In order to explore further the regulatory factors to the potentiality in inducing osteogenesis by fibroblasts, the fibroblasts were isolated, and purified from human skin, and were grown in incubation in the media of EGF, IL-6, TNF-alpha and BMP2 at different concentrations for two weeks, then, the markers for osteogenic features were investigated by biochemistry, histochemistry and electron microscopic observations. It was found that the combined use of TNF-alpha and BMP2 could stimulate fibroblasts to secrete alkaline phosphatase, osteocalcin and collagen, and the morphological changes of the fibroblasts were also very striking. In the extracellular matrix, the collagen fibrils, with or without periodicity, were arranged regularly or randomly oriented, and numerous minute calcium granules were interspersed among them. The fibroblasts were interwoven one on top of another in the form of multilayer structure and on the surface, there were secreting granules and piling up of calcium crystals which coalessed steadily and increased in size in forming bony nodules. It was considered that TNF-alpha and BMP2 were capable of inducing the fibroblasts to form bone.
Ceramiclike xenogeneic bone (CXB) was obtained from the fresh bone of pig ribs being treated by physical and chemical methods to deprive of its organic substance. The CXB possessed the same natural porous network system as that of the human. The CXB was cultured with the bone marrow stromal cells of rabit. When the marrow cells had integrated with the CXB, thus a new material was obtained. (CXB-BM), and was implanted sacro-spinal muscle of rabbit. The specimens were observed under phase microscope, light microscope and electronic scanning microscope. The results showed that: at the 2nd week after the implantation of CBX-BM composite material there began the new bone formation, and the rate of bone formation was increased with time. There was evident new bone formation after 24 weeks. The process of the new bone formation were quite similar to the composite graft of HAP red autogenous and marrow, but the former degraded faster and formed typical cancellous structure earlier. There was no new bone formation when CXB was implanted alone in the control. Both the mechanism of osteogenetic potential and its clinical application were discussed.
Objective To review the progress, controversy and trend in the regulation and mechanism of the microRNAs (miRNAs) during the osteogenesis. Methods Recent l iterature concerning regulation and mechanism of the miRNAs during the osteogenesis was extensively reviewed, summarized and analyzed. Results Recently miRNAs was a hot topic for osteogenesis. More and more materials showed its important role in ossification, but its definite mechanism was notclear. Conclusion Osteogenesis can be strengthened by miRNAs technology, which has a bright future and may also provide the molecular mechanism. The study on miRNAs of osteogenesis can provide a model to analyze and compare the osteogenetic effects of novel drugs.
Objective
To construct recombinant lentiviral vectors of porcine bone morphogenetic protein 2 (BMP-2) gene and to detect BMP-2 gene activity and bone marrow mesenchymal stem cells (BMSCs) osteogenetic differentiation so as to lay a foundation of the further study of osteochondral tissue engineering.
Methods
BMSCs were isolated from bone marrow of 2-month-old Bama miniature porcines (weighing, 15 kg), and the 2nd generation of BMSCs were harvested for experiments. The porcine BMP-2 gene lentiviral vector was constructed by recombinant DNA technology and was used to transfect BMSCs at multiplicity of infection (MOI) of 10, 25, 50, 100, and 200, then the optimal value of MOI was determined by fluorescent microscope and inverted phase contrast microscope. BMSCs transfected by BMP-2 recombinant lentiviral vectors served as experimental group (BMP-2 vector group); BMSCs transfected by empty vector (empty vector group), and non-transfected BMSCs (non-transfection group) were used as control groups. RT-PCR, immunohistochemistry staining, and Western blot were performed to detect the expressions of BMP-2 mRNA and protein. Then the BMSCs osteogenesis was detected by alkaline phosphatase (ALP) staining, ALP activities, and Alizarin red staining.
Results
The recombinant lentiviral vectors of porcine BMP-2 gene was successfully constructed and identified by RT-PCR and gene sequencing, and BMSCs were successfully transfected by BMP-2 recombinant lentiviral vectors. Green fluorescent protein could be seen in the transfected BMSCs, especially at MOI of 100 with best expression. The immunohistochemistry staining and Western blot showed that BMSCs transfected by BMP-2 recombinant lentiviral vectors could express BMP-2 protein continuously and stably at a high level. After cultivation of 2 weeks, the expression of ALP and the form of calcium nodules were observed.
Conclusion
The porcine BMP- 2 gene lentiviral vector is successfully constructed and transfected into the BMSCs, which can express BMP-2 gene and protein continuously and stably at a high level and induce BMSCs differentiation into osteoblasts.
This paper aims to explore the effect of electrical stimulation of triboelectric nanogenerators (TENGs) on the osteogenic and other biological behaviors of mouse embryonic osteoblast precursor cells (MC3T3-E1 cells) on titanium surfaces. First, an origami-type TENG was fabricated, and its electrical output performance was tested. The optimal current of the generator and the feasibility of the experiment were verified by the CCK-8 assay and scratch assay. At the optimal current, the osteogenic conditions of the cells in each group were determined by quantitative analysis of the total protein content, alkaline phosphatase (ALP) activity, and alizarin red staining (ARS) on the titanium surface. Finally, the adhesion and spreading of cells on the titanium surface after electrical stimulation were observed. The results showed that the TENG had good electrical output performance, with an open-circuit voltage of 65 V and a short-circuit current of 42 μA. Compared with the rest of the current, a current strength of 30 μA significantly improved cell proliferation and migration, osteogenesis, and adhesion and spreading capabilities. The above results confirm the safety and operability of TENG in biomedical applications, laying the foundation for future TENG applications in reducing the time of bone integration around titanium implants after surgery.
ObjectiveTo compare the effects on the osteogenesis of bone marrow mesenchymal stem cells (BMSCs) between hypoxia and hypoxia mimetic agents dimethyloxalylglycine (DMOG) under normal oxygen condition.
MethodsBMSCs were isolated and cultured from healthy 3-4 weeks old Kunming mouse. Cell phenotype of CD29, CD44, CD90, and CD34 was assayed with flow cytometry; after osteogenic, adipogenic, and chondrogenic induction, alizarin red staining, oil red O staining, and toluidine blue staining were performed. The passage 3 BMSCs were cultured under normal oxygen in control group (group A), under 1%O2 in hypoxia group (group B), and under normal oxygen and 0.5 mmol/L DMOG in DMOG intervention group (group C). BMSCs proliferation was estimated by methyl thiazolyl tetrazolium assay at 1, 2, 3, and 4 days. Alkaline phophatase (ALP) expression was determined at 7 and 14 days after osteogenic induction. Western blot was employed for detecting hypoxia inducible factor-1α(HIF-1α) at 24 hours. Real time fluorescence quantitative PCR was employed for detecting the mRNA expression of runt-related transcription factor 2 (RUNX2) and Osterix at 3 and 7 days. Alizarin red staining was applied to assess the deposition of calcium tubercle at 21 days.
ResultsThe BMSCs presented CD29(+), CD44(+), CD90(+), and CD34(-); and results of the alizarin red staining, oil red O staining, and toluidine blue staining were positive after osteogenic, adipogenic, and chondrogenic induction. No significant difference in BMSCs proliferation was observed among 3 groups at 1 day (P>0.05); compared with group A, BMSCs proliferation was inhibited in group C at 2, 3, and 4 days, but no significant difference was observed (P>0.05); compared with group A, BMSCs proliferation was significantly promoted in group B (P < 0.05). At each time point, compared with group A, the ALP expression, HIF-1αprotein relative expression, and mRNA relative expressions of RUNX2 and Osterix were significantly up-regulated in groups B and C (P < 0.05); compared with group B, the ALP expression, the RUNX2 and Osterix mRNA relative expression were significantly up-regulated in group C (P < 0.05); compared with group C, the HIF-1αprotein relative expression was significantly up-regulated in group B (P < 0.05). The alizarin red staining showed little red staining materials in group A, some red staining materials in group B, and a large number of red staining materials in group C.
ConclusionHypoxia can promote BMSCs proliferation, DMOG can not influence the BMSCs proliferation; both hypoxia and DMOG can improve osteogenic differentiation of BMSCs, and DMOG is better than hypoxia in improving the BMSCs osteogenesis.
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.
ObjectiveTo observe the ability of osteogenesis in vivo using the injected absorbable polyamine acid/calcium sulfate (PAA/CS) composites and assess their ability to repair bone defects.
MethodWe selected 48 New Zealand white rabbits, and half of them were male with a weight between 2.0 and 2.5 kg. Bone defect models were made at the rabbit femoral condyle using electric drill, and the rabbits were divided into two groups. One group accepted implantation of the material at the defect, while nothing was done for the control group. After four, eight, twelve and sixteen weeks, the animals were killed. The line X-ray and hard tissue slices histological examination (HE, MASSON staining) were observed to assess the situation of degradation, absorption and bone formation of the material.
ResultsFour weeks after operation, bone defect of the experimental group had no obvious callus growth on X-ray imaging. Histology showed that the material began to degrade and new immature trabecular bone grew. The bone defect of the experimental group had a small amount of callus growth on X-ray imaging after eight weeks. And histology showed that the material continued to degrade and new immature trabecular bone grew continually. There was an obvious callus growth after twelve weeks, and the bone defect area had smaller residual low-density shadow on X-ray imaging. Histology showed that most of the materials degraded and parts of woven bone grew into lamellar bone. After sixteen weeks, the composites were absorbed completely, replaced by new bone tissues, and the new bone was gradually changed from woven bone into mature plate of bone. There was no significant change in bone defect in the control group within twelve weeks, and part of bone defect hole became smaller, and partial edge repair could be detected.
ConclusionsThe PAA/CS composites can be completely degraded and absorbed, with a certain activity of bone formation, expected to be used as bone repair materials.
OBJECTIVE To investigate the factors which affect the bone union of distracted region after limb lengthening, so as improve the curative effect and diminish the incidence of complication. METHODS To look up the latest literatures dealing with the bone union in limb lengthening, then review the procedure of osteogenesis and the affecting factors. RESULTS The osteogenesis of distracted region after limb lengthening is a sophisticated procedure. It can be affected by the velocity of lengthening, the period of lengthening, the site and method of osteotomy, the age etiology of patient. CONCLUSION The bone union of distracted region after limb lengthening can be facilitated by following factors: 1. the velocity of lengthening slower than 1.0 mm/day; 2. moderate delay in distraction; 3. axial shortening of distracted region; 4. micromovement stimulation.
Objective To evaluate the osteogenic potential of human bone marrow mesenchymal stem cells (MSCs) transferred with human bone morphogenetic protein 2(BMP 2) gene by adenovirus. Methods The MSCs were isolated from human bone marrow and cultured in vitro. They were divided into 3 groups: Adv hBMP 2 transduced group; Adv βgal transduced group; untransduced group. Western immunoblot analysis, alkaline phosphatase(ALP) staining, Von Kossa staining, and a quantitative ALP activity assay were performed. Nine unde mice received injection into a thigh muscle to test the osteoinductivity of the three types of cells. Results In the Adv-hBMP-2 transprotein; most MSCs were stained positively for ALP activity 9 day after transduction; the MSCs reached the peak of ALP activity 12 day after transduction; the calcified nodes formed 21 days after transduction. The ectopic bones formed in the thigh muscles of the nude mice. Little bone formation was observed in the other groups 4 weeks after cell injection. Conclusion Adenovirus mediated hBMP-2 gene transfection can induce osteogenesis of human bone marrow mesenchymal stem cells.
