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Objective: To investigate the effects of silencing P75 neurotrophin receptor (P75NTR) and nerve growth factor (NGF) overexpression on the proliferative activity and ectopic osteogenesis ability of bone marrow mesenchymal stem cells (BMSCs) combined with demineralized bone matrix for heterotopic osteogenesis. Methods: BMSCs of Sprague Dawley (SD) rats were cultured and passaged by adherent isolation method. The third generation BMSCs were transfected with lentivirus mediated P75NTR gene silencing (group B), NGF overexpression gene (group C), P75NTR silencing and NGF overexpression double genes (group D), respectively, and untransfected cells as control (group A). After 7 days of transfection, the expression of fluorescent protein of the target gene was observed by fluorescence microscope; cell counting kit 8 method was used to detect the cells activity for 8 days after transfection; the expressions of P75NTR and NGF proteins in each group were detected by Western blot. The adhesion of BMSCs to demineralized bone matrix (DBM) was observed by inverted phase contrast microscope and scanning electron microscope after transfection of p75NTR silencing and NGF overexpression double genes. After transfection, BMSCs and DBM were co-cultured to prepare 4 groups of tissue engineered bone, which were respectively placed in the dorsal subcutaneous tissue of 8-week-old SD rats to construct subcutaneous ectopic osteogenesis model ( n=6). HE staining was performed at 4 and 8 weeks after operation. ALP staining was used to observe the formation of calcium nodules at 8 weeks after operation. The expressions of Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) were detected by real-time fluorescent quantitative PCR. Results: At 7 days after transfection, there was no fluorescence expression in group A, red fluorescence expression was seen in group B, green fluorescence expression in group C, and red-green compound fluorescence expression in group D. The fluorescence expression rate of target gene was about 70%. Western blot detection showed that the relative expression of P75NTR protein in groups A and C was significantly higher than that in groups B and D, and the relative expression of NGF protein in groups C and D was significantly higher than that in groups A and B ( P<0.05). With the passage of time, the cell proliferation activity increased in all groups, especially in group D, which was significantly higher than that in group A at 3-8 days ( P<0.05). The results of inverted phase contrast microscope and scanning electron microscope showed that BMSCs could adhere well to DBM. In the subcutaneous ectopic osteogenesis experiment, HE staining showed that at 4 and 8 weeks after operation, the more bone tissue was formed in group D than in the other 3 groups. ALP staining showed that group D had the highest ALP activity and better osteogenic expression. Compared with group A, the relative expressions of Runx2, ALP, and OCN mRNAs in group D were significantly higher than those in group A ( P<0.05). Conclusion: Silencing P75NTR and NGF overexpression double genes co-transfected BMSCs with DBM to construct tissue engineered bone has good ectopic osteogenic ability. By increasing NGF level and closing P75NTR apoptosis channel, it can not only improve cell activity, but also promote bone tissue regeneration.
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Objective: To study the ectopic osteogenesis and biocompatibility of bone morphogenetic protein 2 (BMP-2)-derived peptide P24 loaded chitosan-4-thio-butylamidine (CS-TBA) hydrogel. Methods: First, the CS-TBA/hydroxyapatite (HA) solution was prepared by using chitosan, 2-iminothiolane hydrochloride, and HA. Then, the different amount of P24 peptides were added to the CS-TBA/HA to prepare the CS-TBA/5%P24/HA and CS-TBA/10%P24/HA solutions. Finally, β-glycerophosphate disodium (β-GP) was added to the CS-TBA/HA, CS-TBA/5%P24/HA, and CS-TBA/10%P24/HA to prepare the CS-TBA/HA/β-GP, CS-TBA/5%P24/HA/β-GP, and CS-TBA/10%P24/HA/β-GP hydrogels, respectively. Eighteen Sprague Dawley female rats were randomly divided into 3 groups ( n=6), which were injected into the back muscle pouches with equal volume CS-TBA/HA/β-GP hydrogel (group A), CS-TBA/5%P24/HA/β-GP hydrogel (group B), and CS-TBA/10%P24/HA/β-GP hydrogel (group C). The animals were sacrificed at 4 and 8 weeks and conducted micro-CT. The ability of biodegradation and osteogenesis of hydrogl was detected by trabecular thickness (Tb.Th), trabecular number (Tb.N), bone mineral density (BMD), and histological staining (HE and Masson). Results: All the rats in 3 groups survived to the time point of the harvest. Micro-CT results showed that the new bones gradually increased in each group after operation. At the same time, the new bone formation was more obvious in groups B and C than in group A, and with the increase of P24 concentration, new bone formation in group C was much more than that in group B. The Tb.Th, Tb.N, and BMD increased gradually in 3 groups, and the differences between 4 and 8 weeks were significant ( P<0.05) except the Tb.Th in group A. At different time points, the Tb.Th, Tb.N, and BMD were significantly higher in groups B and C than in group A ( P<0.05), and in group C was higher than in group B ( P<0.05), showing significant differences between groups. Histological staining showed that the materials of groups B and C were biodegradable, and the osteogenic effect was increased with the increase of P24 concentration. Conclusion: P24 peptide can improve the ectopic osteogenesis of CS-TBA hydrogel, and the 10% concentration is more effective.
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OBJECTIVE@#To investigate the synergistic effect and mechanism of the combined application of recombinant human bone morphogenetic protein-2 (rhBMP-2) and basic fibroblast growth factor (bFGF).@*METHODS@#24 KM male mice were randomly divided into 6 groups with 4 mice in each group, namely, Group A (control group), Group B (only treated with collagen), Group C (treated with 2 ng bFGF+collagen), Group D (treated with 4 μ g rhBMP-2+collagen), Group E (treated with 4 μ g rhBMP-2+2 ng bFGF+collagen) and Group F (treated with 4 μ g rhBMP-2+4 ng bFGF+collagen). The composites were implanted into the intermuscular septum of hind legs mice; whereas in control group, intermuscular septum of mice was separated and no implantation was performed. General observation, detection of concentration of calcium content, micro computed tomography (Micro-CT), three-dimensional reconstruction scan, measurement of bone mineral density (BMD), bone volume fraction (BVF) and trabecular thickness (Tb.Th), as well as histological observation with HE staining and ALP and CD34 immumohistochemical staining were performed.@*RESULTS@#Ectopic osteogenesis was found in Groups D, E and F mice. The difference in concentration of calcium contents was statistically significant between Groups D and E (P0.05). Micro-CT and three-dimensional reconstruction revealed continuous newborn bone substance in external surface of ectopic bone formation, and the center of bone formation did not show obvious substantial filling by bone substance. The differences in BMD, BVF and Tb.Th were statistically significant between Groups D and E or F (P<0.01 or <0.05). HE staining showed that in Groups D, E and F, newborn bone substance was mainly located at the edge of ectopic bone formation, and the bone formation in Groups E and F was better than that in Group D. ALP and CD34 immumohistochemical staining revealed the positive expression mainly at the edge of ectopic bone formation, and area of positive expression in Groups E and F was larger than that in Groups D.@*CONCLUSIONS@#rhBMP-2 possesses the capacity to induce ectopic osteogenesis independently, but bFGF does not have this ability; the combined application of rhBMP-2 and bFGF can enhance the synergetic effect on inducing ectopic osteogenesis.
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Objective: To investigate the synergistic effect and mechanism of the combined application of recombinant human bone morphogenetic protein-2 (rhBMP-2) and basic fibroblast growth factor (bFGF). Methods: 24 KM male mice were randomly divided into 6 groups with 4 mice in each group, namely, Group A (control group), Group B (only treated with collagen), Group C (treated with 2 ng bFGF+collagen), Group D (treated with 4 μ g rhBMP-2+collagen), Group E (treated with 4 μ g rhBMP-2+2 ng bFGF+collagen) and Group F (treated with 4 μ g rhBMP-2+4 ng bFGF+collagen). The composites were implanted into the intermuscular septum of hind legs mice; whereas in control group, intermuscular septum of mice was separated and no implantation was performed. General observation, detection of concentration of calcium content, micro computed tomography (Micro-CT), three-dimensional reconstruction scan, measurement of bone mineral density (BMD), bone volume fraction (BVF) and trabecular thickness (Tb.Th), as well as histological observation with HE staining and ALP and CD34 immumohistochemical staining were performed. Results: Ectopic osteogenesis was found in Groups D, E and F mice. The difference in concentration of calcium contents was statistically significant between Groups D and E (. P0.05). Micro-CT and three-dimensional reconstruction revealed continuous newborn bone substance in external surface of ectopic bone formation, and the center of bone formation did not show obvious substantial filling by bone substance. The differences in BMD, BVF and Tb.Th were statistically significant between Groups D and E or F (. P<0.01 or <0.05). HE staining showed that in Groups D, E and F, newborn bone substance was mainly located at the edge of ectopic bone formation, and the bone formation in Groups E and F was better than that in Group D. ALP and CD34 immumohistochemical staining revealed the positive expression mainly at the edge of ectopic bone formation, and area of positive expression in Groups E and F was larger than that in Groups D. Conclusions: rhBMP-2 possesses the capacity to induce ectopic osteogenesis independently, but bFGF does not have this ability; the combined application of rhBMP-2 and bFGF can enhance the synergetic effect on inducing ectopic osteogenesis.
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Objective:To investigate the synergistic effect and mechanism of the combined application of recombinant human bone morphogenetic protein-2(rhBMP-2) and basic fibroblast growth factor (bFGF).Methods:24KM male mice were randomly divided into6 groups with4 mice in each group, namely,GroupA(control group),GroupB(only treated with collagen),GroupC(treated with 2 ng bFGF+collagen),GroupD(treated with4 μg rhBMP-2+collagen),GroupE(treated with4 μg rhBMP-2+2 ng bFGF+collagen) andGroupF(treated with4 μg rhBMP-2+4 ng bFGF+collagen). The composites were implanted into the intermuscular septum of hind legs mice; whereas in control group, intermuscular septum of mice was separated and no implantation was performed. General observation, detection of concentration of calcium content, micro computed tomography (Micro-CT), three-dimensional reconstruction scan, measurement of bone mineral density(BMD), bone volume fraction(BVF) and trabecular thickness(Tb.Th), as well as histological observation withHE staining andALP andCD34 immumohistochemical staining were performed.Results:Ectopic osteogenesis was found inGroupsD,E andF mice.The difference in concentration of calcium contentswas statistically significant betweenGroupsD andE(P0.05).Micro-CT and three-dimensional reconstruction revealed continuous newborn bone substance in external surface of ectopic bone formation, and the center of bone formation did not show obvious substantial filling by bone substance.The differences in BMD,BVF andTb.Th were statistically significant betweenGroupsD andE orF(P<0.01 or <0.05). HE staining showed that inGroupsD,E andF, newborn bone substance was mainly located at the edge of ectopic bone formation, and the bone formation inGroupsE andF was better than that in GroupD.ALP andCD34 immumohistochemical staining revealed the positive expression mainly at the edge of ectopic bone formation, and area of positiveexpression inGroupsE andF was larger than that inGroupsD.Conclusions:rhBMP-2 possesses the capacity to induce ectopic osteogenesis independently, but bFGF does not have this ability; the combined application of rhBMP-2 and bFGF can enhance the synergetic effect on inducing ectopic osteogenesis.
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OBJECTIVE@#To explore the effect of sustained-release recombinant human bone morphogenetic protein-2 (rhBMP-2) on ectopic osteogenesis in the muscle pouches of rats through preparing rhBMP-2 sustained-release capsules by wrapping morphogenesis protein bones-2 (BMP-2) using chitosan nanoparticles, and compositing collagen materials.@*METHODS@#Twenty four Sprague-Dawley rats were randomly divided into four groups with six rats in each group, that is Group A (control group), Group B (only treated with collagen), Group C (rhBMP-2+collagen treated group) and Group D (rhBMP-2/cs+collagen treated group). The composite materials for each group were implanted in the bilateral peroneal muscle pouches in rats. The peroneal muscles were only separated without implanting any materials in control group. Rats were sacrificed 2 weeks and 4 weeks post treatment and samples were cut off for general observation, Micro CT scans and histological observation.@*RESULTS@#General observation showed no new bone formation in Groups A and B mice, while new bones were formed in Groups C and D mice. Two weeks after treatment Micro CT scans showed that The bone volume fraction (BVF), trabecular thickness (Tb.Th), bone mineral density (BMD) in Group C mice were all higher than that in Group D (P<0.05). At the fourth week, the BVF, Tb.Th and BMD were significantly higher than that at the second week (P<0.01).@*CONCLUSIONS@#The slow-release effect of rhBMP-2/cs sustained-release capsules can significantly promote ectopic osteogenesis. Its bone formation effect is better than that of rhBMP-2 burst-release group.
Subject(s)
Animals , Rats , Bone Morphogenetic Protein 2 , Pharmacology , Collagen , Pharmacology , Delayed-Action Preparations , Pharmacology , Drug Carriers , Pharmacology , Intercellular Signaling Peptides and Proteins , Muscle, Skeletal , Nanocapsules , Osteogenesis , Rats, Sprague-Dawley , Recombinant Proteins , Pharmacology , Tissue Engineering , Methods , Transforming Growth Factor beta , Pharmacology , X-Ray MicrotomographyABSTRACT
To experimentally evaluate the ectopic osteogenetic capacity of synthesized BMP2-derived peptide P24 combined with poly lactic-co-glycolic acid (PLGA), Wistar rats were divided into two groups: group A, in which BMP2-derived peptide P24/PLGA complex was implanted,and group B which received simple PLGA implant. The complex was respectively implanted into the back muscles of rats. Samples were taken the 1 st, 4 th, 8 th, and the 12 th week after the implantation.Their bone formation was detected by X-ray examination, and tissue response was histologically observed. Western blotting was used for the detection of the expression of collagen Ⅰ (Col- Ⅰ ) and osteopontin (OPN). There was acute inflammation in the tissue around both types of implants at early stage. The cartilage was found around implant areas 4 weeks after the implantation of BMP2-derived peptide p24/PLGA complex, 8 weeks after the implantation, osteoblasts were found, and 12 weeks after the implantation, typical trabecular bone structure was observed. In group B, after 12 weeks, no osteoblasts were found. It is concluded that PLGA is an ideal scaffold material for bone tissue engineering. BMP2-derived peptide can start endochondral ossification and is more effective in inducing ectopic osteogenesis.