Bone morphogenetic protein (BMPs) has been so far regarded as one of the highly potent osteoinductive growth factors. Recombinant human bone morphogenetic proteins have been utilized extensively in the disciplines of orthopedics, stomatology, etc. For clinical application, BMPs are usually loaded in carriers with a controlled-release system, to maintain concentration to induce de novo bone formation at the desired site. In this article, the research advancements of the carriers and release systems of BMP are reviewed.
Pulmonary hypertension is a disease characterized by pulmonary artery pressure increased, with or without small artery pathological change, which ultimately leads to right heart failure or even death. Pulmonary hypertension seriously threatens to human health, however, the pathogenesis of pulmonary hypertension is unclear. Previous studies have found that bone morphogenetic protein (BMP) signaling system played an important role in the progress of pulmonary hypertension. In the current review, we describe the mechanism of BMP4 in the development of pulmonary hypertension.
Objective To investigate and compare the osteogenic potential of three kinds of calcium phosphate ceramic as carriers for recombinant human bone morphogenetic protein-2(rhBMP-2) in vivo.Methods BCPceramics (HA,TCP,HA/TCP) impregnated with rhBMP-2 (experimental groups) and without rhBMP-2(control groups) were implanted into 6 muscles pockets on the dorsum of 3month-old Wistar rabbits. The rabbits were sacrificed 2, 4 and 8 weeks after implantation and bone induction was estimated by alkaline phosphatase(ALP) activity measurement. The implants were also examined histologically and histomorphometrically by HE staining and computerized graphical analysis. Results The ALPactivity of implants withrhBMP-2 was higher than that of control groups(P＜0.05), but there was no difference between 2 and 4 weeks in experimental groups. In all experimental groups,theimplants exhibited that new bone formation increased with the lapse of time. The amount of new bone formation is more in -HA/rhBMP-2 group than in the other two group in the 2nd and 4th weeks, but there was no difference between them (P＞0.05).In the 8th week, the amount of bone formation was most in HA/TCP with -rhBMP-2, and was more than that in the 2nd and 4th weeks. Whereas in control groups, there was only fibrous connective tissue. Conclusion HA/TCP- is a good carriers of rhBMP-2 and can be used as bone substitutes clinically.
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 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.
Objective To investigate bone regeneration of the cell-biomaterial complex using strategies of tissue engineering based on cells.Methods Hydroxyapatite/collagen (HAC) sandwich composite was produced to mimic the natural extracellular matrix of bone, with type Ⅰ collagen servingas a template for apatite formation. A three-dimensional ploy-porous scaffoldwas developed by mixing HAC with poly(L-lactic acid) (PLA) using a thermally induced phase separation technique (TIPS). The rabbit periosteal cells were treated with 500 ng/ml of recombinant human bone morphogenetic protein 2(rhBMP-2), followed by seeded into pre-wet HAC-PLA scaffolds. Eighteen 3-month nude mice were implanted subcutaneously cell suspension (groupA, n=6), simple HAC-PLA scaffold (group B, n=6) and cell-biomaterial complex(group C, n=6) respectively.Results Using type Icollagen to template mineralization of calcium and phosphate in solution, we get HAC sandwich composite, mimicking the natural bone both in compositionand microstructure. The three dimensional HAC-PLA scaffold synthesized by TIPShad high porosity up to 90%, with pore size ranging from 50 μm to 300 μm. SEMexamination proved that the scaffold supported the adhesion and proliferation of the periosteal cells. Histology results showed new bone formation 8 weeks after implantation in group C. The surface of group A was smooth without neoplasma. Fibrous tissueinvasion occured in group B and no bone and cartilage formations were observed.Conclusion The constructed tissue engineering bone has emerged as another promising alternative for bone repair.
OBJECTIVE To improve the osteoinduction of coral and provide a perfect bone graft substitute for clinical bone defects. METHODS By combining coral with collagen and recombinant human bone morphogenetic protein-2(rhBMP-2), coral/collagen/rhBMP-2 composite was obtained. The composite was implanted into the back muscle pouches of mice, and coral/collagen or coral/rhBMP-2 were implanted as control. The osteoinduction of the composite was assessed by histology and image analysis system. RESULTS The chondrocyte differentiation and matrix formation were observed in local sites after one week, lamellar bone with bone marrow were formed after 4 weeks, and coral were absorbed partially. The quantity of osteoinduction was time-related and rhBMP-2 dose-related(P lt; 0.01). Coral/collagen and coral/rhBMP-2 implants did not show any bone or cartilage formation. CONCLUSION The coral/collagen/rhBMP-2 composite possesses a superior osteoinduction and will be a new type of bone substitute to be used in orthopedic and maxillofacial surgery.
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 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 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.