Agnieszka Korobowicz

Morphology and physiology of the epiphyseal growth plate

The epiphyseal growth plate develops from the cartilaginous-orientated mesenchymal cells that express SOX family genes. This multilayer structure is formed by the proliferation and hypertrophy of cells that synthesize the extracellular matrix composed of collagen (mainly type II, IX, X, XI) and proteoglycans (aggrecan, decorin, annexin II, V and VI). The resting zone is responsible for protein synthesis and maintaining a germinal structure. In the proliferative zone, cells rapidly duplicate. The subsequent morphological changes take place in the transformation zone, divided into the upper and lower hypertrophic layers. In the degenerative zone, the mineralization process becomes intensive due to increased release of alkaline phosphate, calcium and matrix vesicles by terminally differentiated chondrocytes and some other factors e.g., metaphyseal ingrowth vessels. At this level, as well as in the primary and secondary spongiosa zones, chondrocytes undergo apoptosis and are physiologically eliminated. Unlike adult cartilage, in fetal and early formed growth plates, unusual forms such as authophagal bodies, paralysis and dark chondrocytes were also observed. Their ultrastructure differs greatly from apoptotic and normal cartilage cells. Chondrocyte proliferation and differentiation are regulated by various endocrine, paracrine, and autocrine agents such as growth, thyroid and sex hormones, beta-catenin, bone morphogenetic proteins, insulin-like growth factor, iodothyronine deiodinase, leptin, nitric oxide, transforming growth factor beta and vitamin D metabolites. However, the most significant factor is parathyroid hormone-related protein (PTHrP) which is synthesized in the perichondrium by terminally differentiated chondrocytes. Secondary to activation of PTH/PTHrP receptors, PTHrP stimulates cell proliferation by G protein activation and delays their transformation into prehypertrophic and hypertrophic chondrocytes. When proliferation is completed, chondrocytes release Indian hedgehog (Ihh), which stimulates PTHrP synthesis via a feedback loop. Any disturbances of the epiphyseal development and its physiology result in various skeletal abnormalities known as dysplasia.

Congenital ventricular septal defects and prenatal exposure to cyclooxygenase inhibitors

Ventricular septal defects (VSDs) are common congenital abnormalities which have been reported to be associated with maternal fever and various environmental factors. The aim of the present study was to evaluate the effect of prenatal exposure to cyclooxygenase (COX) inhibitors on heart defects. A retrospective statistical analysis was performed using data collected in our laboratory during various teratological studies carried out on albino CRL:(WI)WUBR Wistar strain rats from 1997 to 2004. The observations were compared with concurrent and historic control data, as well as findings from other developmental toxicological studies with selective and nonselective COX-2 inhibitors. Despite the lack of significant differences in the frequency of VSDs between drug-exposed and control groups, statistical analysis by the two-sided Mantel-Haenszel test and historical control data showed a higher incidence of heart defects in offspring exposed to nonselective COX inhibitors (30.06/10,000). Unlike other specific inhibitors, aspirin (46.26/10,000) and ibuprofen (106.95/10,000) significantly increased the incidence of the VSD when compared with various control groups (5.38-19.72/10,000). No significant differences in length or weight were detected between fetuses exposed to COX inhibitors and born with VSD and non-malformed offsprings. However, a statistically significant increase of fetal body length and decrease of body mass index were found in fetuses exposed to COX inhibitors when compared with untreated control. We conclude that prenatal exposure to COX inhibitors, especially aspirin and ibuprofen, increased the incidence of VSDs in rat offspring but was not related to fetal growth retardation.

Prenatal effects of DuP-697—the irreversible, highly selective cyclooxygenase-2 inhibitor

DuP-697 (5-bromo-2-(4-fluorophenyl)-3-[4-(methylsulfonyl)phenyl]-thiophene), like celecoxib and rofecoxib, is a vicinal diaryl heterocycle highly selective cyclooxygenase-2 (COX-2) inhibitor. The aim of the study was to evaluate prenatal tolerability of DuP-697. The drug was administered orally in Tween 80 water suspension once a day to pregnant Wistar rats, on Days 7-18 of gestation. The initial dose, similar to the rat antipyretic dose, was set at 0.05 mg/kg. The middle dose, 3.5 mg/kg, corresponded to the rat anti-inflammatory and analgesic dose. The high dose was set at 35.0 mg/kg. Control animals received Tween 80 water suspension. On Day 21 of gestation, fetuses were delivered by laparotomy and double stained with alcian blue and alizarin red S or examined using the Wilson technique. Intrauterine growth retardation occurred in the groups exposed to the middle and highest dose of DuP-697. Minimal reactive and degenerative hepatic changes were found in both drug-exposed and control groups. Skeletal malformations were seen occasionally in all drug-treated and control groups. A significant increase in skeleton variations, such as delayed and asymmetrical ossification, was observed in fetuses exposed to the highest drug dose when compared with the control. These changes were not increased (P<0.1) in the middle drug-dose group. The experimentally-derived NOAEL for developmental toxicity was 0.05 mg/kg, and the corresponding LOAEL was 3.5 mg/kg.