Objective To study in vitro sustained release behaviour of the recombinant human bone morphogenetic protein 2(rhBMP-2) from the sample which porous calcium phosphate cement (PCPC) was combined with rhBMP-2, and to evaluate the effect of PCPC/rhBMP-2 composite on repairing bone defect in the animalstudy.Methods rhBMP-2 was absorbed into PCPC by vacuum-adsorption and freeze-dried at -40℃, the PCPC/rhBMP-2 enwrapped with chitosan as the experimental group, the pure PCPC/rhBMP-2 as the control group, then the sustained release ofrhBMP-2 from PCPC was determined in simulated body fluid (SBF) by UV-VIS spectrophotometer. At same time, the PCPC/rhBMP-2 composites with chitosan were implanted into the (4.2 mm×5.0 mm femora defects of rabbits, which were considered as the experimental group, whereas in the control group only PCPC was implanted. The effect of repairingbone defect was evaluated in the 4th and 8th week postoperatively by radiograph and histomorphology.Results The PCPC have a high absorption efficiency to rhBMP-2, and the release of rhBMP-2 was sustained release system. The release of rhBMP-2 from PCPC in the experimental group (99% after 350 hours) was slowerthan that in the control group (100% after 150 hours). In the experimental group, the radiological and histomorphological evaluations showed that theinterfaces between the materials and host bones became blurred both at 4th and 8th week. The implanted materials were partially absorbed, and the implanted areas exhibited the formation of new bone. In the control group, a little amount of new bones was observed. Conclusion The PCPC shows great clinical potential as a carrier for rhBMP-2. The PCPC/rhBMP-2 composite possesses much potentialities of osteoinductivity and the ability of repairing bone defect, so it can be used as a novel bone substitute clinically.
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 To evaluate the bone regenerative potential of reconbinant human bone morphogenetic protein 2(rhBMP-2) / collagen on adult rat calvarial bone. Methods A tight subperiosteal pocket was produced under both sides ofthe temporal muscle in rats. rhBMP-2 / collagen was implanted in one side and collagen alone was implanted in the other side as control. The rats were sacrificed 2, 4 and 8 weeks after operation. The specimen was harvested and examined histologically. For morphometric analysis, the thickness of the temporal bone of both sides was measured and compared. Results The rhBMP-2 / collagen onlay implant resulted in active bone formation and the augmented bone was connected directly with the original bone, whereas the collagen alone resulted in neither bone nor cartilage production. The ossification process in the rhBMP-2 / collagen occurred directly through bone formation, similar to intramembranous ossification. Conclusion rhBMP-2 / collagen is an effective material as a biological onlay implant.
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.
ObjectiveTo construct bone morphogenetic protein 2 (BMP-2) gelatin/chitosan hydrogel sustained-release system, co-implant with induced pluripotent stem cells (iPS) derived mesenchymal stem cells (MSCs) to hydroxyapatite (HA)/zirconium dioxide (ZrO2) bio porous ceramic foam, co-culture in vitro, and to explore the effect of sustained-release system on osteogenic differentiation of iPS-MSCs.MethodsBMP-2 gelatin/chitosan hydrogel microspheres were prepared by water-in-oil solution. Drug encapsulation efficiency, drug loading, and in vitro sustained release rate of the microspheres were tested. HA/ZrO2 bio porous ceramic foam composite iPS-MSCs and BMP-2 gelatin/chitosan hydrogel sustained release system co-culture system was established as experimental group, and cell scaffold complex without BMP-2 composite gelatin/chitosan hydrogel sustained release system as control group. After 3, 7, 10, and 14 days of co-culture in the two groups, ALP secretion of cells was detected; gene expression levels of core binding factor alpha 1 (Cbfa1), collagen type Ⅰ, and Osterix (OSX) were detected by RT-PCR; the expression of collagen type Ⅰ was observed by immunohistochemical staining at 14 days of culture; and cell creep and adhesion were observed by scanning electron microscopy.ResultsBMP-2 gelatin/chitosan hydrogel sustained-release system had better drug encapsulation efficiency and drug loading, and could prolong the activity time of BMP-2. The secretion of ALP and the relative expression of Cbfa1, collagen type Ⅰ, and OSX genes in the experimental group were significantly higher than those in the control group at different time points in the in vitro co-culture system (P<0.05). Immunohistochemical staining showed that the amount of fluorescence in the experimental group was significantly more than that in the control group, i.e. the expression level of collagen type Ⅰ was higher than that in the control group. The cells could be more evenly distributed on the materials, and the cell morphology was good. Scanning electron microscopy showed that the sustained-release system could adhere to cells well.ConclusioniPS-MSCs have the ability of osteogenic differentiation, which is significantly enhanced by BMP-2 gelatin/chitosan hydrogel sustained-release system. The combination of iPS-MSCs and sustained-release system can adhere to the materials well, and the cell activity is better.
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.
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.
ObjectiveTo investigate the effect of micro RNA (miR)-335-5p regulating bone morphogenetic protein 2 (BMP-2) on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs).MethodshBMSCs were cultured in vitro and randomly divided into control group (group A), miR-335-5p mimics group (group B), miR-335-5p mimics negative control group (group C), miR-335-5p inhibitor group (group D), and miR-335-5p inhibitor negative control group (group E). After grouping treatment and induction of osteogenic differentiation, the osteogenic differentiation of cells in each group was detected by alkaline phosphatase (ALP) and alizarin red staining; the expressions of miR-335-5p and BMP-2, Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) mRNAs were detected by real-time fluorescence quantitative PCR analysis; the expressions of Runx2, OPN, OCN, and BMP-2 proteins were detected by Western blot.ResultsCompared with group A, the relative proportion of ALP positive cells and the relative content of mineralized nodules, the relative expressions of BMP-2, miR-335-5p, OPN, OCN, Runx2 mRNAs, the relative expressions of Runx2, OPN, OCN, and BMP-2 proteins in group B were significantly increased (P<0.05); the above indexes in group D were significantly decreased (P<0.05); the above indexes between groups C, E and group A were not significantly different (P>0.05).ConclusionmiR-335-5p can up-regulate BMP-2 expression and promote osteogenic differentiation of hBMSCs.
Objective To investigate if the course of intervertebral disc degeneration (IDD) is delayed by injecting lentivirus (Lv) vector carrying bone morphogenetic protein 2 (BMP-2) and inhibitor of differentiation 1 (Id1) genes directly into the nucleus pulposus. Methods Thirty-two New Zealand white rabbits, 2.0-2.5 kg in weight and 4 months in age, were used to establish the IDD models at L3, 4, L4, 5, and L5, 6 discs with annular puncture via transabdominal approach. Thirty rabbits with successful modeling were randomly divided into 5 groups, 6 rabbits every group. At 4 weeks after modeling, rabbits were injected with Lv-BMP-2 (group A), with Lv-BMP-2 and Lv-Id1 (group B), with Lv-Id1 (group C), with Lv-green fluorescent protein (group D), and with PBS (group E). At 2, 4, and 8 weeks after injection, T2-mapping MRI was performed on 2 rabbits each group to obtain the T2 values, and then subsequently the lumbar disc tissues were harvested to test the mRNA expressions and contents of collagen type II and proteoglycan by real-time fluorescent quantitative PCR and ELISA methods. Results T2-mapping MRI demonstrated that there was no significant difference in the T2 value between different groups at immediate and 2 weeks after injection (P>0.05). The T2 value of groups A and B was significantly higher than that of groups C, D, and E at 4 weeks after injection (P<0.05), but no significant difference was observed between group A and group B (P>0.05). The T2 value of group B was significantly higher than that of the other groups at 8 weeks after injection (P<0.05). The real-time fluorescent quantitative PCR and ELISA showed that the expressions and contents of collagen type II and proteoglycan in group B were significantly higher than those in the other groups at 2, 4, and 8 weeks after injection (P<0.05). Conclusion Combined application of Lv-BMP-2 and Lv-Id1 can delay IDD changes in rabbit IDD models.
