Objective To explore the osteogenic potential of cervical intervertebral disc fibroblasts in vitro, to investigate the regulatory factors of recombinant human bone morphogenetic protein 2(rhBMP-2) and tumor necrosis factor α(TNF-α) on osteogenic phenotype of fibroblasts and to discuss the condition that facilitates osteogenesis of fibroblasts. Methods Theannulus fibroblasts cell lines of experiment goats were established in vitro and the biologicspecificity was found. According to different medias, 4 groups were included in this experiment: control group, TNF-α group ( 50 U/ml TNF-α), rhBMP-2 group (0.1 μg/ml rhBMP-2) and TNF-α+rhBMP-2 group (50 U/ml TNF-α+0.1 μg/ml rhBMP-2). Thefibroblasts were incubated in the media for about 3 weeks,and then the markers for osteogenic features were investigated by biochemistry, histochemistry observations. Results rhBMP-2 and TNF-α had no effect on the proliferation of fibroblasts from the experiment goats. rhBMP-2 or TNF-α could stimulate fibroblasts to secrete alkaline phosphatase and collagen type Ⅰ. The combined use of rhBMP-2 and TNF-α or the single use of rhBMP-2 could make fibroblasts to secrete osteocalin and the morphological changes of the fibroblasts were very obvious. Histochemical study of the nodules with specific new bone labeler(Alizarin red S) revealed positive reaction, denoting that the nodules produced by the fibroblasts werebone tissues. There was statistically significant difference(Plt;0.05) inALP activity between 3 experimental groups and control group and in secretion of osteocalcin between rhBMP-2 group, TNF-α+rhBMP-2 group and control group. Conclusion The results point out clearly that rhBMP-2 can induce theosteogenic potential of annulus fibroblasts in vitro.
The osteogenc potential of bone marrow has been proved by experiment. To investigate more in details, bone marrow was obtained from the trochanteric region of femur of NewZealand rabbit in 4 to 8 weeks old. After being cultured in vitro for one week, the hematopoietic component of the bone marrow had disappeared, thus the stromal cells were obtained. Then the stromal cells were subcultured in cultural fluid containing dexamethasone (10-8 mol/L) and natrium glycerophosphate (10mmol/L). Under the phasecontrast microscope, it was found that being cultured for 15 days. The stromal cells were lined up in one layer and late the secretion activity was increased and gradually transformed into multilayer structure and was congregated into diffused opaque clusters in twenty days. During culture, the cells were examined by tetracycline fluorescence label, histochemistry stains, transmission electron microscopy, scanning electron microscopy and energy dispersive X-ray microanalysis. The results showed that the morphological and biological characteristics of the cultured stromal cells derived from the bone marrow were similiar to those of osteoblasts and could synthesized mineralized new bone tissue in vitro.
Objective To investigate early clinical manifestations of osteogenic sarcoma to help establishment of an early diagnosis of the disease.Methods A total of 92 patients with osteogenic sarcoma in the extremities were admitted to our hospital from April 1984 to October 2002. Of the 92 patients, 71 (42 males and 29 females; averaged age 17.4 years, range 666 years; illness course 1-28 weeks) had a complete record of their medical history and examination. From their first medical visits, we obtained their clinical symptoms, physical sings, diagnoses, and duration of the delayed diagnoses. The patients were pathologically confirmed as having osteogenic sarcoma in the extremities, with the lesions located in the distal femur in 38 patients, proximal tibia in 22, proximal femur in 3, proximal fibula in 3, proximal humerus in 2, distal tibia in 2, and distalradius in 1. Results Of the 71 patients, 70 had a local pain and/or a palpable mass, 37 had a persistent pain with no difference between day and night, 23 had an intermittent pain, and 11 had a nocturnal pain. Of the 71 patients, 42 had an initial pain related to trauma, and 3 of the 42 patients had a pathologic fracture. The patients with the local mass had a delayed diagnosis of osteogenic sarcoma with a delayed duration of 1-14 weeks, averaged 4 weeks; however, the patients without the local mass had a delayed diagnosis of this disease, with a delayed duration of 3-30 weeks averaged 14 weeks. In the patients undergoing an X-ray examination at the first medical visit, the duration of the delayed diagnoses was 1-20 weeks, averaged 8 weeks, but in the patients without an X-ray examination at first, the duration was 4-30 weeks, averaged 16 weeks. Conclusion Intermittent and persistent pains and local masses are the most characteristic clinical manifestations in the early stage of osteogenic sarcoma. A history of trauma often helps to make a diagnosis of the disease. Carefulclinical examination and observation should be given to adolescent patients whohave a recurrent pain around the joint.
Objective To investigate the effects of the recombinanthuman bone morphogenetic protein 2 (rhBMP-2) and/or the osteogenic agents on proliferation and expression of the osteoblast phenotype differentiation of the SD rat mesenchymal stem cells（MSCs）. Methods The rat MSCs were cultured in vitro and were randomly divided into the experimental groups(Groups A-I) and the control group. In the experimental group, MSCs were induced by rhBMP2 in different doses (10, 50, 100 and 200 μg/L) in Groups BE, the osteogenic agent alone (Group A) and by the combined use of rhBMP-2 ［in different doses (10,50, 100 and 200 μg/L)］ and the osteogenic agent in Groups F-I. The MTT colorimetric assay was used to evaluate the proliferation, and the activities of alkaline phosphatase (ALP) and osteocalcin (OC) were observed at 3, 6, 9, 12 days, respectively. Results The inverted phase contrast microscopy showed that MSCs by primary culture for 12 hours were adhibited, with a fusiform shape at 48 hours. At 4 days they were polygonal or atractoid, and were spread gyrately or radiately at 6 days. At 10 days, they were spread at the bottom of the bottle.The statistical analysis showed that the expression of the osteoblast phenotype differentiation of MSCs could be induced in the experimental groups. The proliferation of MSCs could be enhanced in a dosedependent manner in GroupsB-E. The expression of the osteoblast phenotype differentiation, which was tested by the activities of ALP and OC, was significantly higher in Groups F-I than in Groups A-E. Conclusion The combined use of rhBMP-2 and the osteogenic agents can enhance the MSC proliferation and induce an expressionand maintenance of the osteoblast phenotype differentiation of the rat MSCs.
ObjectiveTo review the research progress of induced osteogenesis of bone marrow mesenchymal stem cells (BMSCs) transfected by double-gene.
MethodsThe recent literature concerning the comparative research of induced osteogenesis of BMSCs transfected by double-gene was extensively reviewed. The characteristics of BMSCs, the advantage and effect of synergistic inductive osteogenesis, the application prospect and problems of BMSCs transfected by double-gene were summarized.
ResultsThe effect of induced osteogenesis concerning BMSCs transfected by double-gene is far superior to single gene transfection and the activity of osteoblast is also significantly increased. The research used in bone tissue engineering experiment also obtain good effect.
ConclusionInduced osteogenesis of BMSCs transfected by double-gene is able to make up for the lack of a single gene transfection and has great development prospects in the orthopaedic field.
Objective Calcium phosphate bioceramics has a broad appl ication prospect because of good biocompatibil ity, but porous scaffolds with complex shape can not be prepared by the traditional methods. To fabricate porous calcium phosphate ceramics by rapid prototyping and to investigate the in vitro osteogenic activities. Methods The porous calcium phosphate ceramics was fabricated by rapid prototyping. The bone marrow mesenchymal stem cells (BMSCs)were isolated from bone marrow of Beagle canine, and the 3rd passage BMSCs were seeded onto the porous ceramics. The cell/ceramics composite cultured in osteogenic medium were taken as the experimental group (group A) and the cell/ceramics composite cultured in growth medium were taken as the control group (group B). Meanwhile, the cells seeded on the culture plate were cultured in osteogenic medium or growth medium respectively as positive control (group C) or negative control (group D). After 1, 3, and 7 days of culture, the cell prol iferation and osteogenic differentiation on the porous ceramics were evaluated by DNA quantitative analysis, histochemical staining and alkal ine phosphatase (ALP) activity. After DiO fluorescent dye, the cell adhesion, growth, and prol iferation on the porous ceramics were also observed by confocal laser scanning microscope (CLSM). Results DNA quantitative analysis results showed that the number of BMSCs in all groups increased continuously with time. Plateau phase was not obvious in groups A and B, but it was clearly observed in groups C and D. The CLSM observation indicated that the activity of BMSCs was good and the cells spread extensively, showing good adhesion and prol iferation on the porous calcium phosphate ceramics prepared by rapid prototyping. ALP quantitative analysis results showed that the stain of cells on the ceramics became deeper and deeper with time in groups A and B, the staining degree in group A were ber than that in group B. There was no significant difference in the change of the ALP activity among 4 groups at the first 3 days (P gt; 0.05); the ALP activity increased obviously in 4 groups at 7 days, group A was significantly higher than other groups (P lt; 0.05) and groups C, D were significantly higher than group D (P lt; 0.05). Conclusion The porous calcium phosphate ceramics has good cytocompatibil ity and the designed pores are favorable for cell ingrowth. The porous ceramicsfabricated by rapid prototyping has prominent osteogenic differentiation activity and can be used as a choice of scaffolds for bone tissue engineering.
ObjectiveTo investigate the effect of KLD-12 polypeptide complexed with recombinant human bone morphogenetic protein 2 (rhBMP-2) on osteogenic activity of rabbit bone marrow mesechymal stem cells (BMSCs).
MethodsBone marrow was harvested from 3-month-old New Zealand white rabbit, and density gradient method was used to isolate and culture BMSCs. The third generation BMSCs were used for three-dimensional culture of KLD-12 polypetide/rhBMP-2 in vitro (experimental group) and KLD-12 polypeptide (control group). The morphology of the cells in the gel was observed by inverted phase contrast microscope at 7 days; alkaline phosphatase (ALP) and osteocalcin protein content were dectected at 3, 7, 10, 14, and 21 days; collagen type I immunofluorescence staining was done and real-time fluorescent quantitative PCR was performed to detect the relative expression of collagen type I and osteocalcin gene at 14 days.
ResultsUnder the inverted phase contrast microscope, the BMSCs in the gel of the experimental group and the control group showed circular growth, and the distribution was uniform at 7 days. There was no significant difference in the expressions of ALP and osteocalcin protein content between 2 groups at 3 and 7 days (P > 0.05); the above indexes in experimental group were significantly higher than those in the control group at 10-21 days (P < 0.05). Laser scanning confocal microscope observation showed that immunofluorescence staining for collagen type I was positive in the experimental group, and the expression was higher than that in the control group at 14 days. Real-time fluorescence quantitative PCR detection showed that the collagen type I and osteocalcin gene expressions were significantly higher than those in the control group (t=15.902, P=0.000; t=12.998, P=0.000).
ConclusionBMSCs can normally grow and proliferate in the KLD-12 polypeptide, and KLD-12 polypeptide/rhBMP-2 has good biological activity to induce BMSCs differentiation into osteoblasts.
Objective To study the culture and purification of the fetal mouse liver mesenchymal stem cells(MSCs) in vitro and to investigate their differentiation potential and the composite ability with true bone ceramic(TBC). Methods The single cell suspension of MSCs was primarily cultured and passaged, which was prepared from the fetal mouse liver; the flow cytometry was applied to detectCD29, CD34, CD44 and CD45. The osteogenic differentiation was induced in chemical inducing system; the osteogenic induction potency was tested. The purified fetal mouse liver MSCs were compounded with TBC covered with collagen type Ⅰ in vitro and the cell attachment and proliferation to the TBC were observed. Results The primary MSCs of fetal mouse liver were easy to culture in vitro. They proliferated well and were easy to subcultured. The proliferation ability of primary and passaged MSCs was similar. Flow cytometric analysis showed the positive results for CD29, CD44 and the negative results for CD34, CD45. After 7 days of induction, the MSCs expressed collagen type I and alkaline phosphatase(ALP) highly. After 14 days of induction, the fixed quantity of ALP increased significantly. After 28 days of induction, calcium accumulation was observed by Von Kossa’s staining. Many liver MSCs attached to the surface of TBC. Conclusion The MSCs of the fetalmouse liver can be obtained, subcultured and purified easily. After culturing in chemical inducing system, the MSCs of fetal mouse liver can be successfully induced to osteoblast-like cells, attach to the surface of TBC and proliferate well.
Objective To review the progress and clinical application of malleable bone paste/putty. MethodsRecent literature about malleable bone paste/putty was reviewed and analyzed. ResultsThe preparation and clinical application of malleable bone paste/putty have become increasingly mature. Many kinds of malleable bone paste/putty have been applied extensively and the good clinical results have been achieved in the treatment of the irregular bone defects. The materials and methods for preparing malleable bone paste/putty are different. Then they have different bone repair abilities. ConclusionMalleable bone paste/putty provides effective method to treat irregular bone defects. But the malleable bone paste/putty still has some shortage, so further researches should be carried out.
Objective
To review the osteogenic mechanism and osteogenic effects of bone morphogenetic protein 6 (BMP-6) so as to provide the basis for further research of BMP-6.
Methods
The related articles about the osteogenic mechanism and the osteogenic effects of BMP-6 in experimental animals were extensively summarized.
Results
BMP-6 from bone matrix can transduct the osteogenic signal to bone marrow mesenchymal stem cells (BMSCs) by means of Smad protein signal transduction pathway. And the BMSCs which received the signals will differentiate into osteoblasts and chondroblasts. Therefore, BMP-6 plays an important role in the development and maturation of bone and cartilage. In addition, BMP-6 has a close relation with bone diseases, such as fracture, osteoporosis, and bone tumor.
Conclusion
The deep research of BMP-6 is expected to provide a new therapeutic approach for treating bone diseases of nonunion, osteoarthritis, and osteoporosis.
