Objective To study biological rule of recombinant human bone morphogenetic protein 2 (rhBMP-2) in regulating the expression of vascular endothelial growth factor (VEGF) of adipose-derived stem cells (ADSCs) at different induced concentrations and time points at gene level and protein level. Methods ADSCs were separated from adult human adipose tissues and cultured until passage 3. After ADSCs were induced by rhBMP-2 in concentrations of 0, 50, 100, and 200 ng/ mL respectively for 24 hours, and by 100 ng/mL rhBMP-2 for 3, 6, 12, 18, 24, 36, and 48 hours (ADSCs were not induced at corresponding time point as controls) respectively, the VEGF mRNA and protein expressions were detected by RT-PCR and ELISA. Results The VEGF mRNA and protein expressions induced by rhBMP-2 were concentration-dependent; the expressions were highest in a concentration of 100 ng/mL. The VEGF mRNA expression in concentrations of 50, 100, and 200 ng/mL were significantly higher than that in a concentration of 0 ng/mL (P lt; 0.05); and the expression in concentration of 100 ng/ mL was significantly higher than that in concentrations of 50 and 200 ng/mL (P lt; 0.05). The VEGF protein expression in a concentration of 100 ng/mL was significantly higher than that in the other concentrations (P lt; 0.05). The VEGF mRNA and protein expressions induced by rhBMP-2 were time-dependent. The VEGF mRNA and protein expressions at 3 and 6 hours after induction were significantly lower than those of non-induced ADSCs (P lt; 0.05); the expressions were lower at 12 hours after induction, showing no significant difference when compared with those of non-induced ADSCs (P gt; 0.05); the expressions reached peak at 18 and 24 hours after induction, and were significantly higher than those of non-induced ADSCs (P lt; 0.05); the expressions decreased in induced and non-induced ADSCs at 36 and 48 hours, showing no significant difference between induced and non-induced ADSCs (P gt; 0.05). Conclusion rhBMP-2 adjusts VEGF expression of ADSCs in a concentration- and time-dependent manner. The optimum inductive concentration of rhBMP-2 is 100 ng/mL, induced to 18-24 hours is a key period when rhBMP-2 is used to promote tissue engineering bone vascularization.
Objective To explore a novel nanometer biomaterial which could induce the regeneration of tooth tissues intell igently, and to evaluate the feasibil ity of using this kind of biomaterial as the scaffold for tooth tissue engineering by investigating the role it plays in tooth tissue engineering. Methods The scaffold for tooth tissue engineering containing recombinant human bone morphogenetic protein 2 (rhBMP-2) was prepared by mixing nanoscale β tricalcium phosphate (β-TCP)/collagen particles. Forty-six 8-10 weeks old specific pathogen free Sprague Dawley (SD)rats, including 34 females and 12 males, weighing 250-300 g, were involved in this study. Tooth germs were removed under a stereomicroscope from the mandible of newborn SD rat, then digested and suspended. Scanning electronic microscope (SEM), adhesion rate of cells, and MTT assay were used to evaluate the effects of the scaffold on the tooth germ cells cultured in vitro. The tissue engineered tooth germ which was constructed by tooth germ cells and scaffold was transplanted under SD rat’s kidney capsule as the experimental group (n=12); the tooth germ cells (cell-control group, n=12) or scaffold without cells (material-control group, n=4) were transplanted separately as control groups Specimens were harvested to perform general and histological observations at 4 and 8 weeks after transplantation. Results β-TCP/collagen showed a loose and porous appearance with soft texture and excellent hydrophil icity. Tooth germ cells grew well and could attach to the scaffold tightly 3 days after coculture. The adhesion rates of tooth germ cells were 27.20% ± 2.37%, 44.52% ± 1.87%, and 73.81% ± 4.15% when cocultured with scaffold for 4, 8, and 12 hours, respectively. MTT assay showed that the cell prol iferation status of experimental group was similar to that of the control group, showing no significant difference (P gt; 0.05). Some white calcified specimens could be harvested at 4-8 weeks after transplantation. At 4 weeks after transplantation some typical structures of dental cusp and enamel-dentin l ike tissues could be seen in the experimental group. Enamel-dentin l ike tissues also formed in some specimens of cell-control group, but they arranged irregularly. At 8 weeks after transplantation the enamel-dentin l ike tissue of experimental group exhibited a mature appearance and organized structure in comparison with that at 4 weeks. And mature enamel or dentin l ike tissue also could be seen in cell-control group. In contrast, there was no enamel or dentin l ike tissue in material-control group at 4 or 8 weeks after transplantation. Conclusion rhBMP-2 decorated β-TCP/collagen scaffold has good biocompatibil ity and can be used as a novel nanometer biomaterial, so it is a good choice in scaffolds for tooth tissue engineering.
Objective To construct inducible lentiviral vector containing human bone morphogenetic protein 2 (hBMP-2) gene and to study its expression in human umbil ical cord blood mesenchymal stem cells (HUMSCs). Methods hBMP-2 gene was ampl ified by PCR from a plasmid and was cloned into pDown by BP reaction. pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-reverse transactivator (rtTA) were obtained with GATEWAY technology, and then were sequenced and analyzed by PCR. The recombinant vectors were transfected into 293FT cells respectively through l ipofectamine, and the lentiviral viruses were harvested from 293FT cells, then the titer was determined. Viruses were used to infect HUMSCs in tandem. In order to research the influence of induction time and concentration, one group of HUMSCs was induced by different doxycl ine concentrations (0, 10, 100 ng/mL, and 1, 10, 100 μg/mL) in the same induction time (48 hours), and the other by the same concentration (10 μg/mL) in different time points (12, 24, 48, and 72 hours). The expression of target gene hBMP-2 was indentified by ELISA method. After 2-week osteogenic induction of transfected HUMSCs, the mineral ization nodes were detected with Al izarin bordeaux staining method. Results Therecombinant inducible lentiviral vectors (pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-rtTA) were successfully constructed. The lentiviruses were also obtained and mediated by 293FT cells, and the virus titers were 3.5 × 108 TU/mL and 9.5 × 107 TU/mL respectively. HUMSCs could expression hBMP-2 by induction of doxycycl ine. The expression of hBMP-2 reached the peak at 10 μg/mL doxycl ine at 48 hours of induction. After 2-week osteogenic induction, a lot of mineral ization nodes were observed. Conclusion The recombinant inducible lentiviral vectors containing hBMP-2 gene can be successfully constructed, which provide an effective and simple method for the further study of stem cells and animal experiment in vivo.
Objective To construct the eukaryotic expression vector of human bone morphogenetic protein 7 (hBMP-7) gene so as to observe its expression in rabbit adipose-derived stem cells (ADSCs) and its effects on osteogenic phe notype. Methods Several healthy 3-month-old Japanese rabbits of clean grade were chosen, female or male and weighing 3-4 kg. ADSCs were isolated and cultured with collagenase digestion, then were detected and identified by CD44, CD49d, andCD106 immunofluorescence staining. The eukaryotic expression vector of hBMP-7 gene (pcDNA3.1-hBMP-7) was constructed, which was transfected into rabbit ADSCs (3rd passage) by Li pofectamineTM 2000 after identified, then the expression of hBMP-7 in transfected ADSCs was detected. The alkal ine phosphatase (ALP) level and the collagen type I expression were detected by intracellular ALP spectrophotometry and immunofluorescence, respectively to assess the effect of hBMP-7 gene on the osteoblastic differentiation of ADSCs. Results ADSCs mostly presented fusiform and polygon shape with positive expressions of CD44 and CD49d and negative expression of CD106. The eukaryotic expression vector of pcDNA3.1-hBMP-7 gene was successfully constructed and the expression of hBMP-7 was confirmed in ADSCs by immunohistochemical staining. The intracellular ALP quantitative detection showed that the activity of ALP was significantly higher in pcNDA3.1-hBMP-7 transfected group (experimental group) than in pcDNA3.1 transfected group (control group) at 7, 10, and 14 days after transfection (P lt; 0.05). The expression of collagen type I was higher in experimental group than in control group at 7 and 14 days after transfection (P lt; 0.05). Conclusion The eukaryotic expression vector of pcDNA3.1-hBMP-7 gene is successfully constructed, which can express in ADSCs. The expressions of collagen type I and ALP in experimental group are higher than those in control group, which lays a basis for the local gene therapy of skeletal regeneration.
Objective To study the biological activity of recombinant adeno-associated virus vector (rAAV) coexpressing human vascular endothel ial growth factor165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes in vitro so as to provide a new method for the therapeutics of osteonecrosis. Methods The 3rd passage rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with rAAV-hVEGF165-internal ribosome entry site (IRES)-hBMP-7(experimental group) and green fluorescent protein (GFP) labeled rAAV-IRES-GFP (control group). The expressions ofhVEGF165 and hBMP-7 were detected by ELISA assay at the 1st, 2nd, 3rd, 7th, 14th days and Western blot assay at the14th day after transfection. The expression consistencies of hVEGF165 and hBMP-7 were observed by immunofluorescence assay at the 14th day after transfection. The biological activity of hVEGF165 was assessed by angiopoiesis experiment of the 3rd passage human umbil ical vein endothel ial cells (HUVEC). The biological activity of hBMP-7 was assessed by mineral ization of BMSCs detected by ALP staining and al izarin red staining. Results With infecting time, the hVEGF165 and hBMP-7 expressions increased gradually in two groups, showing significant difference between two groups (P lt; 0.05). The expressions of hVEGF165 and hBMP-7 were positive in experimental group and negative in control group, respectively. Immunofluorescence assay showed positive expressions of hVEGF165 and hBMP-7 in the exprimental group and negative expression in the control group, the expression of hVEGF165 and hBMP-7 had good consistencies. hVEGF165 secreted from BMSCs enhanced HUVEC migration, prol iferation and tube formation in experimental group. There was significant difference in the number of blood vessel between two groups (P lt; 0.05). The ALP staining showed more bly stained granules in experimental group than in control group. There was significant difference in the number of the mineral ized nodules between two groups (P lt; 0.05). Conclusion The rAAV-hVEGF165-IRES-hBMP-7 has good biological activity in vitro.
Objective To study the time effect of the gene expression of recombinant adeno-associated virus (rAAV) vector co-expressing human vascular endothel ial growth factor 165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes so as to lay a theoretical foundation for gene therapy of osteonecrosis. Methods The best multipl icity of infection (MOI) of BMSCs transfected with rAAV was detected by fluorescent cell counting. The 3rd generation rabbit bone mesenchymal stem cells (BMSCs) were transfected with rAAV-hVEGF165-internal ribosome entry site (IRES)-hBMP-7 (experimental group) and green fluorescent protein (GFP) labeled rAAV-IRES-GFP (control group), respectively. The expression of GFP was observed by inverted fluorescent microscope. The expressions of hVEGF165 and hBMP-7 were assessed by RT-PCR assay and Western blot assay in vitro. The transfected cells in 2 groups were prepared into suspension with 5 × 106 cells/mL, and injected into the rabbit thigh muscles of experimental group 1 (n=9) and control group 1 (n=9), respectively. The muscle injected with rAAV-IRES-GFP was sl iced by frozen section method and the expression of GFP protein was observed by inverted fluorescent microscope. The expressions of hVEGF165 and hBMP-7 were assessed by Western blot assay and ELISA assay in vivo. Results The best MOI of BMSCs transfected with rAAV was 5 × 104 v.g/cell. In vitro, the expressions of GFP, hVEGF165, and hBMP-7 genes started at 1 day after transfection, the expressions obviously increased at 14 days after transfection, and the expression maintained the b level at 28 days after transfection. In vivo, the expressions of GFP, hVEGF165, and hBMP-7 genes could be detected at 2 weeks after injection, and b expressions were shown at 6 to 8 weeks after injection. The values of hVEGF165 and hBMP-7 were (248.67 ± 75.58) pg/mL and (4.80 ± 0.61) ng/mL respectively in experimental group 1, and were (32.28 ± 8.42) pg/mL and (0.64 ± 0.42) ng/mL respectively in control group 1; showing significant differences between 2 groups (P lt; 0.05). Conclusion The rAAV-hVEGF165-IRES-hBMP-7 has efficient gene expression ability.
Objective To study the effect of recombinant adeno-associated virus (rAAV) vector co-expressing human vascular endothel ial growth factor 165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes on bone regeneration and angiopoiesis in vivo so as to provide a theoretical basis for the gene therapy of avascular necrosis of thefemoral head (ANFH). Methods Twenty-four male adult New Zealand rabbits were made the ischemic hind l imb model and divided into 4 groups (n=6). The 3rd generation rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with the following 4 virus and were administered intramuscularly into the ischemic thigh muscle of 4 groups, respectively: rAAVhVEGF165- internal ribosome entry site (IRES)-hBMP-7 (group A), rAAV-hVEGF165-green fluorescent protein (GFP) (group B), rAAV-hBMP-7-GFP (group C), and rAAV-IRES-GFP (group D). At 8 weeks after injection, the blood flow of anterior tibial artery in the rabbit hind l imb was detected by ultrasonographic image. Immunohistochemical staining for CD34 was performed to identify the prol iferation of capillary. Another 24 male adult New Zealand rabbits were made the femur muscle pouch model and divided into 4 groups (n=6). The above 4 BMSCs transfected with rAAV were administered intramuscularly into the muscle pouch. At 8 weeks after injection, X-ray radiography was used to assess orthotopic bone formation, and von Kossa staining to show mineral ization. Results No symptoms of local or systemic toxicity were observed after rAAV injection. At 8 weeks after injection, the ratio of ischemic to normal blood flow and the number of capillaries in group A were the highest among 4 groups (P lt; 0.05). The ratio of ischemic to normal blood flow and the number of capillaries in group B were significantly higher than those in group C and group D (P lt; 0.05). However, there was no significant difference between group C and group D (P gt; 0.05). At 8 weeks after injection, orthotopic ossification and mineral ization were evidently detected in group A and group C, and group A was ber than group C. No obvious evidence of orthotopic ossification and mineral ization were observed in group B and group D. Conclusion rAAV-hVEGF165-IRES-hBMP-7 vector has the biological activities of inductive bone regeneration and angiopoiesis in vivo.
Objective To investigate the effect of tissue engineering bone compounded in vitro by nanohydroxyapatite/collagen/ polylactic acid (nHAC/PLA) and recombinant human bone morphogenetic protein 2 (rhBMP-2) in repairing rabbit critical calvarial defects. Methods Forty eight New Zealand rabbits, weighting 2.0-2.5 kg, were made the models of critical cranial defects(15 mm in diameter) and divided into 4 groups randomly. Defects were repaired with autoflank bone in the positive control group; with no implant in the blank control group; with nHAC/PLA in the negative control; and with active nHAC/PLA(AnHAC/PLA) in the experimental group(the average quality of each AnHAC/PLA absorbed rhBMP-2 was 1.431 mg). The reapir results were observed through X-ray,HE dyeing and Masson’s trichrism dyeing after 8 and 16 weeks. Results The difference of bone formation was observed by X-ray block degree of skull defect area at 8 and 16 weeks. In the 8 th week and 16 th week, the radiopacities on cranial defect were 67.21%±2.06% and 86.48%±1.73% in the positive control group； 5.84%±1.92% and 9.48%±2.72% in the blank control group; 19.13%±2.51% and 35.67%±3.28% in the negative control group; and 58.84%±2.55% and 8561%±3.36% in the experimental group. There were significant differences between the negative control and the positive control group, and between the experimental group and the positive control group at 8 weeks(Plt;0.05) . There were significant differences between the negative control and blank group, and between the experiment and the blank group at 8 and 16 weeks(P＜0.05). The histology observation showed that the width of bone trabecula at 16 weeks was more than that at 8 weeks and bone defectwas full of bone tissue in positive control group. The bone defect was full of fibrous tissue at 8 and 16 weeks, and there was no new bone in the blank group. The bone defect was full of remnant material and fibrous tissue in the negative control group. The implanted area was replaced by the new bone at 8 weeks and the new bone was lamellar at 16 weeks in the experimental group; the residual material was less in defect area and there were more osteoblasts surrounding. Conclusion The nHAC/PLA is a good scaffoldmaterial of rhBMP-2 and AnHAC/PLA has agood ability in repairing bone defect. So it is hopeful to be applied in the clnical repair of large bone defect.
Objective To investigate the differentiation of theadipose-derived adult stem cell (ADASC) induced by the recombinant adenovirus’s containing fibers derived from B-group serotype 35 (rAd5/F35)mediated human bone morphogenetic protein 7 (hBMP-7) gene and to explore a new cell sourcefor the bone tissue engineering. Methods The hBMP-7 gene wasamplified with the pcDNA1.1/AMP-hBMP-7 plasmid as a formwork. After the purification, the gene fragment was cloned into the pDC316 carrier for the recombination of the plasmid of pDC316-hBMP-7. The 293 cells were cotransfected by the skeleton plasmid of pBHG-fiber5/35 and the shuttle plasmid of pDC316-hBMP-7, and the recombinant plasmid of Ad5/F35-hBMP-7 was obtained; the recombinant plasmid of Ad5/F35enhancd green fluorescent protein(EGFP) was obtained by the similar method. The rat ADASCs were cultured and transfected by the Ad5/F35-hBMP-7plasmid and the Ad5/F35-EGFP plasmid, respectively; the remaining untransfected ADASC were used as the controls. The morphology and the growth pattern of the transfected cells were evaluated. The transcription and the expression of the transfected genes and the steogenic phenotypes such as calcium nodules and osteocalcin were evaluated by ELISA. Results The identification of PCR and enzyme cutting showed that the construction of the recombinant Ad5/F35-hBMP-7 plasmid could be confirmed. The transfection rate of the ADASC by the Ad5/F35-EGFP plasmid was determined to be greater than 90%. The hBMP-7 gene in thetransfected ADASC could express the corresponding protein, and the formation ofthe calcium nodules could be found in the induced group. The electron microscopy showed that there was a calcium element in the cytoplasm, the alkaline phosphatase result was positive, and the expression of osteocalcin was increased. Conclusion The rAd5/F35-hBMP-7 gene can promote the differentiation of the adiposederived adult stem cells to the osteoblasts in the bone tissue engineering.
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.