Functional Effects of Sericin on Bone Health and D-Serine Regulation in Estrogen-Deficient Rats

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The findings support the potential use of sericin-containing dietary supplements as functional foods or nutraceuticals to help preserve bone mineral density and trabecular structure in postmenopausal osteoporosis.

Abstract

Postmenopausal osteoporosis is characterized by progressive bone loss and deterioration of trabecular microarchitecture, yet safe and effective nutritional interventions remain limited. This study investigated the skeletal effects of whole sericin compared to isolated L-serine and calcium-only formulations in an ovariectomized (OVX) rat model. Forty female Sprague–Dawley rats underwent either sham surgery or OVX, followed by 8 weeks of daily oral administration with vehicle (calcium + vitamin D, NS), sericin formulation (S55), or L-serine. Sham and untreated OVX groups served as controls. Serum D-serine concentrations and femoral trabecular microarchitecture were assessed using fluorometric assays and micro-computed tomography (μCT), respectively. OVX significantly decreased bone volume fraction (BV/TV), bone mineral density (BMD), and trabecular number, while increasing trabecular separation. Sericin supplementation markedly improved BV/TV, BMD, trabecular thickness, and trabecular number, and reduced trabecular separation compared to both vehicle and untreated OVX controls. Sericin improved multiple trabecular parameters compared with L-serine. Serum D-serine levels were significantly elevated in the sericin group relative to calcium-only controls, though comparable to sham. These findings suggest that whole sericin exerts skeletal benefits beyond those attributable to its primary amino acid component, supporting its potential as a functional food ingredient for enhancing postmenopausal bone health.

1. Introduction

Menopause, typically occurring in women around the age of 50, is defined as the permanent cessation of menstruation following 12 consecutive months of amenorrhea [1,2]. This physiological transition is accompanied by a marked decline in circulating estrogen levels, which profoundly affects bone metabolism, including cortical and trabecular bone structures [3]. The resulting imbalance between osteoclast-mediated resorption and osteoblast-driven bone formation disrupts normal bone remodeling processes, leading to decreased bone mass and increased susceptibility to fractures [4,5].

Current therapeutic approaches for postmenopausal osteoporosis include bisphosphonates, denosumab, PTH, romosozumab, and estrogen and their receptor modulators, alongside nutritional supplementation with calcium and Vitamin D [6]. While the interventions are widely used as first-line or adjunctive treatments, they are not without limitations. Although rare, bisphosphonates and denosumab have been associated with medication-related osteonecrosis of the jaw (MRONJ), raising concerns about long-term safety [7,8]. Hormone replacement therapy remains an option for managing postmenopausal symptoms and osteoporosis; however, concerns have been raised regarding its potential association with breast cancer and thromboembolic events [9,10]. Although calcium and vitamin D are essential for bone health, supplementation alone yields modest benefits and is generally recommended as supportive rather than a standalone therapy [11].

Sericin, a silk protein derived from Bombyx mori, possesses a unique amino acid profile, comprising approximately 30% L-serine [12], and is further enriched in acidic residues such as glutamate and aspartate [13]. These residues exhibit Ca²⁺/phosphate-chelating properties, potentially facilitating mineral crystal deposition [14,15]. Upon ingestion, a portion of L-serine is enzymatically converted to D-serine via serine racemase, providing a dual pathway for osteogenic and anti-resorptive effects [16,17]. Mechanistically, L-serine has been shown to upregulate osteoblast markers, including alkaline phosphatase (ALP), Runx2, and osterix, thereby promoting bone formation. In contrast, D-serine inhibits osteoclast activation by suppressing cathepsin K expression and reducing tartrate-resistant acid phosphatase (TRAP) activity [18,19].

Recent animal studies have demonstrated that oral sericin supplementation in ovariectomized (OVX) mice or rats significantly improved trabecular bone architecture compared to both OVX controls and amino acid mixture-treated groups, although cortical bone parameters remained unchanged [19,20]. Additionally, incorporation of L-serine into gelatin scaffolds for calvarial defects in OVX rats enhanced bone mass, while downregulating cathepsin K and upregulating serine racemase expression [16]. These findings underscore the potential of serine-via its L- and D-enantiomers to simultaneously stimulate osteoblast activity and suppress osteoclast function, a particularly advantageous dual action for osteoporosis management.

However, the limited cortical response to sericin observed in prior studies [19,20] indicates the need for complementary strategies. In this study, we tested a novel sericin-based formulation (S55) that combines sericin with calcium citrate and vitamin D₃, reflecting the composition of widely used nutraceutical bone health supplements. This design enabled us to evaluate not only whether sericin exerts trabecular benefits beyond its primary amino acid component (L-serine) but also whether its combination with calcium and vitamin D₃ could enhance cortical outcomes. By situating sericin within a clinically relevant formulation, our work provides new translational insight into its potential as a functional food for postmenopausal osteoporosis.

Given the commercial availability of single-ingredient L-serine supplements, it is critical to determine whether whole sericin protein offers superior benefits compared to L-serine alone or conventional calcium-based functional foods. Therefore, this study was designed to compare (i) calcium-based formulations with or without sericin and (ii) an L-serine-only formulation against a sericin-based test food to clarify whether the whole sericin protein offers additive or synergistic effects beyond those of its primary amino acid component.

2. Materials and Methods

2.1. Materials

Experimental diets were formulated by Cefo Bio (Seoul, Republic of Korea) and administered once daily via oral gavage in a fixed volume of 0.20 mL per rat. All formulations were freshly prepared weekly, stored at 4 °C, and equilibrated to room temperature prior to administration to ensure consistency across treatment groups.

• Sericin formulation (S55; Cefo Bio, Seoul, Republic of Korea; not commercially available) contained whole sericin calibrated to deliver 25 mg/kg/day. Each 0.20 mL dose also included calcium citrate (8 mg/kg/day) and vitamin D₃ (≈15 IU/kg/day).
• Vehicle control (NS) was compositionally matched to the S55 formulation but devoid of sericin, serving as the sericin-deficient negative control.
• L-serine comparator (L-serine) consisted of crystalline L-serine (Sigma-Aldrich, St. Louis, MO, USA; Cat. #S4500), formulated to provide 25 mg/kg/day in the same 0.20 mL dose.

The sericin dose (25 mg/kg/day) was selected on the basis of previous OVX rodent studies [19]. L-serine was administered at the same equivalent dose to enable direct comparison with the whole sericin protein.

Based on an average body weight of 350 g, the required per-rat dose was calculated as 8.75 mg (25 mg/kg × 0.35 kg). To achieve this dosage, the solution was adjusted to a final concentration of 43.75 mg/mL. All animals received their respective formulations by oral gavage once daily for the duration of the study.

2.2. Animal Experiment

Forty 6-week-old female Crl:CD (Sprague–Dawley) rats (160–190 g; Samtako Bio Inc., Osan, Republic of Korea) were used in this study. Animals were housed in pairs or individually under standard laboratory conditions (temperature: 20–22 °C; humidity: 40%; light/dark cycle: 12 h/12 h), with ad libitum access to water and a controlled semisynthetic diet. Following a 1-week acclimation period, all experimental procedures were conducted under institutional guidelines and approved by the Gangneung–Wonju National University IACUC (GWNU-2024-25 approved on 27 December 2024).

Postmenopausal osteoporosis was induced via bilateral OVX. Rats were anesthetized by intramuscular injections of Zoletil (Virbac Korea, Seoul, Republic of Korea) and Rompun (Bayer Korea, Seoul, Republic of Korea). In the sham-operated group, skin and muscle were incised and sutured without ovary removal. Postoperative care included subcutaneous administration of gentamycin (5 mg/kg, SC) and meloxicam (2 mg/kg, SC) for two consecutive days. Animals were monitored daily and allowed a 4-week recovery period to permit the development of osteoporotic changes.

After the 4-week post-OVX period, rats were randomly assigned to five groups (n = 8 per group):

• Sham [non-OVX control],
• OVX-control [OVX control without treatment],
• OVX-NS [OVX + vehicle (no sericin; 0.2 mL/day)],
• OVX-S55 [OVX + sericin formulation (0.2 mL/day)],
• OVX-L-serine [OVX + L-serine only (0.2 mL/day)]

The sample size of eight rats per group was determined based on prior OVX rat studies assessing micro-computed tomography (μCT)-derived trabecular parameters, which reported statistically significant differences with group sizes ranging from six to eight under comparable conditions [19]. This number was considered sufficient to detect meaningful changes in bone mineral density and microarchitectural indices while minimizing animal use in accordance with the 3Rs principle. The OVX-control and sham groups received no test formulations beyond the standard diet. Test formulations were administered via oral gavage using a 1.0 mL syringe once daily, 5 days per week, for a total of 8 weeks. The sham and OVX-control groups received no test formulations beyond the standard diet. Body weight was recorded weekly. Brief inhalational isoflurane was used as needed to minimize stress during handling and administration. At the end of the 8-week treatment period, rats were euthanized according to ethical guidelines. Femora and blood samples were collected for subsequent analyses. The schematic overview of the experimental design is provided in Figure 1.

2.3. D-Serine Quantification in Serum Samples

Serum aliquots were thawed on ice and centrifuged at 10,000× g for 5 min to remove particulate matter. The resulting supernatants were transferred to fresh microcentrifuge tubes. To eliminate low-molecular-weight interferents and precipitate macromolecules, each sample was mixed at a 1: 25 (v/v) ratio with a proprietary sample cleanup reagent (Abcam, Cat. ab241027, Cambridge, UK), incubated at 37 °C for 15 min, and subsequently filtered through a 10 kDa molecular weight cut-off spin column (10,000× g, 10 min). The filtrates were stored at −20 °C for up to two months prior to analysis.

Fluorometric quantification of D-serine was performed using black, flat-bottom 96-well microplates. For each sample, 5 µL of the deproteinized filtrate was dispensed into two wells: one designated for selective D-serine detection and the other containing assay buffer alone to measure background fluorescence. Serine Assay Buffer was added to each well to achieve a final volume of 60 µL. Plates were pre-equilibrated at 37 °C for 10 min, followed by the addition of reaction mixes according to the manufacturer’s instructions. Reactions were incubated at 37 °C for 60 min in the dark. Endpoint fluorescence was measured using a SpectraMax 190 plate reader (Molecular Devices, San Jose, CA, USA) with excitation and emission wavelengths set at 535 nm and 587 nm, respectively. Fluorescent signals were background-corrected and converted to D-serine concentrations using standard curves generated in parallel.

2.4. Micro-Computerized Tomogram (μCT)

To ensure blinding, all femoral specimens were anonymized with coded labels prior to imaging. Raw projection data were processed at the Preclinical Center, Daegu Gyeongbuk Advanced Medical Industry Promotion Foundation. Scans were acquired using a Quantum FX μCT system (PerkinElmer, Waltham, MA, USA) under the following parameters: field of view (FOV) of 10 mm, scan duration of 3 min, tube voltage of 90 kV, and tube current of 180 μA. The system was calibrated for bone densitometry using hydroxyapatite (HA) phantom standards (0–1200 mg HA/cm³). Phantom scans were used to establish a linear calibration curve, allowing conversion of grayscale values into absolute BMD (mg HA/cm³). Images were reconstructed with filtered back-projection, incorporating manufacturer-provided beam-hardening correction and ring-artifact reduction. A 3 × 3 × 3 median filter was applied to reduce high-frequency noise. Regions of interest (ROI) were defined in the distal femoral metaphysis, starting at 0.5 mm above the growth plate to exclude the primary spongiosa, and extending 1.5 mm proximally toward the diaphysis. Cortical bone was excluded by manually contouring the endosteal boundary, restricting analysis to the trabecular compartment. A global threshold (≈5500 grayscale units), validated against HA calibration, was applied uniformly across all samples to segment mineralized bone.

Standard trabecular indices were quantified in accordance with ASBMR μCT guidelines, including bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), and structure model index (SMI). Cortical parameters (cortical thickness, cortical area, cortical BMD) were also assessed when appropriate. All analyses were performed using Analyze 12.0 software (AnalyzeDirect, Overland Park, KS, USA) by an operator blinded to group allocation, with identical reconstruction and segmentation parameters applied across groups.

2.5. Statistical Analysis

All statistical analyses were performed using GraphPad Prism 10.6.0 (GraphPad Software, San Diego, CA, USA). Normality of data distribution was assessed using the Shapiro–Wilk test, confirming Gaussian distribution in all groups. Homogeneity of variances was evaluated by Brown–Forsythe and Bartlett’s tests. Where variance equality was not assumed, Welch’s ANOVA with Welch’s correction was applied. For group comparisons, one-way ANOVA was performed, followed by Tukey’s multiple comparison test for all pairwise comparisons under equal variance conditions. In cases where variance equality was not met, Welch’s t-test was used for pairwise comparisons. Multiplicity-adjusted p-values were reported, and a family-wise alpha of 0.05 was applied. Graphs are presented with 95% confidence intervals in addition to mean ± SD. Effect sizes were expressed as mean difference ± standard error of the difference. Compact letter display was applied to summarize multiple comparisons in figures for clarity.

Additionally, complete post hoc results have been included in the Supplementary Material (Table S1). These tables provide mean differences, 95% confidence intervals, multiplicity-adjusted p-values, and effect sizes for all pairwise comparisons.

3. Results

3.1. Differential Effects of Sericin and L-Serin on Body Weight and Circulating D-Serine in OVX Rats

All animals completed the 8-week feeding protocol without mortality or observable complications related to surgery or treatment. As shown in Figure 2a, body weight differed significantly among groups (p < 0.001, one-way ANOVA). Sham-operated rats exhibited the lowest body mass (277.9 ± 19.7 g), consistent with intact estrogen status. Ovariectomy induced a predictable increase in weight, with the vehicle-treated OVX group (NS) and untreated OVX-control group reaching 360.5 ± 13.7 g and 352.1 ± 17.0 g, respectively. Sericin-supplemented rats (S55) showed similar weight gain (363.4 ± 32.1 g), whereas L-serine-treated animals (L-serine) exhibited attenuated weight gain (330.0 ± 18.2 g). Post hoc analysis confirmed that all OVX groups were significantly heavier than the sham group (p < 0.001 for each comparison). Within OVX cohorts, L-serine treatment significantly reduced body weight relative to both NS (p = 0.048) and S55 (p = 0.025), although it did not differ from the untreated OVX control. These findings suggest a weight-sparing effect of L-serine that was not observed with the sericin formulation.

Circulating D-serine levels mirrored the body weight trends (Figure 2b). Ovariectomy significantly reduced plasma concentrations from 2.452 ± 0.545 µmol/L in sham animals to 1.688 ± 0.372 µmol/L in the NS group (p = 0.0064 vs. sham) and 1.785 ± 0.484 µmol/L in the OVX control (p = 0.0217, vs. sham). Sericin supplementation restored D-serine levels to near-baseline values (2.179 ± 0.349 µmol/L), significantly exceeding the NS group (p = 0.0167). L-serine treatment resulted in an intermediate increase (2.037 ± 0.571 µmol/L), which did not reach statistical significance compared to either NS or S55.

Taken together, these findings indicate that sericin effectively reverses the OVX-induced decline in endogenous D-serine, whereas L-serine alone provides only partial restoration. Notably, the weight-sparing effect observed in L-serine was not replicated by the sericin formulation, suggesting divergent metabolic profiles despite shared amino acid content.

3.2. Sericin Preserves Trabecular Architecture in OVX Rats:μCT-Based Morphological Assessment

Representative μCT images of the distal femoral metaphysis are presented in Figure 3. The top row shows transverse 3D reconstructions of cortical and trabecular compartments, the middle row depicts longitudinal 3D renderings through the trabecular-rich metaphyseal region, and the bottom row displays raw μCT cross-sections taken at the same anatomical level with consistent scaling. Sham-operated rats exhibited the expected dense trabecular architecture, characterized by a well-organized network of plate- and rod-like structures that nearly filled the medullary cavity. Thick, interconnected trabeculae extended across the endosteal surface and bridged the cortical shell, reflecting intact bone homeostasis.

In contrast, both vehicle-treated OVX rats (NS) and untreated OVX controls displayed the classic osteoporotic phenotype associated with estrogen deficiency. These groups showed pronounced trabecular rarefaction and fragmentation, with only a few thin, disconnected struts persisting near the endosteal margin, resulting in an expanded marrow cavity and compromised structural integrity. Sericin supplementation (S55) markedly preserved trabecular architecture. Numerous struts and plates were retained, especially along the metaphyseal and endosteal regions, forming a visibly denser and more continuous lattice compared with NS or OVX-control animals. L-serine treatment produced an intermediate phenotype: while trabecular structures were sparser than in the S55 group, they remained more robust and interconnected than in NS or OVX-control, suggesting partial protection against OVX-induced bone loss.

Across groups, cortical thickness appeared largely comparable, indicating that the 8-week interventions primarily influenced trabecular, rather than cortical bone compartments.

3.3. Sericin Significantly Restores BMD and Bone Volume in OVX Rats

The qualitative differences in trabecular architecture were substantiated by quantitative morphometric data, as shown in Figure 4 and Figure 5. BMD was highest in sham-operated femora, averaging 466.9 ± 39.7 mg/cc (Figure 4a). Ovariectomy induced a marked reduction in BMD, with values declining to approximately 60% of sham levels in both the NS vehicle (290.9 ± 23.0 mg/cc) and untreated OVX-control (292.2 ± 14.3 mg/cc) groups (p < 0.001 vs. sham). Treatment with the sericin formulation (S55) significantly mitigated this loss, elevating BMD to 324.3 ± 19.9 mg/cc, representing an 11% increase over NS (p = 0.0079) and a notable improvement over OVX-control (p = 0.0028). L-serine alone yielded an intermediate BMD (299.3 ± 22.4 mg/cc), which was significantly lower than S55 (p = 0.0339) but not statistically different from either NS or OVX-control group.

BV showed a similar pattern across groups (Figure 4b). Sham rats exhibited a BV of 1.166 ± 0.305 mm³, which declined sharply in the NS group (0.096 ± 0.060 mm³; p < 0.001). Treatment with S55 more than doubled BV compared to NS (0.269 ± 0.132 mm³; p = 0.0075) and tripled it relative to OVX controls (0.074 ± 0.059 mm³; p = 0.0037). L-serine provided partial rescue (0.149 ± 0.102 mm³), though this increase was not statistically significant compared to NS. BV/TV ratio mirrored these findings (Figure 4c). Sham animals maintained BV/TV at 8.9 ± 2.3%, consistent with published ranges for healthy rat trabecular bone. In contrast, NS and OVX-control groups fell to 0.7 ± 0.5% and 0.6 ± 0.5%, respectively, reflecting severe trabecular depletion. S55 significantly restored BV/TV to 2.0 ± 0.8%, nearly threefold higher than NS (p = 0.0025) and OVX controls (p = 0.0017). L-serine increased BV/TV to 1.2 ± 0.8%, representing a numerical improvement over untreated OVX animals, though without statistical significance.

3.4. Sericin Preserves Trabecular Microstructure and Mitigates OVX-Induced Architectural Deterioration

Representative three-dimensional reconstructions demonstrated severe trabecular loss in OVX groups compared with sham, with partial restoration after sericin supplementation (Figure 5a). Quantitative analysis confirmed these observations. Tb.Th was significantly lower in NS (67 ± 12 µm) and OVX-control (63 ± 9 µm) compared with sham (100 ± 14 µm; p < 0.001). S55 treatment improved Tb.Th to 82 ± 13 µm (p = 0.014 vs. NS), whereas L-serine (71 ± 11 µm) did not significantly differ from NS.

Tb.Sp increased markedly in NS (412 ± 88 µm) and OVX controls (439 ± 102 µm) compared to sham (221 ± 52 µm; p < 0.001). S55 significantly reduced Tb.Sp (308 ± 77 µm; p = 0.009 vs. NS), whereas L-serine (389 ± 93 µm) showed no statistical difference from NS. Trabecular number (Tb.N) followed a similar pattern, with sham (1.25 ± 0.23 mm⁻¹) higher than NS (0.58 ± 0.17 mm⁻¹; p < 0.001) and OVX-control (0.56 ± 0.19 mm⁻¹). S55 partially rescued Tb.N (0.79 ± 0.21 mm⁻¹; p = 0.018 vs. NS), whereas L-serine (0.63 ± 0.18 mm⁻¹) remained comparable to NS.

3.5. Cortical Morphometry Shows OVX-Related Increases, with Limited Sericin Effect

Cortical BMD did not differ significantly among groups (Figure 6). Interestingly, cortical morphometric indices (volume, thickness, and area) were higher in OVX groups (NS and S55) compared with sham. This pattern is consistent with prior reports that ovariectomy induces transient periosteal apposition, leading to apparent cortical enlargement despite overall skeletal fragility [21,22]. Within this context, S55 treatment further increased cortical volume, thickness, and area relative to sham and L-serine (p < 0.05–0.01). L-serine did not significantly differ from NS. These findings suggest that while cortical BMD remained unchanged, the cortical morphometric changes observed after sericin supplementation likely reflect the periosteal response characteristic of OVX rather than a direct protective effect of sericin on cortical bone. Thus, the beneficial actions of sericin are more clearly supported by its effects on trabecular bone preservation, whereas cortical outcomes should be interpreted with caution.

4. Discussion

This study aimed to evaluate the potential of whole sericin as a safe, dual-acting functional food ingredient for postmenopausal osteoporosis, with particular emphasis on its skeletal benefits relative to isolated L-serine and calcium-only formulations. Using an OVX rat model, we demonstrated that oral administration of sericin significantly improved both trabecular and cortical bone parameters. Notably, sericin elevated circulating D-serine levels compared to calcium-only controls, although values remained statistically indistinguishable from sham-operated animals (Figure 2b). Micro-computed tomography revealed that sericin supplementation preserved trabecular architecture (Figure 3), higher BMD, greater BV (Figure 4), and improved structural indices such as trabecular thickness, separation, and number (Figure 4 and Figure 5). In cortical bone, sericin modestly but significantly increased cortical volume, thickness, and area compared with L-serine, despite no changes in cortical BMD (Figure 6), suggesting structural benefits beyond the trabecular compartment.

The OVX-NS group was included as the vehicle control for S55 to separate the effect of sericin from calcium and vitamin D₃. In contrast, the L-serine group was designed to evaluate the action of free L-serine itself, rather than as part of the formulation. For this reason, L-serine was administered without the vehicle, which creates some asymmetry in study design but allows complementary interpretation of sericin’s effects versus its major amino acid component. Consistent with prior reports, OVX rats exhibited significant weight gain relative to sham control (Figure 2a), likely due to estrogen deficiency-induced hyperphagia, reduced thermogenesis, and increased adiposity [20]. Both sericin and L-serine attenuated this weight gain, with L-serine exerting a more pronounced effect. This disparity may reflect differences in absorption kinetics and metabolic activity. L-serine, as a free-form amino acid, is rapidly absorbed and has been shown to stimulate brown adipose tissue thermogenesis and reduce weight regain following fasting [23,24]. In contrast, sericin—a complex protein—undergoes slower digestion, potentially resulting in more gradual metabolic effects.

Importantly, only sericin group significantly increased serum D-serine compared to calcium-only controls group (Figure 2b), suggesting sustained release and conversion of L-serine during gastrointestinal transit. Crystalline L-serine is rapidly absorbed and exhibits transient plasma levels (within 0.75–2 h) [25], which may limit its conversion to D-serine. Sericin’s resistance to proteolytic degradation and its slow-release profile likely contribute to its superior D-serine bioavailability [26]. The enhanced skeletal outcomes observed in the sericin group may be attributed not only to its sustained release properties but also to the broader amino acid composition. Unlike isolated L-serine, sericin contains multiple non-essential amino acids—including glycine, aspartic acid, threonine, and alanine—that may synergistically support bone metabolism [15]. Glycine and aspartic acid are essential for collagen fibril assembly and bone matrix mineralization [27,28], threonine contributes to extracellular matrix stability and mucin synthesis [29,30], and alanine plays a role in energy metabolism via the glucose–alanine cycle [31]. These bioactive components likely complement the osteogenic and anti-resorptive effects of L-serine [15], providing a mechanistic rationale for sericin’s superior performance in trabecular and cortical bone indices.

Beyond direct skeletal effects, it is increasingly recognized that amino acid metabolism intersects with the gut microbiome in ways that may influence bone health. Gut microbes utilize serine and threonine in mucin O-glycosylation, and mucin turnover releases these amino acids back into the lumen, supporting commensals such as Akkermansia [32,33]. Microbial fermentation of amino acids also produces short-chain fatty acids and other metabolites that can modulate bone remodeling; for instance, short-chain fatty acids have been shown to increase bone mass and protect against ovariectomy-induced bone loss in mice [34]. Importantly, estrogen deficiency is linked to altered gut permeability, bacterial translocation, and low-grade inflammation, which may exacerbate skeletal fragility; this pathway has been demonstrated to mediate bone loss in sex steroid deficiency models and to be mitigated by probiotics [35]. Therefore, the beneficial actions of sericin and L-serine observed in this study might not only reflect direct osteoblast and osteoclast modulation but could also involve microbiome-mediated pathways. Although this mechanism was beyond the scope of the present experiments, future studies should investigate how serine metabolism and gut microbial composition interact to shape bone outcomes in estrogen-deficient states.

Although cortical BMD remained unchanged, sericin group significantly improved cortical volume, thickness, and area compared to L-serine group (Figure 6). However, similar cortical enhancements have been previously documented in OVX models and are generally attributed to periosteal apposition rather than true cortical preservation [21,22]. In a longitudinal in vivo μCT study, Brouwers et al. [21] reported that cortical thickness initially declined after OVX but subsequently increased, with OVX values surpassing controls by 16 weeks. This was interpreted as compensatory periosteal bone formation. Likewise, Omelka et al. [22] observed that OVX rats exhibited increased cortical thickness and periosteal apposition despite marked trabecular bone deterioration. These findings suggest that the cortical changes observed in our study most likely reflect this OVX-induced adaptive response, while the protective effect of sericin is more convincingly supported in the trabecular compartment.

Despite these promising results, several limitations should be acknowledged. First, the OVX rat model, while widely accepted, does not fully replicate the complexity of human bone physiology. Second, only a single dose of sericin and L-serine was tested, precluding dose–response analysis. Third, the 8-week duration may have been insufficient to capture long-term skeletal remodeling, particularly in cortical bone and fracture resistance. Fourth, although μCT and serum assays provided robust structural and biochemical data, histomorphometric and molecular analyses were not performed, limiting insight into cellular mechanisms. Fifth, while sericin’s amino acid diversity likely contributed to its efficacy, individual components such as glycine, aspartic acid, threonine, or alanine were not independently evaluated. Finally, sericin and L-serine were administered five days per week rather than daily, and the potential differences in skeletal outcomes under continuous daily dosing were not considered.

Future studies should incorporate dose-escalation protocols, extended treatment durations, and mechanistic assays to delineate the cellular pathways underlying sericin’s effects. Moreover, translational research in human populations will be essential to validate sericin’s therapeutic potential as a functional food ingredient for postmenopausal bone health.

5. Conclusions

In conclusion, sericin supplementation significantly preserved trabecular microarchitecture and partially restored BMD and bone volume in OVX rats. Importantly, when combined with calcium citrate and vitamin D₃ (S55 formulation), sericin produced additive benefits compared with L-serine or vehicle controls, including modest improvements in cortical indices. These cortical effects most likely reflect OVX-induced periosteal apposition rather than direct preservation, underscoring that the strongest evidence of benefit lies in the trabecular compartment. To our knowledge, this is the first study to demonstrate that sericin, when formulated with calcium and vitamin D₃, provides additive skeletal benefits in a clinically relevant nutraceutical context. This highlights sericin’s translational potential as a functional food ingredient for postmenopausal osteoporosis management, while pointing to future research directions such as dose–response studies, biomechanical validation, and exploration of gut microbiome-mediated mechanisms.