Antibody against RUNX-2 and COL-1 were purchased from Cell Signaling Technology. Antibodies against OSX, NFATc1, RANKL, and CTSK were purchased from Santa Cruz Biotechnology. Antibodies against BMP-2 was purchased from Servicebio (Table 1). Hematoxylin and Eosin (H&E) Staining Kit (C0105S) and BCIP/NBT Alkaline Phosphatase (ALP) Color Development Kit (C3206) were purchased from Beyotime. Masson’s trichrome staining solution (G1006) and tartrate-resistant acid phosphatase (TRAP) staining reagents (G1050) were purchased from Servicebio. Alizarin Red S (ARS) solution (G1452) was purchased from Solarbio, and Oil Red O staining solution (320-06-5) was purchased from Sigma-Aldrich.

Alaskan snow crab (Genus: Chionoecetes, Species: Opilio) caught wild in the United States. Fresh snow crab shells were cut into ∼1 cm pieces, crushed into fragments, washed, pH-adjusted to 10 with 0.2 mol/L KOH, and hydrolyzed at 70°C under constant stirring for 3 h. The hydrolysate was filtered, neutralized with acetic acid, hydrolyzed using papain for 4 h at 37°C, vacuum-concentrated, precipitated using ethanol, and vacuum-dried at 40°C. The molecular weights of SCSP were determined based on viscosity and retention time using a PL aquagel-OH Mixed-H Column (8 μm, 7.5 × 300 mm, Agilent) coupled with a Refractive Index Detector (Agilent) and Multi-Angle Laser Light Scattering Detector (Agilent) at 45°C. Amino acid composition was analyzed by hydrolyzing samples in 6M hydrochloric acid at 110°C for 22 h, followed by chromatography with Sulfonic Acid Cation Exchange Resin Columns (Agilent). Detections were performed at wavelengths of 570 nm and 440 nm.

Five-week-old female C57BL/6 mice (20 ± 5 g) were purchased from Home-SPF Biotechnology Co., Ltd. (Beijing, China). Mice were housed in an SPF-grade facility at Qingdao University under controlled conditions (25°C ± 3°C, 60%–70% humidity, 12-h light/dark cycle). All animal procedures followed ethical guidelines approved by the Shandong Provincial Laboratory Animal Management Committee and the Experimental Animal Center of Qingdao University (QDU-AEC-2024418).

Mice were anesthetized, and the surgical area was shaved and sterilized with iodine. The skin, mucosa, and muscle layers were incised sequentially, and a dorsal incision was made approximately 2 cm lateral to the spine at the level of the last rib. Both ovaries were ligated at the oviduct and excised. A total of 24 female C57BL/6 mice were randomly assigned into 4 groups: Sham-operated (Sham), osteoporotic model (OVX), OVX+50 mg/kg SCSP treatment (Low-SCSP), and OVX+100 mg/kg SCSP treatment (High-SCSP). The SCSP treatment groups received SCSP via oral gavage daily, while the Sham and OVX groups received equivalent volumes of saline. All treatments were continued for 8 weeks before femur collection.

Femurs were fixed in 4% paraformaldehyde (PFA) and scanned using a Micro-CT System (Quantum GX2, PerkinElmer, Japan) at 90 kV and 200 μA. 100 layers at the proximal end of the tibial platform were selected for statistical analysis of cortical layer thickness, trabecular structure, and bone marrow cavity volume using Analyzer 12.0 Software (PerkinElmer). The volume of interest (VOI) was positioned at the proximal tibial metaphysis, starting precisely 0.5 mm distal to the growth plate to exclude the primary spongiosa and epiphyseal tissue. 100 layers = 1 mm: 100 layers × 10 μm = 1000 μm (1 mm).

Femurs were decalcified, embedded in paraffin, and sectioned for histological analysis. For H&E staining, sections were deparaffinized, stained with hematoxylin for 2 min and eosin for 10 s, washed with water, and imaged. For masson trichrome staining, sections were stained with Weigert’s iron hematoxylin for 10 min, sequential stained with acid ethanol, masson blue, aniline blue, and imaged. For TRAP staining, sections were incubated with TRAP solution at 37°C for 30 min in the dark, counterstained with hematoxylin, and imaged. All the histological staining were imaged with a light microscope (Ni-U, Nikon, USA).

Decalcified bone sections were dewaxed, rehydrated, quenched, antigen-retrievaled, blocked with 5% BSA, incubated overnight at 4°C with primary antibodies, washed, incubated with secondary antibodies, counterstained with hematoxylin, and imaged with a light microscope (Nikon, USA).

Urine was collected and centrifuged at 13,000 rpm for 5 min to obtain the supernatant. Blood was collected via orbital puncture and centrifuged at 3,000 rpm for 10 min. The concentrations of calcium (Ca²⁺) and inorganic phosphate (Pi) in serum and urine were measured using Calcium (Ca²⁺) colorimetric Assay Kit (E-BC-K103-M, Elabscience) and Phosphorus (Pi) Colorimetric Assay Kit (E-BC-K245-M, Elabscience) according to the manufacturer’s instructions. Absorbance were recorded at 610 nm (Ca²⁺) and 660 nm (Pi) using a microplate reader (SpectraMax iD3, Molecular Devices, USA).

Bone marrow mesenchymal stem cells (MSCs) (CP-M131) was purchased from Pricella. MSCs were cultured in BC-T4 medium (04304P05, Baso) supplemented with 10% FBS (A5670701, Gibco). Murine MC3T3-E1 preosteoblasts were cultured in α-MEM media supplemented with 10% FBS. Cultures were passaged every 3-4 days by adding 0.25% trypsin (25300054, Gibco) for 5-10 min and re-plating at a 1:4 ratio. Osteogenic differentiation was induced with DMEM medium containing 10 mM β-glycerophosphate (HY-126304, MCE), 100 nM dexamethasone (HY-14648, MCE), and 50 μM L-ascorbic acid (HY-B0166 MCE) for 21 days (Choi et al., 2009). Adipogenic differentiation was induced with DMEM containing 100 μg/mL 3-isobutyl-1-methylxanthine, 1 μM dexamethasone (HY-14648, MCE), and 50 μg/mL ascorbic acid for 12 days (Liu et al., 2023). All cultures were maintained at 37 °C and 5% CO₂. Cell experiments were divided into 4 groups: untreated group (Normal), differentiated group (Control), 100 μg/mL SCSP group (Low-SCSP) and 200 μg/mL SCSP group (High-SCSP).

5 × 10³ cells per well were seeded in a 96-well plate and cultured in complete medium for 24 h. After SCSP treatment for 48 h, 10 μL of CCK-8 (Yeasen) solution was added to each well, and incubated for 2 h at 37°C in the dark. Absorbance was measured at 450 nm using a full-wavelength microplate reader (SpectraMax iD3, Molecular Devices, USA).

Cells were fixed with 4% PFA for 20 min and washed, stained with 1% ARS solution, BCIP/NBT staining solution, or Oil Red O solution, incubated at room temperature in the dark for 30 min, washed, and imaged with a light microscope (Ni-U, Nikon, USA).

Cells or tissues were lysed with RIPA buffer containing protease and phosphatase inhibitors (E-BC-R327, Elabscience), homogenized, and incubated on ice for 10 min. The supernatant protein concentration was determined using a BCA assay. Protein lysate was resolved on SDS-PAGE gel and transferred onto a PVDF membrane (1620177, BIO-RED). Blots were blocked in 5% non-fat dry milk, incubated with primary antibody overnight at 4°C, washed, incubated with secondary antibody for 1 h at room temperature, washed, and developed with Super Excellent Chemiluminescent Substrate Detection Kit (E-IR-R308, Elabscience).

Total RNA was isolated using FreeZol Reagent (R711-01, Vazyme), precipitated, washed with 70% ethanol and dissolved in H₂O. 1 μg of total RNA was reverse transcribed using random hexamers and Hiscript III Reverse Transcriptase (R302-01, Vazyme). 20 ng cDNA was used in each RT-qPCR reaction on a CFX96 instrument using Taq Pro Universal SYBR qPCR Master Mix (Q712-02, Vazyme). The primers used for qPCR were listed in Table 2.

RNA libraries were prepared using the VAHTS^® Universal V8 RNA-Seq Library Prep Kit (NRM605, Vazyme). Sequencing was performed on the MGI-SEQ 2000 platform. Reads were aligned to the mouse genome (GRCm38) using HISAT2, and differential expression analysis was conducted using DESeq2. GO and KEGG pathway enrichment analyses were performed using WebGestalt.

All the data conform to a normal distribution. Data were represented as means ± SEM. Statistical comparisons were performed using GraphPad Prism 9.5, employing one-way ANOVA, two-way ANOVA, or Student’s t-test. Significance was defined as P < 0.05.

To characterize SCSP, we first quantified its yield following enzymatic hydrolysis. After 4 h of hydrolysis, SCSP yield reached 12.2% of the input snow crab shells, significantly higher than the chitin content (6.42%). Molecular weight distribution analysis revealed that the 5,000-10,000 Da fraction constituted the highest proportion (47.58%), followed by the 10,000-20,000 Da fraction (27.56%) (Table 3). These findings indicate that SCSP predominantly consists of low-to medium-molecular-weight peptides, a property potentially linked to its functional stability and biological activity. Amino acid composition analysis (Table 4) showed that glutamic acid (Glu) was the most abundant (5.91 g/100 g), followed by aspartic acid (Asp, 4.59 g/100 g) and lysine (Lys, 3.36 g/100 g). Essential amino acids (EAA) constituted 39.21% of the total, while non-essential amino acids (NEAA) accounted for 60.78%. The presence of both EAA and NEAA, particularly the enrichment in Glu, Asp, and Lys, suggests that SCSP may possess substantial bioactive benefits, such as promoting bone health.

To investigate the therapeutic effects of SCSP on osteoporosis (OP), we utilized an ovariectomy (OVX)-induced osteoporosis mouse model (Figure 1A) and assessed bone morphology, bone mineral density (BMD), and trabecular parameters. Micro-CT scanning revealed significant trabecular degradation and reduced BMD in OVX mice compared to Sham controls, confirming osteoporotic bone loss. Treatment with SCSP at 50 mg/kg and 150 mg/kg markedly improved BMD and trabecular architecture, with the 150 mg/kg group showing bone parameters similar to those observed in the Sham group (Figure 1B). Quantitative analyses confirmed these findings, demonstrating that BMD, trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were significantly reduced in the OVX group compared to the Sham group, while SCSP group showed notably higher as for these parameters, highlighting the protective effects of SCSP on bone quality and morphology (Figure 1C).

Histological analysis using H&E and Masson’s staining further validated these structural improvements. Bone tissue integrity and collagen fiber distribution were severely disrupted in OVX mice, whereas SCSP administration preserved these features, particularly in the 150 mg/kg group (Figure 1D). Importantly, SCSP treatment did not induce histological abnormalities in major organs, including the heart, liver, spleen, lungs, and kidneys, across all experimental groups, as evidenced by H&E staining (Figure 1E).

These findings demonstrate that SCSP alleviates OVX-induced bone loss by improving BMD, restoring trabecular architecture, and preserving bone tissue integrity, without inducing systemic toxicity.

OP is characterized by an imbalance between osteoblast-mediated bone formation and osteoclast-driven bone resorption. To assess whether SCSP modulates this balance, we analyzed markers of osteoblast and osteoclast activity. IHC staining showed significantly reduced expression of osteoblast markers (BMP-2, RUNX-2, and COL-1) in OVX mice, reflecting impaired osteoblast function (Figure 2A). SCSP administration restored these markers in a dose-dependent manner, with the 150 mg/kg group achieving levels comparable to Sham controls. Western blot analysis further confirmed increased expression of RUNX-2 and OSX in SCSP-treated groups (Figure 2B). In contrast, TRAP staining demonstrated a significant increase in osteoclast numbers in OVX mice, indicative of enhanced bone resorption (Figure 2C). SCSP treatment significantly reduced osteoclast numbers, particularly at the 150 mg/kg dose, where levels were comparable to Sham controls. Western blot analysis revealed elevated expression of osteoclast-related proteins (NFATc1, RANKL, and CTSK) in OVX mice, which was markedly reduced by SCSP treatment (Figure 2D).

With these data in hand, we examined the effects of SCSP on calcium homeostasis, which is often disrupted in OP. As expected, OVX mice exhibited reduced urinary calcium excretion (0.18 ± 0.52 mmol/L) and elevated serum phosphorus levels (2.46 ± 0.26 mmol/L). SCSP treatment increased urinary calcium and phosphorus levels above both Sham and OVX groups (Table 5). Suggesting that SCSP modulates calcium and phosphorus metabolism, potentially counteracting the metabolic disruptions induced by ovariectomy.

These results demonstrate that SCSP re-establishes the osteoblast/osteoclast balance by enhancing osteoblast activity, inhibiting osteoclast-driven bone resorption, and normalizing calcium and phosphorus metabolism.

To further elucidate the effects of SCSP on bone regeneration, we examined its influence on osteoblast proliferation, differentiation, and mineralization using MC3T3-E1 preosteoblasts cells. CCK-8 assays revealed a dose-dependent increase in osteoblast viability with SCSP treatment (Figure 3A). ARS staining indicated enhanced mineralized nodule formation and calcium deposition in SCSP-treated groups, particularly at the high dose. ALP staining confirmed enhanced early osteogenic differentiation (Figure 3B). qPCR analysis demonstrated upregulation of osteogenic genes, including Runx-2, Bmp-2, and Opg, and downregulation of Rankl expression (Figure 3C). Western blot analysis corroborated these findings, showing increased protein levels of RUNX-2 and OSX and decreased expression of NFATc1, RANKL, and CTSK (Figure 3D), suggest the role of SCSP in maintaining osteoblast/osteoclast balance.

Similarly, MSCs, which can undergo both osteogenesis and adipogenesis exhibit enhanced osteogenic differentiation capacity in the presence of SCSP. ARS staining showed increased mineralized nodule formation in the SCSP-treated groups, indicating enhanced osteogenesis (Figure 4A). Western blot analysis confirmed these findings, with increased RUNX-2 and OSX expression and decreased NFATc1, RANKL, and CTSK levels in SCSP-treated groups (Figure 4B). In the contrast, SCSP treatment reduced lipid accumulation, suggesting an inhibitory effect on adipogenic differentiation (Figure 4C).

These findings highlight SCSP’s capacity to bias MSCs differentiation toward osteogenesis, promoting bone formation while inhibiting adipogenesis, and osteoclastgenesis.

To obtain molecular insights into the observed above mentioned bias toward to osteogenesis, we performed RNA-Seq analysis on MC3T3-E1 differentiated osteoblast cells treated with and without 200 μg/mL of SCSP. A total of 2,410 genes were upregulated and 1,837 genes were downregulated in SCSP-treated cells compared to controls (Figure 5A). KEGG pathway enrichment analysis identified significant involvement of the Cell Cycle, inflammation, and Motor Proteins pathways (Figure 5B). Key genes involved in the Cell Cycle pathway included Ccnb1, Ttk, Ndc80, Ccnb2, Cdc20, Espl1, Plk1, and Cdc25. Among these, Plk1, Ccnb2, and Ccnb1 are closely associated with the FoxO signaling pathway, a key regulator of osteoblast survival, oxidative stress, and bone remodeling. SCSP treatment also modulated inflammatory responses, altering expression of IL-17 signaling, Toll-like receptor signaling, and rheumatoid arthritis-related genes, including Ccl2, Il17re, Fosl1, Mmp13, Ccl5, Tlr1, Il12b, and Atp6v1b1. Notably, SCSP significantly upregulated genes associated with Motor Protein activity, including Myo5c, Kif20a, Kif18b, Kif4, Kif23, Kif2c, Cenpe, Kif20b, Kif14, and Myh7b.

These findings suggest that SCSP enhances osteoblast activity via cell cycle regulation and immunomodulation, and simultaneously modulating cytoskeletal function through Motor Proteins.