Probiotics Enhance Bone Growth and Rescue BMP Inhibition: New Transgenic Zebrafish Lines to Study Bone Health

Abstract

Zebrafish larvae, especially gene-specific mutants and transgenic lines, are increasingly used to study vertebrate skeletal development and human pathologies such as osteoporosis, osteopetrosis and osteoarthritis. Probiotics have been recognized in recent years as a prophylactic treatment for various bone health issues in humans. Here, we present two new zebrafish transgenic lines containing the coding sequences for fluorescent proteins inserted into the endogenous genes for sp7 and col10a1a with larvae displaying fluorescence in developing osteoblasts and the bone extracellular matrix (mineralized or non-mineralized), respectively. Furthermore, we use these transgenic lines to show that exposure to two different probiotics, Bacillus subtilis and Lactococcus lactis, leads to an increase in osteoblast formation and bone matrix growth and mineralization. Gene expression analysis revealed the effect of the probiotics, particularly Bacillus subtilis, in modulating several skeletal development genes, such as runx2, sp7, spp1 and col10a1a, further supporting their ability to improve bone health. Bacillus subtilis was the more potent probiotic able to significantly reverse the inhibition of bone matrix formation when larvae were exposed to a BMP inhibitor (LDN212854).

A realistic mixture of ubiquitous persistent organic pollutants affects bone and cartilage development in zebrafish by interaction with nuclear receptor signaling

“…Following the assessment of bone mineralization, in a second experiment, the transgenic line Tg(col10a1a : col10a1a-GFP) was used, which contains the coding sequence for the fluorescent protein GFP inserted into the coding region of the endogenous col10a1a gene, thereby causing a fusion protein col10a1a-GFP mRNA to be expressed under the control of the endogenous col10a1a promoter [ 38 ]. The expressed fusion protein is secreted from the cells and then binds to mineralized or un-mineralized (total) bone matrix in the living larvae.…”

“…A decrease in mineralized bone formation, as observed here, may be due to inhibition of osteoblast proliferation, differentiation, inhibition of ECM deposition, or the final step of mineralization ( Fig 8 ) [ 36 , 83 , 84 ]. Here, the transgenic line Tg(col10a1a : col10a1a-GFP) was used to reveal total (i. e. unmineralized and mineralized) bone ECM, combined with live staining of the mineralized ECM [ 38 ]. This experiment revealed that the POP125× more strongly affected deposition of the unmineralized ECM than the subsequent mineralization.…”

“…Adult wild-type zebrafish (WT) of the AB strain and Tg(col10a1a : col10a1a-GFP) [ 38 ] were obtained from breeding facilities at the GIGA-Institute, Liege, Belgium. Fish maintenance, breeding conditions, and egg production were described [ 93 , 94 ] and are in accordance with internationally accepted standards.…”

Interindividual differences contribute to variation in microbiota composition more than hormonal status: A prospective study

“…Lactococcus lactis produces the bacteriocin nisin, which markedly decreases the inflammatory infiltrate and increases the number of fibroblast-like and osteoblast cells in the periodontal complex, which in turn prevents alveolar bone loss in a mouse model of periodontitis (77). It is not clear if these effects may be achieved systematically, but L. lactis upregulates genes responsible for osteoblast formation and bone matrix growth and mineralization in zebrafish (78). This suggests that an increase in B. vulgatus and a decrease in L. lactis may accelerate bone health deterioration after menopause by multiple mechanisms.…”

Oral administration of live combined Bacillus subtilis and Enterococcus faecium alleviates colonic oxidative stress and inflammation in osteoarthritic rats by improving fecal microbiome metabolism and enhancing the colonic barrier

“…Supplementation with B.subtilis reduced bone loss due to periodontitis in rats. Lactobacillus and Bifidobacterium have been shown to prevent ovariectomy-mediated bone loss (Sojan et al, 2022 ). Although the effects of probiotics on osteoarthritis are less well reported at present, there is a growing amount of evidence emerging.…”