| Background Shortening of the unilateral lower limb deformity is one of the reasons for the disabled which can leads to abnormal gait,degenerative weight-bearing joints osteoarthritis and scoliosis.In the process of bone lengthening,the key factors of bone formation is the strength of tensile force and technology of bone osteotomy,and frequency and speed of tensile force must be appropriate,which increases periodically on the basis of continuous strain,otherwise it may lead to the complications of poor bone formation and fracture healing in advance.When the strength of the tensile force is equal,the process and prognosis of bone lengthening,affected by surgical osteotomy procedure,is not the same.It is not known that how to transfer the impetus of distraction to the message of bone regeneration in limb lengthening at present,as well as the molecular mechanism of bone lengthening is not clear.Objective Respectively affected by tensile force with different strength which increased periodically and continuously,to study the proliferation of osteoblast,the effects on the expression of osteocalcin m RNA and the expression of inducible nitric oxide synthase(iNOS)m RNA of osteoblast cultured in vitro.At the same strength of tensile force,to explore the dynamic expression of inducible nitric oxide synthase in human serum during operative bone lengthening.To study the effects of tensile force strength on the proliferation of osteoblast and the molecular mechanism of distraction osteogenesis and bone lengthening.At the same strength of tensile force in bone lengthening,to explore the differences between bone mineral density,mineralization time and healing index of three groups in speed and quality of new bone formation.In group A,it had been implemented the only corticotomy of tibia at the proximal mataphysis after stripping on the outside of periosteum.In group B,it had been implemented the only corticotomy of tibia at the proximal mataphysis after stripping below periosteum.In group C,it had been implemented the whole osteotomy of tibia at the proximal mataphysis after stripping below periosteum.At the same time to provide good technical means for bone lengthening of clinical limb balanced operation.The first part: Effects of tensile force on the proliferation of osteoblast and expression of osteogenesis-related genesThe first experiment: Effects of periodical strain on the proliferation of osteoblast and expression of osteocalcin m RNAMethods The osteoblast was collected from newborn SD rats,skull and cultured,and then randomly divided into 4 groups(8 samples for each group):group A,group B,group C and group D.All osteoblast was cultured for 24 hours which had been adherent.In group A,the cells were treated with a periodical tensile force of 1000mstrain.In group B,the cells were treated with a periodical tensile force of 2000mstrain.In group C,the cells were treated with a periodical tensile force of 3000mstrain.In group D,the cells were cultured not to add any force.The duration of mechanical stimulation was 12 hours and the frequency of periodical tensile force was 0.2Hz in all the four groups.The cells were collected and total RNA were obtained after being treated with strain of 12 hours.The expression of osteocalcin m RNA of osteoblast was analysed with Reverse Transcription-Polymerase Chain Reaction(RT-PCR).The amount of osteocalcin in culture medium was detected by the method of radioimmunoassay.The ratio of proliferation of osteoblast was measured using MTT reduction assay.Results The expression of osteocalcin m RNA in group A,B,C and D was 0.421±0.022,0.446±0.015,0.361±0.018 and 0.415±0.014 respectively.The level of osteocalcin in the culture medium in group A,B,C and D was(3.201±0.216)ng/ml,(3.457±0.096)ng/ml,(2.641±0.019)ng/ml and(3.159±0.322)ng/ml respectively.The ratio of osteoblast proliferation in group A,B,C and D was 0.2869±0.0079,0.2971±0.0105,0.2778±0.0084 and 0.2861±0.0125 respectively.Compard with group D,there were significant differences of the expression of osteocalcin m RNA,the concentration of osteocalcin and ratio of osteoblast proliferation in group B and group C(P<0.05).There was no significant defference between group A and group D(p>0.05).The second experiment: Effects of continuous strain on the proliferation of osteoblast and the expression of inducible nitric oxide synthase m RNAMethods The osteoblast was collected from newborn SD rats,skull and cultured,and then randomly divided into 4 groups(8 samples for each group): group A,group B,group C and group D.All osteoblast was cultured for 24 hours which had been adherent.In group A,the cells were treated with a continuous tensile force of 1000 m strain.In group B,the cells were treated with a continuous tensile force of 2000 m strain.In group C,the cells were treated with a continuous tensile force of 3000mstrain.In group D,the cells were cultured not to add any force.The duration of mechanical stimulation was 48 hours and the frequency of tensile force was 0Hz in all the four groups.The ratio of proliferation of osteoblast was measured using MTT reduction assay.The cells were collected and the total RNA were obtained;The expression of iNOS m RNA was analysed with Reverse Transcription-Polymerase Chain Reaction,and the amount of NO in culture medium was detected by the method of nitric acid reduction enzyme.Results The ratio of osteoblast proliferation in group A,B,C and D was 0.2823±0.0138,0.3751±0.0408,0.2159±0.0154,0.2796±0.0222 respectively.The expression of iNOS m RNA in group A,B,C and D was 0.490±0.011,0.543±0.048,0.457±0.012,0.486±0.009 respectively.The level of NO in the culture medium was(14.47±0.39)μmol/L,(16.47±0.38)μmol/L,(12.28±0.41)μmol/L,(14.32±0.37)μmol/L respectively.Compard with group D,there were significant differences of the ratio of proliferation of osteoblast,the expression of iNOS m RNA,level of NO in group B and group C(P<0.05).There was no significant defference between group A and group D(p>0.05).The second part:Tensile force effects on the concentration of inducible nitric oxide synthase in human serumMethods From October 2012 to June 2015,12 patients with unilateralosteotomy of tibia and fibula and being used circular external fixator were involved in bone lengthing group.Using human iNOS ELISA kit,The serum concentration of inducible nitric oxide synthase were measured respectively by double antibody sandwich method at 3 days preoperatively,at 3,7,8,11,30 days postoperatively,stopping bone lengthing,at 3,15,30 days after stopping bone lengthing,removing the external lengthing apparatus and at 30 days after removing the external lengthing apparatus.In the control group were also 12 patients,whose age were similar to the bone lengthing group and the serum concentration of inducible nitric oxide synthase were measured respectively at the same time as the bone lengthing patients.Results The serum concentration of iNOS in bone lengthing group began to increase at the first day after the distraction and the peak values at the time of stopping bone lengthing.The concentration of iNOS in the bone lengthing group were significantly higher than that of the control group at the every time point from the first day after the distraction to the day of stopping bone lengthing(p<0.01).Compard to that of control group,the serum concentration of iNOS in the bone lengthing group at 3 days after stopping bone lengthing was also higher,There was significant defference between the two groups(P<0.05).While no defference(p>0.05)was observed in two groups at the time of 3 days before operation,3,7 days after operation,15,30 days after stopping bone lengthing,removing the external lengthing apparatus and 30 days after removing the external lengthing apparatus.The third part: The clinical effects of corticotomy and complete osteotomy on the quality of new bone formation at the same strength of tensile force in bone lengtheningMethods Ninety-six patients with unilateral tibia and fibula shortening deformity correction by osteotomy lengthing technique using circular external fixator were randomly divided into three groups: group A,group B and group C.There was all the same in strength of tensile force and pathogenesis,as well as almost the same in osteotomy position at tibia and fibula whose lengthening length was 4.9-5.1cm.The defference was injuries of periosteum,cancellus and bone marrow in the three ways of tibial osteotomy.In group A,it had been implemented the only corticotomy of tibia after stripping on the outside of periosteum.In group B,it had been implemented the only corticotomy of tibia after stripping below periosteum.In group C,it had been implemented the complete osteotomy of tibia after stripping below periosteum.To explore the differences of speed and quality of new bone formation in bone mineral density,mineralization time and healing index.Results1 In comparison with bone mineral density of new bone formation in three groups at 30 days and 60 days of lengthening period,30 days and 90 days of mineralization period,30 days and 60 days of reconstruction period,There was no significant defference between group A and group B(p>0.05).The bone mineral density of group A is higher than that of group C,there was significant defference between the two groups(P<0.05).The bone mineral density of group B is higher than that of group C,there was significant defference between the two groups(P<0.05).2 In comparison with mineralization time of new bone formation in three groups,There was no significant defference between group A and group B(p>0.05).The mineralization time of group A is lower than that of group C,there was significant defference between the two groups(P<0.05).The mineralization time of group B is lower than that of group C,there was significant defference between the two groups(P<0.05).3 In comparison with healing index of new bone formation in three groups,there was no significant defference between group A and group B(p>0.05).The healing index of group A is lower than that of group C,there was significant defference between the two groups(P<0.05).The healing index of group B is lower than that of group C,there was significant defference between the two groups(P<0.05).Conclusions1 It demonstrates that defferent strength of periodical tensile force have defferent results on the proliferation of osteoblast and expression of osteocalcin m RNA.Under right impetus of periodical strain,transfering the higher expression of osteocalcin m RNA to the message of the proliferation of osteoblast may be one of the molecular mechanism of distraction osteogenesis and bone lengthening.2 It demonstrates that defferent strength of continuous tensile force have defferent results on the proliferation of osteoblast and expression of iNOS m RNA.Under right impetus of continuous strain,transfering the higher expression of iNOS m RNA to the message of the proliferation of osteoblast is the other one of the molecular mechanism of distraction osteogenesis and bone lengthening.3 The higher expression of inducible nitric oxide synthase in human serum increased by impetus of mechanical force in distraction osteogenesis is one of the biological mechanisms of bone lengthing.4 At the same strength of tensile force in bone lengthening,the quality of new bone formation in corticotomy patients is better than that of complete osteotomy at the mataphysis of proximal tibia.5.Between stripping on the outside of periosteum and stripping below periosteum,there was no significant defference in new bone formation of tibia corticotomy lengthing technique. |
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