Versican contributes to ligament formation of knee joints.

Versican is a large proteoglycan in the extracellular matrix. During embryonic stages, it plays a crucial role in the development of cartilage, heart, and dermis. Previously, we reported that Prx1-Vcan conditional knockout mice, lacking Vcan expression in mesenchymal condensation areas of the limb bud, show the impaired joint formation and delayed cartilage development. Here, we investigated their phenotype in adults and found that they develop swelling of the knee joint. Histologically, their newborn joint exhibited impaired formation of both anterior and posterior cruciate ligaments. Immunostaining revealed a decrease in scleraxis-positive cells in both articular cartilage and ligament of Prx1-Vcan knee joint, spotty patterns of type I collagen, and the presence of type II collagen concomitant with the absence of versican expression. These results suggest that versican expression during the perinatal period is required for cruciate ligaments' formation and that its depletion affects joint function in later ages.

Surgical treatment of posterior cruciate ligament lesions does not cause growth disturbances in pediatric patients.

PURPOSE: The aim of the present study was to describe epidemiology, management and outcome of pediatric and adolescent patients with posterior cruciate ligament (PCL) injuries. METHODS: Sixteen patients of less than 18 years of age with 7 PCL avulsion fractures and 9 PCL tears were included over a 10-year period. Trauma mechanism, additional injuries and treatment methods were analyzed. Follow-up examination included range of motion and ability to perform squats. Pedi-IKDC and Lysholm score were obtained and posterior shift was measured in kneeling view radiographs and compared to the contralateral side. Patients were grouped into pediatric patients with open physes at the time surgery and adolescent patients with closing or closed physes. In case of open physes, growth disturbances were assessed. RESULTS: Six of the treated patients (median age 12.5 years, range 10-13) had open physes at time of surgery. Five of those sustained avulsion fractures and treatment consisted of open reduction and screw fixation in four cases and graft reconstruction in one case. One patient sustained a PCL tear and underwent graft reconstruction. Follow-up at a median of 71.5 months (range 62-100) did not reveal any growth disturbances. Median Pedi-IKDC was 71.9 (range 51.7-92.1), median Lysholm score was 81.5 (range 66-88) and median posterior shift difference was 2.5 mm (range 0-11). The remaining 10 patients (median age 16 years, range 14-17) had closing/closed physis at the time of operation. Two patients presented with avulsion fractures treated with open reduction and screw fixation and 8 patients sustained PCL tears treated with graft reconstruction. At a median follow-up of 69.5 months (range 11-112), median Pedi-IKDC was 86.8 (range 36.8-97.7), median Lysholm score was 84.0 (range 45-95) and median posterior shift difference was 4 mm (range 0-15). CONCLUSIONS: In our small number of pediatric patients with PCL injuries, open reduction and epiphyseal screw fixation of displaced avulsed fractures and steep tunnel drilling in case of PCL reconstruction did not cause growth disturbances. Nevertheless, long-term functional impairment should be expected and close follow-up has to be recommended. LEVEL OF EVIDENCE: Therapeutic, Level IV.

Orientation changes in the cruciate ligaments of the knee during skeletal growth: A porcine model.

Musculoskeletal injuries in pediatric patients are on the rise, including significant increases in anterior cruciate ligament (ACL) injuries. Previous studies have found major anatomical changes during skeletal growth in the soft tissues of the knee. Specifically, the ACL and the posterior cruciate ligament (PCL) change in their relative orientation to the tibial plateau throughout growth. In order to develop age-specific treatments for ACL injuries, the purpose of this study was to characterize orientation changes in the cruciate ligaments of the Yorkshire pig, a common pre-clinical model, during skeletal growth in order to verify the applicability of this model for pediatric musculoskeletal studies. Hind limbs were isolated from female Yorkshire pigs ranging in age from newborn to late adolescence and were then imaged using high field strength magnetic resonance imaging. Orientation changes were quantified from the magnetic resonance images using image segmentation software. Statistically significant increases were found in the coronal and sagittal angles of the ACL relative to the tibial plateau during pre-adolescent growth. Additional changes were observed in the PCL angle, Blumensaat angle, intercondylar roof angle, and the aspect ratio of the intercondylar notch. Only the sagittal angle of the ACL relative to the tibial plateau experienced statistically significant changes through late adolescence. The age-dependent properties of the ACL and PCL in the female pig mirrored results found in female human patients, suggesting that the porcine model may provide a pre-clinical platform to study the cruciate ligaments during skeletal growth. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2725-2732, 2017.

Copy number variation in CEP57L1 predisposes to congenital absence of bilateral ACL and PCL ligaments.

BACKGROUND: Absence of the anterior (ACL) or posterior cruciate ligament (PCL) are rare congenital malformations that result in knee joint instability, with a prevalence of 1.7 per 100,000 live births and can be associated with other lower-limb abnormalities such as ACL agnesia and absence of the menisci of the knee. While a few cases of absence of ACL/PCL are reported in the literature, a number of large familial case series of related conditions such as ACL agnesia suggest a potential underlying monogenic etiology. We performed whole exome sequencing of a family with two individuals affected by ACL/PCL. RESULTS: We identified copy number variation (CNV) deletion impacting the exon sequences of CEP57L1, present in the affected mother and her affected daughter based on the exome sequencing data. The deletion was validated using quantitative PCR (qPCR), and the gene was confirmed to be expressed in ACL ligament tissue. Interestingly, we detected reduced expression of CEP57L1 in Epstein-Barr virus (EBV) cells from the two patients in comparison with healthy controls. Evaluation of 3D protein structure showed that the helix-binding sites of the protein remain intact with the deletion, but other functional binding sites related to microtubule attachment are missing. The specificity of the CNV deletion was confirmed by showing that it was absent in ~700 exome sequencing samples as well as in the database of genomic variations (DGV), a database containing large numbers of annotated CNVs from previous scientific reports. CONCLUSIONS: We identified a novel CNV deletion that was inherited through an autosomal dominant transmission from an affected mother to her affected daughter, both of whom suffered from the absence of the anterior and posterior cruciate ligaments of the knees.

MRI evaluation of the development of intercondylar notch width in children.

PURPOSE: The purpose of this study was to evaluate the development of intercondylar notch width in children using MRI and assesse the relationship between the cruciate ligaments and the width of the intercondylar notch during growth. METHODS: We reviewed MRIs and medical records of 76 children (age range 3.8-16.9) without derangements of the knee. Cases with knee joint instability and/or major trauma were excluded. On a standardized coronal image, measurements of the width of the intercondylar notch, knee, ACL and PCL were performed using image analysis software. The notch width index (NWI), ACL/notch, ACL/knee width, PCL/notch, and PCL/knee width were calculated. RESULTS: NWI showed statistical correlation with age. The highest values were found in the youngest children, and the lowest in the oldest group. While ACL/knee width, and PCL/knee width ratios remained proportional over time, the ACL/notch, and PCL/notch ratios increased with the age of the patient. CONCLUSION: NWI changes with age and decreases from childhood to adolescence. A relative decrease in NWI accounts for an increase in the ACL/notch and PCL/notch ratios with age. Studies about NWI in children should consider age as a cofactor for analysis.

Postnatal maturation of tendon, cruciate ligament, meniscus and articular cartilage: a histological study in sheep.

Orthopaedic basic science data on immature skeletons are rare in the literature. Since the number of knee injuries in young humans is steadily increasing, studies on immature animals such as sheep, which can be used as model systems are becoming more and more important. However, no baseline data are available on physiologic and morphologic changes during growth in the relevant tissues. In the present study, histomorphometric changes in the tendon of the musculus flexor digitalis superficialis, the cranial cruciate ligament, the medial meniscus and the articular cartilage of the medial femoral condyle were identified in sheep between the ages of 1 and 40 weeks postnatally. Profound changes in tissue composition during growth could be observed. A high cellularity in the early postnatal period decreases to a constant lower level after 18 weeks. Similar changes during postnatal growth could be observed for blood vessel density. Also, staining of alpha-smooth muscle actin (SMA) and vascular endothelial growth factor (VEGF) steadily decreased. In contrast, the number of components of extracellular matrix steadily increased in all tissues. The age of 18 weeks seems to be a threshold after which the tissue composition of the observed structures remains constant in this species.

Postnatal development of the cruciate ligament insertions in the rat knee. morphological evaluation and immunohistochemical study of collagens types I and II.

The postnatal structural remodelling and calcification patterns in the insertions (entheses) of both cruciate ligaments were studied in a rat model with histology and immunohistochemical analysis of collagens types I and II. In the neonate, both ligaments which labelled only for type I collagen attached to epiphyseal cartilage which solely labelled for collagen type II. The entheses calcified between days 20 and 35, and a subchondral bone plate formed under the entheses between days 30 and 55. Thus, within a period of 35 days the tissue to which the ligaments attached increased multifold in stiffness. Interestingly, the process of enthesial calcification and formation of compact bone did not happen simultaneously in both ligaments, not even synchronous at both ends of the same ligament or within a single insertion. This asynchronous calcification of the different knee ligament insertions may make the sudden change in mechanical environment at the entheses less dramatic for the ligaments and knee joint surfaces as anticipated from mechanical models. In addition, a fibrocartilaginous tissue, rich in collagen type II, formed in the ligament at a time when the epiphyseal cartilage was replaced by bone, and grew wider with time. The interposition of a fibrocartilaginous zone in the insertion may diminish the sudden change in stiffness between ligament soft tissue and hard bone.