Influence of Reactive Oxygen Species on Wound Healing and Tissue Regeneration in Periodontal and Peri-Implant Tissues in Diabetic Patients.

Diabetes mellitus (DM) is associated with periodontal disease. Clinically, periodontal treatment is less effective for patients with DM. Oxidative stress is one of the mechanisms that link DM to periodontitis. The production of reactive oxygen species (ROS) is increased in the periodontal tissues of patients with DM and is involved in the development of insulin resistance in periodontal tissues. Insulin resistance decreases Akt activation and inhibits cell proliferation and angiogenesis. This results in the deterioration of wound healing and tissue repair in periodontal tissues. Antioxidants and insulin resistance ameliorants may inhibit ROS production and improve wound healing, which is worsened by DM. This manuscript provides a comprehensive review of the most recent basic and clinical evidence regarding the generation of ROS in periodontal tissues resulting from microbial challenge and DM. This study also delves into the impact of oxidative stress on wound healing in the context of periodontal and dental implant therapies. Furthermore, it discusses the potential benefits of administering antioxidants and anti-insulin resistance medications, which have been shown to counteract ROS production and inflammation. This approach may potentially enhance wound healing, especially in cases exacerbated by hyperglycemic conditions.

Impaired dental implant osseointegration in rat with streptozotocin-induced diabetes.

OBJECTIVE: Few studies have reported on the impact of oxidative stress on the dental implant failure. The aim of this study was to investigate the impact of hyperglycemia-induced oxidative stress on dental implant osseointegration in diabetes mellitus (DM). METHODS: Acid-treated titanium implants were bilaterally placed in the maxillary alveolar ridge of streptozotocin-induced diabetic (DM group) and control rats after extraction of first molars. Histological analysis and micro-push-out test were performed 4 weeks after surgery. Oxidative stress and osteogenic markers in the surrounding bone were quantified by real-time polymerase chain reaction. In the in vitro study, rat bone marrow-derived mesenchymal stem cells (BMMSCs) were cultured on acid-treated titanium discs in a high-glucose (HG) or normal environment. Intracellular reactive oxygen species (ROS), cell proliferation, alkaline phosphatase (ALP) activity, and extracellular calcification were evaluated following antioxidant treatment with N-acetyl-L-cysteine (NAC). RESULTS: The implant survival rate was 92.9% and 75.0% in control and DM group, respectively. Bone-implant contact and push-out loads were significantly lower in the DM group. Expression of superoxide dismutase 1 at the mRNA level and on immunohistochemistry was significantly lower in the DM group. In vitro experiments revealed that the HG condition significantly increased ROS expression and suppressed the proliferation and extracellular calcification of BMMSCs, while NAC treatment significantly restored ROS expression, cell proliferation, and calcification. The ALP activity of both groups was not significantly different. CONCLUSION: In diabetes, high-glucose-induced oxidative stress downregulates proliferation and calcification of BMMSCs, impairing osseointegration and leading to implant failure.

Metformin accelerates wound healing by Akt phosphorylation of gingival fibroblasts in insulin-resistant prediabetes mice.

BACKGROUND: This study aimed to investigate the effects of metformin on gingival wound healing in insulin-resistant prediabetes. METHODS: C57BL/6J mice were fed normal diet (ND) or high-fat diet (HFD) for 10 weeks; half of the HFD mice were treated with metformin (HFD+ Met) for the last 2 weeks. Insulin and glucose tolerance tests were performed. The palatal gingiva (2.0 × 0.5 mm) was surgically removed adjacent to the maxillary molars. Post-surgical wound closure was histomorphometrically evaluated for 1 week. The mRNA expression of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in the tissue were quantified by real-time polymerase chain reaction. In vitro, the proliferation and migration of human gingival fibroblasts (HGFs) cultured under high-glucose or control conditions with/without metformin were analyzed. Akt phosphorylation and VEGF expression following the insulin stimulation were evaluated with/without metformin in high-glucose or control media. RESULTS: HFD mice showed significantly higher plasma glucose levels and insulin resistance than ND mice. Gingival wound healing was delayed in HFD group compared with ND group but significantly improved in HFD + MET group. The decreased expression of VEGF and eNOS in HFD group was significantly elevated in the HFD + MET group. The proliferation and migration of HGFs were significantly impaired in high-glucose conditions compared with control; metformin treatment partially attenuated these effects. Metformin treatment significantly recovered the downregulated Akt phosphorylation and VEGF expression in high-glucose conditions. CONCLUSIONS: Metformin improved delayed gingival wound healing in insulin-resistant prediabetes by accelerating HGFs proliferation and migration via Akt phosphorylation in insulin signaling pathway.

Antioxidant effect of enamel matrix derivative for early phase of periodontal tissue regeneration in diabetes.

BACKGROUND: Diabetes involves metabolic disorders in various tissues via hyperglycemia-induced oxidative stress. This study aimed to investigate the antioxidative effect of enamel matrix derivative (EMD) on periodontal regeneration in diabetes. METHODS: Twenty-two rats were equally divided into streptozotocin (STZ)-induced diabetes or control group. Two months after induction of hyperglycemia, systemic oxidative stress was measured using urinary 8-hydroxy-2'-deoxyguanosine. EMD or saline was applied into the intrabony defects created in the bilateral maxillary molar. mRNA expressions of inflammatory and oxidative stress markers were quantified (n = 6). Histometric analyses and immunohistochemistry of superoxide dismutase-1 (SOD-1) were performed 7 days postoperatively (n = 5). For in vitro experiments, the bone marrow-derived mesenchymal stem cells were isolated from rat femur and cultured in a high glucose (HG) or control medium. Reactive oxygen species (ROS) measurement and alizarin red staining were performed with/without EMD. RESULTS: Systemic oxidative stress was significantly higher in the diabetic group. The connective tissue attachment and cementum formation were significantly increased at EMD-treated sites in both diabetic and non-diabetic groups. The expression of nicotinamide adenine dinucleotide phosphate oxidase two and four was significantly lower at EMD-treated sites than at EMD-untreated sites in both diabetic and non-diabetic rats. Immunohistochemistry showed significantly higher SOD-1 expression at the EMD-treated site. In vitro, HG culture had significantly higher ROS production compared with control, which was downregulated by EMD. EMD treatment significantly recovered the impaired calcification in HG. CONCLUSION: EMD promoted early-phase wound healing and periodontal tissue regeneration in the surgically created bony defect of STZ-induced diabetic rat by suppressing hyperglycemia-induced oxidative stress.

Effects of Antioxidant in Adjunct with Periodontal Therapy in Patients with Type 2 Diabetes: A Systematic Review and Meta-Analysis.

This review investigated whether the adjunctive use of antioxidants with periodontal therapy improves periodontal parameters in patients with type 2 diabetes. A systematic and extensive literature search for randomized controlled trials (RCTs) conducted before April 2021 was performed on the PubMed, Cochrane Library, and Web of Science databases. The risk of bias was assessed using the Cochrane risk-of-bias tool. A meta-analysis was performed to quantitatively evaluate the clinical outcomes following periodontal therapy. After independent screening of 137 initial records, nine records from eight RCTs were included. The risk-of-bias assessment revealed that all RCTs had methodological weaknesses regarding selective bias, although other risk factors for bias were not evident. This meta-analysis of two RCTs showed that periodontal pocket depths were significantly reduced in the groups treated with combined non-surgical periodontal therapy and melatonin than in those treated with non-surgical periodontal therapy alone. The present systematic review and meta-analysis suggest that the adjunctive use of melatonin, resveratrol, omega-3 fatty acids with cranberry juice, propolis, and aloe vera gel with periodontal therapy significantly improves periodontal disease parameters in patients with type 2 diabetes, and melatonin application combined with non-surgical periodontal therapy might significantly reduce periodontal pocket depth. However, there are still limited studies of melatonin in combination with non-surgical periodontal therapy in Type 2 diabetic patients, and more well-designed RCTs are required to be further investigated.

Correction: Periodontal regenerative effect of enamel matrix derivative in diabetes.

[This corrects the article DOI: 10.1371/journal.pone.0207201.].

Periodontal regenerative effect of enamel matrix derivative in diabetes.

The present study aimed to investigate the periodontal regenerative effect of enamel matrix derivative (EMD) in diabetes. Thirty-six rats were assigned to streptozotocin-induced diabetes or control (non-diabetic) groups. Three-wall intrabony defects were surgically generated in the bilateral maxilla molar, followed by application of EMD or saline. Primary wound closure and defect fill were evaluated via histomorphological analysis and micro-computed tomography. mRNA expression levels of inflammatory and angiogenic factors in the defects were quantified via real-time polymerase chain reaction. Gingival fibroblasts were isolated from control animals and cultured in high-glucose (HG) or control medium. The effects of EMD on insulin resistance and PI3K/Akt/VEGF signaling were evaluated. The achievement rate of primary closure and the parameters of defect fill were significantly higher at EMD-treated site than at EMD-untreated sites in both diabetic and non-diabetic rats, although defect fill in the diabetic groups was significantly lower in the control groups on two-way repeated-measures analysis of variance (for both, p<0.05). Newly formed bone and cementum were significantly increased at EMD-treated sites in diabetic rats than at EMD-untreated sites in control rats (for both, p<0.05). Vegf was significantly upregulated at EMD-treated sites in both diabetic and non-diabetic rats (for both, p<0.05). In vitro, insulin or EMD-induced Akt phosphorylation was significantly lower in cells cultured in HG medium (p<0.05). EMD-mediated Vegf upregulation was suppressed by the Akt inhibitor wortmannin, although the effect was significantly lower in HG medium (p<0.01). In conclusion, EMD might promote periodontal tissue regeneration via Akt/VEGF signaling, even in a diabetic condition.

High glucose-induced oxidative stress impairs proliferation and migration of human gingival fibroblasts.

Delayed gingival wound healing is widely observed in periodontal patients with diabetes. However, the molecular mechanisms of the impaired function of gingival fibroblasts in diabetes remain unclear. The purpose of this study was to investigate changes in the properties of human gingival fibroblasts (HGFs) under high-glucose conditions. Primary HGFs were isolated from healthy gingiva and cultured with 5.5, 25, 50, and 75 mM glucose for 72 h. In vitro wound healing, 5-ethynyl-2'-deoxyuridine (EdU), and water-soluble tetrazolium salt (WST-8) assays were performed to examine cell migration and proliferation. Lactase dehydrogenase (LDH) levels were measured to determine cytotoxicity. The mRNA expression levels of oxidative stress markers were quantified by real-time PCR. Intracellular reactive oxygen species (ROS) were also measured in live cells. The antioxidant N-acetyl-l-cysteine (NAC, 1 mM) was added to evaluate the involvement of ROS in the glucose effect on HGFs. As a result, the in vitro wound healing assay showed that high glucose levels significantly reduced fibroblast migration and proliferation at 6, 12, 24, 36, and 48 h. The numbers of cells positive for EdU staining were decreased, as was cell viability, at 50 and 75 mM glucose. A significant increase in LDH was proportional to the glucose concentration. The mRNA levels of heme oxygenase-1 and superoxide dismutase-1 and ROS levels were significantly increased in HGFs after 72 h of exposure to 50 mM glucose concentration. The addition of NAC diminished the inhibitory effect of high glucose in the in vitro wound healing assay. The results of the present study show that high glucose impairs the proliferation and migration of HGFs. Fibroblast dysfunction may therefore be caused by high glucose-induced oxidative stress and may explain the delayed gingival wound healing in diabetic patients.