Rhizoma Drynariae Flavonoids Promote Genetically Engineered Myoblast Osteogenetic Differentiation And The Study Of Guided Bone Regeneration

Objective：To probe into the effects of assemble flavone of rhizome drynaria(AFDR) on myoblast proliferation and osteogenic differentiation of culturingCNTF genes in vitro, and to research the mechanism of AFDR promoting osteogenicdifferentiation;Transfected CNTF myoblasts pretreated by AFDR medicated serumwere given microencapsulation processing to get immune and isolated. Combinedwith sodium hyaluronate of BMP and GBR film—silicone tube to construct a newartificial bone of tissue engineering, the effects of GBR promoting boneregeneration was observed through the experiments on animals in vivo. Thefeasibility of myoblasts as a new type of seed cells and AFDR as a newbone-induction factor, which was applied to tissue engineering, was evaluated.Material and method：PartⅠ: The experiment on transfected CNTF genes promoting the differentiationof rats’ myoblastsFour five-day Wistar clean rats were in vitro isolated, cultured, purifiedinto myoblasts with the improved method, identified and then reserved.CNTFplasmid DNA was extracted with plasmid rapid extraction box. The cationicliposomes method affected the differentiation of rats’ myoblasts transfectionand made it keep the dedifferentiation state. The activity, expression anddifferentiation of transfected cells were detected and carried on the statisticsprocessing.Part Ⅱ: The experimental study on AFDR promoting transfected CNTF todifferentiate into osteogenesis and the discussion on its mechanismTwenty twelve-month healthy female Wistar rats were randomly divided intohigh, medium and low dose treatment groups and the control group of blankserum.According to the equivalent dose rate table of the body surface area ofpeople and animals, rats’ stomachs were AFDR irrigated, blood was drawn from abdominal aortic under aseptic conditions, centrifuged, live complement was putout, and then AFDR medicated serum of different concentrations was prepared withsuction filter degerming; transfected CNTF myoblasts were divided into GroupsA, B, C, D and E, and were pretreated by adding the corresponding cultures.GroupA (AFDR group of high concentration): AFDR medicated serum of high concentration+osteogenesis inducers; Group B (AFDR group of medium concentration): AFDRmedicated serum of medium concentration+osteogenesis inducers; Group C (AFDRgroup of low concentration): AFDR medicated serum of low concentration+osteogenesis inducers; Group D (group of blank serum): blank serum+osteogenesisinducers; Group E (group of standard medium): complete medium.After21days’culture, the proliferation and differentiation of transfected CNTF myoblastsafter pretreatment was observed and determined by MTT. Alkaline phosphatase,alizarin red, collagen type I dyed observation, osteocalcin, calcium depositionweight, ALP specific activity and other osteogenesis markers were detected. Then,osteogenesis and adipogenesis were determined, and statistical analysis wascarried out.Part Ⅲ: The research on microcapsule AFDR pretreating transfected CNTFmyoblasts to promote guided bone regenerationThe medium of Group B in the partⅡ(AFDR medicated serum of mediumconcentration+osteogenesis inducers) was applied to the osteoblast inductionand differentiation of transfected CNTF, and then was set aside after relateddetection.These transfected CNTF myoblasts were microencapsulated and survivalrate of cells was determined.24New Zealand clean rabbits were taken for radiusdefect mode. According to the disposal conditions, they were randomly dividedinto GroupsⅠ, Ⅱ, Ⅲ, Ⅳ with six animals in each group. Silicon tube was usedas GBR membrane and implanted into different transplants.GroupⅠweremicrocapsule of transfected CNTF myoblasts pretreated by AFDR and HA gel grouploaded with BMP. GroupⅡwere microcapsule of transfected CNTF myoblasts and HAgel group loaded with BMP. Group Ⅲ was only HA gel group loaded with BMP. GroupⅣ was only HA gel group. Then, the statistical analysis was conducted after general postoperation observation, X-ray determination and histological dyeingdetection.Results:1.Myoblasts with good growth state can be obtained through ameliorative ways,and the myoblasts were appraised positive through scanning electron microscopy(SEM) and protein antibody staining; In the experiment, CNTF plasmid can besuccessfully extracted to transfect rats’ myoblasts. Transfected CNTFmyoblasts can secrete and express CNTF mRNA with cell differentiation ratedecreasing and in the state of dedifferentiation.2.AFDR medicated serum of high, medium and low concentration and blank serumwere successfully obtained in the experiment. Transfected CNTF myoblasts werepretreated by AFDR medicated serum of different concentrations (including blankserum), observed and compared with those cultured by standard medium.(1) Underinverted phase contrast microscope: Transfected CNTF myoblasts pretreated byAFDR turned into fusiformis or polygon. Some had several bumps and graduallyformed a scattered island-shape structure of dense cells, like whirlpool.Intermediate cells ranged closely and indistinctly, were gradually embedded byhigh refractive cavitation and dark particles, and formed white calcified nodule.Among them, AFDR medicated serum group of low concentration was the mostobvious.(2) MTT results showed that transfected CNTF myoblasts in differenttreatment groups all proliferated as time went by, reaching proliferation peakon the5thor6thday, and declining on the7thday. At the same time, in the AFDRgroups of low concentration, medium concentration and blank serum group, OD valuewas higher than standard medium group (p＜0.05), and AFDR medicated serum groupof lower concentration was the strongest.(3) The results of Alkaline phosphatasedyeing, alizarin red vital staining, and immunohistochemical stains oftypeⅠcollagen: besides standard medium group presenting negative, transfectedCNTF myoblasts in other groups all had atropurpureus granules orcurdyprecipitate, jacinth precipitate, claybank solids precipitation. They showed positive, with the reaction of AFDR group of low concentration thestrongest, AFDR group of high concentration the weakest, AFDR group of mediumconcentration and blank serum group equal.(4) The osteoblast marker test showedthat:①on the7thday of cell culture, there was no statistical significancein the OCN content of the five groups (p＞0.05); on the14thand21stdays of cellculture, the OCN secretion in AFDR groups of low and medium concentration andblank serum group was all higher than that of the standard culture solution group,while the OCN secretion in AFDR group of low concentration was obviously higherthan that of AFDR groups of medium concentration and blank serum group, whichhad significantly statistical significance (p＜0.05); Through the comparisonin each group at different time, except AFDR group of high concentration andstandard medium group, the OCN secretion on the21stday in the rest three groupswas significantly higher than the14thand7thdays, and the OCN secretion on the14thday was also higher than the7thday;②On the3rd,6thand9thdays, there wasno statistical significance in measuring the calcium deposition of the fivegroups (p＞0.05);On the12thday, the calcium deposition of AFDR groups of low,medium and high concentration was higher than that of the blank serum group andthe standard medium group. While, there was no statistical difference in thecalcium deposition among the AFDR groups of low, medium and high concentration,and also between the blank serum group and the standard medium group;On the15th,18thand21stdays, there was significant difference among the five groups(p＜0.01), and through further comparison between each two groups, it was foundthat on the15th,18thand21stdays, the calcium deposition in AFDR groups of low,medium and high concentration was higher than the standard medium group. Inaddition, the blank serum group was also higher than the standard medium group;③Variance analysis by ALP specific activity determination at different timepoints between different treatment groups showed that there was no statisticalsignificance among the five groups on the1stand3rddays of culture (p＞0.05);Onthe7th,11thand14thdays, the ALP specific activity in AFDR groups of low andmedium concentration and blank serum group was obviously higher than AFDR group of high concentration and the standard medium group. Moreover, the ALP specificactivity in AFDR group of low concentration was distinctly higher than that ofAFDR group of medium concentration and blank serum group, and there wasstatistical significance (p＜0.05). While, there was no statisticalsignificance in ALP specific activity between AFDR group of medium concentrationand the blank serum group (p＞0.05);The ALP specific activity in both AFDR groupof high concentration and the standard medium group was extremely low, and thedifference had no statistical significance (p＞0.05).(5) On the14thday, thedetection of Cbfa1mRNA, ALP mRNA, PPARγ mRNA expression of the five groupsshowed the differences among these groups were significant (p＜0.05or p＜0.01).Especially, the effects of AFDR group of low concentration on Cbfa1mRNA、ALPmRNA、PPARγ mRNA expressions in the process of transfected CNTF myoblastsdifferentiating into osteoblasts were the most significant.That is, the expressions of bone related factors Cbfal mRNA, ALP mRNA were thehighest, while bone related factor PPARγ mRN expressed the lowest.3.The microencapsulated cells in vitro lived well. The appearance ofmicro-capsule was complete and without adhesion, size was uniform, and the innercells can be clearly seen; In the experiments on animals in vivo, every indexof the transfected CNTF myoblasts pretreated by the microencapsulated AFDR inGroupⅠand guided bone regeneration in BMP factor group was better than GroupsⅡ,Ⅲ and Ⅳ, and the difference among these groups was significant, p＜0.05.Conclusions:1.The application of transfected CNTF myoblasts can inhibit myoblasts from thedifferentiation of myoblasts in vitro, keep myoblasts dedifferentiating, andprovide the theoretical foundation for myoblasts to be used as seed cells fortissue engineering researches.2.After being induced by osteogenesis inducers in vitro, transfected CNTFmyoblasts turned into osteoblastic progenitor cells.3.AFDR medicated serum could significantly improve the proliferation and osteogenic differentiation of transfected CNTF myoblasts in vitro, which showedthat AFDR had the potential to become the new drug to promote fracture healingand antiosteoporosis.4.AFDR medicated serum of low concentration was the most effective in improvingthe proliferation and osteogenic differentiation of transfected CNTF myoblastsin vitro. On the contrary, AFDR medicated serum of high concentration restrainedtheir proliferation and differentiation. Therefore, the effects of AFDRmedicated serum on proliferation and osteogenic differentiation in vitro oftransfected CNTF myoblasts were related to its concentration.5.The mechanism of AFDR medicated serum improving the osteogenic differentiationin vitro of transfected CNTF myoblasts was related to up-regulating theexpressions of Cbfal mRNA and ALP mRNA and down-regulating the expression ofPPARγ mRNA in the process of osteogenic differentiation.6.A combination of AFDR and myoblasts can be applied to promote the repair ofbone defects and be used in the researches on bone tissue engineering.7.In terms of the innovative application of guided bone regeneration theory,sodium hyaluronate gel and silicon tube can achieve good effects on repairingbone defects, and has been used in clinical practice as favourable bone tissueengineering materials.