Patients with diabetes frequently suffer from periodontitis, which progresses rapidly and is difficult to cure. Mesenchymal stem cell (MSC) transplantation may effectively treat periodontitis, but high glucose limits its therapeutic effect in diabetes. Nerve growth factor (NGF) has the functions of cell protection, anti-apoptosis and immune regulation, and may have potential application in diabetic periodontitis. In the present study, flow cytometry indicated that NGF inhibited MSC apoptosis induced by high glucose. Of note, high glucose promoted the transformation of MSCs into the proinflammatory type. NGF inhibited this transformation of MSCs under diabetic conditions and further decreased the proportion of T cells and monocytes/macrophages among lymphocytes. An animal model of diabetic periodontitis was constructed and MSC transplantation was demonstrated to reduce alveolar bone loss caused by diabetes. NGF enhanced the therapeutic effect of MSCs and maintained transplanted MSC survival in periodontal tissue of diabetic mice. Immunohistochemical analysis of periodontal tissues suggested that in the NGF group, infiltration of T cells and macrophages was reduced. Neurotrophic receptor tyrosine kinase 1 was indicated to have a key role in these effects of NGF. In conclusion, NGF may enhance the therapeutic effect of MSCs on diabetic periodontitis by protecting the cells and promoting the transformation of MSCs into the immunosuppressive type.
Periodontitis is a destructive disease with periodontal tissue inflammation, which seriously affects oral health. In addition to plaque and calculus, certain systemic diseases are also important contributors to periodontitis (
Mesenchymal stem cells (MSCs) may be isolated from bone marrow, fat and umbilical cord. There are numerous immunosuppressive molecules expressed on the MSCs, which have certain immune regulation functions (
Nerve growth factor (NGF) is an important nerve factor that maintains nerve growth, differentiation and axon production. NGF may reduce cerebral ischemia-reperfusion injury and promote neuronal repair (
MSCs used in the present study were human bone marrow MSCs (hMSCs) purchased from Guangzhou Saiye Biotechnology Co. hMSCs were cultured in DMEM/F12 medium (Hyclone; Cytiva) containing 10% fetal bovine serum (Hyclone; Cytiva). MSCs of the 3rd-5th generation were used for cell and animal experiments. For the
MSCs were digested with 0.25% trypsin (Hyclone; Cytiva) and seeded in 6-well plates (Corning, Inc.) at a density of 2x104 cells/well. The treatment of MSCs was as aforementioned. After 48 h of 10 ng/ml NGF stimulation, MSCs were digested with 0.25% trypsin and suspended in 100 µl PBS. Annexin V-FITC and PI (Roche Diagnostics) were added, followed by incubation at room temperature in the dark for 15 min. Binding buffer (400 µl) was added and the ratio of apoptotic cells was detected by flow cytometry (BD LSRFortessa X-20; BD Biosciences). The proportion of apoptotic cells was quantified using FlowJo software (version 7.6; BD Biosciences).
MSCs were digested with 0.25% trypsin and seeded in 96-well plates at a density of 6x103 cells/well. MSCs were treated as aforementioned. After 48 h of 10 ng/ml NGF stimulation, 20 µl MTT solution (5 mg/ml) was added to each well and the culture was continued for 4 h. After discarding the supernatant, 150 µl DMSO (Sigma-Aldrich; Merck KGaA) was added to each well. After 10 min of low-speed oscillation, the absorbance value of each well at 490 nm was measured with an ELISA plate reader.
After 48 h of NGF stimulation, MSCs were digested with 0.25% trypsin and suspended in 100 µl PBS. Toll-like receptor (TLR)4-PE (cat. no. 564215), TLR3-APC (cat. no. 565984), human leukocyte antigen G (HLA-G)-FITC (cat. no. 751657) (all purchased from BD Biosciences) were added and used as provided by the manufacturer, followed by incubation at room temperature in the dark for 15 min. After washing with PBS three times, the mean fluorescence intensity of the fluorophores for TLR4, TLR3 and HLA-G was detected by flow cytometry (BD LSRFortessa X-20; BD Biosciences). Proportion of apoptotic cells was quantified using FlowJo software (version 7.6; BD Biosciences).
After 48 h of NGF stimulation, the supernatant was discarded and fresh medium was added after washing the cells with PBS for three times. Male, eight-week-old C57BL/6 mice (Charles River Laboratories, Inc.) were raised in a suitable environment (temperature, 22˚C; relative humidity, 50%; access to food and water
All experiments were approved by the Ethics Committee of The Air Force Characteristic Medical Center (Beijing, China) and were performed in accordance with the institutional regulations. Male, eight-week-old C57BL/6 mice (Charles River) were raised in a suitable environment (temperature, 22˚C; relative humidity, 50%; access to food and water
Statistical analyses were performed using SPSS 19.0 (IBM Corp.). Values are expressed as the mean ± standard deviation. Data comparisons were performed by ANOVA and Bonferroni's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.
Flow cytometry showed that there was no significant difference in the apoptosis rate between the LG and LG+NGF groups. Compared with that in the LG group, the apoptosis rate of MSCs in the HG group was significantly increased. NGF was able to significantly inhibit the apoptosis of MSC caused by HG; compared with that in the HG group, the apoptosis rate of MSCs in the HG+NGF group was decreased by 60.98%. TrkA inhibition was able to completely block this effect of NGF (
The MTT assay indicated that there was no significant difference in the viability rate between the LG and LG+NGF groups. Compared with that in the LG group, the proliferation rate of MSCs in the HG group was significantly decreased. Compared with that in the HG group, the viability rate in the HG+NGF group was increased to 24.5% and the difference was significant. TrkA inhibition was able to completely block this effect of NGF (
As established by numerous studies, MSCs may be divided into a proinflammatory type and an immunosuppressive type (
Compared with that in the LG group, the concentration of TGF-β and IL-10 in the MSC supernatant was significantly decreased in the HG group, while the concentration of TNF-α and IL-6 was significantly increased. NGF promoted the secretion of immunosuppressive molecules from MSCs under HG conditions; compared with that in the HG group, the concentration of TGF-β and IL-10 in the HG+NGF group was significantly increased, while the concentration of TNF-α and IL-6 was significantly decreased. TrkA inhibition was able to completely block these effects of NGF (
The proportion of CD45+CD3+T cells among lymphocytes of the HG-MSC group was significantly increased compared with that in the LG-MSC group. The proportion of CD45+CD3+T cells in the HG+NGF-MSC group was 56.42%, with a statistically significant difference compared with that in the HG-MSC group. TrkA inhibition was able to completely block these effects of NGF (
In the co-culture experiment, the proportion of CD14+ monocytes/macrophages in the HG-MSC group was significantly increased compared with that in the LG-MSC group. The proportion of CD14+ monocytes/macrophages in the HG+NGF-MSC group was 9.53%, which was significantly lower than that in the HG-MSC group. TrkA inhibition was able to completely block these effects of NGF (
The results of the animal experiment are provided in
Immunohistochemical analysis of periodontal tissue indicated that compared with that in the model group, the number of T cells and macrophages was markedly decreased in the MSC group. NGF further inhibited the infiltration of T cells and macrophages. TrkA inhibition blocked these effects of NGF (
Periodontitis is a chronic inflammatory disease of periodontal tissue, with high incidence and recurrent episodes, bringing great pain to patients (
HG and high oxidative stress may damage the function of transplanted stem cells in patients with diabetes, which makes the effect of MSC therapy unsatisfactory (
Numerous studies have indicated that MSCs may have two different phenotypes. Waterman
TrkA is the major high-affinity receptor of NGF and has an irreplaceable role in the growth and development of the nervous system and the maintenance of neuronal characteristics (
In conclusion, the present study indicated that NGF is able to enhance the therapeutic effect of MSCs in diabetic periodontitis by protecting the cells and promoting the transformation of MSCs into the immunosuppressive type. The present study provided a novel agent for stem cell therapy for diabetic periodontitis.
Not applicable.
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
JP, YL and SW drafted and revised the manuscript. JP and YL designed and performed most of the experiments. SW performed the cell experiments. YX and BL performed the animal experiments. JP and YL confirm the authenticity of all the raw data. All authors discussed the results and approved the final submitted version. All authors read and approved the final manuscript.
The animal protocol was approved by the Ethics Committee of The Air Force Characteristic Medical Center (Beijing, China).
Not applicable.
The authors declare that they have no competing interests.
NGF inhibits MSC apoptosis induced by HG. (A) Flow cytometry was used to detect MSC apoptosis. (B) Compared with that in the HG group, the apoptotic rate of MSCs in the HG+NGF group decreased by 60.98%. TrkA inhibition was able to completely block this effect of NGF. (C) The proliferation rate of the HG+NGF group was 24.5% and the difference compared with the HG group was statistically significant. TrkA inhibition completely blocked this effect of NGF. Values are expressed as the mean ± standard deviation (n=6). *P<0.05 vs. HG; #P<0.05 vs. HG+NGF. TrkA, neurotrophic receptor tyrosine kinase 1; Q, quadrant; MSC, mesenchymal stem cell; NGF, nerve growth factor; HG, high glucose; LG, low glucose.
NGF inhibits the transformation of MSCs into the proinflammatory type under HG conditions. (A) TLR4, (B) TLR3 and (C) HLA-G expression was detected by flow cytometry. Quantified levels of (D) TLR4, (E) TLR3 and (F) HLA-G expression. Compared with that in the HG group, the expression of TLR3 and HLA-G in the HG+NGF group increased by 68.27% and 1.41-fold, and the expression of TLR4 decreased by 40.39%. TrkA inhibition completely blocked these effects of NGF. Values are expressed as the mean ± standard deviation (n=6). *P<0.05 vs. HG; #P<0.05 vs. HG+NGF. TrkA, neurotrophic receptor tyrosine kinase 1; MSC, mesenchymal stem cell; NGF, nerve growth factor; HG, high glucose; LG, low glucose; TLR, Toll-like receptor; HLA-G, human leukocyte antigen G; MFI, mean fluorescence intensity.
NGF promotes the release of paracrine immunosuppressive molecules from MSCs under HG conditions. The concentrations of (A) TGF-β, (B) IL-10, (C) TNF-α and (D) IL-6 were detected by ELISA. Values are expressed as the mean ± standard deviation (n=6). *P<0.05 vs. HG; #P<0.05 vs. HG+NGF. TrkA, neurotrophic receptor tyrosine kinase 1; MSC, mesenchymal stem cell; NGF, nerve growth factor; HG, high glucose; LG, low glucose.
NGF enhances the function of MSCs in inhibiting T cells under HG conditions. The proportion of CD45+CD3+ T cells in lymphocytes of the HG+NGF-MSC group was significantly decreased compared with that of the HG-MSC group. TrkA inhibition completely blocked these effects of NGF. Values are expressed as the mean ± standard deviation (n=6). *P<0.05 vs. HG-MSC; #P<0.05 vs. HG+NGF-MSC. TrkA, neurotrophic receptor tyrosine kinase 1; Q, quadrant; MSC, mesenchymal stem cell; NGF, nerve growth factor; HG, high glucose; LG, low glucose.
NGF enhances the function of MSCs in inhibiting monocytes/macrophages under HG conditions. The proportion of CD14+ monocytes/macrophages in the HG+NGF-MSC group was significantly decreased as compared with that in the HG-MSC group. TrkA inhibition completely blocked these effects of NGF. Values are expressed as the mean ± standard deviation (n=6). *P<0.05 vs. HG-MSC; #P<0.05 vs. HG+NGF-MSC. TrkA, neurotrophic receptor tyrosine kinase 1; SSC, side scatter; MSC, mesenchymal stem cell; NGF, nerve growth factor; HG, high glucose; LG, low glucose.
NGF maintains MSC survival in periodontal tissue of diabetic mice. (A) ANA was used to detect transplanted human bone marrow MSCs in periodontal tissue. Arrows indicate ANA+ cells (magnification, x100). (B) There was no significant difference in body weight between the groups. (C) Compared with the MSC group, alveolar bone loss in the NGF group decreased by 43.62%. (D) Compared with the MSC group, the number of transplanted MSCs in the NGF group increased by 2.11-fold. TrkA inhibition completely blocked these effects of NGF. Values are expressed as the mean ± standard deviation (n=6). *P<0.05 vs. MSC; #P<0.05 vs. NGF. TrkA, neurotrophic receptor tyrosine kinase 1; Q, quadrant; MSC, mesenchymal stem cell; NGF, nerve growth factor; HG, high glucose; ANA, anti-nuclear antibody; HPF, high-power field.
NGF enhances the anti-inflammatory function of MSCs in periodontal tissue of diabetic mice. Compared with that in the diabetes group, the number of CD3+ T cells and CD68+ macrophages was significantly decreased in the MSC group. NGF further inhibited the infiltration of CD3+ T cells and CD68+ macrophages. TrkA inhibition completely blocked these effects of NGF (magnification, x100). TrkA, neurotrophic receptor tyrosine kinase 1; MSC, mesenchymal stem cell; NGF, nerve growth factor.