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 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 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 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 preventing the loss of correction and vertebral defects after thoracolumbar burst fractures treated with recombinant human bone morphogenetic protein 2 (rhBMP-2) and allogeneic bone grafting in injured vertebra uniting short-segment pedicle instrumentation. Methods A prospective randomized controlled study was performed in 48 patients with thoracolumbar fracture who were assigned into 2 groups between June 2013 and June 2015. Control group (n=24) received treatment with short-segment pedicle screw instrumentation with allogeneic bone implanting in injured vertebra; intervention group (n=24) received treatment with short-segment pedicle screw instrumentation combining with rhBMP-2 and allogeneic bone grafting in injured vertebra. There was no significant difference in gender, age, injury cause, affected segment, vertebral compression degree, the thoracolumbar injury severity score (TLICS), Frankel grading for neurological symptoms, Cobb angle, compression rate of anterior verterbral height between 2 groups before operation (P>0.05). The Cobb angle, compression rate of anterior vertebral height, intervertebral height changes, and defects in injured vertebra at last follow-up were compared between 2 groups. Results All the patients were followed up 21-45 months (mean, 31.3 months). Bone healing was achieved in 2 groups, and there was no significant difference in healing time of fracture between intervention group [(7.6±0.8) months] and control group [(7.5±0.8) months] (t=0.336, P=0.740). The Frankel grading of all patients were reached grade E at last follow-up. The Cobb angle and compression rate of anterior verterbral height at 1 week after operation and last follow-up were significantly improved when compared with preoperative ones in 2 groups (P<0.05). There was no significant difference in Cobb angle and compression rate of anterior verterbral height between 2 groups at 1 week after operation (P>0.05), but the above indexes in intervention group were better than those in control group at last follow-up (P<0.05). At last follow-up, there was no significant difference of intervertebral height changes of internal fixation adjacent upper position, injured vertebra adjacent upper position, injured vertebra adjacent lower position, and internal fixation adjacent lower position between 2 groups (P>0.05). Defects in injured vertebra happened in 18 cases (75.0%) in control group and 5 cases (20.8%) in intervention group, showing significant difference (χ2=14.108, P=0.000); and in patients with defects in injured vertebra, bone defect degree was 7.50%±3.61% in control group, and was 2.70%±0.66% in intervention group, showing significant difference (t=6.026, P=0.000). Conclusion Treating thoracolumbar fractures with short-segment pedicle screw instrumentation with rhBMP-2 and allogeneic bone grafting in injured vertebra can prevent the loss of correction and vertebral defects.
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 explore the in vitro osteogenesis of the chitosan-gelatin scaffold compounded with recombinant human bone morphogenetic protein 2 (rhBMP-2). Methods Recombinant human BMP-2 was compounded with chitosan-gelatin scaffolds by freezedrying. 2T3 mouse osteoblasts and C2C12 mouse myoblasts were cultured and seeded onto the complexes at thedensity of 2×104/ml respectively. The complexes were divided into two groups. Group A: 2T3 osteoblasts seeded, consisted of 14 rhBMP-2 modified complexes. Each time three scaffolds were taken on the 3rd, 7th, 14th, and 21st day of the culturing, then the expression of osteocalcin gene (as the marker of bone formation) in adherent cells was detected by semiquantitative RT-PCR with housekeeping gene β-tubulin as internalstandard. The other 2 rhBMP-2 modified complexes were stopped being cultured on 14th day after cell seeding, and the calcification of the complexes was detected by Alizarian Red S staining. Five scaffolds without rhBMP-2 modification as the control group A, they were stopped being cultured on 14th day after cell seeding. Of the 5 scaffolds, 3 were subjected tothe detection of osteocalcin gene expression and 2 were subjected to the detection of calcification. Group B: C2C12 myoblasts seeded, had equal composition andwas treated with the same as group A. Besides these 2 groups, another 2 rhBMP2 modified complexes with 2T3 osteoblasts seeding were cultured for 3 days and then scanned by electron microscope (SEM) as to detect the compatibility of the cell to the complex. ResultsSEM showed that cells attached closely to the complex and grew well. In group A, the expression level(1.28±0.17)of osteocalcin gene in cells on rhBMP-2 modified complexes was higher than that (0.56±0.09) of the control group A, being statistically -significantly different(P＜0.05) control. C2C12 myoblasts which did not express osteocalcin normally could also express osteocalcin after being stimulated by rhBMP-2 for at least 7 days. Alizarian Red S staining showed that there was more calcification on rhBMP-2 modified complexes in both groups. There were more calcification in the group compounded with rhBMP-2, when the groups were seeded with the same cells. Conclusion The complexmade of rhBMP-2 and chitosan-gelatin scaffolds has b osteogenesis ability in vitro.
ObjectiveTo evaluate the combination of lipopolysaccharide-amine nanopolymersomes (LNPs), as a gene vector, with target gene and the transfection in bone marrow mesenchymal stem cells (BMSCs) so as to provide a preliminary experiment basis for combination treatment of bone defect with gene therapy mediated by LNPs and stem cells. MethodsPlasmid of bone morphogenetic protein 2 (pBMP-2)-loaded LNPs (pLNPs) were prepared. The binding ability of pLNPs to pBMP-2 was evaluated by a gel retardation experiment with different ratios of nitrogen to phosphorus elements (N/P). The morphology of pLNPs (N/P=60) was observed under transmission electron microscope (TEM) and atomic force microscope (AFM). The size and Zeta potential were measured by dynamic light scattering (DLS). The resistance of pLNPs against DNase I degradation over time was explored. The viability of BMSCs, transfection efficiency, and expression of target protein were investigated after transfection by pLNPs in vitro. ResultsAt N/P≥1.5, pLNPs could completely retard pBMP-2; at N/P of 60, pLNPs was uniform vesicular shape under AFM; TEM observation demonstrated that pLNPs were spherical nano-vesicles with the diameter of (72.07±11.03) nm, DLS observation showed that the size of pLNPs was (123±6) nm and Zeta potential was 20 mV; pLNPs could completely resist DNase I degradation within 4 hours, and such protection capacity to pBMP-2 decreased slightly at 6 hours. The cell survival rate first increased and then decreased with the increase of N/P, and reached the maximum value at N/P of 45; the cytotoxicity was in grade I at N/P≤90, which meant no toxicity for in vivo experiment. While the transfection efficiency of pLNPs increased with the increase of N/P, and reached the maximum value at N/P of 60. So it is comprehensively determined that the best N/P was 60. At 4 days, transfected BMSCs expressed BMP-2 continuously at a relatively high level at N/P of 60. ConclusionLNPs can compress pBMP-2 effectively to form the nanovesicles complex, which protects the target gene against enzymolysis. LNPs has higher transfection efficiency and produces more amount of protein than polyethylenimine 25k and Lipofectamine 2000.
Objective To construct the recombinant adeno-associated virus vector with human bone morphogenetic protein 4 gene（AAV-hBMP4）. Methods The hBMP-4 gene primer was designed basing on the corresponding gene sequence in GenBank. EcoR I site was introduced into the upstream of the primer and Sal Ⅰ site into downstream. The hBMP-4 gene was amplifiedwith the template of EX-A0242-M01-hBMP-4, then was cloned into pUC18 vectorto construct recombinant plasmid pUC18-hBMP-4. The plasmids pUC18-hBMP-4 and plasmid pSNAV cut by EcoR Ⅰ and Sal Ⅰenzyme, the fragments were collected and linked with T4 DNA ligase at 16℃ over night, recombinant plasmid pSNAVhBMP-4 was obtained. The recombinant plasmid was then transfected into BHK21 cells using Lipofectamine TM2000. The G418 resistant cells were obtained consequently. Thesecells were infected with HSV1-rc/△UL2 which has the function of packaging andcopying the recombinant AAV. After purification, the construction of recombinant AAV-hBMP-4 was completed. Results The construction of the recombinant pSNAV-hBMP-4 was confirmed by PCR electrophoresis and digestion with restriction enzyme. The gene sequence in the recombinant pSNAV-hBMP-4 wascorrect. The virus titer was about 1.5×1012 μg/ml.The purity of the virus was more than 95% using the SDSPAGE method. Conclusion With this method, high virus titers and purity of AAV-hBMP-4 can be acquired successfully and it is useful to bone tissue engineering.
ObjectiveTo investigate the correlation between the content of bone morphogenetic protein 2 (BMP-2) in demineralized bone matrix (DBM) and its osteogenic activity in vitro and in vivo, in order to choose a simple and convenient method to evaluate the osteogenic activity of DBM.MethodsThe left mid-femoral tissues of 9 donors were taken, and DBMs (S1-S9) were prepared by dynamic decalcification process, and inactivated DBM (control group) was prepared at the same time. Protease inhibitor method, collagenase method, guanidine hydrochloride/ethylene diamine tetraacetic acid (EDTA) method, and RIPA lysate method were used to extract BMP-2 in S1-S9 and inactivated DBMs. The BMP-2 content was measured and the differences between DBMs were compared. Then the S1-S9 and inactivated DBMs were co-cultured with mouse embryonic osteoblasts MC3T3-E1, respectively. The cell proliferation was detected by MTT method and fluorescence staining, and alkaline phosphatase (ALP) activity was detected at the same time. Thirty BALB/c male nude mice were divided into 10 groups, namely S1-S9 DBM groups (S1-S9 groups) and inactivated DBM group (control group), with 3 mice in each group. Muscle pockets of the middle thighs were prepared on both hindlimbs of mice in each group, and implanted corresponding DBM materials. At 4 weeks after operation, the samples were taken for HE staining observation and semi-quantitative evaluation, and the new bone formation score was calculated.ResultsThe BMP-2 content of DBM derived from different donor bones was distinct. The BMP-2 content obtained by different extraction methods for DBM prepared from the same donor bone was also different, and the extraction efficiency of the guanidine hydrochloride/EDTA method was the highest. In vitro cell experiments, MTT test displayed that cell proliferations and ALP activity were significantly higher in S4 and S6 groups than in other groups at each time point after co-cultivation (P<0.05). Moreover, the cell proliferation of S4 group was the most significant at 7 days (P<0.05); fluorescence staining demonstrated that the osteoblasts of each group was in good condition, but the osteoblasts of S1, S2, S3, S4, and S6 groups were significantly more than other groups. In vivo ectopic osteogenesis experiments, the cartilage and new bone formation could be seen in the bone graft area of S1-S6 groups at 4 weeks after operation, and with the increase of BMP-2 content, the more new bone formation induced by the material, the higher the score of new bone formation of the material (P<0.05). Among them, S4 and S6 groups contained a large number of chondrocytes and osteoblasts in the osteogenesis area.ConclusionThe osteogenic activity of DBM can be evaluated through BMP-2 quantitative detection combined with in vitro osteoblast proliferation and differentiation experiments.