examined by cell count using trypan blue,and cells growth curve was made.Experiment was divided into control group,chondrogenic differentiation group and BMP4 group,cells were expanded and differentiated in the presence or absence of BMP4 and complete medium.Then toluidine and immunohistochemical staining analyzed proteoglycan and collagenⅡ expression of these cells after 14 and 21 days.The expression of aggrecan,collagen Ⅱ and SOX9 mRNA of these cells using real-time PCR.Results (1) Periosteumderived cells adhered to growth in vitro,the shape of cell presented fibroblast-like morphology changing into polygonal after 1 week and round cell formation after 2 weeks chondrrogenic differemtiation.Growth curve showed that the passage 3 and 9 cells had similar reproductive activity.The passage 3 cells were positive for CD90 (21.07％) and CD105 (25.84％).(2)Toluidine bule staining and type Ⅱ collagen immunohistochemical staining showed BMP4 group (40.29 ± 4.29,56.74 ± 5.12) and chondrogenic differentiated group (19.27 ± 3.71,38.31 ± 4.25) ccould secrete proteoglycan and collagen Ⅱ,control group were negative (10.24 ± 1.21,15.28 ± 2.23),BMP4 group were significantly than chondrogenic differentiated group.(3) The expression of aggrecan,collagen Ⅱ and SOX9 mRNA of BMP4 group(25.76 ±0.57,6.48 ±0.48,2.91 ±0.18)were significantly higher than that of control group(2.37 ±0.24,1.12 ± 0.31,1.07 ± 0.22)and chondrogenic differentiated group(11.12 ± 0.38,2.24 ± 0.41,1.54 ± 0.35)using real-time PCR.Conclusion Periosteum-derived cells have strong proliferative,and have good potentials of differentiating into chondroblasts like mesenchymal stem cells.BMP4 can promote chondrogenic differentiation of periosteum-derived cells in vitro cultures.

BACKGROUND:Study confirms that bone morphogenetic protein can induce osteogenesis;however the ultrastructure of periosteal cells induced by bone morphogenetic protein-7 remains poorly reported.
OBJECTIVE:To study the bioactivity and ultrastructure of periosteal cells induced by bone morphogenetic protein-7 in vitro.
METHODS:The primary periosteal cells isolated from adult tibial bone were in vitro cultured, and then divided into experimental group and control group. In the experimental group, cells were cultured with bone morphogenetic protein-7 and culture adjuvant;while cells in the control group were only cultured with the adjuvant. Three samples in each group were tested at 5, 10, 15 days, respectively. The general structure of cultured cells was observed using von Kossa staining, and the ultrastructure was observed under transmission electron microscopy.
RESULTS AND CONCLUSION:The periosteal cells in the two groups grew wel in vitro, showing uniform morphology. Early cells were spindle-shaped, with strong three-dimensional sense and ful transparency;mitotic cells were short columnar or cubic shaped, there were a lot of rough endoplasmic reticulum and Golgi complex in osteoblasts under electron microscope. Later stage of cells developed from long fusiform into wide shuttle and irregular shape, there were a large number of matrix vesicles within the cells under the electron microscope. The membrane coating, alkaline phosphatase and calcium-binding protein in the cytoplasm, as wel as calcium crystals were found. The osteogenesis basement and lateral sides appeared projections, which were connected with adjacent bone cells. Induction of bone morphogenetic protein-7 in vitro promotes the osteoblasts proliferation, division and bone formation speed. The results suggest that bone morphogenetic protein-7 can significantly enhance the proliferation ability of osteoblasts in vitro.

Objective To study the role of bone morphogenetic protein-7 in the osteogenic differentiation of periosteal cellsin vitro. Methods Periosteal cells, obtained from adult tibial periosteum, were cultured by routine method in vitro, and divided into two groups. One group cultured with BMP7 and the supplements of 100 nmol dexametasone, 10 mmol b-glycerophosphate and 50 mg/mL L-ascorbic acid (BMP7 group), the other cultured with the supplements alone as the control (control group). Ultrastructure and morphological changes of periosteal cells were observed by contrast phase microscope and electron microscope. In order to test the expression of markers of osteoblastic differantiation in periosteal cells, involved mineralized node and alkaline phosphatase. Each group was tested at the time of 5 d, 10 d, 15 d, 20 d, respectively, using ALP kit stain and Von Kossa stain with 3 samples at each time. Results The periosteal cells cultured by routine method and induced into osteoblast differentiation with BMP7 were both growing well, in vitro. Microscope observations showed that the periosteal cells were spindle-shaped, well-stacked, transparent and three-dimensional in the early stage, and cube-shaped or puncheon shaped in the mitotic phase, gradually became wide shuttle and irregular shape with a lot secretion in telophase. The positive cells were visible by the ALP kit staining and Von Kossa staining of calcium nodules at 5 d, 10 d, 15 d and 20 d in both groups.A difference of positive rate at each time point was found between BMP7 group and control group at 5 d, 10 d, 15 d, 20 d, and the difference was statistically significant (P ＜ 0.01). Conclusion It displayed well regeneration and osteogenesis ability in the periosteal cell. BMP7 has definite osteo-inductive activity, which can obviously enhance the proliferation and ossifyng differentiation of periosteal cells.