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.

The effect of pulsed electromagnetic fields on chondrocyte morphology.

Osteoarthritis is a debilitating joint disease where the articular cartilage surface degrades and is unable to repair itself through natural processes. Chondrocytes reside within the cartilage matrix and maintain its structure. We conducted in vitro experiments to investigate the morphological response of cultured human chondrocytes under different pulsed electromagnetic field (PEMF) conditions. In the control experiments, cultured chondrocytes attached to the bottom of a culture dish typically displayed either a stellate or spindle morphology with extended processes. Experimental chondrocyte cultures were placed in a Helmholtz coil to which a ramp waveform was applied. Exposure to PEMFs caused the chondrocytes to retract their processes, becoming spherical in shape. This change in morphology followed a progression from stellate to spindle to spherical. These morphological changes were reflected in an average reduction of 30% in the surface contact area of the chondrocytes to the culture dish. Understanding the mechanisms by which PEMFs affect the morphology of chondrocytes will help lead to new treatments for osteoarthritis.

Is it possible to identify a population in which the incidence of future development of AIS is greatly increased when compared to the normal population?

For future research of predictors of AIS, it would be advantageous to identify a general population in which the development of AIS is greatly increased when compared to the normal population. The probability of predicting future development of AIS among younger relatives of current patients based on the probability of AIS incidence was assessed from the research literature. Although there is considerable literature relating to familial relationships of the probability of developing AIS or having AIS, the probability is relatively low in most cases. Even with the best of predicted probabilities, the identification of patients with a high probability of developing AIS remained low. The identification of people among the general population who have a high probability of developing AIS based on the probabilities expressed in the literature is not possible.

The development of osteoblasts from stem cells to supplement fusion of the spine during surgery for AIS.

Surgical correction in severe cases of AIS is often hampered by insufficient autograft bone to facilitate the fusion. The development of other sources of bone generating cells would greatly enhance the surgical. Bone marrow derived stem cells were harvested from femoral reaming during total hip arthroplasty for the purpose of differentiating into osteoblasts. Stem cells were isolated from the marrow and successfully differentiated into three cell lines (osteoblasts, chondrocytes and adipocytes) to confirm multilineage potential. Osteoblasts were developed from the stem cells and demonstrated the ability to be cultured to possibly provide a source of bone generating cells to augment surgical fusions. It is anticipated that the addition of osteoblasts created from stem cells (combined with appropriate matrix) will have significant influence on the success of AIS surgery through improvement of bone fusion.

Atypical shoulder muscle activation in multidirectional instability.

OBJECTIVE: Surface and intramuscular electromyography was used to investigate shoulder muscle activity in subjects with multidirectional instability (MDI). METHODS: Subjects (seven MDI, 11 control) performed repetitive shoulder abduction/adduction, flexion/extension and internal/external rotation movements on an isokinetic dynamometer. The activity of the deltoid, infraspinatus, supraspinatus, latissimus dorsi, and pectoralis major muscles were recorded using double-differential surface and intramuscular fine-wire electrodes. A repeated measures analysis of variance evaluated group differences in the amplitude, onset, termination and duration of the muscle activity. RESULTS: Significant activation parameter differences for the supraspinatus, infraspinatus, posterior deltoid and pectoralis major muscles were found in the subjects with MDI. The rotator cuff and posterior deltoid muscles demonstrated abbreviated periods of activity when performing internal/external rotation, despite activation amplitudes that were similar to the controls. In contrast, the activation of the pectoralis major differed from the control group in both the amplitude and time domains when performing shoulder extension. CONCLUSIONS: MDI is associated with atypical patterns of muscle activity that occur even when highly constrained movements are used to elicit the activity. SIGNIFICANCE: In addition to glenohumeral hyperlaxity, the results suggest that dysfunctional neuromuscular control of the rotator cuff is also a contributing factor to the pathoetiology of MDI.

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.

Validation and reproducibility of computerised cell-viability analysis of tissue slices.

BACKGROUND: The identification of live cells using membrane integrity dyes has become a frequently used technique, especially with articular cartilage and chondrocytes in situ where tissue slices are used to assess cell recovery as a function of location. The development of a reproducible computerised method of cell evaluation would eliminate many variables associated with manual counting and significantly reduce the amount of time required to evaluate experimental results. METHODS: To validate a custom computerised counting program, intra-person and inter-person cell counts of nine human evaluators (three groups - unskilled, novice, and experienced) were compared with repeated pixel counts of the custom program on 15 digitised images (in triplicate) of chondrocytes in situ stained with fluorescent dyes. RESULTS: Results indicated increased reproducibility with increased experience within evaluators [Intraclass Correlation Coefficient (ICC) range = 0.67 (unskilled) to 0.99 (experienced)] and between evaluators [ICC = 0.47 (unskilled), 0.85 (novice), 0.93 (experienced)]. The computer program had perfect reproducibility (ICC = 1.0). There was a significant relationship between the average of the experienced evaluators results and the custom program results (ICC = 0.77). CONCLUSIONS: This study demonstrated that increased experience in cell counting resulted in increased reproducibility both within and between human evaluators but confirmed that the computer program was the most reproducible. There was a good correlation between the intact cell recovery determined by the computer program and the experienced human evaluators. The results of this study showed that the computer counting program was a reproducible tool to evaluate intact cell recovery after use of membrane integrity dyes on chondrocytes in situ. This and the significant decrease in the time used to count the cells by the computer program advocate its use in future studies because it has significant advantages.

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.

The development of scoliosis following pinealectomy in young chickens is not the result of an artifact of the surgical procedure.

Pinealectomy in young chickens consistently results in scoliosis which has many characteristics similar to those seen in adolescent idiopathic scoliosis. The mechanism underlying this phenomenon remains a mystery and it is not yet entirely clear whether some unidentified aspect of the extensive surgery is the major factor rather than the removal of the pineal gland. Four different types of pinealectomy surgery were performed on young chickens as well as deliberate damage to the cerebral cortex which simulated the extreme of any accidental damage that might occur during surgery. Scoliosis was assessed from weekly radiographs. No differences in incidence of scoliosis, degree of severity or pattern of curve development were observed for any of the experimental groups when compared with controls. In all groups approximately 55% of the chickens developed scoliosis that progressed rapidly. Different pinealectomy procedures and deliberate damage to the cerebral cortex produce scoliosis in young chickens with the same incidence and characteristics. This suggests strongly that the mechanism behind the phenomenon is due to the removal of the pineal gland and not some artifact of the extensive surgery. The pinealectomy model in young chickens is proving to be a good model for studying AIS in humans. An understanding of the mechanism underlying this phenomenon has the potential to provide further insights into the aetiology of AIS and can lead to the development of novel treatement methods.

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.

Development of scoliosis following pinealectomy in young chickens is not the result of an artifact of the surgical procedure.

Pinealectomy in young chickens consistently results in scoliosis, which has many characteristics similar to those seen in adolescent idiopathic scoliosis. The mechanism underlying this phenomenon remains a mystery and it is not yet entirely clear whether some unidentified aspect of the extensive surgery is the major factor rather than the removal of the pineal gland. Four different types of pinealectomy surgery were performed on young chickens as well as deliberate damage to the cerebral cortex which simulated the extreme of any accidental damage that might occur during surgery. Scoliosis was assessed from weekly radiographs. No differences in incidence of scoliosis, degree of severity, or pattern of curve development were observed for any of the experimental groups when compared with controls. In all groups approximately 55% of the chickens developed scoliosis that progressed rapidly. Different pinealectomy procedures and deliberate damage to the cerebral cortex produce scoliosis in young chickens with the same incidence and characteristics. This suggests strongly that the mechanism behind the phenomenon is due to the removal of the pineal gland and not some artifact of the extensive surgery. The pinealectomy model in young chickens is proving to be a good model for studying AIS in humans. An understanding of the mechanism underlying this phenomenon has the potential to provide further insights into the etiology of AIS and can lead to the development of novel treatment methods.

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.

Structure of the primordial diaphragm and defects associated with nitrofen-induced CDH.

The goals of this study were to further our understanding of diaphragm embryogenesis and the pathogenesis of congenital diaphragmatic hernia (CDH). Past work suggests that the pleuroperitoneal fold (PPF) is the primary source of diaphragmatic musculature. Furthermore, defects associated with an animal model of CDH can be traced back to the formation of the PPF. This study was designed to elucidate the anatomic structure of the PPF and to determine which regions of the PPF malform in the well-established nitrofen model of CDH. This was achieved by producing three-dimensional renderings constructed from serial transverse sections of control and nitrofen-exposed rats at embryonic day 13.5. Renderings of left- and right-sided defects demonstrated that the malformations were always limited to the dorsolateral portions of the caudal regions of the PPF. These data provide an explanation of why the holes in diaphragmatic musculature associated with CDH are characteristically located in dorsolateral regions. Moreover, these data provide further evidence against the widely stated hypothesis that a failure of pleuroperitoneal canal closure underlies the pathogenesis of nitrofen-induced CDH.

The effects of melatonin therapy on the development of scoliosis after pinealectomy in the chicken.

The mechanism underlying the development of scoliosis after pinealectomy in young chickens is unknown. However, since the main product of the pineal gland is melatonin, melatonin remains an obvious focus in studies designed to discover this mechanism. One confounding factor is that serum melatonin levels are close to zero after pinealectomy but scoliosis does not develop in all chickens that have had this procedure. Therefore, the role of melatonin in the development of scoliosis in chickens after pinealectomy remains controversial. In the current investigation, two pilot studies demonstrated that a physiological therapeutic dose of melatonin (2.5 milligrams per kilogram of body weight) restored the circadian rhythm of melatonin, as measured by serum assay. In the main study, this dose was administered daily starting either immediately after the pinealectomy or two weeks after it, when scoliosis had developed. Scoliosis was assessed on weekly radiographs, and the Cobb angle was determined for all chickens in which scoliosis developed. Overall, scoliosis developed in only 56 percent (fifty) of the eighty-nine chickens that had had a pinealectomy; this rate was consistent throughout all experimental groups. Scoliosis did not develop in any of the control chickens, which did not have a pinealectomy. On the basis of the average Cobb angles in the chickens in which scoliosis had developed, it was determined that neither the prevalence nor the pattern of the scoliosis was affected by the therapy in any of the experimental groups. It was thus concluded that melatonin therapy after pinealectomy in young chickens has no effect on the development or progression of scoliosis. These results raise doubts regarding the role of melatonin in the development of scoliosis after pinealectomy in the young chicken.