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 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.
ObjectiveTo investigate whether miR-93-5p suppresses osteogenic differentiation of mouse mesenchymal stem cells (C3H10T1/2) by targeting Smad5, a predicted target in silicon. MethodsSmad5 3'-UTRluciferase vector (pmiR-RB-REPORTTM) was constructed and dual-luciferase reporter gene assay was employed to examine the effect of miR-93-5p on Smad5 3'-UTR-luciferase activity to identify whether Smad5 was the target gene of miR-93-5p. miR-93-5p mimics (group M), miR-93-5p inhibitor (group In), miR-93-5p mimics negative control (group MC), and miR-93-5p inhibitor negative control (group InC) were transfected into the C3H10T1/2 cells, respectively, and followed by induction of osteogenic differentiation. After 48 hours, the real-time fluorescent quantitative PCR (qRTPCR) and Western blot assays were performed to detect the relative expressions of Smad5 mRNA and protein. At 14 days, to realize the regulation role of miR-93-5p in osteogenic differentiation, the extracellular calcium deposition during the osteogenesis of C3H10T1/2 cells was tested by Alizarin red staining. ResultsDual-luciferase reporter gene assay showed that miR-93-5p could combine with Smad5 mRNA 3'-UTR specificity, and inhibited its luciferase activity (P<0.05). After 48 hours, no significant difference was shown in the relative expression of Smad5 mRNA between group M and group MC as well as between group In and group InC by qRT-PCR assay (P>0.05); however, the results of Western blot assay showed that the relative expression of Smad5 protein was significantly decreased in group M and increased in group In when compared with groups MC and InC (P<0.05). At 14 days after osteogenic induction, Alizarin red staining showed that the extracellular calcium deposition of group M was obviously less than that of group MC, and it was obviously more in group In than in group InC. ConclusionSmad5 may be the target gene of miR-93-5p. And miR-93-5p can suppress osteogenic differentiation of C3H10T1/2 cells by directly targeting Smad5.
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.
Objective As one of the adult stem cells, adi pose-derived stem cells (ADSCs) have become an important seed cell source for tissue engineering recently. But whether the thawed cryopreserved ADSCs could be used to tissue engineered bone remains unknown. To investigate the effect of cryopreservation on the growth and osteogenesis of ADSCs invitro. Methods The ADSCs were isolated from the adipose aspirates by collagenase digestion method. For the experimental group, the 2nd generation cells were stored with a simple method of cryopreservation by slow cool ing with dimethyl sulphoxide as a cryoprotectant and rapid thawing. After cryopreserved in l iquid nitrogen for 4 weeks, ADSCs were recovered and cultured in osteogenic media, with non-cryopreserved ADSCs as the control group. The osteogenic differentiation was evaluated by alkal ine phosphatase (ALP) staining and Al izarin red O staining at 2 and 3 weeks respectively. The cell growth and osteogenesis of ADSCs were further determined using DNA assay and the ALP activity and calcium content were measured. Results The survival percentage of the cryopreserved cells was 90.44% ± 2.62%. The cell numbers and ALP activity increased with osteogenic induction time, and reach plateaus at 7 days and 11 days, respectively. The ALP staining and Al izarin red O staining results were both positive at 2 weeks and 3 weeks after osteogenic induction, respectively. And no significant difference in the cells number, ALP activity, and calcium content were found between experimental group and control group (P gt; 0.05). Conclusion Cryopreservation does not affect the growth and osteogenesis of ADSCs, and the cryopreserved ADSCs can be used as cell source for tissue engineered bone.
Objective To evaluate the adhesion, prol iferation and osteogenic differentiation of rabbit BMSCs after cultured on freeze-dried demineral ized bone matrix (FDBM) modified with type II cadherin ectodomain (Cad- II). Methods BMSCs isolated from 10 Japanese white rabbits (male and female, 4-week-old, 0.61-0.88 kg) were cultured. The second generation of BMSCs (cell density 1 × 106 /mL) were seeded onto the Cad-II modified allogenic FDBM (experimental group) and only FDBM (control group) respectively, and then cocultured in vitro. The densities of seeded cells, the adhesion rate and their ALP activity were measured. The complex was observed through inverted phase contrast microscope and scanning electron microscope to evaluate the interaction between cells and FDBM. Another group of second generation of BMSCs (cell density 5 × 105 /mL) were seeded onto the Cad-II modified FDBM (experimental group) and only FDBM (control group) respectively, and then cocultured in vitro too. The ALP activity and osteocalcin immunohistochemical was measured. Results There was no significant difference in cell prol iferation between experimental group and control group. The adhesion rate of cells in the experimental group was 87.41% ± 5.19%, higher than that in the the control group 35.56% ± 1.75% (P lt; 0.01); the densities of seeded cells reached 5.0 × 105, showing significant difference compared with the control group (2.6 × 104, P lt; 0.05). Inverted phase contrast microscope showed that in the experimental group, more cultured BMSCs pasted in the hole and edge of the scaffold than that in the control group. HE staining showed the densities of seeded cells in the experimental group was higher than that in the control group. Scanning electron microscope showed that in the experimental group, a lot of cultured BMSCs adhered, spreaded in the scaffold, in the control group only a few BMSCs unevenly distributed in the scaffold. After 7 days of culture, the cultured BMSCs on modified FDBM expressed higher ALP activity; after 14 days of culture, the ALP activity (29.33 ± 1.53) was higher than that cultured on unmodified FDBM (18.31 ± 1.32), the positive rates of osteocucl in were 83% ± 7% in the experimental group and 56% ± 7% in the control group, showing significant difference (P lt; 0.01). Conclusion Cad-II enhanced cell adhesion to FDBM and promoted BMSCs differentiate to osteoblast, but no obvious effects were observed in cell prol iferation.
Abstract：Pulmonary metastasectomy is an important curative option for patients with osteogenic and softtissue sarcoma spread to the lungs. Complete surgical removal of pulmonary metastases can improve survival and is recommended under certain criteria. Specific issues that require consideration when planning pulmonary metastasectomy include: preoperative assessment of the operation index and contraindications, choice of surgical strategies, pulmonary parenchymal preservation, and the role of lymphadenectomy. With the development of iconography and chemotherapy, the emergence of targeted drugs, and the innovation of radiotherapy, the concept of the diagnosis and treatment for pulmonary metastases from osteogenic and softtissue sarcoma is also undergoing great changes.
OBJECTIVE: To prevent the senescence of ’seed cells’ for tissue engineering, the life span of human fibroblasts is extended by reconstitution of telomerase activity, and the osteogenic potential of these fibroblasts are tested. METHODS: The pGRN145 plasmids encoding human telomerase reverse transcriptase (hTERT) were introduced into the normal human primary fibroblasts by electroporation. Telomerase activity was analyzed by TRAP-PCR assay. The beta-galactosidase stain was used to indicate the signs of cell senescence. The hTERT positive fibroblasts were then induced to form bone nodules. The bone nodules were stained by tetracycline and Alizarin Red S. RESULTS: Stable telomerase activity could be detected in the transfected fibroblasts and no signs of cell senescence were found in the fibroblasts cultured for more than 50 doublings. The hTERT positive fibroblasts could form bone nodules when they were cultured in vitro induced by bone morphogentic protein 2 and tumor necrosis factor-alpha. CONCLUSION: The fibroblasts with reconstituted telomerase activity reserve their osteogenic potential.
ObjectiveTo investigate the effect of cyclic stretch stress on the osteogenic differentiation of human cartilage endplate-derived stem cells (CESCs). MethodsCESCs were isolated from the endplate cartilage tissues by the method of agarose suspension culture system. The endplate cartilage tissue was harvested for immunohistochemical staining. Flexercell-4000TM Tension Plus system was used to apply cyclic stretch on CESCs at a frequency of 1 Hz and at a stretch rate of 10% for 1, 6, 12, or 24 hours (experimental group). No stretch stress was performed on CESCs in the same culture condition (control group). After mechanical loading, the protein expression of bone morphogenetic protein 2 (BMP-2) was measured by Western blot, and gene expressions of runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and SOX9 were detected by real-time fluorescent quantitative PCR. ResultsImmunohistochemical staining showed BMP-2 protein expression in chondrocytes. The continuous cyclic stretch stress of 10% can increase the expression of BMP-2 protein in CESCs. Significant differences were observed in the expressions of BMP-2 protein (P<0.05) between 2 groups at the other time points except at 1 hour (P>0.05), in a time-dependent manner. The real-time fluorescent quantitative PCR indicated that the gene expressions of Runx2 and ALP showed an increasing tendency with time in the experimental group when compared with the control group, but there was down-regulated expression of SOX9. Significant difference was found in mRNA expressions of Runx2 and ALP at 12 and 24 hours and in mRNA expressions of SOX9 at 6, 12, and 24 hours between 2 groups (P<0.05), in a time-dependent manner. ConclusionCyclic stretch stress may induce osteogenic differentiation of CESCs by regulating the expressions of some genes related osteogenesis in CESCs.
ObjectiveTo investigate the role of the forkhead/Fox transcription factor 2 (Foxc2) over-expression in regulating osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by Wnt-β-catenin signaling pathways in vitro so as to provide the experimental basis for repairing osteonecrosis of the femoral head. MethodsThe recombinant lentivirus carrying green fluorescent protein (group A) or Foxc2 (group B) were used to transfect the fifth generation rabbit BMSCs, and untransfected BMSCs served as a control (group C). The cell viability was measured with water soluble tetrazolium-1 (WST-1) regent at 72 hours after transfection. After 2 weeks of transfection, the expression of β-catenin in BMSCs was detected by real time fluorescence quantitative PCR, Western blot, and immunofluorescence staining. Meanwhile, the β-catenin inhibitors XAV-939 (0, 0.1, and 1.0 μmol/L) was added in group B; at 2 weeks after osteogenic and adipogenic induction, the gene and protein expressions of collagen type I (COL I), osteocalcin (OCN), and peroxisome proliferator activated receptor gamma 2 (PPARγ-2) were detected by real time PCR and Western blot. ResultsWST-1 results showed that the cell viability of group B (130.85%±0.15%) was significantly higher than that of group A (100.45%±0.35%) (t=7.500, P=0.004) at 72 hours after transfection. At 2 weeks after transfection, the gene and protein expressions of β-catenin in group B were significantly higher than those in group A (P<0.01). After XAV-939 was added in group B, the mRNA and protein expressions of OCN and COL I gradually decreased; the mRNA and protein expressions of PPARγ-2 significantly increased (P<0.05), showing a dose-dependent manner. ConclusionThe over-expression of Foxc2 gene in BMSCs may promote osteogenic differentiation by Wnt-β-catenin signaling pathway.