Fig 1.
Overall concept of antibiotics and nitric oxide (NO) releasing biomimetic nanomatrix gel in tooth revitalization.
(A) Synthesis of peptide amphiphiles (PAs) using Fmoc chemistry: PA-YK was synthesized and self-assembled by mixing PA-YIGSR and PA-KKKKK in a 9:1 ratio. PA-YK was then reacted with NO to synthesize PA-YK-NO. (B) Encapsulation of antibiotics (CF and MN) within PA-YK-NO gel. (C) Application of antibiotics-PA-YK-NO gel for disinfection & pulp-dentin tissue revitalization to the open apex tooth, (C1) Infected tooth with necrotic pulp tissue and periapical lesion, (C2) Injected therapeutic antibiotics-PA-YK-NO gel into the root canal after the root conditioning, (C3) Healed tooth with dentin tissue deposition in canal wall and apex via pulp tissue revitalization. Tooth anatomical nomenclatures were added to the Figure.
Fig 2.
Four conditions of bacterial Transwell culture.
Bottom well contains bacterial sample, Transwell insert contains antibiotics and NO releasing biomimetic nanomatrix gel. (A) NO (-) antibiotics (-). (B) NO (+) antibiotics (-). (C) NO (-) antibiotics (+). (D) NO (+) antibiotics (+).
Fig 3.
Schematic illustration of experimental groups.
The second incisor (I2), the third incisor (I3), the first premolar (P1), and the second premolar (P2) or the third premolar (P3) in jaws were assigned to experimental groups.
Table 1.
Experimental groups according to root canal treatment modalities.
Fig 4.
The effect of antibiotics and NO releasing nanomatrix gel.
(A) Mixed antibiotics (0. 125 μg/mL of CF and MN) and four different concentrations of NO were evaluated on E. faecalis. (B) Mixed antibiotics (0.125 μg/mL of CF and MN) and four different concentrations of NO were evaluated on clinical samples. (C) Effect of antibiotics and NO (100μg/mL) releasing nanomatrix gel using C1. Live/Dead Staining Image of E. faecalis, T. denticola and clinical samples for 24 hours anaerobic culture using fluorescent microscopic image in 40X, C2. Quantitated bar graph using Image J.
Fig 5.
Radiographs and CT images of I3 and P1 in Group 1.
(A)A periapical view of I2 and I3 before treatment with wide-open apices (asterisks) in both incisors. (B) 5-month follow-up radiograph of I2 and I3 after the two-step NG2M treatment with a completed apical formation. (C)CT view of I3 with a thickening of apical section (arrows). (D) Periapical view of P1 before treatment with wide-open apices (asterisks). (E) 5-month follow-up radiograph of P1 after the two-step NG2M treatment with completed apical formation. (F) A CT view of P1 with a thickening of apical section (arrows). AF. Apical Formation; CB, Calcific Barrier; NCM, Newly formed Calcific Mass.
Fig 6.
Histological and IHC images of each I3 and P1 Group 1.
Each histological specimen was prepared: (A) as indicated as a circle insert and (B) as indicated as a rectangular insert. Each section was stained as H&E, MT and DSP. CD31 staining was magnified from the circle insert of in DSP image of I3. CD31 positive was indicated as brown staining. DSP positive was marked as blue arrow. H&E, hematoxylin and eosin; MT, Masson’s trichrome; DSP, dentin sialoprotein; Ce, cementum; De, dentin; NCM, new calcific mass; Bv, blood vessel. Scale bars (white): 500 μm, scale bar (yellow): 50 μm: 20 μm.
Fig 7.
Radiographs and CT images of P1 and P2 in Group 2.
(A) & (B) Periapical view before treatment with wide-open apices (asterisks) in both premolars, P1 and P2. (C) 5-month follow-up of P1 and P2 after the one-step NG2M treatment with a completed apical formation. (D) CT view of P1 and P2 with a thickening of apical section (arrows). AF. Apical Formation; CB, Calcific Barrier; NCM, Newly formed Calcific Mass.
Fig 8.
Histological and IHC images of P1 and P2 in Group 2.
Each histological specimen was prepared: (A) as indicated as a circle insert and (B) as indicated as a rectangular insert. Each section was stained as H&E, MT and DSP. CD31 staining was magnified from the circle insert of in DSP image of P1. CD31 positive was indicated as brown staining. P2 MT staining was magnified as (C). DSP positive was marked as blue arrow. Blood vessels were pointed by arrowhead. Ce, cementum; De, dentin; NCM, newly formed calcific mass. Scale bars (white): 200 μm, scale bar (yellow): 50 μm.
Fig 9.
Radiographs and CT images of P1 and P3 in Group 3.
(A) Periapical view of P1 before treatment with an open apex (asterisk). (B) 5-months follow-up radiograph of P1 after the two-mix antibiotics only treatment. (C) 5-month follow-up CT view with completion of apical formation P1. (D) Periapical view of P3 before treatment with an open apex (asterisk). (E) A 5-month follow-up radiograph of P3. (F) A 5-months follow-up CT view of P3 with completed apical formation, with periapical lesion (PL) around the root. AF, Apical Formation; CB, Calcific Barrier; NCM, Newly formed Calcific Mass; PL, Periapical Lesion.
Fig 10.
Histological and IHC images of P1 in Group 3.
The histological specimen was prepared from P1 (A) and (B). Each section was stained as H&E, MT and DSP. (C) indicated magnification from the circle of DSP image. DSP positive was marked as blue arrow. Ce, cementum; De, dentin; NCM, newly formed calcific mass. Scale bars (white): 200 μm, scale bar (yellow): 50 μm.
Fig 11.
Radiographs and CT images of I2 and I3 in Group 4.
(A) Periapical view of I2 and I3 before treatment. (B) A 5-month follow-up radiograph of I2 and I3 after the apexification with calcium hydroxide. The root apex showed inflammatory root resorption (IR) without apical formation. (C) A 5-months follow-up CT view of I3 with calcific barrier formation in the apical part without newly formed calcified mass. IR, inflammatory root resorption; CB, calcific barrier.
Fig 12.
Histological and IHC images of I3 in Group 4.
The histological specimen was prepared from I3 (A) and (B). Each section was stained as H&E, MT and DSP.. CD31 staining was magnified from the circle insert of DSP image. Ce, cementum; De, dentin; NCM, newly formed calcific mass. Scale bars (white): 500 μm, scale bar (yellow): 100 μm.
Table 2.
Summary of experimental results.