Structural Changes in Trabecular Bone, Cortical Bone and Hyaline Cartilage as Well as Disturbances in Bone Metabolism and Mineralization in an Animal Model of Secondary Osteoporosis in Clostridium perfringens Infection

Abstract

There is no information regarding whether changes in the microbiological balance of the gastrointestinal tract as a result of an infection with Clostridium perfringens influence the development of metabolic bone disorders. The experiment was carried out on male broiler chickens divided into two groups: control (n = 10) and experimental (n = 10). The experimental animals were infected with Clostridium perfringens between 17 and 20 days of age. The animals were euthanized at 42 days of age. The structural parameters of the trabecular bone, cortical bone, and hyaline cartilage as well as the mineralization of the bone were determined. The metabolism of the skeletal system was assessed by determining the levels of bone turnover markers, hormones, and minerals in the blood serum. The results confirm that the disturbed composition of the gastrointestinal microflora has an impact on the mineralization and metabolism of bone tissue, leading to the structural changes in cortical bone, trabecular bone, and hyaline cartilage. On the basis of the obtained results, it can be concluded that changes in the microenvironment of the gastrointestinal tract by infection with C. perfringens may have an impact on the earlier development of osteoporosis.

Keywords: Clostridium perfringens; gastrointestinal; metabolic bone disorders; microflora; osteoporosis; short-term infection.

Figure 1

The effect of C. perfringens infection on hormonal and biochemical analysis in blood serum of animals at the age of 42 days: (A) osteprotegerin; (B) osteocalcin; (C) insulin-like growth factor 1 (IFG-1); (D) growth hormone; (E) insulin; (F) calcitriol; (G) calcium; (H) phosphorus; and (I) alkaline phosphatase. Control—the control group of birds; experimental—the experimental group of birds subjected to Clostridium perfringens infection. Data are mean values ± SD (whiskers) from n = 10 birds. Statistical significance: * p < 0.05; ** p < 0.01; *** p < 0.001 (two-tailed Student’s t-test, t-test with Welch’s correction, or Mann–Whitney U test).

Figure 2

Trabecular bone morphology from proximal part of the tibia bone in examined animals of the age of 42 days. (I) Representative pictures of Goldner Trichome method staining carried out on formaldehyde-fixed samples from the cancellous bone. The yellow arrows show the trabeculae, while the red arrows show the bone marrow. (II) BV/TV-relative bone volume. Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; Tb.N, trabecular number; BTD, bone tissue density; BS/BV, bone surface density. Control group (A); experimental group (B). Data are mean values ± SD (whiskers) from n = 10 birds. Statistical significance: *** p < 0.001 (two-tailed Student’s t-test, t-test with Welch’s correction, or Mann–Whitney U test).

Figure 3

The effect of C. perfringens infection on the bone content of mature collagen in tibia of animals at the age of 42 days: (A) representative photos of the PSR staining carried out on formaldehyde-fixed from the trabecular (upper panels) and cortical bone (lower panels) of: mature, thick collagen, which is red or orange, and immature, thin collagen, which is green. All the scale bars represent 10 µm. (B) Content of mature, thick collagen fibers in the trabecular (upper graph) and cortical bone (lower graph) of tibia. Control, the control group of birds; experimental, the experimental group of birds subjected to C. perfringens infection. Data are mean values ± SD (whiskers) from n = 10 birds. Statistical significance: *** p < 0.001 (two-tailed Student’s t-test, t-test with Welch’s correction, or Mann–Whitney U test).

Figure 4

Representative photos of Safranine O staining carried out on formaldehyde-fixed sections from the growth plate cartilage of tibia of animals at 42 days of age. Control group (A); experimental group (B). Differences were observed in proteoglycan staining in tibial growth plate cartilage. Staining with Safranin O indicates the presence of proteoglycans, the red color in the matrix. All the scale bars represent 8 µm.

Figure 5

Representative photos of Safranine O staining carried out on formaldehyde-fixed sections from the articular cartilage of tibia of animals at 42 days of age. Control group (A); experimental group (B). Differences were observed in proteoglycan staining in tibial articular cartilage. Staining with Safranin O indicates the presence of proteoglycans, the red color in the matrix. All the scale bars represent 8 µm.