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Soft-tissue Wound Healing by Anti-advanced Glycation End-products Agents P.-C. Chang, S.-C. Tsai, Y.-H. Jheng, Y.-F. Lin and C.-C. Chen J DENT RES 2014 93: 388 originally published online 19 February 2014 DOI: 10.1177/0022034514523785 The online version of this article can be found at: http://jdr.sagepub.com/content/93/4/388
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research-article2014
JDR
93410.1177/0022034514523785
RESEARCH REPORTS Biological
P.-C. Chang1,2*, S.-C. Tsai3, Y.-H. Jheng1, Y.-F. Lin4, and C.-C. Chen4 1
Graduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University, Taipei, Taiwan; 2Department of Dentistry, National Taiwan University Hospital, Taipei, Taiwan; 3Faculty of Dentistry, National University of Singapore, Singapore, Singapore; and 4Taiwan Mouse Clinics -National Phenotyping and Drug Testing Center, National Research Program for Biopharmaceuticals, National Science Council, Taipei, Taiwan; *corresponding author, changpc@ ntu.edu.tw
Soft-tissue Wound Healing by Anti-advanced Glycation Endproducts Agents
J Dent Res 93(4):388-393, 2014
Abstract
The blocking of advanced glycation end-products (AGE) has been shown to reduce diabetic complications and control periodontitis. This study investigated the pattern of palatal wound-healing after graft harvesting under the administration of aminoguanidine (AG), an AGE inhibitor, or N-phenacylthiazolium bromide (PTB), a glycated cross-link breaker. Full-thickness palatal excisional wounds (5.0 x 1.5 mm2) were created in 72 SpragueDawley rats. The rats received daily intraperitoneal injections of normal saline (control), AG, or PTB and were euthanized after 4 to 28 days. The wound-healing pattern was assessed by histology, histochemistry for collagen matrix deposition, immunohistochemistry for AGE and the AGE receptor (RAGE), and the expression of RAGE, as well as inflammation- and recovery-associated genes. In the first 14 days following AG or PTB treatments, wound closure, re-epithelialization, and collagen matrix deposition were accelerated, whereas AGE deposition, RAGE-positive cells, and inflammation were reduced. RAGE and tumor necrosis factoralpha were significantly down-regulated at day 7, and heme oxygenase-1 was persistently down-regulated until day 14. The levels of vascular endothelial growth factor, periostin, type I collagen, and fibronectin were all increased at day 14. In conclusion, anti-AGE agents appeared to facilitate palatal wound-healing by reducing AGE-associated inflammation and promoting the recovery process.
KEY WORDS:
free-tissue flaps, guanidines, thiazoles, gene expression.
DOI: 10.1177/0022034514523785 Received October 8, 2013; Last revision January 13, 2014; Accepted January 18, 2014 A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. © International & American Associations for Dental Research
Introduction
O
riginally introduced by Nabers (Nabers, 1966) the free gingival graft (FGG) technique has been recognized as a reliable procedure for increasing the keratinized mucosa around natural teeth (Agudio et al., 2008) and dental implants (Stimmelmayr et al., 2011). The major complication of FGG is the post-operative morbidity associated with the open palatal wound, including acute pain, excessive hemorrhage, and bone exposure (Griffin et al., 2006). These symptoms and signs are not resolved until the completion of wound epithelialization, which usually takes 2 to 4 wks (Silva et al., 2010). Although the acellular dermal allograft has been developed as a substitute for FGG, to prevent the creation of the palatal wound, the treatment outcome is relatively ineffective and unpredictable (Wei et al., 2000). Thus, the development of therapeutics to accelerate the healing and reduce the complications of the palatal wound is still needed. Advanced glycation end-products (AGE), the irreversible adducts from the non-enzymatic glycosylation of proteins or lipids (Lalla et al., 2001; Abbass et al., 2012), have been found to accumulate in the tissues of animals with diabetes, as well as in physiologically healthy animals in which the periodontal tissues are undergoing progressive inflammation or are in the early repair stage (Chang et al., 2012, 2013a). AGE have been reported to interfere with matrix-cell interactions by altering the cross-linking of the extracellular matrix and impairing wound-healing (Mealey and Oates, 2006). By engaging the cellular receptor for AGE (RAGE), the activation of the AGE-RAGE axis may further increase the oxygen tension, leading to the production of proinflammatory cytokines via the NF-κβ pathway (Wei et al., 2013). In the acute wound, the up-regulation of RAGE signaling perpetuates inflammatory responses and exaggerates cellular responses to cause tissue destruction (Sparvero et al., 2009). To control the AGE-RAGE axis, several glycated cross-link breakers and inhibitors have been used to break glycated collagen cross-links, scavenge AGE precursors, and reduce lipid peroxidation or oxidative stress (Engelen et al., 2012). Aminoguanidine (AG), an inhibitor of AGE and inducible nitric oxidase synthase, has been shown to reduce periodontal and periapical inflammation and accelerate osseointegration (Di Paola et al., 2004; Kopman et al., 2005; Farhad et al., 2011). N-phenacylthiazolium bromide (PTB), a glycated cross-link breaker, has also been shown to prevent diabetic complications (Cooper et al., 2000). Our recent investigations demonstrated that AG
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J Dent Res 93(4) 2014 389 Anti-AGE for Soft-tissue Wound-healing inhibited the induction but did not apparently modulate the progression and recovery phases of periodontitis, presumably related to the limited effect of AG on the highly concentrated, pre-existing AGE at the sites of periodontitis (Chang et al., 2014a). In contrast, PTB inhibited the induction and progression phases and facilitated the recovery phase of periodontitis (Chang et al., 2014b). The main mechanisms of these anti-AGE agents were the inhibition of AGE-associated inflammation and the maintenance of tissue integrity. Since the inactivation of RAGEdependent signaling has also been reported to reduce inflammation and increase matrix synthesis to promote wound-healing (Zhang et al., 2012; Sorci et al., 2013), in this study, we hypothesized that controlling the deposition of AGE may facilitate the healing of palatal wounds in rats. We investigated the regulatory mechanisms of AG and PTB by examining the profile of gene expression and the pattern of wound closure.
Materials & Methods Animal Models All the procedures performed on animals followed approved protocol no. 20130054 from the Institutional Animal Care and Use Committee of the National Taiwan University. In total, 72 systemically healthy male Sprague-Dawley rats (250-300 gm, 9-11 wks old), without any pathology or inflammation noted on the palatal mucosa, were utilized. The study design is illustrated in Fig. 1A, and the sample size was determined by Power Analysis (n = 6/group/time-point). In each rat, bilateral 5.0 x 1.5 mm2 and 1.0-mm-depth full-thickness palatal excisional wounds were created, parallel to the gingival margin of the maxillary molars, by means of a double-bladed scalpel (1.5 mm interblade distance) with 2 No. 15 surgical blades (Fig. 1B). All animals were covered with buprenorphine (0.01 mg/kg) for 7 days and fed standard chow ad libitum post-surgery. Based on the effective dose from the previous studies (Chang et al., 2014a, 2014b), the animals were randomly assigned and received daily intraperitoneal injections of normal saline (control), AG (100 mg/kg), or PTB (5 mg/kg), and were euthanized at 4, 7, 14, and 28 days after the creation of the palatal wound. The bilateral maxillae were harvested, and the tissues within the palatal wound of one randomly selected side of the maxillae were excised for the evaluation of gene expression levels, while the other side of the maxillae was fixed in 10% formaldehyde for histological examination.
Histological Staining After fixation, the maxillae were decalcified with 12.5% ethylenediaminetetraacetic acid, embedded in paraffin, and cut from the cross-sectional plane. Sections (5 μm) from across the mid-wound area were selected and stained with hematoxylin and eosin for descriptive histology and histomorphometry, Masson’s trichrome for collagen deposition, and immunohistochemical staining for AGE deposition and RAGE expression, as previously described (Chang et al., 2013). Each stain was performed in 3 consecutive slides, and the images were acquired by the AxioCam® ERc5s system (Carl Zeiss Microscopy GmbH, Munich, Germany).
Figure 1. The study design and palatal wound model. (A) The study design. In total, 72 animals (6 per group per time-point) and 144 wound sites (2 per animal) were evaluated. (B) The illustration of a palatal wound. A standardized defect was created by means of a double-bladed scalpel with a 1.5-mm inter-blade distance. This figure is available in color online at http://jdr.sagepub.com.
Quantitative Histology The healing of the wound was assessed by the fraction of inflammatory cells/total cells, the status of re-epithelialization and wound closure, the scar elevation index (SEI), the fraction of collagen deposition/lamina propria, the fraction of AGE deposition/newly formed tissue and the fraction of RAGE(+) cells/total cells, evaluated with Image-Pro® Plus software (Media Cybernetics Inc., Rockville, MD, USA). All measurements were made and averaged from three consecutive slides by a blinded examiner (TSC), and the definitions of all examined parameters are listed in the Appendix.
Real-time PCR Analysis The RNA was harvested from the wound area without any unwounded tissue and was transcribed with the iScript-cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA) to yield cDNA, and the mRNA levels of genes were quantitatively analyzed by a real-time PCR system (StepOnePlus, Applied Biosystems, Foster City, CA, USA) and sequence-specific TaqMan gene expression assays (Applied Biosystems) for GAPDH (housekeeping gene of carbohydrate metabolism), β-actin (housekeeping gene of intercellular integrity), heme oxygenase-1 (HO-1, oxidative stress marker), the receptor for advanced glycation end-products (RAGE), tumor necrosis factoralpha (TNF-α), vascular endothelial growth factor (VEGF), fibronectin (an extracellular matrix protein required for cell attachment), periostin (a matricellular protein essential for periodontal tissue integrity) (Rios et al., 2008), and type I collagen (a major extracellular matrix protein in the periodontium). All gene expression
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J Dent Res 93(4) 2014 groups. The wound edges were swollen and reddish at day 4, and wound depression was visible at days 4 and 7, in all groups. The inflammation subsided, and the wound edges were apparently closer in most specimens of the AG and PTB groups at day 7. There was no noticeable difference among the groups after day 14.
Descriptive Histology At day 4, the underlying alveolar bone was exposed without obvious signs of necrosis, and the surface was partially covered by the blood clot and degraded tissue mass in all groups (data not shown). Epithelium approximation was noted in all groups at day 7, and the underlying lamina propria was generally composed of a primitive fiber matrix, with prominent inflammatory cell infiltration, AGE deposition, and occasional sequestrum in the control and AG groups (Figs. 2A, 2B, Appendix Figs. 1, 2). In the PTB group, the wound was closed, characterized by the down-grown and folded epithelium (Fig. 2C). The wound was fully covered by the epithelium, and the lamina propria was hypertrophic and infiltrated with inflammatory cells in all groups at day 14 (Figs. 2D-2F). Sequestrum was noted in most speciFigure 2. Histological photographs under 100x magnification. Scale bar: 100 µm. (A) A mens of the control group, with the wound site of NS-treated (control) animals at day 7. (B) A wound site of AG-treated animals at accumulation of degraded collagen day 7. (C) A wound site of PTB-treated animals at day 7. (D) A wound site of NS-treated matrix on the surface (Fig. 2D). The (control) animals at day 14. (E) A wound site of AG-treated animals at day 14. (F) A wound presence of sequestrum and AGE deposite of PTB-treated animals at day 14. (G) A wound site of NS-treated (control) animals at day sition was reduced, and collagen matrix 28. (H) A wound site of AG-treated animals at day 28. (I) A wound site of PTB-treated animals deposition was more intense, in both the at day 28. The asterisks indicate sequestrum, the arrowhead indicates the degraded tissue AG and PTB groups (Figs. 2E, 2F, mass, and the areas with inflammatory cell infiltration are indicated by arrows. This figure is available in color online at http://jdr.sagepub.com. Appendix Figs. 1, 2). However, inflammatory cell infiltration was still noted on the surface of the alveolar crest (Figs. levels were normalized to the housekeeping gene, respectively, 2E, 2F). At day 28, inflammation had subsided, and the rete pegs and all experiments were performed in triplicate. were prominently extended in all groups (Figs. 2G-2I). However, sequestrum was still present in 3 specimens of the control group Statistical Analysis (Fig. 2G). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to compare the difference between control and Quantitative Histology AG- or PTB-treated groups at each time-point. The data are preThe extents of wound closure and epithelialization were apparsented as the mean ± standard deviation (SD) of measurements, and ently facilitated in the AG and PTB groups relative to the control a p value less than .05 was considered statistically significant. at days 7 and 14 (Figs. 3A, 3B), and the complete wound closure and epithelialization were noted in all groups at day 28 (data not Results shown). The SEI was significantly increased in the AG and PTB groups at day 7 (p < .05, Fig. 3C). The wound was progressively Gross Observation swollen in all groups at day 14 but apparently recessed at All animals recovered well from the anesthesia and intervenday 28. However, a milder hypertrophy was still shown in the tions, and there was no obvious difference in food intake among control group, whereas the wound thickness was significantly Downloaded from jdr.sagepub.com at Universitaetsbibliothek Bern on October 1, 2014 For personal use only. No other uses without permission. © International & American Associations for Dental Research
J Dent Res 93(4) 2014 391 Anti-AGE for Soft-tissue Wound-healing
Figure 3. Quantitative histological measurements. (A-D) The measurements from the specimens of hematoxylin and eosin staining. (A) The extent of wound closure by lamina propria. (B) The extent of re-epithelialization. (C) The scar elevation index (SEI). (D) The fraction of inflammatory cells/ total cells. (E) The fraction of collagen-depositing area/lamina propria from the specimens of Masson’s trichrome staining. (F) The fraction of AGEdepositing area/newly formed tissue of the immunohistochemical staining for AGE. (G) The fraction of RAGE(+) cells/total cells from the specimens of the immunohistochemical staining for RAGE. The definitions of all examined parameters are listed in the Appendix. (AG or PTB group compared with control group at the same time-point: *p < .05; **p < .01.)
lower but close to the level of unwounded tissue in the AG and PTB groups (p < .05, Fig. 3C). Inflammatory cell infiltration was significantly reduced in both the AG and PTB groups relative to the control at day 7 (p < .05, Fig. 3D). Collagen deposition was initialized in all groups at day 7. At day 14, the deposition was dramatically increased in all groups, and the deposited area in the AG and PTB groups was significantly greater, relative to the control group (p < .01, Fig. 3E). There was no noticeable difference among the groups at day 28. The AGE deposition was apparently milder in both the AG and PTB groups relative to the control at days 7 to 28, and a significantly lower level of AGE was noted in the AG (p < .05) and PTB (p < .01) groups at day 7 and in the PTB group (p < .05) at day 14 (Fig. 3F). The fraction of RAGE(+) cells gradually decreased with time in all groups and was significantly lower in both the AG and PTB groups relative to the control at days 7 and 14 (Fig. 3G).
Gene Expression Profiles The fluctuations of all examined genes were generally similar relative to GAPDH and β-actin (Fig. 4, Appendix Fig. 3). The markers of AGE-associated inflammation were generally downregulated in the early stage of wound repair under AG or PTB treatment (Figs. 4A-4C). Specifically, RAGE was significantly down-regulated in the AG (p < .05) and PTB (p < .01) groups at day 7 (Fig. 4A), and a significant reduction in the expression level of HO-1 in both the AG and PTB groups was noted at days 7 and
14 (p < .05, Fig. 4B). The significant down-regulation of TNF-α in both the AG and PTB groups was also noted at day 7 (p < .05, Fig. 4C). Conversely, the markers of tissue repair and homoeostasis were up-regulated within the first 14 days under AG or PTB treatment (Figs. 4D-4F). VEGF was significantly elevated at days 7 and 14 in the AG group (p < .05), but only at day 14 in PTB group (p < .01, Fig. 4D). Additionally, significant up-regulation of periostin, type I collagen, and fibronectin was observed in both the AG and PTB groups at day 14 (Figs. 4E-4G).
Discussion To simulate the donor wound of a free gingival graft, in the present study, a strip-shaped palatal mucosal graft was harvested (Fig. 1B). Although the wounds were relatively small, the pattern of wound closure and histological changes in the control group were similar to those of trans-arch wounds previously described (Kahnberg and Thilander, 1982). The wound underwent the healing sequences of blood clot accumulation, inflammation and granulation tissue formation, approximation of epithelium and connective tissue, and the elimination of damaged tissue elements, followed by ongoing tissue maturation in 4 wks. The healing process was facilitated by the administration of AG or PTB (Figs. 2, 3). Since interference of RAGE signaling has been previously shown to reduce the clinical and biochemical signs of inflammation (Yan et al., 2009), in the present study, systemically administrating anti-AGE agents, including
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J Dent Res 93(4) 2014
Figure 4. Gene expression of the palatal wound (GAPDH as the housekeeping gene). (A) Receptor for advanced glycation end-products (RAGE). (B) Heme oxygenase-1 (HO-1). (C) Tumor necrosis factor-alpha (TNF-α). (D) Vascular endothelial growth factor (VEGF). (E) Periostin. (F) Type I collagen. (G) Fibronectin. All gene expression levels were normalized to GAPDH, and the data are presented as the expression levels relative to the averaged levels of the specimens of the control group at the same time-point. Refer to Appendix Fig. 3 for the gene expression levels relative to β-actin. (AG or PTB group compared with control group at the same time-point: *p < .05, **p < .01, ***p < .001.)
AG and PTB, effectively inhibited RAGE-mediated inflammatory signaling and reduced inflammatory cell infiltration at day 7 (Figs. 3D, 4A, 4C). The down-regulation of HO-1 and upregulation of VEGF in the AG and PTB groups (Figs. 4B, 4D) also indicated the recovery of the hypoxic and ischemic status of the wound (Lokmic et al., 2012). The abundance of blood and oxygen supply has been shown to prevent osteonecrosis (Bejar et al., 2005) and triggered the release of growth factors as well as the recruitment of fibroblasts (Diegelmann and Evans, 2004). As a consequence, matrix synthesis was significantly upregulated in the AG and PTB groups at day 14 (Figs. 4D-4G), resulting in the significant recession of swollen lamina propria and the generation of densely aligned fiber matrix at day 28 (Fig. 3C). Therefore, without the administration of anti-AGE agents, greater AGE deposition and the presence of RAGE(+) cells (Figs. 3F, 3G) may still lead to persistent inflammation and the interference of collagen synthesis and expression in the control group at day 14 (Figs. 3D, 3E, 4F). Because AGE formation is triggered by hyperglycemia or oxidative stress (Nedic et al., 2013) and RAGE signaling is able to activate inflammatory pathways (Wei et al., 2013), the blockade of the AGE-RAGE axis has also been considered to control inflammation in non-diabetic conditions. As shown in our previous studies, both AG and PTB significantly retarded the induction of experimental periodontitis in non-diabetic animals (Chang et al., 2014a, 2014b). Since AGE deposition was evident during palatal wound-healing in the control group (Fig. 3F), the reduction of AGE deposition (Fig. 3F) and the down-regulation of
RAGE (Fig. 4A) confirmed the anti-AGE effects of AG and PTB. The down-regulation of TNF-α (Fig. 4C) also confirmed that the activation of the AGE-RAGE axis was in parallel with the progression of inflammation (Chang et al., 2012, 2013). However, without the infection from exogenous pathogens, inflammation was elicited only in the initial stages and gradually receded in the control group (Fig. 3D), implying that AGE deposition was milder relative to the conditions of experimental periodontitis (Chang et al., 2014a), and the inhibition of the AGE-RAGE axis by the anti-AGE agents became negligible after day 14 (Figs. 3G, 3H, 4A). Given that inflammation and AGE deposition were initiated after wound creation, the de-activation of the AGE-RAGE axis occurred in both the AG and PTB groups and consequently promoted wound healing. To further distinguish among the differential mechanisms of these agents, the prescription of AG or PTB, after inflammation is established, might be necessary. The main limitation of the study is that the partial-thickness graft was not achievable because of the limited thickness of the rat palate mucosa, and the exposure of the underlying bone might increase the incidence of wound morbidity or osteonecrosis. Additionally, the healing dynamics observed in animals might not be directly relevant to humans. Although no systemic adverse effects of the administration of the anti-AGE agents were noted in the present study, complications such as glomerulonephritis and cardiovascular symptoms have been reported in previous studies (Goh and Cooper, 2008; Hartog et al., 2011). Thus, the development of a local delivery vehicle to reduce the systematic dosage of anti-AGE agents is still needed.
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J Dent Res 93(4) 2014 393 Anti-AGE for Soft-tissue Wound-healing Within the limitations of the study, we conclude that the antiAGE agents AG and PTB facilitated the healing of palatal wounds via the inhibition of the AGE-RAGE axis. Further investigations in a more clinically relevant model and the development of a local delivery vehicle for anti-AGE agents are warranted.
Acknowledgments The authors acknowledge the technical support from the Histopathological Laboratory of the Department of Pathology, National Taiwan University Hospital, for assisting with the staining of histological slides. The study was supported by research grant 102-2314-B-002-003 from the National Science Council (Taiwan). The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
References Abbass MM, Korany NS, Salama AH, Dmytryk JJ, Safiejko-Mroczka B (2012). The relationship between receptor for advanced glycation end products expression and the severity of periodontal disease in the gingiva of diabetic and non diabetic periodontitis patients. Arch Oral Biol 57:1342-1354. Agudio G, Nieri M, Rotundo R, Cortellini P, Pini Prato G (2008). Free gingival grafts to increase keratinized tissue: a retrospective long-term evaluation (10 to 25 years) of outcomes. J Periodontol 79:587-594. Bejar J, Peled E, Boss JH (2005). Vasculature deprivation-induced osteonecrosis of the rat femoral head as a model for therapeutic trials. Theor Biol Med Model 2:24. Chang PC, Chung MC, Wang YP, Chien LY, Lim JC, Liang K, et al. (2012). Patterns of diabetic periodontal wound repair: a study using microcomputed tomography and immunohistochemistry. J Periodontol 83:644-652. Chang PC, Chien LY, Yeo JF, Wang YP, Chung MC, Chong LY, et al. (2013). Progression of periodontal destruction and the roles of advanced glycation end products in experimental diabetes. J Periodontol 84:379-388. Chang PC, Chong LY, Tsai SC, Lim LP (2014a). Aminoguanidine inhibits the AGE-RAGE axis to modulate the induction of periodontitis but has limited effects on the progression and recovery of experimental periodontitis: a preliminary study. J Periodontol [Epub ahead of print 7/15/2013] (in press). Chang PC, Tsai SC, Chong LY, Kao MJ (2014b). N-phenacylthiazolium bromide inhibits the AGE-RAGE axis to modulate experimental periodontitis in rats. J Periodontol [Epub ahead of print 2/7/2014] (in press). Cooper ME, Thallas V, Forbes J, Scalbert E, Sastra S, Darby I, et al. (2000). The cross-link breaker, N-phenacylthiazolium bromide prevents vascular advanced glycation end-product accumulation. Diabetologia 43:660-664. Di Paola R, Marzocco S, Mazzon E, Dattola F, Rotondo F, Britti D, et al. (2004). Effect of aminoguanidine in ligature-induced periodontitis in rats. J Dent Res 83:343-348. Diegelmann RF, Evans MC (2004). Wound healing: an overview of acute, fibrotic and delayed healing. Front Biosci 9:283-289. Engelen L, Stehouwer CD, Schalkwijk CG (2012). Current therapeutic interventions in the glycation pathway: evidence from clinical studies. Diabetes Obes Metab 15:677-689.
Farhad AR, Razavi S, Jahadi S, Saatchi M (2011). Use of aminoguanidine, a selective inducible nitric oxide synthase inhibitor, to evaluate the role of nitric oxide in periapical inflammation. J Oral Sci 53:225-230. Goh SY, Cooper ME (2008). Clinical review: the role of advanced glycation end products in progression and complications of diabetes. J Clin Endocrinol Metab 93:1143-1152. Griffin TJ, Cheung WS, Zavras AI, Damoulis PD (2006). Postoperative complications following gingival augmentation procedures. J Periodontol 77:2070-2079. Hartog JW, Willemsen S, van Veldhuisen DJ, Posma JL, van Wijk LM, Hummel YM, et al. (2011). Effects of alagebrium, an advanced glycation endproduct breaker, on exercise tolerance and cardiac function in patients with chronic heart failure. Eur J Heart Fail 13:899-908. Kahnberg KE, Thilander H (1982). Healing of experimental excisional wounds in the rat palate. (I) Histological study of the interphase in wound healing after sharp dissection. Int J Oral Surg 11:44-51. Kopman JA, Kim DM, Rahman SS, Arandia JA, Karimbux NY, Fiorellini JP (2005). Modulating the effects of diabetes on osseointegration with aminoguanidine and doxycycline. J Periodontol 76:614-620. Lalla E, Lamster IB, Stern DM, Schmidt AM (2001). Receptor for advanced glycation end products, inflammation, and accelerated periodontal disease in diabetes: mechanisms and insights into therapeutic modalities. Ann Periodontol 6:113-118. Lokmic Z, Musyoka J, Hewitson TD, Darby IA (2012). Hypoxia and hypoxia signaling in tissue repair and fibrosis. Int Rev Cell Mol Biol 296:139-185. Mealey BL, Oates TW (2006). Diabetes mellitus and periodontal diseases. J Periodontol 77:1289-1303. Nabers JM (1966). Free gingival grafts. Periodontics 4:243-245. Nedic O, Rattan SI, Grune T, Trougakos IP (2013). Molecular effects of advanced glycation end products (AGEs) on cell signalling pathways, ageing and pathophysiology. Free Radic Res 47(Suppl 1):28-38. Rios HF, Ma D, Xie Y, Giannobile WV, Bonewald LF, Conway SJ, et al. (2008). Periostin is essential for the integrity and function of the periodontal ligament during occlusal loading in mice. J Periodontol 79:1480-1490. Silva CO, Ribeiro Edel P, Sallum AW, Tatakis DN (2010). Free gingival grafts: graft shrinkage and donor-site healing in smokers and nonsmokers. J Periodontol 81:692-701. Sorci G, Riuzzi F, Giambanco I, Donato R (2013). RAGE in tissue homeostasis, repair and regeneration. Biochim Biophys Acta 1833:101-109. Sparvero LJ, Asafu-Adjei D, Kang R, Tang D, Amin N, Im J, et al. (2009). RAGE (Receptor for Advanced Glycation Endproducts), RAGE ligands, and their role in cancer and inflammation. J Translational Med 7:17. Stimmelmayr M, Stangl M, Edelhoff D, Beuer F (2011). Clinical prospective study of a modified technique to extend the keratinized gingiva around implants in combination with ridge augmentation: one-year results. Int J Oral Maxillofac Implants 26:1094-1101. Wei PC, Laurell L, Geivelis M, Lingen MW, Maddalozzo D (2000). Acellular dermal matrix allografts to achieve increased attached gingiva. Part 1. A clinical study. J Periodontol 71:1297-1305. Wei Q, Ren X, Jiang Y, Jin H, Liu N, Li J (2013). Advanced glycation end products accelerate rat vascular calcification through RAGE/oxidative stress. BMC Cardiovasc Disord 13:13. Yan SF, Yan SD, Ramasamy R, Schmidt AM (2009). Tempering the wrath of RAGE: an emerging therapeutic strategy against diabetic complications, neurodegeneration, and inflammation. Ann Med 41:408-422. Zhang Q, O’Hearn S, Kavalukas SL, Barbul A (2012). Role of high mobility group box 1 (HMGB1) in wound healing. J Surg Res 176:343-347.
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Soft-tissue Wound Healing by Anti-advanced Glycation End-products Agents P.-C. Chang, S.-C. Tsai, Y.-H. Jheng, Y.-F. Lin and C.-C. Chen J DENT RES 2014 93: 388 originally published online 19 February 2014 DOI: 10.1177/0022034514523785 The online version of this article can be found at: http://jdr.sagepub.com/content/93/4/388
Published by: http://www.sagepublications.com
On behalf of: International and American Associations for Dental Research
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research-article2014
JDR
93410.1177/0022034514523785
RESEARCH REPORTS Biological
P.-C. Chang1,2*, S.-C. Tsai3, Y.-H. Jheng1, Y.-F. Lin4, and C.-C. Chen4 1
Graduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University, Taipei, Taiwan; 2Department of Dentistry, National Taiwan University Hospital, Taipei, Taiwan; 3Faculty of Dentistry, National University of Singapore, Singapore, Singapore; and 4Taiwan Mouse Clinics -National Phenotyping and Drug Testing Center, National Research Program for Biopharmaceuticals, National Science Council, Taipei, Taiwan; *corresponding author, changpc@ ntu.edu.tw
Soft-tissue Wound Healing by Anti-advanced Glycation Endproducts Agents
J Dent Res 93(4):388-393, 2014
Abstract
The blocking of advanced glycation end-products (AGE) has been shown to reduce diabetic complications and control periodontitis. This study investigated the pattern of palatal wound-healing after graft harvesting under the administration of aminoguanidine (AG), an AGE inhibitor, or N-phenacylthiazolium bromide (PTB), a glycated cross-link breaker. Full-thickness palatal excisional wounds (5.0 x 1.5 mm2) were created in 72 SpragueDawley rats. The rats received daily intraperitoneal injections of normal saline (control), AG, or PTB and were euthanized after 4 to 28 days. The wound-healing pattern was assessed by histology, histochemistry for collagen matrix deposition, immunohistochemistry for AGE and the AGE receptor (RAGE), and the expression of RAGE, as well as inflammation- and recovery-associated genes. In the first 14 days following AG or PTB treatments, wound closure, re-epithelialization, and collagen matrix deposition were accelerated, whereas AGE deposition, RAGE-positive cells, and inflammation were reduced. RAGE and tumor necrosis factoralpha were significantly down-regulated at day 7, and heme oxygenase-1 was persistently down-regulated until day 14. The levels of vascular endothelial growth factor, periostin, type I collagen, and fibronectin were all increased at day 14. In conclusion, anti-AGE agents appeared to facilitate palatal wound-healing by reducing AGE-associated inflammation and promoting the recovery process.
KEY WORDS:
free-tissue flaps, guanidines, thiazoles, gene expression.
DOI: 10.1177/0022034514523785 Received October 8, 2013; Last revision January 13, 2014; Accepted January 18, 2014 A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. © International & American Associations for Dental Research
Introduction
O
riginally introduced by Nabers (Nabers, 1966) the free gingival graft (FGG) technique has been recognized as a reliable procedure for increasing the keratinized mucosa around natural teeth (Agudio et al., 2008) and dental implants (Stimmelmayr et al., 2011). The major complication of FGG is the post-operative morbidity associated with the open palatal wound, including acute pain, excessive hemorrhage, and bone exposure (Griffin et al., 2006). These symptoms and signs are not resolved until the completion of wound epithelialization, which usually takes 2 to 4 wks (Silva et al., 2010). Although the acellular dermal allograft has been developed as a substitute for FGG, to prevent the creation of the palatal wound, the treatment outcome is relatively ineffective and unpredictable (Wei et al., 2000). Thus, the development of therapeutics to accelerate the healing and reduce the complications of the palatal wound is still needed. Advanced glycation end-products (AGE), the irreversible adducts from the non-enzymatic glycosylation of proteins or lipids (Lalla et al., 2001; Abbass et al., 2012), have been found to accumulate in the tissues of animals with diabetes, as well as in physiologically healthy animals in which the periodontal tissues are undergoing progressive inflammation or are in the early repair stage (Chang et al., 2012, 2013a). AGE have been reported to interfere with matrix-cell interactions by altering the cross-linking of the extracellular matrix and impairing wound-healing (Mealey and Oates, 2006). By engaging the cellular receptor for AGE (RAGE), the activation of the AGE-RAGE axis may further increase the oxygen tension, leading to the production of proinflammatory cytokines via the NF-κβ pathway (Wei et al., 2013). In the acute wound, the up-regulation of RAGE signaling perpetuates inflammatory responses and exaggerates cellular responses to cause tissue destruction (Sparvero et al., 2009). To control the AGE-RAGE axis, several glycated cross-link breakers and inhibitors have been used to break glycated collagen cross-links, scavenge AGE precursors, and reduce lipid peroxidation or oxidative stress (Engelen et al., 2012). Aminoguanidine (AG), an inhibitor of AGE and inducible nitric oxidase synthase, has been shown to reduce periodontal and periapical inflammation and accelerate osseointegration (Di Paola et al., 2004; Kopman et al., 2005; Farhad et al., 2011). N-phenacylthiazolium bromide (PTB), a glycated cross-link breaker, has also been shown to prevent diabetic complications (Cooper et al., 2000). Our recent investigations demonstrated that AG
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J Dent Res 93(4) 2014 389 Anti-AGE for Soft-tissue Wound-healing inhibited the induction but did not apparently modulate the progression and recovery phases of periodontitis, presumably related to the limited effect of AG on the highly concentrated, pre-existing AGE at the sites of periodontitis (Chang et al., 2014a). In contrast, PTB inhibited the induction and progression phases and facilitated the recovery phase of periodontitis (Chang et al., 2014b). The main mechanisms of these anti-AGE agents were the inhibition of AGE-associated inflammation and the maintenance of tissue integrity. Since the inactivation of RAGEdependent signaling has also been reported to reduce inflammation and increase matrix synthesis to promote wound-healing (Zhang et al., 2012; Sorci et al., 2013), in this study, we hypothesized that controlling the deposition of AGE may facilitate the healing of palatal wounds in rats. We investigated the regulatory mechanisms of AG and PTB by examining the profile of gene expression and the pattern of wound closure.
Materials & Methods Animal Models All the procedures performed on animals followed approved protocol no. 20130054 from the Institutional Animal Care and Use Committee of the National Taiwan University. In total, 72 systemically healthy male Sprague-Dawley rats (250-300 gm, 9-11 wks old), without any pathology or inflammation noted on the palatal mucosa, were utilized. The study design is illustrated in Fig. 1A, and the sample size was determined by Power Analysis (n = 6/group/time-point). In each rat, bilateral 5.0 x 1.5 mm2 and 1.0-mm-depth full-thickness palatal excisional wounds were created, parallel to the gingival margin of the maxillary molars, by means of a double-bladed scalpel (1.5 mm interblade distance) with 2 No. 15 surgical blades (Fig. 1B). All animals were covered with buprenorphine (0.01 mg/kg) for 7 days and fed standard chow ad libitum post-surgery. Based on the effective dose from the previous studies (Chang et al., 2014a, 2014b), the animals were randomly assigned and received daily intraperitoneal injections of normal saline (control), AG (100 mg/kg), or PTB (5 mg/kg), and were euthanized at 4, 7, 14, and 28 days after the creation of the palatal wound. The bilateral maxillae were harvested, and the tissues within the palatal wound of one randomly selected side of the maxillae were excised for the evaluation of gene expression levels, while the other side of the maxillae was fixed in 10% formaldehyde for histological examination.
Histological Staining After fixation, the maxillae were decalcified with 12.5% ethylenediaminetetraacetic acid, embedded in paraffin, and cut from the cross-sectional plane. Sections (5 μm) from across the mid-wound area were selected and stained with hematoxylin and eosin for descriptive histology and histomorphometry, Masson’s trichrome for collagen deposition, and immunohistochemical staining for AGE deposition and RAGE expression, as previously described (Chang et al., 2013). Each stain was performed in 3 consecutive slides, and the images were acquired by the AxioCam® ERc5s system (Carl Zeiss Microscopy GmbH, Munich, Germany).
Figure 1. The study design and palatal wound model. (A) The study design. In total, 72 animals (6 per group per time-point) and 144 wound sites (2 per animal) were evaluated. (B) The illustration of a palatal wound. A standardized defect was created by means of a double-bladed scalpel with a 1.5-mm inter-blade distance. This figure is available in color online at http://jdr.sagepub.com.
Quantitative Histology The healing of the wound was assessed by the fraction of inflammatory cells/total cells, the status of re-epithelialization and wound closure, the scar elevation index (SEI), the fraction of collagen deposition/lamina propria, the fraction of AGE deposition/newly formed tissue and the fraction of RAGE(+) cells/total cells, evaluated with Image-Pro® Plus software (Media Cybernetics Inc., Rockville, MD, USA). All measurements were made and averaged from three consecutive slides by a blinded examiner (TSC), and the definitions of all examined parameters are listed in the Appendix.
Real-time PCR Analysis The RNA was harvested from the wound area without any unwounded tissue and was transcribed with the iScript-cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA) to yield cDNA, and the mRNA levels of genes were quantitatively analyzed by a real-time PCR system (StepOnePlus, Applied Biosystems, Foster City, CA, USA) and sequence-specific TaqMan gene expression assays (Applied Biosystems) for GAPDH (housekeeping gene of carbohydrate metabolism), β-actin (housekeeping gene of intercellular integrity), heme oxygenase-1 (HO-1, oxidative stress marker), the receptor for advanced glycation end-products (RAGE), tumor necrosis factoralpha (TNF-α), vascular endothelial growth factor (VEGF), fibronectin (an extracellular matrix protein required for cell attachment), periostin (a matricellular protein essential for periodontal tissue integrity) (Rios et al., 2008), and type I collagen (a major extracellular matrix protein in the periodontium). All gene expression
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J Dent Res 93(4) 2014 groups. The wound edges were swollen and reddish at day 4, and wound depression was visible at days 4 and 7, in all groups. The inflammation subsided, and the wound edges were apparently closer in most specimens of the AG and PTB groups at day 7. There was no noticeable difference among the groups after day 14.
Descriptive Histology At day 4, the underlying alveolar bone was exposed without obvious signs of necrosis, and the surface was partially covered by the blood clot and degraded tissue mass in all groups (data not shown). Epithelium approximation was noted in all groups at day 7, and the underlying lamina propria was generally composed of a primitive fiber matrix, with prominent inflammatory cell infiltration, AGE deposition, and occasional sequestrum in the control and AG groups (Figs. 2A, 2B, Appendix Figs. 1, 2). In the PTB group, the wound was closed, characterized by the down-grown and folded epithelium (Fig. 2C). The wound was fully covered by the epithelium, and the lamina propria was hypertrophic and infiltrated with inflammatory cells in all groups at day 14 (Figs. 2D-2F). Sequestrum was noted in most speciFigure 2. Histological photographs under 100x magnification. Scale bar: 100 µm. (A) A mens of the control group, with the wound site of NS-treated (control) animals at day 7. (B) A wound site of AG-treated animals at accumulation of degraded collagen day 7. (C) A wound site of PTB-treated animals at day 7. (D) A wound site of NS-treated matrix on the surface (Fig. 2D). The (control) animals at day 14. (E) A wound site of AG-treated animals at day 14. (F) A wound presence of sequestrum and AGE deposite of PTB-treated animals at day 14. (G) A wound site of NS-treated (control) animals at day sition was reduced, and collagen matrix 28. (H) A wound site of AG-treated animals at day 28. (I) A wound site of PTB-treated animals deposition was more intense, in both the at day 28. The asterisks indicate sequestrum, the arrowhead indicates the degraded tissue AG and PTB groups (Figs. 2E, 2F, mass, and the areas with inflammatory cell infiltration are indicated by arrows. This figure is available in color online at http://jdr.sagepub.com. Appendix Figs. 1, 2). However, inflammatory cell infiltration was still noted on the surface of the alveolar crest (Figs. levels were normalized to the housekeeping gene, respectively, 2E, 2F). At day 28, inflammation had subsided, and the rete pegs and all experiments were performed in triplicate. were prominently extended in all groups (Figs. 2G-2I). However, sequestrum was still present in 3 specimens of the control group Statistical Analysis (Fig. 2G). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to compare the difference between control and Quantitative Histology AG- or PTB-treated groups at each time-point. The data are preThe extents of wound closure and epithelialization were apparsented as the mean ± standard deviation (SD) of measurements, and ently facilitated in the AG and PTB groups relative to the control a p value less than .05 was considered statistically significant. at days 7 and 14 (Figs. 3A, 3B), and the complete wound closure and epithelialization were noted in all groups at day 28 (data not Results shown). The SEI was significantly increased in the AG and PTB groups at day 7 (p < .05, Fig. 3C). The wound was progressively Gross Observation swollen in all groups at day 14 but apparently recessed at All animals recovered well from the anesthesia and intervenday 28. However, a milder hypertrophy was still shown in the tions, and there was no obvious difference in food intake among control group, whereas the wound thickness was significantly Downloaded from jdr.sagepub.com at Universitaetsbibliothek Bern on October 1, 2014 For personal use only. No other uses without permission. © International & American Associations for Dental Research
J Dent Res 93(4) 2014 391 Anti-AGE for Soft-tissue Wound-healing
Figure 3. Quantitative histological measurements. (A-D) The measurements from the specimens of hematoxylin and eosin staining. (A) The extent of wound closure by lamina propria. (B) The extent of re-epithelialization. (C) The scar elevation index (SEI). (D) The fraction of inflammatory cells/ total cells. (E) The fraction of collagen-depositing area/lamina propria from the specimens of Masson’s trichrome staining. (F) The fraction of AGEdepositing area/newly formed tissue of the immunohistochemical staining for AGE. (G) The fraction of RAGE(+) cells/total cells from the specimens of the immunohistochemical staining for RAGE. The definitions of all examined parameters are listed in the Appendix. (AG or PTB group compared with control group at the same time-point: *p < .05; **p < .01.)
lower but close to the level of unwounded tissue in the AG and PTB groups (p < .05, Fig. 3C). Inflammatory cell infiltration was significantly reduced in both the AG and PTB groups relative to the control at day 7 (p < .05, Fig. 3D). Collagen deposition was initialized in all groups at day 7. At day 14, the deposition was dramatically increased in all groups, and the deposited area in the AG and PTB groups was significantly greater, relative to the control group (p < .01, Fig. 3E). There was no noticeable difference among the groups at day 28. The AGE deposition was apparently milder in both the AG and PTB groups relative to the control at days 7 to 28, and a significantly lower level of AGE was noted in the AG (p < .05) and PTB (p < .01) groups at day 7 and in the PTB group (p < .05) at day 14 (Fig. 3F). The fraction of RAGE(+) cells gradually decreased with time in all groups and was significantly lower in both the AG and PTB groups relative to the control at days 7 and 14 (Fig. 3G).
Gene Expression Profiles The fluctuations of all examined genes were generally similar relative to GAPDH and β-actin (Fig. 4, Appendix Fig. 3). The markers of AGE-associated inflammation were generally downregulated in the early stage of wound repair under AG or PTB treatment (Figs. 4A-4C). Specifically, RAGE was significantly down-regulated in the AG (p < .05) and PTB (p < .01) groups at day 7 (Fig. 4A), and a significant reduction in the expression level of HO-1 in both the AG and PTB groups was noted at days 7 and
14 (p < .05, Fig. 4B). The significant down-regulation of TNF-α in both the AG and PTB groups was also noted at day 7 (p < .05, Fig. 4C). Conversely, the markers of tissue repair and homoeostasis were up-regulated within the first 14 days under AG or PTB treatment (Figs. 4D-4F). VEGF was significantly elevated at days 7 and 14 in the AG group (p < .05), but only at day 14 in PTB group (p < .01, Fig. 4D). Additionally, significant up-regulation of periostin, type I collagen, and fibronectin was observed in both the AG and PTB groups at day 14 (Figs. 4E-4G).
Discussion To simulate the donor wound of a free gingival graft, in the present study, a strip-shaped palatal mucosal graft was harvested (Fig. 1B). Although the wounds were relatively small, the pattern of wound closure and histological changes in the control group were similar to those of trans-arch wounds previously described (Kahnberg and Thilander, 1982). The wound underwent the healing sequences of blood clot accumulation, inflammation and granulation tissue formation, approximation of epithelium and connective tissue, and the elimination of damaged tissue elements, followed by ongoing tissue maturation in 4 wks. The healing process was facilitated by the administration of AG or PTB (Figs. 2, 3). Since interference of RAGE signaling has been previously shown to reduce the clinical and biochemical signs of inflammation (Yan et al., 2009), in the present study, systemically administrating anti-AGE agents, including
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J Dent Res 93(4) 2014
Figure 4. Gene expression of the palatal wound (GAPDH as the housekeeping gene). (A) Receptor for advanced glycation end-products (RAGE). (B) Heme oxygenase-1 (HO-1). (C) Tumor necrosis factor-alpha (TNF-α). (D) Vascular endothelial growth factor (VEGF). (E) Periostin. (F) Type I collagen. (G) Fibronectin. All gene expression levels were normalized to GAPDH, and the data are presented as the expression levels relative to the averaged levels of the specimens of the control group at the same time-point. Refer to Appendix Fig. 3 for the gene expression levels relative to β-actin. (AG or PTB group compared with control group at the same time-point: *p < .05, **p < .01, ***p < .001.)
AG and PTB, effectively inhibited RAGE-mediated inflammatory signaling and reduced inflammatory cell infiltration at day 7 (Figs. 3D, 4A, 4C). The down-regulation of HO-1 and upregulation of VEGF in the AG and PTB groups (Figs. 4B, 4D) also indicated the recovery of the hypoxic and ischemic status of the wound (Lokmic et al., 2012). The abundance of blood and oxygen supply has been shown to prevent osteonecrosis (Bejar et al., 2005) and triggered the release of growth factors as well as the recruitment of fibroblasts (Diegelmann and Evans, 2004). As a consequence, matrix synthesis was significantly upregulated in the AG and PTB groups at day 14 (Figs. 4D-4G), resulting in the significant recession of swollen lamina propria and the generation of densely aligned fiber matrix at day 28 (Fig. 3C). Therefore, without the administration of anti-AGE agents, greater AGE deposition and the presence of RAGE(+) cells (Figs. 3F, 3G) may still lead to persistent inflammation and the interference of collagen synthesis and expression in the control group at day 14 (Figs. 3D, 3E, 4F). Because AGE formation is triggered by hyperglycemia or oxidative stress (Nedic et al., 2013) and RAGE signaling is able to activate inflammatory pathways (Wei et al., 2013), the blockade of the AGE-RAGE axis has also been considered to control inflammation in non-diabetic conditions. As shown in our previous studies, both AG and PTB significantly retarded the induction of experimental periodontitis in non-diabetic animals (Chang et al., 2014a, 2014b). Since AGE deposition was evident during palatal wound-healing in the control group (Fig. 3F), the reduction of AGE deposition (Fig. 3F) and the down-regulation of
RAGE (Fig. 4A) confirmed the anti-AGE effects of AG and PTB. The down-regulation of TNF-α (Fig. 4C) also confirmed that the activation of the AGE-RAGE axis was in parallel with the progression of inflammation (Chang et al., 2012, 2013). However, without the infection from exogenous pathogens, inflammation was elicited only in the initial stages and gradually receded in the control group (Fig. 3D), implying that AGE deposition was milder relative to the conditions of experimental periodontitis (Chang et al., 2014a), and the inhibition of the AGE-RAGE axis by the anti-AGE agents became negligible after day 14 (Figs. 3G, 3H, 4A). Given that inflammation and AGE deposition were initiated after wound creation, the de-activation of the AGE-RAGE axis occurred in both the AG and PTB groups and consequently promoted wound healing. To further distinguish among the differential mechanisms of these agents, the prescription of AG or PTB, after inflammation is established, might be necessary. The main limitation of the study is that the partial-thickness graft was not achievable because of the limited thickness of the rat palate mucosa, and the exposure of the underlying bone might increase the incidence of wound morbidity or osteonecrosis. Additionally, the healing dynamics observed in animals might not be directly relevant to humans. Although no systemic adverse effects of the administration of the anti-AGE agents were noted in the present study, complications such as glomerulonephritis and cardiovascular symptoms have been reported in previous studies (Goh and Cooper, 2008; Hartog et al., 2011). Thus, the development of a local delivery vehicle to reduce the systematic dosage of anti-AGE agents is still needed.
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J Dent Res 93(4) 2014 393 Anti-AGE for Soft-tissue Wound-healing Within the limitations of the study, we conclude that the antiAGE agents AG and PTB facilitated the healing of palatal wounds via the inhibition of the AGE-RAGE axis. Further investigations in a more clinically relevant model and the development of a local delivery vehicle for anti-AGE agents are warranted.
Acknowledgments The authors acknowledge the technical support from the Histopathological Laboratory of the Department of Pathology, National Taiwan University Hospital, for assisting with the staining of histological slides. The study was supported by research grant 102-2314-B-002-003 from the National Science Council (Taiwan). The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
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