| Backgrounds and objectives:Grafts coming from autogenous bone,allograft bone,and heterogeneous bone are usually used to repair bone fracture,bone defect,and other common clinical disease.However,there is certain limitation in such repair method regarding to the suffering of second operation,risk of transmission and infection,difficulty of host integration,etc.Due to the fact that tissue engineering technology can be adopted to construct bone tissue,and its structure and physiological function are similar to the bone in human body,the clinical limitation emerged from bone fracture and bone defect could be remedied or even addressed.More specifically,cell and growth factor,as the core elements to construct compound,play a decisive role in repairing or re-engineering tissue and organs.Mesenchymal stem cells(MSCs),which can be supplied from various sources,become seed cells in various researches because of their powerful proliferative function and multi-directional differentiation.In terms of tissue engineering technology,certain achievement has been made in the clinical test with various MSCs at present.The research of jaw fracture and defect caused by mouth and jaw trauma and tumor showed that MSCs can promote new bone formation and reduce autogenous bone healing time.However,a lot of researches reveal that the biological function when using MSCs only is too simple,while integration of various growth factors is required to better promote bone formation.Bone morphogenetic proteins(BMPs)are one of the most important growth factors in transforming growth factor ?(TGF-?)superfamily.At present,over 20 BMPs members are discovered in human body.The research showed that BMP9 was equipped with strong bone formation ability and its bone formation effect was once superior to BMP2 and BMP7 which have been approved in clinical treatment.However,the research on its bone formation mechanism is still insufficient.In the meantime,which type of carrier material should be combined and how to accurately control drug delivery system to assist BMP9 to form bone tissue comprehensively and effectively are problems demanding prompt solutions in the clinical research.The formation of bone tissue is usually accompanied with complex biological mechanism as well as the joint effect of nerve and blood vessels.Research showed that various nerve fibers in bone tissue can govern bone marrow,periosteum and play a key role in the bone healing process as they can grow in the callus.Nerve growth factor(NGF),as the first neurotrophic factor being discovered,is also able to induce nerve fiber to grow in the immature bone tissue and callus,in addition to its adjustment on the various physiological functions of central nervous and peripheral nerve cell.In recent years,various researches show that NGF can be used to promote bone formation and improve the blood vessel micro-environment formed in bone tissue.Further,in the research of oral and maxillofacial region,such as mandibular defect reconstruction,bone healing of peri-implant,tooth mineralization and development,NGF is usually regarded as the key growth factor of composite scaffold,but whether NGF governs nerve fiber via bone tissue or stimulates cell or polypeptide for bone formation mechanism is still not clear yet.Therefore,how to effectively utilize NGF for bone formation in tissue engineering technology deserves in-depth research.To sum up,by comparing separate and joint application of BMP9 and NGF to stimulate C3H10T1/2 osteogenic differentiation,this research observes the expression pattern on classic markers of osteogenesis in such process,probes into the effect of BMP9 and NGF on the osteogenic differentiation of MSCs,and provides experimental foundation for the multi-cell factors combined application in oromaxillo-facial regeneration research and tissue engineering field.Methods:1.Cell cultureC3H10T1/2 cells were inoculated in 250 ml culture flask with 10% bovine calf serum,100U/ml penicillin and 100?g/ml streptomycin H-DMEM culture for routine culture;293T cells were inoculated in 100 mm culture flask with 10% bovine calf serum,100U/ml penicillin and 100?g/ml streptomycin DMEM:F12 culture for routine culture.The cells were incubated under 37? and 5% CO2 condition.When the growth density reached 80%,0.25% trypsin was adopted for digestion and passage and cell aging with good condition were selected for the experiment.2.Assessment of different concentration of NGF on C3H10T1/2 osteogenic differentiationWhen the C3H10T1/2 growth density reached 70%-80%,osteogenic differentiation was induced,and three types of NGF(final concentration: 10ng/ml,50ng/ml,and 100ng/ml)were added into the experiment group for stimulation.No NGF was processed on the control group.Detection was made on the mRNA transcription level and protein expression of the early phase of osteogenic differentiation,on the ALP staining condition and on the formation of calcium nodule in the later phase of osteogenic differentiation after 3d,7d and 21 d of processing respectively.3.Expression and regulation of exogenous BMP9 on the BMP9 of C3H10T1/2First,recombinant adenovirus Ad-BMP9 and Ad-GFP were applied to infect 293 T for amplification,which was used to transfect C3H10T1/2 after repeat amplification.Fluorescence microscope was used to observe and select virus of higher titer for experiment.When the growth density of C3H10T1/2 reached 50%,appropriate Ad-BMP9 was transfected in the experiment group according to standard program of adenovirus transfection,while appropriate Ad-GFP was transfected in the control group.Close attention was paid to the fluorescent expression of C3H10T1/2 within 24 h.C3H10T1/2 was collected from the two groups after 12 h of transfection,and the relative mRNA expression of BMP9 was detected.4.Influence of BMP9 and NGF on C3H10T1/2 osteogenic differentiation under different phase of osteogenic markers4.1 Marker detection in the early phase of osteogenic differentiation: When the growth density of C3H10T1/2 reached 70%-80%,osteogenic differentiation was induced.According to different treatment factors,the experiment was divided into(1)GFP Group: green fluorescence was adopted as blank control;(2)NGF Group: NGF stimulation cell was adopted separately;(3)BMP9 Group: Ad-BMP9 stimulation cell was adopted separately;(4)NGF+BMP9 Group: joint application of NGF and Ad-BMP9 stimulation cell.After 3h,12 h,24h,and 48 h of processing,detection was made on the mRNA transcriptional level and protein expression of related gene in the early phase of osteogenic differentiation.Detection was made on the ALP activity and staining condition after 3d and 7d of processing.4.2 Marker detection in the later phase of osteogenic differentiation: The GFP Group,NGF Group,BMP9 Group and NGF+BMP9 Group were processed according to the different factor mentioned above so as to stimulate C3H10T1/2 osteogenic differentiation constantly.Detection was made on the mRNA transcriptional level and protein expression of related gene in the later phase of osteogenic differentiation after 9d and 12 d of processing,and on the staining degree of calcium nodule formation after 14 d and 21 d of processing.Results:1.RT-PCR results showed that after 3d of osteogenic differentiation,in the experiment group in which 10ng/ml,50ng/ml,and 100ng/ml NGF were adopted for stimulation,the relative mRNA expression of Runx2 was significantly higher than the control group without NGF stimulation(P<0.001).And when the NGF concentration was 10ng/ml,Runx2 had highest expression contents(P<0.01 or P<0.001).The results of Western blot further showed that after 3d of osteogenic differentiation,the protein expression of Runx2 was detected in each experiment group and control group,and when the NGF concentration was 10ng/ml,Runx2 had the most significant protein expression.ALP staining results showed that after 7d of osteogenic differentiation,deep blue staining was shown in some cells of each experiment group and control group.And when the NGF concentration was 10ng/ml,the staining degree was most intensive.Alizarin red staining results showed that after 21 d of osteogenic differentiation,red calcium nodule formation could be seen in some of the cells of the experiment group and control group,and when the NGF concentration was 10ng/ml,calcium nodules accounted for maximum quantity.2.The recombinant adenovirus after amplification had sound activity.BMP9 and GFP could be successfully expressed in C3H10T1/2 after the transfection of Ad-BMP9 and Ad-GFP,and it was measured that the most suitable infection was 4?l/ml with 30% of infection efficiency.When C3H10T1/2 was transfected by Ad-BMP9 at 12 h,green fluorescence expression in certain cells could be observed via fluorescence microscope.RT-PCR results further showed that after 12 h of infection,the relative mRNA expression of BMP9 of cells in experiment group which was transfected by Ad-BMP9 increased significantly,which was 5.14 times than the BMP9 mRNA of cells in the control group.(P<0.001)3.RT-PCR results showed that after 3h of osteogenic differentiation,the relative mRNA expression of Runx2 and Col I in NGF Group,BMP9 Group and NGF+BMP9 Group was significantly higher than the control group(P<0.05 or P<0.01 or P<0.001),and NGF+BMP9 Group had highest expression(P<0.001);after 12 h of osteogenic differentiation,the relative mRNA expression of Runx2 and Col I in each experiment group was still significantly higher than the control group(P<0.05 or P<0.001),and NGF+BMP9 Group had highest expression(P<0.001).The results of Western blot and Quantity One gray value scan showed that after 24 h of osteogenic differentiation,the protein expression of Runx2 in each experiment group was significantly higher than the control group(P<0.05 or P<0.001),and NGF+BMP9 Group had strongest expression(P<0.001);after 48 h of osteogenic differentiation,the protein expression of Runx2 in each experiment group was significantly higher than the control group(P<0.001),and NGF+BMP9 Group had strongest expression(P<0.001).ALP activity results showed that after 3d of osteogenic differentiation,the ALP activity intensity in each experiment group was significantly higher than the control group(P<0.001),and NGF+BMP9 Group had strongest expression(P<0.001);after 7d of osteogenic differentiation,the ALP activity intensity in each experiment group was still significantly higher than the control group(P<0.001),and NGF+BMP9 Group had the strongest expression(P<0.001).ALP staining results showed that after 3d and 7d of osteogenic differentiation,some of cells presented blue or deep blue staining in each experiment group and control group.NGF+BMP9 Group had the most intensive degree,and the color and scope of deep staining of each group was deepening and expanding.4.RT-PCR results showed that after 9d of osteogenic differentiation,the relative mRNA expression of OPN in each experiment group was significantly higher than the control group(P<0.05 or P<0.01 or P<0.001),and NGF+BMP9 Group had highest expression(P<0.001).The results of Western blot and Quantity One gray value scan showed that after 12 d of osteogenic differentiation,the protein expression of OPN in each experiment group was significantly higher than the control group(P<0.05 or P<0.001),and was fully expressed in NGF+BMP9 Group(P<0.001).Alizarin red staining results showed that after 14 d and 21 d of osteogenic differentiation,red calcium nodule could be seen in certain cells of the experiment group and control group.NGF+BMP9 Group had the most significant signs and as time went by,the quantity of calcium nodules of each group was increasing significantly.Conclusions:1.NGF has promoted the expression of osteogenic markers in the early phase and later phase,indicating that it can enhance the effect of C3H10T1/2 in osteogenic differentiation.When the NGF concentration is 10ng/ml,it has the most significant effect in promoting C3H10T1/2 osteogenic differentiation.It is speculated that NGF has threshold value during the enhancing process of C3H10T1/2 osteogenic differentiation,and too much NGF may inhibit osteogenic differentiation.2.BMP9 and NGF have enhanced the expression of osteogenic markers in the early phase of C3H10T1/2 osteogenic differentiation,indicating that both factors have synergetic effect.Especially Significant up-regulation of mRNA of Runx2 and Col I can be detected after 3h of group processing,indicating that relevant signal path of osteogenesis promoted by BMP9 and NGF may be activated at the beginning of stimulation.3.BMP9 and NGF have enhanced the expression of C3H10T1/2 osteogenic markers in the later phase.The combination can promote the formation of calcified nodules,further indicating that both factors have synergetic effect on the osteogenic differentiation of mesenchymal stem cells. |
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