Human DPSCs were purchased from Lonza Pharma & Biotech (Cat. # PT-5025). MEM-alpha, DMEM, PBS, fetal bovine serum, L-glutamine, and antibiotic–antimycotic solution were procured from Gibco™ Fisher Scientific (Waltham, MA, United States). Poly-D-Lysine (Bio-Coat™, 12 mm)-coated round German glass coverslips were purchased from Corning™ Fisher Scientific (Cat. # 354087; Waltham, MA, United States). RIPA buffer was from Cell Signaling (Danvers, MA, United States), the DMP-1 ELISA kit was from Invitrogen, Thermo Fisher Scientific (Waltham, MA, United States), and the DSPP ELISA kit was from MyBioSource (San Diego, CA, United States). Various antibodies were procured: anti-C5L2 was from BioLegend (San Diego, CA, United States), anti-DMP-1, anti-DSPP, and anti-β-actin were from Fisher Scientific (Waltham, MA, United States), and anti-STRO-1 was from Santa Cruz (Dallas, Texas, United States). Fluorescent secondary antibodies were from Life Technologies (Grand Island, NY, United States). Human recombinant TNFα was from Invitrogen, Thermo Fisher Scientific (Waltham, MA, United States), and a few other chemicals were from Fisher Chemical (Nazareth, PA, United States). C5L2 CRISPR/Cas9 KO plasmid human (sc-410578) and reagent system were purchased from Santa Cruz Biotechnology (Dallas, TX, United States).

Commercially available human DPSCs, which were guaranteed through 10 population doublings, to express CD105, CD166, CD29, CD90, and CD73 and to not express CD34, CD45, and CD133, were further evaluated by immunocytochemistry in cultures with the STRO-1, a stem cell marker. DPSCs were cultured at 37°C and 5% CO2 in regular/osteogenic media for 72 h in regular growth media (α MEM containing 10% fetal bovine serum (FBS) and 1% L-glutamine), and then, cells were transfected with CRISPR/Cas9 C5L2 KO, and successful transfection was confirmed by Western blot. Then, cells were swapped with dentinogenic media (DMEM containing 20% FBS, 1% L-glutamine, and antimycotic/antibiotic, i.e., 100 μg/mL streptomycin and 100 U/mL penicillin, supplemented with 100 μg/mL ascorbic acid, 10 mmol/L β-glycerophosphate, and 10 mmol/L dexamethasone) for further 10-day differentiation and treated with TrkB inhibitor (cyclotraxin-B: CTX; 200 nM) or TNFα (10 ng/mL) every 3 days. All the experiments were conducted with different sets of DPSCs (between the second and fourth passages) three times, and cell proliferation was measured by counting the total number of cells.

Human DPSCs were grown in a six-well plate culture chamber in 2 mL of free-antibiotic medium up to 60% confluence, and then, transient transfection with CRISPR/Cas9 KO was performed using the C5L2 CRISPR/Cas9 KO plasmid (h) (sc-410578), according to the manufacturer’s protocol. Cells were incubated at 37°C in a CO₂ incubator in 2 mL of antibiotic-free and serum-free transfection solution containing a mixture transfection reagent and 0.75 μg/mL of C5L2 plasmid. After an incubation of 2 days, transfection was confirmed by Western blotting. Then, the medium was aspirated and replaced with fresh dentinogenic media every 3 days.

Human DPSCs were cultured in a six-well plate at 5 × 10⁴ cells/well, up to 7 days in dentinogenic media. The total mRNA was extracted using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) and analyzed using the Fisher Scientific NanoDrop 2000 device. The cDNA samples were analyzed using the Applied Biosystems SYBR green reagent system, according to the manufacture’s protocol. Primer sequences were purchased from IDT. The expression levels of target genes were standardized by the housekeeping gene GAPDH using the 2^−ΔΔCt method (Livak and Schmittgen, 2001).

Human DPSCs were seeded at 1 × 10⁴ cells/well on 12-well glass culture chambers overnight before stimulation with TNFα. After required proliferation and differentiation (day 4 or day 16), cells were fixed with 4% paraformaldehyde and permeabilized using the Triton X-100 PBS solution. Then, cells were incubated overnight at 4°C with primary antibodies. The next day, cells were washed and incubated for 2 hours with a mixture of Alexa Fluor-594 anti-mouse IgG, Alexa Fluor-488 anti-rabbit IgG (2 μg/mL), and/or DAPI (1 μg/mL). The coverslips were sealed, and photographs were taken using a Leica DMI6000 B microscope. Fluorescence staining was statistically analyzed by determining the integrated density of each condition using ImageJ 1.49v software. A co-localization analysis was performed using both Colocalization Finder and JACoP plugins on ImageJ software.

The six-well plates were washed two times with distilled water and fixed with 4% PFA for 1 hour at RT. The cells were then washed with distilled water two times, and 1 mL of 40 mM of alizarin red stain (ARS) was added per well; the ARS was provided by ScienCell (#8678). After 1 hour of gentle shaking, the plate was washed with distilled water (pH 4) three times and dried. The cells were inspected using a Leica DMi1 phase microscope, and images were obtained. Then, 10% acetic acid solution was added to the wells for 30 min at RT with gentle shaking, and samples were collected and processed with 10% ammonium hydroxide for ARS quantification. The plot was drawn against standards and respective samples.

To see the effects of dentin sialophosphoprotein (DSPP) and DMP-1, cell lysates were prepared using RIPA buffer (50 mM Tris pH 7.6, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 0.5% sodium deoxycholate, and 0.1% SDS) containing a protease inhibitor (Roche, Indianapolis, IN), and the protein was measured using the 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 DSPP, DMP-1, or β-actin overnight at 4°C and then washed and probed with secondary antibodies for 2 h at RT. The protein bands were visualized by Odyssey CLx.

Human DPSCs were seeded in growth media at 15 × 10³ cells/cm² in 96-well plates. At sub-confluency, cells were incubated in an antibiotic-free medium and treated as mentioned above to KO and then differentiated in dentinogenic media. 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 with anti-DMP-1, anti-DSPP, or anti-β-actin. Cells were then washed (0.05% Tween-20/PBS) and incubated with respective IRDye-680RD or IRDye-800RD secondary antibodies (1 h) at RT. After five washes, plates were dried and scanned at 700 and/or 800 nm (Odyssey CLx).

Supernatants from DPSC culture, incubated with various abovementioned treatments, were collected from cultures after 7, 10, or 14 days of differentiation and assayed using the ELISA kit according to the manufacturer’s protocol. Then, a standard curve was constructed using standards and test samples in duplicate at increasing concentrations, and values were normalized accordingly.

The statistical analyses were performed on at least three 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 different treatments and their respective controls (p-value of 0.05 or less was considered statistically significant). In addition, the data were also analyzed by Tukey’s test for statistical significance between the groups. For the 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 the number or fluorescence intensity of differentiated cells.

Our previous studies have shown that complement C5a and its receptor C5aR had a positive regulatory role in inflammation-induced odontoblastic differentiation of DPSCs (Irfan et al., 2022a; Pasiewicz et al., 2022). To identify the role of C5a’s second and controversial receptor, C5L2 in this process, DPSCs were cultured and treated according to scheme given in Figure 1A, and DPSCs were confirmed by the mesenchymal stem cell marker, i.e., STRO-1 (Figure 1B). These cells have been extensively used and validated in our previous publications (Irfan et al., 2022a; Irfan et al., 2022b; Kim et al., 2023b). It is already known that DPSCs subconstitutively express the C5L2 receptor, as shown in Figure 1C, while stimulation with TNFα enhances its expression (Figure 1D) as previously described by Chmilewsky et al. (2019). The inflammatory cytokine TNFα was used to mimic inflammatory environments (Tiegs and Horst, 2022). Differentiating DPSCs in dentinogenic media are shown at D4 (Figures 1E–G) and at D16 (Figures 1H–J) using a light microscope and fluorescent phase contrast microscope. Cells were transfected with C5L2 KO plasmids and treated with TNFα or TrkB antagonists (cyclotraxin-B; CTX). To determine whether these applications have effects on their proliferation or differentiation, cells were counted, and no significant difference was found among different treatment groups (Figure 1K). A successful C5L2 CRISPR/Cas9 KO was confirmed by Western blot, and bands clearly show the inhibition of C5L2 in DPSCs for further experimentation (Figure 1L). To validate the proposed in vitro model for studying odontoblast-like cell differentiation from DPSCs, alkaline phosphatase (ALP), bone morphogenic proteins 2 (BMP-2), type 1 collagen (Col1A1), and DMP-1 were analyzed using real-time PCR as previously described by Teti et al. (2013), and our results showed significant increment in their expression indicating the odontoblastic differentiation of DPSCs (Figure 1M; p < 0.05 and p < 0.01).

DPSCs were subjected to differentiate in the dentinogenic medium for 10 days after C5L2 KO with TNFα stimulation. Under a light microscope observation, differentiated cells showed mineralization, analyzed by experts blinded to the study, which was further confirmed through various other following experiments. C5L2 KO groups showed more mineralization (Figures 2E–H) compared to the control differentiated group (Figures 2A–D). During stimulation with TNFα, differentiating DPSCs secreted more minerals (Figures 2I–L) than the control and C5L2 KO alone groups. It was further observed in higher magnification that calcium crystals are formed and abundantly found in the C5L2 KO group (Figures 2N, N1) and TNFα alone-treated group (Figure 2O) compared with the control group (Figure 2M), while crystallization significantly increased in the TNFα-stimulated C5L2 KO group (Figures 2P, P1).

Cultured wells with differentiated DPSCs were stained with ARS to analyze the formation of a mineralization matrix surrounding the microenvironment during dentinogenic differentiation. The C5L2 KO group (Figures 3B, B1; p < 0.05) and TNFα group (Figures 3C, C1; p < 0.05) show more mineralization matrix than the control (Figures 3A, A1), while their combined effect drastically enhanced mineralization phenotypes (Figures 3D, D1; p < 0.01).

Our studies demonstrated that BDNF-TrkB receptor signaling affects DPSC odontoblastic differentiation under an inflammatory context (Kim et al., 2023b). In addition, C5L2 directly regulates BDNF modulations on DPSCs (Irfan and Chung, 2023) suggesting the possible link between them. To demonstrate the specific role of TrkB in DPSC differentiation under TNFα/C5L2 KO-treated conditions, cells were treated with a TrkB inhibitor CTX. Interestingly, CTX treatment reversed the effects of TNFα and C5L2 KO (Figures 3E–F1) indicating the involvement of TrkB in TNFα/C5L2 KO-mediated mineralization (Bernar et al., 2022).

Our results clearly show significant differences among the various treatment groups (Figure 3G) indicating that TNFα and C5L2 KO have potentially triggered the mineralization matrix in the surrounding microenvironment.

To further confirm the role of C5L2 in DPSC dentinogenic differentiation, DPSCs were cultured and differentiated for 10 days in dentinogenic media after C5L2 KO. Cells were treated with TNFα or CTX every 3 days as mentioned above. It is known that differentiated cells show odontoblastic-like properties as they express DSPP and DMP-1, which are well-established odontogenic markers (Demarco et al., 2010; Sabbagh et al., 2020). Our results show that the C5L2 KO group (Figures 4E–H1) and TNFα-treated cell group (Figures 4I–L1) show higher expression of DSPP and DMP-1 compared with the control cells (Figures 4A–D1), while their combined effect even more enhanced the expression of mineralization markers (Figures 4M–P1). Consistent with the mineralization assay (Figures 3, 4), CTX treatment significantly abolished the effects of TNFα (Figure 4Q-T1) and C5L2 KO (Figures 4U–X1). The bar graph (Figure 4Y) shows a significant increment in DSPP and DMP-1 expression in TNFα and C5L2 alone (p < 0.05) and their co-treatment (p < 0.001), while CTX treatment reversed the effect of TNFα or C5L2 KO, suggesting the role of TrkB in the modulation of these mineralization marker proteins.

Figure 4Z shows the line graph for the co-localization of DSPP and DMP-1. Figures were analyzed using ZEN (blue) software, and graphs were created. The graphs clearly show the significantly enhanced expression of DSPP and DMP-1 in the co-treated group of TNFα and C5L2 KO compared with other groups, indicating that TNFα and C5L2 KO modulate the expression of odontoblastic mineralization markers.

Similarly, the in-cell Western assay showed an increment in the C5L2 KO and TNFα-treated groups, while there was significant secretion of DSPP and DMP-1 in the co-treated group compared to the control (Figures 5A, B). Figures 5A1, B1 show 2.5D model graphs depicting high peaks in the TNFα and C5L2 KO groups and lower peaks in the CTX-treated groups, indicating the abolishing effect of the TrkB antagonist on DSPP and DMP-1. Similarly, the bar graph shows a significant difference between the TNFα and C5L2 KO groups (p < 0.05) and co-treated groups (p < 0.001) against the control, while CTX reversed the effects of TNFα and C5L2 KO (Figure 5C).

To complement immunocytochemistry and in-cell Western data, we performed ELISA. Our data show C5L2-mediated DSPP and DMP-1 regulations in odontoblastic differentiating DPSCs at different stages (Figure 6). Supernatants were collected at D7, D10, and D14 and analyzed. DSPP and DMP-1 productions were observed increasingly at various stages from day 7 to day 14. The co-treated group showed the most significant production of DSPP and DMP-1 in the TNFα and C5L2 KO (p < 0.001) groups compared with alone (p < 0.05) or control groups, while the CTX treatment significantly abolished the effect of TNFα or C5L2 KO (p < 0.001). These results suggest that C5L2 KO odontoblastic differentiating DPSCs secrete higher levels of mineralization markers via modulating TrkB receptors, and TNFα stimulation enhances the effect of C5L2 KO.