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.
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.
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 investigate the possibility of differentiation of theisolated and cultured adipose-derived adult stem cells into chondrocytes, which is induced by the recombinant human bone morphogenetic protein 2 (rhBMP-2). Methods The rabbit adipose tissue was minced and digested by collagenase Type Ⅰ. The adposederived adult stem cells were obtained and then they were cultured inthe micropellet condition respectively in the rhBMP-2 group, the rhTGF-β1 group, the combination group, and the control group for 14 days. The differentiation of the adiposederived stem cells into chondrocytes was identifiedby the histological methods including HE, Alcian blue, Von kossa, and immunohistochemical stainings. Results After the continuous induction by rhBMP-2 and continuous culture for 14 days, the HE staining revealed a formation of the cartilage lacuna; Alcian blue indicated that proteoglycan existed in the extracellular matrix; the immunohistochemical staining indicated that collagen Ⅱ was in the cellular matrix; and Von kossa indicated that the adipose-derived stem cells couldnot differentiate into the osteoblasts by an induction of rhBMP-2. Conclusion In the micropellet condition, the adipose-derived adult stemcells can differentiate into the chondrocytes, which is initially induced by rhBMP-2. This differentiation can provide a foundation for the repair of the cartilage injury.
In the present research, the effects of sintered bone modified with surface mineralization/P24 peptide composite biomaterials on the adhesion, proliferation and osteodifferentiation of MC3T3-E1 cells were investigated. The experiments were divided into three groups due to biomaterials used: Group A (composite materials of sintered bone modified with surface mineralization and P24, a peptide of bone morphogenetic protein-2); Group B (sintered bone modified with surface mineralization) and Group C (sintered bone only). The three groups were observed by scanning electron microscopy (SEM) before the experiments, respectively. Then MC3T3-E1 cells were cultured on the surfaces of the three kinds of material, respectively. The cell adhesion rate was assessed by precipitation method. The proliferative ability of MC3T3-E1 cells were measured with MTT assay. And the ALP staining and measurement of alkaline phosphatase (ALP) activity were performed to assess the differentiation of cells into osteoblasts. The SEM results showed that the materials in the three groups retained the natural pore structure and the pore sizes were in the range between 200-850 μm. The adhesive ratio measurements and MTT assay suggested that adhesion and proliferation of MC3T3-E1 cells in Group A were much higher than those in Group B and Group C (P<0.05). The ALP staining and ALP activity of MC3T3-E1 cells in Group A were significantly higher than those in Group B and Group C (P<0.05). The sintered bone modified with surface mineralization/P24 composite material was confirmed to improve the adhesion rate and proliferation and osteodifferentiation of MC3T3-E1 cells, and maintained their morphology.
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.
ObjectiveTo explore the possibility that GREM1, a bone morphogenetic protein (BMP) antagonist, is a mechanical explanation for BMP signal suppression in congenital heart disease associated pulmonary arterial hypertension (CHD/PAH) patients.MethodsSystemic-to-pulmonary shunt induced PAH was surgically established in rats. At the postoperative 12th week, right heart catheterization and echocardiography evaluation were performed to evaluate hemodynamic indexes and morphology of right heart system. Right heart hypotrophy index and pulmonary vascular remodeling were evaluated. Changes of BMP signal pathway related proteins and GREM1 in lungs and plasma GREM1 concentration were detected. The effect of GREM1 on the proliferation and apoptosis of pulmonary arterial endothelial cells (PAECs) was also explored.ResultsThe hypertensive status was successfully reproduced in rats with systemic-to-pulmonary shunt model. BMP signal pathway was suppressed but GREM1 was up-regulated with no change in hypoxia inducible factor-1 in lungs exposed to systemic-to-pulmonary shunt, while this trend was reversed by systemic-to-pulmonary shunt correction (P<0.05). Immunohistochemical staining demonstrated enhanced staining of GREM1 in remodeled pulmonary arteries. In vitro experiments found that BMP signal was down-regulated but GREM1 expression and secretion were up-regulated in proliferative PAECs (P<0.05). Furthermore, BMP2 significantly inhibited PAECs proliferation and promoted PAECs apoptosis (P<0.05), which could be antagonized by GREM1. In addition, plasma level of GREM1 in rats with systemic-to-pulmonary shunt was also increased and positively correlated with pulmonary hemodynamic indexes.ConclusionSystemic-to-pulmonary shunt induces the up-regulation of GREM1 in lungs, which promotes pulmonary vascular remodeling via antagonizing BMP cascade. These results present a new mechanical explanation for BMP pathway suppression in lungs of CHD/PAH patients.
ObjectiveTo investigate the correlation between the expressions of bone morphogenetic protein 4 (BMP4) and sma and mad homologue 4 (Smad4) and their clinicopathological features in papillary thyroid carcinoma (PTC).MethodsEighty patients with PTC confirmed by pathology in the Pingdingshan Second People’s Hospital from March 2018 to March 2020 were selected as the research objects, the cancer tissues and adjacent tissues removed during surgery were collected. The mRNA expression levels of BMP4 and Smad4 were detected by real-time quantitative PCR (qRT-PCR). The correlation between BMP4 and Smad4 mRNA expression levels was analyzed by Pearson method. The expressions of BMP4 and Smad4 protein were detected by immunohistochemistry. The correlation between the expressions of BMP4 and Smad4 protein and clinicopathological features of PTC was analyzed.ResultsThe mRNA expression levels of BMP4 and Smad4 in PTC tissues were lower than those in adjacent tissues (P<0.05). Pearson analysis showed that there was a positive correlation between expressions of BMP4 mRNA and Smad4 mRNA in PTC cancer (r=0.660, P<0.05). BMP4 and Smad4 protein were localized in cytoplasm, and the cytoplasm was stained yellow or brown yellow. The results of immunohistochemistry showed that the expression positive rate of BMP4 in cancer tissues of PTC patients was lower than that in adjacent tissues (18.8% vs 97.5%, χ2=101.916, P<0.05), and the expression positive rate of Smad4 protein in cancer tissues of PTC patients was also lower than that in adjacent tissues (11.3% vs 93.8%, χ2=109.173, P<0.05). The expressions of BMP4 and Smad4 protein in PTC patients were correlated with the tumor size, TNM stage, lymph node metastasis, degree of infiltration and multiple foci (P<0.05).ConclusionsThe expression levels of BMP4 mRNA and Smad4 mRNA in PTC tissues are decreased, and the expression of BMP4 protein and Smad4 protein are closely related to tumor size, TNM stage and lymph node metastasis, which may be used as new therapeutic targets.
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 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.