Fate Of The Inverted Limbus In Children After Closed Reduction With Developmental Dysplasia Of The Hip And Its Effect On The Development Of Acetabulum And Femoral Head:Clinical And Experimental Observation

Objective: Developmental dysplasia of the hip(DDH)is a common hip disease in pediatric orthopedics,closed reduction is the preferred treatment option for children aged6 to 18 months clinically."Concentric reduction" of the acetabulum and femoral head is the gold standard for successful closed reduction treatment of DDH and a prerequisite for the normal development of the hip joint.However,due to the soft tissue obstruction,it is often impossible to achieve complete concentric reduction.Among them,limbus inversion is a common pathological change of DDH,and it is also a pathological factor which obstruct the concentric reduction.Is the limbus inversion after closed reduction can be acceptable after closed reduction? That is still in controversy.In the early stage,we conducted a series of magnetic resonance imaging(MRI)follow-up observations on children with DDH treated with closed reduction found that the inverted limbus after closed reduction was not completely absorbed,but evolved into a thin layer of fibrous tissue embedded between the acetabulum and femoral head.Long-term outcome and its effect on acetabular and femoral head development of this phenomenon are still unclear.In addition,the inverted limbus is also one of the causes of primary acetabular dysplasia and early osteoarthritis.The mechanism may be related to the relative reduction of the internal volume of the acetabulum and changes in the pressure on the acetabular and femoral head cartilage.Many studies have confirmed that mechanical stimulation plays an important role in gene expression,extracellular matrix synthesis and transmembrane transduction in bone formation.The transforming growth factor-β(TGF-β)signaling pathway plays an important role in the process of bone formation,remodeling and maintenance.The TGF-β signal is mainly transmitted to the nucleus through the intracellular medium Smads,and then can regulates the expression of Runx.Runx is a multifunctional transcription factor containing Runt homologous region,which can regulate transcription by binding to DNA or protein.Runx2 is a member of its family and plays an important role in the process of cartilage formation and endochondral ossification.Therefore,the purpose of this study is to use prospective clinical observations to evaluate the morphological evolution of the limbus and the effect on the development of the acetabulum and femoral head through a series of MRI in children with limbus inversion after DDH closed reduction.Then an animal model of the inverted limbus was established to observe the changes in the microscopic morphology of the acetabular and femoral head cartilage tissues and the changes in the expression of proteins related to the TGF-β signaling pathway after the limbus inversion.Methods:This research is divided into three parts.1.The fate of the inverted limbus in children after closed reduction with developmental dysplasia of the hip and its effect on the development of acetabulum and femoral head:clinical observation.From March 2014 to June 2015,the follow-up data of children with DDH underwent closed reduction were collected prospectively.Patients showed limbus inversion on MRI after reduction were selected as research object.According to whether the inverted limbus was residual at the last follow-up,patients were divided into residual inversion group and spontaneous resolution group.The IHDI classification,acetabular index(AI)and cartilaginous acetabular index(CAI)were compared between the two groups before reduction.The magnitude of limbus inversion after reduction,the AI,CAI,central edge angle(CEA),height-to-width index(HWI)and avascular necrosis(AVN)at the last follow-up were compared.2.Pathological changes in epiphyseal plate of the acetabulum and femoral head and changes in the expression of TGF-β/Smad signaling pathway related proteins in the animal model of limbus inversion.We interpositioned the meniscus of 5-week-old rabbits into the hip joints to simulate limbus inversion following closed reduction for DDH.Imaging and histological observations were performed at 4 weeks and 8 weeks after modeling.The AI was measured on the anterior and posterior pelvic radiographs to evaluate the development of the acetabulum,and the success of the modeling was evaluated on the MRI.After the imaging examination was completed,the animals were sacrificed,paraffin sections were made,and the proteins were extracted for subsequent experiments.Histological sections were stained with HE to assess the morphology and number of chondrocytes in the epiphyseal plate and measured the width of the epiphyseal plate of the femoral head.Immunohistochemistry and Western blot analyses were used to examine the expression of alkaline phosphatase(ALP),vascular endothelial factor(VEGF),Runx2,Smad2,and TGF-β1.3.Pathological changes in articular cartilage of acetabulum and femoral head and changes in the expression of the markers of early degeneration of the cartilage in the animal model of limbus inversion.The production of the animal models and specimens obtained are the same as in the previous description.Histological sections were stained with Safranin-O-Fast Green and Toluidine Blue and scored in accordance with the modified Mankin system.Scanning electron microscopy(SEM)and transmission electron microscopy(TEM)were used to examine the ultrastructure of the articular cartilage in acetabular and femoral head.TUNEL staining was used to evaluate the rates of chondrocyte apoptosis.Immunohistochemistry and Western blot analyses were used to examine the expression of type X collagen(Col-X),matrix metalloproteinase 13(MMP-13).Results:1.The residual inversion group before reduction was classified into 11 hips of type III and type IV by IHDI;3 hips of type II and 4 hips of type III in the spontaneous resolution group(P<0.001).The preoperative AI and CAI of the two groups were not statistically different(P=0.261,0.843).2.After reduction,the magnitude of limbus inversion was 55.10%±9.60% and43.92%±17.59%(P=0.005)in two groups.There was no significant difference in the magnitude of limbus inversion in the residual inversion group after reduction compared with that at the last follow-up(P=0.183).3.At the last follow-up,the AI was 24.73°±5.72° in the residual inversion group and19.57°±2.23° in the spontaneous resolution group(P=0.029);the CAI was 16.68°±4.05°and 14.71°±2.36°respectively(P=0.236);the CEA were 20.77°±8.82° and18.86°±6.89°respectively(P=0.605);the HWI were 44.13±4.24 and 48.11±6.84,respectively(P=0.074).4.At the last follow-up,5 hips(22.73%)in the residual inversion group had AVN,all of which were Kalamchi-Mac EwenⅠ.There was no AVN in the spontaneous resolution group.5.The animal model of limbus inversion was successfully constructed,and its imaging performance was similar to that in clinical studies.There was no statistically significant difference in AI between the experimental group and the sham group at 4 weeks after modeling(P=0.079),but the AI of the experimental group was increased significantly at8 weeks after the modeling(P<0.001).6.The meniscus in the experimental group gradually became thinner with the extension of the observation time.Compared with the sham group,the acetabular cartilage surface of the experimental group was rougher,the transverse diameter increased,and the acetabular fossa became shallower;the cartilage surface of the femoral head also became thinner and rougher,and the shape of osteoepiphysis becomes flat.7.Compared with the sham group,the expressions of TGF-β1 and Smad2 were up-regulated in the cartilage of epiphyseal plate in acetabulum and femoral head,while the expressions of Runx2,ALP and VEGF were down-regulated.8.Compared with the sham group,experimental group has obvious cartilage degeneration on the surface of the articular cartilage of the acetabulum and femoral head,which is manifested as surface fibrosis;hypertrophy and “clone” of chondrocytes;increased apoptosis rates as well as cartilage matrix disorder.The expressions of Col-Ⅹand MMP-13 were up-regulated.Conclusion:1.After DDH closed reduction,most of the inverted labrum cannot be absorbed in a short period of time.Instead,it evolves into a thin layer of fibrous tissue embedded between the acetabulum and femoral head,which may delay the ossification of the acetabular cartilage.2.Long-term invertion of the limbus will activate the TGF-β/Smad pathway of the hypertrophic chondrocytes in the epiphyseal plate of acetabulum and femoral head,inhibit the expression of Runx2,and reduce the expression of VEGF and ALP in the hypertrophic chondrocytes,leading to the cartilage ossification delayed.3.Long-term invertion of the limbus will destroy the fibrous structure of articular cartilage on the surface of the acetabulum and femoral head,make chondrocytes hypertrophy differentiation and increased the apoptosis rate like early OA manifestations.Besides,the expression of Col-Ⅹ and MMP-13 were up-regulated.