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. 2021 Oct 20;10(21):4808.
doi: 10.3390/jcm10214808.

Femoral µCT Analysis, Mechanical Testing and Immunolocalization of Bone Proteins in β-Hydroxy β-Methylbutyrate (HMB) Supplemented Spiny Mouse in a Model of Pregnancy and Lactation-Associated Osteoporosis

Ewa Tomaszewska  1 , Siemowit Muszyński  2 , Janine Donaldson  3 , Piotr Dobrowolski  4 , Deepesh K P Chand  1 , Agnieszka Tomczyk-Warunek  5 , Monika Hułas-Stasiak  4 , Iwona Puzio  1 , Krzysztof Lamorski  5 , Cezary Sławiński  5 , Mirosław Jabłoński  6 , Tomasz Blicharski  6
Affiliations

Femoral µCT Analysis, Mechanical Testing and Immunolocalization of Bone Proteins in β-Hydroxy β-Methylbutyrate (HMB) Supplemented Spiny Mouse in a Model of Pregnancy and Lactation-Associated Osteoporosis

Ewa Tomaszewska et al. J Clin Med. .

Abstract

A metabolite of leucine, ß-hydroxy-ß-methylbutyrate (HMB), used as a dietary supplement effects muscle tissue gain and bone tissue quality. Since there are no studies on the effects of HMB during pregnancy yet, the aim of the current study was to determine the effects of HMB supplementation during pregnancy on osteoporotic bone quality postpartum and post-lactation using spiny mice (Acomys cahirinus) as the animal models. The six-month-old dams were divided into four groups: pregnant and lactating controls, and pregnant and lactating HMB-treated (during the second trimester of pregnancy) females. The intensity of the immunoreaction of osteocalcin (OC), osteoprotegerin (OPG), bone morphogenetic protein 2 (BMP-2), tissue inhibitor of metalloproteinases 2 (TIMP-2), matrix metalloproteinase 8 and 13 (MMP-8 and MMP-13) and proteins involved in bone turnover, was measured in femoral trabecular and compact bone, as well as in the hyaline and epiphyseal cartilage of the femora. The analysis of the trabecular bone microarchitecture showed that the administration of HMB to pregnant females, by influencing the proteins responsible for bone cell activity and collagen remodeling, can provide protection from bone loss. Based on the results of the current study it can be assumed that HMB administration to pregnant females has a more positive impact on trabecular than compact bone.

Keywords: bone; bone proteins; bone quality; lactation; pregnancy; β-hydroxy β-methylbutyrate.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of HMB supplementation (0.02 g/kg b.w.) during the middle trimester of pregnancy (13–26 d) on (A) weight, (B) length of femora as well as bone mid-diaphysis (C) cross-sectional area, (D) mean relative wall thickness (MRWT) and (E) cortical index of femora of spiny mice at delivery or after the lactation period. Figure shows LSMeans ± standard deviation as well as p-values of a two-way ANOVA with the supplementation (control, HMB) and period (delivery, lactation) and their interaction as fixed effects. Means with different letters in interaction effect plots differ significantly (Tukey’s post-hoc test).
Figure 2
Figure 2
Representative µCT images of sagittal sections of the femur distal diaphysis (A) and transverse sections of the femur mid-diaphysis (B) from pregnant female controls (not receiving HMB) at delivery (CONT-D) or after the lactation period (CONT-L) and from pregnant HMB females (receiving HMB at a dose of 0.02 g/kg b.w. during the middle trimester of pregnancy) at delivery (HMB-D) or after the lactation period (HMB-L). Scale bars represent 1000 µm.
Figure 3
Figure 3
Effect of HMB supplementation (0.02 g/kg b.w.) during the middle trimester of pregnancy (13–26 d) on (A) fracture strength, (B) yield strength, (C) Young’s modulus, (D) work to fracture and (E) stiffness of femora of spiny mice at delivery or after the lactation period. Figure shows LSMeans ± standard deviation as well as p-values of a two-way ANOVA with the supplementation (control, HMB) and period (delivery, lactation) and their interaction as fixed effects. Means with different letters in interaction effect plots differ significantly (Tukey’s post-hoc test).
Figure 4
Figure 4
Effect of HMB supplementation (0.02 g/kg b.w.) during the middle trimester of pregnancy (13–26 d) on histomorphometry of femoral trabeculae in bone (A–C) metaphysis and (a–c) epiphysis of femora of spiny mice at delivery or after the lactation period. Figure shows LSMeans ± standard deviation as well as p-values of a two-way ANOVA with the supplementation (control, HMB) and period (delivery, lactation) and their interaction as fixed effects. Means with different letters in interaction effect plots differ significantly (Tukey’s post-hoc test).
Figure 5
Figure 5
Representative images of the immunohistochemical reactions for osteoprotegerin (OPG), osteocalcin (OC), bone morphogenetic protein 2 (BMP-2), tissue inhibitor of metalloproteinases 2 (TIMP-2), matrix metalloproteinase 8 (MMP-8) and matrix metalloproteinase 13 (MMP-13) in the growth plate cartilage of femora from pregnant female controls (not receiving HMB) at delivery (CONT-D) or after the lactation period (CONT-L) and from pregnant HMB females (receiving HMB at a dose of 0.02 g/kg b.w. during the middle trimester of pregnancy) at delivery (HMB-D) or after the lactation period (HMB-L). All the scale bars represent 40 µm.
Figure 6
Figure 6
Representative images of the immunohistochemical reactions and the percent of immunoreactive cells for osteoprotegerin (OPG), osteocalcin (OC), bone morphogenetic protein 2 (BMP-2), tissue inhibitor of metalloproteinases 2 (TIMP-2), matrix metalloproteinase 8 (MMP-8) and matrix metalloproteinase 13 (MMP-13) in the trabecular bone of femora from pregnant female controls (not receiving HMB) at delivery (CONT-D) or after the lactation period (CONT-L) and from pregnant HMB females (receiving HMB at a dose of 0.02 g/kg b.w. during the middle trimester of pregnancy) at delivery (HMB-D) or after the lactation period (HMB-L). All the scale bars represent 40 µm. Bar plots show the percentage of immunoreactive osteocytes in the trabecular bone in each group.
Figure 7
Figure 7
Representative images of the immunohistochemical reactions and the percent of immunoreactive cells for osteoprotegerin (OPG), osteocalcin (OC), bone morphogenetic protein 2 (BMP-2), tissue inhibitor of metalloproteinases 2 (TIMP-2), matrix metalloproteinase 8 (MMP-8) and matrix metalloproteinase 13 (MMP-13) in the compact bone of femora from pregnant female controls (not receiving HMB) at delivery (CONT-D) or after the lactation period (CONT-L) and from pregnant HMB females (receiving HMB at a dose of 0.02 g/kg b.w. during the middle trimester of pregnancy) at delivery (HMB-D) or after the lactation period (HMB-L). All the scale bars represent 40 µm. Bar plots show the percentage of immunoreactive osteocytes in the trabecular bone in each group.
Figure 8
Figure 8
Representative images of the immunohistochemical reactions and the percent of immunoreactive cells for osteoprotegerin (OPG), osteocalcin (OC), bone morphogenetic protein 2 (BMP-2), tissue inhibitor of metalloproteinases 2 (TIMP-2), matrix metalloproteinase 8 (MMP-8) and matrix metalloproteinase 13 (MMP-13) in the articular cartilage of femora from pregnant female controls (not receiving HMB) at delivery (CONT-D) or after the lactation period (CONT-L) and from pregnant HMB females (receiving HMB at a dose of 0.02 g/kg b.w. during the middle trimester of pregnancy) at delivery (HMB-D) or after the lactation period (HMB-L). All the scale bars represent 40 µm. Bar plots show percentage of immunoreactive chondrocytes in the articular cartilage in each group.
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