Limited resources during early life, including both prenatal and postnatal periods, have been shown to significantly affect the offspring by impairing cognitive development, increasing susceptibility to immune dysfunction, and reducing BMD [5, 40, 41]. CES disrupts various physiological systems, including skeletal development [42, 43]. However, most CES-related bone studies have focused on aging populations [44,45,46,47] or utilized maternal separation models [43]. In contrast, our study employed the LBN model, a minimally invasive paradigm designed to mimic resource scarcity while preserving maternal presence, to evaluate postnatal skeletal development. Compared to maternal separation, LBN may more accurately simulate limited resources in early life [43]. The postnatal timepoints selected, PND 10, 21, and 35, correspond to early infancy, childhood, and preadolescence, respectively, in humans [48], providing a translational framework for developmental evaluation. Our findings support the hypothesis that limited resources act as a significant risk factor for altered bone growth and mineralization.

Tibial BMC, indicative of calcium and phosphorus accumulation, was significantly reduced by CES at PND 21 but not at PND 10 or 35, suggesting a specific vulnerability during this window of development. Interestingly, tibial length was consistently reduced across all ages in CES rats, suggesting potential growth retardation. Since the mother is the primary source of nourishment prior to weaning, it is possible that altered maternal care or milk composition contributed to reduced mineral accrual [49, 50]. However, this remains speculative and warrants further investigation in future studies.

The observed BMD reduction is consistent with published findings on the long-term skeletal consequences of early-life stress [42, 43, 51,52,53]. For instance, maternal separation in rodents resulted in reduced BMD at 8–10 months of age [43], while stress-induced models, such as electric foot shock or PTSD induction, similarly showed BMC/BMD loss in prepubertal mice but not in adults [51]. Importantly, Zupan et al. [54] linked these changes to increased osteoclastogenesis, suggesting stress-driven enhancement of bone resorption pathways. Microarchitectural assessments revealed early effects of CES on cortical bone. At PND 10, CES increased cortical thickness, potentially suggesting compensatory mineral deposition; however, this was coupled with lower bone perimeter, indicating reduced bone size and possibly diminished structural integrity. By PND 21, CES significantly decreased cortical total volume and area, markers of compromised growth. These effects were no longer significant by PND 35, though sex differences emerged, with males showing greater cortical area and volume. These findings highlight the age-specific nature of the impacts of CES with limited resources setting and suggest partial recovery or adaptation after weaning.

Notably, CES caused persistent reductions in vertebral BMC and BMD, particularly in females, across all time points. Given the high trabecular content of the lumbar vertebrae and its metabolic sensitivity, these findings suggest long-term susceptibility of axial skeleton to CES. Increased DA at PND 21 and elevated SMI at PND 35 further suggest altered bone architecture and a shift toward mechanically unfavorable, rod-like trabecular structures. Such changes may reduce peak bone mass accrual and increase fracture risk, emphasizing the clinical relevance of preventing CES.

In terms of molecular mechanisms, gene expression analysis of the L4 vertebra at PND 21 provided insight into stress-induced alterations. We chose the L4 vertebra for transcriptomic analysis due to observed structural and mineralization changes, as well as the role of bone as a sensitive target of stress-regulated hormonal and metabolic pathways. Though RNA-seq in bone presents challenges due to tissue heterogeneity, recent literature supports its utility in identifying relevant biological changes when interpreted in the context of supporting phenotypic data [39]. Our data revealed upregulation of HOXA4 and FOS—genes associated with inhibited cell proliferation and disrupted bone remodeling, respectively [55]. Moreover, we identified downregulation of neutrophil degranulation pathways, which may suggest suppressed immune activity or altered bone marrow niche function. While previous work showed similar suppression in response to physical stress (e.g., exercise) [56,57,58], our findings extend this to early-life limited resources paradigm. Gene set enrichment analysis showed that in standard (STD) conditions, pathways related to cell growth and differentiation were dominant, consistent with increased BMC and BMD. In contrast, CES animals exhibited enrichment of neurotransmitter-related pathways (e.g., glutamate, GABA, acetylcholine release cycles), aligning with previous studies showing that chronic stress perturbs central neurotransmitter systems [52, 59, 60]. Though speculative, the presence of such pathways in bone may reflect peripheral neural-immune interactions or stress hormone signaling, which are known to influence bone remodeling.

Our study has several limitations. First, this is a descriptive study lacking histological, bone turnover, and mechanical strength assessments. These analyses would provide deeper insights into bone cell activity, osteoid formation, and structural resilience, and should be prioritized in future work. Second, shorter bones could reflect developmental delay. Although litter size and survival were similar between groups, body weights were lower in CES pups at PND 21 but normalized by PND 35 (Fig. 2), supporting a transient growth delay rather than a permanent deficit with limited resources paradigm. We did not assess maternal milk production, pup suckling behavior, or locomotion, which are relevant to bone development. However, our goal was to minimize pup handling to avoid confounding stressors during the sensitive LBN period. No observable differences in stomach milk content weights were noted at PND 10 (data not shown). Furthermore, while cross-fostering experiments could help distinguish maternal versus direct pup effects, this was beyond the scope of the current study. We have discussed this as a critical future direction and recognize it as a limitation. Lastly, while bulk RNA-seq lacks cellular resolution, our gene expression changes correspond with phenotypic differences, particularly at PND 21 when bone and body weight changes were also observed, the current transcriptomic data provide an informative molecular snapshot of CES-induced disruption in bone development.

In conclusion, our study provides evidence that early-life CES through limited bedding and nesting alters bone growth, mineralization, and structure during critical developmental windows. These effects appear age- and sex-specific, with persistent vertebral deficits and transient cortical changes. Molecular analyses suggest disrupted growth and immune signaling. Given the potential for long-term skeletal consequences, interventions aimed at maternal support and stress mitigation may be key to improving developmental outcomes in the offspring.