Detection of Genetic Biomarkers of Dental Pulp Stem Cell–Mediated Bone Regeneration Using RUNX2 in a Rat Mandibular Defect Model

2. Materials and Methods

2.1. Isolation and Culture of DPSCs

• Collagenase I: (Sigma-Aldrich, St. Louis, MO, USA).
• Dispase: (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).
• Filter (70 µm): (Falcon, Corning Inc., Corning, NY, USA).
• α-MEM (Minimal essential medium Eagle – alpha modification): (Gibco, Thermo Fisher Scientific).
• Fetal bovine serum (FBS): (Gibco, Thermo Fisher Scientific).
• Penicillin–streptomycin: (Gibco, Thermo Fisher Scientific).

2.2. Phenotypic Characterization of DPSCs

• Bovine serum albumin (BSA): (Sigma-Aldrich).
• Antibodies: Ecto-5'-nucleotidase (CD73), Thy-1 (CD90), Endoglin (CD105), CD34, CD45.
• Flow cytometer: (BD Accuri C6 flow cytometer, BD Biosciences, San Jose, CA, USA).
• Data analysis software: (FlowJo v10, FlowJo LLC, Ashland, OR, USA).

2.3. In Vivo Mandibular Fracture Model

• Isoflurane/O₂ anesthesia: (Baxter Healthcare, Deerfield, IL, USA).
• Micro-saw: (Fine Science Tools, Foster City, CA, USA).
• Biphasic calcium phosphate (BCP): (Berkeley Advanced Biomaterials Inc., Berkeley, CA, USA).
• RUNX2 lentivirus: (VectorBuilder Inc., Chicago, IL, USA).
• Resorbable collagen membrane: (Geistlich Pharma AG, Wolhusen, Switzerland).
• Vicryl Rapide™ sutures: (Ethicon, Johnson & Johnson, Somerville, NJ, USA).
• Buprenorphine (0.05 mg/kg): (Reckitt Benckiser, Slough, UK).

2.4. Genetic Marker Expression Analysis

• RNA extraction kit: (RNeasy Mini Kit, Qiagen, Hilden, Germany).
• cDNA synthesis kit: (High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Thermo Fisher Scientific).
• SYBR Green assay kit: (PowerUp SYBR Green Master Mix, Applied Biosystems).
• SDS-PAGE: (Bio-Rad Laboratories, Hercules, CA, USA).
• PVDF membrane: (MilliporeSigma, Burlington, MA, USA).
• Chemiluminescence substrate: (SuperSignal™ Enhanced Chemiluminescence), (ECL), (Thermo Fisher Scientific).
• Paraformaldehyde (4% PFA): (Electron Microscopy Sciences, Hatfield, PA, USA).
• Triton X-100 (0.1%): (Sigma-Aldrich).
• BSA (5%): (Sigma-Aldrich).
• Primary antibodies (anti-RUNX2/OCN): (Abcam, Cambridge, UK).
• Secondary antibodies (Alexa Fluor): (Thermo Fisher Scientific).
• DAPI (4′,6-diamidino-2-phenylindole): (Sigma-Aldrich).

2.5. Histological and Immunohistochemical Evaluation

• Decalcification solution (10% EDTA): (Sigma-Aldrich).
• Hematoxylin and Eosin (H&E): (Sigma-Aldrich).
• Masson’s trichrome stain: (Polysciences, Warrington, PA, USA).
• Antigen retrieval buffer (citrate buffer pH 6): (Dako, Agilent Technologies, Santa Clara, CA, USA).
• Anti-Osteocalcin (OCN) antibody: (Abcam).
• DAB detection kit: (Dako).
• Hematoxylin counterstain: (same as above, Sigma-Aldrich implied)
• ImageJ software: (NIH, Bethesda, MD, USA).

2.6. Scaffold Preparation and Cell Seeding

• Biphasic calcium phosphate (BCP; 60/40 HA/β-TCP, 0.5–1 mm granules): (Berkeley Advanced Biomaterials Inc., Berkeley, CA, USA).
• α-MEM: (Gibco, Thermo Fisher Scientific).

2.7. Scaffold Design and Fabrication

• Type I collagen: (Corning).
• Polycaprolactone (PCL): (Sigma-Aldrich).

2.8. Methods

2.8.1. Isolation and Culture of DPSCs

Teeth were collected from healthy Male Sprague–Dawley rats (8–10 weeks, 250–300 g). Dental pulp was extirpated, minced into ~1 mm³ fragments, and digested in 3 mg/mL collagenase I (Sigma-Aldrich, St. Louis, MO, USA) and 4 mg/mL dispase (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at 37 °C. The resulting cell suspension was filtered (70 µm, Falcon, Corning Inc., Corning, NY, USA), centrifuged (300×g, 5 min), and plated in a (Gibco’s MEM) minimal essential medium Eagle – alpha modification (α-MEM; Gibco, Thermo Fisher Scientific) with 10% Fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific) and 1% penicillin–streptomycin (Gibco, Thermo Fisher Scientific) at 1×10⁴ cells/cm². Cultures were maintained at 37 °C, 5% CO₂; passages 2–4 were used for all experiments [33].

2.8.2. Phenotypic Characterization of DPSCs

Passage 3 DPSCs were harvested, blocked with 0.5% Bovine serum albumin (BSA; Sigma-Aldrich), and stained for Ecto-5'-nucleotidase (CD73), Thy-1 (Thymocyte differentiation antigen 1) (CD90), Endoglin (CD105), Hematopoietic progenitor cell antigen (CD34), and Protein tyrosine phosphatase, receptor type C, also known as Leukocyte common antigen (CD45). After washing, cells were analyzed on a BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA) and data processed with FlowJo v10 (FlowJo LLC, Ashland, OR USA) to confirm Mesenchymal stem cells (MSC) marker profile (CD73⁺, CD90⁺, CD105⁺; CD34⁻, CD45⁻) [33].

2.8.3. In Vivo Mandibular Fracture Model

Male Sprague–Dawley rats (36 Rats, 8–10 weeks, 250–300 g) were anesthetized with isoflurane/O₂ (Baxter Healthcare, Deerfield, IL, USA). A unilateral mid-body mandibular osteotomy (5 mm defect) was created with a micro-saw (Fine Science Tools, Foster City, CA, USA). Rats were randomized into three groups (n = 12 each):

• Control: defect + BCP only
• DPSC: defect + BCP + DPSCs (1×10⁶)
• RUNX2-modulated DPSC: defect + BCP + DPSCs transduced with RUNX2 lentivirus (MOI 50; VectorBuilder Inc., Chicago, IL, USA)

Defects were covered with a resorbable collagen membrane (Geistlich Pharma AG, Wolhusen, Switzerland) and closed with Vicryl Rapide™ sutures (Ethicon, Johnson & Johnson, Somerville, NJ, USA). Buprenorphine (0.05 mg/kg; Reckitt Benckiser, Slough, UK) and soft diet were provided post-operatively. Animals were sacrificed in weeks 4 and 8; mandibles harvested for analysis [31].

2.8.4. Genetic Marker Expression Analysis

• Real-Time Quantitative Reverse Transcription PCR (qRT-PCR): RNA extraction (RNeasy Mini Kit, Qiagen, Hilden, Germany), cDNA synthesis (High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Thermo Fisher Scientific), and SYBR Green assays (PowerUp SYBR Green Master Mix, Applied Biosystems) for RUNX2, alkaline phosphatase (ALPL), Collagen type 1 alpha 1 (COL1A1), bone morphogenetic protein 2 (BMP2), and Osteocalcin (OCN); bone gamma-carboxyglutamic acid-containing protein (BGLAP) as internal control.
• Western Blot: Protein lysates separated by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad Laboratories, Hercules, CA, USA), transferred to Polyvinylidene fluoride (PVDF; MilliporeSigma, Burlington, MA, USA), probed for RUNX2, COL1A1, and OCN, and visualized via Thermo Fisher Scientific’s SuperSignal™ Enhanced Chemiluminescence (ECL).
• Immunocytochemistry: Cells fixed Paraformaldehyde (4% PFA; Electron Microscopy Sciences, Hatfield, PA, USA), permeabilized (0.1% Triton X-100; Sigma-Aldrich), blocked (5% BSA; Sigma-Aldrich), incubated with anti-RUNX2/OCN (Abcam, Cambridge, UK), then Alexa Fluor secondary antibodies (Thermo Fisher Scientific); nuclei counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) [12,13].

2.8.5. Histological and Immunohistochemical Evaluation

Specimens were decalcified (10% EDTA; Sigma-Aldrich), paraffin-embedded, and sectioned (5 µm). Sections stained with Hematoxylin and Eosin (H&E; Sigma-Aldrich) and Masson’s trichrome (Polysciences, Warrington, PA, USA). For IHC, antigen retrieval (citrate buffer pH 6; Dako, Agilent Technologies, Santa Clara, CA, USA) preceded incubation with anti-OCN (Abcam) and DAB detection (Dako). Sections counterstained with hematoxylin. New bone area quantified via ImageJ (NIH, Bethesda, MD, USA) [34].

2.1.6. Scaffold Preparation and Cell Seeding

Biphasic calcium phosphate (BCP; 60/40 HA/β-TCP, 0.5–1 mm granules; Berkeley Advanced Biomaterials Inc., Berkeley, CA, USA) was pre-wetted in α-MEM overnight at 37 °C. DPSCs were resuspended at 1×10⁶ cells per 30 mg BCP and incubated in 96-well plates at 37 °C, 5% CO₂ for 12 h to allow attachment [31].

2.1.7. Scaffold Design and Fabrication

Scaffolds were designed to mimic the native extracellular matrix (ECM) architecture with high porosity (100–700 µm macropores; <100 µm micropores) to facilitate vascularization and nutrient diffusion. Fabrication techniques included salt leaching, gas forming, phase separation, freeze drying, and electrospinning. Biphasic and multiphasic constructs were produced by layering materials with distinct compositions and pore geometries. Decellularized ECM and polymeric biomaterials collagen (Type I collagen; Corning) and Polycaprolactone (PCL; Sigma-Aldrich) were functionalized with ECM proteins and growth factors [32].

2.1.8. Statistical Analysis

Data (mean ± SD) were analyzed by one-way ANOVA with Tukey’s post hoc, and repeated-measures ANOVA for time-courses; p < 0.05 considered significant. Analyses performed in GraphPad Prism 9 software [34].

3. Results

3.1. Isolation and Phenotypic Characterization Confirms the Mesenchymal Stem Cell Identity of Cultured DPSCs

This study confirms that primary cultures from rat teeth yielded an average of 1.2 × 10⁶ cells per tooth fragment by passage 2 in flow cytometry, with a typical spindle-shaped MSC morphology and >95% viability on Trypan Blue exclusion. Dental pulp stem cells (DPSCs) at P3 expressed high levels of CD73 (98.2 ± 1.1 %), CD90 (97.5 ± 1.3 %), CD105 (96.8 ± 1.5 %) and were negative for CD34 (1.2 ± 0.5 %) and CD45 (0.8 ± 0.4 %), confirming their mesenchymal identity. These phenotypic data establish the MSC profile of the cultured DPSCs and provide the baseline cell characterization for subsequent osteogenic induction and in vivo experiments (see the schematic of the in vivo model in Figure 1).

Figure 1. Schematic representation of the in vivo mandibular defect model and experimental workflow. Male Sprague–Dawley rats underwent standardized 5 mm unilateral mid-body mandibular defects. Groups included: (1) BCP only (Control), (2) BCP + DPSCs, and (3) BCP + RUNX2-modified DPSCs. After scaffold implantation and collagen membrane coverage, wounds were closed with absorbable sutures. Post-operative care included analgesia and soft diet. Animals were sacrificed at 4 and 8 weeks for micro-CT, histology, and biomechanical testing. This schematic illustrates defect creation, treatment allocation, and analysis timeline.

Figure 1. Schematic representation of the in vivo mandibular defect model and experimental workflow. Male Sprague–Dawley rats underwent standardized 5 mm unilateral mid-body mandibular defects. Groups included: (1) BCP only (Control), (2) BCP + DPSCs, and (3) BCP + RUNX2-modified DPSCs. After scaffold implantation and collagen membrane coverage, wounds were closed with absorbable sutures. Post-operative care included analgesia and soft diet. Animals were sacrificed at 4 and 8 weeks for micro-CT, histology, and biomechanical testing. This schematic illustrates defect creation, treatment allocation, and analysis timeline.

Figure 2. Workflow of Histological and Immunohistochemical Evaluation in Mandibular Defect Healing.

Figure 2. Workflow of Histological and Immunohistochemical Evaluation in Mandibular Defect Healing.

3.2. Sequential and Temporal Induction of Key Osteogenic Genes Confirmed by qRT-PCR Analysis

RUNX2 (an early/mid osteogenic transcription factor) rose markedly by day 7 and peaked at day 14 before slightly declining by day 21; ALPL (ALP) and BMP2 also showed early-to-mid induction with highest fold-changes at day 14; COL1A1 increased steadily during the induction period; and OCN (BGLAP), a late mineralization marker, showed the greatest increase by day 21. My results in Table 1 showed that fold-change versus Day 0 and indicate statistical significance versus Day 0. The qRT-PCR findings provide the molecular context for the subsequent protein-level validation.

3.3. Western Blot and Immunocytochemistry Confirm Robust Elevation of RUNX2 and OCN Protein Expression

At the protein level, Western blot densitometry and immunocytochemistry confirmed the transcriptional trends: protein levels of RUNX2 and OCN increased concordantly, with densitometry showing ~4-fold (RUNX2) and ~6-fold (OCN) elevations at day 21. Immunocytochemistry confirmed robust nuclear RUNX2 and cytoplasmic OCN staining by day 14. These protein-level data validate the qRT-PCR results and demonstrate that transcriptional induction translates into increased osteogenic protein expression (see qRT-PCR quantitation in Table 1 and representative images in the Figure 4).

3.4. DPSC-BCP Scaffold Interactions: High Seeding Efficiency and Favorable Cell Adhesion

DPSC scaffold interactions were favorable: seeding efficiency onto BCP granules was 85 ± 4% after 12 h, and SEM imaging showed cells spreading over pore surfaces and establishing filopodia-mediated contacts. The attachment and spreading data document that the BCP scaffold supports efficient DPSC adhesion before implantation, which underpins the in vivo repair results presented below.

3.5. Micro-CT Morphometric Analysis Demonstrates Superior Bone Volume and Microarchitecture in DPSC-Treated Defects

Micro-CT morphometric analysis of mandibular defects showed clear, group-dependent improvements in bone metrics. Bone volume / total volume (BV/TV) at week 4 and week 8, trabecular thickness (Tb.Th) and trabecular number (Tb.N) were all improved in the DPSC + BCP group versus Control (BCP only), with further enhancement in the RUNX2-DPSC + BCP group. These quantitative micro-CT results are presented in Table 2 and indicate statistically significant differences. The micro-CT outcomes provide 3D structural evidence that DPSCs enhance bone formation and that RUNX2 overexpression further potentiates microarchitecture improvements (see Table 2 for the full morphometric dataset).

3.6. Histological and Immunohistochemical Evaluation Corroborates Enhanced Osteogenic Activity in DPSC and RUNX2-DPSC-Treated Defects

Histological and immunohistochemical evaluation corroborated imaging and molecular findings: H&E and Masson’s trichrome revealed sparse, discontinuous bone islands in Control defects, abundant woven bone bridging >60% of the gap in DPSC-treated defects by week 8, and near-complete defect closure with dense lamellar bone in the RUNX2-DPSC group. ImageJ-based area quantification of new bone confirmed these observations (see the quantification in Figure 3 and the representative histology panels in Figure 4). OCN immunostaining showed strong positive staining in DPSC and RUNX2-DPSC groups, with semi-quantitative image analysis indicating OCN-positive area fractions of 48 ± 5% (DPSC) and 62 ± 6% (RUNX2-DPSC) versus 25 ± 4% in controls, further confirming enhanced osteogenic activity in treated groups (quantitative IHC results and visual examples are provided in the figures below — as mentioned in Figure 3 and Figure 4).

Figure 3. Error bars represent ± standard deviation (SD), indicating the variability around the mean.

Figure 3. Error bars represent ± standard deviation (SD), indicating the variability around the mean.

Figure 4. Representative ImageJ-processed histological sections and quantification of new bone area in mandibular defects treated with BCP only (Control), BCP + DPSCs, and BCP + RUNX2-modified DPSCs. Binary threshold masks illustrate bone tissue (white) vs. non-bone areas (black), with corresponding bar graph showing % bone area.

Figure 4. Representative ImageJ-processed histological sections and quantification of new bone area in mandibular defects treated with BCP only (Control), BCP + DPSCs, and BCP + RUNX2-modified DPSCs. Binary threshold masks illustrate bone tissue (white) vs. non-bone areas (black), with corresponding bar graph showing % bone area.

4. Discussion

The restoration of critical-sized mandibular defects continues to pose a considerable clinical challenge in maxillofacial surgery, frequently necessitating bone grafts that present limitations, including donor site morbidity and restricted availability. Our research presents substantial evidence that dental pulp stem cells (DPSCs) implanted onto a biphasic calcium phosphate (BCP) scaffold represent a highly promising tissue engineering approach to tackle this challenge. The primary outcomes of this study indicate that rat dental pulp stem cells (DPSCs) significantly promote bone regeneration in a rat mandibular defect model, and that the osteogenic capacity of these cells is considerably enhanced by the overexpression of the transcription factor RUNX2. A multi-faceted assessment showed that the therapeutic benefit was real. Defects treated with RUNX2-overexpressing DPSCs had not only more bone volume, but also a more mature microarchitecture and better biomechanical strength than defects treated with naive DPSCs or just scaffolds.These results strongly support the idea that DPSCs have an inherent ability to form bone, which has been well-documented in vitro [8], and successfully show that this ability works in a complex in vivo setting.

The BCP scaffold, which is known for its ability to help bones grow, seems to be the perfect three-dimensional structure that keeps cells in place, helps them grow, and eventually leads to the formation of a mineralized matrix. The substantial improvement in regenerative outcomes seen with RUNX2-overexpressing DPSCs highlights an essential principle: the mere presence of stem cells on a scaffold may not suffice for optimal repair. Instead, turning on important genetic programs that control osteoblast lineage commitment ahead of time can have a big impact on the process of regeneration. RUNX2 is very important for the differentiation of mesenchymal stem cells into pre-osteoblasts and their later maturation. It is often called the "master regulator" of osteogenesis. Our findings indicate that increasing RUNX2 expression in DPSCs before implantation effectively primes the cells, expediting their differentiation process and improving their functional performance at the defect site. This resulted in the development of bone tissue exhibiting characteristics—demonstrated by micro-computed tomography (micro-CT) metrics such as bone volume/total volume (BV/TV), trabecular number (Tb.N), and connectivity density—that more closely resemble native mandibular bone.

The in-vivo results shown here are very promising, but they also make us want to learn more about the molecular mechanisms behind them. RUNX2 overexpression probably causes more bone formation by starting a chain reaction of events that happen after it. RUNX2 may not only directly enhance the expression of bone matrix proteins such as osteocalcin and bone sialoprotein but also regulate the secretion of osteotropic factors that facilitate angiogenesis and coordinate the activity of host progenitor cells, thereby establishing a more potent regenerative niche. To transcend correlation and ascertain causation, current investigations in our laboratory are utilizing transcriptomic (e.g., RNA sequencing) and epigenetic profiling [13] of collected constructs. These analyses will elucidate the extensive gene regulatory networks activated by RUNX2 in this particular context and identify potential epigenetic modifications that stabilize the osteogenic phenotype, yielding a system-level comprehension of how genetic priming enhances DPSC-mediated repair. The scaffold–cell interface is another important place to look for ways to improve things. The BCP scaffold used in this study worked well, but its surface is not very reactive, so it may not fully mimic the changing signaling environment of the native extracellular matrix (ECM). Subsequent iterations of this strategy will emphasize the advanced functionalization of biomaterials to establish a more bioactive environment [4]. This includes putting certain ECM-derived peptides (like RGD sequences) or recombinant growth factors (like BMP-2) on the surface to give cells long-lasting, localized signals that work with their genetic programming. Also, looking into multiphasic or gradient scaffold designs that control the timing and location of biochemical and biophysical signals could better mimic the transitional zones of the mandible and help create bone tissue that is anatomically correct. Ultimately, the encouraging short-term outcomes observed in a rodent model must be situated within the broader trajectory toward clinical implementation. An essential phase will involve the evaluation of long-term safety, stability, and immunogenicity in more extensive, immunocompetent preclinical models [33]. Important questions about the possibility of ectopic bone formation, the future of the implanted cells, and the immune response to both the allogeneic cells and the scaffold material over long periods of time still need to be answered. A thorough assessment involving serial imaging, histopathology, and immune cell profiling will be crucial to mitigate risks associated with this therapeutic strategy. In conclusion, this study demonstrates that RUNX2-overexpressing dental pulp stem cells (DPSCs) on a biphasic calcium phosphate (BCP) scaffold constitute a robust combination for bone regeneration in a mandibular defect model. The results show how well a supportive biomaterial platform and the strategic genetic enhancement of stem cells work together. Our current and forthcoming endeavors are focused on elucidating the mechanistic foundations, enhancing the biomaterial component, and conducting thorough safety evaluations. We expect that these coordinated efforts will not only confirm and build on the current findings but also lay the groundwork for the creation of effective and tailored DPSC-based therapies for complicated craniofacial reconstruction.

5. Conclusions

In conclusion, this study demonstrates that RUNX2-overexpressing dental pulp stem cells (DPSCs) on a biphasic calcium phosphate (BCP) scaffold constitute a robust combination for bone regeneration in a mandibular defect model. The results show how well a supportive biomaterial platform and the strategic genetic enhancement of stem cells work together. Our current and forthcoming endeavors are focused on elucidating the mechanistic foundations, enhancing the biomaterial component, and conducting thorough safety evaluations. We expect that these coordinated efforts will not only confirm and build on the current findings but also lay the groundwork for the creation of effective and tailored DPSC-based therapies for complicated craniofacial reconstruction.