Fig 1.
(A) Diabetes was induced via intraperitoneal injection of streptozotocin (STZ). Seventy-two hours after injection, rats with fasting glucose levels above 350 mg/dL were used as diabetic rats. In total, 36 animals (4 per group per time point) and 72 surgically induced defects (2 per animal) were evaluated. (B) Imaging of surgical defects. Intrabony defects were created bilaterally on the mesial surface of the maxillary first molar along the line of the maxillary dentition. (C) Histomorphometric measurement method at surgical sites. The distances from the lowest point of the defect to the most coronal point of the cement-enamel junction (CEJ) (a), junctional epithelium (b), newly formed cementum (c), and newly formed bone (d) were measured. The area of new bone (e) and cementum (f) were also measured. (D) The sections of a micro-computed tomographic image. Newly formed bone was three-dimensionally assessed by focusing on a boxed region of interest (pink-framed rectangles). The volume of bone defects was evaluated in a 1.0 × 1.0 × 2.0-mm box shape.
Fig 2.
Analysis of postsurgical wound healing.
(A) Representative photographs of postsurgical wounds at 7 d after surgery. In the EMD-untreated sites of diabetic rats, wound closure deficiency was observed (arrowheads). (B) Wound closure rates 7 d after surgery differed significantly among the four groups [control without EMD, CE(-); control with EMD, CE(+); diabetes without EMD, DE(-); and diabetes with EMD, DE(+)].*p < 0.05 (chi-squared test, n = 24).
Fig 3.
(A) Representative histological photographs at 28 days after surgery of the four groups [control without the enamel matrix derivative [EMD] and without the cement enamel [CE(-)]; control with EMD, CE(+); diabetes without EMD, DE(-); and diabetes with EMD, DE(+)]. In the mesio-distal section, the defects were filled with newly formed connective tissue, cementum, and bone. Newly formed bone was observed along the roots of surgical defects. Hematoxylin–eosin staining, magnification 10×. Scale bar: 500 μm. (B) Higher magnification of the interface of the original bone (OB), newly formed bone (NB), and newly formed cementum (NC) at the bottom level of the original defect (arrowheads). Hematoxylin–eosin staining, magnification 40×. Scale bar: 100 μm.
Fig 4.
The distance of the cement-enamel junction (CEJ) to the bottom of the defect, length of the junctional epithelium, length of new cementum, length of new bone, area of new bone, and area of new cementum were histomorphometrically compared among groups [control without EMD, CE(-); control with EMD, CE(+); diabetes without EMD, DE(-); and diabetes with EMD, DE(+)]. Measurements were independently obtained by two blinded examiners (T.M. and D.K.), and the data were analyzed. *p < 0.05 (two-way repeated-measures analysis of variance, n = 24). NS, not statistically significant.
Fig 5.
Micro-computed tomographic (CT) analysis.
(A) Representative three-dimensional volume images of new bone formation by micro-CT after 28 d in the four groups [control without the enamel matrix derivative [EMD] and without the cement enamel CE(-); control with EMD, CE(+); diabetes without EMD, DE(-); and diabetes with EMD, DE(+)]. Defects in the control and DM groups were assessed by micro-CT. Less regeneration was observed in the DE(-) and DE(+) groups than in the CE(-) and CE(+) groups. More newly formed bone was observed in CE(+) and DE(+) than in CE(-) and DE(-). (B) Quantitative analysis of new bone formation by micro-CT in each group. (1) Bone volume, BV; (2) cancellous bone volume, CBV; (3) bone mineral density, BMD; and (4) cancellous bone mineral content, CBM were compared between groups. Measurements were independently made by two blinded examiners (T.M. and D.K.), and the data were analyzed. Data are presented as means ± SD (n = 24). *p < 0.05 (two-way repeated-measures analysis of variance). NS, not statistically significant.
Fig 6.
In vivo mRNA expression of inflammatory and angiogenic factors.
(A) The mRNA expression levels of Il-6, Tnf-α, Vegf, Col1, and Runx2 were determined via real-time quantitative polymerase chain reaction at 3 d and (B) 7 d. Inflammatory cytokines; Il-6 and Tnf-α were significantly upregulated in the DE(-) and DE(+) groups compared to those in the CE(-) and CE(+) groups after 7 d (p<0.05). Angiogenesis-related gene; Vegf were significantly upregulated in the CE(+) and DE(+) groups after 3 and 7 d (3 d p<0.01, 7d p<0.05). Col1 was significantly upregulated in the CE(+) and DE(+) groups compared to those in the CE(-) and DE(-) groups after 3 d (p<0.05). These findings were confirmed in three independent experiments. Data are presented as mean ± SD values (n = 12). *p < 0.05 (two-way repeated-measures analysis of variance). NS, not statistically significant.
Fig 7.
Akt and Erk1/2 phosphorylation in fibroblasts in vitro.
(A) Representative immunoblot images of EMD-treated or -untreated groups. Effect of 10 or 60 min of EMD treatment on Akt phosphorylation in gingival fibroblasts maintained at a control (Cont) or high glucose (HG) concentration. (B) Akt phosphorylation was quantified via densitometric analysis and expressed as a percentage of phosphorylation in EMD-untreated groups. Data are presented as mean ± SD values. *p < 0.05 (Tukey-Kramer test). These findings were confirmed in three independent experiments. (C) The mRNA expression level of Vegf was measured via real-time polymerase chain reaction (PCR) in the four groups (control glucose medium without EMD, Cont-; control glucose medium with EMD for 3–12 h, Cont+; high glucose medium without EMD, HG-; and high glucose medium with EMD for 3–12 h, HG+). Data are presented as mean ± SD values. *p < 0.05 (Tukey-Kramer test). These findings were confirmed in three independent experiments. (D) The mRNA expression level of Vegf with/without wortmannin was measured via real-time PCR. Data are presented as mean ± SD values. *p < 0.05 (Tukey-Kramer test). These findings were confirmed in three independent experiments.