Synergistic Wnt/BMP Co-activation accelerates osteogenic differentiation of human pluripotent stem cells via paraxial mesoderm induction

hiPSCs culture

The hiPSCs used in this study was provided by the National Stem Cell Bank of South Korea, a commercial supplier that received initial ethical approval (Cat. CMC-hiPSC-022). These lines were authenticated by STR analysis and found to be free of mycoplasma, and showed normal nucleoid type (46,XX). The hiPSCs line were maintained in Essential 8 (E8) Basal medium (A15169-01, Gibco, USA) on 6-well plates (Corning, USA) pre-coated with recombinant human vitronectin (rhVTN-n; A14700, Gibco, USA). The hiPSCs were passaged every five days, and the culture medium was replaced daily. For subculturing, hiPSCs were washed once with 1 mL of phosphate buffered saline (PBS) (SH30028.02, HyClone, USA) and subsequently incubated with 500 μL of Accutase (SCR005, Millipore, USA) at 37°C for 3 minutes to facilitate dissociation. Following incubation, 1.5 mL of PBS was added to the culture vessel, and the cell suspension was gently triturated to ensure uniform dissociation before being transferred to a 15 mL conical tube. The suspension was then centrifuged at 850 rpm for 3 minutes. For routine maintenance, the cells were reseeded at a split ratio of 1/40 onto VTN-coated dishes. The remaining cell population was collected by an additional round of centrifugation for cryopreservation. After the supernatant was aspirated, the resulting cell pellet was resuspended in 1 mL of Synth-a-Freeze (A12542-01, Gibco, USA) and transferred into a cryogenic vial (368632, Thermo SCIENTIFIC, USA). The vials were then stored at -80°C. For recovery, cryopreserved vials were rapidly thawed in a 37°C-water bath for 3 minutes. The cell suspension was immediately transferred to a 15 mL tube containing 1 mL of pre-warmed E8 medium and centrifuged at 850 rpm for 3 minutes. The cell pellet was resuspended in fresh growth medium and plated onto VTN-coated dishes for further expansion. hiPSCs between passages 15 and 30 were utilized for differentiation experiments.

Induction of paraxial mesoderm cells from hiPSCs

hiPSCs were differentiated into paraxial mesoderm cells following a published protocol with minor modifications. ¹⁸ Briefly, hiPSCs were dissociated using Accutase (SCR005, Millipore, USA) and re-seeded onto Matrigel-coated (354277, Corning, USA) 6-well plates in mesoderm induction medium. The medium consisted of STEMdiff™ APEL™2 Medium (APEL; 05270, STEMCELL, Canada) supplemented with 6 ng/mL StemMACS CHIR99021 (CHIR; 130-103-926, Miltenyi Biotec, Germany) and 50 ng/mL BMP4 (CYT-1093, ProSpec, Korea) for two days. The duration of induction was established based on the temporal expression patterns of T (Brachyury) and TBX6.

To optimize the differentiation efficiency into the paraxial mesoderm lineage, several culture conditions were evaluated using four distinct media formulations. The basal APEL medium was used alone for the MM1 condition, while MM2 was supplemented with CHIR to initiate mesodermal signaling. In MM3, both CHIR and BMP2 (CYT-627, ProSpec, Korea) were added to the APEL medium to assess their synergistic effects, and MM4 involved the combined treatment of CHIR99021 and BMP4 within the same basal medium. These conditions were systematically compared during the early induction phase to identify the most effective signaling combination for robust paraxial mesoderm commitment.

Induction of osteoblasts from paraxial mesoderm

The differentiated paraxial mesoderm was cultured in osteoblast induction medium. This medium comprised Dulbecco’s Modified Eagles Medium (DMEM) high glucose (SH30243.01, HyClone, USA) supplemented with 10% FBS (SH30919.03, HyClone, USA), 1% penicillin/streptomycin (15140-122, Gibco, USA), 0.1 M Dexamethasone (D2915, Sigma-Aldrich, USA), 50 M L-Ascorbic acid (49752, Sigma-Aldrich, USA), and 10 mM beta-glycerophosphate (G9422, Sigma-Aldrich, USA). The medium was replenished every two days.

RNA extraction, cDNA synthesis, and real-time PCR (qPCR) analysis

Total RNA was extracted using the AccuPrep Universal RNA Extraction Kit (K-3140, Bioneer, Korea). Complementary DNA (cDNA) was synthesized from 1 µg of RNA using the MG cDNA Synthesis Kit (MR01611, MGmed, Korea). Quantitative PCR was performed on a QuantStudio 3 system (A28137, Applied Biosystems, USA) using SYBR Green (RR430A, Takara, Japan). Target gene expression levels were normalized to GAPDH and calculated using the ΔΔCt method. Primer sequences are listed in Table S1.

Alizarin Red Staining

Cultured cells were washed twice with PBS and fixed with 4% paraformaldehyde (PFA) (PC2031-100-00, Biosesang, Korea) for 15 min. Fixed cells were stained with 2% ARS solution (0223, ScienceCell, USA) for 30 min at room temperature. After staining, the solution was removed, and the cells were washed five times with distilled water. ARS staining was imaged using a microscope (IX73P2F, Olympus, Japan). Quantitative analysis of mineralization was conducted by eluting the bound stain with a 10% Cetylpyridinium chloride (CPC) solution. The CPC elution protocol was: 10 mL of 10 mM sodium phosphate (BSP-9570, T&I, Korea) mixed with 1 g of CPC (C0732, Sigma-Aldrich, USA). The absorbance of the elevator was measured at 540 nm using an Epoch microplate reader (BioTek, USA).

Von Kossa staining

Cultured cells were washed twice with PBS and fixed with 4% PFA for 30 min at 37°C. Fixed cells were stained using the Von Kossa Stain Kit (KTVKO, MasterTech, USA). After staining, the solution was removed, and the cells were washed three times with distilled water. Von Kossa staining was analyzed using a microscope, and the area of calcium deposition was quantified using ImageJ software (National Institutes of Health, USA).

Immunofluorescence staining

Cells were washed with PBS (SH30028.02, Hyclone, USA) and fixed with 4% PFA at 37°C for 10 min. Permeabilization was performed by treating cells with 0.1% Triton X-100 (1.12298.1001, Merck, USA) at room temperature for 15 min. Non-specific binding was blocked using 2% bovine serum albumin (BSA) (30063-572, Gibco, USA) at room temperature, followed by overnight incubation with primary antibody solution at 4°C. The primary antibodies used were: RUNX2 (D1L7F) Rabbit mAb (1:6400; 12556, Cell Signaling, USA) and COL1A1/Collagen I Recombinant Rabbit Monoclonal Antibody (1:200; Huabio, China). After three washes with PBS, cells were incubated with Alexa Fluor 594 donkey anti-rabbit secondary antibody (1:1000; ET1609-68, Invitrogen, USA) for 1 h at room temperature. Nuclei were counterstained using Antifade Mounting Medium with DAPI (H-2000, Vector Laboratories, USA). Immunofluorescence staining was analyzed and quantified (fluorescently stained areas) using a fluorescence microscope and ImageJ software, respectively.

Chondroitin sulfate cryogel scaffold fabrication

Chondroitin sulfate methacrylate (CSMA) was synthesized by reacting 0.5 g of chondroitin sulfate A sodium salt (GC6763, Glentham, UK) dissolved in PBS with 0.5 mL (74 mM) glycidyl methacrylate (779342, Sigma-Aldrich, Germany) for 10 days. The CSMA solution was purified using a dialysis membrane cassette for 48 h and subsequently dialyzed against deionized water for 24 h. After lyophilization, CSMA powder was stored at -20°C. Chondroitin sulfate (CS) cryogels were prepared by dissolving CSMA in deionized water at 10% w/v at room temperature. Whitlockite was added to the polymer solution at 6% w/v and vortexed to ensure particle dispersion. The mixture was cast into a plastic mold to achieve the desired shape. Cross-linking was initiated using 2.5% v/v thermal initiators (100 mg/mL, APS; 9913, Sigma-Aldrich, USA) and 0.12% v/v accelerators (N, N, N, N-tetramethylethylenediamine, TEMED; T7024, Sigma-Aldrich, USA). The mixture was then lyophilized at -20°C for 20 h to create the cryogel for in vivo experiments.

Cell culture for transplantation

A 4-well plate was covered with cover glass, and both the cover glass and the CSMA cryogel scaffold were coated with Matrigel for 2 h. A total of 1×10⁶ hiPSCs were suspended in Matrigel solution. In parallel, human bone marrow-derived mesenchymal stem cells were prepared via LONZA (PCS-500-012, Switzerland). BMSCs were used in the experiment with cells between passages 4 and 8. They were prepared with BMSCs at the same density as hiPSCs and transplanted under the same in vivo conditions as positive controls. A 100 µL aliquot of the cell-Matrigel mixture was dispensed onto the CSMA cryogel scaffold to facilitate swelling. The scaffold was incubated in a 37°C incubator for 30 min. Subsequently, 1 mL of E8 medium was added, and the culture was stabilized for one day before initiating the differentiation protocol. Upon completion of differentiation, the scaffolds were transplanted subcutaneously into mice and harvested at 1, 4, and 6 weeks.

In vivo osteogenesis differentiation studies

All experimental procedures were carried out in compliance with ethical regulations and were approved by the 4D Biomaterials Animal Care and Use Committee (KNUT IACUC 2024-02). A total of 20 female BALB/c nude mice (CoreTec, Korea) underwent a 1-week acclimatization period in an environmentally controlled facility. Breeding conditions were maintained at a temperature of 23 ± 3°C, a relative humidity of 50 ± 10 %, and a 12-hour light/dark cycle, and feed and water were freely ingested. The experimental groups were divided into: Scaffold only, Bone mesenchymal stem cells (BMSC), CHIR (AC), and CHIR+BMP4 (ACB). Samples were prepared by seeding the cells into the prefabricated CSMA cryogel scaffolds. Before surgery, the animals were treated after obtaining sufficient anesthesia under isoflurane inhalation (657801261, Hana Pharm, Korea) anesthesia, and after the surgery was completed, the incision was treated using a local disinfectant. During the procedure and recovery period, the animal’s condition was closely monitored and managed to meet the institutional welfare standards. At the end of the experiment, euthanasia was performed using the carbon dioxide overabsorption method, and the treatment was performed in a way that gradually increased the carbon dioxide concentration. Scaffolds were taken at 1, 4, and 6, respectively, and fixed with 4% PFA (47608, Sigma-Aldrich, Switzerland).

Micro-computed tomography (Micro-CT) analysis

To evaluate the extent of osteogenic mineralization within the scaffolds, high-resolution micro-computed tomography (micro-CT) imaging was performed using a Quantum GX3 micro-CT imaging system (Revvity, Waltham, MA, USA). Scans were conducted using the standard acquisition mode for 2 minutes at an X-ray tube voltage of 60 kV and a current of 160 µA. An aluminum (Al) filter with a thickness of 0.5 mm was applied. For comprehensive 3D visualization and quantitative analysis, the reconstructed volumetric datasets were imported into Analyze 15.0 software (AnalyzeDirect, Overland Park, KS, USA). The mineralized regions were digitally segmented from the scaffold background by applying a globally uniform radiodensity threshold (Hounsfield Units, HU). Based on the thresholded regions, 3D volumetric models were rendered, and the total volume of the mineralized tissue (mm³) within each scaffold was calculated to quantify the degree of bone-like tissue formation.

Histological staining of CSMA cryogel scaffolds

Scaffolds were processed for tissue staining by embedding in paraffin (CelltisBio, Korea) and sectioning. Paraffin-embedded slides were deparaffinized in xylene (534056, Sigma-Aldrich, USA), gradually rehydrated using a graded series of 70 – 100% Ethanol (NBM203, Duksan, KR), and finally washed with Distilled Water (D.W). For general histological analysis, slides were stained using the Hematoxylin and Eosin (H&E) Stainless Kit (H-3502, Vector Laboratories, USA). Samples were incubated in Hematoxylin solution for 5 min, washed twice with D.W., treated with a Bluing agent solution for 10 – 15 min, washed twice with D.W., briefly exposed to 100% Ethanol, and finally stained with Eosin Y solution for 2 - 3 min. Dehydration was performed by washing three times for 1 – 2 min with 100% Ethanol. To evaluate calcium deposition, fixed samples were incubated with 2% ARS solution (0223, Science Cell™, USA) for 20 min at room temperature, washed three times with D.W. to remove excess dye, and then stored in D.W. to prevent drying. Images were collected using a Zeiss microscope and quantified using ImageJ. For the analysis of calcium salt detection, samples were stained with a Von Kossa staining kit (ab150687, Abcam, UK) according to the manufacturer’s protocol. Image collection and quantification were performed using a Zeiss microscope and ImageJ, respectively.

Immunofluorescence staining of CSMA cryogel scaffolds

Paraffin-removed tissue slides underwent antigen retrieval by incubation with Proteinase K at 37°C for 20 min, followed by cooling at room temperature for 10 min. Permeabilization was achieved by incubating sections with 0.5% Triton-X (T8787, Sigma-Aldrich, USA) solution for 15 min, followed by washing. Blocking was performed using a 1:1 mixture of 0.2% BSA (A7906, Sigma-Aldrich, USA) and 0.1% Triton-X. After washing, primary antibodies were diluted 1:500 in PBS and incubated overnight at 4°C. The primary antibodies used were: COL1A1 (ET1609-68, Huabio), RUNX2 (12556S, Cell Signaling), Osteocalcin (OCN; SC-74495, Santa Cruz), and Osteopontin (OPN; SC-21742, Santa Cruz). Following three washes with 0.1% Triton-X solution, sections were incubated with secondary antibodies (Alexa-Fluor 594, ab150080; Alexa-Fluor 488, ab150113; Abcam, UK) at room temperature for 1 h. After three final washes, the scaffolds were mounted and counterstained with Mounting Medium containing DAPI (AB104139, Abcam, UK) and observed under a Zeiss microscope.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9. Data are presented as means ± SD. Before statistical tests, the normality of the data distribution was assessed using a diagnostic procedure embedded in the Prism. Sample sizes for each experiment are plotted in the corresponding figure legends. n denotes independent biological repeats, and when technical repeats were included, values were averaged within each biological repeat and then used for statistical analysis. One-way ANOVA was applied to assess differences between groups according to one independent variable was used for comparison between three or more groups, and Tukey’s multiple comparison test was used for postmortem tests. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Co-activation of Wnt/BMP signaling directs hiPSC differentiation toward paraxial mesoderm

The initial phase of this study aimed to optimize the conditions for the efficient differentiation of hiPSCs into paraxial mesoderm (PM). hiPSCs were cultured on Matrigel-coated dishes using mesoderm differentiation medium (MM) supplemented with varying activators. Traditionally, PM differentiation is carried out over 4 days. However, to account for potential rapid differentiation into off-target lineages (e.g., vascular cells) within 2 days, a comparative time-course analysis was performed at day 2 (D2) and day 4 (D4) to determine the optimal differentiation time point (Figure 2(a)). For paraxial mesenchymal induction, hiPSC was treated in 6 ng/mL CHIR and 50 ng/mL BMP4 for 2 days. Quantitative polymerase chain reaction (qPCR) analysis of the PM-specific marker Mesogenin 1 (MSGN1) revealed an increase in expression at D2 in groups treated with the Wnt activator CHIR (MM2, MM3, and MM4), with average fold changes of 18.35, 21.42, and 20.8, respectively (Figure 2(b)). Similarly, T-box transcription factor 6 (TBX6), another PM marker, showed increased expression at D2 in MM2, MM3, and MM4, with fold changes of approximately 12.4, 8.72, and 14.85, respectively (Figure 2(c)). Notably, the MM4 group, which received simultaneous treatment with CHIR and BMP4, demonstrated the most significant upregulation of both MSGN1 and TBX6. To assess lineage specificity and potential off-target differentiation, we analyzed the expression of lateral mesoderm markers ISL LIM Homeobox 1 (ISL1) and NK2 Homeobox 5 (NKX2.5) (Figures 2(d) and 2(e)). While a general upregulation in both ISL1 and NKX2.5 was observed across the groups, the differences between the MM2, MM3, and MM4 groups at D2 were statistically insignificant. Consequently, the optimal PM differentiation period was established as 2 days. These results strongly suggest that the co-activation of the Wnt signaling pathway (via CHIR) and the BMP signaling pathway (via BMP4) is crucial for robust PM induction.OPEN IN VIEWER

Next, we compared the efficiency of BMP2 and BMP4 at various concentrations (25, 50, and 100 ng/mL) to further optimize PM differentiation. qPCR analysis of the PM marker MSGN1 showed that BMP4 treatment consistently resulted in higher differentiation efficiency than BMP2, with fold differences of 2.72 times at 50 ng/mL and 3.58 times at 100 ng/mL (Figure 2(f) and S1). Furthermore, the expression of RUNX2, a key transcription factor indicative of osteoblast differentiation potential, was also significantly higher with BMP4 treatment: 2.93 times higher at 50 ng/mL and 2.04 times higher at 100 ng/mL compared to BMP2 (Figure 2(g)). Based on these findings, a concentration of 50 ng/mL BMP4 was selected for further analysis. Comparing the control PM group (PM), the CHIR-only group (AC), and the CHIR/BMP4 co-treated group (ACB), the gene expression of TBX6 was 11.45-fold higher in the AC group and 13.7-fold higher in the ACB group compared to PM. Similarly, RUNX2 expression was 3.82-fold and 7.9-fold higher in the AC and ACB groups, respectively (Figures 2(h) and 2(i)). These results confirm the establishment of a robust protocol for inducing paraxial mesoderm differentiation using 50 ng/mL BMP4 in conjunction with CHIR and validate the use of RUNX2 as an early indicator of osteogenic commitment.

Co-activation of Wnt/BMP signals in paraxial mesodermal differentiation promotes osteogenic commitment

To investigate the effects of the 2-day mesodermal induction on subsequent osteogenic fate, the transcriptional profiles of 75 osteogenesis-associated genes were analyzed in hiPSCs, AC, and ACB groups (Figure 3(a)). Heatmap analysis clearly demonstrated distinct gene expression patterns in the AC and ACB groups compared to the undifferentiated hiPSC group, particularly concerning Wnt/BMP signaling-related genes. Twelve key genes were selected for detailed analysis (Figure 3(b)). Notably, the ACB group showed upregulated expression of mesodermal and BMP-responsive transcription factors, including Dickkopf-1 (DKK1), Msh Homeobox 1 (MSX1), and MSGN1. Conversely, the hiPSC group exhibited low overall expression, and the AC group showed only partial increases in select genes.OPEN IN VIEWER

Gene Ontology (GO) analysis of the differentially expressed genes (DEGs) between the AC and ACB groups revealed significant changes in biological processes associated with major signaling cascades, such as the MAPK cascade and Wnt signaling pathway, as well as cell structure and differentiation-related processes (Figure 3(c)). The highest number of DEGs and the greatest proportional changes were observed in GO terms related to extracellular matrix organization, positive regulation of the ERK1/ERK2 cascade, and skeletal system development. These findings collectively suggest that the simultaneous activation of Wnt signaling (CHIR) and stimulation by BMP4 strongly promotes mesodermal transformation and effectively primes the transcription factor network for subsequent entry into the osteogenic lineage. Therefore, the ACB treatment condition demonstrated superior efficacy in promoting both mesoderm induction and downstream osteogenic commitment compared to existing conditions.

Limited BMP4 activation during the mesoderm period enhances osteoblast maturation

Following the successful establishment of the 2-day paraxial mesoderm differentiation protocol (Stage 1), we varied the subsequent BMP4 treatment duration during the 7-day osteoblast differentiation period (Stage 2) (Figure 4(a)). Cellular morphology observation confirmed stable cell growth throughout the 9-day Stage 2 period (Figure 4(b)). RT-PCR analysis of the early osteoblast marker RUNX2 confirmed that the addition of BMP4 (compared to the control group) enhanced osteoblast differentiation by 2.42-fold (Figure 4(c)). Importantly, the co-treatment of CHIR and BMP4 (at both 50 ng/mL and 100 ng/mL) during Stage 1 further improved RUNX2 expression by 2.73 and 3.57 times, respectively, underscoring the benefits of co-activating the Wnt and BMP pathways during the initial mesodermal phase. When evaluating Collagen Type I (COL1) expression, the group co-treated with CHIR and BMP4 at 50 ng/mL showed a 1.6-fold enhancement compared to the BMP4-only group, whereas the 100 ng/mL co-treatment did not yield significant improvement (Figure 4(d)).OPEN IN VIEWER

Detailed analysis comparing hiPSC, mesoderm-induced media (DAG; Dorsal axial mesoderm, APEL; Albumin, polyvinyl alcohol, essential lipids), AC, and ACB groups confirmed that the AC and ACB groups exhibited higher expressions of key bone formation markers: COL1, osteonectin (ON), and osteocalcin (OCN) (Figures 4E–4(g)). The ACB group showed a significant advantage over the AC group, with expression increases of 1.93-fold (COL1), 1.8-fold (ON), and 2.71-fold (OCN). These results establish that Wnt/BMP co-signaling during the mesodermal period not only promotes osteoblast differentiation but also significantly enhances osteoblast maturation.

Co-activation of Wnt/BMP signaling promotes osteoblast maturation and mineralization

We assessed the terminal outcome of osteoblast differentiation by analyzing calcium deposition and matrix protein expression. Cellular morphology remained stable across all groups over the 9-days (Figure 5(a)). ARS, a classic measure of calcium deposition, revealed the most intense staining in the ACB group (Figure 5(b)). Quantitative spectrophotometric analysis (absorbance at 540 nm) confirmed that the ACB group exhibited a 1.81-fold higher staining level compared to the AC group (Figure 5(c)). Von Kossa staining, which detects calcium phosphate and other mineral salts, similarly showed a greater area of black staining in the ACB group (Figure 5(d)), indicating superior calcium deposition. Quantification of the stained regions confirmed the ACB group was 3.64-fold higher than the AC group (Figure 5(e)).OPEN IN VIEWER

Immunofluorescence staining was used to evaluate osteoblast maturity (Figure 5(f)). The expression of both RUNX2 (early marker) and COL1 (middle/late marker) was more pronounced in the ACB group. Quantification of the expression area showed that the ACB group was 2.29-fold higher in RUNX2 and 1.57-fold higher in COL1 compared to the AC group (Figure 5(g)). These results unequivocally demonstrate that Wnt/BMP co-activation significantly increases calcium deposition and promotes osteoblast differentiation and maturation in vitro.

In vivo bone formation assessment

To validate the in vitro findings, an in vivo bone formation experiment was conducted using a chondroitin sulfate-methacrylate (CSMA) cryogel scaffold (Figure S2) [16]. The CSMA cryogel, known for the biocompatibility of chondroitin sulfate [17], provides a stable microenvironment for cellular activities [18]. In this study, CSMA cryogel scaffold was used as a three-dimensional (3D) cell carrier to help the survival and bone differentiation of hiPSC-derived cells. The scaffold maintains cells at the transplant site and provides an environment suitable for early bone formation, and to evaluate bone formation in vivo, the cells were seeded on a cryogel scaffold and differentiated into osteoblasts that had previously differentiated into osteoblasts through the mesoderm. And the scaffold containing cells was subcutaneously implanted into mice, collected at 1, 4, and 6 weeks, and analyzed (Figure 6(a)). BMSC group served as positive control. The body weight remained overall stable throughout the 6-week observation period, and no statistically significant difference was found between the groups. These results indicate that the scaffold and transplanted cells are well tolerated in vivo, supporting biocompatibility (Figure 6(b)). Observation of the collected scaffolds showed a decrease in size by 6 weeks, confirming the expected biodegradation in vivo (Figure 6(c)). The collected scaffolds were photographed by micro-CT for calciumization (Figure 6(d)). At week 1, it was confirmed that the area of calcium in the ACB group was larger than that of other groups, and the area of calcium compared to the total volume was higher in the ACB at week 6. In the quantification of bone volume to total volume, the ACB group at week 1 was 8.5 times higher than that of the Scaffold group, and at week 6, the bone volume was 4.96 times (Scaffold group), 3.19 times (BMSC group), and 5.28 times (AC group) higher, showing effective calcium (Figure S3). H&E staining at weeks 1, 4, and 6 confirmed that cells were well-localized within the scaffold pores, further demonstrating biocompatibility and cell attachment. Degradation was visibly progressing by week 6 (Figure 6(e)).OPEN IN VIEWER

ARS and Von Kossa staining were performed on the collected scaffolds (Figure 6(f) and S4). Calcium salt deposition was first observed via Von Kossa staining at 4 weeks and became more extensive at 6 weeks (Figure S5). Quantification of the Von Kossa-stained area showed no significant difference among groups at Week 1. However, at Week 4, the expression increased compared to the Scaffold-only control in the order: BMSC (2.8 times), AC (3.4 times), and ACB (3.7 times). At Week 6, the trend continued, with expression in BMSC (1.5 times), AC (1.6 times), and ACB (2.3 times), confirming that the ACB treatment group yielded the highest level of in vivo calcium salt deposition and bone matrix formation. The accelerated mineralization observed in immunodeficiency mice was found to be associated with the initial osteogenic activity of transplanted cells.

Analysis of bone formation markers in the scaffold by immunofluorescence staining

Immunofluorescence staining of the in vivo scaffolds analyzed the expression of key osteogenic markers over time (Figure 7(a)). At Week 1, the AC and ACB groups showed clearer expression of Osteopontin (OPN) (a middle marker) than RUNX2 (an early marker), suggesting rapid commitment. OPN expression intensified at Weeks 4 and 6. Importantly, the ACB group consistently showed a wider range and clearer expression of OPN throughout the subcutaneous implantation period, confirming superior osteogenic differentiation. COL1 expression was observed in the BMSC and AC groups at Week 1 (Figure 7(b)). OCN (a late maturation marker) expression, however, was observed in the ACB group starting from Week 1, suggesting accelerated maturation. By Week 6, OCN expression was increased in both AC and ACB groups, indicative of effective mineralization.OPEN IN VIEWER

Quantitative analysis of the osteogenic markers (Figure 7(c)) revealed a reduction in early marker expression from Week 4 to Week 6: RUNX2 was reduced in AC (0.53 times) and ACB (0.37 times), and COL1 was reduced in AC (0.12 times) and ACB (0.32 times). This sequential increase followed by a decrease in early/middle markers confirms the expected progression toward terminal bone formation and maturation. Crucially, the ACB group demonstrated a significant increase in late-stage maturation markers from Week 4 to Week 6: OPN (5.01 times) and OCN (5.00 times). These results definitively establish that the Wnt/BMP co-activation protocol (ACB) promotes the most effective and accelerated bone formation differentiation in vivo.