The review, conducted by a team at Manipal Academy of Higher Education and registered prospectively with PROSPERO, followed the PRISMA 2020 reporting guidelines and searched PubMed, Scopus, and Embase for studies published between June 2015 and June 2025. From 975 initial records, the researchers whittled the field down to 105 eligible studies, all of which used chemicals to induce osteoporosis-like bone loss in zebrafish. The sheer volume of work is striking: publications rose steadily over the decade, with a notable surge after 2021 and a peak of 24 studies in 2024 alone, signaling rapidly growing interest in the tiny striped fish as a skeletal research platform.

Why zebrafish? The answer lies in a remarkable combination of biology and practicality. Humans and zebrafish share more than 70 percent of their genes, with at least one zebrafish ortholog existing for the vast majority of human genes. Crucially, the molecular machinery that governs bone remodeling, including the Wnt/beta-catenin, BMP, and RANK/RANKL pathways, is conserved between fish and mammals. Zebrafish larvae are optically transparent, which means researchers can watch bone mineralization unfold in real time under a microscope using fluorescent dyes, without sacrificing the animal. Their small size, rapid development, and high fecundity make them dramatically cheaper and faster than mice or rabbits, and they sidestep many of the ethical concerns associated with larger vertebrate models.

The review found that glucocorticoids dominate the field. Of the 105 studies, 92 relied on these steroid drugs to trigger bone loss, with prednisolone used in 54 studies and dexamethasone in 38. The remaining studies employed a colorful menagerie of other compounds: ferric ammonium citrate to model iron overload appeared in seven studies, while doxorubicin, thioacetamide, alloxan, streptozotocin, and aluminum chloride collectively accounted for six more. Each chemical class produces bone loss through a distinct route, yet the review identified a striking convergence in the resulting pathology.

That convergence centers on a well-defined molecular cascade. Glucocorticoids enter cells and bind to cytoplasmic glucocorticoid receptors, which then translocate to the nucleus and reprogram gene expression. One consequence is the suppression of antioxidant defenses such as superoxide dismutase, glutathione, and catalase, flooding cells with mitochondrial reactive oxygen species. This oxidative stress activates NF-kappaB signaling and the RANK/RANKL/OPG axis, driving osteoclasts, the cells that demolish bone, into overdrive. Simultaneously, glucocorticoids block bone-forming pathways including focal adhesion signaling, TGF-beta/Smad, and Wnt/beta-catenin, hobbling the osteoblasts that build new bone. The result is a double hit: resorption accelerates while formation stalls, precisely the imbalance that characterizes human osteoporosis.

Iron overload models tell a parallel story. Excess iron accumulates in bone tissue, generates oxidative stress, and impairs mineralization, with studies documenting depleted levels of calcium, magnesium, potassium, and zinc in the skeleton. Doxorubicin wreaks havoc through overproduction of reactive oxygen and nitrogen species, while alloxan and streptozotocin damage osteoblasts through the buildup of advanced glycation end products. Despite these different triggers, nearly every model reproduced the same core phenotype: reduced mineralization, suppressed osteoblast differentiation, heightened osteoclast activity, and oxidative stress-mediated bone loss.

Methodologically, the field has settled into recognizable patterns. Most studies exposed larvae between 3 and 9 days post-fertilization, with prednisolone typically applied at 10 to 25 micromolar and dexamethasone at 5 to 20 micromolar. Adult fish, used in a smaller subset of studies, received higher concentrations over longer periods, sometimes up to 25 days. Assessment techniques revolve around skeletal staining: Alizarin Red to visualize calcium deposition, Calcein for real-time tracking of bone formation, and Alcian Blue for cartilage. These are complemented by quantitative polymerase chain reaction measuring osteogenic genes such as RUNX2, SP7, ALP, and OCN, alongside osteoclast markers including RANKL, CTSK, and TRAP. A handful of studies pushed the envelope further, using micro-computed tomography to measure bone mineral density or optical coherence tomography to image living bone noninvasively.

But the review also exposes a troubling weakness: methodological inconsistency. Chemical concentrations, exposure windows, developmental stages, and outcome measures vary widely across studies, making direct comparison difficult. The quality assessment was even more sobering. Only 34 of the 105 studies reported randomizing animals to groups, just 15 described random outcome assessment, and not a single study reported blinded outcome evaluation. Allocation concealment was essentially absent from the literature, and no study performed a priori sample size calculations. Many papers omitted animal numbers, ages, or routes of administration. The authors caution that researchers may have followed sound practices without reporting them, but the gap between what is done and what is documented undermines reproducibility and, ultimately, confidence in the models.

There are also inherent translational limits to keep in mind. Zebrafish lack weight-bearing bones, so their skeletons experience mechanical loads very differently from the human hip or spine. Most zebrafish studies measure mineralization and cellular activity rather than bone fragility, biomechanical strength, or fracture susceptibility, which means these models more accurately represent osteopenia-like or osteoporosis-like conditions than the full clinical disease. The review’s authors argue this should temper interpretation without diminishing the models’ genuine value for mechanistic discovery and early-stage drug screening, where zebrafish excel at rapidly identifying candidate compounds before costly validation in mammals.

To close these gaps, the review proposes a standardization framework that could reshape how the field operates. It calls for explicit reporting of developmental stage, with larvae between 3 and 9 days post-fertilization recommended for rapid mineralization screening and juvenile or adult fish reserved for structural bone assessment. Exact chemical concentrations, exposure windows, and administration routes should be specified, alongside quantitative metrics such as integrated optical density, fluorescence intensity, and vertebral counts. A core panel of molecular markers, spanning both osteogenic and osteoclastic genes, would allow results to be compared across laboratories. Transparent reporting of sample sizes, ethical approvals, and statistical methods rounds out the recommendations. If the field embraces these standards, the humble zebrafish, already swimming through hundreds of drug screens, could become a trusted bridge between cell culture and mammalian studies, accelerating the search for therapies that protect the world’s aging bones.

Subject of Research: Chemically induced osteoporosis models in zebrafish

Article Title: Experimental models of chemically induced osteoporosis in zebrafish: A systematic review

Article References: Experimental models of chemically induced osteoporosis in zebrafish: A systematic review. (n.d.). https://doi.org/10.1016/j.heliyon.2026.e45495

Image Credits: AI Generated

DOI: 10.1016/j.heliyon.2026.e45495

Keywords: zebrafish, osteoporosis, bone loss, glucocorticoids, prednisolone, dexamethasone, systematic review, bone mineralization, osteoblasts, osteoclasts, drug screening, oxidative stress