The dimethyloxalylglycine-functionalized nanofibers for in situ regeneration of infected developing dental roots

Abstract

In situ regeneration in restorative dentistry targets the repair of tissues directly at the injury site by utilizing engineered biomaterials to guide endogenous cell activity. This approach aims to simplify treatment procedures and achieve more predictable outcomes, thus to supports the regeneration of damaged tissues and potentially restores tooth vitality, reducing the need for more invasive treatments. This study explores the potential of poly(ε-caprolactone) fibers (PCLF) functionalized with a hypoxia-inducible factor 1-alpha (HIF-1α) stabilizing small molecule dimethyloxalylglycine (DMOG) for in situ regeneration in the context of dental root repair in developing immature teeth. PCLF functionalized with DMOG (PCLF/DMOG) was applied to regenerative endodontic procedure (REP) treatment of infected developing dental roots, and its biologic properties and therapeutic potential were investigated through both in vitro studies and in vivo experiments, focusing on their capacity to promote in situ regeneration. In vivo application demonstrated the effectiveness of PCLF/DMOG in promoting root development, apical closure, and improving infectious lesions, contrasting with contemporary REP treatment controls that showed unpredictable outcomes. Mechanistically, the sustained release of DMOG from PCLF/DMOG significantly enhanced the expression of HIF-1α and upregulated expression of genes associated with angiogenesis and neurogenesis, including VEGF-α and NGF. The PCLF/DMOG upregulated antimicrobial peptides, facilitated efferocytic activities, promoted macrophage polarization to the M2 phenotype, and mobilized mesenchymal stem cells. Taken together, PCLF/DMOG could enhance innate immune responses and foster favorable microenvironment to guide cellular differentiation, promoting in situ regeneration of dental roots in the inflammatory microenvironments.

Keywords: HIF-1α; In situ regeneration; Infected immature dental roots; Macrophages; Mesenchymal stem cells; Nanofibrous scaffolds.

Graphical abstract

Fig. 1

Characterization of PCLF and PCLF/DMOG sheets. The SEM images of PCLF (A) and PCLF/DMOG (B) were observed. Scales represent 1 μm. The fiber diameters were measured from SEM images and the distribution is presented (C). The 3D roughness of the PCL-solid and PCL-fiber (D, E, and F) were measured. Tensile strength and elastic modulus (G), water contact angle (H), and biodegradation (I) were also detected. Data are shown as the mean ± SD (n = 9). (J) Cell viability. Dental pulp stem cells were seeded on PCLF and PCLF/DMOG. Cell viability was assessed at 12, 24, and 48 h post-seeding using the EZ-Cytox cell viability assay kit. Data are presented as mean ± SD from three independent experiments (n = 3). Dental pulp stem cells were seeded on PCLF and PCLF/DMOG, and the number of adhered cells at 1, 24, and 48 h post-seeding was quantified via hematoxylin staining. (K) Quantification of adhered cells within a 2223 μm × 1667 μm field of view. Data are presented as mean ± SD from three independent experiments (n = 3). (L) Phase-contrast images of dental pulp stem cells cultured on PCLF and PCLF/DMOG at 1, 24, and 48 h post-seeding. ∗ Significant differences between PCLF and PCLF/DMOG (p < 0.05).

Fig. 2

Release of DMOG from PCLF/DMOG sheets. (A) Cumulative release of DMOG from PCLF/DMOG sheets submerged in PBS. (B) Representative image of Western blot analyses for HIF-1ɑ in the NIH3T3 cells cultured on each substrate for 4 days. (C) RT-qPCR analyses for the expressions from the tissues surrounding sheets transplanted into murine subcutaneous tissue for a week. Untreated subcutaneous tissues served as a control. Data are shown as mean ± SD (n = 4). ∗Significantly different from control (p < 0.05). ^#Significantly different from PCLF (p < 0.05).

Fig. 3

MicroCT analyses. Representative sagittal images of teeth along the long axis (upper A) and horizontal ones at the apical 30 % of tooth root (lower A). The 3-D images of root apex (B). Quantified apical length (C) and dentin thickness (D) from 3-D constructs by volume-of-interests with (VOIs) specific root length (L_analysis).

Fig. 4

Histology with hematoxylin–eosin staining (A), Periostin immunostaining (B), and Gram stain (C). Representative micrographs of dental roots are shown.

Fig. 4

Histology with hematoxylin–eosin staining (A), Periostin immunostaining (B), and Gram stain (C). Representative micrographs of dental roots are shown.

Fig. 5

Cell mobilization to the PCLF/DMOG implanted into subcutaneous tissues. The PCLF and PCLF/DMOG sheets were implanted to the subcutaneous tissue of mouse dorsal skin, retrieved after 4 days, and stained with DAPI. The cell numbers on the sheets were counted (A). MSCs (SCAP) were seeded on the upper insert, and the retrieved, implanted sheets were placed on the lower bottom. After 24 h, numbers of the cells crossed the trans-well were counted (B). Data are shown as mean ± SD (n = 3). ∗Significant differences between PCLF and PCLF/DMOG (p < 0.05).

Fig. 6

Macrophage phenotypes. Total RNAs were extracted from the retrieved, implanted PCLF and PCLF/DMOG sheets (A, B) and from the BMMs cultured on PCLF and PCLF/DMOG (C). Data are shown as mean ± SD (n = 3). ∗Significant differences between PCLF and PCLF/DMOG (p < 0.05).

Fig. 7

Responses of neutrophils and macrophages to DMOG. Activity of phagocytosis (A) was checked in primary mouse neutrophils using FITC-Zymosan Bioparticle, and efferocytosis (B) was checked in primary mouse macrophages with apoptosis-induced neutrophils as bait cells. The cultures were treated with DMOG (100 nM) for the last 24 h. Data are shown as mean ± SD (n = 3). ∗Significantly different from non-treated control (p < 0.05).

Fig. 8

Effect of DMOG on the expression of anti-microbial peptides. RT-qPCRs were performed to detect the expression levels in the primary bone marrow-derived macrophages. The cells were treated with DMOG (100 nM) in the presence or absence of LPS (10 ng/mL) for 24 h. Data are shown as mean ± SD (n = 3). ∗Significantly different from non-treated control (p < 0.05).

Fig. 9

Odontoblast differentiation of murine dental pulp cells cultured on PCLF and PCLF/DMOG (A) or with DMOG treatment (100 nM). The cells were cultured for 1 or 2 weeks, and analyzed through alizarin red staining, ALP activity, western blot, and RT-qPCR. Data are shown as mean ± SD (n = 3). ∗Significantly different from non-treated control (p < 0.05).