| ObjectiveDistraction osteogenesis（DO）is considered as an endogenous tissue engineering that utilizes the mechanism of callus healing to achieve bone regeneration.It was first used in orthopedics and orthopedic surgery,and later used in craniofacial surgery for the repair of craniofacial microsomia and bone defects,and has become an effective tool for the repair of various craniomaxillofacial deformations and defects.Compared with traditional surgery,DO has some obvious advantages,but its poor osteogenic quality,long treatment cycle,and ensuing related complications seriously restrict its application in clinical practice.How to improve the speed and quality of new bone formation during DO and shorten the treatment cycle has become a hot topic in this field.In the early stage,Ma Dongyang et al.established cell sheets of bone marrow stromal stem cells（BMSCs）without external scaffolds by tissue engineering method and achieved good results in promoting DO.In recent years,researchers at home and abroad have applied BMSCs modified with bone morphogenetic proteins（BMPs）and other growth factors to fast DO,which has also shown obvious advantages.Therefore,this experiment intends to promote DO by combining tissue engineering technology with genetic engineering technology.Firstly,a lentiviral vector was used to introduce rabbit bone morphogenetic protein-1（BMP-1）into rabbit BMSCs,and then the BMSCs polymeric modified by BMP-1 was established.Finally,its fragments were injected into the DO gap.By increasing the local BMSCs concentration of DO,BMSCs were induced to convert to osteogenesis and chondroblast,which ultimately promoted the rate and quality of new bone formation of DO and shortened the treatment cycle of DO.Methods1.The whole bone marrow adherence screening method was used to isolate,culture,and purify BMSCs.After primary culture,BMSCs with relatively high purity was obtained.The morphological characteristics of cells were observed by an inverted microscope,and then BMSCs at different time nodes were selected for counting and growth curves were drawn.Finally,the multidirectional differentiation of BMSCs was observed under osteogenic,chondrogenic,and lipid induction conditions.2.Rabbit bone morphogenetic protein-1（BMP-1）gene fragment was amplified from rabbit ovaries by reverse transcriptase-polymerase chain reaction（RT-PCR）.The gene fragment was recombinant on the lentivirus vector by DNA recombination technology,and the recombinant DNA was identified by q PCR and Western Blot.3.BMSCs were transfected with lentivirus expression plasmids containing rabbit BMP-1 coding sequence by a relatively mature lentivirus transfection technique,and the transfection efficiency of BMP-1 was detected by immunofluorescence observation and western blot.Then,BMSC polymers were obtained by continuous culture without passage of BMSC modified with BMP-1,and they were tested for CCK-8,ALP activity,Ca2+concentration,and HE and immunohistochemical staining,respectively.4.36 healthy New Zealand white rabbits were randomly divided into 3 groups with12 rabbits in each group.The experimental group（BMSCs polymeric fragments modified by BMP-1 gene）,the control group（BMSCs polymeric fragments without modifying by BMP-1 gene）,and the blank group（normal saline）were injected.Transfection modification with the BMP-1 gene was performed after the transplantation of autologous BMSCs into P-3 cells.The animal model of distraction osteogenesis was established with a latency period of 3 days.From the 4th day after the operation,DO was conducted once a day with a length of 2.0mm for 5 days.The total stretch length was 10mm.After the end of DO,the experimental group was injected with 3mlbmp-1 modified BMSCs cell sheets in the DO interval.The Control group was injected with 3ml GFP modified BMSCs cell sheets.The blank group was injected with 3mlof normal saline.At the fixation stage,6animals were sacrificed in each experimental group at the third week and the sixth week,and the formation of new bone in each experimental group was observed by general observation,micro-CT examination,HE,and Masson staining tissue sections.Results:1.The whole bone marrow adherence screening method is a simple and easy way to isolate,culture,and purify BMSCs,both of which primary and subcultured cells maintained high proliferation activity.Under the various induction conditions,bone marrow mesenchymal stem cells can differentiate into osteogenic,chondrogenic and adipogenic cells,and the cells induced in vitro grow well,showing similar morphological and biological characteristics with the corresponding cells,which proves that bone marrow mesenchymal stem cells have the potential of multidirectional differentiation.2.By PCR amplification,enzyme digestion,electrophoresis,and DNA sequencing,the lentivirus vector containing the rabbit BMP-1 target gene was successfully established in this experiment.3.The BMP-1 gene and GFP gene was introduced into rabbit BMSCs by lentivirus transfection vector,and the transfection efficiency was detected by fluorescence microscopy and Western blotting.The expression of BMP-1 histone increased after transfection.Then BMSCs of BMP-1 group,GFP group,and control group were cultured for 2 weeks without continuous subculture to obtain a certain elastic and thick cell sheets,After HE staining,the thickness of the cell sheet in the BMP-1 group（60.3±78.28）was significantly higher than that in the GFP group（23.81±0.77）.Cell viability tests of the three groups at different time points in the cell membrane showed that,from Day-4,cell viability in the BMP-1 group was significantly higher than that in the GFP group and the control group,and there was no significant difference between the GFP group and the control group（P<0.05）.Osteogenic correlation indexes at different time points showed that,compared with GFP and the control group,the ALP activity of the BMP-1 group was significantly up-regulated at each time point,which meant that overexpression of BMP-1could promote the differentiation of osteogenic precursor cells into mature osteoblasts,and there was no significant difference between the GFP group and the control group.The results of Ca2+concentration determination in group 3 suggested that the concentration of Ca2+in the BMP-1 group was significantly higher than that in the GFP group and the control group,indicating that the mineralization in the BMP-1 group was significantly higher than that in the other two groups.Immunohistochemical staining of the BMP-1group and the GFP group showed that compared with the GFP group,the expression of type I collagen in the BMP-1 group was significantly increased,which further indicated that the BMSCs cell sheet transfected with BMP-1 had a stronger osteogenic capacity.4.After 3 and 6 weeks of fixation,the left mandible of all the animals was successfully distracted,the mandibular was significantly skewed to the right,and the upper and lower anterior teeth were slightly inverted.Specimens were taken after fixation of 3w and 6w at the end of DO.Through gross observation,lingual osteogenesis in the distraction area of all animals was significantly better than buccal osteogenesis,and the bone cortex was smooth and continuous with higher hardness.The order of bone mass or hardness from high to low is the experimental group>the control group>blank group;Through micro-ct observation and statistical analysis of relevant indicators,it was found that bone mineral density（BMD）in the distraction area of the experimental group was significantly higher than that of the control group and the blank group at the 3rd and 6th week of fixation period（P<0.01）.After histomorphological observation and quantitative observation,it was found that the experimental group was superior to the control group and the blank group in terms of the degree of new bone maturation and bone mass in the DO area at 3 and 6 weeks.Conclusions1.The whole bone marrow adherence screening method is a simple and easy way to isolate,culture,and purify BMSCs,both of which primary and subcultured cells maintained high proliferation activity.Under the condition of in vitro osteogenesis BMSC can be transformed into bone,cartilage,and lipid cells.2.The lentivirus vector of the rabbit BMP-1 gene was successfully constructed,which laid a foundation for subsequent experiments.3.The BMP-1 gene was introduced into rabbit BMSCs by the lentivirus transfection vector,and the transfection efficiency was detected by fluorescence microscopy and Western blotting.The results indicated that the BMP-1 gene was successfully transfected,and the expression of it in of BMP-1 after transfection was significantly increased compared with that of the control group.The BMSCs transfected with BMP-1 were better than the control group in terms of cell activity and osteogenesis。4.BMSCs polymer modified with BMP-1 gene can effectively promote the formation of new bone in the process of rapid distraction osteogenesis of the mandible,compensate for the poor osteogenesis caused by rapid distraction,and provide a new idea for shortening the treatment cycle of distraction osteogenesis in the clinic. |
