| Objective : The incidence of femoral shaft fractures in children is5.82/100,000-45.5/100,000,which is higher in boys than in girls.Infants and children have a strong ability in fracture healing,which means the fractures can be cured after standardized treatment,and generally leave no serious sequelae.However,overgrowth is a common biological phenomenon in children with femoral shaft fractures,which leads to leg length discrepancy.The discrepancies of the lower limbs reported in the literature are varied,with an average of about 7-10 mm.Large leg length discrepancies may lead to limp in adolescence and increase the incidence of knee arthritis and chronic low back pain in adulthood.C laudication can be corrected with heels in children with mild symptoms.However,some children with large discrepancies need correcting by temporary epiphyseal block or limb lengthening.It is generally believed that the younger the child suffers a femoral shaft fracture,the more obvious the overgrowth phenomenon is,which may be related to its own growth potential,but the specific mechanism is still unclear.Some researchers also believed that overgrowth is related to gender,fracture location,and fixation methods.Radionuclide labeling studies found that the growth plate of the affected limb of fractured animals widened,with cell column arrangement disordered,proliferation accelerating,and calcification rate increas ing.An imaging study confirmed that the acceleration of distal femoral growth plate in children with femoral shaft fractures lead to overgrowth.Our group also observed that the distances between the Harris line of the distal femur and the growth plate were wider than that of the healthy side in clinical cases in children with overgrowth of the femur.Growth plate was a key part leading to overgrowth,but its biological mechanism was still unclear.By establishing an animal model of overgrowth after femoral shaft fracture,the cartilages of distal femoral growth plate were collected and TMT proteomic analysis was performed to find different proteins due to the limitation of human body materials.Next,we intended to explore the biological mechanism of overgrowth after femoral shaft fracture.Methods: Through the construction of animal models,we researched on the phenomenon of overgrowth after fracture and analysis of its molecular biological mechanisms.Ninety male 6-week-old Japanese white rabbits were operated using the method of stripping the femoral shaft periosteum as experimental group.The right side was the operated side and the left side was the sham-operated side.At 2,4,8weeks and 6 months after surgery,the total lengths of the femur on the operated side and the sham-operated side were measured.In addition,twenty 10-week-old Japanese white rabbits of the same sex were taken as a blank control,and the bilateral femur lengths were measured as well.Distal femurs of experimental group were taken,paraffin sections were made,and the width of the growth plates were measured by safranin-O-fast green staining.TUNEL staining was performed to observe the apoptosis of the growth plate chondrocytes.The distal femoral growth plates were taken 4 weeks after the operation for TMT quantitative proteomic analysis.The differential proteins were analyzed by Gene Ontology analysis and KEGG analysis to find the expression changes of related proteins.Proteins in growth plates were extracted to detect the corresponding differentially expressed proteins.Results: The bilateral femur length discrepancy(right minus left)of the blank control group was-0.025±0.26 mm.At 2,4,8 weeks and 6 months after surgery,bilateral femur length discrepancies(operated side minus sham-operated side)in the experimental group were 0.09±0.31 mm(P=0.264),0.62±0.63 mm(P <0.001),0.66±0.26 mm(P <0.001),and 0.68±0.14mm(P <0.001),respectively.Safranin-O-fast green staining showed no obvious morphological changes of the growth plate chondrocytes on the operated sides.The width of the growth plate cartilage on the operation side was 434.9±179.1μm,with the sham-operated side326.4±118.0μm(P=0.018).TUNEL staining showed that hypertrophic chondrocytes in the cartilage hypertrophy layer on the operation side contained an increased apoptosis compared with the sham-operated side.TMT proteomics identified a total of 283 differential proteins for bilateral growth plate cartilage 4 weeks after surgery.GO analysis indicated that the differential proteins were enriched in cell metabolism,intracellular structure,organic cyclic compound binding,etc.KEGG enrichment suggested differential protein pathways focused on ribosomes,protein transcription,spliceosome,apoptosis and protein processing in the endoplasmic reticulum.Western blot detection indicated that type Ⅹ collagen,matrix metal oproteinase-13 and histone deacetylase 1 were up-regulated in the overgrown distal femoral growth plate cartilage.Conclusions: 1.Circular stripping of femoral shaft periosteum can be used to construct an animal model of overgrowth after femoral shaft fracture.2.Growth plates showed active proliferations after long bone fractures,with transformation of hypertrophic chondrocytes into bone accelerated,which was one of the main reasons of overgrowth.3.Overgrowth after long bone fractures was potentially related to the signaling pathways of cell metabolism and development,such as type Ⅹ collagen,matrix metal oproteinase-13 and histone deacetylase 1,may be involved in the overgrowth. |
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