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. 2026 Jun 25;31(3):pnf.2026.043. doi: 10.3746/pnf.2026.043

Deer Antler Extract Promotes Osteoblast Differentiation and Improves Bone Microarchitecture in an Ovariectomy-Induced Osteoporosis Model

Jae-Hee Kwon 1, Sang-Hoon Lee 1, Seung-Ho Lee 2, Sung-Won Kim 2, Young-Eun Cho 1,✉
PMCID: PMC13314397  PMID: 42382026

Abstract

Deer antler has long been recognized for its pronounced influence on bone growth and regeneration via its unique ability to induce rapid repeated ossification. This study aimed to investigate whether deer antler water extract (DWE) can prevent osteoporosis by promoting osteoblast differentiation and preserving bone microarchitecture. In vitro experiments using MC3T3-E1 pre-osteoblast cells demonstrated that DWE enhanced osteoblast differentiation without affecting cell viability. DWE increased alkaline phosphatase (ALP) activity; intensified ALP, Von Kossa, and Alizarin Red S staining; and upregulated the expression of osteogenic markers, including runt-related transcription factor 2, ALP, osteopontin, and procollagen type I, in a concentration- and time-dependent manner. In an ovariectomy (OVX)-induced osteoporosis mouse model, DWE improved serum biochemical parameters. Micro-computed tomography analysis of the femur revealed a marked restoration of bone microarchitecture, as indicated by increased bone volume to total volume and trabecular number, along with reduced trabecular separation. Histological examination using hematoxylin and eosin staining further confirmed morphological improvements in bone tissue. Overall, these findings demonstrate that DWE contributes to bone homeostasis maintenance by enhancing osteoblast differentiation and improving bone microarchitecture both in vitro and in an OVX-induced osteoporosis model. This study suggests that DWE may serve as a promising natural product for osteoporosis prevention.

Keywords: bone tissue, deer antler, deer antler extract, osteoblast, osteoporosis

INTRODUCTION

Deer antler is used as a disease-prevention supplement in the pharmacopoeias of many countries, including Korea, Japan, and China (Sui et al., 2014), and has traditionally been used for osteoporosis management (Choi et al., 2026). Long-term administration of deer antlers or their extracts suppresses bone density loss, improves blood mineral levels, and changes bone metabolism-related hormones in ovariectomized animals; in addition, deer extract or collagen also showed a protective influence by alleviating bone loss in an animal model of osteoporosis induced by glucocorticoid administration or ovariectomy (OVX) (Baek et al., 2023; Pan et al., 2023). Recent research has reported that deer antler extract exerts an anti-osteoporotic effect through a dual mechanism of promoting osteoblast differentiation and inhibiting osteoclast production (Choi et al., 2026), further strengthening the scientific basis for the bone health-promoting effects of deer antler, a traditional medicine.

Osteoporosis is a systemic bone metabolic disease that reduces strength due to decreased bone mass and microstructure deterioration, increasing fracture risk (Marcucci et al., 2023). Post-menopausal osteoporosis is the most common type caused by estrogen deficiency. Decline in female hormone levels promotes resorption in all types of bone cells, disrupting the bone formation-resorption balance and causing rapid bone loss (Black and Rosen, 2016). Pathophysiologically, an imbalance in which bone resorption by osteoclasts gains dominance over bone formation by osteoblasts is at the core of osteoporosis; such a change manifests as loss of connectivity in cancellous bone and reduction in cortical thickness and porosity in compact bones (Huang et al., 2024). As the population ages, the number of patients with osteoporosis and the incidence of fractures are increasing, rendering the development of effective treatment methods an important public health requirement (Plank et al., 2025). Clinical osteoporosis therapies are classified as bone resorption inhibitors or bone formation promoters (Hwang et al., 2023; Huang et al., 2024). However, they exhibit limitations, such as the appearance of various side effects with long-term use and large differences in efficacy among patients (Sözen et al., 2017).

Therefore, safer and more effective new therapeutic strategies are required to replace or supplement existing drugs, and natural products with fewer side effects are attracting attention as alternative drugs for the prevention and treatment of osteoporosis. Studies have reported the bone-protective impacts of specific deer antler-derived fractions, collagen components, and isolated bioactive constituents. Building on these findings, this study aimed to comprehensively evaluate the bioefficacy of deer antler water extract (DWE), a form more directly applicable to functional food and nutraceutical development. In particular, a hot-water-based soluble extract offers practical advantages in terms of industrial scalability, manufacturing reproducibility, and future expansion as a functional natural material.

To achieve this objective, the present study systematically integrated an in vitro osteoblast differentiation assay using MC3T3-E1 cells with in vivo validation of bone microarchitecture preservation in an OVX-induced osteoporosis mouse model. Furthermore, by combining molecular osteogenic marker analysis, mineralization assays, micro-computed tomography (µCT)-based bone structural assessment, and histomorphometry evaluation, this study establishes a comprehensive validation framework for the anti-osteoporotic efficacy of DWE. Collectively, these findings provide integrated scientific evidence supporting the potential application of such an extract as a natural product-based practical candidate for osteoporosis prevention (Fig. 1).

Fig. 1.

Fig. 1

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A schematic of the experimental design and osteogenic effects of deer antler water extract (DWE) in vitro and in vivo. DWE was prepared and applied to in vitro and in vivo experimental models to evaluate its osteogenic effects. In vitro, DWE was administered to osteoblast precursor cells, enhancing cell proliferation and osteogenic differentiation, as indicated by increased matrix mineralization. In vivo, DWE was orally administered to ovariectomized mice, improving bone microarchitecture and enhancing bone formation. Blue arrows indicate the promotive effects of DWE on osteogenesis and bone formation.

MATERIALS AND METHODS

Animal ethics

All animal experiments were conducted in accordance with the ARRIVE guidelines and the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Female C57BL/6J mice, 9 weeks old, were used to establish the OVX-induced osteoporosis model. All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Andong National University, Gyeongsangbuk-do, South Korea (approval number: 2024-4-1111-02-01). The DWE used was provided by Korea Ginseng Corporation. As no live animals were handled, sacrificed, or subjected to experimental procedures, therefore, institutional animal ethics approval was not required for the use of deer antler-derived materials.

Water extraction of deer antlers

The deer antler used was provided by Korea Ginseng Corporation and was originally sourced from Christchurch, the South Island region of New Zealand. The antlers were harvested during the velvet stage within approximately 45－60 days after antler emergence, before complete ossification, which is considered the optimal collection period in New Zealand. Deer antler was extracted with distilled water to prepare the DWE. Briefly, the deer antler material was mixed with distilled water and hot water extracted. The extract was then cooled to room temperature, centrifuged to remove the insoluble debris, and the supernatant was collected and filtered. The filtrate was concentrated under reduced pressure and lyophilized to obtain the DWE powder. The dried extract was stored at −20°C until use and reconstituted in sterile distilled water [or phosphate-buffered saline (PBS)] immediately before experiments.

Materials

Bicinchoninic acid assay (BCA), radio-immunoprecipitation assay (RIPA) buffer, and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) reagents, enhanced chemiluminescence substrate were purchased from Thermo Fisher Scientific. α-Minimum essential medium (α-MEM), PBS at 1x (pH 7.4), fetal bovine serum (FBS), and penicillin-streptomycin (P/S) were bought from Gibco. Ascorbic acid, β-glycerophosphate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide, Alizarin Red S, AgNO3 were purchased from Sigma-Aldrich. Primary antibodies against osteopontin (OPN) (sc-21742), alkaline phosphatase (ALP) (sc-271431), procollagen type I (Pro COL I), and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology, and the runt-related transcription factor 2 (RUNX2) (ab76956-100) antibody was obtained from Abcam.

Cell culture

MC3T3-E1 cells were cultured in α-MEM supplemented with 10% FBS and 1% P/S at 37°C in a humidified incubator containing 5% CO2. Osteogenic differentiation was induced using α-MEM containing 10% FBS, 1% P/S, 100 µg/mL ascorbic acid, and 10 mM β-glycerophosphate under the same conditions.

MTT assay

MC3T3-E1 cells were seeded into 96-well plates at a density of 1×104 cells per well and cultured at 37°C and under 5% CO2. After cell attachment, the medium was replaced with differentiation-inducing medium, and the cells were treated with DWE at concentrations of 10, 100, 250, and 500 µg/mL for 3 or 7 d. Subsequently, MTT solution was added to each well and incubated for 3 h at 37°C. After removing the medium, dimethyl sulfoxide was added, and the plate was gently shaken for 30 min to dissolve the formazan crystals. The A570 was measured with an AU/M200 microplate reader (Infinite M200, Tecan Systems Inc.) to assess cell proliferation. Each experimental condition was performed independently in triplicate at 3 or 7 d (n=3).

Osteogenic and mineralization staining

MC3T3-E1 cells were seeded into a 12-well plate at a density of 1×105 cells per well, and DWE was administered when the cells reached ∼60% confluence. After 7 or 14 days of treatment, the culture medium was removed, and the cells were washed twice with PBS. The cells were then fixed with 70% ethanol at room temperature for 30 min, followed by three washes with distilled water. For ALP staining, fixed cells were stained using NBT/BCIP. For Von Kossa staining, fixed cells were incubated with 3% AgNO3 solution under ultraviolet light for 1 h and then washed with distilled water. For Alizarin Red S staining, fixed cells were incubated with 40 mM Alizarin solution for 30 min at room temperature and washed thoroughly with distilled water. Mineralized nodules stained by all three assays were observed under a microscope. Alizarin Red S-stained cells were quantified by dissolving them with cetylpyridinium chloride and measuring their A570. Alizarin Red S in four independent wells per condition was quantitatively analyzed at each time point (n=4).

Western blots

Cells were lysed in RIPA buffer to extract the total protein, which was quantified using the BCA assay. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Cytiva) in skim milk powder dissolved in Tris-buffered saline with Tween 20 (TBS-T) (5% 20× TBS buffer, 1% Tween-20 in distilled water) and washed with TBS-T. After blocking, the membranes were incubated overnight at 4°C with primary antibodies, and then with HRP-conjugated secondary antibodies. Protein bands were visualized utilizing enhanced chemiluminescence substrate, and band intensities were quantified using the Fusion Solo X imaging system (Vilber). Western blot analyses were repeated independently (RUNX2, ALP, and Pro COL I: n=3 at 3 and 7 d; OPN: n=2 at 3 d and n=3 at 7 d).

Animal experiments

Female C57BL/6J mice were used in this study. Following ovariectomy, the animals were allowed to recover for a week before daily oral DWE administration for 8 weeks. The experimental groups were as follows: sham-operated (Sham) group, subjected to a sham surgery; OVX group, ovariectomized mice; estradiol-treated (EST) group, ovariectomized mice receiving oral estradiol; DWE-65 group, ovariectomized mice receiving DWE at 65 mg/kg; and DWE-500 group, ovariectomized mice receiving DWE at 500 mg/kg. Animals were provided ad libitum access to a standard chow diet and water; body weight was measured daily throughout the experimental period. At the end, the mice were sacrificed, and blood and tissue samples were collected.

Serum biochemical analysis

Serum total cholesterol (TC) levels were measured to assess lipid metabolism. Serum samples were analyzed using FUJI DRI-CHEM SLIDE. The DRI-CHEM NX500 analyzer (Fujifilm) was employed for the measurements per the manufacturer’s instructions. For serum TC analysis, n=6 per group was applied.

µCT analysis of bone parameters

Bilateral femurs were isolated from OVX female mice and scanned with a Skyscan 1276 µCT system (Bruker) at a 9.7 µm resolution. Three-dimensional (3D) images of the trabecular bone were reconstructed with uniform threshold and segmentation parameters (sigma=1.2, support=2, and threshold=180). Bone volume (BV, mm3); bone volume to total volume (BV/TV, %); trabecular thickness (Tb.Th, mm); trabecular number (Tb.N, mm−1); trabecular separation (Tb.Sp, mm) for each group was measured and compared to assess bone loss or preservation. All analyses were performed in a blinded manner at the Chronic and Metabolic Diseases Research Center, Sookmyung Women’s University, Seoul, South Korea. The sample numbers for each analysis were as follows: Sham (n=7), OVX (n=5), EST (n=7), DWE-65 (n=8), and DWE-500 (n=8).

Histological and histomorphometric analyses of bone

Femurs were fixed in 4% paraformaldehyde (Lugen Sci Co.), dehydrated with ethanol, and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin (H&E), and osteoblasts were quantified. Static bone parameters, including the number of osteoblasts per bone perimeter (N.Ob/B.Pm, mm) and osteoblast surface per bone surface (Ob.S/BS, %), were calculated. All analyses were performed in a blinded manner at the Chronic and Metabolic Diseases Research Center, Sookmyung Women’s University. For the histomorphometry of N.Ob/B.Pm and Ob.S/BS, n=5 per group, was employed.

Statistical analysis

All experimental data are presented as mean±standard deviation. Statistical analyses were performed using one-way analysis of variance (ANOVA) to evaluate inter-group differences. Following ANOVA, Tukey’s multiple comparison test was applied as a post hoc analysis. Statistical significance was defined as P<0.05.

RESULTS

DWE promotes osteogenic differentiation in MC3T3-E1 cells

To examine the effects of DWE on osteoblast differentiation, MC3T3-E1 pre-osteoblast cells were treated with increasing concentrations of DWE. As shown in Fig. 2A and 2B, DWE did not induce any cytotoxicity or adversely affect cell proliferation after 3 or 7 d. Instead, DWE-treated cells maintained comparable or slightly increased viability relative to the control group.

Fig. 2.

Fig. 2

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Effects of deer antler water extract (DWE) on osteogenic differentiation in MC3T3-E1 cells. (A and B) Proliferation of MC3T3-E1 cells treated with 10, 100, 250, and 500 µg/mL of DWE for 3 or 7 d was ascertained using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (n=3 for each time point). (C) Alkaline phosphatase staining was evaluated in MC3T3-E1 cells treated with DWE for 7 and 14 d (n=3). All data are presented as mean±standard deviation. Statistical analysis was conducted using one-way analysis of variance, followed by Tukey’s multiple comparison post hoc test. Various letters indicate statistically significant inter-group differences (P<0.05). CON, control.

ALP staining revealed that DWE enhanced early osteogenic differentiation in a concentration-dependent manner at 7 and 14 d (Fig. 2C). These results indicate that DWE promotes osteoblast differentiation without compromising cell viability.

DWE enhances osteogenic mineralization in MC3T3-E1 cells

The effects of DWE on extracellular matrix mineralization were evaluated via Von Kossa and Alizarin Red S staining. As shown in Fig. 3A and 3B, DWE markedly increased nodule mineralization at 7 or 14 d compared with the control group. Quantitative analysis of Alizarin Red S staining data demonstrated a significant enhancement in calcium deposition in DWE-treated cells in a concentration- and time-dependent manner (Fig. 3C and 3D). These findings suggest that DWE effectively promotes late-stage osteogenic mineralization in MC3T3-E1 cells.

Fig. 3.

Fig. 3

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Effects of deer antler water extract (DWE) on osteogenic mineralization in MC3T3-E1 cells. (A) Von Kossa staining was performed to evaluate mineral deposition in MC3T3-E1 cells treated with 10, 100, 250, and 500 µg/mL of DWE for 7 or 14 d. (B) Alizarin Red S staining was conducted to assess calcium deposition in MC3T3-E1 cells following DWE treatment at 10, 100, 250, and 500 µg/mL for 7 or 14 d. (C and D) Mineralization was quantitatively analyzed at 7 or 14 d by measuring Alizarin Red S staining intensity and expression as a percentage of the control (CON) (n=4 for each time point). All data are presented as mean±standard deviation. Statistical analysis was performed using one-way analysis of variance, followed by Tukey’s multiple comparison post hoc test. Various letters indicate statistically significant inter-group differences (P<0.05).

DWE upregulated the expression of osteogenic differentiation-related proteins

To investigate the molecular mechanisms underlying DWE-induced osteogenic differentiation, the levels of key osteogenic proteins were analyzed by Western blotting. As shown in Fig. 4A and 4B, DWE remarkably enhanced the expression of RUNX2, ALP, OPN, and Pro COL I proteins at 3 and 7 d. Densitometric analysis confirmed that the contents of these osteogenic markers increased in a concentration-dependent manner compared with the control group (Fig. 4C－4F). These results indicate that DWE enhances osteoblast differentiation by upregulating osteogenesis-related signaling proteins.

Fig. 4.

Fig. 4

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Effects of deer antler water extract (DWE) on the expression of osteogenic differentiation-related proteins in MC3T3-E1 cells. (A and B) Representative Western blot images showing the levels of runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteopontin (OPN), and procollagen type 1 (Pro COL I) proteins in MC3T3-E1 cells treated with DWE at 10, 100, 250, and 500 µg/mL for 3 (A) or 7 d (B). Beta-actin served as a loading control (CON). (C－F) Quantitative densitometric analyses of RUNX2 (C), ALP (D), OPN (E), and Pro COL I (F) expression at 3 or 7 d of DWE treatment. Protein levels were normalized to those of β-actin and expressed as a percentage of the CON. All data are presented as mean±standard deviation. RUNX2, ALP, and Pro COL I were analyzed with n=3 at 3 and 7 d, whereas OPN was analyzed with n=2 at 3 and n=3 at 7 d. Statistical analysis used one-way analysis of variance, followed by Tukey’s multiple comparison post hoc test. Various letters indicate statistically significant inter-group differences (P<0.05).

DWE improves bone microarchitecture in OVX-induced osteoporotic mice

The in vivo effects of DWE on bone structure were evaluated in an OVX-induced osteoporosis mouse model. Representative 3D µCT images of the distal femur in OVX mice showed severe trabecular bone loss, which was markedly attenuated by DWE (Fig. 5A). Quantitative µCT analysis revealed that DWE-treated OVX mice (65 and 500 mg/kg) exhibited significantly increased BV and BV/TV compared with the OVX group (Fig. 5C and 5D). In addition, Tb.Th and Tb.N were increased, whereas Tb.Sp was markedly reduced following DWE treatment (Fig. 5E－5G). Serum TC levels were also improved in the DWE-treated groups (Fig. 5B).

Fig. 5.

Fig. 5

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Effects of deer antler water extract (DWE) on bone microarchitecture and serum parameters in ovariectomized (OVX) mice. (A) Representative three-dimensional micro-computed tomography (µCT) images of the distal femur trabecular bone from sham-operated (Sham), OVX, estradiol-treated (EST), and DWE-treated OVX mice at 65 or 500 mg/kg. Scale bar=500 µm. (B) Serum total cholesterol (TC) levels were measured in Sham, OVX, EST, and DWE-treated OVX mice (n=6 per group). (C) Bone volume (BV, mm3) and (D) bone volume to total volume (BV/TV, %) were quantified by µCT analysis. (E) Trabecular thickness (Tb.Th, mm), (F) trabecular number (Tb.N, mm—1), and (G) trabecular separation (Tb.Sp, mm) were also evaluated to assess changes in trabecular bone microarchitecture. The sample numbers for panels C－G were Sham (n=7), OVX (n=5), EST (n=7), DWE-65 (n=8), and DWE-500 (n=8). All data are presented as mean±standard deviation. Statistical analysis used one-way analysis of variance, followed by Tukey’s multiple comparison post hoc test. Various letters indicate statistically significant inter-group differences (P<0.05).

DWE protects against OVX-induced bone loss by enhancing osteoblastic activity

Histological analysis via H&E staining demonstrated a marked structural deterioration of the trabecular bone in OVX mice, whereas DWE markedly preserved trabecular integrity and bone morphology (Fig. 6A). Histomorphometry revealed that DWE significantly increased N.Ob/B.Pm and Ob.S/BS compared with OVX mice (Fig. 6B and 6C). These findings indicate that DWE protects against OVX-induced bone loss by enhancing osteoblastic activity and bone formation. DWE promoted osteogenic differentiation and mineralization in osteoblast precursor cells in vitro and improved bone microarchitecture and osteoblastic activity in OVX-induced osteoporotic mice in vivo, highlighting its potential role in bone regeneration and osteoporosis prevention (Fig. 6).

Fig. 6.

Fig. 6

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Protective effects of deer antler water extract (DWE) against ovariectomy (OVX)-induced osteoporosis. (A) Representative hematoxylin and eosin-stained sections of the distal femur from sham-operated (Sham), OVX, estradiol-treated (EST), and 65 or 500 mg/kg DWE-treated OVX mice. The upper panels show low-magnification images (×4; scale bar=500 µm), and the lower panels present higher-magnification views (×10; scale bar=200 µm) of the trabecular bone indicated by red boxes. (B) Number of osteoblasts per bone perimeter (N.Ob/B.Pm, mm) quantified by histomorphometry (n=5 per group). (C) Osteoblast surface per bone surface (Ob.S/BS, %) was quantified to evaluate osteoblastic activity (n=5 per group). Data are presented as mean±standard deviation. Various letters indicate statistically significant inter-group differences (P<0.05), as determined by one-way analysis of variance followed by Tukey’s multiple comparison.

DISCUSSION

Deer antler has long been associated with bone growth and regeneration due to its unique biocharacteristics of rapid and repeated ossification (Pan et al., 2023; Li et al., 2024). Accordingly, increasing scientific attention has been directed toward elucidating the potential role of deer antler-derived materials in bone metabolism and osteoporosis prevention (Li et al., 2024). In this study, a DWE promoted osteogenic differentiation in vitro and improved bone microarchitecture in an OVX-induced osteoporosis model, supporting its potential as a natural agent for maintaining bone homeostasis.

In vitro, DWE enhanced osteoblast differentiation in MC3T3-E1 pre-osteoblast cells, as evidenced by increased ALP activity, enhanced matrix mineralization, and upregulated the expression of osteogenic differentiation-related proteins, including RUNX2, ALP, OPN, and Pro COL I (Wang et al., 2023b). These findings are consistent with previous reports that deer antler extracts or their processed forms stimulate osteogenic activity in osteoblast-like cells and bone marrow-derived mesenchymal stem cells. Prior investigations have demonstrated that deer antler-derived preparations can promote early osteoblast differentiation and matrix maturation, often accompanied by the increased expression of osteogenic markers and enhanced mineral deposition (Gu et al., 2025). In comparison, the present work demonstrates that a water-based extract, more favorable for industrial application and functional food development, is sufficient to induce robust osteogenic responses without inducing cytotoxicity.

The molecular changes observed in this study suggest that DWE may enhance osteogenic differentiation through activation of osteoblast-related signaling pathways. Deer antler-derived components may activate bone morphogenetic protein (BMP)-related signaling cascades, upregulating RUNX2 and downstream osteogenic genes (Zuo et al., 2024; Gu et al., 2025). Although upstream signaling molecules such as BMPs or phosphorylated Smad were not directly examined, the increases in RUNX2 expression, ALP activity, and collagen-related proteins observed were consistent with the activation of osteogenesis-related transcriptional programs. Future studies on upstream signaling pathways may help to further clarify the precise molecular mechanisms underlying DWE-induced osteogenesis.

The in vivo findings further support the osteoprotective role of DWE. In OVX-induced osteoporotic mice, DWE markedly improved the trabecular bone microarchitecture, as indicated by increased BV/TV and Tb.N and reduced Tb.Sp (Widyowati et al., 2021; Wang et al., 2023b). These results agree with previous studies showing that deer antler-derived extracts or protein fractions attenuate estrogen deficiency-induced bone loss and preserve trabecular bone structure. Notably, this study extends earlier observations by demonstrating that DWE enhances bone structural parameters while simultaneously elevating osteoblast number and surface, as confirmed by histomorphometry. This finding suggests that the beneficial impacts of DWE on bone microarchitecture are closely associated with enhanced osteoblastic activity and bone formation.

Several studies have suggested that deer antlers exert a dual modulation of bone remodeling by promoting bone formation and contributing to the regulation of bone resorption (Wang et al., 2023a). Bone remodeling is impacted by the balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. However, the present study primarily focused on osteoblast-mediated bone formation, and direct markers of bone resorption or osteoclast activity were not evaluated. Therefore, the osteoprotective influence of DWE observed in this study is most appropriately interpreted as being predominantly driven by enhanced osteogenic activity rather than a definitive suppression of bone resorption. Additional investigations incorporating comprehensive bone turnover markers and dynamic histomorphometry are necessary to fully elucidate the potential bidirectional effects of DWE on bone remodeling.

Despite its strengths, this study has several limitations. DWE is a complex mixture of multiple bioactive components, and the specific compounds responsible for these osteogenic effects remain to be identified. However, water extracts as potential candidates may include low-molecular-weight peptides, collagen-derived fragments, growth factor-like proteins, and glycosaminoglycan-related components, which may plausibly enhance RUNX2 expression, ALP activity, and matrix mineralization observed in this study. In particular, these components may synergistically promote osteogenic differentiation by activating osteoblast-related signaling pathways and extracellular matrix maturation (Li et al., 2016). In addition, dose optimization, long-term safety evaluation, and human-equivalent dose calculations are required to support future translational applications. Nevertheless, the consistent in vitro and in vivo findings presented here provide strong evidence that DWE promotes osteoblast differentiation and preserves bone microarchitecture under osteoporotic conditions.

In conclusion, this study demonstrates that DWE enhances osteogenic differentiation in osteoblast precursor cells and improves bone microarchitecture in an OVX-induced osteoporosis model. These findings are consistent with and extend previous reports on the bone-protective properties of deer antler, highlighting the potential of DWE as a natural, osteogenesis-promoting agent for preventing osteoporosis.

Footnotes

FUNDING

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2024-00340542). This study was partially supported by a research grant from Korea Ginseng Corporation.

AUTHOR DISCLOSURE STATEMENT

The authors declare no conflict of interest.

AUTHOR CONTRIBUTIONS

Concept and design: JHK, Sang-Hoon L, Seung-Ho L, SWK, YEC. Analysis and interpretation: JHK, Sang-Hoon L, YEC. Data collection: Sang-Hoon L. Writing the article: JHK. Critical revision of the article: JHK, YEC. Final approval of the article: All authors. Statistical analysis: JHK, Sang-Hoon L. Obtained funding: Seung-Ho L, SWK, YEC. Overall responsibility: YEC.

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