The Expression Of Collagen Ⅰ And Ⅲ,Biglycan And Decorin In The Hip Capsule And Ligamentum Teres Of The Patients With Developmental Dysplasia Of The Hip(DDH)

Developmental dysplasia of the hip(DDH) is a spectrum of disorders of development of the hip that present in different forms at different ages. It is a common deformity in pediatric orthopedics with the incidence 1-1.5‰in live birth. The common etiology of DDH is excessive laxity of the hip joint, which fails to maintain the femoral head within the acetabulum. During the open reduction of DDH, the laxity of the capsule and ligament can also be verified. So it is well known that hip laxity is one of the major contributory factor which would lead to DDH.Joint Hypermobility(JHM) or Laxity can occur in a single joint,or it can be more widespread throughout the body. In the main, it is thought genetically determined, those affected having loose connective tissue- in particular, ligaments and articular capsules. However some researchers insisted that joint hypermobility and genetic collagen disorders might be unrelated. Till now , three classic diseases have been known as genetic collagen disorders: 1) Marfan syndrome and congenital contractural arachnodactily: Mutations in the fibrillin genes, FIB 1 and FIB 2; 2) Osteogenesis imperfecta: Over 200 separate mutations in genes encoding type 1 collagen, COL 1a1 and COL1a2; 3) Ehlers- Danlos syndrome: About 80 mutations have been described in COL3a1. Studies of laxity and congenital dislocation of the hip in patients and families have also noted an apparent association.The stability of the hip joint depends upon the shape of the articular surfaces and also on the soft tissue of the capsule and the ligamentum teres. The capsule and ligamentum teres are the main tissues which hold the hip stable in neonates and infants. They are composed of kinds of collagens and proteoglycans which play an important role in maintaining biomechanical force in these connective tissues. Previously there were several reports on the collagen content of hip joint of DDH, but the results were contradictory. Biochemical studies of DDH capsules of patients aged 1-4 year suggest that the amount of type III compared with type I collagen is reduced , but increased amounts of type III collagen were found in the umbilical cords of newborn DDH babies . There is a similar unresolved discrepancy in values of collagen fibril diameters.The aims of this study were at investigating the location and distribution of collagen I and III by immunohistochemistry, expression of collagen,biglycan and decorin at mRNA and(or) protein level. We try to reveal the mechanism of joint laxity in DDH at molecular level and make a further exploration in the etiology of DDH.Materials and Methods1. Sample collection and treatmentCapsule of 33 patients and ligamentum teres of 12 patients were collected from operative resection for open reduction of DDH. Site-matched capsule and ligamentum teres of 10 normal children dead from accident were collected by biopsy. To make paired control of same sex and age between DDH group and Normal Control group , we obtained 9 pairs for hip capsule and 6 pairs for ligamentum teres. The samples were chopped into small pieces ,some were stored under -80℃and others were fixed in 4 % formaldehydum polymerisatum, then were dehydrated embedded in wax within 24 hours and 5-μm slices were made.2. Expermental methods2.1 Immunohistochemistry(SABC method): To detect the location and distribution of COL1a1 and COL3a1 in the capsule and ligamentum teres, furthermore semi-quantify the content of them.2.2 RT-PCR method: To detect the COL1a1,COL3a1,Biglycan and Decorin in the capsule and ligamentum teres at mRNA level. GAPDH was used as a house-keeping gene.2.3 Western-Blot method:To detect the COL1a1 and COL3a1 in the capsule and ligamentum teres at protein level.β-actin was used as a protein loading control.3. Statistical analysisSPSS version 10.0 for Windows was used to perform statistical analysis with all data expressed as x±s. The Pearson method was used for analysis of correlation among patient age, percentage of positive fibroblast and gray-scale density of positive tissues. The Paired T test was conducted for paired data. A p value of <0.05 was considered significant.Results1. The location,distribution and content of COL I andCOLlII1.1 In the capsule(1) COL I : High density expression was observed in the synovial layer with fibroblast and collagen I fibers regularly arranged parallel to the joint surface.There were decreased expression in the fibrous layer of the capsule with wave-like or multipolar collagen I fibers disposed in which fibroblasts dispersed. There were significant difference in the percentage of positive fibroblast and gray-scale density in the fibrous layer between DDH group and Normal Control group (p<0.05).(2) COLIII : Of all the cases, There were only 3 positive staining of 3 elder female children in DDH group.2 cases were pericellular brown staining of fibroblast and 1 case for grid-like staining in the matrix.1.2 In the ligamentum teres(1) COL I : High density expression was observed in the ligament sheath with collagen I fibers regularly arranged parallel to the synovial surface. There were decreased expression within the ligament with wave-like or multipolar collagen I fibers disposed in which fibroblasts dispersed. There were significant difference in the percentage of positive fibroblast and gray-scale density in the fibrous layer between DDH group and Normal Control group (p<0.05).(2) COLIII: For some reason, there were no positive staining for COLIII in either group.2. COL1a1,COL3a1 and Biglycan expression at mRNA level.2.1 In the capsule: COL I a1,COLIIIa1,Biglycan and Decorin expression were decreased at mRNA level in the DDH group compared to the control group(p<0.01).2.2 In the ligamentum teres: COL1a1,Biglycan and Decorin expression were decreased at mRNA level in the DDH group compared to the control group(p<0.01). There were no significant difference for COL3a1 between DDH and control group(p>0.05).3. COL1a1 and COL3a1 expression at protein level.3.1 In the capsule: COL1a1 protein expression were decreased in the DDH group compared to the control group(p<0.01). There were no significant difference for COL3a1 between DDH and control group(p>0.05).3.2 In the ligamentum teres: COL1a1 protein expression were decreased in the DDH group compared to the control group(p<0.01). There were no significant difference for COL3a1 between DDH and control group(p>0.05).Conclusions1. Duringthe period between infant and impuberal child,the percentage of active collagen I -secreting fibroblast in the capsule and ligamentum teres of DDH group was lower than that of Normal Control, suggesting decreased productive ability of collagen I of child with DDH.2. The decreased collagen I expression at mRNA and protein level in the capsule and ligamentum teres of children with DDH may probably lead to hip joint laxity in DDH.3. Hip joint laxity in DDH may be independent of the content of collagen III. 4. The decreased biglycan and decorin expression at mRNA level in the capsule and ligamentum teres of children with DDH may probably take a role in the mechanism of hip joint laxity in DDH.