Histological scoring of articular cartilage alone provides an incomplete picture of osteoarthritic disease progression.

PURPOSE: To ascertain whether molecular subcategories of disease progression exist within established histological grades of articular cartilage (AC). METHODS: Based on H&E and safranin-O staining of AC sections obtained from 18 knee arthroplasty surgeries, 30 samples ranging from Mankin Scoring System grade 1 through 5 were identified. Immunohistochemical (IHC) analysis for collagen type II and aggrecan was performed on serial sections of the paraffin-embedded AC samples. Six AC samples from each of the five Mankin Scoring System grades were examined. RESULTS: Significant IHC differences in collagen type II and aggrecan deposition were seen within AC samples from all five histological grades. The range of IHC differences in collagen type II and aggrecan increased with increasing histological grade. A change in the pattern of collagen type II deposition was observed in MG-3 AC that was consistent with a switch in collagen type II metabolism. CONCLUSIONS: IHC staining of collagen type II and aggrecan can identify differences within histological grades of AC that are consistent with the existence of molecular subcategories. These differences were detectable even within the lowest histological grades; therefore the use of IHC staining can further enhance and refine the scoring of AC deterioration in early osteoarthritis (OA). Furthermore, the changes seen in the deposition pattern for both aggrecan and collagen type II suggest that they could be used to monitor key molecular events in OA progression. These findings also underscore the need for the development of IHC scoring criteria.

[Molecular and genetic aspects of idiopathic scoliosis. Blood test for idiopathic scoliosis]. / Molekulare und genetische Aspekte der idiopathischen Skoliose. Bluttest bei idiopathischer Skoliose.

Spinal deformities, and particularly scoliosis, are the most frequent forms of orthopedic deformities in children and adolescents. About 1-6% of the population has scoliosis. This disorder leads to severe spinal deformities and predominantly affects adolescent girls.Although the multifactorial origin of adolescent idiopathic scoliosis (AIS) is broadly recognized, the genetic causes of AIS are still largely unknown. Our previous studies suggested a generalized dysfunction of melatonin transduction (the hormone that is primarily produced in the brain and epiphysis). In the meantime we have demonstrated that such a defect of signal transduction is caused by chemical alterations, which inactivate the function of the inhibitory G protein-coupled melatonin receptors. This discovery has led to the development of the first blood test to detect children without symptoms who are at risk of developing scoliosis. Since a single function (cellular reaction to melatonin) is determined, the unique advantage of this test is that it can be performed without knowledge of mutations in defective genes that could provoke the onset of AIS.

Examination of the failure properties of the anterior cruciate ligament during the estrous cycle.

The purpose of this study was to determine whether the mechanical properties of the rat anterior cruciate ligament (ACL) vary when tested in vitro at different stages of the estrous cycle. Sixty female rats were allocated to four groups according to their stage of the estrous cycle: diestrus (n=16), proestrus (n=17), estrus (n=13) and metestrus (n=14). Right hindlimbs were harvested for mechanical testing and left hindlimbs were harvested for immunohistochemical staining to confirm the presence of the estrogen receptor. Results from the first relaxation test showed a significant difference between the estrus and proestrus stage, which was not observed in a second subsequent relaxation test. Likewise, no significant differences were found when comparing failure load and stiffness between the different stages of the estrous cycle. These results suggest that normal physiological fluctuations in estrogen during the estrous cycle did not alter the failure properties of the rat ACL.

Stiffness characteristics of hamstring tendon graft fixation methods at the femoral site.

In ACL reconstruction, stiffness and strength of a tendon graft complex are important features for knee stability and rehabilitation. The fixation between tendon and bone is known to be one of the weakest components of the graft complex. We examined the tensile load-displacement characteristics of looped semitendinosus tendons in a porcine femoral tunnel. Two groups of six cadaveric semitendinosus tendons and porcine femurs were tested, secured with either an aperture or non-aperture fixation method. Constructs were tested at 1 mm/s until failure in a materials testing machine, which allowed force and displacement data to be recorded. The non-aperture fixation group was significantly less stiff for the first 4 mm of displacement and had significantly higher ultimate failure loads. Provided that adequate ultimate strength can be achieved, stiffness properties of a tendon graft will be improved by using aperture fixation in femoral-site ACL reconstruction.

Using magnetic resonance imaging to characterize pedicle asymmetry in both normal patients and patients with adolescent idiopathic scoliosis.

STUDY DESIGN: Morphometric analysis of vertebrae from normal patients and patients with adolescent idiopathic scoliosis. OBJECTIVES: To use magnetic resonance imaging to assess pedicle asymmetry in normal patients and patients with adolescent idiopathic scoliosis in the early stages of scoliosis development and to determine if patients with adolescent idiopathic scoliosis exhibited a consistent vertebral morphology. SUMMARY OF BACKGROUND DATA: To date, most studies of vertebral morphology in adolescent idiopathic scoliosis have produced conflicting data, especially on pedicle length, and have been conducted on patients in the late stages of scoliosis development, which may affect the patterns of vertebral morphology detected. Magnetic resonance imaging enables in vivo assessment of curves during development and permits improved acquisition of transverse images. METHODS: Magnetic resonance images of 76 pedicles from 8 normal patients and 80 pedicles from 10 patients with adolescent idiopathic scoliosis were examined retrospectively. Recorded parameters included pedicle lengths, pedicle widths, pedicle areas, pedicle perimeters, and lamina lengths. The extent and direction of asymmetry in vertebrae from normal patients and patients with adolescent idiopathic scoliosis were determined and compared. RESULTS: Normal patients displayed significant neural arch asymmetry, with the left sided measurements being greater. Patients with adolescent idiopathic scoliosis also displayed significant neural arch asymmetry; however, the longer pedicle was not consistently on the convexity or the concavity. CONCLUSIONS: The baseline used to assess adolescent idiopathic scoliosis vertebral morphology must take into consideration the extent and direction of normal vertebral asymmetry. The pattern of vertebral asymmetry seen inadolescent idiopathic scoliosis may depend on the specific cause of the disorder, with no consistent pattern evident when data from different causes are pooled together.

MRI characteristics of the neurocentral synchondrosis.

BACKGROUND AND OBJECTIVES: The neurocentral synchondrosis (NCS) is a cartilaginous growth plate that since the early 1900s has been implicated as a potential cause of adolescent idiopathic scoliosis (AIS). Previous studies have focused only on the closure age without characterizing normal NCS development. Using MRI, the normal development of the NCS image can be characterized, and the stages preceding the disappearance of this image can be specified. METHODS: A total of 405 NCSs were visualized in 11 normal pediatric patients using T1 and T2 transverse and sagittal MRI views. The images were correlated and the variety of images recorded to categorize the NCS into developmental stages. RESULTS: The development of the NCS was categorized into five developmental stages. The image of the NCS became absent in a specific pattern along the vertebral column, first in the cervical region (age 6), then in the lumbar region (age 12), and finally in the thoracic region (age 14). CONCLUSION: The normal development of the NCS at the level of individual vertebrae and also along the vertebral column as a whole was determined using MRI. These patterns of development are valuable and necessary to evaluate the role of the NCS in pathological conditions.

Development of the neurocentral junction as seen on magnetic resonance images.

The neurocentral junction (NCJ) is a cartilaginous growth plate in the vertebra that has been implicated as a potential cause of adolescent idiopathic scoliosis (AIS) since the early 1900s. Studies to date have focused on the age of closure without characterizing normal NCJ development. Using MRI, the normal development of the NCJ image can be determined and the stages preceding the disappearance of the NCJ image can be characterized. 405 NCJs from 11 pediatric patients were examined using MRI and the various images were categorized. NCJ development encompassed five stages, with a specific pattern of absence of the NCJ image noted in each vertebra and in the vertebral column as a whole. The image of the NCJ first became absent in the cervical region (age 6), then in the lumbar region (age 12) and finally in the thoracic region (age 14). These patterns of development serve as a baseline to evaluate NCJ pathology in conditions such as adolescent idiopathic scoliosis (AIS).

The components of the magnetic resonance image of the neurocentral junction.

The neurocentral junction (NCJ) is a cartilaginous growth plate located between the vertebral centrurn and the neural arch. In characterizing the age of closure of this growth plate, anatomic studies have suggested that the NCJ closes before age 10, whereas MRI studies have suggested that the NCJ does not fuse until adolescence In this study, gross anatomic and histologic sections were correlated with MR images to determine the components of the NCJ image. The NCJ image appeared as a thick white line that was shown to encompass the cartilage of the growth plate, the surrounding woven bone and a portion of the trabecular bone of the vertebra. Although the MR pixels were too large to completely resolve the tissues that surround the growth plate, MRI was shown to be a valuable technique of visualizing the NCJ cartilage and further MRI studies of the human NCJ are needed.

Preservation of the morphological and molecular stability of embryonic tissues.

There is presently great interest in using early embryonic tissues, particularly human tissue, for studies of protein and gene expression. Embryonic human tissue is very fragile, and delays often occur before it can be properly prepared for scientific study. Using chick embryos, we have studied the effects of delaying fixation or biochemical isolation on the preservation of cytological characteristics and biochemical molecules. Our study shows that by 60 min post-harvest, tissue morphology and immunofluorescence staining degrades, but the total mRNA profile remains stable. This study suggests that the time between removal of the tissue and fixation is critical to the results and that the critical time is much shorter for embryonic tissues than for more developed tissues. Our results have implications for all research where embryonic tissues are harvested but not processed immediately.

Pineal transplantation after pinealectomy in young chickens has no effect on the development of scoliosis.

STUDY DESIGN: Three experimental groups and one control group of chickens underwent different surgical procedures to determine the effects of pineal gland transplantation on the development of scoliosis. OBJECTIVE: To determine whether transplantation of the pineal gland to the body wall musculature maintains serum melatonin levels at normal values and prevents the development of scoliosis. SUMMARY OF BACKGROUND DATA: Scoliosis occurs consistently after pinealectomy in young chickens. Many characteristics of this scoliosis are similar to those seen in patients with adolescent idiopathic scoliosis. It is not clear whether the underlying mechanism is dependent on reduced levels of serum melatonin or some other aspect of the extensive surgery. METHODS: Four groups of chickens were selected: normal chickens, pinealectomized chickens, chickens that underwent simple cutting of the pineal stalk, and chickens that underwent transplantation of the pineal gland into the body wall. Development of scoliosis was determined from measurement of the Cobb angle from weekly radiographs. RESULTS: All of the experimental groups showed the same levels of incidence and the same patterns of scoliosis development. Serum melatonin levels were reduced to nearly zero in all the experimental groups for the duration of the experiment. Scoliosis developed in none of the normal chickens. CONCLUSIONS: Neither transplantation of the pineal gland into the body wall musculature nor simple cutting of the pineal stalk was able to maintain normal levels of serum melatonin because both procedures reduced levels to nearly zero. The incidence and pattern of scoliosis development in these groups were the same as those for the pinealectomized group. Reduction of serum melatonin levels remains a prerequisite for scoliosis development in young chickens.

The critical stage of pinealectomy surgery after which scoliosis is produced in young chickens.

STUDY DESIGN: Stages of the surgical procedure for pinealectomy in chickens were identified. Groups of chickens were selected for each stage. Scoliosis development was identified from radiographs. OBJECTIVES: To determine the critical stage of surgery for pinealectomy after which scoliosis develops in young chickens. SUMMARY OF BACKGROUND DATA: Pinealectomy in young chickens consistently produces scoliosis in young chickens that has many characteristics similar to those seen in patients with adolescent idiopathic scoliosis. The mechanism behind this phenomenon remains unknown. METHODS: Five distinct stages in the pinealectomy surgery were identified. Groups of chickens were selected to undergo surgery to represent these five stages. Scoliosis was determined from weekly radiographs. RESULTS: Cutting the pineal stalk was identified as the critical stage in the surgery after which scoliosis developed. The incidence of scoliosis did not increase after more extensive surgery in which the pineal bulb was removed from the skull. This stage was also correlated with a significant reduction of serum melatonin levels. CONCLUSIONS: Cutting the pineal stalk was identified as the critical stage of pinealectomy surgery after which scoliosis may develop. This stage was also correlated with the significant reduction of average serum melatonin levels. These results allow the focus of attention into the mechanism behind this phenomenon to center on the consequences of cutting the pineal stalk rather than total removal of the pineal gland.

Transforming growth factor-beta1 is a new form of tumor suppressor with true haploid insufficiency.

Components of the transforming growth factor-beta (TGF-beta) signal pathway function as classic tumor suppressors, but the role of the TGF-betas themselves is less clear. Here we show that mice heterozygous for deletion of the TGF-beta1 gene express only 10-30% of wild-type TGF-beta1 protein levels. Although grossly normal, these mice have a subtly altered proliferative phenotype, with increased cell turnover in the liver and lung. Treatment of these mice with chemical carcinogens resulted in enhanced tumorigenesis when compared with wild-type littermates. However, tumors in the heterozygous mice did not lose the remaining wild-type TGF-beta1 allele, indicating that the TGF-beta1 ligand is a new form of tumor suppressor that shows true haploid insufficiency in its ability to protect against tumorigenesis.

Melatonin levels in idiopathic scoliosis. Diurnal and nocturnal serum melatonin levels in girls with adolescent idiopathic scoliosis.

STUDY DESIGN: Matched pairs of adolescent girls were used to compare serum melatonin levels in adolescent patients and control subjects with idiopathic scoliosis during the day and in the middle of the night. OBJECTIVES: To compare serum melatonin levels in patients with adolescent idiopathic scoliosis and matched control subjects during the day and in the middle of the night. SUMMARY OF BACKGROUND DATA: Recent studies using the chick as the animal model have suggested that the pineal gland and its main product, melatonin, might be involved in the cause of scoliosis. There have been no studies of melatonin levels in patients with adolescent idiopathic scoliosis. METHODS: Blood was collected from seven adolescent girls with idiopathic scoliosis and a group of seven age-matched control subjects. Two samples were collected, one in the middle of the day and one in the middle of the night, to examine the diurnal variation of melatonin production. Serum melatonin levels were measured using a radioimmunoassay technique. RESULTS: No significant differences were found in serum melatonin levels between experimental and control groups either during the day, when melatonin levels were low, or during the night, when melatonin levels were high. CONCLUSIONS: Whereas pinealectomy in young chickens leads to reduced melatonin levels and the development of scoliosis, the results of this study suggest that melatonin levels in mature patients who already have severe scoliosis do not differ from healthy subjects. Whether melatonin levels differ in humans between healthy subjects and patients with scoliosis at the time of onset of the disease remains to be seen.

Regeneration following somite removal in chick embryos.

A technique was developed for ensuring complete removal of single somites with minimal damage to surrounding tissues in 2-day-old chick embryos. Histological examination of the site of somite removal at various time intervals after operation revealed that a regeneration mechanism could be triggered. Replacement of the cells that had been removed could occur, but the extent of the replacement was dependent on the immediate fate of the gap created. If the gap was closed by enlargement of the adjacent somites, no replacement of the cells occurred. If the gap remained, then cells invaded the gap and were able to produce a normal sclerotome and dermomyotome. By labelling adjacent cells with the carbocyanine dye. DiI, it was shown that the replacement cells could come from the adjacent somites, as well as the intermediate mesoderm. Use of an antibody to HNK-1 established that the replacement cells did not come from the neural crest and that the neural crest cell distribution was little affected. Staining with peanut agglutinin showed that the replacement cells were able to adopt the characteristics associated with rostral and caudal halves of the normal sclerotome. These results provide possible explanations for the variety of vertebral anomalies produced by removal of somites and for the production of some congenital vertebral anomalies.

The nature and distribution of the innervation of human supraspinal and interspinal ligaments.

STUDY DESIGN: To use fresh, human supraspinal and interspinal ligaments and document their innervation. OBJECTIVES: To characterize the innervation of the human supraspinal and interspinal ligaments. SUMMARY OF BACKGROUND DATA: The nature and distribution of the innervation of spinal ligaments remains unknown. METHODS: Sections of spinal ligaments were labeled with a fluorescent antibody against neurofilament proteins and observed with a confocal microscope. RESULTS: The ligaments were found to be well innervated. Innervation was equally distributed along the ligament, symmetrically distributed between left and right sides, and more densely distributed in the periphery. Pacinian corpuscles were scattered randomly, close to blood vessels, whereas Ruffini corpuscles were in the periphery, close to the collagen bundles. CONCLUSIONS: Human supraspinal and interspinal ligaments are well innervated. This innervation might form the basis of neurologic feedback mechanisms for the protection and stability of the spine. These mechanisms might also be important in the development of diseases such as scoliosis.

Quantitative morphology of the lateral ligaments of the spine. Assessment of their importance in maintaining lateral stability.

STUDY DESIGN: This study used human cadaveric material to examine the three-dimensional morphology and biomechanics of the superior and lateral costotransverse ligaments and the intertransverse ligament of the spine. OBJECTIVES: To provide descriptive and quantitative data on the morphology of the lateral ligaments of the spine and to assess their importance in maintaining lateral stability, especially regarding the pathogenesis of idiopathic scoliosis. SUMMARY OF BACKGROUND DATA: Ligaments have been reported as being able to stabilize the spine by mechanical constraint and by neurologic feed-back. Midline spinal ligaments have been well studied but do not appear to be effective in maintaining lateral stability because of their sites of attachment. Lateral ligaments of the spine have not been adequately documented in the literature. METHODS: The morphology, sites of attachment, and dimensions of the superior costotransverse ligament, lateral costotransverse ligament, and intertransverse ligament from thoracic level 7 to thoracic level 10 were determined on 32 human cadavers. RESULTS: The intertransverse ligament was found not to be a true ligament. The lateral costotransverse ligament was a true ligament but did not have the characteristics appropriate for involvement in lateral stability. The superior costotransverse ligament also was a true ligament and had all of the characteristics appropriate for involvement in the active lateral balancing of the spine. CONCLUSIONS: In contrast to the midline ligaments of the spine, the superior costotransverse ligament perhaps is the most important ligament for active lateral balancing of the spine and warrants further study, particularly regarding the development of idiopathic scoliosis.

Increases in the number of cells in different areas of epithelial somites related to changes in morphology and development.

There are two distinct groups of cells in the epithelial somite: cells in the epithelial ball that form the periphery, and loose mesenchymal cells found in the central cavity (somitocoele). Recent work has produced evidence to show that these two groups of cells have significant differences (morphology, origin, fibronectin content, reaction to peanut lectin, communication properties) but the significance of these differences has yet to be established. It is not yet clear whether the epithelial somite stage of development is merely a time for cell proliferation, or whether it is a time when significant differences develop which have consequences in subsequent morphogenesis. Certainly, there are indications that the two groups of cells might form different structures related to the vertebral column based on their position in the subsequent sclerotome. In this study, we have examined the number of cells that are present in both the epithelial ball and the somitocoele at various stages of maturity. The results show that later-formed somites contain significantly more cells in both the epithelial ball and the somitocoele. Furthermore, while the density of cells in the epithelial ball remains constant (accounting for an increase in dimensions of the somite), there is a significant increase in density of cells in the somitocoele. This suggests that there is an important distinction being created between the cells of the epithelial ball and those in the somitocoele.(ABSTRACT TRUNCATED AT 250 WORDS)

The immediate fate of cells in the epithelial somite of the chick embryo.

The precise origins and fates of cells forming the epithelial ball and those contained within the somitocoele at the centre of the somite in the chick embryo are unknown. In particular, it is not known whether the progeny of the proliferating cells in both the epithelial ball and the somitocoele simply mix with each other, or whether they represent two separate populations that remain isolated during proliferation. We tested whether cells found in both of these locations are the result of cell migration, cell division or both. To do this, we injected single cells in both the epithelial ball and the somitocoele with lysinated fluorescein dextran, a molecule that can only be transferred to other cells through cell division, and observed their development over the next 24 h. Control experiments were also performed to assess the incidence of error associated with the microinjection method and to provide reliable and valid comparative data. Our results showed that the progeny of the cells in the somitocoele, as well as any non-proliferating cells, appear to remain in the somitocoele. In contrast, the progeny of the cells in the epithelial ball either remain in the epithelial ball or migrate into the somitocoele to mix freely with the other cells present. These results suggest that the cells in the epithelial ball and the somitocoele develop as discrete groups, with the possibility of diverse fates accompanying further development.

Communication compartments in the axial mesoderm of the chick embryo.

Intracellular microinjection of the fluorescent tracer Lucifer Yellow into mesoderm cells along the rostrocaudal axis of the early chick embryo has revealed compartments where the intercellular diffusion of dye, presumably via gap junctions, is restricted at the borders between groups of cells. Cells in the segmental plate were dye-coupled, as were cells forming the epithelial somites. However, dye-coupling was not observed between different somites, nor was it observed between the outer epithelial cells and the cells in the somitocoele. On dispersal of the somite, dermatome cells were dye-coupled. However, sclerotome cells were found to be divided into rostral and caudal compartments separated by a group of cells bordering the intrasclerotomal fissure (of von Ebner) that also exhibited dye-coupling, restricted primarily to cells along the fissure. Some of these compartment borders can be accounted for by the presence of a morphological barrier which reduces cell-cell contact, but others are more difficult to explain, as there appears to be extensive cell-cell contact across the border. This would be analogous to some compartments found in insects. Some of the compartments also have borders similar to those described by cell lineage studies. The results also indicate that dye-coupling becomes restricted in a spatial and temporal manner as the mesodermal cells mature.

The migration and distribution of somite cells after labelling with the carbocyanine dye, Dil: the relationship of this distribution to segmentation in the vertebrate body.

The cells of individual somites in 2-day-old chick embryos were marked by injecting a fluorescent dye into the somitocoele. This procedure permanently marked the cells and allowed their subsequent development and distribution to be followed. The cells were found to remain in close association with each other within limited boundaries and did not mix to any great extent with similar cells from adjacent somites. Fluorescent cells from single somites were found in the intervertebral disc, connective tissue surrounding two adjacent neural arches, all the tissues between the neural arches, the dermatome, and the associated myotome. No fluorescent cells were found in the notochord or in any nervous tissue apart from accompanying connective tissue. Surprisingly, the vertebral bodies and neural arches did not contain any fluorescent cells apart from those in the connective tissue surrounding them, but this absence of fluorescent cells was thought to be due to the dilution of the fluorescence following cell proliferation. These results provide further experimental support for the theory of resegmentation in vertebral formation, and also provide evidence of a compartmental method of development along the rostrocaudal axis in vertebrates, similar to that already discovered in insects. On the basis of cell lineage criteria, the sclerotome might be considered as a developmental compartment.