IRON DEPOSITION IN THE GROWTH PLATE OF LONG BONES OF THE OFFSPRING WHEN GIVEN DURING PREGNANCY IN RAT MODEL

Objective: To evaluate histologically the deposition of iron in the epiphyseal cartilage of offspring’s of dams given iron supplementation during pregnancy and lactation in rat model. Study Design: Laboratory based experimental study. Place and Duration of Study: Department of Anatomy, Army Medical College, Rawalpindi and National Institute of Health (NIH) Islamabad, from Mar to Nov 2016. Methodology: In this study, 16 female and 4 male adult rats were chosen for breading. After confirmation of pregnancy, pregnant rats were separated in two groups. One group was given oral iron supplementation for four weeks till delivery and half of the pups fed by mothers who were given iron during lactation. The other group was kept on normal lab diet. Deposition of iron in the epiphyseal cartilage of newborn rats after four weeks was evaluated histologically in pups. Results: Iron deposition was maximum in group C i.e. 1.30 ± 0.48; in group B it was 0.20 ± 0.44. Statistically significant iron deposition (p<0.001) was observed in the growth plate of off springs when mothers were given iron supplements during pregnancy and lactation. Conclusion: Present study proves that injudicious iron supplementation through pregnancy results in deposition of iron in epiphyseal growth plate of the fetus and it can have damaging effects on bones of fetus.

Sequential Epiphyseal Cartilage Changes of Femoral Heads in C57BL/6 Female Mice Treated with Excessive Glucocorticoids

Objective Excessive use of glucocorticoids (GCs) may cause adverse effects on the skeletal system in children. However, only a few studies have reported the effects of GCs on the epiphyseal cartilage. This study aimed to uncover the subsequent epiphyseal cartilage changes of immature femoral heads after excessive GC treatment in a mouse model and explain the pathological changes preliminarily. Design Female C57BL/6 mice were divided into control and model (excessive GC treatment) groups. The structure of the femoral heads was evaluated by using micro-computed tomography, hematoxylin-eosin staining, and safranin staining analyses. Immunohistochemistry was used to detect angiogenesis and cartilage metabolism. Western blotting and TUNEL staining were used to examine epiphyseal cartilage chondrocyte apoptosis. Primary chondrocytes were isolated from the femoral heads of healthy mice for in vitro studies. The effects of GCs on chondrocyte apoptosis and metabolism were determined by flow cytometry and Western blotting. Results The epiphyseal cartilage ossification had started at 4 weeks posttreatment in a portion of mice; the ossification presented as a sequential process in the model group, while the epiphyseal cartilage maintained an unossified state in the control group. Vascular invasion into the epiphyseal cartilage of the model mice was observed at 4 weeks posttreatment. GCs induced chondrocyte apoptosis and altered chondrocyte metabolism in the epiphyseal cartilage. Conclusions The epiphyseal cartilage ossification accelerated in the femoral heads of female C57BL/6 mice after excessive GC treatment. Increased chondrocyte apoptosis, altered chondrocyte metabolism, as well as increased vascular invasion, are the potential factors influencing epiphyseal cartilage ossification.

Long-Term Exposure to Temozolomide Affects Locomotor Activity and Cartilage Structure of Elderly Experimental Rats

Chemotherapy with temozolomide (TMZ) is an essential part of anticancer therapy of various malignant tumours; however, its long-term effects on patients’ health and life quality need to be further investigated. Here, we studied the effects of TMZ and/or companion drug dexamethasone (DXM) on the locomotor activity and cartilage structure of elderly Wistar rats (n = 40). Long-term TMZ treatment selectively inhibited the horizontal, but not vertical locomotor activity of the rats (6.7-fold, p < 0.01) and resulted in delamination of the superficial epiphyseal cartilage of the femoral epiphysis of knee joints, a 2-fold decrease in mean thickness of epiphyseal cartilage (p < 0.001), and changes in the proliferative and maturation cartilage zones ratio. The simultaneous use of DXM attenuated TMZ-induced changes in cartilage thickness and integrity and compensated the decrease in horizontal locomotor activity of experimental animals. Nevertheless, combined TMZ/DXM treatment still significantly affected the structure of proximal tibial, but not distal femoral epiphysis of knee joints of the rats. These changes were accompanied by the increased content of total glycosaminoglycans (GAGs) and their partial re-localisation from chondrocytes into tissue matrix, as well as the decrease in sulfated GAGs content in both compartments. Taken together, the results demonstrate that long-term treatment with TMZ results in a significant decrease in locomotor activity of elderly Wistar rats and the reorganisation of their knee joint cartilage structure, while DXM treatment attenuates those effects. So, use of DXM or chondroprotective drugs might be beneficial to maintain quality of life for TMZ-treated cancer patients.

Progression of Epiphyseal Cartilage and Bone Pathology in Surgically Treated Cases of Osteochondritis Dissecans of the Elbow

Background: Although a variety of pathologic conditions associated with osteochondritis dissecans (OCD) have been reported, the pathological progression has remained unclear. Hypothesis: Separation of the immature epiphyseal cartilage is an early event in OCD, and osteonecrosis in the articular fragment is a late event. Study Design: Case Series; Level of evidence, 4. Methods: The participants were 26 boys (mean age, 13.8 years; mean skeletal age score for the elbow, 24.6 points) with capitellar OCD who underwent osteochondral autograft transplantation. A total of 28 cylindrical osteochondral plugs, including the articular fragment, an intermediate layer, and proximal epiphyseal bone, were harvested from the central area of the capitellum and were examined histologically. The articular fragments of OCD were independently assessed by 5 observers and divided into 4 pathological variations: IA, nearly normal-cartilaginous; IB, deteriorated-cartilaginous; IIA, cartilage-ossifying; and IIB, cartilage-osteonecrotic. The reliability of assessment and the correlation of the pathological variations with the clinical data were examined. Results: The reliability of the assessment among 5 observers was almost perfect (Cohen kappa value = 0.91). OCD variations of IA, IB, IIA, and IIB were evident in 5, 10, 5, and 6 patients, respectively. OCD-I (cartilaginous) and OCD-II (osteochondral) corresponded significantly to radiographic stage I (radiolucency or slight calcification with open physis) and stage II (delayed ossification or bony fragment), respectively (Cohen kappa value = 0.79; percentage agreement = 81%). The pathological OCD variations were significantly correlated with the clinical data, including the period from symptom onset to surgery, patient age, and the skeletal age score ( P < .01, in each). Conclusion: The present study has revealed that the pathological variations correspond to the progression of OCD, thus proving our hypothesis. OCD-IA was shown to be an early lesion caused by separation of the immature epiphyseal cartilage. OCD-IB appeared to result from ossification arrest over a prolonged period from the onset of OCD-IA, whereas OCD-IIA showed delayed ossification in the epiphyseal cartilage where vascularization from the surrounding bone had been established. Osteonecrosis in OCD-IIB was shown to be a late pathological event caused by disruption of the vascular supply to OCD-IIA.

Effect of intraarticular injection of chondroitin sulfate into synovial joint elements in an experiment

Objective: to study morphological and morphometric changes in the epiphysis bone in a rat during the intra-articular injection of chondroitin sulfate (CS). Mаteriаl аnd methods. The object of the study was the knee joints of 36 Wistar rats. CS injections at a dose of 0.05 ml were performed once a week into one of the knee joints (experimental joint), and isotonic NaCl solution at the same volume was injected into the opposite joint (control joint). The animals in the number of 12 units were withdrawn from the experiment on the 7th, 14th, and 21st days, which corresponded to one week after one-, two-and three-fold intra-articular injections of HC and 0.9 % NaCl. The isolated knee joints were placed in a decalcifying liquid, then were fixed in 10% neutral buffered formalin. 4 micron histological sections were stained with hematoxylin and eosin. The morphometric analysis assessed the thickness of hyaline articular cartilage, the thickness of the epiphysis growth zone cartilage, and the cell content of the subchondral bone. Results. The study of the thickness of the articular cartilage, the growth zone of epiphyseal cartilage and the cellular composition of the subchondral bone has showed a statistically significant dynamic increase in these indicators after the 2nd and 3rd intra-articular injections of CS. The assessment of the thickness of the articular cartilage on the 21st day found some statistically significant differences between the experimental and control groups (p < 0.0001), the thickness of the epiphyseal cartilage had increased significantly by that time (p < 0.0001), and the cell content of bone marrow showed statistically significant differences (p = 0.002). Conclusion. The obtained data testify to a pronounced regenerative effect of CS, injected intra-articularly into articular cartilage.

Histochemical assessment on the cellular interplay of vascular endothelial cells and septoclasts during endochondral ossification in mice

This study was aimed to verify the cellular interplay between vascular endothelial cells and surrounding cells in the chondro-osseous junction of murine tibiae. Many CD31-positive endothelial cells accompanied with Dolichos Biflorus Agglutinin lectin-positive septoclasts invaded into the hypertrophic zone of the tibial epiphyseal cartilage. MMP9 immunoreactive cytoplasmic processes of vascular endothelial cells extended into the transverse partitions of cartilage columns. In contrast, septoclasts included several large lysosomes which indicate the incorporation of extracellular matrices despite no immunopositivity for F4/80—a hallmark of macrophage/monocyte lineage. In addition, septoclasts were observed in c-fos-/- mice but not in Rankl-/- mice. Unlike c-fos-/- mice, Rankl-/- mice showed markedly expanded hypertrophic zone and the irregular shape of the chondro-osseous junction. Immunoreactivity of platelet-derived growth factor-bb, which involved in angiogenic roles in the bone, was detected in not only osteoclasts but also septoclasts at the chondro-osseous junction. Therefore, septoclasts appear to assist the synchronous vascular invasion of endothelial cells at the chondro-osseous junction. Vascular endothelial cells adjacent to the chondro-osseous junction possess endomucin but not EphB4, whereas those slightly distant from the chondro-osseous junction were intensely positive for both endomucin and EphB4, while being accompanied with ephrinB2-positive osteoblasts. Taken together, it is likely that vascular endothelial cells adjacent to the chondro-osseous junction would interplay with septoclasts for synchronous invasion into the epiphyseal cartilage, while those slightly distant from the chondro-osseous junction would cooperate with osteoblastic activities presumably by mediating EphB4/ephrinB2. Mini-abstract: Our original article demonstrated that vascular endothelial cells adjacent to the chondro-osseous junction would interplay with septoclasts for synchronous invasion into the epiphyseal cartilage, while those slightly distant from the chondro-osseous junction would cooperate with osteoblastic activities presumably by mediating EphB4/ephrinB2.(A figure that best represents your paper is Fig. 5c)