Bone regenerative capacity of the newly synthesized material QG3030: an in vitro and in vivo study

Abstract

Purpose

Current bone grafting materials often lack inherent osteoinductive properties, underscoring the need for new therapeutic agents that promote regeneration. In this study, we evaluated the effects of QG3030, a novel osteoinductive derivative of lappaconitine, on the proliferation and osteogenic differentiation of rat bone marrow-derived stem cells (BMSCs) in vitro. Furthermore, we examined its influence on bone regeneration in vivo using a rat calvarial critical-sized defect model.

Methods

The effects of QG3030 on BMSC proliferation and osteogenic differentiation were assessed in vitro using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the alkaline phosphatase (ALP) activity assay, and alizarin red S staining. For in vivo evaluation, 18 Sprague–Dawley rats with 8-mm critical-sized calvarial defects were randomly assigned to 3 groups (n=6 per group): deproteinized bovine bone mineral (DBBM; Bio-Oss) alone (B0, control), DBBM combined with 100 mg/mL QG3030 (B100), and DBBM combined with 300 mg/mL QG3030 (B300). Bone regeneration was analyzed after 8 weeks by micro-computed tomography (micro-CT), histology, and histomorphometry.

Results

In vitro, QG3030 improved BMSC viability, significantly increased ALP activity, and promoted matrix mineralization within the tested non-cytotoxic range. On in vivo micro-CT analysis, the B100 group exhibited significantly higher bone mineral density and new bone volume fraction (new bone volume/total volume) than the B0 group (P=0.043 for both). Furthermore, histomorphometry revealed significantly greater new bone area (P=0.046) and percentage of new bone (P=0.032) in the B300 group compared to the B0 group.

Conclusions

Combining QG3030 with DBBM significantly enhanced bone regeneration in a rat calvarial critical-sized defect model, with QG3030 demonstrating distinct osteogenic effects both in vitro and in vivo. Accordingly, the combination of QG3030 and DBBM represents a promising therapeutic approach for bone regeneration strategies, especially in periodontology and implant dentistry, and warrants further investigation.

Graphical Abstract

INTRODUCTION

Adequate alveolar bone volume is crucial for successful periodontal and implant therapy, yet managing bone deficiencies remains challenging. Although autogenous bone has ideal biological properties, its use is limited by availability, donor site morbidity, and unpredictable volumetric resorption that can compromise long-term outcomes [1]. Alternative grafting materials such as allografts raise concerns regarding biological variability and disease transmission, while synthetic substitutes often exhibit suboptimal biological performance or resorption kinetics compared to native bone [2,3].

Xenografts such as deproteinized bovine bone mineral (DBBM) are widely used in dental and maxillofacial regeneration based on their documented success in complex applications, including guided bone regeneration, sinus augmentation, and periodontal defect filling [4,5,6]. The clinical efficacy of these materials is attributed to their biocompatibility, osteoconductive structure, and slow resorption rate, which helps ensure volume stability [6,7,8]. Despite these advantages, DBBM is biologically passive and lacks the intrinsic osteoinductive signals essential for robust new bone formation, a limitation that can compromise healing at challenging sites [9,10]. Thus, enhancing the osteoinductive capacity of stable scaffolds like DBBM is a key objective in the search for potent bioactive agents [11].

A widely recognized strategy to improve osteoinduction involves growth factors, particularly those from the bone morphogenetic protein (BMP) family. Among these, recombinant human BMP-2 (rhBMP-2) strongly induces osteogenic differentiation and is clinically approved for demanding applications such as spinal fusion and certain oral and maxillofacial procedures [12,13]. The combination of rhBMP-2 with osteoconductive materials like DBBM has been explored for potential synergistic structural and biological advantages [14]. However, this approach has demonstrated inconsistent clinical efficacy, especially for procedures such as maxillary sinus augmentation, in which superiority over DBBM alone has not been consistently observed [15,16]. Moreover, the widespread application of rhBMP-2 is limited by substantial costs; potential adverse effects, including inflammation, edema, and heterotopic bone formation; and challenges related to supraphysiological dosing and rapid in vivo degradation [17]. These limitations highlight the need for continued research into safer, more cost-effective osteoinductive alternatives.

Natural products provide a valuable source for identifying novel therapeutic agents. Alkaloids from Aconitum species, for example, have long been used in traditional medicine due to their analgesic and anti-inflammatory properties [18]. Lappaconitine, one such alkaloid, has demonstrated pro-osteogenic and anti-osteoclastogenic effects, potentially mediated via the adenosine monophosphate-activated protein kinase/Sirtuin 1 (AMPK/SIRT1) pathway [19]. However, its clinical application is constrained by a narrow therapeutic window and notable toxicity, with reported oral median lethal dose (LD₅₀) values of 20–40 mg/kg in animal models [20].

To address these limitations, QG3030, a novel derivative of lappaconitine, was engineered to retain osteogenic activity with an improved safety profile [21]. Preliminary findings by Chang et al. [22] highlighted the osteogenic potential of QG3030, demonstrating its capacity to induce osteogenesis in vitro in human mesenchymal stem cells and to promote bone formation in vivo using a collagen scaffold in a rat ilium defect model. Notably, QG3030 exhibited a substantially wider safety margin, with a reported LD₅₀ exceeding 2,000 mg/kg, compared to its parent compound [23].

A previous in vivo study employed QG3030 with a collagen scaffold; however, this scaffold’s rapid degradation and limited mechanical stability may be suboptimal for dental augmentations that require sustained space maintenance [24]. The present study therefore further investigated the osteogenic potential of QG3030. In vitro experiments were performed to characterize its effects on rat bone marrow-derived stem cell (BMSC) proliferation and osteogenic differentiation. Subsequently, its in vivo efficacy in promoting bone regeneration was assessed using DBBM—a clinically validated scaffold known for volume stability—within a rat calvarial critical-sized defect model, a standard method for evaluation due to its limited self-healing capacity [25]. To our knowledge, this is the first study to investigate the combination of QG3030 and DBBM in this preclinical model. We hypothesized that QG3030 would demonstrate pro-osteogenic effects in vitro and that its combination with DBBM in vivo would significantly enhance bone regeneration compared to DBBM alone.

MATERIALS AND METHODS

Ethics statement and animals

All animal procedures were conducted in accordance with the 3Rs (replacement, reduction, and refinement) and were approved by the Institute of Laboratory Animal Resources (approval No. SNU 230310-8-2) and the Institutional Animal Care and Use Committee of Seoul National University. The study adhered to the ARRIVE 2.0 guidelines [26].

In vitro experiments

Isolation and culture of BMSCs

BMSCs were harvested from rat tibias and cultured according to previously published methods [27]. Briefly, cells obtained through centrifugation were suspended in alpha-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. After subsequent centrifugation at 1000 rpm for 10 minutes, mononuclear cells were isolated. Cultures were maintained in a humidified incubator at 37°C with 5% CO₂, and the growth medium was replaced twice weekly. Cells between passages 3 and 5 were used for all experiments. Before assays, cells were detached with trypsin, washed, and counted using a hemocytometer. For 96-well plate assays, cells were seeded at a density of 5×10³ cells/well.

Cell proliferation assay

BMSC proliferation was assessed on days 1, 4, and 7 after seeding using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cultured BMSCs were treated with 0.01, 0.1, 1, 10, or 100 μM concentrations of QG3030 (provided by Qgenetics Co. Ltd., Seoul, Korea) dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA). The vehicle control contained an amount of DMSO equivalent to the highest QG3030 concentration. At each time point, 10 μL of MTT reagent (Sigma-Aldrich) was added to each well and incubated for 4 hours at 37°C in the dark. The supernatant was then removed, 100 μL DMSO was added to solubilize the formazan crystals, and optical density at 540 nm was measured using a microplate reader (Molecular Devices, San Jose, CA, USA) to determine cell viability. This assay was performed in triplicate.

Alkaline phosphatase activity assay

Alkaline phosphatase (ALP) activity in BMSCs was evaluated on days 1, 3, and 7 following treatment with QG3030 (0.1, 1, and 10 μM). rhBMP-2 (10 μM, Novosis; CGBIO, Seoul, Korea) and DMSO served as positive and negative controls, respectively. ALP activity was quantified using a commercial kit (SensoLyte; Anaspec Co., Fremont, CA, USA) according to the manufacturer’s instructions. The results were normalized to total protein content, determined using the SMART BCA Protein Assay Kit (IntronBio, Seongnam, Korea), and expressed as nmol p-nitrophenol/min/μg protein. All assays were conducted in triplicate.

Mineralization assay by alizarin red S staining and elution

Matrix mineralization was assessed by alizarin red S (ARS) staining after 14 days. BMSCs were cultured in osteogenic differentiation medium (α-MEM containing 10% FBS, 0.2 mM ascorbic acid, 10 mM β-glycerophosphate, and 10 nM dexamethasone; Sigma-Aldrich), supplemented with QG3030 (0.1, 1, or 10 μM), BMP-2 (10 μM), or DMSO vehicle control, with the medium replaced every 3 days. After the culture period, cells were rinsed with phosphate-buffered saline and fixed with 95% ethanol for 15 minutes at 4°C. The cells were then stained with 0.1% ARS solution (pH 4.2; Sigma-Aldrich) for 5 minutes at room temperature. Excess dye was removed by extensive washing with distilled water, and mineralized matrix deposition was examined microscopically. For quantitative analysis, ARS bound to the mineralized matrix was eluted by incubating samples with 10% cetylpyridinium chloride (Sigma-Aldrich) for 1 hour at room temperature under gentle agitation. The optical density of the eluate was measured at 540 nm (Molecular Devices). This assay was performed in triplicate.

In vivo rat experiment

Materials

QG3030 (Qgenetics Co. Ltd.) was prepared at concentrations of 100 mg/mL and 300 mg/mL in sterile distilled water, and DBBM (Bio-Oss particles, small granules of 0.25–1 mm; Geistlich Pharma AG, Wolhusen, Switzerland) was utilized for all experimental groups. Eighteen male Sprague–Dawley rats (8 weeks old, 280–320 g) were used following a 1-week acclimatization period. Animals were randomly allocated to 3 groups (n=6 per group):

- B0: defects were treated with DBBM only (control group).

- B100: defects were treated with DBBM combined with the 100 mg/mL QG3030 solution.

- B300: defects were treated with DBBM combined with the 300 mg/mL QG3030 solution.

Simple randomization was conducted using a computer-generated number sequence. To minimize bias, the investigators performing the surgical procedures and analyses were blinded to group allocation throughout the experiment. As this was a preliminary proof-of-concept investigation, a sample size of 6 animals per group was selected based on established precedent for this experimental model [28].

Surgical procedures

All surgical interventions were conducted under aseptic conditions. Rats were anesthetized via intraperitoneal administration of tiletamine/zolazepam (8 mg/kg Zoletil 50; Virbac, Carros, France) combined with xylazine (2.25 mg/kg Rompun; Bayer, Seoul, Korea). Following aseptic preparation of the surgical site, local anesthesia (lidocaine HCl 2% with 1:100,000 epinephrine; Huons Co. Ltd., Seongnam, Korea) was administered to the calvarial region.

A mid-sagittal incision approximately 15 mm in length was made through the skin and periosteum along the sagittal suture. The calvarial bone was exposed by elevating a full-thickness mucoperiosteal flap. An 8-mm critical-sized defect was created across the sagittal suture in the middle of the calvaria using a sterile 8-mm trephine bur under continuous sterile saline irrigation to minimize thermal damage and protect the underlying dura mater. Immediately prior to implantation, graft materials were prepared. A consistent weight of DBBM (30 mg) was used for each defect. For the experimental groups, the DBBM was hydrated with 30 µL of the 100 mg/mL (B100) or 300 mg/mL (B300) QG3030 solution, while the control group (B0) received an equivalent volume of sterile distilled water. The prepared materials were carefully placed in the defects to ensure complete filling and adaptation to the margins (Figure 1).

Figure 1. Representative image of the surgical model. An 8-mm critical-sized calvarial defect was created, followed by placement of deproteinized bovine bone mineral alone or in combination with QG3030.

The periosteum was repositioned to completely cover the graft and sutured with 5-0 absorbable chromic catgut (Ailee Co. Ltd., Busan, Korea) to achieve tension-free closure, followed by skin closure using 5-0 non-absorbable black silk sutures (Ailee Co. Ltd.). Postoperatively, intramuscular cefazolin (30 mg/kg; Yuhan Co., Seoul, Korea) was administered for 2 days as infection prophylaxis. Pain management consisted of subcutaneous buprenorphine (0.05 mg/kg; Yuhan Co.) administered twice daily for 3 days. Throughout recovery, the animals were monitored daily for signs of pain, infection, or distress, with unrestricted access to food and water.

Micro-computed tomography analysis

At 8 weeks post-surgery, the rats were euthanized, and calvarial specimens containing the defects were harvested and fixed in 10% neutral buffered formalin. Micro-computed tomography (micro-CT) scanning (SkyScan 1173; Bruker, Kontich, Belgium) was performed at a voxel size of 12 μm with settings of 130 kV and 60 μA. Images were reconstructed, and quantitative analyses were conducted using CTAn software (Bruker). A cylindrical region of interest (ROI), corresponding to the original 8-mm defect and similarly spanning the full thickness of the calvaria (from dura to periosteum), was defined for quantitative measurements.

Within this ROI, gray-level thresholds on an 8-bit gray scale (0–255) were applied to distinguish mineralized from non-mineralized tissue. The following parameters were then determined: bone mineral density (BMD; g/cm³); new bone volume fraction (new bone volume/total volume, NBV/TV; %); residual graft volume fraction (residual graft volume/total volume, RGV/TV; %); trabecular thickness (Tb.Th; mm); trabecular number (Tb.N; 1/mm); and trabecular separation (Tb.Sp; mm).

Histological and histomorphometric analyses

Following micro-CT analysis, calvarial specimens were decalcified in 10% EDTA for approximately 2 weeks and then processed for standard paraffin embedding. Coronal sections 5 μm thick were prepared from the central region of each defect. Sections were stained with Masson trichrome (MT) and hematoxylin and eosin (H&E) and examined by light microscopy (Olympus, Tokyo, Japan).

For histomorphometric assessments, digital images of stained sections were analyzed using CaseViewer (version 2.1; 3D Histech, Budapest, Hungary) and Image-Pro Plus (version 4.5.0.29; Media Cybernetics, Inc., Rockville, MD, USA). The ROI encompassed the entire original defect area, extending vertically from the dura mater to the periosteum. Within this ROI, the following were quantified: new bone area (NBA), the total area occupied by newly formed bone in mm²; percentage of new bone (%NB), calculated as (NBA/total ROI area) × 100; residual graft area (RGA), the total area occupied by residual graft particles in mm²; and percentage of residual graft (%RG), computed as (RGA/total ROI area) × 100.

Statistical analysis

Data were analyzed using SPSS Statistics (version 29.0; IBM Corp., Armonk, NY, USA). Normality was assessed with the Shapiro–Wilk test. Non-normally distributed data were analyzed using non-parametric methods. Comparisons across groups were performed with the Kruskal–Wallis test, followed by the Dunn post hoc test with Bonferroni correction for pairwise comparisons. Values are presented as mean ± standard deviation. A P value of less than 0.05 was considered to indicate statistical significance.

RESULTS

In vitro study: cell culture and assays

Cell viability (MTT assay)

The effects of QG3030 (0.01, 0.1, 1, 10, and 100 µM) on BMSC viability were assessed on days 1, 4, and 7 using the MTT assay (Figure 2). Viability increased significantly in all groups from day 1 to day 7 (P<0.05), indicating cell proliferation over the experimental period. Significant between-group differences were noted on day 4 (P=0.042) and day 7 (P=0.033). Notably, cells treated with 0.01 µM QG3030 exhibited significantly greater viability than the DMSO control cells at both day 4 (P=0.044) and day 7 (P=0.032). Collectively, these findings suggest that QG3030, particularly at 0.01 μM, promotes BMSC viability. Importantly, concentrations up to 100 μM did not significantly reduce viability relative to controls throughout the 7-day period.

Figure 2. Effect of QG3030 on BMSC viability assessed by MTT assay. QG was non-cytotoxic at all tested concentrations. Notably, 0.01 µM QG significantly increased BMSC viability relative to the DMSO control at day 4 and day 7.

DMSO: dimethyl sulfoxide, QG: QG3030, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, OD₅₄₀: optical density at 540 nm, BMSC: bone marrow-derived stem cell.

^a)Significant difference between day 7 and day 1 at the same concentration (P<0.05).

^b)Significant difference between the 0.01 μM QG and DMSO groups on day 4 (P<0.05).

^c)Significant difference between the 0.01 μM QG and DMSO groups on day 7 (P<0.05).

ALP activity

ALP activity in BMSCs treated with QG3030 (0.1, 1, or 10 μM), BMP-2 (10 μM), or DMSO control was measured on days 1, 3, and 7 (Figure 3). All groups exhibited a significant time-dependent increase in ALP activity from day 1 to day 7 (P<0.05). Significant intergroup differences were observed on day 3 (P=0.033) and day 7 (P=0.039). Specifically, on day 3, the BMP-2 positive control group displayed significantly higher ALP activity than the DMSO group (P=0.035). By day 7, cells treated with 0.1 μM QG3030 demonstrated significantly greater activity than the DMSO control (P=0.039). Notably, ALP activity did not differ significantly between the BMP-2 group and any QG3030-treated group at any time point (P>0.05).

Figure 3. Effect of QG on ALP activity in bone marrow-derived stem cells. On day 7, the 0.1 µM QG group showed significantly higher ALP activity than the DMSO control. Notably, the ALP activity in all QG-treated groups was comparable to the BMP-2 positive control at all evaluated time points (P>0.05).

DMSO: dimethyl sulfoxide, QG: QG3030, BMP-2: bone morphogenetic protein 2, ALP: alkaline phosphatase.

^a)Significant difference between day 7 and day 1 at the same concentration (P<0.05).

^b)Significant difference between the 10 μM BMP-2 and DMSO groups on day 3 (P<0.05).

^c)Significant difference between the 0.1 μM QG and DMSO groups on day 7 (P<0.05).

Matrix mineralization (ARS staining)

Matrix mineralization was qualitatively assessed by ARS staining after 14 days (Figure 4) and quantitatively analyzed by stain elution (Figure 5). Qualitative evaluation revealed the most intense staining—indicative of robust calcium deposition—for 10 μM BMP-2 and 10 μM QG3030 compared to lower QG3030 concentrations and the DMSO control. Quantitative analysis showed significantly higher mineralization in the BMP-2 group than in the DMSO group (P=0.047). Although a trend toward greater mineralization was observed with increasing QG3030 concentration, differences from the DMSO control were not statistically significant (P>0.05). Similarly, no significant differences were found among QG3030 concentrations or between the QG3030-treated groups and the BMP-2 group (P>0.05).

Figure 4. Mineralization assessment of rat bone marrow-derived stem cells cultured for 14 days in osteogenic medium with different treatments, evaluated by alizarin red S staining. Representative images suggest a dose-dependent trend toward increased calcium deposition (red staining) with QG treatment. The most intense staining was observed in the 10 µM BMP-2 positive control, followed by the 10 µM QG group.

DMSO: dimethyl sulfoxide, QG: QG3030, BMP-2: bone morphogenetic protein 2.

Figure 5. Quantitative analysis of matrix mineralization by alizarin red S elution assay after 14 days. A trend toward increased mineralization was observed with increasing QG concentration; however, only the BMP-2 positive control group displayed a statistically significant increase compared to the DMSO control.

ARS: alizarin red S, DMSO: dimethyl sulfoxide, QG: QG3030, BMP-2: bone morphogenetic protein 2.

^a)Significant difference between the 10 μM BMP-2 and DMSO groups (P<0.05).

In vivo study: rat calvarial critical-sized defect model

Surgical procedures were well tolerated by all animals, with no signs of postoperative complications observed during the 8-week healing period.

Micro-CT analysis

Bone regeneration within the calvarial defects at 8 weeks was quantitatively evaluated using micro-CT. Representative 3-dimensional (3D) reconstructions illustrating external and internal views of the defects in each experimental group are presented in Figure 6.

Figure 6. Representative 3-dimensional micro-computed tomography reconstructions of calvarial defects at 8 weeks post-surgery. External views (A-C) and internal views (D-F) are shown for each group. (A, D) The B0 group displayed minimal bone regeneration. (B, E) The B100 group showed substantial new bone formation, largely filling the defect. (C, F) The B300 group also exhibited considerable bone regeneration, though some areas appeared less radiopaque than in the B100 group.

BMD, NBV/TV, and RGV/TV

Micro-CT analysis revealed statistically significant differences among groups for both BMD and NBV/TV (P=0.045 for each) (Figure 7A and B). Specifically, the B100 group displayed significantly higher BMD (0.235±0.019 g/cm³) and NBV/TV (30.299%±2.663%) than the B0 control group (BMD: 0.007±0.001 g/cm³; NBV/TV: 18.187%±3.524%) (P=0.043 for both). Although the B300 group exhibited higher BMD (0.186±0.064 g/cm³) and NBV/TV (26.894%±4.811%) than the B0 group, these differences were not statistically significant (P>0.05). Additionally, no significant differences in BMD or NBV/TV were observed between the B100 and B300 groups (P>0.05). Importantly, analysis of RGV/TV showed no significant differences among the 3 groups (P>0.05), indicating comparable graft resorption across conditions (Figure 7C).

Figure 7. Quantitative micro-computed tomography analysis at 8 weeks post-surgery. (A) BMD (g/cm³). (B) NBV/TV (%). The B100 group exhibited significantly higher BMD and NBV/TV compared to the B0 control. No significant differences were observed between the B300 and B0 groups. (C) RGV/TV (%). Analysis of this parameter revealed no statistically significant differences among the 3 groups (P>0.05).

BMD: bone mineral density, NBV: new bone volume, TV: total volume, RGV: residual graft volume.

^a)Significant difference between the B100 and B0 groups (P<0.05).

Trabecular parameters

Analysis of trabecular microarchitecture (reflected by Tb.Th, Tb.N, and Tb.Sp) revealed no statistically significant differences among groups at the 8-week time point (P>0.05 for all parameters) (Figure 8). Despite this lack of statistical significance, both QG3030-treated groups (B100 and B300) exhibited a clear trend toward improved bone microarchitecture relative to the control (B0), with greater trabecular thickness and number and reduced trabecular separation.

Figure 8. Microstructural trabecular parameters assessed by micro-computed tomography at 8 weeks. (A) Tb.Th (mm). (B) Tb.N (1/mm). (C) Tb.Sp (mm). Although the B100 and B300 groups showed a trend toward increased Tb.Th and Tb.N and decreased Tb.Sp compared to the B0 group, none of the trabecular parameters differed significantly among groups (P>0.05).

Tb.Th: trabecular thickness, Tb.N: trabecular number, Tb.Sp: trabecular separation.

Histological and histomorphometric analysis

Histological examination at 8 weeks demonstrated clear differences in bone regeneration patterns among groups based on MT and H&E staining (Figure 9). The B0 group displayed limited peripheral new bone formation with poor DBBM integration (Figure 9A-D). The B100 group exhibited moderate new bone formation, improved DBBM integration, and numerous osteocytes (Figure 9E-H). The B300 group displayed extensive new bone formation, characterized by densely regenerated bone substantially filling the defect, as well as excellent integration of DBBM particles and high cellular activity (Figure 9I-L).

Figure 9. Histological evaluation of bone regeneration in the B0, B100, and B300 groups at 8 weeks post-surgery. Representative sections stained with Masson trichrome (A-C, E-G, I-K) and hematoxylin and eosin (D, H, L). Dashed lines indicate the original defect boundaries. The B0 group (A-D) displayed minimal new bone formation with poorly integrated DBBM particles (G) and few osteocytes/osteoblasts. The B100 group (E-H) exhibited moderate new bone formation with integrated DBBM particles and numerous osteocytes. The B300 group (I-L) displayed extensive, dense new bone with well-integrated DBBM particles and high cellularity with abundant osteocytes.

G: DBBM particle, NB: new bone, OB: osteoblast (yellow arrows), OC: osteocyte (black arrows), DBBM: deproteinized bovine bone mineral.

Histomorphometric analysis quantitatively supported these observations (Figure 10). Significant differences were observed among groups for NBA (P=0.046) and %NB (P=0.030). Specifically, the B300 group displayed significantly greater NBA (1.926±0.337 mm²) and %NB (30.492%±10.621%) than the B0 group (NBA: 0.854±0.195 mm²; %NB: 12.256%±2.602%) (P=0.046 and P=0.032, respectively). The B100 group exhibited intermediate NBA (1.307±0.338 mm²) and %NB (19.041%±4.111%), which did not differ significantly from either B0 or B300 values (P>0.05). Crucially, neither the area nor the percentage of residual graft material differed significantly among the 3 groups (P>0.05) (Figure 11).

Figure 10. Histomorphometric analysis of new bone formation at 8 weeks. (A) NBA (mm²). (B) %NB. Quantitative analysis showed that the B300 group exhibited significantly greater NBA and %NB compared to the B0 control group (P<0.05). The B100 group displayed intermediate values that did not differ significantly from either B0 or B300.

NBA: new bone area, %NB: percentage of new bone.

^a)Significant differences between the B300 and B0 groups (P<0.05).

Figure 11. Histomorphometric analysis of residual graft material at 8 weeks. (A) RGA (mm²). (B) %RG. Quantitative analysis confirmed the absence of statistically significant differences in the amount of residual graft material among the B0, B100, and B300 groups (P>0.05).

RGA: residual graft area, %RG: percentage of residual graft.

DISCUSSION

While bone graft substitutes such as DBBM provide excellent osteoconductive scaffolds, their clinical efficacy is limited by a lack of inherent osteoinductivity. This study therefore evaluated whether QG3030, a newly synthesized osteogenic derivative of lappaconitine, could confer this essential bioactivity. Our findings support this potential: QG3030 promoted the osteogenic differentiation of rat BMSCs in vitro and, when combined with DBBM in vivo, significantly enhanced bone regeneration compared to DBBM alone. These results suggest that QG3030 could serve as a valuable adjunct to conventional bone grafting materials, potentially overcoming their limitations by providing essential osteoinductive stimulation.

QG3030 was developed through rational drug design to capitalize on the osteogenic potential of lappaconitine while mitigating its toxicity [22]. As a single, purified small molecule, it offers advantages over complex natural extracts for therapeutic development, including improved standardization and more predictable pharmacokinetics [23,29]. Our in vitro data further indicate a favorable biological profile; QG3030 was non-cytotoxic to BMSCs and promoted proliferation at 0.01 μM. Notably, its effects differed between early (ALP) and late (ARS staining) osteogenic markers. A low concentration (0.1 μM) was found to significantly increase ALP activity on day 7, indicating enhanced osteoblast commitment [30]. In contrast, quantitative ARS staining did not show a statistically significant increase in late-stage mineralization compared to controls, although qualitative observations suggested improved mineralization, particularly at 10 μM, approaching levels seen with the BMP-2 positive control. This apparent discrepancy likely reflects the temporal dynamics of osteogenesis, suggesting that a lower dose is sufficient for initial differentiation, whereas the prolonged mineralization process may require more sustained stimulation. These results therefore imply that QG3030 primarily influences early differentiation, providing a strong rationale for its in vivo evaluation.

The in vivo doses of 100 and 300 mg/mL were determined via dose escalation from a prior study that demonstrated the efficacy of QG3030 in a less challenging 2-mm rat iliac defect model [22]. Higher concentrations were deemed necessary here to account for the substantially larger and more demanding 8-mm critical-sized calvarial defect. At these concentrations, the experiments confirmed the osteogenic potential of QG3030 but also revealed a nuanced dose-dependent effect. One intriguing observation was the differing apparent optimal doses identified by the 2 assessment methods: 100 mg/mL (B100) yielded significantly higher BMD and NBV/TV by 3D micro-CT, whereas 300 mg/mL (B300) produced significantly greater NBA and %NB on two-dimensional (2D) histomorphometry. To interpret this divergence, we considered the potential influence of residual graft material. While BMD encompasses the total mineralized volume of both new bone and graft, parameters such as NBV/TV, NBA, and %NB were quantified from new bone analyzed separately from graft particles. Crucially, subsequent quantitative analysis confirmed that the residual graft parameters (RGV/TV, RGA, and %RG) were statistically comparable across all groups. Having excluded this potential confounder, a plausible explanation for the discrepancy between methods lies in both the inherent differences between evaluation methodologies and a potential biphasic dose-response effect [31]. Specifically, micro-CT quantifies total 3D mineralized volume, providing a measure of bone quality and density. In contrast, histomorphometry produces a 2D assessment of the new tissue area, which includes less mature, cellular osteoid; thus, it reflects tissue quantity [32,33]. These findings underscore the value of employing complementary evaluation methods to achieve a comprehensive understanding of bone regeneration [34]. We therefore hypothesize that 100 mg/mL is optimal for promoting bone maturation and mineralization, leading to higher density that is accurately captured by 3D micro-CT. Conversely, 300 mg/mL appears to stimulate a more robust cellular proliferative response, resulting in a larger total quantity of new tissue, as correctly quantified by 2D histomorphometry.

Importantly, both QG3030 doses yielded superior regeneration compared to the DBBM control, with no significant differences between B100 and B300 for any single parameter. The absence of significant differences in trabecular parameters at 8 weeks may reflect the cortical nature of this non–load-bearing model or indicate that substantial maturation requires a longer follow-up period. This contrasts with prior findings for QG3030 in a rat ilium defect, underscoring the influence of anatomical site, defect type, and scaffold properties on outcomes [22].

The combination of QG3030 and DBBM addresses the primary limitation of DBBM by supplying osteoinductive signals. This concept is supported by our in vitro data, which show modulation of pathways governing osteoblast differentiation. While this initial study did not elucidate precise molecular mechanisms, prior research suggests potential pathways including modulation of runt-related transcription factor 2, extracellular signal-regulated kinase/mitogen-activated protein kinase signaling, or the AMPK/SIRT1 pathway involved in bone cell activity [19,21]. Additionally, given the known anti-inflammatory properties of lappaconitine, modulation of the local inflammatory environment may also contribute to the beneficial in vivo effects [35]. Through such potential mechanisms, QG3030 likely functions synergistically with DBBM. Confirmation of these pathways will require dedicated mechanistic studies.

Contextualizing these findings requires comparison with rhBMP-2, the benchmark for potent osteoinduction [36]. However, its clinical use is constrained by high costs and safety concerns, which largely stem from the rapid burst release of its conventional carrier [37]. Research to overcome these limitations is advancing on several fronts. One approach focuses on engineering novel protein variants, such as the activin A/BMP-2 chimera AB204, to enhance intrinsic bioactivity [38]. Another involves designing advanced delivery systems—using carriers such as injectable hydrogels and microspheres—to provide sustained or delayed release [39,40]. In this context, QG3030, a small molecule with a favorable safety profile and in vitro performance comparable to BMP-2, may offer advantages such as lower manufacturing costs and more predictable pharmacokinetics [21,22,23]. Although direct comparisons were not performed, our in vivo results suggest that QG3030 merits consideration as a promising alternative, especially in clinical scenarios where the extreme potency of rhBMP-2 is unnecessary or its risk-benefit profile is unfavorable. Future comparative studies are warranted.

This preclinical study has several limitations. First, the rat calvarial defect model has translational constraints for human load-bearing defects, and the single 8-week endpoint precluded analysis of early healing kinetics or long-term remodeling. Furthermore, the small sample size (n=6) and absence of a direct positive control such as rhBMP-2 limited the statistical power and assessment of comparative efficacy. Finally, detailed mechanistic, angiogenic, and toxicological evaluations were beyond our scope and should be included in future research.

In conclusion, this study provides strong preclinical evidence that combining the novel small molecule QG3030 with a DBBM scaffold significantly enhances bone regeneration in a challenging rat calvarial defect model. QG3030 represents a promising osteoinductive agent meriting further development, potentially offering a safer, cost-effective alternative for bone augmentation procedures in periodontology and implant dentistry.