Our previous study demonstrated that TNFα augments the odontoblast differentiation of DPSCs in a TrkB-dependent manner (Irfan & Chung, 2023; Kim, Irfan, Hossain, George, et al., 2023). The literature indicates that pro-inflammatory cytokines, such as TNFα, LTA (lymphotoxin-alpha), and LPS (lipopolysaccharides), play significant roles in the formation of tertiary dentin (Irfan, Kim, Marzban, et al., 2022). To further confirm the role of inflammation in this process, various inflammatory mediators were applied during DPSC odontoblastic differentiation. The outline of the experimental timeline is shown (Figure 1A). Human DPSCs were seeded on Day 0, followed by culture in normal growth media. On Day 3, the cells were transferred to dentinogenic media. TNFα, LTA, and LPS treatments were initiated every other three days. The representative image shows the immunofluorescence staining results for TrkB and phosphorylated TrkB (p-TrkB) under various treatments (Figure 1B). The quantification of fluorescence intensity across these different treatment groups was analyzed by percentages of control (Figure 1C). TrkB expression and activation in DPSCs were significantly upregulated during odontogenic differentiation, especially under inflammatory stimulants such as TNFα, LTA, and LPS by 301 ± 17, 320 ± 15.2, and 250 ± 19, respectively vs control 165 ± 12.4 (p < 0.01). The spatial distribution of fluorescence intensity for TrkB and p-TrkB was measured across different treatment groups (Figure 1D). Figures were analyzed using ZEN (blue) software, and graphs were created. This spatial analysis confirms the enhanced and localized expression of TrkB and p-TrkB in response to inflammatory stimuli.

Next, we examined the expression levels of TrkA and TrkB receptors in response to various inflammatory stimuli in normal and dentinogenic media. The results indicate significant differential expression of these receptors under both conditions. TrkB expression was modulated by the media and stimuli (Figure 2C). Under control conditions, no significant differences were observed between the media types. However, LPS, LTA, TNFα, and C5a significantly increased TrkB expression in dentinogenic media compared to normal media (p < 0.001 for LPS, TNFα, and C5a; p < 0.05 for LTA). IL-5 caused a slight but significant increase in TrkB expression in dentinogenic o media (p < 0.05). Conversely, baseline TrkA expression showed no significant difference between normal and dentinogenic media (Figure 2A), and C5a and IL-5 did not induce significant changes in TrkA expression in either medium.

Figures 2B and 2D show the effect of TNFα stimulation on the expression of TrkA and TrkB and their phosphorylation vs control. 2D and 2.5 models show fluorescence spikes to evident the phenomenon. It is obvious that TNFα stimulation caused significant phosphorylation of TrkB (p < 0.01) compared to TrkA (p < 0.05). Graphs (Fig. 2B1 and D1) show significant changes among TNFα treated cells vs control. So, we chose TrkB as a target receptor for our further study.

The dose-dependent response of TrkB to TNFα (Figure 2E) revealed minimal baseline expression at 0 ng/mL TNFα, with significant increases at 0.1 ng/mL, 1 ng/mL, and a pronounced peak at 10 ng/mL in dentinogenic media (p < 0.001). At 20 ng/mL TNFα, TrkB expression remained significantly higher in dentinogenic media compared to normal media (p < 0.001). The time-dependent response to a fixed TNFα concentration (10 ng/mL) showed that TrkB expression in dentinogenic media was significantly higher on day 1, further increased by day 3, and peaked on day 7 (p < 0.001) (Figure 2F). Although TrkB levels slightly decreased on days 10 and 14, they remained significantly elevated compared to normal media. These findings emphasize the importance of the local microenvironment in influencing receptor expression and inflammatory responses.

Regenerative odontogenic differentiation in the context of caries occurs within an inflammatory environment, making it essential to understand the impact of inflammation on DPSCs to comprehend dentin regeneration fully (Kim, Irfan, Hossain, Shin, et al., 2023). Immunofluorescence double staining was employed to analyze the expression of STRO-1, which is a stem cell marker, and TrkB in human carious and normal teeth, as well as in DPSCs (Fig. 3A-K). The in vivo analysis revealed that STRO-1 was primarily localized around blood vessels, while TrkB was more abundantly expressed in the inflamed pulp tissue of carious teeth compared to normal teeth. Quantitative analysis revealed significantly higher TrkB expression in carious teeth (p < 0.001). In vitro, both STRO-1 and TrkB were expressed in control DPSCs, but TNFα treatment markedly increased TrkB expression (Fig. 3L-T). The data showed that TrkB expression was significantly elevated in TNFα-treated DPSCs (p < 0.01). These findings suggest that TrkB is upregulated in response to inflammation in both carious teeth and DPSCs.

Our previous data demonstrated that inflammation and BDNF-induced DPSC odontogenic differentiation increased the expression of odontogenic differentiation markers. (Kim, Irfan, Hossain, George, et al., 2023). To investigate whether BDNF-TrkB signaling controls dentin regeneration following inflammation in vivo, we utilized CRISPR-engineered BDNF-overexpressing DPSCs in a pulp-capping mouse model (Figure 4A). We initially utilized the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique to activate BDNF expression in DPSCs. Transfection of DPSCs with the BDNF CRISPR activation plasmid was confirmed through Western blot analysis, which revealed increased BDNF expression compared to the control group (Figure 4B). Quantification of the integrated intensity relative to beta-actin further indicated a significant increase in BDNF expression in CRISPR-activated DPSCs (Figure 4C). Histological analysis of H&E-stained sections was performed to evaluate tissue morphology and surgery site from the transplantation of CRISPR-engineered BDNF-overexpressing DPSCs in a pulp-capping mouse model (figure 4D).

Following confirmation of successful CRISPR activation, we conducted the transplantation of CRISPR-engineered BDNF-overexpressing DPSCs on dentin regeneration in a pulp-capping mouse model. Micro-computed tomography (micro-CT) imaging was utilized to assess dentin volume in both sham control and experimental groups. The sham control group [Figure 5A(a-h)] exhibited a clear demarcation of the injured dentin area, highlighted by the white arrow indicating the drilling operation and the dotted line specifying the damaged region. In contrast, the experimental group treated with CRISPR-engineered BDNF-overexpressing DPSCs [Figure 5A(i-p)] showed a notable increase in dentin formation at the injury site. A quantitative analysis of dentin density (Figure 5D) revealed a significant increase in the dentin volume in the group treated with CRISPR-engineered BDNF-overexpressing DPSCs compared to the sham control group. Specifically, the dentin volume in the treated group (Figure 5C) was 1241 ± 51 mg HA/ccm, significantly higher than the control group (Figure 5B), which measured at 1169 ± 9 mg HA/ccm (p < 0.05).

To evaluate morphological changes after transplantation, H&E staining was employed to assess cellular and structural changes in the dentin and pulp tissue following the transplantation of CRISPR-engineered BDNF-overexpressing DPSCs in the pulp-capping mouse model. Figure 5 displays representative images of the sham control group and surgery group. The overall morphology of the tooth structure (Figure 4D) and higher magnifications of the injured area highlighted by the box are provided (Figure 5). In contrast to the control group, the BDNF-DPSC transplantation group demonstrated significant dentin regeneration and repair. The newly formed dentin at the injury site appears more organized and closely resembles the native dentin structure.

GFP staining allowed us to identify and localize the transfected areas within the injury site (Figure 5-6). Specifically, GFP expression was observed in areas correlating with the injury compared to the control (Figure 6A). This confirmed the successful transfection and localization of the CRISPR-engineered DPSCs in the mandible of the mouse tooth. line graphs show colocalizations of GFP-tagged DPSCs expressing BDNF and TrkB (Figure 6B). These results indicate that DPSCs overexpressing BDNF, engineered using CRISPR and transplanted into a pulp-capping mouse model, significantly improve dentin regeneration.

In our investigation of the combination of TNFα and CTX-B treatment compared to the control, we further aimed to identify the transcriptomic differences associated with the involvement of the BDNF-TrkB pathway related to inflammation. The gene heatmap and transcripts heatmap revealed distinct high and low gene expression patterns between the control and TNFα + CTX-B groups (Figure 7A-B). The volcano plot of differentially expressed genes (DEGs) illustrated the significant downregulation and upregulation in gene expression between the control and TNFα + CTX-B groups (Figure 7C-D).

We identified significantly enriched Gene Ontology (GO) terms in the categories of biological process, cellular component, and molecular function between these groups (Figure 7E). Notably, there was a distinct difference in membrane-related cellular functions and molecular functions related to protein binding. The GO enrichment pathway analysis revealed that the highest p-values were associated with the extracellular space and region (Figure 8A). Furthermore, Reactome pathway analysis indicated high p-values for pathways related to TNF signaling (Figure 8B).

Sashimi plots comparing control and TNFα + CTX-B treated dental pulp stem cells (DPSCs) highlighted differentially spliced exons in genomic regions of interest (Figure 6D, E, F). The transcriptional response of TNFα + CTX-B treated odontoblast-like differentiated DPSCs showed significant downregulation in TCFs and ATF4 and upregulation in signal transducers and activators of STAT1 and STAT3 (Figure 8C). These findings suggest that TNFα and CTX-B treatment induces significant transcriptomic alterations, particularly affecting pathways involved in cellular signaling and extracellular matrix interactions.

Human DPSCs were purchased from Lonza, Pharma & Biotech (Cat. # PT-5025). MEM-alpha, DMEM, PBS, fetal bovine serum, L-glutamine, and Antibiotic–Antimycotic were procured from Gibco™ Fisher Scientific (Waltham, MA, USA). Poly-D-Lysine coated (BioCoat™, 12 mm) round German glass coverslips were purchased from Corning™ Fisher Scientific (Cat. # 354087; Waltham, MA, USA). RIPA buffer was from Cell Signaling Technology (Danvers, MA, USA). Various antibodies were procured: anti-TrkA, anti-phospho-TrkA, anti-TrkB, anti-phospho-TrkB, anti-BDNF, and β-tubulin from BioLegend (San Diego, CA, USA), and anti-β-actin from Fisher Scientific (Waltham, MA, USA), anti-GFP and anti-STRO-1 from Santa Cruz (Dallas, Texas, USA). Fluorescent secondary antibodies were from Life Technologies (Grand Island, NY, USA). TNFα was from Invitrogen, Fisher Scientific (Waltham, MA, USA), and a few other chemicals were from Fisher Chemical (Nazareth, PA, USA). BDNF CRISPR activation plasmid (h) (Cat# sc-400029-ACT) and Reagent System were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

All experiments were conducted with different sets of human dental pulp stem cells (DPSCs) 3-4 times, using cells at the 2^nd and 3^rd passages and cell proliferation was measured by counting the total number of cells. Commercially available human DPSCs isolated from third molars of adult donors (ages 29-30 years old) collected during the extraction of wisdom teeth, which were guaranteed through 10 population doublings, to express CD105, CD166, CD29, CD90, and CD73, and do not express CD34, CD45, and CD133 106–108; cultured in regular growth media (αMEM containing 10% FBS, 1% L-glutamine, and 1% antimycotic/antibiotic) at 37°C and 5% CO₂ for 3-4 days. After this initial period, the media was swapped to odontogenic media (DMEM containing 10% FBS, 1% L-glutamine and antimycotic/antibiotic, 50 µg/mL ascorbic acid, 10 mM β-glycerophosphate, and 10 nM dexamethasone) at day 4 until day 17. TrkB antagonist (CTX-B, 200nM/ml) was treated every three days until day 17. Cells were treated with LPS, TNFα, LTA, C5a, and IL-5 at days 4 and 7. The CaMKII inhibitor (5 μM/mL), or CaMKII protein (1 μM/mL) were treated with dentinogenic media at days 4, 7, 10, and 14.

DPSCs were cultured in a 6-well plate at 5 × 10⁴ cells per well. The total mRNA was extracted using a RNeasy Mini Kit (74104, Qiagen), and the containing quantity of cDNA was measured using the NanoDrop 2000 (ND2000, Fisher Scientific). The Fast SYBR™ Green Master Mix (4385616, ThermoFisher) was used to identify the cDNA sample according to the manufacture’s protocol. Primer sequences (Integrated DNA Technologies) were used predesigned hGAPDH. (Forward: 5′-GGC ATC CAC TGT GGT CAT GAG-3′, Reverse: 5′-TGC ACC ACC AAC TGC TTA GC-3′), hDSPP (Forward: 5′-CTG TTG GGA AGA GCC AAG ATA AG-3′, Reverse: 5′-CCA AGA TCA TTC CAT GTT GTC CT-3′), and hDMP-1 (Forward: 5′-CAC TCA AGA TTC AGG TGG CAG-3′, Reverse: 5′-TCT GAG ATG CGA GAC TTC CTA AA-3′).

The seeded DPSCs were incubated in a 12-well plate with coverslips at 37°C in a CO₂ incubator until reaching 70-80% cell confluency. The coverslips were then fixed with 4% paraformaldehyde for 2 hours at 4°C. Blocking and permeabilization were performed using 5% normal goat serum and 0.01% Triton X in 0.01 M phosphate buffer solution (PBS) for 1 hour at room temperature. For primary antibody treatment, the specimens were treated overnight at 4°C with the following antibodies diluted in 5% serum: anti-TrkB (1:1000), anti-pTrkB (1:1000), and/or anti-STRO1 (1:1000). After the overnight incubation with primary antibodies, the secondary antibodies were applied for 2 hours with Alexa Fluor-594 anti-mouse IgG, Alexa Fluor-488 anti-rabbit IgG (1 μg/mL), and/or DAPI (1 μg/mL). The coverslips were mounted on glass slides, and images were taken using a Leica microscope. MATLAB (R2022a) software was used to measure fluorescence intensity and perform quantification.

DPSCs were incubated in a 6-well plate with 3 ml of antibiotic-free standard growth medium at 37°C in a 5% CO2 incubator until reaching 40-60% confluency in preparation for CRISPR activation plasmid transient transfection. For the preparation of transfection solutions, 2 µg of plasmid DNA was diluted in Plasmid Transfection Medium (sc-108062) to a final volume of 150 µl (Solution A) and mixed and incubated for 5 minutes at room temperature. In parallel, 10 µl of UltraCruz® Transfection Reagent (sc-395739) was diluted in Plasmid Transfection Medium (sc-108062) to a final volume of 150 µl (Solution B) and incubated for 5 minutes at room temperature. Solutions A and B were then combined by vortexing immediately and incubated for 20 minutes at room temperature. For the transfection procedure, the cell culture medium was replaced with a fresh antibiotic-free growth medium, and 300 µl of the Plasmid DNA/UltraCruz® Transfection Reagent Complex was added to each well. The cells were incubated for 48 hours under standard culture conditions. Protein analysis was conducted to confirm overexpression.

To verify BDNF activation using CRISPR plasmid, cell lysates were prepared using RIPA buffer (50 mM Tris pH 7.6, 150 mM NaCl, 1% Triton X100, 1mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitor (Roche, Indianapolis, IN) and protein was measured using BCA method (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, Lenexa, KS). Equal amounts of protein (35 μg) were loaded and separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose (Bio-Rad, CA). Blots were probed using polyclonal antibodies specific for BDNF or β-actin for overnight at 4°C and then washed and probed with secondary antibodies for 2 hours at RT. Odyssey CLx visualized the protein bands.

Human DPSCs were seeded in growth media at 15 × 10³ cells/cm² in 96-well optical plates. At sub-confluency, cells were incubated in an antibiotic-free medium and treated as mentioned above. Then, cells were immediately fixed with 100% cold methanol (15 min) and saturated with 5% BSA (1.5 h). Cells were incubated overnight at 4 °C, anti-TrkA, anti-TrkB, anti-phospho-TrkA, anti-phospho-TrkB, or anti-β-tubulin. Cells were then washed (0.05% Tween-20/PBS) and incubated with respective IRDye-680RD or IRDye-800RD secondary antibody (1 h) at RT. After 5 washes, plates were dried and scanned at 700 and/or 800 nm (Odyssey CLx).

The Institutional Animal Care and Use Committee (IACUC) at the University of Illinois Chicago reviewed and approved all surgical and experimental procedures. C57BL/6 male mice, 6-8 weeks old (n = 12), were used for the experiments and purchased from Jackson Laboratory (#000461). The animals were housed in three per cage in a temperature-controlled room (23 ± 1°C, 12 h/12 h light/dark cycle) and maintained at the UIC animal facility. For the mouse molars, the mandibles of the C57BL/6 mice were collected for further experimentation (Protocol no.: 21-197).

C57BL/6 male mice aged 6 to 8 weeks were employed for this mouse model. Dentin of the lower left first molar was penetrated using a 0.3 mm rounded carbide burr drill operating at automatic speed. Subsequently, an 11 G needle was utilized to remove any remaining dentin upon exposure. The exposed dentin was then treated with BDNF CRISPR Activation Plasmid-transfected DPSCs embedded in collagen (from rat tail), followed by sealing with mineral trioxide aggregate (MTA) to shield the pulp from further inflammation. Animals were euthanized beginning 4 weeks post-injury, and their mandibles were excised. The molars were subsequently harvested for histological, molecular, and immunohistochemical analyses to evaluate the efficacy of the treatment in promoting dental pulp regeneration.

Mandibles from C57BL/6 male mice were collected from both the sham control group and the group receiving transplantation of CRISPR-engineered BDNF-overexpressing dental pulp stem cells (DPSCs) in the pulp-capping mouse model. Micro-CT (μCT) analyses were conducted using the commercial service at RUSH Hospital to assess the volume of regenerated dentin quantitatively. All animal phenotype analyses were blinded to mitigate potential examiner bias and ensure impartial evaluation.

The mandibles of C57BL/6 mice were harvested and fixed in a 4% formaldehyde solution immediately after extraction. Subsequently, they were placed in decalcification solution within 15 mL Falcon tubes for two days, with daily changes in concentration (starting from 10% sucrose in PBS, progressing to 20% and then 30%). The mandibles were sectioned following decalcification, and histological staining protocols were optimized specifically for dental tissues. Sections underwent a sequence of staining procedures, including bleaching, eosin staining, and dehydration through graded ethanol solutions (ranging from 70% to 100% ethanol). Finally, the sections were cleared in xylene to prepare them for microscopic analysis and further histopathological evaluation.

Human wisdom tooth extractions were conducted by licensed dentists specializing in oral surgery. This study involving human specimens received ethical approval from the Endodontics clinic at the College of Dentistry, University of Illinois Chicago (Protocol no.: 2012-0588). Prior to participation, all patients provided informed written consent detailing the nature and objectives of the experimental procedures. Ethical considerations and patient confidentiality were strictly followed throughout the study, ensuring compliance with established guidelines and protocols for human research.

The extracted mandibles were embedded in an OCT cryostat sectioning medium and stored at −20°C for 2 hours before being cut into 20 µm thick sections. The mounted tissues were fixed with 4% paraformaldehyde for 1 hour at room temperature. Subsequently, blocking and permeabilization were performed using 10% normal goat serum (NGS) and 0.1% Triton X in 0.01 M phosphate buffer solution (PBS) for 1 hour at room temperature. For primary antibody incubation, the specimens were treated overnight at 4°C with mouse anti-CaMKII (1:200), rabbit anti-p-CaMKII (1:200), mouse anti-GFAP (1:200), and/or mouse anti-STRO1 (1:200), diluted in 10% NGS. Following overnight incubation with primary antibodies, the specimens were incubated for 2 hours at room temperature with secondary antibodies: Alexa Fluor-594 anti-mouse IgG, Alexa Fluor-488 anti-rabbit IgG (1 μg/mL), and/or DAPI (3 μg/mL). After secondary antibody treatment, the specimens were mounted on glass slides, and images were acquired using a Leica microscope. Fluorescence intensity was quantified using Image J software. Representative images were exported in TIFF format. Image J software and GraphPad Prism version 10 were employed for fluorescence intensity analysis.

RNA Sequencing was employed to comprehensively analyze the transcriptome, focusing specifically on mRNA, utilizing services provided by LC Biosciences (Houston, Texas). This method capitalizes on the polyadenylated (poly-A) tails present at the 3’ ends of eukaryotic mRNA molecules. Dental pulp stem cells (DPSCs) underwent a 10-day differentiation protocol with or without specific treatments. Post-differentiation, total RNA was extracted from the odontogenic differentiated DPSCs for sample preparation. LC Biosciences conducted rigorous assessments to evaluate the extracted RNA’s quantity and quality, followed by poly-A selection to enrich for mRNA transcripts. Subsequent cDNA synthesis facilitated library preparation, paving the way for high-throughput sequencing to explore the transcriptomic landscape in depth. This approach enabled the identification and characterization of gene expression patterns associated with the differentiation of DPSCs under various experimental conditions.

The statistical analyses were performed on at least 3 independent experiments with duplicates or triplicates, and statistical significance was determined using one-way analysis of variance (ANOVA) followed by post-hoc Dunnett’s test (SAS 9.4) to compare the different treatments and their respective controls (p-value of 0.05 or less was considered statistically significant). In addition, the data were analyzed using Tukey’s test to determine statistical significance between the groups. For quantification of immunofluorescence staining intensity, ImageJ 1.49v software was used. Fixed areas of 1 mm × 1 mm or 2 mm × 2 mm were selected to analyze differentiated cells’ number or fluorescence intensity.