The effect of supplementation of a glutamine precursor on the growth plate, articular cartilage and cancellous bone in fundectomy-induced osteopenic bone

Abstract

The aim of the study was to investigate the effect of 2-oxoglutaric acid (2-Ox) supplementation (a precursor of glutamine and hydroxyproline, the most abundant amino acid of collagen) on cartilage and bone in pigs after fundectomy. Pigs at the age of forty days were subjected to fundectomy and divided into two groups depending on 2-Ox supplementation (at the daily dosage of 0.4 g/kg of body weight). Other pigs were sham operated. Pigs were euthanized at the age of eight months. An analysis of the morphometry of trabeculae, growth plate and articular cartilage in fundectomy-induced osteopenic bone was performed. Moreover, the levels of expression of osteocalcin, osteopontin and osteoprotegerin in trabecular bone and osteocalcin in articular cartilage were evaluated. Articular cartilage was thinnest in fundectomized pigs and thickest in 2-Ox-supplemented animals after fundectomy. Moreover, 2-Ox supplementation after fundectomy enhanced the total thickness of the growth plate and trabeculae in fundectomized pigs. The most evident signal for osteocalcin and osteoprotegerin in trabecular bone was in sham-operated and 2-Ox-supplemented pigs; a low reaction was observed in the fundectomized group. Additionally, as a long-term postoperative consequence, a change was observed in the expression of osteocalcin in articular cartilage. It seems that 2-Ox is suitable for use in preventing the negative effects of fundectomy on cancellous bone and cartilage.

Fig. 1.

Representative pictures of the immunohistochemical analysis of the expression of osteopontin (OPN) and osteoprotegerin (OPG) carried out on formaldehyde-fixed sections from femoral trabeculae of pigs from the Fx group, the fundectomized group; the 2-Ox group, supplemented with 2-Ox after fundectomy; and the SHO group, the sham-operated group. AC, antibody control. Scale bar for all panels: 200 µm.

Fig. 2.

Representative pictures of the immunohistochemical analysis of the expression of osteocalcin (OCN) carried out on formaldehyde-fixed sections from femoral articular cartilage of pigs from the Fx group, the fundectomized group; the 2-Ox group, supplemented with 2-Ox after fundectomy; and the SHO group, the sham-operated group. In normal chondrocyte from the SHO group, the nucleus is stained blue (the negative OCN signal) and indicated by black arrows. The positive OCN signal is indicated in cells from the Fx group by white arrows. Scale bar: 200 µm.

Fig. 3.

Representative images of PSR staining carried out on formaldehyde-fixed sections from femoral articular cartilage of pigs. Vertical section of transitional and radial zones of femoral articular cartilage from the Fx group, the fundectomized group; the SHO group, the sham-operated group; and the 2-Ox group, supplemented with 2-Ox after fundectomy. Magnification ×100.

Fig. 4.

Representative images of Safranin O staining carried out on formaldehyde-fixed sections from femoral articular cartilage of pigs from the Fx group, the fundectomized group; the 2-Ox group, supplemented with 2-Ox after fundectomy; and the SHO group, the sham-operated group. Highly cellular articular cartilage in fundectomized animals with chondrocytes is separated by thin septa of matrix in the Fx group compared with the SHO and 2-Ox groups. Magnification ×200.